Expression Analysis of Genes Associated with Prolificacy in FecB Carrier and Noncarrier Indian Sheep.

The effect of FecB mutation on the gene expression in FecB carrier and noncarrier estrous synchronized ewes, has been analyzed. For this study the whole ovarian tissues and Graafian follicles were collected from estrus synchronized FecB carrier Garole, and non-carrier Deccani Indian sheep, showing remarkable differences in the numbers of preovulatory follicles among two groups. Eleven potential candidate genes (BMP15, GDF9, BMP4, BMP7, BMPR1B, BMPR1A, SMAD9, LHCGR, FSHR, IGF1R, and STAT5) were selected for their expression analysis by SybrGreen based real-time PCR, across ovaries and Graafian follicles of different fecundity groups, for having better insights into the effect of FecB genotypes on follicular development. Variable expression was observed for almost all the genes included in the present study among high and low fecundity groups that was most significant for the BMP7, BMP4, LHCGR, and FSHR transcripts in the ovarian follicles of high and low fecundity ewes, indicating their importance in governing the fecundity in FecB carrier, Indian Garole sheep. BMP4 expression among the genes studied was significantly higher in FecB carrier Garole sheep. This study confirms the changes in mRNA expression of the genes implicated in follicular development in FecB carrier and noncarrier Indian sheep breeds.

Genetic analysis of river, swamp and hybrid buffaloes of north-east India throw new light on phylogeography of water buffalo (Bubalus bubalis).

This study analysed buffaloes from north-east India and compared their nuclear and mitochondrial DNA variations with buffaloes of mainland India, China, Mediterranean and South-East Asia. Microsatellite genotypes of 338 buffaloes including 210 from six north-east Indian buffalo populations and three mainland Indian breeds were analysed to evaluate their genetic structure and evolutionary relationships. Phylogenetic analysis and multidimensional scaling plot of pairwise FST revealed the clustering of all swamp-type buffaloes of north-east India with Lower Assamese (significantly hybrid type) buffaloes in one plane and all the mainland river buffaloes in another plane while the upper Assamese buffaloes being distinct from both these clusters. Analysis of mtDNA D-loop region of 530-bp length was performed on 345 sequences belonging to 23 buffalo populations from various geographical regions to establish the phylogeography of Indian water buffalo. The swamp buffaloes of north-east India clustered with both the lineages of Chinese swamp buffalo. Multidimensional scaling display of pairwise FST derived from mitochondrial DNA data showed clustering of upper Assamese, Chilika and Mediterranean buffaloes distinctly from all the other Indian buffalo populations. Median-joining network analysis further confirmed the distinctness and ancestral nature of these buffaloes. The study revealed north-east region of India forming part of the wider hybrid zone of water buffalo that may probably extend from north-east India to South-East Asia.

MARKER ASSISTED EVALUATION OF MORPHOLOGICAL AND GENETIC ATTRIBUTES OF SUB-POPULATIONS OF NILI-RAVI BUFFALO: A VULNERABLE DAIRY TYPE RIVERINE BREED OF INDIA.

In the present study, we report the distribution of true to type and atypical Nili-Ravi buffalo, a vulnerable dairy type riverine breed of North India and its underlying genetic structure. Out of total investigated buffaloes 73.5% had bilateral wall eyes while 5.4% had unilateral wall eyes and 21.1% had no wall eyes. 41.15% of Nili-Ravi buffaloes maintained in the breeding farm were having typical true to the type characteristics (both eyes walled, white markings in forehead, muzzle/chin, all the four legs and tail) while only 28.5% of Nili-Ravi buffaloes were true to the type under field conditions. Genotypic data were generated in four groups of Nili-Ravi buffalo (FMTNR--Typical Nili-Ravi from farm; FMANR--Atypical Nili-Ravi from farm; FDTNR--Typical Nili-Ravi from field; FDANR--Atypical Nili-Ravi from field) at 16 microsatellite loci. Comparative genetic analysis of various groups of Nili-Ravi buffaloes with Murrah revealed significant between group differences with an estimated global F(ST) of 0.063. Pair-wise F(ST) values ranged from 0.003 (between FDTNR and FDANR) to 0.112 (between FMTNR and FDTNR). Phylogenetic analysis and multi-dimensional scaling revealed clustering of FDTNR and FDANR together while FMTNR and FMANR clustered separately with Murrah in between farm and field Nili-Ravi buffaloes. Based on the results, the paper also proposes three pronged strategy for conservation and sustainable genetic improvement of Nili-Ravi buffalo in India.

Characterization of hsp70 gene promoter for cis- acting elements in Indian zebu cattle of Hariana breed.

The promoter region of hsp70 gene was characterized for cis-acting elements in zebu cattle of Hariana breed. The basal regulatory domain of CAAT box identified as CAAT/enhancer binding protein (C/EBP) and CAAT binding transcription factor (CTF) binding sites, as well as GC box identified as sp1 binding site, were localized in at least two regions in the hsp70 gene promoter. A highly conserved heat shock element was found between position -108 to -95, which exactly matched at all eight positions with the consensus sequence. These cis-acting elements were found to be conserved between Holstein-Friesian and studied zebu breed.

Genetic polymorphisms in the bovine toll-like receptor 4 (TLR4) and monocyte chemo attractant protein-1(CCL2) genes: SNPs distribution analysis in Bos indicus Sahiwal cattle breed.

Toll-like receptor 4 gene (TLR4) that recognizes the Gram negative bacterial ligand LPS was sequenced in the Bos indicus Sahiwal cattle breed. Ninety four single nucleotide polymorphisms (SNPs) were detected within 10.8 kb gene region. Seventeen of the SNPs were in the coding regions and the one at position 9589(A > G) in exon3 resulted in an amino acid change from Valine to Isoleucine. These SNPs led to generation of 27 TLR4 gene haplotypes. All the Sahiwal animals studied presently showed the occurrence of the genotype CC at gene position 9662, which codes for the amino acid threonine at position 674 of the TLR4 protein, and which had been reported to be associated with lower somatic cell score and, therefore, a lower susceptibility to mastitis, in Taurus cattle. This nucleotide configuration of the Toll-like receptor 4 gene of the Bos indicus Sahiwal cattle breed could possibly indicate toward a lower susceptibility to mastitis in the Sahiwal animals. Monocyte chemo-attractant protein-1 (CCL2) gene encoding for small inducible cytokine A2 that belongs to the CC chemokine family was also sequence characterized in these Sahiwal animals. The CCL2 gene was observed to have 12 polymorphic sites in 3.3 kb region of which one SNP at position 2500 (A > G) in exon 3 resulted in amino acid change from Valine to Isoleucine at position 46 of the mature CCL2 peptide. Seventeen haplotypes of the CCL2 gene were predicted corresponding to 12 genotypes detected.

Population structure in Indian sheep ascertained using microsatellite information.

This study attempts to provide a comprehensive insight into the prevailing genetic status of Indian sheep breeds using microsatellite markers. Seventeen Indian sheep breeds from 3 agroecological zones were analysed using a panel of 25 microsatellite markers. All of the sheep breeds investigated were genetically diverse, as evident from the high allele (>6) and gene (>0.6) diversity values. The gene diversity values for all breeds ranged from 0.621 to 0.780. The within-population heterozygote deficit (F(IS)) varied from -0.098 to 0.234, reflecting significant levels for 12 of the 17 breeds investigated. The average genetic differentiation between all breeds (F(ST)) was 11.1%, revealing moderate discrimination between the indigenous sheep breeds. The genetic distance and principal component analysis revealed a separation of sheep breeds based on geographical propinquity. The Bayesian clustering approach suggested poor breed differentiation in the north-western arid and semi-arid region when compared to the breeds from the eastern and southern peninsular regions. The observed results mirror the divergent management strategies in the different agroecological regions, lack of specific selection policies, and intermixing of breeds in close proximity. Immediate steps to curb the intermixing and erosion of breed purity for some of these breeds need to be implemented, for example, by introducing measures like making proven rams available and ensuring their frequent exchange between flocks. The data generated here provides valuable information about the genetic structure of the 17 Indian sheep breeds and this can be used for designating priorities for their conservation.

Analysis of genetic variation at the prolactin-RsaI (PRL-RsaI) locus in Indian native cattle breeds (Bos indicus).

This study assessed the distribution pattern of allelic variants at the prolactin-RsaI locus in 23 Indian native cattle breeds (Bos indicus). PCR-RFLP genotyping of a 156 bp fragment of prolactin (PRL) in exon 3 revealed the predominance of the heterozygous AB genotype (mean frequency 0.58) irrespective of utility type (dairy, dual, draft), geographic region (northern, central, southern), and coat color (red, gray) of the breeds analyzed. The overall frequencies of homozygous AA (0.22) and BB (0.20) genotypes were in a similar range. The PRL (A) and PRL (B) alleles exhibited similar gene frequencies (means 0.52 and 0.48, respectively). The existing profile of the PRL-RsaI gene locus in a large set of Indian native cattle breeds was different from that of Bos taurus and cattle breeds of other countries, where either the BB genotype and PRL (B) allele or the AA genotype and PRL (A) allele have been reported to be more prevalent.

Sequence characterization of S100A8 gene reveals structural differences of protein and transcriptional factor binding sites in water buffalo and yak.

The present study was undertaken to characterize the structure of S100A8 gene and its promoter in water buffalo and yak. Sequence data of 2.067 kb, 2.071 kb, and 2.052 kb with respect to complete S100A8 gene including 5' flanking region was generated in river buffalo, swamp buffalo, and yak, respectively. BLAST analysis of coding DNA sequences (CDS) of S100A8 gene revealed 95% homology of buffalo sequence with cattle, 85% with pig and horse, 83% with dog, 72-73% with murines, and around 79% with primates and humans. Phylogenetic analysis of predicted CDS revealed distinct clustering of murines, primates, and domestic animals with bovines and bubalines forming a subcluster among farm animals. In silico translation of predicted CDS revealed a sequence of 89 amino acids with 7 amino acid changes between cattle and buffalo and 2 changes between cattle and yak. The search for Pfam family revealed the N-terminal calcium binding domain and the noncanonical EF hand domain in the carboxy terminus, with more variations being observed in the N-terminal domain among different species. Two amino acid changes observed in carboxy terminal EF hand domain resulted in altered secondary structure of yak S100A8 protein. Analysis of S100A8 gene promoter revealed 14 putative motifs for transcriptional factor binding sites. Two putative motifs viz. C/EBP and v-Myb were found to be absent in swamp buffalo as compared to river buffalo and cattle. Differences in the structure of S100A8 protein and the transcriptional factor binding sites identified in the present study need to be analyzed further for their functional significance in yak and swamp buffalo respectively.

Genetic evaluation of Ethiopian Boran cattle and their crosses with Holstein Friesian for growth performance in central Ethiopia.

Breed additive and non-additive effects, and heritabilities of birth weight (BWT), weaning weight (WWT), 6 months weight (SMWT), yearling weight (YWT), eighteen months weight (EWT), 2 years weight (TWT) and average daily weight gain from birth to 6 months (ADG1) and from 6 months to 2 years (ADG2) were estimated in Ethiopian Boran (B) cattle and their crosses with Holstein Friesian (F) in central Ethiopia. The data analysed were spread over 15 years. Ethiopian Boran were consistently lighter (p < 0.01) than the B-F crosses at all ages. Ethiopian Boran also gained lower weight than all the crosses. At birth, 50% F crosses were significantly (p < 0.01) lighter than all the other crosses. However, the differences in SMWT, YWT, EWT, TWT, ADG1 and ADG2 were all non-significant among the crosses. The individual additive breed differences between B and F breeds were positive and significant (p < 0.01) for all traits. The individual heterosis effects were significant (p < 0.05) for all traits except WWT for which the effect was non-significant. The maternal heterosis effects were significant (p < 0.01) for BWT (2.5 kg) and WWT (-3.0 kg). The heritability estimates for all traits in B and crosses were generally moderate to high indicating that there is scope for genetic improvement through selection. Selection within B and crossbreeding should be the strategy to enhance the growth performance under such production systems.

Population structure and phylogeography of Toda buffalo in Nilgiris throw light on possible origin of aboriginal Toda tribe of South India.

We report the genetic structure and evolutionary relationship of the endangered Toda buffalo of Nilgiris in South India with Kanarese and two other riverine buffalo breeds. The upgma phylogeny drawn using Nei's distance grouped South Kanara and Toda buffaloes at a single node while Marathwada and Murrah together formed a separate node. Principal component analysis was performed with pairwise interindividual chord distances which revealed clustering of Murrah and Marathwada buffaloes distinctly, while individuals of Toda and South Kanara breeds completely intermingled with each other. Furthermore, there were highly significant group variances (p < 0.01) when the breeds were grouped based on phylogeny, thus revealing the existence of cryptic genetic structure within these buffalo breeds. To know the evolutionary relationship among these breeds, 537-bp D-loop region of mitochondrial DNA was analysed. The phylogenetic analysis of mtDNA haplotypes following NJ algorithm with Chinese swamp buffalo as outgroup revealed a major cluster that included haplotypes from all the four investigated breeds and two minor clusters formed by South Kanara and Toda haplotypes. Reduced median network analysis revealed haplotypes of South Kanara and Toda to be quite distinct from the commonly found haplotypes indicating that these might have been ancestral to all the present-day haplotypes. Few mutations in two of the haplotypes of South Kanara buffalo were found to have contributed to ancestral haplotypes of Toda buffalo suggesting the possible migration of buffaloes from Kanarese region towards Nilgiris along the Western Ghats. Considering the close social, economic and cultural association of Todas with their buffaloes, the present study supports the theory of migration of Toda tribe from Kanarese/Mysore region along with their buffaloes.

Variation of 18 STR loci in Shahabadi sheep of India.

Shahabadi sheep represent an important but uninvestigated source of genetic diversity. Eighteen microsatellite markers were employed to analyze the genetic diversity of Shahabadi sheep population found in Bihar, India with fifty samples. Microsatellites were highly polymorphic with a mean allelic number 5.56 +/- 1.79. The observed heterozygosity, expected heterozygosity and observed and effective number of alleles were used to estimate the genetic variation of this breed. The observed heterozygosity in the population varied from 0.279-0.739 with the mean of 0.501 +/- 0.151, reflecting substantial genetic variation in this population. Population was observed to be heterozygote deficient (21.5%). The results of this study indicated that conservation of genetic variation in Shahabadi population should be considered by breeders, in the interest of long term future of the breed in its native tract.

Novel polymorphisms in UTR and coding region of inducible heat shock protein 70.1 gene in tropically adapted Indian zebu cattle (Bos indicus) and riverine buffalo (Bubalus bubalis).

Due to evolutionary divergence, cattle (taurine, and indicine) and buffalo are speculated to have different responses to heat stress condition. Variation in candidate genes associated with a heat-shock response may provide an insight into the dissimilarity and suggest targets for intervention. The present work was undertaken to characterize one of the inducible heat shock protein genes promoter and coding regions in diverse breeds of Indian zebu cattle and buffaloes. The genomic DNA from a panel of 117 unrelated animals representing 14 diversified native cattle breeds and 6 buffalo breeds were utilized to determine the complete sequence and gene diversity of HSP70.1 gene. The coding region of HSP70.1 gene in Indian zebu cattle, Bos taurus and buffalo was similar in length (1,926 bp) encoding a HSP70 protein of 641 amino acids with a calculated molecular weight (Mw) of 70.26 kDa. However buffalo had a longer 5' and 3' untranslated region (UTR) of 204 and 293 nucleotides respectively, in comparison to Indian zebu cattle and Bos taurus wherein length of 5' and 3'-UTR was 172 and 286 nucleotides, respectively. The increased length of buffalo HSP70.1 gene compared to indicine and taurine gene was due to two insertions each in 5' and 3'-UTR. Comparative sequence analysis of cattle (taurine and indicine) and buffalo HSP70.1 gene revealed a total of 54 gene variations (50 SNPs and 4 INDELs) among the three species in the HSP70.1 gene. The minor allele frequencies of these nucleotide variations varied from 0.03 to 0.5 with an average of 0.26. Among the 14 B. indicus cattle breeds studied, a total of 19 polymorphic sites were identified: 4 in the 5'-UTR and 15 in the coding region (of these 2 were non-synonymous). Analysis among buffalo breeds revealed 15 SNPs throughout the gene: 6 at the 5' flanking region and 9 in the coding region. In bubaline 5'-UTR, 2 additional putative transcription factor binding sites (Elk-1 and C-Re1) were identified, other than three common sites (CP2, HSE and Pax-4) observed across all the analyzed animals. No polymorphism was found within the 3'-UTR of Indian cattle or buffalo as it was found to be monomorphic. The promoter sequences generated in 117 individuals showed a rich array of sequence elements known to be involved in transcription regulation. A total of 11 nucleotide changes were observed in the promoter sequence across the analyzed species, 3 of these changes were located within the potential transcription factor binding domains. We also identified 4 microsatellite markers within the buffalo HSP70.1 gene and 3 microsatellites within bovine HSP70.1. The present study identified several distinct changes across indicine, taurine and bubaline HSP70.1 genes that could further be evaluated as molecular markers for thermotolerance.

The major histocompatibility complex in bovines: a review.

Productivity in dairy cattle and buffaloes depends on the genetic factors governing the production of milk and milk constituents as well as genetic factors controlling disease resistance or susceptibility. The immune system is the adaptive defense system that has evolved in vertebrates to protect them from invading pathogens and also carcinomas. It is remarkable in the sense that it is able to generate an enormous variety of cells and biomolecules which interact with each other in numerous ways to form a complex network that helps to recognize, counteract, and eliminate the apparently limitless number of foreign invading pathogens/molecules. The major histocompatibility complex which is found to occur in all mammalian species plays a central role in the development of the immune system. It is an important candidate gene involved in susceptibility/resistance to various diseases. It is associated with intercellular recognition and with self/nonself discrimination. It plays major role in determining whether transplanted tissue will be accepted as self or rejected as foreign.

Genetic evaluation of Ethiopian Boran cattle and their crosses with Holstein Friesian in central Ethiopia: milk production traits.

Breed additive and non-additive effects, and genetic parameters of lactation milk yield (LYD), 305-day milk yield (305YD), lactation length (LL), milk yield per day of lactation (DM) and lifetime milk yield (LTYD) were estimated in Ethiopian Boran cattle and their crosses with Holstein in central Ethiopia. The data analyzed included 2360 lactation records spread over 15 years. Ethiopian Boran cattle were consistently inferior (P < 0.01) to the Ethiopian Boran-Holstein crosses for the dairy traits studied. When the crosses were compared, LYD, 305YD and DM were higher (P < 0.01) for 75% and 87.5% crosses compared to 50% and 62.5% ones. However, the 50% crosses had higher (P < 0.01) LTYD than the other genetic groups. The individual additive genetic breed differences for milk production traits were all significant (P < 0.01). The estimates, in favor of Holstein, were 2055 ± 192 kg for LYD, 1776 ± 142 kg for 305YD, 108 ± 24 days for LL, 5.9 ± 0.5 kg for DM and 3353 ± 1294 kg for LTYD. Crossbreeding of the Holstein with the Ethiopian Boran resulted in desirable and significant (P < 0.01) individual heterosis for all milk production traits. The heterosis estimates were, 529 ± 98, 427 ± 72 kg, 44 ± 12 days 1.47 ± 0.23 kg and 3337 ± 681 kg, for LYD, 305YD, LL, DM and LTYD, respectively. The maternal heterotic effects were non-significant (P > 0.05) for all traits. Heritabilities of LYD, 305YD, LL, DM and LTYD for Ethiopian Boran were 0.20 ± 0.03, 0.18 ± 0.03, 0.26 ± 0.03, 0.13 ± 0.03 and 0.02 ± 0.04, respectively. The corresponding estimates for crosses were 0.10 ± 0.002, 0.11 ± 0.003, 0.63 ± 0.02, 0.45 ± 1.05 and 0.24 ± 0.11, respectively. Selection within each of the genetic groups and crossbreeding should substantially improve the milk production potential of the Ethiopian Boran breed under such production system.

Study of candida and Trichomonas vaginalis in Leucorrhoea.

Of 150 clinically suspected cases of leucorrhoea, 44 cases (29.33%) showed growth of candida and 30 cases (20%) of trichomonas, by wet preparation. Among the candida species the most frequent isolate was C albicans (56.8%), followed by C tropicalis (20.4%), C krusei (11.4%), C stellatoidea (9.1%) and C parapsilosis (2.3%). The higher incidence was found among the pregnant women than among non-pregnant women. Among pregnant women multipara in third trimester showed higher incidence of candida and trichomonas infection.

Antifungal metabolites (monorden, monocillin IV, and cerebrosides) from Humicola fuscoatra traaen NRRL 22980, a mycoparasite of Aspergillus flavus sclerotia.

The mycoparasite Humicola fuscoatra NRRL 22980 was isolated from a sclerotium of Aspergillus flavus that had been buried in a cornfield near Tifton, Ga. When grown on autoclaved rice, this fungus produced the antifungal metabolites monorden, monocillin IV, and a new monorden analog. Each metabolite produced a clear zone of inhibition surrounding paper assay disks on agar plates seeded with conidia of A. flavus. Monorden was twice as inhibitory to A. flavus mycelium extension (MIC > 28 microg/ml) as monocillin IV (MIC > 56 microg/ml). Cerebrosides C and D, metabolites known to potentiate the activity of cell wall-active antibiotics, were separated from the ethyl acetate extract but were not inhibitory to A. flavus when tested as pure compounds. This is the first report of natural products from H. fuscoatra.

New cyclic peptide and bisindolyl benzenoid metabolites from the sclerotia of Aspergillus sclerotiorum.

Scleramide (1), a new cyclic hexapeptide, and a new oxidized bisindolyl benzenoid derivative (2' '-oxoasterriquinol D methyl ether, 2) were isolated from extracts of the sclerotia of Aspergillus sclerotiorum (NRRL 5167). The structures of these compounds were determined by analysis of 1D and 2D NMR experiments.