Abstract

Introduction

Populations of bottlenose dolphins tend to exhibit bimodal peaks of seasonal reproductive activity or calf production. However, individual animal variations within these populations can include long periods of polyestrous activity throughout the year, long anestrus intervals or pseudopregnancy. Attempts at maximizing the reproductive potential of these populations are difficult when potential breeding females are experiencing anestrus or pseudopregnancy. In addition, unpredictable estrus cycling patterns reduce reproductive managers' control of potential breeding events. Multiple attempts at inducing ovulation with exogenous gonadotropins in dolphins have been performed.^5,6 These attempts have been met with wide variations in apparent responses between females. However, without ovarian observations, it was impossible to determine either the animals' reproductive physiologic state when the protocols were administered or the effect that these protocols had on ovarian activity. Prostaglandin F2α has been commonly used in domestic animals for estrus synchronization.^3,8 The mechanism of action involves the destruction or lysis of the progesterone secreting corpora lutea (CL) resulting in a return to estrus for cycling animals. And finally, progestogens are commonly used to synchronize ovulation in multiple species.⁹ In cetaceans, Regu-Mate® has been used to suppress ovulation, but has never been evaluated for its potential to synchronize estrus. In this preliminary research, we used ultrasound and endocrine data to evaluate the effectiveness of ovulation induction protocols, prostaglandin F2α for CL lysis, and Regu-Mate® for estrus synchronization in delphinids.

Methods and Results

Ovulation Induction

Three dolphins in two states of ovarian activity (with or without follicles greater than 5mm) were initial placed on 1.5 ml per 110 lb. body weight, p.o., s.i.d. of altrenogest (Regu-Mate®, Hoechst Roussel Vet, Melbourne, VIC, 3004, Australia) for 16 days. Animal one had a follicle of 26 mm on its right ovary, while the two other animals had no visible follicular activity. On day 14, they received 1500 IU of PG600® (Intervet America Inc., Millsboro, DE) i.m. and 17.6 mg FSH i.m. Ovaries were evaluated for evidence of follicular growth with ultrasonography. Two of the animals had multiple small cortical follicles of less than 5 mm in diameter. The third animal exhibited no increase in ovarian size or any evidence of folliculogenesis. The two animals that responded were given a second dose of 1800 IU of PG600® on day 22. By day 32, animal 1 had a total (from both ovaries) of eight follicles from > 15 to < 25 mm, and three follicles > 25 mm in diameter. Animal 2 did not exhibit any change in follicular activity. Based on having three preovulatory follicles of > 25 mm, animal 1 was administered 100 µg of GnRH (Cystorelin®) i.v. on day 32. By day 41, in addition to the follicles observed on day 32, two more follicles of 13 and 11 mm were observed. A second dose of 100 µg GnRH (Cystorelin®) i.v. was then administered. No change was observed in follicular size, recruitment or steroid secretion. It was decided to watch animal 1 with sonography and weekly blood samples for evidence of spontaneous ovulation. Animal 1 continued to have multiple follicles that slowly grew until day 96 when all but three regressed. On day 151, only the original 25-mm follicle on the right ovary remained. The animal was administered 3000 IU of hCG (Profasi®, Serono Lab. Inc., Randolph, MA). Serum progesterone rose from 0.6 ng/ml to 4.2 ng/ml in 3 days and remained elevated. The follicle one on the right remained sonographically unchanged. On day 162, with progesterone of 1.7 ng/ml, the animal was administered two doses of 25mg Lutalyse® (Dinoprost trometamol, Pharmacia & Upjohn Pty Limited, Rydalmere, N.S.W. 2116, Australia) q 6 hr. The follicle of the right ovary did not change, and progesterone values did not drop below 1 ng/ml and remain at that level 2 mo later.

Prostaglandin Treatment

Three sonographically diagnosed non-pregnant animals were selected based on the presence of persistently, a minimum of 2 mo, elevated progesterone. Animals were administered an initial dose of 25 mg Lutalyse® b.i.d. or s.i.d. and serum progesterone was determined 1 wk after the initial dose. Two animals responded after the initial dose(s), the other one had to be given two additional doses. The "resistance" dolphin responded after two doses of 25 mg Lutalyse 6 hr apart (Table 1). Two of the animals went on to cycle normally, one of which has become pregnant. Side effects of drug administration generally consisted of apparent abdominal discomfort, nausea, and on two occasions, inappetence for the remainder of the day. All obvious abdominal discomfort was gone within 1 hr, and all animals returned to normal behavior by the following next day.

Estrus Synchronization

Three dolphins and two killer whales were placed on Regu-Mate® for up to 31 days (Table 2). Both of the killer whales and one of the dolphins were cycling prior to administration of the hormone. The time from progesterone withdrawal to estrus in the dolphins and killer whales was a mean 17.6 and 21.3 days, respectively (Table 2).

Discussion

The ovulation induction trials, while a continuance of early attempts at inducing ovulation, produced unsatisfactory results.^4,6 The two animals without any signs of ovarian activity prior to hormone administration did not produce any follicles greater than 5 mm, while the animal with a follicle on her ovary prior to the induction protocols exhibited a tremendous response. It appears that it will be important to change the dosing schedule and amount for follicular recruitment to occur in anestrus animals. For the animal that exhibited follicular recruitment, GnRH administration did not stimulate ovulation despite the presence of follicles similar in size to preovulatory follicles previously characterized for Tursiops truncatus aduncus.¹ This may indicate that either the follicle was not preovulatory and/or that the dose of GnRH was ineffective. Administration of GnRH to animals that have non pre-ovulatory follicles usually results in luteinization.^2,7 However, with our animal neither luteinization nor ovulation occurred. Thus, it indicates an insufficient dose of GnRH was used. Based on the progesterone production and because the original follicle on animal 1 did not change ultrasonographically, it appeared that the hCG caused the follicle to form a luteinized cyst. This cyst was not responsive to Lutalyse®. It is obvious that further investigations evaluating the differential sensitivity of the dolphin hypothalamic-pituitary-ovarian (HPO) axis to exogenous gonadotropins during anestrus or estrus, and at different stages of follicular growth are needed.

In both dolphins and killer whales, Regu-Mate® appears to cause a delay or suppression of follicular growth after the hormone is withdrawn. The mean length of this suppression appears to be similar to the animals' normal luteal phase length. After this interval has been reached, folliculogenesis and ovulation occur. Three of the dolphins placed on Regu-Mate returned to estrus within 1 wk of each other. While this interval is prolonged and too variable compared to traditional estrus synchronization methods, it could provide a useful mechanism for coordinating ovulation in a group of females during intensive natural or artificial insemination trials.

Despite an apparent refractoriness of one of the animals to Lutalyse®, it appears that it can be safely used for lysis of retained CL's in dolphins. Thus, animals with a history of elevated progesterone (greater than 3 ng/ml) should be considered candidates for prostaglandin treatment. The results hint that it may have a similar effect on the HPO axis as progesterone treatment. However, further trials with cycling dolphins need to be conducted to confirm this observation.

Table 1. Lutalyse® administration in bottlenose dolphins.

a. Retained corpora lutea (rCL).
b. Progesterone (P) concentration prior to administration of Lutalyse®.
c. Interval from Lutalyse® administration to ovulations as determined by ultrasonography.
d. Current reproductive status.
e. For animal B, the rCL reflects a nonresponse to the initial Lutalyse® administration.
Once the animal responded to the Lutalyse® it went into anestrus.

Table 2. Regu-Mate® administration in delphinids.

a. For O. orca, estrus was defined as the rise in urinary estrogen conjugates. For T. aduncus, estrus was defined as the presence of a follicle greater than 1 cm in diameter.

Acknowledgments

We thank animal care, training, laboratory and veterinary staff at SeaWorld San Antonio, SeaWorld San Diego, U.S. Navy Marine Mammal Program and Ocean Park, Hong Kong for their support and efforts. This is a SeaWorld technical contribution number 2000-02-T.

References

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