Responses to incremental nutrient supply on energy and protein metabolism in pre-weaning dairy calves

Recently reviewed development objectives and feeding practices in young dairy calves require an adaptation of nutrient recommendations set for milk replacer (MR) composition. Nutrient requirements of calves younger than 21 d of age, and those of calves fed with high levels of milk replacer are insufficiently quantified. The efficiency at which macronutrients are utilized, particularly protein, substantially diminishes with age, and there is little data for the first weeks of life. In addition, in older (pre-)ruminants, protein and energy can be simultaneously limiting for protein gain. Whether this also applies to calves in the first weeks of life is unknown. Therefore, this study aimed to quantify the responses in protein and fat gain to incremental supply of protein, fat, or lactose to MR in very young calves. Thirty-two groups of 3 mixed-sex Holstein-Frisian newborn calves (3.4 ± 1.6 d of age), were randomly assigned to one of 4 dietary treatments applied for 19 d: a basal MR (23.3% CP, 21.2% CF and 48.8% lactose of DM), provided at 550 kJ/kg BW 0.85 per day (CON; n = 24), or the basal MR incrementally supplied with 126 kJ of DE/BW 0.85 per day as milk fat (+FAT; n = 23), lactose (+LAC; n = 24) or milk protein (+PRO; n = 23). Calves were fed MR in 2 daily meals and had ad libitum access to water, but did not have access to calf starter nor any other solid feed. After 2 weeks of adaptation to their respective diets, groups of calves were placed for one week in an open-circuit respiration chamber for nitrogen and energy balance measurements (5 d). The incremental nutrient efficiencies indicate what percentage of extra intake of nutrients is retained. In this study, we observed that with every 100 g increase in protein intake, 52% was converted into protein deposition, while 44% contributed to heat production. Similarly, a 100 g increase in fat intake resulted in 67% being stored as fat, 22% being released as heat, and only 5% being retained as protein. Likewise, a 100 g increment in lactose intake led to 49% being stored as fat, with 38% being released as heat. Additional protein intake was not deposited as fat, extra energy intake (fat, and additional lactose) increased post absorptive N efficiency in young calves.


INTRODUCTION
Traditionally, the amount of whole milk (WM) or milk replacer (MR) fed to dairy calves has been severely restricted compared with their natural voluntary intake.Driven by the higher costs of liquid feeds, this strategy aimed to stimulate starter intake and rumen development and allow for early weaning (Warner, 1991).However, research over the past decade has demonstrated that increasing the supply of milk closer to ad libitum levels improves growth, health, and even long-term milk-production (Soberon et al., 2013, Davis Rincker et al., 2011).While the benefits of increased nutrient supply to dairy calves are now well recognized, the importance of macronutrient composition of MR is insufficiently understood for these feeding levels.
Although current feeding levels, in terms of volume, are very close to natural ad libitum intakes (Jasper and Weary, 2002;Webb et al., 2014) the feeding frequency and meal sizes remain different from the biological standard.As current guidelines were established to suit low planes of nutrition (Diaz et al., 2001;Quigley, 2019), there is a lack of data for nutrient requirements and reference recommendations for calves younger than 21 d of age, fed with high levels (>8 L per day) of MR.The effects of nutrient intake on growth rate and growth composition have been described for young (Diaz et al., 2001;Blome et al., 2003) and older pre-ruminant calves (Gerrits et al., 1996;Labussière et al., 2008).This research has shown that the gross protein efficiency in calves weighing between 65 to 105 kg is around 60% but decreases to below 50% when calves exceed 110 kg Responses to incremental nutrient supply on energy and protein metabolism in pre-weaning dairy calves of BW.Besides, the incremental efficiency of protein has been demonstrated to diminish significantly with age, with values ranging between 20% to 40% for body weights above 150 kg (Gerrits et al., 1996;Labussière et al., 2008;Van den Borne et al., 2006a;Gerrits, 2019).Furthermore, unpublished data from our group has shown that the incremental energy efficiency of protein, lactose, or fat supply in MR for heavy veal calves (125 to 250 kg of BW) was found to be 39%, 74%, and 73%, respectively, regardless of the intake level of solid feed.To our knowledge, there is no such data available for calves in their first few weeks of life.
Both in calves fed with WM or MR, lactose and fat are the most important energy sources.It has been demonstrated that young calves have a high capacity to digest and metabolize lactose, as carbohydrate oxidation remained high (above 90%) when increasing lactose intake (57 kg of lactose; Huber et al.,1964).In the case of fat, Van den Borne et al. (2006a) showed that when feed supply increases, the proportion of dietary fatty acids oxidized drops from 80% to about 30%.Extensive research has also been done investigating the relationship between production (either growth or lactation) with energy and protein supply in various animal species.In ruminants, it has been suggested that energy and protein are often co-limiting factors (Gerrits et al., 1996;Zanton and Heinrichs, 2008).For instance, in Holstein steers, increasing energy intake through abomasal glucose infusions and intraruminal acetate infusions has been found to enhance nitrogen retention and improve the efficiency of amino acid utilization (Schroeder et al., 2006).Similarly, in a metaanalysis (Daniel et al., 2016), an increase in dietary net energy for lactation was shown to increase milk yield and milk protein efficiency in dairy cows, and the increase was independent of level of metabolizable protein fed.In calves, insulin sensitivity is high at birth, and decreases independent of feeding strategy in early age to low levels when compared with other species (Pantoplhet et al., 2016).Consequently, the role of energy is in these young calves, and whether the simultaneously limiting protein and energy also applies to them remain unknown.
Most observations on energy and protein efficiency are performed on individually-housed animals, and it is known that this individual housing affects their energy metabolism, particularly when it can modify behavior and induce stress.These behaviors, such as repetitive oral manipulation of the pen or tongue playing, can impose considerable energetic costs on the animals (Van den Borne et al., 2004Borne et al., , 2006a)).To date, no studies have been conducted on group-housed calves.
Therefore, the objectives of this study were: to quantify protein efficiency, both gross and incremental; to study the effect of energy supplied as lactose or fat on protein efficiency, and to quantify the fate of energy from different sources in group-housed calves up to 21d of age.

MATERIALS AND METHODS
This study was conducted at Carus, Research Facility of the Department of Animal Sciences, Wageningen University and Research (Wageningen, the Netherlands) from April 2018 to June 2018.All procedures described in this article complied with the Dutch Law on Experimental Animals, which meets European guidelines defined by 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) and the Animal Care and Use Committee of Wageningen University.The project application code is AVD2040020173425.

Animals and Housing
Ninety-four Holstein-Frisian calves (37 females, 57 males) were sourced at birth from 2 commercial dairy farms and were selected based on clinical health, age, BW, and sex uniformity.Mean age and BW upon arrival were 3.5 ± 1.4 d and 44.5 ± 0.3 kg, respectively.At the farm of origin, a standardized protocol for colostrum management was followed where calves were fed 4 L within the first hours after birth, followed by one feeding of 2 L for a total of 2 feedings of colostrum in the first 12 h of life.From 24 h onward, calves were fed 5.25 L/d of MR (Sloten B.V., Trouw Nutrition; 48.8% lactose, 21.2% crude fat, and 23.3% CP of DM; 15% solids) in 2 or 3 meals.The MR was 40 to 42°C at feeding and was provided in a clean teat bucket.Calves arrived in 9 batches to the research facilities, and measurement periods were staggered.During the adaptation period, for the first 14 d after arrival, calves were housed in groups of 3 in pens of 3 × 3 m, equipped with rubber-slatted floors and open fences on all sides, with fresh straw as bedding material.Temperature was maintained between 15 and 21°C, relative humidity between 55 and 75%, and air velocity at < 0.8 m/s.Calves were exposed to natural light (400-500 lx) from 0530 to 1930h and to darkness (5 lx) during the remainder of the day.After the adaptation period, the first measurement period, which involved measuring a complete energy balance, including heat production (HP), lasted for 5 d.This was followed by the second measurement period, which involved measuring fasting heat production (FHP), lasting for 2 d.Before the FHP period, one calf from each group was randomly selected and euthanized for the collection of gastrointestinal tract tissues (the results of which have been reported elsewhere).Each group (n = 3 for HP and n = 2 for FHP) was housed in pens measuring 3.0 × 3.0 m, with tenderfoot® (Minneapolis, MN, USA) flooring and open fences only at the front side, without bedding material, in one of 4 identical 80 m3 indirect calorimetric chambers (described by Heetkamp et al., 2015).Temperature was maintained at 18°C, relative humidity at 65%, and air velocity at < 0.2 m/s.Calves were exposed to artificial light (420 lx) from 0530 to 1930h and to darkness (3.5 lx) during the remainder of the day.

Diets and feeding
Calves were fed according to their metabolic body weight (BW 0.85 ), weekly adjusted as they grew at individual animal level.Groups were assigned to one of 4 dietary treatments: a control diet (basal, CON; Trouw Nutrition, Deventer, the Netherlands), or the control diet supplemented with butterfat (+FAT; anhydrous milk fat; Royal VIV Buisman BV, Zelhem, the Netherlands), lactose (+LAC; anhydrous lactose powder; Arla Foods Ingredients Group, Viby, Denmark) or protein (+PRO; Milk Protein Concentrate (MPC) powder 80; Fonterra Ltd., Auckland, New Zealand), and were exposed to the dietary treatments for 14 d before the measurement period started.Supplemented treatments were isoenergetic, meaning that they were equal in estimated digestible energy intake (675 MJ/kg BW 0.85 /d), provided at equal amount 2 times daily.The ingredient and analyzed nutrient composition of the experimental MR, as well as the designed nutrient intakes per treatment are shown in Table 1.Milk replacer was reconstituted with water (150 g/L) and supplied in a teat bucket at a temperature of about 40°C.Feeding times were 0600, and 1800 h.Calves had ad libitum access to water but did not have access to calf starter or any other solid feed.

Measurements
Each calf was weighed at d 0, 7, 14, 19 and 21.The calves were in the respiration chambers for 7 d, for which the first 5 d HP (kJ/kg 0.85 /d) and the last 2 d FHP (kJ/kg 0.85 /d) were measured.From d 14 and 19, gas exchange was measured in 10-min intervals by measuring the exchange of O 2 , CO 2 , and CH 4 , as described by Heetkamp et al. (2015).To check the proper functioning of the chambers, a carbon dioxide recovery test was performed before the start of the experiment, according to procedures described by Heetkamp et al. (2015).In the 4 chambers, 99.8%, 100.4%, 100.5%, and 99.9% of the carbon dioxide released was recovered.A radar device was mounted in the chamber above each pen completely covering the housing area to measure physical activity continuously during the respiration, and fasting period.During the first 5 d (respiration period), manure was quantitatively collected (mix of urine, feces and cleaning water) and stored at −20°C.After cleaning the respiration chambers, 2 of the 3 calves returned to the respiration chambers for 2 d (d 19 and 20) for measuring the FHP.Aerial NH 3 was collected from a quantified sample of the outgoing air in H 2 SO 4 , and NH 4 + in water that condensed on the heat exchanger were collected quantitatively.MR was sampled during each measurement period, and milk refusals were recorded at an individual animal level.Treatments were randomly coded and treatment identity was blinded in the research farm.
At d 12 (2 d before the start of the respiration chamber measurements) a marker cobalt-ethylenediamine tetraacetic acid (Co-EDTA; all batches) was mixed with at 0.5 g/kg MR for 48h.On d 13 and d 14, multiple rectal fecal samples were taken after manual stimulation of the rectum.One pooled sample were made for each experimental unit for the 2 sampling days and were transferred to a freezer (−20°C) awaiting further processing.

Chemical Analysis
Feed refusals, and feces were freeze-dried for determination of DM content.Milk replacer, feed refusals, and feces were ground to pass 1-mm sieve.DM content was determined by drying to a constant weight according to ISO standard 9831 (ISO, 1998).Crude fat content was determined after acid hydrolysis in feed and in freezedried manure according to ISO Standard 6492 (ISO, 1999).Crude ash content was determined in feed and in freeze-dried feces.Samples were carefully incinerated in a muffle furnace by slowly increasing the temperature from 20 to 550°C to prevent foaming, and subsequent incineration took place according to ISO Standard 5984 (ISO, 2002).Lactose content was analyzed enzymatically in feed and in freeze-dried feces (Enzytec; Diffchamb Biocontrol, Nieuwerkerk aan den IJssel, The Netherlands).Nitrogen content was measured in fresh feed, feed refusals, manure, and aerial NH 3 and water that condensed on the heat exchanger according to ISO Standard 5983 (ISO, 1997).Gross energy content was analyzed in feed, freeze-dried manure, using adiabatic bomb calorimetry (model C7000 calorimeter; IKA Werke GmbH & Co. KG, Staufen, Germany) according to ISO Standard 9831 (ISO, 1998).Cobalt was analyzed in feed and freeze-dried fecal samples after ashing and acid hydrolysis as described by Williams et al. (1962), using a SpectrAA 300 atomic absorption spectropho-

Calculations
Gross energy (GE) intake was calculated as feed intake multiplied by the GE content of the MR.Intake of metabolizable energy (ME) was determined by subtracting energy excretion in manure and CH 4 from the gross energy intake.For each balance period, nitrogen retention (NR) of each group of 3 calves was calculated as the difference between ingested nitrogen and nitrogen lost in manure, condensed water and extracted air.Total HP was calculated from gas exchanges according to the formula of Brouwer (1965).Energy retention (ER) was calculated as the difference between ME intake and HP.Protein retained (N retention × 6.25) and energy retained as protein (protein retained × 23.7 kJ/g) were calculated from N retention.Energy retained as fat was calculated from the difference between total energy retained and energy retained as protein in the body.
Apparent total-tract digestibility (ATTD) of nutrients were calculated from the following equation: ATTD = 100 -100 × ((Co feed /Co digesta ) × (Nutrient digesta /Nutrient feed )) where Nutrient digesta is the nutri-ent concentration in the feces (g/kg DM), Co digesta is the cobalt concentration in feces (g/kg DM), Nutrient feed is the nutrient concentration in the MR (g/kg DM), and Co feed is the cobalt concentration in the MR (g/ kg DM).

Statistical Analysis
All statistical analyses were conducted using PROC MIXED in SAS 9.4 (SAS Studio, SAS Institute, Inc., Cary, NC).The experimental unit for all the measurements and both nitrogen and energy balance consisted of a group of calves housed in one respiration chamber, and both dependent variables and covariables were expressed as averages per group.Incremental response differences were calculated by considering the CON treatment as a reference within each batch.To verify model assumptions, we assessed the normal distribution of residuals using the Shapiro-Wilk statistic, and the normality assumption was confirmed.The model incorporated fixed effects of diet, batch, and their interactions.For comparisons across treatments at each significant point, the PDIFF option of the LSMEANS statement in SAS was employed.The chosen covariance structure was compound symmetry based on the lowest AIC.All values are presented as least squares means ± SEM.Significance was declared when P ≤ 0.05, and trends were declared when P < 0.10.

RESULTS
The data set included 34 observations, i.e., 8 replicates per treatment.Two replicates were based on 2, instead of 3 calves.The apparent total-tract digestibility (ATTD) of the experimental diets is shown in Table 2.The ATTD of DM ranged between 88 and 91% and was not different between the groups.The ATTD of crude fat was lower for +LAC calves (−5% for +PRO and −7% for +FAT and CON; P = 0.018).The highest ATTD of crude protein (CP) digestibility was observed in calves fed the +PRO diet, and the lowest was reported for calves fed the +LAC diet (P < 0.01).
Treatment effects on performance are presented in Table 3. Calves in the +PRO treatment had a greater BW (P = 0.01), being 2.8 kg and 2.2 kg heavier than +FAT and +LAC calves, respectively.Consequently, +PRO calves had greater average daily gain (ADG; P < 0.01) than +FAT and +LAC calves (567 g, 420 g, and 449 g, respectively).
The effects of dietary treatments on energy and N balances are reported by the difference with the CON treatment, and shown in Table 3.As result of design, digestible N intake increased with the dietary CP intake from 895 to 1,538 mg of N/kg of BW 0.85 (P < 0.01).Fecal N excretion was affected by the diet being greater in the +LAC calves when compared with +FAT calves (P = 0.03), but no differences were found between +LAC and +PRO treatments.N retained in the body increased from 545 (CON) to 818 (+PRO) mg of N/kg of BW 0.85 per day and differed from +FAT and +LAC treatments (P < 0.01).Calves in the +PRO group retained 131 kJ/ kg of BW 0.85 per day as protein at GE intake of 659 kJ/ kg of BW 0.85 per day, whereas energy retained as fat was the highest for +FAT (148 kJ/kg of BW 0.85 per day) and the lowest for the +PRO group (61 kJ/kg of BW 0.85 per day; P < 0.01).Energy output with feces and urine did not differ between treatments and averaged 44 ± 3.40 kJ/kg of BW 0.85 per day.Methane production was very low and unaffected by the dietary treatments.Total heat production was greater in +PRO than +FAT calves (Table 3; P ≤ 0.01), however, there were no differences observed between +LAC and +PRO or +LAC and +FAT.Activity related heat production (AQ) was not affected by dietary treatment (P = 0.10); however, calves in the +LAC treatment showed a marked numerical increase.The respiratory quotient was higher in the +LAC treatment when compared with +FAT and +PRO treatments.Additionally, the +FAT and +PRO treatments differed slightly but significantly from each other (Table 3; P < 0.01).
Differences in gross and incremental efficiencies are presented in Table 4. Treatment variations in N efficiency, expressed per gram of digested N intake, were 77% for CON calves, which increased for +LAC (89%) and +FAT (83%) calves, and decreased for +PRO calves (68%; P < 0.05).The gross efficiency of retaining GE, represented as a percentage of GE intake, was 24% for CON calves and significantly increased (P < 0.05) with all macronutrient additions.The highest energy efficiency was observed in the +FAT treatment (34%), followed by +LAC (30%), and +PRO (29%).The incremental nutrient efficiencies indicate the percentage of additional nutrient intake that is retained.With every 100 g increase in protein intake, 52% was retained as protein deposition, 44% contributed to heat production, and 2% to fat deposition.Similarly, for every 100 g increase in fat intake, 67% was stored as fat, 22% dissipated as heat, and 5% deposited as protein.
Lastly, for every 100 g increase in lactose intake, 49% was retained as fat, 38% lost as heat, and 0% converted to protein.

DISCUSSION
The present study investigated the incremental efficiencies of energy and protein deposition in young calves (21 d old) fed with a MR topped with either an isoenergetic amount of incremental protein, fat, or lactose.In this study, the apparent total-tract digestibility of DM, crude fat, and crude protein, ranged from 88 to 91, 86 to 93, and 70 to 85%, respectively.In a metaanalysis examining the effects of age on the intestinal digestibility of liquids feeds in young calves conducted by Quigley et al. (2021), apparent total-tract digestibility in calves before one month of age ranged from DM: 88-96%, crude fat: 89-99%, to CP: 85-99%.Our results fall within these ranges, although at the lower end.Nonetheless, our results seem to be consistent with earlier research by Terosky et al. (1997), in which the digestibility of CP in preweaned dairy calves of 2 weeks of age was between 61% to 73%.In that study, the effects of age were documented, and the low CP digestibility at this age was attributed to the presence of diarrhea (Terosky et al., 1997).In our study, the calves in the +LAC group consistently had lower apparent total-tract digestibility of DM, crude fat and CP compared with the other groups.This might be due to the higher concentration of lactose (60.2% in DM) in the +LAC group, which has been suggested to exceed the absorptive capacity of the calves and adversely affecting nutrient digestibility (Lodge and Lister, 1973;Hof, 1980).Additionally, compared with the other treatments (approximately 17%), the +LAC group displayed the lowest fecal DM content at 14%.It is worth noting that diarrhea is typically associated with a fecal DM content below 10% and clinical diarrhea was not observed in this group of calves.The elevated lactose content in the MR could potentially trigger an osmotic effect in the intestines, even without manifesting as loose feces (Hof, 1980).
Throughout the experimental period, calves fed +PRO grew more than calves on the other diets, with a higher BW and ADG (55.2 kg and 567 g/d, respectively).Our results are consistent with previous findings on effects of dietary CP content in calves (Blome et al., 2003), in which calves fed high protein MR (CP = 26%) had an ADG between 560 and 620 g/d.Studies have shown that increasing CP content affects growth and body composition in milk-fed pre-ruminants which is in line with the higher BW and ADG observed in +PRO calves in this study (Gerrits et al., 1996;Blome et al., 2003).Different superscripts between columns denote a significant difference among +FAT, +LAC, and +PRO treatments.
Incremental efficiencies for energy and N retention were calculated based on nutrient digestibility and retention values.This study provides quantitative estimates of the efficiency at which increments in nutrient supply are utilized by very young calves.The incremental efficiency of protein utilization, calculated as the percentage of additional nitrogen deposition in relation to digested nitrogen, was found to be 52% for +PRO calves at 21 d of age.This value is higher than the values reported by Gerrits et al. (1996) for pre-ruminant calves weighing between 80 and 160 kg BW (40%) and by Donelly and Hutton (1976; 45%) for milk-fed calves weighing 40 to 70 kg.However, our results were not higher than the values observed for calves weighing between 50 and 83 kg BW in studies by Labussière et al. (2008;64%) and Blome et al. (2003;66%).Nevertheless, as the calves aged, meaning its live weight increases, the impact of CP concentration on protein deposition diminishes, resulting in a lower marginal efficiency of nitrogen utilization (Labussière et al., 2008) and increased urea excretion (Labussière et al., 2009).Previous studies have shown that as the BW of heavy pre-ruminant calves increases, there is a decline in the efficiency of nitrogen retention.According to Gerrits (2019), these efficiencies decrease from 50 to 65% when the BW is below 70 kg, to values ranging between 20 and 40% when the BW exceeds 150 kg.Similarly, in the NASEM (2021) report, protein efficiency was characterized by an empirical equation, resulting in a decrease from 70% at birth to 55% at 200 kg BW.Consequently, heavy pre-ruminant calves exhibit lower efficiency in utilizing protein for growth compared with very young calves and other species.Van den Borne (2006b) concluded that multiple factors might contribute to the observed low nitrogen efficiency observed in pre-ruminant calves.These factors may include a decrease in the supply of (essential) amino acids post-absorption, possibly due to milk fermentation or preferential use by intestinal tissues, insulin resistance, and a potential mismatch between the total nutrient supply and the total nutrient requirement for growth and maintenance.This mismatch could arise from factors like feeding frequency (twice daily) and differences in nutrient passage rates, ultimately resulting in varying post-absorptive availability of individual nutrients.However, the exact mechanism affecting protein efficiency in young calves remains unknown.
The gross efficiency of energy use, calculated as energy retention divided by gross energy intake, was found to be higher in calves fed with an additional supply of fat (34%), while no significant difference was observed between the +LAC and +PRO calves (30% and 29% respectively).The gross energy retention efficiency of the +FAT treatment was notably greater compared with that observed in calves with a body weight of 56 kg, which were fed an isocaloric whey protein-based MR with increasing CP concentration (14-26%), resulting in values ranging from 27% to 29% (Barlett et al., 2006).These values are more similar to the efficiency obtained in the CON group (26%), and they align with the findings of other studies (Diaz et al., 2001).The incremental efficiency of energy retention, calculated as the additional energy retained per kilojoule of extra ME intake from the MR, was determined to be 76% in +FAT calves, 58% in +LAC calves, and 55% in +PRO calves.According to data collected from various studies (van Es et al., 1969;Neergaard et al., 1976;Vermorel et al., 1979;Aurousseau et al., 1984;Arieli et al., 1995;Blome et al., 2003;Barlett et al., 2006), the average incremental efficiency of ME utilization for growth is estimated to be 69% for milk-fed calves weighing between 45 to 60 kg.Similarly, van den Borne et al. (2006a) and Gerrits et al. (1996) reported incremental efficiencies of 72% and 71%, respectively, for calves fed MR twice daily at 140 kg BW and for calves with a BW ranging from 160 to 240 kg.The efficiency of utilizing ME for energy retention shows a considerable range, from 0.40 to more than 0.70 (NASEM, 2021).This variability could be influenced by factors such as the age of the calf, whether it is undergoing rapid protein accretion with limited fat deposition, or actively gaining fat in addition to protein.Moreover, the dietary fat content relative to total ME and protein also plays a significant role.It is important to note that while protein deposition demands higher energy expenditure, the process of fat deposition is energetically more efficient (NASEM, 2021).
The partitioning of energy between protein and fat differed between treatments.About 52% of total incremental energy was retained as protein in the +PRO group, where this percentage was 5% for +FAT and 0% for +LAC calves.Energy retention as fat accounted for 67% of the total energy retained in +FAT calves and 49% in +LAC calves, whereas only 2% was stored as fat in +PRO calves.Increases in protein supply have been associated with increased fat deposition, even when protein intake is low (Donnelly and Hutton, 1976;Gerrits et al., 1996).In the study conducted by Labussière et al. (2008), it was observed that varying protein intake while maintaining equivalent ME intake did not affect overall HP or energy retention in 70 kg BW calves.However, differing protein intake levels did result in variations in lipid deposition.In contrast, our study showed that the +PRO group exhibited greater total HP and energy retention compared with the +FAT group, with no significant differences observed in comparison to +LAC calves.Calves in the +PRO treatment either utilized the extra protein for growth, resulting in a higher BW and ADG, or lost it as heat (44%).As reported by Roy (1980), increased protein deposition has a greater impact on BW, as it is associated with lean tissue which is highly hydrated.Regardless, our data showed that the energy intake did not limit protein deposition in the young calves.The lack of an increase in fat deposition with elevated protein supply might be related to reduced incremental protein efficiency as calves age, which in turn increases the likelihood of extra protein leading to additional fat deposition (Gerrits et al., 1996).
Furthermore, additional fat and lactose intake did enhance fat deposition as expected, with an increase in protein deposition.This response in protein deposition with increasing energy intake is consistent with observations in young calves (Donnelly and Hutton, 1976) and older (pre) ruminants (Gerrits et al., 1996;van den Borne et al., 2006;Labussière et al., 2008).Altogether, the increase in ME intake, coupled with reduced heat production (HP) and respiratory quotient (RQ) due to increased fat intake, resulted in a significantly increased energy retention in the form of fat.The conversion of dietary fat to body fat has been found to be efficient, leading to increased body fat rather than a significant surplus of additional protein (Bartlett et al., 2006;Bascom et al., 2007).
Additionally, research conducted on preruminant calves has demonstrated that the energetic costs of stereotype behaviors, such as excessive oral manipulation of the pen or tongue playing, can be quite high in individually-housed calves.These behaviors can have a significant impact on the relationship between physical activity and total heat production (Van den Borne et al., 2004Borne et al., , 2006)).In pigs, Gerrits et al. (2015) analyzed the effects of individual vs group housing on activityrelated heat production and found that, even though group-housed animals have more room to move around, single housing increased activity-related heat production by 40 to 60%.Although in our study, the calves were housed in groups, we found similar values for activity related heat production (as a % of total HP) as those reported by van den Borne (2006).These results have been discussed in more detail in a paper reported by Amado et. al. (submitted) about heat partitioning and substrate oxidation with the same data.
Moreover, increasing lactose intake yielded a numerically higher activity related heat production in comparison to the other treatment groups.This observation quantitatively accounts for the elevated total HP in this group.While a link has been established between excess glucose intake and hyperactivity in humans (Wolraich et al., 1995;Schulte et al., 2015), studies investigating the impact of high glucose intake on activity or behavior in young calves remain limited.

CONCLUSIONS
These results describe responses of young calves to incremental supply of protein, fat, or lactose.In this study, we observed that with every 100 g increase in protein intake, 52% was converted into protein deposition, while 44% contributed to heat production.Similarly, a 100 g rise in fat intake led to 67% being stored as fat with 22% being released as heat.Likewise, a 100 g increment in lactose intake resulted in 49% being stored as fat with 38% being released as heat.Additional energy did not significantly increase protein deposition, but when expressed as N efficiency corrected for effects in digestibility, additional energy (fat, but more so additional lactose) increased post absorptive N efficiency anywhere between 5 and 10%.These findings offer an opportunity to revise nutritional recommendations considering responses to nutrients of young calves and to improve milk replacer formulations.
Amado et al.: ENERGY AND PROTEIN METABOLISM IN YOUNG CALVES Amado et al.: ENERGY AND PROTEIN METABOLISM IN YOUNG CALVES tometer (Varian B.V., Middelburg, the Netherlands).All analyses were carried out in duplicate.
Amado et al.: ENERGY AND PROTEIN METABOLISM IN YOUNG CALVES 4

Table 1 .
Amado et al.: ENERGY AND PROTEIN METABOLISM IN YOUNG CALVES Ingredient and analyzed nutrient composition of the experimental milk replacers and design of the nutritional treatments 2Premix, provided per kilogram of milk replacer: 25,000 IU/kg of vitamin A; 5,000 IU/kg of vitamin D3; 90 mg/kg of vitamin E; 158 mg/kg of vitamin C; 94 mg/kg of iron; 47mg/kg of manganese; 124 mg/kg of zinc; 11 mg/kg of cooper; and 0.2 mg/kg of selenium.

Table 2 .
Amado et al.: ENERGY AND PROTEIN METABOLISM IN YOUNG CALVES Apparent total-tract digestibility coefficients of nutrients in young Holstein-Frisian calves as affected by the dietary treatments 1 CON = 23.3%CP,21.2%CF and 48.8% lactose on DM basis, provided at 550 kJ/kg BW 0.85 per day; or the basal MR incrementally supplied with 125 kJ of DE/BW 0.85 per day as milk fat (+FAT), lactose (+LAC) or milk protein (+PRO).2Apparenttotal-tract digestibility.3Different superscripts between columns denote a significant difference between treatments.

Table 3 .
Body weight and body weight development, nitrogen, and energy balance traits of calves receiving the control treatment, and effects of supplementation of 125 kJ of DE/kg of BW 0.85 /d as fat, lactose, or protein on these traits, reported by difference with the control treatment, in calves younger than 21 d of age 1 CON = 23.3%CP, 21.2% CF and 48.8% lactose on DM basis, provided at 550 kJ/kg BW 0.85 per day; or the basal MR incrementally supplied with 125 kJ of DE/BW 0.85 per day as milk fat (+FAT), lactose (+LAC) or milk protein (+PRO). 2 SE CON = Standard Error of Control treatment.3 Pooled SE = Average Standard Error of + FAT, +LAC, and +PRO treatments.

Table 4 .
Amado et al.: ENERGY AND PROTEIN METABOLISM IN YOUNG CALVES Gross and incremental efficiencies of N and energy of young Holstein-Frisian calves fed a milk replacer with increased intake of fat, lactose, or protein 8As heat (GE) = 100 × Total heat production / extra GE intake.9 Energy efficiency (ME) = 100 × Energy retention / ME intake.10 Incremental efficiency of energy retention (ME) = 100 × extra energy retained / extra ME intake.11 As protein deposition (ME) = 100 × extra energy retained as protein / extra ME intake.12 As fat deposition (ME) = 100 × extra energy retained as fat / extra ME intake.13 As heat (ME) = 100 × Total heat production / extra ME intake.