Current reproduction of captive marine mammals, dolphins in particular, has grown utilizing assisted reproduction techniques. The most common being via artificial insemination AI. However, in order to implement an AI protocol, utilization of hormones is needed to synchronize estrus, induce ovulation, and to determine the appropriate timing for AI or natural reproduction (4,2). Hormones like altrenogest have been utilized on domestic species (6) and with some captive dolphins and whales with the intent of avoiding estrus without affecting fertility (3).
This study was to evaluate reproductive activity on females T. truncatus treated with altrenogest over a year. Eleven females ranging from 10 to 15 years in three different groups G1 and G2 = 4 animals and G3 = 3 animals, each groups in different facilities of Dolphin Discovery (Puerto Aventuras, Isla Mujeres and Cozumel) on the Mexican Caribbean. In the tree facilities the animals were in semi captivity on the sea. A frozen thawed fish diet consisting of Clupea arengus, Mallotus villosus, Menidis spp and Loligo vulgaris was given and consumption was approximately 60 to 80 kcal/kg live weight per day. Each animal was given 14 mg altrenogest with its food for a minimum of 12 months prior this study.
Thirty days after the altrenogest administration was removed two samples were taken: A) Vaginal cytology was used to determine any changes in cellular structure. All samples were collected using positive reinforcement training and voluntary behaviors in order to obtain constant samples. A 20 cm cotton swab was introduced in to the vagina to obtain the exfoliative cells. The swab was washed with Hartman solution for 10 seconds to later deposit 25 µl of this solution on a glass slide and stained with Diff/Quick to determinate the parabase cell percentage as well as cornificate and intermediate cells. B) Blood samples were collected every 3rd day and centrifuged 600 G for 10 min to obtain serum to determine utilizing quimoluminescence the following-progesterone levels; estradiol and luteinizing hormone (samples were analyzed at a commercial laboratory).
Regarding the vaginal cytology 25-45% cornificated cells were recorded before stopping the altrenogest. Two peaks were observed, one shortly after treatment (60-70%) which then increased another 10% on days 3 to 9 followed by a decrease to 35-55% cornificated cells with a the second peak showing on days 12 to 20 at 45-65%.
Estradiol values found were between 16 and 114pg/ml showing a clear relationship with the increase of cornificated cells and progesterone with values were between 0.2 to 10 ng/ml. Females which were fertilized showed values of 15 ng/ml luteinizing hormone values were between 0.1 to 0.24 mUI/ ml increasing while observing higher levels of estradiol. This is consistent with prior marine mammal findings (5)
The most relevant finding in this study was numerous sperm in vaginal exudates of two females on days 4 and 5 after altrenogest removed. This confirms sexual activity almost immediately after treatment and showing that altrenogest activity is quickly reversed so as to not interrupt sexual activity. This is contrary previous research in which sexual activity occurred 21 days after treatment (3). In this study which females were monitored and gestation was confirmed with increases in progesterone values of 11 and 17 ng/ml at days 20 and 30 after finding the sperm, and embryonic development was observed using the ultrasound.
Reproductive activity was observed after the fourth day of removal yet it varied amongst each animal but was directly related with estradiol levels. Results of this study proved reproductive activity at different times but always with an increase of cornificated cells and in two occasions with an increase in estradiol
The importance of this study is to find efficient programs for assisted reproduction including in vitro fertilization (1), natural or controlled assisted reproduction in captive animals. This would help broaden the gene pool without a need to more animals (2). With our results, we conclude that it's possible to set up efficient reproductive programs with captive animals without interfering with their health while controlling estrus and reproductive timing.
1. Fukui Y, Mogoe T (1997). In vitro fertilization of in vitro matured minke whales (Balaenoptera acutorostrata) follicular oocytes. Marine mammal science 13:395-404.
2. Robeck T, Curry B, McBain J, Kraemer D (1994). Reproductive biology of the bottlenose dolphin (Tursiops truncates) and potential application of advanced reproductive technologies. Journal of Zoo and Wildlife Medicine 25:321-336.
3. Robeck T, Steinman KJ, Gearhart S, Reidarson (2004). Reproductive physiology and development of artificial insemination. Biology of reproduction En prensa para publicación en abril 28.
4. Schroeder J (2000). Reproductive aspects of marine mammals. En: CRC Handbook of marine mammal medicine: Health, disease and rehabilitation. Pp353-369. Ed. Leslie A D. Washington DC.
5. Walker LA, Cornell L, Dahl K, Czekata N, Dargen C, Joseph B, Hsueh A, Lasley B (1998). Urinary concentrations of ovarian steroid hormone metabolites and bioactive follicle-stimulating hormone in killer whales (Orcinus orchus) during ovarian cycles and pregnancy. Biology of Reproduction 39: 1013-1020.
6. Wright P, Malmo J (1992). Pharamacologic manipulation on fertility. Applied Pharmacology and Therapeutics 8:57-89.