醋酸棉酚对绵羊瘤胃液真菌多样性的影响
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摘要
本试验旨在运用高通量测序方法研究醋酸棉酚对绵羊瘤胃液真菌多样性的影响。选取8只健康、体重为(49.13±4.70) kg、安装有永久性瘤胃瘘管的哈萨克羊母羊,随机分为2组,分别为对照组和试验组,每组4只。试验期对照组绵羊饲喂基础饲粮;试验组绵羊第1～26天在基础饲粮中添加600 mg/(d·只)醋酸棉酚,在第27～47天醋酸棉酚添加量增加至1 200 mg/(d·只)。采集试验期正试期第1、20、37和47天瘤胃液样品,运用Illumina Hiseq平台测序分析绵羊瘤胃液真菌变化。结果显示:1)由样品物种稀释曲线和累积曲线可以看出,测序深度已经覆盖了瘤胃液样品中大部分的真菌。2)第1、20、47天,试验组与对照组相比瘤胃液真菌的Alpha多样性指数差异不显著(P>0.05);第37天时,试验组瘤胃液真菌的Shannon和Simpson指数显著高于对照组(P<0.05)。3)主坐标分析图显示,试验组与对照组瘤胃液真菌样品聚集在一起。4)在属水平上,第20和37天,试验组瘤胃液真菌区系中毛霉菌属(Mucor)相对丰度显著高于对照组(P<0.05)。第47天,试验组绵羊瘤胃液真菌区系中Metschnikowia相对丰度显著高于对照组(P<0.05)。综上所述,低浓度醋酸棉酚对绵羊瘤胃液真菌多样性无明显影响,醋酸棉酚浓度增加至1 200 mg/(d·只)时,Simpson和Shannon指数显著升高;醋酸棉酚对绵羊瘤胃液真菌主要优势菌属组成无显著影响。
This study was aimed to evaluate the effects of gossypol acetic acid on rumen fungi diversity of sheep by high-throughput sequencing. Eight healthy Kazakh female sheep weighted( 49.13±4.70) kg with permanently rumen fistula were randomly divided into control group and trial group,each group contained 4 sheep. Sheep in the control group were fed a basal diet; and sheep in the trial group were fed the basal diet supplemented with 600 mg/d gossypol acetic acid per sheep during 1 to 26 days and 1 200 mg/d gossypol acetic acid per sheep during 27 to 47 days. Rumen fluid samples at days 1,20,37 and 47 were collected to determined fungi communities by Illumina Hiseq platform sequencing. The results showed as follows: 1) rarefaction curve and accumulation curve of sample species showed that all samples were reached the saturation phase,indicating depth of sequencing was reached. 2) On days 1,20 and 47,there was no significant effect on Alpha diversity of fungi in rumen fluid between control group and trial group( P > 0. 05); on day 37,the Shannon and Simpson indexes in rumen fluid of trial group were significantly higher than those of the control group( P<0.05). 3) The principal coordinate analysis diagram showed that rumen fluid fungi samples in control group and trial group clustered together. 4) At the genus levels,on days 20 and 37,the relative abundance of Mucor in rumen fluid fungi flora of trial group was significantly higher than that of the control group( P < 0.05); on day 47,the relative abundance of Metschnikowia in rumen fluid fungi flora of trial group was significantly higher than that of the control group( P<0.05). In summary,the lowconcentration of gossypol acetic acid has no significant effect on fungi diversity of sheep, when gossypol acetic acid concentration increases to1 200 mg/d per sheep,the Simpson and Shannon indexes significantly increase; gossypol acetic acid has no effect on predominant fungi composition in rumen fluid of sheep.[Chinese Journal of Animal Nutrition,2019,31(7):3374-3382]
This study was aimed to evaluate the effects of gossypol acetic acid on rumen fungi diversity of sheep by high-throughput sequencing. Eight healthy Kazakh female sheep weighted( 49.13±4.70) kg with permanently rumen fistula were randomly divided into control group and trial group,each group contained 4 sheep. Sheep in the control group were fed a basal diet; and sheep in the trial group were fed the basal diet supplemented with 600 mg/d gossypol acetic acid per sheep during 1 to 26 days and 1 200 mg/d gossypol acetic acid per sheep during 27 to 47 days. Rumen fluid samples at days 1,20,37 and 47 were collected to determined fungi communities by Illumina Hiseq platform sequencing. The results showed as follows: 1) rarefaction curve and accumulation curve of sample species showed that all samples were reached the saturation phase,indicating depth of sequencing was reached. 2) On days 1,20 and 47,there was no significant effect on Alpha diversity of fungi in rumen fluid between control group and trial group( P > 0. 05); on day 37,the Shannon and Simpson indexes in rumen fluid of trial group were significantly higher than those of the control group( P<0.05). 3) The principal coordinate analysis diagram showed that rumen fluid fungi samples in control group and trial group clustered together. 4) At the genus levels,on days 20 and 37,the relative abundance of Mucor in rumen fluid fungi flora of trial group was significantly higher than that of the control group( P < 0.05); on day 47,the relative abundance of Metschnikowia in rumen fluid fungi flora of trial group was significantly higher than that of the control group( P<0.05). In summary,the lowconcentration of gossypol acetic acid has no significant effect on fungi diversity of sheep, when gossypol acetic acid concentration increases to1 200 mg/d per sheep,the Simpson and Shannon indexes significantly increase; gossypol acetic acid has no effect on predominant fungi composition in rumen fluid of sheep.[Chinese Journal of Animal Nutrition,2019,31(7):3374-3382]
引文
[1]STIPANOVIC R D,LOPEZ J D,Jr,DOWD MK,et al.Effect of racemic and(+)-and(-)-gossypol on the survival and development of Helicoverpa zea larvae[J].Journal of Chemical Ecology,2006,32(5):959-968.
[2]WANG X,HOWELL C P,CHEN F,et al.Gossypola polyphenolic compound from cotton plant[J].Advances in Food and Nutrition Research,2009,58:215-263.
[3]FOMBAD R B,BRYANT MJ.Effects of cottonseed cake-based diets supplemented with blood meal,alone or with lysine,on the growth of pigs[J].Tropical Animal Health and Production,2004,36(4):385-395.
[4]ZENG Q F,YANG G L,LIU G N,et al.Effects of dietary gossypol concentration on growth performance,blood profiles,and hepatic histopathology in meat ducks[J].Poultry Science,2014,93(8):2000-2009.
[5]IMAIZUMI H,DE SOUZA J,BATISTEL F,et al.Replacing soybean meal for cottonseed meal on performance of lactating dairy cows[J].Tropical Animal Health and Production,2016,48(1):139-144.
[6]SMITH G S.Toxification and detoxification of plant compounds by ruminants:an overview[J].Journal of Range Management,1992,45(1):25-30.
[7]石彩霞,侯彩云,王海荣,等.游离棉酚对绵羊瘤胃发酵和微生物区系的影响[J].中国饲料,2013(5):17-19.
[8]ISMARTOYO I,STEWART C S,ACAMOVIC T.The effect of gossypol on the rumen microbial degradation of grass hay under consecutive batch culture(CBC)[C]//Proceedings of winter meeting of the british society of animal production.Scarborough:BSAP,1993:171.
[9]KHALAF MA,MELEIGY S A.Reduction of free gossypol levels in cottonseed meal by microbial treatment[J].International Journal of Agriculture&Biology,2008,10(2):185-190.
[10]赵静.高效降解棉酚菌株的分离、鉴定及其降解机理的研究[D].硕士学位论文.新乡:河南师范大学,2017.
[11]LIN J,WU Y J,YANG D J,et al.Induction of apoptosis and antitumor effects of a small molecule inhibitor of Bcl-2 and Bcl-xl,gossypolacetate,in multiple myeloma in vitro and in vivo[J].Oncology Reports,2013,30(2):731-738.
[12]YILDIRIM-AKSOY M,LIMC,DOWD MK,et al.In vitro inhibitory effect of gossypol from gossypol-acetic acid,and(+)-and(-)-isomers of gossypol on the growth of Edwardsiella ictaluri[J].Journal of Applied Microbiology,2004,97(1):87-92.
[13]EID J E,UENO H,WANG C C,et al.Gossypol-induced death of African trypanosomes[J].Experimental Parasitology,1988,66(1):140-142.
[14]PACHECO L B,CASTRO-GARZA J,PEREZ-EGUA E,et al.Growth inhibitory activity of gossypol against several trichomonas vaginalis isolates[J].Pharmacy and Pharmacology Communications,1996,2(7):333-334.
[15]CHEN C,NIE C X,LIANG J,et al.Validated method to determine(±)-gossypol in Candida tropicalis culture by high-performance liquid chromatography[J].Acta Chromatographica,2018,30(4):269-273.
[16]DUNCAN A J,MILNE J A.Rumen microbial degradation of allyl cyanide as a possible explanation for the tolerance of sheep to brassica-derived glucosinolates[J].Journal of the Science of Food and Agriculture,2010,58(1):15-19.
[17]JONES R J,MEGARRITY R G.Successful transfer of DHP-degrading bacteria from Hawaiian goats to Australian ruminants to overcome the toxicity of Leucaena[J].Australian Veterinary Journal,2010,63(8):259-262.
[18]MAO S Y,HUO W J,ZHU W Y.Microbiomemetabolome analysis reveals unhealthy alterations in the composition and metabolism of ruminal microbiota with increasing dietary grain in a goat model[J].Environmental Microbiology,2015,18(2):525-541.
[19]王佳堃,和文凤.组学技术揭秘草食动物消化道真菌组成和功能[J].浙江大学学报(农业与生命科学版),2018,44(2):131-139.
[20]MARTN-OR U'E S M,BALCELLS J,ZAKRAOUIF,et al.Quantification and chemical composition of mixed bacteria harvested from solid fractions of rumen digesta:effect of detachment procedure[J].Animal Feed Science and Technology,1998,71(3/4):269-282.
[21]REISER R,HC F U.The mechanism of gossypol detoxification by ruminant animals[J].The Journal of Nutrition,1962,76(2):215-218.
[22]ZHANG Y H,ZHANG Z Y,DAI L,et al.Isolation and characterization of a novel gossypol-degrading bacteria Bacillus subtilis strain rumen bacillus subtilis[J].Asian-Australasian Journal of Animal Sciences,2018,31(1):63-70.
[23]MILLER A W,OAKESON K F,DALE C,et al.Effect of dietary oxalate on the gut microbiota of the mammalian herbivore Neotoma albigula[J].Applied and Environmental Microbiology,2016,82(9):2669-2675.
[24]张文举.高效降解棉酚菌种的选育及棉籽饼粕生物发酵的研究[D].博士学位论文.杭州:浙江大学,2006.
[25]MARGALITH P.Inhibitory effect of gossypol on microorganisms[J].Applied Microbiology,1967,15(4):952-953.
[26]MELLON J E,ZELAYA C A,DOWD MK.Inhibitory effects of gossypol-related compounds on growth of Aspergillus flavus[J].Letters in Applied Microbiology,2011,52(4):406-412.
[2]WANG X,HOWELL C P,CHEN F,et al.Gossypola polyphenolic compound from cotton plant[J].Advances in Food and Nutrition Research,2009,58:215-263.
[3]FOMBAD R B,BRYANT MJ.Effects of cottonseed cake-based diets supplemented with blood meal,alone or with lysine,on the growth of pigs[J].Tropical Animal Health and Production,2004,36(4):385-395.
[4]ZENG Q F,YANG G L,LIU G N,et al.Effects of dietary gossypol concentration on growth performance,blood profiles,and hepatic histopathology in meat ducks[J].Poultry Science,2014,93(8):2000-2009.
[5]IMAIZUMI H,DE SOUZA J,BATISTEL F,et al.Replacing soybean meal for cottonseed meal on performance of lactating dairy cows[J].Tropical Animal Health and Production,2016,48(1):139-144.
[6]SMITH G S.Toxification and detoxification of plant compounds by ruminants:an overview[J].Journal of Range Management,1992,45(1):25-30.
[7]石彩霞,侯彩云,王海荣,等.游离棉酚对绵羊瘤胃发酵和微生物区系的影响[J].中国饲料,2013(5):17-19.
[8]ISMARTOYO I,STEWART C S,ACAMOVIC T.The effect of gossypol on the rumen microbial degradation of grass hay under consecutive batch culture(CBC)[C]//Proceedings of winter meeting of the british society of animal production.Scarborough:BSAP,1993:171.
[9]KHALAF MA,MELEIGY S A.Reduction of free gossypol levels in cottonseed meal by microbial treatment[J].International Journal of Agriculture&Biology,2008,10(2):185-190.
[10]赵静.高效降解棉酚菌株的分离、鉴定及其降解机理的研究[D].硕士学位论文.新乡:河南师范大学,2017.
[11]LIN J,WU Y J,YANG D J,et al.Induction of apoptosis and antitumor effects of a small molecule inhibitor of Bcl-2 and Bcl-xl,gossypolacetate,in multiple myeloma in vitro and in vivo[J].Oncology Reports,2013,30(2):731-738.
[12]YILDIRIM-AKSOY M,LIMC,DOWD MK,et al.In vitro inhibitory effect of gossypol from gossypol-acetic acid,and(+)-and(-)-isomers of gossypol on the growth of Edwardsiella ictaluri[J].Journal of Applied Microbiology,2004,97(1):87-92.
[13]EID J E,UENO H,WANG C C,et al.Gossypol-induced death of African trypanosomes[J].Experimental Parasitology,1988,66(1):140-142.
[14]PACHECO L B,CASTRO-GARZA J,PEREZ-EGUA E,et al.Growth inhibitory activity of gossypol against several trichomonas vaginalis isolates[J].Pharmacy and Pharmacology Communications,1996,2(7):333-334.
[15]CHEN C,NIE C X,LIANG J,et al.Validated method to determine(±)-gossypol in Candida tropicalis culture by high-performance liquid chromatography[J].Acta Chromatographica,2018,30(4):269-273.
[16]DUNCAN A J,MILNE J A.Rumen microbial degradation of allyl cyanide as a possible explanation for the tolerance of sheep to brassica-derived glucosinolates[J].Journal of the Science of Food and Agriculture,2010,58(1):15-19.
[17]JONES R J,MEGARRITY R G.Successful transfer of DHP-degrading bacteria from Hawaiian goats to Australian ruminants to overcome the toxicity of Leucaena[J].Australian Veterinary Journal,2010,63(8):259-262.
[18]MAO S Y,HUO W J,ZHU W Y.Microbiomemetabolome analysis reveals unhealthy alterations in the composition and metabolism of ruminal microbiota with increasing dietary grain in a goat model[J].Environmental Microbiology,2015,18(2):525-541.
[19]王佳堃,和文凤.组学技术揭秘草食动物消化道真菌组成和功能[J].浙江大学学报(农业与生命科学版),2018,44(2):131-139.
[20]MARTN-OR U'E S M,BALCELLS J,ZAKRAOUIF,et al.Quantification and chemical composition of mixed bacteria harvested from solid fractions of rumen digesta:effect of detachment procedure[J].Animal Feed Science and Technology,1998,71(3/4):269-282.
[21]REISER R,HC F U.The mechanism of gossypol detoxification by ruminant animals[J].The Journal of Nutrition,1962,76(2):215-218.
[22]ZHANG Y H,ZHANG Z Y,DAI L,et al.Isolation and characterization of a novel gossypol-degrading bacteria Bacillus subtilis strain rumen bacillus subtilis[J].Asian-Australasian Journal of Animal Sciences,2018,31(1):63-70.
[23]MILLER A W,OAKESON K F,DALE C,et al.Effect of dietary oxalate on the gut microbiota of the mammalian herbivore Neotoma albigula[J].Applied and Environmental Microbiology,2016,82(9):2669-2675.
[24]张文举.高效降解棉酚菌种的选育及棉籽饼粕生物发酵的研究[D].博士学位论文.杭州:浙江大学,2006.
[25]MARGALITH P.Inhibitory effect of gossypol on microorganisms[J].Applied Microbiology,1967,15(4):952-953.
[26]MELLON J E,ZELAYA C A,DOWD MK.Inhibitory effects of gossypol-related compounds on growth of Aspergillus flavus[J].Letters in Applied Microbiology,2011,52(4):406-412.