K. E. Webb

BOARD-INVITED REVIEW: Peptide absorption and utilization: Implications for animal nutrition and health

Over the last 50 yr, the study of intestinal peptide transport has rapidly evolved into a field with exciting nutritional and biomedical applications. In this review, we describe from a historical and current perspective intestinal peptide transport, the importance of peptides to whole-body nutrition, and the cloning and characterization of the intestinal peptide transporter, PepT1. We focus on the nutritional significance of peptide transport and relate these findings to livestock and poultry. Amino acids are transported into the enterocyte as free AA by a variety of AA transporters that vary in substrate specificity or as di- and tripeptides by the peptide transporter, PepT1. Expression of PepT1 is largely restricted to the small intestine in most species; however, in ruminants, peptide transport and activity is observed in the rumen and omasum. The extent to which peptides are absorbed and utilized is still unclear. In ruminants, peptides make a contribution to the portal-drained visceral flux of total AA and are detected in circulating plasma. Peptides can be utilized by the mammary gland for milk protein synthesis and by a variety of other tissues. We discuss the factors known to regulate expression of PepT1 including development, diet, hormones, diurnal rhythm, and disease. Expression of PepT1 is detected during embryological stages in both birds and mammals and increases with age, a strategic event that allows for the immediate uptake of nutrients after hatch or birth. Both increasing levels of protein in the diet and dietary protein deficiencies are found to upregulate the peptide transporter. We also include in this review a discussion of the use of dietary peptides and potential alternate routes of nutrient delivery to the cell. Our goal is to impart to the reader the nutritional implications of peptide transport and dietary peptides and share discoveries that shed light on various biological processes, including rapid establishment of intestinal function in early neonates and maintenance of intestinal function during fasting, starvation, and disease states.

Energy, Protein, and Seasonal Influences on White-Tailed Deer Fawn Nutritional Indices

Shortand long-term influences of dietary energy and protein on physiological indices of nutritional status were determined in a factorial experiment with 12 male and 12 female captive whitetailed deer (Odocoileus virginianus) fawns. Blood and urine samples were collected after 1, 2, 4, 8, 16, and 32 weeks on experimental diets. Body weights throughout the experiment were greater (P < 0.001) for male than for female fawns. Concentrations of blood urea nitrogen (BUN) and urinary urea/creatinine (U/C) ratios throughout the experiment were greater (P < 0.001) in fawns on high protein diets than in fawns on low protein diets. Blood urea nitrogen concentrations and urinary U/C ratios also were greater (P < 0.01) in fawns on low energy diets than in fawns on high energy diets over the duration of the experiment. A substantial reduction in BUN concentrations was observed after 8 months in those fawns which initially exhibited elevated BUN concentrations. Significant differences were observed among sample periods during the 8-month experiment for hemoglobin, packed cell volume, mean corpuscular hemoglobin content, albumin, BUN, cholesterol, nonesterified fatty acids, total ketone bodies, and urinary U/C ratios. J. WILDL. MANAGE. 46(2):302-312 Physiological indices for assessing nutritional status of populations of wild animals, most notably cervids, have long been the subject of research in wildlife science. Biochemical analyses of blood and urine hold promise as being reliable nutritional indices. However, assessing nutritional status with these methods must necessarily depend on controlled experiments to evaluate their efficiency and accuracy. Several investigators have examined nutritional indices of whitetailed deer under controlled conditions (Kirkpatrick et al. 1975, Seal et al. 1978a, Bahnak et al. 1979). No reports have been found in which differences in nutritional indices according to energy, protein, sex, and duration of dietary exposure were examined in a single experiment. The objective of the present study was to determine the shortand long-term effects of dietary energy and protein on nutritional indices of male and female white-

Gene Expression of Nutrient Transporters in the Small Intestine of Chickens from Lines Divergently Selected for High or Low Juvenile Body Weight

Nutrient transporters in the small intestine are responsible for dietary nutrient assimilation; therefore, the expression of these transporters can influence overall nutrient status as well as the growth and development of the animal. This study examined correlated responses to selection in the developmental gene expression of PepT1, EAAT3, SGLT1, and GLUT5 in the small intestine of chickens from lines divergently selected for 48 generations for high (HH) or low (LL) 56-d BW and their reciprocal crosses (HL and LH). Duodenum, jejunum, and ileum were collected from male and female chicks on embryonic d 20, day of hatch with no access to feed, and d 3, 7, and 14 posthatch. Total RNA was extracted, and nutrient transporter expression was assayed by real-time PCR using the relative quantification method. In comparing male and female HH and LL chicks, there was a mating combination x age x sex interaction for PepT1 expression (P < 0.001), a main effect of sex for EAAT3 (P < 0.05) and SGLT1 (P < 0.001) expression, and an age x sex interaction for SGLT1 expression (P < 0.001). These results demonstrate a sexual dimorphism in the capacity to absorb nutrients from the intestine, which has implications for the poultry industry with regard to diet formulations for straight-run and sex-separate grow-out operations. Results from comparing male LL, LH, HL, and HH chicks indicate that selection for high or low juvenile BW may have influenced the gene expression profiles of these nutrient transporters in the small intestine, which may contribute to the overall differences in the growth and development of these lines of chickens.

Expression profiling of the solute carrier gene family in chicken intestine from the late embryonic to early post‐hatch stages

Intestinal development during late embryogenesis and early post-hatch has a long-term influence on digestive and absorptive capacity in chickens. The objective of this research was to obtain a global view of intestinal solute carrier (SLC) gene family member expression from late embryogenesis until 2 weeks post-hatch with a focus on SLC genes involved in uptake of sugars and amino acids. Small intestine samples from male chicks were collected on embryonic days 18 (E18) and 20 (E20), day of hatch and days 1, 3, 7 and 14 post-hatch. The expression profiles of 162 SLC genes belonging to 41 SLC families were determined using Affymetrix chicken genome microarrays. The majority of SLC genes showed little or no difference in level of expression during E18-D14. A number of well-known intestinal transporters were upregulated between E18 and D14 including the amino acid transporters rBAT, y(+)LAT-2 and EAAT3, the peptide transporter PepT1 and the sugar transporters SGLT1, GLUT2 and GLUT5. The amino acid transporters CAT-1 and CAT-2 were downregulated. In addition, several glucose and amino acid transporters that are novel to our understanding of nutrient absorption in the chicken intestine were discovered through the arrays (SGLT6, SNAT1, SNAT2 and AST). These results represent a comprehensive characterization of the expression profiles of the SLC family of genes at different stages of development in the chicken intestine and lay the ground work for future nutritional studies.

Dietary protein composition influences abundance of peptide and amino acid transporter messenger ribonucleic acid in the small intestine of 2 lines of broiler chicks

This study evaluated the effect of dietary protein composition on mRNA abundance of a peptide transporter (peptide transporter 1, PepT1), amino acid (AA) transporters [Na(+)-independent cationic and zwitterionic AA transporter (b(o,+)AT), excitatory AA transporter 3 (EAAT3), Na(+)-independent cationic and Na(+)-dependent neutral AA transporter 2 (y(+)LAT2), L-type AA transporter 1 (LAT1), and cationic AA transporter 1 (CAT1)], and a digestive enzyme (aminopeptidase N) in 2 lines (A and B) of broilers that differentially express PepT1 mRNA (line B > line A). From d 8 to 15 posthatch, birds were fed 1 of 3 diets. Protein sources included whey protein concentrate, a whey partial hydrolysate (WPH), or a mixture of free AA (AA) identical to the composition of whey. Quantities of mRNA were assayed by real-time PCR in the small intestine of males at d 8, 9, 11, 13, and 15. For all genes except LAT1, abundance of mRNA was greatest in line B birds that consumed the WPH diet (P < 0.006). When mRNA abundance was normalized to beta-actin quantities, this effect disappeared, demonstrating a generalized effect on gene expression in line B birds that consumed the hydrolysate. There was a greater villus height:crypt depth ratio (P < 0.05) in line B birds fed the WPH diet as compared with line A. In conclusion, line B birds, which express greater PepT1, displayed enhanced intestinal mucosal absorptive surface area and differential regulation of PepT1, AA transporters, and aminopeptidase N in response to dietary protein composition.

Mammary Uptake, Portal-Drained Visceral Flux, and Hepatic Metabolism of Free and Peptide-Bound Amino Acids in Cows Fed Steam-Flaked or Dry-Rolled Sorghum Grain Diets

Our objectives were to measure net fluxes of free AA (FAA) and peptide-bound AA (PBAA) across portal-drained viscera, liver, splanchnic tissues, and mammary tissues, and milk AA output of lactating Holstein cows (n = 8, 86 +/- 8 d in milk). Cows were fed an alfalfa-based total mixed ration containing 40% steam-flaked (SFS) or dry-rolled (DRS) sorghum grain. The total mixed rations were offered at 12-h intervals in a crossover design. Blood samples were obtained from indwelling catheters in portal, hepatic, and mammary veins and from mesenteric or costoabdominal arteries every 2 h from each cow and diet. Intake of dry matter was 17.9 and 18.6 kg/d of the SFS and DRS diets, respectively, but dropped to 16.3 kg/d for cows fed the SFS diet in the last 3 experimental days, sampling day included. Milk and milk crude protein yields (kg/12-h sampling) were 13.85 vs. 13.25 and 0.425 vs. 0.396 for cows fed SFS or DRS, respectively, and were not affected by the considerable drop in dry matter intake of cows fed the SFS diet during the last 3 experimental days. The portal-drained visceral flux of total essential FAA was 417 and 442 g/12 h (SEM 63) in cows fed SFS and DRS, respectively. However, the portal-drained visceral flux of 7 essential PBAA out of the 9 determined was numerically greater in cows fed the SFS diet, and total essential PBAA in that treatment was 77.4 +/- 22.2 compared with 35.4 +/- 50.2 g/12 h for cows fed the DRS diet. This phenomenon was again observed in a greater total splanchnic flux (FAA + PBAA) of 462 and 371 g/12 h in SFS- and DRS-fed cows, respectively. Mammary uptake of essential AA from both pools (free and peptide bound), and recovery of essential AA in milk, was again numerically higher in SFS-fed cows. In addition to FAA, quantifying the contribution of PBAA may improve our understanding of tissue use of AA substrates, and this may ultimately lead to improved diet formulations with respect to intestinal absorption and mammary uptake of AA.

Uptake of zinc from zinc sulfate and zinc proteinate by ovine ruminal and omasal epithelia

Uptake and transport of Zn from (65)Zn-labeled ZnSO(4) and Zn proteinate (ZnProt) by ruminal and omasal epithelia were examined by using a parabiotic chamber system. Uptake was measured during a 4-h incubation with 10, 20, or 200 microM Zn as ZnSO(4) or ZnProt in the mucosal buffer (pH 6.0, Krebs-Ringer phosphate). Zinc uptake and transport were also evaluated after simulated ruminal digestion. Buffered ruminal fluid contained a feed substrate and 10 or 200 microM added Zn as ZnSO(4) or ZnProt. In a preliminary experiment, uptake of Zn by omasal tissue was low; thus, the remaining experiments were conducted solely with ruminal epithelium. Incubations to determine the effect of time on Zn uptake from mucosal buffer containing 20 microM added Zn as ZnSO(4) or ZnProt resulted in increased (P < 0.01) Zn uptake as incubation time increased from 30 to 240 min. Zinc uptake was also greater (P = 0.02) from mucosal buffer containing ZnProt compared with ZnSO(4). Zinc uptake from incubations containing 10 or 200 microM was affected by source x concentration (P = 0.05) and concentration x time (P < 0.01) interactions. With 10 microM Zn, uptake was not influenced by Zn source, whereas when 200 microM Zn was added, Zn uptake from ZnProt was greater than from ZnSO(4). Increasing incubation time resulted in increased Zn uptake with 200 microM Zn in the mucosal buffer; however, with 10 microM Zn, uptake did not change after 30 min. After simulated ruminal fermentation, the proportion of Zn in a soluble form was influenced by a source x concentration interaction (P = 0.03). After 18 h of incubation, the proportion of Zn that was soluble was not different between ZnProt and ZnSO(4) in buffered ruminal fluid that contained 10 microM added Zn, but was greater for ZnProt compared with ZnSO(4) with 200 microM Zn in the incubation. Zinc uptake from the aqueous fractions of simulated ruminal digestions containing 200 microM added Zn was greater (P < 0.01) than from those containing 10 microM added Zn. Zinc transport, based on detection of (65)Zn in serosal buffer, did not occur in any of the experiments. The results of the current experiments suggest that absorption of Zn into the bloodstream does not occur from the ruminant foresto-mach; however, Zn uptake occurs in ruminal tissue and is greater from ZnProt than from ZnSO(4).

Eimeria Species and Genetic Background Influence the Serum Protein Profile of Broilers with Coccidiosis

Background Coccidiosis is an intestinal disease caused by protozoal parasites of the genus Eimeria. Despite the advent of anti-coccidial drugs and vaccines, the disease continues to result in substantial annual economic losses to the poultry industry. There is still much unknown about the host response to infection and to date there are no reports of protein profiles in the blood of Eimeria-infected animals. The objective of this study was to evaluate the serum proteome of two genetic lines of broiler chickens after infection with one of three species of Eimeria. Methodology/Principal Findings Birds from lines A and B were either not infected or inoculated with sporulated oocysts from one of the three Eimeria strains at 15 d post-hatch. At 21 d (6 d post-infection), whole blood was collected and lesion scoring was performed. Serum was harvested and used for 2-dimensional gel electrophoresis. A total of 1,266 spots were quantitatively assessed by densitometry. Protein spots showing a significant effect of coccidia strain and/or broiler genetic line on density at P<0.05−0.01 (250 spots), P<0.01−0.001 (248 spots), and P<0.001 (314 spots) were excised and analyzed by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry. Proteins were identified in 172 spots. A total of 46 different proteins were identified. Of the spots with a corresponding protein identification, 57 showed a main effect of coccidia infection and/or 2-way interaction of coccidia infection×broiler genetic line at P<0.001. Conclusions/Significance Several of the metabolic enzymes identified in this study are potential candidates for early diagnostic markers of E. acervulina infection including malate dehydrogenase 2, NADH dehydrogenase 1 alpha subcomplex 9, and an ATP synthase. These proteins were detected only in Line A birds that were inoculated with E. acervulina. Results from this study provide a basic framework for future research aimed at uncovering the complex biochemical mechanisms involved in host response to Eimeria infection and in identifying molecular targets for diagnostic screening and development of alternative preventative and therapeutic methods.

Nutritional consequences of gastrolith ingestion in blue-winged teal: A test of the hard-seed-for-grit hypothesis

One benefit attributed to retention of mineral grit in avian gizzards is enhanced digestive efficiency, which may be realized through higher metabolizable energy of hard seed diets. The hard-seed-for-grit hypothesis also suggests that hard seeds can substitute for grit in the gizzard as an aid to mechanical digestion. However, neither of these presumed nutritional benefits of gastrolith ingestion have been quantified. We maintained captive blue-winged teal (Anas discors) on 4 experimental dietary supplements (none, grit, milo, and both grit and milo), and conducted true metabolizable energy (TME) feeding trials to demonstrate the nutritional consequences of gastrolith consumption. We measured TME of 3 test diets: millet (Echinochloa crus-galli), milo, and smartweed (Polygonum pensylvanicum), Mean TME did not differ among treatments for any of the test diets, suggesting that pretrial gastrolith ingestion did not appreciably increase metabolizability of these foods. There was little evidence that birds retained either pretrial seed supplements or the experimental diets in the gizzard, suggesting that seed substitution did not occur. Birds frequently regurgitated the test diet. Although we corrected food intake for regurgitated food mass, a significant positive relationship between net intake and TME occurred for smartweed. However, variation in net intake and occurrence of regurgitation did not appear to alter the effects of gastroliths on TME. This study does not provide support for either presumed nutritional benefit of gastrolith ingestion. These results may reflect the lack of energetic constraints on food acquisition in captive birds. A similar study of Canada geese (Branta canadensis) showed a significant effect of grit on TME of some foods, suggesting that digestive responses may vary across a gradient in body size.

Predicting metabolizable energy in the diet of ruffed grouse

The percent metabolizable energy (ME) in 13 of 14 diets fed to captive ruffed grouse (Bonasa umbellus) in metabolism trials was highly related (P = 0.0001, R2 = 0.88) to the percent neutral detergent solubles (NDS) minus total phenol content of the diets. A regression equation based on these data provided good prediction of the ME of 8 diets fed to grouse in a 2nd set of metabolism trials. The ME of diets containing acorn meat was underestimated by the prediction equation; therefore, a multiple regression equation based on NDS minus phenols and percent acorn meat was developed to predict the ME of diets containing acorn meat. The effects of storage and drying treatments of forages on measured NDS, total phenols, and predicted ME levels were determined. Oven-drying fresh, leafy forages and oven-drying after frozen storage consistently resulted in lower levels of NDS and total phenols than freeze-drying fresh forages. Effects on predicted ME were small because changes in NDS and total phenol levels are offsetting to a large extent in the prediction equation. J. WILDL. MANAGE. 51(3):560-567 Ruffed grouse food habits are well documented (Brown 1946, Bump et al. 1947, Korschgen 1966, Phillips 1967, Woehr and Chambers 1975, Stafford and Dimmick 1979, Seehorn et al. 1981), but relationships between food habits and diet quality are largely unknown. Little information is available on the ME of grouse forages or diets. This is due in part to a lack of accurate and efficient methods for measuring the ME in the natural diet of ruffed grouse. Studying all forages individually in conventional metabolism trials is impractical because of the varied diet of grouse. In addition, 1-time measurements of ME from metabolism trials does not account for variation among seasons, years, or locations. Predicting the ME of forages of ruffed grouse from forage chemical composition is a promising alternative method because large numbers of forages could be studied much more efficiently and crop contents could be chemically analyzed, thereby eliminating the bias between hand-picked forages and forages selected by wild birds. The Van Soest method of forage chemical analysis (Goering and Van Soest 1970) has proven to be reliable for predicting forage digestion in both ruminant and monogastric mammals (Van Soest 1967, Mould and Robbins 1982, Servello et al. 1983, MacPherson et al. 1985) and may be useful for avian species also. However, high levels of phenolic compounds in forages may complicate prediction of forage digestion when using the Van Soest analysis. Phenols are extracted as part of the theoretically highly digestible neutral detergent soluble fraction; i.e., primarily soluble carbohydrate, protein, and fat. However, they have little or no nutritional value and also may interfere with digestion and absorption (McLeod 1974; Mould and Robbins 1981a,b, 1982). Also, storage and drying treatments can influence measured chemical constituents of forages, especially highly reactive phenols (Mould and Robbins 1981a). The objectives of the present study were: (1) to determine the relationship between forage chemical composition (as measured by the Van Soest method) and forage ME for ruffed grouse, (2) to develop a predictive equation for estimating ME based upon this relationship, (3) to determine the influence of phenolic compounds on prediction of forage ME, and (4) to determine the effects of a number of commonly used storage and drying treatments on NDS and phenol levels and therefore on prediction of forage ME. We thank W. B. Morehead, S. L. MacPherson, and K. B. Preli for assistance with metabolism trials and laboratory analyses. This research was supported in part by the Natl. Rifle Assoc. and the Dep. Fish. and Wildl. Sci., Virginia Polytechnic Inst. and State Univ. The senior author was supported by a John Lee Pratt Anim. Nutr. Fellowship.