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In conjunction with artificial insemination (AI) and sperm preservation, sperm sexing technology has great potential as a population management strategy for captive bottlenose dolphins. Successful AI using fresh spermatozoa (Robeck TR et al. 2001 CRC Marine Mammal Medicine 193-226) and flow cytometric analysis of bottlenose dolphin spermatozoa (Garner DL and Seidel GE Jr 2002 CSAS Symposium 2-13) support this approach. For sperm sexing, methods for short-term storage of semen in a liquid state are required to enable transport of spermatozoa to the sorting laboratory. In addition, cryopreservation techniques must be optimized for long-term storage of sexed spermatozoa. Our objectives were to assess: (i) 3 cryopreservation methods�_�2 straw sizes�_�3 thawing rates (Exp. 1) and (ii) effects of liquid storage for 24�h (pre-cryopreservation) and sperm concentration at freezing (Exp. 2) on post-thaw characteristics (PT) of bottlenose dolphin spermatozoa. For Exp. 1 and 2, 4 ejaculates (collected by manual stimulation)�_�3 males (aged 14-34�yr)�_�4 replicates were used. For Exp. 1, semen was frozen in 0.25-mL (SM) and 0.5-mL straws (LG) by 3 methods (Mt) (Mt1: lactose, egg yolk, -32�C�min-1; Mt2: lactose, egg yolk, 1.5% Equex STM (Nova Chemical, Calgary, Canada), -19.7�C�min-1; Mt3: Test yolk buffer (TYB), -116�C�min-1). All Mt had 3% glycerol. Samples were thawed using a slow (S: 2.8�C�s-1), medium (M: 8.8�C�s-1) or fast (F: 21�C�s-1) rate. In Exp. 2, ejaculates were divided into 4 aliquots for dilution (1:1) and stored at 4�C with EquiPro� (EP4�C, Minitube, Verona, WI, USA) and TYB (TYB4�C) or at 21�C with Androhep EnduraguardTM (AH21�C, Minitube) or no dilution (NEAT21�C). After 24�h, samples were frozen and thawed using Mt3�_�SM�_�F at 10�_�106 sperm�mL-1 (LOW) or 100�_�106 sperm�mL-1 (STD). PT evaluations of motility (total motility [TM], % progressive motility [PPM], kinetic rating [KR, 0 to 5]) and acrosomal status (Spermac� , Minitube) were performed at 30�min and 6�h after dilution (1:1) with AH at 21�C. For statistical analysis (ANOVA), a sperm motility index (SMI�=�TM�_�PPM�_�KR) was calculated and expressed as % of initial SMI. For all ejaculates, initial TM and PPM were greater than 85% and KR was 5. In Exp. 1, at 6�h PT, %SMI was highest for Mt3�_�LG�_�M (45.5ʱ�8.7) and Mt3�_�SM�_�F (44.8ʱ�11.9). For Exp. 2, %SMI at 0�h PT was higher for samples stored at 4�C than at 21�C (TYB4�C 41.0ʱ�8.4, EP4�C: 36.7ʱ�7.7, NEAT21�C: 23.8ʱ�8.6, AH21�C: 14.8ʱ�8.6, P�<�0.001) and, with the exception of AH21�C, was similar between the LOW and STD concentration. At 6�h PT, %SMI for all treatments was higher for STD than LOW concentration (P�<�0.05). Acrosome integrity was similar across treatments. In summary, a semen cryopreservation protocol maintained high levels of the initial characteristics of ejaculated spermatozoa. Transport of semen for sex pre-selection and cryopreservation within 24�h may be feasible, but impact of storage time on functional capacity of dolphin spermatozoa is unknown. |
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