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Inducing synchronous ovarian maturation in the

crayfish, Procambarus clarkii, via eyestalk

interventional injection as compared with eyestalk

ablation and combined injection of serotonin and

domperidone

Shengli Liu1,2, Shiyuan Gong1,2, Jinmei Li2 & Wenhu Huang2

1Key Lab of Agricultural Animal Genetics, Breeding and Reproduction of the Ministry of Education, Huazhong

Agricultural University, Wuhan, China2College of Fishery, Huazhong Agricultural University, Wuhan, China

Correspondence: S Liu, Key Lab of Agricultural Animal Genetics, Breeding and Reproduction of the Ministry of Education,

Huazhong Agricultural University, Wuhan 430070, China. E-mail: [email protected]

Abstract

We examined the feasibility of inducing synchro-

nous ovarian maturation in the crayfish, Procamb-

arus clarkii, via eyestalk interventional injection

(EI) using a Bletilla striata polysaccharide (BSP)

gelatin containing tranexamic acid (TRA), dom-

peridone (DOM) or serotonin (5-HT). In total, 360

females were randomly assigned to seven experi-

mental groups and a control group. The averagesurvival rate (SR%) and average synchronous

ovarian maturation rate (SMR%) in survivors in

the EI groups, EI-TRA, EI-DOM, EI-5HT were

compared with bilateral eyestalk ablation (BEA),

unilateral eyestalk ablation (UEA), abdominal

injection (AI) of DOM (0.5 mg crayfish1) alone

(AI-DOM) or combined with 5-HT (0.5 mg cray-

fish1) (AI-DOM+5-HT). The experiment covered a

prolonged period of 32 days until two ovigerous

females were observed in BEA. EI-DOM achieved a

SMR (66.67 9.62%) higher than the control

(22.05 3.06%) (P < 0.05), but lower than BEA

(88.89 11.11%), UEA (63.33 18.56) and

AI-DOM+5-HT (59.26 10.14%) (P > 0.05).

EI-DOM also achieved a higher SR (53.33 3.85%)

than BEA (15.56 2.22), UEA (24.44 5.88)

(P < 0.05) and AI-DOM+5HT (51.11 4.44)

(P > 0.05). However, EI-TRA (SR= 28.89 4.44,

SMR= 37.78 2.22) and EI-5-HT (SR= 15.56

4.44, SMR = 22.22 11.11) failed to induce

significantly higher SMR than the control. These

findings suggest that EI methods, such as EI-DOM,

may have positive characteristics for the develop-

ment of inexpensive and less labour-intensive

techniques for induction of ovarian maturation in

decapods.

Keywords: Bletilla striata polysaccharide, Eyestalk

interventional injection, Ovarian maturation,

Procambarus clarkii

Introduction

Currently, at least two methods have been proved to

be effective in accelerating ovarian maturation of

economic decapod crustaceans. One method is eye-

stalk ablation, which was considered to be one of the

determinant factors for mass production of quality

stocking seeds in captive penaeid shrimps (Santiago

1977; Primavera 1978; Choy 1987), and also in spe-

cies such as crab Potamon persicum (Khazraeenia &

Khazraiinia 2009), crayfish Cherax quadricarinatus

(Sagi, Shoukrun, Levy, Barki, Hulata & Karplus

1997), as well as prawnMacrobrachium(Okumura &

Aida 2001; Varalakshmi & Reddy 2010). The other

method is injecting with serotonin (5-HT) and/or a

dopamine (DA) antagonist. Injection with 5-HT

alone was reported to induce ovarian maturation in

red swamp crayfishProcambarus clarkii(Sarojini, Na-

gabhushanam & Fingerman 1995b), white shrimp,

Litopenaeus vannamei (Vaca & Alfaro 2000), black

tiger shrimp, penaeus monodon (Wongprasert,

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Aquaculture Research 2014, 45, 14021414 doi:10.1111/are.12086


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basal level of 5-HT detected by Rodriguez-Sosa

et al. (1997) was 95.4 49.3 pg mg1 wet mass,

which was comparable to the value reported by

Kulkarni et al. (1992) of 102 pg mg1 wet mass.

5-HT was present in all four ganglia of the eye-

stalk and the highest proportion (40.2%) wasfound in the medulla terminalis (MT) (Rodriguez-

Sosa et al. 1997). The DA content in the eyestalks

of P. clarkii was reported by Alvarez et al. (2005)

to be 5.6 0.1 pmol (about 1062 18.9 pg) per

structure; the highest content was also found in

the MT (over 60%). Using electrical stimulation

and high concentrations of K+ and Ca2+,

enhanced levels of DA and 5-HT were also

detected (Alvarez et al. 2005; Rodriguez-Sosa et al.

1997). These studies suggested that eyestalks

might play an important role in the release of DA

and 5-HT to control ovarian development.

However, the safety and effect of eyestalk interven-

tional injection with a DA antagonist or 5-HT

have not yet been documented in vivo. In addition,

an eyestalk intervention method has not previ-

ously been reported.

In general, an interventional injection requires

gelatin as an agent carrier. The gelatin should

be a non-dispersible, sustained-releasing and

harmless material. The polysaccharide extracted

from Bletilla striata (BSP) appears to be a non-

dispersible and sustained-releasing bioadhesive

material and has been used as an embolizing

agent (Chuansheng, Gansheng & Huimin 1998;Feng, Kramann, Zheng & Zhou 1996; Zheng,

Feng & Zhou 1996) and a non-viral gene vec-

tor (Xiang-wen, Xin, Gan-sheng, Yan-bing &

Chuan-sheng 2008) in recent years. This polysac-

charide is the effective constituent of a Chinese

Herbal Medicine, B. striata (Chinese name is Bai

Ji) and is used to stop bleeding caused by trau-

matic injuries, heal wounds, reduce swelling,

and promote regeneration of tissue (Yeung

1985). BSP does not influence the endocrine reg-

ulation of decapods and can prevent excessive

bleeding after injections when is used as a car-

rier of eyestalk interventional injection agents.

The aim of our study was to examine the feasi-

bility of inducing synchronous ovarian maturation

in red swamp crayfish, P. clarkii, via eyestalk inter-

ventional injection with non-dispersible haemol-

ymph coagulant, or sustained-release 5-HT or

DOM. We used BSP as a carrier for tranexamic

acid (TRA, used as a haemolymph coagulant),

5-HT or DOM when injected into the eyestalks of

P. clarkii. The D2-type DA antagonist, domperi-

done, was also evaluated in ovarian maturation

induction ofP. clarkii when injected alone or com-

bined with 5-HT.

Material and methods

Experimental animals

Collection of P. clarkii

Mature female P. clarkii (2530 g body weight

and 7080 mm measured from the base of the

eyestalk to the end of the telson) were obtained

from an aqua farm in Wuhan city, China in late

August, just before the peak season of their ovar-

ian maturation in this area (Gong, Lv, Sun, Li &

He 2008), of 2009 and 2010. The crayfish had

thicker carapace with yellow granules. After selec-

tion, a random sample of 30 females from 925

female crayfish showed that all were in ovarian

stage III to ovarian stage IV (ovarian stage register

see section Sample processes and ovarian develop-

ment stages register).

Maintenance of P. clarkii

All selected crayfish were immediately transferred

into the laboratory after collection. First, they were

placed into a plastic tank with a water depth of

1.5 cm and left for 4 h. The active ones were then

selected and maintained in 120 9 60 9 50 cm

self-circulation aquariums with a temperature reg-ulating device under a photoperiod 12L:12D. Cray-

fish were given surimi (70% in total food weight)

and fresh vegetable (30% in total food weight) and

fed ad libitum. Every aquarium had 35 cm of

water, 15 artificial caves ( = 5 cm, L = 18 cm),

2 polyethylene climbing nets set on a substratum

of gravel and was given constant aeration. The

density of crayfish was 15 females per aquarium.

To acclimatize them to lab conditions, all crayfish

were cultured for 8 days at approximately

20~26C before treatment. During acclimatization,

dead individuals were quietly replaced by active

ones during the 8 days.

Agent preparation

Solutions

(1) A crayfish saline solution (Vans solution):

205 mM Na+, 5.4 mM K+, 13.5 mM Ca2+,

2.5 mM Mg2+, 241.4 mM Cl, 2.5 mM Hepes

(Van Harreveld, 1936).

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(2) A 0.1 g mL1 TRA solution: tranexamic acid

(TRA) (Alfa-Aesar, CAS: 1197-18-8) was dis-

solved in the Vans solution and made into a

0.1 g mL1 solution.

(3) A 2 mg mL1 DOM solution: 200 mg dom-

peridone (Sigma-Aldrich, CAS: 57808-66-9)was dissolved in 2 mL 99.9% (v/v) alcohol

and well ground in a glass tissue homoge-

nizer, then adjust to 100 mL with Vans solu-

tion to make a 2 mg mL1 DOM solution.

(4) A 2 mg mL1 5-HT solution: serotonin hypo-

chloride (Sigma-Aldrich, CAS: 153-98-0) was

dissolved in the Vans solution and made into

a 2 mg mL1 solution.

Interventional injection agent

BSP extract. The pulverized powder of the dried

root of B. striata was purchased from the Tongren

Drugstore in Wuhan, China. To extract BSP, we used

a modified form of the alcohol-precipitation method

used by Xiang-wen et al. (2008). Our extraction

steps included cold infusion (Vwater/MB. striata= 15/1)

at 4C for 1824 h, a water bath at approximately

70C for 1.52 h, centrifugation (1400 g, 30 min)

using a Hettich centrifuge (ROTO SILENTA R/RS),

graded alcohol-precipitation using 85% (v/v) and

95% (v/v) alcohol, filtration using a copper screen

(sieve mesh number was 500), dehydration three

times using 99.9% (v/v) alcohol, sifting using a silk

gauze (sieve mesh number was 300), and then blowdrying using warm air. The dry powder of BSP was

kept in a refrigerator at 4C until the intervention

agent was prepared.

Preparation of the gelatin containing TRA, 5-HT or

DOM using BSP. BSP (6 g) was dissolved in

100 mL of 0.1 g mL1 TRA solution, 2 mg mL1

DOM solution or 2 mg mL1 5-HT solution to

obtain a gelatin containing TRA, 5-HT or DOM for

eyestalk interventional injection. Our steps

included soaking BSP in 0.1 g mL1 TRA solution,

2 mg mL1 DOM solution or 2 mg mL1 5-HT

solution at 4C for 4 h, adjusting to 100 mL with

the same solution, mixing using ultrasonic waves

(for 5 s with an interval time of 10 s [Sonics,

JY92-II]) in an ice bath and addition of the same

solution until the volume reached 100 mL, suc-

tion filtration through a copper screen sieve (mesh

number was 500), mixing again using ultrasonic

waves (as before), and then confirming that the

gelatin could pass through a 30-G needle.

Experimental design

After acclimatization, 30 P. clarkii individuals were

sacrificed to confirm their ovarian development

stages (only 3 of the 30 individuals had reached

ovarian stage IV), and 360 females were randomlyassigned to 24 aquariums (15 females per aquar-

ium). The aquariums were divided into eight

groups (7 experimental groups and one control

group) as follows: Crayfish in the seven experimen-

tal groups were treated with bilateral eyestalk

ablation (BEA); unilateral eyestalk ablation (UEA);

eyestalk intervention with a gelatin containing

0.1 g mL1 TRA (EI-TRA), 2 mg mL1 DOM

(EI-DOM) or 2 mg mL1 5-HT (EI-5-HT); abdomi-

nal injection at 3-day intervals with 2 mg mL1

domperidone and 2 mg mL1 5-HT (AI-DOM+5-

HT), or 2 mg mL1 domperidone (AI-DOM) only.

The control group was untreated.

When two ovigerous females were observed in

BEA, which took a prolonged period of 32 days,

all the females in the eight groups were dissected.

The average survival rate (100Nsurvived/15 = SR%)

and ovarian synchronous maturation rate of the

survivors (100Novary-matured/(15-Ndied)=SMR%) in

the three parallel aquariums in each group as well

as their standard errors (SE) were calculated and

compared. Although the brood size in P. clarkii

ranges from dozens to hundreds (Trimble & Gaude

1988), the average GSI (gonadosomatic index,

100Wovary/(Wbody)=GSI%), average HSI (hepatoso-matic index, 100Wovary/Wbody=HSI%) and the

average oocyte diameter (OD) in each group were

determined for reference.

Eyestalk interventional injection

The interventional agent (gelatin containing

0.1 g mL1 TRA, 2 mg mL1 DOM or 2 mg mL1

5-HT) was injected into the internal base of the

eyestalk (proximal medulla terminalis, see Fig. 1)

of 135 females (45 females for each agent) via a

1-mL insulin syringe with a sterilized, permanently

attached, 29G91/2 inch needle (BD Ultra-Fine,

USA). The injection volume was 0.20.3 mL per

eyestalk.

Eyestalk ablation

Using a sterilized scalpel, 45 females had both eye-

stalks ablated (BEA) and 45 females had one eye-

stalk ablated (UEA) at the proximal end. Following

the application of powdered, hot and dry BSP (kept

at 75~80C before use), the specimens were placed

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in water until the wound healed. The scalpel was

sterilized by dipping in 70% alcohol after eachcrayfish was treated.

Abdominal injection of DOM with or without 5-HT

Forty-five females were injected in the second abdomi-

nal segment with 0.25 mL 2 mg mL1 domperidone

solution (0.5 mg crayfish1, about 1720 lg g1

b.w.). In addition, 45 females were injected in the sec-

ond abdominal segment with 0.25 mL 2 mg mL1

domperidone solution and 0.25 mL 2 mg mL1 5-HT

solution (0.5 mg crayfish1, about 1720 lg g1

b.w.). The crayfish were injected at 3-day intervals.

Sample processes and ovarian development stages

register

The sampling process consisted of two steps: First,

females which had completed spawning and dead

individuals were counted and removed each day.

Spawning crayfish were calculated as ovary-

mature. Second, at the end of the experiment, the

ovaries in surviving individuals which had not

spawned were dissected. Ovarian development

stages were registered based on external observa-

tion of ovarian colour and oocyte size (Table 1) as

described previously by Li and Z. (1999), Ando

and Makioka (1998) and Kulkarni, Glade and Fin-

german (1991a). As the mature oocytes of

P. clarkii were very large (diameter often exceeded

1.4 mm) and it was difficult to obtain histological

sections, oocyte diameter (OD) was measured

under a Leica MZ7.5 high-performance stereomi-croscope in 4% formalin using an ocular microme-

tre. Twenty-five advanced-stage oocytes were

measured in each ovary. The remainder of the

ovary was fixed in Bouins solution to obtain histo-

logical sections for the confirmation of OD. Previ-

tellogenic/primary oocytes were not used for OD

measurement as they reduced to a very small pro-

portion (


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Statistical analyses

The values of survival rate (SR,%) and synchronous

maturation rate (SMR,%) represent as Mean SE

(standard errors of the 3 parallel aquariums in each

group,%). The average gonadosomatic index (GSI,%),average hepatosomatic index (HSI, I%) and the

average oocyte diameter (OD) represent as Mean

SD. The data were analysed with the Sigmaplot.13

program using one-way analysis of variance (ANO-

VA) and Ducans multiple range test. A difference

was considered to be significant at P < 0.05.

Results

Synchronous ovarian maturation rate

Bilateral eyestalk ablation and unilateral eyestalk

ablation

Bilateral eyestalk ablation (BEA) significantly pro-

moted ovarian maturation in P. clarkii (P < 0.05).

When spawning occurred in the BEA group, most

females in the group matured synchronously. A

significantly higher SMR (88.89 11.11) was

detected when compared with the control group

(22.05 3.06). Unilateral eyestalk ablation (UEA)

also induced SMR when compared with the control

group (P < 0.05), but was not statistically significant

in BEA (P > 0.05). In general, SMR in UEA crayfish

(63.33 18.56) was lower than that in BEA crayfish

and higher than that in the control group (Fig. 3).

Eyestalk interventional injections

In the eyestalk interventional injection groups, the

SMR of the EI-TRA group (37.78 2.22) was

lower than that in the UEA, EI-DOM

(66.67 9.62) and the combined abdominal

injection with DOM and 5-HT (AI-DOM+5-HT)

groups (P > 0.05), but higher than the EI-5-HT

(22.22 11.11) and control groups (P > 0.05).

The SMR of the EI-DOM group was significantly

higher than the EI-5-HT and control groups

(P < 0.05), and was similar to the UEA and

AI-DOM+5-HT groups (P > 0.05). However, the

EI-DOM group also exhibited a lower SMR when

compared with the BEA group (P > 0.05). The

SMR in the EI-5-HT group was similar to that in

the control group (P > 0.05) (Fig. 3).

Abdomen injection of DOM with or without 5-HT

The abdominal injection with combined DOM and

5-HT (AI-DOM+5-HT) resulted in a lower SMR

(A) (B) (C)

(E) (F)

(H) (I)

(J) (K)

(L) (M)

(N)

(P)

(O)

(G)

(D)

Figure 2 The gross appearance and histological char-

acter of oocytes during the ovarian development of red

swamp crayfish P. clarkii. Picture A~H show the gross

appearance changes during ovarian maturation and

Picture J~P show the histological changes during ovar-

ian stage I~IV; Oog represents oogonia; Oc1, Oc2, Oc3,

Oc4, mOc represent the 5 stages of oocytes. Fig.2I

shows an ovigerous female carrying big eggs (diameter

reached 2.30 mm) obtained in this study.




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(59.26 10.14) when compared with the BEA

and UEA groups (P > 0.05), but showed a much

higher SMR than the control group (P < 0.05).

However, abdominal injection with DOM solution

alone (AI-DOM) achieved a SMR of 22.69 2.52,

similar to the control group (P > 0.05) (Fig. 3).

Survival rate

Bilateral eyestalk ablation and unilateral eyestalk

ablation

Both bilateral eyestalk ablation and unilateral eye-

stalk ablation led to high mortality in P. clarkii

(P < 0.05). Moreover, in the 32-day experiment

(90 samples), we detected no significant difference

between the SR of the BEA (15.56 2.22) and

UEA (24.44 5.88) groups (P > 0.05) (Fig. 3).

Eyestalk interventional injections

The eyestalk interventional injection groups EI-TRA

(28.89 4.44) and EI-5-HT (15.56 4.44) had amuch lower SR than the control group

(71.11 5.88) and the AI-DOM group

(77.78 5.88) (P < 0.05).Compared with the BEA

and UEA groups, there was no significant increase in

SR (P > 0.05) in the EI-TRA and EI-5-HT groups.

However, EI-DOM showed a SR (53.33 3.85) sig-

nificantly higher than those of the BEA and UEA

groups (P < 0.05), but lower than the AI-DOM and

control groups (71.11 5.88) (P < 0.05) (Fig. 3).

Abdomen injection of DOM with or without 5-HT

The combined injection of DOM and 5-HT

(AI-DOM+5-HT) resulted in a relatively high SR of

51.11 4.44, which was significantly higher

than that in the eyestalk ablation groups

(P < 0.05), but lower than the control group

(71.11 5.88) (P < 0.05). However, injection of

DOM (AI-DOM) resulted in a higher SR than the

control group (P > 0.05) (Fig. 3).

GSI, HSI and OD of different groups

Table 2 shows the GSI (MeanSD,%), HSI

(Mean sd,%) and OD (Mean sd) of each

group when the specimens were dissected at theend of the experiment. The GSI was highest in

the BEA (4.33 2.10) and EI-DOM

(3.26 2.15) groups, followed by the UEA

(2.97 2.61), EI-TRA (1.76 2.00), AI-

DOM+5-HT (1.62 1.10), control (1.24 1.68),

EI-5-HT (1.21 2.24) and AI-DOM (1.17 1.49)

groups. The GSI in the EI-DOM group was lower

than that in the BEA group (P > 0.05), but sig-

nificantly higher than that in the control group

and the abdominal injection groups AI-DOM and

AI-DOM+5-HT (P < 0.05). The GSI in the EI-

DOM group was lower than that in the UEA

group (P > 0.05), but higher than that in the AI-

DOM+5-HT, AI-DOM and the control groups

(P > 0.05). The GSI in the EI-5-HT group was

lower than that in the control group (P > 0.05),

but higher than that in the AI-DOM group

(P > 0.05). The HSI was lowest in the BEA

(4.46 1.08), EI-TRA (5.59 1.64) and

EI-DOM (5.53 1.58) groups, followed by the

UEA (6.37 1.63), control (6.54 1.22),

Figure 3 The survival rate (SR) and synchronous

ovarian maturation rate (SMR) of Procambrus clarkii

performed different treatments (sampled when two

ovigerous females were observed in the bilateral eyestalk

ablation group). BEA: bilateral eyestalk ablation; UEA:

unilateral eyestalk ablation; EI-TRA: eyestalk interven-

tion with TRA; EI-DOM: eyestalk intervention with

DOM; EI-5-HT: eyestalk intervention with 5-HT;

AI-DOM+5HT: combined injection with 2mgml-1 5-HT

and 2mgml-1 DOM solution (0.5 mg/crayfish of each

agent) in the abdomen in 3-day intervals; AI-DOM:

injection with 2mgml-1 DOM solution (0.5 mg/cray-

fish) in the abdomen in 3-day intervals. Control: con-trol group, untreated. SR and SMR were expressed as

Mean SE (%) in the figure. Statistical analyses used

the Sigmaplot.13 program using one-way analysis of

variance (ANOVA) and Ducans multiple range test.

A difference was considered to be significant at P < 0.05.




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group in this study matured synchronously when

two ovigerous females were observed. This sug-

gested that the majority of the females we selected

in late August had mated and carried active sper-

matophores. Third, recent research suggests that

P. clarkii may also be reproduced by parthenogene-sis (Yue, Wang, Zhu, Wang, Zhu & Lo 2008; Li,

Deng, Yang & Wang 2012).

In this study, our experiment using P. clarkii

was carried out over a prolonged period of

32 days. The start time in this ovarian induction

experiment was limited by the need to select speci-

mens carrying active spermatophores. The timing

to close the experiment was decided, for the first

time by us, when at least two ovigerous females

were observed in the bilateral eyestalk ablation

group, which required 32 days. We believe that

this is the preferred method to decide the time

interval before starting this type of ovarian induc-

tion experiment when a new method or dose of

agent is compared with a proved effective method

or dose of agent. In several previous studies, the

evaluation of ovarian maturation induction

depended on comparing the number of days before

spawning, which was known as the ovarian matu-

ration period (OMP) (Tinikul, Soonthornsumrith,

Phoungpetchara, Meeratana, Poljaroen, Duangsu-

wan, Soonklang, Mercier & Sobhon 2009), or the

periodically average maturation index (M.I.)

(Alfaro et al. 2004), or weekly spawning rate

(Vaca & Alfaro 2000) as well as the mean GSI(Radhakrishnan & Vijayakumaran 2011) or Vg

levels (Tinikul et al. 2008) in a given period. These

given periods seem subjective, and lack a uniform

standard. In fact, a comparison of synchronous

maturation rate (SMR) when the method is known

to be effective is preferred to evaluate the effect of

different treatments on ovarian maturation induc-

tion efficiency in P. clarkii, as the P. clarkii speci-

mens were mostly obtained from natural ponds

and it was very difficult to select specimens at the

same ovarian stage. Although a study on the cray-

fish Cherax quadricarinatus detected Vg levels

during its reproductive cycle (Ferre, Medesani,

Garca, Grodzielski & Rodrguez 2012), we did not

include this subject in our study. A comparison of

Vg levels has been reported in prawns such as M.

rosenbergii (Chen et al. 2003; Tinikul et al. 2008),

but has not been reported in the crayfish P. clarkii.

Moreover, achieving synchronous ovarian matura-

tion is enough to break the production bottleneck

ofP. clarkii juveniles for use in aquaculture. In this

study, we also determined the GSI, HSI and OD in

different treatments to support the calculated SMR,

although the brood size of P. clarkii ranges from

dozens to hundreds (Trimble & Gaude 1988) and

the OD of spawned eggs in different females ranged

from 1.1 to 2.3 mm (Fig. 2I).As expected, with mature female P. clarkii, both

bilateral and unilateral eyestalk ablation resulted

in a significantly higher SMR (P < 0.05) and led

to a much higher mortality compared with the

control (P < 0.05). The maturation induction

results were consistent with the findings following

scalding of the eyestalks in P. clarkii (Ruijie 2009),

and the mortality rate was consistent, but lower,

with the trend reported in M. lanchesteri (Var-

alakshmi & Reddy 2010). Compared with eyestalk

ablation, abdominal injection of a proved effective

dosage, 0.5 mg crayfish1 (0.5 mg crayfish1 = 17

20 lg g1 b.w. = 2.5 9 106mol crayfish1 in

this study), of 5-HT combined with DOM, resulted

in a significantly lower SMR (P > 0.05), but much

higher SR (P < 0.05). Abdominal injection of DOM

alone failed to induce accelerated ovarian matura-

tion and only achieved an SMR non-significantly

higher than the untreated control (P > 0.05).

Such results are consistent with the reports by

Chen et al. (2003) using M. rosenbergii and Alfaro

et al. (2004) using L. stylirostris and L. vannamei.

As a D2-type DA antagonist, DOM injection alone,

at a dosage of 1720 lg g1 b.w., seemed to have

limited effectiveness. However, DOM seemed toincrease the SR of P. clarkii, as the SR following

DOM injection was even higher than that in the

control group (P > 0.05), which, to our know-

ledge, has not previously been demonstrated.

Unlike DOM injection alone, eyestalk interven-

tional injection of DOM resulted in a significantly

higher SMR than the control (P < 0.05), but was

non-significantly lower than eyestalk ablation and

combined injections of 5-HT and DOM (P > 0.05).

A comparison of GSI, HSI and OD among the

treatments supported the above results. Eyestalk

interventional injection of DOM also led to a better

SR, which was significantly higher than the eye-

stalk ablations (P < 0.05). Although the SR was

still lower than that in the control group

(P < 0.05), no significant difference was observed

when it was compared with that in the combined

injection of 5-HT and DOM group (P > 0.05).

These results suggest that eyestalk interventional

injection of DOM has the positive characteristics of

a relatively low mortality rate and higher SMR




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and may be a potential technique for inducing

ovarian maturation in P. clarkii as an alternative

technique to traditional eyestalk ablation. The

mechanism to explain the significantly higher

SMR (P < 0.05) achieved in the DOM eyestalk

interventional group compared with the DOMabdominal injection group deserves further study.

We agree that DA is a crucial neurotransmitter,

which inhibits ovarian maturation (Chen et al.

2003); however, we think that the role of eye-

stalks in the effective changes in DA level which

control ovarian development is more important

than other tissues such as the thoracic ganglia,

which was also proposed by Chen et al. (2003).

Disappointingly, eyestalk interventional injection

of TRA resulted in a SMR only slightly higher

than that in the control group (P > 0.05), but

was still lower than that in the BEA ( P < 0.05),

UEA (P > 0.05), as well as the combined injection

of 5-HT and DOM groups (P > 0.05). It also

resulted in a SR significantly lower than that in

the control group and the combined injection of

5-HT and DOM group (P < 0.05), but was slightly

higher than both the BEA and UEA groups

(P > 0.05) (Fig. 3). In addition, the levels of OD

and GSI were non-significantly higher than the

control (P > 0.05), whereas the level of HSI exhib-

ited a significant decreasing trend compared with

the control group (P < 0.05) and was non-signifi-

cantly higher than that in the BEA group

(P > 0.05). These results failed to provide new evi-dence to support the most commonly accepted

theory, which proposed that the sinus gland (SG)

in the eyestalks is the storage and release site of

the haemolymph-borne neurohormones, such as

GIH and MOIH, which inhibit precocious ovarian

maturation in non-breeding seasons (Bray & Law-

rence 1992). Such a vague effect on accelerating

ovarian maturation can be explained by the fact

that eyestalk intervention with gelatin containing

TRA failed to embolize the eyestalk to prevent GIH

and MOIH release into the haemolymph circula-

tion. Another explanation is that the eyestalk tis-

sue does not play a significant role in releasing

haemolymph-borne GIH or MOIH to control ovar-

ian maturation; thus even when both GIH and

MOIH release from the eyestalks was effectively

blocked by the gelatin containing TRA injection,

other inhibiting factors outside the eyestalks pre-

vented precocious ovarian maturation in P. clarkii.

Several previous studies seem to support the latter

explanation. Chen et al. (2003) reported that DA

was able to inhibit Vg synthesis in eyestalk-

ablated M. rosenbergii and proposed that DA exerts

its inhibitory effect on Vg synthesis by inhibiting

the release of GSH from the thoracic ganglia

rather than stimulating the release of GIH from

the eyestalks. Alvarez et al. (2005) reported thatthe tissue distribution of DA in P. clarkii was not

limited to the eyestalks. In our opinion, further

study such as examining the changes in DA,

MOIH, GIH as well as GSH levels after eyestalk

ablation, eyestalk embolization and during ovarian

maturation may cast light on the mechanism

involved.

Unexpectedly and unsatisfactorily, eyestalk

interventional injection of 5-HT achieved the

worst results among all the treatments. It showed

a similar SMR to that of the control ( P > 0.05), a

similar SR to the BEA group (P > 0.05) and a sig-

nificantly lower SR than the control (P < 0.05).

These negative results were unexpected as we

achieved significantly higher SMR, GSI and OD fol-

lowing abdominal injection of DOM with 5-HT

compared with that without 5-HT. 5-HT was also

reported to extensively accelerate ovarian matura-

tion in previous studies in the crayfish P. clarkii

(Sarojini et al. 1995b; Kulkarni et al. 1992) as

well as the shrimp L. vannamei (Vaca & Alfaro

2000), F. merguiensis (Zacharia & Kakati 2011),

the prawn M. rosenbergii (Tinikul et al. 2009) and

several other decapods. Fingerman (1997) and

Kulkarni et al. (1992) suggested that 5-HT mightact indirectly on the gonads by stimulating the

release of a putative gonadotropic factor such as

GSH from the thoracic ganglia, and/or by inhibit-

ing the release of GIH from the optic lobe in the

eyestalk of P. clarkii. The primary target of 5-HT

seems to be the X-organ neurons in the eyestalks

(Tinikul et al. 2009). The mechanism to explain

the difference between the findings following 5-HT

injection and those following the 5-HT eyestalk in-

terventional injection in this report also deserves

further study. From our current research, we are

unable to provide a convincing explanation for the

unsatisfactory effect of the eyestalk interventional

injection of 5-HT on the induction of ovarian mat-

uration. However, as we were concerned with the

safety aspects when we evaluated the efficiency of

5-HT on inducing ovarian maturation, one point

we would like to stress is that 5-HT appears to

increase the mortality rate of P. clarkii (Fig. 3),

which, to our knowledge, has not previously been

demonstrated.




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There is no precedent, in prior studies, to follow

in performing an eyestalk intervention method.

For this study, we selected the BSP gelatin to make

the carrier for TRA, 5-HT or DOM because it is a

biological material that is non-dispersible, sus-

tained-releasing and harmless. Feng et al. (1996)reported its character of non-dispersiblity. Tradi-

tional Chinese medicine has the principle of not

using Bletilla striata with aconite root because it

prolongs the time of aconite root toxicity (Yeung

1985) suggesting that it is a sustained-releasing

material. As an internal medicine for humans, it is

also harmless. Hence, we believe that BSP is basi-

cally a safe and neutral vector for interventional

injection. In this study, all interventional agents

were made of the crude BSP we extracted; the

survival rate and ovarian maturation induction

performance seems to relate to the TRA, DOM or

5-HT; it contains rather than BSP itself. It deserves

further tests and improvement to be applicable to

mass production of interventional injection agents.

In summary, our investigation into the induc-

tion of ovarian maturation in P. clarkii via eyestalk

interventional injection was based on the hypothe-

sis that the eyestalk plays an incomparable role in

releasing neuropeptides such as haemolymph-

borne GIH and MOIH, and is deeply involved in

the antagonistic action of DA and 5-HT. We

induced ovarian maturation by disturbing the eye-

stalks functions via interventional injections of a

gelatin containing TRA, DOM or 5-HT into theeyestalk of P. clarkii. Our findings appear to show

that the eyestalk is deeply involved in the inhibi-

tory action of DA during the ovarian maturation

in P. clarkii. In practice, the positive characteristics

of eyestalk interventional injection with DOM have

implications in the design of a safe, efficient, inex-

pensive, less labour-intensive and time-controllable

method to induce ovarian maturation in decapods.

The traditional eyestalk ablation technique is

cruel, the speed of maturation is uncontrollable,

and leads to permanent damage, whereas injection

of 5-HT and/or a DA antagonist needs to be per-

formed repeatedly and is expensive. These methods

are not widely adopted in the mass production of

stocking seeds in the inexpensive crayfish P. clarkii.

In this study, eyestalk interventional injection was

only performed once, and BSP, which is an abun-

dant material in nature, deserves further assess-

ment as a safe, efficient, inexpensive and less

labour-intensive method for maturation induction

in decapods. Moreover, by regulating the dosage of

the interventional injection agent, it is possible to

control the speed of ovarian maturation induction.

Eyestalk intervention methods also provide other

ways to explore the neuroendocrine function of

the XO-SG of these species in vivo. In the prior

studies, the eyestalk tissue was ablated orimplanted into the abdomen to note or measure its

effect or response.

Acknowledgments

We are grateful to Professor W. Zhao of Shanghai

Ocean University, Professor W. Wang, senior engi-

neer M. Wang and Dr. Z. Luo of Huazhong Agri-

cultural University for their contributions and

assistance. This research was supported by the

program Transformation of Agricultural Science

and Technology Achievements (project number

4002-092062) financed by the ministry of finance

china.

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