Feeding an amino acid formulated milk replacer for Holstein calves

Abstract

The milk-fed calf has a requirement for amino acids (AA) instead of crude protein (CP); however, most milk replacers (MR) are still formulated for CP concentrations. Previous work has demonstrated that feeding a modified MR (24:20; CP:fat @ 0.64 kg/d) improved calf growth performance compared with standard (20:20 @ 0.57 kg/d) and accelerated MR (26:16 @ 0.78 kg/d). The 56-d study objective was to determine if feeding an MR formulated using synthetic AA to achieve the AA concentrations of a 24:20 MR while reducing CP results in similar or enhanced growth performance and/or reduce cost compared with standard MR formulations. Eighty 3- to 5-d-old Holstein bull calves received in two lots (40) within the same week were blocked by body weight (BW) and randomly assigned to one of four MR treatments consisting of 20:20 (20), 22:20 (22), 24:20 (24), and a 22:20 having the AA concentrations of the 24, but with reduced CP (22AA). All MR contain decoquinate and were fed at 0.57 kg/calf daily split into 2×/d feeding for 14 d via bucket, increased to 0.85 kg/calf daily in two feedings until 35 d, and then fed 1×/d at 0.41 kg/calf daily with weaning after day 42. Calves were housed in straw-bedded hutches with ad libitum access to water and pelleted calf starter (CS). All data were statistically analyzed as a randomized complete block design with block considered random with week as a repeated measurement. Initial BW was similar (P > 0.10) across all treatments (42.4 ± 2.2 kg). Calves fed 22AA MR demonstrated greater (P < 0.05) BW compared with calves fed the 24 MR, and calves fed the 20 and 22 MR being intermediate and similar (P > 0.10; 78.7, 78.8, 76.5, and 81.8 kg for 20, 22, 24, and 22AA, respectively). CS intake was greater (P < 0.05) for calves fed 22AA (0.74, 0.78, 0.65, and 0.81 kg/d) compared with calves fed the 20 and 24, but similar (P > 0.10) to calves fed 22. Calves fed 24 MR demonstrated the lowest CS intake. This study demonstrates that similar growth performance can be achieved by feeding an AA fortified MR having a lesser CP concentration, which might reduce feed costs.

Introduction

The goal of a successful calf-rearing program is to achieve optimal growth performance at an economical cost to elicit the maximum return on investment while achieving improved lactational performance upon calving (Trinacty et al., 2006; Huuskonen, 2017). The formulation of all milk replacers (MR) has changed in recent decades by reducing the use of skim milk and/or casein to utilizing more whey protein concentrates, whey protein isolates, and delactosed whey (Davis and Drackley, 1998). However, the increasing human demand for whey protein has resulted in whey proteins becoming more expensive (Morrison et al., 2017). Therefore, the development of new approaches to MR formulation is needed for the future calf MR production (Kertz et al., 2017).

The calf has a metabolic requirement for amino acids (AA) instead of crude protein (CP) (NRC, 2001) even though most commercial MR formulations are still based on a CP concentration. The Dairy NRC (2001) calculated the requirement of apparent digestible protein for calves using a model for predicting protein-limited and energy-limited growth performance. Nevertheless, the Dairy NRC (2001) calf model does not include an AA model for predicting requirements. Prior research work (Strayer, 2014) based on CP concentrations demonstrated that newborn calves fed a modified accelerated calf feeding program, that is, 24:20 (CP:fat) MR, resulted in improved calf performance compared with calves fed other CP concentrations. Those studies, while optimizing the CP energy (fat) ratio, did not consider AA concentrations.

AA play an indispensable role in improving protein utilization efficiency to increase protein deposition (Yasuhiko, 2004). Several experiments have attempted to define an optimal AA concentration for calves (Klemesrud et al., 2000; Hill et al., 2007, 2008a). Calves fed an MR with Met and Lys demonstrated greater BW and improved ADG compared with calves fed an MR without these AA (Jenkins and Emmons, 1983; Kanjanapruthipong, 1998). Hill et al. (2008a) feeding a 26% CP MR reported that 2.34% lysine and 0.72% methionine was optimal while observing no benefit to adding synthetic threonine.

If the calf has an AA requirement, then meeting the AA concentrations of a 24 CP MR should be possible with the use of synthetic AA, while allowing for a reduction in CP concentration and thereby the ingredient cost. The synthetic AA Met, Lys, Thr, Trp, and Val are commercially readily available at economical prices. In theory, the same growth performance should be achieved, but at a reduced cost to the producer.

The study objectives were to determine the growth performance of calves fed commercially available standard MR of 20%, 22%, and 24% CP compared with calves fed an AA formulated MR (22AA). Thus, the hypothesis is that formulating an MR to specific AA concentrations while allowing a CP reduction should result in similar or improved growth performance with a lesser feed cost, that is, 22AA compared with 20%, 22%, and 24% CP.

Materials and Methods

Calf management and feeding

This experiment was conducted at the Casper’s Calf Ranch (Freeport, IL) from September 11 through November 8, 2017. The calves were managed and fed according to the guidelines published in the Care and Use of Agricultural Animals in Research and Teaching (FASS, 2010), as well as the Chinese Standards for the Use and Care of Research Animals (He et al., 2016).

Three to 5-d-old Holstein bull calves were sourced from a commercial calf buyer (Reynolds livestock, Dodgeville, WI) and delivered in two lots of 40 calves during the same week (i.e., Tuesday and Thursday night). Calves were known to be purchased through several livestock auction barns and represented commingled calves from numerous dairy farms. Calves were assumed to have been fed colostrum at the dairy farms. However, a blood sample for the measurement of total serum protein was not collected due to the concern of transportation stress and dehydration biasing the results (Guzelbektes et al., 2007). Calves were vaccinated upon arrival with Inforce 3 (Zoetis Inc. Florham Park, NJ) and placed in a straw-bedded calf hutch (Calf-Tel Deluxe II, Hampel, Germantown, WI) measuring 220 × 122 × 38 cm that was placed on a grass pasture in an open naturally well-ventilated area. Hutches were spaced 0.6 m apart in rows of 10. Each hutch had a 183 cm × 122 cm × 107 cm wire panel attached to the front with two bucket holders (0.65 m high) for 8 liters plastic buckets. One bucket contained ad libitum freshwater and the other bucket contained a pelleted calf starter (CS). The water bucket was emptied at each feeding and filled with the appropriate experimental MR at the designated feeding rate. Upon completion of milk consumption, the bucket was rinsed and filled with fresh water. Calves were fed the control 20 MR upon evening arrival and the next morning. After the morning feeding, calves were weighed and frame measurements collected. Calves were then blocked by BW and randomly assigned to one of four treatments. Treatments were: 1) a standard commercially available 20% CP MR (20); 2) a standard commercially available 22% CP (22); 3) a standard commercially available 24% CP MR (24); and 4) the AA formulated MR having the AA concentrations of a 24 but the CP reduced to 22% (22AA). All MR contained 20% fat and decoquinate (Zoetis, Inc.) was added to the MR at 41.7 mg/kg (as-is basis) for coccidiosis control. The ingredient composition of the experimental MR are given in Table 1.

Table 1.

Ingredient composition of MR containing 20% (20), 22% (22), and 24% CP (24), or AA concentration of the 24, but 22% CP (22AA)

	MR CP, %
Ingredient	20	22	24	22AA
Fat base	36.6	35.7	35.4	35.7
Whey protein	60.1	61.0	61.5	60.8
Drugs & additives	1.59	1.59	1.59	1.59
Minerals & vitamins	1.51	1.51	1.51	1.51
Lysine hydrochloride	0.10	0.10	—	0.25
dl-Methionine	0.10	0.10	0.10	0.20
Retail cost, US $/ton	2,506	2,638	2,810	2,520

The MR feeding time was fixed at 6:30 a.m. and 6:00 p.m. and the feeding sequence was kept consistent. Each MR was weighed (Model ACE110, Smart Weigh Inc., Hurricane, WV) and mixed with the appropriate amount of 46 °C hot water using an Urban milk shuttle (Urban GmbH & Co., Hamburg, Germany). The shuttle with its computerized mixing, heating, and delivery systems was calibrated to ensure an accurate delivery of a homogeneously mixed MR being fed at a temperature of 37.5 °C or greater at the correct volume. Each experimental MR was fed at the rate of 0.57 kg/calf daily for the first 14 d, then increased to 0.85 kg/calf daily divided into two equal feedings (i.e., morning and evening). For 5 d during week 5 (days 30 to 35), Amprolium (Merial, LLC., Duluth, GA) was added at the rate of 10 mg/kg BW to the experimental MR for coccidiosis control. Starting on day 36 through day 42, the MR feeding rate was decreased to 0.41 kg/calf daily fed 1×/d at the morning feeding to facilitate weaning. All experimental MR were fed at 15% solids and if any calf did not consume its milk, the refused volume was recorded. Starting on day 1, a 2.4-mm mini-pellet pelleted 22% CP CS (as-is basis; Table 2) and water were offered ad libitum throughout the study. All experimental MR and CS were sourced from Furst-Mcness (Freeport, IL) and manufactured in sufficient quantities at one time to complete the study using the same lots of ingredients (Tables 1 and 2). At the completion of the 56-d experimental period, calves were then dehorned via hot iron and castrated using a scalpel to remove the scrotum bottom and physically removing the testes by a Veterinarian (Dr. Bardon Scharping) and then sold through a commercial livestock sale barn (Equity Livestock, Monroe, WI).

Table 2.

Ingredient composition of pelleted CS

Ingredient	% of mix
Wheat midds	35.7
Soybean meal, 48%	29.7
Corn, ground	22.5
Molasses	5.50
Calcium carbonate	2.25
Wheat flour	1.25
Salt	1.10
Ameri-Bond 2x lignin	0.50
Soy oil	0.43
Bovine B premix1	0.25
Vitamin A premix2	0.18
Vitamin E premix3	0.16
Yeast, CitriStim4	0.15
Trace mineral premix5	0.13
Vitamin D premix6	0.07
Cherry flavor	0.05
Rumensin 198 g/kg	0.02
Selenium 0.06%	0.02

¹Bovine B contains biotin 10.5 mg/kg, choline 167.6 g/kg, folic acid 50.3 mg/kg, niacin 94 g/kg, pantothenic acid 10.5 g/kg, pyridoxine 4.4 mg/kg, riboflavin 4.4 g/kg, thiamine 1.1 g/kg, and vitamin B12 19.8 mg/kg.

²Vitamin A premix contains 11,050,072 IU/kg.

³Vitamin E premix contains 44,750 IU/kg.

⁴CitriStim is Pichia guilliermondii yeast, ADM Animal Nutrition, Decatur, IL.

⁵Trace mineral premix contains cobalt 1,350 mg/kg, copper 23,500 mg/kg, iodine 2,000 mg/kg, manganese 100,000 mg/kg, selenium 510 mg/kg, and zinc 125,000 mg/kg.

⁶Vitamin D premix contains 8,375,055 IU/kg.

Feed intake and analysis

Starting on day 1, the amounts of CS offered and refusal weights were recorded daily using a digital scale (Model ACE110, Smart Weigh Inc., Hurricane, WV). In the case of those days when feed was wet, that is, rain, that day’s data were eliminated and the remaining days during that week were compiled into weekly means of CS intake. Samples of each experimental MR and CS were collected weekly and stored frozen at—20 °C until composited at the end of the study. Samples collected during weeks 1, 2, and 3 or weeks 4, 5, and 6 were composited into two samples for each experimental MR, while CS samples collected during weeks 1, 2, 3, and 4 or weeks 5, 6, 7, and 8 were composited into two lots. Samples were submitted to Dairyland Laboratories (Arcadia, WI) for nutrient analyses. Samples of MR were analyzed using the following AOAC International (2016) methods for dry matter (DM; 930.15), CP (990.03), acid hydrolysis fat (954.02), neutral detergent fiber (NDF) (Van Soest et al., 1991), ash (942.05), Ca (985.01), P (985.01), Mg (985.01), K (985.01), S (923.01), Na (985.01), Cu (985.01), Fe (985.01), Mn (985.01), and Zn (985.01). CS samples were analyzed for DM (930.15), CP (990.03), soluble protein (Krishnamoorthy et al., 1982), NDF (Van Soest et al., 1991), ADF, acid detergent fiber (ADF) (973.18), lignin (973.18), NDF insoluble protein (2002.04 without sulfite and 976.06), ADF insoluble protein (973.18 and 976.06), sugar (Dubois et al., 1956), starch (Hall, 2009), fat (2003.05), ash (942.05), Ca (985.01), P (985.01), Mg (985.01), K (985.01), S (923.01), Na (985.01), Cu (985.01), Fe (985.01), Mn (985.01), and Zn (985.01). Chloride for both MR and CS samples were extracted with 0.5% nitric acid and analyzed by potentiometric titration with silver nitrate (Metrohm 848 Titrino Plus, Metrohm, Riverview, FL). Nonfiber carbohydrates (NFC) were calculated using the equation of: NFC = [100 – ((NDF – NDF-insoluble protein) + CP + fat + ash)]. Samples of experimental MR and CS were sent to the Agricultural Experiment Station Chemical Laboratories at the University of Missouri, Columbia for AA analysis (AOAC, 2016, method 982.30E). For all AA, except cystine, methionine, and tryptophan, a test portion was hydrolyzed in 6 M HCl at 110 °C in an atmosphere free of oxygen for 24 h. The hydrolyzate was filtered to remove carbon and brought to a specified volume. An aliquot of the filtrate was evaporated to dryness under vacuum. The AA residue was dissolved in a buffer and injected into an AA analyzer. For cystine and methionine, a test portion was mixed with cold performic acid and allowed to stand overnight at 0 to 5 °C to convert methionine to methionine sulfone and cysteine/cystine to cysteic acid. The performic acid mixture was then neutralized with cold hydrobromic acid and evaporated to dryness under vacuum. The residue was then hydrolyzed in acid as described previously. For tryptophan, a test portion was hydrolyzed in 4 M NaOH under vacuum for 22 h at 110 °C. The hydrolyzate was neutralized with HCl and diluted to a specified volume using a buffer. Aliquots of the prepared hydrolyzates were injected onto an AA analyzer (Hitachi Model 8900, Hitachi, Schaumberg, IL) that used the classical ion-exchange resolution and ninhydrin post-column derivatization method developed by Moore and Stein (1958).

Body and health measurements

Body weight (BW) was measured weekly using a Wrangler Jr. digital scale (Digi-Star, LLC, Fort Atkinson, WI) placed on a 1.2 × 2.4 m sheet of a 1.9-cm-thick sheet of plywood towed by a John Deere 825 Gator (John Deere, Moline, IL). BWs were taken after the morning feeding starting at approximately 9:00 a.m. each week. Hip height (HH) and withers height (WH) were measured using a Ketchum Teletape having a level mounted on top (Ketchum Manufacturing Inc., Brockville, ON, Canada); body length (BL) and heart girth (HG) were measured using a Nasco dairy calf weigh tape (Nasco, Fort Atkinson, WI). Body measurements were taken at the same time as BW, but only every 4 wk (i.e., weeks 0, 4, and 8).

Calf health along with fecal, nasal, and ear/eye scores were monitored daily during the study. Health scores were visually assessed before the evening feeding according to the University of Wisconsin Calf Health Scoring Chart (McGuirk, 2013) and were based on a scale of 0 to 3. 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. Nasal scores were 0 = normal serous discharge, 1 = small amount of unilateral cloudy discharge, 2 = bilateral and/or cloudy or excessive mucus discharge, and 3 = copious bilateral mucopurulent discharge. Eye/ear scores were 0 = normal, 1 = small amount of ocular discharge with ear flick or head shake, 2 = moderate amount of bilateral ocular discharge and/or slight unilateral droop, and 3 = heavy ocular discharge and/or head tilt or bilateral droop. In cases of illness, body temperature was measured using a rectal thermometer (Zoe+Ruth, Portland, OR) and appropriate medical treatments as prescribed by a licensed Veterinarian (Dr. David Jeans, Monroe, WI or Dr. Brandon Scharping, Lena, IL) were administered, if needed. All health incidents and treatments were recorded for the length of the study.

Economic analyses

The time point of March 2019 was selected to calculate the cost to raise the calves per kilogram of BW gain. Retail prices of MR and CS were based on the company’s price list to the customer and did not include freight costs. Data on BW gain, MR intake, and CS intake were used with retail product pricing to calculate the feed cost per kg of BW gain. MR prices were US$2.76, US$2.91, US$3.10, and US$2.78 per kilogram, while CS was US$0.43 per kilogram. Per the study’s hypothesis, using synthetic AA to meet the AA specifications in the MR formulation while being able to reduce CP concentration reduced ingredient cost resulting in a more economically priced MR that meets the AA concentrations of a 24% CP MR, that is, US$70.25 vs. US$63.00 for 24 and 22AA, respectively.

Statistical analysis

All data were checked for normality and outliers using the UNIVARIATE procedure of SAS (version 9.4, SAS Institute Inc., Cary, NC) before any statistical analyses were conducted. The box and whisker plots and Shapiro–Wilk test were used to verify that data were normally distributed, (P > 0.10). All data were then subjected to least squares analysis of variance (ANOVA) for a randomized complete block design (Steele and Torre, 1980) having four treatments via the MIXED procedure of SAS with study week as a repeated measure ANOVA. The statistical model used was:

$${\displaystyle \begin{array}{l}{\displaystyle {\mathrm{Y}}_{\mathrm{i}\mathrm{j}\mathrm{k}}=\mu +{\mathrm{B}}_{\mathrm{i}}+{\mathrm{T}}_{\mathrm{j}}+{\mathrm{W}}_{\mathrm{k}}+({\mathrm{T}}_{\mathrm{j}}\times {\mathrm{W}}_{\mathrm{k}})+\mathrm{C}\mathrm{a}\mathrm{l}{\mathrm{f}}_{\mathrm{i}\mathrm{j}\mathrm{k}}+{\mathrm{e}}_{\mathrm{i}\mathrm{j}\mathrm{k}}}\end{array}}$$

Where Y_ijk = dependent variable, µ= overall mean, B_i = Block, T_j = MR treatment, W_k = study week, and (T_i x W_k) = treatment by week, Calf_ijk = Calf, and e_ijk = residual random error. Study week (W_k) was considered a repeated measurement in time having an autoregressive covariance structure. Treatment, week, and treatment × week interactions were considered as fixed effects with block and calf as random effects. Least squares means were separated by PDIFF statement. All other data that were summarized utilizing the same model described above but excluded week. Initial BW was tested as a covariate but did not improve statistical significance (P > 0.15) and, therefore, excluded from the model. Significance was declared at P < 0.05 and trends at 0.05 < P ≤ 0.10. Daily feed intake and orts were compiled as weekly averages and DM and AA intakes were calculated. Each daily fecal, nasal, and eye/ear scores were summarized by tallying by week the number of days having a specific score, that is, # day of score 0, and analyzed as weekly averages and the second way was to total the number of days of a specific score for the entire 6 wk MR phase of the study.

Results and Discussion

Nutrient composition of feeds

The nutrient composition of the four experimental MR fed to the calves in this study indicated that the nutrient composition met or slightly exceeded formulated specifications for CP, fat, and minerals (Table 3). These nutrient concentrations would meet or exceed the nutrient requirement guidelines for neonatal calves (NRC, 2001). The pelleted CS met or exceeded formulation specifications to provide the nutrients to meet or exceed the NRC (2001) nutrient requirement guidelines for growing Holstein dairy calves (Table 4).

Table 3.

Nutrient composition of MR containing 20% (20), 22% (22), and 24% CP (24), or AA concentration of the 24, but 22% CP (22AA)¹

	MR CP, %
Nutrient	20	22	24	22AA	SEM	P-value < MR2
N	2	2	2	2	—	—
DM, %	96.2a	96.2a	96.2ab	95.9b	0.07	0.10
	------------------- % of DM --------------------------
CP, %	21.9c	22.9b	24.7a	23.1b	0.07	0.01
Fat, %	21.1b	21.5b	21.0b	22.8a	0.20	0.02
NDF, %	0.38	0.29	0.24	0.06	0.11	0.32
NFC, %	47.0a	45.1b	44.0c	43.6c	0.22	0.01
Ash, %	9.8c	10.4ab	10.3b	10.6a	0.05	0.01
Ca, %	1.10b	1.23a	1.19ab	1.19ab	0.03	0.09
P, %	0.83b	0.89a	0.88a	0.90a	0.01	0.02
Mg, %	0.15b	0.16a	0.16a	0.16a	<0.01	0.11
K, %	2.59	2.59	2.64	2.66	0.03	0.41
S, %	0.34b	0.37a	0.38a	0.33b	<0.01	0.02
Na, %	0.93c	1.06a	0.97b	1.09a	0.01	0.01
Cl, %	1.52a	1.50a	1.41b	1.57a	0.02	0.02
Cu, ppm	11	18	21	13	7.62	0.76
Fe, ppm	95	123	138	136	19.0	0.46
Mn, ppm	41	55	48	52	5.72	0.45
Zn, ppm	70	88	69	86	5.98	0.16

¹Anaylses conducted by Dairyland Laboratories.

²Probability of F test for MR.

^a–cMeans differ within the row, P < 0.05.

Table 4.

Nutrient composition of pelleted CS¹

Nutrient	CS	SD
N	2	—
DM, %	87.2	0.09
	-------- % of DM ---------
CP, %	24.9	0.50
Soluble protein, % of CP	41.7	0.03
NDIP, %	1.27	0.02
ADIP, %	0.48	0.02
NDF, %	22.6	0.49
ADF, %	7.69	0.05
Lignin, %	1.67	0.08
Sugar, %	11.1	0.95
Starch, %	23.6	0.06
Fat, %	4.15	0.04
NFC, %	45.1	0.38
Ash, %	8.75	0.18
Ca, %	1.66	0.08
P, %	0.83	0.01
Mg, %	0.40	0.01
K, %	1.58	0.03
S, %	0.28	0.01
Na, %	0.58	0.05
Cl, %	0.87	0.03
Cu, ppm	48	4.24
Fe, ppm	201	2.83
Mn, ppm	214	7.78
Zn, ppm	238	2.83

¹Anaylses conducted by Dairyland Laboratories.

Formulating MR to a CP specification can result in variable AA concentrations from batch to batch within each MR due to utilization of different CP ingredients based on costs, ingredient availability, nutrient variability, etc. (Table 5). For example, the lysine concentration was lesser for calves fed the 22 MR compared with calves fed the 20 MR. Thus, formulating an MR to specific AA concentrations (Met, Lys, Thr, Trp, and Val) ensures that the AA requirements of the calf can be met without over or under supplementation of any specific amino acid, with the potential of reducing the CP concentration and ultimately reducing ingredient costs. Methionine and Lys are usually considered the first two limiting AA (Hill et al., 2007); however, given further reductions in CP concentrations could result in other AA becoming limiting (i.e., Thr, Trp, and Val). If these AA are critical for calf growth performance, then formulating for these specific AA will ensure consistent animal growth performance over time, that is, manufacture batch to batch. For example, calves fed the 24 MR would have a greater intake of several essential AA than calves fed the 22AA by formulating to a CP specification.

Table 5.

AA composition (% of DM) of MR containing 20% (20), 22% (22), and 24% CP (24), or AA concentration of the 24, but 22% CP (22 AA) and the pelleted CS¹

	MR						CS
AA	20	22	24	22AA	SEM	P-value < MR2	Mean	SD
N	2	2	2	2	—	—	2	—
Arg	0.52c	0.59b	0.64a	0.61b	0.01	0.01	1.65	0.01
His	0.48	0.40	0.44	0.41	0.06	0.79	0.65	<0.01
Ile	1.27c	1.34bc	1.48a	1.37ab	0.02	0.01	1.07	0.01
Leu	2.05c	2.18bc	2.44a	2.25b	0.04	0.01	1.88	<0.01
Lys	2.02b	1.83c	2.03b	2.29a	0.02	0.01	1.39	0.01
Met	0.56	0.69	0.61	0.89	0.20	0.71	0.32	0.01
Phe	0.69c	0.75b	0.83a	0.78b	0.01	0.01	1.21	<0.01
Thr	1.34c	1.41b	1.55a	1.45b	0.02	0.02	0.91	0.01
Trp	0.40b	0.41ab	0.44a	0.40b	0.01	0.10	0.30	0.01
Val	1.21c	1.29b	1.42a	1.32b	0.02	0.01	1.22	<0.01
Total EAA	10.5 b	10.9 b	11.9 a	11.8 a	0.21	0.01	10.6	0.04
Ala	0.97c	1.04b	1.14a	1.06b	0.01	0.03	1.14	<0.01
Asp	2.08c	2.20bc	2.40a	2.23b	0.03	0.01	2.42	0.01
Cys	0.45c	0.48bc	0.53a	0.50ab	0.01	0.01	0.40	0.01
Gly	0.42c	0.47bc	0.52a	0.48ab	0.01	0.02	1.10	<0.01
Glu	3.28c	3.47b	3.82a	3.54ab	0.04	0.03	4.45	0.06
Pro	1.19	1.22	1.35	1.43	0.06	0.16	1.40	0.01
Ser	0.90c	0.99b	1.07a	1.01a	0.02	0.02	1.04	0.01
Tyr	0.57c	0.60bc	0.66a	0.62b	0.01	0.01	0.74	0.01
Total AA	20.5 b	21.4 b	23.4 a	22.7 a	0.28	0.01	23.3	0.05
CP, %	21.5d	22.4b	24.6c	22.7a	0.06	0.01	25.0	0.11

¹Anaylses conducted by the University of Missouri.

²Probability of F test for MR.

^a–dMeans within the same row for the MR differ, P < 0.05.

Body growth and measurements

Of the 80 calves purchased for the study, 8 calves died during the study’s first week (data removed from analysis) due to scours and dehydration (Table 6). Fecal samples were submitted to the University of Wisconsin Veterinary Diagnostic Laboratory (Madison, WI) and tested positive (PCR) for Salmonella dublin, but individual calves were not necropsied. The Salmonella outbreak resulted in remaining calves being given antibiotics and electrolyte therapy treatments to mitigate disease symptoms. The calf losses of 1, 0, 3, and 4 calves for calves fed the MR treatments of 20, 22, 24, and 22AA MR, respectively, would not be expected to be influenced by CP and/or AA concentrations. While a 20% death loss is not uncommon for Holstein bull calves marketed through sale barns and raised in a commercial calf raising facility (R. Knueppel, Knueppel Livestock, Shawano, WI, personal communication), due to transportation, environmental and physiological stresses, and disease challenges that typically would not be encountered on a commercial dairy operation, the death loss would be expected to be approximately 7% or less (NAHMS-USDA, 2007). Subjecting the data to survival analyses indicated no significant differences among treatments (Chi-square, P > 0.14). Calf health after the first week improved dramatically due to medical treatments and no further interventions were warranted with the remaining calves completing the study. Replacement of calves was not an option. Using the variation for this study (new facility, first study), a post-study power and sample size analysis indicated that only 12 calves per treatment were needed to achieve a statistical difference at the P < 0.05 with a power > 0.95. Thus, even with 16 calves per treatment, enough animal numbers are present for statistical conclusions to be drawn with confidence. The speculation is that serendipity resulted in more stressed calves being randomly assigned to the treatments with death loss combined with the Salmonella outbreak vs. other treatments due to the purchasing of sale barn calves that may have varied due to dairy farm colostrum quality, amount fed, shipping stresses, handling stress, etc. Stressful environments are the main cause of neonatal calf death (Smith, 2017); for instance, transportation, vaccination, castrating, weaning, and dehorning can make calves more susceptible to infection with gastrointestinal diseases and increase the risk of diarrhea.

Table 6.

Death loss, BW, BW gains, and average daily gains for calves fed an MR containing 20% (20), 22% (22), and 24% CP (24), or AA concentration of the 24, but 22% CP (22AA)

	MR CP, %
Measurement	20	22	24	22AA	SEM	P-value < Trt1
No. of calves	20	20	20	20	—	—
Died, #	1	0	3	4	—	—
Death loss, %	5	0	15	20	—	—
Survival, d2	53.5	56.0	48.7	45.8	2.8	—
BW, kg
Initial	42.3	42.5	42.3	42.3	0.53	1.00
Final	79.4a	78.8ab	75.4b	82.1a	1.91	0.04
Average	59.4ab	59.2b	57.2b	60.2a	0.96	0.01
Total gain, kg	37.3a	36.5ab	33.4b	39.4a	1.77	0.09
Average daily gain
0 to 14 d, g/d	266.2b	206.9b	215.3b	330.8a	35.4	0.01
0 to 28 d, g/d	482.6ab	449.6b	425.5b	516.1a	29.8	0.05
0 to 42 d, g/d	547.7ab	538.3ab	490.7b	577.3a	51.6	0.19
0 to 56 d, g/d	664.4ab	652.7ab	600.1b	701.2a	30.2	0.12

¹Probability of F test for treatment.

²Chi-square, P > 0.14.

^a,bMeans with unlike superscripts differ, P < 0.05.

The initial mean calf BW was 42.4 ± 2.2 kg and similar (P > 0.10; Table 6). Calves fed the 22AA MR demonstrated greater (P < 0.05) study average BW, final BW, and BW gains compared with calves fed the 24 MR with calves fed the 20 and 22 MR being intermediate and similar (P > 0.10). The use of initial BW as a covariate did not improve statistical significance (P > 0.15). All calves lost BW during the first week of the study due to the Salmonella outbreak (Figure 1). Calves started to gain BW by the end of week 2, except calves fed the 24 MR treatment, which responded slower. By the end of the study, calves fed the 22AA demonstrated greater (P < 0.05) BW and BW gains compared with calves fed the 24 MR treatment. Getting calves started correctly and growing in the first weeks of life by minimizing or eliminating disease and stress challenges is crucial to calf growth performance.

Figure 1.

Body weight by experimental week by calves when fed an MR containing 20% (20), 22% (22), and 24% CP (24), or AA concentration of the 24, but 22% CP (22AA).

Calves fed the 22AA MR demonstrated greater (P < 0.05) 0 to 14 d ADG compared with calves fed the other treatments. The benefit of formulating an MR with AA vs. CP resulted in calves gaining more BW during the crucial first 14 d of life. Feeding excess CP with an imbalance of AA can result in an AA deficiency limiting the growth, while resulting in the excess AA being catabolized, which is a metabolic costly, energy-intensive process (NRC, 2001), thereby limiting energy for growth. Statistical differences between treatments in the cumulative ADG changed with each successive 14-d growth period. Such that by the end of the 56-d study, calves fed the 22AA had greater (P < 0.05) ADG than calves fed the 24 MR with calves fed the 20 and 22 MR treatments being intermediate and similar (P > 0.10). These data could be interpreted that as calves fed the 22AA transitioning to the CS that these early increases in ADG could not be maintained. Future studies should be conducted to develop a CS that will maintain and/or improve upon the early performance achieved by feeding the 22AA MR. Tzeng and Davis (1980) but found that requirements for Lys and dl-Met in neonatal calves ranged from 0.27 to 0.31 and 0.17 to 0.23 g/d per kilogram of BW, respectively, but in calves fed no starter. Kanjanapruthipong (1998) reported that when supplementing an MR containing soy protein with 0.131% dl-Met, 0.597% l-Lys hydrochloride, and 0.272% Thr, the ADG were greater for calves supplemented with AA than for calves without AA supplementation in the MR. Hill et al. (2007) reported that when supplementing Lys and Met to a 20/20 (CP: fat) MR fed to calves at 0.45 kg/d, the ADG were increased in all whey CP formulas. With respect to Lys and Met in this study, all treatment concentrations ranged from 1.83% to 2.29% and 0.56% to 0.89%, respectively (Table 5). It is noteworthy that the Lys and Met concentrations of the 22AA MR treatment were close to values reported by Hill et al. (2008a), in which calves were fed 0.68 kg/d of a 26% CP and 17% fat whey-based MR with synthetic Lys and Met. Average daily gains were improved by almost 17%; however, feeding calves 4% to 11% more CP and essential AA did not improve ADG, suggesting that raising Met to 0.80% will not further increase ADG (Hill et al, 2008a). Our results indicated that 22AA would be a preferable alternative under the premise of controlling feed costs. The experimental results were also in agreement with the results demonstrated by growing heifers (Gajera et al., 2013).

The greater growth rates for calves fed the greater AA MR (22, 24, and 22AA) appear to correlate with gain increases (P < 0.05) in HH (Table 7) compared with calves fed the 20 MR treatment. These data would support that the improved growth rates resulted in increased frame growth and not body fat deposition. AA are required for frame growth and this is the most efficient time in the animal’s life cycle to grow the skeletal frame (Le Cozler et al., 2008). Strayer (2014) reported that feeding based on a modified accelerated MR program resulted in greater hip width growth rates. In this study, hip width was not measured, but meeting the nutrient and AA requirements for frame growth will be beneficial to the calf for achieving key frame growth points (benchmarks) in its life, that is, breeding and calving. However, calves fed the 22 MR demonstrate greater (P < 0.05) gains in HG compared with calves fed the 24 MR with calves fed 20 and 22AA being intermediate and similar (P > 0.05). No differences were detected for gains in WH and BL among treatments. Gains in HG can be separate from height gains because frame growth is driven mostly by AA. It could be speculated that AA being used for energy would increase HG resulting in differences in frame growth among treatments. However, a lesser concentration of Lys and Met (Table 6) in the 22 compared with the 22AA MR could be expected to limit frame growth due to a shortage of Lys and Met. Thus, it might be plausible that increases in HG may be the result of some body fat deposition due to the excess protein being used as an energy source; however, no such measurements of body fat were collected. Previous studies have indicated that supplementation of MR with Lys and Met demonstrated no differences in frame measurements (Castro et al., 2016; Silva et al., 2018), but in this study, these data demonstrate sufficiently to show that feeding a 22AA MR will enhance calf and frame growth rates compared with calves fed 20 (HH) and 22 (HG).

Table 7.

Frame measurements for calves fed an MR containing 20% (20), 22% (22), and 24% CP (24), or AA concentration of the 24, but 22% CP (22AA)

	MR CP, %
Measurement	20	22	24	22AA	SEM	P-value < Trt1
HH
Initial, cm	82.0	80.7	81.0	80.6	0.85	0.20
Final, cm	91.7	92.4	91.7	92.2	0.98	0.64
Gain, cm	9.75b	11.7a	10.6ab	11.5a	0.74	0.03
WH
Initial, cm	77.7	77.1	77.6	76.1	0.77	0.26
Final, cm	88.0	89.0	87.9	88.1	1.00	0.58
Gain, cm	10.3	11.8	10.3	11.9	0.72	0.13
HG
Initial, cm	78.2	78.4	78.0	78.5	0.60	0.78
Final, cm	98.7	99.6	97.3	99.2	0.92	0.06
Gain, cm	20.5ab	21.2a	19.3b	20.7ab	0.68	0.14
BL
Initial, cm	41.6	41.1	41.1	41.5	0.65	0.83
Final, cm	51.6	52.0	51.1	51.9	0.51	0.24
Gain, cm	10.0	10.9	10.1	10.5	0.70	0.65

¹Probability of F test for treatment.

^a,bMeans with unlike superscripts differ, P < 0.05.

Dry matter intake, feed efficiency, and amino acid intake

Per the study design, the MR dry matter intake (DMI) was the same for all treatments (Table 8); however, CS intake was greater (P < 0.05) for calves fed 22AA compared with calves fed 24 with the other MR treatments being intermediate (Figure 2). Total DMI (MR plus CS) was greater (P < 0.05) for calves fed 22AA compared with calves fed 20 and 24 being the lesser with calves fed 22 being intermediate, but similar (P > 0.05) to calves fed 22AA (Figure 3). The calves fed the 24 MR are speculated to be inhibiting CS intake, which may be due to an imbalance in AA intake (Park, 2006). The speculation is that altering the balance of AA supplied to the calf through the MR may have been allowing for faster ruminal development to enhance ruminal microbial digestion, leading to greater CS and total DMI for calves fed 22AA compared with calves fed the 24 MR program.

Table 8.

DMI of MR, pelleted CS, and feed conversions for calves fed an MR containing 20% (20), 22% (22), and 24% CP (24), or AA concentration of the 24, but 22% CP (22AA)

	MR CP, %
Measurement	20	22	24	22AA	SEM	P-value < Trt1
MR DMI, kg/d
Week 1 & 2	0.548	0.548	0.548	0.548	—	—
Week 3, 4, & 5	0.822	0.822	0.822	0.822	—	—
Week 6	0.411	0.411	0.411	0.411	—	—
CS Intake, kg/d
0 to 56 d, kg	0.73bc	0.78ab	0.67c	0.82a	0.02	0.01
Total DMI
0 to 56 d, kg/d	1.23b	1.27ab	1.17c	1.32a	0.02	0.01
Gain/DMI, kg/kg
0 to 14 d	0.45ab	0.39b	0.35b	0.52a	0.05	0.07
0 to 28 d	0.57a	0.51b	0.55ab	0.57a	0.02	0.08
0 to 42 d	0.55	0.54	0.54	0.53	0.02	0.91
0 to 56 d	0.53	0.53	0.52	0.53	0.01	0.96

¹Probability of F test for treatment.

^a–cMeans with unlike superscripts differ, P < 0.05.

Figure 2.

Calf starter intake by experimental week by calves when fed an MR containing 20% (20), 22% (22), and 24% CP (24), or AA concentration of the 24, but 22% CP (22 AA).

Figure 3.

Total DMI by experimental week by calves when fed an MR containing 20% (20), 22% (22), and 24% CP (24), or AA concentration of the 24, but 22% CP (22 AA).

Feed efficiency, expressed as kg BW gain/total DMI, was greatest (P < 0.05) for calves fed 22AA compared with calves fed 22 and 24, while calves fed 20 were intermediate and similar (P > 0.05) for the growth period of 0 to 14. Calves fed 22AA and 20 demonstrated greater (P > 0.05) feed efficiency compared with calves fed 22 with calves fed 24 being similar (P > 0.05) and intermediate for the 0- to 28-d period. The AA formulation of the MR allowed for greater efficiency of BW gain through the highest MR feeding phase (first 28 d) compared with calves fed 22, which is considered an industry-standard MR and it appears that as rumen development occurred resulting in the establishment of ruminal microbial digestion, then feed efficiency became similar for calves fed all treatments. The improvements in feed efficiency observed in the first 28 d of the study are speculated to be due to an improvement in post absorption efficiency of nutrient utilization. As the calves’ transition from a liquid MR diet to CS, the ruminal development and initiation of ruminal microbial digestion should not have been influenced by nutrient digestion prior to rumen development. Since the same CS was fed to all treatments, the differences in feed efficiency between treatments disappeared with increasing CS intake. One reason for this observation is that a greater digestibility would be expected when feeding calves a liquid MR than CS (Hill et al., 2008b). With increasing CS intake, calves gradually minimized the differences in feed efficiency observed between treatments in the early days of the study. Some studies have reported similar results when supplementing AA to calves. Hill et al. (2007) reported that calves fed a 20% CP MR with added AA compared with calves fed a 20% CP MR without added AA demonstrated no differences in CS intake and gain per feed efficiency from 0 to 42 and 0 to 56 d. Not unexpectedly, Hill et al. (2008a) observed that CS intake did not increase with different Lys or Thr concentrations.

The essential AA intake (MR plus CS) was greatest (P < 0.05) for calves fed the 22AA MR compared with calves fed 24 MR with the other treatments sometimes being intermediate that was either similar or different (Table 9). For Lys and Met, which are usually considered the first two limiting AA for growth (Hill et al., 2007), the intake of these AA was increased approximately 15.5% and 22.0%, respectively, compared with the next highest intake. What is interesting is that calves fed the 24 MR demonstrated similar or lesser intakes of Lys and Met, while consuming more protein compared with calves fed the 20 MR treatment. These AA intakes would explain the results demonstrated in Table 6 and Figure 1 that increasing the essential AA intake results in improved growth performance, that is, g/d. Thus, formulating for only a CP specification could result in an insufficient intake of AA to support BW gains and ADGs that would be expected for feeding a greater CP MR. The use of AA (Met, Lys, Thr, Trp, and Val) as nutrient specifications in formulations would ensure more consistent performance from batch to batch, as ingredients and sources change.

Table 9.

Total amino acid intake (g/d) from MR and CS for calves fed an MR containing 20% (20), 22% (22), and 24% CP (24), or AA concentration of the 24, but 22% CP (22AA)

	MR CP, %
Amino acid	20	22	24	22AA	SEM	P-value < Trt1
Arg	14.7b	15.7a	14.0b	16.4a	0.64	0.01
His	7.2a	7.0ab	6.4b	7.3a	0.25	0.01
Ile	14.2c	14.9b	14.3c	15.5a	0.41	0.01
Leu	24.0c	25.4b	24.4c	26.5a	0.73	0.01
Lys	20.3b	19.8bc	19.2c	22.7a	0.54	0.01
Met	5.1c	5.9b	5.1c	7.0a	0.12	0.01
Phe	11.7b	13.1a	12.0b	13.7a	0.47	0.01
Thr	13.4c	14.0b	13.6bc	14.6a	0.35	0.01
Trp	4.2bc	4.4ab	4.1c	4.4a	0.12	0.01
Val	15.0c	15.9b	15.0b	16.5a	0.47	0.01
Total	130.2c	136.2b	128.3c	144.8a	4.05	0.01

¹Probability of F test for treatment.

^a–cMeans within the same row differ, P < 0.05.

Health performance

In general, health scores as measured by fecal, nasal, and eye/ear scores for calves fed all treatments were similar (P > 0.10; Table 10). No differences were detected (P > 0.10) when scores were expressed as fecal scores for the number of days during the week or total days in the study for the 6 wk when MR treatments were fed. The Salmonella outbreak certainly had an effect on fecal scores and the number of days for greater fecal scores, etc. score 3. However, it would appear that greater CP concentrations could be detrimental to calf health and survivability if diarrhea/scours does occur. Calves fed 22AA had a greater day/week with a score of 3 than calves fed 20 with calves fed 22 and 24 being intermediate. This observation was also detected for a 3 score for eye/ear. However, no differences were detected for total days of scores among treatments. For the remainder of the experiment after the outbreak, fecal scores and health issues were minimal after the first week, and calves were healthy.

Table 10.

Fecal, nasal, and eye/ear scores during the milk feeding phase (6 wk) for calves fed an MR containing 20% (20), 22% (22), and 24% CP (24), or AA concentration of the 24, but 22% CP (22AA)

	MR CP, %
Measurement	20	22	24	22AA	SEM	P-value < Trt1
Fecal scores, d/wk
Score = 0	2.2	2.5	2.2	2.5	0.19	0.24
Score = 1	2.1	1.9	2.0	1.8	0.18	0.34
Score = 2	1.2	1.4	1.3	1.2	0.16	0.68
Score = 3	0.2b	0.3ab	0.4ab	0.4a	0.11	0.09
Total days of fecal score
Score = 0, d	13.2	15.3	13.3	15.2	1.16	0.28
Score = 1, d	12.8	11.2	18.8	11.1	1.20	0.36
Score = 2, d	7.4	8.2	7.9	7.4	1.16	0.86
Score = 3, d	1.5	2.0	1.5	2.6	0.67	0.19
Nasal score, d/wk
Score = 0	2.2	2.5	2.2	2.5	0.19	0.24
Score = 1	2.1	1.9	2.0	1.8	0.18	0.34
Score = 2	1.2	1.4	1.3	1.2	0.16	0.68
Score = 3	0.2	0.3	0.4	0.4	0.11	0.09
Total days of nasal score
Score = 0	13.2	15.3	13.3	15.2	1.25	0.29
Score = 1	12.8	11.2	11.8	11.1	1.21	0.36
Score = 2	7.4	8.2	7.9	7.4	1.16	0.86
Score = 3	1.5	2.0	2.5	2.6	0.67	0.19
Eye/ear score, d/wk
Score = 0	2.2	2.5	2.2	2.5	0.19	0.24
Score = 1	2.1	1.9	2.0	1.8	0.18	0.34
Score = 2	1.2	1.4	1.3	1.2	0.16	0.68
Score = 3	0.2a	0.3ab	0.4ab	0.4a	0.11	0.09
Total days of eye/ear score
Score = 0	13.2	15.3	13.3	15.2	1.25	0.29
Score = 1	12.8	11.2	11.8	11.1	1.20	0.36
Score = 2	7.4	8.2	7.9	7.4	1.16	0.86
Score = 3	1.5	2.0	2.5	2.6	0.67	0.19

¹Probability of F test for treatment.

^a,bMeans within the same row differ, P < 0.05.

Economic analysis

The total cost of MR per calf per the 42-d MR feeding period based on the calculation MR DMI times the US$/kg of each MR (Table 11) was greater (P < 0.05) for calves fed 24 compared with calves fed the remaining treatments. The CS daily cost (DMI CS × US$/kg CS) per calf was similar (P > 0.10) for calves fed all treatments while the total feed (MR + CS) costs per day were greatest (P < 0.05) for calves fed 24 and lowest (P < 0.05) for calves fed 20, and for calves fed 22 and 22AA were intermediate and different (P < 0.05). The total feed cost per calf per the 56-d experiment was US$96.68, US$104.72, US$107.52, and US$102.48, which resulted in a total feed cost per kilogram of BW gain being highest (P < 0.05) for calves fed 24 compared with calves fed the remaining treatments, which may be of interest to a dairy producer.

Table 11.

Cost (US$) per day for MR, pelleted CS, and total cost per day and total cost per kg of BW gain for calves fed an MR containing 20% (20), 22% (22), and 24% CP (24), or AA concentration of the 24, but 22% CP (22AA)

	MR CP, %
Measurement	20	22	24	22AA	SEM	P-value < Trt1
MR, US $/d, 0 to 42 d	1.89b	1.99b	2.12a	1.90b	0.05	0.01
CS, US $/d, 0 to 56 d	0.36	0.38	0.33	0.40	0.04	0.45
Total cost, US $/d,
0 to 56 d	1.78d	1.87b	1.92a	1.83c	0.01	0.01
Total cost, US $/kg BW gain
0 to 56 d	2.77b	2.85b	3.21a	2.68b	0.10	0.01

¹Probability of F test for treatment.

^a–cMeans with unlike superscripts differ, P < 0.05.

Conclusions

The study objective was to evaluate an MR formulated using synthetic AA (22AA) to achieve the AA concentrations of a 24:20 MR with reduced CP demonstrated the improved growth performance compared with the 24:20 when fed to Holstein bull calves. Based on the study hypothesis, calves fed 22AA MR demonstrated similar or greater (first 14 d) growth performance with numerically the lowest feed cost per kilogram BW gain. Our findings indicate that feeding a 22AA MR can improve BW, ADG, and feed conversion during the early period of life, that is, 0 to 14 d. Similar or improved growth performance can be achieved by feeding an AA fortified MR having a lesser CP concentration, which reduces feed costs. In general, calves benefited from being fed MR with an AA formula should result in more consistent performance from batch to batch as the amino acid concentrations of ingredients can vary.

Glossary

Abbreviations

AA

amino acids

ADF

acid detergent fiber

ANOVA

analysis of variance

BL

body length

BW

body weight

CP

crude protein

CS

calf starter

DM

dry matter

DMI

dry matter intake

EAA

essential amino acids

HG

heart girth

HH

hip height

MR

milk replacers

NDF

neutral detergent fiber

NFC

nonfiber carbohydrates

WH

withers height

Conflict of interest statement

At the time of study conception, design, execution, and summary, K.H. and Dr. D.P.C. were employed by the Furst-McNess Company. This study was part of Y.B.’s Ph.D. program at Gansu Agricultural University. All remaining authors declare no conflict of interest.