Ashok Kumar Mohanty

Background and Aim: Postpartum infertility remains a major challenge in tropical dairy systems, particularly in Zebu crossbred cows that experience delayed ovarian cyclicity and increased metabolic and environmental stress. Improving early conception is essential to reducing calving intervals and enhancing productivity. This study evaluated the effectiveness of an Augmented-OvSynch protocol incorporating a post-insemination gonadotropin-releasing hormone (GnRH) injection in improving reproductive performance and assessing associated endocrine and oxidative stress dynamics in postpartum Zebu crossbred cows under field conditions. Materials and Methods: A total of 219 clinically healthy pluriparous Zebu crossbred cows (Holstein-Friesian × Sahiwal), 55–90 days postpartum, were randomly allocated to either a control group receiving the standard OvSynch protocol (n = 82) or a treatment group receiving the Augmented-OvSynch protocol (n = 137), which included an additional GnRH dose on day 5 post-artificial insemination (AI). Estrus response, follicular dynamics, pregnancy rate, and pregnancy loss were monitored through behavioral signs and ultrasonography. Blood samples collected on days 0, 5, 10, and 21 were analyzed for estrogen, progesterone, malondialdehyde (MDA), superoxide dismutase (SOD), and total antioxidant capacity (TAC). Statistical significance was set at p < 0.05. Results: The Augmented-OvSynch protocol significantly improved estrus expression (83.94%) compared with the control group (37.80%). Pregnancy rate per AI was markedly higher in treated cows (68.61%) than in controls (29.27%). Progesterone concentrations increased significantly from day 5 to day 10 in treated cows, indicating enhanced luteal support and probable accessory corpus luteum formation. Estrogen levels declined more sharply in pregnant cows. Oxidative stress markers were more strongly associated with pregnancy status than protocol type: pregnant cows consistently showed lower MDA and higher TAC levels, whereas SOD exhibited moderate variation without strong treatment effects. Conclusion: The Augmented-OvSynch protocol substantially improved reproductive outcomes in postpartum Zebu crossbred cows, primarily by enhancing luteal activity and improving synchronization responses. Although oxidative stress markers were influenced more by pregnancy outcome than treatment, their integration provided valuable insight into physiological determinants of fertility. Owing to its simplicity, low-cost, and field suitability, the protocol offers a practical and scalable reproductive management strategy for tropical smallholder dairy systems. Keywords: Augmented-OvSynch protocol, estrus synchronization, malondialdehyde, oxidative stress biomarkers, postpartum infertility, progesterone profile, reproductive performance, smallholder dairy systems, total antioxidant capacity, Zebu crossbred cows.

Milk fat globule EGF factor 8 (MFGE8) is a glycoprotein which plays a crucial role in mammary gland remodeling. Our group previously identified MFGE8 as a marker associated with high milk yielding cows. Here, we generated MFGE8 knock-out buffalo mammary epithelial cells (BuMEC) via CRISPR-cas9 technology to decipher its role in lactation. gRNA3 reduced MFGE8 expression with good efficiency which was confirmed at transcriptomic and proteomic level and the stable knock-out cells obtained were named mfge8-/-/gRNA3. The amplicon sequencing of the edited region using next generation sequencing (NGS) showed that 54% of total reads showed indels, 3–4 bp upstream to PAM site in 2nd exon. A total 4282 proteins were identified when proteome level changes were examined and 178 were found to be differentially expressed above and below a threshold of ≥ 1.5 and ≤ 0.6. Major DEPs were found to be associated with regulation of hydrolase activity, endopeptidase activity and cytoskeletal organization and some DEPs including FABP3, FABP4, FABP5, KNG1, MT2A, CD82, SLC7A1 and SERPINH1 belonged to genes associated with milk synthesis. To the best of our knowledge, this is the first study which provides a comprehensive proteome profile of MFGE8 knockout BuMEC and explores downstream effects of disruption of MFGE8 gene.

Background Semen sexing is a much sought-after technique in the livestock industry. Current research focuses on selecting DNA aptamers (Synthetic single-stranded DNA) against X-sorted sperm. Methods and results Four rounds of Cell-SELEX were performed, where the DNA aptamer library was incubated with X-sorted semen. Subsequently, affinity-depicting aptamers were sequenced. Fourteen out of thirty-four identified sequences were tested for specificity towards X-sorted sperm. The magnetic bead-based experiment revealed that the aptamers interacted preferentially with the acrosome region (sperm head) of sperm. We investigated the specificity of aptamers for X-spermatozoa by conducting real-time PCR assays targeting sex-specific genes. Aptamers X8, X19, X22 & X27 exhibited higher affinity for X-sorted sperm than Y in bovine semen. Western blot analysis showed an interaction between the aptamer and sperm protein at 35 kDa. The results underwent further analysis using in-gel mass spectrometry (MS) that predicted SPACA1 protein as the potential/putative aptamer binding protein on the sperm surface. Competitive western blot further confirmed aptamers’ differential specificity for X-sperm. Conclusions The current work helps in creating new knowledge for the separation of Y-chromosome-bearing sperm from XY sperm populations that have the potential to produce good-quality cattle (cows). The work also generates proof of concept (POC) of using aptamers, which is cost-effective and could be used in sorting sex-specific spermatozoa without causing any ill effect on sperm.

Proteomic analysis of sperm cells offers significant insights into proteins' structural, functional, and localization aspects within biological systems. Sahiwal, a native Indian cattle breed, is well known for its disease resistance, calving ease, and resilience to drought. This study addressed the gap in Sahiwal's comprehensive sperm proteome profiling data. The research involved the global in-silico quantitative high-resolution mass spectrometry-based protein profiling of Indian Zebu sperm, identifying 4651 sperm proteins. Beyond mere identification, the study characterized these proteins at a sub-organellar level to facilitate a better understanding of their functional attributes. Gene Ontology analysis of sperm proteins facilitated the segregation of proteins based on their function, localization, and mode of action. The study revealed that despite the limited number of organelles, sperm cells encapsulate a wide array of crucial proteins, compensating for the deficiency of organelles through the presence of multifunctional proteins. Most identified sperm proteins actively participate in spermatogenesis, motility, acrosome reaction, capacitation, and seminal plasma binding, directly or indirectly. Notably, the results not only present the highest number of identified bovine sperm proteins but also hold the potential to pave the way for empirical research on sperm functionality, egg-sperm interaction, sperm-sex sorting biomarkers, sperm quality, and bull fertility.

Tissue Inhibitor of Metalloproteinase‐2 (TIMP‐2) is part of the tissue inhibitors of the metalloproteinases (TIMPs) family. Its primary function is to regulate the activity of matrix metalloproteinases (MMPs) across various tissues, including those of the reproductive system. This study aimed to quantify the natural levels of TIMP‐2 in seminal plasma (SP) and sperm membrane (SM) of bulls, explore potential associations between TIMP‐2 levels and semen quality parameters, and examine the relationship between TIMP‐2 levels and sperm cryoresistance in bulls. Thirty semen samples from Frieswal breeding bulls were categorized into two groups based on their initial progressive motility (IPM): Good (IPM ≥ 70%; n = 21) and Poor (IPM ≤ 40%; n = 9). The samples were evaluated for their quality parameters at the fresh stage, and TIMP‐2 levels were measured in SP and SM using a bovine‐specific ELISA kit. Following cryopreservation of Good samples (n = 21), post‐thaw motility (PTM) was used to further classify samples into Freezeable (PTM ≥ 50%; n = 14) and Non‐Freezable (PTM < 50%; n = 7) groups. In frozen–thawed samples, sperm attributes, kinetics, and functional parameters were assessed, and the results were correlated with retrospective TIMP‐2 levels of SP/SM. Our study revealed that the quantified levels of TIMP‐2 ranged from 100.27 to 535.95 ng/L in SP and from 0 to 115.78 ng/10 million spermatozoa in SM. TIMP‐2 levels in both SP and SM were significantly higher in Good ejaculates compared to Poor ejaculates (p < 0.01). Furthermore, total TIMP‐2 levels in the SP/SM of semen samples from bulls showed a positive correlation with fresh semen attributes. However, SP/SM TIMP‐2 levels in the Freezeable group did not show any significant differences compared to the Non‐Freezable group in post‐thaw semen quality attributes, kinetic parameters, and functional tests, except for a significant positive correlation (r = 0.530, p < 0.05) between sperm DNA integrity and SP‐TIMP‐2 levels. In conclusion, the findings suggested that TIMP‐2 can be a positive regulator of semen quality at the neat stage. However, when it comes to the resilience of sperm to cryopreservation, the levels of TIMP‐2 do not seem to exert any significant influence in breeding bulls.

Background: The mammary gland is a crucial organ in dairy animals, responsible for milk production, regulated by intricate hormonal and cellular interactions. Traditional two-dimensional (2D) cell culture systems used in mammary gland research fail to replicate the complex in vivo environment, limiting the understanding of mammary gland biology. The study explores the advantages of three-dimensional (3D) culture systems for Buffalo Mammary Epithelial Cells (BuMECs), aiming to enhance morphological and functional differentiation compared to 2D cultures. Methods: BuMECs were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM/F12) supplemented with 10% FBS and specific growth factors. For 2D culture, BuMECs were grown on a plastic substratum. For 3D culture, cells were cultured on top of and embedded in growth factor-reduced (GFR) Matrigel. Morphological differentiation was observed under phase-contrast microscopy. Total RNA was extracted and cDNA was synthesized to measure β-casein gene expression using quantitative real-time PCR (qPCR). Protein expression was analyzed by Western blotting, targeting total casein. Result: BuMECs grown in 3D cultures exhibited enhanced morphological differentiation, forming alveolar and ductal structures, unlike the monolayer formation observed in 2D cultures. Rapid formation of duct-like structures was noted within 24 hours in 3D cultures. Functional differentiation was significantly improved, evidenced by a five-fold increase in β-casein mRNA expression and higher protein levels of α, β and κ-casein in 3D cultured cells compared to 2D cultures. Western blot analysis confirmed the elevated casein protein expression in 3D cultures.

Urinary proteins have been studied quite exhaustively in the past, however, the small sized peptides have remained neglected for a long time in dairy cattle. These peptides are the products of systemic protein turnover, which are excreted out of the body and hence can serve as an important biomarker for various pathophysiologies. These peptides in other species of bovine have been reported to possess several bioactive properties. To investigate the urinary peptides in buffalo and simultaneously their bioactivities, we generated a peptidome profile from the urine of Murrah Buffaloes (n = 10). Urine samples were processed using S. aureus, E. coli, and S. agalactiae. The urinary peptides observed in the study are the result of the activity of possibly 76 proteases. The GO of these proteases showed the significant enrichment of the antibacterial peptide production. The total urinary peptide showed antimicrobial activity against the aforementioned pathogenic bacterial strains with no significant inhibitory effects against a buffalo mammary epithelial cell line. Just like our previous study in cows, the present study suggests the prime role of the antimicrobial peptides in the maintenance of the sterility of the urinary tract in buffalo by virtue of their amino acid composition.

Infertility is a major problem in farm animals, which has a negative economic effect on farm industries. Infertility can be defined as the inability of animals to achieve a successful pregnancy. Early pregnancy is crucial to establish a successful pregnancy, and it is reported that 70–80% and 20–30% of total embryonic loss occur in cattle and pigs, respectively, during the first month of pregnancy. The advanced high-throughput proteomics techniques provide valuable tools for in-depth understanding of the implantation process in farm animals. In the present review, our goal was to compile, assess, and integrate the latest proteomic research on farm animals, specifically focused on female reproduction, which involves endometrial tissues, uterine fluids, oviductal fluids, and microRNAs. The series of studies has provided in-depth insights into the events of the implantation process by unfolding the molecular landscape of the uterine tract. The discussed data are related to pregnant vs. non-pregnant animals, pregnancy vs. oestrous cycle, different days of the early pregnancy phase, and animals with uterine infections affecting reproduction health. Some of the studies have utilized non-invasive methods and in vitro models to decipher the molecular events of embryo-maternal interaction. The proteomics data are valuable sources for discovering biomarkers for infertility in ruminants and new regulatory pathways governing embryo-uterine interaction, endometrium receptivity, and embryonic development. Here, we envisage that the identified protein signatures can serve as potential therapeutic targets and biomarkers to develop new therapeutics against pregnancy diseases.