We collected semen at Xilingxue Agricultural Development Co., Ltd. (103°17′E, 30°35’N) in Chengdu, Sichuan Province, China, during the breeding season (August–November) in 2023. A total of 10 healthy goats of normal breeding age (2–4 years old) were selected, and each male goat was individually housed and managed. All male goats had free access to water and feed. Semen was collected from the male goats using an artificial vagina method (with water temperature controlled between 40 and 42°C). Collections were performed once a week for 12 weeks, with semen samples taken from five male goats each time. The semen was collected using disposable centrifuge tubes, and the volume was recorded each time. First, the color and viscosity of the semen were observed visually. Then, the collected semen was diluted with a base extender, wrapped in a cotton gauze, and transported in a foam box (18–20°C) to the laboratory within 1 h.

A 3 μL aliquot of diluted semen was transferred into a pre-heated (37°C) disposable sperm test plate. Computer-assisted semen analysis (CASA) (AndroVision®, Minitube, Germany) was used to select the goat species in the software, and the relevant parameters were analyzed and recorded at 200x magnification. For each sample, five fields were counted, and at least 2,500 sperm were analyzed. The CASA system parameters were set as described in published article (8), including curvilinear velocity (VCL; μm/s), velocity of straight line (VSL; μm/s), velocity of average path (VAP; μm/s), beat cross-frequency (BCF; HZ); radius: sperm path radioactivity coefficient. Briefly, sperm trajectory motion were continuously captured within 0.5 s using the following parameters, in situ motility (VSL < 24.0000, VCL < 48.0000), circular motion (radius > 9.00 and radius < 90.00 and rotation >0.70) and slow motion (VCL < 120.0000), respectively. The semen samples used in this study had to meet the following criteria: volume ranging from 0.7 mL to 2 mL, a minimum sperm density of 2.5 × 10⁹ sperm/mL, and total motility of at least 70%.

The semen from each goat after passing the quality test was equally divided into four groups: fresh and frozen groups treated with commercial diluent (CD) and fresh and frozen groups treated with homemade diluent. The CD for the goat was divided into liquid I and liquid II (Beijing Tianyuan Aorui Biotechnology Co., Ltd., China). The commercial fresh group was diluted with liquid I (1:19). When the CD is used as the cryopreservation diluent, 25% (v:v) fresh yolk solution should be added to both liquid I and liquid II before use. In brief, the homemade base extender composition included 19.98 mM Tris (T8060, Solarbio, China), 4.67 mM glucose, 7.18 mM citric acid (C8610, Solarbio, China), 5.84 mM sucrose, and 100,000 IU each of penicillin and streptomycin. This solution was used to dilute fresh sperm at a ratio of 1:19, with 100 mL of dilution solution. Liquid I was prepared by adding 20% egg yolk (v:v) to the homemade base extender. Liquid II was then prepared by adding 6% glycerol (v:v) and 0.6% Equex STM paste (v:v) to liquid I (13,560/0008, Minitube, Germany).

Semen freezing was performed using a two-step dilution method. The specific procedure was as follows: Cryopreservation dilution liquids I and II containing sperm were mixed homogeneously (1:9:10) at isothermal and equal volumes; then, the semen samples were packed in 0.25 mL plastic straws (1.25 × 10⁸ sperm/mL), which was sealed with polyvinyl alcohol powder, wrapped in 8 layers of cotton gauze, and placed in a refrigerator at 4°C for cooling and equilibration for 1.5 to 2 h. The plastic straws were placed 4 cm above liquid nitrogen for 10 min, and the temperature was kept at −130°C, then plunged into liquid nitrogen (− 196°C), and kept for 7 days. The straws were thawed in a water bath at 37°C for 30 s before use. The qualified semen, with sperm motility above 0.35, was first centrifuged at 4,000 rpm and 4°C for 5 min to remove the supernatant. Then, it was resuspended in phosphate buffer saline (PBS) without calcium and magnesium, washed three times, and diluted to a concentration of 1.25 × 10⁸ sperm/mL, The diluted semen was incubated at 37°C for 1 h, followed by centrifugation again (4,000 rpm, 4°C, 5 min) in preparation for the next step of metabolomics analysis. The flowchart of the experimental design is shown in Figure 1.

The Enhanced ATP Assay Kit (S0027, Beyotime, China) was used for the analysis. Before the analysis, the thawed semen was diluted to 1.6 × 10⁶ sperm/mL, washed three times with calcium and magnesium-free PBS, and centrifuged at 4000 rpm for 5 min at 4°C each time; after discarding the supernatant, the precipitate was left. After each group was added with 200 μL lysate, repeated blowing was performed to lyse the cells (placed on ice). After lysis, the cells were centrifuged at 4°C 12000 g, and the supernatant was taken and used for the determination of subsequent tests. The ATP standard solution was diluted to concentrations of 0.01, 0.03, 0.1, 0.3, 1, and 3 μM using ATP assay solution. The ATP assay reagent was then diluted with the ATP assay reagent diluent at a ratio of 1:4. Next, 100 μL of ATP assay working solution was added to the assay wells and left at room temperature for 3–5 min to allow for the consumption of any background ATP. The RLU values were determined using a luminometer (Varioskan LUX, Thermo, USA), and ATP content was calculated.

A volume of 900 μL of fresh semen was injected into a clean EP tube. A volume of 100 μL of Rosup reagent from the reactive oxygen detection kit (CA1410, Solarbio, China) was added to the test tube and then incubated for 20 min at 37°C in an incubator as a positive control sample to be tested. The volume of 2′,7’-Dichlorodihydrofluorescein diacetate (DCFH-DA) working solution (200 μL/tube) was calculated according to the number of samples to be tested, and the DCFH-DA probe was diluted with sterile PBS buffer at a ratio of 1:3000. After that, 200 μL of each semen to be tested was mixed with an equal volume of working solution and then incubated at 37°C for 20 min, during which the samples were inverted several times every 5 min to make full contact between the semen and the probe. Another sample without a probe was set as a blank control. After incubation, the samples were centrifuged (4,000 rpm, 37°C, 5 min), the supernatant was discarded and washed twice with PBS, and 500 μL of PBS was added to the precipitate to be detected by flow cytometry.

Next, 1 mg of JC-1 dry powder (J8030, Solarbio, China) was added to 1 mL of dimethyl sulfoxide (DMSO) and mixed well to form a storage solution. PBS was used for the dilution of JC-1 stock solution to create a final concentration of 250 μg/mL (working solution). An equal volume of semen and working solution was taken and mixed well by gently blowing followed by incubation at 37°C for 20 min and centrifugation (4,000 rpm, 4°C, 5 min). After that, the supernatant was discarded and precipitation was washed twice with PBS (4,000 rpm, 4°C, 5 min), and finally, 600 μL of PBS was added. For the blank control, a sample without JC-1 treatment was used. A Canto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) was used for the evaluation of samples by setting a total of 10,000 events per sample, and then, data were processed using FlowJo software (Becton Dickinson).

Fresh group sperm were centrifuged directly (1,000 rpm, 4°C, 5 min), and the supernatant was discarded. The spermatozoa of the frozen group were thawed and washed three times with calcium and magnesium-free PBS (1,000 rpm, 4°C, 5 min), and the supernatant was discarded after the final time to leave the precipitate. The cells were resuspended by slowly adding 1:5 (3% glutaraldehyde: 0.1 mol/L PBS buffer) diluted fixative along the wall of the tubes with a pipette and left to stand for 5 min at 4°C. High-speed centrifugation (12,000 rpm, 4°C, 10 min) was performed, the supernatant was discarded, and the precipitate was retained. Then, 3% glutaraldehyde fixative (3%, Sinopharm Chemical Reagent Co., Ltd., China) was again added along the wall of the tube (not to be blown out of the cells) and stored at 4°C. The collected samples were fixed, dehydrated, permeabilized, embedded, sectioned, and stained, and then, the mitochondrial ultrastructure of spermatozoa was observed using transmission electron microscopy (JEM-1400FLASH, JEOL, Japan).

Fluorescein isothiocyanate conjugated peanut agglutinin (FITC-PNA; Sigma, L7381) and propidium iodide (PI) were used to detect sperm acrosome integrity according to the manufacturer’s instruction. In brief, after washing three times with PBS, 980 μL of sperm suspension was stained with 20 μL of FITC-PNA stock solution (20 μg/mL) and 5 μL of propidium iodide (PI) stock solution (20 μg/mL) for 25 min in the dark. Aliquots of the stained suspensions were analyzed using the fluorescence microscope.

Membrane integrity was measured using the LIVE/DEAD™ Sperm Viability Kit (L-7011; Thermo Fisher Scientific) according to the manufacturer’s instructions. In brief, the goat sperm samples were stained with SYBR-14/PI. The staining was analyzed using fluorescence microscopy with excitation/emission = 485/517 nm for SYBR-14 fluorescence and excitation/emission = 586/617 nm for PI fluorescence. A total of 2,896 sperm were analyzed.

The sperm precipitation was followed by thawing at 4°C, and then 800 μL of cold methanol/acetonitrile (1:1) was used to remove proteins and extract metabolites. The mixture was collected into a new centrifuge tube and centrifuged at 14000 g for 20 min to collect the supernatant, and then the supernatant was dried in a vacuum. For liquid chromatography–tandem mass spectrometry (LC–MS) analysis, the samples were re-dissolved in 100 μL acetonitrile/water (1:1, v/v) solvent and centrifuged at 14000 g at 4°C for 15 min; then the supernatant was injected.

Analysis was performed using ultra-high-performance liquid chromatography (UHPLC) (1,290 Infinity LC, Agilent Technologies; Shanghai Applied Protein Technology Co., Ltd.; Shanghai, China). For hydrophilic interaction liquid chromatography (HILIC) separation, the samples were analyzed using a 2.1 mm × 100 mm ACQUIY UPLC BEH Amide 1.7 μm column (waters, Ireland). In both positive and negative modes of electrospray ionization (ESI), the mobile phase contained 25 mM ammonium acetate +25 mM ammonium hydroxide in water (labeled A) and acetonitrile (labeled B). The gradient was 95% B for 0.5 min, linearly reduced to 65% in 6.5 min, reduced to 40% in 1 min and kept for 1 min, and then increased to 95% in 0.1 min, with a 3-min re-equilibration period employed. Gradients were at a flow rate of 0.5 mL/min, and column temperatures were kept constant at 25°C. A 2 μL aliquot of each sample was injected.

The mass spectrometry analysis was performed using a quadrupole time-of-flight mass spectrometer (Q-TOF) (AB SCIEX TripleTOF 6,600; Shanghai Applied Protein Technology Co., Ltd.; Shanghai, China). The conditions for the ESI source were set as follows: Ion Source Gas 1 and 2 were both set at 60, the curtain gas was set to 30, the source temperature was set to 600°C, and the ion spray voltage floating (ISVF) was set to ±5,500 V. During MS-only acquisition, the instrument was programmed to acquire data over the m/z range of 60–1,000 Da, with an accumulation time of 0.20s/spectrum for TOF MS scans. For auto MS/MS acquisition, the instrument was set to acquire data over the m/z range of 25–1,000 Da, with an accumulation time of 0.05 s/spectrum for production scans. The production scan was obtained using information-dependent acquisition (IDA) with a high-sensitivity mode selected. The parameters for IDA were as follows: The collision energy (CE) was fixed at 35 V with a ± 15 eV range, the declustering potential (DP) was set to 60 V (+) and − 60 V (−), and isotopes within 4 Da were excluded. In addition, 10 candidate ions were monitored per cycle.

Raw MS data were converted to MzXML files using ProteoWizard MSConvert before free import was available in the XCMS software. For peak pickup, the following parameters were used: centWave m/z = 10 ppm; peak width = c (10, 60), and prefilter = c (10, 100). Peak grouping was performed with the following parameters: bw = 5, mzwid = 0.025, and minfrac = 0.5. The Collection of Algorithms of Metabolite Profile Annotation (CAMERA) was used for isotopes and adducts. Only variables with more than 50% non-zero measurements had at least one set of values that remained constant in the extracted ion features. Compound identification accuracy was assessed by comparing the m/z values (<10 ppm) of metabolites and MS/MS spectra with an in-house database (Shanghai Applied Protein Technology), which includes authentic standards for reliable identification.

Statistical analyses were performed, and data normality was assessed using SPSS (v.27.0, IBM, Chicago, IL, USA) with the Shapiro–Wilk test. After normalization, the processed data were analyzed using the R package (ropls), in which processed multivariate data analyses were carried out using overall sample principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) to differentiate the overall differences in metabolic profiles between the groups and to identify the metabolites that differed between the before and after semen freezing in CBG. To prevent model overfitting, we used a 7-fold cross-validation and response alignment test to assess the robustness of the model. Variable importance in the projection (VIP) obtained from the OPLS-DA model can be used to measure the strength and explanatory power of the influence of the expression pattern of each metabolite on the classification and discrimination of each group of samples and to mine biologically significant differential metabolites. Fold multiplicity of variation and VIP > 1, with p < 0.05, were used to screen for significantly varying metabolites. Student’s t-test was applied to determine the significance of differences between fresh and frozen groups. All metabolite identifications were performed using the in-house database (Shanghai Applied Protein Technology).

ACACA activity was measured according to the instructions of the assay kit (KTB1261, Abbkine, China). Briefly, fresh sperm and frozen–thawed goat sperm were washed with pre-cooled PBS. Briefly, frozen–thawed goat sperm were washed three times with pre-cooled PBS to remove extra homemade extender and then centrifuged to discard the supernatant (4,000 rpm, 4°C, 5 min). One milliliter of extraction buffer from the kit was added, and the sperm were broken by ultrasonic waves in an ice bath, with 30 cycles (200 W, ultrasonic waves for 3 s at 7 s intervals for a total time of 5 min). The mixture was then centrifuged (8,000 g, 4°C, 10 min), and the supernatant was collected and placed on ice to be measured. Then, the protein concentration of the samples was determined using a BCA protein assay kit (PC0020, Solarbio, China). Detection was carried out via a microplate reader with the wavelength parameter set to 660 nm. The ACACA activity was calculated from the sample protein concentration.

FASN activity was carried out according to the manufacturer’s instructions for the assay kit (BC0555, Solarbio, China). In short, 1 mL of the extract from the kit was added to the prepared sperm precipitate, broken by ultrasonic waves in an ice bath, with 30 cycles (power 300 W, ultrasonic waves for 3 s, intervals of 9 s, total time 5 min). The mixture was then centrifuged (12,000 g, 4°C, 20 min), and the protein concentration was detected after the supernatant was taken. Detection was performed via a microplate reader with the wavelength parameter set to 340 nm. The FASN activity was calculated according to the sample protein concentration.

CPT-1 activity was assayed according to the kit instructions (ml076617, Shanghai Enzyme-linked Biotechnology Co., Ltd., China). The wavelength setting parameter of the microplate reader was 412 nm. The CPT-1 activity was calculated by sample protein concentration.

Different concentrations of capric acid were added to homemade semen diluents I and II, respectively, and divided into the following groups: fresh group and capric acid frozen–thawed group treated with different concentrations (0 μM, 125 μM, 250 μM, 500 μM, and 1,000 μM groups), incubated together at room temperature for 1 h, and then frozen by liquid nitrogen fumigation. Fresh and frozen–thawed groups (0 μM) were used as control groups, after at least 2 weeks of preservation in liquid nitrogen. Then, the motility of thawed sperm was evaluated according to the steps described in Section 2.3 of the Materials and Methods section.

All experiments were repeated at least three times, all raw data were counted and organized using Excel 2019 (Microsoft, USA). Statistical analysis was performed using SPSS (v.27.0, IBM, Chicago, IL, USA). Student’s t-test was performed to compare the two groups. A one-way analysis of variance (ANOVA) followed by post-hoc Fisher’s least significant difference (LSD) test was used to compare more than two groups. All data were expressed as mean ± standard error (mean ± SEM). Graphs were made jointly using GraphPad Prism (v.5.0, GraphPad Software LLC, USA) and Adobe Illustrator 2020 (Adobe Inc., USA). A p-value of < 0.05 indicates a statistical difference.

After ultra-low temperature freezing, the total motility (72.21 ± 3.14% vs. 35.15 ± 5.05%, Figure 2A), progressive motility (63.65 ± 5.13% vs. 27.49 ± 4.31%, Figure 2B), VCL (97.16 ± 7.21 vs. 44.45 ± 4.77%, Figure 2C), VSL (43.36 ± 3.99 vs. 16.77 ± 2.30, Figure 2D), VAP (49.75 ± 3.73 vs. 20.57 ± 2.74, Figure 2E), and BCF (12.74 ± 2.86 vs. 6.85 ± 0.60, Figure 2F) of goat sperm were significantly reduced compared to the fresh group when using homemade dilutions (p < 0.05). Similarly, when a commercial diluent was used, the total motility (71.73 ± 3.59% vs. 43.91 ± 0.87%, Figure 2A), progressive motility (63.12 ± 4.48% vs. 35.30 ± 0.58%, Figure 2B), VCL (99.11 ± 9.73 vs. 48.13 ± 2.18, Figure 2C), VSL (42.44 ± 5.78 vs. 18.77 ± 0.76, Figure 2D), and VAP (49.14 ± 5.81 vs. 23.29 ± 0.91, Figure 2E) of goat sperm showed significant decrease after ultra-low temperature freezing compared to the fresh group (p < 0.05). However, there was no significant difference in BCF (11.97 ± 2.07 vs. 6.67 ± 0.35, Figure 2F) (p > 0.05). Furthermore, an analysis of the movement trajectories of fresh sperm diluted with homemade and commercial diluent showed no significant differences (Fresh vs. Fresh CD) (Figures 2G,H). Similarly, when sperm was diluted with homemade and commercial diluent before cryopreservation, there were also no significant differences in the movement trajectories for frozen-thawed sperm (Frozen-thawed vs. Frozen-thawed CD) (Figures 2I,J). These findings suggest both our homemade diluent and the commercial diluent can be used interchangeably for treating and freezing CBG semen without affecting sperm motility or other parameters in either the fresh or frozen–thawed groups (Figures 2A,B) (p > 0.05). Considering factors such as timeliness and cost, we chose to use our homemade diluent for subsequent experiments.

Since mitochondria are closely associated with sperm viability, we subsequently investigated the mitochondrial ultrastructure and function of sperm. Our findings revealed that frozen CBG sperm exhibited vacuolization in the mitochondrial vesicles, as well as a reduction in the density of the mitochondrial matrix (Figure 3A), indicating impaired mitochondrial ultrastructure. Following ultra-low temperature freezing, there was a significant decrease in ATP content (0.92 ± 0.11 vs. 0.40 ± 0.17, Figure 3B), high-MMP ratio (70.83 ± 5.97% vs. 36.97 ± 7.13%, Figure 3C), and ROS level (67.37 ± 1.22% vs. 80.57 ± 2.64%, Figure 3D) of sperm compared to the fresh group (p < 0.05).

To elucidate the effect of cryopreservation on the integrity of the acrosome and plasma membrane of goat sperm, we observed the ultrastructure of the sperm acrosome and plasma membrane using transmission electron microscopy, respectively. The results showed that sperm exhibited acrosomal detachment and plasma membrane damage after freezing (Figure 4A). In addition, we reassessed the sperm acrosome and plasma membrane integrity by fluorescence staining that showed a significant decrease in acrosomal integrity of sperm (40.27 ± 0.99% vs. 52.41 ± 1.11%, p < 0.01) (Figures 4B,D) and the integrity of sperm plasma membrane (50.62 ± 4.56% vs. 69.04 ± 2.71%, p < 0.05) (Figures 4C,E), respectively, in frozen–thawed group compared to the fresh group.

To further evaluate the impact of cryopreservation-induced mitochondrial damage on mitochondrial metabolism, we performed untargeted metabolomics analysis on pre- and post-freezing sperm samples. Our findings demonstrated a clear distinction in metabolite profiles between the fresh and frozen groups (Figures 5A–D), with validation provided by PLS-DA and OPLS-DA analysis models (Tables 1, 2). In total, we identified a total of 1,384 metabolites in goat sperm samples before and after freezing (772 in positive ion mode and 612 in negative ion mode). These metabolites were categorized based on their chemical composition into lipids and lipid-like molecules (30.708%), organic acids and derivatives (20.014%), undefined metabolites (12.283%), organoheterocyclic compounds (9.682%), and organic oxygen compounds (9.538%) (Figure 5E).

Based on the univariate analysis of the variance method, significant differences were observed in the metabolites of frozen and thawed goat sperm (FC > 1.5 or FC < 0.67, p < 0.05). The predominant differential metabolites were identified as lipids and lipid-like molecules (Figures 6A,B). Out of the 342 differential metabolites identified (OPLS-DA VIP > 1, p < 0.05), 246 were upregulated and 96 were downregulated (Figure 6C). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that these differential metabolites were mainly enriched in nine metabolic pathways: β-oxidation of very long-chain fatty acids, fatty acid biosynthesis, glutamate metabolism, steroidogenesis, arginine and proline metabolism, ketone body metabolism, spermidine and spermine biosynthesis, mitochondrial electron transport chain, and butyrate metabolism (Figure 6D). These nine KEGG metabolic pathways can be classified into two categories based on their relevance to freeze-thawed sperm quality (Figure 6E): energy-related metabolites such as capric acid (Figure 7A), glucosamine-6-phosphate (Figure 7B), malonyl-L-carnitine (Figure 7C), creatine (Figure 7J), creatinine (Figure 7K), and antioxidant-related metabolites such as cholesterol sulfate (Figure 6D), probucol (Figure 7E), and saikosaponin A (Figure 7F) showed a significant decrease, while succinate (Figure 7G), cholesterol (Figure 7H), and phytosphingosine (Figure 7I) exhibited a significant upregulation pattern within other categories. Furthermore, our literature review indicated that creatine is highly expressed in sperm cells, with its synthesis and degradation primarily occurring through spontaneous, non-enzymatic processes (29). Within the cell, ATP, adenosine diphosphate (ADP), creatine, and phosphocreatine must diffuse or be transported through the cell membrane for high-energy phosphate transfer between mitochondria and ATP utilization sites (30). Therefore, the downregulated expression of creatinine and creatine in frozen spermatozoa has negative effects on creatine metabolism (Figure 7L).

Among the nine metabolic pathways observed in metabolomic analysis, fatty acid biosynthesis and β-oxidation metabolism play crucial roles in sperm energy production (31, 32). Consequently, we conducted further analysis of the activities of the key rate-limiting enzymes ACACA, FASN, and CPT-1 in these metabolic pathways. The activities of ACACA (0.68 ± 0.07 vs. 0.41 ± 0.05, p < 0.01) (Figure 8A), FASN (20.57 ± 1.12 vs. 8.46 ± 0.66, p < 0.01) (Figure 8B), and CPT-1 (13.32 ± 2.77 vs. 0.81 ± 0.37, p < 0.05) (Figure 8C) were significantly lower compared to those of fresh controls.

Considering that the differentially expressed metabolite capric acid significantly decreased in frozen–thawed sperm and was enriched in the fatty acid synthesis metabolic pathway (Figure 7A), we investigated the effects of various concentrations of capric acid on the motility of frozen–thawed sperm. The results showed that compared to the fresh group, the total motility (85.80 ± 2.51 vs. 34.80 ± 2.29, p < 0.001) (Figure 8D) and progressive motility (76.23 ± 0.91 vs. 26.77 ± 3.12, p < 0.001) (Figure 8E) of sperm significantly decreased after freezing–thawing, while the addition of 500 μM capric acid significantly improved the total motility (63.40 ± 2.67 vs. 34.80 ± 2.29, p < 0.001) (Figure 8D) and progressive motility (52.47 ± 2.62 vs. 26.77 ± 3.12, p < 0.001) (Figure 8E) of goat sperm after cryopreservation.