Influence of Cu-ion doping in NiO NPs and their structural, morphological, optical and magnetic behaviors for optoelectronic devices and magnetic applications.

NiO and Cu-ion doped NiO nanoparticles with various concentrations (0.01-0.04 M) have been effectively synthesized in the current investigation using chemical precipitation method. The following techniques were used to characterized the materials' structural, morphological, elemental analysis, functional group, optical and magnetic properties: XRD, TEM, HR-TEM, SAED, SEM, EDX, FTIR, UV, PL and VSM. According to this Scherrer formula, the average crystalline sizes of the materials of pure NiO and Cu-doped NiO were determined to be 16.37 nm, 15.21 nm, 14.88 nm, 18.35 nm, and 10.88 nm, respectively. The HR-TEM images revealed that the d-spacing values about 0.24 nm, which coincides with the (111) plane of cubic NiO for pure and copper doped NiO nanoparticles. The SEM micrographs of Cu-doped NiO nanomaterials shows tiny agglomerated particles, while that of pure NiO nanoparticles shows spherical structure. Pure NiO and Cu-doped NiO nanoparticles have band gap values of 2.32 eV, 2.29 eV, 2.24 eV, 2.22 eV, and 2.27 eV, respectively. The Cu-doped NiO nanoparticles (0.01-0.03 M) at various concentrations can significantly reduce the band gap without significantly altering the structure, making them a potential material for creating optoelectronic devices. Copper was incorporated into NiO nanoparticles, which had a significant impact on the magnetic properties and changed the material from weakly ferromagnetic to ferromagnetic. In comparison to undoped NiO nanoparticles, the saturation magnetization and coercivity values of the 0.01 M and 0.03 M of Cu-doped nanoparticles is much higher. This outcome demonstrates that such Cu-doped NiO nanoparticles have promising magnetic applications.

Identification of novel polymorphism in mammary-derived growth inhibitor gene of water buffalo and its expression analysis in the mammary gland.

Mammary-derived growth inhibitor (MDGI), a member of the lipophilic family of fatty acid-binding proteins, plays an important role in the development, regulation, and differentiation of the mammary gland. The aim of the study was to identify polymorphism in the MDGI gene and its expression analysis in the mammary gland at various stages of lactation, in Indian buffalo. Nucleotide sequence analysis of MDGI gene in different breeds of riverine and swamp buffaloes revealed a total of 16 polymorphic sites and one Indel. Different transcription factor binding sites were predicted for buffalo MDGI gene promoter sequence, using online tools and in-silico analysis indicating that the SNPs in this region can impact the gene expression regulation. Phylogenetic analysis exhibited the MDGI of buffalo being closer to other ruminants like cattle, yak, sheep, and goats. Further, the expression analysis revealed that buffalo MDGI being highly expressed in well-developed mammary glands of lactating buffalo as compared to involution/non-lactating and before functional development to start the milk production stage in heifers. Stage-specific variation in expression levels signifies the important functional role of the MDGI gene in mammary gland development and milk production in buffalo, an important dairy species in Southeast Asia.

PCR-SSCP analysis of MDGI gene and its association with milk production traits in river buffalo (Bubalus bubalis).

In this study, we investigated the genetic variation within 3'UTR of Mammary-Derived Growth Inhibitor (MDGI) gene of buffalo using PCR-SSCP and sequencing; and also analyzed association of polymorphism with the milk production traits. The study revealed two conformational patterns, 'A' and 'B' among 234 Mehsana buffaloes maintained with their records in the field and at farm. The frequency of SSCP variant 'A' was found to be invariably high in the buffalo population under study. Further, association analysis of SSCP variants with various milk production and milk quality traits indicated no significant effect on any of the traits investigated. Sequencing of SSCP variant 'A' showed homozygous G/G and A/A and 'B' had heterozygous G/C and A/G at positions +124 and +140 respectively, in the 3'UTR of buffalo MDGI. The preliminary results showed the substantial variations in the distribution of SSCP variants' frequencies within Mehsana buffaloes, however these variants had non-significant association with milk yield, fat yield and fat percentage in Mehsana buffaloes.

Molecular Characterization of Buffalo Haptoglobin: Sequence Based Structural Comparison Indicates Convergent Evolution Between Ruminants and Human.

Haptoglobin (Hp) protein has high affinity for hemoglobin (Hb) binding during intravascular hemolysis and scavenges the hemoglobin induced free radicals. Earlier reports indicate about uniqueness of Hp molecule in human and cattle, but in other animals, it is not much studied. In this paper, we characterized buffalo Hp molecule and determined its molecular structure, evolutionary importance, and tissue expression. Comparative analysis and predicted domain structure indicated that the buffalo Hp has an internal duplicated region in α-chain only similar to an alternate Hp2 allele in human. This duplicated part encoded for an extra complement control protein CCP domain. Phylogenetic analysis revealed that buffalo and other ruminants were found to group together separated from all other non-ruminants, including human. The key amino acid residues involved in Hp and Hb as well as Hp and macrophage scavenger receptor, CD163 interactions in buffalo, depicted a significant variation in comparison to other non-ruminant species. Constitutive expression of Hp was also confirmed across all the vital tissues of buffalo, for the first time. Results revealed that buffalo Hp is both structurally and functionally conserved, having internal duplication in α-chain similar to human Hp2 and other ruminant species, which might have evolved separately as a convergent evolutionary process. Furthermore, the presence of extra Hp CCP domain possibly in all ruminants may have an effect during dimerization of molecule in these species.

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.

Association analysis of polymorphism in thyroglobulin gene promoter with milk production traits in riverine buffalo (Bubalus bubalis).

Polymorphism within the promoter region of bovine thyroglobulin has been reported to be associated with milk and meat quality. In this study, we investigated the genetic variation within thyroglobulin promoter region of swamp and riverine buffaloes using PCR-SSCP technique and sequencing, and also analyzing association of polymorphism with the milk production traits. The study revealed four conformational patterns, A, B, C, and D among 323 buffaloes of two riverine breeds and different swamp populations. The frequency of SSCP variant 'A' was found to be invariably high among all buffalo populations. Variant 'C' was found to be absent in pure swamp population and present with higher frequency among riverine dairy breeds Mehsana and Nili Ravi. Frequency of D variant was observed to be highest in buffalo population, representing riverine and hybrid types. Sequencing of three representative PCR products of each of the SSCP variants, revealed three polymorphic sites responsible, 33C > T, 176G > T and 221C > T, in the buffalo TG promoter region. Further, association studies of SSCP variants with various milk production and milk quality traits indicated significant effect on fat percentage in buffaloes belonging to Mehsana and Nili Ravi dairy breeds. The preliminary results also showed the substantial variations in the distribution of SSCP variants' frequencies across swamp and riverine buffaloes, two distinct populations being reared for meat and milk production, respectively.

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.

Caprine Toll-like receptor 8 gene sequence characterization reveals close relationships among ruminant species.

TLR8 mediates antiviral immunity by recognizing ssRNA viruses and triggers potent antiviral and antitumor immune responses. In this study, approximately 3.5 Kb nucleotide sequence data of caprine TLR8 gene were generated from one sample each of twelve different Indian goat breeds belonging to different geographical regions. Cloning and characterization of cDNA synthesized from RNA purified from goat spleen revealed TLR8 ORF to be of 3102 nucleotides long coding for 1033 amino acids similar to other ruminant species, that is sheep, buffalo and cattle. The sequence analysis at nucleotide level revealed goat TLR8 to be closer to buffalo sharing 99.6% homology, followed by cattle and sheep. Simple Modular Architecture Research Tool (SMART) used for the structural analysis of goat TLR8 showed the presence of 16 leucine-rich repeats (LRRs) along with single Toll/interleukin-1 receptor (TIR) domain. TIR domain when compared with other livestock species was found to be conserved in ruminant species goat, sheep, cattle and buffalo. The phylogenetic analysis also revealed grouping of all ruminant species together, goat being closer to buffalo followed by cattle and sheep. Total 4 polymorphic sites were observed in TLR8 gene of one specimen goat representing each of 12 different breeds studied, all of which were synonymous and present within the coding region. Of these 4 SNPs, two were in ectodomains, one in TIR domain and one was found to be present in transmembrane domain. PCR-RFLP genotyping of two of the SNPs indicated variations in allele frequencies among different goat breeds. The expression profiling in 13 tissues of goat showed maximum expression of TLR8 gene in kidney followed by spleen, lung and lymph node. Overall, our results indicate conservation of TLR8 gene among the ruminant species and low variation within Indian goat breeds.

Sequence characterization of river buffalo Toll-like receptor genes 1-10 reveals distinct relationship with cattle and sheep.

The present study was undertaken to characterize the full-length transcripts of Toll-like receptor (TLR) genes 1-10 of river buffalo. The conceptualized amino acid identity of bubaline TLRs ranged between 86% to 100% with ruminants, while it ranged between 45% to 91% with other vertebrate species. Simple modular architecture tool (SMART) analysis revealed the presence of TIR domains and varying numbers of leucine-rich repeat motifs in all the buffalo TLRs. With respect to TIR domains, TLRs 1, 2 and 3 of river buffalo were found to have 99.3% identity with cattle and 100% identity of TLRs 4, 6 and 10 with sheep. Phylogenetic analysis of TLRs of buffalo and different vertebrate species revealed the clustering of major TLR gene subfamilies with high bootstrap values. The evolutionary relationship between buffalo and other ruminant species was found to vary among different TLRs. In order to understand the relationship between TLRs of different ruminant species, multidimensional scaling (MDS) analysis of pairwise amino acid differences between different species within each TLR was performed. Buffalo and cattle were found to be closely related only with respect to TLRs 1, 2 and 7, while buffalo and sheep were found to be clustering together with respect to TLRs 3, 6, 8 and 10. The distinct relationship of bubaline TLRs with cattle and sheep revealed the possible differences in the pathogen recognition receptor systems in these animals and consequently the differences in their susceptibility/resistance to various invading organisms.

Detection of polymorphism and sequence characterization of Toll-like receptor 7 gene of Indian goat revealing close relationship between ruminant species.

In this study, approximately 3.4 kb nucleotide sequence of caprine TLR7 (Toll-like receptor 7) gene was generated from twelve different Indian goat breeds belonging to different geographical regions. Goat TLR7 gene ORF (Open Reading Frame) was found to be 3141 nucleotides long coding for 1046 amino acids similar to sheep. The sequence analysis at nucleotide level revealed goat TLR7 having 99.5% homology with sheep, followed by other livestock species. Simple Modular Architecture Research Tool (SMART) was used for the structural analysis of goat TLR7 that showed the presence of 22 leucine rich repeats (LRRs) along with single Toll/interleukin-1 receptor (TIR) domains. TIR domain, when compared, was found to be similar in ruminant species, goat, sheep, cattle, and buffalo. The phylogenetic analysis also revealed grouping of all ruminant species together, goat being closer to sheep followed by cattle and buffalo. A total of 22 polymorphic sites were observed in TLR7 gene of 24 goats representing 12 different breeds, out of which 19 were present within the coding region and three in 3'UTR. Out of the seven nonsynonymous SNPs, two were in ectodomains and one in TIR domain. Overall our results indicate substantial variation within goat TLR7 gene, which could be exploited for association with disease susceptibility.

Power of exclusion of 19 microsatellite markers for parentage testing in river buffalo (Bubalus bubalis).

In the present study, 19 microsatellite markers were assessed for their power of exclusion to test parentage in river buffalo. Microsatellite genotypes of 216 unrelated buffaloes belonging to five different breeds were utilized for the study. The probabilities of exclusion were calculated for three hypothetical situations viz. paternity testing (PE1), one parental genotype unavailable (PE2) and exclusion of both parents i.e. substituted offspring (PE3). The mean probability of exclusion across 19 investigated markers in buffalo was 0.578 (PE1), 0.405 (PE2) and 0.764 (PE3) respectively. The probability of exclusion for paternity (PE1) ranged between 0.297 and 0.814 across different markers. The exclusion probability for the cases one parent unavailable (PE2) and substituted offspring (PE3) varied from 0.143 to 0.688 and 0.465 to 0.946 respectively. Polymorphism information content and expected heterozygosity were found to have significantly high correlation with probability of exclusion of microsatellite markers. The cumulative PE1 of nine marker loci was estimated to be 0.9999 while in case of absence of one of the parental genotypes, a minimum of 11 markers were required to achieve a cumulative PE2 of 0.999. In conclusion, the present study proposes two multiplex sets with four and five markers respectively for routine parentage testing in buffalo and an additional set of four markers for doubtful cases of paternity.

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.

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.

Protein tyrosine phosphorylation and zona binding ability of in vitro capacitated and cryopreserved buffalo spermatozoa.

Many similarities between the changes associated with normal capacitation and cryocapacitation have been demonstrated. The present study was undertaken to determine whether similarities exist in the protein tyrosine phosphorylation pattern and zona binding ability between in vitro capacitated (heparin induced; 20 µg/ml) and frozen-thawed (cryocapacitated) buffalo spermatozoa. Semen from seven buffalo bulls (eight ejaculates each) was divided into two parts. Part I was used as fresh semen and part II was extended in Tris-egg yolk extender, equilibrated and frozen in liquid nitrogen. Localization of phosphotyrosine-containing protein was determined using an indirect immunoflourescence assay with anti-phosphotyrosine antibody. For zona binding assay, good quality oocytes collected by aspiration technique from fresh buffalo ovaries were used. The bound spermatozoa were stained with Hoechst 33342 dye and observed under fluorescent microscope. The results revealed sperm head associated protein tyrosine phosphorylation in both in vitro capacitated and frozen-thawed spermatozoa. In the zona binding assay, the mean number of bound spermatozoa was 90.6 ± 1.9 and 104.7 ± 2.2 in fresh semen after incubation in non capacitating media at 0 h and 3 h, respectively. But after incubation in capacitating media with heparin for 3 h, the mean number of spermatozoa attached to zona pellucida was 138.4 ± 2.6. The in vitro capacitated spermatozoa had significantly (P < 0.05) higher binding ability than that of fresh spermatozoa. After freezing and thawing, 2.5 fold reductions in the zona binding ability of cryopreserved spermatozoa was observed compared to in vitro capacitated spermatozoa. The binding ability of in vitro capacitated spermatozoa was significantly (P < 0.01) higher than that of frozen-thawed (cryocapacitated) spermatozoa. The study concluded that both in vitro capacitated and frozen-thawed (cryocapacitated) spermatozoa had similar immune-localization of tyrosine phosphorylated protein pattern, however, differed in the zona binding ability.

Objective sperm motion analysis to assess dairy bull fertility using computer-aided system--a review.

Motility is one of the most important characteristics associated with the fertilizing ability of spermatozoa and is an expression of their viability and structural integrity. Computer-assisted semen analyser (CASA) provides precise and accurate information on different sperm motion characteristics. This article reviews various aspects of computer-aided motility analysis of bull sperm like sample preparation, standardization of instrument settings, importance of various motility parameters evaluated by the system and its impact on basic functional studies of spermatozoa. It gives special emphasis to various aspects of bull sperm motion analysis especially sub-populations of spermatozoa, hyper-activation, motion characteristic in different genetic and age groups, etc. and their utility in predicting the fertility of dairy bulls. The need to fill the gap in research and the necessity of universal standardization of the equipment has been discussed.

Short tandem repeat based analysis of genetic variability in Kanarese buffalo of South India.

The goal of the present study was assessing genetic diversity within Kanarese buffalo, the dual purpose breed of South India. A total of 48 unrelated animals were genotyped at 23 short tandem repeat (STR markers) loci. The total number of observed alleles was 180 with a mean of 7.83 per locus, which varied from 3 to 12 across different loci. The mean observed and expected heterozygosity in South Kanara buffaloes was estimated to be 0.518 and 0.712 respectively. Within population inbreeding estimate (F(IS)) was significantly positive in most of the investigated loci which resulted in significant deviation from Hardy-Weinberg equilibrium at 19 of 23 loci analyzed. Evaluation of South Kanara buffalo population for mutation drift equilibrium revealed no significant heterozygosity excess under three different models of evolution viz. infinite alleles model (IAM), stepwise mutation model (SMM) and two phase model (TPM), thus indicating the absence of any recent genetic bottleneck. The results of the present study will help in formulating rational breeding strategies as well as conservation of this important germplasm.

Genetic diversity and bottleneck analysis of Nagpuri buffalo breed of India based on microsatellite data.

In this study, 25 heterologous bovine microsatellite markers have been used for the assessment of genetic diversity in Nagpuri buffalo, an important breed of Central India. For this, 48 DNA samples of unrelated individuals of Nagpuri buffalo were PCR amplified and microsatellite alleles were resolved in 6% denaturing, silver stained Urea-PAGE gel. Genotypic status of individuals at each locus was identified manually and data analysis carried out using POPGENE software. Observed number of alleles varied from 2 (ILSTS073 locus) to 8 (HEL13 & ILSTS058 loci) with a mean of 5.24 alleles per locus. Moderate level of heterozygosity (0.45) indicated sufficient genetic diversity existing in this buffalo population. PIC values for the microsatellite loci analysed, ranged from 0.10 (ILSTS019 locus) to 0.81 (ILSTS058 locus) with a mean of 0.53. No shift in the frequency distribution of alleles and a normal L-shaped curve indicated non-existence of any bottleneck in Nagpuri. The study thus highlights the usefulness of heterologous bovine microsatellite markers to assess the genetic variability in buffalo breeds as well. Also various diversity indices suggest sufficient genetic variability within Nagpuri buffalo that can be utilized as initial guidelines for future breeding strategies and conservation.

Capacitation status of fresh and frozen-thawed buffalo spermatozoa in relation to cholesterol level, membrane fluidity and intracellular calcium.

The present study was conducted to assess the capacitation status of fresh and frozen-thawed buffalo spermatozoa and its relationship with sperm cholesterol level, membrane fluidity and intracellular calcium. Semen from seven buffalo bulls (eight ejaculates each) was divided into two parts. Part I was used as fresh semen and part II was extended in Tris-egg yolk extender, equilibrated (4 degrees C for 4h) and frozen at -196 degrees C in LN(2). The fresh and frozen-thawed spermatozoa were assessed for capacitation status using chlortetracycline (CTC) fluorescent assay, membrane fluidity using merocyanine 540/Yo-Pro-1 assay and intracellular calcium using Fluo-3 AM with flowcytometry. Results revealed a significant (P<0.01) increase in capacitated sperm population in frozen-thawed semen compared to fresh semen (42.21% vs 14.32%). Similarly, a significantly (P<0.01) higher proportion of frozen-thawed live spermatozoa showed high membrane fluidity (53.62% vs 25.67%) and high intracellular calcium (43.68% vs 11.72%) compared to fresh semen. The sperm cholesterol was significantly (P<0.01) reduced after freezing-thawing as compared to fresh semen. The proportion of capacitated spermatozoa (CTC pattern B) was positively correlated with the proportion of sperm with high intracellular calcium (r=0.81) and high membrane fluidity (r=0.65), and negatively correlated with cholesterol level (r=-0.56) in frozen-thawed semen. The membrane fluidity was also strongly associated with the cholesterol level and intracellular calcium. The study concluded that changes in buffalo spermatozoa and established the relationship among capacitation status, sperm cholesterol level, membrane fluidity and intracellular calcium concentration in frozen-thawed spermatozoa.

Riverine status and genetic structure of Chilika buffalo of eastern India as inferred from cytogenetic and molecular marker-based analysis.

The present study aimed at assessing the status of the Chilika buffalo population of eastern India employing cytogenetic and molecular markers. The Chilika buffaloes investigated cytogenetically possess a somatic chromosome count of 50, identical to that of typical riverine buffaloes. Various diversity estimates, viz. observed number of alleles (4.68), effective number of alleles (2.79), and observed (0.487) and expected (0.602) heterozygosity across 25 heterologous microsatellite markers indicated the presence of a moderate level of genetic diversity in Chilika buffaloes, comparable with three other prominent Indian riverine buffalo breeds (Murrah, Nagpuri and Toda) included in this study. Across the four buffalo populations, mean estimates of F-statistics from Jackknifing over loci were significantly different from zero (p < 0.05), with F(IT) (total inbreeding estimate) = 0.315 +/- 0.038, F(IS) (within-population inbreeding estimate) = 0.178 +/- 0.038, and F(ST) (population differentiation) = 0.166 +/- 0.025. Inter-breed analysis reflected Chilika buffaloes to be genetically close to Nagpuri followed by Murrah and Toda buffaloes. Factorial correspondence analysis (FCA) revealed low breed-specific clustering of Chilika and Nagpuri buffaloes. Additionally, the neighbour-joining tree structure of mitochondrial DNA D-loop haplotypes indicated clear grouping of the Chilika haplotypes with the riverine buffalo. Thus the cytogenetic, microsatellite and mitochondrial data analysed in the present study classify Chilika buffalo of eastern India to be of the riverine type and not swamp-type buffalo.

Evaluation of genetic variability and mutation drift equilibrium of Banni buffalo using multi locus microsatellite markers.

The present study was conducted to evaluate genetic diversity of Banni buffalo and its relationship/differentiation with Murrah using genotypic data on 24 heterologus bovine specific microsatellite marker loci. A total of 138 alleles were observed with a mean of 5.75 alleles/locus across two populations. The mean observed and expected heterozygosities were found to be 0.441 and 0.572 respectively in Banni buffaloes while it was 0.464 and 0.610 respectively in Murrah buffaloes. The average heterozygosity deficit was significantly positive with substantially higher values observed in Banni (22.3%) and Murrah (24%) buffalo populations. Banni buffalo population, when evaluated for mutation drift equilibrium revealed significant heterozygosity excess under IAM while no such excess was observed under SMM and TPM. The qualitative graphical test revealed a normal L-shaped distribution of allele frequencies indicating the absence of genetic bottleneck in Banni buffaloes. The mean estimates of F-statistics over all the loci were 0.376 for F(IT), 0.187 for F(ST) and 0.232 for F(IS) respectively. Analysis of molecular variance (AMOVA) revealed 18.95% of the total variation being explained by between breed differences while 14.36% of the variation explained differences between individuals within each breed. Genotype assignment test revealed distinct clustering of Banni and Murrah buffaloes. Genetic distance was estimated using three different methods, the results of which revealed considerable genetic differentiation between these two buffalo populations. The divergence time between Banni and Murrah buffaloes was estimated to be around 7286 years. The results of the present study may be helpful in decision making for conservation programs as Banni buffalo population is on decline.