Eucommia ulmoides (EU) is a traditional medicinal herb in eastern Asia(Reference He, Wang and Li1–Reference Fan, Yin and Li5). It was said that more than forty compounds such as iridoids, phenolics and steroids exist in EU, among them a major bioactive compound called chlorogenic acid endows it with precious medicinal value(Reference Hussain, Tan and Liu2,Reference Liu, Li and Zhao6) . Over the recent years, several researches reported that EU extract has the effects of anti-hypertensive(Reference Kwan, Chen and Deyama7,Reference Luo, Wu and Zhou8) , anti-obesity(Reference Fujikawa, Hirata and Wada9,Reference Hirata, Kobayashi and Wada10) , anti-bacteria(Reference Zhang, Ravipati and Koyyalamudi11), anti-inflammation(Reference Kim, Park and Chang12,Reference Kim, Kim and Kim13) , antioxidation(Reference Yen and Hsieh14–Reference Lin, Fan and Mehl16) and neuroprotection(Reference Lin, Fan and Mehl16,Reference Kwon, Lee and Kim17) .
The leaf of EU showed a higher activity than its cortex, flower and fruit(Reference Yen and Hsieh18,Reference Zhang, Su and Yang19) . In particular, eucommia ulmoides leaves extract (ELE) can enhance the growth performance and improve meat quality in mammalian species(Reference Lee, Kim and Song20–Reference Ding, Cao and Li24). In recent years, EU was used as a new feed addictive in the aquaculture industry to enhance the growth and immunological response. Supplementation of EU in turbot diets could strikingly improve the antioxidant activity and kept an active immune response(Reference Zhang, Li and Wang25). Furthermore, Sun et al. (Reference Sun, Li and Xu26) and Yang et al. (Reference Yang, Li and Xu27) had revealed that chlorogenic acid supplementation (main active components in EU) could enhance the growth performance as well as improve flesh quality of grass carp. It is said that there are relatively few studies on the application of EU in fish larvae, but it is also valuable in marine carnivorous species.
Large yellow croaker (Larimichthys crocea) is a monetarily significant aquaculture species and broadly cultivated in southeastern China(Reference Ai, Zhao and Mai28–Reference Liu, Miao and Xu32). However, the sufficient development and survival are still the key limiting factors in marine fish larviculture(Reference Rojo-Cebreros, Ibarra-Castro and Martinez-Brown33,Reference Vadstein, Bergh and Gatesoupe34) . The immature feeding and intestinal function of larvae limited to absorb the micro-diet, which is crucial for growth and survival of fish larvae(Reference Liu, Miao and Xu32,Reference Abiayad and Kestemont35) . Chinese herbs characterised by abundant bioactive substances, without side effects brought from chemicals, showed a promising potential for application and development in the aquaculture industry. Previous studies have found that supplementation of Chinese herbs at appropriate dosage in diets could enhance fish immunity, improve growth and regulate intestine development(Reference Liu, Zhang and Yang36–Reference He, Xiao and Zhang38). However, there were almost no reports about the regulatory effects of ELE supplementation on the fish larval nutrition and physiological states. Therefore, this study was to focus on the effects of ELE supplementation on the growth, survival, expression of feeding-related genes, activities of digestive enzymes, antioxidant capacity, immunity and cytokines expression of large yellow croaker larvae.
Methods
Animal ethics
In the present study, those conventions for fish larvae were accordance with the Management Rule of Laboratory Animals strictly (Chinese Order No. 676 of the State Council, revised 1 March 2017).
Diets formulation
The experiment lasted 30 d and tested four diets in triplicate: the testing diets were formulated and supplementation with 0 g kg−1 (the control group), 5 g kg−1 (0·5 %), 10 g kg−1 (1·0 %) and 20 g kg−1 (2·0 %) of ELE, respectively (online Supplementary Table 1S). The testing diets were formulated in light of the nutritional requirements of large yellow croaker larvae based on Ai et al. (Reference Ai, Zhao and Mai28) with slight adjustment. The ELE was purchased from Shaanxi Chen-xi Bio Co., Ltd in Shaanxi, China. The micro-diets were made according to the norm procedures in our laboratory and stored at –20°C prior to use in light of Yao et al. (Reference Yao, Huang and Liu39)
Fish and experimental procedures
Large yellow croaker larvae were acquired from Xiangshan Harbor Aquatic Seeds Company, Ningbo, China, and then reared at Marine and Fishery Science and Technology Innovation Base, Ningbo, China. Prior to the initial stocking, the fish larvae were acclimatised for 2–3 d in the laboratory condition by feeding live copepods and artificial experiment diets to adapt to the experimental feed. Larvae were weaned at 16 d of age (4·71 ± 0·27 g) and were randomly distributed into 220 L blue plastic tanks for a density of 3500 fish in each tank. During the trial, water temperature was kept in 21·0–23·0°C, salinity was kept in 25·0–28·0 g/l, dissolved oxygen content was maintained at 6·0 mg/l and maintain 100 % of the water change daily. Fish larvae were fed to the experiment micro-diets seven times a day (06.30, 08.30, 10.30, 13.30, 15.30, 17.30 and 22.30).
Sampling and dissection
A 24-h fasting experiment was conducted after feeding the last meal, and forty-five individuals (15/tank) were sampled at 1, 3, 6, 12 and 24 h, respectively, for the expression of feeding-related genes assay. The rest of fish larvae were sampled after 24 h of fasting, and the number and weight of fish larvae were sampled from each tank for calculating the survival rate and growth. Subsequently, the larval tissue (intestine segment-IS, pancreatic segments-PS, brain and visceral mass) was placed on the glass plate and then dissected under a microscope. The temperature was kept at 0°C for digestive enzyme activity assay as described by Cahu and Infante(Reference Cahu and Infante40). The final body weight of larvae in each tank was measured (forty-five individuals, fifteen/tank) with a microbalance. Initial body length and final body length of fish larvae in each tank were measured (forty-five individuals, fifteen/tank) with a Vernier caliper, and the rest of fish larvae were collected and stored at −20°C for body composition assay.
Proximate composition analysis
The feed samples and fish body were dried until a constant weight at 105°C for moisture assay. The crude protein and crude lipid of diets and fish larvae were determined following the standard methods (AOAC, 1995)(41).
Digestive enzyme activities assay
IS and PS of fish larvae were weighed (0·2–0·3 g) and then homogenised in 2 ml 0°C ultrapure water (from Milli-Qsystem) and centrifuged at 3300 g for 10 min. The supernatant had been utilised to digestive enzyme activities assay. The brush border membranes from fish larvae intestine were purified according to a method for intestinal scrapping(Reference Crane, Boge and Rigal42) and adapted to intestinal segments(Reference Cahu and Infante40). Leucine-p-nitroanolide was used as substrate for leucine-aminopeptidase activity assay(Reference Maroux, Louvard and Baratti43). The enzyme activity was expressed by specific activity (mU/mg·protein). Protein was determined based on Bradford (1976) research that using bovine serum albumin (Sigma A-2153) as a criterion(Reference Bradford44). Activities of α-amylase and trypsin in PS and IS and alkaline phosphatase activity in the BBM were determined by commercial reagents and kits, purchased from Nanjing Jiancheng Bioengineering Insitute (Nanjing) and strictly refer to the operation method on the kit for determination.
Antioxidant and immune enzyme activity assay
The visceral mass of fish larvae was used to antioxidant and immune enzyme activity assay. Activities of catalase, total superoxide dismutase, total antioxidant capacity and the content of malondialdehyde were determined by commercial reagents and kits (Nanjing Jiancheng Bio-Engineering Institute). Besides, activities of lysozyme, total nitric oxide synthase, inducible nitric oxide synthase and the content of nitric oxide in visceral mass of larvae were determined by commercial reagents and kits (Nanjing Jiancheng Bio-Engineering Institute).
Total RNA extraction, cDNA synthesis and real-time quantitative polymerase chain reaction
Total RNA was extracted using TRIzol reagent (Takara). The quantity and concentration of extracted RNA was detected by a Nano Drop®2000 spectrophotometer (Thermo Scientific). Subsequently, reversed the extracted RNA into cDNA use the PrimeScript™ RT reagent Kit (Takara) following the manufacturer’s instructions. The β-actin has been viewed as the housekeeping gene in the present study(Reference Li, Ji and Cui45). The RT-qPCR primers of the candidate genes were designed in light of the nucleotide sequences of large yellow croaker (online Supplementary Table 2S). The RT-qPCR was carried out in a quantitative thermal cycler (CFX96TM Real-Time System, BIO-RAD, USA). The volume of reaction system and the procedure of RT-qPCR program were conducted according to the methods of Huang et al. (Reference Huang, Yao and Liu46). The PCR reaction mixtures with SYBR qPCR Mix (10 μl), forward and reverse primer (1 μl), cDNA (1 μl) and DEPC-treated water (7 μl). The reaction conditions were as follows 95°C for 2 min, followed by 39 cycles of 95°C for 10 s, anneal for 30 s and 72°C for 20 s. The level of gene expression was calculated with the 2−ΔΔCT method as described by Livak and Schmittgen(Reference Livak and Schmittgen47).
Calculations and statistical analysis
Survival rate (SR, %) = 100 × Nt/N0
Specific growth rate (SGR, % d−1) = 100 × (Ln Wt - Ln W0)/d
where Nt and N0 were the fish larval final and initial numbers, respectively; Wt was the larval final wet body weight (g), W0 was the larval initial wet body weight and d was the experimental duration, respectively.
In addition to feeding-related gene expression, all data were analysed using SPSS 23·0 (IBM, America) for one-way ANOVA. Expression of feeding-related genes were analysed by two-way ANOVA (diet × time) to analyse the effects of diet, fasting time and interaction. Statistics with P < 0·05 were regarded to be significant, and the results were shown as means values with their standard errors.
Results
The effect of eucommia ulmoides leaves extract supplementation on growth performance of larvae
The SR of larvae was significantly higher than larvae fed diets with 0 % ELE (14·98 %), 0·5 % ELE (20·27 %) and 2·0 % ELE (14·78 %) after fed the diet with 1·0 % ELE (27·00 %) (P < 0·05). Larvae fed diets with 0·5 % ELE (9·59 % d−1) and 1·0 % ELE (9·52 % d−1) showed significantly higher SGR than larvae fed the diet with 0 % ELE (8·38 % d−1) and 2·0 % ELE (8·58 % d−1) (P < 0·05). The final body weight was significantly increased in larvae fed diets with 0·5 % ELE (84·00 mg) and 1·0 % ELE (81·80 mg) compared with larvae fed the diet with 0 % ELE (58·23 mg) and 2·0 % ELE (62·34 mg) (P < 0·05). The final body length was significantly higher in larvae fed the diet with 1·0 % ELE (16·89 mm) than the control group (13·95 mm) (P < 0·05) (Table 1). There were no significant differences in body composition (protein, lipid and moisture) of larvae among larvae fed different diets (P > 0·05) (Table 2).
Table 1. Growth performance of large yellow croaker larvae fed graded level of eucommia ulmoides leaf extract (ELE) supplementation
(means values with their standard errors)*
* Data are expressed as means values with their standard errors. Mean values with different superscripts are significantly as determined by Tukey’s test (P > 0·05). SEM, standard error of means.
Table 2. Body composition of large yellow croaker larvae fed graded level of eucommia ulmoides leaf extract (ELE) supplementation
(means values with their standard errors)*
* Data are expressed as means values with their standard errors. Mean values with different superscripts are significantly as determined by Tukey’s test (P > 0·05). SEM, standard error of means.
The effect of eucommia ulmoides leaves extract supplementation on feeding-related genes expression of larvae
Two-way ANOVA detected a strongly significant dependence of neuropeptide Y (npy) expression on diets and fasting time (diets: P < 0·001; time: P < 0·001), but not on the interaction of diets and fasting time (interaction: P = 0·169). The transcriptional level of npy in larvae increased with the prolongation of fasting time and reached the highest level after a 24 h of fasting. At the specific time point, mRNA expression of npy was significantly increased in larvae fed diets with 0·5 % ELE and 1·0 % ELE compared with larvae fed the diet with 0 % ELE after fasting for 12 hours (P < 0·05). Moreover, mRNA expression of npy significantly increased in larvae fed the diet with 1·0 % ELE compared with larvae fed the diet with 0 % ELE after fasting for 24 h (P < 0·05) (Fig. 1).
Fig. 1. The transcriptional levels of npy in the brain of large yellow croaker larvae. Two-way ANOVA to determine the effects of short-term fasting on diet, time and interaction. Different letters at the same time point indicate significant differences as determined by Tukey’s test (P < 0·05). The results were shown as means values with their standard errors.
The mRNA expression of ghrelin was related to diets, fasting time and the interactive effects of diets and fasting time (diets: P < 0·05; time: P < 0·001; interaction: P < 0·01). Interestingly, the mRNA expression of ghrelin in larvae fed diets with 0·5 % ELE, 1·0 % ELE and 2·0 % ELE increased with the prolongation of fasting time. At the specific time point, the mRNA expression of ghrelin was significantly higher in larvae fed the diet with 0·5 % ELE than larvae fed diets with 0 % ELE and 2·0 % ELE after fasting for 24 h (P < 0·05) (Fig. 2).
Fig. 2. The transcriptional levels of ghrelin in the visceral mass of large yellow croaker larvae. Two-way ANOVA to determine the effects of short-term fasting on diet, time and interaction. Different letters at the same time point indicate significant differences as determined by Tukey’s test (P < 0·05). The results were shown as means values with their standard errors.
The mRNA expression of leptin was related to by diets and fasting time (diets: P < 0·001; time: P < 0·001), although no interactive effects between fasting time and diets were observed (interaction: P = 0·195). At the specific time point, the mRNA expression of leptin was significantly higher in larvae fed the diet with 2·0 % ELE than the other three dietary treatments at time points of 1, 12 and 24 h during fasting (P > 0·05) (Fig. 3).
Fig. 3. The transcriptional levels of leptin in the visceral mass of large yellow croaker larvae. Two-way ANOVA to determine the effects of short-term fasting on diet, time and interaction. Different letters at the same time point indicate significant differences as determined by Tukey’s test (P < 0·05). The results were shown as means values with their standard errors.
The effects of eucommia ulmoides leaves extract supplementation on digestive enzyme activities of larvae
Both the activity of α-amylase and trypsin in larval PS and IS was independent of ELE supplementation (P > 0·05). Similarly, the ratio of Try-IS/Try-(PS + IS) was independent of ELE supplementation (P > 0·05). The activity of leucine-aminopeptidase was significantly higher in larvae fed the diet with 1·0 % ELE than larvae fed the diet with 0 % ELE (P < 0·05), while alkaline phosphatase was significantly higher in larvae fed the diet with 2·0 % ELE than larvae fed the diet with 0 % ELE (P < 0·05) (Table 3).
Table 3. Activities of trypsin and α-amylase of large yellow croaker larvae fed graded level of eucommia ulmoides leaf extract (ELE) supplementation
(means values with their standard errors)*
* Data are expressed as means values with their standard errors. Mean values with different superscripts are significantly as determined by Tukey’s test (P > 0·05). SEM, standard error of means.
† The unit of enzyme activity is U/mg protein.
‡ PS, pancreatic segments; IS, intestinal segments; BBM, brush border membranes.
The effects of eucommia ulmoides leaves extract supplementation on antioxidation capacity of larvae
The antioxidant capacity of fish larvae visceral mass was assayed. Activity of total antioxidant capacity was significantly higher in larvae fed the diet with 1·0 % ELE than larvae fed the diet with 0 % ELE (P < 0·05). Also, the activity of catalase was significantly higher in larvae fed diets with 1·0 % and 2·0 % ELE than larvae fed the diet with 0 % ELE and 0·05 % ELE (P < 0·05). However, no significant differences were observed in the activity of total superoxide dismutase and malondialdehyde content in the visceral mass of larvae among four dietary treatments (P > 0·05) (Fig. 4(a)–(d)).
Fig. 4. Activities of T-AOC (A) and CAT (B), activities of T-SOD (C), concentration of MDA (D) and LZ (E), content of NO (F), activities of T-NOS (G) and iNOS (H) in the visceral mass of large yellow croaker larvae. T-AOC, total antioxidant capacity; CAT, catalase; MDA, malondialdehyde; T-SOD, total superoxide dismutase; LZ, lysozyme; NO, nitric oxide; T-NOS, total nitric oxide synthase; iNOS, inducible nitric oxide synthase. Columns sharing mean values with different superscripts are significantly as determined by Tukey’s test (P > 0·05).
The effects of eucommia ulmoides leaves extract supplementation on immunity capacity of larvae
The activity of lysozyme was significantly higher in larvae fed the diet with 1·0 % ELE than larvae fed the diet with 0 % ELE (P < 0·05). Similarly, the content of nitric oxide and the activity of total nitric oxide synthase were significantly higher in larvae fed the diet with 1·0 % ELE than larvae fed the diet with 0 % ELE (P < 0·05). Furthermore, the activity of inducible nitric oxide synthase in larvae fed diets with 0·5 % ELE, 1·0 % ELE, and 2·0 % ELE were significantly higher than larvae fed the diet with 0 % ELE (P < 0·05) (Fig. 4(e)–(h)). Besides the enzyme activity, the transcriptional levels of tnfα in larvae showed no significant differences among dietary treatments (P > 0·05). The transcriptional level of ifnγ was significantly lower in larvae fed the diet with 2·0 % ELE than larvae fed the diet with 0 % ELE (P < 0·05). The transcriptional level of il-1β was significantly lower in larvae fed diets with 0·05 % ELE and 1·0 % ELE than larvae fed the diet with 0 % ELE (P < 0·05). Moreover, mRNA expression of cox-2 and il-6 decreased significantly with dietary ELE supplementation. Both cox-2 and il-6 expressions in larvae fed diets with 0·5 % ELE, 1·0 % ELE and 2·0 % ELE were significantly lower than control (P < 0·05) (Fig. 5).
Fig. 5. The transcriptional levels of inflammatory genes in the visceral mass of large yellow croaker larvae. Tnfα, tumor necrosis factor α, ifnγ, interferon γ; cox-2, cyclooxygenase-2; il-1β, interleukin-1β; il-6, interleukin-6. Data are presented as means values with their standard errors. Columns sharing mean values with different superscripts are significantly as determined by Tukey’s test (P > 0·05). 
, 0 % ELE (the control); 
, 0.5 % ELE; 
, 1.0 % ELE; 
, 2.0 % ELE
Discussion
In the present study, we demonstrated that dietary ELE at 1·0 % supplementation could improve survival rate of large yellow croaker larvae, which were probably linked to the immunity-promotion effects of active ingredients in EU(Reference Zhu, Zhang and Zhang48–Reference Feng, Fan and Song50). Meanwhile, dietary ELE at 0·5–1·0 % supplementation could increase the specific growth rate of larvae, which agreed well with several previous studies investigations on weaned piglets(Reference Ding, Cao and Li24), broiler chicks(Reference Wang, Du and Ye51), turbot(Reference Zhang, Li and Wang25) and grass carp(Reference Sun, Li and Xu26,Reference Yang, Li and Xu27) .
Orexigenic stimulation plays an important role in individual viability of the larval stage(Reference Opazo, Plaza-Parrochia and Cardoso dos Santos52). Ghrelin and leptin are key hormones in food intake regulation(Reference Ronnestad, Gomes and Murashita53,Reference Volkoff54) . More specifically, the ghrelin would increase animal food intake via activating NPY/AgRP neurons or inhibiting POMC/CART neurons in the arcuate nucleus(Reference Nakazato, Murakami and Date55–Reference Chen, Trumbauer and Chen57). Conversely, leptin supposedly inhibits the NPY/AgRP neurons or activates the POMC/CART neurons(Reference Robertson, Leinninger and Myers58). In the present study, the mRNA expression of npy and ghrelin increased with the prolongation of fasting time, indicating that npy and ghrelin expression play a role in feeding stimulation. Similar results were also found in other fish species, such as npy expression in goldfish(Reference Narnaware, Peyon and Lin59), coho salmon(Reference Silverstein, Breininger and Baskin60) and yellowtail(Reference Hosomi, Furutani and Takahashi61), ghrelin expression in sea bass(Reference Terova, Rimoldi and Bernardini62), zebrafish(Reference Amole and Unniappan63) and grass carp(Reference Feng, Zhang and Wei64). While the expression of leptin increased at first and then decreased, indicating that leptin expression was involved in feeding inhibition, which was consistent with northern snakehead(Reference Wen, Qin and Wang65). Importantly, compared with the control, a significantly higher npy expression was observed in larvae fed the diet with 1·0 % ELE at 12 and 24 h of fasting, while a remarkably higher ghrelin expression was found in larvae fed the diet with 0·5 % ELE at 24 h of fasting. Additionally, larvae fed the diet with 2·0 % ELE showed a significantly higher leptin mRNA expression than the other three diets at time points of 1, 12 and 24 h during fasting, which suggested that excessive intake of ELE suppressed the appetite of fish larvae. Similarly, Zhang et al. (Reference Zhang, Li and Wang25) had elucidated that an overdose of EU Oliver (10·0–20 g kg−1) supplementation in diets significantly suppressed the feed intake of turbot. Conclusively, these results indicate that ELE supplementation might enhance the appetite of large yellow croaker larvae at appropriate dosage but exert an inhibitory effect at a high dosage.
The imperfect intestinal function of fish larvae usually led to the hyposecretion of enzymes, which affected the growth and survival rate(Reference Liu, Miao and Xu32,Reference Abiayad and Kestemont35,Reference Li, Mai and Xu66) . In particular, larval intestinal brush border membrane enzymes such as those with involvement of alkaline phosphatase and leucine-aminopeptidase can reflect the maturity of intestinal cells(Reference Cahu, Infante and Peres67). In the present study, dietary ELE supplementation improved the activities of alkaline phosphatase and leucine-aminopeptidase in brush border membranes, indicating that ELE played an important role in promoting larval intestinal development. Similar results were also observed in early-weaned piglets with incomplete digestive tract development(Reference Ding, Cao and Li24,Reference Peng, Wang and Peng68) . The possible mechanism is that EU contain a variety of bioactive ingredients, which can stimulate the maturation process of the digestive function of animals(Reference Ding, Cao and Li24). Paradoxically, no significant effects were observed in α-amylase and trypsin activity on PS and IS, as well as the ratio of Try-IS/Try-(PS + IS), which was probably attribute to the allometric growth of fish biomass in the larval stage(Reference Cahu and Infante40).
Marine fish larvae are vulnerable to oxidative stress due to the high energy consumption required for rapid growth(Reference Liu, Mai and Xu69). Early pharmacological studies have shown that EU have a strong anti-oxidation capacity, which can scavenge free radicals to reduce oxidative damage to biomolecules(Reference He, Wang and Li1,Reference Park, Choi and Jung15,Reference Lin, Fan and Mehl16) . In the present study, we found that appropriate dosage of ELE in micro-diets could improve larval antioxidant capacity of large yellow croaker larvae. Especially, total antioxidant capacity activity was significantly higher in larvae fed the diet with 1·0 % ELE than the control group. catalase activity significantly increased in larvae fed diets with 1·0 % ELE end 2·0 % ELE. These results were consistent with the research that ELE supplementation could increase activities of T-AOC and catalase in turbot(Reference Zhang, Li and Wang25).
High mortality is a key limiting factor during fish larviculture, and this project also considered the influence of dietary ELE on immunological enzymes and transcription factors thought to be pivotal in proinflammatory cytokine in particular. In the present study, activities of lysozyme, total nitric oxide synthase and the content of nitric oxide were significantly higher in larvae fed the diet with 1·0 % ELE than the control group, which was consistent with the highest survival rate in fish larvae fed the diet with 1·0 % ELE. Moreover, the activity of inducible nitric oxide synthase was significantly affected by dietary ELE. Purportedly, NO was produced via the NOS pathway and exerted protective roles of immuno-protection against pathogenic infection(Reference Zhou, Li and Han70), and Lee et al. (Reference Lee, Lee and Choi71) had elucidated that ELE enhances NO production in ox-LDL treated human endothelial cells through NOS signalling pathways. Besides the nitric oxide, the inflammatory factors were also an important indicator to evaluate the health status of fish(Reference Li, Chen and Chen31). In the study, the mRNA expression of ifnγ, cox-2, il-1β and il-6 significantly decreased with ELE supplementation. Similarly, Kim et al. (Reference Kim, Park and Chang12) had revealed that EU extracts had higher anti-inflammatory activities than the anti-inflammatory drugs. Additionally, the EU cortex also inhibited inflammatory cytokine production(Reference Kim, Kim and Kim13). Overall, these results could be speculated that proper ELE supplementation could increase immunity in fish larvae by increasing lysozyme activity, regulating NOS signalling pathways and alleviating inflammation.
In summary, the micro-diet supplemented with 10 g kg−1 (1·0 %) ELE could improve the growth performance of large yellow croaker larvae via enhancing expression of feeding-related genes, promoting antioxidant capacity and immunity and inhibiting cytokines expression.
Acknowledgements
This research was financially supported by the China Agriculture Research System (CARS47-11), Scientific and Technological Innovation of Blue Granary (grant number: 2018YFD0900402), Leading Talent of Technological Innovation of Ten-Thousands Talents Program (CS31117200001) and Distinguished Young Scholars of China (grant number: 31525024).
The authors’ contributions were as follows: K. M., Q. A. and W. H. designed the research; H. W., Y. L. and C. Y. conducted the research; H. W., N. X. and Z. Y. analysed the data; W. H. wrote the paper; W. X., M. Y. provided language help. All authors reviewed and approved the final manuscript. We thank Xueshan Li, Yunqiang Zhang and Wencong Lai for their help during the experiment.
The authors declare that there are no conflicts of interests associated with the manuscript.
Supplementary material
For supplementary material/s referred to in this article, please visit https://doi.org/10.1017/S0007114521004621
