手持密度仪检测鸡与鸭精子密度技术的研究
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摘要
精子密度仪在哺乳动物中已推广应用,但在家禽中还研究很少。本文以黑凤鸡和攸县麻鸭精液为实验材料,手持精子密度仪与血细胞计数板为检测工具,对影响精子密度测定方法准确率因素进行分析,建立鸡、鸭精子密度-吸光度函数并进行验证。结果表明:用密度仪测定时的稀释液(简称"测试液")为3%NaCl时,精液稀释后应尽快检测吸光度;样液在比色皿中的混匀方式对吸光度测定有很大影响;精液用3%NaCl以及3种常用家禽精液稀释液进行10倍稀释后测定吸光度,B液吸光度显著高于与其它3组(P<0.05),其它3组间差异不显著(P>0.05)。用测试液为3.0%NaCl,鸡和鸭精子密度-吸光度呈三次函数关系,回归方程分别是Y_1=1.374X~3-0.786X~2+0.945X-0.002(R~2=0.997)、Y_2=1.283X~3-0.899X~2+0.994X-0.009(R~2=0.996);用0.9%NaCl代替3%NaCl作测试液简化精子密度测定方法可行,鸡精子密度-吸光度回归方程为Y_3=-0.264X~3+1.23X~2+0.468X+0.019(R~2=0.999)。鸡精液测试液为0.9%NaCl、3%NaCl时两种函数的吸光度最佳范围分别是0.035～0.692、0.069～0.624,在此范围内计数板与方程两种方法得到的密度差异不显著(P>0.05),以计数法为真实值,两种方法平均相对误差分别为6.95%、3.11%。以3%NaCl为测试液,鸭精液函数所测样品吸光度最佳范围小于鸡精液的,以计数板法为真实值,在吸光度0.054～0.123范围内的,函数与计数板两种方法所得的精子密度的平均相对误差为4.61%。本文将促进家禽手持精子密度仪研发,以及更好的使用哺乳动物精子密度仪。
Sperm densitometer has been widely applied in mammals,whereas little research has been done in poultry.Taking Black Silkies chicken and Youxian Partridge Duck semen as experimental materials,the sperm densitometer and blood cell counting board were used as testing tools,the factors affecting the determination accuracy rate were analyzed,and the roosters and drakes sperm absorbance-density curve were established and verified. The results showed that the absorbance should be measured as soon as possible after the semen was diluted using a diluent of 3% NaCl. There was great influence on the absorbance measurement when the original semen was diluted by different mixing method. The absorbance of B liquid is significantly higher than that of other 3 groups( P < 0. 05) when the same semen was diluted 10 times with 3% NaCl and other three kinds of common semen dilutions,and there were no significant differences within the other 3 groups( P > 0. 05). Using the 3. 0% NaCl as diluents,the relationship of the absorbance-density curve was cubic function for the rooster and ducks semen,and the regression equations were Y_1= 1. 374 X~3-0. 786 X~2+ 0. 945 X-0. 002( R~2= 0. 997) and Y_2= 1. 283 X~3-0. 899 X~2+ 0. 994-0. 009( R~2= 0. 996). It was feasible to simplify the sperm density determination methods using 0. 9% NaCl instead of 3% NaCl,and the regression equations was Y~3=-0. 264 X~3+1. 23 X~2+ 0. 468 X + 0. 019( R~2= 0. 999). The optimal range of absorbance for the curve of roosters semen 0. 9% NaCl and3% NaCl was between 0. 035 ～ 0. 692 and 0. 069 ～ 0. 624. And as for 3% NaCl drakes semen,it might be less than that of 3. 0% NaCl roosters semen. There was no significant difference between the cell count and the curve calculated density within each optimized range( P > 0. 05). The average relative error of the 0. 9% NaCl and 3% NaCl was 6. 95% and 3.11% when using the counting value as the true one which is within each optimized absorbance range,and that of 3% NaCl darkes semen was 4. 61% with the absorbance range of 0. 054 ～ 0. 123. This paper will promote the research and development of hand-held sperm densitometer for poultry and better use of mammalian sperm densitometer.
Sperm densitometer has been widely applied in mammals,whereas little research has been done in poultry.Taking Black Silkies chicken and Youxian Partridge Duck semen as experimental materials,the sperm densitometer and blood cell counting board were used as testing tools,the factors affecting the determination accuracy rate were analyzed,and the roosters and drakes sperm absorbance-density curve were established and verified. The results showed that the absorbance should be measured as soon as possible after the semen was diluted using a diluent of 3% NaCl. There was great influence on the absorbance measurement when the original semen was diluted by different mixing method. The absorbance of B liquid is significantly higher than that of other 3 groups( P < 0. 05) when the same semen was diluted 10 times with 3% NaCl and other three kinds of common semen dilutions,and there were no significant differences within the other 3 groups( P > 0. 05). Using the 3. 0% NaCl as diluents,the relationship of the absorbance-density curve was cubic function for the rooster and ducks semen,and the regression equations were Y_1= 1. 374 X~3-0. 786 X~2+ 0. 945 X-0. 002( R~2= 0. 997) and Y_2= 1. 283 X~3-0. 899 X~2+ 0. 994-0. 009( R~2= 0. 996). It was feasible to simplify the sperm density determination methods using 0. 9% NaCl instead of 3% NaCl,and the regression equations was Y~3=-0. 264 X~3+1. 23 X~2+ 0. 468 X + 0. 019( R~2= 0. 999). The optimal range of absorbance for the curve of roosters semen 0. 9% NaCl and3% NaCl was between 0. 035 ～ 0. 692 and 0. 069 ～ 0. 624. And as for 3% NaCl drakes semen,it might be less than that of 3. 0% NaCl roosters semen. There was no significant difference between the cell count and the curve calculated density within each optimized range( P > 0. 05). The average relative error of the 0. 9% NaCl and 3% NaCl was 6. 95% and 3.11% when using the counting value as the true one which is within each optimized absorbance range,and that of 3% NaCl darkes semen was 4. 61% with the absorbance range of 0. 054 ～ 0. 123. This paper will promote the research and development of hand-held sperm densitometer for poultry and better use of mammalian sperm densitometer.
引文
[1]辛清武.优质中型肉用鸭的精液特性及人工授精方法研究[D].武汉:华中农业大学,2011:1-2.XIN Qingwu.On the characteristics of semen and artificial insemination of quality medium-sized meat of ducksand artificial insemination methods of high quality medium meat ducks[D].Wuhan:Huazhong Agricultural University,2011:1-2.
[2]张勇.鸡精液低温保存稀释液的研究[D].银川:宁夏大学,2014.ZHANG Yong.Investigation on the cryopreservation of cock semen in various diluents[D].Yinchuan:Ningxia University,2014.
[3]朱利琴,刘璐,贺长青,等.分光光度计和手持精子密度仪测定鸡精子密度的比较[J].中国家禽,2017,39(23):60-63.ZHU Liqin,LIU Lu,HE Changqing,et al.The comparison of spectrophotometer and hand-held sperm density detector for determining sperm density[J].China Poultry,2017,39(23):60-63.
[4]张亮,郭宗义,潘红梅.分光光度法快速测算公猪精子密度的研究[J].广东农业科学,2012,39(05):103-105,108.ZHANG Liang,GUO Zongyi,PAN Hongmei.Rapid estimation on the sperm concentration in boar semen by spectrophotometric method[J].Guangdong Agricultural Sciences,2012,39(05):103-105,108.
[5]吴宓.应用7230型分光光度计测定公牛精子密度[J].黄牛杂志,1994,67(02):39-41.WU Mi.The sperm density of bulls was determined by 7230spectrophotometer[J].Journal of Yellow Cattle Science,1994,67(02):39-41.
[6]肉孜汗,热合曼,热孜亚.应用721型分光光度计测定公羊精子密度的试验[J].新疆畜牧业,2002,(4):20-21.ROU Zihan,RE Heman,RE Ziya.The experiment of measuring the semen density of ram by 721 spectrophotometer[J].Xinjiang Animal Husbandry,2002,(4):20-21.
[7]张涛,章龙珍,庄平.分光光度法测定俄罗斯鲟精子密度标准的研究[J].海洋渔业,2009,31(1):87-91.ZHANG Tao,ZHANG Longzhen,ZHUANG Ping.Determination on the sperm concentration standard of russian sturgeon(Acipenser gueldenstaedtii)by means of spectrophotometric method[J].Mariner Fisheries,2009,31(1):87-91.
[8]王坤,蒋隽,朱立军,等.鸡精子密度检测方法研究[J].中国家禽,2014,36(12):12-14.WANG Kun,JIANG Jun,ZHU Lijun,et al.Detection methods of sperm density in roosters[J].China Poultry,2014,36(12):12-14.
[9]严家来,陈雨京,张发苏.《临床检验基础》课程中细胞计数的常用方法[J].包头医学院学报,2016,32(6):157-158.YAN Jialai,CHEN Yujing,ZHANG Fasu.Common methods of cell counting in the course of basic clinical examination[J].Journal of Baotou Medical College,2016,32(6):157-158.
[10]张友清.用血球计数板测定核多角体病毒数量的误差分析[J].微生物学通报,1982,3:142.ZHANG Youqing.Error analysis of nuclear polyhedrosis virus quantities determined by hematology counter plate[J].Microbiological Bulletin,1982,3:142.
[11]韩卫杰,黄晓凤,刘晓华.斑嘴鸭采精技术及精液品质研究[J].湖北农业科学,2013,52(07):1611-1612.HAN Weijie,HUANG Xiaofeng,LIU Xiaohua.Studies on artificial semen collection and semen quality of anas poecilorhyncha[J].Hubei Agricultural Sciences,2013,52(07):1611-1612.
[12]笠井浩司,张鸿译.鸭的人工授精技术[J].国外畜牧科技,1998,25(3):36-37.LIJING Haosi,ZHANG Hong,translation.Artificial insemination of ducks[J].Animal Science Abroad,1998,25(3):36-37.
[13]宋卫涛,李慧芳,徐文娟,等.苏邮1号蛋鸭精液品质评估[J].家畜生态学报,2017,38(5):49-53.SONG Weitao,LI Huifang,XU Wenjuan,et al.Semen quality assessment of SuyouⅠducks[J].Journal of Domestic Animal Ecology,2017,38(5):49-53.
[14]郭海宁,陈开丰,苏红卫,等.笼养山麻鸭适宜采精频率和输精参数的研究[J].江西农业大学学报,2018,40(02):350-357.GUO Haining,CHEN Kaifeng,SU Hongwei,et al.Optimum semen collection frequency and insemination parameters of cagerearing ShanMa ducks[J].Acta Agriculture Universitis Jiangxiensis,2018,40(02):350-357.
[15]陈章言,杜文兴,段修军.番鸭与樱桃谷鸭人工授精的研究[J].安徽农业科学,2009,37(33):16733-16735,16737.CHEN Zhangyan,DU Wenxing,DUAN Xiujun.Artificial insem ination of the Cherry Valley ducks with Muscovy dukes[J].Journal of Anhui Agricultural Sciences,2009,37(33):16733-16735,16737.
[16]魏文栋,张亮,张伟.不同精子密度仪测定猪精子密度的准确性比对试验[J].猪业科学,2012,(10):70-71.WEI Wendong,ZHANG Liang,ZHANG Wei.Accuracy comparison test of pig sperm density measured by different sperm densitometer[J].Swine Industry Science,2012,(10):70-71.
[17]谢杏仪,方健源,何琨仪,等.两种稀释液在高精子密度精液分析中的比较应用[J].实验与检验医学,2014,32(4):438-439.XIE Xingyi,FANG Jianyuan,HE Kunyi,et al.Comparison of two dilutions in semen analysis of high sperm density[J].Experimental and Laboratory Medicine,2014,32(4):438-439.
[2]张勇.鸡精液低温保存稀释液的研究[D].银川:宁夏大学,2014.ZHANG Yong.Investigation on the cryopreservation of cock semen in various diluents[D].Yinchuan:Ningxia University,2014.
[3]朱利琴,刘璐,贺长青,等.分光光度计和手持精子密度仪测定鸡精子密度的比较[J].中国家禽,2017,39(23):60-63.ZHU Liqin,LIU Lu,HE Changqing,et al.The comparison of spectrophotometer and hand-held sperm density detector for determining sperm density[J].China Poultry,2017,39(23):60-63.
[4]张亮,郭宗义,潘红梅.分光光度法快速测算公猪精子密度的研究[J].广东农业科学,2012,39(05):103-105,108.ZHANG Liang,GUO Zongyi,PAN Hongmei.Rapid estimation on the sperm concentration in boar semen by spectrophotometric method[J].Guangdong Agricultural Sciences,2012,39(05):103-105,108.
[5]吴宓.应用7230型分光光度计测定公牛精子密度[J].黄牛杂志,1994,67(02):39-41.WU Mi.The sperm density of bulls was determined by 7230spectrophotometer[J].Journal of Yellow Cattle Science,1994,67(02):39-41.
[6]肉孜汗,热合曼,热孜亚.应用721型分光光度计测定公羊精子密度的试验[J].新疆畜牧业,2002,(4):20-21.ROU Zihan,RE Heman,RE Ziya.The experiment of measuring the semen density of ram by 721 spectrophotometer[J].Xinjiang Animal Husbandry,2002,(4):20-21.
[7]张涛,章龙珍,庄平.分光光度法测定俄罗斯鲟精子密度标准的研究[J].海洋渔业,2009,31(1):87-91.ZHANG Tao,ZHANG Longzhen,ZHUANG Ping.Determination on the sperm concentration standard of russian sturgeon(Acipenser gueldenstaedtii)by means of spectrophotometric method[J].Mariner Fisheries,2009,31(1):87-91.
[8]王坤,蒋隽,朱立军,等.鸡精子密度检测方法研究[J].中国家禽,2014,36(12):12-14.WANG Kun,JIANG Jun,ZHU Lijun,et al.Detection methods of sperm density in roosters[J].China Poultry,2014,36(12):12-14.
[9]严家来,陈雨京,张发苏.《临床检验基础》课程中细胞计数的常用方法[J].包头医学院学报,2016,32(6):157-158.YAN Jialai,CHEN Yujing,ZHANG Fasu.Common methods of cell counting in the course of basic clinical examination[J].Journal of Baotou Medical College,2016,32(6):157-158.
[10]张友清.用血球计数板测定核多角体病毒数量的误差分析[J].微生物学通报,1982,3:142.ZHANG Youqing.Error analysis of nuclear polyhedrosis virus quantities determined by hematology counter plate[J].Microbiological Bulletin,1982,3:142.
[11]韩卫杰,黄晓凤,刘晓华.斑嘴鸭采精技术及精液品质研究[J].湖北农业科学,2013,52(07):1611-1612.HAN Weijie,HUANG Xiaofeng,LIU Xiaohua.Studies on artificial semen collection and semen quality of anas poecilorhyncha[J].Hubei Agricultural Sciences,2013,52(07):1611-1612.
[12]笠井浩司,张鸿译.鸭的人工授精技术[J].国外畜牧科技,1998,25(3):36-37.LIJING Haosi,ZHANG Hong,translation.Artificial insemination of ducks[J].Animal Science Abroad,1998,25(3):36-37.
[13]宋卫涛,李慧芳,徐文娟,等.苏邮1号蛋鸭精液品质评估[J].家畜生态学报,2017,38(5):49-53.SONG Weitao,LI Huifang,XU Wenjuan,et al.Semen quality assessment of SuyouⅠducks[J].Journal of Domestic Animal Ecology,2017,38(5):49-53.
[14]郭海宁,陈开丰,苏红卫,等.笼养山麻鸭适宜采精频率和输精参数的研究[J].江西农业大学学报,2018,40(02):350-357.GUO Haining,CHEN Kaifeng,SU Hongwei,et al.Optimum semen collection frequency and insemination parameters of cagerearing ShanMa ducks[J].Acta Agriculture Universitis Jiangxiensis,2018,40(02):350-357.
[15]陈章言,杜文兴,段修军.番鸭与樱桃谷鸭人工授精的研究[J].安徽农业科学,2009,37(33):16733-16735,16737.CHEN Zhangyan,DU Wenxing,DUAN Xiujun.Artificial insem ination of the Cherry Valley ducks with Muscovy dukes[J].Journal of Anhui Agricultural Sciences,2009,37(33):16733-16735,16737.
[16]魏文栋,张亮,张伟.不同精子密度仪测定猪精子密度的准确性比对试验[J].猪业科学,2012,(10):70-71.WEI Wendong,ZHANG Liang,ZHANG Wei.Accuracy comparison test of pig sperm density measured by different sperm densitometer[J].Swine Industry Science,2012,(10):70-71.
[17]谢杏仪,方健源,何琨仪,等.两种稀释液在高精子密度精液分析中的比较应用[J].实验与检验医学,2014,32(4):438-439.XIE Xingyi,FANG Jianyuan,HE Kunyi,et al.Comparison of two dilutions in semen analysis of high sperm density[J].Experimental and Laboratory Medicine,2014,32(4):438-439.
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