Summary
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.
When fractionation of meat lipids is not required, procedures such as saponification can be used to extract total fatty acids, reducing reliance on toxic organic compounds. However, saponification of ...muscle fatty acids is laborious, and requires extended heating times, and a second methylation step to convert the extracted fatty acids to fatty acid methyl esters prior to gas chromatography. Therefore the development of a more rapid direct methylation procedure would be of merit. The use of freeze-dried material for analysis is common and allows for greater homogenisation of the sample. The present study investigated the potential of using freeze-dried muscle samples and a direct bimethylation to analyse total fatty acids of meat (beef, chicken and lamb) in comparison with a saponification procedure followed by bimethylation. Both methods compared favourably for all major fatty acids measured. There was a minor difference in relation to the C18:1 trans 10 isomer with a greater (P<0.05) recovery with saponification. However, numerically the difference was small and likely as a result of approaching the limits of isomer identification by single column gas chromatography. Differences (P<0.001) between species were found for all fatty acids measured with no interaction effects. The described technique offers a simplified, quick and reliable alternative to saponification to analyse total fatty acids from muscle samples.
Although often investigated within the context of plant growth and development and/or seed composition, plant lipids have roles in responses to environment. To dissect changes in lipid and fatty acid ...composition linked to drought tolerance responses in oats, we performed a detailed profiling of (>90) different lipids classes during a time course of water stress. We used two oat cultivars, Flega and Patones previously characterized as susceptible and tolerant to drought, respectively. Significant differences in lipid classes (mono, di and triacylglycerols; respectively MAG, DAG, and TAG and free fatty acids FFA) and in their fatty acid (FA) composition was observed between cultivars upon drought stress. In Flega there was an increase of saturated FAs, in particular 16:0 in the DAG and TAG fractions. This led to significant lower values of the double bond index and polyunsaturated/saturated ratio in Flega compared with Patones. By contrast, Patones was characterized by the early induction of signaling-related lipids and fatty acids, such as DAGs and linolenic acid. Since the latter is a precursor of jasmonates, we investigated further changes of this signaling molecule. Targeted measurements of jasmonic acid (JA) and Ile-JA indicated early increases in the concentrations of these molecules in Patones upon drought stress whereas no changes were observed in Flega. Altogether, these data suggest a role for jasmonates and specific fatty acids in different lipid classes in coping with drought stress in oat.
Silages prepared from pure stands of ryegrass, alfalfa, white clover, and red clover over two successive year were offered to lactating dairy cows in two feeding experiments. Proportional mixtures of ...all cuts prepared in a yr were used to ensure that the forage treatments were representative of the crop. Additional treatments involved mixtures of grass silage with either white clover silage or red clover silage (50/50, on a DM basis). Silages were prepared in round bales, using a biological inoculant additive, and wilting for up to 48h. Although the legumes were less suited to silage-making than grass, because of their higher buffering capacity and lower water-soluble carbohydrate content, all silages were well-fermented. A standard concentrate was offered at a flat-rate (8 kg/d in yr 1, and 4 or 8 kg/d in yr 2). All of the legume silages led to higher DM intake and milk yields than for the grass silage, with little effect on milk composition. Intake and production responses to legumes were similar at the two levels of concentrate feeding and with forage mixtures they were intermediate to those for the separate forages. An additional benefit of the clover silages, particularly red clover silage, was the increase in levels of polyunsaturated fatty acids, particularly α-linolenic acid, in milk. Legume silages also led to a lower palmitic acid percentage in milk. The efficiency of conversion of feed N into milk N declined with increasing levels of legume silage. White clover silage led to a higher N-use efficiency when the effect of N intake level is taken into account.
Tocopherols are lipid-soluble antioxidants that contribute to plant resistance to abiotic stresses. However, it is still unknown to what extent alterations in tocopherol composition can affect the ...plant response to biotic stresses. The response to bacterial and fungal attack of the vte1 mutant of Arabidopsis thaliana, which lacks both α- and γ-tocopherol, was compared to that of the vte4 mutant (which lacks α- but accumulates γ-tocopherol) and the wild type (with accumulates α-tocopherol in leaves). Both mutants exhibited similar kinetics of cell death and resistance in response to Pseudomonas syringae. In contrast, both mutants exhibited delayed resistance when infected with Botrytis cinerea. Lipid and hormonal profiling was employed with the aim of assessing the underlying cause of this differential phenotype. Although an altered tocopherol composition in both mutants strongly influenced fatty acid composition, and strongly altered jasmonic acid and cytokinin contents upon infection with B. cinerea, differences between genotypes in these phytohormones were observed during late stages of infection only. By contrast, genotype-related effects on lipid peroxidation, as indicated by malondialdehyde accumulation, were observed early upon infection with B. cinerea. We conclude that an altered tocopherol composition in chloroplasts may negatively influence the plant response to biotic stress in Arabidopsis thaliana through changes in the membrane fatty acid composition, enhanced lipid peroxidation and delayed defence activation when challenged with B. cinerea.
•Vitamin E (vte) mutants exhibited reduced resistance when infected with Botrytis cinerea.•An altered vitamin E composition strongly influenced fatty acid accumulation.•Jasmonic acid biosynthesis was delayed in vte mutants to compromise resistance.•Cytokinin contents were also reduced in both vte mutants compared to the wild type upon fungal infection.
Polyphenol oxidase (PPO) activity in red clover (Trifolium pratense) has been reported to reduce both proteolysis and lipolysis, resulting in greater N use efficiency and protection of PUFA across ...the rumen. Although high levels of PPO have been reported in grasses such as cocksfoot (orchard grass; Dactylis glomerata), no in vivo research has determined whether grass PPO elicits the same response as red clover PPO. To test the hypothesis that silage ensiled from grass with high levels of PPO protects N and PUFA across the rumen, 6 steers with ruminal and duodenal cannulas were offered cocksfoot silage (CO; high-PPO grass), perennial ryegrass silage (PR; Lolium perenne; low-PPO grass), or red clover silage (RC; high-PPO control) at 16 g DM/kg BW daily with the experiment consisting of two 3 × 3 Latin squares with 21-d periods, consisting of 12 d of diet adaptation, 6 d of duodenal marker infusion, 2 d of duodenal sampling, and 1 d of ruminal sampling. All silages were well preserved, with DM of 34.4, 55.3, and 45.4% for CO, PR, and RC. Activity of PPO in silages was low due to deactivation but was greater in CO than either PR or RC (0.15 vs. 0.05 and 0.08 μkatal/g DM). Protein-bound phenol (mg/g DM) as a measure of the degree of oxidation and an indication of PPO protection was greatest for RC (15.9) but comparable for PR (10.1) and CO (12.2). Biohydrogenation of C18 PUFA was significantly lower on RC compared to the 2 grass silages with CO greater than PR. Despite lower levels of total fatty acid intake and subsequent duodenal flow, CO resulted in greater levels of phytanic acid and total branched and odd chain fatty acids in duodenal digesta than RC or PR. Ruminal ammonia concentration was greatest for RC, with no difference between the grasses. Duodenal flow of microbial N and efficiency of microbial protein synthesis were lowest for CO and comparable for RC and PR. The CO (high-grass PPO) did not result in elevated levels of C18 PUFA escaping the rumen or improve efficiency of total N transfer through the rumen compared to PR. The RC resulted in a greater flow of N and nonmicrobial N to the duodenum than the 2 grasses with PR greater than CO.
Polyphenol oxidase (PPO) in red clover (RC) has been shown to reduce both lipolysis and proteolysis in silo and implicated (in vitro) in the rumen. However, all in vivo comparisons have compared RC ...with other forages, typically with lower levels of PPO, which brings in other confounding factors as to the cause for the greater protection of dietary nitrogen (N) and C18 polyunsaturated fatty acids (PUFA) on RC silage. This study compared two RC silages which when ensiled had contrasting PPO activities (RC+ and RC−) against a control of perennial ryegrass silage (PRG) to ascertain the effect of PPO activity on dietary N digestibility and PUFA biohydrogenation. Two studies were performed the first to investigate rumen and duodenal flow with six Hereford×Friesian steers, prepared with rumen and duodenal cannulae, and the second investigating whole tract N balance using six Holstein-Friesian non-lactating dairy cows. All diets were offered at a restricted level based on animal live weight with each experiment consisting of two 3×3 Latin squares using big bale silages ensiled in 2010 and 2011, respectively. For the first experiment digesta flow at the duodenum was estimated using a dual-phase marker system with ytterbium acetate and chromium ethylenediaminetetraacetic acid as particulate and liquid phase markers, respectively. Total N intake was higher on the RC silages in both experiments and higher on RC− than RC+. Rumen ammonia-N reflected intake with ammonia-N per unit of N intake lower on RC+ than RC−. Microbial N duodenal flow was comparable across all silage diets with non-microbial N higher on RC than the PRG with no difference between RC+ and RC−, even when reported on a N intake basis. C18 PUFA biohydrogenation was lower on RC silage diets than PRG but with no difference between RC+ and RC−. The N balance trial showed a greater retention of N on RC+ over RC−; however, this response is likely related to the difference in N intake over any PPO driven protection. The lack of difference between RC silages, despite contrasting levels of PPO, may reflect a similar level of protein-bound-phenol complexing determined in each RC silage. Previously this complexing has been associated with PPOs protection mechanism; however, this study has shown that protection is not related to total PPO activity.
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×Friesian dairy cows in mid-lactation were allocated at random to 1 of 3 dietary treatments in a 3×3 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 (18.8kg/d and 550g/d, respectively). Milk yields and total fatty acid content were similar across treatments (32.6kg/d and 34.8mg/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.
Aims
In this experiment, we investigated the effect of ‘green odour’ products typical of those released from fresh forage postabiotic and biotic stresses on the rumen microbiota and lipid metabolism.
...Methods and Results
Hydroperoxyoctadecatrienoic acid (HP), a combination of salicylic and jasmonic acid (T), and a combination of both (HPT) were incubated in vitro in the presence of freeze‐dried ground silage and rumen fluid, under rumen‐like conditions. 16S rRNA (16S cDNA) HaeIII‐based terminal restriction fragment length polymorphism‐based (T‐RFLP) dendrograms, canonical analysis of principal coordinates graphs, peak number and Shanon‐Weiner diversity indices show that HP, T and HPT likely had antimicrobial effects on the microbiota compared to control incubations. Following 6 h of in vitro incubation, 15·3% of 18:3n‐3 and 4·4% of 18:2n‐6 was biohydrogenated in control incubations, compared with 1·3, 9·4 and 8·3% of 18:3n‐3 for HP, T and HPT treatments, respectively, with negligible 18:2n‐6 biohydrogenation seen. T‐RFLP peaks lost due to application of HP, T and HPT likely belonged to as yet uncultured bacteria within numerous genera.
Conclusions
Hydroperoxyoctadecatrienoic acid, T and HPT released due to plant stress potentially have an antimicrobial effect on the rumen microbiota, which may explain the decreased biohydrogenation in vitro.
Significance and Impact of the Study
These data suggest that these volatile chemicals may be responsible for the higher summer n‐3 content of bovine milk.
Polyphenol oxidase (PPO) has been shown to reduce proteolysis and lipolysis in red clover through deactivation of proteolytic and lipolytic enzymes and/or through formation of protein–phenol–lipid ...complexes. This experiment investigated the time course of both lipolysis and proteolysis in two red clover lines with different PPO activities with and without addition of a silage inoculant to help understand the action of PPO in the silo, and its potential effects on protein and glycerol-based lipid conservation, and to determine effects of a more rapid pH reduction with inoculation on PPO activity. Four silages were prepared from high or low PPO precision chopped red clover in 60 test-tube-silos, each containing 120
g fresh weight: (a) high PPO red clover without inoculation (H−), (b) low PPO red clover without inoculation (L−), (c) high PPO red clover with inoculation (H+), and (d) low PPO red clover with inoculation (L+). Each treatment had three replicates for each time point of 1, 2, 4, 8 and 90 days. The inoculant used was
Lactobacillus plantarum strain L54 applied at a rate of 10
6
CFU/g fresh weight. Silage pH was reduced (
P
<
0.001) by inoculation with no effect of PPO. Inoculation had no effect on either lipolysis or free amino acid release, although more (
P
<
0.01) soluble protein and less (
P
<
0.01) ammonia-N was in inoculated silages. H silages had a lower level of both proteolysis (release of free amino acids,
P
<
0.05) and lipolysis (loss of membrane lipid,
P
<
0.01) than L red clover silages. Results indicate that PPO reduced proteolysis and lipolysis in the silo and that inoculation had no adverse effects on PPO activity.
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