MANIPULATION OF INTESTINAL STRUCTURE AND ENZYMES IN ANIMALS

Abstract: A method of decreasing the reduction in digestive enzymes and altering mucosal structure in the intestine of animals is described wherein a probiotic is applied to the animals to achieve such results where the animal may benefit from such a change. Further, a method for maintaining such a benefit after weaning is demonstrated comprising in administering the probiotic to the animal prior to weaning.

Description: MANIPULATION OF INTESTINAL STRUCTURE AND ENZYMES IN ANIMALS BACKGROUND OF THE INVENTION Attempts have increased over the years to try to manipulate the intestinal flora of animals. It has been foun that where one appropriately manipulates the intestinal microflora, there can be a positive effect upon growth rate o the animal. One of the means of affecting the microflora is through the use of antimicrobial agents, and while use of suc agents in feed of animals has been well documented, the effec of changes in the icrobial population of the small and large intestine are not well understood. One antimicrobial compound which has been used includes dietary copper which has been shown to be effective in improving growth rates in pigs. Braude, R. , "Copper is a Growth Stimulant in Pigs.", Animal Production 3:69-75 (1967). Antibiotics have also been used in order to eliminate . pathogenic flora within the digestive tract. However, the agriculture industry has attempted to move away from use of antibiotics since their ultimate effect upon humans is not well established. A widely spreading perception of the public is that antibiotic additives in livestock are undesirable and efforts have turned away from such use and towards other me_-.ns of favorably effecting the intestinal microflora. With both consumer and manufacturer concerned about the side effects of the use of antibiotics as therapeutic agents, probiotics have received more attention in filling this role. A probiotic is generally defined as a live microbial feed supplement which beneficially effects the host by improving its intestinal microbial balance. See, Fuller, R.. "Probiotics in Man and Animals.", J_^ Appl. Bacteriol. 66:365- 378 (1989). A major problem with probiotics is the lack of information on how such substances function. Beneficial affect upon growth is influenced by a wide variety of preparations used, their interaction with other growth- promoting substances in the diet and poor definition of the organisms tested. Therefore, while there is data to suggest that such preparations can be effective, the extent t which they influence animal growth in production and the mechanisms of their actions are still unclear. An understanding of this process would provide a rational basis for the level and frequency of dosing required to optimize beneficial effects. With such knowledge changes in requirement and response to probiotic treatment might be anticipated with the stage of growth and development of the gastrointestinal tract and in animals subject to physiologica or environmental stress which alter the microbial balance of the gut. In many animals there is a phenomena which occurs after weaning of the animal in which it loses weight and is especially susceptible to infection. Such an affect has been frequently studied in porcine animals, since it is not uncommon for there to be considerable livestock losses following weaning. This lag in the growth of pigs after weaning has become a recognized phenomena of "early weaning" production systems. Lecce, J.G., Armstrong, W.D., Crawford, P.C. and Ducharme, G.A., "Nutrition and Management of Early Weaned Piglets: Liquid versus Dry Feeding.", _^ Anim. Sci . 48:1007-1014 (1989). The intestinal structure has been observed to change pos weaning. Transverse sectioning of the gastrointestinal tract shows that all regions of the tract are structurally similar and beneath the outer serous coating are layers of musculature. The innermost layer of the tract has three components: the muscularis mucosae which consists of two layers of smooth muscle, a thick layer of connective tissue, and a mucous membrane. The mucous membrane, luminal surface of the tract, is composed of columnar epithelial cells and al but the oesophagus. The small intestine has depressions throughout which project down into the connective tissue (lamina propria) creating what is termed the crypts of Liberkhun. The small intestinal surface is further increased by folding of the mucosa and sub-mucosa and by mucosa projections which are termed villi. The epithelial cells which cover the villus are continuous with those lining the crypts. Weaning has been noted to have marked effects on thi structure and function of the small intestinal epithelium. Miller, B.G., James, P.S., Smith, N.W. and Bourne, F.J., "Effective Weaning on the Capacity of Pig Intestinal Villus t Digest and Absorb Nutrients.", ^ Agric. Sci. Camb. , 107:579- 589 (1986). Reduced height and increased complexity of the villus structure occur post-weaning. Post weaning reductions in villus height, increases in crypt depth as crypt cell production increases-results in migration onto the villus of younger enterocyte cells which may be present upon its surface for a shorter period of time. This limits the digestive and absorptive capacity of the epithelial tissue. Reductions in the digestive and absorptive ability of intestinal tissue as a consequence of this may be the cause of checks in pig growth seen post-weaning. In addition, impaired intestinal function coupled with alterations in the composition of the gastro¬ intestinal microflora, i.e. particularly increased numbers of enteropathogenic strains of Escherichia coli can result in acute enteric infection after weaning. Thus, there is a clear need for better understanding of the actions of probiotics in the intestine of animals, in order to be able to better use and apply such probiotics. Further, there is also a need for overcoming negative affects on the intestinal tract of animals, and in particular porcine and other livestock animals, which occur post-weaning. This invention relates to application of probiotics to increase digestive enzymes in the intestinal tract, to improve the intestinal epithelial structure for increased digestive and absorptive ability, and to reducing negative effects which occur in the intestinal tract post-weaning which can cause decrease in growth of animals. Thus, it is one object of this invention to provide for a means of altering digestive enzymes in animals that are benefited by such altering. A further object of this invention is to provide for a method of altering the mucosal structure of the intestinal tract in animals, where such animals are benefited by such change. A still further object of this invention is to cause changes to the intestinal tract and the chemical components o the same in order to decrease negative effect on the intestinal tract of animals. A still further object of the invention is to provide fo a method of improving animal health post-weaning. Still further objects of the invention will become apparent through the following disclosure. SUMMARY OF THE INVENTION The invention relates to a method to alter digestive enzymes in animals where the animal can benefit from such alteration comprising administering to the animals probiotic strains of Lactobacillus and Streptococcus. The invention also relates to a method of altering mucosal structure in the intestinal tract of animals where the animal can benefit from such alteration through the application of such probiotic strains. The invention provides for a method of improving animal health post-weaning by applying such strains to the animal. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a graphic depiction showing alkaline phosphatase activity in control, tylosin, and Probios-treated pigs at 7,17,42 and 80 days of age. Fig. 2 is a graphic depiction showing sucrase activity i control, tylosin, and Probios-treated pigs at 7,17,42 and 80 days of age. Fig. 3 is a graphic depiction showing lactase activity i control, tylosin, and Probios-treated pigs at 7,17,42 and 80 days of age. Fig. 4 is a graphic depiction showing tripeptidase activity in control, tylosin, and Probios-treated pigs at 7,17,42 and 80 days of age. Fig. 5 is a graphic depiction showing dipeptidase activity in control, tylosin, and Probios-treated pigs at 7,17,42 and 80 days of age. Fig. 6 is a graphic depiction showing alkaline phosphatase activity in control and Probios-treated pigs at 8,20,28 and 35 days of age. Fig. 7 is a graphic depiction showing lactase activity i control and Probios-treated pigs at 8,20,28 and 35 days of age. Fig. 8 is a graphic depiction showing tripeptidase activity in control and Probios-treated pigs at 8,20,28 and 35 days of age. Fig. 9 is a graphic depiction showing dipetidase activity in.control and Probios-treated pigs at 8,20,28 and 35 days of age. DETAILED DESCRIPTION OF THE INVENTION The mode of operation of probiotics in animals, and in particular how it may affect animal's intestinal tract is not well understood. As has been noted, the increased understanding of such operation would greatly assist in understanding when, how and with what strains to apply probiotics to animals. It is the surprising finding of this invention that probiotics alter digestive enzymes, which enzymes are associated with improved capacity to digest nutrients. The inventors have also discovered that the mucosal structure of the intestinal epithelial surface in the intestinal tract of animals is changed in a manner which exposes more mature cells associated with better capacity to digest nutrients than younger cells. Where such probiotics are applied to an animal prior to weaning, it has further been found that the probiotic will increase such enzymes and mucosal structure associated with improved nutrient digestion and can decrease negative post-weaning effects. The digestive enzymes studied here included sucrase (sucrosefo -D-glucohydrolase; lactase ( f -D-galactoside galactohydrolase); dipeptidase (substrate L-leucylglycine) an tripeptidase (substrate L-leucylglycylglycine) . These enzyme are active in the final processes of digestion prior to the absorption of nutrients into the body. Of these, the carbohydrates are located in the brush border membrane of the enterocyte cell and the dipeptidase in the cytosol. Tripeptidase activity is located primarily, although not exclusively, in the brush border region of the cell. Of thes enzymes studied, it was found that there was a significant affect on all enzymes but tripeptidase when probiotic is applied. As the experiment described below shows, in piglets in which Probios was applied beginning at 17 days after birth, the enzymes were found to be lower in the control group versu the group in which Probios was added, or in which a control antibiotic was added. Further, the typical pre-weaning to post-weaning decline in enzymatic activity was found to be reduced considerably by the application of Probios. Additionally, morphology of the intestine was affected when the probiotic was applied. The villus height and ratio of villus height to crypt depth was not as greatly reduced pre- to post-weaning when the probiotic was applied. The probiotic administration to pigs influenced the pre-weaning changes in mucosal structure typically by preventing the reduction of villus height and crypt elongation between Day 7 and 17 of age. While not wishing to be bound by any theory, it is believed that this result of enhancement effect upon enzyme activities is a consequence of change in epithelia cell turnover, resulting in the presence upon the villus of enterocytes which are more numerous and/or functionally mature. Post-natal changes in villus structure occurring in the pig may be influenced by the composition of the endogenous microbial population. Probiotic administration, it is theorized, influences the balance of microorganisms and the pattern of bacterial metabolism in the gastrointestinal tract resulting in the differences in small intestinal histology and enzyme activity observed in Experiment 1. Applying the probiotic prior to weaning has a favorable affect upon the growth rate of the animal as well. The growth rates of the animals in Experiment 1 increased from Day 26 on when compared to the control group. In the antibiotic comparison, an increase in growth was seen as compared to the control, but not until Day 36. The probiotics of this invention can take a variety of forms as long as they improve enzyme activity and structure of the intestine. The first preparation here is a 12 strain version including Lactobacillus acidophilus (3 strains); L. casei (one strain), L^ plantarium (four strains) and Streptococcus faeciu (2 strains). The five strain version includes _^ acidophilus, L. c. ei, two strains of ^ plantarum and Streptococcus faecium. These are deposited in Germany at the Deutsche Sammlong von Mikroorganismen Und Zell Kulturen G bh. and will be deposited with the American Type Culture Collection to be made available to the public upon allowance of any claims. The form of administration of the probiotic has not been found to be critical. In livestock it typically is administered in the form of granules included in feed. The granules are ordinarily applied at times of stress, arrival or change in ration of feed. A gel composition may be used to administer the probiotic and may supplement the addition of granules to feed. A gel is typically administered at birth, 7 to 10 days of age and at weaning or other times of stress. Since the detrimental enzyme reduction and intestinal structure changes occurring post weaning can be impacted by the probiotic, dosing prior to weaning is most effective. It is known to include other components in such gel compositions and the additions are not critical as long as they do not interfere with the increased enzyme activity or changed intestinal structure. Additions such as those to improve delivery include yeast, vegetable oil, silicon dioxide, titanium dioxide, vitamins, coloring agents and preservatives. The amount of lactic acid bacteria included must be sufficient to cause the desired enzymatic and structural changes to the intestine. The effective amount used in these experiments include about 10 7 viable counts per gram of composition. The following is presented by way of example and is not intended to limit the scope of the inventions. EXPERIMENT 1 Measurement of growth rate occurred from birth to 80 days of age and was compared with the growth rate of animals fed diets containing no growth promoting agent (negative controls), with pigs receiving an antibiotic growth-promoter used in the pig industry (positive controls) and the probiotic described. Aspects of the microbiology and development of the digestive track at 7, 17, 42 and 80 days of age were studied as reflected below. PROBIOTIC PREPARATIONS The probiotic preparations administered during this study were Probios ® brand (Pioneer Hi-Bred International, Inc., Johnston, Iowa, U.S.A.) which contained twelve bacterial strains, including dried Lactobacillus plantarum. Streptococcus faecium, Lactobacillus casei and Lactobacillus acidophilus and their fermentation products. The preparations used in this experiment were of two types: Probios Oral Gel for Swine Composition: Dried fermentation products and selected strains of lactic acid bacteria (7 Lactobacillus spp. and 5 Streptococcus spp. ) together with: yeast culture, vegetable oil, sucrose, silicon dioxide, titanium dioxide, vitamins A,D,E and B,F,D and C yellow No. 6 Lake, Polysorbate 80, TBHQ and ethoxyquin preservatives. Total lactic acid bacteria = 10 7 viable counts/g. Recommended dosage: Newborn pigs - 1 g at birth or at first handling. An additional 1 g given 7 to 10 days later. Weaning/starter pigs - 2 g at weaning, during ration changes and other environmental changes. Probios Granules for Swine Composition: Dried fermentation products and selected strains of lactic acid bacteria (7 Lactobacillus spp. and 5 Streptococcus spp. ) together with yeast culture, calcium carbonate, corn cob fractions, vegetable oil, TBHQ and ethoxyquin preservatives. Total lactic acid bacteria = 10 7 viable counts/g. Recommended dosage: Weaning and starter pigs (up to 18 kg) - granules at a rate of 1 kg/ton of creep feed during periods of environmental changes, upon arrival, or ration change. Growing pigs (18 to 45 kg) - granules at a rate of 500 g/ton of complete feed during periods of environmental changes, upon arrival, or ration change. Finishing pigs (45 kg to market weight) - granules at a -rate of 250 g/ton of complete feed during periods, of environmental changes, upon arrival, or ration change. Sows - granules added to ration at a rate of 10 g/head/day for five days prior to farrowing. Granules at a rate of 500 g/ton of complete feed during lactation. TYLOSIN INCLUSION IN POSITIVE CONTROL DIETS Tylosin is a acrolide antibiotic, isolated from Streptomyces fradiae (Brander, G.L. "Chemicals for Animal Health Control" Taylor and Francis Ltd. London (1986)) and active against Gram- positive organisms. At subtherapeutic levels tylosin is used extensively as a growth-promoter in pigs and poultry. In the present study tylosin was included in 'positive control' diets as Tylamix Premix 100 g/kg (Product License No 0006/4055. Elanco Products Ltd., Basingstoke, England.) each kilogram of which contains tylosin phosphate equivalent to 100 g of tylosin base. TREATMENTS The trial consisted of three treatments: (1) basal diet containing no added growth promoter and with copper at nutritional levels; (2) basal diet and antibiotic growth promoter Tylosin (Elanco Products Ltd, Basingstoke, Hants), a macrolide antibiotic isolated from Streptomyces fradiae and active against Gram positive organisms (Brander, Supra, at 40 mg/kg; (3) basal diet and probiotic preparation Probios ® (Pioneer Hi-Bred International Ltd, Johnston, Iowa, USA) administered as an oral gel 24 hours after birth (1 g), again at 7 days of age (1 g) and at weaning (2 g). The probiotic was included as granules in both the creep feed and follow-on diet at 1 kg/ton. Both forms of the probiotic contained 10 7 colony-forming units/g and comprised a mixture of multiple strains of Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus casei and Streptococcus faecium. EXPERIMENTAL ANIMALS The present study was carried out on litters of cross¬ bred (Large White/Landrace/Duroc) piglets, and because of space limitations in the farrowing house was organized in three phases with two of the treatments represented in each phase. All four sows in each phase farrowed within 48 hours of each other and there was a period of 2 weeks between phases. Sows were paired according to farrowing date, and within each pair of sows each litter was split into two, keeping the sex ratio equal in each half. Each of the sows, therefore, had a cohort of piglets half of her own and half cross-fostered. Runts were included in the cross-fostering process. Selection and allocation occurred on three occasions (i.e. the experiment was conducted in three phases). The treatments represented in a particular phase were randomly allocated to the four cohorts such that within each pair of sows one cohort was allocated to each treatment. While in the farrowing house all cohorts were penned individually, and following weaning animals were penned in treatment groups with the two cohorts from each treatment in each pen. At all time precautions were taken to avoid cross-contamination between treatment groups. DIETS AND FEEDING The creep feed and the follow-on ration were formulated to provide respectively 253 and 200 g crude protein/kg (nitrogen x 6.25) with lysine contents of 0.133 and 0.11 g/kg. The diet formulations and relevant chemical analyses are shown in Table 1. Both diets contained copper at nutritional levels (20-70 mg/kg). Litters were offered creep feed from 7 days of age and piglets were weaned from the farrowing house to flat- deck housing at 21 days of age. All pigs were weighed at weekly intervals. TABLE 1 Components of creep feed and follow-on diet (g/kg fresh weight) and dietary nutrients Wheat Barley Dry skimmed milk Soya-bean meal Fishmeal Vitamin-mineral mix Dry matter (DM:g/kg) Ash (g/kg DM) Oil (g/kg DM) Crude protein (nitrogen x 6.25: g/kg DM) Gross energy (MJ/kg DM) Copper (mg/kg DM) TISSUE SAMPLING On each of days 7,17,42 and 80, one pig was randomly selected from each cohort for tissue sampling. At these times an attempt was made to balance for sex and for natural/foster mother. This process resulted in four pigs per treatment being sampled at each of the four ages. In order to obtain tissues that had not been subjected to mucosal shedding, animals were restrained and anaesthetized with Halothane (4%)-oxygen-nitrous oxide administered via an open mask. Following laparotomy the animals were killed by an injection of sodium barbitone given via the portal vein. The gastrointestinal tract was removed immediately and the small intestine freed from the mesentery. Samples of mucosal tissu were isolated from 50 mm lengths of intestine taken at sites 0.05,0.1,0.3,0.6 and 0.8 along the small intestine. Intestinal sections were opened longitudinally, rinsed with ice-cold saline (9 g sodium chloride/1), blotted dry and placed on a chilled porcelain tile. Mucosa was scraped from the muscle layer using a microscope slide, wrapped in tin foi and snap frozen in liquid N 2 before being stored at -70° . ENZYME ASSAYS All assays were carried out on homogenates of mucosal tissue obtained by thawing approximately 1 g tissue and homogenizing it in 15 ml of a solution containing 50 mM- mannitol and 2 mM-Tris-hydrochloride (pH 7.1) using twelve strokes of a teflon-glass homogenizer. The homogenate was made up to 25 ml with the same buffer and portions stored at -70° before being assayed. Sucrase and lactase were assayed by the method of Dahlqvist, Arne, "Method for assay of intestinal disaccharidases" Analyt. Biochem. 7:18-25 (1964), and dipeptidase (substrate L-leucylglycine) and tripeptidase (substrate L-leucylglyclglycine) by that of Nicholson, J.T. and Kim, Y.S., "A one-step L-aminoacidoxidase assay for intestinal peptide hydrolase activity" Analyt. Biochem. 63:110-117 (1975) incorporated herein by reference. In these assays the hydrolysis of the di- or tripeptide to release amino acids is linked to oxidation of the leucine by L-amino acid oxidase with the resulting formation of hydrogen peroxide. Oxidation of o-dianisidine by hydrogen peroxide following the action of peroxidase results in a specific end- point to the reaction with an absorbance at 530 nm. In using the substrates leucylglycine and leucylglycylglycine, the activities of the two hydrolases could be distinguished from one another. The end product of the tripeptidase assay, i.e. glycylglycine, if hydrolysed by dipeptidase activity does not react in the assay system. All assays were adjusted to ensure linear rates of reaction over time with volume of homogenate added and substrate concentration. SCANNING ELECTRON MICROSCOPY (S.E.M) Changes in overall intestinal morphology with age was assessed from sections viewed under S.E.M.. From tissues fixed in 4% neutral buffered formaldehyde smaller sections (3 mm x 3 mm) were taken and dehydrated in a series of ethanol solutions (25,50,70,90,95 and 100% v/v) , 30 minutes in each. Tissue sections were then processed in the Department of Plant Biology, University of Newcastle upon Tyne. Each section was placed in 50% amylacetate: 50% ethanol for 15 minutes, followed by 100% amylacetate. Finally, tissue sections were critical-point dried (to avoid distortion) and carbon and gold 'splutter-coated' before examination under S.E.M. (Jeol SI S.E.M., Jeol (UK) Ltd., London). LIGHT MICROSCOPY. A quantitative study of changes in intestinal morphology was performed on tissue sections viewed under the light microscope. From tissues fixed initially in 3% glutaraldehyde smaller sections (1 mm x 2 mm) were taken under a Vickers M69 dissecting microscope and stored overnight in phosphate buffer (pH 7.4). Tissue sections were then processed by the Department of Anatomy, Medical School, Newcastle upon Tyne. Intestinal sections were viewed (x50) using a Vickers V80 stereoscopic microscope and accurately drawn using a camera lucida attachment. A scale (0-50 urn) was drawn (x50) from a stage micrometer, copied onto an acetate sheet and used to overlay the camera lucida drawing to allow precise measurement of the tissue section. Measurements taken included villus height (distance from villus tip to villus/crypt junction) and crypt depth (distance from villus/crypt junction to muscularis mucosa). On average 6 measurements of each parameter were made per section, and the ratio of villus height:crypt depth calculate . STATISTICAL ANALYSIS The data were analyzed using the GLM statistical package (Baker, R.J. and Nelder, J.A. , "GLM System Release" 3 Numerical Algorithms Group, Oxford (1978)) which allows for the sequential fitting of all factors which may affect the variable being analysed. The initial analysis was carried ou across all ages studied with the summed data for all sites within the intestine and was designed to test whether there were any significant effects of age and treatment or the 'blocking' factors phase, sex and mothering. The latter factor was included to identify effects that might have arise due to the fact that some pigs were reared on a foster rather than a natural mother. In addition the interactions of all the factors were fitted and the residual sums of squares used to calculate the error term against which all the null hypotheses were tested. As a result of this initial analysis it was determined that the factors sex and mothering had no significant effects on any of the enzyme activities measured and the analysis was consolidated to determine the effects of phase, treatment and age and treatment x age interactions. Phase was retained in the analysis as. it was found to be significant in a number of cases although this may have been related to the unbalanced nature of the experimental design (not all treatments were included in each phase) rather than any biological effect. The analysis also included splitting the age effect into pre- and post-weaning (7 + 17 d v. 42 + 80 d) and the treatment effect into control v. tylosin + Probios and tylosin v. Probios. An outline of the analysis of variance table is shown in Table 2. Data analysed at each of the four ages of pig studied were expanded to include values for each of the five intestinal sites. The factors were fitted in the analysis as phase, treatment, intestinal site and treatment x intestinal site and subsequently the effect of treatment divided to consider the analysis of control v. tylosin + Probios and tylosin v. Probios. Intestinal site was also re-analysed to consider the effect of proximal (0.05, 0.10 and 0.30) sites v. distal (0.60 and 0.80) sites. An outline of the analysis of variance table is shown in Table 3. TABLE 2 Sources of variation and degrees of freedom for analysis of data for enzyme activity summed across all intestinal sites for pigs at 7,17,42 and 80 d age Source of Variation Degrees of Freedom Total 47 Phase 2 C v. T + P 1 T v. P 1 Pre v. Post 1 Other age effects 2 (Pre v. Post) x (C v. T + P) 1 (Pre v. Post) x (T v. P) 1 Other treatment x age effects 4 Residual 34 Treatment: C, basal diet: T, basal diet + tylosin (40 mg/kg): P, basal diet + Probios; age: Pre, pre-weaning (7 d + 17 d): Post, post-weaning (42 d + 80 d) . TABLE 3 Sources of variation and degrees of freedom for analysis of effects of treatment and intestinal site on enzyme activity for pigs at 7,17,42 and 80 d of age Source of Variation Degrees of Freedom _ 2 1 1 1 3 1 1 6 Residual 43 Treatment: C, basal diet: T, basal diet + tylosin (40 mg/kg): P, basal diet + Probios: site, Prox, proximal sites (0.05, 0.10, 0.30): Dist. distal sites (0.60, 0.80). RESULTS The Activities of Mucosal Enzymes The mean values (+ SEM for activities of alkaline phosphates shown in measurements of IU/g mucosal wet weight (Figure 1), sucrase (Figure 2), lactase (Figure 3), tripeptidase (Figure 4) and dipeptidase (Figure 5) at five intestinal sites in pigs is shown on treatment at 7,17,42 and 80 days of age. Table 4 summarizes the affects of treatment and age on each mucosal parameter studied for all intestinal sites and pigs on each treatment at 7,17,42 and 80 days of age. TABLE 4 • Summary of the affect of treatment, age and treatment x age on enzyme activity in the small intestine of the pig summed across all intestinal sites Enzyme activity Treatment Age Treatment x age Sucrase NS Pre<Post*** [EC 3.2.1.48) Lactase NS Pre>Post*** (EC 3.2.1.23) Dipeptidase NS Pre>Post*** (EC 3.14.13.11) Tripeptidase NS NS NS (EC 3.4.11.4) Treatment: C, basal diet: T, basal diet + tylosin (40 mg/kg): P, basal diet + Probios: age: Pre, activity in pre-weaned pig (7 and 17 d of age): Post, activity in post-weaned pigs (42 and 80 d of age): NS, not significant. *P<0.05; **P<0.01; ***P<0.001. Activities of alkaline phosphatase, lactase and dipeptidase were significantly greater in pre-weaned pigs compared to post-weaned animals. Conversely, sucrase activit increased between pre- and post-weaning periods. There was n significant change in tripeptidase activity pre- to post- weaning. There was significant effect at certain ages of the pig from treatment with Probios and antibiotic treatment on all enzymes except for dipeptidase. 7 DAYS OF AGE: At this age alkaline phosphatase activity showed significant interactions between treatment and site, the increase in activity from proximal to distal small intestine being significantly lower in control pigs compared with treated animals (p<0.05 for IU/gMWW and p<0.01 for specific activity) . Other enzymes at this age did not show treatment effect. The activities of alkaline phosphatase, tri- and dipeptides enzymes in proximal tissue were significantly lower than that in the distal intestine (p<0.001 for all except tripeptidase specific activity for which p<0.05). Conversely, the activity of sucrase was significantly greater in the proximal tissue (p<0.001). There were no significant effects of site on lactase activity. 17 DAYS OF AGE: . The activities of all enzymes studied with the exception of dipeptidase were significantly lower, across all sites, in control pigs compared with treated animals (p.0.001 for sucrase IU/gMWW and lactase activity; p<0.01 for alkaline phosphatase IU/gMWW and sucrase specific activity; p<0.05 for alkaline phosphatase specific activity and tripeptidase activity). The activities of all enzymes studied, with the exception of tri- and dipeptidase expressed as IU/gMWW were significantly higher in tylosin-treated pigs than in Probios- treated animals (p<0.001 for lactase activity; p<0.01 for alkaline phosphatase activity; p<0.05 for sucrase activity and tri- and dipeptidase specific activities). Apart from the affects of treatment, sucrase and lactase activities were significantly higher in tissue from the proximal intestine than .. the distal gut (p<0.001 for IU/gMWW of both enzymes and p<0.01 for specific activity of both enzymes). Conversely the activities of all the other enzymes studied were significantly lower in the proximal intestine compared to the distal (p.0.001 for all activities except tripeptidase IU/gMWW for which p<0.05). The magnitudes of change in activity between proximal an distal regions was significantly lower in control pigs compared with treated animals for sucrase, tripeptidase and dipeptidase (p.0.001 for tripeptidase specific activity; p<0.01 for sucrase IU/gMWW, tripeptidase IU/gMWW and dipeptidase specific activity; p<0.05 for sucrase specific activity and dipeptidase IU/gMWW). Sucrase activity increase between the proximal and distal small intestine in control animals while decreasing in the tylosin- and Probios-treated groups. Conversely, tripeptidase activity decreased between these two regions in control animals while increasing in tylosin- and Probios-treated animals. The increase in tylosin-treated pigs was greater than in Probios-treated p.0.05). Dipeptidase activity in control and treated pigs increased between the proximal and distal small intestine, the increase in specific activity of this enzyme for tylosin- treated being (p<0.01) greater than for Probios-treated animals. There, were no significant interactions between site and treatment for alkaline phosphatase or lactase activities. 42 DAYS OF AGE: Activities in tylosin-treated animals at this age were all significantly lower than in Probios-treated (p<0.001 for sucrase IU/gMWW; p.0.01 for sucrase specific activity; p<0.05 for alkaline phosphatase IU/gMWW). At this age, across all sites, there were no significant treatment affects on alkaline phosphatase specific activity, tri- or dipeptidase activities. Alkaline phosphatase IU/gMWW and sucrase activity were not significantly different between control and treated pigs. Lactase activities in control pigs were significantly (p<0.05) greater than in treated animals. Site was found to have no effect on the levels of alkaline phosphatase activity or tripeptidase IU/gMWW. Sucrase activity and tripeptidase specific activity in proximal tissue were significantly lower than in the distal intestine (p.0.001). Conversely, proximal activity was significantly greater than distal for lactase (p<0.001) and dipeptidase IU/gMWW (p<0.05). Dipeptidase specific activity did not differ significantly between proximal and distal regions. The magnitude of increase in tripeptidase specific activity between the proximal and distal intestine was not significantly different for control pigs when compared with treated animals. However, this increase in tylosin-treated animals was significantly (p<0.05) lower than in the Probios- treated group. 80 DAYS OF AGE: Sucrase and dipeptidase activities were significantly lower across proximal sites studied * than distal ones (p.0.001 for sucrase activity; p<0.01 for dipeptidase IU/gMWW and p<0.05 for dipeptidase specific activity). Conversely, lactase activity was significantly greater in proximal tissue compared with that in the distal intestine (p<0.001). There were no significant differences between the proximal and distal small intestine in levels of alkaline phosphatase or tripeptidase activity. For all enzymes studied, except the dipeptidase, activities at 17 days of age in control pigs, across all sites, were shown statistically to be lower than those in other treatment groups and to be greater in the tylosin- than Probios-treated animals (although not for the tripeptidase when expressed on a mucosal wet weight basis). Specific activity of the dipeptidase in 17 day old pigs was greater in tylosin- than Probios-treated animals but in control and Probios groups the activity was similar. Although statistically there was no treatment effect on activity of the dipeptidase (IU/gMWW) it was greater in tylosin- than Probios- treated pigs and lowest in controls. The inclusion of either an antibiotic or probiotic in the diet influenced the pre- to post-weaning changes in enzyme activity. Effects of Treatment and Age on Intestinal Structure Mean values (+ SE) of villus height, crypt depth and villus height:crypt depth ratio at 0.10 and 0.60 intestinal sites for pigs on each treatment at 7,17,42 and 80 days of age are given in Table 5 below. Initial analysis of villus height:crypt depth indicated a significant interaction between treatment and mothering, the latter was therefore included as a "blocking" factor in subsequent analysis. There was a significant effect of treatment on villus height or crypt depth at particular ages of the animal. Tylosin or Probios administration to pigs during the present experiment was associated with prevention of reduction in villus height and the increase in crypt depth at the 0.10 intestinal site seen between 7 and 17 days of age in control animals. At the 0.60 site only antibiotic-treated pigs showed no obvious loss in villus height between 7 and 17 days of age. All animals had reduced villus height from 17 to 42 days of age at the 0.10 site. At the 0.60 site, there were no marked changes in villus height or crypt depth in control pigs between 17 and 42 days of age, however, reductions and increases respectively in these parameters were apparent in both tylosin- and Probios-treated animals. Probiotic or antibiotic administration to pigs altered the pattern of epithelial structural changes preweaning particularly in the proximal intestine. These changes reflect the theory that there is a slower rate of epithelial cell turnover in these treated animals, and therefore an increased population of functionally mature enterocytes. Among potential benefits conferred by such changes in intestinal functions, a result of treatment may be reflected in improved digestion and increased growth rate post weaning. Probiotics also afford the advantage of avoiding consumer concern associated with antibiotic treatment. EXPERIMENT 2 This experiment further demonstrates the effects of probiotic feeding on animals pre- and post-weaning. The methodology used was essentially the same as in Experiment 1, with the primary difference that the probiotic preparation contained strains of only four Lactobacillus sp. and one Streptococcus sp.. Creep and grower diets varied somewhat from the first experiment. An antibiotic group was not included, and instea a control treatment and a Probios treatment were the only one included. They were varied as follows: TABLE 6 Components of Creep and Grower Diets Creep Grower (% fresh weight) (% fresh weight) Complan 25.0 Barley 48.0 Wheat 24.0 Wheat 25.0 Barley 24.0 Soya bean meal 15.0 Soya bean meal 15.0 Fishmeal 10.0 Fishmeal 10.5 Vitamin/mineral mix 2.0 Vitamin/mineral mix 1.5 Calculated dietary nutrients (fresh weight basis): Protein (%) 23.40 20.40 D.E. (MJ/kg) 15.20 13.10 Av. Lysine (%) 1.39 1.07 Cu (mg/kg) 30.40 36.50 Oil (%) 7.81 1.91 Fibre (%) 2.73 3.84 Salt (%) 0.57 0.45 Methionine & Cysteine (%) 0.86 0.69 Threonine (%) 0.72 0.54 Ca (%) 1.19 1.13 P (%) 0.77 0.71 There were four phases. All pigs were subject to the normal unit regimen and were weaned at 21 days of age with th exception of those animals born in the first and second phase which were weaned at Day 28. Samples were taken at 8 days of age, when the piglet was established on the sow and had not yet taken creep feed; 20 days of age, when the piglet had creep feed available but was not yet weaned; 28 days of age, when the piglet had been weaned for one week and was not full adapted to the grower diet; and 35 days of age, where the piglet had recovered from weaning stress and was adapted to the grower diet. RESULTS The five strain probiotic has the effect of greater recovery of both peptidase enzymes studied. The activities o all enzymes fell between 20 and 28 days in the animals weaned at 21 days and by Day 35 there was evidence of a recovery in activity of both dipeptidase enzymes which was greater in treated animals. The fall in enzymes after weaning in the animals weaned at 28 days was not as great as in 21 day weaned animals. The significant effect of treatment seen in the first experiment was not repeated here, however, there was the recovery affect noted with treatment and a significant increase in dipeptidase was seen in the second experiment at Day 8. There was a reduction in weight seen with control animals past weaning not apparent in Probios-treated animals. The table shows that between 8 and 20 days of age, compared with controlled pigs, there was a smaller reduction in villus height in Probios-treated animals. Between 20 and 28 days of age, the loss in villus neight and therefore the change in villus height:crypt depth ratio was greater in Probios treated pigs than control animals. At the 0.6 site the effect of treatment was apparent at 28 and 35 days of age. This observation was made on crypt depth determinations in one pig per treatment. The probiotic treatment did not have a significant effect upon the magnitude of pre- to post-weaning changes in structural parameters studied with the five strain species, however, the reductions in villus height and villus height:crypt depth ratio between and 20 days of age seen in control pigs appeared to have been increased by Probios administration, although crypt elongatio between these two ages was not distinctly affected by treatment. Conversely, between 20 and 28 days of age the los in villus height seen in control pigs was not as evident in Probios-treated animals. * Pigs weane at ays of age The decline between 20 and 28 days in villus height at the 0.10 intestinal site was not apparent with animals weaned at 28 days. The large loss in villus height at the 0.06 site seen between 20 and 28 days in pigs weaned at 21 dyas did not occur in pigs weaned at 28 days. In the first experiment improvements seen in weight gain were not apparent until Day 26 and Day 36 for the Probios and tylosin groups respectively and it is possible that the animals in the latter experiment would have shown a greater correlation across the board of treatment if measurements had continued. It is of interest that in this experiment there was a reduction in weight associated with weaning in the control group that was not apparent in the Probios-treated animals and the probiotic group had a higher body-weight at Day 34. An effect of treatment upon recovery from the reductions in growth rate and enzyme activity seen at weaning was apparent in both animal trials carried out in this study. Thus, the experiments show that Probios is correlated with effects in alleviating post-weaning detrimental changes in intestinal structure and enzyme reduction. The application of Probios has been associated with increased activity of digestive enzymes which can include alkaline phosphatase, sucrase, lactase and tripeptidase. The effect varies across intestinal sites. Reduction in villus height:crypt depth also demonstrates affects of Probios administration. The redirection occurring typically post weaning in enzyme activity and villus height and accompanying increase in crypt depth is decreased with probiotic use. Therefore, it can be seen that the invention accomplishes at least all of its objectives.

Claims: We claim : 1 A method of stimulating activity of digestive enzymes in animals benefited by such alteration comprising administering to the animal a probiotic composition. 2 A method according to Claim 1 wherein the probiotic composition used comprises Lactobacillus and Streptococcus. 3 The method of Claim 2 wherein the composition used comprises Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus plantorium and Streptococcus faecium. 4 A method according to Claim 3 wherein the probiotic composition used comprises Lactobacillus strains identified by ATCC number , , , , , , , and Streptococcus strains identified by ATCC number 5 A method according to Claim 3 wherein the probiotic composition comprises Lactobacillus strains identified by ATCC number , , , , and Streptococcus strain identified by ATCC number . 6 A method according to Claim 1 wherein the digestive enzyme is selected from the group consisting of sucrase, lactase, alkaline phosphatase, dipeptidase and tripeptidase and combinations thereof. 7 A method of altering intestinal mucosal structure in the intestine of an animal benefited by such alteration comprising administering to the animal a probiotic composition. 8 A method according to Claim 7 wherein the probiotic is a composition used comprises Lactobacillus and Streptococcus . 9 The method of Claim 8 wherein the composition comprises Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus plantorium and Streptococcus faecium. 10 A method according to Claim 9 wherein the probiotic composition used comprises Lactobacillus strains identified b ATCC number , , , , , , , and Streptococcus strains identified by ATCC number 11 A method according to Claim 9 wherein the probiotic composition comprises Lactobacillus strains identified by ATC number , , , , and Streptococcus strain identified by ATCC number . 12 A.method according to Claim 7 whereby an increase in villus-height-to-crypt-depth ratio is obtained. 13 A method according to Claim 7 whereby a reduction in the decrease in villus height to crypt depth ratio occurring post weaning is obtained. 14 A method according to Claim 13 wherein the probiotic is administered to the animal prior to weaning. 15 A method of altering animal intestinal structure and enzymatic processing of ingested nutrients post-weaning in an animal benefited by such alteration comprising administering a probiotic composition to the animal. 16 The method of Claim 15 wherein the probiotic composition used comprises Lactobacillus and Streptococcus. 17 The method of Claim 16 wherein the composition comprise Lactobacillus acidophilus, Lactobacillus casei, Lactobacillu plantorium and Streptococcus faecium. 18 A method according to Claim 17 wherein the probiotic composition used comprises Lactobacillus strains identified ATCC number , , , , , , , and Streptococcus strains identified by ATCC number 19 A method according to Claim 17 wherein the probiotic composition comprises Lactobacillus strains identified by ATC number , , , , and Streptococcus strain identified by ATCC number . 20 The method of Claim 15 wherein the probiotic is administered prior to weaning. 21 The metho.d of Claim 16 wherein the probiotic is administered to a porcine animal. 22 A method according to Claim 21 wherein the probiotic comprises a composition of lactic acid bacteria and is administered in doses of about 10 7 viable counts of lactic acid bacteria per gram of composition. 23 A method according to Claim 22 wherein the probiotic composition is administered in the form of a gel. 24 A method according to Claim 23 wherein the compound administered to newborn animals, at about 7 to 10 days of age and at weaning or times of stress. 25 A method according to Claim 22 wherein the probiotic composition is administered in the form of granules in feed. 26 A method according to Claim 25 wherein the probiotic composition is administered to the animals at times of stress arrival or change in ration of feed. 27 A method * of altering animal intestinal structure and chemical composition post weaning, comprising administering t an animal benefited by such alteration prior to weaning a probiotic composition comprising Lactobacillus and Streptococcus. 28 A method according to Claim 27 wherein the composition comprises Lactobacillus strains identified by ATCC number / / / / i i / and Streptococcus strains identified by ATCC number ,. , 29 A method according to Claim 27 wherein the probiotic composition comprises Lactobacillus strains identified by ATCC number , , , , and Streptococcus strain identified by ATCC number . 30 A method according to Claim 27 comprising stimulating digestive enzymes in the animal and reducing post-weaning decrease in villus height to crypt depth ratio.

Inventors: COLLINGTON K GEORGINA, PARKER DAVID, COLLINGTON, K., GEORGINA, PARKER, DAVID
Applicants: PIONEER HI BRED INTERNATINAL I, PIONEER HI-BRED INTERNATINAL, INC.
Application year: 1992
Main classification: A01N63
IPC codes: A01K67, A23K1, A61D7, A61K35, C12N1, C12R1