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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 β-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, TGFβ1) 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., Competing Interests: Declaration of competing interest The authors declare no conflict of interest, financial or otherwise., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors have no competing interests to declare., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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 × 10 3 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Alhussien, Panda and Dang.)

The caprine mesenchymal stem cells (MSCs) derived from fetal adnexa are highly proliferative. These cells possess tri-lineage differentiation potential and express MSC surface antigens and pluripotency markers with a wound-healing potential. This present study was conducted to compare the immunomodulatory potential of caprine MSCs derived from the fetal adnexa. Mid-gestation caprine uteri (2-3 months) were collected from the abattoir to isolate MSCs from amniotic fluid (cAF), amniotic sac (cAS), Wharton's jelly (cWJ) and cord blood (cCB), which were expanded and characterized at the 3 rd passage. These MSCs were then stimulated with inflammatory cytokines (IFN-γ and TNF-α) to assess the percentage of inhibition produced on peripheral blood mononuclear cells (PBMCs) proliferation. The percentage of inhibition on activated PBMCs proliferation produced by cWJ MSCs and cAS MSCs was significantly higher than cCB and cAF MSCs. The relative mRNA expression profile and immunofluorescent localization of different immunomodulatory cytokines and growth factors were conducted upon stimulation. The mRNA expression profile of a set of different cytokines and growth factors in each caprine fetal adnexa MSCs were modulated. Indoleamine 2, 3 dioxygenase appeared to be the major immunomodulator in cWJ, cAF, and cCB MSCs whereas inducible nitric oxide synthase in cAS MSCs. This study suggests that caprine MSCs derived from fetal adnexa display variable immunomodulatory potential, which appears to be modulated by different molecules among sources.

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, 10 5 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 ± 0.20 vs. 3.86 ± 0.23 ng/mL) and milk plasma (3.96 ± 0.13 vs. 2.71 ± 0.10 ng/mL) cortisol, milk SCC (3.05 ± 0.11 vs. 2.12 ± 0.10 × 10 5 cells/mL) and increased (P < 0.05) total milk Ig concentration (18.80 ± 0.11 vs. 23.04 ± 0.57 mg/mL) and the PA of blood neutrophils (0.84 ± 0.03 vs. 1.07 ± 0.03). Similarly, lower blood cortisol concentration (9.69 ± 0.35 vs. 6.02 ± 0.18 ng/mL) and higher (P < 0.05) total Ig (23.26 ± 0.11 vs. 30.34 ± 0.70 mg/mL) and PA of blood neutrophils (0.37 ± 0.02 vs. 0.52 ± 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., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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 TGFβ1), 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 10 th and 18 th 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., Competing Interests: Declaration of Competing Interest The authors reported no declarations of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they don’t have any competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest, financial or otherwise., (Copyright © 2020 Elsevier B.V. All rights reserved.)

This study was conducted to characterize canine bone marrow-derived mesenchymal stem cells (BMSCs); in vivo tracking in mice, and therapeutic evaluation in canine clinical paraplegia cases. Canine BMSCs were isolated, cultured, and characterized in vitro as per International Society for Cellular Therapy criteria, and successfully differentiated to chondrogenic, osteogenic, and adipogenic lineages. To demonstrate the homing property, the pGL4.51 vector that contained luciferase reporter gene was used to transfect BMSCs. Successfully transfected cells were injected around the skin wound in mice and in vivo imaging was done at 6, 12 and 24 hr post MSCs delivery. In vivo imaging revealed that transfected BMSCs migrated and concentrated predominantly toward the center of the wound. BMSCs were further evaluated for allogenic therapeutic potential in 44 clinical cases of spinal cord injuries (SCI) and compared with conventional therapy (control). Therapeutic potential as evaluated by different body reflexes and recovery score depicted significantly better results in stem cell-treated group compared to control group. In conclusion, allogenic canine BMSCs can serve as potent therapeutic candidate in cell-based therapies, especially for diseases like SCI, where the conventional medication is not so promising., (© 2018 Wiley Periodicals, Inc.)

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The aim of the present study was to determine potential role of leptin on in vitro developmental competence of buffalo oocytes and embryos. Slaughterhouse derived culture grade buffalo cumulus oocyte complexes (COCs) were matured in vitro (IVM) with leptin (10 ng/ml) or without leptin (control). In each experiment, a pool of matured COCs was used for further in vitro embryo production and another pool of COCs was used for cumulus cells and mature oocytes isolation to study the relative mRNA expression of developmentally important genes. Presumptive zygotes were cultured in embryo culture (IVC) media supplemented with leptin (10 ng/ml) or without leptin (control). Cleavage rate was higher (p < 0.05) when leptin was supplemented during IVM + IVC, both, as compared to other groups. Higher cleavage rate was observed in leptin-treated groups, though it was non-significant. Blastocyst rate was higher (p < 0.05) in all the leptin treated groups. The relative mRNA expression of LEPR (Ob-Rb), HAS2 and EGFR was significantly (p < 0.05) up-regulated and the expression of CASPASE3 was down-regulated in cumulus cells of leptin-treated groups. The expression of GDF9, BMP15, GLUT1, LEPR and CASPASE3 transcripts in leptin and non-treated oocytes did not differ. The relative mRNA expression of POU5F1and LEPR transcripts in blastocysts was higher (p < 0.05) in leptin-treated groups; the change in expression of GLUT1, INF-τ and CASPASE3 transcripts was not significant (p > 0.05). Thus, it is concluded that leptin promotes developmental competence of bubaline oocytes by modulating cumulus enabling factors and genes regulating pluripotentcy in the blastocysts., (Copyright © 2017 Elsevier Inc. All rights reserved.)

This study was conducted to know the impact of cryopreservation on caprine fetal adnexa derived mesenchymal stem cells (MSCs) on the basic stem cell characteristics. Gravid caprine uteri (2-3 months) were collected from local abattoir to derive (amniotic fluid [cAF], amniotic sac [cAS], Wharton's jelly [cWJ], and cord blood [cCB]) MSCs and expanded in vitro. Cells were cryopreserved at 3rd passage (P3) using 10% DMSO. Post-thaw viability and cellular properties were assessed. Cells were expanded to determine growth kinetics, tri-lineage differentiation, localization, and molecular expression of MSCs and pluripotency markers; thereafter, these cells were transplanted in the full-thickness (2 × 2cm 2 ) rat skin wound to determine their wound healing potential. The post-thaw (pt) growth kinetics study suggested that cWJ MSCs expanded more rapidly with faster population doubling time (PDT) than that of other fetal adnexa MSCs. The relative mRNA expression of surface antigens (CD73, CD90, and CD 105) and pluripotency markers (Oct4, KLF, and cMyc) was higher in cWJ MSCs in comparison to cAS, cAF, and cCB MSCs post-thaw. The percent wound contraction on 7th day was more than 50% for all the MSC-treated groups (pre and post-thaw), against 39.55% in the control group. On day 28th, 99% and more wound contraction was observed in cAF, cAF-pt, cAS-pt, cWJ, cWJ-pt, and cCB, MSCs with better scores for epithelization, neovascularization, and collagen characteristics at a non-significant level. It is concluded that these MSCs could be successfully cryopreserved without altering their stemness and wound healing properties. J. Cell. Physiol. 232: 2186-2200, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Oocyte-secreted factors (OSFs) play an important role in the acquisition of oocyte developmental competence through bidirectional cross-talk between oocyte and cumulus cells via gap junctions. Thus, the present study was designed to investigate the effect of two OSFs, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), on the developmental competence of buffalo oocytes derived from two different follicle sizes. Cumulus-oocyte complexes (COCs) from large follicles (LF, >6 mm) or small follicles (SF, 0.05) between DOs and combination groups. Relative mRNA analysis revealed significantly higher (P > 0.05) expression of the cumulus cell marker genes EGFR, HAS2, and CD44 in LF-derived than SF-derived oocyte; the expression of these markers was significantly higher (P > 0.05) in DOs and combination groups, irrespective of the follicle size. These results suggested that LF-derived oocytes have a higher developmental competence than SF-derived oocytes and that supplementation of GDF9 and BMP15 modulates the developmental competence of buffalo oocytes by increasing the relative abundance of cumulus-enabling factors and thereby increasing cleavage and the quality of blastocyst production.