Hormonal Manipulation of Reproduction

Foreword

The increased interest in camelidae in general, and in the racing camel in particular, has led in the last decade to a tremendous development in the use of new breeding technologies. These include the use of hormones for the manipulation of reproduction, artificial insemination and more recently, embryo transfer and assisted reproduction such as in vitro fertilization. The objective of the present chapter is to present these state-of-the-art breeding technologies and point out the major limitations for their use as well as the areas that need more research.

Hormonal manipulation of reproductive phenomena has been the most important development in reproductive management of all animal species in the last 25 years. The purification of the major reproductive hormones and the development of new and very accurate methods for hormonal assays have led to a more thorough understanding of the regulation mechanisms of reproduction.^{(1, 8, 9, 28-30)} This knowledge has allowed scientists to develop hormonal treatments to manipulate reproduction by mimicking their natural variation in the body and consistently obtaining the desired effect. The most used hormonal treatments in camelidae have two main objectives: the manipulation of ovarian activity and the modification of the normal evolution of pregnancy. The use of hormone therapy for specific purposes such as increasing spermatogenesis and modification of breeding behavior is also possible but is still under study in the camelidae.⁽⁶⁵⁾

Manipulation of ovarian activity

Hormonal manipulation of ovarian activity is probably the most important development in breeding management of the female camelidae. These treatments include the induction of follicular activity and ovulation, as well as the synchronization of these phases in a group of females.

Induction of follicular activity

Follicular activity is largely dependent on the effect of Follicle Stimulating Hormone (FSH). Induction of follicular growth can therefore be obtained by administration of Follicle Stimulating Hormone (FSH) or other hormones with FSH-like activity such as equine Chorionic Gonadotropin (eCG). These treatments are used mainly for superovulation of donors in an embryo transfer program (cf. Embryo transfer). However, in the dromedary, some authors have used FSH or eCG to advance puberty^{(72, 92)} or induce ovarian activity during the postpartum period^{(37, 64)} or seasonal anestrus.^{(11, 33, 38)} All these treatments are based on a single or double intramuscular injection of 1500 to 7000 IU of eCG. Appearance of mature follicles usually occurs between 5 and 10 days post-treatment. We do not support the use of these treatments because they do not deal directly with the underlying cause of anestrus which is, in most cases, nutritional. In addition, most of the females receiving this treatment have more than one follicle which increases the risks of superovulation and embryo mortality.

Other hormonal treatments are currently being investigated as a means for the induction of follicular activity in anestrous females. These include serial low doses of FSH,⁽⁵⁾ hMG and multiple small doses of GnRH delivered by injection⁽⁵³⁾ or osmotic pumps (Anouassi and Tibary, unpublished).

Induction of ovulation

Ovulation is normally induced by copulation in the camelidae female (cf. Physiology). Hormonal induction of ovulation is probably the most widely used type of reproductive management technique in these species. Two principal hormonal compounds are used for this purpose: human Chorionic Gonadotropin (hCG) and Gonadotropin Releasing Hormones (GnRH) or its analogue Busereline.

In the dromedary and Bactrian camels, ovulation can be induced 26 to 48 hours following injection of GnRH (0.5 mg to 1 mg IM or IV)^{(10, 12, 37)} or Buserelin (15 to 20 µg).^{(31, 32, 62, 66, 76, 78)} Response to this treatment depends on the follicular status. The best responses (85 to 90%) are obtained when the females are selected on the basis of follicular size and uterine tone. Follicles become responsive to the LH peak following the GnRH treatment when they reach 9 mm in diameter. However, the maximum response is seen when follicular size is between 10 and 22 mm.^{(10, 78, 84)} Follicles that are in the process of regressing have a poorer response rate. Another criterion that helps differentiate between growing follicles and regressing follicles is the tone and edema of the uterus. The best response is obtained when the follicles have the required size and the uterus has developed maximum tone.^{(10, 84)} The proportion of non-ovulating females (10 to 15%) is comparable to that observed after natural mating
and could be due to inadequate release of LH.^{(10, 84)}

Similarly, in the llama and alpaca, intramuscular or intravenous administration of GnRH (0.5 to 1 mg) (2, 20) or Busereline (4 to 8 µg)^{(17, 21, 60, 70, 80, 81, 87)} induces an LH peak followed by ovulation 24 to 30 hours later if a mature follicle (> 7 mm) is present (cf. Physiology).

Ovulation can also be induced by intravenous or intramuscular administration of Human Chorionic Gonadotropin (hCG). In the dromedary and Bactrian camel, hCG is used at dosages ranging from 2500 to 4000 IU with response rates varying from 85 to 100% in animals selected on the basis of follicular size and uterine tone.^{(7, 10, 24, 26, 31, 32, 54, 62, 76, 78)}

In llamas and alpacas, hCG was not effective at a dose of 10 IU⁽⁴⁰⁾ but a 50% ovulation rate was obtained with doses as small as 25 IU.⁽⁴¹⁾ Doses currently used range between 500 to 750 IU with a response rate of 85 to 100%.^{(2-4, 14, 15, 17, 47, 49, 60)} Ovulation usually takes place 26 hours after injection of hCG.⁽⁷⁴⁾

Many authors prefer hCG to GnRH for induction of ovulation because the former acts directly on the follicle whereas the latter relies on endogenous LH release from the pituitary. A more reliable response may be obtained with hCG rather than GnRH in females suffering from low pituitary response and in superovulated females. In addition, hCG will cause luteinization even in large follicles and prevents formation of large anovulatory or hemorrhagic follicles. However, because hCG is a protein, frequent administration of this hormone may cause immunization of the female and reduce its efficacy.

Synchronization of follicular development and ovulation

Synchronization of ovulation is used as a breeding management tool and is required for any Multiple Ovulation and Embryo Transfer (MOET) program, Synchronization of the cycle in the female camelidae has met with many difficulties due to the peculiar nature of follicular activity in these species. Techniques used in other domestic animals for the synchronization of estrus and ovulation in a group of females, such as treatment with progesterone, prostaglandin, or a combination of both, are not practical or have only limited success in camelidae.

Progestagen treatments with Progesterone Releasing Intravaginal Devices (PRID, CIRD) or subcutaneous implants (Norgestomet) do not seem to completely arrest follicular development and therefore have very limited efficacy in synchronizing follicular development. Natural progesterone in oil seems to give better results, at least in the dromedary camel, if administered daily (100 mg per day) for 10 to 15 days. Synchronization of follicular development and ovulation is improved by administration of eCG (1500 to 2000 IU) one day before, or on the last day of, progestagen treatment (cf. Embryo transfer).

In our laboratory, other techniques have been used to improve synchronization of follicular development and ovulation. These include prostaglandin administration following breeding or the use of GnRH antagonist. In the breeding-PGF2α scheme, a group of females is joined to a male for 3 weeks, pregnancy is detected by behavioral signs (tail cocking) at the end of the joining period, and all pregnant females receive an injection of PGF2α or its analogue to cause abortion (cf. Induction of abortion). This technique is relatively good since the majority of treated females will have a mature follicle 5 to 8 days following prostaglandin treatment and will be ready for an induction of ovulation with hCG or GnRH. However, this synchronization method has two major drawbacks; first it is time-consuming, and second, it presents a risk of venereal transmission of infection.

Induction of parturition and abortion

The two main reasons for induction of abortion in the dromedary female that we encountered in our clinic are accidental mating (young or racing female) or mismatched mating (wrong male). Another medical reason for this treatment is high-risk pregnancy such as when the dam has a chronic debilitating disease. Induction of abortion in the female camelidae is possible at any stage of pregnancy using PGF2α or its analogues. The recommended dosage for the llama are respectively 10 mg, 0.5 mg, and 100 to 150 µg for Dinoprost tromethamine (natural PGF2α) Fenprostalen, and Cloprostenol. Dinoprost tromethamine and Fenprostalen are administered subcutaneously, whereas Cloprostenol is given intra-muscularly.⁽⁵⁵⁾ It is sometimes necessary to administer Dinoprost tromethamine twice at 24-hour intervals in order to obtain the desired effect.⁽⁵⁵⁾ However, this drug has been associated with 4 deaths in the llama following severe colicky reaction after injection. In the dromedary, we have been using both Dinoprost tromethamine (25 to 35 mg, IM) or Cloprostenol (750 µg, IM) in a single injection without any adverse effects on the animals. In a group of 96 females, all the females aborted between 24 to 120 hours after Cloprostenol injection, with the maximum number of abortions occurring between 55 and 80 hours. Natural PGF2α was less efficient in causing abortion than Cloprostenol. If no abortion is observed within 120 hours post-treatment with PGF2α or its analogues, a vaginal examination should be conducted to verify whether the fetus is blocked in the cervix.

Induction of parturition is not commonly used for management but we have been able to induce parturition in a group of 42 dromedary females with the Prostaglandin F2α analogue, Cloprostenol, at the same doses used for induction of abortion. Parturition started between 8 and 72 hours later. There was no effect of the treatment on milk production of the dam or on behavior or survival of the young. There are no scientific studies concerning the criteria to be used for the induction of parturition that will guarantee the survival of the neonate in the camelidae. In the dromedary, we have been using mammary development and a pregnancy stage of at least 370 days as minimal requirements, with good results.

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