Introduction

With the development of duck industry the traditional free-range rearing system resulted in the low nutrient efficiency high incidence of diseases and severe water pollution of wastage in duck production [1, 2]. The duck housing system is being gradually shifted from the non-caged system to the caged system to ensure the better health status and maximal economic benefit in China. However one major issue was the fact that the caged system provided a restricted physical and psychological space and prevented birds from performing natural behaviors compared with the non-caged system [3]. Moreover the caged system exerted some negative effects on the productivity of laying birds due to limited movement such as poor bone strength [4] and egg quality [5, 6]. Poor eggshell quality characteristics could reduce the shelf life and safety of eggs and egg products [7]. For example the decreased eggshell thickness could increase the risk of the bacterial contamination in hatching eggs [8] and then impair hatchability performance of laying breeders [9]. However there is limited systematic information available on eggshell quality of duck breeders in alternative furnished cage system. Some researchers have proposed that the effects of different housing systems on eggshell quality were also dependent on the layer age [10, 11]. Therefore we firstly investigated the effects of bird age and rearing system (floor pen vs furnished cage) on eggshell quality of laying duck breeders

Zinc (Zn) as a component of carbonic anhydrase (CA) enzyme plays a role in eggshell formation [11, 12]. Many studies have showed that dietary Zn supplementation enhanced eggshell quality by increasing eggshell thickness and strength of hen layers and duck layers [13, 14]. Dietary Zn requirement recommendations for Peking duck breeders in NRC (1994) [15] and Muscovy duck breeders in INRA (1989) [16] were sourced from the data based on the non-caged systems. However, caged system that allows less movement of birds could influence feed intake and energy expenditure of birds [17]. Thus, nutrient requirements should be modified as with the changes of physical activity and energy intake of birds under the different housing systems [6]. So far, there has been little research on dietary Zn nutrition for improving eggshell quality of duck breeders reared in furnished cage system. Therefore, the effects of dietary Zn levels on laying performance, egg quality, and eggshell microstructure were investigated in Muscovy duck breeders under furnished cages in the present study.

Materials and Methods

Animals and Diets

All procedures of our experiments were approved by animal care and welfare committee institute of South China Agricultural University. Firstly, the eggshell quality of laying Muscovy duck breeders reared in two rearing technologies, a cage-free system and a furnished cage system, was compared (Exp. 1). Sixty Muscovy ducklings were selected and housed in the plastic floor system (from 0 to 12 weeks of age) and caged-free littered system (from 13 to 27 weeks of age) at the early laying period. After that, sixty laying duck breeders were divided into two groups consisting of thirty birds, which were placed into littered floor system (LF) and furnished cage system (FC), respectively. The littered system had an indoor and outside dirt lot, which both had perches (15 cm2 of area per duck) and nest box (4 ducks per nest). The floor in the indoor area was covered with wood shavings as a litter material. The outside area without grass or forage had a shelter and was enclosed by wire fences to keep out predators. A total of seven and eight ducks per square meter were provided in the indoor and outside areas, respectively. The furnished cage met the requirement of the EU Directive 1999/74/EC. The furnished cage units were made of a wire mesh floor, galvanized metal partitions between the cages, and full-opening fronts at each long side of the cages consisting of spaced horizontal bars. All laying duck breeders’ ducks were fed restrictively with a commercial feed at the same nutritional level (11.82 MJ ME/kg, 180 g CP/kg, 8.0 g lysine/kg, 7.2 g methionine + cysteine/kg, 33.0 g Ca/kg, 3.8 g available P/kg). Water was available ad libitum. The droppings were automatically removed on manure belts. The 20 eggs were collected from the laying birds between at the end of 35 and 40 weeks of age, respectively. A total of 40 eggs from each rearing system were analyzed for eggshell quality. In Exp. 2, a total of 370 26-week-old Muscovy duck breeders obtained from a commercial duck breeder farm (WENS Group, Yunfu, Guangdong) were housed in furnished cages for 4 weeks of an adaptation period. During the early laying period, all ducks were fed restrictively with a commercial feed at the nutritional level (11.32 MJ ME/kg, 180 g CP/kg, 7.0 g lysine/kg, 7.2 g methionine + cysteine/kg, 24.0 g Ca/kg, 3.8 g available P/kg, 40 mg Zn/kg). Water was provided ad libitum. At 30 weeks of age, 324 laying duck breeders were selected, balanced for laying rate, and then randomly allotted into 3 dietary Zn groups with 6 replicates (18 ducks per replicate). The experimental diets included base diet without Zn supplementation (control-Zn group, C-Zn), basal diet + 40 mg Zn/kg (normal-Zn group, N-Zn), and basal diet + 140 mg Zn/kg (high-Zn group, H-Zn) as ZnSO4.7H2O source. The basal diet was formulated to meet or exceed the nutritional requirements of laying duck breeders except Zn. The composition and nutrient level of the basal diet were shown in Table 1. The analyzed values of Zn contents in C-Zn, N-Zn, and H-Zn diets were 29.2, 63.4, and 163.4 mg/kg, respectively. The experimental period lasting for 6 weeks was divided into 3 periods of 30–32, 32–34, and 34–36 weeks of age. All ducks had diets restrictively and access to water ad libitum according to the operation manual and guideline of Muscovy duck breeders. The ducks received 16 h of daily lighting from 04:30 am to 20:30 pm. Room temperature and humidity were controlled by the air conditioner and recorded daily. Manure was removed through an automatic belt system daily. All eggs were collected from each replicate daily and egg production (number of total laid eggs, defective eggs, and average qualified egg weight) were recorded daily. Feed consumption and egg weight were measured weekly. Feed intake was calculated by dividing total feed consumed by numbers of ducks per replicate per day. Four eggs based on the average egg weight each replicate were collected at the last day of each period for the measurements of egg quality and eggshell microstructure.

Table 1 Composition and nutrient levels of the basal diets for laying duck breeders during 30–36 weeks of age (as-fed basis)
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Sample Analyses

The Zn concentrations in diets were measured using atomic absorption spectrophotometry (model Shimadzu AA-6800, Tokyo, Japan). At the end of 32, 34, and 36 weeks of age, four eggs per replicate were used to measure the related indices of egg quality. The color of the yolk and albumen height from the collected eggs were determined using an egg multitester (model EMT-5200, Touhoku Rhythm Ltd., Tokyo, Japan). Haugh unit was calculated based on egg weight and albumen height (Haugh, 1937). Shell strength was determined with an Egg Force Reader (model EFR-01, Orka, Ramat HaSharon, Israel). Eggshell was separated from albumen and yolk, washed to remove residual albumen, and cleaned by a paper towel and then weighed. Eggshell thickness was measured without inner and outer shell membranes at the blunt end (bottom), sharp end (top), and middle using a peacock dial pipe gauge (model P-1, Ozaki MFG Ltd., Tokyo, Japan). Shell thickness was calculated as the average thickness at the three points of eggshell. Eggshell microstructures were measured by scanning electron microscopy as described by Berwanger et al. (2018) [17]. Average values of the mammillary cone width and the thickness of mammillary layer, palisade layer, and eggshell membrane were estimated from 3 measurements (μm) in each photograph.

Statistical Analysis

Data were analyzed by 2-way ANOVA using the PROC GLM procedure of the SAS 9.2 (SAS Inst. Inc., Cary, NC). The models included rearing system and age, and their interactions for Exp. 1, as well as dietary Zn, period, and their interactions for Exp. 2. The replicate served as the experimental unit. Differences among means were tested by the LSD method, and statistical significance was set at P ≤ 0.05.

Results

Eggshell Quality

Shell weight, shell strength, and shell thickness were affected by age (P < 0.01), not by rearing system (P > 0.05). The eggs from laying duck breeders at 40 weeks of age had higher shell weight and weaker shell strength and thickness than those from birds at 35 weeks of age. The interaction between age and system affected the average shell thickness (P = 0.05) and had no effect on other measured indices (P > 0.05). The rearing system did not influence the average shell thickness of duck breeder eggs at 35 weeks of age, whereas FC system had a lower average shell thickness of duck breeder eggs at 40 weeks of age compared with the LF system (Table 2).

Table 2 Effects of age and rearing system on eggshell quality of laying duck breeders at 35 and 40 weeks of age
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Egg Production Performance

Egg weight, laying rate, egg production, egg to feed ratio, and qualified eggs ratio were affected by age (P < 0.0001) and dietary Zn (P < 0.05), not by their interaction (P > 0.13). Egg weight, laying rate, egg production, egg to feed ratio, and qualified eggs ratio were increased as age and dietary Zn level increased (P < 0.04). Egg weight, laying rate, egg production, egg to feed ratio, and qualified eggs ratio of duck breeders during 32–34 weeks and 34–36 weeks of age were greater than birds during 30–32 weeks of age (P < 0.0001), with no differences between the periods of 32–34 weeks and 34–36 weeks of age (P > 0.05). Compared with C-Zn group, N-Zn and H-Zn groups increased egg weight, egg production, and egg to feed ratio of duck breeders (P < 0.02), with no differences between N-Zn and H-Zn groups (P > 0.36). Duck breeder from H-Zn group had higher laying rate and qualified egg ratio than C-Zn group (P < 0.01), and no differences were observed between C-Zn and N-Zn groups as well as N-Zn and H-Zn groups (P > 0.24) (Table 3).

Table 3 Effects of age and dietary Zn on egg production performance of duck breeders at 30 to 36 weeks of age under furnished cage system
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Egg Quality

Shell thickness, Haugh unit, and yolk color were affected by duck age (P < 0.03). Dietary Zn and the interaction between age and dietary Zn influenced (P < 0.04) Haugh unit and shell thickness, respectively. Age, dietary Zn, and their interaction had no effects (P > 0.05) on the shell weight and shell strength of duck breeders. Eggs from ducks at 34 weeks old had higher Haugh unit and lower shell thickness than birds at 32 and 36 weeks old (P < 0.03), while egg yolk color from ducks at 34 and 36 weeks old was higher than birds at 32 weeks old (P < 0.01). Haugh unit in H-Zn group was higher (P < 0.02) than C-Zn group, with no differences between C-Zn and N-Zn groups. Dietary Zn level had no effect on shell thickness of duck breeders at 32 and 34 weeks of age (P > 0.27), whereas dietary supplementation of 40 and 140 mg Zn/kg increased shell thickness of duck breeders at 36 weeks of age compared with the control group (P < 0.05) (Table 4).

Table 4 Effects of age and dietary Zn on egg quality of duck breeders at 32, 34, and 36 weeks of age under furnished cage system
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Eggshell Microstructure

The mammillary cone width and thickness were affected (P < 0.02) by bird age. Dietary Zn level had an effect (P < 0.02) on mammillary cone width and did not affect (P > 0.05) mammillary cone width, palisade layer thickness, and membrane thickness. The interaction between age and dietary Zn influenced (P = 0.05) palisade layer thickness of ducks. Eggs from ducks at 32 and 34 weeks old had greater mammillary cone width than birds at 36 weeks old, while H-Zn group had a lower mammillary cone width and a greater membrane cross-linking structure than C-Zn and N-Zn groups (P < 0.01; Figs. 1 and 2). Dietary Zn supplementation had no effect on palisade layer thickness of duck breeders at 32 and 34 weeks of age (P > 0.48), whereas H-Zn group increased palisade layer thickness of duck breeders at 36 weeks of age compared with C-Zn group (P < 0.04), with no differences between C-Zn and N-Zn groups (P > 0.72) (Table 5).

Fig. 1
figure 1

Scanning electron cross-sections of eggshells from duck breeders fed C-Zn (A, D, G), N-Zn (B, E, F), and H-Zn (C, F, I) diets at 32, 34, and 36 weeks of age (×200). C-Zn, control Zn group with 0 mg Zn/kg diet; N-Zn, normal Zn group with 40 mg Zn/kg diet; H-Zn, high Zn group with 140 mg Zn/kg diet; MT, membrane thickness; MCW, mammillary cone width; MLT, mammillary layer thickness; PLT, palisade layer thickness

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Fig. 2
figure 2

Scanning electron fiber cross-linking structure of eggshell membranes from duck breeders fed C-Zn (A, D, G), N-Zn (B, E, F), and H-Zn (C, F, I) diets at 32, 34, and 36 weeks of age (×600). C-Zn, control Zn group with 0 mg Zn/kg diet; N-Zn, normal Zn group with 40 mg Zn/kg diet; H-Zn, high Zn group with 140 mg Zn/kg diet. The representative defective fiber cross-linking structure of eggshell membranes was indicated in red circle

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Table 5 Effects of age and dietary Zn on eggshell microstructure of duck breeders at 32, 34, and 36 weeks of age under furnished cage system
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Discussion

Broken egg problem is a large financial loss to the poultry industry. The eggshell quality is one of the most important parameters for maintaining the shelf life and the safety of eggs and egg products [7]. For poultry breeders, good eggshell quality could prevent the hatching eggs from bacterial contamination [8] to maintain hatchability performance [9]. Several studies have proved that eggshell characteristics could be affected by bird age and different housing systems in laying hens and breeder hens [10, 18]. In the current study, eggshell parameters such as weight, strength, and thickness were decreased with the increased age of duck breeders at 35–40 weeks of age regardless of rearing systems. Moreover, significant interaction between age and housing system on eggshell thickness demonstrated that no differences were observed on the average shell thickness of duck breeder eggs at 35 weeks of age under two rearing systems, whereas FC system had a lower average shell thickness than LF system in duck breeders at 40 weeks of age. It was suggested that shell thickness of a duck breeder in the FC system was decreased much greater as the age was increased. The declined eggshell quality of duck breeders in the FC system was confirmed by the comparison of eggshell quality in laying hens under the battery cage and deep litter systems [19]. It is probably due to the lack of exercise and poor bone mineral mobilization of birds in the FC system contributing to the poor mineralization during eggshell formation [4]. Another factor was that providing insufficient light intensity for laying birds at the lower tiers in multitier cage systems could induce the less calcium mobilization for eggshell formation resulting in the lower shell thickness [20]. Additionally, compared with the non-caged system, the caged system was characterized with the restricted space that allowed less movement of birds [21], which further affected physical activity, energy expenditure, and nutrient requirement of birds [6]. Therefore, it is very important to change the nutritional strategies to improve eggshell quality when duck breeders were shifted from the non-caged systems to FC systems.

Egg weight, egg production, and egg quality could be influenced by bird age [22, 23]. As the age was increased from 30 to 36 weeks old, egg production performance and yolk color of female breeders were increased linearly. The enhancement in egg production with advancing age was exhibited by the lower ovulation rate and shorter ovulation and oviposition sequence from laying early period to laying peaking period [23, 24]. Zn is a cofactor of more than 200 enzymes functioning in diverse physiological processes to maintain the optimum productive performance of poultry breeders [25]. The observations that dietary Zn deficiency impaired egg production and egg quality in layer and breeder birds were well established [26]. Our results showed that dietary Zn supplementation increased egg weight, egg production, egg to feed ratio, and Haugh unit in duck breeders housed in the FC system during 30–36 weeks old. The positive effects of Zn addition on laying performance were confirmed in laying hens [27,28,29] and laying ducks [14]. Dietary Zn nutrition could improve the epithelium quality via increasing the protein synthesis deposition of albumen, resulting in greater values of egg production and Haugh unit. However, other studies reported that adding Zn to the diets had no effects on the characteristics of egg production of laying hens and broiler breeders [30, 31]. The differences between the studies might depend on the differences in dietary Zn levels, Zn content in basal diets, experimental periods as well as the breed differences, ages, and physiological states of birds under different rearing systems. Moreover, the diets with 140 mg Zn/kg improved laying rate and qualified egg ratio compared with the control diet, whereas no differences were observed between the control diet and diet with 40 mg Zn/kg. It is implied that dietary higher Zn level revealed the greater beneficial effect on egg production and egg quality in laying duck breeders in the FC system. Abd El-Hack et al. (2018) also stated that laying hens fed diet supplemented with 100 mg Zn/kg as Zn-Met source had greater egg production compared with birds fed basal diet supplemented with 50 mg Zn/kg as Zn-Met source [29]. One possible explanation for improvement in egg production may be the interaction of Zn with the secretion of reproductive hormones of the endocrine system during egg formation [32, 33].

Zinc is part of the CA enzyme which is essential in supplying carbonate ions during eggshell formation [34]. Numerous studies have shown that dietary Zn supplementation enhanced eggshell quality by increasing eggshell thickness and strength in laying birds [11, 12]. However, eggshell quality of laying ducks were not affected by dietary Zn level in the present study, which does not agree with results reported in laying hens [13]. The inconsistent results may be due to the differences in the Zn content and supplemental Zn levels in basal diets, experimental periods, age, and rearing systems of birds. In the present study, the basal diet containing about 29 mg Zn/kg diet seem to be sufficient for maintaining the eggshell quality in a shorter period during 30–34 weeks, according to the INRA (1989) recommendation of 20–40 mg Zn/kg of laying Muscovy duck breeders. The lacking effect of Zn addition on eggshell quality was consistent with the previous results in laying ducks fed a corn-soybean meal basal diet containing 37 mg Zn/kg during laying early and peak periods [14]. Although dietary Zn level had no effect on shell thickness of duck breeders at 32 and 34 weeks of age, whereas Zn supplementation in diets improved shell thickness of duck breeders at 36 weeks of age compared with the control diet. The interaction between age and dietary Zn on shell thickness implied that dietary Zn nutrition has a prolonged and accumulative beneficial effect on maintaining eggshell thickness of laying duck breeders. As the feeding time was increased, feeding Zn non-supplemented diet plus the lack of exercise of birds in the FC system resulted in the more severe body Zn depletion and insufficient bone Zn mobilization and then revealed the negative effect on shell thickness. Zhang et al. (2017) also indicated that Zn deficiency impaired eggshell thickness associated with the decreased CA activity during eggshell formation for the 6 weeks of experimental period [11].

The eggshell is structurally combined by organic and inorganic components [35]. The organic components of eggshell consisted of ultrastructural layers divided into shell membrane, mammillary knob, palisade, and cuticle [36]. The assembly and calcitic mineralization of different eggshell compartments are thought to be guided by organic molecules, including non-collagenous proteins and proteoglycans [37]. It has been shown that the enzymatic activity as trace cofactors influenced the formation of the mammillary layer in the region of the oviduct termed the tubular shell gland [12]. In the present study, scanning electron photographs results showed that 140 mg Zn/kg addition in diet reduced the mammillary cone width of eggshell compared with the control diet, which was in agreement with laying hens fed Mn-supplemented diet [38]. The decreased size of mammillary cones due to Zn intake implied that the number of mammillary cones per unit area on the internal shell membrane was increased. It was thought that more mammillary buttons grew into adjacent palisade column by calcitic mineralization and then resulted in a greater resistance of breakage [39]. The palisade layer accounts for about two-thirds of the thickness of the calcified eggshell. Diet supplemented with 140 mg Zn/kg increased the palisade layer thickness of duck breeders at 36 weeks of age compared with the control diet, with no effects observed in duck breeders at 32 and 34 weeks of age, which coincided exactly with the results of shell thickness in the present study. It is suggested that Zn nutrition could contribute to eggshell quality via improving the ultrastructural features. Previous study has demonstrated that proteoglycans plays a key role in the crystallization of the eggshell [40]. The Zn addition in diets could stimulate the proteoglycans synthesis to promote the eggshell nucleation and crystalline palisade growth [41, 42]. In addition, the greater Zn addition could enlarge the negative effect of the presence of high Ca concentration (3.7%) and phytate in basal hen diet due to the formation of an insoluble Ca-phytate-Zn complex [43, 44]. Zn also plays a role in deposition of albumen in the magnum and production of eggshell membranes in the isthmus [26]. Although the outer shell membrane thickness is not affected by dietary Zn levels, the poor membrane fiber formation or cross-linking structure occurred in dietary Zn deficiency and was characterized by abnormal compactness and distribution uniformity of fibers. It is implied that the enhancement of ultrastructural structure of shell membrane may produce resistant eggshells and then prevent the moisture loss of inner egg components due to Zn addition, which contributed to the qualify egg ratio and Haugh unit score in the present study.

In conclusion, dietary Zn supplementation improved the egg production performance and Haugh unit of laying duck breeders reared in the FC system during 30–36 weeks of age, displaying the greater beneficial effect for duck breeders fed a diet containing 140 mg Zn/kg. In addition, dietary high level of 140 mg Zn/kg increased ultrastructural palisade layer thickness contributing to the greater eggshell thickness of duck breeders at 36 weeks of age.