Veerle Fievez

Accumulation of trans C18:1 Fatty Acids in the Rumen after Dietary Algal Supplementation Is Associated with Changes in the Butyrivibrio Community

ABSTRACT Optimization of the fatty acid composition of ruminant milk and meat is desirable. Dietary supplementation of algae was previously shown to inhibit rumen biohydrogenation, resulting in an altered milk fatty acid profile. Bacteria involved in biohydrogenation belong to the Butyrivibrio group. This study was aimed at relating accumulation of biohydrogenation intermediates with shifts in Butyrivibrio spp. in the rumen of dairy cows. Therefore, an experiment was performed with three rumen-fistulated dairy cows receiving a concentrate containing algae (9.35 g/kg total dry matter [DM] intake) for 20 days. Supplementation of the diet with algae inhibited biohydrogenation of C18:2 omega 6 (n-6) and C18:3n-3, resulting in increased concentrations of biohydrogenation intermediates, whereas C18:0 decreased. Addition of algae increased ruminal C18:1trans fatty acid concentrations, mainly due to 6- and 20-fold increases in C18:1trans 11 (t11) and C18:1t10. The number of ciliates (5.37 log copies/g rumen digesta) and the composition of the ciliate community were unaffected by dietary algae. In contrast, supplementation of the diet with algae changed the composition of the bacterial community. Primers for the Butyrivibrio group, including the genera Butyrivibrio and Pseudobutyrivibrio, were specifically designed. Denaturing gradient gel electrophoresis showed community changes upon addition of algae without affecting the total amount of Butyrivibrio bacteria (7.06 log copies/g rumen DM). Clone libraries showed that algae affected noncultivated species, which cluster taxonomically between the genera Butyrivibrio and Pseudobutyrivibrio and might play a role in biohydrogenation. In addition, 20% of the clones from a randomly selected rumen sample were related to the C18:0-producing branch, although the associated C18:0 concentration decreased through supplementation of the diet with algae.

Invasion of Salmonella enteritidis in avian intestinal epithelial cells in vitro is influenced by short-chain fatty acids.

Fermentation reactions in the caeca of chickens, the predominant place for Salmonella colonization, result in high concentrations of short-chain fatty acids (SCFA). Thus Salmonella bacteria are in close contact with SCFA during their life cycle. A study was carried out to analyse the effects of SCFA on invasion of Salmonella enteritidis in an avian intestinal epithelial cell line. Preincubation of S. enteritidis for 4 h in growth media supplemented with various concentrations of propionate or butyrate resulted in decreased invasion compared to bacteria, preincubated in nonsupplemented media, and to bacteria, preincubated in media supplemented with formate or acetate. Incubation of the S. enteritidis bacteria in media supplemented with mixtures of SCFA mimicking the in vivo caecal concentrations resulted in increased invasion compared with butyrate-exposed bacteria, but equal invasion compared with nonexposed bacteria. Increasing the butyrate concentration in these mixtures did not modify invasion compared with the original mixtures.

Effect of dietary starch or micro algae supplementation on rumen fermentation and milk fatty acid composition of dairy cows.

Two experiments with rumen-fistulated dairy cows were conducted to evaluate the effects of feeding docosahexaenoic acid (DHA; C22:6 n-3)-enriched diets or diets provoking a decreased rumen pH on milk fatty acid composition. In the first experiment, dietary treatments were tested during 21-d experimental periods in a 4 x 4 Latin square design. Diets included a control diet, a starch-rich diet, a bicarbonate-buffered starch-rich diet, and a diet supplemented with DHA-enriched micro algae [Schizochytrium sp., 43.0 g/kg of dry matter intake (DMI)]. Algae were supplemented directly through the rumen fistula. The total mixed ration consisted of grass silage, corn silage, soybean meal, and a standard or glucogenic concentrate. The glucogenic and buffered glucogenic diet had no effect on rumen fermentation and milk fatty acid composition because, unexpectedly, no reduced rumen pH was detected. The algae diet had no effect on rumen pH but provoked decreased butyrate and increased isovalerate molar proportions in the rumen. In addition, algae supplementation affected rumen biohydrogenation of linoleic and linolenic acid as reflected in the modified milk fatty acid composition toward increased conjugated linoleic acid (CLA) cis-9 trans-11, CLA trans-9 cis-11, C18:1 trans-10, C18:1 trans-11, and C22:6 n-3 concentrations. Concomitantly, on average, a 45% decrease in DMI and milk yield was observed. Based on these drastic and impractical results, a second animal experiment was performed for 20 d in which 9.35 g/kg of total DMI of algae were incorporated in the concentrate and supplemented to 3 rumen-fistulated cows. Algae concentrate feeding increased rumen pH, which was associated with decreased rumen short-chain fatty acid concentrations. Moreover, a different shift in rumen short-chain fatty acid proportions was observed compared with the first experiment because molar proportions of butyrate, isobutyrate, and isovalerate increased, whereas acetate molar proportion decreased. The milk fatty acid profile changed as in experiment 1. However, the decrease in DMI and milk yield was less pronounced (on average 10%) at this algae supplementation level, whereas milk fat percentage decreased from 47.9 to 22.0 g/kg of milk after algae treatment. In conclusion, an algae supplementation level of about 10 g/kg of DMI proved effective to reduce the milk fat content and to modify the milk fatty acid composition toward increased CLA cis-9 trans-11, C18:1 trans, and DHA concentrations.

Red clover polyphenol oxidase and lipid metabolism.

Increasing the polyunsaturated fatty acid (PUFA) composition of milk is acknowledged to be of benefit to consumer health. Despite the high PUFA content of forages, milk fat contains only about 3% of PUFA and only about 0.5% of n-3 fatty acids. This is mainly due to intensive lipid metabolism in the rumen (lipolysis and biohydrogenation) and during conservation (lipolysis and oxidation) such as drying (hay) and ensiling (silage). In red clover, polyphenol oxidase (PPO) has been suggested to protect lipids against degradation, both in the silage as well as in the rumen, leading to a higher output of PUFA in ruminant products (meat and milk). PPO mediates the oxidation of phenols and diphenols to quinones, which will readily react with nucleophilic binding sites. Such binding sites can be found on proteins, resulting in the formation of protein-bound phenols. This review summarizes the different methods that have been used to assess PPO activity in red clover, and an overview on the current understanding of PPO activity and activation in red clover. Knowledge on these aspects is of major importance to fully harness PPOs lipid-protecting role. Furthermore, we review the studies that evidence PPO-mediated lipid protection and discuss its possible importance in lab-scale silages and further in an in vitro rumen system. It is demonstrated that high (induction of) PPO activity can lead to lower lipolysis in the silage and lower biohydrogenation in the rumen. There are three hypotheses on its working mechanism: (i) protein-bound phenols could directly bind to enzymes (e.g. lipases) as such inhibiting them; (ii) binding of quinones in and between proteins embedded in a lipid membrane (e.g. in the chloroplast) could lead to encapsulation of the lipids; (iii) direct binding of quinones to nucleophilic sites in polar lipids also could lead to protection. There is no exclusive evidence on which mechanism is most important, although there are strong indications that only lipid encapsulation in protein-phenol complexes would lead to an effective protection of lipids against ruminal biohydrogenation. From several studies it has also become apparent that the degree of PPO activation could influence the mode and degree of protection. In conclusion, this review demonstrates that protein-bound phenols and encapsulation in protein-phenol complexes, induced by PPO-mediated diphenol oxidation, could be of interest when aiming to protect lipids against pre-ruminal and ruminal degradation.

Effects of saponins, quercetin, eugenol, and cinnamaldehyde on fatty acid biohydrogenation of forage polyunsaturated fatty acids in dual-flow continuous culture fermenters

Four different plant secondary metabolites were screened for their effect on rumen biohydrogenation of forage long-chain fatty acids, using dual-flow continuous culture fermenters. Treatments were as follows: control (no additive), positive control (12 mg/L of monensin), and plant extracts (500 and 1,000 mg/L of triterpene saponin; 250 and 500 mg/L of quercetin; 250 mg/L of eugenol; 500 mg/L of cinnamaldehyde). Monensin increased propionate, decreased acetate and butyrate proportions, and inhibited the complete biohydrogenation of fatty acids resulting in the accumulation of intermediates of the biohydrogenation process (C18:2 trans-11, cis-15 rather than C18:1 trans-11). Cinnamaldehyde decreased total VFA concentration and proportions of odd and branched-chain fatty acids in total fat effluent. Apparent biohydrogenation of C18:2n-6 and C18:3n-3 was also less, and a shift from the major known biohydrogenation pathway to a secondary pathway of C18:2n-6 was observed, as evidenced by an accumulation of C18:1 trans-10 and trans-10, cis-12 CLA. Quercetin (500 mg/L) increased total VFA concentration, but no shifts in the pathways or extent of biohydrogenation were observed. Eugenol resulted in the accumulation of C18:1 trans-15 and C18:1 cis-15, end products of an alternative biohydrogenation pathway of C18:3n-3. Triterpene saponins did not affect the fermentation pattern, the biohydrogenation pathways, or the extent of biohydrogenation. At the doses tested in this study, we could only show a direct relation between changes in the rumen fatty acid metabolism and the presence of cinnamaldehyde but not for eugenol, quercetin, or triterpene saponins.

Effect of induction of subacute ruminal acidosis on milk fat profile and rumen parameters

High-concentrate diets can lead to subacute ruminal acidosis and are known to result in changes of the ruminal fermentation pattern and mammary secretion of fatty acids. The objective of this paper is to describe modifications in milk fatty acid proportions, particularly odd- and branched-chain fatty acids and rumen biohydrogenation intermediates, associated with rumen parameters during a 6-wk subacute ruminal acidosis induction protocol with 12 ruminally fistulated multiparous cows. The protocol involved a weekly gradual replacement of a standard dairy concentrate with a wheat-based concentrate (610 g of wheat/kg of concentrate) during the first 5 wk and an increase in the total amount of concentrate in wk 6. Before the end of induction wk 6, cows were switched to a control diet because 7 cows showed signs of sickness. The pH was measured continuously by an indwelling pH probe. Milk and rumen samples were taken on d 2 and 7 of each week. Data were analyzed using a linear mixed model and by principal component analysis. A pH decrease occurred after the first concentrate switch but rumen parameters returned to the original values and remained stable until wk 5. In wk 5 and 6, rumen pH values were indicative of increasing acidotic conditions. After switching to the control diet in wk 6, rumen pH values rapidly achieved normal values. Odd- and branched-chain fatty acids and C18:1 trans-10 increased with increasing amount of concentrate in the diet, whereas C18:1 trans-11 decreased. Four fatty acids [C18:1 trans-10, C15:0 and C17:0+C17:1 cis-9 (negative loadings), and iso C14:0 (positive loading)] largely correlated with the first principal component (PC1), with cows spread along the PC1 axis. The first 4 wk of the induction experiment showed variation across the second principal component (PC2) only, with high loadings of anteiso C13:0 (negative loading) and C18:2 cis-9,trans-11 and C18:1 trans-11 (positive loadings). Weeks 5 and 6 deviated from PC2 and tended toward the negative PC1 axis. A discriminant analysis using a stepwise approach indicated the main fatty acids discriminating between the control and acidotic samples as iso C13:0, iso C16:0, and C18:2 cis-9,trans-11 rather than milk fat content or C18:1 trans-10, which have been used before as indicators of acidosis. This shows that specific milk fatty acids have potential in discriminating acidotic cases.

Butyricicoccus pullicaecorum gen. nov., sp. nov., an anaerobic, butyrate-producing bacterium isolated from the caecal content of a broiler chicken

Five isolates that produced large amounts of butyrate were obtained in the course of a study on the butyrate-producing microbiota from the caecal content of a 4-week-old broiler chicken. The five isolates were virtually indistinguishable in biochemical and genetic terms, suggesting that they were derived from a single bacterial clone colonizing this habitat. A phylogenetic analysis based on 16S rRNA gene sequences demonstrated that the five isolates represented a unique lineage within the Clostridium leptum subgroup of the clostridia, with Eubacterium desmolans as the closest phylogenetic neighbour (about 93 % similarity). These data indicate that the five novel isolates represent a single novel species within a novel genus, for which we propose the name Butyricicoccus pullicaecorum gen. nov., sp. nov. The type strain of Butyricicoccus pullicaecorum is 25-3(T) (=LMG 24109(T) =CCUG 55265(T)). The DNA G+C content of strain 25-3(T) was 54.5 mol% .

Effect of Lactation Stage on the Odd- and Branched-Chain Milk Fatty Acids of Dairy Cattle Under Grazing and Indoor Conditions

The pattern of odd- and branched-chain fatty acids (OBCFA) in milk fat reflects rumen microbial activity and proportions of different rumen microbial groups. Therefore, these milk fatty acids (FA) are used to predict rumen proportions of volatile fatty acids, duodenal flow of microbial protein, and occurrence of rumen acidosis. However, current models do not correct for the potential effects of lactation stage on the level of OBCFA in milk fat. Hence, the objectives of this study were 1) to describe progressive changes related to lactation stage in concentrations of milk FA, with emphasis on the OBCFA, using the incomplete gamma function of Wood, and 2) to analyze whether lactation curves of milk FA on the one hand and milk production or milk fat content on the other hand coincide through evaluation of the correlation between the parameters of the Wood functions fitted to individual animal data. Data were collected from 2 trials in which milk FA during lactation were monitored. The first experiment was a stable trial with 2 groups of 10 cows receiving 2 dietary treatments from wk 1 to 40 of lactation. The second experiment was a grazing trial with 9 cows that were followed during the first 18 wk of lactation. Lactation curves of milk production, milk fat content, and individual milk FA were developed using the incomplete gamma function of Wood for each of the 3 dietary strategies separately. For almost all of the milk FA, lactation curve shapes were similar for all 3 dietary treatments. The OBCFA with chain lengths of 14 and 15 carbon atoms followed the lactation curves of the short- and medium-chain milk FA, which increased in early lactation. The OBCFA with chain length of 17 carbon atoms decreased during the early lactation period, following the pattern of milk long-chain fatty acids. The short- and medium-chain milk FA and OBCFA in the early lactation period seemed to be negatively correlated with the starting milk production and milk fat content, but correlations were modest. Information of milk FA lactation curves should be incorporated in predictive and classification models based on these milk FA, to improve their performance.

Effect of dry period length and dietary energy source on energy balance, milk yield, and milk composition of dairy cows

The objective of this study was to evaluate the effects of dry period length and dietary energy source in early lactation on milk production, feed intake, and energy balance (EB) of dairy cows. Holstein-Friesian dairy cows (60 primiparous and 108 multiparous) were randomly assigned to dry period lengths (0, 30, or 60 d) and early lactation ration (glucogenic or lipogenic), resulting in a 3 × 2 factorial design. Rations were isocaloric and equal in intestinal digestible protein. The experimental period lasted from 8 wk prepartum to 14 wk postpartum and cows were monitored for milk yield, milk composition, dry matter intake (DMI), energy balance, and milk fat composition. Prepartum average milk yield for 60 d precalving was 13.8 and 7.7 ± 0.5 kg/d for cows with a 0- and 30-d dry period, respectively. Prepartum DMI and energy intake were greater for cows without a dry period and 30-d dry period, compared with cows with a 60-d dry period. Prepartum EB was greater for cows with a 60-d dry period. Postpartum average milk yield until wk 14 was lower for cows without a dry period and a 30-d dry period, compared with cows with a 60-d dry period (32.7, 38.7, and 43.3 ± 0.7 kg/d for 0-, 30-, and 60-d dry period, respectively). Postpartum DMI did not differ among treatments. Postpartum EB was greater for cows without a dry period and a 30-d dry period, compared with cows with a 60-d dry period. Young cows (parity 2) showed a stronger effect of omission of the dry period, compared with a 60-d dry period, on additional milk precalving (young cows: 15.1 kg/d; older cows: 12.0 kg/d), reduction in milk yield postcalving (young cows: 28.6 vs. 34.8 kg/d; older cows: 41.8 vs. 44.1 kg/d), and improvement of the EB postcalving (young cows: 120 vs. -93 kJ/kg(0.75)·d; older cows: -2 vs. -150 kJ/kg(0.75)·d. Ration did not affect milk yield and DMI, but a glucogenic ration tended to reduce milk fat content and increased EB, compared with a more lipogenic ration. Reduced dry period length (0 and 30 d) increased the proportion of short- and medium-chain fatty acids in milk fat and omitting the dry period decreased the proportion of long-chain fatty acids in milk fat. In conclusion, shortening and omitting the dry period shifts milk yield from the postpartum to the prepartum period; this results in an improvement of the EB in early lactation. An increased energy status after a short dry period can be further improved by feeding a more glucogenic ration in early lactation.

Short Communication: Elevated Concentrations of Oleic Acid and Long-Chain Fatty Acids in Milk Fat of Multiparous Subclinical Ketotic Cows

The objective of this study was to determine whether concentrations of specific fatty acids in milk fat are a candidate for the early detection of subclinical ketosis. The case study included multiparous cows fed a lipogenic diet or a mixed glucogenic:lipogenic diet during the first 9 wk of lactation. Milk fatty acid profiles of cows classified as healthy (n = 8) or as subclinically ketotic (n = 8) based on a blood plasma beta-hydroxybutyrate threshold concentration of 1.2 mmol/L were compared. Subclinically ketotic cows showed an elevated proportion of C18:1 cis-9 in milk fat during the whole registration period.