Optimization of Dietary Zinc Requirement for Broiler Breeder Hens of Chinese Yellow-Feathered Chicken

Simple Summary China is the second-largest global producer of chicken meat, almost half of which is from the Chinese yellow-feathered breed; a systematic program has been initiated to improve its feeding standards. This study evaluated the optimal requirement of dietary zinc for maximal egg production, egg quality, tibial quality, and antioxidant indices of laying broiler breeders. The results revealed several beneficial effects of supplementary zinc on egg production, feed conversion ratio, yolk zinc content, tibial quality and the antioxidant indices in the serum, liver and ovary. The optimal zinc requirement was estimated based on a regression model. Abstract This study aimed to establish the optimal dietary zinc requirement of Chinese yellow-feathered Lingnan broiler breeders. A total of 576 breeder hens aged 58 weeks were randomly assigned to six treatments, each with 6 replicates of 16 birds (n = 96/treatment). The hens were fed either a basal diet (22.81 mg/kg Zn) or the same basal diet supplemented with additional 24, 48, 72, 96, and 120 mg Zn/kg up to 65 weeks of age. Compared to the results of birds fed the basal diet (22.81 mg Zn/kg), the dietary supplementation with additional Zn (mg/kg) showed higher egg laying rate (at 48–120 mg), EM (at 96 mg/kg), yolk Zn content (at 24–120 mg/kg), fertility (at 48–120 mg/kg), hatchability (at 48–96 mg/kg), tibial breaking strength (at 24–48 mg/kg), tibial ash content (at 48 mg/kg), serum CuZnSOD activity (at 72 mg/kg) and T-AOC (at 48 mg/kg), and ovarian CuZnSOD and GSH-Px activities (at 96–120 mg/kg), and lower FCR (at 96 mg/kg). The regression model showed that the optimal supplemental Zn for maximal egg laying rate, yolk Zn content, fertility, and hatchability of Chinese yellow-feathered broiler breeders aged 58 to 65 weeks were 71.09, 92.34, 94.44 and 98.65 mg/kg diet, respectively.


Introduction
Zn is an important trace mineral, which is involved in various biological activities in animals' bodies [1,2]. It is considered to be a coenzyme of more than 240 enzymes in birds' bodies, and plays

Productive Performance
On a replicate basis, the final BW of hens at 65 week of age, feed intake, average daily gain, ELR (%), average daily feed intake, average egg weight, daily egg mass (EM), feed conversion ratio (g feed: g egg, FCR), and sellable egg percentage (%) were determined from 58 to 65 week of age.

Egg Incubation Indices
During wk 64 of age, all birds were inseminated artificially two times, with a three days interval, with 100 µL of fresh semen (diluted 1:1 volume/volume, with 0.9% saline solution) collected from males belonging to the same breed. During the following wk (65th wk), fifty settable eggs (of 50-70 g weight) were collected from each replicate (n = 300 eggs/treatment), weighed and incubated under standard incubation conditions (at 36.5 • C to 38.4 • C, and 55% to 65% relative humidity). At hatch, all unhatched eggs were cracked to identify the unfertile eggs and to calculate the fertility rate (%) [(Number of fertile eggs/number of total incubated eggs) × 100]. The hatchability rate (%) [(Number of hatched chicks/number of incubated eggs) × 100], the sellable chick percentage (%) [(Number of healthy hatched chicks/number of fertilized eggs) × 100], and the average chick weight (g) at birth were calculated.

Tibial Quality Measurements
The length and fresh weight of collected tibias, free of fascia tissues, were measured. The left tibias were used in measuring the breaking strength, which was performed using the tenderness tester (INSTRON 4411, Instron Corporation, Norwood, MA, USA) as described by Chen et al. [18]. Right tibias were boiled in water for 6 min and de-fatted by soaking in diethyl-ether for 96 h, which were then oven dried to a constant weight and ashed using a muffle furnace (550-600 • C for 24 h) to determine the ash content, expressed on the basis of dry-defatted weight. The obtained ash of the right tibias was used in analyzing the content of Zn by flame atomic absorption spectrometry (SpectrAA50/55, Warian Corporation, Palo Alto, CA, USA).

Egg Quality Indices
During the week 64 of age, a total of 504 eggs (2 eggs per replicate/day), were collected, labelled, weighed individually, and used in measuring the egg quality indices. Egg shell strength was determined with an Egg Force Reader (EFR-01, Orka, Ramat HaSharon, Israel). Egg shape index [(width/length) × 100], shell thickness (average thickness at the blunt end, sharp end and middle points of the egg), yolk color, and Haugh unit were measured using an egg multi tester EMT-5200 (Robotmation Co. Ltd., Tokyo, Japan). Yolk, shell (free of membranes), and albumin fractions of the total egg weight (%) were determined.

Biochemical Determinations of Liver, Ovary and Serum
Homogenates of liver and ovary tissues, prepared as described by Zhang et al. [5], were clarified by centrifugation at 3000× g for 10 min at 4 • C and the supernatants were used in enzyme assays. In serum, liver and ovary samples, content of malondialdehyde (MDA), total antioxidant capacity (T-AOC), and glutathione (GSH) were determined. The activity of glutathione peroxidase (GSH-PX), copper-Zn superoxide dismutase (CuZnSOD), and alkaline phosphatase (AKP) were measured using kits purchased from Nanjing Jiancheng Bioengineering Institute, Nanjing, China, and using an automated spectrophotometric analyzer (Cobas FARA II, Roche, Palo Alto, CA, USA) according to the manufacturer's directions.

Zn Content in Diet, Serum, Tibia and Yolk
Concentrations of Zn in diets, serum, tibia and yolk were measured by flame atomic absorption spectrometry (SpectrAA50/55, Warian Company of America) as described by Liao et al. [19].

Statistical Analysis
The effects of dietary treatment were assessed by one-way GLM ANOVA procedures of SAS (version 9.3, SAS Inst., Cary, NC, USA, 2014), and replicates were considered to be as the experimental unit for each variable (n = 6). When not presenting residues with normal distribution, data were transformed with the arcsine square root percentage (z = asin (sqrt (y + 0.5))) [20]. All data were expressed as means and SEM, derived from the ANOVA error mean square. Estimates of Zn requirements were obtained using quadratic polynomial (QP) or exponential asymptotic (EA) regression analysis [21,22]. Where appropriate, quadratic polynomial (Y = c + bX + aX2; where "Y" is the dependent variable as a function of dietary level of Zn, "c" is the intercept, "b" is the linear coefficient, and "a" is the quadratic coefficient.) and exponential asymptotic [Y = a + b × (1 − EXP (−c × (X − d))); where "Y" is the dependent variable as a function of dietary level of Zn, "a" is the relative response to the diet containing the lowest Zn, "b" is the difference between the minimum and the maximum response dietary Zn, "c" is the curve slope coefficient, and "d" is the Zn level of the deficient diet] models were fitted to the responses of the dependent variables to Zn level. The level of dietary Zn at which the response reached 95% at maximum was estimated as the requirement.

Productive Performance
The effects of dietary Zn levels on Lingnan breeder hens' BW and egg production variables are shown in Table 2. The ELR at 48 to 120 mg Zn/kg diets were higher (p < 0.01) than those of 24 mg/kg and controls. The birds fed 96 mg of supplemental Zn/kg diet showed a higher daily EM and lower FCR compared to the controls (p < 0.05). There were no significant differences in the final BW, average daily gain, sellable egg rate, and average egg weight (p > 0.05) due to the dietary Zn concentration. The regression model indicated that the optimal Zn level for maximal ELR was 71.09 mg Zn/kg diet.
In the same row, means not sharing a similar superscript ( a,b,c,d ) differ significantly (p < 0.05), the number of replicates was used as the experimental unit (n = 6). 1 The "0" treatment contained 22.81 mg Zn/kg, and the other zinc levels are additional to that level. 2 Standard error of the mean from ANOVA (n = 6), 3 Y is laying rate, and X is total dietary content of zinc. 4 ER: Estimated from regression analysis.

Egg Quality Indices
The results shown in Table 3 indicate that the egg quality indices, in terms of egg weight, egg shape index, eggshell strength, eggshell thickness, and Haugh unit were not affected (p > 0.05) by the dietary Zn level. The content of Zn in egg yolks increased (p < 0.05) in all treatments received additional dietary Zn than in controls and, according to the regression model, the maximal yolk Zn content was obtained with 92.34 mg of supplemental Zn/kg diet.

Egg Incubation Indices
As shown in Table 4, the fertility, hatchability and salable chick rate values increased significantly (p < 0.05) with 48, 72 and 96 mg of supplemental Zn/kg (p < 0.05) compared to controls. The chick weight at birth was not affected by the supplemental Zn (p > 0.05). According to the regression model, the optimal levels of supplemental Zn for fertility and hatchability were 94.44 and 98.65 mg/kg diet.

Tibia Quality
There was a significant effect of dietary Zn level on the tibial breaking strength and tibial ash content of breeder hens (Table 5). Hens fed diets with an additional 24 and 48 mg/kg Zn both had higher (p < 0.05) tibia breaking strength compared to that of the controls and 72 mg Zn/kg treatment. Ash content of the tibia was higher (p < 0.05) in breeders received 48 mg additional Zn/kg diet than in those fed the basal diet and 72 mg additional Zn/kg.

Ovarian and Oviductal Variables
The results shown in Table 6 reveal that the ovarian weight, oviduct weight, oviduct length, and the numbers and weights of dominant follicles were not affected by the additional Zn levels (p > 0.05).

Biochemical Variables in Serum, Liver and Ovary
As shown in Table 7, the birds fed 72 mg supplemental Zn/kg exhibited significantly increased CuZnSOD activity in serum (p < 0.05) compared with those in the other treatments. The dietary addition of Zn at levels 48 to 120 mg/kg diet showed lower serum MDA content than those of the lower levels. The T-AOC and Zn content of serum in hens supplemented with 48 and 96 mg Zn/kg diet were higher (p < 0.05) than the corresponding values in the other treatments, respectively. The activities of CuZnSOD in the liver of hens fed a diet with 48, 72, 96, and 120 mg of supplemental Zn/kg was higher than those in the controls (p < 0.01). The supplemental Zn levels did not show significant effects on AKP activity, GSH content and GSH-Px activity in serum, or MDA content in the liver tissue (p > 0.05). The results, in Table 8, showed that the biochemical indices in the ovary of Chinese breeder chickens as affected by dietary Zn levels. The birds fed 96 and 120 mg supplemental Zn/ kg diet showed higher (p < 0.01) CuZnSOD activity and GSH-Px activity in the ovary tissues than those in controls. The MDA, GSH and T-AOC of the ovary were not affected by the additional dietary Zn (p > 0.05). In the same row, means not sharing a similar superscript ( a,b ) differ significantly (p < 0.05), the number of replicates was used as the experimental unit (n = 6). 1 The "0" treatment contained 22.81 mg Zn/kg, and the other zinc levels are additional to that level. 2 Standard error of the mean from ANOVA (n = 6). 3 Where Y is yolk zinc, and X is total dietary content of zinc; 4 ER, Estimated from regression analysis. In the same row, means not sharing a similar superscript ( a,b ) differ significantly (p < 0.05), the number of replicates was used as the experimental unit (n = 6). 1 The "0" treatment contained 22.81 mg zinc/kg, and the other zinc levels are additional to that level. 2   In the same row, means not sharing a similar superscript ( a,b,c ) differ significantly (p < 0.05), the number of replicates was used as the experimental unit (n = 6). 1 The "0" treatment contained 22.81 mg zinc/kg, and the other zinc levels are additional to that level. 2 Standard error of the mean from ANOVA (n = 6). 3 On dry matter basis. The number of replicates was used as the experimental unit (n = 6). 1 The "0" treatment contained 22.81 mg zinc/kg, and the other zinc levels are additional to that level. 2 Standard error of the mean from ANOVA (n = 6). In the same row, means not sharing a similar superscript ( a,b,c ) differ significantly (p < 0.05), the number of replicates was used as the experimental unit (n = 6). 1 The "0" treatment contained 22.81 mg zinc/kg, and the other zinc levels are additional to that level. 2 Standard error of the mean from ANOVA (n = 6). 3 CuZnSOD, copper-zinc superoxide dismutase. 4 MDA: malondialdehyde. 5 T-AOC, total antioxidant capacity. 6 AKP, alkaline phosphatase. 7 GSH, glutathione. 8 GSH-PX, Glutathione peroxidase. In the same row, means not sharing a similar superscript ( a,b ) differ significantly (p < 0.05), the number of replicates was used as the experimental unit (n = 6). 1 The "0" treatment contained 22.81 mg zinc/kg, and the other zinc levels are additional to that level. 2 Standard error of the mean from ANOVA (n = 6). 3 CuZnSOD, copper-zinc superoxide dismutase. 4 MDA: malondialdehyde. 5 T-AOC, total antioxidant capacity. 6 GSH, glutathione. 7 GSH-PX, Glutathione peroxidase.

Discussion
The findings of the present study confirm that the tested dietary Zn levels affected the egg production, reproductive performance, biochemical levels, ovary and liver, and tibial quality of Chinese yellow chicken breeders. Supporting results were reported in previous studies. Zhang et al. [5] found that the dietary addition of 80 mg supplemental Zn/kg of a basal diet containing 24 mg Zn/kg (104 mg/kg as a final concentration) improved the FCR, ELR, fertility and hatchability of Lingnan Yellow broiler breeders from 38-57 week of age. Naibi et al. [23] showed that supplementing the Yuehuang broiler breeders with 70.09 mg Zn/kg of corn-soybean diets from 14 to 34 week of age increased their ELR, fertility and hatchability than those in the controls. Mayer et al. [14] found that the Cobb 500 broiler breeder hens fed diets containing 50.3 to 170.6 mg Zn/kg between 37 and 40 week of age showed a higher ELR than in controls. Other studies showed partial consistency with our results. The results of Durmuşg et al. [24] indicated that the brown laying hens fed 180 mg Zn/kg had the highest hatchability, whereas those fed a diet supplemented with 120 mg Zn/kg had the lowest FCR than the controls and other treatments. Sharideh et al. [4] found that the Cobb 500 broiler breeder hens fed diets containing 90 and 120 mg Zn/kg showed a higher fertility rate than those of birds fed 30 and 60 mg Zn/kg between 62 and 72 week of age. In White Leghorn hens, Stahl et al. [25] concluded that the diet with 28 mg Zn/kg was sufficient to prevent decreased egg production, fertility, hatchability and growth of progeny.
The results obtained here, compared to the controls, revealed higher ELR and fertility with the levels from 48 to 120 mg of supplemental Zn/kg (70.81 to 142.81 mg Zn/kg, as final concentrations), higher daily EM and lower FCR with 96 mg of supplemental Zn/kg (118.81 mg/kg, as a final concentration), and a higher hatchability with the levels from 48 to 96 mg of supplemental Zn/kg (70.81 to 118.81 mg Zn/kg, as final concentrations). The results obtained in the present study as well as the aforementioned findings of previous studies therefore show that the response of broiler breeders to dietary Zn is dependent on the supplemental levels, the measured variable, the genetic differences, the physiological status of the bird, and age.
Egg quality has been reported to be an important indicator in the evaluation of the optimal dietary Zn level for laying hens [8,25]. The results here showed that there was no effect of dietary Zn concentration on all egg quality measurements, but the hens fed 48 mg of supplemental Zn/kg diet (70.81 mg Zn/kg, as final concentration), or higher, had a higher yolk Zn content. Our results were consistent with those of Mayer et al. [14], and agreed partially with those of Bahakaim et al. [9], where the addition of 50, 100 and 150 mg of Zn/kg did not affect the egg shape index, shell thickness, shell, yolk and albumen fractions of the egg, but the Haugh unit decreased significantly at 150 mg Zn/kg compared to the controls. Nutritional status of laying breeders ensures adequate nutrient transferee into the egg, which is required for a normal development of embryos [26]. Among the egg components, the yolk is considered to be the major mineral source for the embryo, which contains most of the mineral content of the egg, including the P, Zn, Cu, Mn and Fe [27]. Badawy et al. [28] reported a positive relationship between Zn content in the egg and hatchability values. This could explain the consistency between the obtained increase in yolk Zn content and the corresponding increase in fertility and hatchability in the present study.
Bone status is a common variable used in estimating mineral adequacy in poultry diets [29]. Zn is essential for bone strength; it enters in the formation of bone tissue in the form of alkaline phosphatase, collagenase and aminocly tRNA synthetase [30][31][32][33][34]. The insufficient dietary Zn caused a deterioration of bone formation, which reduced bone density due to its important role in protein synthesis [33,35,36]. The results of the present study showed that the dietary addition of 24 and 48 mg of supplemental Zn (46.81 and 70.81 mg/kg as final concentration) increased the tibial breaking strength (p < 0.01) than those in the control (22.81 mg Zn/kg). The result obtained here is consistent with the previous finding of Olgun et al. [10], who reported that using 75 mg Zn/kg diet significantly increased the shear force of the tibia when compared to values obtained with 50 or 100 mg Zn/kg. Stofaníková et al. [12] found that birds fed 100 mg Zn/kg diet had higher tibia strength compared to chickens fed 50 mg/kg group. The tibial ash content reflects the rate of the mineral density of bones [37], which affects bone strength [38]. Our data showed that the tibial ash content of birds received 48 mg of supplemental Zn/kg diet (70.81 mg Zn/kg, as a final concentration) was higher than those of the control (fed 22.81 mg/kg). This is in agreement with the results reported previously by Ao et al. [11] and Sahraei et al. [13]. The results here, therefore, suggest that the 70.81 mg Zn/kg diet (as a final concentration) is an adequate level for laying breeders, which led to a higher breaking strength and ash content of the tibia.
The enzyme CuZnSOD exerts an important function in maintaining the redox balance of the bird's immune system by eliminating reactive oxygen species [39]. Zn is an important functional constituent of that enzyme (CuZnSOD), which accounts for around 90% of its structure [3,39]. The total antioxidant capacity (T-AOC) contributes mainly to the dynamic balance of active oxygen, which works as an integrative factor reflecting the status of all antioxidants in serum and body fluids [40]. MDA content is used as an indicator of lipid peroxidation and oxidative damage caused by reactive oxygen species [41]. The present study showed that the dietary Zn supplementation (mg/kg diet) increased the activity of serum CuZnSOD (with 72 mg/kg) and T-AOC (at 48 mg/kg) content, and the dietary levels from 48 to 120 mg/kg reduced the serum content of MDA and increased the activity of CuZnSOD in the liver as compared to the controls. Supporting results were reported by Zhang et al. [5], who found that using 104 mg Zn/kg increased the antioxidant status by stimulating the activity of CuZnSOD and T-AOC in the serum, and suppressed the generation of ROS, and therefore decreased MDA content in hen's serum and liver. In a similar manner, Zhao et al. [40] found that the addition of Zn to the feed increased T-AOC capacity and CuZnSOD activity in serum.
In the poultry industry, the age-related decline in reproductive performance of breeder stocks is a common phenomenon [42][43][44]. Jiang et al. [45] and Liu et al. [46] reported that the low antioxidant status in the ovary is associated with age-related decline in hen's reproduction. Liu et al. [46] found that enhancing the antioxidant enzyme activities can effectively prevent the ovarian aging process in hens. This indicates that the results obtained here regarding the enhanced ovarian CuZnSOD and GSH-Px activities due to Zn supplementation has a significant importance, where our experiment was carried out with breeders at the late phase of the laying period. This could imply that the dietary Zn supplementation could contribute in alleviating the decline in reproductive performance of breeders at late ages through increasing the antioxidant activities and, therefore, persist a longer productive season with higher performance; this is confirmed by the improved ELR and EM in the present study.
Evaluating the response of more than one variable to a dietary nutrient makes it difficult to determine a unique requirement value [14]. Pesti et al. [22] reported that the nutritional requirements derived from dose-response experiments are considered physiologically less accurate than regression models, since it hypothesize symmetrical fixed responses to deficiency and excess, but the non-linear models depict the biological responses better than models that force responses to conform to straight lines. Additionally, Mayer et al. [14] reported that using QP, broken line quadratic (BLQ), and EA models was more appropriate for Zn requirements in Cobb 500 broiler breeder hens [14]. In the present study, we therefore used QP Models and EA models in determining the optimal levels of Zn for the most important indices. The recommended Zn requirement according to the Feeding Standard of Chicken [16] for Chinese yellow-feathered laying hens is 72 mg Zn/kg of feed. According to the QP models and EA models, the estimated optimal Zn requirement for broiler breeders were 71.09, 92.34, 94.44 and 98.65 mg/kg, for ELR, yolk Zn content, fertility and hatchability, respectively. The value estimated here for ELR was close to the recommended value of Feeding Standard of Chicken [16] for laying hens, and those estimated for yolk Zn content, fertility and hatchability were higher than the corresponding values in the Feeding Standard of Chickens [16].