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Animal Science Departmental Report 2004-2005 Return to Beef Cattle articles
Effect of Calcium
Propionate on Ruminal Soluble Calcium and Microbial Fermentation V. Fellner and J. W. Spears Introduction
The importance of calcium in ruminant rations is well known. Calcium is ubiquitous and a key cellular mineral involved in several metabolic pathways. However, little is known of the role of calcium on rumen fermentation. Microorganisms in the rumen may have a specific requirement for calcium. In fact, calcium seems to be essential for the growth of Fibrobacter (formerly known as Bacteroides) succinogenes, a cellulolytic anaerobe commonly found in the rumen. Limited data suggest a beneficial effect of calcium on fiber digestion particularly when supplemental fat is included in the diet. In those studies, the improvement in fiber digestion was attributed to the ability of calcium to bind to the fatty acids and remove their inhibitory effect on ruminal microorganisms. In contrast, other researchers reported improved digestion of fiber with additional calcium that could not be attributed to increased formation of fatty acid soaps in the rumen. The process by which cellulose is digested in the rumen is extremely complex and involves a concerted hydrolysis by a number of enzymes. Calcium has been implicated in maintaining optimum enzyme activity as well as increased enzyme stability. The present study was conducted to determine the effect of calcium propionate on rumen fermentation in continuous cultures of ruminal microorganisms. Calcium propionate was provided either in a prilled form or as a powder. Experimental
Procedures
Inoculum and culture conditions Rumen
inoculum was obtained from a nonlactating, ruminally cannulated Holstein cow
fed a predominantly forage diet. The surgery protocol and animal handling
procedures for the cannulated cow were approved by The North Carolina State
University Institution of Animal Care and Use Committee. Whole rumen contents
(approximately 4 L) were obtained 2 h post prandially and squeezed through a
double layered cheesecloth. The strained ruminal fluid (700 mL per fermentor)
was used to inoculate four fermentors (Eun et al., 2004). Anaerobic conditions
in the culture vessels were maintained by infusion of CO2 gas at a
rate of 20 mL/min. A circulating water
bath was used to maintain the temperature of the fermentors at 39°C. Continuous stirring of fermentor contents was achieved
with the aid of a central paddle set at a speed of 10 rpm for the duration of
the experiment. The liquid dilution rate of the cultures was maintained at
6.3%/h by regulating the addition of artificial saliva prepared as described by
Slyter et al. (1966). Experimental diets (14 g DM Basis) were added to
the culture vessels twice daily at 0800 and 1700 h.
Cultures were allowed to stabilize for 48 h to respective diets prior to initiation
of data collection for 3 d. Dietary Treatments Four dietary treatments (Table 1) were tested as follows:1) Control (No added Ca; .24% total dietary calcium); 2) Control + calcium carbonate (0.60% added Ca); 3) Control + calcium propionate (Nutrocal-Prilled) (0.60% added Ca) and, 4) Control + calcium propionate (powder) (0.60% added Ca). Diets were comprised of (DM Basis) corn silage (35%), soybean meal (18%) corn (15%), cottonseed hulls (22%) and whole cottonseed (10%) (Table 1). All diets also received a vitamin-mineral premix at 0.12%. The control diet was formulated to contain 0.24% total calcium. All three calcium sources replaced cottonseed hulls in amounts to result in 0.6% of added calcium. Total dietary crude protein was formulated to be 16.7% (Table 1). Corn silage that had been frozen (-20°C) was thawed, and then processed in a food chopper (approximate length = 1 cm; model FC 19, G. S. Blakeslee & Co., Cicero, IL). All dietary ingredients were weighed and gradually added to the chopper. The vitamin and mineral premix and the calcium sources were preweighed and mixed with the concentrate before they were gradually added into the chopper. All dietary ingredients were allowed to mix thoroughly for 10 minutes before being placed in bags and stored in the refrigerator. Sample Collection and Analytical
Procedures
Samples were analyzed for DM according to the methods of
the AOAC (1999). Concentrations of NDF in ruminal culture samples were
determined using an Ankom 200 fiber extractor (Ankom Technologies, Fairport,
NY) according to the method of Van Soest et al. (1991). Five milliliters of
thoroughly mixed culture contents were collected 2 h after feeding daily,
centrifuged and the supernatant was analyzed for VFA by GLC (model CP-3380;
Varian, Walnut Creek, CA) and for NH3-N using a colorimetric assay
(Beecher and Whitten, 1970). Ten microliters of headspace gas samples from the
fermentor were drawn into a gas tight syringe (Hamilton Co., Reno, NV) 2 h
after feeding and analyzed for methane (CH4)
using GLC. The pH of the ruminal cultures was recorded when samples were taken
for CH4 analysis. Feed and culture contents were analyzed for
soluble calcium content according to the AOAC (1990) procedures. Separate 5 mL samples of the mixed culture contents were
taken on the last day of each period at 2 h after the morning feeding and
frozen (-70°C) for long chain fatty acid (LCFA) analysis. The frozen samples were
thawed, methylated (Kramer et al., 1997), and then analyzed for LCFA by GLC. Statistical
Analysis
Data were analyzed according to a randomized
complete block design with repeated measures using
the PROC MIXED procedures of PC SAS (SAS Inst., Inc., Cary, NC). The model
included the effect of treatment and the random effect of run. Preplanned
contrasts were evaluated. Results and Discussion Total
Ca content in the control diet, based on actual Ca analysis on individual
ingredients was 0.18 mg/100g of diet DM. Calcium concentration of CaCO3,
Ca propionate, prilled and Ca propionate powder, was 38%, 21% and 20%,
respectively. The amount of CaCO3 and Ca propionate (prilled and
powder) added daily to the respective cultures, based on 14 g of total DM fed,
was 0.22g, 0.41g, and 0.42g, respectively. This maintained the amount of
additional calcium at 0.61% and total dietary Ca at 0.79 mg/100g of diet DM. Compared to control, both CaCO3 (P
< 0.04) and Ca propionate (P < 0.0001) increased total volatile fatty
acids in ruminal cultures (Table 2). The increase tended to be greater (P <
0.07) when propionate was provided in the prilled form versus the powder. With
CaCO3 the increase was due mainly to an increase in ruminal butyrate
(P < 0.0002); the greater total volatile fatty acid concentration in
cultures receiving Ca propionate was due primarily to an increase in ruminal
propionate (P < 0.0001) butyrate (P < 0.03) and valerate (P < 0.05).
Prilled Ca propionate resulted in greater concentrations of ruminal butyrate (P
< 0.0006), and the isoacids, isobutyrate and isovalerate, (P < 0.0001)
when compared to the powder. Molar
percentages of acetate were not affected ( P >0.10) and those of propionate
tended (P < 0.10) to be lower with the addition of CaCO3 compared
with the control (Table 3). The increased ruminal propionate in cultures
receiving Ca propionate lowered (P < 0.0001) the molar ratio of acetate and
increased (P < 0.0001) that of propionate (Table 2); prilled Ca propionate
was associated with a lower (P <0.01) proportion of ruminal acetate compared
with the powder. Molar proportions of the isoacids, isobutyrate and
isovalerate, were significantly higher in the control cultures compared with
cultures that received Ca propionate (Table 3). However, the physical form of
Ca propionate had an effect on isoacids with prilled Ca propionate resulting in
much greater (P < 0.0001) concentrations than the powder. Ruminal pH ranged between 5.64 and 5.87 and was lower in cultures that received CaCO3 (P < 0.003) and Ca propionate (P < 0.0001; Table 5). Concentration of methane remained unaffected (P > 0.10) by treatment and ranged between 1245 nmol and 1373 nmol per ml of culture content. Neutral detergent fiber digestibility was generally high across all treatments and ranged between 72% and 82%. Fiber digestibility was similar (P > 0.10) between the control and CaCO3. There was also no difference between the control and the average effect of both prilled and powder Ca propionate, however there was a significant effect of the physical form of Ca propionate; the prilled form increased (P <0.03) fiber digestion compared with the powder. As expected, supplemental dietary calcium resulted in significantly greater soluble calcium content in ruminal cultures (Table 5). Control cultures averaged 9.8 mg/L soluble Ca and that was significantly lower compared with 16.0 mg/L and 19.1 mg/L for CaCO3 and Ca propionate treatments, respectively. Solubility of calcium was greater (P < 0.036) in the prilled form than the powder (20.8 mg/L and 17.4 mg/L, respectively). If we account for the Ca provided in the basal diet, which was similar for all treatments, and the average ruminal soluble Ca in control cultures our calculations show that the Ca in the prilled form was 80% more soluble than the Ca in CaCO3 and almost 50% more soluble than the Ca in the powder. Calcium propionate provided in the prilled form enhanced ruminal fermentation more than any other treatment. It shifted the concentration of individual fatty acids in a manner that is indicative of supporting a predominantly cellulolytic population. The total DM offered to the cultures was 14 g/d and at 3.0%, total amount of NutroCal™ added was 0.42g/d. This would have resulted in 0.34 g or approximately 4.4 mmol of propionate added per day to the cultures. The lower acetate to propionate ratio was due mainly to the additional propionate provided in the additives and not a reduction in total acetate production. Although, both CaCO3 and Ca propionate added as a powder had similar effects on ruminal fermentation, an increase in fiber digestion was observed only when Ca propionate was provided in the prilled form. Since the amount of propionate added to the cultures was similar between the prilled and powder the beneficial effects on fiber digestion would have to be related to ruminal soluble Ca concentrations that were much higher with the prilled form. Prilled Ca propionate also supported a greater production of the isoacids, isobutyrate and isovalerate. Several rumen bacterial species have a specific requirement for these isoacids and they have been shown to also stimulate growth of other species particularly the fibrolytic organisms. Lipid profile in cultures was affected by treatment (Table 6). Calcium carbonate tended to increase (P < 0.07) C18:0 and lower (P <0.02) C18:2 when compared to the control (Table 6). Total C18:1 content was similar (P > 0.10) between the control and CaCO3 treatments however addition of CaCO3 increased (P < 0.03) the proportion of the C18:1 trans isomers and reduced (P < 0.01) the proportion of C18:1 cis isomers. In contrast, Ca propionate had no effect (P > 0.10) on total C18:0 or C18:2 contents but increased (P < 0.03) total C18:1 when compared to the control. The increase in total C18:1 in cultures receiving Ca propionate was due to an increase (P < 0.06) in the C18:1 trans isomers. These results were unexpected. However, they indicate that the addition of CaCO3 seemed to enhance the rate of ruminal biohydrogenation by converting a greater proportion of dietary C18:2 to C18:0. This is difficult to explain since CaCO3 resulted in a lower culture pH and low ruminal pH inhibits lipolysis and biohydrogenation. Similarly, the lack of an effect of Ca propionate on methane concentration suggests that the additional propionate did not interfere with the pool of metabolic hydrogen required for the process of biohydrogenation. The question still remains that the calcium treatments affected biohydrogenation and the effect was not the same. Not all microbes are capable of biohydrogenating unsaturated fatty acids to completeness. It is possible that the calcium treatments caused a shift in the microbial species associated with the completeness of biohydrogenation. Microorganisms capable of fatty acid hydrogenation in the rumen are often divided into Groups A and B based on their end-products and patterns of isomerization during biohydrogenation. Bacterial species in Group A hydrogenate linoleic acid to trans C18:1 but appear incapable of hydrogenating monoenes. Group B bacteria can hydrogenate a wide range of monenes, including trans-11 C18:1, to stearic acid. References
AOAC. 1999. Official Methods of Analysis. 16th ed. Assoc. Offic. Anal. Chem. International, Arlington, VA. AOAC. 1990. Official Methods of Analysis. Offic. Anal. Chem. International, Arlington, VA. Beecher,
G. R., and B. K. Whitten. 1970. Ammonia determination: reagent modification and
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J. K. G., V. Fellner, M. R. Dugan, F. D. Sauer, M. M. Mossob, and M. P.
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Effect of pH on population and fermentation in a continuously cultured rumen
ecosystem. Appl. Microbiol. 14:573-578. Van Nevel, C. J., and D. I. Demeyer. 1996. Influence
of pH on lipolysis and biohydrogenation of soybean oil by rumen contents in
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A. Lewis. 1991. Methods for dietary fiber, neutral detergent
fiber, and non-starch polysaccharides in relation to animal nutrition. J. Dairy
Sci. 74:3583. Table 1. Ingredient composition of diets fed to continuous cultures. 
Table 2. Concentrations of short chain fatty acids
(SCFA) in continuous cultures receiving calcium carbonate or calcium propionate
(n=5). 
Table 3. Molar percents of volatile fatty acids in continuous cultures receiving calcium carbonate or calcium propionate (n=5). 
Table 4. Production of short chain fatty acids (SCFA) in continuous cultures receiving calcium carbonate or calcium propionate (n=5). 
Table 5. Ruminal pH, methane output, and NDF digestibility in continuous cultures receiving calcium carbonate or calcium propionate1. 
Table 6. Fatty acid profile in cultures receiving
calcium carbonate or calcium propionate (n=5). 
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