2.1. In Vivo Experiment

In another study,¹⁵ 10 g of t10c12-CLA was found to exhibit the maximum inhibition rate of milk fat biosynthesis in dairy cows. To ensure efficacy and prevent deficiencies, 15 g of t10c12-CLA was used in this experiment. Since t10c12-CLA is prone to saturation by rumen microbiota, a rumen-protected coating was applied to the raw material. The proportions of each component in the conjugated linoleic acid mixture are listed in Supporting Table 1 (Qingdao Aohai Biotechnology Co., Ltd., Shandong, China), where the proportion of t10c12-CLA was above 36.0%. The coating of the raw materials was performed by Hangzhou Kangdequan Feed Co., Ltd. The coating efficiency and rumen protection rate were determined to be 40 and 85%, respectively. To ensure that 15 g of t10c12-CLA was administered per cow, 136.17 g of RP-CLA was administered orally.

The experiment was conducted at Yanqing Shounong Livestock Breeding Farm in Beijing. The Institutional Animal Care and Use Committee of China Agricultural University reviewed and approved the experimental protocol with approval number AW02113202–2–1.

Primiparous Holstein cows (n = 12) at 7 and 21 days postpartum were allocated into two groups: the control group (n = 6; Body condition score (BCS): 3.25 ± 0.16; mean ± SD) and the CLA group (n = 6; (BCS): 3.29 ± 0.19; mean ± SD), ensuring an even distribution between the groups. Cows in the CLA group were orally administered 136.17 g of RP-CLA daily from days 1 to 7 of the experiment, whereas cows in the control group were provided with a standard diet without CLA. Changes in various parameters were monitored continuously from days 8 to 14 of the experiment. Throughout the experimental period, all cows were provided with a total mixed ration (TMR) twice daily at 7 a.m. and 7 p.m. (see Table 1 for details). Milking was conducted 4 times daily at 6 a.m., noon, 6 p.m., and midnight, and ad libitum access to water was provided.

The data on feeding periods and milk production, including milk yield and milk composition, for all cows within 7 days after cessation of supplementation were collected from the Afifarm Management System and stored until further analysis.

Milk fat accounts for the largest proportion of energy value in milk, as reported by Tyrrell and Reid¹⁶ (total energy output (MJ/kg) = [0.384 × fat percentage + 0.223 × protein percentage + 0.199 × lactose percentage −0.108] × milk yield (kg)).