Improved sperm freezing in the endangered African wild dog (Lycaon pictus) using a two-step dilution TRIS-egg yolk extender containing Equex STM
Van den Berghe, Femke, Paris, Monique C.J., Sarnyai, Zoltan, Briggs, Michael B., Farstad, Wenche K., and Paris, Damien B.B.P. (2016) Improved sperm freezing in the endangered African wild dog (Lycaon pictus) using a two-step dilution TRIS-egg yolk extender containing Equex STM. In: Proceedings of the 18th International Congress on Animal Reproduction. PW820. pp. 319-320. From: ICAR 2016: 18th International Congress on Animal Reproduction, 26-30 June 2016, Nouzilly, France.
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Abstract
Development of assisted breeding techniques can aid conservation and management of the endangered African wild dog (Lycaon pictus). Previous attempts to freeze sperm from this species has proven unsuccessful with sperm motility dropping to nearly 0% within 2 h of thawing. The aim of this study was to improve the freezing success of African wild dog sperm by testing two routinely used canine cryopreservation protocols.
Sperm was frozen from n=3 captive African wild dog males housed at Albuquerque BioPark (Albuquerque, NM, USA) and Binder Park Zoo (Battle Creek, MI, USA) during the breeding season (Aug-Sept 2014). Freshly collected semen samples were evaluated for volume, colour, pH, motility, viability, morphology, sperm number, acrosome status and DNA integrity. Each sample was split and frozen using two different protocols. Protocol 1: semen was diluted with a Tris-egg yolk extender containing 8% glycerol and 20% egg yolk, and slowly cooled from 37°C to 4°C over 2.5 h. The sample was then loaded into 0.25 mL straws, suspended 4 cm over liquid nitrogen vapour for 10 min, then plunged in liquid nitrogen. Protocol 2: semen was first diluted with a Tris-egg yolk extender containing only 3% glycerol and 20% egg yolk, followed by a second extender (same composition) now containing 7% glycerol and 1% Equex STM, added after the 2.5 h refrigeration period. The freezing procedure was the same as Protocol 1. Straws from both protocols were thawed in a 37ᵒC water bath, but Protocol 2 straws were further diluted by with a thawing solution which that consisted of the initial extender solution without glycerol and egg yolk. Sperm were incubated at 37 ᵒC and motility evaluated at 5 min and every 2 h for 8 h after thawing. Viability, morphology and acrosome integrity was evaluated over 6 h and DNA integrity was evaluated immediately post-thaw.
Sperm motility declined significantly for both protocols immediately after thawing (fresh 78.9 ± 2.6%; Protocol 1 24.4 ± 5.0%; Protocol 2 36.7 ± 4.2%; P ≤ 0.05). Motility was significantly higher for Protocol 2 from 2 h after thawing (Protocol 1 1.0 ± 0.8%; Protocol 2 30.8 ± 1.9%; P ≤ 0.05) and sperm remained motile for up to 8 h. Sperm frozen with Protocol 2 also had significantly higher viability (Protocol 1 37.0 ± 5.7%; Protocol 2 65.3 ± 9.9%; P ≤ 0.05) and acrosome integrity (Protocol 1 22.8 ± 8.2%; Protocol 2 69.3 ± 8.8%; P ≤ 0.05) immediately after thawing. There was no difference in the proportion of normal morphology or DNA fragmentation between both protocols.
Our results demonstrate that using a two-step dilution with TRIS-egg yolk extender containing Equex STM yields greatly improved post-thaw quality and longevity in African wild dog sperm; making it suitable for use in artificial insemination.
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