92 EFFECT OF LOW-DENSITY LIPOPROTEIN ON THE QUALITY OF CRYOPRESERVED RAM SEMEN

Abstract

Egg yolk is the most widely used cryoprotectant in the composition of extenders for cryopreservation of mammalian spermatozoa; yet, efforts have been made to find ways to substitute it due to the possibility of transporting pathogenic microorganisms, the lack of standardization, and the presence of substances that inhibit metabolic exchanges or decrease the motility of sperm. The protective effect of egg yolk was attributed to the presence of low density lipoproteins (LDL) that prevent cholesterol efflux by increasing membrane stability and resistance to low temperatures. (Moussa et al. 2002 Theriogenology 57, 1695–1706) demonstrated that the purification of LDL is possible, thereby allowing its use as a replacement for integral egg yolk in extender. Then, this study aimed to evaluate the effect of substitution of egg yolk by different LDL concentrations during cryopreservation of ram semen. A total of 6 sheep, 3 ejaculates per animal, were collected. After collection, the ejaculates were evaluated and diluted in different extenders: E1) Tris, glucose, 15% egg yolk, and 5% glycerol; E2) Tris, glucose, 5% LDL (Moussa et al. 2002 Theriogenology 57, 1695–1706), and 5% glycerol; E3) Tris, glucose, 10% LDL, and 5% glycerol; and E4) Tris, glucose, 20% LDL, and 5% glycerol. The samples were adjusted to a final concentration of 600 × 10⁶ sptz mL^–1 and filled into 0.25-mL straws and frozen in a TK 4000^® machine. After thawing, sperm motility and spermatic vigor were evaluated, and the test of thermo-resistance (TTR) was conducted. Functional and structural integrity of the spermatic membrane were evaluated through the hypoosmotic test and the use of fluorescent dyes. The kinetic parameters of sperm were assessed by the computerized system (CASA). The statistical analysis was performed using the statistical program SAS (Statistical Analysis System), and the averages were compared using the Duncan multiple test. No difference (P > 0.05) was found between extenders for progressive motility after thawing. After 3 h of TTR, E1 showed higher values (P < 0.05) than E2, not differing from E4. The percentage of cells reactive to the hypoosmotic test was lower with the use of E2 (P < 0.05) than with other groups. Regarding the fluorescence technique, the average percentage of cells with intact membrane after thawing was higher in samples preserved in the extenders E1, E3, and E4 (P < 0.05) than in E2. Velocity average pathway (VAP), velocity straight line (VSL), and linearity of cryopreserved ram semen were (P < 0.05) significantly higher in E1 than in E2 and E3. The other kinetic parameters were similar in all groups tested. The results indicate that the extenders containing 10 and 20% of LDL are capable of protecting the spermatic cells during cryopreservation.