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Kerala Veterinary and Animal Sciences University, Wayanad
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ThesisItem Open Access MARKER ASSISTED SELECTION FOR MILK PRODUCTION TRAITS IN VECHUR CATTLE(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2005) SHYMAJA UTHAMAN; K.V. RaghunandananA study was conducted in Vechur cattle of Kerala to study the association of microsatellite marker alleles with the milk production and composition traits and to evaluate seven ancestors using microsatellite markers. Blood and semen was used as the source of DNA in this study. DNA samples were extracted from fifty unrelated animals and thirty three ancestors and progenies. Five microsatellite markers with established linkage to QTL controlling milk production traits like milk yield, fat percentage and protein percentage were used. The DNA samples obtained from the fifty unrelated Vechur cattle were typed using these five microsatellite markers. The five markers were found to be highly polymorphic with the PIC value ranging from 0.82 at the BM1508 locus to 0.89 at the HUJII77 locus. Data regarding the peak yield, days to attain peak yield, AFC and intercalving period were collected from the Vechur Conservation Centre, Mannuthy. Milk samples collected from the Vechur Conservation Centre were used to determine the milk fat percentage, protein percentage, SNF and total solids. The mean fat percentage and AFC were found to differ significantly in different families. Statistical analysis revealed that the V43 and V88 families had a mean fat percentage significantly greater than the population mean. The mean AFC of V43 family was also found to be significantly greater than the population mean. The V44, V88 and VI17 families had a mean AFC significantly lower than that of population mean. The V44 family had the lowest mean AFC and was also significantly different from the V88 and VI17 families. Microsatellite analysis was carried out for the thirty three combinations of ancestors and progeny using the same five markers. The allelic effect of microsatellite markers in the families was studied for fat percentage and AFC. The allelic mean for fat percentage of 223 bp size allele at the HUJII77 locus was found to be significantly higher when compared to those animals without this allele even though there were no earlier reports indicating linkage between this marker and QTL affecting fat percentage. The allelic mean of fat percentage for 205, 209 and 221 bp at the HUJII77 locus was significantly lower when compared to those of animals without this allele. The mean of the animals with 208 bp allele at the ILSTS096 locus was found to be significantly greater for fat percentage when compared to the mean of those without this allele. There are earlier published reports indicating linkage between this marker and QTL affecting fat percentage. Selection for this allele will not be effective as it is already present in high frequency in the population. The 252 bp allele at the BL41 locus had an allelic mean for fat percentage significantly lower than that for animals without this allele. The 238 bp size allele of this locus had an allelic mean for AFC significantly greater than that for animals without this allele. Earlier reports establish linkage between this marker and QTL affecting fat percentage but not AFC. Selection can be applied against this allele to prevent its frequency from increasing in the population. The allelic mean of 158 bp allele at the BM1508 locus had a mean fat percentage significantly lower than that for the animals without this allele. There are earlier reports of linkage of this marker with QTL affecting protein percentage but not fat percentage. Selection against this allele is necessary as this allele is present in high frequency in the population. Animals with 113 bp allele had an allelic mean of fat percentage significantly lower than that for the animals without this allele. This marker has linkage with QTL affecting milk fat percentage. It is advisable to select against this allele to increase the milk fat percentage in the population. Even though the V44 sire can be selected for improving the fat percentage, the mean AFC of this sire was found to significantly greater than that of the population mean hence it is not preferred. The sire V88 can be selected for improving the fat percentage as well as reducing the AFC. There is a possibility to improve the Vechur cattle maintained at the Vechur Conservation Centre, Marmuthy for fat percentage and AFC by adopting line breeding for V88 sireThesisItem Open Access STUDY OF GENETIC DIVERSITY IN MALABARI GOATS (Capra hircus) UTILIZING BIOCHEMICAL AND IMMUNOLOGICAL MARKERS(College of Veterinary and Animal Sciences, Mannuthy., 2006) K. A. BINDU; Dr.K.C.raghavanABSTRACT Goat populations of Tanur, Thalassery and Badagara were studied for biochemical polymorphisms, immunological and microsatellite markers to investigate the similarities and differences between these populations. With regard to biochemical markers tested, polymorphism was observed only for haemoglobin, transferrin and glutathione loci. Two variants were observed for haemoglobin, Hb and Hb with a frequency of 0.987 and 0.012, respectively, suggestive of three phenotypes, viz. Hb aa, Hb ab and Hb bb, and indicating the predominance of Hb in the pooled population. Hb ^ variant was observed only in the Thalassery population (gene frequency 0.038). Two variants for transferrin (Tf^ and Tf were detected with a predominance of TC in the population. All the goats from Thalassery population belonged to Tf aa type. In the present study only two phenotypes as regards transferrin locus could be observed, (Tf AA and Tf bb) with,the notable absence of Tf ab- No polymorphism was observed for albumin, cerruloplasmin, amylase and carbonic anhydrase loci in all the animals tested. The animals were classified as low and high glutathione types based on the values obtained for blood glutathione concentration. In the pooled population, majority of the animals belonged to low GSH type (53.68 per cent). The least square analysis of glutathione concentrations showed significant variation between populations. With regard to potassium loci, all the animals in the present study belonged to low potassium type, with the mean potassium concentration of the pooled population recorded at 4.18 :L0.09 meq/1. The least square analysis of variance of potassium concentrations showed that there existed significant difference between different sub-populations. Genetic distance was calculated as described by Balakrishnan and Sanghvi (1968), using the allelic frequencies of protein polymorphic loci. Genetic distance between Tanur and Badagara was found to be 0.1249 , while that between Tanur and Thalassery was 0.6690 and between Badagara and Thalassery was 0.3351. The only possible conclusion that could be arrived at from the above studies is the existence of a relationship between these populations. Hence an attempt was made to study the different populations at molecular level, using microsatellite markers. Three markers, viz. INRA 063, HUJ 1177and ILSTS 030 were found to be polymorphic. Based on the genetic distances, it was found that Thalssery and Badagara were closely related than Tanur population.. This finding, much in agreement with biometrical traits, reiterates the close relationship between the Thalassry and Badagara populations. On an average, the goats of Thalassery and Badagara were heavier in comparison to Tanur goats, though the prolificacy remained higher in Tanur animals than the other two populations. The different sub-populations under the present study were also screened for the antibody response to SRBC. The highest concentration of antibody was observed on day seventh after primary immunization. The titre gradually reduced by the 15"^ day, reaching the lowest values on IT"^' day of post immunization. The effect of antibody response to SRBC on the 7"^, IS"*" and 2C days post immunization was not found to be significant for the occurrence of diseases like diarrhoea and pneumonia. The cutaneous response to intradermal injection to Phytahaemagluttinin - M (PHA-M) was also studied to find out the differences, if any in and between the various sub- populations under study. The values for skin thickness were maximum at 24 hours post-intradermal injection of PHA-M and were recorded as 3.24±0.05, 3.23±0.06 and 3.33±0.06 mm in Tanur, Thalassery and Badagara, respectively. The skin thickness reduced considerably after 48 hours and reached 1.61±0.02, 1.62±0.02 and 1.65±0.02 mm, respectively at 72 hours. The least square analysis of variance revealed that the values for pre and post immunization skin thickness at 0, 24, 48 and 72 hours were non significant between different sub-populations. Total protein, albumin and globulin concentrations also were estimated. The highest mean concentration for globulin was detected in Badagara population (3.28±0.22g/dl) and the lowest in Tanur (2.340 ± 0.3]g/dl). The least square analysis of variance of globulin concentration revealed significant difference between populations. Though all populations under study had all predominant physical characteristics of the Malabari breed, the Tanur population stood apart as regards the biometrical characteristics, like litter size and body weight and charecteristics perceivable at the molecular level. It could well be inferred that this population might have evolved through mixing up of the local nondescript Tanur goats with original Malabari goats. The study reiterates the need for more research activities directed at exploring the chances of conserving and developing such unique populations within a breed.ThesisItem Open Access GROWTH AND SURVIVABILITY OF GH/Msp I GENOTYPES IN MALABARI GOATS(College of Veterinary and Animal Sciences, Mannuthy., 2004) BINDU MATHEW; Dr. k.C. RaghavanGrowth hormone gene, due to its essential role in lactation and growth processes, is a perfect candidate marker associated with somatotropic axis. Selection of animals based on the growth hormone genotypes can be tntroduced in the animal husbandry sector for better production. The present investigation was undertaken to study the growth and survivability of GH/Mspl genotypes in Malabari goats at different centers of Badagara. Thalassery. Thanur as well as Malabari conservation unit. Regional Agricultural Research Station, Pilicode. DNA was isolated from 32 bucks, 241 does mated to those bucks and 297 of their progeny using phenol - chloroform extraction method. A 768-bp fragment from third exon to fifth exon containing the polymorphic Mspl site was amplified well using bovine primers, indicating species homology. The amplified product on digestion with the Mspl enzyme revealed the GWMspl {+) and (-) alleles. The percentage of incidence of (+/-) genotype was 61.76 and that of (+/+) genotype was 38.24. None of the animals typed were of the GH/Mspl (-/-) genotype. The genotype frequencies of bucks and does were consistent with the general population. In the specific heterozygous mating (+/- x +/-), 28 per cent of the progenies were (+/+) homozygotes and the rest 72 per cent were heterozygotes (+/-). Early embryonic mortality was not found to be a cause for the absence of the GH/Mspl -/- genotype as the kidding percentage in heterozygous mating were not different from other types of ma.rngs. There was no relationship between growth upto SIX months of age and G\l!Msp\ genotypes. The study conf.mrs a strong heterozygotie advantage for the GH/Mspl +/genotype and also the absenee of GH/Mspl -/- genotype in Malaban goats. Presence of duplicate copies of the growth hormone gene in goats may be a possible reason for the above results.ThesisItem Open Access MICROSATELLITE MARKER BASED CHARACTERIZATION OF INDIGENOUS PIGS OF KERALA(College of Veterinary and Animal Sciences, Mannuthy., 2006) ARIPRASATH. K; Dr. A.P. UshaThe study was undertaken to assess the genetic diversity among four indigenous pig population of Kerala using microsatellite markers. The animals were selected from various part of Kerala, population I included the conserved Angamali pigs from university farm, Mannuthy, population II consisted of animals from Koothattukualm, population III were the animals from Ollur and animals from border districts of Kerala formed the population IV. Genetic analysis was carried out using five polymorphic microsatellite markers. Blood samples were collected from 100 unrelated indigenous pigs from all four populations and DNA was isolated. The phenol-chloroform method of extraction yielded 224.35±9.86pg/5ml of blood. PGR conditions were standardized for all five selected markers namely, S0005, SOlOl, SW1026, SW2517 and S0008. The forward primer of each marker was endlabelled with 732 P-ATP as source of radio signal. The Ml3 single strand DNA was sequenced and used as a size standard. Autoradiography was employed to visualize the results. A total of eight alleles were detected in S0005 and SOlOl, five alleles in each of SW1026 and S0008, and six in SW2517. The heterozygosity varied from 0.7747 in SW2517 to as large as 0.8475 for S0005. The heterozygosity values for SOIOI, SW1026 and S0008 were 0.7774, 0.7672, and 0.7424 respectively. The PIC values ranges from 0.6974 for S0008 to 0.8291 for S0005. The PIC values for SOIOI, SW1026 and SW2517 were 0.7483, 0.7284 and 0.7381 respectively. The allele frequencies were used to estimate the Nei's standard genetic distance among the populations. The distance measure ranged from 0.5704 to 0.7161, with the highest value noticed between population II and IV and the lowest between population I and III. A dendrogram was constructed using the POPGENE version 3.2 program which grouped the population I and IV in one cluster and II and III populations in another cluster. >ThesisItem Open Access PORCINE IMMUNE RESPONSE AS MARKER TRAITS FOR SELECTIVE BREEDING(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2002) . RAJAN, M. R; RAGHUNANDANAN, K.V.Survivability and better performance of pigs under tropical stress have been reported to be significantly influenced by immune responses. Immune response traits under genetic control offer potential possibilities for exploited in commercial pig production. The present research project on the utilisation of porcine immune responses by estimating the magnitude of humoral and cell mediated immune responses in Desi and Large White Yorkshire attempted to evaluate the genetics of immune responses and to identify the association between the immune response traits and economic traits. The immune response traits were studied in 150 piglets aged between two to three months, 75 each belonging to Desi and Large White Yorkshire of both sexes and sired by eight sires each. The immune response traits studied were antibody response to sheep red blood cells (SRBC), delayed type hypersensitivity (DTH) to intradermal injection of PHA-M and lymphocyte transformation response to BCG. The economic traits recorded were litter size at birth, litter weight at birth, litter size at weaning, litter weight at weaning, weaning mortality and the occurrence of diarrhoea and pneumonia. Naturally occurring antibodies to SRBC could not be detected in both the breeds. Peak antibody response to SRBC was obtained at day 7 post immunisation with a mean titre of 4.830. Heritability estimates of antibody response to SRBC were 0.8969 + 0.4235. 0.9187 ± 0.4893 and 0.8174 ± 0.4893 respectively at 7*^ day, 15"' day and 21" day post immunisation. Litter size at birth and weaning had no significant influence on antibody response. Similarly, antibody response to SRBC among piglets was not influenced by the incidence of diarrhoea, pneumonia and pre-weaning mortality to a significant level. DTH responses to intradermal injection of PHA peaked at 24 hours post injection with a mean value of 3.39 mm. The mean pre injection skin thickness was 3.508 mm and 3.012 mm among Large White Yorkshire and Desi pigs respectively. This difference was found to be significant (P<0.05) and this difference was due to the significant breed difference confounding with body weight classes. The effect of breed on PHA responses at 24, 48 and 72 hours were not significant. Sex of the pig also did not influence the PHA responses significantly. The body weight classes did not influence the DTH response to PHA significantly. Sire effect was not significant on the pre injection skin thickness. But the DTH response at 24 hours was influenced by the sires in both Large White Yorkshire and Desi pigs to a highly significant level (P<0.01). At 48 and 72 hours post injection also DTH responses were influenced by sires to a significant level (P<0.05). The heritability estimates for pre injection skin thickness and DTH responses at 24, 48 and 72 hours were 0.5173 + 0.4179, 0.8136 + 0.5643, 0.6816 + 0.5187 and 0.7134 ± 0.5283 respectively. The litter size at weaning was not influenced by the initial skin thickness. DTH responses to PHA at 24, 48 and 72 hours had no significant influence on the litter size at birth and weaning. PHA responses at 24, 48 and 72 hours were not influenced significantly by the incidence of diarrhoea, pneumonia and pre weaning mortality. The analysis of lymphocyte transformation and stimulation index to BCG on zero day was around one indicating that there was not marked increase in the lymphocyte multiplication in PPD stimulated samples. The stimulation index on 15"'' day was 6.0161 in Large White Yorkshire and 6.3340 in Desi. This index further increased to 6.1070 and 6.5920 on 30'^' day and began to decline from 45"' day with a mean value of 6.0020 in Large White Yorkshire and 5.9890 in Desi pigs. The effect of breed, sex and body weight class of piglets was not found to influence the stimulation index significantly. Sire effect was not significant on the pre inoculation index while it was highly significant on 15*^. and 45*^ day in Large White Yorkshire and Desi. The estimates of heritability on 30"^ and 45*^ day stimulation index were 0.5171 ± 0.2893, 0.6289 + 0.3817 and 0.4983 ± 0.2583 respectively. Litter size at birth and weaning was not found to have any significant influence on the LT response to BCG. Correlation analysis among different immune response traits revealed that antibody response to SRBC at 7, 14 and 21'' day had highly significant positive correlation. Similarly, the correlation between PHA responses at 24, 48 and 72 hours were also highly significant and positive. PHA responses at 24 hours and LT responses at 15*^ day was also positive and significant. LT responses at 15*^ and 30th day were also significant and positive. The association between LT responses during different time intervals were always positive and significant. PHA responses were always negatively correlated with initial skin thicknesss to a significant level. Antibody response at 7, 15 and 21" day had a significantly high negative influence on the body weight at weaning. There was a significant decrease in pre weaning mortality associated with LT response at 15th day.ThesisItem Open Access GENETIC DIVERSITY ANALYSIS OF GOAT BREEDS USING MICROSATELLITE MARKERS(College of Veterinary and Animal Sciences, Mannuthy., 2005) AMRITA SUSAN JACOB; Dr. T.V. AravindakshanThe study was undertaken to assess the genetic vana^lfly' tSnong four goat breeds of South India using microsatellite markers. Three breeds studied were native to Kerala. These were Malabari, Attappadi Black and non-descript goats of Thrissur. The fourth breed, Salem Black, originated in the Salem district of Tamil Nadu. Microsatellite analysis was carried out using four highly polymorphic bovine markers. Blood samples from 30 genetically unrelated animals of each breed were collected and used as a source of DNA. The phenol-chloroform extraction procedure was used and the mean yield of DNA obtained was 361.43±10.73 pg/five ml blood. The four markers selected for the study were, INRA63, ILSTS030, HUJII77 and BM6121. PGR conditions were standardised for all the primers. The forward primer of each primer pair used in the PGR assay was end labeled with y^^P-ATP prior to setting up of the PGR. Ml 3 DNA was sequenced and used as the size standard. The PGR products were separated by denaturing polyacrylamide gel electrophoresis. Detection of the products was done by autoradiography. Gels after electrophoresis were dried and was set for autoradiography with X-ray film in a cassette fitted with intensifying screen. Allele sizes were obtained by comparing with the sequence of Ml 3 single stranded DNA size standard. A total of eleven alleles were detected at the INRA63 locus. The mean heterozygosity and PIG values obtained were 0.774 and 0.743, respectively. Seventeen alleles were detected at the ILSTS30 locus. The mean values of heterozygosity and PIG were 0.878 and 0.866, respectively. Thirteen alleles were detected at the BM6121 locus with mean heterozygosity and PIG values of 0.851 and 0.833, respectively. The HUJII77 locus was the most polymorphic of all the four loci detecting 21 alleles. The mean heterozygosity and PIG values were 0.899 and 0.88, respectively. The allele frequency measures were used to estimate the Nei's standard genetic distance among the populations using the POPGENE program. The distance measures ranged from 0.388 to 0.224, with the highest value noticed between Salem Black and non-descript goats of Thrissur and the lowest between Malabari and non-descript animals. A dendrogram was constructed using the POPGENE program which grouped the Salem Black and Attappadi Black goats in one cluster and Malabari and the non-descript goats of Thrissur in another.ThesisItem Open Access COMPARATIVE EVALUATION OF LITTER TRAITS IN DESI, LARGE WHITE YORKSHIRE AND THEIR CROSSBRED PIGS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2001) GOPINATHAN., A.; USHA, A. P.The present study was undertaken to compare and evaluate litter traits in Large White Yorkshire, Desi and their Crossbred pigs and to decide a breeding strategy. The data on 20-25 farrowings were collected from Centre for Pig Production and Research, Mannuthy for Large White Yorkshire, Desi and Crossbred pigs. A random sample of eight animals from each genetic group was selected and maintained from weaning to eight month of age to study the growth, feed conversion efficiency and carcass characteristics. The average birth weight and weaning weight, litter size at birth and weaning, litter weight at birth and weaning, pre-weaning mortality for each genetic group were calculated. Large White Yorkshire was found to be superior for all traits followed by Crossbred and Desi pigs. Crossbred pigs had lowest pre-weaning mortality while Desi pigs had highest litter size at birth. Analysis of variance showed that the effect of genetic group was found to be highly significant for all litter trmts except litter size at birth and weaning. The data were analysed using least squares analysis of variance to study the effect of different factors on birth weight and weaning weight in all three genetic groups. Least squares analysis of variance for birth weight revealed that the effect of sire and litter size at birth was highly significant in all three genetic groups. Sex had significant effect only in crossbreds. For weaning weight, the effect of sire and litter size at birth were found to be highly significant while sex did not show a significant effect on weaning weight in all three genetic groups. The effect of genetic group was found to be highly significant for third, fifth and eighth month body weight. But there was no significant effect noticed between Large White Yorkshire and Crossbred pigs during third month. The average daily gain and feed conversion efficiency was highest for Large White Yorkshire followed by Crossbred and Desi pigs from weaning to eight months of age. In carcass traits like back fat thickness, loin eye area, dressing percentage and carcass length. Large White Yorkshire averaged better than Desi and Crossbred pigs. The effect of genetic group was found to be highly significant for all carcass traits. The cost of production per kg of live body weight was calculated in three genetic groups, Large White Yorkshire had lower cost of production when compared to Crossbred and Desi pigs from weaning to eight month of age. The present study revealed that Large White Yorkshire had higher body weight gain, better feed conversion efficiency, average daily gain and higher values for carcass and litter traits when compared to Crossbreds and Desi pigs.ThesisItem Open Access POLYMORPHISM OF GROWTH HORMONE GENE IN MALABARI GOATS {Capra hircus)(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2002) CHITRA, R.; Aravindakshan, T.V.Malaban goats are noted for their high milk yield and meat production qualities. They represent a unique genetic resource by virtue of their adaptability, resistance to many infectious diseases and prolificacy in the humid tropics of Kerala. They also exhibit considerable variation in individual performance m milk production, growth rate and fecundity. The growth hormone (GH) gene is a potential target for studies of molecular variation because of its possible direct or indirect effects upon growth, lactation and mammar>' gland development in dair>' animals. The polymorphism m the third mtron of GH gene was investigated by polymerase chain reaction (PGR) and restriction endonuclease digestion. Genomic DNA was isolated from a random sample of 196 genetically unrelated Malaban does in second lactation belonging to three different population groups based on geographical locations. The mean yields of DNA from 5 ml of whole blood extracted by phenol-chloroform was 231.097±11.65 pu. The ratio of optical densities at 260 and 280 nm was above 1.7 indicating good deprotemisation. Oligonucleotide primers based on bovine sequences were used for amplification of GH gene in goats. The successful amplification of the expected 768 bp fragment indicated the suitability of bovine primers for goats and conservation of DNA sequence in related species. The digestion of the amplified product with Msp\ restriction enzyme revealed two alleles viz., Msp\ (+) and Msp\ (-) with gene frequencies of 0.70 and 0.30, respectively. In the Malabari goats tested in this study only (+/+) and (+/-) GH/Mspl genotypes were encountered where as none of the animal was of the (-/-) genotype. It is suspected that the Msp\ (-) allele in the homozygous condition might be lethal or linked to a lethal gene. The population of the Malabari goats investigated in the present study was not under Hardy-Weinberg equilibrium though it was a large random mating one with no known mutation, or migration. Hence, it is suspected that the selection process acting against the (-/-) genotype through reduced viability or early embryonic death may be a possible reason for the disagreement of the Hardy-Weinberg equilibrium in Malabari goats for this locus. It was observed that the goat population was in genetic equilibrium with respect to growth hormone locus in all population groups. The genetic equilibrium might be indicative of the homogeneity of different population groups though they differed significantly with respect to morphological and other phenotypic characters. Studies on the association of GH/Mspl genotypes with growth and milk traits in Malabari goats revealed the following results. The (+/-) genotype was superior to (+/+) genotype with respect to milk protein percentage in population-1. The (+/-) genotype was found to be associated with significantly high milk SNF percentage in population II and pooled population. The GHIMspl genotypes did not exhibit significant effect on body weight, body measurements, peak milk yield, milk fat and the litter size. The , ^ T. Kil-I ARY ) population groups differed significantly with respect to body weight, body measurements like length, heart girth and height at withers, peak milk yield and litter size. These results revealed the influence of management and other environmental effects on growth traits in Malabari goats. It is concluded that the typing of the Msp\ polymorphism using PCR-based procedure is a very efficient way to identify the GH genotypes in Malabari goatsThesisItem Open Access DELINEATION OF RANDOM AMPLIFIED POLYMORPHIC DNA MARKERS IN CROSSBRED CATTLE(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2003) ANILKUMAR, K.; RagiTunandanan, K. V.A study was conducted to delineate the Random Amplitied PoKmorphic DNA markers of crossbred cattle of Kerala. DNA was isolated from whole blood, fresh semen or frozen semen samples for this study. Three panels of DNA samples were used in this stud)'. The)' were DNA samples of 84 unrelated crossbred cattle, which was used for assessing the RAPD polymrophic patterns. DNA samples from 52 offspring, dam and sire combinations for assessing the possibility of using the technique in parentage identification and DNA samples ot 108 dwarf cattle for microsatellite analysis. The average yields of DNA obtained from whole blood was 256 ± 2.12|ig/ 5 ml. fresh semen was 241.23 ± 8.42pg/ 400 million sperms and trom frozen semen was 91.32 ± 6.01 pg/ 0.25 ml straw: The optical density ratio calculated as an indicator of purity of the sample were 1.72 ± 0.14. 1.81 ± 0.26 and 1.61 ± 0.34 for the DNA obtained from whole blood, frozen semen and fresh semen respectively. Twenty random oligonucleotide primers from Operon kit A and six custom svnthesised primers were used for the stud). Based on intensit). clarity and polymorphism of bands. 19 primers were selected for amplification of DNA samples of crossbred cattle. Number of bands obtained from different primers ranged from 2 to 16. The acerage number of bands produced by different primers ranged from 3.78 ± 0.15 in OPA 4 to 8.15 ± 0.25 in ILO1127. The size of amplified products ranged from -230 base pairs to -3580 base pairs. Percentage of polymorphism represented by indi\ idual primer caried from 66.66 to 100 in different groups. Se\en primers namely 0PA2. OPA 4. OPA 17. OPA 18. OPA 20. ILO 1127 and ILO 876 yielded 100% polymorphic bands. Frequencies of the bands and their allelic frequencies were worked out. The largest average number of bands was produced by the primer ILO 1127 ( 8.15 ± 0.25)-and the lowest average number of bands was observed for the primer OPA 4 (3.78 ±0.15). The bands having frequency less than 0.25 were classified as rare bands. The primer OPA 15 produced 7 rare bands, the primer OPA 2 and OPA 19 five bands each, the primers for OPAl. 0PA4. OPA 8. OPA 9.OPA 16 and ILO 1127 four each and primers 0PA7. OPA 14. OPA 17 and ILO 876 three each.ILO 526oduced two rare bands, four primers namely OPA 10. OPA 12. OPA 18 and OPA 20 produced one rare band each . and no rare bands were observed for the primer Gl. Twelve RAPD primers were selected to study 52 offspring, dam and sire combinations. Non parent bands were observed in offspring to the extent of 2 to 13.6%. Three of these primers nameh' OPA 14. OPA 4 and 01 did not produce any non parent bands in offspring. It was concluded that due to presence of the non parent bands in offspring. RAPD-PCR technique cannot be the method of choice for parentage verification. Phenol chloroform procedure was used to isolate DNA from whole blood of 24 Vechur. 25 Highrange dwarf. 24 Vatakara and 35 Kasargode animals for microsatellite studies. The average yield of DNA obtained per 5 ml ot blood samples was 210.1 ±9.4 pg with the ratio of optical density at 260 nm and 280 nm as 1.86 ± 0.23. PGR conditions for the four microsatellite loci namely DRB3. ETH 131. HEL 6 and FSHp were standardised. The size of the alleles ranged from 176 to 236 bp for DRB3 locus. 160 to 184 bp in ETH 131 locus and 263 to 295 base pairs in HEL-6 locus. The number of alleles identified in different loci were 21. 14 and 13 respectively for DRB3. ETHI31 and HEL 6 loci. The PIC \ alue of the primers, direct count heterozygosity and unbiased heterozygosit) were worked Ill out. The identification ot new alleles in this stud) was attributed to the tact that dwarf cattle of Kerala are Bos indiciis . The possibilit> of using the microsatellite marker analysis for genetic characterization of dwarf cattle ot Kerala and their phylogenic studies are indicated in this work. Parentage \eritication tacilities can be established, where this technique can be employed in disputed cases.