Name: eyestalk ablation to increase ovarian maturation in mud crabs
Uploaded: 2023-03-31T13:30:00
Description: Watch this Scientific Journal Video about Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs at JoVE.com

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

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

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

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

Erratum: Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs

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

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eyestalk ablation to increase ovarian maturation in mud crabs

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

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

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

Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs

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

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

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

eyestalk-ablation-to-increase-ovarian-maturation-in-mud-crabs

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Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are ...

Benefits of Cardiac Resynchronization Therapy in an Asynchronous Heart Failure Model Induced by Left Bundle Branch Ablation and Rapid Pacing

It is now well recognized that heart failure (HF) patients with left bundle branch block (LBBB) derive substantial clinical benefits from cardiac resynchronization therapy (CRT), and LBBB has become one of the important predictors for CRT response. The conventional tachypacing-induced HF model has several major limitations, including absence of stable LBBB and rapid reversal of left ventricular (LV) dysfunction after cessation of pacing. Hence, it is essential to establish an optimal model of chronic HF with isolated LBBB for studying CRT benefits. In the present study, a canine model of asynchronous HF induced by left bundle branch (LBB) ablation and 4 weeks of rapid right ventricular (RV) pacing is established. The RV and right atrial (RA) pacing electrodes via the jugular vein approach, together with an epicardial LV pacing electrode, were implanted for CRT performance. Presented here are the detailed protocols of radiofrequency (RF) catheter ablation, pacing leads implantation, and rapid pacing strategy. Intracardiac and surface electrograms during operation were also provided for a better understanding of LBB ablation. Two-dimensional speckle tracking imaging and aortic velocity time integral (aVTI) were acquired to validate the chronic stable HF model with LV asynchrony and CRT benefits. By coordinating ventricular activation and contraction, CRT uniformed the LV mechanical work and restored LV pump function, which was followed by reversal of LV dilation. Moreover, the histopathological study revealed a significant restoration of cardiomyocyte diameter and collagen volume fraction (CVF) after CRT performance, indicating a histologic and cellular reverse remodeling elicited by CRT. In this report, we described a feasible and valid method to develop a chronic asynchronous HF model, which was suitable for studying structural and biologic reverse remodeling following CRT.

The establishment of a chronic asynchronous heart failure (HF) model by rapid pacing combined with left bundle branch ablation is presented. Two-dimensional speckle tracking imaging and aortic velocity time integral are applied to validate this stable HF model with left ventricular asynchrony and the benefits of cardiac resynchronization therapy.

It is now well recognized that heart failure (HF) patients with left bundle branch block (LBBB) derive substantial clinical benefits from cardiac ...

Reproductive Techniques for Ovarian Monitoring and Control in Amphibians

Ovarian control and monitoring in amphibians require a multi-faceted approach. There are several applications that can successfully induce reproductive behaviors and the acquisition of gametes and embryos for physiological or molecular research. Amphibians contribute to one-quarter to one-third of vertebrate research, and of interest in this context is their contribution to the scientific community&#39;s knowledge of reproductive processes and embryological development. However, most of this knowledge is derived from a small number of species. In recent times, the decimation of amphibians across the globe has required increasing intervention by conservationists. The captive recovery and assurance colonies that continue to emerge in response to the extinction risk make existing research and clinical applications invaluable to the survival and reproduction of amphibians held under human care. The success of any captive population is founded on its health and reproduction and the ability to develop viable offspring that carry forward the most diverse genetic representation of their species. For researchers and veterinarians, the ability to monitor and control ovarian development and health is, therefore, imperative. The focus of this article is to highlight the different assisted reproductive techniques that can be used to monitor and, where appropriate or necessary, control ovarian function in amphibians. Ideally, any reproductive and health issues should be reduced through proper captive husbandry, but, as with any animal, issues of health and reproductive pathologies are inevitable. Non-invasive techniques include behavioral assessments, visual inspection and palpation and morphometric measurements for the calculation of body condition indices and ultrasound. Invasive techniques include hormonal injections, blood sampling, and surgery. Ovarian control can be exercised in a number of ways depending on the application required and species of interest.

The study of amphibian biology provides valuable information on the reproductive, physiological, embryological and developmental processes that drive organisms of many taxonomic groups. Here, we present a comprehensive guide on different methodologies that can be used to study ovarian control and monitoring in amphibians.

Ovarian control and monitoring in amphibians require a multi-faceted approach. There are several applications that can successfully induce reproductive ...

Bovine Ovarian Cortex Tissue Culture

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors from somatic cells and cumulus cell-oocyte communication. Little is known about the ovarian microenvironment and how the cytokines and steroids produced in the surrounding milieu affect follicle progression or arrest. In vitro culture of ovarian cortex enables follicles to develop in a normalized environment that remains supported by adjacent stroma. Our objective was to determine the effect of nutritional Stair-Step diet on the ovarian microenvironment (follicle development, steroid, and cytokine production) through in vitro culture of bovine ovarian cortex. To accomplish this, ovarian cortical pieces were removed from heifers undergoing two different nutritionally developed schemes prior to puberty: Control (traditional nutrition development) and Stair-Step (feeding and restriction during development) that were cut into approximately 0.5-1 mm3 pieces. These pieces were subsequently passed through a series of washes and positioned on a tissue culture insert that is set into a well containing Waymouth&#39;s culture medium. Ovarian cortex was cultured for 7 days with daily culture media changes. Histological sectioning was performed to determine follicle stage changes before and after the culture to determine effects of nutrition and impact of culture without additional treatment. Cortex culture medium was pooled over days to measure steroids, steroid metabolites, and cytokines. There were tendencies for increased steroid hormones in ovarian microenvironment that allowed for follicle progression in the Stair-Step versus Control ovarian cortex cultures. The ovarian cortex culture technique allows for a better understanding of the ovarian microenvironment, and how alterations in endocrine secretion may affect follicle progression and growth from both in vivo and in vitro treatments. This culture method may also prove beneficial for testing potential therapeutics that may improve follicle progression in women to promote fertility.

In vitro culture of bovine ovarian cortex and the effect of nutritional Stair-step diet on ovarian microenvironment is presented. Ovarian cortex pieces were cultured for seven days and steroids, cytokines, and follicle stages were evaluated. The Stair-Step diet treatment had increased steroidogenesis resulting in follicle progression in culture.

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors ...

Megakaryocyte Culture in 3D Methylcellulose-Based Hydrogel to Improve Cell Maturation and Study the Impact of Stiffness and Confinement

The 3D environment leading to both confinement and mechanical constraints is increasingly recognized as an important determinant of cell behavior. 3D culture has thus been developed to better approach the in vivo situation. Megakaryocytes differentiate from hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM). The BM is one of the softest tissues of the body, confined inside the bone. The bone being poorly extensible at the cell scale, megakaryocytes are concomitantly subjected to a weak stiffness and high confinement. This protocol presents a method for the recovery of mouse lineage negative (Lin-) HSPCs by immuno-magnetic sorting and their differentiation into mature megakaryocytes in a 3D medium composed of methylcellulose. Methylcellulose is non-reactive towards megakaryocytes and its stiffness may be adjusted to that of normal bone marrow or increased to mimic a pathological fibrotic marrow. The process to recover the megakaryocytes for further cell analyses is also detailed in the protocol. Although proplatelet extension is prevented within the 3D milieu, it is described below how to resuspend the megakaryocytes in liquid medium and to quantify their capacity to extend proplatelets. Megakaryocytes grown in 3D hydrogel have a higher capacity to form proplatelets compared to those grown in a liquid milieu. This 3D culture allows i) to differentiate progenitors towards megakaryocytes reaching a higher maturation state, ii) to recapitulate phenotypes that may be observed in vivo but go unnoticed in classical liquid cultures, and iii) to study transduction pathways induced by the mechanical cues provided by a 3D environment.

It is now acknowledged that the three-dimensional environment of cells can play an important role in their behavior, maturation and/or differentiation. This protocol describes a three-dimensional cell culture model designed to study the impact of physical containment and mechanical constraints on megakaryocytes.

The 3D environment leading to both confinement and mechanical constraints is increasingly recognized as an important determinant of cell behavior. 3D ...

Cell-Specific Paired Interrogation of the Mouse Ovarian Epigenome and Transcriptome

Assessing cell-type-specific epigenomic and transcriptomic changes are key to understanding ovarian aging. To this end, the optimization of the translating ribosome affinity purification (TRAP) method and the isolation of nuclei tagged in specific cell types (INTACT) method was performed for the subsequent paired interrogation of the cell-specific ovarian transcriptome and epigenome using a novel transgenic NuTRAP mouse model. The expression of the NuTRAP allele is under the control of a floxed STOP cassette and can be targeted to specific ovarian cell types using promoter-specific Cre lines. Since recent studies have implicated ovarian stromal cells in driving premature aging phenotypes, the NuTRAP expression system was targeted to stromal cells using a Cyp17a1-Cre driver. The induction of the NuTRAP construct was specific to ovarian stromal fibroblasts, and sufficient DNA and RNA for sequencing studies were obtained from a single ovary. The NuTRAP model and methods presented here can be used to study any ovarian cell type with an available Cre line.

In this protocol, the translating ribosome affinity purification (TRAP) method and the isolation of nuclei tagged in specific cell types (INTACT) method were optimized for the paired interrogation of the cell-specific ovarian transcriptome and epigenome using the NuTRAP mouse model crossed to a Cyp17a1-Cre mouse line.