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.

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.

Macrophage-activating factor of bovine colostrum promotes phagocytic activity of murine macrophages and bovine phagocytes.

Periparturient dairy cows and their newborn calves are highly prone to health complications. Enhancing the innate immune system of these animals is essential to mitigate the transition period stress and promote their health. Macrophage activating factor (MAF) possess immunomodulatory properties and is believed to enhance immune response. In the present study, the impact of different concentrations (10, 50, 100 ng) of MAF on the phagocytic activity (PA) of murine and bovine phagocytoses was explored. MAF synthesized from IgA of cow colostrum was studied for its effect on the phagocytic index (PI) of cow colostrum macrophages (Mφ) and blood neutrophils (sick and healthy calves) under in vitro conditions. Besides, the impact of MAF on the PI of peritoneal Mφ of healthy and immunocompromised mice was studied. PI of healthy Mφ (mice peritoneal and cow colostrum) and healthy neutrophils (blood calf) increased significantly (P < 0.05) after MAF supplementation. MAF also significantly (P < 0.05) increased the PI of neutrophils and Mφ obtained from sick calf and immunocompromised mice, respectively. Results indicate that colostrum MAF can be used as a potential immune modulator to promote immunity and fight infections in dairy animals.

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.

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.

Interferon-tau stimulated gene expression: A proxy to predict embryonic mortality in dairy cows.

The embryonic mortality in cows is a growing concern for an ever-expanding dairy industry. The current study was an attempt to shorten the open period of dairy cows having suffered embryonic loss by diagnosing them at an earlier stage. The blood samples were collected from the Karan Fries (KF) cows on days 0 (day of AI/estrus), 4, 8, 12, 14, 16, 18, 21, 24, 28, 35 and 42 post insemination. The experimental animals were then categorized into pregnant (P), conception failure/early embryonic mortality (EEM) and late embryonic mortality cows (LEM), based on progesterone assay, ultrasonography and per-rectal palpation. There were 6 animals in each group. The plasma progesterone was higher in pregnant than EEM and LEM cows. Plasma Interferon-tau concentration was significantly (p < 0.05) lower in LEM than pregnant cows where it could be detected from day 14-21 but was non-detectable in EEM cows. The mRNA expression of ISG15, OAS1, MX1 and MX2 in blood neutrophils was significantly (p < 0.05) higher from day 8-42 as against day 0 in pregnant cows. The highest expression was observed around day 18-21 in pregnant cows. The ISG15, OAS1, MX1 and MX2 mRNA expression was significantly (p < 0.05) higher from day 4-42 as compared to day 0 in LEM cows, whereas in EEM cows the expression stayed close to that of day 0 (1.00 ±â€¯0.00). The mRNA expression of ISG15, OAS1, MX1 and MX2 started to decline from day 24 onwards. The degree of expression of Interferon-tau stimulated genes was higher in pregnant and LEM cows than EEM cows. The study reveals that the Interferon tau stimulated gene expression in neutrophils can act as peripheral biomarkers for detecting the embryonic mortality in dairy cows.

Effect of tropical thermal stress on peri-implantation immune responses in cows.

Pregnancy losses during the peri-implantation period cause a negative impact on the reproductive and economic performance of dairy herds. In this study, we investigated the possible immunological factors which may contribute to pregnancy loss during the peri-implantation period under different seasons of tropical conditions. Eighteen Karan Fries cows, six cows in each season (W: winter; HH: hot-humid; HD: hot-dry) were selected. These cows exhibited heat and were brought for artificial insemination (AI; day 0). Blood was collected on days 0, 10, 14, 16, 18, 21 and 28 post-AI. Pregnancy was confirmed by non-return to heat, progesterone assay and ultrasonography. Blood neutrophils were isolated and tested for their number, myeloperoxidase (MPO) concentrations and expression of cell adhesion molecules (CD11b, CD14, CD25, CD47), interferon tau stimulated genes (ISG15, MX1, OAS1) and chemokine receptors (CXCR1, CCL2). Plasma cortisol, progesterone, IL-2 and IL-10 were also estimated. Neutrophil number, MPO levels, the relative expression of various neutrophil receptors and plasma IL-2 were low between days 14-21 post-AI in all seasons. However, plasma cortisol and IL-10 were higher during the same period. The inflammatory activity of neutrophils, plasma IL-2 and cortisol were highest in HH, intermediate in HD and lowest in W season. However, plasma progesterone and IL-10 were highest in W season and lowest in HH season. Our results show that blood neutrophils sense the implanting embryo and downregulate their activity to ensure successful implantation; however, under harsh environmental conditions, it is a great challenge for the immune system to maintain such balance and thus it may negatively affect the outcome of pregnancy.