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.

Peripheral plasma FSH concentration in relation to expression of estrus in Sahiwal cattle (Bos indicus).

An experiment was conducted to investigate the changes in peripheral plasma FSH concentrations in relation to expression of estrus in Sahiwal cows. Out of total five estrus, three were accompanied by overt signs whereas rest two were silent estrus. In cows with overt estrus, plasma FSH concentrations during periestrus, early luteal, midluteal and late luteal phase were 1.65+/-0.34, 1.67+/-0.21, 1.58+/-0.18 and 1.69+/-0.31 ng/ml, respectively and the corresponding values in cows with silent estrus being 0.68+/-0.39, 0.50+/-0.12, 0.75+/-0.13 and 0.46+/-0.12 ng/ml, respectively. The overall plasma FSH levels in cows that exhibited overt estrus was 1.63+/-0.82 ng/ml as against 0.64+/-0.55 ng/ml in silent estrus. It was concluded that FSH levels were higher (P<0.01) in cows that exhibited overt estrus compared to silent estrus.

Effect of naloxone on GnRH-induced LH and FSH release in buffalo, Bubalus bubalis.

The effect of naloxone on GnRH-induced LH and FSH release was measured in buffaloes in luteal phase of estrous cycle. Animals were administered intravenously, naloxone/saline (50 mg/injection) every 15 min for 3 hr followed by GnRH (100 micrograms). Peripheral plasma LH and FSH concentrations were measured in blood samples collected at 15 min intervals from 1 hr prior to beginning of naloxone/saline treatment up to 3 hr post GnRH administration and every 30 min for the subsequent 3.5 hr. Between the animals of Group I administered naloxone and those of Group II given saline, GnRH-induced peak LH and FSH concentrations, the total LH and FSH released in response to GnRH, and the time to peak LH and FSH concentrations were not significantly different. The results of the present study suggest the absence of a direct effect of naloxone on pituitary responsiveness to GnRH.

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.

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.