Mihir Sarkar

Expression of leptin and its receptor in corpus luteum during estrous cycle in buffalo (Bubalus bubalis)

Leptin is supposed to play a crucial role in ovarian luteal dynamics. The present study was aimed to investigate the importance of leptin and its receptors in buffalo corpus luteum (CL) obtained from different stages of the estrous cycle. Real-time RT-PCR (qPCR), western blot and immunohistochemistry techniques were applied to investigate mRNA expression, protein expression and localization of examined factors. Additionally to assess the contribution of leptin in progesterone production the expression profiles of StAR, P450scc and HSD were also investigated. In general, we demonstrated presence of leptin and its receptors in buffalo CL during the estrous cycle. The mRNA levels of leptin and its receptors were significantly up regulated in (P<0.05) in all the stages and highest levels were observed in mid and late luteal stages consistent with in vivo luteinization of buffalo CL and declined coincidental to luteal regression. The expression of StAR, P450scc and HSD factors maintained low in early luteal phase, after that level of expression increased steadily to show a significant rise (P<0.05) in mid luteal phase followed by gradual decline in late luteal phase and regressed CL and this correlates well with the Ob and ObR receptor activity, verifying their key role in progesterone and other steroids production in functional CL. As revealed by immunohistochemistry, leptin protein was localized predominantly in large luteal cells however leptin receptor (Ob-R) was localized in large luteal cells as well as in endothelial cells. It can be concluded from our study that leptin via its autocrine/paracrine effects play a significant role in promoting angiogenesis, steroidogenesis and also acts as key survival factor in bubaline CL.

Divergent development of testosterone secretion in male zebu (Bos indicus) and crossbred cattle (Bos indicus x Bos taurus) and buffaloes (Bubalus bubalis) during growth

Delayed pubertal development and low fertility of Bos indicus x Bos taurus crossbred male cattle and domestic buffaloes is hardly understood hence, a sensitive enzymeimmunoassay (EIA) was developed using the second antibody-coating technique and testosterone-3-O-carboxymethyloxime-horseradish peroxidase conjugate as a label for determination of testosterone in blood plasma. The EIA was validated by standard criteria. Blood samples were collected by venipuncture from growing male cattle (Karan Fries and Sahiwal) and buffalo (Murrah) and testosterone was estimated using the EIA procedure. Plasma testosterone concentrations increased significantly (P < 0.05) with advancing age. Testosterone concentrations were significantly (P < 0.01) higher in Sahiwal males in comparison to Karan Fries males. The low testosterone levels in crossbred than Sahiwal could imply that crossbred males have either not stabilized genetically or not adapted well in Indian climatic conditions resulting in poor libido and poor semen quality. The low testosterone levels in Murrah buffalo males may be the possible reason for delayed maturity in this species. The direct, sensitive EIA validated for estimating the plasma testosterone concentration was reliable for studying the testosterone profile in blood plasma of males. The results suggest that there could be a requirement for higher testosterone secretion by males during early stages of growth for attaining early sexual maturity.

Development and validation of a sensitive enzyme immunoassay (EIA) for blood plasma cortisol in female cattle, buffaloes, and goats

A highly sensitive enzyme immunoassay (EIA) that used the second antibody coating technique and the cortisol-horseradish peroxidase conjugate as a label for determination of free and total cortisol in blood plasma of dairy animals (cows, buffaloes, and goats) was developed. For biological validation of the EIA, blood samples were collected from the animals at 48 and 24 h before and 0, 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, and 132 h after dexamethasone administration. The EIA was performed directly with 20 μL of fresh plasma (for free cortisol) and also with 20 μL of heat-treated plasma (for total cortisol) after 1:5 dilutions with PBS. Cortisol standards ranging from 0.39 to 200 pg/well/20 μL were used, and the sensitivity of the EIA procedure was found to be 0.39 pg/well/20 μL, which corresponded to 0.02 ng/mL. In comparison with RIA the EIA was at least 4 times more sensitive and required 5 times less cortisol antiserum. In female cattle, buffaloes, and goats, the total, free, and bound plasma cortisol before dexamethasone administration was significantly (P < 0.05) higher than the total, free, and bound cortisol after dexamethasone administration. It can be concluded from these studies that the direct, sensitive EIA validated for estimating the free and total cortisol concentrations was sufficiently reliable and quick for studying the dynamics of cortisol distribution in blood plasma of dairy animals.

Modulatory effect of betaine on expression dynamics of HSPs during heat stress acclimation in goat (Capra hircus).

Changing climatic scenario with expected global rise in surface temperature compelled more focus of research over decoding heat stress response mechanism of animals and mitigation of heat stress. Recently betaine, a trimethyl form of glycine has been found to ameliorate heat stress in some species of animals. To overcome deleterious effect of heat stress, an attempt was taken to investigate the effect of betaine supplementation on heat stress mitigation in goats. Eighteen female Barbari goats were taken and randomly divided into 3 groups (n=6) such as control, HS (Heat stressed), HS+B (Heat stressed administered with betaine). Except for the control group, other groups were exposed to repeated heat stress (42 °C) for 6 h for sixteen consecutive days. Blood samples were collected at the end of heat exposure on day 1 (Initial heat stress acclimation - IHSA), day 6 (Short term heat stress acclimation - STHSA) and day 16 (Long term heat stress acclimation - LTHSA). When the groups were compared between different heat stress acclimatory phases, expression of all HSPs (HSP60, HSP70, HSP90 and HSP105/110) showed a similar pattern with a first peak on IHSA, reaching a basal level on STHSA followed by second peak on LTHSA. The messenger RNA (mRNA) and protein expression of HSPs was observed to be higher (P<0.05) in HS group than HS+B group except HSP90 on IHSA and HSP60 on STHSA. HSP105/110 expression was highest (P<0.05) on LTHSA. Immunocytochemical analysis revealed that HSPs were mainly localized both in nucleus and cytoplasm of PBMCs. In conclusion, heat stress increases HSPs expression and betaine administration was shown to have a dwindling effect on expression of HSPs, suggesting a possible role of this chemical chaperone on heat stress amelioration.

Expression and localization of fibroblast growth factor (FGF) family in buffalo ovarian follicle during different stages of development and modulatory role of FGF2 on steroidogenesis and survival of cultured buffalo granulosa cells

The present study investigated the expression and localization of FGF and its functional receptors in the follicle of buffalo and the treatment of FGF2 on mRNA expression of CYP19A1 (aromatase), PCNA, and BAX (BCL-2 associated X protein) in cultured buffalo granulosa cells (GCs). Follicles were classified into four groups based on size and E2 level in follicular fluid (FF): F1, 4-6mm diameter, E2<0.5ng/ml of FF; F2, 7-9mm, E2=0.5-5ng/ml; F3, 10-13mm, E2=5-40ng/ml; F4, >14mm, E2>180ng/ml. The qPCR studies revealed that the mRNA expression of FGF1, FGF2 and FGF7 were maximum (P<0.05) in theca interna (TI) whereas the transcripts of FGFR1, FGFR2, FGFR2IIIB and FGFR2IIIC were up-regulated (P<0.05) in GCs of F4 follicles. Protein expression of most members were maximum (P<0.05) in F4 follicles except FGFR3 and FGFR4. All members were localized in GC and TI with a stage specific immunoreactivity. Primary culture of GCs with treatment of FGF2 at different dose-time combinations revealed that the mRNA expression and immunoreactivity of CYP19A1 and PCNA were maximum (P<0.05) whereas BAX was minimum (P<0.05) with 200ng/ml at 72h of incubation. The findings indicate that FGF family members are expressed in a regulated manner in buffalo ovarian follicles during different stages of development where FGF2 may promote steroidogenesis and GC survival through autocrine and paracrine manner.

Expression analysis of NOS family and HSP genes during thermal stress in goat ( Capra hircus )

Approximately 50 genes other than heat shock protein (HSP) expression changes during thermal stress. These genes like nitric oxide synthase (NOS) need proper attention and investigation to find out their possible role in the adaptation to thermal stress in animals. So, the present study was undertaken to demonstrate the expressions of inducible form type II NOS (iNOS), endothelial type III NOS (eNOS), constitutively expressed enzyme NOS (cNOS), HSP70, and HSP90 in peripheral blood mononuclear cells (PBMCs) during different seasons in Barbari goats. Real-time polymerase chain reaction, western blot, and immunocytochemistry were applied to investigate messenger RNA (mRNA) expression, protein expression, and immunolocalization of examined factors. The mRNA and protein expressions of iNOS, eNOS, cNOS, HSP70, and HSP90 were significantly higher (P < 0.05) during peak summer, and iNOS and eNOS expressions were also observed to be significantly higher (P < 0.05) during peak winter season as compared with moderate season. The iNOS, eNOS, cNOS, HSP70, and HSP90 were mainly localized in plasma membrane and cytoplasm of PBMCs. To conclude, data generated in the present study indicate the possible involvement of the NOS family genes in amelioration of thermal stress so as to maintain cellular integrity and homeostasis in goats.

A comparative study on the expression profile of MCTs and HSPs in Ghungroo and Large White Yorkshire breeds of pigs during different seasons.

Thermal stress has a significant adverse effect on commercial swine production but it is not easy to measure. Animals may adapt to stress conditions by an alteration in the expression of stress-related genes such as heat shock proteins (HSPs) and monocarboxylate transporters (MCTs). The present study presents a comparative analysis of seasonally varied effects on the expression profiles of HSPs (27, 70, and 90) and MCTs (1, 2, and 4) transcripts in thigh muscle and colon tissue of Ghungroo and Large White Yorkshire (LWY) breeds of pig. By real-time polymerase chain reaction, the mRNA expression of HSP27 and HSP90 genes was found to be higher in both thigh muscle and colon tissue in Ghungroo compared to Large White Yorkshire pigs during the summer. However, the relative expression of HSP70 was significantly higher (P < 0.01) in Ghungroo compared to Large White Yorkshire pigs during both seasons in both thigh muscle and colon tissue. The expression of HSP90 was higher in Ghungroo when compared to LWY though the variation was non-significant (P > 0.05) in the colon during different seasons. However, in Ghungroo, the mRNA expression of MCT1 was found to be significantly (P < 0.05) higher in thigh muscle and colon regions during the summer compared to LWY, whereas MCT2 was expressed more in the colon in LWY compared to Ghungroo during the summer. The relative expression of mRNA of MCT4 was found to be significantly (P < 0.05) higher in thigh region in both summer and winter in Ghungroo compared with LWY. Thus, the study demonstrated that both HSPs and MCTs gene expression during thermal stress suggests the possible involvement of these genes in reducing the deleterious effect of thermal stress, thus maintaining cellular integrity and homeostasis in pigs. These genes could be used as suitable markers for the assessment of stress in pigs.

A Comparative Study of Growth Kinetics, In Vitro Differentiation Potential and Molecular Characterization of Fetal Adnexa Derived Caprine Mesenchymal Stem Cells.

The present study was conducted with an objective of isolation, in vitro expansion, growth kinetics, molecular characterization and in vitro differentiation of fetal adnexa derived caprine mesenchymal stem cells. Mid-gestation gravid caprine uteri (2–3 months) were collected from abattoir to derive mesenchymal stem cells (MSCs) from fetal adnexa {amniotic fluid (cAF), amniotic sac (cAS), Wharton’s jelly (cWJ) and cord blood (cCB)} and expanded in vitro. These cultured MSCs were used at the 3rd passage (P3) to study growth kinetics, localization as well as molecular expression of specific surface antigens, pluripotency markers and mesenchymal tri-lineage differentiation. In comparison to cAF and cAS MSCs, cWJ and cCB MSCs showed significantly (P<0.05) higher clonogenic potency, faster growth rate and low population doubling (PDT) time. All the four types of MSCs were positive for alkaline phosphatase (AP) and differentiated into chondrogenic, osteogenic, and adipogenic lineages. These stem cells expressed MSC surface antigens (CD73, CD90 and CD105) and pluripotency markers (Oct4, Sox2, Nanog, KLF, cMyc, FoxD3) but did not express CD34, a hematopoietic stem cell marker (HSC) as confirmed by RT-PCR, immunocytochemistry and flow cytometric analysis. The relative mRNA expression of MSC surface antigens (CD73, CD90 and CD105) was significantly (P<0.05) higher in cWJ MSCs compared to the other cell lines. The mRNA expression of Oct4 was significantly (P<0.05) higher in cWJ, whereas mRNA expression of KLF and cMyc was significantly (P<0.05) higher in cWJ and cAF than that of cAS and cCB. The comparative assessment revealed that cWJ MSCs outperformed MSCs from other sources of fetal adnexa in terms of growth kinetics, relative mRNA expression of surface antigens, pluripotency markers and tri-lineage differentiation potential, hence, these MSCs could be used as a preferred source for regenerative medicine.

Effect of mitotic inducers and retinoic acid blocker on expression of pluripotent genes in ES cells derived from early stage in vitro-produced embryos in buffalo

So far, it has been difficult to generate embryonic stem (ES) cell from early stage preimplantation embryos of buffalo. These ES cells will be more helpful for efficient embryo cloning and generation of body cells as they are more primitive than inner cell mass (ICM)-derived ES cells. The present study was conducted to find the effect of lipopolysaccharide (LPS), melatonin (N-acetyl-5-methoxytryptamine, a pineal gland product), and citral (3,7-dimethyl-2,6-octadienal and a retinoic acid synthesis blocker) on establishment of primary ES cell colonies, the comparative size of the ES cell colonies, and expression of pluripotent genes during extended period of culture in buffalo. Zona-free eight-cell stage in vitro fertilization (IVF) embryos were cultured in ES cell medium supplemented with none (media I as control), LPS (media II), citral melatonin (media III), or melatonin (media IV). The multiplication of blastomere leading to ES cell colony formation and expression of pluripotent genes were assessed up to day 20 of culture. The primary colony formation, the comparative size of the ES cell colonies, and expression of pluripotent genes in these colonies were better in the medium supplemented with melatonin in all days of culture. Within melatonin supplementation, the colony size was comparatively larger on day 8 and day 12 of culture. Further, with this supplementation, the Oct-4 and Nanog expression was comparatively higher on all days of culture. The results indicated that supplementation of melatonin helped in the formation of better primary ES cell colony as well as in the maintenance of pluripotency. The results also indicated that primary colonies developed on day 8 to day 12 of culture may be better for passaging them for establishment of ES cell line from early stage preimplantation IVF embryos of in buffalo.

Expression and localization of locally produced growth factors regulating lymphangiogenesis during different stages of the estrous cycle in corpus luteum of buffalo (Bubalus bubalis)

Recent experiments using expression, immunolocalization, and cell culture approaches have provided leading insights into regulation of luteal angiogenesis by different growth factor systems and its role in the function of corpus luteum (CL) in buffalo. On the contrary, lymphangiogenesis and its regulation in the CL are still poorly understood. The aim of this study was to evaluate the expression and localization of lymphangiogenic factors (vascular endothelial growth factor [VEGF]-C and VEGFD), their receptor (VEGFR3), and lymphatic endothelial marker (LYVE1) in bubaline CL during different stages of the estrous cycle and to investigate functional role of VEGFC and VEGFD in luteal lymphangeogenesis. The mRNA and protein expression of VEGFC, VEGFD, and VEGFR3 was significantly greater in mid and late luteal phases, which correlated well with the expression of LYVE1. The lymphangiogenic factors were localized in luteal cells, exclusively in the cytoplasm. Immunoreactivity of VEGFC was greater during midluteal phase and that of VEGFD was greater during the mid and late luteal phases. Luteal cells were cultured in vitro and treated for different time duration (24, 48, and 72 hours) with VEGFC and VEGFD each at 50, 100, and 150 ng/mL concentration and VEGFC with VEGFD at 100 ng/mL concentration. The temporal increase in LYVE1 mRNA expression was significant (P < 0.05) in VEGFC and VEGFC with VEGFD treatment and no significant change was seen in VEGFD treatment. Thus, it seems likely that VEGFD itself has little role in lymphangiogenesis but along with VEGFC it might have a synergistic effect on VEGFR3 receptors for inducing lymphangiogenesis. In summary, the present study provided evidence that VEGFC and VEGFD, and their receptor VEGFR3, are expressed in bubaline CL and are localized exclusively in the cell cytoplasm, suggesting that these factors have a functional role in lymphangiogenesis of CL in buffalo.