INTEGRAL REPRODUCTION PROGRAM IN DOLPHINARIS:

RELEVANT ASPECTS AND REGISTER OF THE FIRST DOLPHIN

CALF TURSIOSP TRUCANTUS OBTAINED BY ASSISTED
REPRODUCTION TECHNIQUES DEVELOPED BY THE COMPANY.

Cristian Ugaz1, Raúl Fuentes1, Andrés Casarrubias1, Leonardo Ibarra1

Dolphinaris, Cancún, Quintana Roo, México

ABSTRAC

The reproduction in all zoological collection plays a crucial role in conservation of the
species and the sustainability of the populations. In the case of the dolphins that are
maintained under human care in Mexico, the reproduction has evolved significantly to the
sustainability of the population, since it is not allowed to collect dolphins from the wild nor
import or export them.

The characteristics of the captive dolphin population in Mexico, framed in government

restrictive conservation policies, have raised the need for more efficient and responsible
breeding programs. The application of biotechnologies of the reproduction became more
important, because of the fact that small populations need to maintain a constant reproduction
rate, with the purpose of injecting new genetic resources to ensure the variability of the
population.

In 2006 Dolphinaris began an integral reproductive program in the dolphin population; with a
considerable important investment made in to the development of their own proper
technologies. The program covers the development of new techniques and 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, which so far 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
techniques 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 have 800 million sperm per milliliters.

The next phase of the project, was to increase the number of animals by two different ways of
reproductions, natural mating in a group of females and artificial insemination with another
group of females. Within the reproductive characteristics of dolphins, they present long
anestrous periods without apparent causes, which is why the handling of the reproductive
cycles of the females should be profited to the maximum. Dolphinaris developed a protocol
to induce the ovulation and in which the prolonged anestrous can be interrupted; the
treatment is based in luteolytic a compound (Lutalise® Pharmacia Animal Health) and
reproductive hormones (FSH – HCG. PG600 ®, Intervet Labs.) IM., with a subsequent
application of the same treatment. The treatment was given to a female of 20 years old, who
had never showed reproductive activity since she became part of the collection in 1999, she
was induced with the hormonal treatment previously descript and 20 days post treatment,
follicle growth was observed. The ovulation occurred 34 days post injection it was detected
and followed by ultrasound. In this case the female copulated with the male directly and
pregnancy diagnosis was performed at 50 days and it lasted 368 days.

A third stage of the project was to implement and develop our own techniques for artificial
insemination. In order to get it, we implemented the techniques previously described by
Robbeck (2005), and Brook (pers. comm.), those were performed by using a flexible
endoscope and deposit of the 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 mill sperm / ml. From these attempts non pregnancy was obtained,
subsequently 4 attempts were made with similar insemination techniques to those already
described, but with varied in the application site, the semen characteristics that were used
were different in each case (Table 1) without achieving a pregnancy. In the next female
candidate for insemination, follicular growth was tracked based on the animal follicle growth
history, this ovulates with less follicular diameter (<2.0 cm) on a regular basis.

Knowing the previous data of individual follicular growth on this female, prediction of
ovulation was expected to occur at certain follicular size. The ovulation happened in an
interval of 4 hours between ultrasound exams. She was inseminated only once. The technique
used in this attempt was different from those previously published, it was done with a sterile
conical 30 cm vaginoscope with its own light source, and the semen deposition was
performed 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 the first dolphin calf which was
obtained by artificial insemination with frozen semen.

In 2009 Dolphinaris achieved 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 completing the gestation of each of
the females, providing the best care for them. With these results, we could manage the growth
of our population adequately and in each effort achievements were more specific. Moreover,
as the frozen semen that was used come from another population, new genetic material was
introduced to the group without the need to move animals from one location to other. All
trails and techniques described in this paper were developed entirely by the Dolphinaris
medical and research team.
Table 1: Semen Quality and technique of Artificial Insemination in Dolphinaris

Semen sperm
Follicle size %
Date Female volume concentration Status Semen Deposit semen
(cm) Motility
ml x106 ml
08/12/2006 1 4 800 50 4
Post-ovulation Frozen Intra-vaginal
09/12/2006 1 4 800 50 4

23/02/2007 2 2.06 63 900 85 4

Fresh Intrauterine
24/02/2007 2 Post-ovulation 32 900 90 4.5

23/03/2007 3 3 850 70 4
Post-ovulation Frozen Intrauterine
24/03/2007 3 3 850 70 4

01/04/2007 4 2.12 4 800 80 4

02/04/2007 4 4 800 70 4 Frozen Intrauterine
Post-ovulation
03/04/2007 4 4 800 80 4.5

12/06/2007 5 Ov 25 750 90 4.5 Fresh Intrauterine

15/06/2007 6 2.15 25 650 85 4.5

Fresh Intrauterine
16/06/2007 6 Ov 45 850 95 4.5

18/07/2007 7 2.05 7 800 70 4.5

19/07/2007 7 2.12 6 800 70 4.5
19/07/2007 7 2.15 6 800 70 4.5 Frozen Intra-vaginal
20/07/2007 7 2.2 6 800 70 4
20/07/2007 7 Post-ovulation 6 800 80 4

12/08/2007 8 2.3 5 800 70 4

13/08/2007 8 10 800 70 4.5 Frozen Intra-vaginal
Post-ovulation
13/08/2007 8 10 800 80 4.5

16/12/2008 9 Post-ovulation 12.5 390 75 4.5 Frozen Intrauterine

LITERATURED CITED

Robeck, T. R., Steinman, K. J., Yoshioka, M., Jensen, E., O’Brien, J. K., Katsumata, E., Gili,
C., McBain, J. F., Sweeney, J. and Monfort, S. L. (2005). Estrous cycle characterisation and
artificial insemination using frozen-thawed spermatozoa in the bottlenose dolphin (Tursiops
truncatus). Reproduction 129:659-674.

Ugaz Cristian; G.A. Montaño; J.L. Solórzano. The Effect of Equek® STM Paste on the
Quality of Semen Thawing of Tursiops truncatus in the Aquarius of Aragon in Mexico City.
IAAM Proceedings 2006