As expected, the δ13C values of C16:0 and C18:0 fatty acids extracted from cattle milk are more enriched in 13C when cattle are raised on diet containing increasing amounts of C4 plant matter (maize). While C3 plants fix atmospheric CO2 using the ribulose biphosphate carboxylase (RuBisCO) catalysed Calvin cycle (Calvin and Benson, 1948) and discriminate against 13CO2 (Boutton, 1991), C4 plants fix atmospheric CO2 with the Hatch-Slack pathway (Hatch and Slack, 1966) which discriminates less against 13CO2 (Boutton, 1991). Bulk δ13C values of C3 and C4 plants thus range from −34 to −24 ‰ and −19 to −6 ‰, respectively (Smith and Epstein, 1971). Hence, the δ13C values of the fatty acids and carbohydrates are more enriched in C4 plants compared to C3 plants. The carbohydrates and fatty acids are then incorporated into the body fats of the consumer animals, leading to enriched δ13C16:0 and δ13C18:0 values of synthesised milk.
The difference between the raw δ13C values of C18:0 and C16:0 fatty acids (namely Δ13C values) in milk from cattle raised on a pure C3 diet range between −5.9 and −3.6 ‰. Furthermore, milk from cattle raised traditionally on pastures comprising C3 and C4 plants in Africa display Δ13C (= δ13C18:0 - δ13C16:0) values similar to that observed in cattle raised on C3 diet in Britain (Fig. 3). This difference in the isotopic composition of the two main fatty acids reflects the different sources for the C16:0 and C18:0 fatty acids in milk. In fact, while C16:0 fatty acids are biosynthesised from carbohydrates and fatty acids from the diet, C18:0 fatty acid in milk has two distinct contributions. As the mammary gland is unable to biosynthesise the C18:0 fatty acid, it is sourced from: (i) the remobilisation of C18:0 fatty acids from the carcass depot fats and (ii) the direct incorporation of C18:0 from the diet, after rumen biohydrogenation of C18:n fatty acids from dietary plants (Fig. 4a; Copley et al., 2003). The difference in the isotopic composition of these two pools relates to the 8 ‰ difference between carbohydrates and fatty acids in plants when pyruvate is decarboxylated to acetylCoA (DeNiro and Epstein, 1977) with the mass balance leading to the difference of ca. 3 ‰ evident between the C18:0 fatty acids from carcass and dairy fats (Copley et al., 2003).
The consistent Δ13C values arise because the carbon isotopic difference between fatty acids and carbohydrates (ca. 8 ‰) is common to all plants, no matter what photosynthetic fixation mechanism is used. The difference is controlled by fractionation resulting from the pyruvate dehydrogenase enzyme complex involved in the decarboxylation of the pyruvate in forming acetyl CoA (DeNiro and Epstein, 1977). Given the latter and the fact metabolism of ruminant mammals is consistent no matter what region of the world the animal comes from, the arithmetic transformation from raw δ13C values to Δ13C values is valid in removing the influence of varying proportions of C3 and C4 plants in ruminant forages (Mukherjee et al., 2005; Dunne et al., 2012). While this model was devised on the basis of fresh C3 plant consumption, as discussed above, this difference of ca. 3 ‰ holds for cattle, or any other ruminant animal, raised on any combination of traditional C3 and C4 pastures. This is confirmed here by the similarity between Δ13C values of milk from cattle raised on a pure C3 diet (diet A) or the C3/C4 diets from Africa.
In contrast, however, milk from cattle raised on a mixture of grass/maize silage exhibits Δ13C higher values ranging from −3.4 to −1.6 ‰ (Fig. 3). The carbon isotopic compositions of the main saturated fatty acids (C16:0 and C18:0) are thus more similar by ca. 2 ‰, compared to the same fatty acids from cattle grazing pasture (C3 in the UK and C3/C4 in Kenya), suggesting that the C16:0 and C18:0 fatty acids have a more similar metabolic source than when cattle are grazing on herbage.
Changes in the biohydrogenation of polyunsaturated fatty acids in the rumen is known to happen when high starch and high free oil diets are fed to cattle, inducing milk fat depression through a downregulation of de novo synthesised fatty acids in the mammary gland. Remobilisation of C18:0 fatty acids from the adipose fats to milk fats is thus increased when diets fed to cattle are high in starch or free oil (Peterson et al., 2003), leading to the carbon isotopic composition of C18:0 fatty acids from milk fats being more comparable to that of C18:0 fatty acids from the adipose fats (Fig. 4b). The difference between the δ13C values of C18:0 and C16:0 fatty acids in milk is thus diminished, leading to less negative Δ13C values.