Effect of cyclodextrin-loaded cholesterol conjugates on plasma membrane viability of Piau swine breed frozen/thawed spermatozoa.

The objective of this study was to investigate the effect of cyclodextrin-loaded cholesterol conjugates addition to freezing extenders on plasma membrane viability of frozen-thawed spermatozoa of the Piau swine breed. Twenty semen samples were used from five males. The freezing extender was based on lactose-egg yolk extender, added to 2% glycerol, 3% dimethylacetamide. The addition of cyclodextrin-loaded cholesterol conjugates was performed after centrifugation, when semen was diluted with the cooling extender. Four groups were subjected to the following treatment: without addition (group 1); 1.5 mg of cyclodextrin-loaded cholesterol/120 × 10(6) sperm (group 2); 1.5 mg of cyclodextrin-loaded cholestanol/120 × 10(6) sperm (group 3); 1.5 mg of cyclodextrin-loaded desmosterol/120 × 10(6) sperm (group 4). To check post-thawing sperm quality sperm motility and sperm morphology evaluation were used. Additionally, to check sperm viability the hypoosmotic swelling test, supravital staining, and fluorescent assay were used. The mean values recorded for total sperm motility of semen immediately after thawing were 54.5 ± 5.8, 55.5 ± 5.3, 53.7 ± 6.7, and 52.5 ± 6.6% respectively for groups one to four, without difference between themselves (p > 0.05). Regarding fluorescent assay the results were 28.3 ± 13.2, 26.9 ± 12.2, 22.2 ± 11.4, and 32.0 ± 15.3% respectively for groups one to four, also without difference between groups (p > 0,05). Similarly, complementary tests for evaluating the integrity and functionality of the plasma membrane showed no difference between treatments (p > 0.05). In conclusion, use of cyclodextrin-loaded cholesterol conjugates added to the plasma membrane of sperm did not demonstrate any additive effect on increasing and/or maintaining sperm motility.

Effect of different cryo-protectants on the viability of frozen/thawed semen from boars of the Piau breed.

The objective of this study was to evaluate the effect of different cryo-protectants (glycerol, dimethylacetamide and dimethylformamide alone or combined and added to lactose-egg yolk extender) on the viability of frozen/thawed semen from the Piau breed as assessed by in vitro testing. Frozen semen samples (n=20) were used from five male swine. Five different freezing extenders, including 2% glycerol (Group 1 - G), 2% glycerol and 3% dimethylacetamide (Group 2 - GA), 2% glycerol and 3% dimethylformamide (Group 3 - GF), 5% dimethylacetamide (Group 4 - A) and 5% dimethylformamide (group 5 - F), were evaluated. To assess post-thawing sperm quality, sperm motility and morphology were evaluated. Sperm viability was determined using the hypoosmotic swelling test, supravital staining, and a fluorescent assay (carboxyfluorescein diacetate and propidium iodide). The mean total sperm motility of semen immediately after thawing was 46.2±1.3, 57.7±1.5, 53.2±2.1, 51.7±1.2, and 46.5±1.6% for groups 1-5, respectively. Groups 2 (GA) and 3 (GF) had greater motility values (P<0.05). Fluorescent assay values of 22.3±2.3%, 35.2±3.7%, 30.8±3.4%, 36.6±3.7%, and 26.5±3.8% were obtained for Groups 1-5, respectively, showing that Group 4 (A) sperm had greater viability than those from Group 1 (G), although there was no differences between the other treatments (P>0.05). The other complementary tests (hypoosmotic swelling test and supra-vital staining) demonstrated that sperm in Groups 2 (GA), 3 (GF) and 4 (A) had the greatest viability and there were no significant differences among these three groups (P>0.05). The most effective cryo-protectant combinations likely minimized and controlled the deleterious processes that occur in the sperm cell during freezing/thawing, thus improving post-thawing sperm viability. In conclusion, the combination of amides (3%) and glycerol (2%) or dimethylacetamide (5%) alone were more efficient at cryo-protection than glycerol alone for semen freezing in the Piau swine breed.