Effects of weaning and inactivated Lactobacillus helveticus supplementation on dairy calf behavioral and physiological indicators of affective state.

The objectives of this study were to determine if weaning would induce behavioral and physiological indicators of a negative affective state, and if supplementation of inactivated Lactobacillus helveticus (ILH) to dairy calves would reduce those indicators of negative affect during weaning. Male Holstein calves (n = 23) were enrolled in the study on d 1 of life. The calves were housed in individual pens in 1 of 4 rooms for the 42 d study. Calves began a stepdown weaning from 9 L/d of milk replacer (MR), at 150 g of MR powder/L, on d 35 and received 6 L/d on d 35 - 36, 3 L/d on d 37 - 38, and 0.4 L/d on d 39 - 42. The MR was divided between 3 meals/d until the last 0.4 L/d phase which was divided between 2 meals/d. Calves had ad libitum water access throughout the study and calf starter from d 28 onwards. Within room, calves were assigned to 1 of 2 treatments: 1) control (CON; n = 11) and 2) 5 g of ILH/d split over and mixed into the 0800 h and 2000 h milk feedings from d 3-42 (ILH; n = 12). Lying behavior was recorded using HOBO data loggers from d 21-41. On d 33, 37 and 41, infrared eye images were taken to determine maximum eye temperature (MET), saliva samples were collected to determine cortisol concentration, and play assessments were conducted to quantify play behavior. On d 34, 38, and 42, blood samples were collected to determine blood serotonin concentration, whereas on d 38 and 39, calves were tested with a cognitive task. A subset of calves (n = 5/treatment) were euthanized to collect gut and brain tissue samples for serotonin concentration on d 43. Weaning resulted in fewer (d 37-41, tendency: d 36), but longer (d 38-41, tendency: d 37), lying bouts and reduced play (d 41), although no changes in lying time, MET, saliva cortisol, nor blood serotonin were detected with initiation of weaning. Supplementation of ILH was associated with lower lying time throughout the study, and reduced play duration and higher salivary cortisol and MET during weaning. No differences in lying bouts, play count, blood and tissue (colon, ileum, prefrontal cortex and brain stem) serotonin concentration, and time to complete the cognitive task were detected between the treatments. Overall, weaning induced behavioral changes indicative of negative affective state, and some behavioral differences were observed with ILH supplementation both before and during weaning, with some physiological changes observed during weaning.

Developmental adaptations of γδ T cells and B cells in blood and intestinal mucosa from birth until weaning in Holstein bull calves.

This study aimed to characterize the development of systemic and colon tissue resident B and γδ T cells in newborn calves from birth until weaning. At birth, calves have limited capacity to initiate immune responses, and the immune system gradually matures over time. Gamma delta (γδ) T cells are an important lymphocyte subset in neonatal calves that confer protection and promote immune tolerance. A total of 36 newborn calves were enrolled in a longitudinal study to characterize how systemic and colon tissue resident B and γδ T cells develop from birth until weaning. Blood and colon biopsy samples were collected on d 2, 28, and 42 to determine the proportions of various B and γδ T cell subsets by flow cytometry. We classified γδ T cells into different functional subsets according to the level of expression intensity of the coreceptors WC1.1 (effector function) and WC1.2 (regulatory function). Furthermore, naive B cells were classified based on the expression IgM receptor, and activation state was determined based on expression of CD21 and CD32, 2 receptors with opposing signals involved in B cell activation in early life. Additional colon biopsy samples were used for 16S sequencing, and microbial diversity data are reported. At birth, γδ T cells were the most abundant lymphocyte population in blood, accounting for 58.5% of the lymphocyte pool, after which the proportions of these cells declined to 38.2% after weaning. The proportion of γδ T cells expressing WC1.1 decreased by 50% from d 2 to d 28, whereas no change was observed in the expression of WC1.2. In the colon, there was a 50% increase of γδ T cells after weaning and the proportion of WC1.2+ γδ T cells doubled from d 28 to 42. The proportion of IgM+ B lymphocytes in blood increased from 23.6% at birth to 30% after weaning, were the proportion of B cells expressing CD21 increased by 25%, while the proportion of B cells expressing CD32 decreased by 30%. While no changes were observed for the overall proportion of IgM+ B lymphocytes in the colon, there was a 6-fold increase in the proportion of CD21+ B cells from pre- (d 28) to postweaning (d 42). Microbial diversity increased from d 2 of life to 28 and declined abruptly after weaning. The reduction in microbial diversity during weaning was negatively correlated with the increase in all γδ T cell subsets and CD21+ B cells. These data suggest that developmental adaptations after birth coordinate expansion of γδ T cells to provide early systemic protection, as well as to steer immune tolerance, while B cells mature over time. Additionally, the increase of colonic γδ T cells on d 42 suggests a protective role of these cells during weaning.

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.

A novel method for isolation and flow cytometry analysis of intraepithelial lymphocytes from colon biopsies.

Investigating the immune responses of the intestine in response to different insults is predominantly limited to indirect methods such as circulating markers of intestinal health or gene expression from dissections. We describe here a validated protocol for the isolation and subsequent flow cytometry analysis of intestinal intraepithelial lymphocytes (IEL) from colonic biopsy samples. Colon biopsy samples were collected with endoscopy forceps from Holstein dairy bull calves at d 2, 28, and 42 of life. The biopsies were put into an isolation solution of Hanks' balanced salt solution, and fetal bovine serum followed by digestion solution. The solution was filtered and the flow-through, containing IEL, was stained with fluorescent antibodies for flow cytometry analysis. Density gradient separation of the isolate yielded higher viability and cleaner samples for flow cytometry analysis. Anti-bovine γ chain of the T cell receptor was used to identify populations of gamma delta (γδ) T cells via flow cytometry. In addition, γδ T cell subsets were identified using an anti-bovine antibody against the coreceptor workshop cluster 1. This method allowed for the precise identification of lymphocyte populations and evaluation of the proportion of different subsets of γδ T cells from intestinal IEL over time. The technique described here will allow the research community to characterize intestinal immune function over time and improve our understanding of how different management and nutritional strategies affect intestinal health.

Transcriptomic analysis reveals gene expression changes in peripheral white blood cells of cows after embryo transfer: Implications for pregnancy tolerance.

Most embryo losses occur in the first trimester of pregnancy in cows and include losses following embryo transfer. There is a resulting negative economic impact on cattle production systems when this occurs. Cellular and molecular mechanisms behind the maternal immune response to the growing embryo have not been fully characterized. The objective of this study was to examine the gene expression profiles of peripheral white blood cells (PWBCs) from pregnant cows 21 days after an embryo was transferred, and cows that were treated equally but lost the embryo. Specifically, we obtained and compared the transcriptome of PWBC from heifers that became pregnant at day 21 (N = 5) or failed to become pregnant after the embryo transfer (N = 5). Sequencing data can be accessed by Gene Expression Omnibus (GEO) with the accession number GSE210665. A total of 13,167 genes were evaluated for differential expression between groups. A total of 682 genes showed differential expression (p-value <.01), 302 genes were up-regulated while 380 were down-regulated due to pregnancy. The most significant genes were COL1A2, H2AC18, HTRA1, MMP14, CD5L, ADAMDEC1, MYO1A and RPL39, among others. Most of the significant genes are related to the up-regulation of inflammatory chemokine activity and immune defence response. Our findings extend the current knowledge that pregnancy alters the PWBC by promoting immune tolerance, cell chemotaxis, blood coagulation, angiogenesis, inflammatory response, cell adhesion and cytokine secretion. Our data suggest that pregnancy and ectoparasites could trigger poorly described genes in PWBC of cows, and a few previously escribed genes, such as IFI44. These results could shed light on the genes and mechanisms that promote tolerance to pregnancy and allow survival of the developing embryo.

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.

ADSA Foundation Scholar Award: New frontiers in calf and heifer nutrition-From conception to puberty.

Dairy calf nutrition is traditionally one of the most overlooked aspects of dairy management, despite its large effect on the efficiency and profitability of dairy operations. Unfortunately, among all animals on the dairy farm, calves suffer from the highest rates of morbidity and mortality. These challenges have catalyzed calf nutrition research over the past decade to mitigate high incidences of disease and death, and improve animal health, growth, welfare, and industry sustainability. However, major knowledge gaps remain in several crucial stages of development. The purpose of this review is to summarize the key concepts of nutritional physiology and programming from conception to puberty and their subsequent effects on development of the calf, and ultimately, future performance. During fetal development, developmental plasticity is highest. At this time, maternal energy and protein consumption can influence fetal development, likely playing a critical role in calf and heifer development and, importantly, future production. After birth, the calf's first meal of colostrum is crucial for the transfer of immunoglobulin to support calf health and survival. However, colostrum also contains numerous bioactive proteins, lipids, and carbohydrates that may play key roles in calf growth and health. Extending the delivery of these bioactive compounds to the calf through a gradual transition from colostrum to milk (i.e., extended colostrum or transition milk feeding) may confer benefits in the first days and weeks of life to prepare the calf for the preweaning period. Similarly, optimal nutrition during the preweaning period is vital. Preweaning calves are highly susceptible to health challenges, and improved calf growth and health can positively influence future milk production. Throughout the world, the majority of dairy calves rely on milk replacer to supply adequate nutrition. Recent research has started to re-evaluate traditional formulations of milk replacers, which can differ significantly in composition compared with whole milk. Transitioning from a milk-based diet to solid feed is critical in the development of mature ruminants. Delaying weaning age and providing long and gradual step-down protocols have become common to avoid production and health challenges. Yet, determining how to appropriately balance the amount of energy and protein supplied in both liquid and solid feeds based on preweaning milk allowances, and further acknowledging their interactions, shows great promise in improving growth and health during weaning. After weaning and during the onset of puberty, heifers are traditionally offered high-forage diets. However, recent work suggests that an early switch to a high-forage diet will depress intake and development during the time when solid feed efficiency is greatest. It has become increasingly clear that there are great opportunities to advance our knowledge of calf nutrition; yet, a more concentrated and rigorous approach to research that encompasses the long-term consequences of nutritional regimens at each stage of life is required to ensure the sustainability and efficiency of the global dairy industry.

The effect of neomycin inclusion in milk replacer on the health, growth, and performance of male Holstein calves during preweaning.

The prophylactic use of oral antimicrobials, such as neomycin, in milk replacer (MR) or whole milk is a common practice in calf-rearing that is thought to aid in preventing disease. Heavy reliance on antimicrobials is of concern not only because of the development of antimicrobial resistance, but also because of the potentially negative effects on health. The objective of this study was to investigate the effects of neomycin on calf health and growth performance. One hundred and sixty calves (approximately 3-10 d of age), distributed across 2 experimental periods, were stratified by body weight (BW) and serum total protein, and assigned to 1 of 3 treatments: control (CON; nonmedicated MR, n = 60), short-term antibiotic (ST; neomycin mixed in MR from d 1-14, n = 50), or long-term antibiotic (LT; neomycin in ​MR from d 1-28, n = 50). Arrival BW (47.69 ± 0.87 kg) and serum total protein (5.67 ± 0.09 g/dL) were not different between treatment groups. Neomycin in ST and LT was dosed in MR at a rate of 20 mg/kg of BW and was adjusted weekly according to BW. Calf BW was measured weekly for 49 d, and health indicators (fecal score, attitude score, respiratory score, and rectal temperature), MR intake, starter intake, and the use of additional electrolytes and antimicrobials were recorded daily. Calves in the CON group experienced a higher proportion of days with diarrhea (20.32 ± 0.02%) compared with ST (14.70 ± 0.02%) or LT (13.80 ± 0.02%) calves, as well as longer bouts of diarrhea (7.45 ± 0.38 d, 5.69 ± 0.46 d, and 5.62 ± 0.45 d for CON, ST, and LT calves, respectively). Calves in the CON group also experienced higher fecal scores (score of 0.64 ± 0.04) than ST (score of 0.53 ± 0.04) or LT (score of 0.49 ± 0.04) calves, especially at d 7. However, no differences were observed in other health-related measures. The time to reach first diarrhea and first respiratory illness was not different between treatments, nor was the time to recover from respiratory illness. The time to intervention with additional electrolytes or antimicrobials was not different between treatment groups. Furthermore, growth performance, feed intake, and feed conversion ratio were not different. No differences were found when comparing ST and LT, except in the defined daily dose of total antimicrobials received. Calves in the LT group received a higher overall dose than ST calves, and both ST and LT calves received a higher dose than CON calves, which received no prophylactic antimicrobials. Given that there were no differences in performance variables and no additional health benefits aside from reduced fecal scores in calves fed neomycin, current practices involving the use of antimicrobials on dairy and veal operations need to be considered more prudently.