EFFECT OF SODIUM BICARBONATE ON MICROBIAL ACTIVITY IN THE RUMEN 1, 2,,3

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1 EFFECT OF SODIUM BICARBONATE ON MICROBIAL ACTIVITY IN THE RUMEN 1, 2,,3 J. w. LASSITER, M. K. HAMDY AND PRASOB BURANAMANAS 4, 5 University ECENT experimental work has indicated R that concentrated or pelleted complete rations can be successfully fed to ruminants (Matrone et al., 1959; Lassiter et al., 196, I962). Lack of success earlier with such rations might have been caused by failure to buffer the volatile fatty acids produced from carbohydrate fernlentation (Wise et al., 1961). Some ruminants fed concentrated or pelleted rations fail to ruminate normally (Cullison et al., 196). This may curtail the recirculation of the mineral cations of the saliva thus decreasing or changing the buffering capacity of the rumen and the microbial activity. If this is true, incorporation of sodium bicarbonate in the drinking water of ruminants fed such rations might serve to buffer the acids, normally buffered by the mineral cations from the saliva, and thus affect rumen activity. This study was undertaken to investigate the effect of sodium bicarbonate, incorporated in the drinking water of steers fed pelleted complete rations, on the rumen microbial flora, ph and volatile fatty acids. Materials and Methods The data in this investigation were obtained from two groups (I and II) each containing two fistulated Hereford steers. The experimental design was a 2 x 2 latin square reversal design in which the same animals received a rotation of treatments. Both groups were changed, at the beginning of the experiment, from a long hay and grain ration to a pelleted complete ration [5~o ground yellow corn, 3~o ground Coastal hay, 1~o cottonseed meal (417o protein), 97 1 Journal Paper No. 224 of the College Experiment Station of the UniversRy o:f Georgia Cullege of Agriculture Experiment Stations. 2 Departments of Animal Husbandry and Food Technology. Presented at the 53rd annual meeting of the American Society of Animal Production, Chicago, Illinois, November 24-25, Present address: Department of Livestock, Payathai Road, Bangkok, Thailand. s We are grateiui to A. E. Cullison for his suggestions, to J. L. Cannon for the statistical analyses and to Merck and Company, Rathway, New Jersey for supplying the sodium bicarbonate. of Georgia, Athens cane molasses, and.33% each of ground limestone, defluorinated phosphate, and sodium chloride].' During the first 21 days, group I received.5% sodium bicarbonate in the drinking water and group II received water without sodium bicarbonate; during the next 21 days, the treatment of the two groups was reversed. Water was 6ffered twice daily at and immediately after feeding. Feeding and watering were conducted in individual stalls to which the steers were allotted. Four hours following the morning feeding, two representative samples (a and b) were collected through the fistulas. Sample a was used for immediate ph determination using a Model G Beckman ph meter, whereas, sample b was immediately overlaid with two mm. of mineral oil and kept frozen ( 1-3 weeks) at --33 ~ C. for microbial and chemical analyses. Just before analysis, sample b was thawed and the contents strained through muslin into sterile flasks. The filtrate was then thoroughly mixed (under aseptic conditions) and subjected to standard procedures for the counting and isolation of aerobic organisms. The anaerobic bacteria were counted and isolated in pure cultures with the aid of the shake culture method modified by Hamdy et al. (1954) using tubes anaerobically sealed with pyrogallic acid and KOH. Tryptone glucose yeast extract (TGYE) agar medium (Difco) was used and counts were performed after 48 hours incubation at 37 ~ C. Predominant microorganisms were isolated from each sample and were studied for colony type, morphology, motility, Gram reaction, appearance of growth on TGYE liquid and agar media, relation to oxygen and gas production and other physiological characteristics. These characteristics were used to identify the organisms for genera following Bergey's Manual (Breed etal., 1957) and Bryant (1959). For the determination of volatile fatty acids, the same filtrate was centrifuged at 68 x g for 2 minutes, the supernatant rumen juice was strained through eight layers of cheese cloth and assayed for its acetic, propionic, 335

2 336 LASSITER, HAMDY AND BURANAMANAS TABLE 1. DAILY FEED, WATER AND SODIUM BICARBONATE INTAKES OF STEERS (LB.) Period 1 Period 2 Group Feed Water NaHCO.~ Feed Water NaHCO~ adlibitum adlibitum _+.12 butyric and valeric acid contents using the direct chromatographic procedure of Harper (1953) with a slight modification. This modification consisted of applying the rumen juice, acidified to ph 1.7 to 2., with a slightly bent tip pipette on the top of the silicic acid column to avoid disturbing the service of the column. For the determination of total volatile fatty acids, samples were subjected to steam distillation and the results were reported as milliequivalents (meq) per 1 ml. of sample. Results and Discussion The average daily feed, water and sodium bicarbonate intakes are shown in table 1. In this study sodium bicarbonate incorporation in the drinking water affected the anaerobic population in the rumen. It was found (Figure 1) that the number increased during 1 to 11 days in the rumen juice of bicarbonate treated and control animals; thereafter, the anaerobic counts in animals without sodium bicarbonate declined while those in animals receiving sodium bicarbonate continued to increase. Upon reversing the treatments at the end of 21 days, a marked decline was noted in animals without sodium bicarbonate, whereas, a noticeable increase was evident in animals receiving sodium bicarbonate. This may be due to an effect on the rumen environment or to meeting a nutritional need of the microorganisms. Q) o E o).j w 6.6 o.j. /J" o "~" /" 9 NaHCO 3 / o 9 "~-~ ~ o - ^ 7 o/~._ o _ o o~ ~ w z i, z : 9 Z _3 54, I L I * I I l 1 2 3O 4 T I M E- days Figure 1. Effect of sodium bicarbonate on the total number of anaerobes in the rumen.

3 MICROBIAL ACTIVITY IN RUMEN 337 Burroughs et al. (1951) reported varying needs for several of the mineral elements by rumen microorganisms and found that some of them were effective in stimulating digestion. McNaught and Owen (1949) suggested that mineral requirements reflect to a large extent the needs of the rumen microorganisms rather than the host animal. The anaerobic flora may be affected by the carbon dioxide from the sodium bicarbonate. High levels of bicarbonate and/or carbon dioxide are important in the isolation and enumeration of many rumen bacteria (Bryant and Burkey, 1953). It is also possible that sodium bicarbonate increased the buffering capacity of the rumen juice and affected the number of the microorganisms accordingly. The predominant organisms isolated from the rumen juice and studied were found to be members of the genera Ruminococcus, Lacto~acillus and Clostridium. Although sodium bicarbonate affected the total number of these anaerobes, they continued to be the predominant genera found in the rumen. The bacterial counts were of the order of 1 ~ per ml. The problem of total viable counts of bacteria from the rumen is only relatively important because the numbers will be influenced by food and water intake, substrate utilized, sampling technique and other factors. Use of the culture method has resulted in estimates of the total living bacterial population in the rumen of from 1~-19 or more per ml, depending upon the media and the objective (Gall et al., 1947; Hungate, 1946; Johns, 1951). A variety of cultural methods have been employed in studies of rumen bacteria and diverse culture media have been utilized for their isolation and/or enumeration. These media can be divided into non-selective for growth of the largest number and most diverse types of bacteria and selective for certain physiological groups (Bryant, 1959). While all media for non-selective growth of predominant rumen bacteria are selective, some are much more than others. Of the general types of non-selective media, the one used in this study (TGYE) contained sources of growth factors and nitrogen (yeast extract and tryptone) that are relatively reproducible and commonly used in bacteriological analysis. Analyses of variance, partial regression coefficients and multiple correlations indicated that the addition of sodium bicarbonate had a highly significant (P~.1) effect on the anaerobic counts in the rumen juice and regression of anaerobic counts with time. No significant regression of counts on water or feed consumption was observed. Aerobic bacteria are considered of less significance in the rumen than the anaerobes, however, they may, along with the facultarive organisms, and in symbiosis with the anaerobes, play an important role in the overall microbial activity of the rumen. Studies were thus conducted to examine the effect of sodium bicarbonate in the drinking water of steers on the aerobes and facultative micoorganisms that may be present in the rumen. The results obtained showed that the number of these organisms increased rapidly to 21 days in steers receiving sodium bicarbonate followed by a gradual decline when it was withheld. In steers not receiving sodium bicarbonate during the first 21 days there was a slight increase which continued after the sodium bicarbonate was incorporated in their drinking water. However, when these results were statistically examined, it was found that neither the incorporation of sodium bicarbonate in the drinking water nor such factors as time, water or feed consumption had any significant effect on the total number of aerobic organisms. It should be reported that spreaders of spore-forming rods greatly increased the difficulty of securing accurate counts and thus may obscure some of the effects of the sodium bicarbonate on the counts of the aerobic organisms. The incorporation of sodium bicarbonate in the drinking water exerted no effect in changing the predominant aerobic flora, which were isolated, examined and found to be members of the genera Bacillus, Micrococcus and Ramibacterium, with the latter being facultative. One of the objectives of this experiment was to observe whether, over the period studied, there would be a change in rumen ph, measured at the same time after feeding, due to sodium bicarbonate incorporation in the water. Sodium bicarbonate did not affect significantly the rumen ph, however, a significant (P~.5) regression of ph on time was observed. This indicates that the level of sodium bicarbonate used was without effect under these conditions. The conditions were offering water free choice twice daily at and immediately after the time of feeding. The bicarbonate-fed animals consumed the water avidly and appeared very thirsty at each feeding. The average daily feed, water and sodium bicarbonate intakes are shown in

4 338 LASSITER, HAMDu AND BURANAMANAS TABLE 2. EFFECTS OF SODIUM BICARBONATE ON THE TOTAL VOLATILE FATTY ACIDS IN THE RUMEN. RESULTS REPORTED AS MEQ. PER 1 ML. RUMEN JUICE Steers Steers not Time, receiving receiving days NaHCOa NaHCO,~ NaHCO3 NaHCO3 removed added table 1. In period 1 they consumed an average of 8.8 lb. water daily and in period 2, 52.2 lb. daily. Measurements of urine volume and urine sodium were not made, but urination was increased. This may have been due to a need to eliminate sodium in the urine. Cullison et al. (196) fed the same pelleted ration used in this report, without sodium bicarbonate incorporation, and found that rumen ph levels were affected. Their ph measurement, however, was made after a period of 196 days on feed and the feed and water were furnished ad libitum instead of twice daily. Results showing the effects of sodium bicarbonate on the total volatile fatty acids as measured by steam distillation are recorded in table 2. Figure 2 shows this effect on the sum of the concentrations of acetic, propionic, butyric and valeric acids. The increase in concentration of these acids as a result of incorporating sodium bicarbonate in the drinking water and the decease following its withholding, is evident. Table 3 summarizes data showing the effect on the concentrations of the individual volatile fatty acids and again indicating the significant effect on these individual acids in both groups of animals. Butyric and valeric acids were absent in the rulnen juice of steers on both treatments during the early stages of the experiment. Acetic, propionic and butyric acids are the " r 7 E O) M. j o=.,o / ~ o V--,,,./ II~ z F- <._l > 1,.. t, _ I _~- l ~ 1 I TIME- days Figure 2. Effect of sodium bicarbonate on the sum ol acetic, propionic, butyric and valeric acids of the rumen.

5 MICROBIAL ACTIVITY IN RUMEN 339 TABLE 3. EFFECTS OF SODIUM BICARBONATE ON THE INDIVIDUAL VOLATILE FATTY ACIDS IN THE RUMEN OF STEERS WITH AND WITHOUT SODIUM BICARBONATE. RESULTS REPORTED AS MEQ. PER 1 ML. RUMEN JUICE Time, Acetic Propionic Butyric Valeric days With" Without ~ With ~ Without" With ~ Without" With ~ Without " ~ Without ~ With ~ Without ~ With ~ Without a With" Without ~ With" O a With; without sodium bicarbonate, principal products of microbial fermentation in the rumen. The total concentrations of volatile fatty acids in the rumen and the amounts of the individual acids present, however, are dependent on the diet (Annison and Lewis, 1959). Gray (1948) reported that the rates of absorption of the volatile fatty acids are considerably lowered at ph values above 7.. Sodium bicarbonate in the drinking water exerted no change in the ph of the rumen in our study and therefore decreased absorption for this reason must not be the explanation for the increased concentrations of these acids in the rumen. Dobson and Phillipson (1956) showed that the addition of free volatile fatty acids or lowering the ph when volatile fatty acids were present caused an increased blood flow from the rumen capillaries. There is no evidence that an active transport mechanism is involved in the absorption of volatile fatty acids from the rumen (Annison and Lewis, 1959); therefore, if the rate of passage of a substance from the rumen to the surrounding body fluids is largely a function of the concentration gradient, an enhanced blood flow would result in an increased rather than a decreased rate of absorption. It appears that the increased number of the organisms in the rumen may be the important factor affecting the volatile fatty acid concentration rather than decreased rate of absorption since there was an increase in the anaerobic population without any change in the predominant flora in response to sodium bicarbonate addition to the water. Summary Sodium bicarbonate, incorporated in the drinking water of fistulated steers fed a pelleted complete ration, had a highly significant effect on the anaerobic counts in the rumen juice and on the regression of anaerobic counts with time. No significant regression of counts on water or feed consumption was observed but significant negative regressions of acetic, butyric, valeric acids and the sum of these plus propionic acid on water consumption occurred. There was no significant effect on the ph of the rumen juice, and no apparent effect in changing the predominant flora of the rumen as detected on TGYE media. The predominant organisms were found to be members of the genera, Ruminococcus, Lactobacillus, and Clostridiura (anaerobic) and Bacillus, Micrococcus, and Ramibacterium (aerobic) with the latter being facultative. Literature Cited Annison, E. F. and D. Lewis Metabolism in the Rumen. John Wiley and Sons, Inc. New York, N.Y. Breed, R. S., E. D. D. Murray and A. P. Hicthens Bergey's Manual of Determinative Bacteriology (7th ed.) Williams and Wilkins Co., Baltimore, Maryland. Bryant, M. P Bacterial species of the rumen. Bact. Rev. 23:125. Bryant, M. P. and L. A. Burkey Cultural methods and some characteristics of some of the more numerous groups of bacteria in the bovine rumen. J. Dairy Sci. 36:25. Burroughs, W., A. Latona, P. DePaul, P. Gerlaugh and R. M. Bethke Mineral influences upon urea utilization and cellulose digestion by rumen microorganisms using the artificial rumen technique. J. Animal Sci. 1:693. Cullison, A. E., B. M. Campbell and R. W. Walker, 196. Effect of physical form of the ration on steer performance and certain rumen phenomena. J. Animal Sci. 19:1256. Dobson, A. and A. T. Phillipson The influence of the contents of the rumen and of adrenaline upon its blood supply. J. Physiol. 133:76P.

6 34 LASSITER, HAMDY Gall, L. S., C. N. Stark and J. K. Loosli The isolation and preliminary study of some physiological characteristics of the predominating flora of the rumen of cattle and sheep. J. Dairy Sci. 13:891. Gray, F. V The absorption of volatile fatty acids from the rumen, II. The influence of ph on absorption. J. Exp. Biol. 25:135. Hamdy, M. K., E. L. Sherrer, H. H. Weiser and W. D. Sheets Microbiological factors in the treatment of phenol wastes. Appl. Microbiol. 2:143. Harper, W. J Direct chromatographic determination of acetic, propionic and butyric acids in cheese. J. Dairy Sci. 36:88. Hungate, R. E The symbiotic utilization of cellulose. J. Elisha Mitchell Sci. Soc. 62:9. Johns, A. T Isolation of a bacterium producing propionic acid from the tureen of sheep. J. Gen. Microbiol. 5:317. Lassiter, J. W., A. E. Cullison and E. P. Warren. AND BURANAMANAS Effect of sodium bicarbonate on growth and nutrient digestibility in lambs. J. Animal Sci. 21:385. Lassiter, J. W., J. W. White, M. K. Cook, E. P. Warren and R. A. Long Feed efficiencies of young ruminants fed low-roughage rations. J. Animal Sei. 19:1272. McNaaght, M. L. and E. C. Owen The iron requirement of rumen bacteria. Biochem. J. 44: XXIV. Matrone, G., R. A. Ramsey and G. H. Wise Role of sodium and potassium cations in volatile fatty acid metabolism or ruminants. Proc Cornell Nutr. Cont. for Feed Manuf. p. 36. Wise, M. B., T. N. Blumer, G. Matrone and E. R. Barrick Investigations on the feeding of allconcentrate rations to beef cattle. J. Animal Sci. 2:561.