Single embryos or multiple embryos may be collected from naturally ovulating or superovulated buffaloes, respectively. For optimal efficiency two to four donors should be treated and synchronized with their recipients for each attempt; this allows the sharing of the recommended potential of three to four recipients per donor.
Superovulation is the least predictable step in the process of embryo production. In cattle a tremendous variation in response occurs with age, breed, lactational and nutritional status, season and stage of the cycle at which treatment is initiated. Follicle-stimulating hormone (FSH) or pregnant mare serum gonadotrophin (PMSG) may be used. The short half-life of FSH necessitates twice-daily injections over a period of four to five days. Treatment is begun during the mid-luteal phase (day 8 to 12) of the donor's cycle and employs the use of prostaglandins (PGF2 alpha) to synchronize the cycles of the donors and the recipients. Alternatively, treatment may be initiated on day 16 or 17 (day 0= oestrus) of the donor's natural oestrous cycle. Dose levels of FSH vary from decreasing levels of 6, 5, 4, 3 and 2 mg twice daily to a uniform 4 or 5 mg twice-daily injection schedule. Two examples of superovulatory regimens with FSH are given in Table 1. Prostaglandins (PG) (25–35 mg PGF2 alpha or 500 mcg PG-analogue IM) are routinely given at the time of the fifth and sixth FSH injection which is then followed by oestrus and ovulation in two and three days, respectively. This interval from PG to the onset of oestrus is 12–24 hours shorter in superovulated animals than in naturally ovulating buffaloes. Consequently, recipients should be injected with PG 24 hours before the donors if this method of synchronization is used. The response to the foregoing FSH regimens ranges from zero to as many as 15 ovulations, with an average of five or six. There appears to be no difference in response between a four-day and a five-day regimen. Extrapolating from cattle (Lerner et al., 1986) heifers may require a smaller total dose and older animals a higher total dose. When a donor has failed to respond to the superovulatory treatment, the second attempt to superovulate it with the same or a similar regimen is also likely to result in failure.
It is difficult to assess the number of ovulations accurately by palpation of the ovaries per rectum when the number exceeds four to six per ovary or when several anovulatory follicles are present. It is felt that an excessive number of anovulatory follicles in the presence of CLs adversely influences the recovery percentage because of an unfavourable oestrogen/progesterone ratio which allegedly affects gamete and embryo transport.
Pregnant mare serum gonadotrophin (PMSG) has also been used for superovulation. While PMSG has the advantage of requiring only one single injection, its half-life is perhaps too long as measurable levels could still be detected in the blood of cattle 10–15 days after administration of 1 500–3 000 IU PMSG. The FSH/LH ratio of PMSG ranges from 1.9 to 95.5 and depends on the stage of gestation at which the serum was collected. PMSG is a crude product and is therefore difficult to standardize. It is a foreign protein that is antigenic, which may lead to a reduced response after repeated use. PMSG may be injected subcutaneously or intramuscularly on day 16 or 17 of a normal oestrous cycle. Doses range from 1 500 to 3 000 IU, but 2 000 to 2 500 IU are most commonly used. When used in conjunction with prostaglandin, PMSG is administered between days 8 and 15 of an oestrous cycle followed by prostaglandin 48–72 hours later. A sample treatment schedule is presented in Table 2. PMSG injections initially exert a luteotrophic effect that has been related to its luteinizing hormone (LH) activity.
LH is presumed to be responsible for the occasional premature ovulation of a large follicle present at the time of the PMSG injection. The precocious CL is believed to inhibit further ovulations, perhaps because it is not old enough to be lysed by PG given two days later, yet it secretes enough progesterone to block endogenous LH release. A promising new development in the superovulation of cattle with PMSG is the administration of anti-PMSG at the time the donor comes into oestrus. This counteracts the continued stimulation of follicles due to the long half-life (first order half-life, 36 hours; second order half-life, 370 hours) (Dieleman, Bevers and Gielen, 1987; Kim et al., 1987; Moyaert et al., 1985; Saumande and Chupin, 1987; Saumande, Procureur and Chupin, 1984; Wang et al., 1987).
Superovulation treatments with follicle-stimulating hormone in the buffalo
|Day||Treatment I||Treatment II|
|-17||25 mg PGF2 alpha*||25 mg PGF2 alpha*|
|-4||a.m.||5 mg FSH||6 mg FSH|
|p.m.||5 mg FSH||6 mg FSH|
|-3||a.m.||5 mg FSH||5 mg FSH|
|p.m.||5 mg FSH||5 mg FSH|
|-2||a.m.||5 mg FSH||4 mg FSH|
|35 mg PGF2 alpha||35 mg PGF2 alpha|
|p.m.||5 mg FSH||4 mg FSH|
|20 mg PGF2 alpha||20 mg PGF2 alpha|
|-1||a.m.||5 mg FSH||3 mg FSH|
|p.m.**||5 mg FSH||3 mg FSH|
|Insemination 8–10 hours after onset of oestrus, repeated once 10–12 hours later|
* Recipients receive 25 mg PGF2 alpha on day -13 and day -3.
** FSH treatment is discontinued when donor comes into oestrus early. If donor does not come into oestrus, FSHtreatment is continued one additional day.
There are no reports on the use of equine pituitary extract or human menopausal gonadotrophin for superovulation of buffaloes.
In cattle, there is no need to give the donor gonadotrophin releasing hormone or LH at the onset of oestrus in an attempts to precipitate or to group ovulations. There is also no benefit from the injection of a physiological dose of oestradiol (500 mcg) at the anticipated time of onset of oestrus to intensify the expression of heat. No such information is available at present for buffaloes.
Superovulation treatments with pregnant mare serum gonadotrophin in the buffalo
|Day||Treatment I||Treatment II|
|-18||25 mg PGF2 alpha||25 mg PGF2 alpha|
|-5||a.m.||2 500 IU PMSG|
|-4||a.m.||2 500IU PSMG|
|-2||a.m.||35 mg PGF2 alpha||35 mg PGF2 alpha|
|p.m.||20 mg PGF2 alpha||20 mg PGF2 alpha|
|Al 8–10 h after onset of oestrus, repeated once 10–12 h later||Al 8–10 h after onset of oestrus repeated once 10–12 h later|
|Natural service by exposing donor to bull every 8 h for 10 min until no longer receptive||Natural service by exposing donor to bull every 8 h for 10 min until no longer receptive|