Milk fatty acids originating from the rumen as biomarkers to monitor animal health

Changes in the ruminal metabolism of dietary lipids and de novo bacterial FA synthesis might indicate changes and even disturbances in the ruminal microbial population which sometimes initiate a chain of events that eventually might lead to (subacute) ruminal acidosis (SARA). Disturbed rumen (pH) conditions have been correlated with inflammation, as measured by increased acute-phase proteins in the blood (e.g. Gozho et al., 2005, 2007), which further might result in feed-intake depression and production losses (Plaizier et al., 2008). Reduced rumen pH might also result in reduced fibre digestion (Plaizier et al., 2008) and/or in milk fat depression (Dewanckele et al„ 2019). Other consequences of SARA are diarrhoea, laminitis and lameness, and liver abscesses.

Particularly specific trans FA, notably trans-10 intermediates, odd chain and iso-fatty acids have been suggested as candidates for the (early) detection of SARA. Here, we first summarize the most important changes in trans FA and OBCFA associated with shifts in the rumen microbial population. Further, these FA in milk are evaluated as candidates to identify SARA (risk).

Rumen bacteria related to the accumulation of trans-70 intermediates

Situations with greater trans-10 accumulation are often associated with increased ruminal abundance of the lactate-utilizing bacterium Megasphaera elsdenii (Weimer et al., 2010; Dewanckele et al., 2018). Furthermore, this rumen species was increased during grain-induced SARA both in vitro (Mickdam et al„ 2016) and in vivo (Khafipour et al., 2009; Fernando et al., 2010; Plaizier et al., 2017), which is probably linked to the higher lactate production in those situations.

Correlation analysis based on ruminal bacterial populations and milk (Pitta et al., 2018; Dewanckele et al., 2019) or rumen FA profiles (Zened et al„ 2016; Dewanckele et al„ 2018) revealed positive correlations of trans- 10 intermediates with Acidaminococcus spp., Bulleidia spp., Bifidobacterium spp., Carnobacterium spp., Desulfovibrio spp., Dialister spp., Eubacterium spp., Lactobacillus spp., Olsenella spp., Sharpea spp., Syntrophococcus spp. and unclassified Coriobacteriaceae, Lachnospiraceae and Ruminococcaceae. Some of those genera were also increased during a SARA challenge, for example, Bifidobacterium (Mao et al., 2013), Lactobacillus (Petri et al„ 2013; Mickdam et al., 2016; Plaizier et al., 2017), Olsenella (Petri et al., 2013; Zened et al., 2013), Sharpea (Petri et al., 2013; Plaizier et al., 2017) and Syntrophococcus (Petri et al., 2013). Additionally, the genera Lactobacillus and Sharpea are important lactate producers in the rumen (Sharpe et al., 1973; Kamke et al., 2016), which might explain their association with SARA.

Devillard et al. (2007) and McIntosh et al. (2009) observed the formation of trans-10, c/s-12 CLA from 18:2n-6 by Propionibacterium freudenreichii, a bacterial species isolated from the human intestine. In line with this, Wallace et al. (2007) found that Propionibacterium acnes, which was isolated from sheep rumen, converts 18:2n-6 to trans-10, c/s-12 CLA as an end product (McKain et al., 2010; Dewanckele et al., 2017). In our laboratory, a human strain of P. acnes isomerized 18:3n-3 to trans-10, c/s-12, c/s-15 CLnA (L. Dewanckele, J. Jeyanathan, B. Vlaeminck, V. Fievez, unpublished data). Propionibacteria produce propionic acid, whereas some strains additionally ferment lactate (Bryant, 1959). High-grain diets might induce a shift from mainly the production of acetic acid towards increased production of propionic acid and lactate (Balch and Rowland, 1957), which might support a link between Propionibacteria and SARA. Nevertheless, no studies reported on an increased abundance of this genus upon a SARA challenge, which might be due to the rather low abundance of this genus in ruminal content (Kim et al., 2002; Shingfield et al., 2012).

Rumen bacteria and their odd- and branched-chain fatty acids

Microbial formation of OBCFA has been outlined in detail by Vlaeminck et al. (2006). In summary, odd-chain fatty acids (15:0 and 17:0) are formed through elongation of propionate or valerate, whereas precursors of branched-chain fatty acids (iso 13:0, iso 14:0, iso 15:0, iso 16:0, iso 17:0, iso 18:0, anteiso 13:0, anteiso 15:0, anteiso 17:0) are branched-chain amino acids (valine, leucine and isoleucine) and their corresponding branched short-chain carboxylic acids (/sobutyric, isovaleric and 2-methyl butyric acids). The OBCFA profile of the rumen bacteria seems to be largely determined by the fatty acid synthase activity of the microorganism rather than by the precursor availability (Vlaeminck et al., 2006). Hence, variation in the OBCFA profile leaving the rumen is expected to reflect changes in the relative abundance of specific bacterial populations in the rumen rather than an altered bacterial fatty acid synthesis. Accordingly, higher proportions of /so-fatty acids in solid-associated bacteria were suggested to reflect their enrichment in cellulolytic bacteria, whereas higher proportions of anteiso 15:0 in liquid-associated bacteria might indicate their enrichment in pectin and sugar-fermenting bacteria, which seem particularly rich in this fatty acid (Table 3) (Vlaeminck et al., 2006; Bessa et al., 2009).

Detailed information on the OBCFA profile of different rumen bacteria as well as their main fermentation substrates and end products are shown in Table 3. The OBCFA profile of Ruminococcus albus, Butyrivibrio fibrisolvens and

Ruminococcus flavefaciens, which are considered to be representative rumen cellulolytic bacteria, contains relatively high proportions of even and/or odd- r'so-fatty acids (Table 3). These fibre-digesting bacteria mainly ferment cellulose, hemicellulose and pectin to acetate, butyrate, hydrogen (H,) and carbon dioxide (C02). On the other hand, the OBCFA profile of Ruminobacter amylophilus, Selenomonas ruminantium, Streptococcus bovis and Succinomonas amylolytica, considered as representative amylolytic bacteria, shows low proportions of branched-chain fatty acids, particularly /so-branched chains and are rich in linear odd-chain fatty acids and/or anfe/so-branched-chain fatty acids (Table 3). The latter fatty acids seem to be of particular importance in sugar or pectin fermenting bacteria such as Prevotella spp., Lachnospira multiparus and Succinovibrio dextrinosolvens (Table 3). Amylolytic or starch and sugar- digesting bacteria ferment sugar, starch and peptides to propionate, butyrate, acetate, lactate, H2 and C02. Eubacterium ruminantium and Streptococcus bovis are lactate producers and do not synthesize acetate. These bacterial species play an important role in the onset of acidosis. Their most important OBCFA are 15:0 and anteiso 0 5:0. Also Megashpaera elsdennii plays an important role (Nagaraja and Lechtenberg, 2007) with the main FA in its cell wall 15:0 (Table 3).

Rumen bacteria associated with the accumulation of tra ns- 7 0 intermediates, rich in odd-chain fatty acids and poor in iso-fatty acids linked to inflammation?

Many of the bacteria associated with the accumulation of trans-10 intermediates, rich in odd-chain fatty acids and poor in iso-fatty acids, for example, of the genera Acidaminococcus, Desulfovibrio, Dialister and Syntrophococcus as well as the Succinivibrionaceae family, including the genera Succinivibrio, Succinimonas and Ruminobacter are gram-negative. It is well known that gramnegative bacteria contain endotoxins such as lipopolysaccharide (LPS) in the outer membrane of their cell wall, which act as immunogenic compounds in their free form (Hurley, 1995). These endotoxins are extensively shed during the logarithmic and stationary phases of bacterial growth and also released following cell disintegration and lysis (Nagaraja et al., 1978a,b; Hurley, 1995; Plaizieretal., 2012). Nevertheless, the LPS potency varies among gram-negative species, and as such, the proinflammatory response of LPS may differ between species (Ghaffari et al., 2017). Furthermore, M. elsdenii produces material which has many biological and chemical characteristics common to enterobacterial endotoxins (Nagaraja et al., 1979). Although its endotoxic potency is rather low (Nagaraja et al., 1979), a higher rumen concentration upon increased M. elsenii abundance might partially contribute to the onset of an immune response (Plaizier et al., 2008). Nevertheless, further research is required to confirm this hypothesis.

Table 3 Predominant substrate (from Harfoot and Hazlewood, 1997), fermentation end products (from Russell and Rychlik, 2001) and OBCFA profile (g/100 g fatty acids; original references in Vlaeminck et al., 2006) of some major bacteria involved in rumen carbohydrate fermentation

Ferm.

prod.d

Anteiso C13:0

Anteiso C15:0

Anteiso C17:0

Iso

C13:0

Iso

C1 5:0

Iso

C17:0

Iso

C14:0

Iso

C16:0

C13:0

C15:0

C17:0

C17:1

Ruminococcus albuse

A

-

9.4

1.3

-

-

0.7

20.6

11.0

-

10.3

1.4

-

Butyrivibrio fibrisolvens"

A, B, F

6.4

16.2

8.6

6.8

10.4

5.7

10.8

11.1

2.9

7.8

4.3

3.5

Ruminococcus flavefaciens"

A, S

-

2.3

2.9

-

35.7

5.2

2.5

7.3

0.1

3.2

0.5

-

Succinimonas amylolyticab N6

A, P

-

-

-

-

52.6

10.8

1.6

5.3

1.6

5

-

-

Succinimonas amylolyticab B24

A, P

-

-

-

-

0.1

0.3

-

0.6

1.4

ДЗ

1.3

0.6

Prevote llabc

A, S

1.2

36.7

4.2

3.0

14.7

2.3

3.3

3.0

1.2

12.1

2.1

-

Lachnospira multi parusbc

A, L, F

-

4J3

2.6

-

1.1

1.1

1.2

1.8

0.3

2.9

0.8

0.1

Succinivibrio dextrinosolvensc

A, S

0.8

3.6

1.0

-

0.1

-

0.6

1.5

0.5

AH

0.7

-

Ruminobacter amylophilusb

A, S, F

-

1.1

-

-

-

-

-

-

0.5

1.1

0.3

0.1

Fibrobacter succinogenes[1]

A, S

3.9

7.7

1.2

-

0.1

0.2

3.6

3.4

9.0

30.2

2.1

-

Streptococcus bovisb

L

-

0.9

-

-

-

-

0.4

0.2

0.6

17

1.2

0.2

Megasphaera elsdeniic

A, P, В

-

2.8

-

0.1

0.2

0.2

1.5

0.5

1.5

4.5

3.0

Eubacterium ruminantiumb B1C23

В, L, F

-

-

-

-

17.7

1.4

-

-

5.4

49.0

1.5

-

Eubacterium ruminantiumb GA195

B, L, F

-

30.1

1.7

-

0.4

0.2

6.1

3.7

0.4

6.5

0.4

-

Selenomonas rummantiumb

A, R L

-

0.1

-

-

0.2

-

0.3

0.1

1.3

6.0

2.9

2.6

Trans and odd- and branched-chain fatty acids in milk fat to identify subacute ruminal acidosis (risk)

Traditionally, subacute ruminal acidosis has been defined as a rumen condition associated with a depressed pH for several hours per day (Plaizier et al., 2008). Hence, the frequency of observations below an arbitrary pH threshold is used, for example, time spent below pH < 5.6 or 5.8 and pH < 6.0 (when using rumen pH boli residing in the reticulum). Alternatively, the area under the curve (drop below pH-threshold multiplied with the duration of this drop), either or not normalized for feed intake level to obtain an acidosis index (Gao and Oba, 2014), is used as an improved indicator of SARA severity. Some applied signal processing on the raw pH of the daily kinetic data in order to calculate relative pH indicators, which correct for inter-individual variability, sensor drift and sensor noise (Villot et al., 2018). Such relative pH indicators monitor intra-individual changes in pH values. Denwood et al. (2018) also proposed to monitor deviation, but they assessed the deviation from a predictable pattern, describing the temporal variation in rumen pH. In research from our group (Colman et al., 2012), it was proposed to describe diurnal pH variation by fitting a logistic curve, which is described by (81 (reflecting average rumen pH) and (30 (representing the rumen pH range). Trans and OBCFA in milk fat have been explored as biomarkers to diagnose SARA and identify cows at risk for SARA development (early warning and inter-animal susceptibility). For this, identification of SARA cases most often have been based on the daily time below a pH threshold and to a lesser extent by the acidosis index and the parameters of the logistic curve. Milk FA have notyet been linked to deviations in rumen pH from a kinetic pattern nor to SARA-related inflammatory responses.

Vlaeminck et al. (2006) suggested higher proportions of iso-fatty acids in milk to reflect a rumen condition associated with a lower risk for SARA, while higher proportions of anteiso 15:0, 15:0 and 17:0 might indicate a situation of higher risk for SARA. Indeed, increasing levels of milk fat 15:0 have been reported during SARA development (Enjalbert et al., 2008; Colman et al., 2010). This is in accordance with the recent meta-analysis of Prado et al. (2019) who reported that dietary starch and ruminal pH are associated with a positive and negative slope, respectively, in equations predicting milk yield of 15:0 (g/d) as well as 17:0 (g/d). Also anteiso 17:0 (g/d) was negatively correlated with rumen pH, but the determination coefficient of this equation was smaller. Iso-even chain fatty acids were not reported in this meta-analysis. In the study of Colman et al. (2015), combining data of six acidosis induction experiments with rumen-fistulated dairy cows, reduced amounts of/so-branched chain fatty acids seemed more determining than increased concentrations of 15:0 and 17:0 to distinguish SARA cases, while the inverse was true in the study of Jing et al. (2018). Nevertheless, in both papers, trans-10 18:1 and/or the ratio of trans-10

18:1 to trans-11 18:1 were associated with SARA (development) and/or cows at risk for SARA, although in some models of Colman et al. (2015), trans-10 18:1 was replaced by c/s-9 trans-'П 18:2 as a major discriminating variable. Furthermore, Jing et al. (2018) monitored SARA-indicative milk FA(/so even and odd chain FA, anteiso odd chain FA as well as trans-FA) over a 4-week period of concentrate build-up during early lactation. Next to trans-10 18:1 and 15:0, Д trans-11 18:1, defined as the maximum decrease in the proportion of this trans-isomer over the 4-week period, was proposed as an additional parameter to determine inter-animal variability in SARA susceptibility. Indeed, in the work by Jing et al. (2018), milk OBCFA and trans-FA not only showed the potential to diagnose the occurrence of SARA during a SARA-induction experiment, but also to distinguish cows with relatively high or low susceptibility for SARA development within the same herd.

Further, Colman et al. (2012) attempted to distinguish milk FA associated with rumen pH level from those associated with rumen pH fluctuations as described by the logistic curve-parameters (31 and (3, respectively. Based on the two experiments described in this article, milk fat proportions of trans-10 18:1 were suggested to be associated with situations of both low and largely fluctuating pH, whereas situations with low, stable pH did not induce a shift from the formation of mainly trans-11 intermediates towards increased formation of trans-10 intermediates. On the other hand, trans-11 18:1 and c/s-9, trans-11 18:2 were only influenced by pH variation and not by the average pH, whereas iso FA depended on the average pH and were not influenced by diurnal pH variation. The relation between the iso FA (positive) and trans-10 18:1 (negative) with the average rumen pH was confirmed in the extended database of six SARA- induction experiments described in Colman's PhD dissertation (Colman, 2012). In all experiments, the rumen pH range related to the specific milk OBCFA and trans-FA, but the relations were equivocal, which might be related to the large variation in amplitude of the pH range between the six experiments.

A multivariate and robust model should be developed in the future to identify cows at risk for SARA. Furthermore, to be of practical relevance, these diagnostic milk FA should be determined routinely, which is currently not possible. Earlier research by our group indicated Raman spectroscopy showed the potential for the determination of individual and grouped frans-(mono-) UFA in milkfat(Stefanov et al., 2011), but further investment in the development of such a routine technology is required.

  • [1] Bacteria fermenting cellulose and hemicellulose. 6 Bacteria fermenting starch.c Bacteria fermenting sugar and pectin. d Fermentation products: A: Acetate; S: Succinate; B: Butyrate; F: Formate; P: Propionate; L: Lactate.Predominant OBCFA is underlined. Indication of main substrate is given by superscript letter.
 
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