Bacteria colonization and gene expression related to immune function in colon mucosa is associated with growth in neonatal calves regardless of live yeast supplementation.

BACKGROUND: As Holstein calves are susceptible to gastrointestinal disorders during the first week of life, understanding how intestinal immune function develops in neonatal calves is important to promote better intestinal health. Feeding probiotics in early life may contribute to host intestinal health by facilitating beneficial bacteria colonization and developing intestinal immune function. The objective of this study was to characterize the impact of early life yeast supplementation and growth on colon mucosa-attached bacteria and host immune function. RESULTS: Twenty Holstein bull calves received no supplementation (CON) or Saccharomyces cerevisiae boulardii (SCB) from birth to 5 d of life. Colon tissue biopsies were taken within 2 h of life (D0) before the first colostrum feeding and 3 h after the morning feeding at d 5 of age (D5) to analyze mucosa-attached bacteria and colon transcriptome. Metagenome sequencing showed that there was no difference in α and ß diversity of mucosa-attached bacteria between day and treatment, but bacteria related to diarrhea were more abundant in the colon mucosa on D0 compared to D5. In addition, qPCR indicated that the absolute abundance of Escherichia coli (E. coli) decreased in the colon mucosa on D5 compared to D0; however, that of Bifidobacterium, Lactobacillus, and Faecalibacterium prausnitzii, which could competitively exclude E. coli, increased in the colon mucosa on D5 compared to D0. RNA-sequencing showed that there were no differentially expressed genes between CON and SCB, but suggested that pathways related to viral infection such as "Interferon Signaling" were activated in the colon mucosa of D5 compared to D0. CONCLUSIONS: Growth affected mucosa-attached bacteria and host immune function in the colon mucosa during the first 5 d of life in dairy calves independently of SCB supplementation. During early life, opportunistic pathogens may decrease due to intestinal environmental changes by beneficial bacteria and/or host immune function. Predicted activation of immune function-related pathways may be the result of host immune function development or suggest other antigens in the intestine during early life. Further studies focusing on the other antigens and host immune function in the colon mucosa are required to better understand intestinal immune function development.

Immune cells phenotype and bioenergetic measures in CD4+ T cells differ between high and low feed efficient dairy cows.

Identifying sources of variance that contribute to residual feed intake (RFI) can aid in improving feed efficiency. The objectives of this study were to investigate immune cells phenotype and bioenergetic measures in CD4+ T cells in low feed efficient (LE) and high feed efficient (HE) dairy cows. Sixty-four Holstein cows were enrolled at 93 ± 22 days in milk (DIM) and monitored for 7 weeks to measure RFI. Cows with the highest RFI (LE; n = 14) or lowest RFI (HE; n = 14) were selected to determine immune cells phenotype using flow cytometry. Blood was sampled in the same LE and HE cows at 234 ± 22 DIM to isolate peripheral blood mononuclear cells, followed by magnetic separation of CD4+ T lymphocytes using bovine specific monoclonal antibodies. The metabolic function of isolated CD4+ T lymphocytes was evaluated under resting and activated states. An increased expression of CD62L+ cells within CD8+ T lymphocytes and CD21+ B lymphocytes was observed in HE cows compared to LE cows. CD4+ T lymphocytes of HE cows exhibited an increased mitochondrial and glycolytic activity in resting and activated states compared to LE cows. These data suggest that immune cells in HE cows exhibit an increased metabolic function, which might influence nutrient partitioning and utilization and serve as a source of variation in feed efficiency that warrants future investigation.

Saccharomyces cerevisiae boulardii accelerates intestinal microbiota maturation and is correlated with increased secretory IgA production in neonatal dairy calves.

Neonatal calves have a limited capacity to initiate immune responses due to a relatively immature adaptive immune system, which renders them susceptible to many on-farm diseases. At birth, the mucosal surfaces of the intestine are rapidly colonized by microbes in a process that promotes mucosal immunity and primes the development of the adaptive immune system. In a companion study, our group demonstrated that supplementation of a live yeast probiotic, Saccharomyces cerevisiae boulardii (SCB) CNCM I-1079, to calves from birth to 1 week of age stimulates secretory IgA (sIgA) production in the intestine. The objective of the study was to evaluate how SCB supplementation impacts the intestinal microbiota of one-week-old male calves, and how changes in the bacterial community in the intestine relate to the increase in secretory IgA. A total of 20 calves were randomly allocated to one of two treatments at birth: Control (CON, n = 10) fed at 5 g/d of carrier with no live yeast; and SCB (n = 10) fed at 5 g of live SCB per day (10 × 109 CFU/d). Our study revealed that supplementing calves with SCB from birth to 1 week of age had its most marked effects in the ileum, increasing species richness and phylogenetic diversity in addition to expediting the transition to a more interconnected bacterial community. Furthermore, LEfSe analysis revealed that there were several differentially abundant taxa between treatments and that SCB increased the relative abundance the family Eubacteriaceae, Corynebacteriaceae, Eggerthellaceae, Bacillaceae, and Ruminococcaceae. Furthermore, network analysis suggests that SCB promoted a more stable bacterial community and appears to reduce colonization with Shigella. Lastly, we observed that the probiotic-driven increase in microbial diversity was highly correlated with the enhanced secretory IgA capacity of the ileum, suggesting that the calf's gut mucosal immune system relies on the development of a stable and highly diverse microbial community to provide the necessary cues to train and promote its proper function. In summary, this data shows that supplementation of SCB promoted establishment of a diverse and interconnected microbiota, prevented colonization of Escherichia Shigella and indicates a possible role in stimulating humoral mucosal immunity.

Prophylactic feeding of neomycin to Holstein calves alters gut microbiota, bile acid metabolism, and expression of genes involved in immunometabolic regulation.

The objective of this study was to evaluate the effects of prophylactic neomycin administration on Holstein bull calves' intestinal microbiota, bile acid (BA) metabolism, and transcript abundance of genes related to BA metabolism. A total of 36 calves were blocked by body weight and assigned to either non-medicated milk replacer (CTL), or neomycin for 14 days (ST) or 28 days (LT) in their milk replacer. At the end of the study, calves were euthanized to collect tissue and digesta samples from the gastrointestinal tract, liver, and adipose tissue for analysis of intestinal microbial diversity, bile acid concentration and profile in various body tissues, and gene expression related to bile acid, lipid, carbohydrate metabolism, and inflammation. Calves that received prophylactic administration of neomycin for 28 d (LT) had reduced species richness (chao1 index), and tended to have reduced phylogenetic diversity in the ileum tissue. The relative abundance of Lactobacillus, and Bifidobacterium in ileum and colon digesta were decreased in LT compared with CTL. Concentrations of primary, secondary, and total BA were increased by ST in ileal tissue. In plasma, ST and LT treatments had lower concentrations of secondary BA. Gene expression of the BA receptor FXR was increased in ileum and liver by LT compared to CTL. The expression of FXR and TGR5 in the liver was increased in the ST group compared with CTL, and in adipose tissue, 5 genes related to triglyceride, gluconeogenesis, and immune activation were differentially expressed between CTL and ST. In conclusion, we provide evidence that prophylactic administration of neomycin leads to aberrant changes in BA concentration and profile in different compartments of the enterohepatic system through a process that possibly entails antimicrobial disruption of key bacterial groups, which persists even after cessation of neomycin administration. Additionally, we uncovered an apparent link between dysregulated BA metabolism and changes in lipid metabolism and immune activation in adipose tissue and liver.

Triterpenes from Olea europaea modulate in vitro ruminal fermentation.

Bioactive compounds present in Olea europaea have shown promising antimicrobial potential as an alternative to conventional coccidiostats. These effects are exerted by triterpenic acids (TT) present in the olive plant, namely, oleanolic acid (OA), ursolic acid (UA), and maslinic acid (MA). The objective of this study was to determine the effects of OA, UA, and MA on in vitro ruminal fermentation in comparison with monensin (MON). The study consisted of two experiments conducted as randomized complete block designs using bahiagrass hay or a high-concentrate mixed ration as basal substrates. In the first experiment (Exp. 1), a batch culture was performed with increasing doses of OA, UA, or MA. In Exp. 2, to increase the solubility of OA, two chemical forms were evaluated: a sodium salt (OA-NA) or a phyto-phospholipid complex (OA-PHYT) at 0, 4, 40, 100, and 200 mg/L of incubation inoculum. In both experiments, the dose 0 was used as control (CTL) and monensin (MON) as a positive control. Data were analyzed as a randomized complete block design with a factorial arrangement of treatments. For Exp. 2, orthogonal polynomial contrasts, adjusted for unequal spacing were used to determine the linear effects of increasing doses of OA-NA and OA-PHYT. In Exp. 1, OA reduced the concentration of CH4 in the high-concentrate substrate compared with CTL (P = 0.04). In Exp. 2 the total gas production was linearly decreased with increasing doses of OA-NA in both substrates (P ≤ 0.02). Furthermore, OA-NA and OA-PHYT decreased in vitro organic matter digestibility (P < 0.01) in the bahiagrass substrate to the same extent that MON did. However, the concentration of CH4/g of incubated DM was only reduced by the highest doses of OA-NA (P < 0.02). Lastly, no effects were observed for total VFA nor the VFA profile; however, OA-NA linearly decreased the A:P ratio in the bahiagrass substrate (P = 0.03). In conclusion, the acid form of OA as well as the sodium salt and phyto-phospholipid complex of OA were able to modify some fermentation parameters in this study; however, the magnitude of the responses was lower compared with monensin. Future studies should test OA in vivo to determine if the effects on ruminal fermentation observed here can translate into improve production efficiency while reducing carbon emissions.

Impacts of polyclonal antibody preparations from avian origin as a feed additive to beef cattle: immune responses during the step-up transition diets.

This study investigated the effects of feeding an avian-derived polyclonal antibody preparation (PAP; CAMAS, Inc.) against Streptococcus bovis, Fusobacterium necrophorum, and lipopolysaccharides (LPS; 40%, 35%, and 25% of the preparation, respectively) on immune responses (haptoglobin [Hp], serum amyloid A [SAA], rectal temperature [RT], leukocyte counts, and expression of cell adhesion molecules cluster of differentiation [CD] CD11b, CD14, and CD62L) of beef steers during a 21-d step-up adaptation to a high-grain diet. Eight ruminally cannulated Angus crossbred beef steers (658 ± 79 kg of BW) were assigned in a cross-over design and transitioned from a diet containing bermudagrass hay (Cynodon dactylon (L.) Pers.) ad libitum plus 0.45 kg/d of molasses with 0 (CON) or 3 g of PAP to a high-grain diet. Transition consisted of three 7-d steps of increased inclusion of cracked corn (35%, 60%, and 82% of the diet dry matter for STEP1, STEP2, and STEP3, respectively). On each transition day and 7 d after STEP3 (STEP3-7d), RT was obtained every 3 h for a total of 24 h, whereas blood was collected on days 0, 1, and 3, relative to diet transition. There were no effects of PAP inclusion in any of the blood parameters (P > 0.11). However, a tendency for day effect (P = 0.10) was observed for concentrations of Hp, which were greater on days 3 and 7 vs. day 0 relative to the second diet transition (STEP2). Plasma concentrations of SAA were greater on days 1, 3, and 7 compared to day 0 during STEP1 (P = 0.01), while during STEP2 and STEP3, SAA concentrations increased (P < 0.01) from day 0 to 3. During STEP2, PAP steers tended to have lower (P = 0.08) RT than CON steers. Neutrophil and monocyte counts were the least during STEP3 (P < 0.01), whereas expression of CD11b and CD62L was the least through forage feeding (P < 0.01). Concentration of starch in the diet was correlated to all the variables tested (P ≤ 0.01), except for the percentage of B cells (P = 0.22). Yet only ruminal pH, RT, monocyte, and neutrophil counts presented strong correlation coefficients. In conclusion, the step-up transition from forage to high-grain diets triggered systemic inflammation in beef steers as observed by increased plasma concentrations of Hp, SAA, and expression on adhesion molecules in leukocytes. However, feeding polyclonal antibody preparations against S. bovis, F. necrophorum, and LPS did not provide benefits to mitigate inflammation.

Effects of providing artificial shade to pregnant grazing beef heifers on vaginal temperature, growth, activity, and behavior.

This experiment evaluated the effects of providing artificial shade during summer on activity, behavior, and growth performance of pregnant grazing beef heifers. Thirty-six black-hided Angus and Angus crossbred pregnant heifers [418 ± 9 kg body weight (BW); approximately 90 d of gestation] were stratified by breed, blocked by BW, and allocated to 12 "Pensacola" bahiagrass pastures (Paspalum notatum Flüggé; 1.3 ha, n = 3 heifers/pasture) with or without access to artificial shade (SHADE vs. NO SHADE; 6 pastures each) for 7 wk during summer. The shade structures were composed of shade cloth (11 × 7.3 m length, 2.4 m height: 26.8 m2 of shade per heifer). Shrunk BW was recorded on enrollment (day 0) and week 7 (day 47), whereas full BW was obtained on week 2 (day 14), 4 (day 28), and 6 (day 42) to assess average daily gain (ADG). Vaginal temperature was recorded for five consecutive days during weeks 1, 3, 5, and 7 using an intravaginal digital thermo-logger, and individual GPS devices were used to quantify the use of shade for an 8-h period. Activity was monitored using automated monitoring devices (HR-LDn tags SCR Engineers Ltd., Netanya, Israel) through the experimental period. Vaginal temperature was lower (P < 0.01) for heifers in the SHADE compared with heifers in the NO SHADE treatment from 1200 to 1600 h and 1100 to 1900 h for weeks 1 and 3, respectively. Heifers in the SHADE treatment spent 70% of the 8-h period evaluated under the shaded structure. Provision of shade increased (P < 0.01) daily lying time (11.4 ± 0.2 vs. 10.3 ± 0.2 h/d) and standing bouts per day (P < 0.01; 12.6 ± 0.4 vs. 10.8 ± 0.4 bouts/d), whereas it reduced (P < 0.01) standing bout duration (61.6 ± 3.0 vs. 82.9 ± 3.0 min/bout) relative to heifers without access to shade. The interaction between treatment and hour affected (P < 0.01) daily rumination time because heifers with access to SHADE had greater rumination between 1000 and 1200 h. Although ADG tended (P = 0.08) to be greater for the heifers in the SHADE treatment (0.20 vs. -0.02 kg, respectively), the access to shade did not (P = 0.79) affect the final BW. In conclusion, providing artificial shade during summer to pregnant grazing beef heifers was effective in reducing vaginal temperatures and exerted changes in heifer behaviors that translated into slight improvements in growth performance.

Effects of bismuth subsalicylate and encapsulated calcium-ammonium nitrate on enteric methane production, nutrient digestibility, and liver mineral concentration of beef cattle.

Two randomized block designs were performed to evaluate the effects of bismuth subsalicylate (BSS) and encapsulated calcium-ammonium nitrate (eCAN) on enteric methane production, nutrient digestibility, liver mineral concentration, and performance of beef cattle consuming bahiagrass hay (Paspalum notatum; ad libitum) and sugar cane molasses [1.07 kg/d; dry matter basis]. Experiment 1, used 25 crossbred steers [335 ± 46 kg of initial body weight (BW)] with a 2 × 2 + 1 factorial arrangement of treatments for two 20 d periods. Factors were nonprotein nitrogen (NPN) source (350 mg/kg BW of nitrate or 182 mg/kg BW of urea), BSS (0 or 58.4 mg/kg BW), and a negative control (NCTRL; bahiagrass hay and molasses only). Steers were re-randomized for a second period (n = 10/treatment total). Intake, apparent total tract digestibility and enteric methane were evaluated. Experiment 2 used 75 crossbred heifers in 25 pens (3 heifers/pen; 279 ± 57 kg of initial BW), consuming the same diet and treatments as experiment 1, to determine liver mineral concentration and growth performance over 56 d. Orthogonal contrasts were used to evaluate the effects of NPN (NCTRL vs. others), source of NPN (NS; urea vs. eCAN), BSS, and NS × BSS. For experiment 1, no interactions were observed for any variables, nor were there any effects of NPN on total tract digestibility of nutrients, except for crude protein. Digestibility of all nutrients was reduced (P ≤ 0.021) for steers consuming eCAN compared with urea. There was no effect (P > 0.155) of BSS on digestibility of nutrients; however, BSS reduced (P = 0.003) apparent S retention. Enteric CH4 emission (g/kg BW0.75) was decreased (P = 0.051) by 11% with the addition of eCAN compared with urea. For experiment 2, no NS × BSS interactions (P ≥ 0.251) were observed to affect liver mineral concentration; however, the addition of BSS decreased liver concentration of Cu (P = 0.002) while increasing Fe concentration (P = 0.016). There was an NS × BSS interaction (P = 0.048) where heifers consuming eCAN and BSS had lesser final BW compared with heifers consuming urea and BSS. While eCAN may be a viable resource for mitigating enteric CH4 production of forage-fed cattle, the negative effects on digestibility should be considered. Furthermore, BSS, at the amount provided, appears to have no negative effects on digestibility of nutrients in forage-fed cattle; however, there may be deleterious impacts on performance depending upon what nitrogen source is supplied.

Effects of bismuth subsalicylate and encapsulated calcium ammonium nitrate on ruminal fermentation of beef cattle.

A replicated 5 × 5 Latin square design with a 2 × 2 + 1 factorial arrangement of treatments was used to determine the effects of bismuth subsalicylate (BSS) and encapsulated calcium ammonium nitrate (eCAN) on ruminal fermentation of beef cattle consuming bahiagrass hay (Paspalum notatum) and sugarcane molasses. Ten ruminally cannulated steers (n = 8; 461 ± 148 kg of body weight [BW]; average BW ± SD) and heifers (n = 2; 337 ± 74 kg of BW) were randomly assigned to one of five treatments as follows: 1) 2.7 g/kg of BW of molasses (NCTRL), 2) NCTRL + 182 mg/kg of BW of urea (U), 3) U + 58.4 mg/kg of BW of BSS (UB), 4) NCTRL + 538 mg/kg of BW of eCAN (NIT), and 5) NIT + 58.4 mg/kg of BW of BSS (NITB). With the exception of NCTRL, all treatments were isonitrogenous. Beginning on day 14 of each period, ruminal fluid was collected and rectal temperature was recorded 4× per day for 3 d to determine ruminal changes every 2 h from 0 to 22 h post-feeding. Ruminal gas cap samples were collected at 0, 3, 6, 9, and 12 h on day 0 of each period followed by 0 h on days 1, 2, 3, and 14. Microbial N flow was determined using Cr-Ethylenediaminetetraacetic acid, YbCl3, and indigestible neutral detergent fiber for liquid, small particle, and large particle phases, respectively. Data were analyzed using the MIXED procedure of SAS. Orthogonal contrasts were used to evaluate the effects of nonprotein nitrogen (NPN) inclusion, NPN source, BSS, and NPN source × BSS. There was no treatment effect (P > 0.05) on concentrations of H2S on day 0, 1, 2, or 14; however, on day 3, concentrations of H2S were reduced (P = 0.018) when NPN was provided. No effect of treatment (P = 0.864) occurred for ruminal pH. There was an effect of NPN source on total concentrations of VFA (P = 0.011), where a 6% reduction occurred when eCAN was provided. There were effects of NPN (P = 0.001) and NPN source (P = 0.009) on the concentration of NH3-N, where cattle consuming NPN had a greater concentration than those not consuming NPN, and eCAN reduced the concentration compared with urea. Total concentrations of VFA and NH3-N were not affected (P > 0.05) by BSS. There was an effect of BSS (P = 0.009) on rectal temperature, where cattle not consuming BSS had greater temperatures than those receiving BSS. No differences for NPN, NPN source, nor BSS (P > 0.05) were observed for microbial N flow. In conclusion, eCAN does not appear to deliver equivalent ruminal fermentation parameters compared with urea, and BSS has limited effects on fermentation.

A bioactive extract from Olea europaea protects newly weaned beef heifers against experimentally induced chronic inflammation1.

Weaning is one of the most stressful periods in the life of a ruminant. Several factors entrenched within typical management practices pose challenges to the calf gastrointestinal health. Weaning is associated with losses in BW and feed intake. In addition, increasing highly fermentable carbohydrates in the diet at the expense of physically effective fiber after weaning predisposes the development of rumen acidosis and increases the concentration of endotoxin in rumen fluid and the permeability of the lower gut to luminal contents. Endotoxin translocation can elicit immune activation, shifting the metabolic priorities toward the immune system, which if sustained over time can hinder animal health and performance. Strategic supplementation of additives with anti-inflammatory capacity could represent a suitable approach to decrease systemic inflammation, restoring barrier function to luminal contents. Bioactive extracts from Olea europaea have anti-inflammatory activity and have been shown to reduce systemic inflammation in other animal models. A generalized randomized block design was used to evaluate the impact of feeding an olive oil bioactive extract (OBE) to newly weaned heifers injected intravenously with sequentially increasing doses of lipopolysaccharide (LPS). A total of 36 heifers, distributed across 3 experimental periods, were randomly assigned to 1 of 4 treatments that consisted of intravenous injection of either saline (CTL-) or with 6 sequentially increasing doses of LPS (0.10, 0.25, 0.50, 0.75, 1.00, and 1.25 µg/kg of BW) over a 10-d period (CTL+), and CTL+ plus dietary supplementation with a low (OBE-L; 0.04% of diet DM) or a high (OBE-H; 0.16% of diet DM) dose of OBE. Feeding OBE reduced some of the negative effects of prolonged immune activation with LPS, such as improved DMI and decreased intravaginal temperature in some, but not all of the days of LPS challenge (P < 0.05). In addition, feeding OBE reduced circulating concentration of inflammatory markers such as IL-6 and haptoglobin (P < 0.05). Heifers supplemented with OBE had reduced cell surface expression of the cluster of differentiation 14 (CD14) in monocyte cells (P < 0.01), a key receptor for LPS recognition, which was correlated with a faster recovery of immune cell counts in plasma. In conclusion, dietary supplementation with OBE was successful in mitigating the negative effects of sustained immune activation in newly weaned heifers.