Understanding rumen lipid metabolism to optimize dairy products for enhanced human health and to monitor animal health

Introduction

Bovine milk contains approximately 3.5-5.0% milk fat in the form of milk fat globules dispersed in an aqueous phase. The fat globules consist of a relatively large core of triacylglycerides (TAG) enveloped by the milk fat globule membrane, a thin tri-layer membrane of, mainly, phospholipids (PL) (Bernard et al., 2018). As a result, milk fat mainly consists of TAG (97.5-99%), a minority of diglycerides (<1%), PL, non-esterified fatty acids (NEFA), cholesterol and monoglycerides, and nil or only trace amounts (<0.1%) of cholesterol esters (CE) (Castro-Gomez et al., 2014; Glasser et al., 2007).

http://dx.doi.org/10.19103/AS.2020.0067.15 © Burleigh Dodds Science Publishing Limited. 2020. All rights reserved.

Table 1 Fatty acid composition of German summer and winter milk lipids (wt %)’

Fatty acid

Summer

Winter

4:0

3.79

3.85

5:0

0.02

0.02

6:0

2.10

2.37

7:0

0.02

0.03

8:0

1.19

1.39

9:0

0.02

0.04

10:0

2.44

3.03

10:1

0.27

0.27

11:0

0.04

0.06

12:0

2.98

3.57

12:1

0.08

0.09

13:0 iso

0.14

0.13

13:0 anteiso

0.02

-

13:0

0.08

0.10

14:0 iso

0.16

0.10

14:0

9.75

11.1

14:1

1.08

1.07

15:0 iso

0.43

0.29

15:0 anteiso

0.74

0.50

15:0

1.35

1.17

16:0 iso

0.30

0.22

16:0

23.5

30.3

16:1

2.00

2.03

17:0 iso

0.65

0.55

17:0 anteiso

0.55

0.52

17:0

0.72

0.64

17:1

0.39

0.36

18:0 iso

0.05

0.08

18:0

10.6

9.42

trans-4 18:1

0.02

0.01

trans-5 18:1

0.02

0.01

trans-6/7/8 18:1

0.26

0.21

trans-9 18:1

0.27

0.17

trans-10 18:1

0.29

0.25

trans-11 18:1

3.82

1.11

trans-12 18:1

0.33

0.27

trans-13/14 18:1

0.34

0.24

trans-15 18:1

0.51

0.38

Fatty acid

Summer

Winter

trans-16 18:1

0.26

0.19

trans (total) 18:1

6.12

2.84

cis-9 18:1

19.4

17.3

cis-11 18:1

0.55

0.53

cis-12 18:1

0.10

0.14

c/s-13 18:1

0.06

0.08

cis-15 18:1

0.02

0.03

18:2 (total)

2.17

2.18

18:3 n-3

0.61

0.42

19:0

0.09

0.05

20:0

0.16

0.15

20:1

0.31

0.22

20:4

0.09

-

22:0

0.07

0.06

22:1

0.03

-

24:0

0.04

0.04

'Adapted from Jensen (2002).

As a result of the microbial activity in the rumen and of the mammary gland metabolism, bovine milk is characterized by a high variety of milk fatty acids (FA) (Jensen, 2002). Typical bovine milk FA profiles are shown in Table 1. Odd- and branched-chain FA (OBCFA) mainly are the result of microbial de novo synthesis of FA in the rumen, while a series of trans and cis unsaturated FA (UFA) are formed from incomplete biohydrogenation of (poly)-unsaturated FA (PUFA), resulting in milk fat that is high (-70%) in saturated FA (SFA). Both aspects of ruminal lipid metabolism and their implications are elaborately discussed in the current chapter.

For a better understanding of the origin of fatty acids in dairy products, the first sections address the ruminal metabolism, intestinal digestion, transfer to the mammary gland and mammary fatty acid metabolism. The chapter then addresses the potential to improve the fatty acid composition of dairy products for enhanced human health. Here, technologies to protect unsaturated fatty acids from rumen biohydrogenation are particularly emphasized as nonprotected lipid supplements that already have been extensively reviewed in earlier studies (e.g. Kliem and Shingfield, 2016; Gebreyowhans et al., 2019). Finally, the chapter discusses the use of variation in the milk fatty acid profile, induced by changes in the ruminal lipid metabolism, for monitoring animal health.

Ruminal metabolism of dietary lipids and de novo fatty acid synthesis

Lipolysis and biohydroganation of dietary lipids

The diet of lactating dairy cows typically contains 4-5% crude fat (on DM basis). Primary sources of lipid in the ruminant diet are forages and concentrates, which mainly contain 18-carbon UFA (i.e. a-linolenic acid, 18:3n-3; linoleic acid, 18:2n-6; and oleic acid, c/s-9 18:1) (Ferlay et al., 2017). However, the lipid content can be increased by the use of fat supplements. The major lipid class of forages is glycolipids, whereas the majority of lipids in concentrates are present in the form of TAG. Following ingestion, dietary lipids are hydrolyzed and the NEFA are released into the rumen. 18:3n-3, 18:2n-6 and cis-9 18:1 are then converted by the rumen microbial community to SFA first via a cis- trans isomerization to trans FA intermediates followed by hydrogenation of the double bonds (Harfoot and Hazlewood, 1997; Shingfield and Wallace, 2014). This process is called biohydrogenation.

The main members of the rumen microbial community are (per mL of live liquor) anaerobic bacteria (1010), ciliate protozoa (107) and anaerobic fungi (106) (Jenkins et al., 2008; Buccioni et al., 2012). Bacteria, either or not living in symbiosis with protozoa, are known to be mainly responsible for rumen biohydrogenation, while the contribution of protozoa and fungi is negligible (Louremjo et al., 2010; Buccioni et al., 2012). Based on in vitro research, Butyrivibrio spp. seems to be the predominant biohydrogenating bacteria (e.g. Kepler et al., 1966; Maia et al., 2007; Wallace et al., 2007). Nevertheless, in several in vivo studies, a correlation between intermediate or end products of the biohydrogenation process and abundance of Butyrivibrio spp. in the rumen or at the entrance of the omasal canal was virtually absent (e.g. Zened et al., 2016; Zhu et al., 2016; Kairenius et al., 2017). This suggests potentially that also other - as yet uncultured - species might be involved in ruminal biohydrogenation (Huws et al., 2011; Toral et al., 2016). From such association studies, it is suggested that uncultured bacteria phylogenetically classified as Prevotella, Lachnospiraceae incertae sedis and unclassified Bacteroidales, Clostridiales and Ruminococcaceae may play a role in rumen biohydrogenation. The main reason for biohydrogenation of PUFA by bacteria is thought to be reduction of the toxicity of those PUFA (Maia et al., 2007, 2010; Fukuda et al., 2009). The mode of action of PUFA antimicrobial activities is not yet clear, but the prime target seems to be the bacterial cell membrane and the various essential processes that occur within and at the membrane. Bacteria prefer SFA for their membrane synthesis as the double bonds present in UFA alter the shape of the molecule and disrupt the lipid bilayer structure (Keweloh and Heipieper, 1996).

Numerous in vivo and in vitro studies have enabled several ruminal biohydrogenation pathways of 18:2n-6,18:3n-3 and c/s-9 18:1 to be elucidated. Under normal rumen conditions, 18:2n-6 is mainly isomerized to c/s-9, trans-11 conjugated linoleic acid (CLA), which is further hydrogenated to trans-11 18:1, and ultimately to 18:0 (Fig. 1, solid line arrows). The major biohydrogenation pathway of 18:3n-3 involves c/s-9, frans-11, c/s-15 conjugated linolenic acid (CLnA), trans-11, c/s-15 18:2 and trans-11 18:1 as intermediates (Fig. 2, solid line arrows), whereas the major part of c/s-9 18:1 is directly hydrogenated to 18:0 in the rumen (Fig. 3, solid line arrow). Flowever, ruminal biohydrogenation of 18:2n-6, 18:3n-3 and c/s-9 18:1 might also result in the formation of several other minor FA intermediates, for example, trans-9, frans-11 CLA, trans-10 18:1 and c/s-12 18:1 (Figs. 1-3, dashed line arrows). Some of these alternative pathways might become more important under certain rumen conditions

Pathways of ruminal 18:2n-6 metabolism

Figure 1 Pathways of ruminal 18:2n-6 metabolism (adapted from Shingfield and Wallace, 2014). Arrows with solid lines highlight the major biohydrogenation pathway while arrows with dashed lines describe the formation of minor intermediates, under physiologically normal conditions in the rumen.

Pathways of ruminal 18:3n-3 metabolism

Figure 2 Pathways of ruminal 18:3n-3 metabolism (adapted from Ferlay et al., 2017). Arrows with solid lines highlight the major biohydrogenation pathway while arrows with dashed lines describe the formation of minor intermediates, under physiologically normal conditions in the rumen.

Pathways of ruminal c/s-9 18:1 metabolism

Figure 3 Pathways of ruminal c/s-9 18:1 metabolism (adapted from Shingfield and Wallace, 2014). Arrows with solid lines highlight the major biohydrogenation pathway while arrows with dashed lines describe the formation of minor intermediates, under physiologically normal conditions in the rumen.

and might be of interest as indicators to monitor these rumen conditions. For example, when high-starch diets are fed, 18:3n-3 and 18:2n-6 might also be converted via an alternative pathway, resulting in an increased accumulation of trans-10 intermediates in the rumen, particularly of frans-10 18:1 (Shingfield and Griinari, 2007). However, these shifts may vary among ruminant species. While a high-starch diet supplemented with sunflower oil resulted in a trans-11 to trans-10 shift in cows, this shift tends to be less in goats (Toral et al., 2016). On the other hand, marine lipids, rich in very-long-chain n-3 PUFA, efficiently inhibit the last step of C18 FA biohydrogenation in the bovine, ovine and caprine, increasing the outflow from the rumen of trans-11 18:1, c/s-9, trans-11 CLA and trans-10 18:1 (Toral et al., 2016). In addition to the pathways shown in Figs. 1-3, other alternative pathways might also exist, as supported by the identification of numerous additional biohydrogenation intermediates in recent in vitro experiments using stable isotopes (deuterium oxide and ,3C-labelled FA; Honkanen et al., 2016; Toral et al., 2018a, 2019).

De novo fatty acid synthesis by rumen bacteria

Bacterial de novo synthesis of SFA is mediated by two types of FA synthases, being straight-chain and branched-chain FA synthase (Kaneda, 1991). De novo synthesis of straight-chain FA with an even number of carbons is achieved by repeated condensation of malonyl-coenzyme A (CoA) with acetyl-CoA as primer, yielding palmitic acid as the dominant end product(Fulco, 1983). Linear odd-chain FA are formed when propionyl-CoA, instead of acetyl-CoA, is used as primer(Fulco, 1983; Kaneda, 1991). Branched-chain FAcan be distinguished in three series: even iso acids (e.g. iso 14:0, iso 16:0), odd iso acids (e.g. iso 15:0, iso 17:0) and odd anteiso acids (e.g. anteiso 15:0, anteiso 17:0) (Kaneda, 1977), and their primers are isobutyryl-CoA, /sovaleryl-СоА and 2-methylbutyryl-CoA, respectively (Kaneda, 1977; Annous et al., 1997). Bacterial de novo synthesis of

OBCFA is of particular interest as only trace levels occur in most plants (Diedrich and Henschel, 1990) and their presence in animal tissues is particularly limited to those animals with important symbiotic fermentation (Keeney et al., 1962). More importantly, rumen bacteria show a distinct OBCFA profile which seems largely determined by the substrate specificity of the acetyl-CoA acyl-carrier- protein transacylase (Kaneda, 1991). As a result, variations in the profile of ruminal OBCFA are mainly a reflection of changes in the relative abundance of specific bacterial populations in the rumen.

Digestion and transfer of dietary and rumen fatty acids to the mammary gland and fatty acid metabolism in the mammary gland

Dietary FA bypassing rumen metabolism as well as rumen FA flows to the duodenum. The apparent recovery of these duodenal FA in milk fat depends on various factors, including digestibility, FA metabolism (both synthesis and oxidation) in the cow's tissues, the cow's physiological status (positive vs. negative energy balance), the blood lipid classes in which FA are transported and the duodenal FA flow, with higher transfer efficiencies at lower intestinal flows (Chilliard et al., 2000). Finally, some FA are also partially transformed in the mammary gland.

Intestinal digestion of fatty acids

About 70% of the short-chain fatty acids are absorbed in the rumen, while uptake of medium- and long-chain fatty acids before reaching the small intestine is negligible (Noble, 1978). Fatty acids reaching the small intestine are absorbed from the jejunum after solubilization in a micellar phase. Although in ruminants - due to lipolysis in the rumen - mainly free FA flow to the small intestine, these FA still need to be "released" to a certain extent. Indeed, because of their hydrophobic nature, free FA are attached to small particles of the digesta. To make these hydrophobic FA "soluble" in the aqueous environment, micelle formation must take place. In both ruminants and monogastric animals, the efficiency of micelle formation is the limiting factor in the absorption of FA in the small intestine. Micelle formation of long-chain, SFA (such as e.g. stearic acid) is generally less efficient than of short-chain and UFA. Ruminants have therefore developed a number of important differences and characteristics in FA absorption as compared to non-ruminants that allow efficient absorption of SFA (Bauman and Lock, 2006). These include differences in both bile salt composition (more taurine-conjugated bile salts than glycine-conjugated) and the predominant amphiphilic substances involved in micelle formation. Lysolecithin is the most important amphiphilic substance in the small intestine of ruminants, while monoglycerides and bile salts primarily perform this function in monogastric animals. Compared to these two components, lysolecithin is a much better emulsifier of stearic acid. Lysolecithin (lysophosphatidylcholine) is formed in the duodenum from lecithin (a phospholipid) through the action of phospholipases (produced in the pancreas). In addition, the supply to the small intestine is characterized by a slow and continuous release of relatively small amounts of FA (due to the continuous feed intake and passage from the rumen). Consequently, in general, the ability of ruminants to absorb SFAis much higher than that of non-ruminants (Bauman and Lock, 2006). The average apparent intestinal absorption of FA is 0.83, with a tendency for higher absorptions of microbial FA (Schmidely et al., 2008).

Nevertheless, recently, abomasal infusion of exogenous emulsifiers has been shown to improve FA absorption, which implies that solubilization of FA is the primary limiting step for the transfer of FA from the intestinal lumen to the blood stream (Prom and Lock, 2019). Moreover, excessive supplementation of dietary fat will suppress the FA digestibility, and this effect is much greater with increased uptake (or outflow from the rumen) of 18:0 than 16:0 (Boerman et al., 2015,2017; Rico et al., 2017).

Fatty acid transport in blood and transfer to the mammary gland

After absorption, free FA are esterified into TAG and PL in intestinal epithelial cells and transported, first in the lymph and afterwards in the blood. They predominantly occur as chylomicrons (Demeyer and Doreau, 1999), and to a lesser extent in very-low-density lipoproteins (VLDL). Both lipoproteins predominantly consist of TAG, besides PL, CE and NEFA (in decreasing order of importance) (Bruss, 1997). Lipid classes largely differ in composition, due to selective incorporation of specific FA, as well as in effectiveness to deliver FA to the mammary gland. Indeed, FA concentrated in CE and PL are poorly transferred to milk fat, given the low affinity of the mammary gland lipoprotein- lipase for these fractions (Annison et al., 1967; Shennan and Peaker, 2000). Such distribution differences in lipid classes - at least partially - could explain difficulties in enriching milk fat with PUFA as, for example, 18:3n-3, which is predominantly incorporated in blood plasma CE (Loor et al., 2002b; Tyburczy et al., 2009), while, for example, 20:5n-3 (eicosapentaenoic acid, EPA) and 22:6n-3 (docosahexaenoic acid, DHA) are predominantly present in PL. Additionally, mobilization of adipose tissue might contribute to modification of the milk FA profile compared with the duodenal FA profile (Jorjong et al., 2014), particularly because adipose tissue is rich in FA of longer chain length. For the OBCFA, this results in a preferential incorporation during anabolism of OBCFA with a chain length of 17 carbon atoms compared with OBCFA with chain lengths of 13-15 carbon atoms, as supported by the inverse ratio of 17:0

to 15:0 in adipose tissue (2:1) as compared to milk (1:2) (Craninx et al., 2008). As a result, during the first weeks of lactation when the cow is mobilizing fat, milk secretion of OBCFA with 17 carbon atoms was increased. Inversely, shorter OBCFA (with chain lengths of 14-15 carbon atoms) were diluted in milk fat, similarly to other short- and medium-chain FA (Craninx et al., 2008).

 
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