Effect of Dietary Choline Chloride Supplementation on Growth Performance and Carcass Characteristics of Broiler Chickens Reared to 32 Days of Age

: Research on the impact of supplementing additional dietary choline to modern, large-frame broiler chickens is limited, and current choline recommendations are outdated. The objective of this study was to determine the effect of additional dietary choline chloride supplementation on growth performance and carcass characteristics of modern broilers reared to 32 days of age. Corn and soybean meal-based diets were formulated to contain an additional 0, 400, 800, 1200, 1600, or 2000 mg of choline chloride per kg of feed above the choline present in the basal diet ingredients. As-hatched Ross 708 × Yield Plus broiler chicks ( n = 2160; 30 birds per pen; 44 ± 0.2 g initial BW) were randomly allotted to the experimental diets, reared for 31 days, and processed at day 32. Growth performance, carcass yield, and breast myopathy incidence were not impacted by supplemental choline. However, abdominal fat pad weight tended to diminish with increasing dietary choline, and drumstick yield was reduced in birds fed over 800 mg per kg of feed. These results indicate feeding additional choline does not impact growth performance but may result in changes in carcass characteristics of modern, high-yielding broilers reared for 32 days.


Introduction
Choline is a crucial nutrient for broiler chickens.It contributes to various metabolic functions including lipid transport, cell signaling, and biosynthesis of methylated compounds.Due to its role in lipid metabolism, choline deficiency is often associated with fatty liver development [1,2], which has been demonstrated in chicken models [2], and shown to reduce egg production in laying hens [3].Genetic selection to produce heavily muscled and efficient broiler chickens occurs rapidly to satisfy an ever-changing market.Symptoms of choline deficiency appear to be rare in commercial broiler chickens.However, as genetics progress, it is likely that nutrient requirements for modern broilers will change [4].The most recent NRC Nutrient Requirements of Poultry [5] establish required dietary choline concentrations using studies published prior to 1987.The NRC recommends 1300 mg of choline per kg of feed in broiler diets when sulfur amino acids are fed at adequate concentrations.
The interconnected biosynthesis pathways of choline and the sulfur amino acids Met and Cys make it difficult to determine dietary choline requirements [6][7][8].The generation of many methylated compounds relies on the S-Adenosyl Methionine (SAM-e) cycle.Met is converted to SAM-e, which then acts as a cofactor for many methyl group transfer reactions.Choline enters the cycle upon conversion to betaine, which then donates its methyl groups to a folic acid derivative.This results in the formation of Met to regenerate SAM-e [9].When sulfur amino acid availability is low in broiler diets, choline can spare Met to rescue growth performance the same way that Met can spare for low concentrations of choline [10,11].Therefore, dietary choline requirements are dependent on concentrations of sulfur amino acids, betaine, and folic acid in the body, which makes establishing a definitive choline requirement challenging.
Betaine has osmoregulatory qualities, and it has been evaluated as a dietary supplement to potentially alleviate heat stress in broilers [12].It has also been suggested that dietary betaine supplementation may be suitable to partially replace choline in broiler diets [13,14].This is because betaine is a metabolite of choline that enters the SAM-e cycle to contribute to the donation of methyl groups for a multitude of anabolic pathways.However, the oxidation of choline to form betaine is irreversible [15].Therefore, dietary betaine may aid in replenishing SAM-e cycle intermediates, but it cannot be converted back to choline to carry out choline's other important functions such as maintaining cell membrane integrity and lipid metabolism.
In more recent experiments, additional dietary choline supplementation above what is naturally occurring in a corn and soybean meal-based diet has produced conflicting results on broiler growth performance.In studies where Met was fed at or above breeder recommendations, increasing dietary choline resulted in an increase in feed intake that did not impact the feed conversion ratio (FCR) [16], as well as a reduction in feed intake and improvement in FCR [17].While current choline recommendations may be sufficient to prevent deficiency, they are not necessarily adequate for optimizing growth performance and carcass characteristics of modern, large-frame broilers.Therefore, the objective of this study was to determine the effects of increasing dietary choline concentrations on the growth performance and carcass characteristics of broilers grown from 0 to 32 days of age.

Materials and Methods
All procedures regarding live birds were approved by the Auburn University Institutional Animal Care and Use Committee (PRN 2017-3013).

Diet Formulation
Six experimental diets were manufactured in the Auburn University Poultry and Animal Nutrition Center and formulated to contain an additional 0, 400, 800, 1200, 1600, or 2000 mg of choline (supplied as choline chloride) per kg of feed in addition to that provided by the ingredients in the basal diet (Table 1).Initial choline concentrations in the starter and grower basal diets were 1183 and 1171 mg per kg, respectively.The industry standard, antibiotic-free, corn and soybean meal-based basal diet was formulated to meet or exceed the primary breeders' nutrient recommendations [18].All diets were completely antibiotic free, all-vegetable, and formulated to have equal nutrient composition with the exception of differing final choline concentrations.To achieve this, a hand add containing the additional choline chloride was added to the basal diet prior to mixing and pelleting.Broilers were fed in two different dietary phases: starter from day 0 to 15 and grower from day 16 to 32.Diet composition by phase is outlined in Table 1. 1 SBM = soybean meal, DDGS = dried distillers' grains with solubles. 2Choline hand add contained basal diet and added choline chloride.

Broiler Husbandry
As-hatched, Aviagen Ross 708 × Yield Plus broiler chicks (n = 2160) were transported 5 h from a commercial hatchery to the Auburn University Charles C. Miller Jr.Poultry Research and Education Center.Upon arrival, chicks were weighed, randomly allotted to 1 of 6 dietary treatments, and placed into 2.23 m 2 pens bedded with new wood shaving litter (24 replicates per treatment; 30 birds per pen).Both feed and water were provided on an ad libitum basis.Environmental temperature began at 33.3 • C and was gradually reduced according to bird comfort to reach a final temperature set point of 20.0 • C. The lighting program was set according to the breeder management recommendations.

Broiler Growth Performance and Carcass Part Yields
Individual bird and feeder weights were recorded at the end of each dietary phase to determine mortality-corrected BW gain (BWG), feed intake (FI), and feed conversion ratio (FCR) with mortality recorded on a daily basis.On day 32, birds from 6 replicate pens per treatment (n = 1080; 30 birds per pen per treatment) were fasted for 8 h and processed at the Auburn University Pilot Processing Plant.Chilled carcass weight without giblets (WOG) and fat pad weight were measured following a 3 h static chill, and carcasses were deboned after an overnight static chill.Carcasses were deboned by professional deboners and whole boneless, skinless breast, tender, bone-in, skin-on wing, boneless, skinless thigh, and bone-in drumstick weights were recorded.

Wooden Breast and White Striping Scoring
Breast fillets were scored for the Wooden Breast (WB) and White Striping (WS) myopathies on a 4-point scale (0 = normal, 1 = mild, 2 = moderate, and 3 = severe) as previously described [19].WB evaluation was conducted with hand palpation of each fillet to determine the proportion of the fillet affected by palpable hardness, and WS was evaluated by visually determining the proportion of the fillet affected by white striations.Score 0 fillets were free of defects, score 1 fillets were up to 1  4 affected, score 2 fillets were up to 1  2 affected, and score 3 fillets were greater than 1  2 affected.The same trained evaluator scored all fillets for both defects.WB and WS scores are displayed as a proportion of all breasts within each dietary treatment group.

Statistical Analysis
Data were analyzed using the MIXED and GLIMMIX procedures of SAS 9.4.For all data analysis, dietary treatment was the fixed effect, and the Satterthwaite adjustment was used to correct degrees of freedom.The MIXED procedure of SAS was used for all continuous data (e.g., weights and FCR) while GLIMMIX was used to analyze proportional data (e.g., mortality, carcass parts yields).Percentages were derived from proportions generated using the events/possible events syntax with a binomial distribution and R-side covariance structure in SAS.Pens were blocked by location and served as the experimental unit.Means were separated using the PDIFF option and considered different when p ≤ 0.05.Tendencies for differences among treatment means were declared when 0.05 < p ≤ 0.10.

Broiler Growth Performance
Supplementing the diets of large-frame, modern broilers with increasing concentrations of choline chloride did not impact feed intake, BW gain, or FCR under these experimental conditions, as shown in Table 2.These results differ from those of Igwe et al. who saw improvements in FCR of Ross 308 broilers fed up to 2000 mg per kg supplemental choline chloride [4].Betaine, the downstream SAM-e cycle metabolite of choline, has shown promise in alleviating the negative impacts of heat stress on broiler chicken growth performance [12].This suggests increasing dietary choline may not be as beneficial for broilers reared under optimal temperatures, such as in the present study.However, the metabolic relationship between Met and choline may be more important for altering broiler performance than the effects of heat stress alone.Kpodo et al. [20] did not find that supplemental choline or betaine could ameliorate the losses in BW gain associated with heat stress in broilers.Supplemental choline has been shown to partially replace inadequate Met in broiler diets [8,10,21,22].It is possible that providing dietary Met above the primary breeder nutrition specifications in this experiment was responsible for a lack in response in growth performance due to added choline chloride [23][24][25][26].Mortality was not impacted by supplemental choline in the diet in either growth phase (Table 3). 1 Data were subjected to analysis of variance using the MIXED procedure of SAS with pen as the experimental unit and pen location as the blocking factor.The PDIFF option was used to perform pair-wise comparisons on the LS means.Each treatment was represented by 24 replicate pens (30 birds/pen).Broilers received diets provided in two phases: starter (day 1 to 15) and grower (day 16 to 31). 2 AD = average daily, BW = body weight, MC = mortality corrected, BWG = body weight gain, FI = feed intake, FCR = feed conversion ratio (feed:gain).
3 SEM = highest standard error of the LS mean pair-wise comparisons. 1 Data were subjected to analysis of variance using the GLIMMIX procedure of SAS with pen as the experimental unit.Percentages were derived from proportions generated using the events/possible events syntax with a binomial distribution and R-side covariance structure in SAS.The PDIFF option was used to perform pair-wise comparisons on the LS means.Each treatment was represented by 24 replicate pens (30 birds/pen).Broilers received diets provided in two phases: starter (day 1 to 15) and grower (day 16 to 31). 2 SEM = highest standard error of the LS mean pair-wise comparisons.

Carcass Characteristics
Adding increasing concentrations of choline chloride above what was already present in the diet did not impact chilled WOG weight or carcass yield (Table 4).Carcass part weights were similar among treatments with the exception of a trend for reduced abdominal fat pad weight in broilers fed 1600 and 2000 mg of added choline per kg of feed compared with birds not provided additional choline.This is consistent with choline's known functions in lipid metabolism and energy homeostasis [27].Similar reductions in broiler abdominal fat with increasing dietary choline have been observed previously [17], but these observations are inconsistent across studies [16,28].The trend observed in diminishing abdominal fat was not observed when carcass part yields were determined as a proportion of the day 31 live bodyweight, which is in agreement with Igwe et al. [4].Birds fed 800 mg or more of added choline per kg of feed resulted in a reduction in drumsticks as a proportion of the chilled WOG when compared with no added choline.This serves as a potential benefit of choline supplementation as carcass weight may be diverted to other more profitable carcass parts such as breast meat.However, no other impacts of choline supplementation of individual carcass part yields were observed, which may be due to high variability in the flock as both male and female broilers were used in this experiment.Future studies using single sex flocks are likely to capture effects of increased dietary choline on broiler carcass characteristics more precisely. 1Continuous data (e.g., parts weights) were subjected to analysis of variance using the MIXED procedure of SAS with pen as the experimental unit and pen location as the blocking factor.Proportional data (e.g., percent yields) were subjected to analysis of variance using the GLIMMIX procedure of SAS with pen as the experimental unit.Percentages were derived from proportions generated using the events/possible events syntax with a binomial distribution and R-side covariance structure in SAS.The PDIFF option was used to perform pair-wise comparisons on the LS means.Each treatment was represented by 24 replicate pens (30 birds/pen).Broilers received diets provided in two phases: starter (day 1 to 15) and grower (day 16 to 32).

Wooden Breast and White Striping
Table 4 describes the proportions of breasts assigned each WB and WS severity score out of the total breasts scored within each treatment.The incidence of WB and WS as well as average severity scores were similar among added choline chloride treatments.WB development is associated with rapid growth and increased breast yield [29].Therefore, these findings are as expected considering no differences in growth rate, breast weight, or breast yield were observed.
In these low-stress, research conditions where stocking density, temperature, and dietary nutrient composition were maintained within optimal ranges, increasing additions of choline chloride in the diets of modern, large frame broilers reared to 32 days did not impact growth performance.Supplemental dietary choline reduced abdominal fat pad weight and drumstick yield but did but not alter entire carcass yield or breast myopathy incidence.Further investigation is warranted evaluating supplementary choline in birds reared to heavier weights and in rearing conditions that more closely resemble a commercial broiler environment.
2 BW = body weight, WOG = without giblets, chilled carcass weight, WT = weight. 3SEM = highest standard error of the LS mean pair-wise comparisons.a,b Means with different superscripts differ p ≤ 0.05 and are considered different.v,w,x,y,z Means with different superscripts differ 0.0501 ≤ p ≤ 0.10 and are considered tendencies.

Table 1 .
Composition of broiler chicken diets fed for 32 days.

Table 2 .
Effects of choline chloride supplementation on broiler growth performance from 0 to 31 days 1 .

Table 3 .
Effects of choline chloride supplementation on broiler mortality from 0 to 31 days 1 .

Table 4 .
Effects of choline chloride supplementation on broiler carcass characteristics at 32 days of age 1 .