<table><caption>**Table 1.** Effect of diet composition (g/kg DM) on energy digestibility (DCe, %), ME:DE coefficient (%) and efficiency of utilization of ME for NE of mixed diets for growth ($k_g$, %)$^a$</caption><thead><tr><th>Equation</th><th>RSD</th><th>Source<sup>b</sup></th></tr></thead><tbody><tr><td>1 DCe = 98.3 - 0.090 x NDF</td><td>2.0</td><td>1</td></tr><tr><td>2 DCe = 96.7 - 0.064 x NDF</td><td>2.2</td><td>1</td></tr><tr><td>3 ME/DE = 100.3 - 0.021 x CP</td><td>0.5</td><td>1</td></tr><tr><td>4 k<sub>g</sub> = 74.7 + 0.036 x EE + 0.009 x ST - 0.023 x CP - 0.026 x ADF</td><td>1.2</td><td>2</td></tr></tbody></table>

$^a$CP: crude protein, NDF: Neutral Detergent Fibre, EE: ether extract, ST: starch, ADF: Acid Detergent Fibre; RSD: Residual standard deviation.

$^b$1: Le Goff and Noblet, 2001 (n=77 diets; equations 1 and 3 in 60 kg growing pigs and equation 2 in adult sows, respectively); 2: Noblet et al., 1994a (n=61 diets; 45 kg pigs).

Energy digestibility is also affected by animal factors. In growing pigs, DCe increases with increasing BW (Noblet, 2006; Noblet et al., 2013) and the largest effect of BW is observed when adult sows either pregnant or lactating and (close to) ad libitum growing pigs are compared (Le Goff and Noblet, 2001; Le Gall et al., 2009). The difference due to BW increase is the most pronounced for high fiber diets or ingredients. The negative effect of DF on DCe is then lower in adult pigs than in growing pigs (table 1). In the case of adult sows and "60 kg" growing pigs, the DE value is 1.8, 4.2, 6.0, 10.3 and 16.6% higher in sows for wheat, corn, soybean meal, wheat bran and corn gluten feed, respectively (Sauvant et al., 2004). This improvement of energy digestibility with BW is mainly related to an improved digestive utilization of DF (Noblet and Le Goff, 2001). Little information concerning comparative digestibility in piglets and growing pigs is available. Considering that piglets are usually fed low-fiber diets for which the effect of BW is minimized, piglets can, from a practical point of view, be considered as growing pigs concerning the digestive utilization of energy.

The ME content of a feed for pigs is the difference between DE and energy losses in urine and gases (methane). In growing pigs, average energy loss in methane is equivalent to 0.4% of DE intake and is 2-3 times this amount in adult sows. Energy loss in urine represents a variable percentage of DE since urinary energy depends greatly on the urinary nitrogen excretion. At a given stage of production, urinary nitrogen excretion is mainly related to the (digestible) protein content of the diet (table 1). On average, it represents about 4% of the DE value. However, this mean value cannot be applied to single feed ingredients. The most appropriate solution is to estimate urinary energy (kJ/kg feed DM) from urinary nitrogen (g/kg feed DM) according to the following equation (in growing pigs):

$$ \text{Urinary energy} = 192 + 31 \times \text{urinary nitrogen} $$

with urinary nitrogen representing 50% of digestible nitrogen or 40% of total nitrogen (Noblet et al. 2004).

Net energy is defined as ME minus heat increment associated with metabolic utilization of ME and to the energy cost of ingestion, digestion and some physical activity. The NE content, as a percentage of ME content (k) corresponds to the efficiency of utilization of ME for NE. In the studies of Noblet et al. (1994a) conducted on 61 diets fed to growing pigs, the variations in k were due to differences in efficiencies of ME utilization between nutrients with the highest values for fat (~90%) and starch (~82%) and the lowest (~60%) for DF and crude protein; k as obtained in growing pigs is then directly dependent on diet chemical composition (table 1) with positive effects of starch and fat on k and negative effects of DF and protein. Additional measurements conducted in pigs which differed for their BW and the composition of BW gain suggest that the efficiency of ME for NE is little affected by the composition of BW gain, at least under most practical conditions (Noblet et al., 1994b). Similarly, the ranking between nutrients for efficiencies is similar in adult sows fed at maintenance level and in growing pigs (Noblet et al., 1993). Finally, the heat increment associated with protein utilization, either retained as protein or catabolized, is constant (van Milgen et al., 2001) which means that the NE value of dietary CP is not dependent on its final utilization (deposition vs. deamination).
<table><caption>**Table 2.** Equations for prediction of NE in feeds for growing pigs (NEg; 61 diets; MJ/kg dry matter and % of DM; adapted from Noblet et al., 1994a)</caption><thead><tr><th>N°</th><th>Equation<sup>a</sup></th></tr></thead><tbody><tr><td>5</td><td>NEg2 = 0.121 DCP + 0.350 DEE + 0.143 ST + 0.119 SU + 0.086 DRes</td></tr><tr><td>6</td><td>NEg4 = 0.703 DE - 0.041 CP + 0.066 EE - 0.041 CF + 0.020 ST</td></tr><tr><td>7</td><td>NEg7 = 0.730 x ME - 0.028 x CP + 0.055 x EE - 0.041 x CF + 0.015 x ST</td></tr></tbody></table>

<sup>a</sup> CP: crude protein, EE: ether extract, ST: starch, DCP: digestible CP, DEE: digestible EE, DRes: digestible residue (i.e., difference between digestible organic matter and other digestible nutrients considered in the equation).

The NE measurements conducted on 61 diets by Noblet et al. (1994a) were used to establish prediction equations relating dietary NE to predictors such as DE or ME and chemical indicators or digestible nutrients; the most practical ones have been used for the establishment of the INRA & AFZ feeding tables (Sauvant et al., 2004) or the EvaPig software (www evapig.com); they are listed in table 2. Comparable equations have been produced by Schiemann et al. (1972) and further adapted in the Dutch feeding tables (CVB).

## Energy systems for pig feeds

Apart from direct measurement on pigs, the DE and ME values of raw materials can be obtained from feeding tables (Sauvant et al., 2004; EvaPig; NRC, 2012). The utilization of these tabulated values should be restricted to ingredients having chemical characteristics similar or close to those in the tables. But, practically, the chemical composition and therefore the energy value of most raw materials differ from those listed in the feeding tables. It is then advised to correct the energy value for differences in chemical composition between ingredients proposed in the tables and those actually available for least-cost formulation; the EvaPig tool has been proposed for such a purpose (www evapig.com). As illustrated in the previous section, DCe is affected by BW of the animals. It is therefore appropriate to use DE and ME values adapted to each BW class. However, from a practical point of view, it is suggested to use two values, one for "60 kg" pigs which can be applied to piglets and growing-finishing pigs and one for adult pigs applicable to both pregnant and lactating sows. The INRA & AFZ feeding tables propose such a differentiation (Sauvant et al., 2004) and the re-calculation of both energy values according to the chemical characteristics of ingredients is possible from the EvaPig software (www evapig.com). Finally, a rather frequent difficulty in feed evaluation consists in estimating the energy value of compound feeds when their ingredients composition is unknown. The best solution is then to use prediction equations based on chemical criteria (Le Goff and Noblet, 2001; EvaPig) or estimates from near infrared or *in vitro* methods (Noblet and Jaguelin-Peyraud, 2007).

All published NE systems for pigs combine the utilization of ME for maintenance and for growth or for fattening. The system proposed by Noblet et al. (1994a) and applied in the INRA & AFZ feeding tables (Sauvant et al., 2004) is based on NE measurements on 61 diets and the NE prediction equations that have been generated from these measurements (table 2) are applicable to ingredients and compound feeds and at any stage of pig production (Noblet, 2006). It is important to point out that different DE values or digestible nutrient contents should be used in growing-finishing pigs and adult sows with two subsequent sets of NE values. Similarly, reliable information on digestibility of energy or of nutrients is absolutely necessary for an accurate prediction of NE content of pig feeds. In fact, this information represents the most limiting factor for predicting energy values of pig feeds. From that point of view, the lack of comprehensive information on effects of technology (pelleting, extrusion, enzymes addition, etc.) is a major limiting factor for getting accurate estimates of energy values for swine whatever the system used.

Assuming that NE represents the best estimate of the "true" energy value of feeds and according to differences in efficiencies of nutrients ME for NE, the energy value of protein-rich or fibrous feeds is overestimated when expressed on a DE (or ME) basis. On the other hand, fat or starch sources are underestimated in DE and ME systems (Table 3). With regard to NE for pigs, several systems have been proposed over the last 40-50 years. The INRA proposal (Noblet et al., 1994a; 2004) is probably the most advanced system which has been validated by calorimetry measurements (Noblet and van Milgen, 2013) and growth trials (table 4).

As previously mentioned, it is extremely important to use the same energy system for expressing the diet energy values and the animal energy requirements. From that point of view, the only energy system in which the requirements are the most independent on diet characteristics should be the NE system. This is illustrated by several growth trials
conducted with variable dietary fat or CP levels that show that the energy cost of growth or the daily energy requirement are independent on diet composition when expressed on a NE basis. On the other hand, on DE or ME bases, the energy cost is increased when CP content is increased or decreased when fat content is increased (Table 4). In other terms, relative to the NE system, the DE and ME systems are less able to predict the performance of growing pigs and reproductive sows.

<table><caption>**Table 3.** Relative DE, ME and NE values of ingredients for growing pigs<sup>a</sup></caption><thead><tr><th></th><th>DE</th><th>ME</th><th>NE</th><th>NE/ME, %</th></tr></thead><tbody><tr><td>Animal fat</td><td>243</td><td>252</td><td>300</td><td>90</td></tr><tr><td>Corn</td><td>103</td><td>105</td><td>112</td><td>80</td></tr><tr><td>Wheat</td><td>101</td><td>102</td><td>106</td><td>78</td></tr><tr><td><strong>Reference diet</strong></td><td><strong>100</strong></td><td><strong>100</strong></td><td><strong>100</strong></td><td><strong>75</strong></td></tr><tr><td>Pea</td><td>101</td><td>100</td><td>98</td><td>73</td></tr><tr><td>Soybean (full-fat)</td><td>116</td><td>113</td><td>108</td><td>72</td></tr><tr><td>Wheat bran</td><td>68</td><td>67</td><td>63</td><td>71</td></tr><tr><td>Soybean meal</td><td>107</td><td>102</td><td>82</td><td>60</td></tr></tbody></table>

<sup>a</sup> From Sauvant et al. (2004). Within each system, values are expressed as percentages of the energy value of a diet containing 68% wheat, 16% soybean meal, 2.5% fat, 5% wheat bran, 5% peas and 4% minerals and vitamins.

<table><caption>**Table 4.** Performance of growing-finishing pigs according to energy system and diet characteristics<sup>a</sup></caption><thead><tr><th>Energy system</th><th>DE</th><th>ME</th><th>NE</th></tr></thead><tbody><tr><td><u>Trial 1: Added fat (%)</u></td><td></td><td></td><td></td></tr><tr><td>0</td><td><strong>100</strong></td><td><strong>100</strong></td><td><strong>100</strong></td></tr><tr><td>2</td><td>100</td><td>100</td><td>100</td></tr><tr><td>4</td><td>99</td><td>99</td><td>100</td></tr><tr><td>6</td><td>98</td><td>98</td><td>100</td></tr><tr><td><u>Trial 2: crude protein content (30-100 kg)</u></td><td></td><td></td><td></td></tr><tr><td>Normal</td><td><strong>100</strong></td><td><strong>100</strong></td><td><strong>100</strong></td></tr><tr><td>Low</td><td>96</td><td>97</td><td>100</td></tr><tr><td><u>Trial 3: crude protein content (90-120 kg)</u></td><td></td><td></td><td></td></tr><tr><td>Normal</td><td><strong>100</strong></td><td><strong>100</strong></td><td><strong>100</strong></td></tr><tr><td>Low</td><td>97</td><td>98</td><td>100</td></tr></tbody></table>

<sup>a</sup> Energy requirements (or energy cost of BW gain) for similar daily BW gain and composition of BW gain; values are expressed relative to the energy requirement (or energy cost of BW gain) in the control treatment (considered as 100; values in bold characters); from Noblet (2006), Wu et al. (2007) and unpublished data.

Most energy recommendations for pigs were obtained according to DE and ME estimates for feeds and conclusions were expressed as DE or ME values. These recommendations were obtained with rather conventional feeds, i.e. cereals-soybean meal based diets whose efficiency of ME utilization in growing pigs is close to 74%. It is then proposed to estimate the NE recommendations (diet energy density, daily energy requirements, components of energy requirements, etc.) as DE or ME requirements multiplied by 0.71 or 0.74, respectively. This proposal is applicable at any stage of pig production, including pregnant or lactating sows, since NE value is calculated for any stage from one single set of equations obtained in growing pigs (Table 2). However, DE or ME or digestible nutrients may differ between stages of production, i.e. growing-finishing pigs vs reproductive adult sows, with subsequent different NE values.

## Energy utilization and energy systems in poultry

Numerous studies have concerned the digestibility (in fact metabolizability) of energy in broilers or poultry. As for pigs, the main factor of variation of energy metabolizability is the DF content with even more pronounced effects since DF is almost undigested by broilers and the contribution of DF to ME supply is then even slightly negative. The recent paper of Carré et al. (2013) on 28 diets illustrates these observations. In addition, the (apparent) ME value of poultry feeds is frequently corrected for a zero N balance, assuming then that all absorbed amino acids are deaminated; even
such a correction eliminates the differences between ingredients due to their different CP contents, it is not representative of the reality for growing broilers, turkeys, etc. and even laying hens that retain 50% or more of the N that is absorbed. Therefore, this correction is questionable.

With regard to NE, several trials or theoretical assumptions over the last 70 years have been carried out to quantify the utilization of ME and its digestible nutrients for NE in poultry (review of Pirgozliev and Rose, 1999). Some studies were performed on ingredients or unbalanced diets with subsequent limitations in the interpretation of the results (cf. methodological section). The most comprehensive series of measurements were conducted in USA by Fraps (1946) and by the Rostock group (Schiemann et al., 1972), mainly focussing on feed ingredients. Unfortunately, most attention in these studies was focused on starch, DF and CP with little variability in fat content of diets or feedstuffs. A recent study of Carré et al. (2014) was conducted on quite variable complete feeds (n=28; 18.0 to 29.8 % CP, 5.6 to 12.3 % fat, 27.4 to 45.9 % enzymatic starch and 8.1 to 20.6 % NDF; expressed relative to DM) fed to 3 to 5 wk old broilers. The comparative slaughter method was used to quantify energy retention and NE was calculated according to a FHP value (500 kJ/ kg BW<sup>0.60</sup>/d) measured in respiration chambers in comparable birds. A small concern in that study originates from an unexplained trial effect that has been included for interpreting the data and may have biased the final results.

As for pigs, literature values on poultry indicate that the lowest efficiency is observed for CP and the highest for fat (table 5). However, the difference between the most extreme values (i.e., CP and EE) appears to be lower in poultry (65 and 85%) than in pigs (60 and 90%). In addition, the values from Schiemann et al. (1972) were obtained in adult fattening birds which are in a physiologically different stage. From that point of view, the study of Carré et al. (2014) is probably more representative of modern broiler production. Preliminary results obtained in Australia (Barekatain et al., 2014) or in the USA (Cerrate Fernandez et al., 2012; Cerrate et al., 2013) on a limited number of diets would confirm these literature data. In addition, the data of Cerrate et al. (2013) indicate efficiencies of ME for NE in broilers that are remarkably close to those obtained in pigs (Sauvant et al., 2004) for a few typical ingredients used in pigs and broilers diets.

An alternative approach to study the effect of diet on the efficiency of ME for NE was tested in trials conducted at INRA (Noblet et al., 2003; 2007; 2009). The approach consisted in preparing diets focussing each time on one specific nutrient (in exchange for starch). The effects of CP, EE and DF contents were evaluated and the trials were conducted in respiration chambers on group housed and *ad libitum* fed growing broilers over the 3rd to the 6th week of age. The methodology allowed to quantify the HP related to physical activity of the birds and potential differences between diets due to changes in their behaviour. The most important results are presented in Table 6.

<table><caption>**Table 5.** Efficiency of ME utilization of digestible nutrients for NE in poultry (%)</caption><thead><tr><th>Reference</th><th>Production<sup>1</sup></th><th>CP</th><th>EE</th><th>CHO</th><th>Diet</th></tr></thead><tbody><tr><td>Schiemann et al., 1972</td><td>M + fattening</td><td>61</td><td>84</td><td>75</td><td>73<sup>2</sup></td></tr><tr><td>De Groote, 1974</td><td>M + growth</td><td>60</td><td>90</td><td>75</td><td>74<sup>2</sup></td></tr><tr><td>Carré et al., 2014</td><td>M + growth</td><td>68</td><td>85</td><td>78</td><td>76</td></tr></tbody></table>

<table><caption>**Table 6.** Effect of diet composition on heat production and efficiency of ME for NE in broilers: compilation of INRA data<fnref n="1" /></caption><thead><tr><th rowspan="2">Trial</th><th rowspan="2">Diets</th><th colspan="3">kJ/kg BW<sup>0.70</sup>/d</th><th rowspan="2">NE/ME<br>%</th></tr><tr><th>ME</th><th>HP</th><th>AHP</th></tr></thead><tbody><tr><td rowspan="2">1</td><td>18% CP</td><td>1609</td><td>853</td><td>146</td><td>75.1</td></tr><tr><td>22.7% CP</td><td>1609</td><td>846</td><td>153</td><td>74.8</td></tr><tr><td rowspan="2">2</td><td>2.8% EE</td><td>1739</td><td>884</td><td>127</td><td>74.3<sup>2</sup></td></tr><tr><td>9.7% EE</td><td>1728</td><td>875</td><td>143</td><td>75.5<sup>2</sup></td></tr><tr><td rowspan="2">3</td><td>9.5% NDF</td><td>1503</td><td>912</td><td>170</td><td>71.3</td></tr><tr><td>17.7% NDF</td><td>1521</td><td>923</td><td>175</td><td>72.3</td></tr></tbody></table>

<footnote n="1">¹ M: maintenance; CP: crude protein; EE: ether extract; CHO: carbohydrates</footnote>

<footnote n="2">² Assuming that 25, 20 and 55% of ME is provided as CP, EE and CHO</footnote>

<footnote n="1">¹ Measurements carried out in groups of broilers weighing 1.3 to 1.5 kg BW on average for each trial (5 to 8 measurements per diet); the indirect calorimetry method in respiration chambers was used; AHP: Activity heat production; complementary details by Noblet et al. (2007) for trial 1 and Noblet et al. (2009) for trial 2; trial 3: unpublished data. In trials</footnote>
1 and 2, the variation in CP or EE is associated with an inverse variation in starch content; in trial 3, the increased NDF level corresponds to a dilution by dietary fibre provided by wheat bran, maize bran and soybean hulls. In trial 1, data have been adjusted for a similar ME intake while observed values are given for trials 2 and 3. None of the differences between treatments within each trial were significant (P>0.05).

² Values adjusted for the same level of physical activity with the 2 diets

<table><caption>**Table 7.** Comparative effect of dietary crude protein on energy utilisation in growing pigs and broilers (adapted from Noblet et al., 2003)¹</caption><thead><tr><th rowspan="2">Species<br>Dietary protein level</th><th colspan="2">Pigs</th><th colspan="2">Broilers</th><th rowspan="2"></th></tr><tr><th>Normal</th><th>Low</th><th>Normal</th><th>Low</th></tr></thead><tbody><tr><td>Body weight, kg</td><td>57.6</td><td>57.2</td><td>1.47</td><td>1.46</td><td></td></tr><tr><td>Energy balance<sup>2</sup>, kJ/kg BW<sup>0.60</sup></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>ME intake</td><td>2564</td><td>2566</td><td>1626</td><td>1642</td><td></td></tr><tr><td>Heat production</td><td>1402<sup>a</sup></td><td>1346<sup>b</sup></td><td>862</td><td>861</td><td>Fasting heat</td></tr><tr><td>production</td><td>735</td><td>731</td><td>446</td><td>456</td><td>Heat incre-</td></tr><tr><td>ment</td><td>667<sup>a</sup></td><td>614<sup>b</sup></td><td>417</td><td>404</td><td></td></tr><tr><td>NE/ME (x100)</td><td>73.9<sup>a</sup></td><td>75.9<sup>b</sup></td><td>74.8</td><td>75.0</td><td></td></tr></tbody></table>

¹ The reduction of dietary CP consists in a replacement of protein from soybean protein concentrate by maize starch with supplementation of amino acids in order to meet the requirements.

² Energy balance data should be preferably expressed per kg BW<sup>0.70</sup> in broilers (Noblet et al., 2015)

The most surprising result of these trials is the absence of effect of dietary CP on HP and NE/ME in broilers; a parallel study conducted in both pigs and broilers confirmed this major difference between pigs and poultry (Noblet et al., 2003; Table 7). Table 6 also indicates that the replacement of starch by fat increased numerically the NE/ME ratio as it can be expected from similar trials in pigs (van Milgen et al., 2001) but this variation was not significant. Finally, the presence of high levels of undigested DF in broilers diets did not significantly change the HP and the NE/ME ratio of the diet. This latter result does not confirm the hypotheses of Emmans (1994) who suggested attributing an energy cost to undigested organic matter for its excretion in order to calculate the so-called "effective" energy. Overall and unlike our previous results on pigs or the preliminary results of Cerrate et al. (2013) in broilers, our studies indicate that major changes in diet composition do not affect markedly the efficiency of ME for NE in broilers. This result also means that the hierarchy between diets or ingredients for poultry would not be markedly affected by the energy system (ME vs. NE). Overall, additional studies conducted with adequately designed diets and discriminating methodologies and statistics are urgently required to solve these discrepancies between studies and to answer the question: is a NE system justified for poultry?

The NE equations from digestible nutrients contents proposed by Carré et al. (2014) represent a new and reliable basis for implementing a NE system in broilers feeds. Their evaluation on other NE data, either complete feeds or ingredients, is required. In a validation exercise conducted by Pirgozliev and Rose (1999), the NE value of a series of feedstuffs as measured by Fraps (1946) was compared to the values calculated according to different NE prediction equations. None of the literature NE systems for poultry was able to accurately predict the NE values measured on the feedstuffs; the biggest discrepancy was observed with the system proposed by Emmans (1994). This exercise might be repeated with the equations proposed by Carré et al. (2014). One may also question the values proposed by Fraps (1946)! Finally, to our knowledge, there is no clear and convincing study conducted on broilers for illustrating the relationship between their performance and the energy intake, either as ME or NE. Again, the preliminary results of Cerrate et al. (2013) suggest that BW gain is better predicted by NE intake than by ME intake; they deserve a confirmation.

## Conclusions and perspectives

This review indicates that NE is a better predictor than DE or ME of the "true" energy value of poultry or pig feeds. In pigs, the interest of formulating on a NE basis is obvious and NE systems should be implemented for getting a reliable prediction of performance of animals, especially when quite numerous and variable ingredients are available. The system proposed by Noblet et al. (1994a) and implemented in feeding tables by Sauvant et al. (2004) or in the EvaPig software (www evapig com) has been widely used internationally. In the case of poultry, literature is less clear
with no convincing advantage of a NE system over a ME system for predicting the performance of broilers; further in-
vestigations are necessary to evaluate the potential interest of a NE system for poultry. For achieving that, most atten-
tion should be focused on the impact of dietary fat and protein levels with potential consequences on the relative energy
value of fat- or protein-rich ingredients and results in least-cost formulation. Finally, even though NE is the final objec-
tive in energy evaluation of feeds, most attention should be paid to the accurate estimation of DE or ME values, which
are the most important factors of variation of the energy value of poultry or pig feeds. The lack of comprehensive infor-
mation on the effects of technology (e.g., pelleting, extrusion, enzymes addition) or about the differences in digestion
between poultry species or physiological stages (growing vs. adult, etc.) is a major limiting factor for getting accurate
estimates of energy values for pigs or poultry, irrespective of the energy system used. Improvements in energy evalua-
tion will also come from proposals for rapid and non-invasive prediction methods such as in vitro or NIR methods (Bas-
tianelli et al., 2015). Finally, even it has not been considered in detail in this review, the change from DE or ME systems
to a NE system is usually associated with a shift in diet composition with lower crude protein contents and slightly
higher fat levels and therefore different profiles of ingredients used in the diets.

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# EVALUATING THE ENERGY CONTENT OF INGREDIENTS IN POULTRY DIETS – AN UPDATE

G. G. Mateos, L. Cámara, B. Saldaña, P. Guzmán, and R. Lázaro

Departamento de Producción Agraria, Universidad Politécnica de Madrid, 28040 Madrid, Spain;
gonzalo.gmateos@upm.es

## Summary

The methodology currently in use to evaluate the energy content of ingredients, under practical situations, needs improvements to increase the accuracy of the estimation, reduce safety margins, and improve feed cost. Different methods can be used to estimate the energy values of the ingredients and diets. The method most widely used worldwide to estimate the energy content of the ingredients is the use of tabulated values but most companies involved in poultry production are moving towards the use of predictive regression equations based on chemical analyses and in vitro and in vivo data. More research to improve the accuracy of systems in use for the estimation of the energy content of poultry feeds is needed. Also, we need a better knowledge of the many factors that affect energy values. The final objective is to implement an easy method to predict more accurately the energy content of the feeds using on line NIR technology.

## Introduction

Feed energy is one of the major cost in the production of eggs and poultry meat. Accurate evaluation of the energy content of the ingredients is fundamental to reduce feed cost. Apparent metabolizable energy corrected for nitrogen retention (AMEn) is widely used for feed evaluation in most countries (HILL AND ANDERSON, 1958; VOHRA, 1972; CHOCT, 2012) but the system might not work properly under all circumstances (VAN DER KLIS and FLEDERUS, 2007). As a consequence, many scientists advocate for the use of net energy systems to better estimate the energy content of the ingredients (DE GROOTE, 1974; NOBLET et al., 2010; CHOCT, 2012). In practice, the 3 main approaches used to evaluate the energy content of ingredients in poultry diets are based on values obtained from a) tables, b) predictive equations (in vitro studies, wet chemistry, or NIR technology), and c) in vivo experiments (research farms). Each of them has advantages and disadvantages and at present time, it is not easy to make a fair recommendation on which one is best. In practice, many feed mills use table values to estimate the energy value of the ingredients. However, most of the nutritionists and feed mill managers from key European companies and broiler integrators are moving to evaluate ingredients energy by using predictive regression equations. Finally, the in vivo trials are time consuming and expensive, and not always the data are consistent and accurate overtime, especially in the case of soybean meal, wheat, and lipid sources (BOURDILLON et al., 1990; YEGANI and KORVER, 2012; FRIKHA et al., 2012; RAVINDRAN et al., 2014). Consequently, the use of in vivo trials is not common in commercial operations. In any case, none of these systems is free of problems. Many factors, including feed technology (i.e., particle size, heat processing, and feed form) (MCKINNEY and TEETER, 2004; SKINNER-NOBLE et al., 2005), diet composition including fat inclusion (MATEOS and SELL, 1980; MATEOS et al., 1982), type of cereal (YEGANI and KORVER, 2012), use of whole wheat (RAVINDRAN et al., 2006), and additives (ANNISON, 1991; MATEOS et al., 2002) modify in different ways, the energy content of the ingredients in practical diets. In the current presentation we will focus on practical problems encountered by the industry when using Institutional tables, predictive regression equations, and in vivo values for key ingredients, namely cereals, soybean meal, and fats.

## Table values

In spite of the wide criticism, the use of table values is the main used worldwide to evaluate the energy content of ingredients and feeds. Table values provided by recognized, institutional sources, are of value. In fact, the approach might be the most adequate for ingredients with limited information published and not many own lab analyses available. However, the wide range in energy values proposed by the different Institutions for a given ingredient is of concern. Examples for SBM, corn, rapeseed meal, and DDGS are shown in Tables 1 to 4, respectively. In some cases, the variability in energy values can be justified by differences in the CP and antinutritional factors (ANF) content (i.e., rapeseed meal tables), the moisture of the grain (i.e., corn), or the nature of the heating process used (e.g., soybean meal). However, the wide range of values (e.g., 230 kcal AME/kg for SBM and 850 kcal AMEn/kg for corn DDGS) detected in some other cases deserves a thorough revision.
<table><caption>**Table 1. AMEn content of soybean meal**</caption><thead><tr><th>Institution</th><th>Country</th><th>Year</th><th>CP (%)</th><th>AMEn (kcal/kg)</th></tr></thead><tbody><tr><td>NRC</td><td>USA</td><td>1994</td><td>47.2</td><td>2,380</td></tr><tr><td>INRA</td><td>France<sup>2</sup></td><td>2002</td><td>47.2</td><td>2,340</td></tr><tr><td>NARO</td><td>Japan</td><td>2009</td><td>47.1</td><td>2,450</td></tr><tr><td>FEDNA</td><td>Spain</td><td>2010</td><td>47.5</td><td>2,380</td></tr><tr><td>CVB</td><td>Neth.<sup>1</sup></td><td>2011</td><td>47.5</td><td>2,220</td></tr><tr><td>ROSTAGNO</td><td>Brazil<sup>2</sup></td><td>2011</td><td>47.0</td><td>2,320</td></tr><tr><td>MPA<sup>3</sup></td><td>Russia</td><td>2014</td><td>47.0</td><td>2,300</td></tr></tbody></table>

<table><caption>**Table 2. AMEn content of corn for poultry**</caption><thead><tr><th>Institution</th><th>Country</th><th>Year</th><th>Moisture (%)</th><th>CP (%)</th><th>EE (%)</th><th>AMEn (kcal/kg)</th></tr></thead><tbody><tr><td>NRC</td><td>USA</td><td>1994</td><td>11.0</td><td>8.5</td><td>3.8</td><td>3,350</td></tr><tr><td>INRA</td><td>France<sup>2</sup></td><td>2002</td><td>13.6</td><td>8.1</td><td>3.7</td><td>3,200</td></tr><tr><td>FEDNA</td><td>Spain</td><td>2010</td><td>13.8</td><td>7.7</td><td>3.6</td><td>3,280</td></tr><tr><td>Rostagno</td><td>Brazil</td><td>2011</td><td>12.5</td><td>7.9</td><td>3.7</td><td>3,380</td></tr><tr><td>CVB</td><td>Netherlands</td><td>2011</td><td>12.8</td><td>8.2</td><td>3.8</td><td>3,294</td></tr></tbody></table>

<table><caption>**Table 3. AMEn content of rapeseed meal expeller in poultry**</caption><thead><tr><th>Institution</th><th>Country</th><th>Year</th><th>CP (%)</th><th>AMEn (kcal/kg)</th></tr></thead><tbody><tr><td>NRC</td><td>USA</td><td>1994</td><td>38.0</td><td>2,000</td></tr><tr><td>INRA</td><td>France<sup>2</sup></td><td>2002</td><td>33.7</td><td>1,440<sup>2</sup></td></tr><tr><td>Premier</td><td>UK</td><td>2008</td><td>33.9</td><td>1,670</td></tr><tr><td>NARO</td><td>Japan</td><td>2009</td><td>37.3</td><td>1,740</td></tr><tr><td>FEDNA</td><td>Spain</td><td>2010</td><td>33.8</td><td>1,700</td></tr><tr><td>CVB</td><td>Neth.</td><td>2011</td><td>33.5</td><td>1,580<sup>2</sup></td></tr><tr><td>MPA<sup>3</sup></td><td>Russia</td><td>2014</td><td>35.5</td><td>1,800</td></tr></tbody></table>

<table><caption>**Table 4. AMEn content of corn DDGS in poultry**</caption><thead><tr><th>Institution</th><th>Country</th><th>Year</th><th>CP (%)</th><th>EE (%)</th><th>AMEn (kcal/kg)</th></tr></thead><tbody><tr><td>WPSA</td><td>Europe</td><td>1986</td><td>25.0</td><td>6.5</td><td>2,380</td></tr><tr><td>NRC</td><td>USA</td><td>1994</td><td>28.5</td><td>9.0</td><td>2,930</td></tr><tr><td>INRA</td><td>France<sup>2</sup></td><td>2002</td><td>24.6</td><td>5.1</td><td>2,190</td></tr><tr><td>NARO</td><td>Japan</td><td>2009</td><td>30.8</td><td>11.0</td><td>2,170</td></tr><tr><td>FEDNA</td><td>Spain</td><td>2010</td><td>26.6</td><td>10.1</td><td>2,350</td></tr><tr><td>CVB</td><td>Neth.</td><td>2011</td><td>27.0</td><td>10.0</td><td>2,070</td></tr></tbody></table>

**Use of predictive regression equations**

Regression equations are becoming popular for the evaluation of the energy content of feedstuffs and used consistently by major companies involved in animal feeding. The implementation of predictive equations reduces the workload for diet formulation under all circumstances, but it is of primary interest in these companies or integrators with different feed units and high number of recipes. Regression equations are easy to implement, allowing for the updating of feed ma-

<footnote n="1">¹Estimated from offered values</footnote>

<footnote n="2">²Average of broilers and laying hens</footnote>

<footnote n="3">³Russian feed tables</footnote>

<footnote n="1">¹HCl hydrolysis</footnote>

<footnote n="1">¹2.5 to 5.0% ether extract</footnote>

<footnote n="2">²Average</footnote>

<footnote n="3">³Russian feed tables</footnote>

<footnote n="1">¹HCl hydrolysis</footnote>
trixes of the ingredients in use and facilitating the use of lab data into the feed formulation process. However, under practical conditions and certain circumstances, some problems might arise.

1) The regression equations were obtained using samples belonging to a different population or it is of "unknown" origin.

2) The summation of lab analyses of the major dietary components of the ingredient (moisture + ash + CP + EE + NDF + sugar + starch + soluble fiber) do not add to 100%.

3) The equations used include chemical values but potential differences because of effects of processing on nutrient digestibility (i.e., heat processing of SBM or DDGS) or method of analyses applied (i.e., polarimetry rather than enzymatic process for starch and HCl hydrolysis for fat) have not been taken into consideration.

4) Predictive equations in use had a small r and a high RSD values. This situation is frequent when the batches of the ingredient used have a narrow range of values within samples (i.e., trypsin inhibitor content of commercial SBM samples).

The use of predictive equations obtained with a set of samples of a different population is quite common, and will result in inaccurate estimation of the energy value. For example, in many instances the same equation is used for grain by-products that have been obtained by different processes (i.e., temperature and heating conditions). A situation that deserves special attention is the estimation of the energy value of SBM to be received in coming months from the values of samples collected in the previous month. The information needed is not the value of the SBM of the last vessel but to know in advance, the characteristics of the meal at the arrival to the port of the new vessel.

A problem often found, especially in small, local feed mills, relates to the use of chemical lab analyses in which the sum of all proximal analyses vary widely from 100% to predict the energy value of a novel or local ingredient. Under circumstances of poor lab analyses (below or higher, depending on potential errors), the misuse of the ingredient in diets is warranted, with important economic and/or production losses.

## Evaluation of the energy content of cereals

Cereals are the main source of energy in commercial poultry diets worldwide. Consequently, the accurate determination of its energy content is of paramount interest. However, the discrepancies among authors, when estimating the energy content of cereals, such as corn and wheat, by using tabulated values, predictive regression equations, or in vivo data (FARRELL, 1999; YESANI and KORVER, 2012) are wide. For example, moisture content is probably the main constituent affecting the AMEn content of corn (See NRC, 1994 value in Table 2). However, not in all labs, the moisture content of the cereals is analyzed in a correct form. Moreover, not always moisture content is taken into consideration when estimating the energy content of the corn under practical conditions. For wheat, energy data is even more variable, with samples analyzed in the same lab varying often between 50 and 70% (ABDOLLAHI et al., 2011; YEGANI and KORVER, 2012). Factors such as variety (hard vs. soft), length of storage (new crop vs. old crop), ANF content (xylanase level and type), diet composition (level and type of fat), and age of the birds, might impact the energy content of the cereal. Consequently, the practical interest of determining the energy content of the cereals in vivo, is not always justified.

## Evaluation of the energy content of soybean meals

A good example of the advantages and disadvantages of the use of predictive regression equations to evaluate the AMEn content of protein sources is that of the European table of energy values for poultry feedstuffs (WPSA, 1986) and its recommendation for SBM. This equation (AMEn (kcal/kg) = 37.5 x CP + 46.4 x EE + 14.9 x NFE) is widely used and recognized as a good tool to evaluate the energy content of the meals in poultry. However, this equation, created 25 years ago, might not be as precise as needed and thus, the interest of its use might be limited in commercial current situation. For example, the same equation is recommended for all SBM, independent of the origin of the beans. However, several reports (MATEOS et al., 2011; FRIKHA et al., 2012; RAVINDRAN et al., 2014) clearly indicate that the chemical composition, and therefore the energy content, varies with the country of origin of the meal, an effect probably related with day length (latitude), soil characteristics, and growing and storage conditions of the beans. In this respect, MATEOS et al. (2011) and FRIKHA et al. (2012) have reported that for meals with similar CP content, those of USA origin had more sucrose and oligosaccharides and less NDF than those from Brazil origin. Moreover, process conditions applied for the crushing of the beans will also affect the AMEn content of the meal. Under-heating of the beans will reduce the energy content of the corresponding meal, because of the presence of high amounts of TI in the meal,
whereas over-heating will reduce this level of TI but at the expense of a higher incidence of Maillard reactions. However, the European equation equalizes the energy content of SBM from different crushing plants, independent of the heating conditions applied during the processing of the beans and the final quality of the protein fraction.

A second problem related to the use of the WPSA (1986) equation for SBM, relates to the ether extract part of the equation. Energetically, EE is an important constituent of the bean and has to be taken into account when evaluating the energy content of a meal. However, the equation does not take into consideration several points of interest. For example, the real fat content of the EE fraction varies depending on the amount of lipids left in the meal. Also, different lipid fractions (e.g., acid soapstocks, lecithins, gums) might be used to increase the fat content of the SBM. Finally, the use or not of previous HCl hydrolysis by the lab, will give different EE content of the meal (up to 1% higher). A last concern with the use of the WPSA (1986) equation is the use of nitrogen free extract (NFE) as a part of the equation. This fraction does not mean much from a nutritional point of view. It obtained by difference between 100 and the proximal analyses contents. Therefore, it includes 2 sources of variation: a) no distinction among components, giving the same energy value to lignin, pectins, or other more digestible components and b) all mistakes that might occur during the calculation process, including errors in lab analyses will have a positive (or negative) effect on energy evaluation. Thus, NFE should not be included in predictive regression equations to estimate the energy value of any ingredient. Intuitively, a good equation to determine the AMEn of a given batch of SBM should include the amount of digestible protein (not good lab methods available yet) and the amount of sucrose of the sample. Also of interest, could be the inclusion in this equation of real fat content (quite similar for all solvent meals marketed in the EU-28 but of interest for expeller meals or solvent meals that add other lipid fractions) and the content in oligosaccharides (stachyose and raffinose). Oligosaccharides (around 7% of the meal on DM bases) are not digested by the bird but will be fermented at some extent on the cecum, being a good source of energy, especially in laying hens.

## Evaluation of the energy content of lipid sources

The use of lipid sources have increased steadily in poultry diets in the last decade. The main factors affecting the energy content of oils and fats are the chemical quality (including among other variables, the gross energy, moisture, impurity, and unsaponifiable content, and the peroxide value) and the characteristics of the molecule (namely, proportion of free fatty acids (FA), degree of unsaturation, and length of the carbon chain). Most of the data available on the energy content of fats and oils were obtained more than 30 years ago and consequently, the practical application of these values to the new standards of feed formulation and sources of fats (i.e., lecithins and blended fats) might not apply in some occasions.

Recent research conducted in our lab (MANDALAWI et al., 2015, unpublished data) has shown that the inclusion of lecithins in laying hen diets improved the AMEn content of the feed more than expected. In fact, the substitution of animal fat by soy lecithins, increased egg weight and improved feed efficiency (Figure 1). Probably, the lecithin, a lipid source rich in phospholipids and unsaturated FA, improved bile production and micelle formation, facilitating the incorporation of the FA into the yolk.

Fats are the most difficult ingredient to evaluate in poultry diets, the main reason being problems in the vivo determination of their energy content. Fats are incorporated into the experimental diets at low levels (usually less than 6-8%) and consequently, the experimental errors magnifies differences in energy values among lipid sources. Moreover, in many cases the control diet is based on corn that contains appreciable amounts of intracellular oil which might have different digestibility (usually lower) to that of the fat source tested (IRANDOUST et al., 2012). Consequently, the experimental error is large in fat digestibility studies, which in turn might explain, at least in part, the wide difference in values reported by different authors for a given source of fat and AMEn values of a fat beyond its GE content. In addition, it has been shown that the inclusion of fat in the diet might improve the digestibility and utilization of other components of the diet (MATEOS et al., 1980), which adds new confusion to energy determination. Consequently, new approaches are needed to better estimate the energy content of fat sources in poultry diets.
<figure><img src="image_10.png" /><figcaption>**Figure 1.** Effect of inclusion of soy lecithin in the diet on egg weight (a), feed conversion ratio (b), and egg mass (c) from 23 to 51 wk of age.</figcaption></figure>

## Interactions among dietary components

One of the main assumptions of the AMEn system is the additivity of the energy values of the ingredients within a given formula. However, this assumption might not be correct. For example, the inclusion of fat (level and type) and fiber, the presence of contaminants and ANF, and the use of additives, including enzymes (phytases, carbohydrates, and proteases), organic acids, and others might modify the energy value of ingredients and diets

Fat supplementation reduces the rate of passage of the digesta through the GIT, which in turn might favor the utilization of the components of the diets, including the lipid, carbohydrate, and protein fractions (MATEOS et al., 1980). For example, the inclusion of unsaturated fats might improve micelle formation and the utilization of the saturated fat present in the constituents of the diet, contributing to the “so called” extra caloric effects of the fat.

Dietary fiber has been considered as an ANF factor in diet for poultry, because of its negative effects on palatability and nutrient digestibility. However, recent research (HETLAND et al., 2003, 2005; GONZÁLEZ-ALVARADO et al., 2007; JIMÉNEZ-MORENO et al., 2009; MATEOS et al., 2012) has shown that this might not be the case, and that under certain circumstances, the inclusion of small amounts (2-3%) of insoluble fiber sources in diets low in fiber, might improve nutrient digestibility and growth in broilers and young pullets.

The presence of ANF, contaminants (i.e., mycotoxins), or toxic components (i.e., heavy metals) in an ingredient affects nutrient digestibility and therefore, its energy content. The presence of ANF in the diet affects not only the energy of the ingredient “per se” but also that of the remaining components of the diet, because of its negative effects on the digestive mucosa and GIT function.
Additives are widely used in poultry feeding, especially in diets for young chicks (MATEOS et al., 2002). In many instances, the companies responsible for the research on the potential benefits of these products create matrixes for the additive that include “energy equivalent values”. These matrixes facilitate the work of the nutritionist, but are not free of problems. When several additives, each of them with its own matrix are incorporated into the diet, the “matrix approach” is probably not correct, because the potential benefits of the additives (i.e., exogenous enzymes, probiotics, prebiotics, essential oils, organic acids, etc) on energy are not additive.

## Heat processing, mean particle size, and feed form

Grinding and heat processing of the cereals results generally in an improvement in the energy value of ingredients in young chicks but the effects tended to disappear with age (GRACIA et al, 2003; FRIKHA et al, 2013). Pelleting affects the AMEn of the diet, probably by modifying the structure of the fiber fraction (i.e., increasing the solubilization), releasing the lipids inside the cells (i.e., toasted soybeans and corn), and improving carbohydrate digestibility (i.e., pea starch) (ABDOLLAHI et al., 2012; SERRANO et al., 2013). Moreover, feed form of the diet and particle size of the ingredients, affect GIT development and function, mainly that of the gizzard. Also, diet characteristics modifies the rate of passage of the digesta through the GIT, which in turn might alter microbiota profile and the energy content of the feed (AMERAH et al., 2007; SVIHUS, 2011; MATEOS et al., 2012). However, the effects of feed form and particle size are not uniform. In fact, the benefits of fine grinding are less evident (and even negative) with the use of mash diets (ABDOLLAHI et al, 2011; SERRANO et al, 2013). Also, pelleting improves feed intake and broiler growth, but not always nutrient digestibility or AMEn content of the diet (ZELENKA, 2003). Recent research has shown that often the beneficial effects of pelleting reflects a lower feed wastage and feed intake and not necessarily an increase in the AMEn content of the ingredients (ABDOLLAHI et al, 2001; SERRANO et al, 2013). As a result, the AMEn of the diet might not be uniform and will depend not only on the ingredient composition and the physical and chemical characteristics of the diet but also on the health status of the bird. All these components are difficult to evaluate and add uncertainties to the real energy contribution of ingredients in poultry diets.

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ABDOLLAHI, M. R., V. RAVINDRAN, T. J. WESTER, G. REVINDRAN, D. V. THOMAS. 2011. Influence of feed form and conditioning temperature on performance, apparent metabolisable energy and ileal digestibility of starch and nitrogen in broilers starters fed wheat-based diets. Anim. Feed. Sci. Technol. 168: 88-99.

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CHOCT, M. 2012. Feed energy- what system to use and prospects for evaluation. XXIV World's Poult. Congress. Salvador de Bahia, Brazil. 8 pp.

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DE GROOTE, G. 1974. A comparison of a new net energy system with the metabolizable energy system in broiler diet formulation, performance and profitability. Br. Poult. Sci. 15: 75-95.

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# IMPACT OF FEED MYCOTOXINS ON PERFORMANCE AND HEALTH OF POULTRY

## Sven Dänicke

Institute of Animal Nutrition, Friedrich-Loeffler-Institute (FLI), Federal Research Institute for Animal Health, Bundesallee 50, D-38116 Braunschweig, Fax: + 49 531-580-44199, E-mail: sven.daenicke@fli.bund.de

## Introduction

Molds belong to the natural microflora which commonly infests feedstuffs. Under certain gross-environmental conditions, such as temperature, moisture content, air humidity and water availability, oxygen availability and micro-environmental conditions such as microbes competing for ecological niches the molds are stimulated to form secondary metabolites. If these metabolites exert toxic effects against the living environment, they are collectively termed as mycotoxins. Due to the ubiquitous occurrence of molds it is not surprising that mycotoxins belong to natural contaminants often detected in feedstuffs.

Owing to different chemical structures of mycotoxins, their toxicodynamics also varies considerably (Table 1). Mycotoxins might exert toxic effects in farm animals when feedstuffs are contaminated at levels that exceed the tolerance level by the animals. This tolerance is species specific, but is not unique among the mycotoxins. In addition, the clinical manifestation of a particular mycotoxin intoxication might also differ between species (e.g., fumonisins). By comparing the species-specific sensitivity together with the relevance for practical feeding conditions it becomes obvious that the pig is on special risk in spite of different chemical natures and toxicodynamics of different mycotoxins while poultry appears to respond less sensitive (Table 1).

<table><caption>**Table 1.** Overview on selected mycotoxins, their mechanisms and toxic effects and their relevance for practical feeding (literature compilation)</caption><thead><tr><th>Mycotoxins</th><th>Mechanisms and toxic effects</th><th>Predisposed feedstuffs</th><th>Affected livestock and relative sensitivity<fnref n="1" /></th><th>Relevance<fnref n="2" /></th></tr></thead><tbody><tr><td>Zearalenone (ZEN)</td><td>Estrogen-like, endocrine disruptor / reproductive disorders</td><td>Maize, wheat</td><td>Pig > cattle > poultry</td><td>++</td></tr><tr><td>T-2 Toxin</td><td>Inhibition of protein synthesis, immune-modulating / anorectic, cytotoxic, dermatotoxic, hepatotoxic</td><td>Oats, barley, maize, wheat</td><td>Pig > cattle ~ poultry</td><td>+</td></tr><tr><td>Deoxynivalenol (DON)</td><td>Inhibition of protein synthesis, ribotoxic / immune-modulating, anorectic</td><td>Maize, wheat</td><td>Pig > cattle ~ poultry</td><td>++</td></tr><tr><td>Fumonisins (FUM)</td><td>Disruption of sphingolipid metabolism / carcinogenic, neural tube defects, hepatotoxic, pneumotoxic (Porcine pulmonary edema), neurotoxic (Equine leukoencephalomalacia)</td><td>Maize</td><td>Horse ~ pig > cattle ~ poultry</td><td>+</td></tr><tr><td>Aflatoxin B1 (AFB1)</td><td>DNA- and protein adduct formation / carcinogenic cytotoxic, hepatotoxic</td><td>Pea nuts, cotton seed, maize, sunflower seed, soy beans</td><td>Cattle, pig, poultry</td><td>++</td></tr><tr><td>Ergot alkaloids (EA)</td><td>Vessel-active, pharmacological effects, performance depressive</td><td>Rye, triticale, wheat</td><td>Poultry, Cattle, Pig</td><td>+</td></tr></tbody></table>

## Current risk management

The practical relevance of a particular mycotoxin for a specific species results from its sensitivity to this toxin and from the occurrence of the toxin on feedstuffs commonly used for feeding of that species (Table 1). For risk management is it consequently necessary to consider both aspects, i.e., the sensitivity of the animal species and the most pre-disposed feedstuffs (Table 1).

<footnote n="1">¹Farm animals which are not considered are less sensitive or there is insufficient information on toxic effects</footnote>

<footnote n="2">²Relevance for practical feeding (occurrence of the toxins in relation to sensitivity of livestock): "++" highly important, "+" important, "-" less important, "?" relevance unclear.</footnote>
<table><caption>**Table 2.** European Commission recommendation of 17 August 2006 on the presence of mycotoxins in products intended for animal feeding</caption><thead><tr><th>Mycotoxin</th><th>Products intended for animal feed</th><th>Guidance value in mg/kg (ppm) relative to a feedstuff with a moisture content of 12 %</th></tr></thead><tbody><tr><td rowspan="5">Deoxynivalenol</td><td><strong>Feed materials (*)</strong></td><td></td></tr><tr><td>- Cereals and cereal products (***) with the exception of maize by-products</td><td>8</td></tr><tr><td>- Maize by-products</td><td>12</td></tr><tr><td><strong>Complementary and complete feedstuffs with the exception of:</strong></td><td>5</td></tr><tr><td>- Complementary and complete feedstuffs for pigs</td><td>0.9</td></tr><tr><td>Zearalenone</td><td>- Complementary and complete feedstuffs for calves (&lt; 4 months), lambs and kids</td><td>2</td></tr><tr><td rowspan="5">Ochratoxin A</td><td><strong>Feed materials (*)</strong></td><td></td></tr><tr><td>- Cereals and cereal products (***) with the exception of maize by-products</td><td>2</td></tr><tr><td>- Maize by-products</td><td>3</td></tr><tr><td><strong>Complementary and complete feedstuffs</strong></td><td></td></tr><tr><td>- Complementary and complete feedstuffs for piglets and gilts (young sows)</td><td>0.1</td></tr><tr><td rowspan="3">Fumonisin B1 + B2</td><td>- Complementary and complete feedstuffs for sows and fattening pigs</td><td>0.25</td></tr><tr><td>- Complementary and complete feedstuffs for calves, dairy cattle, sheep (including lamb) and goats (including kids)</td><td>0.5</td></tr><tr><td><strong>Feed materials (*)</strong></td><td></td></tr><tr><td></td><td>- Cereals and cereal products (***)</td><td>0.25</td></tr><tr><td></td><td><strong>Complementary and complete feedstuffs</strong></td><td></td></tr><tr><td></td><td>- Complementary and complete feedstuffs for pigs</td><td>0.05</td></tr><tr><td></td><td>- Complementary and complete feedstuffs for poultry</td><td>0.1</td></tr><tr><td></td><td><strong>Feed materials (*)</strong></td><td></td></tr><tr><td></td><td>- Maize and maize products (***)</td><td>60</td></tr><tr><td></td><td><strong>Complementary and complete feedstuffs for:</strong></td><td></td></tr><tr><td></td><td>- Pigs, horses (Equidae), rabbits and pet animals</td><td>5</td></tr><tr><td></td><td>- Fish</td><td>10</td></tr><tr><td></td><td>- Poultry, calves (&lt; 4 months), lambs and kids</td><td>20</td></tr><tr><td></td><td>- Adult ruminants (&gt; 4 months) and mink</td><td>50</td></tr></tbody></table>

<footnote>(*) Particular attention has to be paid to cereals and cereals products fed directly to the animals such that their use in a daily ration should not lead to the animal being exposed to a higher level of these mycotoxins than the corresponding levels of exposure where only the complete feedstuffs are used in a daily ration.</footnote>

<footnote>(**) The term 'Cereals and cereal products' includes not only the feed materials listed under heading 1 'Cereal grains, their products and by-products' of the non-exclusive list of main feed materials referred to in part B of the Annex to Council Directive 96/25/EC of 29 April 1996 on the circulation and use of feed materials (OJ L 125, 23.5.1996, p. 35) but also other feed materials derived from cereals in particular cereal forages and roughages.</footnote>

<footnote>(***) The term 'Maize and maize products' includes not only the feed materials derived from maize listed under heading 1 'Cereal grains, their products and by-products' of the non-exclusive list of main feed materials referred to in the Annex, part B of Directive 96/25/EC but also other feed materials derived from maize in particular maize forages and roughages.</footnote>
The European Commission responded to the risk characterization and evaluation of the presence of DON, ZON, OTA and FUM in feedstuffs by the European Food Safety Authority (EFSA 2004b, c, a, 2005b) with the release of a recommendation for critical orientation (guidance) values for these toxins in feedstuffs (see Table 2) and for monitoring of the simultaneous occurrence of these mycotoxins (CEC 2006).

A distinction must be made between guidance values for individual feedstuffs (feed materials) and for the total diet (complete feedstuffs) or parts of it (complementary feeding stuffs) (Table 2). From a toxicological viewpoint the guidance values for complete feedstuffs needs to be considered as total dietary toxin concentrations, together with the level of feed intake, determine the toxin exposure of the animal.

While the guidance values for critical feedstuff concentrations of DON, ZEN, OTA and FUM were derived based on animal health aspects, the upper limits for AFB1 consider mainly the high carry over into milk, but also animal health effects in young animals including poultry (Table 3).

Up to now, the risk management for ergot remains an unsolved issue. Toxicologically, ergot alkaloids (EA) potentially interact with serotoninergic, dopaminergic and adrenergic receptors depending on their specific chemical structure (EFSA 2005a). Moreover, the combined overall toxic effects of differentially acting individual alkaloids further depend on their absolute concentrations and proportions to each other (EFSA 2005a). In addition, the EA content of sclerotia from C. purpurea varies largely between nearly zero to approximately 10400 mg/kg (=1.04%) as reviewed recently depending on geographic region and harvesting year, cereal species, variety and genotype (EFSA 2012, MIEDANER et al. 2010, MAINKA et al. 2007). Especially this tremendous variation generally questions the reliability of the current regulation regarding the upper limit of 1000 mg ergot (C. purpurea) per kg unground cereal grains (=0.1%, see Table 3) as specified by Directive 2002/32/EC for animal health protection. Dietary levels of 0.3% ergot caused increased mortality in broilers while in another experiment ergot levels of 0.7% were without adverse effects supporting the view that variation in EA content of ergot clearly determines its toxic effects (EFSA 2005a). Consequently, EFSA recommended replacing the physical method by chemical analysis.

<figure><img src="image_11.png" /><figcaption>**Figure 1.** Estimation of the ergot proportion in diets for ducks where the LOAEL of 0.6 mg TA/kg diet is reached in dependence on varying TA contents of ergot.</figcaption></figure>

Especially ducks appear to respond quite sensitive to the presence of EA in feed as the lowest dietary concentrations causing a significant decrease in feed intake and live weight gain (LOAEL) amounted to 0.6 mg/kg (DÄNICKE 2015). This LOAEL further suggests that ducks are not protected by the current EU legislation (see Figure 1).
<table><caption>**Table 3.** Upper limits for Aflatoxin B1 and for rye ergot (Directive 2002/32/EC of the European Parliament and of the Council of 7 May 2002 on undesirable substances in animal feed)</caption><thead><tr><th>Undesirable substance</th><th>Products intended for animal feed</th><th>Maximum content in mg/kg (ppm) relative to a feedstuff with a moisture content of 12 %</th></tr></thead><tbody><tr><td rowspan="4">Aflatoxin B1</td><td>All feed materials,</td><td>0.02</td></tr><tr><td>Complementary and complete feedstuffs excepting:</td><td>0.01</td></tr><tr><td>- Complete feedstuffs for dairy cows and calves, dairy sheep and lambs, dairy goats and kids, piglets and young poultry</td><td>0.005</td></tr><tr><td>- Complete feedstuffs for cattle (excepting dairy cows and calves), sheep (excepting dairy sheep and lambs), goats (excepting dairy goats and kids), pigs (excepting piglets), and poultry (excepting young poultry)</td><td>0.02</td></tr><tr><td>Rye ergot (Claviceps purpurea)</td><td>All feedstuffs containing unground cereals</td><td>1000</td></tr></tbody></table>

### Interpretation of guidance values for critical mycotoxin concentrations in feedstuffs

The compliance with the guiding values for complete feedstuffs ensures that no adverse effects occur in farm animal feeding under normal production conditions. Deviations from “normality” might include the presence of stress factors such as latent or manifest infections of poultry by pathogens (e.g. DÄNICKE et al. 2011, DÄNICKE et al. 2013), poor keeping conditions (too high stocking density, inadequate nutrition, too high concentration of corrosive gases and others) and can result in a decrease of mycotoxin concentrations in the diet to be considered as critical and thus lower than the guidance values.

It needs to be stressed that the current mycotoxin management is based on the free forms of the toxins in feed which are detectable by routine methods. However, in evaluating the overall toxicity of the free form of mycotoxins it needs to be further considered that mycotoxins often exist in feedstuffs in a modified form (RYCHLIK et al. 2014) which is usually not detectable by routine methods. Digestive processes in general, and species-specific differences in the site of microbial digestion in particular, also influence the sensitivity to mycotoxins.

Pre-ingestively modified forms of mycotoxins (e.g., matrix associated; thermally formed; conjugated by plants) might further be exposed to pre-absorptive (i.e., ingestive) structural alterations, as mediated by gastro-intestinal microbes (e.g., DON-3-O-glucoside → free DON, NAGL et al. 2012) or endogenous digestive enzymes (e.g., fumonisins bound to starch → free fumonisins, HUMPF and VOSS 2004), which can result in a reverted toxicity of the originally free mycotoxin (i.e., DON and fumonisins, respectively) or in a further modification giving rise to an altered toxicity (e.g., DON-3-O-glucoside → DON → de-epoxy-DON, NAGL et al. 2012).

Although de-epoxy-DON seems to be of a generally low toxicity for immune cells, the interspecies differences in pre-absorptive modification need to be considered in evaluating the overall toxicity of DON. While DON is nearly completely modified to de-epoxy-DON in cattle by rumen micro-organisms prior to systemic absorption, this modification occurs also in the hindgut of pigs, but only very small amounts of non-absorbed free DON reach this intestinal segment. Therefore, in DON exposed cattle mostly de-epoxy-DON is detectable in blood while in pigs the majority is in the form of free DON (e.g., DÄNICKE and BREZINA 2013). According to recent findings, modification of DON to de-epoxy-DON appears to play a minor role in the chicken while DON seems to be metabolized to sulfate conjugates to a large extent (WAN et al. 2014). Up to now, the sites of these modifications remain obscure but might be principally managed both at the inges-tive and metabolic level.
Conclusions

Compliance with the guiding values for critical mycotoxin concentrations for complete feedstuffs ensures that no adverse effects occur under normal production conditions. Deviations from these assumptions might result in adverse health effects at mycotoxin concentrations lower than the corresponding guidance values. Furthermore, compliance with the guidance values for a particular mycotoxin such as DON assumes that all other modified (non-free) forms of the same toxin as well as other mycotoxins which might be present in the contaminated feedstuff at the same time, but for which no management exists, are controlled at the same time. However, under certain contamination scenarios this assumption might not apply with the result that adverse effects on health are observed at indicator toxin concentrations (e.g. DON) lower than the guidance value for its critical (free) concentration.

These aspects and the occasional exceedance of critical concentrations of mycotoxins in feedstuffs lead to the conclusion that mycotoxins are relevant for health of poultry. Therefore, preventive measures should include both minimization strategies to decrease feedstuff contaminations according to the ALARA (as low as reasonably achievable) principle and all measures to optimize the housing conditions for poultry.

References

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DÄNICKE, S., 2015: Ergot alkaloids in feed for pekin ducks: toxic effects, metabolism and carry over into edible tissues. Toxins **In press**.

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# HOW TO FEED LAYERS UNTIL 100 WEEKS? – A PRACTICAL APPROACH

**Robert Pottgueter**

Lohmann Tierzucht GmbH – Cuxhaven, Germany; [pottgueter@ltz.de](mailto:pottgueter@ltz.de)

## Abstract

Due to ongoing genetic improvement in layer type breeders, farmers and nutritionists are facing an increasing persistency and lifetime production period of the hens. Optimal egg shell quality will be the one and only reason to sign off a layer flock with highly extended lifetime production period. Well working calcium metabolism and health status are of highest priority to ensure saleable egg shell quality under the pre-mentioned circumstances. Basic preconditions will be optimal pullet rearing, management during transfer from rearing to layer house and well-adopted nutrition at the start of the production period. The most important nutritional tool supporting calcium metabolism and egg shell quality is high quality limestone with fine and coarse structure – well balanced when the flocks are aging. As the liver functions as a very important organ in general and for egg production as well, liver health is an additional tool to boost even the egg shell quality, which is regularly proven in practise. Additionally the liver provides the protein in the egg shell, which is mainly responsible for a certain elasticity of the egg shell. Feeding and nutrition targeting gut health proof as further tools when it comes to supporting the egg shell quality. Following the varying demand for calcium over the day is an- other idea to support the egg shell quality, which has mainly been developed in practise and might end up in the mod- ern strategy of split feeding. Control of egg weight throughout extended production cycles of layer flocks is an overall nutritional approach as oversized eggs will always cause downgraded egg shell quality. Last but not least it needs to be mentioned that avoiding heat stress as well as possible will be a basic request to positively influence the egg shell qual- ity in extended lifetime production cycles of layer flocks.

**Keywords:** persistency, long-viability layers, egg shell quality, nutrition layers, calcium supply

## Introduction

Modern layer hybrids are a four-line-cross with a long breeding history and show a very high production potential in different housing systems all around the world. This proofs for white egg producing lines and for brown egg producing hens as well. Due to ongoing genetic work the egg industry and farmers realise steadily increasing persistency and longer liability under all housing conditions. The higher performance has been achieved by an increased efficiency too, which means that the daily feed intake has not been increased over the last ten years. In practice this can be shown by a steadily increasing feed conversion ratio. Based on parent stock performance data from the last four years it can al- ready be expected that the performance on commercial level will be further increased. The main increase in laying per- centage is based on longer clutch length and improved uniformity of layer flocks. The most popular aim of nowadays' layer geneticists is the 500 egg hen – in one cycle and without molting. Genomic selection will even speed up the proc- ess to achieve this aim. Under good conditions this aim has already been reached in quite a lot of flocks all around the world and sometimes farmers start kind of a competition how long they can extend the lifetime production period of layer flocks. By doing this, they realise that the egg shell quality at the end of the production period again deserves much more attention and support in comparison to a one year production cycle (72 weeks of life). Additionally the so called post-peak-dip shows up as an additional challenge at the beginning of the production period. Both topics – a secured start of production together with reaching peak performance and optimal egg shell quality up to 90 weeks (or more) of age of flocks – are highly related to optimal nutrition of nowadays' high prolific layer flocks.

## Materials and methods

### Importance of rearing and pre-lay phase

Optimal layer nutrition for a long liability and a high number of saleable eggs starts directly after hatch. This means to fulfil the nutritional demand for the enormous growth potential during the first half of the rearing period followed by the decreased growth potential and nutritional consequences in the second half of the rearing period. The feeding pro- gramme during the first half of the rearing period needs to focus on an optimal supply of digestible amino acids and even minerals to ensure the basic growth of the inner organs, muscles and even the skeleton in this early stage of life. The second half of the rearing phase is determined by a biologically reduced physiological development of the pullet, which offers the chance of training the pullets for an optimal feed intake capacity respectively feed intake behaviour. This aspect is of enormous importance at the following start of the egg production where all nowadays' layer breeds
tend to have a daily feed intake being way too low. During the second half of the rearing phase a reduced demand for protein and amino acids is offering the chance to include lower dense raw materials with higher content of crude fibre into the diet formulation. If suitable raw materials are available, it can be recommended to reach at least 5.5 % crude fibre in this feeding phase. At the end of the rearing period, just before the start of producing eggs, the bird moves from pullet phase to layer phase, which means that nearly all metabolic processes change to a high extend. Due to hormonal regulation the development of the medullary bones is initiated and all metabolic processes move to the egg production. Nevertheless at the start of the production layer flocks need to show further weight gain. The development of the medullary bones takes place roughly throughout ten days, needs additional calcium supply and can be called the pre-lay period. This physiologically based transition period should be used to carry out a smooth transition between two highly different types of feed – meaning developer feed and layer feed – and encourages layer nutritionists to use a pre-lay feed during this period. A pre-lay feed is mainly determined by a content of calcium of around 2 – 2.5 %; the other nutrients can/should be similar to a layer feed. The major challenge to implement the idea of a pre-lay feed is more a question of feed logistics and correct use on farm side in comparison to a basic nutritional question. It can be suggested to use pre-lay feed for approximately ten days with a maximum amount of maximal 1 kilo per pullet. The biggest mistakes happening regularly in practice on layer farms are too early use of pre-lay feed or too long time of using pre-lay feed. Both practices will always harm layer flocks and might destroy a good peak performance of layer flocks. The advantages of a professional use of pre-lay feed can be summarised as followed: smooth transition between too highly different feed types, adapting the birds to the high calcium-content of layer feed, maintaining daily feed intake during this period, offering protein and amino acids for those birds being behind the average body weight, increasing uniformity of pullet flocks, avoiding decalcification of hens already being ahead of the average flock and producing eggs. As these benefits show up regularly all around the world and under all production systems, it is highly recommended to implement the pre-lay idea into all nutritional programmes of layer flocks.

## Support of liver health

After an optimal start into the laying period the liver will be the most important organ of the bird. The topic of feeding on liver health is not only a veterinarian issue but an important nutritional topic as well. The liver provides nearly all basic nutrients for the development of the yolk and albumen and even supports the development of the egg shell. Additionally the liver provides the protein in the egg shell, which is mainly responsible for a certain elasticity of the egg shell. Feeding layer birds with a certain amount of added fat and oil respectively crude fat in the diets serves as a well-known tool to reduce the incidence of the so called fatty liver syndrome. Furthermore it should be a non questionable standard practice to have a certain amount of added choline chloride in layer diets to support the liver metabolism. Every so called fatty liver supplement will be based on choline chloride together with some important vitamins as there are: K3, E, B12, B1, folic acid. In addition sometimes Methionine and Betaine are used to relieve liver metabolism. A very important aspect in terms of liver health is a possible contamination of layer feeds with mycotoxines. You can never expect a "zero level" of mycotoxines; nevertheless it is well accepted that at a certain level of contamination the liver will be harmed by mycotoxines. It is considered that this level will be reached earlier when causing harm to the liver than veterinarians realise problems with liver health in post-mortem examinations. Recommendations about a more or less harmless contamination with mycotoxines for layer birds vary to a high extend; therefore everybody needs to make his own decision when to use a mycotoxine binder.

## Limestone – as a basic tool

Limestone of optimal quality is the most important and cheapest tool to support the egg shell quality in a high prolific layer bird. As a hen's demand for calcium in the daytime is highly varying, fine and coarse limestone with different solubility should be a standard practice. A further source of calcium for the egg shell development during the night is the complex of medullary bones - already mentioned before. They serve as a fast acting calcium store during the night when the hens normally have no feed intake. To rely on the medullary bones to a too high extend means a loss of phosphorus which needs to be refilled by nutrition. Because of this it should be a feeding strategy to optimise the calcium metabolism by an optimal limestone support from the gut and to reduce the necessity of calcium supply from the medullary bones for egg shell formation as best as possible. The average retention of calcium from limestone in layer breeds varies between 40 - 60% with regard to the amount of calcium being consumed. This means that on average 50% of the limestone being ingested will be excreted by the feces regularly. In addition fine limestone has a higher solubility compared to coarse limestone with an increased particle size. Due to this biologically based pre-condition it is highly recommended to use two sources of limestone in formulating layer diets at all times. Roughly 1/3 should be
added as fine limestone and 2/3 as coarse limestone. This basic idea has proven for decades when layer farmers have been adding oyster shells on top of compound feed in order to improve egg shell quality when the calcium metabolism of layers is aging. In alternative housing systems and non-cage systems practice has shown that the coarse particle size of limestone even has a positive impact on the birds' behaviour, which means that some flocks are more docile and less anxious. The same effect sometimes is being observed when flocks are offered grit which will be small insoluble stones and therefore might even support the gizzard activity as a biologically developed grinder.

## Correct calcium intake and timing

Nutritionists nowadays can easily calculate the daily demand for an optimal egg shell development based on standard scientific knowledge. In addition it is urgently needed to know the daily feed intake of an actual specific flock in order to ensure sufficient daily intake of calcium. When targeting on 4.1 gram of calcium intake per hen per day the amount of calcium in the diet needs to be adjusted up to 4.5% with 90 gram of daily feed intake or to 3.4% of calcium with 120 gram of feed per hen per day. As there is a lack of information quite often concerning daily feed intake, a lot of layer flocks are already harmed in an early stage of the laying period due to the situation described before. If layer birds might be observed on feed intake behaviour during the day, one will realise that they consume roughly 1/3 of the daily amount in the first half of the day and 2/3 in the second half of the day. This is particularly the case in order to fulfil the higher demand for calcium during the night when most of the egg shell formation takes place. Due to this situation a top dressing of compound layer feed at the end of the day respectively with the last feeding with 1 - 2% of coarse limestone is being realised on many layer farms. A further development of this idea is the so called split feeding with a morning feed and an afternoon feed, which differ highly in the content of calcium. From a nutritional point of view this idea works very well; the challenge of implementing it into practice is rather related to feed logistics in general and the feeding equipment in the layer house.

## Controlling egg size

In order to ensure an optimal egg shell quality in extended production cycles, the egg weight/ size needs to be considered. This is due to the aging calcium metabolism and the decreased percentage of the egg shell in relation to the total egg weight in larger eggs. A big data survey with Lohmann Brown in France from around 30 flocks of Lohmann Brown Classic has shown a variation of plus minus 2.5 gram of egg weight based on all flocks – with having the same genetic background. This proofs as a practical example of the importance of the overall management inclusive nutrition in guiding layer flocks in terms of an optimal egg weight. The basic idea of an optimal egg weight might be an egg weight of 60 gram immediately after the start of the production and 60 - 65 gram at the end of the production. As everybody might know that this will be a theoretical idea to some extend, management and nutrition can serve to reach such an aim. The most important nutritional factors to influence the egg weight are the content of Methionine and sulphur amino acids and crude protein in not well balanced amino acid profile. A sometimes neglected tool to influence the egg weight is added fat and oil respectively crude fat in layer diets and additionally on top of this the content of linoleic acid. The basic nutritional demand of layers for linoleic acid can easily be fulfilled with approximately 1% in practical layer diets. Nevertheless scientific data and field experience from a lot of countries have proven a positive effect on the egg weight if linoleic acid is adjusted to 2%, or even 3% in practical layer diets. Due to this basic knowledge it can be recommended to adjust an increased content of linoleic acid in diets at the beginning of the laying period and a decreased content of linoleic acid at the end of the laying period. It is a common practise to reduce the nutrient density of the feed throughout the laying period in order to control the egg size and to reduce feed costs. As layer birds adjust their feed intake mainly with regard to their energy demand, the reduction of the nutrient density and energy of the formula will be counteracted quite often by an increased feed intake and finally an increased intake of amino acids and crude protein. Therefore it can be recommended to maintain the level of energy throughout the laying period and to reduce amino acids and crude protein in order to achieve kind of a qualitative restriction.

## Feeding on gut health

As already mentioned the calcium metabolism of a laying hen is aging and normally the egg size will be increased. The topic feeding on gut health to ensure nutrient and mineral absorption from the gut serves as a further tool to support the overall egg quality. Additionally this topic got a high attraction under the bottom line of a reduced use of feed supplements with antibiotic activity. The feed industry and layer nutritionists are offered a bunch of feed additives to anticipate this idea. Feed enzymes are a well proven tool to support gut health in general. Furthermore: herbs & spices and essential oils, acidifiers and organic acids, probiotics, prebiotics (MOS), medium and short chain fatty acid products, inclusion of non-digestible fibre (Lignin). A further idea of feeding on gut health will be to focus on the biologically given job
of the gizzard – as a grinding machine and start-up of the digestive process. The gizzard basically is a muscle which needs stimulation and needs to be clearly distinguishable from proventriculus. If the gizzard is filled with coarse feed, it will grind and mix feed particles before passing them on into the intestine. If the gizzard is getting too fine feed, it will not work and will not change particle size before passing them on into the intestine. In practise this will result in some kind of diarrhea, wet litter and dirty eggs. As an optimal working gizzard ensures a healthy and stable digestion, a coarse grinded feed structure is recommended. Larger feed particles have an increased retention time in the crop and the gizzard, which stimulates the ph drop and due to this has a bacteriocide effect. An excess amount of feed particles smaller than 1mm causes a direct outflow through the gizzard without utilisation. As layer birds being fed predominately with mash feed on a worldwide view, the physical structure in terms of coarseness and homogeneity needs to be given more attention because it is the basis for a healthy digestion (dry litter, clean eggs). It even influences the birds' behaviour as the birds are searching for structure during the eating activity. Furthermore a good structure improves the palatability of mash feed and even the intake of the fines in crumbled or pelleted feed.

## Recommendations for practice

In order to feed layers until 100 weeks the most important aspects - being outlined in this paper - can be summed up:

- improve overall rearing of pullets

- improve the "conditions" during the transfer period

- emphasize the pre-lay idea

- support daily feed- and nutrient intake at start of production

- emphasize the idea of "feeding on gut health"

- improve overall Ca- metabolism

- take care of and support regularly good liver health

- control egg weight in aging flocks

- never "bad" compromises in feeding of high prolific flocks

## Results and discussion

Egg shell quality will be more and more the one and only reason determining the point when to "sign off" the production period of a layer flock. With regard to the topic of this paper, which is "How to feed layers until 100 weeks?" supporting egg shell quality is already - and will be - the major challenge for nutritionists, veterinarians and the management in total to maintain and increase profitability in egg production. When targeting on an extended production cycle with layer hens, optimal nutrition starts just after hatch to develop a pullet on an optimal body weight, uniformity and feed intake capacity. The transition period from pullet to layer phase together with the transfer from rearing to the layer house have an enormous impact on an optimal and healthy start of the egg production period. Good understanding and implementation of the idea of a pre-lay feed serve in this phase already as a support in extended production cycles. Optimal quality of fine and coarse limestone needs to be mentioned as the most important tool for egg shell quality in general. Feed structure together with the idea of feeding on gut health even will support layer hens when aging.

<table><caption>Table 1: The correct use of pre-lay feed</caption><thead><tr><th colspan="2">Age of transfer</th><th colspan="3">Feeding program</th></tr><tr><th>week</th><th>days</th><th>Developer feed</th><th>followed by</th><th>Pre lay feed</th></tr><tr><th></th><th></th><th>kg feed</th><th>→</th><th>kg feed</th></tr></thead><tbody><tr><td>15</td><td>105</td><td>1.0</td><td>→</td><td>1.0</td></tr><tr><td>16</td><td>112</td><td>0.5</td><td>→</td><td>1.0</td></tr><tr><td>17</td><td>119</td><td>-</td><td>→</td><td>1.0</td></tr><tr><td>18</td><td>126</td><td>-</td><td>→</td><td>0.5</td></tr><tr><td>after 18</td><td>after 126</td><td colspan="3">→ immediately: start lay feed - or layer phase 1</td></tr></tbody></table>
<table><caption>Table 2: Continuous supply of fine and coarse limestone</caption><thead><tr><th rowspan="2">Feed type</th><th>Fine limestone</th><th>Coarse limestone</th></tr><tr><th>0 - 0.5 mm</th><th>1.5 - 3.5 mm</th></tr></thead><tbody><tr><th>Layer phase 1</th><td>30%</td><td>70%</td></tr><tr><th>Layer phase 2</th><td>25%</td><td>75%</td></tr><tr><th>Layer phase 3</th><td>15%</td><td>85%</td></tr></tbody></table>

<figure><img src="image_12.png" /><figcaption>Figure 1: The physiology development of the pullet (source: Ysilevitz 2007 Israel)</figcaption></figure>

References:

Available on request
# INSECTS AS POULTRY FEED

**D. Józefiak<fnref n="1" /> and R. M. Engberg²**

## Abstract

The consumption of poultry meat and eggs is expected to increase considerably in the nearest future, which increases the demand for poultry feed ingredients to support a sustainable intensive production. Moreover, the constant improvement of the genetic potential of poultry has resulted in an increased density of the diets, which limits the inclusion of low quality feed compounds. Therefore, the feed industry needs new sources of highly digestible protein with a desirable amino acid composition to substitute other valuable limited protein sources of animal origin such as fishmeal. The phylogeny of insects includes the largest species variety in the world and suitable species providing high protein and high sulphur containing amino acid concentrations have been identified which can be successfully exploited as feed items for poultry. The aim of this paper is to review the state of the art on the use of insect protein in poultry nutrition and the possibilities of insect mass production for the feed industry. Although, legislative barriers for the inclusion of insect meals still exist, there is no doubt that insects have an enormous potential as feed item for poultry.

## Introduction

The requirement for valuable protein sources of a continuously growing world population and the simultaneously decreasing areas suitable for agricultural production present a serious global challenge. In this context, the global demand for poultry meat and eggs is expected to increase significantly in the future, which is due to the facts that these products have a very high nutritive value, they are relatively cheap and no religious issues are connected to their consumption. Further, compared to other livestock, the production of poultry is considered to be relatively environmentally friendly having a significantly lower CO₂ footprint. An increasing intensive poultry production requires increasing amounts of protein to cover the amino acid requirements of the bird for maintenance, plumage development, growth and egg production. Currently available vegetable protein sources for poultry are extraction meals of soya beans, rape seed, legumes, and maize gluten. However, the amino acid composition of plant proteins for poultry is inferior to that of animal based proteins, specifically with respect to their content of essential sulphur containing amino acid in particular methionine. Therefore, fish meal is still used to quite an extend in poultry diets. However, due to overfishing, fish meal has become a very limited resource which is reflected by increasing marked prices over the last decades. Alternative protein sources of comparable value are therefore urgently needed in order to make poultry production a sustainable production form in the future. In this connection, the potential of insect protein in poultry diets has attracted much attention. Chickens with access to outdoor area pick up insects at all life stages and eat them with great pleasure, which indicates that they are evolutionary adapted to insects as natural part of their menu. Therefore, it is seems reasonable to consider the inclusion of insect proteins as raw material to be used in commercial feed manufacturing and to develop intensive farming systems for a new six-legged livestock. In order to ensure a cost effective insect based protein production, the ideal insect candidate should have a short reproduction cycle and should be nutritious providing high concentrations of protein and sulphur containing amino acids. In order to guarantee a constant insect supply, the ideal insect candidate should further be easy to rear in intensive rearing sites.

## Nutrient composition of insects

Insects at all life stages are rich sources of animal protein. Until now, the main research efforts have focussed on the larvae of the Black soldier fly (Hermetia illucens), the maggot and pupae of the housefly (Musca domestica), the larvae of the mealworm (Tenebrio molitor), and insect families belonging to the order Orthoptera including locusts, grasshoppers, crickets and katylids. However, insects of the order Blattodea, like American, German, and Asian cockroach are also interesting candidates. A comprehensive review on the nutrient content of different insects and insect meals including larvae of the Black soldier fly, meals based on housefly maggot meal and pupae meal, mealworms, as well as meals based on locusts, grasshoppers and crickets and silkworm pupae meal is given by Makkar et al. (2014) and Sánchez-Muros et al. (2014).

As shown in Table 1 and 2, insects are a rich source of protein, essential amino acids and fat. The protein content of insect meals varies considerably from below 40% to over 60% even when the meals are based on the same insect

<footnote n="1">¹Department of Animal Nutrition and Feed Management, Poznan University of Life Science, ul. Wolynska 33, 60-637 Poznan, Poland; ²Department of Animal Science, Aarhus University, Blichers Allé 20, 8830 Tjele, Denmark; damjo@up.poznan.pl</footnote>
species (Table 2). The same holds true for the fat content. However, it is important to note that insect meals compared to fishmeal contain lower concentration of methionine, which has to be considered when formulating diets based on insect proteins. Further, the calcium concentration is usually lower than that of fish meal. Larvae of the Black soldier fly do however provide substantially more calcium than other insects (Table 2). The nutrient concentration of insects depends on the life stage of the insect, the rearing conditions and the composition of the growth media used for insect production. For instance, house fly larvae grown on chicken manure had a lower dry matter content, but a higher methionine content per kg dry matter as compared to larvae grown on a medium providing wheat bran, alfalfa, malt and dried yeast (Engberg et al., unpublished). Mealworm larvae prefer protein based diets rather than starch based diets and they appreciate the inclusion of yeast. The feed conversion ratio of mealworms fed 11.9% crude protein was 6.05 kg/kg and improved significantly to 3.04 kg/kg when feed with a crude protein of 32.7%, was fed. No significant change in protein concentration of the mealworm meal was observed (van Broekhoven et al., 2015). However, the fat content of mealworms fed low protein diets was significantly lower as compared to high protein diets (18.9 vs 26.3 %). The live insects contain an average of 30% dry matter, therefore the processing parameters of the insect meal are essential to obtain high quality raw material, which can be used at commercial feed milling operations. For today in the available literature, there are no studies describing the effect of processing on insect protein quality. However, crude protein and crude fat are the main nutrients in dried insect meal. Therefore, similarly to other meals of animal origin, oxidation processes and microbial deterioration during storage (Awoniyi et al., 2004) determine the shelf life quality, and should be taken into consideration.

## Insect protein in poultry production

For a summary of poultry feeding trials involving diets containing different insect meals, the reader is referred to Makkar et al. (2014) and Sánchez-Muros et al. (2014). Most of the published trials have been carried out with broilers fed housefly larvae meal. The results showed that housefly larvae can be added at approximate dietary levels of 25% (Pretorius, 2011) without any negative effect on weight gain, feed intake and feed efficiency. This suggests that maggot meal can efficiently replace other protein sources, e.g. soy bean meal, fish meal and groundnut cake. With respect to the metabolizable energy (ME) value of maggot meal, only limited data are available, however values of 17.9 MJ/kg DM (Zuidhof et al., 2003) for turkey poults and 14.2 MJ/kg DM (Pretorius, 2011) for broilers have been reported. Due to variations in fat and “fibre”/chitin content of maggot meal, the ME values vary considerably. Both authors observed a high total tract amino acid digestibility (95% and 91%, respectively). Feeding maggot meal at the expense of fish meal at dietary concentrations of 16 % to young pullets in the period from 1-56 days, did not negatively influence growth and feed intake of the birds (Engberg et al., unpublished).

The feeding of Black soldier fly larvae as a substitute for soymeal resulted in a similar weight gain but a lower feed intake as compared to control indicating an improved feed conversion (Makkar et al. (2014).

High concentrations of chitin present in the exoskeleton of insects influence feed intake negatively and interfere with the use of protein (Longvah et al., 2011). Although chickens have been shown to produce chitinase in the proventriculus and hepatocytes (Suzuki et al., 2002), the digestibility of chitin seems to be limited (Hossain and Blair, 2007), particularly in young birds (Ijaiya and Eko, 2009). Whether chitin may be a substrate for microbial fermentation and therefore may have a positive effect on microbial balance in the gastrointestinal tract similar to a probiotic is at the moment a matter of speculation.

Another interesting aspect of insects considered for poultry feed, is their content of antimicrobial peptides which are highly abundant in several species. In recent studies (Józefiak, unpublished), we used relatively small dietary inclusion levels (up to 0.2%) of low temperature (50°C) dried full fat meals from *Tenebrio molitor*, *Hermetia illucens* and *Shelfordella lateralis* and observed an improvement of the body weight gain in broilers, when *Shelfordella lateralis* meal was fed. These results may be explained by antimicrobial effects on bacterial populations in the ileum. In contrast, even low inclusion levels of *Hermetia illucens* full-fat meal impaired broiler performance. It may be speculated that the dietary inclusion of *Shelfordella lateralis* meal rich in antimicrobial peptides (defensin) could have beneficial effects on poultry health and welfare. Although the feeding of fresh larvae to poultry includes potential risks primarily in relation to feed hygiene, in particular when organic waste products or even manure is used as medium for larvae growth, there is no doubt that chicken really enjoy the consumption of fresh larvae. In line with this, the supplementation of living maggots as a supplement on top of a balanced diet for young pullets significantly reduced the fearfulness of the birds (Engberg et al., unpublished).
## Nutritional requirements of insects

Until now, the main research efforts have focussed on the larvae of the Black soldier fly, the maggot and pupae of the housefly, the larvae of the mealworm and insect families belonging to the order Orthoptera including locusts, grasshoppers, crickets and katylids. However, insects of the order Blattodea, like American, German, and Asian cockroach are also interesting candidates. Of special interest is their robustness and ability to grow under extreme conditions (low oxygen, no lighting, high stocking density) as well as their high nutritive value (Table 1 and 2). For instance Orthoptera need relatively high amounts of dietary calcium while larvae of the mealworm grow without any calcium supplementation. For today no nutritional recommendations for the different insects exist, but it is obvious that there is a huge variation in the nutrient requirements of different insect species.

The ability of house fly larvae and Black soldier fly larvae to feed on organic waste material Čičkova, et al., 2015) including manure (Newton et al., 2005; Zhu et al., 2015); with high moisture content (60-80%) and convert it to valuable insect protein. This sounds immediately attractive because the production of these larvae can be used as a biological tool to solve environmental problems. Black soldier fly larvae can reduce the accumulation of poultry manure by 50% and more in the process Newton et al., 2005). Moreover, the feeding of larvae reduce the available phosphorous in the manure by 61-70% and nitrogen by 30-50% (Makkar et al., 2014). A further advantage of larvae is their ability to reduce bacterial growth in the manure which consequently results in a reduced odour development and the growth suppression of significant pathogens, e.g. Salmonella (Erickson et al., 2004). As a further co-product, the waste residue of manure can be recycled and used as fertilizer. Even though fly larvae are capable of utilizing animal waste product, e.g. manure and slaughter house offal, it may be doubted whether these feed items will be approved in the diets for a six-legged livestock and may give rise to biosecurity issues. However, fly larvae particularly the Black soldier fly can also easily grow on plant material. In contrast to fly larvae, mealworms and members of the Orthoptera are unable to utilize chicken manure even when dried (FIG.1) and prefer feeds of plant origin.

The great advantage of insect production is that no additional drinking water has to be applied. As compared to other livestock species, insects utilize water very effectively and in most cases the feed is the main source of water. As the feed supplies both nutrients and water, feed optimization is a very important part of insect production and should be addressed in future research. In our laboratory, work is going on to identify different substrates that are preferred by different insects. In a choice feeding test it was clearly shown that mealworms prefer different roughages rather than wheat middlings or DDGS. However, we are only at the beginning to understand the special requirements of different insect species under intensive production conditions and much work has to be done to optimize the diets to support their optimal performance and their nutrient composition.

<figure><img src="image_13.png" /><figcaption>**Figure 1** Weight gain (g) of mealworm larvae fed on different media (88% dry matter)</figcaption></figure>
## Intensive insect production

Considering insects as a new six-legged livestock, the prerequisite for any commercial production is knowledge on optimal housing conditions (temperature, humidity and ventilation), and feeding both regarding feed composition and structure. Further, it is important to gain knowledge of insect diseases and biosecurity standards for this production form. In contrast to most of the livestock species (excluding fish), insects can be produced in 3D dimension systems, which allows a very effective usage of the buildings. Taking the life cycle and growth rate of insects into consideration, the most effective are larvae. It is for instance possible to produce more than 180 kg of live weight of Black soldier fly larvae in 42 days from 1m² whereas only 30 kg of adult crickets can be produced on the same area. Finally insects are capable to utilize some poor quality raw materials as by-products from food industry for example apple or potato pulps.

The growth rate as well as feed utilization is highly depended on temperature, which for most insects is optimal in the range of 27-30°C. Insects are durable to temperature changes for instance for mealworms optimal rearing temperature is 28°C but they easily survive -15°C for 48h. However under high humidity conditions (>70%) they die very quickly.

Another important issue of insect production is farm biosecurity. Due to size and activity of invertebrates, a very efficient control of the buildings has to be applied. For instance, the usage of plastic nets is effective in the production of Black soldier flies but not in cockroach or cricket rearing. The latter species can easily force most plastic materials. Therefore aluminium or copper nets should be used. Finally intensive insect production should also consider invasive species, which can interfere with the natural environment. This is why insect farming should be considered as "all in all out" system with a separate hatching-brooding area.

## Barriers for the inclusion of insect protein in poultry feed

There is no doubt that insect meals from a nutritional perspective are suitable for the feeding of poultry. However, a barrier for the inclusion of insects in feed for livestock is the present EU legislation (Regulation (EC) No. 1069/2009), where insect meals are defined as processed animal protein (PAP). Insects and other invertebrates are classified as Category 3 material (fit but not intended for human food chains). As such, they are suitable as feed for livestock in particular for fish, poultry and pigs. However, despite Regulation (EC) No. 1069/2009, Regulation (EC) No. 999/2001 ("BSE" regulation) prohibits the feeding of farmed animals with PAPs, with the exception of hydrolysed proteins. The feeding of insect meals to aquaculture species is going to be allowed and a re-authorization of these PAPs for pig and poultry feed is expected in the near future.

At the moment, a significant obstacle for the use of insects in animal feed is the limited quantity of produced insects in particular shortcomings in breeding, which does not guarantee a constant supply. The prices for insects and insect meals are presently very high, and cannot compete with other protein sources in this respect. To overcome this problem, the identification of the most suitable insect species giving rise to a cost efficient protein production at an industrial scale is a prerequisite. For mass production, it is necessary to develop automated process technologies for the rearing, harvest and post-harvest procedures, which certainly include the monitoring of product safety and quality (Rumpold and Schlüter, 2013).

The general acceptance of the inclusion of insects in animal feed has been frequently discussed to be a barrier. However, in a recent study from Belgium, cross sectional data were collected among farmers, agriculture sector stakeholders and citizens (Verbeke et al., 2015). The results of this study indicate a broad acceptance. The perceived benefits such as improved sustainability of livestock production, lower dependence on imported protein sources and lower environmental impact, outweighed the perceived risks, such as microbiological contamination, chemical residues in the food chain and lower consumer acceptance of animal products.

At the moment, significant knowledge gaps in the field of insect production exist, particularly in Europe, where insects are not considered as traditional food item (Vantomme et al., 2012, Van der Huis et al., 2013, Veldkamp et al., 2012). However, most signals seem to be set at green for the use of insect proteins in animal feed, so it is all about pulling on the work gloves.
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<table><caption>Table 1. Amino acid composition of different insect and fish meal</caption><thead><tr><th rowspan="2">Amino acid</th><th>Mealworm</th><th>Tropical house cricket</th><th>Field cricket</th><th>Black soldier fly</th><th>Turkish cockroach</th><th>Housefly</th><th>Fish meal</th></tr><tr><th>(Tenebrio molitor)</th><th>(Gryllodes silligatus)</th><th>(Gryllus assimilis)</th><th>(Hermetia illucens)</th><th>(Shelfordella lateralis)</th><th>(Musca domestica)</th><th></th></tr><tr><th></th><th>Larvae</th><th>Subimago</th><th>Imago</th><th>Larvae</th><th>Subimago</th><th>Larvae</th><th></th></tr><tr><th colspan="8">% of crude protein</th></tr></thead><tbody><tr><td>Hisidine</td><td>2.7</td><td>2.2</td><td>2.1</td><td>2.6</td><td>2.5</td><td>2.8</td><td>2.6</td></tr><tr><td>Arginine</td><td>4.5</td><td>5.7</td><td>5.8</td><td>4.8</td><td>5.6</td><td>4.9</td><td>5.8</td></tr><tr><td>Threonine</td><td>3.6</td><td>3.5</td><td>3.3</td><td>3.6</td><td>3.3</td><td>3.3</td><td>4.3</td></tr><tr><td>Tyrosine</td><td>5.4</td><td>4.2</td><td>4.5</td><td>6.0</td><td>5.6</td><td>5.1</td><td>3.1</td></tr><tr><td>Valine</td><td>5.9</td><td>5.2</td><td>5.3</td><td>5.6</td><td>5.1</td><td>4.4</td><td>4.8</td></tr><tr><td>Methionine</td><td>1.2</td><td>1.6</td><td>1.2</td><td>1.4</td><td>1.3</td><td>2.2</td><td>2.9</td></tr><tr><td>Cysteine</td><td>0.6</td><td>0.9</td><td>0.5</td><td>0.7</td><td>0.7</td><td>0.4</td><td>1.2</td></tr><tr><td>Isoleucine</td><td>4.0</td><td>3.7</td><td>3.4</td><td>4.0</td><td>3.1</td><td>3.2</td><td>4.0</td></tr><tr><td>Leucine</td><td>6.9</td><td>6.9</td><td>6.6</td><td>6.6</td><td>5.8</td><td>5.7</td><td>7.4</td></tr><tr><td>Phenylalanine</td><td>3.2</td><td>3.1</td><td>2.9</td><td>3.8</td><td>3.0</td><td>5.0</td><td>3.6</td></tr><tr><td>Lysine</td><td>4.9</td><td>5.3</td><td>5.0</td><td>5.6</td><td>4.9</td><td>6.9</td><td>7.8</td></tr><tr><td>Tryptophan</td><td>1.0</td><td>0.9</td><td>0.7</td><td>1.1</td><td>0.8</td><td>3.2</td><td>1.2</td></tr><tr><td>Total</td><td>43.9</td><td>43.2</td><td>41.3</td><td>45.8</td><td>41.7</td><td>47.1</td><td>48.7</td></tr><tr><th colspan="8">% relative to lysine</th></tr><tr><td>Lysine</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td></tr><tr><td>Hisidine</td><td>55</td><td>42</td><td>42</td><td>46</td><td>51</td><td>41</td><td>33</td></tr><tr><td>Arginine</td><td>92</td><td>108</td><td>116</td><td>86</td><td>114</td><td>71</td><td>74</td></tr><tr><td>Threonine</td><td>73</td><td>66</td><td>66</td><td>64</td><td>67</td><td>48</td><td>55</td></tr><tr><td>Tyrosine</td><td>110</td><td>79</td><td>90</td><td>107</td><td>114</td><td>74</td><td>40</td></tr><tr><td>Valine</td><td>120</td><td>98</td><td>106</td><td>100</td><td>104</td><td>64</td><td>62</td></tr><tr><td>Methionine</td><td>24</td><td>30</td><td>24</td><td>25</td><td>27</td><td>32</td><td>37</td></tr><tr><td>Cysteine</td><td>12</td><td>17</td><td>10</td><td>13</td><td>14</td><td>6</td><td>15</td></tr><tr><td>Isoleucine</td><td>82</td><td>70</td><td>68</td><td>71</td><td>63</td><td>46</td><td>51</td></tr><tr><td>Leucine</td><td>141</td><td>130</td><td>132</td><td>118</td><td>118</td><td>83</td><td>95</td></tr><tr><td>Phenylalanine</td><td>65</td><td>58</td><td>58</td><td>68</td><td>61</td><td>72</td><td>46</td></tr><tr><td>Tryptophan</td><td>20</td><td>17</td><td>15</td><td>20</td><td>17</td><td>46</td><td>15</td></tr></tbody></table>
<table><caption>Table 2 Nutrient composition of different insect meals</caption><thead><tr><th rowspan="2">Item<br>per kg DM</th><th colspan="2">Field cricket<br>(Gryllus assimilis)</th><th colspan="2">Housefly<br>(Musca domestica)</th><th colspan="2">Black soldier fly<br>(Hermetia illucens)</th><th colspan="2">Mealworm<br>(Tenebrio molitor)</th><th colspan="2">Turkestan cockroach<br>(Blatta lateralis)</th></tr><tr><th>Imago</th><th>Subimago</th><th>Pupae</th><th>Larvae</th><th>Larvae</th><th>Larvae</th><th>Larvae</th><th>Nymph</th><th>Nymph</th><th>Nymph</th></tr></thead><tbody><tr><td>Gross energy, MJ</td><td>21.5</td><td>19.3</td><td>20.1</td><td>20-24</td><td>22.1</td><td>26.8-27.3</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Crude fibre, g</td><td>70</td><td>94</td><td>157</td><td>16-86</td><td>70</td><td>51-88</td><td>86-89</td><td>46-54</td><td>0.6-0.7</td><td>0.2</td></tr><tr><td>Ash, g</td><td>64</td><td>54</td><td>55-98</td><td>31-173</td><td>146-284</td><td>10-45</td><td>4.4-14.2</td><td>543-734</td><td>176-261</td><td>150-350</td></tr><tr><td>Phosphorus</td><td>8.0</td><td>8.6</td><td>-</td><td>9.2-24.0</td><td>6.4-15.0</td><td>0.3-6.2</td><td>451-603</td><td>250-431</td><td>4.6-4.8</td><td>3.9-5.6</td></tr><tr><td>Calcium</td><td>9.9</td><td>3.1</td><td>-</td><td>3.1-8.0</td><td>50.0-86.0</td><td>-</td><td>4.4-5.1</td><td>4.7-5.8</td><td>3.8-5.6</td><td>4.8-8.0</td></tr><tr><td>Crude protein, g</td><td>564</td><td>638</td><td>630-762</td><td>380-604</td><td>411-450</td><td>1.1-2.0</td><td>1.1-1.2</td><td>4.0-4.9</td><td>2.5-3.3</td><td>3.5-4.4</td></tr><tr><td>Crude fat, g</td><td>238</td><td>168</td><td>144-161</td><td>90-260</td><td>150-350</td><td>0.1</td><td>-</td><td>-</td><td>1.1-1.2</td><td>0.9-1.0</td></tr><tr><td>Amino acids per 16 g N</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Glycine, g</td><td>3.0</td><td>2.7</td><td>3.9-4.3</td><td>3.7-5.1</td><td>5.1</td><td>3.9-5.6</td><td>4.6-4.8</td><td>4.7-5.8</td><td>4.6-4.9</td><td>3.9-5.6</td></tr><tr><td>Arginine, g</td><td>3.7</td><td>3.3</td><td>4.2-5.9</td><td>3.7-5.8</td><td>4.8-8.0</td><td>3.8-5.6</td><td>4.4-5.1</td><td>4.7-5.8</td><td>4.6-4.9</td><td>3.8-5.6</td></tr><tr><td>Threonine, g</td><td>2.1</td><td>1.9</td><td>3.0-3.4</td><td>2.0-4.4</td><td>1.3-4.8</td><td>3.5-4.4</td><td>4.4-5.1</td><td>4.7-5.8</td><td>4.6-4.9</td><td>3.5-4.4</td></tr><tr><td>Valine, g</td><td>3.4</td><td>3.0</td><td>3.4-4.6</td><td>1.3-5.1</td><td>5.6-9.1</td><td>5.5-6.6</td><td>1.1-2.0</td><td>4.6-6.1</td><td>4.6-6.1</td><td>3.5-4.4</td></tr><tr><td>Methionine, g</td><td>0.8</td><td>0.8</td><td>1.5-2.6</td><td>1.3-4.6</td><td>1.4-2.4</td><td>1.1-2.0</td><td>0.8-0.9</td><td>6.7-10.6</td><td>4.6-6.1</td><td>3.9-5.6</td></tr><tr><td>Cysteine, g</td><td>-</td><td>-</td><td>0.4</td><td>0.5-1.0</td><td>0.1</td><td>0.8-0.9</td><td>-</td><td>6.7-10.6</td><td>4.6-6.1</td><td>3.9-5.6</td></tr><tr><td>Leucine, g</td><td>4.2</td><td>3.6</td><td>4.9-5.4</td><td>4.5-7.8</td><td>6.6-8.4</td><td>6.7-10.6</td><td>4.7-5.8</td><td>4.7-5.8</td><td>4.6-4.9</td><td>3.9-5.6</td></tr><tr><td>Lysine, g</td><td>3.2</td><td>2.9</td><td>4.8-6.5</td><td>5.0-8.2</td><td>5.5-8.0</td><td>4.6-6.1</td><td>4.0-4.9</td><td>4.7-5.8</td><td>4.6-4.9</td><td>3.9-5.6</td></tr><tr><td>Fatty acids per kg fat</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>SFAs, g</td><td>351</td><td>352</td><td>476</td><td>417</td><td>749</td><td>229-334</td><td>28.7</td><td>46.3-50.2</td><td>138-219</td><td>28.7</td></tr><tr><td>MUFAs, g</td><td>298</td><td>261</td><td>307</td><td>314</td><td>155</td><td>407-536</td><td>138-219</td><td>46.3-50.2</td><td>138-219</td><td>28.7</td></tr><tr><td>PUFAs, g</td><td>336</td><td>369</td><td>291</td><td>399</td><td>74</td><td>254-323</td><td>1-11</td><td>1-11</td><td>1-11</td><td>28.7</td></tr><tr><td>Total Omega 3, g</td><td>22</td><td>17</td><td>-</td><td>-</td><td>2</td><td>2-4</td><td>1-11</td><td>1-11</td><td>1-11</td><td>28.7</td></tr><tr><td>Total Omega 6, g</td><td>314</td><td>352</td><td>-</td><td>-</td><td>23</td><td>81-93</td><td>35-207</td><td>35-207</td><td>1-11</td><td>28.7</td></tr></tbody></table>
# IMPRINTING – PRE/POST HATCH AS A NUTRITIONAL TOOL

**Roselina Angel**¹, **Fernando Escribano**¹, **Yael Noy**², and **Chris Ashwell**³

¹Department of Animal and Avian Sciences, Univ. of Maryland, College Park MD 20742, USA; ²Miloubar Feedmill, MP Ashrat 25201, Israel; ³Department of Poultry Science, North Carolina State University, Raleigh, NC 27695, USA; rangel@umd.edu

## Summary

Most traits of economic importance in animal production undergo continuous phenotypic variations due to multi gene changes and environmental factors. Numerous quantitative trait loci that impact agronomic traits have been identified, but generally the specific set of genes responsible, are not defined. Studies using genome-wide associations have shown that genetic variation only partially explains the variability of complex traits and that, it is not only the DNA sequence that affects phenotype, but that epigenetic changes that can be transmitted from one generation to the next are also involved (JABLONGA and RAZ, 2009).

Non DNA mediated changes have been identified in mammals and birds. Nutritional impacts that appear to elicit “epigenetic” changes have been documented. Published results will be discussed on epigenetic effects associated with protein concentration changes in the maternal line changing protein usage in offspring’s. Effects seen associated with neonatal nutritional changes impacting the ability of broilers to grow well when fed deficient Ca and P containing diets that decrease growth in non challenged broilers will also be discussed

## Introduction

As we look ahead at technologies with the potential to deliver exponential improvements in our ability to produce animal products with lower inputs and at lower production costs as well as with less impact on the environment, application of epigenetic principles in one of them. What the correct term is and how to define it are still in debate. Early on conditioning was used but currently metabolic imprinting, programming and epigenetics are the terms most used.

But what is epigenetics? Numerous and somewhat divergent definitions for epigenetics can be found in the literature and numerous terms are applied to the same “apparent” mechanism. There is no complete agreement on what the term “epigenetics” refers to and this leads to numerous definitions (HOLLIDAY, 2006; BIRD, 2007; JABLONGA and RAZ, 2009; HO and BURGGREN, 2010; FRESARD et al., 2013). Epigenetics comes from the words “epi” greek for over or above and genetics. It involves modifications of the phenotype without changes in DNA sequence. These modifications can be transmitted to the next generations (HO and BURGGREN, 2010). BIRD (2007) defined epigenetics as “the structural adaptation of chromosomal regions so as to register, signal or perpetuate altered activity states”. FRESARD et al., (2013) used the definition of FEIL and FRAGA (2012) where epigenetics was defined as the study of mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence. Epigenetics allows the same genetic information to be used differently by individuals grown in different environments.

Epigenetic changes can be mediated by DNA methylation, chromatin folding and its attachment to the nuclear matrix, packaging of DNA around nucleosomes, non coding RNA and covalent modifications of histone tails to name a few in the growing list of mediating factors. For the purpose of this review the definition of epigenetic changes will involve both phenotypic as well as changes in gene expression that persist through the life of the animal and/or subsequent generations (mainly F1 generation) and that are changes that are greater than the normal adaptations that, for example, occur during nutrient deficiency states.

There is great potential for these types of changes to be used as a tool to improve productivity in animal production while decreasing the need for costly inputs as well as decreasing environmental impacts. Extensive information on epigenetic changes in wild mammals, humans and wild birds is available. In poultry production, work has been done trying to harness this potential both for poultry management and nutrition. The potential has been demonstrated under controlled conditions but application under commercial conduits lags behind. Full understanding of when these “environmental influences” should occur to maximize positive effects later in life or in future generations has not been fully elucidated. In the area of nutrition, work is just beginning to demonstrate the effects but again full understanding of mechanisms and of how to maximize positive effects is lacking.
## Background on Epigenetic Regulation

One of the fundamental assumptions of Mendelian genetics is that a specific allele behaves in the same way no matter from where (which parent) it originates. But as with many assumptions in science this rule does not always hold true. Initial observations in mice looking for the effects of paternal or maternal impacts using genomic translocations indicated that identical chromosomal regions were not equivalent and acted differently in embryo development between maternal and paternal sources (CATTANACH and KIRK, 1985).

The growing incidence of metabolic diseases in humans, such as obesity, diabetes, and cardio-vascular disease has sparked interest and research efforts into both their genetic and environmental (nutritional among others) basis. Fetal programming as it was initially referred to in humans (HALES and BARKER, 1992; BARKER, 2004), encompasses the role of developmental plasticity in response to environmental signals, including nutrition, during early life and its potential adverse consequences (risk of cardiovascular, metabolic and behavioral diseases) in later life. The first studies in this field highlighted an association between poor fetal growth and chronic adult diseases. Adverse long-term effects reflect a mismatch between early (fetal and neonatal) environmental conditions and the conditions that the individual will confront later in life.

Adverse nutritional conditions during fetal development lead to adaptive changes in metabolism that lead to a 'thrifty phenotype' in the offspring (HALES and BARKER, 1992). For example, poor nutrition in early life produces permanent changes in glucose-insulin metabolism, including a reduced capacity for insulin secretion and insulin resistance (HALES and BARKER, 2001). The initiating factor(s) in the case of nutrients for fetal programming may be nutrient(s) interacting directly with genes and their regulatory elements at the cellular level, altering patterns of growth and gene expression. Embryonic and fetal cells have a complex system to integrate nutritional signals from their environment and adapt their development accordingly to ensure survival (FRESARD et al., 2013).

In chickens there are reports where conditioning in early-life imparts long-term effects. The first report of this type of response was to thermal stress. High temperatures during the first week of life were reported to modulate the response to thermal stress later in life (YAHAV and MCMURTRY, 2001). By simply increasing the brooding temperature from 30°C to 37.5°C for 24 hr within the first 5 days post-hatch, broiler chickens were able to tolerate an acute exposure to a temperature of 35°C for 6 hr at 42 days of age, while "unconditioned" birds were unable to acclimate. The mechanism for this conditioned response was not elucidated then. More recently studies have shown the gene expression changes occur as a result of a heat stimulus in the neonatal period (YOSSIFOFF et al., 2008; KISLIOUK and MERI, 2009; KISLIOUK et al., 2010, 2011). These researchers reported that the expression of brain-derived neurotrophic factor, a key regulator of thermo tolerance acquisition in the hypothalamus of the chick, was different between birds grown under normal temperature and those subjected to high temperatures within the first 3 days of life. These changes were associated with increased methylation of CpG sites in the promoter of the brain-derived neurotrophic factor gene.

Although little is known about the underlying molecular mechanisms, there is evidence that feed stress may alter gene expression, in part through epigenetic changes. For example, XU et al. (2012) found that when 3-day old chicks were subjected to a 24-hour fasting, there were methylation changes in histone H3 in the anterior hypothalamus, the area in the brain that controls of body temperature and food intake. Feed stressors such as feeding Ca and P deficient diets in the starter (YAN et al., 2005) or during the first 90 hr post placement (ASHWELL and ANGEL, 2010) change the ability of broilers to utilize diets deficient in Ca and P later in life. These changes were shown to be greater than those associated with normal adaptations that occur when deficient diets are fed. These changes were partially mediated through increased methylation of the NaP cotransporter (ASHWELL and ANGEL, 2010).

There are other examples of potential epigenetic effects mediated by changes in the embryonic or neonatal period of poultry development. For example, exposure of the embryo to green monochromatic LED light appears to improve growth in broilers and turkeys (HALEVY et al., 2006; ZHANG et al., 2012). This can be partially explained by the enhancement that was seen in the differentiation and proliferation of myoblasts (HALEVY et al., 2006).

## Effect of Early Nutrition

Most of our knowledge on the effects of nutrients on gene expression has been acquired in animal models, many examples of which can be found in the overview of the mechanisms by which nutrients interact with their molecular targets to modify gene expression (MULLER and KERSTEN, 2003). In avian species nutrients deposited into the egg by the hen are the only source of nutrients available to the embryo, and this may be the last direct chemical means by which the hen may transfer an epigenetic message to its offspring. Although the digestive capacity begins to
develop after the embryo consumes the amniotic fluid, part of the development occurs post-hatch when the neonatal chick begins consuming feed. Any delay in feed intake initiation will suppress gastro-intestinal development (UNI and FERKET, 2003) and inhibit satellite cell proliferation and muscle growth potential (HALEVY *et al.*, 2003) among others changes. Whether these effects are epigenetically driven has not yet been determined.

A means of manipulating the nutrients deposited in the egg, beyond those that can be changed through material nutrition, is to inject nutrients into the egg at a time when the embryo is most sensitive to epigenetic programming. This timing has not been fully elucidated and may differ for different system. Injecting an isotonic in ovo feeding (IOF) solution into the embryonic amnion, the embryo can naturally consume supplemental nutrients orally before hatching and thus, nutritional manipulations can be implemented inside the egg. In ovo feeding "jump-starts" the ability of the animal to adjust to external feeding. In addition to the increased body weights typically observed at hatch, the positive effects of in ovo feeding may include increased hatchability (UNI *et al.*, 2005); advanced morphometric development of the intestinal tract (TAKO *et al.*, 2004) and mucin barrier; enhanced expression of genes for brush boarder enzymes (sucrase-isomaltase, leucine aminopeptidase) and their biological activities, along with enhanced expression of nutrient transporters, SGLT-1, PEPT-1, and NaK ATPase (TAKO *et al.*, 2005; FOYE *et al.*, 2007); increased liver glycogen status (UNI *et al.*, 2005; TAKO *et al.*, 2004) and increased breast muscle size at hatch (UNI *et al.*, 2005; Foye *et al.*, 2006). In ovo feeding clearly advances the digestive capacity, energy status, and development of critical tissues of the neonate by about 2 days at the time of hatch.

## Early Dietary Adaptation

Adaptation to low nutrient diets has been long recognized. Animals respond to nutrient restriction in general by increasing absorption rates and utilization efficiency, which decreases excretion of the restricted nutrients. Although several reports demonstrate the ability to program chicks through early dietary manipulation to improve Ca and P utilization later on in life, there has been little work reported in the literature applying this concept with dietary protein utilization.

A study by RAO *et al.* (2009) where the effect of feeding a low protein diet (100 g/kg) versus a normal breeder diet (150 g/kg) to females of an inbred indigenous Chinese line (Langshan). Chicks from eggs laid by hens fed the low protein diet were lighter (5%) and had less pectoralis muscle weight (16%) at hatch as compared to chicks from eggs laid by hens fed the normal breeder diet. By 28 days of age these chicks from eggs laid by low protein fed breeders had greater body weight (6%) and pectoralis major muscle weight (51%) then those of for birds hatched from eggs laid by hens fed the normal protein diet. These changes in growth rate were associated with changes in patterns of gene expression in the yolk sac membrane, hypothalamus and muscle in the embryo. While these authors did not look at the persistence of these gene expression changes or the mechanisms that caused them, they clearly demonstrated a long term effect of maternal nutrition challenges.

Adaptation to P and Ca restricted diets has been reported in chickens. BLAHOS *et al.* (1987) reported an increase in duodenal and ileal P absorption in broiler chickens fed a low Ca diet for two weeks and a smaller, but still significant increase in duodenal but not ileal P absorption in chicks fed a low P diet. The adaptation to P or Ca restriction has been associated with an increased level of circulating 1,25-(OH)2-D3 (BLAHOS *et al.*, 1987) and duodenal calbindin content (MONTECUCCOLI *et al.*, 1977). When duodenal calbindin concentrations and changes with age for 1991 and 2001 strains of broilers, BAR *et al.* (2003) concluded that modern broilers exhibit higher adaptation capacity to P or Ca deficiency and this capacity remains high for the whole growth period.

However, the authors could find no published work that evaluate the long-term effects of neonatal P or Ca restriction on growth performance, bone mineralization, and Ca and P absorption in poultry and whether "epigenetic" type of impacts had been documented. Thus, a study was done to determine whether we could elicit a positive epigenetic response to short term neonatal deficiencies in Ca and P. We defined epigenetic responses (phenotypic and in the expression of specific genes) as effects that were significantly above those that occur at the time the animal is exposed to a nutrient deficiency and that persisted through the productive life of the broiler chicken. In the first study, we evaluated the ability of the broiler chicken to adapt to a moderate early life deficiency in P and Ca and characterized this adaptation changes by examining the impact of the previous P and Ca status (starter phase, hatch to 18 days of age) on performance, bone characteristics, and nutrients absorption of broilers in the grower phase (19 to 32 days of age) (YAN *et al*, 2005).
Broilers fed a diet moderately deficient in P and Ca from hatch to 18 days of age demonstrated the ability to adapt to the deficiency, shown in the increased ileal absorption of P and Ca, the increased PP disappearance, greater growth and bone mineralization (tibia ash, tibia and shank bone mineral density and bone mineral content) in a later growth phase (18 to 32 days of age) in broilers fed the deficient diets early on as compared to those fed diets that met requirements (YAN *et al*, 2005). A follow up experiment was done to better define the degree, timing, and length of the early restriction and document gene expression changes of specific genes and further document an increased ability of conditioned broilers to handle P and Ca deficient diets in later growth phases. A main objective was to be able to achieve similar or better productivity with minimal negative impacts on bone mineralization when broilers were fed severely deficient Ca and P diets in the finisher phase.

The second experiment (ANGEL and ASHWELL, 2008) was done to determine if feeding deficient diets for a shorter time after hatch could elicit positive effects on performance and change expression of the chicken intestinal sodium phosphate co-transporter (NaPcoT). Ross 308 male chicks were fed either a prestarter control diet (C) consisting of 11.1 g/kg Ca and 5.0 g/kg non phytate P (nPP) which met or exceeded NRC (1994) recommendations or a deficient or low diet (L) containing 5.9 g/kg Ca and 2.5 g/kg nPP from hatch to 90 hr. Between 90 h and 8 days of age, all birds were fed the prestarter C diet and from 9 to 22 days of age a starter C diet (8 g/kg Ca and 4.5 g/kg nPP). From day 23 to 38 of age, broilers were either maintained on a grower C diet containing 7.0 g/kg Ca and 3.0 g/kg nPP or a grower L diet containing 4.0 g/kg Ca and 1.2 g/kg P. The three treatments, C- C-C, C-C-L, and L-C-L met or exceeded all other nutrient recommendations (NRC, 1994) and were formulated to meet average USA industry use for all nutrients except Ca and nPP. Data collected for each phase included body weight and feed intake, and at the end of each phase all birds in preselected pens were sampled and toe and tibias removed to determine bone ash and ileal content for apparent Ca and P digestibility determinations as well as duodenal, jejunal and ileal mucosa for gene expression determinations. Data for performance, bone ash and digestibility are presented in Table 1 and for gene expression in Table 2.

Broilers fed the prestarter L diet from placement to 90 hr were better able to handle a P deficiency in the grower phase (22 to 38 days of age) than those fed the C diet in the first 90 hr (non conditioned birds) and then fed deficient diets (L) in the grower phase Table 1). Broilers fed the L grower diet from hatch to 90 hr were heavier, had better feed conversion, greater toe ash and P digestibility at 38 days of age, as compared to the none conditioned birds when these two groups of birds were fed the same deficient diet in the grower phase. The improved digestibility was greater than that seen simply by feeding a defiant diet in the grower phase. At 38 days of age, P digestibility was lowest in broilers fed the C diets from hatch to 38 days, a significantly greater P digestibility was seen in broilers fed the L diet from 23 to 38 days and that had been fed the C diets prior to 23 days of age, and greatest for the conditions birds (fed the L diet for the first 90 hr after placement) and the L diet from 23 to 38 d of age. This establishes that there was a "conditioning or imprinting" in these birds meaning that modifications are occurring in these birds that are long term and that allow for improved P utilization when P deficient diets are fed in the grower/finisher phases. Despite this improved ability to handle deficiencies in Ca and P in latter growth phases, these "conditioned" birds were not able to catch up performance wise and in bone ash with those fed the C diets throughout all growth phases.

These data suggest that, in broilers, during the period immediately post hatch there is a phenomenon occurring related to Ca and P digestibilities that allows for changes to be made that can alter the bird's long term ability to handle Ca and P deficiencies. But are these phenotypic changes associated to long term changes in gene expression. The chicken sodium phosphate co transporter (NaPcoT) gene expression was determined using real-time quantitative PCR. The differences in CT (expression) values for gene expression were determined to be different using a student's t-test and relative differences in expression caused by the dietary treatment were determined by correcting for the efficiency of the PCR as calculated by the standard curves of dilutions of pooled cDNA across the experiment (ASHWELL and ANGEL, 2008). Expression levels and N-fold expression changes (changes relative to the control) are reported in Table 2. Feeding the prestarter L diet from hatch to 90 hr had a significant effect on the expression of the NaPcoT as seen by the average n-fold increase of 2.8 in the mRNA levels in the small intestine. Nearly identical stimulatory effects were seen across all segments of the intestine with relative expression decreasing from duodenum to ileum.

These apparent epigenetic changes appear to be mediated through changes in methylation. When methylation determinations were done on the small intestine samples obtained at 90 hr, apparent methylation was observed at 29 positions within the 52 predicted CpGs in the PCR products in samples fed the C diets while 19 CpGs were methyl-
lated in the DNAs extracted from the programmed or condition birds fed the L diet from hatch to 90 h (ASHWELL and ANGEL, 2008). This 43% reduction in methylation of cytosines in this region may be involved in the increased gene expression observed in the programmed birds relative to controls. Further characterization of the differential methylation patterns is needed both post programming as well as later in life to verify these observations.

This work clearly establishes that “imprinting” or modifications are occurring in the animal that are long term and that allow for improved P utilization when P deficient diets are fed in the grower/finisher phases (Angel and Ashwell, 2007; Ashwell and Angel, 2007). These long term effects on performance and gene expression as a result of nutritional neonatal programming in the chick are strong evidence for the role of epigenetics in the regulation of these phenomena.

The challenge now is how to combine neonatal programming diets while minimizing unwanted negative effects. For example, skeletal growth rate is highest in broilers during the first 8 to 10 days post hatch but neonatal programming Ca and P deficient diet concentrations appears to be required for at least 90 hr post hatch. This feeding of low nutrient concentration diets during the neonatal period goes against the long held “dogma” for prestarter diets where nutrient density tends to be high for nutrients that are in high demand during early post-hatch development. Herein lies the challenge and also the rational for neonatal programming. Research must identify “the window of opportunity” and the extent of the deficiency needed to exert these epigenetic changes where short term early life changes in nutrients fed can elicit long term changes in gene expression while minimizing the deleterious effects of feeding a deficient diet on productivity.

## Conclusions

Nutrition is constantly varying and composed of a very large amount of known and unknown bioactive compounds. Furthermore, nutrition touches the core of metabolism by supplying the vast majority of nutrients (both macro- and micronutrients) for maintaining metabolic homeostasis. This homeostasis stretches from gene expression to lipid metabolism and from signaling molecules to enzyme cofactors. Thus, nutrition by its nature *needs* to be studied in an integrated way. So far, most of the tools for this integration have been lacking, thus maintaining an unbridgeable gap between classical nutrition (studying physiology with a focus on biochemical pathways) and biomedical sciences (determination of disease-related molecular mechanisms). In applying Systems Biology to nutritional sciences, these paradoxical extremes are bridged and the complexity of the relationship between nutrition and health can be met in an integrated approach. Many hurdles need to be overcome, before this research area reaches maturity.

The data included in this review along with other preliminary data demonstrating long term effects on performance and gene expression as a result of nutritional manipulation in the chick are evidence for the role of epigenetics in the regulation of these phenomena. Further work must be conducted to elucidate the specifics of neonatal programming in the chicken and the extent of its effects on DNA methylation or other mechanisms that mediate epigenetic changes. Demonstrating these epigenetic effects in the chicken will contribute to the understanding of the impact of the environment during key development in the embryo and neonate on the regulation of the genome and start to elucidate the potential mechanisms that will allow for improving performance and economics of poultry production beyond the incremental improvements that have been seen up to now. Harnessing the potential benefits of these effects for productive purposes will be the challenge.

Nutrition research in the future will increasingly focus on the ways in which genes are affected by what we feed breeders, how these breeders are managed, how eggs are incubated and how we manage and feed the neonatal. If these effects can be better understood and implemented, then dietary “conditioning” offers opportunities for exponential improvements in animal productivity.
<table><caption>**Table 1.** Impact of early dietary deficiencies of phosphorus (P) and calcium (Ca) on performance and apparent digestibility (AD) of P (ANGEL and ASHWELL, 2008)</caption><thead><tr><th>Treatment <sup>1</sup></th><th>C-C-C</th><th>C-C-L</th><th>L-C-L</th><th>SEM</th><th>P values</th></tr></thead><tbody><tr><td><strong>Hatch to 90 hour</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Weight gain, g</td><td>41.2<sup>2</sup></td><td>36.2<sup>2</sup></td><td>0.775</td><td>&lt;0.001</td><td></td></tr><tr><td>Feed to gain ratio</td><td>0.83</td><td>0.88</td><td>0.016</td><td>0.028</td><td></td></tr><tr><td>Toe ash, g/kg</td><td>133.7</td><td>111.7</td><td>0.57</td><td>&lt;0.001</td><td></td></tr><tr><td>90 hour AD P, g/kg P</td><td>576.2</td><td>663.9</td><td>6.67</td><td>&lt;0.001</td><td></td></tr><tr><td><strong>91 hr to 8 days of age</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Body weight, g (8 days)</td><td>150.2</td><td>141.8</td><td>0.248</td><td>&lt;0.001</td><td></td></tr><tr><td>Weight gain, g</td><td>63.5</td><td>64.5</td><td>1.222</td><td>0.557</td><td></td></tr><tr><td>Feed to gain ratio</td><td>1.15</td><td>1.12</td><td>0.027</td><td>0.367</td><td></td></tr><tr><td><strong>9 to 22 days of age</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>22 days Body weight, g</td><td>904.9</td><td>884.7</td><td>6.530</td><td>0.025</td><td></td></tr><tr><td>Weight gain, g</td><td>754.2</td><td>742.2</td><td>6.605</td><td>0.179</td><td></td></tr><tr><td>Feed to gain ratio</td><td>1.40</td><td>1.40</td><td>0.010</td><td>0.967</td><td></td></tr><tr><td>22 day AD P, g/kg P</td><td>514.6</td><td>529.4</td><td>16.12</td><td>0.513</td><td></td></tr><tr><td><strong>23 to 38 days of age<sup>4</sup></strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>38 days Body weight</td><td>2345.9<sup>a</sup></td><td>2235.4<sup>c</sup></td><td>2285.6<sup>b</sup></td><td>20.158</td><td>0.001</td></tr><tr><td>Gain, g</td><td>1450.9<sup>a</sup></td><td>1333.0<sup>c</sup></td><td>1390.6<sup>b</sup></td><td>15.766</td><td>0.03</td></tr><tr><td>Feed to gain ratio</td><td>1.82<sup>ab</sup></td><td>1.89<sup>a</sup></td><td>1.76<sup>b</sup></td><td>0.033</td><td>0.013</td></tr><tr><td>38 day Toe ash, g/kg</td><td>126.2<sup>a</sup></td><td>105.3<sup>c</sup></td><td>118.4<sup>b</sup></td><td>1.71</td><td>&lt;0.001</td></tr><tr><td>38 day AD P, g/kg P</td><td>453.9<sup>c</sup></td><td>515.4<sup>b</sup></td><td>601.1<sup>a</sup></td><td>1.562</td><td>&lt;0.001</td></tr></tbody></table>

<footnote n="1">¹Treatments are: Control (C) – C – C (fed in all phases) diets that met or exceeded NRC (1994) nutrient recommendations (including those for Ca and P); C-C- Low (C-C-L) fed the C diets from hatch to 22 d of age (prestarter from hatch to 8 days and starter from 9 to 22 days) and then the grower L diet from 22 to 38 d of age; L-C-L fed a prestarter L diet from hatch to 90 hr, a C diet from 90 hr to 22 d (prestarter C from 90 hr to 8 days and starter from 9 to 22 days) and the grower L diet from 22 to 38 d. The L diet met or exceeded all NRC (1994) nutrient recommendations except for those of Ca and P. From hatch to 90 hr the L diet contained 5.9 g/kg Ca and 2.5 g/kg non phytate P (nPP) and the C diet 11.1 g/kg Ca and 5.0 g/kg nPP. From 22 to 38 d of age the L diet contained 4.0 g/kg Ca and 1.1 g/kg nPP and the C diet 7.0 g/kg and 3.0 g/kg nPP.</footnote>

<footnote n="2">² Up to 22 days of age, treatment C was replicated 16 times and treatment L eight times. Between 22 and 38 days of age all treatments were replicated 8 times.</footnote>

<footnote n="3">³ Means within row with different superscript letter differ (as specified in the P value column). Where there are only 2 means in a row the P value column shows significance.</footnote>
<table><caption>**Table 2** - Neonatal phosphorus (P) and calcium (Ca) deficiencies on sodium phosphate co-transporter (NaPcoT) gene expression (ASHWELL and ANGEL, 2008)</caption><thead><tr><th rowspan="2">Dietary treatment</th><th rowspan="2">Tissue</th><th colspan="2">Gene expression<sup>1</sup></th><th rowspan="2">n-fold<sup>2</sup><br>change</th></tr><tr><th>18s rRNA</th><th>NaPcoT<br>mRNA</th></tr></thead><tbody><tr><td rowspan="6">90 hr</td><td rowspan="3">Control</td><td>Duodenum</td><td>15.8±0.2a</td><td>23.2±0.2a</td><td>1.0</td></tr><tr><td>Jejunum</td><td>16.1±0.3<sup>a</sup></td><td>24.4±0.3<sup>a</sup></td><td>1.0</td></tr><tr><td>Ileum</td><td>16.2±0.3<sup>a</sup></td><td>25.3±0.4<sup>a</sup></td><td>1.0</td></tr><tr><td rowspan="3">Low P</td><td>Duodenum</td><td>16.0±0.3<sup>a</sup></td><td>24.5±0.3<sup>b</sup></td><td>3.1</td></tr><tr><td>Jejunum</td><td>16.2±0.3<sup>a</sup></td><td>26.1±0.4<sup>b</sup></td><td>2.9</td></tr><tr><td>Ileum</td><td>16.2±0.4<sup>a</sup></td><td>26.9±0.2<sup>b</sup></td><td>2.5</td></tr><tr><td rowspan="6">d 38</td><td rowspan="3">Control</td><td>Duodenum</td><td>16.5±0.4a</td><td>24.1±0.5a</td><td>1.0</td></tr><tr><td>Jejunum</td><td>16.2±0.3<sup>a</sup></td><td>23.7±0.4<sup>a</sup></td><td>1.0</td></tr><tr><td>Ileum</td><td>15.8±0.4<sup>a</sup></td><td>24.9±0.4<sup>a</sup></td><td>1.0</td></tr><tr><td rowspan="3">Low P</td><td>Duodenum</td><td>16.6±0.4<sup>a</sup></td><td>25.4±0.4<sup>b</sup></td><td>2.5</td></tr><tr><td>Jejunum</td><td>16.1±0.5<sup>a</sup></td><td>25.3±0.6<sup>b</sup></td><td>2.7</td></tr><tr><td>Ileum</td><td>16.8±0.5<sup>a</sup></td><td>26.7±0.4<sup>b</sup></td><td>1.8</td></tr></tbody></table>

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<footnote n="1">¹Expression values are presented as the average ± the Std Dev (n=8).</footnote>

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<footnote>abMeans within a column and age with common superscript letter do not differ (P<0.05).</footnote>
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# IMPROVING BROILER PERFORMANCE THROUGH BREEDER NUTRITIONAL MANAGEMENT

Martin J. Zuidhof

410 Agriculture & Forestry Centre, University of Alberta, Edmonton, AB T6G 2P5, Canada;
martin.zuidhof@ualberta.ca

## Abbreviated Title: MATERNAL NUTRITION

## Summary

Recent research on the subject of maternal nutrition has focused largely on the effects of vitamins, antioxidants, and most recently the complex interactions of dietary macronutrients (energy and protein) on economically important broiler traits: primarily growth rate and breast meat yield. Vitamin E and organic Se show great promise for improving hatchability and chick health. Recommendations for ideal maternal levels of dietary protein and energy are not as clear. The concept of ideal protein has greatly advanced the discipline of livestock nutrition, and it is likely that a quest for an optimal ratio of dietary energy to ideal protein will simplify our understanding of maternal nutrition. The fit of published research on maternal dietary energy and protein levels, ratios, and intakes are tested against several hypotheses. The most consistent alignment is with regard to the relationship between low maternal protein intake and progeny fatness. Lack of congruency with other hypotheses underscores a need for further research. It is clear that a more strategic approach is needed to improve understanding of this multifaceted question. Rather than studying offspring performance as an afterthought, experimental designs must prioritize discovery of the impact of maternal nutrition on offspring performance. Combined with carefully executed control of breeder experiments to reduce variation in breeder feed allocation, and thoughtful standardization of hen age, egg handling, incubation, hatching, and brooding conditions, carefully considered experiments will add greater value to our knowledge of transgenerational effects of maternal nutrition.

## Key words: Maternal nutrition, broiler breeder, offspring, progeny, growth, yield, review

## Introduction

Broiler growth rate, yield and efficiency are of key economic importance for chicken meat production. Understandably, most nutrition studies are conducted to understand the direct impact of broiler diet composition on broiler growth, development, and efficiency. Far less information is available on the effects of maternal nutrition on broiler productivity, though minor improvements in broiler productivity achieved through maternal nutrition may have significant economic benefit (Calini and Sirri, 2007). A major challenge with maternal nutrition is that its effects on broiler performance tend to be confounded by a large temporal lag during which many environment factors can influence the results.

Broiler breeder nutrition can directly affect chick health and productivity through egg composition (reviewed by Wilson, 1997; Kidd, 2003). Currently, however, a new and fascinating field of influence is being discovered. There is increasing disparity between the growth potential of broilers and the degree of expression allowed in broiler breeders to maximize reproductive output. In mammals, it is well established that maternal undernutrition can have negative metabolic effects in offspring and even grand offspring. Many of these insights originated from studying the impact of malnutrition on offspring growth, development and health during the 'Dutch famine' at the end of the Second World War (Roseboom et al., 2011). These and subsequent studies with various animal models have led to discovery of epigenetic (non-DNA sequence) moderators of progeny gene expression. The list of potential epigenetic mechanisms is expanding rapidly. Though not well understood, it is probable that aspects of broiler performance may be influenced indirectly by epigenetic mechanisms. Given the severe degree of feed restriction practiced in meat type chicken parent stocks, the importance of maternal nutrition may be increasing.

The objectives of the current paper are 1) to identify maternal nutritional strategies with potential to increase broiler health and performance, 2) to serve as an update on previous reviews, particularly of maternal dietary energy and protein effects on offspring performance, and 3) to outline key considerations for future research aimed at maternal nutrition strategies for optimal broiler productivity.

## Mechanisms for maternal nutrition effects

There are two main ways that maternal nutrition can affect broiler performance. The first is through differential incorporation of nutrients into the egg, which *directly* impacts progeny development. The second involves epigenetic effects that *indirectly* affect progeny growth development by changing the regulation of gene expression in the progeny. Epigenetic mechanisms that impact skeletal muscle growth and development, for example, has been reviewed by Saccone
and Puri (2010). For many years, we have known that the phenotype of an animal is the result of its genome and its environment. This has often been referred to as the G x E interaction. The environment dictates the degree of gene expression that influences among other things, the animal's size and composition. There is increasing evidence that the environment can influence gene expression later in the life of an individual, but also trans-generationally through DNA or chromatin modifications (reviewed by Jirtle and Skinner, 2007) or diffusible RNA factors (Daxinger and Whitelaw, 2012). This provides a new framework for understanding the impact of broiler breeder nutrition on broiler performance. Many studies of maternal nutrition in many species have shown an impact on progeny body weight (BW) and carcass composition. Unfortunately, there is often little in the way of an obvious pattern in these effects. However, since many epigenetic mechanisms are influenced by the availability of nutrients to the parents, ever-increasing degrees of feed restriction in broiler breeders suggests direct relevance of epigenetics to the field of broiler maternal nutrition. Though important to note the significance, a review of possible epigenetic mechanisms is beyond the scope of the current analysis.

## Challenges of measuring impact of maternal nutrition on broiler performance

It is often difficult to determine patterns in the effects of maternal nutrition on progeny performance. Part of the challenge is the lengthy delay between feeding the pullet and the hen and the resulting progeny performance. There is a time lag of months during which many confounding environmental influences can complicate the interpretation of the effect of maternal nutrition on progeny performance. The following effects summarize some of the important research into maternal nutritional management that may be applied to increase broiler health, livability, growth rate, and yield.

## Hatching egg composition, hatchability and chick quality

### Vitamins and antioxidants

There is clear evidence that incorporation of vitamins and minerals, many of which are antioxidants, into hatching eggs is highly influenced by maternal intake. Vitamin and mineral deficiencies, or large excesses can harm or kill the embryo, reducing hatchability (reviewed by Wilson, 1997). In reviewing the subject of maternal vitamin and mineral nutrition, Hocking (2007) concluded that organic forms of minerals, potentially in slightly higher than recommended concentrations appear to be beneficial for progeny performance, and since that time further research has provided more evidence for this hypothesis. Surai (2000) reported that supplementation of broiler breeder diets with selenium (200 and 400 µg/kg) and vitamin E (40 to 200 mg/kg α-tocopherol) increased egg concentrations of these antioxidants. The liver antioxidant system in 1- and 5-day-old chicks, but not in 10-day-old chicks was enhanced, suggesting a direct effect via increased antioxidant levels in the egg. Rajashree et al. (2014) fed up to 500 µg/kg of organic Se, and observed both an increase in hatchability of over 4%, and increased antioxidant status of eggs. Urso et al. (2015) found that 120 mg/kg of vitamin E increased 29 week hatchability by 3.2 chicks per 100 eggs set, and high organic Se increased hatchling weight by approximately 0.4 to 0.7 g until 33 weeks of age. In terms of effects on progeny growth, An et al. (2012) found that high maternal vitamin E (100 mg/kg vs. 20 mg/kg) increased FCR to 1.75 vs. 1.68, respectively, but reduced drip loss from 2.60 to 1.85%. Vitamin E levels of 120 mg/kg and organic selenium at 400 µg/kg are recommended to increase chick quality and hatchability.

### Immune function

Quereshi et al. (1998) exposed broiler breeders to aflatoxin and observed reduced hatchability and compromised antibody response in broiler chicks. Studying maternal supplementation of antioxidants on broiler innate immunity, Johnson (2013) found that maternal dietary replacement of synthetic vitamin E with a natural vitamin E increased in vitro E. coli bactericidal capacity of chicks from older (58 wk old) breeders, and oxidative burst in chicks from 46 and 58 wk old breeders. Supplementation with 6 and 12 ppm of canthaxanthin increased plasma antioxidant capacity, while supplementation with 50 mg/kg L-carnitine decreased oxidative burst in 4-day old chicks (Johnson, 2013). Hen and chick age had large effects on immune status of broilers. The degree of variation due to hen and chick age in progeny responses to maternal nutrition were strongly established by Johnson (2013) and Torres (2013). Inconsistent reports of the effects of maternal nutrition on broiler performance is likely due to lack of consistency in hen age between experiments.

### Omega-3 fatty acids

Supplementation of maternal diets with long-chain omega-3 fatty acids lowered egg weight, and reduced 11- and 28-day BW, and increased both feed conversion ratio and mortality of offspring (Koppenol et al., 2015b). Further, there was little benefit of omega-3 supplementation for the immune response of the progeny (Koppenol et al., 2015a).
# Managing maternal nutrient intake

## Maternal feed restriction

Waaij et al. (2011) conducted a study to investigate the effects of maternal feed intake on broiler growth, and to specifically to match and mismatch the nutritional environments of hens and chicks. These authors fed 60-week-old broiler breeders at recommended or 35% higher intake, then fed the progeny ad libitum or at 70% of ad libitum. Of hens were fed restricted amounts, their male broiler progeny grew less quickly, while their ad libitum-fed female progeny were 8% fatter. This was a short-term hen feeding study, so it is possible that the nutritional status of the hen caused a shift in egg composition, leading in turn to modified performance of the broilers. However, it raises important questions because notably, they identified outcomes of maternal feed restriction that are undesirable commercially.

Among a suite of complex interactions, Eusebio-Balcazar et al. (2015) observed that maternal nutrient restriction at a rate of 89% of during weeks 20 to 26 followed by rapid growth from 26 to 29 weeks of age worsened the walking ability of corn-fed male broilers, but only when maternal feeder space increased 33% at 22 weeks of age.

## Maternal dietary energy and protein

Quantitative restriction of calorie and protein intake is one of the major strategies employed to optimize broiler breeder BW for reproductive success. There is a growing body of information on the subject, of the impact of amino acid or crude protein (CP) and metabolizable energy (ME) levels on offspring performance, but the approach has not been systematic, and the results often paradoxical. To understand the effects of maternal protein and energy nutrition, the ratio of energy to amino acids, total balanced protein, and other nutrients must be taken into account – not simply dietary level. This is because the partitioning of energy to maintenance, growth, and egg production is dictated by the most limiting nutrient. Growth and reproduction follow the organism's first metabolic priority, maintenance (survival). It is critical to understand maternal maintenance energy and amino acid requirements because the residual energy and amino acids remaining after maintenance is taken care of are used for growth and reproduction. Excess or insufficient energy or essential amino acids precedes complex metabolic decision making that dictates the productivity and health of the hen.

Low ME-high CP diets have generally reduced hatchability (Leeson and Summers, 1991; Pearson and Herron, 1982). These effects on hatchability could be explained by altered composition in the egg, directly affecting the developing embryo. Typically, higher maternal CP intake resulting from higher dietary CP or lower ME levels increases egg and chick weights (Joseph et al., 2000; Enting et al. 2007). Though weakly correlated, this typically translates into larger chicks and larger broilers, but these effects will not be the focus of the current review. The following summarizes much of the recent published literature on the effects of maternal dietary energy and protein levels on progeny performance in the context of some classical works. Because of the complexity of the subject, this section is organized as a series of general hypotheses, specifically supported or contradicted by the research that has been conducted in this area. Factors such as hen age, the timing of the maternal dietary treatment (rearing or laying phases, or both), and the sex of the progeny tested have been considered. Several inconclusive reports have been omitted from this classification scheme.

### Hypothesis 1³: Low maternal protein intake leads to lipid accumulation in offspring.

Of all the propositions, this one had the clearest support from published studies. Several studies provided evidence supporting, and none contradicted this hypothesis.

* Higher abdominal fat percentage in female broilers from hens fed low CP diets (10% compared 16% CP; Lopez and Leeson, 1995)

* Maternal dietary ME restriction during lay (20, 30, and 50%) increased abdominal fatpad, subcutaneous fat, liver fat, and breast muscle fat content (Li et al., 2014)

* Moraes et al. (2014) predicted a 4.8% larger fatpad in broilers from hens fed lower CP during their rearing period (13.8 vs. 15.5% maternal CP)

* Male broilers from hens with low maternal ME intake during the laying period (450 vs. 385 or 325 kcal/d) had more carcass fat (Spratt and Leeson, 1987)

<footnote n="3">³ Corollaries of Hypotheses – since studies almost always tested ratios of energy to protein:</footnote>

<footnote>A. High maternal energy intake leads to the same effect</footnote>

<footnote>B. High maternal protein intake leads to the opposite effect</footnote>

<footnote>C. Low maternal energy intake leads to the opposite effect</footnote>
**Hypothesis 2: Low maternal protein intake leads to lower offspring body weight.**

*In support of hypothesis 2:*

* Female broilers from hens with lower maternal CP intake during rearing had reduced BW (Moraes et al., 2014)

* At a low maternal pullet diet ME level (2,528 kcal/kg), a higher CP level (15.3 vs. 13.7%) increased male broiler BW to 22 day of age (Moraes, 2013)

* Male broilers from hens with higher BW (higher feed intake) had higher BW than those from standard BW hens (Emous et al., 2015)

* Broilers had higher BW when maternal diets contained low ME (ME:CP ratio of 18.0 kcal/g; Peebles et al., 2002)

Because the effect of rearing period nutrition was often more important than the laying phase, these findings point to epigenetic programming. Similarly, after the 'Dutch famine' increased neonatal adiposity was observed in children and grandchildren of women exposed prenatally to nutrient shortage (Painter et al., 2008). Heritable, transgenerational mechanisms for maternal nutrition to influence offspring growth and development are currently being explored (Jirtle and Skinner, 2007; Daxinger and Whitelaw, 2012).

*Contradicting hypothesis 2:*

* Ciacciariello and Tyler (2013) fed digestible maternal lysine levels ranging from 0.44 to 0.75% during the laying phase and found that progeny of young breeders (not beyond 38 weeks of age) fed the lowest lysine levels grew dramatically heavier and converted feed more efficiently

* Higher maternal pullet phase ME level (2,736 vs. 2,528 kcal/kg; 15.3% CP) decreased CP intake and increased female broiler BW to 22 day of age (Moraes, 2013)

Suggesting the possibility of a nonlinear relationship, an optimal maternal pullet phase ME:CP ratio of 18.25 kcal/g was proposed for maximizing female broiler BW (Moraes et al., 2014). For chick weight, a similar optimal maternal laying phase ME:CP ratio of 18.0 kcal/g can be inferred from Leeson and Summers (1991).

**Hypothesis 3: Low maternal CP intake leads to lower breast or total carcass yield.**

*In support of hypothesis 3:*

* Chicks from young broiler breeders (26 weeks of age) fed low digestible lysine (0.6 g/d) had lower BW, carcass weight, and breast weight, and higher drumstick and thigh weights (Mejia et al., 2013). This transient effect was not observed in progeny of 31 and 36 week-of-age broiler breeders.

* Li et al. (2013) suggested that myogenin (MYOG) gene expression was delayed in embryos from hens receiving low feed intake (75% of recommended)

*Contradicting hypothesis 3:*

* Emous et al. (2015) found that male broilers from pullets fed lower maternal dietary CP (ME:CP = 21.2 vs. 18.4 kcal/g) had higher breast yield than from pullets fed higher maternal dietary CP.

* Feeding higher levels of CP to broiler breeder pullets during the rearing phase reduced breast yield in male and female broilers (Moraes, 2013). Dietary combinations that increased CP intake of broiler breeders during rearing and lay had sex-specific additive effects on broilers: it decreased male broiler breast yield, but increased female breast and carcass yield.

* Moraes (2013) observed that in pullets fed 15.3% CP during rearing, a higher ME level (2,736 vs. 2,528 kcal/kg), which decreased CP intake increased overall broiler carcass yield at 40 d of age.

Zhu et al. (2012) fed Chinese yellow breeders maternal diets containing ME ranging from 2,650 to 2,850 kcal/kg, with 15.5 to 17.5% CP. Hens were fed fixed amounts of feed daily. Moderate ME:CP ratios tended to optimize live performance, whereas yield was highest with low CP intake, and lowest at moderate CP intake. This result also points to a nonlinear relationship between CP intake and carcass yield.

**Other notable findings**

Several observations did not fit the relatively simple suite of hypotheses, and suggest that more complex nonlinear relationships should be investigated to explain interactions between maternal ME and CP on progeny performance. Leeson and Summers (1991) reported that a laying phase ME to CP ratio of 18 kcal/g maximized chick weight. Enting et al.
(2007) fed normal and 12 or 23% lower maternal ME levels, and reported an increase in 38 d BW in the moderate ME level, which had a pullet phase ME:CP ratio of 16.0 kcal/g, increasing to 16.6 and 17.7 kcal/g during early and late lay periods. Enting et al. (2007) also reported reduced mortality with their lowest ME level. For male broiler breast and carcass yield, optimal maternal ME:CP ratios during rearing and lay were (average of 16.5 and 17.9) and 18.5 kcal/g, respectively (Moraes et al. 2014). For female broiler breast and carcass yield, they reported optimal maternal ME:CP ratios during rearing and lay of 17.2 and 19.4 kcal/g, respectively. Conversely, the worst maternal diet ME:CP for male broiler breast and carcass yield were 19.2 and 18.5 kcal/g, respectively. For female broiler breast and carcass yield, the worst ME:CP ratios during rearing and lay were 17.2 and 18.5 kcal/g, respectively. Interestingly, maternal diets that were optimal for male performance were worst for female broilers. Increasing the dietary ME:CP ratio from 17.2 to 19.4 kcal/kg appeared to be optimal for mixed sex flocks, as the yield performance of male progeny in this treatment was not significantly different from the optimal male scenario. A lower ME:CP ratio during lay should be avoided (Moraes et al., 2014). In other words, *higher (over-?) consumption of CP during the laying period appeared to have the most negative effects on broiler performance*.

Moraes et al. (2014) found that breast yield increased when maternal ME:CP ratio increased during transition from pullet to laying phase diets (17.5 or 19.0 to 19.4; 17.5 to 18.5 kcal/g; Moraes et al., 2014). This is consistent with current recommended practice, where recommended ME levels are maintained and CP levels drop in later dietary phases.

Lopez and Leeson (1995) observed lower feed intake in male broiler offspring of parents fed 10 to 16% dietary CP, but the broilers compensated such that those from hens fed 10 and 12% dietary CP had lower feed conversion rates than those fed 14 or 16%.

## Management of broiler breeder feeding

### Metabolic stress

Reducing metabolic stress may be key to reproductive success. Zuidhof et al. (2007) demonstrated that radical target BW strategies during the pullet phase can yield similar reproductive outcomes. A key success strategy was adherence to the principle: "Don't let the bird's metabolism know you have made a change to the feed allocation". Stable metabolism in that study was accomplished by weighing birds and determining feed allocations twice per week. Liver weight and composition is an indicator of the metabolic status of birds, and it is a key organ in storage and mobilization of energy. de Beer et al. (2007) reported similar baseline liver glycogen stores, which doubled in skip-a-day fed versus every-day fed broiler breeders. A similar pattern was observed for liver weight. Metabolic stress may also affect progeny. Portha et al. (2011) reviewed the effects of maternal metabolic stress on beta-cell programming. In rats, both undernutrition and obesity decreased the development of beta cells and increased the likelihood of diabetes in offspring and grand offspring. In birds, where there is no physiological contact with the mother during embryo development, epigenetic mechanisms are more likely to be involved in transgenerational inheritance than parental physiological mechanisms. The escalating severity of feed restriction in broiler breeders presents a great model to study maternal metabolic stress on the offspring. However, there are large gaps in the study of transgenerational effects of nutrition in meat-type chickens.

### Precision feeding

Given the challenges around metabolic stress in broiler breeders, strategies for achieving more stable metabolism will likely improve breeder performance, and will likely influence progeny performance. A precision feeding system for broiler breeders has been developed at the University of Alberta (Zuidhof, 2014). The system has been used to raise broiler breeder pullets to sexual maturity with a BW CV of less than 3%. The sequential feeding system is a paradigm shift for feeding breeders, as birds can eat multiple small meals per day rather than binging and fasting, which has become the norm for broiler breeder management. Such a system shows promise for accelerating research on maternal nutrition. It is now possible to control previously confounding effects of BW differences and lack of uniformity, and the true effects of nutrition on breeder and progeny performance will become clearer.

### Considerations for future maternal nutrition research

Much research is still required to fill the knowledge gaps in this complex field. Better control of confounding sources of variation in studies of the effects of maternal nutrition on progeny growth and development is needed. The following categories should be strategically considered in the study of transgenerational impacts of maternal nutrition. Standardization within and between studies will aid the interpretation of experimental results.