Induced Breeding, Embryonic and Larval Development of Koi Carp in Recirculatory Aquaculture System

Kalaiselvi Natarajan1, Karal Marx Karuppiah1, Sheena K Baby1, Sakthivel Mohammed2, Shanmugam Seerappalli Aran1 and Suresh Eswaran1* 1Institute of Fisheries Postgraduate Studies, Tamil Nadu Dr. J. Jayalalithaa Fisheries University, OMR Campus, Chennai-603 103, India 2Mandapam Regional Centre of Central Marine Fisheries Research Institute, Mandapam, Tamil Nadu-623 520, India Article Information Received 29 August 2022 Revised 15 September 2022 Accepted 08 October 2022 Available online 14 January 2023 (early access)


INTRODUCTION
O rnamental fishes fetch a higher price than the food fish in international markets because of their aesthetic value and high commercial value in the export trade. Koi carp, Cyprinus carpio, is a well-known decorative fish. It is a variety of the common carp (Cyprinus carpio) species, a native of South East Asia. Koi carp produced from China and Japan fetches high market value for its excellent colour pattern. New varieties of colours and colour patterns in Koi carp have been developed over recent years. The common colours are white, cream, red, blue, yellow and black. Koi carps generally live up to 15-24 years. Being hardy, it is highly suitable for garden pools and aquarium keeping (Hussian et al., 2014).
Aquaculture faces many obstacles related to water quality and waste management. The waste can cause environmental pollution if it is not treated before disposal. Therefore, the development of aquaculture innovation is fundamentally essential to develop into sustainable aquaculture practices. One of the more intriguing strategies for intensifying production while simultaneously reducing wastes is the development of recirculating aquaculture systems (RASs). These systems are designed to collect and remove waste products, uneaten feed, and bacteria from the tank where the fish live so that water can be recycled back into the system. Even though many ornamental fish and invertebrates have been induced to spawn in captivity, the larvae of only a few species have been successfully reared in captivity (Holt, 2003;Olivotto et al., 2003). The bottleneck is the heavy mortality encountered in the larvae during the critical period of development, mostly noticed when the larvae change over from internal yolk storage to exogenous feed (Madhu et al., 2012). Breeding is a complex process which depends on several factors. In addition to the other environmental and biological factors,

O n l i n e F i r s t A r t i c l e
the condition of brooders is the major factor determining the success. Selecting the appropriate inducing agents will enhance the overall reproductive success. Based on the literature, for many fish species synthetic hormone, Ovaprim (M/s Syndel Laboratories, Canada) has been successfully used since 1990. After 1997, Ovatide (M/s Hemmo Pharma, Mumbai), an indigenous synthetic hormone which is more affordable than Ovaprim was used. Nowadays, WOVA-FH (M/s.Biostadt India Limited, Mumbai) is used as an inducing agent for many ornamental fishes. Hence, the present study was conducted using three commercially available GnRH synthetic hormone viz., WOVA-FH, Ovaprim and Ovatide. Literature on the seed production and embryonic development of Koi carp is very scanty. Considering these facts, the present study was undertaken on broodstock development, induced breeding and larval development of Koi carp using RAS for sustainable aquaculture practices.

Collection and conditioning of broodfish
Experiments were conducted at Aquatic Rainbow Technology Park, Madhavaram, Chennai, India. The brooders (Female: 250±50 g; Male: 200±50 g) were collected from commercial ornamental fish market in Kolathur, Chennai and were transported in oxygenated polythene bags.

Brood stock maintenance
The broodfishes were segregated into males of 2+ years and females of 4+ years. They were reared separately in 2 cemented grow out raceway tanks (20-ton capacity) with RAS facility. The brooders were fed with commercial GROWFIN supplementary feed (1.8 mm) containing 38% protein, at 5% of body weight, twice a day. Along with the supplementary diet, brooders were also fed with earthworm and Tubifex tubifex (sludge worm) to enhance maturity under captive condition for 4 months. Then the brooders were separated based on the secondary sexual characteristics (Table I). Matured males were selected by observing freely oozing milt (Fig. 1a). Matured females were observed with bulged belly region, slightly swollen genital aperture, and oozing ova upon gentle pressure on the abdomen (Fig. 1b).

Preparation of spawning tank
For induced breeding of fish, FRP (fibre-reinforced plastic) circular tanks of 1-ton capacity with RAS facility were filled (1ft of height) with water. The tanks surface was laid with artificial polyethylene strips (breeding mops) and nylon fibres. Nylon fibres act as a substrate to attach the eggs because Koi carp eggs are adhesive in nature.

Optimization of dosage for induced breeding
In the present study induced breeding trials were carried out using three different inducing agents to optimize the hormone on breeding performance. Ovatide, ovaprim and WOVA-FH were used as an inducing agent at 0.7 ml per kg of body weight (Table II). The synthetic hormone which showing better performance were administrated at 4 different doses viz., 0.3, 0.5, 0.7 ml, 1 ml per kg body weight intramuscularly for dose optimization. One control unit was also set up. Both males and females were induced by injecting single intramuscular injection, between the base of the dorsal fin and lateral line, in the evening (1730 h). After injecting the hormone, brooders were released at 2:1 (M: F) sex ratio into spawning tanks supplied with continuous aeration. The occurrence of spawning was monitored periodically by checking for the presence of eggs in the breeding hapa. Egg collection was done on the next day morning at 0530 h (Fig. 1e).

Estimation of hormone efficiency
The efficiency of hormone was estimated based on parameters such as number of eggs spawned, fertilization rate and hatching rate. Number of spawned eggs was estimated by random sampling or by counting the eggs in each breeding mop or nylon fibres. The fertilization rate of eggs was determined by counting the egg having intact nucleus with yellowish colour. Fertilization rate and hatching rate was calibrated using the following formulae:

Data analysis
The data on number of ovulated eggs, fertilization, and hatching rates of different treatments were analysed by compared using one way ANOVA method using Statistical Package for the Social Sciences (SPSS) version 20.0. Duncan's multiple range tests was used to compare the significant differences within and between the treatments. The significance level was set at p<0.05.

RESULTS AND DISCUSSION
In the present study, induced breeding trials were carried out using three different inducing agents ovaprim, ovatide and WOVA -FH at 0.7 ml per kg of body weight. Most carp species responded well at a dosage ranging between 0.4 and 0.70 ml per kg (Nandeesha et al., 1990;Das, 2003). Comparatively WOVA-FH at 0.7 ml per kg showed better performance with higher number of ovulated eggs, fertilization and hatching rate than ovaprim and ovatide (Table II). For dose optimization the matured brooders (250-300 g) were administered with the synthetic hormone WOVA-FH (M/s. Biostadt India Limited), Mumbai consisting of Synthetic Gonadotropin Releasing Hormone Analogue (SGnRH), Domperidone and Glycerol at four different doses 0.3, 0.5 and 0.7 ml,1 ml per kg of body weight. The ideal dose of synthetic hormone WOVA-FH for induced breeding of Koi carp was standardized and the results are summarised in terms of number of eggs per female, fertilization and hatching rate are given in Table III. During the entire culture period. the optimum water quality parameters such as pH and total dissolved solids were maintained as required for Koi fish (Table IV). The spawning was observed, in all breeding trials with significantly varied latency period (The time between administration of inducing agent and initiation of spawning) of 7-12 h. Similarly, variation in the latency period with dose and hormone in induced fish breeding was observed in Gonoproktopterus curmuca, Pethia manipurensis and Osteobrama belangeri (Padmakumar et al., 2014;Motilan et al., 2014;Das et al., 2016). This infers the effect of hormone and dose on the breeding performance in terms of pairing, courtship behaviour and spawning. In the present study, spawning activity was quicker at higher doses compared to lower doses; shows the difference in the mode of action and interaction of hormone. Similar observation was reported by Pandey et al. (2002) and Behera et al. (2007) in Labeo rohita and L. bata, respectively.  The significant difference (P<0.05) was observed in egg output of the fishes bred with different hormone and doses. The better spawning was observed in WOVA-FH (0.5 ml per kg) and (0.7 ml per kg). Comparatively WOVA-FH at 0.5 ml per kg yielded (3380.5 ±202.75) higher number of eggs. Further, results showed that Ovatide at 0.7 ml per kg produced lower number of spawned eggs. It was observed that fertilization rate was also varied significantly (P<0.05) with different hormone and doses. The higher percentage of fertilization rate (90%) was observed in WOVA-FH (0.5 ml per kg). Lowest fertilization rate (52%) was observed in Ovatide (0.7 ml per kg). Rath et al. (2007) and Motilan et al. (2014) also reported better performance of breeding of IMC and P. manipurensis while administrating WOVA-FH at 0.4-0.5 ml per kg of body weight. The egg output by the female also depends on the release of the gonadotropin releasing hormone which can be stimulated by synthetic inducing agents, which is evidenced by the several studies such as cherry barb (Sundarabarathy et al., 2004), P. manipurensis (Motilan et al., 2014) and O. belangeri (Das et al., 2016). In the present study dose of different hormone affected the rate of fertilization apparently. Inducement of hormone at higher and lower doses causes poor fertilization that may be due to early milting and late inducement in males, respectively (Pandey et al., 2002;Das et al., 2016).

O n l i n e F i r s t A r t i c l e
The higher percentage of hatching rate (88%) was observed in WOVA-FH (0.5 ml per kg) and lower percentage of hatching rate (42%) was observed in Ovatide (0.7 ml per kg). Further, results showed that hatching rate varied significantly with different hormone and dose administered. In a similar experiment, mature brooder was induced with three different dosages of ovaprim (0.4, 0.7 and 1.0 ml per kg) and it was found that Koi carp responds at all the three dosages but the best response was found at a higher dosage of 1.0 ml per kg (Ghosh et al., 2012). Koi brood fish injected with synthetic hormone ovaprim at 0.2 ml per kg for male and 0.5 ml per kg for female with aquatic macrophytes (Hydrilla verticillata) for attachment and development showed the number of ovulated eggs, fertilization and hatching rate as 100%, 75.2%, and 83.3%, respectively (Malik et al., 2014).
Several methods have been tried for induced spawning in fishes with various levels of success (Harvey and Hoar, 1979). Induced breeding of many Indian fishes was attempted by several workers however, the level of success varied based on the combination of the fish species and inducing agent (Ramaswamy and Sundaraj, 1969;JR et al., 1992;Alok et al., 1998). A study on different species of IMCs in comparison of spawning success with different inducing agents revealed that Catla catla responds more with Ovaprim, whereas, the results were better with Ovatide in L. rohita and Cirrhinus mrigala (Dhawan and Kamaldeep, 2004). Reddy and Mathur (2000) also reported similar results with ovatide in L. rohita and C. mrigala as compared to C. catla. Tiwana and Sudhanshu (2012) proved that Ovaprim gave 5.83 and 12.95% better performances over Ovatide and carp pituitary extract, respectively in terms of hatching rate of L. rohita eggs. The difference in effective dosage among different species was attributed to the varied levels of dopamine activity (Billard et al., 1983;Richard et al., 1986). Devi et al. (2009) attempted induced spawning and hatching of O. belangeri in its natural habitat using Ovatide (0.6 ml per kg for females and 0.3 ml per kg for males) and reported 95.0±1.05% Comparing all the parameters, among the synthetic inducing agents used WOVA-FH at 0.5 ml per kg performed better in terms of number of spawned eggs, fertilization and hatching rate. Hence WOVA-FH at 0.7 ml per kg is the standardized hormone with standardized dose for induced breeding of Koi carp.

Embryonic and larval development
Changes in anatomical or morphological structure gives importance to study the difference between embryonic, larval and post-larval development (Kovac, 2000). The term hatchling, larvae and post larvae are used to indicate different stages of development from onset of hatchling to fingerling stage (Boglinoe et al., 1992) and is further divided into six stages namely embryo, hatchling, larva, post-larva, fry and fingerling and each stage was characterized by typical anatomical and physiological features (Jhingran and Pullin, 1985). In the present study, spawning was noticed 6-7 h after the injection and the eggs were hatched 68-72 h after fertilization at a water temperature of 25-26 o C. The Koi carp eggs were spherical, demersal and adhesive in nature throughout their incubation period which made the observation of developmental stages more difficult. The diameter of the fertilized egg ranged between 0.9 and 1.10 mm while the yolk sphere ranged between 0.6 and 0.8 mm. The incubation period of eggs depends largely on water quality parameters such as salinity, temperature and pH. The embryonic developments were observed in three distinct phases viz., cleavage, embryonic and larval development stages (Tables V and VI). These were observed using trinocular microscope NLCD-120E, Lawrence and Mayo (Fig. 2).

Cleavage phase
Embryonic development lasted up to 12 h after fertilization of eggs. It consisted of the formation of perivitelline space, bipolar differentiation and first multiplication of cells, formation of blastula, early gastrula, late gastrula, beginning of epiboly, germ ring, and embryonic shield. The blastodisc was noticed within 30 mins after fertilization (Fig. 3B) and first two-cell division occurred in the animal pole (posterior side) at 0.55 h post-fertilization (h.p.f). The four-cell division occurred at 1.20 h.p.f. The embryo attained an 8-celled stage, 16 -celled stage, 32-celled stage, 64-celled stage and 128-blastomeres stages within 2.45 h.p.f . The size of the blastomere reduced and appeared as cluster of solid cells called the morula. The blastoderm covered almost 1/3 of the yolk sac and formed dome like structure at 4.25 h.p.f. and entered to early gastrula stage. Germ ring was completed and Blastoderm started enveloping 3/4 of the yolk sphere at 7.45 h.p.f and entered into late gastrula stage. Blastoderm completely covered the yolk plug and epiboly came to an end at11.40 h.p.f.

Embryonic phase
The initiation of the embryonic body (organogenesis) formation was observed when the blastomeres covered the whole yolk. The embryo reached 5 somite stage; the tailbud formed at the posterior end of the yolk mass. A pair of kidney shaped optic capsules was visible in the optic vesicle and the lens started to differentiate. As the embryo increased in size, anterior and posterior region became distinguishable as broader cephalic region with the distinct forehead and tail. When the embryo reached 10-12 somite stages,   occupied the entire space of the capsule, while pigmentation became more apparent during the pre-hatching stage. The wriggling movement of embryos was more visible at this stage, and tail wrapped completely around the egg. The eyes were pigmented. All the embryos were ready to hatch at this stage. The embryo had undergone rapid development and was clearly visible through the transparent egg membrane just prior to hatching with large eyes, and an orange yolk sac. After the 3 rd day of incubation (72 h), the embryo hatched out through the distal end of chorion by breaking the egg capsule with its active wriggling and the hatchlings emerged with tail first. The peak hatching took place between 06.00 to 08.00 h.

Larval metamorphosis
The newly hatched larvae measured 2.7-2.9 mm and were actively swimming near the corner of the water surface; devoid of mouth and anus. On the 3 rd day posthatch, the yolk sac was fully absorbed and the larvae started exogenous feeding. Jameson and Santhanam (1996) also reported that after 3 days, the larvae started exogenous feeding. During the 7 th day post-hatch yellow pigmentation appeared in the body and the mouth size was 1.26 mm. After the 21 st day post-hatch, various colour combinations appeared on the body. From the 35 th day post-hatch, the larvae were fed with GROWFIN feed of size 0.6mm.

CONCLUSION
Koi carp is a colourful species which has numerous strains and it is an economically valuable ornamental species in aquaculture. Availability of quality seed is the basic and critical component of successful aqua culture practices. Induced breeding paves the way for continuous supply of seeds throughout the year. Result of the present study indicates that using WOVA-FH hormone at 0.5 ml per kg of body weight is the ideal dosage for inducing Koi carp using RAS system. This developed induced breeding technology would be helpful in producing year-round seed production with sustainability. The detailed description of embryonic development of Koi carp in this study will serve as baseline information for ornamental hatcheries due to its aquaculture importance.