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ThesisItem Unknown MITOCHONDRIAL DIVERSITY AND PHYLOGENETIC STATUS OF PUNGANUR CATTLE(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-11) DEEPTHI, CHANDAKA; MURALIDHAR, M (MAJOR); VINOO, R; SUDHAKAR, KCattle in India played an important role in the rural economy as supplementing family incomes and generating gainful employment in the rural sector, particularly among the landless laborers, small and marginal farmers. Among all the cattle breeds the dwarf breeds are more preferred among farmers as they can be maintained with less fodder, high disease resistant ability, high survivability in adverse environmental conditions and optimum milk production. Punganur is one such dwarf breed present in Andhra Pradesh. According to the available literature, the Punganur cattle were crossed with Mysore cattle and Kerry breed with time. Punganur cow has become a status symbol in recent years as wealthy livestock farmers began buying it, due to its cultural value. However, the present day Punganur cattle description was not in accordance with the available literature. With this in mind the objective of the present research is to study the genetic variation and phylogenetic status of Punganur cattle. using mitochondrial DNA D-loop region. Blood samples were collected from 28 unrelated Punganur animals which were further grouped as 14 Punganur animals and 14 Punganur type animals based on literature available. Unrelated Blood samples were also collected from Ongole, Hallikar, Vechur, Malnad Gidda and Bargur. Genomic DNA was isolated and 1024 bp mitochondrial D-loop region was amplified and sequenced. Sequence of Indian cattle breeds were obtained from NCBI database and total of 212 sequences were aligned and trimmed to have a final length of 912 bp. The measures like polymorphism, divergence, gene flow and genetic differentiation were estimated using DnaSP software. A phylogenetic tree was constructed using Neighbour joining method by MEGA software. The tree topology was tested using 1000 bootstrap replications. The results of polymorphism and divergence analysis disclosed the existence of high within breed polymorphism compared to between breed polymorphism which is in accordance with other cattle breeds of India. The findings of gene flow and genetic differentiation estimates concluded the presence of low differentiation and presence of gene flow between Punganur and Punganur type animals. The phylogenetic analysis indicated that, there is no differentiation between Punganur and Punganur type animals.ThesisItem Open Access MORPHOMETRIC AND MOLECULAR STUDIES IN PUNGANUR AND PUNGANUR TYPE OF CATTLE(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-11) SURENDRA, KORRAPATI; SUDHAKAR, K (MAJOR); VINOO, R; JAGADEESWAR RAO, SThe present study was conducted on 255 animals belonging to Punganur breed and “Punganur type” cattle in order to establish the morphometric traits and to screen those two populations for known putative dwarf mutations. The coat colour in Punganur cattle was predominantly white (47%) followed by red (19%), combination of colours. Punganur animals were characterized by black muzzle, pigmented eye lids, black coloured hoofs, and flat forehead profile short ears horizontal in orientation with pointed tips. In majority of Punganur cattle horns are curved attaining crescent shape (71.6%) when fully grown, horns usually emerged sideward direction from top of the poll and oriented upwards, slightly forward or backward. Stumpy horns (17.9%), horns upward with sideward orientation (5.2%), and downwards (5.2%) were also observed. Small to medium sized cervico thoracic hump was observed. Udder was small and rounded or bowl in shape and having cylindrical teats. Teat tip was rounded in majority of animals. “Punganur type” animals were predominantly with white coat colors but black and broken colours are not uncommon. The pink and white muzzle and animals with loose horns and horns with irregular pattern are observed in this population. The mean height at withers in adult “Punganur type” animals was 96.75±1.03 cm and is significantly (p<0.05) high compared to Punganur animals (93.93±1.18 cm). The farm bred animals mean height at withers was 94.85±0.80 and in field animals was 95.83±1.44 cm, respectively. Least squares means for chest girth in adult Punganur was 132.28±1.78 and 133.95±1.56 cm in “Punganur type” animals. The corresponding value in farm and field animals were 122.93±1.20 and 143.30±2.17 cm respectively, which differed significantly (p<0.01). The mean tail length with switch in adult Punganur and “Punganur type” animals were 87.35±1.65 and 87.49±1.45 cm respectively. All the animals were screened for mutations in PRKG2, ACAN and EVC2 genes which are responsible for dwarfism in cattle. All the screened animals in both Punganur and “Punganur type” were of wild type without any mutations.ThesisItem Open Access MOLECULAR CHARACTERIZATION OF NELLORE PALLA SHEEP AND ITS' GENETIC DIVERGENCE WITH OTHER VARIETIES OF NELLORE SHEEP(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-11) JAYA MADHURI, BHUMIREDDY; SUDHAKAR, K (MAJOR); MURALIDHAR, M; VENKATA SESHAIAH, Ch.The Indian subcontinent is conferred with a rich livestock genetic diversity. Forty three sheep breeds are registered in India representing abundant genetic resources. Of these, Nellore sheep breed is harboured in Andhra Pradesh and is the only recognised breed of the state. Three phenotypically distinct varieties viz. Palla, Brown and Jodipi are present in the Nellore breed based on their coat colour. The present study is carried out to genetically characterize the Nellore Palla sheep and document the genetic divergence among the three varieties of the Nellore sheep breed. Thirty two unrelated individuals from the hometract of Palla, nine each of Brown and Jodipi were sampled for the molecular studies. Fluroscent microsatellite markers were used to genetically characterize Palla population. The analysis revealed a total of 201 alleles across the 26 loci studied. The mean observed and effective number of alleles per locus was 7.73 and 5.82 respectively. The overall observed and expected heterozygosity across 26 loci was 0.52 and 0.69 respectively. The mean FIS value in the sampled population was 0.24 with a range varying between -0.24 (MAF209) to 0.77 (INRA063). The mean PIC value and the Shannon’s index value across the 26 loci was 0.643 and 1.49 respectively. The study concluded a considerable amount of genetic diversity in the Nellore Palla population. The genetic divergence among the three varieties was analyzed using mitochondrial control region. DNA was isolated from the individuals of three varieties and 885 bp region of the mtDNA control region was amplified and sequenced. The analysis for polymorphism and divergence was done with DnaSP software and the results concluded a high polymorphism with in the genetic groups. The genetic differentiation estimates revealed no genetic differentiation among the three varieties of Nellore sheep breed. The phylogenetic analysis indicated that all the haplotypes of the three varieties of Nellore were clustering with lineage A except for one haplotype of Palla variety which was clustering with lineage B.ThesisItem Open Access CHARACTERIZATION AND EVALUATION OF PHYLOGENETIC STATUS OF KENGURI, MOULI AND YALAGA SHEEP OF KARNATAKA(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-09) SIDDALINGSWAMY HIREMATH; VINOO, R(MAJOR); APPANNAVAR, M.M.; MURALIDHAR, M; VENKATA SESHAIAH, Ch.; RAMANI PUSHPA, R.N.Karnataka state hosts five well adapted indigenous sheep breeds viz. Bellary, Kenguri, Hassan, Deccani and Mandya. Apart from these recognized breeds, there are two lesser known genetic groups viz., Mouli and Yalaga reared by farmers in North Karnataka in Vijayapura and Bagalkote districts, respectively. The objective of the present research is to document and compare the phenotypic traits of three genetic groups, Kenguri, Mouli and Yalaga sheep of Karnataka and to study the genetic diversity existing in these genetic groups, using mitochondrial and Y-chromosome markers. Data on morphological and morphometric traits were recorded in the three genetic groups from farmer's flocks in their breeding tracts. Data was classified based on their age and sex. It was observed that the Kenguri sheep were predominantly brown with white patch on forehead. Males are horned and ewes are polled in this breed. The animals are usually with black hooves, medium long drooping ears, short tail, straight face/fore-head or nose line in males and females. The Mouli sheep were generally white with brown patches all over the body, long tail, polled in both sexes, long drooping ears and typical convex face or bowed fore head with Roman nose. The Yalaga sheep were white in colour, with males being horned and females were polled. They have long tail, long drooping ears, straight forehead and straight nose line. The morphometric traits namely body length, height at withers, flank width and face length were longer in Mouli sheep compared to Kenguri and Yalaga sheep. Body weight is also larger in Mouli sheep compared to Kenguri and Yalaga sheep. The trend also holds good for age wise analysis among the three breeds. The genetic diversity and divergence among the three breeds were assessed using mitochondrial DNA. Metadata of fourteen Indian, Chinese, Caucasian breeds and wild Mouflon were also used for the analysis. The wild ancestor of sheep i.e. urial mitochondrial D-loop region was used as an outgroup for phylogenetic analysis. Mitochondrial DNA analysis indicated that there is high variation within these genetic groups but low differentiation between the genetic groups. Also there is high rate of gene flow among the three genetic groups, particularly between Mouli and Yalaga breeds. The observation is contrasting to the phenotypic differences between the two breeds. The phylogenetic analysis indicated that three genetic groups were predominantly clustered in clade A except one Kenguri sheep which is clustered in clade B. To understand paternal origin of the three breeds, the 611 bp promoter region of SRY gene was amplified and sequenced in the three genetic groups. Orthologous region for a total of 17 sheep representing domestic and wild sheep were obtained from the NCBI database. The three sheep breeds under study didn’t reveal any variation in the promoter region of the SRY chromosome, suggesting they are likely to have same paternal origin. Phylogenetic analysis of sequences indicated a plausible paternal lineage from European or Armenian Mouflon for the three genetic groups. The molecular clock analysis based on mitochondrial control region indicated that the Karnataka breeds must have diverged in the lineage A about 20000 yrs ago. A similar number of years ago the Kenguri individual in clade B also might have diverged. This estimate is slightly higher than the time of sheep domestication suggesting possibility of multiple domestication events in the Indian subcontinent. The results in the present study indicate that phenotypically the three genetic groups are different. At molecular level the Mouli and Yalaga sheep are not differentiated based on the mitochondrial DNA analysis. The paternal source for the three genetic groups is likely from the European or Armenian Mouflon.ThesisItem Unknown GENETIC POLYMORPHISM AND EXPRESSION PROFILE OF BOVINE TRANSMEMBRANE PROTEIN 95 GENE (TMEM95)(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-09) SIDDHARTHA, BATCHU; SUDHAKAR, K(MAJOR); MURALIDHAR, M; ANITHA, ATMEM95 gene is known to be involved in idiopathic male subfertility in cattle. The present study was aimed at identifying polymorphism of TMEM95 gene in Ongole, Jersey, HF, Jersey crossbred, HF crossbred and Murrah bulls and to study tissue specific expression profile of the gene in buffalo. A total of 270 blood samples were collected from bulls across all the genetic groups maintained at different Frozen Semen Bull Stations maintained in Andhra Pradesh and University Livestock Research Station. The available sequences of Murrah buffalo, Mediterranian buffalo, Ongole cattle, Gir cattle, Herford cattle were used for identification of putative SNP variants. Three SNP loci namely, SNP1100 (rs209226692), SNP1275 and SNP2192 (rs41904693) were identified for genotyping. RFLP was employed to detect the polymorphic patterns to genotype the bulls for each locus. TMEM95/NlaIII digestion of 600 bp amplicon in Jersey crossbred and Ongole bulls resulted in all the three genotypes, whereas in Jersey, HF, and HF crossbred resulted in two genotypes for the C/T polymorphism. In none of the exotic breed cattle bulls studied CC genotype was observed. TMEM95/AvaII polymorphism at rs41904693 in all the cattle genetic groups revealed three genotypes i.e. TT, GG and TG. The frequency of G allele was observed to be more except in HF bulls. The TMEM95/TaqI genotyping revealed that all the cattle genetic groups are monophorphic (TT). The fixation of T allele at rs209226692 and rs41904693 loci in Murrah bulls was observed, but polymorphic at SNP1275. The effective number of alleles (Ne) across the studied genetic groups ranged between 1.069 (Jersey) and 1.471 (HF). In Ongole bulls the same value was much higher 1.957. The PIC values varied from 0.057 (Jersey) to 0.370 (Ongole) for SNP1100 and 0.269 (HF) to 0.374 (Jersey) for SNP2192 among the different cattle populations. The PIC value for SNP1275 estimated in Murrah was 0.360. The FIS values were negative ranging from – 0.0345 to – 0.2875 for SNP1100 in the cattle genetic groups except Jersey crossbred cattle. At SNP2192 the HF crossbred and Ongole were with negative FIS values. The only estimated locus in Murrah bull population was SNP1275 which showed a negative FIS value. Expression of TMEM95 gene was studied in testis and brain to understand the functional role of TMEM95 gene in bovine and bubaline male fertility. Seven primers were designed spanning various intron exon boundaries, that could differentiate the isoform that is being expressed in those tissues. Total RNA was isolated from testis and brain tissues. The successful amplification by all the primer combinations indicated the expression of all the isoforms (X1 to X7) in testis of cattle, but not in brain tissue. In buffalo, the primer combinations were amplified in testis. No amplification is observed in the brain tissue of buffalo with any of the primer combinations. However, the expression profile has not matched with any of the isoforms that are predicted in buffalo using orthologous information from cattle.ThesisItem Open Access GENETIC ANALYSIS OF PRODUCTIVE AND REPRODUCTIVE TRAITS IN FARM BRED NELLORE JODIPI SHEEP(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-07) BHAKTHAVATCHALAM, S; PUNYA KUMARI, B (MAJOR); SUDHAKAR, K; JAGADEESWARA RAO, S; RAMANI PUSHPA, R.N.The data collected on 5008 production records and 2965 breeding records of Nellore Jodipi sheep maintained at the Network Project on Sheep Improvement, Livestock Research Station Palamaner, Chittoor district was utilized in the present investigation to study the effect of various factors including inbreeding on productive and reproductive traits and also to predict and compare the breeding values by using Least Squares (LS), Best Linear Unbiased Prediction (BLUP) and Restricted Maximum Likelihood (REML) methods. The overall least squares means for BW (Birth Weight), 3M (3 Months), 6M (6 Months), 9M (9 Months) and 12 M (12 Months) body weights were 3.03 ± 0.01, 13.29 ± 0.07, 17.86 ± 0.10, 21.34 ± 0.12, 24.96 ± 0.15 kg, respectively. Period, season, sex and parity had significant influence (P<0.01) on all the growth traits under study. The overall least square means for AGR i.e. from birth to 3, 3 to 6, 6 to 9, 9 to 12 and 3 to 12 months were 114.95 ± 0.76, 50.01 ± 0.65, 38.50 ± 0.64, 38.00 ± 0.72 and 40.19 ± 0.46 gms, respectively. Period and sex had significant influence on AGR for all age intervals. Both pre-weaning AGR and post-weaning AGR’s were significantly (p < 0.01) influenced by season of birth except post-weaning AGR 3-12 months. The overall least squares means for age at first lambing (AFL) in Nellore Jodipi sheep was 794.2 ± 6.9 days. Period of birth of ewe but not season of birth was found to be significant on AFL. The overall least squares means for lambing interval was 371.6 ± 11.9 days. Parity of ewe at lambing was found to be significant. The overall least squares mean lambing percentage (ewe covered) was 63.1 ± 2.30. Contemporary Group (CG) at breeding, but not parity of ewe at lambing had a highly significant influence on lambing percent. The mean generation interval, inbreeding coefficient (F), proportion of population inbred, individual increase in inbreeding, equivalent number of generations (EqGi) and effective population size based on birth year were 3.37y, 0.97 %, 31 %, 0.48 %, 2.54 and 1260.52, respectively. The regression coefficients of most of the traits on inbreeding coefficient (F), were negative and non-significant. Maximum and significant effect (p < 0.01) of ‘F’ was noticed on 9M, with a decrease of 0.075 kg of mean value per every 1 per cent increase in inbreeding. Effect of inbreeding was significant on post-weaning AGR 3-6 months. There was a decrease of 0.411 grams mean value in pre-weaning AGR, for every 1 per cent increase in ‘F’. The regression of age at first lambing on F was positive and significant. There was an increase of 5.565 days in AFL per every 1 per cent increase in F. The regression of lambing interval on F was negative (-1.223 days) and insignificant. The regression of lambing percent on F was positive and non-significant (0.001%). When inbreeding coefficient (F), included as fixed class in the analysis it exhibited highly significant effect on BW (p < 0.01), 3-6 months and 6-9 months post-weaning AGR; while significant (p < 0.05) on 3M, 12M body weights and 3-12 months post-weaning AGR. Effect of level of inbreeding on all reproductive traits was also found to be insignificant. The direct heritability estimates for BW, 3M, 6M, 9M and 12M from the best models in Nellore Jodipi sheep were 0.076, 0.194, 0.208, 0.199 and 0.136, respectively. The maternal heritability (m2), estimated from best models for body weights in which it was important, were 0.046, 0.038, 0.063 and 0.082 at 3M, 6M, 9M and 12M of age, respectively in Nellore Jodipi sheep. The estimates of h2 were moderate for pre-weaning (0.210) and all post weaning AGR (0.158 to 0.263) traits. The maternal heritability (m2), estimated from best models for the AGR traits in which it was important were 0.032, 0.025 and 0.029 for pre-weaning; post weaning 6-9 and 3-12 months AGR, respectively. The heritability estimates obtained for age at first lambing (0.082) and lambing percent (0.013) were low. For lambing interval, the estimated heritability was near to zero (0.0002). Repeatability for repeatable reproductive traits like lambing per cent (per ewe covered) was low (0.059) and for lambing interval, the value was near to zero (0.00018). All the estimates of genetic correlations among body weight traits were positive. Estimates of genetic correlations for birth weight with weight at later ages were medium (0.212 to 0.508). The correlation coefficients for 3M, 6M and 9M, with weights at later ages were medium to high (0.449 to 0.894). All the estimates for phenotypic correlations among body weight traits were positive. Birth weight had the lowest phenotypic correlation with the other traits (0.260 to 0.371), whereas, the remaining weights had moderate correlations (0.577 to 0.873). High estimates greater than 0.7 were observed for WW, 6M and 9M with subsequent ages. All the body weight traits (6M, 9M and 12M) had strong positive relationship with pre weaning AGR with medium to high range of values (0.393 to 0.855). Phenotypic correlations also followed the similar trend as genetic correlations (0.545 to 0.778). Genetic association between birth weight with age at first lambing (AFL) was positive and low in magnitude (0.07). Whereas, all body weight traits with lambing interval (LI) showed negative trend. Lambing interval recorded highest and lowest correlations with birth weight (-0.211) and 3 months body weight, respectively (-0.502). Phenotypic association between birth weight and age at first lambing (AFL) was negative and low in magnitude (-0.036). Highest value was noticed between birth weight with lambing interval (-0.031) and lowest between 3 months with lambing interval (-0.153) on phenotypic scale. The estimated overall average breeding values of sires for BW, 3M, 6M, 9M and 12 M body weights were found to be 3.09, 14.36, 19.04, 22.78, 24.73 kg by LSM; 2.96, 13.20, 18.12, 21.59 and 24.62 kg by BLUP method and 2.96,13.19, 18.11, 21.60 and 24.63 kg by DFREML methods, respectively. BLUP method was adjudged to be the most efficient method followed by LS and DFREML methods, because of its least error variance. LSM was best fitted model over the other two methods of sire evaluation based on coefficient of determination (R2), but the accuracy of BLUP was much closer to LSM than DFREML. The rank correlations among breeding values of sires estimated from different methods of sire evaluation were all high, and highly significant (P<0.01). The values ranged from 0.487 (LSM with DFREML for 9M) to 0.999 (BLUP with DFREML for BW). The rank correlations between BLUP and DFREML were greater than 0.9 for all the traits. BLUP or DFREML could select the same set of sires with almost similar ranking for unbiased estimates of breeding values for production traits. The direct genetic trends for BW, 3M, 6M, 9M and 12M were 0.003, 0.067, 0.084, 0.038 and 0.030, respectively in Nellore Jodipi sheep and the corresponding phenotypic trends were 0.010, 0.044, 0.083, 0.201 and 0.248, respectively.ThesisItem Unknown MOLECULAR GENETIC ANALYSIS OF VIZIANAGARAM SHEEP USING MICROSATELLITE MARKERS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-04) PRASANNA, BARATAM; VINOO, R(MAJOR); MURALIDHAR, M; VENKATA SESHAIAH, Ch.India is bestowed with 43 recognized sheep breeds and several unrecognized genetic groups exists in different geographic locations which are yet to be documented and registered. So far Nellore is the only sheep breed that is recognized in Andhra Pradesh. Apart from this breed, other locally adopted genetic groups exist and one popular sheep among them is in north coastal region of Andhra Pradesh, called Vizianagaram sheep. The present study was carried out to establish breed characteristics of Vizianagaram sheep and also to assess the genetic diversity at molecular level using thirty microsatellite markers recommended by FAO for genetic diversity studies in sheep. A total of 451 sheep of different age groups were used to study the morphometric characters of Vizianagaram sheep and 56 blood samples from unrelated sheep were used for molecular studies. The predominant color of these animals is white with tan/brown/black color patches on nose line, lateral face, neck/mane, perineum, back and lower extremities. The ears in this genetic group of sheep are observed to be either pendulous or tubular. Few of the animals are with rudimentary ears. Prominent occipital bone, black patches around eyes, hair on thigh, dewlap and neck/mane region are the important features of this genetic group. The overall least squares means of body weights (kg), height at withers (cm), chest girth (cm) and body length (cm) were 22.10±0.76, 64.96±0.48, 68.67±0.64 and 62.30±0.42 at 2 teeth age, 26.03±0.75, 66.86±0.74, 72.33±0.78 and 65.13±0.55 at 4 teeth age, 28.89±1.21, 67.20±0.69, 74.75±1.07 and 65.76±0.81 at 6 teeth age and 28.09±0.62, 67.22±0.40, 76.76±0.51 and 65.89±0.37 at full mouth respectively. DNA isolated from 56 Vizianagaram and 40 Nellore Jodipi blood samples was utilized for microsatellite analysis. A total of 377 and 321 alleles were observed across 30 loci in Vizianagaram and Nellore Jodipi respectively. On an average about 11-12 alleles per locus were observed in both the genetic groups. Effective number of alleles range from 1.999 to 11.314 and 1.838 to 11.220 in Nellore Jodipi and Vizianagaram sheep respectively. The allele sizes ranged from 70bp (OARFCB20) to 293bp (ILSTS11) and 70bp to 283bp (ILSTS11) in Vizianagaram and Nellore Jodipi sheep, respectively. The mean observed and expected heterozygosity value was 0.685 and 0.757 in Vizianagaram population while it was 0.689 and 0.744 in Nellore Jodipi population. PIC of the markers ranged between 0.457 (OARCP38) and 0.861 (OARFCB304) in Vizianagaram sheep while it was 0.345 (MAF214) and 0.866 (ILSTS11) in Nellore Jodipi sheep. The mean FIS estimates among the two populations, were 0.084 for Nellore Jodipi and 0.086 for Vizianagaram sheep. The Pairwise Nei’s genetic distance and identity estimates were 1.6% and 98.5% respectively. The study concluded that Vizianagaram sheep were phenotypically distinct from other breeds with low genetic differentiation at molecular level.