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Lala Lajpat Rai University of Veterinary & Animal Sciences, Hisar
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ThesisItem Restricted Genetic diversity between Murrah and Bhadawari breeds of Indian buffalo using RAPD-PCR(LUVAS, 2006) Barwar, Anurag; Sangwan, M.L.The present study was conducted to estimate genetic diversity within and between Murrah and Bhadawari breeds using RAPD-PCR. Genomic DNA was isolated from 20 unrelated animals of each breed. DNA was evaluated for quality, purity and concentration. Optimization of PCR was carried out using various concentrations of different components of reaction mixture. Out of 40 random primers of series OPU and OPV, only 9 were found to be informative and were used further for amplification of genomic DNA. From the amplification profile of these primers, values of band frequency, genetic similarity, band sharing frequency, genetic distance, average percentage difference and mean average percentage difference were calculated. From 9 random a total of 84 bands were amplified between breeds and out of these 51 were polymorphic (60.72%). In Murrah, overall percentage polymorphism of 56.36 was observed, while in Bhadawari it was 57.14. Average number of bands in Murrah was 5.04, while in Bhadawari were 5.03. Higher genetic similarity of 0.81 and 0.80 in Murrah and Bhadawari breeds, respectively, was observed as compared to between breed genetic similarity of 0.31. Genetic distance between breeds was 1.20. MAPD values of 10.72 and 16.10 were observed in Murrah and Bhadawari breeds, respectively, while MAPD of 60.73 was observed between breeds, indicating high genetic diversity between breeds. Six primers (OPU-01, OPU-02, OPU-05, OPU-07, OPU-14 and OPV-14) in Murrah and five primers (OPU-05, OPU-07, OPU-14, OPU-19 and OPV-14) in Bhadawari were found to be specific for these breeds.ThesisItem Restricted Development of non radioactive DNA probe for diagnosis of bovine herpes virus-1(LUVAS, 2006) Kapgate, Sunil; MinakshiBovine herpesvirus-l (BHV-1), a member of the family Herpesviridae and subfamily Alphaherpesvirinae is an important cause of respiratory and genital diseases in cattle. Infection with BHV-1 occurs worldwide and causes serious economic losses due to mortality, abortions, decreased milk production and loss of weight. The current techniques for detection of BHV- 1 in the diagnostic laboratories include virus isolation, and antigen detection by ELISA. These techniques lack sensitivity and are both time consuming and expensive. Keeping this point in view the present study was undertaken to develop and evaluate the non radioactive probe for detection of BHV-1. The non radioactive DNA probe was successfully evaluated for detection of BHV-1 in biological field samples. By using DNA probe total a 4 Nasal swabs, 8 vaginal swabs, lung, liver and cotyledons of aborted material were detected positive. Efficiency of two different primer pairs belonging to gI and gB glycoproteins, for detection of BHV-1 was compared. The gI specific PCR detected upto 1fg of viral DNA from purified BHV-1. Whereas the gB gene specific PCR detected upto 10fg of nucleic acid The gI gene specific PCR was used to confirm the results of probe hybridization assay. Probe positive all the samples also detected positive by PCR. None of the probe negative samples was found positive by PCR. The techniques could be useful for screening of large number of biological samples. The chelex method of DNA isolation from clinical samples included a fast, safe and convenient extraction procedure. The study clearly indicated that, the novel extraction method with non radioactive DNA probe hybridization assay provide simple, rapid and sensitive diagnostic tool for detection of BHV-1 infection in biological samples especially when large number have to be processed.ThesisItem Restricted Apoptosis related gene transcripts in buffalo cumulus oocytes complex in relation to its morphology and in vitro maturation conditions(LUVAS, 2007) Yadav, Ramprakash; Trilok, NandaThe present study was carried out on Cumulus Oocyte Complex (COCs) aspirate from buffalo ovarian follicles obtained from slaughter house and investigated for the presence of Bcl-2 and Bax gene transcript in poor and good quality COCs graded mainly on the basis of cumulus cells layers enclosing oocyte and was further related with outcome of in vitro fertilization (IVF) rates in these groups. The overall recovery rate of COCs per ovary was 0.84 with 1667 COCs recovered from 1965 ovaries. After their grading the average recovery rate of grade-I COCs was 0.38, lower as compare to grade-II with 0.48. The grade-I COCs had homogenous ooplasm with hazy zona pellucida, visible only as a dark structure enclosing ooplasm and oocyte completely enclosed inside several layers of cumulus cells. In poor grade COCs, the ooplasm was heterogeneous, and oocyte was completely or partially denuded with clearly visible zona pellucida and the cumulus layers were already expanded and detached from oocyte while handling. Good quality COCs showed maximum maturation percent 79.48 and minimum 68.57 with expanded cumulus cell layers and single polar body formation after in vitro maturation (IVM). In poor quality COCs this percentage varied between 8.69 and 4.83 percent in addition there was nuclear chromatin condensation, ooplasm shrinkage and increase in perivitelline space. Maturation was observed only in partially denuded COCs under grade-II COCs. Percent maturation was very less in poor quality COCs as compare with good quality. The percent cleavage rate after IVF under good quality COCs was found to vary between 64.44 percent and 55.31 with overall average of 61.57 %. In poor quality oocytes cleavage percentage ranged between 5.55 and 2.77 with overall 4.91 percent. This result clearly indicates that the cleavage rate in good quality COCs is much higher as compared to poor quality COCs graded on the basis of their morphology. Furthermore, zygote obtained from grade-I COCs developed beyond two celled stage in contrast from grade-II COCs were unable to develop beyond two celled stage and degenerated gradually. The blastomeres in such incompetent embryos were also not identical in size. The fertilized COCs showed formation of secondary polar body and unfertilized in both grade were found to degenerate gradually. For the first time our study showed the presence of Bcl-2 and Bax transcripts in COCs of buffaloes, which was detectable in COCs of both good and poor grade. No significant difference was observed for transcript level of Bcl-2 in grade-I COCs before and after maturation but the transcripts for Bax was up-regulated after maturation. Under grade-II COCs transcript level for Bax was relatively more as compare Bcl-2 before maturation but after maturation no transcripts were evident for both genes but β-actin, positive control was amplified. The genomic DNA isolated from COCs after IVM showed the presence of high molecular weight DNA along with diffuse pattern in gel indicating presence of low molecular weight fragmented nucleic acid, possibly generated due to action of caspase activated DNAse, but fragmentation of DNA giving ladder like pattern was not clearly evident which is hallmark for apoptosis. Such changes were not present in the DNA isolated from immature COCs in which only DNA of high molecular weight was evident possibly due to less number of apoptotic cells at the start of IVMThesisItem Restricted Studies on growth differentiation factor-9 transcripts in in vitro matured buffalo cumulus oocyte complexes(LUVAS, 2009) Malik, Hrudananda; Nanda, TrilokThe present study was carried out on buffalo ovarian follicles. Ovaries were obtained from slaughter house. Follicles present on the ovarian surface were counted and classified as small, medium and large follicles based on their diameter size. The follicles with diameter less than 5 mm were placed under the category of small follicles; those of diameter 5-9 mm were classified as medium follicles, whereas follicles with diameter more than 9 mm were placed under the category of large follicles. From these graded follicles cumulus oocyte complexes were aspirated and in vitro matured in TCM-199 supplemented with 10 percent FCS and 5 percent NBS and in only TCM-199 (control). In vitro matured cumulus oocyte complexes were investigated for the presence of GDF-9 gene transcript using RT-PCR analysis. After the RT-PCR analysis the amplified product was purified and cloned. The cloned product was further sequenced and analyzed to study the phylogenetic relationship of GDF-9 gene of buffalo with other species. The classification of follicle into different categories showed that the average surface count per ovary of individual class of follicle was found to be 6.80, 1.10 and 0.29 for small, medium and large follicles respectively. The overall mean COCs recovery rate was 1.3 per ovary. Based on cumulus-cell expansion, the overall maturation rate in 10 percent FCS, 5 percent NBS and control are 86.60, 76.47 and 54.79 percent respectively. RT-PCR analysis it revealed that GDF-9 transcripts were expressed in in vitro matured cumulus oocyte complexes from all grades of follicles in spite of variation in size. Upon nucleotide sequence it revealed that the sequences of GDF-9 gene is 99 percent similarity to that of Bubalus bubalis and this gene (Bubalus bubalis) clustered together with Bus taurus and separately clustered from Rattus norvegicus.ThesisItem Restricted Vitrification of buffalo oocytes and their post-thaw potential for in vitro fertilization(LUVAS, 2007) Yosef Deneke Belachew; Nanda, TrilokAvailability of developmentally competent oocytes, especially cryopreserved ones, is critical for in vitro embryo production and application of related biothecniques. Reportedly, matured oocytes are better vitrified than their immature counterparts. The objective of the present study was to assess the effect of BSA in place of FCS as maturation media supplement on vitrification of in vitro matured buffalo oocytes, to study the effect of different concentrations of ethylene glycol as a CPA on the vitrification of buffalo oocytes and their post-thaw potential for in vitro fertilization. Oocytes were aspirated from abattoir ovarian follicles of 2-8mm diameter. Oocytes were matured in TCM-199 supplemented with hCG, PMSG and containing either 0.4% BSA (group-I) or 10% FCS (group-II). Matured oocytes, denuded by vortexing, were equilibrated for 2 min each either in 2.5M ethylene glycol (EG) or 3.6M EG and vitrified either in 5M EG or 7.2M EG, before loading in 0.25ml French straws which were then plunged directly in liquid nitrogen. After at least one week of preservation, thawing was performed with a serial dilution of EG in 0.5M, 0.25M, 0.125M sucrose solutions. Post-thaw viability of oocytes was assessed either by morphological appearance of uniformly spread cytoplasm within intact zona, or staining of dead oocytes with trypan blue or by ultimate ability of the thawed oocytes to undergo in vitro fertilization as determined by cleavage. Post-thaw percentage of morphologically normal oocytes was higher in group-I oocytes vitrified in 7.2M EG (76.8%, 146/190) than in group-II oocytes (61.6% 98/ 159). A significantly higher percentage of morphologically normal oocytes were also recovered when oocytes were vitrified in 5 M EG. Namely, 81.8% for group-I (123/151) and 41.4% (79/191) for group-II, respectively.Thirty four in 7.2M EG vitrified warmed oocytes were randomly selected from group-I and 30 from group-II and were subjected to trypan blue vital staining, of which 24 (70.6%) from group-I and 6 (20%) from group-II were found to be viable. The same high survivability was also observed in group-I oocytes (64.6%, 31/48) than group-II oocytes (11.8%, 4/34), when oocytes were vitrified in 5M EG. Viability assessment by post-thaw IVF revealed that only 7 of 68 oocytes randomly selected in group-II (10.3%) got fertilized, in comparison to 22/112 oocytes (19.8%) in group-I as 7.2M EG was used for vitrification. Whereas 8.0% of group-I oocytes (6/75) and 4.4% group-II oocytes (2/45) got fertilized when the EG concentration was 5 M. These results suggested that for successful vitrification, BSA supplementation has a positive influence on post-thaw survival and maintenance of developmental competence of in vitro matured buffalo oocytes, in comparison to FCS supplement. Possible reason may include hardening of zona pellucida in FCS matured oocytes, which may affect permeation of cryoprotective agent into the oocytes for successful vitrification and also affect ability of sperm penetration in post-thaw oocyte. The overall low percentage of morphologically normal, viable oocytes and the low post-thaw IVF rate in oocytes vitrified in 5 M EG may be due to improper dehydration of the oocytes during vitrification.ThesisItem Restricted RAPD-PCR based characterization of Murrah and Nili Ravi breeds of buffalo(LUVAS, 2005) Ahlawat, Sonika; Sangwan, M.L.The present study was conducted to estimate genetic relatedness within and between Murrah and Nili Ravi breeds using RAPD-PCR. Genomic DNA was isolated from 20 animals of each breed, evaluated for purity, concentration and quality. Optimisation of PCR was carried out by varying the concentration of different components of the reaction mixture. A total of 11 decamer primers were employed for estimating band frequency, genetic identity index, band sharing frequency, genetic distance and mean average percentage difference between and within breeds. All the 11 primers amplified the genomic DNA, generating bands ranging from 150- 3000 bp. From the 11 random primers, a total of 110 bands were amplified and 78 of these (about 71%) were found to be polymorphic in Murrah breed. The number of polymorphic loci ranged from 2 to 13 in Murrah breed with an average of 7.10. From the 8 polymorphic random primers, a total of 57 bands were amplified and 34 of these (about 60%) were found to be polymorphic in Nili Ravi breed. The number of polymorphic loci ranged from 1 to 10 in -xxxvi- Nili Ravi breed with an average of 4.86. A total of 114 bands were amplified from the 11 random primers and 56 of these (about 49%) were found to be polymorphic between Murrah and Nili Ravi breeds. The number of polymorphic loci ranged from 2 to 11 between the breeds with an average of 5.82. Higher genetic similarity of 0.79 and 0.85 in Murrah and Nili Ravi breeds, respectively, was obtained as compared to between breeds genetic similarity of 0.62±0.06. Low overall genetic distance of 0.26 and 0.18 was observed in Murrah and Nili Ravi breeds, respectively, in comparison to between breeds genetic distance of 0.53±0.11. The MAPD in Murrah breed was 21.29% and in Nili Ravi breed was 14.57%. Between breeds MAPD was 36.64±5.85% indicating medium level of genetic diversity. Four random primers showed Murrah breed specific amplicons.ThesisItem Restricted Nucleic acid based characterization of field isolates of avian adenovirus from chickens(LUVAS, 2008) Gupta, Om Narayan; MinakshiAvian adenoviruses (AAV) are diverse group of pathogens causing variety of problems for poultry production. Most of them are regarded as complicating organisms in diseases primarily induced by other agents or as component of multifactorial problems. However A few AAVs are considered exceptionally highly pathogenic for chickens such as fowl adenovirus (FAV) causing hydropericardium syndrome (HPS) and inclusion body hepatitis (IBH). These diseases have posed a serious threat to poultry industry by causing severe economic losses through out the country in recent year. Antigenically, FAV is very complex and is reflected by the presence of 12 different serotypes. The major virus surface protein of adenovirus is the “hexon protein” on which, type, group and subgroup antigenic determinants are located. With a need for more rapid, sensitive and specific detection procedures, PCR assays of variable region of hexon gene combined with restriction endonuclease analysis have been proved to be very suitable. PCR-RFLP and nucleotide sequencing of locally prevalent AAV strains circulating in poultry population would eventually help in formulation of comprehensive disease diagnosis and control strategies. To address these concerns, the present study was undertaken to characterize AAVs circulating in this area. A total of 245 tissue samples from, chickens were screened 31 (12.4%) were found positive by PCR. Twenty-nine Positive samples were from 100 liver tissues suspected for IBH (29%). One tissue from trachea of chicken diagnosed with respiratory disease, and one from intestine of apparently healthy chicken was found positive. PCR-RFLP of hypervariable region hexon gene revealed presence of three FAV serotypes (FAV-4, FAV-9, and FAV-12) in Haryana state. Based on RFLP pattern, Out of 31 positive samples 25 (80%) were found to be FAV-12 indicating it as a major cause of IBH in this state. Representative of all three serotypes were also isolated in CEL cell culture and tested by PCR. Amplified partial length hexon gene hypervariable region were cloned and screened by touch PCR. The positive recombinant bacterial colonies were subjected to sequencing. The sequences were analysed using BlastN and aligned by Clustal X. The percent identity and distance matrices of the partial hexon gene sequences of Haryana isolates with representatives of all FAV serotypes from other countries were generated by MEGA version 4. Phylogenetic trees were constructed by analyzing nucleotide sequences by MEGA 4 software. Sequence comparision studies revealed that Indian sequences might have originated from Canada, U.S.A. or Belgium. Hence, it can be concluded that hypervariable L1 loop region of hexon gene of FAV might be useful in determining the evolution of virus. In conclusion, the vaccine strain used in Haryana is specific for HPS but not effective against various other serotypes associated with IBH cases in this region. Since incidence of IBH is several times more than HPS and so are the economic losses pertaining to it, hence there is strict need to formulate a vaccine which takes in to account multiple serotypes circulating in this region.ThesisItem Restricted Nsp4 gene based genetic diversity among diarrheic bovine and human group A rotaviruses(LUVAS, 2008) Atul Kumar Singh; MinakshiRotaviruses are the major cause of severe gastroenteritis and mortality in young children and animals. The rotavirus genome is composed of eleven segments of double-stranded RNA. Due to segmented nature of the RNA genome and wide host range, vast genetic and antigenic diversity exists amongst different isolates of rotavirus. Genetic diversity based on NSP4 gene sequence analysis of local bovine and human group A rotaviruses may help in improvement of further control strategies. Keeping these perspectives in the view, the present study was undertaken to know the genetic diversity among bovine & human group A rotaviruses by phylogenetic analysis based on NSP4 gene sequences. A total of 150 faecal samples from human infants and bovine calves from organized livestock farms & hospitals in north India, were screened and 16 samples were found positive by PAGE and 6 were positive by RT-PCR. All the samples revealed long electropherotypes by RNA-PAGE. NSP4 gene of BRV & HRV was amplified and yielded PCR product of 725bp. vp7 and VP4 genes of BRV & HRV were also amplified and yielded PCR product of 1011bp (VP7of BRV&HRV), 864bp (VP4 of BRV) and 877bp(vp4 of HRV). PCR Amplified NSP4 gene amplicons were cloned and screened by touch PCR. The positive recombinant bacterial colonies were subjected to sequencing. The sequences were analysed using BlastN and aligned by Clustal X. The percent identity and distance matrices of the partial NSP4 gene sequences with representatives of representative NSP4 genotypes were generated by MEGA version 4. Phylogenetic trees were constructed by analyzing nucleotide sequences by MEGA 4 software. By phylogenic and evolutionary analysis the genotype of buffalo, cattle, and human rotaviruses were classified as genotype A, A and B. By Phylogenetic analysis it was also confirmed that buffalo and cattle rotaviruses were clustered with Simian, equine, human and lamb rotaviruses. By sequence analysis it may be stated that there is interspecies transmission of rotaviruses among different host species. These results may have better implications for the design and implementation of successful h rotavirus vaccine strategies.ThesisItem Restricted Study on LH receptor transcripts in buffalo ovarian follicles(LUVAS, 2008) Lail, Najmul; Trilok NandaThe present study was carried out on buffalo ovarian follicles. Ovaries were obtained from slaughter house in summer and winter. Follicles present on the ovarian surface were counted and classified as small, medium and large follicles based on their diameter size. The follicles with diameter less than 5 mm were placed under the category of small follicles; those of diameter 5-9 mm were classified as medium follicles, whereas follicles with diameter more than 9 mm were placed under the category of large follicles. These graded follicles were isolated mechanically and looked for the presence of LHr transcript in each season. Each class of follicles was investigated for the presence of Lhr gene transcript in the two seasons using RT-PCR analysis. Further the concentration of LHr cDNa was checked in each category of follicles in the two different seasons using spectrophotometric estimation. The classification of follicles into different categories showed that the presence of higher proportion of small size follicles constituting 80.01% as compared to 16.37% of medium size and 3.6%of large size follicles during winter season. Where as in summer it was 83.28% for small follicles 14.1% of medium size and 8.5% for large sized follicles. The overall average number of follicles present on a single ovary was found to be 8.99 and 9.35 in summer and winter respectively. The average number of small, medium and large follicles was found to be 7.49, 1.27 and 0.23 respectively in summer. There was not much difference in the average count of small and medium follicles per ovary in the winter than that of summer. But there was a significant increase in the average number of large follicles per ovary in winter compared to that of summer season. It was found to be 0.33 in winter. Our study showed the presence of LHr gene transcripts in all the categories of follicles, LHr transcript expresses itself in all follicles irrespective of variation in their diameter size. LHr expression was also found to be continuous irrespective of variation in season that is LHr expression in ovarian follicles was found to be both in summer and winter season. A marked variation was found in the transcript level of LHr gene with increase in follicular size. It was observed that LHr transcript level increases with the increase in follicular size. There was also a notable variation in the transcript level of LHr in summer and winter. It was found that LHr expression decreases in the summer season for the corresponding follicles. This decrease in LHr transcript level during summer may be due to effect of heat stress on LHr expression in ovarian follicles.
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