In the present study, both measurement periods were characterized by elevated concentrations of the supplemented isomers in milk lipids following supplementation with 5.7 g/d c 9,t 11-CLA and 6 g/d t 10,c 12-CLA (Tables 1 and 2). According to Baumgard et al. , elevated concentrations of t 10,c 12-CLA are linked to reductions in milk fat content and/or milk fat yield in a dose-dependent manner. We have previously shown that the given dose of CLA was potent towards reducing the content of milk fat significantly by 14.1% in period 1 and by 25.4% in period 2. Further, CLA led to a decrease in the milk fat yield by 17.1% in period 2 in early lactation . This is in accordance with data from the study by Moallem et al. , in which 4.7 g/d t 10,c 12-CLA and 4.7 g/d c 9,t 11-CLA of the same lipid-encapsulated supplement was used and showed similar reductions in milk fat content (13%) and milk fat yield (9%, insignificant) compared to period 1 of the present study. Further, our results are also supported by observations from Giesy et al. , who reported a reduction in milk fat content of approximately 14% by supplementing four CLA isomers as a mixture that, amongst other CLA isomers, contained 4.4 g/d or 8.6 g/d of t 10,c 12-CLA. Although the main effect on the milk fat-lowering effect is ascribed to t 10,c 12-CLA, a possible participation or interaction of the other isomers (e.g. c 9,t 11-CLA, c 8,t 10-CLA or c 11,t 13-CLA) cannot be excluded.
The reduction in milk fat content and milk fat yield in the present study is marked by a significant decrease in single DSFA (8:0, 10:0 and 12:0) as well as 16:0. The latter fatty acid originated to about 50% each from de novo- synthesis and from circulating fatty acids. Castaneda-Gutierrez et al.  showed similar results on single DSFA, ΣC16, and fatty acid ratios with the exception that the latter were unaffected when provided with 9.2 g/d t 10,c 12-CLA as part of a rumen-protected CLA-mix supplement as Ca-salts. Interestingly, Bernal-Santos et al.  used a CLA-mix consisting of four CLA isomers (c 9,t 11-CLA; t 10,c 12-CLA; t 8,c 10-CLA and c 11,t 13-CLA) that provided 8.8 g/d t 10,c 12-CLA as Ca-salts, and reported no significant effect on DSFA, except for ΣC16 fatty acids. In the study , the fatty acid ratios also remained unaffected. As proposed by Baumgard et al.  and Peterson et al. , low doses of t 10,c 12-CLA show no effect on Δ9-desaturase activity despite significant reduction of milk fat content and/or milk fat yield. In contrast, abomasal infusions of several doses of t 10,c 12-CLA resulted in alterations of fatty acid ratios indicating inhibition of Δ9-desaturase activity [7, 14, 34, 35]. The c 9-14:1/14:0 ratio provides a suitable estimation of Δ9 desaturase activity that is specific for the mammary gland and thus for milk lipids, given that 14:0 and c 9-14:1 are almost exclusively derived by de novo-synthesis in the mammary gland . In the present study, we could show a significant reduction of the c 9-14:1/14:0 ratio in period 1, indicating inhibition of Δ9 desaturase activity. This is in accordance with our observations on DSFA and ΣC16 in the present study. In addition, because the amount of c 12-18:1 was significantly elevated in period 1, it might have further enhanced the inhibition of Δ9 desaturase activity .
MFD, either diet induced or mediated by t 10,c 12-CLA, is caused by down-regulation of several transcription factors [13, 38] and enzymes involved in de novo-synthesis, desaturation, and elongation [14, 39] processes. Based on our gene expression analysis, we did not observe a treatment-related effect on genes coding for relevant enzymes or transcription factors in lipid metabolism in the mammary gland that could explain the reduction of the c 9-14:1/14:0 ratio.
T-18:1 acids, intermediates of partial hydrogenation of unsaturated fatty acids, especially of linoleic acid are often linked to diets that cause MFD . Several research groups using rumen-protected CLA, observed a significant increase of single 18:1 fatty acids (e.g., t 9-, t 10-, t 11-18:1) in milk fat [32, 33, 41]. The results of our study concur with these findings as we observed similar elevations in single t-18:1 acids which were close to significance in both periods (Tables 1 and 2).
Increased concentrations of t-18:1 acids, especially t 10-18:1 and t 11-18:1, suggest a poor protection rate of the supplemented CLA. Investigating the duodenal availability of the same lipid-encapsulated CLA supplement, Pappritz et al.  showed protection rates of 16% and 5% after providing 3 and 8 g/d of t 10,c 12-CLA, respectively, and assumed an impaired rumen protection due to the pelleting process. Because we used the same conditions for preparing of the CLA supplement, it is most likely that unprotected CLA serve as potential substrates for microbial alterations and, consequently, lead to elevated concentrations of t-18:1 acids. Since Shingfield et al.  recently showed that a mixture of t-18:1 acids, in particular t 10-18:1 and t 11-18:1, are capable of inducing MFD, it is most likely that the reduction of milk fat yield and milk fat content in the present study is caused by both t 10,c 12-CLA and, to a lesser extent by t 10-18:1.
CLA, originally identified in fried ground beef, gained considerable attention because of its anticarcinogenic effects . Other studies with CLA supplemented in feed either as a mixture or as pure isomers showed specific changes in body composition that are almost exclusively due to the t 10,c 12-CLA isomer [44, 45]. Jahreis et al.  reported in their summary that liver and adipose tissue, and to a certain extent muscle tissue are most affected by CLA. However, these changes are not consistent among species. The majority of studies investigating the effects of CLA on body composition and/or fatty acid distribution have mainly been conducted in rodents and pigs. The few studies in dairy cattle, concentrated on the mammary gland as the principal object of research.
In the present study, supplementation with CLA showed only marginal effects on the fatty acid distribution of the above mentioned tissues. Despite supplementing a mixture with equal amounts of both isomers, t 10,c 12-CLA was detected only in the mammary gland (Table 3) and retroperitoneal adipose tissue (Additional file 2), and not in the lipids of liver and M. longissimus. Kramer et al.  showed that the distribution of the different CLA isomers incorporated into liver and heart lipids in pigs is by no means equal for all lipid classes but rather specific for each individual CLA isomer. Furthermore, Kramer et al.  showed that the percentage of t 10,c 12-CLA is generally low in liver lipids compared to c 9,t 11-CLA. This observation has been previously reported [19, 48] and is explained as a preferable conversion of t 10,c 12-CLA through elongation and desaturation or elevated beta-oxidation in liver and muscle which finally results in lower concentrations than the supplementation might predict [44, 49]. Further, we have to concede that emerging isomerization products due to transesterification with BF3 may have reduced the already low concentration of t 10,c 12-CLA.
Mice fed diets with different amounts of CLA, in particular pure t 10,c 12-CLA or in combination with c 9,t 11-CLA, exhibited decreased concentrations of linoleic acid and increased accumulation of oleic acid in liver lipids which, in turn, led to significantly higher liver weights [16, 19, 50, 51]. This is in contrast to the results of the present study, where liver weights increased in a fashion typical during early lactation but independent of CLA supplementation . Furthermore, changes of the fatty acid distribution in liver lipids were limited to linoleic acid and c 12-18:1 in both periods, and to t 10-18:1 and DHA in period 1 (Table 4). Increased percentages of linoleic acid and DHA in liver lipids in the present study could be explained by a shift in the triglyceride:phospholipid ratio because phospholipids contain higher portions of long-chain fatty acids . Based on an average dry matter intake of 15.3 kg in both periods, the dose of the two supplemented CLA isomers at 0.04% is low in the present study compared to approximately 0.5% – 1% used in rodent or pig models . C 9,t 11- and t 10,c 12-CLA, administered as pure isomers are described to have opposite effects on body composition and fatty acid distribution in mice. For example, Kelley et al.  showed a decrease of oleic acid in total lipids of liver, retroperitoneal adipose tissue, and spleen after administration of 0.5% c 9,t 11-CLA, whereas 0.5% t 10,c 12-CLA increased oleic acid in liver and heart lipids, and decreased linoleic and arachidonic acid in liver lipids.
Studies in rodents showed that the retroperitoneal adipose tissue was most sensitive to CLA [54–56]. Although we observed a significant reduction of the retroperitoneal adipose tissue as part of the empty body weight in dairy cattle , this effect is not necessarily linked to a changed fatty acid distribution as the present study demonstrated.