Shaowu Ll，Hongbai LlP

Heilongjiang River Fisheries Research lnstitute/Chinese AcademY of FisherY Sciences，Harbin 150070，China

Screening of SaProlegniasis Preventive Drugs for Golden Trout（Oncorhynchus mykiss）Fertilized Eggs During Hatching

Shaowu Ll，Hongbai LlP*

Heilongjiang River Fisheries Research lnstitute/Chinese AcademY of FisherY Sciences，Harbin 150070，China

ln order to screen the effective preventive drugs for saprolegniasis occurred during hatching of the fertilized eggs of golden trout，effects of two kinds of self-made Chinese herbal compounds （A&B）and two kinds of chemical bactericides（peroxYacetic acid andhYdrogen peroxide，C&D）with three disinfection concentrations were compared in this studY.The fertilized eggs of golden trout were randomlY divided into 13 groups，each group with two parallels of 1 000 eggs.Drugs at high，medium and low concentration were respectivelY used to immerse the fertilized eggs 35 min for disinfection once a daY.The numbers of live and dead eggs were recorded each daY before disinfection and the numbers of survival eYed eggs were counted in each experimental group after finishing the whole test.The results indicated that all of four kinds of drugs used here had a certain effect on prevention and control of saprolegniasis during hatching of fertilized eggs of golden trout，and also showed a correlation between the concentrations and effects.After the selection of several drugs，we found that group B-medium，C-medium and D-high exhibited the most remarkable effects on control of saprolegniasis.However，careful consideration should be given to chemical drugs residues and their potential hazard to the eYed eggs.Therefore，Chinese herbal compound B was suggested to be used for prevention and control of saprolegniasis to achieve the healthY aquaculture.And the concentration and operation time of Chinese herbal compound B should be strictlY controlled when used in disinfection of fertilized eggs.

Golden trout （Oncorhynchus mykiss）；Fertilized eggs；Saprolegniasis；Preventive drugs

O ncorhynchus mykiss， also named as the rainbow trout or golden trout［1］，is a species of salmonid native to cold-water tributaries of the Pacific Ocean in Asia and North America［2］.TheY are stream lined，with 8 to 12 spines in the anal fin and lack teeth at the base of the tongue in general［3］.Since there is no cholesterol in the trout muscle，the golden trout is of high nutritional value［4］.Because theY have to live in an extremelY clean water environment，theY are considered as natural green marine products.At present，the cultivation of Oncorhynchus mykiss has been promoted to more than 20 provinces such as Qinghai，Xinjiang and Liaoning，and so on.The cultivation of golden trout has become the second essential species to raise，onlY after Oncorhynchus mykiss［5］.

Saprolegniasis is of stramenopila，oomYcetes，saprolegniales and sprolegniaceae［6］.Reports have pointed out that the saprolegina parasitica，S.ferax and S.diclina are dangerous pathogenic bacteria that can infect all fresh water fisheries［7-9］and their eggs［10］.Saprolegniasis spreads widelY［11］，and fishes can be infected throughout the Year，especiallY the late winter and earlY spring，which brings about huge economic loss to the freshwater aquaculture，and seriouslY threatens the sustainable development of freshwater aquaculture［12］.

ln order to screen the effectivepreventive drugs forsaprolegniasis occurred during hatching of the fertilized eggs of golden trout，effects of two kinds of self-made Chinese herbal compounds （A&B）and two kinds of chemicalbactericides （peroxYacetic acid andhYdrogen peroxide，C&D）with three disinfection concentrations were compared in this studY.The purpose of this studY is to provide scientific statistics for the prevention of saprolegniasis.

Materials and Methods

Fertilized eggs and incubation condition

The hatching of the fertilized eggs of golden trout is carried out in the fisherY experiment station of Heilongjiang River Fisheries Research lnstitute，Chinese AcademY of FisherY Sciences.The fertilized eggs of golden trout were randomlY divided into 13 groups，each group with 2 parallels of 1000 eggs.

The fertilized eggs were put into the horizontal tank for incubation，and during the incubation period，the average water temperature was 10.3℃and the dissolved oxYgen was 6.0 to 9.0 mg/L.

PreParation of Chinese herbal com-Pounds and its agents

Two Chinese herbal compounds were selected forthis experiment，namelY Compound A （composed bY radix sophorae flavescentis，Chinese gall，and pseudolarix）and Compound B （composed bY agastache rugosus，rhizoma coptidis，pericarpium granati）. Those Chinese herbalcompounds were purchased from the ShiYitang pharmaceutical shop in Harbin.

The Chinese herbal compound extracted its effective components in two forms，alcohol leaching and aqueous extraction method.At first，the compound medicine was put into the 80%ethanol for 30 minutes.After that，the filtered medicine was extracted again bY the traditional pot.The 1 ml extracted medical solution was as effective as 1 g raw compound［13］.The alcohol fluid and water extract were put together in the proportion of 1:1.

Two kinds of chemical bactericides（peroxYacetic acid andhYdrogen peroxide）were used in this experiment，and was marked as C and D.

Disinfection of fertilized eggs and calculation

The prepared Chinese herbal compound and chemical bactericides were injected into the fertilization bucket before mixing with flowing water for 1.5 min，and then was soaked for 35 min.The fertilized eggs of golden trout were randomlY divided into 13 groups，each group with two parallels of 1 000 eggs.Drugs at high，medium and low concentration were respectivelY used to immerse the fertilized eggs 35 min for disinfection once a daY （Ahigh-low，Bhigh-low，Chigh-low，Dhigh-low）.The high，medium and low disinfection concentration of A and B was 3，1 and 0.35 g/L，and the high，medium and low disinfection concentration of C and D was 2.5，0.5 and 0.1 g/L and 1.2，0.6 and 0.3 g/L.

The numbers of live and dead eggs were recorded each daY before disinfection and the numbers of survival eYed eggs were counted in each experimental group after finishing the whole test.

The survivaleYed eggsratio，dead eggs ratio，the ratio of dead eggs caused bY saprolegniasis and the ratio of dead eggs caused bY other reasons were the proportion of the number of survival eYed eggs，deed eggs，dead eggs caused bY saprolegniasis and dead eggs caused bY other reasons to the total number of fertilized eggs.

Results and Analyses

Disinfection result of Chinese herbal comPound A

According to the methods mentioned above，we disinfected the fish eggs at a specific time everY daY，and recorded the status and number of fish eggs.The statistics of Chinese herbal compound A was shown in Fig.1.

According to Fig.1，compared with the control，the eYed eggs and dead eggs of the Ahigh-lowgroup did not exhibit significant differences to the control group，but the eYed eggs ratio ofAhigh-lowincreased，as Ahigh＞Amedium＞Alow＞the control.The ratio of dead eggs caused bY saprolegniasis was sequenced as the control＞ Alow＞Ahigh＞Amedium，and Ahigh was similar to Amedium（40.98% and 40.31%respectivelY）.

Disinfection of Chinese herbal comPound B

According to the methods mentioned above，we disinfected the fish eggs at a specific time everY daY，and recorded the status and number of fish eggs.The statistics of Chinese herbal compound B was shown in Fig.2.

According to Fig.2，compared with the control，the Chinese herbal compound B can effectivelY lower the ratio of dead eggs caused bY saprolegniasis，and its disinfection result was obvious.The saprolegniasis prevention effect of Bhighwas the best，and the eYed eggs ratio of Bmediumwas the highest.The eYed eggs ratio was sequenced as the Bmedium＞ Bhigh＞Blow＞control，and the ratio of dead eggs caused bY saprolegniasis was ranked as the control＞Blow＞Bmedium＞Bhigh.The ratio of dead eggs caused bY saprolegniasis and the ratio of dead eggs caused bY other reasons in the control group were 56.28%and 21.11%，whilethe ratio of dead eggs caused bY saprolegniasis of the Bhighgroup was 24.68%，which was significantlY lower than the control group.The dead eggs caused bY saprolegniasis of the Bhighgroup onlY accounted for 39.72%of the number of total dead eggs，which was significantlY lower than the control group 72.72%.The eYed eggs ratio of the Bmediumgroup was 45.60%，which was dramaticallY higher than the control group 22.61%.

The disinfection result of Peracetic acid

According to the methods mentioned above，we disinfected the fish eggs at a specific time everY daY，and recorded the status and number of fish eggs.The statistics of peracetic acid was shown in Fig.3.

According to Fig.3，compared with the control，the eYed eggs and dead eggs of the high，medium and low concentration of peracetic acid C differed significantlY.However，the ratio of eYed eggs of Chigh-lowwas significantlY higher than the control group，but the percentage caused bY saprolegniasis was lower than the control group.The eYed eggs ratio of Cmediumwas 52.58%，remarkablY higher than the control group.The ratio of dead eggs caused bY saprolegniasis was sequenced as the control＞Cmedium＞Clow＞Chigh.lt is worth noticing that the ratio of dead eggs caused bY other reasons declined significantlY，bY 9.14% and 16.22%，respectivelY，both of which where lower than the control group 21.11%.

The disinfection result of hydrogen Peroxide

According to the methods mentioned above，we disinfected the fish eggs at a specific time everY daY，and recorded the status and number of fish eggs.The statistics of hYdrogen peroxide was shown in Fig.4.

According to Fig.4，compared with the control，the eYed eggs of the high，medium and low concentration of hY-drogen peroxide D were higher than the control group，but the dead eggs caused bY saprolegniasis was lower than the control group.The ratio of eYed eggs of Dhighreached 55.75%，while the ratio of dead eggs caused bY saprolegniasis was onlY 20.11%.The eYed eggs ratio was sequenced as Dhigh＞Dmedium＞Dlow＞the control.The ratio of dead eggs caused bY saprolegniasis was as the control＞Dlow＞Dmedium＞Dhigh.The dead eggs ratio caused bY saprolegnia and the ratio of dead eggs caused bY other reasons accounted for 56.28%and 21.11%，while the dead eggs ratio of saprolegniasis was obviouslY lower than the control group.

Discussions

ln the earlYproduction stage，malachite green is often applied as the medicine to prevent fish eggs from infecting saprolegniasis，since it is cheap and effective［14］.However，the residual of malachite green is difficult to remove and it is verY likelY to result in abnormal fishes.China has listed malachite green into the List of lnhibited Animal Nedicine and its compounds［15］in NaY，2002.FormaldehYde was also listed as one of the medicine that cannot be used［16］.Researchers have later studied the performance of iodophor，saline solution，and heavY metal ion，and the Chinese herbal compound caught their attention.As an effective and environmentallY-friendlY medicine，Chinese herbalcompounddidnot have anY side effects［17］.

Prevention and control of saProlegniasis during the hatching of fertilized eggs of golden trout by Chinese herbal comPound

Chinese herbal compound is effective and affordable，as well as cheap［18］.ln recent Years，experts have started to select effective Chinese herbal compounds and found that the oil of litsea cubeba［19］，atractYlodes rhizome［20］，chinaberrY［16］and rhizome coptidis can inhibitthe growth of saprolegnia.However，there are not anY reports about the effects of Chinese herbal compounds on saprolegniasis during the hatching of fertilized eggs.

The two kinds of Chinese herbal compounds chosen in this experiment can effectivelY increase the ratio of eYed eggs and bring the number of dead eggs caused bY saprolegniasis under control，but the ratio of dead eggs caused bY other reasons of both kinds of compounds was consistent with the control group，even slightlY higher.The possible reasons need further investigation.

Prevention and control of saProlegniasis during the hatching of fertilized eggs of golden trout by chemical Pesticides

Gao Peng et al found that besides formaldehYde， potassium permanganate， trichloroisocYanuric acid，Benzalkonium Bromide Solution，PolY VinYl PYrrolidone，and glutaric dialdehYde also have effects on the prevention of saprolegniasis［22］.Zhang Shiqi et al.also pointed out the Kathon biocide and ethYlparaben reached a certain concentration，and can prevent the bioactivitY of 80%saprolegniasis.The prevention concentration of calcium propionate，potassium sorbate，Sodium diacetate，Sodium dehYdroacetate was much higher than the applicable concentration in actual production［23］.

This experiment suggested that the peracetic acid and hYdrogen peroxide can effectivelY prevent and control the saprolegniasis during the hatching of fertilized eggs of goldentrout，and increase the eYed eggs ratio.

Effects of different disinfection concentration on the Prevention results

All of four kinds of drugs used here had a certain effect on prevention and control of saprolegniasis during hatching of fertilized eggs of golden trout，and also showed a correlation between the concentrations and effects.

ln terms of the Chinese herbal compound，the ratio of dead eggs caused bY saprolegniasis rose graduallY along with the decreasing of inhibition concentration，which lowered the eYed eggs ratio and increased the total death ratio.That was to saY the Chinese herbal compound can be effective onlY if it reached certain disinfection concentration，but the high concentration maY also lead to the increase of the dead eggs ratio caused bY other reasons （Such as Bhigh group）.Therefore，choosing the effective Chinese herbal compound，and determining the appropriate disinfection concentration and time were the keY to guarantee the prevention results.ln this experiment，the data of Bmedium group were suitable，and can be practiced in future.

Both peracetic acid and hYdrogen peroxide also showed a correlation between the concentrations and effects，but there were also special effects.As for the Chighgroup，although the dead eggs ratio caused bY saprolegniasis was lower than that of Cmediumand Clow，the ratio caused bY other factors was higher than other group，which gave rise to low eYed eggs ratio.With regard to the Dhighgroup，although the percentage of eYed eggs was high，and the percentage of saprolegniasis infection was low，the ratio of dead eggs caused bY other reasons was higher than the control group.The influence of chemical pesticides exerted significant effects on the growth of fertilized eggs，and there were reports about abnormal fish.Therefore，it is necessarY to make medical plan cautiouslY when using such medicine.

Conclusions

ln order to screen the effective preventive drugsforsaprolegniasis occurred during hatching of the fertilized eggs of golden trout，effects of two kinds of self-made Chinese herbal compounds （A&B）and two kinds of chemicalbactericides （peroxYacetic acid andhYdrogen peroxide，C&D）with three disinfection concentrations were compared in this studY.The results indicated that all of four kinds of drugs used here had a certain effect on prevention and control of saprolegniasis during hatching of fertilized eggs of golden trout，and also showed a correlation between the concentrations and effects.After the selection of several drugs，we found that group B-medium，C-medium and D-high exhibited the most remarkable effects on control of saprolegniasis.However，careful consideration should be given to chemical drugs residues and their potential hazard to the eYed eggs. Therefore，Chinese herbal compound B was suggested to be used for prevention and control of saprolegniasis to achieve the healthY aquaculture. And the concentration and operation time of Chinese herbal compound B should be strictlY controlled when used in disinfection of fertilized eggs.

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金鱒受精卵孵化期間水霉病防治藥物的篩選

李紹戊，劉紅柏*（中國水產(chǎn)科學(xué)研究院/黑龍江水產(chǎn)研究所，黑龍江哈爾濱150070）

為了篩選金鱒受精卵孵化期間水霉感染的有效防治藥物，比較研究了2種自組中藥復(fù)方（A和B）及2種化學(xué)除菌劑（過氧乙酸和過氧化氫，C和D）3種消毒濃度下的作用效果。將金鱒發(fā)眼卵隨機(jī)分為13組，每組2個平行，每個平行1 000粒卵，選取適當(dāng)?shù)母摺⒅?、低濃度對魚卵進(jìn)行浸泡消毒，每天1次，每次持續(xù)35 min，每日消毒前進(jìn)行活卵、死卵（霉菌感染死亡及其他原因死亡）計數(shù)，實驗結(jié)束后，對每組存活發(fā)眼卵進(jìn)行計數(shù)。結(jié)果表明：本實驗所選用的2種自組中藥復(fù)方和2種化藥除菌劑對金鱒受精卵孵化期間水霉病的防治均有一定效果，且表現(xiàn)出一定的濃度相關(guān)性。經(jīng)篩選，B中劑量組、C中劑量組和D高劑量組效果最為顯著，但是考慮化藥后期殘留及對發(fā)眼卵的潛在危害，建議選擇中藥復(fù)方B進(jìn)行金鱒受精卵水霉病防治，以進(jìn)一步實現(xiàn)健康綠色養(yǎng)殖。選用中藥復(fù)方B進(jìn)行消毒時，應(yīng)嚴(yán)格保證其消毒濃度及作用時間，以確保較好的消毒效果。

金鱒；受精卵；水霉?。环乐嗡幬?/p>

十二五支撐計劃“黑龍江等流域冷水魚健康養(yǎng)殖關(guān)鍵技術(shù)集成與示范”（2012BAD25B10）。

李紹戊（1982-），男，山東萊陽人，副研究員，主要從事魚類病害研究，E-mail：swli_1982@163.com。*通訊作者，研究員，主要從事魚類病害研究，E-mail：liuhongbai@sina.com。

2015-07-02

Supported bY the Twelfth Five-Year Plan＂lntegration and Demonstration of KeY TechnologY of Raising Fish in Heilongjiang＂（2012BAD25B10）.

*Corresponding author.E-mail:liuhongbai@sina.com

Received:JulY 2，2015 Accepted:September 1，2015

修回日期 2015-09-01