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10.3390/ani11061505

PMC8224732

The present study was designed to investigate the role of three different light-emitting diode (LED) light color temperatures (Neutral, Cool, and Warm) on the growth performance, carcass characteristics, and breast meat quality of broilers. No significant differences were observed in carcass yield in any of the experimental conditions. The changes observed in physical and chemical properties of breast meat samples suggest that LED light was not able to modify the quality of the products; therefore, it could represent a good alternative technology to traditional light sources.

The present study was designed to investigate the role of three different light-emitting diode (LED) light color temperatures on the growth performance, carcass characteristics, and breast meat quality of broilers. In our experimental condition, 180 chicks were randomly distributed into four environmentally controlled rooms (three replicates/treatment). The experimental design consisted of four light sources: neon (Control), Neutral (Neutral LED; K = 3500–3700), Cool (Cool LED; K = 5500–6000), and Warm (Warm LED; K = 3000–2500). Upon reaching the commercial weight (3.30 ± 0.20 kg live weight), 30 birds from each group were randomly selected, and live and carcass weight were evaluated to determinate the carcass yield. Following the slaughtering, samples of hemibreast meat were collected from each group and analyzed for physical and chemical properties, fatty acids composition, and volatile compounds. Live weight and carcass weight were negatively influenced by the Warm LED; however, no significant differences were observed in carcass yield in any of the experimental conditions. Higher drip loss values were detected in breast meat samples obtained by broilers reared under Neutral and Cool LEDs. In regard to the meat fatty acids profiles, higher polyunsaturated fatty acids (PUFA) values were detected with the Warm LED; however, the ratio of PUFA/saturated fatty acids (SFA) did not change in any group. The evaluation of volatile profiles in cooked chicken meat led to the identification of 18 compounds belonging to the family of aldehydes, alcohols, ketones, and phenolic compounds, both at 0 (T0) and 7 (T7) d after the cooking. The results of the present study suggest that the LED represents an alternative technology that is cheaper and more sustainable than traditional light sources, since it allows economic savings for poultry farming without significant alterations on the production parameters or the quality of the product.

1. IntroductionIn addition to animal diet and genetics, the farming system is one of the factors that most influences the conversion efficiency of feed and therefore the production performance of poultry. The light (intensity, photoperiod, and wavelength) has direct effects on the behavior, physiology, immunity, and performance of poultry [1,2]. Artificial lighting is a fundamental tool used in poultry production that aims to improve food and water intake and consequently the growth and the economic yield of poultry [3]. The Council of the European Union has established that in poultry farms, the lighting must follow a 24 h rhythm and include periods of darkness lasting at least 6 h in total, with at least one uninterrupted period of darkness of at least 4 h, excluding dimming periods [4]. The impact of various intensities and wavelengths on the production performance of chickens has been extensively studied in recent decades; this has led to testing in commercial farms of various lighting systems, such as incandescent and fluorescent lamps. Recently, light-emitting diode (LED) lamps have gained increasing interest in poultry businesses due to their high energy efficiency, long operating life, availability in different wavelengths, low electricity consumption with consequent reduction of CO2 emissions into the atmosphere, and low farming costs [5]. Many studies have been conducted to evaluate the effect of monochromatic light produced by LED lamps on the production performance and quality of chicken meat, and although a wide range of colors is available, there are conflicting reports on the impact of different colors on production performance. It has been shown that LEDs, compared to other light sources, are able to reduce stress and fear in broilers [6]. Broilers reared under green or blue light showed significantly more body weight gain and muscle development compared to those reared under white and red light [1,7,8]. Cao et al. [7] correlated the increase of body growth with the stimulation of testosterone secretion and myofiber growth. In addition, an increase in body weight and carcass yield has been found when breeding poultry in the presence of LEDs, possibly due to muscle hypertrophy induced by the production of testosterone. According to a study of Parvin et al. [5], LEDs that emit blue, green, and yellow wavelengths are able to improve the immune system and meat quality in broilers. Blue and green light helps promote higher antibody production than red light. Poultry reared under mixed yellow and green-blue lights showed softer breast musculature, while white light resulted in increased amino acid content [5]. In a study carried out on chickens subjected to two-color green-blue LED lights for 81 d, chicks exposed to LED (green-blue) light gained weight compared to chicks exposed to normal artificial light without resulting changes in blood biochemical parameters. Moreover, in chickens under mixed green-blue light, the carcass yield and food conversion index were significantly higher than in chickens under single LED light [2]. Different light spectra produced by LED lights of 2700 K (Warm) or 5000 K (Cold) can influence the production, stress, and behavior of broilers; cold LEDs can reduce stress and fear, increasing weight and food conversion index [9].At present, nutrient composition and meat quality characteristics of poultry products are widespread concerns by consumers. The quality of poultry products varies with growth rate and body composition [10]. Light has an influence on growth, since it can alter the structure of muscle myofiber, enhancing myoblast proliferation [11,12]. There is illimited information regarding LEDs and their impact on meat quality. However, there is no information in the literature on whether monochromatic light stimuli that showed growth-promoting effects can also influence breast meat composition and subsequent meat quality. Thus, the aim of this study was to investigate the effect of LED lights with three different color temperatures, Neutral (K = 3300–3700), Cool (K = 5500–6000), and Warm (K = 3000–2500), on production performance and breast muscle meat quality of broilers. It was concluded that the three light sources evaluated in this study may be suitable to replace the traditional light source in poultry facilities to reduce energy costs and optimize production efficiency.2. Materials and Methods2.1. Experimental Design and Samples CollectionThe experimental test was carried out on a farm located in the Abruzzo region (Italy) that had adopted LED technology in breeding practices. All the procedures concerning the animals’ management were carried out in accordance with the European directive 2007/43/EC for the protection of chickens kept for meat production [4]. During the trial, no breeding practices other than those normally adopted were introduced; therefore, approval by the ethics committee was not considered necessary. The company made available 4 boxes, in one of which the conventional lighting system based on the use of neon was maintained (Control), and in the other three of which LED lights were installed in Neutral (Neutral LED; K = 3500–3700), Cool (Cool LED; K = 5500–6000), and Warm (Warm LED; K = 3000–2500) shades, respectively. The broilers (Ross 508, Aviagen Group, Huntsville, AL, USA) were exposed to light according to the European directive (6 h of darkness and 18 h of light). Male chicks, offspring of hens around 35 weeks of age, were vaccinated by local application for Marek’s disease, infectious bronchitis, Newcastle disease, and Gumboro disease. A total of 180 chicks were randomly divided into 4 groups (45 chicks per group) in 4 environmentally controlled rooms. Each room was divided in 3 replicates with 15 birds in each replicate. The production cycle lasted until the animals reached the commercial weight (3.30 ± 0.20 kg live weight). During the entire period, the animals were reared in accordance with the protocols normally applied by the company for the production of heavy chickens. All birds were fed the same diet throughout the study. Birds were provided a 3-phase feeding program (starter: 1 to 12 d; grower: 13 to 21 d; finisher: 22 to 48 d of age), the chemical characterization of which is reported in Table 1. Feed conversion ratio (FCR) could not be calculated because the boxes were equipped with a single silo from which the food was evenly distributed. At the end of the normal production cycle, 30 chickens from each experimental group were randomly selected, weighed, and slaughtered in the presence of the responsible veterinarian and in accordance with current regulations in terms of animal welfare. After evisceration, the carcasses were weighed in order to determine the yield. After 24 h, during which the carcasses were stored at 4 °C and covered by a synthetic film in order to avoid exposure to the surrounding environment, the hemibreast meat was sampled. Part of the meat was immediately used for the determination of pH, moisture, color, and drip loss, while the remainder was frozen at −20 °C for subsequent analysis (total lipids and fatty acid profile). The same samples used for drip loss, after 24 h, were used for cooking loss. The cooked meat was sampled at defined intervals to evaluate the volatile profile immediately after cooking (T0) and after 7 d (T7) stored at 4 °C.2.2. Evaluation of Breast Meat ColorThe pH evaluation on chicken breast samples 24 h (pH24) after slaughtering was performed by using a portable pH meter equipped with an electrode (Crison, Barcelona, Spain) that was inserted about 1–1.5 cm into the tissue, adjusting each evaluation in relation to the muscle temperature. Before the analysis, a calibration of the instrument was performed by using standard phosphate buffers (pH 4.00 and 7.00), and at the end of each measurement, the electrode was carefully rinsed in distilled water before the next evaluation. Color measurements were determined according to the procedure reported by Bennato et al. [13]. Briefly, the analysis was performed on the transverse section of the chicken breast muscle by using the Minolta CR-5 reflectance colorimeter. All evaluations were carried out taking into account the CIELAB system, which exploits the chromaticity coordinates L* (lightness), a* (redness), and b* (yellowness). Before each series of measurements, the colorimeter was calibrated by using a white tile (L* = 100) and black glass (a* = 0).2.3. Drip Loss, Cooking Loss, and Chemical Composition of Breast MeatIn order to determinate the capability of meat to retain water, meat samples of about 2–2.5 cm of thickness and approximate weight of 100 g were inserted inside an expanded and closed bag and stored at 4 °C for 24 h. The meat was weighed at the beginning and end of this time period, and the drip loss was expressed as a percentage of the initial sample weight. The cooking loss, useful to characterize the ability of meat to retain water during cooking, was evaluated on the same meat samples used for drip loss. Meat samples were weighed and cooked in a water bath until the core temperature of 70 °C was reached. Samples were then cooled at room temperature and weighted. The cooking loss was expressed as a percentage of the initial raw sample weight. The evaluation of meat moisture and fat content was made according AOAC methods (2000) [14].2.4. Fatty Acids Profile of Breast MeatFatty acids were extracted by using the Folch method (1957) [15]. Approximately 5 g of meat was homogenized by using Ultra-turrax-T25 with 45 mL of Folch solution (Chloroform: methanol, 2:1). Subsequently, the homogenized samples were transferred into a flat bottom flask and stirred in the dark for 7 h at room temperature. All samples were transferred into a separating funnel with the addition of 15 mL of 1% NaCl and left overnight. Total fat for each sample was obtained through the chloroform phase evaporation to dryness by using a Strike-Rotating Evaporator set at 40 °C. For each sample, the formation of fatty acid methyl esters (FAME) was induced by mixing 60 mg of fat with 1 mL of hexane and 500 µL of sodium methoxide. Detection of FAMEs was performed by a gas chromatograph (Focus GC; Thermo Scientific, Waltham, MA, USA) equipped with a capillary column (Restek Rt-2560 Column fused silica 100 m × 0.25 mm highly polar phase; Restek Corporation, Bellefonte, PA, USA) and a flame ionization detector (FID). Hydrogen was used as carrier gas. The initial holding temperature was 55 °C for 1 min; then it was increased to 170 °C at a rate of 10 C/min and held for 30 min. The final temperature of 215 °C was reached at a rate of 2 °C/min and held for 4 min. Peak areas were quantified using ChromeCard (Thermo Fisher Scientific, Milan, Italy) software, and the relative value of each individual FA was expressed as a percentage of the total FAME. The value of each FA was used to calculate the sum of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acid (PUFA).2.5. Determination of Volatile Components of Cooked Breast MeatVolatile compound (VOC) evaluation was performed with a gas chromatograph (Clarus 580; Perkin Elmer, Waltham, MA, USA) coupled with a mass spectrometer (SQ8S; Perkin Elmer, MA, USA) and equipped with an Elite-5MS column (length × internal diameter: 30 × 0.25 mm; film thickness: 0.25 µm; Perkin Elmer). Briefly, 3.5 g of minced cooked meat was mixed with 10 mL of an aqueous solution of NaCl (360 g/L) and 10 µL of internal standard (3-methyl-2-heptanone; 10 mg/kg in ethanol) and exposed to SPME fiber (divinylbenzene-carboxy-polydimethylsiloxane in solid phase; length: 1 cm, film thickness: 50/30 µm; Sigma-Aldrich, Milan, Italy) for 60 min at 60 °C. Then, the extracted VOCs were thermally desorbed in GC–MS. The thermal program and the recognition of the individual VOCs were performed as previously described by Ianni et al. [16].2.6. Statistical AnalysisAll the analyses were performed on 15 animals per group (5 samples for each replicate), randomly selected, and the analysis on the single sample was performed in triplicate. Results were reported as mean values with the correspondent standard deviations (SD). The statistical analysis was performed by using SigmaPlot 12.0 Software (Systat software Inc., San Jose, CA, USA) for windows operating system. The ANOVA model was applied, and the post-hoc comparison was performed through Tukey’s test; p values lower than 0.05 were considered statistically significant.3. Results3.1. Production and Physical ParametersIn Table 2, the productive parameters and the chemical characteristic of breast meat samples are reported. The live weight evaluations carried out at the end of the production cycle showed overlapping values among the Control, Neutral, and Cool LEDs. Significant variations were observed between Control and Warm LED (p < 0.01) and Cool and Warm LED (p < 0.05). A lower carcass weight was detected in the Warm LED group than in the Control group (p < 0.05). However, no significant variations among the groups were found in evaluations of carcass yield. Compared to the Control, the exposure to Neutral (p < 0.05) and Cool LEDs (p < 0.05) increased the ability of fresh meat samples to retain water. On the contrary, no significant differences were detected in meat samples exposed to Warm LED compared to the other treatment. The cooking loss values closed to 15–17% in all samples. Color evaluations showed a lower brightness in samples of meat belonging to chickens exposed to Warm LED compared to the other groups. Samples obtained from chickens reared under Neutral LED showed higher a* values compared to those from the Control. Within the groups exposed to different LEDs, significant chromatic variations in redness were observed between Neutral and Warm LEDs. A higher b* value was found in Neutral LED samples than in those from the other groups. Significant variations were observed in moisture between Neutral LED and Control. The lipid content of breast meat was found to be unaffected in all LED groups.3.2. Fatty Acid ProfileThe total lipid percentage is reflected in scarce variation in fatty acids profile (Table 3). The use of LEDs did not induce significant changes in the total content of SFA and monounsaturated fatty acids (MUFA). On the contrary, the exposure to Warm LED induced an increase of PUFA (p < 0.05) respect to the Control. In the meat samples belonging to the Neutral LED group, a significant increase compared to the Control was observed in cis-vaccenic acid (C18:1, cis11).3.3. Volatile Profile of Cooked MeatThe evaluation of volatile profile in cooked chicken meat led to the identification of 18 compounds (both at T0 and T7) belonging to the family of aldehydes, alcohols, ketones, and phenolic compounds (Table 4). In all groups, the most represented compound was hexanal, the concentration of which did not significantly change between the groups at either T0 or to T7. Among alcohols, a significant decrease in 1-Octen-3-ol was recorded in T0 samples obtained from chickens reared under Neutral LED compared with the Control (p < 0.01). After 7 d of storage, compared to the Control, a significant increase of 1-Pentanol (p < 0.05) was detected in Neutral LED samples; conversely, in Cool LED samples, 1-Octanol (p < 0.05) and 1-Octen-3-ol (p < 0.05) decreased. Regarding ketones, significant decreases related to LED light exposure were observed for all identified compounds. In particular, significant variations were observed in 2-Heptanal (p < 0.05) between the Cool and Warm groups, in 1-Octenal (p < 0.05) between the Neutral and Warm groups, in 2-Hexanone, 4-methyl (p < 0.05) between the Control and Neutral groups, and in 3-Octanone, 2-methyl (p < 0.05) between the Control and Warm groups. On the contrary, after 7 d, 1-Octenal differed between Cool and the other groups, and 3-Octanone, 2-methyl differed between Control and Cool and Control and Warm. No significant changes within the groups were observed for the phenolic compounds.4. DiscussionOne of the biggest challenges in broiler production is to achieve maximum production while reducing energy consumption and production costs. Artificial lighting is a fundamental tool used in poultry production that aims to improve food and water intake and consequently the growth and the economic yield of poultry. It has been shown that the use of LED light in poultry farms has numerous advantages linked to greater energy savings, greater luminous efficiency, and a reduction in environmental impact that can nevertheless improve the production parameters of broilers.Our study showed that Neutral and Cool LED light did not determine changes in broiler growth; on the contrary, broilers reared under Warm LED had a lower live weight than those reared under the Control and Cool LED, though there was no influence on the carcass yield. Previous studies carried out on broiler growth performance and development have showed discordant results [9,17,18]. Archer (2017) [9] observed that broilers raised under Cool LED (K = 5000 K) grew to a heavier weight at the end of 42 d than birds raised under Warm LED (K = 2700). Moreover, Cool birds had better FCR than Warm birds. These results demonstrate that raising broilers under 5000 K LED lights can reduce their stress and fear and increase weight gain when compared with 2700 K lights. In Arbor Acres broilers, blue monochromatic light increased body weight and carcass yield compared with red, white, and green light [19]. On the contrary, ducks reared under red light showed an increase of body weight, body weight gain, and feed intake compared to those reared under yellow light [20]. In broilers exposed for two weeks to LED with different wavelengths, it has been demonstrated that green light increased the mRNA and protein levels of growth hormone-releasing hormone (GHRH) in the hypothalamus as well as plasma growth hormone (GH) concentrations by activating the secretion of plasma melatonin, which plays a key role in photoelectric conversion [21]. These findings suggest the capability of light to modulate gene expression. The differences observed in our experimental condition may be due to the use of differing LED light sources, varying light intensities, time of exposure, and animal species.Beyond broiler performance and meat yield, an important aspect, especially for consumers, is meat quality. Therefore, in our study, we evaluated the possible effects of LED light on quality properties of breast meat such as water-holding capacity, cooking loss, lightness, fat content, and the fatty acids profile and volatile profile of cooked meat.The exposure to LED affected the ability of raw meat to retain water (drip loss); Neutral and Cool LED meat samples showed a greater weight loss compared to both the Control and Warm LED samples. In a study conducted by Wang et al. [22], an association was demonstrated in goat longissimus dorsi muscle between high drip loss values and low levels of metabolic enzymes (α-enolase, NADH dehydrogenase, pyruvate dehydrogenase), stress response factors (Hsp27), and structural proteins (myosin) that affected glycolysis, oxidation, and muscle contraction. The authors showed that the correlation between the declines in α-enolase, NADH dehydrogenase, and pyruvate dehydrogenase levels and higher drip loss may be due to an increase of glycolysis and accumulation of lactic acid in muscle tissues with consequent lowering of pH. The pH decline to the pI value of myosin reduces the distance between thick and thin myofilaments and the sarcomere length [23], allowing water in the myofibril gap to flow out with the consequence of an increase of drip loss values. However, in our experimental condition, the increase of drip loss values observed in Neutral and Cool LED was not associated to a decrease in pH, which may owe to different characteristics of the myofiber of breast muscle. The relationship among muscle fiber characteristics, size, types with post-mortal biochemical processes, and meat quality is well documented in different animal species [24,25]. In the longissimus dorsi muscle of bulls, a correlation has been observed between muscle fiber area and water-holding capacity indicating that muscles with larger fiber areas had a lower drip and ageing loss but a higher cooking and grilling loss [26]. In a study by Cao et al. [7] carried out on broilers reared under different light spectra, it was observed that the cross-section area and density of myofibers changed among various groups, and the myofiber area of blue-light-exposed broilers was larger than that of the other group, suggesting that light could influence the myofiber growth in broiler skeleton muscle and consequently water holding capacity. No significant change among the groups was observed in cooking loss, and it may be correlated to the same fat content in breast muscle samples of the different treatment. In duck breast meat, higher cooking loss values have been observed in breast muscle containing high lipid levels [27].Meat color greatly influences choices made by the consumer. The factors that can influence the color are the content of pigments (myoglobin and hemoglobin), the time elapsed since slaughter, the processing and storage conditions, the muscle section considered, the cutting direction, the fat, the state of hydration, and the species, sex, and age of the animal. In this study, significant differences were evidenced among the different treatments for lightness (L*) and for chromaticity coordinates a* (redness) and b* (yellowness). The normal color of raw chicken breast is slightly pinkish but can also appear bluish-white to yellow; it depends on the concentration and the chemical and physical state of myoglobin and also on the structure of the meat surface. In all the groups, meat samples had low values of lightness with respect to values reported in literature; however, as reported by Petracci et al. [28], color variation of broiler breast meat could be affected by the age and season of broiler slaughter. Our data showed a lower L* value in Warm LED breast meat samples than in other groups. Our data are in contrast with other studies that showed higher L* values in the breast of broilers exposed to red light than in white, green, and blue light [19]. Napper et al. [29] found heat stress to cause a significant increase in the lightness of broiler chicken meat compared with a cold stress treatment. Schneider et al. [30] reported that meat from the hot and thermo-neutral treatments was lighter in color than that from the cold group. An increase in the L* value is associated to the denaturation of myofibrillar proteins, followed by aggregation, consequently changing the surface reflectance and increasing lightness [31]. Therefore, a lower lightness in the Warm breast sample may be associated to a different composition in myofibrillar proteins. Regarding the a* and b* parameters, differences were observed among all the groups. Meat redness is associated to the oxidation state of hemoglobin; a decrease of redness is attributed to a large degree to the oxidation of the bright red oxymyoglobin or the purplish deoxymyoglobin into the brownish metmyoglobin, as well as to the denaturation of myoglobin. In research by Lindahl et al. [32] on pork meat, it was evidenced that about 86–90% of variations in meat lightness, redness, yellowness, and chroma (saturation) can be explained by variations of pigment content, myoglobin forms, and reflectance of internal surface. Specifically, lightness, redness, and chroma appear to be influenced to a similar extent by both the pigment content and the myoglobin forms, while yellowness seems to vary mostly in relation to the myoglobin forms and less to internal reflectance, without significant effects induced by pigment content. For this reason, the observed finding deserves further and more specific assessments. Broilers reared under Neutral LED had a significant increase of cis-vaccenic acid (C18:1, cis 11). Cis-vaccenic acid can derive from the diet or biosynthetic pathways, but its role in metabolism is largely unknown. In mammalians, it has been confirmed that 18:1, cis-11 is an elongation product of 16:1 by the ELOVL6 isoform of the mammalian elongase enzyme, which adds an acetate molecule to the carboxylic acid end of the fatty acyl chain [33]. An increase of total PUFA was observed in meat of broilers reared under Warm light; however, the ratio of PUFA/SFA did not change in any of the different experimental conditions. There are few reports of the effects of light color on muscular fatty acid composition. Kim et al. [34] reported that red light LED increased the concentration of MUFA, SFA, and the SFA/PUFA ratio, but reduced the concentration of PUFA, n-3 fatty acid, and n-6 fatty acid, and it is still unclear exactly how light color alters meat fatty acid composition. It is possible to hypothesize an influence of light on gene expression; in fact, exposure to light can alter metabolic function, and many genes involved in nutrient metabolism display rhythmic oscillations. Therefore, further study is required to evaluate the influence of light on fatty acid metabolism in broilers.The characterization of volatile compounds in foods represents an aspect of pivotal interest for understanding the biochemical mechanisms responsible for the accumulation of flavor determinants. These mechanisms are mostly associated with the degradation of the lipid and protein components and are very often mediated by endogenous enzymatic forms or by oxidative events induced by the heating treatments used for cooking. With specific regard to cooked meat, the accumulation of volatile flavor compounds is generally due to reactions that concern above all thermal lipid degradation and Maillard–lipid interactions [35]. Obviously, the accumulation of certain VOC depends on the type of substrate present in raw meat, which in turn is a function of several pre- and post-slaughter factors, including breed, feeding strategy, post-mortem carcass conservation, and cooking method.In this study, 18 VOC were detected in cooked meat samples obtained from all the experimental groups. The majority of these compounds were aldehydes, alcohols, and ketones, i.e., chemical families mainly deriving from the lipolytic process. Such findings are therefore inserted in a context of compliance with respect to what has been reported in other studies, in which the main substrate for the production of flavor compounds in cooked poultry meat is highlighted in the lipid component [36]. Immediately after cooking and after 7 d of storage of the cooked product, the most represented compound was hexanal. This finding was quite expected, since hexanal is a volatile compound characteristic of cooked poultry meat [37]. Furthermore, hexanal is also generally considered a marker of lipid oxidation, and the lack of variations between the different analyzed conditions testifies to the fact that the lighting program did not induce effects from this point of view. Specifically, this may be due to the presence of similar contents of linoleic acid (C18:2), which is the main substrate for hexanal production. A compound to which significant differences were associated among the various experimental groups was 1-octen-3-ol, belonging to the alcohols group. This compound provided fishy, fatty, mushroom, grassy odors, and was mainly derived from enzymatic reactions mainly catalyzed by lipoxygenases and hydroperoxide lyases. High values of this compound have been associated with marked oxidative processes, so it is telling that immediately after cooking, the samples obtained following the breeding with Neutral and Warm LED were found to be poorer in this compound. In a study conducted by Mielnik et al. [38] on cooked turkey meat, a direct proportionality was highlighted between the 1-octen-3-ol concentration and the presence of thiobarbituric acid-reactive substances (TBARS). Therefore, cooked meat low in this compound was associated with better resistance to lipolytic processes, with a consequent improved chemical stability of the product over the storage period. With regard to meat samples analyzed after storage, the data was confirmed only for samples obtained from the treatment with Warm LED, which therefore tends to confirm itself as the best condition for limiting the oxidative processes.Exposure to Neutral and Warm LEDs also proved effective in limiting the accumulation of ketones immediately after cooking, while the treatment with Cool LED produced values comparable to those obtained from the Control group. These compounds are reported to directly derive from hydroperoxides, which are considered to represent the first oxidation products [39,40]. Hydroperoxides are described to be odorless, with a slight possibility of contributing to meat aroma; however, the ketone accumulation was instead associated with the onset of off-flavors and off-odors. The data obtained in the presence of treatment with Neutral and Warm LED were therefore confirmed of potential interest, although it should be considered that after 7 d of cooked meat storage this evidence disappeared, obtaining values comparable to those of the Control group.5. ConclusionsThe application of three shades of LED lights in broilers did not result in significant changes in carcass yield. Even the parameters evaluated on the breast muscle did not reveal any changes that could justify a qualitative alteration of the product. The most interesting data concerns the volatile profile characterized on cooked meat samples. This approach indicated an improvement, albeit very slight, in the oxidative stability of the samples obtained from the Neutral LED group. These changes in the volatile profile are also generally associated with variations in the aroma and taste of food products, so it would be desirable to develop sensory assessments, based above all on the organization of panel tests, in order to better characterize this aspect. Overall, therefore, the application of LED light in broiler breeding has led to production that is qualitatively in line with the company high standards but more sustainable both from an economic and environmental point of view, since LED is a technology with less impact on energy consumption and therefore on the release of CO2.

animals : an open access journal from mdpi

10.3390/ani11113110

PMC8614460

Hot weather is associated with reduced milk yield of dairy cows. Supplementing the diet of lactating cows with ingredients that increase dietary energy density or that reduce internal heat production, may reduce some of the negative impacts of hot weather on milk yield. We used controlled-climate chambers to simulate a short hot-weather event and measured changes in milk yield, feed intake, and body temperature of cows fed either a fat supplement, betaine or a combination of both. Feeding cows fat resulted in improved milk production but also increased body temperature and caused a decrease in feed intake. Feeding betaine did not affect milk yield but did reduce cow body temperature at times. Contrary to our expectations, the combination of fat and betaine supplements did not result in a clear benefit in terms of milk production or body temperature. Further work is warranted to understand the interactions between dietary fat type and betaine supplements when offered to cows during periods of hot weather.

Supplementing the diet of lactating cows with ingredients that increase energy density, or reduce internal heat production, may reduce some of the negative impacts of hot weather on milk yield. Thirty-two dairy cows were assigned either: (1) basal diet only, (2) basal diet plus canola oil, (3) basal diet plus betaine, or (4) basal diet plus canola oil and betaine. The basal diet was lucerne hay, pasture silage, and grain. Cows were exposed to a four-day heat challenge (temperature-humidity index 74 to 84) in controlled-environment chambers. Canola oil supplementation increased milk production (22.0 vs. 18.7 kg/d) across all periods of our experiment and increased body temperature (39.6 vs. 39.0 °C) during the heat challenge. Betaine supplementation reduced maximum body temperature during the pre-challenge period (39.2 vs. 39.6 °C) but not during the heat challenge (40.3 °C). Cows fed canola oil had greater declines in dry matter intake (5.4 vs 2.7 kg DM) and energy corrected milk (1.3 vs. 1.0 kg) from the pre-challenge to the heat challenge than other cows. Contrary to our expectations, the combination of fat and betaine supplements did not result in a clear benefit in terms of milk production or body temperature. Further work is warranted to understand the interactions between diet and hot weather.

1. IntroductionHigh ambient temperature and humidity can result in both short- and long-term effects on the milk production, reproduction and health of dairy cattle [1,2,3]. This is important for the dairy industry given the intensity, frequency and duration of heat waves are forecast to increase [4].Feed intake of dairy cows is driven in part by their milk production [5]. High-producing cows are more susceptible to hot weather than low-producing cows due to greater internal heat production resulting from the digestion and metabolism of large amounts of feed eaten [3]. Hot weather causes a reduction in milk yield, with up to 50% of this reduction attributed to reduced dry matter intake (DMI) [6,7]. Increasing the fat concentration in the diet of dairy cows has been proposed as a way of reducing the heat load of cows by enabling them to have lower body temperatures during periods likely associated with heat stress [8]. Compared to other feeds, fat has a lower heat increment—the heat produced as a result of its digestion and metabolism [3,8]. The gross-energy density of fat (~39 MJ/kg DM) is greater than that of either protein (~24 MJ/kg DM) or carbohydrate (~18 MJ/kg DM) [9]. The gross-energy density of most grazed and conserved forages in Australia during summer is ~18 MJ/kg DM, with fat concentration in forages generally being less than 40 g/kg DM [10]. In contrast, canola oil contains approximately 990 g fat/kg [11]. Thus, supplementing a forage-based diet with a fat supplement can increase the energy density of the diet without reducing ruminal fermentation or voluntary DMI, providing the dietary fat concentration does not exceed 70 g/kg DM [12]. Furthermore, dairy cows offered high-fat diets during hot weather produced more energy-corrected milk (ECM) than cows offered low-fat diets [13,14]. This suggests that diets containing a high concentration of fat may reduce the negative impact that heat events have on milk production, while also limiting increases in body temperature due to lower heat of fermentation, digestion and metabolism of feed. Addition of betaine to the diet of animals can increase their resilience to heat exposure, with several modes of action on metabolism having been proposed [15]. Betaine (trimethylglycine) is a modified amino acid with three methyl groups that can act as methyl donors in various metabolic pathways. Observed effects when added to the diets of animals include reducing the incidence or severity of dehydration, reducing osmotic damage to tissues, reducing intracellular accumulation of Na+, reducing energy depletion within cells, and reducing protein denaturation. A reduction in each of these mechanisms reduces cellular damage and, perhaps most importantly, reduces the chance of endotoxins escaping from the gut into the body [15]. Betaine has been fed to sheep [16], dairy cattle [17,18,19], rabbits [20], and beef cattle [21], experiencing heat events with a variety of responses. There is little evidence that betaine directly offsets the physiological effects of hot conditions, but an animal with reduced organ and cellular damage is expected to recover faster once the heat load is removed. No reports were found detailing the effects of feeding betaine in the recovery period after heat exposure. Feeding fat and betaine together could be one way to maintain the milk production of cows during hot weather and minimize the physiological effects of heat stress. We were unable to find any reports of feeding a fat supplement in combination with betaine to ruminants. However, there is evidence from studies using mice that offering betaine in a high-fat diet is beneficial [22,23]. Offering cows a fat supplement during periods of heat exposure is expected to improve milk production [13,14] and reduce body temperature [8]. Offering betaine is also expected to improve milk production [17] and reduce body temperature [16]. Therefore, it is plausible that the effects of feeding fat and betaine together are additive. The objectives of this research were to determine the effects of adding canola oil (as an example fat) or betaine or canola oil plus betaine to the diet, on the DMI, milk production and body temperature of lactating dairy cows exposed to heat.We hypothesized that compared to a non-supplemented diet, (1) supplementing the diet of dairy cows with fat before and during a heat challenge would result in smaller declines in DMI and ECM yield and a smaller increase in body temperature during the heat challenge, and (2) supplementing the diet of dairy cows with betaine before and during a heat challenge would result in smaller declines in DMI and ECM yield and a smaller increase in body temperature during the heat challenge. We also hypothesized that (3) during a heat challenge, cows given fat and betaine in combination would exhibit greater DMI and ECM yield and lower body temperature than cows given either alone. 2. Materials and MethodsThe experiment was conducted at the Agriculture Victoria Research, Ellinbank Research Centre, Victoria, Australia (38°14′ S, 145°56′ E).2.1. Cows and DietsThirty-two multiparous, lactating Holstein-Friesian cows producing 18.6 ± 2.37 kg milk/d (mean ± standard deviation) with 566 ± 47.1 kg body weight (BW), 216 ± 18.5 days in milk (DIM), 2.7 ± 0.79 parity and 101 ± 2.9 heat tolerance breeding value (DataGene, Bundoora, Victoria, Australia; 100 = national breed mean) were assigned one of 4 diets on a daily basis (1) BASE—basal diet only, (2) CAN—basal diet plus 0.7 kg canola (Brassica napus L.) oil, (3) BET—basal diet plus 16 g betaine (trimethylglycine as a powder; Feedworks, Romsey, Victoria, Australia), or (4) CB—basal diet plus 0.7 kg canola oil and 16 g betaine (trimethylglycine). The daily basal diet was a total mixed ration (TMR) comprised of 7 kg DM lucerne hay (Medicago sativa L.), 6 kg DM pasture silage (predominantly perennial ryegrass, Lolium perenne L.), 5.0 kg DM grain mix (500 g/kg wheat grain (Triticum aestivum L.), 500 g/kg barley grain (Hordeum vulgare L.)), 1.5 kg DM solvent-extracted canola meal, 0.2 kg DM of minerals and vitamins (Ca 134 g/kg, Mg 110 g/kg, P 60 g/kg, Zn 6.4 g/kg, Mn 2.4 g/kg, Cu 1.2 g/kg, I 80 mg/kg, Co 100 mg/kg, Se 24 mg/kg, Vitamin A 165 IU/g, Vitamin D3 24 IU/g, Vitamin E 800 mg/kg), 0.1 kg DM salt, and 42 mL of Bloat Drench (271 g/L alcohols, C12-15 ethoxylated; VicChem, Coolaroo, Victoria, Australia). The compositions of the main dietary ingredients are shown in Table 1. Betaine doses were wrapped in ~50 g DM of silage, then offered by hand to individual cows on betaine treatments prior to the bulk of the ration being offered. Canola oil was incorporated into the CAN and CB rations by pouring it over the feed and mixing the ration by hand. 2.2. Experiment DesignAll cows were familiarized with the experimental environments prior to the experiment by feeding them in each of those environments for seven days. The cows were managed in four cohorts of eight cows. Each treatment was assigned at random to two cows within each cohort, while simultaneously balancing treatment groups for BW, milk yield, DIM and heat tolerance breeding value using covariate design software in Genstat 19 (VSN International Ltd., Hemel Hempstead, UK). Six of the eight cows in each cohort were assigned to six controlled-climate chambers for heat challenge according to a row-column design with cohorts as rows and chambers as columns. Each diet treatment appeared once in each chamber and once or twice in each cohort. The two cows not exposed to the heat challenge in each cohort were selected as a different pair of treatments in each cohort and were available as spares for use in the event of loss for any extraneous reason, such as mastitis or lameness. Cohorts were staggered in time to ensure that all animals entered the heat challenge after the same number of days on the diet.During the covariate period (days 1 to 7) cows were managed as a single group under ambient conditions in a paddock with measurements made of milk yield and composition, and BW. During this time, cows were offered 5 kg DM/day of crushed wheat grain during milking and approximately 15 kg DM/day of lucerne hay per day in a paddock with negligible pasture available.The adaptation period was commenced on day 8. Cows were moved to the experiment facilities where they were fed in individual feed stalls for two periods of 3.5 h each within a well-ventilated animal house [24] and rested on a loafing pad covered with wood chips. Cows were transitioned to their treatment diets over three days (days 8 to 10), then adapted to those diets for a further 11 days (days 11 to 21). Pre-challenge measurements were recorded for three days (days 22 to 24) while cows were housed in ambient conditions. The heat challenge was conducted for up to four days (days 25 to 28). Six cows in each block were individually exposed to the heat challenge in controlled-climate chambers [25]. During the heat challenge, the targeted conditions were 30 °C and 50% RH (temperature-humidity index, THI = 80) from 0601 to 1200 h; 33 °C and 50% RH (THI = 84) from 1201 to 1800 h; and 25 °C and 60% RH (THI = 74) between 1801 to 0600 h. If an individual cow’s rectal temperature was greater than 40.9 °C (the limit approved by the animal ethics committee), the cow was cooled by opening the chamber doors and adjusting the conditions in the chamber to thermoneutral (17 °C, 60% RH). Cows were also cooled if, in the opinion of the investigators, continuing in the heat challenge would be detrimental to the health of the cow. Individual cows thus cooled stayed within their chamber at thermoneutral conditions for the remainder of their scheduled time in the chamber. Only data from cows completing two or more days of the heat challenge were included in the analysis.Cows that were cooled had their recovery monitored from the day chamber temperature was reduced. The recovery of cows completing the four-day heat challenge was monitored under ambient conditions for 7 d (days 29 to 35). 2.3. Feeding and Feed AnalysisFeed was offered in two equal portions immediately following the morning and afternoon milkings. Dry matter concentration was determined on samples of grain mix, canola meal and minerals collected on two consecutive days of each week; samples of lucerne hay were collected every morning and silage at every feeding. Refusals of the TMR were collected, weighed and sampled. Dry matter concentration was determined by drying in a forced draft oven at 105 °C for 24 h. The nutritional composition was determined on samples of the grain mix, canola meal, lucerne hay, and silage offered that were collected daily, a 100 g sub-sample was taken, bulked by week for each feed type, and stored at −18 °C. Bulked samples were subsequently freeze-dried and ground to pass through a 0.5-mm screen then analyzed for crude protein (CP), soluble protein, acid detergent fiber (ADF), neutral detergent fiber (NDF), lignin, non-fiber carbohydrate, starch, ash, total digestible nutrients (TDN), crude fat (ether extract, EE), sodium, potassium, calcium, magnesium, phosphorous, sulfur and chloride by chemical analytical methods according to the procedures of Dairy One [26]. Water was offered to all animals at least once during each 3.5 h feeding period and was available ad libitum when cows were on the loafing pad or in the controlled-climate chambers.2.4. Milk Production and Composition Cows were milked twice daily, at ~0600 h and ~1500 h. Throughout the experiment, milk yield was measured for each cow at each milking. When cows were not housed in the chambers, milk yields were recorded automatically using a DeLaval milk metering system (MM25; DeLaval International, Tumba, Sweden). Milk samples for composition analysis were collected on days 1 to 7 (covariate), 1 of days 22 to 24 (pre-challenge, day closest to thermoneutral), and days 29 to 35 (recovery). During the heat challenge in the chambers, milk yield measurements were made by collecting and weighing the milk from individual cows and milk samples were collected at every milking. Fat and protein in milk samples were measured by means of a near-infrared milk analyzer (model 2000, Bentley Instruments, Chaska, MN, USA). Energy-corrected milk, standardized to 4.0% fat and 3.3% protein, was calculated using Equation (1) [27]:ECM (kg/d) = (milk yield (kg) × (376 × fat% + 209 × protein% + 948))/3138,(1)2.5. PhysiologyBody temperature was measured intravaginally (vaginal temperature) and recorded every 15 min during the covariate, pre-challenge, heat challenge and recovery periods using temperature loggers (iButton DS1922L; Maxim Integrated, San Jose, CA, USA) as described by Garner et al. [25]. Rectal temperature was only measured during the heat challenge at 0600 h and 1500 h using an EcoScan Temp 5 with 100 K thermistor temperature probe (Eutech Instruments, Singapore, Singapore).2.6. Calculations and Statistical AnalysesTemperature-humidity index (THI) was calculated using Equation (2) [28]. THI = Tdb + (0.36 × Tdp) + 41.2,(2) where: Tdb is dry bulb temperature (°C); Tdp is dew point temperature (°C),Tdp = (237.3 × b)/(1.0 − b); b = [log(RH/100.0) + (17.27 × Tdb)/(237.3 + Tdb)]/17.27.RH = relative humidity (%) Temperature and relative humidity data were collected every 10 min during the pre-challenge and recovery periods using Minnow 1.0 loggers (Senonics LLC, Arvada, CO, USA). During the heat challenge, temperature and relative humidity data were recorded every 1 min by the control system of the controlled-climate chambers. A THI threshold of 68 was used to determine if weather conditions were imposing heat stress on the animals as this is the point that has previously been identified as when weather conditions start affecting milk production [29].Pre-challenge data for analysis were intended to represent thermoneutral conditions and were recorded under ambient conditions on a day just prior to the heat challenge. The proportion of cows completing all four days of the heat challenge was tested using a generalized linear model with change in deviance χ2 tests [30]. The model used binomial distribution for the number completing out of the number eligible, with log-link function and factorial effects of fat by betaine. There were two types of statistical analyses conducted on experimental data: (1) an analysis of the main effect of the heat challenge or time, (2) the factorial effects of canola oil and betaine diet within periods or on changes between periods, (1) Effect of heat challenge: Trends over time in DMI, milk production and body temperature averaged across diet treatments were assessed for all cows that completed the heat challenge. This was achieved by calculating summary statistics for each cohort since each cohort provided an independent replication with respect to time, and using them as data in a t-test on degrees of freedom. Summary statistics were calculated as mean changes between pre-challenge and heat challenge, heat challenge and recovery, or pre-challenge and recovery, and as linear regression slopes from the last day of pre-challenge to day 4 of heat challenge, and from the last day of heat challenge to day 7 of recovery. (2) Factorial effects of diet: Only data from cows that had completed two or more days of the heat challenge were included in the analysis of treatment effects. Data from 22 cows (BASE, n = 6; CAN, n = 4; BET, n = 6; CB, n = 6) from day 2 of the heat challenge were used to represent the effects of the heat challenge. This was a compromise between the heat challenge having an effect on the cows and retaining sufficient animals in the analysis to enable conclusions to be drawn. Variables were constructed to address hypotheses, with one datum per animal. To achieve this for each animal, raw data within the pre-challenge period were averaged, and day 2 of the heat challenge was taken to represent the heat challenge. In addition, change variables were calculated for each individual cow by taking the difference between the mean value of a specific variable measured during the pre-challenge period and the value of the same variable measured during day 2 of the heat period. Similar change variables were calculated between the first and second days of recovery (initial recovery), and between the pre-challenge period and the final day (day 7) of the recovery period (extent of recovery). Each of these constructed variables was subjected to statistical analysis using the following linear model: y = μ + βycov + F + B + FB + C + Κ + ε,(3) where y was the outcome variable of interest, ycov was the same variable if available from the covariate period, F was a factor for the effect of added fat, B was a factor for the effect of added betaine and FB their interaction, C was an effect of chamber, K as an effect of cohort and ε a random error for individual animal. The model was applied using ReML software in GenStat 20 (VSN International Ltd., Hemel Hempstead, UK) with fixed effects for the covariate, cohort, chamber, and factorial fat by betaine diet treatments, and with animal as a random effect. Residuals were examined graphically to check distributional assumptions of normality and constant variance.3. Results3.1. Effect of Heat ChallengeMean daily THI during the pre-challenge period was 65 ± 7.3 (mean ± standard deviation, Figure 1). Measurements were made and samples were collected only on days when maximum THI was less than 68. All cohorts experienced at least one day of ambient conditions with maximum THI greater than 68 in the pre-challenge period. During the heat challenge, mean daily THI in the controlled-climate chambers was 75 ± 4.1. Mean daily THI during the recovery period was 62 ± 5.6. All cohorts experienced at least two days of ambient conditions with a maximum THI greater than 68 during the recovery period.Across all treatments, the conditions during the heat challenge induced heat stress in all cows. This was evidenced by a reduction in daily DMI (3.9 ± 1.22 kg DM, p = 0.008) from the pre-challenge period to day 2 of the heat challenge (Figure 2) and an increase in maximum vaginal temperature (1.0 ± 0.24 °C, p < 0.004). For those cows that completed the 4-day heat challenge, DMI declined to a minimum on day 4 of the heat challenge then increased during the recovery period to 99% of pre-challenge at recovery day 7. Both milk yield and ECM were lowest on day 1 of the recovery, increased to around 90% of pre-challenge on recovery day 2 then increased to pre-challenge by recovery day 7.3.2. Challenge CompletionNot all cows completed all four days of the heat challenge (Table 2). Two cows on the CAN diet were excluded from the analysis, one due to injury sustained on day 1 of the heat challenge, and one due to equipment failure that resulted in no heating on day 1 of the heat challenge. Of the remaining 22 cows, two cows (1 BASE, 1 CAN) were cooled on day 3, and seven cows (2 BASE, 1 CAN, 1 BET, 3 CB) were cooled on day 4 of the heat challenge because their body temperature exceeded the predetermined limit of 40.9 °C. One cow (CB) was cooled on day 4 of the heat challenge at the discretion of the investigators as she exhibited an extended period of the elevated panting score. There was no difference due to treatment on the number of cows completing the heat challenge (p = 0.234). However, it must be noted that the two cows on the CAN treatment lost on day 1 of the heat challenge were not due to treatment (1 physical injury, 1 equipment failure) and the remaining low number of animals meant the resultant power of this analysis was low. 3.3. Main Effects-Dry Matter IntakeDry matter intake during the pre-challenge period (Table 3) was not affected by the feeding of canola oil (p = 0.081) nor betaine (p = 0.181). Intake of metabolizable energy was greater in cows fed canola oil than in cows not fed canola oil (p < 0.001), which was in accordance with the experiment design. On day 2 of the heat challenge, DMI was 15% lower in cows given canola oil than other cows but this only tended towards being different (p = 0.071, Table 3). Feeding betaine had no effect on DMI (p = 0.489). There was no difference in intake of ME between treatments. From the pre-challenge period to day 2 of the heat challenge, the decline in DMI was greater for cows offered canola oil than those not offered canola oil (p = 0.035) and betaine had no effect (p = 0.68). The reduction in ME intake was greater for cows given canola oil compared with those not (p = 0.029). Offering betaine had no effect on the decline in ME intake (p = 0.875). During the initial recovery, DMI increased for all cows (p = 0.005). However, neither canola oil (p = 0.11) nor betaine (p = 0.765) had any effect. By day 7 of the recovery period, there was a tendency (p = 0.07) for a decrease in DMI of 3.8 kg/d for all cows relative to the pre-challenge period. This change was unaffected by the canola oil (p = 0.45) or betaine (p = 0.49) diets.3.4. Main Effects-Milk YieldDuring the pre-challenge period, milk yields (Table 4) were greater from cows fed canola oil than from cows not fed canola oil (p < 0.001) and similarly for ECM (p = 0.032). Cows fed canola oil also had greater yields of protein (p = 0.007) but not fat (p = 0.138) compared to the yields from cows not fed canola oil. Cows fed canola oil produced milk with lower concentrations of milk fat (p = 0.022) compared to those not fed canola oil. Cows fed betaine tended to have lower milk yield than those not fed betaine (p = 0.073).On day 2 of the heat challenge, milk yield was greater from cows given canola oil than those not (p = 0.026), but ECM yield was only numerically greater (p = 0.105). From the pre-challenge period to day 2 of the heat challenge, the decline in milk yield was not affected by feeding canola oil (p = 0.429) or betaine (p = 0.540). The decline in ECM yield was unaffected by either canola oil (p = 0.778) or betaine (p = 0.429). During the initial recovery, milk yield increased for all cows. The increase in milk yield was not affected by the feeding of either canola oil (p = 0.823) or betaine (p = 0.610). Yield of ECM also increased for all cows but feeding neither canola oil nor betaine had an effect. Recovery of ECM yield to seven days post-heat challenge was unaffected by treatment and there was no interaction between canola oil and betaine. 3.5. Main Effects-Body TemperatureDuring the pre-challenge period, the mean vaginal temperature was not affected by the addition of canola oil to the diet, or the supplementation with betaine (Table 5). However, the maximum vaginal temperature of cows fed betaine was lower than that of cows not fed betaine (p = 0.014). On day 2 of the heat challenge, cows fed canola oil had greater mean (p = 0.016) and maximum vaginal temperatures (p = 0.039) than other cows. Feeding betaine had no effect on mean (p = 0.31) or maximum (p = 0.37) vaginal temperatures. From the pre-challenge period to day 2 of the heat challenge, cows given canola oil tended to have a greater increase in mean vaginal temperature (p = 0.074) than other cows but there was no effect on maximum vaginal temperature (p = 0.127). Supplementing the cows’ diet with betaine had no effect on the change in mean (p = 0.679) or maximum (p = 0.825) body temperature.During the recovery period, the mean vaginal temperature decreased in all cows from day R1 to R2 (Table 5) with the change tending to be greater in cows fed canola oil than other cows (p = 0.07). The change in maximum vaginal temperature from day R1 to R2 was not affected by either canola oil (p = 0.38) or betaine (p = 0.60). Neither canola oil nor betaine had any effect on the difference in vaginal temperature between day R7 and pre-challenge (p > 0.38), and this small difference in vaginal temperature was not significantly different to zero (p > 0.16).3.6. Treatment Effects—Dry Matter IntakeOn day 2 of the heat challenge, the DMI of cows given the CAN diet was less than that of cows given the BASE diet (p < 0.05), but there was no difference in DMI between cows given the CB and BASE diets. Similarly, the decline in DMI from the pre-challenge period to day 2 of the heat challenge in cows offered the CAN diet was greater than that in cows offered the BASE diet (p < 0.05), but there was no difference in decline of DMI between cows given the CB and BASE diets.3.7. Treatment Effects—Milk YieldFrom the pre-challenge period to day 2 of the heat challenge, the change in milk yield of cows on the BET diet was +0.3 kg/day, but this was not statistically different (p > 0.05) from the change in milk yield of cows on the other diets (−1.1 kg/day). Dietary treatment had no effect on the declines in yield of fat (p > 0.330), or protein (p > 0.490). Moreover, dietary treatment did not influence (p > 0.300) the change in composition of milk fat or milk protein.During the initial recovery, there was a tendency for an antagonistic interaction (p = 0.054) between canola oil and betaine for milk yield such that the increase in milk yield for cows given the CB diet was 2.5 kg/day compared with 4.2 ± 0.82 kg/day for each of the CAN and BET treatments and 1.3 kg/day for cows on the BASE diet.3.8. Treatment Effects—Body TemperatureOn day 2 of the heat challenge, mean vaginal temperature of cows offered the BET treatment was lower than that of cows offered the CAN and CB treatments. However, none of these treatments resulted in a mean vaginal temperature different to that of the cows offered the BASE diet.4. DiscussionThe temperature and humidity settings applied in the controlled-climate chambers successfully induced a state of heat stress in all cows. This was observed as an 18% decrease in DMI and a 1 °C increase in maximum vaginal temperature of the cows. However, the heat challenge was not completed by all cows. While challenge completion may have been influenced by treatment, we were unable to detect any treatment effect. Ten of our 24 cows were removed from the heat challenge on either day 3 or 4. For this reason, we chose to analyze the effects of treatment at the completion of day 2 of the heat challenge using data from the 22 cows that completed the heat challenge to this point. 4.1. Canola Oil Main EffectCows offered diets supplemented with canola oil had a greater decline in DMI, a similar decline in ECM, and a greater increase in body temperature than cows not offered canola oil. Thus, we reject our first hypothesis. During the pre-challenge period, our cows offered diets containing canola oil had a 7% greater intake of metabolizable energy compared to cows offered diets not containing canola oil. However, during the heat challenge, the large decrease in DMI of the cows given canola oil meant that their resulting intake of metabolizable energy was 7% less than that of the other cows. Thus, the greater concentration of energy in the canola oil diets did not offset the reduction in intake of metabolizable energy due to the reduction in DMI. Why the DMI of cows offered canola oil declined so dramatically is not known. Fat supplementation has been shown to reduce voluntary DMI due to the depression of ruminal fiber digestion [31]. However, in our experiment the canola oil supplement was added to the basal diet instead of being substituted for the existing components of the diet, and the total concentration of fat in the canola oil supplemented diets was below the threshold of 70 g/kg DM where DMI can be depressed [12]. Furthermore, the fatty acids in canola mainly comprise oleic and linoleic acids [32], and almost none of the medium chain fatty acids (lauric and myristic) that have generally been associated with inhibition of dry matter intake [33,34]. The effects of dietary fat supplementation on ECM during hot weather are generally positive. In agreement with our results, dairy cows fed fat during hot weather generally have greater production of milk and ECM relative to those cows not receiving the supplement [13,14,35]. Experiments using controlled-environment chambers to create a heat challenge also found greater milk yields in cows fed a diet supplemented with fat [36,37]. However, some studies report nil effect of dietary fat on milk production during hot weather [38]. This could be due to a number of reasons, such as the type of fat supplement [39], the severity and duration of heat exposure, and in the case of Moallem et al. [38], the use of cooling amenities five times per day. Our cows offered canola oil produced 3.5 kg more milk (2.8 kg ECM) per cow per day during the pre-challenge period than those cows not offered canola oil. The increase in milk is explained by the additional energy intake derived from the canola oil supplement, and the difference between milk yield and ECM yield is explained by the observed decrease in milk fat concentration during the pre-challenge period. This can occur in cows offered a diet with a fat supplement due to the formation of fatty-acid isomers in the rumen that cause milk fat depression [31].Some previous reports agree with our findings on body temperature in which dairy cows fed a dietary fat supplement during hot weather had greater body temperature than the cows that were not fed the fat supplement [38]. This may have contributed to the reduction in feed intake in the CAN treatment. Conversely, Drackley et al. [13] and Huber et al. [35] reported no effect of dietary fat supplementation on rectal temperature of dairy cows during hot summer conditions, and studies with cows subjected to heat-stress conditions in environmental chambers also showed no difference [36,37]. These findings, and ours, are contrary to the expectation that feeding fat to cows would result in lower body temperature due to the lower heat increment of fat compared to other feeds [3,8]. We could not find evidence to suggest that these differences might be due to the fatty acid profile, or specific fatty acids, of the fats reported. However, Wang et al. [39] found that offering a fat supplement with a high proportion of saturated fatty acids resulted in lower body temperatures compared with no fat supplementation, but only when measurements were made during the hottest part of the day. It is possible that body temperature is more closely related to energy intake than feed type. Results from our pre-challenge period agree with a previous report that dietary addition of fat had no effect on the rectal temperature of lactating cows during thermoneutral conditions [37]. We note that previous studies that have examined the effects of dietary supplementation with fat [13,17,37] have generally not continuously measured body temperature.4.2. Betaine Main EffectCows offered a diet supplemented with betaine did not have smaller declines in DMI or ECM than cows not offered betaine, and the increase in body temperature was also not different during a heat challenge. Thus, we reject our second hypothesis. Our DMI results are consistent with previous research in which betaine was offered to dairy cows at low inclusion rates [40]. In addition, betaine fed in the range of 50 to 150 mg/kg BW0.75 to beef heifers in ambient conditions had no effect on DMI [41]. Similarly, the DMI of sheep fed betaine during a heat challenge was not different from other animals [16]. In contrast, one study reported the DMI of dairy cows fed betaine during a heat challenge was lower than those not fed a betaine supplement [17]. However, the betaine dose of 563 mg/kg BW0.75 used [17] was much greater than the 132 mg/kg BW0.75 used in our experiment and greater than the optimum dose of 126 mg/kg BW0.75 reported for sheep [16]. While there was no increase in milk yield during our short-term heat challenge, greater yields of milk have been reported previously in cows offered similar doses of betaine to that used in our experiment [42,43]. It has been suggested that the greatest effect of betaine may be observed when the overnight temperature remains high [43]. Our betaine dose was in the range suggested by Dunshea et al. [43] to be optimal for beneficial effects but it is possible the overnight temperature during the heat challenge in our experiment (25 °C) was too low, or the heat challenge too short, for the benefits of betaine supplementation to be realized. The body temperatures of our cows during the heat challenge are similar to those in previous research where the diet of lactating dairy cows was supplemented with betaine at 57 and 114 mg/kg BW0.75 for 14 days before cows were exposed to a heat challenge [17], and 17 to 35 mg/kg BW0.75 for three months of summer [18]. However, supplementing the diet of sheep with betaine at 126 mg/kg BW0.75 during heat exposure for three weeks has been found to reduce rectal temperature [16]. Furthermore, a study in buffalo heifers under heat-stress conditions showed that dietary inclusion of betaine at approximately 420 mg/kg BW0.75 was associated with decreased rectal temperature, but the effect was not statistically significant until the fifth week of the experiment [44]. With the exception of the report by Zhang et al. [18], who pooled their results across time, there appears to be a correlation between the duration of feeding of betaine and the extent of the treatment effect. This concept is further supported by a report of the effect of betaine increasing with time when fed to lactating dairy cows over summer [43]. In our experiment, betaine was fed to cows for 14 days prior to the heat challenge. Therefore, we speculate that in our experiment, the short duration of inclusion of betaine in the diet may be one reason why we did not observe an effect on body temperature during the heat challenge.During the initial recovery (day R1 to R2), cows given betaine did not show a greater increase in DMI compared to other cows but their increase in ECM was numerically double that observed in the other cows. However, not all cows completed the heat challenge. This means that the heat challenge was of different lengths for different cows, thereby confounding our assessment of the recovery. Previous reports indicated that betaine should have reduced the effects of the heat challenge on our cows [15] leaving them better positioned to recover once conditions returned to thermoneutral. Reports of animals fed betaine performing better during a heat challenge [42,44] also support the idea that the effects of the heat challenge are lessened for those animals, and as a corollary they should be better positioned to recover. No reports of dairy cow performance immediately following a heat challenge were found in the scientific literature. 4.3. Supplement Interaction–Treatment EffectCows given the fat and betaine in combination did not exhibit greater DMI and ECM yield, and lower body temperature during a heat challenge compared to cows given either alone. Thus, we reject our third hypothesis. However, the variability in results during the heat challenge did reduce our ability to detect differences. On day 2 of the heat challenge the DMI of cows given the CAN diet was less than that of cows given the BASE diet, but there was no difference in DMI between cows given the CB and BASE diets. This suggests that betaine could offset some of the negative impact that canola oil had on DMI in our experiment. A similar effect on body temperature was not observed. Cows given the BET diet did have lower mean and maximum body temperature than the cows given the CAN diet, but those given the CB diet had temperatures close to those given the CAN diet. We speculated that the concentration of fat in the diet could have influenced the reported animal responses to dietary betaine. However, a re-examination of the scientific literature used to determine the betaine dose for our experiment found that only four of the 10 reports specified the concentration of fat in the basal diets, and therefore we were unable to draw a conclusion. In our experiment, the small number of cows in each individual treatment meant that an in-depth investigation of the interactions was not possible, so further work is warranted to untangle the interactions between dietary fat and betaine supplements.5. ConclusionsCows subjected to a heat challenge had decreased DMI and milk production with a concomitant increase in vaginal temperature. Neither canola oil nor dietary betaine supplements had the expected effects on cow milk production or body temperature during a heat challenge. Supplementing the diet of dairy cows with fat resulted in a positive milk production response, and the magnitude of the response was similar before, during and after a heat challenge. However, dietary fat supplementation also resulted in greater body temperature across all periods of our experiment. The declines in DMI and ECM from pre-challenge to the heat-challenge were greater in cows fed canola oil, in contrast to expectations that the declines would be reduced when canola oil was fed. Contrary to our expectations, the combination of fat and betaine supplements did not result in any clear benefit in terms of milk production or body temperature. Due to the short duration of the heat challenge and a low number of cows in our experiment, further research is required to fully understand the interactions between dietary fat type and betaine supplements when offered to cows during periods of hot weather.

animals : an open access journal from mdpi

10.3390/ani12070901

PMC8996832

Chicken meat is a popular food item all over the wrld. Improving the nutrition of broilers is important for producing high-quality broiler meat. The inclusion of natural effective ingredients, such as ginger, in the diet of broilers did not adversely affect the palatability of the diet, nor did it cause anemia in the broilers. Rather, ginger enhanced the oxidative status and growth rate of broiler chickens.

The effect of dietary ginger powder on the production performance, digestibility, hematological parameters, antioxidant status, dietary oxidation stability, and plasma cholesterol content of broiler chickens was investigated. Ginger powder was included in the diet at 0, 5, 10, or 15 g/kg. Total antioxidant capacity and malondialdehyde in sera samples, superoxide dismutase activity, glutathione peroxidase, catalase, and malondialdehyde in liver samples, and the peroxide value and acid value of the stored diets were evaluated. The results showed that ginger inclusion significantly improved antioxidation indices in broiler sera and liver. Total body weight gain in ginger-supplemented birds was higher than that of control birds (p < 0.048). Supplementing the broiler chickens with ginger powder reduced total feed consumption (p < 0.031). White blood cell counts and the percentage of heterophils in the blood were increased in birds that received ginger supplementation (p < 0.001). The inclusion of ginger in the diet improved dry matter digestibility, crude protein utilization, crude fiber utilization, and ether extract utilization. In addition, blood cholesterol, triglyceride, and very low-density lipoprotein levels were decreased (p < 0.001), and high-density lipoprotein and levels were increased, following the inclusion of ginger in the diet (p < 0.001).

1. IntroductionThere is interest in elevating the production performance of broiler chickens using effective nutritional additives in the feed rations, especially after the COVID-19 crisis. Medicinal plants are used as natural feed additives in poultry diets to enhance the performance, anti-oxidative status, and immune response of chickens [1,2,3,4,5,6,7,8]. One of these additives is ginger powder. Ginger is the rhizome of the plant Zingiber officinale. It belongs to the family Zingibeaceae, which includes aromatic herbs with fleshy, tuberous or non-tuberous rhizomes, that often have tuber-bearing roots [9]. It has long served as a popular culinary and traditional medicinal herb. Ginger contains several effective compounds, such as gingerol and gingerdione that exert strong antioxidant activity. In addition, it has antibacterial properties and is immunomodulatory in laboratory animals [10,11,12,13]. Plant-derived additives used in animal feed to improve production performance are known as phytogenic feed additives, and ginger is one such additive [14]. Ginger powder has lipid-reducing effects and can also be used as a growth promoter. When included in chicken feed, it has properties similar to those of antibiotics. These natural feed additives lower enteric pathogen microbial loads and improve nutrient digestion and absorption, which improve poultry production and broiler performance [15].Antioxidants can impact the health status of poultry [16]. The inclusion of ginger in the diet at 5–6 g/kg is thought to increase total protein and lower cholesterol concentration. Studies have also found that ginger in chicken feed enhances immunity against Newcastle disease and bacterial bursal infections [17]. Because of its antioxidant properties, and ability to enhance immune function and inflammatory responses, ginger can improve both chicken production performance and the immune system [18]. Furthermore, Sahoo, Mishra [19] reported that feed rations supplemented with ginger, either alone or with 1% turmeric, significantly enhanced the antioxidative status and gut health of broiler chickens. An, Liu [18] investigated the effect of ginger supplementation on antioxidative indices in broiler chickens. The results of their study revealed that neither the activity of glutathione peroxidase nor total antioxidant capacity were affected by ginger extract supplementation, but plasma dismutase activity and malondialdehyde (MDA) content were significantly decreased. In addition, the antioxidant stability of the feed ration was increased. The effects of ginger have also been investigated in laying hens. For example, Wen, Gu [20] reported increased superoxide dimutase (TSOD) activity, and decreased yolk MDA and cholesterol content in hens supplemented with ginger, compared to control hens. These findings were consistent with those of Zhao, Yang [21], who found that layers fed a diet rich in ginger powder had a reduced concentration of MDA and increased TSOD activity in the yolk. In addition, Yang, Ding [22] observed increased blood antioxidant enzymes and improved egg quality in ginger root-fed laying hens.In addition to its effect as an antioxidant, several research studies have reported that poultry diets supplemented with ginger powder have positive effects on broiler performance. As a natural feed additive, ginger may have great benefit and value in poultry nutrition—especially for broilers—due to its antibacterial, anti-inflammatory, antioxidant, antiseptic, antiparasitic, and immunomodulatory properties [23]. Ginger is a natural plant that can be used as a phytobiotic to improve the performance of broilers. This improved performance may be attributed to two types of digestive enzymes found in ginger—protease and lipase—which are part of the plant’s natural protective mechanisms [24]. Diets enriched with ginger may have the potential to improve production performance and modulate the biological properties of the blood in broiler chickens. Sa’aci, Alabi [25] studied the effect of aqueous ginger extract (AGE) on growth, nutrient digestibility, and the economy of Marshal broiler chicks. The AGE supplementation of the diet had no effect on dry matter, crude protein, ether extract, or nitrogen-free extract, but crude fiber digestibility was significantly affected. Shewita and Taha [17] reported that ethanolic extract of ginger significantly lowered serum total cholesterol and triglyceride levels and increased high-density lipoprotein (HDL) cholesterol levels, preventing tissue damage due to lipid peroxidation. It also showed lipid-lowering activity. In their study on broiler chicks, the authors fed the chicks diets supplemented with 2, 4, and 6 g/kg ginger powder and observed that serum very low-density lipoprotein (VLDL) and triglyceride levels were reduced significantly in all ginger-supplemented groups. Another study used ginger (5, 10, and 15 g/kg ginger powder) and thymol (200 and 400 mg/kg) as feed supplements in the diet of broiler chicks, which led to significantly decreased levels of serum total cholesterol and triacylglycerol [26]. The aim of this study was to investigate the effects of ginger on the antioxidant status, production performance, and hematological parameters of broiler chickens fed a ginger-enriched diet, as well as the oxidation stability of ginger-supplemented feed. The hypothesis was that ginger powder would improve the aforementioned parameters but would have dose-dependent effects. Although previous studies have investigated the effect of ginger on productive performance parameters in broiler chickens, relatively limited data exist in the literature on the direct effects of ginger on the antioxidative status of blood and liver tissues in these birds and the effect on the oxidative stability of the stored diets at different ginger levels.2. Materials and Methods2.1. Chickens, Experimental Design, and DietThis research study was approved by the department committee of the Environment and Life Sciences Research Center in Kuwait Institute for Scientific Research under project No. FA157K (2017). These procedures and protocols followed the official animal welfare guidelines and regulations encoded with reference No. PMO/PV/RP/032/2017. This protocol recommends humane treatment of experimental animals with no pain, stress, or harm. The vitality rate was 100% and no abnormal signs were observed during the experiment. Fresh ginger (Zingiber officinale) roots were purchased from a reliable local supermarket, originated from India. The roots were washed, sliced, freeze-dried, and milled into a powder that was used in the broiler feed rations. This study used 1-day-old, male, Cobb 500 broiler chicks that were vaccinated against infectious bronchitis and Newcastle disease. Water and feed were provided ad libitum. Four experimental diets/treatments were used, with 0, 5, 10, or 15 g/kg ginger in the diet. For each treatment, 340 birds were randomly housed in a battery cage with five levels. Each level contained 17 birds, for a total of 85 birds in the battery. This density provided a space of 0.05 m2/bird. Each level was considered as a replicate (total of 5 replicates). The broiler chicks were fed a corn/soy-based diet that met Cobb 500 guidelines [27]. The chicks received a starter diet from hatching until 7 days of age, a grower diet from 8 to 21 days of age, and a finisher diet from 22 to 35 days of age. All diets were prepared as needed. Diet formulation, as well as chemical analyses of the control diet and ginger supplement are shown in Table 1. The control birds received no ginger. The ambient temperature for the broilers was kept at 30 °C for 14 days, and then gradually reduced to 21 °C by 21 days. A proximate analysis of the ginger was performed for crude protein, ash, dry matter, and crude fiber.2.2. Sample CollectionAt 5 weeks of age, blood samples were collected from the branchial vein of birds in vacutainer tubes (K2EDTA). Blood samples were collected from five chickens from each treatment, and 8–10 mL of blood was collected in each tube. Meat tissue and liver samples were also collected from five chickens per treatment. Analysis was completed in triplicates.2.3. Production Performance Parameters Body weight and feed consumption were recorded; broiler chicks were weighed at hatch, at one week of age, and at the end of every two weeks afterward until 35 days [28]. The temperature and relative humidity were recorded daily and adjusted to minimize stress surroundings in the poultry house. Mortality was recorded daily. 2.4. Apparent Digestibility Coefficient The apparent digestibility coefficient was evaluated, using a biological assay, as affected by the different levels of ginger powder in the experimental diets. Dry matter digestibility (DMD), crude protein utilization (CPU), crude fiber utilization (CFU), and ether extract utilization (EEU) were determined according to Horwitz [29]. The apparent digestibility coefficient was estimated and expressed as a percentage [30,31].The experimental birds were individually raised in cages and fed ad libitum on diets with ginger solution for four days as the adjusting period. During this period, the excreta was discarded. Water was available ad libitum throughout the experimental period. After the adjusting period, birds were fasted for 24 h to empty all remaining contents in the alimentary canal. In one group, birds were continuously starved for 24 h to obtain data on endogenous nitrogen excretion. Birds in the other group were force-fed with the ginger solution at a dose rate of 60 g/day/bird for three consecutive days. The excreta was collected using clean, rigid trays placed under the clean wire cages in which the birds were housed. Droppings retained on the wire screen floor of the cage were also collected. The contamination of excreta with feathers, fed, scale, and vomit was avoided. The excreta of the first group was collected after the starving period for determination of endogenous nitrogen content. Excreta of the other group was individually collected every 6 h during the three subsequent days and for approximately 24 h after the birds were fed for three subsequent days. The excreta samples were individually weighed and sprayed with 2% boric acid solution to fix the nitrogen in the samples and stored in the freezer at 20 °C until analyses. Excreta was then dried in the freeze drier for 2 days, weighed, homogenized, and ground for the estimation of nutrients. Crude protein was detected using Macrok-jeldahl method that involves the transformation of nitrogen into ammonium sulphate by acid digestion using boiling sulphuric acid. The ammonia was trapped by boric acid and was titrated using standard hydrochloric acid solution. The percentage of nitrogen was calculated from the percentage of crude protein. The crude fiber was determined using the Weende method and the Fiber Tec system. A moisture-free and fat-free sample was first digested with a weak acid solution, and then with the weak base solution. The digested residue was then collected in a filter crucible, dried and ignited. The loss of weight on ignition was the crude fiber. For crude fat determination, an organic solvent was used to extract the crude fat from a known weight of the sample. The dissolved fat was then recovered by the evaporation and condensation of the solvent. The fat extracted was a representative of the crude fat of the sample. The digestibility coefficients of the nutrients were calculated as follows:Digestibility (%) = (NF − NE + NENC) × 100 where NF = nutrient in feed, NE = Nutrient in Excreta, and NENC = Nutrient in Excreta of negative control.2.5. Antioxidant StatusAntioxidant status was investigated by measuring the antioxidant indices in the sera and livers of broiler chickens supplemented with different concentrations of ginger powder. The TSOD activity in the liver was measured using a Ransod kit from Randox Laboratories, UK, as described by Habibi, Sadeghi [32]. Liver glutathione peroxidase (Gpx) was indicated based on the protocol used by Paglia, Valentine [33]. Catalase (CAT) enzyme activity in the liver was determined using the method described by Aebi [34]. Sera MDA and total antioxidant capacity (TAC) were measured as described by Habibi, Sadeghi [32].2.6. Oxidation Stability of the Feed RationsLipid peroxidation of the dietary feed rations, including the different concentrations of ginger powder, was determined by measuring the peroxide value (PV) and the acid value (AV) of the feed for a period of 50 days, starting from day 10. Measurements were recorded every 10 days thereafter, until day 50. The PV and the AV were measured using the methods described in AOCS [35] and Rao, Xiang [36], respectively.2.7. Hematological MeasurementsThe samples were analyzed using a Cell-Dyn 3500 Hematology system (Abbott Laboratories, Abbott Park, IL, USA) to measure total and differential WBC and blood quality parameters, including red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW), and platelet count (PLT).2.8. Plasma Cholesterol Content Blood cholesterol, triglyceride, and high- and low-density lipoprotein contents were measured calorimetrically using a commercial kit from Bioassay Technology Laboratory, China. Serum was first collected by centrifugation at 2000–3000 rpm for 20 min. All reagents and standard solutions were used at room temperature. In total, 50 µL of standard solution were added to a standard well, and 40 µL of sample were added to the sample wells. Then, the corresponding LDL and HDL antibodies were added to the sample wells. Streptavidin-HRP was added to both the sample and standard wells. The plate was then incubated for 60 min at 37 °C, and 50 µL of substrate solutions A and B were added to all wells. After incubation for 10 min at 30 °C in the dark, 50 µL of stop solution were added to each well. The optical density (OD) of each well was then determined.2.9. Statistical AnalysisFour experimental diets/treatments were used. For each treatment, 340 birds were randomly housed in four multi-floor batteries, each of which had five levels. Each level contained 17 birds, for 85 birds in the battery. Each level was considered as a replicate (5 replicates per treatment). Overall differences among the dietary treatments were evaluated using one-way ANOVAs via the general linear model procedure in Minitab. Differences among treatments were considered statistically different at p ≤ 0.05. Data were arcsine transformed before analysis to improve normality. Where significant differences occurred, pairwise Tukey post-hoc comparisons were made to identify significant differences between groups.3. Results 3.1. Growth PerformanceAll broilers appeared to be healthy, and no significant mortality occurred during the experiment. Table 2 shows the effects of different levels of ginger powder on body weight, feed consumption, feed efficiency, and weekly body weight gain of broiler chickens. The results showed that addition of ginger powder at all levels improved the body weight of the broiler chickens at 5 weeks of age (p < 0.001). Feed consumption of broilers fed 0, 5, 10, or 15 g/kg of ginger powder is shown in Table 2. The results showed that supplementing broiler chickens with ginger powder significantly reduced feed consumption (p < 0.031). Results in Table 2 showed that there was no significant effect of ginger powder on the feed efficiency of broilers. Results in Table 2 showed that the total body weight gain of birds supplemented with ginger was significantly higher than that of control birds (p < 0.048).3.2. Apparent Digestibility CoefficientThe effects of different levels of ginger powder on nutrient digestibility are shown in Table 3. The results showed that there were significant (p ≤ 0.05) differences among the dietary treatment groups for DMD, CPU, CFU, and EEU. 3.3. Antioxidative IndicesTable 4 shows the effect of ginger on serum and liver antioxidant indices in broiler chickens. The results in Table 4 show that broiler chickens fed diets rich in ginger powder had increased serum TAC (p < 0.021). However, MDA concentrations were decreased with ginger supplementation (p < 0.038). For the liver parameters, the results of Table 4 show that broiler chickens fed a diet supplemented with 5 g/kg ginger did not have changes in liver TSOD. However, broilers fed a diet supplemented with 10 or 15 g/kg of ginger had increased liver TSOD compared to the control group and the 5 g/kg ginger-supplemented group (p < 0.049). Consumption of feed rations supplemented with ginger did not affect the liver GPX level (p < 0.949). The concentration of CAT was increased by ginger supplementation, compared to the control group (p < 0.050). In contrast, broiler chickens that consumed different concentrations of ginger had decreased liver MDA (p < 0.020) compared to the control group.3.4. Antioxidative Capacity of Feed RationsTable 5 shows the PV of lipids extracted from stored ginger-supplemented dietary feed rations over 50 days of oxidation. From Table 5, it is evident that there was an interaction between PV value and the storage duration of ginger-supplemented feed rations. The control diet remained stable over the 50 days of storage. However, the diet supplemented with ginger at a concentration of 5 g/kg showed stability in PV value until 40 days but approached the control value by day 50. The PV value of the diet supplemented with ginger at a concentration of 10 g/kg of diet increased until day 30 but decreased to the initial value by day 40. In addition, the PV value of the feed ration supplemented with ginger at a concentration of 15 g/kg increased until day 30, started to decrease at day 40, and reached the control level at day 50 (Table 5). Table 6 shows the AV of lipids extracted from stored, ginger-supplemented dietary feed rations over 50 days of oxidation. The results of Table 6 also indicate an interaction between AV and storage time. The AV value increased with increasing storage duration. At all days of storage, the AV value of the control group was significantly higher than that of the groups supplemented with ginger at different concentrations.3.5. Hematological MeasurementsThe effects of ginger powder on the blood composition of broiler chickens are shown in Table 7. The results in Table 7 show that providing broiler chickens with feed rations supplemented with ginger at 5, 10, or 15 g/kg of diet enhanced WBCs of broilers, compared to the control (p < 0.001). In addition, the percentages of heterophils in birds from groups fed ginger powder at 10 or 15 g/kg of diet were significantly higher than that of the control group, and lower than that of the group supplemented with ginger powder at a concentration of 15 g/kg. The results in Table 6 show that ginger supplementation had no effect on any other blood parameter.3.6. Plasma Cholesterol Content The effects of different levels of ginger powder on blood cholesterol, triglyceride, HDL, and VLDL levels are shown in Table 8. A significant decrease in total cholesterol (TC) was observed in treatment groups supplemented with 5–15 g/kg ginger powder. A similar trend was also observed in total glycerides (TG). The HDL levels were found to be increased (p ≤ 0.05) in treatment groups fed 5–15 g/kg ginger powder, compared to the control group. However, LDL levels in the supplemented birds were decreased (p ≤ 0.05).4. Discussion The current study was conducted to investigate the effects of increased concentrations of dietary ginger powder on the antioxidation status, production performance, and hematological parameters of broiler chickens. In this study, both the control and the treatment diets were consumed equally by chickens, indicating that the inclusion of ginger in the diet did not adversely affect the palatability of the diet. The significant increase in the total body weight gain observed in this study is in agreement with some previous studies that investigated the same variable [23,37]. However, other studies have reported that the inclusion of ginger in the diet of broiler chickens did not improve weight gain [38,39,40]. Zhang, Yang [24] investigated the effect of dried ginger root on the growth performance of broilers and found that supplementation with ginger powder led to better production performance. The positive effect of phytobiotics on the production performance of broilers was also investigated by Hashemi and Davoodi [41]. Moorthy, Ravi [42] reported that dried ginger powder increased the body weight of broilers when it was included in the diet at a level of 2%. In addition, Tekeli, Zengin [43] reported that supplementing broiler feed rations with 120, 240, and 360 ppm of ginger significantly enhanced the broiler body weight gain. Similar enhancement results in the body weight gain were reported by Onu [23], Kausar, Rizvi [44], Javandel, Navidshad [39], Herawati [37], and Ademola, Farinu [38]; however, no significant effect of ginger was observed in the average broiler daily weight gain in broilers when using 5 g of ginger/kg of diet [24] or 1 g of ginger/kg of diet [45]. In contrast, a reduction in the starter broiler growth rate was reported by Al-Homidan [46] when ginger was supplemented at a level of 60 g/kg of diet. The authors explained this reduction as a result of the toxic action of ginger. Interestingly, Zhang, Yang [24] reported a better carcass yield (p < 0.014) of the ginger-supplemented broilers, compared to the control group and attributed this effect to the antioxidant effect of ginger that stimulates protein and fat metabolic pathways. Conversely, Moorthy, Ravi [42] and Onu [23] suggested that supplementing broiler feed rations with ginger does not affect carcass quality.Results of the current study showed that supplementing the broiler chickens with ginger powder reduced total feed consumption (p < 0.031). This result is in line with that of Herawati [37] who reported that broilers fed a 2% ginger-supplemented diet had significantly lower feed consumption than the control group. These results are in contrast with the findings of Onu [23], who reported no significant differences in the feed consumption of birds provided with different ginger treatments versus the control. In addition, Ademola, Farinu [47] observed significantly higher feed consumption in broilers fed a ginger-supplemented diet compared to the control group. The results of the analysis of feed efficiency showed that there were no significant differences across the treatments. This finding agrees with some studies that have investigated the same variable [25,39,48]. However, Onimisi, Dafwang [49], Moorthy, Ravi [42], Onu [23], and Onimisi, Dafwang [49] reported significantly lower feed efficiency in ginger-supplemented groups compared to the control group. These authors suggested that this outcome may be due to improved gut micro-flora, which inhibited microbial fermentation and improved feed efficiency. Conversely, Ademola, Farinu [38] reported a significant, 5% increase in feed efficiency in birds supplemented with ginger compared to control birds. There was a significant effect of ginger powder supplementation on nutrient digestibility in the chickens. This result could be attributed to stimulation of digestive enzymes by bioactive compounds of ginger, and thus improvement of overall digestion. The active compound gingerol contributes to the secretion of digestive enzymes, which aids in the digestive process and helps provide nutrients. The active compounds in herbs stimulate the pancreas to produce digestive enzymes in larger amounts, which leads to increased nutrient digestibility and absorption to support growth [48]. Biochemical studies of the blood of ginger-fed broiler chicken have previously been undertaken by several authors. Interestingly, there is a debate regarding the effect of ginger on lipid profile and blood parameters among different studies. This could be attributed to differences in strain, age, ginger level, genetics, and experiment circumstances. For example, Rehman, Durrani [50] fed broiler chickens on 10 mL of therapeutic plants (garlic, mberberine, and aloe vera)/L of drinking water alongside ginger. The authors reported that serum glucose, alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase levels were significantly reduced and the serum protein increase was significantly enhanced in supplemented broilers. Results of the same study revealed a significant reduction in the total cholesterol, triglyceride, LDL, and VLDL and significant enhancement of the HDL level in the supplemented broilers. Similarly, Zhang, Yang [24] reported that ginger inclusion increased the broiler total protein concentration and reduced cholesterol concentration at 21 and 42 days of broilers. On the other hand, Kausar, Rizvi [44] reported no effect of ginger inclusion at dosages of 2 or 4 mL/L of drinking water on serum albumin, globulin, or total protein. Al-Homidan [46] revealed a reduction in the total protein and globulin in the plasma of broilers after supplementing ginger at 60 g/kg, which could have been due to the toxic effect of ginger at that dose. However, Ademola, Farinu [47] reported that ginger supplementation at concentrations of 5, 10, or 15 g/kg did not affect total protein or albumin in the serum of broilers. The current effects of different levels of ginger powder on blood cholesterol, triglycerides, HDL, and LDL showed significant results. Our findings are in line with those of Shewita and Taha [17], who showed that lipid profile parameters such as total cholesterol, total triglycerides, HDL, LDL, and VLDL were found to be significantly modulated in the ginger-supplemented groups. These findings could be due to the anti-hypercholesterolemia and hypolipidemic activity of ginger. Dietary ginger acts on total serum cholesterol by inhibition of hydroxylmethyl-glutaryl-coenzyme-A reductase (HMG-CoA), or by increasing the excretion of bile acid and fecal cholesterol. However, Hayajneh [15] observed no significant differences in total protein, albumin, total cholesterol, or triglyceride levels after dietary treatment, but plasma cholesterol was found to be higher in broilers fed a diet supplemented with ginger powder. Ginger-supplemented diets administered over short terms and at low doses have been implicated in lower plasma cholesterol levels. Similarly, Barazesh, Boujar Pour [51] studied the effect of 0%, 0.5%, 1%, and 1.5% ginger powder on the blood parameters of broiler chickens. They found significant effects of ginger on blood parameters, and cholesterol and triglyceride levels. Zomrawi, Abdel Atti [52] studied the effects of 0%, 0.5%, 1%, and 1.5% ginger root powder on the blood and serum constituents of broiler chickens. They observed significant differences in serum triglyceride and cholesterol levels. In addition, inclusion of ginger root powder in the diet at levels of 0.5% and 1% lowered the cholesterol level. Notably, inclusion of ginger in the diet did not cause anemia in the broilers, as evidenced by the lack of a significant effect on RBC counts and hemoglobin concentration. White blood cells and their sophisticated interactions are essential for developing and stimulating immune responses [53]. Tan and Vanitha [54] concluded that essential oil constituents from the rhizomes of Z. officinale exert immune-stimulating effects by enhancing the phagocytic activity of heterophils. Furthermore, Vattem, Lester [55] found that dietary supplementation of Zingiberaceae spices significantly increased the number and viability of coelomcytes, in addition to promoting differentiation into neutrophil-like cells, thus increasing phagocytic activity. Ginger consumed at a level of 100 mg/kg of diet was found to be effective for stimulating innate immunity by increasing the phagocytic capacity of heterophils, and for humoral immunity by increasing the production of antibodies; consequently improving the immunological profile of broiler chickens [56]. Additionally, Azhir, Zakeri [57] found that adding ginger rhizome powder at a concentration of 10 g/kg improved the humoral immunity of broilers at 35 days of age. Nidaullah, Durrani [58] observed that an aqueous extract of ginger rhizome mixed with water acted as an immune stimulant against Newcastle disease and coccidiosis. Ademola, Farinu [38] observed that ginger provided to chickens at a concentration of 1.0% caused a significant decrease in the total number of WBCs. The authors also found that ginger failed to affect the RBCs of broiler chickens. According to Nasiroleslami and Torki [59], the differential count of WBCs was similarly not affected by the dietary inclusion of ginger essential oil. In the current study, the inclusion of ginger in the diet of broiler chickens did not affect the hematological parameters of the birds, except for the total WBC and percentage of heterophils. The total WBC count increased significantly with an increased level of ginger in the diet. This indicates an enhanced immune response of cells involved in the innate and/or specific immune system.Oxidative stress in broilers is associated with a high concentration of MDA and fatty acid peroxidation, due to increased free radicals [60]. Ginger has been shown to enhance antioxidative status. The results of the current study are in agreement with those reported in the literature. For example, Safiullah, Chand [61] reported that the inclusion of ginger powder and ginger essential oil in the feed rations of broiler chickens decreased MDA in liver and sera samples compared to birds fed a control diet. Wen, Gu [20] reported that the addition of ginger extract significantly increased the total antioxidant potential, decreased MDA content, and increased glutathione peroxidase activity in the serum and breast muscles. Interestingly, Mountzouris, Paraskeuas [62] showed that inclusion of a phytogenic premix including ginger and other natural herbs elevated the expression of cytoprotective genes against oxidation. In particular, the cytoprotective genes opposing oxidation were upregulated generally in the duodenum and ceca, and secondarily in the jejunum [62].5. Conclusions Ginger inclusion in the broiler diet can be safely used to enhance the production performance, immune response, and antioxidative status of broiler chickens.

animals : an open access journal from mdpi

10.3390/ani11030708

PMC8002130

The use of research animals is regulated within the EU through Directive 2010/63/EU on the protection of animals used for scientific purposes, as well as through national legislations and guidelines. However, the ethical review process, which all animal research must undergo, has been heavily criticized. This pilot study has analyzed the ethical review process in Sweden, focusing on how well legislative demands are fulfilled by researchers and animal ethics committees. After developing a score sheet, 18 documents (including both applications and decisions) were thoroughly reviewed, and the requests in the application form were compared to legal demands. The results revealed a number of issues concerning how HBA (harm–benefit analysis) was conducted by the committees, application, and review of the 3Rs (Replace, Reduce, Refine), as well as how humane end-points, severity assessment, and the “upper limit” of suffering were implemented and assessed. The study further indicates disconcerting discrepancies between the Swedish application forms for project evaluation, national legislation, and the directive as well as a lack of transparency throughout the review process. These findings risk compliance with the directive, animal welfare, research validity, and public trust. Therefore, a number of suggestions for improvements are provided, and the need for further research is emphasized.

The use of animals in research entails a range of societal and ethical issues, and there is widespread consensus that animals are to be kept safe from unnecessary suffering. Therefore, harm done to animals in the name of research has to be carefully regulated and undergo ethical review for approval. Since 2013, this has been enforced within the European Union through Directive 2010/63/EU on the protection of animals used for scientific purposes. However, critics argue that the directive and its implementation by member states do not properly consider all aspects of animal welfare, which risks causing unnecessary animal suffering and decreased public trust in the system. In this pilot study, the ethical review process in Sweden was investigated to determine whether or not the system is in fact flawed, and if so, what may be the underlying cause of this. Through in-depth analysis of 18 applications and decisions of ethical reviews, we found that there are recurring problems within the ethical review process in Sweden. Discrepancies between demands set by legislation and the structure of the application form lead to submitted information being incomplete by design. In turn, this prevents the Animal Ethics Committees from being able to fulfill their task of performing a harm–benefit analysis and ensuring Replacement, Reduction, and Refinement (the 3Rs). Results further showed that a significant number of applications failed to meet legal requirements regarding content. Similarly, no Animal Ethics Committee decision contained any account of evaluation of the 3Rs and a majority failed to include harm–benefit analysis as required by law. Hence, the welfare may be at risk, as well as the fulfilling of the legal requirement of only approving “necessary suffering”. We argue that the results show an unacceptably low level of compliance in the investigated applications with the legal requirement of performing both a harm–benefit analysis and applying the 3Rs within the decision-making process, and that by implication, public insight through transparency is not achieved in these cases. In order to improve the ethical review, the process needs to be restructured, and the legal demands put on both the applicants and the Animal Ethics Committees as such need to be made clear. We further propose a number of improvements, including a revision of the application form. We also encourage future research to further investigate and address issues unearthed by this pilot study.

1. IntroductionAnimal research procedures are defined in Article 3 p. 1 of Directive 2010/63/EU (henceforth known as “the Directive”). The definition includes the creation of animals through breeding or genetic manipulation but excludes animals euthanized solely for organ or tissue harvest, as well as all procedures less invasive than what corresponds to a needle stick. In Sweden, the definition is wider and dependent on purpose of use rather than level of invasiveness or suffering [1] (Chapter 1: Sections 3 and 4). If the purpose of use is gained knowledge through scientific research, disease diagnosis, drug development, teaching, or other purposes of similar nature, it is considered animal research by Swedish standards, and the animals being used are deemed research animals. There is no requirement of level of invasiveness, and animals for instance observed through behavioral studies, euthanized solely for their organs, or captured and/or killed for species conservation purposes or wildlife studies are included in the definition [1] (Chapter 1: Sections 3 and 4). In the following, we mainly refer to the Directive for general issues, since this is the basis of the Swedish legislation, but also since the Swedish text is not understandable to all readers.Throughout the EU member states, 9,581,741 animals were used in research during 2017 [2]. In Sweden, the most recent statistics are from 2018 and show that 274,655 animals were used that year according to the European definition and 5,801,463 were used that year according to the broader Swedish definition, including test fishing [3].Although the definitions may differ in detail, one pillar upon which they rest remains the same: animals have intrinsic value that should be respected and they should be treated as sentient beings [4] (Article 2 p. 21), [5] (Recital 12), [1] (Chapter: 1 Section 1). According to the globally adopted “Five Freedoms”, animals under human care should be spared the negative emotional or physical states of: hunger, malnutrition, and thirst; fear and distress; discomfort; pain, injury, and disease; as well as the inability to express natural behavior [6]. The knowledge that many animal species feel both positive and negative emotions is central to the animal welfare movement and the regulation of animal welfare through laws and guidelines [7]. However, even though the five freedoms and animal welfare legislation aim to prevent animal suffering, animals can be subjected to pain and potential suffering if deemed necessary, for example in animal research. The Directive states (Article 3 p. 1), and has its parallel in Swedish legislation [1] (Section 36 p. 2), that animal research is justified only if ethically approved by a competent authority. However, some animal research, such as observing wild animals from afar, is exempt from this requirement [8] (Chapter 2: Sections 17–25). Pain, suffering, distress, or lasting harm that cannot be considered necessary is not permitted by the Directive (Article 24 p. 2a). Furthermore, the scientific quality in terms of likelihood of success shall be assessed, in order to ensure the relevance, i.e., benefit, of the procedure (Article 38 p. 1a, 2a, 3a). Hence, it is the task of appointed authorities within the member states to determine which research can be condoned and which cannot (Article 36, 38, 40 p. 1a). It is worth mentioning that expressions of values and ethical stances found in the so-called recitals in the Directive are not in themselves legally binding but serve as guidance and ethical background on which the legal requirements of the Directive are based. In the following, we refer to both recitals and articles for the sake of clarity.1.1. Ethical Review of Animal ResearchAnimal research encompasses many societal and ethical issues and ideals, and there is a global widespread consensus that harm done to animals in the name of science should be regulated and undergo ethical approval before being performed [9]. Within the EU, the Directive sets the ultimate goal of full replacement of procedures on live animals (Recital 10). However, until this goal becomes reality, an impartial ethical project evaluation has to -Include a full harm–benefit analysis (HBA) (Recital 39, Article 38 p. 2d);-Address the 3Rs (the principles of Replace, Reduce and Refine) (Recital 38, Article 38 p. 2b);-Determine the degree of severity (Recital 22, Article 15 p. 1); and-Ensure that no projects are approved beyond an upper threshold of suffering (Recital 23, Article 15 p. 2). The Directive further emphasizes consideration of ethical concerns of the general public (Recital 12) and transparency within the review process (Article 38 p. 4). In Sweden, the contents of the directive have been implemented into national animal welfare legislation (the Animal Welfare Act and the Animal Welfare Ordinance) and a detailed guidance document (SJVFS 2019:9/SJVFS 2012:26), known as the L150, from the Swedish Board of Agriculture (SBA) concerning the use of animals for research purposes. An investigation by the Swedish government was in 2011 established to propose a strategy for national consideration of the 3Rs within the sector of alternative research methods, alongside the implementation of the then newly drafted Directive [10]. Furthermore, in 2017, the Swedish 3Rs Center was founded to function as a resource for knowledge and progress concerning the 3Rs and with the goal of minimizing animal use and suffering in research in Sweden.1.2. Past Critique of the Ethical ReviewHowever, fulfilling the aims of the ethical review has proved challenging, and inadequate ethical reviews are a global concern [11,12,13]. Problems brought to light by these international studies can be categorized into two main issues. The first concerns the content of the ethical review process and the way in which it is conducted, including the inherent difficulty in weighing harms and benefits [14,15,16,17,18] and struggles ensuring that the 3Rs are properly taken into account [19,20,21]. The second issue concerns the transparency and legality of how the process is documented and presented, and the unavoidable subjective elements of the weighing process, which have been discussed as a source of uncertainty [16,22,23].1.3. The Ethical Review System in SwedenIn Sweden, as in many other countries, ethical project evaluations are conducted by animal ethics committees (AECs). Sweden’s first AECs were established by researchers in 1979, and their decisions have been legally binding since 1989. There are six regional committees in Sweden today, each comprised of 14 members, whereof six are researchers or research technicians and six are lay persons to enable public insight. At least one lay person, and maximum two, should represent an animal welfare organization. Additionally, to ensure legal certainty, the chairperson and vice chairperson of each committee are former or practicing lawyers.For this study, we have chosen to analyze the Swedish ethical review process for several reasons. First, multiple previous studies have shown that Swedish AECs may not be as efficient and reliable as intended and that the Swedish method for ethical review may in itself be flawed and difficult to apply [17,24,25,26,27,28]. Second, with a long tradition of including lay persons in their ethical review committees, Sweden makes for an interesting case of the connection between scientific research and public trust. Furthermore, the Directive has been in place for seven years, and the member states are expected to have adjusted well to its framework and requirements. Finally, neither the structure nor the task of the Swedish committees has been revised for many years, and there is a need for insight into how the Directive has been implemented into the work of the Swedish AECs.Based on the requirements set in the Directive, the above-mentioned challenges of conducting an ethical review, and the current international discourse on what a proper HBA entails and the core role of the 3Rs, we have chosen to put special emphasis on the following dimensions: -The amount and quality of information being provided by applicants-HBA performance-The 3Rs-The cluster of humane end-points, severity assessment, and the “upper limit” of suffering-The connection between ethical review and public trust Therefore, these dimensions will be introduced in said order in the sections to follow.1.4. The Importance of InformationFor the appointed authorities to fulfill their task of ethical review, the information provided by applicants needs to be complete, correct, current, and relevant [29]. Without the necessary information on which to base their discussions and decisions, the committees simply cannot do what they have been assigned. A report from the Swedish Board of Agriculture [10] addresses the concern (amongst others) of insufficient information in applications resulting in difficulties ensuring the requirements of the 3Rs are met. Already before the implementation of the Directive, poor application content was argued to cause the Swedish AECs to base decisions on insufficient grounds [30]. A study of how the AECs reviewed proposals concerning the use or creation of genetically modified animals further supported this statement. The applications often lacked information of the animals’ situation and yet they were reviewed, and often approved, by the AECs [31]. Correspondingly, problems with project applications observed in European member states include failing to adequately explain benefits, failing to sufficiently address likelihood of success, failing to sufficiently address the application of the 3Rs, and failing to adequately estimate harms [29] (p. 7–8).1.5. Harm–Benefit AnalysisAll project evaluations within the EU must contain a harm–benefit analysis aiming at approving only projects where the expected benefit, for humans, animals, or the environment, is in balance with the estimated harm to the research animals [5] (Recital 39, Article 38 p. 2d), [32]. As defined by the Expert Working Group for Project Evaluation and Retrospective Assessment established by the Commission in 2013, a proper HBA requires “a good understanding of the nature and impact of the potential benefits, of all of the expected harms to the animals, taking into account all Refinement measures, and the likelihood of achieving the projected benefits.” [29] (p. 4). The same document further specifies the following guidelines for those performing the review: -“Should not automatically assume that claims of potential scientific benefit are always correct;-Should understand all the potential harms to the animals;-Should be prepared to challenge the status quo and to reject poorly designed and ill thought through projects and-Be prepared to challenge cultural/social/political issues e.g., outdated methodologies or views that animals do not need pain relief.” The problems associated with conducting HBAs have been discussed for some time, and ethical advice for addressing its complexity is surprisingly hard to come by [14]. The FELASA Working Group on Ethical Evaluation of Animal Experiments, wrote in 2005 that, “[T]here can be no straightforward ‘algorithm’ for ethical weighing, nor any other quantitative approach that can remove the need for sensitive ethical judgment” [33]. The vagueness of existing guidelines has been welcomed by some authors due to its allowance for interpretation, whilst others have resolved to provide more explicit guidance [14]. In addition to a “heat map model” suggested by the FELASA Working Group on harm–benefit analysis [32], a number of different models for HBA have been proposed over the years in attempts to provide framework or guidance. Among the more renowned worth mentioning are the algorithm models suggested by Mellor and Reid in 1994 [34] (revised in 2004 [35]), Stafleu et al. in 1999 [36], and graphic models such as the “Bateson square” and “Bateson cube” [37,38]. A less well known, albeit more recent, model is Ringblom et al.’s weighting of ethical costs [16].Several years before the implementation of the Directive, the Swedish AECs were considered to be approving applications too light-heartedly and that ethical debate was not given enough space within the ethical review process [30]. A framework for ethical discussion was proposed as well as suggestions of changes to be made to the Swedish Animal Welfare Act and Animal Welfare Ordinance. In 2004, 40 participants from nine countries at a Nordic European Workshop on Ethical Evaluation of Animal Experiments concluded that the weighing that needs to be done is in itself problematic as it requires balancing very different entities against one another [39], which is a conclusion several studies have since agreed upon [14,22,40,41,42]. Another problem discussed was the fact that the two entities do not share a common time interval. The harms may be immediate whereas benefits, if at all achieved, may arise at different times in the future [39].Ethical considerations by Swedish AECs have been deemed too shallow [25], and it has been proposed that committees, in Sweden as well as other countries, may regard information concerning potential benefits as more important than information about the harm and suffering of the animals [23,27]. According to Ideland [26], Swedish AEC members had a tendency to focus on discussing experimental methodology rather than the weighing of harms and benefits during committee meetings. There was also disagreement amongst the interviewees on how to define the ethical questions discussed and who was to gain from the ethical evaluation: the animals, the patients, or science. According to recent studies, Swedish AECs still do not fulfill the inherent criteria of HBA and hence, it is argued, ethical justification of animal experiments is not reached through the current review system [17,43].1.6. Replace, Reduce, RefineThe principles of Replace, Reduce, and Refine were coined by researchers Russell and Burch in 1959 [44] and have since been widely accepted and integrated into the planning, execution, and evaluation of animal research globally. According to the Directive, all EU member states are to promote alternative methods (Article 47 p. 1) and ensure prioritization of the 3Rs (Recital 10, Article 4) by ensuring that sufficient information be provided by researchers upon application (Article 37 p. 1) and that the AECs, or their equivalents, include in their project evaluation an assessment of how Replacement, Reduction, and Refinement requirements have been met (Article 38 p. 2b). An HBA requires the consideration of proposed Refinement measures [29]. A mandatory way of achieving Refinement is through the use of humane end-points whereby euthanasia or amelioration of suffering is carried out as early as possible during a study [5] (Recital 14). Information about the humane end-points chosen is to be specified by the researcher upon application [5] (Article 37 p. 1c).Critique aimed at the implementation of the 3Rs is nothing new. In 1998, a Swedish government-sanctioned report on the use of animals in research concluded that the AECs lacked the competence and resources to determine themselves if alternative methods for proposed research projects were available or not [45]. This left the AECs reliant on information provided by the applicants, which, according to the report, meant that the project evaluation was as such based on insufficient grounds. The report concluded that this dependability would passivate the AECs, jeopardizing the goal of reducing the use of animals in research. Four years later, critique remained and the AECs were criticized of approving projects without having enough information to back their decisions, and the suggestion was made that the AECs should be able to set higher demands on the information submitted by applicants [30].The lack of sufficient understanding and implementation of the 3Rs amongst AECs has also been identified globally. Curzer et al. [14] argue that the biggest problem of the 3Rs is that they fail to explain why researchers should try to minimize harm to animals and that even though there have been discussion of the application of the 3Rs, very little has been published about their key concepts and principles. Graham [46] found in a survey of U.S. Institutional Animal Care and Use Committees (IACUCs) that the majority of participants believed the search for alternatives is most important for research causing more than slight pain to the animals. However, some researchers thought alternatives were not sought for or simply did not exist. Schuppli and Fraser [19] concluded from a survey of members of Canadian ACEs that the 3Rs were rarely mentioned, although some aspects of the concept were applied. Some of the factors hindering the implementation of the 3Rs were incomplete understanding of the 3Rs, a belief that the researchers themselves apply the 3Rs satisfyingly, and lack of consensus on key issues, for example the nature and moral significance of animal pain and suffering. Houde et al. [21] found in interviews that Canadian AEC members mention the 3Rs but that there was still a lack of an understanding of the principles, and they pointed out a need for emphasis on the 3Rs in the AEC members’ instructional training. In a study analyzing membership of IACUCs at U.S. research institutions, Hansen et al. [47] suggested the inclusion of a larger number of scientists who do not conduct animal research, animal welfare experts, and non-affiliated laypeople in the ethical committees to increase attention to and consideration of the 3Rs.1.7. Humane End-Points, Severity Assessment, and the “Upper Limit” of SufferingAfter reviewing the proposed project, including applied Refinement measures such as humane end-points, and the described resulting suffering or harm for each group of animals, it is the AEC’s task to classify any approved project’s level of severity as non-recovery, mild, moderate, or severe [5] (Recital 22, Article 15 p. 1), [1] (Chapter 7: Section 10). Guidelines for the severity assessment are found in Annex VIII of the Directive.Furthermore, it is stated that any proposed procedures involving “severe pain, suffering or distress that is likely to be long-lasting and cannot be ameliorated” are not to be granted approval unless a member state specifically applies for and is granted exemption from this “upper limit” [5] (Recital 23, Article 15 p. 2, Article 55 p. 3), [48] (Section 41c), [49] (Chapter 7: Section 9). This limit could be seen as a special form of Refinement but without allowing for compromise in the pursuit of research goals. Instead, this restriction is absolute [50]. Furthermore, the Directive states that any violation of the ban where experiments have turned out to cause more suffering than legally allowed are to be reported retroactively by the researchers [51] (Annex II p. 7), [8] (Chapter 13: Sections 1 and 3). Up until the writing of this paper, no exemptions from the “upper limit” have been requested by any member state, and as such, no relaxation of the prohibition has been made [52].1.8. Public Interest and TransparencyPublic interest in animal research is generally low [53]. Nevertheless, the public’s views on animal welfare and using animals for human benefit are important influences on how animal research is conducted and what projects are granted approval [54]. In a recent survey of attitudes amongst Europeans toward animal welfare, nearly half of those questioned considered animal welfare to be “the duty to respect all animals”, and 94% of the participants believed it important to protect the welfare of farmed animals [55]. In Sweden, 99% found it important. Despite research animals not being specifically mentioned in the survey, its results indicate a general high regard across all member states for the wellbeing of animals. However, opinions on animal research are often far from unanimous. In a recent investigation of the public’s attitudes toward animal research in Sweden, 55% of participants found animal use for medical research acceptable. If ensured the animals were well cared for and not subjected to unnecessary suffering, acceptance reached 82% [56]. This effect of public opinion varying depending on the circumstances of the research, together with the extensive general interest in animal welfare presented above, indicate the need for deeper and more nuanced ethical reasoning regarding animal research.The Directive promotes public confidence related to the use of animals for research purposes and calls for transparency within the ethical review process (Recital 22, 41, Article 38 p. 4) and the animal research field as a whole (Recital 4, 36). In Sweden, two-thirds (66%) of people questioned trust that animal research scientists correctly abide by the laws and regulations governing animal research, while one-tenth (11%) claim they have little or very little faith that this is the case [56].In order to achieve transparency, it is crucial that the basis of the approval or disapproval is clearly motivated, and hence the authority responsible for the ethical review must describe in detail the facts and considerations that led up to the decision made [57]. The current Swedish Administrative Procedure Act [58] states that “[a] decision which can be expected to affect a person’s situation in a way that is not insignificant shall contain a clarifying motivation, unless this is without a doubt unnecessary” (our translation). It further concludes that the motivation must include applied regulations and decisive circumstances [58] (Section 32). At the time during which the documents reviewed in this study were composed, the law had a slightly different formulation albeit similar purpose. According to the Swedish administrative law in use in 2017, if a governmental decision is made that impacts an individual (such as a researcher applying for ethical approval of a planned research project), the reasoning behind said decision should be made available for scrutiny. However, only decisions that go against the interests of a party need motivating without exceptions [59] (Section 20). Importantly though, this does not mean that favorable decisions never require motivating. Contrarily, decisions that can be viewed as presidential or that may benefit one party at the cost of another may also require motivating [60] (p. 244). It is evident that when, how, and why motivations are required by authorities is not always easily decipherable.2. Materials and MethodsThe documents to be analyzed were obtained through the district courts of the cities where the six Swedish AECs are located: Stockholm, Uppsala, Umeå, Linköping, Göteborg, and Malmö-Lund. The district courts were contacted by email, and all documents were received in digital form. The documents requested consisted of applications that had been sent in to the AECs during the months of March and October 2017 (with one exception, see the first bullet point below) together with their corresponding decisions. These particular dates were chosen for the analysis for the following reasons: One member of the research group was appointed committee member of Göteborg AEC in November 2017. Therefore, to rule out bias, no documents analyzed were to have been received by the AECs later than October 2017. Before that date, the member in question had no involvement in the committee’s work. For this reason, documents were requested from Göteborg AEC from March and September, so as to ensure that these had finished being processed when said researcher began their assignment. As the AECs meet once a month, this was deemed an appropriate time margin.Applications and decisions dated after the implementation of Directive 2010/63/EU had to be chosen, and preferably from as recently as possible so as to allow for any initial “breaking-in period” regarding implementation of the Directive to have passed and the AECs to be as comfortable and settled in their assignment as possible.March and October have been shown to be two months of the year during which a large number of applications usually reach the AECs (Karin Gabrielson Morton, Expert and Senior policy advisor at the Swedish Fund for Research without Animal Experiments, personal communication, 12 January 2020), thereby allowing for a greater starting population to select from. We analyzed three documents per AEC. These were selected through a randomized and blinded process for each AEC, i.e., applications were not grouped in relation to content or any other category. First, the total number of documents deemed appropriate for review from each AEC was counted. Then, each document was assigned a number within that range, and three numbers were randomly selected by a third party for analysis.Then, the 18 documents selected were analyzed with regard to how well their content responded to legal requirements of the L150 (SJVFS 2012:26). An Excel sheet was used with the legal requirements listed whereby each document was revised to determine if it could be ascertained from the contents of the application or decision that the requirements had been met (Table S1). Depending on how well the contents fulfilled the requirements, this was categorized as Y (Yes), I (Incomplete/Insufficient/Indeterminable), or N (No). To ensure continuity and transparency of the analysis, a guide of how the requirements were to be judged and categorized was conducted before the review started (Document S1). For cases where it was unclear how certain criteria put forth by the Directive should be interpreted, the research group detailed their chosen interpretation in the aforementioned guide.The performance of an HBA by the applicant is encouraged by the PREPARE guidelines but not specified by the Directive or the L150 document [61]. However, it is asked for in the Swedish application form. Hence, this has been included for analysis by the present research group, as it was considered necessary for the interpretation of the rest of the results and conclusions of this study to know the full extent of the information provided for the AECs by the applicants.The extent of argumentation (or justification) of AEC decisions is, other than the requirement for transparency (Article 38 p. 4), not detailed in the Directive but in administrative legislation. Therefore, we have analyzed the decisions according to the Administrative Procedure Act of 2017 [59] (Section 20) with the guidance of Hellners’s and Malmqvist’s book on the subject [60]. There is a clearly labeled section within the decision template where the AEC’s justification and decision are to be specified, and so, this part of the decision has been analyzed for this particular requirement.To increase scientific validity, the selected documents were analyzed a second time regarding a selection of legal requirements deemed especially important for the study (concerning HBA, the 3Rs, and scientific and humane end-points) by another scientist. So as to avoid influencing the outcome, this person was not privy to the other’s observations, but they were familiar with animal-based research. Then, the two sets of results were compared, and any dissimilarities were discussed and reviewed again using the aforementioned guide (Document S1) until consensus was reached. The final results can be seen in full in (Table S1).Seeing as this study was based on documents from 2017, their contents have been compared to the legal requirements of legislation and guidelines present and in use at the time of application. Thus, unless stated otherwise, the legal documents and guidelines referenced throughout this paper are the versions valid between September and November 2017. Any subsequent changes of importance for the discussion or conclusions of this paper will be clearly stated and referenced.3. ResultsA total of 131 documents were obtained: 13 from Göteborg, 16 from Linköping, 32 from Malmö-Lund, 26 from Stockholm, eight from Umeå, and 36 from Uppsala. The annual registers for 2017 from each AEC were also received from all district courts except from Linköping, who gave no reason despite numerous requests.All documents were revised upon procurement to guarantee that only those including both applications and decisions were ultimately selected. A total of 13 documents missing pages or judged otherwise incomplete were discarded, in spite of several requests to obtain complete review documentation. Decisions both with and without the “The AECs Decision—Non-Technical Project Summary” form supplied by the SBA were included for analysis. To ensure the applications and decisions would be as similar in structure to each other as possible and thereby comparable despite being randomly selected, only initial applications (in Swedish: grundansökningar) were included in the draw. For clarity and scientific validity, an initial application was deemed as an application which the AEC upon review had categorized as belonging to category three or four as defined by SJVFS 2008:19 Section 2a, namely that they were self-standing new applications and not changes to pre-existing approvals. A total of 39 documents were excluded for not fulfilling this criterion. Thus, a total of 78 documents were included in the draw. Out of these, all but one research proposal had been approved by the AECs.3.1. Analysis of the Ethical ReviewFor the sake of clarity, the results from the 18 closely analyzed documents are presented separately for the analyses of applications and decisions. The results for the applications are divided into those obtained from the main “technical body” of the application followed by those from the non-technical project summary (NTPS). For the sake of clarity, the order in which the legal requirements observed are presented here differs from the order in which they appear in the L150 (SJVFS 2012:26) and Excel sheet. Instead, they have been regrouped to facilitate for the reader.3.2. Analysis of Applications3.2.1. The Main “Technical” Body: Content Provided by the ApplicantsA description and motivation of scientific end-points were satisfactorily provided (Y) in all but one application, which was instead judged as I (inadequate) for this criteria.Description and motivation of humane end-points were included satisfactorily (Y) in 12 out of 18 applications, judged insufficient (I) in five, and completely lacking (N) in one. The demand for clear criteria of when said humane end-points are to be considered reached was fulfilled (Y) in nine out of 18 applications, whilst criteria were deemed indeterminable or insufficient (I) in seven and not provided at all (N) in two.How the animals’ pain, discomfort, or other suffering is to be observed and determined was only sufficiently described and motivated (Y) in three out of 18 applications. In six out of 18, it was judged insufficient (I) and it was not mentioned at all (N) in half of the applications (nine out of 18).Five out of 18 applications clearly described the need for monitoring of the animals (Y), while seven out of 18 were insufficient in their descriptions and/or motivations (I), and six applications failed to mention it at all (N).The application form further requests that the applicant provides an account of how they have reflected when deciding that the benefit of their proposed study surmounts the harm inflicted on the animals: in essence, their HBA. For practical reasons, we have divided this one point in the application form into two, judging the mention and deliberation of benefit and harm separately. In seven out of 18 applications information on harm was judged satisfactory (Y), whilst it was unsatisfactory (I) in five and not mentioned at all (N) in six. The reasoning around benefit was deemed satisfactory (Y) in nine applications, unsatisfactory (I) in six, and not included (N) in the remaining three.3.2.2. The Non-Technical Project Summary: Content Provided by the ApplicantsCertain legal requirements specifically regard the NTPS section of the application. For example, it is within the frames of the NTPS that the researcher is requested to describe their application of the 3Rs. For two of the 18 applications, a non-technical project summary was not included at all. Hence, it should be kept in mind that these will represent two of the N for each of the requirements below.The non-technical project summary (NTPS) should state the number and type of animals requested for use. In 16 out of 18 applications, this was done as required (Y).Within the realms of the NTPS, the applicant is further required to inform about the aim and benefit of the project as well as the suffering of the animals. In the vast majority of all applications (16 out of 18), the aim of the proposed project was well defined (Y). Half of all analyzed applications satisfactorily informed about the benefit of the project (Y), seven informed but unsatisfactorily (I), and two lacking an NTPS did not share any information of this at all (N). Regarding the harm done to the animals, seven out of 18 applications gave sufficient information (Y), another seven gave insufficient information (I), and the remaining four did not inform about the harm at all (N).Regarding the 3Rs, half (nine out of 18) of the applications contained satisfactory accounts (Y) of how Replace had been achieved, whilst seven only gave unclear or insufficient accounts (I), and two failed to mention this at all (N). For Reduce, seven applications contained satisfactory accounts (Y), eight contained unsatisfactory accounts (I), and three had no information regarding this at all (N). Similarly, seven applications gave satisfactory information (Y) about Refine, six gave insufficient information (I), and five gave no information at all. There were no patterns indicating that the satisfactory inclusion of one R in an application was consistent with the other Rs having been deemed satisfactory for said application as well, nor that applications failing to account for one R would do so to a greater extent for the other Rs, too.Furthermore, Reduce and Refine were occasionally confused with each other by the applicants. In three out of the 15 applications describing Reduce (graded as Y or I), Reduce was described under the Refine section, and in two out of 13 applications (likewise graded Y or I), Refine was described under the Reduce section. Replace was, if graded as Y or I, described under the correct section throughout.3.2.3. The Non-Technical Project Summary: Content Provided by the AECsThe AEC is required to amend the NTPS submitted by the applicant with a definite degree of severity, any changes or additions needed for project approval, and a detailed account of whether the proposed project is to be subject to retrospective assessment or not. This can, but must not, be performed in a template designed for this purpose. The results were the same for all of these three requirements respectively. Four out of 18 applications were categorized as Y, none were categorized as I, and the remaining 14 were categorized as N. All decisions marked N were so labeled as they did not contain the form “The AECs Decision—Non-Technical Project Summary” nor any other document indicating that the criteria listed above had been met.3.3. Analysis of DecisionsIn a vast majority of the decisions (16 out of 18), the principal investigator and director were specified (Y). The remaining two, both from the same AEC, failed to disclose this information altogether (N). In addition to this, the same decisions by the same AEC also failed to disclose the location of the proposed study (N), as did the third decision by said AEC. All remaining 15 decisions from other AECs contained the location information (Y).All 18 decisions contained a specific section labeled “Terms of decision” and were thereby, regardless of what terms were listed therein, considered to live up to the requirement to disclose the terms of approval specified by the AECs (Y). Similarly, all 18 decisions contained a final assessment of the level of severity (Y).To analyze the presence, or lack thereof, of differences of opinion (meaning that one or more of the committee members disagree with the committee decision), the categorization of Y, I, and N had to be carefully defined and differed somewhat from the demands set for other requirements (see Document S1). Nonetheless, one decision contained a difference of opinion clearly stated (Y). Three decisions, all from the same AEC (not the AEC mentioned in the first paragraph of this section), referred to an appendix of the decision protocol for differences of opinion but did not include this in the documents initially requested by the research group, whereby these were marked as I. The district court did not supply the protocols at a later point of the study despite being asked specifically and repeatedly to do so. The remaining decisions did not include or indicate the presence of any differences of opinion (N).Replace, Reduce, or Refine were not included in any of the 18 decisions analyzed. Consequently, all 18 decisions failed to fulfill this criteria (N).As for the applications, harm and benefit in the decisions were judged separately. In four out of 18 decisions, harm was satisfactorily mentioned and described (Y), whilst in another four decisions, harm was not mentioned at all (N). The remaining 10 decisions simply included a boilerplate stating that, “The committee considers the importance of the project to outweigh the suffering of the animals” (our translation). This was marked as insufficient (I), as it was considered a statement of the outcome of the decision rather than the reasoning behind the weighing of harms and benefits. Likewise, four out of 18 decisions satisfactorily mentioned and described the benefit (Y), four did not mention it at all (N), and the remaining 10 included only the aforementioned boilerplate (I). The decisions that fulfilled the criteria (Y) for the description of harm were also the ones that fulfilled the criteria (Y) for benefit. The same correlation was true for the decisions that failed to describe harm or benefit.Out of the 18 decisions analyzed, four satisfactorily motivated the decisions in accordance with administrative law (Y), whilst three included no motivation at all (N). Three out of the four satisfactory motivations were provided by the same AEC (meaning that all reviewed decisions by said AEC fulfilled this criteria), and the fourth decision provided by another AEC was judged satisfactory due to the inclusion of terms in the approval and as such there being more information given than the standard boilerplate. Out of the remaining 11 (I), eight only included the aforementioned standard boilerplate, whilst three did not contain a motivation per se but a reference to the review panel documents. The review panels are to submit a written and motivated proposition, or if more appropriate, a written decision basis for the AEC to base their final decision on. In 16 out of 18 cases, a written document was produced and submitted (Y), and in two, there was no record of such (N). Half of the 16 written documents (eight out of 16) included a satisfactory motivation (Y), one included a brief standard motivation deemed unsatisfactory/incomplete (I), and seven contained no motivation at all (N).3.4. Additional ResultsBased on the material obtained for the study, we gained results outside the pre-established foci, whereof themes relevant for a more comprehensive understanding of the results presented above are presented in the following.3.4.1. Discrepancy between Law and Application FormWhen creating the Excel sheet used for the analysis, it became clear that there is a discrepancy between the demands set by legislation and the questions asked in the application form. For example, that the applicant accounts for how the animals’ pain, discomfort, or other suffering is to be observed and determined is a specific requirement detailed in the L150 [62] to be considered, described, and motivated. However, even if a description of the potential impact on animal welfare is requested by the form, there is no question or section in the application form regarding method for welfare assessment or scoring of potential impairments (aka the how in question). Similarly, the form does not include a request for information about the specific judgment criteria for humane end-points, nor the consideration, description, and motivation of the need for monitoring—although this is requested by the L150 [62]. In addition, as mentioned above, how the applicant has taken the 3Rs into consideration is only given specific space to be described within the NTPS, and not asked for elsewhere by the form, i.e., risking omitting details relevant to the AEC. In neither the Directive nor the L150 [62] is this placement referenced. Thus, there are gaps between the requirements of the legislation and the information requested in the application form in several points related to core values of the ethical evaluation and decision-making process.3.4.2. Inconsistencies in Documentation and Archiving of DocumentsAs stated in Article 45 of the Directive, “all relevant documentation” concerning the ethical review should be kept for at least three years from the expiry date of the project authorization. All AECs seemingly fulfill this time frame criteria at a first glance, as the research group was in the spring of 2020 able to request and receive documents from 2017. However, exactly what documents had been saved and were made available for our study differed between committees. For example, in only two out of the six committees was the form “The AECs Decision—Non-Technical Project Summary” included amongst the saved documents. This form is specifically tailored by the SBA to enable the committee to add to the NTPS a degree of severity, requested changes (if any), and the possible need for retrospective assessment. Since the form was not included and as such seemingly not used by the other committees, they were judged as failing to live up to these demands because it was not ascertainable whether or not they had revised the NTPS. As no other documentation was provided to show that revision had taken place, the legal requirements were not met. Furthermore, the filing order of the different sections of the documents was inconsistent both between AECs and between handled cases within one and the same AEC. Early and final versions of applications, additions, or alterations made per email, decisions to postpone or to approve, review panel proposals, and the few “AECs Decision—Non-technical project summaries” received by the research group were inconsistently arranged by the different district courts. Due to this, a thorough review was needed in order to know for sure whether the documents received were in fact complete. As demonstrated by the need for an initial review of the documents described under “Methods” above, individual or multiple pages had on occasion been left out, rendering these documents useless for analysis. One district court was asked by the research group to provide the protocols from two AEC meetings as references had been made in decisions that differences of opinion could be found there. However, the district court did not grant this request and gave no reason. Therefore, it is unclear if said notes were saved or not, assuming that the AECs had kept minutes as stated and legally required [62] (Chapter 7: Sections 12 and 13).3.4.3. Confusion Around the “Upper Limit” of SufferingDuring the study, we came across the discussion of how the “upper limit” of suffering [5] (Article 15 p. 2), [48] (Section 41c), [49] (Chapter 7: Section 9) should be interpreted and applied. Statistical documents as well as correspondence with the National Animal Ethics Committee (NAEC) revealed that Sweden up until the writing of this article had not once handled a case where a project had been considered to exceed the upper limit [3].The Working document on a severity assessment framework supplied by the Commission [63] includes only one brief mention of the upper limit. The document provides an example of a study of severe long-lasting suffering caused by arthritis in mice, which could be “authorised subject to an otherwise positive project evaluation including review of the harms and benefits” [63]. Apart from this example whereby “a number of weeks” is evidently considered prolonged (for this particular scenario), no definition or precedent of what constitutes “long-lasting” or “severe” with regard to Article 15 p. 2 exist as far as the research group have been able to find out. Nor is “cannot be ameliorated” defined and so to what degree the suffering has to be lessened is unclear.3.4.4. Unclear Rules of Motivation of Authoritarian Decisions and Transparency RequirementsAs mentioned in the introduction, the reasoning behind decisions made by authorities should be made clear, at least when the authority has ruled against the interests of a party, the outcome benefits one party at the cost of another, or when the decision could be considered especially important for the ruling of future cases of similar nature [59] (Section 20), [60] (p. 244). However, exactly how this should be applied to the decisions made by AECs seems unclear, since there was no consistency amongst the documented decisions analyzed in our study as to what extent a motivation was included or what it should contain. In addition, the SBA has presented no guidelines regarding this point. Correspondingly, there exists no clear definition or guidelines determining how the transparency required by the Directive should be achieved.4. Discussion4.1. Harm–Benefit AnalysisIn order for an HBA to be performed correctly and live up to its purpose, all harm needs to be known by its performers [23], [29] (p.4). Correspondingly, so do the benefits of the proposed research and the probability of their occurrence [23,64,65,66]. To aid in assessing the level of harm of a project, the reviewing authorities may look to the classification scheme in Annex VIII of the Directive. However, the Directive does not provide guidance for assessing proposed benefits [42]. As it was already in 2004 found that costs (today “harms”) were in general easier to identify and define than benefits [39], this is surprising. However, contrarily, in human clinical studies, harm has instead been regarded as the more difficult of the two to identify [23]. To our knowledge, the reason for this difference is unknown. One way to facilitate the weighing of harms and benefits could be to develop and ensure the use of proper professional terminology for benefits similar to that which already exists for harm, as suggested by Brønstad et al. [23]. This would avoid discussions of benefits from becoming too general and allow for a more nuanced and exhaustive ethical debate. The risk of the HBA being constrained by “situated ethics”, whereby ethics is viewed as something that is contextually and situationally negotiable [26], would also decrease if the committee members use the same language, so to speak.In addition, a further point of discussion lies inherent in the requirement to perform an HBA, namely how to handle the general claim of performing an ethical evaluation, which can be regarded to either cover all but only measurable points usually considered in an HBA, or, on the other hand, understood as an expectation to build the ethical analysis on a wider understanding of harms and benefits. This can at times be expressed as a need to go beyond the HBA to ensure that a proper ethical evaluation is performed. As put forth by Alzmann [22], a weighing of competing interests can only be carried out if all aspects relevant for the case are considered, and so, a comprehensive analysis of considerations should always precede the weighing. The parties responsible for assessing the ethics of a proposed research project should according to Alzmann “employ a comprehensive and thorough approach that includes but is broader than harm–benefit analysis” [22]. Correspondingly, Röcklinsberg et al. [27] concluded that the authorities responsible for conducting the ethical review must widen the aspects included in the decision-making process to be able to live up to the intent of the legislation. Alzmann [22] further argues that there are ethically relevant aspects beyond the procedural suffering, pain, distress, and harm of the animals and the benefits for humans, animals, or the environment as formulated by the Directive, and that there is need for the development of a catalogue also encompassing such things as husbandry conditions and non-physical suffering. Since the HBA should take all harms into account, it is reasonable that the possible pain, stress, or suffering caused not only by the procedures themselves but also by handling of the animals, cage conditions, social isolation, gene modification, and other parts of husbandry be considered. A number of catalogues have been suggested, and all include the same and/or similar criteria to be evaluated, but a catalogue that is both practical and exhaustive is yet to be developed [22].Furthermore, it is important to note that harm or suffering does not have to be physical [14]. On the contrary, the Directive repeatedly emphasizes “distress” in animals and the definition of suffering provided by the L150 (Chapter: 1 Section 8) clearly states that psychological harm or suffering is included in the concept [8,62]. Despite this, we found it rarely mentioned by the applicants and seemingly often overlooked by the AECs.According to the PREPARE (Planning Research and Experimental Procedures on Animals: Recommendations for Excellence) guidelines [61], the researcher should perform a harm–benefit assessment and justify any likely animal harm. However, since our study shows that the harms were not always properly accounted for by the applicants and applications have been approved nonetheless, this indicates that the AECs might not have the knowledge or guidance as to what constitutes the concept of harm and what information they should be receiving from the applicants. Additionally and likewise alarmingly, seeing as the manner in which the ethical weighing is performed is of great importance [22], studies have shown that how harms versus benefits are balanced is inconsistent and that there are discrepancies amongst the performers as to which benefits may justify which harms [26,67]. As a consequence of the lack of visible HBAs in the decisions we have analyzed, our study can neither confirm nor deny that this might be occurring amongst Swedish AECs. Seeing as the present review system in Sweden can be argued as failing to achieve ethical justification for animal research [17,43] and applications are continuously approved on insufficient grounds, as revealed by the results of our study, the need for further review of the process and suggestions for how to improve and facilitate the HBA process is pressing.One may debate if the inclusion of ethical reasoning by the applicant is solely positive or if it may in fact also be counterproductive. If the applying researcher includes an ethical reasoning and it is viewed as comprehensive enough by the AEC, as was the case in the majority of applications in our study both regarding harm and benefit, this on the one hand proves that the applicant is aware of existing ethical dilemmas, is able to reason around them, and reach a conclusion of ethical justification. However, on the other hand, if the AEC is too reliant on the information given and too readily accepts the applicant’s arguments and viewpoints as true, the committee is at risk of passivation of a similar kind to that discussed in 1998 [45]. The Swedish AECs were already almost two decades ago criticized for not placing enough emphasis on the ethical debate [30]. Hence, we argue that there is a risk that they may base their own ethical reasoning and justification off of the ethical standpoint of the applicant. In line with the urging by the Expert Working Group for Project Evaluation and Retrospective Assessment [29] (p. 4) that the AECs “should not automatically assume that claims of potential scientific benefit are always correct”, we argue that they should avoid accepting the ethical reasoning of the applying researchers too light-heartedly. The inclusion of a full HBA by the AECs in the decisions of project evaluation should be self-evident. However, the theory of passivity is contradicted in our study by the fact that the AEC, which had included satisfactory HBAs in all their decisions, had reviewed applications containing generally very satisfactory ethical reasoning by the applicants.Even if all the needed information for the HBA is supplied, the weighing process in itself is far from straightforward, and the ethics involved in balancing harms versus benefits are not clearly identified [14]. According to Schuppli [67], neither harms nor benefits are really quantifiable nor in commensurable units, which is highly problematic. To balance interests against each other, one has to consider the principle of proportionality whereby the weight or significance of the conflicting values is determined so that one may know how to balance the scales [42]. However, the weighing of harms and benefits in animal research can, as previously mentioned, be likened to the comparing of apples and oranges [14,22,23,39]. Within the ethical review of animal research, non-human interests have to be dealt with in a legal context more or less solely catering to the interests of humans [14,42]. That is, the animals’ situation is never in its raw form truly comparable to the other side, as non-humans do not have the same legal status as humans [40] and, as emphasized by Orlans, almost all the harm is on the animals’ side whilst all the benefit is on the humans’ [68]. As animals are not seen as worthy of equal consideration to humans, the weighing of animal interests against human interests is troublesome [40]. Not only does the weighing thereby metaphorically concern different kinds of fruit, it is arguably inherently biased, as an apple will always weigh less than an orange. Furthermore, HBA means balancing harms that are more or less guaranteed, perhaps not regarding their extent but their occurrence, against benefits that may well be anticipated but cannot truly be defined until after the experiments have been conducted [66]. Similarly, harms will occur at certain calculated moments and last for fixed periods of time, whilst benefits may occur at unknown points in the future or never at all [39]. This imbalance is most relevant when assessing proposals of basic research seeing as possible benefits of the acquired knowledge are unknown until the research has ended [42,66]. Curzer et al. [14] illuminated the difficulties of classifying knowledge as a benefit by raising questions such as “What counts as knowledge?”, “How are different bits of knowledge to be compared?” and “How are the bits of knowledge interconnected?”. They argue that the meaning of knowledge is as vague as that of harm, and that it is inevitable that different appointed authorities will evaluate the terms differently from each other.Another argument put forth against the usefulness of the HBA is that placing an absolute cap on the amount of suffering allowed contradicts the consequentialist system of harm–benefit analysis as a tool for ethical evaluation [50]. Although, theoretically, the concept of HBA should mean that any amount of suffering could be outweighed by even greater benefits, we do not agree that this is a valid argument for abandoning the use of HBA altogether because: (a) if clearly defined and harmoniously interpreted, the “upper limit” should not pose a threat to the weighing of harms and benefits but rather work as a beneficial complement to improve Refinement; (b) some actions simply cannot be justified no matter the benefits reaped due to the grave moral or ethical consequences (such as public distain). However, the question of whether an “upper limit” can be applied to the amount of harm/suffering allowed matters little if the process for ethical review in itself does not work (as demonstrated above). For example, if the AECs are not provided access to all aspects of harm, they will not be able to determine the degree of severity and will hence for especially invasive procedures not be able to tell if the upper limit should in fact be considered breached and an HBA not be performed at all. Therefore, it is vital that the ethical review process, including methods for HBA and applicability of the upper limit, is thoroughly revised sooner rather than later and preferably on an international level.It is unknown from our study if the AECs that did not include HBAs failed to do so due to ignorance, the aforementioned possible passivity, lack of knowledge about what was required of them with regard to transparency, or perhaps as a direct result of not having included sufficient ethical discussion of harms and benefits during the reviewing process. To know this, further research is needed, perhaps including in-depth surveys or interviews of the AEC members and/or auscultation of committee meetings.4.2. Replace, Reduce, Refine4.2.1. Content Provided by the ApplicantsNot more than half of the applications included satisfactory descriptions on how the different Rs had been achieved (within the NTPS where this was specifically requested). This result suggests that the applying researchers inadequately considered the 3Rs throughout the planning of their projects and hereby did not fulfill the intentions of the legislation. There may be several explanations for this.First, the applicants may not have enough knowledge of the 3Rs and how to apply these principles throughout the research process, or perhaps they underestimate the importance of their inclusion. The ultimate goal of the EU Directive is a complete replacement of research animals. In line with this, Curzer et al. [14] and Franco et al. [69] stressed the importance of the hierarchal application of the 3Rs proposed by Russell and Burch [44] where first priority should be to Replace, followed by Reduce, and lastly Refine. Animal researchers have an overall good knowledge and a positive attitude toward the 3Rs [70,71,72,73]. The 3Rs should be considered throughout the entire research process, including application writing and ethical review [74] and according to Curzer et al. [14], the “should” expresses a moral obligation of researchers to minimize harm to animals. In a survey amongst Dutch researchers in 2011, the majority of the 46 respondents answered that the 3Rs play a role in the application process [72]. Approximately half emphasized Replacement, while Refinement and Reduction were considered relevant by nine out of 10. Schuppli and Fraser [19] found that the majority of questioned Canadian researchers considered the 3Rs equally important. More recent studies show that researchers seem to be least in favor of Replacement whilst finding Refinement the most feasible or important to apply [69,72]. However, our results do not seem to mirror this attitude, as half of the applications contained satisfactory accounts of Replacement and approximately one-third described Refinement satisfactorily. However, to be able to say with scientific certainty if such is truly the case, a larger sample population is needed. In addition, the possible impact on the results of the 3Rs being confused with one another by some researchers, as seen in our study as well as others, would need to be investigated [70,73]. Interestingly, in our study, these mix-ups have not been corrected by the AECs in the documentation of their decisions. Schuppli and Fraser [19] suggested that Refinement may be difficult to understand because of its various applications. Proper definitions are crucial for an appropriate review, and these findings highlight the need for increased understanding of the 3Rs among both applicants and AECs. In what way knowledge and attitude among Swedish researchers today may affect the description of the 3Rs in the AEC applications warrants further research.Second, the applicants may not be aware of how to describe the application of the 3Rs in their research. Guidelines on how to plan research with animal use, such as PREPARE [61], can aid both the application and ethical review process, and thorough planning by researchers can assist AECs in their assessments of research projects. This will contribute to less wasteful use of animals, which is an important part of the implementation of the 3Rs [61,74,75]. However, if the researchers are not adequately guided as to how to provide the needed information to the AECs, or what constitutes said information, for example through application forms not corresponding to legal demands, their task is not an easy one.Third, the NTPS provides information adapted to the layman, and the applicant may therefore intentionally not include a detailed, technical description of the 3Rs in this section. Hence details are instead often spread out under different headings in the “technical body” of the application form, however without a clear reference to the 3Rs. This leads to a more difficult evaluation by the AEC and challenges the validity of the ethical review as the AEC risks missing important information. As studies have shown, the AECs are often short on administrative staff for the preparation of documents as well as meeting time, and hence inconsistencies as to where in the applications information of certain importance may be found can only be expected to strain their resources further.4.2.2. Content Provided by the AECsThe AECs are responsible for ensuring that the 3Rs have been duly considered and applied throughout the proposed study by the applying researchers. Varga [41] suggested that the prevention of harm, i.e., Refinement, is the most important duty of the AEC and that animal suffering is a potential measure for outcome assessment of the performance of AECs. However, as previously mentioned, the AECs cannot fulfill their task without access to the information on which they are to base their decisions, and as shown, this information is sometimes lacking or scattered due to the structure of the application form, which increases the difficulty of gaining a good overview of the researcher’s account of harm and the 3Rs. The AECs need to understand the inflicted harm, i.e., having an understanding of the 3Rs, particularly Refinement [29] (p. 4). The unsatisfactory descriptions of harm in the HBAs, together with omission of the 3Rs in the decisions by the Swedish AECs included in our study, give the impression that in general, this may often not be the case. However, in a previous study, Hagelin et al. [76] found that the majority of modifications of applications requested by Swedish AECs were in fact related to Refinement. Furthermore, it is clear that Refinement and Reduction often go hand in hand [77], but in some circumstances, Refinement measures minimizing animal suffering may only be only feasible if a larger number of animals are used [69,78,79,80]. Thus, evaluating the 3Rs is not an easy feat and requires balancing the Rs against each other. Nonetheless, it needs to be done. Not in any of the decisions analyzed in our study have the AECs mentioned Replace, Reduce, or Refine. Consequently, all 18 decisions failed to fulfill these criteria. Both Houde et al. [20] and Schuppli and Fraser [19] noted that AEC members bring up the 3Rs during AEC meetings, and it cannot be ruled out that this is occurring also in the AECs participating in our study. However, it was not documented in the decisions and the lack of headings and space reserved for the 3Rs in the decision template do not facilitate the inclusion.It may be possible that the AECs, similar to the applying researchers, may lack the proper understanding of how to apply the 3Rs. Alternatively, the AECs may not be clear as to which extent the 3Rs need to be included in the decisions and HBAs. Schuppli and Fraser [19] found that some AEC members trusted that researchers considered and applied Reduction and Replacement sufficiently themselves, which may hinder the consideration of the 3Rs by the AECs. Incomplete understanding of the 3Rs, lack of consensus on key issues such as the nature and moral significance of animal pain and suffering, were also considered to impede the 3Rs’ implementation. Furthermore, studies have shown that AEC members may have doubts about the applicability of Replacement, e.g., when alternatives are sought for full body models [19,81], creating obstacles for its implementation by the AECs [19]. The authors also described five aspects limiting the discussion of Reduction by AECs during the review: emphasis on sample size rather than experimental design; lack of expertise to critically evaluate numbers of animals; confidence that researchers apply Reduction adequately themselves; confidence in the scientific peer review by granting agencies; and the concern of harm over numbers. The lack of inclusion of the 3Rs within the decision is not only an omission of the 3Rs per se, but it may mirror an inadequate consideration of the 3Rs throughout the review process.Despite our choice to address them separately in this article, there is no clear line separating the 3Rs from the HBA. Rather, they are very much entwined [14]. The 3Rs shall, in addition to improved animal welfare, provide a high scientific quality with benefits for society. Graham and Prescott [79] gave examples from research using animal models of disease on how appropriate model selection, proper study design, questioning the value of translational research, and using less stressed animals provided more reliable results, ultimately contributing to the anticipated benefits for society. As such, approaching Refinement from a broad perspective that considers the animal’s interest as well as promotes the scientific objective both decreases harm and increases benefit [79]. However, this balance is difficult and requires skills in modern research, animal science, animal welfare, and applied ethics, because defining “unnecessary suffering” depends on the interpretation of a series of complex factors [27]. Thus, if the AECs are discussing technical and medical terms rather than animal welfare and ethical issues [26,27,82], this may be hindered. Clearly, similarly to the applying researchers, the AECs need to not only ensure that the 3Rs have been applied but consider them in their harm analysis, as well as in their benefit analysis.4.3. Humane End-Points, Severity Assessment and the “Upper Limit” of SufferingDirectly linked to the level of harm, the application (or lack thereof) of the 3Rs and HBA are the use of humane end-points, severity assessment, and the “upper limit” of suffering.The severity assessment “shall be based on the most severe effects likely to be experienced by an individual animal after applying all appropriate refinement techniques”, and additional factors such as humane end-points shall also be taken into account [5] (Annex VIII Section II). The Directive proposes the use of “early and humane end-points” (Recital 14, Article 13 p. 3), and an account of planned humane end-points as a Refinement strategy is explicitly requested by the L150 [62] (Chapter 3: Section 4). Despite this, progress in implementation of humane end-points has been slow in several areas of research concerning severe diseases such as sepsis, cancer, and sclerosis [50]. Amongst the reasons given for this reluctance to change is peer pressure to comply with established norms. However, arguments have also been made that earlier and more humane end-points results in more precise scientific results as data is collected before the animals observed deteriorate, become moribund, or die [50,79,83]. In our study, humane end-points were only satisfyingly described and motivated in two-thirds of analyzed applications, and clear criteria for end-points were provided for just three quarters of those. Hence, not all applicants fulfill their task of completing the form as requested, and in turn, the AECs determine levels of severity on the basis of insufficient knowledge about the animals’ situation. Even though the reasons behind the lack of sufficiently described humane end-points are unclear and there may be several, the consequences are obvious and grave. Humane end-points are a mandatory part of Refinement and, as previously mentioned, of great importance both to avoid unnecessary animal suffering and to ensure reliable scientific results. Therefore, their exclusion from the ethical review can be expected to have a negative impact on animal welfare, research validity, and in the long run, public trust in animal research.When determining the appropriate degree of severity for a proposed project, the Directive states that, “The severity of a procedure shall be determined by the degree of pain, suffering, distress or lasting harm expected to be experienced by an individual animal during the course of the procedure.” (Annex VIII). Thus, being aware of the full extent of the harm inflicted on the animals is pivotal, something we found that the documentation of the AECs decisions could not show was the case. As a result, there is a risk that some degrees of severity determined by the AECs may have proven to have been incorrectly anticipated once the research was conducted. However, to determine if this was in fact the case, one would have to cross-reference AEC decisions with retrospective reviews by the NAEC, which is something we have not been able to do within the confines of this project but would highly recommend be done. However, it is worth noting that only projects of the highest degree of severity are currently mandatorily reviewed by the NAEC and would therefore be the only ones that could be scrutinized in this way. It is our opinion that comparing the decisions with the researchers’ own reporting of “actual level of severity” would not be scientifically just, because there is a risk of bias in the researchers’ reporting. Additionally, as will be further discussed below, we believe the reporting of animal research statistics by researchers is unreliable and in need of revision, specifically concerning severity assessment.There is an absolute “upper limit” to the amount of pain, suffering, or distress an animal may be subjected to in the name of research [5] (Article 15 p. 2, Article 55 p. 3), [48] (Section 41c). However, what value does a strict “upper limit” hold if those abiding by it do not know how to apply it? Unfortunately, we received no answer from the NAEC or the SBA as to how the AECs are to interpret the “upper limit” in Sweden. However, this is not surprising, as seemingly very little guidance on the subject can be found, and the words “severe”, “long-lasting” and “ameliorated” used by the directive may be interpreted in a number of ways.Olsson et al. [50] state that “severe” suffering is “more than merely a quantitative increase in negative state” and that it occurs “when negative experiences dominate attention; there is limited capacity for distraction or compensation; normal life cannot be pursued; full recovery cannot occur even if the external situation improves; or (in humans) one’s own life is judged not to be worth living.” They claim that existing methods of assessing animal welfare fail to focus on these qualitative features of severe suffering and that there is need for “insight into how animals are affected by the total load of aversive experiences (including a consideration of additive, multiplicative, and cumulative effects) to which they may or may not habituate.” As such, biomarkers or visible changes such as cortisol levels or bruising are according to Olsson et al. [50] not reliable enough beyond a certain point of suffering. A FELASA Working Group Report by Fentener van Vlissingen et al. [84] similarly conclude that an overall assessment and the duration of a condition needs to be taken into account when determining severity and that severity is comprised of the clinical condition of the animal as one aspect and the procedures it undergoes as another. Suffering involves “complex, subjective experiences” [85]. Thus, we ask the question if commonly used criteria such as weight loss, porphyrin staining, movement and posture deviations, or piloerection could in fact be questioned for use when the proposed projects are expected to cause severe suffering? Furthermore, when severe suffering takes place, pain or stress will in itself interfere with the animal’s ability to store and recall information [50]. Thus, “the ability of the animal to take control and ‘tell’ us anything about its own state becomes limited.” This means that conditioned place preference tests and other behavioral tests of similar nature could be deficient for measuring suffering regarded as severe [50]. Hence, an assessment model taking into consideration the extent to which damage to one functional system, for example caused by great pain, can impact another may be preferable. Violence or other disrupted social behavior, chronic fatigue or sleep loss, reduced appetite, or atrophy of brain regions may all be observable signs that an animal is no longer able to mount an adaptive response or cope with its situation [86]. As shown by Lindl et al. [87], there exists an inverse correlation between the severity of animal research projects and the ultimate applicability and value of their results for humans. Hence, higher levels of severity could arguably, apart from being larger harms themselves, be considered as directly detrimental for the weight of the proposed benefits. Bearing this in mind, we believe it is of outmost importance for a correct ethical evaluation that severity can be reliably assessed at all levels and that the “upper limit” is enforced as intended.According to Beauchamp and Morton [85], the Directive lacks information about what both intensity and duration of harm constitutes. They argue that these two components require detailed conceptual analysis and that any “upper limit” they are applied to must be both practically measureable and avoidable. When it comes to defining what constitutes “long-lasting”, looking at the term through the lens of human experience and opinion is probably of little help (Paul Flecknell, personal communication, 25 August 2020). Similar to the practice of determining animal sentience based on typically human characteristics or behaviors, there are risks associated with applying an anthropomorphic way of thinking when determining animal suffering and in assuming that what affects humans will have similar impact on other animals [22]. Instead, Flecknell toys with the idea of translating time intervals of pain from man to mouse or vice versa by placing them in relation to the average life span of their species. A similar approach does exist in the Directive where the “lifetime experience” is to be considered when contemplating the reuse of research animals [5] (Preamble 25) but nowhere else. Hypothetically, if a human is in extreme pain for one month of their life, how many days, hours, or minutes would this correspond to in a mouse? The idea of such a method is certainly interesting, albeit difficult if not impossible to apply; however, it does not provide an answer to the question at the base of the issue: How much time or how large a proportion of an individual’s life could be allowed to consist of severe pain or suffering? Especially when said individual is most likely not the one reaping the fruits of its sacrifice. In addition, consideration must be given to the fact that the extent and nature of prior harm may impact the experience of subsequent harm [85]. Flecknell concludes that, based on the Directive and associated documents together with his suggestion of looking at time as a proportion of one’s life span, severe pain or distress that lasts 24–48 h in a mouse should, in his opinion, be seen as long-lasting. However, he does point out that he believes the often automatic assumption that duration increases severity is flawed. Instead, he argues that, “severity is something that requires careful assessment in an individual animal” and that “duration of the adverse effects is something to consider, but not as a reason to automatically increase the severity category.” Finally, he adds that if an animal has little or no sense of “future” but rather “lives in the moment”, then the concept of long-lasting pain would be very different for such an animal, and this should be taken into consideration when estimating duration of suffering.Although the aforementioned lack of consensus regarding the “upper limit” is hardly surprising, it is nonetheless alarming. Without consensus amongst member states and responsible authorities as well as national AECs (or their equivalents) regarding its interpretation and implementation, the prohibition is all bark and no bite, and its purpose ultimately toothless. A robust and comprehensible prohibition of inflicting suffering above a certain threshold would according to Olsson et al. [50] lead to an increased motivation amongst researchers to find alternative methods. Thus, establishing a firm “upper limit” of suffering and enforcing it would not only spare animals from suffering but encourage application of the 3Rs and, as previously mentioned, possibly result in more reliable scientific results. Limiting the suffering of research animals in this way could also lead to increased public support for animal-based research as severe suffering inflicted on animals, be it for the sake of research or otherwise, is perceived by many as unacceptable [50,88].As is evident, information about how suffering is to be observed and graded, including clear judgment criteria and/or assessment templates, needs to be provided by the applying researchers. If not, the AECs are not able to correctly ascertain whether or not Refinement strategies have been sufficiently considered nor if there is a danger that the suffering will not be judged correctly, possibly risking exceeding the “upper limit”. Without this information, especially painful or otherwise detrimental projects and procedures risk slipping through the system as “severe” when they should never have been approved at all. Thus, the fact that only 17% of applicants in our study fulfilled the criteria of describing how the animals’ pain, discomfort, or other suffering was to be observed is beneath criticism.Regrettably, the lack of knowledge as to how and when the “upper limit” should be applied may allow, or already have allowed for, projects to be labeled as “severe” when in fact they may have transgressed the “upper limit” [89]. As a result, the welfare of an unforeseeable number of research animals would be endangered as they risk being subjected to unnecessary and unlawful suffering, directly contradicting the aim of the Directive and the “Five Freedoms”. Furthermore, the AEC would then not only have failed its task as a committee, but such an approval could be seen as in breach with both legislation safeguarding animal welfare as well as the administrative laws outlining the AECs’ authority. In Sweden, a person found guilty of disregarding the rules of a certain task when acting on behalf of an authority may be subject to fines or up to two years in prison for misconduct, be it through intent or negligence, unless the misconduct is judged as slight [90] (Chapter 20: Section 1). However, how such possible mistakes by the AECs would come to result in any form of legal consequence for the committees or their chairpersons is unclear, as it is currently the role of the NAEC to review all projects categorized as “severe” and any other that the AECs may have demanded a review of. That same NAEC has admitted to not knowing how the “upper limit” should be applied.A related issue concerns the reporting of national animal research statistics to the EU—namely, that possible transgressions of the “upper limit” of suffering are lost in the system. If the “severe” classification is found to have been exceeded, the responsible researchers are to report this “normally like any other use” under the “severe” category, and the reasons as to why the “severe” classification was exceeded should be included as commentary [51] (Annex II p. 7). However, this is where the system falters. The documents used by researchers in Sweden to report animal use, as well as the accompanying instructions, do not contain any mention of how to report cases where animals have been subjected to harm or suffering above the “upper limit” [91,92]. When contacted by the authors of this article, the Swedish 3Rs Center confirmed that the researchers are not informed or reminded of the “upper limit” and how transgressions of it are to be reported, but that they are able to add such information as comments should they wish (and know) to do so (Cecilia Bornestaf and Per Ljung, the Swedish 3Rs Center, personal communication, 14 April 2020). However, it may not always lie in the researcher’s interest to admit that a project for which they have been responsible has resulted in severe and unjustifiable suffering. Therefore, we believe it to be problematic that information requested for statistical purposes it is less nuanced than what would be needed to reflect compliance with legal requirements, risking the transparency of the ethical review process as well as the public’s trust in the system. The possibility, however small, that research animals may be subjected to severe prolonged suffering and that this is invisible in publically accessible statistics provides a false sense of comfort that animal research is better regulated and in some cases less harmful than is really the case. Additionally, this system of statistical gathering prohibits the SBA, via the NAEC, from knowing to the full extent how projects have turned out regarding degree of severity. We suggest a careful review of approved research projects given the “severe” classification by AECs but having transgressed the “upper limit”. This would guide the SBA as to where efforts should be directed to improve the ethical review and the severity assessment guidelines in particular. Are there patterns to which species’ suffering is most often underestimated? What kind of research has transgressed the limit? Are there certain AECs or researchers that seemingly lack the ability to correctly anticipate animal suffering? Seeing as there is no separate categorization or assembly of these projects today, this possibly beneficial information is lost.4.4. Discrepancies between Law and Application Form/Insufficient InformationAs our study has shown, the demands set by the Directive and their transcription into the L150 guidelines differ in several regards, as does the application form compared to the two aforementioned documents. As a result, what the Directive demands of the applicant could not, even if the application form was filled out to perfection, be satisfactorily provided by the applying researcher. In other words, the information requested by the application form and thus provided for the project evaluation is incomplete by nature and the AECs do thereby not receive enough information on which to base their decisions. As neither an HBA nor an overview of the 3Rs can be accurately performed without sufficient information [19,21], this risks the credibility of the Swedish ethical review process, public trust in the system, and the welfare of animals used in research when the Directive is not implemented and followed as is mandatory by all member states.4.5. Public Interest and TransparencyAs we discovered in our study, the AECs did not once include any mention in their decisions of having discussed the 3Rs. As such, even though one may hope and assume that the 3Rs were discussed, there is no evidence to support this. Neither did the AECs give satisfactory accounts of having conducted HBAs in more than four out of 18 cases. This is troubling. Similarly, the template “AECs Decision—Non-technical project summaries” is provided by the SBA for use by the AECs but was only included in four out of the 18 analyzed decisions. Using the template is not mandatory, but its inclusion would promote both constancy and transparency and in turn public insight. The reason(s) behind why the template was not included in more or all of the decisions has not been determined in our study. It is possible that the template is in fact used at committee meetings but for some reason is not incorporated amongst the final documents or that the AECs choose not to include it, nor any other document of corresponding content, unless the determined degree of severity differs from the one proposed by the researcher. Regardless, it is problematic from a transparency viewpoint that this step of the ethical review is not visible. Since applications submitted to, as well as decisions made by, AECs in Sweden are public documents, it could be anticipated that the lack of description of the 3Rs and HBA by the AECs may negatively impact public trust in animal-based research.Seeing as the AECs are a part of, and therefore act in the name of, the Swedish Board of Agriculture, their decisions require motivating as specified by the Administrative Procedure Act [59] (Section 1, 20), [58] (Section 1, 32), Anders Elmström, Department of Animal Research at SBA, personal communication, 20 August 2020). What is less clear is how such a motivation should be constructed and how in depth it need be. This current uncertainty and allowance for interpretation as to how AEC decisions are to be motivated risks the integrity and transparency of the ethical review process, as well as the public’s insight and thereby trust in the system.5. ConclusionsAs is evident from our study, there are several areas within the ethical review of animal research in Sweden that are not working as intended.Despite the limited number of applications included in this study, the revelation that not one single AEC included any mention of the 3Rs in their documented decisions is cause for great concern. So is the fact that the majority of the decisions did not contain a documented HBA. This is, we believe, largely due to the structure of the application form. By revising the application form (as further described below), the AECs would receive sufficient information to be able both to control that the 3Rs have been fulfilled by the applicants and to conduct a proper HBA. Furthermore, a clear expectation of a description of the 3Rs within the “technical body” of the application form would increase the awareness of the relevance of Replace, Reduce, and Refine for a proper HBA among both applicants and members of the ACEs. We further see a need of a revised common mandatory template for the AECs decisions where the inclusion of the 3Rs and a full HBA are clearly requested. This would not only facilitate the work of the AECs but also ensure increased transparency and public trust.Furthermore, given the inadequate information provided by many researchers regarding the 3Rs and the description of potential or known harm due to housing and/or research procedures, we strongly recommend a review of the current education of researchers in Sweden. Judging by our results, it would be especially important to increase focus on the 3Rs, HBA, the “upper limit”, severe suffering and general awareness of precisely what information is to be provided in an application. This could be done through improving and strengthening the current mandatory laboratory animal science (LAS) courses and their subsequent continuing professional development (CPD) courses, and by ensuring competence is regularly updated, as required by the Directive [5] (Article 23, p. 2). To be concrete, all employees with function A and B eligibility could be required to demonstrate their competence and skills every third year so as to ensure good animal welfare, high quality research, and compliance with the Directive’s strive to continuously improve and streamline animal research procedures in line with the 3Rs. Additionally, research organizations and the scientific community as a whole could benefit greatly from adopting new approaches to increase and inspire 3R awareness and application among researchers, for example through revised organization and management strategies [77,93,94]. As it has been reported that researchers may prefer to consult other colleagues to increase their 3R knowledge rather than literature or databases [72], focusing on increasing the competence within the research community is highly recommendable. In addition, in line with the EU Directive, 3R support from animal welfare bodies may further strengthen researchers’ awareness and willingness to apply the 3Rs, although this is not yet fully implemented [95].However, it is not only the researchers who would benefit from more and improved education judging by our results. AEC members evidently need more information on how to fulfill legal and ethical criteria through review of the 3Rs and performance of HBA. In addition to this, a better understanding among committee members of how to assess (and Refine) severe cases of suffering as well as how to apply the “upper limit” would ensure better protection for research animals, increased public trust and research quality, as well as, we believe, greater job satisfaction amongst AEC members. We also suggest that the AECs’ competence could be strengthened by adding expertise to knowledge and implementation of the different Rs through the employment or consultation of for example statisticians, system modelers, engineers, etc., and that the roles and impact of animal welfare bodies are strengthened. We further believe, similar to Hansen et al. [47], that the work of the AECs to ensure the application of the 3Rs could be improved and facilitated further through the inclusion of researchers in the committees who work with non-animal research and/or alternative methods.Our study has also shed light on the difficulty of interpreting the “upper limit” of pain, suffering, or distress specified by the Directive. As such, we believe that it is urgent for all member states to create and adopt common guidelines and/or assessment templates specifically tailored to assess severe suffering and ensure that exceeding the “upper limit” is avoided at all costs. In addition, we believe it would be beneficial for animal welfare as well as the quality and transparency of animal research to place higher demands on researchers’ abilities to guarantee that suffering will not exceed the “upper limit” in studies that are categorized as severe. One possibly helpful way of regulating this could be to include in the application form a question of how long lasting the proposed suffering is expected to be. This is today already common practice in, for example, Germany where the categories “<1 day”, “1–7 days”, “7–30 days”, and “>30 days” are used [33]. Ideally, this would be accompanied by a consensus amongst member states concerning how to define “long-lasting” suffering and how the duration of suffering, pain, or distress should influence the severity assessment of a procedure.In line with Alzmann [22], we suggest revisiting the idea of creating a comprehensive catalogue for harms (psychological and physical, present, past, and future) to be used when implementing the 3R and performing an HBA. Such a catalogue should preferably include but not be limited to, pain, suffering and distress due to the following factors: handling; genetic modification; social isolation or group size/dynamics; unnatural diurnal rhythms; room temperature, noise levels and other environmental factors; abrupt environmental changes; lack of environmental stimulation and/or inability to perform natural behavior and fulfill behavioral needs; anticipatory fear; and negative emotional states such as boredom, stress, and depression. For transparency and uniformity, the catalogue should be one and the same for all member states. Likewise, constructing common guidelines for all member states on how to better assess benefits would greatly improve and facilitate the work and task of the AECs, in turn ensuring trustworthy HBAs. However, achieving this, if at all possible, would warrant additional research.As seen, there exist discernible discrepancies between the legal requirements of Directive 2010/63/EU, its implementation in Swedish legislation (SJVFS 2012:26), and the application form for project evaluation. As such, applying researchers are unable to provide the legally requested information concerning their proposed studies, and the AECs are forced to work with fragmented facts. In an effort to resolve this, a thorough comparison of the Directive and the L150 together with an exhaustive evaluation of which legal demands are and are not met by the current application form format will be conducted by the research group during 2021. This upcoming project will provide suggestions for how the application form may be revised to ensure that it corresponds to legal demands and is clear and easy to both fill out and read.Additionally, due to the risk of violations of the “upper limit” getting lost in the statistics, we further believe that the system for reporting of actual degree of severity needs to be revised. One resolution could be to adopt an additional category of severity, higher than severe, solely for statistical use, so that any and all research whereby the “upper limit” has been exceeded is clearly visible, and strategies for addressing the causes for this can be applied. However, as long as it is the researchers themselves who are responsible for reporting the actual level of severity, we realize it may be difficult to motivate the use of such a category, especially if there would be reprimands involved due to the cause of unnecessary suffering and thereby the possible violation of both animal research and welfare legislation. This brings us to our next suggestion; in order to avoid bias and ensure transparency, we would like to suggest the implementation of an independent system for review of how the AECs have made and motivated their decisions, how they fulfilled the required points of analysis (3R, HBA, degree of severity, etc.), and how well the estimated degrees of severity correspond to the observed pain, distress, and suffering. This would motivate researchers to thoroughly plan for, implement, and demonstrate their use of the 3Rs (Refinement in particular). It would also stimulate the AECs to carry out a careful review of how the 3Rs have been applied and to perform in-depth HBAs. We would also like to open up the possibility for parties other than the researchers to contest decisions made by the AECs so as to ensure transparency, public trust, and that the welfare and intrinsic value of animals is safeguarded, valued, and respected.It is today unclear exactly what is required for transparency to be fulfilled according to the Directive and to what extent the AECs’ decisions need to be motivated to live up to Swedish administrative law. What is clear is that the vast majority of the analyzed decisions of our study lacked transparency, as they did not include an account of how the 3Rs had been reviewed nor of a proper HBA. Thus, there is a need for in-depth interdisciplinary research into how the ethical aspects of the review process are to be applied and expressed so as to fulfill the transparency demands of the Directive and administrative legislation. Based on the results of such research, guidelines for motivating decisions could be formulated.

animals : an open access journal from mdpi

10.3390/ani11061546

PMC8228274

Keel bone damage is an important welfare issue for laying hens. Four lines of laying hens, differing in phylogenetic origin and laying rate kept in single cages or a floor housing system were weighed and deformities of the keel bone were evaluated regularly between 15 and 69 weeks of age. Deformities, fractures and the bone mineral density of the keels were assessed after hens were euthanized. We analyzed the relationship between bone mineral density and total egg number as well as body growth. Hens kept in cages showed more deformities, but fewer fractures and a lower bone mineral density of the keel bone than did floor-housed hens. Keel bones of white-egg layers had a lower mineral density and were more often deformed compared with brown-egg layers. Keel bones were more often broken in hens of the layer lines with a high laying rate compared to the lines with a moderate laying rate. Laying rate and adult body weight had an effect on the keel bone mineral density. The study contributes to the understanding of factors causing keel bone damage in laying hens. It showed that the bone mineral density greatly affects keel bone deformities.

Keel bone damage is an important animal welfare problem in laying hens. Two generations of four layer lines, differing in phylogenetic background and performance level and kept in single cages or floor pens were weighed and scored for keel bone deformities (KBD) during the laying period. KBD, keel bone fractures (KBF) and the bone mineral density (BMD) of the keels were assessed post mortem. For BMD, relationships to laying performance and body growth were estimated. Caged hens showed more deformities, but fewer fractures and a lower BMD of the keel bone than floor-housed hens. White-egg layers had a lower BMD (0.140–0.165 g/cm2) and more KBD than brown-egg layers (0.179–0.184 g/cm2). KBF occurred more often in the high-performing lines than the moderate-performing ones. However, in the high-performing lines, BMD was positively related to total egg number from 18 to 29 weeks of age. The adult body weight derived from fitted growth curves (Gompertz function) had a significant effect (p < 0.001) on keels’ BMD. The study contributes to the understanding of predisposing factors for keel bone damage in laying hens. It showed that the growth rate has a rather subordinate effect on keels’ BMD, while the BMD itself greatly affects KBD.

1. IntroductionKeel bone damage is an important animal welfare issue, as an alarmingly high number of laying hens show keel bone deformities (KBD) and/or -fractures (KBF) (reviewed by [1]). Moreover, keel bone damages are of economic importance. KBF are often accompanied by a lower egg production [2,3] and poor egg quality including reduced shell thickness and breaking strength [4] as well as lower shell weights [2]. Chargo et al. [5] attributed the decreased production to changes in the hens’ behavior due to pain or musculature changes as a result of keel bone damage. KBF are likely to induce chronic stress and a depression-like state in laying hens [6] and also increase flock mortality [7]. Keel bone damage is a complex trait and there are numerous factors, including nutritional, environmental and genetic ones, which influence the risk and possibility of developing such disorders [8]. KBF often occur as a result of falls within alternative housing systems [9]. In this context, collisions with housing equipment, especially perches, or due to social interactions between hens were identified as a main source of keel bone damage in laying hens [9]. Nevertheless, KBF can also be a result of non-collision events such as wing-flapping [8]. In contrast to these short-duration traumata responsible for KBF, KBD are a result of a protracted process of bone remodeling due to a continued mechanical pressure load during perching [10,11] and therefore increase when giving hens access to perches [12,13].As selection of laying hens mainly focused on a high number of saleable eggs for many years, skeletal problems, including keel bone disorders, are often considered a direct consequence of the decades-long breeding work. A gradual but persistent loss of structural bone tissue in favor of meeting the calcium requirement for eggshell formation is thought to be the main cause of the increased KBF frequency in layers [14]. However, the assumption that selection for high egg numbers is solely responsible for bone weakness is viewed critically and recent studies suggested, for example, that early puberty is a decisive factor [15,16]. Leyendecker et al. [17] concluded that the lack of exercise laying hens experience in furnished cages has a higher impact on the development of weak bones than calcium depletion from eggshell formation. Rennie et al. [18] calculated a low negative correlation (−0.16) between egg production and trabecular bone volume in hens at the end of the laying period, leading to the assumption that the number of eggs has little or no effect on bone quality.The current study compared the keel bone status of four lines of laying hens differing in two dimensions, laying performance and phylogenetic origin. Furthermore, we aimed to examine whether the bone mineral density of the keel bone is affected by hens’ individual egg production and growth rate.2. Materials and Methods2.1. Chicken LinesAn animal model consisting of four purebred layer lines differing in laying intensity and phylogenetic background was used. While the two white egg layers, WLA and R11, were descended from White Leghorn, the brown egg strains, BLA and L68, originated from Rhode Island Red and New Hampshire, respectively [19]. Within each of the phylogenetic groups, a high performing line (WLA, BLA) was contrasted to a moderate performing ones (R11, L68). WLA and BLA originated from breeding program of Lohmann Breeders GmbH (Cuxhaven, Germany). They have been maintained in a sire rotation program since 2012 and lay approximately 320 eggs per year. R11 and L68 are part of the resource populations of the Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut (Neustadt, Germany) and achieve a laying performance of about 200 eggs per year [20]. For further information about performance data of caged hens examined in the present study, see Jansen et al. [16].2.2. Housing System, Management and FeedingThe first generation was kept from September 2015 to January 2017 and the second from November 2016 to March 2018. After hatch, sex was determined visually by inverting the cloaca of day-old chicks. Female chicks were individually weighed and wing-tagged for identification. During the 15-week rearing period, hens were kept line-separated in four floor housing pens, located within one room. Each pen had a floor space of 24 m2, was littered with wood shavings and was equipped with a feeding line and nipple drinkers. Moreover, pullets had access to straw bales and two wooden ladders per pen that were placed in an inclined position at one of the walls. Each ladder had three crossbars at a height of 35 cm, 80 cm and 130 cm and a width of 110 cm and 150 cm, respectively. The perch space per pullet was approximately 6 cm. For further information on lighting regime, climatic conditions and the composition of feeding stuffs, see Tables S1 and S2 in Jansen et al. [16]. At the end of the 15th week of age, pullets were transferred to the layer house.In each of the generations, a total of 288 pullets, 72 per layer line, were distributed equally throughout the single cages of a three-tiered system arranged in two rows separated by a grid. The floor space per cage was 2400 cm2, and each cage was equipped with a feeding through, a nipple drinker and a round plastic perch (3 cm in diameter), which was slightly elevated at a height of approximately 6.5 cm and arranged transversely to the feeding through.In addition, another 384 pullets of the same four lines and sire families in each generation were kept line-separated in a floor housing system, which was located in an adjacent barn room. The floor housing system consisted of two side-by-side rows with eight pens each. Half of the pens had a floor space of 4 m2 (2 m × 2 m; small pens) and the other half measured 8 m2 (4 m × 2 m; large pens). They were arranged in alternate order and comprised 24 hens each, given a floor space of 1667 cm2/hen (small pens) and 3333 cm2/hen (large pens), respectively. A droppings pit of 94 cm depth and 67 cm height was located at the longitudinal side of the pens and could be accessed by the hens via jumping or a ramp. One automatic feeder and a line of eight and 13 nipple drinkers was installed on the droppings pit of the small and large pens, respectively, and provided ad libitum food and water supply throughout the whole laying period. Composition of layer diets is given in Table S3 in Jansen et al. [16]. Each pen was equipped with a round metal perch (3.5 cm in diameter) lengthwise over the droppings pit, given approximately 16.5 cm and 8.5 cm perch space pen hen in the large and small pens, respectively. In the large pens, the perch was fixed at a height of 61 cm, and, in the small pens, it was arranged 5 cm above the ground of the dropping pits. To induce further movement, one plastic box was located in the litter area of the large pens. In each pen, hens had access to one nest box of approximately 120 cm length and 80 cm width, which was accessible from the droppings pit via a balcony.When the hens were moved to the layer house, the lightning period was gradually stepped up from nine hours to 14 h per day in the course of six weeks and remained constant until the end of the laying period.2.3. Data CollectionChickens of the first generation were weighed every second week from hatch to week 14 on a digital table scale with a weighing accuracy of 0.1 g (Sartorius CPA 16001S, Sartorius Group, Göttingen, Germany). After another weighing immediately before the birds were transferred to the layer house at the end of the 15th week of age, the weighing interval during the laying period was four weeks. In the case of the second generation, the birds were weighed at four-week intervals during the rearing period and at seven time points during the laying period (at the end of week 15, 17, 21, 25, 29, 49, and 69).Keel bone status of all of the hens of the first generation was assessed in regular intervals, at the end of the 15th, 21st, 29th, 37th, 45th, 57th, 61st, and 69th week of age. According to Habig and Distl [21], the severity of KBD was recorded on a three-scale scoring system (4 = no deformity, 3 = slight deformity, 2 = moderate to severe deformity). The hens were securely held upside down by their legs with one hand, and the keel bone was palpated using the thumb and forefinger of the other hand. Scoring was made by the same experienced person at any time, which was not blinded to the treatment.In both generations, the laying hens were euthanized by carbon dioxide inhalation after 70 weeks within five consecutive days, with hens randomly selected each day from both housing systems and all genetic lines. The keel bones were dissected, with adherent muscle tissue being removed, and stored frozen at −20 °C for further analysis. The ’small animal’ mode of a GE Lunar iDXA scanner (enCORE® software version 13.5, GE Healthcare GmbH, Solingen, Germany) was used to measure bone mineral density (BMD) of the keel bones by dual energy X-ray absorptiometry (DXA). Therefore, the whole keel bones were scanned in a latero-lateral direction. The dissected keel bones were scored by palpation for KBD and KBF. Deformities were evaluated according to the scale mentioned above. Additionally, the direction of the deviation was noted as transverse and/or sagittal. In order to improve detection of fractures, keel bones were placed on a light box. If fractures occurred, the type of fracture (transversal or longitudinal) as well as the number of fractures (1 to 3 or ≥4), and their location in the cranial, intermediate or caudal third of the carina sterna were recorded. Scoring was done by the same experienced examiner, blinded to the housing system.2.4. Statistical AnalysesStatistical analyses were performed using JMP v14.0 (Statistical Analysis System Institute, Cary, NC, USA, 2018) and R v4.0.2 (R Core Team, Vienna, Austria, 2018).2.4.1. Deformities and Fractures of the Keel BoneIntra vitam KBD scores of the first generation and post mortem KBD and KBF scores of both generations, including the direction of KBD and the type, number and location of fractures, were analyzed by Chi-square tests towards an effect of the generation, layer line and housing system. Statistical significance was set at p < 0.05.2.4.2. Approximation of Growth CurvesGrowth curves were fitted to the body weight records of the individual hens applying the Gompertz function [22], computing the parameters of asymptotic final weight at the 69th week of age (a), the maximal growth rate (slope of the growth curve (b)) and the time of inflection, when growth rate is starting to decrease (c). We then analyzed the effect of the generation, housing system, layer line and their interactions on the growth parameters according to the following model:(1)γijklm=μ+Gi+HSj+LLk+GiHSj+GiLLk+HSjLLk+Sl+εijklm where γijklm is the respective growth parameter (a, b, or c); μ is the general mean; Gi is the fixed effect of the generation (i = 1, 2); HSj is the fixed effect of the housing system (j = 1, 2); LLk is the fixed effect of the layer line (k = 1 to 4); GiHSj, GiLLk and HSjLLk are the interactions of the respective variables; Sl is the random effect of the sire (l = 1 to 145); and εijklm is the random error variance. Tukey’s HSD (honestly significant difference) test was performed for multiple comparisons of means. Statistical significance was set at p < 0.05.2.4.3. Bone Mineral Densities Measured in Floor Pens and CagesWe applied a linear mixed model to study the variation of the keel BMD considering main factors and the growth curve parameters as covariates. The model was as follows:(2)γijklmnopqr=μ+Gi+HSj+LLk+KBDl+KBFm+GiHSj+GiLLk+HSjLLk+GiKBDl+GiKBFm+LLkKBDl+LLkKBFm+HSjKBDl+HSjKBFm+KBDlKBFm+an+bo+cp+Sq+εijklmnopqr where γijklmnopqr is the observation of BMD; μ is the general mean; Gi is the fixed effect of the generation (i = 1, 2); HSj is the fixed effect of the housing system (j = 1, 2); LLk is the fixed effect of the layer line (k = 1 to 4); KBDl is the effect of post mortem keel bone deformities (l = 1 to 3); KBFm is the effect of post mortem keel bone fractures (m = 1, 2); GiHSj, GiLLk, HSjLLk, GiKBDl, GiKBFm, LLkKBDl, LLkKBFm, HSjKBDl, HSjKBFm and KBDlKBFm are the interactions of the respective variables; an is the effect of the asymptotic final weight of the growth curve (a); bo is the effect of the slope of the growth curve (b); cp is the effect of the inflection point of the growth curve (c); Sq is the random effect of the sire (q = 1 to 145); and εijklmnopqr is the random error variance. Tukey’s HSD (honestly significant difference) test was performed for multiple comparisons of means. Statistical significance was set at p < 0.05.2.4.4. Bone Mineral Densities Measured in Single CagesAs an extension of the basic model (2), the individual laying performance was included as additional factor in model (3), which is why it was only applicable to the single cage data. The laying performance was divided into two laying periods (LP), with LP1 covering weeks 18 to 29 (pre-peak) and LP2 covering weeks 30 to 69 (post-peak). We cleaned the dataset by removing hens (i) with total egg numbers outside the line specific threefold interquartile range (IQR) (<X0.25 − 3 x IQR; >X0.75 + 3 x IQR) and those who did not lay an egg during the last three consecutive weeks of the study (n = 52) [16], and (ii) for which no Gompertz growth function could be fitted (n = 5). After filtering, a total of 519 individuals (BLA: n = 130; L68: n = 128; R11: n = 133; WLA: n = 128) remained for further analyses. The BMD measurements of these hens were analyzed considering the factors given in model (2) as well as the performance data including the age at first egg and the egg number within the two laying periods. The model was as follows:(3)γijklmnopqrst=μ+Gi+LLj+KBDk+KBFl+GiLLj+GiKBDk+GiKBFl+LLjKBDk+LLjKBFl+KBDkKBFl+am+bn+co+Sp+FEq+LP1r+LP2s+εijklmnopqrst where γijklmnopqrst is the observation of BMD; μ is the general mean; Gi is the fixed effect of the generation (i = 1, 2); LLj is the fixed effect of the layer line (j = 1 to 4); KBDk is the effect of post mortem keel bone deformities (k = 1 to 3); KBFl is the effect of post mortem keel bone fractures (l = 1, 2); GiLLj, GiKBDk, GiKBFl, LLjKBDk, LLjKBFl and KBDkKBFl are the interactions of the respective variables; am is the effect of the asymptotic final weight of the growth curve (a); bn is the effect of the slope of the growth curve (b); co is the effect of the inflection point of the growth curve (c); Sp is the random effect of the sire (p = 1 to 145); FEq is the effect of the age at first egg laid; LP1r is the effect of the number of eggs laid within weeks 18 to 29; LP2s is the effect of the number of eggs laid within weeks 30 to 69; and εijklmnopqrst is the random error variance. Tukey’s HSD (honestly significant difference) test was performed for multiple comparisons of means. Statistical significance was set at p < 0.05.As the pre- (LP1) and post-peak (LP2) egg number as well as the asymptotic final body weight at the 69th week of age had a significant effect on keels BMD, these traits were analyzed within the layer lines, applying a univariate regression approach. The linear relationships between egg number and adult body weight, respectively, and the keel BMD was modelled as follows:(4)γi=β0+β1xi+εi where γi is the observation of BMD; β0 is the intercept; β1 is the slope; xi is the pre-peak (week 18 to 29) or post-peak (week 30 to 69) egg number or the asymptotic final weight of the growth curve (a); and εi is the random error variance.3. Results3.1. Keel Bone Deformities Assessed Intra Vitam in the First GenerationFigure 1 shows the KBD prevalence assessed intra vitam in the course of the laying period. The corresponding significant differences in KBD frequencies between layer lines and housing systems within the single weeks of age are given in Table 1. After the rearing period at the end of 15 weeks of age (Figure 1, Part A), none of the L68- and R11-hens showed KBD and only one BLA-pullet had a slight deformity. In contrast, WLA hens showed a significantly higher frequency of KBD of six hens (approx. 3%) affected by a slight to severe deformity. In single cages (Figure 1, Part C), L68 hens were significantly less affected by KBD than BLA hens at any time of examination (Table 1). The same tendency was observed within the floor housing system (Figure 1, Part B), but significant results were only detected for the 69th week of age. While KBD occurred significantly more often and more severe in hens of the white-egg layer lines compared to the brown ones at any time of examination within the cage system, in the floor housing system, the same trend was seen from the 29th week of age onwards, without reaching a significant value at any day of scoring. With the exception of the 21st week of age, where WLA (45.83%) hens kept in cages showed less deformities than R11 (76.39%), no significant differences were detected between the two white-egg layers within the same housing system. In all layer lines, KBD appeared significantly more often with a higher degree of severity in hens housed in cages (Figure 1, Part C) than in floor pens (Figure 1, Part B). The smallest differences between the two housing systems were recorded for L68 and reached a significant level in the 57th and 61st week of age. The compartment size of the floor housing system had no significant effect on the occurrence of KBD.3.2. Post Mortem Examination of Keel Bone StatusResults of post mortem keel bone status of laying hens of the first and second generation are presented in Table 2 and Table 3, respectively. As the effect of generation was significant for all of the keel bone traits (data not shown), with the exception of KBF, the post mortem keel bone status of the first and second generation were analyzed separately.In all layer lines, the number of hens with KBD as well as the severity of deformation was significantly higher in cages compared to the floor housing system. Accordingly, the direction of deformities that occurred in floor pens was primarily transverse, while most of the very severe deformities due to a transverse and at the same time sagittal deviation were found in hens kept in cages. Only one R11-hen showed a KBD that was directed sagittal.In contrast to these findings, hens kept in floor pens were significantly more often affected by KBF (BLA: 61.82%; L68: 28.40%; R11: 39.27%; WLA: 74.83%) than their conspecifics in cages (BLA: 38.79%; L68: 5.15%; R11: 20.71%; WLA: 36.03%), with the exception of R11 in the first generation, where KBF were equally distributed among housing systems. Overall, 38.77% of all scored keel bones (both generations) showed at least one fracture. More than 89% of these fractures were found in the caudal part of the keel bone (carinal apex). In contrast, fractures solely located in the intermediate (4.24%) and cranial (0.42%) part of the keel bone occurred less often. Approximately 6% of the hens with a broken keel bone showed fractures in at least two different locations.In the first generation, none of the effects were significant regarding the number of fractures at the caudal part of the keel. In contrast, the second generation was different, with WLA hens kept in floor pens having significantly more KBF at the caudal keel portion compared to L68 within the same system and R11 in both housing systems. However, within cages, fractures accounted at the keels’ tip were lower for WLA than for R11, whereas in floor pens the number was higher for BLA compared to R11.Only one L68- and one R11-hen had a longitudinal fracture in the caudal part of the keel bone, the rest of the fractures detected in this area were transversely directed.In floor pens as well as in cages, the keel bones of white-egg layers were significantly more often deformed than those of brown-egg lines, with the exception of the comparison of BLA and WLA kept in cages in the second generation. Hens of the BLA line showed significantly more KBD as well as KBF than L68 in both housing systems except for deformities detected in floor pens of the first generation. Similarly, the frequency and/or degree of KBD was significantly higher for the high-performing white-egg strain compared to their moderate-performing counterpart, with the exception of hens kept in cages the second generation. However, within the phylogenetic groups, KBF were more common in the high-performing strains than the moderate-performing ones in both husbandry systems.The compartment size of the floor housing system had a significant effect on the post mortem keel bone status (Supplementary Tables S1 and S2). With the exception of L68 hens kept in the second generation, keel bones of all lines of laying hens were significantly more frequently broken in hens out of the large compartments compared to the small ones. Furthermore, in the two high-performing layer lines BLA (first generation) and WLA (second generation), the number of fractures located in the caudal third of the keel bone was significantly higher and keel bone deformities occurred significantly more often and more severely when hens were kept in the large pens rather than the small pens.3.3. Fitting the Gompertz Function to the Growth DataThe results of fitting the Gompertz function to growth data are shown in the Supplementary Tables S3 and S4. The main factors of generation, layer line, and housing system as well as the interaction between layer line and housing system, had a significant effect on all of the parameters of the Gompertz growth curve. The interaction effect of generation and layer line was significant for the curves’ point of inflection, while the interaction of generation and housing system significantly influenced the slope of the curve.3.4. Bone Mineral Density of the Keel Bone in both Housing SystemsTable 4 shows the effects of the main factors and their interactions on the BMD of the keel bone. The BMD was significantly affected by the fixed effects of generation, layer line, housing system and KBD, the layer line × housing system and housing system × KBD interactions, and the adult body weight at 69 weeks of age. The corresponding least squares means (LSM) for the keels’ BMD are given in Table 5. Across layer lines, the BMD of keel bones examined in hens of the first generation was significantly higher compared to those measured in the second generation. In hens of both generations, a significant effect of the housing system, but not compartment size (data not shown), on BMD of the keel bone was detected. All layer lines showed significantly higher values of keel BMD when they were kept in floor pens than in cages. Across both generations, WLA hens had a significantly lower BMD of the keel bone compared to the other layer lines. Within housing systems, this was also the case in cages, while, within floor pens, R11 and WLA had similar values. While no significant differences have been detected between BLA and L68, the moderate-performing layer line R11 had a significantly higher BMD of the keel bone than their high-performing counterpart WLA when they were kept in cages.The BMD of the keel bone differed significantly between the three deformity scores. Across all layer lines and housing systems, the BMD of severe deformed keel bones (score 2) was significantly lower compared to slightly (score 3) or undeformed keel bones (score 4). While the BMD did not differ significantly between the three deformity scores when hens were housed in floor pens, all of the scores differed significantly from each other in cages, i.e., the higher the keels’ BMD, the less severe its deformity. In contrast, no significant relationship between KBF and BMD was shown.Regarding the effects of the Gompertz growth curve parameters, the analysis revealed no significant effect of the slope of the curve and its point of inflection on keels’ BMD, but a significant positive relationship between BMD of the keel bone and the adult body weight in white-egg layers, i.e., heavier birds had a higher BMD of the keel bone.3.5. Bone Mineral Density Measured in Single CagesFurther analyses of the cleaned dataset (519 individuals) of hens kept in cages for relationships between BMD, laying performance, and growth rate showed significant effects of the main factors of generation and KBD as well as the total number of eggs laid during the pre- (LP1) and post-peak (LP2) period and the adult body weight on keels’ BMD (Table 6).In accordance with the results presented for the keels’ BMD in both housing systems (Table 5), laying hens with severe deformed keel bones showed lower BMD values compared to their conspecifics with slightly or undeformed keel bones (Table 6).Regarding laying hens’ performance, it could be shown that the age when the first egg was laid had no effect on the BMD of the keel bone. In contrast, the number of eggs laid during the early laying period from 18 to 29 weeks of age was significantly related to the BMD of the keel bone in BLA, whereas the laying performance from 30 to 69 weeks of age, i.e., from the peak until the end of the laying period significantly influenced keel BMD in R11.Results of univariate regression analysis, which was only performed for those covariates found to significantly influence keels’ BMD (Table 6), are shown in Figure 2. Regression coefficients (β1) of the total number of eggs laid during the early laying period and from 30 to 69 weeks of age in relation to the BMD of the keel bone are presented in Figure 2A and Figure 2C, respectively. While, in the high-performing layer lines, significant positive regression coefficients were obtained for the total number of eggs laid from 18 to 29 weeks of age (WLA: β1 = 4.39 × 10−4, BLA: β1 = 0.141 × 10−4), regression coefficients for the moderate-performing lines were not significant. In contrast, a significant negative regression coefficient (β1 = 4.16 × 10−4) was observed for the laying rate from 30 to 69 weeks of age and keels’ BMD in R11. However, further significant relationships between the BMD of the keel bone and laying rate have not been observed. The trends of BMD with increasing egg number from 18 to 29 weeks of age and from 30 to 69 weeks of age, respectively, are shown in Figure 2B and Figure 2D.While the slope of the Gompertz growth curve and its point of inflection had no significant effect on the BMD of the keel (Table 6), a significant positive relationship was found for the adult body weight in white-egg layers, i.e., heavier birds had a higher BMD of the keel bone. Figure 2E shows the regression coefficients (β1) of the adult body weight at 69 weeks of age in relation to the BMD of the keel bone and Figure 2F illustrates the trend of BMD with increasing body weight. A positive, but not significant, relationship was found between the BMD of the keel bone and the adult body weight, with the exception of L68, where the trend was rather negatively directed.4. DiscussionThe objective of the present study was to assess keel bone damage in laying hens and its relationship to keel BMD, laying performance, and body growth rate. We detected KBD more often in the white-egg layer lines WLA and R11 compared to the brown-egg layer lines BLA and L68. This is in accordance with the findings of Eusemann et al. [23], who scored nine to ten hens per layer line (BLA, WLA, L68, R11, G11) and housing system (single cages vs. floor housing system) repeatedly in the 35th, 51st, and 72nd week of age for keel bone damage using X-ray images. One possible explanation for the observed differences between white- and brown-egg layers might be that, in white-egg layers, the frequency of perching is higher compared to brown-egg layers [24], in particular when kept in alternative housing systems, rather than in cages. Regarding KBF, the risk-promoting factors causing differences between the two phylogenetic groups are contradictory. On the one hand, white hens’ risk for collisions with the housing equipment and resulting KBF seem to be lower due to better flight and 3D-movement skills than brown hens [25,26]. On the other hand, white-egg layers are more fearful and more flighty than brown-egg layers [27,28], which can cause more panic reactions at the group and individual level [29], and consequently KBF due to collisions with the housing system [8,25]. Although R11-hens kept in the current study were also very nervous and got quite agitated and panicky when persons passed or entered the cages or floor pens (personal observations), their keel bones were significantly less often broken than those of BLA- and WLA-layers. Heerkens et al. [25] assumed that the keel bones of white hens, known to produce more eggs than brown hens, become progressively weaker, raising its vulnerability for deformities. Candelotto et al. [4] examined the likelihood of experimental KBF of four specific cross-bred and one pure line, differing in egg production and quality. Their results implied a strong propensity for genetic regulation of fracture susceptibility, as one of the lines, which has not been selected for egg production for several years had less than 20% of experimental fractures, while more than 90% of the keel bones of hens of another commercial line fractured due to impact testing. Our results are in accordance with the observations made by Candelotto et al. [4,30], as we found less KBD and KBF in the moderate-performing line L68 compared to its high-performing counterpart BLA as well as more severe deformities and a higher fracture prevalence at necropsy in WLA than R11. This is consistent with observations on KBF occurrence from a previous study with hens of the same genetic line [31]. Furthermore, regression analyses revealed a significant negative relationship for keels’ BMD and the number of eggs laid from 30 to 69 weeks of age in R11 hens kept in cages. This agrees with the findings of Jansen et al. [16], who observed decreasing BMDs of the humerus and tibiotarsus with increasing total eggshell production in R11. However, for the pre-peak production, a significant positive relationship was found for the high-performing layer lines, i.e., the higher the number of eggs laid until the 29th week of age, the higher the hens’ keel BMD. In contrast, the moderate-performing lines showed negative regression coefficients for laying performance from 30 to 69 weeks of age, although not significant. These results suggest that, alongside increased egg production, the BMD of laying hens might be also increased following selection and support early findings that a high laying performance does not necessarily adversely affect bone quality [15,16].The majority of fractures observed in the present study were located in the caudal third, i.e., the tip, of the carina sterna. In accordance with this finding, other researchers [5,28,31] observed high prevalence’s of fractures located at the tip of the keel bone assessed through a three-dimensional model, radiography and CT scans, respectively. This might be due to the fact that this is the last part to be ossified [32,33] and its higher susceptibility to impacts and consequently resulting damages depending on the position of the caudal keel bone portion in the hen’s body [34].In contrast to the results of the present study, Eusemann et al. [23] did not find a significant difference in the prevalence of KBD and KBF between the housing systems, with the exception of week 72, when the number of hens with fractured keel bones was higher in the floor housing system compared to cages. A possible explanation for this contrary result might be seen in the different methodologies used for the detection of keel bone damages, the lower sample size examined by Eusemann et al. [23] as well as the fact that the housing systems were not totally equal in the two studies. Overall, the housing system is seen as one of the main factors influencing keel bone damage in laying hens. The more complex a housing system is, the higher is the probability of hazardous events resulting in KBF [35]. By reducing falls and collisions, the installation of ramps in aviary systems can limit the damaging consequences for laying hens [36]. As the prevalence of fractures was low compared to several previous studies (reviewed by [37]), it might be assumed that the number of KBF would be higher if floor pens examined in the present study had not been equipped with ramps to give access to the droppings pit. However, a recent study of Thøfner et al. [38] raises doubts about whether collisions are the main cause of KBF. Moreover, it has to be considered that results might be confounded due to the different compartment sizes and perch heights within the floor housing system and the different perch types in the two housing systems examined in the present study.Apart from the described hazards of a large degree of freedom of movement in alternative husbandry systems, the higher motion activity can lead to an increased bone quality too [17]. Even a higher breaking strength [39] or shear strength of the tibiotarsus can contribute to a reduced number of hens with KBF, likely due to the positive correlation of total bone mineral content of the keel bone and the tibiotarsus [40]. In accordance with previous reports [41], keel bones with a high BMD were less severe deformed when hens were kept in cages. The same tendency was shown for layers housed in floor pens but did not reach significance. Similarly, Candelotto et al. [30] found laying hens being most resistant to experimental KBF having the greatest keel BMD, while the two lines most susceptible to fractures had the lowest values, indicating this property as important in affecting keel bones’ breaking strength. The higher the BMD, the lower the prevalence of keel bone damages, most probably arising from a higher bone breaking strength, as could be shown previously [41,42]. This confirms the results drawn from several other studies. Gebhardt-Henrich et al. [40] detected a greater cortical and trabecular bone mineral content and calcium content in intact or only slightly deformed keel bones than in broken ones. Moreover, they found lower keel BMDs in white-egg compared to brown-egg layers, which follows our findings. Toscano et al. [43] described the BMD of the keels’ lateral surface as an effective predictor of the likelihood of KBF, with an increasing BMD decreased the likelihood of a fracture. Fleming et al. [42] and Stratmann et al. [44] compared keel bone damages in hens of a pure line selected for high bone strength (H line), characterized by a greater BMD, with an line selected for low bone strength (L line) and found fewer KBF and KBD in hens of the H line.Regarding the effect of the parameters of the Gompertz growth curve on keel BMD, the analysis of variance of data assessed in both housing systems (model 2) and single cages (model 3), respectively, revealed a significant positive relationship between the adult body weight and keels’ BMD, i.e., the heavier the laying hen is, the higher is her keel BMD. However, this significant effect disappeared when within-line univariate regression analyses (model 4) were performed.5. ConclusionsThe study contributes to the understanding of predisposing factors for keel bone damage in laying hens. Based on our results, we can confirm that the housing system and age of laying hens affect the occurrence of keel bone alterations. Both performance level and phylogenetic background seem to play a role regarding the development of keel bone damage. The current experiment showed that the adult body weight had an effect on the BMD of the keel bone in laying hens. However, the keels’ BMD was shown to be decisive and greatly affects the susceptibility of the keel bone to be injured.

animals : an open access journal from mdpi

10.3390/ani13050794

PMC10000047

Back pain in Thoroughbred racehorses is frequent and significantly decreases their athletic performance. The most common thoracolumbar alteration in Thoroughbreds is Kissing Spines Syndrome (KSS). The objective of the current study was to evaluate and compare soft tissue response to high-intensity laser therapy (HILT) by measuring changes in skin surface temperature and longissimus dorsi muscle tone, located in the thoracolumbar back area, in Thoroughbreds with back pain diagnosed with and without KSS. The Thoroughbreds were divided into two groups, those with KSS (n = 10) and those without KSS (n = 10). A single laser treatment of the longissimus dorsi muscle (on the left side, between the fifteenth thoracic and the second lumbar vertebrae) was performed. Thermographic examination and palpation were repeated before and after HILT to assess changes in skin surface temperature, muscle tone and pain response. In both groups, HILT was associated with an average skin surface temperature increase of 2.5 °C and a palpation score reduction of 1.5 points, without any differences between the groups. In conclusion, HILT was found to be a safe and supportive treatment method for longissimus dorsi muscle pain and discomfort as assessed by digital palpation in Thoroughbreds. The results of the present study are encouraging, but further studies with larger samples, a longer follow-up period and comparisons with placebo control groups are needed to draw a more valid conclusion.

The reason for undertaking this study was to investigate soft tissue response to high-intensity laser therapy (HILT) by measuring changes in skin surface temperature and longissimus dorsi muscle tone in the thoracolumbar back area in Thoroughbreds with back pain and diagnosed with and without Kissing Spines Syndrome (KSS). Thoroughbreds aged 3–4 years with clinically presented back pain underwent a radiological examination (to assess a lack or presence of KSS) and longissimus dorsi muscle palpation (to assess muscle tone and pain degree). The subjects were divided into two groups, those with KSS (n = 10) and those without KSS (n = 10). A single HILT treatment on the longissimus dorsi muscle, on the left side, was performed. Thermographic examination and palpation were repeated before and after HILT to assess changes in skin surface temperature and muscle pain response. In both groups, HILT caused a significant increase in skin surface temperature of 2.5 °C on average and a palpation score reduction of 1.5 degrees on average (p = 0.005 for both measurements), without differences in any outcome measures between the groups. Furthermore, the correlation between changes in the average skin surface temperature and the average palpation scores in horses with and without KSS were negative (rho = 0.071 and r = −0.180, respectively; p > 0.05). The results of the present study are encouraging, but further studies with larger samples, a longer follow-up period and comparisons with placebo control groups are needed to draw a more valid conclusion.

1. IntroductionThoracolumbar back pain is an important factor causing poor performance in ridden horses [1,2]. A horse’s back protects and stabilizes the body frame and is composed of both soft tissue, such as the muscles and ligaments, and the rigid skeletal elements, which create functional consistency. Equine back disorder may be the result of their individual conformation. Soft tissue injuries are often found in long-backed horses, while osseus lesions are diagnosed in short-backed animals [3]. The longissimus dorsi muscle and supraspinous ligament are commonly found to be the soft tissue cause of thoracolumbar pain [3]. The main cause of epaxial muscle and ligament pain is improper saddle and girth fitting [4], the animal’s emotional tension or physiological stress [5], and the rider’s load on the horse’s back [6,7]. One of the most common osseus causes of back pain is dorsal spinous process impingement, which is a part of overriding dorsal spinous processes, often referred to as “Kissing Spines Syndrome” (KSS) [8]. KSS can be diagnosed in all breeds, at any age, and any sex [1], but racing Thoroughbreds have been shown to have a higher prevalence when compared with other breeds [9]. KSS is mostly localised between the T10-T18 vertebrae, but it can also affect the lumbar spine [10]. One of the major clinical sings of KSS is chronic longissimus dorsi muscle soreness and increased muscle tone. The pathway of secondary epaxial muscle pain, associated with primary osseus lesions in horses, is not understood in detail. One possible explanation, based on human medicinal knowledge, is that spinal vertebrae diseases cause local ipsilateral multifidus muscle atrophy, resulting in spinal instability. The musculoskeletal system then compensates for the disability by epaxial muscle tension and shortening [11,12].The clinical signs of back pain are highly variable [9], but orthopedic evaluation of muscle pain in horses is traditionally assessed by palpation [13]. Deep palpation allows for a general evaluation of back health, based on spinal mobilization and palpable muscle hypertonicity trigger points [14,15]. Pain is detected based on the evaluation of “pain reactions”, which are measured in terms of several scoring systems and scales used by both scientists and practitioners [13,16,17].Longissimus dorsi pain, whether of primary or secondary origin, reduces thoracolumbar spine flexibility and, thus, disrupts its proper biomechanics [10]. The elimination of epaxial muscle spasms and pain is therefore an important part of treating back diseases in horses.Several treatment modalities are available for treating epaxial muscle pain in horses. Medical therapies include the use of general administered non-steroidal anti-inflammatory drugs [3,8,10] or local corticosteroid injections [8,10]. Alternative treatments include manual therapy such as massage, stretching or chiropractic, and physical therapies like magnetic field therapy, extracorporeal shock wave therapy and hydrotherapy [18,19,20]. Currently, laser therapies such as high-intensity laser therapy (HILT) are very popular [21]. HILT is a non-invasive and safe therapy based on the application of focused light generated by class IV (power > 0.5 W) laser devices [22]. It utilises high peak power (1–3 kW), infrared wavelengths (600–1000 nm) and short single pulse durations (>150 ms). As such, HILT can reach deep tissue without excessive thermal effects or cellular damage and can be used in the treatment of bigger joints and large muscle groups, which are difficult to reach with low power lasers [22,23]. Only a few equine studies have demonstrated the positive results of HILT in the treatment of musculoskeletal disorders. HILT is effective in the treatment of soft tissue injuries, such as tendinopathies and desmopathies [24,25,26,27,28]. In an earlier article, we described eleven clinical cases of horses with tarsal osteoarthritis, treated with HILT as a monotherapy, where we found that HILT reduced joint pain and lameness grade, but poorly limited joint discomfort after a flexion test in short-term outcomes [29]. Studies describing the impact of laser therapy on muscle pain and tension have been conducted in both experimental animals and humans. Lopes-Martins et al. [30] have found that 0.5 J/cm2 and 1 J/cm2 (655 nm and 2.5 mW) doses of low-level laser therapy prevent the development of muscle fatigue in rats after repeated tetanic contractions. Ramos et al. [31] have reported that 3 J doses of low-level laser therapy at 810 nm and 100 mW are effective for improving functional outcomes in the early phase following tibialis anterior muscle strain injury in rats. In humans, studies have shown that the photothermal effects of HILT may lead to improved muscle relaxation which, thus, reduces pain [32]. Moreover, HILT (808 nm wavelength, continuous wave mode, average power of 4.28 W/cm2) has resulted in positive effects in relieving muscle tension in patients with hemiplegia [33]. The only research which evaluated the clinical effectiveness of laser therapy in treating thoracolumbar pain in horses was performed by Haussler et al. [34]. They found that laser treatment produced significant reductions in back pain, epaxial muscle hypertonocity and trunk stiffness in 61 competitive western performance horses. However, this research involved the use of low-level laser therapy. A review of the published literature was carried out using the following databases: Scopus, Web of Science and Google Scholar. Additionally manual searches were performed in the indices of the Equine Veterinary Journal and Journal Equine Veterinary Science. The key words for the literature search included “HILT”, “High Intensity Laser Therapy”, “musculoskeletal pain”, “muscle pain”, “Kissing Spines Syndrome”, and “Thoroughbreds”. To the best of our knowledge, this study is the first clinical examination of the effectiveness of HILT in treating muscle tissue in horses with back muscle pain. The objectives of the study were to evaluate and compare soft tissue responses to HILT by measuring changes in skin surface temperature and longissimus dorsi muscle tone in the thoracolumbar back area in Thoroughbreds presenting back pain (diagnosed with or without KSS). It was hypothesised that HILT would increases skin surface temperature and reduce longissimus dorsi muscle tone in both groups of horses but that better effects would be achieved in horses without KSS.2. Materials and MethodsThe Animal Welfare Advisory Team at Wroclaw University of Environmental and Life Sciences approved the study design, which is in compliance with Polish and European Union legislation on animal experimentation (no 1/2023). The procedures used in this study were deemed not to cause pain, suffering, distress or lasting harm equivalent to or higher than that caused by the introduction of a needle (article 1.5f EU directive 2010/63/EU). Ethical approval was granted without a formal application. Written consent was obtained from Partynice Racecourse in Wroclaw for all the racehorses participating in this study.2.1. Horses and Inclusion CriteriaThe horses were selected from the Partynice Racecourse in Wroclaw (Poland) in September 2021. A total of 20 3–4 year-old racehorses (11 stallions and 9 mares) were selected after fulfilling the inclusion criteria. The criteria were as follows: (1) present longissimus dorsi muscle pain in thoracolumbar region palpation; (2) be healthy according to basic clinical examination and to have no clinical lameness in walking and trotting on hard ground in a straight line; (3) maintain the same race training programme with the same trainer; (4) be free from any systemic and local administration of anti-inflammatory drugs or analgetic drugs during the 8 weeks before the study; (5) be free from any manual or physical therapy, including acupuncture, massage, osteopathy or magnetotherapy in the 4 weeks prior to the study; (6) have the presence or lack of radiographic signs of KSS (radiological assessment and KSS criteria are described below); (7) have the presence of pigmented skin in the thoracolumbar back area (black, bay or chestnut coat color).2.2. Study DesignThe horses were divided into two groups, those with KSS (n = 10) and those without KSS (n = 10). On the examination day, each horse underwent the same examination protocol. The horses were inspected early in the morning and before any activity. All the measurements were conducted with the animal standing equally on all weight bearing limbs in a stable corridor. Thermographic examination was performed to determine the skin surface temperature of the thoracolumbar back, followed by longissimus dorsi muscle palpation, to assess tone and pain degree. A single laser treatment of the longissimus dorsi muscle was then performed. The thermographic examination followed by palpation were then repeated immediately after HILT to assess changes in skin surface temperature and muscle pain response. 2.3. Radiographic Examination and PalpationLaterolateral projections of the thoracolumbar spine were performed for the purposes of radiological KSS assessment. The horses stood square and weightbearing on hard ground, with the head and neck in a natural position to avoid false changes in the distance between the dorsal spinous processes [35]. The images were blindly and retrospectively evaluated by one veterinarian (P.Z.). According to Turner [9], a diagnosis of KSS can be made when two or more vertebrae touch or overlap. Non-instrumental palpation, i.e., conventional palpation, was performed unilaterally (left side) on the longissimus dorsi muscle in the area between the fifteenth thoracic and the second lumbar vertebrae. A palpation scoring system for horse muscle tone and pain reaction was used, according to Varcoe-Cocks et al. [13], which is as follows: (0) soft, with low muscle tone; (1) normal tone; (2) stiff muscle but not painful; (3) stiff and/or painful muscle with slight associated spasms but without horse movement; (4) painful muscle with associated spasms and local horse movement, i.e., pelvic tilt and extension response; (5) very painful muscle with spasm and behavioural response, i.e., ears flat back and kicking. The horses were examined and scored subjectively for longissimus dorsi muscle pain by a qualified equine physiotherapist (M.S.D.) 2.4. Thermographic Examination The thermographic examination was performed with a VarioCam HR infrared camera (uncooled microbolometer focal plane array; resolution, 640 × 480 pixels; spectral range, 7.5–14 mm; accuracy ±1 °C, sensitivity 0.02 °C InfraTec, Dresden, Germany). Prior to the study, the thermal camera was quality assured using an Isotech 988 blackbody calibration source (Isothermal Technology, Stockport, United Kingdom). The examination protocol was the same as previously described by Soroko et al. [36]. To reduce the effect of environmental factors, like air draughts and sunlight, the images were obtained within an enclosed stable with closed windows. Horses were examined with the acclimatization period of 30 min prior the imaging and had a brushed back area, approximately 1 h before examination. The distance between the horse’s back and the equipment was set at 1.5 m for all images, with the emissivity set at 1 for all measurements [37]. The average ambient temperature in the stable was 19 °C and humidity 45% at the time that the images were taken, as measured by a TES 1314 thermometer (TES, Taipei, Taiwan). Regions of interest (ROIs) were determined for each thermographic image. The average skin surface temperature of the square treatment area (10 × 10 cm2) in the region under investigation was determined using IRBIS 3 Professional software (InfraTec, Dresden, Germany) (Figure 1). The thermographic examination was performed by the same therapist (M.S.D.).2.5. High Intensity Laser TherapyThe laser therapy was performed with a class IV Polaris HPS laser (Astar, Bielsko-Biała, Poland), a commercial laser light source used in human physiotherapy and in veterinary medicine. The Polaris HPS has two synchronised sources of different wavelength laser light in the near-infrared spectrum. The first wavelength is 808 nm (AlGaAs laser with 8 W of output power), while the second is 980 nm (InGaAs/AlGaAs laser with 10 W of output power). The two wavelengths are emitted simultaneously with the propagation axes of the two laser beams being coincident. The same parameters were used at both wavelengths. The energy density was 20 J/cm2, power was 5 W, frequency was 100 Hz and duty cycle was 80%. The total energy delivered over a period of 500 s was 2000 J. The square treatment area was the same as the region previously identified as an ROI in the thermographic examination and was localised over the longissimus dorsi muscle in the thoracolumbar junction on the horse’s left side (in the area between the fifteenth thoracic and the second lumbar vertebrae). The treatment area was not shaved, and no other skin preparation was performed. The laser treatment was administered using a handpiece held in firm contact with the tissue and manually moved during treatment while maintaining even irradiation of the treatment surface. The handpiece spot size was 5 cm2. Both the person holding the horse and the therapist wore laser goggles. The laser scanning in all horses was performed by the same veterinarian (P.Z.).2.6. Statistical AnalysisStatistical analysis of the results obtained was performed using STATISTICA v. 13.3 (TIBCO Software Inc., Palo Alto, CA, USA) and a MS EXCEL template (Microsoft, Redmond, Washington, USA). The data were first plotted for normal distribution analysis using the Shapiro–Wilk test. The significance level was set at p < 0.05. For the quantitative continuous and discrete variables, the following basic descriptive statistics were estimated: medians (Me), lower (Q1) and upper (Q3) quartiles, extreme lowest values (Min) and extreme highest values (Max). Depending on the test results, the non-parametric Wilcoxon signed-rank test was used to compare baseline and post-treatment within a group, while Mann–Withney’s U test was used for comparison between the groups. Correlations between differences in skin surface temperatures and palpation score after the application of HILT in the two groups were calculated using Spearman’s rank correlation coefficient (rho).3. ResultsThe average skin surface temperatures and average palpation scores in horses diagnosed with and without KSS, both before and after HILT, did not differ significantly (p > 0.05; Table 1). In both groups, HILT treatment caused average skin surface temperature to increase by 2.5 °C (Z = 2.803, p = 0.005, Table 1, Figure 2) and a palpation score reduction of 1.5, which gave a highly significant difference (Z = 2.803, p = 0.005, Table 1, Figure 3).There was no correlation between changes in the average skin surface temperature and average palpation scores in either group (rho = 0.071 for group without KSS and r = −0.180 for with KSS, p > 0.05; Table 2).4. DiscussionHorses are predisposed to general back pain and back disorders because of the type and intensity of their work [38]. Furthermore, a study carried out on 572 horses showed that Thoroughbreds have a 76% greater prevalence as well as a higher KSS grading system than other breeds [39]. This is probably because the tips of their spinous processes are close to each other [40]. The elimination of epaxial muscle spasms and pain (whether it is primary or secondary) plays an important role in the welfare of racehorses.In this small trial, the hypothesis that HILT increases skin surface temperature and reduces longissimus dorsi muscle sensitivity in horses with and without KSS was confirmed, but the groups did not differ in terms of the parameters measured, as we expected. Moreover, there was no correlation between changes in the average skin surface temperature and the average palpation score in either group.Similar findings, regarding skin surface temperature increases after HILT, have been reported in our previous study performed on clinically healthy tissue. A single HILT irradiation of the dorsomedial aspect of the tarsal joint in 16 racing Thoroughbreds caused a significant increase in skin surface temperature in the treatment area [41]. In both this study and the previous one, the mean temperature increase was 2.5 °C. The laser parameters used in our previous study were lower compared to those used in the current study. A possible explanation for the similar thermal effect in both studies might be found in regard to the preparation of the irradiated area. In our previous research, the treatment area was additionally clipped. It is known that when laser light passes through a coat it can cause reflection, absorption and a scattering of photons, thus reducing photon penetration in the target tissue [42,43,44]. The photothermal effect of HILT induces mild local hyperthermia sufficient enough to accelerate cellular activity and vasodilatation, thus leading to enhanced local blood circulation in irradiated tissue [45]. This results in a hastened removal of inflammatory cytokines, improvement in the mitochondrial oxidation process, production of adenosine triphosphate, and more efficient absorption of tissue swelling [46]. In the current study, we did not control the presence and degree of vasodilatation, although our previous research confirmed vessel diameter increase in irradiated tissue immediately after HILT [47,48].Mild inflammation is reduced when tissue temperature increases by more than 1 °C, and an analgesic effect and muscle relaxation is obtained when it increases by 2–3 °C. Changes in tissue extensibility can be reached with a temperature increase of 3–4 °C [49]. Muscle sensitivity is one of the main causative factors in decreased muscle flexibility in horses diagnosed without KSS. Conversely, both bone tissue and inflamed muscles with the connective tissue are the main targets in the treatment of horses diagnosed with KSS. The present study showed a palpation score reduction in the longissimus dorsi muscle of 1 to 2 degrees in both groups of horses. In their prospective study, Alayat et al. [32] also confirmed that the photothermal effect leads to improved fascia extensibility and a muscle-relaxing effect and, thus, a reduction in muscle sensitivity. Similar results have been found in human hemiplegic patients, where HILT rapidly decreased muscle tension during laser irradiation, with low muscle tension maintained following treatment [34]. Muscle relaxation and muscle sensitivity reduction are also the outcome of the photomechanical effect of HILT [22]. A particular waveform with regular peaks of elevated values of amplitude and distances (in time) between them can rapidly induce in the deep tissue a photomechanical effect [50]. HILT can provide extremely brief beats at a maximum repetition rate, thus creating real pressure. Heat waves travel through the tissue, stimulating the free nerve endings and causing their inhibition [51]. A possible explanation for the lack of differences between the groups in longissimus dorsi muscle sensitivity reduction is the short-term HILT efficacy control. It can be assumed that, in horses without KSS, the myorelaxation effect can be longer than in horses with KSS, which (as a chronic disease) causes constant tissue irritation. Lee et al. [33] have pointed out that in humans with hemiplegia, treatment efficacy decreases exponentially as a function of illness duration. Moreover, many other studies have shown that the effectiveness of treatment decreases with the duration of the disease [30,52]. In our study, we did not clarify the underlying cause of pain in horses without KSS. Furthermore, in the horses with KSS, we have not established the real cause of back pain. Further studies are needed to clarify this matter. None of the horses included in the study experienced skin burns, swelling or pain reactions during or after HILT application. Moreover, all horses had no interruption in their daily race training due to complications after HILT. The maximum temperature reached after HILT was 34.5 °C, while superficial thermal injuries are noted after topical application of heat at 50 °C [53]. The use of different HILT parameters, with different protocols, may be necessary in future research investigating the maximum positive effects of laser therapy, without tissue overheating.The main limitation of this study was the lack of long-term assessment, especially in horses diagnosed with KSS. The other limitation included a lack of control or laser-sham group, and a blinding test was not included for the post-treatment data collection. There is need for follow-up data to investigate the number and frequency of treatments needed to achieve the best therapeutic effects. The relatively small sample size in both groups of horses was also a study limitation. It would be ideal to have a larger number of Thoroughbreds presenting with back pain, regardless of whether they have KSS. However, from a clinical point of view, it was possible to indicate the positive impact of HILT in the case of treatment performed on a large muscle, such as the longissimus dorsi muscle. Automatic laser scanning is also recommended in future research to overcome the limitations of manual scanning, although the same laser operator was used in all applications.5. ConclusionsIn conclusion, HILT is a safe and supportive treatment method for longissimus dorsi muscle pain and discomfort in Thoroughbreds under the conditions of this study. Furthermore, the photothermal and muscle-relaxing effects were similar in horses suffering from longissimus dorsi muscle pain, regardless of the presence of KSS. The results of the present study are encouraging, but further blinded studies with larger samples, longer follow-up periods and possible comparisons with placebo control groups are needed to make a more valid conclusion.

animals : an open access journal from mdpi

10.3390/ani12070812

PMC8996831

Chronic disorders of the intestinal tract (CID) are characterized by signs of inflammation of the intestine for a period of at least three weeks. Both humans and pets can be affected by these disorders. Different therapeutic approaches can be selected to treat patients and the use of natural products has been increased in the last decade, since oxidative stress plays a key role in the progression of the chronic intestinal disorders. In this review, the antioxidant proprieties of several natural products with potential for treatment of CID in human and veterinary medicine are highlighted. Unfortunately, few clinical trials report the use of these products for treating CID in humans and none in animals.

Chronic intestinal disorders (CID) are characterized by persistent, or recurrent gastrointestinal (GI) signs present for at least three weeks. In human medicine, inflammatory bowel disease (IBD) is a group of chronic GI diseases and includes Crohn’s disease (CD) and ulcerative colitis (UC). On the other hand, the general term chronic enteropathies (CE) is preferred in veterinary medicine. Different therapeutic approaches to these diseases are used in both humans and pets. This review is focused on the use of traditional therapies and nutraceuticals with specific antioxidant properties, for the treatment of CID in humans and animal patients. There is strong evidence of the antioxidant properties of the nutraceuticals included in this review, but few studies report their use for treating CID in humans and none in animals. Despite this fact, the majority of the nutraceuticals described in the present article could be considered as promising alternatives for the regular treatment of CID in human and veterinary medicine.

1. IntroductionChronic intestinal disorders (CID) are a common cause of persistent or recurrent gastrointestinal (GI) signs extended for more than three weeks.Inflammatory bowel disease (IBD) in humans is a group of chronic diseases of the gastrointestinal (GI) tract. They include Crohn’s disease (CD) and ulcerative colitis (UC). The peak of onset is at the age of around 20–40 years old, but they can occur at all ages, last for a lifetime, and severely affect the quality of life [1]. CD can involve the entire GI tract from the mouth to the anus and is characterized by deep lesions of the GI wall. Symptoms include diarrhea, abdominal pain, and weight loss. CD is burdened by complications like stenosis, abscess, and perianal involvement [2]. On the other hand, UC typically affects the rectum and can involve the colon in a continuous way. It is characterized by lesions of the mucosa (erythema, erosions, and ulcers) and clinically followed by bloody diarrhea [3]. Unfortunately, the etiological factors triggering IBD are not yet fully elucidated, but genetic predisposition, gut microbiota dysbiosis, dysregulated immune response, and environmental factors (including diet) are thought to be involved in the pathophysiology mechanisms [4].In veterinary medicine, the use of the term chronic enteropathies (CE) is preferred instead of IBD to identify a group of idiopathic intestinal disorders with evident GI signs (recurrent or chronic), and inflammation in the lamina propria of the small intestine, large intestine, or both [5]. Nonetheless, several phenotypes of IBD have been identified in dogs [6]. In fact, IBD in dogs has different forms when compared to humans, where more standardized clinical, endoscopic, and pathologic aspects can be found [7,8]. CE can be classified retrospectively based on the response to treatment into (1) food-responsive enteropathy, (2) antibiotic-responsive enteropathy, (3) immunosuppressant-responsive enteropathy, and (4) protein-losing enteropathy [9]. Most forms of CE involve a complex interplay among host genetics, the intestinal microenvironment (including bacteria and dietary patterns), and the immune system [10]. The clinical signs often overlap, but they can be distinguished into large intestinal localization (dyschezia, tenesmus, increased frequency of defecation, small volume of faeces, mucus, and blood), small intestinal localization (large volume diarrhoea, weight loss, and vomiting), melaena (upper GI bleeding/ulceration), and abdominal pain, which is uncommon in chronic enteropathy and raises suspicion of pancreatic disease, structural disorders, or perforation. A breed predisposition for CE in dogs has also been described [11]. Different CE phenotypes may reflect different disease severity affecting the intestinal immune system and varied response to treatment over time. This makes it difficult to standardize a treatment plan for these animals. With respect to CE treatment for dogs and cats, there is strong evidence that controlled, elimination, and hydrolyzed diets are beneficial [12]. In addition, the traditional choice of drugs in dogs and cats with CE includes anti-inflammatories, antibiotics, immunosuppressive, and other medications [9]. The use of complementary and alternative medicine is gaining increasing evidence in both humans and pets with digestive disorders. For instance, some commonly used treatments include probiotics, prebiotics, omega-3-fatty acids, vitamins, minerals, bioactive peptides, colostrum, aloe vera, and turmeric. These novel approaches may improve human and animal medical conditions in case of chronic intestinal disorders [9,12].This review will be focused on both traditional therapies and nutraceuticals with specific antioxidant properties that possess established or promising effectiveness for the treatment of CID in humans and animals. 2. Oxidative Stress-Induced Damage in Chronic Intestinal DisordersCell inflammation and oxidative reactions caused by activated leukocytes producing excessive reactive oxygen species (ROS) can overpower the tissue’s antioxidant defenses, resulting in a dysfunction of the enteric mucosa. As part of basal metabolic function, ROS are produced by numerous enzymatic reactions in various cell compartments, including the cytoplasm, endoplasmic reticulum, cell membrane, peroxisome, and mitochondria. ROS plays a variety of physiological roles, including the control of cell differentiation and development, apoptosis, and inflammatory processes via cell signaling [13]. A complex, dynamic mechanism, where different molecules undergo well-established oxidation–reduction reactions, maintains the homeostasis of the intestinal ecosystem, which represents the intestinal mucosa’s response to prevent oxidative damage. The ROS generated by unstable types of oxygen—superoxide anion, hydrogen peroxide (H2O2), and hydroxyl radicals—are the key pro-oxidant molecules. The pathogenesis of CE has been linked to oxidative stress, which may be a key effector mechanism leading to molecular/cellular damage and tissue injury. ROS promote cell damage by preventing the accumulation of antioxidant defenses in cells. For example, oxidative damage is observed in CD patients’ intestinal mucosa as well as their peripheral blood leukocytes [14]. Immune cells that enter the mucosa produce a number of ROS that can be harmful to tissue integrity. Patients with CD have lower levels of antioxidant vitamins A, C, E, and beta-carotene in their blood and mucosa, as well as lower activity of the major cellular antioxidant enzymes glutathione peroxidase (GPx) and superoxide dismutase (SOD) [15]. Oxidative stress and redox signaling are intimately involved in the upregulation of inflammatory cytokines as well as in the increased infiltration of inflammatory cells, through the stimulation of signaling pathways (especially the redox-sensitive transcription factor, nuclear factor kappa-light-chain-enhancer of activated B cells). Inflammation also enhances oxidative stress by inducing the development of ROS and the release of myeloperoxidase from inflammatory cells [16]. In the literature, there is evidence indicating the role of oxidative stress in humans with IBD and recent studies suggest that it could also be a relevant factor in the pathogenesis of dogs affected by IBD [17].3. Therapeutic Management of Chronic Intestinal DisordersCurrent therapies for IBD in humans are based on shared guidelines, particularly those published in the United States and Europe [18,19,20]. Patients affected by IBD are normally treated with a step-up approach, starting with mesalazine and gradually adding higher-level drugs until a complete clinical, laboratory, and endoscopic remission is achieved. The step-up approach was the one classically used also for CD, but in recent years the top-down paradigm has been established, starting the most effective drugs available in patients with predictive factors of more aggressive disease (Figure 1).In veterinary medicine, new approaches to the management of CID in dogs and cats have been developed over the last 30 years. However, therapy for CE is difficult to establish, since the pathogenesis of the disease is not easily understood. The choice depends on the seriousness of the disease and the response to drugs. The most used therapies are antimicrobials, immunosuppressants, and anti-inflammatory drugs. Unfortunately, the scientific evidence of the efficacy and effectiveness of these drugs in animals is lacking and variable across studies. There is stronger evidence for the use of controlled, elimination, and hydrolyzed diets, which are the first-choice approach for pets [5] (Section 4, Figure 1). When deciding which treatment is the most appropriate, adding symptomatic measures (gastroprotectors, antiemetics, motility modulators, etc.) would be beneficial to correct any imbalance.3.1. Humans3.1.1. Conventional TherapyMesalamine represents the cornerstone of therapy of UC. Mesalamine is able to induce and maintain remission. It is most prescribed for UC with mild or moderate disease symptoms. Topical (rectal) plus oral mesalamine is the most efficacious mode of administration. Mesalamine’s exact function is unclear, however the most accepted theory is that it reduces the synthesis of prostaglandins and leukotrienes by modulating the inflammatory response. Mesalamine is also thought to be an antioxidant and a free radical scavenger [21].Systemic corticosteroids (particularly prednisone and methylprednisolone) are the therapy of choice in patients with severe disease activity or with mesalamine failure in UC. In case of mild or moderate disease activity, low-absorption corticosteroids are available: budesonide for CD, beclomethasone, or budesonide multi matrix (MMX) for UC [22]. Steroids have a good rate of effectiveness (around 80%), but unfortunately they cannot be used for prolonged times (no more than 3–4 months) as they are characterized by numerous side effects and lose effectiveness over time [23].After initial steroid-induced remission, immunosuppressants are used to achieve long-term steroid-free remission [24]. The immunosuppressants used in IBD include thiopurine (azathioprine, 6-mercaptopurine), methotrexate (mainly in CD), and cyclosporine (mainly in UC). Another approach to improve gut health and to treat IBD includes the use of probiotics, and prebiotics [25].3.1.2. Advanced TherapyBiological drug therapy (target therapy) has revolutionized therapy for IBD and other autoimmune and neoplastic diseases over the last 25 years.Anti-tumor necrosis factor (TNF) therapy has been widely used for the treatment of IBD in the last two decades as a new approach to the disease’s management. Anti-TNF therapies approved for the treatment of both CD and UC, include infliximab and adalimumab. Golimumab has also been evaluated for use in UC [26]. Unfortunately, one-third of patients are primary non-responders, and dose intensification is needed in 23 to 46% of responders, with drug discontinuation occurring in 5% to 12% of patients per year [27].Vedolizumab is a humanized monoclonal antibody and is the first biological drug created to be selectively effective on the bowel [28].Ustekinumab is a human monoclonal antibody and it is the latest biological drug introduced for the treatment of IBD, borrowed from the excellent experience in psoriasis [29].Tofacitinib (anti-JAK) is the latest drug available to treat IBD, in particular UC. Blocking the JAK-STAT pathway, it interferes with the signaling pathways of several cytokines [30]. 3.2. Dogs and CatsConventional TherapyAntimicrobials are used to fight the microbial dysbiosis which may initiate and drive host inflammatory responses in animals with CE. This therapy is often associated with diet and other drugs, leading to difficulties in interpreting the effectiveness of a single antimicrobial product. For example, studies have supported the efficacy of rifaximin and oxytetracycline or of metronidazole and tylosin with an additional anti-inflammatory action [9,31,32,33]. If an antimicrobial treatment is not successful within two weeks, a new therapy should be considered, with the aim of achieving long-term control of the disease. This would also avoid the risk of developing antimicrobial resistance. In addition, interest in new antimicrobial alternatives to manipulate the GI microbiome is growing [5].A small percentage of animals affected by CE need administration of immunosuppressants. Following the failure of diet and antibiotic treatments, the administration of immunosuppressants could be successful. When a good response to this treatment exists the pathological condition is defined as immunosuppressant-responsive CE. On the other hand, dogs not responding to this treatment are categorized as having non-responsive enteropathy. A treatment plan with immunosuppressive drugs usually includes the use of these medications and modification of the diet [34]. Commonly used immunosuppressant drugs are azathioprine and cyclosporine [33,35].The use of anti-inflammatory drugs helps control the inflammation of the intestine in animals with CE. They usually work together with other therapeutic approaches such as diet and antimicrobial agents. The most commonly used anti-inflammatory drugs are glucocorticoids, 5-aminosalicylates [33]. As reported before, there are a few antimicrobials with anti-inflammatory properties like metronidazole and tylosin [33].In addition, the use of prebiotics and probiotics improves the gut microbiota of animals, especially when affected by GI diseases [25].4. Dietary Interventions in Chronic Intestinal DisordersIn the medical community, there is currently no consensus on dietary recommendations for adult patients with IBD. It is difficult to make strong recommendations due to the lack of randomized controlled trials investigating specific diets and eating habits. Exclusive enteral nutrition (EEN) is an exception since it is prescribed as a first-line treatment for children and adolescents with acute active CD to promote remission [36]. The European Society for Clinical Nutrition and Metabolism (ESPEN) advises that during remission periods, no particular diet should be undertaken because it does not seem to be successful in maintaining remission [37]. Several dietary compounds have been identified as influencing the development and maintenance of IBD, while others appear to be protective.In veterinary medicine, the dietary approach is the first choice to control the symptoms of CE in pets [12]. Several studies demonstrated the efficacy of diet manipulation that in many cases results in a promising long-term outcome (>6 months). In some more severe cases, where a long-term positive effect is not observed, antibiotic or immunomodulant treatments should be added [5]. The change in diet alone has been reported to be effective in over 50% of CE cases as reported in a previous review [38]. Despite the fact that most of the study protocols have low-quality designs, long-term response seems to be supported by diet when used as first-line treatment. The GI tract can be damaged when a subject is affected by CE (microbiota, intestinal permeability and motility, and mucosal immune system). As a consequence, choices in nutrition can influence the equilibrium of GI components and functions. However, well-designed studies are still needed to determine whether specific dietary elements could represent risk factors for the development of CE in dogs and cats. A recent review by Kathrani [12] exhaustively reported the use of diet to manage CE in dogs and cats.4.1. Humans4.1.1. Fiber ContentHoweler created the term “dietary fiber” to characterize a complicated group of non-digestible components of cell walls [39]. Non-starch polysaccharides (e.g., pectin, cellulose), non-carbohydrate-based polymers (e.g., lignan), resistant oligosaccharides (e.g., galatooligosaccharides, fructooligosaccharides), and animal-derived carbohydrates (e.g., chitin) have all been lumped together under the term [40]. Unlike most dietary components, non-digestible dietary carbohydrates (resistant starch and fiber) can withstand stomach acidity and do not degrade in the human small intestine; instead, they are fermented by the gut microbiota consortium within the large intestine, where one microbe initiates the fermentation process and others continue it, resulting in a systematic process [41].Dietary fiber is made up of a variety of connected monosaccharides that form a variety of molecules with different side chains and physical properties, such as solubility and physical organization. While dietary fibers can be classified in a variety of ways, the most prevalent method for nutritional purposes in humans divides them into water-soluble and insoluble fibers [42]. The degree of fermentation by gut bacteria is related to water solubility in the gastrointestinal system. Soluble dietary fiber can reduce glycemic response by increasing digesta viscosity, which delays stomach emptying and nutrient release. Easily digestible fibers (Arabinoxylan, pectin, inulin, b-glucans, fructo-oligosaccharides, xyloglucans, and galactooligosaccharides) are examples of soluble dietary fibers and are degraded to volatile fatty acids, which serve as a nutrient substrate for the microbiota as well as the enterocytes [43]. Insoluble dietary fibers, such as lignin and cellulose, are thought to be less beneficial to gut microorganisms because their strong hydrogenbinding networks restrict the amount of surface area available for fermentation. Fibers that are difficult to digest stimulate intestinal peristalsis and thus the expulsion of pathogenic microorganisms [44].Although the literature on the relationship between dietary components and the onset of IBD is still uncertain, several studies showed that dietary fiber intake has a positive impact and plays an important role in the prevention of CD [45]. Anti-inflammatory action through butyrate’s protective effects, reduction in colonic permeability, and prevention of pro-inflammatory cytokine transcription are some of the mechanisms proposed in the literature [46]. Furthermore, dietary fibers have shown an effect on the microbiome, influencing immunological homeostasis in a regulatory manner [47]. Fiber has a prebiotic function by increasing the growth of beneficial bacteria [12]. Patients with IBD, on the other hand, often complain that high-fiber foods aggravate their symptoms [48] and fiber is contraindicated in patients with stenosing CD.4.1.2. Specific DietsIn the existing scientific literature, complex diets for IBD treatment have been suggested.Exclusive Enteral Nutrition (EEN) EEN relies on the administration of a liquid nutrient formula orally or via a feeding tube for 4–12 weeks. Individual amino acids are found in elemental formulas; semi-elemental peptides of different chain lengths are found in semi-elemental formulas; and intact proteins are found in polymeric formulas. Food reintroduction data is still sparse and inconclusive after this time frame. Most centers, on the other hand, suggest a 2–3 week gradual reintroduction of the normal diet [49]. This type of diet is useful in children with CD, but it is not useful in UC [50]. However, since this dietary therapy requires children to abstain from eating for many weeks, adherence is challenging and unpredictable. This type of diet can have a big impact on the child’s family as well. In addition, essential amino acids have been shown to activate mucosal immunity while maintaining intestinal homeostasis and forming the intestinal mucosal barrier [51]. During EEN therapy, there could be a decrease in the development of metabolites that may be involved in the immunological attack on gut microbiota [52]. Carbohydrate Diet (SCD)SCD is a diet for patients affected by IBD. This diet consists of a modified carbohydrate diet that allows monosaccharides but prohibits disaccharides and most polysaccharides. Fruits and vegetables with more amylose than amylopectin, dry-curd cottage cheese, butter, nuts, nut-derived flours, meats, eggs, and oils are allowed in the SCD. Sucrose, isomaltose, maltose, lactose, both real and pseudo-grains and grain-derived flours, okra, potatoes, fluid milk, corn, soy, lactose-rich cheeses, and most food additives are not permitted. To avoid lactose, the diet is supplemented with entirely fermented yogurt [53]. SCD is focused on the ingestion of complex carbohydrates with limited digestive requirements. Although the mechanism of action is unknown, it is hypothesized that changes in the fecal microbiome reduce intestinal inflammation. A very restrictive diet necessitates significant lifestyle changes, and patients need follow-up to ensure proper nutrition, taking into account that specific dietary deficiencies can occur as a result of specific food restrictions, especially of dairy products that contain vitamin D and calcium. The limited intake of grains, fruits, and vegetables can also result in folate, thiamine, and vitamins B6, C, and D deficiencies.Anti-Inflammatory Diet for IBD (IBD-AID) IBD-AID was developed by a team at the University of Massachusetts Medical School and is based on the SCD. This diet was created for patients who had failed to respond to pharmacological treatment [54]. The IBD-AID is made up of five parts: the first is the modification of carbohydrates, such as refined or processed complex carbohydrates and lactose; the second is the ingestion of probiotics and prebiotics; the third is the modification of dietary fatty acids; the fourth is the detection of the overall dietary pattern and missing nutrients, as well as the identification of intolerances; and the fifth is the modification of dietary fat acids. Likewise, this is a very restrictive diet, that can lead to nutritional deficiencies due to the lack of nutrients, especially micronutrients. Its mechanism of action has yet to be discovered.4.2. Dogs and Cats4.2.1. Fiber ContentThe dietary use of high fibers has been found to have numerous health benefits including anti-inflammatory properties and helping to maintain the intestinal barrier function. Fiber has a prebiotic function in promoting the growth of beneficial microorganisms in the intestine [12]. 4.2.2. Specific DietsIn the scientific literature, different diets as treatments for CE in dogs and cats have been suggested.Hydrolyzed DietsHydrolyzed diets have been successfully used in the management of CE in dogs and cats. In this type of diet, proteins are broken down by enzymes and the organisms do not recognize them as proteins. This type of diet can help to reduce the level of dysregulation of the immune system and is considered highly digestible [55].Limited-Ingredient DietsA limited-ingredient diet should ideally provide a single carbohydrate and a single protein source. Considering the exposure of dogs and cats to multiple ingredients in their diets, a limitation of the number of ingredients could be beneficial to limiting the antigen load in the GE tract in order to reduce intolerances [55].Fat Reduced DietsReduction of fat in the regular diet reduces the passage of the fat in the colon, reducing dysbiosis and epithelial cell damage. A fat restriction diet has been shown to be effective in dogs affected by PLE-lymphangiectasia but it could also be considered as an option in cases of CE [55]. The fat content of the dry matter must be no more than 15% in dogs and 25% in cats [56].Gluten-Free DietsA gluten-free diet seems to be quite effective in reducing GE symptoms even though no specific trials have been performed yet. The effectiveness of this diet has been hypothesized because some of the commercial diets are already gluten-free. A novel protein diet has been found to be more effective in pathologies involving the large intestine [49]. Parenteral Nutrition (PN)This type of approach is an option that is required only in rare cases, being very expensive and of difficult management for the pet owners. This type of nutrition keeps under control the daily intake of the different nutrients and contributes to bowel rest. This technique is used in human medicine even if the enteral nutrition results in a more physiological intervention for the intestine [12].5. Role of Nutraceuticals as Antioxidants in Chronic Intestinal Disorders: Phytocomplex, Trace Elements, and Vitamins Functional foods and bioactive natural compounds have become relevant research topics when discussing new approaches to treat intestinal disorders in human and veterinary medicine, as the long-term use of traditional drugs causes complications. Nutraceuticals by definition, are food or food supplements that have been formulated or processed to enhance the pharmacological properties of functional bioactive ingredients, providing health and medical benefits, including the prevention and treatment of diseases [57]. Phytochemicals of nutraceutical importance, are non-nutritive plant chemicals found in fruits and vegetables [50,58,59]. Plants produce phytochemicals as part of their defense mechanisms against pathogens, which are derived from their primary and secondary metabolisms. These chemicals play a significant role in the body’s defense against oxidative stress and inflammation together with other potential health benefits.Complementary and alternative medicine (CAM) use has been found to be more common in human patients with IBD than in healthy individuals, with some studies reporting values as high as 72% [60]. CAM can assist in the treatment of chronic intestinal disorders and prolong the clinical remission in human patients because of the antioxidant and anti-inflammatory properties of the used plants. Several studies in dogs and cats reported the use of these natural ingredients as promising to manage diseases. Nutritional supplements described in this section include phytocomplex, vitamins, and minerals. Here, available evidence of their proven or promising effectiveness in human and veterinary medicine is presented (Table 1).5.1. Phytocomplex5.1.1. Curcuma longaCurcuma longa is a perennial herb which, when dried, becomes the source of the spice turmeric. Turmeric’s active part is the flavonoid curcumin. Water and fat-soluble turmeric and curcumin show strong antioxidant properties. Since curcumin has autophagy-regulating properties it helps in the improvement of colitis. Curcumin also inhibits the development of autophagosomes in colonic epithelial cells and has also an anti-inflammatory effect in acute and chronic inflammation status [61].The administration of curcumin was found to be more effective than placebo in keeping human patients with quiescent UC in remission [62] and with various pro-inflammatory diseases [63]. A dosage range of 1500–3000 mg/day demonstrated a significant difference between clinical remission and endoscopic remission rates in the intervention and placebo groups [62]. Only Hanai et al. reported mild side effects in a Japanese population, such as abdominal distension, nausea, and an increased number of bowel movements [64].The veterinary use of curcumin has also shown promise in intestinal diseases in dogs with stimulation of the antioxidant system and evidence of anti-inflammatory effects [65,66]. The findings of a study suggested that curcumin and the commercial product Meriva curcumin phytosome® reduced inflammation in canine IBD but no specific antioxidant effect was tested [67]. No data on cats is available.5.1.2. Aloe veraAloe vera is a tropical, drought-resistant succulent plant. The leaves are filled with brown or yellowish milky juice that contains the most bioactive compounds. Not all species are therapeutic, others can be toxic or neutral. It has been shown to have antioxidant, antibacterial, anti-inflammatory, immune-boosting, anti-cancer, healing, and anti-diabetic effects. The 75 biologically active compounds (i.e., flavonoids, terpenoids, lectins, magnesium, zinc, vitamins) present in Aloe vera show synergic effects [68,69]. A randomized, double-blind, placebo-controlled trial of oral Aloe vera in human patients affected by UC has been performed [70]. The volume was 100 mL twice daily, which is the greatest quantity that may be tolerated and is widely used by people who use aloe vera gel for a variety of purposes. To ensure tolerance and reduce the risk of adverse effects, patients were advised to start with 25–50 mL twice daily for up to 3 days. Oral Aloe vera was found to produce a clinical response more frequently than placebo, as well as a reduction in histological disease activity. Among the 30 patients randomized to Aloe vera gel, no serious adverse events were registered; however, nine patients reported of abdominal bloating, one of foot pain, one of sore throat, one of temporary ankle swelling, one of acne, and one of worsening eczema.One study reported that Aloe vera juice was used as a stomach tonic for vomiting and irritation in dogs [71]. To our knowledge, no specific studies on the use of this nutraceutical in the treatment of CE have been performed in veterinary medicine.5.1.3. Boswellia serrataThe gum resin obtained from the Boswellia serrata (B. serrata) tree, a species of the Burseraceae family, is known as frankincense. The major active derivatives are 11-keto-β-boswellic acid, boswellic acid, and acetyl-11-keto-β-boswellic acid, all of which are believed to have antioxidant and anti-inflammatory properties [72]. In India, preparations made from the gum resin of B. serrata have been used as a common remedy for inflammatory diseases in Ayurvedic medicine. For example, B. serrata extract was successfully used in maintaining the remission phase over a prolonged period of time in human patients with UC [73]: Casperome® is a delivery form containing a 1:1 ratio of highly standardized B. serrata extract and soy lecithin, as well as around half a part of microcrystalline cellulose to improve the physical condition and standardize the product to a content of triterpenoid acids of at least 25%. Remission or improvement in one or more of the parameters was achieved by administering Boswellia gum resin [74]. This nutraceutical is largely demonstrated to be effective as an anti-oxidant, anti-inflammatory, and anti-diabetic agent in dogs in both in vitro and in vivo studies [75,76,77]. No studies on the use of B. serrata in the management of CE in dogs and cats have been performed so far.5.1.4. Triticum aestivumWheatgrass juice (Triticum aestivum) is an extract made from wheatgrass pulp. Wheatgrass extracts showed antioxidant activity by scavenging free radicals in conjunction with phenolic and flavonoid material. In a study, for one month, 100 mL wheat grass juice was consumed. The juice was to be consumed right away by the patients. The doses were gradually raised, starting with a 20 mL initial dose and increasing by 20 mL every day. It was proven to be an efficient and safe treatment for active distal UC as a single or adjuvant treatment [78]. This natural product has been demonstrated to be an effective antioxidant in reducing senile cataracts in dogs [79]. The biologically active substances can be partially absorbed during digestion and future use in treating GI conditions in animals can be considered. 5.1.5. Plantago spp.Plantago ovata is an annual herb, local to the Mediterranean region. It is a source of soluble fiber and has been reported to treat intestinal disorders like diarrhea, constipation, IBD, and hemorrhoids [80]. Polysaccharides, flavonoids, phenolic compounds (caffeic acid derivatives), terpenoids, alkaloids, and vitamins are among the bioactive components [81].Its seeds (10 g b.i.d.) have been shown to be as effective as mesalazine in preventing UC relapse. These treatment choices may be appealing to human patients who are unable to tolerate mesalazine [82]. Plantago major seems to be effective in the complementary management of UC [81]. Plantago spp. can be used to treat endoparasites and stomach problems in animals [71,83], but no specific studies on CE in animals have been carried out.5.1.6. Serpylli herbaSerpylli herba is a European Pharmacopeia officinal medicine made up of the aerial portions of wild thyme (Thymus serpyllum). It has been tested in rodent colitis experimental models [84]. In two separate experimental models of colitis in rats (TNBS) and mice (DSS), S. herba extract showed intestinal anti-inflammatory properties, indicating that it could be used to treat human IBD [84]. The antioxidant qualities are most likely attributable to the high polyphenol content. Given the in vitro results, its efficacy on intestinal disorders in human and veterinary medicine cannot be excluded even though no in vivo trials have been performed to date.5.1.7. Vaccinium myrtillusBilberries (Vaccinium myrtillus) contain one of the highest levels of natural anthocyanins that exert the most effective antioxidant activity [85]. It inhibits protein and lipid oxidation. Human patients with mild to moderate UC were treated with an anthocyanin-rich bilberry preparation in addition to their regular medicine in an open-label pilot study [86]. The bilberry preparation was made specifically for this investigation under highly standardized settings, with the primary ingredients being dried, sieved bilberries (59.63%) and concentrated bilberry juice (25.90%). Small metal trays containing 40 g of the preparation were used to package it. For a total of six weeks, patients were given a daily bilberry preparation dose of 160 g (4 trays per day), equal to 95 g dry weight (similar to about 600 g fresh fruit, assuming an 80–85 percent water content in fresh bilberries). Patients were instructed to avoid eating or drinking for one hour before and after ingesting bilberries. Endoscopic and histologic disease activity, as well as fecal calprotectin levels, were considerably reduced in the study participants after six weeks, suggesting that anthocyanins could be used as an alternative treatment human IBD patients. Both the feces and the tongue of all individuals had a dark bluish to black staining (one patient furthermore reported slight discoloration of the teeth). Mild dyspeptic symptoms were noted by one patient (heartburn). Furthermore, 33% of patients complained of mild to moderate flatulence. There were no major clinical adverse events or changes in the safety laboratory indicators that we noticed. Studies performed in healthy dogs confirmed the strong anti-oxidant activity of bilberry extract [87] but no data on the effects on dogs and cats affected by CE is available. 5.1.8. Camellia sinensisCamellia sinensis (C. sinensis) is a species of evergreen shrubs or small trees. The leaves and leaf buds are used to produce tea (yellow tea, green tea, oolong tea, white tea, dark tea, and black tea). It has been demonstrated to have anti-inflammatory effects on lipopolysaccharide-stimulated macrophages and DSS-induced colitis in mice, reducing the oxidative stress. The researchers hypothesized that C. sinensis could be used as a safe and effective dietary strategy in preventing and treating human UC [41,88]. One study reports an anti-diarrheal effect of C. sinensis in children suffering from nonbacterial diarrhea [89,90]. Studies in dogs confirm the antioxidant properties of this plant [91,92] but no data on its effect on dogs and cats affected by CE is available in literature.5.1.9. CitrusCitrus fruit and juices are among the most common phenolic rich dietary sources. For example, diosmetin is a natural flavonoid molecule found in citrus plants. It has a number of pharmacological properties, but little is known about its impact on CID. The therapeutic effects of diosmetin on mice models of chronic and acute colitis were investigated in a recent study [93]. They discovered that diosmetin treatment significantly reduced colon oxidative damage by regulating intracellular and mitochondrial reactive oxygen species levels. No information on the effects on dogs and cats affected by CE is available.5.1.10. PomegranatePomegranates are not citrus fruits, despite their citrus flavor. They do not come from the same plant family and cannot be considered cousins. Their juice, however, can be blended to make a refreshing drink that is high in critical vitamins.In mouse models of IBD, pomegranate fruit administration reduced colon tissue damage, antioxidant status, and inflammation [94]. In a DSS-induced colitis model, pomegranate extract was demonstrated to lessen the severity of colitis by modulating the gut microbiota and down-regulating COX-2, PTGES, iNOS, and PGE2 expression [94]. We found no in vivo studies on the use of citrus in the management of CE in humans, dogs, and cats.5.2. Trace ElementsTwo relevant trace elements, zinc, and selenium have been discussed in this review because of their antioxidant and anti-inflammatory effects. Overall, trace element status in CID in human and veterinary medicine, appears to be a neglected subject, and further clinical trials are needed to investigate this issue more thoroughly [32,95,96].5.2.1. ZincZinc is an essential mineral that is naturally present in some foods (e.g., beans, nuts, seafood). Zinc influences oxidative stress, immune response, and inflammation [97]. It appears that zinc deficiency is widespread among human patients with IBD, with a prevalence of 15 to 45% [98]. Provided that IBD is a chronic inflammatory disease linked to immune system function, controlling and maintaining normal zinc levels in IBD human patients appears to be important. Reduced serum zinc concentrations have been shown in preclinical models, human translational studies, and animal model studies to exacerbate inflammation; this effect may be mediated by a variety of pathophysiological mechanisms, including the increased production of pro-inflammatory cells, modulation of the inflammatory cytokine response, aggravation of mucosa leakage, and disruption of the epithelial barrier [99]. Zinc is a cofactor for many enzymes and is involved in a variety of important processes, including the protection against free radicals and the control of innate immunity by regulatory cells [100]. Zinc deficiency has been shown to cause oxidative stress in a variety of cells and tissues, and zinc supplementation may help prevent oxidative harm [101]. Zinc, as a cofactor of the antioxidant enzyme SOD1, is integrated into the cellular antioxidant protection mechanism and protects cells from oxidative stress by increasing GPx biosynthesis, inducing metallothionein synthesis, and inhibiting NADPH oxidase [102], which is one of the most important sources of free radical activity [103]. The zinc-cytoprotective enzymes metallothioneins are up-regulated in response to an inflammatory stimulus as direct oxidant scavengers. These proteins belong to a group of cysteine-rich small proteins that play a role in reducing ROS development during oxidative stress [104]. Zinc also seems to be involved in the mucosal barrier function of the intestine. Zinc deficiency increases occludin proteolysis and decreases claudin-3 expression. Since these proteins are involved in the formation of tight junctions between intestinal epithelial cells, it appears that low zinc levels will weaken the intestinal mucosal barrier [105]. Due to zinc’s anti-inflammatory and antioxidant properties, as well as its protective role in the pathogenesis of IBD and the high prevalence of zinc deficiency among IBD human patients, providing adequate zinc levels is likely to be beneficial for more successful treatment of this chronic disease.Vomiting, diarrhea, fever, lethargy, muscle discomfort, and stiffness are all symptoms of zinc toxicity, as are anemia, copper insufficiency, and kidney injury. In humans, the fatal dose of intravenous zinc is unknown. The National Institute of Health’s (USA) upper safe daily oral intake limit for elemental zinc is 40 mg/d, while the European Food Safety Authority’s lower limit is 25 mg/d. Zinc has been used as a parenteral nutrition component at levels ranging from 5 to 22 mg/d without any known negative effects [106]. In dogs and cats, zinc is known to be necessary for several normal functions linked to metabolic, enzymatic, and transcription factor activities, due to its antioxidant activity. Recommended zinc maximum level for complete dog food is 22.7 mg per 100 g dry matter (DM) [107]. Findings of a recent research demonstrated low levels of zinc in serum of dogs suffering from LPE (lymphocytic-plasmacytic enteritis), a kind of IBD. Thus, concentration of serum zinc could act as an indicator of LPE prognosis [108]. However, studies on zinc deficiency and the supplementation with this trace element in veterinary medicine are scarce [109,110]. Promising positive effects of the combination of zinc and other antioxidant elements on the oxidative stress caused by different diseases could be hypothesized. In one study, dogs fed with a diet supplemented with a preparation of selenium/zinc-enriched probiotics showed an increased total antioxidant capacity in the blood compared to the control group, supporting the antioxidant capacity of zinc [111]. Zinc has been used as a treatment in rats with experimentally induced liver cirrhosis and in copper toxicosis in dogs [112,113]. In another study, strong therapeutical potential of ZnO Nanoparticles has been reported to treat IBD in mice [114]. The synergic effect of zinc, silymarin, and vitamin E in a supplement tested in dogs has been shown to improve liver function [115]. In a group of dogs with IBD, the levels of zinc and magnesium were found to be lower compared to control dogs but no supplementation was tested. In addition, zinc was recommended for inflammatory and malabsorptive intestinal diseases in dogs where zinc absorption may be compromised [109]. Although preliminary studies on dogs have demonstrated lower zinc levels in various disorders i.e., skin, hepatic, renal, neurological, and behavioral disorders, therapeutic effects of zinc supplementation can be only hypothesized [116]. Unfortunately, the role of zinc deficiency and the usefulness of dietary supplementation with zinc in CE are yet to be investigated in dogs and cats. Minimum but not maximum zinc supplementation doses for dogs and cats have been established by the National Research Council (NRC, [117]) and safe doses for improving liver conditions have been tested in in vivo trials [115]. 5.2.2. SeleniumSelenium is a member of the sulfur family of elements and is naturally present in some foods (e.g., seafood, liver, and cereals). Selenium is primarily known for its antioxidant properties. The selenium-dependent enzyme glutathione peroxidase (GPX) is an essential antioxidant enzyme involved in the elimination of peroxides and hydroxyl free radicals produced during metabolism [118]. Selenium deficiency has been identified in human IBD patients in some studies [119]. Various selenoproteins are abundant in the intestine and may play a role in redox homeostasis control. The nuclear factor erythroid 2-related factor 2 (Nrf2)/Keap1 signaling pathway controls redox balance in cells. Nrf2 is a transcription factor that helps the body’s antioxidant defense system. The inflammatory processes in IBD appear to be weakened by Nrf2, and some studies have indicated that increasing its activity with compounds like polyphenols can help protect against intestinal inflammation [120]. LPS-induced oxidative stress and NO elevation can be decreased by selenium: NO is involved in the pathogenesis of IBD and has the ability to stimulate TNF-alfa development [95]. Furthermore, selenium may be able to minimize the severity of IBD inflammation by inhibiting prostaglandin E2 (PGE2) pro-inflammatory activity [95]. Selenium has been shown to protect against lipid peroxidation, and selenium supplements have also been shown to be helpful in this regard [121]. It is unclear if selenium supplements will help human patients with IBD stay in remission but, given the important roles of selenium and selenoproteins in reducing oxidative stress, inhibiting inflammatory signaling pathways, and increasing the population of anti-inflammatory M2 macrophages, it is conceivable that appropriate selenium levels could help keep them in remission. A study was carried out to see if a combination of selenium and vitamin E could protect rats against experimental colitis caused by acetic acid [122]. In plasma and colon samples, researchers measured the activities of prolidase (PRS), catalase (CAT), total antioxidant capacity (TAC), myeloperoxidase (MPO), oxidative stress index (OSI), total oxidant status (TOS), and total thiol (T-SH). Selenium and vitamin E treatment reduced MPO activity in the colon (p < 0.05). Selenium and vitamin E enhanced TAC and T-SH in the colon (p < 0.05). According to these findings, selenium and vitamin E may play a significant role in the prevention of oxidative damage caused by acetic acid-induced inflammation.Selenium consumption levels are typically between 11 and 280 g per day. In the typical population, selenium levels in plasma and blood are around 100 g/L. In healthy adults, urine concentrations range from 10 to 85 g/L. It has been discovered that human doses of 10 mg/kg and higher are linked to an increased risk of death. The intake of gun bluing compounds, which often contain selenous acid as well as other potentially poisonous substances, is linked to the majority of human fatal instances. The administration of organic selenium in the form of selenocysteine or selenomethionine was not linked to any cases of acute toxicity [123].For a long time, selenium was considered solely as a toxic element in animals. Recommended selenium maximum level for complete dog food is 56.80 µg per 100 g dry matter (DM) [107]. Cases of intoxication in horses, hogs, cattle, and chicken were reported for several years but not in dogs where the no-observed-adverse-effect level (NOAEL) of organic selenium was set in beagle dogs [124]. Probably, carnivores are able to maintain a high level of selenium compared to other species of animals, like most farm animals [96]. An interest in studying this element in the lab started to highlight its importance for health. At the moment, research on this trace element mainly focuses on humans and farm animals [125]. There is still a need for further studies on selenium targeting different species, including dogs, cats, and other carnivores. In a study, dogs were fed with a diet supplemented with a preparation of selenium/zinc-enriched probiotics, the biomass of the final product was 26.83 g/L, organic Se concentration was 173.35 µg/g, organic Zn concentration was 4.38 mg/g, Candida utilis biomass was 8.69 lg colony-forming units (CFU)/mL, and Lactobacillus biomass was 9.61 lg CFU/mL). The study showed total antioxidant capacity in the blood increased, supporting the antioxidant capacity of selenium [111]. A recent review reported interesting results on the effects of a diet supplemented with selenium in dogs with cancer, reproductive problems, renal disease, and parasitical diseases [96]. Unfortunately, no specific studies on the effect of selenium alone or in combination with other nutraceuticals on CE in dogs or cats are available. 5.3. Vitamins5.3.1. Vitamin AThe most notable member of the group of carotenoids found in the human diet is b-carotene (b-CAR), a naturally occurring provitamin A that is a significant source of vitamin A for humans. Dogs and cats should get enough vitamin A from their normal diet. Vitamin A is essential for maintaining the integrity of the gastrointestinal tract’s epithelial cell lining as well as regulating immune activity. Reduced carotenoid levels have been linked to increased indicators of inflammation and oxidative stress [17,18]. Both these issues play a role in the pathophysiology of UC in humans. When compared to persons with normal mucosa, human patients with UC have significantly low serum (b-CAR) values. A study found that b-CAR had a protective effect in a DSS-induced UC animal model by reducing inflammation, oxidative stress, fibrosis, and DNA damage. It also reduced colonic mucosal damage and prevented occludin, a tight junction protein, from being reduced in the colons of mice with DSS colitis [126]. Vitamin A toxicity is uncommon in humans, however, it can occur as a result of increased vitamin A intake or after retinoid injection for therapeutic purposes. When the blood concentration of retinol in the plasma exceeds 2.09 µM, hypervitaminosis is diagnosed. The overuse of dietary supplements is frequently linked to toxicity. Chronic toxicity can develop with a long-term ingestion of 10 mg/day of vitamin A in adults and 7.5–15 mg/day in children for several months. Intakes of less than 30 mg/day (25,000–30,000 IU/day) are unlikely to cause toxicity [127] Vitamin A has functions in supporting vision, bone growth, reproduction, cellular differentiation, and immune response in dogs [128]. Dogs and especially cats have no capacity to synthesize vitamin A from b-CAR, as happens in humans. The required amounts of Vitamin A have been described in a study, given that Vitamin A is necessary for growth, maintenance, and lactation in dogs and cats [129]. Recommended Vitamin A maximum level for complete dog food is 40,000 IU (nutritional) per 100 g dry matter (DM) [107] No specific trials assessing the effect of Vitamin A in dogs and cats with CE were found in the literature.5.3.2. Vitamin EVitamin E is an essential vitamin, and a-tocopherol is the most active available form. Vitamin E is a lipophilic antioxidant that protects cellular membrane lipids from peroxidation, reduces the production of free radicals, and has anti-inflammatory properties. Enemas containing vitamin E reduced the disease symptoms of mild and moderately active human patients affected by UC in a clinical investigation [130]. Normally, cats and dogs experience oxidative damage that leads to chronic diseases. Vitamin E aids in preventing free radical damage. Findings of a study demonstrated that enhanced dietary levels of Vitamin E promote anti-oxidation and reduce oxidative damage in dogs and cats [131]. Dogs and cats should get enough vitamin E from their normal diet. Vitamin E can often be associated with Vitamin C in pets’ diet in order to increase antioxidant activity. No specific trials assessing the effect of Vitamin E in dogs and cats with CE were found in the literature [131].5.3.3. Vitamin CVitamin C is a group of related water-soluble substances (ascorbic acid, L-ascorbic acid, ascorbate, L-ascorbate). It is an antioxidant-rich natural substance that is utilized as a health supplement. The effects of intraperitoneal injection of high-dose vitamin C (4 g/kg) on DSS-induced UC were investigated in a study. High-dose vitamin C delivery reduced interleukin-6, hydrogen peroxide (H2O2), tumor necrosis factor-alfa, and iron levels in the blood. High-dose vitamin C delivery, on the other hand, raised the levels of H2O2 and iron in the colon, as well as the amount of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling-positive cells. In mice given high doses of vitamin C, the expression of collagen type I, fibroblasts, and collagen type III increased. These findings imply that taking a high-dose vitamin C supplement can improve UC inflammation [132]. Vitamin C deficiency has also been linked to an increased risk of osteoporosis in human patients suffering from IBD [133].Research suggests that both healthy dogs and cats are able to synthesize vitamin C independently from diet, so it is not recommended to add this vitamin in the diet [134]. Studies on dogs and cats have assessed vitamin C levels in patients with various medical disorders. The majority of these studies are focused on diseases associated with increased oxidative stress, including cancer, cardiovascular, renal, and infectious diseases. Evaluation of the level of Vitamin C in sick animals and studies on the effects of diets supplemented with vitamin C are limited in the veterinary literature [134]. As reported in a recent review, vitamin C was used in cases of severe burn injury in sheep, ischemic-reperfusion injury in a model of renal transplant in dogs, shock/traumatic injury in pigs [134]. The effect of vitamin C as an antioxidant has been confirmed in several studies suggesting a promising use in animals with severe oxidative damage. No specific trials assessing the effect of this vitamin in dogs and cats with CE have been found in the literature. animals-12-00812-t001_Table 1Table 1Main human trials testing the antioxidant effects of nutraceuticals in CID patients.NutraceuticalHuman Trial [Reference]Proved Effectiveness in Human CID PatientsPhytocomplex Curcuma longa Yes [62]More effective than placebo in keeping human patients with quiescent UC in remission Aloe vera Yes [70]Produces clinical response more frequently than placebo and reduction in histological disease activity Boswellia serrata Yes [73,74]Maintains remission over a prolonged period in UC patients Triticum aestivum Yes [78]Efficient treatment for active distal UC as a single or adjuvant treatment Plantago ovata Yes [82]Its seeds have been shown to be as effective as mesalazine in preventing UC relapse Serpylli herba noNA Vaccinium myrtillus Yes [86]Endoscopic and histologic disease activity, as well as fecal calprotectin levels, were considerably reduced in UC Camellia sinensis noNA Citrus noNATrace elementsZincnoNASeleniumnoNAVitaminsVitamin AnoNAVitamin EYes [130]Enemas containing vitamin E reduced the disease symptoms of mild and moderately active UCVitamin CnoNANA: no data available, UC: ulcerative colitis.6. Concluding RemarksAlthough an increasing number of new drugs are entering the market, especially to treat IBD in humans and serious CE in pets, the efficacy and the safety profiles of the current medications are far from optimal. Because CID is similar in pets (cats and dogs) and humans, the best of both experiences can benefit both worlds. Animals can represent a model for humans (they have a shorter lifespan and diseases have a faster course), while the human medicine field has a cutting-edge approach useful for the veterinary sector.This review highlights the strong evidence for the antioxidant effect of all the selected nutraceuticals in both human and veterinary medicine. Unfortunately, in vivo studies where nutraceuticals are specifically used for treating CID are lacking in humans and absent in animals. However, given their unquestionable antioxidant and anti-inflammatory properties, most of these substances can be considered as a promising alternative for regular treatments of CID. In this perspective, the use of dietary interventions and complementary feeds could be beneficial for our patients in terms of efficacy and safety. New randomized controlled trials are needed to confirm their usefulness.

animals : an open access journal from mdpi

10.3390/ani11123388

PMC8698186

Probiotics are microorganisms that can interact with the host and with the other microbiota present in the gastrointestinal tract of the host. Rabbits’ digestive characteristics are based on highly specialised colonies of intestinal microorganisms, which makes them vulnerable to metabolic disorders. Probiotics can balance gut microbiota and have several positive effects on the health status of the animal, leading also to the increase of the growth performance and meat quality.

The rabbit’s complex microbiota of the gastrointestinal tract (GIT) plays a critical role in feed digestion, in vitamin production, in fermentative activity with production of volatile fatty acids, and stimulation of immune response, as well as in the infection defence against pathogens and countering environmental stresses. To prevent digestive disorders of this fragile ecosystem, rabbit breeders adopt suitable diets supplemented with additives such as probiotics. Probiotics can interact with the host and with the other microflora leading to an increased health status. A review on the effects of probiotics on rabbit growth performance, health status, and meat quality was conducted to reduce the incidence of digestive diseases and enhance productive performance. Some authors observed that the supplementation of probiotics to the diet improved feed conversion ratio and growth and digestion coefficients, while other authors reported a lack of effect on the live performance. Benefits derived from the use of probiotics were observed on the mortality and the morbidity. The studies performed, to evaluate the effects of probiotic supplementation in diets on the immune response, showed variations in the results. Some authors reported no significant effect on haematological parameters, such as total protein, immunoglobulins, and IgG, while others observed a significant increase or decrease of the same parameters. Most of the research reported significant modifications of intestinal morphology and positive effects on the GIT microbiota, supporting the host’s natural defences. Regarding the carcass and meat quality, the studies reported only partial and opposing results.

1. IntroductionRabbit represents one of the most interesting production animals as, theoretically, it is an ideal meat-producing animal. Indeed, rabbit has a short life cycle, it is very prolific, has a short gestation period, and it has a high feed conversion capacity (2–2.3 on high grain diets, and 3–3.8 on high forage, grain-free diets) [1,2]. The rabbit is a monogastric hindgut fermenter, as via caecotrophy its digestive physiology allows it to obtain proteins and vitamins. Despite all these important features, rabbit consumption is decreasing worldwide, mostly in relation to the consumers’ acceptance and the requested cooking time [3]. Thus, rabbit farmers, also in Mediterranean countries in which rabbit meat was popular in the last decades (France, Italy, and Spain), are facing a market severity and a decrease of the meat request [4]. On the other hand, rabbit meat could be “re-discovered” by food producers as a healthy food as it is rich in protein while low in fat, cholesterol, and sodium [5]. It could be also proposed as an alternative to the conventional meat-based products (mostly containing beef and pork), especially for children and the elderly [3]. Furthermore, ready-to-cook products could be well accepted by consumers and meet new market trends [6,7].Despite this regression in European countries, rabbit farming is becoming an important emerging business in the developing countries, mostly in relation to the abovementioned productivity and to the already established highly specialised farming procedures, the technically advanced and unique livestock industry [8]. Farming stress is very critical in rabbit farming, mostly in hot environments. Rabbit farming faces a very critical step in the weaning period, as kits are separated from the mothers and solid feed replaces maternal milk [9]. During this period, as a consequence of environmental and physiological changes, rabbits are easily stressed and subject to non-specific enteritis and gastrointestinal infections, normally linked to dietary stresses, to parasites (Coccidia) and bacteria (Clostridia spp. and enteropathogenic Escherichia coli), leading to a multifactorial gastrointestinal syndrome (epizootic rabbit enteropathy, ERE) [10].2. ProbioticsThe term probiotic derives from two words: pro of the Latin language and βίος of the Greek language and literally means “for life” [11]. The history of probiotics began with the civilisation of humans: since the times of the Greeks and the Romans, the properties of fermented products were known, and their consumption was recommended both in children and in convalescents [12]; they are mentioned also in the Bible and in the sacred books of Hinduism. Particularly, they were identified in cultured dairy products. As reported in some studies, it seems that “fermented dairy products” were known also by Sumerians and that pictorial finds on the treatment of milk emerged during excavation in ancient Mesopotamia [11]. Moreover, archaeologists found evidence of fermented products derived from rice, fruits, honey, and cereals in Neolithic villages in China, Egypt, and Mesopotamia [11]. Moreover, Homer and Plinio tell of fermented milk and suggest its use in the treatment of gastrointestinal infections [11]. At the origin, these products were considered part of “folklore” until the healthy properties began to make their way and they were used as functional food linked to the presence of probiotic bacteria [11]. Considering the literature, probiotic is a relatively new word used to indicate microorganisms being able to provide healthy effects in humans and animals [12].At the beginning of the 20th century, the role of gut flora was unknown and in 1908 it was Metchnikoff who began to study the role of bacteria involved in fermentation of the intestinal flora and laid the foundation for the development of what we actually name “probiotic microorganism” [12,13]. At the time, many researchers were sceptical about the use and the healthy promoting effect of bacteria on the intestinal tract and opposed the bacterial therapy [12]. It was only in the first decades of 1900 that the therapeutic effects of Lactobacillus and Bifidobacterium were documented, and researchers began to believe that these microorganisms were essential to keep the digestive tract healthy [12,14,15].Only at the end of the century the role of gut flora and the protective function of bacteria against pathogens were cleared and some fermented foods were considered probiotic products due to the presence of one or more probiotic bacteria [12].The first study that proposed the term “Probiotika” was Kollath in 1953 to indicate substances opposite to antibiotics and “connected with vital processes” [13]. Rusch [16], in a short overview, reported the definitions of the term probiotic used by different authors and observed that the term is controversial. In most cases, the authors agree with the definition proposed in 1974 by Parker [17], who described the probiotic as “Organism and substance which contributes to intestinal microbial balance” [13,18]. Subsequently, in 1989, Fuller proposed another definition and considered the probiotic as “a live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance” [19]. The US FDA (Food and Drug Administration) uses the term “direct feed microbial” (DFM) instead of probiotics and suggests the following definition: “DFM is a source of live naturally occurring micro-organism” and included bacteria, fungi, and yeast [13,18].The EU legislation does not report a definition of probiotics but particularly the Regulation (EC) No 1831/2003 establishes the additives for use in animal nutrition and included the microorganism as “feed additives” and established the conditions for authorisation. Among the conditions, the Regulation reports the capacity of feed additives “to affect favourably animal production, performance or welfare, particularly by affecting the gastro-intestinal flora or digestibility of feeding stuffs”. The same Regulation establishes the conditions to obtain the authorisation for the use of feed additives. The request for authorisation must be sent to the European Commission that forwards the application to the European Food Safety Authority that shall give an opinion regarding the application. EFSA reports that the probiotics are substances that improve the equilibrium of the intestinal tract microflora [20].Different bacterial strains have different probiotic potential and differences are within the same species. The different strains have specific areas of adherence (site-specific), exact immunological effects, and different modes of actions if in the presence of a healthy or inflamed gastrointestinal tract. The goal of the researchers involved in the studies of probiotic microorganisms was to understand the interactions between the supplemented microorganisms and the microbiota of the host and define possible probiotic bacteriotherapy applications.However, not all the research studies agreed on the role of probiotics. Despite the numerous definitions, some authors consider the problem of probiotics a question that needs further clarification and in particular suggest the use of strict criteria before considering a substance as probiotic or not [12]. For example, Havenaar et al. [21] established a new concept of probiotics, basing their evaluation on strict criteria such as the resistance to gastric acidity and pancreatic secretions, the adhesion to epithelial cells, the presence of antimicrobial activity, the inhibition of adhesion of pathogenic bacteria, the evaluation of resistance to antibiotics, the tolerance to feed additives, and the stability in the feed matrix.To describe the mechanism of action of probiotics, non-specific terms are generally used. To indicate biological effects of probiotics, authors report terms such as colonisation resistance or competitive exclusion. However, probiotic microorganisms might be able to control commensal and/or pathogenic ones, neutralising the toxic effect of pathogens, increasing the host’s defences from the immune system, and showing antioxidant activity [12,21,22].The microbiota, represented by a great number of microbial agents which colonise specific ecological niches, contribute to the health status of the gastrointestinal tract due to its multiple functions.The animals’ complex microbiota of the gastrointestinal tract (GIT) plays a critical role in feed digestion, in vitamin production, in fermentative activity with production of volatile fatty acids, and in the stimulation of immune response, as well as in the defence against pathogen infection and a hostile environment. Moreover, modifications in villus height and crypt depth, which are considered the major markers of gut development, health, and functionality, can be influenced by gut bacteria.The control and the balance of rabbit microbiota contribute to produce positive effects on productive performance and health. In rabbits, the alteration of microbiota equilibrium can determine pathological effect, such as enteritis producing economic losses in rabbit farms. In general, the aetiopathogenesis of intestinal inflammatory processes is caused by multifactorial factors, such as environmental, nutritional, age, management, and pathogenic agents (Escherichia coli, Clostridium spiroforme, Lawsonia intracellularis, Clostridium piliforme, Salmonella spp., rotaviruses, coronaviruses, parvoviruses, and astroviruses) [23].To prevent digestive disorders of this fragile ecosystem, breeders adopt suitable diets supplemented with additives such as probiotics.In general, probiotic properties are evaluated in vitro by testing their antimicrobial potential, ability to adhere to the host’s intestinal mucin, and resistance to the gastrointestinal environment. Meanwhile, other probiotic properties, expressed in vivo, are more difficult to be evaluated, such as the ability to stimulate the development of the intestinal immune system and the ability to regulate intestinal innate immune and inflammation homeostasis.Moreover, different mechanisms of action have been ascribed to probiotics, even if some of them are hypothetical. The probiotics contribute to the maintenance of the gut habitat in eubiosis, probably preventing the entry to and the gut colonisation of pathogenic bacteria, increasing the activities of commensal bacteria, producing inactivation of toxins, and detoxification of host and nutrients in the gut.3. Effects of Probiotics on RabbitsDuring the last decades, several research studies were published about the effects of probiotics on rabbit farming. As the research articles deal with several different factors, such as rabbit breeds, sex, ages, type of basal diets, type of probiotics administration, duration of the trials, environmental conditions (rearing climate), etc., we report here a review of the main results focusing our attention on the productive performance of rabbits (i.e., studies about the use of rabbits intended as experimental animals were not taken into account) and how probiotics could contribute, due to their capacity to interact with feedstuffs, with other microorganisms, and with their host, to affect rabbits’ health status and production. Experimental designs of the reviewed research articles can be found in Table 1.3.1. Live PerformanceProbiotics could have a role in rabbit weight gain and in the capacity of the animals to assimilate the nutritional value of the feedstuffs and positively convert them into body mass. Feed conversion ratio (FCR) could be positively affected by probiotic metabolisms that might contribute to a better use of feeds, as also metabolised probiotic cells could be part of the assimilated nutrition.Abdel-Wareth et al. [24], who tested supplementation of mixes of fenugreek seeds and probiotics (AmPhi-Bact, American Pharmaceutical Innovations Company®, containing a mix of lactic acid bacteria culture, Lactobacillus acidophilus, Lactobacillus plantarum, Bifidobacterium bifidum, Bacillus subtilis fermentation extract, and Aspergillus niger fermentation extract) in 45-day-old New Zealand White rabbits for 6 weeks, reported a positive effect on FCR and a higher digestibility of crude protein. Probiotics and digestive enzymes (amylase, cellulase, beta-glucanase, and hemicellulose) present in the added product might also have worked synergistically, and as a result, the gut’s health and environment improved, supporting an improvement in nitrogen utilisation and positively affecting growth. Indeed, the authors also reported an increased weight gain in the final part of the trial in the rabbits fed with higher concentrations of probiotics [24]. Similarly, Lam Phuoc and Jamikorn [26] highlighted that the addition of B. subtilis and L. acidophilus complex (at, respectively, 0.5 × 106 CFU/g feed and 0.5 × 107 CFU/g feed) increased the FCR in 28-day-old rabbits fed for two weeks (42 days old). This positive effect was then lost, and the results were no longer different from the control diet (tested period 42 days to 70, and also total period 28 days to 70 days old). Then, effects of probiotics could be also related to a specific time of the rabbit’s life and their positive effects could be ascribed only in relation to the physiological status of the animal. Indeed, the FCR data were only partially in line with those of the digestibility trial (took place at day 63 for 5 days), which highlighted better digestibility coefficients of dry matter, organic matter, crude protein, neutral detergent fibre, and gross energy for the rabbits fed the mix of the probiotics and also the diet with only one probiotic strain (L. acidophilus at 1 × 107 CFU/g feed). Nonetheless, FCR data also highlighted a synergistic effect of the microorganisms, as the single use of B. subtilis (at 1 × 106 CFU/g feed) or L. acidophilus did not show the same results as their mix [26].On the contrary, it must be taken also into consideration the possibility of an antagonistic effect of the microorganisms employed. About this eventuality, El-Badawi et al. [40] reported a negative association between Bacillus subtilis and Saccharomyces cerevisiae. The authors tested the bacteria alone (0.1% of bacterial dry media of 3 × 107 CFU/g, Enviva PRO, Dupont, USA) and the yeast (0.1% of dry live yeast of 108 CFU/g, RUMI YEAST Sc47, Neovia, France) along with their mix (0.05%, respectively) in comparison to a control diet in 8-week-old New Zealand White rabbits for 10 weeks. The results of the FCR highlighted that the rabbits fed the mix of microorganisms did not differ from the control, whereas the employment of the two probiotics alone increased it. Similarly, digestion coefficients of most measured nutrients (dry matter, organic matter, crude protein, and nitrogen-free extract) were higher in rabbits fed the two diets with the single cell type than their mix and the control (mix similar to control) [40].Despite the beneficial effects reported by the abovementioned publications, other authors showed a lack of efficiency of probiotics in modifying the live performance of rabbits treated with these types of microorganisms. Some examples of the lack of effects of yeasts used as probiotics could be found in Emmanuel et al. [39], Tag El Din [29], and Rotolo et al. [43]. In the first study, the authors fed rabbits (16-week-old New Zealand White rabbit, 12-week trial) with Saccharomyces cerevisiae (Agro-Chemical Company, Nsukka, Nigeria) at the concentration of 0.12 g per kg, highlighting a lack of variations in FCR, final body weight, and average daily weight gain [39]. Similarly, Tag El Din reported that 6-week-old Californian x New Zealand White rabbits fed 0.5, 1.0, and 2.0% dry live yeast inclusion (10 CFU/g, RUMI YEAST Sc 47, Neovia, France) for 5 weeks did not show any statistical differences to the control in the FCR and digestibility coefficient [29]. Noteworthily, inclusion of 1.5% of dry yeast induced a positive variation in FCR. The authors hypothesised that these results may be due to the reduction of toxins or antimicrobial substances produced by other microorganisms, the competition for adhesion to epithelial cells, the increased resistance to colonisation, the stimulation of the immune system of the host, and the reduction of stress in rabbits. Likewise, Rotolo et al. [43] reported no variations in performance parameters by live yeast addition (37-day-old New Zealand White rabbits fed for 47 days with 300 and 600 mg/kg of Saccharomyces cerevisiae boulardii, LSB, LEVUCELL® SB10 ME TITAN, Lallemand Sas, Blagnac, France). A lack of effects on live performance (final weight and FCR) was also reported for the employment of some bacteria, as reported by Fathi et al. [25], which tested two supplementation levels (200 or 400 g of probiotic/t feed) of Bacillus subtilis (4 × 109 CFU/g) in 8-week-old Jabali breed rabbits (Egyptian local breed) reared at 35 °C for 8 weeks.3.2. Health Status and Gastrointestinal Tract MicrobiotaSeveral authors reported that during the experiments all animals remained in good health condition, with no symptoms of disorders; therefore, no mortality and morbidity were noted [24,28,30,31,32,43]. As reported in the paragraph above, Cunha et al. [34] interrupted the administration of E. coli strains isolated from rabbit faeces after morbidity signals such as diarrhoeic faeces with a decrease in feed intake associated with an increase in water consumption. Dimova et al. [42] recorded a lower mortality rate (16.67%) in fattening rabbits (White New Zealand, 14 days old to 101 days old) fed 0.5% of probiotics (Zoovit, no details given) than animals fed the control diet (27.78%). A similar reduction of mortality (−10.8%) was also highlighted in weaned rabbits derived from does fed the probiotic. Similarly, Kimsé et al. [44] reported that growing rabbits (INRA hybrid line, UMR 1289 INRA TANDEM, from day 35 to day 70) fed a supplementation of S. cerevisiae strain NCYC Sc 47 (Actisaf: S. cerevisiae NCYC Sc 47 coated with saccharides, Lesaffre Feed Additives, Marquette-Lez-Lille, France; at 10 g/kg of basal diet) significantly decreased mortality (22.5%) compared to the control (45.0%). These improvements of digestive health might be associated with the possibility of the probiotic strains to colonise the caecum and then modify the caecal physico-chemical characteristics, such as an increase in redox potential [44].To evaluate the effect of probiotic supplementation in diets on the immune response, some authors took into account specific and non-specific immune response. For this reason, different haematological parameters were analysed, mainly total protein, immunoglobulins, white blood count (WBC), and lymphocytes. The studies reported variations in the results, and the reasons for that could be related to the use of different types and doses of probiotics, as well as differences in feed composition and mechanism of action which characterised different probiotics.El-Shafei et al. [37], who tested supplementation of probiotic Lactobacillus plantarum in growing New Zealand White rabbits, reported no significant effect of probiotics on total protein, immunoglobulins, and on IgG. The results reported by Mohamed et al. [27] partially agreed with the abovementioned, as no significant effect was reported on globulin, while a significant increase in WBC and total protein in growing rabbits fed with diets supplemented with Lactobacillus acidophilus were observed. An increase of total protein along with an increase in globulins was observed by other authors [25,41]. Fathi et al. [25], in local breed growing rabbits fed with two different doses of probiotic containing Bacillus subtilis, reported a slight increase of total protein level and a similar trend in globulins with the highest dose of probiotic. Similarly, Abdel-Azeem et al. [41], who employed a commercial probiotic product, obtained higher levels of these parameters in treated groups than the control.An opposite trend was reported by other authors [33,36]. Beshara et al. [36] reported a decrease of total protein, globulin, and WBC in treated groups with probiotics; likewise, Wlazło et al. [33], who studied the effect of fermented rapeseed meal in rabbit diets on immune status, determining the class A, G, and M immunoglobulins in blood plasma, observed a lower level of class G immunoglobulins in the treated group than the control and a decrease of the IgG values as the level of fermented rapeseed meal increased. The same author also observed a correlation between the IgG values and the numbers of microorganisms in the GIT.As reported above, the different results obtained probably may be related to different mechanisms of action of probiotics. In fact, the probiotic might be able to (i) modulate the host’s defences, increasing the immunity response or (ii) can exercise a direct effect on microorganisms, e.g., pathogens, reducing the host requirement of immune defences.Some authors analysed the effects of probiotics on the intestinal morphology. Shen et al. [35] reported that oral administration with Lactobacillus casei significantly increased the length of the vermiform appendix in suckling rabbits, but did not change the intestinal morphological indices, including villus height, crypt depth, and the ratio of villus height to crypt depth. As development of the vermiform appendix is associated with the immune capacity of the intestine of a rabbit, the results of this study indicated that the development of the special intestinal immune organ in rabbits needs bacterial stimulation. Moreover, Shen et al. [35] reported an increase in the percentage of degranulated Paneth cells in the duodenum and jejunum of suckling rabbits orally administered with Lactobacillus casei RABX1. Degranulation is the way that a Paneth cell secretes its synthesised antimicrobial substances to the intestinal lumen, such as defensin and lysozyme. The increased percentage of degranulated Paneth cells and the expression of toll-like receptors (the first barrier in host defence against pathogen infection that induces production of type I interferons and inflammatory cytokines) in the duodenum and jejunum indicated that the probiotic was involved in the regulation of Paneth cell function in the rabbits.Liu et al. [28] tested three doses of a probiotic strain of Clostridium butyricum (CCTCC AB: 2017089; low dose, 1.0 × 103 CFU/g; medium dose, 1.0 × 104 CFU/g; high dose, 1.0 × 105 CFU/g) in 5-month-old primiparous female rabbits (Sichuan white rex rabbit) until new-born weaning (35 days), and then also fed the weaning rabbits themselves with the same diet of the mothers (4-week feeding trial). The authors reported that compared to the control, rabbits supplemented with a high dose of probiotic elongated the length of the villi of small intestinal tissues, while the medium dose group showed longer villi in the duodenum and ileum. On the other hand, probiotic treatments decreased the crypt depth of weaning rex rabbits. Therefore, the ratios of villus length to crypt depth (VL/CD) were greater in the high dose group than in the control and low dose group. Probiotics can increase villus length and decrease crypt depth in the small intestine, which is beneficial for the digestion and absorption of nutrients, thus directly affecting mucosa morphology, digestive enzyme activity, and consequently growth performance.El-Shafei et al. [37] tested, in four-week-old male New Zealand White rabbits, two concentrations of a Lactobacillus plantarum strain (0.25 g and 0.5 g per kg of 1 × 106 CFU/g) for 8 weeks. Results showed that the goblet cells appeared in the duodenum and caecum epithelia of the treated groups, suggesting an improvement in production of mucus compared with the control group. Meanwhile, the group fed 0.5 g probiotic/kg diet showed improvement in goblet cells and crypts in the base of the tissue or surface compared to the control group. These results confirmed the increased health status of treated rabbits as the enhanced mucus layer covering the epithelial lining of the gut can serve as an antibacterial shield that prevents the binding of enteric pathogens, and goblet cells have a role in defence at the intestinal mucosa.Not all the studies about the dietary administration of probiotics in rabbits reported modification in the intestinal morphology. For instance, Pogány Simonová et al. [32] and Oso et al. [45] did not report modification in jejunal morphometry or morphological parameters in the rabbit ileum after probiotic inclusion (E. faecium and a mix of Pediococcus acidilactici and Bacillus cereus, respectively).All the research studies that evaluate GIT microbiota found modification in microorganisms’ populations of GIT in relation to probiotic addition. Wlazło et al. [33] tested the effects of the administration of a fermented rapeseed meal with Bacillus subtilis as the probiotic (strain 87Y from the collection of InventionBio Ltd., Bydgoszcz, Poland) in 35-day-old New Zealand White × Popielno White rabbits for 85 days. The authors enumerate few microbial species in the duodenum, small intestine, caecum, and colon sections. Duodenum, small intestine, and colon lactic acid bacteria were increased due to probiotic addition, as well as small intestine mesophilic aerobic bacteria. No variation was detected in number of total fungi in all the sections. Noteworthily, a number of coliforms and Escherichia coli decreased in the small intestine and colon sections in relation to the probiotic diets [33]. A Bacillus subtilis strain, alone and in association with Lactobacillus acidophilus strain (also tested alone), were also tested by Lam Phuoc and Jamikorn [26]. The addition of the Bacillus subtilis probiotic strain (alone and in association with Lactobacillus acidophilus probiotic strain) increased the numbers of bacilli in the ileum and colon, and generally, an increment of the numbers of bacilli were observed in all segments in the rabbits supplemented with either one of the probiotics. Similarly, the average number of lactobacilli in all intestinal segments of the rabbits were increased after the probiotic diets. The authors hypothesise a synergistic effect between B. subtilis and L. acidophilus. On the other hand, no difference was observed in the ileum coliform number, even if L. acidophilus showed an effect on coliform numbers in the cecum and colon, and an average number in all segments. These variations led, in rabbits fed L. acidophilus probiotics, to an increase in Gram-positive bacteria (lactobacilli) and a reduction in Gram-negative bacteria (coliforms).An increment in lactobacilli, due to probiotic diets, was also reported by Shen et al. [35] and Abdel-Azeem et al. [41]. New Zealand White rabbits were fed Lactobacillus casei RABX1 (accession number: KT944253) resuspended in the milk (5–6 × 108 CFU/mL) and orally administered from 5 to 13 days of age. The probiotic milk feeding was suspended two days before slaughtering (slaughtered at 15 days old) [35]. Shen et al. reported that Lactobacillus casei significantly increased the length of the vermiform appendix in suckling rabbits without modifying the intestinal morphological indices, villus height, crypt depth, and the ratio of villus height to crypt depth. The content of the small intestine showed to be affected by the diet; suckling rabbits fed the “probiotic milk” presented a higher relative proportion of Lactobacilli in total intestinal bacteria and a lower relative proportion of Escherichia–Shigella than rabbits fed the control milk. Abdel-Azeem et al. [41] employed a commercial probiotic product (ZAD®, Academy of Scientific Research and Technology, Egypt) in an oral gavage administration in 6-week-old New Zealand White rabbits for 56 days. The probiotic product (mix of enzymes and bacteria) increased the concentration of Lactobacillus spp. in the caecum and decreased the total coliform and total anaerobic bacteria. On the other hand, Beshara et al. [36] reported a lack a variation in lactic acid bacteria in rabbits fed probiotics, whereas an increase of the total bacterial count was detected.Some research studies were also carried out on probiotic strains isolated from rabbits itself. Cunha et al. [34] tested, in 38-day-old New Zealand White rabbits, the administration of enterococci and three E. coli strains isolated from rabbit faeces. Interestingly, the administrations of E. coli were interrupted as the animals showed, between the second and fifth day of the trial, diarrhoeic faeces and gastrointestinal signs with a decrease in feed intake and an increase in water consumption. On the other hand, Enterococcus spp. showed a positive interaction with the hosts even if the authors highlighted those probiotic bacterial strains did not remain in the gastrointestinal tract longer than one week after the administration ended. Cunha et al. hypothesised that this might be caused by an impaired probiotic persistence in the rabbits’ intestinal microbiota due to a lack of (re)inoculation, a dietary change, a different water source, and/or a new household.The use of Enterococcus spp. was also taken into account by Pogány Simonová et al. [32] using a bacteriocin-producing strain (Enterococcus faecium EF9a) with probiotic properties isolated from the faeces of the Hungarian Pannon White rabbit breed [46]. The probiotic strain Enterococcus faecium EF9a was added to the drinking water and induced a significant decrease in the coliforms, coagulase-positive staphylococci, pseudomonads, and coagulase-negative staphylococci in the rabbit faeces, as well as showing antimicrobial effects in the caecum against coliforms, coagulase-negative staphylococci, and pseudomonads and in the appendix versus the coliforms [32]. Furthermore, Lauková et al. [30] tested an Enterococcus faecium strain. The employed strain, an environmental isolate, was also an enterocin M producer (strain AL41, registered in Czech Culture Collection, Brno, Czech Republic—CCM8558), which was tested alone and in association with Eleutherococcus senticosus (Siberian ginseng), a herb with adaptogenic, anti-stress, and immunomodulatory properties. The authors reported that E. faecium AL41 colonised the rabbits’ GIT better when employed alone than in association with the herb, and it remained present in the animals after 3 weeks of its cessation. Significant reductions of coagulase-negative staphylococci, coagulase-positive staphylococci, Clostridia, coliforms, and/or pseudomonads were also highlighted in relation to E. faecium AL41 administration. Changes in microbiota were associated both to the antibacterial effect of the produced enterocin M as well as to the production of lactic acid from the probiotic strain.3.3. Carcass and Meat QualityAs reported for the live performance, carcass traits and meat quality were also partially affected by probiotic supplementation.Some authors reported that probiotics ameliorate the carcass traits, even increasing the edible parts. Fathi et al. [25] showed that rabbits fed Bacillus subtilis supplementation (200 and 400 g/t) increased the carcass weight (only 400 g/t), dressing percentage, and cuts of mid part and hind part as a percentage of live body weight. Interestingly, the authors reported that rabbits fed 400 g/t of Bacillus subtilis at 4 × 109 CFU/g for 8 weeks increased the carcass weight, on average by 130 g, corresponding to an increase of 12% of the weight of the carcasses derived from the rabbits fed the control diet. These data are even more outstanding if we take into consideration that the final live weight was not statistically different between the two diet groups and that rabbits were reared under a hot climate (35 °C). Likewise, Mohamed et al. [27] reported that most of the carcass traits studied were affected in rabbits fed probiotics compared with the control group (two breeds, New Zealand White and local Egyptian breed called Baladi Black; five different probiotic diets, dose per day: 1 mL fresh culture suspension of Bifidobacterium bifidum 1 × 107 CFU, 1 mL fresh culture suspension of Lactobacillus acidophilus 7 × 106 CFU, 1 mL fresh culture suspension of bacterial mixture of Bifidobacterium bifidum and Lactobacillus acidophilus at 3.5 × 107 CFU, and 1 mL of Saccharomyces cerevisiae). The authors also highlighted that the local breed, Baladi Black, fed Bifidobacterium bifidum supplementation recorded the highest values of carcass weight, carcass percentage, and heart, liver, kidneys, lungs, and giblets (absolute weights) in comparison with the other groups.Carcass traits were not affected in other research trials, such as the data reported by Beshara et al. [36], who tested 0.4 g/kg of a thermo stable probiotic (Lactobacillus lactis 2.5 × 108 CFU/kg, Bacillus subtilis 1.8 × 109 CFU/kg—calcium carbonate up to 1 g as carrier, Saltose Ex, Pic-Bio Inc. Company, Shinagawa, Japan) in the same local Egyptian breed (Baladi Black) employed by the former authors. Moreover, the employment of bacteria or yeast alone or in combination did not affect the carcass traits also, as reported by El-Badawi et al. [40] in New Zealand White rabbits. No differences in carcass characteristics among treatments were also reported in Rotolo et al. [43].Interestingly, Abdel-Wareth et al. [24] reported even a negative effect on the carcass yield percentage in relation to the probiotic administration. Noteworthily, it is important to highlight that Abdel-Wareth et al. tested three different diets containing increasing concentrations of probiotics in relation to increasing dietary dry fenugreek seeds. It is reported that some cultivars of fenugreek (Trigonella foenum-graecum) could also bring some antinutritional factors, such as phytic acid, also present in its seeds [47]. Abdel-Wareth et al. also reported that rabbits fed the highest concentration of dry fenugreek seeds/probiotics (diet with 15 g/kg dry fenugreek seeds and 450 mg/kg probiotic—AmPhi-Bact®) showed lower caeca weight (percentage of slaughter weight) than the control diet. On the contrary, Rotolo et al. [43] reported that caecum weight was not affected by treatment in weaning rabbits upon dietary inclusion of a probiotic (live Saccharomyces cerevisiae boulardii).Meat quality of rabbits fed probiotics showed as well mixed results. No variations in the pH48, colour, proximate composition, and water holding capacity was reported by Pogány Simonová et al. [31], who tested in 5-week-old Hyplus breed rabbits for 28 days a strain of Enterococcus faecium (EF9a isolated from Pannon White rabbit, 1 × 109 CFU/mL, in a dose 500 µL/animal/day) in the drinking water. Moreover, El-Badawi et al. [40] and Islamov et al. [38] did not find, respectively, significant differences in rabbit meat quality (proximate composition) with the use of alone or combined bacteria yeast supplements (Bacillus subtilis and Saccharomyces cerevisiae) and a probiotic preparation called “Rescue Kit” (1 kg of preparation contains 800 × 109 CFU Bacillus subtilis and 800 × 109 Bacillus licheniformis; inclusion level of 10 g of preparation per 1 kg of feed; White Giant breed; from 70 days old to 120). No significant effects of live Saccharomyces cerevisiae boulardii supplementation were observed also by Rotolo et al. [43] in pH24, colour, cooking loss, and proximate composition of longissimus dorsi muscle.Fathi et al. [25] reported instead modifications of the proximate composition of rabbits fed Bacillus subtilis as a probiotic. The authors reported higher percentages of dry matter, organic matter, protein, and fat in rabbits fed a diet containing 400 g/t probiotics than the control one and probiotics at 200 g/t. As well, Abdel-Wareth et al. [24] reported a decrease in water holding capacity (estimated by centrifuging the muscle) and cooking loss in a group of rabbits fed combinations of fenugreek seeds and probiotics (diet with 5 g/kg dry fenugreek seeds and 150 mg probiotic—AmPhi-Bact®). As reported by Pogány Simonová et al. [31], no variation was detected in meat pH values (in this case measured 24 h post-mortem).4. ConclusionsSome evidence suggests that probiotics can play several roles in rabbit farming, from biological control against pathogenic microorganisms, to growth enhancer or active compounds to increase the quantity and quality of the final product. The positive effects determined in rabbits fed the experimental diets are in general ascribed to the capacity of probiotics to interact with the host and with all the microbiota present in the different parts or organs of the GIT, modifying the entire production process. From the analysis of the different research studies emerges the findings that probiotics have different modes of action and that many factors can modify the responses, confirming that probiotic properties have bacterium–host specificity. Research of this type is especially important in terms of reducing the use of antibiotics for therapeutic purposes through nutritional prevention in animals.Attention must be taken to the correct time of administration and also in relation to the probiotic activities and amounts. Optimisation of the microbiota composition leads to an increase in digestive efficiency, directly improving nutrient digestibility and stimulating immune processes, increasing the profitability of production.Future research must be focused also on the technologies of the administration (microencapsulation, cell immobilisation, and continuous fermentation) to ensure that these beneficial microorganisms will reach, in high numbers, the target site of action.

animals : an open access journal from mdpi

10.3390/ani12010073

PMC8749834

Regular exercise is a stressful stimulus that elicits physiological responses in systolic and diastolic functions in human athletes, the so-called “athlete’s heart”. The present study reports findings obtained from echocardiography to measure the ventricular dimensions at rest in beagle dogs undergoing an endurance training program carried out on a treadmill with the intensity set at 70–80% of the velocity corresponding to the lactate threshold. Echocardiography was performed with routine measurements of the left ventricular systolic and diastolic function by the two-dimensional and Doppler techniques. After the training, the principal component analysis of echocardiographic variables was conducted to evaluate dimensional changes in left ventricular function. Principal components analysis was able to capture the qualitative echocardiographic changes produced by the endurance training. Eight weeks of the lactate-guided endurance training program could lead to concomitant left ventricular dilation without hypertrophy of the ventricular walls, emphasizing the left ventricular systolic and diastolic functions. These results suggest that submaximal aerobic training may induce physiological cardiac remodeling, improve the left ventricular functions, promote health, and minimize any injuries produced during heart disease, although its effectiveness for the latter effect must be confirmed in future studies.

This research focuses on the adjustments in systolic and diastolic functions that are not fully understood in dogs submitted to athletic training. Beagle dogs carried out an endurance training program (ETP) prescribed from the external training load, corresponding to 70–80% of the lactate threshold (VLT) velocity. Eighteen dogs were randomly assigned to two groups: control (C, n = 8), active dogs that did not perform any forced exercise, and trained (T, n = 10), submitted to the ETP during eight weeks. All dogs were evaluated before and after the ETP period using two-dimensional echocardiography, M-mode, Doppler, and two-dimensional speckle tracking. A principal component analysis (PCA) of the echocardiographic variables was performed. The ETP improved the left ventricular internal dimension at the end of diastole (LVDd), the left ventricular internal dimension at the end of diastole to aorta ratio (LVDd: Ao), and the strain rate indices. PCA was able to capture the dimensionality and qualitative echocardiography changes produced by the ETP. These findings indicated that the training prescribed based on the lactate threshold improved the diastolic and systolic functions. This response may be applied to improve myocardial function, promote health, and mitigate any injuries produced during heart failure.

1. IntroductionProlonged physical conditioning is often associated with inducing morphological and functional cardiac changes in human athletes, the so-called “athlete’s heart”. Cardiac remodeling in human athletes is well recognized. However, in veterinary medicine the approach has received less consideration [1]. Assessed by echocardiography, ventriculography, or nuclear magnetic resonance, this morphofunctional alteration may indicate an increase in the parasympathetic tone associated with an increase in the left and right ventricles. These physiological changes are also described in dogs used in various activities such as rescue, agility, or mushing [2,3,4]. Like the previous research in human athletes, evidence of large hearts in athletic horses antedate many years. Horses have been an important animal model for exercise physiology studies. Analyzing the association between heart size and aerobic performance in horses, the literature has shown intense positive relationships between several measures of cardiac function and V˙O2max, a key decisive of aerobic capacity [1,5].The cardiac remodeling response described in athletes such as cyclists [6], marathoners, and weightlifters [2,7] has been recently reported in dogs that practice bikejöring [2]. However, it must be distinguished from deleterious changes. This remodeling is characterized by an increase in the left ventricle internal dimension (LVID) in diastole, left ventricle (LV) hypertrophy, and left atrium increase (LA). LV adjustments are induced by volume overload associated with high cardiac output during acute and intense exercise. The increase in LA volume and dilation is caused by the compensatory response of the LA for maintaining the sound volume in the LV [8].In recent years, considerable progress has been made in echocardiography techniques for detecting cardiac changes induced by regular exercise [2,9,10]. Noninvasive auxiliary diagnostic methods can determine these changes. In the literature, several studies revealed that the systolic function of rowers, marathoners, fighters, as well as active and healthy individuals improved with conditioning after being evaluated using myocardial deformation techniques such as speckle tracking [10,11,12]. Recently, echocardiography has been performed to assess the effects of overtraining syndrome in high-level male human athletes [13]. As a complex system, the organism acts as an inseparable and integrated whole that cannot be constrained only to the univariate quantitative statistical analysis of its physiological functions separately [14,15]. The cardiovascular system and the cooperative intrinsic function of its multiple subsystems are interdependent and interact in a dynamic and nonlinear way. Thus, the subsystems need to be approached as a nonlinear statistical design. These multivariate methodologies, focusing on the integrated aspect of cardiovascular variables in dogs, should consider the aerobic training effect on cardiovascular function. To this end, researchers have evaluated cardiorespiratory coordination in humans undergoing different training modalities by analyzing all the major components using a principal component analysis (PCA) approach. PCA diminishes the data dimensionality of associated systems, withdrawing the narrowest range of components that account for most of the variation in the seminal multivariate data and summarizing it with a minor deficit of information. This analysis may be used to monitor the integration of multiple variables in a physiological system and a wide range of biological research fields [15].Prolonged physical exercise, if well prescribed, establishes forms of adjustment that enhance performance and may be called a “eustress”. “Eustress” suggests that an “acceptable or desirable stress degree” may have a positive influence on welfare and fitness [16]. The training load in the background of athletic training has been characterized as the input guideline manipulated to elicit the desirable physical conditioning response. Studies addressing the sport training protocols of human athletes often apply the external or internal load concept when elaborating and prescribing the training programs by considering the exercise’s volume, duration, and intensity. External load is defined as the variables that induce internal physiological responses such as duration, the distance covered, the average and maximum speeds, and the treadmill slope. Internal load indicates an effective physiological response promoted by the athlete to adjust to the stimuli induced by the external load. Both heart rate and plasma lactate concentration can be used as indicators to measure the internal load during running [17].In studies with human athletes, exercise physiologists employ fitness protocols that were prescribed based on maximum oxygen consumption (V˙O2max) and lactate threshold (LT) for specifying the training program load aimed at improving systolic and diastolic function in healthy or sick individuals [18,19,20,21]. In veterinary medicine, relatively few studies [22,23,24] have used the individualized prescription of internal or external load for elaborating training protocols while evaluating their effects on the echocardiographic variables. Moreover, prolonged submaximal exercise to prevent or treat cardiovascular disease is still relatively limited, notably in dogs. The benefits of prolonged training as a therapeutic measure to treat ventricular dysfunction diseases are practically not described in the specific literature on dogs [25]. Therefore, studies are needed to verify the safety and effects of the conditioning protocols performed with a refined control of the training load on healthy dogs. The scientific findings of these studies can be extrapolated to dogs with cardiovascular diseases.Our research group recently determined the LT in dogs subjected to the incremental exercise testing (IET) [23,24,26] to obtain the velocity corresponding to the LT (VLT). This variable was the reference used for establishing the external training load since it is considered more reliable compared to both the percentage of maximum oxygen consumption (%O2MAX) and the maximum heart rate (%HRMAX) [16]. The training protocol improved aerobic capacity and increased heart rate variability after eight weeks of training [23,24].To the best of the authors’ knowledge, no studies determining the cardiac function response of dogs submitted to the aerobic training prescribed from the VLT are available in the literature. Herein, we expand these findings by evaluating whether the endurance-type training, prescribed based on VLT, can improve healthy dogs’ diastolic and systolic functions. We also conducted a principal component analysis (PCA), a robust analysis tool for exploratory assessment and which consists of gaining observation in the degree of co-relatedness, or its absence, among the echocardiographic variables under examination.2. Materials and Methods2.1. DogsThis was a prospective case–control study. The experiment was conducted in the Laboratory of Pharmacology and Physiology of Equine Exercise (LAFEQ) and the Cardiology Laboratory of “Governor Laudo Natel” Veterinary Hospital of São Paulo State University (UNESP), School of Agricultural and Veterinarian Sciences, Jaboticabal (FCAV), Brazil. The study followed the Ethical Principles in Animal Experimentation adopted by the Brazilian College of Animal Experimentation and approved by the Ethics Committee on Animal Use (CEUA), protocol 008272/17.Eighteen healthy beagles (10 males and 8 females) aged 12 to 24 months were used in the study. The dogs belong to the kennel of the Laboratory of Nutrition and Nutritional Diseases of the School of Agricultural and Veterinarian Sciences (FCAV), Sao Paulo State University (UNESP), Jaboticabal, Brazil. All dogs lived in 1.5 × 4.0 m kennels with a solarium and 4 h daily access to the 1000 m2 outdoor area for spontaneous and accessible physical activity, recreation, and socialization. It is highlighted that the dogs were not submitted to any forced training on the treadmill before the experiment. The dogs were considered healthy based on their clinical history, physical examination, blood test, as well as a two-dimensional echocardiogram, M-mode, and Doppler. Exclusion criteria included any changes in blood tests and possible clinical abnormalities that could affect exercise performance, cardiovascular, neuromuscular, and orthopedic conditions.The dogs were fed enough commercial food to satisfy their individual energy needs equivalent to the active dogs, according to the NRC [27]. Water was available ad libitum. During the trial, the dogs were fasted for a 12 h interval before training sessions or exercise mats but were continuously fed 20 min after the exercise sessions. On training days, food was given 20 min after the exercise period. During the experimental period, the dogs were weighed every fifteen days so that, when deemed necessary, the food amount was adjusted to maintain the body weight.2.2. Experimental GroupsEnrolled dogs were randomly assigned to two groups: control (C, n = 8; 4 males, 4 females) consisting of active dogs that were not submitted to any training exercise, and the trained group consisting of dogs (T, n = 10; 6 males, 4 females) that were submitted to submaximal training on the treadmill three times a week for eight weeks. The control group was used to verify possible interference from environmental stimuli, such as behavioral and emotional factors, which may interfere with the echocardiography exam used to study systolic and diastolic ventricular function. The following is a detailed description of the general management of both groups. 2.3. Adaptation, Incremental Exercise Test (IET), and Endurance Training Program (ETP)An overview of the study design is shown in Figure 1. The dogs were adapted to the laboratory, where exercise testing was performed on the treadmill and the ETP. The adaptation protocol followed has been previously published by our research group [23,26]. A motorized treadmill (Galloper® 5500, Sahinco) was used to perform the two incremental exercise tests (IETs) before and after (IET-1 and IET-2) the ETP, like the weekly submaximal exercise sessions during the conditioning period.The IET (Figure 1A) was performed to determine the lactate–velocity curve (LVC) and the visual LT, establishing the lactate threshold velocity (VLT) and prescribing the individual submaximal conditioning program for group T. This protocol was adjusted from Restan et al. [25]. Once adapted to the treadmill, the dogs started the exercise test at 0% slope and 1.5 m/s initial speed during warm-up; subsequently, treadmill slope was increased to 7.5%, with 0.5 m/s speed increments, every 5 min. Between each speed increase (effort step), the mat was stopped for 2 min to collect blood samples. The velocity was increased incrementally until the dogs showed signs of fatigue, such as the inability to follow the speed of the treadmill mat. IETs were always performed in the morning when temperatures varied between 19 and 21 °C. All dogs underwent 3 h fasting, but the water was offered ad libitum. All dogs in group T were submitted to this procedure. After the conditioning period, the IET was repeated to compare the VLTs obtained in the initial and final tests and to quantify the anaerobic power and the maximum velocity (Vmax) reached in both IETs.The ETP was performed between the IET-1 and IET-2 (Figure 1B) and lasted eight weeks. Only dogs in group T carried out the ETP, with conditioning intensity set at 70% and 80% of VLT and 7.5% slope. Thus, in the first four weeks, velocity was set at 70% of VLT and, from the 5th week on, increased to 80% of VLT. The dogs trained three times a week (alternate days) in 30 min sessions consisting of 5 min warm-up at 50% of the established VLT, 20 min exercise session at the stipulated speed (70% or 80% of the VLT), and 5 min cooling down after daily exercise at 50% of the determined speed). The dogs belonging to group C were familiarized with the researchers and the daily handling by positive reinforcements such as offering cookies and toys in the stall without forcing exercise. These activities that took place three times a week aimed at eliminating possible behavioral and emotional interferences during containment while keeping the dogs in lateral decubitus in the echocardiographic exams. Both groups had access to the identical recreation and socialization periods mentioned in item 2.1. Such kennel management is often used to maintain and promote the well-being of dogs.2.4. Lactate Threshold (LT)Between the IET’s speed increments, blood samples (2 mL) were collected from the dogs at rest in the quadrupedal position through venous catheterization of the left jugular vein, using a 16G Insyte™ catheter (Becton Dickinson and Company Belliver Industrial Estate, Plymouth, UK) that was maintained in place with a drop of Super Bonder®. The area was previously shaved and scrubbed with an applicator that contained 2% chlorhexidine gluconate, followed by application of topical and superficial skin anesthesia, 2.5% lidocaine and 2.5% prilocaine (EMLA®). Between each incremental step, the dogs rested for 2 min, and the blood sample was collected at 90 s. This procedure is based on a study previously carried out on dogs by our laboratory [26].Venous blood samples were stored in BD Vacutainer® Fluoride/EDTA tubes containing EDTA (12 mg) and sodium fluoride (6 mg) until analysis (Becton Dickinson and Company Belliver Industrial Estate, United Kingdom). The lactate concentration analysis followed the electro-enzymatic methodology (YSI 2300, Yellow Springs Instrument, Yellow Springs, OH, USA), previously validated for use in dogs [28]. LT was identified by visual inspection of LVC. Three evaluators experienced in exercise physiology examined the curves and determined the velocity at which the lactate concentration showed an abrupt and exponential increase. The inflection point represents the beginning of an imbalance between lactate production and removal/metabolism [24,25]. The VLT obtained in IET-1 was used to prescribe the individual training routine.2.5. EchocardiographyThe possible morphological changes and systolic and diastolic functions were evaluated by echocardiographic examination using an ultrasound system (Acuson X300 Ultrasound System, Premium Edition, Siemens, Munich, Germany) in the 5.0–7.5 MHz range to allow for both resolution and sufficient penetration. After shaving the right and left thoracic regions, the dogs were restrained in the lateral decubitus position to obtain the standard images following recommendations previously established by Boon [29] and the American Society of Echocardiography [30] for conventional and tissue echocardiography.2.6. Morphological and Volumetric EvaluationThe evaluations included two-dimensional, M-mode, pulsed, continuous Doppler color, and tissue flow. The M-mode image was obtained in the chordal plane in the cross-section of the right parasternal window by positioning the cursor perpendicularly to the interventricular septum and equidistant from the papillary muscles. From this image, echocardiographic variables such as left ventricular internal diameter at the end of diastole (LVDd), left ventricular internal dimension at the end of systole (LVDs), left ventricular free wall thickness (LVW), and interventricular septum (IVS) were obtained during diastole and systole. Lastly, the fractional shortening (FS%) was determined by the Teichholz method.The diameters of the left atrium (LA) and aorta (Ao) were measured from the 2D image at the level of the aortic valve, in the transversal image of the right parasternal window, to determine the left atrium to aorta ratio (LA/Ao). The left ventricular internal diameter at the end of diastole to aorta ratio (LVDd/Ao) and the left ventricular internal diameter at the end of systole to aorta ratio (LVDs/Ao) were determined. The 2D measurements also included left ventricular end-systolic (LVVs) and end-diastolic (LVVd) volumes, as well as ejection fraction (EF) and systolic volume (SV), which were calculated by the Simpson uniplanar method.2.7. Systolic and Diastolic EvaluationThe flow rate in the pulmonary artery was determined using a pulsed Doppler. The Doppler velocities of the mitral and aortic flow were acquired in the left parasternal apical windows using cross-sections of four and five chambers, respectively. The flow velocity in the aortic artery was determined using pulsed Doppler to quantify the LV ejection time (LVE) and the LV pre-ejection period (PEP). The mitral flow was assessed in the apical section of four chambers with a sample placed at the height of the tips of the mitral leaflet. The early (E) and late (A) left ventricular filling velocities, as well as the ratio of the early and late (E/A) left ventricular filling velocities, were determined in the same way. The isovolumetric relaxation time (IVRT) was measured as the time interval between the end of the aortic flow and the beginning of the mitral influx using a pulsed Doppler. Tissue Doppler images (TDI) obtained at the site of septal and lateral insertion of the mitral annulus were used to determine the systolic excursion velocities (S’), early diastolic excursion velocities (E’), and late diastolic excursion velocities (A’) [29].2.8. Speckle TrackingThe indices representing the percentage of deformation and myocardial deformation speed, the strain (SR), and strain rate (SRT), respectively, were obtained using the 2D-STE methodology, previously described by Chetboul et al. [31]. For this procedure, two-dimensional images were acquired in the right parasternal cross-section, at the height of the papillary muscles, and the images were analyzed using the optical flow algorithm in the software Syngo Velocity Vector Imaging (VVI) (SIEMENS®). Three consecutive cardiac cycles were collected, using continuous ECG monitoring, with a sampling rate between 50 to 90 rams/s. For myocardial screening, the endocardial border was manually marked at the end of the systole and then the epicardial border was automatically delimited by the software, being manually adjusted when necessary. Point tracking was only accepted if the software inspection and visual inspection were deemed adequate. All measurements were made at week 0 (W0) and after the conditioning period, being reassessed at week 8 (W8).2.9. Intra and Interobserver VariabilityA repeatability study was conducted on six animals assessed the intra and interobserver variability of the speckle tracking evaluation. These dogs were randomly reassessed using images obtained previously with a minimum of fifteen days from the first evaluation. The same observer was evaluated to calculate the intraobserver variability. The same studies were examined by a researcher blinded to the results of the first investigation to measure interobserver variability. 2.10. Heart RateThe heart rate was evaluated by a 24 h Holter test using a Cardioflash® (digital-Cardios Sistemas-São Paulo, Brazil) to capture the signals of cardiac electrical activity for 24 h, following the methodology previously published by our laboratory [24,26]. The electrocardiographic tracings were processed by a specific software (Cardio Manager S540, Cardios Sistemas, Brazil). The dogs remained in the stalls during the 24 h test to minimize environmental interferences to obtain the heart rate. Therefore, HR was determined during rest before and after the conditioning period.2.11. Statistical AnalysisAll raw data were assessed using the Shapiro–Wilk normality test. The Mann–Whitney and Student t-test for unpaired samples were applied to evaluate the physical and cardiac variables at the beginning of the experiment. Moreover, the Student’s t-test was applied to evaluate trained dogs’ aerobic and anaerobic fitness variables, such as VLT and Vmax, respectively, body weight and the 24 h resting HR. The echocardiographic variables were analyzed using two-way analysis of variance (ANOVA), with the factors, and group ((two levels: control and trained dogs) vs. conditioning period (two moments: W0, W8)). Post hoc comparisons of the data were then performed using the Tukey–Kramer multiple comparison test. Moreover, the coefficients of variation were calculated to assess intra and interobserver measurements. The principal component analysis was performed to identify the principal components and explore the echocardiographic variables associated with the differentiation between the control and trained groups. Principal components analysis (PCA) of sample replicates were performed using the prcomp function, setting the argument scale = TRUE, from the ‘stats’ package in R. This was generated using ‘factoextra’ (v.1.0.7) and pca3d (v.0.10.2) packages. The heat map method is a practical way of evaluating grouping distances and was plotted using the R ‘pheatmap’ package (version 1.0.12) implemented in the R (v.3.6.3) using values of echocardiographic variables. Other statistical analyses were performed using the SigmaPlot software (v.12.0) at 5% significance.3. ResultsTable 1 summarizes the physical and cardiac variables correlated with the physical characteristics and the cardiac health of the dogs in the experimental groups before the ETP. It is noteworthy that the raw data regarding both sexes were grouped and analyzed together since their physiological characteristics were not significantly different at the beginning of the ETP. The body weight of the dogs in groups C and T did not differ during the experimental period. The cardiac physiological and echocardiographic variables revealed that the dogs of both groups were healthy. None of the dogs displayed changes in rhythm (ventricular or supraventricular arrhythmias) during the study. It is essential to inform that, at the end of the ETP, a male dog of the trained group presented trivial mitral valve regurgitation, and this condition has been monitored weekly by the research team ever since. After the tenth week, mitral regurgitation was no longer evidenced by echocardiographic examination.The body weight of the dogs did not change at the end of the study for groups T (p = 0.57) and C (p = 0.45), as well as in the intergroup comparison (p = 0.108). At the end of the conditioning period, HR, obtained at rest during the 24 h test, was lower in group T (p < 0.001) in the intragroup check compared to group C (p = 0.034). This study used the velocity corresponding to the inflection point of plasma lactate concentration (VLT) to prescribe and evaluate the effect of training (Table 2). The intragroup comparison showed that group T aerobic and anaerobic fitness improved while VLT, Vmax, and VLT:Vmax were higher than IET-1, with p = 0.016, 0.017, and 0.003, respectively. The active control group consisted of an “untrained group” of dogs used in research previously published by our group that followed the same experimental design. Both VLT and Vmax did not differ between the IETs (Please see [23,24]).Cardiac morphology was altered with the ETP. A significant interaction was observed between the conditioning period (moments W0 and W8) and groups (C and T) for LVDd (p = 0.005), DIVEd/Ao (p = 0.005), and LA/Ao (p = 0.025). DIVs and DIVs/Ao did not change significantly during the study. However, there was a statistical tendency for the conditioning period (p = 0.059 and p = 0.06, respectively) (Figure 2). The thicknesses of the interventricular septum and free wall did not change significantly.Moreover, the TEVE and PPE did not change after the conditioning period. The FS% had a group effect (p = 0.01), since the T and C groups differed before the study, but this difference disappeared after the conditioning period. The conditioning period significantly affected the EF and LVVd (p = 0.039 and p = 0.003, respectively) since EF (p = 0.032) increased in group T after the conditioning period while LVVd increased over eight weeks (p = 0.004). Figure 3 reveals that VS affected the interaction (p = 0.039), increasing in group T at the end of the study (p < 0.001).The tissue Doppler did not change in the study (Table 3). Diastolic function increased with conditioning. Wave E did not change but wave A had a group effect (p = 0.03), decreasing in group T after the conditioning period (p = 0.04). A significant interaction (p = 0.026) between the conditioning period and group was observed for the E/A ratio, which was higher in the trained group compared to group C, in W8 (p < 0.001), and at this moment, the intragroup comparison also indicated that this variable improved in the dogs of group T (p = 0.002). IVRT and E/IVRT had an effect in the period (p = 0.049 and p = 0.035, respectively). IVRT decreased in the trained group after the conditioning period (p = 0.012). On the other hand, E/IVRT increased in group T (Figure 4) after eight conditioning weeks (p = 0.005). E/E’ was also affected by the conditioning period (p = 0.02) and increased in group T in W8 (p = 0.006).Speckle tracking was used for precisely evaluating myocardial function from the two-dimensional echocardiogram curves by the objective quantification of myocardial deformation and left ventricular systolic and diastolic dynamics. The SR and radial SRT indexes changed significantly with the ETP. The SR had a significant interaction between the conditioning period and the group (p = 0.001). Figure 5 shows that SR increased significantly in the trained dogs compared to the control group after the ETP (p < 0.001). A significant interaction between the conditioning period and group (p = 0.03) was observed for SRT, which increased significantly at W8 in the trained group (p = 0.009). Finally, the inter and intraobserver repeatability analyses showed acceptable coefficients. The SR and SRT indices demonstrated reliable intraobserver analyses, with coefficients of variation (CV) determined as 7.5% and 5.45%, respectively. The interobserver analysis had a coefficient of variation of 6% and 4.42% for SR and SRT, respectively.PCA characterizes and compiles a dataset to mitigate the dimensionality of the nineteen measured echocardiographic variables into three components (58.35% of the total variance). A biplot was performed to observe the variability and similarities of echocardiographic variables among the studied dogs, revealing distinct profiles for C and T groups, and their association with each variable (Figure 6A,B). In addition, from this PCA we obtained the contribution of each variable on components 1 and 3, which were selected to clearly distinguish the trained dogs (Figure 6C). The variables with a contribution above the reference value, i.e., the expected value if the contribution were uniform, could be considered as important for the related dimensions.The heatmap visualization technique is one of the most practical ways of evaluating cluster distances. The map shows the grouping of scores obtained after evaluating the echocardiographic variables of the groups before and after ETP. The black and white gradients show the contrasting scores, that is, the observed response intensities, where the white color indicates a minimal or absent response, and the black color suggests a higher response. In this sense, Figure 7 reveals that dogs trained for eight weeks were clustered in the center of the heatmap (green rectangles) with the highest intensity of black areas.4. DiscussionOur study was the first to evaluate the effect of chronic submaximal exercise prescribed from the velocity corresponding to the LT on the diastolic and systolic function in beagle dogs. The main finding is that this type of aerobic conditioning improved cardiac function, with physiological adjustments characterized by ventricular dilation, increased early diastolic relaxation, and improved LV radial systolic mechanics. To date, no study has reported such changes in cardiac function at rest due to an endurance training program in dogs guided by the external load corresponding to the lactate threshold.To study the correlation between the multiple echocardiographic variables, some complex system approaches propose detecting the coordination variables, also known as the order or collective variables, using the principal component analysis (PCA), a multivariate statistical technique for identifying the coordination variables [15]. The PCA discriminated against echocardiographic variables used for evaluating morphological changes (LVDd), probably induced by the increased volume load (LVVd, SV, and LVVs), as well as diastolic (E/A, A, E and E/E’) and systolic (ST) adjustments. Thus, the PCA and the heatmap quantified the effect induced by the ETP while capturing the resulting qualitative changes. Our method to evaluate the ETP effects provided qualitative information that complemented the traditionally used submaximal performance indexes (HRrest and VLT) and maximal index Vmax. The VLT, Vmax, and VLT:Vmax of trained dogs increased at the end of the eight-week training period. Likewise, previous studies with horses [32] and dogs [23,24] reported similar findings. Moreover, the VLT increase suggests less need to use the anaerobic glycolytic pathway for producing ATP, indicating an improvement of aerobic fitness. This probably resulted from the increase in the number of mitochondria and oxidative enzymes in muscle fibers, which maximized the metabolic pathway of oxidative phosphorylation [32]. Besides, the Vmax increase in dogs of the trained group observed in IET-2 represented a positive anaerobic metabolic adjustment in response to the conditioning program [23]. This latter study reported that VLT and Vmax remained unchanged in dogs not submitted to the same conditioning protocol used here compared with trained dogs. VLT-based submaximal training prescription protocol improved the oxidative functional capacity in dogs. Another aspect being addressed is the decreasing HR detected by the 24 h electrocardiography at rest. This response is expected after aerobic conditioning programs, with bradycardia used as a marker of cardiovascular improvement [24]. These results confirm that the ETP improved the aerobic and cardiovascular capacity of the trained dogs.The increase of the LVDd, LVDd/Ao, and LA/Ao variables are consistent with other results obtained in human endurance athletes [11,20] and sled dogs after training [33]. The LA and left ventricular sizes of the trained dogs increased significantly, consistent with increased preload, where LV end-diastolic volume was the major determinant of LA volume increase in athletes [7,34]. During ventricular diastole, the LA is exposed to LV systolic pressure. Consequently, when the LV pressure increases, the LA pressure rises to maintain adequate LV filling. This increasing tension in the atrial wall can lead to dilation of the chamber and stretching of the myocardium of the LA [7,35]. In our study, both LV and LA diameters increased in the trained dogs, with a proportional increase in LV systolic volume caused by volume overload without eccentric LV hypertrophy. This finding is compatible with short-term conditioning periods and changes observed due to physical conditioning [9]. Therefore, the increased LA and LV dimensions of dogs submitted to aerobic conditioning can be considered a physiological consequence and included in the dog’s athlete’s heart context.Adaptations induced by exercise in LV function have been relatively well studied. Studies have examined the LV systolic function of human athletes at rest [11,20,36]. In the present study, FS% did not change with regular exercise, a result compatible with a previous study with sled dogs, indicating that this cardiac variable remains unchanged after training [33]. Additionally, FS% is not commonly used to determine improvement in systolic function in humans, as it measures only radial contractility. However, in most cases, the longitudinal increments of the systolic function appear before the radial function. Thus, FS% must be interpreted from the hemodynamic point of view. It can be affected not only by the degree of myocardial contractility but also by the conditions of preload and afterload, and HR [37].On the other hand, LVDd, SV, and EF increased in the trained dogs. The systolic function assessed by EF is generally normal among athletes of the human species [36]. This finding contradicts other studies [11,20,31,36] that reported that the practice of long-term exercise training did not cause these variables to change. However, there are reports of increased systolic function in Olympic athletes who have undergone intense and uninterrupted endurance training [38].Still on this topic, the increase in LVDd and SV are classic findings in trained individuals [11,20,36]. SV can increase significantly with training during exercise and at rest. The increase in the cardiac chamber and the ability to generate a higher systolic volume are the direct results of the athlete’s physical training and the increase in the LV end-diastolic volume [8]. This LV end-diastolic volume is determined by diastolic filling, a complex process that is affected by a variety of variables, including heart rate, intrinsic myocardial relaxation, ventricular compliance, ventricular filling pressures, atrial contraction, among others [36].The E/A ratio of the transmitral flow increased in the trained group, probably induced by the A wave decrease. Additionally, other parameters such as E/IVRT and E/E’ also increased in the trained dogs, while the IVRT values decreased after the conditioning period. These findings are compatible with increased LV and LA pressure, an effect induced by an increase in the final diastolic volume [39,40,41]. The decrease in IVRT to less than <45 ms, as well as the increase in the E/IVRT (values > 2.3), E/E (values > 12), and E/A (>2) ratios are strongly correlated with the increased pressure of LV filling in dogs [40,42,43]. It is highlighted that shortened IVRT, together with increased E/A, E/IVRT, and E/E (Figure 2) is, by definition, an integral part of restrictive diastolic dysfunction, a flow pattern considered specific to advanced diastolic dysfunction and high LV pressure [40,43,44]. Although, after the conditioning period, the trained group had increased E/A and reduced IVRT compared to the beginning of training (W0), while the E/IVRT and E/E’ parameters tended to the upper limit of normality, this finding cannot be interpreted as a diastolic dysfunction of the LV but as a normal finding in dogs submitted to physical conditioning.The TDI parameters (S’, E’, and A’), measured in the septal and lateral mitral annulus, remained similar in both experimental groups. Although conventional Doppler echocardiographic variables are useful for distinguishing between physiological or pathological LV diastolic changes, the TDI results were ineffective for detecting cardiac characteristics/parameters in human athletes [11].LV diastolic function has also been extensively assessed in trained individuals using pulsed and tissue Doppler. It is well recognized that regular exercise promotes an increase in LV early diastolic filling assessed by the E wave velocity, the velocity of the mitral annular tissue, and IVRT [10,34,45,46]. Human studies have shown that hemodynamic changes occurring during acute exercise are the primary stimulus for cardiac remodeling. Aerobic training can increase LV early diastolic filling [47] and faster heart filling during intense exercise [48]. This rapid filling is due to the mechanical forces resulting from the heart remodeling that leads to a noticeable increase in the intraventricular pressure gradient, whereby blood is rapidly sucked from the LA to the LV apex [8,49]. Therefore, this diastolic suction and rapid LV filling are mainly due to the LV’s ability to quickly relax at high heart rates during physical exercise, being an essential mechanism for preserving systolic volume during exercise [36].Considering that the FS% of the LV remained unchanged in the dogs of the T group, more direct LV contractility indexes obtained through speckle tracking increased significantly. Specifically, the SR and SRT increased in the trained dogs while speckle tracking data revealed increased radial systolic function, despite an FS% without LV changes.Recent advances in quantifying LV strain can help characterize myocardial contractility in a wide range of experimental and clinical settings [31,50,51,52]. The increase in SR and radial SRT resulting from fitness protocols, even when EF remains unchanged, has been elucidated in humans [11]. This methodology has been demonstrated to be more sensitive for determining systolic function changes than traditional echocardiographic indexes and is not influenced by the HR of trained individuals [11]. Similarly, previous studies on dogs have shown that speckle tracking was little or not influenced by HR [52]. Certainly, the data from the present study revealed that ETP was able to improve systolic function, as determined by speckle tracking more sensitively, even without changes observed in the conventional echocardiogram.4.1. Study LimitationsSome limitations of our study must be recognized. First, the failure to perform the initial and final stress test in the active control group to demonstrate the influence of training on VLT and Vmax. However, a previous study showed that our conditioning protocol was responsible for the improved aerobic fitness of dogs when comparing active and untrained dogs [23]. Second, the lack of measurements of systolic pressure in dogs before and after the conditioning period. This variable could have complemented the results obtained here. Additionally, the lack of measurements of parameters evaluating the longitudinal systolic function since such data could have reinforced the present study’s findings. It has been shown that changes in systolic function may occur first in the longitudinal deformation parameters of the myocardium and then in the radial function of the heart [53]. Nevertheless, the findings obtained here showed an improvement in systolic function. Further studies are needed to confirm whether the use of variables to determine longitudinal systolic function effectively determines changes in the systolic function of dogs undergoing regular exercise.4.2. Clinical ImplicationsSystolic and diastolic function is adversely altered in many pathological conditions, especially heart disease [43,54]. In cardiac patients, physical conditioning is used as nonpharmacological treatment, since they have been demonstrated to increase the quality of life and survival while decreasing cardiac remodeling, maintaining the systolic function, and improving diastolic function [55]. However, to date, the prescription of regular exercise programs in Veterinary Cardiology is still limited, especially as a treatment for diseases such as mitral valve degeneration [25]. Besides, there are clinical implications for human patients after mitral valve repair. Following mitral repair, exercise training was safe and associated with an improvement in exercise capacity, not deteriorating the outcome of recent surgery, and effective in increasing peak oxygen consumption and the anaerobic threshold [56,57]. Physical conditioning improves cardiac function (diastolic and systolic function), has positive vascular effects (angiogenesis, increase in nitric oxide production), and neurohormonal and autonomic effects such as the increase in the parasympathetic system and antiarrhythmic effects, and the decrease in angiotensin II concentrations, which are beneficial for the quality of life and survival of human patients [58]. Thus, the results of this study with healthy animals are essential for future studies, since the literature expresses the need for several controlled clinical studies using healthy patients to evaluate the results, efficacy, and safety of several training modalities [59].It is still necessary to discuss the male dog that presented reversal of mitral valve regurgitation diagnosed at the end of the experimental period. Mitral, aortic, or pulmonary regurgitation is a relatively common clinical finding in retired human athletes, horses, and greyhounds [60,61]. This regurgitation is considered physiological, generally induced by morphofunctional adjustments caused by regular exercise, and does not represent a pathological finding in trained individuals [62]. It is important to emphasize that the alteration diagnosed here was no longer detected by conventional echocardiography after a ten-week detraining period. A study in rats showed that cardiac adjustments resulting from regular exercise could be reversed after a similar period of detraining [63]. Further studies are needed to quantify the incidence of mitral regurgitation in dogs undergoing training and to characterize the time of reversibility of these physiological adjustments.5. ConclusionsEight weeks of continuous exercise prescribed with submaximal external load promoted concomitant LV dilation without hypertrophy of the ventricular walls, emphasizing LV systolic and diastolic function. This conditioning protocol can be applied to improve systolic and diastolic functions in dogs. These findings together imply the need for future studies to confirm that the protocol used here has similar effects in dogs affected by chronic diseases that alter the cardiac function and could pave the way for new treatments for dogs with heart failure.

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