95 THE EFFECT OF FREEZING TECHNIQUES ON QUALITY OF CAT TESTICULAR SPERM

Abstract

Cryopreservation of testicular tissue is beneficial for valuable animals that die unexpectedly or when elective castration is required. Until recently, knowledge regarding cryopreservation of testicular tissue/sperm in the domestic cat has been limited. The objective of this study was to determine the effects of freezing techniques and cryoprotectants on quality of testicular sperm. In Experiment 1, each testis was cut into 10 equal small pieces (∼2 × 3 × 5 mm) and cryopreserved in freezing medium containing with 5% (v/v) glycerol using conventional (10 min in liquid nitrogen vapors) or controlled-rate freezing techniques. In Experiment 2, testicular tissues were conventionally frozen with different types of 5% (v/v) cryoprotectants [glycerol (Gly), dimethylsulphoxide (DMSO), 1,2-propanediol (PrOH), or ethylene glycol (EG)]. Non-cryopreserved testicular sperm was used as a control. After thawing, testicular sperm were extracted and examined for viability and DNA integrity using non-membrane-permeable DNA staining (Ethidium homodimer-1) and TUNEL assay, respectively. Viability of testicular sperm cryopreserved by controlled-rate cryopreservation (45.9 ± 3.7) was significantly lower than non-frozen control (60.3 ± 0.9) and conventional freezing technique (55.0 ± 2.7). Gly (58.2 ± 2.6) and EG (53.3 ± 2.3) yielded a similar viability compared with non-frozen control (P > 0.05), whereas DMSO and PrOH demonstrated an inferior cryoprotectant for feline testicular sperm (% viability for DMSO and PrOH: 46.3 ± 3.3 and 44.3 ± 2.9, respectively). In both experiments, DNA integrity of frozen–thawed testicular sperm did not significantly differ from the control group. In conclusion, cat testicular tissue can be frozen as small pieces using both conventional technique and controlled rate freezing. However, freezing technique and type of cryoprotectant markedly affect the post-thawed quality of testicular sperm. Further study requires examination of the optimal cooling rate during cryopreservation and also the fertilizability of frozen–thawed testicular sperm.