ples, standard values for protein per bacterial cell and protein per micrometer cubed of protozoal cell volume were

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1984, p /84/ $02.00/0 Vol. 47, No. 3 Negative Correlation Between Protozoal and Bacterial Levels in Rumen Samples and Its Relation to the Determination of Dietary Effects on the Rumen Microbial Populationt R. M. TEATHER,* S. MAHADEVAN, J. D. ERFLE, AND F. D. SAUER Animal Research Centre, Agriculture Canada, Ottawa, Ontario KIA 0C6, Canada Received 1 September 1983/Accepted 2 December 1983 The bacterial protein content and protozoal protein content of unfractionated samples from the liquidsmall particle phase of the rumen were determined on the basis of direct microscopic measurement of bacteria numbers and protozoa numbers and cell volumes. Standard values of 8.7 x mg of protein per bacterial cell and 5.9 x 10- mg/p.m3 of protozoa cell volume, obtained from analysis of isolated cells, were used to convert the microscopic measurements to an estimate of the protein content of the rumen sample. When the correlation between bacterial and protozoal protein levels was examined within groups of animals, a highly significant negative correlation between these two parameters was found (P < 0.001). The variation among animals for total (bacterial plus protozoal) microbial protein was smaller than the variation among animals for bacterial or protozoal protein alone. There was also a highly significant positive correlation (P < 0.001) between protozoal protein level and total microbial protein level. The variation found among animals in total microbial protein level could be reduced by using a regression equation determined for bacterial versus protozoal protein to correct for the different population dynamics of the two groups. Studies on dietary effects on the rumen ecosystem commonly deal only with the bacterial or the protozoal population of the rumen, most commonly with the former. The protozoal population tends to be ignored for a variety of reasons. A number of studies have shown that there is little difference between faunated and defaunated animals in either rumen function or animal performance (3, 5-8, 12, 17, 21, 26). The protozoa are also difficult to enumerate directly (1, 13, 24, 39, 40; D. B. Purser, Ph.D. dissertation, University of Western Australia, Perth, 1961), and no reliable biochemical marker which would allow an indirect quantitation is known (4, 16). However, comparison of either bacterial or protozoal populations alone within a group of animals generally shows a high level of variation among animals (e.g., three- to fivefold for protozoa [14, 38] and two- to fivefold for bacteria [31, 38]; for review see references 13, 23, 39), which makes meaningful comparisons between experimental treatments difficult. Because these populations interact through both competition and predation, some of this apparent variation may be due to an altered balance between the bacterial and protozoal populations rather than to variation among animals in total rumen biomass. The interdependency of these populations is apparent in in vitro studies and in cases in which faunated and defaunated animals have been compared (10, 15, 17, 18, 20, 23, 29). The objective of this study was to investigate the relationship between bacterial and protozoal protein levels in samples from the liquid-small particle phase of the rumen of faunated animals, first to determine whether the levels were significantly related to each other and second to compare animal-to-animal variation in total microbial protein level with animal-to-animal variation in bacterial or protozoal protein levels. To facilitate separate estimation of protozoal and bacterial protein levels in unfractionated rumen sam- * Corresponding author. t Animal Research Centre Contribution no ples, standard values for protein per bacterial cell and protein per micrometer cubed of protozoal cell volume were determined by analysis of isolated bacterial and protozoal cell fractions. These values were used to convert bacterial numbers per milliliter and micrometers cubed of protozoal cell volume per milliliter, measured by microscopic examination of the unfractionated sample, to an estimate of the bacterial and protozoal protein content of the sample. MATERIALS AND METHODS Animals and feeding. Four mature nonlactating Holstein cows fitted with rumen fistulae were housed in a tie-stall barn and fed a corn silage-concentrate ration premixed on a 60:40 dry matter basis. The concentrate consisted of (percentage of dry matter): soybean meal, 33.5; barley, 53.2; molasses, 8.0; dicalcium phosphate, 2.3; limestone, 1.4; Dynamate (a sulfur, magnesium, and potassium supplement from International Minerals and Chemical Corp., Mundelein, Ill.), 0.6; sodium chloride, 1.0. The ration was supplemented with (per ton) 107 IU of vitamin A, 3 x 106 IU of vitamin D, and 20,000 IU of vitamin E. The animals were offered feed 10% in excess of ad libitum intake, with 90% of the daily allotment being offered at 1100 h and the remainder offered at 0630 h. Water was continually available. The ration contained 15.2% crude protein. To extend the results obtained with the fistulated animals, we examined a group of 22 lactating Holstein cows maintained for the purpose of experimentation in the Animal Research Centre dairy cattle herd. These animals were housed and fed as described above and maintained on a concentrate similar to that described above, fed on a 50:50 ratio with corn silage (crude protein content of the ration was ca. 13.4%). Preparation and analysis of protozoal and bacterial fractions for determination of standard values for protein content per cell. Samples (600 ml) from the mixed liquid-small particle phase of the rumen were obtained from the fistulated cows as previously described (30). To examine the effect of

2 VOL. 47, 1984 PROTOZOAL AND BACTERIAL LEVELS IN RUMEN SAMPLES 567 diurnal variation, we collected samples at hourly intervals from 0830 to 1500 h. Each sample was immediately transported to the laboratory in a sealed vacuum flask and placed in an anaerobic hood. The sample was then mixed and allowed to settle for 1 min to remove very heavy particles before the supernatant was poured into a 1-liter separatory funnel. The sample was incubated for 1 h at room temperature (ca. 25 C), during which time a slow fermentation proceeded resulting in flotation of the feed particles while the protozoa settled to the bottom as a white layer (32). The bottom layer (50 ml) was drawn off (protozoal fraction). After removing small heavy particles from the protozoal fraction by repeated gentle mixing and immediate decantation (six cycles), the material was filtered through one layer of cheesecloth, diluted to 200 ml with 0.9% NaCl, and centrifuged at 164 x g for 5 min. The supernatant was discarded, and the dilution and centrifugation were repeated once more. The pellet was then suspended in 300 ml of 0.9% NaCl at 4 C and layered on a 1-liter 5 to 20% (wt/vol) polyethylene glycol (Carbowax 6000) (PEG 6000) gradient (in 0.9% NaCl) in a 1-liter graduated cylinder. The gradient separation and all subsequent steps were performed at 4 C; rewarming of the material resulted in lysis of the protozoa. Sedimentation was allowed to proceed until the most rapidly sedimenting cells approached the bottom of the cylinder (ca. 50 min). Eight 125-ml fractions were then removed with a siphon tube which was fixed in the cylinder before the sample was applied. Each fraction was centrifuged for 5 min at 164 x g. The pellets were resuspended in 2.0 ml of 0.9% NaCl. Samples from each fraction were mixed with an equal volume of 10% formaldehyde in 0.9% NaCl for microscopic examination as described below, and the remainder was stored at -20 C for protein determinations. Bacteria were isolated from the material remaining in the separatory funnel after removal of the protozoal fraction. The sample was filtered through two layers of cheesecloth and centrifuged at 164 x g for 6 min. The pellet was discarded. The supernatant was centrifuged at 27,000 x g for 20 min to sediment the bacteria. The pellet was then resuspended in 500 ml of cold (4 C) 0.9% NaCl, and all subsequent steps were performed at 4 C. The resuspended cells were again centrifuged at 164 x g for 5 min, the pellet was discarded, and the bacteria were pelleted by centrifugation at 27,000 x g for 20 min. This washing cycle was repeated twice, and the final pellet was resuspended in ca. 10 ml of 0.9% NaCl. A sample of the resuspended pellet was mixed with an equal volume of 10% formaldehyde in 0.9% NaCl for counting, and the remainder was frozen at -20 C for protein determination. Bacteria were counted as described previously (30). Protozoa were counted in a hemacytometer counting slide with a depth of 0.1 mm, using a Zeiss Universal microscope equipped with a x 16 phase-contrast objective. Cell size was measured with an ocular micrometer. For the purpose of this study, approximate protozoal cell volumes were calculated by using the empirical formula: volume = length(length/4)2'rr. The use of this equation results in an approximately linear relation between the calculated volume and the measured cell protein content. Two methods were compared for determining microbial protein in the isolated microbial fractions. In the first method, the samples were mixed with a 0.1 volume of 3 M trichloroacetic acid. The precipitated protein was dissolved in a 0.5 volume of 1 M NaOH by heating at 90 C for 5 min. In the second method, the sample was dissolved in 0.5% Triton X-100 in 1.43 N NaOH, and sodium dodecyl sulfate was used to prevent precipitation of the Triton X-100 (37). Protein in the solubilized suspensions was determined by the method of Miller (33), using bovine serum albumin as a standard. Both methods gave identical values. The results of the protein determinations and microscopic counts and size measurements were used to calculate standard values for protein per cell (bacteria) and protein per micrometer cubed of cell volume (protozoa). Determination of bacterial and protozoal protein content of rumen samples. Samples for the determination of bacterial and protozoal protein levels were obtained from the fistulated animals as previously described (30). The lactating cows were sampled by stomach tube (sample size, 1 to 2 liters). Each animal was sampled four times over a 7-week period. All samples were taken between 1000 and 1100 h. Five milliliters was immediately taken from the well-mixed sample with a wide-bore pipette and mixed with an equal volume of 10% formaldehyde in 0.9% NaCl. The samples were not fractionated. Bacterial numbers and protozoal numbers and size were measured microscopically in these fixed samples as described above for the isolated cell fractions. The number of bacteria per milliliter and the volume of protozoa (micrometers cubed per milliliter) in the rumen sample were calculated. The standard values obtained above were used to calculate the bacterial and protozoal protein content of the samples. RESULTS Determination of standard values for cell protein content by using the isolated protozoal and bacterial fractions. Among the eight protozoal fractions isolated from the PEG 6000 gradient, four distinct size classes were obtained (Fig. 1). Fraction A consisted almost exclusively of small entodinia, ca. 50 p,m in length. Fraction B contained mainly small holotrichs, together with a small proportion of Entodinium spp. and Eudiplodinium spp., which ranged from 50 to 90,um in length. Fraction C contained almost exclusively Isotricha spp., ranging from 70 to 120,um in length. Fraction D consisted of large isotrichs, large diplodinia, and Ophryoscolex spp., which ranged in length from 120 to 240 p.m. All fractions were essentially free of visible feed particles and free bacteria. The cell concentration, size distribution, and protein content were determined for each fraction (Table 1). Whereas protein content per protozoal cell varied with cell size, protein content per micrometer cubed of cell volume (calculated as described) was essentially constant. Given the limits of accuracy of the counting method (ca. 10%) and considering the assumptions inherent in the cell volume calculations, an average value of 5.9 x 10"1 mg of protein per p.m3 of cell volume was used for estimation of protozoal protein in all subsequent measurements on rumen samples. This value is in general agreement with the results of Gutierrez (19) and Holler and Harmeyer (22). The discrepancy in absolute value can largely be attributed to the inclusion of all organic nitrogen in their measurements. The use of direct microscopic counting and sizing offers a comparatively quick and effective method of estimating protozoal protein in rumen samples. Protein content per cell is less likely to vary with growth conditions than other parameters such as dry weight or carbohydrate content (14, 15), but although the range of variation encountered in this study was small, it may be necessary to prepare appropriate standards if radically different diets are to be compared. The bacterial preparations were free of any visible particulate contamination when examined microscopically. The

3 568 TEATHER ET AL. average protein content per cell for all preparations was x mg of protein per cell (data not shown). There were no significant differences (P < 0.1) in protein content per cell either with time of sampling relative to feeding or among animals with different bacterial population levels. Thus, although protein content per bacterial cell is known to vary with growth rate (27), the range of growth rates encountered in the rumen is not sufficient to affect the protein content per cell of the bacterial population as a whole under these conditions. Leedle et al. (25), in fact, found only small diurnal variations in protein content per cell even when animals were fed only once daily. Relationship between protozoal and bacterial protein levels in rumen samples. The first group of animals compared consisted of the same four fistulated Holstein cows used to determine standard values for cell protein content. Over a 3- year period, these animals have maintained a consistent ranking in terms of the number of bacterial cells per milliliter of rumen fluid, with animal number 995 < 998 < 993 and 999 (30, 31). The difference in bacterial numbers between cows FIG. 1. Rumen protozoa fractions used as standards to determine the relationship between cell size and cell protein content. The fractions were separated by sedimentation through a PEG 6000 density gradient as described in the text. Each microscope field shown is 440 by 300 plm. TABLE 1. APPL. ENVIRON. MICROBIOL. Analysis of protozoa fractions Frac- Mean Mean vol Protein per Protein per tin (>m) (,um ) 10(gxl-6) X (Mg x lo-,, tin length (I..M3 X 10-4) (gcell'ia3 A ± 0.5 B ± 0.2 C ± ± 0.8 D ± a Standard deviations given represent the variation between replicate analyses. Variation within fractions in protein per cell due to the range of cell sizes present is not included. The average amount of protein per micrometer cubed (milligrams x 10-") is 5.9 ± and 999 has been greater than 2.5-fold in all samples taken over this period. When protozoal and bacterial protein levels were determined, however, a clear inverse relationship between bacterial and protozoal protein levels in these animals emerged, so that total microbial protein levels varied over a smaller range than bacterial or protozoal protein levels (Table 2). The correlation coefficient between bacterial and protozoal protein (r = -0.51) was highly significant (P < 0.02). A closer fit was obtained (r = -0.63; P < 0.001) for protozoal protein versus log10 bacterial protein. The calculated equations for the relationship were: y = x and y = loglox, where x = bacterial protein (milligrams per milliliter) and y = protozoal protein (milligrams per milliliter). The second group of animals examined consisted of 22 lactating Holstein cows. Each animal was sampled four times over a 7-week period. During this time, most animals maintained a consistent level of protozoal and bacterial protein in the rumen samples (the average standard deviation for four samples from each animal was 28%). The variation between animals, however, was very large, ranging from <0.01 to 5.73 mg/ml for protozoal protein and from 0.58 to 5.76 mg/ml for bacterial protein. In contrast, total microbial protein levels ranged only from 1.33 to 6.42 mg/ml. The relationship between bacterial and protozoal protein levels was similar to that found for the fistulated cows: r = (P < 0.001) for bacterial versus protozoal protein and r = (P < 0.001) for protozoal protein versus log10 bacterial protein. The equations calculated for this relation were: y = x and y = log1ox. DISCUSSION The results of this study show that the variation among animals in total microbial protein level in rumen samples from the liquid-small particle phase is less than the variation among animals in either bacterial or protozoal protein levels. TABLE 2. Microbial protein in rumen fluid' Animal Bacterial protein Protozoal protein Total protein no. (mg/ml) (mg/ml) (mg/ml) ± 0.60' 2.52 ± 0.53' ± 3.33b 5.32 ± b 1.65 ± ± ± 0.55c 1.64 ± 0.47' 3.30 ± 0.91c a Each value represents an average of duplicate analyses of four independent samples. For each sample, a minimum of 100 bacterial and 100 protozoal cells were counted. Differences between cows were tested for significance by Duncan's multiple range test. Means in columns with different superscripts are significantly different (P < 0.01).

4 VOL. 47, 1984 PROTOZOAL AND BACTERIAL LEVELS IN RUMEN SAMPLES 569 Regression analysis demonstrated a highly significant negative correlation between bacterial and protozoal protein levels and suggested a nonlinear relationship between these two parameters. The curvilinear relationship is most probably the result of the competition between bacteria and protozoa for limiting nutrients in the rumen. Given conditions that tend to favor one group, that group is able to effectively limit the growth of the competing group to that part of the ecological space where the two groups do not overlap. The animal-specific factors which affect the balance between these two competing populations are not known, but the resulting high animal-to-animal variation in the level of either population considered in isolation requires that both bacteria and protozoa be considered when dietary effects on the rumen ecosystem are investigated. Otherwise, the high animal-to-animal variation encountered will preclude statistical significance for all but very major effects on rumen biomass or effects on the bacteria/protozoa ratio. The results also showed that biomass in the liquid-small particle phase of the rumen is affected by the bacteria/protozoa protein ratio, which ranged in the fistulated animals from 0.14 to 1.05 and in the lactating cows from 0.08 to >1,000. The total microbial protein level in the samples increased as the protozoa protein level increased (r = 0.96 and r = 0.70 for the fistulated and lactating groups, respectively; P < 0.001). This is presumably the result of the tendency of the protozoa to remain sequestered in the rumen (23, 24, 40); the bacterial population of the rumen is maintained largely by growth, whereas the protozoal population can be maintained to a large extent by the energy required for cell maintenance. Rumen biomass (bacteria plus protozoa) and, consequently, biomass in the liquid-small particle phase, then, will differ from animal to animal, depending on the relative sizes of the bacterial and protozoal populations. This does not imply that the production of microbial protein necessarily differs; this would depend on the rumen turnover rate and on the relative turnover rates of the bacterial and protozoal populations. However, to compare microbial protein levels in rumen samples among animals without making some correction for the bacteria/protozoa ratio will leave a high level of animalto-animal variation due to the tendency of individual animals to maintain different bacteria/protozoa ratios. The effect of the bacteria/protozoa ratio on the variation among animals in the estimated microbial protein level can be minimized by using a regression calculated between the bacterial and protozoal protein levels in the rumen samples to correct the measured values to a common population ratio; e.g., the protozoal value could be converted to a bacterial equivalent value so that the microbial protein value is equivalent to that expected for a defaunated animal. The population ratio chosen is not important, but correcting to a common population ratio is necessary to minimize the apparent animal-toanimal variation. For example, if the data from the lactating cow group is treated in this way, the standard deviation of the mean for total microbial protein is reduced from to ±0.90. The existence of specific animal effects on microbial numbers found in rumen samples has been known for some time (38). This variation among animals has often caused difficulty in evaluating feed effects on rumen bacterial or protozoal populations, even when large treatment effects are found (2, 9, 11, 28, 30, 31, 34-36). The results of this investigation suggest that a large part of the variation among animals in the numbers of bacteria or protozoa in rumen samples is due to the tendency of individual animals to maintain different bacteria/protozoa ratios in the rumen. To minimize the apparent animal-to-animal variation, both bacterial and protozoal biomass must be determined, the regression between bacterial and protozoal biomass must be calculated, and the regression equation must be used to estimate total biomass at a standard bacteria/protozoa ratio. The use of this method for determination of microbial protein in rumen samples from the liquid-small particle phase in combination with measurement of rumen volume and turnover rate should allow estimation of microbial protein production in the rumen. 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