Arjava Sharma

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.

Genetic variants of beta-lactoglobulin gene and its association with milk composition traits in riverine buffalo.

A study was carried out to determine genetic variants of beta-lactoglobulin gene and to explore associations between these and milk composition traits in riverine buffalo. Single strand conformation polymorphism was employed to detect the genetic variants of the gene. Two fragments of this gene i.e. 119 bp of exon I and 400 bp spanning exon IV and intron IV were included in the study. For 119 bp fragment, three alleles namely, A, B and C were observed in all the buffalo breeds whereas four alleles (A, B, C and D) were detected for 400 bp fragment. The frequency distribution of alleles was different in different breeds of buffaloes for both the fragments. For exon I fragment, the milk composition traits such as total SNF, protein, solid, fat and whey protein yield were found to be significantly (P<0.05) associated with genotypes in Murrah and Bhadawari buffalo whereas in Mehsana breed genotypes were significantly (P<0.05) co-related with total SNF, solid and fat yield. Genotypes of 400 bp fragment, only total fat yield in Mehsana buffalo was found to be significantly (P<0.05) associated with genotypes.

A RAPID METHOD FOR THE FUNCTIONALISATION OF POLYMER SUPPORTS FOR SOLID PHASE OLIGONUCLEOTIDE SYNTHESIS

Abstract A rapid method is described for the covalent anchoring of appropriately protected 2′-deoxyribonucleoside-3′-O-succinates to LCAA-CPG, widely used support for solid phase oligonucleotide synthesis. The method involves the reaction of nucleoside-3′-O-succinates with aminoalkyl functions of the support in the presence of improved and commercially available condensing reagent, TBTU or TPP-DTNP to generate fully functionalised polymer supports with excellent nucleoside loadings.

Allelic diversity at MHC class II DQ loci in buffalo (Bubalus bubalis): evidence for duplication.

The genetic diversity of MHC class II DQ genes was investigated in riverine buffalo (Bubalus bubalis) by PCR-RFLP and sequencing. Highly variable regions (exons 2-3) of DQ genes were amplified from 152 buffaloes and genotyped by PCR-RFLP. Alleles identified by differential restriction patterns were sequenced for the characterization. PCR-RFLP was a rapid method to discriminate between DQA1 and duplicated DQA2 genes in buffalo, however, the method appeared to be inadequate for determining the more complicated DQB genotypes. A total of 7 and 10 alleles were identified for DQA and DQB loci, respectively. Nucleotide as well as amino acid variations among DQ alleles particularly at peptide binding regions were high. Such variations were as expected higher in DQB than DQA alleles. The phylogenetic analysis for both genes revealed the grouping of alleles into two major sub-groups with higher genetic divergence. High divergence among DQ allelic families and the isolation of two diverse DQA and DQB sequences from individual samples indicated duplication of DQ loci was similar in buffalo to other ruminants.

Isolation of two cDNAs encoding MHC-DQA1 and -DQA2 from the water buffalo, Bubalus bubalis.

In the present study, we explored structural and functional variations and possible duplication of the major histocompatibility complex (MHC)-DQA gene in water buffalo (Bubalus bubalis). Two cDNA sequences, amplified from one individual water buffalo, were designated as Bubu-DQA1 (DQA*0101) and -DQA2 (DQA*2001). The percentage of nucleotide and amino acid similarity between Bubu-DQA1 and -DQA2 revealed that these sequences display more similarity to alleles of respective DQA1 and DQA2 genes from other ruminant species than to each other. The phylogenetic analysis also revealed a considerably larger genetic distance between these two genes than between homologous genes from other species. The larger genetic distance between DQA*0101 and DQA*2001, and the presence of different bovine DQA putative locus specific amino acid motifs, suggests these sequences are non-allelic. This finding is consistent with DQA gene duplication in other ruminants.

Genetic Relationship Among Four Indian Breeds of Sheep Using RAPD-PCR

Abstract Kumar, K.G., Kumar, P., Bhattacharya, T.K., Bhushan, B., Patel, A.K., Choudhary, V. and Sharma, A. 2003. Genetic relationship among four Indian breeds of sheep using RAPD-PCR. J. Appl. Anim. Res., 24: 177–183. The study was carried out in four Indian sheep breeds namely, Marwari, Mandya, Madras Red and Muzaffarnagari maintained at different organised herds of the country. A total of six oligonucleotide primers were subjected to perform random amplified polymorphic DNA (RAPD) fingerprinting to estimate genetic relationship (relatedness) among different breeds of sheep. All the six primers showed polymorphism in producing bands. The average number of bands in different breeds ranged from 6 to 15 with size of 0.3 to 1.9 kb. Out of six primers, OPM-2 and ILO-17 produced comparatively large number of bands in agarose gel. The intrabreed relatedness in terms of band sharing ranged from 0.66 to 1.00, the highest being in Marwari, whereas, lowest in Muzaffarnagari breed. The interbreed relatedness in terms of mean average percentage difference (MAPD) was highest (9.44±1.66) between Marwari and Muzaffarnagari breed while lowest value (4.90±1.54) was found between Mandya and Mujaffarnagari. It may be concluded that the Mandya breed is closest to Marwari while Marwari breed is too distant from Muzaffamagari breed.

Association of BoLA DRB3 alleles with variability in immune response among the crossbred cattle vaccinated for foot-and-mouth disease (FMD).

Polymorphism of bovine leukocyte antigen (BoLA) DRB3 gene is being intensively investigated for potential association with economically important diseases of cattle. Accordingly, we investigated the association of DRB3 Exon 2 polymorphism as evidenced by the variation in the binding pockets with variability in immune response to inactivated trivalent (O, A and Asia1) foot and mouth disease virus (FMDV) vaccine in a closed population of crossbred cattle. Antibody titer of ≥ 1.8 was set as the cut off value to distinguish the protected (≥ 1.8) and unprotected (<1.8) animals. Eleven different alleles of over 3% frequency were detected in the population. We found that DRB3 alleles 0201, 0801 and 1501 always ranked high for protective immune response whereas alleles 0701, 1103 and 1101 consistently ranked low for unprotected immune response for all the three serotypes. Rank correlation of DRB3 alleles among the three serotypes was positive, high in magnitude and statistically significant (P<0.05). Logistic regression analysis revealed that odds of protection from the vaccine were highest for all the three serotypes if allele (∗)1501 was present and strengthened the results of allele ranking. Predicted amino acid substitution in the peptide binding pockets revealed that all the important sites had high Wu-Kabat index. Similarly, specific residues in pockets were crucial for immune response to FMD vaccine. There were specific substitutions in un-protected alleles such as absence of acidic amino acids substituted by basic amino acid at β71, presence of non-polar cysteine or basic histidine at β30 and presence of polar tyrosine at β37. From the observations, we hypothesize that the substitutions lead to unique conformational changes in the protein products of the studied alleles that would associate with the protective or unprotective antibody response to FMDV vaccine. The knowledge has potential implications in future selection programs if integrated with the complete BoLA haplotype details and production traits of the herd.

Effect of platelet activating factor on the motility and acrosome reaction of buffalo (Bubalus bubalis ) spermatozoa.

Platelet activating factor (PAF; 1-0-alkyl-2 acetyl-sn-glycerol-3 phosphocholine) has been shown to have a wide range of biological activities. In this study, PAF was used to induce acrosome reactions in fresh as well as frozen-thawed buffalo spermatozoa at different incubation periods and PAF levels. As the period of incubation increased, there was a gradual decrease in motility and increase in acrosome reaction in both fresh and frozen-thawed spermatozoa. With increasing PAF levels, the motility of fresh spermatozoa decreased and acrosome reaction increased whereas in frozen-thawed semen, motility remained almost constant, and the increase in acrosome reaction was not pronounced. Differences in motility and acrosome reaction among different bulls, types of semen, periods of incubation and PAF levels were significant (P < 0.01). A PAF level of 100 microM and an incubation period of 15 min were found to be optimum for inducing acrosome reaction in buffalo spermatozoa, since at this combination acrosome reaction increased significantly (P < 0.01) over that of the control without much loss of motility.

Polymorphism of ZuBeCa3 microsatellite and its association with mammary tumor in dogs

A study was conducted to explore the genetic polymorphism at ZuBeCa3 microsatellite and to estimate the association between microsatellite polymorphism and mammary cancer in dogs. Three genotypes namely AA, AB and BB were observed both in dogs affected by mammary cancer and in non‐affected dogs. In the affected group, the frequency of genotype and allele was 0.63 for AA, 0.25 for AB and 0.12 for BB genotype, and 0.76 for A and 0.24 for B allele. In the nonaffected group, the frequency of AA, AB and BB genotype was 0.39, 0.38 and 0.23 and the frequency of A and B allele was 0.58 and 0.42, respectively. Histopathological observation classified the cancer‐affected animals into three groups namely, malignant solid mammary carcinoma, malignant papillary adenocarcinoma and benign papillary adenoma in which the frequency of A allele was relatively more predominant in benign tumor group, which is more than 80%. Statistical tests showed significant differences (P < 0.01) of allelic distribution between tumor‐affected and non‐affected group, which reveals the effect of polymorphism on occurrences of mammary cancer in dogs. Besides, χ2 test also reflected the significant effect of genotypes on occurrences of three groups of mammary cancer in dogs.