WWW.WAS.ORG • WORLD AQUACULTURE • DECEMBER 2025 23 (CONTINUED ON PAGE 24) for water clarity and for avoiding the buildup of harmful compounds in a closed system where water exchange is limited. By comparing the tightly controlled environment of Batch 1 with the more ecologically integrated system of Batch 2, the study generated valuable insights into the role of natural microbial networks in maintaining oyster health. The findings suggest that while engineered systems can provide precision, bio-integrated systems — through natural microbial interactions and self-regulating dynamics — may offer comparable, and in some respects, superior conditions for pearl formation and oyster survival. This comparative approach reinforces the importance of both technological precision and ecological balance in the design of sustainable onshore aquaculture systems. Anesthesia and Pre-Surgical Handling To ensure humane, non-traumatic handling during surgical implantation and mantle tissue manipulation, all oysters underwent pre-surgical anesthesia using Magnesium sulfate (MgSO4). This non-lethal anesthetic has been widely validated in molluscan research for its neuromuscular relaxing properties and its safety across various bivalve species, including Pinctada margaritifera and Pteria penguin. Magnesium ions (Mg2+) function as competitive inhibitors at calcium-binding sites, suppressing synaptic transmission at neuromuscular junctions. In oysters, this action prevents adductor muscle contraction, inducing a relaxed, passive state. This state is characterized by valve opening, exposure of the mantle edge and gill lamellae, and reduced stress and tissue tension — factors crucial for successful graft acceptance and post-operative healing. To achieve this anesthetic effect, a solution was prepared using a concentration of 100 grams of MgSO4·7H2O per liter of filtered, UV-sterilized seawater. The preparation method involved first dissolving the magnesium sulfate in a small volume of freshwater to ensure complete dissolution, after which it was diluted with seawater to attain full homogeneity and physiological compatibility. During exposure, water temperature was carefully maintained between 26 to 28°C to optimize ion uptake and reduce metabolic stress. The oysters were immersed in the solution for a duration of 40 to 45 minutes within insulated 100-liter holding tanks. These tanks were gently aerated and covered to minimize light, vibration, and external disturbances. In some trials, oysters underwent an optional pre-treatment involving air exposure for 30 to 45 minutes prior to immersion. This step, based on established field protocols for Ostrea lurida, was found to enhance the oysters’ responsiveness to the anesthetic. Anesthesia was considered successful when specific physiological markers were observed. These included partial to full opening of the shell valves without forced gaping, an absence of clamping or retraction response upon gentle tactile probing of the mantle edge, stable exposure of the gill lamellae and pallial tissues, and minimal mucus secretion with no shell closure reflex during handling. Once these criteria were met, the anesthetized oysters were carefully transferred onto sterile surgical trays, placed on moistened pads to prevent desiccation, and subjected to implantation procedures under aseptic conditions. Surgical Implantation and Recovery Post-anesthesia observations revealed that recovery from magnesium sulfate-induced anesthesia was spontaneous, rapid, and non-lethal in all treated oysters. After surgical implantation, oysters were returned to clean, aerated seawater at ambient temperature. Within 1 to 2 hours, most individuals resumed normal valve closure, pumping and filtration behavior, and feeding activity. No signs of prolonged stress, abnormal behavior, or mortality attributable to anesthesia were noted in either Batch 1 or Batch 2 throughout the experimental period. The use of magnesium sulfate as a noninvasive neuromuscular relaxant significantly improved surgical outcomes by minimizing tissue trauma and supporting rapid physiological stabilization. This method played a crucial role in achieving low graft rejection, high implant retention, and ethical handling, forming an essential component of the onshore rearing approach demonstrated in this study. Surgical implantation was conducted under sterile, cooled conditions to minimize the risk of microbial contamination and to reduce inflammation at the graft site. This sterile environment was essential to ensure a high rate of successful graft acceptance and to prevent post-operative complications. A total of 240 oysters were selected for the procedure and were evenly distributed across four separate experimental tanks to maintain consistent environmental conditions and minimize stress from overcrowding. The surgical procedure began with the careful extraction of mantle tissue from donor oysters. A small section, approximately 2 mm by 2 mm in size, was aseptically excised from the posterior end of healthy donor individuals using precision micro-dissection scissors. This donor tissue, rich in epithelial cells, is critical for the subsequent formation of the pearl sac around the implanted nucleus. Once the graft tissue was prepared, each recipient oyster underwent nucleus implantation. A sterilized spherical nucleus, 6 mm in diameter, was gently inserted into the gonadal pocket of the recipient oyster. The mantle graft was positioned adjacent to the nucleus with great care to maintain the correct epithelial polarity, which is crucial for successful pearl sac development and nacre secretion. Given the natural structure of oysters as bivalves, no suturing was required after implantation. Instead, the natural valve closure behavior of the oysters was relied upon to seal the surgical site. Valve closure was closely monitored during the immediate recovery phase to ensure that the oysters could reestablish a secure seal, which is indicative of a successful surgical recovery. Following the surgical procedure, the implanted oysters were gently transferred to separate recovery tanks specifically designed to provide an optimal healing environment. These tanks were equipped with continuous aeration and gentle water circulation to maintain oxygen levels and prevent sedimentation or water stagnation, which could interfere with the healing process. The post-operative recovery period lasted for 48 hours. During this time, each oyster was monitored for several physiological and behavioral indicators of recovery. Particular attention was paid to valve closure behavior, as consistent and firm shell closure suggests the restoration of muscular control and internal pressure regulation. The oysters were also observed for byssus thread reattachment, which serves as a marker of resumed substrate attachment and behavioral normalization. No spawning activity was recorded during this period, which is desirable, as spawning can introduce physiological stress and divert metabolic energy from healing
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