日粮精粗比对奶牛瘤胃厌氧细菌和挥发性脂肪酸及血液指标的影响
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摘要
日粮精粗比对奶牛的的生产性能和机体健康有着显著的影响,长期饲喂高精料饲粮易引起奶牛营养代谢病的发生,导致奶牛生产性能的下降。本试验选用5头健康的干奶期荷斯坦奶牛(体重为502±25㎏),以玉米、豆粕、麦麸、青贮、甘薯蔓及羊草等为主要原料,配制精粗质量比分别为30:70、35:65、40:60、45:55、50:50、55:45、60:40、70:30和100:0的9种日粮,连续进行9期不同精粗比日粮的饲喂试验。前8期试验每期30d,最后一期试验为3d,研究不同日粮精粗比对奶牛瘤胃厌氧菌和挥发性脂肪酸及血液相关指标的影响,分别检测了瘤胃pH,挥发性脂肪酸(乙酸、丙酸、丁酸)浓度,血清乙酸、丙酸、丁酸浓度,血脂(甘油三酯、总胆固醇、高密度脂蛋白、低密度脂蛋白)浓度,血清游离脂肪酸浓度,血清Na+,K+,Cl-,TCO2,HCO3-离子浓度,同时从瘤胃中分离出6株优势厌氧产乳酸细菌,进行了16Sr DNA序列分析和系统发育树的构建。结果表明:
1随精料水平的提高奶牛瘤胃pH逐渐降低,其中饲喂精粗比100:0日粮组的奶牛瘤胃pH显著低于饲喂其它精粗比日粮组(P<0.05);精粗比为55:45日粮组的奶牛瘤胃乙酸、丙酸和丁酸浓度,显著低于粗精比为30:70、35:65、40:60、45:55、50:50日粮组(P<0.05);50:50日粮组奶牛血清乙酸浓度显著低于粗精比为30:70、35:65和100:0日粮组(P<0.05);不同精粗比日粮条件下,奶牛血清丙酸和丁酸浓度组间差异不显著(P>0.05);血清乙酸和丁酸浓度与瘤胃中乙酸、丁酸浓度无明显相关性(P>0.05),但血清丙酸浓度与瘤胃丙酸浓度组间呈极显著负相关性(P<0.01)。
2随精料水平的提高,血清甘油三脂(TG)含量逐渐升高,100:0日粮组TG含量显著高于30:70日粮组(P<0.05);55:45日粮组血清高密度脂蛋白(HDL)含量显著高于30:70、35:65、40:60、45:55日粮组(P<0.05),55:45日粮组血清低密度脂蛋白(LDL)含量显著高于30:70、40:60日粮组,其余各日粮组差异不显著(P>0.05),血清总胆固醇(CHO)各日粮组之间差异不显著(P>0.05);月桂酸(C12:0)、棕榈酸(C16:0)含量在55:45日粮组达到最大,显著高于30:70日粮组(P<0.05),豆蔻油酸(C14:1)、亚油酸(C18:2)含量55:45日粮组最小,显著小于30:70、35:65、40:60日粮组(P<0.05),豆蔻酸(C14:0)、油酸(C18:1)含量各组间差异不显著(P>0.05),硬脂酸(C18:0)含量55:45日粮组显著高于30:70、60:40、70:30、100:0日粮组(P<0.05),其余各日粮组差异不显著(P>0.05);血清饱和脂肪酸含量先下降后上升,不饱和脂肪酸含量先上升后下降。
3随精料水平的提高,血清Na+浓度呈逐渐上升趋势,且各日粮组之间差异不显著(P>0.05);血清K+,Cl-,TCO2浓度呈先下降后上升趋势,各日粮组之间K+浓度差异不显著(P>0.05),100:0日粮组血清Cl-浓度显著高于40:60、45:55、50:50日粮组(P<0.05),其余各日粮组之间Cl-浓度差异不显著(P>0.05),55:45日粮组血清TCO2浓度显著低于30:70、35:65、40:60、45:50、50:50日粮组(P<0.05) ;血清HCO3-浓度先逐渐上升,在55:45日粮组后虽略有下降,但其后各日粮组之间差异不显著(P>0.05)。
4随精料水平的提高,瘤胃中产乳酸菌的含量呈逐渐上升趋势,且100:0日粮组显著高于其他日粮组(P<0.05)。分离的6株产乳酸菌,以细菌总DNA为模板,采用细菌16SrDNA的通用引物,经PCR扩增到一条约为1.5Kb左右的16Sr DNA片段,连接到PMD-18-T克隆载体上,并用化学法转化E.coli DH5α。用HindШ和EcoRⅠ对转化子进行酶切鉴定,DNA测序表明PCR扩增到的16Sr DNA片段长为1 540～1 615核苷酸。与Genbank上已提交的16SrDNA进行比较(BLAST)表明菌株8502,9101,1501,4101,5101,6601归属于链球菌属、盐单胞菌属、肠球菌属、不动杆菌属、肠杆菌属。由MEGA4.0软件构建的系统发育树表明菌株8502,9101,1501,4101,5101,6601分别同牛链球菌JB1(Streptococcus bovis strain JB1)、牛链球菌B315(Streptococcus bovis strain B315 )、盐单胞菌F22157(Halomonas sp. F22157)、粪肠球菌SL5(Enterococcus faecalis strain SL5)、不动杆菌TCCC11051(Acinetobacter sp. TCCC11051)、肠杆菌J11(Enterobacter sp. J11)的亲缘性最近。
5综上述结果可知:不同精粗比日粮对奶牛瘤胃pH、VFA和血清丙酸产生了显著影响;高精料日粮可导致血脂水平的升高和血清饱和脂肪酸含量上升,不饱和脂肪酸含量下降;随日粮精料比例的提高,瘤胃产乳酸菌的数量逐渐升高,奶牛在饲喂过程中逐渐对精料产生了耐受性,获得了相对稳定的瘤胃生态系统。日粮精粗比为55:45时奶牛的生产性能最佳,高精料日粮水平是导致奶牛营养代谢病(瘤胃酸中毒、酮病、脂肪肝综合症等)发生的重要原因,因此合理的日粮精粗比是奶牛饲养的关键。
Different forage to concentrate ration has a significant impact on production and health of cow,high-forage diet can result in nutrition metabolism disease and reduce production frequently.Five healthy cows(502±25㎏) during the dry period were used to study the effects of forage to concentrate ration on rumen anaerobic bacterial and volatile fatty acids,some blood indexes. Cows were fed continuously in nine periods and experiment with 30 d each periods except last period that only keeping 3 d.The main ingredients were corn,wheat bran,soybean meal,pachyrhizus vine,corn silage and so on.Dietary forage to concentrate ration were 30:70, 35:65, 40:60, 45:55, 50:50, 55:45,60:40, 70:30, 100:0 concentration respectively.The indexes of rumen pH、VFA(acetate,butyrate and butyric acid ) and the concentration of serum VFA,lipide(TG,CHO,HDL,LDL),Na+,K+,Cl-,HCO3- and free fatty acids were tested. We separated 6 strains anaerobic bacterial from rumen fluide ,analysed 16SrDNA sequence and phylogenic tree construction. The result indicate that:
1 Rumen pH was down gradually with the increase of forage to concentrate ration and tended to be the lowest in 100:0 concentration(P<0.05). Serum acetate were different lower (P<0.05) in 50:50 compared with 30:70, 35:65 and 100:0 concentration. Serum propionate and butyrate were no significant different(P>0.05). No relationships were observed between rumen acetate, butyrate and acetate, butyrate in serum(P>0.05), while the relationships between rumen propionate and serum propionate reached extremely significant(P<0.01).
2 The serum content of TG was up gradually with the increase of Forage to concentrate ration and tended to be the highest in 100:0(P<0.05) compared with 30:70 concentration.HDL and CHO were significantly higher(P<0.05) in 55:45 compared with 30:70 concentration,others were no significantly different(P>0.05).No significant different(P>0.05) were observed in CHO concentration.The serum content of Lauric acid and Palmitic acid in 55:45 concentration were significantly higher(P<0.05) than that in 30:70. Linoleic acid and nutmeg acid in 55:45 concentration were significantly lower(P<0.05) than that in 30:70,35:65,40:60.Oleic acid and Myristic acid concentration were no significantly different(P>0.05).The serum content of Stearic acid in 55:45 concentration were significantly higher(P<0.05) than that in 30:70,60:40,70:30,100:0.The serum content of saturated fatty acid increased first and then gradually decreased but the content of unsaturated fatty acid was opposite with the increase of Forage to concentrate ration.
3 The serum content of Na+ was up gradually with the increase of dietary forage to concentrate ration and no significantly different in every group(P>0.05). The serum content of K+, Cl-, TCO2 decreased first and then gradually increased, there is no siginficant difference in serum concent of K+ among every group. The serum content of Cl- in 100:0 were significantly higher than that in 40:60,45:55,50:50(P<0.05), serum TCO2 in 55:45 concentration were significantly lower than that in 30:70,35:65,40:60,45:50,50:50(P<0.05). The serum content of HCO3- increased first and then gradually decreased after 55:45, but there is no siginficant difference in serum concent of HCO3- after 55:45 group(P>0.05).
4 The content of rumen bacterial which produce lactic acid was up gradually with the increase of dietary forage to concentrate ration and tended to be the highest in 100:0(P<0.05) compared with other groups. A 1.5Kb of 16SrDNA fragment amplified through general PCR with the template of bacterium total DNA, and bacteria 16SrDNA universal primers, was ligated into PMD-18-T vector which is classic cloning vector for PCR products, and then was taken to transform chemically competent cell of E.coli DH5α.Restriction enzyme analysis of the transformants, mediated by HindⅢand EcoRⅠ.The BLAST of the 1540～1615 by of 16SrDNA sequence obtained by DNA sequencing, with all 16SrDNA sequences in GenBank indicated that the bacterium 8502,9101,1501,4101,5101,6101 belong to Streptococcus ,Halomonas, Enterococcus、Acinetobacter、Enterobacter. The phylogenic tree of the field bacterium 8502,9101,1501,4101,5101,6101 which were generated by MEGA4.0 software,basing on 16SrDNA full-length sequence,showed that it is most close to Streptococcus bovis strain JB1、Streptococcus bovis strain B315、Halomonas sp. F22157、Enterococcus faecalis strain SL5、Acinetobacter sp. TCCC11051、Enterobacter sp. J11 respectively.
5 Based on the result,the conclusion is differents diet forage to concentrate ration have notable effects on rumen pH, VFA and propionate in serum, high forage to concentrate ration increase the concentration of serum lipid and saturated fatty acid,decrease the concentration of un saturated fatty acids, the content of rumen bacterial which produce lactic acid was up gradually with the increase of forage to concentrate ration, the cow obtained relatively tolerance and established stable rumen ecosystem with the increase of forage to concentrate ration.The best production was obtained in 55:45 concentration of the dairy cow,high forage to conentrate ration can result in nutrion and metabolize disease of the cow,such as rumen acidosis,ketosis in dairy cows ,fatty liver syndrome and so on.So the appropriate forage to concentrate ration is very important in the process of cattle breeding.
1随精料水平的提高奶牛瘤胃pH逐渐降低,其中饲喂精粗比100:0日粮组的奶牛瘤胃pH显著低于饲喂其它精粗比日粮组(P<0.05);精粗比为55:45日粮组的奶牛瘤胃乙酸、丙酸和丁酸浓度,显著低于粗精比为30:70、35:65、40:60、45:55、50:50日粮组(P<0.05);50:50日粮组奶牛血清乙酸浓度显著低于粗精比为30:70、35:65和100:0日粮组(P<0.05);不同精粗比日粮条件下,奶牛血清丙酸和丁酸浓度组间差异不显著(P>0.05);血清乙酸和丁酸浓度与瘤胃中乙酸、丁酸浓度无明显相关性(P>0.05),但血清丙酸浓度与瘤胃丙酸浓度组间呈极显著负相关性(P<0.01)。
2随精料水平的提高,血清甘油三脂(TG)含量逐渐升高,100:0日粮组TG含量显著高于30:70日粮组(P<0.05);55:45日粮组血清高密度脂蛋白(HDL)含量显著高于30:70、35:65、40:60、45:55日粮组(P<0.05),55:45日粮组血清低密度脂蛋白(LDL)含量显著高于30:70、40:60日粮组,其余各日粮组差异不显著(P>0.05),血清总胆固醇(CHO)各日粮组之间差异不显著(P>0.05);月桂酸(C12:0)、棕榈酸(C16:0)含量在55:45日粮组达到最大,显著高于30:70日粮组(P<0.05),豆蔻油酸(C14:1)、亚油酸(C18:2)含量55:45日粮组最小,显著小于30:70、35:65、40:60日粮组(P<0.05),豆蔻酸(C14:0)、油酸(C18:1)含量各组间差异不显著(P>0.05),硬脂酸(C18:0)含量55:45日粮组显著高于30:70、60:40、70:30、100:0日粮组(P<0.05),其余各日粮组差异不显著(P>0.05);血清饱和脂肪酸含量先下降后上升,不饱和脂肪酸含量先上升后下降。
3随精料水平的提高,血清Na+浓度呈逐渐上升趋势,且各日粮组之间差异不显著(P>0.05);血清K+,Cl-,TCO2浓度呈先下降后上升趋势,各日粮组之间K+浓度差异不显著(P>0.05),100:0日粮组血清Cl-浓度显著高于40:60、45:55、50:50日粮组(P<0.05),其余各日粮组之间Cl-浓度差异不显著(P>0.05),55:45日粮组血清TCO2浓度显著低于30:70、35:65、40:60、45:50、50:50日粮组(P<0.05) ;血清HCO3-浓度先逐渐上升,在55:45日粮组后虽略有下降,但其后各日粮组之间差异不显著(P>0.05)。
4随精料水平的提高,瘤胃中产乳酸菌的含量呈逐渐上升趋势,且100:0日粮组显著高于其他日粮组(P<0.05)。分离的6株产乳酸菌,以细菌总DNA为模板,采用细菌16SrDNA的通用引物,经PCR扩增到一条约为1.5Kb左右的16Sr DNA片段,连接到PMD-18-T克隆载体上,并用化学法转化E.coli DH5α。用HindШ和EcoRⅠ对转化子进行酶切鉴定,DNA测序表明PCR扩增到的16Sr DNA片段长为1 540～1 615核苷酸。与Genbank上已提交的16SrDNA进行比较(BLAST)表明菌株8502,9101,1501,4101,5101,6601归属于链球菌属、盐单胞菌属、肠球菌属、不动杆菌属、肠杆菌属。由MEGA4.0软件构建的系统发育树表明菌株8502,9101,1501,4101,5101,6601分别同牛链球菌JB1(Streptococcus bovis strain JB1)、牛链球菌B315(Streptococcus bovis strain B315 )、盐单胞菌F22157(Halomonas sp. F22157)、粪肠球菌SL5(Enterococcus faecalis strain SL5)、不动杆菌TCCC11051(Acinetobacter sp. TCCC11051)、肠杆菌J11(Enterobacter sp. J11)的亲缘性最近。
5综上述结果可知:不同精粗比日粮对奶牛瘤胃pH、VFA和血清丙酸产生了显著影响;高精料日粮可导致血脂水平的升高和血清饱和脂肪酸含量上升,不饱和脂肪酸含量下降;随日粮精料比例的提高,瘤胃产乳酸菌的数量逐渐升高,奶牛在饲喂过程中逐渐对精料产生了耐受性,获得了相对稳定的瘤胃生态系统。日粮精粗比为55:45时奶牛的生产性能最佳,高精料日粮水平是导致奶牛营养代谢病(瘤胃酸中毒、酮病、脂肪肝综合症等)发生的重要原因,因此合理的日粮精粗比是奶牛饲养的关键。
Different forage to concentrate ration has a significant impact on production and health of cow,high-forage diet can result in nutrition metabolism disease and reduce production frequently.Five healthy cows(502±25㎏) during the dry period were used to study the effects of forage to concentrate ration on rumen anaerobic bacterial and volatile fatty acids,some blood indexes. Cows were fed continuously in nine periods and experiment with 30 d each periods except last period that only keeping 3 d.The main ingredients were corn,wheat bran,soybean meal,pachyrhizus vine,corn silage and so on.Dietary forage to concentrate ration were 30:70, 35:65, 40:60, 45:55, 50:50, 55:45,60:40, 70:30, 100:0 concentration respectively.The indexes of rumen pH、VFA(acetate,butyrate and butyric acid ) and the concentration of serum VFA,lipide(TG,CHO,HDL,LDL),Na+,K+,Cl-,HCO3- and free fatty acids were tested. We separated 6 strains anaerobic bacterial from rumen fluide ,analysed 16SrDNA sequence and phylogenic tree construction. The result indicate that:
1 Rumen pH was down gradually with the increase of forage to concentrate ration and tended to be the lowest in 100:0 concentration(P<0.05). Serum acetate were different lower (P<0.05) in 50:50 compared with 30:70, 35:65 and 100:0 concentration. Serum propionate and butyrate were no significant different(P>0.05). No relationships were observed between rumen acetate, butyrate and acetate, butyrate in serum(P>0.05), while the relationships between rumen propionate and serum propionate reached extremely significant(P<0.01).
2 The serum content of TG was up gradually with the increase of Forage to concentrate ration and tended to be the highest in 100:0(P<0.05) compared with 30:70 concentration.HDL and CHO were significantly higher(P<0.05) in 55:45 compared with 30:70 concentration,others were no significantly different(P>0.05).No significant different(P>0.05) were observed in CHO concentration.The serum content of Lauric acid and Palmitic acid in 55:45 concentration were significantly higher(P<0.05) than that in 30:70. Linoleic acid and nutmeg acid in 55:45 concentration were significantly lower(P<0.05) than that in 30:70,35:65,40:60.Oleic acid and Myristic acid concentration were no significantly different(P>0.05).The serum content of Stearic acid in 55:45 concentration were significantly higher(P<0.05) than that in 30:70,60:40,70:30,100:0.The serum content of saturated fatty acid increased first and then gradually decreased but the content of unsaturated fatty acid was opposite with the increase of Forage to concentrate ration.
3 The serum content of Na+ was up gradually with the increase of dietary forage to concentrate ration and no significantly different in every group(P>0.05). The serum content of K+, Cl-, TCO2 decreased first and then gradually increased, there is no siginficant difference in serum concent of K+ among every group. The serum content of Cl- in 100:0 were significantly higher than that in 40:60,45:55,50:50(P<0.05), serum TCO2 in 55:45 concentration were significantly lower than that in 30:70,35:65,40:60,45:50,50:50(P<0.05). The serum content of HCO3- increased first and then gradually decreased after 55:45, but there is no siginficant difference in serum concent of HCO3- after 55:45 group(P>0.05).
4 The content of rumen bacterial which produce lactic acid was up gradually with the increase of dietary forage to concentrate ration and tended to be the highest in 100:0(P<0.05) compared with other groups. A 1.5Kb of 16SrDNA fragment amplified through general PCR with the template of bacterium total DNA, and bacteria 16SrDNA universal primers, was ligated into PMD-18-T vector which is classic cloning vector for PCR products, and then was taken to transform chemically competent cell of E.coli DH5α.Restriction enzyme analysis of the transformants, mediated by HindⅢand EcoRⅠ.The BLAST of the 1540～1615 by of 16SrDNA sequence obtained by DNA sequencing, with all 16SrDNA sequences in GenBank indicated that the bacterium 8502,9101,1501,4101,5101,6101 belong to Streptococcus ,Halomonas, Enterococcus、Acinetobacter、Enterobacter. The phylogenic tree of the field bacterium 8502,9101,1501,4101,5101,6101 which were generated by MEGA4.0 software,basing on 16SrDNA full-length sequence,showed that it is most close to Streptococcus bovis strain JB1、Streptococcus bovis strain B315、Halomonas sp. F22157、Enterococcus faecalis strain SL5、Acinetobacter sp. TCCC11051、Enterobacter sp. J11 respectively.
5 Based on the result,the conclusion is differents diet forage to concentrate ration have notable effects on rumen pH, VFA and propionate in serum, high forage to concentrate ration increase the concentration of serum lipid and saturated fatty acid,decrease the concentration of un saturated fatty acids, the content of rumen bacterial which produce lactic acid was up gradually with the increase of forage to concentrate ration, the cow obtained relatively tolerance and established stable rumen ecosystem with the increase of forage to concentrate ration.The best production was obtained in 55:45 concentration of the dairy cow,high forage to conentrate ration can result in nutrion and metabolize disease of the cow,such as rumen acidosis,ketosis in dairy cows ,fatty liver syndrome and so on.So the appropriate forage to concentrate ration is very important in the process of cattle breeding.
引文
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