Genome analyses revealed genetic admixture and selection signatures in Bos indicus.

The genomic diversity and relationship among seven diverse cattle breeds viz. Sahiwal, Tharparkar, Gir, Vechur, Ongole, Kangayam and Hariana were investigated in 132 random samples based on high density SNP array comprising > 777 K SNPs. A total of 1993 SNPs (0.25% of the total) having greater power (FST ≥ 0.20) to differentiate these cattle populations were identified, and utilized to partition genome of each animal into a predefined number of clusters. The structure of these cattle indicated shared ancestry of dairy breeds viz. Gir, Tharparkar and Sahiwal. Most of the animals (> 76%) of different populations under study except Vechur clustered into their own group of animals called breed. Vechur population retained highest rate of admixture, consistent with its crossing with other breeds. Ongole, Kangayam and Hariana shared comparatively less of their genome (≤ 15%) with other breeds. The study indicated that all seven breeds evolved from their independent ancestry but there was intermixing of these breeds in the recent past. The selection signatures identified between draft (Kangayam) and dairy breeds included several genes like FAM19A2, RAB31P, BEST3, DGKA, AHCY, PIGU and PFKP which are involved in immune response, metabolic pathway, transportation of glucose and sugars, signaling pathways, cellular processes, cell division and glycolysis regulation, respectively. Moreover, these genomic regions also harbour QTLs affecting milk performance traits. The signatures were also identified even between the dairy breeds. In comparison to large-sized cattle, there were significant differences in the number of QTLs affecting production (body weight, growth rate etc.) and morphological traits (height) in short-statured Vechur breed. The presence of HMGA2 gene in the selection signature on chromosome 5 may explain the variations in stature between these cattle.

Implantation associated divergence in neutrophil glucocorticoid and cysteine-X-cysteine receptor genes in cattle.

The differential roles of Interferon-tau (IFNT) and Progesterone (P4) in triggering pro- and anti-inflammatory responses during peri-implantation period were assessed in cows. An ex vivo study was done by isolating neutrophils from the animals post artificial insemination (AI) at defined intervals and analyzing the expression changes of Glucocorticoid receptor-α (GRα) and Interleukin-8 receptor (CXCR1) genes. An in vitro study was also done isolating neutrophils from prepubertal heifers subjecting them to IFNT and Progesterone P4 supplementation separately. We observed that from day 14 post Artificial Insemination, there is a significant up-regulation of neutrophil GRα and CXCR1 in pregnant and non-pregnant cows respectively. The in vitro study showed that IFNT significantly up-regulates GRα whereas P4 significantly up-regulates CXCR1. The total leukocyte count and neutrophil count were also significantly higher in non-pregnant cows. We conclude that IFNT efficiently mediates neutrophil immunosuppression during peri-implantation period in cows by interacting with Interleukin 8 receptor and can also exert ligand independent actions on GRα.

Exploring the concentration-dependent actions of interferon-τ on bovine neutrophils to understand the process of implantation.

Interferon-τ (IFNT) is a major cytokine produced by the ruminant trophoectoderm during the peri-implantation period exerting immunomodulatory actions on various cells including neutrophils. The current in vitro study was undertaken to analyze the concentration-dependent effects of IFNT on neutrophil gene dynamics to understand its possible role in implantation process. The neutrophils were isolated from the blood of heifers and were cultured subjecting them to different IFNT concentrations (1, 5, or 10 ng/mL). The gene-expression patterns of different interferon-stimulated genes, l-selectin, CD31, CD11b, and progesterone-induced blocking factor (PIBF) were analyzed by quantitative real-time PCR. It was observed that at lower concentrations of IFNT, the IFI16, l-selectin, ISG15, and PIBF were upregulated, whereas at higher concentrations the same were down regulated. At all the experimental concentrations, IFI44, OAS1, MX genes were significantly upregulated and CD31, CD11b were significantly downregulated. At lower concentrations of IFNT, the neutrophil activity with respect to chemoattraction is stimulated, whereas at higher concentrations the same is reduced. Hence, it can be concluded that IFNT exerts concentration-dependent actions on neutrophil gene-expression dynamics indicating a fine modulation of its activity depending upon the temporal variation in its destined functions ultimately leading to successful implantation.

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 feed supplement and additives on stress mitigation in Karan Fries heifers.

AIM: The objective of this study was to evaluate the effects of protected fat plus yeast, niacin, zinc, and chromium dietary supplementation on the reduction of heat stress in Karan Fries (KF) heifers during hot humid months. MATERIALS AND METHODS: The basal ration for both the control and treatment groups was the same, containing maize as green fodder and concentrate mixture. However, the treatment group was supplemented with protected fat (2.5% of dry matter intake [DMI]), yeast (10 g/animal/day), niacin (6 g/animal/day), zinc (40 mg/kg DMI), and chromium (1.5 mg/kg DMI). RESULT: The overall mean value of afternoon rectal temperature for control and treatment group was 103.17±0.09 and 102.72±0.10°F, respectively, and was significantly (p<0.01) lower in the treatment group. The overall mean value of afternoon respiration rate for control and treatment group was 76.35±0.56 and 73.13±0.58 breaths/min, respectively, and was also significantly (p<0.01) lower in the treatment group. The overall mean value of afternoon pulse rate for control and treatment group was 97.09±0.63 and 94.67±0.67 beats/minute, respectively, and was also significantly (p<0.01) lower in the treatment group. Finally, the mean cortisol concentration for control and treatment group was 3.94±0.05 ng/ml and 3.70±0.06 ng/ml, respectively, and was significantly (p<0.01) lower in the treatment group. CONCLUSION: The present study shows that supplementation with the above feed additives could serve as a heat stress abatement strategy in growing KF heifers during extreme conditions in summer months.

Alterations in cortisol concentrations and expression of certain genes associated with neutrophil functions in cows developing retention of fetal membranes.

Elevated cortisol concentrations have been reported to impair the functions and alter the life span of neutrophils in cows. The present study assessed the cortisol concentrations and expression of few genes related to longevity (Fas, Caspase 3, Bcl2) and margination (CD 62L, CD 18/11b) of neutrophils in relation to retention of fetal membranes (RFM) in dairy cows. Cortisol concentrations were significantly higher on the day of calving and day 2 postpartum in RFM cows than normal cows. Expression of CD 62L was significantly lower on the day of calving and day 2 postpartum in RFM cows than normal cows. While expression of Fas and GR was significantly lower on the day of calving, expression of Bcl2 was lower on day 7±2 pre-partum in RFM cows compared to normal cows. No significant difference was observed in the expression of CD 18/11b and Caspase 3 between RFM and normal cows. Cortisol concentration was negatively correlated with expression of GR, Fas, CD 62L, CD18/CD11b and Caspase 3, while positively correlated with immature neutrophil percentage and expression of Bcl2. It may be inferred that cortisol concentrations and expression of certain genes associated with lifespan and margination of neutrophils were altered in cows developing RFM compared to those expelled the fetal membranes normally.

Modulation of post-partum reproductive performance in dairy cows through supplementation of long- or short-chain fatty acids during transition period.

Thirty-six cross-bred cows were used to study the effect of long-chain (flaxseed) or short-chain (butyric acid) fatty acid supplementation on metabolic status, ovarian function and reproduction performance during transition period. Control cows received a routine feed of transition diet, while the cows in two treatment groups were supplemented with either 750-g crushed flaxseed or 250 g butyric acid per cow per day. Ovarian activity was monitored by transrectal ultrasonography on 10th, 20th and 30th days post-partum. Blood samples were collected by jugular venipuncture into heparinized polystyrene tubes; plasma was prepared and stored under -20 °C until analysis. Results indicated that cows in flaxseed group were in positive energy balance as indicated by lower NEFA and Beta hydroxy Butyrate and higher glucose concentrations. Uterine involution was completed well within 30 days post-partum in all the cows in flaxseed fed group compared to 76.9% in butyric acid supplemented and 61.5% in control groups. The size of dominant follicle and corpus luteum was significantly higher (p < 0.05) for flaxseed group compared to control group, which in turn resulted in higher concentrations of plasma progesterone. Cows fed on diets supplemented with flaxseed exhibited post-partum heat earlier and bred sooner (p < 0.05) than control cows. It has been noticed that supplementation of flaxseed and butyric acid enhanced involution of uterus, early resumption of cyclicity and thereby early breeding. However, in view of the encouraging results obtained for flaxseed supplemented group, its organic nature and easier availability at farmer's gate, we concluded that flaxseed can be safely included in transition diet to modulate reproductive performance of dairy cattle.

Comparative expression profiling of insulin-like growth factor binding protein-5 in milk of Bos indicus and Bubalus bubalis during lactation.

Insulin-like growth factor binding protein-5 (IGFBP-5) is a key molecule in mammary gland development, which facilitates the removal of mammary epithelial cells (MECs) by apoptosis that takes place during remodeling of the mammary gland during involution. IGFBP-5 binds with IGFs for their bioavailability. IGFBP-5 has been reported to perform pleiotropic roles such as cellular apoptosis, proliferation and differentiation. To understand the role of IGFBP-5 during lactation and clinical mastitis, expression profiling of IGFBP-5 at the protein level was performed in both indigenous cows (Bos indicus) and buffaloes (Bubalus bubalis) belonging to two different breeds - Sahiwal cows and Murrah buffaloes. Reverse-transcriptase PCR (RT-PCR) of IGFBP-5 mRNA confirmed its expression in milk somatic cells and MECs of Sahiwal cows. ELISA was performed for quantitative measurement of IGFBP-5 concentrations in milk during different days (0, 50, 100, 150, 200, 250 and 300) of lactation, during the involution period and in animals exhibiting short lactation and clinical mastitis. The highest concentration of IGFBP-5 in milk was observed during the involution period followed by colostrum, late and early lactation, respectively, in both cattle and buffaloes. No significant difference in the concentration of IGFBP-5 was observed during the first 150 days of lactation between cows and buffaloes. However, higher concentration of IGFBP-5 was observed in cows during late lactation (200 to 300 days) in comparison with buffaloes. To validate the ELISA data, quantitative real-time PCR was performed in MECs of Sahiwal cows. The relative mRNA abundance of IGFBP-5 was found to be significantly (P<0.05) higher on day 15 than between 50 and 150 days of lactation in case of Sahiwal cows. Highest mRNA expression of IGFBP-5 was observed around 300 days of lactation followed by 200 and 250 days (P<0.05), respectively. Murrah buffaloes showed low levels of IGFBP-5 protein in milk as compared with Sahiwal cows during lactation in ELISA. Animals having history of short lactation length (short lactating animals) showed higher levels of IGFBP-5 expression (at protein level) in comparison with normal lactating animals. We propose that higher level IGFBP-5 expression may have functional significance in lactation persistency. As a pro-apoptotic molecule, higher expression of IGFBP-5 was observed to be inversely related to lactation length and milk production.

Effect of supplementation of vitamin E, copper and zinc on the in vitro phagocytic activity and lymphocyte proliferation index of peripartum Sahiwal (Bos indicus) cows.

To study the effect of vitamin E (VE), copper (Cu) and zinc (Zn) supplementation on the in vitro phagocytic activity (PA) and lymphocyte proliferation response (LPR) of blood neutrophils and lymphocytes, thirty Sahiwal pregnant cows (six in each group) in their late gestation at 30 days before the expected date of calving were selected from the NDRI experimental herd and supplemented with various micronutrients from 30 days before calving to 45 days after calving. Cows were supplemented individually with VE (1000 IU/cow/day), Cu (20 ppm/cow/day) and Zn (80 ppm/cow/day) and also with a combination of VE, Cu and Zn to study cumulative effect of all micronutrients. One group without any supplementation acted as a control. Blood neutrophils and lymphocytes were isolated and studied for their PA and LPR. Supplementation of micronutrients like VE, Cu, Zn and a combination of all these nutrients significantly (p < 0.01) increased the PA of experimental cows as compared to control (unsupplemented) cows during the pre-partum period. During post-partum, all the micronutrients (VE, Cu, Zn and their combination) showed a significant (p < 0.01) increase in the PA of experimental cows as compared to control cows. Of all the groups, significant (p < 0.01) and maximum PA was observed in the combination group followed by Zn-supplemented group during both the pre- and post-partum period. A significant (p < 0.01) increase in LPR of B lymphocytes was observed in combination-supplemented group during the pre-partum period and during both the pre- and post-partum period in the Cu-supplemented group.

Selection of suitable reference genes for quantitative gene expression studies in milk somatic cells of lactating cows (Bos indicus).

We assessed the suitability of 9 internal control genes (ICG) in milk somatic cells of lactating cows to find suitable reference genes for use in quantitative PCR (qPCR). Eighteen multiparous lactating Sahiwal cows were used, 6 in each of 3 lactation stages: early (25 ± 5 d in milk), mid (160 ± 15 d in milk), and late (275 ± 25 d in milk) lactation. Nine candidate reference genes [glyceraldehyde 3-phosphate dehydrogenase (GAPDH), protein phosphatase 1 regulatory subunit 11 (PPP1R11), ß-actin (ACTB), ß-2 microglobulin (B2M), 40S ribosomal protein S15a (RPS15A), ubiquitously expressed transcript (UXT), mitochondrial GTPase 1 (MTG1), 18S rRNA (RN18S1), and ubiquitin (UBC)] were evaluated. Three genes, ß-casein (CSN2), lactoferrin (LTF), and cathelicidin (CAMP) were chosen as target genes. Very high amplification was observed in 7 ICG and very low level amplification was observed in 2 ICG (UXT and MTG1). Thus, UXT and MTG1 were excluded from further analysis. The qPCR data were analyzed by 2 software packages, geNorm and NormFinder, to determine suitable reference genes, based on their stability and expression. Overall, PPP1R11, ACTB, UBC, and GAPDH were stably expressed among all candidate reference genes. Therefore, these genes could be used as ICG for normalization of qPCR data in milk somatic cells through lactation.

Purification, sequence characterization and effect of goat oviduct-specific glycoprotein on in vitro embryo development.

Oviduct-specific glycoprotein (oviductin) plays an important role during fertilization and early embryonic development. The oviductin cDNA was successfully cloned and sequenced in goat, which possessed an open reading frame of 1620 nucleotides representing 539 amino acids. Predicted amino acid sequence showed very high identity with sheep (97%) followed by cow (94%), porcine (77%), hamster (69%), human (66%), rabbit (65%), mouse (64%) and baboon (62%). The bioinformatics analysis of the sequences revealed the presence of a signal sequence of 21 amino acids, one potential N-linked glycosylation site at position 402, 21 potential O-linked glycosylation sites and 36 potential phosphorylation sites. The native oviductin was purified from the oviductal tissue, which showed three distinct bands on SDS-PAGE and western blot (MW ~60-95 kDa). The predicted molecular weight of goat oviductin was 57.5 kDa, calculated from the amino acid sequences. The observed higher molecular weight has been attributed to the presence of large number of potential O-linked glycosylation sites. The lower concentration (10 µg/mL) of oviductin increased the cleavage rate, morula and blastocyst yield significantly (P < 0.05) as compared to higher concentration (100 µg/mL). Goat oviductin retarded the activity of pronase (0.1%) on zona solubility of oocytes significantly (P < 0.01).

In vitro phagocytic activity of milk neutrophils during lactation cycle in Murrah buffaloes of different parity.

Milk samples were collected from 34 lactating Murrah buffaloes on days 0, 5, 15, 30, 45, 60, 90, 120, 150, 180, 210, 240, 270 and day 300 after calving. Milk somatic cell counts (SCC) were highest in multiparous buffaloes. Milk SCC were significantly lower in buffaloes of third and fourth parity during early lactation and than increased significantly (p < 0.01) by the end of lactation. Milk neutrophils were significantly lower in all the buffaloes during early lactation, but increased significantly (p < 0.01) afterwards. Milk lymphocytes were significantly higher during early lactation, but decreased significantly (p < 0.01) by the end of lactation. Phagocytic activity (PA) was highest in day 1 colostrum and then decreased significantly (p < 0.01) by the fourth milking in buffaloes of second, third and fourth parity. Phagocytic index (PI) was also highest in colostrums of primiparous buffaloes. Irrespective of parity, maximum PA and PI was observed during mid lactation. In terms of in vitro phagocytic activity, early lactation is the most critical period followed by late and mid lactation.

Changes in colostrum of Murrah buffaloes after calving.

Colostrum samples were collected from 8 Murrah buffaloes on days 1, 2, 3, 4 and 5 after calving. Levels of IgG averaged 54.0 mg/ml at calving, then decreased significantly (P < 0.01). IgA and IgM on day 1 were 3.22 mg/ml and 5.22 mg/ml, respectively; both decreased during the first five days after calving. Values of IgA and IgM were higher than those reported in cows. SCC values, which were high at calving (500,000 per ml), reduced significantly (P < 0.01) on day 2, then decreased slightly until day 5 (180,000 per ml). At calving, macrophages were the most prominent cells in buffalo colostrum, followed by lymphocytes and neutrophils. Phagocytic activity was 23% at calving and reduced significantly (P < 0.01) to 14% on day 5. Phagocytic index was highest in the first colostrum, and then decreased non-significantly.

Sperm motion characteristics of Garole rams raised for a prolonged period in a semi-arid tropical environment.

The Garole is a prolific but less well known and rare breed of small sheep found in the hot and humid Sunderban region of West Bengal. An ability to breed throughout the year and to graze in knee-deep water, resistance to foot rot and a strong mothering instinct are some of the special features of this breed. Garole rams could provide germplasm to incorporate prolificacy traits by artificial insemination of the nonprolific sheep breeds found in abundance in the semi-arid and arid tropical climates of India. The aim of the present study was to evaluate the semen production by Garole rams maintained in a semi-arid climate for three years and to objectively assess their semen quality by a computer-assisted sperm analysis technique. The donor rams were randomly selected each year from the original flock procured from their natural habitat or from the offspring born at the Institute farm. Semen was collected weekly for three weeks each autumn for three consecutive years from 8 rams each year. The overall means (SD) of the traits that did not differ significantly with age or year were volume, concentration, curvilinear velocity, average path velocity, amplitude of lateral head displacement, beat frequency, motility and the percentages of rapid motile sperms and of slow motile sperms. The age of the rams had a significant effect (p<0.05) on the straight-line velocity but this was not significantly affected by the length of exposure to the semi-arid climate. However, the age and year had significant effects (p<0.05) on linearity, straightness and the percentage of medium motile sperms. It was concluded that Garole rams are capable of producing good-quality semen even after a prolonged period of exposure to a semi-arid tropical climate.