G. Sai Kumar

Expression of mRNA encoding leukaemia inhibitory factor (LIF) and its receptor (LIFRβ) in buffalo preimplantation embryos produced in vitro : markers of successful embryo implantation

Summary The objective of this study was to evaluate the effect of supplementation of recombinant leukaemia inhibitory factor (LIF) in culture media on blastocyst development, total cell number and blastocyst hatching rates and the reverse transcription-polymerase chain reaction analysis of preimplantation buffalo embryos to determine whether they contain the LIF-encoding mRNA and its beta receptor (LIFRβ) genes in different stages of preimplantation buffalo embryos. Cumulus-oocyte complexes retrieved from slaughterhouse buffalo ovaries were matured in vitro and fertilized using frozen buffalo semen. After 18 h of co-incubation with sperm, the presumptive zygotes were cultured in modified synthetic oviductal fluid without (control) or with rhLIF (100 ng/ml). There was no significant difference in the overall cleavage rate up to morula stage however the development of blastocysts, hatching rate and total cell numbers were significantly higher in the LIF-treated group than control. Transcripts for LIFRβ were detected from immature, in vitro-matured oocytes and in the embryos up to blastocyst stage, while transcripts for the LIF were detected from 8-16-cell stage up to blastocyst, which indicated that embryo-derived LIF can act in an autocrine manner on differentiation process and blastocyst formation. This study indicated that the addition of LIF to the embryo culture medium improved development of blastocysts, functional (hatching) and morphological (number of cells) quality of the blastocysts produced in vitro. The stage-specific expression pattern of LIF and LIFRβ mRNA transcripts in buffalo embryos indicated that LIF might play an important role in the preimplantation development and subsequent implantation of buffalo embryos.

Localization and Expression of Follicle-Stimulating Hormone Receptor Gene in Buffalo (Bubalus bubalis) Pre-Antral Follicles

Follicle-stimulating hormone (FSH) stimulates antral follicles to grow, but its role in earlier stages (pre-antral) of follicle development, if any, is obscure. Aim of this study was to study the expression of follicle-stimulating hormone receptor (FSHR) gene in different sizes of pre-antral follicles (PFs) (<150, 200, 250, 300, 350, 400 μm) and to find out an optimum dose of FSH for better growth, development and steroidogenesis of PFs in vitro. Buffalo ovaries were collected from a local abattoir, and PFs were isolated by mechanical method. A semi-quantitative RT-PCR amplification strategy was used for mRNA expression, while FSHR protein was localized by immunohistochemistry. Isolated pre-antral follicles (80-85 μm) were cultured in TCM-199 supplemented with 10% foetal bovine serum, 1% ITS and 30 ng/ml EGF served as control medium. Addition of three different doses of FSH (0.5, 1.0, 2.0 μg/ml) in control medium was considered as treatment groups. A single 2.184-kb receptor mRNA transcript was present in all sizes (<150-400 μm) of follicles. Follicle-stimulating hormone receptor was also localized immunohistochemically in granulosa cells of all sizes of follicles. Survival and growth rate of follicles significantly (p<0.05) increased following supplementation of FSH at a concentration of 1.0 μg/ml and the culture medium also showed a significantly (p<0.05) greater accumulation of oestradiol and progesterone. In conclusion, FSHR is expressed in all sizes of PFs and in vitro survival, growth and steroidogenesis of follicles are optimally stimulated by 1.0 μg/ml FSH. These findings demonstrate that FSH has an important role during the recruitment, growth and development of buffalo ovarian PFs.

Expression Profile of Connexin 43 and Poly A Polymerase Genes in Buffalo (Bubalus bubalis) Oocytes and Developing Embryos Produced in vitro

Abstract Mishra, A., Sharma, G.T. and Kumar, G.S. 2010. Expression profile of connexin 43 and poly A polymerase genes in buffalo (Bubalus bubalis) oocytes and developing embryos produced in vitro. J. Appl. Anim. Res., 38: 29–32. To find out the expression pattern of Connexin 43 (Cx43) a gap junction protein and Poly A polymerase (PAP) necessary for synthesis of poly (A) tail in mRNA, genes in buffalo (Bubalus bubalis) oocytes and developing embryos produced in vitro, pools of immatured oocytes (n=200), in vitro matured oocytes (n=200), embryos of 2–4 cell (n=83), 8–16 cell (n=80), morula (n=77) and blastocyst (n=40) were collected in vitro from slaughter house ovaries for mRNA isolation. RT-PCR was carried out by using mRNA as the template and PCR was carried out by using cDNA as template for amplification of 425 bp Cx43 and 252 bp PAP. Cx43 was expressed in all except blastocysts stage, whereas, PAP was expressed in all. It is concluded that expression pattern of Cx43 and PAP in oocytes and developing embryos can be used as a marker of developmental potential for embryos derived from oocytes.

Neurocysticercosis in free roaming pigs – a slaughterhouse survey

To investigate the prevalence of neurocysticercosis among free ranging pigs and to study the type of pathomorphological lesions in affected brains, a total of 200 brains were collected from pigs slaughtered at a local abattoir, between August, 2005 to March, 2006. Gross and histopathological examination revealed 3 % (6/200) occurrence of neurocysticercosis in pigs. Taenia solium cysticercosis is an under-rated zoonosis and is a leading cause of epilepsy due to neurocysticercosis in human population of India. The prevailing situation warrants immediate implementation of effective control measures for this dreaded disease.

Therapeutic Potential of Canine Bone Marrow Derived Mesenchymal Stem Cells and its Conditioned Media in Diabetic Rat Wound Healing

Present study was conducted to explore the possibility of treating the diabetic rat wounds by canine bone marrow derived mesenchymal stem cells (BMSCs) and its conditioned media (CM). Canine bone marrow stem cells were isolated ascetically from dog and cultured in vitro. Stem cells conditioned media was collected from third passage cells at 120 hours of culture. Streptozotocin was used for induction of diabetes in rats. Six groups were made for the wound healing therapy in which group I, II and III were non-diabetic while group IV, V and VI were diabetic with six animals in each group and one wound was created in each animal. Group II and V received stem cells in culture media and group III and VI received conditioned media while group I and IV were kept as respective controls given only stem cell culture media. Stem cells and its conditioned media were injected at the periphery of wounds. Wound healing was assessed by wound contraction, photographic evaluation at different time interval (0, 3rd, 7th, 14th, 21st and 28th day) and histomorphological examination on day 28. The outcomes of wound healing experiment suggested that canine bone marrow stem cells as well as its conditioned media can be exploited xenogenically very well for diabetic rat wound healing.

Collagen-IV supported embryoid bodies formation and differentiation from buffalo (Bubalus bubalis) embryonic stem cells

Embryoid bodies (EBs) are used as in vitro model to study early extraembryonic tissue formation and differentiation. In this study, a novel method using three dimensional extracellular matrices for in vitro generation of EBs from buffalo embryonic stem (ES) cells and its differentiation potential by teratoma formation was successfully established. In vitro derived inner cell masses (ICMs) of hatched buffalo blastocyst were cultured on buffalo fetal fibroblast feeder layer for primary cell colony formation. For generation of EBs, pluripotent ES cells were seeded onto four different types of extracellular matrices viz; collagen-IV, laminin, fibronectin and matrigel using undifferentiating ES cell culture medium. After 5 days of culture, ESCs gradually grew into aggregates and formed simple EBs having circular structures. Twenty-six days later, they formed cystic EBs over collagen matrix with higher EBs formation and greater proliferation rate as compared to other extracellular matrices. Studies involving histological observations, fluorescence microscopy and RT-PCR analysis of the in vivo developed teratoma revealed that presence of all the three germ layer derivatives viz. ectoderm (NCAM), mesoderm (Flk-1) and endoderm (AFP). In conclusion, the method described here demonstrates a simple and cost-effective way of generating EBs from buffalo ES cells. Collagen-IV matrix was found cytocompatible as it supported buffalo EBs formation, their subsequent differentiation could prove to be useful as promising candidate for ES cells based therapeutic applications.

An allogenic therapeutic strategy for canine spinal cord injury using mesenchymal stem cells: BHAT et al.

This study was conducted to characterize canine bone marrow‐derived mesenchymal stem cells (BMSCs); in vivo tracking in mice, and therapeutic evaluation in canine clinical paraplegia cases. Canine BMSCs were isolated, cultured, and characterized in vitro as per International Society for Cellular Therapy criteria, and successfully differentiated to chondrogenic, osteogenic, and adipogenic lineages. To demonstrate the homing property, the pGL4.51 vector that contained luciferase reporter gene was used to transfect BMSCs. Successfully transfected cells were injected around the skin wound in mice and in vivo imaging was done at 6, 12 and 24 hr post MSCs delivery. In vivo imaging revealed that transfected BMSCs migrated and concentrated predominantly toward the center of the wound. BMSCs were further evaluated for allogenic therapeutic potential in 44 clinical cases of spinal cord injuries (SCI) and compared with conventional therapy (control). Therapeutic potential as evaluated by different body reflexes and recovery score depicted significantly better results in stem cell‐treated group compared to control group. In conclusion, allogenic canine BMSCs can serve as potent therapeutic candidate in cell‐based therapies, especially for diseases like SCI, where the conventional medication is not so promising.

Expression and secretory profile of buffalo fetal fibroblasts and Wharton's jelly feeder layers

The present study examined the comparative expression and secretory profile of vital signaling molecules in buffalo fetal fibroblasts (BFF) and Whartons jelly (BWJ) feeder layers at different passages. Both feeder layers were expanded up to 8th passage. Signaling molecules viz. bone morphogenetic protein 4 (BMP4), fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF) and transforming growth factor beta 1 (TGFB1) and pluripotency-associated transcriptional factors (POU5F1, SOX2, NANOG, KLF4, MYC and FOXD3) were immunolocalized in the both feeder types. A clear variation in the expression pattern of key signaling molecules with passaging was registered in both feeders compared to primary culture (0 passage). The conditioned media (CM) was collected from different passages (2, 4, 6, 8) of both the feeder layers and was quantified using enzyme-linked immunosorbent assay (ELISA). Concomitant to expression profile, protein quantification also revealed differences in the concentration of signaling molecules at different time points. Conjointly, expression and secretory profile revealed that 2nd passage of BFF and 6th passage of BWJ exhibit optimal levels of key signaling molecules thus may be selected as best passages for embryonic stem cells (ESCs) propagation. Further, the effect of mitomycin-C (MMC) treatment on the expression profile of signaling molecules in the selected passages of BFF and BWJ revealed that MMC modulates the expression profile of these molecules. In conclusion, the results indicate that feeder layers vary in expression and secretory pattern of vital signaling molecules with passaging. Based on these findings, the appropriate feeder passages may be selected for the quality propagation of buffalo ESCs.