epithelium spreads around the nucleus until it encloses the nucleus. This has been Ž
. reported to take from 3 days Wada, 1968 in the Japanese pearl oyster P. imbricata
Ž .
Ž .
refer Shirai, 1994 to 23 days Scoones, 1996 in P. maxima. An organic periostracal Ž
. layer s conchiolin and a prismatic layer of calcite crystals are then laid down. After
Ž about 40 days, the nacreous layer of aragonite crystals begins to be secreted Kawakami,
. 1952 . However, this process of deposition may become defective and result in
Ž .
abnormal pearls Scoones, 1996 . Details of the three main types of pearl sac epithelium and the pearl layers produced by these sacs have been documented for P. imbricata
Ž .
Ž .
Aoki, 1966 and P. maxima Dix, 1972 . The three layers correspond to the three layers of pearl oyster shell, secreted by the mantle.
However, despite the relative success of this pearl formation method, substantial failures continue to occur, including mortalities from the surgery, bead rejection and
Ž .
poor quality pearls Cabral, 1990; Scoones, 1990; Wada, 1991; Scoones, 1996 . Virtu- ally no studies of factors influencing the outcome of the insertion operation in pearl
Ž .
oysters have been published. Meng and Xing 1991 is one example of a preliminary Ž
. study of factors influencing cultured pearls in P. margaritifera. Scoones 1996 also
studied the development of the pearl sac in P. maxima with reference to pearl defects. This study is part of a broader investigation into the application of surgical techniques
used in modern veterinary science to improve the quality and yield of cultured pearls. In particular, this experiment examined the effects of relaxation, antiseptic application and
incision closure on the round pearl formation technique, when these treatments were used in association with the bead insertion procedure in P. margaritifera.
2. Materials and methods
2.1. Oysters The blacklip pearl oysters, Pinctada margaritifera, used in this study had been
Ž
X
collected as spat on commercial spat collectors in the lagoon of Manihiki atoll 11824 S,
X
. 161801 W , one of the northern Cook Islands in the southern Pacific Ocean. They were
reared on subsurface long-lines in the lagoon, some 3 to 5 m below the surface, prior to use in the experiments. Most were about 2 years old at the time of the experiment.
Oysters were collected from the long-lines in early morning on the day of bead insertion. They were transported a short distance to the work area, Takaniko Islet, at the
southern end of the atoll, where they were held in shallow water. The oysters were scrubbed and washed clean of any loose debris, algae, etc. Just before operating on each
group of eight oysters, each oyster was forced open about 5 to 10 mm with a pair of pearl oyster openers and then chocked with a plastic wedge. They were held in the air in
the shade prior to the various treatment combinations and the pearl seeding operation. The oysters were not ‘conditioned’ or weakened prior to the seeding operation.
2.2. Treatment combinations Oysters were subjected to one of eight treatment combinations together with the usual
Ž . insertion of a bead and piece of mantle tissue. The treatment combinations were: 1
Ž . Ž .
Ž . Ž .
relaxant, 2
relaxant and antiseptic, 3
relaxant and adhesive, 4
antiseptic, 5
Ž . Ž .
Ž . antiseptic and adhesive, 6 adhesive, 7 relaxant and antiseptic and adhesive, and 8
Ž .
Ž .
no treatment control . The bead-insertion technician Ian Turner selected from a range of bead diameters based on his subjective assessment of oyster and gonad size. After
Ž .
being operated on, each oyster was enclosed in a mesh bag a ‘ vomit’ bag with mesh fine enough to catch the bead if it was subsequently rejected by the oyster.
For the relaxant treatment, groups of eight oysters were placed in 30 L of seawater in a large plastic bin. Aeration was used to circulate the water containing propylene
Ž .
phenoxetol at 2 mlrl Norton et al., 1996 . The solution was renewed after every 32 to 48 oysters. Oysters were held in the solution for approximately 15 min. Water
temperature ranged from 25 to 288C. The antiseptic, a 1:50 aqueous dilution of 10 Ž
povidone iodine solution Betadine, Faulding Pharmaceuticals, Salisbury, South Aus- .
tralia , was applied to the incision site using a soft artist’s brush for a minimum of 30 s prior to surgery. Incisions were closed using a small amount of flexible cyanoacrylate
Ž .
adhesive Loctite 454, Loctite Australia, Carringbah, New South Wales, Australia on Ž
. the end of a thin metal spatula. Excess fluid mainly haemolymph and gonad products
were removed from the incision site with a sterile gauze swab before application of the adhesive. More than one application of adhesive was often needed to seal the incision.
Often an oyster with its opening clamp in place had to be removed from the operating stand and inverted so that excess haemolymph could be drained from the incision site
before swabbing.
The commercial beads inserted into the oyster’s gonad were produced from freshwa- Ž
. Ž ter mussel shell and ranged in size from 2.1 to 2.6 bu 6.3–7.8 mm diameter . The
. Japanese unit of measurement, ‘bu’, is equivalent to 3 mm . Each oyster was tagged
Ž .
with a length of coloured plastic tubing 5 mm diameter , with eight colours to identify the eight different treatment combinations. Each tag was tied to the oyster with
monofilament fishing line inserted through a hole drilled in the posterior-dorsal corner of the shell. A second hole was drilled in this region through which a piece of stainless
steel wire was inserted. This wire was used to attach each oyster to an approximately 2 m long vertical rope, with eight oysters per rope at about 20 cm intervals. Each vertical
Ž .
rope drop line was individually identified with a large plastic tag and number, and it was attached to a subsurface long-line with a stainless steel clip. The oysters were thus
suspended about 3 to 5 m below the surface of the lagoon. Ž
. The times taken to insert the beads were recorded for a run of eight oysters on 5
randomly chosen occasions for each treatment combination. However, since the time to relax the oysters was not part of the actual insertion process, only four treatment
combinations were recorded: control, antiseptic, adhesive, and antiseptic plus adhesive.
2.3. Groups of oysters Group I consisted of 96 oysters, derived from the eight treatment combinations
imposed in random order on eight oysters and repeated 12 times to form a randomised block design. The oysters were hung on 12 vertical ropes, each with eight oysters, one of
each treatment per rope in random order from top to bottom. Each oyster was measured Ž
. for size shell height . The bead size that the technician chose to be inserted was also
recorded.
Group II consisted of 768 oysters. The treatment combinations were imposed in 12 blocks of 64 oysters. Groups of 8 oysters were given the same treatment, and the
treatment combinations were applied in a random order within each block of 64 oysters. Ž
. This occurred over a period of 4 days Sept. 27 to Oct. 1, 1996 . The 64 oysters of each
block were then hung on 8 vertical ropes with 8 oysters of the same treatment on each rope. The oysters on 22 randomly selected vertical ropes were measured for size.
Six weeks after the beads were implanted, both groups of oysters were examined for Ž
. deaths and bead rejections bead in mesh bag . The treatment combination, vertical rope
Ž .
number and depth oyster’s position on each vertical rope, from top s 1 to bottom s 8 were recorded for each dead oyster or bead rejection. All surviving Group I oysters were
sacrificed. Their gonad tissue was removed and fixed in 10 seawater formalin. The Group II oysters were inspected for deaths and rejected beads. Then, eighteen months
after the pearl operation, the pearls from the Group II oysters were harvested, counted
Ž .
and graded for quality April 1998 . The numbers of deaths and bead rejections were also recorded at this time.
2.4. Pearl classification The basis of pearl classification is to assess their market value. Some pearl attributes,
such as diameter, are quantitative and readily measured; others are more qualitative and not as readily measured. In seeking to try to quantify those more qualitative attributes of
pearls that affect their value, e.g., lustre, main colour, tint, surface perfection and shape Ž
. Rambaud, 1991 , a classification was developed scoring each pearl for these attributes
Ž .
Ž .
on a 5 s best to 1 s poorest scale Table 1 . As well as these, diameter mm , length Ž
. Ž .
s longest dimension, mm and weight g of each pearl were measured. Pearls are graded according to the combination of their attributes; and, to produce something
equivalent to a commercial assessment, the scores for each pearl for lustre, surface perfection, main colour, tint and shape were pooled. Size is an important factor, so a
Ž score for diameter - 9.0, 9.0, 10.0, 11.0, 12.0 mm s scores of 1, 2, 3, 4 and
. 5, respectively was included.
Table 1 Ž
. Ž
. Numerical grading of cultured pearls from P. margaritifera with scores of 5 best to 1 poorest for various
attributes Pearl parameter
Score 5
4 3
2 1
Lustre Very good
Good Fair
Poor Nil
Surface No more
Several Numerous
Ringed No clear
perfection than 1 or 2
scattered or small or
face small, close,
grouped extensive
defects defects
defects Main
Black White, yellow
Grey Silver
Other colour
or blue Tint
Green Purple
Blackrblue Red
Nil Shape
Completely Near spherical
Symmetrical Symmetrical,
Baroque spherical
in one plane elongate
2.5. Histopathology The preserved gonads from the Group I oysters were examined histologically to
identify any variations in the pearl sacs or their process of formation that may have resulted from the different treatments. Where the shell bead had been retained for 6
weeks, an incision was made partially around the circumference of the sac that enclosed the bead. The incision was large enough to allow the bead to be removed. The spherical
cavity was filled with warm 1 agarose agar so as to exclude air bubbles. After the agar had set, the oyster tissue was returned to 10 seawater formalin for 24 h. The original
incision in the pearl sac was continued so as to divide the sac into two hemispheres that were placed cut-side down, processed and sectioned. This produced two circles of pearl
sac tissue per oyster. The heights of the pearl sac epithelium of both circles were measured at the 08, 908, 1808 and 2708 positions to give eight readings.
The ‘tails’ from six of the beads that had large ‘tails’ were removed, decalcified and processed by routine procedures for histopathology. Haematoxylin and eosin slides of
the ‘tail’ tissue were examined. 2.6. Statistical analyses
Group I and II experiments were arranged in separate randomised block designs with the three treatments in factorial combinations. Hence data from each experiment were
analysed by standard factorial analysis of variance, estimating and testing the individual factors and all interactions. For percentage response data arcsine transformations were
applied prior to analysis, and equivalent means, with indications of statistical differ- ences, presented in result tables.
3. Results
