Optimization of a comprehensive metabolomic analysis system for characterizing metabolic alterations in the cauda epididymis of mature and juvenile mice

Abstract

The cauda epididymis protects and stores mature sperm in mammals. Recently, comprehensive transcriptomic and proteomic analyses have been conducted to understand its molecular functions; however, fundamental information on its metabolites has not been reported. In this study, we optimized a system for the comprehensive metabolic analysis of the cauda epididymis in mature and juvenile mice. This system identified 116 and 92 metabolites in mature and juvenile mice, respectively. Comparative analysis revealed that 44 and 13 metabolites were upregulated and downregulated, respectively, in the cauda epididymis of mature and juvenile mice. Based on the identified metabolites, 34 metabolic and unique pathways (mature: four pathways and juvenile: one pathway) were determined. In conclusion, the levels of certain metabolites in the cauda epididymis differed between mature and juvenile mice. These results contribute to understanding of the unique functions of the cauda epididymis based on dynamic changes in metabolites.

The epididymis is an important reproductive organ that determines sperm quality in developmental, healthy, and pathological states [[] and performs crucial sperm maturation, transportation, concentration, protection, and storage functions. The epididymis is anatomically divided into four regions: the initial segment, caput, corpus, and cauda. Sperm undergo maturation and acquire motility during transit from the initial segment to the cauda epididymis, where mature sperm are stored in a fertile quiescent state until ejaculation [3].

Recently, the cauda epididymis has been noted for its unique ability to maintain sperm fertility for long periods until ejaculation. Biochemical studies have revealed that the cauda epididymis lumen duct has a low sodium, hyperosmotic, and acidic microenvironment and a characteristic cell profile [4,5,6]. In addition, several omics analyses, including proteome [7, 8], transcriptome [9, 10], and metabolome [11] approaches, have been employed to investigate age-dependent or region-specific gene expression and protein and cellular profiles. These comprehensive studies are helpful for understanding the functions and diseases of the cauda epididymis and for developing effective treatments.

To date, comprehensive metabolomic investigations have focused solely on the lumen fluid in the cauda epididymis [11], leaving the metabolic features of the entire tissue largely uncharacterized. Therefore, elucidating functional alterations in the cauda epididymis requires metabolomic analysis of the entire tissue.

In this study, we optimized a protocol for metabolomic analysis of the cauda epididymis and obtained comprehensive metabolite data using gas chromatography–mass spectrometry (GC-MS/MS). To examine the efficacy of the metabolic analysis system of the cauda epididymis, we performed a comparative analysis using samples of different ages before (juvenile) and after (mature) sexual maturation. These metabolites were compared between juvenile (30–31 days old) and mature (12–14 weeks old) male mice. Metabolic pathways associated with differentially increased or decreased metabolites identified in the comparative analysis between juvenile and mature mice were also identified.

Materials and Methods

Animals

Mature (12–14 weeks old) and juvenile (30–31 days old) male C57BL/6J mice were purchased from CLEA (Tokyo, Japan). Samples of immature cauda epididymides, which did not contain sperm, were collected from juvenile male mice [12, 13]. Mature male mice (n = 5) were used to optimize the protocol for metabolomic analysis of the cauda epididymis. Cauda epididymides collected from mature (n = 5) and juvenile male (n = 5) mice were prepared in parallel. All animals were maintained under a 12-h/12-h dark/light cycle (lights-on: 0700–1900 h) at a constant temperature of 22°C ± 2°C with free access to food and water. All animals were maintained in healthy conditions and none required early euthanasia based on predefined humane endpoints. All animal experiments were approved by the Animal Care and Use Committee of the Kumamoto University (ID: A2023-077).

Preparation of the cauda epididymides

The sample preparation methods were modified from a previous liver metabolomics study [14] and optimized for the cauda epididymis. Cauda epididymis samples (mature group: n = 10; juvenile group: n = 5) were collected from the mice after euthanasia by cervical dislocation. A pair of cauda epididymides from each mouse was stored in a 1.5-ml centrifuge tube. Given the smaller size of the cauda epididymis, metabolic substances were extracted from the entire tissue without dissecting into smaller weights. For the data alignment, the area between each sample was adjusted based on the weight of the cauda epididymis. Samples were frozen and preserved in liquid nitrogen before preparation for metabolic analyses.

Extraction of metabolites from the cauda epididymides

A mixed extraction solvent (250 µl, water:methanol:chloroform ratio of 1:2.5:1) was added to each cryopreserved sample tube in liquid nitrogen. The samples were crushed with a bead crusher (30 sec, 3,200 rpm), then 750 µl of the mixed extraction solvent was added to the sample tubes, bringing the total volume to 1,000 µl. Afterward, the tubes were mixed using a vortex mixer and shaken in a heated shaker for 30 min (37°C, 1,200 rpm). After shaking, the mixtures were centrifuged (4°C, 16,000 g) for 3 min. To prevent peak saturation, the supernatant (100 µl) was collected in a new 1.5 ml centrifuge tube as the optimal concentration. Subsequently, 150 µl of the mixed extraction solvent and 200 µl of ultrapure water were added. This new mixture was centrifuged for 3 min (4°C, 16,000 g), then 250 µl of the supernatant was collected in a new centrifuge tube. Afterward, 10 µl of 2-isopropyl malicacid (0.05 mg/ml), which was not present in the sample, was added as an internal standard. The solution was then treated in a centrifugal evaporator for 25 min to vaporize the methanol. After treatment, the samples were placed in a freezer at −80°C for 15 min, then dried overnight using vacuum freeze-drying equipment. The following day, the dried samples were collected, then 80 µl of a 20 mg/ml methoxyamine– pyridine solution containing 20 mg methoxyamine hydrochloride dissolved in 1.0 ml of pyridine was added. The mixtures were dissolved completely in a sonicator, then shaken in a heated shaker for 90 min (30°C, 1,200 rpm). Afterward, 40 µl of N-methyl-N-trimethylsilyltrifluoroacetamide was added, then the mixture was shaken again in a heated shaker for 30 min (37°C, 1,200 rpm). After shaking, the samples were centrifuged for 3 min (4°C, 16,000 g). Lastly, 80 µl of the supernatant from each sample was added to different vials for measurement. The reagents used for the extraction procedures are listed in Supplementary Table 1.

GC-MS/MS analysis

Each 1-µl sample was injected into a GC-MS-TQ8050 NX (Shimadzu, Kyoto, Japan) in splitless mode using a nonpolar DB-5 capillary column (length: 30 m, ID: 0.25 mm, film thickness: 1.00 µm). The GC conditions were set to a constant flow rate of 1.10 ml/min with high-purity helium as the carrier gas, pressure of 83.7 kPa, total flow rate of 17.1 ml/min, linear velocity of 39.0 cm/sec, purge flow of 5.0 ml/min, and split ratio of 10.0. The temperature of the vaporization chamber was set at 280°C. The oven temperature was initially set at 100°C for 4 min, increased to 320°C for 67 min, and finally maintained at 320°C for 8 min. The MS conditions were set in the MRM (Multiple Reaction Monitoring) mode with an ion source temperature of 200°C, interface temperature of 280°C, and solvent elution time of 3.5 min. The scan mode was set from 45.00 m/z to 600.00 m/z. The detector voltage was set at 0.1 kV.

To test the sample detection accuracy, all samples were mixed with an internal standard, 2-isopropyl malicacid, and four sets of measurements were independently conducted. All detected metabolites were identified based on retention time, and target and modified ions. Data were processed using the Smart metabolites database ver.1 (Shimadzu). After acquiring the MS data, the mismeasured peak areas were manually corrected using GC-MS solution software v4.50 (Shimadzu), which was provided along with the GC-MS-TQ8050 NX (Shimadzu). The measurement data were exported to a CSV file that included a dataset containing sample information, peak areas, and retention times.

Data alignment

The MS data were sorted using Excel (Microsoft, Redmond, WA, USA). To calculate the peak area calibrated using an internal standard, all detected metabolites were divided by the peak area of the internal standard (2-isopropyl malicacid) in the same sample. To account for the GC-MS/MS instrumentation measurement error, metabolites with peaks identified in at least 50% of the samples were selected (5/10 and 3/5 samples from the mature and juvenile groups, respectively). In some cases, the same metabolite was detected as a different metabolite (Lysine-3TMS and Lysine-4TMS) due to differences in the number of conversions to the functional group (the trimethylsilyl [TMS] group) in the detected data. In such cases, a metabolite with a clearer waveform was selected for statistical analysis.

Metabolite identification and statistical analysis

Statistical analyses of the metabolites identified in the cauda epididymis of the mature and juvenile groups were performed using MetaboAnalyst 6.0 [15]. Principal component analysis (PCA) was used to identify global metabolic differences [16]. Metabolites were considered significantly different based on a P-value < 0.05 (Student’s t-test) and were categorized by fold change (FC) value. Partial least squares regression discriminant analysis (PLS-DA) was used to compare the two groups.

Metabolic pathway analysis

The metabolic pathways involving metabolites that were common or different between mature and juvenile mice were identified by analyzing the concentration table of the pathway analysis using MetaboAnalyst 6.0 [15]. The detection data were normalized by autoscaling to standardize the variables. Mus Musculus (KEGG) was selected as the pathway library [17].

Results

Identification of metabolites in the mouse cauda epididymis

The cauda epididymides of mature (n = 10) and juvenile (n = 5) male mice were subjected to metabolomic analyses. In the PCA score plot, different samples were scattered into two distinct regions (Fig. 1A), suggesting metabolic differences between the two groups in the cauda epididymis. A total of 116 and 92 metabolites were identified in the mature and juvenile groups, respectively (Fig. 1B). There were 34 and 10 metabolites unique to the mature and juvenile groups, respectively, whereas 82 metabolites were common to both groups (Supplementary Tables 2 and 3).

Fig. 1.

Metabolomic analysis of the cauda epididymis in mature and juvenile male mice. A) Principal component analysis of the normalized metabolomic analysis data. Each dot represents a cauda epididymide pair sample from mature (n = 10) or juvenile (n = 5) male mice. B) The number of identified metabolites in mature and juvenile mice is shown, represented by the red and blue circles, respectively. The overlapping section indicates the number of common metabolites between the groups.

Comparison of the abundant metabolites of cauda epididymides between the mature and juvenile groups

Differential metabolites between the mature and juvenile groups were compared using MetaboAnalyst 6.0 with Statistical Analysis (one-factor Student’s t-test). Forty-four metabolites (Table 1) were more abundant (P < 0.05, FC > 1.0) in the mature group (Fig. 2). Thirteen metabolites (Table 2) were more abundant (P < 0.05, FC < 1.0) in the juvenile group (Fig. 2).

Table 1. Abundant metabolites in the cauda epididymis of mature male mice.

No	Compound	FC (> 1.0)	P-value (< 0.05)	No	Compound	FC (> 1.0 )	P-value (< 0.05)
1	Urea	199490.00	0.0031	23	Margaric acid	14.48	0.0026
2	Sarcosine	76614.00	0.0004	24	Mannitol	11.39	0.0018
3	Palmitic acid	46682.00	0.0031	25	1,6-Anhydroglucose	9.57	0.0374
4	Glycerol	35898.00	0.0021	26	Hydroxylamine	8.13	0.0364
5	N-Acetylmannosamine	10433.00	0.0001	27	Glycerol 2-phosphate	6.32	0.0036
6	Galactose	7149.20	0.0000	28	Lactitol	5.91	0.0389
7	Succinic acid	2327.60	0.0023	29	2-Hydroxyglutaric acid	5.23	0.0003
8	3-Hydroxybutyric acid	2265.10	0.0000	30	Threonic acid	5.10	0.0044
9	3-Hydroxypropionic acid	1436.30	0.0000	31	Taurine	4.41	0.0009
10	Dimethylglycine	1307.50	0.0000	32	Phenylalanine	3.62	0.0201
11	Glutamic acid 5-methylester	507.78	0.0033	33	Orotic acid	3.43	0.0117
12	Adenine	431.00	0.0105	34	Niacinamide	3.41	0.0199
13	4-Hydroxyphenyllactic acid	406.33	0.0273	35	Ribose	3.41	0.0372
14	Ribonic acid	337.34	0.0001	36	Fructose 1-phosphate	3.31	0.0008
15	Glutamine	261.32	0.0179	37	Glucose 6-phosphate	2.98	0.0216
16	2-Hydroxyisovaleric acid	242.30	0.0010	38	3-Aminopropanoic acid	2.75	0.0015
17	Arabinose	190.22	0.0493	39	4-Aminobutyric acid	2.68	0.0212
18	Octadecanol	178.01	0.0148	40	Adenosine	2.53	0.0430
19	Pantothenic acid	159.39	0.0084	41	3-Hydroxyisovaleric acid	2.02	0.0100
20	Xylitol	37.49	0.0009	42	Putrescine	1.82	0.0308
21	Dihydroxyacetone phosphate	23.38	0.0041	43	2-Aminoethanol	1.67	0.0417
22	Allose	22.61	0.0044	44	Maltose	1.5891	0.0377

Metabolites that were more abundant in the cauda epididymis of mature male mice (n = 10). The metabolites are arranged by increasing fold-change (FC > 1.0) (P < 0.05, versus the juvenile group).

Fig. 2.

Volcano plot showing significant quantitative differences in metabolites between mature and juvenile male mice. The plots indicate the identified metabolites. The size of the circle indicates the p-value, with larger circles indicating lower P-values. The colors of the dots represent the FC values in the blue, grey, and red spectra. The metabolites that were more abundant in the mature and juvenile groups are represented in red and blue, respectively, whereas those common to both groups are represented in gray.

Table 2. Abundant metabolites in the cauda epididymis of juvenile male mice.

No	Compound	FC (< 1.0)	P-value (< 0.05)
1	Pyruvic acid	0.620	0.0414000
2	Fructose	0.539	0.0021000
3	Oleic acid	0.473	0.0366000
4	Sorbose	0.417	0.0129000
5	4-Hydroxyproline	0.391	0.0008000
6	Uridine	0.331	0.0010000
7	Xylulose	0.330	0.0193000
8	Mannose 6-phosphate	0.216	0.0001000
9	Azelaic acid	0.144	0.0000000
10	Cholesterol	0.025	0.0000000
11	Oxalic acid	0.012	0.0000000
12	3-Hydroxyisobutyric acid	0.0032862	0.0000000
13	Phosphoric acid	0.00000198	0.0000000

Metabolites that were more abundant in the cauda epididymis of juvenile male mice ([). The metabolites are arranged by decreasing fold-change (FC < 1.0) (P < 0.05 vs. the mature group).

Related metabolic pathways based on identified metabolites in the mature and juvenile groups

Metabolic pathways were identified by analyzing the concentration table of the MetaboAnalyst 6.0 Pathway analysis. The impact values of the metabolic pathways calculated from the topology analysis were used to evaluate the importance of the pathways regarding the differential metabolites between the two groups. A total of 34 metabolic pathways (impact > 0 and P < 0.05; Supplementary Table 4) were determined based on the total identified metabolites in mature and juvenile cauda epididymides (Fig. 3). From Supplementary Table 4, the six major metabolic pathways are listed in Table 3 (impact > 0.5 and P < 0.05).

Fig. 3.

Differential metabolic pathways of the cauda epididymides between mature and juvenile male mice. The plots indicate the metabolic pathways of the identified metabolites in the cauda epididymides of the mature and juvenile groups. The size of the circle indicates the impact score level of the pathway, with larger circles representing greater relative impact scores. The colors indicate the level of significance (white < yellow < orange < red). The red circles represent the lowest P-values. (1) Taurine and hypotaurine metabolism (2) Glycine, serine and threonine metabolism (3) Alanine, aspartate and glutamate metabolism (4) Starch and sucrose metabolism (5) Arginine and proline metabolism (6) Glyoxylate and dicarboxylate metabolism (7) Phenylalanine, tyrosine and tryptophan biosynthesis (8) Fructose and mannose metabolism (9) Galactose metabolism (10) Pentose and glucuronate interconversions (11) beta-Alanine metabolism (12) Glycerolipid metabolism (13) Phenylalanine metabolism (14) Arachidonic acid metabolism (15) Amino sugar and nucleotide sugar metabolism (16) Glycolysis / Gluconeogenesis (17) Arginine biosynthesis (18) Nicotinate and nicotinamide metabolism (19) Pyrimidine metabolism (20) Inositol phosphate metabolism (21) Glycerophospholipid metabolism (22) Purine metabolism (23) Primary bile acid biosynthesis (24) Pantothenic acid and CoA biosynthesis (25) Butanoate metabolism (26) Steroid biosynthesis (27) Valine, leucine and isoleucine degradation (28) Steroid hormone biosynthesis (29) Propanoate metabolism (30) Fatty acid degradation (31) Fatty acid elongation (32) Biosynthesis of unsaturated fatty acids (33) Nitrogen metabolism (34) Histidine metabolism.

Table 3. Major common pathways of identified metabolites in the mature and juvenile cauda epididymides.

Pathways	P-value	Impact	No of identified metabolites	Metabolites
Taurine and hypotaurine metabolism	0.008370	0.82857	3	Taurine Cysteine Hypotaurine
Glycine, serine and threonine metabolism	0.000027	0.69379	9	Sarcosine Glyoxylic acid Pyruvic acid N,N-Dimethylglycine Glycine Serine Threonine Glycerate Cysteine
Alanine, aspartate and glutamate metabolism	0.007326	0.67148	11	Aspartate Alanine Glutamate 4-Aminobutanoate Glutamine Citrate Fumarate Pyruvate N-Carbamoyl-L-aspartate Succinate 2-Oxoglutarate
Starch and sucrose metabolism	0.009869	0.64698	8	Fructose Sucrose Glucose 6-phosphate Glucose alpha,alpha-Trehalose Maltose Isomaltose Fructose 6-phosphate
Arginine and proline metabolism	0.001058	0.62790	9	Putrescine Glyoxylic acid Pyruvic acid 4-Aminobutanoate Arginine Hydroxyproline Ornithine Proline Spermidine
Glyoxylate and dicarboxylate metabolism	0.017718	0.53440	9	Glutamine Glyoxylic acid Pyruvic acid Citric acid Glycine Glyceric acid Isocitric acid Serine Malate

Metabolites identified in the pathways (P < 0.05, impact > 0.5).

Among the 34 pathways, the identified metabolites of the cauda epididymides in the mature (Supplementary Table 2) and juvenile (Supplementary Table 3) groups were identified in 29 pathways (Supplementary Table 5). Two unique pathways were observed in the juvenile group (steroid and steroid hormone biosynthesis), whereas four unique pathways were observed in the mature group (butanoate, histidine, nitrogen, and propanoate metabolism).

Discussion

This study revealed a comprehensive profile of the metabolites in the cauda epididymis of both mature and juvenile mice. In total, 116 and 92 metabolites were identified in the mature (12–14 weeks old) and juvenile (30–31 days old) groups, respectively. Differentially abundant metabolites were identified by comparing both groups. In addition, 34 metabolic pathways were identified by analyzing the identified metabolites and their levels.

Previous metabolomic analyses of the cauda epididymis have been limited to the lumen fluid [11]; therefore, we compared our results with those reported in that study to validate the accuracy of the identified metabolites and aid in their biological interpretation. A previous study identified 236 metabolites in the lumen duct fluid of the cauda epididymis of mature male mice (10 weeks old) [11], of which 31 were detected in the mature group in our study. Furthermore, 10 of these 31 metabolites were differentially abundant in the mature group compared to those in the juvenile group. Pathway analysis revealed that six of these 10 metabolites were present in the taurine, sarcosine, putrescine, urea, glutamine, and orotic acid pathways (Supplementary Table 5).

Taurine is a sulfur-containing β-amino acid that plays an important role in osmotic regulation and acts as an antioxidant [18]. In male reproduction, taurine exerts protective effects in both the physiological and pathological states [19]. Notably, a key enzyme of cysteine sulfinate decarboxylase related to the taurine biosynthesis pathway is expressed in the testis, epididymis, and vas deferens [20, 21]. In the epididymis, cysteine dioxygenase, which is involved in taurine production by synthesizing cysteine sulfinate from cysteine, also contributes to post-testicular maturation and osmoadaptation of sperm [22]. This study demonstrated that taurine is more abundant in the cauda epididymis of mature male mice than in juveniles. In addition, a previous study showed that taurine concentrations differed in certain parts of the epididymis, being greatest in the cauda, followed by the corpus, caput, and initial segment [23]. These age- and location-dependent changes in taurine concentrations can help elucidate the function and maturation process of the cauda epididymis.

Sarcosine is an endogenous amino acid that is generated from glycine or dimethyl glycine by glycine-N-methyltransferase (GNMT) or dimethylglycine dehydrogenase (DMGDH), respectively; it is metabolized to glycine by sarcosine dehydrogenase (SARDH) [[]. Sarcosine exists in the luminal fluid of the cauda epididymis [11]; however, its function in the epididymis has not been reported [26]. In a previous report, the sarcosine-regulated enzymes GNMT and SARDH were upregulated and downregulated, respectively, via androgen receptors in cancer cells [26]. In mice, androgen receptors in the epididymis maintain its structure and functions [27, 28]. Higher sarcosine levels were observed in the mature group than in the juvenile group. This suggests that androgens promote sarcosine production in the cauda epididymis during sexual maturation.

Putrescine is a major biogenic polyamine, along with spermidine and spermine, which regulate cell growth and proliferation in the reproductive organs [29]. Putrescine is produced from ornithine by the ornithine decarboxylase (ODC) enzyme [30]. ODC are expressed in the seminiferous epithelia of mice and rats [31]. Putrescine regulates stimulatory and inhibitory DNA synthesis during spermatogenesis [32]. Moreover, epididymal ODC activity is regulated in an androgen-dependent manner in rats [33]. Consistent with this, we noted an abundance of putrescine in the cauda epididymides of the mature group. Putrescine suppresses sperm motility and induces the acrosome reaction by inhibiting cAMP production in mouse sperm [34]. Thus, putrescine may play a role in maintaining the dormant state of sperm in the cauda epididymis.

Urea is an end product of protein metabolism in mammalian cells [35]. In rats, urea transporter 3 (UT3) is found in the Sertoli cells of seminiferous tubules, suggesting the involvement of urea in spermatogenesis [36]. Urea transporter B (UT-B) is also expressed in the Sertoli cells in rats [37, 38]. A previous study found that UT-B knockout mice have higher testicular urea concentrations than wild-type mice, demonstrating early maturation [39]. The abundance of urea in the cauda epididymides of the mature group in this study was likely derived from the lumen of the testes during spermatogenesis. Nevertheless, further studies are required to understand the physiological roles of urea in the cauda epididymis and epididymal sperm.

Glutamine is produced from glutamic acid through acid hydrolysis and from ammonia by glutamine synthetase, and is used for the de novo synthesis of pyrimidine metabolism [40]. In rats, high glutamine synthetase activity has been observed in the caput epididymis [41], and LC-MS analysis in mice has confirmed the presence of glutamine in the epididymal lumen fluids [42]. Orotic acid is produced from dihydroorotate by dihydroorotate dehydrogenase (DHODH) and is an essential intermediate in de novo pyrimidine synthesis [43]. DHODH has been previously detected in the mitochondria of sperm in bulls, boars, and humans. The functions of orotic acid and glutamine in spermatogenesis and sperm maturation have been discussed but have not been thoroughly examined. In this study, both compounds were identified as unique pathways in the mature group (Supplementary Table 5). Further studies are required to understand the functions of glutamine and orotic acid in the cauda epididymis.

During maturation of the cauda epididymis, epididymis gene expression profiles associated with maturation, protection, antiviral immunity, and inflammatory response regulation were identified in juvenile (5 weeks old) and mature (10 weeks old) C57BL/6J mice [5]. However, the relationship between changes in gene expression in epididymal epithelial cells and metabolism is unknown. Analysis of sexual maturation from a metabolic perspective may provide deeper insights into age-dependent epididymal maturation.

The metabolic analysis system for the cauda epididymis used in this study could facilitate the development of new assisted reproductive technologies. Previously, we reported that cold storage of cauda epididymides with dimethyl sulfoxide and quercetin maintains sperm fertility in mice and rats [44, 45]; however, the underlying molecular and pharmacological mechanisms are not fully understood. Therefore, comprehensive metabolic data from the cauda epididymis can be a powerful tool for understanding the molecular mechanisms of cold storage agents and potentially improve strategies for developing cold-storage technology for sperm.

A technical limitation of this study is that the detected compounds were primarily soluble organic acids owing to the GC-MS/MS analysis extraction procedures and settings. In the epididymis, the composition of phospholipids, fatty acids, and cholesterol changes during sperm maturation [1]. Androgen-dependent transcriptional changes occur when the epididymis matures [46]. Therefore, further studies that include multi-omics analyses using our data on metabolites, coupled with in vitro and in vivo assays, are necessary to fully understand the functions of the cauda epididymis.

In summary, we conducted metabolomic analysis of the entire cauda epididymis of mature and juvenile mice using GC-MS/MS. Differentially abundant metabolites and related metabolic pathways were identified in mature and juvenile mice. These comprehensive metabolite data reveal the function of the cauda epididymis and aid in the development of new assisted reproductive technologies.

Conflict of interests

The authors declare no conflicts of interest associated with this manuscript.

Availability of data and materials

The authors declare that the data supporting the findings of this study are available in this study and supplementary files.