Graduate Student Literature Review: Developmental adaptations of immune function in calves and the influence of the intestinal microbiota in health and disease.

This graduate student literature review provides an examination of the ontological adaptations of the calf's immune system and how the intestinal microbiota influences calf immune function in health and disease. Within dairy rearing systems, various nutritional and management factors have emerged as critical determinants of development influencing multiple physiological axes in the calf. Furthermore, we discuss how multiple pre- and postnatal maternal factors influence the trajectory of immune development in favor of establishing regulatory networks to successfully cope with the new environment, while providing early immune protection via immune passive transfer from colostrum. Additionally, our review provides insights into the current understanding of how the intestinal microbiota contributes to the development of the intestinal and systemic immune system in calves. Lastly, we address potential concerns related to the use of prophylactic antimicrobials and waste milk, specifically examining their adverse effects on intestinal health and metabolic function. By examining these factors, we aim to better understand the intricate relationship between current management practices and their long-term effect on animal health.

Heat stress-associated changes in the intestinal barrier, inflammatory signals, and microbiome communities in dairy calves.

Recent studies indicate that heat stress pathophysiology is associated with intestinal barrier dysfunction, local and systemic inflammation, and gut dysbiosis. However, inconclusive results and a poor description of tissue-specific changes must be addressed to identify potential intervention targets against heat stress illness in growing calves. Therefore, the objective of this study was to evaluate components of the intestinal barrier, pro- and anti-inflammatory signals, and microbiota community composition in Holstein bull calves exposed to heat stress. Animals (mean age = 12 wk old; mean body weight = 122 kg) penned individually in temperature-controlled rooms were assigned to (1) thermoneutral conditions (constant room temperature at 19.5°C) and restricted offer of feed (TNR, n = 8), or (2) heat stress conditions (cycles of room temperatures ranging from 20 to 37.8°C) along with ad libitum offer of feed (HS, n = 8) for 7 d. Upon treatment completion, sections of the jejunum, ileum, and colon were collected and snap-frozen immediately to evaluate gene and protein expression, cytokine concentrations, and myeloperoxidase activity. Digesta aliquots of the ileum, colon, and rectum were collected to assess bacterial communities. Plasma was harvested on d 2, 5, and 7 to determine cytokine concentrations. Overall, results showed a section-specific effect of HS on intestinal integrity. Jejunal mRNA expression of TJP1 was decreased by 70.9% in HS relative to TNR calves. In agreement, jejunal expression of heat shock transcription factor-1 protein, a known tight junction protein expression regulator, decreased by 48% in HS calves. Jejunal analyses showed that HS decreased concentrations of IL-1α by 36.6% and tended to decrease the concentration of IL-17A. Conversely, HS elicited a 3.5-fold increase in jejunal concentration of anti-inflammatory IL-36 receptor antagonist. Plasma analysis of pro-inflammatory cytokines showed that IL-6 decreased by 51% in HS relative to TNR calves. Heat stress alteration of the large intestine bacterial communities was characterized by increased genus Butyrivibrio_3, a known butyrate-producing organism, and changes in bacteria metabolism of energy and AA. A strong positive correlation between the rectal temperature and pro-inflammatory Eggerthii spp. was detected in HS calves. In conclusion, this work indicates that HS impairs the intestinal barrier function of jejunum. The pro- and anti-inflammatory signal changes may be part of a broader response to restore intestinal homeostasis in jejunum. The changes in large intestine bacterial communities favoring butyrate-producing organisms (e.g., Butyrivibrio spp.) may be part of a successful response to maintain the integrity of the colonic mucosa of HS calves. The alteration of intestinal homeostasis should be the target for heat stress therapies to restore biological functions, and, thus highlights the relevance of this work.

Induction of leaky gut by repeated intramuscular injections of indomethacin to preweaning Holstein calves.

This study was designed to develop a protocol for repeated intramuscular indomethacin injections to replicate leaky-gut-like symptoms in male Holstein calves to model and study the detrimental effects of leaky gut on gut tissue function and inflammatory response. A generalized randomized block design was used to evaluate how repeated indomethacin intramuscular injections affected the development of leaky gut in 18 male Holstein calves. Animals were enrolled at 3 ± 1 d of life, and after 21 d of adaptation, they were randomly assigned to 1 of 3 treatments consisting of intramuscular saline or indomethacin injections every 12 h for 48 h: (1) control (CTL), saline injection, (2) low intramuscular indomethacin (INDO-L) dosed at 1.2 mg/kg of body weight (BW), and (3) high intramuscular indomethacin (INDO-H) dosed at 2.4 mg/kg of BW. During the challenge, milk intake, starter intake, fecal scores, and rectal temperature were measured daily, and BW was measured at the beginning and at the end of the challenge. Plasma samples were used to measure the recovery of markers of intestinal permeability before and after the challenge by dosing lactulose, d-mannitol, and chromium-EDTA. In addition, several cytokines were measured in plasma during the challenge. Calves were dissected at the end of the challenge to obtain tissue and digesta samples from the gastrointestinal tract and liver. No treatment differences were observed for starter and milk intakes, fecal scores, BW, and rectal temperature. The difference in marker concentrations between pre and post challenges was higher for INDO calves compared with CTL calves in the case of lactulose and chromium-EDTA. In addition, chemokine ligand 2 and 4 and IL-6 were higher for INDO-H calves compared with CTL. Both doses of indomethacin resulted in reductions in villus length and surface area in the distal jejunum and ileum and reductions in crypt depth and width in the colon. We showed that repeated indomethacin injections over a 48-h period induced leaky-gut-like symptoms in a region-specific manner, affecting mainly the distal section of the intestine. This outcome was characterized by histomorphological changes in the distal jejunum, ileum, and colon and by increased gut permeability. Interestingly, changes in liver morphology and immune function also occurred, possibly due to the increased translocation of foreign antigens breaching the epithelial cell wall. The leaky gut challenge model described here could be used to improve understanding of the pathogenesis of intestinal disorders in cattle and provide a reliable alternative for testing feed additives with intestinal health benefits.

Short communication: Promotion of glucagon-like peptide-2 secretion in dairy calves with a bioactive extract from Olea europaea.

Diarrhea episodes in dairy calves involve profound alterations in the mechanism controlling gut barrier function that ultimately compromise intestinal permeability to macromolecules, including pathogenic bacteria. Intestinal dysfunction models suggest that a key element of intestinal adaptation during the neonatal phase is the nutrient-induced secretion of glucagon-like peptide (GLP)-2 and associated effects on mucosal cell proliferation, barrier function, and inflammatory response. Bioactive molecules found in Olea europaea have been shown to induce the release of regulatory peptides from model enteroendocrine cells. The ability to enhance GLP-2 secretion via the feeding of putative GLP-2 secretagogues is untested in newborn calves. The objectives of this study were to determine whether feeding a bioactive extract from Olea europaea (OBE) mixed in the milk replacer (1) can stimulate GLP-2 secretion beyond the response elicited by enteral nutrients and, thereby, (2) improve intestinal permeability and animal growth as well as (3) reduce the incidence of diarrhea in preweaning dairy calves. Holstein heifer calves (n = 60) were purchased, transported to the research facility, and blocked by body weight and total serum protein and assigned to 1 of 3 treatments. Treatments were control (CON), standard milk replacer (MR) and ad libitum starter; CON plus OBE added into MR at 30 mg/kg of body weight (OBE30); and CON plus OBE added into MR at 60 mg/kg of body weight (OBE60). The concentration of GLP-2 was measured at the end of wk 2. Intestinal permeability was measured at the onset of the study and the end of wk 2 and 6, with lactulose and d-mannitol as markers. Treatments did not affect calf growth and starter intake. Compared with CON, administration of OBE60 increased the nutrient-induced response in GLP-2 by about 1 fold and reduced MR intake during the second week of study. Throughout the study, however, all calves had compromised intestinal permeability and a high incidence of diarrhea. The GLP-2 response elicited by OBE60 did not improve intestinal permeability (lactulose-to-d-mannitol ratio) and incidence of diarrhea over the course of the preweaning period. The response in GLP-2 secretion to the administration of OBE reported herein warrants further research efforts to investigate the possibility of improving intestinal integrity through GLP-2 secretion in newborn calves.

Secretion of glucagon-like peptide-2 responds to nutrient intake but not glucose provision in milk-fed calves.

Glucagon-like peptide 2 (GLP-2) is a peptide released by the lower gut that has potent trophic and restorative effects on the intestinal epithelium. Two experiments were conducted to assess the effects of feeding rate and either metabolizable or nonmetabolizable glucose supplementation on GLP-2 concentrations in plasma and intestinal development in Holstein calves. In the first experiment, 48 newborn calves were assigned to 12 treatments (n=4) corresponding to the factorial combination of 4 milk feeding amounts [1.75, 1.32, 0.88, and 0.44% of body weight (BW) as dry matter (DM)] and 3 oral supplementation treatments (nonsupplemented, glucose-supplemented, and 3-O-methyl glucose-supplemented). In the second experiment 30 newborn calves (n=10) were fed milk at a fixed rate of 1.75% of BW as DM and assigned to the same glucose supplementation treatments used in experiment 1 to investigate effects on intestinal development. In the first experiment, we found a saturating response of plasma GLP-2 to increasing feeding levels. The feeding rate at which 50% of the maximal GLP-2 release occurred was estimated to be 0.53% of BW as DM or 30.3% of the maximum feeding rate (1.75% of BW as DM), whereas maximal secretion was estimated to be about 98.6 pmol/L. In turn, feeding 75, 50, or 25% of the maximal feeding rate (i.e., 1.75% BW as DM) resulted in plasma GLP-2 concentrations 87, 72, and 49% of that in fully fed calves, respectively. Neither metabolizable nor nonmetabolizable glucose supplementation affected GLP-2 secretion and no interaction with feed intake level was detected. In the second experiment, no effect of glucose supplementation was observed on intestinal growth, mucosal cell proliferation, or expression of genes related to the actions of GLP-2. Nonetheless, we observed that a pool of genes of the GLP-2 signaling pathway was more abundantly and coordinately regulated in the colon than in the ileum of these animals, indicating an opportunity for dietary induction of the peptide in this organ. In conclusion, during this experiment, plasma GLP-2 concentrations responded in a diminishing return fashion to milk intake but not to glucose supplementation, even at milk consumption levels of only 0.4% of BW as DM.

Dietary preference in dairy calves for feed ingredients high in energy and protein.

In 3 experiments, we assessed preference of recently weaned dairy calves for (1) 8 high-energy feed types [barley meal, corn meal, corn gluten feed (CGF), oat meal, rice meal, sorghum meal, wheat meal, and wheat middlings meal]; (2) 6 high-protein feed types [corn gluten meal (CGM), wheat distillers dried grains, rapeseed meal, soybean meal (SBM), sunflower meal, and pea meal]; and (3) 4 mixtures (50:50) of the highest- and lowest-ranked high-energy and high-protein feeds, to assess whether calves maintain preference for feed ingredients that are included in a mixture. In all experiments, pairwise preference tests were conducted between all feed types (28 different pairwise preference tests in experiment 1, 15 tests in experiment 2, and 6 tests in experiment 3). Each pairwise preference test was conducted by offering ad libitum access to both feed types for 6h. All tests were repeated with 20 Holstein calves. Before this study, calves were offered milk replacer at a rate of 4 L/d and a pelleted starter feed ad libitum. After weaning at 62 d of age, each calf was involved in a pairwise preference test at 3 and 5d postweaning. A preference ratio was calculated for each calf in each test as (intake of feed type A)/(intake of feed type A + intake of feed type B). Preference for feed types was ranked across tests in each experiment using pairwise comparison charts. In experiment 1, the highest-ranked high-energy feed type was wheat meal and the lowest ranked were rice meal and CGF. In experiment 2, the highest-ranked high-protein feed type was SBM and the lowest ranked was CGM. According to the preference rankings from experiments 1 and 2, experiment 3 evaluated (50:50) mixtures of SBM + wheat meal, SBM + CGF, CGM + wheat meal, and CGM + CGF. The mixture of SBM + wheat meal was highest ranked, CGM + CGF was lowest ranked, and the mixtures containing one high-ranked and one low-ranked feed ingredient (SBM + CGF and CGM + wheat meal) were ranked equally. The results of this study indicate that young calves exhibit clear preferences for certain high-energy and high-protein feeds that may be considered highly palatable. Further, preference ranking of feed types provided as 50:50 mixtures was consistent with ranking of individual feed types, suggesting that palatability of mixed starter rations can be improved by inclusion of a preferred feed type.

Blocking opioid receptors alters short-term feed intake and oro-sensorial preferences in weaned calves.

Opioid peptides may participate in the control of feed intake through mechanisms involving pleasure reward linked to consumption of palatable feed. The objective of this study was to determine whether blocking opioid receptors might void oro-sensorial preferences of calves, and affect circulating glucose, insulin, and anorexigenic hormones in fasted and fed calves. Two experiments involved 32 Holstein calves [body weight (BW)=86.5±1.73 kg, age=72±0.6 d]. In experiment 1, all calves received an ad libitum choice of the same feed either unflavored or flavored with a sweetener (Luctarom SFS-R, Lucta, Montornès del Vallès, Spain). Feed consumption was recorded every 2 h from 0800 to 1400 h for 3 consecutive days to verify the establishment of an oro-sensorial preference for sweet feed (SF). The SF was preferred over the control feed (CF) at all recorded times. In experiment 2, calves were subjected to a 2 × 2 factorial design to study the interaction between opioid activity and metabolic state. Half of the calves were fasted for 14 h (FAS), whereas the other half remained well fed (FED). Within each of these groups, at feeding time (0800 h), half of the calves received an i.v. injection of naloxone (NAL, an opioid receptor antagonist; 1 mg/kg of BW) and the other half was injected with saline solution (SAL; 0.9% NaCl). Therefore, treatments were FED-NAL, FED-SAL, FAS-NAL, and FAS-SAL. Blood samples were taken at -10, 20, 180, and 240 min relative to NAL or SAL injections. As expected, cumulative consumption of starter feed was greater in FAS than in FED calves. Total feed consumption 2 h after feeding was lower in NAL than in SAL calves. Calves in the FAS group did not discern between CF and SF during the first 4 h after feed offer. Preference for SF was greater in SAL than in NAL calves. Calves in the FED-SAL treatment preferred SF at 2 and 6 h after feed offer and tended to prefer SF at 4 h after feeding. However, FED-NAL calves did not discern between SF and CF during the first 4 h after feeding and tended to prefer SF only after 6 h from feeding. Plasma glucose, insulin, and cholecystokinin concentrations were greater in FED than in FAS calves. Injection of naloxone decreased plasma glucagon-like peptide-1 (GLP-1) in NAL calves. Blocking opioid receptors reduced intake the first 2 h after naloxone injection in FED calves, altered oro-sensorial preferences, and reduced plasma GLP-1 concentration. In conclusion, the opioid peptide system may control short-term feed intake by modulating the oro-sensorial response triggered by feed consumption, especially when calves are fed ad libitum.

Physiological responses of Holstein calves to heat stress and dietary supplementation with a postbiotic from Aspergillus oryzae.

Increased ambient temperature causes heat stress in mammals, which affects physiological and molecular functions. We have recently reported that the dietary administration of a postbiotic from Aspergillus oryzae (AO) improves tolerance to heat stress in fruit flies and cattle. Furthermore, heat-induced gut dysfunction and systemic inflammation have been ameliorated in part by nutritional interventions. The objective of this study was to characterize the phenotypic response of growing calves to heat stress compared to thermoneutral ad libitum fed and thermoneutral feed-restricted counterparts and examining the physiologic alterations associated with the administration of the AO postbiotic to heat-stressed calves with emphasis on intestinal permeability. In this report, we expand previous work by first demonstrating that heat stress reduced partial energetic efficiency of growth in control (45%) but not in AO-fed calves (62%) compared to thermoneutral animals (66%). While heat stress increased 20% the permeability of the intestine, AO postbiotic and thermoneutral treatments did not affect this variable. In addition, AO postbiotic reduced fecal water content relative to thermoneutral and heat stress treatments. Heat stress increased plasma concentrations of serum amyloid A, haptoglobin and lipocalin-2, and administration of AO postbiotic did not ameliorate this effect. In summary, our findings indicated that heat stress led to reduced nutrient-use efficiency and increased systemic inflammation. Results suggest that the AO postbiotic improved energy-use efficiency, water absorption, and the intestinal permeability in heat stress-mediated increase in gut permeability but did not reduce heat stress-mediated rise in markers of systemic inflammation.

A postbiotic from Aspergillus oryzae attenuates the impact of heat stress in ectothermic and endothermic organisms.

Heat stress is detrimental to food-producing animals and animal productivity remains suboptimal despite the use of heat abatement strategies during summer. Global warming and the increase of frequency and intensity of heatwaves are likely to continue and, thus, exacerbate the problem of heat stress. Heat stress leads to the impairment of physiological and cellular functions of ectothermic and endothermic animals. Therefore, it is critical to conceive ways of protecting animals against the pathological effects of heat stress. In experiments with endothermic animals highly sensitive to heat (Bos taurus), we have previously reported that heat-induced systemic inflammation can be ameliorated in part by nutritional interventions. The experiments conducted in this report described molecular and physiological adaptations to heat stress using Drosophila melanogaster and dairy cow models. In this report, we expand previous work by first demonstrating that the addition of a postbiotic from Aspergillus oryzae (AO) into the culture medium of ectothermic animals (Drosophila melanogaster) improved survival to heat stress from 30 to 58%. This response was associated with downregulation of genes involved in the modulation of oxidative stress and immunity, most notably metallothionein B, C, and D. In line with these results, we subsequently showed that the supplementation with the AO postbiotic to lactating dairy cows experiencing heat stress decreased plasma concentrations of serum amyloid A and lipopolysaccharide-binding protein, and the expression of interleukin-6 in white blood cells. These alterations were paralleled by increased synthesis of energy-corrected milk and milk components, suggesting enhanced nutrient partitioning to lactogenesis and increased metabolic efficiency. In summary, this work provides evidence that a postbiotic from AO enhances thermal tolerance likely through a mechanism that entails reduced inflammation.

Effects of dietary gamma-cyclodextrin on voluntary activity and muscle strength in mice.

Gamma-cyclodextrin (γCD) is a cyclic oligosaccharide consisting of eight α-(1,4)-linked glucopyranose subunits, which is often used in the food and pharmaceutical industries. However, little is known regarding the metabolic activity of "empty" γCD per se. Therefore, in the present study young C57BL/6 male mice received a control diet (CON) or an experimental diet that was supplemented with 12.88% γCD exchanged against corn starch. After 6 weeks of treatment, the voluntary wheel running activity was monitored and the muscle strength of mice was measured by employing Kondziela's inverted screen test and forelimb grip strength assay. The γCD-treated mice covered a significantly larger distance per night (CON 8.6 km, γCD 12.4 km) and were significantly longer active (CON 340 min, γCD 437 min). Moreover, γCD-treated mice significantly performed better at the inverted screen test indicated by an enhanced Kondziela score (CON 3.10, γCD 4.63). These data suggest that dietary γCD leads to an increased endurance. We also found a slightly anti-glycemic effect of γCD during oral glucose tolerance test. However, our mice from the γCD group exhibited no difference in terms of GLUT2 protein level in ileum tissue nor increased muscle glycogen storage. Furthermore, γCD exhibited no DPP-4 inhibitory activity in vitro. By analysing candidate muscle genes and proteins related to endurance and muscle performance we did not observe any differences in terms of Sirt1, Pgc1α, Cpt1b, Mef2c, Myh1 and Myh2 gene expression levels as well as total oxidative phosphorylation (OXPHOS), mtTFA and GLUT4 protein expression levels in skeletal muscle in response to γCD. We could not fully establish the exact underlying molecular mechanisms of the fitness improvement by dietary γCD which warrants further investigations.

Effects of a bioactive olive pomace extract from Olea europaea on growth performance, gut function, and intestinal microbiota in broiler chickens.

The present study aims to investigate the effects of supplementing broiler diets with a bioactive olive pomace extract (OE) from Olea europaea on growth performance, digestibility, gut microbiota, bile acid composition, and immune response. To this end, three hundred and six 1-day-old broiler chickens (Ross 308) were housed in floor pens (6 pens/treatment, with 17 birds/pen). Animals were fed with a standard non-medicated starter diet for 21 D, and from 22 to 42 D of age with their respective experimental diet: a negative control with no additives (Control), a positive control with 100 ppm of monensin (Monensin) and the basal diet supplemented with 750 ppm of an OE (Lucta S.A., Spain). Feed intake and growth rate were monitored weekly throughout the trial. From 21 to 42 D of age, no significant differences in feed intake were observed among dietary treatments; however, lower average daily gain and higher feed conversion ratio (P < 0.05) was observed in birds fed the Control compared to Monensin and OE groups. Performance of birds fed OE or Monensin was similar throughout the trial. The apparent ileal digestibility of crude protein was higher in birds fed Monensin than Control treatment (P < 0.05). No significant changes on bacterial composition at a family level were observed in the caeca of birds fed the experimental diets. Moreover, no significant differences on plasma and intestinal bile acid composition were observed among treatments. Birds fed the OE showed a significant decrease of IL-8 expression in the ileum (P < 0.05). Additionally, the expression of TGF-ß4, and Bu-1 was significantly upregulated (P < 0.01) in broilers fed the OE and Monensin diets compared to those fed the Control. In conclusion, the inclusion of 750 ppm of a bioactive olive pomace extract from Olea europaea in broiler chicken diets improved animal growth likely as result of its anti-inflammatory properties.

Dietary resveratrol impairs body weight gain due to reduction of feed intake without affecting fatty acid composition in Atlantic salmon.

Recent studies suggest that the use of vegetable oils at expense of fish oil in aquaculture feeds might have potential negative effects on fish redox homeostasis and adiposity. Resveratrol (RESV) is a lipid-soluble phytoalexin present in fruits and vegetables with proven in vivo antioxidant function in animals. The present study aims to assess the potential use of RESV in Atlantic salmon feeds. To this end, post-smolt salmons with an initial BW of 148±3 g were fed four experimental diets for 15 weeks. A diet low in fish oil served as a control and was supplemented with 0, 0.5, 1.5 and 2.5 g/kg of RESV, respectively. The effect of the experimental diets on animal performance, tissue fatty acid composition, and the expression of genes encoding proteins involved in antioxidant signalling, lipid peroxidation, and metabolism were studied. Resveratrol significantly reduced feed intake and final BW of the salmon. Feeding RESV did not affect the sum of saturated and monounsaturated fatty acids or total lipids in the fillet. While the content of total polyunsaturated fatty acids was not affected, the percentages of some fatty acids in the liver and fillet were changed by RESV. Furthermore, in liver, the relative expression of glutathione peroxidase 4b, nuclear factor-like 2, and arachidonate 5-lipoxygenase remained unchanged across treatment groups. In conclusion, the negative impact of dietary RESV on FI and hence reduction of the BW discourages its inclusion in low fish oil diets for Atlantic salmon.

Omasal flow of soluble proteins, peptides, and free amino acids in dairy cows fed diets supplemented with proteins of varying ruminal degradabilities.

Three ruminally and duodenally cannulated cows were assigned to an incomplete 4 x 4 Latin square with four 14-d periods and were fed diets supplemented with urea, solvent soybean meal, xylose-treated soybean meal (XSBM), or corn gluten meal to study the effects of crude protein source on omasal canal flows of soluble AA. Soluble AA in omasal digesta were fractionated by ultrafiltration into soluble proteins greater than 10 kDa (10K), oligopeptides between 3 and 10 kDa (3-10K), peptides smaller than 3 kDa (small peptides), and free AA (FAA). Omasal flow of total soluble AA ranged from 254 to 377 g/d and accounted for 9.2 to 15.9% of total AA flow. Averaged across diets, flows of AA in 10K, 3-10K, small peptides, and FAA were 29, 217, 50, and 5 g/d, respectively, and accounted for 10.3, 71.0, 17.5, and 1.6% of the total soluble AA flow. Cows with diets supplemented with solvent soybean meal had higher flows of Met, Val, and total AA associated with small peptides than those whose diets were supplemented with XSBM, whereas supplementation with corn gluten meal resulted in higher total small peptide-AA flows than did XSBM. Averaged across diets, 27, 75, and 93% of soluble AA in 10K, 3-10K, and peptides plus FAA flowing out of the rumen were of dietary origin. On average, 10% of the total AA flow from the rumen was soluble AA from dietary origin, indicating a substantial escape of dietary soluble N from ruminal degradation. Omasal concentrations and flows of soluble small peptides isolated by ultrafiltration were substantially smaller than most published ruminal small peptide concentrations and outflows measured in acid-deproteinized supernatants of digesta.

A comparison of sampling sites, digesta and microbial markers, and microbial references for assessing the postruminal supply of nutrients in dairy cows.

This study evaluated the impact of some methodological factors on the flows of nutrients at the omasal canal and duodenum of dairy cows fed corn-based diets. Three ruminally and duodenally cannulated cows were assigned to an incomplete 4 x 4 Latin square with four 14-d periods and fed diets formulated to contain different amounts and ruminal degradabilities of crude protein. Samples from the omasal canal and duodenum were obtained and processed according to methodologies routinely used in our laboratories and elsewhere. Methodological factors that were evaluated included microbial references and markers, digesta markers, and sampling sites (techniques). Considerable variation was found for the compositions of microbial references and their impact on the intestinal supply of microbial nonammonia nitrogen. Likewise, it appears that variation in measuring the ruminal outflow of nitrogen fractions of microbial and dietary origin could be reduced by using 15N rather than purines as microbial markers. Sampling from the omasum and duodenum resulted in differences for ruminal outflow and site of digestion as well as digestibility of some nutrients, particularly nitrogen fractions and starch. A sizable portion of this variation was associated with deviations from the assumed ideal behavior of digesta markers and collection of samples that were unrepresentative of true digesta. Collectively, outcomes from this study indicate that more research will be required to determine the accuracy of nutrient flows and the agreement between measurements at the omasal canal and duodenum when dairy cows are fed a variety of diets under different feeding systems. Therefore, caution is recommended when extrapolating or interpreting the underlying biology of published results as well as the results of their application (e.g., model parameters and predictions).

NutriDense corn grain and corn silage for dairy cows.

Twenty multiparous Holstein cows, 4 of them surgically fitted with ruminal cannulas, were used in a replicated 4 x 4 Latin square to compare the effects of whole-plant silage and grain produced from NutriDense (ND), leafy NutriDense (LND), or a conventional yellow dent (YD) hybrid on ruminal fermentation, total tract nutrient digestibility, and performance of lactating dairy cows. On a DM basis, diets contained 30.6% corn silage and 27.7% corn grain provided from the 3 hybrids according to the following combinations: 1) YD grain and YD silage, 2) YD grain and LND silage, 3) ND grain and YD silage, and 4) ND grain and LND silage. The average concentrations of crude protein, neutral and acid detergent fiber, and ether extract of LND silage and ND grain were higher, but the contents of nonfibrous carbohydrates and starch were lower than those of their YD counterparts. Although DM intake was similar among treatments, feeding ND grain, LND silage, or both reduced the intakes of nonfibrous carbohydrates and starch but increased the intake of ether extract. Apparent digestibility of starch in the total tract was highest for the diet that contained LND silage and YD grain, whereas the amount and percentage of ether extract that were apparently digested in the total tract was increased and tended to be increased, respectively, by the addition of ND grain, LND silage, or both to the diets. Ruminal fermentation parameters were unaffected by treatments except for the concentration of ammonia nitrogen in the ruminal fluid, which tended to be increased by the feeding of ND grain, LND silage, or both. Production of milk, crude and true protein, fat, lactose, and total solids did not differ among diets. Concentration of milk urea nitrogen increased when the ND grain, LND silage, or both were fed to the cows. Results indicate that ND grain and LND silage were similar to the conventional grain and silage for the feeding of lactating dairy cows.

Varying protein and starch in the diet of dairy cows. II. Effects on performance and nitrogen utilization for milk production.

The main objective of this experiment was to examine the effects of the percentage and source of crude protein (CP) and the amount of starch in the diet of dairy cows on the lactational performance and use of N for milk production. Sixty multiparous Holstein cows were used in a 210-d lactational trial with a completely randomized design with a 2 x 3 factorial arrangement of treatments. Two sources of CP [solvent-extracted soybean meal (SBM) and a mixture of SBM and a blend of animal-marine protein supplements plus ruminally protected Met (AMB)] and 3 levels of dietary CP (means = 14.8, 16.8, and 18.7%) were combined into 6 treatments. On a dry matter (DM) basis, diets contained 25.0% corn silage, 20.0% alfalfa silage, 10.0% cottonseed, 26.7 to 37.0% corn grain, and 4.8 to 13.5% protein supplement, plus minerals and vitamins. Across the 210 d of lactation, the productive response of dairy cows to the source of supplemental CP depended on the concentration of CP in the diet. At 18.7% CP, cows fed SBM consumed more DM and produced more milk, 3.5% fat-corrected milk, fat, and true protein, but had lower efficiency of feed use and body condition score than cows fed AMB. At 16.8% CP, cows fed AMB produced more 3.5% fat-corrected milk, fat, and true protein than cows fed SBM. At 14.8% CP, cows fed SBM consumed more DM but produced less true protein and had lower feed efficiency than cows fed AMB. Across CP sources, cows fed 14.8% CP produced less fat-corrected milk and true protein than cows fed 16.8 and 18.7% CP. Across CP percentages, cows fed AMB produced more fat-corrected milk per kilogram of DM consumed than cows fed SBM. Despite these interactions, improvements in the gross efficiency of N use for milk production were achieved through reductions in the intake of N independently of the source of CP. Data suggest that the intake of N by high-producing dairy cows that consume sufficient energy and other nutrients to meet their requirements can be decreased to about 600 to 650 g daily if the source of RDP and RUP are properly matched with the source and amount of carbohydrate in the diet.

Impacts of the source and amount of crude protein on the intestinal supply of nitrogen fractions and performance of dairy cows.

The objective of this article was to review and summarize the significance of the amount and source of dietary crude protein supplements on the supply of nitrogen fractions passing to the small intestine and the performance of lactating dairy cows. A meta-analysis was used to evaluate 2 data sets, one for nitrogen flow to the small intestine and one for performance of cows. The response of dairy cows to rumen-undegradable protein supplements is variable. A portion of this variable response from research trials is explained by the source of crude protein in the control diet, the proportion and source of rumen-undegradable protein in the experimental diet, the effect of rumen-undegradable protein on microbial protein outflow from the rumen, the degradability and amino acid content of the rumen-undegradable protein, and the crude protein percentage of the diet. Compared with soybean meal, the mean milk production responses to feeding rumen-undegradable protein supplements ranged from -2.5 to +2.75%. Because of the large variation and small magnitude of response when rumen-undegradable protein supplements are fed compared with soybean meal, efficiency of nitrogen utilization and the cost to benefit ratio for these crude protein supplements may determine the source and amount of crude protein to feed to dairy cows in the future.

Rumen fermentation and intestinal supply of nutrients in dairy cows fed rumen-protected soy products.

Four multiparous lactating Holstein cows that were fistulated in the rumen and duodenum and that averaged 205 d in milk were used in a 4 x 4 Latin square design to evaluate the practical replacement of solvent-extracted soybean meal (SSBM) with soy protein products of reduced ruminal degradability. On a dry matter (DM) basis, diets contained 15% alfalfa silage, 25% corn silage, 34.3 to 36.9% corn grain, 19.4% soy products, 18.2% crude protein, 25.5% neutral detergent fiber, and 35.3% starch. In the experimental diets, SSBM was replaced with expeller soybean meal (ESBM); heated, xylose-treated soybean meal (NSBM); or whole roasted soybeans (WRSB) to supply 10.2% of the dietary DM. Intakes of DM (mean = 20.4 kg/d), organic matter, and starch were unaffected by the source of soy protein. Similarly, true ruminal fermentation of organic matter and apparent digestion of starch in the rumen and total tract were not altered by treatments. Intake of N ranged from 567 (WRSB) to 622 g/d (ESBM), but differences among soy protein supplements were not significant. Compared with SSBM, the ruminal outflow of nonammonia N was higher for NSBM, tended to be higher for ESBM, and was similar for WRSB. The intestinal supply of nonammonia nonmicrobial N was higher for NSBM and WRSB and tended to be higher for ESBM than for SSBM. However, no differences were detected among treatments when the flow to the duodenum of nonammonia nonmicrobial N was expressed as a percentage of N intake or nonammonia N flow. The ruminal outflow of microbial N, Met, and Lys was not altered by the source of soy protein. Data suggest that partially replacing SSBM with ESBM, NSBM, or WRSB may increase the quantity of feed protein that reaches the small intestines of dairy cows. However, significant improvements in the supply of previously reported limiting amino acids for milk production, particularly of Met, should not be expected.

Varying protein and starch in the diet of dairy cows. I. Effects on ruminal fermentation and intestinal supply of nutrients.

The main objective of this experiment was to examine the effects of the percentage and source of crude protein (CP) and the amount of starch in the diet of dairy cows on ruminal fermentation, nutrient passage to the small intestine, and nutrient digestibility. For this purpose, 6 multiparous Holstein cows fistulated in the rumen and duodenum that averaged 73 d in milk were used in a 6 x 6 Latin square design with a 2 x 3 factorial arrangement of treatments. Two sources of CP [solvent-extracted soybean meal (SBM) and a mixture of SBM and a blend of animal-marine protein supplements plus ruminally protected Met (AMB)] and 3 levels of dietary protein (about 14, 16, and 18%) were combined into 6 treatments. On a dry matter (DM) basis, diets contained 25% corn silage, 20% alfalfa silage, 10% cottonseed, 26.7 to 37% corn grain, and 4 to 13.5% protein supplement. Intakes and digestibilities in the rumen and total tract of DM, organic matter, acid and neutral detergent fiber were unaffected by treatments. Increasing dietary CP from 14 to 18% decreased the intake and apparent ruminal and total tract digestion of starch, but increased the proportion of starch consumed by the cows that was apparently digested in the small intestine. At 14% CP, starch intake and total tract digestion were higher for the AMB diet than for the SBM diet, but the opposite occurred at 16% CP. Across CP sources, increasing CP in the diet from 14 to 18% increased the intakes of N and amino acids (AA), and ruminal outflows of nonammonia N, nonammonia nonmicrobial N, each individual AA except Met, total essential AA, and total AA. Across CP percentages, replacing a portion of SBM with AMB increased the intake of Met and Val and decreased the concentration of ammonia N in the rumen, but did not affect the intake of other essential AA or the intestinal supply of any essential AA and starch. The ruminal outflow of microbial N, the proportional contribution of Lys and Met to total AA delivered to the duodenum, and milk yield were unaffected by treatments. Data suggest that the intake of N by high-producing dairy cows that consume sufficient energy and other nutrients to meet their requirements can be decreased to about 600 to 650 g daily without compromising the supply of metabolizable protein if the source and amount of dietary CP and carbohydrate are properly matched.