This study was supported by the Ministry of Education, Malaysia, under the Higher Institution Centre of Excellence (HICoE) program, Malaysia, accredited to the Institute of Tropical Aquaculture and Fisheries, Universiti Malaysia Terengganu (Vot No. 63933 &#38; Vot No. 56048). We acknowledge the support of Universiti Malaysia Terengganu and Sayap Jaya Sdn. Bhd. via the Private Partnership Research Grant (Vot. No. 55377). An adjunct Academic Fellow position from Universiti Sains Malaysia to Khor Waiho and Hanafiah Fazhan is also acknowledged.

This protocol follows the Malaysian Code of Practice for the Care and Use of Animals for Scientific Purposes outlined by the Laboratory Animal Science Association of Malaysia. The sacrifice of the experimental samples was done according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978). Sexually pre-mature female mud crabs (orange mud crab S. olivacea) were collected from the local market (5&#176;66&#8242;62&#8242;&#8242;N, 102&#176;72...

This protocol was developed for the eyestalk ablation of the mud crab, Scylla spp., and can be applied as an efficient method to induce gonad maturation. This protocol can be easily replicated for the commercial ovary maturation of mud crabs and can be implemented to reduce the latent period (time from one spawning to another) in mud crab seed production.
The eyestalk ablation of crustaceans (i.e., freshwater prawn, marine shrimp) is typically done to induce gonad maturation and out-o...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/65039">Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs</a>. The Introduction, Protocol, Discussion and References were&#160;updated.

Erratum: Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs

All four mud crab species belonging to the genus Scylla are commercially important crustacean species in aquaculture1,2. The growth of crustaceans, including mud crabs, and their transformation from the pre-mature (sub-adult or pubertal) phase to the sexually mature (adult) phase occur through a molting process that involves the periodic shedding of older and smaller exoskeletons. Carapace width (CW), chelipeds, and abdominal flap morphologies are widely used to determine the sexual maturity of Scylla spp.3,4,5. The process of molting is regulated by the action of various hormones and requires a huge amount of energy6. In addition to the normal molting process, the loss of limbs, either voluntarily or induced by external factors, expedites the molting of crabs without affecting their survival rate7,8,9. Therefore, limb autotomy is commonly used for molt induction in the soft-shell mud crab farming industry7,9.
Unilateral or bilateral eyestalk ablation is mostly popular in freshwater prawn and marine shrimp for gonad maturation and seed production10,11,12,13. Common eyestalk ablation techniques in crustaceans include the following: (i) ligation at the base of the eyestalk using a string14,15; (ii) cauterization of the eyestalk using hot forceps or electrocautery devices16; (iii) removal or direct pinching of the eyestalk to leave an open wound12; and (iv) removal of the eyestalk contents through incision after slicing the distal portion of the eye with a razor17. The eyestalk X-organs are important endocrine organs in crustaceans as they regulate crustacean hyperglycemic hormones (CHH), molt-inhibiting hormones (MIH), and vitellogenesis-inhibiting hormones (VIH)6,18,19,20,21,22. Eyestalk X-organs (or the sinus gland complex) synthesize and release gonad-inhibiting hormones (GIH), also known as vitellogenesis-inhibiting hormones (VIH), belonging to the neuropeptide hormone family6. Unilateral or bilateral eyestalk ablation reduces GIH synthesis, resulting in the dominance of stimulating hormones (i.e., gonad stimulating hormones, GSH) and the acceleration of the ovarian maturation process in crustaceans23,24,25,26. Without the influence of GIH after eyestalk ablation, female crustaceans devote their energy to ovary development27. It has been found that unilateral eyestalk ablation is sufficient for the induction of ovarian maturation in crustaceans11 and that the ablated eyestalk of shrimps and crabs can regenerate after several moltings28. There are four ovarian development stages recorded in Scylla spp.: i) immature (stage-1), ii) early maturing (stage-2), iii) pre-maturing (stage-3), and iv) fully mature (stage-4)29,30. The immature ovarian stage is found in immature females. After pubertal molting and mating, the immature ovary starts developing and finally matures (stage-4) before spawning31.
An eyestalk ablation protocol is essential for mud crab broodstock development and seed production. In the global food market, mature mud crabs with fully mature ovaries (stage-4) rather than crabs with higher muscle content are preferred by consumers and, thus, have a higher commercial value, even higher than large males. There is no complete protocol for the eyestalk ablation of mud crabs. The eyestalk ablation protocol in this work minimizes stress by using fully anesthetized&#160;crabs and minimizes physical injury to personnel from crab bites. This protocol is easy and cost-effective. Here, we present a protocol for the eyestalk ablation of Scylla spp. that can induce the maturation of the gonad. Two techniques of eyestalk ablation (cauterization and surgery) were tested and their efficiencies compared based on the gonadal development rate of female mud crabs.

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			<td>MR D.I.Y. Group Berhad</td>
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			<td>Shopee Malaysia</td>
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			<td>Thermo Scientific Chemicals</td>
			<td>033361.M1</td>
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			<td>N/A</td>
			<td>Research Center of Universiti Malaysia Terengganu</td>
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			<td>Fine sand</td>
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			<td>First Aid Kits</td>
			<td>Watsons Malaysia</td>
			<td>N/A</td>
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			<td>Flat head nickel steel metal rod (Screw driver)</td>
			<td>MR D.I.Y. Group Berhad</td>
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			<td>Formaldehyde</td>
			<td>Thermo Scientific Chemicals</td>
			<td>119690010</td>
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			<td>Gas cylinder (butane gas) for blow&#160; torch</td>
			<td>MR D.I.Y. Group Berhad</td>
			<td>N/A</td>
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			<td>Gas lighter gun (long head)</td>
			<td>MR D.I.Y. Group Berhad</td>
			<td>N/A</td>
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			<td>Glass beaker (100 mL))</td>
			<td>Corning Life Sciences</td>
			<td>1000-100</td>
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			<td>Watsons Malaysia</td>
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			<td>Eco-Shop Marketing Sdn. Bhd.</td>
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			<td>Bina Plastic Industries Sdn Bhd (HQ)</td>
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			<td>ATAGO CO.,LTD.</td>
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			<td>Sharp Corporation Japan</td>
			<td>N/A</td>
			<td>Chest Freezer SHARP 110L - SJC 118</td>
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			<td>Scoop net</td>
			<td>MR D.I.Y. Group Berhad</td>
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			<td>Hatcheri AKUATROP&#160;</td>
			<td>N/A</td>
			<td>Research Center of Universiti Malaysia Terengganu</td>
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			<td>Siphoning pipe</td>
			<td>MR D.I.Y. Group Berhad</td>
			<td>N/A</td>
			<td>N/A</td>
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			<td>Spray bottle</td>
			<td>Mr. DIY Sdn Bhd</td>
			<td>N/A</td>
			<td>N/A</td>
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			<td>Stainless surgical forceps&#160;</td>
			<td>N/A</td>
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			<td>Stainless surgical scissors&#160;</td>
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			<td>Submersible water pump&#160;</td>
			<td>AS</td>
			<td>N/A</td>
			<td>model: Astro 4000</td>
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			<td>Tincture of iodine solution&#160; (Povidone Iodine)</td>
			<td>Farmasi Fajr Sdn Bhd</td>
			<td>N/A</td>
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			<td>Tissue paper&#160;</td>
			<td>N/A</td>
			<td>N/A</td>
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			<td>Transparent plastic aquarium</td>
			<td>Ming Yu Three</td>
			<td>N/A</td>
			<td>aquarium and pet shop</td>
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			<td>Waterproof table</td>
			<td>Hatcheri AKUATROP&#160;</td>
			<td>N/A</td>
			<td>Research Center of Universiti Malaysia Terengganu</td>
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eyestalk ablation to increase ovarian maturation in mud crabs

Mud crabs (Scylla spp.) are commercially important crustacean species that can be found throughout the Indo-West Pacific region. During culture, the induction of ovarian maturation is important to meet the consumer demand for mature mud crabs and hasten seed production. Eyestalk ablation is an effective tool to enhance ovarian maturation in mud crabs. However, there is no standard protocol for the eyestalk ablation of mud crabs. In this study, two eyestalk ablation techniques are described: cauterization (the use of hot metal to ablate the eyestalk of an anesthetized crab) and surgery (the removal of the eyestalk using surgical scissors). Before eyestalk ablation, sexually mature females (CW &#62; 86 mm) were anesthetized using an ice bag (&#8722;20 &#176;C) with seawater. When the water temperature reached 4 &#176;C, the ice bag was removed from the water. Flowing seawater (ambient temperature: 28 &#176;C) was used for recovery from the anesthesia immediately after eyestalk ablation. Mortality did not occur during or after the process of eyestalk ablation. The eyestalk ablation protocol presented here accelerated the ovarian maturation of the mud crabs.

Gonad maturation 
Creamy white ovarian tissues (immature ovaries, stage-1) were found in 100% of the dissected females (n = 6) before performing the eyestalk ablation (Figure 2). The gonad maturation rate of the eyestalk-ablated female crabs (n = 63; 31 females with the cauterization technique and 32 females with the surgery technique) was higher compared to female crabs that were not subjected to eyestalk ablation (n = 31) after 30 days of individual rearing (<strong c...

Watch this Scientific Journal Video about Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs at JoVE.com

Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs

Two eyestalk ablation protocols (i.e., cauterization and surgery&#160;approaches) were performed on anesthetized female crabs. The eyestalk ablation of mud crabs hastened the ripening of ovaries without decreasing the survival rate.

Mud crabs (Scylla spp.) are commercially important crustacean species that can be found throughout the Indo-West Pacific region. During culture, the ...

The described protocol minimizes stress during eyestalk ablation of mud crabs, thereby reducing the mortality occurrence due to eyestalk ablation. Time efficiency is the key to minimizing stress to the animals. Make sure everything is prepared before performing the procedure.Begin by washing the healthy, active, and premature female mud crabs with chlorinated tap water to remove any debris and parasites. Soak the crabs in formaldehyde for 30 minutes. Ensure continuous and gentle aeration with air stones during the formaldehyde treatment.Next, use flowing seawater to wash the crabs to remove any residual formaldehyde. To perform cauterization of the crab eyestalk, begin by heating the tip of a metal rod with a blowtorch until the rod is bright red. Cover an anesthetized crab with a wet cotton towel and hold one eye of the crab with sterilized forceps.Press the red hot metal tip onto the eye of the crab for about 10 to 15 seconds until the eyestalk turns an orange or reddish orange color. Disinfect the forceps with 70%ethanol and clean them with sterile tissue paper to ensure no cross-contamination between crabs. After performing ablation on all crabs, dip the metal rod into tap water.To perform surgical ablation, hold the anesthetized crab using thick cotton gloves and cover it with a wet cotton towel. Use sterile forceps to hold one eye of the crab and swiftly cut the eyestalk with surgical scissors. Post-surgery, prepare an overhead tank with 20-parts-per-thousand filtered seawater with continuous aeration.Connect a flexible pipe with the tank for gravitational water flow. Place the crab into a basket and subject it to flowing seawater from the overhead tank. Monitor the crab under flowing seawater until it can move spontaneously.Place the crabs individually in another aquarium with seawater for 30 minutes for further observation. Randomly select newly mature females that have not undergone eyestalk ablation. After removing the top carapace and the hepatopancreas, observe and identify the maturation stage of the crab ovary.The ovarian maturation stages can be identified based on their coloration, with the immature stage one being translucent or creamy white, the early maturing stage two being pale to light yellow, pre-maturing stage three being yellow to light orange, and the fully mature stage four being a dark orange to reddish color. The gonad maturation rate was higher in the eyestalk-ablated females relative to the ones that were not ablated. The highest percentage of stage-three ovaries were found in eyestalk-ablated female crabs.One-way analysis of variance showed significant differences among the experimental female crabs'ovarian maturation stages. The control group had a higher prevalence of immature female crabs relative to the ablated groups. Only the ablated groups were able to produce stage-four female crabs from an immature stage 30 days post-treatment.It is important to ensure that the eyestalk of mud crab has been fully ablated following either cauterization or surgery technique. Eyestalk ablation allows crustacean farmers to induce ovarian maturation and molting easily. It also allows researchers to investigate further the role of eyestalk in regulating mud crab reproduction.

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1.2K 조회수.  Universiti Malaysia Terengganu. 설명된 프로토콜은 진흙 게의 안자 절제 중 스트레스를 최소화하여 안자 절제로 인한 사망 발생을 줄입니다. 시간 효율성은 동물에 대한 스트레스를 최소화하는 열쇠입니다. 절차를 수행하기 전에 모든 것이 준비되었는지 확인하십시오.건강하고 활동적이며 조산한 암컷 머드 크랩을 염소 처리된 수돗물로 세척하여 파편과 기생충을 제거하는 것으로 시작하십시오. 게?...