Abstract

Reproduction in zoological collections plays a crucial role in conservation and sustainability. In the case of the dolphins that are maintained under human care in Mexico, reproduction has evolved significantly to assure the sustainability of the population. Collection, importation, and exportation are not permitted. Due to government restrictive conservation policies, there is a need for more efficient and responsible dolphin breeding programs. The application of reproductive biotechnology is important to manage small populations in need of a constant reproduction rate and new genetic resources to ensure variability of the population.

In 2006, Dolphinaris began an integral dolphin reproductive program. This program focuses on considerable efforts to develop in house technologies. The program covers the development of procedures to register the tracking of the follicular dynamics, the detection of ovulations, selection and storage of semen, artificial insemination, ovulation induction and cycle synchronization.

The first phase of the breeding program focused in the creation of a dolphin frozen semen bank. So far, this bank has genetic material from three species of dolphins that are maintained under human care in Mexico (Atlantic Bottlenose, Pacific Bottlenose, Aduncus dolphins). The semen is obtained by training through operant conditioning techniques. The freezing technique is manual (Ugaz et al., 2006). In summary, fructose-based extender was used, freezing rate of 0.1° Celsius/min, from ambient temperature to 4° Celsius and a second freezing rate of the 1.5° Celsius/second. Each straw has 800 million sperm per milliliters.

The next phase was to increase the number of animals by two reproductive strategies, natural mating and artificial inseminations. Dolphins present long anestrous periods without apparent causes; management of the female reproductive cycles should be maximized. Dolphinaris developed a protocol to induce ovulation and interrupt prolonged anestrous using a luteolytic compound (Lutalyse^® Pharmacia Animal Health) and reproductive hormones (FSH--HCG, PG600^®, Intervet Labs.) IM. The treatment was given to a 20 year old female who had not demonstrated reproductive activity since she became part of the collection in 1999. She was induced with the hormonal treatment and 20 days post treatment, follicle growth was observed. Ovulation occurred 34 days post injection; it was detected and followed by ultrasound. In this case, pregnancy resulted from natural mating. Pregnancy was identified at 50 days and it lasted 368 days.

A third stage of the project was to implement and develop in house artificial insemination. We implemented the techniques previously described by Robeck (2005) and Brook (pers. comm.). These were performed using a flexible endoscope and depositing semen into the uterine horns. In 2006 the first two attempts were unsuccessful, females were inseminated when pre-ovulatory follicles presented a size > 20 mm, the procedure was performed twice in each case 3 ml of semen dose was used, with a concentration of 800 million sperm/ml. Subsequently 4 attempts were made with similar insemination techniques, but with variations in the application site, the semen characteristics that were used were different in each case (Table 1). No pregnancy was achieved.

In the next candidate for insemination, follicular growth was tracked based on the individual's follicle growth history. This animal typically ovulates with a follicular diameter less than 2.0 cm on a regular basis. Prediction of ovulation was based on follicular size. The ovulation occurred within an interval of 4 hours between ultrasound exams. The animal was inseminated only once. The technique used in this attempt was performed with a sterile conical 30 cm vaginoscope with its own light source; semen was deposited through a polypropylene catheter 55 cm long and 3 mm in diameter, which was introduced into the body of the uterus. In this case, 12.5 ml of semen thawed at 36° Celsius for 45 seconds was used (Table 1). The donor was from another city, and the semen was preserved for a period of 1.5 years. The result of this trail was our first dolphin calf obtained by artificial insemination with frozen semen.

In 2009 Dolphinaris had 5 viable calves: 3 from natural ovulation and mating, 1 from induced ovulation and natural mating, and one by artificial insemination and natural ovulation. The pre and post delivery management allowed full gestation and best care for dams and babies. With these results, we can manage the growth of our population adequately. The use of frozen semen from another population affords new genetic material without the need to move animals from one location to other. All trails and techniques described in this paper were developed by the Dolphinaris medical and research team.

Table 1. Semen quality and technique of artificial insemination in dolphins.

References

1.  Robeck TR, Steinman KJ, Yoshioka M, Jensen E, O'Brien JK, Katsumata E, Gili C, McBain JF, Sweeney J, Monfort SL 2005. Estrous cycle characterisation and artificial insemination using frozen-thawed spermatozoa in the bottlenose dolphin (Tursiops truncatus). Reproduction 129:659-674.

2.  Ugaz Cristian M, Montaño GA, Solórzano JL The Effect of Equek^® STM Paste on the quality of semen thawing of Tursiops truncatus in the Aquarius of Aragon in Mexico city. 2006 IAAAM Proceedings.