Effects of a Maternal Essential Fatty Acid and Conjugated Linoleic Acid

animals

Article Effects of a Maternal Essential Fatty Acid and Conjugated Linoleic Acid Supplementation during Late Pregnancy and Early Lactation on Hematologic and Immunological Traits and the Oxidative and Anti-Oxidative Status in Blood Plasma of Neonatal Calves

Wendy Liermann 1,† , Katrin Lena Uken 1,†, Christine Schäff 1, Laura Vogel 1, Martina Gnott 1, Armin Tuchscherer 2, Erminio Trevisi 3 , Tadeusz Stefaniak 4 , Helga Sauerwein 5 , Arnulf Tröscher 6 and Harald Michael Hammon 1,*

1 Institute of Nutritional Physiology “Oskar Kellner”, Leibniz Institute for Farm Animal Biology (FBN), 18196 Dummerstorf, Germany; [email protected] (W.L.); [email protected] (K.L.U.); [email protected] (C.S.); [email protected] (L.V.); [email protected] (M.G.) 2 Institute of Genetics and Biometry, FBN, 18196 Dummerstorf, Germany; Citation: Liermann, W.; Uken, K.L.; [email protected] Schäff, C.; Vogel, L.; Gnott, M.; 3 Department of Animal Sciences, Food and Nutrition (DIANA), Università Cattolica del Sacro Cuore, Tuchscherer, A.; Trevisi, E.; 29122 Piacenza, Italy; [email protected] Stefaniak, T.; Sauerwein, H.; 4 Department of Immunology, Pathophysiology and Veterinary Preventive Medicine, Faculty of Veterinary Tröscher, A.; et al. Effects of a Medicine, Wroclaw University of Environmental and Life Science, 50-375 Wroclaw, Poland; Maternal Essential Fatty Acid and [email protected] 5 Conjugated Linoleic Acid Institute of Animal Science, Physiology and Hygiene Unit, University of Bonn, 53115 Bonn, Germany; Supplementation during Late [email protected] 6 BASF SE, 68623 Lampertheim, Germany; [email protected] Pregnancy and Early Lactation on * Correspondence: [email protected]; Tel.: +49-38208-68670 Hematologic and Immunological † Authors contributed to a similar value to the manuscript. Traits and the Oxidative and Anti-Oxidative Status in Blood Simple Summary: Fatty acids play an important role in the regulation of inﬂammation and oxidative Plasma of Neonatal Calves. Animals stress. The birth and the neonatal period are characterized by a high risk of inﬂammation and 2021, 11, 2168. https://doi.org/ 10.3390/ani11082168 an increased production of reactive oxygen species in the calf. The present study deals with the effects of a different maternal fatty acid supply including the supplementation of saturated fatty

Academic Editors: Ingrid Lorenz and acids by coconut oil, essential fatty acids, conjugated linoleic acid or a combination of essential fatty Florian Trefz acids and conjugated linoleic acid on the immunological and oxidative as well as anti-oxidative status of neonatal calves. Maternal essential fatty acid as well as conjugated linoleic acid supply Received: 10 June 2021 affected the inflammatory response and the oxidative and anti-oxidative status of the neonatal Accepted: 15 July 2021 offspring. Essential fatty acids might have beneficial effects on the prevention of dysregulated Published: 22 July 2021 inflammation after birth and reduced the plasma bilirubin concentrations in this period. Conjugated linoleic acid and saturated fatty acids might increase the inflammatory response. Similarly, plasma Publisher’s Note: MDPI stays neutral bilirubin increased, which in part might serve as a protector against oxidative stress in the early with regard to jurisdictional claims in phase after birth. published maps and institutional affil- iations. Abstract: Fatty acids are known for their regulatory role in inflammation and oxidative stress. The present study investigated 38 calves born from dams, abomasally supplemented with coconut oil, essential fatty acids (EFA), conjugated linoleic acid (CLA) or EFA + CLA, according to immunological traits and the oxidative and anti-oxidative status for the first 5 days of life. On day 2 of life, plasma Copyright: © 2021 by the authors. total bilirubin, cholesterol, interleukin 1-β and ferric ion reducing anti-oxygen power (FRAP) were Licensee MDPI, Basel, Switzerland. lower in calves with than without maternal EFA supplementation, and FRAP additionally on day 4. This article is an open access article On day 3, the concentrations of reactive oxygen metabolites were higher in calves with than without distributed under the terms and conditions of the Creative Commons maternal EFA supplementation and additionally on day 5 together of retinol. Total leucocyte counts Attribution (CC BY) license (https:// were decreased in the EFA group compared to the CLA group on day 5. Lymphocyte proportions creativecommons.org/licenses/by/ decreased from day 1 to 5 only in the EFA + CLA group. On day 2, plasma total protein was 4.0/). higher in CLA and EFA + CLA than in EFA calves. Similarly, CLA calves had higher interleukin 1-β

Animals 2021, 11, 2168. https://doi.org/10.3390/ani11082168 https://www.mdpi.com/journal/animals Animals 2021, 11, 2168 2 of 23

concentrations compared to EFA + CLA calves. FRAP was decreased by CLA on day 4. Overall, the maternal fatty acid supply affected the inﬂammatory response and the oxidative and anti-oxidative status of the neonatal offspring.

Keywords: calf; essential fatty acid; conjugated linoleic acid; inﬂammation; anti-oxidants; bilirubin

1. Introduction Neonatal calves are at risk of sickness because of their immature immune system and the elevated stress level due to the birth process itself and the exposure to an oxygen-rich environment leading to an increased generation of reactive oxygen species [1,2]. Oxidative stress is defined as an imbalance between the oxidative and the anti-oxidative system of the organism, characterized by an excessive production of reactive oxygen species and the incapacity to neutralize them, leading to cellular DNA, protein and lipid damage [3,4]. Providing the newborn with adequate colostrum intake immediately after birth is essential for reducing calf mortality and morbidity [5,6]. Metabolic stress in dairy cows leads to lipid mobilization, inflammation and oxidative stress and metabolic stress in the prenatal period, which has an impact on the metabolic and inflammatory response of calves [7–11]. Thus, an elevated maternal oxidant status index increased serum concentrations of reactive oxygen and nitrogen species in calves [8]. Beside immunoglobulins, immunomodulatory factors and anti-oxidative substances, colostrum contains long-chain fatty acids that may play a crucial role in inflammation and immune regulation and affect the oxidative status of neonatal calves [12]. In studies of Trevisi et al. [7], it was shown that a supplementation of n-3 fatty acids in the transition period mitigates the impact of subclinical inflammation and has beneficial effects on the energy balance of cows. An influence of maternal fatty acid supplementation on the neonatal immunoglobulin and inflammatory as well as anti-oxidative status was shown in a previous study of Garcia et al. [13] in calves. The supplementation of n-3 fatty acid by milk replacer affected phagocytosis of monocytes, neutrophil oxidative burst capacity and the secondary humoral response in calves [14]. Furthermore, maternal fatty acid supplementation and especially dietary n-6 to n-3 fatty acid ratio can influence the immunoglobulin and cytokine production in piglets [15]. Conjugated linoleic acids (CLA) are produced by the natural biosynthesis of unsatu- rated fatty acids in the rumen of cows and also enter the milk in higher concentrations [16]. They are involved in cytokine modulation and immunoglobulin synthesis; they are able to influence the anti-oxidative status and the proliferation of bovine peripheral blood mononuclear cells (PBMC) [16–18]. In cows, CLA influenced the fibrinogen release in the transition period [19]. Furthermore, CLA have inhibitory effects on the production of reactive oxygen species and the transcription of pro-inflammatory cytokines in bovine mammary epithelial cells [20]. The knowledge of the effects of these fatty acids on the inflammatory and oxidative as well as anti-oxidative status in calves is scanty. However, studies of Dänicke et al. [21] gave evidence that stimulation ability of PBMCs in calves is influenced by maternal CLA supplementation. Elevated n-3 FA and CLA concentrations are known from cows on pasture or fed fresh grass [22–24]. During gestation and via the intake of colostrum and milk, the maternal supply of essential fatty acids (EFA) can be transferred to the calf [13,25]. The first results of the present study showed increased n-3 fatty acid and CLA concentrations, as well as a decreased n-6: n-3 fatty acid status in colostrum and in the blood plasma of calves when dams were supplemented with EFA (mainly n-3 fatty acids provided by linseed oil) and CLA (cis-9, trans-11 CLA and trans-10, cis-12 CLA) during late gestation and early lactation [26,27]. Therefore, the present study aimed to investigate the inflammatory and oxidative status as well as anti-oxidative response of calves born from cows, supplemented abomasally with coconut oil as a saturated fatty acid source, EFA and CLA during late Animals 2021, 11, x FOR PEER REVIEW 3 of 24

Animals 2021, 11, 2168lactation [26,27]. Therefore, the present study aimed to investigate the inflammatory and 3 of 23 oxidative status as well as anti-oxidative response of calves born from cows, supplemented abomasally with coconut oil as a saturated fatty acid source, EFA and CLA during late pregnancypregnancy and early and lactation, early lactation, within the within first the5 days ﬁrst after 5 days birth. after We birth. hypothesized We hypothesized that that a combineda combined maternalmaternal EFA and EFACLA and supply CLA would supply promote would promotethe immune the immuneas well as as well as the the inflammatoryinﬂammatory and anti-oxidative and anti-oxidative status in calves status immediately in calves immediately after birth. after birth.

2. Material and2. MaterialMethods and Methods The current studyThe current was conducted study was at conductedthe experimental at the experimentalstation of the stationLeibniz of Institute the Leibniz Institute for Farm Animalfor FarmBiology Animal (FBN), Biology Dummerstorf (FBN), Dummerstorf from December from 2015 December to September 2015 to2017, September in 2017, in accordance withaccordance the guidelines with the of guidelinesthe German of Animal the German Prot Animalection Law. Protection The housing Law. The con- housing condi- ditions and tionsexperimental and experimental procedures procedures were approved were approved by the by Landesamt the Landesamt für Land- für Landwirtschaft wirtschaft LebensmittelsicherheitLebensmittelsicherheit und und Fischerei Mecklenburg-Vorpommern,Mecklenburg-Vorpommern, Rostock, Rostock Germany, (regis- Germany (registrationtration number number 7221.3-1-052/15). 7221.3-1-052/15). 2.1. Animals, Experimental Design, and Husbandry 2.1. Animals, Experimental Design, and Husbandry In total 38 Holstein calves (14 male calves, 23 female calves; birth weight: 42.1 ± 4.9 In total 38 Holstein calves (14 male calves, 23 female calves; birth weight: 42.1 ± 4.9 (means ± SD)) were investigated during the first 5 days of life in the current experiment. (means ± SD) were investigated during the first 5 days of life in the current experiment. The calves were born from 37 Holstein cows (36 single born calves and 1 twin pair), The calves were born from 37 Holstein cows (36 single born calves and 1 twin pair), which which were fed a total mixed ratio with corn silage as main forage component. Cows were fed a total mixed ratio with corn silage as main forage component. Cows were abo- were abomasally supplemented with coconut oil (control group, CON), essential fatty masally supplemented with coconut oil (control group, CON), essential fatty acids (EFA), acids (EFA), CLA or a combination of essential fatty acids and conjugated linoleic acid CLA or a combination of essential fatty acids and conjugated linoleic acid (EFA + CLA) (EFA + CLA) from d 63 before calving until early lactation (week 9 postpartum; 2nd to 3rd from d 63 before calving until early lactation (week 9 postpartum; 2nd to 3rd lactation) as lactation) as represented in Table1 and described in detail in the study of Vogel et al. [ 27]. represented in Table 1 and described in detail in the study of Vogel et al. [27]. For technical For technical reasons, the study was subdivided into 5 consecutive blocks including 7−8 reasons, the study was subdivided into 5 consecutive blocks including 7−8 calves born per calves born per block. block. Table 1. Experimental design and animals. Table 1. Experimental design and animals. Group Number of Calf Sex Group MaternalMaternal Fatty Fatty Acid Acid Supplementation Supplementation (MFAS) Number * of Calf Sex Calves (n) (MFAS) * Calves (n) Male FemaleMale Female Control (CON) Control (CON) 7676 g/dayg/day coconut oil †† 9 5 94 5 4 7878 g/dayg/day linseed oil ‡ andand 4 g/d g/dayay EFA EFA 9 4 95 4 5 safflowersafflower oiloil ⸸ CLA 38 g/day Lutalin §38(CLA; g/daycis Lutalin-9, trans §-11, (CLA;trans cis-10,-9, cistrans-12-11 CLA,, 10 g/day each) 9 1 8 CLA 9 1 8 EFA + CLA 78 g/day linseedtrans oil-10, + 4cis g/day-12 CLA safflower, 10 g/day oil each) + 38 g/day Lutalin 11 $ 4 7 78 g/day linseed oil + 4 g/day safflower * abomasal supplementationEFA + CLA of dams starting 63 days before expected calving until early11 lactation, $ amounts4 of supplement7 were halved during dry period starting 6 weeks before calving;oil + 38† Bio-Kokosöl g/day Lutalin #665, Kräuterhaus Sanct Bernhard KG, Bad Ditzenbach, Germany; ‡ DERBY Leinöl* abomasal #4026921003087, supplementation DERBY Spezialfutter of dams GmbH, starting Münster, 63 days Germany; before expectedGEFRO Distelöl, calving GEFRO until early Reformversand lactation, Frommlet KG, Memmingen,amounts Germany; of supplement§ BASF SE, Ludwigshafen, were halved Germany; during dry$ includes period 9 singlestarting born 6 calvesweeks and before one twincalving; pair. † Bio-Ko- kosöl #665, Kräuterhaus Sanct Bernhard KG, Bad Ditzenbach, Germany; ‡ DERBY Leinöl #4026921003087, DERBYImmediately Spezialfutter after GmbH, birth, calvesMünster, were Germany separated; ⸸ GEFRO from their Distelöl, dams, GEFRO weighed, and sub- Reformversandsequently Frommlet suppliedKG, Memmingen, with first Germany colostrum,; § BASF which SE, wasLudwigshafen, fed within Germany the first; 2.5$ in-± 1.7 h of life. cludes 9 singleWithin born calves the firstand one 24 htwin calves pair received. an amount of 10% of body weight (BW) (provided in two meals). The feed allowance on day 2 of life amounted to only 6% of BW to ensure that Immediatelyall calves after receivedbirth, calves thesame wereamount separated of colostrumfrom theirand dams, transition weighed, milk and during sub- the first 48 h sequently suppliedof life irrespectivewith first colostrum, of their birthwhich time. was Fromfed within day 3 the onwards, first 2.5calves ± 1.7 h were of life. fed an amount Within the firstof 24 12% h calves of BW received per day, an which amount was of provided 10% of body in 2 meals. weight Details (BW) (provided on feeding in management two meals). Theincluding feed allowance the milk on composition day 2 of life were amounted recently to published only 6% of in BW a companion to ensure that paper [26]. all calves receivedIn the general, same amount colostrum of colostrum and transition and milktransition fed to milk the calves during were the derived first 48 fromh their own of life irrespectivedam. of The their first birth 2 meals time. consisted From day of 3 firstonwards, colostrum. calves If were the quantity fed an amount of first of colostrum was 12% of BW perinsufficient, day, which required was provided amounts in were 2 meals. complemented Details on feeding by milk management from the second in- milking after cluding the milkparturition. composition In the were following recently days, published the morning in a companion milking, was paper fed to[26] the. calves, respectively. In general,Ifthe colostrum morning and milking transition was insufficient,milk fed to the the calves milk gainedwere derived from the from evening their milking was own dam. Thealso first considered. 2 meals consisted Only if colostrumof first colostrum. and transition If the quantity milk quantity of first of colostrum a dam was insufficient, colostrum or transition milk from another cow of the respective group was added [26]. Calves were fed by nipple bottle and refused milk was tube fed. All calves had free access Animals 2021, 11, 2168 4 of 23

to water. During the whole experiment, calves were housed individually in boxes, which were integrated in a climate-controlled room (19 ◦C) and littered with chopped straw. Calves were monitored daily to determine their general health status including the determination of body temperature and macroscopic assessment of feces. Two calves of the CON group, one calf of the EFA group and one calf of the EFA + CLA group showed a temporary elevated body temperature (≥39.6 ◦C). Four animals (CON = 2 calves, EFA = 1 calf, EFA + CLA = 1 calf) transiently suffered from diarrhea. Calves were medically treated according to the care instructions of the veterinarian (CON = one calf with analgesic and antibiotic agent; 1 calf with antibiotic and 2 calves with analgesic agent; CLA = one calf with analgesic).

2.2. Sample Collection Samples of first colostrum and subsequent transition milk as fed to the calves were taken and stored at −20 ◦C until further analyses. Blood samples were taken before colostrum feeding and daily before the morning feeding from the jugular vein, using commercial K3EDTA (day 1–2: 9 mL; day 3–5: 4.9 mL), lithium heparin (2 mL) and sodium fluoride/potassium oxalate (2 mL) tubes (1.2–2 mg of K3EDTA/mL; 12–13 IU heparin/mL; 2–4 mg/L sodium fluoride and 1–3 mg/L potassium oxalate; Greiner Bio-One International GmbH, Kremsmünster, Austria). Blood samples were placed on ice before centrifugation at 2700× g and 4 ◦C for 20 min. The obtained plasma samples were stored at −20 ◦C until analyses.

2.3. Milk Analyses Thawed milk samples were homogenized by warming (41 ◦C) and pivoting before further analyses. Concentrations of immunoglobulin (Ig) G1, IgG2, and IgM in milk were determined by ELISA as previously described by Gerbert et al. [28]. The intra-assay coefﬁcients of variation (CV) were 9.9, 5.5, and 8.2% and the inter-assay CV were 2.7, 10.6, and 7.1% for IgG1, IgG2, and IgM, respectively. The Ig concentrations in milk (wt/wt) were computed by correction for the density of milk from the respective milking according to data from Madsen et al. [29].

2.4. Blood Analyses The hematocrit and hemoglobin concentration in whole blood as well as the specific blood cell traits (total counts of erythrocytes, red cell distribution width (RDW), mean corpuscular volume of erythrocytes (MVC), mean corpuscular hemoglobin of erythrocytes (MCH), mean corpuscular hemoglobin concentration of erythrocytes (MCHC), total counts of leucocytes, proportions of atypical lymphocytes, proportions of basophilic granulocytes, proportions of thrombocytes, mean thrombocyte volume (MTV), thrombocrit, and immature cells) were determined by an automatic hematology analyzer (ABX Pentra 60, Horiba ABX SAS, Montpellier, France). Plasma IgG was analyzed in diluted lithium heparin plasma (1:300,000) using a bovine specific sandwich ELISA (#E10-118; Bethyl Laboratories Inc., Montgomery, TX, USA). Absorbance was measured at 450 nm with a multi-detection microplate reader (BioTek Synergy 2, Winooski, VT, USA) and was analyzed with Gen5 software (BioTek). The intra- and inter-assay CV for IgG measurement were 8 and 12%, respectively. Sodium fluoride/potassium oxalate containing plasma was analyzed for total protein, albumin, cholesterol and total bilirubin using an automatic spectrophotometer (ABX Pentra 400, Horiba ABX SAS) and the following kits: total protein (#553–412) and cholesterol (#553- 126) from mti-diagnostics (Idstein, Germany); albumin (#A11A01664) and total bilirubin (#LT-BR 0500) from LABOR + TECHNIK Eberhard Lehmann GmbH, Berlin, Germany). Haptoglobin was analyzed in K3EDTA plasma by ELISA as published by Hiss et al. [30] with an intra- and inter-assay CV of 9.5 and 11.8%. Animals 2021, 11, 2168 5 of 23

Interleukins were measured in twofold diluted lithium heparin plasma using bovine- specific commercially available colorimetric sandwich ELISA kits for interleukin 1-β (Cat. No. ESS0027; Thermo Scientific, Frederick, MD, USA) and interleukin 6 (Cat. No. ESS0029; Thermo Scientific). The intra- and inter-assay CV were 4.5 and 17.0% for interleukin 1-β and 3.5 and 13.4% for interleukin 6. Concentrations of retinol, β-carotene and tocopherol were determined in lithium heparin plasma after extraction with hexane by reverse-phase HPLC (LC4000; JASCO Europe Ltd., Cremella, LC, Italy) with a Zorbax Eclipse Plus C18 column (150 × 4.6 mm, 3.5 µm; Agilent Technologies, Santa Clara, CA, USA), an UV detector set at 325 nm (for retinol), 460 nm (for β-carotene), and 290 nm (for tocopherol), using methanol: tetrahy- drofuran (80:20) as mobile phase. A spectrophotometric method modified by Regenhard et al. [31] was used to determine derivatives of reactive oxygen metabolites (dROM) in K3EDTA plasma. Intra- and inter-assay CV were 6.3 and 10.0%, respectively. The ferric ion reducing antioxidant power (FRAP) in K3EDTA plasma was analyzed according to Benzie and Strain [32] with intra- and inter-assay CV of 2.7 and 2.6%, respectively. Oxygen radical absorbance capacity (ORAC) was measured in lithium heparin plasma according to the procedure of Cao and Prior [33].

2.5. Statistical Analyses For statistical analyses the MIXED procedure of SAS 9.4 for Windows (SAS Institute Inc., Cary, NC, USA) was used. The model included the fixed effects of the maternal fatty acid supplementation of EFA (level: yes, no), CLA (levels: yes, no), time (levels: day relative to birth) and their interactions. Furthermore, the block and the sex of the calf were considered as fixed factors. The duration of the maternal supplementation and gestation length were added as covariates and the calf was considered as subject for the repeated factor time. The Tukey–Kramer test was used to analyze pairwise differences of least-squares means (LSMeans). The SLICE statement of the MIXED procedure was considered for the separated analyses of LSMeans for interactions. Effects were defined as significant if p < 0.05. Pearson correlations according were determined by the CORR procedure of SAS 9.4 and were assessed as significant if p < 0.05 and if the correlation power > 0.8. The correlation power was determined by the POWER procedure of SAS 9.4.

3. Results None of the analyzed milk or blood parameter was affected by sex (p > 0.05). Therefore, sex will not be further considered in the following descriptions and discussions.

3.1. Hematology While the values of hematocrit, hemoglobin, erythrocyte cell counts, MCV and number of atypical lymphocytes decreased with age, the MCHC and basophilic granulocytes increased with increasing age in all calves (p < 0.05; Table2). Total cell counts of leucocytes were inﬂuenced by EFA and time (p < 0.05). EFA and EFA + CLA calves showed lower total leucocyte counts on day 5 of life compared to CLA calves (p < 0.05). Proportions of lymphocytes decreased with time after birth in all calves except for EFA calves, which were slightly increased from day 1 to 5 of life. A signiﬁcant CLA-by-time-interaction was detected in case of lymphocyte proportions (p = 0.029). Animals 2021, 11, x FOR PEER REVIEW 3 of 24

lactation [26,27]. Therefore, the present study aimed to investigate the inflammatory and oxidative status as well as anti-oxidative response of calves born from cows, supplemented abomasally with coconut oil as a saturated fatty acid source, EFA and CLA during late pregnancy and early lactation, within the first 5 days after birth. We hypothesized that a combined maternal EFA and CLA supply would promote the immune as well as the inflammatory and anti-oxidative status in calves immediately after birth.

2. Material and Methods The current study was conducted at the experimental station of the Leibniz Institute for Farm Animal Biology (FBN), Dummerstorf from December 2015 to September 2017, in accordance with the guidelines of the German Animal Protection Law. The housing conditions and experimental procedures were approved by the Landesamt für Land- wirtschaft Lebensmittelsicherheit und Fischerei Mecklenburg-Vorpommern, Rostock, Germany (registration number 7221.3-1-052/15).

2.1. Animals, Experimental Design, andAnimals Husbandry2021, 11, 2168 6 of 23 In total 38 Holstein calves (14 male calves, 23 female calves; birth weight: 42.1 ± 4.9 (means ± SD) were investigated during the first 5 days of life in the current experiment. The calves were born from 37 Holstein cows (36 single born calves and 1 twin pair), which Table 2. Hematology of calves depending on maternal fatty acid supplementation (MFAS) and time after birth. were fed a total mixed ratio with corn silage as main forage component. Cows were abomasally supplemented with coconut oil (control group, CON), essential fatty acidsMFAS (EFA), p-Values Variable Time # EFA × EFA × CLA × CLA or a combination of essential fatty acids and conjugatedCON linoleicα acidEFA (EFβ A + CLA)CLA γ EFA + CLA EFA CLA Time from d 63 before calving until early lactation (week 9 postpartum; 2nd to 3rd lactation) as CLA Time Time Hematocrit, % 1 30.9 ± 3.8 26.6 ± 3.6 30.1 ± 3.7 27.1 ± 3.2 0.21 0.95 0.38 <0.001 0.37 0.49 represented in Table 1 and described in detail in the study5 of 23.0 Vogel± 3.6 ***et al. [27] 19.7 ±. For3.3 *** technical 23.0 ± 3.5 *** 20.6 ± 2.9 *** reasons, the study was subdivided intoHemoglobin, 5 consecutive blocks including 7−8 calves born per 1 8.48 ±1.01 7.31 ± 0.94 8.47 ± 0.97 7.57 ± 0.83 0.17 0.81 0.85 <0.001 0.44 0.81 block. g/dL 5 6.64 ± 0.95 *** 5.69 ± 0.87 *** 6.72 ± 0.91 *** 5.98 ± 0.77 *** Erythrocytes, 1 7.20 ± 0.77 6.54 ± 0.72 7.37 ± 0.75 6.66 ± 0.63 0.22 0.73 0.98 <0.001 0.85 0.78 Table 1. Experimental design and animals10. 6/mm3 5 5.77 ± 0.75 *** 5.16 ± 0.69 *** 5.92 ± 0.72 *** 5.39 ± 0.60 *** Group Maternal Fatty Acid Supplementation Number of Calf Sex RDW †,% 1 16.9 ± 0.6 16.7 ± 0.6 15.8 ± 0.6 16.8 ± 0.5 0.49 0.19 0.12 0.05 0.19 0.55 (MFAS) * 5Calves 16.9 (n±) 0.7Male 16.4 ± 0.6Female 15.6 ± 0.6 16.5 ± 0.6 Control (CON) 76 g/day coconutMCV ‡, µoilm 3† 19 42.8 ± 1.65 41.2 ± 1.64 40.9 ± 1.6 41.0 ± 1.4 0.72 0.45 0.47 <0.001 0.050 0.29 78 g/day linseed oil ‡ and 4 g/day 5 40.2 ± 1.5 *** 39.2 ± 1.4 *** 38.6 ± 1.5 *** 39.3 ± 1.3 *** EFA 9 4 5 safflowerMCH oil ⸸ , pg 1 11.8 ± 0.4 11.4 ± 0.4 11.5 ± 0.4 11.5 ± 0.3 0.36 0.71 0.37 0.016 0.62 0.82 5 11.7 ± 0.4 11.2 ± 0.3 11.4 ± 0.3 11.3 ± 0.3 38 g/day Lutalin § (CLA; cis-9, trans-11, CLA MCHC §, 9 1 8 1 27.5 ± 0.6 27.8 ± 0.6 28.5 ± 0.6 27.9 ± 0.5 0.54 0.25 0.21 <0.001 0.33 0.91 trans-10, cis-12 CLA, 10g/dL g/day each) 78 g/day linseed oil + 4 g/day safflower5 28.7 ± 0.7 *** 29.0 ± 0.7 *** 29.9 ± 0.7 *** 28.8 ± 0.6 *** EFA + CLA 11 $ 4 7 oil + 38 g/dayLeucocytes, Lutalin 3 3 1 9.4 ± 1.4 8.9 ± 1.4 10.8 ± 1.4 9.4 ± 1.2 0.029 0.14 0.44 0.01 0.09 0.61 * abomasal supplementation of dams starting10 /mm 63 days before expected calving until early lactation, 5 8.4 ± 1.4 ab 6.2 ± 1.3 b 10.7 ± 1.4 a 6.9 ± 1.2 b amounts of supplement were halved during dry period starting 6 weeks before calving; † Bio-Ko- Lymphocytes, 1 34.3 ± 8.3 33.0‡ ± 7.9 35.2 ± 8.0 42.6 ± 6.9 0.44 0.84 0.98 0.002 0.68 0.029 kosöl #665, Kräuterhaus Sanct Bernhard %KG, Bad Ditzenbach, Germany; DERBY Leinöl #4026921003087, DERBY Spezialfutter GmbH, Münster,5 Germany 25.6; ±⸸ 8.8GEFRO 35.1Distelöl,± 8.1 GEFRO 22.5 ± 8.3 23.7 ± 7.1 * Reformversand Frommlet KG, Memmingen,Atypical Germany; § BASF SE, Ludwigshafen, Germany; $ in- Lymphocytes, 1 4.29 ± 0.54 4.19 ± 0.52 4.05 ± 0.52 3.90 ± 0.46 0.80 0.95 0.74 <0.001 0.85 0.27 cludes 9 single born calves and one twin pair%. 5 2.30 ± 0.57 * 2.04 ± 0.53 ** 2.38 ± 0.53 * 2.57 ± 0.46 Immediately after birth, calves wereBasophile separated from their dams, weighed, and sub- Granulocytes, 1 0.21 ± 0.17 0.10 ± 0.17 0.21 ± 0.17 0.47 ± 0.15 0.81 0.67 0.44 <0.001 0.25 0.09 sequently supplied with first colostrum,% which was fed within the first 2.5 ± 1.7 h of life. Within the first 24 h calves received an amount of 10%5 of body 0.90 ± weight0.16 ** (BW) 0.82 ± (provided0.15 ** 0.83 in± 0.15 * 0.67 ± 0.13 Thrombocytes, two meals). The feed allowance on day 2 of life amounted1 to only 482 ± 996% of BW 471 to± ensure93 that 645 ± 95 586 ± 82 0.49 0.13 0.50 0.33 0.76 0.18 103/mm3 all calves received the same amount of colostrum and5 transition 486 ± milk103 during 490 ±the95 first 48 598 h ± 98 486 ± 83 of life irrespective of their birth time.MTV From$, µm day3 3 onwards,1 6.36calves± 0.51 were fed 6.30 an± 0.47 amount 6.80 of ± 0.49 6.68 ± 0.42 0.59 0.36 0.85 0.052 0.29 0.27 12% of BW per day, which was provided in 2 meals.5 Details 6.71 on± feeding0.52 management 6.52 ± 0.48 7.01in-± 0.50 6.63 ± 0.42 Thrombocrit, cluding the milk composition were recently published1 in a companion 0.32 ± 0.10 paper 0.36 ± [26]0.09. 0.48 ± 0.10 0.39 ± 0.08 0.54 0.24 0.36 0.14 0.26 0.32 % In general, colostrum and transition milk fed to5 the calves 0.33 ± 0.11were derived 0.33 ± 0.10 from their 0.45 ± 0.10 0.33 ± 0.09 Immature own dam. The first 2 meals consisted of first colostrum.1 If the 25.4 quantity± 11.7 of 17.1 first± 11.8 colostrum 16.6 ± 11.5 3.5 ± 11.8 0.16 0.68 0.77 0.55 0.78 0.12 cells, % 5 16.2 ± 8.5 2.6 ± 7.8 16.2 ± 7.9 14.6 ± 6.7 α CON, control; β EFA, essential fatty acids; γ CLA, conjugated linoleic acid; † RDW, red cell distribution width; ‡ MCV, mean corpuscular volume of erythrocytes; MCH, mean corpuscular hemoglobin of erythrocytes; MCHC §, mean corpuscular hemoglobin concentration of erythrocytes; MTV $, mean thrombocyte volume; # Day of life; ab Different superscripts mark signiﬁcant differences between groups (p < 0.05). * Asterisk show signiﬁcant differences between days of life within groups (* p < 0.05; ** p < 0.01; *** p < 0.001).

3.2. Immunoglobulins in Colostrum and Transition Milk

The concentrations of IgG1 and IgM decreased in colostrum and transition milk after calving (p < 0.05) but did not differ between groups with different maternal fatty acid supply (Figure1A,C). Colostral IgG 2 concentrations decreased after calving as well but the decrease was slower in CON cows leading to a higher IgG2 concentration in transition milk of CON cows compared to EFA cows on day 2 after calving (p < 0.05; Figure1B). Animals 2021, 11, 2168 7 of 23 Animals 2021, 11, x FOR PEER REVIEW 7 of 24 A)

IgG1 (g/kg) 10

1 2 3 4 5 B) Days(A) postnatum 5

a 2 ab

IgG2 (g/kg) 1

0 ab b

1 2 3 4 5 Days(B) postnatum Figure 1. Cont. Animals 2021, 11, x FOR PEER REVIEW 8 of 24

Animals 2021, 11, 2168 8 of 23 C)

IgM (g/kg) IgM 2

1 2 3 4 5 Days postnatum

(C)

Figure 1. Immunoglobulin (Ig) G1 (A), IgG2 (B) and IgM (C) concentration in colostrum and transition milk of cows receiv- Figure 1. Immunoglobulin (Ig) G1 (A), IgG2 (B) and IgM (C) concentration in colostrum and transition milk of cows ing fatty acid supplementation (○ control, CON n = 9; ▲ EFA, essential fatty acids, n = 9; ▼, CLA, conjugated linoleic acid, receiving fatty acid supplementation ( control, CON n = 9; N EFA, essential fatty acids, n = 9; H, CLA, conjugated linoleic n = 9; ♦, EFA + CLA, n = 11) and time after calving. Day 1 correspond to the day of birth. ab Different superscripts mark acid, n = 9; , EFA + CLA, n = 11) and# time after calving. Day 1 correspond to the day of birth. ab Different superscripts significant differences between groups at similar time points (p < 0.05). mark signiﬁcant differences between groups at similar time points (p < 0.05). 3.3. Immunoglobulin G, Metabolites, Interleukins, Vitamins and Oxidative and Anti-Oxidative Traits3.3. Immunoglobulin in Plasma of Calves G, Metabolites, Interleukins, Vitamins and Oxidative and Anti-Oxidative Traits in Plasma of Calves Plasma IgG concentrations increased from day 1 to day 2 of life in all calves (p < 0.001) Plasma IgG concentrations increased from day 1 to day 2 of life in all calves (p < 0.001) and remained elevated until the end of the study (Figure 2A). Total protein concentrations and remained elevated until the end of the study (Figure2A). Total protein concentrations increased from day 1–2 in all calves (p < 0.001; Figure 2B). Total protein tended to be ele- increased from day 1–2 in all calves (p < 0.001; Figure2B). Total protein tended to be elevated vated due to maternal CLA treatment (p = 0.072) and after birth tended to increase faster due to maternal CLA treatment (p = 0.072) and after birth tended to increase faster in CLA inthan CLA non-CLA than non calves-CLA (CLA calves x time(CLA interaction, x time interaction,p = 0.090). p = Total 0.090). protein Total was protein higher was on higher day 2 onof lifeday in 2 CLA of life and in EFA CLA + CLAand EF thanA + in C EFALA calvesthan in (p EFA< 0.05). calves Plasma (p < albumin0.05). Plasma concentrations albumin concentrationsin plasma decreased in plasma independently decreased fromindependently maternal fatty from acid maternal supplementation fatty acid supplemen- from day 1 tationto day from 2 of life day (p 1< to 0.001) day 2 and of life increased (p < 0.001) thereafter and increased until day thereafter 4 (p < 0.05; until Figure day2 4C). (p < 0.05; FigurePlasma 2C). cholesterol concentration increased (p < 0.001) from d 1 to d 5 of life, whereas plasma total bilirubin increased (p < 0.001) from day 1 to day 2 of life and decreased (p < 0.001) thereafter (Figure3A,B). The increase of bilirubin was much more pronounced in CON calves leading to a signiﬁcantly higher bilirubin concentration in CON calves compared to EFA and EFA + CLA calves on day 2 of life (p < 0.05). On d 2, plasma total bilirubin (p < 0.01) and cholesterol (p = 0.04) were lower in calves with maternal EFA-supplementation than in the calves whose dams did not receive EFA. Animals 2021, 11, x FOR PEER REVIEW 9 of 24

Animals 2021, 11, 2168 9 of 23 A)

IgG(g/l) 10

1 2 3 4 5 B) (ADay) of life 80 Y 70 a a

(g/L) 60

50 ab protein b

40 Total Total

Total Protein (g/L) Total Protein 30

1 2 3 4 5

(BDay) of life Figure 2. Cont. Animals 2021, 11, x FOR PEER REVIEW 10 of 24

Animals 2021, 11, 2168 10 of 23 C)

Albumin (g/L) Albumin 18

1 2 3 4 5

Day of life (C)

FigureFigure 2. 2. PlasmaPlasma immunoglobulin immunoglobulin G G ( (AA),), total total protein protein ( (BB)) and and albumin albumin ( (CC)) concentrations concentrations in in calves calves dependent dependent on on maternal maternal fatty acid supplementation (○ control, CON n = 9; ▲ EFA, essential fatty acids, n = 9; ▼, CLA, conjugated linoleic acid, n fatty acid supplementation ( control, CON n = 9; N EFA, essential fatty acids, n = 9; H, CLA, conjugated linoleic acid, = 9; ♦, EFA + CLA, n = 10) and time after birth. Day 1 corresponded to the day of birth. n = 9; , EFA + CLA, n = 10)# and time after birth. Day 1 corresponded to the day of birth. ab Different superscripts mark significant differences between groups at similar time points (p < 0.05). Y between calves with and without maternal CLA Plasma cholesterol concentration increased (p < 0.001) from d 1 to d 5 of life, whereas supplementation (p < 0.05). plasma total bilirubin increased (p < 0.001) from day 1 to day 2 of life and decreased (p < 0.001)While thereafte ther plasma (Figure interleukin 3A,B). The 1-increaseβ concentration of bilirubin in was CLA much calves more increased pronounced from day in CON1 to day calves 2 of leading life (p < to 0.001), a significantly the concentrations higher bilirubin showed concentration only marginal in CON alterations calves incom- the paredother groupsto EFA and (Figure EFA4A). + C Consequently,LA calves on day CLA 2 of calves life (p had < 0.05). higher On interleukind 2, plasma 1-totalβ concen- biliru- bintration (p < compared0.01) and cholesterol to EFA + CLA (p = calves0.04) were on day lower 2 of in life calves (p = 0.009).with maternal Furthermore, EFA-supple- plasma mentationinterleukin than 1-β inwas the lower calves in whose calves dams with thandid not without receive maternal EFA. EFA treatment on day 2 of life (p = 0.017). Interleukin 1-β correlated with the bilirubin concentration in plasma of calves (r = 0.263; p = 0.001; Table3). Plasma haptoglobin concentration indicated a trend for an EFA x CLA (p = 0.091) and EFA x time (p = 0.086) interaction, respectively, and tended to increase during the first 24 h of life in calves with maternal EFA treatment (p = 0.08; Figure3C ). On day 2 of life, plasma haptoglobin tended to be higher in calves with than without EFA treatment (p = 0.056). Considering calves from dams treated with CLA, interleukin 6 was affected in a time dependent manner similar to interleukin 1-β (p < 0.001); however, there were no significant differences between groups within time points (Figure4B). The plasma concentration of β- carotene increased from day 1 to day 5 of life in all calves (p < 0.001; Figure5A). A trend for a CLA-by-day interaction was detected for β- carotene concentration (p = 0.083), but there were no individual treatment effects in certain time points. Plasma retinol concentration increased in a time dependent manner (p < 0.001) and concentration on day 5 of life was higher in calves from dams treated with EFA than in calves without maternal EFA treatment (p = 0.048; Figure5B). AnimalsAnimals2021, 1120,21 2168, 11, x FOR PEER REVIEW 11 of 24 11 of 23

3.0

2.5

2.0

1.5 X

1.0

Cholesterol (mmol /L) (mmol Cholesterol 0.5

0.0

1 2 3 4 5 B) X Day of life a 20 ab

10 b b

Bilirubin (µmol/L) Bilirubin

1 2 3 4 5 Day of life

Figure 3. Plasma cholesterol (A) and bilirubin (B) concentration in calves dependent on maternal fatty acid supplementa- Figure 3. Plasma cholesterol (A) and bilirubin (B) concentration in calves dependent on maternal fatty acid supplementation tion (○ control, CON n = 9; ▲ EFA, essential fatty acids, n = 9; ▼, CLA, conjugated linoleic acid, n = 9; ♦, EFA + CLA, n = ( control,11) and CON time nafter= 9; birth.N EFA, Day essential 1 corresponded fatty acids, to then day= 9; ofH ,birth. CLA, ab conjugated Different superscripts linoleic acid, markn significant= 9; , EFA differences + CLA, n = 11) and# time after birth. Day 1 corresponded to the day of birth. ab Different superscripts mark signiﬁcant differences between groups at similar time points (p < 0.05). X designates signiﬁcant differences between calves with maternal EFA supplementation and without maternal EFA supplementation within a time point (p < 0.05). Animals 2021, 11, x FOR PEER REVIEW 3 of 24

2. Material and Methods

AnimalsThe2021 current, 11, 2168 study was conducted at the experimental station of the Leibniz Institute 12 of 23 for Farm Animal Biology (FBN), Dummerstorf from December 2015 to September 2017, in accordance with the guidelines of the German Animal Protection Law. The housing conditions and experimental procedures were approved by the Landesamt für Land- wirtschaft LebensmittelsicherheitTable 3.undCorrelations Fischerei between Mecklenburg selected plasma-Vorpommern, variables. Rostock, Germany (registration number 7221.3Variable-1-052/15). Correlated Parameters Correlation Coefﬁcient (r) p-Value

2.1. Animals, Experimental Design,Plasma and Husbandry traits Animals 2021, 11, x FOR PEER REVIEW Bilirubin 0.255 <0.00112 of 24 In total 38 Holstein calves (14 male calves, 23 female calves; birth weight: 42.1 ± 4.9 Interleukin 6 0.278 0.001 (means ± SD) were investigated during the first 5 days of life in the current experiment. β- carotene 0.447 <0.001 Ig † G The calves were born from 37 Holstein cows (36 single bornTocopherol calves and 1 twin pair), which 0.245 0.001 between groups at similar time points (p < 0.05). X designates significant differences between calves with maternal EFA were fed a total mixed ratio with corn silage as main foragedROM component. Cows were abo- 0.491 <0.001 masallysupplementation supplemented and withoutwith coconut maternal oil EFA (control supplementation group, CON),ORAC within essential§ a time fattypoint acids (p < 0.05). (EFA), 0.378 <0.001 CLA or a combination of essential fatty acids and conjugatedInterleukin linoleic 1-β acid (EFA + CLA)0.263 0.001 WhileBilirubin the plasma interleukin 1-β concentration in CLA calves increased from day 1 from d 63 before calving until early lactation (week 9 postpartum;Retinol 2nd to 3rd lactation) as− 0.388 <0.001 to day 2 of life (p < 0.001), the concentrations showed only marginal alterations in the other represented in Table 1 and described in detail in the study of Vogel et al. [27]. For technical groupsInterleukin (Figure 64A). Consequently, ORAC CLA calves had higher interleukin 0.270 1-β concentration 0.001 reasons, the study was subdivided into 5 consecutive blocks including 7−8 calves born per compared to EFA + CLA calvesRetinol on day 2 of life (p = 0.009). Furthermore, 0.371 plasma interleu- <0.001 block. Tocopherol 0.648 <0.001 kin 1-ββ- was carotene lower in calves with than without maternal EFA treatment on day 2 of life (p = 0.017). Interleukin 1-β correlateddROM with the bilirubin concentration 0.610 in plasma of <0.001calves (r Table 1. Experimental design and animals. ORAC 0.155 0.047 = 0.263; p = 0.001; Table 3). Plasma haptoglobin concentration indicated a trend for an EFA Group Maternal Fatty Acid Supplementation NumberTocopherol of Calf Sex 0.602 <0.001 x CLA Retinol(p = 0.091) and EFA x time (p = 0.086) interaction, respectively, and tended to increase(MFAS) during * the first 24 h Cofalves lifedROM in(n) calvesMale with maternalFemale EFA 0.338 treatment (p = 0.08; <0.001 Figure Control (CON) 76 3C).g/day OnTocopherol coconut day 2 oil of †life, plasma haptoglobin dROM9 tended5 to be higher4 0.520 in calves with than <0.001without 78 g/day linseeddROM oil ‡ and‡ 4 g/day ORAC 0.238 0.002 EFA EFA treatment (p = 0.056). 9 4 5 safflowerFRAP oil ⸸ ORAC 0.245 0.002 38 g/day Lutalin† Ig, immunoglobulin; § (CLA; cis-9, trans‡ dROM,-11, derivates of reactive oxygen metabolites; FRAP, ferric ion reducing anti-oxygen CLA 9 1 8 transA)-10, cispower;-12 CLA ORAC, 10 §g/d, oxygenay each) radical absorbance capacity. 78 g/day linseed oil + 4 g/day safflower EFA + CLA 11 $ 4 7 oil + 38 g/day Lutalin X * abomasal supplementation of dams starting 63 days before expected calving until early lactation, amounts of supplement140 were halved during dry perioda starting 6 weeks before calving; † Bio-Ko- kosöl #665, Kräuterhaus Sanct Bernhard KG, abBad Ditzenbach, Germany; ‡ DERBY Leinöl #4026921003087, DERBY120 Spezialfutter GmbH, Münster, Germany; ⸸ GEFRO Distelöl, GEFRO Reformversand Frommlet KG, Memmingen, Germany; § BASF SE, Ludwigshafen, Germany; $ includes 9 single born100 calves and one twin pair. Immediately after birth, calves were separated from their dams, weighed, and sub- sequently supplied80 with first colostrum, which was fed within the first 2.5 ± 1.7 h of life. Within the first 24 h calves received an amount of 10% of body weight (BW) (provided in two meals). The feed60 allowance on day 2 of life amounted to only 6% of BW to ensure that all calves received the same amount of colostrum and transition milk during the first 48 h of life irrespective of40 their birth time. From day 3 onwards, calves were fed an amount of 12% of BW per day, which was provided in 2 meals. Details on feeding management in-

IL-1 beta (pg/mL) IL-1 beta cluding the milk composition20 were recently published in a companion paper [26]. In general, colostrum and transition milkab fed to the calves were derived from their own dam. The first 20 meals consisted of first colostrum.b If the quantity of first colostrum

1 2 3 4 5 (ADay) of life Figure 4. Cont. Animals 2021, 11, x FOR PEER REVIEW 13 of 24 Animals 2021, 11, 2168 13 of 23 B)

1200

1000

800

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400

IL-6 (pg/mL)

200

1 2 3 4 5 C) (B) 800 Day of life

600

400

200

Haptoglobin(µg/mL) 0

1 2 3 4 5

Day of life (C)

FigureFigure 4. 4.Plasma Plasma interleukininterleukin (IL) 1 1--ββ (A(A),), IL IL-6-6 (B ()B and) and haptoglobin haptoglobin (C) ( Cconcentration) concentration in calves in calves dependent dependent on maternal on maternal fatty acid supplementation (○ control, CON n = 9; ▲ EFA, essential fatty acids, n = 9; ▼, CLA, conjugated linoleic acid, n = 9; fatty acid supplementation ( control, CON n = 9; N EFA, essential fatty acids, n = 9; H, CLA, conjugated linoleic acid, ♦, EFA + CLA, n = 11) and time after birth. Day 1 corresponded to the day of birth. ab Different superscripts mark signifi- n = 9; , EFA + CLA, n = 11)# and time after birth. Day 1 corresponded to the day of birth. ab Different superscripts mark cant differences between groups at similar time points (p < 0.05). X designates significant differences between calves with X signiﬁcantmaternal differencesEFA supplementation between groups and without at similar maternal time points EFA supplementation (p < 0.05). designates within signiﬁcanta time point differences (p < 0.05). between calves with maternal EFA supplementation and without maternal EFA supplementation within a time point (p < 0.05). Animals 2021, 11, x FOR PEER REVIEW 15 of 24

Animals 2021, 11, 2168 14 of 23 A)

0.030 0.05 ) 0.025

mL 0.04 0.020 0.03

(mg/100 0.015

0.020.010

carotene 0.005

- 0.01 β

beta-Carotene (mg/100 mL) (mg/100 beta-Carotene 0.000 0.00 1 2 3 4 5 B) 1 2 3 4 5 (A)Day of life 30 X 25

Retinol (µg/100Retinol mL) 5

1 2 3 4 5

(BDay) of life Figure 5. Cont. Animals 2021, 11, x FOR PEER REVIEW 16 of 24

Animals 2021, 11, 2168 15 of 23 C)

2.0

1.5

1.0

0.5

Tocopherol (µg/mL) Tocopherol

0.0

1 2 3 4 5

Day of life (C)

FigureFigure 5. 5. PlasmaPlasma β-β carotene- carotene (A (),A retinol), retinol (B), (B and), and tocopherol tocopherol (C) (concentrationsC) concentrations in calves in calves dependent dependent on maternal on maternal fatty acid fatty supplementation (○ control, CON n = 9; ▲ EFA, essential fatty acid, n = 9; ▼, CLA, conjugated linoleic acid, n = 9; ♦, EFA acid supplementation ( control, CON n = 9; N EFA, essential fatty acid, n = 9; H, CLA, conjugated linoleic acid, n = 9; + CLA, n = 11) and time after birth. Day 1 corresponded to the day of birth. X marks differences between calves with , EFA + CLA, n = 11) and# time after birth. Day 1 corresponded to the day of birth. X marks differences between calves maternal EFA supplementation and without maternal EFA supplementation within the time points (p < 0.05). with maternal EFA supplementation and without maternal EFA supplementation within the time points (p < 0.05).

TheThe plasma concentration concentration of of tocopherol tocopherol increased increased after after birth birth until until the end the of end the of study the study(p < 0.001), (p < 0.001), but showed but showed no differences no differences between between groups groups (Figure (Figure5C). 5C). TheThe concentration concentration of of dROM dROM was was significantly significantly increased increased from from day day 1 to 1 today day 5 of 5 age of age (p <( p0.<001; 0.001 Figure; Figure 6A).6A). On On day day 3 and 3 and 5 5of of life, life, plasma plasma dROM dROM was was higher higher in in calves calves with than withoutwithout maternal maternal EFA EFA supplementation supplementation ( (pp << 0.05). 0.05). dROM dROM was was positively positively correlated correlated with with concentrationsconcentrations of of vitamins vitamins in in the the plasma plasma (r (r > > 0.338; 0.338; pp << 0.05; 0.05; Table Table 3).3). FRAPFRAP was was influ influencedenced by by time time (p ( p< <0.001) 0.001) and and increased increased from from day day 1 to 1 day to day 4 of 4 life of lifein CONin CON animals animals (p < (p0.05;< 0.05; Figure Figure 6B).6 AB). significant A significant EFA EFA effect effect on FRAP on FRAP was wasdetected detected (p = 0.007),(p = 0.007 but), also but a also significant a significant EFA-by EFA-by-CLA-CLA interaction interaction (p = 0.009). (p = 0.009). Additionally, Additionally, calves calves with maternwith maternalal EFA EFAsupplementation supplementation had hadlower lower plasma plasma FRAP FRAP on onday day 2 of 2 of life life (p (p == 0.014) 0.014) and and tendedtended to to be lowerlower onon dayday 3 3 of of life life in in these these calves calves compared compared to calvesto calves without without maternal maternal EFA EFAsupplementation supplementation (p = 0.071). (p = 0.071). On day On 4 ofday life, 4 CONof life, calves CON showed calves ashowed higher FRAPa higher compared FRAP compareto all otherd to groups all other (p < groups 0.05). At(p the< 0.05). same At time the point, same FRAPtime point, was lowered FRAP was by maternal lowered EFA by maternaland by CLA EFA treatment and by CLA (p = treatment 0.037 and (pp == 0.019).0.037 and There p = was0.019). a positive There was correlation a positive between corre- lationFRAP between and bilirubin FRAP concentrations and bilirubin concentrations in plasma on day in plasma 2 of life on (r =day 0.637; 2 of plife< 0.001).(r = 0.637; p < 0.001).The ORAC was higher in EFA + CLA calves on day 2 and day 5 of life compared to the initialThe ORAC level (wasp < 0.05)higher (Figure in EF6AC). + C OnLA day calves 2 of on life day plasma 2 and ORAC day 5 tendedof life compared to be higher to thein calvesinitial withlevel maternal(p < 0.05) CLA(Figure supplementation 6C). On day 2 comparedof life plasma to calves ORAC without tended maternal to be higher CLA insupplementation calves with maternal (p = 0.070) CLA leadingsupplementation in a significant compared CLA-by-time to calves interactionwithout matern (p = 0.050).al CLA supplementation (p = 0.070) leading in a significant CLA-by-time interaction (p = 0.050). Animals 2021, 11, x FOR PEER REVIEW 17 of 24

Animals 2021, 11, 2168 16 of 23 A)

40 X X

/mL)

2 30

dROM (µg H dROM

1 2 3 4 5 B) (ADay) of life 350 X Y 300 a

/ L) / 250 X 2+

Fe 200

mol 150 b 100 b b

FRAP (µmol Fe2+/L) (µmol FRAP FRAP (µ FRAP 50

1 2 3 4 5

(BDay) of life Figure 6. Cont. Animals 2021, 11, x FOR PEER REVIEW 18 of 24

Animals 2021, 11, 2168 17 of 23

14,00014000

12,00012000

10,00010000

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1 2 3 4 5 Day of life

(C)

Figure 6. PlasmaPlasma derivates derivates of of reactive reactive oxygen oxygen metabolites metabolites (dROM) (dROM) (A (),A ferric), ferric ion ion reducing reducing anti anti-oxygen-oxygen power power (FRAP) (FRAP) (B) and oxygen radical absorbance capacity (ORAC) (C) in calves dependent on maternal fatty acid supplementation (○ con- (B) and oxygen radical absorbance capacity (ORAC) (C) in calves dependent on maternal fatty acid supplementation trol, CON n = 9; ▲ EFA, essential fatty acids, n = 9; ▼, CLA, conjugated linoleic acid, n = 9; ♦, EFA + CLA, n = 11) and ( control, CON n = 9; EFA, essential fatty acids, n = 9; , CLA, conjugated linoleic acid, n = 9; , EFA + CLA, n = 11) time after birth. Day 1 correspN onded to the day of birth. ab DifferentH superscripts mark differences between groups at sim- # ab andilar time time points after birth. (p < 0.05). Day 1 X corresponded designates significant to the day differences of birth. betweenDifferent calves superscripts with maternal mark differences EFA supplementation between groups and X atwithout similar maternal time points EFA ( psupplementation< 0.05). designates at simila significantr time points differences and Y between calves calves with with and maternal without EFA maternal supplementation CLA sup- Y andplementation without maternal (p < 0.05). EFA supplementation at similar time points and between calves with and without maternal CLA supplementation (p < 0.05). 4. Discussion 4. DiscussionIn a companion paper of Uken et al. [26] the effects of the maternal fatty acid supple- mentationIn a companion on the colostrum paper ofand Uken milk et composition al. [26] the effects of the ofdams the from maternal the calves fatty acid used supple- in the presentmentation study on thewere colostrum demonstrated. and milk The composition results indicated of the damslower from dry matter the calves content used in the firstpresent colostrum study weremilking demonstrated. after calving The of CLA results- than indicated non-CLA lower-treated dry mattercows and content lower in pro- the teinfirst concentration colostrum milking in the after first calving colostrum of CLA- of CLA than- non-CLA-treatedtreated cows. Lower cows protein and lower concentra- protein tionsconcentration seemed to in be the not first related colostrum with the of CLA-treated immunoglobulin cows. concentrations Lower protein in concentrations the milk pre- sentedseemed in to the be notcurrent related publication. with the immunoglobulin EFA treatment resulted concentrations in lower in thefat concentrations milk presented in colostrumthe current of publication. EFA supplemented EFA treatment cows but resulted CLA did in lower not reduce fat concentrations milk fat during in colostrumfirst milk- ingsof EFA after supplemented calving [26]. cows but CLA did not reduce milk fat during first milkings after calvingPlasma [26]. Ig and total protein concentrations in calves reflected the Ig concentrations in colostrumPlasma of Igtheir and dams total and protein indicated concentrations the absorption in calves of Ig reflected from colostrum the Ig concentrations and successful in passivecolostrum immunization of their dams of and calves indicated in the first the absorptiondays of life of [5,34 Ig from]. The colostrum maternal anddiet successfulcan influ- encepassive the immunization Ig concentration of calves in colostrum in the first of sows days of[15] life and [5,34 the]. Therelation maternalship between diet can influenceneonatal absorptionthe Ig concentration of polyunsaturated in colostrum fatty ofacid sows and [Ig15 was] and recently the relationship discussed in between piglets neonatal[35]. The absorption of polyunsaturated fatty acid and Ig was recently discussed in piglets [35]. The maternal EFA and CLA supplementation in the present study affected the EFA and CLA status in the calves [26] but did not influence the total IgG absorption in calves. Animals 2021, 11, 2168 18 of 23

Recent studies in calves showed positive as well as negative effects of maternal EFA supply on neonatal IgG absorption [25,36]. The lower IgG2 concentration in the transition milk of the 2nd day in EFA cows might not explain the differences in plasma total protein concentration, as total plasma IgG on day 2 after birth was not different among groups. Plasma albumin and haptoglobin concentrations, which are also an integral part of total proteins, were not a plausible explanation for the lower total protein concentration in EFA calves because of no or opposite influences of the fatty acid treatments on these plasma traits in the calves. Therefore, we currently cannot explain the up to 10 g/L lower plasma concentration of total protein in the EFA calves compared with the other groups on d 2 of life and we cannot exclude other causes such as an altered protein accretion. However, because of the slightly higher haptoglobin concentrations in EFA calves compared to CON calves we speculated that lower plasma total protein concentrations in EFA calves might indicate an altered liver activity. In contrast to studies of Panousis et al. [37], the current study revealed no differences between hematological traits of male and female calves. Nevertheless, the values of calves in the present study were in the reference intervals described by Knowles et al. [38], Brun-Hansen et al. [39] and Panousis et al. [37], except for hemoglobin, RDW, MCHC and thrombocytes, which were slightly lower in the mentioned studies. The age-dependent effects on HCT, hemoglobin, RDW, erythrocyte cell counts and MCV were also described in studies of Knowles et al. [38] and Panousis et al. [37]. In both studies, it was demonstrated that leucocyte cell counts decrease until day 3 or 4 of life. In comparison with the present results, it might be assumed that EFA supports the period of physiological decrease of leucocyte counts after birth while CLA shortened or inhibited this period. The biological relevance of this aspect has to be studied in further trials. According to Knowles et al. [38] proportions of lymphocytes increase physiologically in calves after birth but proportions of neutrophils decrease. Interestingly, only in EFA calves there was a trend for this physiological increase of lymphocyte proportions on day 5 of life. In contrast, the combined EFA + CLA treatment seemed to intensify the decrease of the lymphocyte proportion, leading to the assumption that other leucocyte populations increase such as neutrophilic granulocytes. Because only proportions of basophilic granulocytes were additionally determined we can only exclude a marked increase of this cell population in the EFA + CLA calves. Essential fatty acids such as linoleic acid are able to increase the mitogen-stimulated proliferation of PBMCs at lower concentrations in cattle [18,40]. At higher concentrations, this fatty acid inhibited mitogen-stimulated PBMC proliferation but also unstimulated lymphocyte proliferation [18,40]. The continuous increase of plasma cholesterol in the calves was in accordance with the increase in plasma cholesterol after parturition in dams [19]. The lower plasma cholesterol concentration on day 2 of life in the EFA calves was probably a consequence of lower milk fat content in the colostrum of the dams supplemented with EFA [26]. It might also reflect a lower liver activity in EFA calves. Furthermore, EFA might have cholesterol clearing effects via the activation of PPARα, which corresponds to lower cholesterol levels found previously in the plasma of calves supplemented with essential fatty acids [13,41]. The increase of bilirubin in the first 24 h after birth is a physiological phenomenon that has already been shown in the study of Kurz and Willet [42] with neonatal calves [42]. The increase from day 1 to day 2 of life might indicate an enhanced degradation of fetal erythrocytes and the increased synthesis of bilirubin in calves [43]. Regarding the lower postnatal increase in plasma bilirubin in the EFA calves, we speculate that maternal EFA treatment may decelerate erythrocyte decay in newborn calves, probably because of stabilization of the fetal erythrocyte membrane by EFA incorporation. The erythrocyte membranes of the dams were enriched, especially with α-linolenic acid and other n-3 FA, due to EFA supplementation [19]. However, the number of erythrocytes in calf blood did not differ among the groups. In addition, the plasma bilirubin concentration was lower at parturition in the dams treated with EFA only [19]. A decrease in the n-6/n-3 fatty acid ratio reduced the plasma bilirubin concentration and prevented liver diseases in preterm infants [44] and Animals 2021, 11, 2168 19 of 23

in neonatal piglets [45] fed parenteral nutrition. The stabilization of the erythrocyte membrane by reducing the n-6/n-3 fatty acid ratio in the membrane may lead to less iron release from fetal erythrocytes. Free iron is able to generate harmful oxygen species by producing oxidant hydroxyl radicals through the Fenton reaction [46,47]. Unfortunately, plasma iron was not measured in the present study but plasma FRAP was lower in EFA calves and there was a close correlation between plasma bilirubin and FRAP on day 2 of life. In studies of Opgenorth et al. [48] it was shown that n-3 fatty acids supplemented by colostrum are able to improve the anti-inflammatory state of neonatal calves. In the present study the maternal EFA supplementation decreased and CLA increased the secretion of the pro-inflammatory cytokine interleukin 1-β on day 2 of life. Similarly, interleukin 6 production was increased in the CLA group with time, which might be the result of the increased interleukin 1-β concentration. Interleukin 1-β is able to induce the release of prostaglandin E2, which is a potent stimulator of interleukin 6 [49–51]. Plasma interleukin 1-β may have additionally contributed to the increased bilirubin plasma concentration in CLA calves on day 2 of life. Bilirubin is known as an inflammatory marker in cattle [52]. Elevated levels of bilirubin are toxic for the organism and have to be cleared rapidly, however, it was shown that bilirubin is also a potent anti-oxidant [53,54] and an effective inhibitor of inducible nitric oxide synthase (iNOS) [55]. This enzyme is known as a potent stimulus of reactive oxygen species production. The production of iNOS is upregulated by interleukin 1-β and iNOS are elevated in the liver and ileum 24 h after birth in calves [49,56]. An increase in the production of reactive oxygen species can lead to oxidative stress. Therefore, the upregulation of bilirubin in non-EFA calves might be a response and clearing mechanism of increased interleukin 1-β production and resulting increased reactive oxygen species release. However, according to dROM no differences in reactive oxygen metabolites in plasma were found between EFA and non-EFA calves. The anti-oxidative potential of bilirubin might be supported by the significant correlation between bilirubin and FRAP on day 2 of life and might be an explanation for the higher FRAP of non-EFA groups compared to EFA groups on this day. Relationships between FRAP and bilirubin were already shown in rats [57]. Whether the increase of interleukins is a response to increased bilirubin concentrations because of its toxicity which was possibly induced by erythrocyte degradation or whether this is a clearing mechanism of the enhanced pro-inflammatory interleukin release might be investigated in further studies. However, in the present study clear relations between these factors were shown and that these relationships depended on the maternal fatty acid supply which in turn altered the colostral and plasma fatty acid composition of the offspring [26,27]. Wiedemann et al. [54] found relationships between the plasma oxidation rate and bilirubin as well as the polyunsaturated fatty acid concentrations in plasma of newborns. They reported improved protection against oxidation immediately after birth by higher bilirubin but lower polyunsaturated fatty acid concentration. Compared to bilirubin, other measured plasma anti-oxidants increased more slowly after birth which might be an evidence of the importance of bilirubin especially during the first hours after birth. With regard to these aspects, CLA and CON calves might be superior compared to EFA calves because of the provoked increase of bilirubin concentrations. Indeed, in the case of EFA, plasma FRAP was reduced on day 2, which corresponded to the increased dROM plasma concentration on day 3 of life. In studies of Ballou and DePeters [14], n-3 fatty acid supplementation resulted in a higher percentage of oxidative burst-producing polymorphonuclear leucocytes in calves. Similar to the oxidative system as indicated by dROM, the anti-oxidative blood components such as β- carotene, retinol and tocopherol, FRAP and ORAC increased age- dependently after birth in all calves. Boosting of the anti-oxidative system in calves after birth leading to a better protection against oxidative stress in this period compared to cows was also demonstrated by Gaál et al. [58]. Because the oxidative and anti-oxidative status of calves did not differ immediately after birth, it was suggested that treatment-dependent differences between calves where more related to colostral supply. Abuelo et al. [2,59] Animals 2021, 11, 2168 20 of 23

pointed at the importance of the redox balance of the colostrum on the oxidative status of the calf. Blum et al. [60] already demonstrated the importance of colostrum on the vitamin status of newborn calves and emphasized the relationships between the absorption of fatty acids and fat-soluble vitamins such as retinol. According to the present results, the fatty acid composition in colostrum, which was influenced by the maternal fatty acid supply, did not influence the absorption of fat-soluble vitamins few days after birth in a consistent manner. An elevated retinol plasma concentration was found with respect to EFA treatment on day 5 of life, which might have boosted the anti-oxidant status in these calves. Correlations between dROM and plasma vitamins indicate their role to balance the age-dependent increases in dROM concentrations. In turn, EFA calves might have a more efficient protection against pathogens by increased radical production and similarly improved anti-oxidative status, which prevents against oxidative stress in a later stage after birth. Additionally, in studies of Opgenorth et al. [61], n-3 fatty acid supplementation by colostrum improved the oxygen status of newborn calves.

5. Conclusions The maternal fatty acid supplementation influenced the inflammatory response shortly after birth. Especially, EFA might have beneficial effects on the prevention of inflammation after birth and against oxidative stress in a later stage after birth. Furthermore, EFA reduced the plasma bilirubin concentrations in an early period. CLA and saturated fatty acids might increase the inflammatory response. Similarly, bilirubin was increased by this maternal supplementation, which might serve as a protector against oxidative stress in this early stage. In general, calves have potent instruments for the clearance of inflammatory or oxidative stress after birth in which bilirubin seemed to be involved. In further studies, the relationships between bilirubin and the interleukin response as well as the anti-oxidative and the oxidative status in the neonatal calf should be studied in more detail to support the present findings.

Author Contributions: Conceptualization, H.M.H. and A.T. (Arnulf Tröscher); methodology, A.T. (Armin Tuchscherer), E.T., T.S. and H.S.; investigation, W.L., K.L.U., C.S., L.V. and M.G.; resources, H.M.H. and A.T. (Arnulf Tröscher); writing—original draft preparation, W.L. and K.L.U.; writing— review and editing, H.M.H., H.S. and E.T.; supervision, H.M.H.; project administration, H.M.H. and A.T. (Arnulf Tröscher); funding acquisition, H.M.H. and A.T. (Arnulf Tröscher). All authors have read and agreed to the published version of the manuscript. Funding: The present study was supported by the BASF SE (Ludwigshafen, Germany). Institutional Review Board Statement: The animal study was in accordance with the guidelines of German Animal Protection Law (LALLF) and reviewed and approved by the Landesamt für Land- wirtschaft Lebensmittelsicherheit und Fischerei Mecklenburg-Vorpommern, Rostock (registration number 7221.3-1-052/15). Data Availability Statement: The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Acknowledgments: The authors gratefully acknowledge the staff of the Experimental Animal Facility Cattle as well as Susanne Haubold and Jeannine Gruse for the support and animal care during the experimental period. Furthermore, we thank Paulina Jawor (Wroclaw University of Environmental and Life Science), Annarita Ferrari and Giogia Lovotti (DIANA, Università Cattolica S. Cuore), Heike Pröhl, Claudia Reiko (FBN, Dummerstorf) and Barbara Heitkönig (Uni Bonn) for the excellent laboratory work. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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