Sodium alginate and bovine serum albumin co-combined improved the cryopreservation quality of boar sperm through the PI3K-AKT pathway

Animals

Semen samples were collected from 18 healthy, sexually mature Yorkshire boars, aged 8–10 months, at a commercial breeding farm (Fujian Yongcheng Agriculture and Animal Husbandry Technology Co., China). Each set consisted of six samples, supported by a replicate of the experiment. Additionally, six samples were randomly mixed in pairs to form three sets of parallel experiments. All animal experiments were approved by the Animal Ethics Committee of Fujian Agriculture and Forestry University (PZCASFAFU22001). The reagents required for this experiment are listed in Supplementary Table S1.

Experimental design

Different concentrations of SA (0, 0.5, 1, 2, 4 mg/mL) (Shanghai Yuanye Bio-technology, S11053) were added to the filler to determine the optimal concentration of sodium alginate (SA) on the basis of the combined levels of sperm motility, plasma membrane integrity, acrosome integrity, mitochondrial membrane potential, and antioxidant capacity. Each treatment wass repeated three times. Additionally, the optimal concentration of BSA (0, 1, 3, 5, 7 mg/mL) (Shanghai Yuanye Bio-technology, S12012) was screened using the same procedure. Then, the optimal concentration of the combination was screened by the optimal concentrations of SA and BSA as the center points (Control, S1-B4, S1-B5, S1-B6, S2-B4, S2-B5, S2-B6, S3-B4, S3-B5, S3-B6). Here S1-B4 is defined as SA 1 mg/mL + BSA 4 mg/mL, and the remaining combined concentrations are calculated similarly. Finally, the mechanism of action of the filler was combined from the motility performance, antioxidant performance, protein molecular concentrations, and proteomic analysis of spermatozoa.

Semen collection

Boar semen was collected using the traditional hand-held method (Yánez-Ortiz et al. 2022). A thermostatic semen collection cup maintained at 37°C was utilized to obtain the sample. The sample was then mailed to the laboratory by mail at 17°C for subsequent sperm-quality testing. To eliminate gelatinous proteins, the semen sample was filtered through three layers of gauze. After quality control, the viability of the sperm sample exceeded 80%, and the concentration surpassed 2 × 10⁸ sperm/mL, ensuring adequate support for subsequent experiments.

Cryopreservation and thawing of semen

The Beltsville thawing solution (BTS) was prepared by dissolving 37.0 g of glucose, 1.25 g of EDTA, 6.0 g of sodium citrate, 0.65 g of potassium chloride, 1.25 g of sodium bicarbonate, 0.65 g of penicillin, and 0.6 g of streptomycin sulfate in 1 L of distilled water. Different concentrations of SA or/and BSA were added as experimental additives to the BTS buffer solution to create the final diluent. The final dilution was mixed with egg yolk in a ratio of 4:1 to produce a Freezer Solution I, which was temporarily stored at 17°C. This final dilution had an osmolality of 300–320 mOsm/kg and a pH of 7.2–7.4. To prepare Refrigerant Solution II for temporary storage at 4°C, 6% glycerol and 0.6% SDS were added to Solution I. Once the original semen was cooled to 17°C, the supernatant was removed by centrifugation at 800g for 10 min at 17°C. The semen was then diluted to 3 × 10⁸ sperm/mL by adding Solution I. Subsequently, it was slowly cooled to 4°C and an equal volume of Solution II was added to make its final density 1.5 × 10⁸. Semen is usually filled with a straw, then placed in −140°C for fumigation for 15 min, and then put in liquid nitrogen for long-term storage. To thaw sperm, you need to take the frozen sperm preservation tube in a 50°C water bath for 15 s, add BTS thawing solution at a ratio of 1:1 and incubate at 37°C for 15 min.

Sperm-motility parameters

Our study previously reported a method for measuring sperm-motility parameters by using a computer-assisted sperm analysis system (CASA) (Hamilton IVOS V3, USA) (Lan et al. 2021). The HT-CASA II system can determine the concentration and displacement of spermatozoa by utilizing sequential frames captured at a set acquisition frequency of 60 Hz. Additionally, the first 10 frames are collected to ascertain the motile sperm count. Briefly, 0.2 mL of a diluted semen sample (1 × 10⁶ sperm/mL) is pipetted at 37°C onto a pre-warmed disposable chamber slide (Leja Sperm Counting Slide, Netherlands). Four fields, containing a total of no fewer than 200 spermatozoa, are randomly selected for observation to increase the accuracy of the results. The parameters of CASA are set according to the manufacturer’s instructions, and the system automatically calculates sperm motility capacity and condition.

Plasma membrane integrity, acrosome integrity, and mitochondrial activity

The thawed semen was diluted to a concentration of 8 × 10⁷ cells/mL. The integrity of the sperm plasma membrane was assessed using the sperm tail hypotonic expansion test (HOST). The main step involved preparing a fructose–sodium citrate hypotonic solution, which was then mixed with semen and incubated in a water bath at 37°C for 1 h, followed by observation under light microscope. Second, the integrity of sperm acrosomes was detected by Coomassie Brilliant Blue staining. This involved fixing the samples with 4% formaldehyde, staining with Coomassie Brilliant Blue, and observing them under a ×1000 immersion-oil microscope after natural drying. To evaluate mitochondrial membrane potential, spermatozoa were treated with JC-1 staining solution and incubated at 37°C for 20 min. Following incubation, the supernatant was removed by centrifugation, and the samples were washed twice with 1 × JC-1 staining buffer. The spermatozoa were then re-suspended and measured using a microplate reader. Values were recorded four times with the microplate reader and then normalized.

Determination of redox state

The detection of ROS (reactive oxygen species) was to suspend the sperm in the DCFH-DA probe and incubating it at 37°C for 20 min. Subsequently, the sperm was washed three times with PBS, and the ROS content in the sperm was measured using a fluorescence microplate reader. To detect MDA (malondialdehyde), and MDA detection solution was added to the sperm samples. The mixture was thoroughly combined and heated at 100°C heat for 15 min, then allowed to cool to room temperature in a water bath. After centrifuging the supernatant, the absorbance was measured with a microplate reader at 532 nm to detect the change in MDA content. For the total detection of T-AOC (total antioxidant capacity) measurement, pre-cooled PBS was added to the sperm sample, breaking the sample to release antioxidants. The sample was then centrifuged at 4°C. The T-AOC Working Solution was added to the resulting supernatant liquid. The mixture was allowed to react at room temperature for 6 min before measuring the absorbance at 405 nm with a microplate reader, to determine the T-AOC content. SOD (superoxide dismutase) is detected by first centrifuging the sample to remove the supernatant, followed by ultrasonic disruption. The SOD working solution was then added, mixed, and incubated at 37°C for 20 min. Finally, the absorbance was measured at 450 nm using a microplate reader to detect SOD viability. The reagents required for the above tests are listed in Table S1.

Western blotting

To prepare sperm samples, break them using sonication, Next, lyse the samples on ice with RIPA and PSMF, and then centrifuge the samples at 4°C for 2 min to collect the supernatant. Protein quantification was performed using the BCA Protein Assay Kit. The protein samples were mixed with a protein loading buffer for denaturation at 100°C. The following proteins were separated using SDS-PAGE (20 μg/sample): CAT, SOD1, SOD2, Caspase-3, Caspase-9, BAX, Nrf2, Cytc, GSTA2, NME5, AKT, p-AKT, p-PI3K, PI3K, and GAPDH. The target proteins Were then transferred the target protein to a nitrocellulose (NC) membrane, which was sealed with a 5% skim milk buffer. The membrane was washed three times with Tris-buffered saline containing 1% Tween-20 (TBST). Then, the NC membrane was incubated with the primary antibody overnight at 4°C. After three additional washes, the membrane was incubated with the secondary antibody (Goat anti-Rabbit IgG Secondary Antibody) for 2 h at room temperature. Finally, the blot was then visualized using a LI-COR image analyzer. The antibodies used are listed in Table S2.

Proteome sequencing

Raw data were obtained using the TMT peptide labeling method with the Easy nLC 1200 chromatography system (Thermo Scientific) (Casey et al. 2017). We utilized the Maggi self-authoring platform for quality control of the machine’s raw reads. Differentially expressed proteins were identified on the basis of a false discovery rate (FDR) of less than 0.05 and a fold change of >2. Additionally, we conducted functional annotation and enrichment analyses for the differentially expressed genes in both the blank and treatment groups. The gene ontology (GO) enrichment analysis was conducted using the Goatools software (ver. 0.6.5; Shanghai Majorbio Bio-Pharm Technology Co., Ltd), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed using Python software. Finally, protein–protein interaction network analysis was performed by comparing the significantly different proteins with the STRING database (ver. 11.5). All of the above analyses were processed using the Majorbio cloud platform (www.majorbio.com).

Statistical analysis

SPSS 26.0 software (IBM, Chicago, IL, USA) was used for statistical analysis. Each measurement was performed at least three times, and the results were reported as the average of these replicates. The results are expressed as mean ± s.e. (s.e.m.). Utilizing a two-tailed Student’s t-test was employed to determine the significance of the comparisons between two groups. Subsequently, a one-way ANOVA was performed to assess total sperm motility, progressive motility, plasma membrane integrity, mitochondrial activity, acrosome integrity, antioxidant capacity, and marker protein expression across different experimental groups. P-values of <0.05 and <0.01 were considered to indicate significant and extremely significant differences respectively.

Ethical statement

All animal experiments were approved by the Animal Ethics Committee of Fujian Agriculture and Forestry University (PZCASFAFU23001).

Effects of SA supplements on sperm-quality parameters, mitochondrial membrane potential, and oxidative stress after cryogenic storage

In this study, alterations in SA concentration were found to lead to significant increases in various sperm parameters, including motility, vitality, curvilinear velocity (VCL), average path velocity (VAP), straight-line velocity (VSL), beat cross-frequency (BCF), average path distance (DAP), curve path distance (DCL), straight path distance (DSL), linearity (LIN), straightness (STR), and head swing proportions (WOB), when compared with the control group (P < 0.05 or P < 0.01). The highest values for motility and vitality were recorded in the group treated with 2 mg/mL of SA (Table 1). Furthermore, the supplementation of SA resulted in significantly higher index values for sperm plasma membrane integrity, mitochondrial motility, and acrosome integrity than in the control group, with the most notable improvements observed in the 2 mg/mL SA group (P < 0.05 or P < 0.01) (Table 1, Supplementary Fig. S1a). Evaluation of sperm oxidative stress indicated elevated concentrations of SOD and T-AOC in the SA supplementation group, in contrast to the control group, whereas the concentrations of ROS and MDA were lower in the SA supplementation group (P < 0.05 or P < 0.01). Notably, the group treated with 2 mg/mL of SA exhibited the most significant changes (Fig. S1a). In summary, the addition of 2 mg/mL of SA as a sole supplement to the filler was deemed the optimal concentration for providing comprehensive protection during frozen sperm storage.

Effects of BSA on sperm-quality parameters, mitochondrial membrane potential, and oxidative stress after cryostorage

This study showed a positive impact of BSA on pig sperm. It demonstrated that varying concentrations of BSA lead to increased motility, vitality, VAP, VSL, BCF, DAP, DSL, LIN, STR, and WOB parameters of sperm when compared with the control group (P < 0.05 or P < 0.01). The group treated with 5 mg/mL of BSA exhibited the highest values for motility, vitality, VSL, and BCF (Table 2). Additionally, the BSA supplementation group showed significantly higher values in sperm plasma membrane integrity, mitochondrial viability, and acrosome integrity than did the control group, with the 5 mg/mL BSA group displaying the most notable results (P < 0.05 or P < 0.01) (Table 2, Fig. S1b). The evaluation of sperm oxidative stress showed that the concentrations of SOD and T-AOC in the BSA supplementation group were significantly higher than those in the control group. Conversely, the concentrations of ROS and MDA were lower in the BSA supplementation group than in the control group (P < 0.05 or P < 0.01). Notably, the 5 mg/mL BSA group exhibited the most significant results (Fig. S1b). In conclusion, the addition of 5 mg/mL of BSA alone to the filler is recommended as the optimal concentration for achieving a comprehensive protective effect during the storage of frozen sperm.

The combined effect of SA and BSA on sperm-quality parameters, mitochondrial membrane potential, and oxidative stress after frozen storage in liquid media

The optimal concentrations of BSA and SA, when considered individually, cannot be directly extrapolated to determine the optimal concentration of their combination. Therefore, the combined concentration was screened in proximity to the identified optimal concentrations. On varying the concentrations of BSA and SA, we observed a significant increase in the sperm motility, vitality, and various parameters such as VCL, VAP, VSL, DAP, DCL, and DSL in the combined group compared with the control group (P < 0.05 or P < 0.01) (Table 3). The motility and vitality of the S2-B4 and S2-B5 treatment groups surpassed those of the other combination groups. Notably, the S2-B5 group demonstrated superior performance in VCL, VAP, VSL, and DCL indicators compared with the S2-B4 group (P < 0.05 or P < 0.01) (Table 3). Regarding sperm plasma membrane integrity and acrosome integrity, the index values for the S2-B4 and S2-B5 groups were significantly higher than those of the other groups. Furthermore, the acrosome integrity of the S2-B5 group exceeded that of the S2-B4 group (Table 3). On the basis of these outcomes, the control group, and SA, BSA, S2-B4, and S2-B5 groups were selected for the assessment of mitochondrial activity and oxidative stress. The findings showed that the S2-B5 treatment group was the most effective in mitigating the decline in membrane potential and oxidative stress, induced by frozen sperm preservation (P < 0.05 or P < 0.01) (Table 3, Fig. 1a–e).

Fig. 1.

The effect of the combined treatment with SA and BSA on sperm cryopreservation. (a) Thawed-sperm enzyme activity of MDA after the preservation of SA and BSA combined additives. (b) SOD enzyme activity. (c) T-AOC enzyme activity. (d) ROS activity. (e) Mitochondrial membrane potential. (f) Western blot analysis of oxidative stress-related protein markers. (g) Western blot analysis of apoptosis-related protein markers. (h) Western blot analysis of the PI3K-Akt signaling pathway. (i) The ratio of P-PI3K to total PI3K. (j) The ratio of P-AKT to total AKT. Data are expressed as the mean ± s.e. (s.e.m.). Significant (P < 0.05) differences between the groups are indicated by different lowercase letters, and extremely significant (P < 0.01) differences are indicated by uppercase letters. Control, control group; SA, 2 mg/mL SA; BSA, 5 mg/mL BSA; S2-B4, 2 mg/mL SA and 4 mg/mL BSA; S2-B5, 2 mg/mL SA and 5 mg/mL BSA.

We measured protein concentrations in the S2-B5 and control groups. Among these, the oxidative stress proteins SOD1, SOD2, and CAT demonstrated that the S2-B5 group significantly alleviated the down-regulation of oxidative stress caused by frozen sperm preservation. The Nrf2 protein, a central target in the Nrf2-Keap1 pathway, and its protein concentrations also indicated that the S2-B5 group has alleviated the up-regulation of the protein during frozen sperm preservation (Figs 1f, S1c). The protein concentration of Cytc also indicated that the S2-B5 group could significantly alleviate the up-regulation during frozen sperm preservation. The apoptotic proteins Casp3, Casp9, and BAX indicated that the S2-B5 group inhibited the up-regulation of apoptosis caused by frozen sperm preservation (Figs 1g, S1d). In summary, the combined effect of adding 2 mg/mL SA and 5 mg/mL BSA into the filler resulted in an excellent comprehensive protective effect at both the apparent and protein concentrations during storage. Finally, the activities of AKT, p-AKT, PI3K, and P-PI3K at the end of the preservation period were evaluated using Western blot analysis. The results showed that the combination of SA and BSA significantly alleviated the down-regulation of phosphorylation activity in the PI3K-AKT pathway (P < 0.05 or P < 0.01) (Fig. 1h–j).

Quality control and differential protein analysis in sperm proteomics

Check the identified peptide quality error rate to assess the accuracy of omics data. The distribution of mass error rates was close to zero, with most homologous peptides falling within 5 ppm (Fig. S2a), indicating that the mass accuracy of the data meets the standard requirements. Most peptides typically consist of 8–20 amino acid residues, which is characteristic of trypsin peptides. Therefore, the sample preparation meets the technical standards (Fig. S2b). In addition, the molecular weight of the proteins is mainly distributed between 1 and 150 kDa (Fig. S2c), and the protein coverage is comprehensive (Fig. S2d), meeting the criteria for further proteomics analysis.

The total number of proteins detected in this experiment was 966, including 947 ptoteins shared between the two groups, including three proteins specific to the control group, and 16 proteins specific to the S2-B5 group (Fig. 2a). The criteria for the differential screening analysis of significantly different proteins included a P-value of <0.05 and a fold change of >2. In total, 41 differential proteins were identified in comparison between the S2-B5 and control groups, with 31 proteins being up-regulated and 10 proteins down-regulated (Fig. 2b). In addition, the differential protein enrichment analysis also showed that the two experimental groups statistically categorized the 41 differential proteins into five detailed module branches (Fig. 2c).

Fig. 2.

Differential protein screening and protein cluster analysis. (a) Presentation of common and endemic proteins among different groups. (b) Differential protein expression between the sample groups. (c) Cluster analysis of protein expression patterns, based on differential protein concentrations, was visualized using heat maps.

Functional enrichment analysis and interaction network analysis of differential proteins

On the basis of the functional enrichment and protein–protein interaction analyses of GO and KEGG pathways, the characteristics of differentially identified proteins in the S2-B5 group were shown. Molecular function (MF) annotation showed that these proteins are primarily involved in cation binding. Cellular component (CC) annotations predominantly identified intracellular organelles and membrane-bound organelles. The main biological process (BP) annotations included the regulation of cellular processes, regulation of multicellular organismal processes, and negative regulation of biological processes (Fig. S3). The up- and down-regulated proteins were enriched separately. In the enrichment results of up-regulated proteins, the primary enrichment was related to lipid metabolism, whereas the KEGG pathway analysis showed significant enrichment in the AMP-activated protein kinase (AMPK) signaling pathway (Fig. 3a, b). The top five results of down-regulated protein enrichment were spermatid differentiation, sperm mitochondrion organization, positive regulation of cell division, and endoplasmic reticulum organization. The KEGG pathway was mainly enriched in cytochrome P450-substance metabolism (Fig. 3c, e). Finally, an interaction analysis of the differentially expressed proteins was preformed, showing a low degree of linkage among the 41 differential proteins, with NME5 and HSPA13 being identified as the core proteins (Fig. 3d).

Fig. 3.

Differential protein enrichment analysis, protein interaction, and verification between the S2-B5 group and the control group. (a) The top five GO terms enriched by 31 up-regulated proteins. (b) The 31 up-regulated proteins were enriched in the top five KEGG terms. (c) Five KEGG pathways were identified before enriching 10 down-regulated proteins. (d) Interactions between important proteins. (e) Ten down-regulated proteins were enriched in five KEGG pathways. (f) Validation of the core differential proteins NME5 and GSTA2. (g) Quantification of GSTA2 protein concentrations. (h) Quantification of NME5 protein concentrations. Data are expressed as the mean ± s.e. (s.e.m.). Differences between groups indicated by different lowercase letters are significant (P < 0.05); different uppercase letters indicate an extremely significant (P < 0.01) difference between the values.

Verification of differential core proteins involved in oxidative stress

This study primarily investigated the protective effect of the interaction between SA and BSA on frozen sperm, with a focus on oxidative stress. Down-regulated protein enrichment was selected for verification, specifically the cell-differentiated oxidative protein GSTA2 and the closely associated key protein NME5. The protein concentrations suggest that the expression of NME5 and GSTA2 in the S2-B5 treatment group is lower than that in the control group (Fig. 3f–h).