66 EFFECT OF ADDITION OF CHOLESTEROL-LOADED CYCLODEXTRIN BEFORE FREEZING ON QUALITY AND FERTILITY OF STALLION FROZEN SEMEN

Abstract

The aim of the present study was to evaluate the effect of cholesterol-loaded cyclodextrin on the quality and fertility of stallion frozen semen. Three ejaculates from each of 4 stallions were used. The semen was diluted (1 : 1) with a skim milk-based extender (Botu-Semen^TM, Botupharma, Brazil). The samples were divided into 2 groups: control group (CG), composed of semen diluted only with extender, and treated group (TG), composed of semen diluted with extender plus 750 mg mL^–1 of cholesterol-loaded cyclodextrin. Both groups were incubated for 15 min at 20°C. The semen was frozen with Botu-Cryo^TM (Botupharma, Brazil) extender according to the manufacturer's protocol in 0.5-mL straws containing 100 × 10⁶ of total sperm. The sperm kinetic parameters were analysed by computer-assisted semen analysis, and plasma membrane integrity by flow cytometer (propidium iodide and fluorescein isothiocyanate -PSA) on post-thaw. The fertility trial was carried out inseminating 2 cycles of 20 mares (total of 40 cycles) immediately post-ovulation using 4 straws of CG or TG (400 × 10⁶ total sperm), one from each stallion in a randomised design. Comparison of sperm parameters was performed by t-test and fertility by Fisher's exact test. No difference (P > 0.05) was observed in total motility (%, CG = 57.9 ± 6.5 v. TG = 60.2 ± 6.7), progressive motility (%, CG = 26.9 ± 4.8 v. TG = 28.5 ± 4.8), percentage of rapid sperm (%, CG = 43.5 ± 8.8 v. TG = 45.7 ± 7.6), membrane integrity (%, CG = 20.1 ± 5.1 v. TG = 20.3 ± 6.3), and fertility (CG = 60% v. TG = 70%) between the groups. The results of this study showed that the use of cholesterol-loaded cyclodextrin did not affect sperm kinetic parameters and fertility in stallion with good quality in post-thaw semen. Further studies must be performed with stallions sensitive to freeze-thawing process.