棉籽壳对雄性新疆细毛羊繁殖机能的影响
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摘要
棉花的副产品棉籽壳数量极大并富含粗纤维和一定量的蛋白质与脂肪,是可利用的饲料资源。但因其含有有毒物质游离棉酚,长期饲喂可使动物机体产生毒害危害,从而影响动物的生长发育和繁殖能力。本试验以雄性细毛羊为试验对象,着重探讨饲料中添加不同水平的棉籽壳对雄性细毛羊繁殖机能的影响。并通过分离培养三月龄公羊的睾丸间质细胞,研究醋酸棉酚对睾丸间质细胞的影响。旨在为棉籽壳在反刍动物饲料中的应用提供科学依据。本论文的主要研究内容和结果如下：
1棉籽壳对雄性细毛羊抗氧化能力和内脏器官的影响
选择3月龄断奶体重为19.22±0.82kg雄性细毛羊48只,随机分为3组,每组16只。对照组(每日饲喂1000g芦苇+无棉粕精料),试验1组(每日饲喂500g棉籽壳+500g芦苇+无棉粕精料),试验2组(每日饲喂1000g棉籽壳+无棉粕精料)。预试期7天,正试期8个月,分别于5、7、9和11月龄每组屠宰3只,共36只,取不同组织样品,进行相关指标测定。主要研究结果如下：
1)对照组平均日增重为157g,试验1组为163g,试验2组为176g,组间差异不显著(P>0.05)。
2)组织器官棉酚残留量的测定结果表明,随着棉籽壳饲喂时间的延长,试验羊组织中棉酚残留量均呈上升趋势,棉酚在体内的蓄积分布具有不同的组织亲和性,其残留量由高到低依次为：睾丸>肝脏>肾脏>脾脏>背长肌>臀肌。
3)饲喂棉籽壳致血清指标发生了明显变化,其中11月龄后,血清T-AOC、GSH、CAT和GSH-PX活性,与对照组相比,试验1组的分别降低了3.12%(P<0.05)、25.17%(P<0.05)、20.45%(P<0.05)和28.50%(P<0.05)；试验2组活性降低了9.37%(P<0.05)、55.82%(P<0.05)、34.09%(P<0.05)和42.27%(P<0.05)。但血清MDA试验1组提高了29.50%(P<0.05)；试验2组提高了80.09%(P<0.05)。血清代谢酶活性测定结果表明,11月龄(试验8个月)后,与对照组相比,试验2组血清ALT和AST的活性分别提高了31.25%和12.72%(P<0.05)；与试验1组相比,试验2组血清中ALT和AST活性显著增高了22.91%和3.63%(P<0.05)。与对照组相比,试验1组和试验2组血清中LDH的活性分别降低了5.21%和46.40%(P<0.05);与试验1组相比,试验2组降低了45.43%(P<0.05)。与对照组相比,试验1组和试验2组血清中Na, K-ATPase活性降低了70.9%和77.41%(P<0.05)。
4)肝脏相关抗氧化指标测定结果表明,11月龄后,与对照组相比,试验1组肝脏T-AOC、GSH、CAT和GSH-PX的活性分别降低了24.84%(P<0.05)、29.17%(P<0.05)、18.35%(P<0.05)和20.23%(P<0.05)；试验2组肝脏酶活性降低了31.84%(P<0.05)、51.00%(P<0.05)、20.37%(P<0.05)和31.42%(P<0.05)；与对照组相比,试验2组肝脏MDA活性提高了22.09%(P<0.05)。与试验1组相比,试验2组肝脏T-AOC、GSH、CAT和GSH-PX的活性分别降低了9.32%(P<0.05)、30.82%(P<0.05)、2.49%(P<0.05)和14.02%(P<0.05)；肝脏MDA活性提高了19.31%(P<0.05)。
2棉籽壳对雄性细毛羊血清性激素、睾丸显微结构和精液品质的影响
1)棉籽壳对雄性细毛羊精液品质的影响结果表明,试验组的精子活力、活精子率、pH值和精液浓度等指标与对照组相比都显著降低(P<0.05)。
2)血清性激素水平的结果表明,与对照组相比,试验1组和试验2组血清睾酮水平分别提高了38.70%和290.32%(P<0.05)。血清胰岛素和T3、T4水平均明显下降趋势(P<0.05)。
3)显微观察可见,与对照组相比,试验1组睾丸小管之间距离增大,表现血肿变,有的曲精小管内细胞明显减少；试验2组睾丸曲精小管破裂,细胞数量显著减少,坏死。
3棉酚对体外培养睾丸间质细胞的影响
1)MTT法检测棉酚对睾丸间质细胞增殖的影响：1.消旋棉酚：棉酚处理48小时后,64、128、256、512mg/L浓度的消旋棉酚产生了细毛羊睾丸间质细胞增殖起到显著的抑制作用(P<0.05)。2.左旋棉酚：棉酚处理48小时后,64、128、256、512mg/L左旋棉酚均显著抑制了睾丸间质细胞的增殖(P<0.05),并随左旋棉酚浓度增加抑制效果越明显。
2)左旋棉酚和消旋棉酚处理细胞24小时后,棉酚能影响睾丸间质细胞的Na-K-ATP酶,当左旋棉酚和消旋棉酚达到512 mg/L,细胞内Na-K-ATP酶活性显著降低(P<0.05)。
3)不同浓度消旋棉酚对睾丸间质细胞处理48小时后,随着棉酚浓度的增加,坏死细胞的数量也随之增加。
4)通过流式细胞仪检测细胞凋亡和细胞周期结果表明：消旋棉酚作用于睾丸间质细胞后,S期细胞减少,GO/G1期细胞比例增加,细胞进入增殖期减少,DNA合成速度减慢。醋酸棉酚能抑制睾丸间质细胞的增殖,改变细胞周期。
Cotton seed shell is the by-product of cotton, it is rich in crude fiber and has a certain amout of fat and protein which can be used as available feed resources. However, it contains toxic substances free gossypol and long-term animal feeding can produce poisoning hazards that affect the growth and reproduction. In this experiment, we selectd fine wool male sheep as the test subject to disscuss the influence of different amount of cotton seed shell in reproduction of fine wool male sheep. We also isolated and cultured Leydig cells of age ram march to study the effects of gossypol acetic acid on it.The purpose of the experiment is to provide the Scientific Basis for its application in Ruminant animal feed. The main contents of this paper and the results are as follows:
1 Effects of the cotton seed shell on fine wool male sheep antioxidant capacity and the internal organs
Forty eight three months weaned fine wool male sheep at an average initial body weight of 19.22kg were randomly allocated into 3 groups (sixteen sheep in each group). The dietary treatments were as follows:
1) Control group (fed basal diet),2) Group 1:(basal diet+500g cotton seed shell+500g reed,3) Group 2:(basal diet+1000g cotton seed shell). Experiment test period for 7 days, trial period for eight months.At the age of 5、7、9、11 mouth, Thirty six sheep with 3 pigs per group were randomly selected and humanely slaughtered.We selected different organ samples to determine the related indicators. Separately in May age, in July age, in September age and in November age takes the Main research results were as follows:
1) The average daily gain(ADG) of Control group was 157g, Group 1 was 163g and 176g in group 2. No significant difference was found in every group(P>0.05).
2) Results of gossypol residues in tissues and organs showed that with the time extended, gossypol residues in sheep tissues improved. The accumulation of gossypol residues in different organs had different tissue compatibility.Its residual quantity from high to low was as follows:Testicle> liver> kidney> spleen> longissimus dorsi muscle> gluteus.
3) The blood serum oxidation index changed when sheep fed Cotton seed shell. At the age of 11 month, serum T-AOC, GSH, CAT and GSH-PX of Group 1 and 2showed to be less. Compared with control, serum T-AOC, GSH, CAT and GSH-PX of Group 1 decreased 3.12%(P<0.05)、25.17%(P<0.05)、20.45%(P<0.05) and 28.50%(P<0.05),respectively. Group 2 decreased 9.37%(P<0.05)、55.82%(P<0.05)、34.09%(P<0.05) and 42.27%(P<0.05) respectively. We also found that serum MDA of test experiment increased. Compared with control, serum MDA of Group 1 increased 29.50% and Group 2 increased 80.09%(P<0.05)。Results of serum enzyme activities indicated that with 8 mouths of experiment serm ALT and AST greatly improved(P<0.05). Compared with Group 1, they increased by 22.91% and 3.63% (P<0.05). Compared with control, serm LDH of Group 1 and 2 had decreased 5.21% and 46.40%(P<0.05), respectively. Compared with Group 1, serm LDH decreased 45.43%(P<0.05); serm Na, K-ATPase of Group 1 and 2 decreased 70.9% and77.41%(P<0.05).
4) The results of liver oxidation resistance index analysis:At the age of 11 mounth, liver oxidation resistance index significantly decreased. Compared with control, liver T-AOC、GSH、CAT and GSH-PX of Group 1 decreased 24.84%(P<0.05)、29.17%(P<0.05)、18.35%(P<0.05)、20.23%(P<0.05) and Group 2 decreased 31.84%(P<0.05)、51.00%(P<0.05)、20.37%(P<0.05) and 31.42%(P<0.05); Compared with control, Liver MDA improved 22.09%(P<0.05);Compared with Group 1, liver T-AOC、GSH、CAT and GSH-PX of Group 2 decreased 9.32%(P<0.05)、30.82%(P<0.05)、2.49%(P<0.05) and 14.02%(P<0.05),respectively. Liver MDA improved by 19.31%(P<0.05)。
2 Effects of cotton seed shells on blood serum sex hormone, testicle microstructure and seminal fluid quality in male fine-wool sheep
1) Through the experiment, we found that sperm motility, live sperm rate, pH and sperm concentration greatly reduced (P<0.05).
2) Results of the blood serum sex hormone indicated that serum testosterone and blood serum insulin, T3 and T4 changed.Compared with control, There were higher serum testosterone in group 1 and 2.They had improved by 38.70% and 290.32% (P<0.05),separately.Besides, serum insulin, T3 and T4 greatly reduced (P<0.05).
3) Results of light microscope were as follows:compared with the control group, testicle acorn tube distance of Group 1 increased and the performance haematoma changed, some tubulary acorn tube cell obvious reduced; Testicle tubular of Group 2 crackered, cell quantity of Group 2 obviously decreased, some cell necrosised.
3 Effects of Gossypol on testicle intersititial cell in virto
1) MTT results of gossypol on testicle intersititial cell were as followes:1. Laevo-rotatory gossipol function:after 48 hours of treatment, laevo-rotatory gossipol greatly inhibit the intersititial cell increment when the concentration of Laevo-rotatory gossipol was 64,128,256,512 mg/L.2. Racemization gossipol function:after 48 hours of treatment, racemization gossipol greatly inhibit the intersititial cell increment when the concentration of Laevo-rotatory gossipol was 64,128,256,512mg/L.With the increase of racemization gossypol, the inhibition ehchanced.
2) After 24h of laevo-rotatory gossipol and racemization gossipol treatments, gossypol could affect the Na-K-ATP enzyme activity in intersititial cell.When the concentration attained 512 mg/L, Na-K-ATP enzyme activity in intersititial cell greatly decereased (P<0.05)
3) After 48h of laevo-rotatory gossypol treatment,with the increasement of laevo-rotatory gossypol, the quantity of dead cells also increased.
4) Results of apoptosis and cell cycle showed that S phase cells of intersititial cell reduced, G0/G1 phase cells increased, Cells into the proliferative phase reduced and DNA synthesis slowed down when using racemization gossypol. Gossypol acetic acid could inhibit the proliferation of intersititial cell and changed the cell cycle.
1棉籽壳对雄性细毛羊抗氧化能力和内脏器官的影响
选择3月龄断奶体重为19.22±0.82kg雄性细毛羊48只,随机分为3组,每组16只。对照组(每日饲喂1000g芦苇+无棉粕精料),试验1组(每日饲喂500g棉籽壳+500g芦苇+无棉粕精料),试验2组(每日饲喂1000g棉籽壳+无棉粕精料)。预试期7天,正试期8个月,分别于5、7、9和11月龄每组屠宰3只,共36只,取不同组织样品,进行相关指标测定。主要研究结果如下：
1)对照组平均日增重为157g,试验1组为163g,试验2组为176g,组间差异不显著(P>0.05)。
2)组织器官棉酚残留量的测定结果表明,随着棉籽壳饲喂时间的延长,试验羊组织中棉酚残留量均呈上升趋势,棉酚在体内的蓄积分布具有不同的组织亲和性,其残留量由高到低依次为：睾丸>肝脏>肾脏>脾脏>背长肌>臀肌。
3)饲喂棉籽壳致血清指标发生了明显变化,其中11月龄后,血清T-AOC、GSH、CAT和GSH-PX活性,与对照组相比,试验1组的分别降低了3.12%(P<0.05)、25.17%(P<0.05)、20.45%(P<0.05)和28.50%(P<0.05)；试验2组活性降低了9.37%(P<0.05)、55.82%(P<0.05)、34.09%(P<0.05)和42.27%(P<0.05)。但血清MDA试验1组提高了29.50%(P<0.05)；试验2组提高了80.09%(P<0.05)。血清代谢酶活性测定结果表明,11月龄(试验8个月)后,与对照组相比,试验2组血清ALT和AST的活性分别提高了31.25%和12.72%(P<0.05)；与试验1组相比,试验2组血清中ALT和AST活性显著增高了22.91%和3.63%(P<0.05)。与对照组相比,试验1组和试验2组血清中LDH的活性分别降低了5.21%和46.40%(P<0.05);与试验1组相比,试验2组降低了45.43%(P<0.05)。与对照组相比,试验1组和试验2组血清中Na, K-ATPase活性降低了70.9%和77.41%(P<0.05)。
4)肝脏相关抗氧化指标测定结果表明,11月龄后,与对照组相比,试验1组肝脏T-AOC、GSH、CAT和GSH-PX的活性分别降低了24.84%(P<0.05)、29.17%(P<0.05)、18.35%(P<0.05)和20.23%(P<0.05)；试验2组肝脏酶活性降低了31.84%(P<0.05)、51.00%(P<0.05)、20.37%(P<0.05)和31.42%(P<0.05)；与对照组相比,试验2组肝脏MDA活性提高了22.09%(P<0.05)。与试验1组相比,试验2组肝脏T-AOC、GSH、CAT和GSH-PX的活性分别降低了9.32%(P<0.05)、30.82%(P<0.05)、2.49%(P<0.05)和14.02%(P<0.05)；肝脏MDA活性提高了19.31%(P<0.05)。
2棉籽壳对雄性细毛羊血清性激素、睾丸显微结构和精液品质的影响
1)棉籽壳对雄性细毛羊精液品质的影响结果表明,试验组的精子活力、活精子率、pH值和精液浓度等指标与对照组相比都显著降低(P<0.05)。
2)血清性激素水平的结果表明,与对照组相比,试验1组和试验2组血清睾酮水平分别提高了38.70%和290.32%(P<0.05)。血清胰岛素和T3、T4水平均明显下降趋势(P<0.05)。
3)显微观察可见,与对照组相比,试验1组睾丸小管之间距离增大,表现血肿变,有的曲精小管内细胞明显减少；试验2组睾丸曲精小管破裂,细胞数量显著减少,坏死。
3棉酚对体外培养睾丸间质细胞的影响
1)MTT法检测棉酚对睾丸间质细胞增殖的影响：1.消旋棉酚：棉酚处理48小时后,64、128、256、512mg/L浓度的消旋棉酚产生了细毛羊睾丸间质细胞增殖起到显著的抑制作用(P<0.05)。2.左旋棉酚：棉酚处理48小时后,64、128、256、512mg/L左旋棉酚均显著抑制了睾丸间质细胞的增殖(P<0.05),并随左旋棉酚浓度增加抑制效果越明显。
2)左旋棉酚和消旋棉酚处理细胞24小时后,棉酚能影响睾丸间质细胞的Na-K-ATP酶,当左旋棉酚和消旋棉酚达到512 mg/L,细胞内Na-K-ATP酶活性显著降低(P<0.05)。
3)不同浓度消旋棉酚对睾丸间质细胞处理48小时后,随着棉酚浓度的增加,坏死细胞的数量也随之增加。
4)通过流式细胞仪检测细胞凋亡和细胞周期结果表明：消旋棉酚作用于睾丸间质细胞后,S期细胞减少,GO/G1期细胞比例增加,细胞进入增殖期减少,DNA合成速度减慢。醋酸棉酚能抑制睾丸间质细胞的增殖,改变细胞周期。
Cotton seed shell is the by-product of cotton, it is rich in crude fiber and has a certain amout of fat and protein which can be used as available feed resources. However, it contains toxic substances free gossypol and long-term animal feeding can produce poisoning hazards that affect the growth and reproduction. In this experiment, we selectd fine wool male sheep as the test subject to disscuss the influence of different amount of cotton seed shell in reproduction of fine wool male sheep. We also isolated and cultured Leydig cells of age ram march to study the effects of gossypol acetic acid on it.The purpose of the experiment is to provide the Scientific Basis for its application in Ruminant animal feed. The main contents of this paper and the results are as follows:
1 Effects of the cotton seed shell on fine wool male sheep antioxidant capacity and the internal organs
Forty eight three months weaned fine wool male sheep at an average initial body weight of 19.22kg were randomly allocated into 3 groups (sixteen sheep in each group). The dietary treatments were as follows:
1) Control group (fed basal diet),2) Group 1:(basal diet+500g cotton seed shell+500g reed,3) Group 2:(basal diet+1000g cotton seed shell). Experiment test period for 7 days, trial period for eight months.At the age of 5、7、9、11 mouth, Thirty six sheep with 3 pigs per group were randomly selected and humanely slaughtered.We selected different organ samples to determine the related indicators. Separately in May age, in July age, in September age and in November age takes the Main research results were as follows:
1) The average daily gain(ADG) of Control group was 157g, Group 1 was 163g and 176g in group 2. No significant difference was found in every group(P>0.05).
2) Results of gossypol residues in tissues and organs showed that with the time extended, gossypol residues in sheep tissues improved. The accumulation of gossypol residues in different organs had different tissue compatibility.Its residual quantity from high to low was as follows:Testicle> liver> kidney> spleen> longissimus dorsi muscle> gluteus.
3) The blood serum oxidation index changed when sheep fed Cotton seed shell. At the age of 11 month, serum T-AOC, GSH, CAT and GSH-PX of Group 1 and 2showed to be less. Compared with control, serum T-AOC, GSH, CAT and GSH-PX of Group 1 decreased 3.12%(P<0.05)、25.17%(P<0.05)、20.45%(P<0.05) and 28.50%(P<0.05),respectively. Group 2 decreased 9.37%(P<0.05)、55.82%(P<0.05)、34.09%(P<0.05) and 42.27%(P<0.05) respectively. We also found that serum MDA of test experiment increased. Compared with control, serum MDA of Group 1 increased 29.50% and Group 2 increased 80.09%(P<0.05)。Results of serum enzyme activities indicated that with 8 mouths of experiment serm ALT and AST greatly improved(P<0.05). Compared with Group 1, they increased by 22.91% and 3.63% (P<0.05). Compared with control, serm LDH of Group 1 and 2 had decreased 5.21% and 46.40%(P<0.05), respectively. Compared with Group 1, serm LDH decreased 45.43%(P<0.05); serm Na, K-ATPase of Group 1 and 2 decreased 70.9% and77.41%(P<0.05).
4) The results of liver oxidation resistance index analysis:At the age of 11 mounth, liver oxidation resistance index significantly decreased. Compared with control, liver T-AOC、GSH、CAT and GSH-PX of Group 1 decreased 24.84%(P<0.05)、29.17%(P<0.05)、18.35%(P<0.05)、20.23%(P<0.05) and Group 2 decreased 31.84%(P<0.05)、51.00%(P<0.05)、20.37%(P<0.05) and 31.42%(P<0.05); Compared with control, Liver MDA improved 22.09%(P<0.05);Compared with Group 1, liver T-AOC、GSH、CAT and GSH-PX of Group 2 decreased 9.32%(P<0.05)、30.82%(P<0.05)、2.49%(P<0.05) and 14.02%(P<0.05),respectively. Liver MDA improved by 19.31%(P<0.05)。
2 Effects of cotton seed shells on blood serum sex hormone, testicle microstructure and seminal fluid quality in male fine-wool sheep
1) Through the experiment, we found that sperm motility, live sperm rate, pH and sperm concentration greatly reduced (P<0.05).
2) Results of the blood serum sex hormone indicated that serum testosterone and blood serum insulin, T3 and T4 changed.Compared with control, There were higher serum testosterone in group 1 and 2.They had improved by 38.70% and 290.32% (P<0.05),separately.Besides, serum insulin, T3 and T4 greatly reduced (P<0.05).
3) Results of light microscope were as follows:compared with the control group, testicle acorn tube distance of Group 1 increased and the performance haematoma changed, some tubulary acorn tube cell obvious reduced; Testicle tubular of Group 2 crackered, cell quantity of Group 2 obviously decreased, some cell necrosised.
3 Effects of Gossypol on testicle intersititial cell in virto
1) MTT results of gossypol on testicle intersititial cell were as followes:1. Laevo-rotatory gossipol function:after 48 hours of treatment, laevo-rotatory gossipol greatly inhibit the intersititial cell increment when the concentration of Laevo-rotatory gossipol was 64,128,256,512 mg/L.2. Racemization gossipol function:after 48 hours of treatment, racemization gossipol greatly inhibit the intersititial cell increment when the concentration of Laevo-rotatory gossipol was 64,128,256,512mg/L.With the increase of racemization gossypol, the inhibition ehchanced.
2) After 24h of laevo-rotatory gossipol and racemization gossipol treatments, gossypol could affect the Na-K-ATP enzyme activity in intersititial cell.When the concentration attained 512 mg/L, Na-K-ATP enzyme activity in intersititial cell greatly decereased (P<0.05)
3) After 48h of laevo-rotatory gossypol treatment,with the increasement of laevo-rotatory gossypol, the quantity of dead cells also increased.
4) Results of apoptosis and cell cycle showed that S phase cells of intersititial cell reduced, G0/G1 phase cells increased, Cells into the proliferative phase reduced and DNA synthesis slowed down when using racemization gossypol. Gossypol acetic acid could inhibit the proliferation of intersititial cell and changed the cell cycle.
引文
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