1. Lipid Metabolism In Ruminants G. R. Ghorbani

2. Overview Herbivores diets are normally quiet low in lipid because of the small quantity (2-5%) contained in most plant food sources. These dietary characteristics have required both metabolic adaptations and methods for conserving essential fatty acids (EFA). Plant lipids are altered extensively by the rumen fermentation, and the lipid actually received and absorbed by the animal differs from that ingested.

3. Overview The rumen is intolerant to high levels of fat, which may upset the fermentation. This situation in functioning ruminant contrast with that in the newborn ruminant, which ingests milk at about 30% or more fat in the DM, representing 50% or more of its caloric intake.

4. Overview In most metabolic systems FA’s are derived from glucose. Dietarily derived glucose is scarce in ruminant metabolism, however and ruminants have evolved mechanisms for its conservation, the most important of which is the lack of pathways for converting glucose into FA’s.

5. Overview About 90% of fat synthesis in ruminants occurs in the adipose tissue. The liver, which is the major lipogenesis site in many non-ruminants species, accounts for only 5% in ruminants.

7. Plant Lipids Lipids can be grouped into storage compounds in seeds (TG), leaf lipids (galactolipids and phospholipids), and a miscellaneous assortment of waxes, carotenoids, chlorophyll, essential oils and other ether-soluble substances. TG are negligible in forages. Leaf lipids are mainly galactolipids involving glycerol, galactose, and unsaturated FA’s.

8. Plant Lipids The leaf lipids are generally more polar than TG’s and have a lower energy value than would be estimated by the 2.25 factor used to calculate TDN. The FA’s associated with GL and many of the TG’s of the seed organs are relatively unsaturated and contain high amounts of linoleic and linolenic acids (Table 1).

9. Table-1. content and composition of EE from forage leaves % Of DM% of EE Ether extract5.3100 Fatty acids2.343 Non fatty acid Wax0.917 Chlorophyll0.234 Galactose0.418 unsaponifable fat 1.019

11. Triacylglycerol Triglycerides R-COO-CH 2 R-COO-CH R-COO-CH 2 Triglycerides found in seeds and animal adipose. Diglycerides found in plant leaves, one fatty acid is replaced by a sugar (galactose).

12. Triglyceride Containing Linoleic Acid Omega-6

13. Linolenic Acid Omega-3

14. Fatty Acid Isomers

15. Lipolysis Shortly after esterified plant lipids are consumed, they are hydrolyzed extensively by microbial lipases, causing the release of constituent FA’s. Anaerovibrio lipolytica, which is best known for its lipase activity, produces a cell bound esterase and a lipase. The lipase is an extracellular enzyme packaged in membranous particles composed of protein, lipid, and nucleic acid.

16. Lipolysis + 3H 2 0 + Esterified Plant Lipid Lipases Free Fatty Acids

17. Lipid Digestion Rumen DigalDiglyMonogalDigly Galactose PropionateDiglyceride Glycerol TriglyerideFatty acids Saturated FA CaFACa ++ Feed particles  -galactosidase  -galactosidase Lipase Anaerovibrio lipolytica H + Reductases Lipase

18. Lipolysis The lipase hydrolyzes acylglycerols completely to FFA, glycerol and galactose with little accumulation of mono or diglycerides. Glycerol and galactose are fermented rapidly, yielding propionic and butyrate acid as a major end product. Despite its high lipase activity, the general esterase activity in A.lipolytica is lower than in many non lipolytic bacteria.

19. Hydrolysis of lipids in the rumen (Bath & Hill 1969) Total Lipid %TGDGMGPLFFA Diet*6.372.413.71.71.211 Rumen digesta, 0 h5.1.1.0.115.285 Rumen digesta, 1 h6.230.4 1.7.0 7.161 Rumen digesta, 5 h6.411.1.0.012.476 *Diet consisted of 1 kg chopped hay + 50 g of palm oil % of Total Lipid

20. Lipolysis Fay et al identified 74 strains of ruminal bacteria that were capable of hydrolyzing the ester bond. Known lipolytic strains, including A lipolytic, and Butyrivibrio fibrosolvens, had low hydrolysis in that assay. Also, bacteria with general esterase activity are not necessarily capable of hydrolyzing lipid esters. Hespell and O’Bryan-shah found a wide variety of ruminal bacteria with esterase activity, including 30 strains of B. fibrosolvens, but only a few bacteria could hydrolyze long chain fatty acids (LCFA)

21. Lipolysis The extent of hydrolysis is very high for most unprotected lipids: 85-95%. This % is higher for diets rich in fats than for conventional diets, in which most lipids are in cellular structures. Hydrolysis seems to be highest for diets rich in protein.

22. Lipolysis Gerson et al. have shown that lipase was more active with diets rich in fiber than for diets rich in starch, but that a short-term supply of starch in a fiber diet could increase lipolysis. This suggests either that the rate of lipolysis could depend on the microbial ecosystem, or that variations of ruminal pH control lipase activity.

25. Lipolysis Protozoa are not involved to any great extent in hydrolysis, except for that of phospholipids. Salivary lipase present in ruminants has a very low activity, whereas in monogastric animals it plays a more important role

26. Biohydrogenation Unsaturated FFA have relatively short half lives in ruminal contents because they are rapidly hydrogenated by microbes to more saturated end products. The initial step in biohydrogenation (BH) is an isomerization reaction that converts the cis- 12 double bond in unsaturated FA’s to a trans-11 isomer.

27. Biohydrogenation Reduction of double bonds Result: fatty acids that are more saturated with hydrogen Saturated Unsaturated

28. Linolenic Acid Omega-3

29. Hydrogenation of Fatty Acids in the Rumen Polyunsaturated fatty acids (all cis) Isomerase (from bacteria) Needs free carboxyl group and diene double bond Shift of one double bond (cis & trans) Hydrogenation Hydrases (from bacteria, Hydrogenated fatty acid mostly cellulolytic) (stearic and palmitate)

30. Hydrogenation of Fatty Acids in the Rumen All unsaturated fatty acids can be hydrogenated Monounsaturated less than polyunsaturated 65 to 96% hydrogenation Numerous isomers are produced Biohydrogenation is greater when high forage diets fed Linoleic acid depresses hydrogenation of FA

31. Biohydrogenation The isomerase is not functional unless the FA has a free carboxyl group, and in the case of PUFA;s such as C18:2, a cis-9, cis-12 diene double bond configuration is present. The requirement of a free carboxyl group establishes lipolysis as a prerequisite for biohydrogenation.

32. Biohydrogenation Once the trans-11 bond is formed by action of the isomerase, then hydrogenation of the cis-9 bond in C18:2 occurs by a microbial reductase. The extent to which trans-11 C18:! Is hydrogenated to C18:0 depends on conditions in the rumen. For example, complete hydrogenation to stearic acid is promoted by the presence of cell-free ruminal fluid and feed particles, but it is inhibited irreversible by large amounts of linoleic acid.

33. Biohydrogenation Linolenic acid is often completely hydrogenated in stearic acid. The hydrogenation of linleic acid is not complete.. It provides stearic acid and different monounsaturated isomers, of which trans- vaccenic acid is characteristic of ruminal metabolism

34. Biohydrogenation Time (h) 18:2 converted (%) (adapted from Harfoot et al., 1973)

38. Conjugated Linoleic Acid - Rumen Most Common Pathway (High Roughage) Linoleic acid (cis-9, cis-12-18:2) Conjugated linoleic acid (CLA, cis-9, trans-11- 18:2) Vaccenic acid (Trans-11-18:1) Stearic acid (18:0) Cis-9, trans-12 isomerase Butyrivibrio fibrosolvens At low rumen pH, trans-10, cis-12 isomer of CLA is produced.

41. CLA absorbed from the intestines available for incorporation into tissue tryglycerides. Reactions from linoleic acid to vaccinic acid occur at a faster rate than from vaccinic acid to stearic acid. Therefore, vaccinic acid accumulates in the rumen and passes into intestines where it is absorbed. Quantities of vaccinic acid leaving the rumen several fold greater than CLA.

42. Conversion of Vaccinic Acid to CLA In mammary gland and adipose Trans-11-18:1 CLA, cis-9, trans-11 18:2 Stearoyl CoA Desaturase ‘  9 -desaturase’ This reaction probably major source of CLA in milk and tissues from ruminants. Also transforms PalmiticPalmitoleic StearicOleic

43. CLA Isomers - Rumen (High Concentrate) Low Rumen pH Linoleic acid (cis-9, cis-12-18:2) Cis-9, trans-10 isomerase CLA Isomer (trans-10, Cis-12-18:2) This isomer is inhibitory to milk fat synthesis. Trans-10-18:1

44. Effect of CLA isomers on milk fat % 1.5 2 2.5 3 3.5 -212345678 Day Milk Fat, percentage c/t 10,12 CLA c/t 9,11 CLA Control Infusion Baumgard et al. (2000)

49. Potential Value of CLA in Foods of Ruminant Origin Anticarcinogenic effects in lab animals given chemicals to cause cancer Reduce atherosclerosis Direct evidence with rabbits Indirect evidence with humans Reduce fat accumulation in the body Laboratory animals and pigs Evidence not conclusive with humans

51. CLA Content of Foods CLA isomers cis 9, trans 11 Foodmg/g fat% Beef4.385 Pork0.682 Chicken0.984 Milk5.592 Colby cheese6.192 Corn oil0.239

53. Partially Hydrogenated Vegetable Oil (PHVO)

62. Microbial Fatty Acid Synthesis Total lipid content of bacterial dry mass in the rumen ranges from 10 to 15%. Bacterial lipids originate from exogenous sources (uptake of dietary LCFA) and endogenous (de novo synthesis) sources; the contribution of each source depends on lipid content of the diet and bacterial species. Increasing lipid concentration in the diet enhance exogenous uptake by some microbes.

64. Microbial Fatty Acid Synthesis FA’s synthesized de novo consist mainly of C18:0 and C16:0 in an approximate ratio of 2:1 Significant amounts of radioactivity from [ 14 C] acetate or [ 14 C] glucose are incorporated into microbial lipid as straight chain, even-numbered carbon fatty acids. Propionate or valerate substitute for acetate yields straight chain, odd numbered carbon LCFA in ruminal microbes.

65. Microbial Fatty Acid Synthesis Branched chain FA’s (Iso) can be accounted for by utilization of isobutyrate, isovalerate, and 2- methylbutyrate as primers. Monounsaturated FA’s that constitute 15 to 20% of bacterial FA’s are synthesized by the anaerobic pathway.

66. Microbial Fatty Acid Synthesis Polyunsaturated FA’s are not commonly synthesized by bacteria. Therefore, PUFA’s reported to exist in ruminal microbes are likely the result of exogenous uptake of preformed FA’s. Odd numbered FA can be obtained by reducing the chain length through alpha-oxidation or from propionyl-CoA.

67. Fatty Acid Composition (% by weight) of Lipids of Mixed Rumen Bacteria % Composition CattleSheep Fatty Acid 11:0 0.1 n.d. 12:0 0.4 4.6 12:0br 0.7 n.d. 13:0 0.3 n.d. 13:0br 0.7 n.d. 14:0 2.3 3.7 14:0br 2.4 n.d. 15:0 4.4 n.d. 15:0br10.1 n.d. 16:035.2 25.4

68. Fatty Acid Composition (% by weight) of Lipids of Mixed Rumen Bacteria % Composition of CattleSheep Fatty Acid 16:0br 1.0 n.d. 16:1 -- tr 17:0 1.8 n.d 17:0br 1.7 n.d. 18:032.020.8 18:0br n.d n.d 18:1 3.919.7 18:2 3.5 5.6 18:3 tr 20:0 -- 20.2

69. Lipid Balance Across The Rumen Fatty acids loss from ruminal content was negligible in many studies that examined LCFA absorption across the ruminal epithelium or their catabolism to VFA or CO 2. 85 to 96% of radioactive linoleic acid added to the rumen of sheep was recovered from ruminal contents after 48 h.

70. Lipid Balance Across The Rumen Likewise, radioactivity was minimal in blood plasma of sheep given a ruminal dose of labeled unsaturated LCFA. Degradation of LCFA to CO 2 and VFA was less than 1% when acids were incubated with ruminal microbes in vitro or in vivo, demonstrating that LCFA have little energy-sparing effect on growth of ruminal microbes. Protozoa, especially holotriches, ingest LCFA mainly for direct incorporation into cellular lipids, but few LCFA are catabolized.

72. Possible Routes of FA Loss From Ruminal Fluid Goosen incubated [ 14 C]oleic acid with ruminal epithelium and reported 31.5% uptake by the tissue and 8.2% transport. Plamitate was metabolized readily to KB’s by ruminal epithelium and to C15 acids by  oxidation and then to C13 and C11 acids by ß oxidation. Palmitate oxidation, and its conversion to KB’s also occurred in epithelial cells isolated from the rumen of sheep.

73. Possible Routes of FA Loss From Ruminal Fluid Wu reported greater than 90% disappearance from the rumen of FA’s shorter than C 14. Table 1 summarized data from 15 published studies that examined lipid flow to the small intestine of cattle or sheep. Of the 47 animal groups, 15 had net loss of lipid from the mouth to duodenum. Lipid loss across the rumen was more common for diets with added fat (11 out of 15) than for control diets (4 out of 15).

75. Possible Routes of FA Loss From Ruminal Fluid Regression of dietary lipid flow against lipid intake (Figure 3) gave a slope of.92, indicating loss of dietary lipid in the rumen equal to 8 g/100 g of lipid intake.