2.1 Semen processing
The experiment has been performed as part of routine activities during the current semen production in the reproductive station and did not require the approval of the ethics committee.
These experiments were performed on the Breeding and Insemination Centre ‘MCB’ (Krasne, Poland). The study included 36 healthy Simmental bulls with an average age of 2 ± 0.5 years housed individually in pens. Two ejaculates were collected from each bull using an artificial vagina at 7 a.m. The semen was held in a water bath at 37°C, where the sperm concentration and initial percentage of motile spermatozoa were estimated. Sperm concentration was assessed using a digital photometer (Dr Lange, LP 300 SDM; Minitube, Tiefenbach b. Landshut, Germany) at 560 nm.
Semen samples were immediately after collection transferred into graduated test tubes, placed in a water bath at 37°C. The fresh undiluted semen was then evaluated microscopically (Nikon E 200, China) for mass motility. Subsequently, the semen was extended with animal protein–free commercial BIOXcell® extender (IMV Technologies, L’aigle, France) to a final concentration of 160x106 spermatozoa/mL, and rated in terms of motile sperm percentage, progressive motility, viability and abnormality of spermatozoa. Semen samples that showed more than 60% motility and 60% viability, were selected for the experiment. After a positive evaluation, semen samples were pooled to eliminate individual differences.
The fresh semen was then divided into five equal fractions. The first fraction was left for the control group (without Elamipretide), to the next were added in succession 0.1; 1; 5; and 10 μM of Elamipretide TFA (Trifluoroacetic) (MedChemExpress, USA). Semen was automatically packed (Bloc Machine FIN, IS 4, France) into polyvinyl chloride (PVC) straws (0.25 mL) (IMV, France) which were filled and equilibrated for 1.5 h at 4°C. After equilibration, the straws were frozen in liquid nitrogen vapour using a computer controlled automatic freezer from 4°C to -15°C at the rate of -3°C/min and from -15°C to -80°C at the rate of -10°C/min (IMV Technologies, France). After reaching -80°C, semen straws were plunged into liquid nitrogen and packaged in plastic goblets for 24 hours of storage in the liquid nitrogen container. After one day, the straws were thawed in a water bath at 38°C for 20 sec and then were examined.
2.2 Computerized assessment of sperm motility
Sperm motility was examined using a Sperm Class Analyzer (SCA, version 5.1, Microptic, Barcelona, Spain), a light microscope (Nikon Eclipse E200). Just prior to analysis, semen was diluted 1:10 in a warm (25°C) physiological solution (sodium chlorate 0.9%). Then, 2 lL of the prepared sample was placed in a Leja 4 analysis chamber (Leja Products B.V., Holland) of a thickness of 20.0 lm. The slide was placed on a stage warmer (38°C). The following motility parameters were included in this study: percentage of motile sperm, curvilinear velocity (VCL), straight-line velocity (VSL), path velocity (VAP), linearity (LIN) and amplitude of lateral head displacement (ALH). Minimum 500 cells were evaluated, and depending on sperm concentration, five analyses were performed per sample.
2.3 Viability
The double stain SYBR-14 with propidium iodide (L-7011 LIVE/DEAD Sperm Viability Kit; Invitrogen, Molecular Probes, Barcelona, Spain) using flow cytometer was applied (CytoFlex Beckman Coulter, B3-R1-V0, China). For this purpose, 50 mL of thawed semen was measured (37°C for 20 sec) and 940 ml NaCl (0.9%) and 5 mL SYBR14 were added. The whole was thoroughly mixed and then incubated (36°C for 10 min) without light access. Subsequently, 5 ml of PI was remixed and incubated 3min without light, followed by a test.
2.4 Biochemical assays
Semen samples were centrifuged at 750 rpm for 10 min to remove the supernatants, obtained cell pellets were washed three times with PBS and then incubated with 0.2% Triton X-100 on ice for 20 min. The supernatants were pipetted for subsequent biochemical analyses. Superoxide dismutase (SOD) and Catalase (CAT) activities were examined using the spectrophotometric method by commercially available enzyme activity test kits (Jiancheng Bioengineering Institute, China), Malondialdehyde (MDA) content was determined using the thiobarbituric acid (TBA) method by chemical reaction kits (Jiancheng Bioengineering Institute, China), which performed following manufacturer’s instructions as described previously [11].
2.5 Statistical analysis
Data are presented as mean ± standard error of the mean (SEM). Analysis of variance (ANOVA) was used to assess differences between concentrations of Elamipretide supplementation on all semen characteristics. When the F ratio was significant (p < 0.05), Duncan’s multiple range test was used to compare treatment means. The statistical analysis of the results was performed with Statistica 12.0 (StatSoft, Poland).