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Kerala Veterinary and Animal Sciences University, Wayanad
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ThesisItem Open Access POLYMORPHISM OF CANDIDATE GENES CONTROLLING TEMPERAMENT TRAITS AND THEIR ASSOCIATION ON REPRODUCTION TRAITS IN VECHUR CATTLE(Kerala Veterinary and Animal Sciences Mannuthy, Thrissur, 2018-09-30) ANANDAKUMAR PONNALA; Manoj M.Temperament in cattle plays an important role in animal safety, production and reproduction performance. Bos indicus species are known for their aggressive behaviour than Bos taurus. The present study was conducted to identify the polymorphism of candidate genes controlling temperament traits and their effects on reproductive performance of Vechur cattle. The study comprised of 68 Vechur cattle that were divided into four temperament groups based on their subjective temperament score, social separation test, docile test and handler’s/milker’s score test. Available reproductive performance data of animals were collected from Vechur cattle conservation project to study the following traits - age at first service, age at first calving, service period, conception rate and number of services per conception. Six candidate genes - Tyrosinase (TYR), Dopamine receptor D4 (DRD4), Monoamine Oxidase A (MAO A), Cannabinoid receptor 1 (CNR1), Regulator of G-protein signaling 2 (RGS2) and Plexin A2 (PLXA2) - for temperament were selected. Three selected SNPs from each gene were screened using PCR-RFLP/ PCR-SSCP and further confirmed by sequencing. Out of these 18 SNPs, 9 were present in the Vechur herd. The genes MAOA, TYR, CNR1 and PLXA2 contain two, two, three and two SNPs, respectively. Except TYR 6416322 SNP remaining eight SNPs, coat colour (p=0.003) and sex (p=0.001) were significantly associated with handler’s/milker’s temperament score of handler three (H3). MAOA 105380513 SNP C to T transition was fixed in Vechur population. The average service period of four temperament classes (docile, restless, nervous and aggressive) were 251.02 ± 24.04, 168.57 ± 19.28, 230.59 ± 21.75 and 243.05 ± 32.26 days, respectively. Second restless group had significantly (p=0.039) lower average service period in comparison to all other groups. However, age at first service, age at first calving, conception rate and number of services per conception were not significantly associated with temperament score in the present study. There were significant correlations among different reproductive traits. Results of this study revealed that the selected genes were having significant effect on temperament and reproduction traits, which may be confirmed by further research using larger population and more accurate temperament score. This would enable us to incorporate temperament as an important trait in the selection programmes for genetic improvement of Vechur cattle.ThesisItem Open Access RUMEN METAGENOME PROFILES AND METHANE EMISSION LEVELS IN VECHUR AND CROSSBRED CATTLE UNDER DIFFERENT DIETARY REGIMENS(Kerala Veterinary and Animal Sciences Mannuthy, Thrissur, 2019-09-30) TINA SADAN; T. V. AravindakshanA study was conducted to assess the rumen metagenome profiles and methane emission levels in Vechur and crossbred cattle under different dietaryregimens. Feeding patterns were designed to have an increasing proportion of forage and a decreasing proportion of the concentrate mix. A total of ten adult cows comprising five in each genetic group maintained on standard ration (Forage: concentrate ratio of 50:50) formed the material for the whole metagenomic study. Out of twelve adult cows, six adult cows each of Vechur and crossbred cattle fed with forage: concentrate ratio of 75:25 and 100:0 for a period of three weeks were selected as the experimental animals for 16S rRNA based metagenome study. Rumen liquor and rumen gas samples were collected from all the experimental animals. DNA samples isolated from rumen liquor using standard procedure were pooled genetic group wise and subjected to metagenome sequencing using Illumina HiSeq 2500 platform and further bioinformatics analysis. The concentrations of methane (per cent) in the gas samples were determined using a methane analyser. Research findings from whole metagenomic study revealed that bacteria followed by Archaea and Eukaryota dominated in the Vechur as well as the crossbred rumen samples. 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 two genetic groups. Diversity indices displayed a higher microbial diversity in Vechur cows compared to crossbred cows and also there was a significant difference in diversity between genetic groups. Functional annotation of contigs carried out by SEED classification revealed sequence reads related to carbohydrate and protein metabolism were the most abundant in rumen of both genetic groups. A significant difference in genes associated with different metabolic pathway as revealed by KEGG pathway analysis was found between two genetic groups. The 16S rRNA based metagenome sequencing study showed that Bacteroidetes and Firmicutes were the predominant phyla in both genetic groups. An increase in the Firmicutes to Bacteroidetes ratio observed with the increase in roughage proportion. There was highly significant difference in bacterial diversity indices and no significant difference in total bacterial phylum abundance between diets and genetic groups. Comparison of methane emission levels in Vechur and crossbred cattle under different diets confirmed the effect of genetic group and diet on methane emission levels. Significantly higher methane emission levels were observed for Vechur cattle and 100 per cent forage dietary regimen in both genetic groups.The 16S rRNA based metagenome sequencing study showed that Bacteroidetes and Firmicutes were the predominant phyla in both genetic groups. An increase in the Firmicutes to Bacteroidetes ratio observed with the increase in roughage proportion. There was highly significant difference in bacterial diversity indices and no significant difference in total bacterial phylum abundance between diets and genetic groups. Comparison of methane emission levels in Vechur and crossbred cattle under different diets confirmed the effect of genetic group and diet on methane emission levels. Significantly higher methane emission levels were observed for Vechur cattle and 100 per cent forage dietary regimen in both genetic groups.ThesisItem Open Access ASSOCIATION OF SINGLE NUCLEOTIDE POLYMORPHISMS IN THE QUANTITATIVE TRAIT LOCI WITH RESISTANCE OR SUSCEPTIBILITY TO TICK INFESTATION IN CATTLE(COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE WAYANAD, 2019-10-05) BHAGYASHREE MUKUND, KAMBLE; Dinesh, C.N.The present study was aimed to identify the polymorphisms in five QTLs that were reported to have an association with resistance/susceptibility to tick infestation in cattle and to find out the association of SNPs with tick infestation in cattle. It was also intended to study the effect of season on the association between the SNPs and tick infestation in cattle. The population under study comprised 45 Vechur and 74 crossbred cattle from the farms of Kerala Veterinary and Animal Sciences University. The QTLs and SNPs were selected from the QTL database, Animal QTLdb. The SNPs and the QTLs (in parenthesis) used in this study were: rs41661020 (#101148), rs109162468 (#101169), rs43708490 (#135798), rs41577070 (#135800) and rs29009970 (#135801). Blood samples were collected from the animals and the genomic DNA was isolated and used for genotyping and polymorphism study by PCR-RFLP assay and confirmed by sequencing. Genotyping was done by direct counting and the allelic and genotypic frequencies were estimated by POPGENE 1.32. The results revealed a monomorphic pattern for the SNP rs109162468 and polymorphic pattern for the SNPs rs41577070, rs41661020, and SNP rs43708490 in the population under study. The SNP rs29009970 was monomorphic in the crossbred cattle, whereas, dimorphic in Vechur. The Chi-square test revealed that crossbreds and Vechur were in HWE for SNPs rs41577070, rs41661020, and rs43708490, whereas, the SNPs rs29009970 showed a significant departure from the equilibrium. For the association study, tick counts were taken during summer (March to May) and rainy (June to August) seasons from the animals using Wharton and Utech (1970) method. However, no tick infestation could be seen in any animals during the period of study. Hence, all the 119 animals were grouped in the class: no infestation and the analysis to find the association of SNPs with tick infestation and the effect of the season on the SNP-tick load relationship in cattle could not be carried out.ThesisItem Open Access SINGLE NUCLEOTIDE POLYMORPHISM ANALYSIS AND EXPRESSION PROFILE OF INHIBIN ALPHA GENE IN MALABARI GOATS(College of Veterinary and animal Science,Mannuthy, 2019) HARITHA B. PILLAI; R. Thirupathy VenkatachalapathyThe present study was carried out to identify single nucleotide variations in inhibin alpha (INHA) gene to evaluate its association with production traits and find out the tissue expression profile in Malabari goats. A total of 207 Malabari adult goats were screened for polymorphism using PCR-SSCP. Representative samples of each SSCP pattern were sequenced to ascertain the genotypes. Five fragments, 274 bp, 220 bp, 160 bp, 287 bp and 253 bp covering the two exons of INHA were analysed. Similar monomorphic band pattern was observed for 220 bp fragment of exon 2. The 274 bp fragment showed two diplotypes (AA and AB) and sequencing revealed one synonymous SNP (c.129G>A) in exon 1. The AA genotype showed significant (p≤0.05) association with body length. The 160 bp fragment exhibited two genotypes DD and DE with a synonymous SNP c.567G>A in exon 2. Goats carrying DD genotype had significantly (p≤0.05) higher adult body weight and body length compared to DE genotype. Three diplotypes, HH, HJ and JJ with a synonymous SNP, c.651G>A were observed at 287 bp fragment. Significantly (p<0.01) higher body length was noticed in goats with HJ and JJ genotypes. The 253 bp fragment of exon 2 exhibited three genotypes PP, PQ and QQ with a non synonymous SNP, c.911T>C. Genotypes of INHA 253 bp (exon 2) locus had significant (p≤0.05) influence on body weight and trunk circumference. The PP and PQ genotypes showed a significantly higher trunk circumference (p≤0.05) compared to QQ. Tissue samples of ovary, uterus and fallopian tube were collected from seven Malabari adult goats to study INHA gene expression pattern. The relative tissue expressions of INHA mRNA were normalised with the reference gene (GAPDH). Relative expression was significantly (p<0.01) higher in ovary, followed by uterus and fallopian tube. The results obtained from this study indicate the potential role of INHA gene on reproduction and growth; hence it can be used as a candidate gene for the genetic improvement of Malabari goats.ThesisItem Open Access POLYMORPHISM STUDY ON MILK PRODUCTION TRAITS IN GOATS USING DOUBLE DIGEST RESTRICTION ASSOCIATED DNA SEQUENCE(College of Veterinary and animal Science,Mannuthy, 2019) K.MATHIVATHANI; Radhika G.Native goat breeds of Kerala; namely Attappady Black and Malabari, apparently varied in milk production capacity. Milk production data was collected from 100 each of Malabari and Attappady Black goats and the data analysed. Parity (p≤0.01), breed (p≤0.05) and herd (p≤0.01) showed significant influence on milk production. Fat per cent showed significant (p≤0.05) breed differences during early, middle and late lactation. Double digest Restriction Associated DNA Sequencing (ddRADseq) is a modern approach for identification of Single Nucleotide Polymorphisms (SNP) across populations. The ddRADseq technique was performed in three pooled samples (ten each) of high and low milk producing Malabari and Attappady Black goats. The SNPs discovered were analysed using bioinformatics tool Venny 3. A total of 4141 and 2192 variants were observed in Malabari and Attappady Black goats respectively. A panel of 42 candidate genes affecting milk production were screened and 26 SNPs were discovered in 22 genes comprising of 19 intron, five downstream and two upstream variants. From a set of 11 milk production related genes, 11 SNPs were identified based on the difference in allelic distribution of SNPs between the two groups. NRXN3, LPL, GLI2 were selected for validation studies. Intronic variant c.301+155748C>T of NRXN3 was validated using High Resolution Melting analysis and showed significant (p≤0.01) association, with mean daily milk yield (mL) of 558.17±4.58, 462.21 ±16.53 and 416.62 ±11.13 for CC, CT and TT genotypes, respectively. In Attappady Black goats, CC genotype was absent as indicated by ddRADseq results. Intronic variant c.243+1082C>T LPL, was validated using Polymerase Chain Reaction-Single Strand Conformation Polymorphism and further sequencing. It had significant (p≤0.05) association with mean daily milk yield (mL) of 437.12±12.85, 494.72±11.60 and 467.63 ± 19.86 for CC, CT and TT genotypes respectively. The GLI2 variant c.64T>G revealed monomorphic pattern in PCR-RFLP. Expression study of BTN1A1 revealed significantly (p≤0.05) higher expression (6.74 fold) in Malabari than Attappady Black. Current study emphasized the relevance of ddRADseq in identifying novel SNPs in candidate and unexplored genes influencing economic traits.ThesisItem Open Access MODELLING OF BEST FIT LACTATION CURVE IN CROSSBRED CATTLE OF KERALA(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2018) G. PRAKASH; K. AnilkumarThe main objectives of the present study was modelling of best fit lactation curve in crossbred cattle of Kerala by using monthly test day milk yields (MTDMYs). For developing lactation curve models, data of 936 crossbred cattle sired by 188 bulls spread over a period of 16 years (2002-2017) in seven different field centers of ICAR-Field Progeny Testing Scheme, Kerala were used. Least squares means for ten monthly test days starting from 20th day of calving at 30 days interval and first lactation milk yield (FLMY) were 10.35±0.15, 10.71±0.14, 10.25±0.14, 9.73±0.13, 9.11±0.13, 8.44±0.13, 7.80±0.12, 7.03±0.0.11, 6.16±0.12, 5.41±0.13 and 2510.71±30.49 kg, respectively. Period of calving and batches of sires had significant effect on some of the MTDMYs and FLMY. Season of calving had significant effect only on first MTDMY. Genetic group, sources of sires and age at first calving had non-significant effect on MTDMY and FLMY. Heritability estimates ranged from 0.004±0.107 in 9th MTDMY to 0.531±0.136 in 4th MTDMY. Lactation curves using different MTDMY were modelled with Inverse Polynomial Function (IPF), Mixed Log Function (MLF), Parabolic Exponential Function (PEF), Wilmink function (WIF) and Woods function (WOF). Among these, Wilmink Function showed best fitness of good criteria. WIF had maximum R2 (86.45%), low RMSE (0.009949 kg), minimum AIC (-58059.79) and minimum BIC (-9656.57) values. Stepwise backward multiple regression method was used to predict FLMY using different combination of MTDMYs. Prediction equation with 3 variables viz. MTDMY-1, MTDMY-2 and MTDMY-5 was found to predict of FLMY with 84.08 per cent accuracy. Lactation curve parameters were validated with 60 crossbred cattle of a different herd. The average deviations of predicted FLMY of IPF, MLF, PEF and WOF from actual FLMY were significantly different. For animals with initial MTDY more than 11 liters, prediction with WOF was effective as it was not significantly different from observed value. Wilmink function is adjudged as the most effective function in predicting MTDMYs and FLMY of crossbred cattle of KeralaThesisItem Open Access RUMEN METAGENOME PROFILES AND METHANE EMISSION LEVELS IN VECHUR AND CROSSBRED CATTLE UNDER DIFFERENT DIETARY REGIMENS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2018) TINA SADAN; T. V. AravindakshanA study was conducted to assess the rumen metagenome profiles and methane emission levels in Vechur and crossbred cattle under different dietary regimens. Feeding patterns were designed to have an increasing proportion of forage and a decreasing proportion of the concentrate mix. A total of ten adult cows comprising five in each genetic group maintained on standard ration (Forage: concentrate ratio of 50:50) formed the material for the whole metagenomic study. Out of twelve adult cows, six adult cows each of Vechur and crossbred cattle fed with forage: concentrate ratio of 75:25 and 100:0 for a period of three weeks were selected as the experimental animals for 16S rRNA based metagenome study. Rumen liquor and rumen gas samples were collected from all the experimental animals. DNA samples isolated from rumen liquor using standard procedure were pooled genetic group wise and subjected to metagenome sequencing using Illumina HiSeq 2500 platform and further bioinformatics analysis. The concentrations of methane (per cent) in the gas samples were determined using a methane analyser. Research findings from whole metagenomic study revealed that bacteria followed by Archaea and Eukaryota dominated in the Vechur as well as the crossbred rumen samples. 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 two genetic groups. Diversity indices displayed a higher microbial diversity in Vechur cows compared to crossbred cows and also there was a significant difference in diversity between genetic groups. Functional annotation of contigs carried out by SEED classification revealed sequence reads related to carbohydrate and protein metabolism were the most abundant in rumen of both genetic groups. A significant difference in genes associated with different metabolic pathway as revealed by KEGG pathway analysis was found between two genetic groups. The 16S rRNA based metagenome sequencing study showed that Bacteroidetes and Firmicutes were the predominant phyla in both genetic groups. An increase in the Firmicutes to Bacteroidetes ratio observed with the increase in roughage proportion. There was highly significant difference in bacterial diversity indices and no significant difference in total bacterial phylum abundance between diets and genetic groups. Comparison of methane emission levels in Vechur and crossbred cattle under different diets confirmed the effect of genetic group and diet on methane emission levels. Significantly higher methane emission levels were observed for Vechur cattle and 100 per cent forage dietary regimen in both genetic groups.ThesisItem Open Access POLYMORPHISM OF CANDIDATE GENES CONTROLLING TEMPERAMENT TRAITS AND THEIR ASSOCIATION ON REPRODUCTION TRAITS IN VECHUR CATTLE(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2018) ANANDAKUMAR PONNALA; Manoj. MTemperament in cattle plays an important role in animal safety, production and reproduction performance. Bos indicus species are known for their aggressive behaviour than Bos taurus. The present study was conducted to identify the polymorphism of candidate genes controlling temperament traits and their effects on reproductive performance of Vechur cattle. The study comprised of 68 Vechur cattle that were divided into four temperament groups based on their subjective temperament score, social separation test, docile test and handler’s/milker’s score test. Available reproductive performance data of animals were collected from Vechur cattle conservation project to study the following traits - age at first service, age at first calving, service period, conception rate and number of services per conception. Six candidate genes - Tyrosinase (TYR), Dopamine receptor D4 (DRD4), Monoamine Oxidase A (MAO A), Cannabinoid receptor 1 (CNR1), Regulator of G-protein signaling 2 (RGS2) and Plexin A2 (PLXA2) - for temperament were selected. Three selected SNPs from each gene were screened using PCR-RFLP/ PCR-SSCP and further confirmed by sequencing. Out of these 18 SNPs, 9 were present in the Vechur herd. The genes MAOA, TYR, CNR1 and PLXA2 contain two, two, three and two SNPs, respectively. Except TYR 6416322 SNP remaining eight SNPs, coat colour (p=0.003) and sex (p=0.001) were significantly associated with handler’s/milker’s temperament score of handler three (H3). MAOA 105380513 SNP C to T transition was fixed in Vechur population. The average service period of four temperament classes (docile, restless, nervous and aggressive) were 251.02 ± 24.04, 168.57 ± 19.28, 230.59 ± 21.75 and 243.05 ± 32.26 days, respectively. Second restless group had significantly (p=0.039) lower average service period in comparison to all other groups. However, age at first service, age at first calving, conception rate and number of services per conception were not significantly associated with temperament score in the present study. There were significant correlations among different reproductive traits. Results of this study revealed that the selected genes were having significant effect on temperament and reproduction traits, which may be confirmed by further research using larger population and more accurate temperament score. This would enable us to incorporate temperament as an important trait in the selection programmes for genetic improvement of Vechur cattle.ThesisItem Open Access DOUBLE DIGEST RESTRICTION ASSOCIATED DNA SEQUENCING FOR IDENTIFICATION OF PROLIFICACY RELATED SINGLE NUCLEOTIDE POLYMORPHISM MARKERS IN GOATS(College of Veterinary and animal Science,Mannuthy, 2018) SHALU ELIZABETH SIMON; Radhika G.Goats, commonly called as ‘poor man’s cow’ forms the basis of livelihood for economically weaker sections of society. India has 23 well defined goat breeds, of which Kerala has two native breeds, Malabari and Attappady Black, which vary significantly in prolificacy. Double digest restriction associated DNA sequencing (ddRADseq) is a modern reduced representation sequencing technique which is a powerful and inexpensive approach for identification of Single Nucleotide Polymorphisms (SNP) across populations. In this study ddRADseq technique was applied to highly prolific Malabari and low prolific Attappady Black goats to unravel the genetic variations existing between them. ddRADseq discovered 4140 SNPs and 126 INDELS in Malabari and 2192 SNPs and 59 INDELS in Attappady Black goats of which 55 per cent variants were common between the groups. A panel of 38 candidate prolific genes were screened and 25 SNPs were discovered in 20 genes which included one exonic variant and 18 intronic variants. Another set of 11 genes were identified based on the difference in allelic distribution of SNPs between the two groups, which might have an effect on prolificacy. Based on in silico studies, variant c.614-32789C>T in BMP6 was chosen for validation studies in 100 Malabari and 50 Attappady Black goats. PCR-RFLP revealed all the three genotypes TT, TC and CC, thus confirming the robustness of SNPs discovered by ddRADseq. SERPIND1 of super family Serpins is reported to have a major role in placenta formation in humans. Variant c.1259T>C identified in exon three of SERPIND1, showed significant association with litter size with a mean value of 1.348±0.071 for TT genotype and 2.27 ±.0.132 for TC genotype. This emphasized the relevance of this polymorphism and usefulness of ddRADseq in identifying novel SNPs in unexplored genes.RGS 14 gene which affects early embryonic development, harboured a missense mutation c.1624G>A resulting in p.Pro542Ser, suggesting a variation in protein quality. Expression study of RGS 14gene in endometrial tissues revealed a 3.35 fold higher expression in Malabari than Attappady Black goats which was statistically significant. Current study emphasized the relevance of ddRADseq in identifying novel SNPs in candidate genes responsible for economic traits and unexplored genes affecting complex traits.
