摘要
选取24头年龄和体重相近、采精正常、生理状态基本一致的健康荷斯坦成年种公牛,采用两因子有重复试验设计,根据前期采精记录中射精量、精液密度、鲜精活力等指标进行分组,分别设30、150、300和600 IU/kg4个添加水平和春、(早)夏、秋、冬4个不同季节(阶段),每个处理6个重复,每个重复1头牛。
本文研究了VE不同添加水平在不同季节(阶段)对荷斯坦种公牛精液品质、血清抗氧化性能及血液超氧化物歧化酶(SOD)基因mRNA表达的影响。结果表明:
1、对精液品质的影响
在不同季节(阶段),VE添加水平对种公牛精液品质主要指标的影响有一定的差异。随着添加水平的提高,在春、早夏、秋三个季节(阶段),精液品质显著改善,尤其秋季表现最为明显,精子畸形率极显著下降(P<0.01),鲜精活力显著提高(P<0.05)。VE添加水平对种公牛全年精液品质有着显著的影响,其中对射精量和总精子数影响极显著(P<0.01),对鲜精活力和精液密度影响显著(P<0.05)。本试验条件下,根据拟合曲线模式,VE添加水平在340~380 IU/kg范围时可最大程度地降低因季节因素造成的精液品质下降,使全年精液品质保持较高的水平。
2、对血清抗氧化性能的影响
VE添加水平可极显著提高种公牛血清中的GSH-Px、T-AOC和抑制·OH能力(P<0.01)、显著提高SOD活性(P<0.05)、极显著降低MDA含量(P<0.01)。MDA含量的变化受季节因素影响较小,主要是VE添加水平的影响;在夏、秋季节,种公牛血清中GSH-Px、SOD活性、T-AOC和抑制·OH能力等抗氧化指标明显高于春、冬季节;根据拟合曲线模式,当VE添加范围为350~410IU/kg时,抗氧化指标受季节因素影响最小,机体抗氧化能力处于较高水平,基本上可满足各季节种公牛对VE的需求。
3、对血液SOD基因mRNA表达的影响
VE添加水平可极显著提高荷斯坦种公牛春季血液SOD1基因(P<0.01)、显著提高SOD3基因的mRNA表达水平(P<0.05)。随着添加水平的提高,SOD1和SOD3基因的mRNA表达水平呈先升高后下降的趋势。在本试验条件下,根据拟合曲线模式,330~380 IU/kg添加水平时达到高峰。
综合分析表明,成年荷斯坦种公牛精料补充料中VE适宜的添加水平为330~410 IU/kg,此时精料补充料中VE水平为385~445IU/kg。
Twenty-four breeding Holstein bulls assigned randomly to four groups were conducted to study the effects of different additional levels of vitamin E on semen quality, serum antioxidant properties and cloning and mRNA expression of SOD gene in Holstein bulls. The experimental bulls were divided into in 4 dietary treatments by two factors design, consisting of four additional levels of vitamin E which was 30, 150,300,600 IU/kg within 12 months. Each group included four stages for spring, (early) summer, autumn and winter. It showed that:
1 The effects on semen quality
The effects of different additional levels of vitamin E on semen quality in different stages were significant. With the increase of additional levels of vitamin E, semen quality in spring, early summer and autumn were significantly improved(P<0.05). And the greater improvement of semen quality was showed in autumn than the orther seasons. Sperm abnormalities rate decreased significantly(P<0.01), motility increased significantly(P<0.05) in autumn. According to quadratic curve fitting models, the optimal additional level of vitamin E to semen quality was 340~380 IU/kg in this experiment. In this additional level, it had the higher levels of semen quality the whole year and the low lost because of the season.
2 The effects on serum antioxidant properties
In different additional levels of vitamin E, it showed the extremely significant difference in serum GSH-Px, T-AOC, inhibiting OH ability and MDA(P<0.01), and significant in SOD(P<0.05). The main effect of MDA parameter was the different additional levels of vitamin E, not the season. Other antioxidant parameters including GSH-Px, T-AOC, SOD, inhibiting OH ability in summer and autumn were higher than in spring and winter. According to quadratic curve fitting models, in 350~410 IU/kg additional level of vitamin E, it could keep serum antioxidant capacity at a high level, and could meet the Stud bulls' requirement for whole year.
3 The effects of cloning and expression of SOD gene in blood
The supplemental of vitamin E could enhance the mRNA expression of SOD1 extremely (P<0.01), and SOD3 significantly(P<0.05) in Holstein bulls blood in spring. As the additional level added, the mRNA expression of SOD1 and SOD3 were both increased firstly and then decreased. According to quadratic curve fitting models, the mRNA expression of SOD1 and SOD3 gene were both reached the peak in 330~380 IU/kg additional level in this experiment.
It seemed that the optimal additional level of vitamin E of Holstein bulls was 330-410 IU/kg in this experiment.Total levels of vitamin E in the concentrate supplement is 385~445 IU/kg.
引文
[1]中华人民共和国农业部.NY/T 34-2004[S].中国人民共和国农业行业标准-奶牛饲养标准.北京:中国农业出版社,2004-08-25.
[2]孟庆翔主译.奶牛营养需要第7次修订版[M].北京:中国农业大学出版社,2001:232-234.
[3]Evans H M, Bishop K S. On the existence of a hitherto unrecognized dietary factor essential for reproduction[J]. Science,1922,56:650-651.
[4]Hidiroglou M, Jenkins K J, Wauthy J M, et al. Changes in plasma and milk tocopherol levels in beef cattle turned out to pasture[J]. Animal Production,1973,16:179.
[5]Alderson N E, Mttchell G E, Little C O, et al. Preintestinal disappearance of vitamin E in ruminants[J]. The Journal of Nutrition,1971,101:655-660.
[6]Leedle S K, Tatum J D, Smith G C, et al. Quality gains-due to vitamin E supplementation-from cattle in the Strategic Alliance Field Study[J]. Final Report,1993,12:35-49.
[7]Weiss W P, Hogan J S, Todhunter D A, et al. Effect of vitamin E supplementation in diets with a low concentration of selenium on mammary gland health of dairy cows[J]. Journal of Dairy Science.1997. 80:1728-1737.
[8]于芳.维生素E、维生素C与基因的稳定性[J].国外医学卫生学分册,2002,29(3):141-145.
[9]孙永叶.不同剂量维生素C、E抗氧化作用研究[D].硕士学位论文.青岛:青岛大学医学院营养研究所,2003.
[10]Kennedy T, Ghio A, Reed W, et al. Copper-dependent inflammation and nuclear factor activation by particulate air pollution[J]. American Journal of Respiratory Cell and Molecular Biology,1998,19: 366-378.
[11]陈东升,杨家驹,朱俊尔,等.急性缺氧对大鼠脂质过氧化的影响及补充维生素E的效应[J].营养学报,1997,19(1):28-31.
[12]祝素珍,李福吕,李强,等.维生素E与家兔生产[J].饲料广角,2002,8:25-28.
[13]任善茂,陶勇,张牧.维生素对母畜繁殖力的影响[J].饲料博览,2001,3:12-15.
[14]何河,方热军.日粮中添加VE对牛肉品质的调控[J].湖南饲料,2007,7:29-31.
[15]Konopacha M, Widel M, Rzeszowska-Wolny J. Modifying effect of vitamin C, E and beta-carotene against gamma-ray-induced DNA damage in mouse cells[J]. Mutation Research,1998,417(2-3): 85-94.
[16]Rotruck J T. Selenium:Biochemical role as a component of glutathione peroxidase[J].Science,1972, 179(2):588.
[17]陈瑾.血清T-SOD、GSH-Px、MDA、NO、OS水平与衰老的关系及康寿益胶[D].硕十学位论文.重庆:重庆医科大学,2003.
[18]张世珍,王兴亚,齐志明,等.硒和维生素E在硒缺乏动物自由基代谢中作用机制的研究[J].畜牧兽医学报,1997,28(4),311-317.
[19]Buckley J A, Wulf D M, Morgan J B, et al. Dietary supplementation of vitamin E to feedlot cattle affects beef retail display properties[J]. Food Sci,1995,60:250-255.
[20]Monahan F J, Buckley D J, Morrissey P A, et al. Influence of dietary fat and alpha-tocopherol supplementation on lipidoxidation in pork[J]. Meat Science,1992,31:229.
[21]Asghar K, Jakemanl P, Maxwell S. Effect of antioxidant vitamin supplementation on muscle function after eccentric exercise[J]. European Journal of Applied Physiology and Occupational Physiology, 1991,11(67):426-430.
[22]Cheah G, Schaefer D M, Williams S N, et al. Improvement of pigmentand lipid stability in Holstein steer beef by dietary supplementation with vitamin E[J]. Food Sci,1995,54:858-862.
[23]Asghar K, Maxwell S, Branen A L. Interaction of fat oxidation and microbial spoilage in muscle foods[J]. Reciprocal Meat Conf,1992,31:156-158.
[24]Munozt R N, Schaefer D M, Williams S N, et al. Tissue equilibration and subcellular distributionof vitamin E relative to myoglobin and lipid oxidation indisplayed beef[J]. Anim Sci,1996,71:105-111.
[25]刘丽玲,李涛,徐世文,等.VE对奶牛抗氧化功能的影响[J].黑龙江畜牧兽医,2000,3:6-7.
[26]汪求真,马爱国,孙永叶,等.大剂量VE对大鼠抗氧化和DNA损伤的影响[J].营养学报,2005,27(6):467-470.
[27]王福娣,蔡梅雪,祝寿高.硒、维生素E对血脂及脂质过氧化作用的影响[J].营养学报,1997,19(1):16.
[28]黄素珍,孟雪燕,古少鹏,等.维生素E对鸡体脂质过氧化的影响[J].中国兽医学报,1998,18(2):191.
[29]王晓霞,滑静.VC及VE对热应激蛋鸡生产性能及抗氧化功能的影响[J].北京农学院报.2000,4:37-40.
[30]Meydani S N, Meydani M, Bluberg J B, et al. Assessment of the safety of supplementation with different amounts of vitamin E in healthy older adults[J]. American Journal of Clinical Nutrition,1998, 68(2):311-318.
[31]黄海雄,张锦周,庄志雄,等.VE对三氯乙烯急性染毒大鼠肝脏脂质过氧化与抗氧化酶的影响[J].中国实验动物学杂志,1999,9(3):129-132.
[32]Nochcls P S, Inna K, Graham W, etal. Effect of vitamin E and selenium supplementation ofcockerel diets on glutathione peroxidase activity and lipid peroxidation susceptibility in sperm, testes, and liver[J]. Biological Trace Element Research,1998,64:119-132.
[33]邓守恒,孙各琴.高硒情况下VE对幼龄大鼠抗氧化作用的研究[J].广东微量元素科学,2001,8(6):23-26.
[34]Marin M, Lucille S, Hurley D, et al. Effect of large amounts of vitamin E during pregnancy and lactation[J]. The American Journal of Clinical Nutrition,1994,30:1629-1637.
[35]覃敬东,张桂荣.母猪补硒和维生素E预防仔猪水肿病试验[J].广西畜牧兽医,2004,20(4):167-168.
[36]张庚华.母猪补充维生素E和硒的研究现状[J].中国饲料,1995,11:28.
[37]梁远东,冯娟,吴林舅,等.日粮维生素E水平对后备种母鸡生长发育的影响[J].畜牧与兽医,2005,37(2):10-12.
[38]Lee B E, Hong Y C, Lee K H, et al. Influence of maternal serum levels of vitamins C and E during the second trimester on birth weight and length[J]. European Journal of Clinical Nutrition,2004,58: 1365-1371.
[39]李瑞文,李向臣,安志,等.维生素E提高牛孤雌胚胎的体外发育研究[J].畜牧兽医学报,2005,36(11):1135-1138.
[40]刘德义,吴庆婷,陈会良.血清硒、维生素E水平对奶牛胎衣不下影响的研究[J].中国奶牛,2006,09:47-49.
[41]刘宇,冯淇辉,刘纯传,等.维生素E和硒防治奶牛乳房炎及繁殖疾病的研究[J].中兽医医药杂志,1995,05:6-8.
[42]张春有,杨旭华,王芳,等.VE对动物健康及营养影响的分析[J].太原科技,2003,1:46-49.
[43]梁明振,卢克焕,李琼华,等.饲粮维生素E水平对公猪繁殖及精浆营养生化参数的影响[J].养猪,2003,04:7-9.
[44]Marin G J, Mahan D C, Chung Y K, et al. Effects of dietary selenium and vitamin E on boar performance and tissue responses, semen quality, and subsequent fertilization rates in mature gilts[J]. Journal of animal science,1997,75:2994-3003.
[45]李志芳,江永辉.饲料中VE和VA含量对种公鸡精液品质及受精率的影响[J].养禽与禽病防治,2002,2:16.
[46]Yousef M I, Abdallah G A, Kamel K I. Effect of ascorbic acid and Vitamin E supplementation on semen quality and biochemical parameters of male rabbits[J]. Animal Reproduction Science,2003,76: 99-111.
[47]武秀库,吴广安.丙酸睾丸素与维生素C配合治疗种公羊死精、无精的效果报告[J].黑龙江畜牧科技,1995,1:24.
[48]罗海玲,贾志海,朱十恩,等.VE对绵羊鲜精及冻精品质的影响[J].中国畜牧杂志,2004,11:7-9.
[49]张坚中,徐铁铮.家畜冷冻精液[M].北京:中国农业科技出版社,1988:222-245.
[50]刘乾,李云.VE对畜禽免疫功能的影响[J].饲料研究,2002,1(12):22-23.
[51]石娇,于维军,王洪海,等.硒和VE协同添加对肉鸡机体抗氧化能力的影响[J].黑龙江畜牧兽医,2006,12:42-43.
[52]Peplowski M A, Mahan D C, Moxon A L, et al. Effect of dietary and injectable vitamin E and seleniumon weanling swine antigenically challenged with sheep red blood cells[J]. Journal of Animal Science,1981,51:344.
[53]Meydani M, Jeffrey S C, John B M, et al. Postprandial changes in the plasma concentration of a-and β-tocopherol in human subject fed a fat-rich meal supplemented with fat-soluble vitamins[J]. The Journal of Nutrition,1989,119:125.
[54]Hatfield P G, Robinson B L, Mininkhiem D L, et al. Serum a-tocopoherol and immune function in yearling ewes supplemented with zinc and vitamin E[J]. Journal of Animal Science,2002,80:1329-1334.
[55]文杰,林济华,王和民.日粮VE、抗坏血酸水平对肉仔鸡生长及免疫功能的影响[J].畜牧兽医学报,1996,27(6):481-488.
[56]Politis A D, Cipriano J E, Morrill J L, et al. Effect of dietary vitamin E on immune responses of calves[J]. Dairy Sci,1995,65:2357-2361.
[57]Reddy P G, Morrill J L, Minocha H C, et al. Effect of supplemental vitamin E on the immune system of calves[J]. Journal of Dairy Science,1986,69:164.
[58]李泰万,苏松辉,罗卫,等.硒和维生素E对鸡新城疫油乳苗免疫效果的影响[J].中国兽医科技,1995,02:22-23.
[59]王治华,杨志飞,王连仲,等.日粮中硒和VE对奶牛泌乳性能的影响[J].中国草食动物,2005,25(2):33~34.
[60]张桂芝,黄保华,石天虹,等.不同维生素水平对蛋种鸡生长期影响的研究[J].山东农业科学,2001,6:32-34.
[61]冯焱,佟建明.21世纪营养学研究的新课题-免疫营养学的研究[J].中国农业科技导报,2004,6(1):44-48.
[62]Mnydani S N, Barklund M P, Liu S, et al. Vitamin E supplementation enhances cell mediated immunity in healty elderly subjects[J]. American Journal of Clinical Nutrition,1990,52:557-563.
[63]Romach E H, Kidao S, Sanders B G, et al. Effects of RRR-alpha-tocopherol succinate on IL-1 and PGE2 production by macrophages[J]. Nutrition and Cancer,1993,20:205-214.
[64]Erf G F, Bottjc W G, Bersi T K, et al. Effects of dietary vitamin E on t he immune system in broilers: altered proportions of CD4+ T cells in the thymus[J]. Poultry Science,1998,77:529-537.
[65]高树新,王国赋,庞瑞雪.添加VE、VC对鸡抗热应激力的影响[J].内蒙古民族大学学报(自然科学版),2002,17(4):313~317.
[66]刘铀,刘艳芬,林红英,等.维生素E对热应激肉用鸡免疫及内分泌功能的影响[J].中国兽医科技,1998,09:8-9.
[67]唐现文,张响英.维生素E在畜禽生产上的应用[J].畜禽业,2001,04:40-41.
[68]刘铀,刘艳芬,林红英,等.维生素E防治肉鸡热应激机理的初步研究[J].中国家禽,1999,06:40-41.
[69]王胜林.动物VE的营养研究进展[J].兽药与饲料添加剂,1999,4(2):19-21.
[70]杨凤.动物营养学[M].北京:中国农业出版社,2002:pp 130-131.
[71]吴晋强,何茂龙.微量元素营养配方试验研究[J].安徽农学院学报,1989(3):185-190.
[72]孙克年.维生素E在禽业生产中的应用[J].湖南饲料,1999,03:13-14.
[73]冯仰廉主编.反刍动物营养学[M].北京:科学出版社.2004:493-497。
[74]徐雁,李舜伟,左萍萍,等.维生素E对慢性间断性缺氧大鼠学习记忆的作用[J].中国行为医学,2003,12:146-152.
[75]Privalle C, Fridovich T. Transcriptional and maturational effects of manganese and iron on the biosynthesis of manganese-superoxide dismutase in E. coli[J]. The Journal of Biological Chemistry, 1992,267(13),9140-9145.
[76]魏俊发.锰超氧化物歧化酶及其化学模拟研究[J].化学进展,1997,9(1):56-58.
[77]卜友泉,罗绪刚,等.动物锰营养中含锰超氧化物歧化酶研究进展[J].动物营养学报,2002,1:1-7
[78]张艳红,廖晓全,袁勤生.人锰超氧化物歧化酶的研究进展[J].药物生物技术,2001,8(6):352-356.
[79]罗绪刚,李素芬,邝霞,等.鸡含锰超氧化物歧化酶cDNA克隆及序列分析[J].2001,17(4):463-467.
[80]李素芬.有机锰源的化学特性及其对肉仔鸡的相对生物学利用率研究[D].博士学位论文.北京:中国农科院,2002.
[81]林月霞.以含锰超氧化物歧化酶基因表达评价肉仔鸡实用饲粮中锰的适宜水平[D].硕士学位论文.江苏:扬州大学,2005.
[82]Oberley L W. Increase in manganese superoxide dismutase activity in the mouse heart after X-irradition[J]. Archives of Biochemistry and Biophysics,1987,54:69
[83]Daret K S, John C H. Complementary DNA encoding human colon cancer manganese superoxide dismutase and the expression of its gene in human cells[J].Cancer Research,1991,51:939.
[84]中国农业大学.家畜繁殖学(第二版).北京:中国农业出版社,2003:72-77.
[85]中华人民共和国国家质量监督检验检疫总局.GB4143-2008[S].中华人民共和国国家标准-牛冷冻精液.北京:中国农业出版社,2008.06.27.
[86]刘洪波,罗海玲,刘志宏,等.日粮维生素E水平对山羊精液品质的影响[J].中国畜牧杂志,2005,41(10):12-14.
[87]肖雪梅,任冬仁,李琳.营养供给对种公猪精液品质的影响[J].中国畜牧兽医,2006,33(16):24-26.
[88]Robbins K R. Estimation of nutrient requirements from growth data. Nutr.,1979,109:1710-1714.
[89]Hoeketra W G. Biochemical function of selenium and its relation to vitamin E[J]. Fed Proc,2000,34: 2083-2086.
[90]Dowwell L R, Williams S N, Hidiroglou N, et al. Vitamin E supplementation for ruminant[J]. Anim Feed Sci Tech,1996,60:273-296.
[91]李彦,杨在宾,杨维仁,等.日粮维生素A和维生素E水平对肉鸡抗氧化和免疫性能的影响[J].物营养学报,2008,20(4):417-422.
[92]史大永,李良智,邱龙辉,等.不同来源SOD的制备及作用机理[J].青岛化工学院学报,2001,22(1):50-52.
[93]滑静,王晓霞,杨佐君,等.维生素E、C对热应激期蛋鸡血液生化指标的影响[J].动物营养学报,2001,13(4):59-62.
[94]张世珍,王兴亚,李建喜,等.VA在硒缺乏的动物自由基代谢中的作用[J].中国兽医学报,1998,18(1):15-17.
[95]李权超,王锡锋,詹纯列,等.补充VE和Vc对鸡胴体脂质过氧化物的保护作用[J].肉品卫生,1997,9:4-6.
[96]Sambrook J, Fritsh E F, Maniatis T.分子克隆实验指南(第二版)[M].北京:科学出版社,1992.
[97]Ramaehandran S, Bell R B. Heat shock protein70 gene polymorphisms and multiple scleroses[J]. Tissue Antigens,1995,46:140-141.
[98]Caseino I, Galeazzi M, Salvetti M, et al. HSP-1 promoter region polymolphism tested in three atttoimnlme diseases[J]. Inmmnogenetics,1994,39:291-293.
[99]Heringstand B, Klemetsdal G, Ruane J. Selection for mastitis resistance in dariry cattle:a review with focus on the Nordic countries[J]. Li vest Prod Sci,2000,64:95-106
[100]Shook G E. Approaches to summarizing somatic cell counts which improve interpretability[J]. Proc. Natl. Mastitis Countil,1982,150-166.
[101]Shook G E. Genetic Improvement of mastitis through selection on somatic cell count, veterinary clinics of North America[J]. Food Animal Practice,1993,9(3):563-581.
[102]Krystyna M. Relationship between transferrin and globulin antigen polymorphism and sheep resis-tance to mastitis[J]. Appl Genet,1996,37(2): 161-172.
[103]Oddgeirsson S, Simpson S P, et al. Relationship between the bovine major histocompatibility complex, erythrocyte markers and susceptibility to mastifs in Icelandic cattle[J]. Animal Genetics, 1988,19:11-16.
[104]Stasio L D, Sartotore S, Albera A. Lack of Association of GH1 and POU1F1 gene variants with met production traits in Piemontese cattle[J]. Animal Genetics.2002,33:61-64.
[105]Rogerio A, Curil, Dario A Palmieril, Liliane Suguisawa, et al. Growth and carcass traits associated with GHl/ALu and POUIFI/Hinfl gene polymorphisms in Zebu and crossbred beef cattle[J]. Genetics and molecular biology,2006,29(1):56-61.
[106]Stasio L D, Sartore S, Albera A. Lack of association of GH and POU1F1 gene variants with meat production traits in piemontese cattle[J]. Animal Genetics.2006,33(1),61-64.
[107]Hong J M, Chae S H, Oriero N. Identification and chromosomal localization of repeat sequences through BAC and sequence analysis in Korean cattle[J]. Journal of genetics,2005,84(3):329-335.
[108]张金波,罗佳滨,张春斌,等.荧光定量PCR技术原理及在分子诊断中的应用进展[J].中国优生与遗传杂,2006,14(12):13-15.
[109]罗绪刚,李素芬,刘彬,等.以含锰超氧化物歧化酶基因表达评价肉鸡对不同形态锰源的生物学利用率[J].卫生研究,2004,11(6):681-686.