Semen collection
Semen from sexually mature male giant pandas housed in Shaanxi Wild Animal Research Center (SWARC; Louguantai, Zhouzhi County, Xi’an city, 34°06′ N, 108°32′ E) was collected via electrostimulation during the breeding season. The giant pandas were anesthetized by injection with ketamine hydrochloride (8 mg/kg, IM) and halothane after 12 h of fasting. The pandas were then given 10–90 mA electroejaculation of semen collection for 5 s with an interval of 2 s. Fresh semen was collected into sterile cups after a 37 °C pre-warmed process and with a diluted 1:1 with a diluent (Irvine Scientific 90129). This work involved in giant pandas semen collection were in compliance with the Wildlife Protection Law of the Peoples Republic of China and Regulations of Shaanxi Province on the Protection of Wild Animals and Plants. Furthermore, all the protocols were approved by Comittee for the Ethics on Animal Care and Experiments in Northwest A&F University.
Experimental design
The semen was aliquoted into eight pre-heated tubes before freezing. The original cryoprotectant was added to one tube prior to the freezing process and was used as the control. Prior to cryopreservation, three tubes were diluted in a freezing medium with 50 μM, 100 μM, or 150 μM RSV. Two tubes received LBP (2 mg/mL and 4 mg/mL); LJP (1 mg/mL) was added to the freezing medium in one tube, and the remaining tube of semen received a combination of LJP (1 mg/mL), RSV (100 μM), and LBP (2 mg/mL).
Fresh semen samples were diluted with cryopreservation agents at a 1:1 ratio in a 15 mL tube placed in a beaker of water at 37°C. Subsequently, the tubes were incubated into 4 °C for 3 h. After that, glycerin was added to the cryopreservation semen at a final concentration of 400 × 106 spermatozoa/mL. Diluted semen was equilibrated at 4 °C for 1 h. Finally, diluted semen aliquots were packed into prelabeled 250 mL straws, frozen in liquid nitrogen vapor for 2 min, and stored in nitrogen for long-term preservation.
For thawing, frozen straws were removed from liquid nitrogen and immediately placed in a water bath at 37 °C. After shaking back and forth for 60 s, the thawed semen was transferred into a 2.5 mL tube for subsequent experiments.
Semen evaluation
Sperm viability and motility tests
Spermatozoa motility parameters of thawed semen were examined using an automatic analyzer (Naturegene Sperm Tracker).
Sperm plasma membrane integrity testing
As per previous evidence, the integrity of the sperm plasma membrane was assessed using HOST staining. Briefly, hypotonic solutions were mixed with the rapidly thawed giant panda semen, and the mixture was incubated at 37 °C for 30 min. A total of 200 spermatozoa were counted under an inverted microscope to identify the expansion percentage of sperm tails from a 10 μL mixture.
Acrosome integrity assessment
Acrosome integrity was assessed by fluorescein isothiocyanate-labeled peanut agglutinin (FITC-PNA) staining (Vector Laboratories, United States). Thawed semen with a final concentration of 105/mL was prepared for sperm smears and kept dry. The cells were then fixed with methanol for 10 min and incubated with 30 μL FITC-PNA at 37 °C for 30 min under an environment of light avoidance. Then, sperm smears was sealed with a colorless nail polish after washing with PBS in triplicate. Finally, more than 200 sperms were evaluated using fluorescence microscopy.
Mitochondrial activity analysis
Tubes containing 1 μL PI (0.02 mg/mL in PBS solution) (Solarbio, Beijing, China) and 1 μL Rh123 (0.2 mg/mL in DMSO) (Solarbio, Beijing, China).were protected against light at room temperature for 10 min and carefully added to the 50 μL suspension of sperm which was freeze-thawed for 30 min. After sperm smears were prepared with 10 μL of the incubated sample, estimation of sperm mitochondrial activity parameter was performed using an inverted microscope at 400× magnification. Nonviable in head sperm were identified as live sperm with high mitochondrial membrane potential (ΔΨ m). Therefore, the percentage of spermatozoa with intact mitochondria was also calculated.
Evaluation of DNA integrity
DNA integrity was assessed by acridine orange (AO) staining (Solarbio, Beijing, China). The sperm were washed three times with PBS and suspended in PBS at a concentration of 2 × 104/mL. The suspension of sperm was cased on clean glass slides and air-dried for the preparation of smears. The smears were then rinsed in a fixative solution (3:1, absolute ethanol: glacial acetic acid) for 5 min. After fixation, the slides were stained with AO solution for 10 min and thoroughly washed with distilled water. Finally, the air-dried smears were sealed with paraffin oil and analyzed by fluorescence microscopy. At least 300 sperm were counted per smear.
ELISA estimation of ROS, MDA, SOD and GSH-PX levels
The reagent kits were equilibrated at room temperature for 30 min. ROS, MDA), SOD, and GSH were determined via ELISA (Jining, Shanghai, China) in triplicate following the manufacturer’s instructions. The optical density of each sample was measured using a Spectra MR microplate reader (Dynex Technologies) at a wavelength of 450 nm. Subsequently, the levels of ROS, MDA, SOD, and GSH were calculated against each standard curve and expressed as pg concentration.
ELISA detection of HAase and ACE level
The levels of HAase and ACE in semen were assessed using ELISA kits (Jining, Shanghai, China). Each sample in triple well was performed at 450 nm according to the manufacturer’s manufacturer’s instructions. Finally, HAase and ACE contents were analyzed using each standard curve.
Statistical analysis
All experiments were performed in triplicates. Sperm quality metrics for antioxidant capacity, including CASA value, HOST, DNA integrity, acrosomal integrity, and ROS and MDA were analyzed using GraphPad Prism 7.00 software. Normality of the data was checked using the Kolmogorov–Smirnov normality test. All final data were shown as mean ± standard deviation. Statistical significance values was set at p ≤ 0.05.