Rumen enzyme profile and fermentation characteristics in sheep as affected by treatment with sodium lauryl sulfate as defaunating agent and presence of ciliate protozoa

The efficiency of sodium lauryl sulfate as a defaunating agent and effect of rumen protozoa on nutrient utilization, fermentation characteristics and enzyme profile were evaluated in adult sheep maintained on a mixed ration containing 65:35% Pala (Ziziphus numularia) leaf: concentrate. Twenty-one adult Malpura sheep divided into three equal groups (DF, RF and F) were either defaunated by oral administration of sodium lauryl sulfate at the rate of 8 g/100 kg body weight (DF), or defaunated and again refaunated (RF), or maintained faunated (F). Daily dry matter intake was similar in defaunated, refaunated and faunated sheep. However, digestibility of cell wall and cell wall contents (NDF, ADF and cellulose) were lower (P < 0.01) in defaunated than refaunated and faunated sheep. Irrespective of the presence or absence of rumen protozoa, daily intake of DCP and DE were similar in the three experimental groups. Even with similar DM, DCP and DE intake, N-retention, blood glucose level, ruminal concentration of total VFA and total-N were higher (P < 0.01), while rumen pH and NH₃-N concentration were lower (P < 0.01) in defaunated sheep. Ruminal activity of amylase, xylanase, protease and urease enzymes were not influenced by presence or absence of ciliate protozoa. However, carboxymethyl cellulase enzyme activity was lower (P < 0.01) in the rumen of defaunated sheep. The total and differential counts of rumen protozoa were similar in refaunated and faunated sheep indicating lack of residual toxic effect of sodium lauryl sulfate. It is concluded that absence of ciliate protozoa increased ruminal TVFA, total-N with lower NH₃-N concentration and fibre digestibility in sheep. Moreover, sodium lauryl sulfate was fully effective for complete removal of rumen ciliate protozoa and successfully defaunated the sheep.     

Effect of ciliate protozoa on the activity of polysaccharide-degrading enzymes and fibre breakdown in the rumen ecosystem

The effect of ciliate protozoa on the activity of polysaccharide-degrading enzymes in microbial populations from the digesta solids and liquor fractions of rumen contents was examined after the refaunation of ciliate-free sheep with an A-type rumen protozoal population. Although the culturable rumen bacterial population was reduced after refaunation the number of fibrolytic micro-organisms detected was higher; the xylanolytic bacterial population and numbers of fungal zoospores were increased after refaunation. The proportion of propionic acid was lower in the refaunated animals, whereas the concentration of ammonia and the acidic metabolites acetate, butyrate and valerate were all increased. The range of enzyme activities present in the digesta subpopulations were the same in defaunated and refaunated animals. The activities of the polysaccharide-degrading enzymes, however, were increased in the microbial populations associated with the digesta solids after refaunation, and at 16 h after feeding the activities were 4–8 times (β-d-xylosidase 20 times) higher than the levels detected in the adherent population from defaunated sheep. The protozoa, either directly through their own enzymes or indirectly as a consequence of their effects on the population size and activity of the other fibrolytic micro-organisms present, have an important role in determining the level of activity of polysaccharide-degrading enzymes in the rumen ecosystem. Although the extent of ryegrass ( Lolium perenne ) hay digestion was similar after 24 h in the absence or presence of protozoa, the initial ruminal degradation was higher in refaunated sheep.     

Effect of ciliate protozoa on the activity of polysaccharide-degrading enzymes and fibre breakdown in the rumen ecosystem.

The effect of ciliate protozoa on the activity of polysaccharide-degrading enzymes in microbial populations from the digesta solids and liquor fractions of rumen contents was examined after the refaunation of ciliate-free sheep with an A-type rumen protozoal population. Although the culturable rumen bacterial population was reduced after refaunation the number of fibrolytic micro-organisms detected was higher; the xylanolytic bacterial population and numbers of fungal zoospores were increased after refaunation. The proportion of propionic acid was lower in the refaunated animals, whereas the concentration of ammonia and the acidic metabolites acetate, butyrate and valerate were all increased. The range of enzyme activities present in the digesta subpopulations were the same in defaunated and refaunated animals. The activities of the polysaccharide-degrading enzymes, however, were increased in the microbial populations associated with the digesta solids after refaunation, and at 16 h after feeding the activities were 4-8 times (beta-D-xylosidase 20 times) higher than the levels detected in the adherent population from defaunated sheep. The protozoa, either directly through their own enzymes or indirectly as a consequence of their effects on the population size and activity of the other fibrolytic micro-organisms present, have an important role in determining the level of activity of polysaccharide-degrading enzymes in the rumen ecosystem. Although the extent of ryegrass (Lolium perenne) hay digestion was similar after 24 h in the absence or presence of protozoa, the initial ruminal degradation was higher in refaunated sheep.     

Contribution of protozoa to lysine synthesis in the in vitro rumen microbial ecosystem

Isotopic tracer experiments were conducted in vitro to determine contribution of protozoa toward the biosynthesis of lysine in the rumen microbial ecosystem. The presence of protozoa in a rumen microbial suspension always increased lysine synthesis from aspartate. Rumen contents from a faunated goat produced a higher amount of lysine than did those from a defaunated one.     

Contribution of protozoa to lysine synthesis in the in vitro rumen microbial ecosystem.

Isotopic tracer experiments were conducted in vitro to determine contribution of protozoa toward the biosynthesis of lysine in the rumen microbial ecosystem. The presence of protozoa in a rumen microbial suspension always increased lysine synthesis from aspartate. Rumen contents from a faunated goat produced a higher amount of lysine than did those from a defaunated one.     

Influence of ciliate protozoa on biochemical changes and hydrolytic enzyme profile in the rumen ecosystem

AIMS: To assess the effect of presence or absence of rumen protozoa on fermentation characteristics and enzyme profile in growing lambs. METHODS AND RESULTS: Weaner lambs (G1, G2, G3, G4, G5 and G6 groups) were defaunated by oral administration of sodium laurel sulphate (at 8 g 100 kg(-1) body weight). The lambs of G4, G5 and G6 groups were refaunated. The roughage and concentrate ratio in the diet of G1 and G4, G2 and G5, and G3 and G6 were 50:50 (R1), 65:35 (R2) and 80:20 (R3), respectively. Daily dry matter intake was similar in defaunated and faunated lambs. However, digestibility of organic matter (OM), cellulose and gross energy were lower in defaunated lambs while crude protein (CP) digestibility was similar in both defaunated and faunated lambs. The rumen pH and NH3-N were lower (P < 0.01) while TVFA, total-N and TCA-ppt-N were higher (P < 0.01), in defaunated lambs. Ruminal activity of carboxymethyl cellulase was lower (P < 0.01) in defaunated lambs and amylase, xylanase, protease and urease were similar in faunated and defaunated lambs. Nutrient utilization, rumen metabolites and ciliate protozoal count were higher, whereas digestibility of fibre fractions was lower in high rather than low concentrate fed lambs. The rumen protozoa present before defaunation were B-type and the protozoa which re-established on refaunation were also B-type. CONCLUSIONS: Absence of ciliate protozoa decreased nutrient digestibility and increased ruminal TVFA and total-N with lower NH3-N concentration, indicating better energy and protein utilization in defaunated lambs. SIGNIFICANCE AND IMPACT OF THE STUDY: Defaunation improved energy and protein utilization in lambs.     

Omasal ciliated protozoa in cattle, bison, and sheep.

Omasal contents were collected from slaughtered cattle (n = 54), bison (n = 15), and sheep (n = 40) to determine numbers and generic distribution of ciliated protozoa. Total protozoan numbers were significantly lower in omasal contents than in ruminal contents of all three species, but the percent composition of all protozoan genera was similar between omasal and ruminal populations. The highest numbers of omasal protozoa found were 7.61 X 10(5)/g in cattle, 7.01 X 10(5)/g in bison, and 1.29 X 10(6)/g in sheep. Omasal dry matter was significantly higher than ruminal dry matter in all species and ranged up to 51.5% in cattle fed high-concentrate diets. The omasal pH was similar to the ruminal pH in all species. The number of omasal laminae averaged 149, 145, and 74 for cattle, bison, and sheep, respectively. Although protozoan concentrations in omasal contents were approximately 80% lower than those in ruminal contents, the omasum harbored relatively high numbers of ciliated protozoa. The resident omasal protozoa are extremely difficult to remove, particularly in cattle, and apparently are responsible for reinoculating transiently defaunated rumens.     

Omasal ciliated protozoa in cattle, bison, and sheep

Omasal contents were collected from slaughtered cattle (n = 54), bison (n = 15), and sheep (n = 40) to determine numbers and generic distribution of ciliated protozoa. Total protozoan numbers were significantly lower in omasal contents than in ruminal contents of all three species, but the percent composition of all protozoan genera was similar between omasal and ruminal populations. The highest numbers of omasal protozoa found were 7.61 X 10(5)/g in cattle, 7.01 X 10(5)/g in bison, and 1.29 X 10(6)/g in sheep. Omasal dry matter was significantly higher than ruminal dry matter in all species and ranged up to 51.5% in cattle fed high-concentrate diets. The omasal pH was similar to the ruminal pH in all species. The number of omasal laminae averaged 149, 145, and 74 for cattle, bison, and sheep, respectively. Although protozoan concentrations in omasal contents were approximately 80% lower than those in ruminal contents, the omasum harbored relatively high numbers of ciliated protozoa. The resident omasal protozoa are extremely difficult to remove, particularly in cattle, and apparently are responsible for reinoculating transiently defaunated rumens.     

Symbiotic nitrogen fixation and nitrate reduction in two peanut cultivars with different growth habit and branching pattern structures

We have investigated the response of two peanut cultivars (TEGUA and UTRE) with different growth habits and branching pattern structures to different nitrogen (N) sources, namely, N-fertilizer or N₂ made available by symbiotic fixation, and analysed the pattern of nitrate reductase (NR) activity in these cultivars. Nitrate and amino acid contents were also examined under these growth conditions. In terms of nitrogen source, cv. TEGUA showed a better response to inoculation with Bradyrhizobium sp. SEMIA 6144 at 40 days after planting, while cv. UTRE responded better to N-fertilizer (5 mM KNO₃). Both cultivars showed different patterns of NR activity in the analyzed plant organs (leaves, roots, and nodules), which were dependent on the N source. When nitrogen became available to the plant through symbiotic N₂ fixation, the patterns of NR activity distribution were different in the two cultivars, with cv. TEGUA showing a higher NR activity in the nodules than in the leaves and roots, and cv. UTRE showing no difference in terms of NR activity among organs. The nitrate and amino acid contents showed a similar trend between the two cultivars, with the highest nitrate content in the leaves of fertilized plants and the highest amino acid content in the nodules. The high nitrate content of the leaves of cv. UTRE indicated the better response of this cultivar to N-fertilizer.     

Rhizobium leguminosarum strains forming nitrogen-rich nodules inPisum sativum

The nodules which developed on the roots ofPisum sativum after inoculation withRhizobium leguminosarum bv.viciae strains showing a Hup⁺ symbiotic phenotype and/or a high relative efficiency of electron transfer to dinitrogen (RE) had a high nitrogen content of their tissue. In comparison with the nodules initiated by a strain possessing the Hup⁻ symbiotic phenotype or by an indigenous soilRhizobium population, the nitrogen-rich root nodules contained up to four times more nitrogen (9.2% of dry mass). In the nitrogen-rich nodules, total amino acid, and especially Ala, Lys and Phe contents were significantly increased. The nitrogen-rich nodule symbiotic phenotype (Nrn) was well expressed, irrespective of pea cultivars and host plant age. If a strain showing the Nrn phenotype was applied in double-strain inocula, a significant correlation was found between increasing rates of the strain and increasing percentage nitrogen content of nodule tissue in the nodulated plants.     

Simple Method to Remove Completely Ciliate Protozoa of Adult Ruminants

Dioctyl sodium sulfosuccinate (Aerosol OT) has been used in a remarkably short period to obtain rumen ciliate-free cows. Two 30-g doses of Aerosol OT given on consecutive days appeared to effectively eliminate all types of rumen ciliate protozoa without harming the health of the host animal. Measurement of the rumen metabolic parameters of gas production, carbon dioxide to methane ratio, pH value, volatile fatty acids, ammonia, and in vitro cellulose digestion, along with total amylolytic streptococci counts of the rumen contents, showed that conditions in rumens of defaunated animals are normal 4 days after such treatment. It appears that such animals may be used in ruminal studies that require defaunated animals.     

Relationships between nitrogen,amino acids and storage proteins in Lupinus albus seeds

Lupinus albus seeds (20 samples from 10 different lines or cultivars) with protein contents distributed from 23.8 to 48.4% were analysed for their amino acid composition with great accuracy (from 6 hydrolysates per sample). Amino acid levels in seeds increased as linear functions of nitrogen content with correlation coefficients close to unit in all the lupin genotypes and phenotypes. Hence the composition of any lupin seed sample can be predicted from its nitrogen content. Amino acid in total seed protein varied as hyperbolic functions of nitrogen content. The same was true for nitrogen-to-protein conversion factors. By controst, the composition of storage proteins accumulated in seeds remained constant and independent of seed protein content.     

稻田温室气体减排措施对稻米氨基酸含量的影响

为探索稻田中温室气体减排措施对稻米氨基酸含量的影响,用10个不同方法对双季稻田进行处理,并使用高效液相色谱分别测定了各处理稻田中所产稻米中的16种氨基酸的含量,其中色谱柱为Agilent Zorbax AAA分析柱,柱前衍生使用邻苯二甲醛(OPA)和9-芴甲基氯甲酸酯(FMOC-CL)为衍生试剂。结果发现:1)10个处理中的稻米16种氨基酸种类齐全,施氮肥+添加生物质炭48 t/hm2+间歇灌溉(NPK+HBC+IF)处理中所得氨基酸总量为6520.7 mg/100g,效果最佳;对照组处理(不施加氮肥+无稻草还田+间歇灌溉)所得氨基酸含量4338.0 mg/100g为最低。以对照组处理所得必需氨基酸百分含量36.8%为最高值;无稻草还田+长期淹水(NPK+CF)处理方法所得必需氨基酸百分含量33.1%为最低值。10个处理中16种氨基酸中含量较高的氨基酸均为天冬氨酸、谷氨酸和精氨酸,含量最低的均为甲硫氨酸;2)施氮肥量相同时,长期淹水与间歇灌溉相比,氨基酸总量增加185.1 mg/100g,非必需氨基酸百分含量增加3%,谷氨酸、组氨酸和丝氨酸含量明显升高,但亮氨酸含量显著降低;3)施用氮肥能提高稻米中的氨基酸含量,且随着氮肥使用量的增加,氨基酸含量也随之增加,组氨酸含量增加显著;4)供氮量相同时,添加猪粪使氨基酸总含量升高了286.0 mg/100g,此结果表明,在供氮量相同的情况下,施用猪粪更有利于稻米氨基酸含量的提高;5)灌溉模式相同时,稻草还田配施氮肥对必需氨基酸和氨基酸总量均有提高,天冬氨酸、谷氨酸和组氨酸的含量增加较多,甲硫氨酸含量略有下降;随着稻草还田量的增加,对非必需氨基酸影响较为明显;当稻草半量还田(还田量为3 t/hm2)时,稻米中氨基酸总量增加最多;稻草全量还田+长期淹水(NPK+HRS+CF)与稻草半量还田+间歇灌溉(NPK+LRS+IF)处理中的氨基酸含量基本接近,但必需氨基酸含量前者略高于后者,说明稻草还田与水肥管理对氨基酸含量影响可能存在交互作用;6)添加生物质炭配施氮肥提高了稻米必需氨基酸与非必需氨基酸含量,且随着生物质炭添加量的增加而增加;与稻草还田、添加猪粪处理相比,生物质炭的添加对氨基酸总含量提升的效果最为显著,对稻田实际生产具有指导意义且具有一定的环境效益。     

Relationship Between Rumen Ammonia Levels and the Microbial Population and Volatile Fatty Acid Proportions in Faunated and Defaunated Sheep

Cheviot wethers were defaunated by using dioctyl sodium sulfosuccinate and were constantly infused with urea to provide 2.87% of the daily N intake. Defaunation resulted in higher rumen dry matter and lower rumen pH. The molar per cent propionate was higher in defaunated sheep, whereas the molar per cent butyrate and acetate was lower. Apparent nitrogen digestibility, nitrogen utilization, and nitrogen balance were higher in defaunated sheep when compared with faunated animals. Urea infusion resulted in lower apparent nitrogen digestibility, nitrogen utilization, and nitrogen balance in faunated sheep, but did not affect nitrogen metabolism in defaunated sheep. Rumen ammonia-N levels in defaunated sheep were lower than those observed for faunated animals, and urea infusion into faunated sheep increased rumen ammonia-N levels to a greater extent than did the urea infusion into defaunated animals. Significant correlations were demonstrated between rumen ammonia-N levels and C(2)/C(3), C(3)/C(4) and C(2)/C(4) volatile acid ratios. From this it was concluded that, as rumen ammonia-N levels increased, there was a shift from propionate to higher proportions of butyrate and acetate.     

Two-Step Freezing Procedure for Cryopreservation of Rumen Ciliates, an Effective Tool for Creation of a Frozen Rumen Protozoa Bank

The present study aimed at the long-term storage of rumen protozoa as living cells in liquid nitrogen. The two-step or interrupted slow freezing procedure was used to cryopreserve six of the dominant species of rumen ciliates isolated from monofaunated animals, Dasytricha ruminantium, Entodinium caudatum, Epidinium ecaudatum caudatum, Eudiplodinium maggii, Isotricha prostoma, and Polyplastron multivesiculatum. We optimized the first step in the interrupted slow freezing procedure, from the extracellular ice nucleation temperature to the holding temperature, and studied the effects of the cooling rates on survival. In addition to the nature of the cryoprotectant (dimethyl sulfoxide), the equilibration temperature and equilibration time (25°C and 5 min, respectively), and the holding time at subzero temperature (45 min) recommended previously (S. Kišidayová, J. Microbiol. Methods 22:185-192, 1995), we found that a holding temperature of −30°C, a cooling rate from extracellular ice nucleation temperature to holding temperature of between 1.2°C/min and 2.5°C/min, depending on the ciliate, and rumen juice as the freezing and thawing medium markedly improved the survival rate. Survival rates determined after 2 weeks in liquid nitrogen were 100% for Isotricha, 98% for Dasytricha, 85% for Epidinium, 79% for Polyplastron, 63% for Eudiplodinium, and 60% for Entodinium. They were not significantly modified after a period of 1 year in liquid nitrogen. Four of the five ciliate species cryopreserved for 8 months in liquid nitrogen successfully colonized the rumen when inoculated into defaunated animals. These results have made it possible to set up a bank of cryopreserved rumen protozoa.     

Methane production and substrate degradation by rumen microbial communities containing single protozoal species in vitro

AIMS: To assess the effect of protozoal species on rumen fermentation characteristics in vitro. METHODS AND RESULTS: Entodinium caudatum, Isotricha intestinalis, Metadinium medium, and Eudiplodinium maggii from monofaunated wethers and mixed protozoa from conventional wethers were obtained by centrifugation, re-suspended at their normal densities in rumen fluid supernatants from defaunated or conventional wethers and incubated in vitro. The presence of protozoa increased the concentration of ammonia and altered the volatile fatty acids balance with more acetate and butyrate produced at the expense of propionate. Differences among species were observed, notably in the production of methane, which increased with E. caudatum as compared to other ciliates and to defaunated and mixed protozoa treatments (P < 0.05). The increased methanogenesis was not correlated to protozoal biomass indicating that the metabolism of this protozoan and/or its influence on the microbial ecosystem was responsible for this effect. CONCLUSIONS: Entodinium caudatum stimulated the production of methane, a negative effect that was reinforced by a concomitant increase in protein degradation. SIGNIFICANCE AND IMPACT OF THE STUDY: Comparison of individual species of protozoa highlighted the particular influence of E. caudatum on rumen fermentation. Its elimination (targeted defaunation) from the rumen could reduce methane production without affecting feed degradation.     

Planthopper "adaptation" to resistant rice varieties: changes in amino acid composition over time

The brown planthopper, Nilaparvata lugens, shows considerable geographic and temporal variability in its response to varieties of cultivated rice. N. lugens has repeatedly “adapted” to resistant rice varieties; however, the physiological changes underlying planthopper adaptation are poorly understood. Endosymbionts within planthoppers, such as yeast-like endosymbionts (YLS) could play a role as they produce essential amino acids for planthoppers. We used a full factorial study to determine how natal rice variety, exposed rice variety, YLS presence, and the number of reared generations affected nymphal development, planthopper total nitrogen content, and planthopper hydrolyzed amino acid profiles. Nymphal development was strongly influenced by a four-way interaction between the exposed rice variety, natal rice variety, number of reared generations, and YLS presence. While symbiosis improved nymphal performance in the 8th generation, it appeared to be a drain on nymphs in the 11th generation, when the aposymbiotic nymphs actually showed higher performance than the symbiotic nymphs. This suggests that the symbiotic relationship may be acting beneficially in one generation while acting as a drain during another generation. Aposymbiotic planthoppers reared for 11 generations had a higher proportional concentration of rare amino acids than those reared for 8 generations, indicating that the planthopper itself appears to improve its ability to acquire rare amino acids. Therefore, the change in amino acid composition of planthoppers suggests an underlying change in protein expression or amino acid metabolism over time.     

The importance of methanogens associated with ciliate protozoa in ruminal methane production in vitro

The importance of methanogenic bacteria associated with ciliate protozoa was estimated either by removing protozoa from whole rumen fluid (using defaunated rumen fluid to correct for the effects of centrifugation on bacteria) or by isolating the protozoa. Rumen fluid was withdrawn from sheep inoculated with either Polyplastron multivesiculatum , a co-culture of Isotricha prostoma plus Entodinium spp. or a mixed type B fauna of Entodinium, Eudiplodinium and Epidinium spp. Methanogenesis was highest in rumen fluid containing a mixed protozoal population of the following genera: Entodinium, Eudiplodinium and Epidinium , was lower in defaunated rumen fluid and lowest in rumen fluid containing either I. prostoma plus Entodinium or P. multivesiculatum . Methanogenic bacteria associated with rumen ciliates were apparently responsible for between 9 and 25% of methanogenesis in rumen fluid.