The interaction of essential fatty acids and conjugated linoleic acid on regulation of milk synthesis and the formation of milk ingredients from [13C6]-glucose during early lactation in dairy cows

1 SUPPLEMENTARY DATA 1 Supplemental Table S1. Amounts of daily abomasally infused supplements1 (Vogel et al., 2020) 2 Treatments CTRL2 EFA CLA2 EFA+CLA Supplementation Coconut oil3 Linseed oil4 Safflower oil5 Lutalin®6 Linseed oil4 Safflower oil5 Lutalin®6 Daily infused oils (g/d) Dosage lactation 76 78 4 38 78 4 38 Dosage dry period 38 39 2 19 39 2 19 Daily infused fatty acids (g/d) at dosage lactation7 18:3 cis-9, cis-12, cis-15 0.00 39.9 0.01 0.00 39.9 0.01 0.00 18:2 cis-9, cis-12 1.39 12.4 2.48 1.34 12.4 2.48 1.34 18:2 cis-9, trans-11 0.00 0.00 0.01 10.3 0.00 0.01 10.3 18:2 trans-10, cis-12 0.00 0.02 0.01 10.2 0.02 0.01 10.2 1Cows were supplemented daily with coconut oil (CTRL), linseed and safflower oil (EFA), Lutalin® (CLA, cis-9, trans-11 and trans-10, cis-12 CLA), or EFA+CLA. 2 2Addition of Vitamin E (0.06 g/d), Covitol 1360 (BASF, Ludwigshafen, Germany), to compensate for Vitamin E in linseed oil (0.07%) and safflower oil (0.035%). 3Sanct Bernhard, Bad Ditzenbach, Germany 4DERBY, Derby Spezialfutter GmbH, Münster, Germany 5GEFRO, Memmingen/Allgäu, Germany 6BASF, Ludwigshafen, Germany 7The dosage lactation was halved during dry period. 3 3 Supplemental Table S2. Fatty acid composition of the daily infused supplements during lactation1 (Vogel et al., 2020) 4 CTRL2 EFA3 CLA4 EFA+CLA5 Fatty acid (%) Coconut oil Linseed oil Safflower oil Total Lutalin® Total SFA 89.4 10.4 12.3 10.5 11.1 10.7 6:0 0.93 − − − − − 8:0 9.85 − − − − − 10:0 6.10 − − − − − 12:0 45.5 − − − − − 14:0 16.9 0.02 0.09 0.02 − 0.02 16:0 6.87 5.78 7.63 5.87 6.23 5.98 17:0 − 0.03 − 0.03 − 0.02 18:0 3.10 4.11 3.71 4.10 4.19 4.13 20:0 0.11 0.19 0.42 0.20 0.22 0.21 22:0 − 0.13 0.25 0.14 0.37 0.21 24:0 − 0.09 0.16 0.10 0.10 0.10 4 MUFA 8.35 21.5 23.5 21.6 29.5 24.1 16:1, cis-9 − 0.07 0.12 0.07 0.09 0.08 18:1, cis-9 8.35 20.7 22.4 20.8 28.3 23.1 18:1, cis-11 − 0.61 0.66 0.61 0.67 0.63 20:1, cis-11 − 0.16 0.23 0.16 0.51 0.27 24:1, cis-15 − − 0.11 0.01 − < 0.01 PUFA 1.83 67.1 62.5 66.9 3.75 46.9 18:2, cis-9, cis-12 1.83 15.9 62.0 18.2 3.52 13.5 18:2, cis-9, trans-12 − − − − 0.08 0.03 18:2, trans-9, cis-12 − − − − 0.07 0.02 18:3, cis-6, cis-9, cis-12 − − 0.05 < 0.01 0.07 0.02 18:3, cis-9, cis-12, cis-15 − 51.1 0.24 48.7 − 33.3 20:2, cis-11, cis-14 − 0.06 − 0.06 − 0.04 20:3, cis-11, cis-14, cis-17 − 0.02 − 0.02 − 0.02 22:5, cis-7, cis-10, cis-13, cis-16, cis-19 − − 0.28 0.01 − 0.01 CLA < 0.05 0.02 0.32 0.04 54.4 17.2 5 18:2, cis-9, trans-11 CLA − − 0.12 0.01 27.2 8.61 18:2, trans-10, cis-12 CLA − 0.02 0.20 0.03 27.0 8.56 18:2, cis-9, cis-11 CLA − − − − 0.23 0.07 1Dosage of oil supplementation was halved during the dry period in all groups, respectively. 5 2Control (CTRL, n=9): 76 g/d coconut oil (Bio-Kokosöl #665, Kräuterhaus Sanct Bernhard KG, Bad Ditzenbach, Germany) and 0.06 g/d Vitamin E 6 (Covitol®1360, BASF SE, Ludwigshafen, Germany), 1.48 MJ NEL/d 7 3Essential fatty acids (EFA, n = 9): 78 g/d linseed (DERBY® Leinöl #4026921003087, DERBY Spezialfutter GmbH, Münster, Germany) and 4 g/d 8 safflower oil (GEFRO Distelöl, GEFRO Reformversand Frommlet KG, Memmingen, Germany), comprised 0.06 g/d Vitamin E, 1.57 MJ NEL/d 9 4Conjugated linoleic acid (CLA, n = 10): 38 g/d Lutalin® (BASF SE, Ludwigshafen, Germany) and 0.06 g/d Vitamin E (Covitol®1360, BASF SE, 10 Ludwigshafen, Germany), 0.69 MJ NEL/d 11 5Essential fatty acids and conjugated linoleic acid (EFA+CLA, n = 10): 78 g/d linseed (DERBY® Leinöl #4026921003087, DERBY Spezialfutter 12 GmbH, Münster, Germany),4 g/d safflower oil (GEFRO Distelöl, GEFRO Reformversand Frommlet KG, Memmingen, Germany) and 38 g/d 13 Lutalin® (BASF SE, Ludwigshafen, Germany), comprised 0.06 g/d Vitamin E, 2.26 MJ NEL/d 14 6 Supplemental Table S3. Ingredients and chemical composition of the diets (Vogel et al., 15 2020) 16 Diet Item (g/kg of DM) Dry period1 Lactation Ingredients Corn silage 421 457 Straw 223 97 Compound feed DEFA2 (granulated) − 446 Dried sugar beet pulp 163 − Extracted soybean meal 99 − Rye grain 75 − Minerals and vitamins3 10 − Urea4 9 − Chemical composition NEL (MJ/kg DM)5 6.5 7.1 Crude fat 21 23 Crude fiber 219 173 Crude protein 141 146 Utilizable protein5 141 143 NFC 379 432 NDF 423 346 ADF 249 197 RNB56 0.0 0.5 7 1The dry period diet was fed from wk 6 to 1 before calving. The actual dry period started 40 ± 17 6 d days before birth. 18 2Ceravis AG, Malchin, Germany 19 Ingredients: 46.5% dried sugar beet pulp, 25.3+ extracted soybean meal, 23.8% grain of rye, 20 1.4% urea, 1.1% premix cow, 1.00% calcium, 0.37% phosphorus, 0.42% sodium, vitamins A, 21 D3, E, copper, ferric, zinc, manganese, cobalt, iodine, selenium 22 Chemical composition: 44.4% NFC, 24.1% crude protein, 21.6% NDF, 12.4% ADF, 9.3% 23 crude fiber, 8.2% crude ash, 1.8% crude fat, 7.9 MJ NEL/kg DM 24 3Concentrations of minerals and vitamins (KULMIN®MFV Plus, Bergophor 25 Futtermittelfabrik Dr. Berger GmbH & Co., KG, Kulmbach, Germany): 8.5% magnesium, 26 7.5% phosphorus, 6.5% sodium, 3.5% HCl-insoluble ash, 1.5% calcium; vitamins and trace 27 minerals per kg: 1,000,000 I.E. vitamin A, 200,000 I.E. vitamin D3, 10,000 mg vitamin E, 180 28 mg vitamin B1, 90 mg vitamin B2, 90 mg vitamin B6, 200 mg vitamin B5, 2500 mg vitamin 29 B3, 675 mg vitamin B12, 12 mg vitamin B9, 100 mg vitamin H, 2500 mg zinc, 3500 mg 30 manganese, 500 mg copper, 20mg cobalt, 75 mg iodine, 30 mg selenium as sodium selenite, 31 15 mg Se from Saccharomyces cerevisiae. 32 4Piarumin® (SKW Stickstoffwerke Piesteritz GmbH, Lutherstadt Wittenberg, Germany): 33 99% urea, 46.5% total nitrogen 34 5German Society of Nutrition Physiology (2001, 2008, 2009) and Deutsche 35 Landwirtschaftliche Gesellschaft (DLG, 2013) 36 6RNB = ruminal nitrogen balance 37 38 39 8 40 Supplemental Table S4. Fatty acid composition of the experimental diets (Vogel et al., 2020) 41 Diet Fatty acid (g/kg of DM) Dry period1 Lactation 10:0 0.01 0.01 12:0 0.03 0.04 14:0 0.18 0.12 15:0 0.04 0.04 16:0 4.53 4.73 16:1, cis-9 0.05 0.06 17:0 0.08 0.09 17:1, cis-9 0.01 0.01 18:0 0.60 0.63 18:1, cis-9 3.84 4.82 18:1, cis-11 0.21 0.28 18:2, cis-9, cis-12 9.32 9.63 18:3, cis-9, cis-12, cis-15 1.37 1.35 18:4, cis-6, cis-9, cis-12, cis-15 0.02 0.04 20:0 0.16 0.15 20:1, cis-11 0.06 0.08 20:2, cis-11, cis-14 0.02 0.05 21:0 0.02 0.01 22:0 0.25 0.18 22:1, cis-13 − 0.01 9 22:2, cis-13, cis-16 0.04 0.01 23:0 0.02 0.05 24:0 0.29 0.23 SFA2 6.21 6.27 MUFA3 4.17 5.27 PUFA4 10.77 11.08 Sum of n-3 fatty acids5 1.39 1.39 Sum of n-6 fatty acids6 9.38 9.69 Ratio n-6 to n-3 6.76 7.00 1The Dry period diet was fed from wk 6 to 0 before calving. 42 2Sum of 10:0; 12:0; 14:0; 15:0; 16:0; 17:0; 18:0; 20:0; 21:0; 22:0; 23:0 and 24:0 43 3Sum of 16:1 cis-9; 17:1 cis-9; 18:1 cis-9; 18:1 cis-11; 20:1 cis-11 and 22:1 cis-13 44 4Sum of 18:2 cis-9, cis-12; 18:3 cis-9, cis-12, cis-15; 18:4 cis-6, cis-9, cis-12, cis-15; 20:2 cis-45 11, cis-14 and 22:2 cis-13, cis-16 46 5Sum of 18:3 cis-9, cis-12, cis-15 and 18:4 cis-6, cis-9, cis-12, cis-15 47 6Sum of 18:2 cis-9, cis-12; 20:2, cis-11, cis-14 and 22:2 cis-13, cis-16 48 16 Supplemental Figure S2. Protein expression (western blot) of fatty acid binding protein 4 (FABP4; 15 kDa) in the mammary gland of cows. A) Test for linearity using 25, 30, 35, 40, 45 and 50 µg tissue protein. B) Regression analysis of the linearity blot. C) Protein expression in cows abomasally infused daily with coconut oil (CTRL), linseed and safflower oil (EFA), conjugated linoleic acid (CLA) or the combination of EFA and CLA (EFA+CLA in wk 9 postpartum. A) B) C) FABP4 (15 kDa) 17 Supplemental Figure S3. Protein expression (western blot) of sterol regulatory element-binding protein 1 (SREBP; 68 kDa) in the mammary gland of cows abomasally infused daily with coconut oil (CTRL), linseed and safflower oil (EFA), conjugated linoleic acid (CLA) or the combination of EFA and CLA (EFA+CLA in wk 9 postpartum. 18 Supplemental Figure S4. Milk yield immediately before and 2, 4, 6, 8, 10, 24, 36, 48, 60, and 72 h after i. v. infusion of [13C6]-glucose infusion for 4 h in cows abomasally infused daily with coconut oil ( CTRL; n = 9), linseed and safflower oil ( EFA; n = 9), CLA (▼ n = 10), or the combination of EFA and CLA ( EFA+CLA; n = 10) during late gestation and early lactation. Data are presented as LSM ± SE. 19 Supplemental Figure S5. Enrichment of 13C-glucose (mol % excess; MPE) in blood plasma at 60, 120, 150, 180, 210, and 240 min after [13C6]- glucose administration in cows abomasally infused daily with coconut oil ( CTRL; n = 9), linseed and safflower oil ( EFA; n = 9), CLA (▼ n = 10), or the combination of EFA and CLA ( EFA+CLA; n = 10) on d 21 postpartum. Data are presented as LSM ± SE. Sample collection was described by Vogel et al. (2021). The values from 150 min to 240 min after the start of the tracer infusion show the plateau of 13C-glucose enrichment. The area under the curve (AUC; LSM ± SE were 58.5 ± 2.5, 57.1 ± 2.2, 59.9 ± 2.3, and 60.3 ± 2.1 MPE × min for groups CTRL, EFA, CLA, and EFA+CLA, respectively) calculated from this showed no group effects: P = 0.9 for EFA treatment, P = 0.3 for CLA treatment, and P = 0.7 for EFA × CLA interaction. The statistical model is described in the paper. 20 Supplemental Figure S6. Relationship between 13C enrichment in milk fat (TG; triglycerides) and in glycerol of the TG in milk. For the comparative measurement, 20 cows (Hötger, K., H. M. Hammon, C. Weber, S. Görs, A. Tröscher, R. M. Bruckmaier, and C. C. Metges. 2013. Supplementation of conjugated linoleic acid in dairy cows reduces endogenous glucose production during early lactation. J. Dairy Sci. 96:2258-2270. http://dx.doi.org/10.3168/jds.2012-6127.) were used and measurements were taken at 6, 16, and 26 hours after the start of the i. v. [13C6]-glucose infusion.