Fat composition of milk replacer influences postprandial and oxidative metabolism in dairy calves fed twice daily.

Milk replacers (MR) for calves contain alternative fat sources as substitute for milk fat. This substitution leads to differences in fat properties, such as the fatty acid profile and the triglyceride structure. This study evaluated how fat composition in MR affects gastrointestinal health, blood redox parameters, and postprandial metabolism in calves fed twice daily. Forty-five individually housed male Holstein-Friesian calves (2.3 ± 0.85 d of age) were assigned to 1 of 15 blocks based on the age and the day of arrival. Within each block, calves were randomly assigned to one of 3 experimental diets and received their respective diet from arrival until 35 d after arrival. The 3 experimental diets (n = 15 per treatment group) consisted of a MR with a blend of vegetable fats containing rapeseed and coconut (VG), a MR with only animal fats from lard and dairy cream (AN), and a MR containing a mixture of animal and vegetable fats including lard and coconut (MX). The fatty acid profile of each MR was formulated to resemble that of bovine milk fat while using only 2 fat sources. All MR contained 30% fat, 24% crude protein, and 36% lactose. Chopped straw and water were available ad libitum from arrival onwards but no starter feed was provided. Daily milk allowances were 6.0 L from d 1 to 5, 7.0 L from d 6 to 9, and 8.0 L from d 10 to 35, divided into 2 equal meals and prepared at 135 g/L (13.5% solids). Fecal appearance was scored daily, calves were weighed, and blood was drawn on arrival and weekly thereafter. Urine and feces were collected over a 24 h-period at wk 3 and 5 to determine apparent total-tract digestibility and assess gastrointestinal permeability using indigestible markers. Postprandial metabolism was evaluated at wk 4 by sequential blood sampling over 7.5 h, and the abomasal emptying rate was determined by acetaminophen (Ac) appearance in blood. Fat composition in MR did not affect growth, MR intake, gastrointestinal permeability, nor nutrient digestibility. The percentage of calves with abnormal fecal scores was lower at wk 2 after arrival in calves fed VG than those fed MX, whereas AN did not differ from the other treatments. Calves fed AN and MX had higher thiobarbituric acid reactive substances measured in serum than VG, whereas plasma ferric-reducing ability was greater in calves fed MX than VG. Postprandial Ac concentrations did not differ across treatment groups, but the area under the curve was smaller in calves fed VG than in other 2 treatments, which is indicative of a slower abomasal emptying. Postprandial serum TG concentration was greater in calves fed AN than VG, whereas MX did not differ from the other treatments. Based on these outcomes, all 3 fat blends can be considered suitable for inclusion in MR for calves.


INTRODUCTION
In recent years, it has been proposed to increase the fat content of milk replacer (MR) for calves to align with the macronutrient profile of bovine whole milk (WM; Echeverry-Munera et al., 2021;Welboren et al., 2021;Wilms et al., 2022a).This nutritional approach may underscore the importance of considering fat composition which modulates fat absorption and digestion in neonates (Hageman et al., 2019).The fatty acid (FA) profile and triglyceride (TG) structure in MR differ from those of milk fat due to the use of alternative fat sources.In Europe and other regions, primarily vegetable fats are used (Wilms et al., 2022a;Mellors et al., 2023), while in North America, blends of animal and vegetable fats are commonly used (Huuskonen et al., 2005;Drackley, 2008).There might be opportunity to further optimizing fat composition in MR to enhance fat digestion and utilization and to promote optimal health and development of neonatal calves.
Ensuring that the FA profile of milk substitutes resembles that of milk fat allows for optimal digestion and provides FA that are essential for gastrointestinal development and immune functions (Guilloteau et al., 2010;Garcia et al., 2015;McDonnel et al., 2019).In addition to the FA profile of MR, the TG structure is also an important component of fat digestibility and absorption in neonates (Innis, 2011;Yao et al., 2014).Upon digestion, FA in the sn-1 and sn-3 positions are preferentially cleaved off as free FA, whereas the sn-2 FA remains attached to the glycerol backbone (Hageman et al., 2019).In bovine WM, about 40 to 45% of all palmitic acid (Bracco, 1994) is positioned at the sn-2 position on the glycerol backbone.In contrast, only 10 to 20% of palmitic acid is located at the sn-2 position in vegetable fat blends containing palm and coconut oils (Bracco, 1994;Sun et al., 2018).When palmitic acid and other long chain saturated FA (LCSFA) are released from sn-1 and sn-3 positions, they tend to form complexes with Ca which are absorbed and digested in a lower extent in infants (Petit et al., 2017).Hill et al. (2011) showed that in MR with 15 to 19% fat, the inclusion of 1% of a blend containing butyrate, coconut oil, and flax oil reduced diarrhea incidence and improved growth rate and feed efficiency in calves fed 1 kg DM of MR per day.Hamilton and Raven (1975) showed that the apparent total-tract digestibility of fat was greater with butterfat than with tallow, in MR with 20% fat (% DM) and fed 4.0 L daily at 125 g/L.In contrast, a study from Huuskonen et al. (2005) found no differences in apparent total-tract digestibility of MR (18% fat), including either 100% lard or blends of nonhydrogenated vegetable oils (palm, coconut, and rapeseed) in bull calves fed at 10% birth BW (as volume fed).Furthermore, Jenkins et al. (1986) showed that a 1:1 mixture of corn oil and tallow in MR (20% fat) resulted in increased diarrhea and poor growth rates as compared with canola oil in calves fed 4.0 L/d at 140 g/L.
Most of the previous experiments investigating fat composition in MR for calves have included relatively low-fat inclusion levels (15-20% DM) and were fed at low feeding rates (~10% birth BW; Jenkins et al., 1986;Huuskonen et al., 2005;Hill et al., 2011).This deeply contrasts with current recommendations for calves, where animals are fed as much as 20% of their birth BW as volume (Echeverry-Munera et al., 2021;Wilms et al., 2022a).In addition, the FA profile and TG structure are usually not balanced across MR treatments (Jenkins et al., 1986;Huuskonen et al., 2005), making it difficult to compare the effects of fat composition in MR on growth and digestibility in calves.Although dietary fat composition affects gastric emptying rates in humans and rats (Meyer et al., 1998;Feltrin et al., 2004) and gastrointestinal integrity in rats (Benoit et al., 2015), the effects of fat composition on these parameters have to the best of our knowledge not been studied in calves.
Overall, fat characteristics related to FA profile and TG structure are expected to affect digestion and absorption of dietary fats and metabolic responses in calves, but this has not been adequately described at currently proposed fat contents and feeding levels.The current study evaluated the effects of fat composition in MR on total apparent tract digestibility, gastrointestinal permeability, and postprandial metabolism in male dairy calves fed MR high in fat twice daily.The hypothesis was that a MR containing a blend of lard and dairy cream would show benefits on digestibility and gastrointestinal health, compared with MR formulations containing vegetable fats.The benefits of the animal fat treatment are expected based its FA profile and a TG structure close to that of milk fat.

MATERIALS AND METHODS
This study was conducted at the Calf and Beef Research Facility of Trouw Nutrition Research and Development (Sint Anthonis, the Netherlands) between July and December 2020.All procedures complied with the Dutch law on Experimental Animals, which complied with ETS123 (Council of Europe, 1985 and the 86/609/ EEC Directive), and were approved by the animal welfare authority (Centrale Commissie Dierproeven, CCD, the Netherlands).The project application code is AVD2040020173425.

Animals and Experimental Design
This experiment was conducted in a randomized complete block design.A total of 45 Holstein-Friesian male calves between 0 to 3 d of life were acquired from 7 collaborating dairy farms located within 30 km from the research facility.Calves were purchased in successive batches for the study, and measurement periods were staggered accordingly.The recruitment of calves took place between the 20th of July and the 26th of October 2020.At the farm of origin, calves were handled according to a standardized protocol for colostrum feedings including a first meal between 3.0 and 4.0 L within the first 3 h after birth followed by 2 feedings of 2.0 L. Colostrum quality was monitored and required a Brix value of 22% or greater, indicating an immunoglobin content of 50 mg/L or greater.Thereafter, meals of 3.0 L of MR (Sprayfo Delta, Trouw Nutrition, the Netherlands) were offered twice daily until calves were transported to the research facility.Upon arrival at the research facility, blood IgG was measured within 48 to 72 h after birth.The mean BW at arrival was 45.6 ± 4.0 kg (mean ± SD) and the age at arrival was 2.3 ± 0.85 d.On the day of arrival, calves were assigned to one of 15 blocks based on arrival day and age to minimize age differences within the same block of 3 calves.Within each block, calves were randomly assigned to one of 3 experimental diets (n = 15 per treatment group) and were exposed to their respective diet from arrival at the research facility until d 35 after arrival.The randomization was performed using the random function [RANDBETWEEN (0,100000)] in Microsoft® Excel® (Microsoft 365 MSO, Version 2212, Build 16.0.15928.20278).Experimental diets consisted of a MR that included different fat blends (Trouw Nutrition, Deventer, the Netherlands) formulated with different fat sources.The 3 experimental diets consisted of: 1) a MR with only vegetable fats composed of 40% of Racomelt fat blend (Cargill, Minnesota, US) mixed with 60% unhardened rapeseed oil (VG); 2) a MR formulated with only animal fats, including 65% of packers lard and 35% of liquid dairy cream (AN); and 3) an MR with a mix of animal and vegetable fats, including 80% of packers lard and 20% of coconut oil (MX).The Racomelt product from Cargill (Minnesota, US) was a mixture of 50% coconut oil interesterified with 50% fully hydrogenated rapeseed oil.While using only 2 fat sources, the VG, and MX MR treatments represent 2 rather distinct approaches to fat composition in MR.The AN treatment represented an attempt to align with the FA profile of milk fat without including solely milk fat, as this would defeat one of the main purposes of MR, which is increasing the net dairy product yields of dairy farms.All MR were composed of 63.0% skim milk powder, 30.0%crude oils and fats, 5.0% sweet whey powder, and 2% premix (vitamins, minerals, and additives; Trouw Nutrition, Deventer, the Netherlands).This led to the 3 MR formulas with the same nutrient content (30% fat, 24% crude protein, and 36% lactose; DM basis).Despite slight variations in analytical values (Table 1), it can be considered that these diets were isonitrogenous and isoenergetic.During the process of MR production, the fats were sprayed dried.All 3 diets were not only formulated based on crude fat percentage but aimed to be close to the FA profile of bovine milk fat (Moate et al., 2007), while remaining within formulation constraints and using only 2 fat sources (Table 2).However, it should be noted that the entire FA profile of milk fat was not considered, as some FA only exist in milk fat.Calves were fed their respective MR diet twice daily via teat buckets at 0630 and 1730 h.The daily milk allowance was 6.0 L from d 1 to 5 after arrival, 7.0 L from d 6 to 9, and 8.0 L from d 10 to 35, divided over 2 equal meals.The concentration of the MR in water was 135 g/L (13.5% solids) across all treatments and was based on general recommendations for MR high in fat and to get closer to the percentage of solids of WM.The farm staff responsible for scoring and sampling the calves were unaware of the treatment group to which each calf belonged as a letter (A, B, or C) was assigned to each treatment, thus ensuring a blinded approach.
Housing.Calves were housed indoors in individual pens (2.34 m × 1.16 m) separated by galvanized bar fences and equipped with a rubber-slatted floor in the front area (50% of the total pen area) and a laying area in the back, which contained a mattress covered with flax straw.During total urine and fecal collection, calves were tethered to the front of the pen and an elevated plateau covered with rubber was placed at the front of the pen to elevate the animals and facilitate urine collection.The temperature in the calf pen was maintained at a minimum of 12°C and a maximum of 28°C.Relative humidity was held between 60 and 85%.Calves were exposed to daylight and artificial light from 0530 to 2130 h.

Measurements
General health was monitored daily by caretakers and administration of medical treatments and oral rehydration solution (Sprayfo OsmoFit, Deventer, Netherlands) was recorded daily.Calves were weighed at 1300 h on the day of arrival and once weekly thereafter until 35 d after arrival.Individual MR, water, and straw intakes were recorded daily throughout the experimental period by weighing refusals.Photos of feces were taken daily for all calves until d 21 after arrival, and fecal scores were evaluated at the end of the study by a single observer who was blinded to the treatments.Feces were scored on a 3-point scale, ranging from 0 (normal) to 1 (loose) to 2 (very watery).Feces were collected over a 24 h period in wk 3 (21.8± 0.84 d after arrival, means ± SD) and 5 (35.8 ± 0.84 d) from 1200 h to 1200 h on the following day to measure apparent total-tract digestibility.In brief, feces were collected using fecal bags glued to the hindquarters of the calves, as described by Wilms et al. (2020a).Gastrointestinal permeability was assessed by measuring the recovery of indigestible markers in urine over a 24-h collection period performed on the same days as total fecal collection.A spot urine sample was collected on the day before the marker dosing in wk 3 for background measurement.On the day of urine collection, calves received a combination of lactulose (0.2 g/kg of BW; Sigma-Aldrich, Zwijndrecht, the Netherlands), D-mannitol (0.12 g/kg of BW; Sigma-Aldrich, Zwijndrecht, the Netherlands) dissolved in 100 mL warm water.A solution containing 8.3 g/L of chromium (Cr) was prepared by Masterlab (Boxmeer, the Netherlands) and the volume of the solution was adjusted to provide a dose of 0.1 g Cr-EDTA per kg of BW.Both marker solutions were successively orally pulse dosed at 0630 h instead of the morning meal to avoid interactions with digestion of the MR.A urine bag was glued to the underside of the calf, and bags were connected to a bucket with a tube, as described by Wilms et al. (2020a).Subsequently, urine was collected over 2 periods, from 0 to 6 h and 6 to 24 h relative to marker administration.On the day of the gut permeability assessment, the morning MR meal was fed at 1230 h to allow having a 6 h urine collection without interference from the MR meal.
Blood samples were collected weekly at the same time as weighing and took place at 1300 h by jugular venepuncture at arrival and weekly thereafter until d 35.At each sampling time point, blood samples were collected in one 9 mL tube with gel for serum (BD Vacutainer, Becton Dickinson), one 9 mL and one 4 mL lithium heparin (LH, BD Vacutainer, Becton Dickinson), and one 4 mL NaF vacutainer (BD Vacutainer, Becton Dickinson).For evaluation of postprandial dynamics, all calves were sedated by intramuscular (IM) injection of an anesthetic (Sedamun; Xylazine 2%/20mg; 23.32 mg xylazine hydrochloride) into the neck at 1400 h on d 27 to alleviate the stress associated with catheter placement.The procedure was performed at least one hour after the 1200 h milk feeding and water was withdrawn until the end of the procedure.Throughout the procedure, calves were kept in the sternal position, and the sedation effect was reversed with an IM injection of an anti-sedative (Antisedan; atipamezole hydrochloride; 5 g/mL).Intakes of MR from the subsequent evening meal were not affected by the procedure.On d 28, sequential blood sampling was performed to evaluate postprandial dynamics.To evaluate abomasal emptying rates, a dose of acetaminophen (150 mg Ac/kg BW) was mixed into the morning milk meal at 0600h.Notwithstanding, postprandial responses have been successfully performed in previous experiments with lower doses of acetaminophen (0.13 g/kg of BW 0.75 ;  MacPherson et al., 2016; Stahel et al., 2019; Welboren  et al., 2021).Blood samples were collected in one 9.0 mL LH monovette, one 5.0 mL NaF monovette, and one 9.0 mL serum monovette at −30 min and 30,60,90,120,150,180,210,240,300,360, and 420 min relative to the morning meal.For the weekly and postprandial blood collection, serum tubes were set for 30 min at room temperature and centrifuged at 1,500 g for 10 min at 20°C.Plasma and NaF tubes were immediately placed on ice and centrifuged at 1,500 g for 15 min at 4°C within 10 min after blood collection (Rotina 380 R, Hettich, Tuttlingen, Germany).Serum, plasma, and NaF samples were stored in 1.5-mL cryotubes.All samples were transported in boxes with cooling elements and stored at −20°C until the analyses were performed.

Laboratory Analysis
Milk replacer and straw were sampled for analyses at the start of the study.All feed samples were analyzed for moisture content (EC 152/2009;EC, 2009), crude protein (CP) content (Dumas' method: Etheridge et al., 1998), crude fat content using hydrochloric acid pre-treatment and petroleum extraction (EC 152/2009;EC, 2009), crude ash content (EC 152/2009;EC, 2009), and macro-minerals by inductively coupled plasma mass spectrometry (PerkinElmer ICP-MS 300D), according to NEN-EN 2017(2017).Crude fiber in straw was analyzed via intermediate filtration (NEN-EN-ISO 6865:2000;NEN, 2001).Milk replacer samples were also analyzed for lactose using the titrimetric method according to 1971 71/250/EEG.All feed samples were processed and analyzed at Masterlab (Boxmeer, the Netherlands).The FA profile of the MR diets was analyzed by QLIP (Zutphen, the Netherlands) by gas chromatography according to NEN-ISO 15885.
Urine samples were analyzed for lactulose, D-mannitol, and Cr, as described in Mellors et al. (2023).Fecal samples were analyzed for DM, CP, crude fat, crude ash, and calcium.Oxidative status was assessed at the University of Bonn (Bonn, Germany) by determination of reactive oxygen species.The detection of reactive oxygen metabolites test (dROM) was performed according to Alberti et al. (2000) with modifications (Regenhard et al., 2014) using N, N-diethyl-paraphenylendiamine as chromogenic substrate.The dROM values are expressed as H 2 O 2 equivalents (intra-assay 7.53% and inter-assay 4.55%).The antioxidative capacity was measured as the ferric-reducing ability of plasma (FRAP) expressed in µmol Fe 2+ /L according to Benzie and Strain (1996).For FRAP, the intra-assay was 1.69% and the inter-assay was 3.51%.The oxida-tive damage of lipids in plasma was evaluated via the TBARS (thiobarbituric acid reactive substances) assay according to Gutteridge and Quinlan (1983).The results are expressed as malondialdehyde (MDA) concentration (intra-assay 5.51% and inter-assay 9.77%).
The activity of the selenium-dependent antioxidant selenoenzyme glutathione peroxidase (GSH-Px) in plasma was determined by spectrophotometry according to Paglia and Valentin (1967).One unit (U/mL) is equal to the amount of enzyme necessary for catalyzing the oxidation of 1 µmol NADPH per min at 25 C, pH 7.0.For GSH-Px, the intra-assay was 2.63% and the inter-assay was 12.6%.The dROM and FRAP values were used to calculate an oxidative stress index (OSi),

Calculation and Statistical Analysis
Based on outcomes of an experiment conducted in the same research facility (Wilms et al., 2023a, in press) investigating macronutrient composition of MR, and performing the same measurements as in the present study, the projected sample size was 15 calves per treatment group.The apparent total-tract digestibility of DM and nutrients (fat, protein, ash, minerals) was calculated using MR intakes and fecal output over a 24 h period on d 21 as follow: Digestibility (fat) = 100 -fat in feces (g DM) / fat in feed ingested (g DM) × 100 Continuous variables were analyzed using a mixedeffects model with PROC MIXED in SAS (SAS 9.4M6, SAS Institute Inc., Cary, NC).The calf was the experimental unit, and the statistical model was as follows: where: Y ijkl is the dependent variable; µ is the overall mean; T i is the fixed effect of the ith treatment; V j is the random effect of the jth block; W k is the fixed effect of the kth day entering the model as a repeated measure; TW ik is the fixed effect interaction between the ith treatment and the kth day; and ε ijk is the random error associated with the jth block at the kth day with the ith treatment.In the case of BW and ADG, arrival BW was used as a baseline covariate (µ 0 ).For variables with equally spaced time points, the covariance structure (autoregressive covariance [AR(1)] or the heterogeneous autoregressive covariance [ARH(1)]) with the lowest corrected Akaike Information Criterion was used.For variables with unequally spaced time points, toeplitz (TOEP) or the heterogeneous Toeplitz (TOEPH) covariance structure was used.Data that did not meet the assumptions of normality of residuals had to be log-transformed (base 10).A significant effect of treatment was contrasted with the LSMEANS statement using the PDIFF option of the MIXED procedure of SAS.Results in tables and figures are presented as LSM with the SEM.Discrete variables (e.g., therapeutic interventions, and fecal scores) were analyzed using mixed effects logistic regressions.These analyses were conducted with PROC GENMOD in SAS.Significance was declared at P ≤ 0.05, and the trend threshold was set at 0.05 < P ≤ 0.10.Model Parametrization of Abomasal Emptying.For each calf, the differential equations describing the disappearance of Ac from the abomasum and serum Ac concentrations were solved using Wolframaplha (Wolfram Research, 2010) and then were entered in Microsoft® Office Excel® (2007) to predict values at each sampling time point.The Z value representing the occurrence of abomasal emptying, and its speed (Z = 0 for no gastric emptying occurring, Z = 1 being slow, and Z = 2 being fast) was determined based on the slope of the observed successive Ac postprandial concentrations.The best-fit parameters of Ac kinetics (slow emptying rate, fast emptying rate, and utilization rate) were estimated with the Solver function of Microsoft Excel to minimize the residual sum of squares between observed and predicted serum Ac.

Feed Intakes and Blood Redox Parameters
The concentration of blood IgG measured between 48 to 72 h after birth (20.3 ± 1.86 g/L; mean ± SD; Table 3), BW at arrival (45.6 ± 0.59 kg), and age at arrival (2.29 ± 0.13 d) did not differ between treatment groups.All calves completed the entire experimental period, and the administration of medical treatments did not differ across treatment groups.However, the percentage of days with a high fecal score was significantly lower in VG-fed calves (score 1: 27%, score 2: 10%) than MX (score 1: 35%, score 2: 25%) in wk 2 (P < 0.01), whereas AN (score 1: 23%, score 2: 22%) did not differ from the other treatment groups.Fat composition in MR did not affect growth (Supplemental Figure 1A) nor MR intakes (Supplemental Figure Figure 1B).Serum GSHPx, dROM, and OSi were also not affected by treatments, whereas serum TBARS (Supplemental Figure 2A) was lower for calves fed VG compared with calves fed AN and MX (P < 0.01).Similarly, plasma FRAP (Supplemental Figure 2B) was lower for VG-fed calves than MX (P = 0.03), whereas AN did not differ from other treatments.

Apparent Total-tract Digestibility and Gastrointestinal Permeability
Apparent total-tract digestibility (Table 4) did not differ across treatment groups.Fecal DM was higher in calves fed VG than AN and MX (P = 0.05) in fecal samples collected in wk 3 and 5, which is consistent with the lower fecal scores in that group in wk 2. However, the total fecal DM output expressed as % live BW did not differ across groups.Similarly, gastrointestinal permeability assessed by recovery of indigestible markers (Cr-EDTA, lactulose, and D-mannitol) was not affected by fat composition in MR.

Postprandial Dynamics
Postprandial TG concentration was elevated in calves fed AN compared with VG (P = 0.05), whereas MX did not differ from the other treatment groups (Supplemental Figure 3A).The time to reach the maximal concentration (T max ) was shorter in calves fed AN than other treatments (P = 0.01).In contrast, postprandial NEFA did not differ across treatment groups.Although postprandial Ac concentrations were not different across treatment groups (Supplemental Figure 3B), the AUC for Ac was lower in calves fed VG than AN and MX (P = 0.04).Besides, parameters describing Ac kinetics (slow emptying rate, fast emptying rate, and utilization rate) did not differ across treatment groups.

DISCUSSION
In this study, the effect of fat composition in MR on postprandial and oxidative metabolism was investigated in calves.Three types of fat composition in MR were compared: exclusively vegetable sources, exclusively animal sources, and a mixture of both.As expected from the equal macronutrient profile of the diets and the restricted feeding scheme, treatments did not have a significant effect on intake and growth.Formulating the fat fraction of MR based on the FA profile of milk fat resulted in marginal differences in gastrointestinal health; however, specific serum redox parameters and postprandial metabolism was altered in calves fed VG.
In previous work, the inclusion of corn oil (Adams et al., 1959), cottonseed oil (Jarvis and Waugh, 1949), and soybean oil (Wiese et al., 1947;Jacobson et al., 1949) led to poorer performance compared with coconut oil, tallow, lard, or butterfat in calves (as reviewed by Raven, 1970).When the FA profile and the TG structure substantially differ from milk fat, fat absorption and utilization by neonates may be impaired (Delplanque et al., 2015).Corn, cottonseed, and soybean oils typically have high levels of unsaturation (about 55% of total FA as PUFA) which has been shown to reduce calf performance due to a possible reduced capacity of calves to metabolize high dietary PUFA from MR (Barrows et al., 1980;Richard, 1980;Wilms et al., 2023b, in review).In the current study, the 3 fat concentrates were formulated to have a similar FA profile and TG structure to milk fat, which may explain why no growth differences were detected.In addition, the VG fat was partly inter-esterified, which lowered the amount of long chain SFA in the sn-1 and sn-3 positions on the glycerol backbone (as reviewed by Hunter, 2001).Upon digestion, sn-1 and sn-3 FA are preferentially cleaved off as free FA, and sn-2 FA remains attached to the glycerol backbone (Hageman et al., 2019;Jenkins et al., 1985).When long chain SFA are released from sn-1 and sn-3 positions, they tend to form complexes with Ca which are poorly absorbed and digested by infants (Petit et al., 2017).Nevertheless, this aspect may not be as critical in calves, as bovine WM contains approximately 45% of long-chain SFA such as palmitic and myristic acids in the sn-2 position, compared with 75% in human breast milk (Delplanque et al., 2015).In the current study, fat composition in MR did not affect apparent total-tract digestibility of dietary fat and Ca.Huuskonen et al. (2005) reported that total apparent tract digestibility in calves fed MR with vegetable oil mixtures, including a combination of palm, coconut, and rapeseed oils compared with lard did not differ across treatments.Digestibility values in the current study were substantially higher than those described by Huuskonen et al. (2005).This may be related to differences in fat inclusion level, in processing of dietary fats, or in the experimental method.Indeed, the total collection periods of 24 h in the current experiment might have been too short to accurately determine the apparent total-tract digestibility.
Gastrointestinal permeability was measured by orally pulse dosing solutions of lactulose-D-mannitol and Cr-EDTA instead of the morning milk.These tests rely on the principle that D-mannitol passes the intestinal epithelia paracellularly and transcellularly, whereas lactulose and Cr-EDTA pass the epithelia exclusively paracellularly through the tight junctions (Araujo et al., 2015;Wilms et al., 2023a, in press).Changes in gut permeability are expected to be reflected by an increase in urinary excretion of lactulose and Cr-EDTA and an increase of the intestinal permeability index (Pardal et al., 1995;Klein et al., 2008).In the current study, the lack of differences in the recovery of lactulose and Cr-EDTA across treatments does not suggest an effect of the experimental dietary fats in MR on gut integrity.This is consistent with a study from Liu et al. (2014) in which feeding corn oil, canola oil, poultry fat, or tallow in solid feed did not influence gut permeability in young pigs.In contrast, Benoit et al. ( 2015) reported that feeding 6-wk-old rats with dairy cream from pasture-fed cows improved gut barrier function compared with cream from cows fed a total mixed ration.The increase in gut integrity, as indicated by an increase in Paneth and goblet cells in the small intestine and colon, was attributed to the higher proportions of α-linolenic acid (ALA) and conjugated linoleic acid (CLA) in the dairy cream from pasture-fed cows.The VG treatment contained 4.6-fold greater ALA than AN and MX.The AN treatment contained some CLA (0.23% total FA) due to the presence of dairy cream, whereas VG and MX did not.However, differences in the experimental method in assessing gut barrier integrity could explain why no differences in gut barrier function were detected in the current experiment.Alternatively, it is possible that formulating based on FA profile and TG structure to closely resemble that of milk fat might have limited the possibility to detect differences in gut permeability, considering that all diets were well digested by calves.
When MR oil composition largely differs from that of milk fat, diarrhea has been observed in calves (Jenkins et al., 1985;Jenkins, 1988).High level of unsaturated FA from vegetable oils causes diarrhea in calves, although the mechanisms are not well-known, but are attributed to the FA unsaturation as hydrogenated fats are well digested (Raven, 1970;Jenkins, 1988).Although the fat composition in MR did not result in a different number of medical treatments for diarrhea and respiratory diseases, the percentage of days with abnormal fecal scores were lower in calves fed VG than MX in wk 2 after arrival and this was associated with a higher fecal DM in VG calves than in other treatment groups in wk 3 and 5. Despite the lack of differences in total apparent fat digestibility, these results may indicate enhanced digestibility of the VG diet in relation to the FA composition.Indeed, short, and medium chain FA are highly digestible (Toullec and Matthieu, 1969;Benito-Gallo et al., 2015), and the VG diet contained a larger proportion of C12:0 as compared with other treatment groups.
The PUFA to SFA ratio was higher in the VG treatment (0.25) compared with AN and MX (0.17), due to the high unsaturation level of rapeseed oil, which can increase oxidative stress because PUFA are more susceptible to peroxidation than SFA given the higher number of double bonds (Lenox and Bauer, 2013).In a study where calves were fed the same MR diets as in the present experiment, calves fed VG had substantially higher PUFA concentrations in the plasma than AN and MX (Wilms et al., 2023b, in review).This was associated with a marked increase of total plasma cholesterol with a higher proportion of low-density lipoprotein (LDL) cholesterol in calves fed VG.This aligns with previous findings showing that feeding MR (14% fat) with a high proportion of PUFA from the vegetable origin (soybean oil, corn oil) resulted in accumulation of cholesterol in the blood and body tissues compared with calves fed MR with tallow only (Wiggers et al., 1977;Barrows et al., 1980;Richard, 1980).In humans, oxidation of LDL has been identified as a contributing factor to the development of atherosclerosis (Steinberg et al., 1989;Parthasarathy et al., 1992).In the current study, the antioxidant activity as reflected by the serum FRAP results was lower in calves fed VG compared with calves fed AN and MX.Consistently, calves fed MR enriched in n-3 PUFA had lower FRAP values than calves fed the control MR (Welboren et al., 2023).Nonetheless, there were no differences observed in either the dROM values or the OSi between dietary treatments.This was also indicated by the stable activity of the antioxidant selenoenzyme glutathione peroxidase (GSH-Px) in serum across the 3 treatments.
Serum TBARS are produced through the reaction of malondialdehyde, a lipid peroxidation product with TBA.However, it should be noted that various chemically reactive carbonyl-containing organic molecules, including those derived from oxidized biomolecules other than lipids, can react with TBA and are thus counted as TBARS (De Leon and Borges, 2020).Increased levels of TBARS were reported in various pathological conditions, such as cardiovascular disease, atherosclerosis, and diabetes (Ito et al., 2019).In humans, dietary n-3 PUFA supplementation has cholesterol lowering effects (Goodnight et al., 1982) and reduced blood TBARS values in patients with chronic renal failure (Bouzidi et al., 2010), although there are conflicting findings in the literature.This is consistent with the present study in which serum TBARS were lower in VG calves throughout the experimental period.This suggests a lower lipid peroxidation, indicating lower oxidative damage to lipids in VG calves.These results contradict the serum FRAP outcomes showing a reduced antioxidant activity in VG calves.A possible explanation could be that in response to higher PUFA oxidation, calves upregulated their enzymatic antioxidant response which in turn lowered TBARS in blood.Alternatively, this could also be related to the higher levels of antioxidant in the Racomelt fat blend used in the VG treatment compared with other oils used in this experiment.
Regulation of satiety and energy intake is highly influenced by the interaction of nutrients within the stomach (Barber and Burks, 1983) and small intestine (Lieverse et al., 1994;Randich et al., 2000), as it plays a Treatments (n = 15 per treatment group) included three milk replacers: VG was a MR with vegetable oils (20% coconut and 80% rapeseed oils), in AN, the MR contained animal fats (65% lard, 35% dairy cream), and in MX, a mixture of 80% lard and 20% coconut oil was used.
2 Expressed in % oral dose.Markers were orally pulse dosed instead of the morning meal in wk 3. Lactulose (0.2 g/kg BW; Sigma-Aldrich®, Zwijndrecht, the Netherlands) and D-mannitol (0.12 g/kg BW; Sigma-Aldrich®, Zwijndrecht, the Netherlands) were dissolved separately in 100 mL of warm water.The amount of liquid Cr-EDTA (concentration; Masterlab, Boxmeer, the Netherlands) was adjusted for each calf to provide an individual dose of 0.1 g/kg BW.
crucial role in inhibiting gastric emptying and activating neural and humoral signals.When feeding ad libitum levels of the same MR as in the present experiment, Wilms et al. (2023b, in review) reported higher MR intake in calves fed AN than MX and VG.In the current study, abomasal emptying was slower in calves fed VG than calves fed AN and MX.Some FA, including C12:0 and C18:0, have been demonstrated to regulate appetite by influencing gastric emptying and hormone secretion, such as cholecystokinin (CCK; Meyer et al., 1998;Feltrin et al., 2004).Although the proportion of C18:0 was similar across treatment groups, the VG fat contained relatively more C12:0 (16.8% total FA) than AN (4.2%) and MX (9.0%).Overall, the VG MR (21.9% total FA) contained more medium-chain FA (MCFA) as AN (6.5%) and MX (11.7%) due to the higher inclusion of coconut oil (Odle, 1997).After ingestion, dietary MCFA are mainly transported to the liver as free FA, whereas the enterocytes secrete LCFA as chylomicrons (Schmitz and Ecker, 2008).The MCFA are thus a rapidly available energy source that can influence satiety signaling in calves.In rats, in-testinally perfused lauric acid (C12:0) inhibited energy intake, whereas isocaloric amounts of decanoic (C10:0) or octanoic (C8:0) acid did not (Meyer et al., 1998).
Other experiments reported a slower gastric emptying with FA having 12 or more carbon atoms than FA with shorter chains (Hunt and Knox, 1968).However, AN and MX had proportionally more FA with a chain length longer than 12 carbon atoms.This suggests that the effect of C12:0 may have been more important than that of longer chain FA in determining the abomasal emptying rate.One limitation of research on dietary fat composition is the inability to determine the isolated effects of individual FA, primarily due to variations in FA profiles among different fat sources.Furthermore, there is limited knowledge regarding the synergistic effects that certain FA may have.Postprandial serum TG concentrations were higher in calves fed AN than VG, whereas MX did not differ from the other treatment groups.Consistently, the time to reach the maximal serum TG concentration was shorter in calves fed AN than in VG and MX.The slower abomasal emptying may have resulted in  , 20, 30, 45, 60, 90, 120, 150, 180, 240, 330, and 420  a slower release of fat to the intestine, reducing the appearance of TG in serum.Nevertheless, this contradicts Wilms et al. (2023a, in press), where postprandial TG were higher in calves fed a WM powder despite a slower abomasal emptying rate.Animal fats contain a higher proportion of SFA as compared with vegetable fats.In the present study, AN and MX contained about 54% SFA (% total FA), whereas VG contained 52% SFA.Previous research in humans showed that higher inclusion of n-3 PUFA in the diet reduces postprandial TG concentrations, independently of the type of fat ingested (Harris and Connor, 1980;Harris et al., 1988).Bermudez et al. (2014) reported that butter fat leads to a greater postprandial TG than oils with a higher degree of unsaturation.More generally, lower postprandial TG concentrations following fat tolerance tests are observed with meals rich in PUFA (as reviewed by Monfort-Pires et al., 2016).This is consistent with the higher proportion of n-3 PUFA and total PUFA in the VG MR diet.Following a meal, dietary TG are hydrolyzed by the lipase into FA and monoacylglycerol, which are subsequently absorbed by enterocytes (Lambert and Parks, 2012).Within the enterocyte, FA undergo various processes.These include partitioning into cholesteryl ester or phospholipid, or oxidation, reesterification to form TG for incorporation into chylomicrons or storage in an intracellular TG storage pool (Lambert and Parks, 2012).Polyunsaturated FA such as linoleic acid (LA) and ALA are more incorporated into cholesteryl esters and phospholipids as compared with C16:0 and C18:1 (Burdge et al., 2002;Hodson et al., 2009), which may explain lower postprandial TG after ingestion of fats with a higher PUFA proportion.However, in Wilms et al. (2023a, in press), the proportion of n-3 PUFA was similar between the WM (0.41%) and MR treatments (0.16%, Mellors et al., 2023), but the postprandial TG was higher in calves fed the WM powder.Is it likely that other factors related to the FA profile and the TG structure are the main determinants of postprandial TG.Indeed, the FA chain length may influence the magnitude of postprandial TG concentration (Neumann and Egert, 2021).For instance, coconut oil elicits a lower postprandial lipemic response when compared with cocoa butter, butter, and lard (Karupaiah et al., 2011;Panth et al., 2018).Although all these fats have a high saturation level, these differences may be explained by the FA chain length.However, it is worth noting that conflicting findings exist, and variations in FA profile of dietary fats should be considered when interpreting the results.

CONCLUSIONS
The hypothesis that feeding a MR containing lard and dairy cream would improve digestibility and gastrointestinal health compared with a MR including only vegetable or a mix of vegetable and animal fats was not confirmed by the present findings.In contrast, specific redox parameters were altered in calves fed the VG treatment, but no conclusions could be drawn from the current outcomes as there were contradictory results.In addition, abomasal emptying was slower in calves fed VG than in other treatments, and postprandial TG concentration was lower in calves fed VG than AN, likely due to differences in the FA profile and TG structure of the dietary treatments.Further research is necessary to fully understand the metabolic implications of fat composition in MR for calves.
Wilms et al.: FAT COMPOSITION OF MILK REPLACER Wilms et al.: FAT COMPOSITION OF MILK REPLACER

TABLE 1 .
Wilms et al.:FAT COMPOSITION OF MILK REPLACER Nutrient composition of milk replacers differing in fat composition and fed twice daily (4.0 L per meal) to calves housed in individual pens(n = 45) 1Expressed in % DM unless specified otherwise.2Treatments(n = 15 per treatment group) included three milk replacers: VG was a MR with vegetable oils (20% coconut and 80% rapeseed oils), in AN, the MR contained animal fats (65% lard, 35% dairy cream), and in MX, a mixture of 80% lard and 20% coconut oil was used.

TABLE 2 .
Urh et al. (2019)COMPOSITION OF MILK REPLACER Fatty acid profiles of milk replacers differing in fat sources fed ad libitum to group-housed calves (n = 59) .e., dROM/FRAP, as described byUrh et al. (2019).Postprandial serum samples were analyzed at Masterlab (Boxmeer, the Netherlands) for acetaminophen using the Paracetamol Assay Kit-K8002 (Cambridge Life Sciences Ltd., Ely, UK;MacPherson et al., 2016).The same serum samples were also processed by SYNLAB. i Wilms et al.: FAT COMPOSITION OF MILK REPLACER Wilms et al.: FAT COMPOSITION OF MILK REPLACER

TABLE 3 .
Wilms et al.:FAT COMPOSITION OF MILK REPLACER Parameters describing calves before and after treatment initiation.Calves were fed milk replacers differing in fat composition 4.0 L twice daily (n = 45).Blood samples were collected on d 7, 14, 21, 28, and 35 IgG, immunoglobulins; MR, milk replacer; FRAP ferric reducing ability of plasma; OSi, oxidative stress index; TBARS, thiobarbituric acid reactive substances; GSH-Px, antioxidant selenoenzyme glutathione peroxidase. 1 Treatments (n = 15 per treatment group) included three milk replacers: VG was a MR with vegetable oils (20% coconut and 80% rapeseed oils), in AN, the MR contained animal fats (65% lard, 35% dairy cream), and in MX, a mixture of 80% lard and 20% coconut oil was used. 2 Total number of calves treated for diarrhea and respiratory diseases (calves treated / total calves). 3Calves were weighed on the day of arrival and once weekly thereafter until 35 d of age.Body weight measured at arrival entered the statistical model as baseline covariate for BW and ADG.
a,bMeans with a different superscript are significantly different (P ≤ 0.05).*SEM expressed as log.

TABLE 4 .
Wilms et al.:FAT COMPOSITION OF MILK REPLACER Total apparent tract digestibility measured in wk 3 and 5 arrival by total collection of feces over 24 h in calves fed milk replacers differing in fat composition twice daily (n = 45)

TABLE 5 .
Wilms et al.: FAT COMPOSITION OF MILK REPLACER Postprandial dynamics measured at 4 wk of age in calves fed milk replacers differing in fat composition twice daily (n = 45).Blood was collected at −30 min and at 10 min relative to the morning milk meal of 4.0 L SP,3 , first-order, fast gastric emptying rate constant; k Ac,UAc , first-order, Ac utilization rate constant; rMSPE In , root mean squared prediction error for insulin.
1Treatments (n = 15 per treatment group) included three milk replacers: VG was a MR with vegetable oils (20% coconut and 80% rapeseed oils), in AN, the MR contained animal fats (65% lard, 35% dairy cream), and in MX, a mixture of 80% lard and 20% coconut oil was used.a,bMeans with a different superscript are significantly different (P ≤ 0.05).*SEM expressed as log.