Impact of Maternal and Offspring Dietary Zn Supplementation on Growth Performance and Antioxidant and Immune Function of Offspring Broilers

The current study investigated the effects of the maternal Zn source in conjunction with their offspring’s dietary Zn supplementation on the growth performance, antioxidant status, Zn concentration, and immune function of the offspring. It also explored whether there is an interaction between maternal Zn and their offspring’s dietary Zn. One-day-old Lingnan Yellow-feathered broilers (n = 800) were completely randomized (n = 4) between two maternal dietary supplemental Zn sources [maternal Zn–Gly (oZn) vs. maternal ZnSO4 (iZn)] × two offspring dietary supplemental Zn doses [Zn-unsupplemented control diet (CON), the control diet + 80 mg of Zn/kg of diet as ZnSO4]. oZn increased progeny ADG and decreased offspring mortality across all periods, especially during the late periods (p < 0.05). The offspring diet supplemented with Zn significantly improved ADG and decreased offspring mortality over the whole period compared with the CON group (p < 0.05). There were significant interactions between the maternal Zn source and offspring dietary Zn with regards to progeny mortality during the late phase and across all phases as a whole (p < 0.05). Compared with the iZn group, the oZn treatment significantly increased progeny liver and serum Zn concentrations; antioxidant capacity in the liver, muscle, and serum; and the IgM concentration in serum; while also decreasing progeny serum IL-1 and TNF-α cytokine secretions (p < 0.05). Similar results were observed when the offspring diet was supplemented with Zn compared with the CON group; moreover, adding Zn to the offspring diet alleviated progeny stress by decreasing corticosterone levels in the serum when compared to the CON group (p < 0.05). In conclusion, maternal Zn–Gly supplementation increased progeny performance and decreased progeny mortality and stress by increasing progeny Zn concentration, antioxidant capacity, and immune function compared with the same Zn levels from ZnSO4. Simultaneously, Zn supplementation in the progeny’s diet is necessary for the growth of broilers.


Introduction
Zinc (Zn) is an essential mineral that is usually added to the diets of the breeder to obtain the best reproductive performance. It ensures the development of bones and feathers in birds, as well as fertility and scavenging of oxygen free radicals in animals [1,2]. Additionally, Zn is one of the most important active elements for transforming protein, carbohydrate, nucleic acid, and activating enzyme, which is essential for the proper operation of the immune system [3][4][5].
Maternal effects are one of the most important epigenetic inheritance phenomena, and can directly influence the offspring's growth, development, nutrient metabolism, and physiological characteristics [6]. Previous studies reported that maternal stress not only influences the birth weight and growth of offspring, but also changes the metabolic This study was approved by the Animal Care and Use Committee of Zhejiang University. Experimental design and treatments for the maternal broiler breeder phase were discussed in detail in our previous study [18]. A completely randomized design was performed with two maternal dietary supplemental Zn sources [maternal Zn-Gly (oZn) vs. maternal ZnSO 4 (iZn)] × two offspring dietary supplemental Zn doses [Zn-unsupplemented control diet (CON), the control diet + 80 mg of Zn/kg of diet from ZnSO 4 (80 mg zinc/kg was derived from crystal standard zinc, provided by PANCOSMA, Switzerland). Firstly, a total of 200 39-week-old Lingnan Yellow broiler breeders were randomly divided into two groups-the oZn group and iZn group-with each group containing 5 replicates with 20 birds each. All broiler breeders from the two groups were fed a corn-soybean meal basal diet with 24 mg Zn/kg for four weeks pre-test to consume excess Zn in the body; in the formal feeding period of eight weeks, the iZn group and oZn group were supplemented with 80 mg Zn/kg from ZnSO 4 and 80 mg Zn/kg from the Zn-Gly-based basal diet, respectively. All eggs from the last seven days were collected from two breeder groups (oZn group and iZn group) for offspring incubation. Then, 400 healthy offspring chicks from each group were selected and divided into two groups with two offspring dietary supplemental Zn doses (0 or 80 mg Zn/kg). Figure 1 shows the entirety of the experimental design. Table 1 displays the composition and nutrient content of the basal diet for the broiler breeders. The control group received the basal diet without Zn supplementation (negative control), and (iZn-iZn) and Zn-Gly (oZn-oZn) received a basal diet supplemented with 80 mg of Zn/kg of diet from ZnSO 4 and Zn-Gly, respectively. Then, all hatched chicks were reared for the 60 day offspring broiler phase. Table 2 shows the composition and nutrient level of the basal diet for the offspring broiler. On days 21 and 60, three male broilers from each replicate were randomly selected and slaughtered. Serum samples from the blood were isolated by centrifuging at 3000× g for 10 min and stored at −80 • C until analysis. Liver and left breast muscle samples were immediately stored at −80 • C after slaughter for further analysis.
for the broiler breeders. The control group received the basal diet without Zn supplementation (negative control), and (iZn-iZn) and Zn-Gly (oZn-oZn) received a basal diet supplemented with 80 mg of Zn/kg of diet from ZnSO4 and Zn-Gly, respectively. Then, all hatched chicks were reared for the 60 day offspring broiler phase. Table 2 shows the composition and nutrient level of the basal diet for the offspring broiler. On days 21 and 60, three male broilers from each replicate were randomly selected and slaughtered. Serum samples from the blood were isolated by centrifuging at 3000× g for 10 min and stored at −80 °C until analysis. Liver and left breast muscle samples were immediately stored at −80 °C after slaughter for further analysis.

Measurement of Growth Performance and Sample Collection
On days 1, 21, and 60 of the experiment, the birds and their diet were weighed per the whole replicate. The variables of the growth performance (final body weight (BW)), average daily feed intake (ADFI), average daily gain (ADG), and feed:gain ratio (F:G) were measured.

Zn Concentration Determination
As described in our previous study [17], inductively coupled plasma atomic emission spectrometry (Iris Intrepid; Thermo Elemental, Waltham, MA, USA) was used to determine the concentrations of Zn in serum, liver, and breast muscle after microwave wet digestion in nitric acid (MARS 5; CEM Corp., Matthews, NC, USA) and dilution with distilled water. Calibration of Zn content determination was performed using a series of mixtures containing Zn standard solutions of graded concentrations [19].

Antioxidant Status Analysis
After combining the liver and muscle of each replicating broiler into a separate sample, the sample was weighed and then homogenized into nine volumes of sodium chloride solution (0.86%) (4000 rpm, 4 • C, 15 min) with Ultra Turrax homogenizer (Tekmar Co., Cincinnati, OH, USA). The homogenates were then centrifuged at 3500 rpm at 4 • C for 15 min and the supernatant was collected for further analysis [18].
As demonstrated in our previous study, the total superoxide dismutase (T-SOD) activities, copper zinc superoxide dismutase (Cu-Zn SOD) activities, total antioxidant capacity (T-AOC), and malondialdehyde (MDA) concentrations were evaluated using a commercial analytical kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The results were standardized with the total protein for internal comparison. The concentrations of liver metallothionein (MT) was determined by the commercial ELISA kit purchased from Shanghai Elisa Technology Co., Ltd. All procedures were carried out according to the instructions of the assay kits [18].

Chemical and Cytokine Secretions Analyses
Immunoglobulins (IgA, IgG and IgM) and pro-inflammatory cytokines (IL-1, IL-2, and TNF-α) were determined using the commercial ELISA kit purchased from Shanghai Elisa Technology Co., Ltd. All procedures were carried out according to the instructions of the assay kits.

Statistical Analysis
All data were analyzed by two-way ANOVA using the general linear model procedure of SAS 9.2 (SAS Institute, 2010, Cary, NC, USA). This model combines the main effects of maternal dietary Zn, progeny dietary Zn, and their interaction. The treatment comparisons for significant differences were tested by the LSD method. Each replicate served as the experimental unit for all statistical analyses. Significant differences were set at p ≤ 0.05.

Growth Performance
During days 1-60, oZn increased the progeny ADG and decreased the mortality (p < 0.05), but no significant differences were observed for the ADFI and F/G parameters. The offspring diet supplemented with Zn improved the ADG by 2.55% and significantly decreased mortality compared with the CON group (p < 0.05). Significant interactions between maternal Zn and offspring's dietary Zn supplementation were observed in progeny mortality during the entire phase (Table 3). ADG, average daily gain. ADFI, average daily feed intake. F/G, feed/gain. Different letters (a-c) represent significant differences between the groups. 1 Each value represents the mean ± SE of 5 replicates (n = 200). 2 Each value represents the mean ± SE of 10 replicates (n = 400). 3 Each value represents the mean ± SE of 10 replicates (n = 400).

Zn Concentrations
The effects of maternal Zn in conjunction with their offspring's dietary Zn supplementation on the serum, liver, and muscle Zn concentrations of offspring broilers are shown in Table 4. Maternal dietary Zn as Zn-Gly rather than ZnSO 4 supplementation resulted in higher Zn concentrations in the liver of the offspring at 21 days of age and increased progeny Zn concentrations in serum, liver, and muscle at 60 days of age (p < 0.05). Additionally, the broiler offspring's dietary 80 mg/kg of Zn supplementation increased the Zn concentrations in the serum, liver, and muscle during the entire phase compared to the CON group (p < 0.05). Significant interactions (p = 0.031) between maternal Zn and offspring's dietary Zn supplementation were observed in the progeny's muscle at 60 days of age.

Antioxidant Status in Serum
The effects of maternal Zn in conjunction with their offspring's dietary Zn supplementation on the serum antioxidant status of offspring broilers are shown in Table 5. Maternal dietary oZn supplementation increased the activities of T-AOC (by 5.66%), T-SOD (by 8.58%), and CuZn-SOD (by 6.24) compared to iZn at 21 days of age, and similar results were also observed in the broiler's offspring dietary 80 mg Zn/kg supplementation compared with the CON group (p < 0.05). At 60 days of age, T-AOC activity increased by 7.99% in the case of oZn compared to iZn; and the activity of T-AOC (by 21.49%) and CuZn-SOD (by 6.43%) significantly increased when the broiler's offspring dietary 80 mg Zn/kg supplementation (p < 0.05) compared with the CON group. The MDA content decreased in offspring broilers at 21 and 60 days of age compared to maternal ZnSO 4 (p < 0.05). Significant interactions between maternal Zn and offspring dietary Zn supplementation were observed in progeny MDA content at 21 days of age and activity of T-SOD at 60 days of age (p < 0.05).

Antioxidant Status in Liver
The effects of maternal Zn in conjunction with their offspring's dietary Zn supplementation on the liver antioxidant status of offspring broilers are shown in Table 6. Maternal dietary Zn-Gly supplementation increased the T-SOD activity and MT content in offspring broilers at 21 and 60 days of age and CuZn-SOD activity in offspring broilers at 21 days of age compared with that of maternal ZnSO 4 (p < 0.05), whereas maternal Zn supplementation did not affect their offspring regarding the activity of T-AOC in the liver of the progeny (p > 0.05). Additionally, compared with the CON group, offspring broilers dietary 80 mg/kg of Zn supplementation increased progeny T-AOC, T-SOD, and CuZn-SOD activities at 21 days of age, and decreased progeny MDA content and increased MT content at 21 and 60 days of age (p < 0.05). Significant interactions (p < 0.001) between maternal Zn and offspring dietary Zn supplementation were observed in the progeny's MDA content at 21 days of age and the progeny's T-AOC, T-SOD, and CuZn-SOD activities at 60 days of age (p < 0.05).

Antioxidant Status in Muscle
The effects of maternal Zn in conjunction with their offspring's dietary Zn supplementation on the muscle antioxidant status of broiler offspring are shown in Table 7. Maternal dietary Zn as Zn-Gly rather than ZnSO 4 supplementation increased T-AOC activity and decreased MDA content at both 21 and 60 days of age (p < 0.05). Furthermore, their offspring broiler's dietary 80 mg/kg of Zn increased the activities of T-SOD and CuZn-SOD at 21 days of age, and increased the T-AOC activity and decreased MDA content at both 21 and 60 days of age (p < 0.05). Meanwhile, no significant interactions between maternal Zn

Cytokine Secretion
The effects of maternal Zn in conjunction with their offspring's dietary Zn supplementation on the cytokine secretion of offspring broilers are shown in Table 8. Maternal dietary Zn as Zn-Gly rather than ZnSO 4 supplementation had no significant effects on progeny IL-1, IL-2, and TNF-α concentrations at 21 days of age while significantly decreasing the IL-1 and TNF-α concentrations of progeny at 60 days of age (p < 0.05). Similar results were observed for offspring broilers dietary 80 mg/kg of Zn. No significant interactions between maternal Zn and dietary Zn supplementation were observed in progeny cytokine secretions at 21 days of age (p > 0.05), whereas significant interactions were observed in progeny IL-1 (p = 0.012) and TNF-α (p = 0.030) concentrations at 60 days of age.

Immunoglobulin Content
The effects of maternal Zn in conjunction with their offspring's dietary Zn supplementation on the immunoglobulin content of offspring broilers are shown in Table 9. Maternal dietary Zn-Gly rather than ZnSO 4 supplementation increased progeny IgM concentrations at both 21 and 60 days of age (p < 0.05). Additionally, offspring dietary supplementation of 80 mg/kg of Zn increased the IgA content of progeny at 21 days and the IgG and IgM concentrations of progeny at 60 days of age (p < 0.05); moreover, no significant interactions between maternal Zn and offspring dietary Zn supplementation were observed in progeny immunoglobulin content (p > 0.05). Table 8. Effects of maternal Zn in conjunction with their offspring dietary Zn supplementation on cytokine secretion in serum of offspring broilers (mean ± S.E.). IL-1, interleukin-1. IL-2, interleukin-2. TNF-α, Tumor necrosis factor-α. Different letters (a-b) represent significant differences between the groups. Superscripts referring to 1, 2, and 3 have the same meaning as Table 4. Immunoglobulin A. IgG, Immunoglobulin G. IgM, Immunoglobulin M. Different letters (a-c) represent significant differences between the groups. Superscripts referring to 1, 2, and 3 have the same meaning as Table 4.

Stress Index
The effects of maternal Zn in combination with their offspring's dietary Zn supplementation on the stress index of the broiler's offspring are shown in Table 10. Maternal Zn-Gly rather than ZnSO 4 supplementation had no significant effects on progeny CORT, HSP70, and CK content at 21 days of age; however, it significantly decreased the CORT content of progeny at 60 days of age. Additionally, their broiler's offspring dietary supplementation of 80 mg/kg of Zn significantly decreased the CORT content at 21 days of age (p < 0.05), and Zn-Gly rather than ZnSO 4 supplementation significantly increased the HSP70 content at 60 days of age (p < 0.05). Significant interactions between maternal Zn and offspring's dietary Zn supplementation were observed in progeny CORT (p = 0.033) and HSP70 (p = 0.021) concentrations at 60 days of age.

Discussion
Maternal Zn, in combination with their offspring's dietary Zn supplementation, had no significant effects on the progeny growth performance during the stage of days 1-21. Furthermore, supplementation of 80 mg Zn/kg from Zn-Gly increased progeny BW at 60 days as well as ADG during the stage of days 22-60, and had a lower mortality rate compared to the same Zn levels from ZnSO 4 , thereby indicating that maternal dietary organic Zn in conjunction with their offspring's dietary Zn supplementation improved the growth performance of progeny in the final period. Consistent with our findings, several studies reported that dietary organic Zn supplementation increased growth rate, feed intake, and feed efficiency in broiler chicks [20][21][22][23] however, reports showed that low Zn diets will lead to loss of appetite, feed intake, and weight gain [24,25], resulting in decreased hatchability and increased embryonic mortality [26,27] and even further depressed the growth performance of progeny [8]. It is implied that in the current study, maternal dietary Zn supplementation may be transferred to progeny broilers during the stage of embryonic development and showed in the progeny growth performance of the final period.
Dietary Zn supplementation in broiler breeders markedly enhanced the Zn concentrations of eggs [9,28]. In our previous studies, the Zn concentrations in muscle and eggs [17] increased with dietary organic Zn supplementation compared with inorganic Zn and even increased in the livers of developing embryos and one-day-old chicks [18] moreover, we found that maternal 80 mg Zn/kg from Zn-Gly increased the Zn concentration in progeny serum, liver, and muscle compared to the same Zn levels from ZnSO 4 . This may be because in broilers, dietary organic Zn supplementation had a higher absorption rate and lower excretion rate than inorganic Zn [29], thereby resulting in higher Zn concentrations. Interestingly, the broiler offspring's dietary Zn supplementation increased Zn concentrations in their liver at 21 days of age and remained stable over time. On the contrary, it returned to initial values or was even lower in serum and muscle at 60 days of age. This may be because broilers were at different growth stages and had different nutritional requirements. The increased deposition of Zn in egg yolk leads to higher Zn availability, which facilitates the growth of chick embryos [30]. Consistent with the results in our present study, Kwiecień et al. [1] indicated that organic Zn addition enhanced the Zn concentrations in the liver of broilers compared to inorganic Zn supplementation, implying that the results of increased egg, progeny tissue, and serum Zn concentrations were from the increased maternal Zn.
Given the better antioxidant properties of Zn, therefore, dietary Zn status is closely related to the antioxidant system [31] it influences antioxidant status by a different mechanism: First, protecting proteins and enzymes against free radical attack or oxidation, and second, preventing free radical formation produced by other metals, such as iron and copper [32]. T-AOC, T-SOD, and MDA are marks of oxidative stress as they participate in the antioxidant defense systems that result in potential damages induced by oxidative stress [33]. Zn is an essential component of CuZn-SOD, and its potential antioxidant effects could be associated with its role in the mechanism of the structural integrity of CuZn-SOD [34]. Additionally, its presence in the Zn metallothionein protects organs and tissues against immune-mediated attacks by ROS [35].
Zn deficiency might cause an increase in the production of free radicals [36], resulting in decreased SOD activity and increased MDA content [37]. However, Zn supplementation enhanced the activities of CuZn-SOD and GSH-Px and decreased the MDA levels in broilers' livers [38]. Our previous experiment showed that maternal Zn supplementation significantly reduced the content of MDA and enhanced the activity of T-SOD, CuZn-SOD, and T-AOC in the livers of both chick embryos and one-day-old chicks [18]. Further experiments showed that maternal Zn, in conjunction with their offspring's dietary Zn supplementation, markedly reduced the MDA concentrations and enhanced the activity of T-AOC and CuZn-SOD in progeny serum, liver, and muscle, and enhanced MT concentrations in the liver of progeny. In addition, maternal organic Zn had better effects on the antioxidant defense of progeny serum and tissue than inorganic Zn. It is implied that maternal Zn, in combination with their offspring's dietary Zn supplementation, had better effects on antioxidant capacity; it reduces the oxidative stress of offspring broilers by increasing the activity of CuZn-SOD and inducing the production of MT, thereby improving the production performance of broilers.
Zn can interact with the immune systems' components and is thought to be essential for immune response [39]. It binds to enzymes, proteins, and peptides with different binding affinities [40]. A previous study reported that the immune organs, including the thymus, spleen, and bursa of fabricius of three-week-old broilers, were significantly influenced by Zn-Gly levels in their diets [41]. Additionally, Zn supplementation is an adjunctive therapy for treating chronic and inflammatory diseases; modulating TNF-α, IL-6, and IL-10; and modifying the gene expression of MT in white blood cells. This highlights the importance of Zn in gene transcription and cell metabolism [42,43]. It can also prevent Salmonella-induced liver injury by reducing pro-inflammatory cytokine levels [44]. In the current study, the results indicated that maternal organic Zn and their offspring's dietary Zn supplementation significantly reduced the concentrations of IL-1 and TNF-α, suggesting that the protective effects of Zn on the broiler may be through downregulating the inflammation of the host; moreover, the severity of the inflammatory response is generally inversely proportional to the serum Zn concentration [45]. Hence, the additional possible mediator of Zn, which alleviates the host's inflammatory response, is caused by the enhanced antioxidant defense and higher Zn concentration.
In the present study, maternal organic Zn and their offspring's dietary Zn supplementation had significantly higher immunoglobulin concentrations and complements. Consistent with our present results, ample Zn supplementation improves the ability to produce antibodies [46,47]. Fraker et al. [48] showed that supplementing Zn as ZnSO 4 to a diet deficient in Zn was effective for total serum IgG concentration, and diets supplemented with Zn as Zn-Gly had higher levels of immunoglobulins (IgA, IgM, and IgG) in the serum of broilers [41], moreover, Wellinghausen et al. [49] indicated that Zn status in Zn methionine and Zn sulfate affected the total non-specific IgM and IgG concentrations in seven-day-old broilers.
Stressors (such as heat stress and oxidative stress) can increase plasma corticosterone concentration in different tissues of poultry with a concomitant increase in creatine kinase levels [50]. In the present study, offspring dietary Zn supplementation significantly decreased the corticosterone levels, indicating that Zn supplementation can reduce oxidative stress during broiler production, as Zn supplementation increased broiler tissue Zn content-thereby improving its antioxidant status and reducing oxidative stress. However, we also found that Zn supplementation increased the levels of HSP70, which may be because HSP70 is the most important family of heat shock proteins and can produce nonspecific tolerance to various stressors [51]. The increased levels of HSP70 in vivo alleviates the stress of environmental stressors on chickens to a certain extent.
In the current study, as this experiment was a 2 × 2 factor experimental design, the offspring broilers were only supplemented with inorganic Zn with different doses (0 or 80 mg Zn/kg). In our future experiments, we will explore the effects of adding different forms of Zn sources (including inorganic Zn and organic Zn) to the offspring broilers.

Conclusions
In conclusion, maternal Zn-Gly supplementation increased progeny performance and decreased progeny mortality and stress by increasing the progeny Zn concentration, antioxidant capacity, and immune function at the same Zn levels as ZnSO 4 . At the same time, Zn supplementation in the progeny's diet is necessary for the growth of broilers.