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Mammary fatty acid synthesis and secretion in the milk

Sixty percents (on molar basis) of the milk FA secreted are coming from de novo synthesis, while 40% are coming from direct plasma uptake (origins are diet, RBH, or body reserves mobilization). The milk FA composition depends on plasma uptake, de novo synthesis, and desaturation (Chilliard et al., 2007).
Plasma fatty acid uptake. Milk long-chain FA originate mainly from dietary lipid absorption from the digestive tract (with the dietary FA undergoing total or partial RBH) and from body reserves mobilization (especially at the beginning of lactation with negative energy balance). Commonly, mobilization of body fat reserves accounts for less than 10% of milk FA, with this proportion increasing when ruminants were in negative energy balance (Bauman and Griinari, 2001).
Mammary de novo synthesis. Rumen major VFA (C2, C3, and C4) are taken up from the blood stream by the mammary gland, as well as β-hydroxybutyrate (BHB). FA are imported from plasma, where they are either released by the enzyme lipoprotein lipase (LPL) (Barber et al., 1997) from TG circulating in chylomicra or Very Low Density Lipoprotein (VLDL) or derived from the plasma non-esterified fatty acids (NEFA) that circulate bound to albumin (Bernard et al., 2008). This plasma FA come from feedstuffs and/or body reserves mobilization.
From the C2 and BHB, which comes from the plasma C4, and lead to 15% of the de novo synthesized FA in the milk, the mammary gland synthesized 40% of the milk FA (on molar basis). Two enzymes are involved in the de novo FA synthesis: acetyl-CoA carboxylase (ACC) and FA synthase (FAS). Both C2 and BHB have their active forms in the mammary epithelial cells, acetyl-CoA and butyryl-CoA, respectively, and are the precursors of the de novo synthesis. The enzyme ACC is first activating acetyl-CoA into malonyl-CoA. The FA are then synthesized by repetitive condensations of 2-carbon units derived from malonyl-CoA, upon reaching a carbon chain length of 14 to 16 carbons. Growing FA is released by the cleaving action of a thioesterase enzyme leading to the production of short- and medium-chain saturated FA. The de novo synthesis leads to the total production of SFA from C4:0 to C12:0, 95% of the C14:0 and 50% of the C16:0 in the milk fat (Bernard et al., 2008). The inhibitory effect of FA against the de novo synthesis is increased as the FA chain is longer, polyunsaturated and contained the trans-10 bond (Chilliard et al., 2000; Bauman and Griinari, 2003). Shingfield et al. (2010) reported an inhibitory effect of trans-10 C18:1, trans-10,cis-12, trans-9,cis-11 and cis-10,trans-12 CLA on de novo synthesis of FA. These FA have an inhibitory effect on ACC enzyme activity that decreases the proportions of de novo synthesized FA (8 to 14 carbon) (Chilliard et al., 2000). Furthermore, the mechanisms involved in this inhibition relate to a reduction in the genes expression of several enzymes involved in milk FA synthesis, such as FAS, acetyl-CoA carboxylase, lipoprotein lipase, or Δ9-desaturase (Bauman et al., 2011).
Desaturation. Some of the medium-chain (C10:0, C12:0, C14:0, C16:0) or long-chain FA (C18:0, trans-11 C18:1, C20:0 up to C24:0) can be desaturated on the 9th carbon, by the enzyme Δ9-desaturase present in the endoplasmic reticulum of mammary epithelial cell (Palmquist et al., 2005). The enzyme activity depends on the carbon-chain length of the FA (Shingfield et al., 2010) in order to lower the fusion point of the milk fat. Stearic acid is the preferred substrate for the Δ9-desaturase (Bernard et al., 2008) with 49 to 60% of the C18:0 being desaturated to cis-9 C18:1in the mammary gland, which represent 60% of the cis-9 C18:1 secreted in milk. Furthermore, 90% of the milk cis-9 C14:1, 50 to 56% of the milk cis-9 C16:1 and more than 60% of the milk cis-9,trans-11 CLA come from the desaturation of C14:0, C16:0 and trans-11 C18:1, respectively (Ferlay et al., 2017). This desaturation is the principal source of cis-9 trans-11 CLA in milk (Mosley et al., 2006). Polyunsaturated FA, such as C18:2n-6, C20:4n-6, and C20:5n-3 and the trans-10,cis-12 CLA, have an inhibitory effect on Δ9-desaturase activity (Ntambi and Miyazaki, 2004; Bernard et al., 2008).
Furthermore, endogenous chain elongation of propionyl-CoA as precursor leads to the formation of C5:0, C7:0, C9:0, and C11:0 in milk and these add up to the odd-chain FA C13:0, C15:0 and C17:0 transferred from the duodenum (Fievez et al., 2012). These odd-chain FA can further be desaturated by Δ9-desaturase, but only the conversion of C17:0 to cis-9 C17:1 seems quantitatively important as reported by Fievez et al. (2012). These authors also suggested that C15:0 and C17:0 could be synthesized in the mammary gland.
Free FA are esterified in the reticulum of the mammary gland cells, thanks to three specific enzymes (acyl-transferase). Free FA are successively added on a molecule of glycerol-3-phosphate to obtain a TG. New formed TG are transferred into fat globule before being secreted in milk via exocytose.

General milk fatty acid composition and variations according to nutritional factors

Triglycerides represent on average 98% of milk fat, of which around 95% is FA and more than 400 FA have been identified in milk (Jensen, 2002). Among milk FA, even SFA represent a majority with 69% of total milk FA, ranging from 47 to 78%. Milk C14:0, 16:0 and C18:0 represent 12.0 and 10% of total milk FA, respectively, followed by 29% of MUFA with 19% of cis-9 C18:1, and only 3% of PUFA with, notably, 1.3% of C18:2 n-6 and 0.5% of C18:3 n-3. Milk trans FA represent 4% of total milk FA with 1.5% of trans-11 C18:1, and 0.5% of cis-9,trans-11 CLA (Ferlay et al., 2008). Milk is composed by 5% of OBCFA (Jensen, 2002; Ferlay et al., 2008; Shingfield et al., 2008). The ruminant diet is an important determinant of milk FA profile. Indeed, changes in feeding practices, with higher proportions of concentrates and corn silages in diets and less grazing (Elgersma et al., 2006), decrease concentrations of MUFA (cis-9 C18:1 and trans-11 C18:1) and PUFA – (n-3 and cis-9,trans-11 CLA) and increase concentrations of C12:0, C14:0 and C16:0, when compared with TMR fed (Chilliard et al., 2007). It has been proven that grazing cows have increased milk content of UFA when compared to silage-based diets (Elgersma et al., 2003). Additionally, it has been reported that milk fat from grazing cows had lower C14:0 and C16:0 and higher cis-9 C18:1, trans-11 C18:1, cis-9,trans-11 CLA and C18:3n-3 contents in comparison to milk from cows fed preserved forages (hay or silage; Dewhurst et al., 2006; Ferlay et al., 2006, 2008). Feeding oilseed-supplemented diets largely increased PUFA and decreased SFA contents in milk fat (Chilliard and Ferlay, 2004; Glasser et al., 2008). Glasser et al. (2008) carried out a meta-analysis on the effects of the four major dietary oilseed supplements and their form on milk FA composition. They reported that feeding linseed, rapeseed, sunflower, or soybean, whatever the form, consistently led to an increase in C18 FA content at the expense of SMCFA, and especially C6:0 to C16:0. Milk trans C18:1, total CLA and cis-9,trans-11 CLA contents were also increased by all oilseed supplements, apart from rapeseed when given as seeds or protected or oils (Glasser et al., 2008). Linseed or grazed grass at earlier vegetative stage had more of an effect on milk C18:3n-3 content than other lipid supplement because of their richness in C18:3 content (Ferlay et al., 2013).

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Analytical methods for milk fatty acid determination

Lipids are first extracted and isolated from the other milk components by several methods, most commonly based on the use of organic solvents (Christie, 1993). A mixture of chloroform and methanol (2:1, v:v) is used to extract the lipids fraction from the milk, followed by a washing step with a salt solution (Folch et al., 1957). The gas chromatography (GC) technique has revolutionized the study of lipids by allowing a complete FA composition determination in a relatively short time (Christie, 1993). The FA from fat fraction are first converted to methyl esters [See Appendix 3 for detailed information on fatty acid methyl ester (FAME) preparation], in order to derivate FA on volatile compounds as described above. The GC with flame ionisation detector is the most widely used method for FA analysis (Juanéda et al., 2007). Flexible fused-silica capillary columns coated with highly polar cyanosilicone stationary phases are required for determining the cis/trans FA composition of lipids (Juaneda et al., 2007); with long-length columns (100 and 120 m) recognized to perform better than shorter ones (50 and 60 m). There are other chromatographic techniques, notably high-performance liquid chromatography (HPLC), where alternative derivatives, such as those with UV chromophores, are used and show better performances (for details information on this technique, see Appendix 3).
The GC analysis is the reference method to quantify the milk FA concentrations but it requires high expertise, and is expensive and time-consuming. Therefore, researchers have developed alternative techniques such as the mid-infrared (MIR) spectroscopy, which has the advantages of having very high throughput (up to 500 samples/h; FOSS, 2005), being easy to use, or the near-infrared reflectance (NIR) spectroscopy. These 2 methods are non-destructive, rapid, cheap and multiparametric. These infrared methods are alternative techniques to the GC method used for quantification of milk FA (Andueza et al., 2013; Ferrand-Calmels et al., 2014). The infrared spectrum is caused by the absorption of electromagnetic radiations at frequencies that are correlated to the vibrations of specific chemical bonds within a molecule (Coates, 2006). The spectrum therefore illustrates these absorptions at different wavenumbers (cm−1) for a specific chemical composition (Smith, 2011). The MIR spectroscopy (400 to 4,000 cm−1) is particularly interesting because it is very highly sensitive to the chemical environment, as the fundamental absorptions of molecular vibrations occur in this region (Belton, 1997), and is already implemented in laboratories of Milk Recording Organisation to quantify major milk components used for milk payment. MIR spectroscopy technique can be used to estimate various milk FA based on calibration equations. In the past decades, it has been successfully used to determine the FA composition of oils, butters and margarines (Safar et al., 1994) and to predict the cis and trans content of fats and oils (van de Voort et al., 1995). More recently, MIR spectroscopy has been successfully used to estimate C12:0, C14:0, C16:0, cis-9 C16:1, cis-9 C18:1 and SFA and MUFA in cow milk (Soyeurt et al., 2006; Ferrand-Calmels et al., 2014). NIR spectrometry has been successfully used to quantify FA concentrations in foods such as meat products (González-Martıń et al., 2005; Pla et al., 2007) or cheese (Lucas et al., 2008). Coppa et al. (2010) and Andueza et al. (2013) have shown that NIR spectrometry can be used to satisfactorily predict milk FA from dairy cows and goats, such as sums (SFA, MUFA, PUFA, total trans FA, total trans C18:1 and total cis C18:1, total CLA) and some individual milk FA present with medium-to-high concentrations (C4:0 to C18:0, cis-9 C18:1, trans-11 C18:1 and cis-9,trans-11 CLA; Coppa et al., 2010). It can also accurately predict milk sums from goat (SFA, MUFA, UFA, total trans FA) and cis-9,trans-11 of CLA, cis9-, trans-10, and trans-11 C18:1 (Andueza et al., 2013). The quality of prediction decreased when FA were present in low to very-low concentrations.

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