The effect of conjugated linoleic acid (CLA) supplements on the oxidative and antioxidative status of periparturient and lactating dairy cows

Enhanced metabolism, as present in the periparturient period in dairy cows, might result in an increase of reactive oxygen species (ROS), which are thought to play an important role in various production diseases, such as mastitis. Since antioxidative properties of conjugated linoleic acids (CLA) are discussed controversially, the aim of the presented study was to investigate effects of supplementation of conjugated linoleic acids (CLA) to dairy cows and heifers on their antioxidative and oxidative status. Conjugated linoleic acids are widely used in dairy cattle feeding, to stabilise metabolism in the transition period, by reducing milk fat synthesis. This study was carried out with German Holstein cows (n = 32) and heifers (n = 13), randomly assigned to three groups, receiving a control fat (n = 14), 50 g (n = 15) or 100 g (n = 16) of a mixture of CLA isomers, containing 10 % trans‑10,cis‑12 CLA and 10 % cis‑9,trans‑11 CLA. Stearic acid was used as control fat. The CLA mixture was encapsulated with palmitic acid and stearic acid, both linked to glycerine to protect CLA supplements from rumen biohydrogenation. Blood samples were taken from the jugular vein at day -21, 1, 21, 70, 105, 140, 182, 224 and 252 relative to calving. Animals were supplemented between d 1 and d 182. Statistical analysis between groups was done separately for period one (d -21 until d 105) and period two (d 105 until d 252). Differences with days in milk (DIM) and between cows and heifers of all groups were also evaluated. In the obtained blood samples the antioxidative and oxidative statuses of these animals were determined. For identification of the antioxidative status the ferric reducing ability of plasma was determined, as well as serum concentrations of α‑tocopherol and retinol. The latter were determined to rule out the possibility that differences in antioxidative or oxidative statuses were caused by nutritional antioxidants. Serum cholesterol concentrations were determined for the calculation of the α‑tocopherol:cholesterol ratio. The oxidative status was determined by measurement of the amount of hydroxide radicals in the serum and lipid peroxidation end products, using the thiobarbituric acid (TBA) test. Peroxidative damage to proteins was also determined by means of N′‑formylkynurenine and bityrosine. Plasma sulfhydryl groups (SH groups) were determined as an indicator of oxidative stress. Stage of lactation influenced parameters of the antioxidative status over both periods one and two. During period one the antioxidative parameters significantly decreased towards parturition with a subsequent increase; FRAP (p < 0.001), retinol (p < 0.001), α‑tocopherol (p < 0.001) and α‑tocopherol:cholesterol mass ratio (p < 0.001). During period two, the antioxidative status significantly decreased towards the end of the trial, including FRAP (p < 0.001), retinol (p < 0.001) and α‑tocopherol (p < 0.001), but not the α‑tocopherol:cholesterol mass ratio, which remained constant (p = 0.35). Oxidative parameters were significantly influenced by stage of lactation over both periods. During period one, the amount of hydroperoxides (p < 0.001), TBARS (p < 0.001) and plasma SH groups (p < 0.001) showed the inverse pattern of the antioxidative substances, with a significant increase towards parturition and a subsequent decrease. The decrease continued through to period two, with an increase during depletion period for amount of hydroperoxides (p < 0.001), TBARS (p < 0.001) and plasma SH groups (p < 0.001). In period one protein peroxidation parameters, bityrosine (p < 0.001) and N′‑formylkynurenine (p < 0.001) declined towards parturition and increased during early lactation, whereas in period two both parameters declined towards the end of the trial; bityrosine (p < 0.001) and N′‑formylkynurenine (p < 0.001). There were no significant differences in the determined antioxidative or oxidative parameters between animals of different groups, apart from SH group concentrations. The cows of the CLA 100 group tended to have higher SH group concentrations than the other groups during period one (p = 0.085) and had significantly higher SH group concentrations during period two (p = 0.021). Since animals of the CLA 100 group exhibited the highest average plasma concentrations of SH groups before the CLA supplementation started, it is unlikely that the observed group effect for SH groups was due to the CLA supplementation. Differences between cows and heifers were detected for serum α‑tocopherol concentrations during periods one (p = 0.012) and period two (p < 0.001), with heifers exhibiting higher α‑tocopherol concentrations and a higher α‑tocopherol:cholesterol mass ratio (both periods p < 0.001). During period two, heifers were detected with lower serum concentrations of hydroperoxides in the sample (p = 0.005) and TBARS (p = 0.013). Plasma SH group concentrations were significantly higher in heifers than in cows in periods one (p = 0.024) and two (p = 0.004). A likely explanation for the observed differences is the longer pasture feed period before the study start for the heifers of the present study (about three months) compared to multiparous cows, which were only on pasture for a few weeks, between dry off and enrolment for the present study, leading to a higher vitamin E supply in heifers. Overall, antioxidants were diminished during the periparturient period in dairy cows and heifers of this trial, while oxidants, such as hydroxides, and lipid peroxidation were markedly increased. Even though antioxidants were reduced, the antioxidant defence system could effectively protect proteins from peroxidation. The results of this study also revealed that long term supplementation of a commercial mixture of CLA isomers, containing equal amounts of trans‑10,cis‑12 CLA and cis‑9,trans‑11 CLA, had no effect on the antioxidative and oxidative status of dairy cows during the periparturient period and on-going lactation. This was possibly due to the too low CLA dosage combined with insufficient protection of the CLA supplement against ruminal biohydration, leading to poor bioavailability of the CLA isomers. Results suggest further that vitamin E requirements of pregnant and lactating dairy cows exceed the German recommendations published by GFE (2001) by far.