Improvement of broodstock and seed production technologies Quality seed for sustainable aquaculture

seahorses. Trials on the acclimation of juvenile seahorses are currently conducted in hanging net cages prior to their release in the pilot site. Information, education and communication activities were conducted to promote the conservation and protection of seahorses through the conduct of lectures in schools, distribution of posters, and conduct of interviews with the local community.

One of the most important missions of AQD is to transfer the latest technologies and information on aquaculture to the ASEAN Member States through training courses. Two training programs: Marine Fish Hatchery Training Program and Rural Aquaculture Program were carried out under JTF6. The training on marine fish hatchery which includes breeding, hatchery and seed production, nutrition and health management of grouper, sea bass, and snapper, was conducted in 2018 with 16 participants, four of whom received funding through the JTF. The rural aquaculture program includes a 10-day training focusing on the promotion of community-based freshwater aquaculture for remote rural areas of Southeast Asia, which was conducted before the end of 2018 at AQD’s Binangonan Freshwater Station.

2.3 Improvement of broodstock and seed production technologies

and survival during hatchery phase, no statistically significant stock differences were noted in all the parameters, e.g. genetic variability, growth and reproductive performance. This could mean that wild-sourced abalones from any of the studied sites could be used for local aquaculture. However, to maintain the genetic integrity of wild abalone populations, abalone seedstock production for re-seeding natural populations should use breeders from areas near the restocking sites. Finally, to maintain good quality in hatchery produced seedstock, regular monitoring of the growth performance should be made on the seedstock when reared in the grow out phase since the growth of hatchery-bred stocks appeared to be poor compared to those bred from newly-acquired wild parents. As such, broodstock management through replenishment or replacement of breeders should be made when growth performance is noted to decline.

In previous studies on abalone given formulated diets with varying ratios of protein and energy levels, the broodstock had shown markedly improved fecundity, length of conditioning period, spawning frequency, and egg hatchability when given the diets at higher protein and energy level ratios (up to 42% protein/3750 kcal kg^-1 energy) compared to those given natural food (18% protein/2200 kcal kg^-1 energy). However, the effect of the formulated diets on larval competency in terms of settlement success and post-larval survivorship has not been monitored. The study in 2018 is therefore aimed at verifying the two best performing diets (protein/energy level ratios of 37% /3570 kcal kg^-1 and 42% /3750 kcal kg^-1) on a longer period involving higher number of animals to determine whether further refinements are necessary or if the diets are ready for commercial application. In addition, monitoring of the parameters for larval quality including larval growth, development and survival would also be carried out to complete the existing information. Potential broodstock from Palawan (PA) and Sagay in Negros Occidental (SY) are being grown to reach mean SL of five-six cm, to be used in the verification study. These will be conditioned for two months until these are ready to spawn.

Feed ingredients have been procured and will be prepared two weeks before the feeding trials while gonad maturation will be monitored and the breeding performance as well as larval quality will be evaluated.

Genetic characterization of mangrove crab Scylla serrata stocks based on three novel and three existing short tandem repeat (STR) markers was also carried out to maintain high genetic variability and to check for the negative impacts of domestication in several generations of selected and control stocks from Camarines and Surigao. Thus far, raw data based on uncorrected estimates of the number of alleles (A) showed that the parental stocks from Camarines and Surigao had 10.17 and 10.33 number of alleles, respectively while two batches of the first generation Camarines control stocks had lower alleles at 6.67 and 6.50, respectively, and one batch of the first generation Surigao control stock had A = 6.33. Slightly lower A estimates were noted in the first generation selected stocks from Camarines (6.17 and 6.50) and Surigao (4.0). Expected heterozygosity estimates were not significantly different between the stocks and across generations based on the existing batches that were screened.

Molecular marker data were correlated with the parameters for selected beneficial traits to determine if the markers could be used as preliminary indicators of genetic improvement.

Collection of the last batch of samples from parental, selected, and control batches was completed in 2017. The results of this study, which was extended until the middle of 2018, are currently being consolidated in an article for submission to a scientific journal.

A study of the Philippine native Clariid catfishes (mainly Clariid macrocephalus and C.

batrachus) that focuses on broodstock development and management was initiated in 2018.

Renewed interest of stakeholders on the propagation of the native catfishes is due to the fact that these commodities are disease resistant, can be stocked at high densities, and thrive in areas where water quality is not optimal. As such, the native catfish could be an ideal culture species especially at times when climate change seems to pose numerous challenges to freshwater fish farming. In 2018, stocks from Zambales, Quezon, and Iloilo, were collected for broodstock development and broodstock diet experiments. The stocks were maintained and bred through induced spawning to comprise the founder stocks. Offspring with known ages produced from each stock are now being reared for stock evaluation in 2019 and comparison by looking at possible differences in breeding efficiency and response to broodstock diets. The broodstock diets that have been tested initially on the Zambales founder stocks contained 0.5% mango peel, 0.5% paprika and a combination of both. Preliminary results using mature C. batrachus from Iba, Zambales showed higher relative fecundity (28.3 and 28.6 eggs/g body weight in female, respectively) from those given feeds containing either 0.5% mango peel or the combination of mango peel and paprika. However, hatching rate was noted to be higher (86.3%) in the treatment fed the diet with paprika alone.

In the study on the reproductive performance of captive silver therapon (Leiopotherapon plumbeus) broodstock, three broodstock diets with varying levels of dietary protein (30%

or 30CP; 40% or 40CP; and 50% or 50CP) were formulated to examine the effect of artificial diets on the broodstock. Another set up was established to assess the performance of captive broodstock fed test diet with 50CP and reared in a biofloc technology (BFT) condition.

Broodstock diets were supplemented with dried earthworm meal at 4-12 g/100 g diet. The initial results have shown that all females in the 40CP and 50CP groups spawned, whereas only 89% and 78% of females spawned in the BFT for 50CP and 30CP groups. Gonadosomatic index (GSI) was highest in female broodstock in the 50CP group (12.53), but were comparable with other treatment groups. Male broodstock in the 40CP group showed the highest GSI (9.48) among all the treatment groups. Relative fecundity (313-390 eggs per gram female), fertilization (68-75%) and hatching rates (48-90%) increased with increasing dietary protein level. High larval production (418-421 larvae per gram female) was observed in the 40CP and 50CP groups.

Effect of dietary protein levels on growth, fecundity and egg quality in female silver

therapon broodstock

For better understanding of the conditions that encourage and facilitate mating in the tiger shrimp Penaeus monodon, experiments that aim to determine the differences and problems in breeding performance of male and female spawners from captive and wild environments – stocked separately and/or their combination as mates – were conducted.

Video documentation was made on the reproductive behavior, including the pursuit of females by males and vice versa, and number of mating episodes of adult P. monodon during trials separately exposing spawners to varying depth and temperature. From the video recordings, wild males were observed to spend more time near wild females compared to captive female broodstock. Likewise, wild male pursued the molted female broodstock more frequently compared to the captive males. In terms of molting time and duration of the female shrimp to exit the molt, wild female shrimp was observed to molt earlier compared to captive shrimp. The recorded time, in seconds, in which the females exit the molt was similar for both wild and captive females. The male shrimp touched the discarded female molt shell or stayed within the area of the molt shell for some time. In some of the video recordings, it can be seen that the male shrimp ate the female molted shell or the male killed or ate the newly-molted female. Molting was not observed in the captive female and wild male combination and mating was not observed for the different trial combinations. Histological and gonad morphology evaluation of the captive and wild male breeders indicated that the gonad of wild male is slightly opaque white in color compared to that of the captive male, which is translucent in color. The experiment using a hormone methyl farnesoate to induce female crustacean molting and reproduction, will be continued in 2019.

Activities were implemented in 2018 on the development of technologies for giant grouper aquaculture. Giant groupers have been observed to directly undergo male sexual maturity contrary to the general idea that groupers are protogynous hermaphrodites wherein all individuals mature first as female and then reverse to male. Furthermore, females were noted to mature at 22-25 kg body weight (BW) and 95-99 cm total length (TL) while males mature at 15-20 kg BW and 81-92 cm TL. The reproductive cycle of giant groupers peaks during full moon, thus, induced spawning activity is best performed at this time. Spontaneous spawning of giant groupers in the floating sea cage was achieved through hormonal manipulation using slow-release Gonadotropin-releasing hormone (GnRH) implant and human chorionic gonadotropin (hCG) injection but fertilization rates have been highly variable. Treatment with hCG (500 IU/kg BW) was not effective for induced spawning of the giant groupers.

Spermiation was induced in males through treatment of hCG (1000 IU/kg BW) although better results could be obtained when slow release GnRH pellet (60-80μg/kg BW) was implanted 48 hours prior to hCG treatment. For females, GnRH treatment alone is effective in inducing final oocyte maturation and ovulation. Hence, timing of GnRH and hCG treatment can be modified

Wild male (M) chasing molted wild female (F) shrimp

between the two sexes so that final maturation of female and male gamete would occur at the same time, either for spontaneous spawning or stripping and artificial fertilization.

This study likewise looked at the appropriate larval food for giant groupers. Inclusion of the rotifer Proales during the first 10 days of rearing results in significantly higher larval survival rate. Recently, giant grouper larvae were reared in three-ton circular tanks and Proales- supplement was given starting day 1 and Brachionus was fed on day 2 and onwards. Spines were observed at day 16, however, only the shooters metamorphosed to juvenile stage.

Given Proales’ benthic nature, the increased frequency of direct feeding and the utilization of dripping method yielded successful rearing (day 35 survival rate: 0.3%) compared to the trials previously done in giant groupers with no survival at day 35. As of December 2018, the three-month-old giant groupers have been stocked in one-ton fiberglass tank at AQD’s Marine Finfish Hatchery.

Another component of the study is focused on sperm cryopreservation to prolong the viability of grouper sperm through cryopreservation in liquid nitrogen or in a -80°C biofreezer.

In terms of motility, the tiger grouper sperm is better retained in cryopreserved sperm while long-term fertilization capacity was only confirmed in giant groupers. Generally, viability remains despite a total loss in sperm motility. Initial results in evaluating the suitability of Ficoll 70 as additive to the MPRS-DMSO (9:1 v/v) sperm extender at -80°C storage showed a dose-dependent effect in terms of preserving viability. However, addition of Ficoll 70 does not positively affect motility conservation, which only lasted up to one month of cryopreservation.

Further trials tested the effects of the extender pH (5.0, 6.0, 7.0, 8.0) on sperm motility for sperms stored at 4°C. For giant groupers, highest motility was observed in the treatment with pH 7 followed by pH 6. A 15-day refrigerated storage was the maximum duration resulting in observable motility.

Identification of the critical stage during early development of Marphysa mossambica was carried out in 2018 by refining the rearing and feeding techniques for its mass production. A significant decrease in the survival of the polychaetes after 45 days of rearing indicated that the extension of rearing period on biofloc from one month to another 15 days is no longer favorable. At this period, it is probable that polychaetes already need suitable substrate (mud) and food (formulated diet) for further development until reaching the adult stage. This is therefore a critical stage for the polychaetes as it is a transition period from early juvenile to juvenile stage. Moreover, the egg hatchability and larval development of M. mossambica subjected to varying irradiance and photoperiod treatments have also been investigated.

Results showed that survival of polychaetes were significantly low in one-hour, three-hour, six-hour photoperiods of 747 lux and 1,747 lux compared to no light treatment. The no light treatment had the highest survival but statistically similar to the three-hour and six-hour photoperiods of 42 lux light intensity. In determining their optimum stocking density and sediment depth requirement during their nursery and grow-out phases, polychaetes were stocked at densities of 20 (10,000/m²), 40 (20,000/m²), 30 (30,000/m²), and 80 (40,000/m²) individuals per container with four replicates, each containing 200 ml of seawater. Replicate containers of each treatment were added with one gram biofloc (fresh) at the start of the experiment. Although not significant, the survival of Marphysa sp. declined sharply at a stocking density of 40,000 trochophores/m² (18 + 3%). In terms of growth, density of 10,000 trochophores/m² gave the highest number of segments (21 + 1) than the other stocking density treatments (P < 0.05).

o Refining hatchery and nursery management protocols to improve seedstock production

The feasibility of nursing Philippine native glass eels in captivity was initiated by AQD in late 2017 and continued in 2018, while the appropriate rearing protocol of this fish species is being developed by providing suitable feeding scheme and/or formulated diets for nursing the glass eels and young elvers. Moreover, identification of anguillid eels based on morphological and genetic characterization and potential pathogens in nursery eel systems was also carried out.

In the initial feeding trials of glass eel samples from Aparri, Cagayan, conducted for 24 weeks with six treatments (Table 1), results showed that the glass eels in Treatment II had the highest weight gain of 3,679% and survival was also highest at 77%. The lowest survival was observed in Treatment III at 2%, while glass eels in Treatment IV did not survive beyond week 16.

Table 1. Diet used in initial feeding trials of glass eel samples

Weaning trials were also conducted in 2018 where the glass eels (0.11-0.15 g initial body weight [BW]) from General Santos City in Mindanao were fed Tubifex sp. (50% BW) alone until week 2, tuna eggs (7% BW) and Tubifex sp. (37.5% BW) from week 2 to 8, and formulated diets (1.5 to 3% BW) and Tubifex sp. (from 25% to 12.5% BW) from week 8 to 22. Formulated diets were moist (Treatment I, MF), semi-moist (Treatment II, SMF) and dry feeds (Treatment III, DF). Results showed that Treatment II (SMF) had the highest weight gain of 1171%. Survival after 22 weeks of rearing was highest in Treatment III (MF) at 89% and lowest in Treatment II (SMF) with only 84%. The weaning duration was also determined by analyzing their growth in terms of final body weight, percent weight gain and specific growth rate. Results indicated that growth was significantly higher in Tubifex-fed glass eels (control group) compared to those gradually weaned onto paste diet for 28 and 42 days. No significant difference was observed in survival rates of eel groups gradually weaned onto paste diet and that of the control group.

Meanwhile, glass eels pre-sorted and identified as Anguilla bicolor pacifica from Mindanao were stocked at 1.96 individuals per liter in nine, 500-L polyethylene tanks with 250 L low saline water at 3 ppt. Glass eels were fed Tubifex sp. four times (0900, 1100, 1300, and 1500 h) daily at 50% of estimated body weight. Tubifex sp. were placed in plastic baskets which served as feeding and resting stations for the eels. After two weeks, survival rates ranged from 93.3% to 99.8%.

The stocks used for the feeding trials which came from batches of glass eels collected in 2017 and 2018 were morphologically and genetically identified. Analysis of the 2017 Aparri samples enabled the identification of 77 pieces of Anguilla luzonensis among the stock, aside

Treatment Diet

I Artemia nauplii II Tubifex sp.

III Formulated Diet (FD; 52% crude protein [CP]; 10% crude fat[CFat]) IV Commercial Diet (CD; 47% CP; 6% CFat)

V FD + Artemia VI FD + Tubifex

from 19 tails of A. marmorata. Based on cytB sequence alignments, the samples from General Santos City in Mindanao were composed of 95 A. marmorata and one A. bicolor pacifica. The genetic characterization and identification of the 2018 samples (comprising samples used in selected nursery farms and wild sourced eels) were carried out at the Onagawa Field Science Center of Tohoku University, Japan from 30 November to 14 December 2018. Analysis on the DNA barcoding data using cytB is ongoing, although another COI marker was tried but with little success. Apart from mtDNA sequence analysis, seven microsatellite primers used in Anguillid species were successfully tried on the Philippine Anguillid eel samples and the protocols for cross-amplification and microsatellite analysis were optimized.

The glass eels and rearing water from surveyed eel nursery farms were monitored for the presence of pathogens, and bacterial analyses were conducted by making use of serial dilution and plating method. Results showed that Trichodina and monogenetic trematodes were found in the eel samples, of which the highest prevalence of Trichodina was found in a Zambales farms at 90%, which also had the highest intensity as well as mean abundance. In the case of monegeneans, the incidence with 50% prevalence was found only in the Zambales farm.

For the hatchery rearing of mangrove crab seedstock, the use of algal paste in rotifer cultures for mangrove crab seed production was evaluated. Initially, Nanochlorum paste was used in rotifer culture but Tetraselmis paste proved to be a better option. Hence, further establishment of protocol for the use of Tetraselmis paste was done to improve the growth and density of rotifer compared to the live Nanochlorum batch culture. The results indicated that algal paste acclimated and activated for four hours prior to feeding to rotifers gave better results than feeding rotifers with Tetraselmis batch culture. An assessment comparing the commercially available algal paste used as feed to rotifers with the paste made in AQD through electrolytic flocculation of the live algae from batch culture, in terms of the performance of mangrove crab larvae fed with rotifers is being assessed.

Genetic characterization and identification of samples carried out

at Onagawa Field Science Center

Survival and growth of mangrove crab larvae with rotifers fed with Tetraselmis algal paste and control or Nanochlorum batch culture