title: Deep intra-uterine artificial inseminations using cryopreserved spermatozoa in beluga (Delphinapterus leucas).
abstract: Artificial insemination (AI) with liquid-stored spermatozoa and sperm cryopreservation using directional freezing (DF) have been successful in the beluga. This study built on this foundation to develop a deep intra-uterine AI technique with frozen-thawed semen in beluga. Forty-two ejaculates from one male were cryopreserved using DF technology and subsequently used for 10 insemination attempts with seven females. Percentage pre- and post-thaw progressive motility and viability were (mean +/- SD) 73.0 +/- 12.2, 38.4 +/- 8.8, 88.0 +/- 0.1, and 59.3 +/- 15.7%, respectively. A series of GnRH injections (3 x 250 microg, IV, 1.5 to 2 h apart) were used to induce ovulation, once a growing follicle >2.5 cm in diameter was visualized via trans-abdominal ultrasonography. Artificial insemination was performed at 30.1 +/- 3.8 h post-initial GnRH injection with semen deposited in the uterine horn, 92.6 +/- 16.2 cm beyond the genital opening using a flexible endoscope. The external cervical os (cEOS) was located beyond a series of 5 to 10 vaginal rings, 44.8 +/- 9.3 cm from the external genital opening. The internal bifurcation of the uterus was 27 +/- 6.8 cm beyond the cEOS. Ovulation occurred at 8.5 +/- 7.6 h post-AI. Two of 10 inseminations (20%) resulted in pregnancy. The first pregnancy resulted in twins; both calves were born 442 d after AI, with one surviving. The second pregnancy is ongoing. These findings represent the first successful application of AI using frozen-thawed semen in beluga, and are important examples of how assisted reproductive technologies can provide tools for the global management of threatened species.
authors.affiliation: SeaWorld and Busch Gardens Reproductive Research Center, SeaWorld Parks and Entertainment Corporation, San Diego, CA 92109, USA. todd.robeck@seaworld.com
authors.forename: T R
authors.initials: TR
authors.lastname: Robeck
authors.forename: K J
authors.initials: KJ
authors.lastname: Steinman
authors.forename: G A
authors.initials: GA
authors.lastname: Montano
authors.forename: E
authors.initials: E
authors.lastname: Katsumata
authors.forename: S
authors.initials: S
authors.lastname: Osborn
authors.forename: L
authors.initials: L
authors.lastname: Dalton
authors.forename: J L
authors.initials: JL
authors.lastname: Dunn
authors.forename: T
authors.initials: T
authors.lastname: Schmitt
authors.forename: T
authors.initials: T
authors.lastname: Reidarson
authors.forename: J K
authors.initials: JK
authors.lastname: O'Brien
mesh_terms: D000818:Animals; D050643:Beluga Whale / Q000502:physiology; D015925:Cryopreservation / Q000662:veterinary; D004973:Estrus Synchronization / Q000379:methods; D005260:Female; D007315:Insemination, Artificial / Q000379:methods / Q000662:veterinary*; D008297:Male; D011247:Pregnancy; D018873:Pregnancy Rate; D012661:Semen / Q000166:cytology / Q000502:physiology*; D055101:Semen Analysis / Q000662:veterinary; D012662:Semen Preservation / Q000662:veterinary; D053625:Sperm Retrieval / Q000662:veterinary; D014599:Uterus