Milk odd- and branched-chain fatty acids (OBCFA) have been suggested as potential biomarkers for rumen function. The potential of milk OBCFA as a biomarker depends on whether their profile reflects the profile observed in the duodenum. The objective of this study was to evaluate whether the OBCFA profile in duodenum samples is reflected in plasma and milk. For this, 2 dairy cattle experiments were used. In experiment 1, 4 Holstein cows fitted with rumen and proximal duodenum cannulas were used in a 4×4 Latin square design. The treatments consisted of 2 nitrogen levels (143 vs. 110g of crude protein/kg of dry matter for high and low N, respectively) combined with either 1 of the 2 energy sources (i.e., starch from barley, corn, and wheat or fiber from soybean hulls and dehydrated beet pulp). In experiment 2, 4 Holstein cows fitted with rumen and proximal duodenum cannulas were used in a 3×3 Latin square design, with the treatments consisting of 3 diets: (1) RNB-, a diet with a crude protein content of 122g/kg of dry matter, predicted to provide protein digested in the small intestine according to the requirement of the animals, but with a shortage of rumen degradable protein; (2) RNB- to which 6g/d of niacin was added through inclusion in the mineral and vitamin premix, and (3) RNB- to which urea was added to balance rumen degradable N supply resulting in a CP content of 156g/kg of dry matter. In both experiments, samples of duodenal digesta, plasma, and milk were collected and analyzed for fatty acids. Additionally, lipids in plasma samples were separated in lipid classes and analyzed for fatty acids. The OBCFA profile in milk was enriched in 15:0, iso-17:0, anteiso-17:0, and cis-9-17:1 as compared with duodenal samples, and milk secretions even exceeded duodenal flows, which suggests occurrence of postruminal synthesis, such as de novo synthesis, desaturation, and elongation. The postruminal modification of the OBCFA profile might hamper the application of OBCFA as diagnostic tools of rumen function.

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Polyunsaturated fatty acids given to ruminants are to a large extent hydrogenated to more saturated forms by microbial metabolism. Numerous protection technologies have been developed to overcome this hydrogenation process in order to increase the amount of unsaturated fatty acids bypassing the rumen and resulting in an effective transfer to the peripheral tissues. This review gives an overview of the current state of the art in rumen lipid bypass technologies, with the focus on both patent-described protection mechanisms, possible advantages or drawbacks of the technologies, and protection results being described in recent scientific literature. Lipid bypass techniques which are dealt with include calcium salts, fatty acyl amides, aldehyde treatment, non-enzymatic browning, lipid composite gels, and encapsulation within lipids. Further, the potential of a novel rumen lipid protection technology, based on interfacial cross-linking of emulsions, is explored. Therefore, an overview is given on current knowledge of different types of enzymatically induced cross-linking of protein at emulsion interfaces, both for existing food and possible ruminal bypass applications. Practical applications: Both from a human and animal health care as well as from a resource-saving and economic perspective, protection of PUFA from ruminal BH is of interest. Protection, release, and absorption principle of a lipophilic rumen bypass product: As polyunsaturated fatty acids (P) are hydrogenated to more saturated fatty acids (S) upon ruminal passage (panel A), P sources administered to ruminants have to be protected against microbial turnover to transfer them to the peripheral tissues (panel B)

Most often, farmers consider red clover an unattractive forage because of its low ensilability. Nevertheless, several in vivo and in vitro experiments also showed advantages of red clover silages such as decreased rumen biohydrogenation of polyunsaturated fatty acids. This has been attributed to a possible protective role of protein-bound phenols, with polyphenol oxidase playing a key role in their formation. This enzyme is active in red clover, but not in other green forages, such as, for example, perennial ryegrass. Therefore, the aim was to study the lipid metabolism within red clover/ryegrass mixtures in lab scale silages and during in vitro rumen batch incubations. Ensilability of red clover increased with higher proportions of ryegrass in the silage mixture. However, the lipid-protecting mechanism of red clover does not seem to occur in the co-ensiled ryegrass as lipolysis of polar lipids linearly increased with increasing proportions of ryegrass (86.0%, 91.6%, 89.9%, 93.1% and 95.6% in 60-day-old silages with 100/0, 75/25, 50/50, 25/75 and 0/100 red clover/ryegrass, respectively). Rumen lipolysis and biohydrogenation of C18:3n-3 and C18:2n-6 were negatively related to red clover proportions in the silage mixtures. The lipid-protective mechanism in red clover silages is confirmed, but it seems not to be transferred to lipids in co-ensiled forages.Keywords: lipid metabolism, red clover, in vitro rumen, silage ImplicationsLipids in red clover silages are less degraded as compared with lipids in ryegrass silages, which have been attributed to a lipid-protective mechanism in red clover. This implicates that less oilseeds would be required to reach milk with a healthier fatty acid profile when feeding red clover instead of ryegrass silages. However, the lipid protection is not transferred to grass lipids in mixed grass-red clover silages.

Although forage lipid is generally rich in polyunsaturated fatty acids (PUFA), recovery of these fatty acids (FA) in milk and meat of ruminant origin is generally low, due to microbial biohydrogenation (BH) taking place in the rumen. Since lipolysis is a prerequisite for BH, the latter process is expected to be enhanced when (conserved) forages contain lower levels of esterified FA (particularly polar lipids; PL). However, this was not observed in former studies with red clover (Trifolium pratense L.). Furthermore, red clover inclusion in the herbivore's diet was associated with decreased rumen BH as compared with other forages. Differences in plant lipase activity during wilting and ensiling has been attributed to changes in disappearance from the PL fraction, but a potential role of microbial lipases in silo has not yet been elucidated. Therefore, the aims of the present study were to assess whether BH of red clover FA is linked with PL levels of the (conserved) starting material and to clarify the possible role of in silo microbial activity on PL disappearance. In order to obtain sufficient variation in forage PL and microbial activity, laboratory-scale silages were made by wilting and ensiling damaged or undamaged red clover using molasses or formic acid as ensiling additive, while perennial ryegrass (Lolium perenne L.) was used as a control. Distribution of lipids within three lipid fractions (PL, free FA and neutral lipids) in forages was determined and BH calculated after 24 h in vitro rumen incubation. Results indicated microbial lipases in silages did not enhance FA disappearance from the PL fraction. A gradual decrease of FA in the PL fraction upon conservation was found, both in red clover and ryegrass, irrespective of the degree of damage. In red clover PL losses started from the wilting phase, while substantial PL disappearance from ryegrass only started upon ensiling. Proportions of PUFA remaining in the PL fraction after wilting and ensiling of red clover were positively correlated with PUFA BH, while this effect was not observed for ryegrass. Red clover PUFA seemed to be partially protected against ruminal BH, while disappearance of FA from the PL fraction did not seem to be hampered. Results indicated the encapsulation mechanism as a consequence of protein-bound phenol formation induced by polyphenol oxidase is still the most probable hypothesis to explain red clover's increased flow of PUFA across the rumen