Correlation of udder thermogram and somatic cell counts as a tool for detection of subclinical mastitis in buffaloes.

This study aimed to monitor the mammary health of 37 multiparous Murrah buffaloes through infrared thermography (IRT). Based on the California Mastitis Test (CMT) and milk somatic cell counts (SCC), buffaloes were grouped into healthy (H, n = 16), subclinical mastitis (SCM, n = 10), and clinical mastitis (CM, n = 11). Buffaloes were milked twice daily in the morning (5:00-6:00 AM) and evening (5:00-6:00 PM). Rectal temperature and respiratory rates were recorded, CMT was performed and thermal images of the mammary gland of all the buffaloes were taken before and after each milking. Milk samples were analysed after each milking for SCC, fat, Solids-Not-Fat (SNF), density, protein, lactose, salts, conductivity, and pH immediately in the laboratory from fresh milk samples. The surface temperature of the periocular region of both the eyes, muzzle, flank, and vagina were also taken. Thermal images were used to assess the surface temperature of the udder (USST), teat apex (TAT), teat barrel (TB1T), teat base (TB2T), and teat skin surface (TSST). Eye and USST showed significantly higher temperatures (p < 0.05), whereas skin surface temperatures (SST) of different body parts were non-significant in both SCM and CM animals than buffaloes in the H group. Milk SCC showed a positive correlation with conductivity (r > 0.7), salts, and pH (r < 0.6) and a negative correlation with fat, SNF, density, protein, and lactose. TAT, TB1T, TB2T, TSST, and USST were positively correlated with milk SCC. Receiver Operating Characteristic (ROC) analysis of H and SCM groups showed that USST before milking had optimum sensitivity (Se = 0.80) and specificity (Sp = 0.906) among the various skin temperatures recorded. Thermal images captured during the morning showed higher sensitivity compared to images taken in the evening. Results indicate IRT can be used to monitor the mammary health of buffaloes but using IRT in conjunction with milk SCC can help in the accurate prediction of SCM in dairy buffaloes.

Reliability of udder infrared thermography as a non-invasive technology for early detection of sub-clinical mastitis in Sahiwal (Bos indicus) cows under semi-intensive production system.

The present study focused on Sahiwal cows, a prominent milch breed in tropical India, to correlate udder temperature with physiological markers of stress and inflammation during subclinical mastitis (SCM). The primary goal was to assess the potential of udder infrared thermography for the early detection of SCM under the semi-intensive production. Cows were categorized based on milk somatic cell counts (SCC), with healthy (H) cows having SCC <2 × 105 cells/mL and no history of mastitis, and cows with subclinical mastitis (SCM) and initial stages of clinical mastitis (CM) having quarter milk SCC of 2-5 × 105 and >5 × 105 cells/mL, respectively. Firstly, udder thermograms were analysed for udder skin surface temperature (USST), teat skin surface temperature (TSST), and teat apex temperature (TAT) using Fluke software to determine the optimal site for temperature measurement during intramammary infection. Secondly, milk samples were collected for automatic estimation of compositional changes, electrical conductivity, and pH. Thirdly, milk whey was separated for quantifying stress and inflammatory indicators, including cortisol, prolactin, and acute-phase proteins (APPs): milk amyloid A and milk haptoglobin using bovine-specific ELISA kits. Significant increases (p < 0.01) in USST, TSST, TAT, cortisol, and APPs were observed in SCM and CM compared to healthy cows, while prolactin levels decreased (p < 0.01). The correlation matrix revealed strong positive correlations of SCC with USST (r = 0.84, p < 0.01). In ROC analysis, USST demonstrated cut-off values of 37.74 and 39.58 °C, with accuracy (p < 0.05) of 98% for SCM and 95% for CM, surpassing both TAT and TSST. Therefore, the combination of these non-invasive methods increases the reliability and accuracy of infrared thermography for early detection of SCM, providing valuable insights for the development of a protocol for routine screening and udder health monitoring in indigenous dairy cows.

Pre-clinical safety and toxicity assessment of Limosilactobacillus fermentum NCDC 400 in murine model.

Comprehensive safety assessment of potential probiotic strains is crucial in the selection of risk-free strains for clinical translation. This study aimed to evaluate the biosafety of Limosilactobacillus fermentum NCDC 400, a potential probiotic strain, using oral toxicity tests in a Swiss albino mouse model. Mice were orally gavaged with low (108 CFU/mouse/day) and high (1010 CFU/mouse/day) doses of NCDC 400 for 14 (acute), 28 (subacute), and 90 (subchronic) days to assess behavioral, hematological, biochemical, immunological, and histological effects. The administration of NCDC 400 did not result in any observable adverse effects on general health parameters, including body weight, feed and water intake, and organ indices. Hematological and biochemical parameters, such as glucose, serum enzymes, urea, creatinine, serum minerals, total serum proteins, and lipid profile, remained largely unaffected by the test strain. Notably, NCDC 400 administration led to a significant reduction in harmful intestinal enzymes and improvement in gut health indices, as indicated by fecal pH, lactate, ammonia, and short-chain fatty acids. There were no instances of bacterial translocation of NCDC 400 to blood or extra-intestinal organs. Immune homeostasis was not adversely affected by repeated exposure to NCDC 400 in all three oral toxicity studies. Histopathological examination revealed no strain-related changes in various tissues. Based on these findings, a dose of 1010 CFU/mouse/day was considered as the No Observable Effect Level (NOEL) in healthy mice. In conclusion, this study demonstrates the safe and non-toxic behavior of L. fermentum NCDC 400. The results support and ensure the safety and suitability for clinical trials and eventual translation into clinical practice as potential probiotic.

Indoleamine 2, 3-dioxygenase 1 mediated alterations in the functionality of immune cells, deciphers the pregnancy outcomes in crossbred dairy cows.

Pregnancy establishment in bovines requires maternal immune cell modulation. Present study investigated possible role of immunosuppressive indolamine-2, 3-dioxygenase 1 (IDO1) enzyme in the alteration of neutrophil (NEUT) and peripheral blood mononuclear cells (PBMCs) functionality of crossbred cows. Blood was collected from non-pregnant (NP) and pregnant (P) cows, followed by isolation of NEUT and PBMCs. Plasma pro-inflammatory (IFNγ and TNFα) and anti-inflammatory cytokines (IL-4 and IL-10) were estimated by ELISA and analysis of IDO1 gene in NEUT and PBMCs by RT-qPCR. Neutrophil functionality was assessed by chemotaxis, measuring activity of myeloperoxidase and ß-D glucuronidase enzyme and evaluating nitric oxide production. Changes in PBMCs functionality was determined by transcriptional expression of pro-inflammatory (IFNγ, TNFα) and anti-inflammatory cytokine (IL-4, IL-10, TGFß1) genes. Significantly elevated (P < 0.05) anti-inflammatory cytokines, increased IDO1 expression, reduced NEUT velocity, MPO activity and NO production observed only in P cows. Significantly higher (P < 0.05) expression of anti-inflammatory cytokines and TNFα genes were observed in PBMCs. Study highlights possible role of IDO1 in modulating the immune cell and cytokine activity during early pregnancy and may be targeted as early pregnancy biomarkers.

Repeated injection of multivitamins and multiminerals during the transition period enhances immune response by suppressing inflammation and oxidative stress in cows and their calves.

Periparturient dairy cows undergo major physiological and metabolic changes as well as immunosuppression, associated with decrease in plasma concentrations of various minerals and vitamins. The present study was conducted to investigate effects of repeated injections of vitamins and minerals on oxidative stress, innate and adaptive immune response in periparturient dairy cows and their offspring. Experiment was carried out on 24 peripartum Karan-Fries cows, randomly divided into four groups (n=6): control, Multi-mineral (MM), Multi-vitamin (MV) and Multi-minerals and Multi-vitamin (MMMV). Five ml of MM (Zinc 40 mg/ml, Manganese 10 mg/ml, Copper 15 mg/ml, Selenium 5 mg/ml) and five ml of MV (Vitamin E 5 mg/ml, Vitamin A 1000 IU/ml, B-Complex 5 mg/ml, and Vitamin D3 500 IU/ml) were injected intramuscularly (IM) to the MM and MV groups. MMMV group cows were injected with both. In all treatment groups, injections and blood sampling were carried out on 30th, 15th, 7th days before and after expected date of parturition and at calving. In calves, blood was collected at calving and on 1, 2, 3, 4, 7, 8, 15, 30 and 45 days post-calving. Colostrum/milk were collected at calving and at days 2, 4, and 8 post-calving. A lower percentage of total neutrophils and immature neutrophils, higher percentage of lymphocytes together with increased phagocytic activity of neutrophils and proliferative capacity of lymphocytes found in blood of MMMV cows/calves. Lower relative mRNA expression of TLRs and CXCRs and higher mRNA expression of GR-α, CD62L, CD11b, CD25 and CD44 found in blood neutrophils of MMMV groups. Total antioxidant capacity was higher, activity of antioxidant enzymes (SOD and CAT), TBARS levels were lower in the blood plasma of treated cows/calves. In both cows/calves, plasma pro-inflammatory cytokines (IL-1α, IL-1ß, IL-6, IL-8, IL-17A, IFN-γ and TNF-α) increased, whereas anti-inflammatory cytokines (IL-4 and IL-10) decreased in MMMV groups. Total immunoglobulins increased in colostrum/milk of MMMV injected cows and plasma of their calves. Results indicate that repeated injections of multivitamins and multiminerals to peripartum dairy cows could be a major strategy to improve immune response and decrease in inflammation and oxidative stress in transition dairy cows and their calves.

Allelic diversity at BoLA DRB3 locus and association with predisposition to clinical mastitis in indicus and crossbred cattle.

Bovine lymphocyte antigen (BoLA) DRB3 locus in healthy and mastitis affected cattle has been genotyped by a polymerase chain reaction and restriction fragment length polymorphisms (PCR-RLFP) using RsaI restriction enzyme, followed by sequencing. In 130 farm animals, 25 BoLA DRB3 alleles have been detected by PCR-RFLP. Three distinct allelic patterns significantly associated with mastitis in Karan Fries crossbred and Sahiwal indicus cattle have been identified, whereas, four other allelic patterns were significantly high in frequency among healthy animals. Sequencing of RFLP genotypes revealed 25 and 47 alleles among healthy Sahiwal and Karan Fries, respectively, while 17 and 38 patterns observed in mastitis affected Sahiwal and Karan Fries animals, respectively. From Tajima's D-test of neutrality, it was concluded that alleles associated with mastitis were expanding in the population, whereas those of healthy were under contraction. Phylogenetic analysis carried out to delineate the evolutionary relationship of the farm and field animals at DRB3 locus, differentiating allelic patterns into six different clusters. Among the phylogenetic lineages, five patterns DRB3*028:01, DRB3*011:03, DRB3*031:01, DRB3*001:01 and DRB3*043:01, were previously reported, whereas one novel allelic variant was observed in indicus and crossbred cattle. This information will help in further exploring the association between BoLA-DRB3 genetic diversity and disease resistance in distinct cattle breeds, important in designing breeding strategies for increasing the distribution of favorable alleles.

Pregnancy stage-dependent modulation of neutrophil function may impact embryo survivability and pregnancy outcome in crossbred cows.

Pregnancy is a complicated physiological process that involves synchronized coordination between immune and endocrine systems. Neutrophils have been suggested as a critical immune cell for embryo implantation and pregnancy maintenance. The present study was conducted to evaluate the dynamic changes in the mRNA expressions of the cluster of designation (CD11b, CD31, CD44 and CD62L) molecules and interferon-stimulated genes (ISG15, MX1 and OAS1) in blood neutrophils throughout pregnancy in dairy cows and correlate them with the outcome of pregnancy. Blood samples were taken from negative control (NC) group, and non-pregnant (NP) group at the time of artificial insemination (AI, day zero) and on days 10, 14, 16, 18, and 21 post-AI. In pregnant (P) cows, samples were taken as described above and after every 30 days until the time of parturition. In aborted cows, samples were collected until the time of the abortion. Comparison between pregnant, non-pregnant and aborted cows revealed that the expression of CD molecules increased (p < 0.05) on days 14, 16, 18 and 21 post-AI only in NP cows as compared to other groups. Although the expression of CD molecules remained constant throughout the study period in pregnant and aborted cows, the expression of CD11b, CD31 and CD62L increased (p < 0.05) on the day of abortion and parturition. Unlike CD molecules, the expression of CD44 decreased significantly (p < 0.05) at the time of abortion. There was a significant (p < 0.05) increase in the expression of interferon-stimulated genes including MX1, OAS1 and ISG15 during the peri-implantation period in pregnant cows, and at the time of abortion in aborted cows. However, the expression of ISGs was lower (p < 0.05) in non-pregnant cows as compared to the other groups. The results revealed the critical role played by neutrophils during pregnancy and form the basis to unravel the underlying mechanism for neutrophil associated immunological infertility in bovines.

A Comparative Study on Changes in Total and Differential Milk Cell Counts, Activity, and Expression of Milk Phagocytes of Healthy and Mastitic Indigenous Sahiwal Cows.

Milk somatic cell counts (SCCs) have been used as a gold standard to monitor mammary health as well as an indicator of raw milk quality. The present work was undertaken to compare the changes in the milk SCC, milk differential leukocyte counts (DLCs), phagocytic activity (PA) of milk neutrophils and macrophages (by nitroblue tetrazolium assay), extracellular trap formation (PicoGreen assay) and mRNA expression of various genes in milk neutrophils and macrophages (reverse transcription-polymerase chain reaction), and milk plasma cortisol concentration (enzyme-linked immunosorbent assay) in healthy, subclinical mastitis (SCM), and clinical mastitis (CM) cows. Milk was collected from healthy, SCM, and CM cows grouped based on their SCCs and California mastitis test with eight cows in each group. Milk SCC was estimated by SCC counter, and DLC was done after staining the milk slide under a microscope at 100×. Total SCCs in healthy, SCM, and CM cows were on an average of 128.30, 300.3, and 694.40 × 103 cells/mL, respectively. Milk DLCs indicated a lower percentage of macrophage and lymphocytes and a higher (p < 0.05) percentage of neutrophils in SCM and CM compared to healthy milk. The percentage of mature segmented neutrophils was lower, whereas immature band neutrophils were higher (p < 0.05) in the SCM and CM groups as compared to healthy cows. The viability, in vitro PA, and extracellular trap formation of neutrophils were lower (p < 0.05) in SCM and CM milk samples as compared to healthy samples. However, the PA of macrophage remained unchanged in all the studied groups. The relative mRNA expression of Toll-like receptors (TLR2, TLR4), myeloperoxidase, and interleukin 2α (IL-2α) receptor (CD25) were minimum in healthy samples and increased (p < 0.05) with the progress of mammary inflammation. However, CD44 decreased (p < 0.05), and CD62L remained unchanged in mastitis as compared to healthy cows. Plasma cortisol concentrations were higher (p < 0.05) in mastitis as compared to healthy cows and were negatively correlated with the number of milk macrophages and the functions of milk phagocytes. Estimation of total SCC, milk DLC, and activity of milk phagocytes is essential for effective control and prevention of incidence of mastitis in dairy cows.

JAK3 and PI3K mediates the suppressive effects of interferon tau on neutrophil extracellular traps formation during peri-implantation period.

Interferon tau (IFNτ) is the main maternal signal for pregnancy in ruminants and modulates the functions of various immune cells, including neutrophils. Neutrophil extracellular traps (NETs) are one of the main defence mechanisms of neutrophils. In this study, we observed higher (p < 0.01) ex-vivo NETs extrusion by blood neutrophils from day 16-18 post artificial insemination (AI) in non-inseminated and inseminated non-pregnant cows compared to pregnant cows. In vitro study also showed that IFNτ hampers NETs formation in dose and time dependent manner. The lowest (p < 0.01) NETs formation and the highest (p < 0.01) mRNA expression (RT-PCR) of IFNτ stimulated genes (ISG15, OAS1, MX1) were observed when neutrophil incubated with 9 ng/mL IFNτ for 3.5 h. Signalling cascades mediating IFNτ impairment of NETs formation were identified using inhibitors of JAK2, JAK3, p38, PI3K/Akt and MAPK/Erk. IFNτ reduced (p < 0.01) the mRNA expression (RT-PCR) and concentration (ELISA) of genes and proteins that mediate NETs formation in blood neutrophils including histones (H1, H2), neutrophil elastase (NE) and myeloperoxidase (MPO). However, the effects of IFNτ on these genes and proteins were eliminated in the presence of JAK3 or PI3K inhibitors. Immunocytochemistry study also showed strong MPO signal in the presence of JAK3 or PI3K inhibitors as compared to positive control (PC, IFNτ alone). The results indicate that IFNτ impairs NETs formation using JAK3 and PI3K and thus essential for successful implantation and establishment of pregnancy in cows.

Supplementation of antioxidant micronutrients reduces stress and improves immune function/response in periparturient dairy cows and their calves.

BACKGROUND: Periparturient period induces stress in cows which fluctuates hormonal and metabolic function and causes immune suppression. Apart from impairing the health, production, and reproduction of cows, it also influences the well-being of newborn calves by decreasing the colostrum quality. Micronutrients are known for optimal health and production and their effects on parturition stress, immune response in both cow and its calf need to be explored. AIM: The aim of this study was to see the effect of oral supplementation of micronutrients during the prepartum period on the health status of crossbred dairy cows and subsequently on their newborn calves. METHODS: A total of 42 healthy multiparous cows were selected and randomly divided into five groups with seven cows in each group, i.e. control (Basal Diet, BD), VA group (BD + vitamin A, 105 IU), Zn group (BD + zinc sulphate, 60 ppm), VE group (BD + vitamin E, 2500 IU), and combined supplementation (CS) group (BD + combination of VA, Zn, and VE). The supplements were offered in compounded concentrate DM (100 g) to individual cows once daily before the morning feeding and the remaining portion was incorporated in the TMR. Feeding was started one month before the expected days of calving till calving. Blood samples were collected from cows at days -15, -7, -3, 0, +3, +7, and +15 relative to the day of calving. Blood samples from newborn calves and milk samples of cows were collected at days 0, +3, +7, and +15. Milk somatic cell counts (SCC) were estimated using a cell counter. Cortisol was estimated by ELISA kit in blood and milk plasma of cows and in the blood plasma of their calves. Total immunoglobulins (Ig) were estimated in milk of cows and serum of calves using zinc sulphate turbidity method. Blood neutrophils from cows and calves were studied for phagocytic activity (PA) using nitro blue tetrazolium (NBT) assay.Data were analysed by repeated-measures two-way ANOVA using the mixed procedure of SAS, and the pairwise comparison was performed using a multiple comparison test (Tukey). RESULTS: Combined supplementation of micronutrients decreased (P < 0.05) maternal blood plasma (control vs. CS group, 5.98 ±â€¯0.20 vs. 3.86 ±â€¯0.23 ng/mL) and milk plasma (3.96 ±â€¯0.13 vs. 2.71 ±â€¯0.10 ng/mL) cortisol, milk SCC (3.05 ±â€¯0.11 vs. 2.12 ±â€¯0.10 × 105 cells/mL) and increased (P < 0.05) total milk Ig concentration (18.80 ±â€¯0.11 vs. 23.04 ±â€¯0.57 mg/mL) and the PA of blood neutrophils (0.84 ±â€¯0.03 vs. 1.07 ±â€¯0.03). Similarly, lower blood cortisol concentration (9.69 ±â€¯0.35 vs. 6.02 ±â€¯0.18 ng/mL) and higher (P < 0.05) total Ig (23.26 ±â€¯0.11 vs. 30.34 ±â€¯0.70 mg/mL) and PA of blood neutrophils (0.37 ±â€¯0.02 vs. 0.52 ±â€¯0.02) were observed in the calves born to CS group of cows as compared to the control. Highest (P < 0.05) positive effects (lower stress levels and higher immune response) of treatment were noticed in CS group followed by VE group and then Zn group. However, VA group didn't differ from the control group. CONCLUSION: Our results indicate that micronutrient interventions during the prepartum period can improve the health status of dairy calves and subsequently the well-being of their calves.

Peripartum changes in the activity and expression of neutrophils may predispose to the postpartum occurrence of metritis in dairy cows.

Metritis is a postpartum uterine pathology that causes a huge economic loss due to increased culling risk and impaired milk yield and reproduction in cows. The present study was carried out to study the changes in the activity and expression of blood neutrophils in crossbred dairy cows with and without metritis. Collection of blood samples was done at -3, -2 and - 1 weeks before calving, at calving and during the first day of metritis diagnosis in metritis group (n = 8) or at day 8-10 post calving in healthy group (n = 8). Neutrophils were studied for its percentage (microscopically), respiratory burst (nitro blue tetrazolium assay), myeloperoxidase (MPO) concentrations (sandwich ELISA) and expression of CXCR1, CXCR2, TLR2, TLR4, GRα, CD11b, CD14, CD25, CD44, CD47 and CD62L (RT-PCR). Immunocytochemistry was used to investigate MPO concentration and CD14 activity, and western blotting was used for estimating MPO. Although most of these parameters changed in the cows that developed metritis one week before calving, MPO and CD14 got altered much earlier. Myeloperoxidase concentrations and expression of CD14 were considerably lower starting from -2 weeks before calving in cows that developed metritis compared to healthy cows. Further studies are warranted to study the possible use of MPO and CD14 to identify transition cows more vulnerable to develop metritis several weeks before disease occurrence.

Interaction between stress hormones and phagocytic cells and its effect on the health status of dairy cows: A review.

Dairy cows are exposed to various stressors during their production cycle that makes them more susceptible to various diseases. Phagocytes (neutrophils and macrophages) are important soldiers of the innate immune system. Neutrophils are the first responders to an inflammatory response and stress and kill pathogens by generating reactive oxygen species and by the release of various antimicrobial peptides, enzymes, neutrophil extracellular trap formation, etc. Macrophages, the other phagocytes, are also the cleanup crew for the innate immune system that removes debris, pathogens, and dead neutrophils later on after an inflammatory response. The neuroendocrine system along with phagocytes exhibits an immunomodulatory potential during stressful conditions. Neuroendocrine system directly affects the activity of phagocytes by communicating bidirectionally through shared receptors and messenger molecules such as hormones, neurotransmitters, or cytokines. Different immune cells may show variable responses to each hormone. Short time exposure to stress can be beneficial, but repeated or extended exposure to stress may be detrimental to the overall health and well-being of an animal. Although some stresses associated with farming practices in dairy cows are unavoidable, better understanding of the interactions occurring between various stress hormones and phagocytic cells can help to reduce stress, improve productivity and animal welfare. This review highlights the role played by various stress hormones in modulating phagocytic cell performance of dairy cattle under inflammatory conditions.

Implantation associated changes in expression profile of indoleamine-2, 3-dioxygenase 1, Th1-Th2 cytokines and interferon-stimulated genes on neutrophils and peripheral blood mononuclear cells of crossbred cows.

Effective bidirectional communication between the embryo and dam improves the reproductive efficiency of dairy cows. Possible role of immunosuppressive indolamine-2, 3-dioxygenase 1 (IDO1) enzyme in the regulation of maternal systemic cytokine balance/shift during early pregnancy establishment along with various interferon-stimulated genes (ISGs) expression in neutrophils and peripheral blood mononuclear cell (PBMCs) were investigated in crossbred cows. Blood was collected on days 0 i.e. day of Artificial Insemination (AI), 10, 18 and 36 post-AI followed by isolation of neutrophils and PBMCs for gene expression study of IDO1, anti-inflammatory cytokines (IL-4, IL-10 and TGFß1), pro-inflammatory cytokines (IFNγ and TNFα) and ISGs (ISG15, MX1, MX2, OAS1) in pregnant and non-pregnant cows. Cows were grouped as pregnant and non-pregnant after pregnancy confirmation by non-return to heat, ultrasonography, per rectal examination along with progesterone and IFNτ assay. Significantly (P < 0.05) higher relative mRNA expression of IDO1 and anti-inflammatory cytokines on days 10 and 18 post-AI were observed in both neutrophils and PBMCs of pregnant cows. Pregnant cows showed significantly (P < 0.05) higher mRNA transcripts of IFNγ and TNFα genes on days 18 post-AI in both neutrophils and PBMCs. Expression of ISGs was higher (P < 0.05) on day 10th and 18th post AI in both the neutrophils and PBMCs of pregnant cows. The study indicates that systemic immune regulation by IDO1 (through cytokine shift) and ISGs in peripheral immune cells are essential for the establishment of pregnancy and may be targeted in future as biomarkers for pregnancy diagnosis.

A comparative study on various immunological parameters influencing embryo survivability in crossbred dairy cows.

Since long embryonic mortality has remained an area of concern affecting the reproduction, production, and profitability of dairy cows. We investigated the possible interaction between interleukins, hormones, and neutrophil associated CD markers during the implantation window in Karan Fries (KF) cows naturally coming to heat. Blood collection was done on days 0 i.e. day of Artificial Insemination (AI), 10, 18, 21, 30 and on day 40 post-AI. Total leucocyte count (TLC) and neutrophil to lymphocyte (N:L) ratio were recorded. Blood neutrophils were isolated and their number, phagocytic activity (PA), myeloperoxidase (MPO) concentration and relative mRNA expression of cell adhesion molecules (CD-11b, CD-31, CD-44, CD-62L) as well as progesterone-inducing-blocking-factor (PIBF) and glucocorticoid receptor alpha (GRα) were examined. Plasma progesterone, cortisol, IL-2, IL-8, IL-6, and IL-10 were also measured. Pregnancy was confirmed by non-return to heat, ultrasonography and per rectal examination along with progesterone assay. Cows were further divided into pregnant (P), early embryonic mortality (EEM) and late embryonic mortality (LEM) groups. Embryonic losses cows showed lower plasma concentration of IL-10 (<100 pg/ml) and a higher concentration of IL-2 (>500 pg/ml). Also, a 4 fold increase in the relative mRNA expression of CD-11b and 2.5 fold changes in CD-44 expression were observed in embryonic mortality. We observed a 1.5 fold increase in the relative mRNA expression of PIBF and a 0.5 fold increase in GRα expression in pregnant cows compared to EEM (on day 21) and LEM (on days 30 and 40) cows. Our results depicted that the hyperimmune status of the dam which could be due to multifactorial events that led to the pregnancy failure. The above basic values may be used for checking the immune status and thus timely management strategies can be taken to prevent embryonic losses.

Sensitive and rapid lateral-flow assay for early detection of subclinical mammary infection in dairy cows.

Detection of subclinical mastitis (SCM) in its initial stage can save great economic losses, improve milk quality and animal welfare. We have developed a semiquantitative lateral flow assay for the detection of SCM in dairy cows targeting myeloperoxidase (MPO) enzyme of milk neutrophils. A competitive immunoassay format was used, and colloidal gold nanoparticles (GNP) were prepared and used as a labelling agent. Monoclonal anti-MPO antibodies were used and assessed for its quality by enzyme-linked immunosorbent assay and dot blot. Conjugation method for GNP and anti-MPO antibodies was standardised, and the conjugate was placed over the conjugate pad. MPO coupled with a carrier protein (OVA) and the species-specific secondary antibodies were placed on test and control lines, respectively. The developed assay was verified with 75 milk samples collected from healthy, SCM and clinical mastitis cows. It displayed a high sensitivity as it could detect MPO as low as 1.5 ng/ml, an accuracy greater than 97% and showed no crossreactivity when crosschecked with other milk proteins. The developed assay can be used as an alternative for SCM diagnostic tests where lab structure are available for obtaining the lysate of milk SCC.

Proteomics and transcriptomics study reveals the utility of ISGs as novel molecules for early pregnancy diagnosis in dairy cows.

An early and precise diagnosis of pregnancy in cows is critical to short the calving interval and to improve their reproductive efficiency. Neutrophils are the first blood cells to sensitize the embryo in the uterus and participate in maternal recognition of pregnancy after getting induced by interferon tau (IFNτ). To study the protein abundance ratio, blood samples were collected on 0th, 10th, 18th and 36th day post-artificial insemination (AI) from crossbred Karan Fries cows. Neutrophils were isolated through density gradient centrifugation and studied for protein abundance by high-performance liquid chromatography coupled with mass spectrometry (LC-MS). Protein abundance ratios for Myxovirus resistance (MX1 and MX2) were found to be higher (P < 0.05) on day 10 and day 18 post-AI, whereas Oligoadenylate synthetase-1 (OAS1) and Interferon stimulated gene-15 ubiquitin-like modifier (ISG15) proteins were more abundant on day 18 post-AI. The relative mRNA expressions of these molecules were also studied by qPCR. The gene expression of ISG15, MX1, MX2 and OAS1 was found to be higher (P < 0.05) on day 10th, 18th and 36th post-AI compared to day 0. The study indicates that ISGs on blood neutrophils are essential for the establishment of pregnancy and may be targeted as potential biomarkers for pregnancy diagnosis in cows.

Genome-Wide Runs of Homozygosity Revealed Selection Signatures in Bos indicus.

Genome-wide runs of homozygosity (ROH) are suitable for understanding population history, calculating genomic inbreeding, deciphering genetic architecture of complex traits and diseases as well as identifying genes linked with agro-economic traits. Autozygosity and ROH islands, genomic regions with elevated ROH frequencies, were characterized in 112 animals of seven Indian native cattle breeds (B. indicus) using BovineHD BeadChip. In total, 4138 ROH were detected. The average number of ROH per animal was maximum in draft breed, Kangayam (63.62 ± 22.71) and minimum in dairy breed, Sahiwal (24.62 ± 11.03). The mean ROH length was maximum in Vechur (6.97 Mb) and minimum in Hariana (4.04 Mb). Kangayam revealed the highest ROH based inbreeding (FROH > 1Mb = 0.113 ± 0.059), whereas Hariana (FROH > 1Mb = 0.042 ± 0.031) and Sahiwal (FROH > 1Mb = 0.043 ± 0.048) showed the lowest. The high standard deviation observed in each breed highlights a considerable variability in autozygosity. Out of the total autozygous segments observed in each breed except Vechur, > 80% were of short length (< 8 Mb) and contributed almost 50% of the genome proportion under ROH. However, in Vechur cattle, long ROH contributed 75% of the genome proportion under ROH. ROH patterns revealed Hariana and Sahiwal breeds as less consanguineous, while recent inbreeding was apparent in Vechur. Maximum autozygosity observed in Kangayam is attributable to both recent and ancient inbreeding. The ROH islands were harbouring higher proportion of QTLs for production traits (20.68% vs. 14.64%; P≤ 0.05) but lower for reproductive traits (11.49% vs. 15.76%; P≤ 0.05) in dairy breeds compared to draft breed. In draft cattle, genes associated with resistant to diseases/higher immunity (LYZL1, SVIL, and GPX4) and stress tolerant (CCT4) were identified in ROH islands; while in dairy breeds, for milk production (PTGFR, CSN1S1, CSN2, CSN1S2, and CSN3). Significant difference in ROH islands among large and short statured breeds was observed at chromosome 3 and 5 involving genes like PTGFR and HMGA2 responsible for milk production and stature, respectively. PCA analysis on consensus ROH regions revealed distinct clustering of dairy, draft and short stature cattle breeds.

Potential roles of neutrophils in maintaining the health and productivity of dairy cows during various physiological and physiopathological conditions: a review.

Neutrophils represent the first line of innate immunity and are the most prominent line of cellular defence against invading microorganisms. On stimulation, they can quickly move through the walls of veins and into the tissues of the body to immediately attack or monitor the foreign antigens. Neutrophils are highly versatile and sophisticated cells which are endowed with highly sensitive receptor-based perception systems. They were traditionally classified as short-lived phagocytes actively involved during infection and inflammation, but recently, it has been seen that neutrophils are capable of detecting the presence of sperms during insemination as well as an implanting embryo in the female reproductive tract. These specialised phagocytes play a major role in tissue remodelling and wound healing, and maintain homeostasis during parturition, expulsion of placenta, folliculogenesis, corpus luteum formation and luteolysis. Here, we review the role played by neutrophils in maintaining homeostasis during normal and inflammatory conditions of dairy cattle. We have summarised the alteration in the expression of some cell adhesion molecules and cytokines on bovine neutrophils during different physiological and physiopathological conditions. Some emerging issues in the field of neutrophil biology and the possible strategies to strengthen their activity during the period of immunosuppression have also been discussed.

Impact of different seasons on the milk somatic and differential cell counts, milk cortisol and neutrophils functionality of three Indian native breeds of cattle.

The present study was undertaken to compare the effect of different seasons on the mammary immunity of three Indian native breeds of cows (Tharparkar, Gir and Sahiwal) well adapted to the tropical region. For this milk samples were collected from cows in winter (THI=57, comfortable zone), hot-dry (HD; THI=76, heat stressful zone) and hot-humid (HH; THI = 82, severe heat stress) and estimated for milk somatic cell counts (SCC), phagocytic activity (PA) of milk neutrophils, milk cortisol and heat shock proteins and function associated genes in milk neutrophils. Milk SCC was evaluated using a cell counter and differential cell counts measured microscopically. Cortisol was quantified in skimmed milk by competitive ELISA. Milk PA was estimated using nitro blue tetrazolium assay, and for gene expression studies, milk neutrophils were isolated and studied for heat shock proteins (HSP40, HSP70, HSP90α) and cell adhesion molecules (CD11b, CD25, CD44) using real-time polymerase chain reaction. All the studied parameters increased in HD and HH seasons with highest values observed in Sahiwal cows. However, PA of neutrophil was highest in Tharparkar cows in winter and decreased gradually at higher THI values during hot seasons. Milk cortisol was positively correlated with expression of various CD molecules and HSPs (p < 0.05) in milk neutrophils but negatively correlated (p < 0.05) with PA during HH season in all breeds. The study revealed that Indian native cows were at considerable risk in HH season and Sahiwal cows were more heat stressed followed by Gir and Tharparkar cows, respectively, and thus may require managemental interventions. Also, the higher expression of HSP70 and CD25 with increasing THI levels in hot seasons makes them suitable biological markers for quantifying heat stress.