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Method Article
Due protocolli di ablazione del peduncolo oculare (cioè approcci di cauterizzazione e chirurgia) sono stati eseguiti su granchi femmina anestetizzata. L'ablazione del gambo oculare dei granchi del fango ha accelerato la maturazione delle ovaie senza diminuire il tasso di sopravvivenza.
I granchi del fango (Scylla spp.) sono specie di crostacei commercialmente importanti che possono essere trovati in tutta la regione del Pacifico indo-occidentale. Durante la coltura, l'induzione della maturazione ovarica è importante per soddisfare la domanda dei consumatori di granchi di fango maturi e accelerare la produzione di semi. L'ablazione del gambo oculare è uno strumento efficace per migliorare la maturazione ovarica nei granchi del fango. Tuttavia, non esiste un protocollo standard per l'ablazione del gambo oculare dei granchi del fango. In questo studio vengono descritte due tecniche di ablazione del gambo oculare: cauterizzazione (l'uso di metallo caldo per ablare il gambo oculare di un granchio anestetizzato) e chirurgia (la rimozione del gambo oculare usando forbici chirurgiche). Prima dell'ablazione del peduncolo oculare, le femmine sessualmente mature (CW > 86 mm) sono state anestetizzate usando una borsa del ghiaccio (-20 ° C) con acqua di mare. Quando la temperatura dell'acqua ha raggiunto i 4 °C, la borsa del ghiaccio è stata rimossa dall'acqua. L'acqua di mare corrente (temperatura ambiente: 28 °C) è stata utilizzata per il recupero dall'anestesia immediatamente dopo l'ablazione del peduncolo oculare. La mortalità non si è verificata durante o dopo il processo di ablazione del peduncolo oculare. Il protocollo di ablazione del gambo oculare qui presentato ha accelerato la maturazione ovarica dei granchi del fango.
Tutte e quattro le specie di granchio del fango appartenenti al genere Scylla sono specie di crostacei commercialmente importanti in acquacoltura 1,2. La crescita dei crostacei, compresi i granchi del fango, e la loro trasformazione dalla fase prematura (sub-adulta o pubertale) alla fase sessualmente matura (adulta) avviene attraverso un processo di muta che comporta lo spargimento periodico di esoscheletri più vecchi e più piccoli. La larghezza del carapace (CW), i chelipedi e le morfologie dei lembo addominale sono ampiamente utilizzati per determinare la maturità sessuale di Scylla spp. 3,4,5. Il processo di muta è regolato dall'azione di vari ormoni e richiede un'enorme quantità di energia6. Oltre al normale processo di muta, la perdita degli arti, volontariamente o indotta da fattori esterni, accelera la muta dei granchi senza influire sul loro tasso di sopravvivenza 7,8,9. Pertanto, l'autotomia degli arti è comunemente usata per l'induzione della muta nell'industria dell'allevamento di granchi di fango a guscio molle 7,9.
L'ablazione unilaterale o bilaterale del gambo oculare è per lo più popolare nei gamberi d'acqua dolce e nei gamberetti marini per la maturazione delle gonadi e la produzione di semi10,11,12,13. Le tecniche comuni di ablazione del gambo oculare nei crostacei includono quanto segue: (i) legatura alla base del gambo oculare usando una corda14,15; ii) cauterizzazione del peduncolo oculare mediante pinze calde o dispositivi di elettrocauterizzazione16; iii) rimozione o pizzicamento diretto del gambo oculare per lasciare una ferita aperta12; e (iv) rimozione del contenuto del gambo oculare attraverso l'incisione dopo aver tagliato la porzione distale dell'occhio con un rasoio17. Gli organi X del gambo oculare sono importanti organi endocrini nei crostacei in quanto regolano gli ormoni iperglicemici dei crostacei (CHH), gli ormoni che inibiscono la muta (MIH) e gli ormoni inibitori della vitellogenesi (VIH)6,18,19,20,21,22. Gli organi X del gambo oculare (o il complesso della ghiandola del seno) sintetizzano e rilasciano ormoni che inibiscono le gonadi (GIH), noti anche come ormoni che inibiscono la vitellogenesi (VIH), appartenenti alla famiglia degli ormoni neuropeptidici6. L'ablazione unilaterale o bilaterale del parlato oculare riduce la sintesi di GIH, determinando la dominanza degli ormoni stimolanti (cioè gli ormoni stimolanti le gonadi, GSH) e l'accelerazione del processo di maturazione ovarica nei crostacei23,24,25,26. Senza l'influenza di GIH dopo l'ablazione del gambo oculare, le femmine di crostaceo dedicano la loro energia allo sviluppo ovarico27. È stato riscontrato che l'ablazione unilaterale del gambo oculare è sufficiente per l'induzione della maturazione ovarica nei crostacei11 e che il gambo oculare ablato di gamberetti e granchi può rigenerarsi dopo diverse mute28. Ci sono quattro stadi di sviluppo ovarico registrati in Scylla spp.: i) immaturo (stadio-1), ii) maturazione precoce (stadio-2), iii) pre-maturazione (stadio-3) e iv) completamente maturo (stadio-4)29,30. Lo stadio ovarico immaturo si trova nelle femmine immature. Dopo la muta puberale e l'accoppiamento, l'ovaio immaturo inizia a svilupparsi e infine matura (stadio-4) prima di deporre leuova 31.
Un protocollo di ablazione del gambo oculare è essenziale per lo sviluppo dei riproduttori di granchi del fango e la produzione di semi. Nel mercato alimentare globale, i granchi di fango maturi con ovaie completamente mature (stadio-4) piuttosto che granchi con un contenuto muscolare più elevato sono preferiti dai consumatori e, quindi, hanno un valore commerciale più elevato, persino superiore ai maschi di grandi dimensioni. Non esiste un protocollo completo per l'ablazione del gambo oculare dei granchi del fango. Il protocollo di ablazione del gambo oculare in questo lavoro riduce al minimo lo stress utilizzando granchi completamente anestetizzati e riduce al minimo le lesioni fisiche al personale da morsi di granchio. Questo protocollo è facile ed economico. Qui presentiamo un protocollo per l'ablazione del peduncolo oculare di Scylla spp. che può indurre la maturazione della gonade. Sono state testate due tecniche di ablazione del peduncolo oculare (cauterizzazione e chirurgia) e le loro efficienze sono state confrontate in base al tasso di sviluppo gonadico delle femmine di granchi del fango.
Questo protocollo segue il Codice di condotta malese per la cura e l'uso di animali per scopi scientifici delineato dalla Laboratory Animal Science Association of Malaysia. Il sacrificio dei campioni sperimentali è stato fatto secondo la National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978). Granchi del fango femmina sessualmente prematuri (granchio del fango arancione S. olivacea) sono stati raccolti dal mercato locale (5 ° 66 ′ 62 ′ N, 102 ° 72 ′ 33 ′ E) nelle zone umide di Setiu in Malesia. La specie di granchio del fango è stata identificata in base alle caratteristiche morfologiche1.
1. Raccolta e disinfezione dei campioni
Figura 1: Morfologia addominale delle femmine di granchi del fango utilizzate per identificare le fasi di maturazione sessuale. Fare clic qui per visualizzare una versione ingrandita di questa figura.
2. Acclimatazione
3. Muta indotta per la maturità sessuale
4. Anestesia
5. Ablazione del gambo oculare
6. Cura post-anestesia
7. Osservazione della maturazione ovarica
Maturazione delle gonadi
Tessuti ovarici bianco crema (ovaie immature, stadio 1) sono stati trovati nel 100% delle femmine sezionate (n = 6) prima di eseguire l'ablazione del gambo oculare (Figura 2). Il tasso di maturazione delle gonadi dei granchi femmina ablati con gambo oculare (n = 63; 31 femmine con la tecnica di cauterizzazione e 32 femmine con la tecnica chirurgica) era più alto rispetto ai granchi femmina che non sono stati sottoposti ad ablazione del gambo ocul...
Questo protocollo è stato sviluppato per l'ablazione del peduncolo oculare del granchio del fango, Scylla spp., e può essere applicato come metodo efficace per indurre la maturazione delle gonadi. Questo protocollo può essere facilmente replicato per la maturazione ovarica commerciale dei granchi del fango e può essere implementato per ridurre il periodo di latenza (tempo da una deposizione all'altra) nella produzione di semi di granchio del fango.
L'ablazione del gambo oculare de...
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Questo studio è stato sostenuto dal Ministero della Pubblica Istruzione, Malesia, nell'ambito del programma Higher Institution Centre of Excellence (HICoE), Malesia, accreditato presso l'Istituto di acquacoltura tropicale e pesca, Universiti Malaysia Terengganu (Vot No. 63933 & Vot No. 56048). Riconosciamo il sostegno di Universiti Malaysia Terengganu e Sayap Jaya Sdn. Bhd. attraverso il Private Partnership Research Grant (Vot. No. 55377). Viene inoltre riconosciuta una posizione di Academic Fellow aggiunto da Universiti Sains Malaysia a Khor Waiho e Hanafiah Fazhan.
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 |
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
to:
4. Anesthesia
Section 5 of the Protocol has been updated from:
5. Eyestalk ablation
to:
5. Eyestalk ablation
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:
to:
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