Rajib Deb

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.

Identification of SNP in HSP90AB1 and its Association with the Relative Thermotolerance and Milk Production Traits in Indian Dairy Cattle

Heat shock proteins (Hsp) play crucial role in cellular thermotolerance and heat stress response. In the present work, Allele specific PCR (AS-PCR) was standardized to detect the nucleotide polymorphism within the HSP90AB1 gene (SNP g.4338T>C) in Indian breeds of dairy cattle. The identified genotypes were associated with relative thermotolerance in terms of physiological parameters and milk production traits. The results of the experiments revealed that the genotype frequency of CC, CT, and TT for Sahiwal were 0.05, 0.78, and 0.17, respectively, and in Frieswal, the frequencies were 0.20, 0.70, and 0.10, respectively. The average rectal temperature (ART) and average respiration rates (ARR) were recorded during peak summer stress and heat tolerance coefficient (HTC) was calculated. The association studies indicated that TT genotypes had significantly (P < 0.01) higher HTC and lower ARR values than CT and CC in both the breeds. The TT genotype animals also had better production parameter in terms of total milk yield (TMY) (P < 0.01). These findings may partly suggest the role of HSP90AB1 polymorphisms in the regulation of heat stress response and consequent effect on production traits. Nevertheless, involvement of other regulatory mechanisms cannot be overruled.

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.

Application of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Cow Components Adulterated in Buffalo Milk/Meat

Loop-mediated isothermal amplification (LAMP) is a diagnostic method for amplification of DNA with rapid and minimal equipment requirement. In the present study, we applied the LAMP assay for rapid detection of cow components adulteration in buffalo milk/meat samples. The test can be completed within around 1xa0h 40xa0min starting from DNA extraction and can be performed in water bath without requirement of thermocycler. The cow DNA in buffalo samples were identified in the developed LAMP assay by either visualizing with SYBR Green I/HNB dyes or observing the typical ladder pattern on gel electrophoresis. The test can detect up to 5xa0% level of cow milk/meat mixed in buffalo counterparts. Due to the simplicity and specificity, the developed LAMP test can be easily adapted in any laboratory for rapid detection of cow species identification in livestock by products.

Understanding the mechanisms of ATPase beta family genes for cellular thermotolerance in crossbred bulls

Na+/K+-ATPase is an integral membrane protein composed of a large catalytic subunit (alpha), a smaller glycoprotein subunit (beta), and gamma subunit. The beta subunit is essential for ion recognition as well as maintenance of the membrane integrity. Present study was aimed to analyze the expression pattern of ATPase beta subunit genes (ATPase B1, ATPase B2, and ATPase B3) among the crossbred bulls under different ambient temperatures (20–44xa0°C). The present study was also aimed to look into the relationship of HSP70 with the ATPase beta family genes. Our results demonstrated that among beta family genes, transcript abundance of ATPase B1 and ATPase B2 is significantly (Pu2009<u20090.05) higher during the thermal stress. Pearson correlation coefficient analysis revealed that the expression of ATPase Β1, ATPase B2, and ATPase B3 is highly correlated (Pu2009<u20090.01) with HSP70, representing that the change in the expression pattern of these genes is positive and synergistic. These may provide a foundation for understanding the mechanisms of ATPase beta family genes for cellular thermotolerance in cattle.

Bovine Circadian Locomotor Output Cycles Kaput (CLOCK) and Clusterin (CLU) mRNA Quantitation in Ejaculated Crossbred Bull Spermatozoa

Mammalian circadian locomotor output cycles kaput (CLOCK) gene encodes a transcription factor that affects both the persistence and the period of circadian rhythms. Earlier reports suggested that CLOCK gene might be associated with male infertility in human. Present investigation, for the first time, reports that CLOCK gene expresses differentially between good and poor quality crossbred bull semen. The relative expression of CLOCK was significantly (pxa0<xa00.05) higher among good quality bull semen than motility-impaired ones. Clusterins (CLU) are series of genes associated with a variety of physiological activities including spermatogenesis, apoptosis and degenerative disease conditions. In the present context, we also investigated that the expression of CLU gene was significantly (pxa0<xa00.05) higher among motility-impaired crossbred bull semen compared to the good quality one.

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.