The straws were plunged into liquid N2 for storage. After 1 month, samples were transported to the Integrated Center for Biotechnology (NIB/UECE – Fortaleza, CE, Brazil) for thawing and further analysis. The straws were removed from the liquid nitrogen and randomly thawed on a water bath at 37 °C/1 min 7 days after freezing. Finally, straws were removed, dried, the plug cut off and the contents pushed out into a glass vial that stood in a water bath at 37 °C. Semen samples (two straws per treatment) were immediately evaluated for sperm progressive motility,

morphology and membrane integrity. Thawed semen LDK378 cell line was also evaluated by CASA in accordance with previous recommendations. Briefly, a 10 μL aliquot of semen sample was placed on a pre-warmed Makler counting chamber (Sefi Medical Instruments Ltd., Haifa, Israel), allowed to settle for 1 min, maintained at 37 °C and examined in a phase-contrast microcopy system (Olympus BH-2, Tokyo, Japan), with stroboscopic illumination

coupled to a video camera adapted to the see more Sperm Class Analyzer (SCA version 3.2.0; Microptic S.L., Barcelona, Spain). The settings of the instrument were temperature, 37 °C; frame rate, 25 frames/s; minimum contrast, 75; straightness threshold, 80%; low velocity average pathway (VAP) cutoff, 10; and medium VAP cutoff, 45. Three nonconsecutive randomly selected microscopic fields were scanned. The parameters analyzed were number of counted 3-mercaptopyruvate sulfurtransferase cells, total motility (%), progressive motility (%), velocity average pathway (VAP; μm/s), velocity straight line (VSL; μm/s), curvilinear velocity (VCL; μm/s), amplitude of lateral head (ALH; μm), beat cross frequency (BCF; Hz), straightness (STR; %), and linearity (LIN; %) [12]. Twenty-one replicates were performed for each treatment. The results were expressed as mean ± SEM. Data were checked for normality by Shapiro–Wilk test, and for homoscedasticity by Levene’s test using the univariate procedure of the Statistical Analysis System (SAS 6.10,

SAS Institute Inc., Cary, NC, USA). Data were analyzed by General Liner Model (GLM). Comparisons among different cryoprotectants on seminal parameters were analyzed by Tukey test. To evaluate the individual effect of the animals and its interactions with cryoprotectants effect on studied variables, data were evaluated by Fisher’s PSLD test. For all statistical analysis, a significant difference of 5% was considered. Fresh goat semen was yellowish in color and milky in aspect. Total volume of ejaculates was 1.1 ± 0.1 mL, with a sperm concentration of 2.4 ± 0.2 × 109 spermatozoa/mL. Sperm progressive motility of fresh semen was 95.0 ± 2.0%, and mass activity was 3.9 ± 0.2. Percentage of sperm presenting intact membrane was 90.7 ± 3.5% and sperm with normal morphology was 76.1 ± 1.7%, being 33.0 ± 1.8% with functional membrane integrity. Total morphological defects were found in 23.9 ± 1.7%, being 0.6 ± 0.2% classified as primary and 23.