Successful embryo transfer requires manipulation of oestrus and ovulation of the donor and synchronization of these events with those of the recipients. A brief review of the physiological and endocrinological aspects of the oestrous cycle will contribute to more effective use of gonadotrophins (FSH-P or PMSG) for superovulation, and the use of prostaglandin F2 alpha or its analogues for oestrus synchronization.
The oestrous cycle of the female buffalo is approximately 21 days in length, ranging from 17 to 24 days. The duration of oestrus ranges from 19 to 21 hours (Jainudeen, 1986). Overt signs of oestrus are less obvious than in cattle (see Oestrus detection and insemination). Considerable variation exists according to breed, nutritional status, climate, season, stress and geographical location.
The oestrous cycle is divided into four phases based on physiological and endocrinological events: oestrus (day 0), metoestrus (day 1–4), di-oestrus (day 5–18) and pro-oestrus (day 19 to oestrus). This may be simplified by dividing the cycle into the follicular stage representing the time of greatest follicle growth and includes behavioural oestrus and ovulation, and the luteal phase, characterized by maximum growth and maintenance of the corpus luteum (CL) and production of progesterone. A major role of progesterone during the cycle is to induce and maintain growth of the glandular epithelium of the endometrium for production of the histotroph for potential nourishment of the conceptus. Progesterone also inhibits development and ovulation of large follicles (Figure 1). High levels of progesterone produced by the CL serve as a negative feedback inhibiting the hypothalamic pituitary axis from producing gonadotrophins necessary for the stimulation of follicular growth.
At the time of ovulation, which occurs approximately 30–35 hours after the beginning of behavioural oestrus, the cells of the follicle have already undergone luteinization and are actively secreting progesterone. This newly formed CL is bloody (corpus haemorrhagicum) and soft in consistency and plasma levels of progesterone resulting from this newly formed CL rise from 0.5 ng/ml on day 1–2 to a peak of 2.4 ng/ml by day 8 (Jainudeen, 1986). Progesterone production continues until luteal regression on or about day 18 (Figure 1).
Two approaches can be used to manipulate the oestrous cycle hormonally. One involves the use of exogenous PGF2 alpha to regress the CL whereby the buffalo's own natural luteolysin is mimicked. A dose of 25 mg of Lutalyse will prematurely regress the CL (after day 5) and bring the buffalo into oestrus in approximately three days. This approach is used to synchronize the oestrous cycles of the donors and the recipients. A second method is to maintain elevated plasma progesterone levels beyond day 18, preventing the animal from returning to oestrus. This can be mimicked with a progestational implant such as Synchromate B (Ceva Laboratories, Inc., Overland Park, Kansas) which serves as an artificial CL, releasing a progesterone-like steroid (norgestomet). When the implant is removed the endocrine profile follows that of a naturally regressing CL that leads to follicular growth and ovulation. It is important to note that the progestational implant does not maintain the existing CL.
Follicles continue to grow throughout the oestrous cycle including during the luteal phase. In Figure 1 (upper panel) this is represented by the series of continuous lines that rise (growth) from the horizontal axis only to fall back (atresia) in the same direction. The average number of primordial follicles for both ovaries is approximately 12 000 (range 1 300 to 40 000) in buffaloes, which is much smaller than in cattle where an average of 50 000 (range 7 000 to 100 000) has been reported (Danell, 1987). Thus there is an adequate reservoir to sustain a continual loss through atresia. Although several follicles do grow to a large size, only one ovulates during a 21-day cycle (Figure 1). The gonadotrophins LH and FSH are necessary for follicular growth and eventual ovulation. Note in Figure 1 the low levels of LH that are seen during the luteal phase of the oestrous cycle at a time of high plasma levels of progesterone. Progesterone inhibits the hypothalamus from producing the gonadotrophin releasing hormone responsible for release of LH and FSH from the anterior pituitary gland.
Schematic representation of the oestrous cycle
A schematic representation of the oestrous cycle showing a continual flux of follicular growth and atresia (upper panel). One follicle ovulates approximately one day after the beginning of oestrus forming a corpus luteum that produces progesterone. Note that LH levels and follicular growth are highest when progesterone levels are lowest (lower panel). Superovulation rescues those follicles normally destined for atresia.
Pituitary FSH or PMSG are used to superovulate embryo donors. The exogenous gonadotrophins rescue a number of follicles which otherwise would have undergone atresia. Specific superovulatory treatment regimens are presented in Donor treatment.