Animal Science Departmental Report
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Dose Response of an Enzyme Feed Supplement on Equine Cecal Microbial Fermentation
V. Fellner and P. M. Yocum
The economic impact of colic and laminitis on the horse industry in the United States has been estimated to be over $115 million/year (USDA, 2001). Colic and laminitis are not diseases, but a combination of symptoms that are related to gastrointestinal abnormalities. Although there are several factors that can predispose the animal to colic and laminitis, the etiologies are still not well understood. It has been shown that the large intestine, in particular the cecum, is capable of digesting large amounts of feed resulting in greater production of volatile fatty acids (VFA) as compared to the small intestine or the colon (Kern et al., 1974). Volatile fatty acids produced in the cecum can account for 46% of the energy required for maintenance of the horse (Stevens and Hume, 1998). In addition to VFA production, copious amounts of gas, namely methane, is also produced as a result of microbial digestion. Much of the acid produced during fermentation is absorbed or passed along the tract with the gas.
With increasing amounts of starch in the diet the ability of the small intestine to digest starch can be limited thereby increasing the flow of undigested starch into the cecum (Potter et al., 1992). Increased cecal fermentation will result in greater lactic acid production (Garner et al., 1978; Clarke et al. 1990) that can compromise the integrity of the mucosal lining (Krueger et al., 1986). Some studies have shown that the amount of acid produced varies with the type of diet and proportion of concentrate included in the diet (Argenzio et al., 1974; Glinsky et al., 1976) but there is no data on the effect of enzyme feed supplementation on gas production. A ‘carbohydrate overload’ is one of the most common causes for symptoms related to colic and laminitis.
Since the absorptive capacity for acids in the lower tract is limited, feed additives that moderate fermentation can optimize the use of high carbohydrate diets. The Equigest supplement contains a combination of cellulase, lipase, protease and amylase enzymes, which are indigenous to the intestinal tract of equine. These enzymes are stable within a broad range of pH (2.5 to 7.5). The concentration of the enzymes in Equigest, when compared with other commercial preparations, is high.
To determine the effect of Equigest enzyme supplement, added at two levels, on microbial fermentation by equine cecal bacteria offered a low or high starch diet.
An in vitro batch culture study containing equine cecal microorganisms was performed. Equine cecal contents were removed from an aged (>20 years) gelding after euthanasia and were transported to the laboratory in a pre-heated enclosed container. Cecal contents were filtered through double-layered cheesecloth and 10mL of the culture were placed into incubation bottles containing 20ml of a buffer solution, and 0.5g of the respective feed. Carbon dioxide gas was purged into the bottles to displace O2 gas to maintain anaerobic conditions. The bottles were sealed and incubated at 39ºC within a water bath for either 0, 12, 24 or 48 hours. At the end of the incubation period the bottles were removed from the water bath and placed into the refrigerator.
A total of six dietary treatments were tested and the treatments were arranged in a 2 x 2 factorial (two levels of Equigest at two levels of dietary starch). Diets were incubated in replicates of three (n=3) at four different time periods (0, 12, 24 and 48 h). Diet formulations are shown in Table 1. The Equigest enzyme supplement was added to the concentrate portion of the diet, mixed thoroughly, and then included with the remaining dietary ingredients. The complete diet was mixed thoroughly prior to being added into the bottles. Data were analyzed by SAS® as a completely randomized block design with a factorial arrangement of treatments.
Results and Discussion
Effect of diets on fermentation at the end of 12 h. (Table 2).
The main effects of starch and enzyme are reported in Table 2. As expected, diets that were low in starch content resulted in a higher acetate:propionate ratio and a significantly higher methane production.
Including the enzyme supplement at either 50 or 100 mg lowered the acetate:propionate ratio. There was a numerical decrease in methane concentration with increasing levels of the enzyme supplement but the difference was not significant.
There were several starch x enzyme interactions. Including 50 mg of the enzyme supplement in low starch diets resulted in the greatest increase in total SCFA, acetate and propionate. However, with the high starch diet, addition of 100 mg of the enzyme increased total SCFA, acetate and propionate. Cecal cultures fed high starch had lower pH; the addition of enzyme supplement, irrespective of the level, resulted in lower culture pH with both the high and low starch diets.
Effects of diets on fermentation at the end of 24 h (Table 3).
High starch diets increased total SCFA, propionate and butyrate concentrations. An increase in propionate resulted in a significantly lower acetate:propionate ratio with the high starch diet. A lower concentration of methane and a lower culture pH was also observed in high starch diets compared to the low starch diets.
Addition of the enzyme, at 50 mg and 100 mg, resulted in an increase in total SCFA concentration. Enzyme supplements also increased the concentrations of propionate and butyrate but decreased culture pH when compared to no enzyme.
There was a significant starch x enzyme interaction for acetate. Feeding diets high in starch content reduced cecal acetate but the addition of enzyme at both the 50 and 100 mg levels increased cecal acetate when compared to all other diets.
Effects of diets on fermentation at the end of 48 h (Table 4).
Following 48 of fermentation there was no difference in total SCFA concentration between the low and high starch diets. However, propionate was increased with the high starch diet resulting in a lower acetate:propionate ratio. The isoacids, isobutyrate and isovalerate, were significantly greater in the low starch compared to the high starch diet. The low starch diet had a greater concentration of methane as well as higher culture pH compared to high starch diets.
Addition of 50 mg of the enzyme supplement resulted in the greatest increase in total SCFA concentration when compared to 0 or 100 mg. Similarly, diets receiving 50 mg of the enzyme supplement had higher acetate and propionate concentrations. Both, butyrate and isovalerate were also increased in cultures receiving 50 mg of the enzyme supplement when compared to 0 or 100 mg. Neutral detergent fiber digestibilities ranged between 7% and 15% and were not significantly different across treatments.
Data from the current study show that the effect of enzyme supplementation differs over time and can vary depending upon the level of starch in the diet. At shorter incubation times (12 h) 50 mg of the enzyme proved to be effective in promoting fermentation of diets having low starch content. However, 100 mg of the enzyme had a more favorable effect on fermentation of high starch diets. Typically, a reduction in pH is indicative of acid stress and indeed the greater acid production observed with both the 50 mg and 100 mg enzyme supplementations (low and high starch diets, respectively) resulted in a lower culture pH but there did not seem to be a negative effect on fermentation. The enzyme-induced fermentation was associated with an increase in acetate concentration. During prolonged periods of fermentation (48 h) addition of 50 mg of the enzyme supplement seemed to be most beneficial irrespective of the dietary starch content. Improvement in fermentation was attributed to an increase in total SCFA as well as individual concentrations of acetate and propionate. In addition, there was an increase in cecal isovaleric acid; an important growth factor for cellulolytic bacteria. The pattern of fermentation in the hindgut of horses is similar to that observed in the foregut of ruminants. The major site of fermentation in horses is the colon however all feed must first enter the cecum prior to the colon. Microbial fermentation occurs in the cecum similar to the colon but the solid and liquid phase dynamics are different between the two anatomical regions. The retention time of particulate matter in the cecum is lower hence majority of the fibrous material passes into the colon rapidly. Rapidly soluble material may be more extensively fermented in the cecum compared to the fibrous portion of the diet. The Equigest enzyme supplement ameliorated the negative impact of the rapidly fermentable dietary starch on equine cecal fermentation.
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Table 1. Diet formulation (% of Dry Matter)
Table 2. Main effects of starch and an enzyme supplement on short chain fatty acids (SCFA), methane and pH after 12 hours of fermentation in microbial cecal cultures
Table 3. Main effects of starch and an enzyme supplement on short chain fatty acids (SCFA), methane and pH after 24 hours of fermentation in microbial cecal cultures
Table 4. Main effects of starch and an enzyme supplement on short chain fatty acids (SCFA), methane, pH and neutral detergent fiber disappearance (NDFD) after 48 hours of fermentation in microbial cecal cultures