Reproductive performance of asian catfish ( Hemibagrus wyckii Bleeker, 1858), a candidate species for aquaculture

Background: Hemibagrus wyckii Bagridae is one of the important economic fish species that lives in the rivers and reservoir in Riau Province, Indonesia. The present study aimed to determine the reproductive performance of H.wyckii under culture conditions. Method s: A total of 10 female and 10 male fish were selected, and weight, length, characteristics of egg and sperm, and hatchery performance were measured. Eggs were fertilized using the dry method. Egg weight and egg diameters were measured for 50 eggs per female. Egg size (50 eggs for each fish) was measured using Labo microscope model L-711 using software camera 3. Then, saline solution was added over the eggs, followed by the addition of pooled sperm from 10 males. Results: Average relative fecundity, egg weight and egg diameter were 2060±512 eggs/kg fish, 29.86±1.21 mg and 2.67±0.26 mm, respectively. The fertilization rate and hatching rate were 60.91±4.68% and 42.91±2.92% respectively. Sperm characteristics such as volume per fish (mL), pH, concentration (per mL), motility (%) and duration of motility (second) were 0.82±0.20, 7.15±0.12, 3.68±0.15, 72.77±1.46 and 47.5±4.84, respectively. Conclusion: The study results and scientific observations regarding reproductive performance suggest that H. wyckii can be considered a new candidate species for aquaculture.


Introduction
In Indonesia, the fisheries sector plays an important economic role through income generation, diversification of livelihoods, supply of animal proteins, and foreign exchange earnings 1 . During recent decades, in the freshwater aquaculture sector, the prioritized species for culture were Clarias, Pangasius, Tilapia, Common carp and Giant gourami. Wild fish species in rivers, reservoirs and lakes have not been prioritized for aquaculture operations.
In the Riau Province, there are three rivers, the Kampar Kanan, Kampar Kiri, and Siak rivers, and the Koto Panjang Reservoir. The Kampar Kanan river hosts up to 34 fish species 2 , the Kampar Kiri river hosts up to 86 fish species 3 , the Ukai river, a branch of Siak River hosts up to 31 fish species 4 and Koto Panjang Reservoir hosts up to 26 fish species 5 .
Hemibagrus wyckii (Bagridae) is one of the most important economic fish species that lives in the rivers and reservoir in Riau Province. H.wyckii (its local name is geso) is a carnivorous freshwater finfish native to Indonesia 2,6 . These fish live in Kampar Kanan and Kampar Kiri rivers with the water temperature between 28 to 29°C, pH 5.20 to 7.20, water transparency 0.50 to 2.30 m, dissolved oxygen 4.87 to 4.97 mg L -1 , alkalinity 42.97 to 67.53 mg L -1 and hardness 53.32 to 104.83 mg L -16 . Furthermore, the average standard lengths of H.wyckii from the Koto Panjang Reservoir, Kampar Kanan and Kampar Kiri rivers were 428±15.78, 432.52±66.11 and 425.86±50.41 mm, respectively with the growth model was isometric 7 . H. wyckii has been categorized as of "least concern" by the International Union for Conservation of Nature (IUCN). However, H.wyckii in the Kampar Kanan river was categorized as a vulnerable to endangered species 7,8 .
Due to the endangered population of H. wyckii, it is necessary to domesticate this species as an aquaculture candidate in the future. Therefore, the present study aimed to determine the reproductive performance of H. wyckii as a potential species under culture conditions to provide preliminary scientific information and evaluation.

Ethical considerations
While H. wyckii is classified as vunerable to endangered in the Kampar Kanan river, the Government of the Republic of Indonesia does not require licenses to obtain, capture and rear this species. Hense, no licenses are applicable to this study. There no suffering animal activity in this study. H. wyckii was transported to the pond farm for rearing, injection, ovulation, stripping and sperm production. In the end of the experiment the H. wyckii still in good condition until return to the pond.

Rearing and selection of breeders
Broodfish of H. wyckii were collected from upstream areas of the Kampar Kanan river in the Kouk village (0° 19' 23.44" N and 100° 56' 40.05" E), Kampar Regency, Riau Province. The broodfish kept in oxygenated polythene bag and transported by truck to Sadarlis Green Catfish Farm, Sungai Paku, Kampar Regency, Indonesia. Then, the broodstock of H. wyckii had been adapted and reared to maturation under the farm conditions. Prior to stocking, female and male fish were weighed using balance scale (OHAUS model CT 6000-USA), and their lengths were measured using a meter ruler with 0.01mm accuracy. During the grow-out period, fish were cultured in two ponds (4 × 4 × 3 m) separated by sex. The depth of water in each pond was 2.0 m. The inlet of the pond water come from Sungai Paku reservoir at a rate of 2.0 m 3 per sec. The broodfish fed with freshwater seashell meat (Pilsbryoconcha exilis; Unionidae) collected from local fisherman near to Sungai Paku Reservoir. The seashell meat was kept at cold box with the temperature 5°C prior given to broodfish. According to Aryani et al. 6 the proximate composition (% wet weight base) of the seashell meat was 89.37% moisture, 7.08% crude protein, 0.82% fat, 0.29% crude ash and 2.44% carbohydrate. The total of seashell meat given to broodfish everyday were 2.500g per pond (equivalent with 9% body weight of population). The feeding time at 17:00 pm due to carnivorous broodfish. The average weight and length from ten (10) of the female broodfish were 2,669.4±486.917 g and 62.84±8.20 cm, respectively. Meanwhile, the ten (10) of male broodfish were 1,769.1±401.10 g and 54.52±7.17 cm, respectively.
The fish were checked monthly for ovulation and semen production from mid November, 2017 onwards. The broodfish were captured with a gillnet formed into a net bag with the appropriate mesh size and anesthetized by orally with Tricaine methanesulfonate (MS-222, ethyl 4-aminobenzoate methanesulfonate 98%, Sigma Aldrich Co, USA, MO; 50 mg L -1 ), based on the dosage used for Solea senegalensis 9 . Oocyte maturation was assessed for each individual. The fish were returned to their pond after evaluation, and no mortality occurred. Fish were fasted 48 h prior to the evaluation. Oocytes sampled in vivo were taken from females using the method described by Nowosad et al. 10 and were placed in Serra's solution (6:3:1 of 70% ethanol, 40% formaldehyde and 99.5% acetic acid) for clarification of the cytoplasm. After 5 min, the position of the stage IV oocyte nucleus was determined using criteria by Krejszeff et al. 11 and was classified as germinal vesicle in the periphery or germinal vesicle breakdown (GVBD).
Female reproductive performance H. wyckii was categorized as endangered species and difficult to obtain in Kampar Kanan river. A total of 10 mature have been eligible for the experiment. 10 mature females that had oocyte stage IV were sampled from broodstock from 3 rd week of February to March 2018 in the same farm, and live weights (FeW) and total lengths (FeL) were measured after anesthetization with 0.50 mg L -1 MS-222 9 . For ovulation, each female broodfish received two injections of GnRH analogs with a

Amendments from Version 1
We have been added some information regarding Asian catfish and their biological, physiological, and ecological aspects. We also added some information of the relationship between water quality and biological aspect of Asian catfish. The title was revised to include 'Bleeker, 1858' in the species name.

REVISED
dopamine antagonist (Ovaprim) (manufactured for Syndel Laboratories Ltd, 2595 McCullough Rd. Nanaimo, B.C.V9S 4M9 Canada) applied intraperitoneally under the left pectoral fin. The first injection was 0.2 mL kg BW -1 and the second was 0.6 mL kg BW -1 (total 0.8 mL kg BW -1 ) at 12 h intervals. These dosages refer to the previous doses for ovulation of H. wyckii 6 . At 18 to 20 h after injection, eggs were stripped into a plastic vessel. Eggs were fertilized using the "dry method" as described by Dabrowski et al. 12 Egg weights of each female were by determined weighing 50 eggs to the nearest 0.01 g, and egg diameters were measured to the nearest 0.01 mm. Egg size (50 eggs for each fish) was measured using Labo microscope model L-711 using software camera 3. Then, a balanced saline solution (7.5 g of NaCl, 0.2 g of KCl, 0.2 g of CaCl 2 2H 2 O, and 0.02 g of NaHCO 3 , in 1000 mL distilled water) was added over the eggs 13 , followed by an addition of pooled sperm from 10 males. The eggs were then gently mixed for fertilization and left for three minutes. The fertilized eggs were rinsed several times with incubation water to remove sperm remnants as well as dead and broken eggs. The eggs were left for an additional 25 minutes to facilitate egg hardening by water absorption and disinfected with 100 ppm iodine for 10 minutes. Then, eggs were transferred to incubation trays placed in a vertical hatching system. The water flow rate to each vertical incubator was 3 L minˉ¹. Fifty eggs were randomly sampled at 15 h after fertilization to determine the fertilization rate (FR). The hatching rate (HR) was determined by counting all hatched fry.
Determination of sperm quality Males were stimulated with a half-dose of the same hormonal preparations used to stimulate the females. Semen samples were obtained from 10 fish randomly selected from the farm. The male fish were anesthetized with 50 mg Lˉ¹ of MS-222. The doses of the anaesthetic agents were prepared a few minutes before each experiment based on the methods of Weber et al. 9 , and then, weights (MaW) and total lengths (MaL) were measured. Special care was taken to avoid any contamination of semen with urine, feces, mucus and water. Semen samples were collected using plastic syringes in 3 mL aliquots, then placed in an insulated ice-cooled container, transported to the laboratory and analyzed within 2 h.
The sperm evaluation included gross (visual) and microscopic examination (as reviewed by Rurangwa et al. 14 , and Cabrita et al. 15 . The gross examination was based on visual and physical observation of parameters such as the semen volume by collecting the semen in a graduated cylinder and determining the level in milliliters. The microscopic examination was carried out using an Olympus model CX40, with magnification between X 10 and X 25 to determine other parameters such as: motility (duration and percentage). Motility (MO) percentage and duration were determined by observing water activated semen placed on a glass slide under a microscope. Motile sperm were observed and expressed as a percent of non-moving sperm. Motility duration (DMO) was determined as the period between movements of the sperm to cessation of any progressive movement expressed in seconds. Sperm concentration (SC) was measured under a microscope using Neaubeaur's hemocytometer and calculated as the number of sperm mlˉ¹ 16 . Semen pH was determined with a hand pH meter (HI8424 Hanna Instruments, USA).

Water quality
The water temperature of the farm was measured with a thermometer (Celsius scale), and water samples were collected to determine the dissolved oxygen (DO) concentrations. An oxygen meter (YSI model 52, Yellow Spring Instrument Co., Yellow Springs, OH, USA) was used in situ, and pH values were determined with a pH meter (Digital Mini-pH Meter, 0-14PH, IQ Scientific, Chemo-science Thailand Co., Ltd, Thailand). Alkalinity and hardness levels of the water were measured according to standard procedures 17 . The water quality parameters were measured once per month.

Statistical analysis
Results were given as the means ± SD. Simple linear regression analyses were performed using SPSS software (version 16.0 for Windows; SPSS Inc., Chicago, IL). The standard deviation of each parameter was determined. For linear regression analysis, significant correlations were considered at p<0.05.

Results
Descriptive measurements and the reproductive performance of female H.wyckii are presented in Table 1. Fifty percent of eggs hatched at 60 h (29-30 °C water temperature). The fertilization rate varied between 53.2 and 68.3%, whereas the hatching rate was between 39.5 and 48.3%.
Characteristics of male fish and sperm samples are presented in Table 2. The average live weight of the males is 1,769.1±401.1g. Male H.wyckii are found to be slightly smaller than females. In the genital maturation stage, the papilla is not prominent for all male fish as the second sexual characteristic of the other Hemibagrus.
According to the analysis of the linear relationship (r 2 ) between variables of H. wyckii females shown in Table 3, there was a strong linear relationship between AF and FeW, AF and RF, HEW and EW, HW and EW, HW and HEW, and HR and FR. In contrast, the analysis of the linear relationship (r 2 ) between variables of H. wyckii males shown in Table 4, show a strong linear relationship between MaW and MaL, MaW and GW, and MaL and GW.
The temperature of the pond water ranged from 28°C to 29°C, oxygen ranged from 6.5 mg L -1 to 6.7 mg L -1 , pH ranged from 6.5 to 6.8, alkalinity ranged from 42.97 mg L -1 to 57.33 mg L -1 and hardness ranged from 104.83 mg L -1 to 110.51 mg L -1 .    wyckii had an effective fertilizing ability according to the correlation analysis, which did not detect any significant relationship between FR and sperm parameters. However, there was a positive relationship between MO and DMO (r 2 = 0.49). Sperm morphology, density, volume, motility and fertilizing capacity, as well as the composition and osmolality of the seminal plasma are parameters commonly measured to assess sperm quality in fish 14,44 . In this study, we did not investigate the ionic composition of the semen, but this phenomenon could be related to the ionic composition of semen, which has a significant influence on motility and duration of motility 45-47 .

Conclusion
In conclusion, there was a positive linear relationship between absolute fecundity with body weight and total length. H.wyckii

Introduction
The introduction should be extended by providing more information about Asian catfish and their biological, physiological, and ecological aspects.

Methods
: Please revise, it is seems there is a typo: Ethical consideration : Please revise, it is seems there is a typo: Ethical consideration "[...]the Government of the Republic of Indonesia does not require licences to be obtain to capture and rear this species, hense no licences are applicable to this study." This sentence should be rewritten: "[...] the Government of the Republic of Indonesia does not require licenses to obtain, capture and rear this species. Hence, no licenses are applicable to this study." "In the end of the experiment the still in good condition until return back to the pond." The word H. wyckii pond should be changed to river, because the fish is returned back to the original (River not pond).
"Then, the broodstock of had been adapted and grown to Rearing and selection of breeders: H. wyckii maturation under the farm conditions". The word grown should be changed to reared, because this experiment is not about growth.
: "Alkalinity and hardness levels of the water were measured in each replicate according to Water quality standard procedures". It is not clear what "each replicate" is referring to. Does it mean that the measurement was done in a few replications? Why only alkalinity and hardness?

Results
Results are well presented.

Discussion
The discussion can be extended by explaining the relationship between the results of water quality and the biological reproduction aspect, since the purpose of this study is under culture condition.

Conclusion
Needs slight revision based on all aspects of the findings.

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Partly
No competing interests were disclosed.

2.
Referee Expertise: Fish physiology, Nutrition I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. I think the title should be revised as "Reproductive biology and breeding of the Asian catfish ( ) after domestication process.

Hemibagrus wyckii
In the title: (Hemibagris wyckii Bagridae) is missed understanding, the correct written should be : Bagridae), because Bagridae is a Family level, or you can write in as (

Hemibagrus wyckii
Bleeker, 1858) Hemigarus wyckii Abstract: Background: ..... I think "most" should be deleted because sound so emotional or hyperbolic, just say ".... is one of the important economic fish species...." Methods: Please mention where the brood fish is come from?
Conclusion: The conclusions is out of the context. You have to make conclusion just based on the data (findings), forget for candidate species for aquaculture, because, there was no detail discussion about this issue, so just focus on your present findings.
Keywords: I suggest don't use similar words which already exist in the title.

Introduction:
I think the state of the arts about the asian catfish is still shallow or unclear, so please provide more information about the previous studies on this species related to bio ecology, feeding, aquaculture etc.
Please add some information what is the advantages of the species compared to other freshwater species?

Methods:
Please cite these references to enhance your methods: Muchlisin ., 2010 et al Muchlisin 2011 et al.,

Results:
The results have been explained in several short paragraphs, please extend the paragraph by combining 1 2 The results have been explained in several short paragraphs, please extend the paragraph by combining paragraph 1, 2 in one paragraph and paragraph 3 and 4 in one paragraph.

Discussion:
There was no comprehensive discussion to justify why this species is potential for aquaculture. The justification is not only based on biological aspects but also on economic consideration (see Muchlisin, 2013 ).
Unfortunately, there was no economic evaluation performed.Therefore, I suggest to focus only on the finding as already presented.