Between May and June 2018, 22 patients with asthenospermia, aged 22-45 (mean age 31.9±8.8) years and with a semen volume of 2.4±0.6 ml were selected from the reproductive center of Ningxia Medical University General Hospital. The selection criteria were in accordance with the WHO Human Semen Examination and Processing Laboratory Manual (Fifth Edition) (23) and were as follows: Semen volume, ≥1.5 ml; total sperm number, ≥39 (10⁶/ml); sperm forward progressive motility (PR) <32%; sperm [PR + non-progressive motility (NP)] <40%. Samples with a sperm concentration range of 6-7x10⁷/ml were used in further experiments. In order to eliminate dysliquefaction and severe oligospermia, peroxidase staining was used to eliminate leukocytospermia (peroxidase-positive leukocytes; <1.0x10⁶/ml). To perform peroxidase staining 125 mg benzidine was dissolved in 50 ml 95% methanol. A 150 mg quantity of rose red B was dissolved in 50 ml of distilled water and the two solutions were mixed. A 1 ml volume of this mixed solution had 2 drops of H₂O₂ added and the prepared solution formed the benzidine dye. A drop of semen was placed on a slide with a drop of benzidine dye solution and mixed well. The slide was covered, placed at 37˚C for 20 min and observed under a light microscope (x1,000). White blood cells were brown after staining. The number of leukocytes were observed in 200 sperm visual fields. The present study was approved by the ethics committee of the General Hospital of Ningxia Medical University (Yinchuan, China) and written informed consent was obtained from the patients.

All the subjects were abstinent for 3-5 days. The semen samples were collected by masturbation into aseptic seminal cups. Each semen sample was first placed in a 37˚C water bath for 30 min. After the sample was completely liquefied and evenly mixed, 1,500 µl of each semen sample was divided into three groups: Fresh group (unfrozen sperm), GEYC group (frozen sperm with standard protectant) and LBP group (frozen group with standard protectant and added LBP; 500 µl semen in each group). After liquefication, the semen was diluted with 500 µl Biggers, Whitten and Whittingham (BWW) sperm culture solution (Beijing Solarbio Science & Technology Co., Ltd.; cat. no. G2586), as recommended by the WHO guidelines (23), and centrifuged at 500 x g for 10 min at room temperature. The supernatant was then discarded, and 1 ml sperm culture solution was added (Fresh: 1 ml BWW; GEYC: 1 ml BWW, LBP: 1 ml BWW containing 1,000 µg/ml LBP), followed by incubation at 37˚C for 20 min.

The sperm was then frozen; the cryoprotectant base fluid was prepared according to the protocol for the GEYC protectant recommended by the WHO guidelines (23).

The manual two-step freezing method was employed (9). Semen and protectant were added to the cryopreservation tube at a ratio of 2:1 and the cryopreservation tube was placed at a height of ~5 cm from the liquid nitrogen surface for 10 min. Subsequently, it was lowered to 0.1 cm above the liquid level (no contact with the liquid level) for 5 min and subsequently immersed in liquid nitrogen for preservation.

The cryopreservation tube was taken out of the liquid nitrogen and placed in a 37^˚C water bath for rewarming for 3 min. The sample was then centrifuged 300 x g for 5 min at room temperature and placed into the BWW culture medium for 10 min at 37^˚C. Subsequently, the suspension was gently reversed 10 times and evenly mixed and then sperm was then subsequently tested for viability.

Computer-aided sperm analysis technology (CASA) was used to analyse the relevant parameters. Prior to the assessment of sperm viability, the counting plate [Sefi-Medical Instruments (9), Ltd.] was placed on the temperature control plate of the microscope in advance. This was a single chamber system. A total of six representative fields were examined under light microscopy (x200), at least 200 spermatozoa were assessed for each sample. The test was repeated twice. The color sperm quality detection system (WLJY-9DOO; version 2.0; Beijing Weili New Century Science & Tech. Deve. Co., Ltd.) was used to determine the sperm vitality [percentage of sperm forward movement (PR)] and other relevant parameters [curvilinear velocity (VCL), straight linear velocity (VSL), sperm average path velocity (VAP), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF)] sperm concentration was 2-50x10⁶. Fast forward motion with a speed ≥25 µm/sec was classified as class A; if the velocity of forward movement was <25 µm/sec, it was classified as class B (PR=A+B); if the velocity of non-forward movement or forward movement was ≤5 µm/S, it was classified as class C (NP=C); Total immobility was classified as level D (immobility=D). The spermatozoa were incubated 37^˚C for 10 min prior to measuring motility. If the sperm concentration was >50x10⁶, considering the high frequency of sperm collision producing errors, the semen was diluted with BWW to 2-50x10⁶ before assessment.

The survival rate was assessed using eosin aniline black (Beijing Solarbio Science & Technology, Co., Ltd.; cat. no. G2581), as recommended by the WHO guidelines (23). The sperm concentration was adjusted to 2-50x10⁶ using normal saline and 25 µl of semen and an equal volume of eosin aniline black suspension were added to a 1.5 ml centrifuge tube, mixed with micropipette at room temperature and left to stand for 30 sec. A sperm smear was made with each sperm suspension, dried and checked immediately after drying. Each glass slide was examined using a light-field microscope under an oil immersion objective (magnification, x1,000). Finally, with the help of a sperm counter (cat. no. XK06-6J; Jiangsu Xinkang Medical Devices, Co., Ltd.), the stained and non-stained sperm were counted. Each sample was evaluated twice and 200 sperm from each sample were evaluated to achieve an acceptably low sampling error. If the difference was too large, re-sampling and re-evaluation were performed. Fig. 1 presents the evaluation criteria used.

The sperm morphology was observed using a diff Quik rapid staining kit (cat. no. G1541; Beijing Solarbio Science & Technology Co., Ltd.) First, the sperm concentration was adjusted to (2-50x10⁶) using normal saline, and a sperm smear was then prepared and immersed in fast dye solution I at room temperature for 10 sec, followed by fast dye solution II at room temperature for 5 sec. The slide was then rinsed under running water to remove excess dye solution. A total of 200 sperms were evaluated after sealing under a light microscope (x1,000). The numbers of normal and abnormal spermatozoa were recorded, and the percentage was calculated. This staining method is recommended by the WHO laboratory manual for human semen examination and processing (Fifth Edition) (23).

The DFI of sperm was assessed using a sperm chromatin structure assay (SCSA) (24). A DFI cell flow cytometer test kit (cat. no. CP0101-10T; Cellpro; Zhejiang Xingbo Biological Technology Co., Ltd.) was selected for testing; this kit has been approved by the State Food and Drug Administration (SFDA) for clinical semen examination (approval no. 20160051). Solution A contained: 0.1 mol/l NaCl, 0.4 mmol/l KH₂PO₄, 2.7 mmol/l C₆H₆O₆, 4.7 mmol/l KCl, 0.2 mmol/l MgSO₄•7H₂O, 2 mmol/l CaCl₂•2H₂O, 0.02 mol/l HEPES-Na, 4 mmol/l NaHCO₃, 0.3 mmol/l C₃H₃O₃Na, 0.34% (v/v) C₃H₅O₃Na, 40.5% (v/v) Percol. Solution B contained 0.1 mol/l NaCl, 0.4 mmol/l KH₂PO₄, 2.7 mmol/l C₆H₆O₆, 4.7 mmol/l KCl, 0.2 mmol/l MgSO₄•7H₂O, 2 mmol/l CaCl₂•2H₂O, 0.02 mol/l HEPES-Na, 4 mmol/l NaHCO₃, 0.3 mmol/l C₃H₃O₃Na, 81% (v/v) Percol. 1% (v/v) Triton X-100 (Beijing Solarbio Science & Technology Co., Ltd.; T8200); Solution C contained 6 µg/ml acridine orange, 37 mmol/l citric acid, 126 mmol/l Na₂HPO₄, 1 mmol/l disodium EDTA, 0.15 mol/l NaCl. The specific operation was as follows: Sperm 1-2x10⁶/ml was diluted with solution A to a final volume of 100 µl. Subsequently, 200 µl of solution B was added, followed by incubation at room temperature for 30 sec (accurate timing was required). A total of 600 µl solution C was added and a BD accuri C6 (BD Biosciences) flow cytometer was used to select the cell groups to be studied on the working interface. The sperm flow rate was controlled at 100-300/sec and after two minutes of stable operation, at least 5,000 sperm were collected, and each sample was assessed at least twice. After data collection, the flow cytometry (FCS) files were copied into DFI-View software (DFIView; version 1.10; Cellpro; Zhejiang Xingbo Biological Technology Co., Ltd.) for analysis.

The sperm tail interruption staining kit (cat. no. CP01014-10T; Cellpro; Zhejiang Xingbo Biological Technology Co., Ltd.) was used for the detection of MMP. The kit has been approved by the SFDA for clinical semen examination (zyxb no. 20160052). The specific operation was as follows: The semen was diluted to 2x10⁶/ml and 0.5 ml JC-1 working solution was added, followed by incubation at 37˚C for 20 min and centrifugation at 4˚C and 300 x g for 5 min. The supernatant was removed, and the precipitated cells were washed twice with JC-1 buffer solution, and the cells were analyzed using a BD Accuri C6 (BD Biosciences) with an excitation wavelength of 488 nm and emission wavelength was 530 nm. The sperm flow rate was controlled at 100-300/sec, and at least 5,000 sperm were collected, and each sample was detected at least twice. Following data collection, the FCS files were copied into MMP-test software (MMP-Test; Version 1.0; Cellpro; Zhejiang Xingbo Biological Technology Co., Ltd.) for analysis. For sperm with a high MMP, JC-1 spontaneously forms JC-1 aggregates with intense red fluorescence, which is measured on the FL2 channel. For sperm with low MMP, JC-1 remains in the monomer form and exhibits green fluorescence, which is measured on the FL1 channel (25).

The Sperm Acrosome Staining kit (acrosome test; cat. no. CP01011-10T; Cellpro; Zhejiang Xingbo Biological Technology Co., Ltd.) was selected for detection. The kit has been approved by the SFDA for clinical semen examination (approval no. 20160054). The specific operation was as follows: The sperm concentration was adjusted to 4-5x10⁶ live sperm using solution A; two 1.5-ml centrifuge tubes were prepared and 10 µl solution B and 10 µl solution C was added. The sample was then mixed and incubated at 37˚C for 15 min, and 100 µl pre-cooled 70% ethanol was added into each tube, followed by incubation for 2 min at room temperature. The mixture was centrifuged at 500 x g for 5 min at room temperature, the supernatant was discarded and 500 µl staining working solution (mixture of solution D and solution E) was added, followed by shaking and mixing. Subsequently, the mixture was incubated for 15 min at 37˚C. On the BD Accuri C6 equipment the voltage of the FL1 channel was adjusted so that the major group was in the specified gates. The sperm flow rate was controlled at 100-300/sec, and at least 5,000 sperm were collected each sample was analyzed at least twice. (Solution A, NaCl, KH₂PO₄, C₆H₆O₆, KCl, NaHCO₃; Solution B, Ca²⁺ carrier A23187, DMSO, Solution C, DMSO; Solution D, NaCl, KH₂PO₄, KCl, NaH₂PO₄; Solution E, Isothiocyanate-coupled pea agglutinin).

MDA was detected using thiobarbituric acid (TBA) colorimetry. The MDA test kit of Nanjing Jiancheng Biological Preparation Co., Ltd. was selected for testing. The operation was performed according to the manufacturer's protocol and the absorbance value at 532 nm wavelength. Was read using a spectrophotometer (1410101 Thermo Scientific™ Multiskan™ FC; Thermo Fisher Scientific, Inc.).

The sperm cell oxidative stress activity superoxide anion flow cytometry (cat. no. GMS10096; GenMed, Ltd.) test kit was used for the detection of ROS, and the operation was strictly in accordance with the manufacturer's protocol. In this assay, 2',7'-dichlorofluorescein diacetate is a completely free dye that passes through the cell membrane. Once it is oxidized by molecules including hydrogen peroxide, peroxides and peroxynitrite anions, it produces fluorescence (26). Using this assay, the concentration of ROS in spermatozoa were measured. The specific steps were as follows: Fresh semen (1 ml) was added to a 1.5-ml centrifuge tube, followed by centrifugation at room temperature for 10 min at a speed of 300 x g. After discarding the supernatant, 500 ml of reagent C was added, followed by thorough mixing. Subsequently, 25 µl of dye A and 25 µl of dye B was added to the aforementioned sperm cells. Following thorough mixing, the cells were incubated at room temperature (25˚C) for 40 min under the exclusion of light. The cells were then centrifuged at room temperature for 5 min at a speed of 300 x g. The supernatant was carefully removed and 500 µl of storage solution (reagent C) was added. Following mixing, dual-channel analysis was immediately performed using the BD Accuri C6 (Beckton, Dickinson and Company) with an excitation wavelength at 488 nm. The voltage of the FL1 and FL2 channels was regulated to make the major group appear in the specified gate. After the experiment, the data was imported into FlowJo version 10 (FlowJo LLC) for analysis. The sperm flow rate was controlled at 100/sec, and at least 10,000 sperms were collected, and each sample was analyzed at least twice.

The sperm smears were fixed in 4% paraformaldehyde at room temperature for 20 min and then washed with PBS three times, for 5 min/wash. The slides were placed in a humidified chamber and 0.30% Triton-X 100 (Beijing Solarbio Science & Technology Co., Ltd.; T8200) was used to penetrate the membrane at room temperature for 10 min. PBS was used to wash the slides thrice, for 5 min/wash. Subsequently, two drops of 3% peroxidase inhibitor were added to each slide to block the endogenous peroxidase at room temperature. The slides were incubated at room temperature for 10 min and then washed by PBS three times, for 5 min/wash. To block endogenous biotin and avidin, normal goat serum (Beijing Solarbio Science & Technology Co., Ltd.; cat. no. SL038) was added to the sample, followed by incubation at room temperature for 10 min. The samples were then incubated with primary antibodies in blocking solution overnight at 4˚C. The primary antibodies used were as follows: Rabbit anti-human Bcl-2 (cat. no., 12789-1-AP; ProteinTech Group, Inc.), Bax (cat. no. 50599-2-Ig; ProteinTech Group, Inc.), CytC (cat. no. 12245-1-AP; ProteinTech Group, Inc.), and caspase-3 (cat. no. 19677-1-AP; ProteinTech Group, Inc.) antibody (1:200 dilution; 50 µl/tablet. BS was used instead of the primary antibody in the negative control group.

The slides were then incubated with secondary antibody (goat anti-rabbit IgG-H&L (cat. no. ab6721; 1:1,000; Abcam) for 1 h at room temperature and after washing with PBS, samples were mounted with DAPI (cat. no. ZLI-9557; OriGene Technologies, Inc.). Confocal scanning images were captured using a DM 2000 microscope (x1,000; Leica Microsystems) with a digital camera (cat. no. DFC450C; Leica Microsystems).

Protein was extracted with a whole protein extraction kit (cat. no. KGP2100; Nanjing KeyGen Biotech Co., Ltd.) and protein was then quantified using the BCA method (cat. no. E162-01; Nanjing KeyGen Biotech Co., Ltd.). In each lane, 18-20 µg protein lysate was separated by 10% SDS-PAGE and proteins were transferred to PVDF membranes (Merck KGaA) and the membranes were blocked in 5% non-fat milk (blocking solution) at room temperature for 1 h. The membrane was then incubated with primary antibodies overnight at 4˚C. The membranes were washed with Tris-buffered saline containing Tween-20 three times and then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG-H&L (cat. no. ab6721; 1:5,000; Abcam) secondary antibody at room temperature for 1 h. The PVDF membranes were then visualized using an enhanced chemiluminescence (ECL) detection system reagents and images were captured with the ChemiDocXRS (Bio-Rad Laboratories, Inc.). After the experiment, the images were assessed using ImageJ version 1.8.0 (National Institutes of Health). The primary antibodies were as follows: Caspase-3 (1:10,000; cat. no. 19677-1-AP; ProteinTech Group, Inc.), Bax (1:5,000; cat. no. 50599-2-Ig; ProteinTech Group, Inc.), Bcl-2 (1:10,000; cat. no., 12789-1-AP; ProteinTech Group, Inc.), CytC (1:1,000; cat. no. 12245-1-AP; ProteinTech Group, Inc.), GAPDH (1:10,000; cat. no. 10494-1-AP; ProteinTech Group, Inc.).

Total RNA from each group was extracted with a total RNA preparation kit (cat. no. AP-MN-MS-RNA-520G; Corning, Inc.) and reverse transcribed (42˚C for 15 min, then 85˚C for 5 sec) into complementary (c)DNA using transcript all in one first-strand cDNA synthesis super mix (cat. no. AT341-02; TransGen Biotech Co., Ltd.). The SYBR premix ex taq II dye fluorescence quantitative kit (cat. no. RR820A; Takara Biotechnology Co., Ltd.) was used for PCR amplification in an ABI 7500 fast quantitative PCR instrument (Thermo Fisher Scientific, Inc.). After 30 sec of denaturation at 95˚C, 40 cycles of amplification were performed: 5 sec of denaturation at 95˚C, annealing/elongation at 60˚C for 30 sec. The primer sequences were as follows: Caspase-3 forward, 5'-TGGAATGTCAGCTCGCAATG-3' and reverse, 5'-CAGGTCCGTTCGTTCCAAAA-3'; Bax forward, 5'-AGATCATGAAGACAGGGGCC-3' and reverse, 5'-ATCCTCTGCAGCTCCATGTT-3'; Bcl-2 forward, 5'-AACTCTTCAGGGATGGGGTG-3' and reverse, 5'-GCTGGGGCCATATAGTTCCA-3'; CytC forward, 5'GCCTCAATGTCCCTGCTTCT-3' and reverse, 5'-TCGTTTCCAGCATGAGTGCT-3'; GAPDH forward, 5'-AGTCTACTGGCGTCTTCACC-3' and reverse, 5'-CCACGATGCCAAAGTTGTCA3'.

After liquefaction, the semen samples were washed twice with 0.85% NaCl and centrifuged at room temperature 1,000 x g for 10 min. The supernatant was then discarded and then 2.5% glutaraldehyde was added slowly, and samples were fixed in a 4˚C refrigerator for 2-4 h. Subsequently, the samples were fixed with osmium acid (1%), washed with distilled water, embedded for 2 h at room temperature in Epon812 epoxy resin, cut into semi thin section (1 nm), stained at room temperature for 1 h by toluidine blue and positioned under a light microscope. The sperm cell area was subjected to semi thin section (1 nm) and a JEM1200-EX TEM (JEOL, Ltd.) was used to observe the changes of mitochondria in the sperm tail. An experienced electron microscopist blinded to the origin of the samples tested and recorded the information. The specific evaluation methods were as follows: Under an electron microscope, at least 100 sperm from each group of samples was evaluated, focusing on evaluation of the microstructure of mitochondria in the tail of the sperm, observing whether the arrangement of mitochondria was orderly, whether there was oedema and the severity of oedema in mitochondria and whether there was damage to the structure of the mitochondrial membrane.

Values are expressed as the mean ± standard error of the mean unless otherwise noted in the figure legends. Statistical analysis was performed using a SPSS 24.0 statistical package (IBM Corp.), differences among multiple groups were analyzed by Welch's one-way ANOVA, following by Tukey's HSD test. P<0.05 was considered to indicate a statistically significant difference.

To assess the DFI, a minimum of 5,000 cells from each sample were acquired and analyzed using fluorescence-assisted cell sorting interfaced with a data analysis software. Sperm DFI was assessed using a SCSA. The green fluorescence was that of acridine orange dye binding to double-stranded DNA, while the red fluorescence was that of acridine orange dye binding to single-stranded DNA. As presented in Fig. 2A, following sperm cryopreservation, the DFI rose and the DFI of the LBP group (27.3±5.1%) was lower compared with the GEYC group (38.2±5.6%) but significantly higher compared with the Fresh group (17.5±3.1%).

JC-1 dye molecules accumulate in the mitochondrial matrix and produce red fluorescence; however, if the MMP is low, the JC-1 dye is not able to accumulate in the mitochondrial matrix and green fluorescence is then produced. As presented in Fig. 2B, the MMP of cryopreserved spermatozoa was increased. The MMP of the LBP group (39.2±6.1%) was higher compared with the GEYC group (28.8±7.0%) but significantly lower compared with the Fresh group (49.8±9.5%).

After the acrosome reaction was induced, the relative fluorescence intensity was weaker compared with the control, indicating that the fluorescence peak shifted to the left. The AR of cryopreserved sperm was reduced and there was no significant difference between the LBP group (17.7±3.3%) and the GEYC group (14.7±2.5%); The AR of the GEYC group was lower than that of the Fresh group (24.6±4.3%; Fig. 3A and B).

The results of the immunofluorescence analysis indicated that the expression of Bcl-2, Bax, CytC and caspase-3 was concentrated in the head and middle part of the sperms (Fig. 4A). Western blot analysis suggested that the LBP group had a higher content of Bcl-2 protein compared with the GEYC group, while the expression levels of Bax, CytC and caspase-3 protein were lower compared with the GEYC group. Bcl-2, CytC, Caspase-3 in the LBP group and Fresh group differed significantly, while the differences in Bax were not significant, as presented in Fig. 4C. The results of the reverse transcription PCR analysis indicated that, compared with the GEYC group, Bax, CytC and caspase-3 were significantly decreased in the LBP group. Bcl-2 in the LBP group was higher compared with the GEYC group, Bcl-2, Bax, CytC, Caspase-3 in the LBP group and Fresh group differed significantly, as presented in Fig. 4D.

TEM was used to detect the microstructure of sperm. It was indicated that the mitochondrial structure in the middle part of the sperm tail was severely damaged after freezing, the mitochondrial cristae structure was disorganized and normal mitochondrial cristae disappeared. By contrast, there was a significant improvement in the LBP group. Specifically, the mitochondria at the sperm neck were orderly arranged and abnormal mitochondrial structures were significantly less frequent, as presented in Fig. 5A-B.

The TBA colorimetric method was used for MDA detection. The results suggested that the LBP group (18.0±3.6 nmol/ml) had a significantly lower sperm MDA content than the GEYC group (23.9±4.1 nmol/ml), which was higher than the fresh group (16.3±3.4 nmol/ml), but there was no significant difference between the LBP group and the Fresh group. ROS detection based on flow cytometry indicated that the rate of ROS-positive cells in the LBP group (38.1±6.7%) was significantly lower compared with the GEYC group (58.3±10.8%), and this rate in the LBP group increased as compared with the Fresh group (17.4±5.2%; P<0.05; Table II; Fig. 5C).