Impacts of feeding milk replacer supplemented with increasing concentrations of choline on feed intake, growth, and scouring incidence for 7 weeks preweaning and 1-week postweaning.

: The objective of this study was to evaluate the effect of increasing choline supplementation on the intake of milk replacer (MR) and calf starter (CS), growth performance, and fecal scores of calves over 8 weeks. One hundred calves from commercial herds were transported to the University of Minnesota Southern Research and Outreach Center (SROC) calf facility randomized to one of 4 treatments differing in choline supplementation within MR including: 0 ( C0 ; n = 24), 700 ( C700 ; n = 26), 1400 ( C1400 ; n = 25), and 2100 mg/kg choline ( C2100 ; n = 25). MR contained a basal concentration of 1650 mg/kg MR resulting in total choline concentrations in the MR of 1650 mg/kg for C0 was, 2350 mg/kg for C700 was, 3050 mg/kg for C1400 , and 3750 mg/kg for C2100 . Calves were weaned at 49 d, and the trial ended at 56 d. Body weight (BW) was measured at d 0, 14, 28, 42, 49 and 56. Fecal scores were measured weekly. Feed intake was measured daily and calculated bi-weekly. All feeding, body weight measurements, and fecal scoring were conducted by the research staff at the University of Minnesota SROC. Growth, feed intake, and gain to feed ratio were analyzed using a linear mixed effects model with choline concentration as a fixed effect and source herd and room as random effects. Initial body weight was included as a covariate for average daily gain. Fecal scores were analyzed as a chi-squared test of significance. Feeding 700 mg/kg supplemental choline increased MR intake throughout the preweaning period and increased ADG in both the preweaning and postweaning periods compared with the other treatments. No differences were observed for fecal scores across treatments. Results suggest that calf performance can be improved with MR containing 2350 mg/kg choline, contrasting with current NASEM (2021) requirements of 1000 mg/kg.


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Abstract:
The objective of this study was to evaluate the effect of increasing choline supplementation on the intake of milk replacer (MR) and calf starter (CS), growth performance, and fecal scores of calves over 8 weeks.One hundred calves from commercial herds were transported to the University of Minnesota Southern Research and Outreach Center (SROC) calf facility randomized to one of 4 treatments differing in choline supplementation within MR including: 0 (C0; n = 24), 700 (C700; n = 26), 1400 (C1400; n = 25), and 2100 mg/kg choline (C2100; n = 25).MR contained a basal concentration of 1650 mg/kg MR resulting in total choline concentrations in the MR of 1650 mg/kg for C0 was, 2350 mg/kg for C700 was, 3050 mg/kg for C1400, and 3750 mg/kg for C2100.Calves were weaned at 49 d, and the trial ended at 56 d.Body weight (BW) was measured at d 0, 14, 28, 42, 49 and 56.Fecal scores were measured weekly.Feed intake was measured daily and calculated bi-weekly.All feeding, body weight measurements, and fecal scoring were conducted by the research staff at the University of Minnesota SROC.Growth, feed intake, and gain to feed ratio were analyzed using a linear mixed effects model with choline concentration as a fixed effect and source herd and room as random effects.Initial body weight was included as a covariate for average daily gain.Fecal scores were analyzed as a chi-squared test of significance.Feeding 700 mg/kg supplemental choline increased MR intake throughout the preweaning period and increased ADG in both the preweaning and postweaning periods compared with the other treatments.No differences were observed for fecal scores across treatments.Results suggest that calf performance can be improved with MR containing 2350 mg/kg choline, contrasting with current NASEM (2021) requirements of 1000 mg/kg.C holine is a metabolically-important quaternary ammonium cation that can be synthesized endogenously in the liver.However, in most animals, including dairy cows, hepatic synthesis is insufficient to meet requirements for the animal (Zeisel et al., 2009).Choline has multiple functions and supports production, reproduction, health and growth in cattle.It is a major component of phospholipids and acetylcholine, which affects membrane integrity, and is involved in DNA methylation pathways (Pinotti et al., 2002;McFadden et al., 2020).Additionally, Abdelmegeid et al. (2017) observed in vitro that choline supplementation reduces pro-inflammatory activity and improves antioxidant balance in leukocytes from neonatal Holstein calves.
Although choline supplementation has been extensively researched in lactating dairy cows, specifically during the transition period, limited information on the role of choline supplementation in calves is available, especially in vivo.The current NASEM (2021) recommendation for choline requirements in calves is 1000 mg/kg DM.To our knowledge, few studies have considered the impact of increasing choline supplementation on the intake of milk replacer (MR) and calf starter (CS), weight gain, and fecal scores of young pre-weaned calves.Thus, the objective of this study was to determine the effect of increasing choline supplementation on the intake of MR and CS, growth performance, and health of calves over 7 weeks prepartum and 1 week postpartum.We hypothesized that increased supplementation of choline would increase MR and CS intake, body weight gain, and reduce fecal scores of dairy calves.
To test this hypothesis, Holstein heifer calves (n = 100) aged at 2 to 5 d were sourced from 4 commercial dairy farms and transported to the University of Minnesota Southern Research and Outreach Center (SROC) Calf and Heifer Research Facility (Waseca, MN).Commercial farms were all located in SE Minnesota within a 60-mile radius of SROC, and were selected based on well-documented health records, the cleanliness of maternity facilities, and their compliance with appropriate colostrum feeding guidelines.Enrollment criteria required calves to be at least 48 h of age, have received 3 feedings of colostrum within 48 h of age, be free from apparent health issues, have a serum total protein concentration greater than 5, and have an initial body weight (BW) between 34.0 and 47.6 kg.Calves were delivered to SROC twice per week, blocked by source herd, and randomly assigned to treatment upon arrival using Microsoft Excel (Redmond, WA).
All calves were enrolled on trial between July 12, 2022 and August 16, 2022, with the trial ending on October 14, 2022.Sample size was determined based on a > 80% power of observing P < 0.05 difference in our primary outcome measure of ADG based on a standard deviation of 0.02 kg and an expected difference of 0.1 kg observed in previous experiments (Chester-Jones et al., 2016;Jaeger et al., 2020b).Calves were housed in individual pens (2.3 × 1.2 m) with plastic side panels in one of 4 naturally-ventilated nursery rooms with 2-curtain sidewalls.Calves were placed into nursery rooms sequentially by arrival date.All procedures for animal care and handling were approved by the University of Minnesota Institutional Animal Use and Care Committee (Protocol number: 2110-39487A).Feeding, body weight measurements, and fecal consistency scores were conducted by the previously trained research staff at the University of Minnesota SROC.
Upon arrival (d1 of the trial), body weight was measured using a digital scale (VS-660; A and A Scales LLC; Wyckoff, NJ), hip height was measured using a wooden measuring stick with sliding bar (Nasco Education; Fort Atkison, WI), and 10 mL of blood was collected via jugular venipuncture into serum vacutainer tubes (BD; Franklin Lakes, NJ), allowed to clot, centrifuged at 1500 x g and analyzed using a Brix refractometer (Spartan Refractometer, model A 300 CL, Spartan, Tokyo, Japan).Serum protein at arrival was 6.31 g/dL in C0, 6.11 g/dL in C700, 6.25 g/dL in C1400, and 6.38 g/dL in C2100 (P = 0.55).All calves with d 1 BW of less than 35.0 kg or greater than 47.6 kg were excluded from the study.Initial BW was 38.9 kg for C0, 40.3 kg for C700, 39.4 kg for C1400, and 39.7 kg for C2100 (P = 0.51).
Body weight was measured on d 14, 28, 42, 49, and 56 of the experiment at 1500.Gain to feed ratio (G:F) was calculated as kg of BW gain divided by kg of total feed intake.Hip height was measured on d 1 and d 56, and hip height gain during the experiment was calculated.Daily fecal consistency scores were visually determined daily at 0500 by SROC research staff using a 1 to 4 scale with 1 = normal, 2 = loose, pudding, 3 = very loose, no watery separation, and 4 = very loose, watery separation (Larson et al., 1977).Scouring frequency was determined as the number of days per week that the calves had a fecal score ≥3.Milk replacer intake, CS intake, total feed intake, ADG, G:F, and average fecal score were determined for each of the intervals between d 1 to 14, d 15 to 28, d 42 to 49, and d 49 to 56, for the preweaning period (d 1 to 49) and for the trial period (d 1 to 56).One calf was removed from treatment group C0 due to dramatic weight loss from d 1 to d 7.
A linear mixed model was performed using the MIXED procedure of SAS 9.4 to determine effects of choline concentration on MR intake, CS intake, total intake, BW, ADG, G:F, hip height and hip height gain during each of the biweekly or weekly time intervals.The model included the fixed effect of choline concentration and random effects of source herd and nursery room.Effects of choline concentration on daily MR intake, CS intake, total intake, ADG, and G:F over the preweaning period (d 1 to 49) and the entire trial period (d 1 to 56) were analyzed using repeated measures in the MIXED procedure of SAS with the fixed effect of choline concentration, the random effects of source herd and nursery room, and the repeated effect of time interval.Initial BW was used as a covariate for ADG in the repeated measures analysis.Effects of choline concentration on the discrete variables of fecal consistency score (1, 2, 3, or 4) and scouring frequency (# of days with fecal score ≥3 during each weekly period) were determined using a chi-squared test in PROC GLIMMIX.Linear and quadratic orthogonal contrasts of effects of choline concentration were tested for all responses and the heterogeneous autoregressive (ARH1) covariance structure was used in all analyses.Preplanned contrasts between individual treatment differences were also determined.For all analyses, data points with studentized residuals outside ± 3.5 were removed as outliers.Statistical significance was declared at P ≤ 0.05 and trends acknowledged at 0.05 < P ≤ 0.10.Heteroscedasticity was determined by generating a histogram and normal Q-Q plot of residuals.
A linear decrease in the MR intake during d 1 to 14 was observed due to increased choline inclusion with C0 and C700 having greater MR consumption compared with C1400 and C2100 (P = 0.01).During d 15 to 28 MR intake was lowest in C1400, followed by C2100 (P < 0.01).From d 29 to 42 MR intake tended to be greatest for C700 followed by C2100 (P = 0.08) Finally, from d 1 to 49 the MR intake in C700 was greater compared with that of the other treatments (P < 0.01; Table 1).For CS intake on d 50 to 56, C700 had greater intake compared with that of C0 and C1400 (P = 0.01).When considering total feed intake by day for d 50 to 56, C700 had greater intake compared with C0 and C1400 (P = 0.01).
Classical research suggests that dairy calves are born choline deficient and are reliant on choline from maternal colostrum to meet choline requirements (Waugh et al., 1947;Johnson et al., 1951).However, minimal research has focused on direct supplementation of pure choline to dairy calves on feed intake or performance of dairy calves.Molano et al., (2021) fed a blend of choline and Bvitamins in calf starter from 4 to 13 weeks of age.They observed no differences in DMI as a response to treatment.Recently, several studies have examined the impact of maternal rumen-protected choline (RPC) on growth and feed intake of their offspring.Swartz et al., (2022) observed no effect of feed intake on offspring of

Ravelo et al. | Choline supplementation in dairy calves
RPC-supplemented cows during the first 3 weeks of life.Brown et al., (2023) observed that male Angus-Holstein calves had greater DMI when dams were fed RPC, but the effects appeared to be sexspecific because similar results were not observed in female calves.In mid-lactation adult Holstein cows, feeding unprotected choline at rates of 10 or 20 g/kg diet DM decreased DMI by approximately 8% (Sharma and Erdman, 1988), but similar results have not been observed in cows fed RPC (Sales et al., 2010).In the current study, the choline provided was not encapsulated.Therefore, the unprotected form of choline may have similarly decreased intake, especially when large amounts of MR were consumed during early life, perhaps due to decreased palatability.
During weaning, stress from the transition from liquid to solid feed can decrease feed intake of calves (Weary et al., 2008).In this study, it was observed that the calves that were supplemented with C700 of choline had greater CS intake after weaning compared with the other treatment groups.In lactating dairy cows, it has been observed that choline supplementation can increase postpartum DMI due to reduced inflammation and better lipid export from the liver (Humer et al., 2019).Our results indicate that choline supplementation may increase intake of MR and CS in calves as well, especially during the stressful weaning period.However, our results suggest providing choline above 2350 mg/kg DM does not increase intake in calves.
The BW for calves in C700 tended to be greater compared with calves in C0 and C1400 (P = 0.10; Table 2).The difference in BW became more prominent at d 49 with the C700 group maintaining greatest BW (P = 0.01).On d 56, C700 had the greatest BW followed by C2100, and the lowest weight was the C1400 group (P < 0.01).Average daily gain differed among treatments in the pre-weaning period (P = 0.05).Specifically, during d 43 to 49, the ADG of C700 tended to be greater than that of the other treatments (P = 0.08).Likewise, over the entire 56-d trial period ADG was decreased in C0 and C1400 compared with C700 and C2100 (P < 0.01).Additionally, C2100 had greater d 56 hip heights compared with C0 and C1400 (P = 0.02).
Calves in the C700 group had greater ADG and greater hip height compared with the C0 supplementation treatment group (P < 0.05).Additionally, C700 had improved gains and maintained increased intake compared with the C1400 calves.Including basal choline concentrations in MR, calves supplemented with 700 mg/ kg received a total of 2350 mg/kg DM which is 1350 mg/kg DM greater than the recommended 1000 mg/kg DM.Although our results suggested that some choline supplementation helped in calf growth, Molano et al. (2021) did not observe any growth differences between calves fed a B vitamin and choline blend compared with calves that did not receive the blend during the transition and post-weaning phase.Discrepancy in results could have been due to the time in which supplementation began.In the current study supplementation began within the first few days of life, whereas Molano et al., (2021) supplemented beginning at 4 wks.Likewise, even though the calves in the current study were fed for a longer duration before weaning compared with Molano et al., (2021), differences in growth and ADG were not observed until wk 6 or 7 of treatment implementation.Thus, there may be a delay associated with observed differences.
The C2100 tended to decrease feed efficiency by 12.1%, 10.5%, and 13.5% during d 43 to 49 compared with C0, C700, and C1400, respectively (P = 0.01; Table 3).However, from d 50 to 56, the C1400 had a 11.5% and 14.8% decreased G:F compared with C0 and C700 (P < 0.01).The C700 treatment group had greater gain per feed on d 50 to 56, corresponding to the greatest ADG for that time frame compared with the calves in the other treatments.In Sharma and Erdman, (1988), cows supplemented with greater cho- Treatments included milk replacer with choline included at 0 (C0, n = 24), 700 (C700, n = 26), 1,400 (C1400, n = 25) or 2,100 (C2100, n = 25) mg/kg.Treatments with different superscripts were considered different at (P < 0.05; a, b, c), or exhibited a tendency for difference (0.05 < P < 0.10; x, y, z).line concentrations not only had reduced feed intake but also had decreased milk production.In the current study greater supplementation of choline did not improve feed efficiency or growth in the calves assigned to that treatment.To the authors' knowledge, there have not been studies to consider adverse effects of choline supplementation in MR on intake and growth of dairy cows, however, it seems that over supplementation does not improve feed efficiency.Overall, even though C700 consumed more MR and CS during this period they were more efficient than the calves in the other groups as they gained more kgs per kg of feed than they consumed.Average fecal score did not differ among treatments throughout the entire preweaning period (C0: 1.34, C700: 1.41, C1400: 1.40; C2100: 1.37; χ 2 = 2.83; P = 0.42) or during the postweaning period (C0: 1.02, C700: 1.04, C1400: 1.00; C2100: 1.03; χ 2 = 3.02; P = 0.39).Furthermore, no difference was observed during the entire 56-d experiment (C0: 1.30, C700: 1.37, C1400: 1.35; C2100: 1.33; χ 2 = 2.52; P = 0.47).Scouring frequencies also did not differ among the groups during any week of the experiment.Our results agree with those by Swartz et al., (2022), who observed no differences in risk of neonatal calf diarrhea in calves born from dams that were supplemented with different levels of RPC.
Due to high choline concentrations within basal MR, all treatments provided greater daily choline supply (1650 mg/kg DM) than current NASEM (2021) recommendations of 1000 mg/kg DM.Nevertheless, even with these high basal concentrations, additional benefits to MR intake and ADG were observed with further supplementation of 700 mg/kg (2350 mg/kg DM).These effects were particularly strong immediately post-weaning.No additional increases in feed intake or gain were observed at choline concentrations above 2350 mg/kg.However, it is important to note that the calves in the current study were fed concentrations of MR that are lower than the current NASEM recommendations (>680 g/d) and in the current study conditions 2350 mg/kg supplementation appeared to be closer to the optimal requirement for growth.Future research should continue to revisit choline requirements for young dairy calves.Treatments included milk replacer with choline included at 0 (C0, n = 24), 700 (C700, n = 26), 1,400 (C1400, n = 25) or 2,100 (C2100, n = 25) mg/kg.Treatments with different superscripts were considered different at (P < 0.05; a, b, c), or exhibited a tendency for difference (0.05 < P < 0.10; x, y, z).Treatments included milk replacer with choline included at 0 (C0, n = 24), 700 (C700, n = 26), 1,400 (C1400, n = 25) or 2,100 (C2100, n = 25) mg/kg.Treatments with different superscripts were considered different at (P < 0.05; a, b, c), or exhibited a tendency for difference (0.05 < P < 0.10; x, y, z).

2P-
Value of main effects of choline concentration (Trt) with linear and quadratic (QUAD) contrasts.

2P-
Value of main effects of choline concentration (Trt) with linear and quadratic (QUAD) contrasts.

Table 1 .
Ravelo et al. | Choline supplementation in dairy calves Effect of increasing concentrations of choline in milk replacer on milk replacer and starter intake of dairy calves

Table 2 .
Effect of increasing concentrations of choline in milk replacer on growth of dairy calves

Table 3 .
Effect of increasing concentrations of choline in milk replacer on feed efficiency of dairy calves