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Biology

Ablación del tallo ocular para aumentar la maduración ovárica en cangrejos de fango

Published: March 31, 2023 doi: 10.3791/65039
Automatic Translation
1, 1, 1,2,3, 2,4, 5, 1,2,3
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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Summary

Se realizaron dos protocolos de ablación del tallo ocular (es decir, enfoques de cauterización y cirugía) en cangrejos hembra anestesiados. La ablación ocular de los cangrejos de barro aceleró la maduración de los ovarios sin disminuir la tasa de supervivencia.

Abstract

Los cangrejos de fango (Scylla spp.) son especies de crustáceos comercialmente importantes que se pueden encontrar en toda la región del Indo-Pacífico occidental. Durante el cultivo, la inducción de la maduración ovárica es importante para satisfacer la demanda de los consumidores de cangrejos de fango maduros y acelerar la producción de semillas. La ablación del tallo ocular es una herramienta eficaz para mejorar la maduración ovárica en cangrejos de fango. Sin embargo, no existe un protocolo estándar para la ablación ocular de los cangrejos de fango. En este estudio, se describen dos técnicas de ablación del tallo ocular: cauterización (el uso de metal caliente para extirpar el tallo ocular de un cangrejo anestesiado) y cirugía (la eliminación del tallo ocular con tijeras quirúrgicas). Antes de la ablación del tallo ocular, las hembras sexualmente maduras (CW > 86 mm) fueron anestesiadas usando una bolsa de hielo (-20 ° C) con agua de mar. Cuando la temperatura del agua alcanzó los 4 °C, la bolsa de hielo se retiró del agua. El agua de mar que fluye (temperatura ambiente: 28 ° C) se utilizó para la recuperación de la anestesia inmediatamente después de la ablación del tallo ocular. La mortalidad no ocurrió durante o después del proceso de ablación del tallo ocular. El protocolo de ablación del tallo ocular presentado aquí aceleró la maduración ovárica de los cangrejos de fango.

Introduction

Las cuatro especies de cangrejo de fango pertenecientes al género Scylla son especies de crustáceos comercialmente importantes en la acuicultura 1,2. El crecimiento de crustáceos, incluidos los cangrejos de fango, y su transformación de la fase prematura (subadulta o pubertal) a la fase sexualmente madura (adulto) ocurre a través de un proceso de muda que implica el desprendimiento periódico de exoesqueletos más viejos y más pequeños. El ancho del caparazón (CW), los quelípedos y las morfologías del colgajo abdominal se usan ampliamente para determinar la madurez sexual de Scylla spp. 3,4,5. El proceso de muda está regulado por la acción de varias hormonas y requiere una gran cantidad de energía6. Además del proceso normal de muda, la pérdida de extremidades, ya sea voluntaria o inducida por factores externos, acelera la muda de los cangrejos sin afectar su tasa de supervivencia 7,8,9. Por lo tanto, la autotomía de extremidades se utiliza comúnmente para la inducción de muda en la industria de cultivo de cangrejo de fango de caparazónblando 7,9.

La ablación unilateral o bilateral del tallo ocular es más popular en camarones de agua dulce y camarones marinos para la maduración de las gónadas y la producción de semillas10,11,12,13. Las técnicas comunes de ablación del tallo ocular en crustáceos incluyen las siguientes: (i) ligadura en la base del tallo ocular usando una cuerda14,15; (ii) cauterización del tallo ocular utilizando fórceps calientes o dispositivos de electrocauterio16; (iii) extracción o pellizco directo del tallo ocular para dejar una herida abierta12; y (iv) extracción del contenido del tallo ocular a través de la incisión después de cortar la porción distal del ojo con una navaja de afeitar17. Los órganos X del tallo ocular son órganos endocrinos importantes en los crustáceos, ya que regulan las hormonas hiperglucémicas (CHH) de los crustáceos, las hormonas inhibidoras de la muda (MIH) y las hormonas inhibidoras de la vitelogénesis (VIH)6,18,19,20,21,22. Los órganos X de Eyestalk (o el complejo de glándulas sinusales) sintetizan y liberan hormonas inhibidoras de las gónadas (GIH), también conocidas como hormonas inhibidoras de la vitelogénesis (VIH), pertenecientes a la familia de hormonas neuropeptídicas6. La ablación unilateral o bilateral del tallo ocular reduce la síntesis de GIH, resultando en el predominio de hormonas estimulantes (es decir, hormonas estimulantes de gónadas, GSH) y la aceleración del proceso de maduración ovárica en crustáceos23,24,25,26. Sin la influencia de la GIH después de la ablación ocular, las hembras de crustáceo dedican su energía al desarrollo de los ovarios27. Se ha encontrado que la ablación unilateral del tallo ocular es suficiente para la inducción de la maduración ovárica en crustáceos11 y que el tallo ocular ablacionado de camarones y cangrejos puede regenerarse después de varias mudas28. Hay cuatro etapas de desarrollo ovárico registradas en Scylla spp.: i) inmadura (etapa-1), ii) maduración temprana (etapa-2), iii) premaduración (etapa-3) y iv) completamente madura (etapa-4)29,30. La etapa ovárica inmadura se encuentra en mujeres inmaduras. Después de la muda puberal y el apareamiento, el ovario inmaduro comienza a desarrollarse y finalmente madura (etapa 4) antes de desovar31.

Un protocolo de ablación del tallo ocular es esencial para el desarrollo de reproductores de cangrejo de fango y la producción de semillas. En el mercado mundial de alimentos, los consumidores prefieren los cangrejos de fango maduros con ovarios completamente maduros (etapa 4) en lugar de cangrejos con mayor contenido muscular y, por lo tanto, tienen un valor comercial más alto, incluso más alto que los machos grandes. No existe un protocolo completo para la ablación ocular de los cangrejos de fango. El protocolo de ablación del tallo ocular en este trabajo minimiza el estrés mediante el uso de cangrejos completamente anestesiados y minimiza las lesiones físicas al personal por mordeduras de cangrejo. Este protocolo es fácil y rentable. Aquí, presentamos un protocolo para la ablación ocular de Scylla spp. que puede inducir la maduración de la gónada. Se probaron dos técnicas de ablación del tallo ocular (cauterización y cirugía) y se compararon sus eficiencias en función de la tasa de desarrollo gonadal de las hembras de cangrejo de fango.

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Protocol

Este protocolo sigue el Código de Prácticas de Malasia para el Cuidado y Uso de Animales con Fines Científicos descrito por la Asociación de Ciencia de Animales de Laboratorio de Malasia. El sacrificio de las muestras experimentales se realizó de acuerdo con la Guía de los Institutos Nacionales de Salud para el Cuidado y Uso de Animales de Laboratorio (NIH Publications No. 8023, revisada en 1978). Los cangrejos de fango hembra sexualmente prematuros (cangrejo de fango naranja S. olivacea) fueron recolectados del mercado local (5°66′62′′N, 102°72′33′′E) en los humedales de Setiu en Malasia. La especie de cangrejo de fango fue identificada con base en características morfológicas1.

1. Recogida y desinfección de muestras

  1. Recolecte cangrejos de fango hembras sanas, activas y prematuras (Figura 1).
    NOTA: Los cangrejos hembras prematuros tienen colgajos abdominales triangulares y de color claro junto con un rango CW de 80-85 mm.
  2. Lave los cangrejos con agua del grifo clorada (agua dulce) para eliminar los desechos y los parásitos osmófilos.
  3. Remoje los cangrejos en 150 ppm de formaldehído con 20 ppt de salinidad durante 30 min.
  4. Mantener una aireación continua y suave con piedras de aire durante el tratamiento con formaldehído. La fuente de aireación puede ser de una línea de aireación central o de una bomba de aireación de acuario.
  5. Lave los cangrejos con agua de mar que fluye para eliminar cualquier formaldehído residual.

Figure 1
Figura 1: Morfología abdominal de las hembras de cangrejos de fango utilizadas para identificar las etapas de maduración sexual. Haga clic aquí para ver una versión más grande de esta figura.

2. Aclimatación

  1. Transfiera cada hembra desinfectada a un tanque circular separado de 32 L.
  2. Criar a las hembras durante 3 días en 20 ppt de salinidad y continuar alimentándose dos veces al día (mañana 09:00 am y noche 20:00 pm) con peces marinos picados a aproximadamente 4% −5% del peso corporal del cangrejo.
  3. Elimine el exceso de alimento y el alimento no consumido mediante un sifón antes de la alimentación de la mañana.
  4. Intercambie el 10% del agua de mar de cría de cangrejos (20 ppt) diariamente.

3. Muda inducida para la madurez sexual

  1. Corte todas las piernas excepto las piernas de natación con tijeras esterilizadas.
    1. Atrape el cangrejo con una red de cuchara y sostenga el cangrejo con cuidado. Corte primero ambos quelípedos y luego las piernas para caminar en la segunda articulación con tijeras. El cangrejo autotomizará automáticamente los apéndices dañados. No se requiere anestesia para la autotomía de las extremidades.
  2. Lave el cangrejo en agua dulce inmediatamente después de la autotomía de la extremidad.
  3. Transfiera individualmente los cangrejos autotomizados a cestas de plástico perforadas (28 cm L x 22 cm W x 7 cm H) y colóquelos en un tanque de fibra de vidrio (305 cm L x 120 cm W x 60 cm H).
    NOTA: Se pueden atar y recortar dos canastas juntas. La cesta superior se utiliza como cubierta para que el cangrejo no pueda escapar de la canasta.
  4. Utilice un sistema de acuicultura de recirculación (RAS) con 20 ppt de salinidad y una profundidad de agua de al menos 10 cm para garantizar que toda la canasta de plástico esté sumergida.
  5. Continúe alimentando al cangrejo hembra autotomizado con peces marinos picados dos veces al día al 5% −7% del peso corporal del cangrejo.
  6. Criar los cangrejos hasta que maduren sexualmente a través de la muda (35 días).
    NOTA: La muda inducida se puede omitir para la maduración ovárica comercial y la producción de semillas con cangrejos de fango hembras maduras silvestres. Las hembras maduras cosechadas de la naturaleza deben aclimatarse y someterse directamente a anestesia de choque frío y posterior ablación del tallo ocular.

4. Anestesia

  1. Seleccione hembras sexualmente maduras con un colgajo abdominal ovalado de color oscuro con una CW >86 mm (Figura 1).
  2. Atrape los cangrejos con una red de cuchara y manténgalos individualmente en pequeños acuarios para anestesia.
  3. Después de 5 minutos de período de aclimatación, agregue 2-fenoxietanol (2-PE) a 2 ml / L en cada acuario y permita 15 minutos de tratamiento anestésico.
  4. Asegúrese de que los cangrejos estén completamente anestesiados por la falta de movimiento espontáneo.

5. Ablación del tallo ocular

  1. Técnica de cauterización
    1. Realice todos los procedimientos encima de una mesa y en un área abierta.
    2. Tome una varilla de metal de níquel-acero de cabeza plana (por ejemplo, un destornillador) con un mango de madera o plástico, y cubra el mango con una toalla de algodón húmeda.
    3. Esterilice dos pinzas quirúrgicas inoxidables en un autoclave.
    4. Prepare etanol al 70% en una botella rociadora y manténgalo alejado de cualquier fuente relacionada con el fuego, como el soplete y el destornillador al rojo vivo. Tenga papel de seda listo para usar.
      NOTA: El etanol es altamente inflamable. Mantenga una distancia segura de las fuentes de incendio.
    5. Conecte un soplete a un cilindro de gas (butano) de forma segura.
      PRECAUCIÓN: Siga las instrucciones del soplete y del cilindro de gas. Asegúrese de que el soplete esté apagado cuando se conecte con el cilindro de gas. Lea y siga todas las precauciones de seguridad contra incendios mencionadas en el cilindro de gas.
    6. Use guantes gruesos de algodón para evitar lesiones por objetos calientes.
    7. Sujete la punta de la varilla de metal al fuego del soplete hasta que la varilla de metal esté de color rojo brillante.
    8. Cubra el cangrejo anestesiado con una toalla de algodón húmeda.
      NOTA: Cubra las antenas del cangrejo para evitar daños innecesarios.
    9. Sostenga un ojo del cangrejo con fórceps esterilizados.
      NOTA: Esterilice los fórceps en un autoclave para su primer uso y desinfecte con etanol al 70% para su uso posterior en otros cangrejos.
    10. Sostenga la punta plana de metal al rojo vivo sobre el ojo del cangrejo y presione ligeramente durante aproximadamente 10-15 s hasta que el tallo ocular se vuelva de color naranja o naranja rojizo. Tenga cuidado al realizar este paso para evitar daños a las estructuras adyacentes.
      NOTA: Se necesitan dos personas para ejecutar la ablación de tallo ocular siguiendo el método de cauterización: una para sostener el cangrejo y otra para realizar el procedimiento de ablación.
    11. Desinfecte las pinzas con aerosol de etanol al 70% para garantizar que no haya contaminación cruzada entre cangrejos.
      NOTA: Solo realice este paso al menos esperando 5 minutos después del procedimiento de ablación del tallo ocular para asegurarse de que los fórceps se enfríen antes de la desinfección con etanol al 70% para evitar posibles riesgos de incendio.
    12. Después de realizar la ablación del tallo ocular en todos los cangrejos, sumerja la varilla metálica de acero al níquel caliente (destornillador) en agua del grifo.
    13. Desinfecte la toalla antes de volver a utilizarla. Se pueden usar varias toallas para ahorrar tiempo.
      NOTA: Lave la toalla con agua del grifo y sumérjala en 30 ppm de agua clorada durante 5 minutos. Luego, lave la toalla con agua del grifo nuevamente y sumérjala en una solución de tiosulfato de sodio de 1 g / L.
    14. Mantenga el soplete en un lugar seguro después de apagarlo y espere hasta que vuelva a la temperatura ambiental (aproximadamente 30 minutos) antes de desconectarlo.
  2. Técnica quirúrgica
    1. Realice el procedimiento en un área bien ventilada.
    2. Esterilice dos tijeras quirúrgicas y fórceps en un autoclave.
    3. Vierta 50 ml de etanol al 70% en un vaso de precipitados de vidrio de 100 ml.
    4. Use guantes gruesos de algodón.
    5. Sostenga el cangrejo anestesiado y cúbralo con una toalla de algodón húmeda.
    6. Sostenga un ojo del cangrejo con fórceps esterilizados.
    7. Corte rápidamente el tallo ocular con tijeras quirúrgicas esterilizadas.
      NOTA: La hemolinfa puede perderse de la parte herida del cangrejo.
    8. Sumerja las tijeras y las pinzas en etanol al 70% después de cada uso, y séquelas con papel de seda antes de volver a utilizarlas.

6. Cuidados postanestésicos

  1. Prepare 20 ppt de agua de mar filtrada y manténgala en un tanque superior con aireación continua.
  2. Conecte una tubería flexible con el tanque superior para el flujo de agua gravitacional.
  3. Inmediatamente después de la ablación del tallo ocular, coloque el cangrejo en la canasta y someta el cangrejo al agua de mar que fluye (temperatura del agua ambiente: 28 ° C) desde el tanque superior.
  4. Mantenga el agua de mar fluyendo y controle el cangrejo hasta que pueda moverse espontáneamente, lo que indica la recuperación de la anestesia.
    NOTA: El agua de mar se puede preparar en un tanque de tierra, y se puede usar una bomba de agua sumergible para el flujo de agua.
  5. Mantenga los cangrejos individualmente en 20 ppt de agua de mar con aireación en un acuario durante 30 minutos para una mayor observación.
    NOTA: Los cangrejos recuperados se cultivarán individualmente en el proceso posterior de cultivo de reproductores.

7. Observación de la maduración ovárica

  1. Cría de reproductores
    1. Transfiera los cangrejos maduros a tanques circulares individuales de 32 L.
    2. Continúe alimentando con pescado marino picado (congelado a -20 ° C) dos veces al día (mañana 09:00 am y noche 20:00 pm), y retire el alimento no consumido antes de la alimentación de la mañana.
    3. Criar los reproductores individualmente durante 30 días en 20 ppt de salinidad.
    4. Retire las heces e intercambie el 10% del agua de mar (20 ppt) diariamente.
  2. Disección
    1. Limpie una bandeja de disección, tijeras y pinzas con etanol al 70%.
    2. Anestesiar a las hembras individualmente con el método de anestesia de inmersión 2-PE.
    3. Seleccione al azar hembras recién maduras (después de la muda de hembras prematuras) que no hayan pasado por la ablación del tallo ocular para confirmar sus etapas gonadales.
    4. Sacrifique todas las hembras experimentales ablacionadas por el tallo ocular individualmente e identifique las etapas de maduración de las gónadas. Destruya los ganglios torácicos del cangrejo con un punzón estéril afilado. Retire primero el caparazón superior y luego el hepatopáncreas para hacer visible el ovario. Observe el color del ovario e identifique la etapa de maduración ovárica (Figura 2).
  3. Identificación de etapas de maduración ovárica
    1. Observe el color del ovario a simple vista o bajo un microscopio estereoscópico.
    2. Identificar las etapas de maduración ovárica basadas en la coloración30: la inmadura (etapa-1) muestra un color blanco translúcido o cremoso; La maduración temprana (etapa-2) muestra un color pálido a amarillento claro; (iii) la premaduración (etapa-3) muestra un color amarillo a naranja claro; y (iv) el completamente maduro (etapa-4) muestra un color naranja oscuro a rojizo.

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Representative Results

Maduración de las gónadas
Se encontraron tejidos ováricos blancos cremosos (ovarios inmaduros, estadio-1) en el 100% de las hembras disecadas (n = 6) antes de realizar la ablación del tallo ocular (Figura 2). La tasa de maduración de las gónadas de los cangrejos hembras ablacionados (n = 63; 31 hembras con la técnica de cauterización y 32 hembras con la técnica quirúrgica) fue mayor en comparación con las hembras de cangrejo que no fueron sometidas a ablación ocular (n = 31) después de 30 días de crianza individual (Figura 3). Los mayores porcentajes de ovarios premaduros (estadio-3) se encontraron en las hembras de cangrejo ablacionado (Figura 3; técnicas de cauterización y cirugía), y un análisis unidireccional de varianza (ANOVA) mostró diferencias significativas (p < 0,05) entre las etapas de maduración ovárica de las hembras experimentales (Tabla 1). El grupo control tuvo una mayor prevalencia de cangrejos hembras inmaduras en comparación con los grupos de cauterización y tratamiento quirúrgico (prueba HSD de Tukey, p < 0,001). Los tratamientos de cauterización y cirugía no mostraron diferencias significativas en cuanto al porcentaje de cangrejos hembras en todas las etapas de maduración (prueba HSD de Tukey, todos p > 0,1). Tanto los tratamientos de cauterización (prueba HSD de Tukey , p = 0.004) como la cirugía (prueba HSD de Tukey, p = 0.006) tuvieron porcentajes significativamente más altos de cangrejos hembras en etapa 3 de premaduración que el tratamiento de control, y solo los tratamientos de cauterización y cirugía pudieron producir cangrejos hembras en etapa 4 de una etapa inmadura dentro de los 30 días posteriores al tratamiento (Tabla 2).

Figure 2
Figura 2: Cuatro etapas de maduración ovárica de cangrejos de fango hembras. Las diferencias en la coloración y el volumen de los ovarios entre las etapas están claramente señaladas por las flechas negras. Haga clic aquí para ver una versión más grande de esta figura.

Figure 3
Figura 3: Etapas de maduración ovárica de cangrejos hembras sometidas a ablación del tallo ocular (cirugía y cauterización) y control después del período de crianza de 30 días (n = 94). La barra de error representa la desviación estándar. Las letras superíndice indican diferencias significativas entre los tratamientos en cada etapa de maduración a p < 0,05. Haga clic aquí para ver una versión más grande de esta figura.

Etapa de maduración Suma de cuadrados Df Cuadrado medio F P
Inmaduro (etapa-1) Entre grupos 3755.556 2 1877.778 169 <0,001
Dentro de los grupos 66.667 6 11.111
Total 3822.222 8
Maduración temprana (etapa 2) Entre grupos 1355.556 2 677.778 8.714 0.017
Dentro de los grupos 466.667 6 77.778
Total 1822.222 8
Premaduración (etapa 3) Entre grupos 4688.889 2 2344.444 17.58 0.003
Dentro de los grupos 800 6 133.333
Total 5488.889 8
Completamente maduro (etapa-4) Entre grupos 822.222 2 411.111 9.25 0.015
Dentro de los grupos 266.667 6 44.444
Total 1088.889 8
Nota: La diferencia de medias es significativa a nivel p = 0,05.

Tabla 1: Comparación de las etapas de maduración gonadal de los cangrejos hembra (cauterización y cirugía) y control de cangrejos hembras después de una prueba ANOVA unidireccional. La diferencia de medias fue significativa en p = 0,05.

Etapa de maduración Tratamiento Tratamiento de comparación P
Inmaduro (etapa-1) Cauterización Cirugía 1
Cauterización Control <0,001
Cirugía Control <0,001
Maduración temprana (etapa 2) Cauterización Cirugía 0.129
Cauterización Control 0.014
Cirugía Control 0.232
Premaduración (etapa 3) Cauterización Cirugía 0.934
Cauterización Control 0.004
Cirugía Control 0.006
Completamente maduro (etapa-4) Cauterización Cirugía 0.109
Cauterización Control 0.012
Cirugía Control 0.237
Nota: La diferencia de medias es significativa a nivel p = 0,05.

Tabla 2: Prueba HSD post-hoc de Tukey de las diferencias entre las etapas de maduración gonadal de los tallos oculares (cauterización y quirúrgica) y las etapas de maduración gonadal de las cangrejos hembras. La diferencia de medias fue significativa en p = 0,05.

Tasa de supervivencia
La tasa de supervivencia promedio de las hembras de cangrejo ablacionado fue de 95.45% ± 4.98% (media ± desviación estándar) en el período de crianza de 30 días. No hubo mortalidad dentro de los primeros 7 días después de la ablación y manipulación del tallo ocular. Durante el período de crianza de 30 días después de la ablación del tallo ocular, la tasa de mortalidad no fue significativamente diferente (prueba de Kruskal-Wallis, p = 0,67) entre los tratamientos. La tasa de éxito de la muda de las hembras de cangrejo autotomizadas fue del 80% ± del 2,86% (n = 115).

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Discussion

Este protocolo fue desarrollado para la ablación del tallo ocular del cangrejo de fango, Scylla spp., y se puede aplicar como un método eficiente para inducir la maduración de las gónadas. Este protocolo se puede replicar fácilmente para la maduración comercial de los ovarios de los cangrejos de fango y se puede implementar para reducir el período latente (tiempo de un desove a otro) en la producción de semillas de cangrejo de fango.

La ablación del tallo ocular de los crustáceos (es decir, camarón de agua dulce, camarón marino) se realiza típicamente para inducir la maduración de las gónadas y el desove fuera de temporada11,12,13. La ablación del tallo ocular en cangrejos brachyuran también se ha realizado para estudiar la muda 25,32,33, la regulación hormonal18, la maduración de las gónadas 34 y la reproducción inducida y el rendimiento reproductivo 35,36,37,38,39 . Se utilizó anestesia por inmersión en 2-fenoxietanol ya que es comparable al uso de metanosulfonato de tricaína (MS-222) en artrópodos, pero más barato y no requiere el uso de tampón adicional40. La ablación unilateral o bilateral del tallo ocular influye en la fisiología del crustáceo. La ablación del tallo ocular siguiendo el protocolo establecido en este estudio también influye en la tasa de maduración ovárica de los cangrejos de fango. En el tratamiento control (sin ablación del tallo ocular), el 43,33% ± el 5,77% de las hembras de cangrejo tenían un ovario inmaduro (estadio-1). Sin embargo, en el mismo período de crianza (30 días), las hembras de cangrejo ablacionadas por el tallo ocular tenían ovarios premaduros (etapa 3; 56.67% ± 11.55% y 53.33% ± 15.28% con las técnicas de cauterización y cirugía, respectivamente), lo que muestra que la ablación del tallo ocular puede aumentar la maduración de la gónada de los cangrejos de fango. Estudios previos también han reportado que el desarrollo ovárico de cangrejos intactos (sin ablación del tallo ocular) es más lento que el de los cangrejos ablacionados por el tallo ocular25,31. Debido al desarrollo gonadal más lento en crustáceos intactos, la ablación del tallo ocular se realiza ampliamente en criaderos comerciales de camarones y camarones. En este protocolo, las hembras de cangrejo ablacionadas por el tallo ocular alcanzaron mayores porcentajes de maduración ovárica en comparación con las cangrejos hembras sin el tratamiento de ablación del tallo ocular (Figura 3).

La maduración de las gónadas del cangrejo de fango está regulada por las hormonas21,41,42. El tallo ocular contiene importantes glándulas endocrinas (es decir, el complejo X-órgano-sinusula) que desempeñan un papel vital en el proceso de maduración gonadal de los cangrejos de fango18,21. La ablación unilateral del tallo ocular, ya sea por cauterización o cirugía, daña una de las principales glándulas endocrinas que está involucrada en la síntesis y liberación de hormonas inhibidoras (por ejemplo, VIH), lo que resulta en un mayor nivel de hormonas estimulantes de la gónada (es decir, VSH).

Las etapas de maduración ovárica de Scylla spp. se pueden diferenciar observando la coloración del tejido ovárico a simple vista 29,30,43. Los tejidos ováricos blancos translúcidos o cremosos son indicios de ovarios inmaduros 29,30,43,44. En este estudio, los ovarios inmaduros (etapa 1) todavía se encontraron en el grupo de cangrejos hembras sin ablación del tallo ocular debido al proceso de maduración ovárica más lento. Sin embargo, los cangrejos en los grupos ablacionados por tallo ocular (tanto por las técnicas de cauterización como por cirugía) mostraron principalmente ovarios premaduros (etapa 3), y algunos individuos exhibieron ovarios completamente maduros (etapa 4). Por lo tanto, el protocolo de ablación del tallo ocular descrito aquí se puede utilizar para aumentar la maduración ovárica en cangrejos de fango hembras. Este protocolo también se puede aplicar directamente a los cangrejos de fango hembras maduras recolectadas en la naturaleza para acelerar su producción de semillas. Para evaluar la efectividad de los métodos de cauterización y cirugía en la maduración de las gónadas del cangrejo de fango y para asegurar la estimación precisa de la duración de la muda, se utilizaron cangrejos sexualmente prematuros. Después de la muda (inducida) de cangrejos hembras sexualmente prematuras, notamos que sus ovarios todavía estaban en las etapas inmaduras o de desarrollo temprano29,45. Después de 30 días de criar a los cangrejos hembras recién maduros (ya sea con tallo ocular o sin ablación ocular), las etapas de desarrollo ovárico (etapa 1 a etapa 4) se determinaron por el color de los tejidos ováricos. Este protocolo fomenta el uso de la técnica de cauterización para realizar la ablación del tallo ocular en cangrejos de fango para evitar cualquier pérdida de hemolinfa y prevenir la infección en los sitios ablacionados. La cauterización sella inmediatamente la herida, mientras que la técnica quirúrgica toma tiempo para que la herida sane y esto permitiría la posibilidad de infección. Para fines comerciales, los cangrejos maduros más grandes, preferiblemente en una etapa posterior de la maduración ovárica, deben seleccionarse para la ablación del tallo ocular para acortar el tiempo para alcanzar la etapa de ovario completamente madura para el comercio posterior o el cultivo de reproductores. Además de la ablación del tallo ocular, la cría individual con sustrato de arena y alimentación suficiente, preferiblemente con alimento vivo, puede aumentar la tasa de maduración de las gónadas de los cangrejos de fango en cautiverio 30,35,46,47.

La sangre del crustáceo se llama hemolinfa y se puede perder durante la ablación del tallo ocular. Una pérdida excesiva de hemolinfa puede llevar a la muerte de los cangrejos ablacionados por el tallo ocular, especialmente cuando se realiza una cirugía para extirpar el tallo ocular. La hemolinfa puede coagularse en la parte herida para evitar la pérdida. Sin embargo, en comparación con la técnica quirúrgica, la técnica de cauterización sella la parte herida inmediatamente, evitando así la pérdida de hemolinfa y una posible infección.

La mortalidad del cangrejo de fango después de la ablación unilateral del tallo ocular con cauterización o cirugía no se encontró dentro de los primeros 7 días. Por lo tanto, la ablación del tallo ocular se puede hacer con una mayor tasa de supervivencia. La ablación unilateral del tallo ocular no dificulta la tasa de supervivencia del cangrejo33.

El estrés durante el manejo del cangrejo y la ablación del tallo ocular pueden contribuir a la mortalidad del cangrejo. Se necesita anestesia adecuada para minimizar el estrés de manejo durante la ablación del tallo ocular. En la ablación del tallo ocular de los crustáceos, los anestésicos químicos (es decir, xilocaína, lidocaína) se utilizan en la base del tallo ocular antes de la ablación del tallo ocular14,15,17,48. Sin embargo, debido a la naturaleza agresiva y el gran tamaño de los cangrejos de fango, el uso de anestesia solo en la base del tallo ocular no es suficiente y podría provocar estrés adicional para los animales durante la inyección. Por otro lado, la anestesia sometiéndolas a una temperatura del agua más baja es más económica y segura. El uso de agua fría para la anestesia en cangrejos de fango es común y ha sido utilizado en otros estudios debido a su eficiencia, simplicidad y mínimo impacto en la recuperación y supervivencia 37,49,50. Además, se recomienda la investigación futura sobre la evaluación del dolor después de la ablación del tallo ocular en cangrejos de fango para resaltar el cambio en los comportamientos asociados con el dolor y el estrés, como es evidente en el camarón de agua dulce Macrobrachium americanum51.

Aunque la ablación del tallo ocular utilizando métodos de cauterización y cirugía tiene un efecto mínimo sobre la supervivencia del cangrejo y mejora la maduración ovárica, realizar la ablación del tallo ocular requiere el dominio profesional de las técnicas. El tiempo entre los pasos es crítico ya que cualquier retraso entre protocolos agrega estrés adicional para los cangrejos. A diferencia de la técnica quirúrgica, la técnica de cauterización es peligrosa porque implica el uso de equipos inflamables (es decir, un soplete y gas butano). Por lo tanto, se necesita precaución adicional al realizar la técnica de cauterización.

Los cangrejos son de naturaleza caníbal, y se sabe que se aprovechan de otros que acaban de completar su muda y todavía están en sus condiciones de caparazón blando 7,52,53. Por lo tanto, criar los cangrejos individualmente puede evitar la mortalidad innecesaria debido al canibalismo. El uso de la cría individual en el cultivo de cangrejo de fango se practica comúnmente, tanto en el cultivo de alta densidad como en el cultivo en estanques, con fines de engorde y cultivo de cangrejo de caparazón blando 8,53. Este protocolo también utilizó la cría y el mantenimiento individual. Durante el transporte de los cangrejos para la cría o el comercio, los quelípedos de cangrejo se atan de forma segura (o incluso se autotomizan) para evitar peleas, lesiones innecesarias y pérdida de extremidades34.

El protocolo descrito para la ablación del tallo ocular debe realizarse con varias personas. Después de completar la ablación del tallo ocular, el equipo no desechable (por ejemplo, el acuario, la bandeja, la toalla, etc.) debe desinfectarse con 30 ppm de cloro. Los cangrejos deben ser monitoreados al menos dos veces al día. Cualquier cangrejo muerto, alimento no consumido, extremidades ablacionadas o conchas de cangrejo mudadas deben eliminarse rápidamente (es decir, enterrarse en el suelo con polvo blanqueador) para evitar cualquier potencial de propagación de enfermedades.

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Disclosures

Ninguno de los autores tiene ningún conflicto de intereses.

Acknowledgments

Este estudio fue apoyado por el Ministerio de Educación, Malasia, bajo el programa del Centro de Excelencia de la Institución Superior (HICoE), Malasia, acreditado ante el Instituto de Acuicultura y Pesca Tropical, Universiti Malaysia Terengganu (Vot No. 63933 & Vot No. 56048). Reconocemos el apoyo de Universiti Malaysia Terengganu y Sayap Jaya Sdn. Bhd. a través de la Beca de Investigación de Asociación Privada (Vot. No. 55377). También se reconoce un puesto de becario académico adjunto de la Universidad Sains de Malasia a Khor Waiho y Hanafiah Fazhan.

Materials

Name Company Catalog Number Comments
Aeration tube  Ming Yu Three N/A aquarium and pet shop
Airstone Ming Yu Three N/A aquarium and pet shop
Autoclave machine HIRAYAMA MANUFACTURING CORPORATION N/A MADE IN JAPAN
Bleaching powder (Hi-Chlon 70%) Nippon Soda Co.Ltd,Japan N/A N/A
Blow torch  MR D.I.Y. Group Berhad N/A N/A
Circular tank (32L) BEST PLASTIC INDUSTRY SDN. BHD.  N/A N/A
Cotton hand gloves (thick)  MR D.I.Y. Group Berhad N/A N/A
Cotton towel MR D.I.Y. Group Berhad N/A N/A
Digital thermometer Hanna Instrument HI9814 Hanna Instruments GroLine Hydroponics Waterproof pH / EC / TDS / Temp. Portable Meter HI9814
Digital Vernier Caliper INSIZE Co., Ltd. N/A
Dissecting tray Hatcheri AKUATROP  N/A Research Center of Universiti Malaysia Terengganu
Dropper bottle/Plastic Pipettes Dropper Shopee Malaysia N/A N/A
Ethanol 70% Thermo Scientific Chemicals 033361.M1 Diluted to 70% using double distilled water
Fiberglass tank (1 ton) Hatcheri AKUATROP  N/A Research Center of Universiti Malaysia Terengganu
Fine sand N/A N/A collected from Sea beach of Universiti Malaysia Terengganu
First Aid Kits Watsons Malaysia N/A N/A
Flat head nickel steel metal rod (Screw driver) MR D.I.Y. Group Berhad N/A N/A
Formaldehyde Thermo Scientific Chemicals 119690010
Gas cylinder (butane gas) for blow  torch MR D.I.Y. Group Berhad N/A N/A
Gas lighter gun (long head) MR D.I.Y. Group Berhad N/A N/A
Glass beaker (100 mL)) Corning Life Sciences 1000-100
Ice bag  Watsons Malaysia N/A N/A
Perforated plastic baskets  Eco-Shop Marketing Sdn. Bhd. N/A N/A
PVC pipe 15mm Bina Plastic Industries Sdn Bhd (HQ) N/A N/A
Refractometer ATAGO CO.,LTD.
Refrigerator Sharp Corporation Japan N/A Chest Freezer SHARP 110L - SJC 118
Scoop net MR D.I.Y. Group Berhad N/A
Seawater Hatcheri AKUATROP  N/A Research Center of Universiti Malaysia Terengganu
Siphoning pipe MR D.I.Y. Group Berhad N/A N/A
Spray bottle Mr. DIY Sdn Bhd N/A N/A
Stainless surgical forceps  N/A N/A N/A
Stainless surgical scissors  N/A N/A N/A
Submersible water pump  AS N/A model: Astro 4000
Tincture of iodine solution  (Povidone Iodine) Farmasi Fajr Sdn Bhd N/A N/A
Tissue paper  N/A N/A
Transparent plastic aquarium Ming Yu Three N/A aquarium and pet shop
Waterproof table Hatcheri AKUATROP  N/A Research Center of Universiti Malaysia Terengganu

DOWNLOAD MATERIALS LIST

References

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Erratum

Formal Correction: Erratum: Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs
Posted by JoVE Editors on 05/26/2023. 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

  1. Select sexually mature females with a dark-colored oval-shaped abdominal flap with a CW >86 mm (Figure 1).
  2. Catch the crabs with a scoop net, and keep them individually in small aquariums for cold shock anesthesia.
  3. 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.
  4. Immerse the crab in the 4 °C seawater until sedated (about 3−5 min).
  5. 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

  1. Select sexually mature females with a dark-colored oval-shaped abdominal flap with a CW >86 mm (Figure 1).
  2. Catch the crabs with a scoop net, and keep them individually in small aquariums for anesthesia.
  3. 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.
  4. Ensure the crabs are fully anesthetized by the lack of spontaneous movement.

Section 5 of the Protocol has been updated from:

5. Eyestalk ablation

  1. 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.
  2. 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

  1. 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.
  2. 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:

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. 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.

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 ablation14,15,17,48. 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.

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 conditions7,52,53. 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 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.

The References have been updated from:

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  2. Fazhan, H. et al. Morphological descriptions and morphometric discriminant function analysis reveal an additional four groups of Scylla spp. PeerJ. 8, e8066 (2020).
  3. 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).
  4. 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).
  5. 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).
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  10. 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).
  11. 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).
  12. Primavera, J. H. Induced maturation and spawning in five-month-old Penaeus monodon Fabricius by eyestalk ablation. Aquaculture. 13 (4), 355-359 (1978).
  13. 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).
  14. 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).
  15. 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).
  16. 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).
  17. 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).
  18. 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).
  19. Nagaraju, G. P. C. Reproductive regulators in decapod crustaceans: an overview. Journal of Experimental Biology. 214 (1), 3-16 (2011).
  20. 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).
  21. 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).
  22. 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).
  23. 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).
  24. 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).
  25. 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).
  26. 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).
  27. 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).
  28. Desai, U. M., Achuthankutty, C. T. Complete regeneration of ablated eyestalk in penaeid prawn, Penaeus monodon. Current Science. 79 (11), 1602-1603 (2000).
  29. 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).
  30. 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).
  31. Farhadi, A. et al. The regulatory mechanism of sexual development in decapod crustaceans. Frontiers in Marine Science. 8 (2021).
  32. 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).
  33. 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).
  34. 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).
  35. 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).
  36. 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).
  37. 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).
  38. 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).
  39. 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).
  40. 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).
  41. 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).
  42. 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).
  43. 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).
  44. 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).
  45. 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).
  46. 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).
  47. 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).
  48. 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).
  49. 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).
  50. 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).
  51. 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).
  52. 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:

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  4. 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).
  5. 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).
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  10. 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).
  11. 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).
  12. Primavera, J. H. Induced maturation and spawning in five-month-old Penaeus monodon Fabricius by eyestalk ablation. Aquaculture. 13 (4), 355-359 (1978).
  13. 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).
  14. 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).
  15. 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).
  16. 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).
  17. 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).
  18. 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).
  19. Nagaraju, G. P. C. Reproductive regulators in decapod crustaceans: an overview. Journal of Experimental Biology. 214 (1), 3-16 (2011).
  20. 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).
  21. 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).
  22. 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).
  23. 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).
  24. 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).
  25. 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).
  26. 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).
  27. 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).
  28. Desai, U. M., Achuthankutty, C. T. Complete regeneration of ablated eyestalk in penaeid prawn, Penaeus monodon. Current Science. 79 (11), 1602-1603 (2000).
  29. 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).
  30. 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).
  31. Farhadi, A. et al. The regulatory mechanism of sexual development in decapod crustaceans. Frontiers in Marine Science. 8 (2021).
  32. 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).
  33. 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).
  34. 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).
  35. 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).
  36. 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).
  37. 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).
  38. 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).
  39. 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).
  40.  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).
  41. 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).
  42. 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).
  43. 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).
  44. 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).
  45. 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).
  46. 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).
  47. 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).
  48. 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).
  49. 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).
  50. 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).
  51. 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). 
  52. 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).
  53. 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).
  54. 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).
Ablación del tallo ocular para aumentar la maduración ovárica en cangrejos de fango
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Asmat-Ullah, M., Rozaimi, R.,… More

Asmat-Ullah, M., Rozaimi, R., Fazhan, H., Shu-Chien, A. C., Wang, Y., Waiho, K. Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs. J. Vis. Exp. (193), e65039, doi:10.3791/65039 (2023).

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