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
3.1. Oyster and bead sizes The mean shell height of the Group I oysters, used in the 6-week experiment, was
Ž .
Ž .
114.6 8.5 mm S.D. and it was 119.3 8.2 mm for the Group II oysters Fig. 1 . An analysis of the bead diameter chosen for insertion into the 96 Group I oysters
showed no significant difference in mean diameters chosen for the different treatment Ž
. combinations
range in mean diameter from 6.81 to 7.29 mm , nor between the Ž
. successive replicates. There was no significant correlation r s 0.19, P 0.05 between
Ž .
bead diameter and oyster shell height for these oysters Fig. 2 . This lack of correlation between bead diameter and oyster shell height suggests that the bead diameter selected
for a particular oyster was based more on gonad size than on shell size.
3.2. Time taken to apply treatment combinations Mean time for bead and mantle insertion was 1.22 min, i.e., about 50 insertions per
Ž .
hour Table 2 . Including antiseptic treatment of the incision site did not increase the
Fig. 1. A histogram of the numbers of pearl oysters, P. margaritifera, in shell height size classes for Group 1 Ž
. and Group II see text .
insertion time significantly. However, incision closure with adhesive significantly extended the mean time for each insertion to 1.91 min. The combination of antiseptic
Ž Fig. 2. Diameters of beads inserted into pearl oysters, P. margaritifera, vs. shell heights of the oysters Group
. 1 oysters .
Table 2 Ž
. Mean times for simple bead insertion control into pearl oysters P. margaritifera and bead insertions together
with additional treatments
a
Ž .
Treatment Average time min
Control 1.22 a
Antiseptic 1.35 a
Adhesive 1.91 b
Antisepticqadhesive 2.30 c
a
Ž .
Values followed by the same letter do not differ significantly P 0.05 .
and adhesive treatments further extended the mean insertion time to 2.30 min, almost twice the mean time for simple bead and mantle insertions.
Part of the extra time in applying the adhesive was often taken up in ridding the incision site of haemolymph. Many oysters bled profusely from the incision and it was
necessary to remove this haemolymph before the adhesive would adhere to the incision site. As noted in Section 2, oysters often had to be removed from the operating clamp
and inverted, causing haemolymph to pour copiously from the oyster.
3.3. Treatment effects The depths at which the oysters were suspended, i.e., positions on the vertical ropes,
had no significant effect on mortality or bead rejection rates in the data collected at 6 weeks either as a main effect or in interactions with the treatment combinations. The
lack of any depth effect on mortality or bead rejection is not surprising. It is unlikely that environmental conditions in the surface waters of the lagoon vary enough over 3 to
5 m below the surface of the water column to distinctly impact the oysters.
Factorial analysis of deaths, bead rejection and total failure data showed no signifi- Ž
. cant interactions between any of the 3 main factors relaxant, antiseptic, adhesive , and
Ž .
hence only main effects of each factor are presented Tables 3 and 4 .
Table 3 Ž
. Percentage mortalities, bead rejections, total failures deaths and bead rejections and beads with ‘tails’ in two
Ž .
Ž .
groups of P. margaritifera, Group I 96 oysters and Group II 768 oysters , 6 weeks after the pearl operation. Ž
. Arcsine transformations were used in statistical analyses of percentage data
Treatment Deaths
Bead rejection Total failure
With tails Gp I
Gp II Gp I
Gp II Gp I
Gp II Gp I
UU UU
U UU
Relaxant 22
18 3
8 26
30 64
Non-relaxant 1
12 14
12 18
81 Antiseptic
5 7
5 9
16 21
81 Non-antiseptic
6 7
9 13
21 26
65
UU U
U
Adhesive 8
11 12
12 23
28 53
Non-adhesive 4
4 3
10 14
20 89
U
Significantly different from no treatment at P - 0.05.
UU
Significantly different from no treatment at P - 0.01.
Table 4 Ž
. Percentage mortalities, bead rejections, total failures
deaths and bead rejections in pearl oysters, P.
Ž . Ž
margaritifera, at harvest 18 months after the pearl operation Group II oysters . Arcsine transformations were .
used in statistical analyses of percentage data Treatment
Deaths Bead rejection
Total failure Pearls
UU UU
UU UU
Relaxant 24
6 39
58 No relaxant
2 16
21 74
Antiseptic 10
9 27
69 No antiseptic
12 12
32 63
UU
Adhesive 15
10 33
61 No adhesive
7 10
26 71
U
Significantly different from no treatment at P - 0.05.
UU
Significantly different from no treatment at P - 0.01.
The use of the relaxant, propylene phenoxetol, significantly increased mortality in both groups of oysters. However, it resulted in a lower bead rejection rate, which was
Ž .
significant P - 0.05 for the Group I oysters at 6 weeks. Taking account of total failure Ž
. rates deaths and bead rejections , the higher mortality rates outweighed the lower bead
rejection rates and the net effect of propylene phenoxetol was adverse. This greater percentage of total failures was significant for the Group II oysters both at 6 weeks and
Ž .
at 18 months P - 0.01 . Following from the high percentage of total failures, the
percentage of oysters yielding pearls was significantly lower for those treated with the Ž
. Ž .
relaxant P - 0.01 Table 4 .
Application of an antiseptic, povidone iodine solution, had no significant effects on Ž
. mortality, bead rejection or total failure rates in either group of oysters Tables 3 and 4 .
Interestingly, however, antiseptic was the only treatment that resulted in lower percent- age total failures compared to no treatment both at 6 weeks and at 18 months. There is a
suggestion of marginal improvement in total failure rate with application of this antiseptic.
The use of an adhesive, cyanoacrylate adhesive, to close the incision resulted in Ž
. increased mortality, which was significant P - 0.01 for Group II oysters at 6 weeks
Ž .
and at 18 months Tables 3 and 4 . Bead rejections were also higher for oysters treated Ž
. with adhesive significant, P - 0.05, for Group 1 oysters at 6 weeks . Total failures
were thus greater for adhesive treatment than for no treatment, although the levels did Ž
. not differ significantly Tables 3 and 4 .
3.4. Pearl sac and secretions Of the 78 Group I oysters that retained their shell beads, 76 had an epithelial pearl
sac. The beads in the remaining two oysters were surrounded by an inflammatory zone and no epithelial sac was present. Of the 76 pearl sacs, only two had isolated areas of
inflammation superimposed on the sacs. There were large variations in the epithelial cell height of the pearl sacs. This applied to the 8 measurements taken on each sac
Ž
Ž .
. coefficient of variation CV s 46 and between oysters given the same treatment
Ž .
Ž CV s 39 . Hence, the differences between the 8 treatment combinations mean cell
. heights ranging from 7.7 to 12.9 mm were not significant. Of the 78 beads retrieved, the
Ž .
Ž .
predominant colour was brown 74 , a combination of brown and silverrgrey 4
Ž . Ž .
Fig. 3. A
Pearl sac epithelium E
of P. margaritifera. This was associated with a bead that was Ž .
Ž . predominantly brown in colour. The scale bar is 22 mm. B Pearl sac epithelium E P. margaritifera. This
Ž . was associated with a bead that was predominantly silverrgray in colour. The scale bar is 22 mm. C A
longitudinal section of a conical ‘tail’ structure associated with an inserted shell bead in the pearl oyster, P. Ž
. margaritifera. The ‘tail’ is encased by a calcified layer CL and encloses a large number of degenerating
Ž .
inflammatory cells IC . The bead is above the top of the photo. The scale bar is 44 mm.
Ž .
and silverrgrey 22 . The brown beads had a mean pearl sac epithelial height of Ž
. 10.23 4.94 mm S.D. , while the silverrgrey beads had a mean pearl sac epithelial
Ž .
height of 6.75 1.31 mm. These mean heights were significantly different P - 0.01 . In addition, the variation between the eight measurements made on each pearl sac
epithelium was greater for the brown than for the silverrgrey beads. The standard deviation was 4.03 compared to 2.31. The brown colour is presumably the periostracal
layer, while the silverrgrey is presumably the prismatic andror early nacreous layers. The appearance of the pearl sac epithelium associated with the brown and the silverrgrey
beads is presented in Fig. 3A and B, respectively.
Many of the beads had ‘tails’, cone-shaped extensions of the initial secreted layers that extend into the passageway down which the shell bead had been inserted during the
Ž .
pearl operation. Of the 78 beads recovered, 55 71 had ‘tails’ that were equal to or greater than 0.1 mm long. There were differences in percentages of ‘tails’ in treated vs.
non-treated oysters, with relaxant and adhesive treatments reducing the percentage of oysters with tailed beads, while antiseptic increased the percentage with tailed beads
Ž
. Table 3 . However, only the adhesive treatment resulted in a significant difference
Ž .
P - 0.01 compared to non-treatment. There were large numbers of dead inflammatory Ž
. cells within the ‘tail’ structure of all six ‘tails’ examined Fig. 3C .
The use of the relaxant significantly reduced the pearls harvested from 74 to 58 Ž
. Table 4 . However, use of the antiseptic or the adhesive had no significant effect on
pearls harvested. 3.5. Pearl quality
There were no significant differences between the mean scores for pearls from treatments and no treatments for any of the five qualitative criteria: lustre, surface
Ž .
perfection, main colour, tint and shape Table 5 . In fact, there was little variation between mean scores for each of these criteria over all treatments and no treatments. The
Table 5 Mean classification scores and dimensions of pearls harvested from pearl oysters, P. margaritifera, 18 months
Ž .
after the pearl operation Group II oysters Treatment
Pearl classification Lustre
Surface Main
Tint Shape
Diameter Length
Lengthr Weight
Ž .
Ž .
Ž . score
score colour
score score
mm mm
diameter g
score ratio
U
Relaxant 2.97
2.56 3.71
3.42 2.60
10.59 12.53
1.184 1.72
No relaxant 3.01
2.70 3.88
3.44 2.74
10.72 12.72
1.187 1.83
Antiseptic 3.00
2.56 3.72
3.50 2.67
10.65 12.73
1.195 1.78
No antiseptic 2.98
2.70 3.87
3.36 2.68
10.66 12.52
1.175 1.77
U UU
Adhesive 2.99
2.70 3.74
3.50 2.70
10.68 12.34
1.156 1.76
No adhesive 2.99
2.56 3.85
3.36 2.64
10.63 12.91
1.214 1.79
U
Significantly different from no treatment at P - 0.05.
UU
Significantly different from no treatment at P - 0.01.
mean scores for lustre, surface perfection, main colour, tint and shape were about 3.0, 2.6, 3.8, 3.4 and 2.6 out of 5, respectively, in all cases. Thus, there were no effects of
Ž relaxation, antiseptic nor adhesive on these five criteria of pearl quality. The pearls
were not polished, by the usual procedure of rubbing in salt, at the time of grading and the mean lustre values would be improved by this procedure. The absence of polishing
. is, however, a uniform effect.
In terms of quantitative criteria, pearl diameter, length, lengthrdiameter ratio and weight, there were significant differences. Use of propylene phenoxetol was associated
with the smallest diameter pearls, although not significantly smaller than pearls from oysters not treated with the relaxant. Pearl weight, which is proportional to diameter
3
, Ž
. was significantly lighter in oysters that had been treated with relaxant P - 0.05 .
The other significant results on quantitative criteria from a treatment were the effects Ž
. of adhesive use in reducing the mean length of pearls P - 0.05 and the mean ratio of
Ž . Ž
. pearl lengthrdiameter P - 0.01
Table 5 . The pearls from oysters that were treated with adhesive tended to be less elongate.
The scores for the pearls harvested from the experiment are presented in Fig. 4 as a frequency vs. pearl quality score histogram. Commercial grading of pearls is a complex
process and different attributes are not weighted equally as has been done in this assessment. Furthermore, different markets may not rank the pearl colours in the same
order of priority. Fig. 4, however, gives a good indication of the nature of the pearl harvest. It is clearly a normal distribution in pearl quality. No pearls achieved the perfect
30 score and approximately 6 of the pearls were in the top 20 of scores, 24 q out of 30.
Fig. 4. A histogram of the frequencies of pearl quality scoresr30 for pearls harvested from this experiment with P. margaritifera.
4. Discussion
