Surgical procedures in pearl culture

Mantle Cavity Insertion

In this method, appropriate nuclei up to 1.0 cm in size are placed in the mantle cavity of pearl mussels ( L. marginalis and L. corrianus)of the size 8 to the 10 cm in shell length and the implanted mussels are reared for a period of one year in pond culture environment.The products are generally shell- attached, half round or design pearls, depending upon the shape of the nucleus implanted.

Mantle cavity insertion method is a simple technique. Prior to surgery, the indigenous freshwater mussel species Lamellidens marginalis, L. corrianus and Parreysta corrugata of required shell length and wet weight are collected. They are carefully opened by the means of speculum, 1 cm wide , without causing injury to the adductor mussles and soft parts of the mussel. A small area of the mantle from the anterior side is detached carefully from the upper shell valve and a nucleus of desired size and shape (upto 1 cm in size)is inserted slowly into the mantle cavity and is further pushed deep to avoid rejection. The mussel is now turned over and similar implantations are made in the opposite mantle cavity.

After implantation the mussels are kept in post operation care units. These units consists of Ferro- cement tanks (200 liter) filled with aged tap water and 50 nylon bags(12 sq.cm) arranged in two rows,suspended at 0.2 m depth. The units are daily examined: the dead mussels and the ones that reject the nucleus are removed.

After post operation care these mussels are then stocked in the ponds. The implanted mussels are placed in nylon bags (1 cm mesh- 12 sq.cm) at the rate of 2 mussels per bag suspended at 1.0 m depth in culture ponds. The mussels can be placed at deeper zones (upto 2.0 m) during hot summer months to avoid surface heating. The stocking density of implanted mussels can be 25000/0.4 ha.

Mantle Tissue Implantation

This procedure involves placement of the donor mantle graft (2 to 5 sq. mm) in the space and inner epithelial layers of the left and right mantle lobes of the recipient mussels (preferably of 9 to 10 cm in shell length). A small nucleus (below 2mm dia) may also be placed along with the graft depending on the size and development of the mantle tissue of the recipient mussels. The implanted mussels are transferred to the pond environment for culture for a period of one year. The products are un-attached irregular to oval graft pearl or nucleated, round cultured pearls.

The freshwater pearl mussel has a complicated internal structure and virtually there is no space left for the insertion of a foreign body. However, the mantle is the only structure that occupies the bulk, having two folds on either side of the shell valves. Hence in China and Japan this method of implantation is adopted for production of mode pearls per mussels and also the stress on the animal is minimum in this method of implantation.

Pre-operative Condition

This indigenous pearl mussel species Lamellidens marginalis and L.corrianus are collected from the freshwater bodies and are subjected to pre-operative conditioning for 2 to 3 days. They are kept in Ferro cement tanks (200 liter) with aged tap water at a stocking density of 1 muss l/liter of water. Pre-operative conditioning ensures proper relaxation of adductor muscles in preparation for surgery. This aspect is important in view of limited application of narcotizing procedures as followed in marine pearl culture operations.

Surgery

The mussels before surgery are segregated into two groups, the mussels to be operated upon ‘the operation mussel’ or ‘recipient mussel’ and those to be sacrificed, and the ‘cell mussels’ or the ‘donor mussels'. The live donor mussel is sacrificed and the pallial mantle ribbon of 0.5mm long is wide and 7.0cm is collected on a pre­ cleared moist wooden board. The strip is• then cut into appropriate sized graft pieces (2-4 mm) and implanted alone or along with small nucleus (2m dia)into the mantle tissue of the recipient mussel. Using a shell opener, the recipient mussel is carefully opened (1.0 cm wide).The inner side of the mantle is exposed by gently pushing aside the gills and the foot. By means of a specialized needle few pockets are cut in the inner mantle. The previously prepared live graft pieces are inserted with care into these pockets (one graft piece/pocket) with the outer side of the graft facing the inner side of the operation mussel shell. Such grafting is done on both the side of the mantle lobes. It has been observed that the implanted mantle graft epithelium leads to enveloping the nucleus in the form of a pearl sac in about 15 days and the microvillus of the pearl sac epithelium constituted the. cellular basis for crystallization of aragonite calcium carbonate, the first step in pearl formation (Janaki Ram & Gayatri Misra 1997). The number of implantations can vary between 2-8 depending upon the size and mantle thickness of the recipient mussel.In nucleated operations a small nucleus (less than 2 mm)is inserted along with the live graft piece in close association into these mantle pockets.

Post- operation care

Immediately after implantation, the mussels are kept in post operation care units consisting of a series of Ferro- cement tanks(200 1 capacity each)filled with aged tap water and 50 nylon bags (12 sq.cm)suspended at 0.2m depth. The implanted mussels are placed at the rate of 2 mussels per bag with ventral side up position for a period of 10 days. The units are daily examined the dead mussels and the ones that rejected the nucleus and graft are removed. Treatment of the water in post-operative care units with broad spectrantibiotic. Chloramphenicol at the rate of 1-2 ppm as a prophylactic measure is beneficial for the survival and wound healing of the implanted mussels. It is desirable to add green water(algae enriched)into these units after 3 to 4 days of post operation care.

Gonadal Implantation

In this method of surgery, the donor mantle grafts (2 sq.mm) along with a nucleus (3 to 6 mm dia) are implanted into the gonad of the recipient mussels. The gonad implanted mussels are maintained in post­ operative care units with antibiotic supplements for a period of 7 to 1e days to minimize the rejection rate of the implanted graft and nuclei before transferring to pond culture environment. The products are un­attached, regular, round culture pearls.

The implantation of graft and nucleus is a concept followed in pearl culture operations in the marine oysters. However Japan has successfully employed this method for freshwater culture pearl production. In India, gonadal implantation can also be done in species such as L. marginalis and L. corrianus exercising caution and avoiding damages to the intestine lying deep inside the gonad. The riverine species Parreysia corrugata cannot withstand intricate surgical intervention required for gonadal implantation and thus is unsuitable for this method of implantation.

Post- operative conditioning

Before implantation the common freshwater mussels L.marginalis and L. corrianus and L.corrugata of 8.0cm or above in size and 50g or above in wet weight are collected from the natural environment. The live mussels are transferred for shorter distances in split bamboo or cane baskets or in open plastic buckets. However for longer duration of transport) it is desirable to provide moist pond soil in between layers of mussels. These mussels are maintained in Ferro- cement tanks with aged tap water for a period of 2 to 3 days at a stocking density of 1 mussel/1 liter of water. Crowding of mussels in such a ways for shorter periods ensures proper relaxation of the adductor muscles in preparation for surgery.

Surgery

After pre-operative conditioning the mussels are segregated into two groups. The larger, 10.0 cm and above constitutes the (donor while the rest forms the recipient stock. The donor mussel is cut open through the adductor mussels with a sharp knife following­ the ventral gap between the two. Shell valves without causing damages to the mantle on both the valves. The• margin mantle just above and along the pallial line is then cut using a pair of scissors or a graft knife. The cut out pallial mantle is gently lifted from the posterior en.d with a pair of forceps and transferred to a pre-cleaned moist wooden block ensuring that the inner side of the cut out mantle ribbon faces up wards. The mantle strip is gently wiped clean with a wet sponge. The moist mantle strip is trimmed length wise. on both the sides employing the graft scalpel to obtain a 2 to 3 mm wide pallial mantle ribbon. The trimmed pallial ribbon: is turned up side down exposing the outer side of the pallial mantle. The ribbon is again wiped clean with a wet sponge. The ribbon is then cut into a number of small pieces of 2 to 3 mm₂ with the graft scalpel.The live graft pieces are kept moist till implantation. The mantle on other side of the sacrificed donor can also be processed as above and used for implantations. Once the live graft pieces ready,the recipient mussels are carefully opened with the shell opener inserted through the posterior aspect of the ventral margin of the shell. By using the regulator ring of the shell opener the recipient mussels is opened 1.0 cm wide and is positioned on the clamped recipient mussel are gently pushed up with a spatula and the operating gonad area is exposed. By means of a pair of forceps the foot is stretched to elevate the gonad. A measured small incision is made by means of a special knife placed at the other end of the graft needle; under the outer membrane of the gonad Care is to be taken not to cut deep into the gonadal tissue to avoid damage to the intestine. A live graft piece is picked up and is inserted slowly and carefully through the incision making sure that the outer side of the graft ids facing the entry point. Nucleus implantation is a delicate procedure following graft insertion, the nucleus is picked up with the moistened nuclear of appropriate size and is pushed through the same incision cut for the graft, till it comes in contact with the implanted graft. The nucleus is released and the nucleus cup is then withdrawn. Care must be taken to ensure that the nucleus is in close contact with the outer epithelium of the implanted graft. The margins of the incision are smoothened with the nucleus needle cup to minimize the gap at the incision sit. The middle area of the gonad is the possible site for single implantation.

Post- operative care

Post- operative care is an important step in fresh pearl culture operation which is required for the implanted mussels to recover. Immediately after surgery restricted movement of mussel is essential for the retention of the implanted graft and nucleus. Thus after implantation, the mussels are kept post operation care units. These units consists of rectangular Ferro- cement tanks (200 liter) filled with aged tap water and 50 nylon bags (12 sq. em)suspended at 0.m depth in two rows. Implanted mussels are placed at made in CMFRI, Tuticorin to produce shell beads from the sacred chank shell (Xancus pyrum) which are obtained from the coastal waters of Tamil Nadu, Kerala and Gujarat that requires vigorous follow up action.In CIFA, Bhubaneswar two types of nuclear material have been identified that has given successful results. The preparation procedure is simple and is easy to adopt, cost is low and importantly its acceptance and quality of pearl produced by these materials are fairly comparable with the imported shell beads.

Preparation procedures

The first type of nucleus (stelon beads), is prepared by using commercially available self-cure acrylic repair material (powder and solvent) mostly required in dentistry. The second type of nuclear material (shell bead) is made from the dead shell pieces of the freshwater mussel Lamellidens marginalis.

For the preparation of stelon nuclear material, required amount of acrylic powder is taken in a glass container and slowly the solvent is added to it. (preferably through a syringe) which is then mixed thoroughly to prepare a dough. Immediately, nuclear material of desired shape and size are prepared and allowed to air dry. Later, they are stored in dust free close containers.The nuclear material is boiled in water, air dried and cooled few hours prior to implantation.

For the preparation of the shell bead nucleus,dead shell of Lamellidens marginalis are collected and subjected to thorough washing in water to remove dirt and sand particles. The dry flesh materials, if present are scraped out. The shells are then dipped in 5000 ppm of chlorine solution (50 gms of bleaching power containing 10% chlorine in 0.1 liter of water) for twenty four hours or forty eight hours if required. The Completely lye- peeled shells are sorted out and are then continuously washed in tap water. They are then kept in an oven maintained at 60°C, for more than two hours or can even be sun dried, for a longer duration to ensure the complete removal of chlorine from the treated shells. The dried shells are made into small pieces by using a mortar and pestle and are then finely powdered by means of an electric grinder. The powdered shells are then processed through a sieve of 0.01 - 0.05 mm mesh size. The commercial glue Araldite hardener and resin (that acts as a binder) are mixed in a ratio of 1:1 to prepare a paste. To this paste the sieved shell powder is added gradually to prepare dough of thick consistency. The ratio of the shells powder to the paste should be 5:1. Immediately, nuclei of desired shape and size are prepared an then are air dried till they become hard. Prior to implantation, the nuclei is boiled in water and cooled.

The mussels can now be suspended in the ponds for pond culture.

Source : Mr Ravikiran Bhat, Karnataka