Effect of dietary protein, vitamin E, and vitamin C levels on the growth and gonad development of hybrid male abalone (Haliotis fulgens (cid:0)×H. discus hannai (cid:0)) by the orthogonal array design

A natural feed of fresh seaweed and sixteen diets designed by L16 (43) orthogonal array were formulated to contain differentlevels of protein (200, 250 g, 300, 350 g/kg), α - Vitamin E (VE) (0, 0.1, 0.3, 0.5 g/kg) and Vitamin E (VC) (0, 0.5, 1, 1.5 g/kg) to evaluate the effects of dietary protein, VE, VC and their combination on growth and gonadal development of hybrid male abalone (Haliotis fulgens (cid:0)×H. discus hannai (cid:0)). Each diet was randomly assigned to triplicate groups of 18 abalones (34.27±2.93 g) that were stocked in sea cages for eight weeks. Results showed that the maximum weight gain (WG) and speci�c growth rate (SGR) were obtained in hybrid male abalone fed D10 diets. The highest gonad somatic index (GSI) value was found in the D14 diet. Moreover, the ANOVA results showed that only dietary protein could signi�cantly affect the GSI of hybrid male abalones (P < 0.05). The mRNA expression of vasa in the testis of D14 abalone was the lowest. Abalones fed the D14 diet had higher testosterone level and sperm in testis than those fed other diets. In conclusion, protein of 300 g/kg, VE of 0.1 g/kg and VC of 1.5 g/kg (D10) and protein of 350 g/kg, VE of 0.1 g/kg and VC of 1 g/kg (D14) might be the optimal combinations in the diet for growth and gonadal development of hybrid male abalones respectively.


Introduction
It is well known that abalone cultivation has been very important for many countries because of its high pro t (Cook, 2014).China is one of the most important abalone producing countries, with the farming yield of abalone reaching 217831 tons in 2021 (Liu, 2022).Among a variety of abalone species, the hybrid abalone bred by Haliotis fulgens × H. discus hannai has been accredited as a potential candidate for the diversi cation of China aquaculture, primely due to its heterosis in hypoxia tolerances, heat and stress resistance, and high commercial value (You et al., 2015).At present, hybrid male abalone is mainly used to produce backcrossing of H. fulgens × H. discus hannai to solve the problem of paternity H. fulgens limit.Though the progeny obtained by backcrossing of H. fulgens × H. discus hannai is not as good as those produced by the cross of H. fulgens × H. discus hannai , still shows some heterosis.However, the testicles of the progeny of H. fulgens × H. discus hannai are not well developed, which leads to low prodection, vitality and even reduced fertility (Guo, 2015).Thus, the reproduction and seedling rearing of this hybrid male abalone are faced with serious challenges.It is signi cant to promote the reproductive performance of hybrid abalone.
Previous studies demonstrated that dietary nutrients were vital in ordinary abalone breeding for they could regulate the early development of gonads (Acuña and Mercedes, 1999; Setyono, 2004).For example, protein is one of the most important nutrients related to the growth and gonad development of most aquatic animals (Cottier- Cook and Kelly, 2007;Xu et al., 2016).It was well demonstrated that high protein diets could improve the gonadal development and reproduction of juvenile abalone (H.midae), adult sea urchin (Psammechinus miliaris), brood shes (Channa marulius) and sea urchin (Lytechinus variegatus) (Hafeez-ur-Rehman et al., 2017; He in et al., 2012; Pantazis et al., 2000;Roussel et al., 2019).However, protein de ciency could lead to a small size of the egg and its cytoplasm bulk (Bilbao et al., 2012).Excessive dietary protein may increase nitrogenous excretion, resulting in a water quality descent (Lu et al., 2020).Thus, it is imperative to formulate arti cial abalone feeds that balance the protein requirement and the environmental impact (Hammer et al., 2006;Zhou et al., 2011).
Besides protein, vitamins also play a signi cant role in every animal stage.Vitamin E (α-tocopherol, VE) is a fat-soluble antioxidant.The major function of VE is to prevent the peroxidation of polyunsaturated fatty acids of phospholipids and cholesterol in cellular and sub-cellular membranes (Traber and Atkinson, 2007).VE has been described as a dietary factor in animal nutrition and was considered especially important for normal reproduction (Azzi and Stocker, 2000).Furthermore, VE can protect sperm from damage and improve reproductive performance by increasing serum androgen levels (Sadeghi et al., 2020).It was reported that optimum dietary VE content improved the growth performance and gonadosomatic index (GSI) of female red swordtail (Xiphophorus helleri), sand sh (Holothuria scabra) and Nile tilapia (Oreochromis niloticus) (Ratinasamy et al., 2011).However, the de ciency of VE could result in the slow movement and poor resistance in rats (Noreini et al., 2020) and poor reproductive performance in aquatic animals (Izquierdo et al., 2001), such as female shrimp (Macrobrachium nipponense), yearling rice eld eel (Monopterus albus) (Tan et al., 2007).Vitamin C (L-Ascorbic acid, VC), a water-soluble vitamin and polyhydroxy compound is the most important antioxidant substance in the extracellular uid of organisms (Bendich et al., 1986;Carr and Maggini, 2017).Previous studies demonstrated that dietary VC played an important role in improving the weight gain (WG) of large yellow croaker (Pseudosciaena crocea), reproduction of guppy (Poecilia reticulata), and gonadal maturation of milk sh (Chanos chanos) (Ai et al., 2006;Emata et al., 2000;Mehrad and Sudagar, 2010).However, the information about the effects of dietary protein and vitamins on the growth and gonad development of hybrid male abalone is limited so far.
The orthogonal array design was considered as an e cient and convenient testing method by arranging many variables with a minimum of experiments (Lan et al., 1994a).A derivative algorithm was used to nd the optimum values in the experimental intervals pre-selected for each input variable (Lan et al., 1994b;Sharma et al., 2006).It was valued to note that some authors have previously announced some viable orthogonal array design applications in biological research (Guo et al., 2022;Jin et al., 2018;Lan et al., 1995;Silveira et al., 1991;Tan et al., 2007;Xu et al., 2010).
Therefore, taking the effects of dietary protein, VE and VC contents on growth and reproduction into consideration, it was worth using an orthogonal array design to explore the combined effects of three factors in hybrid male abalone which could provide the scienti c basis for its production and promote the sustainable development of the abalone farming.

Diets and preparation procedure
This study refers to a three-factor, four-level orthogonal experiment (Table 1).Sixteen isolipidic (35 g/kg) diets were made by adding various levels of protein (200, 250, 300 and 350 g/kg), VE (0, 0.1, 0.3 and 0.5 g/kg) and VC (0, 0.5, 1 and 1.5 g/kg) at equilibrium of the microcrystalline cellulose.Fresh seaweed was used as the control diet.In the experimental diets, soybean meal, sh meal and gluten meal were prime protein sources, sh oil and soybean lecithin were lipid sources.VE and VC in the form of DL-α-tocopherol acetate (Herrera and Barbas, 2001;Niki and Traber, 2012) and ascorbic acid acetate (Davies et al., 1991;Padayatty et al., 2003) respectively were added to basic diet.The approximate composition of the dietary formulation and basal diets is shown in Table 2.
Insert Table 1 here Insert Table 2 here All diets were prepared by mixing the ingredients carefully in a horizontal mixer and then pressed into 1.5-2.5 mm akes.After being steamed at 90 ℃, all akes were dried at 55 ℃ and then placed in a -20 ℃ refrigerator.

Animals and culture conditions
Twelve-month hybrid abalones were obtained from the grading station in ZhongXin Yongfeng (Fuzhou, China) in early April 2022.Studies have shown the color of male and female abalone gonads was milky white and grayish green, respectively (Shepherd and Laws, 1974;Vélez-Arellano et al., 2015).Abalones were acclimated and fed a commercial diet for two weeks in the sh steak of abalone farm in Lianjiang Country, Fujian Province before the experiment.A total of 306 hybrid male abalones ( 34.27 ± 2.93 g) were randomly divided into fty-one sea cages (40 cm × 20 cm × 10 cm, length × width × height, six abalones per cage), and three replicates were randomly assigned to each treatment including control.The temperature of natural seawater used in the experiment ranged from 18 to 22 ℃.The concentrations of dissolved oxygen and ammonia nitrogen were measured to be 6.1-7.5 mg/L and 0.01-0.05mg/L by a water quality meter (W-I water quality meter; OCTADEM, China) twice a week.Experimental abalones were hand-fed one time every two days at 06:00 with a ration of approximately 5% total biomass for 56 days.

Sampling collection and data calculation
At the end of trial, all abalones were fasted for 24 hours before being sampled.The number of individuals and body weight were then measured separately for each cage.Hemolymph was aspirated from the foot muscle of the hybrid male abalone using a sterile spear tip and then was kept on ice and centrifuged at 5,000 rpm for 10 min at 4 ℃.Supernatants were carefully collected and then stored at -80 ℃ until assay.The testis and hepatopancreas samples dissected from three random animals in each cage were weighed to calculate GSI and then stored at -80 ℃ until analysis.Partial testis tissues were xed with 4% paraformaldehyde for histological evaluation.

Histological observation of testicular
For histological observation, the testis tissues of the control group and experimental groups with high and low GSI were dehydrated through graded alcohol, then xylene, and nally embedded in the specimen.
The specimen was then para n-embedded and cut into 4-6 µm thin sections, stained with hematoxylin and eosin, and nally observed and photographed with a biomicroscopy (Murzider MSD1125, China).

Testosterone hormone measurement
The hemolymph samples were thoroughly mixed with assay reagent according to the instruction of the testosterone (T) ELISA kit .The T concentrations were measured at 450 nm using an In nite M Nano (TECAN Trading AG, Switzerland).The absorbance (OD) was measured by microplate reader at 450 nm wavelength, and then the levels of T in samples of range from 10 pg/ml to 320 pg/ml were calculated by a standard curve.

Total RNA extraction and cDNA synthesis
Total RNA was extracted from testis samples using a total RNA Kit (Omega, Bio-Tek, China).The quality and concentration of the RNA were assessed by agarose gel electrophoresis and A260/A280 ratio through a NanoDrop microspectrophotometer (NanoDrop™ 2000, Thermo Fisher, Germany).First-strand cDNAs were synthesized from total RNA (1 µg) using the HRbio™ 1st Strand cDNA Synthesis SuperMix for qPCR (Heruibio, Fujian, China).The entire experimental procedures of RNA extraction and cDNA synthesis were in accordance with the manufacturer's instructions.

Quantitative Real-Time PCR Analysis of vasa
Quantitative real-time PCR (qRT-PCR) analysis was detected using the HRbio™ qPCR SYBR Green Master Mix kit (Heruibio, Fujian, China) and Roche Light cycler 480 II System (Roche, Germany).Brie y, the qRT-PCR reaction mixture contained 0.5 µL of cDNA template, 0.25 µL of both forward and reverse primers, 5 µL of SYBR Green Mix, and dH 2 O with an ultimate system of 10 µL under the following PCR ampli cation condition: 95°C for 5 min followed by 40 cycles at 95°C for 10 s, 55-60°C for 20 s and 72°C for 20 s.
Triplicate reactions were performed for target and reference genes in each testis sample.Gene primers (Table 3) for vasa and rpl5 were obtained from a previous study (Guo, 2015).The speci city and e ciency of the primers for housekeeping and target genes were determined by constructing a standard curve using serial dilutions of cDNA.Relative gene expression was quanti ed based on cycle threshold using the 2 −ΔΔCT method with the rpl-5 gene (Livak and Schmittgen, 2001).
Insert Table 3 here

Statistical analysis
The in uence of the protein, VC, and VE on the growth performance and gonad development of hybrid male abalone was evaluated on the basis of the analysis of each factor by the range value.The maximal range value was related to the extremely vital affecting factor.Signi cant differences between groups were analyzed by one-way ANOVA followed by Tukey's multiple range test.The statistical analyses were proceded using SPSS 26.0 for Windows.The data were shown as mean and standard error (mean ± SE, n = 3) and a signi cance level was expressed as P < 0.05.

Growth performance
The SR is shown in Table 4. Hybrid male abalones in D5, D6, and D10 had the lowest SR among all sixteen groups.According to the range value in Table 5, the order of protein, VE and VC that mostly in uenced SR of hybrid male abalones was A > C >B.However, analysis of variance showed that three factors had no signi cant effect on the SR of abalones (P > 0.05) (Table 6).The impact of the combined three factors also showed no signi cant effects on SR among all treatments (P > 0.05; Figure 1A).
Based on the range analysis, the best combination of dietary protein, VE, and VC levels for WG of abalone was A 3 B 2 C 4 , namely, 300 g/kg protein, 0.1 g/kg VE and 1.5 g/kg VC.The order of protein, VE and VC mostly in uenced WG of hybrid male abalones was A > B > C (Table 7).Table 8 showed that WG was not affected by all three factors (P > 0.05).
According to the WG of abalone shown in Table 4, the effect of three factors on WG of hybrid male abalones is presented in Figure 1B.Compared with the fresh feed, the combined experiment diets did not signi cantly affect the WG (P > 0.05).Abalones fed D10 showed the highest WG among all the experimental diets.
A similar pattern with WG was observed for the SGR of hybrid male abalones (Table 4).According to the range analysis in Table 9, the optimal combination for maximum SGR of hybrid male abalones was A 3 B 2 C 4 (300 g/kg protein, 0.1 g/kg VE and 1 g/kg VC).The order of three nutrients which in uenced the SGR of hybrid male abalones was B > C >A. Table 10 shows that three factors at four levels did not signi cantly affect the SGR of hybrid male abalones.As presented in Figure 1C, the SGR of abalone showed a signi cant difference between the experimental and control groups.The SGR of hybrid male abalones fed D6 and D10 were 0.83 %/d and 0.99 %/d (Table 4), respectively, which were signi cantly higher than that in abalones fed on other diets (P < 0.05).
Based on the mean GSI of three factors at four levels in Table 4, D14 showed a signi cantly higher GSI (4.15%), while D4 showed a lower GSI (2.83%) compared with other groups.According to the result of range values (Table 11), three factors in uenced the GSI of hybrid male abalones adhering to the order of A > B > C. The topgallant value of GSI for three factors was A 4 B 2 C 3 , representing the diet containing 350 g/kg protein, 0.1 g/kg VE and 1 g/kg VC.Table 12 showed that the GSI of abalones was signi cantly affected by dietary protein (P < 0.05).As shown in Figure 1D, the highest GSI value occurred in D14 compared with the control treatment.

Histological comparison
Testicles of abalones in D14 have reached the mature stage full of sperm cell (Figure 2, D14).Compared with D14, the histologic stage of the control group and D4 showed the obvious distinction (Figure 2, D4, control group).There were a lot of spermatogonia and a small amount of sperm in both D4 and control groups.
Insert Figure 2 here

Hemolymph hormone levels
The T levels in hemolymph of abalones fed D6, D10, D13, D14, and D16 diets were signi cantly higher than the control group (P < 0.05, Figure 3).In contrast, T levels in the hemolymph of the D4 group were lower than the control group (P < 0.05).
Insert Figure 3 here

The mRNA expression of the vasa gene in the testis of hybrid male abalone
The relative expression of vasa in D4 was signi cantly higher than that in the other groups (P < 0.05).The lowest expression of vasa was shown in hybrid male abalone fed D10 and D14 diets (Figure 4).Insert Figure 4  ) which ignored the important role of dietary nutrients.This study conducted a feeding trial to investigate the effects of the combination of dietary protein, VC and VE levels on the growth and gonad development of hybrid male abalone by using the method of orthogonal array design.In the current study, compared with natural algae kelp, arti cial diets containing different protein, VE and VC levels showed a signi cant promotion effect on the gonadal development and growth of hybrid male abalone which provided a new method for exploring the qualitative and quantitative requirements of the mixed nutrient sources in abalone.
Growth performance (e.g., SR, WG and SGR) were the most common criteria for determining aquatic animal dietary nutrient requirements (Bullon et al., 2022).In the present study, the ANOVA analysis showed that dietary protein level did not affect SR, WG and SGR, which indicated that hybrid male abalone could tolerate wide range of dietary protein levels (200-350 g/kg).Despite the difference in species, the high tolerance of wide dietary protein range (300-350 g/kg) was also observed in carp (Osteobrama belangeri) (Basudha and Vishwanath, 2001), female cat sh (Clarias gariepinus) (Sotolu and Kigbu, 2011) and rohu fry (Labeo rohita) (Prasad et al., 2017).These may be due to their omnivorous nature.Interestingly, abalones fed D10 (A 3 B 2 C 4 , 300 g/kg protein, 0.1 g/kg VE and 1 g/kg VC) had the signi cantly higher WG and SGR than those fed other diets, and rang analysis indicated that 300 g/kg protein was optimum level in diet for hybrid male abalone, which agreed with previous studies in abalones (Fleming et al., 1996), such as H. discus hanni and juvenile hybrid abalone (H.discus hannai × H. fulgens ) (Baek and Cho, 2021;Ma et al., 2022).The results of our study also suggested that there were some interaction and synergistic effects between protein and vitamin levels on growth performance, which was worth further exploring.
Here, it was VE (Factor B) that mostly affected the growth performance of abalone according to range analysis.The WG and SGR increased rstly and then decreased with the VE levels increasing from 0 to 0.1 g/kg and from 0.1 to 0.5 g/kg.When consistent with the result that abalone fed diet D10 showed the maximum growth, 0.1 g/kg of VE in diets could be appropriate for hybrid male abalone.A similar trend of WG and SGR response to dietary VE levels was observed in zebra sh (Danio rerio), juvenile Korean rock sh (Sebastes schlegeli), rohu fry (Labeo rohita) and soft-shelled turtle (Pelodiscus sinensisfed) (Bahareh et al., 2012; Bai and Lee, 1998; Huang and Lin, 2004;Sau et al., 2004).These studies showed that a certain concentration of VE was bene cial to animal body, but excessive VE in diet may result in growth retard.These might be due to the imbalances between VE and cumulated radicals, which acted as pro-oxidants (Fu et al., 2007;Peng et al., 2009).
With the increase of dietary VC levels, the SGR and WG of hybrid male abalone decreased rst and then increased in the study.We observed that dietary VC level exceeding 1.5 g/kg could lead to e cacious growth from range analysis.In previous studies, the growth performance of freshwater prawn (Macrobrachium rosenbergii) (Asaikkutti et al., 2016), sub−adult turbot (Scophthalmus maximus) (Niu et al., 2014), benni ngerlings (Barbus sharpeyi) (Youse et al., 2013) and tilapia (O.niloticus) (Abd and Abd, 2018) were similar to the results of this experiment.In addition, VC could protect against growth inhibition of animals fed a diet lacking vitamin E and promote a sparing impact on VE.Interestingly, previous studies reported that 0.1 g/kg of vitamin E could prevent growth inhibition in a diet lacking vitamin C too (Nguyen et al., 2012;Sealey and Gatlin III, 2002).Therefore, the interaction between vitamin E and vitamin C need to be deeply studied.Above all, in all experiment diets, D10 (300 g/kg protein, 0.1 g/kg VE and 1.5 g/kg VC) was the optimal level for the growth of abalone.

Reproduction performance
Studies have shown that VE de ciency result in low fecundity and gonadal immaturity (Panda and Cherian, 2014; Volkoff and London, 2018).The appropriate dietary VE content could signi cantly improve the GSI and promote sexual maturation of male Japanese quail (Coturnix) (Hooda et al., 2007).However, our study showed that dietary VE did not signi cantly affect the GSI of hybrid male abalone.The result was consistent with those of female shrimp (Macrobrachium nipponense) (Li et al., 2018), male Japanese quail (Coturnix coturnix Japonica) (Biswas et al., 2013), white shrimp broodstock (Litopenaeus vannamei) (Maulana et al., 2017) and female brood sh (Clarias batrachus) (Roy and Mollah, 2010).Besides, the range value showed content of VE at 0.1 g/kg was the optimal level for gonad development of abalone.The different VE effects on GSI may be due to the different ages and species of cultured animals or the ingredients of diets.Notably, the value of GSI showed the rst increase and subsequently decrease trend with the increasing of dietary VE levels, which may suggest that excess dietary VE were intoxication with adverse effects on reproduction and growth (Steinberg, 2022).Similarly, a high concentration of VC in semen could protect sperm from oxygen-free radical damage (Ciereszko and Dabrowski, 1995).In this study, the GSI of hybrid male abalone was not signi cantly affected by dietary VC inclusion levels, which agreed with the studies in other species, such as zebra sh (D. rerio) (Mehrad et al., 2011), red swamp cray sh (Procambarus clarkia) (Xu et al., 2010) and gold sh (Carassiuss auratus) (James and Vasudhevan, 2011).However, according to the range value in the current study, 1 g/kg of VC in diet D10 were recommended for hybrid abalones to gain high GSI and production.This may be due to the sparing effects between VE and VC, and/or the interaction of varied dietary protein, VE and VC levels.
The previous study has shown that the development of abalone gonads was highly subject to nutrient and energy intake, which was associated with the high value of GSI (Akinola et al., 2012).Numerous studies demonstrated that lower protein levels in diet could inhibit the development of the gonad of abalone (Bahareh et al., 2012;Ciereszko and Dabrowski, 1995;He in et al., 2012).Similar studies in green sea urchins (Stonglycentrus droebachiensis), rice eld eel, (Monopterus albus) and Nile tilapia (O.niloticus) demonstrated that an increase in protein level promoted the maturation rate and development of gonads (Al-Hafedh et al., 1999;Kling, 2009;Yuan et al., 2011).Similarity, the range analysis in the present study showed that the GSI was signi cantly increased by dietary protein levels, and was much higher in abalone fed diets containing 350 g/kg protein than that in abalone fed other diets, which was supported by the testis structure (Fig. 2).It is generally known that the spermatozoa are thickening, follicles are swelling, and pigmentation is deepening with the maturation of abalone testicles.The lumen in mature testicles of abalone is full of radially arranged spermatozoa, which occupy the most space of the narrow follicular cavity.Individual spermatocytes were still observed in the germ epithelium.In the later stage, the reproductive epithelium is thinner, and the gonad has reached the thickest (Najmudeen, 2007;Yu et al., 2018).In the study, the sperm cells in the gonad of abalone fed diet D14 were full of mature spermatozoa in the bullet shape with elongated agella, which were much more mature than those fed control and D4 diet.When taking WG and GSI into consideration, the optimum inclusion level for the growth performance and gonadal development of hybrid male abalone were 300 and 350 g/kg, respectively.This may indicate that abalone needs more protein in the duration of gonadal development, which may be related to the development of nutritive phagocytes (Lourenco et al., 2020).
Gonadal development depends on the androgen, such as T (Hanai and Esashi, 2007).Several previous studies have shown that the concentration of T in testis increased up to the highest value at the mature  Schroeder and Keller, 1989;Shin et al., 2008;Turek et al., 1976).In this present result, the T levels in hemolymph of abalone fed D6 and D14 were the highest compared with other groups, which provided evidence for the high GSI value in group D14.This may be because that T was directly involved in the regulation of gametogenesis and thus accelerated the maturity of abalone gonad (Gauthier-Clerc et al., 2006).Additionally, the expression level of vasa was higher in D4, and lower in D10 and D14 than in the other groups in our study.Previous study in tilapia (O.niloticus) reported that vasa signal was high in spermatogonia (germ cells), moderately present in primary spermatocytes while no signal was detected in secondary spermatocytes, spermatid and spermatozoa (Raghuveer and Senthilkumaran, 2010).The expression of vasa gene was high in the early stage and decreased with the maturation of sperm in testis of gibel carp (Carassius auratus gibelio) and Asian seabass (Lates calcarifer) (Xu et al., 2005;Xu et al., 2014).Therefore, the optimum combination of dietary protein, VE and VC levels could effectively promote the gonad development of hybrid male abalone by inhibiting vasa expression and increasing hemolymph T content.Under the experimental condition, D14 (350 g/kg protein, 0.1 g/kg VE and 1g/kg VC) was the optimal diet for gonad development of hybrid male abalone.

Conclusion
In conclusion, this experiment rst investigated the growth and gonad development of hybrid abalone by adjusting nutrient levels in the diet.The combination of dietary protein, VE and VC could signi cantly improve the growth performance, inhibit vasa expression and increase hemolymph T content and thus promote the gonadal development of hybrid male abalone.Among the three factors involved in our experiment, it was dietary protein content that could effectively improve the GSI of hybrid male abalone.Based on GSI, hemolymph T level and vasa expression, D14 (350 g/kg protein, 0.1 g/kg VE and 1g/kg VC) was the optimal diet for gonad development of hybrid male abalone.Declarations 94.Youse P, Yavari V, Zakeri M, Salati AP, Keyvanshokooh S ( 2013  Optimal level means the level at which the average SR of the abalone is highest.R (range) refers to the Dvalue between the maximum and lowest mean SR of hybrid male abalone.ELF imports the most predominant level factors in the arguments.BCm means the best level constitution for every argument.Optimal level means the level at which the average WG of the abalone is highest.R (range) refers to the D-value between the maximum and lowest mean WG of hybrid male abalone.ELF imports the most predominant level factors on the arguments.BCm means the best level constitution for every argument.Note: K1, K2, K3, and K4 imply the means of SGR at levels 1, 2, 3, and 4, respectively.
Optimal level means the level at which the average SGR of the abalone is highest.R (range) refers to the D-value between the maximum and lowest mean SGR of hybrid male abalone.ELF imports the most predominant level factors in the arguments.BCm means the best level constitution for every argument.Optimal level means the level at which the average GSI of the abalone is highest.R (range) refers to the D-value between the maximum and lowest mean GSI of hybrid male abalone.ELF imports the most predominant level factors in the arguments.BCm means the best level constitution for every argument.
Histological analysis of testes in hybrid male abalone in D4, control and D14 groups after 8 weeks (D4: Hypogonadal group; C: Control group; D14: Highest GSI group).The inset picture is at a higher magni cation of sperm.Sp, sperm; Scale bar: 60 µm.
The concentration of testosterone (T) of hybrid male abalone fed 17 experimental diets for 8 weeks.Bars with different superscript letters are signi cantly different (P < 0.05).

Figures Figure 1
Figures

Table 1 Table of
) Effect of dietary supplementation of vitamin C on growth performance feed utilization and carcass composition of Barbus sharpeyi ngerlings.Journal of the Persian Gulf 4: 23-31.95.Yu L, Xu D, Ye H, Yue H, Ooka S, Kondo H, Yazawa R, Takeuchi Y (2018) Gonadal transcriptome analysis of Paci c abalone Haliotis discus discus: Identi cation of genes involved in germ cell

Table 3
Primers used for vasa mRNA expression analysis in the testis of hybrid male abalone(Guo,  2015).

Table 4
Orthogonal matrix of the L16 (4 3 ) indicating the impact of combinatorial Protein, VE and VC on GSI, SR, WG and SGR of hybrid male abalone.

Table 5
Range analysis for the impact of Protein, VE and VC on the SR of hybrid male abalone.

Table 6
The consequence of ANOVA of three compositions on SR of hybrid male abalone.

Table 7
Range analysis for the impact of Protein, VE and VC on the WG of hybrid male abalone.

Table 8
The consequence of ANOVA of three compositions on WG of hybrid male abalone

Table 9
Range analysis for the impact of Protein, VE and VC on the SGR of hybrid male abalone.

Table 10
The consequence of ANOVA of three compositions on SGR of hybrid male abalone

Table 11
Range analysis for the impact of Protein, VE and VC on the GSI of hybrid male abalone.K2, K3, and K4 imply the means of GSI at levels 1, 2, 3, and 4, respectively.

Table 12
The consequence of ANOVA of three compositions on GSI of hybrid male abalone.