22 EFFECT OF CHOLESTEROL ADDITION TO EQUINE SPERM MEMBRANE ON FERTILITY

Abstract

Artificial insemination with cooled-shipped semen has been widely used in horse breeding. However, some stallions, referred to as poor coolers, present abrupt fertility decrease when their semen is processed, cooled, and transported. Cholesterol incorporation into sperm membranes improves the quality of cryopreserved semen by increasing the sperm membrane stability and fluidity at low temperatures. Despite the beneficial effect of cholesterol addition on sperm quality, studies demonstrate that the presence of large amounts of cholesterol in the plasma membrane interferes with the physiological process of sperm capacitation and is detrimental to frozen equine sperm fertility. The aim of this study was to assess the fertility of cooled semen from good-cooler and poor-cooler stallions after adding cholesterol to sperm membranes. Two stallions were used and classified as good cooler (n = 1) and poor cooler (n = 1) based on the ability to maintain sperm progressive motility after 24 h of cooling at 5°C. For classification of the stallions, the fertility history was also taken into account through the results of pregnancy per cycle using inseminations with cooled semen (<50% for poor cooler and >70% for good cooler). Ejaculates of these stallions were subjected to 2 treatments: control (CON) and cholesterol (CLC). In the CON group, the semen was extended to 30 × 10⁶ sperms mL^–1 with skim milk-based extender (BotuSemen™). In the CLC group, the semen was extended as in the CON group plus 0.25 µL/1 × 10⁶ sperms of 6.1 mM cholesterol-loaded cyclodextrin was added. Afterwards, both treatments were cooled at 5°C for 24 h. To test the fertility of poor-cooler and good-cooler stallions, 2 cycles from 25 mares and 2 cycles from 10 mares were respectively used. For both stallions, randomly for each mare, the inseminations were performed by alternating both treatments. If the mare was first inseminated with the CLC treatment, in the next cycle the CON treatment was used and vice versa. After 24 h of ovulation induction, the inseminations were done in the uterine body with 1 × 10⁹ viable cells. Statistical analyses were performed using the Fisher exact test and significance was set at P < 0.05. For the poor cooler, the CON treatment presented 44% pregnancy/cycle compared to 76% for the CLC treatment (11/25^a v. 19/25^b). For the good cooler, both treatments presented 80% (8/10) pregnancy/cycle. The results suggest that the fertility capability of stallions is not affected by incorporation of cholesterol-loaded cyclodextrin to the sperm plasma membrane. Additionally, the utilization of cholesterol-loaded cyclodextrin may be an option to enable the utilization of cooled-shipped semen from poor cooler stallions for AI programs.