Superovulation is a very inefficient method of obtaining oocytes from bovine ovaries and is likely to be replaced by other approaches within the next decade. However, superovulation results in about ten times more embryos than single ovum recovery. Without superovulation, a usable embryo can be recovered about 60 percent of the time from normal donors by skilled technicians. Under similar conditions, superovulation usually yields an average of six usable embryos, although the variation is astounding (Figure 1). Normally, no embryos are recovered from 20–30 percent of superovulated donors and only one to three embryos are obtained from another 20–30 percent (see Table 11). An ideal response of five to 12 embryos is obtained from about one-third of the donors. However, a small percentage of donors yield more than 20 good embryos and, very rarely, more than 50.
Superovulated bovine ovary photographed during a surgical recovery procedure. Also note opening to fimbria
The two generally accepted methods of superovulating cattle are based on two different gonadotrophins, although there are many minor variations of these methods. The simplest is to give an intramuscular (i.m.) injection of 1 800–3 000 IU (usually 2 000–2 500 IU) of pregnant mare's serum gonadotrophin (PMSG), more correctly designated equine chorionic gonadotrophin (eCG), followed by a luteolytic dose of prostaglandin F2 alpha or an analogue i.m. two to three days later. A second prostaglandin injection is often given 12–24 hours after the first, and seems to improve embryo production.
The second method of superovulation is to give eight to ten injections of follicle stimulating hormone (FSH) subcutaneously (s.c.) or i.m. at half-day intervals. Intramuscular injection is more reliable under field conditions. As with PMSG, prostaglandin F2 alpha is given 48–72 hours after initiation of treatment with the fifth, sixth, or seventh FSH injection. The most common FSH regimen is 6,6,4,4,2,2,2, and 2 mg at half-day intervals with prostaglandin F2 alpha given with the sixth or seventh FSH injection. About 20 percent more gonadotrophin should be given to cows weighing over 800 kg. Sometimes, higher doses are used for the first two days; others give 5 mg for each injection. There are few studies with adequate numbers of donors per treatment group in which constant and decreasing doses have been compared, so reliable conclusions cannot be drawn regarding efficacy of such regimens.
A special problem with most commercially available FSH products is that they are quite impure, frequently with less than 5 percent biologically active FSH. The most commonly used product is currently marketed by Schering (see Chapter 17); over the last 25 years, essentially the same product has been marketed under various brand names, including Burns-Biotech, Reheis and Armour. It is obtained from swine pituitaries. Potency is determined relative to an “in-house” Armour standard, so the actual weight of the material in the bottle has little to do with the weight equivalent on the label. Recently, other FSH products of various purities have become available. Most feature low contamination with luteinizing hormone (LH). Although addition of large amounts of LH to FSH reduces its efficacy for superovulation of cattle, there are few convincing studies that the small amount of LH found in commercial batches of FSH decreases efficacy greatly. In fact, some LH may be needed for optimal superovulatory responses. The other impurities in FSH seem to be of little consequence, other than making it difficult to compare dosages among products, which may range from 1 to 50 percent pure. This results in widely different weights of product per dose. Production of bovine FSH either by cells in tissue culture or in milk of transgenic animals from genes cloned by recombinant DNA techniques will result in a more standardized preparation and eliminate much of the confusion in both commercial and experimental applications.
An annoying feature of most commercial FSH products is that insufficient sterile diluent is supplied for practical use. Usually FSH should be diluted at 1 or 2 mg/ml in saline so that a reasonable volume can be injected under field conditions.
In recent years, FSH has surpassed PMSG as the method of choice for superovulating cattle. In most studies comparing the two procedures, the FSH treatment has resulted in slightly higher numbers of usable embryos. However, PMSG works nearly as well. Everyone agrees that PMSG results in a much larger ovary, generally double the volume of one treated with FSH. This is probably related to its very long half-life (five days) in cattle (that of FSH is several hours) which results in continued recruitment of follicles after ovulation, very high progesterone levels and, probably, abnormalities in ovum transport. These problems have been ameliorated by injection of a commercially available antibody to PMSG given at the conclusion of the superovulatory treatment when the donor comes into oestrus. This results in a response more similar to that of FSH, including a small ovary at the time of embryo collection.
Other products that have been used for superovulating cows include equine anterior pituitary extract and human menopausal gonadotrophin (which also contains considerable LH). The former generally is not available commercially and the latter is too expensive for routine use.
It is possible to superovulate without giving prostaglandin F2 alpha by starting gonadotrophin treatment on day 15 or 16 of the cycle of heifers and on day 16 or 17 of cows (oestrus = day 0). This method, which depends on natural luteolysis, is not recommended because the mean number of embryos recovered is reduced, the response is more variable and timing of the onset of oestrus is less predictable. Furthermore, most flexibility in scheduling donors is lost.
An alternative to using prostaglandin F2 alpha is based on progestin withdrawal rather than luteolysis. The progestin can be injected, implanted subcutaneously, given via a vaginal coil or fed orally. Implants or vaginal coils are preferred because systemic concentrations of progestin drop rapidly when these devices are removed. Probably the most widely used progestin withdrawal system for superovulating cows is Syncromate-B (norgestomet) (see Chapter 8). However, for superovulation, the implant is often used without the injection of progestin and oestrogen. The implant (two implants in large cows) is generally given on day 10–12 of the oestrous cycle and removed on day 18–20. Sometimes prostaglandin is given as well. FSH or PMSG injections are initiated two and a half to three days before implant removal. This system is generally not used for normal donors because of the time and expense. However, it is particularly suited to superovulating cows with cystic ovaries or for pre-puberal heifers.
When prostaglandin F2 alpha-induced luteolysis is used for superovulation, it is best to initiate gonadotrophin treatment between days 9 and 14 of the oestrous cycle. Mean response is usually lower if treatment begins later or earlier, especially prior to day 5.
Another factor to consider is the optimal interval between superovulations when donors are treated repeatedly. Repeating superovulation at 15–20-day intervals works poorly. The most common recommendation is 45–60-day intervals, although one study gives reasonably convincing data from large numbers of animals that shorter intervals work well (Looney, 1986).
The treatments discussed so far should not strictly speaking be termed “superovulatory treatments” since their effect is production of additional mature follicles. These extra follicles actually ovulate in response to an endogenous LH surge, which in turn is triggered by secretion of gonadotrophin-releasing hormone (GnRH) in the presence of the high concentrations of oestradiol-17-beta resulting from follicular growth. Some workers inject GnRH or human chorionic gonadotrophin (hCG) 48 hours after prostaglandin or at the beginning of oestrus to augment endogenous LH. This is necessary for good superovulation for some species, but not for cattle. Exogenous LH or GnRH is useful to induce ovulation under special circumstances, e.g. superovulation of pre-puberal calves or timing the LH surge for experiments. However, most studies using FSH for routine superovulation indicate that this treatment does not result in more transferable embryos (Prado-Delgado et al., 1989), probably because the LH surge is frequently induced too early or too late for individual cows. When PMSG is used for superovulation, GnRH injection at the time of oestrus may yield slightly more embryos, but this requires further replication.
Following superovulatory treatment, the donor should be observed closely for signs of oestrus. Superovulated cows sometimes do not display oestrous behaviour as clearly as untreated cows, therefore such oestrous detection aids as KaMaR indicators (see Chapter 17) are helpful. About 10 percent of donors never show behavioural oestrus. These animals should not be bred.
The time when the donor is first observed in standing oestrus is the reference point for insemination treatment. Because the multiple follicles ovulate over a period of time and transport of sperm and ova is altered by superovulatory treatment, it is wise to breed more often and use more semen than normal. Freshly collected liquid semen is slightly superior to high quality frozen semen since unfrozen spermatozoa probably remain viable in the female reproductive tract longer.
If liquid semen is available, between 10 and 50 × 106 motile spermatozoa are inseminated 12 hours after the donor is observed in oestrus, and a similar quantity 12 hours later. If frozen semen is used, one ampoule or straw each time is inseminated 12 and 24 hours after the donor was first noticed in oestrus. Some people recommend two doses per insemination, particularly at the second insemination. Semen is thawed in a water bath at 35°C (95°F) and inseminated immediately. Some workers recommend more frequent inseminations beginning, for example, immediately after first observation of standing oestrus. We are not aware of scientific data indicating that such early insemination is appropriate. If only one insemination is to be done, it should be at 24 hours after oestrus was first detected.
Altered hormonal patterns make the reproductive tract of a superovulated donor a more hostile environment than that of an untreated cow; therefore, semen must be of the highest quality. Use of poor or mediocre semen often results in collection of unfertilized ova or degenerate embryos. Proper semen handling techniques are beyond the scope of this manual, but they cannot be overemphasized (see, for example, Pickett and Olar, 1980).
The inseminator must be gentle and must use hygienic techniques because the stress of superovulatory treatment makes a cow's upper reproductive tract extremely sensitive. Excessive manipulation of the tract could result in adhesions and the failure of the fimbriae to pick up all ova. In addition, with high numbers of ovulations and enlarged ovaries, more haemorrhaging than normal occurs and the relative size of the organs changes. This also increase the likelihood that adhesions will form. Infection introduced at the time of insemination could reduce rates of fertilization and recovery. Moreover, many follicles may not have ovulated by the time of the first insemination; therefore, the reproductive tract should be manipulated as little as possible to reduce risk of follicular rupture. For these reasons, ovaries definitely should not be palpated at the time of insemination.