Abstract：The testis is an important reproductive organ in male animals, capable of secreting hormones and producing sperm. Cellulase is a key factor in breaking down plant cell walls and enhancing the nutrient absorption ability of ruminants. The level of energy supply directly affects testicular development, so it is hypothesized that the addition of cellulase may have an effect on the testicular development of Hu sheep (Ovis aries) lambs. In order to study the effect of adding cellulase to starter feed on testicular development and spermatogenesis of male lambs of Hu sheep before sexual maturity, 24 10-day-old male Hu sheep lambs of similar body weight were selected. They were randomly divided into 2 groups of 12 lambs each. 0.1% cellulase was added to the feed of the experimental group, and 6 lambs were slaughtered at random in each group at 60 and 120 d of age. Testicular tissues were collected for gross dissection, histological observation, hormone determination, and the expression of hormone secretion-related genes and reproductive marker genes detection. The results showed that the testicular indices of Hu sheep lambs at 60 and 120 d of age were extremely significantly higher than those of the control group after the addition of cellulase to the starter diet (P<0.01), and the diameters of the seminiferous tubules increased extremely significantly (P<0.01), and the lumens of the fine tubules appeared by the 120 d of age, with a lumen area that was significantly larger than the lumen area of the control group (P<0.05). Spermatogenic epithelial thickness was extremely significantly (P<0.01) higher than that of the control group at 60 d of age, and the difference was not significant by 120 d of age. Androgen levels in the testes of lambs in the 60 and 120 d of age test groups were extremely significantly higher than those in the control group (P<0.001), and the mRNA expression levels of androgen synthesis-related genes (steroidogenic acute regulatory protein (StAR) and cytochrome P450 family 17 subfamily A member 1 (CYP17A1)) showed the same trend; and the mRNA expression levels of the germ cell marker genes of testis of lambs at 60 and 120 d of age DEAD-box helicase 4 (DDX4) and the gene of deleted in azoospermia like (DAZL) were the same as that of the other genes. Both genes exhibited significantly higher protein and mRNA expression levels compared to the control group (P<0.05). The above results showed that the addition of cellulase to the starter diet could promote the testicular development of male lambs of Hu sheep before sexual maturity, promote the development of the varicose fine tube, increase the level of androgen secretion, and then be able to improve the ability of spermatogenesis. This study provides a theoretical basis for the application of cellulase in open diets for young ruminants.
[1] 白曼, 刘兴旺, 王美桀, 等. 2024. 小尾寒羊公羊性机能发育及精液品质研究[J]. 中国畜牧杂志, 60(07): 226-231. (Bai M, Liu X W, Wang M J, et al.2024. Development of sexual function and semen quality of Small-tailed Han sheep rams[J]. Chinese Journal of Animal Science, 60(07): 226-231.) [2] 薄东东. 2016. 山羊睾丸生长向精子发生阶段过渡的分子遗传机理[D]. 博士学位论文, 福建农林大学, 导师: 姜勋平, pp. 16-18. (Bo D D.2016. Molecular genetic mechanisms of the transition from testicular growth to the spermatogenesis stage in goats[D]. Thesis for Ph.D., Fujian Agriculture and Forestry University, Supervisor: Jiang X P, pp. 16-18.) [3] 池圣清, 吴丽云, 吴家荣, 等. 2017.中草药添加剂对公猪精液品质、血清激素及基因表达量的影响[J]. 黑龙江畜牧兽医, (21): 138-140. (Chi S Q, Wu L Y, Wu J R, et al. 2017. Effects of herbal additives on semen quality, serum hormones and gene expression in boars[J]. Heilongjiang Animal Science and Veterinary Medicine, (21): 138-140.) [4] 付永. 2012. DAZL基因mRNA表达水平与犏牛雄性不育关系的研究[J].青海畜牧兽医杂志, 42(6): 9-11. (Fu Y.2012. Study on the relationship between mRNA expression level of DAZL gene and male sterility of Pian cattle[J]. Chinese Qinghai Journal of Animal and Veterinary Sciences, 42(6): 9-11.) [5] 胡湘云, 张村宇, 易显凤, 等. 2024. 4种粗饲料经纤维素酶发酵后产物的瘤胃降解特性研究[J]. 饲料研究, 47(09): 23-30. (Hu X Y, Zhang C Y, Yi X F, et al.2024. Ruminal degradation characteristics of the products of four types of roughages fermented by cellulase[J]. Feed Research, 47(09): 23-30.) [6] 蒋金书. 1991. 兔病学[M]. 北京农业大学出版社, 北京, pp. 32-33. (Jiang J S.1991. Rabbit Disease[M]. Beijing Agricultural University Press, Beijing, pp. 32-33.) [7] 孔令莹, 岳耀敬, 郑琛, 等. 2023. 湖羊及其与南丘羊杂交后代屠宰性能和肉质特性[J]. 食品科学, 44(23): 64-70. (Kong L Y, Yue Y J, Zheng C, et al.2023. Slaughtering performance and meat quality characteristics of Hu sheep and their offspring crossed with Nanqiu sheep[J]. Food Science, 44(23): 64-70.) [8] 李江源, 郭爱岩. 1992. 睾酮对下丘脑-垂体的反馈调节作用[J]. 军医进修学院学报,(02): 154. (Li J Y, Guo A Y. 1992. Feedback regulation of hypothalamus-pituitary by testosterone[J]. Journal of Chinese PLA Postgraduate Medical School, (02): 154.) [9] 李讨讨, 马友记, 赵兴绪, 等. 2018. DAZL基因在绵羊不同发育期睾丸组织中的表达与细胞定位[J].农业生物技术学报, 26(03): 421-428. (Li T t, Ma Y J, Zhao X X, et al.2018. Expression and cellular localization of DAZL gene in testicular tissues of sheep at different developmental stages[J]. Journal of Agricultural Biotechnology, 26(03): 421-428.) [10] 梁艺馨, 刘超, 牟希东, 等. 2024. 天然和合成虾青素对红白锦鲤生长性能的影响[J]. 水产养殖, 45(11): 21-27. (Liang Y X, Liu C, Mou X D, et al.2024. Effects of natural and synthetic astaxanthin on the growth performance of Cyprinus carpiohaematopterus[J]. Journal of Aquaculture, 45(11): 21-27.) [11] 庞静. 2019. 基于AMPK-ULK1自噬通路研究能量限制对公羊睾丸发育及HT-2毒素对精原干细胞增殖的影响[D]. 硕士学位论文, 南京农业大学, 导师: 张艳丽. pp. 29-43. (Pang J.2019. Study on the effects of energy restriction on testicular development and HT-2 toxin on spermatogonial stem cell proliferation in rams based on AMPK-ULK1 autophagy pathway[D]. Thesis for M.S., Nanjing Agricultural University. Supervisor: Zhang Y L, pp. 29-43.) [12] 任春燕, 郝志云, 邴睿, 等. 2025. 开食料添加纤维素酶对羔羊生产性能、器官发育、肌肉脂肪酸组成及血清抗氧化指标的影响[J/OL]. 草业学报, 1-12 (2025-01-17). http://kns.cnki.net/kcms/detail/62.1105.S.20250116.1717.002.html. (Ren C Y, Hao Z Y, Bing R, et al. 2025. Effects of adding cellulase to open feed on production performance, organ development, muscle fatty acid composition and serum antioxidant indexes in lambs[J/OL]. Acta Prataculturae Sinica, 1-12(2025-01-17). http://kns.cnki.net/kcms/detail/62.1105.S.20250116.1717.002.html [13] 苏杰. 2022. 出生后湖羊睾丸发育图谱、组织结构及X染色体剂量补偿研究[D]. 博士学位论文, 内蒙古农业大学, 导师: 曹贵方, 宋永利, pp. 58-61. (Su J.2022. Developmental mapping, organization and X-chromosome dosage compensation of testis in postnatal Hu sheep[D]. Thesis for Ph.D., Inner Mongolia Agricultural University, Supervisors: Cao G F, Song Y L, pp. 58-61.) [14] 孙胜祥, 张建明, 江小帆. 2024.不同生长阶段湖羊体重与体尺指标相关性研究[J]. 山东畜牧兽医, 45(04): 4-6. (Sun S X, Zhang J M, Jiang X F.2024. Correlation between body weight and body size indexes of Hu sheep at different growth stages[J]. Shangdong Journal of Animal Science and Veterinary Medicine, 45(04): 4-6.) [15] 孙伟. 2020.湖羊高效养殖生产技术[M]. 中国农业出版社, 北京, pp. 7. (Sun W. 2020. Efficient Breeding Production Technology of Hu Sheep[M]. China Agricultural Press, Beijing, pp. 7.) [16] 孙鑫铭, 柳杭, 孙丽敏, 等. 2024. PPARγ在性成熟前公羊生殖器官的表达差异分析[J/OL]. 经济动物学报, 1-5(2024-06-30). http://kns.cnki.net/kcms/detail/22.1258.S.20240618.1207.004.html. (Sun X M, Liu H, Sun L M,et al. 2024. Differential analysis of PPARγ expression in the reproductive organs of rams before sexual maturity[J/OL]. Journal of Economic Zoology, 1-5(2024-06-30). http://kns.cnki.net/kcms/detail/22.1258.S.20240618.1207.004.html [17] 田奎. 2023. 日粮中添加刺芋粉对小鼠和长白公猪生精功能影响的研究[D]. 硕士学位论文, 广西大学, 导师: 廖玉英, pp. 20-31. (Tian K.2023. Effects of dietary addition of taro powder on spermatogenesis in mice and long white boars[D]. Thesis for M.S., Guangxi University, Supervisor: Liao Y Y, pp. 20-31.) [18] 王根林. 1997. 下丘脑-垂体-性腺轴在动物生殖中的作用[J]. 畜牧与兽医, (04): 36-38. (Wang G L. 1997. Role of hypothalamic-pituitary-gonadal axis in animal reproduction[J]. Animal Husbandry and Veterinary Medicine, (04): 36-38.) [19] 王震. 2017. 能量限饲和补偿对湖羊生产性能和繁殖性能的影响[D]. 硕士学位论文, 南京农业大学, 导师: 王锋, 张艳丽, pp. 73-74. (Wang Z.2017. Effects of energy restriction and compensation on production performance and reproductive performance of Hu sheep[D]. Thesis for M.S., Nanjing Agricultural University, Supervisor: Wang F, Zhang Y L, pp. 73-74.) [20] 吴水德, 黄志勇, 邱思锋, 等. 2015.中草药制剂神农精粉对种公猪精液品质的影响[J]. 中兽医学杂志, (06): 6-9. (Wu S D, Huang Z Y, Qiu S F, et al. 2015. Effects of Chinese herbal preparation Shennong Jing Powder on semen quality of breeding boars[J]. Chinese Journal of Traditional Veterinary Science, (06): 6-9.) [21] 杨泽坤. 2017. 反刍动物专用复合酶制剂对奶牛瘤胃发酵、血液指标及生产性能的影响[D]. 硕士学位论文, 西北农林科技大学, 导师: 姚军虎, pp. 24. (Yang Z K. 2017. Effects of ruminant-specific compound enzyme preparation on rumen fermentation, blood indexes and production performance of dairy cows[D]. Thesis for M.S., Northwest Agriculture and Forestry University, Supervisor: Yao J H, pp. 24.) [22] 叶均安, 何世山. 2018.规模湖羊场精细化饲养管理技术及装备[M]. 中国农业科学技术出版社, 北京, pp. 1-40. (Ye J A, He S S.2018. Fine Feeding Management Technology and Equipment for Large-scale Hu Sheep Farms [M]. China Agricultural Science and Technology Press, Beijing, pp. 1-40.) [23] 尹鹏飞, 王继卿, 郝志云, 等. 2024. 开食料中添加纤维素酶对羔羊生长性能、瘤胃发酵和血清生化指标的影响[J].动物营养学报, 36(05): 3170-3182. (Yin P F, Wang J Q, Hao Z Y, et al.2024. Effects of adding cellulase to starter feed on growth performance, rumen fermentation and serum biochemical indexes of lambs[J]. Chinese Journal of Animal Nutrition, 36(05): 3170-3182.) [24] 张远, 陈祥, 张艳,等. 2023. CYP17A1基因敲低对山羊睾丸间质细胞睾酮合成和睾丸发育相关基因表达的影响[J].农业生物技术学报, 31(12): 2535-2544. (Zhang Y, Chen X, Zhang Y, et al.2023. Effect of CYP17A1 knockdown on the expression of genes related to testosterone synthesis and testicular development in testicular mesenchymal stromal cells of goats[J]. Journal of Agricultural Biotechnology, 31(12): 2535-2544.) [25] 赵健, 桂士良, 崔腾腾, 等. 2020.睾酮的生理作用及临床应用进展[J]. 中国性科学, 29(01): 20-24. (Zhao J, Gui S L, Cui T T, et al.2020. Physiological role and clinical application of testosterone[J]. Chinese Journal of Human Sexuality, 29(01): 20-24.) [26] 赵沛迪, 杨可, 蒋玉奇, 等. 2024. 日粮能量水平对绵羊睾丸发育和相关基因表达的影响[J]. 草业学报, 33(06): 219-226. (Zhao P D, Yang K, Jiang Y Q, et al.2024. Effects of dietary energy levels on testicular development and gene expression in sheep[J]. Acta Prataculturae Sinica, 33(06): 219-226.) [27] 周阳, 骆骅, 李伯江, 等. 2013. 牦牛和犏牛睾丸组织DDX4基因mRNA表达水平与启动子区甲基化[J]. 中国农业科学, 46(3): 630-638. (Zhou Y, Luo H, Li B J, et al.2013. Methylation of DDX4 gene mRNA expression level and promoter region in testicular tissues of yak and Pzekin[J]. Scientia Agricultura Sinica, 46(3): 630-638.) [28] 左建军. 2010. 科学自配猪饲料[M]. 化学工业出版社, 北京, pp. 212-214. (Zuo J J.2010. Scientific self-formulated pig feed[M]. Chemical Industry Press, Beijing, pp. 212-214.) [29] Abid K, Jabri J, Ammar H, et al.2020. Effect of treating olive cake with fibrolytic enzymes on feed intake, digestibility and performance in growing lambs[J]. Animal Feed Science and Technology‚ 261: 114405. [30] Aghazadeh Y, Zirkin B R, Papadopoulos V.2015. Pharmacological regulation of the cholesterol transport machinery in steroid ogenic cells of the testis[J]. Vitam Horm, 98: 189-227. [31] Alsersy H, Salem A Z M, Borhami B E, et al.2015. Effect of mediterranean saltbush (Atriplex halimus) ensilaging with two developed enzyme cocktails on feed intake, nutrient digestibility and ruminal fermentation in sheep[J]. Animal Science Journal, 86(1): 51-58. [32] Anderson R A, Fulton N, Cowan G, et al.2007. Conserved and divergent patterns of expression of DAZL, VASA and OCT4 in the germ cells of the human fetal ovary and testis[J]. BMC developmental biology, 7: 136. [33] Beattie M C, Adekola L, Papadopoulos V, et al.2015. Leydig cell aging and hypogonadism[J]. Experimental Gerontology, 68: 87-91. [34] Crichton S, Lishman A W, Hundley M, et al.1991. Role of dihydrotestosterone in the control of sexual behaviour on castrated male sheep[J]. Reproduction, 93(1): 9-17. [35] Elliott D J, Bourgeois C F, Klink A, et al.2000. A mammalian germ cell-specific RNA-binding protein interacts with ubiquitously expressed proteins involved in splice site selection[J]. Proceedings of the National Academy of Sciences of the USA, 97(11): 5717-5722. [36] Erba O, Sima Y A, Kebede A, et al.2020. Isolation of cellulose degrading bacteria from rumen and evaluation of cellulase production potential using lignocellulosic substrate[J]. Ethiopian Journal of Sciences and Sustainable Development, 7(1): 8-17. [37] Fonty G, Jouany J P, Chavarot M, et al.1991. Establishment of a limited rumen microflora in gnotobiotic lambs[J]. Reproduction, Nutrition, Development, 31(5): 521-528. [38] Hickford Danielle E, Frankenberg S, Pask A J, et al.2011. DDX4 (VASA) is conserved in germ cell development in marsupials and monotremes[J]. Biology of Reproduction, 85(4): 733-743. [39] Hu R X, Jiang X P, Liu G Q, et al.2022. Polygenic co-expression changes the testis growth, hormone secretion and spermatogenesis to prompt puberty in Hu sheep[J].Theriogenology, 194: 116-125. [40] Huang Z W, Xie L, Feng H, et al.2024. DAZL regulate germline, pluripotency, and proliferation related genes in chicken PGCs and cooperate with DDX4[J]. Theriogenology, 222: 22-30. [41] Jiang Z Y, Wan Y J, Li P, et al.2019. Effect of curcumin supplement in summer diet on blood metabolites, antioxidant status, immune response, and testicular gene expression in Hu sheep[J]. Animals, 9(10): 720. [42] Kang Z L, Qiao N, Liu G Y, et al.2019. Copper-induced apoptosis and autophagy through oxidative stress-mediated mitochondrial dysfunction in male germ cells[J]. Toxicol In Vitro, 61: 104639. [43] Lysiane D, Philippe R, Yacine L, et al.2023. Effects of rearing mode on gastrointestinal microbiota and development, immunocompetence, sanitary status and growth performance of lambs from birth to two months of age[J]. Animal Microbiome, 5(1): 34. [44] Montoto L G, Arregui L, Sánchez N M, et al.2012. Postnatal testicular development in mouse species with different levels of sperm competition[J]. Reproduction, 143(3): 333-346. [45] Redouane A, Alexandra E K, Natalia A,et al.2020. CYP17A1 deficient XY mice display susceptibility to atherosclerosis, altered lipidomic profile and atypical sex development[J]. Scientific Reports, 10(1): 8792. [46] Tanaka S S, Toyooka T, Akasu R, et al.2000. The mouse homolog of Drosophila Vasa is required for the development of male germ cells[J]. Genes & Development, 14(7): 841-853. [47] Vangompel M J W, XU E Y.2011. The roles of the DAZ family in spermatogenesis:More than just translation?[J]. Spermatogenesis, 1(1): 36-46. [48] Vijayaraghavan P, Arun A, Al-Dhabi N A, et al.2016. Novel Bacillus subtilis IND19 cell factory for the simultaneous production of carboxy methyl cellulase and protease using cow dung substrate in solid-substrate fermentation[J].Biotechnology for Biofuels, 9(1): 73-85. [49] Xu X, Holmes B, Bhangoo A, et al.2021. Chiari 1 malformation as complication of homozygous mutation of steroidogenic acute regulatory protein (StAR), seventeen years' follow up of 46 XY female[J]. Hormone Research in Paediatrics, 94(2): 10.
