Animals, Collection and Processing of Semen
Experiments were done with 5 Kivircik rams (2 to 4 years old) throughout the season of breeding. Animals were at a farm in Bursa (lat. 40°11’N, long. 29°04’E, altitude 155 m asl.), Türkiye with same sheltering conditions. Samples were obtained via electro-ejaculator (Minitüb GmbH, Germany) with at least three days apart (Toker and Alcay, 2022). After gathering, the samples were immersed into a warm water bath (32–34°C) within sterile tubes for evaluations. Upon collection, samples went through a series of quality control and were analysed for volume (measured were done with conical tubes), density, mass motion (3–5 on a 0–5 scale), sperm number by haemocytometer (at least 1.5 x 109 spermatozoa/mL) and motility (at least 75%) and pooled afterwards (Alcay et al., 2017).
Preparing Extenders And Semen Dilution
Freezing solutions were prepared freshly in the study day with the principal of two-step dilution method. Extender A consisted of 223.7 mmol/L Tris, 66.6 mmol/L citric acid, 55.5 mmol/L fructose, 4 g/L penicillin G, 3 g/L dihydrostreptomycin and 20% egg yolk. Extender B consisted with the same ingredients and also 100.4 mmol/L Trehalose, 4.03 mmol/L EDTA, and 6% of glycerol were added (Onder et al., 2022). Whole ingredients were mixed in distilled water after weighing and relevent concentrations of antioxidants were added to the groups. Experiment groups were arranged as; antioxidant free control, 2.5 and 5 mM of methionine, 1 and 2 mM of cysteine, 1 and 2mM of BHT. After pooling the samples, seven equal aliquots were created in every iteration and diluted with the appropriate extender for the final concentration of 150 x 106 spermatozoon/mL.
Semen Freezing And Evaluations
Semen samples that were diluted with extender A were gradually cooled in a water bath with ice cubes in an hour to 4°C. At that temperature, extender B was added by 5 equal aliquots in an hour of time. After that process, diluted sperm cells were held 2 hours at the same temperature for equilibration (Ustuner et al., 2018). At the end of time, semen samples were loaded into French straws (0.25 mL). Freezing procedure was conducted by Nicool Plus PC gamete freezing machine (+ 4°C to -8°C with cooling 3°C/min and − 8°C to -120°C with the rate of 15°C/min cooling by the aid of liquid nitrogen) (Air Liquide, Marne-la-Vallée Cedex 3, France). After cooling to -120°C temperature, straws plunged into − 196°C liquid nitrogen for long time storage (Alcay et al., 2017). At least ten straws for each experiment group were thawed (37°C, 30 sec) in a water bath. At the same time with the sampling procedure, thawed semen samples were placed in a chamber with 5% CO2 at 37°C for the examinations of incubation effects over semen characteristics (6 h) (Toker et al., 2016).
Laboratorial analyses were done by one person at both time points (0 and 6 h). Microscopic evaluations were conducted with a fluorescent attachment specified phase-contrast microscope (Olympus BX51-TF - Olympus Optical Co., Ltd., Japan).
Hypo-osmotic swelling test (HOST) was carried out to explore the integrity of sperm membrane function. Curled and swollen tails of sperm cells were examined after incubation of 10 µL of semen sample for 60 min at 37°C with 100 µL HOST solution (9 g fructose and 4.9 g sodium citrate per liter of distilled water). After incubation process, 20µL of mixture over a warm slide that was covered with a slip was examined. At least 200 cells were counted and evaluated for integrity under 1000x magnification and results were recorded as percentiles (Alcay et al., 2021).
FITC conjugated Pisum sativum agglutinin (PSA-FITC) test was conducted to evaluated the acrosomal integrity of sperm cells. The examination was conducted as the instructions of Toker et al. (2016). At least 200 cells were examined and the results were presented as percentiles.
In Situ Cell Death Detection Kit (Roche Diagnostics GmbH, Mannheim, Germany) used to apply the procedure of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method. DNA damage rates were obtained by using with fluorescein microscopy that Toker et al. (2022) indicated.
Statistical evaluations were analysed with the aid of SPSS. (SPSS 20.0 for Windows; SPSS, Chicago, IL, USA). Normality of the distribution was tested by using Shapiro Wilk. For parametric and homogenously distributed values One-Way ANOVA was used. Tukey was chosen as Post-Hoc test. Non parametrically distributed parameters were analysed by Kruskal Wallis test then Mann Whitney U test used to determine the significances between study groups. Statistical significance level (P) was chosen as 0.05.