Surgical procedures in pearl culture

1. Mantle Cavity Insertion

2. Mantle Tissue Implantation
3. Pre-operative Condition
4. Surgery
5. Post- operation care
6. Gonadal Implantation
7. Post- operative conditioning
8. Surgery
9. Post- operative care
10. Preparation procedures

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 mussels 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 mm 2 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 60C, 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 and
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.