Michael R. F. Lee

As yet uncultured bacteria phylogenetically classified as Prevotella, Lachnospiraceae incertae sedis and unclassified Bacteroidales, Clostridiales and Ruminococcaceae may play a predominant role in ruminal biohydrogenation

Microbial biohydrogenation of dietary poly-unsaturated fatty acids (PUFA) to saturated fatty acids (SFA) in the rumen results in the high ratio of SFA/PUFA in ruminant products, such as meat and milk. In vitro, Butyrivibrio proteoclasticus-related bacteria extensively biohydrogenate PUFA to SFA, yet their contribution in the rumen has not been confirmed. The aim of this study was to evaluate the role of Butyrivibrio proteoclasticus group bacteria in ruminal biohydrogenation and to assess the possible role of other bacteria. Fish oil at 0%, 1.5% and 3% dry matter intake was fed to eight Holstein × Friesian steers, in order to elicit changes in the extent of PUFA biohydrogenation. Fatty acid and B. proteoclasticus group 16S rRNA concentrations in rumen digesta were determined. Correlation between digesta 18:0 concentration and B. proteoclasticus group 16S rRNA concentration was low. Terminal restriction fragment length polymorphism and denaturing gradient gel electrophoresis (DGGE) coupled with multivariate statistics revealed that many terminal restriction fragments (T-RFs) and DGGE bands were linked to cis-9, trans-11 conjugated linoleic acid (CLA), 18:1 trans-11 and 18:0 ruminal concentrations. MiCA T-RF predictive identification software showed that these linked T-RFs were likely to originate from as yet uncultured bacteria classified as Prevotella, Lachnospiraceae incertae sedis, and unclassified Bacteroidales, Clostridiales and Ruminococcaceae. Sequencing of linked DGGE bands also revealed that as yet uncultured bacteria classified as Prevotella, Anaerovoax (member of the Lachnospiraceae incertae sedis family), and unclassified Clostridiales and Ruminococcaceae may play a role in biohydrogenation.

The effect of clover silages on long chain fatty acid rumen transformations and digestion in beef steers

Lee, M. R. F., Harris, L. J., Dewhurst, R. J., Merry, R., Scollan, N. D. (2003). The effect of clover silages on long chain fatty acid rumen transformations and digestion in beef steers. Animal Science, 76, (3), 491-501.

Forage type and fish oil cause shifts in rumen bacterial diversity

Despite evidence supporting improved incorporation of beneficial polyunsaturated fatty acids (PUFA) into ruminant products, such as meat and milk, following red clover and fish oil (FO) inclusion in the ruminant diet, little is known regarding the concomitant bacterial diversity. We evaluated the effects of feeding grass vs. red clover silage with incremental FO inclusion on known lipolytic, biohydrogenating, cellulolytic and proteolytic rumen bacterial communities of steers. Following 14 days of dietary adaptation, liquid-associated (LAB) and solid-associated (SAB) bacterial communities were harvested, DNA extracted and bacterial denaturing gradient gel electrophoresis (DGGE) and specific-bacterial quantitative PCR (QPCR) were undertaken. DGGE-derived dendrograms showed that diet caused the greatest change in LAB and SAB bacterial diversity, with FO inclusion at the 2% and 3% dry matter intake also causing some changes. QPCR revealed that diet resulted in changes in the DNA concentration of Anaerovibrio lipolytica, the Butyrivibrio proteoclasticus group, Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens. FO inclusion caused changes in A. lipolytica, F. succinogenes and R. flavefaciens DNA concentration only. In the B. proteoclasticus group, which are the only known bacteria with the capacity to biohydrogenate PUFA to 18:0, DNA concentration did not correlate to 18:0 flow to the duodenum, however, suggesting that other bacteria may play a role in biohydrogenation. A greater understanding of microbial changes that accompany beneficial dietary changes will lead to novel strategies to improve ruminant product quality.

The effects of fish oil supplementation on rumen metabolism and the biohydrogenation of unsaturated fatty acids in beef steers given diets containing sunflower oil

Lee, M. R. F., Tweed, J. K. S., Moloney, A. P., Scollan, N. D. (2005). The effect of fish oil supplementation on rumen metabolism and the biohydrogenation of unsaturated fatty acids in beef steers given diets containing sunflower oil. Animal Science, 80, (3), 361-367

A comparison between red clover silage and grass silage feeding on fatty acid composition, meat stability and sensory quality of the M. Longissimus muscle of dairy cull cows

Sixteen Holstein-Friesian dairy cull cows were offered either ad libitum grass (G) or red clover (RC) silage for 12 weeks. Dry matter (DM), total nitrogen, and pH was higher for the RC than the G silage and organic matter, water-soluble carbohydrate, fibre, DM digestibility, ammonia-N, vitamin E and acetic acid higher for the G silage (P<0.05). Fatty acid compositions were different (P<0.05) with G silage having higher levels of C12:0, C14:0, C16:1cis-9, C18:3n-3 and total fatty acids whereas RC had higher levels of C18:0, C18:2n-6 and C20:0. Daily liveweight gain was high and not different between groups (average 1.22 kg/d). Body condition score and back fat thickness at slaughter along with conformation, fat grade and slaughter weight were not different between groups. Animals offered the G silage produced larger M. longissimus length (P<0.01) and a trend (P<0.1) for width. RC fed animals had higher proportions of C18:3n-3 (P<0.001), total n-3 fatty acids (P<0.01) and total PUFA compared to animals offered the G silage despite greater intakes of these fatty acids on G (P<0.001). Vitamin E concentration and stability of aged meat during simulated retail display, were lower (P<0.05) from animals offered RC than animals offered G. Shear force and ultimate pH, however were not different in steaks and sensory attributes were similar; the only difference being a higher score (P<0.01) for fishy in the RC steaks. The results suggest that feeding high DMD silage to dairy cull cows can result in a high standard of finish and liveweight gain. Furthermore RC silage as opposed to G silage can increase the beneficial fatty acid profile of the resultant meat.

Latent and Active Polyphenol Oxidase (PPO) in Red Clover (Trifolium pratense) and Use of a Low PPO Mutant To Study the Role of PPO in Proteolysis Reduction

Polyphenol oxidase (PPO) activity in leaf extracts of wild type (WT) red clover and a mutant line expressing greatly reduced levels of PPO (LP red clover) has been characterized. Both latent and active forms of PPO were present, with the latent being the predominant form. PPO enzyme and substrate (phaselic acid) levels fluctuated over a growing season and were not correlated. Protease activation of latent PPO was demonstrated; however, the rate was too low to have an immediate effect following extraction. A novel, more rapid PPO activation mechanism by the enzymes own substrate was identified. Rates of protein breakdown and amino acid release were significantly higher in LP red clover extracts compared with WT extracts, with 20 versus 6% breakdown of total protein and 1.9 versus 0.4 mg/g FW of free amino acids released over 24 h, respectively. Inclusion of ascorbic acid increased the extent of protein breakdown. Free phenol content decreased during a 24 h incubation of WT red clover extracts, whereas protein-bound phenol increased and high molecular weight protein species were formed. Inhibition of proteolysis occurred during wilting and ensilage of WT compared with LP forage (1.9 vs 5 and 17 vs 21 g/kg of DM free amino acids for 24 h wilted forage and 90 day silage, respectively). This study shows that whereas constitutive red clover PPO occurs predominantly in the latent form, this fraction can contribute to reducing protein breakdown in crude extracts and during ensilage.

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.

Effect of fish oil on ruminal biohydrogenation of C18 unsaturated fatty acids in steers fed grass or red clover silages

Red clover and fish oil (FO) are known to alter ruminal lipid biohydrogenation leading to an increase in the polyunsaturated fatty acid (PUFA) and conjugated linoleic acid (CLA) content of ruminant-derived foods, respectively. The potential to exploit these beneficial effects were examined using eight Hereford × Friesian steers fitted with rumen and duodenal cannulae. Treatments consisted of grass silage or red clover silage fed at 90% of ad libitum intake and FO supplementation at 0, 10, 20 or 30 g/kg diet dry matter (DM). The experiment was conducted with two animals per FO level and treatments formed extra-period Latin squares. Flows of fatty acids at the duodenum were assessed using ytterbium acetate and chromium ethylene diamine tetra-acetic acid as indigestible markers. Intakes of DM were higher (P < 0.001) for red clover silage than grass silage (5.98 v. 5.09 kg/day). There was a linear interaction effect (P = 0.004) to FO with a reduction in DM intake in steers fed red clover silage supplemented with 30 g FO/kg diet DM. Apparent ruminal biohydrogenation of C18:2n-6 and C18:3n-3 were lower (P < 0.001) for red clover silage than grass silage (0.83 and 0.79 v. 0.87 and 0.87, respectively), whilst FO increased the extent of biohydrogenation on both diets. Ruminal biohydrogenation of C20:5n-3 and C22:6n-3 was extensive on both silage diets, averaging 0.94 and 0.97, respectively. Inclusion of FO in the diet enhanced the flow of total CLA leaving the rumen with an average across silages of 0.22, 0.31, 0.41 and 0.44 g/day for 0, 10, 20 or 30 g FO/kg, respectively, with a linear interaction effect between the two silages (P = 0.03). FO also showed a dose-dependent increase in the flow of trans-C18:1 intermediates at the duodenum from 4.6 to 15.0 g/day on grass silage and from 9.4 to 22.5 g/day for red clover silage. Concentrations of trans-C18:1 with double bonds from Δ4-16 in duodenal digesta were all elevated in response to FO in both diets, with trans-11 being the predominant isomer. FO inhibited the complete biohydrogenation of dietary PUFA on both diets, whilst red clover increased the flow of C18:2n-6 and C18:3n-3 compared with grass silage. In conclusion, supplementing red clover silage-based diets with FO represents a novel nutritional strategy for enhancing the concentrations of beneficial fatty acids in ruminant milk and meat.

Effect of feeding fresh or conditioned red clover on milk fatty acids and nitrogen utilization in lactating dairy cows

Polyphenol oxidase (PPO) in conditioned red clover (ensiled or cut and crushed) reduces both proteolysis and lipolysis in the herbage, which has led to increases in N use efficiency and polyunsaturated fatty acid (PUFA) content of milk when offered to dairy cows. In damaged plant cells, PPO is activated and binds protein through the formation of protein-bound phenols. This study investigated a) whether freshly cut red clover could increase N use efficiency and milk PUFA concentrations in dairy cows or whether PPO enzymes require prior activation before feeding to elicit a response, and b) apparent whole-tract amino acid digestibility to help determine the effect of PPO on amino acid utilization. Six multiparous Holstein x Friesian dairy cows in mid-lactation were allocated at random to 1 of 3 dietary treatments in a 3 x 3 Latin square: a control treatment of grass (low PPO, G); red clover (high PPO, RC), and conditioned red clover (high fully activated PPO, CRC). The CRC herbage was cut and chopped in the field and then transported with the G and RC herbages to the animal house. Each period consisted of a 2-wk adaptation to diet and a week of measuring dietary effects (N balance and milk collection). The PPO activity was greatest in the RC treatment as fed, whereas activation of latent PPO enzyme and protein-bound phenol levels were greatest in the CRC diet. Dry matter and total fatty acid intakes were comparable across treatments (18.8 kg/d and 550 g/d, respectively). Milk yields and total fatty acid content were similar across treatments (32.6 kg/d and 34.8 mg/mL, respectively). Cows offered either RC or CRC had greater levels of protein, C18 PUFA and total long-chain PUFA in their milk than animals offered grass with no difference between RC and CRC. Nitrogen intakes, and output in milk, urine, and feces were greater in cows offered the 2 red clover treatments than G, with no difference between RC and CRC. However, there were no differences in N use efficiency among diets as measured by the proportion of feed N converted into milk N, possibly as the result of the excessive supply of N with the red clover diets. Amino acid apparent whole-tract digestibilities were greater when on RC than G diets and intermediate when on CRC for all amino acids, with the exception of Met, which was reduced in cows on both red clover diets compared with G. It is proposed that the PPO trait could show more benefit to ruminants if red clover was fed in combination with lesser N-containing forages or if red clover was bred to contain less N.

The influence of plant polyphenols on lipolysis and biohydrogenation in dried forages at different phenological stages: in vitro study

BACKGROUND It is known that forage legumes show a higher transfer efficiency of polyunsaturated fatty acids (PUFA) to ruminant dairy products in comparison with grasses. Legumes are usually characterised by moderate levels of plant secondary metabolites, which can have an effect on lipolysis and biohydrogenation in the rumen. An in vitro study was carried out to compare two species with different plant phenol compositions, Vicia sativa (VS, common vetch, cv. Jose) and Trifolium incarnatum (TI, crimson clover, cv. Viterbo) cut at the vegetative (Veg) and reproductive (Rep) stages, on lipolysis and PUFA biohydrogenation in the rumen. RESULTS The study showed that forage species and phenological stage affected the levels of bound phenols (BP) and tannic polyphenols (TP). VS was characterised by a higher level of TP than TI at both Veg and Rep stages, whereas BP levels were low in both forages. BP and TP had a negative effect on lipolysis and biohydrogenation, but TP showed a greater negative correlation than BP for both forages. CONCLUSION These results showed that lipolysis and biohydrogenation of PUFA could be affected by plant phenols, particularly TP.