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ThesisItem Open Access Genetic polymorphism of major histocompatibility complex class II genes(Department of Animal Breeding, Genetics and Biostatistics, College of Veterinary and Animal Sciences, Mannuthy, 2010) Remya John, v; KAU; Raghavan, K CThesisItem Open Access Beta lactoglobulin polymorphism in goats of Kerala(Department of Animal Breeding, Genetics and Biostatistics, College of Veterinary and Animal Sciences, Mannuthy, 2010) Sudina, K; KAU; Bindu, K.AΒeta-lactoglobulin gene polymorphism and its association with various milk production traits were investigated in DNA samples isolated from blood of 20 Malabari, 25 Attappady black 30 crossbred goats of University goat farm and 45 Malabari goats from the native tract of Malabari breed. Study was conducted by PCR-RFLP using Sac II restriction endonuclease. Digestion of the amplified PCR product with Sac II restriction endonuclease revealed three genotypes (S1S1, S1S2 and S2S2) in Malabari, Attappady black and crossbred goats, indicating the presence of S1 (349 and 77 bp fragments) and S2 (426 bp fragment) alleles. The S1/S2 allele frequencies were 0.37/0.63, 0.34/0.66 and 0.18/0.82, respectively in Malabari, Attappady black and crossbred goats. The genotypes of β-LG/Sac II polymorphism were distributed according to Hardy-Weinberg equilibrium with frequencies 0.04 (S1S1), 0.60 (S1S2) and 0.36 (S2S2) in Attappady black and 0.10 (S1S1), 0.17 (S1S2) and 0.73 (S2S2) in crossbred goats under study. But the Malabari goat population under study with genotype frequencies 0.14 (S1S1), 0.46 (S1S2) and 0.40 (S2S2), was found to violate H-W equilibrium. So this population was further classified and genotypic frequencies of subpopulations like Tellicherry (0.14, 0.50, 0.36), Badagara (0.2, 0.25, 0.55) and University farm (0.09, 0.61, 0.30) populations were in Hardy-Weinberg equilibrium. From the present study a significant association could be detected between β-LG polymorphism and peak milk yield in Malabari breed but not in other groups. Malabari goats carrying S2 allele showed a significantly higher average (p<0.05) (420±74.24 ml and 501.79±46.47 ml) compared to animals homozygous for S1 allele (250±13.36 ml). A statistically significant association could not be established between β-LG polymorphism and other milk production traits. So the introduction of this polymorphism can complement current selection programme.ThesisItem Open Access Cloning and sequence analysis of the growth hormone gene in Indian elephants(Department of Animal Breeding, Genetics and Biostatistics, College of Veterinary and Animal Sciences, Mannuthy, 2009) Bhosale, R A; KAU; Aravindakshan, T VThe study was undertaken with the objectives of cloning and sequence analysis of the growth hormone gene of the Indian elephants. The growth hormone is a peptide hormone produced in the anterior pituitory. It stimulates the growth of vertebates. It is a protein hormone of about 190 amino acids, synthesized and secreted by cells called somatotrophs. Growth hormone is a major participant in the control of several complex physiologic processes, including growth and metabolism and it is also of considerable interest as a drug used in both humans and animals. The genomic DNA was isolated from blood samples and a 1712 bp fragment of the entire transcriptional unit of the GH was amplified by PCR using synthetic oligonucleotide primer pair designed based on the 5′ and 3′ flanking sequences of goat growth hormone gene. The gel purified PCR product was ligated in to the pGEM®-T Easy cloning vector and was transformed by giving heat shock to competent E. coli cells prepared by CaCl2 treatment. The recombinant clones among the transformed cells were identified by Blue–White Screening and the recombinant plasmid carrying the insert gene was isolated from the white clones by a modified SDS-alkaline lysis method. The 1.712 kb GH gene insert in the vector was sequenced by the dideoxynucleotide sequencing method with primer walking using an automated DNA sequencer. The nucleotide sequence showed 75 to 96 per cent homology with pig and 77 to 95 per cent with that of Dolphin GH genes, respectively. The exon-intron boundaries in the porcine gene occur at the codons of the amino acid residues, Gly-4 (intron 1), Phe-57 (intron 2), Ser-96 (intron 3) and Arg-150 (intron 4). The all four residues are conserved in both species and also in African elephants. This strict homology in the sites of insertion of introns suggests that the exon-intron organization of these genes was established before the divergence of these species. The positions of the exon-intron boundaries are also conserved as evidenced from similar sizes of the exons. Evidence for some homology was also seen in intron 1, which showed maximum 84 per cent similarity with giraffe. In contrast, intron 2, 3 and 4 showed no significant similarity both in length and in sequence with other animal species. The Indian elephant GH gene has an open reading frame of 648 nucleotides encoding a signal peptide of 26 amino acid residues and a mature protein of 190 amino acid residues with both NH2- and COOH- terminal phenylalanine. Alignment of this sequence with African elephant counterpart showed that 189 amino acid residues are identical with only one variant while, with pig sequence it showed 186 identical residues with four variants. The predicted secondary structure showed that the larger α-helical lobe is formed by four sections of the polypeptide chain (residues 3-34, 47-80, 110-126 and 148-172) while the smaller lobe, which encompasses a small antiparallel beta-sheet and a small irregular structure formed the remaining structure of the polypeptide chain. The predicted tertiary structure of the Indian elephant GH showed high homology with the human GH structures. Overall, the structures of Indian elephant GH gene was found to be very similar to that of African elephant and porcine reflecting their high degree of amino acid sequence identity (99 – 97 per cent).ThesisItem Open Access Evaluation and comparison of polymorphism of beta casein gene in vechur and crossbred cattle of Kerala(Department of Animal Breeding, Genetics and Biostatistics, College of Veterinary and Animal Sciences, Mannuthy, 2010) Muhammed, E M; KAU; Stephen MathewBeta casein (β-CN) is the major milk protein which imparts biological, technological and physical properties to the milk. Recently, the polymorphism of the gene at codon 67 has attracted much public health attention. A single nucleotide polymorphism (SNP) from CCT to CAT leads to an amino acid change in the mature protein from proline to histidine. Among the twelve β-CN variants identified A1 and A2 are the common types and others are very rare. A1 variant has histidine at position 67 of the amino acid sequence while A2 possess proline at this position. This single amino acid change causes the release of bioactive peptides upon gastro intestinal digestion. Morphine like opioid beta casomorphine-7 (BCM-7) thus released from A1 milk is reported to cause various illness like diabetes mellitus, heart diseases, atherosclerosis, schizophrenia and sudden infant death syndrome (SIDS). The original variant A2 does not produce BCM-7 and thus is safe for human consumption. The major taurine milch breeds such as Holstein Friesian and Ayrshire have a high frequency of A1 allele where as Channel Island breeds such as Guernsey and Jersey have more of A2 allele. It is also interesting to note that most of the Indian breeds of animals have only β-CN A2 allele. The present study was undertaken to characterise the β-CN gene polymorphism in Vechur and crossbred cattle of Kerala considering its public health importance. Blood samples were collected from 72 Vechur cattle, and 14 Kasargode Dwarf cattle available in the KAU farm and 100 crossbred cattle randomly selected from different parts of Kerala. Genomic DNA was isolated by standard phenol chloroform procedure. Beta casein A1 A2 polymorphism was analysed by Allele Specific Polymerase Chain Reaction (AS-PCR). The genotypic frequencies of A1A1, A1A2 and A2A2 were, respectively, 0, 0.40 and 0.60 in Vechur cattle, 0.32, 0.28 and 0.40 in crossbred cattle and 0, 0.79 and 0.21 in Kasargode cattle. Genotypic frequencies were not found to be in Hardy-Weinberg equilibrium in crossbred cattle while in Vechur and Kasargode cattle frequencies were found to be in equilibrium. The A1 and A2 allele frequency was 0.20 and 0.80 in Vechur cattle, 0.46 and 0.54 in crossbred cattle and 0.39 and 0.61 in Kasargode cattle. Comparison of allele frequencies revealed that there is significant variation in allele frequencies among these three groups of animals. The relationship between β-CN A1/A2 polymorphism with milk yield was also assessed. The average daily milk yield was 1.27±0.05 kg in Vechur cattle. The peak yield of crossbred cattle was 10.71±1.046 kg. The average daily milk yield for A1A2 genotype in Vechur cattle was 1.52±0.08 kg and 1.14±0.04 kg for A1A2 genotype. The t-test showed the difference in the milk yield of the two genotypes was significantly different at 5% level of significance. In crossbred cattle the peak yield (kg) of A1A1 genotype was 14.64±3.181, 8.54±0.194 for A1A2 genotype and 9.09±0.125 for A2A2 genotype. The A1A1 genotype in crossbred cattle showed significantly higher average peak yield compared to other genotypes and difference between A1A2 and A2A2 genotypes were not significantly different. From the present study it can be concluded that selection for enhancing milk production may increase the frequency of harmful A1 allele in our bovine population. So efforts should be taken to enhance the A2 allele with a view of transforming our cattle population capable of producing A2 variant of β-CN which has a global demand. The information generated in this study on the genotypes of Vechur and Kasargode dwarf cattle can be very effectively used for developing a herd/breed of cattle with A2A2 β-CN genotype.ThesisItem Open Access Construction and comparison of the efficiency of different selection indices for malabari goats(Department of Animal Breeding, Genetics and Biostatistics, College of Veterinary and Animal Sciences, Mannuthy, 2009) Rani, Alex; KAU; Raghavan, K CThe study was conducted with the objective of optimizing the genetic improvement of Malabari goat population in North Kerala by adopting an index method of selection incorporating various growth traits and peak yield of dam. Present study was undertaken on 1055 records of animals born from August 2005 to August 2008 from the farmers’ flocks of AICRP on goats for Malabari goats. Effect of non-genetic factors on body weight and body measurements at below one, three and six months of age was analyzed. Effect of centre and sex had significant influence on body weight and body measurements of all the ages studied. Type of birth had significant effect on body weight and measurements at below one and three months of age, but the same was non significant at six months of age except for body length. Year of birth had significant effect on body weights at third and sixth month, where as the effect of season was significant for body weight at below one and three months of age. But the effects of these factors on body measurements were significant at all the ages studied. Milk production in Malabari goats was recorded as peak yield and the mean peak yield recorded was 1237.62± 75.44 ml. The effects of centre, year and season of kidding had significant influence on peak yield. A high percentage of multiple births (75.71%) was recorded in the in the total population under study during the period for Malabari goats. The percentage of multiple births was higher in Tanur (79.29%) than in other centres. Heritability estimates were high for body weight and measurements at below one and six months of age and for peak yield also, whereas for three months of age they were low to moderate. Correlations between the traits were positive in almost all cases. The relative economic values of various traits were derived. Taking into consideration the heritability, genetic and phenotypic correlations as well as relative economic value, selection indices were constructed. Selection indices were developed by incorporating body weight and body measurements at below one, three and six months of age. Index with all the traits under study (I11) was expected to achieve more genetic gain in body weight at six months of age in all the groups. By taking into consideration, the rIH and aggregate economic genetic gain also, the same index was found to be the best in each group. Among the groups, the index with pooled data was considered to be most reliable and expected to achieve more gain in body weight at six months (1.490 kg) of age. Restricted selection indices by imposing restriction on body weight at below one month were also constructed. Effective restriction was possible in all the cases. The genetic advancement in all other characters due to selection of goats based on this index was comparatively less to that obtained with the index without restriction. To increase milk production along with body weight, a selection index was constructed with dam’s peak yield as a trait along with the growth traits of progeny.ThesisItem Open Access Leptin gene polymorphism in vechure and crossbred cattle of Kerala(Department of Animal Breeding, Genetics and Biostatistics, College of Veterinary and Animal Sciences, Mannuthy, 2009) Lali F, Anand; KAU; Bindu, K ARepresentative population comprising 74 heads of Vechur and 108 numbers of crossbred cattle of Kerala were investigated for leptin gene polymorphisms and their associations with milk production traits. LEP/BspE1 and BM1500 are the polymorphic markers used for PCR-RFLP and microsatellite analysis, respectively. For PCR-RFLP analysis, the amplified PCR product was digested with restriction enzyme BspE1 which revealed three genotypes in Vechur (CC, CT and TT) and two in crossbred cattle (CC and CT) indicating the presence of C (75 bp and 19 bp fragments) and T (94 bp fragment) alleles. The C/T allele frequencies were 0.82/0.18 and 0.89/0.11, respectively in Vechur and crossbred cattle. The genotypes of LEP/BspE1 polymorphism were distributed according to Hardy-Weinberg equilibrium with frequencies 0.71 (CC), 0.24 (CT) and 0.05 (TT) in Vechur and 0.79 (CC) and 0.21 (CT) with the absence of TT genotype in crossbred cattle under study. The microsatellite polymorphism at BM1500 locus was analyzed in Vechur and crossbred cattle and two novel alleles of size 126 and 132 bp were detected. Allele frequency was highest for 132 bp and in Vechur (0.40) as well as crossbred cattle (0.46) under study. The polymorphic information content of BM1500 marker in Vechur was 0.6595 while 0.656 in crossbred cattle. In the present study, the significant influence of LEP/BspE1 polymorphism on milk production traits. The T allele bearing Vechur and crossbred cattle showed 13.08% (89.77 kg) and 10.7% (466.63 kg) increase for 305 day milk yield, respectively whereas 9.96% (0.31 kg) and 8.38% (1.22 kg), respectively for average daily milk yield. A nonsignificant reduction for milk fat percentage was also observed associated with T allele in both populations. Increased body fat reserves, adipose tissue reserves, feed intake and favorable change in partitioning of nutrients in T allele possessing animals might have contributed to increased milk yield in cattle. For BM1500 marker, highest averages of 305 day milk yield and daily milk yield were observed for Vechur and crossbred cattle possessing 136 bp allele. The highest milk fat percentages were recorded in 136 bp allele bearing Vechur animals and crossbreds possessing 138 bp allele. The association of BM1500 marker with milk production traits can be established only by expanding the research to an extensive cattle population. The selection of Vechur and crossbred cattle, heterozygous for C and T alleles for LEP/BspE1 polymorphism can significantly improve milk yield. The use of molecular techniques provides new opportunities and challenges for building and using more accurately predictive and efficient selection procedures for livestock improvement.ThesisItem Open Access Genetic resistance to gastro-intestinal nematodes in goats(College of Veterinary and Animal Sciences, Mannuthy, 2010) Aparna, Sankar; KAU; Radhika, GThe present study aimed at exploring the possibility of incorporating Gastro Intestinal Nematode (GIN) resistance as a criterion for selection in goat breeding. Here Faecal Egg Count (FEC) was taken as an indicator of host resistance. Heritability estimate for FEC and correlation of FEC with Packed Cell Volume (PCV) were calculated. While assessing resistance at phenotypic level using FEC as indicator, the possibility of using PCV as an alternative to FEC was also considered. Using IFNγ intron 1 as the locus of study, an attempt was made to identify a genetic marker for GIN resistance in goats. University Livestock Farm, Mannuthy and two field centers under All India Co-ordinated Research Project (AICRP), namely - Tellichery and Badagara, formed the venue for study. In the present investigation, data from three genetic groups - Malabari, Attappady Black and Malabari crossbreds were used. Representative population in the study comprised of 150 goats. The nematode eggs encountered during the study belonged to Strongyle and Strongyloides sp. Mean FEC was 764.39 eggs per gram of the faeces (epg) with values ranging from 0.00 to 16700 epg. The value of PCV estimated ranged between 19 and 48 per cent. Effect of non-genetic factors on FEC was analyzed after logarithmically transforming the FEC. University Goat & Sheep farm, Mannuthy recorded the least FEC. This might be due to the improved immune status of the animal through careful selection along with scientific managemental practices followed in the University farm. The non-significant influence of sex, birth weight and type of birth might be due to uniform managemental conditions given to kids irrespective of sex, birth weight or type of birth, which do not favour GIN prevalence. The reason for failure of season in influencing FEC could be due to the fact that, not much demarcation was observed between seasons during the study. The genetic similarity between Malabari purebreds and Malabari crossbreds, lesser sample size for Attapady Black breed and similar management given to the breeds led to non significant effect of breed on FEC. A negative phenotypic correlation estimate of -0.1791 was observed between LFEC and PCV. The heritability estimated for FEC was 0.39 ± 0.3630 as per paternal half sib correlation method and it was moderate. This moderate heritability estimate for FEC shows that there is significant genetic variation within the population for the trait, which makes selection for nematode resistance possible. Genomic DNA was isolated from blood samples of 150 goats maintained in the farm and two field centers of Kerala using phenol chloroform extraction procedure. Through PCR, a 202bp product of IFN γ intron 1 was amplified. On restriction digestion of the amplified product, the restriction-endonuclease, BspHI cut PCR product into two fragments of 180 bp and 22 bp sizes. Monomorphic pattern was observed on the particular RFLP analysis. The 202bp PCR product was sequenced by the dideoxynucleotide sequencing method with M13 universal reverse primer using an automated DNA sequencer. The sequence included a tetra nucleotide repeat, (GT4)5 and an Adenine present at a position, 49 bp downstream of this microsatellite. The sequence had 100 per cent homology with sheep, 89 per cent with European cattle, 73 per cent with pig and 70 per cent with human. On submission of the nucleotide sequence data of goat-IFNγ intron1 to the GenBank DNA database, an Accession Number: HM047072 was obtained. As per available literature, this work seems to be the first of its kind in establishing the DNA sequence data of intron1 of IFN γ gene in goats. It is recommended that study of this gene in a larger population with greater genetic base might bring out the polymorphism and help in marker assisted selection for GIN resistance in goats. Overall, the study aided in establishing nucleotide sequence of IFN γ intron1 in goats and showed that there is immense scope for selection of animals for nematode resistance. This study emphasizes the fact that along with other production traits, host genetic resistance should also be included as a criterion for selecting goats, producing genetically superior stock.