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Sri Venkateswara Veterinary University, Tirupati

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  • ThesisItemOpen Access
    GENOMIC EXPRSSION OF CERTAIN PRO INFLAMMATORY CYTOKINES DURING TRANSITION PERIOD IN ONGOLE CATTLE (Bos indicus)
    (SRI VENKATESWARA VETERINARY UNIVERSITY, TIRUPATI - 517 502. (A.P.) INDIA, 2024-02) GNAPIKA .G; IQBAL HYDER (MAJOR); SRINIVAS PRASAD .CH; MUTHA RAO .M
    The present study was designed to evaluate the effect of transition stress in ongole cows during transition period. Twelve animals (n=12) were randomly selected from livestock research station (LRS), Lam farm, Guntur, Andhra Pradesh of which 6 are multiparous pregnant animals (transition group) and 6 are clinically healthy heifers (control group). From gravid females, samples were collected during transition period week-3, week-2, week-1, on the day of parturition, week+1, week+2, week+3. Physiological parameters like RT, RR and PR were recorded daily at 9:00 AM throughout the study period. Three aliquots of blood sample (whole blood with anticoagulants in two vacutainers and one vacutainer of blood with clot activators for serum isolation) were collected. One aliquot of whole blood was directly used for analysis of haematological parameters (TEC, TLC, Hb, PCV, Platelets, DLC, Erythrocyte indices) and blood glucose, while another aliquot was used for separation of PBMC and subsequent RNA isolation for gene expression studies of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α), HSP90. Blood with clot activators was processed to separate serum, for estimation of biochemical parameters (total protein, albumin, cholesterol, ALT, AST, Creatinine, BUN, Calcium, Phosphorous). The mean±SE values of RT, RR and PR vary significantly (p<0.05) between control and transition group. The mean±SE values of RT, RR and PR didn’t vary significantly (p>0.05) in between weeks of transition group. The mean±SE values of haematological parameters vary significantly (p<0.05) between control and certain weeks in transition group. The mean±SE values of haematological parameters like platelets, MCHC, granulocytes and monocytes vary significantly (p<0.05) within transition group and also vary significantly between the control and transition group and significant (p<0.05) difference was observed within the weeks of transition group and between control and transition group. The mean±SE values of albumin and creatinine did not vary significantly (p>0.05) between control and transition group and no significant (p>0.05) difference was observed. The blood glucose levels vary significantly (p<0.05) between control and transition group when compared to control group the levels of glucose decreased in transition weeks and the blood glucose also vary significantly (p<0.05) within the weeks of transition group, the blood glucose levels decreased significantly from prepartum to on the day of parturition and then increased significantly from the day of parturition to postpartum and significant (p<0.05) difference was observed between control and transition group and within the weeks of transition group. The mean±SE values of ALT were significantly (p<0.05) higher in postpartum when compared to control. The mean±SE values of AST vary significantly (p<0.05) within the weeks of transition group and significant (p<0.05) difference was noticed within the weeks in transition group. The mean±SE values of AST were significantly (p<0.05) higher in prepartum week-2, week-1, 0day and postpartum weeks in comparison to control. The mean±SE values of total protein and globulin were significantly (p<0.05) higher in week-3, week-2 and week+1 of transition group in comparison to control. The mean±SE values of BUN, calcium, phosphorous were significantly (p<0.05) lower on week-2 in transition group when compared to control. The mean±SE values of BUN vary significantly (p<0.05) within the weeks in transition group and significant (p<0.05) difference was within the weeks of transition group. The mean±SE values of cholesterol were significantly (p<0.05) lower on week-2, week-1, 0day, week+1 in transition group in comparison to control. The relative mRNA expression of IL-1β was significantly upregulated in transition group on prepartum 0 day and postpartum week+1, week+2, week+3 compared to control and the expression of IL-1β was Significantly (p<0.05) downregulated in transition group compared to control group on week-3. The relative mRNA expression of IL-6 was not significantly (p>0.05) different between control group and animals in week 3, week-2, week-1, 0day, week+1, week+2 and week+3. The relative mRNA expression of TNF-α was significantly(p<0.05) upregulated in transition group on prepartum week 2, 0 day and postpartum week+3 compared to control. The relative mRNA expression of TNF-α was significantly (p<0.05) downregulated in transition group on week+1 compared to control. The relative mRNA expression of HSP90 was significantly (p<0.05) downregulated in transition group compared to control group in prepartum week-3, week 1, and postpartum week+1, week+2, week+3. No significant (p>0.05) difference was observed between the control group and week-2, 0 day of transition group.
  • ThesisItemOpen Access
    GENE EXPRESSION PATTERNS OF SELECTIVE MONOCARBOXYLATE TRANSPORTERS IN BUBALINE SPERMATOZOA
    (SRI VENKATESWARA VETERINARY UNIVERSITY, TIRUPATI - 517 502. (A.P.) INDIA, 2023-10) NIRUPAMA .M.U.L; SRINIVASA PRASAD .CH (MAJOR); IQBAL HYDER; ASWANI KUMAR .K; MUTHA RAO .M
    The objective of the present study was to evaluate the gene expression of certain monocarboxylate transporters in bubaline spermatozoa. Eight adult healthy buffalo bulls between three to six years of age were randomly selected from ABC Semen Station, Veeravalli, Krishna District, Andhra Pradesh. The fresh semen samples were collected from eight buffalo bulls once in a week for four weeks by AV method. As a preliminary quality check, fresh semen samples (4 samples from each bull, n=32) were evaluated at the semen station for volume, concentration, mass motility, individual progressive motility, viability, HOST, acrosomal integrity and the mean±SE values were found to be 4.09±0.26mL, 1243.875±65.621 million/mL, 3.86±0.34 and 70.04±9.92%, 73.42±0.49%, 73.98±0.56% and 87.75±0.52% respectively. With respect to evaluation of gene expression of monocarboxylate transporters, the fresh semen samples were transported at 37°C. The samples were divided in to two groups. Fresh uncapacitated as control (4 samples from each bull n=32) and fresh capacitated (n=32). It was taken care that fresh semen samples had sperm concentration of 50-60 million spermatozoa. In vitro capacitation of fresh semen samples was performed in BO media supplemented with heparin for 2 hrs. Genes such as SLC16A1 and SLC16A7 were selected for the study. The mRNA isolated from respective samples were converted to cDNA and subjected to q-PCR analysis. The target genes were normalized to endogenous control GLUT5. The mRNA expression of genes in fresh capacitated was analyzed relative to fresh uncapacitated as control. Fold change in gene expression was calculated using ∆∆Cq method. Significant difference between groups for fold change was analyzed using t test and multiple comparison was performed by Duncan multiple range test. No significant difference (p<0.05) was observed in the mRNA expression of MCT1and MCT2 between fresh capacitated and fresh uncapacitated (control) semen samples. It was concluded that, in vitro capacitation induces no significant change in the mRNA expression of both SLC16A1 and SLC16A7 genes which encode for MCT1 and MCT2 respectively and MCTs exert minimal role in capacitation related biochemical changes in bubaline spermatozoa.
  • ThesisItemOpen Access
    EFFECT OF EXOGENOUS MELATONIN ADMINISTRATION ON EXPRESSION PATTERNS OF SELECTIVE AQUAPORIN GENES IN HEAT STRESSED BUBALINE PERIPHERAL BLOOD MONONUCLEAR CELLS
    (SRI VENKATESWARA VETERINARY UNIVERSITY, TIRUPATI - 517 502. (A.P.) INDIA, 2023-08) CHAITANYA .G; IQBAL HYDER (MAJOR); SRINIVASA PRASAD .CH; ASWANI KUMAR .K
    The present study was designed to evaluate the effect of exogenous melatonin administration on heat stress (HS) amelioration in buffaloes during summer season. Fifteen adult Murrah buffaloes were randomly selected from Livestock Farm Complex (LFC), NTR College of Veterinary Science, Gannavaram, Andhra Pradesh. The study was conducted during the months of May - July 2022. The animals were divided into three groups, Group-1: (GI, Control n=5), Group-2: (GII, n=5 Melatonin @18mg/50 kg bwt. Subcutaneously single administration) and Group-3: (GIII, n=5 Melatonin@18mg/50 kg b wt. subcutaneously twice at fortnight interval. The AT and RH were recorded throughout the study period and THI was calculated. Physiological parameters like RT, RR and PR were recorded daily at 1:00 PM throughout the study period. Two aliquots of blood sample (whole blood with anticoagulants and blood with clot activators for serum isolation) were collected from all the 15 animals in weekly interval for seven weeks. An aliquot of whole blood was directly used for analysis of haematological parameters (TEC, TLC, Hb and PCV) and blood glucose, while the remaining aliquot was used for separation of PBMC and subsequent RNA isolation for gene expression studies of HSP90 and selective aquaporins (AQP1, AQP8 and AQP11). Blood with clot activators was processed to separate serum, for estimation of biochemical parameters (total protein, albumin, cholesterol, ALT and AST). The THI value above 78 was observed throughout the study period indicating that the animals were under HS. The mean±SE values of RT, RR and PR didn’t vary significantly (p>0.05) between control and melatonin treated buffaloes The mean±SE values of haematological parameters didn’t vary significantly (p>0.05) between control and melatonin treated buffaloes and no significant difference (p>0.05) was observed among all the weeks of our study period. The mean±SE values of total protein and albumin did not vary significantly (p>0.05) between control and melatonin treated buffaloes and no significant (p>0.05) difference was observed among all the weeks. The blood glucose levels were significantly (p<0.05) higher in GIII compared to control in week 6 and in overall mean. The mean cholesterol concentration was significantly (p<0.05) higher in GIII compared to control during week 5, 6 and in overall mean. The AST levels were significantly (p<0.05) lowered in GIII compared to control in all the weeks and also in overall mean. The ALT levels were significantly (p<0.05) lowered in GIII during week 5, week 6 and in overall mean compared to control. The mean cortisol levels were significantly (p<0.05) lowered in GIII compared control during week 4, 5, 6 and also in overall mean. The relative mRNA expression of HSP90 and AQP8 were significantly (p<0.05) upregulated on week 2 and 3 in GII and on week 2, 3, 4 and 5 in GIII compared to control. Further, the overall expression of HSP90 was significantly (p<0.05) upregulated in melatonin treated animals compared to control. Whereas for AQP8 overall expression was significantly (p<0.05) upregulated in GIII compared GII to control. There was no significant (p>0.05) difference in the mRNA expression of AQP1 and AQP11 between control and melatonin treated groups (GII and GIII) during all the weeks of the study and also in overall. Increased expression of HSP90 in melatonin treated group compared to control in our study suggests the role of melatonin in prevention of abnormal protein aggregation and misfolding of proteins caused due to oxidative stress. The increase in expression of AQP8, a peroxyporin in melatonin treated group establishes the role of melatonin in facilitating H2O2 diffusion and efflux of other free radicals generated due to oxidative stress induced by heat stress. A THI of above 78 throughout the study period is indicative of heat stress in animals. It can be concluded from our study that Melatonin administration can substantially ameliorate the effects of heat stress in buffaloes as by altering biochemical parameters (glucose, cholesterol, AST, ALT and cortisol) and further dynamically altering expression of HSP90 and AQP8 genes.
  • ThesisItemOpen Access
    EFFECT OF EXOGENOUS MELATONIN ADMINISTRATION ON EXPRESSION PATTERN OF CERTAIN MONOCARBOXYLATE TRANSPORTER GENES IN HEAT STRESSED BUBALINE PERIPHERAL BLOOD MONONUCLEAR CELLS
    (SRI VENKATESWARA VETERINARY UNIVERSITY, TIRUPATI - 517 502. (A.P.) INDIA, 2023-08) SUJANA GONTLA; SRINIVASA PRASAD .CH (MAJOR); VASANTHA SESHU KUMARI .I; ASWANI KUMAR .K
    The objective of the present study was to know the effect of exogenous melatonin administration on physiological, oxidant, antioxidant and cortisol profiles along with expression pattern of MCTs and HSP70 in heat stressed Murrah buffaloes. Fifteen non pregnant adult Murrah buffaloes of 5-8 years of age were selected randomly and divided into three groups Control (n=5), T1 (n=5) melatonin@18mg/50 kg b.wt single administration and T2 (n=5) melatonin@18mg/50 kg b.wt twice at fortnight interval. The blood samples were collected by jugular vein puncture into EDTA and serum vials at weekly intervals for a period of seven weeks during study period. Oxidant, antioxidant, cortisol profiles were estimated. The physiological parameters and THI were also recorded during the period of study. THI of the present study recorded was >80 except during 5th week (THI-78). RT, RR and PR were significantly (p<0.05) lower in melatonin treated buffaloes compared to control. Significant decrease (p<0.05) was observed in RT of T2 compared to T1 while no significant (p>0.05) difference was found in RR and PR in between these groups. MDA and cortisol levels were significantly (p<0.05) decreased in T1 and T2 compared to control, while significant (p>0.05) decrease was observed T2 compared to T1. Variation in both parameters was observed in all three groups with respect to THI. The mean values of SOD, CAT and GPx were significantly higher (p<0.05) in both treatment groups compared to control, with highest values obtained (p<0.05) in T2 among all three groups. The mRNA expression of MCT 1, 2 and 8 were significantly (p<0.05) downregulated in melatonin treated groups compared to control. HSP70 expression was upregulated in treatment groups compared to control. No significant (p>0.05) difference in expression of MCT1 and HSP70 was observed between T1 and T2. The expression of MCT2 and MCT8 was significantly (p<0.05) higher in T1 than T2. In conclusion, the present study demonstrated the effective role of melatonin in ameliorating heat stress which is evident through the study on various heat stress markers like physiological parameters, estimation of oxidant and antioxidant enzyme activity, estimation of stress hormone cortisol and gene expression studies on monocarboxylate transporters MCT 1, 2 and 8 along with HSP 70. Hence, we can conclude that melatonin could be effective in ameliorating heat stress in buffaloes.
  • ThesisItemOpen Access
    GENE EXPRESSION PATTERNS OF SELECTIVE AQUAPORINS IN BUBALINE SPERMATOZOA
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2022-07) UDAY SIVA NAGA SANKAR, A; SRINIVASA PRASAD, CH(MAJOR); IQBAL HYDER; MUTHA RAO, M; ASWANI KUMAR, K
    The present study was designed to assess the gene expression patterns of specific aquaporins (AQP’s) in bubaline spermatozoa. Eight healthy Murrah bulls aged between three to six years were selected from ABC Semen Station, Veeravalli, Krishna District, Andhra Pradesh. Fresh and frozen semen samples were collected for the study. As a preliminary quality evaluation, fresh semen samples (4 samplings from each bull, n=32) were examined for semen volume, sperm concentration, mass motility, individual progressive motility, abnormalities, viability, hypo-osmotic swelling test (HOST) and acrosomal integrity. The corresponding mean±SE values were found to be 4.337±0.29mL, 1147±3.10million/mL, 3.637±0.14, 71.5±0.18%, 9.36±0.82, 74.42±0.49%, 74.92±0.56% and 86.40±0.70%, respectively. Similarly, frozen semen samples (four straws per sample, n=32) were also evaluated for concentration, post thaw motility, viability, HOST and acrosomal integrity and the mean±SE values were found to be 19.68±0.28million/straw, 51.82±0.23%, 62.15±0.45%, 51.10±1.17% and 74.12±1.043%, respectively. The functional parameters were compared between fresh and frozen samples using unpaired t-test and it was observed that percent viability, HOST and acrosomal integrity were significantly (p0.05) difference was noticed between the down regulated groups. The expression of AQP7 showed no significant (p>0.05) difference between control and frozen non capacitated group, while the expression was significantly (p<0.05) upregulated in frozen capacitated compared to fresh capacitated group. The mRNA expression of AQP8 was significantly (p<0.05) down-regulated in fresh capacitated and frozen non-capacitated groups compared to fresh non-capacitated control. Further, a significant (p<0.05) increase in the expression of AQP8 was noticed in frozen capacitated compared to fresh capacitated group. The mRNA expression of AQP11 was significantly (p<0.05) upregulated in frozen capacitated compared to fresh capacitated and frozen non capacitated groups. The expression pattern of AQP3 is indicative of intense oxidative stress during capacitation and cryopreservation, while the expression pattern of aquaglyceroporins AQP7 and 11 is suggestive of its protective role in cryopreservation (AQP7) and freeze thaw process (AQP7 and 11) via influx and efflux of glycerol which was added to tris-yolk citrate extender. Similarly, AQP8 being a peroxiporin, its protective role in combating oxidative damage by excess ROS formed due to cumulative effect of both cryopreservation and capacitation has been noticed. The present study confirmed the presence of AQP3, 7, 8 and 11 in Murrah buffalo spermatozoa. Further, the study revealed that the studied genes have potential role in cryotolerance (AQP7), capacitation induced oxidative stress (AQP8) and freeze thaw process (AQP 7 and 11) of frozen semen.
  • ThesisItemOpen Access
    GENOMIC EXPRESSION OF CAPACITATION RELATED FERTILITY BIOMARKERS IN BUBALINE SPERMATOZOA
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2021-04) LAVANYA, SIRIGIRI; SRINIVASA PRASAD, CH (MAJOR); SIVA KUMAR, A.V.N; ASWANI KUMAR, K
    The objective of the present study was to evaluate the genomic expression of capacitation related fertility biomarkers in Bubaline spermatozoa. Eight adult healthy buffalo bulls between three to six years of age were randomly selected from ABC Semen Station, Veeravalli, Krishna District, Andhra Pradesh. The fresh semen samples were collected from eight buffalo bulls once in a week for four weeks by AV method. The frozen semen straws were also obtained from the same bulls semen. As an preliminary quality check, fresh semen samples (4 samplings from each bull, n=32) were evaluated for semen volume, sperm concentration, mass motility, individual progressive motility, viability, HOST, acrosomal integrity and the mean±SE values were found to be 4.48±0.24 mL, 909.28±82.29 million/mL, 3.79±0.12, 69.92±9.46%, 73.37±0.38%, 74.47±0.37% and 87.14±0.52% respectively. The frozen semen samples (n=32) were also evaluated for concentration, post thaw motility, viability, HOST and acrosomal integrity with mean±SE values of 19.28±0.24 million/straw, 48.13±0.66%, 61.20±0.37%, 52.82±0.68% and 76.37±0.41% respectively. Comparison was made between fresh and frozen samples for parameters like viability, HOST and acrosomal integrity using unpaired t-test. The percent viability, HOST and acrosomal integrity was significantly (p<0.05) higher in fresh compared to frozen semen samples. In order to evaluate the gene expression of capacitation related fertility biomarkers, the fresh and frozen samples were divided in to four groups. Fresh non-capacitated as control (four samples from each bull, n=32), fresh capacitated (n=32), frozen non-capacitated (4 samples from each bull by pooling 4 straws each, n=32) and frozen capacitated (n=32). In vitro capacitation of fresh and frozen semen samples was performed in BO media supplemented containing heparin. Genes such as ADCY10, PRKACA, AKAP4, CatSper2, CRISP2 and HSP90 were selected for the study. The mRNA isolated from respective samples were converted to cDNA and subjected to q-PCR analysis. The target genes were normalized to endogenous control GLUT5 and mRNA expression of fresh capacitated, frozen non-capacitated and frozen capacitated was analyzed relative to fresh non-capacitated as control. Relative fold change was calculated using ΔΔCq method. A significant difference between groups for fold change was analyzed using ANOVA and multiple comparison was performed by Duncan multiple range test. The mRNA expression of ADCY10, PRKACA and AKAP4 was significantly (p<0.05) up-regulated in fresh capacitated, frozen non-capacitated and frozen capacitated compared to fresh non-capacitated control. The mRNA expression of CATSPER2 and CRISP2 was significantly (p<0.05) up-regulated in fresh capacitated and frozen non-capacitated compared to fresh non-capacitated control. The mRNA expression of HSP90 was significantly (p<0.05) higher in frozen non-capacitated compared to fresh non-capacitated control. The mRNA expression of ADCY10, PRKACA, AKAP4, CATSPER2, CRISP2 and HSP90 was significantly lower in frozen capacitated compared to frozen non- capacitated group. From the present study it was concluded that the genes studied in the present study could be used as capacitation related biomarkers. We further state that cryopreservation induces capacitation like changes and these changes have much more impact on expression of capacitation related genes.
  • ThesisItemOpen Access
    GENOMIC EXPRESSION OF ATP SYNTHESIS ASSOCIATED FERTILITY BIOMARKERS IN BUBALINE SPERMATOZOA
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2021-03) SAI KIRAN, B.V.S; SRINIVASA PRASAD, CH (MAJOR); RAMBABU NAIK, B; ASWANI KUMAR, K
    The present study was designed to study the genomic expression of ATP synthesis associated fertility biomarkers in Bubaline spermatozoa. The present research was conducted on both fresh and frozen semen, collected from eight Murrah buffalo bulls of age (4-7 years) maintained at ABC semen bank, Veravalli, A.P. Fresh semen samples (n=32) were collected using a sterilized artificial vagina (AV) over a male dummy bull once in a week for four weeks from eight bulls. The samples were transferred to the laboratory for physio-morphological evaluation i.e. volume, sperm concentration (millions/ml), mass-motility, individual motility %, live and dead %, HOST reactive % and acrosome integrity % and the Mean±SE were found to be 4.46±0.23 mL, 921.63±77.65 million/mL, 3.8±0.14, 71.48±0.7%, 73.73±0.70%, 74.53±0.85 and 87.05±0.75 % respectively. In the case of frozen semen (n=32), straws from same selected eight bulls were obtained and assessed for sperm concentration, post thaw motility %, live and dead %, HOST reactive % and acrosome integrity % and the Mean±SE were found to be 19.15±0.23 million/straw,51.17±0.81 %, 62.15±0.45 %, 53.78±0.85 % and 75.31±0.83 % . Comparison between fresh and frozen samples was performed for viability, HOST and acrosome integrity and found that the percent was significantly (p<0.05) higher in fresh compared to frozen semen samples. For gene expression study the fresh and frozen semen samples were grouped into four groups, fresh non capacitated (control, four samples from each bull, n=32), fresh in vitro capacitated (32), frozen non capacitated (four samples from each bull by pooling four straws each, n=32) and frozen in vitro capacitated (n=32). qPCR was performed to evaluate the relative gene expression of GAPDHS, PGK2, ENO4, MDH2, ATP5F1B, AK1 and HSP 70 and normalized to GLUT5 in all the four groups. The mRNA expression of GAPDHS, ENO4, PGK2, MDH2 and ATP5F1B were significantly (p<0.05) up-regulated in frozen non-capacitated compared to fresh non capacitated group (control). The mRNA expression of ENO4 was significantly (p<0.05) up-regulated in fresh capacitated and frozen non capacitated groups compared to the frozen capacitated group. The mRNA expression of PGK2 was significantly (p<0.05) up-regulated in frozen non-capacitated compared to rest of the three groups. Whereas significant (p<0.05) upregulation in the mRNA expression levels was also found in frozen capacitated compared to the fresh non capacitated group (control). The mRNA expression of MDH2 was significantly (p<0.05) up-regulated in frozen non-capacitated compared to rest of the three groups. Whereas significant (p<0.05) upregulation in the mRNA expression levels was also found in fresh capacitated and frozen capacitated groups compared to the fresh non capacitated group (control), however there was no significant variation seen between fresh capacitated and frozen capacitated groups. The mRNA expression of ATP5F1B was significantly (p<0.05) up-regulated in fresh capacitated and frozen non-capacitated compared to rest of the other two groups. On the contrary, the mRNA expression of AK1 was significantly (p<0.05) up-regulated in fresh capacitated compared to rest of the three groups. The mRNA expression of HSP 70 was significantly (p<0.05) down-regulated in frozen non capacitated and frozen capacitated compared to fresh non capacitated (control), group. From the present study, it was concluded that evaluation of critical metabolic enzymes such as Adenylate Kinase 1 which was down regulated during cryopreservation would help us to understand the handling efficiency of semen post collection to ensure a proper fertility after it is thawed/ when it is actually intended to AI or in vitro fertilization.
  • ThesisItemOpen Access
    LEPTIN RECEPTOR EXPRESSION IN CULTURED OVARIAN FOLLICLES OF SHEEP
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2017-05) SRAVANI PRAGNA, K; Siva Kumar, A.V.N(MAJOR); Rambabu Naik, B; Punya Kumari, B
    ABSTRACT : The expression of Leptin receptor (LepR) mRNA and protein was studied in sheep using Real time quantitative PCR and Immunohistochemistry in the cumulus cells and oocytes from : (i) In vivo grown preantral, early antral, antral, large antral follicles and Cumulus Oocyte Complexes (COCs) obtained from large antral follicles and subjected to 24h of In Vitro Maturation ( IVM) and (ii) Preantral follicles (PFs’) exposed to TCM199B, TCM199BL (10ng/mL) and TCM199BHGL (10ng/mL) media for 3min, cultured in vitro for two, four or six days and subsequently matured in vitro for 24h in respective cultures separately. LepR was observed at all stages of in vivo grown and in vitro cultured ovarian follicles in both cumulus cells and oocytes. Expression levels and patterns of LepR mRNA in PFs’ cultured in TCM199BHGL was similar to in vivo in all the stages except in cumulus cells from COCs after in vitro maturation for 24h, where the expression was significantly higher which was a positive effect. The expression of LepR in cumulus cells among the three different in vitro culturing conditions was significantly higher in PFs’ cultured in TCM199BHGL medium. In the oocytes, the Leptin receptor expression was similar or significantly higher in PFs’ cultured in TCM199BHGL medium than in vivo grown follicles except in PFs’ exposed to 3min stage which could suggest the synergistic action of growth factors and hormones with Leptin. Immunohistochemistry studies revealed highest intensity of Leptin receptor protein expression in the oocytes. Mild to moderate staining was observed in cumulus cells with good intensity in large antral stages. This protein expression coincided with gene expression. From our studies it is concluded that Leptin along with other growth factors and hormones supplementation stimulated the expression of Leptin receptor mRNA and protein on par with that of in vivo /even higher than that of in vivo condition.
  • ThesisItemOpen Access
    INFLUENCE OF LEPTIN ON THE EXPRESSION OF p53 and BAK GENES IN CULTURED OVARIAN FOLLICLES IN SHEEP
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2015-11) PRABHAVATHI, T; RAO, V.H(MAJOR); SIVA KUMAR, A.V.N; VEERABRAHMAIAH, K
    ABSTRACT : The present study was conducted to know the influence of Leptin on the expression of p53 and BAK (apoptotic) genes in the sheep preantral follicles (PFs’) cultured in vitro. Ovaries of sheep were collected from the local slaughter house. Intact PFs’ (250-400μm) were isolated and placed individually in 20μl droplets of standard medium (TCM 199 B + 2 μg/ml FSH, 0.1μg/ml T4 + 10 ng/ml IGF-I + 1 mIU/ml of GH) supplemented with Leptin (10ng/ml) for 2, 4 or 6 days. Quantitative expression of Test genes (p53 and BAK) and Reference genes (18s RNA, RPLPO, HPRT1) were studied in the cumulus cells and oocytes isolated at different developmental stages (preantral, early antral, antral and large antral follicles) of the in vivo and in vitro grown follicles. The entire experiment was repeated twice. Duplicate samples of cDNA from each replicate of the experiment were subjected to relative-RT-qPCR. The pattern of expression of p53 gene in the cumulus cells from ovarian follicles grown in vivo and cultured in Leptin was similar from preantral to large antral follicle stage. In the oocytes, however, such similarity was restricted up to the antral follicle stage only. It is concluded that Leptin in culture medium mimiced the pattern of p53 expression in in vitro as in in vivo to some extent. The BAK gene expression was undetected in the cumulus cells and oocytes isolated from all the stages of both in vivo and in vitro developed ovarian follicles. Accordingly it is concluded that (i) Leptin has no influence on the expression of BAK gene in cultured ovarian follicles in sheep (ii) BAK may not be an important regulator of apoptosis in ovarian follicles and (iii) p53 plays a relatively more significant role than BAK in the regulation of apoptosis in ovarian follicles.