Varij Nayan

Heterologous Expression and Characterization of Indian Sahiwal Cattle (Bos indicus) Alpha Inhibin

Inhibin is a non-steroidal glycoprotein hormone of gonadal origin with major action as negative feedback control of the production of FSH by the anterior pituitary gland. The physiological role of inhibin has led to the development of inhibin immunogens for fertility enhancement in farm animals. It is envisaged that a reduction of endogenous inhibin secretion would increase FSH concentrations and thus offers a potential for increasing the number of ovulatory follicles in the ovary. The present work was carried out to produce recombinant bovine (Indian Sahiwal Cattle; Bos indicus) alpha inhibin (bINH-α) in E. coli by optimizing its expression and purification in biologically active form and to study its immunological characterization. A bacterial protein expression vector system based on the phage T5 promoter was used. The bINH-α encoding gene was successfully cloned and expressed in E. coli and the purified recombinant bINH-α was characterized. Recombinant bINH-α (25 µg mL−1) immunized guinea pigs had a significant increase in litter size compared to the control group. These results indicate a role for recombinant bINH-α as a fecundity vaccine to enhance the ovulation rate and litter size in animals.

Exploration of Luteinizing hormone in murrah buffalo (Bubalus bubalis) urine: Extended surge window opens door for estrus prediction

Estrus detection in buffaloes has been a major concern for decades, and lack of reliable methods affects their effective reproductive management. Luteinizing hormone (LH) detection in urine is in practice for several mammals for timed insemination, whereas very few reports are available on buffalo urinary LH. The focus of this study is to detect the presence of LH in buffalo urine, quantitate variation in urinary LH during different estrous cycle phases and examine the duration of mid-cycle LH window. Nearly hundred buffaloes were examined, longitudinal urine samples (n=42) were collected from seventeen animals and classified into respective phases based on several estrus detection parameters. The urinary LH was detected using bovine LH ELISA kit validated for serum/plasma/tissue homogenate. Detection of buffalo LH in the neat urine convincingly proved the competence of the bovine LH kit. Variation in the LH range was observed between different phases of estrous cycle and significant fold variation (P<0.05) was noticed during estrus phase (1.01±0.23) with average baseline value of 46.73±3.36mIU/mL. Interestingly, an extended window (A1-A3) of mid-cycle LH surge was observed due to its lingering excretion in urine. The results, altogether, revealed that LH can be detected in buffalo urine with noticeable fold variation during estrus phase and the extended LH window intensifies the chance of ovulation prediction for timed insemination.

Direct saliva transcript analysis as a novel non-invasive method for oestrus marker detection in buffaloes.

Abstract Salivary RNA-based biomarkers are not available for any physiological condition in farm animals. Hence, an objective of this study was to perform salivary transcript analysis in buffaloes. Saliva, after removal of the cells and particulate matter, was directly used for RT-PCR without RNA isolation. Direct saliva transcript analysis (DSTA) showed a suggestively significant higher expression of the Heat shock protein 70 (HSP70) and Toll-like receptor 4 (TLR4) at oestrus than the diestrous period in buffaloes by a non-parametric Mann-Whitney U test. Therefore, DSTA without RNA isolation is an easy method to identify salivary RNA markers for oestrus detection in buffaloes.

Molecular characterization, modeling, in silico analysis of equine pituitary gonadotropin alpha subunit and docking interaction studies with ganirelix

Equine pituitary gonadotropins (eLH, eFSH, eCG) are heterodimeric glycoprotein hormones with alpha (α) and beta (β) subunits. It is responsible for maintenance of pregnancy in mares during early gestation and fairly valuable for inducing superovulation in animals other than equines. The alpha subunit is common, while beta subunit is species-specific in all glycoprotein hormones. In the present investigation, molecular cloning and in silico characterization including homology modeling and molecular docking analysis of the equine chorionic gonadotropin (eCG) alpha subunit was carried out for gaining structural and functional insights into the eCG alpha subunit and its possible interaction with ganirelix, a gonadotropin-releasing hormone (GnRH) antagonist. The equine chorionic gonadotropin (eCG) alpha subunit expressed in pituitary gland was selected, amplified from total RNA, cloned and sequenced. The in silico analyses were made for homology modelling, structural details, epitope identification and chromosomal localization. Molecular docking studies of eCG alpha were undertaken with a drug ganirelix which is used to control ovulation and has antagonistic activity against GnRH. The protein sequence corresponding to selected open reading frame (ORF) was 99–100% similar with domesticated horse, Przewalski’s horse, and 92–93% with Burchell’s zebra and donkey. Molecular docking studies revealed the possible interaction of eCG alpha with ganirelix. The possible drug-macromolecule interactions were visualized between eCG alpha and ganirelix. The study will provide structural insight into unique sites and an alternate route of gonadotropin suppression applicable to assisted reproductive technologies.

Haematological Profiles of Lactating Nili-Ravi Buffaloes Under Heat Stress-Alleviated Conditions During Sub-tropical Summer Season

The experiment was conducted using forty two Nili-Ravi lactating buffaloes of Central Institute for Research on Buffaloes, Regional Station-Bir Dosanjh, Nabha, Patiala, Punjab to investigate the effect of heat stress-alleviated conditions on haematological profile in Nili-Ravi buffaloes during hot-dry (HD) and hot-humid (HH) season. All buffaloes were uniformly divided into two groups of twenty one in each group considering their lactation number, stage of lactation, body weight, dam’s milk yield and milk yield in current lactation. Out of which ten buffaloes from each group were selected for blood sampling considering those above factors. The control (T 0 ) group were kept in a separate shed without any extra nutrient supplementation and modification in microclimate and management. The treatment (T 1 ) group was supplemented with niacin@6 gm/buffalo/day, yeast@10 gm/buffalo/day and mustard oil @150gms/buffalo/day. They were provided curtains and mist fans in the shed. Feeding time, frequency and type of ration were also altered. During HD period, T 0 group exhibited higher (P<0.002, P<0.05) WBC (17.2 x 10 9 /L), and lymphocyte (12.52 x 10 9 /L) counts and lymphocyte per cent (69.46 %) than T 1 group (12.32 x 10 9 /L, 7.32 x 10 9 /L and 59.68%, respectively). Similar results were recorded during hot humid season in T 0 and T 1 group. The neutrophil per cent differed significantly between T 0 and T 1 group under HD (P<0.001) and HH (P<0.006) season. The study indicated that heat stress during sub-tropical summer months affected the haematology of lactating buffaloes and the stress could be reduced through the change (use of fans and curtains, nutritional supplementations, and feeding alterations together in the form of one package) of microclimate.