160 FREEZABILITY OF ELECTROEJACULATED AND EPIDIDYMAL SPERMATOZOA FROM BLUE WILDEBEEST (CONNOCHAETES TAURINUS)

Abstract

Maintenance of population viability, especially endangered species, requires preservation of as much genetic variability as possible, therefore genetic resource banks are very important. For male gametes, preservation of all available sources (ejaculates and epididymal) are useful. Information regarding sperm characteristics of most wild ruminant species is limited compared to that from domestic species. The objective of this work was to characterize the freezability of electroejaculated and epididymal spermatozoa from a wildebeest (5 year old; housed in Cabárceno Nature Park, Cantabria, Spain) that was castrated because of behavioral problems. After general anesthesia (ethorfine + xilazine, 1.8 mL + 0.5 mg kg^–1) with dart, semen was collected by electroejaculation (3 V and 75 mA). Sperm concentration was 250 × 10⁶ mL^–1 (total spermatozoa: 1128.6 × 10⁶). After castration, epididymides were disected and spermatozoa were collected by making several incisions in the caudal epididymis. Concentration was 12 441 × 10⁶ spermatozoa mL^–1 (total spermatozoa: 24 882 × 10⁶). Samples were diluted to 200 × 10⁶ spermatozoa mL^–1 (TesT-Fructose-Egg yolk-Glycerol-Antibiotics) and chilled to 5°C during 2 h. Diluted semen was packaged in 0.25-mL straws and frozen from 5°C to –100°C (–20°C min^–1) in a programmable cell freezer (Kryo 10, Planer). Straws were plunged into liquid nitrogen until analysis and thawed in a water bath (65°C, 6 s). Fresh, pre-freezing and post-thawed samples were analysed for motility (total motility TM, %; progressive motility PM, %; path velocity VAP, μm s^–1; track speed VCL, μm s^–1; progressive velocity VSL, μm s^–1) using a CASA (ISAS, Proiser, Valencia, Spain). Viability (VIAB %) (SYBR-14 and propidium iodide) and mitochondrial membrane potential (MIT %) (JC1) were assessed by flow cytometry. Post-thawing results for electroejaculated v. epididymal samples were, respectively: TM: 87.0 v. 64.6%; PM: 68.7 v. 33.4%; VAP: 95.9 v. 49.8 μm s^–1; VCL: 108.3 v. 71.6 μm s^–1; VSL: 86.7 v. 40.2 μm s^–1; VIAB: 57.0 v. 73.9%; MIT: 59.5 v. 77.5%. Motility parameters were higher for the electroejaculated sample; however, viability was higher for the epididymal sample. Recovery rates (post-thawed value/pre-freezing value × 100) for electroejaculated v. epididymal samples were: TM: 97.2 v. 93.4%; PM: 113.3 v. 103.2%; VAP: 88.9 v. 122.0 μm s^–1; VCL: 87.5 v. 126.0 μm s^–1; VSL: 93.4 v. 125.3 μm s^–1; VIAB: 75.0 v. 97.7%; MIT: 69.2 v. 95.9%). These rates suggest a good freezability of electroejaculated and epididymal spermatozoa in blue wildebeest.

This work was supported in part by Cantur. ³ Supported by Juan de la Cierva program (MICINN, Spain).