CRYOPRESERVATION OF CANINE SPERMATOZOA IN A SIMPLE EXTENDER COMPOSED OF SKIM MILK, GLUCOSE AND GLYCEROL

Abstract

Cryopreservation of canine spermatozoa offers the potential to exchange genetic materials between and among countries, and improve breeding management programs of working dogs, including guide dogs for the blind. The conventional diluents for canine semen have successfully used egg yolk (EY) to preserve canine spermatozoa against cold shock during the freezing and thawing process. However, due to outbreak of avian influenza in the world, transportation of frozen semen with EY has become difficult. Thus, it is an urgent subject to develop a novel cryoprotectant without EY for canine spermatozoa. The objective of the present study was to evaluate the efficacy of skim milk and sugar (SS) based extender without EY for cryopreservation of canine spermatozoa. A total of 6 ejaculates from 4 Labrador retrievers were collected by digital manipulation into sterile plastic tubes. Only the sperm-rich fractions of the ejaculates were collected. Collected ejaculates were extended in the first extenders at a sperm concentration of 200×106 sperms/ml. SS based extender (30 mg/ml skim milk and 0.3 M glucose) or EY based extender (20% [v/v] EY, 24 mg/ml Tris, 14 mg/ml citric acid monohydrate, 0.8 mg/ml glucose, 0.65 mg/ml penicillin G potassium and 1 mg/ml streptomycin sulphate) were used for the first extenders. After 3h equilibration at 4 °C, the equivalent volume of the second extenders which has same composition as that of first extender except that it contained 14% (v/v) glycerol, was added to the semen aliquots, and the semen samples were left at 4 °C for 15 min. Final sperm concentration after the addition of the second extender to the semen samples were 100 × 106 sperms/ml. The extended semen was packed in a 0.25 ml straw, and then was frozen in a closed styrofoam box (24.5 × 17.5 × 17.5 cm) on a rack 6 cm above the surface of LN2 for 15 min. The straws were plunged into LN2 and stored until evaluation. After thawing by immersing the straws in a water bath at 37 °C for 60 s, the content of each straw was emptied into a 1.5 ml eppendorf tube, and the each sample was evaluated using a light microscope supplied with a Computer Assisted Sperm Analysis (CASA) system. The proportion of total motile spermatozoa (%; TMS) and some motility parameters (straight-line velocity, amplitude of lateral head displacement, etc.) were determined. TMS of each samples were 25–85% (average: 50.2 ± 8.8) in the SS group and 13–83% (average: 48.0 ± 11.0) in the EY group. The percentages of TMS in the SS group to the EY group were 67.3–215.0 in each sample, and there was no significant difference between two groups. Moreover, other motility parameters in the SS group were similar to the corresponding parameters in the EY group. These results suggest that the simple extender composed of skim milk, glucose and glycerol is available for cryopreservation of canine spermatozoa, and it may contribute to efficient production of guide dogs for the blind. (poster)