摘要
本试验以杜洛克猪为研究对象,用0.5ml细管精液冷冻剂型,对猪精液冷冻中的过氧化损伤,以及加入抗氧化剂后对精液质量的影响进行了研究。旨在进一步揭示猪精液冷冻过程中的过氧化损伤机理,筛选出较好的抗氧化剂加入冷冻稀释液,提高冷冻-解冻后精液质量。
用活性氧初级荧光测定试剂盒检测精子冷冻过程中活性氧含量的变化,平衡后,活性氧含量有轻微升高,冷冻-解冻后则急剧升高(P＜0.01)。分别检测鲜精、平衡后精液及冷冻-解冻后精液质量指标:活率、活力、顶体完整性、线粒体活性,精液质量指标极显著下降(P＜0.01)。用SPSS 13.0分析发现精子中活性氧的含量与精子质量指标呈极显著强负相关,活率、活力、顶体完整性、线粒体活性与ROS含量相关系数分别为-0.879、-0.958、-0.819、-0.960。
比较不同的离心力、时间:2400×g,离心3min,800×g,离心10min。2400×g,离心3min能显著提高精子活率、活力、线粒体活性(P＜0.05)。
首次在猪精液冷冻稀释液中加入褪黑素,浓度分别为0.125mg/ml,0.25mg/ml,0.5mg/ml。加入0.25mg/ml褪黑素能显著提高精子活力、顶体完整率、线粒体活性(P＜0.05)。
在猪精液冷冻稀释液中分别加入谷胱甘肽1mmol/L,5mmol/L,及10mmol/L,结果5mmol/L的谷胱甘肽能极显著提高精子冻后活率、活力、线粒体活性(P＜0.01)。1mmol/L、5mmol/L的谷胱甘肽均能显著提高冻后精子顶体完整性(P＜0.05),但是两浓度之间差异不显著。
首次对于褪黑素、谷胱甘肽及维生素C的联合添加采取正交设计的试验方法,用SPSS 13.0分析,褪黑素与谷胱甘肽的交互作用显著,而其余两两组合之间交互作用不显著。同时发现维生素C的添加应避免稀释液中亚铁离子和亚铜离子的存在,以免催化维生素C的氧化,产生过氧化氢而损害质膜。
进一步分析褪黑素和谷胱甘肽的联合添加效应,加入0.125mg/ml的褪黑素及1mmol/L的谷胱甘肽能显著提高解冻后精子活率、活力、顶体完整性和线粒体活性(P＜0.05)。褪黑素与谷胱甘肽的联合添加比谷胱甘肽及褪黑素单独添加效果好。
综上所述,本试验推荐以2400×g,离心3min,联合添加0.125mg/ml的褪黑素及1mmol/L的谷胱甘肽能取得较好的试验效果。
The present research studied the peroxidation damage of boar semen and the effect of addition of antioxidants on the semen quality in cryopreservation and intend to reveal the mechanism of peroxidation damage and screening better antioxitants added to the extender thus improve the quality of boar semen after freezen-thawed.
The change of ROS in cryopreservation was detected by primary fluorescence detection kit. After equilibrium,the content of ROS slightly increased;after freezen-thawed,the content of ROS significantly increased(P＜0.01).
Detect the quality of fresh、after equilibrium、freezen-thawed semen respectively,we found that the quality of semen decreased significantly(P＜0.01).In addition,the negative correlation between the content of ROS and quality of boar semen was significantly through the use of SPSS 13.0,the correlation coefficient of viability,motility,acrosomal integrity,and mitochondrial activity were -0.879、-0.958、-0.819、-0.960 respectively.
The centrifugation tested were 2400×g for 3min and 800×g for 10min.,and we recommend the use of short-term centrifugation with a relatively high g-force(2400 3 g for 3 minutes) in boar sperm cryopreservation protocol,it can improve the motility、viability、and mitochondrial activity significantly(P＜0.05).
The addition of melatonin(0.125mg/ml,0.25 mg/ml,0.5 mg/ml) to the boar semen extender was the first time,when added 0.25 mg/ml melatonin can improve the motility、mitochondrial activity、and acrosomal integrity significantly(P＜0.05).
Added the readuced glutathione(lmmol/L,5 mmol/L,10 mmol/L)to boar semen extenders,5 mmol/L can improve motility、viability and mitochondrial activity significantly (P＜0.01).both the 1 mmol/L and 5 mmol/L can improve the acrosomal integrity significantly(P＜0.05),but there was no significant difference between the two concentration.
Use the orthogonal design first time to study the associated addition of melatonin、readuced glutathione and vitamine C.Through analysis,wo found that the interaction between melatonin and readuced glutathione was notablely(P＜0.05),and the interaction of other combination was not noteblely(P＜0.05).Morever,we should avoid the presense of Fe~(2+)and Cu~+ when add vitamine C to the extenders,in order to avoid catalysis the oxidation of vitamine C to produce H_2O_2 and damage plasmalemma.
Study the interaction between melatonin and readuced glutathione fuether,we found that added melatonin 0.125mg/ml,readuced glutathione lmmol/L can improve the motility、viability mitochondrial activity、and acrosomal integrity notablely(P＜0.05).
The effect of associated addition of melatonin、readuced glutathione was better than added independently.
After all,we recommend use centrifugation:2400×g for 3min,and add melatonin 0.125mg/ml,readuced glutathione 1mmol/L can obtain a good result.
引文
[1]McIntosh KA,Harding JC,Parker S,Ellis JA,Appleyard GD.Nested polymerase chain reaction detection and duration of porcine circovirus type 2 in semen with sperm morphologicalanalysis from naturally infected boars[J].Vet Diagn Invest2006;18:380-4.
[2]季维智,宿兵.遗传多样性研究的原理与方法[M].杭州:浙江科学技术出版社,1999,144.
[3]张志文编译.中国畜牧杂志,1999,35(1):55-60
[4]Polge C,Smith AU,Parkes AS.Revival of spermatozoa after vitrification and dehydration at low temperatures[J].Nature,1949,164:666.
[5]Polge C.Artificial insemination in pigs[J].Vet Pet,1956,68:62-76.
[6]Polge C,Salamon S,Wilmut I.Fertilizing capacity of frozen boar semen following surgical insemination[J].Vet Rec,1970,87:424-428
[7]Crabo B G,Einarsson S.Fertility of deep frozen boar spermatozoa[J].Acta vetscand,1971,12:125-127.
[8]Graham EF,Rajamannan AHJ,Schmel MKL,Maki-Laurila M.Bower RE.Preliminary report on a procedure and rationale for freezing boar semen[J].A I Digest,19718 19:12-14.
[9]Pursel VG,Johnson LA.Fertility with frozen boar semen[J].Anim Sci,1971,33:265.
[10]Salamon S,Visser D.Fertility test of frozen boar spermatozoa[J].Aust J Biol Sci.1973,26(1):291-293.
[11]Pursel VG,Johnson LA.Freezing of boar spermatozoa:Fertilizing capacity with concentrated semen and a new thawing procedure[J].Anim Sci,1975,40:99-102.
[12]Yi Y J,Im G S,Park C S.Lactose-egg yolk diluter suppamented with N-acetyl-D-glucosamine affect acrosome morphology and motility of frozen-thawed boar sperm[J].Animal Rep roduction Science,2002,74(3/4):187-194.
[13]秦鹏春,惠芝.猪精子冷冻损伤的研究[J].猪与禽,1997,(1):33-36.
[14]De Leeuw FE,Colenbrander B,Verkleij AJ.The role membrane damage plays in cold shock and freezing injury[J].Reprod.Domest.Anim.,1990,1:95-104.
[15]Parks JE.Hypothermia and mammalian gametes[M].In:Karow AM,Critser JK(eds),Reproductive Tissue Banking.San Diego:Academic Press,1997:229-261.
[16]Darin-Bennett A,White AG.Influence of the cholesterol content of mammalian spermatozoa on susceptibility to cold shock[J].Cryobiology 1977; 14:466-70.
[17] Parks JE, Lynch DV. Lipid composition and thermotropic phase behavior of boar, bull, stallion, and rooster sperm membranes[J].Cryobiology 1992;29:255-66.
[18] Drobnis EZ, Crowe LM, Berger T, Anchordoguy TJ, Overstreet JW, Crowe JH. Cold shock damage is due to lipid phase transitions in cell membranes: a demonstration using sperm as amodel[J]. Exp Zool 1993;265:432-7.
[19] Janice L. Bailey Christian Lessard, Joannie Jacques,et al. Cryopreservation of boar semen and its future importance to the industry[J]. Theriogenology xxx (2008) xxx-xxx
[20] Petrunkina AM, Volker G, Weitze KF, Beyerbach M, To¨pfer-Petersen E, Waberski D. Detection of cooling-induced membranechanges in the response of boar sperm to capacitating conditions[J]. Theriogenology 2005;63:2278-99.
[21] Bailey JL, Bilodeau JF, Cormier N. Semen cryopreservation indomestic animals: a damaging and capacitating phenomenon[J]. Androl 2000;21:l-7.
[22] Aitken RJ. Free radicals, lipid peroxidation and sperm function[J].Reprod Fertil Dev 1995;7:659-68.
[23] Storey BT. Biochemistry of the induction and prevention oflipoperoxidative damage in human spermatozoa[J]. Mol HumReprod 1997;3:203-13.
[24] Chatterjee S, de Lamirande E, Gagnon C. Cryopreservation alters membrane sulfhydryl status of bull spermatozoa: protection by oxidized glutathione[J]. Mol Reprod Dev 2001;60:498-506.
[25] Joaqui'n Gadea, Elena Selle's, Marco Antonio Marco,et,al. Decrease in glutathione content in boar spermafter cryopreservation. Effect of the addition of reduced glutathione to the freezing and thawing extenders[J]. Theriogenology 62 (2004) 690-701.
[26] H.D. Guthrie, G.R. Welch, J.A. Long. Mitochondrial function and reactive oxygen species action in relation to boar motility[J]. Theriogenology 70 (2008) 1209-1215
[27] Chandel NS, Budinger GR. The cellular basis for diverse responses to oxygen[J]. Free Radic Biol Med 2007;42:165-74.
[28] Mishra DP, Shasha C. Estrogen induced spermatogenic cell apoptosis occurs via the mitochondrial pathway[J]. Biol Chem2005;280:6181-96.
[29] Wronsky R, Golob N, Grygar E, Winsdish M. Two colorfluorescence based microplate assay for apoptosis detection[J].Biotechniques 2002;32:666-8.
[30]J.M.Moran,L.Madejo'n,C.Ortega Ferrusola,et,al.Nitric oxide induces caspase activity in boar spermatozoa[J].Theriogenology 70(2008) 91-96.
[31]Desrosiers P,Legare C,Leclerc P,et al.Membranous andstructural damage that occur during cryopreservation of human sperm may be time-related events[J].Fertil Steril,2006,85(6):1744-1752.
[32]胡建宏.猪精液冷冻保存研究.西北农林科技大学博士论文[D],杨凌,,2006,3-16.
[33]Huang S Y,Kuo Y H,LeeW C,et al.Substantial decrease ofheat 2shock protein 90 precedes the decline of sperm motilityduring cooling of boar spermatozoa[J].Theriogenology,1999,51:1007-1016.
[34]Zeng W X,Shimada M,Isobe N,et al.Survival of boarspermatozoa in diluents of varying osmolality[J].Theriogenology,2001,56:447-458
[35]金一,魏世宝,王晓明等.精液冷却过程中应激蛋白90对猪精子活动力的影响.黑龙江畜牧兽医,2006,8:36-37.
[36]Martin G,Cagnon N,Sabido O,et al.Kinetics of occurrence ofsome features of apop tosis during the cryop reservation process of bovine spermatozoa[J].Hum Rep rod,2007,22(2):380-388.
[37]CaoW L,Xiang Z Q,Wang Y X.Protein changes of humandemonstrated by two 2 dimensional electrophoresis before and after cryop reservation[J].National Journal of Andrology,2003,17(1):18-21.
[38]San-Yuan Huang,Csaba Pribenszky,You-Hai Kuo d,et,al.Hydrostatic pressure pre-treatment affectsthe protein profile of boar sperm beforeand after freezing-thawing[J].Animal Reproduction Science xxx(2008) xxx-xxx.
[39]Thurston LM,Siggins K,Mileham AJ,Watson PF,Holt WV.Identification of amplified restriction fragment length polymorphism markers linked to genes controlling boar sperm viability following cryopreservation[J].Biol Reprod 2002;66:545-54.
[40]J.Roca,M.Hernandez,G.Carvajal,J,et,al.Factors influencing boar sperm cryosurvival[J].Anim Sci 2006.84:2692-2699.
[41]李青旺,江中良,王立强等.冻前预处理对猪精液冷冻效果的影响[J].动物生物技术学报,2004,1:502-506.
[42]Gema Carvajal,Cristina CuellO,Ariaruiz,et al.Effects of centrifugation before freezing on boar sperm cryosurvival[J].Journal of Andrology,2004,25:389-396.
[43]C.Matas,G.Decuadro,S.Martinez-Miro,J.Gadea.Evaluation of a cushioned method for centrifugation andprocessing for freezing boar semen[J].Theriogenology 67(2007) 1087-1091
[44]Fraser,A.Dziekon'ska,R.Strzez'ek,et,al.Dialysis of boar semen prior to freezing-thawing:Its effects on post-thaw sperm characteristics[J].Theriogenology 67(2007) 994-1003.
[45]Ignacio Caballero,Juanm.Vazquea,Mariaa.Gil,et,al.Does Seminal Plasma PSP-Ⅰ/PSP-ⅡSpermadhesin Modulatethe Ability of Boar Spermatozoa to PenetrateHomologousOocytes In Vitro?[J].Journal of Andrology,Vol.25,No.6,November/December 2004
[46]L.Ramio-Llucha,S.Balasch,S.Bonet,et,al.Effects of filtration through Sephadex columns improve overall quality parameters and "in vivo" fertility of subfertile refrigerated boar-semen[J].Animal Reproduction Science xxx(2009) xxx-xxx.
[47]Fiser PS,Fairfull RW.Combined effect of glycerol concentration and cooling velocity on motility and acrosomal integrity of boar spermatozoa frozen in 0.5 ml straws[J].Molecular Reproduction Development,1996,25:123-129.
[48]A.Medrano,P.F.Watson and W.V.Holt.Importance of cooling rate and animal variability for boar spermcryopreservation:insights from the cryomicroscope.[J]Reproduction(2002) 123,315-322
[49]Cabrita E.,Robles V.,Alvarez R.,et al.Cryopreservation of rainbow trout sperm in large volume straws:application to large scale fertilization[J].Aquaculture,2001,201(3-4):301-314.
[50]Cordova-Izquierdo,Oliva,Lleo,Garcia-Artiga,Corcuera,Perez-Gutierrez.Effect of different thawing temperatureson the viability,in vitro fertilizing capacityand chromatin condensation of frozenboar semen packaged in 5ml straws[J].Animal Reproduction Science 92(2006) 145-154.
[51]Marta Hernandez.The cryopreservation of boar semen in 0.5ml straws[J].Animal Reproduction Science 23(2006) 212-120.
[52]Bianchi,Calderam,Maschio,et al.Evaluation of amides and centrifugation temperaturein boar semen cryopreservation[J].Theriogenology,2008,69:632-638.
[53]胡建宏,李青旺,江中良.海藻糖对猪精液冷冻保存效果的影响[J].畜牧兽医学报,2006,37(12),297-1303.
[54]Graham,J.K.,Foote,R.H.,1987.Effect of several lipids fatty acyl chain length and degree of unsaturation on the motility of bull spermatozoa after cold shock and freezing[J].Cryobiology 24,42-52.
[55]Demianowicz,W.,Strezek,J.,1996.The effect of lipoprotein fraction of egg yolk on some of the biological properties of boar spermatozoa during storage of the semen in liquid state.Reprod.Domest[J].Anim.31,279-280.
[56]Moussa,M.,Martinet,V.,Trimeche,A.,Tainturier,D.,Anton,M.,2002.Low density lipoproteins extracted from hen egg yolk by an easy method:cryoprotective effect on frozen-thawed bull semen[J].Theriogenology 57,1695-1706.
[57]Hu,J.-H.,Li,Q.-W.,Li,G.,Chen,X.-Y.,Yang,H.,Zhang,S.-S.,Wang,L.-Q.,2006.The cryoprotective effect on frozen-thawed boar semen of egg yolk low density lipoproteins.Asian-Aust[J].Anim.Sci.19,486-490.
[58]WenXian Zeng,Takato Terada.Freezability of boar spermatozoa is improved by exposure to 2-hydroxypropyl-beta-cyclodextrin[J].Reprod.Fertil.Dev.,2000,12,223-228.
[59]WenXian Zeng,Takato Terada.Effects of Methyl-Beta-Cyclodextrin on Cryosurvival of Boar Spermatozoa[J].Journal of Andrology,Vol.22,No.1,January/February 2001
[60]Satorre,Breininger,Beconi,Beorlegui.a-Tocopherol modifies tyrosine phosphorylation andcapacitation-like state of cryopreserved porcine sperm[J].Theriogenology 68(2007) 958-965.
[61]He L,Bailey JL,Buhr MM.Incorporating lipids into boar sperm decreases chilling sensitivity but not capacitation potential[J].Biology of Roprod,2001,64:69-79.
[62]任俊玲.冷冻稀释液中添加抗氧化剂对猪精液冷冻保存效果的影响[D].硕士学位论文,雅安:四川农业大学,2007.
[63]商学军,黄宇烽,叶章群等.褪黑素在活性氧致精子线粒体功能损伤中的保护作用[J].中华男科学杂志,2004,10(8):604-607.
[64]马忠兰.不同稀释液对猪精液保存效果的研究[J].黑龙江畜牧兽医,2004,(4):31-32.
[65]Bathgate,Maxwell,Evans.Effects of platelet-activating factor and platelet-activating factor:acetylhydrolase on in vitro post-thaw boar sperm parameters[J].Theriogenology 67(2007)886-892.
[66]Pena,Johannisson,Wallgren,Rodriguez-Martinez.Effect of hyaluronan supplementation on boarsperm motility and membrane lipid architecturestatus after cryopreservation[J].Theriogenology 61(2004) 63-70.
[67]Rodriguez-Gil,J.E.,Rigau,T.,1995.Effects of slight agitation on the quality of refrigerated boar semen[J].Anim.Reprod.Sci.39,141-146.
[68]Suzuki,K.,Asano,A.,Eriksson,B.,Niwa,K.,Nagai,T.,Rodriguez-Martinez,H.,2002.Capacitation status and in vitro fertility of boar spermatozoa:effects of seminal plasma,cumulus-oocyte-complexes-conditioned medium and hyaluronan[J].Int.Androl.25,84-93.
[69]M.Yeste,M.Briz,E.Pinart,S.Sancho.Hyaluronic acid delays boar sperm capacitation after 3days of storage at 15℃[J].Animal Reproduction Science 109(2008) 236-250
[70]周佳勃,岳奎忠,孙兴参.猪精液冷冻技术的研究[J].中国兽医学报,2002,22(3):295-298.
[71]项智峰,王清华,张金洲等.稀释液·清洁剂 冷冻程序对猪精液冷冻效果的影响[J].安徽农业科学,2005,33(8):1874-1875.
[72]Joaquin Gadea,David Gumbao,Carmen mata's.Supplementation of the Thawing Media With ReducedGlutathione Improves Function and the In Vitro FertilizingAbility of Boar Spermatozoa After Cryopreservation[J].Journal of Andrology,2005,26:749-756.
[73]Johnson LA.Fertility results using frozen boar sperma-tozoa 1970-1985.In:Johnson LA,Larsson K.(Eds.),Deep Freezing of Boar Semen,SLU,Uppsala,Sweden,1985,pp.199-220.
[74]江中良.猪精液冷冻保存技术研究[D].硕士学位论文.杨凌:西北农林科技大学,2003
[75]Woelders H.,Matthijs A.,Zuidberg C.A.,et al.Cryopreservation of boar semen:equilibrium freezing in the cryomicroscope and in straws[J].Theriogenology,2005,63(20):383-395.
[76]Tuempong Wongtawan,Fernando Saravia,Margareta Wallgren.Fertility after deep intra-uterine artificial insemination of concentrated low-volume boar semen doses[J].Theriogenology,2006,65(4):773-787.
[77]李林林,猪精液5ml细管冷冻保存技术研究[D].硕士学位论文.雅安:四川农业大学,2006
[78]采克俊,司维,李亚辉等.流式细胞术在哺乳动物精液质量检测中的应用[J].动物学研究2003,Aug.24(4):311 317
[79]Garner DL,Thomas CA,Joerg HW,et al.Fluorometric assess2ments of acrosomal integrity and viability in cryopreserved bovinespermatozoa[J].Biology of Reproduction,1997,56(4):1401-1407.
[80]Garner DL,Thomas CA,Joerg HW,et al.Fluorometric assess2ments of mitochondrial function and viability in cryopreservedbovine spermatozoa[J].Biol Reprod,1997,57(6):1401-1406.
[81]Yanagimachi R,Lopata A,Odom CB,et al.Retention of biologic characteristics of zona pellucida in highly concentrated salt solution:the use of salt2stored eggs for assessing the fertilizing capacity of spermatozoa[J]. Fertil Steril, 1979 , 31 (5): 562-574.
[82] H. D. Guthrie2 and G. R. Welch. Determination of intracellular reactive oxygen species and high mitochondrialmembrane potential in Percoll-treated viable boar sperm using fluorescence-activated flow cytometry[J]. Anim Sci 2006. 84:2089-2100.
[83] Mennella MR, Jones R. Properties of spermatozoal superoxidedismutase and lack of involvement of superoxides in metal-ioncatalysedlipid-peroxidation and reactions in semen[J]. Biochem 1980;191:289-97
[84] Roca J, Maria J, Guez R, et al. Survival and in vivo fertility ofboar spermatozoa frozenin the presence of superoxide dismutase and/or catalase[J]. J Androl, 2005, 26(1): 15- 24.
[85] Katkov II, Mazur P. Influence of centrifugation regimes on motility, yield and cell associations of mouse spermatozoa[J]. Androl. 1998;19:232-241.
[86] Alvarez JG, Lasso JL, Blasco L, Nunez RC, Heyner S, Caballero P, Storey BT. Centrifugation of human spermatozoa induces sublethal damage; separation of human spermatozoa from seminal plasma by a dextranswim-up procedure without centrifugation extends their motile life[J].Hum Reprod. 1993;8:1087-1092.
[87] Abidor IG, Li LH, Hui SW. Study of cell pellets. I. Electrical propertiesand porosity[J]. Biophys . 1994;67:418-426.
[88] Eriksson BM, Petersson H, Rodri'guez-Marti'nez H. Field fertility with exported boar semen frozen in the new flatpack container[J]. Theriogenology.2002;58:1065-1079.
[89] Garcia EM, Vazquez JM, Parrilla I, Calvete JJ, Sanz L, Caballerol, et al. Improving the fertilizing ability of sex sorted boarspermatozoa[J]. Theriogenology 2007;68:771-8.
[90] Miguel Martin, Manuel Maci'AS, Germaine Escames,et al. Melatonin but not vitamins C and E maintainsglutathione homeostasis in t-butyl hydroperoxideinduced mitochondrial oxidative stress[J]. FASEB . 14,1677-1679.
[91] Li, X.J., Zhang, L.M., Gee, J., Zhang, A.Z., Sun, F.Y., 1997.Melatonin decreases production of hydroxyl radical during ischemia-reperfusion. Acta Pharmacol[J]. Sinica 18,394-396.
[92] Blanchard, B., Pompon, D., Ducrocq, C, 2000. Nitrosation of melatonin by nitric oxide and peroxynitrite[J]. Pineal Res. 29, 184-192.
[93] Ianas O, Olivescu R, Badescu I. Melatonin involvement in oxidative processes[J]. Rom Endocrinol29:117-123;1991.
[94]Tan,D.X.,Manchester,L.C.,Reiter,R.J.,Plummer,B.F.,Limson,J.,Weintraub,S.T.,Qi,W.,2000b.Melatonin directly scavenges hydrogen peroxide:a potentially new metabolic pathway of melatonin biotransformation[J].Free Radic.Biol.Med.29,1177-1185.
[95]易康乐,谢英,黄卫红等.水牛冷冻精液稀释液中添加SOD、GSH对精子品质的影响[J].广西畜牧兽医,2005,21(5):201-204.
[96]张文举,杨军祥,黄全威等.谷胱甘肽提高牛冻精活力及受胎率的研究[J].甘肃畜牧兽医,1997,27(2):12-13.
[97]R.Groβfeld,B.Sieg,C.Struckmann,et al.New aspects of boar semen freezing strategies[J].Theriogenology 70(2008) 1225-1233.
[98]赵晓娥,王光亚,王希朝.布尔山羊精液保存条件的试验研究.动物医学进展[J],1999,20(2):31-33.
[99]Foote RH,Brockett CC,Kaproth MT.Motility and fertility of bull sperm in whole milk extender containing antioxidants[J].Anim Reprod Sci,2002,71(1-2):13-23.
