Leptin supplementation in vitro improved developmental competence of buffalo oocytes and embryos.

The aim of the present study was to determine potential role of leptin on in vitro developmental competence of buffalo oocytes and embryos. Slaughterhouse derived culture grade buffalo cumulus oocyte complexes (COCs) were matured in vitro (IVM) with leptin (10 ng/ml) or without leptin (control). In each experiment, a pool of matured COCs was used for further in vitro embryo production and another pool of COCs was used for cumulus cells and mature oocytes isolation to study the relative mRNA expression of developmentally important genes. Presumptive zygotes were cultured in embryo culture (IVC) media supplemented with leptin (10 ng/ml) or without leptin (control). Cleavage rate was higher (p < 0.05) when leptin was supplemented during IVM + IVC, both, as compared to other groups. Higher cleavage rate was observed in leptin-treated groups, though it was non-significant. Blastocyst rate was higher (p < 0.05) in all the leptin treated groups. The relative mRNA expression of LEPR (Ob-Rb), HAS2 and EGFR was significantly (p < 0.05) up-regulated and the expression of CASPASE3 was down-regulated in cumulus cells of leptin-treated groups. The expression of GDF9, BMP15, GLUT1, LEPR and CASPASE3 transcripts in leptin and non-treated oocytes did not differ. The relative mRNA expression of POU5F1and LEPR transcripts in blastocysts was higher (p < 0.05) in leptin-treated groups; the change in expression of GLUT1, INF-τ and CASPASE3 transcripts was not significant (p > 0.05). Thus, it is concluded that leptin promotes developmental competence of bubaline oocytes by modulating cumulus enabling factors and genes regulating pluripotentcy in the blastocysts.

Selection of appropriate isolation method based on morphology of blastocyst for efficient derivation of buffalo embryonic stem cells.

The efficiency of embryonic stem cell (ESC) derivation from all species except for rodents and primates is very low. There are however, multiple interests in obtaining pluripotent cells from these animals with main expectations in the fields of transgenesis, cloning, regenerative medicine and tissue engineering. Researches are being carried out in laboratories throughout the world to increase the efficiency of ESC isolation for their downstream applications. Thus, the present study was undertaken to study the effect of different isolation methods based on the morphology of blastocyst for efficient derivation of buffalo ESCs. Embryos were produced in vitro through the procedures of maturation, fertilization and culture. Hatched blastocysts or isolated inner cell masses (ICMs) were seeded on mitomycin-C inactivated buffalo fetal fibroblast monolayer for the development of ESC colonies. The ESCs were analyzed for alkaline phosphatase activity, expression of pluripotency markers and karyotypic stability. Primary ESC colonies were obtained after 2-5 days of seeding hatched blastocysts or isolated ICMs on mitomycin-C inactivated feeder layer. Mechanically isolated ICMs attached and formed primary cell colonies more efficiently than ICMs isolated enzymatically. For derivation of ESCs from poorly defined ICMs intact hatched blastocyst culture was the most successful method. Results of this study implied that although ESCs can be obtained using all three methods used in this study, efficiency varies depending upon the morphology of blastocyst and isolation method used. So, appropriate isolation method must be selected depending on the quality of blastocyst for efficient derivation of ESCs.

Expression of mRNA encoding leukaemia inhibitory factor (LIF) and its receptor (LIFRß) 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 (LIFRß) 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 LIFRß 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 LIFRß mRNA transcripts in buffalo embryos indicated that LIF might play an important role in the preimplantation development and subsequent implantation of buffalo embryos.

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.

Detection of classical swine fever virus in archival formalin-fixed tissues by reverse transcription-polymerase chain reaction.

Classical swine fever (CSF) is an economically important Office International des Epizooties list A disease of swine. Inadequate diagnostic infrastructure in developing countries may sometimes make the conduit of viable samples to diagnostic laboratories difficult and at times formalin-fixed tissues may be the only morbid materials available which are not amenable to most laboratory tests. The potential diagnostic abilities in such situations would be enhanced if a highly sensitive and specific method as reverse transcription-polymerase chain reaction (RT-PCR) could be applied to such samples. In the present study, archival formalin-fixed CSF samples were subjected to successful RT-PCR amplification of a 172-bp fragment from E2/NS2 region of CSFV genome. The fidelity of amplification was established by a dot-blot hybridization assay employing a homologous biotinylated cDNA probe. The optimized procedure may be resorted to as a diagnostic tool if viable samples are unavailable and may have the potential to facilitate retrospective studies on archival samples.