Polymerase chain reaction(PCR) was used to amplify 5S rRNA spacer from wild rice(Oryza rufipogon and O.nivara) and cultivated rice(indica and japonica varieties of O.sativa L).The results show that there is spacer len...Polymerase chain reaction(PCR) was used to amplify 5S rRNA spacer from wild rice(Oryza rufipogon and O.nivara) and cultivated rice(indica and japonica varieties of O.sativa L).The results show that there is spacer length variation within and between species,and the typical indica and japonica varieties have their unique banding patterns of amplified 5S rRNA spacers,whereas intermediate showed no specific amplification profile of spacer regions.The 5S rRNA genes in intermediate are either identical with that of indica variety or that of japonica variety.These data suggest that the spacer length polymorphisms can be used to distinguish between closely ralated species and subspecies.展开更多
We have used the polymerase chain reaction to analyze variation in the size of 5S ribosomal gene spacer sequence. Eighty accessions, including 65 cultivated rice and 15 wild rice, were analyzed. Among them seven size ...We have used the polymerase chain reaction to analyze variation in the size of 5S ribosomal gene spacer sequence. Eighty accessions, including 65 cultivated rice and 15 wild rice, were analyzed. Among them seven size classes of 5S DNA spacer were observed. Classification asindica orjaponica on the basis of 5S DNA spacer patterns generally agrees with classification based on morphological studies, indicating that the length polymorphism of 5S DNA spacer could be used as a molecular marker for taxonomic and phylogenetic analysis.展开更多
Objective:To evaluate microscopy,OptiMAL<sup>?</sup> and multiplex PCR for the identification of Plasmodium falciparum（P.falciparum） and Plasmodium vivax（P.vivax） from the field isolates of Bikaner,Raj...Objective:To evaluate microscopy,OptiMAL<sup>?</sup> and multiplex PCR for the identification of Plasmodium falciparum（P.falciparum） and Plasmodium vivax（P.vivax） from the field isolates of Bikaner,Rajasthan（Northwest India）.Methods:In this study,a multiplex PCR（P.falciparum and P.vivax） was further developed with the incorporation of Plasmodium malariae（P.malariae） specific primer and also a positive control.The performance of microscopy,plasmodium lactate dehydrogenase（pLDH） based malaria rapid diagnostic test OptiMAL<sup>?</sup> and 18S rRNA gene based multiplex PCR for the diagnosis of P.falciparum and P.vivax was compared.Results:The three species multiplex PCR if.falciparum,P.vivax and P.malariae) with an inbuilt positive control was developed and evaluated.In comparison with multiplex PCR,which showed the sensitivity and specificity of 99.36%（95%CI,98.11%-100.00%） and 100.00%（95%CI,100.00%-100.00%）,the sensitivity and specificity of microscopy was 90.44%（95%CI,88.849-95.04%） and 99.22%（95% CI,97.71%-100.00%）,and OptiMAL<sup>?</sup> was 93.58%（95%CI,89.75%-97.42%） and 97.69%（95%CI, 95.10%-100.00%）.The efficiencies were 99.65%,95.10%and 95.45%for multiplex PCK.microscopy and OptiMAL<sup>?</sup>.respectively.Conclusions:Our results raise concerns over the overall sensitivities of microscopy and OptiMAL<sup>?</sup>,when compared to the multiplex PCR and thus stress the need for new molecular interventions in the accurate detection of the malarial parasites.This further highlights the fact that further developments are needed to improve the performance of rapid diagnostic tests at field level.展开更多
粪肠球菌和屎肠球菌是引起猪感染发病的优势肠球菌种,以肠球菌的16 S rRNA基因设计属特异性引物,利用SodA基因多态性设计种特异性引物,同时优化反应条件,建立了能同时测定猪源粪肠球菌和屎肠球菌多重PCR方法。通过对来源于猪的临床菌株...粪肠球菌和屎肠球菌是引起猪感染发病的优势肠球菌种,以肠球菌的16 S rRNA基因设计属特异性引物,利用SodA基因多态性设计种特异性引物,同时优化反应条件,建立了能同时测定猪源粪肠球菌和屎肠球菌多重PCR方法。通过对来源于猪的临床菌株、粪便菌株和鲜猪肉菌株进行测定,均能成功扩增出属特异性片段和种特异性片段。经过与快速生化鉴定试剂盒(Vitek-32)和16 S rRNA测序方法比较,多重PCR与16 S rRNA测序方法对猪的临床菌株、粪便菌株和鲜猪肉菌株的鉴定符合率100%;与Vitek-32鉴定符合率为62.3%,其中,与分离于感染猪的临床菌株符合率仅有46.7%,特别是感染猪的屎肠球菌,符合率仅为22.3%。展开更多
To analyze the microflora in fermented rice bran product, bacterial colonies were grown under various conditions. Although cultivation temperature did not affect the number of bacterial colonies formed on agar plates,...To analyze the microflora in fermented rice bran product, bacterial colonies were grown under various conditions. Although cultivation temperature did not affect the number of bacterial colonies formed on agar plates, twice as many colonies formed under aerobic as under anaerobic conditions. All colonies appearing on the plates showed acid production. Based on 16S rRNA sequence analysis, nearly all of the bacteria in the fermented product were highly similar (>99%) to Lactobacillus johnsonii. In addition, several Bacillus cereus and unidentified Lactobacillus strains that grew only under anaerobic conditions at 30℃?were seen. Random Amplified Polymorphic DNA (RAPD)-PCR analysis showed the amplified patterns of all isolates to differ substantially from the reference strain L. johnsonii. We conclude that L. johnsonii-related strains predominate in fermented rice bran product, and that these bacteria produce lactic acid to decrease the pH of the fermented product. Several novel Lactobacillus strains may also occur in this environment.展开更多
On the basis of a long-term（30 years） field experiment that involved four rotation systems, rice-rice-winter fallow（RRF）, rice-rice-ryegrass（RRG）, rice-rice-rape（RRP）, and rice-rice-milk vetch（RRV）, this stu...On the basis of a long-term（30 years） field experiment that involved four rotation systems, rice-rice-winter fallow（RRF）, rice-rice-ryegrass（RRG）, rice-rice-rape（RRP）, and rice-rice-milk vetch（RRV）, this study described the effects of green manure on the microbial communities in the red paddy soils using 454 pyrosequencing for the 16 S r RNA gene. The Chao1 richness and non-parametric Shannon＇s index increased in all soil samples that received green manure treatments. The communities＇ structures with the green manure applications were significantly dissimilar from that under the winter fallow. Using Metastats tests, many genera in the RRG, RRP and RRV soils were significantly different from those in the RRF soil, including a number of genera that functioned in the nitrogen and sulfur cycles. Analyses of the genera with these functions revealed the shifts in microbial ecosystem functions after long-term green manuring. Changes in the microbial communities increased the ammonium supply and decreased the soil acidification in green-manure-amended soils. Together, these data suggested powerful effects of green manure on both the microbial communities and the biogeochemical cycle driven by the shifts in bacterial functional groups.展开更多
In order to establish a quick and specific method which could identify the avian-derived ingredients,this study used 16 S rRNA gene sequence as target site,and designed the specific primers of chicken,pigeon meat and ...In order to establish a quick and specific method which could identify the avian-derived ingredients,this study used 16 S rRNA gene sequence as target site,and designed the specific primers of chicken,pigeon meat and quail meat. The DNA of common livestock and poultry meat( including mutton,beef,pork,rabbit meat,pigeon meat,quail meat,chicken,duck and goose) was used as template. Though PCR amplification and specific detection,a quick determination method was established to identify the avian-derived ingredients. The results showed that the selected primers could identify the ingredients of animal origin effectively and quickly. The method was convenient and concise,and could detect the chicken-derived,pigeon-derived,quail-derived ingredients in livestock and poultry food quickly and accurately.展开更多
A photoperiod-sensitive genic male sterile (PGMS) rice was found in 1973 as a spontaneous mutant of Nongken 58, a japonica variety. Pollen fertility of Nongken 58s (N58s) is completely sterile when grown under long-da...A photoperiod-sensitive genic male sterile (PGMS) rice was found in 1973 as a spontaneous mutant of Nongken 58, a japonica variety. Pollen fertility of Nongken 58s (N58s) is completely sterile when grown under long-day conditions, whereas fertile under short-day conditions. This PGMS was found to be controlled by one or two recessive gene(s), of which one gene(pms)was linked to a marker gene(d-1) on chromosome 5. In order to identify a more precise location of the pms, we analyzed the populations of BCFand BCFof N58s//N58s/KL211(v-10, virescent) and N58s//N58s/KL520 (gh-1, gold hull). The marker genes v-10 and gh-1 are located on the flanking region of d-1. The F, plants of two crosses were fertile. The number of fertile and sterile individuals in BCFfit展开更多
Biological nitrogen (N) fixation (BNF) plays a significant role in maintaining soil fertility in paddy field ecosystems. Rice variety influences BNF, but how different rice varieties regulate BNF and associated diazot...Biological nitrogen (N) fixation (BNF) plays a significant role in maintaining soil fertility in paddy field ecosystems. Rice variety influences BNF, but how different rice varieties regulate BNF and associated diazotroph communities has not been quantified. Airtight, field-based 15N2-labelling grow th chamber experiments were used to assess the BNF capac 辻 y of different rice varie ties. In addition, both the 16S rRNA and nifH genes were sequenced to assess the influence of different rice varieties on bacterial and diazotrophic communities in paddy soils. After subjecting a rice-soil system to 74 d of continuous airtight, field-based 15N2 labelling in pots in a growth chamber, the amounts of fixed N were 22.3 and 38.9 kg ha^-1 in inbred japonica (W23) and hybrid indica (IIY) rice cultivars plan ted in the rice-soil systems, respectively, and only 1%—2.5% of the fixed N was allocated to the rice plants and weeds. A greater abundance of diazotrophs was found in the surface soil (0-1 cm) under IIY than under W23. Sequencing of the 16S rRNA gene showed significantly greater abundances of the cyanobacterial genera Nostoc, Anabaena, and Cylindrospermum under IIY than under W23. Sequencing of the nifH gene also showed a significantly greater abundance of Nostoc under IIY than under W23. These results indicate that the hybrid rice cultivar (IIY) promoted BNF to a greater extent than the inbred rice cultivar (W23) and that the increase in BNF might have been due to the enhanced heterocystous cyanobacteria Nostoc.展开更多
Objective:To identify the prevalence of Cryptosporidium from goats in three types of farm management systems in Terengganu,Malaysia and to determine the Cryptosporidium species infecting goats by using 18 S r RNA.Meth...Objective:To identify the prevalence of Cryptosporidium from goats in three types of farm management systems in Terengganu,Malaysia and to determine the Cryptosporidium species infecting goats by using 18 S r RNA.Methods:A total of 478 fecal samples were randomly collected from goats in three farms;199 samples were collected from intensive farm,179 samples from semi-intensive farm and 100 samples from extensive farm.The samples were processed by using formolether concentration technique and stained by using modified Ziehl–Neelsen.Positive samples were performed by using nested PCR analysis by using 18 S r RNA.Results:Out of 478 goats,207(43.3%) were found to be infected with Cryptosporidium.Goats reared under the intensive farm management system reported the highest prevalence of infection(49.7%),followed by intensive farm management system(41%) and the lowest prevalence was reported in the goats reared under semi-intensive management system(37.4%).Conclusions:The identified species found in goat was Cryptosporidium parvum.Future study on the zoonotic transmission of Cryptosporidium parvum in goats needs to be done in order to find the source of transmission of this parasite.展开更多
Background： The number and diversity of uncultured ruminal bacterial and archaeal species revealed by 16S rRNA gene firs） sequences greatly exceeds that of cultured bacteria and archaea. However, the significance of...Background： The number and diversity of uncultured ruminal bacterial and archaeal species revealed by 16S rRNA gene firs） sequences greatly exceeds that of cultured bacteria and archaea. However, the significance of uncultured microbes remains undetermined. The objective of this study was to assess the numeric importance of select uncultured bacteria and cultured bacteria and the impact of diets and microenvironments within cow rumen in a comparative manner. Results： Liquid and adherent fractions were obtained from the rumen of Jersey cattle fed hay alone and Holstein cattle fed hay plus grain. The populations of cultured and uncultured bacteria present in each fraction were quantified using specific real-time PCR assays. The population of total bacteria was similar between fractions or diets, while total archaea was numerically higher in the hay-fed Jersey cattle than in the hay-grain-fed Holstein cattle. The population of the genus Prevotello was about one log smaller than that of total bacteria. The populations of Fibrobocter succinogenes, Ruminococcus flovefociens, the genus Butyrivibrio, and R. albus was at least one log smaller than that of genus Prevotello. Four of the six uncultured bacteria quantified were as abundant as F. succinogenes, R. flovefociens and the genus Butyrivibrio. In addition, the populations of several uncultured bacteria were significantly higher in the adherent fractions than in the liquid fractions. These uncultured bacteria may be associated with fiber degradation. Conclusions： Some uncultured bacteria are as abundant as those of major cultured bacteria in the rumen. Uncultured bacteria may have important contribution to ruminal fermentation. Population dynamic studies of uncultured bacteria in a comparative manner can help reveal their ecological features and importance to rumen functions.展开更多
文摘Polymerase chain reaction(PCR) was used to amplify 5S rRNA spacer from wild rice(Oryza rufipogon and O.nivara) and cultivated rice(indica and japonica varieties of O.sativa L).The results show that there is spacer length variation within and between species,and the typical indica and japonica varieties have their unique banding patterns of amplified 5S rRNA spacers,whereas intermediate showed no specific amplification profile of spacer regions.The 5S rRNA genes in intermediate are either identical with that of indica variety or that of japonica variety.These data suggest that the spacer length polymorphisms can be used to distinguish between closely ralated species and subspecies.
文摘We have used the polymerase chain reaction to analyze variation in the size of 5S ribosomal gene spacer sequence. Eighty accessions, including 65 cultivated rice and 15 wild rice, were analyzed. Among them seven size classes of 5S DNA spacer were observed. Classification asindica orjaponica on the basis of 5S DNA spacer patterns generally agrees with classification based on morphological studies, indicating that the length polymorphism of 5S DNA spacer could be used as a molecular marker for taxonomic and phylogenetic analysis.
文摘Objective:To evaluate microscopy,OptiMAL<sup>?</sup> and multiplex PCR for the identification of Plasmodium falciparum（P.falciparum） and Plasmodium vivax（P.vivax） from the field isolates of Bikaner,Rajasthan（Northwest India）.Methods:In this study,a multiplex PCR（P.falciparum and P.vivax） was further developed with the incorporation of Plasmodium malariae（P.malariae） specific primer and also a positive control.The performance of microscopy,plasmodium lactate dehydrogenase（pLDH） based malaria rapid diagnostic test OptiMAL<sup>?</sup> and 18S rRNA gene based multiplex PCR for the diagnosis of P.falciparum and P.vivax was compared.Results:The three species multiplex PCR if.falciparum,P.vivax and P.malariae) with an inbuilt positive control was developed and evaluated.In comparison with multiplex PCR,which showed the sensitivity and specificity of 99.36%（95%CI,98.11%-100.00%） and 100.00%（95%CI,100.00%-100.00%）,the sensitivity and specificity of microscopy was 90.44%（95%CI,88.849-95.04%） and 99.22%（95% CI,97.71%-100.00%）,and OptiMAL<sup>?</sup> was 93.58%（95%CI,89.75%-97.42%） and 97.69%（95%CI, 95.10%-100.00%）.The efficiencies were 99.65%,95.10%and 95.45%for multiplex PCK.microscopy and OptiMAL<sup>?</sup>.respectively.Conclusions:Our results raise concerns over the overall sensitivities of microscopy and OptiMAL<sup>?</sup>,when compared to the multiplex PCR and thus stress the need for new molecular interventions in the accurate detection of the malarial parasites.This further highlights the fact that further developments are needed to improve the performance of rapid diagnostic tests at field level.
文摘粪肠球菌和屎肠球菌是引起猪感染发病的优势肠球菌种,以肠球菌的16 S rRNA基因设计属特异性引物,利用SodA基因多态性设计种特异性引物,同时优化反应条件,建立了能同时测定猪源粪肠球菌和屎肠球菌多重PCR方法。通过对来源于猪的临床菌株、粪便菌株和鲜猪肉菌株进行测定,均能成功扩增出属特异性片段和种特异性片段。经过与快速生化鉴定试剂盒(Vitek-32)和16 S rRNA测序方法比较,多重PCR与16 S rRNA测序方法对猪的临床菌株、粪便菌株和鲜猪肉菌株的鉴定符合率100%;与Vitek-32鉴定符合率为62.3%,其中,与分离于感染猪的临床菌株符合率仅有46.7%,特别是感染猪的屎肠球菌,符合率仅为22.3%。
文摘To analyze the microflora in fermented rice bran product, bacterial colonies were grown under various conditions. Although cultivation temperature did not affect the number of bacterial colonies formed on agar plates, twice as many colonies formed under aerobic as under anaerobic conditions. All colonies appearing on the plates showed acid production. Based on 16S rRNA sequence analysis, nearly all of the bacteria in the fermented product were highly similar (>99%) to Lactobacillus johnsonii. In addition, several Bacillus cereus and unidentified Lactobacillus strains that grew only under anaerobic conditions at 30℃?were seen. Random Amplified Polymorphic DNA (RAPD)-PCR analysis showed the amplified patterns of all isolates to differ substantially from the reference strain L. johnsonii. We conclude that L. johnsonii-related strains predominate in fermented rice bran product, and that these bacteria produce lactic acid to decrease the pH of the fermented product. Several novel Lactobacillus strains may also occur in this environment.
基金supported by the Special Fund for Agro-scientific Research in the Public Interest of China(201103005)the Science and Technology Innovation Project of Chinese Academy of Agricultural Sciences(2013–2017)
文摘On the basis of a long-term（30 years） field experiment that involved four rotation systems, rice-rice-winter fallow（RRF）, rice-rice-ryegrass（RRG）, rice-rice-rape（RRP）, and rice-rice-milk vetch（RRV）, this study described the effects of green manure on the microbial communities in the red paddy soils using 454 pyrosequencing for the 16 S r RNA gene. The Chao1 richness and non-parametric Shannon＇s index increased in all soil samples that received green manure treatments. The communities＇ structures with the green manure applications were significantly dissimilar from that under the winter fallow. Using Metastats tests, many genera in the RRG, RRP and RRV soils were significantly different from those in the RRF soil, including a number of genera that functioned in the nitrogen and sulfur cycles. Analyses of the genera with these functions revealed the shifts in microbial ecosystem functions after long-term green manuring. Changes in the microbial communities increased the ammonium supply and decreased the soil acidification in green-manure-amended soils. Together, these data suggested powerful effects of green manure on both the microbial communities and the biogeochemical cycle driven by the shifts in bacterial functional groups.
基金Supported by New Agricultural Variety,Technology and Model Project in Jiangsu Province(SXGC2015298)Project on Prospective Study of Social Development in Yangzhou City(YZ2014188)+1 种基金Science and Technology Public Service Platform Construction Project in Yangzhou City(YZ2015162)National Agricultural Product Quality and Safety Risk Assessment Project in 2016(GJFP2016007)
文摘In order to establish a quick and specific method which could identify the avian-derived ingredients,this study used 16 S rRNA gene sequence as target site,and designed the specific primers of chicken,pigeon meat and quail meat. The DNA of common livestock and poultry meat( including mutton,beef,pork,rabbit meat,pigeon meat,quail meat,chicken,duck and goose) was used as template. Though PCR amplification and specific detection,a quick determination method was established to identify the avian-derived ingredients. The results showed that the selected primers could identify the ingredients of animal origin effectively and quickly. The method was convenient and concise,and could detect the chicken-derived,pigeon-derived,quail-derived ingredients in livestock and poultry food quickly and accurately.
文摘A photoperiod-sensitive genic male sterile (PGMS) rice was found in 1973 as a spontaneous mutant of Nongken 58, a japonica variety. Pollen fertility of Nongken 58s (N58s) is completely sterile when grown under long-day conditions, whereas fertile under short-day conditions. This PGMS was found to be controlled by one or two recessive gene(s), of which one gene(pms)was linked to a marker gene(d-1) on chromosome 5. In order to identify a more precise location of the pms, we analyzed the populations of BCFand BCFof N58s//N58s/KL211(v-10, virescent) and N58s//N58s/KL520 (gh-1, gold hull). The marker genes v-10 and gh-1 are located on the flanking region of d-1. The F, plants of two crosses were fertile. The number of fertile and sterile individuals in BCFfit
基金the National Natural Science Foundation of China (Nos. 31870500, 40871146, and 41501273)the Special Project on the Basis of National Science and Technology of China (No. 2015FY110700)+1 种基金the Knowledge Innovation Program of Chinese Academy of Sciences (No. KZCX2-EW-409)the Technology Supporting Project of Jiangsu Province (No. BE2013451)
文摘Biological nitrogen (N) fixation (BNF) plays a significant role in maintaining soil fertility in paddy field ecosystems. Rice variety influences BNF, but how different rice varieties regulate BNF and associated diazotroph communities has not been quantified. Airtight, field-based 15N2-labelling grow th chamber experiments were used to assess the BNF capac 辻 y of different rice varie ties. In addition, both the 16S rRNA and nifH genes were sequenced to assess the influence of different rice varieties on bacterial and diazotrophic communities in paddy soils. After subjecting a rice-soil system to 74 d of continuous airtight, field-based 15N2 labelling in pots in a growth chamber, the amounts of fixed N were 22.3 and 38.9 kg ha^-1 in inbred japonica (W23) and hybrid indica (IIY) rice cultivars plan ted in the rice-soil systems, respectively, and only 1%—2.5% of the fixed N was allocated to the rice plants and weeds. A greater abundance of diazotrophs was found in the surface soil (0-1 cm) under IIY than under W23. Sequencing of the 16S rRNA gene showed significantly greater abundances of the cyanobacterial genera Nostoc, Anabaena, and Cylindrospermum under IIY than under W23. Sequencing of the nifH gene also showed a significantly greater abundance of Nostoc under IIY than under W23. These results indicate that the hybrid rice cultivar (IIY) promoted BNF to a greater extent than the inbred rice cultivar (W23) and that the increase in BNF might have been due to the enhanced heterocystous cyanobacteria Nostoc.
基金Supported by IIUM Research Initiative Grant,RIGS grant no.16-301-0465
文摘Objective:To identify the prevalence of Cryptosporidium from goats in three types of farm management systems in Terengganu,Malaysia and to determine the Cryptosporidium species infecting goats by using 18 S r RNA.Methods:A total of 478 fecal samples were randomly collected from goats in three farms;199 samples were collected from intensive farm,179 samples from semi-intensive farm and 100 samples from extensive farm.The samples were processed by using formolether concentration technique and stained by using modified Ziehl–Neelsen.Positive samples were performed by using nested PCR analysis by using 18 S r RNA.Results:Out of 478 goats,207(43.3%) were found to be infected with Cryptosporidium.Goats reared under the intensive farm management system reported the highest prevalence of infection(49.7%),followed by intensive farm management system(41%) and the lowest prevalence was reported in the goats reared under semi-intensive management system(37.4%).Conclusions:The identified species found in goat was Cryptosporidium parvum.Future study on the zoonotic transmission of Cryptosporidium parvum in goats needs to be done in order to find the source of transmission of this parasite.
基金partially supported by an OARDC award(2010-007)to Z.Y
文摘Background： The number and diversity of uncultured ruminal bacterial and archaeal species revealed by 16S rRNA gene firs） sequences greatly exceeds that of cultured bacteria and archaea. However, the significance of uncultured microbes remains undetermined. The objective of this study was to assess the numeric importance of select uncultured bacteria and cultured bacteria and the impact of diets and microenvironments within cow rumen in a comparative manner. Results： Liquid and adherent fractions were obtained from the rumen of Jersey cattle fed hay alone and Holstein cattle fed hay plus grain. The populations of cultured and uncultured bacteria present in each fraction were quantified using specific real-time PCR assays. The population of total bacteria was similar between fractions or diets, while total archaea was numerically higher in the hay-fed Jersey cattle than in the hay-grain-fed Holstein cattle. The population of the genus Prevotello was about one log smaller than that of total bacteria. The populations of Fibrobocter succinogenes, Ruminococcus flovefociens, the genus Butyrivibrio, and R. albus was at least one log smaller than that of genus Prevotello. Four of the six uncultured bacteria quantified were as abundant as F. succinogenes, R. flovefociens and the genus Butyrivibrio. In addition, the populations of several uncultured bacteria were significantly higher in the adherent fractions than in the liquid fractions. These uncultured bacteria may be associated with fiber degradation. Conclusions： Some uncultured bacteria are as abundant as those of major cultured bacteria in the rumen. Uncultured bacteria may have important contribution to ruminal fermentation. Population dynamic studies of uncultured bacteria in a comparative manner can help reveal their ecological features and importance to rumen functions.
