1. Introduction
Fertility function is critical for the successful production of healthy offspring in poultry. However, oxidant stress and immunological imbalance may hasten the decline of reproductive performance in breeding poultry of advancing age [
1,
2]. Supplementation of antioxidants in diets has become a key strategy to solve this problem in poultry reproduction.
For one hundred years, vitamin E (VE) has been extensively studied as a substance that is necessary in reproduction [
3]. Poultry does not synthesize enough VE and depends on dietary sources to meet daily requirements [
4]. The influence of VE deficiency and the nutritional demand for VE has been comprehensively investigated in breeding poultry, including chickens, ducks, and quails [
5,
6,
7]. Specifically, an appropriate VE supplementation can maintain reproductive performance and egg quality, which are the essential criteria for eventual profitability. In recent years, geese production has grown rapidly and created substantial economic benefits [
8]. By summarizing the relevant data in China, Wang [
9] concluded that 30 IU/kg to 50 IU/kg of VE could meet the basic VE requirement of breeding geese. In the latest studies, 40 IU/kg of VE was supplemented to formulate the diets of breeding geese [
10,
11].
However, to achieve higher performance, VE supplementation in poultry diets has been tentatively increased in intensive commercial production. Higher dietary VE additions, including 60, 100, 120, 160, and 200 IU/kg [
5,
12,
13,
14,
15,
16], were reported to obtain better reproductive performance and antioxidant capacity in breeding chickens, compared with basic additions. However, the effects of VE addition beyond the basic demand have not been thoroughly identified in breeding geese. A basic range is still lacking for investigating and optimizing dietary VE formulation. In addition, an exceptionally high intake of VE (approximately 2000 IU/kg) was indicated to harm animal fertility, as reported in a study on sheep [
17]. We noted that some managers have tried to supply geese diets with a high quantity of VE to maintain late laying performance, but achieved decreased egg production and fertility. Based on such issues, we hypothesized that an appropriate range of VE supplementation (40 IU/kg to 200 IU/kg) would improve the reproductive performance of geese. Thus, further exploration needs to be conducted on how a high dose of VE inclusion induces a decline in the performance of breeding geese. Clarifying the effects of dietary VE levels (VE deficiency to high dose VE) on reproductive performance is necessary and practically meaningful for geese production.
Egg characteristics are closely related to a bird’s age, diet, and nutrient composition [
16]. Dietary VE supplementation alters the utilization of protein, lipids, vitamins, and minerals in breeding poultry and affects the nutrient deposition in eggshells, yolk, and albumen [
18]. An increased resistance of chicken embryonic and postnatal tissues to oxidative stress and the establishment of chick immune defense was observed to result from increased VE that was transferred from the maternal diet to the yolk [
14,
19,
20]. In addition, a very high level of dietary VE (10,000 IU/kg and 20,000 IU/kg) could severely decrease the transfer of maternal carotenoids to egg yolk [
21]. However, little research has been reported on the effect of dietary VE on geese egg characteristics, especially the antioxidant capacity in egg components; this warrants further investigation.
Dietary VE protects semen quality by preventing the breakdown of polyunsaturated fatty acids from oxidation, thereby improving fertility functions in males [
15,
22]. The addition of VE acts as a chain-breaking antioxidant to defend cellular membranes against reactive oxygen species and free radical generation [
23,
24]. Meanwhile, VE indirectly strengthens a body’s antioxidant defense by enhancing the activities of antioxidant enzymes, including superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and glutathione (GSH) [
25,
26]. For female geese, however, whether and how dietary VE addition can improve reproductive performance by enhancing antioxidant capacity is unclear and needs to be proven by further correlation analysis. Maternal breeders that received VE supplementation were also found to maintain immune status by increasing serum immunoglobulins and cytokines concentrations and promoting immune organ development [
13,
16]. Nevertheless, either positive or negative antioxidant and immunity-related potential resulting from VE supplementation need more scientific evaluation in breeding geese.
This study aimed to tentatively evaluate the physiological response of geese to dietary VE (from VE deficiency to high-dose VE) during the late laying period and explore a basic VE range for further studies of the nutrient requirements and optimal doses in breeding geese. Therefore, four dietary levels of VE (0, 40, 200, and 2000 IU/kg) were selected from previous studies [
5,
16,
17], representing VE deficiency, basic-dose VE, middle-dose VE, and high-dose VE, respectively. The evaluation focused on the effects of VE on reproductive performance, egg characteristics, antioxidant capacity, and immune status.
2. Materials and Methods
2.1. Birds, Experimental Design, and Management
This study used 480 female and 96 male Jiangnan White breeding geese at 48 weeks of age. The geese were hatched from the same batch offered by a commercial hatchery (Jiangsu Lihua Animal Husbandry Co., Ltd., Changzhou, China). All procedures of this study were permitted by the Institutional Animal Care and Use Committee (IACUC) of the Yangzhou University Animal Experiments Ethics Committee, with permit number SYXK (Su) IACUC 2020-0910.
All breeding geese had the same initial laying rate and similar body weight and were assigned to four treatment groups consisting of four replicates (pens) of 30 female and six male birds each. The four groups were fed a basal diet supplemented with 0 IU/kg, 40 IU/kg, 200 IU/kg, and 2000 IU/kg dietary VE in DL-α-tocopherol acetate form (effective content ≥ 50.0%) purchased from a commercial manufacturer (Xincheng Co., Ltd., Xinchang, China). The basal diet was formulated to meet the nutrient requirements of geese, according to the NRC (1994) [
27] and prior research results [
28] from our laboratory. All of the experimental diets were pelleted and analyzed for α-tocopherol concentration. The composition and nutrient levels of the diets are listed in
Table 1. The feeding experiment was conducted in the experimental pasture of Yangzhou University from December 2020 to the following January. The laying period lasted 6 weeks, from 48 to 54 weeks of age, after 2 weeks of adaptive feeding. During the feeding period, the geese were kept in separate hard-plastic-floored pens that were laid 70 cm above the ground, and they mated naturally. Each pen was equipped with a wooden nest of the same size for laying eggs. The geese were housed under 14 h (4:00 a.m. to 6:00 p.m.) of light per day, and the room temperature was 2 ± 3 °C. All of the geese had access to feed and water ad libitum throughout the trial. The health status and feed intake of the geese were checked daily. No goose died during the experiment.
2.2. Determination of Reproductive Performance
Geese eggs were collected, marked, and weighed daily at 5:00 a.m. and 2:00 p.m. Eggs with damage, deformity, frosted eggshell surface, and extreme weight (≤120 g or ≥180 g) were unqualified. At 53 weeks of age, the qualified eggs from each group were collected for 5 consecutive days before hatching. The results were expressed as the mean of four replicates per group and calculated as follows:
2.3. Determination of Egg Characteristics
At the end of the experiment, three qualified eggs were collected randomly from each replicate (48 eggs in total). The length and width of the eggs were measured using an FHK egg shape determinator (Fujihira Industry Co., Ltd., Tokyo, Japan), and the egg shape index was calculated (i.e., length:width). After weighing, the eggshell, albumen, and yolk were separated and weighed. The eggshell percentage, the albumen percentage, and the yolk percentage were calculated. Eggshell thickness (μm) was determined by measuring the average thickness taken at three locations on the eggshell (sharp end, blunt end, and middle section of the eggshell). Yolk color was measured using the Yolk color chart (Robotmation Co., Ltd., Tokyo, Japan) at three places (blunt, equatorial, and sharp regions), with the average used for analyses. Yolk samples were taken and stored at −80 °C to determine the α-tocopherol concentration and antioxidant capacity indices.
2.4. Sample Collection and Procedure
At the end of 54 weeks of age, all geese were weighed individually after fasting for 6 h. Then, three female breeding geese were randomly selected from each replicate. Blood samples were collected in sterile procoagulant tubes via wing venipuncture, centrifuged immediately at 4500 rpm at 4 °C for 15 min to obtain plasma, then stored at −80 °C for further analyses. Following this, the geese were exsanguinated by severing the jugular vein and carotid artery on one side of the neck. After bleeding and eviscerating, the percentage content of the heart, liver, spleen, and ovary in live body weight was calculated. Follicles were separated from the ovaries and classified according to diameter (DM; i.e., DM ≤ 3 mm, 3 mm < DM ≤ 6 mm, 6 mm < DM ≤ 9 mm, 9 mm < DM ≤ 12 mm, and DM > 12 mm) and counted. Liver and ovarian tissue samples were removed carefully and stored at −80 °C to determine the antioxidant index. The final result of each index for further determination was expressed as the mean value of three geese (or three eggs) from each replicate.
2.5. Determination of VE (α-Tocopherol) in Diets and Egg Yolk
Feed samples were smashed twice by an FW-100 grinder (Taisite Instrument Co., Ltd., Tianjing, China). Then, the feed powder was sieved through a 0.28 mm sieve, harvested in a sealing bag, and stored at −20 °C until analysis. Six replicated samples of each diet were prepared with 35 g per replicate. Samples of egg yolk were prepared with 15 g per each egg in the above way after drying with a freeze dryer (SCIENTZ-12N, Xinzhi Freeze Drying Equipment Co., Ltd., Ningbo, China) at −70 °C for 72 h.
Feed and egg yolk extractions were prepared by the method described in the national standard (GB 5009.82-2016) [
29]. Briefly, that method was as follows. Sample powder (1.5 g) was added to an anaerobic tube; 6 mL ethanol, 1 mL 10% L-ascorbic acid, and 2 mL KOH solution (KOH/water = 1:1, g/mL) were then added. The tube was filled with nitrogen, sealed, and mixed using a vortex. A water bath (70 °C) was carried out for 30 min, followed by cooling on ice. The saponification solution was transferred to a 50 mL tube with 2% NaCl (20 mL). Ten mL of anhydrous ether was added. The solution was shaken at 2500 rpm for 2 min, then kept still for layering. The upper organic phase was carried out in a 50 mL centrifuge tube. Anhydrous ether (5 mL) was added once again to extract the α-tocopherol. The upper organic phase (extracting solution) was mixed in a 50 mL centrifuge tube from the two extraction processes. Ten mL of water was added to wash the ether. The solution was centrifuged at 2500×
g at 4 °C for 10 min. Five mL of the upper organic phase was dried with nitrogen, dissolved with methanol to 1 mL, and filtered via membrane (0.45 μm) for further analysis.
The α-tocopherol content in the feed and muscle extractions was measured by a Waters E2695 high performance liquid chromatography system (HPLC) (Waters Co., Ltd., Milford, CT, USA), equipped with a chromatographic column (Zorbox SB-C18, 250 mm × 4.6 mm, 5 μm) with a working temperature of 30 °C. The mobile phase was methanol/water at 98%: 2%, with a flow rate of 1 mL/min. The detection was carried out at 300 nm wavelength with an injection of 50 μL extraction. The standard curve (1.56, 3.12, 6.24, 15.59, 31.183, and 62.366 μg/mL) and the working curve were established according to GB 5009.82-2016 [
29]. The retention time of the α-tocopherol was approximately 21 min to 22 min. The concentration of the α-tocopherol was expressed as μg/g dry matter.
2.6. Determination of Plasma Parameters
The α-tocopherol and VA in plasma were extracted by the filter paper method and n-hexane method. In detail, that method was as follows. Plasma, calibrator, and a quality control solution (in the commercial kit from Nuomingzhetian Co. Ltd., Yangzhou, China) were permeated through special filter paper and dried naturally to make a dry circle paper sample with 6 mm DM. The paper sample and 150 μL of 10% methanol solution were added together in a 2 mL EP tube and shake for 10 min. Then, 450 μL of internal standard solution (1%; standard, 800 ng/mL VE in methanol, 800 ng/mL VA in methanol; 300 ng/mL butylated hydroxytoluene in acetonitrile) was added, vortexed for 30 s, and shaken at 2500 rpm for 10 min. One1 mL of n-hexane was added, shaken at 2500 rpm for 10 min, and centrifuged at 12,000 rpm for 10 min. Eight hundred μL of the sample extraction was blown dry with nitrogen at 25 °C (flow rate, 20 L/min) and dissolved with 100 μL of 0.1% formic acid in methanol; then, it was vortexed at 1000 rpm for 5 min and centrifuged at 4000 rpm for 3 min. The prepared sample extractions were stored at 4 °C and determined within 24 h.
Concentrations of α-tocopherol and VA were together determined by a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system consisting of a Shimadzu LC20AD HPLC (Shimadzu Co. Ltd., Kyoto, Japan) equipped with a chromatographic column (Bonshell C18 Plus, 3 mm × 50 mm, 2.7 μm, 35 °C of working temperature), and an API 3200MD triple quadrupole mass spectrometer (Sciex Pte. Ltd., Toronto, ON, Canada).The mobile phase A was water/formic acid at 1000:1. The mobile phase B was methanol/formic acid at 1000:1. The flow rate was 1 mL/min. The injection of extraction was 50 μL. The MS adopted atmospheric pressure chemical ionization ion source (APCI) and positive ion MRM scanning analysis mode. The atomizing gas was 60 psi; the curtain gas was 20 psi; the collision gas was 5 psi; the needle current was 5 mA; and the ion source temperature was 400 °C. The ratio between the actual peak area of the sample to be tested and the internal standard peak area was substituted into the standard curve equation (based on peaks of seven standards) to calculate the concentration of the compound to be tested in the sample to be tested.
Determination of plasma Vitamin D3 (VD3) concentration was similarly extracted by the filter paper method and n-hexane method and used the same LC-MS/MS system, as follows. The major difference was in nitrogen blowing (60 °C, 0.02 MPa, 10 min). Then, derivative agent PTAD was added for derivatization, and shaken at 500 rpm for 30 min. After that, sample extractions were transferred to a protein filter plate and filtered by shaking at 4000 rpm for 2 min. The mobile phase A was water/formic acid at 1000:1. The mobile phase B was methanol/formic acid at 1000:1. The flow rate was 0.5 mL/min. The injection of extraction was 20 μL. The working temperature of the chromatographic column was 40 °C. The MS atomizing gas flow was 3 L/min; the dry gas flow was 10 L/min; the heating gas flow was 10 L/min; the interface temperature was 300 °C; the DL temperature was 250 °C; the heating block temperature was 400 °C; and thecollision-induced dissociation (CID) gas was 230 kPa.
Plasma reproductive hormones, including follicle-stimulating hormone (FSH, cat. No. H101-1-2), luteinizing hormone (LH, cat. No. H206-1-2), estradiol (E
2, cat. No. H102-1), progesterone (P, cat. No. H089), prolactin (PRL, cat. No. H095-1-1), either plasma immunoglobulins including immunoglobulin A (IgA, cat. No. H108-1-2), immunoglobulin M (IgM, cat. No. H109-1-2), and immunoglobulin G (IgG, cat. No. H106-1-1), were determined by the method of enzyme-linked immunosorbent assay (ELISA), using commercial kits following the protocols provided by the manufacturer (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) as described by Amevor et al. [
30] and Sun et al. [
31].
2.7. Determination of Antioxidant Enzymes Activity
Each liver, ovarian tissue, and egg yolk sample was accurately weighed to 0.5 g, and a nine-fold volume of specified diluent by weight was added. The samples were homogenized under ice bath conditions by a JXFSTPRP-I-02 fast homogenizer (Shanghai Jingxin Co., Ltd., Shanghai, China) until no particles were visible in the homogenate solution (approximately 60 s). The prepared homogenized solution was centrifuged at 2500 rpm at 4 °C for 10 min to remove debris. One portion of the acquired supernatant was retained for analysis, and the others were immediately stored at −80 °C for further determination. All tissue samples were determined for total antioxidant capacity (T-AOC, cat. No. A015-2-1), superoxide dismutase activity (SOD, cat. No. A001-1), catalase (CAT, cat. No. A007-1-2), glutathione peroxidase activity (GSH-Px, cat. No. A005-1-2), as described by Sun et al. [
31], using the commercial assay kits following the protocols of the manufacturer (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). By using the specific assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), all yolk samples were determined for the total antioxidant capacity (T-AOC, cat. No. A015-2-1), the superoxide dismutase activity (SOD, cat. No. A001-1), and glutathione (GSH, cat. No. A006-2-1), as described by Sun et al. [
31] and Amevor et al. [
30] Total protein (TP, cat. No. A045-3-2) was determined by the method of bicinchoninic acid (the BCA method). The results were normalized against the TP concentration of each sample for intersample comparison.
2.8. Lipid Peroxidation Analysis
Lipid peroxidation was expressed as malondialdehyde (MDA) concentration. Liver, ovarian tissue, and egg yolk were determined by a commercial assay kit (MDA, cat. No. A003-1) following the protocols of the manufacturer (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) as described by Amevor et al. [
30]. Briefly, the MDA level of each tissue supernatant was measured by the method of thiobarbituric acid (TBA). The MDA-TBA mixture produced during the reaction of MDA in tissue with TBA was measured at 535 nm by a UV-1780 ultraviolet spectrophotometer (Shimadzu Suzhou Instruments Mfg. Co., Ltd., Suzhou, China). The results were normalized against the TP concentration of each sample for intersample comparison.
2.9. Statistical Analysis
All of the data were initially processed using Excel 2019 and analyzed using SPSS 20.0 (SPSS Inc., Chicago, IL, USA). One-way ANOVA with a post hoc test was used to elucidate significant differences. All of the data were checked for normality. Duncan’s test was used for multiple comparisons when a significant difference was detected. The correlation between the variables was analyzed with bivariate Pearson’s correlation coefficients. The results were expressed as means and SEM. The difference was significant when p ≤ 0.05, and extremely significant when p < 0.01.