Association of genomic prediction for twin pregnancies with the incidence of double ovulation in primiparous dairy cows.

Twin pregnancies compromise the health and well-being of dairy cattle. A recent genomic prediction model for twin pregnancies has been developed based on twin calving or abortion. However, the incidence of double ovulation is significantly higher than that of twin births. This study aimed to evaluate whether genomic prediction values for twin pregnancies are associated with the incidence of double ovulation in primiparous dairy cows. Factors influencing the double ovulation rate were analysed using binary logistic regression on 676 cows: 475 (70.3%) inseminated at spontaneous estrus and 201 following one of two different estrus synchronization protocols for fixed-time artificial insemination (FTAI). The odds ratio for double ovulations was 0.92 (p = .002) per unit increase in prediction value and 2 (p = .01) for cows subjected to an FTAI protocol. Our findings suggest that genomic prediction values for twin pregnancies can effectively identify the risk of double ovulation at the herd level.

Development of B cell epitopes-based enzyme linked immune sorbent assay for detection of bovine anti-Mullerian hormone.

The present study aimed to generate antibodies against predicted B cell epitopic peptides encoding bAMH for developing different ELISA models. Sandwich ELISA was determined to be an excellent technique for assessing bAMH in bovine plasma based on sensitivity tests. The assay's specificity, sensitivity, inter- and intra-assay CV, recovery %, Lower limit of quantification (LLOQ), and Upper limit of quantification (ULOQ) were determined. The test was selective since it did not bind to AMH-related growth and differentiation factors (LH and FSH) or non-related components (BSA, progesterone). The intra-assay CV was 5.67%, 3.12%, 4.94%, 3.61% and 4.27% for 72.44, 183.11, 368.24, 522.24 and 732.25 pg/ml AMH levels, respectively. At the same time, the inter-assay CV was 8.77%, 7.87%, 4.53%, 5.76% and 6.70% for 79.30, 161.27, 356.30, 569.33 and 798.19 pg/ml AMH levels, respectively. The average (Mean ± SEM) recovery percentages were 88-100%. LLOQ was 5 pg/ml and ULOQ at 50 µg/ml (CV < 20%). In conclusion, we developed a new highly sensitive ELISA against bAMH using epitope specific antibodies.

Pre-treatment but not co-treatment with vitexin alleviates hyperthermia induced oxidative stress and inflammation in buffalo mammary epithelial cells.

This study investigated if in vitro supplementation of vitexin could mitigate the adverse effects of hyperthermia on buffalo mammary epithelial cells (BuMECs). Immortalized BuMECs were divided into seven groups (n = 3): (1) a negative control group at 37 °C; (2) BuMECs exposed to heat stress as a positive control at 42 °C for 1 h; (3-7) heat stressed BuMECs pre-treated or co-treated with different concentrations of vitexin (5 µM, 10 µM, 20 µM, 50 µM, and 100 µM), respectively. Hyperthermia was induced by exposing the cells to 42 ºC for 1 h. For the pre-treatment experiment, BuMECs were treated with vitexin for 2 h before hyperthermia exposure. For co-treatment, vitexin was added simultaneously with hyperthermia for 1 h. Subsequently, the cells were allowed to recover for 12 h at 37 °C. Results showed that pre-treatment with vitexin was more effective than co-treatment in protecting BuMECs from hyperthermia in a dose-dependent manner, with higher concentrations (50 µM and 100 µM) being the most effective. Pre-treatment with vitexin maintained cellular viability and prevented inflammation by inducing the expression of the anti-apoptotic gene (BCL-2) and reducing the expression of the pro-apoptotic gene (Bax) and pro-inflammatory mediators (IL-1ß, IL-6) in heat-stressed BuMECs. Pre-treatment with vitexin reduced oxidative stress and induced thermotolerance by increasing the expression of antioxidants mediators such as SOD, GPx and CAT at mRNA and protein levels, and modulating the expression of heat shock proteins. The findings suggest that vitexin has the potential as a therapeutic agent to protect the mammary gland from the negative impact of hyperthermia in dairy cows.

Differentially expressed microRNAs in biochemically characterized FrieswalTM crossbred bull semen.

In addition to the transmission of paternal genome, spermatozoa also carry coding as well as noncoding microRNAs (miRNAs) into the female oocyte during the process of biological fertilization. Based on RNA deep sequencing, a total 28 number of differentially expressed miRNAs were cataloged in categorized FrieswalTM crossbred (Holstein Friesian X Sahiwal) bull semen on the basis of conception rate (CR) in field progeny testing program. Validation of selected miRNAs viz. bta-mir-182, bta-let-7b, bta-mir-34c and bta-mir-20a revealed that, superior bull semen having comparatively (p < .05) lower level of all the miRNAs in contrast to inferior bull semen. Additionally, it was illustrated that, bta-mir-20a and bta-mir-34c miRNAs are negatively (p < .01) correlated with seminal plasma catalase (CAT) activity and glutathione peroxidase (GPx) level. Interactome studies identified that bta-mir-140, bta-mir-342, bta-mir-1306 and bta-mir-217 can target few of the important solute carrier (SLC) proteins viz. SLC30A3, SLC39A9, SLC31A1 and SLC38A2, respectively. Interestingly, it was noticed that all the SLCs were significantly (p < .05) expressed at higher level in superior quality bull semen and they are negatively correlated (p < .01) with their corresponding miRNAs as mentioned. This study may reflect the role of miRNAs in regulating few of the candidate genes and thus may influence the bull semen quality traits.

Development of CD163 receptor-based enzyme-linked immunosorbent assay for diagnosis of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome (PRRS) is an important economical disease in the global swine industry. The accurate detection of the PRRS virus (PRRSV) antigen is essential for the disease control and prevention programme. In this study, an indirect enzyme-linked immunosorbent test (PRRSVCD163-iELISA) was developed for the detection of the PRRSV antigen in samples of post-mortem swine tissue using the recombinant pig CD163 receptor protein as the capture ligand. The test was found to be specific for PRRSV, with no cross-reactions with other prevalent pig viral pathogens. The assay was validated by testing 217 post-mortem porcine tissue samples and the results were found to be satisfactory with a relative accuracy of 88.88%. Our assay is also quite precise, with intra- and inter-assay CVs of 6% and 10%, respectively. These findings imply that the PRRSVCD163-iELISA developed is capable of detecting the PRRSV antigen in swine post-mortem tissue samples. This research showed that porcine CD163, the PRRSV cellular receptor, can be exploited to build a diagnostic technique for the detection of PRRSV antigen. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03376-z.

Curcumin induces thermotolerance by reducing oxidative stress, apoptosis, and inflammation in buffalo mammary epithelial cells under heat shock conditions.

The epithelial cell is the main basic unit of the udder in which milk synthesis takes place. Curcumin is well known for its antioxidant, anti-apoptotic, and anti- inflammatory properties. The present study was performed to test whether in vitro curcumin supplementation can alleviate the unfavorable impact of hyperthermia on buffalo mammary epithelial cells (BuMECs). The spontaneously immortalized BuMECs were divided into 7 groups (n = 9); 1) unstressed BuMECs (negative control, 37 °C); 2) BuMECs exposed to hyperthermia without curcumin treatment (positive control); 3-7) BuMECs cultured with different concentrations of curcumin (5, 10, 20, 40 and 60 µM), respectively, followed by hyperthermic exposure (42ºC) for 1 h and then returned to 37ºC. Changes in viability (MTT assay), proliferation (BrdU colorimetric immunoassay) and concentrations of antioxidant enzymes, CAT, and SOD (ELISA) of BuMECs were recorded. The gene expression study was performed using qRT-PCR. Lower concentrations of curcumin (5, 10 µM) maintained viability, enhanced proliferation, and content of antioxidant enzymes of heat stressed BuMECs. Curcumin induced thermotolerance and antioxidant status by upregulating the expression of antioxidants genes, anti-apoptotic genes and heat shock proteins in heat stressed BuMECs compared to the positive control group. Besides, curcumin reduced apoptosis and inflammation in BuMECs exposed to hyperthermia by downregulating the expression of genes and transcriptional factors associated with apoptosis and inflammatory immune response. The results reveal the potential roles of curcumin in eliminating the negative impact of hyperthermia on BuMECs by regulating the pathways of apoptosis, inflammation, and oxidative stress.

Characterization of Anti-Müllerian Hormone (AMH) Gene in Buffaloes and Goats.

The Anti-Müllerian Hormone (AMH) is a member of the transforming growth factor beta (TGF-ß) superfamily, playing a significant role in cell proliferation, differentiation and apoptosis. In females, AMH is secreted throughout their reproductive life span from ovaries, whereas in males it is secreted by gonadal cells at a very early stage of testicular development. AMH is a promising marker of ovarian reserve in women and can be used to measure the female reproductive lifespan. In the present study, we cloned and sequenced the GC rich AMH gene from Indian riverine buffalo (Bubalus bubalis) and goat (Capra hircus). Obtained sequences were compared to the AMH sequences of other mammals, and corresponding amino acid sequences revealed that the caprine and bovine AMH sequences are more closely related to each other than to those of other mammals. Furthermore, we analyzed the chromosomal localization of AMH genes in mammalian species to understand potential syntenic relationship. The AMH gene is localized between the sequences for the SF3A and JSRP1 genes and maintains this precise location in relation to other nearby genes. The dN/dS ratio of AMH gene did not indicate any pressure for either positive or negative selection; thus, the physiological function of the AMH gene in the reproduction of these two ruminant species remains very vital. Similar to other mammals, the AMH gene may be an important indicator for regulating female reproductive biology function in bovine, cetacean, caprine, and camelidae.