Effect of prostaglandin treatment on the estrus behaviour, follicular and luteal morphometry and serum hormone profile in sub-estrus buffaloes during non-breeding season.

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 (PGF2α) 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 PGF2α 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 PGF2α treatment group. At the same time, CL diameter was reduced significantly at a higher regression rate in the PGF2α 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 PGF2α 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.

Milk exosomal microRNA profiling identified miR-375 and miR-199-5p for regulation of immune response during subclinical mastitis of crossbred cattle.

BACKGROUND: The dairy industry has experienced significant economic losses as a result of mastitis, an inflammatory disease of cows, including both subclinical and clinical cases. Milk exosome microRNAs have gained attention due to their stable and selective wrapping nature, offering potential for the prognosis and diagnosis of bovine mastitis, the most common pathological condition of the mammary gland. METHODS AND RESULTS:  In the present investigation, the microRNA profile of milk exosomes was explored using high-throughput small RNA sequencing data in sub-clinical mastitic and healthy crossbred Vrindavani cattle. In both groups, 349 microRNAs were identified, with 238 (68.19%) microRNAs co-expressed; however, 35 and 76 distinct microRNAs were found in subclinical mastitic and healthy cattle, respectively. Differential expression analysis revealed 11 microRNAs upregulated, and 18 microRNAs were downregulated in sub-clinical mastitic cattle. The functional annotation of the target genes of differentially expressed known and novel microRNAs including bta-miR-375, bta-miR-199-5p and bta-miR-12030 reveals their involvement in the regulation of immune response and inflammatory mechanisms and could be involved in development of mastitis. CONCLUSIONS: The analysis of milk exosomal miRNAs cargos hold great promise as an approach to study the underlying molecular mechanisms associated with mastitis in high milk producing dairy cattle. Concurrently, the significantly downregulated miR-375 may upregulate key target genes, including CTLA4, IHH, IRF1, and IL7R. These genes are negative regulators of immune response pathways, which could be associated with impaired inflammatory mechanisms in mammary cells. According to the findings, bta-miR-375 could be a promising biomarker for the development of mastitis in dairy cattle.

Genetic analysis of first lactation and lifetime performance traits in composite Vrindavani cattle: important considerations for higher milk production.

The present study was aimed to evaluate the influence of non-genetic factors on several first lactation and lifetime performance traits and elucidate their genetic parameters in an organized Vrindavani cattle population. Data on eight first-lactation and thirteen lifetime traits were collected on 2400 cows with pedigree records that were reared during 33-year period (1989-2021). The first-lactation traits included age at first calving (AFC), total milk yield (FTMY), standard milk yield (FSMY305), peak yield (FPY), lactation length (FLL), dry period (FDP), service period (FSP) and calving interval (FCI). Whereas, the lifetime traits mainly included total lifetime milk yield (TLMY), total standard milk yield (TSMY), number of lactations completed (NL), total lactation length (TLL), herd life (HL), productive life (PL), average milk yield per day of herd life (TLMY/HL), average milk yield per day of productive life (TLMY/PL), average milk yield per day of productive life (TLMY/TLL). Other lifetime production traits included average service period (ASP), average dry period (ADP), average calving interval (ACI) and unproductive days (UD). The heritability estimates of first-lactation traits ranged between 0.026 and 0.228 and were found to be low for AFC (0.180 ± 0.042), FCI (0.191 ± 0.125), FSMY305 (0.145 ± 0.061), FTMY (0.165 ± 0.048), FDP (0.052 ± 0.049) and FSP (0.026 ± 0.033); however, FLL (0.229 ± 0.044) and FPY (0.202 ± 0.046) showed moderate heritability. Positive phenotypic correlation (p < 0.001) was revealed among FTMY, TLMY, TLL, HL and PL. The AFC produced a significant effect (p < 0.05) on several traits i,e, TLL, TLMY/HL, FSMY305, FPY, TLMY, HL and TLMY/PL. Lower AFC was associated with higher TLMY, TLL and TLMY/HL; while FSMY305, FPY, HL and TLMY/PL were higher in heifers that calved late in their life. The results revealed that AFC may be optimized with first lactation and lifetime traits for this population.

The regulatory role of miR-20a in bovine cumulus cells and its contribution to oocyte maturation.

Dynamic changes in microRNAs in oocyte and cumulus cells before and after maturation may explain the spatiotemporal post-transcriptional gene regulation within bovine follicular cells during the oocyte maturation process. miR-20a has been previously shown to regulate proliferation and differentiation as well as progesterone levels in cultured bovine granulosa cells. In the present study, we aimed to demonstrate the function of miR-20a during the bovine oocyte maturation process. Maturation of cumulus-oocyte complexes (COCs) was performed at 39°C in an humidified atmosphere with 5% CO2 in air. The expression of miR-20a was investigated in the cumulus cells and oocytes at 22 h post culture. The functional role of miR-20a was examined by modulating the expression of miR-20a in COCs during in vitro maturation (IVM). We found that the miR-20a expression was increased in cumulus cells but decreased in oocytes after IVM. Overexpression of miR-20a increased the oocyte maturation rate. Even though not statistically significant, miR-20a overexpression during IVM increased progesterone levels in the spent medium. This was further supported by the expression of STAR and CYP11A1 genes in cumulus cells. The phenotypes observed due to overexpression of miR-20a were validated by BMP15 supplementation during IVM and subsequent transfection of BMP15-treated COCs using miR-20a mimic or BMPR2 siRNA. We found that miR-20a mimic or BMPR2 siRNA transfection rescued BMP15-reduced oocyte maturation and progesterone levels. We concluded that miR-20a regulates oocyte maturation by increasing cumulus cell progesterone synthesis by simultaneous suppression of BMPR2 expression.

Engineering interventions in enzyme production: Lab to industrial scale.

Commercial enzyme production has gained popularity due to its extensive applications in traditional and modern industrial sectors. Rigorous research activities are being conducted worldwide to make the enzyme production system more efficient, cost-effective and hence, sustainable. To overcome the lacunae in earlier enzyme production methods, new engineering interventions are being introduced to meet the growing demand for industrial enzymes. This review focuses initially on the current global scenario of the enzyme market followed by a discussion on different bioreactor design approaches. The use of novel membrane based, airlift and reciprocating plate bioreactors along with the emergence of micro-reactors have also been discussed. Further, the review covers different modelling and optimization strategies for the enzyme production process including advanced techniques like neural networks, adaptive neuro-fuzzy inference systems and genetic algorithms. Finally, the required thrust areas in the enzyme production sector have been highlighted with directions for future research.

Genome-wide DNA methylation patterns of bovine blastocysts derived from in vivo embryos subjected to in vitro culture before, during or after embryonic genome activation.

BACKGROUND: Aberrant DNA methylation patterns of genes required for development are common in in vitro produced embryos. In this regard, we previously identified altered DNA methylation patterns of in vivo developed blastocysts from embryos which spent different stages of development in vitro, indicating carryover effects of suboptimal culture conditions on epigenetic signatures of preimplantation embryos. However, epigenetic responses of in vivo originated embryos to suboptimal culture conditions are not fully understood. Therefore, here we investigated DNA methylation patterns of in vivo derived bovine embryos subjected to in vitro culture condition before, during or after major embryonic genome activation (EGA). For this, in vivo produced 2-, 8- and 16-cell stage embryos were cultured in vitro until the blastocyst stage and blastocysts were used for genome-wide DNA methylation analysis. RESULTS: The 2- and 8-cell flushed embryo groups showed lower blastocyst rates compared to the 16-cell flush group. This was further accompanied by increased numbers of differentially methylated genomic regions (DMRs) in blastocysts of the 2- and 8-cell flush groups compared to the complete in vivo control ones. Moreover, 1623 genomic loci including imprinted genes were hypermethylated in blastocyst of 2-, 8- and 16-cell flushed groups, indicating the presence of genomic regions which are sensitive to the in vitro culture at any stage of embryonic development. Furthermore, hypermethylated genomic loci outnumbered hypomethylated ones in blastocysts of 2- and 16-cell flushed embryo groups, but the opposite occurred in the 8-cell group. Moreover, DMRs which were unique to blastocysts of the 2-cell flushed group and inversely correlated with corresponding mRNA expression levels were involved in plasma membrane lactate transport, amino acid transport and phosphorus metabolic processes, whereas DMRs which were specific to the 8-cell group and inversely correlated with corresponding mRNA expression levels were involved in several biological processes including regulation of fatty acids and steroid biosynthesis processes. CONCLUSION: In vivo embryos subjected to in vitro culture before and during major embryonic genome activation (EGA) are prone to changes in DNA methylation marks and exposure of in vivo embryos to in vitro culture during the time of EGA increased hypomethylated genomic loci in blastocysts.

Oxidative and endoplasmic reticulum stress defense mechanisms of bovine granulosa cells exposed to heat stress.

In most mammalian species including cattle, heat stress has detrimental effects on ovarian function through disturbing estradiol production and viability of granulosa cells. However, effect of heat stress and underlying cellular defense mechanisms of bovine granulosa cells is not fully understood. Here, we aimed to investigate the effect of heat stress on granulosa cells function and the associated defense mechanism. For this an in vitro granulosa cell model was used to investigate the role of elevated temperature (41 °C) on granulosa cell functions at 24 h and 48 h exposure compared to the control cultured at 37 °C. The results showed that reactive oxygen species level was higher in cells under 41 °C at 24 h compared to control. In response to increased reactive oxygen species level, the expression of NRF2 and its antioxidant genes, CAT and PRDX1 were higher in bovine granulosa cells exposed to heat stress. Interestingly, heat stress markedly increased expression of endoplasmic reticulum stress marker genes; GRP78 and GRP94, in cultured bovine granulosa cells at 24 h, and higher protein accumulation of GRP78 accompanied by increased expression of apoptotic genes, BAX and CASPASE-3. Moreover, heat stress significantly decreased the bovine granulosa cells proliferation, which was supported by decreased in the expression of proliferation marker gene PCNA. All in all heat stress induce reactive oxygen species accumulation, apoptosis and reduced proliferation, which trigger the NRF2 mediated oxidative stress and endoplasmic reticulum stress response by bovine granulosa cells.

Potential role of microRNAs in mammalian female fertility.

Since the first evidence for the involvement of microRNAs (miRNAs) in various reproductive processes through conditional knockout of DICER, several studies have been conducted to investigate the expression pattern and role of miRNAs in ovarian follicular development, oocyte maturation, embryo development, embryo-maternal communication, pregnancy establishment and various reproductive diseases. Although advances in sequencing technology have fuelled miRNA studies in mammalian species, the presence of extracellular miRNAs in various biological fluids, including follicular fluid, blood plasma, urine and milk among others, has opened a new door in miRNA research for their use as diagnostic markers. This review presents data related to the identification and expression analysis of cellular miRNA in mammalian female fertility associated with ovarian folliculogenesis, oocyte maturation, preimplantation embryo development and embryo implantation. In addition, the relevance of miRNAs to female reproductive disorders, including polycystic ovary syndrome (PCOS), endometritis and abnormal pregnancies, is discussed for various mammalian species. Most importantly, the mechanism of release and the role of extracellular miRNAs in cell-cell communication and their potential role as non-invasive markers in female fertility are discussed in detail. Understanding this layer of regulation in female reproduction processes will pave the way to understanding the genetic regulation of female fertility in mammalian species.

The role of apoptosis in immunosuppression of dogs with demodicosis.

The aim of the present study was to evaluate the status of apoptosis in peripheral blood leukocytes of dogs with demodicosis. A total of 26 dogs suffering from demodicosis, and positive for Demodex canis mites by skin scraping, participated in the study, 13 with localized demodicosis (LD) and 13 with generalized demodicosis (GD). A further 13 clinically healthy dogs, all of whom were negative for mites upon skin scraping, were used as controls. The dogs with GD revealed significantly higher (P ≤ 0.0001) percentage of leukocytes with externalization of phosphatidylserine (PS) and depolarized mitochondrial membrane potentials (ΔΨm) as compared with the dogs with LD and healthy controls. These dogs also revealed significantly lower values (P ≤ 0.0001) of hematological parameters viz. hemoglobin, total erythrocytes count total leukocytes count, lymphocytes, monocytes and neutrophils. Significantly higher (P ≤ 0.0001) percentages of leukocytes with externalization of PS and depolarized ΔΨm were also found in dogs with LD as compared with the healthy controls. These dogs also revealed significantly lower values of Hb (P ≤ 0.0001), TEC (P=0.025), TLC (P ≤ 0.0001), lymphocytes (P=0.008), monocytes (P ≤ 0.0001) and neutrophils (P=0.03). It is concluded that premature apoptosis of PBL may be implicated in the immunosuppression of the dogs with demodicosis.