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Three-month-old sheep were housed in controlled environmental conditions and divided into two groups. One group of ten sheep were fed control feed (1050 kcal); the other group were fed high-concentrate feed (2010 kcal). The mouse was kept in controlled environmental conditions that were free from specific pathogens and divided into two categories of dietary conditions. One group were fed a standard control diet (4.37 kcal); The other group were fed a high-concentrate diet (16.45 kcal). The animals were euthanized by CO₂ inhalation. During the procedure, CO₂ gas was introduced into the euthanasia chamber at a rate of 10% of the chamber volume per minute (corresponding to a flow rate of 5.8 L/min). The gas flow rate was precisely controlled by adjusting the flow control valve to minimize stress in animals prior to loss of consciousness. CO₂ gas was continuously supplied until the animals completely stopped moving, ceased spontaneous breathing, and exhibited dilated pupils. Once these signs were confirmed, the CO₂ gas supply was turned off, and the animals were observed for an additional 2–3 min to ensure death. All animal experiments were conducted in accordance with the ethical policies and procedures approved by the Inner Mongolia University Animal Care and Use Committee (IMU-MOUSE-2023-013, IACUC Issue No. IMU-SHEEP-2021-011), Inner Mongolia University, China.

Single-cell library and sequencing

Testicular tissues were dissected into small fragments and digested with collagenase IV (1 mg/ml, 37 °C, 15 min). After filtration through a 40 μm cell strainer (BD Falcon, USA, 352340), the resulting single-cell suspension was assessed for viability (>90%) and concentration (>1000 cells/μl). Single-cell libraries were prepared using the Chromium Single Cell 3’ Kit v3 (10 × Genomics, USA) and sequenced (PE150) on an Illumina NovaSeq 6000 platform following the manufacturer’s protocol. The sheep reference genome was processed using cellranger mkgtf and cellranger mkref, and raw data were aligned using cellranger count. Subsequent analysis was performed in Seurat, including data normalization, integration, and differential gene expression analysis (FindAllMarkers, thresholds: |log2FC| ≥ 0.25, P < 0.05). Functional enrichment was conducted using g: Profiler based on Gene Ontology (GO) terms.

Metabolome detection and analysis

Metabolomic profiling was performed on sterilely collected small intestinal contents using a Waters Acquity I-Class PLUS UPLC system coupled with a Xevo G2-XS QTof high-resolution mass spectrometer (Waters). Separation was achieved on an Acquity UPLC HSS T3 column (1.8 μm, 2.1 × 100 mm). Raw data were normalized prior to statistical analysis. Differential metabolites were identified based on significance thresholds of P < 0.05 (Student’s *t*-test), |log₂FC| ≥ 1, and VIP (Variable Importance in Projection) > 1 (derived from multivariate cross-validation). Metabolite enrichment analysis was conducted using MetaboAnalyst 5.0, and visualization was performed in R Studio (v4.2).

16S rDNA amplicon sequencing and analysis

After collecting small intestinal contents under sterile conditions, genomic DNA was extracted and purified using the QIAGEN kit. Subsequently, the samples were sequenced using the Illumina platform.

Subsequent data analysis was conducted utilizing R Studio (v4.2) in conjunction with other relevant software tools. Initially, the data underwent alpha diversity and beta diversity analysis using QIIME2. Subsequently, Metastats (v2020.6) was employed to conduct a t-test on the species abundance data between the groups. Differential species were identified using a stringent dual-filter criterion: unadjusted P value < 0.05 and Q value < 0.05. Species with a p-value less than 0.05 were deemed significantly different. To assess the correlation between genera exhibiting distinct abundances, Spearman correlation analysis was performed, and correlation values with a significance level of P value < 0.05 were retained. Following this, the resulting correlation network was visualized using Cytoscape (v3.9.0). The functional prediction of differential genera is carried out using the Linear discriminant analysis Effect Size (LEfSe) method and the PICRUSt2 tool.

Hematoxylin–eosin (H&E) staining

The testis was placed in Bouin’s solution (Sigma, HT10132, Germany) at 4 °C overnight after the dehydration and embedding of tissue blocks. For this study, serial sections with a thickness of 5 μm were utilized. Following drying at 37 °C, the samples were stained using Lillie-Mayer hematoxylin (Solarbio, G1080, China) and eosin (Zsbio, ZLI-9613, China). The tissue sections were subsequently dehydrated with alcohol and sealed with Neutral Balsam Mounting Medium (ZSGB-BIO, 96949-21-2, China). Finally, the samples were left to dry naturally, facilitating easy observation and statistical analysis.

Immunohistochemistry

The testis was placed in Bouin’s solution (Sigma, HT10132, Germany) at 4 °C overnight after the dehydration and embedding of tissue blocks. The tissue sections were dewaxed and incubated in a blocking solution (3% BSA, 10% normal donkey serum in TBST) for 30 min. The primary antibody (Table S1) was incubated overnight at 4 °C. Subsequently, the corresponding secondary antibody was applied, followed by color development using DAB (ZSGB-BIO, ZLI-9013, China). Finally, counterstaining was performed using Lillie-Mayer hematoxylin (Solarbio, G1080, China), and the samples were dehydrated and sealed for further analysis.

Immunofluorescence

The testis was placed in 4% paraformaldehyde solution at 4 °C overnight after the dehydration and embedding of tissue blocks. The experimental procedures for dewaxing, blocking, and the application of primary and secondary antibodies followed the standard protocols for immunohistochemistry (Immunohistochemistry). Following the completion of the secondary antibody incubation, the nuclei were stained with DAPI. Finally, the samples were sealed using Fluoromount™ Aqueous (Sigma, F4680, Germany) Mounting Medium for further analysis.

RNA extraction and RT-qPCR

Total RNA from testicular tissue and mouse TM4 Sertoli cell line was extracted using the EasyPure RNA kit (TRANS, ER101-01, China). Subsequently, cDNA was synthesized through reverse transcription using an RNA reverse transcription kit. RT-qPCR was performed on a Roche Light Cycler 480II System (Roche, Germany) using TB Green Premix Ex TaqII (Takara, RR820A, China). The primer sequences for this study were provided in Table S2.

Fecal microbiota transplantation

Use 3-week-old male ICR mice as recipients for fecal microbiota transplantation. Prepare a solution by mixing 1 g of sheep small intestinal contents with 1 ml of sterile glycerol (20%) and diluting it with normal saline to a concentration of 0.05 g/ml. Mice are treated with a combination of antibiotics, including vancomycin (0.5 g/L), neomycin sulfate (1 g/L), metronidazole (1 g/L), and ampicillin (1 g/L), for two consecutive weeks prior to microbiota transplantation. After the antibiotic treatment, fecal transplantation experiments were conducted daily for a duration of eight weeks.

Cells culture and treatment

Mice TM4 Sertoli cell line were cultured in DMEM medium containing 10% fetal bovine serum (Gibco, 10099-141, Australia) and 1% penicillin-streptomycin (HyClone, SV30010, China). The cells were cultured in a cell incubator at 37 °C and 5% CO₂, and the medium was changed every 24 h. Cells were passaged when the cell density reached 80–90%, and cells were treated 24 h later (5 mM L-Citrulline (MCE, HY-N0391, China); 35 μM Sucrose; 35 μM Sucrose + 5 mM L-Citrulline).

Cell proliferation assay

Cell proliferation was detected using MTT Detection kit (Beyotime, C0009, Nantong, China). After 48 h of treatment, discard the medium, add 10 μl/well of MTT working solution into a 96-well plate, and incubate at 37 °C for 4 h in the dark. Next, add 100 μl per well of the Formazan solution from the MTT kit and incubate for 1 h in the dark. Finally, the absorbance of the 96-well plates at 570 nm was measured using a Microplate Reader (Bio-Rad, iMark™, USA).

ROS evaluation

For TM4 cell line, ROS staining reagent (Beyotime, S0033S, China) was added to a 96-well plate. Then, the plate was incubated at 37 °C for 30 min in a dark environment. Following this, the cells were washed twice with PBS and the fluorescence intensity of each group was immediately observed using a Nikon fluorescence microscope. For tissue ROS assessment, tissue homogenates were prepared in liquid nitrogen and centrifuged to obtain the supernatant. ROS levels were measured using a mouse ROS ELISA kit (JILID, J25190, China). Following the addition of enzyme conjugate reagent, the plates were incubated at 37 °C for 60 min. After washing with the provided buffer, the wells were blotted dry and developed with substrate at 37 °C for 15 min, protected from light. Absorbance was measured at 450 nm.

Sertoli cell isolation and culture

Testes from 7-day-old mice were decapsulated and digested sequentially. The first digestion used 0.1% collagenase IV at 37 °C with shaking (85 rpm) for 20 min. After sedimentation, the supernatant was discarded, and the tissue was washed twice with PBS. The second digestion was with 0.1% collagenase IV and 0.1% hyaluronidase at 37 °C for 10 min, followed by centrifugation (500 rpm, 1 min) and PBS washing (x3). The third digestion involved 0.1% collagenase IV, 0.1% hyaluronidase, and 0.25% trypsin at 37 °C for 20 min. After terminating the digestion, the solution was centrifuged (1000 rpm, 3 min), and the pellet was resuspended in culture medium. The single-cell suspension was seeded onto Matrigel-coated dishes and cultured at 34 °C with 5% CO₂ for 48 h. Cells were treated with 20 mM Tris for 2.5 min, washed with DMEM/F12 (x3), and culture was resumed. Drug treatments followed the same protocol as for TM4 cells.

Western blot

The TM4 Sertoli cells were lysed using RIPA buffer (Beyotime, P00113B, China). Proteins were separated by SDS-PAGE and transferred onto PVDF membranes (Millipore, ISEQ00010, Germany). After blocking with 5% BSA for 1 h, the membranes were incubated overnight at 4 °C with primary antibodies (Table S1), followed by a 2 h incubation with HRP-conjugated secondary antibodies at room temperature. Protein bands were visualized and quantified using ImageJ software.

Enzyme-linked immunosorbent assay (ELISA)

The concentrations of L-citrulline in the small intestinal contents and testis were quantified using enzyme-linked immunosorbent assay (ELISA) kits (J43384 and J25562, Giled Biotechnology, Wuhan, China), following the manufacturer’s protocols.

Statistical analysis

In this study, GraphPad Prism (v 8.0) software was used for statistical analysis. Bioassay data were shown as mean ± SEM. Data analysis was performed using two-tailed Student’s t test or one-way analysis of variance (ANOVA). Significant difference was *P < 0.05, **P < 0.01, ***P < 0.0001. Two-tailed Student’s t-test was used to compare the mean differences between two independent samples. One-way ANOVA was used to compare the mean differences among four independent samples, with post-hoc analysis performed using the Tukey’s test.

Tokyo, October 30, 2025 – Mitsubishi Heavy Industries, Ltd. (MHI) and NIPPON SHOKUBAI CO., LTD. (Nippon Shokubai) have received approval for their jointly submitted proposal to Japan’s New Energy and Industrial Technology Development Organization (NEDO) for its “Development of Technologies for Building a Competitive Hydrogen Supply Chain” project.

The selected project aims to develop technology for the construction of a hydrogen supply chain using ammonia as a hydrogen carrier (hydrogen storage and transport medium), promoting development of medium-scale, decentralized ammonia cracking technology near hydrogen demand sites. The project advances ammonia cracking technology using steam and exhaust gases, employing an independently developed, low-temperature, highly active and highly durable ammonia cracking catalyst without the use of noble metals typically used in conventional low-temperature active catalysts. This innovative technology will be used to verify challenges toward practical application.

Following the project selection, the two companies will conduct the following activities during the project period through fiscal 2027, with long-term testing in mind, using a commercial-scale demonstration plant. MHI will leverage its extensive experience in ammonia plant construction and its knowledge of ammonia handling to carry out the basic design (Front End Engineering Design: FEED) of the demonstration plant. MHI will finalize the demonstration plant specifications aiming to resolve technical challenges necessary for commercialization, with support from Hokkaido Electric Power Co., Inc. (HEPCO). Nippon Shokubai will promote the development of elemental technologies focused on verifying the durability of ammonia cracking catalysts, utilizing its abundant experience and expertise in catalyst development and practical application, including process catalysts such as acrylic acid catalysts and environmental catalysts for automotive and exhaust gas treatment.

MHI and Nippon Shokubai will promote the development of ammonia cracking systems that contribute to building hydrogen and ammonia supply chains, aiming for early establishment and social implementation of decarbonization technologies, and contributing to the realization of a sustainable carbon-neutral society.

HEPCO, aiming to achieve carbon neutrality in Hokkaido by 2050 across the entire energy sector, is expanding the introduction of renewable energy and decarbonizing power sources, while promoting initiatives related to ammonia, hydrogen, and carbon capture utilization and storage (CCUS) in the Tomakomai region and other areas of Hokkaido.

Foreigners working in China now benefit from clearer legal recognition under Judicial Interpretation II. The Supreme People’s Court expands the conditions under which foreign nationals can be confirmed as employees, including those with permanent residency, valid work permits, or other lawful procedures. It also affirms the role of representative offices in labor disputes, strengthening legal accountability. (Also see our series article: China’s New Judicial Interpretation II on Labor Disputes: Key Themes at a Glance)

On August 1, 2025, China’s Supreme People’s Court (SPC) released the long-awaited Judicial Interpretation II on the Application of Law in Labor Dispute Cases (Fa Shi [2025] No. 12, hereinafter “Judicial Interpretation II on Labor Disputes” or “Judicial Interpretation II”), along with a set of illustrative cases. Both came into effect on September 1, 2025. These developments mark a significant update to labor adjudication standards, especially for foreign nationals working in China.

Of particular relevance to foreign-invested enterprises (FIEs), Article 4 of Judicial Interpretation II sets out clearer criteria for recognizing employment relationships involving foreign nationals. This is the SPC’s latest response to legal ambiguities that have emerged since the partial invalidation of Judicial Interpretation I in 2021. The new rules aim to unify judicial practice and provide more predictable outcomes in labor disputes involving expatriates.

Judicial confirmation of employment relationships for foreign nationals in China

Article 4 of Judicial Interpretation II explicitly lists three qualifying scenarios and introduces a catch-all clause, which provides courts with clearer criteria and greater flexibility in adjudicating labor disputes involving foreign employees.

Scenario 1: Obtaining permanent residency

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Foreign nationals who have obtained permanent residency in China, formally recognized through the Foreigner’s Permanent Residence Permit, are entitled to employment rights that closely mirror those of Chinese citizens. According to the Measures on the Enjoyment of Relevant Benefits by Foreigners with Permanent Residency in China, holders of this permit are exempt from the requirement to obtain a separate employment certificate. They may also be prioritized for other talent-related documentation, such as the Foreign Expert Certificate or local talent residence permits. This legal status grants foreign residents the right to work in China without additional administrative hurdles.

Consequently, the SPC’s inclusion of permanent residency as a qualifying condition under Article 4 of Judicial Interpretation II is not a new entitlement, but rather a formal judicial acknowledgment of existing legal norms. It provides courts with a clear basis to support the recognition of labor relationships involving permanent residents, reinforcing consistency in adjudication and reducing ambiguity in cross-border employment disputes.

Scenario 2: Obtained a work permit and legally residing in China

Obtaining a work permit and legally residing in China refers to a foreign national who has followed the official procedures to both work and live in China in compliance with national laws. This typically includes:

• Obtaining a Notification Letter of Foreigner’s Work Permit (《外国人工作许可通知》) before entering China;
• Entering China on a Z visa or R visa, which corresponds to the intended purpose of stay—namely, employment;
• Registering accommodation with the local public security bureau within 24 hours of arrival (if not staying in a hotel);
• Applying for a Foreigner’s Work Permit (《外国人工作许可证》) within 15 days of entry; and
• Obtaining a residence permit from the Exit-Entry Administration of the Public Security Bureau within 30 days of entry, which serves as the legal basis for residence in China.

Together, these steps establish both the right to work and the legal status to reside in China.

Notably, certain categories of foreign talent may be exempt from obtaining the Notification Letter and Work Permit. These include individuals holding a Foreign Expert Certificate, a Permit for Offshore Petroleum Operations, or a Temporary Commercial Performance Permit. Such exemptions are recognized under existing regulations and reflect the flexibility built into China’s foreign employment system.

Scenario 3: Completing other procedures in accordance with national regulations

The third scenario under Article 4 introduces a flexible clause that accommodates foreign nationals who may not fall under the standard employment categories but have nonetheless followed lawful procedures to work in China. This provision acknowledges that, in addition to permanent residency and conventional work permits, there are special employment types governed by specific national regulations.

This catch-all clause serves as a legal safety net, allowing courts to recognize labor relationships in cases where foreign nationals have complied with alternative but legitimate employment procedures. It reflects the SPC’s intent to adapt to the evolving landscape of foreign employment in China, where new talent programs, bilateral arrangements, and industry-specific permits are increasingly common.

For employers, especially FIEs, this provision offers greater clarity and flexibility. It ensures that lawful employment, regardless of the specific permit type, can be judicially recognized, provided the foreign national has followed the relevant national procedures.

Judicial support is not automatic

While Judicial Interpretation II expands the scenarios under which courts may support the recognition of employment relationships involving foreign nationals, it’s important to note that such support is not automatic. Article 4 uses the phrase “the people’s court may support in accordance with the law”, rather than “shall support”, indicating that judicial confirmation is discretionary and subject to further evaluation.

This means that even if a foreign national meets one of the listed conditions, such as holding permanent residency or a valid work permit, the court will still assess the substantive elements of the employment relationship. These include the existence of a signed contract, the nature of the work performed, the degree of control and supervision by the employer, and other indicators of a genuine labor relationship.

For HR professionals and legal teams, this highlights the importance of maintaining clear documentation and ensuring that employment arrangements with foreign nationals comply with both procedural and substantive legal standards. Courts will take a holistic view, and compliance with administrative requirements alone may not suffice if the underlying employment facts are unclear or inconsistent.

Representative offices can be parties to labor disputes

Article 5 of Judicial Interpretation II introduces another clarification: a duly registered representative office (RO) of a foreign enterprise in China may now be named as a party in labor dispute proceedings. This marks a significant shift from prior judicial practice, where representative offices, due to their lack of legal personhood and employer qualifications, were often excluded from direct litigation and could only participate indirectly through labor dispatch agencies.

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Under China’s legal framework, representative offices are non-profit liaison entities established by foreign companies to conduct business-related activities such as market research, coordination, and promotion. They are not permitted to engage in profit-making activities or directly hire employees. Instead, staffing must be arranged through authorized third-party labor dispatch agencies, which formally sign employment contracts with the workers.

Despite these structural limitations, Judicial Interpretation II acknowledges the practical reality that representative offices often function as the de facto employer in daily operations. By affirming their eligibility to be named as parties in labor disputes, the SPC strengthens legal accountability and provides greater protection for employees working under such arrangements.

Moreover, the interpretation allows courts to support applications to add the foreign parent company to the litigation, especially in cases where the representative office lacks independent assets or has been dissolved. This provision reflects a broader trend toward tightening cross-border labor compliance and preventing foreign enterprises from evading liability through corporate structuring.

For FIEs operating representative offices in China, this development underscores the need for robust HR compliance. Even without formal employer status, representative offices must ensure that labor dispatch arrangements, workplace management, and employee rights are handled in full accordance with Chinese labor laws. Failure to do so may expose both the RO and its parent company to legal risk and reputational damage.

Key takeaway

Judicial Interpretation II marks a significant step forward in clarifying and expanding the legal framework for foreign nationals working in China. By broadening the scenarios under which employment relationships can be judicially recognized and affirming the role of representative offices in labor disputes, the SPC provides FIEs with clearer compliance pathways and stronger legal safeguards. HR teams must ensure that employment arrangements meet both procedural and substantive legal standards to mitigate risks and uphold labor rights.