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RESEARCH ARTICLE
Contents Vol 22(4)

Preservation of beluga (Delphinapterus leucas) spermatozoa using a trehalose-based cryodiluent and directional freezing technology

J. K. O’Brien A B C and T. R. Robeck A
+ Author Affiliations
- Author Affiliations

A SeaWorld and Busch Gardens Reproductive Research Center, 2595 Ingraham Street, San Diego, CA 92109, USA.

B Centre for Advanced Technologies in Animal Genetics and Reproduction, Faculty of Veterinary Science, University of Sydney, NSW 2006, Australia.

C Corresponding author. Email: justine.o’brien@seaworld.com

Reproduction, Fertility and Development 22(4) 653-663 https://doi.org/10.1071/RD09176
Submitted: 24 July 2009  Accepted: 9 October 2009   Published: 11 March 2010

Abstract

A beluga (Delphinapterus leucas) sperm preservation method was developed for use in genome banking and AI. In Study 1, glycerol-based cryodiluents (modified BF5F and modified Platz Diluent Variant (PDV)) were unable to maintain adequate progressive motility using straws (fast and slow freezing rate (FR)) or pellets (slow FR). Neither freezing method nor FR affected in vitro sperm characteristics (P > 0.05), but retention of prefreeze progressive motility following thawing was greater (P < 0.05) for BF5F (21%) than PDV (15%). In Study 2, examining the effects of straw freeze–thawing using BF5F with glycerol (1 and 3%, v/v) or trehalose (46 and 91 mM) on sperm characteristics, samples cryopreserved in trehalose exhibited superior (P < 0.05) in vitro parameters compared with their glycerol-treated counterparts. In Study 3, compared with a straw method, directional freezing using 91 mM trehalose enhanced (P < 0.05) sperm characteristics, with samples retaining 38%, 75% and 61% of their prefreeze progressive motility, curvilinear velocity and viability, respectively. A higher (P < 0.05) proportion of motile spermatozoa displayed rapid velocity after directional (21 ± 1%) compared with straw (12 ± 3%) freezing. Systematic development of a cryodiluent and the use of directional freezing resulted in beluga spermatozoa exhibiting adequate post-thaw quality for genome banking and use in AI.

Additional keywords: artificial insemination, cetacean, gamete rescue, genome resource bank, sperm cryopreservation.


Acknowledgements

Animal care, animal training, curatorial and veterinary staff at SeaWorld California are thanked for assistance with animal handling and sample collection. Mrs Mitzi Synnott and Mrs Nicole Grovhoug are thanked for the initial training that led to the successful collection of beluga semen, and the authors are grateful to all Wild Arctic Staff for ongoing collections. Animal Care Laboratory staff (SeaWorld San Diego), including Ms Whitney Coleman, Mrs Leigh Ann Smith, Mrs Pam Thomas and Mrs Melinda Tucker, are thanked for technical support, as are SeaWorld and Busch Gardens Reproductive Research Center staff Ms Karen Steinman and Ms Michelle Morrisseau. Mr Brad Andrews (Busch Entertainment Corporation) is thanked for his invaluable support of this research. This project was funded by SeaWorld Corporation and is a SeaWorld Technical Contribution no. 2009–02-T.


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