Publications by authors named "Ajay Kumar Dang"

23 Publications

  • Page 1 of 1

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.

Front Vet Sci 2021 21;8:670811. Epub 2021 Jun 21.

Lactation and Immuno-Physiology Laboratory ICAR-National Dairy Research Institute, Karnal, India.

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 cells/mL, respectively. Milk DLCs indicated a lower percentage of macrophage and lymphocytes and a higher ( < 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 ( < 0.05) in the SCM and CM groups as compared to healthy cows. The viability, PA, and extracellular trap formation of neutrophils were lower ( < 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 ( < 0.05) with the progress of mammary inflammation. However, CD44 decreased ( < 0.05), and CD62L remained unchanged in mastitis as compared to healthy cows. Plasma cortisol concentrations were higher ( < 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.
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http://dx.doi.org/10.3389/fvets.2021.670811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8255372PMC
June 2021

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

J Reprod Immunol 2021 06 9;145:103321. Epub 2021 Apr 9.

Lactation and Immuno-Physiology Laboratory, ICAR-National Dairy Research Institute, Karnal, Haryana, 132 001, India. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jri.2021.103321DOI Listing
June 2021

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

J Trace Elem Med Biol 2021 May 14;65:126718. Epub 2021 Jan 14.

Lactation and Immuno-Physiology Laboratory, ICAR-National Dairy Research Institute, Karnal, Haryana, 132001, India. Electronic address:

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 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 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.
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http://dx.doi.org/10.1016/j.jtemb.2021.126718DOI Listing
May 2021

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

Res Vet Sci 2021 Mar 10;135:456-468. Epub 2020 Nov 10.

Lactation and Immuno-Physiology Laboratory, ICAR-National Dairy Research Institute, Karnal, Haryana 132001, India.

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.
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http://dx.doi.org/10.1016/j.rvsc.2020.11.003DOI Listing
March 2021

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

Vet World 2020 Sep 10;13(9):1837-1848. Epub 2020 Sep 10.

Lactation and Immuno-Physiology Laboratory, ICAR-National Dairy Research Institute, Karnal, Haryana, India.

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.
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http://dx.doi.org/10.14202/vetworld.2020.1837-1848DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7566244PMC
September 2020

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.

J Reprod Immunol 2020 11 18;142:103188. Epub 2020 Aug 18.

Lactation and Immuno-Physiology Laboratory, ICAR-National Dairy Research Institute, Karnal, Haryana, 132001, India. Electronic address:

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 and 18 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.
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http://dx.doi.org/10.1016/j.jri.2020.103188DOI Listing
November 2020

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

Theriogenology 2020 Nov 24;157:140-148. Epub 2020 Jul 24.

Lactation and Immuno-Physiology Laboratory, ICAR-National Dairy Research Institute, Karnal, Haryana, 132001, India. Electronic address:

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.
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http://dx.doi.org/10.1016/j.theriogenology.2020.05.041DOI Listing
November 2020

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

Sci Rep 2020 07 7;10(1):11161. Epub 2020 Jul 7.

Lactation and Immuno-Physiology Laboratory, ICAR-National Dairy Research Institute, Karnal, Haryana, 132 001, India.

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.
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http://dx.doi.org/10.1038/s41598-020-68174-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7341798PMC
July 2020

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

J Reprod Immunol 2020 08 19;140:103148. Epub 2020 May 19.

Lactation and Immuno-Physiology Laboratory, ICAR-National Dairy Research Institute, Karnal, Haryana 132001, India. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jri.2020.103148DOI Listing
August 2020

Genome-Wide Runs of Homozygosity Revealed Selection Signatures in .

Front Genet 2020 21;11:92. Epub 2020 Feb 21.

Animal Genetics Division, ICAR-National Bureau of Animal Genetic Resources, Karnal, India.

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 () 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 ( = 0.113 ± 0.059), whereas Hariana ( = 0.042 ± 0.031) and Sahiwal ( = 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 ( and ) and stress tolerant () were identified in ROH islands; while in dairy breeds, for milk production ( and ). Significant difference in ROH islands among large and short statured breeds was observed at chromosome 3 and 5 involving genes like and responsible for milk production and stature, respectively. PCA analysis on consensus ROH regions revealed distinct clustering of dairy, draft and short stature cattle breeds.
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http://dx.doi.org/10.3389/fgene.2020.00092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7046685PMC
February 2020

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

Immunol Res 2019 02;67(1):21-38

Lactation and Immuno-Physiology Laboratory, ICAR-National Dairy Research Institute, Karnal, Haryana, 132 001, India.

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.
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http://dx.doi.org/10.1007/s12026-019-9064-5DOI Listing
February 2019

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

J Therm Biol 2018 Dec 25;78:27-35. Epub 2018 Aug 25.

Lactation and Immuno-Physiology Laboratory, ICAR-National Dairy Research Institute, Karnal 132 001, Haryana, India. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jtherbio.2018.08.020DOI Listing
December 2018

Development of enzyme-linked immunosorbent assay for early pregnancy diagnosis in cattle.

Anim Reprod Sci 2018 Oct 17;197:126-133. Epub 2018 Aug 17.

Lactation and Immunophysiology Laboratory, Animal Physiology Division, ICAR-National Dairy Research Institute, Karnal, 132001, Haryana, India.

The prerequisite for enhancing reproductive efficacy and successful calving is an early and accurate diagnosis of pregnancy. A highly sensitive enzyme-linked immunosorbent assay (ELISA) utilizing the second antibody coating technique and the horseradish peroxidase conjugate as a label for the determination of Interferon-stimulated protein, 15 kDa (ISG15) in dairy cows was developed. To validate a neutrophil lysate based ELISA for early pregnancy diagnosis, blood samples from healthy multiparous Karan Fries (KF) cows were collected on day 0 (day of AI), 10, 14, 16 and 21 post artificial insemination. The pregnancy and non-pregnancy in cows were confirmed by plasma progesterone assay, ultrasonography and per rectal palpation. The detection range of the assay was 0.312 to 25 ng/ml with a sensitivity of 0.13 ng/ml. The inter-assay and intra-assay coefficient of variation was 12.1% and 10.1%, respectively. The parallelism for measured and expected concentrations had an r value of 0.90 and 0.93, respectively. It was observed that ISG15 concentration was greater (P < 0.01) in pregnant as compared to non-pregnant cows, the greatest being 9.36 ± 0.50 ng/ml on day 16 in pregnant cows. With the use of immunocytochemistry, there was a positive staining for ISG15 with greater staining in the neutrophils of day 16 pregnant cows. The study reveals that ISG15 protein in a neutrophil lysate can be utilized as a biomarker for early pregnancy diagnosis in cows.
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http://dx.doi.org/10.1016/j.anireprosci.2018.08.020DOI Listing
October 2018

Milk somatic cells, factors influencing their release, future prospects, and practical utility in dairy animals: An overview.

Vet World 2018 May 2;11(5):562-577. Epub 2018 May 2.

Lactation and Immuno-Physiology Laboratory; ICAR-National Dairy Research Institute, Karnal, Haryana - 132 001, India.

Milk somatic cells (SCs) are a mixture of milk-producing cells and immune cells. These cells are secreted in milk during the normal course of milking and are used as an index for estimating mammary health and milk quality of dairy animals worldwide. Milk SC is influenced by cow productivity, health, parity, lactation stage, and breed of an animal. Any change in environmental conditions, poor management practices, and also stressful conditions significantly increases the amount of SC coming in milk. Better hygiene and proper nutrition help in reducing milk SC. Milk with low SC means better milk products with a longer shelf life. The present review describes the role of SCs (both secretory and immune) in milk, their role in maintaining the integrity of the mammary gland, and factors affecting their release in milk. This information may help to reduce milk somatic cell counts (SCCs) and to establish differential SCC standards.
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http://dx.doi.org/10.14202/vetworld.2018.562-577DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5993762PMC
May 2018

Pathogen-dependent modulation of milk neutrophils competence, plasma inflammatory cytokines and milk quality during intramammary infection of Sahiwal (Bos indicus) cows.

Microb Pathog 2018 Aug 19;121:131-138. Epub 2018 May 19.

Lactation and Immuno-Physiology Laboratory, ICAR-National Dairy Research Institute, Karnal 132001, Haryana, India. Electronic address:

The aim of the current study was to investigate the responses of milk neutrophils and plasma inflammatory cytokines to various mastitis pathogens and subsequently on milk composition. Milk was collected from healthy (n = 10) and clinical mastitis indigenous Sahiwal cows naturally infected either with gram-positive bacteria mainly S. aureus (n = 10) and Strep. agalactiae (n = 10) or with gram-negative bacteria, E. coli (n = 10). Phagocytic activity of milk neutrophils decreased in all mastitis cows with the lowest values recorded during gram-positive bacterial infections. Maximum plasma cortisol levels were observed in cows infected with gram-positive bacteria and were positively correlated with the milk neutrophils percentage and negatively correlated with the phagocytic activity of neutrophils and expression of glucocorticoid receptor. The plasma concentrations of IL-2 and IL-8 increased in all mastitis groups with maximum values recorded during E. coli infections. Unlike gram-negative bacterial infections, gram-positive bacterial infections evoked a minimal tumor necrosis factor-α (TNF-α), and IL-6 response. Milk somatic cell counts, fat, protein, pH and electrical conductivity increased in mastitis cows with the highest values exhibited by Strep. agalactiae infection. The expression of chemokine receptors (CXCR1, CXCR2), IL-8 and CD11b was maximum in mastitis neutrophils infected with E. coli. The expression of glucocorticoid receptor (GRα) decreased in all mastitis groups with the lowest values were found in S. aureus infection. Among the various mastitis pathogens, Strep. agalactiae showed maximum adverse effect on milk quality. Attenuated neutrophils, TNF-α and IL-6 response in cows infected by gram-positive bacteria may contribute to the establishment of chronic mastitis.
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http://dx.doi.org/10.1016/j.micpath.2018.05.029DOI Listing
August 2018

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

Theriogenology 2018 Jul 29;114:149-158. Epub 2018 Mar 29.

Lactation and Immuno-Physiology Laboratory, ICAR-National Dairy Research Institute, Karnal, 132 001, Haryana, India. Electronic address:

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.
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http://dx.doi.org/10.1016/j.theriogenology.2018.03.036DOI Listing
July 2018

JAK3 and PI3K mediate bovine Interferon-tau stimulated gene expression in the blood neutrophils.

J Cell Physiol 2018 06 9;233(6):4885-4894. Epub 2018 Jan 9.

Lactation and Immunophysiology Laboratory, Animal Physiology Division, ICAR-National Dairy Research Institute, Karnal, Haryana, India.

Interferon tau, a 23 kDa trophoblast derived protein diffuses out from the uterus into the circulation and leads to the expression of IFNτ stimulated genes viz. ISG15 and OAS1 in blood neutrophils. The IFNτ pathway is species as well as tissue specific. To unsnarl the IFNτ downstream signaling pathway, the blood neutrophils were incubated simultaneously with 10 ng/ml of recombinant bovine interferon tau and the inhibitors of JAK2 (AG490), JAK3 (CP690550), p38 (SB202190), PI3K/Akt (LY294002), and MAPK/Erk (U0126) at specific doses for 4-hr duration. The IFNτ pathway was determined through real-time gene expression of ISG15 and OAS1; immunocytochemistry of ISG15; and Western blotting of ISG15, OAS1, pJAK3 and PI3K. The ISG15 and OAS1 expression decreased significantly (p < 0.001) in the presence of pJAK3 and PI3K inhibitors as compared to a positive control where only interferon tau was used. Immunocytochemistry revealed an attenuated ISG15 response while stimulating blood neutrophils with pJAK3 inhibitor (CP690550) and PI3K inhibitor (LY294002). Similarly, Western blot analysis of neutrophil protein fraction showed weak signals of ISG15, OAS1, pJAK3 and PI3K in the presence of pJAK3 and PI3K inhibitors. The expression profile, immunocytochemistry and western blot analysis revealed a JAK3 and PI3K mediated interferon-tau stimulated gene expression in blood neutrophils.
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http://dx.doi.org/10.1002/jcp.26296DOI Listing
June 2018

Integrated effect of seasons and lactation stages on the plasma inflammatory cytokines, function and receptor expression of milk neutrophils in Sahiwal (Bos indicus) cows.

Vet Immunol Immunopathol 2017 Sep 29;191:14-21. Epub 2017 Jul 29.

Lactation and Immuno-Physiology Laboratory, ICAR-National Dairy Research Institute, Karnal, Haryana 132 001, India. Electronic address:

Mastitis is a highly prevalent and one of the costliest diseases of dairy cows affecting the mammary gland. Milk neutrophils present in the mammary gland serve as an integral part of the mammary immunity, and their performance is influenced by different environmental conditions and lactation stages. To investigate the combined effects of seasons and lactation stages on the mammary immunity, milk and blood samples were collected from three groups of high producing indigenous Sahiwal cows. Function and receptor expression of milk neutrophils together with cortisol and inflammatory interleukins concentration in blood were studied. The first group of cows started their lactation in winter and completed their lactation in hot-humid season; the second group started their lactation in hot-dry season and completed it in winter. The third group started their lactation in hot-humid and completed by the hot-dry season. Plasma cortisol levels were very high during early lactation in all seasons. An inverse relationship was observed between cortisol levels and glucocorticoid receptor. Elevated phagocytic activity and plasma interleukin-2 levels were seen in winter and during mid lactation of all seasons. A positive correlation was noticed between plasma IL-8, the percentage of milk neutrophils and expression of chemokine receptors (CXCR1 and CXCR2). The highest expression of toll-like receptors (TLR2 and TLR4) and chemokine receptors was in hot-humid season. Reduction in the phagocytic activity of neutrophils, pro-inflammatory cytokines and elevated levels of cortisol in cows which started their lactation and attained peak lactation during hot-humid season indicated more stress in them. Integrated influence of both seasons and lactation stages on the activity of milk neutrophils along with plasma interleukins and cortisol levels may be used to develop suitable managemental strategies to improve mammary health and increase milk production in indigenous dairy breeds experiencing harsh environmental conditions.
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http://dx.doi.org/10.1016/j.vetimm.2017.07.010DOI Listing
September 2017

Neutrophil gene dynamics and plasma cytokine levels in dairy cattle during peri-implantation period.

Vet Immunol Immunopathol 2016 May 1;173:44-9. Epub 2016 Apr 1.

Dairy Cattle Physiology Division, National Dairy Research Institute, Karnal, Haryana, India. Electronic address:

Neutrophils being the first line of cellular defense show significant difference in their expression in pregnant (P) and non-pregnant (NP) cows during the peri-implantation period. To study these changes, blood samples were collected from cows coming in heat and brought for artificial insemination (AI) on day 0 (day of AI), 4, 8, 12, 14, 16, 18, 21, 24, 30 and 40 day post-AI. Pregnancy was confirmed by observing non-return of heat, progesterone assay and ultrasonography. Cows were then categorized into pregnant (n=10) and non-pregnant (n=10) groups. Blood neutrophils were isolated and plasma samples collected from both P and NP cows during this period. RNA from neutrophils was isolated and studied for gene expression of adhesion molecules (CD62L, CD11b), chemokine (IL-8), and interferon stimulated genes (ISG15, OAS1, MX1, MX2, IFI16 and IFI44) using qRT-PCR. Adhesion molecules along with IL-8 showed a higher expression in NP cows. Expression of IFI16 was up-regulated as early as day 8, whereas, that of ISG15, OAS1, MX1 and MX2 were up-regulated on days 12-21 post-AI in P cows. Highest expression was shown by OAS1 on day 18 and by ISG15 and MX2 on day 21 post-AI in P cows. Pro-inflammatory cytokines (TNFα and IL-8) were higher, whereas, anti-inflammatory cytokine (IL-10) levels were lower in plasma samples isolated from NP cows. Our study indicates that blood neutrophils are sensitive to implantation signals received from the conceptus and may play an important role in implantation of the developing conceptus.
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http://dx.doi.org/10.1016/j.vetimm.2016.03.017DOI Listing
May 2016

Functional characterization of Mammary Gland Protein-40, a chitinase-like glycoprotein expressed during mammary gland apoptosis.

Apoptosis 2016 Feb;21(2):209-24

Animal Biotechnology Centre, National Dairy Research Institute (NDRI), Karnal, 132001, India.

MGP-40 is a chitinase-like protein which is over expressed during mammary gland involution. However, its physiological function in the mammary gland is poorly understood. In the present investigation, we have reported the functional significance of buffalo specific MGP-40 in the mammary gland by using an in vitro model of the buffalo mammary epithelial cell (BuMEC) line. MGP-40 was highly up regulated in BuMECs in serum starved condition as well as after treatment with prolactin suggesting its role in the stress response. Subsequently, to study the effect of MGP-40 on BuMECs, the cells were transfected with a mammalian expression construct of pCI neo harboring MGP-40 gene. It was observed that over expression of MGP-40 enhanced proliferation of BuMECs and protected the cells from apoptosis under serum free condition. In contrast, MGP-40 attenuated the mitogenic effect of insulin in BuMECs. Besides, over expression of the MGP-40 reduced dome formation, acinar polarization and casein synthesis in BuMECs in the presence of lactogenic hormones, it also induced Stat3 phosphorylation and epithelial to mesenchymal transition (EMT) -like features. Together, our data suggest that MGP-40 is involved in protection of BuMECs under stress conditions, inhibits cellular differentiation and induces EMT-like features. A schematic diagram depicting possible association of MGP-40 in various molecular pathways has been presented.
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http://dx.doi.org/10.1007/s10495-015-1196-zDOI Listing
February 2016

Effect of micronutrient supplementation on the immune function of crossbred dairy cows under semi-arid tropical environment.

Trop Anim Health Prod 2014 Jan 27;46(1):203-11. Epub 2013 Sep 27.

Physiology and Biochemistry Division, Central Sheep and Wool Research Institute, Avikanagar, India,

This study assessed the effect of micronutrient supplementation around peripartum period on immune function, reproductive performance, milk yield and milk quality of crossbred cows. Thirty pregnant crossbred cows in their late gestation were selected and randomly divided into five groups for study. Six cows in each group were supplemented with vitamin E (VE) (2000 IU/cow/day), vitamin A (VA) (100,000 IU/cow/day), copper (Cu) (20 ppm/cow/day), zinc (Zn) (80 ppm/cow/day) individually from 45 days pre-calving to 45 days post-calving and one group without any supplementation served as control. Immune function was studied by in vitro phagocytic activity (PA) of blood neutrophils, lymphocyte proliferation response (LPR) and plasma interleukin-8 (IL-8) concentration. Supplementation of VA significantly (P < 0.05) increased the in vitro PA of blood neutrophils and decreased milk somatic cell counts (SCC). Zn supplementation significantly (P < 0.05) increased the T lymphocyte proliferation response, whereas B lymphocyte LPR was significantly (P < 0.05) increased with both VA and Zn supplementation as compared to the control cows. Plasma IL-8 concentration was significantly (P < 0.05) higher in all supplemented cows. Supplementation of VE, VA and Zn significantly (P < 0.05) reduces days open, whereas VA significantly (P < 0.05) reduced the service per conception. In this study, it is concluded that VE, VA and Zn supplementation around peripartum period can boost the immunity and improve the reproductive performance of crossbred cows in a semi-arid tropical environment.
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http://dx.doi.org/10.1007/s11250-013-0477-1DOI Listing
January 2014

Effect of feeding inorganic chromium on growth performance, endocrine variables, and energy metabolites in winter-exposed buffalo calves (Bubalus bubalis).

Biol Trace Elem Res 2013 Dec 7;155(3):352-60. Epub 2013 Sep 7.

Animal Nutrition Department, College of Veterinary Science and Animal Husbandry, DUVASU, Mathura, 281001, India,

We investigated the effect of chromium (Cr) supplementation on the growth performance, energy metabolites, and hormonal variation in winter-exposed buffalo calves. Twenty-four female buffalo calves were randomly allotted to four dietary treatments (n = 6) for a period of 120 days. Feeding regimen was the same in all the groups, except the animals in the four respective groups were additionally supplemented with 0.0, 0.5, 1.0, and 1.5 mg of Cr/kg DM in the form of CrCl3.6H2O. Calves were monitored daily for physiological variables and dry matter intake (DMI). Blood samples were collected at fortnightly intervals from each buffalo calves to measure concentrations of hormones (insulin, cortisol, and growth hormone), energy metabolites (glucose and non-esterified fatty acids), and plasma mineral levels. After 120 days of feeding trial, buffalo calves fed with Cr had lower (P < 0.05) circulating plasma concentrations of glucose, insulin, and cortisol hormones, whereas plasma thyroid hormone and non-esterified fatty acids concentrations were found similar (P > 0.05) among all the treatments. The results suggested that dietary Cr supplementation influenced plasma Cr levels without affecting the plasma concentrations of other trace minerals. However, physiological variables, nutrient intake, and growth performance of buffalo calves did not differ among all treatments (P > 005). In summary, the current study showed that supplementation of Cr at the level of 1.0 and 1.5 mg of Cr/kg DMI was more effective in improving glucose utilization by increasing potency of insulin hormone and reducing concentration of cortisol hormone. Results also suggested that supplemental Cr also improves blood plasma Cr levels.
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http://dx.doi.org/10.1007/s12011-013-9808-3DOI Listing
December 2013

Effect of micronutrient supplementation around calving on the plasma cortisol levels of Murrah buffaloes and Sahiwal and Karan Fries cows.

Trop Anim Health Prod 2013 Apr 2;45(4):1047-50. Epub 2012 Nov 2.

National Dairy Research Institute, Karnal, 132 001, Haryana, India.

Micronutrients when fed around peripartum may reduce the stress induced by cortisol. With this objective, 24 Sahiwal (SW) and 24 Karan Fries (KF) cows and 24 Murrah (Mu) buffaloes were taken and divided into four groups of six each. Vitamin E (VE), zinc (Zn) and copper were supplemented from 30 days pre- to 30 days postcalving in groups 1, 2 and 3. Animals without supplementation served as control. Blood sampling was done on days 30, 15, 7 and 3 precalving; at calving; and on days 3, 7, 15 and 30 postcalving. Plasma cortisol levels were measured by ELISA. Supplementation of VE and Zn significantly (P<0.05) reduced plasma cortisol levels at calving and at 30 days postcalving. KF cows exhibited best results with VE, whereas SW cows and Mu buffaloes responded best to Zn. Copper increased the peripartum cortisol levels. Reduction in cortisol levels may help in keeping the animal healthy and stronger to fight immuno-suppression generally observed around the period of peripartum.
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http://dx.doi.org/10.1007/s11250-012-0302-2DOI Listing
April 2013
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