The effect of supplementation of a white clover or perennial ryegrass diet with grape seed extract on indole and skatole metabolism and the sensory characteristics of lamb

Condensed tannin in the form of a grape seed extract (GSE) was dosed to weaned wether lambs fed white clover (WC) or perennial ryegrass (PRG) over a 9‐week period to determine whether the ‘pastoral’ flavour and odour of meat could be altered. The concentrations of the pastoral flavour compounds indole and skatole were determined in the rumen fluid, blood plasma and intermuscular fat. The odour and flavour of fat and meat from the slaughtered lambs was assessed by a trained panel. The rumen fluid and blood plasma concentrations of indole and skatole were higher in those lambs fed WC compared to PRG (P < 0.05) and the overall meat flavour intensity was greater when feeding WC (P < 0.01). The observed concentration of indole and skatole in the fat between WC and PRG feeding treatments was not statistically different. Power analysis indicated that increasing the number of lambs per treatment group from 20 to 65 would result in a higher fat skatole concentration (P < 0.05) being detected in lambs fed WC compared to PRG. Dosing with GSE gave a small reduction in skatole concentration in the rumen fluid and reduced plasma concentration of indole and skatole (P < 0.001). Odour and flavour scores of the fat and meat samples were not particularly high however, dosing with GSE lowered the overall and sweet odour and the sheepy, camphor, faecal and barnyard flavour (P < 0.05). Although the plasma concentration of indole and skatole suggests that GSE reduced indole and skatole formation, the intermittent supply of the GSE to the rumen environment was not sufficient to reduce their concentration in the fat. Hence, the small difference in the scores for pastoral odour and flavour attributes associated with GSE treatment may arise from other unknown factors. From a primary investigation, there was no difference in the concentration of indole and skatole in fat samples collected from carcasses before and after chilling. Further investigations into meat pastoral flavour are warranted through feeding condensed tannin‐containing forages. Copyright © 2007 Society of Chemical Industry     

Controlling the formation of indole and skatole in in vitro rumen fermentations using condensed tannin

Indole and skatole are formed in the rumen from the fermentation of tryptophan and have been correlated to the presence of undesirable pastoral flavours in meat from grazing ruminants. A series of four in vitro rumen fermentation experiments was carried out to determine the effectiveness of condensed tannin (CT) for reducing the formation of indole and skatole. Experiment 1 utilised fresh white clover (WC; Trifolium repens) in ratios with the CT‐containing forage Lotus pedunculatus (LP; 97 g CT kg^?1 dry matter (DM)). Increasing the ratio of LP to WC decreased the formation of indole and skatole. Experiments 2 and 3 used extracts of CT from LP and grape seed added to incubations of fresh non‐CT‐containing WC or perennial ryegrass (PRG; Lolium perenne). Including the CT extracts in incubations at 40 and 80 g kg^?1 DM was more effective at reducing indole and skatole formation than lower concentrations of CT extract (P < 0.05). Including fresh LP in ratios with WC gave a linear decrease in indole and skatole concentration (indicating dilution), while including a CT extract in the incubations gave an exponential decrease in indole and skatole concentration (suggesting binding). Experiment 4 elucidated the mechanisms behind the action of CT by delayed addition of tryptophan and polyethylene glycol (PEG) to incubations of LP. Rumen microbes that were exposed to CT of LP in planta for up to 6 h and then provided with tryptophan were still able to convert tryptophan to indole and skatole. Adding PEG to incubations of LP after 6 h inhibited the activity of plant CT and increased the availability of substrate for indole and skatole formation. These studies have shown that a higher concentration of CT is more efficient for reducing indole and skatole formation and that CT contained within plants acts differently in in vitro rumen fermentations than additions of extracted CT. Under the conditions of these experiments, there was no evidence that CT contained in LP affected the protein present in WC in a mixed fermentation. Copyright © 2007 Society of Chemical Industry     

Effect of different condensed tannin‐containing forages,forage maturity and nitrogen fertiliser application on the formation of indole and skatole in in vitro rumen fermentations

The objectives of this work were to establish the effect of seven different forages with a varied condensed tannin (CT) content, plant maturity and nitrogen fertiliser application on the ruminal formation of skatole and indole using an in vitro method designed to mimic rumen fermentation conditions. After 10 h of incubation, the concentration of indole and skatole was highest when incubating white clover (P < 0.05). Polyethylene glycol addition, to inhibit CT, showed that CT significantly reduced the formation of indole and skatole when incubating Lotus corniculatus, sulla, Dorynium rectum and Lotus pedunculatus (P < 0.01). Mature forage growth resulted in a significantly lower concentration of indole and skatole being formed in vitro compared to the incubation of new spring growth (P < 0.001). A higher application of nitrogen fertiliser to perennial ryegrass‐based pasture resulted in a higher crude protein concentration in the plant and a significantly higher concentration of skatole formed in vitro (P < 0.001). Forages containing CT reduced the conversion of endogenous protein to indole and skatole and plants containing a higher CT concentration tended to be more effective, but compositional differences of CT between forages may also have had an influence. New forage growth or swards that had a high application of nitrogen fertiliser promoted the formation of indole and skatole. Copyright © 2007 Society of Chemical Industry     

Simple phenols and indoles in anaerobically stored piggery wastes

Phenol, p-cresol, 4-ethylphenol, indole and skatole, which contribute to a large extent to the bad smell of piggeries, were estimated in anaerobically stored piggery wastes. p-Cresol, indole and skatole were also found in freshly voided faeces. Phenol, p-cresol and 4-ethylphenol are present in the urine as their glucuronides. It was shown that upon contact of urine and faeces, the urinary glucuronides are hydrolysed almost instantly liberating phenols. This hydrolysis is caused by the high β-glucuronidase activity of faeces. Protein degradation followed by transformation of tyrosine and tryptophane were found to be additional processes contributing to the accumulation of phenol, p-cresol, indole and skatole during the anaerobic storage of piggery wastes.     

Protein degradation in the rumen of sheep and cattle

Sheep and steers were given, at a maintenance level of feeding, four diets consisting of either poor quality dried grass, good quality dried grass or separate mixtures (63:35) of each of the dried grasses and barley. Ammonia and total N concentrations in rumen liquor were significantly higher in sheep than in steers whereas total volatile fatty acid concentration was significantly lower and protein N concentration, pH and rumen fluid dilution rate did not differ significantly between species. For all rumen measurements, except total volatile fatty acid concentration, there were significant differences between diets with the dietary responses being similar in both species. Protein degrading activity in the rumen was measured in vitro with casein as the substrate and in situ by measuring N disappearance when soyabean meal, cotton seed meal, groundnut meal, meat and bone meal, fish meal and dried grass were incubated in polyester bags in the rumen. Casein degrading activity of rumen liquor did not differ significantly between species, whereas rumen in-situ degradation of all feedstuffs, except fish meal, was significantly higher in sheep. In both species, ruminal casein degrading activity was higher when the good quality forage was given than when the poor quality forage was given and also increased when part of each forage was replaced by barley. In contrast, rumen in-situ degradability of feedstuffs did not differ when the two all-forage diets were given and the inclusion of barley in the diet reduced the rate of degradation. In both species and with all diets the rumen in-situ degradability ranking of the feedstuffs was similar.     

Screening of indoles in cheese

 Indole and skatole (3-methylindole) are formed from tryptophan by microbial activity. Depending on their concentration, they may either contribute to an unpleasant odour, as described for pig meat, or alternatively to a positive aroma profile, as during ripening of cheese. In a screening study, the two indoles in various types of cheese were determined, by both HPLC and GC. It was found that the two compounds mainly occur in mould cheese, such as Camembert and are even more pronounced in blue-veined cheese. The concentrations in unripened cheese were up to 700 ng/g fat for indole and 50 ng/g fat for skatole. The formation of the two indoles increases when proteolysis during ripening provides more tryptophan. Skatole formation appears to be specifically favoured by a low pH and anaerobic conditions. Received: 14 May 1997     

Screening of indoles in cheese

 Indole and skatole (3-methylindole) are formed from tryptophan by microbial activity. Depending on their concentration, they may either contribute to an unpleasant odour, as described for pig meat, or alternatively to a positive aroma profile, as during ripening of cheese. In a screening study, the two indoles in various types of cheese were determined, by both HPLC and GC. It was found that the two compounds mainly occur in mould cheese, such as Camembert and are even more pronounced in blue-veined cheese. The concentrations in unripened cheese were up to 700 ng/g fat for indole and 50 ng/g fat for skatole. The formation of the two indoles increases when proteolysis during ripening provides more tryptophan. Skatole formation appears to be specifically favoured by a low pH and anaerobic conditions. Received: 14 May 1997     

Effect of Heat Treatment Upon the Chemical Composition of Cottonseed Meal and Upon the Reactivity of Cottonseed Condensed Tannins

The effects of heat treatment on the chemical composition of cottonseed meal (CSM), with or without the addition of cottonseed hulls (containing condensed tannins; CT), and upon reactivity of the CT were studied. Heat was applied in a forced draught oven at 100°C for 2 h. Fluorodinitrobenzene (FDNB)-available lysine, free gossypol, extractable- and bound-CT concentrations, in vitro total nitrogen (N) solubility and the in vitro rumen degradation of the two major seed proteins (52 and 48 kDa) present in cottonseed kernel (which does not contain CT) were determined. The reactivity of CT was assessed by determining N solubility and rumen degradation of cottonseed kernel proteins in the presence or absence of polyethylene glycol (PEG; molecular weight (MW) 3500), which binds and inactivates CT. Heat treatment reduced the concentrations of free gossypol and FDNB-available lysine by small amounts, reduced measurable total CT content by 13%, reduced the solubility of total N, and reduced potential degradability of the 52 and 48 kDa cottonseed storage proteins by mixed rumen microorganisms. Addition of hulls further depressed solubility of total N and ruminal degradation of the two major storage proteins in cottonseed kernel. The action of PEG in vitro indicated that only part of the depression caused by hull addition could be explained by the presence of CT in the hulls, and that the effects of CT upon N solubility and potential degradability in heated CSM were similar to that in unheated CSM. Addition of hulls also substantially reduced FDNB-available lysine. In commercially produced materials, CSM from the Brisbane mill had a lower total CT content, lower N solubility and lower ruminal protein degradation rate than CSM from the Narrabri mill, but a similar level of FDNB-available lysine. Although application of heat inactivated 13% of the total CT, such that it could no longer be extracted and detected with butanol/HCl, it did not seem to change the overall effects produced by CT in reducing N solubility and protein degradation. The effect of hull addition in reducing available lysine has considerable relevance for feeding CSM to monogastric livestock. Interactions involving heat, hulls and CT need to be further studied.     

Evaluation of the effect of formic acid and level of formaldehyde application before ensiling on silage protein degradability

The effect of the application, before ensiling, of formic acid alone, and together with increasing levels of formaldehyde, on the degradability of the protein of ryegrass and red clover silages has been assessed on the basis of nitrogen solubility in mineral buffer; susceptibility of N to degradation during in-vitro incubation with either rumen microorganisms, acid pepsin or neutral protease; and N disappearance when the silages were incubated in situ in Dacron bags in the rumen of sheep receiving dried grass. The relative effects of the additives were generally consistent for both crops and with all procedures: formic acid either had no effect or reduced degradability by only a small amount, whereas a mixture of formic acid and formaldehyde was more effective than formic acid alone in protecting protein from degradation, and degradability decreased in a curvilinear manner with increasing levels of formaldehyde application. Absolute values for protein degradability based on buffer solubility and in-vitro degradation by rumen microorganisms were very similar but lower than those based on digestion with proteolytic enzymes which in turn were lower than those obtained with the rumen in situ procedure. Buffer solubility and in-vitro incubation with rumen microorganisms also showed much bigger differences between the formic acid-treated and the formaldehyde-treated silages than the other methods.     

Threshold Detection of Boar Taint Chemicals Using Parasitic Wasps

Abstract: Surgical castration has been long used to prevent consumers from experiencing taint in meat from male pigs, which is a large problem in the pig husbandry industry. Due to obvious animal welfare issues, the EU now wants an alternative for castration, suggesting an urgent need for novel methods of boar taint detection. As boar taint is only a problem when taint chemicals exceed a well‐defined threshold, detection methods should be concentration‐specific. The wasp, Microplitis croceipes’ ability to learn and respond to particular concentrations of the boar taint compounds, skatole, androstenone, and indole was tested. Also tested was the wasps’ ability to discriminate between known concentrations of indole, skatole, and androstenone in real boar fat samples at room temperature. Wasps were trained using associative learning by providing food‐deprived wasps with sucrose–water in the presence of specific odor concentrations. Trained wasps’ responses were tested to a range of concentrations of 3 compounds. Wasps showed unidirectional generalization of learned concentration responses, whereby the direction of concentration generalization was shown to be chemical‐dependent. Through both positive (sucrose) and negative feeding experiences (water only) with varying compound concentrations, the wasps can also be conditioned to respond to concentrations exceeding a defined threshold, and they were successful in reporting low, medium, and high concentrations of indole, skatole, and androstenone in boar fat at room temperature. The need for threshold detection rather than simple detection of absence/presence applies to many food quality issues, including the detection of spoilage or pest damage in crops or stored foods. Practical Application: An inexpensive and reliable means of detecting boar tainted pork at slaughter to avoid tainted meat on the market and dissatisfied consumers.     

Boar taint in pigs selected for components of efficient lean growth rate

Genetic and nutritional effects on the boar taint traits of androstenone, skatole and indole and the eating quality traits associated with boar taint were examined by testing animals from four selection lines and a control line on isoenergetic diets, which differed in ileal digestible lysine: digestible energy (0.40, 0.76 and 1.12 g lysine/MJ DE). The selected lines resulted from seven generations of selection for high daily food intake, lean food conversion ratio (LFC) and lean growth rate on ad libitum (LGA) or restricted (LGS) feeding regimes in a Large White population. During performance test, from 30 to 90 kg, boars were fed on either ad libitum or restricted (0.75 g/g ad libitum daily food intake) feeding regimes. A sensory panel assessed heated fat samples for androstenone odour, skatole odour and abnormal odour. There were no significant differences between the selection and control lines or diets for log transformed fat content of androstenone, skatole, indole. The significant diet with feeding regime interaction for log transformed fat content of skatole and indole were essentially due to significantly higher log transformed fat contents with ad libitum feeding of the high lysine diet compared to restricted feeding (skatole: -1.94 vs -3.06, s.e.d. 0.43; indole: -3.44 vs -4.22, s.e.d. 0.28), as differences between feeding regimes on diets A and C were not significantly different from zero. There were no significant differences between selection and control lines for sensory panel score for abnormal odour or androstenone odour, but the LFC and LGA selection lines had a significantly higher skatole odour score than the LGS selection line. Neither diet nor feeding regime had any significant effect on sensory panel assessment of odour. Log transformed fat content of androstenone and skatole were significantly correlated with sensory panel score for skatole odour (0.37 and 0.46, s.e. 0.12), but not with sensory panel score for androstenone odour (0.06 and 0.09), such that they would not be useful predictors of androstenone odour.     

Skatole and indole concentrations in Longissimus dorsi and fat samples of pigs

A HPLC-NP (normal phase-high performance liquid chromatography) method for determining the concentration of skatole and indole in Longissimus dorsi samples is described. Lipids containing skatole and indole were extracted in chloroform:methanol (2:1) at room temperature and dehydrated by liquid-liquid extraction with an aqueous solution saturated with 10% of sodium chloride. The organic phase was evaporated to dryness and redissolved in 10 ml of hexane:2-propanol (92:8). Indolic compounds were separated on a Hypersil aminopropylsilica column (5 μm) (250×4.6 mm i.d.). The mobile phase was hexane:2-propanol (92:8) and detection was by fluorescence (excitation at 280 nm and emission at 360 nm). Linearity was found in the range of 0.05-0.4 μg/g and the coefficient of correlation was above 0.99 for both compounds. The within day (n=5) variation was at 0.05, 0.2 and 0.4 μg/g and the CV (coefficient of variation) values for relative areas determined at these concentrations were less than 13%. This method was used to compare the concentrations of skatole and indole in different samples: L. dorsi muscle, the fat covering the L. dorsi and subcutaneous fat. A correlation was observed between the concentration of indole and skatole in the back fat and fat covering the L. dorsi samples (P<0.001, r=0.99). No significant correlation was obtained in L. dorsi samples, between skatole and indole levels. In spite of the correlation shown between skatole and indole concentrations in the back fat and L. dorsi samples, the mean concentrations of these compounds were significantly higher (P<0.05) in the back fat samples.     

Effects of raw potato starch and live weight on fat and plasma skatole,indole and androstenone levels measured by different methods in entire male pigs

A total of 96 entire male pigs were used to evaluate the effects of feeding raw potato starch (RPS) and live weight (LW; 90, 100 or 115 kg) on skatole, indole and androstenone levels in fat and plasma. Different methods to analyse the compounds were compared. Fat levels of skatole analysed by HPLC and colorimetric methods, and androstenone analysed by HPLC and ELISA, were highly correlated. However, androstenone values obtained by ELISA were overestimated. Feeding RPS reduced skatole levels in plasma and fat but did not affect indole levels. Androstenone levels in plasma measured by direct ELISA were not altered by diet, whereas the levels measured by ELISA with extraction were lower in the pigs fed RPS. Feeding RPS did not reduce androstenone levels in fat. Androstenone levels in fat were higher at 115 than at 90 kg, whereas skatole and indole levels were not affected by LW.     

Effect of monensin on the performance and nitrogen utilization of lactating dairy cows consuming fresh forage

We conducted a lactation trial with a fresh forage diet in order to evaluate 1) the effects of monensin on nitrogen metabolism, and 2) the Cornell Net Carbohydrate and Protein System (CNCPS). Thirty Holstein cows in midlactation (eight fitted with ruminal fistulas) were gradually introduced to a fresh forage diet. A concentrate mix based on corn meal was fed before the a.m. and p.m. milking times 0730 and 1730 h, then the fresh forage was fed at 0830 and 1830 h. Fifteen cows each were allocated to a control (no monensin) and a treatment group receiving 350 mg/cow per day of monensin in the p.m. concentrate feeding. A 7-d fecal and urine collection period and a 3-d rumen sampling period were conducted with the fistulated cows. After the lactation study was concluded, the fistulated cows were fed forage regrowth and a 3-d rumen sampling period was repeated. Monensin increased milk production by 1.85 kg. Milk fat and protein concentrations decreased and milk fat and protein yields increased, but the effects were nonsignificant. Monensin did not significantly affect DMI. Ruminal ammonia and the acetate-to-propionate ratio decreased with the addition of monensin in both fed forages. Monensin decreased fecal N output, and increased apparent N digestibility by 5.4%. Because of the decrease in ruminal ammonia and increase in apparent N digestibility, we concluded monensin was sparing amino acids from wasteful rumen degradation with a fresh forage diet. The precision of the CNCPS in predicting performance was high (r2 = 0.76), and the bias was low (overprediction of 3.6%). These results indicate that the CNCPS can be used for dairy cows consuming fresh forage and gives realistic predictions of performance.     

Relationships between milk urea concentrations and nutritional management, production, and economic variables in Ontario dairy herds

The objectives of this study were to describe the relationships between milk urea concentrations and nutritional management, production, and economic variables in commercial dairy herds. Dairy Herd Improvement (DHI) test-day milk urea data, production data, and information on ration nutrient composition and feeding management programs were collected over a 13-mo period from 53 commercial Ontario dairy herds. Economic variables included gross milk revenue, feed costs, and income over feed costs. Herd mean milk urea concentrations had a positive relationship with dietary levels of crude protein (CP), rumen degradable protein (RDP), and rumen undegradable protein (RUP) and a negative relationship with dietary levels of nonfiber carbohydrates (NFC), forage:concentrate (F:C) ratio, NFC:CP ratio, and NFC:RDP ratio. These findings are consistent with experimental studies that used chemical methods of milk urea analysis. Herd mean milk urea concentration was not associated with feeding management (e.g., total mixed rations, component feeding, feeding frequency, or synchrony of forage and concentrate feeding). Herd mean milk urea was not associated with either mean milk yield or linear score. Herd mean milk urea had a positive relationship with feed costs per cow per day but was not associated with gross milk revenue per cow per day. Herds with a high mean milk urea concentration tended to have lower income over feed costs per cow per day. High herd mean milk urea concentrations were associated with higher feed costs per kilogram of milk fat but lower gross milk revenue and lower income over feed costs per kilogram of milk fat. The results of this study demonstrate that DHI milk urea measurements produced by an infrared test method offer a useful tool for monitoring the efficiency of nitrogen utilization in commercial dairy herds. The results also suggest that diets may be balanced to achieve greater efficiency of nitrogen utilization, lower milk urea concentrations, and lower feed costs, while still achieving high milk production. This may lead to improved income over feed costs.     

Effects of dietary components including garlic on concentrations of skatole and indole in subcutaneous fat of female pigs

The results reported here showed that threshold concentrations of skatole and indole in rice-bran oil for Singaporean consumers were 0.028 μg/g and 0.051 μg/g, respectively, and that skatole and indole levels in subcutaneous fat of pigs can be affected by diet. In Experiment A, 31 female pigs were fed with diets based on plant products only (P) or plant plus animal by-products (AP), with added levels of garlic essential oil from zero to 2.15 g/kg feed. Concentrations of skatole and indole increased with increasing garlic concentration (P < 0.001). In Experiment B, P and AP diets were fed to 47 female pigs with different dietary lipid sources (fish oil, tallow, and a mix of linseed oil and soya oil). Skatole and indole concentrations were higher in backfat of pigs fed with the AP diet (P < 0.05), but were unaffected by the type of lipid.     

Formation of lysinoalanine following alkaline processing of soya bean meal in relation to the degradability of protein in the rumen

Effects of sodium hydroxide (NaOH) treatment on the formation of lysinoalanine (LAL) and on in-situ and in-vivo utilisation of soya bean meal (SBM) protein were investigated. Defatted SBM was sprayed with 0 (water), 25, 50 and 100 g NaOH (kg^?1 dry matter), dried at 80°C for 2 h and then fed to four rumen cannulated goats in a 4 × 4 Latin square as a mixed diet (9: 1 w/w) of sudangrass hay and the SBM. LAL was not detected in non-alkaline SBM, but the cross-linked amino acid increased according to the level of NaOH addition. The maximum concentration of LAL was 1.26 g per 16 g nitrogen when the highest level of NaOH was applied. The NaOH treatment did not alter the digestibility or the retention of nitrogen of the mixed diet. Addition of NaOH at 25 and 50 g (kg^?1 dry matter) lowered the solubility of protein, while the highest level of treatment had little effect. Lag time and rate of degradation of protein decreased due to the increasing level in NaOH treatment. A significant correlation (r = —0.860, P < 0.01) between LAL content and rate of protein degradation suggested that the formation of cross-linked amino acid would render protein more resistant to microbial degradation in the rumen.     

Method for the determination of indole and skatole in pig fat

A quantitative method was developed to determine by gas-liquid chromatography the concentrations of indole and skatole in pig fat. Methanolic extracts of subcutaneous adipose tissue were de-fatted by cooling, concentrated using solid-phase extraction columns, and analysed in a gas chromatograph fitted with a polar capillary column and a flame ionisation detector. The method detects indole and skatole to below 0-002 mg kg^?1 fat.