Effects of milk replacer powder added to pasteurized whole milk over different durations on dairy calves fed ground starter diet with alfalfa hay

Adding milk replacer powder ( MRP ) to whole milk during the entire preweaning period can increase growth but raises concern because of low starter feed intake and slumps in average daily gain ( ADG ) at weaning and postweaning. In the current study, effects of adding MRP to pasteurized whole milk ( PWM ) during d 10–41 or d 10–59 of age were investigated in comparison with PWM. Calves [24 females and 21 males; 39.8 ± 1.85 kg body weight ( BW )] were randomly allocated to 1 of 3 treatments: 1) conventional protocol, 5 L/d PWM from d 3–56, and 2.5 L/d from d 57–59 of age ( CONV ; TS intake = 31.9 kg), 2) short duration of adding MRP to PWM protocol, 5 L/d PWM from d 3–9, 5 L/d PWM + MRP (18% TS) from d 10–41, 5 L/d PWM from d 42–56, and 2.5 L/d PWM from d 57–59 ( SD ; TS intake = 42.3 kg), 3) long duration of adding MRP to PWM protocol, 5 L/d PWM from d 3–9, 5 L/d PWM + MRP from d 10–56, 2.5 L/d PWM + MRP form d 57–59 ( LD ; TS intake = 47.7 kg). The osmolality of PWM and PWM + MRP was 278 and 519 mOsm/L, respectively. Calves were weaned on d 60, and the study terminated on d 75. There was a treatment × time interaction for starter intake, where intake was greater for CONV than other treatments from d 14–41 and was greater in CONV than LD during d 42–48 and d 56–62 of age. Final BW was lower in CONV calves than LD calves. Weaning BW and overall hip height were lower in CONV calves than other treatments. The CONV calves had lower ADG at d 14–27 and 35–41 and SD calves had lower ADG at d 42–48 than other treatments. Calves fed CONV treatment had lower ruminal acetate and greater propionate than SD calves during preweaning. Calves fed LD treatment had lower total volatile fatty acids and tended to have greater ruminal pH than other treatments. Calves fed CONV had greater neutrophils and neutrophils/ lymphocytes ratio and lower lymphocytes than other treatments. Glucose concentration was greater for LD versus other treatments at d 56, and lower for SD versus other treatments at d 70 of study. Insulin concentration and homeostatic model assessment of insulin resistance index were greater in LD compared with other treatments during preweaning but were not different postweaning. Serum BHB was greater in CONV than other treatments. Albumin was greater for CONV versus other treatments at d 56, however, it was greater in LD-fed calves at d 70 of study. Results indicate that feeding a PWM + MRP to the calves during the entire preweaning period resulted in lower starter feed intake around weaning, but overall starter intake was similar with a greater final BW and fewer health related issues throughout the study. Shifting a PWM + MRP to the conventional whole milk at d 40 of age decreased the ADG of calves.


INTRODUCTION
Research shows that early life nutrition is crucial for the survival and future productivity of replacement heifers (Soberon et al., 2012).Calves rely heavily on liquid feed for their nutrition during the pre-ruminant phase (Drackley, 2008) and generally are raised on either conventional (CONV) or higher amounts of liquid feeding programs (>900 g/d milk or MR solids; NASEM, 2021).In the CONV method, calves are fed a restricted amount of liquid feed (usually 8 to 10% of their birth BW, which translates to approximately 3 to 6 L/d) during the pre-weaning period to encourage them to start eating solid feed (Drackley, 2008).On the other hand, enhanced liquid feeding programs provide calves with more nutrition (usually 12 to 20% of their birth BW, which is greater than 8 L/d; NASEM, 2021) during early life than CONV programs, improving body and skeletal growth and overall wellbeing in calves compared with conventional limit feeding programs (Geiger et al., 2016;Schäff et al., 2016;Rosenberger et al., 2017).Furthermore, higher growth rate in response to intensive feeding of milk or milk replacer to neonatal calves could improve subsequent milk production (Soberon et al., 2012;Gelsinger et al., 2016).
However, intensive milk feeding programs delay starter diet intake, which can impair ruminal development and functionality during weaning and postweaning growth (Appleby et al., 2001;Jasper and Weary, 2002), leading to growth slumps after weaning.Gelsinger et al. (2016) reported a strong negative correlation between liquid and starter feed intakes (r = −0.82)because of limited capacity of calves for daily DMI.Also, in a meta-analysis study, Hu et al. (2020) reported that while feeding a higher plane of milk replacer can increase ADG and structural development in the pre-weaning period (d 0 to 56), it may have a negative impact on postweaning growth performance (d 56 to 112) due to reduced nutrient digestibility.
Although increasing liquid feed intake frequently decreases starter feed intake (Khan et al., 2007;Silper et al., 2014;Hu et al., 2020) in dairy calves, Azevedo et al. (2016a) indicated that offering a liquid feed with greater concentration of TS (up to 20.4%) increased ADG and skeletal growth without negative effect on starter feed intake during pre-and post-weaning periods.In a recent study, Shiasi Sardoabi et al. (2021) showed that offering liquid feed with 17% TS compared with 12% TS increased feed efficiency and improved skeletal growth of dairy calves.Despite positive effects when TS of liquid feed are increased, the final osmolality must be considered.McGuirk (2003) indicated that caution should be applied when the osmolality of offered liquid feeds was greater than 600 mOsm/L.An increase in liquid feed osmolality could lead to an increase in incidence of diarrhea in dairy calves due to greater water flux in the intestines (Glosson et al., 2015).Glosson et al. (2015) and Azevedo et al. (2016a) fed calves with diets containing 17.6 to 18% solids without issues related to osmolality, and so we chose to limit TS to 18% in this study.
Research has shown beneficial effects of increasing liquid feed TS on calf health, performance, and nonnutritive oral behaviors (Azevedo et al., 2016a); however, the optimum time of increasing liquid feed TS has not been defined.It is essential to find a balance between liquid feeding and starter diet intake to ensure the best outcomes for calves.To our knowledge, no study has investigated the effects of time of feeding liquid feed with higher TS concentration to calves.A shorter period of enhanced liquid feeding might lead to weaning at an earlier age, whereas a longer period of milk feeding would increase growth and starter intake with time and perhaps allow easier weaning.Because differences in growth are often accompanied by changes in blood immune cells and metabolites (e.g., de Paula et al., 2017;Orellana Rivas et al., 2020), it is important to measure those variables to provide a complete understanding of the effects of greater TS intake.
The current study aimed to compare the effects of increasing milk TS concentration (18 vs. 12%) by the addition of milk replacer powder (MRP) to pasteurized whole milk (PWM) in different periods with CONV milk feeding on the performance, rumen and blood parameters, and health of dairy calves.Our hypothesis was that feeding MRP + PWM during different periods would increase performance, alter metabolism, and improve health traits compared with the CONV method.Through this study, we attempted to determine the appropriate period to feed MRP + PWM, with the aim to promote ruminal development and performance of calves without a growth slump after weaning.

Animals, Management, and Experimental Treatments
The experiment was conducted on a commercial dairy farm (FKA Animal Husbandry and Agriculture Co.) located in Isfahan, Iran, from December 2020 to March 2021.The animals were cared for according to the guidelines of the Iranian Council of Animal Care (1995).In this farm, all Holstein calves were removed from their dams immediately after birth, and were weighed and placed in a clean individual pen (2 × 1.1 m) bedded with fresh wheat straw.Then calves were fed 6 L of good-quality maternal colostrum (Brix values ≥22%) in 2 equal meals using nipple bottles within 6 h of birth.Calves were screened for serum total protein concentration (6.4 ± 0.58 g/dL) as a condition for trial enrollment.Forty-five calves that appeared healthy and had a serum protein concentration >6 g/dL (24 females and 21 males; 39.8 ± 1.85 kg of BW) were included in the study.Based on our previously published study (Shiasi Sardoabi et al., 2021), a power test analysis with α = 0.05 and power (1 -β) = 0.80 gave an expected sample size of 15 calves per treatment for ADG, which was considered the most reliable variable for determining power.
After colostrum feeding, the calves were fed 4 L of transition milk on d 2 of life.From d 3 onward, calves were transferred to outdoor individual pens (2 × 1.1 m) bedded with sawdust.Calves were blocked by sex, balanced for BW, and randomly allocated to 1 of 3 dietary treatments (n = 15 calves, 8 female and 7 male calves per treatment; Figure 1).The 3 treatments were 1) conventional protocol, 0.575 kg/d PWM from d Pasteurized whole milk containing 3.27 ± 0.21% fat, 3.14 ± 0.11% CP, 4.79 ± 0.05% lactose, and 11.5 ± 0.21% TS was warmed to 39 ± 1.0°C using a water bath and provided to calves in steel buckets individually in 2 meals of equal volumes at 0800 and 1600 h (2.5 L each meal) from d 3 to 56, and 1 meal at 0800 h (2.5 L) from d 57 until weaning.Milk replacer powder used to increase milk TS content to 18% consisted of whey protein, dried skim milk, and vegetable fat (98% DM, 22% CP, 17% ether extract, 8% ash, 41.8% lactose, < 0.1% crude fiber, 20,000 mg/kg Ca, 10,000 mg/kg Mg, 25,000 mg/kg Na, and 9,000 mg/kg Cl; Novin Roshd Shahran Foudeh Co, Isfahan, Iran).The osmolality of the liquid feed was measured using an osmometer (OS-MOMAT 030, Gonotec, Germany).Calves were weaned on d 60, and the study was terminated on d 76 of age.
The starter diet was formulated for calves in the 60 to 70 kg BW range to achieve target ADG of 0.750 kg/d (Table 1) according to the National Research Council recommendations (NRC, 2001).A hammer mill with a 2-mm screen size (model 5543 GEN, Isfahan Dasht, Isfahan, Iran) was used to grind grains (corn and barley) included in the starter feed.Calves received a blend of starter feed plus 8% chopped second-cut alfalfa hay as a TMR throughout the study.Calves had free access to fresh water via individual nipple drinkers mounted on the side wall of the pens, and starter diet via individual steel buckets mounted on the outside throughout the experimental period.

Data Collection, Sampling, and Analyses
The offered starter feed and refusals were measured daily to determine starter feed intake.Starter feed was sampled monthly and kept frozen (−20°C) for subsequent analyses.Dry matter content of starter diet and refusals was determined after oven-drying samples for 48 h at 65°C, and then samples were ground through a 1-mm screen using a Wiley mill (Arthur Thomas Co., Philadelphia, PA).The ground samples were analyzed for N using Kjeldahl (method 988.05;AOAC, 1990), ether extract (method 920.39;AOAC, 1990), and ash (method 942.05;AOAC, 1990).The NDF and ADF contents were determined with the methods described by Van Soest et al. (1991) using heat stable α-amylase.Weekly samples of milk were preserved with potassium dichromate and analyzed for concentrations of fat, CP, lactose, and TS by Milkoscan (Foss Electric, Hillerød, Denmark).Calves were weighed at birth, and d 3, 13, 20, 27, 34, 41, 48, 55, 62, 69, and 76 of age before the morning milk feeding.Starter diet, total DMI (milk plus starter diet), ADG, and feed efficiency (FE; kg BW gain per kg of total DMI) were calculated throughout the study period (d 3 to 76 of age).Skeletal growth parameters including withers height, hip height, hip width, body length, heart girth, and belly girth were measured as described previously by Valehi et al. (2022) at d 3, 60, and 76 of age.Fecal score data were recorded daily before the morning milk feeding, on a scale of 0 to 3 according to the School of Veterinary Medicine calf health scoring chart, University of Wisconsin-Madison.Fecal scores were established as 0 = normal, 1 = semi-formed and/or pasty, 2 = loose but stays on top of bedding, and 3 = watery and/or sifts through the bedding.Rectal temperature was measured daily during the study using a thermometer (Qingdao Dacon Trading Co. Ltd., Shandong, China).All sick calves were diagnosed according to the standard operating procedures at the FKA Agriculture and Animal Husbandry Facility (Isfahan, Iran) and were treated with standard procedures prescribed by the veterinarian.
Ruminal fluid was sampled 4 h after the morning feeding with a stomach tube fitted to a vacuum pump on d 45 and 76 of age; the first 100 mL was discarded to avoid saliva contamination.The pH of the fresh ruminal fluid samples was measured immediately using a handheld pH meter (HI 8318, Hanna Instruments, Cluj-Napoca, Romania).The samples were squeezed through 2 layers of cheesecloth, and then 4 mL of the ruminal fluid was acidified with 1 mL of 25% metaphosphoric acid and stored (−20°C) until analysis for VFA by gas chromatography as described by Hashemzadeh-Cigari et al. (2014).For ammonia-N determination, a 2-mL subsample of filtered fluid was acidified with 2 mL of 0.2 mol/L HCl and frozen for subsequent analysis as described by Broderick and Kang (1980).
On d 63 of age, blood samples were obtained in tubes coated with EDTA/K3, thoroughly mixed several times, maintained on ice, and promptly transported to the laboratory for hematologic analysis (complete blood count; CBC).Some CBC indices, such as white blood cell count and differential white blood cell count, were measured using an automated hematology analyzer (Sysmex XN-1000, Japan).Moreover, neutrophils/ lymphocytes ratio was calculated as inflammatory indicators.
Blood samples were taken from the jugular vein on d 56 and 70 of life approximately 3 h after the morn-ing milk feeding and placed in 10 mL evacuated tubes coated with tripotassium EDTA for biochemical analysis.Following blood collection, samples were placed on ice and transported to the laboratory within 30 min, centrifuged (1,006 × g for 20 min at 4°C), and plasma samples were kept at −20°C until analysis.Concentrations of glucose, urea-N, total protein, albumin, cholesterol, triglyceride, creatinine, total bilirubin, aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), and alanine aminotransferase (ALT) were measured using an autoanalyzer (Abbott Alycon 300, USA) and commercial kits (Pars Azmoon Co., Tehran, Iran) according to the manufacturer's instructions.The autoanalyzer was calibrated before use with the control sera N and P (TrueLab N and TrueLab P, respectively; Pars Azmoon Co., Iran) and a calibrator solution (TrueCal U, Pars Azmoon Co., Iran).The BHB concentration of plasma was determined using the same autoanalyzer and a commercial colorimetric kit (Randox Laboratories Ltd., Ardmore, UK).Serum insulin concentration was measured using enzyme linked immunosorbent assay kit (Monobind Inc., CA, USA).Intra and inter-assay coefficients of variation for measuring insulin were 6.9 and 8.2%, respectively.Globulin concentrations were calculated by subtracting albumin content from total protein content.The Homeostasis Model Assessment (HOMA) index was determined by the formula: glucose (mmol/L) × insulin (μU/mL) as described by Matthews et al. (1985).

Statistical Analysis
Calf was the experimental unit.Before analysis, data were screened for normality using the UNIVARIATE procedure in SAS (SAS 9.2, SAS Institute Inc., Cary, NC).The data that were not normally distributed including starter diet intake, medication days, and disease incidence were transformed logarithmically.Starter feed intake and total DMI were determined daily and averaged weekly, and then starter, total DM and ME intakes, BW, ADG, feed efficiency, skeletal growth, ruminal fermentation parameters, blood metabolites, and rectal temperature data were analyzed using Proc MIXED in SAS (version 9.4, SAS Institute Inc., Cary, NC) as repeated measures, with period (7-d periods or sampling times) as the repeated variable.The model included the fixed effects of treatment, time, interaction between treatment and time, and sex, with the random effect of block.Initial BW and skeletal measurements served as covariates for their respective variables of interest.The data of blood hematological parameters, and medication days were analyzed using the above model without the effect of time.The effect of dietary treatment on the categorical data related to daily health scores was analyzed using the GLIMMIX procedure of SAS version 9.4.A covariance structure (unstructured, compound symmetry heterogeneous, autoregressive order 1, or ante-dependence order 1) was chosen based on the lowest Akaike information criterion and Bayesian information criterion indices.
Significance was declared at P < 0.05 and tendencies were considered when 0.05 < P < 0.10.

RESULTS
Starter DMI, TDMI, ADG, BW, FE, and structural growth results are shown in Table 2.As programed, calves consumed 30.9, 41.1, and 46.6 kg DM from milk through the CONV, SD, and LD treatments, respectively.Osmolality was 278 and 519 mOsm/L for the PWM and PWM + MRP, respectively.There were treatment × time interactions for starter feed intake (P < 0.01 Figure 2A) and TDMI (P < 0.01; Figure 2B) due to greater starter feed intake and lower TDMI for calves receiving CONV versus other treatments during the preweaning period.Furthermore, the interaction of treatment × time was significant for total ME intake, and this interaction was due to greater ME intake for SD calves during d 14-35 and for LD calves during d 14-49 versus CONV calves (P < 0.01, Figure 2C).Also, the treatment × time interaction for total CP intake was significant, where CONV calves had lower CP intake during d 3-41 than other treatments (P < 0.01, Figure 2D).
Final BW was lower in calves receiving milk through CONV treatment than in those on the LD treatment, and SD treatment was intermediate (P = 0.04, Figure 3A).Weaning BW was lower in CONV calves than other treatments (P < 0.01, Figure 3A).Also, there was interaction between treatment × time for ADG (P < 0.01; Figure 3B), whereby the CONV calves had lower ADG at d 14-27 and 35-48 than other treatments and SD calves had lower ADG at d 42-48 than other treatments.Furthermore, treatment × time interaction was significant for FE (P = 0.01; Figure 3C), where FE was lower at d 14-20 for CONV treatment compared with SD and LD treatments, but SD calves had lower FE during d 42-48 than other treatments.As expected, calf intake and growth increased but FE decreased with calf age.
The treatments had no effect on withers height, hip width, and belly girth; however, hip height was lower in CONV calves than other treatments (P < 0.01).Also, body length was greater in LD calves than other treatments (P = 0.02) and LD calves had greater heart girth than CONV calves with the SD treatment intermediate for this trait (P < 0.01).As expected, skeletal growth parameters increased as calves aged (P < 0.01).However, no interactions were observed for treatment × time (P > 0.10) with respect to skeletal growth measurements.
There was a treatment × time interaction regarding cough score (Table 3; P < 0.01), and this difference was mainly due to greater cough score for calves receiving CONV versus other treatments during the postweaning period.Also, the interaction between treatment × time tended to be significant for nasal discharge score (P = 0.06); all groups had similar nasal discharge score at the preweaning period, but LD calves had lower nasal discharge score at postweaning period.Additionally, the interaction between treatment × time for eye score tended to be significant (P = 0.08) and for ear score was significant (P = 0.02), because the eye and ear scores were lower for LD-fed calves during the preweaning period than other treatments.Furthermore, CONV-treated calves had a slightly lower daily rectal temperature than other treatments (P < 0.01).Fecal score was not affected by treatments.
The effects of treatments on ruminal fermentation are shown in Table 4.There were interactions between treatment × time for acetate (P = 0.04; Figure 5A), propionate (P = 0.03; Figure 5B) and acetate to propionate ratio (P = 0.03; Figure 5C).Calves fed CONV treatment had lower acetate and acetate to propionate ratio and greater propionate than SD calves only during the preweaning period, but not at postweaning period.Calves fed LD treatment had lower total VFA (P = 0.03) and tended to have greater ruminal pH (P = 0.08) than other treatments.As calves aged, the ruminal pH, NH 3 -N, isobutyrate, butyrate, and isovaleric proportions were decreased, while total VFA and propionate concentrations were increased.
The effect of treatments on complete blood count is reported in Table 5. Calves fed CONV had greater neutrophils (P = 0.01) and neutrophils/lymphocytes ratio (P = 0.02) and lower lymphocytes (P < 0.01) than other treatments.
The treatment × time interaction was significant for albumin (P < 0.01; Figure 4A) as follows: albumin differed among all treatments where LD > SD > CONV at d 70 of age, and was greater for CONV versus other treatments at d 56 of age.The treatment × time interaction effect was significant for globulin (Figure 4B) and A/G ratio (Figure 4C) due to lower globulin and higher A/G ratio in LD-fed calves at d 70 of age.There was an interaction between treatment × time where the LD group had lower AST (Figure 4D) than CONV calves at d 70 but not at d 56.The glucose concentration was greater for LD versus SD treatment (Table 6; P = 0.08).Total serum protein, urea-N and HOMA index were greater in LD treatment than other groups, and BHB was greater in CONV versus other treat- ments.Moreover, SD-fed calves had lower cholesterol concentration than other treatments.As calves aged, HOMA index and cholesterol concentrations decreased and GGT and creatinine tended to decrease while BHB, urea-N, total protein, and ALT concentrations were increased.

DISCUSSION
During the preweaning period, calves receive most of their nutrients from milk or MR; therefore, increasing the allowance of liquid feed or the concentration of TS in liquid feed to calves would be an effective strategy to increase available nutrients for growth (Silber et al., 2014).However, information regarding accelerated milk feeding programs at different periods during the preweaning period is scarce.There are concerns with feeding dairy calves with high TS milk during the entire preweaning period because of low starter feed intake and slumps in ADG at weaning and postweaning periods.In the current study, the effects of adding MRP to PWM during d 10 to 41 or d 10 to 59 were investigated in dairy calves.This study aimed to determine the optimal time period for accelerated milk feeding to maximize calf growth and development.
The starter feed intake was greater in calves receiving CONV until d 42 of age but similar among treatments during the postweaning period.In agreement with our finding, Hu et al. (2020) in a meta-analysis study reported that feeding calves with higher amounts of MR led to lower starter feed intake during the preweaning period and similar starter feed intake during the postweaning period.Also, Shiasi Sardoabi et al. ( 2021) showed that feeding milk with higher TS resulted in lower starter feed intake during preweaning period but similar intake during the postweaning period.The low- er starter feed intake during preweaning in calves that received LD and SD treatments probably was related to meeting most of their nutrient requirements due to ingestion of high TS by the addition of MRP to PWM, leading to lower appetite for starter feed intake (Khan et al., 2011).In our experiment, LD-fed calves had lower starter feed intake than other treatments from d 56 to 62 of age.These results indicated that the negative effects of feeding PWM + MRP on starter feed intake were observed only at the first week after weaning compared with CONV and SD treatments.In our experiment the weaning period of LD calves lasted only 3 d and it could be concluded that calves fed PWM + MRP need more time for weaning.However, starter feed intake was similar between treatments from d 63 to 76 of age.This means that despite lower starter feed intake of LD and SD -fed calves during the preweaning period, they increased starter feed intake after weaning, which allowed them to compensate for the lower starter feed intake early in life.Shiasi Sardoabi et al. ( 2021) in an experiment with a 14-d weaning period reported similar starter feed intake at first week after weaning in calves fed milk with 12 or 17% TS during preweaning period.
Interestingly, SD calves increased starter feed intake and decreased TDMI, ADG, and FE at d 42 to 48 of age after milk TS was decreased from 18.5 to 12% at d 41.These results indicated that calves experienced a situation similar to abrupt weaning and they need a longer time, similar to an extended gradual weaning protocol, to help them to adapt and successfully transition from PWM + MRP to PWM without any negative effect on performance.
As programmed, liquid feed solids intake during the preweaning period was in the following order: LD > SD > CONV with means of 46.5 kg, 40.0 kg, and 30.4 kg, respectively.In contrast to starter feed intake and due to greater milk TS intake, TDMI was lower in CONV calves than other treatments during the first 42 d of age.In agreement with our finding, greater TDMI was reported in calves fed a higher amount of liquid feed with greater TS during the preweaning period (Azevedo et al. 2016a,b).Despite the equal intake of starter feed and TDMI, ME and CP intakes were greater in LD and SD calves than CONV calves due to greater TS intake by the addition of MRP to PWM during preweaning period.This result indicated that LD and SD calves consumed sufficient starter feed after weaning to compensate for the loss of energy from the milk.In agreement with our finding, calves fed milk with high TS (18%) had greater ME intake than calves fed milk with normal (12%) TS (Shiasi Sardoabi et al., 2021).
Before weaning, the most important factor in ADG is the amount of TS in liquid feed that is consumed by calves.Body weight and hip height were affected by treatments, so that calves that received PWM + MRP with 18% TS had greater BW and hip height than those fed CONV treatment.These results are in good agreement with other researchers, who reported that BW and some skeletal growth parameters were enhanced in response to increasing TS concentration in liquid feed (Azevedo et al., 2016ab).Because similar growth responses during postweaning were observed between calves fed different treatments, our results indicated that growth promotion during the preweaning period could improve growth measurements during the postweaning period.Higher growth rate during liquid feeding period in dairy calves could improve subsequent milk production (Gelsinger et al., 2016).
Calves fed CONV had lower FE from d 14-20 compared with other calves.These results are probably related to the higher digestibility of nutrients from milk in comparison to starter feed and was in agreement with other reports (Dennis et al., 2018).
During the current experiment, mean rectal temperature of calves was 38.45°C, which is in the normal range of body temperature (Collier et al., 2019).In previous studies negative effects of intensive liquid feeding programs on calf health were reported (Quigley et al., 2006;Orellana Rivas et al., 2020).Also, Shiasi Sardoabi et al. ( 2021) noted increasing liquid feed TS concentration resulted in higher fecal score and diarrhea occurrence from d 4 to 18 d of age.Khan et al. (2011) concluded that increased milk consumption was unlikely to have a specific immunomodulatory effect and that the effect depended on total nutrient availability.Indeed, there is good evidence linking impaired immune function directly to reduced nutrient availability.Therefore, calves with higher nutrient intake are likely to have greater immune performance.This is supported by some studies showing an immunomodulatory effect of low-milk feeding (Hulbert et al., 2011;Ollivett et al., 2012).Means within a row with different non-capital superscript letters significantly differ (P < 0.05) and with different capital superscript letters tend to significantly differ (0.05 < P < 0.10).However, in our experiment, cough score and nasal discharge score decreased in calves fed PWM + MRP, indicating that calves' health status improved.In agreement with our results, other studies reported no difference in diarrhea occurrence in calves fed higher levels of liquid feed compared with restricted fed calves (Jasper et al., 2002;Khan et al., 2007;Valehi et al., 2022).The higher rectal temperature without any effects on fecal score in calves fed PWM + MRP might be related to digestion and metabolism of greater TS consumed.In agreement with our results, the fecal score of dairy calves was not affected when they were fed liquid feed with osmolality between 265 to 533 mOsm/L by increasing TS concentrations from 13.5 to 20.4%, respectively (Azevedo et al., 2016a).The observed results might be related to osmolality of MRP + PWM lower than 600 mOsm/L, the level above which should be offered with caution, as reported by McGuirk (2003).
In the present study, SD-fed calves had lower propionate and greater acetate proportion than CONV calves during preweaning period.In agreement with our finding, Shiasi Sardoabi et al. (2021) reported that calves fed 17% TS liquid feed had greater acetate proportion than those fed liquid feed with 12% TS.We do not have an explanation for this change in acetate concentration between treatments during preweaning.Moreover, ruminal total VFA concentration decreased while ruminal pH increased in LD-fed calves compared with CONV-fed calves.Calves fed LD had lower starter feed intake during d 42-48 and 56-62 of study, resulting in lower total VFA production and hence greater ruminal pH.Calves fed SD had ruminal total VFA and pH similar to CONV calves, indicating that duration of receiving PWM + MRP could affect ruminal fermentation and feeding PWM + MRP for a short time had no effect on ruminal characteristics.In contrast with our results, Azevedo et al. (2016b) showed that rumi- nal pH and total VFA concentration were not affected by liquid feed TS concentrations.Ruminal total VFA and propionate concentrations increased, while ruminal pH, NH 3 , acetate, butyrate, and isobutyrate concentrations decreased as calves aged, which could be related to higher starter feed intake of calves over time and gastrointestinal tract development (Vi et al., 2004;Hill et al., 2016).
Calves fed CONV had greater neutrophil count and neutrophils/lymphocytes ratio and lower lymphocytes count than other calves.Neutrophils are the first cellular defense line against pathogens and increase in stress conditions (Lynch et al., 2010).Critical functions of lymphocytes that are altered during the neonatal period in calves include, among others, activation capacity, cytokine production, and antibody production (Cuervo et al., 2021), and a decreased lymphocyte count will reduce the animals' ability to resist inflammation (Guan et al., 2020).Moreover, blood neutrophils/lymphocytes ratio has been used as one of the indicators to evaluate the body's immune system, a strong prognostic factor of certain cancers, and as a predictor of inflammatory or infectious lesions and postoperative complications (Guan et al., 2020).These results, together, indicate that CONV-fed calves had a lower immune status, and feeding PWM + MRP in different duration preweaning may strengthen immune function in calves.
In the present study, glucose concentration was greater for LD versus other treatments at d 56, and lower for SD versus other treatments at d 70 of study.Higher plasma glucose concentration in LD calves at d 56 was consistent with previous studies (Yunta et al., 2015;Mirzaei et al., 2018).Higher levels of lactose intake are likely able to increase glucose concentration of plasma (Palmquist et al., 1992).At d 56 of study, LD calves were fed greater amounts of TS by the addition of MRP to PWM than CONV and SD calves, resulting in greater concentration of plasma glucose.With advancing age, calves experience a normal physiological shift in the energy source from glucose to VFA metabolism, which is triggered by a transition from the liquid feed (milk or MR) to solid feed and increased microbial fermentation of carbohydrates, thus contributing to the initiation of a metabolically functional ruminal fermentation.These events may help explain the decreasing blood concentration of glucose with calf age (Daniels et al., 2008;Terré et al., 2015).However, an explanation for the lower glucose concentration of SD calves at d 70 is difficult, because intake of starter feed was similar between treatments at this time.
At 56 d of age, LD calves needed to release more insulin to control glycemia, but at 70 d of age, the insulin concentration was similar among treatments.In agreement with our finding, calves fed greater MR had greater insulin concentration during preweaning (Yunta Means within a row with different non-capital superscript letters significantly differ (P < 0.05) and with different capital superscript letters tend to significantly differ (0.05 < P < 0.10)., 2015).The HOMA index is used in animals as an index of insulin resistance, and higher HOMA values are indicative of decreased insulin sensitivity (Guyot et al., 2017).The HOMA index directly reflects the insulin and glucose concentrations with higher values in LD-fed calves.In the current experiment, greater insulin concentration and HOMA index during preweaning period are indicative of increased insulin resistance in LD-fed calves.Yunta et al. (2015) reported when calves were fed 8 L/d of MR, insulin sensitivity tended to decrease in comparison with calves fed 4 L/d.Yunta et al. (2015) and our results indicated that the differences in insulin sensitivity disappeared as calves became older.
As calves aged, concentrations of plasma BHB, a marker of rumen epithelial development (ruminal ketogenesis), increased as a result of increased starter feed intake (De Paula et al., 2017).In line with previous studies (De Paula et al., 2017;Jafari et al., 2021), higher plasma BHB concentration was found in CONV-fed calves, which is an indicator of a more highly developed rumen.In the current experiment, the difference between BHB concentration between treatments was due to differences in the preweaning stage.Independent of treatments, plasma BHB concentration increased about 40% from d 56 to 70 in calves, indicating similar ruminal development.
In this study, lower urea-N concentration for LD-fed calves compared with CONV and SD fed calves were probably due to lower starter feed intake during the first week after weaning.Schäff et al. (2016) and Jafari et al. (2021) reported a decrease in urea-N concentration for calves fed higher MR or milk compared with CONV-fed calves.In contrast to our finding, Quigley et al. (2006) andTerre et al. (2006) reported greater urea-N concentration in calves fed high TS MR, which suggests that a portion of the added CP in the MR consumed by calves was used for energy with subsequent deamination and increased urea N concentration (Terre et al., 2006;Quigley et al., 2006).Our results indicate that CP from added MRP to PWM was not deaminated for producing energy and probably was used for skeletal growth as evidenced by greater hip height, body length, and hearth girth in LD-fed calves.Increasing urea-N concentration after weaning in all groups was likely due to ruminal fermentation of starter feed protein and absorption of ammonia from the rumen (Quigley et al., 2006).
Albumin is a negative acute-phase protein and a decrease in blood concentration can result from reduced liver synthesis due to inflammatory status (Bertoni et al., 2008).Globulin includes the main positive acutephase proteins and the blood concentrations of globulin increase due to inflammatory conditions (Ceciliani et al., 2012).Blood globulin and albumin concentrations have been considered as biomarkers for inflammation in dairy cows (Cattaneo et al., 2021).Cattaneo et al. (2021) reported that cows with high albumin to globulin ratio before dry-off showed an improved adaptation to the new lactation, as demonstrated by a reduced systemic inflammatory response and increased milk yield compared with cows with low albumin to globulin ratio.Calves fed LD had lower globulin and higher albumin concentrations on d 70 of study, resulting in higher albumin to globulin ratio than with the other treatments.Jafari et al. (2021) observed an equal concentration of total plasma protein and greater albumin concentration in calves that were fed greater amount of milk than CONV-fed calves.Moreover, AST activity in the serum is a sensitive marker of liver damage, and increasing AST activities in dairy cows is most often connected with low appetite, fatty liver, and ketosis syndrome (Du et al., 2017).The AST concentration was lower for LDfed calves during postweaning, indicating greater liver health.These results, together, indicate that LD-fed calves had lower inflammatory state and greater liver health, suggesting that feeding PWM + MRP during a longer time preweaning decreased systemic inflammation and increased liver health during postweaning.Limitations of our study include very short weaning process and the lack of behavior evaluation.

CONCLUSIONS
Overall, the study highlights the importance of optimizing milk feeding strategies during the preweaning period to improve calf growth and development.Our findings suggest that increasing milk TS by the addition of MRP to PWM during specific time periods can lead to significant improvements in calf growth, without negatively affecting weaning or calf health.In our experiment, feeding PWM + MRP to the calves during the entire preweaning period resulted in greater performance than feeding PWM + MRP during d 10 to 41.Further research is needed to determine the optimal milk feeding strategies for different times preweaning in dairy calves and to investigate the long-term effects of accelerated milk feeding on cow milk production.

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Table 1 .
Ingredients and chemical composition of starter diet

Table 3 .
Effects of milk replacer powder (MRP) added to pasteurized whole milk (PWM) over different durations on health indices in Holstein calves (n = 15 per treatment) 2 Tr = treatment effect; Per: time effect; Tr × Per = the interaction between treatment and time.

Table 4 .
Effects of milk replacer powder (MRP) added to pasteurized whole milk (PWM) over different durations on ruminal fermentation variables in Holstein calves (n = 15 per treatment)

Table 5 .
Effects of milk replacer powder (MRP) added to pasteurized whole milk (PWM) over different durations on complete blood count in Holstein calves (n = 15 per treatment)

Table 6 .
Fouladi et al.: Effects of milk replacer… Effects of milk replacer powder (MRP) added to pasteurized whole milk (PWM) over different durations on blood parameters in Holstein calves (n = 15 per treatment)