Preparation of 50 µM metformin solution
Metformin was procured as 500 g powder (Sigma Aldrich®, St Louis, USA) and the final concentration used was 50 µM. A stock solution of 1 M was prepared from which a second stock of 500 µM was further prepared. Final dilution was achieved by diluting 10 ml of the second stock with 90 ml of diluent (Tris egg yolk fructose) to make the final volume of 100 mL. All dilutions were made in normal saline as indicated by
(Bencharif et al., 2008). The final concentration of 50 µM was chosen based on the results of previous reports who claimed that 50 µM concentration of metformin exhibits activity without adverse effects on germ cells
(Bertoldo et al., 2016; Faure et al., 2018).
Semen collection and processing
The study involved ten male dogs of different breeds which were referred to the Small Animal Gynaecology Unit (SAC-OP-OG) of the Madras Veterinary College Teaching Hospital, Chennai, Tamil Nadu, India for evaluation of their semen during the period of June 2020 to December 2022. Semen ejaculates were collected by digital manipulation technique in absence of oestrus bitch. The collected semen samples were divided into 2 groups
viz. Group I, Group II with each group comprising of 10 samples. The first group was designated as control and was extended with TRIS-fructose egg yolk extender and the second group was supplemented with 50µM of metformin in TRIS-fructose egg yolk extender. The final concentration was 150 to 200 ×10
6 spermatozoa per mL of diluted semen. The dilution was done at 37°C and cooled down to proceed for equilibration. The diluted semen samples were cooled to 4°C in 1 hour. The equilibration was performed in cold handling cabinet at 5°C for 30 minutes. The equilibrated semen was filled in French mini straws (0.25 mL) and the laboratory end of straws were filled with polyvinyl alcohol (PVA) powder for sealing the sealed ends. The straws were then dipped into ice chilled water bath for 2 minutes for firm sealing. Post equilibration, the samples were checked for pre-freeze motility before proceeding for racking. After racking, the straws were placed horizontally at a height of 4 cm above the liquid nitrogen level in a styrofoam box and exposed to the vapours of liquid nitrogen for 10 minutes and the straws were plunged into liquid nitrogen at -196°C till further use.
Evaluation of semen
The different gross and microscopic examination of the semen is evaluated in the study. Grossly, volume, colour and consistency has been assessed while, the microscopic analysis included progressive motility, concentration, viability, acrosomal integrity and abnormalities as described by
(Patti et al., 2021).
Flow cytometric evaluation of sperm functional attributes
Sperm functional attributes were assessed flow cytometrically to understand the protective effect of metformin on the spermatozoa during cryopreservation. We assessed plasma membrane integrity, acrosomal status, sperm mitochondrial reactive oxygen species (ROS) and DNA integrity in prefreeze and post thaw semen of both control and treatment groups.
Preparation of the spermatozoa
The frozen semen straws were thawed at 37°C for 30 seconds. The samples were washed twice with Sp-TALP medium by centrifugation at 2000 rpm for 5 min. The sperm pellet was then resuspended in 200 µl of Sp-TALP medium. The sperm preparation procedure for all flow cytometric assays remains same unless and otherwise mentioned. Flow cytometric analysis was performed by using Beckman Coulter (MoFloTMXDP) fitted with a bevelled tip. A total of 10,000 spermatozoa events were assessed for each sample. After gating out to exclude debris, spermatozoa were categorized as applicable to the individual tests.
Plasma membrane integrity
Plasma membrane integrity of the spermatozoa was assessed using the combination of SYBR-14 (Invitrogen, Thermo Fisher scientific, USA) and Propidium iodide (Invitrogen, Thermo Fisher scientific, USA) dyes as described by
Nag et al., (2021). Stock solution of the SYBR14 (1mM) was diluted 50 times in dimethyl sulfoxide to prepare the working solution. For 2 million spermatozoa in 200 mL of sp TALP, 1.2 µL was added and incubated at 37°C for 10 min in the dark. This was followed by the addition of 2 µL of propidium iodide (2.4 mM) and further incubated for 2 min and analysed by flow cytometry. Excitation of the dyes were made using blue laser (488 nm). Fluorescence from SYBR14 was detected with a fluorescence channel (FL) 1 band-pass filter (525/40 nm) and PI fluorescence was measured using a FL3 band-pass filter (585/42 nm). The spermatozoa was classified as membrane intact and membrane compromised spermatozoa.
Acrosomal integrity
Acrosomal integrity of the spermatozoa was assessed by using the lectins
i.e. peanut agglutinin conjugated with fluorescein isothiocyanate (FITC) as described by
Nag et al., (2021) and
Kumaresan et al., (2020). After the sample preparation, 1 µL of FITC-PNA (1 mM) was added to the sample and incubated for 15 min at room temperature in the dark following which the exclusion
i.e. propidium iodide (2.4 mM) was added (2 µL) and further incubated for 2 min. Excitation of the dyes were made using blue laser (488 nm). Fluorescence from FITC was detected with a fluorescence channel (FL) 1 band-pass filter (525/40 nm) and PI fluorescence was measured using a FL3 band-pass filter (585/42 nm). The samples were analysed and classified as 2 populations
viz. acrosome intact and acrosome reacted populations.
Sperm chromatin structure assay
Sperm chromatin structure assay was performed as described by
Nag et al., (2021). The spermatozoa after the final wash with TALP, will be dissolved in TNE buffer (0.01 mol/L of Tris-HCl, 0.15 mol/L of NaCl and 1 mmol/L of EDTA, pH 7.4). Detergent solution (0.08 N HCl, 0.1% Triton X-100; pH 1.2) was added to the sperm suspension and incubated for 30 second following which 600 µL of Acridine Orange (Sigma-Aldrich, Germany) staining solution (200 mmol/L of Na
2HPO
4; 0.1 mol/L of citric acid buffer, pH 6.0; 1 mmol/L of EDTA; 150 mmol/L of NaCl; and 6 mg/mL of AO) was added and further incubated for 3 minutes. FL1 (green fluorescence) and FL3 (red fluorescence) were measured after excitation with a blue laser (488 nm).
Sperm mitochondrial ROS
Reactive oxygen species (ROS)/ oxidative stress (OS) in sperm was assessed by using CELLROX
TM Deep Red reagent fluorogenic probe (Invitrogen, Thermo Fisher scientific, USA). Briefly, 2.5 mM CELLROX
TM Deep Red reagent fluorescent probe was diluted in dimethyl sulphoxide for a final concentration of 1 mM (working solution) and stored at -20°C in dark. During use, this working solution was kept in the dark at 37°C. Two hundred microlitres of semen samples diluted in Sp TALP (25 X 10
6 sperms/ml) were added to 0.5 µl of CELLROX
TM and incubated for 30 min at 37°C. After incubation the solution was centrifuged for 5 min at 2000 rpm, the supernatant was removed and the pellet re-suspended in one ml of sheath fluid, filtered through a 40 µm pore size cell strainer to remove any large debris and the cells were analysed by flow cytometry to evaluate the oxidative stress. The percentage of ROS/OS was assessed using the density plot
(Rangasamy et al., 2021).
Statistical analysis
The differences in between the groups was done using independent student t test after arcsine transformation whereas the differences within the groups was done using paired t test after arcsine transformation and the results were back transformed. The significant difference was considered when P value was <0.05.
