Manas Kumar Patra

The present study aimed to assess the presence of humanin in the sperm of crossbred cattle bulls using immunocytochemistry and to evaluate its potential as a semen quality marker. For this, a total of 49 ejaculates were collected from three Vrindavani crossbred bulls and were evaluated for volume, individual progressive motility (IPM), abnormality, and sperm concentration. The ejaculates were grouped into four categories, that is normozoospermia, oligozoospermia, asthenozoospermia and oligoasthenozoospermia, based on sperm concentration and IPM. Furthermore, normozoospermic ejaculates were cryopreserved and retrospectively subdivided into two groups (good‐freezable and poor‐freezable) based on post‐thaw motility (PTM). Immunofluorescence assays revealed humanin in the neck and upper midpiece regions of sperm. Humanin levels in seminal plasma were significantly higher ( p < 0.05) in normozoospermic individuals than in other groups. Humanin levels in seminal plasma were positively and significantly correlated with sperm concentration and individual progressive motility ( p < 0.05). Out of 28 normozoospermia ejaculates, 19 ejaculates (67.86%) were categorized as good freezable, and nine ejaculates (32.14%) were grouped as poor freezable. Humanin level in the seminal plasma of good freezable ejaculates was significantly ( p < 0.05) higher than that of poor freezable ejaculates. In conclusion, our research demonstrated, the presence of the humanin peptide in the sperm and seminal plasma of Vrindavani bulls. Additionally, the role of humanin as a potential semen‐quality marker in crossbred bull semen was established to identify high‐quality, freezeable ejaculates.

Sub‐estrus buffaloes do not exhibit estrus signs despite being cyclic contributing to extended service periods and inter‐calving intervals causing significant economic loss. The present study described the effect of synthetic prostaglandin (PGF_2α) on estrus behaviour, follicular and luteal morphometry, and serum estradiol (E₂) and progesterone (P₄) profile in sub‐estrus buffaloes during the non‐breeding season. The incidence of sub‐estrus was 38.4% during the non‐breeding season. The sub‐estrus buffaloes (n = 33) were divided into two groups, viz., Control (n = 16) and PGF_2α treatment (Inj. Cloprostenol 500 μg, i.m., n = 17). Estrus induction response was significantly greater in the treatment (100 vs. 18.75%, p < .001), and a relatively greater proportion of animals conceived in the treatment group (29.41 vs. 6.25%, p = .08). The time elapsed to induction of estrus and insemination following treatment was significantly lower in the treatment group than control. A significant increment in the follicle diameter (9.72 ± 0.45 vs. 13.00 ± 0.45 mm, P < .0001) and serum estradiol (E₂) concentration (66.01 ± 11.92 vs. 104.9 ± 13.21 pg/mL, p = .003) observed at the post‐treatment period in the PGF_2α treatment group. At the same time, CL diameter was reduced significantly at a higher regression rate in the PGF_2α treated buffaloes than those of control. Of the responded buffaloes, only 30% showed high‐intensity estrus attributed to the expulsion of cervico‐vaginal mucus (CVM), uterine tonicity, micturition, and mounting response by a teaser bull. From this study, it can be concluded that the administration of PGF_2α could induce estrus in the sub‐estrus buffaloes during the non‐breeding season. Behavioural changes, along with sonographic observation of POF, regressing CL, and serum E₂ and P₄ concentration would be useful to determine the right time of insemination in sub‐estrus buffaloes during non‐breeding season.

Kisspeptin (Kp), an upstream regulator of GnRH release, is essential for the development and function of reproductive axis. Previously, we demonstrated the localization of Kp and its receptor (Kiss1r) in the active follicle in the bubaline ovary. Present study aimed to determine the effect of Kp on granulosa cell (GCs) functions, especially oestradiol (E₂) and progesterone (P₄) production, and differential expression of genes regulating the proliferation, apoptosis and steroidogenesis in the buffalo. The ovaries with 6–10 mm size follicles obtained from the cyclic buffaloes after slaughtering were used for isolation of GCs for in vitro study. The primary GCs culture was treated with Kp (0, 10, 50 and 100 nM) and incubated for 48 h. Production of E₂ and P₄ was estimated in the culture supernatant by ELISA. The expression of gonadotropin receptors (FSHR and LHR), steroidogenic genes (STAR, 3β‐HSD, CYP19A1), proliferation marker (PCNA), apoptotic factors (CASP3 and BCL2) and Kp signalling molecule (extracellular signal‐regulated kinase 1/2, ERK1/2 and p‐ERK1/2) was studied in the GCs by qPCR. Significant E₂ production was found in the Kp 50 and 100 nM groups (p < .05), whereas P₄ production was reduced in Kp 100 nM group (p < .05). There was concomitant upregulation of FSHR, ERK1/2, STAR and CYP19A1 in the Kp 100 nM treated GCs. In addition, Kp at 100 nM stimulated the proliferation of GCs by upregulating the expression of BCL2 (5.0 fold) and PCNA (94.9 fold). Further, high immunoreactivity of p‐ERK1/2 was observed in the Kp‐treated GCs. It was concluded that Kp at 100 nM concentration stimulated E₂ production by upregulating the steroidogenic pathway through ERK1/2, STAR and CYP19A1 and modulating PCNA and BCL2 expressions in the GCs. Further experiments are warranted using Kp antagonist in different combinations to establish the signalling pathway in Kp‐mediated steroidogenesis in the GCs for developing strategies to control ovarian functions.

Sexed semen facilitates additional female calf production for the expansion of a herd at a faster rate and also curtails the surplus production of unwanted male calves. A study was conducted to evaluate the performance of sexed semen in indigenous Tharparkar cows based on 114 artificial inseminations (AI) performed at natural oestrus using two protocols i.e., single AI (n = 48) and double AI (n = 66). Overall, the first service conception rate (CR) was significantly higher in double (53.0%) than single (33.3%) AI protocol. The odds ratio of conception rate in double AI was 2.26 (?(2) = 4.4, df = 1, p = .04) with respect to single AI. The time that elapsed since the detection of oestrus to insemination was also analysed. In a single AI protocol, the CR was higher (p < .05) at 16 h (54.6%) than insemination at 8 h (27.0%) following the onset of oestrus. Yet, the CR using double AI protocol did not differ (p = .73) significantly when AIs were performed either at 8 h and 24 h (51.9%) or 16 h and 24 h (57.1%) post onset of oestrus. Besides, like the single AI protocol, the parity of the animals also influenced the CR, being higher in heifers (n = 22) than those of parous (n = 92) cows (72.73 vs. 40.43%, ?(2) = 7.48, df = 1, p = .006) in the present study. The odds ratio of conception in heifers was 3.93 with respect to parous cows. Overall, the birth of female calf was 91.7%. In conclusion, the present study indicates a future promise of the sexed semen for the production of more female offspring from Tharparkar cattle.

Pyometra is a potentially life‐threatening condition that affects intact female dogs in their middle to advance age. Timely diagnosis and appropriate treatment are critical for the survival of patients, especially when pyometra advances to sepsis. This study aimed to investigate the prognostic potential of certain haematology, serum biochemical and inflammatory biomarker, secretory leucocyte protease inhibitor (SLPI) for pyometra in bitches (n = 41). Blood samples were collected after clinical diagnosis of pyometra for haematology and serum biochemistry. Based on the prognosis following medical/surgical treatment, animals were retrospectively categorized into survivor (n = 29) and dead (n = 12). Endometrial tissue sections were obtained from the bitches undergoing ovariohysterectomy (n = 21). Serum concentration of SLPI was quantified using sandwich ELISA and its expression in the endometrium was investigated using RT‐qPCR. A marked increase in the total leucocyte count (TLC), neutrophils, blood urea nitrogen (BUN) and serum creatinine was observed in the female dogs that did not survive. Significant elevation in the serum SLPI concentration (3.49 ± 0.44 vs. 2.38 ± 0.13 ng/mL) was observed in the bitches those died after the treatment, in comparison to those survived (p < .01). Additionally, there was a notable upregulation of SLPI in the endometrium in the bitches those died due to pyometra. Based on the ROC analysis results, it was observed that a cut‐off concentration of 2.93 ng/mL for SLPI, 27.77 mg/dL for BUN and 16.3 × 10³/μL for TLC could effectively distinguish the prognosis of pyometra‐affected dogs. From this study, it can be concluded that upregulation of SLPI in the endometrium and its elevated concentration in peripheral circulation along with TLC and BUN concentration could serve as valuable indicators for predicting the prognosis of pyometra in bitches.

As onset of sepsis adversely affects the prognosis of canine pyometra, finding biomarkers that would distinguish sepsis status would be useful in the clinical management. Accordingly, we hypothesized that differential expression of endometrial transcripts and circulating concentration of certain inflammatory mediators would discriminate pyometra‐led sepsis (P‐sepsis+) from those of pyometra without sepsis (P‐sepsis−). Bitches with pyometra (n = 52) were classified into P‐sepsis+ (n = 28) and P‐sepsis− (n = 24) based on vital clinical score and total leukocyte count. A group of non‐pyometra bitches (n = 12) served as control. The relative fold changes in the transcripts of IL6, IL8, TNFα, IL10, PTGS2, mPGES1 and PGFS, SLPI, S100A8, S100A12 and eNOS were determined by quantitative polymerase chain reaction. Furthermore, the serum concentrations of IL6, IL8, IL10, SLPI and prostaglandin F_2α metabolite (PGFM) were assayed by ELISA. The relative fold changes in S100A12 and SLPI and mean concentrations of IL6 and SLPI were significantly (p < .05) higher in P‐sepsis+ than that of P‐sepsis− group. Receiver operating characteristic analysis revealed that serum IL6 had a diagnostic sensitivity of 78.6% and a positive likelihood ratio (LR+) of 2.09, at a cut‐off value of 15.7 pg/mL to diagnose P‐sepsis+ cases. Similarly, serum SLPI had a sensitivity of 84.6% and an LR+ of 2.23, at a cut‐off value of 2.0 pg/mL. It was concluded that SLPI and IL6 would serve as putative biomarkers for pyometra‐led sepsis in bitches. Monitoring SLPI and IL6 would be a useful adjunct to the established haemato‐biochemical parameters in customizing the treatment strategies and arriving at the decision for management of pyometra bitches with critical illness.