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A 2 × 3 two-factor experiment was designed to assess the effects of dietary cholesterol (CHO) and phospholipids (PLs) on growth and lipid metabolism in early juvenile mud crabs (0.01 g crab−1). The experimental diets were designed with two CHO levels: 0.40 % (LCH) and 0.80 % (HCH), and three PLs levels: 1.80 % (LPL), 2.50 % (MPL), and 3.20 % (HPL). The 58-day aquaculture trial demonstrated that the LCH-HPL group achieved the best growth performance in mud crabs, characterized by the highest final body weight, weight gain, and specific growth rate. Regarding whole-body composition, dietary PLs increased total cholesterol (T-CHO), PLs, triglyceride (TG), and crude lipid content in the LCH groups. Mud crabs in the HCH groups had a higher proportion of saturated fatty acids, a lower proportion of monounsaturated fatty acids, and increased gene expression of sterol regulatory element binding protein 1c (srebp-1c) and fatty acid synthase (fas) compared to those in the LCH groups. As the dietary PLs increased, mud crabs in the LCH groups exhibited up-regulation in the expression of genes, including srebp-1c, fas, elongation of very long-chain fatty acid protein 4 (elovl4), elovl6, and Δ9-fatty acid desaturase (Δ9-fad). In the HCH groups, elevated dietary PLs resulted in the down-regulation of fatty acid binding protein (fabp) gene expression. In addition, high levels of dietary CHO and PLs (HCH-HPL) inhibited the catalase activity of mud crabs, resulting in a significant increase in the malondialdehyde content. Correlation analysis revealed that dietary CHO was positively correlated with the expression of long-chain polyunsaturated fatty acids synthesizing genes, and dietary PLs were positively correlated with the whole-body lipid content of mud crabs. Additionally, a positive correlation was found between the growth performance and whole-body content of crude protein, crude lipid, and antioxidant capacity in mud crabs.

IntroductionScreening excellent bacterial strains for fermentation is the key to improving the nutritional value and bioavailability of soybean meal (SBM). This study investigated the application of Bacillus subtilis-fermented soybean meal (FSBM) on the feed of Pacific white shrimp (Litopenaeus vannamei).MethodsFSBM was used to replace 0%, 25%, 50%, 75%, and 100% fish meal, and the feeding trial was lasted for 8 weeks (initial weight: 0.9 g). Results and discussionThe amino acid profile in the whole shrimp body was tested. FSBM substitution only significantly reduced the content lysine in whole shrimp body, but increased the content of arginine. Fatty acid data showed that the content of n-6 PUFAs in whole shrimp was significantly increased by FSBM substitution. In muscle, FSBM substitution significantly reduced the content of MUFAs, but increased the content of PUFAs including C18:3n-3, C18:2n-6 and C20:4n-6. No hepatopancreas structure modifications appeared in the 25% group compared with the control group. Subsequently, we investigated the response patterns of different organs to FSBM substitution from antioxidant, endoplasmic reticulum stress and immunity. A high proportion of FSBM significantly reduced the content of GSH in hemolymph and hepatopancreas, while increased the mRNA expression of cat. FSBM substitution did not affect the activities of antioxidant enzymes in the intestine. However, the mRNA expression level of hsp70 in the intestine was significantly reduced by FSBM. In terms of immunity, the mRNA expression levels of lgbp and penaeidin in the hepatopancreas showed a significant linear increase trend. In muscle, high proportion of FSBM significantly increased the mRNA expression of imd. FSBM substitution did not significantly affect the expression of immune genes in the intestine. In terms of endoplasmic reticulum stress, FSBM substitution significantly increased the mRNA expression of eif2α in the hepatopancreas. In muscle, FSBM substitution inhibited the mRNA expression of bip. In the intestine, FSBM replacing 75% of fish meal significantly increased the mRNA expression of bip and ire1. In summary, this study indicated that when the fish meal content account for 40% in diets (dry weight), the screened Bacillus subtilis-FSBM can replace 25% of fish meal protein without reducing the antioxidant and immune abilities of shrimp.

This study examined the effects of feeding largemouth bass (Micropterus salmoides) with diets containing different doses of astaxanthin (0, 50, 100, 150, and 200 mg/kg) for 8 weeks. The results showed that the values of weight gain significantly increased from 620.32 ± 50.38% to 826.14 ± 33.49% as dietary astaxanthin levels increased from 0 mg/kg to 100 mg/kg. When the astaxanthin level exceeded 150mg/kg, the weight gain rate showed a downward trend, but there was no significant difference among of the 100, 150 and 200 mg/kg groups. The feed conversion ratio (FCR) and protein efficiency ratio (PER) were also improved by adding astaxanthin to diets (P < 0.05). Meanwhile, adding astaxanthin to the feed increased the length and thickness of intestinal villus and muscle layer thickness (P < 0.05). The astaxanthin supplementation increased the expression of the NF-E2-related factor (Nrf2) gene and reduced malondialdehyde (MDA) content and the expression of apoptosis genes Caspase-9 and Caspase-3 (P < 0.05), indicating that it has a good antioxidant ability. Furthermore, adding astaxanthin increased the content of non-specific immune markers and decreased the expression levels of the inflammatory factors interleukin-15 (IL-15) and tumor necrosis factor-α (TNF-α). Moreover, fish fed diets with astaxanthin exhibited lower blood cortisol levels (P < 0.05). The proportions of C20:4n6 (ARA) and C20:5n3 (EPA) in the liver decreased with increasing dietary astaxanthin levels. Based on WGR and SGR values, the optimal addition level of astaxanthin in largemouth bass feed is 134.8 mg/kg ~ 135.75 mg/kg. In summary, the appropriate dietary astaxanthin enhanced the antioxidant capacity and immune response of largemouth bass and had a positive effect on its intestinal health.

The effects of digestible carbohydrate levels (0–25 %) on growth, body composition, feed efficiency, and biochemical parameters of juvenile Oplegnathus punctatus (IBW, 12.51 ± 0.02 g) were studied. By adjusting the ratio of corn starch to cellulose, six iso-nitrogenous and iso-lipid diets were formulated. Each diet was divided into three groups (18 fish per replication) for 8 weeks. The results showed that specific growth rate (SGR) and weight gain (WG) were significantly improved (P

Soybean meal (SM) is considered an ideal substitute for fish meal; however, its application is mainly limited because of its antigen proteins, glycinin and β-conglycinin. To improve the value of SM in the aquaculture industry, we employed an aerobic bacterial strain (LX-6) with protease activity of 1,390.6 ± 12.5 U/mL. This strain was isolated from soil samples and identified as Bacillus amyloliquefaciens based on morphological and physiological biochemical characteristics and 16S rDNA gene sequence analyses. Subsequently, we quantified the extent of glycinin and β-conglycinin degradation and the total protein and water-soluble protein content after SM fermentation with B. amyloliquefaciens LX-6. At 24 h of fermentation, the macromolecular antigen proteins of SM were almost completely degraded; the maximum degradation rates of glycinin and β-conglycinin reached 77.9% and 57.1%, respectively. Accordingly, not only did the concentration of water-soluble proteins increase from 5.74% to 44.45% after 48 h of fermentation but so did the concentrations of total protein and amino acids compared to those of unfermented SM. Field emission scanning electron microscopy revealed that the LX-6 strain gradually disrupted the surface structure of SM during the fermentation process. In addition, B. amyloliquefaciens LX-6 exhibited broad-spectrum antagonistic activity and a wide pH tolerance, suggesting its application in SM fermentation for fish meal replacement.

The application of fermented soybean meal (FSBM) is an effective strategy to alleviate the shortage of fish meal (FM) in aquaculture. However, an excessive substitution ratio often reduces fish growth and induces liver oxidative stress, while the mechanism remains poorly understood. Here, an 8-week feeding trial was conducted in largemouth bass (initial weight: 6.82 ± 0.09 g) to establish an oxidative stress model by replacing 50% of FM with FSBM (fermented by Bacillus subtilis). The results showed that FSBM substitution significantly reduced the growth performance of largemouth bass, including the weight gain rate and specific growth rate. Moreover, FSBM significantly reduced the contents of essential amino acids and total free amino acids in muscle, along with the mRNA expression of amino acids and small peptide transporters. Enzyme activity detection and liver sections showed that FSBM substitution caused liver oxidative stress, indicating the successful construction of an oxidative stress model. An integrated analysis of transcriptomic and metabolomic data revealed that FSBM substitution impaired glycine, serine and threonine metabolism, as well as glutathione metabolism. In addition, the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) was decreased in the FSBM group, which may explain the mechanism of oxidative stress caused by FSBM substitution. Considering that glycine is an important component of glutathione synthesis, key genes involved in glycine metabolism (glya, gnmt and agxt) and dietary glycine supplementation should be valued to improve the availability of FSBM. This study reveals for the first time the importance of non-essential amino acids in improving the utilization of plant-based protein sources and provides original insight for the optimization of aquatic feeds.

The swimming crab Portunus trituberculatus is one of the most important economic species in China and its mature ovary often determines its commercial value and production. Although the ovary maturation of crustaceans is generally affected by exogenous nutrition, the specific nutritional needs of ovary maturation of P. trituberculatus are poorly understood. To this end, we collected the P. trituberculatus samples with five ovarian maturation stages and measured their biochemical composition of the ovary, hepatopancreas, and muscle at each ovarian developmental stage. We further analyzed their relation to the ovarian developmental stage of P. trituberculatus by principal components analysis (PCA). We found the levels of branched-chain amino acids, long-chain polyunsaturated fatty acids (LC-PUFA), and monounsaturated fatty acids (MUFAs) in the ovary and hepatopancreas increased during the ovary maturation process, and also passively correlated with ovarian developmental stage, which highlights the necessity of these specific nutrients for oogenesis and for improving the nutrient quality of crabs. In addition, we found an increasing tendency of carotenoid content and phosphatidylcholine in phospholipid in the ovary from the pre-developmental stage to the proliferative stage, but not in the hepatopancreas and muscle, which highlights the possible involvement of carotenoids during the rapid oocyte development process. Our study may provide valuable information for developing a suitable broodstock diet that promotes the ovarian maturation of adult P. trituberculatus and ensures high-quality larval production.

During 8-weeks feeding trial, four experimental diets were designed to assess the effects of different lipid sources, including fish oil (FO), soybean oil (SO), rapeseed oil (RO) and linseed oil (LO) on growth performance, body composition and tissue fatty acid composition of Oplegnathus punctatus. Each diet was fed to triplicate groups (initial body weight 12.06 ± 0.09 g per fish). The weight gain (WG), specific growth rate (SGR), feed efficiency (FE) and protein efficiency ratio (PER) of fish fed LO diets were not significantly different from those fed FO diets, but significantly higher than those fed SO and RO diets. Besides, although the contents of EPA or DHA in different vegetable oil diets were same, the EPA content in muscle and liver and DHA content in liver of the LO group were significantly higher than that of the SO and RO groups. In addition, the highest gene expression of fads2 presented in fish fed LO diets, whereas the gene expression of elovl5 was not significantly different in three vegetable oil groups. Fish fed the SO diet had the highest liver lipid content, hepatosomatic index (HSI), viscerosomatic index (VSI), aspartate transaminase (AST) and alanine transaminase (ALT) values. In conclusion, under experimental conditions, the complete substitution of LO for FO had not negative effect on the feed utilization and growth performance of O.punctatus.

This study investigated the effects of different dietary arginine (Arg) levels on the growth, protein synthesis, antioxidant capacity, and immunity of postlarval mud crab Scylla Paramamosain. Six isonitrogenous and isolipidic diets were formulated to contain 1.51%, 1.81%, 2.16%, 2.35%, 2.73%, and 3.07% dietary Arg levels (dry matter). There were four replicates for each diet treatment (26 crabs per replicate, initial body weight: 7.40 ± 0.15 mg). After eight weeks of feeding trial, the survival and molting frequency (MF) of crabs were not affected by the experimental treatment (P>0.05). Crabs fed the 2.50% Arg diet achieved the highest weight gain (WG) and specific growth rate (SGR) (P

An eight-week feeding trial was conducted to evaluate the influence of dietary arachidonic acid (20:4n-6) on growth performance, fatty acid composition and antioxidant capacity on postlarva mud crab (Scylla paramamosain). Five isonitrogenous and isoenergetic diets with 0.34% (the control diet), 0.48%, 0.84%, 1.18% and 1.81% ARA levels were formulated by adding ARA rich oil in the basal diet. There were triplicate groups of 28 postlarval crabs (initial weight 8.15 mg) for each diet treatment. The results indicated that crabs fed diets with 1.18% ARA showed significantly higher final body weight (FBW), weight gain (WG) as well as specific growth rate (SGR) than the other groups. Survival, molting frequency (MF) and intermoult period were not significantly affected by different dietary ARA addition. Compared with the stable DHA content in crab, the EPA content decreased significantly with increase of dietary ARA addition. In crabs, superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) activities generally increased with dietary ARA supplementation, and were highest in 1.18% dietary ARA group. The MDA (malondialdehyde) concentrations showed the opposite trend. In addition, with the increase of dietary ARA content, the relatively increased lipid content in body composition was observed. The gene expression of fatty acid synthase (fas) and fatty acid binding protein-3 (fabp-3) in crabs increased with increasing ARA level in diets (0.34–1.18%) and then down-regulated expression levels with further ARA addition (1.81%). These results suggested that moderate dietary ARA level (1.18%) contributed to the improvement of growth performance and antioxidant capacity, and regulated the fatty acid composition and gene expression of some lipid metabolism in crabs, while excessive ARA addition in diets may cause oxidative stress of postlarval Scylla paramamosain. Moreover, the dietary ARA/EPA ratio of 1.82 may be suitable for the growth of postlarval Scylla paramamosain.

An 8-week feeding trial was conducted to investigate the effects of dietary protein levels on growth performance, feed utilization, and energy retention of juvenile dotted gizzard shad Konosirus punctatus based on the variation of fish meal. Fish meal was used as the sole protein source; five semi-purified diets were formulated with varying crude protein (CP) levels of 22.52%, 28.69%, 34.85%, 38.84%, 45.78% (CP1-CP5 diets). A total of 300 uniform juveniles with initial body weight 3.61 ± 0.20 g fish−1 were randomly divided into five groups with three replicates in each group. The results showed that different CP levels did not significantly affect the survival of juvenile K. punctatus (p > 0.05). The values of weight gain (WG) and specific growth ratio (SGR) showed a general enhancing trend and then weakened with increasing dietary CP levels (p > 0.05). Feed utilization also improved with increasing dietary CP levels (p > 0.05), and the optimal feed conversion ratio (FCR) value was found in fish fed the diet with CP3 (p > 0.05). The rise of dietary CP from 22.52% to 45.78% enhanced the daily feed intake (DFI) and protein efficiency ratio (PER) values of K. punctatus (p < 0.05). With the increase of dietary CP levels, daily nitrogen intake (DNI), energy retention (ER), and lipid retention (LR) elevated, while retention (NR), daily energy intake (DEI), and daily lipid intake (DLI) reduced (p < 0.05). No statistical differences in the content of water, crude protein, and crude lipid were observed among different treatments (p > 0.05). The activity of lipase in CP3 and CP4 diets was significantly higher than that of the CP1 diet (p < 0.05). Fish fed CP2 and CP3 diets had significantly higher amylase activity than that of the CP5 diet (p < 0.05). The levels of alanine aminotransferase (GPT) first enhanced and then decreased as dietary CP levels raised. The second-order polynomial regression model analysis of the WG and FCR indicated that the optimal dietary protein level for K. punctatus is about 31.75–33.82% based on the variation of fish meal.

An 8-week feeding trial was carried out with various lipid level (2.30 %, 6.31 %, 8.98 %, 12.93 %, 16.08 %, and 18.99 %) diets to investigate the effects on growth performance, body composition, and feed utilization of juvenile spotted knifejaw Oplegnathus punctatus. After the feeding experiment, the weight gain (WG) and specific growth rate (SGR) significantly elevated with raising the dietary lipid level to 12.93 %, and then decreased when dietary lipid further increased (p < 0.05). The tendencies of feed efficiency (FE), protein efficiency ratio (PER), nitrogen retention (NR), and energy retention (ER) were consistent with the growth performance (p < 0.05). The lipid retention (LR) significantly decreased with dietary lipid increasing (p < 0.05). The fish consuming 2.30 % and 6.31 % lipid diets had significantly elevated hepatosomatic index (HSI) (p < 0.05). The intraperitoneal fat ratio (IPF) increased with the increase of dietary lipid levels (p < 0.05). The elevation of dietary lipid levels significantly increased the lipid concentrations in the whole body, dorsal muscle, and liver (p < 0.05). The highest liver glycogen deposition was found in the 2.30 % dietary lipid treatment (p < 0.05). The fish fed the diets added with 12.93 % lipid diet had the highest serum protein concentration (p < 0.05). The lowest total cholesterol (TC) content in the serum was detected in 2.30 % dietary lipid treatment (p < 0.05). The fish fed diets with 2.30 % or 18.99 % lipid obtained elevated serum AST and ALT activities (p < 0.05). The regression analysis in second order polynomial and two slope broken-line mode based on SGR and FE indicated that diet containing 10.46–12.83 % lipids was optimal for juvenile O. punctatus to obtain best growth performance and feed efficiency.

A dietary strategy was conducted with graded crude protein (CP) levels (various from 27.08% to 58.10%) in six semi-purified diets to investigate the appropriate dietary CP supplement for juvenile spotted knifejaw (Oplegnathus punctatus). Experimental diets were respectively offered to the juvenile fish with three replicates (18 individuals each replicate, 19.91 g fish-1) for 8-week. After the feeding trial, the weight gain (WG) was improved by increment of dietary CP levels, but deteriorated slightly when dietary CP over 45.87%. The daily feed intake (DFI) elevated as dietary CP decreasing. Besides, the lowest protein efficiency ratio (PER) and feed efficiency (FE) were observed in 27.08% CP treatment. However, the PER and energy retention (ER) were not significantly different among 32.97%, 39.71% and 45.87% CP groups. In the current study, fish fed diet with 58.10% CP obtained slightly lower PER and nitrogen retention (NR), but relatively higher lipid retention (LR) value compared to those fed a diet with 45.87% CP. And the daily nitrogen intake (DNI) in the fish fed on the low CP levels (27.08 to 39.71%) were significantly lower than that in 45.87% CP treatment. The various dietary treatments did not differ in the visceral index (VSI), hepatosomatic index (HSI) and serum glucose, but fish in the lowest CP group had a decreased intraperitoneal fat ratio (IPF). The protein content in the whole body and dorsal muscle of the fish significantly elevated with dietary protein increasing. The aspartate transaminase (AST) in serum increased with increasing dietary CP from 27.08%-52.65 and then decreased. The regression analysis in second order polynomial and two slope broken-line mode based on NR and WG indicated that the optimal protein supplement for juvenile O. punctatus is 42.92%-46.44%. The present study also indicated that O. punctatus had poor ability to utilization dietary carbohydrate.

To determine the impact of replacing fish meal (FM) in the diet with various levels of soybean meal (SBM) on the spotted knifejaw Oplegnathus punctatus, a 56 day feeding trial was done. Seven diets were formulated with SBM to replace 0% (SBM0), 30% (SBM30), 40% (SBM40), 50% (SBM50), 60% (SBM60), and 70% (SBM70) of FM protein, and SBM50 + T was developed on the basis of SBM50 with the addition of 1.2% taurine. There were triplicate groups of 18 fish (initial weight: 14.62 ± 0.02 g). The weight gain (WG), specific growth rate (SGR), feed efficiency (FE), and protein efficiency ratio (PER) values of the SBM0, SBM30, and SBM50 + T groups were found to be significantly higher than those of the SBM60 and SBM70 groups (p < 0.05). The daily energy gain (DEG), daily nitrogen gain (DNG), daily lipid gain (DLG), energy retention (ER), nitrogen retention (NR), and lipid retention (LR) values decreased significantly with increasing dietary SBM levels (p < 0.05). The highest retention of most amino acids (except lysine) was observed in the SBM30 group (p < 0.05). The lipid content of the whole body and dorsal muscle decreased significantly as dietary SBM levels increased (p < 0.05). Fish fed the SBM70 diet had the lowest serum triglyceride (TG) concentrations (p < 0.05). The effects of different treatments on total cholesterol (T-CHO) were not significant (p > 0.05). Fish fed the SBM0 and SBM30 diets had the highest amylase (AMS) and lipase (LPS) activities (p < 0.05). The lowest liver superoxide dismutase (SOD) and catalase (CAT) activities were observed in the SBM70 group. The malondialdehyde (MDA) concentration of the SBM50 to SBM70 groups were significantly higher than that of other groups (p < 0.05). The levels of interleukin 8 (il-8) mRNA were highest in fish fed the SBM0, SBM30, and SBM50 + T diets (p < 0.05), while the level of transforming growth factor β1 (tgf-β1) was the opposite (p < 0.05). According to the broken line regression of WG and FE, the highest level of FM substitution by SBM for Oplegnathus punctatus was 24.07–25.31%.

The purpose of this study was carried out to elucidate the effects of replacing fish meal (FM) with soybean meal (SBM) on growth performance, feed utilization, and physiological status of juvenile redlip mullet Liza haematocheila. Five test diets were formulated to replace FM protein by SBM at the levels of 0% (SBM0), 25 %(SBM25), 50 %(SBM50), 75 %(SBM75), or 100 % (SBM100). Each treatment was triplicated (20 fish per tank, initial average weight 5.08 ± 0.11 g fish−1). No significant differences were observed in weight gain (WG), feed efficiency (FE), and protein efficiency ratio (PER) between fish fed with SBM0 and SBM75 diets. However, the WG, FE, and PER values showed a decreased trend with fish fed diet containing more than 75 % SBM protein. The value of HSI decreased significantly with increasing dietary SBM levels. An increase of dietary SBM level linearly decreased energy retention (ER) and lipid retention (LR). Concentrations of serum triacylglycerol (TG) and total cholesterol (T−CHO) decreased significantly with increasing the dietary SBM levels. FM substitution with different levels of SBM had no statistical differences in the activities of trypsin, lipase (LPS), and amylase (AMS) on the intestine. Activities of superoxide dismutase (SOD), catalase (CAT), and glutamyl oxaloacetic transaminase (GOT) in the liver increased significantly with increasing dietary SBM levels. The content of malondialdehyde (MDA) in the liver was significant higher in fish fed SBM100 diet than other treatments. Based on the model of broken-line model of WG and FE, it showed that SBM can replace 51.18–52.46% FM in juvenile L. haematocheila.

Enhancing the production of aquatic animals is crucial for fishery management and aquaculture applications. Ovaries are specialized tissues that play critical roles in producing oocytes and hormones. Significant biochemical changes take place during the sexual maturation of Portunus trituberculatus, but the genetics of this process has not been extensively studied. Transcriptome sequencing can be used to determine gene expression changes within specific periods. In the current study, we used transcriptome sequencing to produce a comprehensive transcript dataset for the ovarian development of P. trituberculatus. Approximately 100 million sequencing reads were generated, and 126,075 transcripts were assembled. Functional annotation of the obtained transcripts revealed important pathways in ovarian development, such as those involving the vitellogenin gene. Also, we performed deep sequencing of ovaries in phases III and IV of sexual maturation in P. trituberculatus. Differential analysis of gene expression identified 506 significantly differentially expressed genes, which belong to 20 pathway, transporters, development, transcription factors, metabolism of other amino acids, carbohydrate and lipid, solute carrier family members, and enzymes. Taken together, our study provides the first comprehensive transcriptomic resource for P. trituberculatus ovaries, which will strengthen understanding of the molecular mechanisms underlying the sexual maturation process and advance molecular nutritional studies of P. trituberculatus.

In the current study, the therapeutic effects of zeaxanthin dipalmitate (ZD) on a rat alcoholic fatty liver disease (AFLD) model were evaluated. After-treatment with ZD from the 5th week to the 10th week in a 10-week ethanol intragastric administration in rats significantly alleviated the typical AFLD symptoms, including reduction in rat body weight, accumulation of hepatic fat droplets, occurrence of oxidative stress, inflammation, chemoattractive responses and hepatic apoptosis in the liver. The reduction of liver function abnormalities by ZD was partly through lower expression level of cytochrome P450 2E1 (CYP2E1), diminished activity of nuclear factor kappa B (NF-κB) through the restoration of its inhibitor kappa B alpha (IκBα), and the modulation of MAPK pathways including p38 MAPK, JNK and ERK. ZD treatment alone did not pose obvious adverse effect on the healthy rat. In the cellular AFLD model, we also confirmed the inhibition of p38 MAPK and ERK abolished the beneficial effects of ZD. These results provide a scientific rationale for the use of zeaxanthin and its derivatives as new complementary agents for the prevention and treatment of alcoholic liver diseases.