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ThesisItem Open Access A COMPARATIVE STUDY ON CYTOGENETIC PROFILE OF CROSSBRED AND INDIGENOUS PIGS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2017-02) HARSHINI, V; SAKUNTHALA DEVI, K(MAJOR); PUNYA KUMARI, B; SURESH, JABSTRACT: The present analysis was carried out on 30 blood samples of LWY crossbred pigs (15 females and 15 males) maintained at All India Coordinated Research Project on pig (AICRP), College of Veterinary Science, Tirupati, Chittor District of Andhra Pradesh and on 30 blood samples of indigenous pigs (15 females and 15 males) maintained by the farmers in and around Tirupati to study the cytogenetic profile of both the breeds and their morphometric measurements using short term leukocyte culture technique. The diploid chromosome number (2n) in both the breeds was 38 with XX and XY complement in females and males respectively and a fundamental number of 64 as in exotic pigs. Among 18 pairs of autosomes, first 5 pairs were sub-metacentric followed by 6th and 7th pair as sub-telocentric, next 5 pairs (8th to 12th pairs) were metacentric and remaining six pairs (13th to 18th) were telocentric. Among these one pair of allosomes i.e. X-chromosome was large metacentric, while Y-chromosome was metacentric and smallest chromosome of the karyotype. The least squares mean relative length of autosomes in LWY crossbred pigs ranged from 2.79±0.02 to 10.83±0.05 in females and 2.81±0.05 to 10.87±0.07 in males. The least squares mean relative length values for non-descript pigs ranged from 2.84±0.03 to 11.01±0.07 in females and 2.86±0.03 to 11.04±0.05 in males. The relative contribution of X-chromosome in LWY crossbred female and male pig was 4.67±0.01 and 4.65±0.04, whereas the values were 4.82±0.03 and 4.80±0.04 in non-descript pigs respectively. The mean relative contribution of Y-chromosome were 2.05±0.10 and 2.19±0.13 in LWY crossbred and non-descript pigs respectively. Genetic group had significant effect on the relative length of all autosomes and allosomes except 17th and 18th chromosomes, whereas sex had no significant effect on relative length of all chromosomes in both the breeds. The overall mean arm ratio of 12 pairs of autosomes ranged from 1.01±0.03 to 2.45±0.03 and 1.02±0.01 to 2.43±0.02 in LWY crossbred females and males respectively, whereas these values varied from 1.03±0.02 to 2.51±0.06 in females and 1.04±0.06 to 2.52±0.03 in males of non-descript pigs. The mean arm ratio values of X-chromosome was 1.15±0.04 in females and 1.13±0.03 in males of LWY crossbred pigs, while the respective mean values in non-descript pigs were 1.14±0.03 and 1.12±0.03 respectively. The arm ratio values of Y-chromosome were 1.17±0.02 and 1.18±0.02 in LWY crossbred and non-descript pigs. The mean centromeric indices of autosomes (1 to 12 pairs) differed from 0.50±0.04 to 0.71±0.02 and 0.52±0.03 to 0.72±0.03 in LWY crossbred female and male pigs, whereas it ranged from 0.51±0.01 to 0.82±0.01 and 0.53±0.05 to 0.83±0.02 respectively in non-descript pigs. The X-chromosome had mean centromeric index values of 0.53±0.03 and 0.52±0.03 in LWY crossbred female and male pigs, while the value for Y-chromosome was 0.56±0.02. The mean centromeric index values of X-chromosome in non-descript pigs were 0.52±0.02 and 0.54±0.02 in females and males, whereas for Y-chromosome, it was recorded as 0.55±0.01 respectively. The average morphological index value of autosomes ranged from 2.43±0.06 to 7.29±0.11 in females and 2.41±0.05 to 7.31±0.12 in males of LWY crossbred pigs, while X-chromosome had 5.85±0.05 and 5.84±0.05 respectively. The mean values for autosomes ranged in non-descript pigs were 2.42±0.02 to 7.96±0.14 and 2.44±0.04 to 7.98±0.13 in females and males, whereas X-chromosome had an index value 6.01±0.02 and 5.99±0.03 respectively. The values for Y-chromosome were 2.01±0.02 and 2.09±0.01 in LWY crossbred and non-descript pigs. In the present investigation with 128 metaphase spreads, a total of 311 NORs were detected in LWY crossbred and non-descript pigs. The mean number of NORs per metaphase was 2.67 and 2.20 in both breeds respectively. The highest frequency of metaphases (47.61% and 81.54%) had 2 number of NORs, whereas lowest frequency of metaphases (7.94% and 3.07%) had 4 number of NORs. The percentage share of silver deposits on 10th pair of chromosome accounts for 96.67 and 68.33 in LWY crossbred and non-descript pigs, whereas on 8th chromosome pair the percentage share of deposits were 44.16 and 51.67 respectively. The outcome of present study have revealed that the morphology, number and various morphometric measurements of the chromosomes in both the breeds were analogous to those reported in literature for various pig breeds like Canadian Lacombe, Landrace, Duroc, Thai wild boar, Nigerian indigenous pig, Niang Megha pig, Korean native pig, Polish Landrace, Hampshire, Large White Yorkshire, Turkish sus scrofa. Therefore, the efforts should be undertaken to recognize the non-descript pigs as a distinct breed of Andhra Pradesh.ThesisItem Open Access A STUDY ON MORPHOLOGICAL AND GENETIC VARIATION IN NELLORE PALLA SHEEP(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2017-01) SAI HARINI, K; Punya kumara, B(MAJOR); Vinoo, R; Suresh Babu, DABSTRACT: The southern peninsular region of India is home to fourteen descriptive mutton breeds, of which Nellore is the country's tallest sheep, predominantly distributed in areas of Nellore and Prakasam districts of Andhra Pradesh. Based on coat colour three varieties were seen: Jodipi, Brown (Dora) and Palla. Nellore Jodipi is predominant in its population followed by Brown and Palla. Nellore palla sheep are confined to only certain areas of Nellore district and very scanty information is available on the performance of the breed. The population of Nellore Palla is under threat (<1500). Hence, genetic and phenotypic evaluation of the breed is important not only from conservation point of view, but also to assess the level of inbreeding in the population. The present study was carried out to figure out productive and reproductive performance in Nellore Palla sheep under field conditions and also to assess the genetic structure of Nellore Palla sheep at molecular level using twenty four microsatellite markers recommended by ISAG/FAO for genetic diversity studies in sheep. A total of 347 sheep of different age groups were used to study the morphometric measurements of Nellore palla sheep and 50 blood samples from unrelated sheep flocks were used for molecular studies. The overall least squares means of body weights (kg), height at withers, chest girth, paunch girth and body length were 35.18 ± 0.27, 78.47 ± 1.55, 80.82 ± 0.66, 80.93 ± 1.37 and 69.90 ± 0.83 at 2- teeth age, 39.65 ± 0.33, 82.45 ± 0.98, 84.57 ± 0.72, 84.11 ± 1.34 and 72.23 ± 1.02 at 4- teeth age, 41.18 ± 0.56, 83.63 ± 0.64, 86.32 ± 0.74, 85.88 ± 1.23 and 74.55 ± 0.81 at 6- teeth age, 45.00 ± 0.46, 85.22 ± 1.97, 87.17 ± 0.58, 86.14 ± 0.52 and 76.17 ± 0.33 at 8- teeth of age respectively. Villages and sex showed a significant (p<0.01) effect at all ages and the parameters studied. The overall least squares means of age at first mating in males, age at first mating in females, age at first lambing, tupping and lambing percentages were 598.03 ± 3.02 days, 361.54 ± 1.09 days, 509.32 ± 1.00 days, 81.06 ± 1.61 and 73.37 ± 1.53 percent, respectively. The coefficient of correlations between body weights with the height at withers, chest girth, paunch girth and body length varied from 0.58(PG) to 1.00(CG) in males and 0.16(PG) to 0.69(HW) in females. A perfect phonotypic correlation (1.00) was observed for body weights and height at withers at in 8- teeth age males and for body weights and chest girth for males at 6- teeth age. All the correlations were positive and highly significant (p<0.01) in majority of the age groups studied. The standard Phenol-Chloroform method was used for isolation of DNA from 50 blood samples. The quality and quantity of DNA was checked by agarose gel electrophoresis and Nanodrop spectrophotometer, respectively. The allele frequencies, observed number of alleles (Na), effective number of alleles (Ne), observed (Ho) and expected (He) heterozygosity, within population inbreeding estimates (FIS) were computed using POPGENE32 software. A total of 189 alleles were found across the 24 microsatellite loci studied. The number of alleles at each locus ranged from five (BM6506, CSSM31 and HUJ616) to a maximum of thirteen (BM827 and OARHH64) with a mean of 7.8 alleles across all loci. The allele sizes ranged from a minimum of 81 bp (OarJMP8) to a maximum of 258 bp in MAF214, while, the allele frequencies ranged from a minimum of 0.010 (BM827, INRA63, OarFCB48 and OarHH64) to a maximum of 0.40 (OarFCB128). The mean effective number of alleles was 6.6452 ± 0.3933 which was less than the observed number of alleles and ranged from 3.4916 (CSSM31) to 11.574 (BM827). The observed heterozygosity (Ho) and expected heterozygosity (He) ranged from 0.00 (BM1314, BM6506, BM8125, HUJ616, OARCP20, OARFCB128, OARJMP8) to 0.16 (OARHH64) and from 0.7208 (CSSM31) to 0.9228 (BM827) with an overall value of 0.0626 ± 0.010 and 0.8361 ± 0.009, respectively. All the twenty four microsatellite loci were found to be highly polymorphic and the PIC values ranged from 0.6616 (CSSM31) to 0.9136 (BM827) with a mean of 0.8171. The FIS values were found positive and ranged from 0.8201 (OARHH64) to 1.000 (BM1314, BM6506, BM8125, HUJ616, OARCP20, OARFCB128, OARJMP8). The mean FIS value across the twenty four loci observed was 0.9257. The χ2 test revealed that all the twenty four loci deviated significantly from Hardy- Weinberg Equilibrium.ThesisItem Open Access AMPLIFICATION, SEQUENCING AND BIOINFORMATIC CHARACTERIZATION OF TRANSMEMBRANE PROTEIN 95 GENE (TMEM95) IN MURRAH BUFFALO (Bubalus bubalis)(2017-01) SHIREESHA, SAMPATHIRAO; SUDHAKAR, K(MAJOR); VINOO, R; VENKATA SESHAIAH, ChABSTRACT: TMEM95 gene is known to be involved in idiopathic male subfertility in cattle. It is required for the integrity of sperm plasma membrane and thus has a role in fertilization. The present study was aimed at bioinformatic characterization of the Transmembrane protein 95 (TMEM95) gene in Murrah buffalo. The TMEM95 gene was amplified and sequenced in Murrah. Analysis with CodonCode Aligner revealed three heterozygous positions in Murrah sequence at positions 1284 (T and G), 1460 (C and A) and 1897 (G and A) with respect to the 2631 bp reference fragment from the Hereford cattle genome assembly. A 2 bp deletion (at 937 bp) was observed in Murrah buffalo which is causing frame shift mutation. The isoforms were predicted using GeneWise, a comparative gene annotation tool and found that the 2bp deletion resulted in the truncation of isoforms 1, 3 and 4 and were unlikely to form. The transmembrane topology and signal peptide were predicted using Phobius program, and it was observed that the isoforms 1, 2 and 3 doesn’t have any transmembrane domains but the isoform 5 has two transmembrane domains. Analysis of isoforms with MotifScan predicted the presence of Casein kinase II phosphorylation site, which has an important role in sperm morphology. Leucine zipper pattern, N-myristoylation site, protein kinase C phosphorylation site, CHRD domain profile, N-glycosylation site and HIT zinc finger motifs were also predicted. Divergence analysis was carried out for the TMEM95 region across different mammalian species and less divergence was observed among cattle, bison, yak and mithun. Buffalo and sheep are moderately divergent from cattle. Based on the sequence analysis and functional prediction, it was concluded that the TMEM95 gene in Murrah buffalo is likely to have function in male fertility. However there may be some species-specific differences with respect to their function between the two speciesThesisItem Open Access A STUDY ON GENETIC POLYMORPHISM OF MYOSTATIN (GDF8) GENE IN NELLORE AND MACHERLA BROWN SHEEP(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2017-01) VENKATA PRANEETH, D; Vinoo, R(MAJOR); Muralidhar, M; Jagadeeswara Rao, SABSTRACT : The primary objective in small animal production is to have higher meat production. The present study is aimed at understanding natural variation of GDF8 locus in Nellore and Macherla Brown sheep genetic groups by using PCR-RFLP. DNA was isolated from 100 sheep blood samples using a modified high salt method. Two sets of primers were designed to amplify regions of exon 3 and intron 1 of GDF8 gene. A total of 100 blood samples from the three sheep populations were used for amplification of exon 3 and 65 samples were used for amplification of intron 1. The PCR products were subjected to RFLP with HaeIII restriction enzyme for exon 3 and HpyCH4V restriction enzyme for intron 1. Based on different genotypes obtained the genotypic and allelic frequencies were determined. In the present study exon 3 is monomorphic in all the samples and hence, further analysis could not be performed on this locus. The intron 1 region was polymorphic representing HH, Hh, hh genotypes. The heterozygosity values for intron 1 region were 0.48, 0.49 and 0.47 for Nellore Jodipi, Nellore Brown and Macherla Brown genetic groups respectively. The PIC values for Nellore Jodipi, Nellore Brown and Macherla Brown are 0.37, 0.37 and 0.36 respectively, suggesting considerable amount of variation exist in these populations. The allelic frequencies of Nellore Jodipi, Nellore Brown and Macherla Brown were 0.59 and 0.41, 0.56 and 0.44, 0.62 and 0.38 for H and h alleles, respectively. Diversity estimates (FIS) were negative for the three populations indicating that there is no differentiation among the three populations. The test for Hardy Weinberg Equilibrium indicated that the three populations are departing from the equilibrium assumptions. No significant (P>0.05) association of a genotype with body weights at different ages was observed in the present study in both the genetic groups of Nellore Jodipi and Nellore Brown. A new PCR-RFLP marker is designed for intron 1 in this study which is found to be polymorphic and useful in population studies.ThesisItem Open Access AMPLIFICATION, SEQUENCING AND BIOINFORMATIC CHARACTERIZATION OF TRANSMEMBRANE PROTEIN 95 GENE (TMEM95) IN MURRAH BUFFALO (Bubalus bubalis)(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI – 517 502. (A.P) INDIA, 2017-01) SHIREESHA, SAMPATHIRAO; SUDHAKAR, K (Major); VINOO, R; VENKATA SESHAIAH, ChABSTRACT: TMEM95 gene is known to be involved in idiopathic male subfertility in cattle. It is required for the integrity of sperm plasma membrane and thus has a role in fertilization. The present study was aimed at bioinformatic characterization of the Transmembrane protein 95 (TMEM95) gene in Murrah buffalo. The TMEM95 gene was amplified and sequenced in Murrah. Analysis with CodonCode Aligner revealed three heterozygous positions in Murrah sequence at positions 1284 (T and G), 1460 (C and A) and 1897 (G and A) with respect to the 2631 bp reference fragment from the Hereford cattle genome assembly. A 2 bp deletion (at 937 bp) was observed in Murrah buffalo which is causing frame shift mutation. The isoforms were predicted using GeneWise, a comparative gene annotation tool and found that the 2bp deletion resulted in the truncation of isoforms 1, 3 and 4 and were unlikely to form. The transmembrane topology and signal peptide were predicted using Phobius program, and it was observed that the isoforms 1, 2 and 3 doesn’t have any transmembrane domains but the isoform 5 has two transmembrane domains. Analysis of isoforms with MotifScan predicted the presence of Casein kinase II phosphorylation site, which has an important role in sperm morphology. Leucine zipper pattern, N-myristoylation site, protein kinase C phosphorylation site, CHRD domain profile, N-glycosylation site and HIT zinc finger motifs were also predicted. Divergence analysis was carried out for the TMEM95 region across different mammalian species and less divergence was observed among cattle, bison, yak and mithun. Buffalo and sheep are moderately divergent from cattle. Based on the sequence analysis and functional prediction, it was concluded that the TMEM95 gene in Murrah buffalo is likely to have function in male fertility. However there may be some species-specific differences with respect to their function between the two species.ThesisItem Open Access A STUDY ON GENETIC POLYMORPHISM OF MYOSTATIN (GDF8) GENE IN NELLORE AND MACHERLA BROWN SHEEP(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI – 517 502. (A.P) INDIA, 2017-01) VENKATA PRANEETH, D; Vinoo, R(Major); Muralidhar, M; Jagadeeswara Rao, SABSTRACT : The primary objective in small animal production is to have higher meat production. The present study is aimed at understanding natural variation of GDF8 locus in Nellore and Macherla Brown sheep genetic groups by using PCR-RFLP. DNA was isolated from 100 sheep blood samples using a modified high salt method. Two sets of primers were designed to amplify regions of exon 3 and intron 1 of GDF8 gene. A total of 100 blood samples from the three sheep populations were used for amplification of exon 3 and 65 samples were used for amplification of intron 1. The PCR products were subjected to RFLP with HaeIII restriction enzyme for exon 3 and HpyCH4V restriction enzyme for intron 1. Based on different genotypes obtained the genotypic and allelic frequencies were determined. In the present study exon 3 is monomorphic in all the samples and hence, further analysis could not be performed on this locus. The intron 1 region was polymorphic representing HH, Hh, hh genotypes. The heterozygosity values for intron 1 region were 0.48, 0.49 and 0.47 for Nellore Jodipi, Nellore Brown and Macherla Brown genetic groups respectively. The PIC values for Nellore Jodipi, Nellore Brown and Macherla Brown are 0.37, 0.37 and 0.36 respectively, suggesting considerable amount of variation exist in these populations. The allelic frequencies of Nellore Jodipi, Nellore Brown and Macherla Brown were 0.59 and 0.41, 0.56 and 0.44, 0.62 and 0.38 for H and h alleles, respectively. Diversity estimates (FIS) were negative for the three populations indicating that there is no differentiation among the three populations. The test for Hardy Weinberg Equilibrium indicated that the three populations are departing from the equilibrium assumptions. No significant (P>0.05) association of a genotype with body weights at different ages was observed in the present study in both the genetic groups of Nellore Jodipi and Nellore Brown. A new PCR-RFLP marker is designed for intron 1 in this study which is found to be polymorphic and useful in population studies.