Two novel SNPs identified in STAT1 gene adjoining a QTL for milk production in Holstein Friesian crossbreds of Kerala.

We analyzed the effect of a single nucleotide polymorphism, g. C3141T in the 3' UTR of Signal transducer and activator of transcription-1 gene (STAT1) on milk production traits in the Holstein Friesian crossbred cattle of Kerala (n = 144) by association analysis and expression study. The population was genotyped by restriction fragment length polymorphism using Pag1. Association study using the General Linear Model-Analysis of Variance revealed that none of the yield or composition traits analyzed were significantly differed. The expression profile of STAT1 gene in leucocytes of animals bearing homozygous genotypes was compared by quantitative real time PCR using SYBR green chemistry with and relative expression was not found to be significantly differed. The second stage of the study, the STAT1 mRNA spanning 3213 bp was amplified from leucocytes and sequenced (GenBank: MT459802.1). Two novel SNPs were identified; one synonymous mutation in the coding region (g.A1212G) and the other in the 3'UTR (g.T3042C). The novel SNPs might contribute to STAT1 gene regulation mediated by alternate spicing or binding sites for regulatory molecules. The results reiterate the importance of extensive studies of STAT1 gene variants to substantiate the presence of a quantitative trait loci for dairy traits in the vicinity of STAT1 gene.

Genetic diversity analysis of cattle genetic groups of Kerala state using microsatellite data.

Cattle belonging to seven different genetic groups in Kerala state, India were chosen for the study to find out the genetic diversity between the groups, which would aid in their sustainable improvement and conservation of native cattle. They included the native groups namely, Vechur, Kasaragod, Vadakara dwarf and Vilwadri, along with three different grades of crossbred cattle, based on milk production. Genomic DNA was isolated from 20 to 30 unrelated animals of each group and a panel of 25 microsatellite markers as suggested by FAO-ISAG, were amplified by multiplex PCR. The PCR amplicons were genotyped and the allelic data analyzed using suitable Bioinformatics softwares. The present study showed that the observed number of alleles was much more than the expected, in all populations. The mean PIC value obtained for the present study was 0.8912 and increased number of private alleles were observed, especially in Vilwadri and Kasaragod groups. Negative value of FIS (-0.055) indicated that the level of inbreeding was less. The FST value was 0.1442 indicating that the populations showed good genetic differentiation. The results of Structure analysis revealed admixture only in Vadakara population. The results obtained from the present study showed that Vilwadri and Kasaragod cattle showed distinct differences from other groups.

Estimation of co-variance components and genetic parameters of fertility and production traits in crossbred cattle of Kerala.

Estimation of co-variance components and genetic parameters of fertility and production traits will help to find out the relative importance of genetic and environmental components of each trait and to develop a genetic evaluation system for overall improvement in performances of crossbred cattle of Kerala. In the present study, major fertility trait considered was daughters pregnancy rate (DPR), measures the percentage of non-pregnant animals that become pregnant during each oestrous cycle. Data pertaining to 1180 crossbred cattle sired by 208 Frieswal bulls, spread over a period of 16 years from 2003 to 2019, maintained at different farms of Kerala Veterinary and Animal Sciences University and field centres of ICAR- Filed Progeny Testing Scheme were analysed in the study. Estimates of covariance components and genetic parameters were obtained using restricted maximum likelihood (REML) approach using average information (AI) algorithm. It was observed that DPR had low heritability (0.092 ± 0.03), compared to 305 days milk yield (MY) (0.170 ± 0.094) and fat percent (FP) (0.173 ± 0.072). Phenotypic (rp), genetic (rg) and residual (re) correlation indicated unfavourable association of fertility with production traits. The estimates of variance and (co)variance components were computed by multivariate animal model. The results indicated that DPR was having lower direct additive (σ2a) 0.046 and environmental variance (σ2c) 0.063 compared to other traits. Highest additive genetic variance (σ2a) 27035.8 was obtained for MY. The study estimates the magnitude of correlations and covariances of fertility and production traits. Since DPR had lower additive genetic variance and negative association with milk production traits in cattle, it would be included as an indirect measure in the evaluation and breeding programs of crossbred cattle of Kerala.

Deltamethrin resistant alleles predominate in Rhipicephalus sanguineus sensu lato in South India.

Brown dog tick, Rhipicephalus sanguineus sensu lato, an important ectoparasite transmitting several pathogens, is the most common tick species infesting dogs. Control of ticks being central to the control of fatal tick-borne diseases, this study attempted to assess the susceptibility/resistance of brown dog ticks to synthetic pyrethroids, the commonly used acaricides against ticks. Larval packet assay revealed 60% of isolates tested to be resistant and tolerant to deltamethrin as per the resistance factor that ranged from 1 to 53.7. Sequence analysis of the PCR amplified product of domain II S4-5 linker of sodium channel gene in R. sanguineus revealed novel polymorphisms, viz., C190A, G215T and T270C. In domain III S6 region of the gene, a T2134C mutation was observed. Genotyping with allele-specific PCR targeting domain II S4-5 linker region using single larvae revealed that most R. sanguineus larvae in the study population were homozygous resistant (RR) genotypes, followed by heterozygous (RS) and homozygous susceptible (SS) genotypes. A higher proportion of RS genotypes was also observed in domain III S6 region. This first report of genotyping of Indian R. sanguineus to analyse synthetic pyrethroid resistance highlights the need to devise alternate control strategies to reduce the brown dog tick population.

Genotyping of Deltamethrin Resistance in Rhipicephalus (Boophilus) microplus Population in Kerala, South India.

PURPOSE: Genotyping of Rhipicephalus (Boophilus) microplus for polymorphisms in deltamethrin-resistant loci of sodium channel gene by allele-specific PCR (AS-PCR). METHODS: Adult R. (B.) microplus ticks were collected from naturally infested cattle in Kerala. The larval packet test (LPT) was performed with deltamethrin and dose response data were analysed by probit method. Adult and larval tick DNA were amplified by PCR and later sequenced to identify mutations, if any, in the domain II S4-5 linker and domain III S6 regions in para voltage-gated sodium channel gene, at loci that were previously documented to be associated with deltamethrin resistance. Allele-specific PCR was performed for the amplification of target gene locus (C190A and T2134C) to genotype 1000 larvae each, at these loci. Genotype frequency was statistically analysed by Chi-square test. RESULTS: Bioassay using LPT revealed that LC50 and LC95 values of all the R. (B.) microplus isolates in this study were more than double the reported values of reference susceptible strain. Sequence analysis of the PCR amplicons of domain II S4-5 linker of voltage-gated sodium channel gene revealed C190A mutation, A271G mutation as well as A-G mutation at 217th position. AS-PCR done to genotype C190A mutations revealed a frequency of 6%, 15% and 64%, respectively for homozygous-susceptible (SS), heterozygous (RS) and homozygous-resistant (RR) genotypes. In domain III S6 region of the gene, C2121T and A2102T mutations were observed. AS-PCR to genotype the previously reported T2134C mutation revealed 100% SS genotype in R. (B.) microplus isolates of Kerala. CONCLUSIONS: Genotyping of R. (B.) microplus isolates of Kerala for target site mutations reportedly associated with deltamethrin resistance revealed a significantly high frequency of resistant genotypes at II S4-5 linker of voltage-gated sodium channel gene. This study forms the first report of such mutations in Kerala, south India and demands serious attention in the light of increased prevalence of tick-borne haemoparasitic infection.

Ovarian expression, polymorphism identification and association of SIRT3 gene with reproduction traits in goats.

The NAD+-dependent protein deacetylase, Sirtuin3 (SIRT3), plays a role in fertility by preventing the activation of reactive oxygen species (ROS). A novel study was conducted on caprine SIRT3, to study its ovarian expression, explore the sequence variability in exon 7 and analyze its association with prolificacy in two native goat breeds of Kerala, Malabari and Attappady Black. The mRNA isolated from ovaries of six Malabari and Attappady Black goats were subjected to quantitative PCR (qPCR) using GAPDH and ß-actin as reference genes. Genomic DNA was isolated from 185 goats (99 Malabari and 86 Attappady Black) and subjected to PCR-SSCP to identify polymorphism in exon 7 of SIRT3 and association with litter size was analyzed. The ovarian expression of caprine SIRT3 was significantly higher (p ≤ 0.01) in Malabari than low prolific Attappady Black. PCR-SSCP analysis revealed, exon 7 of SIRT3 was polymorphic with three genotypes namely, AA, AB and BB with a novel SNP, g.154C > T in the 3'UTR. A significant association (p ≤ 0.05) was noticed between the genotypes of SIRT3 and litter size. The results obtained from this study highlight the role of SIRT3 in reproduction and hence SIRT3 may be considered as a potential candidate gene for genetic improvement in goats.

Metagenomic analysis exploring taxonomic diversity of rumen microbial communities in Vechur and crossbred cattle of Kerala state, India.

Rumen, one of the most productive diverse microbial habitats plays a vital role in the breakdown of feed to produce energy for maintenance and milk production in cattle. Culture-based procedures could identify only 10% of microbial species present in the rumen. Kerala, one of the southern states of India, owns only one native cattle breed, the Vechur cattle, which is noted for its short stature, disease resistance and adaptability to hot humid climate. Lower population density and decreased milk production potential of Vechur cattle led to the development of crossbred cattle of Kerala, with higher milk yield. A study was conducted to assess the rumen microbial profile of low productive Vechur cattle and high productive crossbred cattle for a better understanding of the relationship between the host and microbial community. DNA isolated from rumen liquor of five cattle each from both genetic groups maintained on standard ration (forage, concentrate ratio of 50:50) was subjected to whole metagenome sequencing. Bioinformatics and statistical analysis revealed that bacteria followed by archaea and eukaryota dominated in the Vechur cattle as well as the crossbred cattle rumen. Bacterial community was dominated by Bacteroidetes and Firmicutes phyla in both genetic groups with a higher Firmicutes to Bacteroidetes ratio of 0.45 in Vechur cattle. Among archaea, Euryarchaeota was more abundant, which constitute methanogens, contributing 98% of total archaeal reads. Prevalent protozoal genus found in the Vechur cattle rumen was Entodinium and in crossbred cattle rumen was Entamoeba. In Vechur and crossbred cattle rumen, 1086 and 1262 microbial species were observed exclusively and 4731 species were shared between habitats. There was a significant difference in total microbial species abundance between the two genetic groups and Vechur cattle displayed significantly higher microbial diversity compared to crossbred. As per literature, this is presumably the first report of rumen metagenome profile of Vechur cattle, a unique short breed of India.

An in silico evaluation of non-synonymous single nucleotide polymorphisms of mastitis resistance genes in cattle.

Recognition of deleterious non-synonymous single nucleotide polymorphism (SNPs) aids in the assessment of genetic basis of diseases and prediction of clinical phenotypes. In this study, data obtained from whole exome sequencing of Vechur cow using Illumina HiSeq 2500 platform is compared with that of crossbred cattle of Kerala. Sequence analysis of selected 18 mastitis resistant genes, evaluated the consequence of non-synonymous SNPs in these genes from both Vechur and crossbred cattle of Kerala, using sequence and structure-based computational tools such as SIFT, PROVEAN and I-MUTANT 2.0. Compared to Vechur cattle, incidence of missense deleterious mutation to effect protein functioning were relatively higher in crossbred cattle. These results on the type of genetic variants and its impact on normal functioning of a protein will assist to predict and enhance the disease resistance in cattle breeds.

Seroprevalence of bovine intestinal schistosomosis in different agro- ecological zones of south India using excretory-secretory antigen based ELISA.

Intestinal schistosmosis caused by Schistosoma spindale and S. indicum is an important snail borne trematode infection that adversely affects the production and productivity of bovines in India. The present communication reports the high seroprevalence of infection among dairy cattle and buffaloes, under field conditions, utilizing a sensitive and specific excretory-secretory antigen based ELISA. Comparison of ELISA with copro-PCR, microscopy and post-mortem mesentery examination revealed the diagnostic superiority of ELISA. Seroprevalence of infection was mapped for the first time in India with special emphasis to agro- ecological zones. Anti-schistosome antibodies were detected in 34.96% of dairy cattle and buffaloes in the state with the evidence of significant influence of topography on the prevalence of infection. The study also highlighted the need to trace endemic pockets of infection in the country through efficient ante-mortem surveillance and to initiate anti-schistosome therapy prior to animal transport.

Sirtuin3 (SIRT3) gene molecular characterization and SNP detection in prolific and low prolific goat breeds.

Sirtuin3 (SIRT3) is a member of the Sirtuin family of NAD+-dependent deacetylase. They have evolved as a vital protein in preventing the activation of reactive oxygen species (ROS) in oocytes. A novel study on caprine SIRT3 was conducted, to characterize caprine SIRT3, to detect potential polymorphisms in SIRT3 and to analyze their association with litter size in the two indigenous goat breeds of India viz., the prolific Malabari and low prolific Attappady Black goats. A 1070 bp mRNA sequence of SIRT3 cDNA comprised of an ORF of 1002 bp encoding 333 amino acids, having 96% identity with bovine SIRT3. The genomic DNAs from the goats (n = 222) were subjected to PCR and single strand conformation polymorphism (SSCP) of exon 5 fragment (213 bp) of caprine SIRT3. On analysis, two genotypes viz., DD and DE were observed with frequencies of 0.63 and 0.37 respectively. Further sequencing of the PCR products of the respective genotypes revealed a novel synonymous SNP (MF176159:c.691C > T). Genotypes of this fragment had a significant influence on number of kids born (P < 0.05) with DD genotype being superior to DE genotype. These results highlight the role of SIRT3 in reproduction traits and the detected novel SNP would aid in the Marker Assisted Selection programmes and thus SIRT3 can considered as a potential candidate gene for reproduction traits in goats.

Evaluation of Genetic Diversity, Population Structure, and Relationship Between Legendary Vechur Cattle and Crossbred Cattle of Kerala State, India.

The legendary Vechur cattle of Kerala, described as a very short breed, and the crossbred (CB) Sunandini cattle population exhibited great phenotypic variation; hence, the present study attempted to analyze the genetic diversity existing between them. A set of 14 polymorphic microsatellites were chosen from FAO-ISAG panel and amplified from genomic DNA isolated from blood samples of 30 Vechur and 64 unrelated crossbred cattle, using fluorescent labeled primers. Both populations revealed high genetic diversity as evidenced from high observed number of alleles, Polymorphic Information Content and expected heterozygosity. Observed heterozygosity was lesser (0.699) than expected (0.752) in Vechur population which was further supported by positive FIS value of 0.1149, indicating slight level of inbreeding in Vechur population. Overall, FST value was 0.065, which means genetic differentiation between crossbred and Vechur population was 6.5%, indicating that the crossbred cattle must have differentiated into a definite population that is different from the indigenous Vechur cows. Structure analysis indicated that the two populations showed distinct differences, with two underlying clusters. The present study supports the separation between Taurine and Zebu cattle and throws light onto the genetic diversity and relationship between native Vechur and crossbred cattle populations in Kerala state.

Molecular evidence of haemoparasites in ixodid ticks of dogs- first report in India.

The distribution of ixodid ticks of the genera Rhipicephalus and Haemaphysalis spp. in tropical regions of India contributes to many serious tick-borne parasitic and rickettsial infections in domestic and wild canines. A preliminary molecular survey of the most prevalent haemoparasites in ixodid ticks of carnivores in Kerala, South India was undertaken using multiplex PCR. Babesia vogeli, B. gibsoni and Ehrlichia canis could be detected in R. sanguineus ticks, while H. bispinosa harboured B. gibsoni alone. Future investigations including transmission trials are to be undertaken to prove the vector potentiality of these ticks in this geo-climatic zone.

Association of a Cac8I polymorphism in the IGF1 gene with growth traits in Indian goats.

The Insulin-like Growth Factor 1 (IGF1) gene is a member of somatotropic axis and plays a key role in proliferation of cells, mitosis, myogenesis, meiosis, differentiation in foetal development and post natal growth. The objectives of this study were to verify the single nucleotide polymorphisms (SNPs) in IGF1 gene and their association with growth traits in two indigenous native goat genetic groups of Kerala, viz., Malabari and Attappady Black. A total of 277 goats were genotyped using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using the restriction enzyme Cac8I. One SNP, A224G was detected in the 5' non-coding region of the IGF1 gene, and accordingly two genotypes were revealed, GG and AG. This SNP was significantly associated with growth traits in Attappady Black goats, which is maintained as meat breed in Kerala. Results from this study demonstrated higher performance of GG animals for growth traits. The association of IGF1 gene with these traits emphasizes the importance of caprine IGF1 as a candidate gene for growth traits in goat breeding.

Relative abundance of tissue mRNA and association of the single nucleotide polymorphism of the goat NGF gene with prolificacy.

Nerve Growth Factor (NGF) promotes the development of pre-antral ovarian follicles through ovarian innervations and regulation of ovarian response to gonadotropins. The present study was conducted to study the tissue gene expression profile, to characterize the genetic variants, find associations of the NGF gene with prolificacy in the prolific Malabari and less prolific Attappady Black goats because NGF has an important role in reproduction by augmenting ovarian folliculogenesis. Relative abundance of NGF mRNA was greatest in reproductive tissues signifying its role in reproduction. The PCR-SSCP analysis of a 251bp fragment of Exon 3 of the NGF gene from the 277 goats revealed four diplotypes (EE, EF, FF and EG) with respective frequencies of 0.76, 0.22, 0.01 and 0.01. Sequencing of the representative samples revealed one synonymous and one novel non synonymous mutations (g.705G>A and g.715C>T). Statistical analysis indicated that the SNP g.705G>A was associated with litter size in Attappady Black goats (P<0.05) and a PCR-RFLP was designed using the restriction enzyme, BpiI, for rapid screening of the SNP. The results of the present study suggest that the NGF gene is a primary candidate gene affecting prolificacy in goats and may be used for Marker Assisted Selection (MAS) in goats, especially in lowly prolific Attappady Black goats.

Nerve Growth Factor gene ovarian expression, polymorphism identification, and association with litter size in goats.

The Nerve Growth Factor (NGF) plays an important role in reproduction by augmenting folliculogenesis. In this study, the coding regions of caprine NGF gene were analyzed to detect single-nucleotide polymorphisms (SNPs), their association with litter size, and the relative ovarian expression of NGF gene in the two indigenous goat breeds of South India viz., the prolific Malabari and less-prolific Attappady Black. The sequence analysis of the third exon containing the entire open reading frame of NGF gene was observed to be of 808 bp with one nonsynonymous mutation at 217th position. Later, polymerase chain reaction (PCR) was performed to amplify a region of 188 bp covering the region carrying the detected mutation. The genomic DNAs from the goats under study (n = 277) were subjected to PCR and single strand conformation polymorphism (SSCP). On analysis, four diplotypes viz., AA, AB, AC, and AD were observed with respective frequencies of 0.50, 0.22, 0.27, and 0.01. Sequencing of the representative samples revealed an additional synonymous mutation, i.e., g.291C>A. Statistical analysis indicated that NGF diplotypes and the SNP g.217G>A were associated with litter size in goats (P < 0.05). Relative expression of NGF gene was significantly higher in the ovaries of goats with history of multiple than single births (P < 0.05). The results of the present study suggest the significant effect of the NGF gene on litter size in goats and identified SNPs would benefit the selection of prolific animals in future marker-assisted breeding programs. The two novel PCR-restriction fragment length polymorphisms designed, based on the detected SNPs, would help in the rapid screening of large number of animals in a breeding population for identifying individual animals with desired genetic characteristics.

Characterization of promoter sequence of toll-like receptor genes in Vechur cattle.

AIM: To analyze the promoter sequence of toll-like receptor (TLR) genes in Vechur cattle, an indigenous breed of Kerala with the sequence of Bos taurus and access the differences that could be attributed to innate immune responses against bovine mastitis. MATERIALS AND METHODS: Blood samples were collected from Jugular vein of Vechur cattle, maintained at Vechur cattle conservation center of Kerala Veterinary and Animal Sciences University, using an acid-citrate-dextrose anticoagulant. The genomic DNA was extracted, and polymerase chain reaction was carried out to amplify the promoter region of TLRs. The amplified product of TLR2, 4, and 9 promoter regions was sequenced by Sanger enzymatic DNA sequencing technique. RESULTS: The sequence of promoter region of TLR2 of Vechur cattle with the B. taurus sequence present in GenBank showed 98% similarity and revealed variants for four sequence motifs. The sequence of the promoter region of TLR4 of Vechur cattle revealed 99% similarity with that of B. taurus sequence but not reveals significant variant in motifregions. However, two heterozygous loci were observed from the chromatogram. Promoter sequence of TLR9 gene also showed 99% similarity to B. taurus sequence and revealed variants for four sequence motifs. CONCLUSION: The results of this study indicate that significant variation in the promoter of TLR2 and 9 genes in Vechur cattle breed and may potentially link the influence the innate immunity response against mastitis diseases.

Copro-PCR based detection of bovine schistosome infection in India.

Schistosomosis and amphistomosis are the two economically important and widely prevalent snail-borne trematode infections in grazing cattle of southern India. Acute infections are symptomatically similar and difficult to detect by routine microscopy for eggs. The present study was directed towards the development of a copro-polymerase chain reaction (copro-PCR) for detection of bovine schistosome species, using custom-designed primers targeting 18S and 28S ribosomal RNA as well as mitochondrial DNA. The study demonstrated the enhanced diagnostic specificity of mitochondrial DNA markers over ribosomal RNA genes as genus-specific probes to detect schistosomes. We developed a sensitive PCR assay using primers designed from mitochondrial DNA sequences targeting the partial rrnl (16S rRNA), tCys (transfer RNA for cysteine) and partial rrnS (12S rRNA) genes of Schistosoma spindale to specifically detect schistosome infection from faecal samples of naturally infected bovines. The salient findings of the work also throw light on to the high similarity of the ribosomal RNA gene sequences of schistosomes with those of Gastrothylax crumenifer and Fischoederius elongatus, the most prevalent pouched amphistomes of the region. Further investigation has to be directed towards unravelling the complete gene sequences of 18S and 28S ribosomal RNA as well as mitochondrial DNA sequences of amphistome isolates from India.

High salt method: a simple and rapid procedure for isolation of genomic DNA from buffalo (Bubalus bubalis) white blood cells.

Three different methods, namely guanidine hydrochloride, phenol-chloroform extraction and high salt method, were compared in order to develop a simple method for the extraction of high yield of DNA from buffalo blood suitable for genome analysis. Both phenol-chloroform and high salt methods produced good yields of high molecular weight DNA as determined by agarose gel electrophoresis. The yield and quality of DNA extracted by high salt method was comparable to that of phenol-chloroform method. The mean yields of DNA from 10 ml of whole blood extracted by either the phenol-chloroform or the high salt methods were 446.16 micrograms (SE = 26.68) and 432.83 micrograms (SE = 19.34) respectively. The DNA obtained from both methods was suitable for conventional as well as polymerase chain reaction (PCR) based restriction fragment length polymorphism (RFLP) studies. Extraction using the guanidine hydrochloride method resulted in a gelatinous material that failed to resuspend in TE buffer. The high salt method is quick and reliable and can be routinely used for the extraction of DNA from buffalo samples instead of phenol-chloroform extraction which is hazardous and time-consuming.

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.

Induction of acrosome reaction in fresh and frozen-thawed bovine spermatozoa by platelet activating factor.

In vitro capacitation has been induced in fresh and frozen spermatozoa of Karan Swiss (KS) and Karan Fries (KF) crossbred cattle by using a phospholipid-platelet activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine). The capacitation was monitored by examining acrosome reaction (AR) and motility at various levels of platelet activating factor (PAF) and at the end of each incubation time. On an average, with the increase in incubation time, there was a gradual decrease in motility and increase in acrosome reaction in both fresh as well as frozen spermatozoa. As PAF level increased, the motility of fresh sperms decreased and their acrosome reaction increased dramatically. However, in frozen-thawed semen, the motility remained almost the same and increase in AR of frozen spermatozoa was not pronounced. PAF level of about 100 microM was observed to be most optimum as at this level AR improved significantly without much loss of motility.