Significance of combined nutritional and morphological precaecal parameters for feed evaluations in non-ruminants

In this thesis the hypothesis is tested that the nutritional evaluation of dietary formulations in non-ruminants requires both functional-nutritional and functional-morphological parameters. The functional-nutritional parameters provide data on the outcome of the digestive process. Additionally, the functional-morphological parameters provide information about the effects of feed components on the small intestinal mucosa.Part I (chapters 2 - 4) considers the apparent digestibility as a functional-nutritional parameter for feed evaluation in pigs and roosters, whereas Part II (chapters 5 - 8) presents studies with functional-morphological parameters of the small intestinal mucosa of chickens, calves and piglets in relation to feed composition and additives.FUNCTIONAL-NUTRITIONAL PARAMETERS (PART I)The amount of protein and amino acids, which disappears in the large intestine of pigs, is not available for animal body maintenance and production (Zebrowska, et al ., 1978). Degradation of protein in the large intestines is mainly fermentative resulting in non-amino acid N end products, which are not available to the animal. This finding implies that precaecal digestion rather than whole tract digestion provides a more accurate parameter for the estimation of protein availability (Dierick et al., 1987). The in vivo determination of precaecal protein digestion relies on quantifying the ratio between the amount of the ingested protein to that which disappears proximal to the caecum. In digestibility experiments the diets and digesta, collected immediately after the ileum, are analysed on their protein contents. But digesta also contain undigested dietary protein of endogenous origin. Therefore, this ratio is determined as the apparent digestibility. Apparent digestibility is a quantitative parameter providing information on the digestive progress measured by nutrient disappearance at a defined site.Quantitative studies concerning the digestive processes in the small intestine require reproducible collection of digesta from the small intestine. Present procedures can be divided into techniques by which digesta are collected after sacrifying the animals and techniques based on a surgical intervention. Collection of digesta from animals after euthanasia is often used in experiments with broilers (Ravindran et al., 1999). This method, however, requires a large number of animals and for this reason is not commonly used in pigs. There are different surgical techniques described in literature for precaecal digesta collection. It is generally concluded that flexible (silicone) rubber is preferable to rigid materials. Regarding surgical techniques for intestinal studies in pigs, there is a consensus that simple T-shaped cannulae in the ileum and ileo-rectal anastomose (IRA) may not provide representative samples of digesta and/or may interfere with the animal's physiology (Köhler, 1992), whereas collection of digesta from re-entrant cannulae is considered to be hampered by technical difficulties (van Leeuwen et al ., 1987).In part I of the thesis surgical techniques and procedures for digesta collection in pigs and roosters are described and results of digestibility determinations are given.Chapter 1 describes a surgical procedure, which is called the Post Valve T-Caecum (PVTC) cannulation and is considered to be an alternative to the existing digesta collection methods. The prerequisites of this technique are that there is minimal hinder of the animal's physiology. Moreover, digesta samples should be representative, and the surgical technique acceptable in terms of animal welfare. The PVTC technique relies on partial caecectomy followed by placement of a wide flexible silicone T-cannula in the caecum. A considerable advantage of this technique is that the region of the intestine to be studied is not surgically treated. Gargallo and Zimmerman (1981) studied the possible effects of caecectomy on digestion in pigs. They observed small effects on overall digestibility of cellulose and nitrogen. Their final conclusion was that the absence of the caecum in pigs did not significantly alter digestive function. Darragh and Hodgkinson (2000) commented that the PVTC cannulation procedure appears to be the preferred method for the collection of ileal digesta.Chapter 2 describes digesta collection procedures and implications when using PVTC cannulated pigs. Collection of digesta after PVTC cannulation necessitates the use of an inert marker in the diets, to quantify the amounts of nutrients present in ileal digesta for determination of diet digestibility. Two experiments were conducted to evaluate chromic oxide (Cr 2 O 3 ) and HCl-insoluble ash as digestive markers by determining the apparent digestibility of dry matter (DM) and crude protein (CP). In addition, studies were performed of the effects of age (i.e. three different body weight (BW) classes) on apparent ileal DM and CP digestibilities. In experiment 1, barrows were fitted with PVTC cannulae to determine apparent ileal DM and CP digestibility of a wheat gluten/wheat bran ration and a soybean meal ration. Immediately after the morning feeding ileal digesta were collected on an hourly basis for a period of 12 hours. Subsequently, nitrogen (N) and marker contents were determined in these samples. The postprandial Cr/N ratio was more constant than the HCl-insoluble ash/N ratio. Therefore, chromic oxide is considered more suitable as a marker than HCl-insoluble ash when apparent digestibility of protein is the parameter to be studied. In experiment 2, apparent ileal DM and CP digestibilities were determined in 18 rations using twelve barrows fitted with PVTC cannulas (BW from 40 - 100 kg). The protein sources for these rations were derived from feedstuffs of different origin. Apparent precaecal digestibility differed significantly (P < 0.05) on the marker in four rations for DM and in three rations for CP. Digestibility coefficients were not systematically higher or lower for either marker. Besides these methodological aspects, a slight increase in apparent ileal CP digestibility was observed with an increase in body weight.Chapter 3 examines precaecal digestion of protein and amino acids (AA) in roosters. Similar to pigs, undigested AA which reach the caeca are deaminated by the microflora and the end- products have no nutritional value (McNab, 1989). Moreover, Parsons (1986) observed a closer relationship between amino acid availability measured in chick growth assays, and digestibility determined in caecectomised rather than in intact birds. This means that, in poultry, digestion in the distal region of the intestines, more specifically the caeca, is mainly fermentative and that the AA synthesized in, or disappearing from the caeca, are not available for protein synthesis by the animal. Therefore, a procedure for ileostomy in adult roosters has been described with the use of flexible silicon cannulae. Apparent ileal digestibility coefficients for dry matter (aDC DM), crude protein (aDC CP) and amino acids (aDC AA) were determined in diets formulated with maize/wheat gluten meal, wheat gluten meal, faba beans, lupins, soybean meal and casein as the main protein sources. These determinations were performed in ileostomised roosters fitted with silicon cannulae. In addition, aDC data determined using roosters (present study) were correlated with previously published aDC data of the same diets determined with pigs (van Leeuwen et al., 1996a, 1996b).The ileal aDC CP in roosters significantly (P < 0.05) differed in aDC CP and aDC AA between diets. Over diets significant linear relationships were found for the digestibility data determined with roosters and pigs and inturn explained 85 % of the variation in ileal aDC CP between the six diets evaluated in roosters and pigs. Variation between roosters and pigs in ileal aDC AA could be explained for 62-90%, for the individual amino acids, with the exception of aDC of arginine. The standard errors of prediction of the models for aDC AA in roosters using aDC AA in pigs were < 0.04 percentage units. Although, more work is needed to validate these correlations, it is likely that this approach can be used for the prediction of aDC values for roosters from values determined in pigs. The results showed a similarity in the level of digestibility coefficients for protein and amino acids in both species. This means that, despite the differences in anatomy between pigs and poultry (Moran Jr., 1982) the differences in apparent precaecal digestibility of CP and AA were limited. The two animal species with their differences in intestinal structures, differences in amounts and activity of the endogenous components were both capable of digesting protein to a similar extent suggesting a similar precaecal digestive capacity.Regarding methodological aspects the study showed comparable aDC CP and AA for soybean meal determined in the present experiment with the cannulated roosters and data from literature using adult caecectomised roosters. Secondly, the roosters provided with cannulae introduced after ileostomy can be used for periods up to a year after surgery.FUNCTIONAL-MORPHOLOGICAL PARAMETERS (PART II)The qualitative functional-morphological parameters of the small intestinal mucosa are examined in the chapters 5 - 8.Chapter 5 considers the morphology of the mucosal surface of the small intestine of broilers and the relationship with age, diet formulation, small intestinal microflora and growth performance. The villi of the small intestine were examined with a dissecting microscope and the surface was described using a morphological scoring scale. As illustrated by pictures, zigzag oriented ridges were observed in the broilers, which seem to be characteristic for poultry.The results showed that in clinically healthy broilers the shape and orientation of the small intestine villi were related to the age of the animal and the intestinal location. Effects of dietary composition and microflora are also demonstrated. Fermentable pectin as dietary component decreased the zigzag villus orientation and reduced performance. Addition of glutamin to a soybean diet limited the decrease of the zigzag villus-orientation caused by pectin and had a beneficial effect on performance. An oral challenge with a non-virulent Salmonella typhimurium increased the effects of dietary pectin on the small intestine morphology and performance.Chapter 6, contains a study of the functional-morphological effects of virginiamycin (VM), used as feed additive in piglets. The objective of this study was to determine the effects of VM on morphological parameters of the small intestinal mucosa, animal growth and feed conversion ratio (feed intake/weight gain) in piglets. The study comprised three trials: two experiments to study the morphological effects of VM on the small intestinal mucosa, whereas the third experiment was a performance study. Each experiment comprised a control group fed a diet without VM, and a VM group fed a diet containing 40 mg/kg VM. In the first experiment, the piglets were individually kept and an oral dose of K88 positive enterotoxigenic Escherichia (E.) coli (ETEC) was given as a sub-clinical challenge. The housing conditions in experiments 2 and 3 were according to practical standards. The results showed that the VM decreased feed conversion ratio and increased villus heights in conventionally kept piglets. Crypt depths were decreased in the individually kept piglets seven days after the ETEC challenge. Corpet (1999) and Anderson et al. (2000) reviewed the mode of action of antibiotics as feed additives and suggested that the antibiotics suppress bacterial activity and decomposition of bile salts resulting in a more slender villus structure. Increased villus heights indicated an increased mucosal surface and absorption capacity, which is in agreement with the improved precaecal nutrient digestibility of diets with VM, as observed by Decuypere et al . (1991). The difference in morphological response to the VM illustrated variation in the morphological characteristics between clinically healthy piglets.In chapter 7 the effect of the use of the combination of two bioactive proteins, lactoperoxidase- system (LP-s) and lactoferrin (LF), on a milk replacer diet were investigated. This study examined the severity of diarrhoea, morphology of the small intestinal mucosa and the microbiology of digesta and faeces in young weaned calves.Following weaning, the incidence of diarrhoea and mortality of calves is usually higher than that for unweaned calves (Reynolds et al ., 1981). In conventional calf production, antibiotics are added to the milk replacer to reduce gastrointestinal disorders caused by pathogenic bacteria in the gut. Recent legislation restricts the addition of antibiotics in diets for calves (EC, 1998) because of possible repercussions on human health (Van den Boogaard and Stobberingh, 1996).LP and LF are both specific protein constituents of colostrum. These naturally occurring proteins are probably at least partly inactivated during the processing of milk because of their thermo-instability, and the remaining levels are not constant. Moreover, in dairy milk replacers a significant part of the protein is of vegetable origin and therefore lacks LP and LF.The experiment with calves comprised the first two weeks post weaning. One group received a control diet and a second group a diet with LP-s/LF. Results showed that faecal consistency of the LP-s/LF group, as assessed by faecal consistency scores, was significantly improved compared to the control group. The numbers of E. coli in faeces were significantly lower and the villi in the distal jejunum more finger shaped and longer in those of the LP-s/LF group compared to the control group. These findings showed that the effects of LP-s/LF are mainly located in the distal region of the gastrointestinal tract. Reiter and Perraudin (1991) also showed positive effects of LP-s on live weight change in field trials. Still et al . (1989) studied the effects of a combination of LP-s and LF on the severity of diarrhoea in calves for a period 0 to 6 days after an experimental E. coli infection. They concluded that LP-s/LF had preventive and curing effects after the E. coli challenged infection. The results of the present experiment were in agreement with their observations.Chapter 8 considers the functional-morphological implications of condensed tannins in faba beans (Vicia faba L.). The nutritional value of faba beans is limited by the presence of these tannins (Marquardt et al., 1977). Jansman et al. (1993) studied the effects of tannins on the apparent faecal digestibility of a control diet, a diet containing hulls of white flowering, low-tannin faba beans, and a diet with hulls of coloured flowering, high-tannin faba beans. They concluded that whole tract crude protein digestibility of the high-tannin diet was significantly (P < 0.05) lower than the control and low-tannin diets. This effect was partly explained by an increase of the endogenous fraction in the faeces and by an increase of the undigested tannin-feed complexes. In addition, the present study investigated samples of the proximal-, mid- and distal jejunum were investigated histologically and biochemically. The histological differences between the diets were not significant. However, differences in aminopeptidase activity were observed in the proximal small intestine. The amino-peptidase activity of the high tannin group was significantly (P < 0.05) depressed compared to the control and low-tannin groups. Furthermore, a correlation was calculated within the three groups between amino peptidase activity, as a functional parameter of the brush border, and the apparent faecal digestibility of CP, as a quantitative nutritional characteristic. No significant correlations were found between apparent CP digestibility and the aminopeptidase activity in the animals fed the control or low-tannin diet. But when the high tannin diet was fed, the correlation was significantly positive (P < 0.002; R = 0.91). This correlation indicated that a decreased aminopeptidase activity of the small intestine mucosa explained, at least in part, the effects of tannins on CP digestibility.