Hand-made Cloning: A Guide for Cloning Water Buffaloes.

The present review is a detailed discussion on comparable benefits of hand-made cloning (HMC) technique than micro-manipulation based conventional cloning and developed in the author’s laboratory. Hand-made cloning technique does not require micromanipulators, because the manipulations required for both enucleation and nucleus transfer are performed by hand. The HMC technique includes manual bisection of zona-free oocytes and the simultaneous fusion of the somatic cell with two cytoplasts to produce a cloned embryo. The benefits of HMC include low setup costs for limited equipment, no requirement of highly trained expertise and in vitro effciency comparable to traditional somatic cell nuclear transfer technology. Embryos produced by HMC can be cryopreserved and capable of producing live births. The HMC technique is now applied to different species and can be used in large scale nuclear transfer programs.

Follicular fluid immunoreactive-inhibin concentrations in relation to follicular diameter and estradiol-17 beta, progesterone and testosterone concentrations in individual ovarian follicles in buffalo, Bubalus bubalis.

The objective of this study was to measure the concentrations of immunoreactive inhibin (ir-inhibin) in follicular fluid from individual ovarian follicles and relate them to follicular diameter and follicular fluid concentrations of estradiol-17 beta, progesterone and testosterone in buffalo. Follicular size was measured with an ultrasound machine and follicles were categorized as small (4 to 5 mm diam.), medium (6 to 9 mm diam.) and large (10 mm and above in diam.). Ir-inhibin concentrations varied markedly between and within follicles of same size category. Follicular fluid ir-inhibin concentrations (microgram/ml) were positively related to follicular diameter (R = 0.32, n = 262, P < 0.001) and were significantly higher (P < 0.05) in large (8.32 +/- 0.56) in comparison to medium (7.02 +/- 0.31) follicles which, in turn had inhibin concentrations significantly higher (P < 0.001) than those in small follicles (5.13 +/- 0.48). Ir-inhibin and estradiol-17 beta concentrations were positively related in medium (R = 0.38, n = 128, P < 0.001) and large (R = 0.64, n = 35, P < 0.001) but not in small follicles. There was a negative relationship between ir-inhibin and progesterone concentrations in large follicles (R = 0.46, n = 33, P < 0.01), with no relationship between the two hormones in small and medium follicles. Ir-inhibin was positively related to molar ratios of estradiol-17 beta to progesterone in medium (R = 0.30, n = 124, P < 0.01) and large (R = 0.49, n = 24, P < 0.01) but not in small follicles. There was no relationship between ir-inhibin and testosterone concentrations in follicles of all size categories. The results of the present study suggest that follicular inhibin production is related to follicular size as well as intrafollicular estradiol-17 beta and progesterone concentrations.

Micromanipulation and cloning studies on buffalo oocytes and embryos using nucleus transfer.

An investigation for testing the viability of production of cloned buffalo embryos through nucleus transfer has been made. Matured buffalo oocytes, after zona cutting to an extent of 60 degrees near polar body, were enucleated using a new approach. Instead of aspirating the cytoplasm contents in a pipette, the half of cytoplasm of oocyte was pushed out, thereby also taking away the nuclear material of the oocyte, leaving the demi-oocyte with the zona pellucida enucleated. The absence of fluorescence confirmed the success of the enucleating process. For enucleating, the oocytes which had intact plasma membrane were eligible for bisectioning. There was no significant difference in oocytes having intact membrane among grade I (33.9%) and grade II (31.4%) oocytes, whereas lower percentage of grade III oocytes had a very low percentage having intact plasma membranes (8.5%). The hours of maturation for 32, 37 and 42 did not influence the per cent oocytes which had intact membranes. All the bisected or demi-oocytes tested with fluorescence screening yielded successful enucleation in 88.2% demi-oocytes. The temporal effect of three maturation hours of 32, 37, and 42 hr; two electrical pulse numbers of 2 and 3 pulses and two magnitudes of electric pulses of 15 and 20 V were studied for their effect on the percentage of successful fusion of demi-oocyte blastomere complexes and the rate of complexes undergoing cleavages. The time period for which the oocytes were subjected to the process of maturation significantly affected the per cent fusions and per cent cleavage of the demi-oocyte blastomere complexes and 32 hr maturation yielded less fusions (38.5%) compared to maturation for 37 and 42 h (53.2 and 57.8%, respectively). The treatment of either 2 or 3 electrical pulse numbers resulted in significantly different fusion (45.6 and 54.1%) as well as cleavage rates (18.2 and 26.1%) of demi-oocyte-blastomere complexes electrofused. The treatment of two levels of magnitude of 15 and 20 V of an electric current resulted in similar per cent fusion (48.0 and 51.6%) and cleavage rates (21.0 and 23.2%). Fortified TCM with either 10 or 20% FBS for culturing freshly electrofused complexes for 1 hr did not differ significantly with respect to per cent complexes fused and cleaved, giving a fusion rate of 46.2 and 53.8% and cleavage rate of 21.2 and 23.2% for 10% and 20% FBS, respectively. Production of cloned embryos through the process of nuclear transfer has been accomplished. The successful cleavages of the nuclear transferred oocytes demonstrated the viability of enucleation procedures of the oocytes and technology implementation of electrofusion in buffalo oocytes.

Increased capacitation of buffalo sperm by heparin as confirmed by electron microscopy and in vitro fertilization.

Capacitation of buffalo sperm was evaluated by induced acrosome reaction (AR) upon the exposure of 10 mM Ca2+. Culture of sperm for 8 hr in BO medium supplemented with 10 micrograms/ml heparin significantly (P < 0.01) increased the percentage of AR and confirmed by transmission electron microscopy. Vesiculization of outer acrosomal membrane and plasma membrane was observed significantly higher (P < 0.01) following 8 hr of sperm culture with heparin. Culture of sperm with heparin also increased rate of fertilization of in vitro matured oocytes and their subsequent development up to morula/blastocyst stage (P < 0.01). The study demonstrates that capacitation of buffalo sperm by heparin required at least 8 hr exposure of sperm to heparin for maximum acrosome reaction.

Inhibin in individual buffalo ovarian follicles in relation to size.

A sensitive and specific radioimmunoassay was validated and applied for measurement of inhibin in follicular fluid (bFF) obtained from individual buffalo ovarian follicles. Follicular size was measured with an ultrasound machine and follicles were categorized as small, medium and large. Presence of inhibin was detected in all the antral follicles above 3 mm diameter. Inhibin concentration showed a positive relationship (R = 0.27, P < 0.01) with follicular diameter and was significantly higher (P < 0.05) in bFF from medium and large follicles (8.44 +/- 0.54 and 7.70 +/- 0.45 micrograms/ml, respectively) in comparison to that from small follicles (5.74 +/- 0.80 micrograms/ml). Total inhibin content was highly correlated (R = 0.92, P < 0.001) with follicular diameter and the inhibin content was higher (P < 0.001) in large > medium > small follicles.