R. Blank

The concept of digestible amino acids in diet formulation for pigs

There is sufficient evidence that ileal rather than fecal amino acid digestibility values provide a more reliable estimate of protein digestion and amino acid absorption. In addition to differences in ileal amino acid digestibility values between feedstuffs there are large differences in ileal amino acid digestibility values within the same feedstuff. Furthermore, in addition to different processing conditions and inherent differences among samples of the same feedstuff, a large proportion of this variation can be attributed to different methodological approaches. In order to reduce the within variation associated with different methods for determination, methods specifically suitable for different feedstuffs are recommended. Differences in dietary amino acid levels are likely to be the largest single contributor to the variation in ileal amino acid digestibility values. Therefore, it is suggested to determine their plateau values, also referred to as dietary threshold levels, after which apparent digestibility values become independent of the dietary amino acid levels. The correction for non-specific endogenous protein and amino acid recoveries in ileal digesta allow for the transformation of apparent digestibility to standardised ileal digestibility values. The non-specific recoveries are related to the dry matter intake but independent of the type of feedstuff. In principle, standardised digestibility values should be the preferred approach in protein evaluation because these values reflect a fundamental property of the feedstuffs being independent of experimental conditions. However, estimates of endogenous recoveries are still confounded by the method used for determination, and further research is warranted in this area

Bioavailability of the flavonol quercetin in cows after intraruminal application of quercetin aglycone and rutin.

The bioavailability of quercetin has been intensively investigated in monogastric species, but knowledge about its bioavailability in ruminants does not exist. Thus, the aim of the present study was to determine the bioavailability of quercetin in nonlactating cows equipped with indwelling catheters placed in one jugular vein after intraruminal and additionally after i.v. application, respectively. Quercetin was administered intraruminally in equimolar amounts, either in the aglycone form or as its glucorhamnoside rutin, each at 2 dosages [10 and 50 mg of quercetin/kg of body weight (BW)]. In a second trial, 0.8 mg of quercetin aglycone/kg of BW was applied i.v. Blood samples were drawn 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, and 24 h after intraruminal application and every 5 min (first hour), every 10 min(second hour), and at 3 and 6h after i.v. bolus application, respectively. Quercetin and quercetin metabolites with an intact flavonol structure (isorhamnetin, tamarixetin, and kaempferol) in plasma samples were analyzed by HPLC with fluorescence detection. After intraruminal application of quercetin and rutin, respectively, quercetin and its methylated (isorhamnetin, tamarixetin) and dehydroxylated (kaempferol) derivatives were present in plasma mainly as conjugated forms, whereas free quercetin and its derivatives were scarcely detected. For rutin, the relative bioavailability of total flavonols (sum of conjugated and nonconjugated quercetin and its conjugated and nonconjugated derivatives after intake of 50 mg/kg of BW) was 767.3% compared with quercetin aglycone (100%). Absolute bioavailability of total flavonols was only 0.1 and 0.5% after quercetin aglycone and rutin applications, respectively. Our data demonstrate that bioavailability of quercetin from rutin is substantially higher compared with that from quercetin aglycone in cows after intraruminal (or oral) application, unlike in monogastric species.

Dietary green tea polyphenols do not affect vitamin E status, antioxidant capacity and meat quality of growing pigs.

Supplementation of pigs with vitamin E, the most important lipid-soluble antioxidant, has been shown to improve meat quality and animal health. Previous studies in cultured cells and laboratory animals indicate synergistic effects between polyphenols and vitamin E. The present feeding trial was undertaken to investigate the effects of dietary green tea polyphenols (GTP) on vitamin E status, antioxidative capacity and parameters of meat quality in growing pigs. Eighteen castrated, crossbred, male pigs received a flavonoid-poor diet based on corn starch, caseinate and rapeseed oil with a total vitamin E content of 17 IU/kg diet over a period of 5 weeks. This basal diet was supplemented with green tea extract to provide daily doses of 0 (control), 10 and 100 mg GTP/kg body weight. Dietary supplementation of growing pigs with GTP did not affect serum, liver, lung and muscle vitamin E (alpha- and gamma-tocopherol) concentrations, plasma antioxidant capacity (ferric reducing ability of plasma, trolox equivalent antioxidant capacity) or parameters of meat quality including meat temperature, pH, conductivity, colour and drip loss. In conclusion, supplementation of pig diets with green tea catechins is not associated with improved antioxidant status and meat quality under practice-oriented conditions.

Ochratoxin A in Ruminants–A Review on Its Degradation by Gut Microbes and Effects on Animals

Ruminants are much less sensitive to ochratoxin A (OTA) than non-ruminants. The ruminal microbes, with protozoa being a central group, degrade the mycotoxin extensively, with disappearance half lives of 0.6–3.8 h. However, in some studies OTA was detected systemically when using sensitive analytical methods, probably due to some rumen bypass at proportions of estimated 2–6.5% of dosage (maximum 10%). High concentrate proportions and high feeding levels are dietary factors promoting the likeliness of systemic occurrence due to factors like shifts in microbial population and higher contamination potential. Among risk scenarios for ruminants, chronic intoxication represents the most relevant.

Effect of fumaric acid supplementation and dietary buffering capacity on the concentration of microbial metabolites in ileal digesta of young pigs

Blank, R., Sauer, W. C., Mosenthin, R., Zentek, J., Huang, S. and Roth, S. 2001. Effect of fumaric acid supplementation and dietary buffering capacity on the concentration of microbial metabolites in ileal digesta of young pigs. Can. J. Anim. Sci. 81: 345‐353. Experiments were conducted to determine the effect of different levels of fumaric acid supplementation to diets with a low or high buffer capacity on the concentrations of microbial metabolites and lipopolysaccharides, as an indicator of gram neg ative bacteria in ileal digesta of young pigs. In two experiments, 12 pigs each were weaned at 14 d of age and fitted with a si mple T-cannula at the distal ileum between 15 and 17 d of age. In experiment 1, the pigs were fed wheat‐soybean meal diets without or with inclusion of 1, 2 or 3% fumaric acid according to a balanced two-period changeover design. In experiment 2, the same diets were fed, except that the dietary buffering capacity was increased by inclusion of 3% sodium bicarbonate to all diets. Th e pigs were fed three times daily, equal amounts at 8 h intervals. The diets were supplied at a rate of 5% (wt/wt) of body weight . The inclusion of fumaric acid to the diet with a low buffering capacity (exp. 1) decreased ( P 0.05) the concentrations of fermentation products in ileal digesta, but there was a decrease (P < 0.05) in the concentration of lipopolysaccharides. Furthermore, in both experiments, the concentration of most fermentation products decreased (P < 0.05) with increasing age after weaning. These results give further evidence that supplementation of fumaric acid to diets for young pigs during the first 3 ‐ 4 wk after weaning reduces the metabolic activity and the concentrati ons of bacteria in the gastrointestinal tract. The magnitude of this effect, however, is dependent on the buffering capacity and th e inclusion level of fumaric acid in the diets.

Effects of live yeast cell supplementation to high concentrate diets on the toxicokinetics of ochratoxin A in sheep

Previous studies have indicated that high concentrate feeding reduces the ruminal degradation of the mycotoxin ochratoxin A (OA) to the less-toxic ochratoxin α (Oα) in ruminants. This is due to a pH-induced decrease in ruminal protozoa and subsequent increasing transfer of OA into the systemic circulation. The present study investigated whether stabilization of rumen pH by the live yeast cell supplementation to high concentrate diets affects the toxicokinetics of OA in sheep. Sheep were fed diets consisting of 70% concentrates and 30% grass silage (dry matter basis) supplemented without or with live yeast cells (Saccharomyces cerevisiae CNCM I-1077). After an adaptation period of 3 weeks, animals were given a single dose of OA (2.46 mg) in the form of contaminated wheat. Even though live yeast cells accelerated the recovery of ruminal pH from the decrease in pH induced by feeding, no effect on ruminal degradability and systemic availability of OA was recorded. Based on in vitro studies, live yeast cells and extracts of live yeast cell walls have been suggested as a mycotoxin-binding agent. However, supplementation with live yeast cells had no effect on the excretion pattern of OA in sheep, indicating that binding of OA to yeast components may be limited in ruminants. With respect to the toxicokinetics of OA, our results are in agreement with earlier results demonstrating that the hydrolysis of OA in the gastrointestinal tract of sheep is substantially less than previously described, especially if OA is ingested in combination with concentrate-rich diets. Our study demonstrates that feeding a live yeast cells product, registered as a feed additive for improving zootechnical performance, had no impact on the toxicokinetics of OA under the chosen conditions.

Effect of quercetin on the toxicokinetics of ochratoxin A in rats

Previous studies indicate that the intestinal absorption of the nephrotoxic mycotoxin ochratoxin A (OTA) occurs mainly through passive diffusion of the undissociated form. However, several in vitro studies have shown that OTA is partly re-secreted into the intestinal lumen by the multi-drug resistance associated protein (MRP2) and breast cancer resistance protein (BRCP). In vitro studies using Caco-2 cells have shown that some polyphenols (quercetin, genistein, resveratrol) may impair OTA efflux through competitive inhibition of MRP2, possibly resulting in an increased systemic availability of OTA. Among the tested polyphenols, quercetin showed the highest potential as efflux pump inhibitor; therefore, the aim of the present in vivo study was to investigate possible effects of quercetin on the toxicokinetics of OTA in rats. Eighteen growing male F344 Fisher rats (body weight: 200 g) were allocated to two dietary treatments consisting of (1) a commercial, flavonoid-free balanced diet containing 10 mg OTA/kg derived from inoculated wheat and (2) the same diet supplemented with 100 mg quercetin/kg. The animals were fed restrictively (~0.7 of ad libitum intake, 13 g/d) to avoid differences in OTA intake. Animals were kept in metabolism cages to facilitate total urine and faeces collection. After 6 days on trial, rats were euthanised and blood, liver, kidney, muscle and brain samples were taken from each animal. Faeces, urine and tissue samples were analysed for OTA and its main metabolite ochratoxin α by high-performance liquid chromatography using fluorescence detection. Quercetin supplementation had no effect (P > 0.05) on feed consumption, OTA-intake, water intake and body weight gain. Faecal and urinary excretion of OTA and ochratoxin α and concentrations of OTA in all tissues were not affected by quercetin supplementation. Based on the total excretion and tissue concentrations of OTA, it is concluded that the polyphenol quercetin has no impact on the toxicokinetics of OTA in vivo.

Liquid chromatographic methods for biotransformation studies of ochratoxin A

Liquid chromatographic methods were used for the detection of ochratoxin A (OTA) and its metabolites ochratoxin alpha (OTalpha), 10-hydroxy OTA (10-OHOTA), 4R-hydroxy OTA (4R-OHOTA) and the ethyl ester of OTA (OTC) in in vitro samples, obtained with Caco-2 cell culture experiments and in in vivo urine samples from sheep. A high-performance liquid chromatography method with fluorescence detection (HPLC-FLD) and a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method were developed and validated for the detection of OTA and its metabolites OTalpha, 10-OHOTA, 4R-OHOTA and OTC, which was used as internal standard. The LOD/LOQ values for OTalpha, 4R-OHOTA and OTA were 0.63/2.11, 0.99/3.31 and 0.84/2.81 microg/L, respectively, for the HPLC-FLD method and 0.98/3.28, 1.11/3.72 and 0.88/2.96 microg/L, respectively for the LC-MS/MS method. Within-day and between-day precision were both <12% for the HPLC-FLD method, and <10% for the LC-MS/MS method. The recovery of OTA and its metabolites ranged between 71 and 111% for the HPLC-FLD method and between 79 and 110% for the LC-MS/MS method. In the first experiment only OTA was added to the Caco-2 cells while in the second experiment 3-methylcholanthrene (3MC) was also present in the cell culture systems. Besides OTA, which was recovered in all the samples, an unknown compound was also observed in the second experiment. When 3MC was added, the results showed that the OTA concentration in the basolateral samples was decreased by 50%. The methods were also implemented for the analysis of urine samples of sheep, fed increasing amounts of OTA. With the HPLC-FLD method it could be concluded that the concentration of OTA and OTalpha increased according to ingested amounts of OTA, with OTalpha being the most abundant compound. The results obtained with the LC-MS/MS method confirmed these results.

Standardized ileal digestibility of amino acids in alfalfa meal, sugar beet pulp, and wheat bran compared to wheat and protein ingredients for growing pigs

A total of 11 (8 + 3 for replacement) barrows with an initial BW of 23 kg and fitted with a simple T-cannula at the distal ileum were used in 2 consecutive experiments (Exp. 1 and Exp. 2) to determine the standardized ileal digestibility (SID) of AA in 7 assay feed ingredients according to 2 consecutive duplicated 4 × 4 Latin square designs. In Exp. 1, 3 corn starch-based assay diets were formulated to contain 170 g CP/kg (as-fed basis) from either soybean meal (SBM), canola meal (CM), or meat-and-bone meal (MBM) and 1 assay diet that contained 136 g CP/kg (as-fed basis) from wheat as commonly used feed ingredients for pigs. In Exp. 2, the pigs were fed 4 assay diets formulated to contain 170 g CP/kg (as-fed basis) from either the same SBM as in Exp. 1 or a combination of this SBM and alfalfa meal (AM), sugar beet pulp (SB), or wheat bran (WB) to compare the SID of AA in these feed ingredients with those used in Exp. 1. The SID of AA in CM was lower compared to SBM (P < 0.05) with intermediate values for MBM and wheat. Among fiber rich feed ingredients, SID values were lower in SB compared to WB (P < 0.05) with intermediate values for AM. In AM, SID values ranged between 29 and 45% for Lys, Cys, Thr, and Phe and between 51 and 71% for Arg, His, Ile, Leu, Met, and Val. In SB, SID values ranged between -21 and 46% for Cys, Thr, Phe, and Val and between 51 and 61% for Arg, His, Ile, Leu, Lys, and Met. In WB, SID values were between 55 and 64% for Lys, Cys, Phe, Thr, and Val and between 68 and 80% for Arg, His, Ile, Leu, and Met. The SID values in WB, SB, and AM provided in the present study may improve diet formulation when these feed ingredients are used in diet formulation for pigs.

Systemic Absorption of Catechins after Intraruminal or Intraduodenal Application of a Green Tea Extract in Cows

Green tea catechins have various potential health benefits in humans including anti-inflammatory, anti-oxidative and hepato-protective effects. If present in the circulation, they might have similar effects in ruminants, which are exposed to oxidative stress and fatty liver disease such as dairy cows during the periparturient phase. However, the bioavailability of a substance is a prerequisite for any post absorptive effect in vivo. This study aimed to investigate the appearance of catechins from a green tea extract (GTE) in cattle plasma after intraruminal and intraduodenal administration because absorption is of major importance regarding the bioavailability of catechins. The studies were performed in 5 rumen-fistulated non-lactating heifers and 6 duodenally fistulated lactating dairy cows, respectively, equipped with indwelling catheters placed in a jugular vein. The GTE was applied intraruminally (10 and 50 mg/kg BW, heifers) or duodenally (10, 20 and 30 mg/kg BW, dairy cows) in a cross‐over design with a 2 d washout period between different dosages. Blood samples were drawn following the GTE administration at various pre-defined time intervals. The concentration of the major GTE catechins (gallocatechin, epigallocatechin, catechin, epicatechin, epigallocatechin-gallate, epicatechin-gallate) in plasma samples were analysed by HPLC with electrochemical detection. Irrespective of the dose, almost none of the catechins originally contained in the GTE were detected in plasma samples after intraruminal application. In contrast, intraduodenal administration of GTE resulted in increased plasma concentrations of epicatechin, epigallocatechin, epigallocatechin gallate in a dose‐dependent manner. Thus, we can conclude that intraruminally or orally administered catechins are intensively metabolized by ruminal microorganisms.