Gyanendra Sengar

Effect of heat stress on the expression profile of Hsp90 among Sahiwal (Bos indicus) and Frieswal (Bos indicus × Bos taurus) breed of cattle: a comparative study.

We evaluated the effect of thermal challenge on the expression profile of heat shock protein 90 (Hsp90) among Sahiwal (Bos indicus) and Frieswal (Bos indicus × Bos taurus) breeds of cattle. The present investigation was focused on the comparative studies on Hsp90 expression among Frieswal and Sahiwal under in vitro and environmental heat stress. Measured immediately after the in vitro heat shock to the peripheral blood mononuclear cells (PBMCs), the relative expression of Hsp90 mRNA was significantly (P<0.05) higher in Sahiwal compared to those in Frieswal. In later intervals of time, the differences in the expression levels between the two breeds become negligible coming down towards the basal level. A similar pattern was observed in the protein concentration showing significantly (P<0.05) higher levels in Sahiwal compared to those in Frieswal. The second sets of experiments were undertaken during summer months (March to May) when temperature peaked from 37 to 45 °C. During these months, Frieswal cows consistently recorded higher rectal temperatures than the Sahiwal breed. Further during this peak summer stress, Sahiwal showed significantly higher levels of mRNA transcripts as well as protein concentration compared to the Frieswal breed. Our findings also interestingly showed that, the cell viability of PBMC are significantly higher among the Sahiwal than Frieswal. Taken together, the experiments of both induced in vitro and environmental stress conditions indicate that, Sahiwal may express higher levels of Hsp90 then Frieswal to regulate their body temperature and increase cell survivality under heat stressed conditions.

Promoter variants at AP2 box region of Hsp70.1 affect thermal stress response and milk production traits in Frieswal cross bred cattle

Heat shock proteins (Hsp) are known to play major role in protection of cells from thermal stress. Nucleotide polymorphisms within the promoter of Hsp affect degree of expression and inducibility of Hsp mRNA. The present study aimed to investigate the effect of polymorphism within promoter region on the cellular expression of Hsp70.1 mRNA and association of identified polymorphisms with the physiological parameters during summer stress and milk production traits in dairy cattle. Two hundred Frieswal cows were genotyped using double PCR-RFLP to identify deletion of cytosine within the Hsp70.1 promoter AP2 box at base position 895. Homozygous wild type genotypes (CC) were found in lower frequency (39.29, n=78) than heterozygous cytosine deletion mutant genotypes (C-) (60.71, n=122). In the observed physiological parameters (rectal temperature, respiration rate and heat tolerance coefficient), cows that were homozygous wild types had better significant (P<0.05) summer tolerance than the heterozygous deletion genotypes. Cytosine deletion mutation in the promoter region negatively affected (P<0.01) the expression of Hsp70.1 mRNA in peripheral bovine mononuclear cells (PBMC) subjected to in vitro heat stress. Further association of observed polymorphism with the milk production traits was significant as the heterozygous cytosine deletion cows had lower total milk yield, peak yield, yield at 300 days, protein% (P<0.01) and fat% (P<0.05) than the native wild type promoter cows. The results from the present study suggest that the promoter region of bovine hsp70.1 gene is polymorphic and may be useful in selection of dairy cows for relatively better thermotolerance and higher milk production.

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.

Genotypic to Expression Profiling of Bovine Calcium Channel, Voltage-Dependent, Alpha-2/Delta Subunit 1 Gene, and Their Association with Bovine Mastitis Among Frieswal (HFX Sahiwal) Crossbred Cattle of Indian Origin

Calcium channel, voltage-dependent, alpha-2/delta subunit 1 (CACNA2D1) gene is considered to be an important noncytokine candidate gene influencing mastitis. Scanty of reports are available until today regarding the role play of CACNA2D1 gene on the susceptibility of bovine mastitis. We interrogated the CACNA2D1 G519663A [A > G] SNP by PCR-RFLP among two hundreds Frieswal (HF X Sahiwal) crossbred cattle of Indian origin. Genotypic frequency of AA (51.5, n = 101) was comparatively higher than AG (35, n = 70) and GG (14.5, n = 29). Association of Somatic cell score (SCS) with genotypes revealed that, GG genotypes showing lesser count (less susceptible to mastitis) compare to AA and AG. Relative expression of CACNA2D1 transcript (in milk samples) was significantly higher among GG than AG and AA. Further we have also isolated blood sample from the all groups and PBMCs were cultured from each blood sample as per the standard protocol. They were treated with Calcium channel blocker and the expression level of the CACNA2D1 gene was evaluated by Real Time PCR. Results show that expression level decline in each genotypic group after treatment and expression level of GG are again significantly higher than AA and AG. Thus, it may be concluded that GG genotypic animals are favorable for selecting disease resistant breeds.

A SNP (g.358A > T) at intronic region of CD9 molecule of crossbred bulls may associate with spermatozoal motility

The surface expression of CD9 (cluster-of-differentiation antigen-9) in sperms of certain mammalian species has been attributed to its fusion with the egg and thereby dictating the fertility of species. In the present study, we investigated the association of CD9 with crossbred bull sperm quality and quantity trait was analyzed using a total of 96 Frieswal (HF × Sahiwal) crossbred. A single nucleotide polymorphism (g.358A > T) in intron 6 was significantly associated with sperm concentration (P < 0.05) and motility percentage (P < 0.01). mRNA was extracted from good (progressive motility > 50%) and motility impaired (progressive motility < 50%) bull semen. The mRNA expression and seminal plasma protein concentration of CD9 was significantly (P < 0.05) higher among good quality bull semen than motility impaired ones. Our results thus may indicate that, mutation in the intronic region may be responsible for the instability of RNA and the subsequent functional protein expression.

Evaluation of three bovine Y specific microsatellite loci as an alternative biomarkers for semen quality traits in crossbred bull.

Although some of the studies earlier reported that bovine semen parameters are associated with some candidate markers genes, but scanty of reports available regarding the effect of allelic variation in Y specific microsatellite markers on semen quality parameters in bulls. In the present study we have targeted three Y specific microsatellite markers (INRA126, INRA 189 and BM861) for their association ship analysis with some semen quality parameters among Frieswal (HF × Sahiwal) crossbred bulls of Indian origin. The polymorphic loci of INRA 126, bulls with 182 and 184 alleles had significantly (P<0.01) higher semen volume as compared to 186 allele, however, 186 allele showed significantly (P<0.01) higher concentration per ml of semen compared to 182 and 184. Interestingly our study also revealed that number of sperm/ejaculate is also significantly (P<0.05) higher in 184 allele compared to 182 and 186. Similarly, association analysis of INRA 189 major three alleles also revealed a significant difference in semen volume and concentration. Allele 89 and 96 having significantly (P<0.01) higher volume compared to 86, whereas allele 86 having significantly (P<0.01) higher concentration per volume of semen than 89 and 96. Again after association of two major alleles (160 and 164) of BM861 loci with semen parameters revealed no significant difference with any of the semen quality parameters chosen here. Therefore the present study may be for the first time revealed that the Y chromosomal microsatellite alleles are important male reproductive biomarkers for improving semen quality traits in bulls.

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.