荷斯坦公牛X和Y精子核形态差异及其影响因素分析

doi:10.11843/j.issn.0366-6964.2022.03.010

摘要/Abstract

摘要： 旨在分析荷斯坦公牛X和Y精子核形态(包括精子大小和形状)及其差异,探究流式分选及分选后冷冻对公牛X和Y精子核形态的影响。本研究使用荧光显微镜拍摄15头年龄相当、健康状况良好的荷斯坦公牛X和Y精子冷冻前后及新鲜精液分选前后共6类精子核图像,利用Image J插件Nuclear Morphology Analysis分析3.7万个精子核图像与形态相关的8个指标信息。结果显示,X和Y精子核形态具有显著性差异(P<0.05)的公牛数量比例少于50%。流式分选后8头(8/11)公牛的精子核面积、周长和长度发生显著性变化,6头(6/11)公牛的精子核宽度发生显著性变化;流式分选后5头(5/11)公牛的精子核椭圆度、伸长度和规则度和3头(3/11)公牛的精子核圆度发生显著性变化。冷冻后11头(11/14)公牛的精子核面积和周长存在显著性差异,9头(9/14)公牛的精子核长度、10头(10/14)公牛的精子核宽度存在显著性差异;5头(5/14)公牛的精子核圆度有显著性差异,6头(6/14)公牛的精子核规则度、伸长度和椭圆度有显著性差异。冷冻前X和Y精子核大小具有显著差异的只有4头公牛,冷冻后精子核大小具有显著性差异的公牛增加到了8头。本研究分析了大量精子核图像的形态信息,从而明确了流式分选和冷冻对荷斯坦公牛精子核大小有显著影响,冷冻对荷斯坦公牛Y精子核大小的影响程度高于X精子,该研究结果将为性控精子分选、精子冷冻等生产技术改进提供参考,对公牛生产性能和奶牛经济效益的提高具有重要意义。

关键词: 精子核大小, 精子核形态, 精子流式分选, 精子冷冻, 荷斯坦公牛

Abstract: This study was conducted to analyze the differences of nuclear morphology (including sperm size and shape) between Holstein bull X and Y sperm, clarifying the effects of freezing and flow sorting on the nuclear morphology of bull X and Y sperm. Six types of sperm nuclei images from 15 healthy Holstein bulls with the same age were captured by fluorescence microscopy for fresh and frozen non-sexed sperm, and fresh and frozen X and Y sperm. The Image J plug-in Nuclear Morphology Analysis was used to analyze the 8 indices information related to the morphology of 37 000 sperm nuclei images. The results showed that the proportion of bulls with significant difference in sperm nucleus morphology between X and Y (P<0.05) was less than 50%. After flow sorting, the sperm nucleus area, perimeter and length of 8 (8/11) bulls, the width of 6 (6/11) bulls changed significantly|The ellipticity, elongation and regularity of sperm nuclei in 5 (5/11) bulls and the circularity of sperm nuclei in 3 (3/11) bulls changed significantly. After freezing, there were significant differences in sperm nuclear area and perimeter among 11 (11/14) bulls, the length of sperm nuclei among 10 (10/14) bulls, width of sperm nuclei among 9 (9/14) bulls changed significantly|And there were significant differences in sperm nucleus circularity among 5 (5/14) bulls, regularity, elongation and ellipticity of sperm nuclei among 6 (6/14) bulls changed significantly. Only 4 bulls had significant differences in the size of X and Y sperm nuclei before freezing, but added to 8 bulls after freezing. Based on the analysis of morphological information from a large number of sperm nuclear images, it could be clarified that flow sorting and freezing had the significant impact on the nuclear size of Holstein bull sperm, and the impact of freezing on the nuclear size of Holstein bull Y sperm was higher than that of X sperm. The research will provide a reference for the improvement of sex-controlled sperm sorting and sperm freezing, and are of great significance to the improvement of bull production performance and cow economic benefit.

Key words: size of sperm nuclei, sperm nuclei morphology, sperm flow sorting, sperm freezing, Holstein bull

中图分类号:

S823.3

丁凤玲, 上官爱哨, 周扬, 丁瑞, 孙伟, 李喜和, 张淑君. 荷斯坦公牛X和Y精子核形态差异及其影响因素分析[J]. 畜牧兽医学报, 2022, 53(3): 766-777.

DING Fengling, SHANGGUAN Aishao, ZHOU Yang, DING Rui, SUN Wei, LI Xihe, ZHANG Shujun. Morphological Differences of X and Y Sperm Nuclei and Their Influencing Factors in Holstein Bulls[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(3): 766-777.

参考文献 41

[1]	VICENTE-FIEL S,PALACÍN I,SANTOLARIA P,et al. In vitro assessment of sperm quality from rams of high and low field fertility[J].Anim Reprod Sci,2014,146(1-2):15-20.
[2]	HILTPOLD M,KADRI N K,JANETT F,et al.Autosomal recessive loci contribute significantly to quantitative variation of male fertility in a dairy cattle population[J].BMC Genomics,2021,22(1):225.
[3]	MAROTO-MORALES A,RAMÓN M,GARCÍA-ÁLVAREZ O,et al.Sperm head phenotype and male fertility in ram semen[J]. Theriogenology,2015,84(9):1536-1541.
[4]	RAMÓN M,SOLER A J,ORTIZ J A,et al.Sperm population structure and male fertility:an intraspecific study of sperm design and velocity in red deer[J].Biol Reprod,2013,89(5):110.
[5]	ANDRASZEK K,BANASZEWSKA D,SZELESZCZUK O,et al.Morphometric characteristics of the spermatozoa of blue fox (Alopex lagopus) and silver fox (Vulpes vulpes)[J].Animals,2020,10(10):1927.
[6]	FILIMBERTI E,DEGL'INNOCENTI S,BORSOTTI M,et al.High variability in results of semen analysis in andrology laboratories in Tuscany (Italy):the experience of an external quality control (EQC) programme[J].Andrology,2013,1(3):401-407.
[7]	CZUBASZEK M,ANDRASZEK K,BANASZEWSKA D,et al.The effect of the staining technique on morphological and morphometric parameters of boar sperm[J].PLoS One,2019,14(3):e0214243.
[8]	侯胜奎,陶晨雨,胡慧艳,等.动物性控精液的分离及纯度评价方法的研究进展[J].中国畜牧杂志,2019,55(4):12-16,24.HOU S K,TAO C Y,HU H Y,et al.Advances in separation and purity evaluation methods of sex-controlled of animal semen[J].Chinese Journal of Animal Science,2019,55(4):12-16,24.(in Chinese)
[9]	何琪富,王宇飞,张康,等.性控奶山羊精液X和Y精子分离比例检测方法的建立[J].畜牧兽医学报,2021,52(1):107-115.HE Q F,WANG Y F,ZHANG K,et al.Establishment of assay for detecting the ratio of X and Y sperm separation in gender controlled dairy goat[J].Acta Veterinaria et Zootechnica Sinica,2021,52(1):107-115.(in Chinese)
[10]	侯胜奎,陶晨雨,胡慧艳,等.牛性控精液纯度快速评价方法研究[J].中国畜牧杂志,2019,55(8):62-66.HOU S K,TAO C Y,HU H Y,et al.Study on rapid evaluation method of bovine sex-controlled semen purity[J].Chinese Journal of Animal Science,2019,55(8):62-66.(in Chinese)
[11]	熊显荣,张雁,熊燕,等.利用流式细胞仪建立牦牛X、Y精子分选体系的研究[J].畜牧兽医学报,2021,52(2):399-407.XIONG X R,ZHANG Y,XIONG Y,et al.The establishment of X,Y sperm sorting system in yak by flow cytometer[J].Acta Veterinaria et Zootechnica Sinica,2021,52(2):399-407.(in Chinese)
[12]	SPINACI M,DE AMBROGI M,VOLPE S,et al.Assessment of some morphofunctional characteristics of flow-cytometrically sorted and stained boar spermatozoa[J].Vet Res Commun,2005,29(2):207-209.
[13]	SEIDEL JR G E.Update on sexed semen technology in cattle[J].Animal,2014,8(S1):160-164.
[14]	BALAO DA SILVA C M,ORTEGA FERRUSOLA C,MORILLO RODRIGUEZ A,et al.Sex sorting increases the permeability of the membrane of stallion spermatozoa[J].Anim Reprod Sci,2013,138(3-4):241-251.
[15]	SPINACI M,VOLPE S,BERNARDINI C,et al.Sperm sorting procedure induces a redistribution of Hsp70 but Not Hsp60 and Hsp90 in boar spermatozoa[J].J Androl,2006,27(6):899-907.
[16]	DA SILVA C B,ORTEGA-FERRUSOLA C,MORRELL J M,et al.Flow cytometric chromosomal sex sorting of stallion spermatozoa induces oxidative stress on mitochondria and genomic DNA[J].Reprod Domest Anim,2016,51(1):18-25.
[17]	王娜,张洁,海超,等.牛精子冷冻损伤及其改善方法[J].中国畜牧兽医,2021,48(6):2101-2112.WANG N,ZHANG J,HAI C,et al.Cryo-damage of bovine sperm and its improvement[J].China Animal Husbandry&Veterinary Medicine,2021,48(6):2101-2112.(in Chinese)
[18]	UGUR M R,ABDELRAHMAN A S,EVANS H C,et al.Advances in cryopreservation of bull sperm[J].Front Vet Sci,2019,6:268.
[19]	GRÖTTER L G,CATTANEO L,MARINI P E,et al.Recent advances in bovine sperm cryopreservation techniques with a focus on sperm post-thaw quality optimization[J].Reprod Domest Anim,2019,54(4):655-665.
[20]	WOJTUSIK J,WANG Y,PUKAZHENTHI B S.Pretreatment with cholesterol-loaded cyclodextrins prevents loss of motility associated proteins during cryopreservation of addra gazelle (Nanger dama ruficollis) spermatozoa[J].Cryobiology,2018,81:74-80.
[21]	YOON S J,RAHMAN M S,KWON W S,et al.Proteomic identification of cryostress in epididymal spermatozoa[J].J Anim Sci Biotechnol,2016,7(1):67.
[22]	ZHANG X G,HU S,HAN C,et al.Association of heat shock protein 90 with motility of post-thawed sperm in bulls[J]. Cryobiology, 2015,70(2):164-169.
[23]	SHANGGUAN A,ZHOU H,SUN W,et al.Cryopreservation induces alterations of miRNA and mRNA fragment profiles of bull sperm[J].Front Genet,2020,11:419.
[24]	ZHOU H,LIU J J,SUN W,et al.Differences in small noncoding RNAs profile between bull X and Y sperm[J].PeerJ,2020,8:e9822.
[25]	PABÓN D,MESEGUER M,SEVILLANO G,et al.A new system of sperm cryopreservation:evaluation of survival,motility, DNA oxidation, and mitochondrial activity[J].Andrology,2019,7(3):293-301.
[26]	CHEN X L,WANG Y G,ZHU H B,et al.Comparative transcript profiling of gene expression of fresh and frozen-thawed bull sperm[J].Theriogenology,2015,83(4):504-511.
[27]	OBLETTE A,RONDEAUX J,DUMONT L,et al.DNA methylation and histone post-translational modifications in the mouse germline following in-vitro maturation of fresh or cryopreserved prepubertal testicular tissue[J].Reprod BioMed Online,2019,39(3):383-401.
[28]	YADAV S K,GANGWAR D K,SINGH J,et al.An immunological approach of sperm sexing and different methods for identification of X-and Y-chromosome bearing sperm[J].Vet World,2017,10(5):498-504.
[29]	VICENTE-FIEL S,PALACÍN I,SANTOLARIA P,et al.A comparative study of sperm morphometric subpopulations in cattle, goat, sheep and pigs using a computer-assisted fluorescence method (CASMA-F)[J].Anim Reprod Sci,2013,139(1-4):182-189.
[30]	MARCO-JIMÉNEZ F,VIUDES-DE-CASTRO M P,BALASCH S,et al.Morphometric changes in goat sperm heads induced by cryopreservation[J].Cryobiology,2006,52(2):295-304.
[31]	QUAN G B,MA Y,LI J,et al.Effects of Hoechst33342 staining on the viability and flow cytometric sex-sorting of frozen-thawed ram sperm[J].Cryobiology,2015,70(1):23-31.
[32]	GARNER D L.Flow cytometric sexing of mammalian sperm[J].Theriogenology,2006,65(5):943-957.
[33]	CAMARA PIREZ M,STEELE H,REESE S,et al.Bovine sperm-oviduct interactions are characterized by specific sperm behaviour,ultrastructure and tubal reactions which are impacted by sex sorting[J].Sci Rep,2020,10(1):16522.
[34]	RATH D,BARCIKOWSKI S,DE GRAAF S,et al.Sex selection of sperm in farm animals:status report and developmental prospects[J].Reproduction,2015,149(3):X1.
[35]	BARBAS J P,LEAHY T,HORTA A E,et al.Sperm kinematics and subpopulational responses during the cryopreservation process in caprine ejaculates[J].Cryobiology,2018,82:137-147.
[36]	D'AMOURS O,FRENETTE G,BOURASSA S,et al.Proteomic markers of functional sperm population in bovines:comparison of low-and high-density spermatozoa following cryopreservation[J].J Proteome Res,2018,17(1):177-188.
[37]	DE CANIO M,SOGGIU A,PIRAS C,et al.Differential protein profile in sexed bovine semen:shotgun proteomics investigation[J]. Mol BioSyst,2014,10(6):1264-1271.
[38]	SCOTT C,DE SOUZA F F,ARISTIZABAL V H V,et al.Proteomic profile of sex-sorted bull sperm evaluated by SWATH-MS analysis[J].Anim Reprod Sci,2018,198:121-128.
[39]	AITKEN R J,BAKER M A,NIXON B.Are sperm capacitation and apoptosis the opposite ends of a continuum driven by oxidative stress?[J].Asian J Androl,2015,17(4):633-639.
[40]	WESTFALEWICZ B,DIETRICH M A,CIERESZKO A.Impact of cryopreservation on bull (Bos taurus) semen proteome1[J].J Anim Sci,2015,93(11):5240-5253.
[41]	NARESH S.Effect of cooling (4℃) and cryopreservation on cytoskeleton actin and protein tyrosine phosphorylation in buffalo spermatozoa[J].Cryobiology,2016,72(1):7-13.