
==== Front
Poult Sci
Poult Sci
Poultry Science
0032-5791
1525-3171
Elsevier

S0032-5791(24)00771-5
10.1016/j.psj.2024.104192
104192
METABOLISM AND NUTRITION
Determination of optimal dietary calcium levels under different sources of zinc in Jing tint 6 layer chicks from 15 to 42 d of age
Gong Chengyan *†
Shi Hongpeng †§
Liu Shuan *†
Gao Xinyi *‡
Zhang Shoujun *‡
Liu Hao *‡
Liu Xin *‡
Li Rui *
Wan Dan w.dan@isa.ac.cn
*1
⁎ Laboratory of Animal Nutritional Physiology and Metabolic Process, Key Laboratory of Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125, China
† University of Chinese Academy of Sciences, Beijing 101408, China
‡ College of Animal Science and Technology, Hunan Agricultural University, Key Laboratory for Quality Regulation of Livestock and Poultry Products of Hunan Province, Changsha 410128, China
§ State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
1 Corresponding author: w.dan@isa.ac.cn
16 8 2024
11 2024
16 8 2024
103 11 1041927 6 2024
5 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
An experiment was conducted to investigate the optimal dietary calcium (Ca) levels in Jing Tint 6 layer chicks fed different sources of zinc (Zn). The diets were formulated using 2 different Zn sources: organic Zn (80 mg/kg Zn as HMZn) and inorganic Zn (80 mg/kg Zn as sulfate). For each Zn source, 5 diets were formulated to contain Ca levels of 0.80, 0.90, 1.03, 1.10, and 1.20%. Results showed that dietary Ca levels had a significant effect on body weight gain (BWG) and feed conversion ratio (FCR) (P < 0.05). In addition, BWG was significantly enhanced by the organic Zn diets (P < 0.05). Dietary Ca levels significantly affected tibia length (P < 0.05) and serum Ca and P contents (P < 0.05) but did not affect serum total protein (TP), albumin (ALB), or alkaline phosphatase (ALP) levels (P > 0.05). The apparent total tract retention coefficients (ATTRC) of Ca showed a quadratic trend (P < 0.05) with increasing Ca levels. Furthermore, organic Zn diets reduced excreta Ca output and enhanced the ATTRC of Ca in birds on d 42 compared with inorganic Zn diets. The optimal dietary Ca levels were estimated as 0.93, 0.94, and 0.96% for birds fed organic diets and 1.07, 0.99 and 0.94% for birds fed inorganic diets using nonlinear models based on the criteria of BWG, tibial length, and serum P, respectively. In general, organic Zn supplementation improved growth performance and reduced the calcium requirements of birds on d 42.

Key words

Layer chick, calcium requirement
organic zinc
inorganic zinc
nonlinear model
==== Body
pmcINTRODUCTION

Calcium (Ca) is one of the most important minerals and is vital for maintaining a wide range of physiological functions, particularly in layer chicks. (Ribeiro, et al., 2019; Zhang et al., 2019; David, et al., 2021). In recent years, remarkable progress has been made in the growth, feed conversion, and egg production rates of laying hens owing to genetic improvements and selection (Buzala, et al., 2015; Li, et al., 2023; Shi, et al., 2023). In addition, Ca absorption and metabolism in birds are not isolated processes and are closely related to the interactions of phosphorus (P), vitamin D, and other trace elements (Wan and Yin, 2023). For example, the available P level and the ratio of Ca to P can significantly affect Ca absorption and utilization in laying hens (Zhang, et al., 2023b; Gong, et al., 2024). In recent years, researchers have suggested that the P requirements of birds should be lower (Jiang et al., 2016; Liu et al., 2017). Both Ca deficiency and excess can damage bone development, eggshell formation, and immunity in laying hens (Li, et al., 2020; Bai, et al., 2022). Therefore, redefining Ca requirements to achieve an optimal nutrient supply balance for modern laying hen breeds is particularly important.

Zinc (Zn) is another micronutrient that is critical for chicken health and performance. It plays a key role in many biological processes, such as DNA replication, enzyme catalysis, and protein synthesis (Mwangi, et al., 2017; Ma, et al., 2023). Zinc deficiency can lead to growth retardation, decreased immune function, and reduced production performance (Jing, et al., 2015; Sacan, et al., 2016; Selvaraj, 2020). Previous studies have reported that organic Zn is more bioavailable than inorganic Zn and is less likely to antagonize other minerals (Mwangi, et al., 2017; Sauer, et al., 2017). The interactions between Zn and Ca in the body are complex (Lin, et al., 2020; Zhang, et al., 2022). Organic Zn may enhance Ca absorption and utilization by modulating the intestinal environment, influencing microbial communities, and improving mineral transport mechanisms (Dong, et al., 2023; Wang, et al., 2023; Xiao, et al., 2023). Thus, different sources of Zn may have different effects on the absorption and utilization of Ca. Therefore, studying the effects of different sources of Zn on the dietary Ca requirements of birds to develop rational and nutritional guidelines is important.

Jing Tint 6, a ternary hybrid laying hen breed, is based on Rhode Island Red and White Leghorns with red feather and pink eggs (Zhang, et al., 2023a; Liu, et al., 2024; Zhao, et al., 2024). The nonphytate P requirements of Jing Tint 6 layer chicks from hatching to d 42 have been well assessed (Gong, et al., 2024); however, Ca requirements still need to be evaluated. Growth performance and serum parameters are common criteria for evaluating calcium requirements in birds (Bai, et al., 2022; Walk, et al., 2024). In addition, tibial characteristics, such as breaking strength and length, are frequently used to assess the Ca requirements in birds (Xia, et al., 2015). Studies have found that high Ca to P ratios impair growth and bone mineralization in Pekin ducklings (Zhu, et al., 2018). Birds have the fastest growth rate and relatively high Ca requirements during the growth period of 15 to 42 d of age (Bai, et al., 2022; Wang, et al., 2022). Bones and muscles develop rapidly during this period, and failure to meet calcium requirements may lead to lifelong growth and production problems (Walk, et al., 2024). Therefore, the objective of the current study was to investigate the optimal dietary Ca level of Jing Tint 6 layer chicks from 15 to 42 d of age using diets with graded levels of Ca. Furthermore, diets were formulated to contain organic or inorganic Zn supplements to emulate their effects on Ca requirements. Nonlinear models based on the criteria of tibia length, growth performance, and serum parameters were used to obtain as accurate estimates as possible for the Ca requirements of the Jing Ting 6 layer chicks.

MATERIAL AND METHODS

Animal Ethics

This study was conducted in accordance with the guidelines of the National Act on the Use of Experimental Animals (People's Republic of China). All animal procedures were approved by the Animal Welfare Committee of the Institute of Subtropical Agriculture, Chinese Academy of Sciences (IACUC # 201302).

Experimental Diets

All birds received one of the experimental diets, organized in a 2 × 5 factorial format, from the age of 15 to 42 d. Diets based on corn–soybean meal and wheat were developed using 2 types of Zn: organic Zn (80 mg/kg Zn as HMZn, Xing Jia Bio-engineering Co., Ltd., Changsha, Hunan, China) or inorganic Zn (80 mg/kg Zn as Zn sulfate, Merck, Darmstadt, Germany). Five distinct diets were developed for every Zn source to contain Ca concentrations of 0.80, 0.90, 1.03, 1.10, and 1.20% of the total diet. Mono-calcium P was incorporated to keep nonphytate P level at 0.46%. Diets based on corn–soybean meal–wheat were developed to cater to the dietary needs of birds aged 15 to 42 d in line with the NRC guidelines, as shown in Table 1.Table 1 Ingredient composition and nutrient contents of the diets.

Table 1Ingredients, %	Levels of Ca, % (Supplementation of HMZn)	Levels of Ca, % (Supplementation of Zn sulfate)	
0.80	0.90	1.03	1.10	1.20	0.80	0.90	1.03	1.10	1.20	
Corn	58.89	58.89	58.89	58.89	58.89	58.89	58.89	58.89	58.89	58.89	
Soybean meal	32.30	32.30	32.30	32.30	32.30	32.30	32.30	32.30	32.30	32.30	
Soybean oil	2.90	2.90	2.90	2.90	2.90	2.90	2.90	2.90	2.90	2.90	
Wheat bran	0.80	0.80	0.80	0.80	0.80	0.80	0.80	0.80	0.80	0.80	
Limestone	1.54	1.54	1.54	1.54	1.54	1.54	1.54	1.54	1.54	1.54	
Mono-calcium phosphate	2.55	2.55	2.55	2.55	2.55	2.55	2.55	2.55	2.55	2.55	
Salt	0.30	0.30	0.30	0.30	0.30	0.30	0.30	0.30	0.30	0.30	
DL-methionine	0.14	0.14	0.14	0.14	0.14	0.14	0.14	0.14	0.14	0.14	
L-lysine hydrochloride	0.14	0.14	0.14	0.14	0.14	0.14	0.14	0.14	0.14	0.14	
Premix1	0.34	0.34	0.34	0.34	0.34	0.34	0.34	0.34	0.34	0.34	
Total amount	100	100	100	100	100	100	100	100	100	100	
Calculated nutritional level										
ME2, Mcal/kg	2.90	2.90	2.90	2.90	2.90	2.90	2.90	2.90	2.90	2.90	
Crude fat, %	5.55	5.55	5.55	5.55	5.55	5.55	5.55	5.55	5.55	5.55	
Crude protein, %	20.00	20.00	20.00	20.00	20.00	20.00	20.00	20.00	20.00	20.00	
Total calcium, %	0.80	0.91	1.03	1.10	1.21	0.80	0.91	1.03	1.10	1.21	
Available phosphorus, %	0.46	0.46	0.46	0.46	0.46	0.46	0.46	0.46	0.46	0.46	
Lysine, %	2.63	2.63	2.63	2.63	2.63	2.63	2.63	2.63	2.63	2.63	
Methionine, %	0.59	0.59	0.59	0.59	0.59	0.59	0.59	0.59	0.59	0.59	
Tryptophan, %	0.02	0.02	0.02	0.02	0.02	0.02	0.02	0.02	0.02	0.02	
Threonine, %	1.65	1.65	1.65	1.65	1.65	1.65	1.65	1.65	1.65	1.65	
Analyzed nutritional level											
Crude fat, %	6.00	6.00	6.00	6.00	6.00	6.20	6.20	6.20	6.20	6.20	
Crude protein, %	19.60	19.60	19.60	19.60	19.60	19.60	19.60	19.60	19.60	19.60	
Total phosphorus, %	0.88	0.88	0.88	0.88	0.88	0.86	0.86	0.86	0.86	0.86	
Calcium, %	0.90	0.96	1.08	1.22	1.24	0.88	0.95	1.00	1.08	1.17	
Zinc, mg/kg	108	108	108	108	108	105	105	105	105	105	
1 Vitamin and mineral premix provided (per kilogram of diet): vitamin A 10,000 IU; vitamin D3 3,000 IU; vitamin E 40 mg; vitamin K 33 mg; vitamin B1 2.5 mg; vitamin B2 8 mg; vitamin B6 5 mg; vitamin B12 0.025 mg; biotin 0.15 mg; folic acid 1.5 mg; niacinamide 50 mg; Pantothenic acid 12 mg; Fe 60 mg; Cu 15 mg; Mn 80 mg; I 1.2 mg; Se 0.297 mg.

2 ME = metabolizable energy.

Birds and Housing

Birds, supplied by Beijing Huadu Yukou Poultry Industry Co., Ltd. (Beijing, China), were randomly placed into groups of 24 in cages (90 cm deep × 600 cm wide × 40 cm high). Each cage was treated as a duplicate of the experiment and 6 replicates were provided for each diet. The photoperiod gradually decreased from 24 h to 12 h with increasing age. The temperature was slowly decreased from 35 to 34°C for d 1 to 7, and from 34 to 30°C for d 8 to 14, and from 30 to 24°C for d 15 to 42 (experimental stage). Maintaining a consistent 65% relative humidity was maintained through a 12-h cycle of light and darkness. All birds had unrestricted access to feed and water throughout the experiments. Body weight and feed consumption were recorded weekly.

Growth Performance

Weekly records were conducted on the feed intake and body weight of each cage, followed by the calculation of the feed conversion ratio (FCR), which is the ratio of daily feed intake (DFI) to BWG. Mortality was recorded daily to correct feed consumption and to adjust feed: gain data.

Serum Biochemical Analysis

A syringe was used to extract 3 mL of blood from a wing vein of 2 randomly selected birds in each cage on d 42. Serum was collected through centrifugation at 3000 rpm and 4°C for 10 min, followed by biochemical analysis using an automated analyzer (Roche, Basel, Switzerland) and commercial reagent kits (Lidman Biotech, Beijing, China). The biochemical indices included serum Ca, P, alkaline phosphatase (ALP), total protein (TP), and albumin (ALB).

Bone Characteristics

The same birds used for blood sampling were sacrificed, and their left tibias were gathered and preserved at −20°C pending further analysis. Tibial length was determined directly using a Vernier scale (Merck, Darmstadt, Germany), and tibial strength was measured using an electronic universal testing machine (Weidu Analytical Instrument Manufacturing Ltd., Wenzhou, China).

Calcium Utilization

Titanium (Ti) dioxide was incorporated into the diets (3 g/kg, as-fed) to calculate nutrient utilization using the index method. Excreta collection trays were introduced on d 38, and total excreta samples were collected during the last 3 d. Feathers and debris in the excreta were removed, sprayed with 10% hydrochloric acid, mixed well, and stored at −20°C. The collected excreta samples were thawed and mixed, dried to a constant weight at 56°C, and ground to pass through a 0.5-mm screen before analysis. The collected excreta were analyzed for Ca and Ti, which were then used to determine the apparent total tract retention coefficients (ATTRC) using the Ti marker ratios in the diet and excreta (Liu, et al., 2013).

Chemical Analysis

Inductively coupled plasma mass spectrometry (Thermos, Schaumburg, IL) was used according to the method described by AOAC International (2016) to determine the Ti concentration in the feed and fecal samples. The Ca content of the feed and fecal samples was determined using an inductively coupled plasma optical emission spectrometer (Perkin Elmer, Waltham, MA), according to the method described by AOAC International (2016). Dry matter contents were measured by drying the samples for 24 h at 105°C via an electric forced ventilation oven (Zhicheng Analytical Instrument Manufacturing Ltd., Shanghai, China). The diets were also analyzed for Zn, P, crude protein, and crude fat (968.06; AOAC, 2016).

Statistical Analyses

All data were subjected to a general linear model (GLM) procedure using IBM SPSS Statistics 22, followed by Duncan's multiple range tests, with the cage serving as the experimental unit for all statistical analyses. Regression analyses of nonlinear models were performed using Origin 2023, and the best-fit models between the response criteria and dietary Ca levels were used to determine the dietary Ca requirements of birds. The level of statistical significance was set at P < 0.05.

RESULTS

Growth Performance

The effects of dietary Ca and Zn on growth performance are presented in Table 2. In both organic and inorganic Zn diets, body weight gain (BWG) increased linearly (P < 0.05) as dietary Ca levels increased, and FCR decreased linearly (P < 0.05) as dietary Ca levels increased; however, dietary Ca levels did not affect daily feed intake (DFI) (P > 0.05). Moreover, compared with the inorganic zinc diets, BWG was significantly improved in birds fed organic zinc diets from 15 to 42 d of age (P < 0.05).Table 2 Effects of dietary calcium and Zn on growth performance of birds from 15 to 42 d.

Table 2Item	Final BW1, g/bird	BWG2, g/bird	DFI3, g/bird/d	FCR4, g/g	
Organic Zn diets	
 0.80% Ca	354.05c	8.47c	29.02	3.44a	
 0.91% Ca	446.23a	11.56a	25.44	2.21c	
 1.03% Ca	454.84a	11.93a	23.31	1.97c	
 1.10% Ca	456.05a	12.08a	25.39	2.11c	
 1.21% Ca	438.62a	11.26a	25.68	2.28c	
Inorganic Zn diets	
 0.80% Ca	357.06c	8.47c	24.74	2.92b	
 0.91% Ca	401.46b	9.92b	26.10	2.62bc	
 1.03% Ca	427.62a	11.21a	25.82	2.31c	
 1.10% Ca	436.27a	11.55a	25.39	2.20c	
 1.21% Ca	430.72a	11.37a	24.16	2.12c	
 SEM	5.26	0.19	0.54	0.07	
Main effects	
 Ca level, %					
 0.8	355.55c	8.47c	26.88	3.18a	
 0.91	423.84b	10.74b	25.77	2.42b	
 1.03	441.23ab	11.57a	24.57	2.14b	
 1.1	447.81a	11.86a	25.39	2.15b	
 1.21	434.67ab	11.31ab	24.92	2.20b	
 SEM	5.96	0.22	1.22	0.12	
 Zn source					
 Organic Zn	430.00a	11.05a	25.95	2.42	
 Inorganic Zn	411.24b	10.53b	25.06	2.42	
 SEM	3.77	0.14	0.77	0.07	
P-value for main effects and interaction	
 Zn source	0.001	0.009	0.422	0.977	
 Ca level	0.000	0.000	0.709	0.000	
 Zn source X Ca level	0.065	0.048	0.358	0.043	
P-value for contrasts of Ca level	
 Organic Zn diets linear P	0.000	0.000	0.288	0.000	
 Organic Zn diets quadratic P	0.000	0.000	0.096	0.000	
 Inorganic Zn diets linear P	0.000	0.000	0.620	0.001	
 Inorganic Zn diets quadratic P	0.000	0.000	0.502	0.253	
1 Final BW = final body weight.

2 BWG = body weight gain.

3 DFI = daily feed intake.

4 FCR = feed conversion ratio.

a,b,c Values within a column with different superscripts differ significantly at P < 0.05.

Tibial Characteristics

As presented in Table 3, the dietary Ca level in both organic and inorganic Zn diets had a significant effect on tibial length (P < 0.05), which increased linearly (P < 0.05) as the dietary Ca level increased. Tibial breaking strength was not affected by Ca levels in either organic or inorganic Zn diets (P > 0.05).Table 3 Effects of dietary calcium and zinc on tibial characteristics of birds on d 42.

Table 3Item	Length, mm	Breaking strength, kg	
Organic Zn diets	
 0.8% Ca	49.40c	7.61	
 0.91% Ca	54.03ab	6.94	
 1.03% Ca	55.64a	8.12	
 1.1% Ca	53.59ab	7.56	
 1.21% Ca	54.79ab	8.75	
Inorganic Zn diets	
 0.8% Ca	48.56c	7.49	
 0.91% Ca	54.02ab	7.86	
 1.03% Ca	56.08a	8.10	
 1.1% Ca	50.62bc	8.36	
 1.21% Ca	56.64a	8.38	
 SEM	0.47	0.21	
Main effects	
 Ca level, %			
 0.8	48.98c	7.55	
 0.91	54.03ab	7.40	
 1.03	55.89a	8.11	
 1.1	52.11b	7.89	
 1.21	55.72a	8.56	
 SEM	0.73	0.47	
 Zn source			
 Organic Zn	53.48	7.92	
 Inorganic Zn	53.12	7.88	
 SEM	0.46	0.30	
P-value for main effects and interaction	
 Zn source	0.644	0.930	
 Ca level	0.000	0.489	
 Zn source X Ca level	0.218	0.846	
P-value for contrasts of Ca level	
 Organic Zn diets linear P	0.003	0.667	
 Organic Zn diets quadratic P	0.008	0.546	
 Inorganic Zn diets linear P	0.003	0.917	
 Inorganic Zn diets quadratic P	0.144	0.780	
a,b,c Values within a column with different superscripts differ significantly at P < 0.05.

Serum Parameters

Table 4 shows serum parameters. The TP, ALB and ALP contents of serum were not affected by Ca levels in either organic or inorganic Zn diets (P > 0.05). However, dietary Ca levels had a significant effect on Ca and P contents of serum (P < 0.05). In both organic and inorganic Zn diets, serum P content increased linearly (P < 0.05) as dietary Ca levels increased. In addition, the interaction between Ca and Zn had a significant effect on the serum P content (P < 0.05).Table 4 Effects of dietary calcium and zinc on serum parameters of birds on d 42.

Table 4Item	TP1, g/L	ALB2, g/L	ALP3, U/L	Ca, mmol/L	P, mmol/L	
Organic Zn diets	
 0.8% Ca	32.00	17.62	1397.67	2.80ab	2.08c	
 0.91% Ca	34.20	18.13	1082.33	3.39a	2.89a	
 1.03% Ca	35.08	19.38	1100.80	2.83ab	2.38b	
 1.1% Ca	32.19	17.87	1217.86	3.43a	2.96a	
 1.21% Ca	34.27	18.72	1553.00	2.87ab	2.63ab	
Inorganic Zn diets	
 0.8% Ca	34.03	18.43	2405.50	2.86ab	2.44b	
 0.91% Ca	32.60	18.17	1028.83	3.01ab	2.97a	
 1.03% Ca	35.37	20.13	1194.33	2.85ab	2.74ab	
 1.1% Ca	32.48	17.46	897.00	3.14ab	2.74ab	
 1.21% Ca	31.25	18.25	1932.17	2.63b	2.64ab	
 SEM	0.43	0.25	130.75	0.05	0.05	
Main effect	
 Ca level, %						
 0.8	33.02	18.03	1901.58	2.83b	2.21c	
 0.91	33.40	18.15	1055.58	3.20a	2.91a	
 1.03	35.22	19.76	1147.57	2.84b	2.54b	
 1.1	32.31	17.70	1084.17	3.31a	2.86a	
 1.21	32.76	18.48	1742.58	2.75b	2.63b	
 SEM	0.95	0.55	282.33	0.10	0.07	
 Zn source						
 Organic Zn	33.76	18.45	1279.96	3.07	2.59	
 Inorganic Zn	32.92	18.40	1492.63	2.90	2.71	
 SEM	0.61	0.35	180.34	0.06	0.05	
P-value for main effects and interaction	
 Zn source	0.330	0.916	0.408	0.057	0.079	
 Ca level	0.278	0.114	0.106	0.000	0.000	
 Zn source X Ca level	0.365	0.601	0.504	0.398	0.032	
P-value for contrasts of Ca level	
 Organic Zn diets linear P	0.380	0.322	0.558	0.617	0.003	
 Organic Zn diets quadratic P	0.309	0.384	0.194	0.037	0.009	
 Inorganic Zn diets linear P	0.283	0.073	0.123	0.117	0.001	
 Inorganic Zn diets quadratic P	0.202	0.040	0.659	0.165	0.002	
1 TP = Total protein.

2 ALB = Albumin.

3 ALP = Alkaline phosphatase.

a,b Values within a column with different superscripts differ significantly at P < 0.05.

Calcium Utilization

The excreta Ca output and ATTRC are presented in Table 5. Both the dietary Ca level and Zn source significantly affected the excreta Ca output and Ca ATTRC (P < 0.05). Overall, organic Zn diets reduced excreta Ca output and enhanced the Ca ATTRC of birds on d 42 in comparison with inorganic Zn diets.Table 5 Effects of dietary calcium and zinc on excreta Ca output and apparent total tract retention coefficients of birds on d 42.

Table 5Item	Excreta Ca output, g/Kg DMI	ATTRC of Ca1, %	
Organic Zn diets	
 0.8% Ca	5.67c	50.93ab	
 0.91% Ca	5.45c	52.55a	
 1.03% Ca	5.69c	50.59ab	
 1.1% Ca	6.35bc	46.54abc	
 1.21% Ca	6.47bc	42.22bc	
Inorganic Zn diets	
 0.8% Ca	8.04a	35.06de	
 0.91% Ca	6.93b	39.91cd	
 1.03% Ca	7.82a	35.85de	
 1.1% Ca	8.06a	34.57de	
 1.21% Ca	8.55a	32.85e	
 SEM	0.21	1.47	
Main effect	
 Ca level, %			
 0.8	6.63bc	44.20a	
 0.91	6.30c	45.25a	
 1.03	6.87bc	42.75ab	
 1.1	7.20ab	40.80ab	
 1.21	7.51a	37.54b	
 SEM	0.38	2.75	
 Zn source			
 Organic Zn	5.93b	48.56a	
 Inorganic Zn	7.90a	35.65b	
 SEM	0.26	1.54	
P-value for main effects and interaction	
 Zn source	0.000	0.000	
 Ca level	0.001	0.034	
 Zn source X Ca level	0.150	0.434	
P-value for contrasts of Ca level	
 Organic Zn diets linear P	0.021	0.045	
 Organic Zn diets quadratic P	0.296	0.276	
 Inorganic Zn diets linear P	0.002	0.005	
 Inorganic Zn diets quadratic P	0.001	0.002	
1 ATTRC = apparent total tract retention coefficients.

a,b,c,d,e Values within a column with different superscripts differ significantly at P < 0.05.

Estimations of Dietary Ca Requirements of Birds Fed Different Zinc Sources

The calcium requirements of birds, as estimated using nonlinear regression analyses, are shown in Table 6. The results indicated that BWG, tibial length, and serum P were suitable criteria for evaluating the Ca requirements of birds aged 15 to 42 d. For birds fed organic Zn diets, the estimated Ca for BWG (0.86–1.02%) was similar to that estimated using the criteria of tibial length (0.88–1.06%) and serum P (0.88–1.09%). However, for birds fed inorganic Zn diets, the estimated Ca for BWG (1.05–1.11%) was higher than those estimated using the criteria of tibial length (0.92–1.12%) and serum P (0.91–0.99%). Therefore, the optimal Ca requirement of Jing Tint 6 layer chicks from 15 to 42 d of age was estimated as 0.93% to 0.96% or 0.94% to 1.07% for organic or inorganic Zn diets, respectively, based on the average estimates from 3 response parameters.Table 6 Estimations of dietary calcium requirements of birds fed different zinc sources based on the best fitted broken-line or quadratic models.

Table 6Zn source	Dependent variable	Model	Regression equation	R2	P-value	Estimated requirement, %	
Organic Zn diets	BWG	Straight-broken line	y = −39.06217+59.41641X	0.672	0.000	0.856	
Quadratic-broken line	y = −5.72298+5.1664X+15.72002 X2	0.660	0.000	0.904	
Quadratic	y = −52.10059+122.96374X-58.60834X2	0.632	0.000	1.017	
Tibial length	Straight-broken line	y = −1.52082+63.64477X	0.399	0.001	0.880	
Quadratic-broken line	y = −19.89081+20.32911X-20.68891X2	0.373	0.004	0.893	
Quadratic	y = −21.77457+142.09741X-65.64421X2	0.354	0.003	1.055	
Serum P	Straight-broken line	y = −4.43679+ 8.14349X	0.343	0.002	0.880	
Quadratic-broken line	y = −0.26896+1.06158X-2.45248X2	0.308	0.008	0.907	
Quadratic	y = −7.65684+19.79376X-9.35684X2	0.232	0.014	1.085	
Inorganic Zn diets	BWG	Straight-broken line	y = −1.01932+11.42267X	0.803	0.000	1.05	
Quadratic-broken line	y = −7.65684+32.25117X-11.10072X2	0.797	0.000	1.064	
Quadratic	y = −23.62053+61.58463X-26.96641X2	0.802	0.000	1.110	
Tibial length	Straight- broken line	y = 8.80114+49.69545X	0.317	0.008	0.923	
Quadratic broken line	y = 21.57355+19.69433X+17.54452X2	0.281	0.026	0.923	
Quadratic	y = −11.34215+117.85105X-52.19177X2	0.228	0.029	1.120	
Serum P	Straight-broken line	y = 0.33934 + 2.68825X	0.308	0.017	0.907	
Quadratic-broken line	y = 0.87679+1.03627X- 1.17036X2	0.277	0.043	0.908	
Quadratic	y = −4.56718+14.74448X-7.32855 X2	0.271	0.031	0.992	

DISCUSSION

Ca and Zn, essential mineral elements in animals, play key roles in many biological processes. Rational supplementation of calcium and zinc is important for healthy growth (Swiatkiewicz, et al., 2014; Li, et al., 2017; Proszkowiec-Weglarz, et al., 2019). In this study, it was found that dietary Ca levels influenced BWG and FCR of 15 to 42 d birds. Compared to birds supplied with 0.80% Ca, the BWG of birds provided with 0.91% Ca was increased, and the FCR was decreased in both organic and inorganic Zn diets. Previous studies have evaluated the effects of calcium on bird growth. High levels of dietary Ca negatively affect BWG in birds from hatching to d 21 (Bai, et al., 2022). A moderate increase in calcium intake has also been found to significantly increase chicken feed intake and contribute to weight gain (Hamdi, et al., 2015). Gautier, et al. (2017) also found that high Ca levels (1.00%) in diets positively affected bird growth, which was similar to the current results where 0.91% to 1.20% Ca resulted in higher BWG of 15 to 42 d birds, compared with 0.80% Ca. However, the effect of different dietary Ca to P ratios on bird growth may vary; for example, the BWG and DFI of birds at 0.6% phosphorus levels decreased with increasing calcium levels (0.55–1.15%) (Zhu, et al., 2018). In addition, it was found that dietary MHA-Zn (90 mg/kg) increased BWG of birds at wk 4 (Dong, et al., 2023), which was similar to this study. Dietary organic Zn supplementation increased organ weight, such as the liver, breast meat yield, and carcass efficiency (Bueno, et al., 2020), which may explain why dietary organic Zn increased the BWG of birds. In the present study, considering only the growth parameters, the recommended levels of Ca in the diets of 15 to 42 d birds were 0.93 and 1.07% for organic and inorganic Zn diets, respectively.

Tibial characteristics, such as breaking strength and length, are traditionally used to evaluate bone mineralization in birds (Liu, et al., 2017). Hamdi, et al. (2015) found a linear increase in tibial weight as dietary Ca increased from 0.50% to 0.90%. In this study, we discovered that the tibial length increased with increasing Ca levels in both organic and inorganic Zn diets. Organic Zn prevents antagonism between Zn and other minerals, such as Cu and Ca, and chelates with phytate (Lu, et al., 2020). Therefore, increased Zn in the tibia enhances carbonic anhydrase activity, promoting calcium carbonate deposition and improving tibia quality (Min, et al., 2019). In this study, considering only the tibia parameters, the optimal levels of Ca in the diets of 15 to 42 d birds were 0.94 and 0.99% for organic and inorganic Zn diets, respectively.

Serum Ca and P contents are considered the traditional criteria for evaluating Ca nutrition in birds (Liu, et al., 2017). Previous studies have indicated that the Ca and P contents in the serum are sensitive to the Ca concentration in the diet (Bai, et al., 2022; Wang, et al., 2022). Our data also indicate that dietary Ca levels affect the serum Ca and P contents of birds from 15 to 42 d of age. The P content in the serum increased linearly with an increase in dietary Ca levels and can be used to evaluate the dietary Ca requirement of birds from 15 to 42 d of age, both in organic and inorganic diets. Gong, et al. (2024) found that serum P content was higher when birds received 1.00% Ca compared to 0.80 and 0.90% Ca levels, which was similar to our present study. Zn plays a crucial role in the enzymatic system responsible for synthesizing vitamin D3, which in turn regulates the absorption of calcium and phosphorus in the intestines, kidneys, and bones (Dukare, et al., 2021). Considering only the serum parameters, the optimal levels of Ca in the diets of 15 to 42 d birds were 0.94 and 0.96% for organic and inorganic Zn diets, respectively.

Animal physiology and diet composition can affect Ca absorption and utilization (Anwar, et al., 2017; David, et al., 2020, 2021). In the present study, the ATTRC of Ca was affected by the Ca and Zn sources. The ATTRC of Ca was reduced in a linear trend when supplemented with 1.20% Ca, which indicated that Ca ATTRC was highly dependent on dietary Ca levels. Hamdi, et al. (2015) found that increasing the dietary Ca concentration resulted in a lower utilization of Ca, leading to an increase in mineral excretion. Gautier, et al. (2017) found similar results in which dietary 1.0% and 1.6% Ca significantly reduced apparent Ca retention of birds on d 23 compared to 0.4% Ca. In addition, a previous study found that dietary organic minerals can reduce fecal Ca content compared to inorganic minerals, and this is dependent on level of organic minerals added (Yenice, et al., 2015). Organic chelates may have reduced the amount of free trace mineral ions in the small intestine, preventing Ca from forming insoluble compounds with these minerals and thus increasing Ca absorption. Our current study found that 80 mg/kg dietary organic Zn reduced fecal Ca content and increased the ATTRC of Ca compared to inorganic Zn, suggesting that organic Zn resulted in higher Ca bioavailability. Although ATTRC and excreta output were significantly affected by calcium and zinc. However, in the organic zinc group, P and R2 values of quadratic-broken line and quadratic models that based on the criteria of excreta output and ATTRC of Ca were poor (results not shown). Therefore, when evaluating the calcium requirements using nonlinear models, we did not consider the criteria of excreta output and ATTRC.

CONCLUSION

In conclusion, this study showed that dietary organic Zn supplementation improved the growth performance of birds on d 42 without decreasing Ca utilization. Furthermore, for birds fed organic Zn diets, 0.96% Ca was sufficient to achieve optimum growth speed without reducing dietary Ca utilization. For birds fed inorganic diets, the Ca requirement would be approximately 1.07% to meet all of the P metabolisms.

It should be noted that the Ca requirements established both the organic and inorganic diets were obtained at the 0.46% dietary available P level recommended by the NRC (1994). Alternatively, the available P levels may result in varying Ca requirements.

DISCLOSURES

The authors declare that there is no conflict of interest.

Appendix Supplementary materials

Image, application 1

Supplementary material associated with this article can be found in the online version at doi:10.1016/j.psj.2024.104192.
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