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Lala Lajpat Rai University of Veterinary & Animal Sciences, Hisar
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ThesisItem Restricted Comparative studies on urinary metabotypes and scrotal thermal signatures in normal and cloned Murrah buffalo bulls(Lala lajpat rai university Hisar, 2022-10) Krishan Kumar; Malik, Rakesh Kumar; Balhara, Ashok KumarThe present study conducted on fourteen Murrah buffalo bulls - ten growing (five each, cloned and normal) and four adult (two each, cloned and normal), evaluated urinary metabolites, skin surface temperatures of different body parts (with particular focus on scrotum) and hemato-biochemical parameters in scrotal thermal signatures in normal and cloned buffalo bulls across four seasons (summer, rainy, autumn and winter) over one year. There was no insignificant difference in body surface temperature of normal and cloned animals except winter season. The scrotal skin surface temperature was significantly high in cloned animals as compare to normal growing (p<0.05) animals. Among different body parts recorded for surface temperatures, the eye temperature had highest correlation with rectal temperature and was also found closed to it at any point of time, suggesting its use as an alternate, non – touch method for recording internal body temperatures. The growth rate and blood-biochemical parameters recorded insignificant differences between normal and cloned animals. 1H-NMR spectroscopy suggested that there are qualitative and quantitative differences in the identified 136-140 urine metabolites, between cloned and normal animals in both groups (i.e. adult and growing). The differences were marked in summer and winter seasons in adult cloned animals. Of the 25 most abundant metabolites identified nine viz. L-arabinitol, xylose, glucose-1-phosphate, uridine diphosphate glucose (UDP-glucose), L-isoleucine, L-leucine, methylmalonic acid, keto-leucine and L valine were found in higher concentration in urine of cloned animals. Contrarily, four metabolites of these abundant metabolites viz. myo-inositol, glucuronic acid, taurine and glycine metabolite showed high relative abundance in the normal animals. The functional bioinformatics suggest significant differences in urinary metabolites between cloned and normal animals w.r.t. thermoregulation, energy metabolism, hormone synthesis and degenerative changes. Study also points towards higher scrotal temperature in growing cloned animals. The thermal screening conducted in the study also suggest that ocular temperature can serve as an alternate, non-touch method for estimating internal body temperature in buffaloesThesisItem Restricted Immune-endocrine interaction in regulation of reproduction in bovines(LUVAS, 2005) Rose, Manoj Kumar; Puri, J.P.The study on the secretion of lymphocytic growth hormone (GH) and nitric oxide (NO) during various stages of estrous cycle and pregnancy, and in different age groups in cattle was carried out. In this study the effects of leptin, N-nitro-L-arginine methyl ester (L-NAME), sodium nitroprusside (SNP), steroids (estradiol, progesterone and tstosterone), cytokines leukaemia inhibitory factor (LIF) and interleukin-10 (IL-10) and incubation of lymphocytes at different temperatures 37.2°C (hypothermia), 38.6°C (normal cow temperature), 41.0°C (hyperthermia) on lymphocytic GH and NO secretion were studied. Total lymphocytic RNAs in cyclic and pregnant cows were also measured. The lymphocytic GH secretion was highest during luteal phase and lowest during mid-pregnancy. During mid-pregnancy lymphocytic NO secretion was higher than other stages of pregnancy and estrous cycle. There was no effect of incubation of lymphocytes without any treatment at different temperatures on lymphocytic GH and NO secretion during pregnancy. But incubation of lymphocytes treated with leptin in hypothermic condition decresed lymphocytic GH secretion without affecting lymphocytic NO secretion. There was no effect of steroids (estradiol, progesterone and testosterone) on lymphocytic GH and NO secretion during estrous cycle in cattle. In pregnant cows and in 6 months old calves incubation of lymphocytes with SNP significantly decresed the NO secretion. SNP treatment significantly increased lymphocytic GH secretion only in cyclic non-pregnant cows. Incubation of lymphocytes with LIF and IL-10 increased lymphocytic GH secretion during pregnancy, without affecting lymphocytic NO secretion. Lymphocytic GH secretion was higher in young (2 weeks old) and growing (6 months old) calves than adult cyclic cow. Plasma GH levels were higher during luteal phase in cow than other stages of estrous cycle and pregnancy. Plasma GH levels in young and growing calves were higher than adut cyclic cows. Total lymphocytic RNAs were more in pregnant than cyclic non-pregnant cows. In conclusion, the lowest level of lymphocytic GH in cattle is at a time when lymphocyric ACTH is very high (previously published work), suggesting a negative correlation among lymphocytic GH and ACTH secretion. Threrefore lymphocytes provide a fine control on immuno-stimulatory and immuno-inhibitory factors during pregnancy. The lymphocytic GH and NO secretion is independent of disease condition involving high fever. But the action of leptin on lymphocytic GH secretion is sensitive to decrease in temperature without affecting the lymphocytic NO secretion. The decrease in NO secretion with SNP treatment in pregnant cows and in 6 months old calves indicates that the control mechanisms for GH secretion is different in cow from pig where SNP treatment results in an elevation of NO levels.The increase in lymphocytic GH secretion with LIF and IL-10 treatments indicates the involvement of cytokines in immunomodulation during pregnancy independent of NO dependent mechanism. Higher lymphocytic GH levels in young and growing calves than adult cyclic animals indicates a possible age dependent role before puberty. Higher lymphocytic RNAs in pregnant cows than cyclic non-pregnant cows indicate changes at molecular level in immunomodulation during pregnancy.ThesisItem Restricted Effect of dietary fluoride levels on rumen ecosystem and growth in buffaloes(LUVAS, 2005) Madan, Jyotsana; Puri, J.P.The investigation was carried out in two phases to study the effect of dietary fluoride levels on rumen ecosystem and growth in buffaloes. In phase I, experiment was conducted on three male, rumen fistulated buffaloes in 3x3 latin square design. The three groups (A, B and C); were fed similar diets consisting of concentrate mixture and roughages, except the fluoride (F), which was added in the diet of group B and C at 30 and 60 ppm level (DM basis). After one month, the rumen liquor samples were collected at 0 hour 2, 4 and 6 hours (post feeding) intervals for two consecutive days. Analysis of rumen liquor samples revealed that there was no treatment effect on ruminal pH, total-nitrogen, ammonia-nitrogen and total volatile fatty acids. A significant increase in acetate (%) and decrease in propionate (%) was observed in group C. Fluoride at 60 ppm level caused a significant decrease in viable bacterial number as well as cellulase activity, where as protease activity was not affected by fluoride supplementation. In phase-II, the effect of dietary fluoride on growth, feed efficiency and thyroid hormones was studied. Twelve, male buffalo calves (10-12 months) were divided into three groups (A, B, and C). The diet in the three groups was similar to phase I. Feeding was done for 3 months. Body weight of each animal was recorded at start of experiment and at fortnightly interval. Dry matter intake and water intake was recorded daily. Blood samples were collected at 0 day, 30, 60 and 90 days. Dry matter intake decreased significantly in group C. The body weight gain decreased non-significantly in group B and C. Fluoride at both levels did not affect the water intake and feed conversion efficiency. Analysis of blood revealed that hemoglobin values decreased significantly in group C at 90 days. Neutropenia and Eosinophillia was observed in group C. A non significant decrease in blood glucose and serum protein in group B and C was observed. Hypocalcemia and hyperphosphatemia was observed in group C. Fluoride @ 60 ppm level resulted in higher serum ALP activity, whereas non significant effect on SGOT and SGPT. Thyroid hormones (T3 and T4) were not affected by supplementation of fluoride at both levels in the diet. Thus, it is clear that 30 ppm level is safe level where as 60 ppm F had some adverse effect on rumen ecosystem and growth performance.ThesisItem Restricted Genetic polymorphism of HSP70, LHR, LEPR genes and selective minerals status in postpartum anestrous buffaloes(LUVAS, 2013) Rajesh Kumar; Gupta, MeenakshiThe present study was planned to obtain the functional information on genetic polymorphism of Leptin receptor gene (LEPR), Luteinizing hormone gene (LHR) and Heat shock protein genes (Hsp70), status of anti-oxidants, minerals and hemogram. To fulfill the present objectives twenty postpartum anestrous (PPA) and twenty normal cyclic (NC) Murrah buffaloes were selected on the basis of their reproductive history and current status of reproductive organs. The animals were maintained as per the standard feeding and management. Venous blood sample was collected from each experimental animal for DNA isolation, plasma and hemolysate separation. The 275 bp fragment of the LHR gene, 413 bp fragment of the LEPR gene and 513bp fragment of the HSP70 gene present in the genomic DNA of Murrah breed was amplified by using the primers pair. These partially amplified segment were then subjected to restriction enzyme digestion and sequencing. Plasma and hemolysates stored at -20°C were used for estimation of antioxidants status, Protein and Minerals. Blood hematology was done with automatic hematology analyser (Vetscam 5HM, ABaxis Ltd.). The RE analysis by AluI and MobII of partially amplified LEPR gene, by HphI of partially amplified LHR gene and by HphI and MobII of partially amplified Hsp70 gene showed only one band pattern in both groups animals under the study, indicates no polymorphism at these RE sites in the population studied. After sequencing and their alignment, Single nucleotide polymorphism (SNP) was detected at 231, 247 and 347 in aligned sequence of LEPR gene and at 243 nucleotide position of LHR gene. Statistical analysis by chi square test did not reveal any significant association of these polymorphism in relation to PPA. After sequencing and their alignment, polymorphism at seven nucleotide positions were found to be associated with nucleotide variations across the various samples studied (192, 256,251,258, 367, 438 and 512). Out of these six variations were consider for further analysis as one variation had have very little representation in the samples. On applying Chi-square test, five out of six nucleotide positions (192, 256, 258, 367 and 438) revealed no significant association between these nucleotide variations and the health status of animal i.e. PPA and NC animals. However, a significant (P<0.05) association was found with relation to nucleotide position 251 and, it can be inferred that nucleotide C at position 251 is associated with postpartum anestrous condition. Since this nucleotide position is not included in the recognition sequence of any RE, we cannot differentiate between normal and PPA animals on the basis of RE analysis.ThesisItem Restricted “Genetic polymorphism of HSP70, LHR, LEPR genes and selective minerals status in postpartum anestrous buffaloes(LUVAS, 2013) Rajesh Kumar; Gupta, MeenakshiThe present study was planned to obtain the functional information on genetic polymorphism of Leptin receptor gene (LEPR), Luteinizing hormone gene (LHR) and Heat shock protein genes (Hsp70), status of anti-oxidants, minerals and hemogram. To fulfill the present objectives twenty postpartum anestrous (PPA) and twenty normal cyclic (NC) Murrah buffaloes were selected on the basis of their reproductive history and current status of reproductive organs. The animals were maintained as per the standard feeding and management. Venous blood sample was collected from each experimental animal for DNA isolation, plasma and hemolysate separation. The 275 bp fragment of the LHR gene, 413 bp fragment of the LEPR gene and 513bp fragment of the HSP70 gene present in the genomic DNA of Murrah breed was amplified by using the primers pair. These partially amplified segment were then subjected to restriction enzyme digestion and sequencing. Plasma and hemolysates stored at -20°C were used for estimation of antioxidants status, Protein and Minerals. Blood hematology was done with automatic hematology analyser (Vetscam 5HM, ABaxis Ltd.). The RE analysis by AluI and MobII of partially amplified LEPR gene, by HphI of partially amplified LHR gene and by HphI and MobII of partially amplified Hsp70 gene showed only one band pattern in both groups animals under the study, indicates no polymorphism at these RE sites in the population studied. After sequencing and their alignment, Single nucleotide polymorphism (SNP) was detected at 231, 247 and 347 in aligned sequence of LEPR gene and at 243 nucleotide position of LHR gene. Statistical analysis by chi square test did not reveal any significant association of these polymorphism in relation to PPA. After sequencing and their alignment, polymorphism at seven nucleotide positions were found to be associated with nucleotide variations across the various samples studied (192, 256,251,258, 367, 438 and 512). Out of these six variations were consider for further analysis as one variation had have very little representation in the samples. On applying Chi-square test, five out of six nucleotide positions (192, 256, 258, 367 and 438) revealed no significant association between these nucleotide variations and the health status of animal i.e. PPA and NC animals. However, a significant (P<0.05) association was found with relation to nucleotide position 251 and, it can be inferred that nucleotide C at position 251 is associated with postpartum anestrous condition. Since this nucleotide position is not included in the recognition sequence of any RE, we cannot differentiate between normal and PPA animals on the basis of RE analysis.ThesisItem Restricted Proteomic analysis of pregnancy-specific serum proteins in buffalo(LUVAS, 2012) Ashok Kumar; Gupta, MeenakshiModern proteomics technique offers an exciting opportunity in solving some of the most challenging problems of livestock sector, including development of diagnostics for early pregnancy. In the present study, sera samples obtained at weekly intervals from early pregnant (Day 0 to Day 42 post-AI) buffaloes, and on days 0, 7 and 14 from non-pregnant cyclic buffaloes, were subjected to depletion of high abundant proteins followed by 2-dimensional gel electrophoresis and densitometric analysis, which revealed the presence of at least 65 2-D gel spots exhibiting up-regulation, down-regulation or specific appearance at a specific stage during early buffalo pregnancy, except for the spots correlating with the high abundant proteins’ location. Comparison with ExPASy and NCBI databases matched 48 of these spots with known proteins, but with varying degrees of confidence in terms of Mascot score and the species. Although high abundant proteins were depleted before 2-D electrophoresis, yet in some of the picked spots isoforms of common abundant proteins viz. Serum Albumin, IgG, Serrotransferrin, Complement and MHC molecules, were found. Some of these identified spots appear promising pregnancy bio-markers, especially synaptojanin-1, apolipoprotein A-1, apolipoprotein B, Keratin 10 and Von Willebrand factors, which are documented to have role in embryogenesis and early pregnancy. Gene ontology based functional analysis of the identified proteins suggested that most of these were involved in regulatory, catalytic, cell differentiation and transporter functions. Involvement of identified proteins in 23 pathways, including the embryogenesis specific Wnt signalling pathway, confirms the essentiality of these proteins in early pregnancy. The result on the protein-protein interaction data from current experiment, described by network of 9 proteins, strengthens our belief that the significantly affected early pregnancy associated proteins are closely related. Considering that proteomics in animal research is still in its infancy, it is a long way before practical applications of such experiments could actually be realized.
