眼柄消融促进泥蟹卵巢成熟

Mohammad Asmat-Ullah; Rusydi Rozaimi; Hanafiah Fazhan; Alexander Chong Shu-Chien; Youji Wang; Khor Waiho

doi:10.3791/65039

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眼柄消融促进泥蟹卵巢成熟

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DOI：

10.3791/65039

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2023年3月31日

Mohammad Asmat-Ullah¹ , Rusydi Rozaimi¹ , Hanafiah Fazhan¹^,²^,³ , Alexander Chong Shu-Chien²^,⁴ , Youji Wang⁵ , Khor Waiho¹^,²^,³

¹Higher Institution Centre of Excellence (HICoE), Institute of Tropical Aquaculture and Fisheries, Universiti Malaysia Terengganu, ²Centre for Chemical Biology, Universiti Sains Malaysia, ³Department of Aquaculture, Faculty of Fisheries, Kasetsart University, ⁴School of Biological Sciences, Universiti Sains Malaysia, ⁵International Research Center for Marine Biosciences, Ministry of Science and Technology, Shanghai Ocean University

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摘要

对麻醉后的雌性螃蟹实施了两种眼柄消融方案（即灼烧法和手术法）。对泥蟹进行眼柄消融可加速卵巢成熟，且不会降低存活率。

摘要

泥蟹（Scylla spp.）是广泛分布于印度-西太平洋地区的重要经济甲壳类物种。在养殖过程中，诱导卵巢成熟对于满足市场对成熟泥蟹的需求以及加快苗种生产具有重要意义。眼柄消融是一种促进泥蟹卵巢成熟的有效手段。然而，目前尚无针对泥蟹眼柄消融的标准化操作规程。本研究介绍了两种眼柄消融技术：灼烧法（使用加热的金属工具对麻醉后的蟹进行眼柄消融）和手术法（使用手术剪切除眼柄）。在实施眼柄消融前，选择性成熟雌蟹（甲宽 > 86 mm）使用含海水的冰袋（−20 °C）进行麻醉。当水温降至 4 °C 时，将冰袋从水中移除。眼柄消融后，立即用流动的海水（环境温度：28 °C）使蟹恢复。在整个眼柄消融过程中及操作后均未出现死亡现象。本研究所提供的眼柄消融方案可显著加快泥蟹的卵巢成熟进程。

引言

属于梭子蟹属的四种泥蟹 Scylla 是水产养殖中具有重要商业价值的甲壳类物种¹^,²甲壳类动物（包括泥蟹）的生长及其从未成年（亚成体或青春期）阶段向性成熟（成体）阶段的转变，是通过蜕壳过程实现的，该过程涉及周期性地蜕去较旧且较小的外骨骼。头胸甲宽度（CW）、螯足以及腹部附肢的形态特征被广泛用于判断其性成熟状态。 Scylla 种（复数形式）.³^,⁴^,⁵蜕皮过程受多种激素的调控，并需要消耗大量能量⁶除了正常的蜕壳过程外，蟹类肢体的丢失（无论是自发还是由外界因素引起）均可加速其蜕壳，且不影响其存活率⁷^,⁸^,⁹因此，断肢自切法在软壳泥蟹养殖业中常被用于诱导蜕皮⁷^,⁹.

单侧或双侧眼柄切除术在淡水虾和海水虾的性腺成熟及苗种生产中应用较为广泛¹⁰^,¹¹^,¹²^,¹³。甲壳动物常见的眼柄切除技术包括以下几种：(i) 使用细线在眼柄基部进行结扎¹⁴^,¹⁵；(ii) 使用热镊子或电灼设备灼烧眼柄¹⁶；(iii) 切除或直接夹闭眼柄，使其形成开放性伤口¹²；以及 (iv) 用剃刀切开眼部远端后，通过切口挤出眼柄内容物¹⁷。眼柄X器官是甲壳动物重要的内分泌器官，可调控甲壳动物高血糖激素（CHH）、蜕皮抑制激素（MIH）和卵黄生成抑制激素（VIH）⁶^,¹⁸^,¹⁹^,²⁰^,²¹^,²²。眼柄X器官（或称窦腺复合体）合成并释放属于神经肽激素家族的性腺抑制激素（GIH），也称为卵黄生成抑制激素（VIH）⁶。单侧或双侧眼柄切除可减少GIH的合成，从而使促性腺激素（即性腺刺激激素，GSH）占主导地位，加速甲壳动物的卵巢成熟过程²³^,²⁴^,²⁵^,²⁶。眼柄切除后，在GIH影响消除的情况下，雌性甲壳动物将能量集中用于卵巢发育²⁷。研究表明，单侧眼柄切除已足以诱导甲壳动物的卵巢成熟¹¹，且虾类和蟹类被切除的眼柄在经历数次蜕皮后可再生²⁸。锯蟹属（Scylla spp.）的卵巢发育可分为四个阶段：i）未成熟期（第1阶段），ii）早期发育期（第2阶段），iii）前期成熟期（第3阶段），iv）完全成熟期（第4阶段）²⁹^,³⁰。未成熟卵巢阶段见于未成熟的雌体。在性成熟蜕皮并交配后，未成熟卵巢开始发育，最终在产卵前达到成熟状态（第4阶段）³¹。

眼柄消融方案对于泥蟹亲本的培育和苗种生产至关重要。在全球食品市场中，消费者更青睐卵巢完全成熟（第4期）的成年泥蟹，而非肌肉含量更高的个体，因此这类泥蟹具有更高的商业价值，甚至高于大型雄蟹。目前尚无完整的泥蟹眼柄消融操作方案。本研究所提出的眼柄消融方案通过使用完全麻醉的螃蟹，最大限度地减少动物应激，并降低操作人员被蟹钳伤的风险。该方案简便且成本低廉。本文提供了一种适用于Scylla spp.的眼柄消融操作流程，可有效促进性腺成熟。我们测试了两种眼柄消融技术（烧灼法与手术切除法），并根据雌性泥蟹性腺发育速率比较了两者的效率。

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方案

本实验方案遵循马来西亚实验动物科学协会（Laboratory Animal Science Association of Malaysia）发布的《马来西亚实验动物用于科研目的的护理与使用规范》。实验样本的处死操作依据美国国立卫生研究院（National Institutes of Health）《实验动物护理与使用指南》（NIH Publications No. 8023，1978年修订版）进行。性未成熟雌性泥蟹（橙色泥蟹 S. olivacea）采自马来西亚实兆远湿地（Setiu Wetlands）当地市场（北纬5°66′62′′，东经102°72′33′′）。泥蟹物种根据形态学特征进行鉴定¹。

1. 样品采集与消毒

采集健康、活跃且未成熟的雌性泥蟹（图1）。
注意：未成熟的雌蟹具有三角形、颜色较浅的腹肢，头胸甲宽度（CW）范围为80−85 mm。
用含氯自来水（淡水）清洗螃蟹，以去除碎屑和嗜渗寄生虫。
将螃蟹浸泡在盐度为20 ppt、浓度为150 ppm的甲醛溶液中30分钟。
在甲醛处理期间，使用气石持续提供轻柔的曝气。曝气源可来自中央供气管道或水族箱曝气泵。
用流动的海水冲洗螃蟹，以去除残留的甲醛。

雌性蟹类未成熟与成熟标本的比较解剖图，比例尺为20 mm。
图1：用于鉴定雌性泥蟹性成熟阶段的腹部形态。请点击此处查看此图的放大版本。

2. 驯化

将每只消毒后的雌蟹分别放入独立的32 L圆形水箱中。
在20 ppt盐度条件下培育雌蟹3天，并每天投喂两次（上午09:00和晚上20:00），投喂切碎的海水鱼，投喂量约为蟹体重的4%−5%。
在早晨投喂前，通过虹吸法清除残余及未食用的饲料。
每天更换10%的育蟹海水（20 ppt）。

3. 诱导换羽以达到性成熟

使用无菌剪刀剪除除游泳足以外的所有步足。
1. 用捞网捕获螃蟹，并小心握住。先剪去双侧螯足，然后用剪刀在第二关节处剪去步足。螃蟹会自动自切受损的附肢。附肢自切过程无需麻醉。
附肢自切后立即用淡水清洗螃蟹。
将完成附肢自切的螃蟹分别转移至带孔塑料篮（28 cm 长 × 22 cm 宽 × 7 cm 高）中，并将塑料篮放入玻璃钢水槽（305 cm 长 × 120 cm 宽 × 60 cm 高）内。
注：两个塑料篮可绑在一起并用夹子固定，上方的篮子作为盖子，防止螃蟹逃出。
使用盐度为 20 ppt、水深至少 10 cm 的循环水养殖系统（RAS），确保整个塑料篮完全浸没于水中。
继续每天两次投喂附肢自切后的雌蟹，饲料为切碎的海水鱼，投喂量为蟹体重的 5%−7%。
养殖螃蟹直至其通过蜕壳达到性成熟（约 35 天）。
注：对于野生成熟雌性泥蟹的商业化卵巢成熟与苗种生产，可跳过诱导蜕壳步骤。从野外捕获的成熟雌蟹必须先进行驯化，随后直接实施冷击麻醉及眼柄切除术。

4. 麻醉

选择性成熟的雌性个体，其腹部瓣膜呈深色、椭圆形，且具有 CW >86 mm (图1).
用捞网捕获螃蟹，并将其单独置于小型水族箱中进行麻醉。
经过5分钟的适应期后，向每个水族箱中加入2-苯氧乙醇（2-PE），使其终浓度为2 mL/L，进行15分钟的麻醉处理。
通过观察螃蟹是否缺乏自发运动，确保其已完全麻醉。

5. 眼柄消融

烧灼法
1. 所有操作均应在桌面上并在开放区域进行。
2. 取一根带木柄或塑料手柄的平头镍钢金属棒（例如螺丝刀），并将手柄部分用湿棉布包裹。
3. 将两把不锈钢外科镊子在高压灭菌器中灭菌。
4. 准备一瓶70%乙醇喷雾剂，并远离任何与火源相关的设备，如喷灯和烧红的螺丝刀。准备好纸巾以备使用。
  注意：乙醇高度易燃。请与火源保持安全距离。
5. 将喷灯牢固连接至燃气罐（丁烷）。
  警告：请遵循喷灯和燃气罐上的使用说明。连接燃气罐时，务必确保喷灯处于关闭状态。仔细阅读并遵守燃气罐上注明的所有防火安全注意事项。
6. 佩戴厚棉手套，以防被高温物体烫伤。
7. 将金属棒的尖端置于喷灯火焰中加热，直至金属棒变为亮红色。
8. 用湿棉布覆盖已麻醉的螃蟹。
  注意：应覆盖螃蟹的触角，以避免不必要的损伤。
9. 用已灭菌的镊子夹住螃蟹的一只眼睛。
  注意：首次使用时，镊子需在高压灭菌器中灭菌；后续对其他螃蟹操作时，应使用70%乙醇进行消毒。
10. 将烧红的金属平头端压在螃蟹的眼睛上，轻轻按压约10−15秒，直至眼柄变为橙色或红橙色。操作时需小心，避免损伤周围结构。
  注意：采用烧灼法进行眼柄切除时需要两人配合：一人固定螃蟹，另一人执行切除操作。
11. 使用70%乙醇喷雾对镊子进行消毒，以防止螃蟹之间的交叉污染。
  注意：眼柄切除操作完成后，至少等待5分钟，待镊子冷却后再用70%乙醇消毒，以防引发火灾危险。
12. 完成所有螃蟹的眼柄切除后，将灼热的镍钢金属棒（螺丝刀）浸入自来水冷却。
13. 重复使用前应对毛巾进行消毒。可准备多块毛巾以节省时间。
  注意：先用自来水清洗毛巾，然后将其浸入30 ppm含氯水中5分钟，再用自来水再次冲洗，最后浸入1 g/L硫代硫酸钠溶液中。
14. 关闭喷灯后，将其放置于安全位置，并等待其冷却至环境温度（约30分钟）后再行拆卸。
手术法
1. 在通风良好的环境中进行操作。
2. 将两把外科剪刀和镊子在高压灭菌器中灭菌。
3. 将50 mL 70%乙醇倒入100 mL玻璃烧杯中。
4. 佩戴厚棉手套。
5. 握住已麻醉的螃蟹，并用湿棉布覆盖。
6. 用已灭菌的镊子夹住螃蟹的一只眼睛。
7. 迅速使用已灭菌的外科剪刀剪除眼柄。
  注意：螃蟹伤口部位可能会流失血淋巴。
8. 每次使用后，将剪刀和镊子浸入70%乙醇中消毒，并用纸巾擦干后方可再次使用。

6. 麻醉后护理

制备20 ppt的过滤海水，并将其置于高位水箱中持续曝气。
将一根软管连接至高位水箱，以实现重力驱动的水流。
在切除眼柄后立即把蟹放入篮子中，并用高位水箱流出的海水（环境水温：28 °C）冲洗蟹体。
保持海水持续流动，并观察蟹直至其能够自主活动，表明已从麻醉状态恢复。
注意：海水也可在地面水箱中配制，可使用潜水泵来驱动水流。
将蟹单独置于含曝气的20 ppt海水的水族箱中，继续观察30分钟。
注意：恢复后的蟹将在后续的亲本培育过程中进行单独养殖。

7. 卵巢成熟过程的观察

亲本培育
1. 将成熟的蟹转移至单独的32 L圆形水箱中。
2. 每天两次（上午09:00和晚上20:00）投喂切碎的海洋鱼类（于−20 °C冷冻保存），并在次日早晨投喂前清除未摄食的残饵。
3. 在20 ppt盐度条件下，将亲本单独培育30天。
4. 每日清除粪便，并更换10%的海水（20 ppt）。
解剖
1. 使用70%乙醇清洁解剖托盘、剪刀和镊子。
2. 采用2-PE浸浴麻醉法对雌蟹进行个体麻醉。
3. 随机选取尚未进行眼柄消融的新生成熟雌蟹（在前成熟雌蟹蜕皮后），以确认其性腺发育阶段。
4. 单独处死所有经过眼柄消融处理的实验雌蟹，并鉴定其性腺成熟阶段。使用无菌锋利探针破坏蟹的胸神经节。首先移除头胸甲，然后移除肝胰腺，以暴露卵巢。肉眼或在体视显微镜下观察卵巢颜色，并根据颜色判定卵巢成熟阶段（图2）。
卵巢成熟阶段鉴定
1. 通过肉眼或体视显微镜观察卵巢颜色。
2. 根据颜色特征鉴定卵巢成熟阶段³⁰：未成熟期（第1阶段）呈透明或乳白色；早期发育期（第2阶段）呈浅黄色至淡黄；前期成熟期（第3阶段）呈黄色至浅橙色；完全成熟期（第4阶段）呈深橙色至红褐色。

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结果

性腺成熟
在进行眼柄切除术之前，所有解剖的雌性个体（n = 6）均发现具有乳白色卵巢组织（未成熟卵巢，第1阶段）（图2）。在单独饲养30天后，经眼柄切除的雌性蟹（n = 63；其中31只为电灼法处理，32只为手术法处理）的性腺成熟率高于未经眼柄切除处理的雌性蟹（n = 31）（图3）。眼柄切除的雌性蟹中，处于卵巢前成熟阶段（第3阶段）的比例最高（图3；包括电灼法和手术法两种技术），单因素方差分析（ANOVA）显示各实验组雌性蟹卵巢成熟阶段之间存在显著差异（p < 0.05）（表1）。对照组中未成熟雌性蟹的比例显著高于电灼法和手术法处理组（Tukey's HSD检验，p < 0.001）。在所有成熟阶段中，电灼法与手术法处理组之间雌性蟹所占比例无显著差异（Tukey's HSD检验，所有 p > 0.1）。与对照组相...

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讨论

本方案专为锯缘青蟹（Scylla spp.）的切除眼柄操作而设计，可作为一种有效诱导性腺成熟的方法。该方案可轻松复制，用于泥蟹商业化卵巢成熟生产，并可用于缩短泥蟹苗种生产中的潜伏期（即从一次产卵到下一次产卵之间的时间）。

甲壳动物（例如淡水虾、海虾）的眼柄切除通常用于诱导性腺成熟和非季节性产卵¹¹^,¹²^,¹³。对短尾蟹类进行眼柄切除也已被用于研究蜕皮²⁵^,³²^,³³、激素调控¹⁸、性腺成熟³⁴以及诱导繁殖和繁殖性能³⁵^,

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披露

所有作者均无利益冲突。

致谢

本研究由马来西亚教育部卓越高等学府（HICoE）计划资助，该计划授予马来西亚登嘉楼大学热带水产与渔业研究所（项目编号：63933）。 & Vot 编号 56048）。我们感谢马来西亚登嘉楼大学和赛亚柏佳有限公司的支持。通过感谢马来西亚理科大学（Universiti Sains Malaysia）为Khor Waiho和Hanafiah Fazhan提供的兼聘学术研究员职位。同时感谢私人合作研究基金（Vot. No. 55377）的支持。

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高压灭菌器	HIRAYAMA MANUFACTURING CORPORATION	N/A	MADE IN JAPAN
漂白粉（Hi-Chlon 70%）	Nippon Soda Co.Ltd,Japan	N/A	N/A
喷灯	MR D.I.Y. Group Berhad	N/A	N/A
圆形水箱（32L）	BEST PLASTIC INDUSTRY SDN. BHD.	N/A	N/A
棉质手套（厚款）	MR D.I.Y. Group Berhad	N/A	N/A
棉质毛巾	MR D.I.Y. Group Berhad	N/A	N/A
数字温度计	Hanna Instrument	HI9814	Hanna Instruments GroLine Hydroponics Waterproof pH / EC / TDS / Temp. Portable Meter HI9814
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70% 乙醇	Thermo Scientific Chemicals	033361.M1	使用双蒸水稀释至 70%
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参考文献

Keenan, C. P., Davie, P. J. F., Mann, D. L. A revision of the genus Scylla de Haan, 1833 (Crustacea: Decapoda: Brachyura: Portunidae). Raffles Bulletin of Zoology. 46 (1), 217-245 (1998).
Fazhan, H., et al. Morphological descriptions and morphometric discriminant function analysis reveal an additional four groups of Scylla spp. PeerJ. 8, e8066(2020).
Ikhwanuddin, M., Bachok, Z., Hilmi, M. G., Azmie, G., Zakaria, M. Z. Species diversity, carapace width-body weight relationship, size distribution and sex ratio of mud crab, genus Scylla from Setiu Wetlands of Terengganu coastal waters Malaysia. Journal of Sustainability Science and Management. 5 (2), 97-109 (2010).
Ikhwanuddin, M., Bachok, Z., Mohd Faizal, W. W. Y., Azmie, G., Abol-Munafi, A. B. Size of maturity of mud crab Scylla olivacea (Herbst, 1796) from mangrove areas of Terengganu coastal waters. Journal of Sustainability Science and Management. 5 (2), 134-147 (2010).
Waiho, K., et al. On types of sexual maturity in brachyurans, with special reference to size at the onset of sexual maturity. Journal of Shellfish Research. 36 (3), 807-839 (2017).
Mykles, D. L., Chang, E. S. Hormonal control of the crustacean molting gland: Insights from transcriptomics and proteomics. General and Comparative Endocrinology. 294, 113493(2020).
Fujaya, Y., et al. Is limb autotomy really efficient compared to traditional rearing in soft-shell crab (Scylla olivacea) production. Aquaculture Reports. 18, 100432(2020).
Waiho, K., et al. Moult induction methods in soft-shell crab production. Aquaculture Research. 52 (9), 4026-4042 (2021).
Rahman, M. R., et al. Evaluation of limb autotomy as a promising strategy to improve production performances of mud crab (Scylla olivacea) in the soft-shell farming system. Aquaculture Research. 51 (6), 2555-2572 (2020).
Okumura, T., et al. Expression of vitellogenin and cortical rod proteins during induced ovarian development by eyestalk ablation in the kuruma prawn, Marsupenaeus japonicus. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology. 143 (2), 246-253 (2006).
Pervaiz, P. A., Jhon, S. M., Sikdar-bar, M. Studies on the effect of unilateral eyestalk ablation in maturation of gonads of a freshwater prawn Macrobrachium dayanum. World Journal of Zoology. 6 (2), 159-163 (2011).
Primavera, J. H. Induced maturation and spawning in five-month-old Penaeus monodon Fabricius by eyestalk ablation. Aquaculture. 13 (4), 355-359 (1978).
Shyne Anand, P. S., et al. Reproductive performance of wild brooders of Indian white shrimp, Penaeus indicus: Potential and challenges for selective breeding program. Journal of Coastal Research. 86 (sp1), 65(2019).
Diarte-Plata, G., et al. Eyestalk ablation procedures to minimize pain in the freshwater prawn Macrobrachium americanum. Applied Animal Behaviour Science. 140 (3-4), 172-178 (2012).
Vargas-Téllez, I., et al. Impact of unilateral eyestalk ablation on Callinectes arcuatus (Ordway, 1863) under laboratory conditions: Behavioral evaluation. Latin American Journal of Aquatic Research. 49 (4), 576-594 (2021).
Chu, K. H., Chow, W. K. Effects of unilateral versus bilateral eyestalk ablation on molting and growth of the shrimp, Penaeus chinensis Osbeck, 1765) (Decapoda, Penaeidea). Crustaceana. 62 (3), 225-233 (1992).
Taylor, J. Minimizing the effects of stress during eyestalk ablation of Litopenaeus vannamei females with topical anesthetic and a coagulating agent. Aquaculture. 233 (1-4), 173-179 (2004).
Wang, M., Ye, H., Miao, L., Li, X. Role of short neuropeptide F in regulating eyestalk neuroendocrine systems in the mud crab Scylla paramamosain. Aquaculture. 560, 738493(2022).
Nagaraju, G. P. C. Reproductive regulators in decapod crustaceans: an overview. Journal of Experimental Biology. 214 (1), 3-16 (2011).
Kornthong, N., et al. Characterization of red pigment concentrating hormone (RPCH) in the female mud crab (Scylla olivacea) and the effect of 5-HT on its expression. General and Comparative Endocrinology. 185, 28-36 (2013).
Kornthong, N., et al. Molecular characterization of a vitellogenesis-inhibiting hormone (VIH) in the mud crab (Scylla olivacea) and temporal changes in abundances of VIH mRNA transcripts during ovarian maturation and following neurotransmitter administration. Animal Reproduction Science. 208, 106122(2019).
Liu, C., et al. VIH from the mud crab is specifically expressed in the eyestalk and potentially regulated by transactivator of Sox9/Oct4/Oct1. General and Comparative Endocrinology. 255, 1-11 (2018).
Chen, H. -Y., Kang, B. J., Sultana, Z., Wilder, M. N. Variation of protein kinase C-α expression in eyestalk removal-activated ovaries in whiteleg shrimp, Litopenaeus vannamei. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology. 237 (300), 110552(2019).
Rotllant, G., Nguyen, T. V., Aizen, J., Suwansa-ard, S., Ventura, T. Toward the identification of female gonad-stimulating factors in crustaceans. Hydrobiologia. 825 (1), 91-119 (2018).
Supriya, N. T., Sudha, K., Krishnakumar, V., Anilkumar, G. Molt and reproduction enhancement together with hemolymph ecdysteroid elevation under eyestalk ablation in the female fiddler crab, Uca triangularis (Brachyura: Decapoda). Chinese Journal of Oceanology and Limnology. 35 (3), 645-657 (2017).
Wilder, M. N. Advances in the science of crustacean reproductive physiology and potential applications to new seed production technology. Journal of Coastal Research. 86 (sp1), 6-10 (2019).
Arcos, G. F., Ibarra, A. M., Vazquez-Boucard, C., Palacios, E., Racotta, I. S. Haemolymph metabolic variables in relation to eyestalk ablation and gonad development of Pacific white shrimp Litopenaeus vannamei Boone. Aquaculture Research. 34 (9), 749-755 (2003).
Desai, U. M., Achuthankutty, C. T. Complete regeneration of ablated eyestalk in penaeid prawn, Penaeus monodon. Current Science. 79 (11), 1602-1603 (2000).
Wu, Q., et al. Growth performance and biochemical composition dynamics of ovary, hepatopancreas and muscle tissues at different ovarian maturation stages of female mud crab, Scylla paramamosain. Aquaculture. 515, 734560(2020).
Ghazali, A., Azra, M. N., Noordin, N. M., Abol-Munafi, A. B., Ikhwanuddin, M. Ovarian morphological development and fatty acids profile of mud crab (Scylla olivacea) fed with various diets. Aquaculture. 468 (Part 1), 45-52 (2017).
Farhadi, A., et al. The regulatory mechanism of sexual development in decapod crustaceans. Frontiers in Marine Science. 8, (2021).
Sukardi, P., Prayogo, N. A., Harisam, T., Sudaryono, A. Effect of eyestalk-ablation and differences salinity in rearing pond on molting speed of Scylla serrata. AIP Conference Proceedings. 2094, 020029(2019).
Stella, V. S., López Greco, L. S., Rodríguez, E. M. Effects of eyestalk ablation at different times of the year on molting and reproduction of the estuarine grapsid crab Chasmagnathus granulata (Decapoda, Brachyura). Journal of Crustacean Biology. 20 (2), 239-244 (2000).
Jang, I. K., et al. The effects of manipulating water temperature, photoperiod, and eyestalk ablation on gonad maturation of the swimming crab, Portunus trituberculatus. Crustaceana. 83 (2), 129-141 (2010).
Millamena, O. M., Quinitio, E. The effects of diets on reproductive performance of eyestalk ablated and intact mud crab Scylla serrata. Aquaculture. 181 (1-2), 81-90 (2000).
Zeng, C. Induced out-of-season spawning of the mud crab, Scylla paramamosain (Estampador) and effects of temperature on embryo development. Aquaculture Research. 38 (14), 1478-1485 (2007).
Rana, S. Eye stalk ablation of freshwater crab, Barytelphusa lugubris: An alternative approach of hormonal induced breeding. International Journal of Pure and Applied Zoology. 6 (3), 30-34 (2018).
Yi, S. -K., Lee, S. -G., Lee, J. -M. Preliminary study of seed production of the Micronesian mud crab Scylla serrata (Crustacea: Portunidae) in Korea. Ocean and Polar Research. 31 (3), 257-264 (2009).
Azra, M. N., Abol-Munafi, A. B., Ikhwanuddin, M. A review of broodstock improvement to brachyuran crab: Reproductive performance. International Journal of Aquaculture. 5 (38), 1-10 (2016).
Archibald, K. E., Scott, G. N., Bailey, K. M., Harms, C. A. 2-phenoxyethanol (2-PE) and tricaine methanesulfonate (MS-222) immersion anesthesia of American horseshoe crabs (Limulus polyphemus). Journal of Zoo and Wildlife Medicine. 50 (1), 96-106 (2019).
Muhd-Farouk, H., Abol-Munafi, A. B., Jasmani, S., Ikhwanuddin, M. Effect of steroid hormones 17α-hydroxyprogesterone and 17α-hydroxypregnenolone on ovary external morphology of orange mud crab, Scylla olivacea. Asian Journal of Cell Biology. 9 (1), 23-28 (2013).
Muhd-Farouk, H., Jasmani, S., Ikhwanuddin, M. Effect of vertebrate steroid hormones on the ovarian maturation stages of orange mud crab, Scylla olivacea (Herbst, 1796). Aquaculture. 451, 78-86 (2016).
Ghazali, A., Mat Noordin, N., Abol-Munafi, A. B., Azra, M. N., Ikhwanuddin, M. Ovarian maturation stages of wild and captive mud crab, Scylla olivacea fed with two diets. Sains Malaysiana. 46 (12), 2273-2280 (2017).
Aaqillah-Amr, M. A., Hidir, A., Noordiyana, M. N., Ikhwanuddin, M. Morphological, biochemical and histological analysis of mud crab ovary and hepatopancreas at different stages of development. Animal Reproduction Science. 195, 274-283 (2018).
Amin-Safwan, A., Muhd-Farouk, H., Mardhiyyah, M. P., Nadirah, M., Ikhwanuddin, M. Does water salinity affect the level of 17β-estradiol and ovarian physiology of orange mud crab, Scylla olivacea (Herbst, 1796) in captivity. Journal of King Saud University - Science. 31 (4), 827-835 (2019).
Wu, X., et al. Effect of dietary supplementation of phospholipids and highly unsaturated fatty acids on reproductive performance and offspring quality of Chinese mitten crab, Eriocheir sinensis (H. Milne-Edwards), female broodstock. Aquaculture. 273 (4), 602-613 (2007).
Azra, M. N., Ikhwanuddin, M. A review of maturation diets for mud crab genus Scylla broodstock: Present research, problems and future perspective. Saudi Journal of Biological Sciences. 23 (2), 257-267 (2016).
Maschio Rodrigues, M., López Greco, L. S., de Almeida, L. C. F., Bertini, G. Reproductive performance of Macrobrachium acanthurus (Crustacea, Palaemonidae) females subjected to unilateral eyestalk ablation. Acta Zoologica. 103 (3), 326-334 (2022).
Zhang, C., et al. Changes in bud morphology, growth-related genes and nutritional status during cheliped regeneration in the Chinese mitten crab, Eriocheir sinensis. PLoS One. 13 (12), e0209617(2018).
Zhang, C., et al. Hemolymph transcriptome analysis of Chinese mitten crab (Eriocheir sinensis) with intact, left cheliped autotomy and bilateral eyestalk ablation. Fish & Shellfish Immunology. 81, 266-275 (2018).
Diarte-Plata, G., Sainz-Hernandez, J. C., Aguiñaga-Cruz, J. A., Fierro-Coronado, J. A., Polanco-Torres, A., Puente-Palazuelos, C. Eyestalk ablation procedures to minimize pain in the freshwater prawn Macrobrachium americanum. Applied Animal Behaviour Science. 130 (3-4), 172-178 (2012).
Mirera, D. O., Moksnes, P. O. Comparative performance of wild juvenile mud crab (Scylla serrata) in different culture systems in East Africa: Effect of shelter, crab size and stocking density. Aquaculture International. 23 (1), 155-173 (2015).
Ut, V. N., Le Vay, L., Nghia, T. T., Hong Hanh, T. T. Development of nursery cultures for the mud crab Scylla paramamosain (Estampador). Aquaculture Research. 38 (14), 1563-1568 (2007).
Fazhan, H., et al. Limb loss and feeding ability in the juvenile mud crab Scylla olivacea: Implications of limb autotomy for aquaculture practice. Applied Animal Behaviour Science. 247, 105553(2022).

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重印与许可

勘误

Formal Correction: Erratum: Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs
Posted by JoVE Editors on 5/26/2023. Citeable Link.

An erratum was issued for: Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs. The Introduction, Protocol, Discussion and References were updated.

The forth sentence in the third paragraph of the Introduction has been updated from:

The eyestalk ablation protocol in this work minimizes stress by using fully sedated crabs and minimizes physical injury to personnel from crab bites.

to:

The eyestalk ablation protocol in this work minimizes stress by using fully anesthetized crabs and minimizes physical injury to personnel from crab bites.

The start of the Protocol has been updated from:

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°66′62′′N, 102°72′33′′E) at the Setiu Wetlands in Malaysia. The mud crab species was identified based on morphological characteristics¹.

to:

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 and was approved by the Universiti Malaysia Terengganu's Research Ethics Committee (Animal ethics approval number: UMT/JKEPHMK/2023/96). The sacrifice of the experimental samples was done according to the AVMA Guidelines for the Euthanasia of Animals: 2020 Edition. Sexually pre-mature female mud crabs (orange mud crab Scylla olivacea) were collected from the local market (5°66′62′′N, 102°72′33′′E) at the Setiu Wetlands in Malaysia. The mud crab species was identified based on morphological characteristics¹.

Section 4 of the Protocol has been updated from:

4. Cold-shock anesthesia

Select sexually mature females with a dark-colored oval-shaped abdominal flap with a CW >86 mm (Figure 1).
Catch the crabs with a scoop net, and keep them individually in small aquariums for cold shock anesthesia.
Prepare 2 L of 4 °C to 1 °C seawater (20 ppt) in a transparent plastic aquarium. Maintain the temperature using (−20 °C) ice bags for cold shock anesthesia.
NOTE: Check the temperature with a digital thermometer.
Immerse the crab in the 4 °C seawater until sedated (about 3−5 min).
Ensure the crabs are fully anesthetized by the lack of spontaneous movement. The legs and chelipeds joints will still show minor movements when touched with forceps.

to:

4. Anesthesia

Select sexually mature females with a dark-colored oval-shaped abdominal flap with a CW >86 mm (Figure 1).
Catch the crabs with a scoop net, and keep them individually in small aquariums for anesthesia.
After 5 min of acclimatization period, add 2-phenoxyethanol (2-PE) at 2 mL/L into each aquarium and allow 15 min of anesthesia treatment.
Ensure the crabs are fully anesthetized by the lack of spontaneous movement.

Section 5 of the Protocol has been updated from:

5. Eyestalk ablation

Cauterization technique
1. Perform all procedures on top of a table and in an open area.
2. Take a flat head nickel-steel metal rod (e.g., a screwdriver) with a wooden or plastic handle, and cover the handle with a wet cotton towel.
3. Sterilize two stainless surgical forceps in an autoclave.
4. Prepare 70% ethanol in a spray bottle. Have tissue paper ready for use.
  NOTE: Ethanol is highly flammable. Maintain a safe distance from fire sources.
5. Connect a blowtorch to a gas cylinder (butane) securely.
  CAUTION: Follow the instructions on the blowtorch and gas cylinder. Make sure that the blowtorch is switched off when connecting with the gas cylinder. Read and follow all the fire safety precautions mentioned on the gas cylinder.
6. Wear thick cotton gloves to avoid injury from hot objects.
7. Subject the tip of the metal rod to the fire of the blowtorch until the metal rod is bright red.
8. Cover the anesthetized (sedated) crab with a wet cotton towel.
  NOTE: Cover all the tentacles of the crab to avoid unnecessary damage.
9. Hold one eye of the crab with sterilized forceps.
  NOTE: Sterilize the forceps in an autoclave for first-time use, and disinfect using 70% ethanol for subsequent use on other crabs.
10. Hold the red-hot metal flat tip onto the eye of the crab and press slightly for about 10−15 s until the eyestalk turns an orange or reddish-orange color.
  NOTE: Two people are needed to execute eyestalk ablation following the cauterization method: one to hold the crab and another to perform the ablation procedure.
11. Disinfect the forceps with 70% ethanol spray to ensure no cross-contamination between crabs.
12. After performing the eyestalk ablation on all crabs, dip the hot nickel steel metal rod (screwdriver) into tap water.
13. Disinfect the towel before reuse. Multiple towels can be used to save time.
  NOTE: Wash the towel with tap water, and dip it into 30 ppm chlorinated water for 5 min. Then, wash the towel with tap water again, and dip it in a 1 g/L sodium thiosulphate solution.
14. Keep the blowtorch in a safe place after turning it off, and wait until it returns to environmental temperature (about 30 min) before disconnecting.
Surgery technique
1. Perform the procedure in a well-ventilated area.
2. Sterilize two surgical scissors and forceps in an autoclave.
3. Pour 50 mL of 70% ethanol into a 100 mL glass beaker.
4. Prepare the tincture of iodine solution in a dropper bottle.
  NOTE: Tincture of iodine (iodine tincture or weak iodine solution) is made up of 2%-7% elemental iodine and potassium iodide, or sodium iodide, dissolved in ethanol and water.
5. Wear thick cotton gloves.
6. Hold the sedated crab, and cover it with a wet cotton towel.
7. Hold one eye of the crab with sterilized forceps.
8. Swiftly cut off the eyestalk using sterilized surgical scissors.
  NOTE: Hemolymph may be lost from the wounded part of the crab.
9. Dip the scissors and forceps in 70% ethanol after every use, and dry them using tissue paper before reuse.
10. Apply two to three drops of iodine tincture to the wounded part of the eyestalk immediately after cutting it off.
  NOTE: Tincture of iodine is used for healing and to prevent infection.

to:

5. Eyestalk ablation

Cauterization technique
1. Perform all procedures on top of a table and in an open area.
2. Take a flat head nickel-steel metal rod (e.g., a screwdriver) with a wooden or plastic handle, and cover the handle with a wet cotton towel.
3. Sterilize two stainless surgical forceps in an autoclave.
4. Prepare 70% ethanol in a spray bottle and keep it away from any fire-related sources, such as blow torch and red hot screwdriver. Have tissue paper ready for use.
  NOTE: Ethanol is highly flammable. Maintain a safe distance from fire sources.
5. Connect a blowtorch to a gas cylinder (butane) securely.
  CAUTION: Follow the instructions on the blowtorch and gas cylinder. Make sure that the blowtorch is switched off when connecting with the gas cylinder. Read and follow all the fire safety precautions mentioned on the gas cylinder.
6. Wear thick cotton gloves to avoid injury from hot objects.
7. Subject the tip of the metal rod to the fire of the blowtorch until the metal rod is bright red.
8. Cover the anesthetized crab with a wet cotton towel.
  NOTE: Cover the antennae of the crab to avoid unnecessary damage.
9. Hold one eye of the crab with sterilized forceps.
  NOTE: Sterilize the forceps in an autoclave for first-time use, and disinfect using 70% ethanol for subsequent use on other crabs.
10. Hold the red-hot metal flat tip onto the eye of the crab and press slightly for about 10−15 s until the eyestalk turns an orange or reddish-orange color. Be careful when conducting this step to avoid damage to adjacent structures.
  NOTE: Two people are needed to execute eyestalk ablation following the cauterization method: one to hold the crab and another to perform the ablation procedure.
11. Disinfect the forceps with 70% ethanol spray to ensure no cross-contamination between crabs.
  NOTE: Only perform this step at least waiting for 5 min after the eyestalk ablation procedure to ensure the forceps are cooled down before disinfection using 70% ethanol to prevent potential fire hazards.
12. After performing the eyestalk ablation on all crabs, dip the hot nickel steel metal rod (screwdriver) into tap water.
13. Disinfect the towel before reuse. Multiple towels can be used to save time.
  NOTE: Wash the towel with tap water, and dip it into 30 ppm chlorinated water for 5 min. Then, wash the towel with tap water again, and dip it in a 1 g/L sodium thiosulphate solution.
14. Keep the blowtorch in a safe place after turning it off, and wait until it returns to environmental temperature (about 30 min) before disconnecting.
Surgery technique
1. Perform the procedure in a well-ventilated area.
2. Sterilize two surgical scissors and forceps in an autoclave.
3. Pour 50 mL of 70% ethanol into a 100 mL glass beaker.
4. Wear thick cotton gloves.
5. Hold the anesthetized crab, and cover it with a wet cotton towel.
6. Hold one eye of the crab with sterilized forceps.
7. Swiftly cut off the eyestalk using sterilized surgical scissors.
  NOTE: Hemolymph may be lost from the wounded part of the crab.
8. Dip the scissors and forceps in 70% ethanol after every use, and dry them using tissue paper before reuse.

Step 7.2.2 of the Protocol has been updated from:

Sedate the females individually with the cold shock anesthesia method.

to:

Anesthetize the females individually with the 2-PE immersion anesthesia method.

The Discussion has been updated from:

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-of-season spawning¹¹^,¹²^,¹³. Eyestalk ablation in brachyuran crabs has also been done to study molting²⁵^,³²^,³³, hormonal regulation¹⁸, gonad maturation³⁴, and induced breeding and reproductive performance³⁵^,³⁶^,³⁷^,³⁸^,³⁹. Unilateral or bilateral eyestalk ablation influences the physiology of the crustacean. Eyestalk ablation following the protocol stated in this study also influences the ovarian maturation rate of mud crabs. In the control treatment (without eyestalk ablation), 43.33% ± 5.77% of female crabs had an immature ovary (stage-1). However, in the same rearing period (30 days), eyestalk-ablated female crabs had pre-maturing ovaries (stage-3; 56.67% ± 11.55% and 53.33% ± 15.28% with the cauterization and surgery techniques, respectively), which shows that eyestalk ablation can increase the gonad maturation of mud crabs. Previous studies have also reported that the ovarian development of intact crabs (without eyestalk ablation) is slower than that of eyestalk-ablated crabs²⁵^,³¹. Due to the slower gonadal development in intact crustaceans, eyestalk ablation is widely done in commercial prawn and shrimp hatcheries. In this protocol, the eyestalk-ablated female crabs achieved higher percentages of ovarian maturation compared to the female crabs without the eyestalk ablation treatment (Figure 3).

The gonad maturation of the mud crab is regulated by hormones²¹^,⁴⁰^,⁴¹. The eyestalk contains important endocrine glands (i.e., the X-organ-sinus gland complex) that play vital roles in the gonadal maturation process of mud crabs¹⁸^,²¹. Unilateral eyestalk ablation, either by cauterization or surgery, damages one of the major endocrine glands that is involved in the synthesis and release of inhibiting hormones (e.g., VIH), thereby resulting in a higher level of gonad-stimulating hormones (i.e., VSH).

The ovarian maturation stages of Scylla spp. can be differentiated by observing the ovarian tissue coloration with the naked eye²⁹^,³⁰^,⁴². Translucent or creamy white ovarian tissues are indications of immature ovaries²⁹^,³⁰^,⁴²^,⁴³. In this study, immature ovaries (stage-1) were still found in the group of female crabs without eyestalk ablation due to the slower ovarian maturation process. However, the crabs in the eyestalk-ablated groups (both by the cauterization and surgery techniques) mostly showed pre-maturing ovaries (stage-3), with some individuals exhibiting fully matured ovaries (stage-4). Therefore, the protocol of eyestalk ablation described here can be used to increase ovarian maturation in female mud crabs. This protocol can also be applied directly to wild-collected mature female mud crabs to hasten their seed production. To evaluate the effectiveness of cauterization and surgery methods on mud crab gonad maturation and to ensure the accurate estimation of molting duration, sexually pre-mature crabs were used. After the (induced) molting of sexually pre-mature female crabs, we noticed that their ovaries were still in the immature or early developing stages²⁹^,⁴⁴. After 30 days of rearing the newly mature female crabs (either eyestalk-ablated or without eyestalk ablation), the ovarian development stages (stage-1 to stage-4) were determined by the color of the ovarian tissues. This protocol encourages the use of the cauterization technique to perform eyestalk ablation in mud crabs to avoid any hemolymph loss and prevent infection at the ablated sites. Cauterization immediately seals the wound, whereas the surgery technique requires an additional step of disinfection using iodine. For commercial purposes, larger mature crabs, preferably at a later stage of ovarian maturation, should be selected for eyestalk ablation to shorten the time to reach the fully matured ovary stage for subsequent commerce or brood stock culture. In addition to eyestalk ablation, individual rearing with sand substrate and sufficient feeding, preferably with live feed, can increase the gonad maturation rate of mud crabs in captivity³⁰^,³⁵^,⁴⁵^,⁴⁶.

Crustacean blood is called hemolymph and can be lost during eyestalk ablation. An excessive loss of hemolymph may lead to the death of eyestalk-ablated crabs, especially when performing surgery to remove the eyestalk. The hemolymph can coagulate in the wounded part to prevent loss. The application of a tincture of iodine can prevent infection of the wounded part. However, in comparison to the surgery technique, the cauterization technique seals the wounded part immediately, thereby preventing the loss of hemolymph and possible infection.

Mud crab mortality after unilateral eyestalk ablation with either cauterization or surgery was not found within the first 7 days. Thus, eyestalk ablation can be done with a higher survival rate. Unilateral eyestalk ablation does not hamper the survival rate of the crab³³.

Stress during crab handling and eyestalk ablation may contribute to crab mortality. Proper anesthesia is needed to minimize handling stress during eyestalk ablation. In crustacean eyestalk ablation, chemical anesthetics (i.e., xylocaine, lidocaine) are used at the base of the eyestalk before eyestalk ablation¹⁴^,¹⁵^,¹⁷^,⁴⁷. However, due to the aggressive nature and large size of mud crabs, the use of anesthesia only at the base of the eyestalk is not sufficient and might result in additional stress to the animals during the injection. On the other hand, anesthesia by subjecting them to a lower water temperature is more economical and safer. The use of cold water for anesthesia in mud crabs is common and has been used in other studies due to its efficiency, simplicity, and minimal impact on recovery and survival³⁷^,⁴⁸^,⁴⁹.

Although eyestalk ablation using both cauterization and surgery methods has a minimal effect on crab survival and enhances ovarian maturation, performing eyestalk ablation requires professional mastery of the techniques. The timing between the steps is critical as any delay between protocols adds additional stress for the crabs. Unlike the surgery technique, the cauterization technique is dangerous because it involves the use of flammable equipment (i.e., a blow torch and butane gas). Thus, extra caution is needed when performing the cauterization technique.

Crabs are cannibalistic in nature, and they are known to prey on others that have just completed their molt and are still in their soft-shell conditions⁷^,⁵⁰^,⁵¹. Thus, rearing the crabs individually can avoid unnecessary mortality due to cannibalism. The use of individual rearing in mud crab culture is commonly practiced, both in high-density culture and pond culture, for fattening and soft-shell crab farming purposes⁸^,⁵². This protocol also utilized individual rearing and maintenance. During the transportation of the crabs for rearing or commerce, the crab chelipeds are tied up securely (or even autotomized) to prevent fighting, unnecessary injury, and limb loss³⁴.

The described protocol for eyestalk ablation should be performed with multiple persons. After completing the eyestalk ablation, non-disposable equipment (e.g., the aquarium, tray, towel, etc.) should be disinfected with 30 ppm chlorine. The crabs must be monitored at least twice per day. Any dead crabs, uneaten feed, ablated limbs, or molted crab shells should be swiftly disposed of (i.e., buried in soil with bleaching powder) to prevent any potential for disease spread.

to:

The eyestalk ablation of crustaceans (i.e., freshwater prawn, marine shrimp) is typically done to induce gonad maturation and out-of-season spawning¹¹^,¹²^,¹³. Eyestalk ablation in brachyuran crabs has also been done to study molting²⁵^,³²^,³³, hormonal regulation¹⁸, gonad maturation³⁴, and induced breeding and reproductive performance³⁵^,³⁶^,³⁷^,³⁸^,³⁹. Anesthesia via immersion in 2-phenoxyethanol was used as it is comparable to the use of tricaine methanesulfonate (MS-222) in arthopods but cheaper and does not require the use of additional buffer⁴⁰. Unilateral or bilateral eyestalk ablation influences the physiology of the crustacean. Eyestalk ablation following the protocol stated in this study also influences the ovarian maturation rate of mud crabs. In the control treatment (without eyestalk ablation), 43.33% ± 5.77% of female crabs had an immature ovary (stage-1). However, in the same rearing period (30 days), eyestalk-ablated female crabs had pre-maturing ovaries (stage-3; 56.67% ± 11.55% and 53.33% ± 15.28% with the cauterization and surgery techniques, respectively), which shows that eyestalk ablation can increase the gonad maturation of mud crabs. Previous studies have also reported that the ovarian development of intact crabs (without eyestalk ablation) is slower than that of eyestalk-ablated crabs²⁵^,³¹. Due to the slower gonadal development in intact crustaceans, eyestalk ablation is widely done in commercial prawn and shrimp hatcheries. In this protocol, the eyestalk-ablated female crabs achieved higher percentages of ovarian maturation compared to the female crabs without the eyestalk ablation treatment (Figure 3).

The gonad maturation of the mud crab is regulated by hormones²¹^,⁴¹^,⁴². The eyestalk contains important endocrine glands (i.e., the X-organ-sinus gland complex) that play vital roles in the gonadal maturation process of mud crabs¹⁸^,²¹. Unilateral eyestalk ablation, either by cauterization or surgery, damages one of the major endocrine glands that is involved in the synthesis and release of inhibiting hormones (e.g., VIH), thereby resulting in a higher level of gonad-stimulating hormones (i.e., VSH).

The ovarian maturation stages of Scylla spp. can be differentiated by observing the ovarian tissue coloration with the naked eye²⁹^,³⁰^,⁴³. Translucent or creamy white ovarian tissues are indications of immature ovaries²⁹^,³⁰^,⁴³^,⁴⁴. In this study, immature ovaries (stage-1) were still found in the group of female crabs without eyestalk ablation due to the slower ovarian maturation process. However, the crabs in the eyestalk-ablated groups (both by the cauterization and surgery techniques) mostly showed pre-maturing ovaries (stage-3), with some individuals exhibiting fully matured ovaries (stage-4). Therefore, the protocol of eyestalk ablation described here can be used to increase ovarian maturation in female mud crabs. This protocol can also be applied directly to wild-collected mature female mud crabs to hasten their seed production. To evaluate the effectiveness of cauterization and surgery methods on mud crab gonad maturation and to ensure the accurate estimation of molting duration, sexually pre-mature crabs were used. After the (induced) molting of sexually pre-mature female crabs, we noticed that their ovaries were still in the immature or early developing stages²⁹^,⁴⁵. After 30 days of rearing the newly mature female crabs (either eyestalk-ablated or without eyestalk ablation), the ovarian development stages (stage-1 to stage-4) were determined by the color of the ovarian tissues. This protocol encourages the use of the cauterization technique to perform eyestalk ablation in mud crabs to avoid any hemolymph loss and prevent infection at the ablated sites. Cauterization immediately seals the wound, whereas the surgery technique takes time for the wound to heal and this would allow for chance of infection. For commercial purposes, larger mature crabs, preferably at a later stage of ovarian maturation, should be selected for eyestalk ablation to shorten the time to reach the fully matured ovary stage for subsequent commerce or brood stock culture. In addition to eyestalk ablation, individual rearing with sand substrate and sufficient feeding, preferably with live feed, can increase the gonad maturation rate of mud crabs in captivity³⁰^,³⁵^,⁴⁶^,⁴⁷.

Crustacean blood is called hemolymph and can be lost during eyestalk ablation. An excessive loss of hemolymph may lead to the death of eyestalk-ablated crabs, especially when performing surgery to remove the eyestalk. The hemolymph can coagulate in the wounded part to prevent loss. However, in comparison to the surgery technique, the cauterization technique seals the wounded part immediately, thereby preventing the loss of hemolymph and possible infection.

Stress during crab handling and eyestalk ablation may contribute to crab mortality. Proper anesthesia is needed to minimize handling stress during eyestalk ablation. In crustacean eyestalk ablation, chemical anesthetics (i.e., xylocaine, lidocaine) are used at the base of the eyestalk before eyestalk ablation¹⁴^,¹⁵^,¹⁷^,⁴⁸. However, due to the aggressive nature and large size of mud crabs, the use of anesthesia only at the base of the eyestalk is not sufficient and might result in additional stress to the animals during the injection. On the other hand, anesthesia by subjecting them to a lower water temperature is more economical and safer. The use of cold water for anesthesia in mud crabs is common and has been used in other studies due to its efficiency, simplicity, and minimal impact on recovery and survival³⁷^,⁴⁹^,⁵⁰. In addition, future research on pain assessment following eyestalk ablation on mud crabs is recommended to highlight the change in behaviours associated with pain and stress, as evident in freshwater prawn Macrobrachium americanum⁵¹.

Crabs are cannibalistic in nature, and they are known to prey on others that have just completed their molt and are still in their soft-shell conditions⁷^,⁵²^,⁵³. Thus, rearing the crabs individually can avoid unnecessary mortality due to cannibalism. The use of individual rearing in mud crab culture is commonly practiced, both in high-density culture and pond culture, for fattening and soft-shell crab farming purposes⁸^,⁵³. This protocol also utilized individual rearing and maintenance. During the transportation of the crabs for rearing or commerce, the crab chelipeds are tied up securely (or even autotomized) to prevent fighting, unnecessary injury, and limb loss³⁴.

The References have been updated from:

Keenan, C. P., Davie, P. J. F., Mann, D. L. A revision of the genus Scylla de Haan, 1833 (Crustacea: Decapoda: Brachyura: Portunidae). Raffles Bulletin of Zoology. 46 (1), 217-245 (1998).
Fazhan, H. et al. Morphological descriptions and morphometric discriminant function analysis reveal an additional four groups of Scylla spp. PeerJ. 8, e8066 (2020).
Ikhwanuddin, M., Bachok, Z., Hilmi, M. G., Azmie, G., Zakaria, M. Z. Species diversity, carapace width-body weight relationship, size distribution and sex ratio of mud crab, genus Scylla from Setiu Wetlands of Terengganu coastal waters, Malaysia. Journal of Sustainability Science and Management. 5 (2), 97-109 (2010).
Ikhwanuddin, M., Bachok, Z., Mohd Faizal, W. W. Y., Azmie, G., Abol-Munafi, A. B. Size of maturity of mud crab Scylla olivacea (Herbst, 1796) from mangrove areas of Terengganu coastal waters. Journal of Sustainability Science and Management. 5 (2), 134-147 (2010).
Waiho, K. et al. On types of sexual maturity in brachyurans, with special reference to size at the onset of sexual maturity. Journal of Shellfish Research. 36 (3), 807-839 (2017).
Mykles, D. L., Chang, E. S. Hormonal control of the crustacean molting gland: Insights from transcriptomics and proteomics. General and Comparative Endocrinology. 294, 113493 (2020).
Fujaya, Y. et al. Is limb autotomy really efficient compared to traditional rearing in soft-shell crab (Scylla olivacea) production? Aquaculture Reports. 18, 100432 (2020).
Waiho, K. et al. Moult induction methods in soft-shell crab production. Aquaculture Research. 52 (9), 4026-4042 (2021).
Rahman, M. R. et al. Evaluation of limb autotomy as a promising strategy to improve production performances of mud crab (Scylla olivacea) in the soft-shell farming system. Aquaculture Research. 51 (6), 2555-2572 (2020).
Okumura, T. et al. Expression of vitellogenin and cortical rod proteins during induced ovarian development by eyestalk ablation in the kuruma prawn, Marsupenaeus japonicus. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology. 143 (2), 246-253 (2006).
Pervaiz, P. A., Jhon, S. M., Sikdar-bar, M. Studies on the effect of unilateral eyestalk ablation in maturation of gonads of a freshwater prawn Macrobrachium dayanum. World Journal of Zoology. 6 (2), 159-163 (2011).
Primavera, J. H. Induced maturation and spawning in five-month-old Penaeus monodon Fabricius by eyestalk ablation. Aquaculture. 13 (4), 355-359 (1978).
Shyne Anand, P. S. et al. Reproductive performance of wild brooders of Indian white shrimp, Penaeus indicus: Potential and challenges for selective breeding program. Journal of Coastal Research. 86 (sp1), 65 (2019).
Diarte-Plata, G. et al. Eyestalk ablation procedures to minimize pain in the freshwater prawn Macrobrachium americanum. Applied Animal Behaviour Science. 140 (3-4), 172-178 (2012).
Vargas-Téllez, I. et al. Impact of unilateral eyestalk ablation on Callinectes arcuatus (Ordway, 1863) under laboratory conditions: Behavioral evaluation. Latin American Journal of Aquatic Research. 49 (4), 576-594 (2021).
Chu, K. H., Chow, W. K. Effects of unilateral versus bilateral eyestalk ablation on molting and growth of the shrimp, Penaeus chinensis (Osbeck, 1765) (Decapoda, Penaeidea). Crustaceana. 62 (3), 225-233 (1992).
Taylor, J. Minimizing the effects of stress during eyestalk ablation of Litopenaeus vannamei females with topical anesthetic and a coagulating agent. Aquaculture. 233 (1-4), 173-179 (2004).
Wang, M., Ye, H., Miao, L., Li, X. Role of short neuropeptide F in regulating eyestalk neuroendocrine systems in the mud crab Scylla paramamosain. Aquaculture. 560, 738493 (2022).
Nagaraju, G. P. C. Reproductive regulators in decapod crustaceans: an overview. Journal of Experimental Biology. 214 (1), 3-16 (2011).
Kornthong, N. et al. Characterization of red pigment concentrating hormone (RPCH) in the female mud crab (Scylla olivacea) and the effect of 5-HT on its expression. General and Comparative Endocrinology. 185, 28-36 (2013).
Kornthong, N. et al. Molecular characterization of a vitellogenesis-inhibiting hormone (VIH) in the mud crab (Scylla olivacea) and temporal changes in abundances of VIH mRNA transcripts during ovarian maturation and following neurotransmitter administration. Animal Reproduction Science. 208, 106122 (2019).
Liu, C. et al. VIH from the mud crab is specifically expressed in the eyestalk and potentially regulated by transactivator of Sox9/Oct4/Oct1. General and Comparative Endocrinology. 255, 1-11 (2018).
Chen, H.-Y., Kang, B. J., Sultana, Z., Wilder, M. N. Variation of protein kinase C-α expression in eyestalk removal-activated ovaries in whiteleg shrimp, Litopenaeus vannamei. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology. 237 (300), 110552 (2019).
Rotllant, G., Nguyen, T. V., Aizen, J., Suwansa-ard, S., Ventura, T. Toward the identification of female gonad-stimulating factors in crustaceans. Hydrobiologia. 825 (1), 91-119 (2018).
Supriya, N. T., Sudha, K., Krishnakumar, V., Anilkumar, G. Molt and reproduction enhancement together with hemolymph ecdysteroid elevation under eyestalk ablation in the female fiddler crab, Uca triangularis (Brachyura: Decapoda). Chinese Journal of Oceanology and Limnology. 35 (3), 645-657 (2017).
Wilder, M. N. Advances in the science of crustacean reproductive physiology and potential applications to new seed production technology. Journal of Coastal Research. 86 (sp1), 6-10 (2019).
Arcos, G. F., Ibarra, A. M., Vazquez-Boucard, C., Palacios, E., Racotta, I. S. Haemolymph metabolic variables in relation to eyestalk ablation and gonad development of Pacific white shrimp Litopenaeus vannamei Boone. Aquaculture Research. 34 (9), 749-755 (2003).
Desai, U. M., Achuthankutty, C. T. Complete regeneration of ablated eyestalk in penaeid prawn, Penaeus monodon. Current Science. 79 (11), 1602-1603 (2000).
Wu, Q. et al. Growth performance and biochemical composition dynamics of ovary, hepatopancreas and muscle tissues at different ovarian maturation stages of female mud crab, Scylla paramamosain. Aquaculture. 515, 734560 (2020).
Ghazali, A., Azra, M. N., Noordin, N. M., Abol-Munafi, A. B., Ikhwanuddin, M. Ovarian morphological development and fatty acids profile of mud crab (Scylla olivacea) fed with various diets. Aquaculture. 468 (Part 1), 45-52 (2017).
Farhadi, A. et al. The regulatory mechanism of sexual development in decapod crustaceans. Frontiers in Marine Science. 8 (2021).
Sukardi, P., Prayogo, N. A., Harisam, T., Sudaryono, A. Effect of eyestalk-ablation and differences salinity in rearing pond on molting speed of Scylla serrata. AIP Conference Proceedings. 2094, 020029 (2019).
Stella, V. S., López Greco, L. S., Rodríguez, E. M. Effects of eyestalk ablation at different times of the year on molting and reproduction of the estuarine grapsid crab Chasmagnathus granulata (Decapoda, Brachyura). Journal of Crustacean Biology. 20 (2), 239-244 (2000).
Jang, I. K. et al. The effects of manipulating water temperature, photoperiod, and eyestalk ablation on gonad maturation of the swimming crab, Portunus trituberculatus.Crustaceana. 83 (2), 129-141 (2010).
Millamena, O. M., Quinitio, E. The effects of diets on reproductive performance of eyestalk ablated and intact mud crab Scylla serrata. Aquaculture. 181 (1-2), 81-90 (2000).
Zeng, C. Induced out-of-season spawning of the mud crab, Scylla paramamosain (Estampador) and effects of temperature on embryo development. Aquaculture Research. 38 (14), 1478-1485 (2007).
Rana, S. Eye stalk ablation of freshwater crab, Barytelphusa lugubris: An alternative approach of hormonal induced breeding. International Journal of Pure and Applied Zoology. 6 (3), 30-34 (2018).
Yi, S.-K., Lee, S.-G., Lee, J.-M. Preliminary study of seed production of the Micronesian mud crab Scylla serrata (Crustacea: Portunidae) in Korea. Ocean and Polar Research. 31 (3), 257-264 (2009).
Azra, M. N., Abol-Munafi, A. B., Ikhwanuddin, M. A review of broodstock improvement to brachyuran crab: Reproductive performance. International Journal of Aquaculture. 5 (38), 1-10 (2016).
Muhd-Farouk, H., Abol-Munafi, A. B., Jasmani, S., Ikhwanuddin, M. Effect of steroid hormones 17α-hydroxyprogesterone and 17α-hydroxypregnenolone on ovary external morphology of orange mud crab, Scylla olivacea. Asian Journal of Cell Biology. 9 (1), 23-28 (2013).
Muhd-Farouk, H., Jasmani, S., Ikhwanuddin, M. Effect of vertebrate steroid hormones on the ovarian maturation stages of orange mud crab, Scylla olivacea (Herbst, 1796). Aquaculture. 451, 78-86 (2016).
Ghazali, A., Mat Noordin, N., Abol-Munafi, A. B., Azra, M. N., Ikhwanuddin, M. Ovarian maturation stages of wild and captive mud crab, Scylla olivacea fed with two diets. Sains Malaysiana. 46 (12), 2273-2280 (2017).
Aaqillah-Amr, M. A., Hidir, A., Noordiyana, M. N., Ikhwanuddin, M. Morphological, biochemical and histological analysis of mud crab ovary and hepatopancreas at different stages of development. Animal Reproduction Science. 195, 274-283 (2018).
Amin-Safwan, A., Muhd-Farouk, H., Mardhiyyah, M. P., Nadirah, M., Ikhwanuddin, M. Does water salinity affect the level of 17β-estradiol and ovarian physiology of orange mud crab, Scylla olivacea (Herbst, 1796) in captivity? Journal of King Saud University - Science. 31 (4), 827-835 (2019).
Wu, X. et al. Effect of dietary supplementation of phospholipids and highly unsaturated fatty acids on reproductive performance and offspring quality of Chinese mitten crab, Eriocheir sinensis (H. Milne-Edwards), female broodstock. Aquaculture. 273 (4), 602-613 (2007).
Azra, M. N., Ikhwanuddin, M. A review of maturation diets for mud crab genus Scylla broodstock: Present research, problems and future perspective. Saudi Journal of Biological Sciences. 23 (2), 257-267 (2016).
Maschio Rodrigues, M., López Greco, L. S., de Almeida, L. C. F., Bertini, G. Reproductive performance of Macrobrachium acanthurus (Crustacea, Palaemonidae) females subjected to unilateral eyestalk ablation. Acta Zoologica. 103 (3), 326-334 (2022).
Zhang, C. et al. Changes in bud morphology, growth-related genes and nutritional status during cheliped regeneration in the Chinese mitten crab, Eriocheir sinensis. PLoS One. 13 (12), e0209617 (2018).
Zhang, C. et al. Hemolymph transcriptome analysis of Chinese mitten crab (Eriocheir sinensis) with intact, left cheliped autotomy and bilateral eyestalk ablation. Fish & Shellfish Immunology. 81, 266-275 (2018).
Mirera, D. O., Moksnes, P. O. Comparative performance of wild juvenile mud crab (Scylla serrata) in different culture systems in East Africa: Effect of shelter, crab size and stocking density. Aquaculture International. 23 (1), 155-173 (2015).
Ut, V. N., Le Vay, L., Nghia, T. T., Hong Hanh, T. T. Development of nursery cultures for the mud crab Scylla paramamosain (Estampador). Aquaculture Research. 38 (14), 1563-1568 (2007).
Fazhan, H. et al. Limb loss and feeding ability in the juvenile mud crab Scylla olivacea: Implications of limb autotomy for aquaculture practice. Applied Animal Behaviour Science. 247, 105553 (2022).

to:

Keenan, C. P., Davie, P. J. F., Mann, D. L. A revision of the genus Scylla de Haan, 1833 (Crustacea: Decapoda: Brachyura: Portunidae). Raffles Bulletin of Zoology. 46 (1), 217-245 (1998).
Fazhan, H. et al. Morphological descriptions and morphometric discriminant function analysis reveal an additional four groups of Scylla spp. PeerJ. 8, e8066 (2020).
Ikhwanuddin, M., Bachok, Z., Hilmi, M. G., Azmie, G., Zakaria, M. Z. Species diversity, carapace width-body weight relationship, size distribution and sex ratio of mud crab, genus Scylla from Setiu Wetlands of Terengganu coastal waters, Malaysia. Journal of Sustainability Science and Management. 5 (2), 97-109 (2010).
Ikhwanuddin, M., Bachok, Z., Mohd Faizal, W. W. Y., Azmie, G., Abol-Munafi, A. B. Size of maturity of mud crab Scylla olivacea (Herbst, 1796) from mangrove areas of Terengganu coastal waters. Journal of Sustainability Science and Management. 5 (2), 134-147 (2010).
Waiho, K. et al. On types of sexual maturity in brachyurans, with special reference to size at the onset of sexual maturity. Journal of Shellfish Research. 36 (3), 807-839 (2017).
Mykles, D. L., Chang, E. S. Hormonal control of the crustacean molting gland: Insights from transcriptomics and proteomics. General and Comparative Endocrinology. 294, 113493 (2020).
Fujaya, Y. et al. Is limb autotomy really efficient compared to traditional rearing in soft-shell crab (Scylla olivacea) production? Aquaculture Reports. 18, 100432 (2020).
Waiho, K. et al. Moult induction methods in soft-shell crab production. Aquaculture Research. 52 (9), 4026-4042 (2021).
Rahman, M. R. et al. Evaluation of limb autotomy as a promising strategy to improve production performances of mud crab (Scylla olivacea) in the soft-shell farming system. Aquaculture Research. 51 (6), 2555-2572 (2020).
Okumura, T. et al. Expression of vitellogenin and cortical rod proteins during induced ovarian development by eyestalk ablation in the kuruma prawn, Marsupenaeus japonicus. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology. 143 (2), 246-253 (2006).
Pervaiz, P. A., Jhon, S. M., Sikdar-bar, M. Studies on the effect of unilateral eyestalk ablation in maturation of gonads of a freshwater prawn Macrobrachium dayanum. World Journal of Zoology. 6 (2), 159-163 (2011).
Primavera, J. H. Induced maturation and spawning in five-month-old Penaeus monodon Fabricius by eyestalk ablation. Aquaculture. 13 (4), 355-359 (1978).
Shyne Anand, P. S. et al. Reproductive performance of wild brooders of Indian white shrimp, Penaeus indicus: Potential and challenges for selective breeding program. Journal of Coastal Research. 86 (sp1), 65 (2019).
Diarte-Plata, G. et al. Eyestalk ablation procedures to minimize pain in the freshwater prawn Macrobrachium americanum. Applied Animal Behaviour Science. 140 (3-4), 172-178 (2012).
Vargas-Téllez, I. et al. Impact of unilateral eyestalk ablation on Callinectes arcuatus (Ordway, 1863) under laboratory conditions: Behavioral evaluation. Latin American Journal of Aquatic Research. 49 (4), 576-594 (2021).
Chu, K. H., Chow, W. K. Effects of unilateral versus bilateral eyestalk ablation on molting and growth of the shrimp, Penaeus chinensis (Osbeck, 1765) (Decapoda, Penaeidea). Crustaceana. 62 (3), 225-233 (1992).
Taylor, J. Minimizing the effects of stress during eyestalk ablation of Litopenaeus vannamei females with topical anesthetic and a coagulating agent. Aquaculture. 233 (1-4), 173-179 (2004).
Wang, M., Ye, H., Miao, L., Li, X. Role of short neuropeptide F in regulating eyestalk neuroendocrine systems in the mud crab Scylla paramamosain. Aquaculture. 560, 738493 (2022).
Nagaraju, G. P. C. Reproductive regulators in decapod crustaceans: an overview. Journal of Experimental Biology. 214 (1), 3-16 (2011).
Kornthong, N. et al. Characterization of red pigment concentrating hormone (RPCH) in the female mud crab (Scylla olivacea) and the effect of 5-HT on its expression. General and Comparative Endocrinology. 185, 28-36 (2013).
Kornthong, N. et al. Molecular characterization of a vitellogenesis-inhibiting hormone (VIH) in the mud crab (Scylla olivacea) and temporal changes in abundances of VIH mRNA transcripts during ovarian maturation and following neurotransmitter administration. Animal Reproduction Science. 208, 106122 (2019).
Liu, C. et al. VIH from the mud crab is specifically expressed in the eyestalk and potentially regulated by transactivator of Sox9/Oct4/Oct1. General and Comparative Endocrinology. 255, 1-11 (2018).
Chen, H.-Y., Kang, B. J., Sultana, Z., Wilder, M. N. Variation of protein kinase C-α expression in eyestalk removal-activated ovaries in whiteleg shrimp, Litopenaeus vannamei. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology. 237 (300), 110552 (2019).
Rotllant, G., Nguyen, T. V., Aizen, J., Suwansa-ard, S., Ventura, T. Toward the identification of female gonad-stimulating factors in crustaceans. Hydrobiologia. 825 (1), 91-119 (2018).
Supriya, N. T., Sudha, K., Krishnakumar, V., Anilkumar, G. Molt and reproduction enhancement together with hemolymph ecdysteroid elevation under eyestalk ablation in the female fiddler crab, Uca triangularis (Brachyura: Decapoda). Chinese Journal of Oceanology and Limnology. 35 (3), 645-657 (2017).
Wilder, M. N. Advances in the science of crustacean reproductive physiology and potential applications to new seed production technology. Journal of Coastal Research. 86 (sp1), 6-10 (2019).
Arcos, G. F., Ibarra, A. M., Vazquez-Boucard, C., Palacios, E., Racotta, I. S. Haemolymph metabolic variables in relation to eyestalk ablation and gonad development of Pacific white shrimp Litopenaeus vannamei Boone. Aquaculture Research. 34 (9), 749-755 (2003).
Desai, U. M., Achuthankutty, C. T. Complete regeneration of ablated eyestalk in penaeid prawn, Penaeus monodon. Current Science. 79 (11), 1602-1603 (2000).
Wu, Q. et al. Growth performance and biochemical composition dynamics of ovary, hepatopancreas and muscle tissues at different ovarian maturation stages of female mud crab, Scylla paramamosain. Aquaculture. 515, 734560 (2020).
Ghazali, A., Azra, M. N., Noordin, N. M., Abol-Munafi, A. B., Ikhwanuddin, M. Ovarian morphological development and fatty acids profile of mud crab (Scylla olivacea) fed with various diets. Aquaculture. 468 (Part 1), 45-52 (2017).
Farhadi, A. et al. The regulatory mechanism of sexual development in decapod crustaceans. Frontiers in Marine Science. 8 (2021).
Sukardi, P., Prayogo, N. A., Harisam, T., Sudaryono, A. Effect of eyestalk-ablation and differences salinity in rearing pond on molting speed of Scylla serrata. AIP Conference Proceedings. 2094, 020029 (2019).
Stella, V. S., López Greco, L. S., Rodríguez, E. M. Effects of eyestalk ablation at different times of the year on molting and reproduction of the estuarine grapsid crab Chasmagnathus granulata (Decapoda, Brachyura). Journal of Crustacean Biology. 20 (2), 239-244 (2000).
Jang, I. K. et al. The effects of manipulating water temperature, photoperiod, and eyestalk ablation on gonad maturation of the swimming crab, Portunus trituberculatus.Crustaceana. 83 (2), 129-141 (2010).
Millamena, O. M., Quinitio, E. The effects of diets on reproductive performance of eyestalk ablated and intact mud crab Scylla serrata. Aquaculture. 181 (1-2), 81-90 (2000).
Zeng, C. Induced out-of-season spawning of the mud crab, Scylla paramamosain (Estampador) and effects of temperature on embryo development. Aquaculture Research. 38 (14), 1478-1485 (2007).
Rana, S. Eye stalk ablation of freshwater crab, Barytelphusa lugubris: An alternative approach of hormonal induced breeding. International Journal of Pure and Applied Zoology. 6 (3), 30-34 (2018).
Yi, S.-K., Lee, S.-G., Lee, J.-M. Preliminary study of seed production of the Micronesian mud crab Scylla serrata (Crustacea: Portunidae) in Korea. Ocean and Polar Research. 31 (3), 257-264 (2009).
Azra, M. N., Abol-Munafi, A. B., Ikhwanuddin, M. A review of broodstock improvement to brachyuran crab: Reproductive performance. International Journal of Aquaculture. 5 (38), 1-10 (2016).
Archibald, K. E., Scott, G. N., Bailey, K. M., Harms, C. A. 2-phenoxyethanol (2-PE) and tricaine methanesulfonate (MS-222) immersion anesthesia of American horseshoe crabs (Limulus polyphemus). Journal of Zoo and Wildlife Medicine. 50 (1), 96-106 (2019).
Muhd-Farouk, H., Abol-Munafi, A. B., Jasmani, S., Ikhwanuddin, M. Effect of steroid hormones 17α-hydroxyprogesterone and 17α-hydroxypregnenolone on ovary external morphology of orange mud crab, Scylla olivacea. Asian Journal of Cell Biology. 9 (1), 23-28 (2013).
Muhd-Farouk, H., Jasmani, S., Ikhwanuddin, M. Effect of vertebrate steroid hormones on the ovarian maturation stages of orange mud crab, Scylla olivacea (Herbst, 1796). Aquaculture. 451, 78-86 (2016).
Ghazali, A., Mat Noordin, N., Abol-Munafi, A. B., Azra, M. N., Ikhwanuddin, M. Ovarian maturation stages of wild and captive mud crab, Scylla olivacea fed with two diets. Sains Malaysiana. 46 (12), 2273-2280 (2017).
Aaqillah-Amr, M. A., Hidir, A., Noordiyana, M. N., Ikhwanuddin, M. Morphological, biochemical and histological analysis of mud crab ovary and hepatopancreas at different stages of development. Animal Reproduction Science. 195, 274-283 (2018).
Amin-Safwan, A., Muhd-Farouk, H., Mardhiyyah, M. P., Nadirah, M., Ikhwanuddin, M. Does water salinity affect the level of 17β-estradiol and ovarian physiology of orange mud crab, Scylla olivacea (Herbst, 1796) in captivity? Journal of King Saud University - Science. 31 (4), 827-835 (2019).
Wu, X. et al. Effect of dietary supplementation of phospholipids and highly unsaturated fatty acids on reproductive performance and offspring quality of Chinese mitten crab, Eriocheir sinensis (H. Milne-Edwards), female broodstock. Aquaculture. 273 (4), 602-613 (2007).
Azra, M. N., Ikhwanuddin, M. A review of maturation diets for mud crab genus Scylla broodstock: Present research, problems and future perspective. Saudi Journal of Biological Sciences. 23 (2), 257-267 (2016).
Maschio Rodrigues, M., López Greco, L. S., de Almeida, L. C. F., Bertini, G. Reproductive performance of Macrobrachium acanthurus (Crustacea, Palaemonidae) females subjected to unilateral eyestalk ablation. Acta Zoologica. 103 (3), 326-334 (2022).
Zhang, C. et al. Changes in bud morphology, growth-related genes and nutritional status during cheliped regeneration in the Chinese mitten crab, Eriocheir sinensis. PLoS One. 13 (12), e0209617 (2018).
Zhang, C. et al. Hemolymph transcriptome analysis of Chinese mitten crab (Eriocheir sinensis) with intact, left cheliped autotomy and bilateral eyestalk ablation. Fish & Shellfish Immunology. 81, 266-275 (2018).
Diarte-Plata, G., Sainz-Hernandez, J. C., Aguiñaga-Cruz, J. A., Fierro-Coronado, J. A., Polanco-Torres, A., Puente-Palazuelos, C. Eyestalk ablation procedures to minimize pain in the freshwater prawn Macrobrachium americanum. Applied Animal Behaviour Science. 140 (3-4), 172-178 (2012).
Mirera, D. O., Moksnes, P. O. Comparative performance of wild juvenile mud crab (Scylla serrata) in different culture systems in East Africa: Effect of shelter, crab size and stocking density. Aquaculture International. 23 (1), 155-173 (2015).
Ut, V. N., Le Vay, L., Nghia, T. T., Hong Hanh, T. T. Development of nursery cultures for the mud crab Scylla paramamosain (Estampador). Aquaculture Research. 38 (14), 1563-1568 (2007).
Fazhan, H. et al. Limb loss and feeding ability in the juvenile mud crab Scylla olivacea: Implications of limb autotomy for aquaculture practice. Applied Animal Behaviour Science. 247, 105553 (2022).

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