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ANIMAL NUTRITION |
* Departments of Animal Sciences,
and
Food Science and Human Nutrition,
and
Statistics, and
and
¶ Veterinary Science, University of Florida-IFAS, Gainesville 32611 and
and
Roche Vitamins, Inc., Parsippany, NJ 07054
Abstract
The objective of this study was to test the hypothesis that serum biotin concentration and biotin balance (consumed - [urinary output + fecal output]) measured as total avidin-binding substances (biotin + biotin metabolites) are responsive to changes in the proportions of dietary alfalfa meal and concentrate fed to sheep. Eight sheep (initial BW = 40 kg) consumed a pelleted alfalfa meal-based diet that had 95:5, 48:52, 23:77, or 9:91% alfalfa meal:concentrate ratios (DM basis) in a replicated 4 x 4 Latin square design with 20-d periods (10 d of acclimation, 7 d of adaptation, and a 3-d collection period with jugular blood drawn on the last day). Replacing alfalfa meal with concentrate in the pelleted diets decreased dietary concentrations of biotin proportionally. As the percentage of alfalfa meal in the diet decreased, there was a linear decrease in daily DM intake (1,128 to 901 g of DMI/d; P < 0.01), with a linear (P < 0.01) and quadratic (P < 0.01) increase in the apparent total-tract DM digestibility of diets (51.0 to 80.0%). The biotin consumed decreased with alfalfa meal proportion in the diet (linear, P < 0.01). Both fecal biotin concentration (linear, P < 0.01) and fecal biotin output (quadratic, P < 0.05) increased, reaching peaks at 23% alfalfa meal. Fecal biotin output was not correlated with biotin intake, DMI, or intake of digestible DM. Mean urinary output, urinary biotin concentration, urinary biotin output, and serum biotin concentration were not affected by treatments. Means of biotin balance were negative and revealed the same trends among treatments as did fecal output. Biotin balance was a quadratic (P < 0.05) function of decreasing alfalfa meal in the diet, with more negative values at the alfalfa meal:concentrate ratio of 23:77. Results suggest that the greatest synthesis of biotin in the total digestive tract occurs with diets of either 52 or 77% concentrate for sheep; however, research addressing the significance of biotin metabolites on biotin balance and plasma biotin pool is needed.
Key Words: Biotin Balance • Concentrate • Diet • Sheep • Total Avidin-Binding Substances
Introduction
It is generally assumed that ruminal microbes are a rich source of water-soluble vitamins, including biotin, to the host; however, the small intestine has appeared as the major site of biotin synthesis in duodenally and ileally cannulated steers (Miller et al., 1986a
,b). Moreover, the contribution of ruminal biotin synthesis to the supply of biotin in ruminants has been questioned (Frigg et al., 1993a). The anabolism of biotin by ruminal microbes is relatively unknown compared to that in models of enteric bacteria, such as Escherichia coli (Chapman-Smith and Cronan, 1999). In a recent in vitro continuous culture study, the production of ruminal biotin decreased by half as the proportion barley to hay increased from 50:50 to 83:17%, likely as a result of an imbalance among microbial species caused by reduced pH (Abel et al., 2001). Contrary in vitro data from our laboratory suggest that low pH (5.3) may decrease the utilization instead of the synthesis of ruminal biotin due to a decrease in cellulolytic microbial growth (Rosendo et al., 2003). Diets having a high concentration of grains can create an acidic rumen or can change the site of starch fermentation, which alters the ruminal and intestinal flora and could in turn affect the synthesis or degradation of microbial biotin. In an earlier study using beef steers, the concentrate:forage ratio in corn grain-alfalfa meal-based diets affected the apparent synthesis (ruminal and small and large intestinal) and absorption of biotin; however, only diets with 30 or 90% corn grain were evaluated (Miller et al., 1986a). The purpose of this study was to evaluate the effect of increasing the level of concentrate in alfalfa-based diets (increasing energy and crude protein densities) on biotin balance (consumed - [urinary + fecal outputs]) and serum biotin concentration quantified as total avidin-binding substances using sheep. It was expected that diets would create differences in the sites and extent of nutrient fermentation.
Materials and Methods
Eight yearling crossbred wether sheep averaging 40 kg were used in a replicated 4 x 4 Latin square design with 20-d periods. Periods were divided into an initial acclimation (d 1 through 10), adaptation (d 11 through 17) for adjustment to diets, and data collection (d 18 through 20). On d 1 through 10, all sheep were housed in a holding pen with free access to water and fed a 48:52 alfalfa-to-concentrate diet. On d 11 through 20, each sheep was placed in a metabolic crate (1.4 m2) and fed one of the following four pelleted diets during each period: 1) 95:5, 2) 48:52, 3) 23:77, and 4) 9:91 alfalfa meal-to-concentrate (DM basis) ratio (Table 1). Diets were formulated based on values published in the NRC feed composition tables (NRC, 1985; NRC, 1996) and to meet or exceed NRC (1985) recommendations for energy and crude protein. The protocol for animal care was approved by the University of Florida Animal Use Committee.
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Blood samples (jugular venipucture) were taken on the final day of each treatment. Blood was centrifuged at room temperature for 25 min at 2,400 x g. Serum was removed and placed in a -20°C freezer until analyzed. Biotin in feed, feces, urine, and serum was quantified as total avidin-binding substances using a modified (Lewis et al., 2001) avidin-binding assay (ABA; Mock, 1997) without metabolite separation by HPLC because this two-part assay (HPLC/ABA) is labor intensive and time consuming. A direct ABA without prior chromatography would be desirable for a large number of samples if results are in agreement with either microbiological or the streptavidin-binding assay (SABA) used in previous studies. This ABA measures the ability of biotin to compete with biotinylated protein absorbed to plastic for the binding site of avidin linked to horseradish peroxidase.
Data on DMI, biotin intake, fecal and urinary biotin outputs, and biotin balance were subjected to ANOVA for a replicated Latin square design using the Proc MIXED procedure of SAS (SAS User’s Guide: Statistics, Version 8.2. Edition; SAS Inst. Inc., Cary, NC) and the following model.
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where µ = overall mean; Pi = random effect of period (i = 1 to 4); Sj(i) = random effect of sheep within period (j = 1 to 8); Dk = fixed effect of diet (k = 1 to 4); and eijk = random residual error, assumed to be normally distributed.
The covariance structure was variance components. Single-df contrasts were used to test the linear, quadratic, and cubic effects of dietary alfalfa concentrations. Contrasts were considered to be significant if P 
0.05. Correlations among variables were determined using the Proc CORR procedure of SAS.
Results and Discussion
This study tested the hypothesis that the abundance of total avidin-binding substances (representing biotin and/or its metabolites) in feces, urine, and serum is responsive to changes in the proportions of dietary alfalfa meal and concentrate (nutrient composition as well) fed to sheep. In cattle, three major approaches have been used for the quantification of biotin, as follows: the microbiological assay based on biotin-requiring strains (Lactobacillus plantarum, Lactobacillus arabinosus) for feed, ruminal fluid, feces, urine, and milk (Frigg et al., 1993b; Steinberg et al., 1995; Abel et al., 2001), the streptavidin-binding assay (SABA) for serum and milk (Frigg et al., 1993b; Midla et al., 1998; Higuchi et al., 2003), and various avidin-binding assays (ABA) for plasma and milk (Zimmerly and Weiss, 2001; Rosendo, 2003). Although these methods measure biotin plus biotin metabolites all together, variability on estimated biotin values may be expected. Replacing alfalfa meal with concentrate in the pelleted diets decreased concentrations of biotin proportionally (Table 1
). Values of biotin content as assayed by a microbiological method (Lactobacillus plantarum) are lower for corn than for soybean meal (Frigg et al., 1993b; Steinberg et al., 1995) and variable in forages depending on stage of maturity and protein content (Weiss and Zimmerly, 2000). By using a similar assay, the dietary concentration of biotin increased from 0.07 to 0.15 µg/g of DM (Miller et al., 1986a) as the proportions of corn grain:alfalfa meal in the diet for steers decreased from 90:10 to 30:70%, whereas the biotin concentration of a conventional diet of corn silage, alfalfa silage, alfalfa hay, and concentrates for lactating dairy cows was 0.41 µg of biotin/g DM (Midla et al., 1998). It is unknown how biotin values from the microbiological assay compare to values from the ABA within feedstuffs or diets, but these results suggest that they are in agreement with those previously reported.
Daily means of DMI, urinary and fecal output, and biotin consumed as well as the biotin excreted in feces and urine are summarized in Table 2. Daily DM intake decreased linearly (P < 0.01) but fecal DM output decreased both linearly (P < 0.01) and quadratically (P < 0.01) as the percentage of alfalfa meal in the diet decreased. As a result, total-tract apparent digestibility of DM increased both linearly (P < 0.01) and quadratically (P < 0.01) with concentrate in the diet.
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reported daily biotin intakes of 69 µg and 150 µg/kg of DMI for steers fed 90:10% and 30:70% corn grain to alfalfa meal diets, respectively. Steinberg et al. (1995) reported a biotin intake of between 157 to 198 µg/kg DMI for lactating dairy cows fed a diet of hay, corn grain, soybean meal, and concentrate. By decreasing alfalfa meal in the diet, both fecal biotin concentration (linear, P < 0.01) and fecal biotin output (quadratic, P = 0.01) increased, reaching peaks at 23% alfalfa meal. Differences in biotin intakes are confounded with differences in DMI, digestible DMI, and diet composition; however, fecal biotin output was not correlated with biotin consumed (r = -0.02; P = 0.93), DMI (r = 0.14; P = 0.46), or digestible DMI (r = 0.15; P = 0.41) in the current study (data no shown). In contrast, Steinberg et al. (1995) found a high correlation between biotin fecal excretion and biotin consumed (r = 0.86) or intake of digestible OM (r = 0.96) in lactating dairy cows without supplemental biotin.
Fecal biotin output did not answer the question as to the location (i.e., the rumen and/or the intestine) of biotin synthesis. A high fecal biotin output in the present study could be interpreted as a high microbial biotin synthesis from the total digestive tract. We are unaware of intestinal flow data of biotin or fecal biotin excretion by sheep. Regardless of diet fed to ewes and preweaning lambs, ruminal biotin concentration decreased as the age of lamb increased from 3 to 21 d, suggesting that the synthesis of biotin by ruminal microbes did not progress with ruminal development (Poe et al., 1972). Apparent biotin synthesis was greater in the small intestine (1.4 to 4.2 mg/d) than in the rumen (0.1 to 1.3 mg/d) for duodenally and ileally cannulated crossbred steers fed different grain-based diets with an 86:13 grain-to-alfalfa meal ratio (Miller et al., 1986a). In a different trial with steers fed either a 90:10% or a 30:70% corn grain-to-alfalfa meal diet, the same authors also found a greater apparent daily biotin synthesis in the small intestine than in the rumen. Fecal amount of biotin in steers fed the 90:10% diet was significantly lower than those fed the 30:70% corn grain-to-alfalfa meal diet. In some studies, the proportion of OM intake digested in the small intestine increased linearly as corn intake increased (Elizalde et al., 1999). However, apparent biotin synthesis in the small and large intestine appeared constantly high in the study of Miller et al. (1986a) in spite of changes in apparent ruminal digestibility of organic matter as percentages of total-tract digestibility. As discernible from the studies of Miller et al. (1986a,b), fecal biotin output includes the microbial synthesis of biotin in the intestines that may occur beyond the absorption sites for biotin.
Biotin is also a required nutrient for several species of ruminal cellulolytic and saccharolytic bacteria (Baldwin and Allison, 1983). In a recent in vitro continuous culture study, the replacement of 83% of hay with barley decreased biotin synthesis (calculated as the difference between biotin in feed minus biotin in solid digesta after incubation plus biotin in the liquid compartment) by 22%, likely due to an imbalance among microbial species (cellulolytic and saccharolytic bacteria) caused by reduced pH (Abel et al., 2001). The question is whether ruminal microbes utilized more or synthesized less biotin with increasing concentrate diets in that experiment. In vitro data from our laboratory suggest that ruminal biotin is less utilized at low pH (5.3), likely due to a decrease in the growth of cellulolytic microbes (Rosendo et al., 2003). Biotin can be degraded by several microorganisms (e.g., Pseudomonas spp.) by ß-oxidation at the valeric acid chain forming bisnorbiotin, tetranorbiotin, and other related intermediates with the consecutive removal of two carbon units (Kazarinoff et al., 1972; McCormick, 1975), but the fate of biotin presented to ruminal microbes has not been demonstrated.
In the present study, neither mean urinary output (2,070 ± 159 mL/d), urinary biotin concentration, nor urinary biotin output was affected by treatments. Mean urinary biotin concentrations in lactating cows were 0.057 µg/g and 0.223 µg/g before and after supplementing 20 mg of biotin/d, respectively (Steinberg et al., 1995). In the same study, nonlactating dairy cows fed 20 mg of supplemental biotin per day had higher urinary biotin concentrations (0.878 µg/g) than supplemented lactating cows.
Serum biotin concentrations were not affected by treatments, with mean concentrations of 4.7, 3.6, 5.7, and 5.4 nmol/L, for diets with 95:5, 48:52, 23:77, and 9:91 alfalfa meal-to-concentrate ratios, respectively. First-lactation cows without supplemental biotin had mean biotin concentrations of 1.9 nmol/L, whereas cows supplemented with 20 mg of biotin per day had 3.1, 7.1, and 7.0 nmol/L serum biotin at 25, 108, and 293 DIM, respectively (Midla et al., 1998). Recently, Zimmerly and Weiss (2001) observed mean plasma biotin of 2.7 nmol/L in control cows. Treated cows with 10 and 20 mg of biotin per day from 14 d before calving had mean plasma biotin concentrations of 9.4 and 18.5 nmol/L, respectively, at calving. Mean plasma biotin concentrations from 30 to 100 DIM were 3.4 and 5.0 nmol/L for cows supplemented with 10 and 20 mg biotin per day (Zimmerly and Weiss, 2001). Theoretically, both the SABA and the ABA would produce similar results. However, the direct ABA would be preferable over the SABA because of the errors attributed to unknown interfering substances not related to biotin metabolites as demonstrated in human urine (Mock et al., 2001). Microbial intestinal synthesis of biotin far beyond the absorption sites for biotin may also give an explanation of why a high-fecal biotin output was not accompanied by either an increased biotin urinary output or serum biotin concentration in the present study. Substantial amounts of biotin metabolites (bisnorbiotin, biotin sulfoxide, biotin sulfone, bisnorbiotin methylketone, and biocytin) are present in urine from rats, pigs, and humans (Mock, 1997; Zempleni et al. 1997; Zempleni and Mock, 1999). Biotin metabolites in human urine originate from biotin catabolism in tissues rather than biotin oxidation by intestinal microorganisms (Zempleni and Mock, 1999). Therefore, the ABA overestimates the true concentration of biotin in human urine (Mock et al., 2001). Data on the presence of avidin-binding substances other than biotin in ruminal fluid, ruminant feces, urine, or serum have not been published. If biotin metabolites from microbial degradation are present in feces of sheep, the analytical procedure (direct ABA) used in the present experiment does overestimate fecal biotin excretion and it is unsuitable for detecting any effect of the treatment diets on the synthesis of true biotin by intestinal and ruminal microbes and so does the microbiological method used in previous studies. Conversely, these results could be interpreted as a greater degradation of biotin in the total digestive tract as dietary alfalfa meal decreased from 95 to 23%. Similarly, the true concentrations of biotin in serum, plasma, and urine from ruminants as measured either by a microbiological method in previous studies or by direct ABA in the present study are overestimated if biotin metabolites from ruminal microbial catabolism of biotin (ruminal degradation or destruction of biotin) are absorbed. Either synthetic biotin given orally or biotin in basal diets fed to cattle had an overall bioavailability between 48 and 60% (Frigg et al., 1993a,b). These values, however, could have been overestimated because they were determined by using kinetics of serum biotin (Frigg et al., 1993b).
Means of biotin balance (consumed - [urinary output + fecal output]) were negative and revealed the same trends among treatments as did fecal output. Biotin balance was a quadratic (P = 0.01) function of decreasing alfalfa meal in the diet. Biotin fecal output did not increase proportionally with increased supplementary biotin given to lactating dairy cows, which suggested a greater bioavailability for synthetic biotin than for natural biotin (Steinberg et al., 1995). Milk biotin concentrations decreased with the number of days supplemental biotin was fed, suggesting that perhaps ruminal destruction of biotin was increasing (Zimmerly and Weiss, 2001). To answer those questions, biotin metabolites in biological samples from ruminants need to be identified. Separation of biotin metabolites from true biotin by HPLC followed by quantification using ABA would give more accurate estimates of true biotin balance and biotin bioavailability, as well as contributing to a greater understanding of biotin metabolism in ruminants.
Implications
Supplemental biotin has been shown to be beneficial for hoof health and milk production in cattle. Large amounts of biotin are synthesized in the digestive tract of ruminants. The need for supplemental biotin may depend on dietary factors that influence biotin synthesis and destruction. The present experiment suggests that the greatest synthesis of biotin in the total tract occurs with concentrate levels of 52 to 77%.
Footnotes
1 Florida Agric. Exp. Stn. No. R-09537. 
2 Appreciation is expressed to Roche Vitamins, Inc., Parsippany, NJ, and to the Florida Dairy Checkoff for partial funding support of this study.
3 Correspondence: P.O. Box 110910 (phone: 352-392-7561; fax: 352-392-7652; e-mail: mcdowell{at}animal.ufl.edu).
Received for publication June 13, 2003. Accepted for publication November 25, 2003.
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