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Effects of abomasal infusion of essential fatty acids together with conjugated linoleic acid in late and early lactation on performance, milk and body composition, and plasma metabolites in dairy cows

Open ArchivePublished:May 28, 2020DOI:https://doi.org/10.3168/jds.2019-18065

      ABSTRACT

      Rations including high amounts of corn silage are currently very common in dairy production. Diets with corn silage as forage source result in a low supply of essential fatty acids, such as α-linolenic acid, and may lead to low conjugated linoleic acid (CLA) production. The present study investigated the effects of abomasal infusion of essential fatty acids, especially α-linolenic acid, and CLA in dairy cows fed a corn silage–based diet on performance, milk composition, including fatty acid (FA) pattern, and lipid metabolism from late to early lactation. Rumen-cannulated Holstein cows (n = 40) were studied from wk 9 antepartum to wk 9 postpartum and dried off 6 wk before calving. The cows were assigned to 1 of 4 treatment groups. Cows were abomasally supplemented with coconut oil (CTRL, 76 g/d), linseed and safflower oil (EFA, 78 and 4 g/d; linseed/safflower oil ratio = 19.5:1; n-6/n-3 FA ratio = 1:3), Lutalin (CLA, 38 g/d; BASF SE, Ludwigshafen, Germany; isomers cis-9,trans-11 and trans-10,cis-12 each 10 g/d) or EFA+CLA. Milk composition was analyzed weekly, and blood samples were taken several times before and after parturition to determine plasma concentrations of metabolites related to lipid metabolism. Liver samples were obtained by biopsy on d 63 and 21 antepartum and on d 1, 28, and 63 postpartum to measure triglyceride concentration. Body composition was determined after slaughter. Supplementation of CLA reduced milk fat concentration, increased body fat mass, and improved energy balance (EB) in late and early lactation, but EB was lowest during late lactation in the EFA group. Cows with CLA treatment alone showed an elevated milk citrate concentration in early lactation, whereas EFA+CLA did not reveal higher milk citrate but did have increased acetone. Milk protein was increased in late lactation but was decreased in wk 1 postpartum in CLA and EFA+CLA. Milk urea was reduced by CLA treatment during the whole period. After calving, the increase of nonesterified fatty acids in plasma was less in CLA groups; liver triglycerides were raised lowest at d 28 in CLA groups. Our data confirm an improved metabolic status with CLA but not with exclusive EFA supplementation during early lactation. Increased milk citrate concentration in CLA cows points to reduced de novo FA synthesis in the mammary gland, but milk citrate was less affected in EFA+CLA cows, indicating that EFA supplementation may influence changes in mammary gland FA metabolism achieved by CLA.

      Key words

      INTRODUCTION

      Essential fatty acids, particularly α-linolenic acid (ALA) and linoleic acid (LA) and their metabolites eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid, as well as arachidonic acid (ARA), are important for several biological functions, such as immune functions, blood coagulation, vascular resistance, enzyme activities, cell proliferation and differentiation, and receptor expression (
      • Moallem U.
      Invited review: Roles of dietary n-3 fatty acids in performance, milk fat composition, and reproductive and immune systems in dairy cattle.
      ). Mammals, including ruminants, are not able to synthesize essential fatty acids; therefore, they must be obtained from food (

      Palmquist, D. L. 2010. Essential fatty acids in ruminant diets. Pages 127–141 in Proc. 21st Annu. Ruminant Nutr. Symp., Gainesville, FL. Institute of Food and Agricultural Sciences, University of Florida, Gainesville.

      ;
      • Spector A.A.
      • Kim H.Y.
      Discovery of essential fatty acids.
      ). In addition, the isomer-specific, health-promoting effects of CLA in humans are well known (
      • Nagao K.
      • Yanagita T.
      Conjugated fatty acids in food and their health benefits.
      ;
      • Shokryazdan P.
      • Rajion M.A.
      • Meng G.Y.
      • Boo L.J.
      • Ebrahimi M.
      • Royan M.
      • Sahebi M.
      • Azizi P.
      • Abiri R.
      • Jahromi M.F.
      Conjugated linoleic acid: A potent fatty acid linked to animal and human health.
      ), and ruminant products are a natural source for CLA isomers (
      • Bauman D.
      • Baumgard L.
      • Corl B.
      • Griinari D.J.
      Biosynthesis of conjugated linoleic acid in ruminants.
      ). Conjugated linoleic acids are produced in the rumen by essential fatty acid transformation, and therefore rumen CLA production depends on essential fatty acid intake (
      • Chilliard Y.
      • Ferlay A.
      • Doreau M.
      Effect of different types of forages, animal fat or marine oils in cow’s diet on milk fat secretion and composition, especially conjugated linoleic acid (CLA) and polyunsaturated fatty acids.
      ;
      • Shingfield K.J.
      • Bernard L.
      • Leroux C.
      • Chilliard Y.
      Role of trans fatty acids in the nutritional regulation of mammary lipogenesis in ruminants.
      ). Some CLA isomers reveal metabolic effects in dairy cows, such as milk fat reduction and glucose-sparing effect (
      • Bauman D.
      • Baumgard L.
      • Corl B.
      • Griinari D.J.
      Biosynthesis of conjugated linoleic acid in ruminants.
      ;
      • Hötger K.
      • Hammon H.M.
      • Weber C.
      • Gors S.
      • Troscher A.
      • Bruckmaier R.M.
      • Metges C.C.
      Supplementation of conjugated linoleic acid in dairy cows reduces endogenous glucose production during early lactation.
      ). These effects are able to improve the energy status of dairy cows, especially in the transition period (
      • Trevisi E.
      • Ferrari A.
      • Piccioli-Capelliand F.
      • Bertoni G.
      Energy balance indexes and blood changes of dairy cows supplemented with rumen protected CLA in late pregnancy and early lactation.
      ;
      • von Soosten D.
      • Meyer U.
      • Piechotta M.
      • Flachowsky G.
      • Dänicke S.
      Effect of conjugated linoleic acid supplementation on body composition, body fat mobilization, protein accretion, and energy utilization in early lactation dairy cows.
      ). In recent studies, lower milk protein and urea levels, which are possibly related to higher body protein accretion and nitrogen retention, were found following CLA supplementation (
      • von Soosten D.
      • Meyer U.
      • Piechotta M.
      • Flachowsky G.
      • Dänicke S.
      Effect of conjugated linoleic acid supplementation on body composition, body fat mobilization, protein accretion, and energy utilization in early lactation dairy cows.
      ;
      • Haubold S.
      • Kröger-Koch C.
      • Starke A.
      • Tuchscherer A.
      • Tröscher A.
      • Kienberger H.
      • Rychlik M.
      • Bernabucci U.
      • Trevisi E.
      • Hammon H.M.
      Effects of abomasal infusion of essential fatty acids and conjugated linoleic acid on performance and fatty acid, antioxidative, and inflammatory status in dairy cows.
      ).
      Over the last few decades, diets for dairy cows have changed dramatically. With increasing milk yield, it has become a common practice to feed TMR containing high amounts of corn silage rather than pasture-based feeding systems (
      • Barkema H.W.
      • von Keyserlingk M.A.G.
      • Kastelic J.P.
      • Lam T.J.G.M.
      • Luby C.
      • Roy J.-P.
      • LeBlanc S.J.
      • Keefe G.P.
      • Kelton D.F.
      Invited review: Changes in the dairy industry affecting dairy cattle health and welfare.
      ). Therefore, the fatty acid (FA) supply and the intake of essential fatty acids and level of rumen and tissue CLA production have also changed. (
      • Chilliard Y.
      • Ferlay A.
      • Doreau M.
      Effect of different types of forages, animal fat or marine oils in cow’s diet on milk fat secretion and composition, especially conjugated linoleic acid (CLA) and polyunsaturated fatty acids.
      ;
      • Shingfield K.J.
      • Bernard L.
      • Leroux C.
      • Chilliard Y.
      Role of trans fatty acids in the nutritional regulation of mammary lipogenesis in ruminants.
      ). Cows on pasture take up high amounts of essential fatty acids, especially ALA (
      • Chilliard Y.
      • Ferlay A.
      • Doreau M.
      Effect of different types of forages, animal fat or marine oils in cow’s diet on milk fat secretion and composition, especially conjugated linoleic acid (CLA) and polyunsaturated fatty acids.
      ;
      • Khiaosa-Ard R.
      • Kreuzer M.
      • Leiber F.
      Apparent recovery of C18 polyunsaturated fatty acids from feed in cow milk: A meta-analysis of the importance of dietary fatty acids and feeding regimens in diets without fat supplementation.
      ), and CLA production in the rumen and mammary gland tissue increases with pasture feeding (
      • Kelly M.L.
      • Kolver E.S.
      • Bauman D.E.
      • Van Amburgh M.E.
      • Muller L.D.
      Effect of intake of pasture on concentrations of conjugated linoleic acid in milk of lactating cows.
      ;
      • Ferlay A.
      • Martin B.
      • Pradel P.
      • Coulon J.B.
      • Chilliard Y.
      Influence of grass-based diets on milk fatty acid composition and milk lipolytic system in Tarentaise and Montbeliarde cow breeds.
      ;
      • Lahlou M.N.
      • Kanneganti R.
      • Massingill L.J.
      • Broderick G.A.
      • Park Y.
      • Pariza M.W.
      • Ferguson J.D.
      • Wu Z.
      Grazing increases the concentration of CLA in dairy cow milk.
      ). The importance of n-3 FA in dairy production has already been reviewed (

      Palmquist, D. L. 2010. Essential fatty acids in ruminant diets. Pages 127–141 in Proc. 21st Annu. Ruminant Nutr. Symp., Gainesville, FL. Institute of Food and Agricultural Sciences, University of Florida, Gainesville.

      ;
      • Moallem U.
      Invited review: Roles of dietary n-3 fatty acids in performance, milk fat composition, and reproductive and immune systems in dairy cattle.
      ). Corn silage is rich in LA but contains low levels of fat and ALA (
      • Chilliard Y.
      • Ferlay A.
      • Doreau M.
      Effect of different types of forages, animal fat or marine oils in cow’s diet on milk fat secretion and composition, especially conjugated linoleic acid (CLA) and polyunsaturated fatty acids.
      ;
      • Khan N.A.
      • Yu P.
      • Ali M.
      • Cone J.W.
      • Hendriks W.H.
      Nutritive value of maize silage in relation to dairy cow performance and milk quality.
      ). In high-yielding herds, fat supplementation is a common feeding strategy to improve energy intake. However, commercial ruminal inert fats containing palmitic acid are usually used (

      Palmquist, D. L. 2010. Essential fatty acids in ruminant diets. Pages 127–141 in Proc. 21st Annu. Ruminant Nutr. Symp., Gainesville, FL. Institute of Food and Agricultural Sciences, University of Florida, Gainesville.

      ), and low levels of n-3 FA are available to cows (
      • Chilliard Y.
      • Ferlay A.
      • Doreau M.
      Effect of different types of forages, animal fat or marine oils in cow’s diet on milk fat secretion and composition, especially conjugated linoleic acid (CLA) and polyunsaturated fatty acids.
      ;
      • Khan N.A.
      • Yu P.
      • Ali M.
      • Cone J.W.
      • Hendriks W.H.
      Nutritive value of maize silage in relation to dairy cow performance and milk quality.
      ). Therefore, the forage type strongly affects the intake of essential fatty acids and the n-6/n-3 FA ratio in the diet, as well as the CLA status of dairy cows (
      • Chilliard Y.
      • Ferlay A.
      • Doreau M.
      Effect of different types of forages, animal fat or marine oils in cow’s diet on milk fat secretion and composition, especially conjugated linoleic acid (CLA) and polyunsaturated fatty acids.
      ;
      • Shingfield K.J.
      • Bernard L.
      • Leroux C.
      • Chilliard Y.
      Role of trans fatty acids in the nutritional regulation of mammary lipogenesis in ruminants.
      ;
      • Khan N.A.
      • Yu P.
      • Ali M.
      • Cone J.W.
      • Hendriks W.H.
      Nutritive value of maize silage in relation to dairy cow performance and milk quality.
      ).
      It is not known how administration of combined essential fatty acids and CLA administration affects performance and lipid metabolism in high-yielding dairy cows around calving, when cows are subjected to significant metabolic stress. In addition, most studies on either essential fatty acid or CLA supplementation are short-term, and no long-term studies are available with a combined essential fatty acid and CLA supplementation from late gestation through subsequent lactation. It is obvious that the nutrient supply during late gestation affects early lactation performance and metabolism (
      • Drackley J.K.
      ADSA Foundation Scholar Award. Biology of dairy cows during the transition period: The final frontier?.
      ). Therefore, the aim of the present study was to investigate the long-term effects of a combined essential fatty acid and CLA supplementation on lactation performance, milk and body composition, and lipid metabolism in dairy cows when starting the supplementation late in the previous lactation. The cows received a corn silage–based TMR with low fat and especially low n-3 FA intake. The treatments focused on the supply of FA that provide essential fatty acids (mainly ALA), CLA, or the combination of both. Such a treatment model refers to the supply of essential fatty acids and related rumen and tissue CLA production in dairy cows receiving fresh grass or on pasture (
      • Kelly M.L.
      • Kolver E.S.
      • Bauman D.E.
      • Van Amburgh M.E.
      • Muller L.D.
      Effect of intake of pasture on concentrations of conjugated linoleic acid in milk of lactating cows.
      ;
      • Ferlay A.
      • Martin B.
      • Pradel P.
      • Coulon J.B.
      • Chilliard Y.
      Influence of grass-based diets on milk fatty acid composition and milk lipolytic system in Tarentaise and Montbeliarde cow breeds.
      ;
      • Lahlou M.N.
      • Kanneganti R.
      • Massingill L.J.
      • Broderick G.A.
      • Park Y.
      • Pariza M.W.
      • Ferguson J.D.
      • Wu Z.
      Grazing increases the concentration of CLA in dairy cow milk.
      ). To avoid rumen degradation of the supplemented FA, all FA were infused into the abomasum. We hypothesized that an elevated combined intake of essential fatty acids and CLA would change performance, milk composition including FA pattern, and lipid metabolism of dairy cows during the transition from late pregnancy to early lactation. Doses for the supplied essential fatty acids (linseed and safflower oil in a ratio of 19.5:1; providing an n-6/n-3 FA ratio of 1:3 in the supplement mixture) and CLA were recently evaluated in a companion dose-response study in mid-lactating dairy cows (
      • Haubold S.
      • Kröger-Koch C.
      • Starke A.
      • Tuchscherer A.
      • Tröscher A.
      • Kienberger H.
      • Rychlik M.
      • Bernabucci U.
      • Trevisi E.
      • Hammon H.M.
      Effects of abomasal infusion of essential fatty acids and conjugated linoleic acid on performance and fatty acid, antioxidative, and inflammatory status in dairy cows.
      ).

      MATERIALS AND METHODS

      Animals, Husbandry, and Fatty Acid Supplementation

      All experimental procedures were carried out in accordance with the German Animal Welfare Act and were approved by the relevant Department for Animal Welfare Affairs of the state of Mecklenburg-West Pomerania (Landesamt für Landwirtschaft, Lebensmittelsicherheit und Fischerei Mecklenburg-Vorpommern, Germany; LALLF M-V/TSD/7221.3–1-038/15).
      Forty Holstein cows were purchased in blocks of 8 cows from a local farm in approximately wk 18 of gestation in their second lactation. The cows were kept in a freestall barn at the Leibniz Institute for Farm Animal Biology (FBN), Dummerstorf, Germany. During the preparation time of the study, cows were adapted to the new environmental conditions and diet and were surgically fitted with rumen cannulas (#2C or #1C 4-inch, Bar Diamond Inc., Parma, ID) 9 to 8 wk before beginning of the experiments and abomasal infusion lines [Teflon tube (inner diameter 6 mm) with 2 perforated Teflon flanges (outer diameter 120 mm)] 3 to 2 wk before beginning of the experiments, as previously described (
      • Haubold S.
      • Kröger-Koch C.
      • Starke A.
      • Tuchscherer A.
      • Tröscher A.
      • Kienberger H.
      • Rychlik M.
      • Bernabucci U.
      • Trevisi E.
      • Hammon H.M.
      Effects of abomasal infusion of essential fatty acids and conjugated linoleic acid on performance and fatty acid, antioxidative, and inflammatory status in dairy cows.
      ). Two cows per block were assigned to 1 of 4 treatment groups with comparable projected milk production (11,101 ± 1,118 kg milk/305 d in second lactation, mean ± SD), BW (662 ± 56 kg, mean ± SD), and predicted calving interval (395 ± 39 d, mean ± SD). Two cows calved prematurely and had to be excluded from the study. Cows were daily abomasally supplemented from d 63 antepartum (AP) until d 63 postpartum (PP) with coconut oil, providing no ALA or CLA (CTRL, n = 9; Bio-Kokosöl #665, Kräuterhaus Sanct Bernhard KG, Bad Ditzenbach, Germany); a combination of linseed oil (Derby Leinöl #4026921003087, Derby Spezialfutter GmbH, Münster, Germany) and safflower oil (Gefro Distelöl, Gefro Reformversand Frommlet KG, Memmingen, Germany) to provide an n-6/n-3 FA ratio of 1:3 in the supplement mixture (EFA, n = 9); Lutalin (CLA treatment, n = 10; cis-9,trans-11 and trans-10,cis-12 CLA, 10 g/d each; BASF SE, Ludwigshafen, Germany); or a combination of EFA and CLA (EFA+CLA, n = 10). The amounts of daily infused supplements are given in Table 1. Treatments were infused using 60-mL catheter-tip syringes twice a day (2 equal portions) at 0700 and 1630 h. All supplements were liquified by heating to 38°C to allow infusion. The fatty acid compositions of the added lipids are shown in Supplemental Table S1 (https://doi.org/10.3168/jds.2019-18065). During the dry period, each dose was halved. Sampling started at wk 10 AP and was terminated at wk 9 PP.
      Table 1Amounts of daily abomasally infused supplements
      Cows were supplemented daily with coconut oil (CTRL), linseed and safflower oil (EFA), Lutalin (CLA, cis-9,trans-11 and trans-10,cis-12; BASF SE, Ludwigshafen, Germany), or EFA+CLA.
      SupplementationTreatment
      CTRL
      Addition of vitamin E (0.06 g/d), Covitol 1360 (BASF SE), to compensate for the vitamin E in linseed oil (0.07%) and safflower oil (0.035%).
      EFACLA
      Addition of vitamin E (0.06 g/d), Covitol 1360 (BASF SE), to compensate for the vitamin E in linseed oil (0.07%) and safflower oil (0.035%).
      EFA+CLA
      Coconut oil
      Sanct Bernhard, Bad Ditzenbach, Germany.
      Linseed oil
      Derby, Derby Spezialfutter GmbH, Münster, Germany.
      Safflower oil
      Gefro, Memmingen/Allgäu, Germany.
      Lutalin
      BASF.
      Linseed oil
      Derby, Derby Spezialfutter GmbH, Münster, Germany.
      Safflower oil
      Gefro, Memmingen/Allgäu, Germany.
      Lutalin
      BASF.
      Daily infused oils (g/d)
       Lactation dosage767843878438
       Dry period dosage383921939219
      Daily infused fatty acids (g/d) at the lactation dosage
      The lactation dosage was halved during the dry period.
       18:3 cis-9,cis-12,cis-150.0039.90.010.0039.90.010.00
       18:2 cis-9,cis-121.3912.42.481.3412.42.481.34
       18:2 cis-9,trans-110.000.000.0110.30.000.0110.3
       18:2 trans-10,cis-120.000.020.0110.20.020.0110.2
      1 Cows were supplemented daily with coconut oil (CTRL), linseed and safflower oil (EFA), Lutalin (CLA, cis-9,trans-11 and trans-10,cis-12; BASF SE, Ludwigshafen, Germany), or EFA+CLA.
      2 Addition of vitamin E (0.06 g/d), Covitol 1360 (BASF SE), to compensate for the vitamin E in linseed oil (0.07%) and safflower oil (0.035%).
      3 Sanct Bernhard, Bad Ditzenbach, Germany.
      4 Derby, Derby Spezialfutter GmbH, Münster, Germany.
      5 Gefro, Memmingen/Allgäu, Germany.
      6 BASF.
      7 The lactation dosage was halved during the dry period.

      Feeding, Feed Samples and Analyses, and Body Condition

      Cows were fed with corn silage–based TMR during lactation (wk −22 to −7 AP and wk 1 to 9 PP) and during the dry period (wk −6 to −1 AP). Diets were fed ad libitum at 0600 h, and the cows had free access to water as well as trace-mineralized salt blocks. After calving, a calcium bolus (Rumin CaDL; Wirtschaftsgenossenschaft Deutscher Tierärzte eG, Garbsen, Germany) as well as 300 mL/d of 1,2-propanediol (Propylenglykol USP; Dr. Pieper Technologie- und Produktentwicklung GmbH, Wuthenow, Germany) were administered intraruminally on 3 consecutive days. Feed samples of TMR and corn silage were taken weekly, and samples from concentrates and straw were taken every 2 mo for the determination of DM content. Additional samples of single components were stored at −20°C, and nutrient compositions were determined at the Agricultural Analysis and Research Institute (LUFA), Rostock, Germany. Based on analysis of the individual TMR components, the compositions of the lactation and dry period diets were formulated and calculated according to the feeding standards of the German Society of Nutrition Physiology (
      • Gesellschaft für Ernährungsphysiologie (German Society of Nutrition Physiology)
      ,

      Gesellschaft für Ernährungsphysiologie (German Society of Nutrition Physiology). 2008. New equations for predicting metabolisable energy of grass and maize products for ruminants. Communications of the Committee for Requirement Standards of the Society of Nutrition Physiology. Proc. Soc. Nutr. Physiol. 17:191–198.

      ,
      • Gesellschaft für Ernährungsphysiologie (German Society of Nutrition Physiology)
      New equations for predicting metabolisable energy of compound feeds for cattle. Communications of the Committee for Requirement Standards of the Society of Nutrition Physiology.
      ) and the German Agricultural Society (Deutsche Landwirtschaftliche Gesellschaft;

      DLG (Deutsche Landwirtschafts-Gesellschaft, German Agricultural Society). 2013. Leitfaden zur Berechnung des Energiegehaltes bei Einzel-und Mischfuttermitteln für die Schweine-und Rinderfütterung (Guidelines for calculation of energy content of single and mixed feedstuff for pigs and cattle). Stellungnahme des DLG-Arbeitskreises Futter und Fütterung.

      ). The ingredients and chemical compositions of the diets with a planned low fat content are shown in Table 2. The fatty acid compositions of the diets were determined via GC and are shown in Table 3. For extraction and direct fatty acid methylation of diets, a modified method from
      • Sukhija P.S.
      • Palmquist D.L.
      Rapid method for determination of total fatty acid content and composition of feedstuffs and feces.
      using 5% methanolic HCl and 6% K2CO3 solution was applied. The fatty acid analysis of the FAME was performed using capillary GC with a CP-Sil 88 CB column (100 m × 0.25 mm; Agilent, Santa Clara, CA;
      • Kalbe C.
      • Priepke A.
      • Nürnberg G.
      • Dannenberger D.
      Effects of long-term microalgae supplementation on muscle microstructure, meat quality, and fatty acid composition in growing pigs.
      ). Individual daily feed intake was recorded as disappearance of feed from troughs connected to an electronic scale to which access was controlled by an individual transponder (Institute for Agricultural Engineering and Animal Husbandry ILT, Bavarian State Research Center for Agriculture LfL, Freising, Germany).
      Table 2Ingredients and chemical compositions of the diets
      Item (g/kg of DM)Diet
      LactationDry period
      The dry period diet was fed from wk 6 to wk 1 before calving.
      Ingredients
       Corn silage457421
       Straw97223
       Compound feed DEFA (granulated)
      Ceravis AG, Malchin, Germany. Ingredients: 46.5% dried sugar beet pulp, 25.3% extracted soybean meal, 23.8% grain of rye, 1.4% urea, 1.1% premix cow, 1.00% calcium, 0.37% phosphorus, 0.42% sodium, vitamins A, D3, E, copper, ferric, zinc, manganese, cobalt, iodine, selenium. Chemical composition: 44.4% NFC, 24.1% CP, 21.6% NDF, 12.4% ADF, 9.3% crude fiber, 8.2% crude ash, 1.8% crude fat, 7.9 MJ of NEL/kg of DM.
      446
       Dried sugar beet pulp163
       Extracted soybean meal99
       Grain of rye75
       Mineral-vitamin mixture
      Kulmin MFV Plus (Bergophor Futtermittelfabrik Dr. Berger GmbH & Co. KG, Kulmbach, Germany): 8.5% magnesium, 7.5% phosphorus, 6.5% sodium, 3.5% HCl-insoluble ash, 1.5% calcium, additives: vitamins A, D3, E, B1, B2, B6, B5, B3, B12, B9, H, zinc, manganese, copper, cobalt, iodine, selenium, and Saccharomyces cerevisiae.
      10
       Urea
      Piarumin (SKW Stickstoffwerke Piesteritz GmbH, Lutherstadt Wittenberg, Germany): 99% urea, 46.5% total nitrogen.
      9
      Chemical composition
       NEL
      German Society of Nutrition Physiology (2001, 2008, 2009) and DLG (2013).
      (MJ/kg DM)
      7.16.5
       Crude fat2321
       Crude fiber173219
       Crude protein146141
       Utilizable protein
      German Society of Nutrition Physiology (2001, 2008, 2009) and DLG (2013).
      143141
       NFC432379
       NDF346423
       ADF197249
       RNB
      German Society of Nutrition Physiology (2001, 2008, 2009) and DLG (2013).
      RNB = ruminal nitrogen balance.
      0.50.0
      1 The dry period diet was fed from wk 6 to wk 1 before calving.
      2 Ceravis AG, Malchin, Germany. Ingredients: 46.5% dried sugar beet pulp, 25.3% extracted soybean meal, 23.8% grain of rye, 1.4% urea, 1.1% premix cow, 1.00% calcium, 0.37% phosphorus, 0.42% sodium, vitamins A, D3, E, copper, ferric, zinc, manganese, cobalt, iodine, selenium. Chemical composition: 44.4% NFC, 24.1% CP, 21.6% NDF, 12.4% ADF, 9.3% crude fiber, 8.2% crude ash, 1.8% crude fat, 7.9 MJ of NEL/kg of DM.
      3 Kulmin MFV Plus (Bergophor Futtermittelfabrik Dr. Berger GmbH & Co. KG, Kulmbach, Germany): 8.5% magnesium, 7.5% phosphorus, 6.5% sodium, 3.5% HCl-insoluble ash, 1.5% calcium, additives: vitamins A, D3, E, B1, B2, B6, B5, B3, B12, B9, H, zinc, manganese, copper, cobalt, iodine, selenium, and Saccharomyces cerevisiae.
      4 Piarumin (SKW Stickstoffwerke Piesteritz GmbH, Lutherstadt Wittenberg, Germany): 99% urea, 46.5% total nitrogen.
      5 German Society of Nutrition Physiology (2001, 2008, 2009) and

      DLG (Deutsche Landwirtschafts-Gesellschaft, German Agricultural Society). 2013. Leitfaden zur Berechnung des Energiegehaltes bei Einzel-und Mischfuttermitteln für die Schweine-und Rinderfütterung (Guidelines for calculation of energy content of single and mixed feedstuff for pigs and cattle). Stellungnahme des DLG-Arbeitskreises Futter und Fütterung.

      .
      6 RNB = ruminal nitrogen balance.
      Table 3Fatty acid composition of the experimental diets
      Fatty acid (g/kg of DM)Diet
      LactationDry period
      The dry period diet was fed from wk 6 to 0 before calving.
      10:00.010.01
      12:00.040.03
      14:00.120.18
      15:00.040.04
      16:04.734.53
      16:1, cis-90.060.05
      17:00.090.08
      17:1, cis-90.010.01
      18:00.630.60
      18:1, cis-94.823.84
      18:1, cis-110.280.21
      18:2, cis-9,cis-129.639.32
      18:3, cis-9,cis-12,cis-151.351.37
      18:4, cis-6,cis-9,cis-12,cis-150.040.02
      20:00.150.16
      20:1, cis-110.080.06
      20:2, cis-11,cis-140.050.02
      21:00.010.02
      22:00.180.25
      22:1, cis-130.01
      22:2, cis-13,cis-160.010.04
      23:00.050.02
      24:00.230.29
      SFA
      Sum 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.
      6.276.21
      MUFA
      Sum 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.
      5.274.17
      PUFA
      Sum 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-11,cis-14; and 22:2 cis-13,cis-16.
      11.0810.77
      Sum of n-3 fatty acids
      Sum of 18:3 cis-9,cis-12,cis-15 and 18:4 cis-6,cis-9,cis-12,cis-15.
      1.391.39
      Sum of n-6 fatty acids
      Sum of 18:2 cis-9,cis-12; 20:2, cis-11,cis-14; and 22:2 cis-13,cis-16.
      9.699.38
      Ratio of n-6/n-37.006.76
      1 The dry period diet was fed from wk 6 to 0 before calving.
      2 Sum 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.
      3 Sum 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.
      4 Sum 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-11,cis-14; and 22:2 cis-13,cis-16.
      5 Sum of 18:3 cis-9,cis-12,cis-15 and 18:4 cis-6,cis-9,cis-12,cis-15.
      6 Sum of 18:2 cis-9,cis-12; 20:2, cis-11,cis-14; and 22:2 cis-13,cis-16.
      The feed efficiency for milk production (FEMY) was calculated as kilograms of milk per kilograms of DMI and feed efficiency for ECM production (FEECM) as kilograms of ECM per kilograms of DMI (
      • Moallem U.
      Future consequences of decreasing marginal production efficiency in the high-yielding dairy cow.
      ). According to the German Society of Nutrition Physiology (2001), the following formula was used to calculate the energy balance (EB):
      EB MJ NEL/d = NEL intake − NEL maintenance − NEL gestation − NEL milk production,


      NEL intake (MJ of NEL/d) = kg of DMI × MJ of NEL/kg of DM + energy content provided by the supplements,


      NEL maintenance (MJ of NEL/d) = 0.293 MJ of NEL × kg of BW0.75,


      NEL gestation (MJ of NEL/d) = 0.044 × e0.0165 × t MJ of NEL, where t is the day of gestation, and


      NEL milk production (MJ of NEL/d) = kg of ECM × 3.14 MJ of NEL.


      Body weight, BCS, and back fat thickness (BFT) were measured after the morning milking once per week. The BCS was scored based on a 5-point scale according to
      • Edmonson A.J.
      • Lean I.J.
      • Weaver L.D.
      • Farver T.
      • Webster G.
      A body condition scoring chart for Holstein dairy cows.
      , and BFT was determined via ultrasonic measurements (SonoSite Titan; Fujifilm SonoSite Inc., Bothell, WA) described by
      • Schröder U.J.
      • Staufenbiel R.
      Invited review: Methods to determine body fat reserves in the dairy cow with special regard to ultrasonographic measurement of backfat thickness.
      .

      Milk Sampling and Analyses

      Cows were milked twice daily at 0630 and 1800 h, and milk yield was recorded electronically after each milking. Colostrum samples from the first milking and pooled milk samples from one evening and the successive morning milking were taken weekly during late and early lactation and analyzed by the Landeskontrollverband für Leistungs- und Qualitätsprüfung Mecklenburg-Vorpommern e.V. (Güstrow, Germany). Determination of milk protein, milk fat, and milk lactose was performed using an infrared spectrophotometric method (MilkoScan FT6000, Foss GmbH, Hamburg, Germany) and SCC by a fluorescence-optical counting system (Fossomatic FC, Foss GmbH). According to
      • Reist M.
      • Erdin D.
      • von Euw D.
      • Tschuemperlin K.
      • Leuenberger H.
      • Delavaud C.
      • Chilliard Y.
      • Hammon H.M.
      • Kuenzi N.
      • Blum J.W.
      Concentrate feeding strategy in lactating dairy cows: Metabolic and endocrine changes with emphasis on leptin.
      , the following formula was used to calculate the ECM:
      ECM (kg) = (0.038 × g of crude fat + 0.024 × g of CP + 0.017 × g of lactose) × kg of milk/3.14.


      Colostrum and milk were centrifuged at 50,000 × g (4°C, 20 min), and milk fat was removed. In colostrum samples protein was precipitated with 1.5 M perchloric acid and 2 M calcium carbonate and centrifuged at 13,000 × g (4°C, 10 min). The whey was stored at −20°C until the milk urea content was determined weekly during lactation by photometric measurements (ABX Pentra 400; Horiba ABX SAS, Montpellier, France) using the kit #LT-UR 0010 from Labor+Technik, Eberhard Lehmann GmbH (Berlin, Germany). The citrate concentration in milk samples was determined at wk −10 and −7 AP and weekly PP at the Institut für Analytik, Hygiene und Produktqualität (MQD, Güstrow, Germany) using a commercial enzymatic kit (#10139076035) from R-Biopharm AG (Darmstadt, Germany). Milk acetone was determined weekly PP at MQD by the Skalar method using a continuous flow analyzer (SAN++, Skalar Analytic GmbH, Erkelenz, Germany), following the procedure described by
      • de Roos A.P.
      • van den Bijgaart H.J.
      • Horlyk J.
      • de Jong G.
      Screening for subclinical ketosis in dairy cattle by Fourier transform infrared spectrometry.
      . The fatty acid composition of milk fat was determined in milk samples from wk −10 and −7 AP, as well as in first colostrum milking after calving and in milk samples from wk 4 and 8 PP at the Bavarian Center for Biomolecular Mass Spectrometry (BayBioMS), Technical University of Munich (Freising, Germany). Single fatty acids in milk fat were determined via lipid extraction and gas chromatography (Agilent CP7420, select FAME 100 × 0.25-mm × 0.25-μm column) with flame ionization detection, as described by
      • Firl N.
      • Kienberger H.
      • Rychlik M.
      Validation of the sensitive and accurate quantitation of the fatty acid distribution in bovine milk.
      . Methylation was performed by the trimethylsulfonium hydroxide method. The apparent transfer efficiencies of ALA and cis-9,trans-11 and trans-10,cis-12 CLA were estimated by dividing the amount of FA in milk fat (minus the CTRL yields) by the amounts of the infused FA (
      • Moallem U.
      • Vyas D.
      • Teter B.B.
      • Delmonte P.
      • Zachut M.
      • Erdman R.A.
      Transfer rate of α-linolenic acid from abomasally infused flaxseed oil into milk fat and the effects on milk fatty acid composition in dairy cows.
      ). Fatty acids representing the product and substrate for Δ9-desaturase were used to calculate the desaturase indexes (
      • Castañeda-Gutiérrez E.
      • Overton T.R.
      • Butler W.R.
      • Bauman D.E.
      Dietary supplements of two doses of calcium salts of conjugated linoleic acid during the transition period and early lactation.
      ).

      Blood and Liver Sampling and Analyses

      Blood samples were taken on d 63, 42, 35, 28, 21, and 10 before expected calving, on d 1 PP, and then once weekly up to d 56 immediately after morning milking before feeding, via jugular vein puncture using the Vacuette system (Greiner Bio-One International GmbH, Kremsmünster, Austria). Samples were immediately placed on ice and centrifuged within 30 min (at 1,565 × g for 20 min at 4°C), and the harvested plasma was stored at −20°C until analysis. Evacuated tubes containing sodium fluoride in combination with potassium oxalate as an anticoagulant were used to measure the plasma concentrations of nonesterified fatty acids (NEFA), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and total cholesterol (TC). Plasma metabolites were analyzed using an automatic spectrophotometer (ABX Pentra 400; Horiba) and respective kits: NEFA, #434 91795 (acyl-CoA synthetase − acyl-CoAoidase method) from Wako Chemicals GmbH (Neuss, Germany); TG, #A11A01640 (lipoprotein lipase–glycerin kinase−glycerin-phosphate-oxidase method), LDL-C, #A11A01638 (direct measurement of cholesterol in LDL by the cholesterol esterase and cholesterol oxidase and LDL cleavage), and HDL-C, #A11A01636 (direct measurement of cholesterol in HDL by accelerator selective detergent method with cholesterol esterase) from Horiba; and TC, #553-126 (cholesterol oxidase method) from mti Diagnostics GmbH (Idstein, Germany).
      Liver tissue samples were obtained by needle biopsy on d −63 and −21 AP, on d 1 and 28 PP, and during slaughter on d 63 PP, as previously described (
      • Weber C.
      • Hametner C.
      • Tuchscherer A.
      • Losand B.
      • Kanitz E.
      • Otten W.
      • Singh S.P.
      • Bruckmaier R.M.
      • Becker F.
      • Kanitz W.
      • Hammon H.M.
      Variation in fat mobilization during early lactation differently affects feed intake, body condition, and lipid and glucose metabolism in high-yielding dairy cows.
      ), to measure the TG concentration. Liver TG concentration was determined using a Triglyceride Quantification Fluorometric Kit (#MAK266, Merck, Darmstadt, Germany).

      Slaughtering and Body Composition

      The cows were slaughtered on d 63 PP in the experimental abattoir of the FBN, which was approved by the EU and the German quality management system QS (QS Qualität und Sicherheit GmbH, Bonn, Germany). After morning milking, cows were weighed, transported to the slaughter facilities, stunned with a bolt gun, and exsanguinated. The head, mammary gland, feet, and skin with the tail were detached first. Thereafter, the mammary gland was weighed. The full gastrointestinal tract was removed, and the liver, kidneys, spleen, pancreas, and retroperitoneal adipose depot were dissected and weighed. Adherent mesenteric fat at the intestine and the omental adipose depot were cut off and weighed. Subcutaneous adipose tissue depots (from the sternum and perineal fat) were manually dissected and weighed. The hot and cold carcass weights were measured as described by
      • Pfuhl R.
      • Bellmann O.
      • Kühn C.
      • Teuscher F.
      • Ender K.
      • Wegner J.
      Beef versus dairy cattle: A comparison of feed conversion, carcass composition, and meat quality.
      . Total fat was calculated as the sum of the omental, mesenteric, retroperitoneal, and subcutaneous fat.

      Statistical Analyses

      Statistical analyses were performed using SAS for Windows, release 9.4 (SAS Institute Inc. Cary, NC). Performance data and plasma concentrations of metabolites were analyzed using the MIXED procedure by repeated-measures ANOVA containing EFA (level: yes, no), CLA (level: yes, no), time (levels: day or week relative to calving), block (levels: 1 to 5), and the respective interactions (EFA × CLA; EFA × time; CLA × time; EFA × CLA × time) as fixed effects. The calving interval and projected milk yield during the second lactation were used as covariates. Repeated measures on each cow were considered by using the repeated statement of the MIXED procedure with compound symmetry (timeline d or wk) covariance structure. The levels of the repeated variable time for performance data were late lactation (wk −10 to −7 AP), dry period (wk −6 to −1 AP), transition period (wk −3 AP to 4 PP), postpartum or early lactation (wk 1 to 8 PP), and entire period (wk −10 AP to 8 PP). Alternatively, for the metabolites, the AP period (d −63 to −1 AP) was evaluated. Data were analyzed for each observation period separately. The least squares means (LSM) and their standard errors (SE) were computed for each fixed effect in the ANOVA model to display the results. Additionally, group differences in these LSM were tested using the Tukey-Kramer procedure. The SLICE statement of the MIXED procedure was used to assess partitioned analyses of the LSM for interactions. All differences with P < 0.05 were considered significant.

      RESULTS

      Animal Performance

      In late lactation, DMI declined (P < 0.05) in CLA-treated cows from wk −10 to wk −8 AP by 7% and tended to be lower (P < 0.1) in wk −8 in CLA- than non-CLA-treated cows. (CLA × time interaction: P = 0.06; Figure 1A). After drying off, DMI decreased (P < 0.001) and after calving increased (P < 0.001) in all groups. At wk 7 and 8 PP, DMI was lower (P < 0.05) in CLA than in non-CLA-treated groups. The NEL intake (Table 4) showed a similar time pattern to that of DMI, and PP NEL intake was lower (P < 0.05) in CLA than that in EFA (wk 7) and in CTRL (wk 8). In late lactation, milk yield (Figure 1B) declined (P < 0.001) in all groups but not in EFA, and after parturition, milk yield increased (P < 0.001) without significant group differences. In late lactation, EB was higher (P < 0.05) in the EFA+CLA cows (wk −8 AP) and CLA cows (wk −7 AP; CLA effect, P < 0.05) than that in EFA cows. During early lactation, EB increased (P < 0.001) in all groups and was less negative (P < 0.01) in both CLA-treated groups than in CTRL and EFA (Figure 1C). Energy-corrected milk decreased during late lactation (P < 0.05) in CLA and EFA+CLA cows, was affected by CLA treatment (P < 0.05), and was lower in wk −8 AP in EFA+CLA than in EFA and CTRL cows and, in wk −7 AP, was lower in CLA and EFA+CLA cows than in EFA cows (Figure 1D). In early lactation, ECM increased (P < 0.05) from wk 1 to wk 2 in all groups and was reduced (P ≤ 0.01) in CLA and EFA+CLA compared with the CTRL and EFA groups. The FEMY showed a similar time pattern to that of milk yield and FEECM as ECM (Table 4). In late lactation, FEMY declined more (CLA × time interaction P < 0.001) in CLA-treated groups compared with non-CLA-treated groups, and FEECM was higher in EFA (P < 0.05) compared with EFA+CLA (wk −8 and −7 AP) and CLA (wk −7 AP). In early lactation, FEECM was lower (P < 0.001) in CLA-treated than in non-CLA-treated groups.
      Figure thumbnail gr1
      Figure 1DMI (A), milk yield (B), energy balance (EB; C), and ECM yield (D) in cows supplemented abomasally daily with coconut oil (○ CTRL; n = 9), linseed and safflower oil (▴ EFA; n = 9), Lutalin (▾ CLA cis-9,trans-11 and trans-10,cis-12; BASF SE, Ludwigshafen, Germany; n = 10), or EFA+CLA (♦; n = 10) from wk 9 antepartum until wk 8 postpartum. Data are presented as LSM ± SE; LSM with different lowercase letters (a–c) differ (P < 0.05) at the respective time point. Y = CLA effect at respective time point. Significant (P < 0.05) effects for DMI during late lactation (time), dry period (time), transition (time; EFA × CLA × time interaction), postpartum (time; CLA), and during the entire study (time). Significant (P < 0.05) effects for milk yield during late lactation (time; CLA × time interaction) and early lactation (time). Significant (P < 0.05) effects for energy balance during late lactation (time; CLA × time interaction), dry period (time), transition (time; CLA; CLA × time interaction), postpartum (time; CLA), and during the entire study (time; CLA; CLA × time interaction). Significant (P < 0.05) effects for ECM during late lactation (time; CLA; EFA × time interaction; CLA × time interaction) and early lactation (time; CLA; CLA × time interaction).
      Table 4Performance data during late lactation, dry and transition periods, postpartum or early lactation, and over the entire study of cows supplemented abomasally daily with coconut oil (CTRL; n = 9), linseed and safflower oil (EFA; n = 9), Lutalin
      Conjugated linoleic acid, cis-9,trans-11 and trans-10,cis-12; BASF SE, Ludwigshafen, Germany.
      (CLA; n = 10), or the combination (EFA+CLA; n = 10) from wk 9 antepartum until wk 8 postpartum
      Variable
      Values are presented as LSM ± SE. FEMY = feed efficiency for milk production; FEECM = feed efficiency for ECM production; BFT = back fat thickness.
      TimeTreatmentFixed effect, P-value
      CTRLEFACLAEFA+ CLAEFACLAEFA × CLATimeEFA × timeCLA × time
      NEL intake, MJ of NEL/dLate lactation120.2 ± 4.6116.7 ± 4.2114.2 ± 3.9113.8 ± 3.90.70.30.70.120.60.05
      Dry period80.6 ± 3.181.9 ± 1.981.4 ± 2.884.1 ± 2.80.50.60.80.0010.70.5
      Transition period93.9 ± 3.393.6 ± 3.191.2 ± 2.993.4 ± 2.90.80.60.70.0010.90.6
      Postpartum120.8 ± 3.8119.4 ± 3.7109.8 ± 3.5115.2 ± 3.50.60.040.30.0010.50.4
      Entire study106.6 ± 3.3106.4 ± 3.1101.2 ± 3.0104.6 ± 2.90.60.20.60.0010.90.05
      FEMY, kg of milk/kg of DMILate lactation0.96 ± 0.111.10 ± 0.101.11 ± 0.090.98 ± 0.090.90.90.190.0010.060.01
      Early lactation2.25 ± 0.102.25 ± 0.102.37 ± 0.092.43 ± 0.090.70.130.70.0010.50.9
      FEECM, kg of ECM/kg of DMILate lactation1.08 ± 0.101.19 ± 0.091.05 ± 0.080.95 ± 0.090.90.130.20.0010.040.001
      Early lactation2.31 ± 0.11
      Means within a row with different lowercase superscripts differ (P < 0.05).
      2.28 ± 0.10
      Means within a row with different lowercase superscripts differ (P < 0.05).
      1.84 ± 0.10
      Means within a row with different lowercase superscripts differ (P < 0.05).
      1.95 ± 0.10
      Means within a row with different lowercase superscripts differ (P < 0.05).
      0.70.0010.50.0011.00.6
      BW, kgLate lactation701 ± 21666 ± 20676 ± 19670 ± 190.30.60.50.0010.90.12
      Dry period742 ± 22700 ± 21710 ± 20718 ± 200.40.70.20.0010.30.4
      Transition period690 ± 20654 ± 19664 ± 18672 ± 180.50.80.30.0010.70.4
      Postpartum634 ± 18604 ± 18622 ± 17621 ± 170.40.90.40.0010.80.16
      Entire study685 ± 20649 ± 19663 ± 18665 ± 180.40.90.30.0010.80.04
      BCSLate lactation3.62 ± 0.113.50 ± 0.113.48 ± 0.103.29 ± 0.100.160.100.70.0010.40.9
      Dry period3.72 ± 0.123.73 ± 0.123.62 ± 0.113.62 ± 0.111.00.40.90.0010.10.02
      Transition period3.54 ± 0.123.55 ± 0.113.50 ± 0.113.50 ± 0.111.00.71.00.0010.70.8
      Postpartum3.12 ± 0.113.13 ± 0.113.15 ± 0.103.10 ± 0.100.81.00.80.0010.70.19
      Entire study3.43 ± 0.113.41 ± 0.103.38 ± 0.103.31 ± 0.100.70.50.80.0010.110.02
      BFT, mmLate lactation13.4 ± 1.012.2 ± 0.912.0 ± 0.911.3 ± 0.90.30.20.80.0010.180.6
      Dry period15.3 ± 1.114.3 ± 1.015.8 ± 1.014.6 ± 1.00.30.70.90.0010.50.4
      Transition period14.7 ± 1.114.2 ± 1.015.5 ± 1.014.5 ± 1.00.50.60.80.0010.90.5
      Postpartum12.1 ± 1.011.7 ± 0.913.5 ± 0.912.6 ± 0.90.50.20.80.0010.90.001
      Entire study13.5 ± 1.012.7 ± 0.914.0 ± 0.913.0 ± 0.90.30.70.90.0010.80.001
      a,b Means within a row with different lowercase superscripts differ (P < 0.05).
      1 Conjugated linoleic acid, cis-9,trans-11 and trans-10,cis-12; BASF SE, Ludwigshafen, Germany.
      2 Values are presented as LSM ± SE. FEMY = feed efficiency for milk production; FEECM = feed efficiency for ECM production; BFT = back fat thickness.
      During the last 10 wk of gestation, all groups gained similar BW (time: P < 0.001), BFT (time: P < 0.001), and BCS (time: P < 0.05; Table 4), reaching the same levels at calving. After calving, BCS and BFT decreased (P < 0.001) continuously, but BW rapidly decreased (P < 0.001) in all groups up to wk 3 and declined to a lesser extent until the end of the study. In wk 6 PP, we detected a CLA effect on BW reduction, and the decline of BW relative to wk −1 AP was less (P < 0.05) in the CLA group compared with CTRL. In wk 7 and 8 PP, BFT was higher (P < 0.05) in CLA- than in non-CLA-treated groups.

      Milk Composition

      Cows receiving CLA showed reduced milk fat concentration (P < 0.001; Figure 2A) compared with the concentration in the CTRL and EFA groups, by an average reduction of 40% AP and 50% PP. After calving, a decrease in milk fat concentration was found until wk 2 in all groups (P < 0.001), and the milk fat concentration continued to decrease in both CLA-treated groups until wk 4 PP and remained at that low concentration until the end of the study. Milk fat yield declined (P < 0.001) in the CLA groups and was reduced (P < 0.001) in both CLA groups in late lactation (wk −9 to −7 AP) by more than 50% and in early lactation on average by 50% compared with the CTRL and EFA groups (Table 5). The milk citrate concentration increased (P < 0.05) during late lactation in CLA groups and showed higher concentration in CLA than non-CLA-treated groups (P < 0.05; Figure 2B). During early lactation, milk citrate decreased (P < 0.05) in cows not treated with CLA. Milk citrate was higher (P < 0.05) in CLA than in non-CLA-treated groups during the whole PP period, was higher (P < 0.05) in CLA-treated than in EFA and CTRL cows in wk 2, 5, 7, and 8 PP, and was highest in CLA cows in wk 6 PP. Milk acetone indicated a CLA effect (P < 0.05) and increased the highest (P < 0.001) in EFA+CLA at 2 wk PP (Table 5). Milk protein concentration during late lactation increased in both CLA groups more than in CTRL and EFA (CLA × time, P < 0.05), and in wk −7 AP the milk protein concentration was higher (P < 0.05) in EFA+CLA and CLA cows than in EFA cows (Figure 2C). After calving we detected a CLA effect (P < 0.05) for the whole period, and in wk 1 protein concentration was lower (P < 0.05) in both CLA groups than in EFA. In addition, we detected a CLA effect with lower milk protein concentration in CLA- than in non-CLA-treated groups in wk 7 PP. The milk urea concentration peaked before drying off (P < 0.001) in all groups (Figure 2D). During early lactation, urea in milk decreased (P < 0.05) in all groups but not in EFA cows. Milk urea was reduced (P < 0.05) by CLA treatment (P < 0.05) during the whole period and was affected by EFA × time interaction (P < 0.05). Milk urea concentration in EFA was higher than in both CLA groups in wk 4 PP, was higher than in CLA in wk 7 PP, and was higher than in CLA and CTRL in wk 8 PP. Lactose concentration and yield (kg/d) decreased in late lactation (P < 0.01) and increased after the onset of lactation until wk 3 PP (Table 5). In late lactation, lactose yield declined more distinctly (CLA × time interaction: P < 0.001) in CLA than in non-CLA-treated groups, and lactose concentration in wk −7 AP was lower (P < 0.05) in CLA- than in non-CLA-treated groups. During early lactation, the lactose concentration in wk 8 was higher (P < 0.05) in EFA- than in non-EFA-treated groups. The SCC increased (P < 0.05) in the CLA-treated groups before drying off, was higher (P < 0.05) in CLA-treated than in non-CLA-treated groups at wk −7, and remained unchanged in early lactation in all groups (Table 5).
      Figure thumbnail gr2
      Figure 2Milk fat concentration (A), milk citrate concentration (B), milk protein concentration (C), and milk urea concentration (D) in cows supplemented abomasally daily with coconut oil (○ CTRL; n = 9), linseed and safflower oil (▴ EFA; n = 9), Lutalin (▾ CLA cis-9,trans-11 and trans-10,cis-12; BASF SE, Ludwigshafen, Germany; n = 10), or EFA+CLA (♦; n = 10) from wk 9 antepartum until wk 8 postpartum. Data are presented as LSM ± SE; LSM with different lowercase letters (a, b) differ (P < 0.05) at the respective time point. X = EFA effect at respective time point. Y = CLA effect at respective time point. Significant (P < 0.05) effects for milk fat concentration during late lactation (time; CLA; CLA × time interaction) and early lactation (time; CLA; CLA × time interaction). Significant (P < 0.05) effects for milk citrate concentration during late lactation (CLA × time interaction) and early lactation (time; CLA; EFA × time interaction). Significant (P < 0.05) effects for milk protein concentration during late lactation (time; CLA × time interaction) and early lactation (time; CLA). Significant (P < 0.05) effects for milk urea concentration during late lactation (time) and early lactation (time; CLA; EFA × time interaction).
      Table 5Milk components during late and early lactation of cows supplemented abomasally daily with coconut oil (CTRL; n = 9), linseed and safflower oil (EFA; n = 9), Lutalin
      Conjugated linoleic acid, cis-9,trans-11 and trans-10,cis-12; BASF SE, Ludwigshafen, Germany.
      (CLA; n = 10), or the combination (EFA+CLA; n = 10) from wk 9 antepartum until wk 8 postpartum
      Variable
      Values are presented as the LSM ± SE.
      TimeTreatmentFixed effect, P-value
      CTRLEFACLAEFA+CLAEFACLAEFA × CLATimeEFA × timeCLA × time
      Milk fat, kg/dLate lactation0.72 ± 0.06
      Means within a row with different lowercase superscripts differ (P < 0.05).
      0.78 ± 0.06
      Means within a row with different lowercase superscripts differ (P < 0.05).
      0.54 ± 0.05
      Means within a row with different lowercase superscripts differ (P < 0.05).
      0.51 ± 0.05
      Means within a row with different lowercase superscripts differ (P < 0.05).
      0.80.0010.50.0010.150.001
      Early lactation1.48 ± 0.08
      Means within a row with different lowercase superscripts differ (P < 0.05).
      1.41 ± 0.08
      Means within a row with different lowercase superscripts differ (P < 0.05).
      0.78 ± 0.07
      Means within a row with different lowercase superscripts differ (P < 0.05).
      0.86 ± 0.07
      Means within a row with different lowercase superscripts differ (P < 0.05).
      1.00.0010.30.0010.60.001
      Milk acetone, mmol/LEarly lactation0.05 ± 0.020.06 ± 0.020.07 ± 0.020.13 ± 0.020.180.040.20.110.60.2
      Milk protein, kg/dLate lactation0.67 ± 0.050.64 ± 0.050.72 ± 0.050.64 ± 0.050.30.70.60.0010.130.18
      Early lactation1.17 ± 0.051.15 ± 0.051.07 ± 0.041.17 ± 0.040.40.30.190.0010.90.9
      Milk lactose, %Late lactation4.71 ± 0.114.61 ± 0.104.51 ± 0.094.45 ± 0.090.40.060.80.0020.50.3
      Early study4.78 ± 0.044.85 ± 0.044.73 ± 0.044.79 ± 0.040.10.20.80.0010.70.3
      Milk lactose, kg/dLate lactation0.75 ± 0.080.81 ± 0.070.79 ± 0.070.71 ± 0.070.90.60.40.0010.050.001
      Early study1.76 ± 0.091.75 ± 0.081.67 ± 0.081.81 ± 0.080.40.90.40.0010.060.8
      SCC × 1,000/mLLate lactation248 ± 193400 ± 172502 ± 163454 ± 1660.80.40.60.0020.70.18
      Early study167 ± 82224 ± 79222 ± 75203 ± 740.80.80.60.60.40.6
      a–c Means within a row with different lowercase superscripts differ (P < 0.05).
      1 Conjugated linoleic acid, cis-9,trans-11 and trans-10,cis-12; BASF SE, Ludwigshafen, Germany.
      2 Values are presented as the LSM ± SE.

      Milk Fatty Acid Pattern

      The concentration of ALA in milk fat increased 5-fold in EFA and 12-fold in EFA+CLA after beginning of supplementation and was higher in both EFA groups than in CTRL and CLA (P < 0.001) before drying off and in early lactation. Enrichment of ALA was higher in EFA+CLA than in EFA at wk −7 AP and at wk 4 and 8 PP (P < 0.001; Figure 3A). During the whole supplementation period, the EPA and DPA concentrations in milk fat were higher (P < 0.05) in EFA and EFA+CLA than in CTRL and CLA (Figure 3B and C). The EPA concentration was higher (P < 0.05) in wk −7 AP and wk 4 and 8 PP in EFA than in EFA+CLA; the DPA concentration was higher (P < 0.05) in wk −7 AP but lower in wk 1 (P < 0.05) in EFA than in EFA+CLA. The LA concentration in milk fat increased the most in EFA+CLA (P < 0.001; 1.9-fold from wk −10 to wk −7 AP) and was lowest in CTRL (P < 0.01) in late and early lactation (Figure 3D). The concentrations of ARA but not of dihomo-γ-linolenic acid (DGLA) in milk fat were higher (P < 0.001) before drying off in the CTRL and EFA groups than those in the CLA and EFA+CLA groups (Figure 3E and 3F). The highest concentration of ARA was reached in the colostrum sample (P < 0.001), and the concentrations of ARA and DGLA were higher in CTRL and CLA (not significant for DGLA) than those in EFA and EFA+CLA (not significant for ARA) in colostrum (P < 0.05). In addition, DGLA concentrations were lower (P < 0.05) in EFA- than in non-EFA-treated groups in wk −7 AP and wk 4 PP, and ARA concentrations were lower in CLA- than in non-CLA-treated groups in wk 8 PP. Milk fat concentrations of cis-9,trans-11 and trans-10,cis-12 CLA increased (P < 0.001) in late lactation (2.1-fold for cis-9,trans-11 and 3.4-fold for trans-10,cis-12) and after calving (3.4-fold for cis-9,trans-11 and 2.8-fold for trans-10,cis-12) in both CLA-treated groups, and concentrations were higher in CLA-treated than in non-CLA-treated groups (P < 0.001; Figure 3G and 3H). The FA composition in milk fat (%) and milk fatty acid yield (g/kg of milk) for all analyzed FA are presented in Supplemental Tables S2 and S3 (https://doi.org/10.3168/jds.2019-18065).
      Figure thumbnail gr3
      Figure 3Milk fat concentrations of α-linolenic acid (ALA; A), eicosapentaenoic acid (EPA; B), docosapentaenoic acid (DPA; C), linoleic acid (LA; D), dihomo-γ-linolenic acid (DGLA; E), arachidonic acid (ARA; F), cis-9,trans-11 CLA (G), and trans-10,cis-12 CLA (H) in cows supplemented abomasally daily with either coconut oil (○ CTRL; n = 9), linseed and safflower oil (▴ EFA; n = 9), Lutalin (▾ CLA cis-9,trans-11 and trans-10, cis-12; BASF SE, Ludwigshafen, Germany; n = 10), or EFA+CLA (♦; n = 10) from wk 9 antepartum until wk 8 postpartum. Data are presented as the LSM ± SE. LSM with different superscripts (a–c) differ (P < 0.05) at the respective time point. X = EFA effect at respective time point. Y = CLA effect at respective time point. Significant (P < 0.05) effects for ALA concentration during late and early lactation (time; EFA; CLA; EFA × CLA; EFA × time; CLA × time interactions). Significant (P < 0.05) effects for EPA concentration during late and early lactation (time; EFA; CLA; EFA × CLA; EFA × time; CLA × time interactions). Significant (P < 0.05) effects for DPA concentration during late lactation (time; EFA; CLA; EFA × time; CLA × time interactions) and early lactation (time; EFA; EFA × time interaction). Significant (P < 0.05) effects for LA concentration during late and early lactation [time; EFA; CLA; EFA × CLA (only early lactation); EFA × time; CLA × time interactions]. Significant (P < 0.05) effects for DGLA concentration during late lactation (time) and early lactation (EFA). Significant (P < 0.05) effects for ARA concentration during late lactation (time; CLA; CLA × time interaction) and early lactation (time; EFA; EFA × time interaction). Significant (P < 0.05) effects for cis-9,trans-11 CLA and trans-10,cis-12 CLA concentrations during late and early lactation, respectively (time; CLA; CLA × time interaction).
      The apparent transfer efficiencies of ALA and cis-9,trans-11 and trans-10,cis-12 CLA changed with time (P < 0.001), and the lowest enrichment of these FA was detected in wk 1 PP (P < 0.001; ALA 26 ± 5.6% in EFA+CLA and 15 ± 5.6% in EFA cows; trans-10,cis-12 CLA 4.3 ± 4.1% in CLA cows and 4.3 ± 3.5% EFA+CLA cows; cis-9,trans-11 CLA was close to 0 in both CLA groups). In wk −7 AP, the apparent transfer efficiency of ALA was lower (P < 0.01) in EFA+CLA (30 ± 5.6%) than in EFA (58 ± 5.9%), whereas in early lactation, the efficiencies were quite similar for EFA (wk 4: 66 ± 5.9%; wk 8: 60 ± 5.9%) and for EFA+CLA (wk 4: 65 ± 5.6%; wk 8: 51 ± 5.6%). The apparent transfer efficiency of trans-10,cis-12 CLA in the EFA+CLA and CLA groups was lower (P < 0.05) in wk −7 AP (12.9 ± 3.5 and 9.4 ± 3.5%; P < 0.05) compared with that in early lactation (wk 4: 33 ± 3.5 and 25 ± 3.5%; wk 8: 27 ± 3.5 and 22 ± 3.5%, respectively). The apparent transfer efficiency of cis-9,trans-11 in EFA+CLA and CLA groups did not differ between wk −7 AP and wk 4 and 8 PP (17.3 ± 5.7% for EFA+CLA and 10.6 ± 5.7% for CLA).
      The n-6/n-3 FA ratio in milk fat decreased with the start of supplementation in EFA and EFA+CLA (P < 0.001) and was lower in both EFA groups than in CTRL and CLA at wk 7 AP (P < 0.001). In early lactation, the ratio changed from 8.3 in CTRL to 1.9 in EFA and 1.4 in EFA+CLA (P < 0.001; Supplemental Table S2, https://doi.org/10.3168/jds.2019-18065). The concentration of FA synthesized de novo (<16 carbons) decreased (P < 0.001), and in accordance, the content of preformed FA (>16 carbons) increased (P < 0.001) in milk fat of cows supplemented with CLA (Supplemental Table S2). In late lactation, the desaturase indexes of 14:1, 16:1, and 18:1 were higher (P < 0.05) in CLA than in EFA, whereas the same desaturase indexes were reduced in CLA and EFA+CLA (P < 0.05) compared with those in the CTRL group in early lactation (Supplemental Table S2). The 18:2 cis-9,trans-11 CLA index was higher in the CLA and EFA+CLA groups (P < 0.05) than in CTRL and EFA in both lactation periods.

      Plasma Metabolites and Liver Triglycerides

      Plasma concentration of NEFA increased rapidly (P < 0.001; Figure 4A) with the onset of lactation; showed a CLA effect and a CLA × time interaction during the transition, PP, and entire period; and was lower (P < 0.05) in both CLA-treated groups than in CTRL (d 21 and 28 PP) and EFA (d 21 PP). Concentration of NEFA at d −42 AP was lower (P < 0.05) in cows of both CLA groups than in CTRL and EFA cows. The plasma TG concentration was highest during the dry period and decreased after calving with ongoing lactation in all groups (P < 0.001; Figure 4B). We detected a CLA effect at d 14, 28, and 35 PP with lower (P < 0.05) TG concentration in CLA- than in non-CLA-treated groups. Liver TG increased in all groups after calving and decreased again until d 63 PP (P < 0.001; Figure 4C). The increase in liver TG was less pronounced following CLA and EFA+CLA supplementation on d 28 PP compared with the CTRL (P < 0.01) and EFA cows (P < 0.05).
      Figure thumbnail gr4
      Figure 4Plasma concentrations of nonesterified fatty acids (NEFA; A), triglycerides (TG; B), and liver triglycerides (LTG; C) in cows supplemented abomasally daily with coconut oil (○ CTRL; white bars in panel C; n = 9), linseed and safflower oil (▴ EFA; light gray bars in panel C; n = 9), Lutalin (▾ CLA cis-9,trans-11 and trans-10,cis-12; BASF SE, Ludwigshafen, Germany; dark gray bars in panel C; n = 10), or EFA+CLA (♦; black bars in panel C; n = 10) from d 63 antepartum until d 56 postpartum. Data are presented as LSM ± SE. LSM with different lowercase letters (a, b) differ (P < 0.05) at the respective time point. Y = CLA effect at respective time point. Significant (P < 0.05) effects for NEFA concentration during antepartum (time), transition (time; CLA; CLA × time interaction), postpartum (time; CLA; CLA × time interaction), and during the entire study (time; CLA; CLA × time interaction). Significant (P < 0.05) effects for TG concentration in plasma during antepartum, transition, postpartum, and during the entire study (time), respectively. Significant (P < 0.05) effects for TG concentration in liver during the entire study (time; CLA × time interaction).
      Plasma concentrations of TC, LDL-C, and HDL-C decreased (P < 0.001; Figure 5) after drying off and rose in all groups (P < 0.001) after calving, with the highest concentrations seen at the end of the study. Time changes in TC concentration were affected by CLA treatment (CLA × time interaction AP, P < 0.05; CLA × time interaction PP, P < 0.1). The increase in plasma TC PP tended to be more pronounced in EFA+CLA than in EFA (EFA × CLA × time interaction; P < 0.1) and tended to be higher (P = 0.06) in EFA+CLA than in EFA on d 56 PP (Figure 5A). We detected a significant CLA effect (P < 0.05) for plasma LDL-C, and the concentration in EFA+CLA was higher (P < 0.05) than in EFA on d −42 AP, higher (P < 0.05) than in CTRL at 28 PP, and higher (P < 0.05) than in CTRL and EFA cows from d 42 to 56 PP (Figure 5B). The plasma concentration of HDL-C in CTRL was higher (P < 0.05) than in CLA on d 35, 42, and 56 and was higher (P < 0.05) than in EFA+CLA on d 42 (Figure 5C).
      Figure thumbnail gr5
      Figure 5Plasma concentrations of total cholesterol (TC; A), low-density lipoprotein cholesterol (LDL; B), and high-density lipoprotein cholesterol (HDL; C) in cows supplemented abomasally daily with coconut oil (○ CTRL; n = 9), linseed and safflower oil (▴ EFA; n = 9), Lutalin (▾ CLA cis-9,trans-11 and trans-10,cis-12; BASF SE, Ludwigshafen, Germany; n = 10), or EFA+CLA (♦;n = 10) from d 63 antepartum until d 56 postpartum. Data are presented as LSM ± SE. LSM with different lowercase letters (a, b) differ (P < 0.05) at the respective time point. Y = CLA effect at respective time point. Significant (P < 0.05) effects for total TC antepartum (time; EFA × time interaction; CLA × time interaction), transition (time), postpartum (time; CLA × time interaction), and during the entire study (time). Significant (P < 0.05) effects for LDL concentration antepartum (time; CLA × time interaction), transition (time), postpartum (time; CLA × time interaction), and during the entire study (time; CLA × time interaction). Significant (P < 0.05) effects for HDL antepartum (time), transition (time), postpartum (time), and during the entire study (time; EFA × CLA × time interaction).

      Body Composition

      The data regarding body composition and dissected fat depots are shown in Table 6. Body weight, hot carcass weight, and cold carcass weight, and weights of liver, kidneys, spleen, pancreas, and mammary gland did not differ between treatments. Body fat (absolute and relative to BW) was higher (P < 0.05) in CLA-treated than in non-CLA-treated cows. Omental and retroperitoneal fat (absolute weight and weight relative to BW) were higher (P < 0.05; trend for absolute retroperitoneal fat, P < 0.1) in CLA-treated than in non-CLA-treated groups, and omental fat relative to BW was higher (P < 0.05) in CLA than in CTRL cows. Subcutaneous fat (weight relative to BW and relative to total fat) was higher (P < 0.05 relative to total fat; P < 0.1 relative to BW) in EFA-treated than in non-EFA-treated groups.
      Table 6Body weight, hot carcass weight (HCW), cold carcass weight (CCW), organ weights, adipose depot weights, and their proportion of BW and total fat at slaughter, in cows daily abomasally supplemented either with coconut oil (CTRL; n = 9), linseed and safflower oil (EFA; n = 9), Lutalin
      Conjugated linoleic acid, cis-9,trans-11 and trans-10,cis-12; BASF SE, Ludwigshafen, Germany.
      (CLA; n = 10), or the combination (EFA+CLA; n = 10) from wk 9 antepartum until slaughter on d 63 postpartum
      Variable
      Values are presented as LSM ± SE.
      TreatmentP-values
      CTRLEFACLAEFA+CLAEFACLAEFA × CLA
      BW, kg624 ± 19584 ± 19617 ± 18618 ± 170.30.50.3
      HCW, kg260 ± 9249 ± 9266 ± 9263 ± 90.40.20.6
      CCW, kg254 ± 9243 ± 9260 ± 9258 ± 90.50.20.6
      Liver, kg11.0 ± 0.510.0 ± 0.510.3 ± 0.510.6 ± 0.50.51.00.2
      Kidney (left and right), kg1.89 ± 0.151.85 ± 0.141.58 ± 0.131.81 ± 0.130.50.20.3
      Spleen, kg0.98 ± 0.041.02 ± 0.041.00 ± 0.041.04 ± 0.040.30.61.0
      Pancreas, kg0.71 ± 0.050.62 ± 0.050.65 ± 0.050.65 ± 0.050.40.80.4
      Mammary gland, kg26.1 ± 1.424.7 ± 1.324.3 ± 1.226.1 ± 1.20.80.90.2
      Subcutaneous fat
       Weight, kg0.72 ± 0.120.92 ± 0.120.85 ± 0.110.97 ± 0.110.20.50.8
       Proportion of BW, %0.11 ± 0.020.16 ± 0.020.13 ± 0.020.15 ± 0.020.080.60.5
       Proportion of total fat, %4.92 ± 0.746.68 ± 0.714.37 ± 0.675.80 ± 0.670.030.30.8
      Retroperitoneal fat
       Weight, kg5.85 ± 0.855.43 ± 0.827.21 ± 0.787.22 ± 0.770.80.060.8
       Proportion of BW, %0.92 ± 0.120.91 ± 0.121.15 ± 0.111.15 ± 0.111.00.050.9
       Proportion of total fat, %39.8 ± 3.137.5 ± 2.936.5 ± 2.842.1 ± 2.80.60.80.18
      Omental fat
       Weight, kg4.62 ± 0.964.86 ± 0.927.17 ± 0.876.30 ± 0.870.70.030.5
       Proportion of BW, %0.72 ± 0.14
      Means within a row with different lowercase superscripts differ (P < 0.05).
      0.83 ± 0.14
      Means within a row with different lowercase superscripts differ (P < 0.05).
      1.14 ± 0.13
      Means within a row with different lowercase superscripts differ (P < 0.05).
      1.01 ± 0.13
      Means within a row with different lowercase superscripts differ (P < 0.05).
      0.90.030.4
       Proportion of total fat, %30.4 ± 1.932.9 ± 1.835.4 ± 1.733.7 ± 1.70.80.10.3
      Mesenteric fat
       Weight, kg3.75 ± 0.653.98 ± 0.674.78 ± 0.594.76 ± 0.630.90.160.8
       Proportion of BW, %0.59 ± 0.100.67 ± 0.100.75 ± 0.090.75 ± 0.090.70.190.7
       Proportion of total fat, %25.0 ± 2.726.1 ± 2.824.1 ± 2.521.3 ± 2.70.80.30.5
      Total fat
      Sum of subcutaneous, retroperitoneal, omental, and mesenteric fat.
       Weight, kg14.9 ± 2.416.6 ± 2.220.1 ± 1.918.5 ± 2.20.70.050.9
       Proportion of BW, %2.35 ± 0.292.44 ± 0.343.16 ± 0.322.96 ± 0.320.90.050.7
      a,b Means within a row with different lowercase superscripts differ (P < 0.05).
      1 Conjugated linoleic acid, cis-9,trans-11 and trans-10,cis-12; BASF SE, Ludwigshafen, Germany.
      2 Values are presented as LSM ± SE.
      3 Sum of subcutaneous, retroperitoneal, omental, and mesenteric fat.

      DISCUSSION

      Animal Performance and Body Composition

      An effect of essential fatty acids on DMI has been shown by previous investigations, which could not be observed in the present study (
      • Drackley J.K.
      • Klusmeyer T.H.
      • Trusk A.M.
      • Clark J.H.
      Infusion of long-chain fatty acids varying in saturation and chain length into the abomasum of lactating dairy cows.
      ;
      • Bremmer D.R.
      • Ruppert L.D.
      • Clark J.H.
      • Drackley J.K.
      Effects of chain length and unsaturation of fatty acid mixtures infused into the abomasum of lactating dairy cows.
      ). The reduction in DMI known to occur with PUFA probably could not be recorded because of the moderate doses of linseed oil applied. Furthermore, effects of essential fatty acids on DMI were less when abomasal infusion of fat was provided as triglycerides instead of as free FA (
      • Litherland N.B.
      • Thire S.
      • Beaulieu A.D.
      • Reynolds C.K.
      • Benson J.A.
      • Drackley J.K.
      Dry matter intake is decreased more by abomasal infusion of unsaturated free fatty acids than by unsaturated triglycerides.
      ). In contrast, we detected a hypophagic effect of CLA at the end of the study; DMI and NEL intake were lower in CLA-supplemented cows in early lactation. A reduction in DMI after CLA treatment has already been observed in other studies (
      • Baumgard L.H.
      • Corl B.A.
      • Dwyer D.A.
      • Sæbø A.
      • Bauman D.E.
      Identification of the conjugated linoleic acid isomer that inhibits milk fat synthesis.
      ;
      • Moallem U.
      • Lehrer H.
      • Zachut M.
      • Livshitz L.
      • Yacoby S.
      Production performance and pattern of milk fat depression of high-yielding dairy cows supplemented with encapsulated conjugated linoleic acid.
      ;
      • Schäfers S.
      • von Soosten D.
      • Meyer U.
      • Drong C.
      • Frahm J.
      • Kluess J.
      • Raschka C.
      • Rehage J.
      • Tröscher A.
      • Pelletier W.
      • Dänicke S.
      Influence of conjugated linoleic acid and vitamin E on performance, energy metabolism, and change of fat depot mass in transitional dairy cows.
      ). An important reason for the decrease in DMI and energy intake is certainly the reduction in energy requirement due to lower milk fat production and ECM in the CLA-treated cows, which additionally leads to a significantly improved energy balance in these cows. Voluntary feed intake decreased during CLA-induced milk fat depression (
      • Harvatine K.J.
      • Perfield 2nd, J.W.
      • Bauman D.E.
      Expression of enzymes and key regulators of lipid synthesis is upregulated in adipose tissue during CLA-induced milk fat depression in dairy cows.
      ). The CLA effect on EB during early lactation is inconsistent, and enhancing as well as lowering effects on EB were observed (
      • Bernal-Santos G.
      • Perfield II, J.W.
      • Barbano D.M.
      • Bauman D.E.
      • Overton T.R.
      Production responses of dairy cows to dietary supplementation with conjugated linoleic acid (CLA) during the transition period and early lactation.
      ;
      • Moallem U.
      • Lehrer H.
      • Zachut M.
      • Livshitz L.
      • Yacoby S.
      Production performance and pattern of milk fat depression of high-yielding dairy cows supplemented with encapsulated conjugated linoleic acid.
      ;
      • Schäfers S.
      • von Soosten D.
      • Meyer U.
      • Drong C.
      • Frahm J.
      • Kluess J.
      • Raschka C.
      • Rehage J.
      • Tröscher A.
      • Pelletier W.
      • Dänicke S.
      Influence of conjugated linoleic acid and vitamin E on performance, energy metabolism, and change of fat depot mass in transitional dairy cows.
      ). The variation in DMI, milk production, and calculated EB due to CLA feeding might depend on the study design (e.g., the amount and time of trans-10,cis-12 CLA isomer fed). In the present study, the long infusion period of 18 wk certainly contributed to the hypophagic CLA effect.
      Calculations of EB do not consider the CLA effects on body composition, presumably via reduced fat mobilization, or on inflammatory status that may result in changes in maintenance requirements and an improved tissue energy level after CLA supplementation (
      • Trevisi E.
      • Ferrari A.
      • Piccioli-Capelliand F.
      • Bertoni G.
      Energy balance indexes and blood changes of dairy cows supplemented with rumen protected CLA in late pregnancy and early lactation.
      ;
      • von Soosten D.
      • Meyer U.
      • Piechotta M.
      • Flachowsky G.
      • Dänicke S.
      Effect of conjugated linoleic acid supplementation on body composition, body fat mobilization, protein accretion, and energy utilization in early lactation dairy cows.
      ). In the current study, improved EB in the CLA and EFA+CLA groups resulted in less BW reduction postpartum and more body and omental fat in CLA-supplemented cows at the end of the study. On the other hand, CLA supplementation caused reduced body fat accretion in growing pigs (
      • Ostrowska E.
      • Suster D.
      • Muralitharan M.
      • Cross R.F.
      • Leury B.J.
      • Bauman D.E.
      • Dunshea F.R.
      Conjugated linoleic acid decreases fat accretion in pigs: Evaluation by dual-energy X-ray absorptiometry.
      ). However, in dairy cows an inhibitory effect of CLA on body fat accretion was not observed, and energy spared from reduction of milk fat synthesis is partitioned toward adipose tissue fat storage during short-term milk fat depression (
      • Baumgard L.H.
      • Corl B.A.
      • Dwyer D.A.
      • Bauman D.E.
      Effects of conjugated linoleic acids (CLA) on tissue response to homeostatic signals and plasma variables associated with lipid metabolism in lactating dairy cows.
      ;
      • Harvatine K.J.
      • Perfield 2nd, J.W.
      • Bauman D.E.
      Expression of enzymes and key regulators of lipid synthesis is upregulated in adipose tissue during CLA-induced milk fat depression in dairy cows.
      ;
      • von Soosten D.
      • Meyer U.
      • Piechotta M.
      • Flachowsky G.
      • Dänicke S.
      Effect of conjugated linoleic acid supplementation on body composition, body fat mobilization, protein accretion, and energy utilization in early lactation dairy cows.
      ). Therefore, the huge milk fat depression and reduction in body fat mobilization due to CLA treatment supports enhanced accretion of body fat in these cows. Interestingly, the proportion of subcutaneous fat relative to total fat was higher and the subcutaneous fat relative to BW tended to be higher in EFA-treated groups. This may indicate less mobilization of subcutaneous fat compared with other fat depots after calving in cows supplemented with EFA. These findings were not supported by different changes in BFT or BCS among treatment groups PP. Therefore, EFA treatment may have affected the relative degree of fat mobilization in different fat depots but not the overall body fat mobilization.
      Despite the reduction in milk fat and ECM in late lactation, we found only a weak effect of CLA feeding on EB due to a reduction of DMI in CLA-supplemented cows. Furthermore, we detected no increase in EB in the EFA group during late lactation, as ECM did not decline in this group in late lactation. The effects of linseed oil treatment on milk production and EB are inconsistent and may depend on the dosage, method of administration, and method of linseed processing; lactation stage could also bias the results (
      • Zachut M.
      • Arieli A.
      • Lehrer H.
      • Livshitz L.
      • Yakoby S.
      • Moallem U.
      Effects of increased supplementation of n-3 fatty acids to transition dairy cows on performance and fatty acid profile in plasma, adipose tissue, and milk fat.
      ;
      • Moallem U.
      Invited review: Roles of dietary n-3 fatty acids in performance, milk fat composition, and reproductive and immune systems in dairy cattle.
      ). However, linseed oil supplementation has possibly improved the persistence of milk production. Several studies have shown higher milk production according to linseed oil feeding (
      • Petit H.V.
      • Germiquet C.
      • LeBel D.
      Effect of feeding whole unprocessed sunflower seeds and flaxseed on milk production, milk composition, and prostaglandin secretion in dairy cows.
      ;
      • Hurtaud C.
      • Faucon F.
      • Couvreur S.
      • Peyraud J.L.
      Linear relationship between increasing amounts of extruded linseed in dairy cow diet and milk fatty acid composition and butter properties.
      ;
      • Moallem U.
      Invited review: Roles of dietary n-3 fatty acids in performance, milk fat composition, and reproductive and immune systems in dairy cattle.
      ). However, to our knowledge, no study has addressed the effects of EFA supplementation on milk production before the onset of the dry period.

      Milk Composition

      The trans-10,cis-12 CLA isomer is responsible for milk fat reduction in the CLA and EFA+CLA groups (
      • Baumgard L.H.
      • Corl B.A.
      • Dwyer D.A.
      • Sæbø A.
      • Bauman D.E.
      Identification of the conjugated linoleic acid isomer that inhibits milk fat synthesis.
      ). Milk fat reduction was particularly obtained by reduced de novo FA synthesis in the mammary gland, as indicated by the decrease in FA < C16 (
      • Mackle T.R.
      • Kay J.K.
      • Auldist M.J.
      • McGibbon A.K.
      • Philpott B.A.
      • Baumgard L.H.
      • Bauman D.E.
      Effects of abomasal infusion of conjugated linoleic acid on milk fat concentration and yield from pasture-fed dairy cows.
      ;
      • Harvatine K.J.
      • Bauman D.E.
      Characterization of the acute lactational response to trans-10, cis-12 conjugated linoleic acid.
      ). Because mammary epithelium is impermeable to citrate in both directions (
      • Linzell J.L.
      • Mepham T.B.
      • Peaker M.
      The secretion of citrate into milk.
      ), increased citrate in milk underlines the CLA-inhibiting effect on FA synthesis in the mammary gland. The citrate-isocitrate pathway is responsible for generating NADPH for de novo milk FA synthesis and is indirectly associated with FA synthesis in the mammary glands of ruminants; hence, elevated citrate concentrations in milk represent a decline in de novo fat synthesis (
      • Mackle T.R.
      • Kay J.K.
      • Auldist M.J.
      • McGibbon A.K.
      • Philpott B.A.
      • Baumgard L.H.
      • Bauman D.E.
      Effects of abomasal infusion of conjugated linoleic acid on milk fat concentration and yield from pasture-fed dairy cows.
      ;
      • Garnsworthy P.C.
      • Masson L.L.
      • Lock A.L.
      • Mottram T.T.
      Variation of milk citrate with stage of lactation and de novo fatty acid synthesis in dairy cows.
      ). The increased milk citrate in the CLA group confirmed the results of
      • Haubold S.
      • Kröger-Koch C.
      • Starke A.
      • Tuchscherer A.
      • Tröscher A.
      • Kienberger H.
      • Rychlik M.
      • Bernabucci U.
      • Trevisi E.
      • Hammon H.M.
      Effects of abomasal infusion of essential fatty acids and conjugated linoleic acid on performance and fatty acid, antioxidative, and inflammatory status in dairy cows.
      . However, in contrast to the study of
      • Haubold S.
      • Kröger-Koch C.
      • Starke A.
      • Tuchscherer A.
      • Tröscher A.
      • Kienberger H.
      • Rychlik M.
      • Bernabucci U.
      • Trevisi E.
      • Hammon H.M.
      Effects of abomasal infusion of essential fatty acids and conjugated linoleic acid on performance and fatty acid, antioxidative, and inflammatory status in dairy cows.
      , elevation of milk citrate was weak in EFA+CLA despite significant milk fat reduction in this group. Furthermore, milk acetone was elevated in EFA+CLA. Milk acetone is positively correlated with BHB in plasma (
      • Klein M.S.
      • Almstetter M.F.
      • Nürnberger N.
      • Sigl G.
      • Gronwald W.
      • Wiedemann S.
      • Dettmer K.
      • Oefner P.J.
      Correlations between milk and plasma levels of amino and carboxylic acids in dairy cows.
      ). However, plasma BHB was not higher in the EFA+CLA group compared with levels in the other groups (data not shown). Whether and how the low citrate and high acetone in milk after EFA+CLA supplementation are connected is somewhat speculative. Due to the influence of CLA, milk fat is low after EFA+CLA supplementation, and generating NADPH through the citrate-isocitrate pathway could not explain the lower milk citrate in this group compared with that in the CLA group. However, enhanced synthesis of amino acids or carbohydrates in the mammary gland is able to reduce citrate and to increase acetone in milk, but further studies are necessary to strengthen this hypothesis.
      In previous studies, abomasal or duodenal infusion of linseed, free ALA, or PUFA (mainly oleic acid and LA) in dairy cows resulted in higher milk fat compared with control groups (
      • Benson J.A.
      • Reynolds C.K.
      • Humphries D.J.
      • Rutter S.M.
      • Beever D.E.
      Effects of abomasal infusion of long-chain fatty acids on intake, feeding behavior and milk production in dairy cows.
      ;
      • Khas-Erdene Q.
      • Wang J.Q.
      • Bu D.P.
      • Wang L.
      • Drackley J.K.
      • Liu Q.S.
      • Yang G.
      • Wei H.Y.
      • Zhou L.Y.
      Short communication: Responses to increasing amounts of free alpha-linolenic acid infused into the duodenum of lactating dairy cows.
      ;
      • Côrtes C.
      • Kazama R.
      • da Silva-Kazama D.
      • Benchaar C.
      • Zeoula L.M.
      • Santos G.T.
      • Petit H.V.
      Digestion, milk production and milk fatty acid profile of dairy cows fed flax hulls and infused with flax oil in the abomasum.
      ). In contrast, in most studies where linseed was fed as extruded flaxseed, the milk fat content declined due to diet-induced milk fat depression (
      • Petit H.V.
      • Palin M.F.
      • Doepel L.
      Hepatic lipid metabolism in transition dairy cows fed flaxseed.
      ;
      • Zachut M.
      • Arieli A.
      • Lehrer H.
      • Livshitz L.
      • Yakoby S.
      • Moallem U.
      Effects of increased supplementation of n-3 fatty acids to transition dairy cows on performance and fatty acid profile in plasma, adipose tissue, and milk fat.
      ;
      • Mach N.
      • Zom R.L.
      • Widjaja H.C.
      • van Wikselaar P.G.
      • Weurding R.E.
      • Goselink R.M.
      • van Baal J.
      • Smits M.A.
      • van Vuuren A.M.
      Dietary effects of linseed on fatty acid composition of milk and on liver, adipose and mammary gland metabolism of periparturient dairy cows.
      ). However, the lack of changes in milk fat in EFA in the present study was consistent with findings in mid-lactating cows using the same EFA dose (
      • Haubold S.
      • Kröger-Koch C.
      • Starke A.
      • Tuchscherer A.
      • Tröscher A.
      • Kienberger H.
      • Rychlik M.
      • Bernabucci U.
      • Trevisi E.
      • Hammon H.M.
      Effects of abomasal infusion of essential fatty acids and conjugated linoleic acid on performance and fatty acid, antioxidative, and inflammatory status in dairy cows.
      ), and
      • Moallem U.
      • Vyas D.
      • Teter B.B.
      • Delmonte P.
      • Zachut M.
      • Erdman R.A.
      Transfer rate of α-linolenic acid from abomasally infused flaxseed oil into milk fat and the effects on milk fatty acid composition in dairy cows.
      indicated only a trend for an increasing milk fat content after infusing higher doses of linseed oil than in the present study. In late lactation, milk protein was higher in CLA-supplemented cows. An increase in milk protein during CLA feeding was also mentioned by
      • Bauman D.E.
      • Perfield II, J.W.
      • Harvatine K.J.
      • Baumgard L.H.
      Regulation of fat synthesis by conjugated linoleic acid: Lactation and the ruminant model.
      . In contrast, in early lactation, milk protein in the CLA groups was reduced. Milk protein reduction in early lactation was also determined by others (
      • Moallem U.
      • Lehrer H.
      • Zachut M.
      • Livshitz L.
      • Yacoby S.
      Production performance and pattern of milk fat depression of high-yielding dairy cows supplemented with encapsulated conjugated linoleic acid.
      ;
      • von Soosten D.
      • Meyer U.
      • Weber E.M.
      • Rehage J.
      • Flachowsky G.
      • Dänicke S.
      Effect of trans-10, cis-12 conjugated linoleic acid on performance, adipose depot weights, and liver weight in early-lactation dairy cows.
      ). The different results of CLA supplementation on the milk protein concentration in late and early lactation might be a consequence of the lactation stage. The protein balance was positive during late lactation but turned to negative results during early lactation, which could have affected CLA responses to milk protein content. Because cows in early lactation received CLA supplementation for a much longer time than did cows in late lactation, the differences in milk protein content in early and late lactation due to CLA treatment were confounded by time of treatment. Therefore, further studies are needed to clarify whether CLA effects on milk protein content depend on lactation stage.
      In accordance with the study of
      • Haubold S.
      • Kröger-Koch C.
      • Starke A.
      • Tuchscherer A.
      • Tröscher A.
      • Kienberger H.
      • Rychlik M.
      • Bernabucci U.
      • Trevisi E.
      • Hammon H.M.
      Effects of abomasal infusion of essential fatty acids and conjugated linoleic acid on performance and fatty acid, antioxidative, and inflammatory status in dairy cows.
      , milk urea was diminished in CLA groups. Moreover, we also found a reduction in milk urea in CTRL. The urea concentration in milk reflects the efficiency of protein utilization and is, in general, positively correlated with crude protein intake and, to a lesser extent, negatively correlated with available energy (
      • Nousiainen J.
      • Shingfield K.J.
      • Huhtanen P.
      Evaluation of milk urea nitrogen as a diagnostic of protein feeding.
      ). Therefore, the slight reduction in DMI in the CLA group cannot explain the reduction in urea, because milk urea is also lowered in the CTRL group. Reduced milk protein and urea concentrations in early lactation after CLA administration have previously been reported by
      • Moallem U.
      • Lehrer H.
      • Zachut M.
      • Livshitz L.
      • Yacoby S.
      Production performance and pattern of milk fat depression of high-yielding dairy cows supplemented with encapsulated conjugated linoleic acid.
      and
      • von Soosten D.
      • Meyer U.
      • Weber E.M.
      • Rehage J.
      • Flachowsky G.
      • Dänicke S.
      Effect of trans-10, cis-12 conjugated linoleic acid on performance, adipose depot weights, and liver weight in early-lactation dairy cows.
      . Higher body protein accretion and nitrogen retention after CLA supplementation are supposed to cause milk protein reduction (
      • von Soosten D.
      • Meyer U.
      • Piechotta M.
      • Flachowsky G.
      • Dänicke S.
      Effect of conjugated linoleic acid supplementation on body composition, body fat mobilization, protein accretion, and energy utilization in early lactation dairy cows.
      ). Other studies could not confirm an effect of CLA on milk protein and urea or showed an increase in these parameters (
      • Bauman D.E.
      • Perfield II, J.W.
      • Harvatine K.J.
      • Baumgard L.H.
      Regulation of fat synthesis by conjugated linoleic acid: Lactation and the ruminant model.
      ). The reduction in milk urea in CTRL also provides evidence that urea reduction might not be a factor in CLA treatment. Further studies are needed to clarify whether there are direct effects of CLA on protein synthesis in the mammary gland or on whole-body protein accretion in cows.

      Milk Fatty Acid Pattern

      According to previous research, the milk FA pattern in response to CLA changed as expected (
      • Chouinard P.Y.
      • Corneau L.
      • Barbano D.M.
      • Metzger L.E.
      • Bauman D.E.
      Conjugated linoleic acids alter milk fatty acid composition and inhibit milk fat secretion in dairy cows.
      ;
      • Perfield II, J.W.
      • Bernal-Santos G.
      • Overton T.R.
      • Bauman D.E.
      Effects of dietary supplementation of rumen-protected conjugated linoleic acid in dairy cows during established lactation.
      ). The altered milk FA composition with CLA supplementation was characterized by lower de novo synthesized FA (<16 carbons), resulting in a shift to longer-chain FA. The differences in the proportions of n-3 and n-6 FA in milk fat are reflected by the composition of the infused FA in EFA and EFA+CLA (
      • Petit H.V.
      Digestion, milk production, milk composition, and blood composition of dairy cows fed whole flaxseed.
      ;
      • Kazama R.
      • Côrtes C.
      • da Silva-Kazama D.
      • Gagnon N.
      • Benchaar C.
      • Zeoula L.M.
      • Santos G.T.
      • Petit H.V.
      Abomasal or ruminal administration of flax oil and hulls on milk production, digestibility, and milk fatty acid profile of dairy cows.
      ;
      • Moallem U.
      • Vyas D.
      • Teter B.B.
      • Delmonte P.
      • Zachut M.
      • Erdman R.A.
      Transfer rate of α-linolenic acid from abomasally infused flaxseed oil into milk fat and the effects on milk fatty acid composition in dairy cows.
      ). The accumulation of ALA and LA was higher in EFA+CLA than in EFA due to the lower milk fat content and reduction of de novo FA synthesis in the mammary gland following CLA supplementation. Therefore, an increase in the LA content in CLA (26% in early lactation) was also measurable. Nevertheless, EPA and DPA as well as ARA were higher AP in the EFA group than in the EFA+CLA group, which points to a trans10,cis12 CLA–related inhibition of FA desaturation in dairy cows (
      • Harvatine K.J.
      • Bauman D.E.
      Characterization of the acute lactational response to trans-10, cis-12 conjugated linoleic acid.
      ;
      • Haubold S.
      • Kröger-Koch C.
      • Starke A.
      • Tuchscherer A.
      • Tröscher A.
      • Kienberger H.
      • Rychlik M.
      • Bernabucci U.
      • Trevisi E.
      • Hammon H.M.
      Effects of abomasal infusion of essential fatty acids and conjugated linoleic acid on performance and fatty acid, antioxidative, and inflammatory status in dairy cows.
      ). Other studies have determined an inhibition of ARA synthesis from LA but not an inhibition of EPA from ALA due to trans10,cis12 CLA treatment (
      • Loor J.J.
      • Herbein J.H.
      Reduced fatty acid synthesis and desaturation due to exogenous trans10,cis12-CLA in cows fed oleic or linoleic oil.
      ). Correspondingly, ARA decreased due to CLA treatment (CLA and EFA+CLA) in late lactation and was higher in CTRL and EFA. In early lactation, ARA decreased equally in all groups. However, because cows in early lactation had a much longer treatment time than those in late lactation, the present study does not allow us to conclude an effect of the lactation stage on ARA in milk fat. The lower transfer efficiencies of ALA and of the infused CLA isomers after parturition were probably a consequence of the enrichment of these FA in colostrum during the dry period that has reached a plateau at the end of colostrogenesis. With ongoing milk production, efficiency rates of the infused FA increased again. Whether differences in transfer efficiencies between late and early lactation were a consequence of lactation stage or of infusion time cannot be ascertained by the present study. The transfer efficiency for ALA was comparable to efficiency rates in early studies with infused linseed oil (
      • Hagemeister H.
      • Precht D.
      • Franzen M.
      • Barth C.A.
      α-Linolenic acid transfer into milk-fat and its elongation by cows.
      ;
      • Moallem U.
      • Vyas D.
      • Teter B.B.
      • Delmonte P.
      • Zachut M.
      • Erdman R.A.
      Transfer rate of α-linolenic acid from abomasally infused flaxseed oil into milk fat and the effects on milk fatty acid composition in dairy cows.
      ). The transfer efficiency in our study during early lactation of trans-10,cis-12 CLA was lower than the transfer efficiency published recently (
      • Urrutia N.L.
      • Toledo M.
      • Baldin M.
      • Ford J.L.
      • Green M.H.
      • Harvatine K.J.
      Kinetics of trans-10, cis-12-conjugated linoleic acid transfer to plasma and milk following an abomasal bolus in lactating dairy cows.
      ). According to the FA composition of EFA and EFA+CLA, supplementation led to a higher content of n-3 FA compared with CTRL and CLA. Such a shift in the n-6/n-3 ratio in the milk FA profile has also been shown several times before (
      • Moallem U.
      Invited review: Roles of dietary n-3 fatty acids in performance, milk fat composition, and reproductive and immune systems in dairy cattle.
      ). As food enriched with n-3 FA is in high demand for human nutrition due to its beneficial health effects (
      • Simopoulos A.P.
      The importance of the ratio of omega-6/omega-3 essential fatty acids.
      ), the supplementation of dairy cows with a combination of CLA and EFA improves both the energy status of the dairy cow due to CLA supplementation and the nutrient value of the milk fat due to EFA supplementation. Keeping animals on pasture provides cows with EFA and CLA, as in the present study (
      • Lahlou M.N.
      • Kanneganti R.
      • Massingill L.J.
      • Broderick G.A.
      • Park Y.
      • Pariza M.W.
      • Ferguson J.D.
      • Wu Z.
      Grazing increases the concentration of CLA in dairy cow milk.
      ), and this is important for consumer acceptance of both dairy production and dairy products (
      • Kühl S.
      • Gassler B.
      • Spiller A.
      Labeling strategies to overcome the problem of niche markets for sustainable milk products: The example of pasture-raised milk.
      ).

      Metabolites in Plasma and Liver

      A reduced severity of negative EB should reduce the mobilization of adipose tissue and, therefore, leads to a lower increase of plasma NEFA concentration around calving (
      • Bauman D.E.
      • Currie W.B.
      Partitioning of nutrients during pregnancy and lactation: A review of mechanisms involving homeostasis and homeorhesis.
      ;
      • Weber C.
      • Hametner C.
      • Tuchscherer A.
      • Losand B.
      • Kanitz E.
      • Otten W.
      • Singh S.P.
      • Bruckmaier R.M.
      • Becker F.
      • Kanitz W.
      • Hammon H.M.
      Variation in fat mobilization during early lactation differently affects feed intake, body condition, and lipid and glucose metabolism in high-yielding dairy cows.
      ). Indeed, in the present study, improved EB and reduced BW loss in the CLA groups led to a reduction in circulating NEFA concentration, as also noted previously (
      • Kay J.K.
      • Roche J.R.
      • Moore C.E.
      • Baumgard L.H.
      Effects of dietary conjugated linoleic acid on production and metabolic parameters in transition dairy cows grazing fresh pasture.
      ;
      • Odens L.J.
      • Burgos R.
      • Innocenti M.
      • VanBaale M.J.
      • Baumgard L.H.
      Effects of varying doses of supplemental conjugated linoleic acid on production and energetic variables during the transition period.
      ;
      • Galamb E.
      • Faigl V.
      • Keresztes M.
      • Csillik Z.
      • Tröscher A.
      • Elek P.
      • Kulcsár M.
      • Huszenicza G.
      • Fébel H.
      • Husvéth F.
      Effect of pre- and post-partum supplementation with lipid-encapsulated conjugated linoleic acid on milk yield and metabolic status in multiparous high-producing dairy cows.
      ). Fatty acids in the liver can be oxidized but also esterified into triglycerides. Re-esterified FA and newly synthesized triglycerides can either be packed into very low density lipoproteins and delivered into blood or stored as cytosolic lipid droplets. In dairy cows, release of very low density lipoproteins by the liver is relatively low (
      • Drackley J.K.
      ADSA Foundation Scholar Award. Biology of dairy cows during the transition period: The final frontier?.
      ). Therefore, in CTRL and EFA, higher plasma NEFA leads to increased liver TG at d 28. Several studies have shown that elevated liver TG is associated with lipomobilization and high plasma NEFA levels (
      • Bobe G.
      • Young J.W.
      • Beitz D.C.
      Invited review: Pathology, etiology, prevention, and treatment of fatty liver in dairy cows.
      ;
      • Overton T.R.
      • Waldron M.R.
      Nutritional management of transition dairy cows: Strategies to optimize metabolic health.
      ;
      • Weber C.
      • Hametner C.
      • Tuchscherer A.
      • Losand B.
      • Kanitz E.
      • Otten W.
      • Singh S.P.
      • Bruckmaier R.M.
      • Becker F.
      • Kanitz W.
      • Hammon H.M.
      Variation in fat mobilization during early lactation differently affects feed intake, body condition, and lipid and glucose metabolism in high-yielding dairy cows.
      ). However, the decrease in NEFA concentration in the current experiment is not in accord with other CLA trials during the transition period (
      • Bernal-Santos G.
      • Perfield II, J.W.
      • Barbano D.M.
      • Bauman D.E.
      • Overton T.R.
      Production responses of dairy cows to dietary supplementation with conjugated linoleic acid (CLA) during the transition period and early lactation.
      ;
      • Hötger K.
      • Hammon H.M.
      • Weber C.
      • Gors S.
      • Troscher A.
      • Bruckmaier R.M.
      • Metges C.C.
      Supplementation of conjugated linoleic acid in dairy cows reduces endogenous glucose production during early lactation.
      ;
      • Schäfers S.
      • von Soosten D.
      • Meyer U.
      • Drong C.
      • Frahm J.
      • Kluess J.
      • Raschka C.
      • Rehage J.
      • Tröscher A.
      • Pelletier W.
      • Dänicke S.
      Influence of conjugated linoleic acid and vitamin E on performance, energy metabolism, and change of fat depot mass in transitional dairy cows.
      ), probably a consequence of the long-lasting CLA treatment in the current study.
      Very low density lipoproteins are processed in circulation into intermediate-density lipoproteins, which can be further metabolized to LDL. From extrahepatic tissues, cholesterol is returned to the liver in HDL (
      • Drackley J.K.
      ADSA Foundation Scholar Award. Biology of dairy cows during the transition period: The final frontier?.
      ). In accordance with the present data, research has demonstrated that the total cholesterol concentration and individual lipoprotein-associated cholesterol fractions in plasma were dramatically decreased at the onset of lactation and steadily increased thereafter (
      • Kessler E.C.
      • Gross J.J.
      • Bruckmaier R.M.
      • Albrecht C.
      Cholesterol metabolism, transport, and hepatic regulation in dairy cows during transition and early lactation.
      ). The presented results contribute to the minor effects of EFA supplementation on total cholesterol and associated fractions in blood plasma. The higher plasma LDL cholesterol concentration after EFA+CLA supplementation possibly indicated a lower mammary uptake of cholesterol and not an enhanced export rate of cholesterol from the liver, particularly because liver TG was diminished in the CLA groups. The lower HDL cholesterol concentration in CLA at the end of the trial may be a consequence of reduced reverse cholesterol transport from extrahepatic tissues, as hormone-sensitive lipase and perilipin 1 are decreased by CLA supplementation (
      • Urrutia N.
      • Harvatine K.J.
      Effect of conjugated linoleic acid and acetate on milk fat synthesis and adipose lipogenesis in lactating dairy cows.
      ). In addition, the highest HDL cholesterol concentration at the end of the study was observed in the CTRL group, and lauric acid that is enriched in coconut oil has been shown to stimulate HDL cholesterol in humans (
      • Mensink R.P.
      • Zock P.L.
      • Kester A.D.
      • Katan M.B.
      Effects of dietary fatty acids and carbohydrates on the ratio of serum total to HDL cholesterol and on serum lipids and apolipoproteins: A meta-analysis of 60 controlled trials.
      ).

      CONCLUSIONS

      Our results indicate a reduced milk fat content after CLA with or without EFA supplementation during late and early lactation. An elevated milk protein content after CLA supplementation was observed only in late lactation, whereas the energy status of the cows was improved, especially during early lactation in both CLA-supplemented groups. The different degrees of CLA effects on milk performance during late and early lactation were probably not only a consequence of the different lactation stage but also due to the fact that cows in early lactation received the treatments for much longer time. Increased milk citrate concentration in cows in the CLA group points to reduced de novo FA synthesis in the mammary gland, but milk citrate was less affected by combined EFA and CLA treatment, indicating that EFA supplementation may influence changes in mammary gland FA metabolism achieved by CLA. However, very few effects of the EFA treatment alone were evident with regard to milk performance and fat metabolism, indicating low importance of an enhanced EFA supply for milk production.

      ACKNOWLEDGMENTS

      The authors express their gratitude to the staff of the Experimental Animal Facility Cattle and the “Tiertechnikum” of the Leibniz Institute for Farm Animal Biology (FBN; Dummerstorf, Germany) for their contribution to the present study and animal care. We especially thank Claudia Reiko, Heike Pröhl, and Christa Fiedler for excellent laboratory work, as well as Matthias Kaiser and Anne Kaiser of the Clinic for Ruminants and Swine (University of Leipzig, Leipzig, Germany) for surgical rumen cannulation. We express our gratitude to Ralf Pfuhl and his team (FBN, Dummerstorf, Germany) for providing the carcass data. We further acknowledge the cattle breeding association (RinderAllianz GmbH, Woldegk, Germany) and Agrarprodukte Dedelow GmbH (Prenzlau, Germany) for the assortment of cows. The project was supported by BASF SE (Ludwigshafen, Germany) and by funds from the Federal Ministry of Food and Agriculture (BMEL; Bonn, Germany) based on a decision of the Parliament of the Federal Republic of Germany via the Federal Office for Agriculture and Food (BLE) under the innovation support program (grant number 313-06.01-28-1-79.003-15). The authors declare that there are no conflicts of interest.

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