Rani Alex

Differential effect of thermal stress on HSP70 expression, nitric oxide production and cell proliferation among native and crossbred dairy cattle.

In a tropical country like India, thermal stress is one of the major factors which significantly affects the productivity of dairy cattle. The present study was aimed to identify the effect of heat and cold stress on cell viability, mitogen stimulation indices, nitric oxide production and HSP70 expression in Sahiwal and Holstein crossbred (Frieswal) population in India. The results indicated that the Sahiwal breed can better withstand the effect of heat and cold stress significantly (P<0.05) when compared to the crossbred cattle due to the higher survivability of the Peripheral Blood Mononuclear Cells (PBMCs) and Phytohemagglutinin (PHA-P) mitogen based stimulation indices. The study also revealed the significant differences (P<0.05) in the level of nitric oxide (µM) production amongst the pre and post thermal stressed samples of Sahiwal and Frieswal crossbred samples. Further, the expression of HSP70 was significantly (P<0.05) higher in Sahiwal compared to Frieswal immediately after heat/cold shock to 6h of recovery as indirect ELISA analysis showed gradual rise in the Hsp70 protein concentration (ng/ml) immediately after heat and cold stress (0h) and reached the peak at 6h of recovery. Western blot and immune fluorescent assay results were also corroborated with the findings of indirect ELISA. In Sahiwal cattle the mRNA expression of HSP70 and its protein concentration were higher (P<0.05) during peak summer (44°C) and winter (10°C) as compared to Frieswal cattle. This investigation supports the earlier information on the higher adaptability of indigenous cattle breeds to hot and humid conditions compared to the crossbreds of temperate cattle breeds.

Development of a tetra-primer ARMS PCR-based assay for detection of a novel single-nucleotide polymorphism in the 5′ untranslated region of the bovine ITGB6 receptor gene associated with foot-and-mouth disease susceptibility in cattle

ITGB6 is known to be one of the major receptor components involved in host tropism of foot-and-mouth disease (FMD) virus in cattle. A competitive PCR technique called ARMS PCR was adapted to identify a single-nucleotide polymorphism (SNP), G29A, db SNP Id: rs109075046, in the 5′ untranslated region (5′UTR) of the bovine ITGB6 gene. Genotype profiling identified three kinds of genetic variation within the targeted SNP among Frieswal crossbred cattle. The occurrence of FMD in the three genotypes was further evaluated, revealing a clear role in the incidence of FMD in the studied population.

RT-LAMP assay: an alternative approach for profiling of bovine heat shock protein 70 gene in PBMC cultured model

The purpose of this study is to develop a novel Reverse Transcriptase Loop-mediated isothermal amplification (RT-LAMP) based assay for in vitro profiling of heat shock protein 70 (Hsp70) in bovine peripheral blood mononuclear cell (PBMC) culture model utilizing the absorbance level of magnesium pyrophosphate—a by-product of LAMP reaction. A set of bovine Hsp70 specific RT-LAMP primers were designed to detect the differential absorbance level of magnesium pyrophosphate by-product which signifies the degree of Hsp70 amplification from cDNA of thermally induced cultured cells at different recovery periods. The study revealed significant (P < 0.05) correlation between absorbance level and the fold change of Hsp70 transcripts at different kinetic intervals of heat stress recovery in bovine PBMC cell culture models. RT-LAMP based absorbance assay can be used as an indicator to measure the degree of bovine Hsp70 transcripts produced during thermal stress and can be used as an alternative to the traditional Real time PCR assay. Developed RT-LAMP assay can be used as a cost-effective method for profiling of bovine HSP70 gene.

Cytosine deletion at AP2-box region of HSP70 promoter and its influence on semen quality traits in crossbred bulls

Present investigation identified the effect of genetic polymorphism within the AP2 box region 12 located at bovine HSP70 promoter on certain semen quality traits among crossbred bulls revealed the fact that superior capability of wild type promoter genotype over the deletion mutation at HSP70 promoter may partly elucidate better semen quality traits for their contribution in selection of better quality bulls. Although artificial insemination (AI) is now extensively used in cattle breeding, there is about 40–50% loss in bull semen quality during the freezing–thawing process, including sperm motility, effective survival time and antioxidant enzymes activity (Hu et al. 2011). Recently, studies have been carried out in our laboratory to unveil the genetic basis of semen quality traits in cattle (Kumar et al. 2014, 2015a, b). It is well known fact that mammalian spermatogenetic cells are sensitive to heat stress (Yin et al. 1997). Studies have established that heat shock proteins (HSPs), particularly, the abundantly expressed 70 kDa HSP (HSP70), plays an important role in acquired thermotolerance (Nover 1991) and HSP70 has been suggested to function as an indicator of thermotolerance in cells (Leung et al. 1996). HSP70 is known to play an important role in sperm function after ejaculation and was identified in proteins extracted from sperm of bull (Kamaruddin et al. 1996) and boar (Huang et al. 1999). Huang et al. (1999) reported that both constitutive and inducible forms of HSP70 are present in ejaculated spermatozoa of boars and both the levels of HSP70 in spermatozoa and semen quality traits of boars significantly declined during the hot season.

Designing of an artificial neural network model to evaluate the association of three combined Y-specific microsatellite loci on the actual and predicted postthaw motility in crossbred bull semen

The freezing of bull semen significantly hamper the motility of sperm which reduces the conception rate in dairy cattle. The prediction of postthaw motility (PTM) before freezing will be useful to take the decision on discarding or freezing of the germplasm. The artificial neural network (ANN) methodology found to be useful in prediction and classification problems related to animal science, and hence, the present study was undertaken to compare the efficiency of ANN in prediction of PTM on the basis of the number of ejaculates, volume, and concentration of sperms. The combined effect of Y-specific microsatellite alleles on the actual and predicted PTM was also studied. The results revealed that the prediction accuracy of PTM based on the semen quality parameters was comparatively lower because of higher variability in the data set. The ANN gave better prediction accuracy (34.88%) than the multiple regression analysis models (32.04%). The root mean square error was lower for ANN (8.4353) than that in the multiple regression analysis (8.6168). The haplotype or combined effect of microsatellite alleles on actual and predicted PTM was found to be highly significant (P < 0.01). On the basis of results, it was concluded that the ANN methodology can be used for prediction of PTM in crossbred bulls.

Single tube tetraplex PCR based screening of a SNP at exon 14 region of bovine ITGB6 among different Zebu breeds

The present study was aimed to screen genetic variation within a SNP (rs136500299) located at exon 14 region of bovine ITGB6 gene among different Zebu cattle breeds. The genotyping method describe in the present study is a tetraplex ARMS PCR, which offers extremely fast, economical, and simple detection tool. The distribution of the ITGB6 genotypes among the different breeds studied suggested that the populations were under Hardy–Weinberg equilibrium. Our findings revealed that TT genotypes are widely distributed among different Zebu cattle breeds, which can be associated with the resistance to FMD virus, as the Bos indicus are more resistant to FMD virus in comparison to Bos taurus.

Identification of differentially expressed microRNAs in Sahiwal ( Bos indicus) breed of cattle during thermal stress

AbstractmicroRNAs (miRNAs) are a class of small non-coding RNAs that play key roles in post transcriptional gene regulation that influence various fundamental cellular processes, including the cellular responses during environmental stresses. However, perusal of literatures revealed few reports on the differential expression of miRNA during thermal stress in Indian native (Bos indicus) cattle breeds. The present investigation aimed to identify differentially expressed miRNAs during thermal stress in Sahiwal (Bos indicus) dairy cattle breed of India, adapted with tropical climate over a long period of time. Stress responses of the animals were characterized by determining various physiological as well as biochemical parameters and differential expression profile of major heat shock protein genes. Ion Torrent deep sequencing and CLC-genomic analysis identified a set of differentially expressed miRNAs during summer and winter seasons. Most of the identified differentially expressed miRNAs were found to target heat shock responsive genes especially members of heat shock protein (HSP) family. Real-time quantification-based analysis of selected miRNAs revealed that bta-mir-1248, bta-mir-2332, bta-mir-2478, and bta-mir-1839 were significantly (p < 0.01) over expressed while bta-mir-16a, bta-let-7b, bta-mir-142, and bta-mir-425 were significantly (p < 0.01) under expressed during summer in comparison to winter. The present study enlists differentially expressed miRNAs at different environmental temperatures in Sahiwal (Bos indicus) that may be importance for further understanding the role of miRNAs on thermo-regulatory mechanisms.

Comparative expression profile of NOD1/2 and certain acute inflammatory cytokines in thermal-stressed cell culture model of native and crossbred cattle

Thermotolerance depends mainly on the health and immune status of the animals. The variation in the immune status of the animals may alter the level of tolerance of animals exposed to heat or cold stress. The present study was conducted to investigate the expression profile of two important nucleotide binding and oligomerization domain receptors (NLRs) (NOD1 and NOD2) and their central signalling molecule RIP2 gene during in vitro thermal-stressed bovine peripheral blood mononuclear cells (PBMCs) of native (Sahiwal) and crossbred (Sahiwal X HF) cattle. We also examined the differential expression profile of certain acute inflammatory cytokines in in vitro thermal-stressed PBMC culture among native and its crossbred counterparts. Results revealed that the expression profile of NOD1/2 positively correlates with the thermal stress, signalling molecule and cytokines. Present findings also highlighted that the expression patterns during thermal stress were comparatively superior among indigenous compared to crossbred cattle which may add references regarding the better immune adaptability of Zebu cattle.

Molecular markers, BM1500 and UMN2008, are associated with post-thaw motility of bull sperm

Cryopreservation is one of the most important aspects of frozen semen technology and livestock breeding. Uses of candidate molecular markers in selection strategies for male fertility are well recognized. The present investigation targeted two microsatellite markers (BM1500 and UMN 2008) for association with semen quality variables and freezing capacity in Frieswal (HF×Sahiwal) crossbred bulls of Indian origin. Of the different alleles at the polymorphic locus BM1500, the 136bp allele was associated with greater (P<0.05) post-thaw motility percentage (PTM) while the 134 allele was associated with less (P<0.05) PTM. The 134/134 genotype at the polymorphic locus, UMN2008 was associated with greater (P<0.05) post-thaw motility while there was no allele effect on PTM. When combined genotypes UMN2008/BM1500 were analyzed, the 134/134-136/136genotype had the greatest (P<0.05) association with PTM. The present study is an initial report on the potential use of these markers as male reproductive biomarkers for improving semen freezing capacity in bulls.