INTRODUCTION
The recommended level of chloride (Cl) and sodium (Na) in the diet for laying hens is 0.15% [1], mainly provided by sodium chloride (NaCl) addition. Additionally, the use of hydrochloride additives may cause excessive Cl in the diet. When Cl intake exceeds a certain level, the quality of the eggshell might be negatively affected [2,3]. There is an increasing interest in feeding laying hens with sodium sulfate (Na2SO4) to reduce the intake of Cl and the consumption of sulfur-containing amino acids [4]. The implications of dietary Cl-free Na source replacement and addition in laying hens had been the subject of our investigations. It had been reported that the addition of dietary Na2SO4 could improve antioxidant capacity and intestinal morphology [4,5]. Nevertheless, an excessive intake of Na2SO4 in the diet could disrupt renal, hepatic, and intestinal functions, thereby affecting laying performance in poultry [4]. Various tolerance levels had been identified, including 3.0% [4] and 1.2% [6]. Previous research demonstrated that the administration of dietary supplementation containing 0.15% Cl by substituting sodium bicarbonate (NaHCO3) or Na2SO4 for NaCl can improve eggshell quality and laying performance [7]. In addition, it could also increase eggshell breaking strength, thickness, and the eggshell ratio. Compared with NaHCO3, Na2SO4 has demonstrated superior production performance [7]. Fu et al [7] and Liu et al [4] reported that feeding laying hens a diet containing 0.6% or 0.71% Na2SO4 could improve the breaking strength, thickness, and eggshell ratio of eggshells in layers. However, there has been no clear information regarding the mechanism of dietary Na2SO4 supplementation on eggshell quality.
The formation of eggshell is an ion exchange process, which requires the involvement of ions and ion transporters. It has been previously reported that uterine Na+, potassium (K+) and calcium (Ca2+) exchanges under the action of ion transporters to form eggshells [8,9], such as sodium channel epithelial 1 subunit α (SCNN1A), solute carrier family 8-member a1 (SLC8A1), ATPase Na+/K+ transporting subunit β 1 (ATP1B1), potassium calcium-activated channel subfamily m α 1 (KCNMA1). Glycosaminoglycan (GAG) is a type of proteoglycan that can regulate the ultrastructure and dominate the process of biomineralization [10–12]. Liu et al [13] reported a significant correlation between the GAG contents of eggshell membranes and breaking strength. The transit of SO42− is a key factor in GAG synthesis [14]. It was found that 64% of the SO42− used in the isthmus was synthesized into chondroitin sulfate (CS), which is a type of GAG, and that SO42− can regulate the content of the CS/dermatan sulfate (DS) copolymer, further improving eggshell quality [13,15]. Therefore, we hypothesized that Na2SO4 would improve eggshell quality by affecting uterine ion transportation and GAG synthesis.
The present study aimed to determine the optimal supplemental level of Na2SO4 based on the corn-soybean basal diet (containing 0.15% Cl), and the variation in eggshell mechanical quality, ultrastructure, and composition in response to dietary Na2SO4 were also observed. The possible mechanism for Na2SO4 improved eggshell quality observed in this study will provide reference data for the application of dietary Na2SO4 in the production of laying hens.
MATERIALS AND METHODS
The experimental use of animals and related procedures were approved by the Animal Care and Use Committee of the Institute of Feed Research of the Chinese Academy of Agricultural Sciences (ACE-CAAS-20210903).
Experimental design and diets
A total of 432 healthy Hy-line Brown laying hens at 48 wk of age were allocated into 6 groups, with 8 replicates and 9 birds per replicate. The basal diet was formulated according to the Chinese Feeding Standard of Chicken [16] and National Research Council [1] to meet the nutritional requirements (Table 1). The 6 groups of experimental laying hens were fed with the basal diets supplemented with Na2SO4 (≥95.50%) at 0.22%, 0.37%, 0.52%, 0.68%, 0.83%, and 0.99%, respectively. The Cl and Na contents of the basal diets not including NaCl and Na2SO4 were 0.06% and 0.02%, respectively. The Cl level of the basal diet was set to be 0.15% by addition of 0.15% NaCl. The total dietary Na levels of the 6 groups were 0.15%, 0.20%, 0.25%, 0.30%, 0.35%, and 0.40%. The analyzed sodium and chloride contents in each group were shown in Table 2. The diets and water were supplied ad libitum. There was no significant difference in water intake among treatments in the last trial [7], so the water intake was not reported. The feeding trial lasted for 12 wks (49 to 60 wk of age) after an acclimation period of 1 wk.
The management of laying hens was consistent with the Hy-Line guidelines. All the laying hens were fed in a fully enclosed chicken house with natural ventilation combined with longitudinal negative pressure ventilation. The temperature was controlled at 18°C to 22°C. Laying hens were housed in 3-tier battery cages and exposed to a 16 h light/8 h dark photoperiod each day. Three adjacent cages with three birds per cage (40 cm×40 cm×35 cm) were assigned as one experimental unit.
Laying performance and eggshell quality
The number and total weight of eggs in each replicate were recorded daily during the feeding trial. The feed intake was recorded, and the egg production, average egg weight, average daily feed intake (ADFI), and feed conversion ratio (FCR) were calculated in each replicate every 2 weeks. In addition, 12 eggs were collected from each replicate for 3 consecutive days at wk 52, 56, and 60 randomly. Breaking strength was measured by an Egg Force Reader (EFR-01; Israel Orka Food Technology Ltd., Ramat Hasharon, Israel), and eggshell thickness was measured using an Egg Shell Thickness Gauge (ESTG-1; Israel Orka Food Technology Ltd.) at the equator and both poles; the values were subsequently averaged at those 3 points. The eggshell weight was measured after drying at room temperature for 48 h. The major and minor axes were measured with a Vernier caliper. The eggshell weight (%) was calculated as (eggshell weight/egg weight)×100, and the egg shape index was calculated as the major axis/minor axis.
Minerals contents in eggshell
Four eggshells were selected randomly from each replicate as samples for measuring the Ca, phosphorus, Na, and sulfur) contents in the eggshells at the end of the trial. These eggshells were washed with distilled water to remove impurities. After drying at room temperature, the samples were mixed and ground into powder. The samples were digested by a microwave digestion instrument (MDS-10; Shanghai Xinyi Instrument Technology Ltd., Shanghai, China). Approximately 0.5 g of eggshell powder was placed in a tetrafluoroethylene digestion tank, 3 mL nitric acid and 3 mL H2O2 were added, mixed and left for 24 h. The samples were transferred to a 50 mL conical flask after digestion. The acid was removed by heating on a temperature-controlled heating plate (T<180°C), and then evaporated and concentrated to 1 to 2 mL. The liquid in the conical flask was transferred to a 25 mL volumetric flask and set aside. The Ca and Na contents were analyzed via atomic absorption spectroscopy (Zeenit700 P; Analytik Jena Ltd., Jena, Germany), the phosphorus content was analyzed via spectrophotometry (UV-2000; Shimadzu Ltd., Kyoto Japan), and the sulfur content was analyzed via inductively coupled plasma-mass spectrometry (Agilent 7700x; Agilent Technology Ltd., Beijing, China) [17,18].
Ion concentrations and pH values in serum and uterine fluid
At 60 wks of age, two hens were selected from each replicate (same as the hens were selected in Laying performance and Eggshell quality), one hen was housed in one cage, and the laying time was recorded for 7 consecutive days. We selected and euthanised hens at 18.5 h post oviposition to collect serum and uterine fluid samples after the trial. The Na+, Cl−, K+, Ca2+, and HCO3− concentrations and pH values in the serum and uterine fluid were determined instantly using an automatic blood analyzer (PL2000; Perlong Medical Ltd., Jiangsu, China).
RNA isolation and real-time polymerase chain reaction
Two hens were selected and euthanized from each replicate at 18.5 h post oviposition to collect uterus randomly. Before RNA isolation, the thawed samples were ground in liquid nitrogen. Total RNA was extracted from tissues using TRIzol reagent (DP419; Tiangen Biotech Ltd., Beijing, China) according to the manufacturer’s instructions, and RNA purity and concentration were determined using a spectrophotometer (ND5000; Thermo Fisher Scientific Ltd., Portland, OR, USA). The integrity of the ribosomal RNA bands was confirmed on agarose gels. The quantification of mRNA consisted of a two-step reaction of reverse transcription and polymerase chain reaction (PCR). Reverse transcription was performed using 2 μg of RNA and a FastQuant RT Kit (KR106; Tiangen Biotech Co. Ltd., Portland, OR, USA). Real-time quantitative PCR was carried out with a CFX96 Touch Real-time PCR detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and SuperReal PreMix Plus (SYBR Green, FP205, Tiangen Ltd.). The PCR cycling program was as follows: initial denaturation for 15 min at 95°C, followed by 40 cycles of 95°C for 10 s, annealing and extension for 30 s at 60°C, each sample with 3 replicates. The results were normalized to that of β-actin, and the relative gene expression levels were calculated via the 2−ΔΔCT method by Livak and Schmittgen [19]. The primer sequences for SCNN1A, SLC8A1, ATP1B1, KCNMA1, and β-actin are given in Table 3. Sequences based on Jonchère [20].
Sulfated glycosaminoglycan in eggshell
At the end of wk 60, 4 eggshells from each replicate were immersed in 5% ethylenediaminetetraacetic acid (EDTA) to separate the calcified eggshell from the membrane and mixed as a sample. Approximately 1 g of calcified eggshell was used to measure the content of sulfated GAG. GAG content was determined using the methods of Ha et al [11] and Xiao et al [21].
Determination of ATP-S, SULT, CS, and DS in the isthmus portion
Two hens were selected and euthanised at 9 h post oviposition to collect the isthmus portion randomly. The contents of ATP-sulfurylase (ATP-S) and sulfotransferase (SULT) were determined by a chicken enzyme-linked immunoassay (ELISA) kit (Shanghai Enzyme-linked Biological Technology Ltd., Shanghai, China). The contents of CS and DS were determined by chicken ELISA kits (Shanghai Xin Yu Biotech Ltd., Shanghai, China).
Eggshell ultrastructure
We collect eggshell samples at 18.5 h post oviposition from 0.22%, 0.68%, or 0.99% Na2SO4 groups for eggshell ultrastructure observation. Four eggshell samples were collected from each replicate randomly, then two pieces of eggshell approximately 0.5 to 1 cm2 in length were selected from the equatorial section of each eggshell sample, a total of 8 pieces from each replicate were selected. Both the inside and outside of the eggshell were cleaned with distilled water to remove albumen and dirt, dried without affecting the vertical profile of the eggshell, fixed in a copper block with conductive glue, and sprayed with gold powder. To calculate the mammillary knob density, the sample was soaked in 1.0 N sodium hydroxide for 72 h to remove the albumen before being washed with the method of Gongruttananun [22]. The samples were imaged by scanning electronic microscopy (FEI Quanta 600; Thermo Fisher Scientific Ltd.). The effective thickness (covering cuticle, vertical crystal layer, and palisade layer), mammillary thickness, mammillary knob width, and total thickness were determined and defined as described by Zhang et al [23]. All the standard error of the mean (SEM) images were taken at 200× magnification.
Statistical analysis
Normality was assessed before the data analysis of the egg production rate was performed. All the data were analyzed using one-way analysis of variance (ANOVA), and the means were compared using Duncan’s multiple range test in SAS (SAS Institute Inc., Cary, NC, USA). The linear and quadratic effects of the supplemental Na2SO4 concentration were assessed using regression analysis. Differences were considered statistically significant at p≤0.05. The data are presented as the mean and pooled SEM.
The regression model was as follows:
Yij was the response variable, α was the intercept, β1 and β2 were regression coefficients, Xi was the studied factor effect that included Na2SO4 (i = 0.22%, 0.37%, 0.52%, 0.68%, 0.83%, and 0.99%), and eij was the observational error for (ij)th observation.
RESULTS
Laying performance
No mortality was observed during the trial. No significant differences were observed in laying performance by various supplemental Na2SO4 concentrations. As shown in Table 4, there were no significant differences in average egg weight, egg production, ADFI, or FCR among the groups (p>0.05).
Eggshell quality
The eggshell quality was improved by 0.52% and 0.68% supplementation with Na2SO4 (Table 5). The eggshell quality improved notably in the 0.68% Na2SO4 group. The breaking strength was greater in the 0.68% Na2SO4 group than in the other groups (p<0.05). Similarly, compared with those in the 0.22% and 0.37% Na2SO4 groups, the eggshell ratio in the 0.68% Na2SO4 group increased at the end of wk 56 (p<0.05). At the end of wk 60, the breaking strength, eggshell thickness, and eggshell weight increased in the 0.52% and 0.68% Na2SO4 groups compared with those in the 0.22%, 0.83%, and 0.99% groups, respectively (p<0.05). There was no significant difference in the egg shape index among the experimental groups (p>0.05).
Minerals contents in the eggshell
In this study, the 0.68% Na2SO4 group had a greater Ca concentration than the other groups (Table 6). There was a quadratic response in the Ca content among the different treatments. In contrast to that in the 0.22% and 0.99% Na2SO4 groups, a higher concentration of Ca was observed in the 0.68% group (p<0.001). Dietary Na2SO4 increased eggshell Na content linearly (p<0.05).
Serum and uterine fluid ion concentrations
Changes in the ion concentrations in the serum and uterine fluid induced by dietary Na2SO4 were found in this study (Table 7). In particular, the changes in Ca2+ concentration in uterine fluid were in accordance with the changes in eggshell Ca content. With increasing Na2SO4 supplementation, the serum Na+ concentration significantly increased (p<0.001). The Ca2+ concentration increased linearly, while the K+ concentration decreased linearly (p<0.05). The Na+ concentrations in the 0.83% and 0.99% Na2SO4 groups were greater than those in the 0.22% and 0.37% Na2SO4 groups (p<0.05). For uterine fluid, the K+ concentrations in the 0.83% and 0.99% Na2SO4 groups were significantly increased, while the Na+ concentrations in the 0.83% and 0.99% Na2SO4 groups were significantly decreased (p<0.05). The Ca2+ concentration was markedly greater in the 0.68% Na2SO4 group than in the other groups (p<0.05).
Expression of ion transfer and transporter genes in the uterus
A greater expression was found in the 0.68% Na2SO4 group than in the other groups, which indicated that ion transport could be promoted by this level (Figure 1). In this study, SLC8A1, SCNN1A, and ATP1B1 expression in the 0.52% and 0.68% Na2SO4 groups was greater than that in the other groups (p<0.05). KCNMA1 expression in the 0.68% Na2SO4 group was significantly greater than that in the other groups (p<0.05).
The regulation of glycosaminoglycan
The organic components of the eggshell increased significantly with increasing dietary Na2SO4 supplementation (Figure 2). The contents of GAG and ATP-S in the 0.68%, 0.83%, and 0.99% Na2SO4 groups were greater than those in the 0.22% Na2SO4 group (p<0.05). Compared with those in the 0.22% and 0.37% Na2SO4 groups, dramatically greater SULT were observed in the 0.83% and 0.99% Na2SO4 groups (p<0.05). The CS and DS contents increased dramatically (p<0.05; Table 8).
Eggshell ultrastructure
Dietary supplementation with 0.68% Na2SO4 improved eggshell ultrastructure in this study (Supplement 1). We scanned the eggshell ultrastructure electron micrographs of laying hens fed diets containing 0.22%, 0.68%, and 0.99% Na2SO4. Compared with those of the other groups, the mammillary knob width, mammillary thickness, and percentage of the mammillary layer significantly decreased, whereas the mammillary knob density, effective thickness, and total thickness noticeably increased in the 0.68% Na2SO4 group (p<0.05; Table 9).
DISCUSSION
Appropriate supplementation of diets with Na2SO4 could improve eggshell quality [24]. However, potential risks must be considered, including reductions in laying rate and feed efficiency [4]. Previous research has indicated that there is no significant impact on daily egg weight or laying rate when dietary Na2SO4 levels range from 0.3% to 0.6% [5]. Likewise, no adverse effects on average egg weight and ADFI were observed when Na2SO4 was supplemented at levels ranging from 0.22% to 0.99% during the 49 to 60 weeks age group. It is worth mentioning that, although statistically not different, the egg production decreased from 0.37% to 0.99% Na2SO4 groups compared with dietary 0.22% Na2SO4 group. It might be that dietary Na levels in these groups exceed the recommended 0.15% [16]. Excessive Na+ may be caused the hyperkalemia which lead to laying performance [25], which further resulted in the increment of FCR. Besides, egg production did not decrease with further increased in dietary Na2SO4 levels. All in all, this result indicated that Na2SO4 supplementation at 0.22% to 0.99% is safe.
A higher breaking strength of eggshells can reduce economic losses in the layer industry by reducing egg breakage [26]. Na2SO4 supplementation of 0.52% and 0.68% was found to increase the breaking strength and thickness of laying hens from 49 to 60 wk of age, which was consistent with the findings of previous studies conducted on younger layers (29 to 40 wk of age [7] and 21 to 28 wk of age [4]). These findings indicate that Na2SO4 is an effective additive for improving eggshell quality. The breaking strength of the eggshell improved notably at a Na2SO4 supplementation level of 0.68% in our study, which aligns with the recommendation (0.71% Na2SO4) of Fu et al [7] but is more than the recommended level (0.3% to 0.6% Na2SO4) reported by Liu et al [4]. This disparity may be due to the differences in the basal dosage of dietary Na and Cl, as the levels and interactions of total Na and Cl in diets should be taken into consideration when supplementing dietary Na [27]. Poor eggshell quality could be caused by higher dietary Na2SO4 levels, which disturb the balance between Na and Cl, further leading to metabolic alkalosis and changing the secretion of substances necessary for eggshell formation [28]. Overall, the addition of 0.52% and 0.68% dietary Na2SO4 improved eggshell quality.
CaCO3 is the major component of eggshells [29]. In our study, the eggshell weight and eggshell ratio were found to increase with increasing Ca content in the eggshell, suggesting that the increase in thickness may be due to the increase in CaCO3 deposition. During eggshell formation, Ca2+ transfers from the blood to uterine epithelial cells via the Ca2+ channel and then is exchanged with Na+ in uterine fluid through SLC8A1. Additionally, Na+ can also enter uterine epithelial cells through SCNN1A and is exchanged with K+ in the blood via ATP1B1. K+ subsequently transported into the uterine fluid via KCNMA1 [20]. All of them are involved in ion transportation. Ultimately, Ca2+ and HCO3-combine to form CaCO3 in uterine fluid. The transfer of Na+ from uterine fluid to serum has a positive relationship with Ca2+ transfer from serum to uterine fluid [30]. Therefore, the increased expression of SLC8A1 and SCNN1A led to increased uterine fluid Ca2+ levels accompanied by decreased Na+ levels in this study. These results indicated that 0.68% Na2SO4 supplementation could improve the exchange of Ca2+ and Na+. Additionally, in the 0.68% Na2SO4 groups, ATP1B1 and KCNMA1 expression increased significantly compared with that in the other groups. The serum Na+ concentration and uterine fluid K+ concentration increased linearly with Na2SO4 supplementation, ranging from 0.22% to 0.68%. This finding suggested that the transportation of K+ from the serum to the uterine fluid could be improved by dietary 0.68% Na2SO4 supplementation. Furthermore, the Ca content of the eggshell, ion concentration, and gene expression of ion transporters showed the opposite trend with increasing Na2SO4 supplementation from 0.68% to 0.99%. These indicated that excessive Na+ decreases the absorption of Ca2+ and leads to a decrease in eggshell quality [28]. These two treatments (0.83% and 0.99% Na2SO4) may exceed their tolerance, which is consistent with the recommendation of Fu et al [31]. The process by which sodium enters the blood occurs against a concentration gradient, while ATP1B1 consumes energy [32]. An elevated serum Na+ concentration may depress the transfer of other ions, and elevated dietary Na2SO4 levels could disrupt the balance between Na+ and Ca2+, leading to a decreased Ca2+ concentration [4]. Excessive Na+ can also reduce the affinity of SCNN1A [33], ultimately leading to a weakened ability for subsequent ion exchange. In general, the Na+ concentration in the 0.68% group was lower than that in the 0.83% and 0.99% groups, which corresponded to the changes in breaking strength and thickness. Based on these results, the changes in ion concentrations and transporter levels could partly explain the increase in breaking strength in the 0.68% Na2SO4 group.
Most of the eggshell matrix is considered to be a part of the eggshell structure or a regulator of eggshell mineralization [10]. We measured the eggshell matrix and its associated proteins. Consistent with previous findings [7], our study showed an increase in GAG content with increasing dietary Na2SO4. Moreover, the contents of CS, DS, ATP-S, and SULT on the isthmus markedly increased with increasing dietary Na2SO4. There are four types of GAG, namely, heparan sulfate, CS, keratan sulfate (KS), and hyaluronic acid; eggshell GAG contains approximately 70% CS [34–36]. KS is involved in the formation of the first mammillary core, while CS, together with DS and OC-116 (where DS is considered a type of CS), regulates the growth of the palisade layer [36,37]. In addition, SO42− is converted to 3′-phosphoadenosine-5′-phosphosulfate (PAPS) by ATP-S. PAPS is subsequently converted to sulfate GAG by SULT [38]. Crystallization has been reported to occur at calcium, which contains a high concentration of sulfur. Additionally, the effect of GAG on the morphology and size of CaCO3 crystals is concentration-dependent [39]. This might also explain why eggshell GAG contents increased. The eggshell CS/DS copolymer was regarded as the major GAG involved in maintaining eggshell strength. It has a particular affinity for Ca2+ and can modify the shape of calcite crystals formed by CaCO3. This difference may be related to the increase in eggshell quality. However, the 0.83% and 0.99% Na2SO4 groups did not exhibit a corresponding change in breaking strength. A previous study demonstrated that certain GAG contents did not have any additional effect on eggshells [39]. This difference may account for the difference in eggshell strength and weight between the 0.83% and 0.99% Na2SO4 groups compared with the 0.68% Na2SO4 group. Therefore, we suggest that the eggshell GAG contents in the 0.68% Na2SO4 treatment group could improve eggshell quality and that higher concentrations of GAG would not produce further effects. On the other hand, we speculated that ion concentrations and transporter levels may be the main factors affecting eggshell quality, while GAG content play an auxiliary role in eggshell quality.
The ultrastructure, which consists of the palisade layer, vertical crystal layer, and cuticle layer, is a primary determinant of eggshell quality [40,41]. Alterations in eggshell ultrastructure are crucial factors for improving physical structure, including increasing effective thickness and knob density, as well as total calcification layer thickness and decreasing mammillary thickness [42]. An effective layer strongly affects the breaking strength [43]. In the 0.68% group, both the effective thickness and total calcification layer thickness were significantly greater than those in the other groups. This variation might result from the increased Ca content in the eggshell, which also appears to increase eggshell thickness and subsequently decrease eggshell quality. Wu et al [39] reported that CS/DS proteoglycans from the eggshell membrane could cause the formation of smaller and more rounded CaCO3 crystals. An increased mammillary knob density and reduced mammillary knob width and thickness might lead to greater binding between mastoid processes and enhanced resistance to external forces [44]. These changes were consistent with the increase in breaking strength, suggesting that the increase in eggshell quality may be related to the improvement in eggshell ultrastructure.
CONCLUSION
In conclusion, our results indicate that supplemental 0.22% to 0.99% Na2SO4 in the corn-soybean basal diet containing 0.15% Cl did not have adverse effects on performance in laying hen. Dietary 0.52% and 0.68% Na2SO4 supplementation could regulate uterine ion transport and increase GAG contents, which contribute to better eggshell quality. In addition, dietary supplementation with 0.68% Na2SO4 resulted in higher eggshell breaking strength, thickness, eggshell weight, and eggshell ratio.
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