M.L. Madan

In vitro production and transfer of embryos in buffaloes

Abstract The buffalo has been identified as a domestic animal of great economic importance for Asia and Mediterranean. Although births of calves from embryos produced in vitro and transferred to recipient buffaloes have been recorded, only a few reports are available on the technology. Inherent problems of low follicular oocyte population coupled with fragility of buffalo semen result in low cleavage rate and morula/blastocyst production. Buffalo estrus serum has been observed to enhance oocyte maturation. Oviductal cell co-culture significantly affected the cleavage and embryo production. Four calves have been produced from buffalo follicular oocytes fertilized and developed in vitro. The procedures for in vitro maturation, fertilization and culture of embryos in buffaloes must be improved and optimized.

Application of reproductive technology to buffaloes

This paper focuses on recent advances in the application of embryo transfer technologies to buffalo. The early recovery rate of 0.15 transferable embryos has now increased to 2.0, with altered superovulation protocols. Lower follicular population and poor follicular development, with adverse seasonal influences explain the lower ovulatory responses. Unovulated follicles at superovulation contribute high quantities of oestrogen, altering the uterine milieu. The birth of calves using in vitro fertilisation technology and frozen-thawed embryos and determination of embryonic sex using a Y-specific DNA probe, are recent milestones achieved using these technologies.

HEAT STRESS IN MURRAH BUFFALO CALVES

Abstract Six young Murrah male buffalo calves, 7 to 9 months old, weighing 165–200 kg, were exposed to solar radiation during summer (Dbmin. 27.1°C, Dbmax. 44.1°C) . The surface temperature at forehead, back middle, pinna, neckmiddle, rump, foreleg and hindlegs were recorded using a non-contact temperature measuring instrument. Respiration rate and rectal temperature were recorded coincidently at hourly intervals. The difference between mean of minimum and maximum temperature, at forehead (20.09°C), back middle (22.33°C), foreleg (lower, 18.50°C), pinna (18.21°C) were higher than the temperature gradient of neck middle (13.96°C), rump (16.13°C), foreleg upper (14.75°C), hindleg upper (14.58°C), hindleg lower (15.84°C). The surface temperature at all sites increased in relation to the length of radiation exposure. During summer when ambient temperature and solar radiation was maximum, young buffaloes were not able to maintain their normal rectal temperature and increased pulmonary frequency by 5–6 times, protruded tongue, and increased salivary activity. Therefore, young buffaloes should be protected from extreme hot conditions and direct sun exposure be avoided.

Pregnancies established from water buffalo (Bubalus bubalis) blastocysts derived from in vitro matured, in vitro fertilized oocytes and co-cultured with cumulus and oviductal cells

Buffalo ovaries were collected immediately after slaughter and were transported to laboratory in sterile saline at 37 degrees C. Follicular oocytes with the cumulus mass aspirated from 2 to 6 mm in diameter follicles were cultured in TCM-199 medium supplemented with 10% buffalo estrus serum (BES) in 5% CO(2) at 38.5 degrees C. After 20 to 24 h of incubation, the oocytes were inseminated with precapacitated frozen thawed spermatozoa for 6 h. The fertilization rate was 78.15% of the matured oocytes. Over an in vitro culture period of 3 to 9 d, 4.02% of the inseminated oocytes developed to the morula stage when cultured with cumulus cells alone and 17.83% when cumulus cells plus oviductal epithelial cells were used. The percentage of developed blastocysts was very low (0.57%) when the oocytes were co-cultured with cumulus cells from the original oocytes. However, 8% of the inseminated oocytes that were denuded 3 d after insemination developed to the blastocyst stage when they were co-cultured with cumulus and oviductal epithelial cells. Sixteen early/expanded blastocysts were transferred non-surgically to 16 recipients. Four of the 16 recipients became pregnant, of which 2 delivered normal buffalo male calves.

Chromosome configuration during in vitro maturation of goat, sheep and buffalo oocytes.

The competence of meiotic chromosome configuration at the time of co-culture of oocytes with spermatozoa is an essential prerequisite for successful in vitro fertilization (IVF). Although this technology has been used in several livestock species, various intrinsic and extrinsic factors affecting the high repeatablity of IVF have yet to be understood. The present study was conducted to determine the appropriate time for coculture of oocytes and spermatozoa in order to optimize the fertilization rate in sheep, goats and buffalo. Oocytes were collected from the ovaries of slaughtered animals. The oocytes were divided into 10 groups and cultured for maturation in TCM-199 supplemented with estrous cow serum for different durations at 38.5 x 0.5/C in a CO(2) incubator. Sheep and goat oocytes were removed from culture medium after 0,6,12,22,24,26,28,30,32 and 36 and buffalo oocytes after 0,6,12,16,20,22,24,26,28, and 36 h. The oocytes were treated with hypotonic solution (0.75 M KCl) and fixed in Caronys fixative on glass slides. The fixed oocytes were stained with Giemsa solution, and the meiotic chromosomes were evaluated under a compound microscope at x 1000 magnification. Observations were recorded on a total of 1328 oocytes (sheep, 409; goat, 727 and buffalo, 192). The sequential configurations of diffused chromatin, pachytene, diplotene (along with nucleoli), diakinesis and metaphase II (MII) were analyzed at different durations of culture. Control oocytes (fixed at 0 h without incubation) were mostly at the pachytene stage, and as the duration of culture increased the instances of diplotene, diakinesis and finally MII increased. Oocytes at the MII stage of meiosis are known to be at the optimal stage of development for co-culture with spermatozoa and successful in vitro fertilization. On the basis of sequential configuration of chromosomes, it was found that the optimal duration of in vitro maturation of oocytes is 32, 30 and 24 h for sheep, goats and buffalo, respectively.

PERIPHERAL INHIBIN LEVELS IN RELATION TO CLIMATIC VARIATIONS AND STAGE OF ESTROUS CYCLE IN BUFFALO (BUBALUS BUBALIS)

The present study investigated the peripheral plasma inhibin levels in relation to 1) the stage of estrous cycle and the effect of climatic variations. Blood samples were collected from cyclic buffalo (n=5) once daily for 32 consecutive days during the tropical hot humid (summer) and cold (winter) seasons. Estrus was recorded by parading a vasectomized bull as well as by plasma progesterone determination. In the winter season, peripheral inhibin concentrations which were lowest (0.35 +/- 0.02 ng/ml) during the mid-luteal phase of estrous cycle (Day 6 to Day 14, Day 0 = day of estrus) increased significantly (P < 0.02) to 0.47 +/- 0.04 ng/ml during the late luteal phase (Day -4 to Day -2) and then further to 0.52 +/- 0.03 ng/ml (P< 0.02) during the periestrus phase (Day -1 to Day 1). Inhibin concentrations then decreased significantly (P < 0.02) to 0.40 +/- 0.03 ng/ml during the early luteal phase (Day 2 to Day 5). In the summer season the differences in peripheral inhibin concentrations among different phases of estrous cycle were found to be nonsignificant. A comparison of the circulating inhibin concentrations between the two seasons indicated that inhibin concentrations were significantly higher in the late luteal phase (P < 0.01) and periestrus phase (P < 0.05) during the winter season compared with corresponding periods during the summer season. The present study suggests that peripheral inhibin concentrations change in the estrous cycle during cooler breeding season and that environmental heat stress can cause a reduction in peripheral inhibin concentrations.

Effect of epidermal growth factor on the cumulus expansion, meiotic maturation and development of buffalo oocytes in vitro.

SEVERAL growth factors have been shown to have a possible role in oocyte maturation and postfertilisation embryonic development in cattle (Heyner and others 1993). The addition of insulin-like growth factor-II (IGF-II) to the in vitro maturation (IvM) medium has been recently shown to stimulate nuclear maturation and postfertilisation embryonic development of cumulus-enclosed buffalo (Bubalus bubalis) oocytes (Chauhan and others 1998). Although buffalo embryos produced by in vitro maturation, fertilisation and culture (IVMFC) produce pregnancies and live calves following transfer to recipients, the production oftransferable blastocysts and the pregnancies obtained is very low in the buffalo (Madan and others 1994, 1996, Chauhan and others 1997). The present study was undertaken to examine the effects of the addition of epidermal growth factor (EGF) to the ivM medium on cumulus expansion, meiotic maturation, fertilisation and subsequent embryonic development of cumulusenclosed buffalo oocytes in an IVMFC system to explore the possibility of improving embryo yields. Buffalo cumulus-oocyte complexes (cocs) were collected as described by Chauhan and others (1997). Only those cocs which had an unexpanded cumulus mass with five or more layers of cumulus cells, and with homogeneous cytoplasm were used in the study. The maturation media consisted of TCM-l99 supplemented with: 1) 10 per cent fetal bovine serum (FBS) (control medium); 2) 10 per cent FBS + 5,g/ml follicle stimulating hormone-P (FSH-P); and 3) 10 per cent FBS + 20 ng/ml EGF. ivM and evaluation of nuclear maturation were carried out as described by Chauhan and others (1998). The criteria used for assessing the degree of cumulus expansion after 24 hours of IVM were as follows.

Alterations in hypophysial responsiveness to synthetic GnRH at different postpartum intervals in Murrah buffalo ( Bubalus bubalis )

The objective of this study was to investigate the hypophysial responsiveness to GnRH at different intervals post partum in Murrah buffalo. Plasma LH and FSH levels were measured at 1 h before and upto 6 h subsequent to the administration of GnRH (1 ug/kg body weight) or saline on Days 2, 20 and 35 post partum in 2 groups of buffalo (n=4 each). Plasma progesterone levels were measured in samples collected once daily from Day 3 to Day 46 post partum. Pretreatment basal LH levels exhibited a progressive increase from Day 2 through Day 35 post partum, while the basal FSH levels increased only until Day 20 post partum. Following a highly subdued LH response to GnRH on Day 2 post partum, a 408% increase (P < 0.01) was observed in the total LH released in response to GnRH on Day 20 post partum, followed by a 20% reduction (non-significant) over Days 20 to 35 post partum. The interval from parturition was highly correlated with total LH released (r = 0.711, P < 0.01). Unlike LH, a substantial amount of FSH was released following GnRH treatment on Day 2 post partum, which was not significantly different from the FSH response on Days 20 and 35 post partum. The LH and FSH response to GnRH was not significantly different between animals in which luteal activity resumed and in those which showed no luteal activity post partum. While pointing to a dramatic enhancement in the hypophysial responsiveness to GnRH between Days 2 and 20 post partum, these results suggest that pituitary responsiveness to GnRH does not appear to be the limiting factor for resumption of estrous cycles by Day 35 post partum in Murrah buffalo.

Peripheral inhibin levels during estrous cycle in buffalo (Bubalus bubalis).

A sensitive, specific RIA was validated and used for measurement of peripheral plasma immunoreactive inhibin (irinhibin) levels during the estrous cycle in Murrah buffalo. The RIA employed an 125-I iodinated inhibin as tracer and an antiserum against dimeric inhibin. The procedure had a sensitivity of 16 pg/tube, and the nonspecific effects of buffalo plasma were compensated for by including 200 ul bullock plasma in the standards. Separation of free and bound inhibin was affected by the use of a second antibody and precipitation with polyethylene glycol. Blood samples were collected once daily for 30 d from Murrah buffalo (n = 6) during the hot month of July. Cyclic activity and estrus were confirmed by plasma progesterone determination. Peripheral plasma concentrations of ir-inhibin fluctuated between 0.40 +/- 0.07 and 0.67 +/- 0.13 ng/ml during the estrous cycle in buffalo. During the same period, plasma progesterone levels increased from 0.21 +/- 0.01 ng/ml at Day 0 to a peak of 3.30 +/- 0.72 ng/ml on Day 13, declining sharply by Day -5. Ir-inhibin levels exhibited an increase during the follicular phase, with the maximum concentration of 0.65 +/- 0.01 ng/ml occuring on the day of estrus, a decline thereafter, and no pattern during the luteal phase. The differences, however, were not statistically significant throughout the estrous cycle.

Effect of gestation on GnRH-induced LH and FSH release in buffalo (Bubalus bubalis)

This study examined the effect of gestation on the hypophyseal responsiveness of buffalo to GnRH-induced LH and FSH release. Peripheral plasma LH and FSH concentrations were measured at 1 h before and upto 6 h after administration of GnRH (1 ug/kg body weight) or saline at Days 60, 150 and 240 of gestation in 2 groups of buffalo (n = 4 each). Basal LH concentrations did not vary at the 3 stages of gestation, while basal FSH concentrations exhibited a significant reduction (P < 0.05) from Day 60 to Day 150 of gestation. There was a significant reduction in the total LH (P < 0.05) and FSH (P < 0.01) released in response to GnRH from Day 60 to Day 240 of gestation. The duration of LH and FSH peaks and the time to attain peak concentration was not affected by the stage of gestation. The results of the present study point to a progressive decline in LH and FSH release responses to GnRH during the advancement of gestation in the buffalo.