Abstract

Background

Heat stress (HS) can impair boar testicular function, leading to reproductive issues. However, chlorogenic acid (CGA) has been shown to mitigate HS-induced damage in various livestock and poultry species. Prepuberty is an important stage of testicular development in boars after birth. However, the protective effect of CGA on testicular HS injury during prepuberty boars and the underlying mechanisms are still not fully understood.

Results

In vivo, a total of 30 healthy boars with similar body weights and ages were obtained and randomly divided into 3 groups, which were fed a basal diet supplemented with CGA 0 (the ND_TN group), 0 (the ND_HS group) or 1,000 (the CGA_HS group) mg/kg. After being fed for 28 d, all the groups, except the ND_TN group, were treated with high temperature for 7 d, after which samples were collected from the boars and analysed. The results showed that CGA significantly mitigated the HS-induced reduction in T-AOC content in testicular tissue and sperm density. Mechanistically, multiomics analysis revealed that the genes differentially expressed by CGA and HS were predominantly associated with the glutathione metabolism pathway. The combined analysis of transcriptomics and proteomics revealed that onlyBLVRAwas affected by both HS and CGA when the mRNA and protein levels of a gene showed differential expression with the same trend. In vitro studies confirmed that CGA modulated GPX3 expression viaBLVRA, affected GPx activity, and attenuated HS-induced ROS accumulation.

Conclusions

In conclusion, prepubertal HS impairs the spermatogenic capacity of boars.BLVRAmay mediate the testicular protective effect of CGA, although in vivo validation of this pathway is needed. This study contributes to elucidating the mechanisms underlying the effects of HS on prepubertal boar testicular development using multiomics approaches, laying a foundation for the potential utilization of CGA in swine production.

Data Availability

The raw RNA-seq data generated in this study have been deposited in the NCBI Sequence Read Archive (SRA, https://www.ncbi.nlm.nih.gov/sra ) under the accession number PRJNA1246667. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the iProX partner repository (https://proteomecentral.proteomexchange.org) under the accession number PXD062793. The 1 H-NMR spectroscopy data have been deposited in the OMIX, China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences ( https://ngdc.cncb.ac.cn/omix ) under the accession number OMIX012729. Apart from the datasets included in the article/supplementary materials, all other data generated in this study are available from the corresponding author upon request.

Abbreviations

  • 1H-NMR:: One-dimensional nuclear magnetic resonance hydrogen spectrum
  • CGA:: Chlorogenic acid
  • GO:: Gene Ontology
  • HS:: Heat stress
  • KEGG:: Kyoto Encyclopedia of Genes and Genomes
  • ND:: Normal diet
  • ROS:: Reactive oxygen species
  • T-AOC:: Total antioxidant capacity
  • TMT:: Tandem mass tag
  • TN:: Thermoneutral

References

  1. 1.Matthews HD, Wynes S. Current global efforts are insufficient to limit warming to 1.5 degrees C. Science. 2022;376(6600).(2022)1126/science.abo3378.: 1404.
    10.1126/science.abo3378
    Article|CAS|PubMed|Google Scholar
  2. 2.Kim B, Park K, Rhee K. Heat stress response of male germ cells. Cell Mol Life Sci. 2013;70(15).(2013)org/10.1007/s00018-012-1165-4.: 2623.
    10.1007/s00018-012-1165-4
    Article|CAS|PubMed|Google Scholar
  3. 3.Hedia MG, El-Belely MS, Ismail ST, Abo El-Maaty AM. Seasonal variation in testicular blood flow dynamics and their relation to systemic and testicular oxidant/antioxidant biomarkers and androgens in rams. Reprod Domest Anim. 2020;55(7).(2020)1111/rda.13696.: 861.
    10.1111/rda.13696
    Article|CAS|PubMed|Google Scholar
  4. 4.Mete F, Kilic E, Somay A, Yilmaz B. Effects of heat stress on endocrine functions & behaviour in the pre-pubertal rat. Indian J Med Res. 2012;135(2).(2012)233–9.: 233.
    Article|CAS|PubMed|Google Scholar
  5. 5.Sabes-Alsina M, Lundeheim N, Johannisson A, Lopez-Bejar M, Morrell JM. Relationships between climate and sperm quality in dairy bull semen.(2019)a retrospective analysis.J Dairy Sci.: 5623.
    10.3168/jds.2018-15837
    Article|CAS|PubMed|Google Scholar
  6. 6.Lugar DW, Proctor JA, Safranski TJ, Lucy MC, Stewart KR. In utero heat stress causes reduced testicular area at puberty, reduced total sperm production, and increased sperm abnormalities in boars. Anim Reprod Sci. 2018;192.(2018)126–35. https://doi. org/10. 1016/j.anireprosci.: 126.
    10.1016/j.anireprosci.2018.02.022
    Article|CAS|PubMed|Google Scholar
  7. 7.Flowers WL. Factors affecting the production of quality ejaculates from boars. Anim Reprod Sci. 2022;246.(2022)2021.106840.: 106840.
    10.1016/j.anireprosci.2021.106840
    Article|CAS|PubMed|Google Scholar
  8. 8.Shakeel M, Yoon M. Changes in characteristics of spermatogonial stem cells in response to heat stress in stallions. Theriogenology. 2024;224.(2024)74–81. https://doi. org/10. 1016/j.theriogenology.: 74.
    10.1016/j.theriogenology.2024.05.007
    Article|CAS|PubMed|Google Scholar
  9. 9.Raoofi A, Omraninava M, Javan R, Maghsodi D, Rustamzadeh A, Nasiry D, et al. Protective effects of epigallocatechin gallate in the mice induced by chronic scrotal hyperthermia. Tissue Cell. 2023;84.(2023)102165. https://doi. org/10. 1016/j.tice.: 102165.
    10.1016/j.tice.2023.102165
    Article|CAS|PubMed|Google Scholar
  10. 10.Ko SH. Effects of heat stress-induced sex hormone dysregulation on reproduction and growth in male adolescents and beneficial foods. Nutrients. 2024;16(17).(2024)org/10.3390/nu16173032.: 3032.
    Article|CAS|PubMed|Google Scholar
  11. 11.Voigt AL, Dardari R, Lara NLM, He T, Steele H, Dufour A, et al. Multiomics approach to profiling Sertoli cell maturation during development of the spermatogonial stem cell niche. Mol Hum Reprod. 2023;29(3).(2023)org/10.1093/molehr/gaad004.
    Article|CAS|PubMed|Google Scholar
  12. 12.Zheng Y, Gao Q, Li T, Liu R, Cheng Z, Guo M, et al. Sertoli cell and spermatogonial development in pigs. J Anim Sci Biotechnol. 2022;13.(2022)org/10.1186/s40104-022-00687-2.: 45.
    10.1186/s40104-022-00687-2
    Article|CAS|PubMed|Google Scholar
  13. 13.Hu H, Dai S, Li J, Wen A, Bai X. Glutamine improves heat stress-induced oxidative damage in the broiler thigh muscle by activating the nuclear factor erythroid 2-related 2/Kelch-like ECH-associated protein 1 signaling pathway. Poult Sci. 2020;99(3).(2020)11.001.: 1454.
    10.1016/j.psj.2019.11.001
    Article|CAS|PubMed|Google Scholar
  14. 14.Zhang P, Zheng Y, Lv Y, Li F, Su L, Qin Y, et al. Melatonin protects the mouse testis against heat-induced damage. Mol Hum Reprod. 2020;26(2).(2020)org/10.1093/molehr/gaaa002.: 65.
    10.1093/molehr/gaaa002
    Article|CAS|PubMed|Google Scholar
  15. 15.Hu Y, Luo NJ, Gan L, Xue HY, Luo KY, Zhang JJ, et al. Heat stress upregulates arachidonic acid to trigger autophagy in Sertoli cells via dysfunctional mitochondrial respiratory chain function. J Transl Med. 2024;22(1).(2024)org/10.1186/s12967-024-05182-y.: 501.
    10.1186/s12967-024-05182-y
    Article|CAS|PubMed|Google Scholar
  16. 16.Shahat AM, Rizzoto G, Kastelic JP. Amelioration of heat stress-induced damage to testes and sperm quality. Theriogenology. 2020;158.(2020)84–96. https://doi. org/10. 1016/j.theriogenology.: 84.
    10.1016/j.theriogenology.2020.08.034
    Article|CAS|PubMed|Google Scholar
  17. 17.Wang S, Li Y, Meng X, Chen S, Huang D, Xia Y, et al. Antioxidant activities of chlorogenic acid derivatives with different acyl donor chain lengths and their stabilities during in vitro simulated gastrointestinal digestion. Food Chem. 2021;357.(2021)129904. https://doi. org/10. 1016/j.foodchem.: 129904.
    10.1016/j.foodchem.2021.129904
    Article|CAS|PubMed|Google Scholar
  18. 18.Zhang S, Sun B, Wang D, Liu Y, Li J, Qi J, et al. Chlorogenic acid ameliorates damage induced by Fluorene-9-Bisphenol in porcine Sertoli cells. Front Pharmacol. 2021;12.(2021)678772. https://doi. org/10.3389/fphar.: 678772.
    10.3389/fphar.2021.678772
    Article|CAS|PubMed|Google Scholar
  19. 19.Hu R, Yang X, Wang L, Su D, He Z, Li J, et al. Gut microbiota dysbiosis and oxidative damage in high-fat diet-induced impairment of spermatogenesis.(2024)role of protocatechuic acid intervention.Food Frontiers.: 2566.
    10.1002/fft2.484
    Article|CAS|PubMed|Google Scholar
  20. 20.Hu R, Yang X, Gong J, Lv J, Yuan X, Shi M, et al. Patterns of alteration in boar semen quality from 9 to 37 months old and improvement by protocatechuic acid. J Anim Sci Biotechnol. 2024;15.(2024)org/10.1186/s40104-024-01031-6.: 78.
    10.1186/s40104-024-01031-6
    Article|CAS|PubMed|Google Scholar
  21. 21.Zhang SX, Wang DL, Qi JJ, Yang YW, Sun H, Sun BX, et al. Chlorogenic acid ameliorates the heat stress-induced impairment of porcine Sertoli cells by suppressing oxidative stress and apoptosis. Theriogenology. 2024;214.(2024)10.018.: 148.
    10.1016/j.theriogenology.2023.10.018
    Article|CAS|PubMed|Google Scholar
  22. 22.Kim H, Pan JH, Kim SH, Lee JH, Park JW. Chlorogenic acid ameliorates alcohol-induced liver injuries through scavenging reactive oxygen species. Biochimie. 2018;150.(2018)131–8. https://doi. org/10. 1016/j.biochi.: 131.
    10.1016/j.biochi.2018.05.008
    Article|CAS|PubMed|Google Scholar
  23. 23.Zhou Y, Zhou L, Ruan Z, Mi S, Jiang M, Li X, et al. Chlorogenic acid ameliorates intestinal mitochondrial injury by increasing antioxidant effects and activity of respiratory complexes. Biosci Biotechnol Biochem. 2016;80(5).(2016)2015.1127130.: 962.
    10.1080/09168451.2015.1127130
    Article|CAS|PubMed|Google Scholar
  24. 24.Li H, Zhao J, Deng W, Li K, Liu H. Effects of chlorogenic acid-enriched extract fromEucommia ulmoidesOliver leaf on growth performance and quality and oxidative status of meat in finishing pigs fed diets containing fresh or oxidized corn oil. J Anim Physiol Anim Nutr (Berl). 2020;104(4).(2020)1111/jpn.13267.: 1116.
    10.1111/jpn.13267
    Article|CAS|PubMed|Google Scholar
  25. 25.Chen J, Li Y, Yu B, Chen D, Mao X, Zheng P, et al. Dietary chlorogenic acid improves growth performance of weaned pigs through maintaining antioxidant capacity and intestinal digestion and absorption function. J Anim Sci. 2018;96(3).(2018)org/10.1093/jas/skx078.: 1108.
    10.1093/jas/skx078
    Article|CAS|PubMed|Google Scholar
  26. 26.Zhang Y, Qu H, Pan H, Xiang D, Choi S, Liang S. Effects of supplementation with chlorogenic acid-rich extract fromEucommia ulmoidesoliver during peri-implantation on the reproductive performance and gut microbiota of sows. Vet Sci. 2025;12(9).(2025)org/10.3390/vetsci12090857.: 857.
    Article|CAS|PubMed|Google Scholar
  27. 27.Consolo NRB, Silva JD, Buarque VLM, Higuera-Padilla A, Barbosa L, Zawadzki A, et al. Selection for growth and precocity alters muscle metabolism in Nellore cattle. Metabolites. 2020;10(2).(2020)org/10.3390/metabo10020058.: 58.
    Article|CAS|PubMed|Google Scholar
  28. 28.Chen H, Buhler K, Zhu Y, Nie X, Liu W. Proteomics analysis reveals the effect of 1α,25(OH)2VD3-glycosides on development of early testes in piglets. Sci Rep. 2021;11.(2021)org/10.1038/s41598-021-90676-8.: 11341.
    Article|CAS|PubMed|Google Scholar
  29. 29.Yang CX, Chen L, Yang YW, Mou Q, Du ZQ. Acute heat stress reduces viability but increases lactate secretion of porcine immature Sertoli cells through transcriptome reprogramming. Theriogenology. 2021;173.(2021)183–92. https://doi. org/10. 1016/j.theriogenology.: 183.
    10.1016/j.theriogenology.2021.06.024
    Article|CAS|PubMed|Google Scholar
  30. 30.Nguyen N, Jennen D, Kleinjans J. Omics technologies to understand drug toxicity mechanisms. Drug Discov Today. 2022;27(11).(2022)103348. https://doi. org/10. 1016/j.drudis.: 103348.
    10.1016/j.drudis.2022.103348
    Article|CAS|PubMed|Google Scholar
  31. 31.Chen H, Zhang L, Liu M, Li Y, Chi Y. Multi-omics research on angina pectoris.(2024)A novel perspective.Aging Dis.: 3381.
    10.14336/AD.2024.1298
    Article|CAS|PubMed|Google Scholar
  32. 32.Long JA. The “omics” revolution.(2020)use of genomic, transcriptomic, proteomic and metabolomic tools to predict male reproductive traits that impact fertility in livestock and poultry.Anim Reprod Sci.: 106354.
    10.1016/j.anireprosci.2020.106354
    Article|CAS|PubMed|Google Scholar
  33. 33.Fan X, Xi H, Zhang Z, Liang Y, Li Q, He J. Germ cell apoptosis and expression of Bcl-2 and Bax in porcine testis under normal and heat stress conditions. Acta Histochem. 2017;119(3).(2017)09.003.: 198.
    10.1016/j.acthis.2016.09.003
    Article|CAS|PubMed|Google Scholar
  34. 34.Mader TL, Johnson LJ, Gaughan JB. A comprehensive index for assessing environmental stress in animals. J Anim Sci. 2010;88(6).(2010)2527/jas.2009-2586.: 2153.
    10.2527/jas.2009-2586
    Article|CAS|PubMed|Google Scholar
  35. 35.Ding H, Luo Y, Liu M, Huang J, Xu D. Histological and transcriptome analyses of testes from Duroc and Meishan boars. Sci Rep. 2016;6.(2016)org/10.1038/srep20758.: 20758.
    10.1038/srep20758
    Article|CAS|PubMed|Google Scholar
  36. 36.Franca LR, Silva VA Jr, Chiarini-Garcia H, Garcia SK, Debeljuk L. Cell proliferation and hormonal changes during postnatal development of the testis in the pig. Biol Reprod. 2000;63(6).(2000)6.1629.: 1629.
    10.1095/biolreprod63.6.1629
    Article|CAS|PubMed|Google Scholar
  37. 37.Yu J, Xiang JY, Xiang H, Xie Q. Cecal butyrate (not propionate) was connected with metabolism-related chemicals of mice, based on the different effects of the twoInonotus obliquusextracts on obesity and their mechanisms. ACS Omega. 2020;5(27).(2020)1021/acsomega.0c01566.: 16690.
    10.1021/acsomega.0c01566
    Article|CAS|PubMed|Google Scholar
  38. 38.Ebrahimi F, Ibrahim B, Teh CH, Murugaiyah V, Chan KL. Urinary NMR-based metabolomic analysis of rats possessing variable sperm count following orally administeredEurycoma longifoliaextracts of different quassinoid levels. J Ethnopharmacol. 2016;182.(2016)80–9. https://doi. org/10. 1016/j.jep.: 80.
    10.1016/j.jep.2016.02.015
    Article|CAS|PubMed|Google Scholar
  39. 39.Griffin JL, Troke J, Walker LA, Shore RF, Lindon JC, Nicholson JK. The biochemical profile of rat testicular tissue as measured by magic angle spinning1H NMR spectroscopy. FEBS Lett. 2000;486(3).(2000)org/10.1016/s0014-5793(00)02307-3.: 225.
    10.1016/s0014-5793(00)02307-3
    Article|CAS|PubMed|Google Scholar
  40. 40.Haritwal T, Maan K, Rana P, Parvez S, Singh AK, Khushu S, et al. Trichostatin A, an epigenetic modifier, mitigates radiation-induced androphysiological anomalies and metabolite changes in mice as evident from NMR-based metabolomics. Int J Radiat Biol. 2019;95(4).(2019)2018.1524989.: 443.
    10.1080/09553002.2018.1524989
    Article|CAS|PubMed|Google Scholar
  41. 41.Islam R, Melvin SD, Yu RMK, O'Connor WA, Tran TKA, Andrew-Priestley M, et al. Exposure to estrogenic mixtures results in tissue-specific alterations to the metabolome of oysters. Aquat Toxicol. 2021;231.(2021)2020.105722.: 105722.
    Article|CAS|PubMed|Google Scholar
  42. 42.Straadt IK, Young JF, Bross P, Gregersen N, Oksbjerg N, Theil PK, et al. NMR-based metabonomic investigation of heat stress in myotubes reveals a time-dependent change in the metabolites. J Agric Food Chem. 2010;58(10).(2010)org/10.1021/jf904197u.: 6376.
    10.1021/jf904197u
    Article|CAS|PubMed|Google Scholar
  43. 43.Gujar G, Tiwari M, Yadav N, Monika D. Heat stress adaptation in cows - Physiological responses and underlying molecular mechanisms. J Therm Biol. 2023;118.(2023)103740. https://doi. org/10. 1016/j.jtherbio.: 103740.
    10.1016/j.jtherbio.2023.103740
    Article|CAS|PubMed|Google Scholar
  44. 44.Zhou J, Zhao JH, Li YH, Bai YY, Wu Y, Xiang B, et al. The hottest center.(2022)characteristics of high temperatures in midsummer of.: 151.
    10.1007/s00704-023-04609-8
    Article|CAS|PubMed|Google Scholar
  45. 45.Li Y, Li Z, Cao Y, Zhou X, Li C. Chronic excessive Zn intake increases the testicular sensitivity to high ambient temperature in Bama miniature pigs. Environ Pollut. 2020;257.(2020)2019.113629.: 113629.
    10.1016/j.envpol.2019.113629
    Article|CAS|PubMed|Google Scholar
  46. 46.Shen H, Fan X, Zhang Z, Xi H, Ji R, Liu Y, et al. Effects of elevated ambient temperature and local testicular heating on the expressions of heat shock protein 70 and androgen receptor in boar testes. Acta Histochem. 2019;121(3).(2019)297–302. https://doi. org/10. 1016/j.acthis.: 297.
    10.1016/j.acthis.2019.01.009
    Article|CAS|PubMed|Google Scholar
  47. 47.Wang K, Li Z, Li Y, Li X, Suo Y, Li C. Impacts of elevated temperature on morphology, oxidative stress levels, and testosterone synthesis in ex vivo cultured porcine testicular tissue. Theriogenology. 2023;212.(2023)181–8. https://doi. org/10. 1016/j.theriogenology.: 181.
    10.1016/j.theriogenology.2023.09.015
    Article|CAS|PubMed|Google Scholar
  48. 48.Gong S, Miao YL, Jiao GZ, Sun MJ, Li H, Lin J, et al. Dynamics and correlation of serum cortisol and corticosterone under different physiological or stressful conditions in mice. PLoS ONE. 2015;10(2).(2015)pone.0117503.
    10.1371/journal.pone.0117503
    Article|CAS|PubMed|Google Scholar
  49. 49.Michel V, Peinnequin A, Alonso A, Buguet A, Cespuglio R, Canini F. Decreased heat tolerance is associated with hypothalamo-pituitary-adrenocortical axis impairment. Neuroscience. 2007;147(2).(2007)522–31. https://doi. org/10. 1016/j.neuroscience.: 522.
    10.1016/j.neuroscience.2007.04.035
    Article|CAS|PubMed|Google Scholar
  50. 50.Bouchama A, Knochel JP. Heat stroke. N Engl J Med. 2002;346(25).(2002)org/10.1056/NEJMra011089.: 1978.
    10.1056/NEJMra011089
    Article|CAS|PubMed|Google Scholar
  51. 51.Veilleux JC, Zielinski MJ, Moyen NE, Tucker MA, Dougherty EK, Ganio MS. The effect of passive heat stress on distress andself-control in male smokers and non-smokers. J Gen Psychol. 2018;145(4).(2018)342–61. https://doi. org/10.1080/00221309.: 342.
    10.1080/00221309.2018.1494127
    Article|CAS|PubMed|Google Scholar
  52. 52.Garolla A, Torino M, Sartini B, Cosci I, Patassini C, Carraro U, et al. Seminal and molecular evidence that sauna exposure affects human spermatogenesis. Hum Reprod. 2013;28(4).(2013)org/10.1093/humrep/det020.: 877.
    10.1093/humrep/det020
    Article|CAS|PubMed|Google Scholar
  53. 53.Wettemann RP, Bazer FW. Influence of environmental temperature on prolificacy of pigs. J Reprod Fertil Suppl. 1985;33.(1985)199–208.: 199.
    Article|CAS|PubMed|Google Scholar
  54. 54.Lervik S, Kristoffersen AB, Conley LN, Oskam IC, Hedegaard J, Ropstad E, et al. Gene expression during testis development in Duroc boars. Animal. 2015;9(11).(2015)org/10.1017/S1751731115000907.: 1832.
    10.1017/S1751731115000907
    Article|CAS|PubMed|Google Scholar
  55. 55.Chen F, Zhang H, Zhao N, Yang X, Du E, Huang S, et al. Effect of chlorogenic acid on intestinal inflammation, antioxidant status, and microbial community of young hens challenged with acute heat stress. Anim Sci J. 2021;92(1).(2021)1111/asj.13619.
    10.1111/asj.13619
    Article|CAS|PubMed|Google Scholar
  56. 56.Ji R, Chen J, Xu J, Zhang L, Liu L, Li F. Protective effect of chlorogenic acid on liver injury in heat-stressed meat rabbits. J Anim Physiol Anim Nutr (Berl). 2024;108(5).(2024)1111/jpn.13966.: 1203.
    10.1111/jpn.13966
    Article|CAS|PubMed|Google Scholar
  57. 57.Nguyen TV, Do LTK, Somfai T, Otoi T, Taniguchi M, Kikuchi K. Presence of chlorogenic acid during in vitro maturation protects porcine oocytes from the negative effects of heat stress. Anim Sci J. 2019;90(12).(2019)1111/asj.13302.: 1530.
    10.1111/asj.13302
    Article|CAS|PubMed|Google Scholar
  58. 58.Zhao JS, Deng W, Liu HW. Effects of chlorogenic acid-enriched extract fromEucommia ulmoidesleaf on performance, meat quality, oxidative stability, and fatty acid profile of meat in heat-stressed broilers. Poult Sci. 2019;98(7).(2019)org/10.3382/ps/pez081.: 3040.
    10.3382/ps/pez081
    Article|CAS|PubMed|Google Scholar
  59. 59.Ma F, Liu J, Li S, Sun P. Effects ofLonicera japonicaextract with different contents of chlorogenic acid on lactation performance, serum parameters, and rumen fermentation in heat-stressed Holstein high-yielding dairy cows. Animals (Basel). 2024;14(8).(2024)org/10.3390/ani14081252.: 1252.
    Article|CAS|PubMed|Google Scholar
  60. 60.Crisostomo L, Jarak I, Rato LP, Raposo JF, Batterham RL, Oliveira PF, et al. Inheritable testicular metabolic memory of high-fat diet causes transgenerational sperm defects in mice. Sci Rep. 2021;11.(2021)org/10.1038/s41598-021-88981-3.: 9444.
    10.1038/s41598-021-88981-3
    Article|CAS|PubMed|Google Scholar
  61. 61.Oliveira PF, Martins AD, Moreira AC, Cheng CY, Alves MG. The Warburg effect revisited–lesson from the Sertoli cell. Med Res Rev. 2015;35(1).(2015)1002/med.21325.: 126.
    10.1002/med.21325
    Article|CAS|PubMed|Google Scholar
  62. 62.Hamed MA, Akhigbe TM, Akhigbe RE, Aremu AO, Oyedokun PA, Gbadamosi JA, et al. Glutamine restores testicular glutathione-dependent antioxidant defense and upregulates NO/cGMP signaling in sleep deprivation-induced reproductive dysfunction in rats. Biomed Pharmacother. 2022;148.(2022)112765. https://doi. org/10. 1016/j.biopha.: 112765.
    10.1016/j.biopha.2022.112765
    Article|CAS|PubMed|Google Scholar
  63. 63.Zhang G, Guo J, Yang H, Li Q, Ye F, Song Y, et al. Metabolic profiling identifies Qrich2 as a novel glutamine sensor that regulates microtubule glutamylation and mitochondrial function in mouse sperm. Cell Mol Life Sci. 2024;81(1).(2024)org/10.1007/s00018-024-05177-4.: 170.
    10.1007/s00018-024-05177-4
    Article|CAS|PubMed|Google Scholar
  64. 64.Awad EA, Idrus Z, Soleimani Farjam A, Bello AU, Jahromi MF. Growth performance, duodenal morphology and the caecal microbial population in female broiler chickens fed glycine-fortified low protein diets under heat stress conditions. Br Poult Sci. 2018;59(3).(2018)340–8. https://doi. org/10.1080/00071668.: 340.
    10.1080/00071668.2018.1440377
    Article|CAS|PubMed|Google Scholar
  65. 65.Deng C, Zheng J, Zhou H, You J, Li G. Dietary glycine supplementation prevents heat stress-induced impairment of antioxidant status and intestinal barrier function in broilers. Poult Sci. 2023;102(3).(2023)2022.102408.: 102408.
    10.1016/j.psj.2022.102408
    Article|CAS|PubMed|Google Scholar
  66. 66.Ruan Z, Yang Y, Zhou Y, Wen Y, Ding S, Liu G, et al. Metabolomic analysis of amino acid and energy metabolism in rats supplemented with chlorogenic acid. Amino Acids. 2014;46(9).(2014)org/10.1007/s00726-014-1762-7.: 2219.
    10.1007/s00726-014-1762-7
    Article|CAS|PubMed|Google Scholar
  67. 67.Xu B, Chen M, Ji X, Yao M, Mao Z, Zhou K, et al. Metabolomic profiles reveal key metabolic changes in heat stress-treated mouse Sertoli cells. Toxicol In Vitro. 2015;29(7).(2015)1745–52. https://doi. org/10. 1016/j.tiv.: 1745.
    10.1016/j.tiv.2015.07.009
    Article|CAS|PubMed|Google Scholar
  68. 68.Cai XQ, Yang H, Liang BQ, Deng CC, Xue HY, Zhang JJ, et al. Glutamate rescues heat stress-induced apoptosis of Sertoli cells by enhancing the activity of antioxidant enzymes and activating the Trx1-Akt pathway in vitro. Theriogenology. 2024;223.(2024)1–10. https://doi. org/10. 1016/j.theriogenology.: 1.
    10.1016/j.theriogenology.2024.04.005
    Article|CAS|PubMed|Google Scholar
  69. 69.Chen N, Huang Z, Lu C, Shen Y, Luo X, Ke C, et al. Different transcriptomic responses to thermal stress in heat-tolerant and heat-sensitive Pacific abalones indicated by cardiac performance. Front Physiol. 2018;9.(2018)1895. https://doi. org/10.3389/fphys.: 1895.
    10.3389/fphys.2018.01895
    Article|CAS|PubMed|Google Scholar
  70. 70.Wang X, Liu F, Gao X, Liu X, Kong X, Wang H, et al. Comparative proteomic analysis of heat stress proteins associated with rat sperm maturation. Mol Med Rep. 2016;13(4).(2016)3547–52. https://doi. org/10.3892/mmr.: 3547.
    10.3892/mmr.2016.4958
    Article|CAS|PubMed|Google Scholar
  71. 71.Wu X, Ji P, Zhang L, Bu G, Gu H, Wang X, et al. The expression of porcinePrdx6gene is up-regulated by C/EBPβ and CREB. PLoS ONE. 2015;10(12).(2015)pone.0144851.
    10.1371/journal.pone.0144851
    Article|CAS|PubMed|Google Scholar
  72. 72.Moulder R, Hirvonen MK, Valikangas T, Suomi T, Overbergh L, Peakman M, et al. Targeted serum proteomics of longitudinal samples from newly diagnosed youth with type 1 diabetes affirms markers of disease. Diabetologia. 2025.https.(2025)org/10.1007/s00125-025-06394-7.
    10.1007/s00125-025-06394-7
    Article|CAS|PubMed|Google Scholar
  73. 73.Xu J, Wang XL, Zeng HF, Han ZY. Methionine alleviates heat stress-induced ferroptosis in bovine mammary epithelial cells through the Nrf2 pathway. Ecotoxicol Environ Saf. 2023;256.(2023)114889. https://doi. org/10. 1016/j.ecoenv.: 114889.
    10.1016/j.ecoenv.2023.114889
    Article|CAS|PubMed|Google Scholar
  74. 74.Chen H, Lin X, Yi X, Liu X, Yu R, Fan W, et al. SIRT1-mediated p53 deacetylation inhibits ferroptosis and alleviates heat stress-induced lung epithelial cells injury. Int J Hyperthermia. 2022;39(1).(2022)977–86. https://doi. org/10.1080/02656736.: 977.
    10.1080/02656736.2022.2094476
    Article|CAS|PubMed|Google Scholar
  75. 75.Yang H, Cai X, Qiu M, Deng C, Xue H, Zhang J, et al. Heat stress induces ferroptosis of porcine Sertoli cells by enhancing CYP2C9-Ras- JNK axis. Theriogenology. 2024;215.(2024)11.027.: 281.
    10.1016/j.theriogenology.2023.11.027
    Article|CAS|PubMed|Google Scholar
  76. 76.Zhao Y, Wang C, Yang T, Wang H, Zhao S, Sun N, et al. Chlorogenic acid alleviates chronic stress-induced duodenal ferroptosis via the inhibition of the IL-6/JAK2/STAT3 signaling pathway in rats. J Agric Food Chem. 2022;70(14).(2022)jafc.2c01196.: 4353.
    10.1021/acs.jafc.2c01196
    Article|CAS|PubMed|Google Scholar
  77. 77.Li LY, Wang Q, Deng L, Lin Z, Lin JJ, Wang XY, et al. Chlorogenic acid alleviates hypoxic-ischemic brain injury in neonatal mice. Neural Regen Res. 2023;18(3).(2023)4103/1673-5374.350203.: 568.
    10.4103/1673-5374.350203
    Article|CAS|PubMed|Google Scholar
  78. 78.Tang Z, Ju Y, Dai X, Ni N, Liu Y, Zhang D, et al. HO-1-mediated ferroptosis as a target for protection against retinal pigment epithelium degeneration. Redox Biol. 2021;43.(2021)101971. https://doi. org/10. 1016/j.redox.: 101971.
    10.1016/j.redox.2021.101971
    Article|CAS|PubMed|Google Scholar
  79. 79.Guo W, Yang H, He W. Paeonol alleviates ox-LDL-induced endothelial cell injury by targeting the heme oxygenase-1/phosphoinositide 3-kinase/protein kinase B pathway. Naunyn Schmiedebergs Arch Pharmacol. 2025;398(1).(2025)org/10.1007/s00210-024-03307-0.: 591.
    10.1007/s00210-024-03307-0
    Article|CAS|PubMed|Google Scholar
  80. 80.Oka S, Shiraishi K, Fujimoto M, Katiyar A, Takii R, Nakai A, et al. Role of heat shock factor 1 in conserving cholesterol transportation in Leydig cell steroidogenesis via steroidogenic acute regulatory protein. Endocrinology. 2017;158(8).(2017)2648–58. https://doi. org/10.1210/en.: 2648.
    10.1210/en.2017-00132
    Article|CAS|PubMed|Google Scholar
  81. 81.Zheng HX, Xu YM, Fan SC, Qi SS, Jia FF, Wu W, et al. Potential protective role of chlorogenic acid against cyclophosphamide-induced reproductive damage in male mice. Toxicol Res. 2024;13(5).(2024)org/10.1093/toxres/tfae176.
    10.1093/toxres/tfae176
    Article|CAS|PubMed|Google Scholar
  82. 82.Kim SJ, Shin MJ, Kim DW, Yeo HJ, Yeo EJ, Choi YJ, et al. Tat-biliverdin reductase A exerts a protective role in oxidative stress-induced hippocampal neuronal cell damage by regulating the apoptosis and MAPK signaling. Int J Mol Sci. 2020;21(8).(2020)org/10.3390/ijms21082672.: 2672.
    Article|CAS|PubMed|Google Scholar
  83. 83.Zhang H, Bao S, Zhao X, Bai Y, Lv Y, Gao P, et al. Genome-Wide Association Study and Phenotype Prediction of Reproductive Traits in Large White Pigs. Animals (Basel). 2024;14(23).(2024)org/10.3390/ani14233348.: 3348.
    Article|CAS|PubMed|Google Scholar
  84. 84.Sullivan R. Epididymosomes.(2015)a heterogeneous population of microvesicles with multiple functions in sperm maturation and storage.Asian J Androl.: 726.
    10.4103/1008-682X.155255
    Article|CAS|PubMed|Google Scholar
  85. 85.D’Amours O, Frenette G, Caron P, Belleannee C, Guillemette C, Sullivan R. Evidences of biological functions of biliverdin reductase A in the bovine epididymis. J Cell Physiol. 2016;231(5).(2016)1002/jcp.25200.: 1077.
    10.1002/jcp.25200
    Article|CAS|PubMed|Google Scholar

Acknowledgements

We sincerely thank Prof. Hongyu Xiang and Dr. Chao Shi from the School of Life Sciences, Jilin University, for their valuable guidance and support in metabolite extraction. We are also grateful to Researcher Chunyu Wang from the State Key Laboratory of Supramolecular Structure and Materials, Jilin University for his technical assistance in metabolite detection. Additionally, we acknowledge the Figdraw platform for providing tools to support Fig. 7 preparation.

Funding

This research was supported by The Program of Science and Technology Development Plan of Jilin Province (20230202066NC) and Graduate Innovation Research Project of Jilin University (2024CX151).

Ethics Declaration

Ethics approval and consent to participate

The animal use protocols were reviewed and approved by the Care and Use of Animals prepared by the Institutional Animal Care and Use Committee of Jilin University (ethical lot number: SY202107001).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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