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Estrous cycle characterisation and artificial insemination using frozen–thawed spermatozoa in the bottlenose dolphin (Tursiops truncatus)

SeaWorld Texas, 10500 SeaWorld Drive, San Antonio, TX 78251, USA, ¹ Conservation and Research Center, National Zoological Park, Smithsonian Institution, Front Royal, VA 22630, USA, ² Laboratory of Fish Culture, Faculty of Bioresources, Mie University, Tsu, Mie 514-8507, Japan, ³ US Navy Marine Mammal Program, San Diego, CA 92152, USA, ⁴ Centre for Advanced Technologies in Animal Genetics and Reproduction, Faculty of Veterinary Science, University of Sydney, Sydney, NSW 2006, Australia, ⁵ Kamogawa SeaWorld, Kamogawa, Chiba 296-0041, Japan, ⁶ Acquario di Genova, Ponte Spinola 16128, Genova,Italy, ⁷ SeaWorld California, 500 SeaWorld Drive, San Diego, CA 92109, USA and ⁸ Dolphin Quest Oahu, 5000 Kahala Ave, Honolulu, HI 96816, USA

Correspondence should be addressed to Todd R Robeck; Email: Todd.Robeck{at}SeaWorld.com

The reproductive endocrinology of the bottlenose dolphin, Tursiops truncatus, was characterized to facilitate the development of artificial insemination using cryopreserved spermatozoa. Specific objectives were: (i) to determine the excretory dynamics of urinary luteinizing hormone (LH) and ovarian steroid metabolites during the estrous cycle; (ii) to evaluate the effect of an exogenously administered synthetic progesterone analog (altrenogest) on reproductive hormone excretion; (iii) to correlate follicular growth and ovulation (as determined by transabdominal ultrasound) to urinary LH and ovarian steroid metabolites; (iv) examine the in vivo fertilisation capacity of cryopreserved semen, and (v) to develop an intrauterine insemination technique. Based on urinary endocrine monitoring of natural estrous cycles (2 consecutive cycles) and nine post altrenogest cycles in ten females, estrous cycles were found to be 36 days long and comprised of an 8 day and 19 day follicular and luteal phase, respectively. Peak estrogen conjugates (EC; 5.4 ± 3.8 ng/mg creatinine (Cr)) occurred 8 h prior to the LH surge (70.9 ± 115.7 ng/mg Cr). The time of ovulation, as determined by ultrasonography, occurred 32.1 ± 8.9 h and 24.3 ± 7.0 h after the onset of the LH surge and LH peak, respectively. Mean preovulatory follicular diameter and circumference were 2.1 ± 0.5 cm and 6.5 ± 1.5 cm, respectively. Of the 27 estrous synchronisation attempts, 13 resulted in an ovulatory cycle, with ovulation occurring 21 days post-altrenogest treatment. Intrauterine (4 of 5) and intracornual (1 of 3) inseminations conducted across eight estrous cycles resulted in five pregnancies (63%), one pregnancy resulted from the use of liquid stored semen, whereas four were achieved using cryopreserved semen. These data provide new information on female bottlenose dolphin reproductive physiology, and demonstrate that the combination of endocrine monitoring and serial ultrasonography contributed to successful AI using liquid-stored and cryopreserved semen.