| The most important sources of AA at the duodenum of ruminants are the microbial crude protein (MCP) synthesized in the rumen, any change in microbes or MCP, such as, microbial flora, microbial AA-N ratio, and AA profile of microbes etc., is bound to affect AA supply to small intestine, and consequently influences animal performance. For a long time, it is well accepted that ruminal microbial AA is constant; moreover, it is common to use certain AA to direct ruminant practice. However, later studies reveal that, microbial AA is not constant. A review by Clark et al. (1992) indicated great differences in the AA composition of rumen bacteria, and followed by a continuous debate about this subject. Unfortunately, whether the microbial AA change or not, are still controversial issues till now. Therefore, in vitro and in vivo experiments were conducted using varied sources in this study, and aimed to illustrate whether microbial AA change, and how microbial AA change with diets. The Fluorescence-Labeled Rumen Bacteria Techniqe (FLRB) was developed to determine grazing rate of protozoa on bacteria in rumen, and the molecular technique including SSCP, cloning and sequencing etc., were also introduced into this paper. This dissertation was described in the following nine sections.Trail 1 Establishment of Fluorescence-labeled Rumen Bacteria Technique and Study on the Grazing Rate of ProtozoaStudies on the bacterial predation rate by rumen protozoa were carried out under laboratory conditions using a technique of fluorescence-labeled rumen bacteria. Four Xuhuai goats were used in this experiment to obtain rumen protozoa and bacteria. Two groups were designed as follows: one group was the whole bacteria which were labeled using fluorescence through removing free bacteria from rumen fluid (WFLB); the other group was bacteria which were labeled using fluorescence without removing free bacteria from rumen fluid (FLB). The result showed that, the bacterial predation rates of rumen protozoa were 398.40 cells/(cell h) for the group WFLB, 230.40 cells/(cell h) for the group FLB; When the corresponding values expressed as bacteria-N were: 2.15 pg N/(cell h) for the group WFLB and 1.24 pg N/(cell h) for the group FLB respectively. Extrapolating the assimilation quantity of nitrogen by ciliates on bacteria of Xuhuai goat, there were 103.20 mg N/(d head) for the group WFLB, 59.50 mg N/(d head) for the group FLB, respectively. It was estimated that protein recycling were 0.645 g Pr/(d head) for the group WFLB and 0.372 g Pr/(d head) for the group FLB, respectively. And finally, the fluorescence-labeled rumen bacteria technique (FLRB) would be a potential assay for determination of bacterial predation rate by rumen protozoa.Trail 2 Effects of Dietary Concentrate to Forage Ratio on Microbial Protein Recycling in the Rumen of GoatsIn this study, the main aims were to investigate effects of dietary concentrate to forage ratio on microbial protein recycling in the rumen of goats using a technique of FLRB, developed in trail 1. 4×4 Latin squares were conducted by using 4 Xuhuai goats with permanent cannulas, and diets were divided into A (10:90), B (30:70), C (50:50), and D (70:30) by varying concentrate to forage ratios, which concentrated food were corn-soybean meal, while forage were straw in this experiment. The result showed that, rumen micro-ecosystem was shifted heavily by concentrate to forage ratio. The highest protozoal density were recorded by group C which dietary concentrate to forage ratio was set as 50:50, whereas densities of both protozoa and bacteria were lowest for the group A; grazing rates of rumen protozoa on bacteria were, respectively: 429.50 cells/(cell h), 366.74 cells/(cell h), 389.48 cells/(cell h), and 402.20 cells/(cell h) for group A, B, C, and D. When the corresponding values expressed as bacteria-N were: 2.319 pg N/(cell h), 1.98 pg N/(cell h), 2.103 pg N/(cell h), and 2.172 pg N/(cell h) respectively. Extrapolating the assimilation quantity of nitrogen by ciliates on bacteria of Xuhuai goat with different diets, there were 136.49 mg N/(d head), 369.02 mg N/(d head), 485.99 mg N/(d head), and 440.56 mg N/(d head) for group A, B, C, and D respectively. It was estimated that, protein recycling of rumen bacteria were 0.853 g Pr/(d head) , 2.306 g Pr/(d head), 3.370 g Pr/(d head), and 2.754 g Pr/(d head) respectively, with group C recording the highest protein recycling of rumen bacteria and turnover rates (3.07 %).Trail 3 Effects of Dietary Concentrate Levels on Rumen Fermentation, Protozoal Profiles and Grazing RatesThe objectives of present studies were to demonstrate the effects of dietary concentrate to forage rate on rumen fermentation, protozoal structure and grazing rates in goats'rumen. The experimental animal and desigh were the same as trial 2. The result showed that, rumen fermentation was shifted heavily by concentrate to forage ratio. High microbial activity and high NDF degradability were observed in group B, and pH was stable in this group, comparatively. It was also observed that, protozoal dynamics was modulated by dietary structure. Percentages of Entodiniinae and Isotrichidae were higher for diets which concentrate level were high, whereas percentages of Diplodiniinae and Ophryoscolecinae were higher for diets which forage level were high. The predation rates were 361.90, 606.30, and 607.50 cells/(cell h) for Entodiniinae, Diplodiniinae, and Ophryoscolecinae respectively, and marked difference was found among genus (P=0.000), however, the change rule of grazing rate with dietary structure seemed to be similar across genus. Regression analysis revealed that, there were strong cubic relationship between predation rates and dietary structure (Y=-724.53X+563.15X2-124.666X3+646.833, R2=0.97864). Additionally, multivariate regression analysis revealed the existence of linear correlations between predation rates and cell densities (protozoa and bacteria) (R2=0.839). The equation was presented bellow: Y=445.514-3.078X1+1.864X2 (Y- predation rate; X1 - bacteria density; X2 - protozoa density).Trail 4 Effects of Different Structure of Carbhyborate on Rumen Fermentation and Microbes characteristics in vitroThree goats fisted with cannulas were used to investigate the effects of rations in different starch to cellulose ratio on rumen fermentation and microbial characteristics in vitro. Substrates were designed by varying the level of starch/cellulose ratio as follows: 100:0, 70:30, 50:50, 30:70, 0:100. The results showed that: Cellulose degradability and microbial biomass were highest when starch/cellulose ratio in the culture was set to 30:70; The regression analysis between microbial protein and substrate were: Bacteria: Y=0.2410+0.0855X-0.0371X2 +0.0029X3 (R=0.7397); Protozoa: Y=0.2276+0.0853X-0.0380X2+0.0030X3 (R=0.7370) (Y: microbial protein, mg/ml; X: Starch/Cellulose), respectively. Significant differences were found in microbial AA-N ratio between groups (P<0.05); Furthermore, protozoa to bacteria ratio had a tendency to increase firstly, followed by decline when starch/cellulose ratio decreased, and the highest record falling in the group provided ration containing starch to cellulose ratio of 50:50; SSCP analysis further revealed that microbial structure was shifted by substrates; and also, cell-counting of protozoa showed that, Entodinium and Isotricha decreased, whereas Diplodinium and Ophryoscolecinae increased according to the decrease of starch/cellulose ratio, and revealed that the profile of protozoa was subjected to substrates, which agreed with SSCP. Additionally, the average recovery rate of microbes after detaching was 53.29 %; the DNA extraction ratio from bacteria (45.40 %) were significantly less than protozoa (56.10 %); the size of DNA fragment extracted from all the samples were larger than 20 kb; and fit for advanced research. It was therefore concluded that carbohydrate structure influenced both rumen fermentation and microbial characteristics. And finally SSCP has a potential for research on characterizing rumen mixed microbes, ITS1 is also good at research on protozoal diversity. So far, a measurement system was developed for the research on microbial characteristics in this trail. Trail 5 Effects of Different Nitrogen Compounds MCP Yields and Microbial Diversity of RumenIn this study, the main aims were to investigate effects of different N compounds on rumen fermentation, MCP yields and microbial diversity in vitro, using rumen liquor provided by 3 goats. Four treatments were NH4Cl, mixed oligo-peptide (<3, 000 Da), mixed oligo-peptide (<500 Da), and free amino acid respectively. Results showed that, the recorded pH-value ranged between 6.40 and 6.90, the average pH-value was lowest for the group with free amino acid, and highest for the group with NH4Cl in the culture. The varied range of NH3-N concentration was from 11.60 to 29.45 mg/100ml. The mean concentration of NH3-N was lowest in mixed oligo-peptide, and the highest was found in free amino acid. It was also observed that, yields of microbial protein varied with substrates, with lowest record dropping in NH4Cl, and microbial yields of both two peptides groups were comparatively higher. The protozoa to bacteria ratio was also shifted by substrates, and oligo-peptide recorded the highest peak, while the opposite was found for NH4Cl (P<0.01). Additionally, significant differences were found in microbial AA-N ratio between groups, with the lowest record falling in the group whose substrate was free amino acid (P<0.05). Furthermore, microbial diversity was demonstrated in SSCP fingerprint clearly, revealed that the structure of bacteria or protozoa community was altered by substrates, and the diversity in NH4Cl was much low. It could be concluded that, both rumen MCP yields and microbial structure were modified by different N compounds.Trail 6 Study on Effects of Certain Amino Acids-Removal on the Growth of Rumen Microbe in vitroThe objectives of this study were to determine the effects of certain amino acids on rumen fermentation and microbial growth in vitro. Three goats fitted with cannula were used to provide rumen liquor, and the removal method was introduced into current experiment. Treatments were, respectively, total essential amino acid (A), His-removal (B), Lys-removal (C), Met-removal (D), branch chain amino acid (BCAA)-removal (E). Results showed that, the pH-value ranged between 5.90 and 6.80, and the highest mean value was observed in the group E (6.54). Concentration of NH3-N ranged between 10.99 to 30.51 mg/100ml, with the highest mean value dropping in the group A (17.85 mg/100ml). It was also observed that, yields of microbial protein and limiting degree on microbial growth were varied with treatments, with the group E demonstrating the lowest record (0.1389, 0.1772 and 0.3161 mg/ml for bacteria, protozoa, and microbes respectively) (P<0.01). The microbial yield of this group, comparing with the group A, decreased by 44.52 %. And also, the response to the substrate of protozoa differed from bacteria, and significant differences were found in the protozoa to bacteria ratio between groups. The group C interpreted the lowest data (89.12 %), and the reverse was true for the group E, displayed the highest value (127.60 %) (P<0.01). Additionally, microbial AA-N ratio was shaped by substrates, and lowest in the group A (16.89), while highest in the group E (18.76) (P<0.01). Further genetic fingerprint analysis revealed that, microbial profile was modified by substrates within bacteria or protozoa community. In conclusion, the rumen fermentation and microbial growth responded differently to certain amino acid, based on current in vitro trail.Trail 7 Effects of Different Protein Supplement on Rumen Fermentation and Microbial Community in vitroCertain protein supplement were used as substrates in this trail, and aimed to detect the characteristics of rumen fermentation and microbial community in vitro, by three goats fitted with cannula. Treatments: A (Plume meal), B (Corn gluten meal), C (Soybean meal), D (Fish meal). Results showed that, the pH-value ranged between 5.80 and 6.80, and the lowest mean value was found in the group C (6.19), while the highest occurred in the group A (6.60). NH3-N concentration ranged between 3.68 to 12.01 mg/100ml, with the group A recording the lowest average NH3-N concentration (5.49 mg/100ml), while the group C (9.95 mg/100ml) writing the highest value. Also, yields of microbial protein were varied with treatments (P<0.05, P<0.01), and the lowest record was observed in the group of A (0.5289 mg/ml), the other way round, the highest was found in the group of D (0.6513 mg/ml), it should be noted that the group C showed the highest bacteria yield (0.3309 mg/ml). It was further observed that, the protozoa to bacteria ratio was lowest in group C (84.30 %), the opposite was found for group A (107.00 %), interpreting the highest peak. Further SSCP analysis revealed that, profiles of both bacteria and protozoa subjected to substrates. Additionally, significant differences were found in microbial AA-N ratio between groups, and AA-N ratio was lowest (or highest) in the group of C (or D) (P<0.01). In a word, protein stuff brought out the changes, which were not just in microbial fermentation and flora, but in microbial AA-N ratio.Trail 8, 9 Effects of Dietary Protein on Microbial Community and AA Composition of RumenTrail 8 The objectives of these parts were to investigate how rumen fermentation, microbial community, microbial protein (MCP) yields, and AA composition of MCP changed with dietary protein. Four goats fitted with rumen cannula, were used in a 4×4 Latin square design. And formula diets were divided into 4 groups according to their nitrogen source, which was, respectively, plume meal (A), corn gluten meal (B), soybean meal (C), and fish meal (D). At the beginning of first experimental period, rumen liquor was collected from each goat, and used for in vitro culture with the corresponding mixed diet, before the animal experiment. This pre-experiment aimed to ensure the necessity and validity of the complex and expensive animal experiment. The results showed that, marked differences were found in fermentation parameters, microbial diversity, and AA-N ratio etc. across groups, although the degree of variation had a somewhat decline, compared with the results of sole culture of protein supplement (trail 7). And these results indicated that, it was necessary and significant to carry out animal experiment using these mixed diet.Trail 9 The results of the following 104-day in vivo experiment were described here. The recorded pH-value ranged between 5.60 and 6.80, and mean pH value of group A and C were high, the reverse was true for group B and D (P<0.05), the most interesting thing was that, although the mean pH value of group A and C (B and D) seemed to be similar, the change patterns of pH with time differed from each other; Concentration of NH3-N ranged between 6.77 to 21.67 mg/100ml, with the group of A interpreting the lowest average NH3-N concentration (11.08 mg/100ml), while highest peak droping in the group of C (15.04 mg/100ml). Yields of microbial protein were also varied with diets; microbial protein of the group C and D were much higher than that of the group A and B comparatively (P<0.05). And surprisingly, the microbial protein yield of group C and D were similar, but the bacterial protein yield of group C was significantly higher than that of group D. As for microbial community, protozoa to bacteria ratio had notable differences across groups, with group C recording the lowest value of 81.27 %, through a comparison with 3 other groups (P<0.05). Further cloning and sequence analysis revealed that, significant differences were found in 8 kinds of bacteria, such as: R.flavefaciens, R.bromii, and Roseburia faecalis etc., and also, there were marked differences in 4 protozoal genuses except Diplodinium. The microbial AA-N ratio was also alerted by dietary protein. Group C recorded the lowest valve, and the opposite was found for group D, recorded the highest peak. Furthermore, multiple regression analysis showed that AA-N ratio had a negative (positive) correlation with bacteria (protozoa) protein. It was also observed that, significant differences were found between protozoa and bacteria in AA content for some, but not all, amino acids. Within each microbial fraction, additionally, significant differences in the content of AA were found between rations. Protozoal Valine content was higher than bacterial, while Lysine content was high in bacteria. For bacteria, significant difference was detected in Arginine only; for protozoa, Methionine, Leusine, and Histidine showed significant differences. And the variations of AA were related to the changes of microbial fractions in some degree. All in all, rumen fermentation, microbial profile, AA-N content, and AA composition of ruminal MCP were all modified by dietary protein.
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