Can milk replacer allowance affect animal performance, body development, metabolism, and skeletal muscle hypertrophy in pre-weaned dairy kids?

We aimed to evaluate performance, body development, metabolism, and expression of genes related to skeletal muscle hypertrophy in non-castrated male dairy kids fed with different levels of MR during the pre-weaning period. Sixty newborn male kids, not castrated, from Saanen and Swiss Alpine breeds, with an average body weight (BW) of 3.834 ± 0.612 kg, were distributed in a randomized block design. Breeds were the block factor in the model (random effect). Kids were allocated into 2 nutrition plans (n = 30 kids per treatment) categorized as follows: low nutritional plan ( LNP ; 1L MR/kid/day) or high nutritional plan ( HNP ; 2L MR/kid/day). All kids were harvested at 45 d of life. The majority of nitrogen balance variables were affected by the nutritional plan ( P < 0.050). Morphometric measures and body condition score (2.99 - LNP vs. 3.28 - HNP) were affected by nutritional plan ( P < 0.050), except hip height, thoracic depth and hip width. The nutritional plan affected the body components ( P < 0.050), except esophagus and trachea. Animal performance and carcass traits were influenced by nutritional plan ( P < 0.050), except carcass dressing (48.56% on average). Nutritional plan affected ( P < 0.050) some blood profile variables as the total cholesterol (141.35 vs. 113.25 mg/dL), triglycerides (60.53 vs. 89.05 mg/dL), LDL (79.76 vs. 33.66 g/mL) and IGF-1 (17.77 vs. 38.55 ng/mL) for LNP and HNP respectively. Hypertrophy was greater in HNP than LNP animals ( P < 0.050), being represented by the proportion of sarcoplasm (39.76 vs. 31.99%). LNP had a greater mTOR abundance than HNP ( P = 0.045), but AMPK was not affected by the nutritional plan. Our findings show that a higher milk replacer allowance enhances animal


INTRODUCTION
Dairy kids on farms worldwide are still fed a low offer of liquid diet (Oderinwale et al., 2020), ranging between 1 to 1.2 L of milk or milk replacer (MR) until weaning.The primary objective of these farms is to optimize commercialized milk by minimizing the expense of feeding young animals but addressing maternal nutrition deficits during late gestation and early lactation.This is particularly crucial for farms located in tropical climates, where such factors can significantly impact milk production and quality (Nørgaard et al., 2008;Flores-Najera et al., 2021).In the pre-weaning phase, kids have an increasing demand of energy for cell growth, promoting body development, especially in the last days before weaning (close to 45 d of age; Ghulam Bugti et al., 2016).During pre-weaning the main source of nutrients comes from milk or MR.Hence, providing a high MR allowance can mitigate the challenges caused by nutrient deficiencies experienced by these young animals on a limited milk diet.This is especially crucial as they undergo rapid and intense growth nearing the weaning stage.
High MR allowance promotes a greater flow of glucose and proteins into the blood plasma, improvingthe health and performance of animals, as observed in calves (Silper et al., 2014).Glucose is an immediate energy source for cellular metabolism, which can also be related to postnatal growth (Costello et al., 2008).The increased energy availability resulting from glucose metabolism triggers the liver to ramp up the production of Insulin-Like Growth Factor 1 (IGF-1; Jeyapalan et al., 2007).Elevated levels of IGF-1 facilitate the utilization of dietary protein, enhancing its efficiency in synthesizing myofibrillar protein.This protein synthesis is crucial for Can milk replacer allowance affect animal performance, body development, metabolism, and skeletal muscle hypertrophy in pre-weaned dairy kids?cellular hypertrophy (Ithurralde et al., 2023).The high allowance of liquid diet also increased the development of the components of the gastrointestinal tract (GIT) and body development in pre-weaning calves (Schäff et al., 2018).The development of the GIT is positively correlated with high nutrient uptake, which directly influences smooth and skeletal muscle development.Furthermore, evaluating the high offer's influence on the weight of non-carcass components, offal, blood, and others is important.Since these require maintenance energy expenditure (Ortigue, 1991), which could be directed toward muscle hypertrophy.
On the other hand, providing calves with limited quantities of liquid diet (4 L/day in dairy calves; approximately 10% of birth weight) during the entire preweaned period leads to a reduction in feed efficiency (Davis and Drackley, 1998), compromising muscle hypertrophy and body development (Gandra et al., 2019).A similar effect may occur in kids receiving reduced MR allowance before weaning.Nutrient restriction can reduce the expression of genes related to muscle synthesis, such as the mammalian target of rapamycin (mTOR), and increase the 5′ adenosine monophosphate-activated protein kinase (AMPK) expression.This occurs due to insufficient dietary energy and protein (Zhou et al., 2021), suppressing hypertrophy and reducing weight and body development.A complex network of molecular signaling pathways regulates muscle energy metabolism.The mTOR pathway, which is highly conserved throughout evolution, functions as a pivotal controller of cell growth and proliferation.mTOR manages intracellular protein production while preventing protein degradation andlays a pivotal role in skeletal muscle development (Saxton and Sabatini, 2017).AMPK pathway monitors energy levels and maintains muscle tissue homeostasis (Cui et al., 2017).This pathway shares a close association with mTOR pathway via internal nutritional signals.This interplay effectively synchronizes the processes of energy sensing and protein synthesis at the cellular level, particularly when energy deficiency is present (Hart et al., 2019).This mechanism has been evidenced in piglets (Jeyapalan et al., 2007), cows (Appuhamy et al., 2014), and rats (Shi et al., 2020).However, the literature has not demonstrated the effect of lower MR allowance (liquid diet restriction) on the synthesis of mTOR and AMPK in pre-weaned dairy kids.
Most studies assessing various levels of liquid diet, especially MR, and their effects on the metabolism have focused on calves during their early life stages (Stiles et al., 1974;Silper et al., 2014).These findings in the literature might not be similar to other species (e.g., goats).Additionaly, the few studies with pre-weaned kids basically focus on evaluating performance without mentioning the mechanisms involved in the animals' body development and hypertrophy (Kumar et al., 2017;Oderinwale et al., 2020).There is a lack of detailed assessment of the interactive effects on performance, metabolism, and growth physiology of dairy kids receiving high MR allowances.Therefore, in the current study, we proposed a detailed trial to evaluate the performance, body development, metabolism, and expression of genes related to skeletal muscle hypertrophy in non-castrated male dairy kids fed with different levels of MR in the pre-weaned period.We hypothesized that animals receiving the highest MR allowance would have better performance, body development and metabolism parameters.We also hypothesized that the highest MR allowance would influence hypertrophy by increasing the gene expression of the mTOR, and decreasing the expression of the AMPK compared with animals receiving a lower MR allowance.

MATERIALS AND METHODS
The experiment was conducted at the Teaching, Research, and Extension Unit in Dairy Goat at the Universidade Federal de Viçosa (Viçosa, Minas Gerais, Brazil; 20°773 S and 42°853 W), from February 25 until June 25, 2022.The environment temperatures ranged from 10°C to 32°C, averaging 21°C throughout our study.The Institutional Ethics Committee approved the experiment (protocol no.016/2022).

Animals, Experimental Design, and Housing
Sixty male newborn kids, not castrated, from Saanen (n = 34) and Swiss Alpine breeds (n = 26), with an average age of 2 ± 1 d and an average body weight (BW) of 3.834 ± 0.612 kg, were included in the study.A power analysis was tested (Hintze, 2008;Ryan, 2013) to estimate sample size for the preliminary response variables, including feed intake, ADG, and feed efficiency, according to prior published literature values (Silva et al., 2015;Marcondes et al., 2016;Rodrigues et al., 2016).Several 30 kids per treatment was projected as a sufficient sample size based on the power test analysis with α = 0.05 and power = 0.80 for the above-addressed variables.The experiment employed a randomized block design, with animals allocated to 2 nutrition plans (n = 30 kids per treatment; low nutritional plan -LNP or high nutritional plan -HNP).Breed served as the blocking factor (1 ~Saanen and 2 ~Swiss Alpine) in the model (random effect).
The liquid diet levels were categorized as follows: 1) LNP, which resembles a standard offering for pre-weaning kids in commercial dairy goat production systems [providing 1 L of MR (0.125 kg of DM/L) per kid per day], and 2) HNP at 2 L of MR (0.125 kg of DM/L) per kid per day, which is set at twice the LNP amount.On the day of birth, the animals remained in the maternity pen (8.0 m × 5.0 m × 1.5 m; length × width × height) with their dams for one day for postpartum care.Briefly, the kids were weighed and received 10% of their weight in colostrum via nipple bottle.Animals were colostrized within 2 h of birth to ensure the maximum transfer of passive immunity.The colostrum quality was measured using a Reichert AR 200 Digital Hand-held Refractometer (Reichert Inc., Depew, NY, USA), with a mean of 25% ± 1.50 on the Brix scale.All animals were colostrized with colostrum from their respective dams.After colostrum procedures, kids were identified, and umbilical cord care was performed for 3 d using a solution with 10% iodine.Then, they received a single dose of Ivomec® Gold (0.02 mL/ kg of BW) as a preventive treatment against ectoand endoparasites.
On the second day of age, they were separated from their dams and allocated in individual cages (1.0 m x 0.6 m x 1.0; length × width × height) with polyethylene buckets for starter feed and water.All cages were located inside the research barn, with adequate lighting and ventilation.The facility was cleaned daily during the morning hours.

Diets and Feeding management
The blue sprayfor® MR (Trouw Nutrition) was used to feed the kids from d 2 to harvest.From the fifth day of birth until the end of the trial, all animals received fresh water and starter feed ad libitum.
Following the first day of life, MR was gradually introduced into the diet at a rate of 200 mL per day until the complete replacement of goat's whole milk was completed.Therefore, on d 2 of the experiment, kids began receiving a 400 mL liquid diet (~10% BW).Due to the daily addition of 200 mL, kids in LNP reached their maximum intake around 8 d of age, while those in HNP reached their maximum intake around 20 d of age.Additionally, during the first week of the experiment, MR was administered in 4 equal portions via bottles at 6 h, 10 h, 14 h, and 18 h.After the first week of experiment, the total volume of MR was offered individually in only 2 portions per day (6 h and 18 h).The adaptation to experimental diets was performed to avoid refusals and to reduce the risks of diarrhea and bloat in the first days of life.This protocol was done according to farm guidelines and veterinary guidance (as detailed below).All the kids consumed the total offered liquid feed, and no MR refusals were observed throughout the study.
Animals presenting any metabolic diseases were measured for body temperature, and fever diagnosis was recorded when the temperature was above 37°C.In this situation, animals were treated via intramuscular injection with 0.1 mL/10 kg BW of dipyrone from IBASA® every 8 h until body temperature was regulated.The protocol for kids who presented diarrhea was oral application of Baycox (Bayer ®) at a dosage of 7 mg/kg BW once daily.The diarrhea protocol was carried out within 5 d of the appearance of liquid feces.Animals that persisted with diarrhea after 5 d of treatment were excluded from the trial.For animals presenting bloat, 0.2 mL/kg BW of Ruminol from Vetoquinol® was administered orally once a day for 3 d.Animals that exceeded 3 d of treatment were excluded from the trial.The sick animals which did not exceed the days of treatment were not removed from the experiment.All protocols were previously established and carried out by the veterinarian at the Teaching, Research, and Extension Unit in Dairy Goat at the Universidade Federal de Viçosa.
The MR was prepared according to the manufacturer's instructions (Trouw Nutrition).Briefly, 125 g of MR was weighed for each 1 L of water.The water was heated in a water bath (1.0 m x 0.30 m; length x width) to 36°C, and then the MR was solubilized until a homogeneous solution.
The starter feed was formulated according to NRC (2007) guidelines and made on the farm in mashed form.However, as the kids did not intake the starter feed, all information about it was removed from the study.Table 1 shows the ingredients and chemical composition of the starter feed and MR.

Feed Intake and Digestibility trial
Intake of MR, and starter feed were recorded during all experiment periods to estimate dry matter (DMI) and nutrient intake.The MR offered were measured using a graduated cylinder, while the starter feed was measured on a digital scale.The starter was fed ad libitum for both nutritional plans.However, the starter feed intake was negligible (less than 3 g/kid/day on average).Thus, we removed the data from the study, and only MR intake was considered.
Then the DMI (g/day) and nutrient intake were considered only the total MR allowance since there were no MR refusals or starter feed intakes.Total nutrient intake was calculated by the proportion (%) of each chemical component (CP, EE, CNF, Ash, ME) by the total DMI of MR per kid/day.MR offered was collected and stored weekly for further analysis.
A digestibility period was performed with a total collection of feces and urine between the 35 and 38 d of life.Six animals from each treatment (3 animals of each breed) were randomly distributed in metabolic cages, totaling 12 animals for the digestibility trial.These animals were confined in individual metabolic cages made of galvanized iron and with the same dimensions as those used before the digestibility test.All cages contained polyeth-de Souza Pinheiro et al.: Influence of milk replacer allowance on performance, metabolism, and skeletal muscle hypertrophy in pre-weaned dairy kids ylene buckets for starter feed and water.After this period, the animals returned to their original cages.During the 3 d of collection, the total feces were sampled and weighed daily in aluminum trays to be dried in a forced circulation oven and ground, as described above.At the end of the experimental period, a composite sample was mixed to be proportional to the amount excreted daily (DM basis).Urine was collected in buckets of 5 L containing 50 mL of 20% (vol/vol) sulfuric acid to preserve the nitrogen, and its volume was measured and sampled daily.Subsequently, a sample based on each day's volume was obtained for chemical analyses (quantification of the total N).Water intake was measured during digestibility by the difference between water supply and surplus.The water intake was corrected using water evaporation quantified using a blank bucket located near the animals.At the end of the digestibility, blood collection was performed 4 h after feeding to characterize blood metabolites and the animals were moved back to their original cages.

Performance and Morphometric Measures
All animals were weighed and morphometrically measured every 15 d during the experiment at 7 h immediately before feeding to estimate the initial (iSBW) and final shrunk body weight (fSBW).The BW was measured using an electronic scale Michelleti Inc.,Guarulhos,São Paulo,Brazil) calibrated by the manufacturer's agent.Average daily gain (ADG; g of BW gain/ day) was calculated by the difference between fSBW and iSBW and divided by the days in experiment.To compute feed efficiency (g of BW gain/g of DM intake), ADG was divided by total DM intake.Body condition score (BCS) was measured while we weighed the kids to evaluate body reserves at the spinous and transverse process of the spine on a scale of 1 to 5 with 0.25-point increments (Ferguson et al., 1994).Thus, the BCS was the average value given by 2 trained technicians.
Heart girth (chest circumference), chest width (measured between both shoulder joints), withers height (distance from the base of the front feet to the withers), thoracic depth (distance between the withers and the sternum), body length (distance between the points of shoulder and rump), hip height (distance from the base of the rear feet to hook bones), and hip width (distance between the points of hook bones) were measured as the body skeletal size using a graduated hypometer (cm) following the method described by Sahlu et al. (1992).

Blood Sample Collection and Analyses
Blood samples from the 12 kids in the digestibility trial were taken on the last day of the digestibility (38th day of life) from the jugular vein into 10-mL vacuum tubes containing separation gel and clot activator.Tubes were kept on ice until centrifugation (3,000 × g at 4°C for 20 min) to obtain blood serum.The serum was pipetted into Eppendorf tubes and stored (−20°C) until analysis.

Harvest procedures and sample collection
All kids were harvested at the experimental slaughterhouse of Universidade Federal de Viçosa, Minas Gerais, Brazil.All kids were harvested at 45 d of life to represent the high activity and changes in the intestinal tract and muscle metabolism during an animal's transition from 30 to 60 d.
The animals were stunned using a pneumatic gun in the frontal region of the head (CONCEA, 2015b), bled, and opened from the bottom (belly) with the aid of a surgical scalpel.The carcass of each animal was divided into 2 halves, which were weighed to estimate carcass dressing (%).
After bleeding and carcass opening, the digestive tract of each kid was emptied and washed, and each organ and viscera were weighed separately on a digital scale (model, US 20/2 POP-S, Urano Pop, São Paulo, Brazil).The non-carcass component weight (NCC) was composed of the sum of the weights of the heart, lungs, liver, spleen, kidneys, internal fat, diaphragm, mesentery, tail, trachea, esophagus, reproductive tract, washed GIT, head, hides, hooves and blood.The NCC was added to the carcass weight to determine the final empty body weight (fEBW).
Additionally, the muscle tissue samples for histomorphometric analysis (2 cm × 3 cm sections) were placed in 50 mL vials with 4% paraformaldehyde in a 0.1 M phosphate buffer solution and incubated overnight at room temperature for 24 h.After this period, the samples were preserved in 70% ethanol until processing and histological cross-sections.A subset (5 g) of muscle and liver samples was collected at harvest, immediately after bleeding, and snap-frozen in liquid nitrogen.Frozen samples were then stored at −80°C until the RNA extraction process.

Histomorphometric Analysis of Skeletal Muscle Tissue -Hypertrophy
Muscle tissue samples (Longissimus lumborum dorsis) previously fixed and stored in 70% ethanol were embedded in 2-hydroxyethyl methacrylate (Historesina®-Leica Biosystems, Buffalo Grove, IL, USA), according to the manufacturer's recommendations.The tissue samples were first prepared by embedding and cross-sectioning at a thickness of 3 µm, followed by staining with hematoxylin-eosin (HE) solution and mounted with Entellan® (Merck, Frankfurt, Germany).A total of 8 images of the slides of each tissue per kid were captured using a light microscope (BX53; Olympus, Tokyo, Japan), at 20 × magnification, with a CMOS 1.3 MP BioCAM camera (Takachiho, Japan).The images were used to determine the volumetric proportion of muscle tissue components and the number of muscle fibers.These variables were measured using the Image J ® program (version 1.50i; National Institutes of Health, Bethesda, MD, USA).
The number of muscle fibers in the tissue samples was assessed by manually counting each histological image, with each image covering an area of 0.131 mm 2 .Hypertrophy was quantified through the percentage of sarcoplasm calculated in the volumetric proportion of muscle tissue (Rodrigues et al., 2021).To determine the volumetric proportion of muscle tissue components, 266 intersections (points) were projected onto each histological image.Coinciding points were categorized as nucleus, sarcoplasm, and connective tissue.The percentage of points representing muscle tissue was calculated using the formula: volumetric proportion (%) = (number of points in each structure / total points in muscle tissue) × 100.

Gene expression involved in muscle synthesis
Muscle (Longissimus dorsis) and liver tissue were used to evaluate the abundance of the mTOR and AMPK genes, respectively.The frozen samples were powdered in liquid nitrogen for total RNA extraction and quantification of the mRNA of the samples.
Total RNA extraction and mRNA abundance analysis.The total RNA was extracted from 0.1 g of tissue using MagMAX Viral/Pathogen II (MVP II) Nucleic Acid Isolation Kit (Thermo Fischer Scientific) in the KingFisher Flex Purification System following the manufacturer's instructions.Total RNA was quantified using a NanoDrop spectrophotometer (GE Healthcare Life Sciences Inc.) and integrity was assessed using 1% agarose gel.The RNA samples were reverse transcribed Real-time quantitative PCR was performed in the thermal cycler ABI Prism 7300 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) using the detection method SYBR Green (Applied Biosystems -Foster City, CA, USA) and GoTaq® qPCR Master Mix kit (Promega Corporation, Madison, WI, USA) using the following cycle parameters: 95°C for 2 min and 40 cycles at 95°C for 15 s and 60°C for 60 s.Melting curve analysis was applied to confirm the specificity of each amplification product.Negative controls were performed by substituting water for cDNA.The threshold cycle (Ct) values obtained were later normalized using the delta-ct method (∆Ct) based on the Ct values obtained for the endogenous control gene [Glyceraldehyde-3-Phosphate Dehydrogenase -GAPDH; F (5′ CACGAGAGGAAGAGA-GAGTT 3′) and R (5′ GGGATGGAAATGTGTGGAG 3′)].The calculation of relative gene expression levels was developed according to the 2 −∆∆Ct method, described by Livak and Schmittgen. (2001), for each sample.

Laboratory Analysis and Calculations
Samples of MR, and feces were analyzed for DM (AOAC, 2012; method 934.01),OM (AOAC, 2012; method 930.05), and TN (AOAC, 2012, method 981.10).The crude protein (CP) was obtained by the product between total nitrogen and factor 6.25.The ether extract (EE) analysis was performed according to AOAC, (2006; method 2003.05) after acid hydrolysis with HCl in feed samples, and feces.The analysis of neutral detergent fiber (NDF) in the feces samples was performed according to the techniques described by (Mertens et al., 2002), without the addition of sodium sulfite, but with the addition of thermostable α-amylase to the detergent.The NDF was corrected for protein and ashes (NDFap).However, due to irrelevant starter feed intake, the quantified NDF concentration was very low (values close to zero).Therefore, the NDF was disregarded in the sample composition and calculation.Non-fiber carbohydrates (NFC) was adapted from the Detmann and Valadares Filho, (2010), where the NDFap was considered zero.
The N balance (g/day) was estimated with the N intake, and N excreted in the feces and urine.Apparent absorbed N was estimated by the difference between N intake (g/ day) and fecal output (g/day), while the N retained was obtained by the following equation: The proportion of N retained by N intake (NR:NI, %) was calculated by the relationship between nitrogen retained and nitrogen intake and multiplied by 100.
The digestibility coefficients (%) of the chemical components were calculated by the difference between the intake and excretion of each component in the feces, divided by the intake and multiplied by 100.
The absorbed mineral (g/day) was calculated as individual mineral intake (g/day) minus the mineral concentration in the feces (g/day) and multiplied by 100.
Data of water intake (mL/day), feces (g DM/day), and urine output (L/day), body components (g), and nitrogen balance (g/day) were used for relativization calculations with the fEBW (kg) of the kids.

Statistical analyses
DMI and nutrient intake were summarized as descriptive statistics with the MEANS and FREQ proc of SAS.9.4.
Digestibility, variables relative to fEBW (water intake, feces, and urine output, and nitrogen balance), mineral absorption, nitrogen balance, performance carcass traits, blood profile, histomorphometric, and gene abundance data were analyzed considering initial shrunk body weight (iSBW) variable as the covariate for body components and body components relative to fEBW, animal performance and carcass traits data, respectively, according to the following model: Where, Y ijkl = dependent variable; μ = overall mean; NP i = fixed effect of nutritional plan (1 or 2 L of MR) i; B j = random effect of breed (block) j: (1 ~Saanen or 2 ~Swiss Alpine); β (Xi − ͞ X) designates the covariate variable, where the β is the regression coefficient relating covariate factor to the variable measured, Xi is the covariate factor for the ith factor, and ͞ X is the overall mean of covariate factor; δ ijk is the sampling error (variance among animals within treatment); and ε ijkl = random error with zero mean and variance σ 2 .When the covariate was not significant, it was removed from the model.Morphometric and body condition score data were analyzed in a repeated measure scheme, according to the following model: Where, Y ijkl = dependent variable; μ = overall mean; NP i = fixed effect of nutritional plan, i: (1 or 2 L of MR); D j = fixed effect of day, j: (15d, 30d and 45d); B k = random effect of breed (block), k: (1 ~Saanen or 2 ~Swiss Alpine); (NP x D) ij fixed effect of interaction between nutritional plan and day; δ ijk is the sampling error (variance among animals within treatment); and ε ijkl = random error with zero mean and variance σ 2 .Several variance-covariance structures (AR1, CS, UN, TOEP, VC, ARH1, TOEPH) were tested, and an autoregressive type 1 covariance structure was fitted and subjected based on the lowest Akaike and Bayesian information criteria.
All data were evaluated by PROC GLIMMIX, version 9.4 of SAS, except nutrient intake data.In summary, the random effect was used to capture the proportions of the total variation aiming to reduce the residual variance (Wang and Goonewardene, 2004).All residuals were tested for normality using the SHAPIRO-WILK test, least squares means were compared using the Tukey test and statistical differences were declared when P < 0.05 with probability for type I error.

Descriptive outcomes
As designed, the intake of all nutrients was greater for HNP than LNP (Table 2).Animals at HNP had the greatest dry matter intake (DMI; g/day) as well as for other chemical components of the diet.Four kids in LNP and 3 HNP had diarrhea lasting a maximum of 2 d.Furthermore, 5 kids in HNP presented bloat lasting an average of 3 h, and no kids in the LNP treatment presented bloat.
Starter feed intake of animals in the LNP was 2.78 g DM/day and for HNP was 0. Twenty-two g DM/day, averaging 1.51 g DM/day for all animals (Supplementary Figure 1; dx .doi.org/ 10 .6084/m9 .figshare.26327197).Kids in the LNP group started starter feed intake around the 22nd day of age, while the animals in the HNP started consuming it around the 40th day of age, respectively.

Digestibility, mineral absorption, and nitrogen balance in dairy kids
The kids ' nutritional plan affected water intake, feces, and urine output (P < 0.001; Table 3).However, the nutritional plan affected only water intake and urine output relative to fEBW (P < 0.050; Supplementary Table 1; dx .doi.org/ 10 .6084/m9 .figshare.26327197).The water intake of animals at HNP was 90.82 mL/day, which was much higher than that of animals at LNP (5.20 mL/day; LNP).Fecal (g/day) and urine (L/day) output of kids in HNP doubled compared with LNP, being 97.15% and 85.71% greater than the group receiving 1 L of MR/kid/ day, respectively.Treatments did not affect total apparent digestibility and mineral absorption (P > 0.050; Table 3).The total apparent digestibility was similar between the groups receiving different levels of MR.The average values of digestibility coefficients were 94.51%, 95.89%, 90.57%, 97.95%, and 97.18% for the DM, OM, CP, EE and NFC, respectively.The absorbed minerals were similar between the nutritional plans, with higher absorption rates for potassium (average of 99.22%), sodium (average of 97.70%), manganese (average of 95.40%) and calcium (average of 92.56%), and lower absorption rates for zinc (average of 66.56%), magnesium (average of 83.21%) and phosphorus (average of 87.80%), respectively.
All nitrogen balance variables were greater for kids at HNP (P < 0.001; Table 4).However, when nitrogen balance variables were relativized with fEBW, the nutritional plan affected only nitrogen intake, absorbed nitrogen, and retained nitrogen (P < 0.050; Supplementary Table 1).Nitrogen intake (g/day), absorbed nitrogen (g/day), and retained nitrogen were, on average, 95.39% greater for animals at the greatest level of MR (HNP) compared with LNP.Nitrogen excretion in feces and urine of animals in LNP was, on average, 70.0% (~0.28 g/day) and 41.50% (~0.44 g/day) lower than in HNP, respectively.

Morphometric and body components measure, performance and carcass traits in dairy kids
Morphometric measurements and body condition score (BCS) were not affected by the interaction between nutritional plans and days (P > 0.050).However, all variables were affected by the nutritional plan and days (P < 0.050), except for hip height, thoracic depth, and hip width, which were not affected by the nutritional plan (P > 0.050; Table 5).The animals in the HNP were morphologically, on average, 2.12 cm larger in most variables compared with the LNP.Body length, hip height, heart girth, and thoracic depth grew linearly over the days.Wither height was similar between d 15 and 30 but lower on d 45.On the contrary, chest width was greater on d 30 and 45 compared with d 15.Body condition scores were greater in animals on HNP and 15 d than in LNP, 30 and 45 d of age.
All body components showed significant differences based on the MR level (P < 0.050), except for the esophagus and trachea (P = 0.202 and P = 0.070, respectively; Table 6).Notably, liver, small and large intestine, mesenteric fat, and hides exhibited the most significant growth  1).Animal performance and carcass traits were notably influenced by the nutrition plan (P < 0.001), except for the dressing carcass variable (P = 0.346; Table 7).Animals at HNP had an fSBW of approximately 2.78 kg heavier (approximately 42.31% greater) than those at LNP.When comparing the fEBW between HNP and LNP, this difference was reduced to 2.02 kg.Carcass and non-carcass weights of kids at LNP were 69.51% and 61.11% of the HNP ones, respectively.

Blood profile in dairy kids
Total cholesterol (141.35 vs. 113.25

Histomorphometric and gene expression in dairy kids
The percentage of nuclear cells and connective tissue were not affected by MR level (P > 0.050; Figure 1).Nonetheless, sarcoplasm was affected by the nutritional plan (P = 0.009).The percentage of sarcoplasm was 7.77% higher for HNP kids than those at LNP.The abundance of mTOR and AMPK transcripts was not influenced by MR level.(P > 0.050; Figure 2).

DISCUSSION
Milk replacers are commonly employed globally to feed newborn kids until weaning, to save dam's milk for sale while providing kids with a more nutrient-rich diet to enhance their growth and body development.The present study was carried out to investigate the influence of different MR allowances on performance, body development, metabolism, and hypertrophy related to the expression of mammalian target of rapamycin (mTOR) and 5′ adenosine monophosphate-activated protein kinase (AMPK).
Kids at HNP who were fed approximately 5% of their birth weight in DM of MR achieved better overall performance than LNP.Similar effects on performance and carcass traits of animals subjected to high nutrient offer were also observed in calves (Wang et al., 2017).Studies with early-weaned animals show that higher allowances and different types of feedstuffs stimulate animal protein, energy, and minerals intake during the pre-weaned phase, leading to higher weight gain.This occurs mainly due to the high demand for nutrients for body development, especially carcass, organs, and viscera.The abovementioned components demand intense metabolic and physiological processes during this phase (De Palo et al., 2015).
The results show that the kids' performance was in accordance with the gain strategy assigned to the groups.Therefore, kids at HNP had the greatest results in MR intake, weight gain and feed efficiency.The ADG of kids in the HNP can be considered high according to studies in the literature (>120g/day, Yeom et al., 2004;Tacchini et al., 2006;Delgado-Pertíñez et al., 2009).However, although the ADG in LNP was positive, it can be considered low, which is not desired in a production system.We expected a weight gain in kids receiving 2 L of MR/ day proportional to the MR allowance.Surprisingly, the HNP animals had a BW gain 3x higher than their birth weight (3.8 kg BW at birth) and 42.31% greater than the final weight of LNP animals.These animals had a high conversion of dietary nutrients into energy for their development; this is evidenced by the higher feed efficiency index.Feed efficiency can be influenced by dietary restrictions, stress, illnesses, and inadequate management, on the other hand, this indicator can be improved through high nutrition and genetics (Chacko Kaitholil et al., 2024).In this case, the greater feed efficiency of HNP animals was influenced by the dilution of the maintenance requirement that these animals reach as they gain greater weight (Nie et al., 2015).
Our results indicate that animals receiving high MR allowance stored considerable energy in their bodies, mainly as evidenced by visceral fat in the HNP group.Evolutionarily, the goat species has become more rustic and efficient in retaining water due to water restrictions and environmental challenges experienced in the past in its place of origin (Silanikove, 1992).Mainly in breeds that produce milk with greater energy and protein requirements.This fact is proven by the anatomical structure of goats, which have several loops in the colon to increase water uptake by the animals (Silanikove, 2000).Perhaps the evolutionary mechanism of water retention in these animals applies to body energy storage.When animals retain the maximum amount of nutrients as a preventive measure against possible nutritional restrictions (Silanikove, 2000).The energy storage can be noted in the high visceral fat accumulation, which represented approximately 4% of the fSBW of HNP animals compared with 2.2% LNP animals.In addition, kids at HNP had a greater Nr:Ni than LNP, demonstrating greater nutrient storage efficiency.
It is speculated that higher levels of liquid diet allowance can increase the passage rate through the GIT (Welboren et al., 2021a), leading to the incidence of diseases such as bloating, mainly diarrhea in pre-weaning animals.Research demonstrates that MR osmolarity can affect GIT permeability, compromising the kinetics of the animals' GIT, increasing its flow, reducing the digestibility and absorption of nutrients (Chapman et al., 2016), and increasing the diarrhea risk in early young calves (Wilms et al., 2019).However, animals in this study that received 2 L MR/day (HNP) had the same digestibility coefficients and mineral absorption as those that received the lowest offer (LNP).These results indicate that kids can increase their capacity to metabolize and absorb nutrients as they increase their demand for energy, protein, and minerals for their growth (Quigley et al., 2019).
In addition, we highlight that the higher liquid feed offer did not increase the incidence of sick animals (only 4 kids in LNP and 3 in HNP presented diarrhea).This further indicates that kids are less sensitive to high MR osmolarity than calves.In another way, the increase in cases of diarrhea must be linked mainly to the poor immunological status of animals fed with low feed offer, making animals more susceptible to pathological environment viruses, as reported by Khan et al. (2007b), andJasper andWeary, (2002).Kids in the HNP also had the highest water intake, which may be associated with the higher metabolizable energy intake (0.75 Mcal/day) of these animals (NRC, 2007), but also with the greater physical capacity (size) of the rumen and abomasum (Table 6).We should note that the significant differences in water intake between LNP and HNP were unexpected.Kids can interact with water, leading to its loss, which is an error that should be considered when measuring water intake.Therefore, readers should carefully evaluate these variables.Additionally, the higher MR allowance resulted in greater urine output, as 87% of 1 L of MR is water (Abe et al., 1999).
Growing animals are more efficient than adult animals in converting dietary nitrogen into protein used for body development, with lower energy costs (BR-CORTE, 2023).The conversion of dietary nitrogen is mediated by enzymes, cofactors, and hormones, especially IGF-1.
IGF-1 has the potential to enhance dietary nitrogen retention by minimizing protein oxidation while simultaneously boosting protein synthesis in body tissues (Wang et al., 2017).In this aspect, we noticed that kids receiving 2 L of MR/day had higher IGF-1 concentration, which influenced a better nitrogen balance, retaining almost 70% of the total nitrogen intake.This result reflected a more significant morphometric development, comprising mainly body length, withers height, heart girth, hip width, and chest width.Furthermore, body growth happened synergistically with the growth of organs and viscera (same proportion of carcass and NCC, Table 7).HNP Kids had all body components (g) ranging between 30 to 100% heavier than LNP animals.It is worth mentioning that the liver was the body component that developed the most in the HNP animals.This organ represented approximately 5% of the fEBW.The rapid development of the liver is due to greater metabolic activity that this organ carries out under conditions of overfeeding (Welboren et al., 2021b).On the other hand, LNP animals had greater energy expenditure with the growth of NCC (e.g., skin, paws, and head; Supplementary Table 1).
Lipid component values in the kids' blood in our study were higher than those in adult goats (Li et al., 2024).High concentration of these components is related to the greater amount of fat in the feeds (e.g., milk and MR) of newborn and pre-weaned animals, unlike the feed of adult animals (e.g., roughage and concentrates; Li et al., 2024).Regarding the nutritional strategy of the trial, a higher concentration of triglycerides was observed in animals at HNP.This is associated with their higher fat intake through the doubled MR allowance.Animals receiving high concentrations of dietary fat, carbohydrates, and sugars typically increase their plasma fat levels, mainly in the form of triglycerides, cholesterol, and their transporters, LDL and HDL (Berends et al., 2020).On the other hand, the age effect and lower nutrient offers have been related to an accumulation of endogenous cholesterol through reduced body metabolism (Fouladi et al., 2024).Interestingly, LNP animals receiving the lowest nutritional intake had the highest levels of cholesterol and LDL.Young goats need enough energy to support increased body mass, muscle and bone development, which makes it essential to adjust dry matter intake as these animals grow.Low volumes or fixed offers of liquid diet can lead to a reduced ADG of animals, as observed at LNP, consequently reducing the cell metabolism of kids in the final offspring phase, close to weaning.
Although the AMPK abundance and glucose results indicate no energy restriction among the treatments, as evidenced by the positive ADG of all animals, the lower ADG in the LNP group suggests a reduction in cellular metabolism.Studies have reported that the accumulation of cellular and blood cholesterol, as well as the accumulation of its transporter (LDL) in the blood are related not only to excessive fat intake but also to a lower cellular metabolic activity of the individual (Feingold, 2024), which can be caused by a reduced feed supply.The accumulation of LDL occurs due to an interactive problem caused by an inefficiency in the cell mechanism of an animal with reduced metabolism.In general, the cell stops absorbing cholesterol due to its low use in the cytoplasm of peripheral tissues, leading to the accumulation of LDL (Feingold, 2024).Cholesterol accumulated in the cytoplasm leads to negative feedback of the transcription factor SREBP-2 in the Golgi complex, leading to blood accumulation.Negative feedback on SREBP-2 stimulates the expression of LDL receptors in the cell nucleus of tissues, especially in the liver, aiming to reduce cellular and blood cholesterol concentrations (Feingold, 2024).High LDL concentration in the blood is also related to a reduced synthesis of their receptors in the liver due to a low metabolism, and this process may occur in animals in the LNP.We want to emphasize that we only had one blood collection throughout the experiment.Therefore, it requires a cautious interpretation of the kids blood variables and their possible associations with other studies.Future studies may evaluate the effects on the transcription of SREBP-2 and LDL receptors in response to cholesterol and LDL accumulation in blood.Furthermore, we also suggest measuring other blood parameters, such as acetone, acetoacetate, and β-hydroxybutyrate, for the possibility of mobilizing body fat in kids receiving 1 L of MR/day.Finally, we recommend that future studies assess blood metabolites at different ages and times after MR feeding, aiming for a more detailed diagnosis.
Postnatal muscle mass increase stems from either the hypertrophy of current myocytes or the activation of satellite cells.These processes hinge on the interaction between IGF-1 and its receptor (IGF1R), as elucidated by Chargé and Rudnicki (2004).Kids receiving the highest nutritional plan had a higher plasma concentration of IGF-1, influencing a higher sarcoplasmic percentage (an indicator of greater hypertrophy; Figure 3).Furthermore, the higher carcass weight of these animals was also mediated by increased IGF-1 plasma concentration, which is related to higher N retention of these animals.Synthesis of IGF-1 is strongly influenced by increase de Souza Pinheiro et al.: Influence of milk replacer allowance on performance, metabolism, and skeletal muscle hypertrophy in pre-weaned dairy kids and availability of nutrients (Wang et al., 2017), mainly proteins and highly digestible amino acids that make up the MR.A diet rich in protein and energy combined with more active behavior of young goats (e.g., locomotion and jumping) can increase IGF-1 plasmatic levels and its receptors in their body.Nutritional level, hormones (especially IGF-1), and physical activity interaction can stimulate gene expression involved in skeletal muscle pathway, especially mTOR, leading to greater hypertrophy in animals.In contrast, due to feed restriction, less developed animals express genes such as AMPK that indicate an energy deficit, causing a downregulation in mTOR synthesis.In our study, mTOR mRNA abundance in animals with greater nutritional intake was opposite to that found in most studies in the literature (Peng et al., 2018;Kim and Kim, 2023).HNP kids, which had the greatest carcass weight and body components, were lower in mTOR abundance, even though they had greater hypertrophy than LNP animals.We suspect that some post-transcriptional process may be acting on the translation of mTOR, influencing its phenotype but without reflecting on the abundance of its mRNA.Alternatively, we suspect another molecular pathway may be modulating the hypertrophy of preweaing dairy kids in HNP.The pattern of transcriptional alterations in skeletal muscle appears to be different for neonate animals.Studies by Suryawan et al. (2007Suryawan et al. ( , 2006)), Naeem et al. (2012), andWang et al. (2014) suggested that the enhanced protein synthesis and hypertrophy was paralleled by an activation of the insulin and IGF-1 signaling cascade.Insulin receptor (INSR), insulin receptor substrate 1 (IRS1), phosphatidylinositol 3-kinase (PI3K), 3-phosphoinositide-dependent protein kinase (PDPK1), and protein kinase B (PKB or AKT) have been mentioned as the main genes in the regulation of skeletal muscle synthesis (especially PKB/AKT).These genes influence hypertrophy in young animals; such pathways might be implicated in the phenotypes described here.Naeem et al. (2012) and Wang et al. (2014) assessed the effect of nutrient intake on gene expression involved in rumen cellular proliferation and signaling pathways in the skeletal muscle of calves, respectively.These authors noticed a decrease in the abundance of mTOR mRNA in animals with high nutrient intake over time.Such signaling behavior matches with other mediators of insulin (e.g., AKT1 and IGF-1), regulating the same aspects of cell development and growth performed by mTOR.In the study by Naeem et al. (2012), the relatively higher abundance of AKT1 mRNA by higher milk offer suggests a potentially important role in the observed reticulorumen cell proliferation and growth response, mediated by higher insulin and IGF-1 in the calves.As reported by Naeem et al. (2012), the reduction in the relative expression of mTOR mRNA at HNP group in the current study suggests that other genes mediate muscle synthesis.These results highlight the potential effect of AKT1 on the hypertrophy process in preweaning kids as observed in calves.
AKT1 is also known as protein kinase B (PKB).This gene plays a crucial role in the activation (phosphorylation) of transcription factors and proteins involved in the regulation of protein synthesis, such as transcription factor GSK3β (glycogen synthase kinase 3 β) and TSC2 protein (tuberin), which are mTOR inhibitors.The activation of AKT1 reduces these inhibitors, thus promoting protein synthesis and muscle hypertrophy.This gene is the most abundant AKT isoform in rodent muscle, being activated in response to IGF-1 (Sandri, 2008).
Similar to mTOR, AKT1 (a downstream effector of IGF-1 signaling) participates in cell survival, metabolism, maintenance and growth in response to growth factors, hormones and nutrients (Kim et al., 2003).Therefore, the lower abundance of mTOR mRNA does not mean that this protein is less active but that other mechanisms must be acting on the expression of this protein.Thus, as described by Naeem et al. (2012), we suspect that the concentration of IGF-1 in the HNP group must have triggered the AKT1 upregulation pathway, influencing the activation of the mTORC1 complex.This mechanism leads to greater skeletal muscle development and hypertrophy, as was observed phenotypically in greater performance, development of body components, and carcass traits of kids at high MR allowance.Nonetheless, this hypothesis requires further investigation.
In summary, all kids had a high mineral absorption, corresponding to the bone and tissue growth period, intensified in the offspring phase until close to 10 mo of life for these animals.Furthermore, these animals had almost total MR digestibility, which maximized nutrient utilization.We highlight that both mineral absorption coefficients and MR digestibility of HNP were similar to those of animals with lower MR intake (LNP), showing that the possible highest pass rate of animals receiving a higher level of liquid diet (HNP) was not decisive in compromising these parameters.In agreement, the incidence of metabolic diseases such as diarrhea and bloating was not influenced by the MR level.However, the greatest benefits of mineral digestibility and absorption were observed in animals at high MR allowance (HNP).The double supply of feed associated with its high digestion and absorption allowed a greater flow of energy (e.g., serum triglycerides), stimulating an intense hepatic metabolism as observed in the greater development of this organ in HNP.This phenomenon triggered a physiological cascade in kids, leading to the synthesis of hypertrophic regulators and modulators in skeletal muscle.
Overall, the greater flow of nutrients in HNP stimulated greater serum concentrations of IGF-1 produced by the liver, which reflected in greater morphometric development, weight of organs, viscera, and other components, as well as the carcass traits.Furthermore, the high circulating IGF-1 in HNP kids made these animals more efficient in retaining dietary nitrogen (e.g., nitrogen balance variables).The greater nitrogen retention in HNP animals was directed toward metabolic processes, especially in skeletal muscle, leading to a greater sarcoplasmic proportion in muscle fibers (greater hypertrophy), evidenced in the greater weight gain of these animals.
In conclusion, our results demonstrate that higher MR allowance lead to better animal performance, body development, and metabolism parameters.Additionally, the higher MR allowance increased cellular hypertrophy but reduced the abundance of mTOR and did not affect the AMPK transcripts.Our findings differ from the conventional results in most species or adult animals.This leads us to suspect that post-transcriptional modulation or activation of other pathways influences the preweaning dairy kids hypertrophy.This is a prospective study that presented new evidence and therefore boosts future studies evaluating the influence of high levels of MR on the post-transcriptional process of mTOR mRNA synthesis, or even the description of other regulators is necessary to elucidate the muscle hypertrophy pathway in non-castrated male kids during pre-weaning.

Notes
Declaration of interest We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.
Financial support statement This study was funded by the National Council for Scientific and Technological Development (CNPq), the Minas Gerais State Research Support Foundation (FAPEMIG), and the Higher Education Personnel Improvement Coordination (CAPES/ PROEX 88887.844747/2023-00).The funders had no role in study design, data collection, analysis, decision to publish, or manuscript preparation.

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Souza Pinheiro et al.: Influence of milk replacer allowance on performance, metabolism, and skeletal muscle hypertrophy in pre-weaned dairy kids

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Souza Pinheiro et al.: Influence of milk replacer allowance on performance, metabolism, and skeletal muscle hypertrophy in pre-weaned dairy kids

deFigure 3 .
Figure 3. Histological images from the skeletal muscle of the of dairy kids.(A) LNP = low nutritional plan (1L of MR/kid/day); (B) HNP = high nutritional plan (2L of MR/kid/day; (→) black arrow indicates cell size.

Table 1 .
Proportions of ingredients in the starter feed, chemical composition of the starter feed, and milk replacer based on dry matter offered to dairy kids de Souza Pinheiro et al.: Influence of milk replacer allowance on performance, metabolism, and skeletal muscle hypertrophy in pre-weaned dairy kids

Table 2 .
Descriptive outcomes of the effect of different levels of milk replacer on individual nutrient intake in dairy kids de Souza Pinheiro et al.: Influence of milk replacer allowance on performance, metabolism, and skeletal muscle hypertrophy in pre-weaned dairy kids 1 DM = dry matter; OM = organic matter; CP = crude protein; EE = ether extract; NFC = non-fibrous carbohydrate.LPN = low plan nutrition; HPN = high plan nutrition.SD = standard deviation.

Table 3 .
Effect of different levels of milk replacer on water intake, feces and urine output, total apparent digestibility coefficients and mineral absorption in dairy kids

Table 4 .
de Souza Pinheiro et al.: Influence of milk replacer allowance on performance, metabolism, and skeletal muscle hypertrophy in pre-weaned dairy kids Effect of different levels of milk replacer on nitrogen balance parameters in dairy kids

Table 5 .
Effect of different levels of milk replacer on morphometric measures and body condition score in dairy kids de Souza Pinheiro et al.: Influence of milk replacer allowance on performance, metabolism, and skeletal muscle hypertrophy in pre-weaned dairy kids

Table 6 .
Effect of different levels of milk replacer on body components measures in dairy kids

Table 7 .
Effect of different levels of milk replacer on animal performance and carcass traits in dairy kids

Table 8 .
Effect of different levels of milk replacer on the blood profile of dairy kids HDL = high density lipoprotein, LDL = low density lipoprotein, VLDL = very low-density lipoprotein, IGF-1 = insulin growth factor 1. LPN = low plan nutrition; HPN = high plan nutrition.SE = standard error.