Heat Stress Induced Testicular Impairment is Related to Orchitis and Complement Activation in Rongchang Boars

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Testis serves as an immune-privileged organ to keep its spermatogenesis undisturbed, while immunity is thermo-sensitive, especially for macrophages who present as the most abundant testicular immune cell. Our study aims to deeply unveil the underlying immune responses and assess its consequences to semen quality under heat stress, seeking for understanding and solutions to obstruction in boar reproductivity under heat stress. Methods We conducted the experiment using 26 8-week-old Rongchang male pigs who were assigned into thermal neutral pair-feed group and heat stress group for a 3-week experiment with the last two weeks as heat stress period. During heat stress period, pigs of heat stress group were subjected to 14-day 35 ± 1℃ heat stress, while pigs of thermal neutral pair-feed group were kept at 26 ± 1℃. Pigs were sampled at the end of heat stress period to obtain gonads tissue for assessing and measurement. Results Our findings confirmed heat stress reactions by elevated respiration rate ( P < 0.05) and expression of heat shock proteins 60 ( P < 0.05) and heat shock proteins 90 (P < 0.05). Tendencies to reduce sperm motility ( P = 0.06) and progressive sperms (P = 0.08) under HS were observed as well as significant reduction in average straight-line velocity ( P < 0.05) and total abnormality were also recorded ( P < 0.05). Visualized fibrosis, caspase-3 expression and accumulation of tumor necrosis factor-α ( P < 0.05) and Interleukin-1β ( P < 0.05) along with elevated macrophage composition ( P < 0.05) characterized the orchitis under heat stress. Accordingly, single cell RNA sequencing revealed fluctuation in engulfing and inflammatory signals in testicular macrophages, especially promotion in complement cascades by CD163 + macropahegs, resulting in final membrane attack complex assembling ( P < 0.05). Linear regression further exhibited a negative correlation between the membrane attack complex and sperm motility ( P < 0.05) a long with near-negative correlation between the membrane attack complex and progressive motility (P = 0.08) or velocity straight line (P = 0.06). Conclusions Our findings highlighted the relationship between heat stress, the onset of orchitis, and the activation of the complement system, all of which caused obstruction in semen quality of boars. Heat stress Boar Semen quality Testis Orchitis Macrophage Complement Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Boars who are capable of impacting a large number of sows and corresponding offsprings due to the universal use of artificial insemination technique are fundamental to modern pig industry. However, during the global climate alternation, heat stress (HS) is becoming an unignorable threat to boars as the susceptibility of semen quality to excessive environmental temperature. It was early reported that only 72h under 33℃ and 50% humidity dampened the semen quality that would remain lame for the next 2 months[ 1 ]. Recent research of our colleagues also verified the continuous obstruction in sperm viability and promotion in sperm apoptosis rate after 2 weeks HS at 33℃[ 2 ]. More importantly, Cameron and colleagues have proved semen from heat stressed boar would directly impact the reproductivity of sow by lowering the pregnant rate, normal embryo number and embryo immortal rate, which would directly dampen the output of piglets[ 3 ]. Testis as the only site for spermatogenesis is key to semen quality, in spite of the thermos-regulating mechanisms of the scrotum prompt the testis to appropriate temperature for spermatogenesis, the scrotum was also more likely to be heat stressed as it was located outside the body core[ 4 ]. Scrotal temperature disruption like HS, varicocele and cryptorchidism have been reported to severely dampen male fertility, yet their mechanism was not fully elucidated[ 5 – 9 ]. Testis is regarded as an immune-privileged organ to keep tolerance to self-antigens and spermatogenesis undisturbed[ 10 , 11 ]. Unfortunately, heat is naturally related to immune activity[ 12 ]. When taking macrophage who is the most abundant testicular immune cells into consideration, heat could directly trigger expression of inflammatory cytokines and engulfment activity of macrophages[ 13 – 15 ]. Within all the inflammatory cytokines originate from macrophages, it is recently noticed that complement C1Q induced complement cascade and its down-stream membrane attacking complex (C5b-9) that leads to membrane damage is responsible for testicular impairment, while early reports have already unveiled the connection between C1Q and HS in other models [ 16 – 19 ]. Finally, we hypothesized that HS would facilitate testicular inflammation characterized by complement cascade, which directly dampened semen quality of boars. With assistance of omics technology, we devoted to clarify the effect of HS on the immunity of testis and its possible connection to HS induced reproductivity changes in Rongchang boars after 14 days at 33℃. Material and Methods Animals and Experimental Design At the beginning, a total of 26 8-week-old male Rongchang pigs were selected and individually housed in stainless-steel metabolism cages at the room temperature 26℃±1 for adaption. Pigs were freely access to water and feed was supplied 4 times per day at 8.00, 12.00, 16.00 and 20.00. The feed was formulated referring to Nutrient requirement of swine, so it provides 3.15 Mcal/kg metabolizable energy and contains 15% of crude protein. After 3 weeks of adaption, 25 pigs at the age of 80 days old were randomly assigned to HS group or thermal neutral pair-feed (TN-PF/PF) group 1 week before the HS treatment, while 1 pig was excluded for cryptorchidism. The experiment was carried out as one way treatment design and randomized complete block experimental design with 1 pig as 1 experiment unit. Besides the ventilation system of the house, warm air blower and heat lamps were applied in controlling the room temperature at 35±1℃ for 14 days for the HS group, the room temperature of TN-PF group was maintained at 26℃±1 in the meantime. HS and temperature measurement Room temperature and humidity were recorded by the auto-thermometer every hour (Renke, Jinan, China) during the whole experiment period while temperature-humidity index (THI) was calculated based on THI = (1.8 × T + 32) - (0.55 - 0.55 × RH × 0.01) × (1.8 × T - 26). Scrotal temperature was measured by infrared thermometer (Hikvision, Hangzhou, China) and respiration rate was counted manually. Sample collection At the end of the experiment, pigs were anaesthetized by 2ml intramuscular injected Zoletil™50 (Virbac, Nice, France) before cutting along the ventral midline to expose the abdominal cavity, afterwards, blood from spermatic vein and portal vein was collected by vacuum blood collection tube with heparin sodium and disposable blood collection needle. The plasma was separated and stored at -20℃ after centrifuging at 3000g for 15 minutes,. The pigs were then slaughtered unconsciously before testis and epididymis were acquired. A part of the testis and intact epididymis was subsequently sent to laboratory within 5 minutes for further operation, while the rest of the was canned and quickly frozen by liquid nitrogen. Semen quality analysis After epididymis was transported to the laboratory, the cauda epididymis was separated and cut to small piece in a tube containing 10ml of semen extender. After 37℃ water bath for 15 minutes, supernatant was collected and 5-fold diluted before analyzing by computer assisted sperm analysis system (CASA) system (Minitube, Tiefenbach, German). The supernatant was also used in preparation of morphology slides of sperms. Generally, 20ul of the supernatant was pipetted to the slide and averagely dispersed, the slide was next air dried and fixed by Immunol Staining Fix Solution (Beyotime, Shanghai, China) for 15 minutes. Then it was washed by ultrapure water and air dried before staining by Crystal Violet - Gentian Violet Stain Solution (Beyotime, Shanghai, China) for 15 minutes. Finally, the prepared slides were washed and dried again before packing up by sealing bags for later observation. Flow cytometry analysis Testis tissue was firstly sliced and incubated in HBSS (Solarbio, Beijing, China) containing 1mg/mL collagenase I (Sigma, Missouri, USA) and 100U/mL DNase I (Solarbio, Beijing, China) at 37℃ water bath for 15 minutes. 1mL fetal bovine serum (Clark, Virginia, USA) was added to the supernatant to stop digestion, the suspension was then filtered by 70 um cell strainer (Nest, Wuxi, China) and centrifuged at 4℃ 350g for 5 minutes. After removal of supernatant, cells were resuspended by DPBS (Gbico, California, USA) containing fixable viability stain 780 (BD, New Jersey, USA) at 4℃ for 30 minutes. The suspension was subsequently centrifuged at 4℃ 350g for 5 minutes before removal of supernatant, the remaining cells were resuspended by 100ul stain buffer (BD, New Jersey, USA) containing mouse anti-pig CD45:FITC (Bio-rad, Virginia, USA) , mouse anti-pig CD163:RPE (Bio-rad, Virginia, USA) and BV421 rat anti-CD11b (BD, New Jersey, USA) at 4℃ for 30 minutes. The suspension was centrifuged at 4℃ 350g for 5 minutes to remove the supernatant. Afterwards, 250ul of cytofix (BD, New Jersey, USA) was used to fix the cells at 4℃ for 20 minutes. The suspension was then centrifuged at 4℃ 350g for 5 minutes before removal of supernatant, cells were later resuspended by 500ul of cytoperm (BD, New Jersey, USA) to permeate the cell membrane for 5 minutes. After centrifuged at 4℃ 350g for 5 minutes and disposal of cytoperm, 100ul of cytoperm with mouse anti-pig macrophages: Alexa Fluor 647(Bio-rad, Virginia, USA) was added to resuspended the cells at 4℃ for 40 minutes. Lastly, cell suspension was centrifuged at 4℃ 350g to remove the supernatant and washed once by 100ul stain buffer, 350ul of stain buffer was then used to resuspended the cells for analysis by BDverse flow cytometer（BD, New Jersey, USA）. ELISA assays ELISA assays were carried out according to the manufacturer’s instruction, including heat shock protein 60 (HSP60) (BIM, California, USA), heat shock protein 90 (HSP90) (BIM, California, USA), 8-hydroxydeoxyguanosine (8-OHG) (BIM, California, USA), Testosterone (BIM, California, USA), interleukin-1β (IL-1β) (BIM, California, USA), tumor necrosis factor-α (TNF-α) (R&D, Minnesota, USA), C1Q (BIM, California, USA), C3 (BIM, California, USA), C5b-9 (BIM, California, USA), IgG (BIM, California, USA). Masson sections Masson sections were prepared by Hubei BIOSSCI Biotech Co., Ltd. Tissue sections were immersed in clearer for 10 minutes. Repeat this step two times, gently shaking off excess liquid between each step. Tissue sections were immersed in progressively more dilute ethanol solutions. The dehydrated tissue sections were immersed in Bouin’s solution or Zenker’s solution overnight, then, rinsed with running water. Sections were stained with hematoxylin solution (Harris) or iron hematoxylin for 5-10 minutes and slightly washed with running water. Sections were differentiated with 0.8% - 1% hydrochloric acid alcohol and washed with running water for several minutes. Sections can also be treated with lithium carbonate solution to be bluer and washed with running water and ultimately immersed in distilled water to rehydrate the tissue: Absolute ethanol for 5min, 95% ethanol for 5min, 85% ethanol for 5min, 75% ethanol for 5min. Rinsing with distilled water for 1min. Sections were stained with ponceau acid fuchsin solution for 5-10 minutes and washed with running water. Sections were treated with phosphomolybidic acid solution for about 5 minutes and then stained with aniline blue solution for 5 minutes without washing. Sections were treated with 1% glacial acetic acid for 1 minute and dehydrated with 95% alcohol for several times. The tissue sections were dehydrated with absolute alcohol and transparent with xylene, then mounted with neutral balsam Immunohistochemistry section Immunohistochemistry sections were prepared by Hubei BIOSSCI Biotech Co., Ltd. In brief, deparaffinization and rehydration were carried out. Then EDTA antigen retrieval solution was then used for antigen retrieval. Afterwards, the section was immersed in 3% H 2 O 2 to block the innate peroxidase. Then the section was blocked by 10% rabbit serum solution. The section was subsequently stained by Caspsae-3 (Cell Signaling Technology, Massachusetts, USA) antibody solution at 1:200. Finally, the immunohistochemistry section was stained by goat anti rabbit IgG-HRP secondary antibody (Abcam, Cambridge, UK) at 1:2000 before 3,3'-Diaminobenzidine (Maxim, Fuzhou, China) chromogenic reaction. Immunofluorescence section Immunofluorescence sections were prepared by Hubei BIOSSCI Biotech Co., Ltd. In brief, deparaffinization and rehydration were carried out. Then EDTA antigen retrieval solution was then used for antigen retrieval. Afterwards, the section was blocked by 10% goat serum solution. The section was subsequently stained by goat anti-pig IgG antibody (Thermofisher, Massachusetts, USA) solution at 1:20. Non-targeted metabolomics Non-targeted metabolomics was performed by LC-Bio Technology CO. The collected spermatic vein plasmas were thawed on ice, and metabolite were extracted with 80% methanol Buffer. Briefly, 100 μl of sample was extracted with 400 μl of precooled methanol. The extraction mixture was then stored in 30 min at -20°C. After centrifugation at 20,000 g for 15 min, the supernatants were transferred into new tube and vacuum dried. The samples were then redissolved with 100μL 80% methanol and stored at -80°C prior to the Liquid Chromatograph Mass Spectrometer analysis. Transcriptomics (RNA-seq) RNA-seq was performed by LC-Bio Technology CO. In brief, total RNA of testis was isolated and purified using TRIzol reagent (Invitrogen, California, USA) following the manufacturer's procedure before it was reverse-transcribed to cDNA by SuperScript™ II Reverse Transcriptase (Invitrogen, California, USA), the cDNA was next processed and sequenced on an illumina Novaseq™ 6000 (LC-Bio Technology CO., Ltd., Hangzhou, China) following the vendor's recommended protocol. In data analysis of RNA-seq, we used HISAT2 (https://ccb.jhu.edu/software/hisat2) to map reads to the reference genome of Sus_scrofa.Sscrofa11.1.(http://ftp.ensembl.org/pub/release-107/fasta/sus_scrofa/dna/). The mapped reads of each sample were assembled using StringTie (https://ccb.jhu.edu/software/stringtie) with default parameters. StringTie was subsequently used to perform expression level for mRNAs by calculating FPKM (FPKM = [total_exon_fragments / mapped_reads(millions) × exon_length(kB)]). The differentially expressed mRNAs were selected with fold change > 2 or fold change < 0.5 and with parametric F-test comparing nested linear models (p value < 0.05) by R package edgeR (https://bioconductor.org/packages/release/bioc/html/edgeR.html). Afterwards, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment were performed based on the differentially expressed genes (DEGs). Single cell transcriptomics (scRNA-seq) scRNA-seq was performed by LC-Bio Technology CO. Firstly, testis tissue was lysed to get cell suspension, then the mouse anti-pig CD45: FITC (Bio-rad, Virginia, USA) and FACS sorting were applied in preparing testicular CD45+ cells who were used in later generation of nanoliter-scale Gel Bead-In-EMulsions (GEMs) (supplemental Fig.3A). After releasing of mRNA from cells in GEMs, cDNA was produced by reverse transcription and was later amplified by PCR to construct single-cell libraries. Afterwards, libraries were sequenced on NextSeq 500 sequencing system. In data analysis of scRNA-seq, quantification and quality control were finished using Cell Ranger to produce post-QC data. Then R package Seurat was applied in filtering abnormal data, preliminary clustering and visualization. Afterwards, cells were identified by repeatedly comparing the cell makers of clusters on Cell Taxonomy database (https://ngdc.cncb.ac.cn/celltaxonomy/) before labeling them. By bimod P=0.26, DEGs were revealed between groups and pathway enrichment were performed based on the DEGs. Re-clustering was carried out based on the identified clusters, DEGs identification and KEGG enrichment were also performed. Conventional statistical analysis Data was analysis with procedure mixed by SAS (9.4), using the following model: Y=mean + treatment + block + error Where mean=overall means, treatment=treatment effect, block=random effect=maternal origin, error=residual. Outlier would be identified if its standard residual (residual/standard deviation) is larger than 2.5. Normality and homogeneity of variances were evaluated by Shapiro-wilk test and Levene test, respectively, data would be transformed if it did not meet normal distribution and unequal variance analysis by procedure Mixed would be applied if variances were significantly unequal. Repeated measured data would be analyzed using repeated measure by procedure Mixed. Grouping letters were assigned by SAS Macro program. All data was presented as mean±standard error mean. P<0.05 was regarded as significance and 0.05<P<0.10 was regarded as a tendency. In addition, linear regression and correlation analysis and was performed by OmicStudio tools at https://www.omicstudio.cn/tool. Results Heat stress and semen quality of boars The curves of room temperature and the THI were presented in Fig.1A, the THI was remained above 86 and the room temperature was kept at 35±1℃ during HS period. There are typical heat-stress reactions in boars as for not only their respiration rate (RR) tremendously elevated and but the scrotal temperature was also promoted (Supplemental Fig. 1A-C). Aside from that, plasma heat shock protein 60 (HSP60) and heat shock protein 90 (HSP90) from spermatic vein of heat stressed boars were significantly promoted comparing to the TN-PF group (Fig. 1B and C). Therefore, HS reaction is confirmed both in the boars and their scrotums. Obviously, HS dampened the semen quality of boars. There were tendencies to reduce percentage of motile sperms and progressive sperms under HS (Fig. 1D and E). In addition, there was significant reduction in average straight-line velocity (VSL) (Fig. 2F). Moreover, tendency to promote sperm head abnormality as well as significant elevation in tail abnormality and total abnormality were also observed (Fig.2H-J). Together, our finding depicted dampened porcine semen quality under HS. HS facilitated orchitis and testicular impairment Results of RNA-seq hinted at changes in testicular immunity, such as MAPK pathway, T cell receptor pathway, TNF-α and hematopoietic cell lineage in KEGG and GO enrichment based on Gene Set Enrichment analysis (GSEA) (Fig.2A and B). Along with that, protein-protein interaction network analysis uncovered‌‌ the key role of immunity in testicular changes as genes associated to macrophage and T cell were regarded as key nodes with high degree level in the network (Fig.2C). The results above remined us of orchitis characterized by testicular fibrosis, accumulation of inflammatory cytokines and invasion of immune cells. Coincidentally, not only Masson sections exhibited enhanced fibrosis but elevation in TNF-α and tendency to promote IL-1β in spermatic vein plasma were also recorded (Fig.2D-F). Promotion in CD68+ cells and tendency to elevate CD68+CD163+ cells observed by flow cytometry also supported invasion of immune cells (Fig.2G-H, Supplemental Fig.2). Finally, testicular tissue impairment was confirmed by upregulation in 8-OHG concentration, lowered testosterone concentration and Caspase-3 expression in seminiferous tubules (Supplemental Fig.1D-F). All in all, our results above verified the appearance of testicular impairment and orchitis under HS. Changes in complement and engulfment activity in testicular macrophage (TM) under HS For further insight into testicular immunity, flow cytometry sorting was next applied to purify the testicular CD45+ immune cells for scRNA-seq as shown in supplemental Fig.3A, while gating was shown in supplemental Fig.3B. A total of 29 clusters of cells with their makers were observed in scRNA-seq (supplemental Fig.3C and D). Most of clusters were then identified based on the cell makers from online databases (Fig.3A and supplemental Fig.3E). As expected, macrophage took a big proportion in the testicular immune cells and their DEGs were enriched in cell death, engulfing and inflammatory pathways (Fig.3B and C). Although we are not capable of fully verifying cell death and engulfing activity, metabolomics demonstrated upregulation of lyso-phosphatidylserine 18:1 (LPS 18:1), phosphatidylserine (18:1(9Z)/0:0) (PS (18:1(9Z)/0:0)) and lyso-phosphatidylserine 18:2 (LPS 18:2) who were associated to cell death and engulfment signal, supporting the perspective HS activated engulfment signal in TMs (Supplemental Fig.4A-D). Within all the enriched inflammatory pathways, complement and coagulation cascades were drawing attention as complement activation has recently been reported to be responsible for poor male fertility under viral stress[17]. Significant elevation in expression of C1QA, C1QB and C1QC both in testis and TMs was confirmed in both RNA-seq and scRNA-seq (Fig.3D, Supplemental Fig.4E and F). It was also exhibited C1Q genes were almost only expressed by TMs (Fig.3D). Re-clustering of TMs identified 16 sub-clusters, which depicted the onset of dramatic shifts in TM subclusters during HS (Fig. 3E and F). In the meantime, re-clustering pointed out the exact sub-cluster responsible for C1Q expression, as KEGG enrichment of sub-cluster 5 maker genes noted complement cascades and expression dotplot showed that sub-cluster 5 TMs were mainly responsible for C1Q expression, which was also accelerated by HS (Fig3.G-I). In previous report, C1Q activation was related to CD163+macrophage infiltration in kidney[20]. We next applied Addmodulescore to identify sub-cluster 5, it was noticed that sub-cluster 5 expressed high level of M2 macrophage genes by a M2 macrophage gene set from Mossadegh-Keller et al. with a little modification[21] (Fig.3J). In agreement with Addmodulescore, tSNE graph of C1QA, C1QB, C1Qc and CD163 represented a large overlapping area (Fig.3K). In addition, significant positive linear regression effects were observed between C1Q genes and CD163 gene or CD68+ CD163+ cells, corresponding to the results of TM composition by flow cytometry (Supplemental Fig.4G-L). Collectively, those results indicated the onset of TM engulfment activity and potential in CD163+ TM related complement activation. Complement cascades is associated to obstruction in semen quality. C1Q is the key component for classical activation of complement cascades leading to final assemble of C5b-9 who specialized in cell membrane breaking and cell lysis with C3 as key signal transduction component[22]. Unsurprisingly, C1Q, C3 and C5b-9 protein were all significantly facilitated in the testis of heat stressed boar (Fig.4A-C). GO enrichment analysis of RNA-seq repeatedly emphasized membrane changes, echoing a key role of complements in testicular changes under HS (Fig.4D). As C1Q is key to classical complement activation whose up-stream activator is antigen-antibody complex, we visualized IgG distribution in the testis[17]. As shown in Fig.4F and E, HS prompted IgG secretion, making a proper cause for classical complement activation. Most importantly, linear regression analysis revealed notable or near-significant linear correlations between the levels of C5b-9 and semen quality (Fig.4G-I). Analogically, C3 and C1Q also exhibit linear associations to semen quality (Supplemental Fig.5A-H). Conclusively, our finding demonstrated negative association between complement activation and semen quality, emphasizing the role of complement in HS related dampened semen quality. Discussion HS has become a tremendous threat with the global warming, it is believed pigs would be heat stressed at 79 THI and the HS would be severe at 84 THI[ 23 , 24 ]. We therefore kept the room temperature above 35 and THI above 85 to ensure HS reactions of pigs during HS period. Due to the absence of swell gland, RR of pigs is susceptible to HS[ 25 , 26 ]. In our experiment, elevation in RR was observed, which could be regarded as the hallmark of HS reaction. Data have shown obvious elevation in scrotal temperature of mice and pig under HS, indicating the possibility of direct impact of HS on scrotum[ 27 , 28 ]. Although scrotal temperature is not numerically strongly altered by HS in our experiment, it was statistically significantly promoted in analysis with environment temperature as main effect. We also observed higher level of HSP60 and HSP90 in the spermatic vein plasma as biological confirmation of scrotal HS reactions. Studies have already unveiled the detrimental impact of scrotal HS on male fertility, emphasizing the importance of scrotal temperature maintaining in spermatogenesis[ 5 , 7 , 29 ]. The semen quality in our HS group was similarly dampened by not only lowering velocity and motility but promoting abnormality as well, which could also be regarded as the phenotype of boars being heat stressed like it was previously reported[ 1 ]. In agreement with these, high room temperature lowers the velocity and motility of mice after 38℃ for 2 hours in 14 days[ 3 , 30 ]. Yaeram and colleagues also revealed obstructed inseminating ability of sperms from mice who were heat stressed at 36℃ for 12 hour per day[ 31 ]. All those data highlighted the detrimental effect of HS and elevated scrotal temperature on male reproductivity. To the best of our knowledge, there was only a little information about the connection between HS and orchitis[ 32 ]. Although it was demonstrated that macrophages as the major testicular immune cells were sensitive to hyperthermia characterized by elevated pro-inflammatory and engulfing activity though NF-kB pathway, TMs were also reported to be less pro-inflammatory for its poor capacity in activating NF-kB pathway comparing to peritoneal macrophage [ 13 , 15 , 33 ]. However, In human model, scrotal HS not only obstructed reproductivity but promoted the level of macrophage migration inhibitory factor who is responsible for macrophage recruitment and inflammation as well, corresponding to the elevation in TM number we observed[ 7 , 34 ]. Koch and colleagues also discovered HS induced infiltration of macrophages with active inflammatory and phagocytosis response into the mucosa and submucosa of the jejunum in bovine model[ 35 ]. Similar to their results, our data have elucidated accumulation of inflammatory cytokines with changes in inflammatory and engulfing pathway in testis, supporting the appearance of HS induced orchitis. The orchitis related testicular impairment could be exerted to Leydig cells for TNF-α could serve as an histone deacetylase 7 activator, in doing so, inhibition of the testosterone secretion took place, which in return blunt the immunosuppressive environment of the testis[ 11 , 36 , 37 ]. When taking engulfing activity into consideration, TMs were capable of engulfing early apoptotic Leydig cells characterized by exposure of phosphatidylserine, corresponding to promotion in phosphatidylserine analogues in our metabolomics. Thus, we also suspected there was engulfing of apoptotic Leydig cells under HS, giving a rise to reduction of testosterone level [ 38 ]. In addition, both HS and inflammation would impair the integrity of blood-testis barrier that is regarded as the guardian of spermatogenetic environment and isolation of spermatogenetic antigens, resulting in possibility of exposing the spermatogenetic antigens and inflammation acceleration[ 39 – 41 ] Complement cascade was rarely focused in all kinds of immune responses, yet it was recently noted it was related to viral stress induced obstruction in male reproductivity[ 17 ]. Our result documented not only accumulation in complement components, but also elevated level of testicular IgG along with heat shock proteins as identified self-antigens, which is necessary to C1Q related classical activation of complement cascade, leading to the possibility of antigen-antibody complex construction [ 17 , 42 , 43 ]. Despite there is little solid evidence about the relationship between HS and C1Q, C1Q was often mentioned in anoxic and ischemic models [ 19 , 20 , 44 ]. Coincidentally, HS is associated to redistribution of blood flow that would result in organic ischemic anoxia to facilitate heat ejection [ 45 , 46 ]. Moreover, it was previously illustrated C1 inhibitor is capable of alleviating empyrosis induced tissue impairment[ 18 ]. The information above illustrated the potential in connecting HS to complement reaction and its consequences, our data verified the onset of C1Q-ralated complement reaction in testis under HS, yet its impact on semen was only speculated by linear regression analysis. Conclusion Collectively, present results support the opinion boar suffered HS induced orchitis, in which TMs underwent dramatic alternations in engulfment and inflammatory activity. Of all those inflammatory pathways, our attention was caught by C1Q promotion by CD163 + TMs and its down-stream complement cascade that was associated to poor semen quality in linear regression. Abbreviations THI Temperature humidity index HS Heat stress TN-PF/PF Thermal neutral pair-feed RR Respiration rate HSP60 Heat shock protein 60 HSP90 Heat shock protein 90 CASA Computer assisted sperm analysis system VSL Average straight-line velocity KEGG Kyoto Encyclopedia of Genes and Genomes GO Gene Ontology GSEA Gene Set Enrichment analysis GEMs Gel Bead-In-Emulsions TM Testicular macrophage 8-OHG 8-hydroxydeoxyguanosine TNF-α Tumor necrosis factor-α IL-1β Interleukin-1β DEGs Differentially expressed genes LPS 18 1:Lyso-phosphatidylserine 18:1 PS (18 1(9Z)/0:0):Phosphatidylserine (18:1(9Z)/0:0) LPS 18 2:Lyso-phosphatidylserine 18:2 Declarations Acknowledgment This work was supported by the Projects of The National Natural Science Foundation of China (U21A20255), National Modern Agricultural Industry Technology System Sichuan Pig innovation team (SCSZTD-2024-08) and Sichuan Province “14th Five-Year” Sichuan Pig Major Science and Technology Project (2021ZDZX0009). Consent for publication All authors consent to this publication. Author contributions Experimental execution: W.S., J.N and X.M.; data analysis and preservation: L.C., B.F. and L.H; manuscript preparation and revision: Y.Z., Z.F., S.X. and J.L.; supervision: X.J., Y.L. and D.W. Competing interests The authors declare no competing interests. Data availability The raw data of transcriptomics is available at NCBI (Accession: PRJNA1236216), and the raw data of single cell transcriptomics is available at NCBI (Accession: PRJNA1237295), any additional information will be available from the De Wu ( [email protected] ) upon request. Ethics The animal protocol for this experiment was approved by the Animal Care and Use Committee of Sichuan Agricultural University. References McNitt JI, First NL. Effects of 72-hour heat stress on semen quality in boars. International journal of biometeorology. 1970;14(4):373-80 http://doi.org/10.1007/bf01462914. Yu C, Li H, Hua L, Che L, Feng B, Fang Z, et al. 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Animals (Basel). 2021;11(1) http://doi.org/10.3390/ani11010215. Brandt P, Bjerg B, Pedersen P, Sørensen KB, Rong L, Huang T, et al. The effect of air temperature, velocity and humidity on respiration rate and rectal temperature as an expression of heat stress in gestating sows. J Therm Biol. 2022;104:103142 http://doi.org/10.1016/j.jtherbio.2021.103142. Li Y, Huang Y, Piao Y, Nagaoka K, Watanabe G, Taya K, et al. Protective effects of nuclear factor erythroid 2-related factor 2 on whole body heat stress-induced oxidative damage in the mouse testis. Reprod Biol Endocrinol. 2013;11:23 http://doi.org/10.1186/1477-7827-11-23. Li Y, Cao Y, Zhou X, Wang F, Shan T, Li Z, et al. Effects of zinc sulfate pretreatment on heat tolerance of Bama miniature pig under high ambient temperature. J Anim Sci. 2015;93(7):3421-30 http://doi.org/10.2527/jas.2015-8910. Parrish JJ, Willenburg KL, Gibbs KM, Yagoda KB, Krautkramer MM, Loether TM, et al. Scrotal insulation and sperm production in the boar. 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Differential activation of inflammatory pathways in testicular macrophages provides a rationale for their subdued inflammatory capacity. J Immunol. 2015;194(11):5455-64 http://doi.org/10.4049/jimmunol.1401132. Kang I, Bucala R. The immunobiology of MIF: function, genetics and prospects for precision medicine. Nat Rev Rheumatol. 2019;15(7):427-37 http://doi.org/10.1038/s41584-019-0238-2. Koch F, Thom U, Albrecht E, Weikard R, Nolte W, Kuhla B, et al. Heat stress directly impairs gut integrity and recruits distinct immune cell populations into the bovine intestine. Proc Natl Acad Sci U S A. 2019;116(21):10333-8 http://doi.org/10.1073/pnas.1820130116. Fijak M, Schneider E, Klug J, Bhushan S, Hackstein H, Schuler G, et al. Testosterone replacement effectively inhibits the development of experimental autoimmune orchitis in rats: evidence for a direct role of testosterone on regulatory T cell expansion. J Immunol. 2011;186(9):5162-72 http://doi.org/10.4049/jimmunol.1001958. Sadasivam M, Ramatchandirin B, Balakrishnan S, Prahalathan C. HDAC7 modulates TNF-α-mediated suppression of Leydig cell steroidogenesis. Mol Cell Biochem. 2015;406(1-2):83-90 http://doi.org/10.1007/s11010-015-2426-y. Yu W, Zheng H, Lin W, Tajima A, Zhang Y, Zhang X, et al. Estrogen promotes Leydig cell engulfment by macrophages in male infertility. J Clin Invest. 2014;124(6):2709-21 http://doi.org/10.1172/jci59901. Pérez CV, Sobarzo CM, Jacobo PV, Pellizzari EH, Cigorraga SB, Denduchis B, et al. Loss of occludin expression and impairment of blood-testis barrier permeability in rats with autoimmune orchitis: effect of interleukin 6 on Sertoli cell tight junctions. Biol Reprod. 2012;87(5):122 http://doi.org/10.1095/biolreprod.112.101709. Hu SQ, Liu DL, Li CR, Xu YH, Hu K, Cui LD, et al. Wuzi-Yanzong prescription alleviates spermatogenesis disorder induced by heat stress dependent on Akt, NF-κB signaling pathway. Sci Rep. 2021;11(1):18824 http://doi.org/10.1038/s41598-021-98036-2. Li MW, Xia W, Mruk DD, Wang CQ, Yan HH, Siu MK, et al. Tumor necrosis factor {alpha} reversibly disrupts the blood-testis barrier and impairs Sertoli-germ cell adhesion in the seminiferous epithelium of adult rat testes. J Endocrinol. 2006;190(2):313-29 http://doi.org/10.1677/joe.1.06781. Nagahori K, Hirai S, Hatayama N, Kuramasu M, Omotehara T, Kawata S, et al. Heat shock protein A4L is a potent autoantigen for testicular autoimmunity in mice. J Reprod Immunol. 2021;145:103318 http://doi.org/10.1016/j.jri.2021.103318. Fijak M, Iosub R, Schneider E, Linder M, Respondek K, Klug J, et al. Identification of immunodominant autoantigens in rat autoimmune orchitis. J Pathol. 2005;207(2):127-38 http://doi.org/10.1002/path.1828. Ziabska K, Gewartowska M, Frontczak-Baniewicz M, Sypecka J, Ziemka-Nalecz M. The Impact of the Histone Deacetylase Inhibitor-Sodium Butyrate on Complement-Mediated Synapse Loss in a Rat Model of Neonatal Hypoxia-Ischemia. Molecular neurobiology. 2025;62(4):5216-33 http://doi.org/10.1007/s12035-024-04591-w. Chen F, Dworak M, Wang Y, Cham JL, Badoer E. Role of the hypothalamic PVN in the reflex reduction in mesenteric blood flow elicited by hyperthermia. American journal of physiology Regulatory, integrative and comparative physiology. 2008;295(6):R1874-81 http://doi.org/10.1152/ajpregu.90384.2008. Chen F, Wang Y, Lee Cham J, Badoer E. Inhibition of nitric oxide synthase in the paraventricular nucleus prevents the hyperthermia-induced reduction of mesenteric blood flow in rats. American journal of physiology Regulatory, integrative and comparative physiology. 2010;299(2):R596-602 http://doi.org/10.1152/ajpregu.00003.2010. Supplementary Files SupplementalFig.1.png Supplemental Fig.1 HS and testicular impairment. A Respiration rate under HS. B Scrotal temperature under HS. C Scrotal temperature within 1-5h at 35℃. D Representative photos of Caspase-3 immunohistochemistry. E Level of 8-OHG in the plasma from spermatic vein blood. F Level of testosterone in the plasma from spermatic vein blood. SupplementalFig.2.png Supplemental Fig.2 The gating of TMs. SupplementalFig.3.png Supplemental Fig.3 Procedures and cell identification in scRNA-seq. A Graphic procedure of Single cell RNA-seq for CD45+ cells. B Gating of flow cytometry sorting. CtSNE graph of automatically defined clusters. D Makers of automatically defined clusters. E Cell markers of cell types. SupplementalFig.4.png Supplemental Fig.4 Change in testicular profile and relationship between CD163 and C1Q. A Log2 intensity of lyso-phosphatidylserine 18:1. B Log2 intensity of phosphatidylserine (18:1(9Z)/0:0). C Log2 intensity of lyso-phosphatidylserine 18:2. D KEGG enrichment of significantly altered metabolites. E Expression of C1QA, C1QB and C1QC in testis from RNA-seq. F KEGG enrichment of DEGs from RNA-seq. G Linear regression of C1QA and CD163. H Linear regression of C1QB and CD163. I Linear regression of C1QC and CD163. J Linear regression of C1QA and CD68+CD163+cells. K Linear regression of C1QB and CD68+CD163+cells. LLinear regression of C1QC and CD68+CD163+cells. SupplementalFig.5.png Supplemental Fig.5 Linear regression of semen quality and complement members. A Linear regression of C1Q and sperm motility. B Linear regression of C1Q and sperm progressive motility. C Linear regression of C1Q and sperm VSL. DLinear regression of C1Q and sperm abnormality. E Linear regression of C3 and sperm motility. F Linear regression of C3 and sperm progressive motility. G Linear regression of C3 and sperm VSL. H Linear regression of C3 and sperm abnormality. Cite Share Download PDF Status: Published Journal Publication published 17 Dec, 2025 Read the published version in Journal of Animal Science and Biotechnology → Version 1 posted Editorial decision: Major revision 26 Aug, 2025 Reviewers agreed at journal 30 Jul, 2025 Reviewers invited by journal 13 Jul, 2025 Editor assigned by journal 09 Jul, 2025 First submitted to journal 08 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7072366","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":484794407,"identity":"7f52355d-01b5-4331-9f56-2fc4e62570b4","order_by":0,"name":"Xiangyuan Ma","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xiangyuan","middleName":"","lastName":"Ma","suffix":""},{"id":484794408,"identity":"004a8776-ef71-430e-8287-d14ab53267b9","order_by":1,"name":"Wenxue Shen","email":"","orcid":"","institution":"Sichuan Agricultural 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sperms. \u003cstrong\u003eE\u003c/strong\u003eProgressive motility of sperms. \u003cstrong\u003eF \u003c/strong\u003eVelocity straight line of sperms. \u003cstrong\u003eG\u003c/strong\u003eHead abnormality ratio of sperms. \u003cstrong\u003eH \u003c/strong\u003eHead abnormality ratio of sperms. \u003cstrong\u003eI \u003c/strong\u003eTotal abnormality ratio of sperms. \u003cstrong\u003eJ \u003c/strong\u003eRepresentative photos of abnormal sperms.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-7072366/v1/94b96e40b29e9caf3b1da4a0.png"},{"id":86872965,"identity":"ae471ca1-95a7-452e-9f90-2736c9614b6f","added_by":"auto","created_at":"2025-07-16 14:45:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":6442611,"visible":true,"origin":"","legend":"\u003cp\u003eTesticular immunity and orchitis under HS. \u003cstrong\u003eA\u003c/strong\u003e GO enrichment based on GSEA. \u003cstrong\u003eB \u003c/strong\u003eKEGG enrichment based on GSEA. \u003cstrong\u003eC \u003c/strong\u003eProtein-Protein interaction network. \u003cstrong\u003eD \u003c/strong\u003eRepresentative photos of collagen fibers stained by Masson in the testis. \u003cstrong\u003eE\u003c/strong\u003e Level of TNF-α in the plasma from spermatic vein. \u003cstrong\u003eF\u003c/strong\u003e Level of IL-1β in the plasma from spermatic vein. \u003cstrong\u003eG\u003c/strong\u003eRepresentative photos of flow cytometry quantification of macrophages in the testis. \u003cstrong\u003eH\u003c/strong\u003e Percentage of CD68+CD163+ cells in CD45+ cells in the testis. \u003cstrong\u003eI \u003c/strong\u003ePercentage of CD68+ cells in CD45+ cells in the testis.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-7072366/v1/9cc019c59171d3248794bdd8.png"},{"id":86871783,"identity":"f5b24f8c-69b0-4b12-85ad-ecee28d9a04c","added_by":"auto","created_at":"2025-07-16 14:37:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2595505,"visible":true,"origin":"","legend":"\u003cp\u003eChange in TM profile and complement pathway under HS. \u003cstrong\u003eA\u003c/strong\u003e tSNE graph of identified cells. \u003cstrong\u003eB\u003c/strong\u003e Percentage of cells in each group. \u003cstrong\u003eC\u003c/strong\u003e KEGG enrichment based on the DEGs of TMs. \u003cstrong\u003eD\u003c/strong\u003e Expression of C1QA, C1QB and C1QC by testicular cells from scRNA-seq. \u003cstrong\u003eE\u003c/strong\u003e tSNE graph of TM sub-clusters in each group. \u003cstrong\u003eF\u003c/strong\u003e Percentage of sub-clusters in each group. \u003cstrong\u003eG\u003c/strong\u003e KEGG enrichment of maker genes of sub-cluster 5 \u003cstrong\u003eH\u003c/strong\u003e Expression of C1QA, C1QB and C1QC by TM sub-clusters from scRNA-seq. \u003cstrong\u003eI\u003c/strong\u003e KEGG enrichment of DEGs from TM sub-cluster 5. \u003cstrong\u003eJ\u003c/strong\u003e Addmodulescores of sub-clusters 5. \u003cstrong\u003eK\u003c/strong\u003etSNE graph of C1QA, C1QB, C1QC and CD163 from scRNA-seq.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-7072366/v1/ab7cb748449af977bbe76804.png"},{"id":86871778,"identity":"941c4326-b1a7-4764-adc7-608b21709d74","added_by":"auto","created_at":"2025-07-16 14:37:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4304657,"visible":true,"origin":"","legend":"\u003cp\u003eRelationship between complement activation and semen quality. \u003cstrong\u003eA \u003c/strong\u003eLevel of C1Q in the testis. \u003cstrong\u003eB \u003c/strong\u003eLevel of C3 in the testis. \u003cstrong\u003eC \u003c/strong\u003eLevel of C5b-9 in the testis. \u003cstrong\u003eD \u003c/strong\u003eGO enrichment of DEGs from RNA-seq.\u003cstrong\u003e E\u003c/strong\u003e Level of IgG in testis. \u003cstrong\u003eF \u003c/strong\u003eRepresentative photos of IgG immunofluorescence in testis. \u003cstrong\u003eG \u003c/strong\u003eLinear regression of C5b-9 and sperm motility.\u003cstrong\u003e H \u003c/strong\u003eLinear regression of C5b-9 and sperm progressive motility. \u003cstrong\u003eI\u003c/strong\u003e Linear regression of C5b-9 and sperm VSL.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-7072366/v1/724fcb43af2280a01040b673.png"},{"id":98814132,"identity":"70671a87-19af-4540-86c6-10673f55f2ea","added_by":"auto","created_at":"2025-12-22 16:11:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":18047401,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7072366/v1/d0a9f853-60dd-41f2-9455-7b5e9f31215b.pdf"},{"id":86871777,"identity":"f37f4df3-eb51-4c27-bbcf-48da10107b6c","added_by":"auto","created_at":"2025-07-16 14:37:30","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5406289,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental Fig.1 HS and testicular impairment.\u003cstrong\u003e A \u003c/strong\u003eRespiration rate under HS. \u003cstrong\u003eB \u003c/strong\u003eScrotal temperature under HS. \u003cstrong\u003eC\u003c/strong\u003e Scrotal temperature within 1-5h at 35℃. \u003cstrong\u003eD\u003c/strong\u003e Representative photos of Caspase-3 immunohistochemistry. \u003cstrong\u003eE\u003c/strong\u003e Level of 8-OHG in the plasma from spermatic vein blood. \u003cstrong\u003eF \u003c/strong\u003eLevel of testosterone in the plasma from spermatic vein blood.\u003c/p\u003e","description":"","filename":"SupplementalFig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-7072366/v1/9f50662e4326bb81a7430c8c.png"},{"id":86871788,"identity":"8100915d-5302-46ad-be54-6b5495d40413","added_by":"auto","created_at":"2025-07-16 14:37:30","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":387819,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental Fig.2 The gating of TMs.\u003c/p\u003e","description":"","filename":"SupplementalFig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-7072366/v1/8c5d3112d45b5f07179e7e36.png"},{"id":86871798,"identity":"662e3584-764c-453b-b8f4-8d418c4ec0f9","added_by":"auto","created_at":"2025-07-16 14:37:30","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2607325,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental Fig.3 Procedures and cell identification in scRNA-seq. \u003cstrong\u003eA\u003c/strong\u003e Graphic procedure of Single cell RNA-seq for CD45+ cells. \u003cstrong\u003eB \u003c/strong\u003eGating of flow cytometry sorting.\u003cstrong\u003e C\u003c/strong\u003etSNE graph of automatically defined clusters. \u003cstrong\u003eD\u003c/strong\u003e Makers of automatically defined clusters. \u003cstrong\u003eE\u003c/strong\u003e Cell markers of cell types.\u003c/p\u003e","description":"","filename":"SupplementalFig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-7072366/v1/1051e94b111322f2c3342d5c.png"},{"id":86871786,"identity":"2d335228-3acc-48ac-817e-be5788dcdaa4","added_by":"auto","created_at":"2025-07-16 14:37:30","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1108426,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental Fig.4 Change in testicular profile and relationship between CD163 and C1Q. \u003cstrong\u003eA \u003c/strong\u003eLog2 intensity of lyso-phosphatidylserine 18:1. \u003cstrong\u003eB\u003c/strong\u003e Log2 intensity of phosphatidylserine (18:1(9Z)/0:0). \u003cstrong\u003eC\u003c/strong\u003e Log2 intensity of lyso-phosphatidylserine 18:2. \u003cstrong\u003eD \u003c/strong\u003eKEGG enrichment of significantly altered metabolites. \u003cstrong\u003eE\u003c/strong\u003e Expression of C1QA, C1QB and C1QC in testis from RNA-seq. \u003cstrong\u003eF\u003c/strong\u003e KEGG enrichment of DEGs from RNA-seq. \u003cstrong\u003eG\u003c/strong\u003e Linear regression of C1QA and CD163. \u003cstrong\u003eH\u003c/strong\u003e Linear regression of C1QB and CD163. \u003cstrong\u003eI\u003c/strong\u003e Linear regression of C1QC and CD163. \u003cstrong\u003eJ\u003c/strong\u003e Linear regression of C1QA and CD68+CD163+cells. \u003cstrong\u003eK\u003c/strong\u003e Linear regression of C1QB and CD68+CD163+cells. \u003cstrong\u003eL\u003c/strong\u003eLinear regression of C1QC and CD68+CD163+cells.\u003c/p\u003e","description":"","filename":"SupplementalFig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-7072366/v1/586730e88fabc7b9e7ad81d0.png"},{"id":86872967,"identity":"a2260cc4-e1d5-46f3-b0e0-000d758298bc","added_by":"auto","created_at":"2025-07-16 14:45:30","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":368320,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental Fig.5 Linear regression of semen quality and complement members. \u003cstrong\u003eA\u003c/strong\u003e Linear regression of C1Q and sperm motility. \u003cstrong\u003eB \u003c/strong\u003eLinear regression of C1Q and sperm progressive motility. \u003cstrong\u003eC\u003c/strong\u003e Linear regression of C1Q and sperm VSL. \u003cstrong\u003eD\u003c/strong\u003eLinear regression of C1Q and sperm abnormality. \u003cstrong\u003eE\u003c/strong\u003e Linear regression of C3 and sperm motility. \u003cstrong\u003eF \u003c/strong\u003eLinear regression of C3 and sperm progressive motility. \u003cstrong\u003eG\u003c/strong\u003e Linear regression of C3 and sperm VSL. \u003cstrong\u003eH\u003c/strong\u003e Linear regression of C3 and sperm abnormality.\u003c/p\u003e","description":"","filename":"SupplementalFig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-7072366/v1/c1818370512bce2f6f937437.png"}],"financialInterests":"","formattedTitle":"Heat Stress Induced Testicular Impairment is Related to Orchitis and Complement Activation in Rongchang Boars","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBoars who are capable of impacting a large number of sows and corresponding offsprings due to the universal use of artificial insemination technique are fundamental to modern pig industry. However, during the global climate alternation, heat stress (HS) is becoming an unignorable threat to boars as the susceptibility of semen quality to excessive environmental temperature. It was early reported that only 72h under 33℃ and 50% humidity dampened the semen quality that would remain lame for the next 2 months[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Recent research of our colleagues also verified the continuous obstruction in sperm viability and promotion in sperm apoptosis rate after 2 weeks HS at 33℃[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. More importantly, Cameron and colleagues have proved semen from heat stressed boar would directly impact the reproductivity of sow by lowering the pregnant rate, normal embryo number and embryo immortal rate, which would directly dampen the output of piglets[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTestis as the only site for spermatogenesis is key to semen quality, in spite of the thermos-regulating mechanisms of the scrotum prompt the testis to appropriate temperature for spermatogenesis, the scrotum was also more likely to be heat stressed as it was located outside the body core[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Scrotal temperature disruption like HS, varicocele and cryptorchidism have been reported to severely dampen male fertility, yet their mechanism was not fully elucidated[\u003cspan additionalcitationids=\"CR6 CR7 CR8\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Testis is regarded as an immune-privileged organ to keep tolerance to self-antigens and spermatogenesis undisturbed[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Unfortunately, heat is naturally related to immune activity[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. When taking macrophage who is the most abundant testicular immune cells into consideration, heat could directly trigger expression of inflammatory cytokines and engulfment activity of macrophages[\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Within all the inflammatory cytokines originate from macrophages, it is recently noticed that complement C1Q induced complement cascade and its down-stream membrane attacking complex (C5b-9) that leads to membrane damage is responsible for testicular impairment, while early reports have already unveiled the connection between C1Q and HS in other models [\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFinally, we hypothesized that HS would facilitate testicular inflammation characterized by complement cascade, which directly dampened semen quality of boars. With assistance of omics technology, we devoted to clarify the effect of HS on the immunity of testis and its possible connection to HS induced reproductivity changes in Rongchang boars after 14 days at 33℃.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003ch2\u003eAnimals and Experimental Design\u003c/h2\u003e\n\u003cp\u003eAt the beginning, a total of 26 8-week-old male Rongchang pigs were selected and individually housed in stainless-steel metabolism cages at the room temperature 26℃\u0026plusmn;1 for adaption. Pigs were freely access to water and feed was supplied 4 times per day at 8.00, 12.00, 16.00 and 20.00. The feed was formulated referring to Nutrient requirement of swine, so it provides 3.15 Mcal/kg metabolizable energy and contains 15% of crude protein. After 3 weeks of adaption, 25 pigs at the age of 80 days old were randomly assigned to HS group or thermal neutral pair-feed (TN-PF/PF) group 1 week before the HS treatment, while 1 pig was excluded for cryptorchidism. The experiment was carried out as one way treatment design and randomized complete block experimental design with 1 pig as 1 experiment unit. Besides the ventilation system of the house, warm air blower and heat lamps were applied in controlling the room temperature at 35\u0026plusmn;1℃ for 14 days for the HS group, the room temperature of TN-PF group was maintained at 26℃\u0026plusmn;1 in the meantime.\u003c/p\u003e\n\u003ch2\u003eHS and temperature measurement\u003c/h2\u003e\n\u003cp\u003eRoom temperature and humidity were recorded by the auto-thermometer every hour (Renke, Jinan, China) during the whole experiment period while temperature-humidity index (THI) was calculated based on THI = (1.8 \u0026times; T + 32) - (0.55 - 0.55 \u0026times; RH \u0026times; 0.01) \u0026times; (1.8 \u0026times; T - 26). Scrotal temperature was measured by infrared thermometer (Hikvision, Hangzhou, China) and respiration rate was counted manually.\u003c/p\u003e\n\u003ch2\u003eSample collection\u003c/h2\u003e\n\u003cp\u003eAt the end of the experiment, pigs were anaesthetized by 2ml intramuscular injected Zoletil\u0026trade;50 (Virbac, Nice, France) before cutting along the ventral midline to expose the abdominal cavity, afterwards, blood from spermatic vein and portal vein was collected by vacuum blood collection tube with heparin sodium and disposable blood collection needle. The plasma was separated and stored at -20℃\u0026nbsp;after centrifuging at 3000g for 15 minutes,. The pigs were then slaughtered unconsciously before testis and epididymis were acquired. A part of the testis and intact epididymis was subsequently sent to laboratory within 5 minutes for further operation, while the rest of the was canned and quickly frozen by liquid nitrogen.\u003c/p\u003e\n\u003ch2\u003eSemen quality analysis\u003c/h2\u003e\n\u003cp\u003eAfter epididymis was transported to the laboratory, the cauda epididymis was separated and cut to small piece in a tube containing 10ml of semen extender. After 37℃ water bath for 15 minutes, supernatant was collected and 5-fold diluted before analyzing by computer assisted sperm analysis system (CASA) system (Minitube,\u0026nbsp;Tiefenbach, German). The supernatant was also used in preparation of morphology slides of sperms. Generally, 20ul of the supernatant was pipetted to the slide and averagely dispersed, the slide was next air dried and fixed by Immunol Staining Fix Solution (Beyotime, Shanghai, China) for 15 minutes. Then it was washed by ultrapure water and air dried before staining by Crystal Violet - Gentian Violet Stain Solution (Beyotime, Shanghai, China) for 15 minutes. Finally, the prepared slides were washed and dried again before packing up by sealing bags for later observation.\u003c/p\u003e\n\u003ch2\u003eFlow cytometry analysis\u003c/h2\u003e\n\u003cp\u003eTestis tissue was firstly sliced and incubated in HBSS (Solarbio, Beijing, China) containing 1mg/mL collagenase I (Sigma, Missouri, USA) and 100U/mL DNase I (Solarbio, Beijing, China) at 37℃ water bath for 15 minutes. 1mL fetal bovine serum (Clark, Virginia, USA) was added to the supernatant to stop digestion, the suspension was then filtered by 70 um cell strainer (Nest, Wuxi, China) and centrifuged at 4℃ 350g for 5 minutes. After removal of supernatant, cells were resuspended by DPBS (Gbico,\u0026nbsp;California, USA) containing fixable viability stain 780 (BD, New Jersey, USA) at 4℃ for 30 minutes. The suspension was subsequently centrifuged at 4℃ 350g for 5 minutes before removal of supernatant, the remaining cells were resuspended by 100ul stain buffer (BD, New Jersey, USA) containing mouse anti-pig CD45:FITC (Bio-rad, Virginia, USA) , mouse anti-pig CD163:RPE (Bio-rad, Virginia, USA) and BV421 rat anti-CD11b (BD, New Jersey, USA) at 4℃ for 30 minutes. The suspension was centrifuged at 4℃ 350g for 5 minutes to remove the supernatant. Afterwards, 250ul of cytofix (BD, New Jersey, USA) was used to fix the cells at 4℃ for 20 minutes. The suspension was then centrifuged at 4℃ 350g for 5 minutes before removal of supernatant, cells were later resuspended by 500ul of cytoperm (BD, New Jersey, USA) to permeate the cell membrane for 5 minutes. After centrifuged at 4℃ 350g for 5 minutes and disposal of cytoperm, 100ul of cytoperm with mouse anti-pig macrophages: Alexa Fluor 647(Bio-rad, Virginia, USA) was added to resuspended the cells at 4℃ for 40 minutes. Lastly, cell suspension was centrifuged at 4℃ 350g to remove the supernatant and washed once by 100ul stain buffer, 350ul of stain buffer was then used to resuspended the cells for analysis by BDverse flow cytometer（BD, New Jersey, USA）.\u003c/p\u003e\n\u003ch2\u003eELISA assays\u003c/h2\u003e\n\u003cp\u003eELISA assays were carried out according to the manufacturer\u0026rsquo;s instruction, including heat shock protein 60 (HSP60) (BIM, California, USA), heat shock protein 90 (HSP90) (BIM, California, USA), 8-hydroxydeoxyguanosine (8-OHG) (BIM, California, USA), Testosterone (BIM, California, USA),\u0026nbsp;interleukin-1\u0026beta; (IL-1\u0026beta;)\u0026nbsp;(BIM, California, USA),\u0026nbsp;tumor necrosis factor-\u0026alpha; (TNF-\u0026alpha;)\u0026nbsp;(R\u0026amp;D, Minnesota, USA), C1Q (BIM, California, USA), C3 (BIM, California, USA), C5b-9 (BIM, California, USA), IgG (BIM, California, USA).\u003c/p\u003e\n\u003ch2\u003eMasson sections\u003c/h2\u003e\n\u003cp\u003eMasson sections were prepared by Hubei BIOSSCI Biotech Co., Ltd. Tissue sections were immersed in clearer for 10 minutes. Repeat this step two times, gently shaking off excess liquid between each step. Tissue sections were immersed in progressively more dilute ethanol solutions. The dehydrated tissue sections were immersed in Bouin\u0026rsquo;s solution or Zenker\u0026rsquo;s solution overnight, then, rinsed with running water. Sections were stained with hematoxylin solution (Harris) or iron hematoxylin for 5-10 minutes and slightly washed with running water. Sections were differentiated with 0.8% - 1% hydrochloric acid alcohol and washed with running water for several minutes. Sections can also be treated with lithium carbonate solution to be bluer and washed with running water and ultimately immersed in distilled water to rehydrate the tissue: Absolute ethanol for 5min, 95% ethanol for 5min, 85% ethanol for 5min, 75% ethanol for 5min. Rinsing with distilled water for 1min. Sections were stained with ponceau acid fuchsin solution for 5-10 minutes and washed with running water. Sections were treated with phosphomolybidic acid solution for about 5 minutes and then stained with aniline blue solution for 5 minutes without washing. Sections were treated with 1% glacial acetic acid for 1 minute and dehydrated with 95% alcohol for several times. The tissue sections were dehydrated with absolute alcohol and transparent with xylene, then mounted with neutral balsam\u003c/p\u003e\n\u003ch2\u003eImmunohistochemistry section\u003c/h2\u003e\n\u003cp\u003eImmunohistochemistry sections were prepared by Hubei BIOSSCI Biotech Co., Ltd. In brief,\u0026nbsp;\u003c/p\u003e\n\u003cp\u003edeparaffinization and rehydration were carried out. Then EDTA antigen retrieval solution was then used for antigen retrieval. Afterwards, the section was immersed in 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to block the innate peroxidase. Then the section was blocked by 10% rabbit serum solution. The section was subsequently stained by Caspsae-3 (Cell Signaling Technology, Massachusetts, USA) antibody solution at 1:200. Finally, the immunohistochemistry section was stained by goat anti rabbit IgG-HRP secondary antibody (Abcam, Cambridge, UK) at 1:2000 before 3,3\u0026apos;-Diaminobenzidine (Maxim, Fuzhou, China) chromogenic reaction.\u003c/p\u003e\n\u003ch2\u003eImmunofluorescence section\u003c/h2\u003e\n\u003cp\u003eImmunofluorescence sections were prepared by Hubei BIOSSCI Biotech Co., Ltd.\u0026nbsp;In brief, deparaffinization and rehydration were carried out. Then EDTA antigen retrieval solution was then used for antigen retrieval. Afterwards, the section was blocked by 10% goat serum solution. The section was subsequently stained by goat anti-pig IgG antibody (Thermofisher, Massachusetts, USA) solution at 1:20.\u003c/p\u003e\n\u003ch2\u003eNon-targeted metabolomics\u003c/h2\u003e\n\u003cp\u003eNon-targeted metabolomics was performed by LC-Bio Technology CO. The collected spermatic vein plasmas were thawed on ice, and metabolite were extracted with 80% methanol Buffer. Briefly, 100 \u0026mu;l of sample was extracted with 400 \u0026mu;l of precooled methanol. The extraction mixture was then stored in 30 min at -20\u0026deg;C. After centrifugation at 20,000 g for 15 min, the supernatants were transferred into new tube and vacuum dried. The samples were then redissolved with 100\u0026mu;L 80% methanol and stored at -80\u0026deg;C prior to the Liquid Chromatograph Mass Spectrometer analysis.\u003c/p\u003e\n\u003ch2\u003eTranscriptomics\u0026nbsp;(RNA-seq)\u003c/h2\u003e\n\u003cp\u003eRNA-seq\u0026nbsp;was performed by LC-Bio Technology CO. In brief, total RNA of testis was isolated and purified using TRIzol reagent (Invitrogen, California, USA) following the manufacturer\u0026apos;s procedure before it was reverse-transcribed to cDNA by SuperScript\u0026trade; II Reverse Transcriptase (Invitrogen, California, USA), the cDNA was next processed and sequenced on an illumina Novaseq\u0026trade; 6000 (LC-Bio Technology CO., Ltd., Hangzhou, China) following the vendor\u0026apos;s recommended protocol.\u003c/p\u003e\n\u003cp\u003eIn data analysis of RNA-seq, we used HISAT2 (https://ccb.jhu.edu/software/hisat2) to map reads to the reference genome of Sus_scrofa.Sscrofa11.1.(http://ftp.ensembl.org/pub/release-107/fasta/sus_scrofa/dna/). The mapped reads of each sample were assembled using StringTie (https://ccb.jhu.edu/software/stringtie) with default parameters. StringTie was subsequently used to perform expression level for mRNAs by calculating FPKM (FPKM = [total_exon_fragments / mapped_reads(millions) \u0026times; exon_length(kB)]). The differentially expressed mRNAs were selected with fold change \u0026gt; 2 or fold change \u0026lt; 0.5 and with parametric F-test comparing nested linear models (p value \u0026lt; 0.05) by R package edgeR (https://bioconductor.org/packages/release/bioc/html/edgeR.html). Afterwards, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment were performed based on the differentially expressed genes (DEGs).\u003c/p\u003e\n\u003ch2\u003eSingle cell transcriptomics (scRNA-seq)\u003c/h2\u003e\n\u003cp\u003escRNA-seq was performed by LC-Bio Technology CO. Firstly, testis tissue was lysed to get cell suspension, then the mouse anti-pig CD45: FITC (Bio-rad, Virginia, USA) and FACS sorting were applied in preparing testicular CD45+ cells who were used in later generation of nanoliter-scale Gel Bead-In-EMulsions (GEMs) (supplemental Fig.3A). After releasing of mRNA from cells in GEMs, cDNA was produced by reverse transcription and was later amplified by PCR to construct single-cell libraries. Afterwards, libraries were sequenced on NextSeq 500 sequencing system. In data analysis of scRNA-seq, quantification and quality control were finished using Cell Ranger to produce post-QC data. Then R package Seurat was applied in filtering abnormal data, preliminary clustering and visualization. Afterwards, cells were identified by repeatedly comparing the cell makers of clusters on Cell Taxonomy database (https://ngdc.cncb.ac.cn/celltaxonomy/) before labeling them. By bimod P\u0026lt;0.01 and log2FC\u0026gt;=0.26, DEGs were revealed between groups and pathway enrichment were performed based on the DEGs. Re-clustering was carried out based on the identified clusters, DEGs identification and KEGG enrichment were also performed.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eConventional statistical analysis\u003c/h2\u003e\n\u003cp\u003eData was analysis with procedure mixed by SAS (9.4), using the following model:\u003c/p\u003e\n\u003cp\u003eY=mean + treatment + block + error\u003c/p\u003e\n\u003cp\u003eWhere mean=overall means, treatment=treatment effect, block=random effect=maternal origin, error=residual. Outlier would be identified if its standard residual (residual/standard deviation) is larger than 2.5. Normality and homogeneity of variances were evaluated by Shapiro-wilk test and Levene test, respectively, data would be transformed if it did not meet normal distribution and unequal variance analysis by procedure Mixed would be applied if variances were significantly unequal. Repeated measured data would be analyzed using repeated measure by procedure Mixed. Grouping letters were assigned by SAS Macro program. All data was presented as mean\u0026plusmn;standard error mean. P\u0026lt;0.05 was regarded as significance and 0.05\u0026lt;P\u0026lt;0.10 was regarded as a tendency. \u0026nbsp;In addition, linear regression and correlation analysis and was performed by OmicStudio tools at https://www.omicstudio.cn/tool.\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003eHeat stress and semen quality of boars\u003c/h2\u003e\n\u003cp\u003eThe curves of room temperature and the THI were presented in Fig.1A, the THI was remained above 86 and the room temperature was kept at 35\u0026plusmn;1℃ during HS period. There are typical heat-stress reactions in boars as for not only their respiration rate (RR) tremendously elevated and but the scrotal temperature was also promoted (Supplemental Fig. 1A-C). Aside from that, plasma heat shock protein 60 (HSP60) and heat shock protein 90 (HSP90) from spermatic vein of heat stressed boars were significantly promoted comparing to the TN-PF group (Fig. 1B and C). Therefore, HS reaction is confirmed both in the boars and their scrotums.\u003c/p\u003e\n\u003cp\u003eObviously, HS dampened the semen quality of boars. There were tendencies to reduce percentage of motile sperms and progressive sperms under HS (Fig. 1D and E). In addition, there was significant reduction in average straight-line velocity (VSL) (Fig. 2F). Moreover, tendency to promote sperm head abnormality as well as significant elevation in tail abnormality and total abnormality were also observed (Fig.2H-J). Together, our finding depicted dampened porcine semen quality under HS.\u003c/p\u003e\n\u003ch2\u003eHS facilitated orchitis and testicular impairment\u003c/h2\u003e\n\u003cp\u003eResults of RNA-seq hinted at changes in testicular immunity, such as MAPK pathway, T cell receptor pathway, TNF-\u0026alpha; and hematopoietic cell lineage in KEGG and GO enrichment based on Gene Set Enrichment analysis (GSEA) (Fig.2A and B). Along with that, protein-protein interaction network analysis uncovered\u0026zwnj;\u0026zwnj; the key role of immunity in testicular changes as genes associated to macrophage and T cell were regarded as key nodes with high degree level in the network (Fig.2C). The results above remined us of orchitis characterized by testicular fibrosis, accumulation of inflammatory cytokines and invasion of immune cells. Coincidentally, not only Masson sections exhibited enhanced fibrosis but elevation in TNF-\u0026alpha; and tendency to promote IL-1\u0026beta; in spermatic vein plasma were also recorded (Fig.2D-F). Promotion in CD68+ cells and tendency to elevate CD68+CD163+ cells observed by flow cytometry also supported invasion of immune cells (Fig.2G-H, Supplemental Fig.2). Finally, testicular tissue impairment was confirmed by upregulation in 8-OHG concentration, lowered testosterone concentration and Caspase-3 expression in seminiferous tubules (Supplemental Fig.1D-F). All in all, our results above verified the appearance of testicular impairment and orchitis under HS.\u003c/p\u003e\n\u003ch2\u003eChanges in complement and engulfment activity in testicular macrophage (TM) under HS\u003c/h2\u003e\n\u003cp\u003eFor further insight into testicular immunity, flow cytometry sorting was next applied to purify the testicular CD45+ immune cells for scRNA-seq as shown in supplemental Fig.3A, while gating was shown in supplemental Fig.3B. A total of 29 clusters of cells with their makers were observed in scRNA-seq (supplemental Fig.3C and D). Most of clusters were then identified based on the cell makers from online databases (Fig.3A and supplemental Fig.3E). As expected, macrophage took a big proportion in the testicular immune cells and their DEGs were enriched in cell death, engulfing and inflammatory pathways (Fig.3B and C). Although we are not capable of fully verifying cell death and engulfing activity, metabolomics demonstrated upregulation of lyso-phosphatidylserine 18:1 (LPS 18:1), phosphatidylserine (18:1(9Z)/0:0) (PS (18:1(9Z)/0:0)) and lyso-phosphatidylserine 18:2 (LPS 18:2) who were associated to cell death and engulfment signal, supporting the perspective HS activated engulfment signal in TMs (Supplemental Fig.4A-D).\u003c/p\u003e\n\u003cp\u003eWithin all the enriched inflammatory pathways, complement and coagulation cascades were drawing attention as complement activation has recently been reported to be responsible for poor male fertility under viral stress[17]. Significant elevation in expression of C1QA, C1QB and C1QC both in testis and TMs was confirmed in both RNA-seq and scRNA-seq (Fig.3D, Supplemental Fig.4E and F). It was also exhibited C1Q genes were almost only expressed by TMs (Fig.3D). Re-clustering of TMs identified 16 sub-clusters, which depicted the onset of dramatic shifts in TM subclusters during HS (Fig. 3E and F). In the meantime, re-clustering pointed out the exact sub-cluster responsible for C1Q expression, as KEGG enrichment of sub-cluster 5 maker genes noted complement cascades and expression dotplot showed that sub-cluster 5 TMs were mainly responsible for C1Q expression, which was also accelerated by HS (Fig3.G-I).\u003c/p\u003e\n\u003cp\u003eIn previous report, C1Q activation was related to CD163+macrophage infiltration in kidney[20]. We next applied Addmodulescore to identify sub-cluster 5, it was noticed that sub-cluster 5 expressed high level of M2 macrophage genes by a M2 macrophage gene set from Mossadegh-Keller et al. with a little modification[21] (Fig.3J). In agreement with Addmodulescore, tSNE graph of C1QA, C1QB, C1Qc and CD163 represented a large overlapping area (Fig.3K). In addition, significant positive linear regression effects were observed between C1Q genes and CD163 gene or CD68+ CD163+ cells, corresponding to the results of TM composition by flow cytometry (Supplemental Fig.4G-L). Collectively, those results indicated the onset of TM engulfment activity and potential in CD163+ TM related complement activation.\u003c/p\u003e\n\u003ch2\u003eComplement cascades is associated to obstruction in semen quality.\u003c/h2\u003e\n\u003cp\u003eC1Q is the key component for classical activation of complement cascades leading to final assemble of C5b-9 who specialized in cell membrane breaking and cell lysis with C3 as key signal transduction component[22]. Unsurprisingly, C1Q, C3 and C5b-9 protein were all significantly facilitated in the testis of heat stressed boar (Fig.4A-C). GO enrichment analysis of RNA-seq repeatedly emphasized membrane changes, echoing a key role of complements in testicular changes under HS (Fig.4D). As C1Q is key to classical complement activation whose up-stream activator is antigen-antibody complex, we visualized IgG distribution in the testis[17]. As shown in Fig.4F and E, HS prompted IgG secretion, making a proper cause for classical complement activation. Most importantly, linear regression analysis revealed notable or near-significant linear correlations between the levels of C5b-9 and semen quality (Fig.4G-I). Analogically, C3 and C1Q also exhibit linear associations to semen quality (Supplemental Fig.5A-H). Conclusively, our finding demonstrated negative association between complement activation and semen quality, emphasizing the role of complement in HS related dampened semen quality.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHS has become a tremendous threat with the global warming, it is believed pigs would be heat stressed at 79 THI and the HS would be severe at 84 THI[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. We therefore kept the room temperature above 35 and THI above 85 to ensure HS reactions of pigs during HS period. Due to the absence of swell gland, RR of pigs is susceptible to HS[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In our experiment, elevation in RR was observed, which could be regarded as the hallmark of HS reaction. Data have shown obvious elevation in scrotal temperature of mice and pig under HS, indicating the possibility of direct impact of HS on scrotum[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Although scrotal temperature is not numerically strongly altered by HS in our experiment, it was statistically significantly promoted in analysis with environment temperature as main effect. We also observed higher level of HSP60 and HSP90 in the spermatic vein plasma as biological confirmation of scrotal HS reactions. Studies have already unveiled the detrimental impact of scrotal HS on male fertility, emphasizing the importance of scrotal temperature maintaining in spermatogenesis[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The semen quality in our HS group was similarly dampened by not only lowering velocity and motility but promoting abnormality as well, which could also be regarded as the phenotype of boars being heat stressed like it was previously reported[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In agreement with these, high room temperature lowers the velocity and motility of mice after 38℃ for 2 hours in 14 days[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Yaeram and colleagues also revealed obstructed inseminating ability of sperms from mice who were heat stressed at 36℃ for 12 hour per day[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. All those data highlighted the detrimental effect of HS and elevated scrotal temperature on male reproductivity.\u003c/p\u003e\u003cp\u003eTo the best of our knowledge, there was only a little information about the connection between HS and orchitis[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Although it was demonstrated that macrophages as the major testicular immune cells were sensitive to hyperthermia characterized by elevated pro-inflammatory and engulfing activity though NF-kB pathway, TMs were also reported to be less pro-inflammatory for its poor capacity in activating NF-kB pathway comparing to peritoneal macrophage [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. However, In human model, scrotal HS not only obstructed reproductivity but promoted the level of macrophage migration inhibitory factor who is responsible for macrophage recruitment and inflammation as well, corresponding to the elevation in TM number we observed[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Koch and colleagues also discovered HS induced infiltration of macrophages with active inflammatory and phagocytosis response into the mucosa and submucosa of the jejunum in bovine model[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Similar to their results, our data have elucidated accumulation of inflammatory cytokines with changes in inflammatory and engulfing pathway in testis, supporting the appearance of HS induced orchitis.\u003c/p\u003e\u003cp\u003eThe orchitis related testicular impairment could be exerted to Leydig cells for TNF-α could serve as an histone deacetylase 7 activator, in doing so, inhibition of the testosterone secretion took place, which in return blunt the immunosuppressive environment of the testis[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. When taking engulfing activity into consideration, TMs were capable of engulfing early apoptotic Leydig cells characterized by exposure of phosphatidylserine, corresponding to promotion in phosphatidylserine analogues in our metabolomics. Thus, we also suspected there was engulfing of apoptotic Leydig cells under HS, giving a rise to reduction of testosterone level [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In addition, both HS and inflammation would impair the integrity of blood-testis barrier that is regarded as the guardian of spermatogenetic environment and isolation of spermatogenetic antigens, resulting in possibility of exposing the spermatogenetic antigens and inflammation acceleration[\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eComplement cascade was rarely focused in all kinds of immune responses, yet it was recently noted it was related to viral stress induced obstruction in male reproductivity[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Our result documented not only accumulation in complement components, but also elevated level of testicular IgG along with heat shock proteins as identified self-antigens, which is necessary to C1Q related classical activation of complement cascade, leading to the possibility of antigen-antibody complex construction [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Despite there is little solid evidence about the relationship between HS and C1Q, C1Q was often mentioned in anoxic and ischemic models [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Coincidentally, HS is associated to redistribution of blood flow that would result in organic ischemic anoxia to facilitate heat ejection [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Moreover, it was previously illustrated C1 inhibitor is capable of alleviating empyrosis induced tissue impairment[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The information above illustrated the potential in connecting HS to complement reaction and its consequences, our data verified the onset of C1Q-ralated complement reaction in testis under HS, yet its impact on semen was only speculated by linear regression analysis.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eCollectively, present results support the opinion boar suffered HS induced orchitis, in which TMs underwent dramatic alternations in engulfment and inflammatory activity. Of all those inflammatory pathways, our attention was caught by C1Q promotion by CD163\u0026thinsp;+\u0026thinsp;TMs and its down-stream complement cascade that was associated to poor semen quality in linear regression.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTHI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTemperature humidity index\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHeat stress\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTN-PF/PF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eThermal neutral pair-feed\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eRespiration rate\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHSP60\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHeat shock protein 60\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHSP90\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHeat shock protein 90\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCASA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eComputer assisted sperm analysis system\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eVSL\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAverage straight-line velocity\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eKEGG\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eKyoto Encyclopedia of Genes and Genomes\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGO\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGene Ontology\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGSEA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGene Set Enrichment analysis\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGEMs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGel Bead-In-Emulsions\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTesticular macrophage\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e8-OHG\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e8-hydroxydeoxyguanosine\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTNF-α\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTumor necrosis factor-α\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIL-1β\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eInterleukin-1β\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDEGs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDifferentially expressed genes\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLPS 18\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e1:Lyso-phosphatidylserine 18:1\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePS (18\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e1(9Z)/0:0):Phosphatidylserine (18:1(9Z)/0:0)\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLPS 18\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e2:Lyso-phosphatidylserine 18:2\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Projects of The National Natural Science Foundation of China (U21A20255),\u0026nbsp;National Modern Agricultural Industry Technology System Sichuan Pig innovation team (SCSZTD-2024-08) and Sichuan Province \u0026ldquo;14th Five-Year\u0026rdquo; Sichuan Pig Major Science and Technology Project (2021ZDZX0009).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors consent to this publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperimental execution: W.S., J.N and X.M.; data analysis and preservation: L.C., B.F. and L.H; manuscript preparation and revision: Y.Z., Z.F., S.X. and J.L.; supervision: X.J., Y.L. and D.W.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw data of transcriptomics is available at NCBI (Accession: PRJNA1236216), and the raw data of single cell transcriptomics is available at NCBI (Accession: PRJNA1237295), any additional information will be available from the De Wu ([email protected]) upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal protocol for this experiment was approved by the Animal Care and Use Committee of Sichuan Agricultural University.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMcNitt JI, First NL. Effects of 72-hour heat stress on semen quality in boars. International journal of biometeorology. 1970;14(4):373-80 http://doi.org/10.1007/bf01462914.\u003c/li\u003e\n\u003cli\u003eYu C, Li H, Hua L, Che L, Feng B, Fang Z, et al. Deciphering the microbiome, lipopolysaccharides, and metabolome interplay: Unveiling putrescine\u0026apos;s mechanism for enhancing sperm quality in heat-stressed boars. Theriogenology. 2025;236:60-73 http://doi.org/10.1016/j.theriogenology.2025.01.027.\u003c/li\u003e\n\u003cli\u003eCameron RD, Blackshaw AW. The effect of elevated ambient temperature on spermatogenesis in the boar. Journal of reproduction and fertility. 1980;59(1):173-9 http://doi.org/10.1530/jrf.0.0590173.\u003c/li\u003e\n\u003cli\u003eWaites GM. Thermoregulation of the scrotum and testis: studies in animals and significance for man. Adv Exp Med Biol. 1991;286:9-17 http://doi.org/10.1007/978-1-4684-5913-5_2.\u003c/li\u003e\n\u003cli\u003eLue YH, Hikim AP, Swerdloff RS, Im P, Taing KS, Bui T, et al. Single exposure to heat induces stage-specific germ cell apoptosis in rats: role of intratesticular testosterone on stage specificity. Endocrinology. 1999;140(4):1709-17 http://doi.org/10.1210/endo.140.4.6629.\u003c/li\u003e\n\u003cli\u003eJensen CFS, \u0026Oslash;stergren P, Dupree JM, Ohl DA, S\u0026oslash;nksen J, Fode M. Varicocele and male infertility. Nat Rev Urol. 2017;14(9):523-33 http://doi.org/10.1038/nrurol.2017.98.\u003c/li\u003e\n\u003cli\u003eZhang MH, Zhai LP, Fang ZY, Li AN, Xiao W, Qiu Y. Effect of scrotal heating on sperm quality, seminal biochemical substances, and reproductive hormones in human fertile men. 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Testis serves as an immune-privileged organ to keep its spermatogenesis undisturbed, while immunity is thermo-sensitive, especially for macrophages who present as the most abundant testicular immune cell. Our study aims to deeply unveil the underlying immune responses and assess its consequences to semen quality under heat stress, seeking for understanding and solutions to obstruction in boar reproductivity under heat stress.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe conducted the experiment using 26 8-week-old Rongchang male pigs who were assigned into thermal neutral pair-feed group and heat stress group for a 3-week experiment with the last two weeks as heat stress period. During heat stress period, pigs of heat stress group were subjected to 14-day 35\u0026thinsp;\u0026plusmn;\u0026thinsp;1℃ heat stress, while pigs of thermal neutral pair-feed group were kept at 26\u0026thinsp;\u0026plusmn;\u0026thinsp;1℃. Pigs were sampled at the end of heat stress period to obtain gonads tissue for assessing and measurement.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOur findings confirmed heat stress reactions by elevated respiration rate (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and expression of heat shock proteins 60 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and heat shock proteins 90 \u003cem\u003e(P\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Tendencies to reduce sperm motility (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.06) and progressive sperms (P\u0026thinsp;=\u0026thinsp;0.08) under HS were observed as well as significant reduction in average straight-line velocity (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and total abnormality were also recorded (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Visualized fibrosis, caspase-3 expression and accumulation of tumor necrosis factor-α (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and Interleukin-1β (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) along with elevated macrophage composition (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) characterized the orchitis under heat stress. Accordingly, single cell RNA sequencing revealed fluctuation in engulfing and inflammatory signals in testicular macrophages, especially promotion in complement cascades by CD163\u0026thinsp;+\u0026thinsp;macropahegs, resulting in final membrane attack complex assembling (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Linear regression further exhibited a negative correlation between the membrane attack complex and sperm motility (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) a long with near-negative correlation between the membrane attack complex and progressive motility (P\u0026thinsp;=\u0026thinsp;0.08) or velocity straight line (P\u0026thinsp;=\u0026thinsp;0.06).\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOur findings highlighted the relationship between heat stress, the onset of orchitis, and the activation of the complement system, all of which caused obstruction in semen quality of boars.\u003c/p\u003e","manuscriptTitle":"Heat Stress Induced Testicular Impairment is Related to Orchitis and Complement Activation in Rongchang Boars","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-16 14:37:25","doi":"10.21203/rs.3.rs-7072366/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2025-08-26T06:45:25+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-07-30T08:20:47+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-14T01:13:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-09T04:05:29+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Animal Science and Biotechnology","date":"2025-07-08T04:20:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-animal-science-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jasb","sideBox":"Learn more about [Journal of Animal Science and Biotechnology](http://jasbsci.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jasb/default.aspx","title":"Journal of Animal Science and Biotechnology","twitterHandle":"@animalplantsci","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3f7d7630-ec3b-4323-8461-21482a0e5c17","owner":[],"postedDate":"July 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-22T16:05:48+00:00","versionOfRecord":{"articleIdentity":"rs-7072366","link":"https://doi.org/10.1186/s40104-025-01296-5","journal":{"identity":"journal-of-animal-science-and-biotechnology","isVorOnly":false,"title":"Journal of Animal Science and Biotechnology"},"publishedOn":"2025-12-17 15:57:59","publishedOnDateReadable":"December 17th, 2025"},"versionCreatedAt":"2025-07-16 14:37:25","video":"","vorDoi":"10.1186/s40104-025-01296-5","vorDoiUrl":"https://doi.org/10.1186/s40104-025-01296-5","workflowStages":[]},"version":"v1","identity":"rs-7072366","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7072366","identity":"rs-7072366","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}