FREE SHIPPING over $40
OVER 9000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Home / Equine Research Bank / Biology (Basel) / Transcriptome Sequencing and Differential Analysis of Testes...
Biology2026; 15(1); 100; doi: 10.3390/biology15010100

Transcriptome Sequencing and Differential Analysis of Testes of 1-Year-Old and 3-Year-Old Kazakh Horses.

Abstract: The Kazakh horse is an outstanding dual-purpose dairy and meat breed in China, characterized by early maturity, tolerance to coarse feed, and strong stress resistance. Previous studies have examined gene expression patterns in the testicular tissues of Kazakh horses at different age stages, but the molecular mechanisms regulating testicular sexual maturation remain unclear. To address this gap, this study conducted HE staining and in-depth transcriptome sequencing analysis of Kazakh horse testicular tissue before and after sexual maturity. HE staining showed that the G3 group had well-formed seminiferous tubule lumens, dense interstitial cells, and visible early spermatocytes and spermatozoa, indicating structural maturation. (G1 group: pre-sexual maturity; G3 group: post-sexual maturity), with four biological replicates per group ( = 4). Differentially expressed genes (DEGs) were called using the criteria of |log(fold change)| ≥ 1.5 and adjusted -value ≤ 0.05. A total of 3054 differentially expressed genes (DEGs), including , , , , and , were identified in the G1 and G3 groups. Among these, 402 genes showed upregulation and 2652 genes showed downregulation. GO annotation and KEGG enrichment analysis of DEGs revealed their predominant enrichment in the following categories: signaling pathways such as Focal adhesion, Pathways in cancer, and the PI3K-Akt signaling pathway. RT-qPCR validation confirmed the accuracy of the transcriptomic sequencing data. This study further elucidates the differentially expressed genes and associated signaling pathways in Kazakh stallion testes tissue before and after sexual maturity, providing a theoretical foundation and data reference for enhancing reproductive efficiency in equids and promoting biological processes such as testes development and spermatogenesis.
Get Full Text
Publication Date: 2026-01-04 PubMed ID: 41514941PubMed Central: PMC12784889DOI: 10.3390/biology15010100Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Overview

  • This study analyzed the changes in gene expression in the testes of Kazakh horses before and after sexual maturity to better understand the molecular processes regulating testicular development and function.

Background

  • The Kazakh horse is a notable dual-purpose breed used for both dairy and meat in China.
  • It is known for early sexual maturity, the ability to consume coarse feed, and strong resistance to environmental stress.
  • Prior research has studied gene expression in testicular tissues at various ages, but the molecular mechanisms of sexual maturation remained unclear.

Objectives

  • To investigate the differential gene expression in the testes of Kazakh horses at pre-sexual maturity (1-year-old, G1 group) and post-sexual maturity (3-year-old, G3 group).
  • To elucidate the biological pathways and genes involved in testicular sexual maturation and spermatogenesis.
  • To provide insights that could improve reproductive efficiency in horses.

Methods

  • Histological Analysis: Hematoxylin and eosin (HE) staining was performed on testicular tissues from both age groups.
  • Transcriptome Sequencing: RNA sequencing was carried out to analyze gene expression profiles, with four biological replicates per group.
  • Differential Gene Expression Analysis: DEGs were identified using thresholds of |log(fold change)| ≥ 1.5 and adjusted p-value ≤ 0.05.
  • Functional Annotation: Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted to determine involved biological functions and pathways.
  • Validation: Quantitative real-time PCR (RT-qPCR) was used to validate the sequencing results.

Results

  • Histology:
    • In the 3-year-old group (G3), seminiferous tubules were well-formed with visible lumens.
    • Dense interstitial cells and early spermatocytes/spermatozoa were evident, indicating structural and functional maturity.
    • The 1-year-old group (G1) showed immature testicular architecture.
  • Differential Expression:
    • A total of 3,054 DEGs were identified between the G1 and G3 groups.
    • 402 genes were upregulated in the post-maturity testes, while 2,652 genes were downregulated.
  • Functional Enrichment:
    • GO and KEGG analyses showed enrichment in signaling pathways related to testicular development and function.
    • Key pathways included focal adhesion, cancer pathways, and PI3K-Akt signaling pathway, which are important for cell communication, proliferation, and survival.
  • Validation:
    • RT-qPCR confirmed the expression patterns observed in the transcriptome sequencing, supporting data reliability.

Conclusions and Implications

  • The study identified critical genes and pathways differentially expressed before and after sexual maturity in Kazakh horse testes.
  • These findings enhance understanding of molecular mechanisms underlying testicular maturation and spermatogenesis in horses.
  • The data serve as a valuable foundation for improving reproductive performance in equine species.
  • The elucidated pathways might be targeted to promote testicular development and biological functions related to fertility in breeding programs.

Cite This Article

APA
Liu J, Yang Y, Wen L, Wen M, Zeng Y, Ren W, Yao X. (2026). Transcriptome Sequencing and Differential Analysis of Testes of 1-Year-Old and 3-Year-Old Kazakh Horses. Biology (Basel), 15(1), 100. https://doi.org/10.3390/biology15010100

Publication

Biology
ISSN: 2079-7737
NlmUniqueID: 101587988
Country: Switzerland
Language: English
Volume: 15
Issue: 1
PII: 100

Researcher Affiliations

Liu, Jiahao
  • College of Animal Science, Xinjiang Agricultural University, Urumqi 830052, China.
Yang, Yuting
  • College of Animal Science, Xinjiang Agricultural University, Urumqi 830052, China.
Wen, Liuxiang
  • College of Animal Science, Xinjiang Agricultural University, Urumqi 830052, China.
  • Xinjiang Key Laboratory of Equine Breeding and Exercise Physiology, Urumqi 830052, China.
Wen, Mingyue
  • College of Animal Science, Xinjiang Agricultural University, Urumqi 830052, China.
  • Xinjiang Key Laboratory of Equine Breeding and Exercise Physiology, Urumqi 830052, China.
Zeng, Yaqi
  • College of Animal Science, Xinjiang Agricultural University, Urumqi 830052, China.
  • Xinjiang Key Laboratory of Equine Breeding and Exercise Physiology, Urumqi 830052, China.
Ren, Wanlu
  • College of Animal Science, Xinjiang Agricultural University, Urumqi 830052, China.
  • Xinjiang Key Laboratory of Equine Breeding and Exercise Physiology, Urumqi 830052, China.
Yao, Xinkui
  • College of Animal Science, Xinjiang Agricultural University, Urumqi 830052, China.
  • Xinjiang Key Laboratory of Equine Breeding and Exercise Physiology, Urumqi 830052, China.

Conflict of Interest Statement

All authors declare that they have no conflicts of interest.

References

This article includes 44 references
  1. Ren W, Zhou J, Zhu J, Zhang J, Zhao X, Yao X. Exploring the Abnormal Characteristics of the Ovaries During the Estrus Period of Kazakh Horses Based on Single-Cell Transcriptome Technology. Biology 2025;14:1351.
    doi: 10.3390/biology14101351pmc: PMC12562243pubmed: 41154754google scholar: lookup
  2. Lu Z, Wen M, Yao X, Meng J, Wang J, Zeng Y, Li L, Ren W. Differential analysis of testicular LncRNA in Kazakh horses of different ages. Int. J. Biol. Macromol. 2025;321:146228.
    doi: 10.1016/j.ijbiomac.2025.146228pubmed: 40706934google scholar: lookup
  3. Wubulikasimu M, Liu J, Yao X, Meng J, Wang J, Zeng Y, Li L, Ren W. Transcriptomic sequencing and differential analysis of Kazakh horse muscles from various anatomical locations. Front. Vet. Sci. 2025;12:1633786.
    doi: 10.3389/fvets.2025.1633786pmc: PMC12330291pubmed: 40777832google scholar: lookup
  4. Wubuli A, Su Y, Yao X, Meng J, Wang J, Zeng Y, Li L, Ren W. Transcriptome Analysis of Muscle Tissue from Three Anatomical Locations in Male and Female Kazakh Horses. Biology 2025;14:1216.
    doi: 10.3390/biology14091216pmc: PMC12467203pubmed: 41007361google scholar: lookup
  5. Chen H, Murray E, Sinha A, Laumas A, Li J, Lesman D, Nie X, Hotaling J, Guo J, Cairns B.R.. Dissecting mammalian spermatogenesis using spatial transcriptomics. Cell Rep. 2021;37:109915.
    doi: 10.1016/j.celrep.2021.109915pmc: PMC8606188pubmed: 34731600google scholar: lookup
  6. Murat F, Mbengue N, Winge S.B., Trefzer T, Leushkin E, Sepp M, Cardoso-Moreira M, Schmidt J, Schneider C, Mößinger K. The molecular evolution of spermatogenesis across mammals. Nature 2023;613:308–316.
    doi: 10.1038/s41586-022-05547-7pmc: PMC9834047pubmed: 36544022google scholar: lookup
  7. Zhang X, Cao Q, Rajachandran S, Grow E.J., Evans M, Chen H. Dissecting mammalian reproduction with spatial transcriptomics. Hum. Reprod. Update 2023;29:794–810.
    doi: 10.1093/humupd/dmad017pmc: PMC10628492pubmed: 37353907google scholar: lookup
  8. Yan Z, Wang P, Yang Q, Gun S. Single-cell rna sequencing reveals an atlas of hezuo pig testis cells. Int. J. Mol. Sci. 2024;25:9786.
    doi: 10.3390/ijms25189786pmc: PMC11431743pubmed: 39337274google scholar: lookup
  9. Han G, Hong S.H., Lee S.J., Hong S.-P., Cho C. Transcriptome analysis of testicular aging in mice. Cells 2021;10:2895.
    doi: 10.3390/cells10112895pmc: PMC8616291pubmed: 34831115google scholar: lookup
  10. Zhang M, An X, Yuan C, Guo T, Xi B, Liu J, Lu Z. Integration analysis of transcriptome and metabolome revealed the potential mechanism of spermatogenesis in Tibetan sheep (Ovis aries) at extreme high altitude. Genomics 2024;116:110949.
    doi: 10.1016/j.ygeno.2024.110949pubmed: 39389270google scholar: lookup
  11. Zhang Y, Liu Z, Yun X, Batu B, Yang Z, Zhang X, Zhang W, Liu T. Transcriptome profiling of developing testes and first wave of spermatogenesis in the rat. Genes 2023;14:229.
    doi: 10.3390/genes14010229pmc: PMC9859615pubmed: 36672970google scholar: lookup
  12. Chen W.B., Zhang M.F., Yang F, Hua J.-L.. Applications of single-cell RNA sequencing in spermatogenesis and molecular evolution. Zool. Res. 2024;45:575.
    doi: 10.24272/j.issn.2095-8137.2024.010pmc: PMC11188606pubmed: 38766742google scholar: lookup
  13. Du Z, Li W.T., Liu C, Wang C, Wang D, Zhu S, Kang X, Jiang R, Deng L, Li D. Transcriptome analysis of the testes of male chickens with high and low sperm motility. Poult. Sci. 2022;101:102183.
    doi: 10.1016/j.psj.2022.102183pmc: PMC9554828pubmed: 36215742google scholar: lookup
  14. Zhao S, Wang H, Hu Z, Sahlu B.W., Heng N, Gong J, Wang H, Zhu H. Identification of spermatogenesis-related lncRNA in Holstein bull testis after sexual maturity based on transcriptome analysis. Anim. Reprod. Sci. 2022;247:107146.
    doi: 10.1016/j.anireprosci.2022.107146pubmed: 36371860google scholar: lookup
  15. Zhang X, Huo H, Li H, Liu Y, Qiao F, Li C, Huo J. An integrated analysis of second-and third-generation transcriptome sequencing technologies reveals the DAZAP1 function in pig testis. Anim. Reprod. 2025;22:e20240141.
    doi: 10.1590/1984-3143-ar2024-012pmc: PMC12203886pubmed: 40584277google scholar: lookup
  16. Chang X., Yao X., Meng J., Wang J., Zeng Y., Li L., Ren W. Whole Transcriptome Sequencing and Differential Analysis of Testes in Pre-and Post-Sexual Maturity Bactrian Camels (Camelus bactrianus) Biology. 2025;14:1254. doi: 10.3390/biology14091254.
    doi: 10.3390/biology14091254pmc: PMC12467276pubmed: 41007398google scholar: lookup
  17. La Y., Ma X., Bao P., Chu M., Yan P., Liang C., Guo X. Genome-wide Landscape of mRNAs, lncRNAs, and circRNAs during Testicular Development of Yak. Int. J. Mol. Sci. 2023;24:4420. doi: 10.3390/ijms24054420.
    doi: 10.3390/ijms24054420pmc: PMC10002557pubmed: 36901865google scholar: lookup
  18. Xi B., Zhao S., Zhang R., Lu Z., Li J., An X., Yue Y. Transcriptomic study of different stages of development in the testis of sheep. Animals. 2024;14:2767. doi: 10.3390/ani14192767.
    doi: 10.3390/ani14192767pmc: PMC11475124pubmed: 39409717google scholar: lookup
  19. Zhao Y., Qin G., Fan W., Zhang Y., Peng H. TF and TFRC regulate ferroptosis in swine testicular cells through the JNK signaling pathway. Int. J. Biol. Macromol. 2025;307:142369. doi: 10.1016/j.ijbiomac.2025.142369.
    doi: 10.1016/j.ijbiomac.2025.142369pubmed: 40120870google scholar: lookup
  20. Su J., Yang Y., Wang D., Su H., Zhao F., Zhang C., Zhang M., Li X., He T., Li X., et al. A dynamic transcriptional cell atlas of testes development after birth in Hu sheep. BMC Biol. 2025;23:1–17. doi: 10.1186/s12915-025-02186-y.
    doi: 10.1186/s12915-025-02186-ypmc: PMC11905504pubmed: 40075363google scholar: lookup
  21. Lin W., Zhang X., Liu Z., Huo H., Chang Y., Zhao J., Gong S., Zhao G., Huo J. Isoform-resolution single-cell RNA sequencing reveals the transcriptional panorama of adult Baoshan pig testis cells. BMC Genom. 2025;26:459. doi: 10.1186/s12864-025-11636-4.
    doi: 10.1186/s12864-025-11636-4pmc: PMC12063418pubmed: 40340725google scholar: lookup
  22. Santos J.R.L., Sun W., Befus A.D., Marcet-Palacios M. SEQSIM: A novel bioinformatics tool for comparisons of promoter regions—A case study of calcium binding protein spermatid associated 1 (CABS1) BMC Bioinform. 2025;26:156. doi: 10.1186/s12859-025-06160-x.
    doi: 10.1186/s12859-025-06160-xpmc: PMC12150522pubmed: 40490738google scholar: lookup
  23. Zhang X., Zhou W., Zhang P., Gao F., Zhao X., Shum W.W., Zeng X. Cabs1 maintains structural integrity of mouse sperm flagella during epididymal transit of sperm. Int. J. Mol. Sci. 2021;22:652. doi: 10.3390/ijms22020652.
    doi: 10.3390/ijms22020652pmc: PMC7827751pubmed: 33440775google scholar: lookup
  24. Huang Y., Wang Y., Li L., Gong F., Lin G., Dai J. Identification of nonfunctional CABS1 causing fertilization failure and male infertility in humans: A case report. J. Assist. Reprod. Genet. 2025;42:2411–2419. doi: 10.1007/s10815-025-03516-3.
    doi: 10.1007/s10815-025-03516-3pmc: PMC12356792pubmed: 40407971google scholar: lookup
  25. Zhong Z., Wang F., Xie X., Wang Z., Pan D., Wang Z., Xiao Q. Integration of multi-omics resources reveals genetic features associated with environmental adaptation in the Wuzhishan pig genome. J. Therm. Biol. 2025;132:104264. doi: 10.1016/j.jtherbio.2025.104264.
    doi: 10.1016/j.jtherbio.2025.104264pubmed: 40921116google scholar: lookup
  26. Zhao X., Zhou W., Nie J., Zhang X., Zeng X., Sun X. CABS1 is essential for progressive motility and the integrity of fibrous sheath in mouse epididymal spermatozoa. Mol. Reprod. Dev. 2024;91:e23776. doi: 10.1002/mrd.23776.
    doi: 10.1002/mrd.23776pubmed: 39526486google scholar: lookup
  27. Zhang W., Liu L., Zhou M., Su S., Dong L., Meng X., Li X., Wang C. Assessing population structure and signatures of selection in Wanbei pigs using whole genome resequencing data. Animals. 2022;13:13. doi: 10.3390/ani13010013.
    doi: 10.3390/ani13010013pmc: PMC9817800pubmed: 36611624google scholar: lookup
  28. Salehi N., Totonchi M. The construction of a testis transcriptional cell atlas from embryo to adult reveals various somatic cells and their molecular roles. J. Transl. Med. 2023;21:859. doi: 10.1186/s12967-023-04722-2.
    doi: 10.1186/s12967-023-04722-2pmc: PMC10680190pubmed: 38012716google scholar: lookup
  29. Xu N., Qin Y., Liu Y., Guan Y., Xin H., Ou J., Wang Y. An integrated transcriptomic analysis unveils the regulatory roles of RNA binding proteins during human spermatogenesis. Front. Endocrinol. 2025;16:1522394. doi: 10.3389/fendo.2025.1522394.
    doi: 10.3389/fendo.2025.1522394pmc: PMC11872710pubmed: 40034235google scholar: lookup
  30. Sang J., Ji Z., Li H., Wang H., Quan H., Yu Y., Yan J., Mao Z., Wang Y., Li L., et al. Triclosan inhibits testosterone biosynthesis in adult rats via inducing m6A methylation-mediated autophagy. Environ. Int. 2024;190:108827. doi: 10.1016/j.envint.2024.108827.
    doi: 10.1016/j.envint.2024.108827pubmed: 38908274google scholar: lookup
  31. Yang W., Li H., Wang S., Huang R., Zhang Y., Guo M., Huang L., Li S., Yang R., Zhao D., et al. Zika virus disrupts steroidogenesis and impairs spermatogenesis by stalling the translation of CYP17A1 mRNA. Nat. Commun. 2025;16:6756. doi: 10.1038/s41467-025-62044-x.
    doi: 10.1038/s41467-025-62044-xpmc: PMC12284141pubmed: 40695837google scholar: lookup
  32. Lea R.G., Mandon-Pépin B., Loup B., Poumerol E., Jouneau L., Egbowon B.F., Bowden A., Cotinot C., Purdie L., Zhang Z., et al. Ovine fetal testis stage-specific sensitivity to environmental chemical mixtures. Reproduction. 2022;163:119–131. doi: 10.1530/REP-21-0235.
    doi: 10.1530/REP-21-0235pmc: PMC8859917pubmed: 35015698google scholar: lookup
  33. Di-Luoffo M., Pierre K.J., Robert N.M., Girard M.-J., Tremblay J.J. The nuclear receptors SF1 and COUP-TFII cooperate on the Insl3 promoter in Leydig cells. Reproduction. 2022;164:31–40. doi: 10.1530/REP-22-0109.
    doi: 10.1530/REP-22-0109pubmed: 35666805google scholar: lookup
  34. Chang H., Lu Y., Yamamoto K., Sun J., Shimada K., Hiradate Y., Fujihara Y., Ikawa M. Mouse genome engineering uncovers 18 genes dispensable for male reproduction. Andrology. 2025 doi: 10.1111/andr.70088. Epub ahead of printing .
    doi: 10.1111/andr.70088pubmed: 40572022google scholar: lookup
  35. Cannarella R., Mancuso F., Arato I., Lilli C., Bellucci C., Gargaro M., Curto R., Aglietti M.C., La Vignera S., Condorelli R.A., et al. Sperm-carried IGF2 downregulated the expression of mitogens produced by Sertoli cells: A paracrine mechanism for regulating spermatogenesis? Front. Endocrinol. 2022;13:1010796. doi: 10.3389/fendo.2022.1010796.
    doi: 10.3389/fendo.2022.1010796pmc: PMC9744929pubmed: 36523595google scholar: lookup
  36. Chen H., Miao X., Xu J., Pu L., Li L., Han Y., Mao F., Ma Y. Alterations of mRNA and lncRNA profiles associated with the extracellular matrix and spermatogenesis in goats. Anim. Biosci. 2021;35:544. doi: 10.5713/ab.21.0259.
    doi: 10.5713/ab.21.0259pmc: PMC8902208pubmed: 34530511google scholar: lookup
  37. Yu B., Yang Y., Li Y., Gao R., Ma M., Wang X. Transcriptomic study of testicular hypoxia adaptation in Tibetan sheep. Reprod. Domest. Anim. 2025;60:e70037. doi: 10.1111/rda.70037.
    doi: 10.1111/rda.70037pubmed: 40099575google scholar: lookup
  38. Fu X., Yang Y., Yan Z., Liu M., Wang X. Transcriptomic study of spermatogenesis in the testis of Hu sheep and Tibetan sheep. Genes. 2022;13:2212. doi: 10.3390/genes13122212.
    doi: 10.3390/genes13122212pmc: PMC9778047pubmed: 36553478google scholar: lookup
  39. Wang Y., Pan Y., Wang M., Afedo S.Y., Zhao L., Han X., Liu M., Zhao T., Zhang T., Ding T., et al. Transcriptome sequencing reveals differences between leydig cells and sertoli cells of yak. Front. Vet. Sci. 2022;9:960250. doi: 10.3389/fvets.2022.960250.
    doi: 10.3389/fvets.2022.960250pmc: PMC9449347pubmed: 36090173google scholar: lookup
  40. Chen K.Q., Wei B.H., Hao S.L., Yang W.-X. The PI3K/AKT signaling pathway: How does it regulate development of Sertoli cells and spermatogenic cells? Histol. Histopathol. 2022;37:621–636.
    pubmed: 35388905
  41. Deng C.Y., Lv M., Luo B.H., Zhao S.-Z., Mo Z.-C., Xie Y.-J. The role of the PI3K/AKT/mTOR signalling pathway in male reproduction. Curr. Mol. Med. 2021;21:539–548.
    pubmed: 33272176
  42. Wang X., Pei J., Xiong L., Guo S., Cao M., Kang Y., Ding Z., La Y., Liang C., Yan P., et al. Single-cell RNA sequencing reveals atlas of yak testis cells. Int. J. Mol. Sci. 2023;24:7982. doi: 10.3390/ijms24097982.
    doi: 10.3390/ijms24097982pmc: PMC10178277pubmed: 37175687google scholar: lookup
  43. Liu H., Shi M., Li X., Lu W., Zhang M., Zhang T., Wu Y., Zhang Z., Cui Q., Yang S., et al. Adipose mesenchymal stromal cell-derived exosomes prevent testicular torsion injury via activating PI3K/AKT and MAPK/ERK1/2 pathways. Oxidative Med. Cell. Longev. 2022;2022:8065771. doi: 10.1155/2022/8065771.
    doi: 10.1155/2022/8065771pmc: PMC9225846pubmed: 35757503google scholar: lookup
  44. Wang D., Lu H., Bin B., Lin S., Wang J. Quantitative proteomics reveal the protective effects of Qiang Jing decoction against oligoasthenospermia via modulating spermatogenesis related-proteins. Transl. Androl. Urol. 2024;13:2268. doi: 10.21037/tau-24-327.
    doi: 10.21037/tau-24-327pmc: PMC11535736pubmed: 39507870google scholar: lookup

Citations

This article has been cited 0 times.
Subscribe to our newsletter
Join 99,101 horse owners like you who receive our email updates on horse health and nutrition.