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.
