Heat Shock and Its Consequences on Early Life Performance of Stripped Catfis (Pangasianodon hypophthalmus)

Researchers have widely applied heat shock on zygote stadia of domesticated aquatic animals especially for the purpose of ploidy manipulation in fish to accelerate growth, intermediate products for the production of infertile seeds, providing prospective brood stock as well as the monosex population. This is certainly an interesting study to see how far the impact of heat shock on the early development stages. This experiment used a 42 C heat shock for 2.5 minutes on the zygote at different initial time i.e. 28.5; 29; 29.5; 30 and 30.5 minutes after fertilization. All treated larvae and control were reared in a 25 liters plastic container at density of 5 larvae per liter. The results showed that high percentage of fertilization rate were 92.61 % to 96.32 % and hatching rate was 85.33 % without any abnormalities were achieved in the control. While in all treatments had a very low hatching rate of 4.12 % to 8.93 % with relatively high abnormalities of 12.36 % to 27.60 % and a significant differences (P<0.05) compared to controls, but survival rate, specific growth rate and feed conversion ratio after 30 days rearing period showed no significant difference (P>0,05). In terms of seed production, the heat shock treatment will limit 78 % to 82 % of larvae production.


Introduction
Striped catfish is one of the most developed aquaculture commodities in Indonesia especially in Java and Sumatera with relatively high production level of 437,111 tons in 2016, and 2018 is targeted to reach 604,587 tons (KKP 2017). Thus making it as cultivated organism that has prospectively to be developed on a large scale. It is also one of the commodities used as raw material of the fillet industry with optimum size when harvest is 1.25 kg/ individual (Islami et al., 2014;Akter et al., 2014). This implies the length of time of cultivation and many feeds consumption, So it is necessary to improve the performance of growth and feed efficiency towards better, like improving genetic quality through heat shock technique.
The application of physical shock (temperature and pressure) as well as chemical in the manipulation of chromosome set were usually performed on the zygote stadia both at the time of meiosis II (shortly after fertilization) and before the first mitotic (first cleavage). The superiority of manipulation technique of chromosomes set in fish is to increase growth, feed efficiency, environmental resistance, and infertile fish production (Thorgaard 1983), have better texture and meat quality (Lefevre et al., 2015) and more appealing form (Kadi 2007). Another advantage of polyploid fish is that it generally does not cause a decrease in genome diversification as in plants (Zhan et al., 2014). Individual polyploids especially triploid and tetraploid are particularly adaptable and can be used as environmental control (control of growth) for other individuals (Kadi 2007).
Research on polyploid fish production has been done by researchers in various species with variations in yield. In general, the researchers have performed ploidy analysis and separated the organism (polyploid and diploid) to compare their growth rate. On the other hand, post-shock studies of larvae that are successfully hatched and have not been analyzed the level of their ploidies are not widely practiced. So that authors need to know how the impact of the heat shock on the growth performance of zygote striped catfish that successfully hatched before known the ploidy level of the larvae. However, this study is limited only to a heat shock trial on a zygote prior to the first mitotic.

Design of Experiments
The heat shock treatments were performed based on the best results of previous researchers in inducing triploid (Ibrahim et al., 2016) and tetraploid (Buulolo et al., 2017)

Heat shock treatment
Fertilization was conducted at 29 °C by mixing semen and ovulated eggs. Prior to first cleavage i.e. 28; 28.5; 29; 29,5; 30; 30,5 min after fertilization, the zygotes were shocked at 42 °C for 2.5 min by transferring into prepared water bath. After heat shocking 6800 treated eggs were incubated in aquaria at normal temperature of 28-30 °C until hatched (20-24 hours after fertilization).

Larvae rearing management
The larvae of treatments and control groups were reared in a 200 x 100 x 40 cm 3 fiber tank equipped with aeration system for seven days. The larvae were fed on Artemia naupli during first three days and mixed with tubificid worm during the rest four days.

Nursery management
Nursery management was conducted for 30 days using a 25 liters plastic container at density of 5 larvae per liter. The fish were fed on 26 % protein content commercial feed three times a day at 8 % feeding rate of total biomass. Sampling simultaneously for body weight and body length measurements every 10 days. During the nursing period, 70 % water exchange was conducted at the same time of sampling. In addition waste and feces were removed daily by syphoning the media culture to maintain an optimum condition. At the same time, dead fish were checked and collected for survival rate and feed efficiency analysis.

Data analysis
Collected data especially hatching rate, survival rate and abnormality were determined according to Effendie (2002); specific growth rate and feed conversion ratio were calculated referring to De Silva and Anderson (1995).Those data were than analyzed statistically by applying F-Test (Anova) with the best response at 95 % confidence range and the Tukey Multiple Range Test to compare the values between treatments. The software used was Minitab 16 th and Ms. Excel 2013.

Fertilization, hatching and survival rate
Observation on the zygotes at 7 hours after fertilization indicated that the ovulated eggs and sperm that used in the experiment have a good qualities, this can be interpreted from the high fertilization of 94.61 %. The percentage value was obtained from three replicates of observations using 218 samples as shown in Table I. Based on Table 2, the highest hatching rate of 86.09 % was obtained in control. While, very low hatching rate values with a range of 3.20 -7.50 % were obtained in all treatment groups. The very low hatching rate in these groups were strongly influenced by physical shocks as also reported by other researchers (Ding et al., (2007;Lebeda and Flajshans, 2015), resulting lethal embrios prior to hatching (Lagler et al., 1977). Furthermore, this phenomenon explains that the effects of physical and chemical Furthermore this phenomenon explains that the effects of physical and chemical shocks cause damage to the cytoplasm (Tave, 1993), as well as damage to the spindle fibers during cell division (Gill et al., 2016). Hersberger and Hostuttler (2005) mentioned that there were various result of physically induced zygote that can survive to hatch with low hatching rate of 8 -40 %. Moreover, Fetherman et al. (2016) state that hatching success depends on several factors including innitial time, duration, and temperature of shock, gamets quality, and egg incubation conditions. The results of statistical analysis for hatching rate showed that control were significantly different (P<0.05) when compared with all treatments but not significant different among treatments (P>0.05).
During 30 days of nurshing, it was seen that reared fries boths control and treatment groups had a good survival rate more than 85.33 ±3.06 %. However, statistical analysis showed that there is no significant effect (P>0.05) of heat shocks treatments on the survival rate (Table 2). This result is better when compared with previous experiments where many larvae failed to survive or dead prior to first feeding (Pandian and Vardaraj, 1987;Haniffa et al., 2004), or only a few larvae (0.2 -16.9 %) were survive (Hersberger and Hostuttler, 2005). According to Pandian and Koteeswaran (1998) the deaths of larvae due to physical shocks that occur in the early stages of rearing activity are caused by several factors: mosaic chromosomes, aneuploid, cell shrinkage, high cytological process failure and homozigosity.  Table 3 shows that larvae abnormalities were found in all heat shock treatments but it was not found in control. The heat shock of 42 0 C for 2.5 minutes at innitial time of 30.5 and 30 minutes after fertilization were the treatments that tend caused the higher abnormality i.e. 20.96 % and 27.60 %. While the other heat shock treatments were caused lower abnormality ranged between 12.36 % -15.51 %, however in control was not found any abnormal larvae. Nevertheless, the statistical test showed no significant effect (P>0.05) on abnormality. Higher abnormalities were observed by Mukti (2005) on carp (Cyprinus carpio Linn) treated with heat shock before the first mitosis resulted in an abnormality of 24.86 ±8,37 %.

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Omni-Akuatika Vol. 14 No. 2 July 2018 : 52 -58  12.49 ±3.80 12.89 ±16.14 15.51 ±4.63 12.36 ±7.61 27.60 ±14.18 20.96 ±14.01 0.00 Observations on abnormal larvae that most of dead occured post hatching and only a few can survived until the end of the experiment as shown in Figure 1. Characteristic of abnormal larvae that was found in this experiment were varied such as spinal bent (Figure 1), curved tail and unproportional body shape. According to Haniffa et al. (2004) and Varadaraj and Pandian (1989) some eggs that hatched after being given a heat shock treatment show morphological abnormalities such as short and curved tails, egg yolk deformities, spinal deformities and head shape abnormalities. Furthermore, Carman (1992) states that the heat shock given in the early phases of embryonic development can cause damage on embryonic membrane so that it can produce abnormal individuals.

Specific growth rate (SGR)
Observation on both body length and body weight showed that SGR of treatment groups tend higher than control. Lenght SGR and weight SGR in treatment groups were 5.60 % -6.56 % and 15.82 % -18.62 % respectively. While in control were 4,58 % and 13,70 % (Table 4). Nevertless, statistical analysis indicated that heat shock treatments had no significant effect on both lenght and weight specific growth rate (P>0.05).  Don and Avtalion, (1988) in blue tilapia that applied physical shock treatment caused growth retardation. Similarly, Sparrow (1979) in Kadi (2007) that organisms from mechanical shocks before the first mitotic cause growth delays compared with no shock. Not only in fish, in some plants according to Sleper et al., (2006) explains that this no-different growth phenomenon caused by shock only increases the size of the merismatic cells, but the total number of cells does not increase. However, in common carp (C. carpio Linn) according to Mukti et al. (2005) that heat shock applied before the first mitosis can increase the fish growth.

Feed conversion ratio
Calculation of feed conversion ratio based on 10 days sequencial sampling are presented in Table 5. The data show that feed conversion ratio values in heat shock treatment groups varied between 1.17 -1.26 and control of 1.47. However, there is no significant differences between treatment groups and control (P>0.05). This result is no different from that obtained by Handayani et al. (2014) in djambal fish fed with commercial, giving the best feed conversion value of 1.15. Moreover, higher feed conversion results were obtained by Da et al. (2016) using various compositions of feed and commercial feed as a benchmark for striped catfish ranging from 1.42 ±0.2 to 2.10 ±0.1. When compared with the results of previous research, the value of feed conversion in control and heat shock treatments is still quite good and in the normal range for the growth of striped catfish. This results indicated that physically heat shock treatments did not affect on feed efficiency. This phenomenon is same as the statement of Chourrout et al. (1986) and Cui et al. (2013) that heat shock that applied before first mitosis tends to lead to poor growth both in fish and shellfish (Dunham 2004). According to Nordrum et al. (2000) both growth performance and FCR of aquatic organisms are strongly influenced by the performance of the intestines in utilizing the substances of feed source including the presence of anti-nutrients in the feed that was given. Furthermore, Da et al. (2016) adds that the presence of low amino acids in feed also has a significant effect on growth performance and FCR. However, there are many other factors such as age, species, maintenance quality of water as media and genetic aspect that also affect the FCR performance.

Conclusions
There is no evidence that heat shock treatment before the first mitosis affects specific growth rate and feed conversion ratio but has a major impact on zygote survival and abnormalities in hatched stripped catfish larvae.