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. 2024 Mar 19;19(3):e0295488. doi: 10.1371/journal.pone.0295488

Choline supplementation: Impact on broiler chicken performance, steatosis, and economic viability from from 1 to 42 days

Matheus Ramalho de Lima 1,*,#, Isabelle Naemi Kaneko 2,‡, Adiel Vieira de Lima 2,‡, Lucas Nunes de Melo 2,‡, Mario Cesar de Lima 2,‡, Anna Neusa Eduarda Ferreira de Brito 2,‡, Fernando Guilherme Perazzo Costa 2,#, Andreia D C Vilas Boas 3,#, Ana Louise Toledo 3,#, Sigfrido Lopez Ferrer 4,#, Saravanakumar Marimuthu 5,#
Editor: Ewa Tomaszewska6
PMCID: PMC10950236  PMID: 38502648

Abstract

This study was carried out to compare the impact of choline supplementation (available from two sources synthetic and natural) on various dosages in broilers. The mode of choline supplementation, via diet and additional sources, synthetic and natural, and the data of performance, carcass quality, blood parameters, and hepatic steatosis were compared. A total of 1050 day-old male Cobb 500 broiler chicks were randomly assigned to 10 treatments, using a completely randomized design model in a factorial scheme, with 6 replicates per treatment and 25 birds per replicate. Choline was supplemented using three sources: synthetic choline chloride 60% (CC), and two sources of natural choline A (NCA), and B (NCB). The Control treatment did not receive any choline supplementation. The diets were supplemented with low, intermediate and high doses of choline sources (400g/t, 800g/t, and 1200g/t of CC; 100g/t, 200g/t, and 300g/t of both NCA and NCB). Data analysis was performed using a factorial model to investigate the effects of choline supplementation (CC, NCA, NCB) and doses on the measured variables. Overall, the results indicated that the the performance of NCA was better than CC & NCB, specifically the dose of 100g/t of NCA outperformed MAR at 100g/t & CC at 400g/t, leading to a significant increase in body weight gain (85.66g & 168.84g respectively), and a noteworthy (9- & 12-point respectively) improvement in feed conversion ratio. Furthermore, NCA contributed to a reduction in steatosis when contrasted with various NCB & CC doses, likely due to the presence of curcumins and catechins in the natural choline source. These findings demonstrated that NCA supplementation yielded superior results compared to CC and NCB across both performance and liver health aspects in broilers aged 1 to 42 days. In conclusion, NCA can be used to replace the CC 60% without compromise on the zootechnical performance in broilers.

Introduction

The use of choline in commercial broiler feed is an important aspect in the feed industry to enhances the growth performance and regulates the lipid metabolism as well. Typically, choline plays a significant role in many important metabolic pathways, being crucial for the structural maintenance of cell membranes and organelles, acting as a constituent of phospholipids [1, 2] synthesis of methionine from homocysteine as a labile methyl donor; and a precursor molecule for the formation of acetylcholine in the nervous system. In addition, the choline is a component of very-low-density lipoproteins (VLDL) which involves in triglyceride transport out of the liver and thereby reduces the accumulation of fat in the liver [3–6]. It also inhibits the fatty acid synthesis by downregulating fatty acid synthase gene expression as well as attenuating its activity [7, 8]. Furthermore, choline is referred to as a "lipotropic" factor due to its role in increasing fat utilization, which results in the reduction of fat deposition in the body [5, 9].

However, the absence of choline, or levels below the recommendation, increases deleterious effects on meat chickens, such as reduction in growth and perosis, especially in younger birds [5, 10]. Choline supplementation is commonly done by synthetic sources, but high hygroscopicity and oxidation with loss of vitamins, its corrosive nature, as well as the trimethylamine formation in the intestinal tract of broilers (the common reason less than half of the choline chloride is absorbed), allowed th te scientist to look for an alternative [11, 12].

In this respect, research has been developed to unveil potential substitutes for synthetic sources, and formulations with herbs have been gaining ground with consistent results in hepatoprotective capacity and improvement of performance parameters. These natural products, produced from selected plants and blends of herbs, which can mimic the function of choline [12]. Chandrasekaran et. al [13] when investigating the lipotropic activity of herbal formulations containing Acacia nilotica and Curcuma longa, demonstrated the lipotropic effect of this natural source which was highlighted by low fat accumulation and anti-lipogenic activity. The formulations of natural choline using herbs such as Acacia nilotica and Curcuma longa paved a way to get the associating benefits and thus enhancing the effects of hepatoprotective action which was being reported discretely [14, 15].

Based on these infromations, it is possible to say the natural choline supplementation, specifically with Acacia nilotica and Curcuma longa, will yield superior results compared to synthetic choline supplementation. This superiority will be evidenced by improved performance parameters, reduced hepatic steatosis, and enhanced hepatoprotective action. This hypothesis is based on the premise that natural sources of choline not only mimic the function of synthetic choline but also offer additional benefits such as hepatoprotective action. Hence the current study was carried out to compare the impact of choline supplementation (available from two sources synthetic source and natural sources) on various dosages in broilers. The mode of choline supplementation, via diet and additional sources, synthetic and natural, and the data of performance, carcass quality, blood parameters, and hepatic steatosis were compared.

Material and methods

Housing conditions and management

Housing, feeding regimens, and rearing conditions used at the Federal University of Paraiba (UFPB) are thought to be representative of modern commercial broiler operations in Brazil. This trial was carried out at an experimental farm located in Areia–Paraiba, Brazil (6 degrees, 57 minutes, and 48 seconds south of the equator (latitude), 35 degrees, 41 minutes, and 30 seconds west of the prime meridian (longitude), and 618 meters above sea level (altitude).

Farm facilities comprise 88 identical floor pens (2.25m2). Of these, 42 pens were used. The trial was carried out with 1050 day-old male Cobb 500 broiler chicks, 39.66 ± 0.20g, organized in a factorial experiment, 3 x 3 + 1. The effect size was calculated using Cohen’s d. A total of 1050 day-old male Ross 500 broiler chicks were randomly assigned to 10 treatments, using a completely randomized design model in a factorial scheme, with 6 replicates per treatment and 25 birds per replicate. Individual pens measuring 1.5 x 1.5 m served as experimental units. Pens were consecutively numbered with the respective treatment number using pen cards.

Birds were kept in appropriate (i.e., optimum) environmental conditions (temperature, from ±32°C from 1-7d; ±30°C from 8-14d; ±26°C from 15 to 42d) for age during the trial. Lighting was provided using fluorescent bulbs, To maintain an intensity of 23 lux, with 24 hours for 7 days, and 23 hours of light up to 42 days. The experimental period totalled 42 days. A 3-phase feeding program was used as follows: starter (days 1–21), grower (days 22–35), and finisher (days 36–42). Feed was provided in a pan feeder (capacity, 20 kg). Water was offered ad libitum using drinkers.

Standard floor pen management practices were used throughout the experimental period. Birds and facilities were inspected twice daily. The following data were recorded: general health status of broilers, environmental temperature, and constant feed and water supply. Dead birds were removed, and unexpected events were identified. Deaths, including pen number, date of death, bird weight, and potential diagnosis, were recorded using a Daily Mortality Record.

Day-old male Cobb chicks hatched from eggs produced by 30 weeks-old breeders and weighing 40g on average were obtained from Frango Dourado hatchery (Pernambuco, Brazil). Twenty-five male broiler chicks were housed in each pen. Birds were sexed at the hatchery by feather sexing method.

Dietary treatments

Choline was supplemented using three sources: synthetic choline chloride 60% (CC), and two sources of natural choline A (NCA), and B (NCB). The Control treatment did not receive any choline supplementation. The diets were supplemented with low, intermediate and high doses of choline sources (400g/t, 800g/t, and 1200g/t of CC; 100g/t, 200g/t, and 300g/t of both NCA and NCB). Natural Choline A (NCA) is indexed as Kolin Plus (M/s Natural Remedies Pvt Ltd, Bengaluru, India), is a polyherbal formulation containing a combination of Acacia nilotica (A. nilotica) and Curcuma longa (C. longa) belonging to the families of Mimosaceae and Zingiberaceae, respectively. Natural Choline B (NCB) contains mainly Achyranthes aspera, Trachyspermum ammi, Azadirachta indica, and others.

Feed

Mash feeds were manufactured at UFPB Agrarian Center—Campus II, located in Areia, Paraiba, Brazil. Broiler diets were formulated with feedstuffs widely used in Brazil. Diets were representative of local commercial feed formulations and designed to meet or exceeded nutritional requirements for broiler strain and age. Feed batches were independently mixed and bagged. Feed bags were labeled with a trial number, mixing date, type of feed, dietary treatment, and replicate number. Detailed records of feed mixtures and test product inventories were kept. Test feeds were mixed for 4 minutes at 28 rev/min in a 100 kg capacity mixer. The experimental diets were described in Tables 1–3.

Table 1. Experimental diets from 1 to 21 days.

Investment Basal CC CC CC NCA NCA NCA NCB NCB NCB
kg 400 g/t 800 g/t 1,200 g/t 100 g/t 200 g/t 300 g/t 100 g/t 200 g/t 300 g/t
Macro Ingredients
Corn 7.8% BRL 1.50 575.1 574.69 574.29 572.89 575 574.9 574.79 575 574.9 574.79
Soybean meal 45% BRL 3.00 334 334 334 334 334 334 334 334 334 334
Meat meal 38% BRL 3.00 29 29 29 29 29 29 29 29 29 29
Degummed soybean oil BRL 6.00 37 37 37 38 37 37 37 37 37 37
Limestone 36% BRL 0.25 8.2 8.2 8.2 8.2 8.2 8.2 8.2 8.2 8.2 8.2
Common salt BRL 0.60 4.6 4.6 4.6 4.6 4.6 4.6 4.6 4.6 4.6 4.6
Mineral and vitamin premix 0.4%1 BRL 1.00 4 4 4 4 4 4 4 4 4 4
Micro Ingredients
Choline chloride 60%, CC BRL 13.68 0.4 0.8 1.2
Natural Choline, NCA BRL 27.35 0.1 0.2 0.3
Natural Choline, NCB BRL 32.82 0.1 0.2 0.3
L-Lysine HCL BRL 8.00 3.15 3.15 3.15 3.15 3.15 3.15 3.15 3.15 3.15 3.15
DL-Methionine 99% BRL 18.00 3.7 3.7 3.7 3.7 3.7 3.7 3.7 3.7 3.7 3.7
L-Threonine BRL 7.00 1.25 1.26 1.26 1.26 1.25 1.25 1.26 1.25 1.25 1.26
Total Mixed (kg) 1,000.00 1,000.00 1,000.00 1,000.00 1,000.00 1,000.00 1,000.00 1,000.00 1,000,00 1,000,00
End Price (BRL/kg) BRL 2.28 BRL 2.29 BRL 2.29 BRL 2.29 BRL 2.28 BRL 2.28 BRL 2.29 BRL 2.29 BRL 2.29 BRL 2.30
Crude protein % 21.22 21.22 21.22 21.22 21.22 21.22 21.22 21.22 21.22 21.22
Crude fiber % 3.35 3.35 3.35 3.35 3.35 3.35 3.35 3.35 3.35 3.35
Calcium % 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99
Available phosphorus % 0.47 0.47 0.47 0.47 0.47 0.47 0.47 0.47 0.47 0.47
Metabolizable energy kcal/kg 3.140.00 3.140.00 3.140.00 3.140.00 3.140.00 3.140.00 3.140.00 3.140.00 3.140.00 3.140.00
Digestible lysine % 1.26 1.26 1.26 1.26 1.26 1.26 1.26 1.26 1.26 1.26
Digestible methionine % 0.65 0.65 0.65 0.65 0.65 0.65 0.65 0.65 0.65 0.65
Digestible methionine + cystine % 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93
Digestible threonine % 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83
Choline mg/kg 1.162.65 1.162.49 1.162.54 1.162.60 1.162.60 1.162.54 1.162.49 1.162.60 1.162.54 1.162.49
Phosphatidylcholine g/kg - - - - 25 50 75 16 32 48
Sodium % 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.22
Chlorine % 0.41 0.41 0.41 0.41 0.41 0.41 0.41 0.41 0.41 0.41
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Dollar equivalent to BRL 5.47, July 20, 2022. 1Minimum premix per kilogram of feed: Mn: 60 g; Fe: 80 g; Zn: 50 g; Cu: 10 g; Co: 2 g; I: 1 g; Se: 250 mg.; Vitamin per kilogram of feed: vitamin A: 15,000,000 IU; vitamin D3: 1,500,000 IU; vitamin E: 15,000 IU; vitamin B1: 2.0 g; vitamin B2: 4.0 g; vitamin B6: 3.0 g; vitamin B12: 0.015 g; nicotinic acid: 25 g; pantothenic acid: 10 g; vitamin K3: 3.0 g; folic acid: 1.0 g.

Table 3. Experimental diets from 36 to 42 days.

Investment Basal CC CC CC NCA NCA NCA NCB NCB NCB
kg 400 g/t 800 g/t 1,200 g/t 100 g/t 200 g/t 300 g/t 100 g/t 200 g/t 300 g/t
Macro Ingredients
Corn 7.8% BRL 1.50 639.33 637.93 637.53 637.12 638.24 638.14 638.03 638.24 638.14 638.03
Soybean meal 45% BRL 3.00 264 265 265 265 265 265 265 265 265 265
Meat meal 38% BRL 3.00 19 19 19 19 19 19 19 19 19 19
Degummed soybean oil BRL 6.00 55 55 55 55 55 55 55 55 55 55
Limestone 36% BRL 0.25 8.5 8.5 8.5 8.5 8.5 8.5 8.5 8.5 8.5 8.5
Common salt BRL 0.60 3.8 3.8 3.8 3.8 3.8 3.8 3.8 3.8 3.8 3.8
Mineral and vitamin premix 0.4%1 BRL 1.00 4 4 4 4 4 4 4 4 4 4
Micro Ingredients
Choline chloride 60%, CC BRL 13.68 0.4 0.8 1.2
Natural Choline, NCA BRL 27.35 0.1 0.2 0.3
Natural Choline, NCB BRL 32.82 0.1 0.2 0.3
L-Lysine HCL BRL 8.00 2.75 2.74 2.74 2.74 2.74 2.74 2.74 2.74 2.74 2.74
DL-Methionine 99% BRL 18.00 2.77 2.78 2.78 2.78 2.77 2.77 2.78 2.77 2.77 2.78
L-Threonine BRL 7.00 0.85 0.85 0.85 0.86 0.85 0.85 0.85 0.85 0.85 0.85
Total Mixed (kg) 1,000.00 1,000.00 1,000.00 1,000.00 1,000.00 1,000.00 1,000.00 1,000.00 1,000.00 1,000.00
End Price (BRL/kg) BRL 2.22 BRL 2.23 BRL 2.23 BRL 2.23 BRL 2.23 BRL 2.23 BRL 2.23 BRL 2.23 BRL 2.23 BRL 2.24
Crude protein % 18.09 18.08 18.08 18.08 18.09 18.09 18.08 18.09 18.09 18.08
Crude fiber % 3.02 3.02 3.01 3.01 3.02 3.02 3.02 3.02 3.02 3.02
Calcium % 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83
Available phosphorus % 0.39 0.39 0.39 0.39 0.39 0.39 0.39 0.39 0.39 0.39
Metabolizable energy kcal/kg 3,330.00 3,330.00 3,330.00 3,330.00 3,330.00 3,330.00 3,330.00 3,330.00 3,330.00 3,330.00
Digestible lysine % 1.04 1.04 1.04 1.04 1.04 1.04 1.04 1.04 1.04 1.04
Digestible methionine % 0.52 0.52 0.52 0.52 0.52 0.52 0.52 0.52 0.52 0.52
Digestible methionine + cystine % 0.77 0.77 0.77 0.77 0.77 0.77 0.77 0.77 0.77 0.77
Digestible threonine % 0.69 0.69 0.69 0.69 0.69 0.69 0.69 0.69 0.69 0.69
Choline mg/kg 1,007.18 1,185.78 1,364.38 1,542.98 1,007.13 1,007.08 1,007.03 1,007.13 1,007.08 1,007.03
Phosphatidyl choline g/kg 25 50 75 16 32 48
Sodium % 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18
Chlorine % 0.34 0.34 0.34 0.34 0.34 0.34 0.34 0.34 0.34 0.34
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Dollar equivalent to BRL 5.47, July 20, 2022. 1Minimum premix per kilogram of feed: Mn: 60 g; Fe: 80 g; Zn: 50 g; Cu: 10 g; Co: 2 g; I: 1 g; Se: 250 mg.; Vitamin per kilogram of feed: vitamin A: 15,000,000 IU; vitamin D3: 1,500,000 IU; vitamin E: 15,000 IU; vitamin B1: 2.0 g; vitamin B2: 4.0 g; vitamin B6: 3.0 g; vitamin B12: 0.015 g; nicotinic acid: 25 g; pantothenic acid: 10 g; vitamin K3: 3.0 g; folic acid: 1.0g.

Table 2. Experimental diets from 22 to 35 days.

Investment Basal CC CC CC NCA NCA NCA NCB NCB NCB
kg 400 g/t 800 g/t 1,200 g/t 100 g/t 200 g/t 300 g/t 100 g/t 200 g/t 300 g/t
Macro Ingredients
Corn 7.8% BRL 1.50 604.29 603.88 602.48 602.08 604.19 604.09 603.98 604.19 604.09 603.98
Soybean meal 45% BRL 3.00 299 299 299 299 299 299 299 299 299 299
Meat meal 38% BRL 3.00 24 24 25 25 24 24 24 24 24 24
Degummed soybean oil BRL 6.00 49 49 49 49 49 49 49 49 49 49
Limestone 36% BRL 0.25 8.3 8.3 8.3 8.3 8.3 8.3 8.3 8.3 8.3 8.3
Common salt BRL 0.60 4.2 4.2 4.2 4.2 4.2 4.2 4.2 4.2 4.2 4.2
Mineral and vitamin premix 0.4%1 BRL 1.00 4 4 4 4 4 4 4 4 4 4
Micro Ingredients
Choline chloride 60%, CC BRL 13.68 0.4 0.8 1.2
Natural Choline, NCA BRL 27.35 0.1 0.2 0.3
Natural Choline, NCB BRL 32.82 0.1 0.2 0.3
L-Lysine HCL BRL 8.00 2.93 2.93 2.93 2.93 2.93 2.93 2.93 2.93 2.93 2.93
DL-Methionine 99% BRL 18.00 3.23 3.24 3.24 3.24 3.23 3.23 3.24 3.23 3.23 3.24
L-Threonine BRL 7.00 1.05 1.05 1.05 1.05 1.05 1.05 1.05 1.05 1.05 1.05
Total Mixed (kg) 1,000.00 1,000.00 1,000.00 1,000.00 1,000.00 1,000.00 1,000.00 1,000.00 1,000.00 1,000.00
End Price (BRL/kg) BRL 2.27 BRL 2.28 BRL 2.27 BRL 2.27 BRL 2.27 BRL 2.27 BRL 2.27 BRL 2.27 BRL 2.28 BRL 2.28
Crude protein % 19.64 19.63 19.63 19.63 19.64 19.63 19.63 19.64 19.63 19.63
Crude fiber % 3.18 3.17 3.17 3.17 3.18 3.18 3.18 3.18 3.17 3.17
Calcium % 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92 0.92
Available phosphorus % 0.43 0.43 0.43 0.43 0.43 0.43 0.43 0.43 0.43 0.43
Metabolizable energy kcal/kg 3,250.00 3,250.00 3,250.00 3,250.00 3,250.00 3,250.00 3,250.00 3,250.00 3,250.00 3,250.00
Digestible lysine % 1.15 1.15 1.15 1.15 1.15 1.15 1.15 1.15 1.15 1.15
Digestible methionine % 0.58 0.58 0.58 0.58 0.58 0.58 0.58 0.58 0.58 0.58
Digestible methionine + cystine % 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85
Digestible threonine % 0.76 0.76 0.76 0.76 0.76 0.76 0.76 0.76 0.76 0.76
Choline mg/kg 1,083.45 1,262.05 1,440.65 1,619.24 1,083.40 1,083.35 1,083.30 1,083.40 1,083.35 1,083.30
Phosphatidyl choline g/kg 25 50 75 16 32 48
Sodium % 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2
Chlorine % 0.37 0.37 0.37 0.37 0.37 0.37 0.37 0.37 0.37 0.37
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Dollar equivalent to BRL 5.47, July 20, 2022. 1Minimum premix per kilogram of feed: Mn: 60 g; Fe: 80 g; Zn: 50 g; Cu: 10 g; Co: 2 g; I: 1 g; Se: 250 mg.; Vitamin per kilogram of feed: vitamin A: 15,000,000 IU; vitamin D3: 1,500,000 IU; vitamin E: 15,000 IU; vitamin B1: 2.0 g; vitamin B2: 4.0 g; vitamin B6: 3.0 g; vitamin B12: 0.015 g; nicotinic acid: 25 g; pantothenic acid: 10 g; vitamin K3: 3.0 g; folic acid: 1.0 g.

Measurements

Performance

In this study, the daily mortality rate of the chicks was recorded. The body weight (BW, g/broiler) of each chick was measured on a scale on Day 0, Day 21, Day 35, and Day 42 (end of the experimental period), and these measurements were used to calculate the body weight gain (BWG, g/broiler). The feed intake (FI, g/broiler) was determined by measuring and recording the amount of feed consumed by the chicks on Day 21, Day 35, and Day 42. The difference between the initial and final weights for each period provided the amount of feed intake. The body weight gain (BWG) was calculated by subtracting the initial body weight (on Day 0) from the final body weight (on Day 42). The feed conversion ratio (FCR, g/g) was then calculated by dividing the total feed intake by the total body weight gain. This calculation was adjusted for mortality, meaning the weight of the chicks that died was subtracted from the total body weight gain before the FCR was calculated.

Carcass

On Day 42, after performance data collection, 5 broilers from each replicate, selected by average weight, were euthanized for assessment of various traits. The carcass characteristics were evaluated, providing insight into the physical and qualitative attributes of the broiler carcasses. The amount of abdominal fat, an indicator of the overall health and condition of the broilers, was determined. The yields of the whole carcass, breast meat, thigh meat, and drumstick meat were also determined. These yields, calculated as a percentage of the broiler’s total body weight.

Fatty liver data

On D42, liver samples (1 cm2) were collected and coated with talc, immediately frozen in liquid nitrogen, and stored in an ultra-freezer. Frozen samples were cut into 20 μm thick sections using a cryostat-microtome for historical slide preparation. Slides were then stained with Sudan IV for lipid quantification. Areas of lipid deposition were stained in red. Samples were scored as follows: 1—no lipid quantification, 2—few areas of lipid quantification, 3—larger areas of lipid quantification, and 4—lipid quantification over all tissue.

Economic analysis

Combined analysis of liveability (i.e., the final number of birds per treatment), performance, carcass yield, and economic data, which can be adjusted according to natural variations, revealed relative gross margin fluctuations across different scenarios. Using the Control treatment as a reference, the relative gross income derived from breast, thigh, drumstick, and whole chicken sales is 100%. The same applies to the remaining treatments in the same order. Hence, 100 can be used as a benchmark to estimate the difference between treatments.

Liver cholesterol, triglycerides, and lipids

On D42, liver samples (1 cm2) were collected. One set (Triplicates of ~1 g of tissue) of samples were collected from dispersed zones of the frozen left lobe. The exact wet weight of each sample was determined after thawing and dehydrating the excess moisture on a Whatman filter paper for 10 min at 25°C. Then the total fat was extracted from the liver samples followed by liver triglycerides and cholesterol were quantified using the [16], and lipids content using [17].

Statistical analysis

The statistical analysis of the characteristics studied was performed using the R software, version 4.2.1. The analysis of variance (ANOVA) assumptions (error normality, random and independent errors, and variance homoscedasticity) was met.

Data analysis was performed using a factorial model to investigate the effects of choline supplementation (CC, NCA, NCB) and doses on the measured variables. The factorial model can be represented as Yijk = μ + αi + βj + (αβ)ij + εijk, where: Yijk is the dependent variable (the measured variables in the study), μ is the overall mean, αi is the effect of the level of choline supplementation (CC, NCA, NCB), βj is the effect of the level of dose, (αβ)ij is the interaction effect between choline supplementation and dose, and εijk is the random error term.

A two-way analysis of variance (ANOVA) was conducted to assess the main effects of choline supply and doses, as well as any interactions between them. The significance level was set at α = 0.05 for all tests. Post hoc Tukey tests were conducted to compare the means of each choline source and dose to identify specific differences.

Results

There was a significant interaction observed in body weight (BW) (P = 0.0405), body weight gain (BWG) (P = 0.0394), and feed intake (FI) (P = 0.0139), as shown in Table 4. In the overall effect, birds exhibited higher weights with CC supplementation compared to the Without group and NCB (P = 0.033), but similar to NCA (P>0.05). Weight gain significantly improved with the intermediate dosage compared to the control (P = 0.164), but was similar to the low and high dosages (P>0.05). The BWG data were consistent with the findings regarding final body weight in all aspects. There was no significant effect observed on feed consumption, feed conversion ratio, and survival rate.

Table 4. Effect of choline supplementation on zootechnical parameters of broilers at 21 days of age.

Supplementation IBW BW BWG FI FCR LIVE %
Without 39.605 866.131b 826.525b 1320.004 1.596 99.123
CC 39.626 894.498a 854.872a 1357.403 1.588 99.415
NCA 39.661 880.030ab 840.370ab 1304.835 1.553 98.538
NCB 39.716 870.052b 830.335b 1336.707 1.610 99.415
Dosage 1 IBW BW BWG FI FCR LIVE
Control 39.605 866.131b 826.525b 1320.004 1.596 99.123
Low 39.649 874.488ab 834.839ab 1335.071 1.600 99.123
Intermediate 39.675 892.914a 853.239a 1353.100 1.586 98.538
High 39.678 877.178ab 837.500ab 1310.773 1.565 99.708
Source 0.5521 0.0033 0.0031 0.3046 0.3764 0.4819
Dosage 0.88 0.0164 0.0162 0.4921 0.7422 0.3534
Interaction 1.000 0.0405 0.0394 0.0139 0.1377 0.097
SEM 0.05 4.7622 4.7572 19.5552 0.02424 0.4623
C.V. (%) 0.54 2.36 2.46 6.29 6.22 1.94
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IBW–Initial body weight; BW–Body weight; BWG–Body weight gain; FI–Feed intake; FCR–Feed conversion ratio; LIVE–Liveability; 1Dosage means the dose of each choline source, being Low (100g/t for NCA and NCB and 400g/t for CC), Intermediate (200g/t for NCA and NCB and 800g/t for CC), and High (300g/t for NCA and NCB and 1200g/t for CC). a,b Means followed by different letters in the same column differ significantly Tukey test, P<0.05.

Higher body weight (BW) was observed with choline supplementation via CC at the intermediate dosage (P = 0.034) and high dosage (P = 0.0032), while no significant difference was found at the low dosage (P = 0.1232), as evidenced by the data presented in Table 5. Notably, there was no observable effect on the incremental doses with CC supplementation (P = 0.0948) or NCA supplementation (P = 0.7947). However, NCB supplementation resulted in a significantly higher final weight at the intermediate dosage compared to the low and high dosages (P = 0.0150). The BWG data exhibited similar patterns to those observed in BW. Regarding feed intake, significantly higher values were obtained with NCB supplementation compared to NCA (P = 0.0161), although no significant difference was observed between NCB and CC (P>0.05). Moreover, NCB showed greater feed intake at the intermediate dosage compared to the high dosage (P = 0.0029), while no significant difference was found between the intermediate and low dosages (P>0.05).

Table 5. Effect of interaction between the supplementation sources of broilers at 21 days of age.

BW Control CC NCA NCB P value
Control 866.131
Low 880.526 882.831 860.106B 0.1232
Intermediate 907.010a 875.366b 896.365abA 0.034
High 895.957a 881.893ab 853.683bB 0.0032
P value 0.0948 0.7947 0.0015
SEM 5.5422 4.4332 3.2703
BWG Control CC NCA NCB P value
Control 826.525
Low 840.921 843.182 820.413B 0.1232
Intermediate 867.378a 835.699b 856.637abA 0.0333
High 856.316a 842.226ab 813.955bB 0.003
P value - 0.094 0.793 0.0015
SEM 4.7253 4.2941 3.8713
FI Control CC NCA NCB P value
Control 1320.000
Low 1356.491 1309.424 1339.298AB 0.6188
Intermediate 1357.958ab 1278.326b 1423.014aA 0.0161
High 1357.758 1326.754 1247.807B 0.0739
P value 0.9995 0.601 0.0029
SEM 20.7263 19.1435 18.1241
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BW–Body weight; BWG–Body weight gain; FI–Feed intake; a,b Means followed by different letters in the same line differ significantly Tukey test, P<0.05. A, B Means followed by different letters in the same column differ significantly Tukey test, P<0.05.

There was a significant interaction observed in BW (P = 0.0164), BWG (P = 0.0164), FI (P = 0.0339), and FCR (P = 0.0097), as presented in Table 6. In the overall effect, feed conversion ratio (FCR) showed higher values with NCB supplementation compared to NCA (P = 0.008) but was similar to CC and the Without supplementation (P>0.05).

Table 6. Effect of choline supplementation on zootechnical parameters of broilers at 35 days of age.

Supplementation BW BWG FI FCR LIVE
Without 2147.639 2108.034 3586.367 1.704ab 96.491
CC 2199.488 2159.862 3648.010 1.690ab 96.491
NCA 2184.211 2144.550 3560.725 1.661b 97.076
NCB 2154.361 2114.645 3671.365 1.736a 97.368
Dosage 1 BW BWG FI FCR LIVE
Control 2147.639 2108.034 3586.367 1.704 96.491
Low 2167.512 2127.863 3606.538 1.696 96.491
Intermediate 2188.708 2149.032 3690.273 1.718 96.491
High 2181.841 2142.162 3583.288 1.673 97.953
Source 0.2937 0.2929 0.1809 0.008 0.8704
Dosage 0.6818 0.6831 0.2054 0.2035 0.5254
Interaction 0.0164 0.0164 0.0339 0.0097 0.2538
SEM 12.6839 12.6845 41.7221 0.0018 1.0065
C.V. (%) 3.67 3.74 4.44 3.73 3.56
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IBW–Initial body weight; BW–Body weight; BWG–Body weight gain; FI–Feed intake; FCR–Feed conversion ratio; LIVE–Liveability; 1Dosage means the dose of each choline source, being Low (100g/t for NCA and NCB and 400g/t for CC), Intermediate (200g/t for NCA and NCB and 800g/t for CC), and High (300g/t for NCA and NCB and 1200g/t for CC. a,b Means followed by different letters in the same column differ significantly Tukey test, P<0.05.

Higher BW was observed with choline supplementation via CC at intermediate and high dosages compared to low dosages (P = 0.099), as indicated by the data presented in Table 7. The BWG data showed similar effects to those obtained in BW. NCB exhibited higher feed intake at the intermediate dosage compared to the high dosage (P = 0.0039) but was similar to the low dosage (P>0.05). The FCR demonstrated better results with NCA compared to CC and NCB at the low dosage (P = 0.0425). At the intermediate dosage, the FCR showed higher values with the NCB source compared to the CC and NCA sources (P = 0.003). The FCR with the use of CC (P = 0.2294) or NCA (P = 0.5825) is similar to the evaluated dosages, whereas, with NCB, there is a decrease in effectiveness at the intermediate dosage compared to the other dosages of this additive (P = 0.0026).

Table 7. Effect of interaction between the supplementation sources of broilers at 35 days of age.

BW Control CC NCA NCB P value
Control 2147.639
Low 2115.493B 2216.527 2170.515 0.1008
Intermediate 2251.508A 2153.38 2161.234 0.0708
High 2231.462A 2182.726 2131.332 0.1053
SEM 10.3563 10.4372 10.6345
P value 0.0099 0.3982 0.6765
BWG Control CC NCA NCB P value
Control 2108.034
Low 2075.888B 2176.878 2130.822 0.101
Intermediate 2211.876A 2113.713 2121.506 0.0706
High 2191.822A 2143.059 2091.604 0.105
SEM 10.7265 10.4523 10.6294
P value 0.01 0.3981 0.6763
FI Control CC NCA NCB P value
Control 3586.367
Low 3579.563 3577.258 3662.793AB 0.58
Intermediate 3679.007 3552.885 3838.927A 0.0128
High 3685.459 3552.031 3512.373B 0.1592
SEM 40.7253 35.0561 34.0662
P value 0.4475 0.9536 0.0039
FCR Control CC NCA NCB P value
Control 1.704
Low 1.725a 1.643b 1.719aB 0.0425
Intermediate 1.664b 1.680b 1.810aA 0.0003
High 1.681 1.657 1.679B 0.7663
SEM 0.0153 0.0184 0.0177
P value 0.2294 0.5825 0.0026
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BW–Body weight; BWG–Body weight gain; FI–Feed intake; FCR–Feed conversion ratio; a,b Means followed by different letters in the same line differ significantly Tukey test, P<0.05. A, B Means followed by different letters in the same collum differ significantly Tukey test, P<0.05.

There was a significant interaction observed in BW (P = 0.0166), BWG (P = 0.0167), and FCR (P = 0.0457), as shown in Table 8. In the overall effect, FCR showed better results with NCA compared to NCB and CC (P = 0.0432), and was similar to the Without supplementation (P>0.05).

Table 8. Effect of choline supplementation on zootechnical parameters of broilers at 42 days of age.

Supplementation BW BWG FI FCR LIVE %
Without 2898.869 2859.263 4756.740 1.665ab 94.737
CC 2938.525 2898.899 4954.271 1.710a 95.029
NCA 2978.035 2938.374 4770.241 1.624b 96.491
NCB 2916.491 2876.775 4884.642 1.699a 96.491
Dosage 1 BW42 BWG42 FI42 FCR42 LIVE42
Control 2898.869 2859.263 4756.740 1.665 94.737
Low 2950.548 2910.899 4856.164 1.670 95.029
Intermediate 2952.020 2912.345 4912.025 1.686 95.322
High 2930.483 2890.805 4840.964 1.677 97.661
Supplementation 0.3214 0.3216 0.2392 0.0432 0.6298
Dosage 0.7367 0.7374 0.7143 0.9541 0.249
Interaction 0.0166 0.0167 0.1926 0.0457 0.8955
SEM 46.343 46.3591 120.5903 0.391 1.8105
C.V. (%) 3.86 3.92 6.08 5.72 4.63
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IBW–Initial body weight; BW–Body weight; BWG–Body weight gain; FI–Feed intake; FCR–Feed conversion ratio; LIVE–Liveability; Means followed by different letters in the same column differ significantly; Tukey test, P<0.05.

Higher BW was observed with choline supplementation via NCA at low dosages compared to CC and NCB (P = 0.0443), as shown in Table 9. The CC supplementation resulted in higher BW with intermediate and high doses compared to the low dose (P = 0.326), while NCA showed better results with the low dose compared to the others (P = 0.0203). The NCB was not influenced by the doses (P = 0.6306). Low dose weight gain was higher with NCA than with CC (P = 0.0445), but similar to NCB. FCR showed better results with NCA compared to NCB (P = 0.0405), but similar to CC.

Table 9. Effect of interaction between the supplementation sources of broilers at 42 days of age.

BW Control CC NCA NCB P value
Control 2898.869
Low 2866.541bB 3035.380aA 2949.721ab 0.0443
Intermediate 2999.664a 2943.436B 2912.958 0.4127
High 2949.368AB 2955.287B 2886.793 0.5159
SEM 20.4835 19.5264 20.4273
P value 0.0326 0.0203 0.6306
BWG Control CC NCA NCB P value
Control 2859.263
Low 2826.936b 2995.730ª 2910.028ab 0.0445
Intermediate 2960.033 2903.77 2873.23 0.4122
High 2909.728 2915.62 2847.065 0.5154
SEM 16.7247 15.7255 17.9958
P value 0.1329 0.3204 0.6305
FCR Control CC NCA NCB P value
Control 1.665
Low 1.7196 1.6 1.6911 0.0889
Intermediate 1.682ab 1.618b 1.757ª 0.0405
High 1.729 1.652 1.647 0.2577
SEM 0.0213 0.0261 0.02196
P value 0.6611 0.6302 0.1446
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BW–Body weight; BWG–Body weight gain; FI–Feed intake; FCR–Feed conversion ratio; a,b Means followed by different letters in the same line differ significantly Tukey test, P<0.05. A, B Means followed by different letters in the same column differ significantly Tukey test, P<0.05.

NCA demonstrates similarity to NCB, however, FCR significantly differs between them in the 1–42 day phase (P = 0.0405). The similarity of NCA with CC in this variable indicates that they are equivalent in providing choline satisfactorily. However, NCA shows better results with doses of 100g/t to 200g/t, while CC shows better results with doses of 800g/t to 1200g/t.

The breast yield was lower when NCB was used (P = 0.0036), as shown in Table 10. On the other hand, this source promoted higher thigh yield (P = 0.0251). The leg yield was better with the low dose of choline supplementation (P = 0.0065).

Table 10. Effect of choline supplementation on carcass characteristics of broilers at 42 days of age.

Supplementation CY BY LY FY TY DY
Without 28.472 31.283a 21.247 0.647 9.853b 11.239
CC 28.467 31.388ab 21.383 0.680 9.935ab 11.440
NCA 29.057 31.259ab 21.352 0.680 10.041ab 11.473
NCB 28.936 30.707b 21.548 0.646 10.133a 11.542
Dosage 1
Control 28.472 31.283 21.247 0.647 9.853b 11.239
Low 29.109 31.213 21.606 0.689 10.150a 11.510
Intermediate 28.676 31.065 21.273 0.665 10.048ab 11.484
High 28.674 31.077 21.405 0.652 9.910ab 11.461
Supplementation 0.0544 0.0036 0.5018 0.623 0.0251 0.1225
Dosage 0.1278 0.07729 0.1832 0.6674 0.0065 0.1938
Interaction 0.5198 0.02 0.031 0.1141 0.5095 0.1
SEM 0.291 0.261 0.201 0.04 0.437 0.499
C.V. (%) 5.53 4.25 5.15 32.54 5.3 5.29
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CY–Carcass yield %; BY–Breast yield %); LY–Leg yield %; FY—Fat yield %; TY–Thigh yield %; DY–Drumstick yield %; Means followed by different letters in the same column differ significantly; Tukey test, P<0.05.

The breast yield was influenced by the supplementation of the high dosage, with a superiority (P = 0.0002) of CC and NCA sources compared to NCB, Table 11. Regarding thighdrums yield, there was a better performance with NCB compared to the others (P = 0.0383). NCB performed better with the low dosage than the intermediate (P = 0.0017) but was similar to the high in ThighDrums yield.

Table 11. Effect of interaction between the supplementation sources of broilers at 42 days of age.

Breast Yield % Control CC NCA NCB P value
Control 31.283  
Low   31.184 31.627 30.827 0.0656
Intermediate   31.251 30.962 30.98 0.6401
High   31.728a 31.188a 30.313b 0.0002
SEM 0.188 0.154 0.172
P value   0.2233 0.1429 0.1257
Thighdrums Yield % Control CC NCA NCB P value
Control 21.247  
Low   21.482ab 21.317b 22.017aA 0.0383
Intermediate   21.428 21.394 20.998B 0.249
High   21.242 21.343 21.628AB 0.3752
SEM 0.131 0.101 0.164
P value   0.6804 0.9626 0.0017  
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a,b Means followed by different letters in the same line differ significantly Tukey test, P<0.05. A, B Means followed by different letters in the same collum differ significantly Tukey test, P<0.05.

A higher cholesterol content was obtained with the control group compared to the intermediate (P = 0.0591), while it was similar in the low and high choline supplementation dosages, as shown in Table 12. Meanwhile, the low dosage promoted a higher liver lipid content compared to the other dosages (P = 0.0394). In this context, the supplemented choline sources reduced the steatosis score compared to the diet without choline supplementation (P<0.001), with a reduction observed in all three evaluated dosages compared to the control (diet origin; P<0.001).

Table 12. Effects of treatments on liver parameters of broilers at 42 days of age.

Supplementation LC LT LL SS
Without 187.254 240.118 2.207 3.311a
CC 162.090 305.788 2.719 2.970b
NCA 144.885 215.012 3.151 2.259d
NCB 152.819 238.510 2.659 2.733c
Dosage 1
Control 187.254a 240.118 2.207b 3.311a
Low 159.284ab 241.614 3.295a 2.867b
Intermediate 131.476b 261.172 2.578ab 2.741b
High 169.034ab 256.524 2.656ab 2.356c
Supplementation 0.3138 0.052 0.1442 <0.001
Dosage 0.0591 0.9167 0.0394 <0.001
Interaction 0.6476 0.0395 0.0623 <0.001
SEM 20.006 39.204 0.370 0.0909
C.V. (%) 25.54 31.14 26.63 22.44
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LC–Liver cholesterol (mg/dL); LT–Liver triglycerides (mg/dL); LL–Liver lipids (%); SS–Steatosis score; Means followed by different letters in the same column differ significantly; Tukey test, P<0.05.

In the interaction, steatosis showed that it is reduced with the supplementation of an additional source in the diet. However, better results were obtained with NCA and NCB compared to CC (P<0.001), as shown in Table 13. NCA showed a better result than NCB and CC even at the low dosage (P<0.001), consistently achieving better results at the intermediate and high dosages (P<0.001). A lower score was observed at the high dosage of NCA (P<0.001). The liver triglyceride content was higher with CC supplementation compared to NCA (P<0.001), but similar to NCB (P>0.05) at the intermediate dosage.

Table 13. Effect of interaction between supplementation sources of broilers at 42 days of age.

Steatosis Control CC NCA NCB P value
Control 3.311
Low 3.288aA 2.488cB 2.82b <0.001
Intermediate 3.066aA 2.355cA 2.80a <0.001
High 2.555aB 1.933bA 2.577a <0.001
SEM 0.632 0.472 0.516
P value <0.001 <0.001 0.1112
Triglycerides Control CC NCA NCB P value
Control 240.118
Low 300.428 242.694 181.718 0.1184
Intermediate 361.959a 159.404b 262.154ab 0.004
High 254.976 242.938 271.657 0.874
SEM 18.383 15.010 17.652
P value 0.1708 0.2375 0.2209
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a,b Means followed by different letters in the same line differ significantly Tukey test, P<0.05. A, B Means followed by different letters in the same column differ significantly Tukey test, P<0.05.

Findings from this trial suggest NCA outperformed other sources evaluated. Aside from superior performance, NCA was also able to provide satisfactory results at lower doses. To support use recommendations, economic estimates accounting for the effects of additive and dose on production were presented in Table 14 (Investment per kg of feed per phase and weighted average in days) and Table 15 (Relative Gross Income calculation).

Table 14. Investment per kg of feed per treatment (BRL/kg of feed/treatment) at the different stages of production in broilers aged 1 to 42 days.

Age Control Choline Chloride 60% NCA NCB
400 g/t 800 g/t 1,200 g/t 100 g/t 200 g/t 300 g/t 100 g/t 200 g/t 300 g/t
1–21 d 2.283 2.288 2.293 2.302 2.286 2.288 2.291 2.286 2.289 2.292
22–35 d 2.267 2.272 2.278 2.283 2.270 2.272 2.275 2.270 2.273 2.277
36–42 d 2.224 2.231 2.236 2.240 2.228 2.231 2.234 2.229 2.232 2.235
1–42 d 2.268 2.273 2.278 2.286 2.271 2.273 2.276 2.271 2.274 2.278
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Price estimates based on values listed in feed Tables, July 2022.

Table 15. Economic assessment.

Control Choline Chloride, g/t NCA g/t NCB, g/t
400 800 1,200 100 200 300 100 200 300
Liveability, % 94.74 93.86 94.74 96.49 94.74 95.61 99.12 96.49 95.61 97.37
Weight gain, kg 2.86 2.83 2.96 2.91 3.00 2.90 2.92 2.91 2.87 2.85
Feed conversion ratio, kg/kg 1.67 1.72 1.68 1.73 1.60 1.62 1.65 1.69 1.76 1.65
Production Factor 386.21 367.30 397.44 386.40 422.34 408.04 417.02 395.59 371.63 400.03
Birds Final Adjusted 142.00 141.00 142.00 145.00 142.00 143.00 149.00 145.00 143.00 146.00
Feed Intake, kg/treat 675.46 685.04 707.76 727.93 680.45 672.14 717.96 713.14 722.38 683.89
Cost of diet, BRL/kg BRL 2.27 BRL 2.27 BRL 2.27 BRL 2.28 BRL 2.27 BRL 2.27 BRL 2.27 BRL 2.27 BRL 2.28 BRL 2.28
Cost of feed, BRL/kg BRL 1,531.84 BRL 1,555.31 BRL 1,608.81 BRL 1,657.90 BRL 1,543.97 BRL 1,525.76 BRL 1,630.53 BRL 1,620.70 BRL 1,645.00 BRL 1,560.54
Broiler production, kg/treat 411.64 404.18 425.95 427.66 431.02 420.91 440.34 427.71 416.55 421.47
Cost of Broiler production, BRL/kg BRL 3.72 BRL 3.85 BRL 3.78 BRL 3.88 BRL 3.58 BRL 3.62 BRL 3.70 BRL 3.79 BRL 3.95 BRL 3.70
Price of broiler, BRL/kg BRL 7.00 BRL 7.00 BRL 7.00 BRL 7.00 BRL 7.00 BRL 7.00 BRL 7.00 BRL 7.00 BRL 7.00 BRL 7.00
Breast production, kg/treat 128.76 126.02 133.11 135.70 136.33 130.31 137.34 131.86 129.05 127.75
Cost/Breast, BRL/kg BRL 11.90 BRL 12.34 BRL 12.09 BRL 12.22 BRL 11.33 BRL 11.71 BRL 11.87 BRL 12.29 BRL 12.75 BRL 12.22
Price of breast, BRL/kg BRL 13.00 BRL 13.00 BRL 13.00 BRL 13.00 BRL 13.00 BRL 13.00 BRL 13.00 BRL 13.00 BRL 13.00 BRL 13.00
Thigh production, kg/treat 40.55 40.54 42.38 42.00 43.79 42.81 43.11 43.84 41.78 42.61
Cost/Thigh, BRL/kg BRL 37.78 BRL 38.37 BRL 37.96 BRL 39.48 BRL 35.26 BRL 35.64 BRL 37.82 BRL 36.97 BRL 39.37 BRL 36.62
Price of the thigh, BRL/kg BRL 12.00 BRL 12.00 BRL 12.00 BRL 12.00 BRL 12.00 BRL 12.00 BRL 12.00 BRL 12.00 BRL 12.00 BRL 12.00
Drumstick, kg/treat 46.27 46.12 49.03 48.75 49.74 48.19 50.33 49.53 47.86 48.72
Cost/Drumstick, BRL/kg BRL 33.11 BRL 33.73 BRL 32.81 BRL 34.01 BRL 31.04 BRL 31.66 BRL 32.40 BRL 32.72 BRL 34.37 BRL 32.03
Price of drumstick, BRL/kg BRL 12.00 BRL 12.00 BRL 12.00 BRL 12.00 BRL 12.00 BRL 12.00 BRL 12.00 BRL 12.00 BRL 12.00 BRL 12.00
Gross income, BRL (Breast + Thigh +Drumstick) BRL 5,597.15 BRL 5,507.47 BRL 5,809.00 BRL 5,846.65 BRL 5,911.88 BRL 5,732.48 BRL 5,989.08 BRL 5,828.61 BRL 5,669.21 BRL 5,707.02
Gross margin, BRL (Gross Income—Cost of feed) BRL 4,065.30 BRL 3,952.15 BRL 4,200.19 BRL 4,188.74 BRL 4,367.91 BRL 4,206.72 BRL 4,358.55 BRL 4,207.90 BRL 4,024.21 BRL 4,146.48
Relative Gross margin, % 100.00 97.17 103.23 102.89 107.42 103.42 107.13 103.53 99.04 102.07
100.00 106.23 105.89 110.54 106.43 110.24 106.55 101.92 105.04
100.00 99.68 104.06 100.19 103.78 100.30 95.95 98.88
100.00 104.40 100.52 104.11 100.62 96.25 99.20
100.00 96.28 103.58 96.64 95.66 103.06
100.00 103.58 100.10 95.76 98.69
100.00 96.64 92.45 95.28
100.00 95.66 98.59
100.00 103.06
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Color scale: color changes indicate a 10% change in value. Red: lowest value. Dark green: highest value. Yellow, orange, light green: 10% value difference intervals. Benchmark: 100.

Combined analysis of liveability (i.e., the final number of birds per treatment), performance, carcass yield, and economic data, which can be adjusted according to natural variations, revealed relative gross margin fluctuations across different scenarios (Table 14).

Using to the Control treatment as a reference, the relative gross income derived from breast, thigh, drumstick, and whole chicken sales is 100%. The same applies to the remaining treatments in the same order. Hence, 100 is a used benchmark to estimate the delta between treatments.

Based on comparative analysis between the control and remaining treatments, NCA fed at 100 g/t yields the highest relative gross income (107.42, i.e., 7.42 p.p higher the than control). Margins derived from NCA at 100 g/ton are also higher relative to the three different doses of Choline Chloride or the NCB product (400 g/t Choline Chloride vs. 100 g/t NCA; 10.54 points; 800 g/t Choline Chloride vs. 200 g/t NCA, 4.06 p.p.; 1,200 g/t Choline Chloride vs. 300 g/t NCA, 4.40 points). NCA fed at 200 g/t, or 300 g/t outperformed the control and Choline Chloride fed at three different doses.

The NCB product was economically superior to the control treatment when fed at 100 g/t and 300 g/t doses, However, 0.6 p.p. losses were estimated at the 200 g/t dose. The NCB product outperformed Choline Chloride fed at 400g/t. In contrast, when fed at 200 g/t, the economic performance of the NCB product was poorer (4 to 5 points less) compared to Choline Chloride fed at 800 g/t or 1,200 g/t (Table 10; highlights in red). Assessment estimates suggest NCA was more efficient than the control treatment, Choline Chloride or NCB at doses used in this trial.

Discussion

During the total period of this trial (1–42 days), statistical differences were observed for the performance variables evaluated by Tukey analysis. Furthermore, employing orthogonal contrasts, it was evident that NCA exhibited superior performance compared to the NCB product. This could be due to the higher bioavailability or better absorption of NCA in the body. Specifically, NCA demonstrated notable enhancements in terms of body weight (BW) with an increment of 61.54 g/bird (P = 0.0143), body weight gain (BWG) with an increment of 61.59 g/bird (P = 0.0142), and feed conversion ratio (FCR) with an improvement of 7.6 points (P = 0.0190). These improvements indicate that NCA is more effective in promoting growth and improving feed efficiency in birds. Furthermore, birds fed with NCA displayed a trend of lower feed intake by 114.4 g/bird, although this difference was not statistically significant. Suggest that NCA might be more satiating or more efficiently utilized by the birds, leading to lower feed intake.

This aligns with a study by [18] that suggested herbal choline supplementation at 0.350 and 0.500 kg/ton of feed can effectively substitute synthetic choline chloride-60%, typically used at 1 kg/ton of broiler feed. Indicates that natural sources of choline can be just as effective, if not more so, than synthetic sources. Similarly, [12] evaluated the use of a vegetal source of choline in five levels of supplementation as a replacement of choline chloride at 60% and did not find any difference between the sources on the performance parameters, but they concluded that the use of 100 mg.kg-1 of a vegetal source of choline to replace the use of choline chloride in corn-soybean meal diets for broilers from 1 to 42 days of age, further supports the idea that natural sources of choline can effectively replace synthetic sources without compromising performance. Likewise, [19] reported comparable body weight outcomes for both natural and synthetic choline-supplemented groups, suggests that the source of choline, whether natural or synthetic, does not significantly affect body weight outcomes. Correspondingly, [5] demonstrated that the NCA at 400 g/ton replaced the function of 1 kg/ton of synthetic choline (choline chloride 60%) in broilers, showing the potential to mimic biological activities of synthetic choline through the restoration of negative effects caused by a choline-deficient diet, so indicates that NCA can be used at lower doses than synthetic choline while still providing the same benefits. On the other hand, [20] investigated the effects of replacing synthetic choline with a natural choline source and demonstrated no significative difference on zootechnical performance and nutrition utilization and carcass characteristics, suggests that the source of choline does not significantly affect these parameters. However, the current results clearly demonstrates the importance of choline supplementation and additionally, the results demonstrate the superiority of natural sources over synthetic ones, with significant results, especially with NCA, and because of that, using natural sources of choline, such as NCA, can provide superior results in terms of performance and economic efficiency.

Exploring carcass characteristics, the study revealed that at 200g/t, NCA led to an increase in leg weight, while at 300g/t, it caused a decrease in thigh weight and an increase in breast weight compared to NCB at 300g/t, suggests that NCA dosage can influence the distribution of muscle mass in broilers. Additionally, NCA at 200g/t exhibited a higher carcass yield compared to Choline chloride at 800g/t. These findings were corroborated by [4], which demonstrated the advantages of herbal choline supplementation at 0.5 kg/t of feed in terms of enhanced liver protection, carcass traits, and economic viability in broiler production, supports the potential benefits of using herbal alternatives to traditional supplements. Moreover, a comparative analysis was conducted among NCA, choline chloride, and NCB. Significant distinctions emerged between NCA versus Choline chloride and NCA versus NCB in terms of controlling fatty liver. NCA exhibited greater efficacy in mitigating fatty liver incidence in broilers compared to choline chloride and NCB, showing a dose-dependent effect, and because of that, that NCA can be a promising supplement for liver health in broilers. In this study, NCA at 200 g/t or 300 g/t outperformed the Control and Choline Chloride at various doses. In a similar vein, [21] reported reduced fatty liver incidence in broilers through herbal choline feeding. NCA contains certain compounds with proven hepatoprotective effects, such as polyphenols & curcuminoids. Therefore, findings from this trial, particularly the significant reduction in fatty liver development, may be attributed to curcumin and catechin contained in the natural source of choline employed. Studies have shown that curcumin supplementation can improve the growth performance of broiler chickens [22–25]. Moreover, the phytoconstituents (polyphenols and curcuminoids) present in NCA modulates the liver genes accounts for fat metabolism/catabolism and lipogenesis [26]. This might enhance the energy availability at the muscular level which inturn enhanced fat utilization. Because of that, the NCA outperformed CC and NCB, as shown by better animal performance parameters and best carcass yields. In summary, the study suggests that NCA, particularly at doses of 200 g/t or 300 g/t, outperforms both the Control and Choline Chloride at various doses in terms of carcass characteristics and liver health in broilers.

As anticipated before, combined analysis of performance data and economic estimates suggest a beneficial effect of NCA compared to remaining treatments, and that NCA given at the 100 g/t dose yields the highest relative gross margin, in this terms, NCA is not Only effective in imporving broiler performance but also costo-effective, and more, NCS is more effective than NCB and CC in promoting growth and yield in broilers. Because of these effects, economic assessment findings estimate to support the superiority of NCA relative to NCB and CC in this trial in terms of animal performance as well as carcass and cut yield in broilers aged 1 to 42 days, particularly when fed at 100 g/t. In this study’s conditions, the dosage of 100g/ton probably was enough to attend to the broilers’ requirements due to the low oxidative stress conditions and the use of a corn-soy-based diet. In field conditions with challenges such as low quality of ingredients, heat stress, and high density of animals, higher doses of the NCA evaluated in this study (200g/t or 300g/t) can be recommended. From a practical standpoint (i.e., product storage and feed logistics) the lower dose requirement is another advantage of NCA relative to Choline Chloride, the commercial doses recommended of CC (400, 800, or 1,200 g/t) can involve several space-related and logistics concerns. Therefore, the incorporation of NCA at a dosage of 100g/t in broiler feed could serve as a viable substitute for choline chloride and NCB.

Conclusion

Natural Choline A is a superior source of supplemental choline, offering enhanced hepatoprotective effects and improved animal performance and carcass quality. It has greater economic efficiency compared to Chloride Choline and Natural Choline B. For optimal results, a dose of 100 g/t of Natural Choline A is recommended under low oxidative stress conditions. In conditions of higher heat stress, doses of 200g/t or 300g/t of Natural Choline A are recommended for broilers from 1 to 42 days of age.

Supporting information

S1 Data

(XLSX)

pone.0295488.s001.xlsx (30.3KB, xlsx)

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

The author(s) received no specific funding for this work.

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Decision Letter 0

Ewa Tomaszewska

23 Jan 2024

PONE-D-23-39073Choline through diet or supplementation? Synthetic or natural source supplementation? Improving performance, reducing steatosis, and economic viability of broiler chickens from 1 to 42 days.PLOS ONE

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PLoS One. 2024 Mar 19;19(3):e0295488. doi: 10.1371/journal.pone.0295488.r003

Author response to Decision Letter 0

1 Feb 2024

Dear Reviewers,

Thank you for reviewing our manuscript.

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Decision Letter 1

Ewa Tomaszewska

13 Feb 2024

Choline supplementation: Impact on Broiler Chicken Performance, Steatosis, and Economic Viability from from 1 to 42 days

PONE-D-23-39073R1

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Acceptance letter

Ewa Tomaszewska

5 Mar 2024

PONE-D-23-39073R1

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