JOURNAL OF THE Volume 36,No.

1
WORLD AQUACULTURE SOCIETY March 2005

Preservation of Black Tiger Shrimp Penaeus monodon

Spermatophores by Chilled Storage

SUBUNTITH
NIMRAT
Department of Microbiology and Environmental Science Program, Faculty of Science, Burapha University,
Chonburi 20131 Thailand

TEERAPAT AND VERAPONC

SANGNAWAIUJ VUTHIPHANDCHAI'
Department of Aquatic Science, Faculty of Science, Burapha University, Chonburi 20131 Thailand

Abstract
Chilled storage of spermatozoa in fish has been extensively investigated for many years, but
limited research was focused on crustacean species. Chilled storage of spermatophores of black
tiger shrimp Penaeus monodon is needed to generate consistent and reliable supply of sperma-
tozoa for subsequent use. The objective of this study was to develop a protocol for the chilled
storage of black tiger shrimp spermatophores and to evaluate bacterial propagation during
chilled storage of spermatophores. In the first experiment, spermatophores were selected and
preserved using four differentextenders, namely mineral oil, Ringer's solution, phosphate buffer
and 0.85% sodium chloride, and stored at low temperature (2-4 C) for 42 d without antibiotic
supplementation. Results showed that mineral oil was the best extender for chilled storage of
spermatophores, since the highest percentage of viable sperm (58.3 f 2.9%) was observed with
this extender at the end of experiment (day 42). Bacillus sp., Staphylococcussp., and Pseudomom
aeruginosa were identified as the predominant bacteria occurring during chilled storage, and
the total bacteria count gradually increased during the experiment. In the second experiment,
spermatophores were preserved in the mineral oil with four concentrations of the antibiotic,
penicillin-streptomycin(O.l%, 1%,2%, and 3%).There was no significant difference (P>0.05)
in the percentage of viable sperm among treatments with 0.1%, 1%, 2%, and 3%antibiotics. The
total count of Bacillus sp., Staphylococcussp., and f? aeruginosa in the antibiotic treated groups
significantly decreased (P< 0.05) to undetectable levels by day 14 of the experiment. Fertility
studies from artificial insemination indicated that l? monodon spermatophores preserved with
mineral oil for 7-8 d at 2-4 C were capable of fertilizing eggs with hatching rates similar to the
controls. This study suggests that chilled storage of spermatophores is a feasible approach for
the management and spawning of black tiger prawn broodstock or other invertebrate species
that produce spermatophores.

Black tiger shrimp Penueus monodon is an eco-
nomically important crustacean species in Thai-
land, generating foreign income of about US$1
billion (89,230 million baht) with the production
of 304,987 tons in the year 2000 (Department of
Fisheries 2000). One of the major obstacles in the
development of the black tiger shrimp industry is
the inadequate and inconsistent supply of good
quality seed. Currently, production of shrimp seed
is still dependent on wild stocks collected from
estuaries, which is unpredictable and seasonal.
Although the domesticationof shrimp broodstocks
has been recently developed to minimize the reli-
'Correspondingauthor
ance on wild stocks, most studies have focused on
the reproductivephy siofogy of females. Neverthe-
less, the nonsynchronous final sexual maturation
between male and female stocks in a hatchery often
creates a problem regarding the management of
male broodstock, since fertilization requires mat-
ing to augment seed production. Mating in penaeid
shrimp involves the transfer of spermatophores
from males to the thelycum of females. Moreover,
problems sometimes arise with the quality of males
in that they may not have mature spermatophores
or their sperm may have deteriorated. Access of
good quality sperm via spermatophore preserva-
tion would provide an alternativesource of reliable
and high quality spermatophores.

8 Copyright by the World Aquaculture Society 2005

76
 PRESERVATION OF BLACK TIGER SHIMP SPERMATOPHORES
Successful preservation of crustacean sper-
matophores was first reported in freshwater
shrimp Macrobrachiurn rosenbergii, in which
sperm viability and successful fertilization were
maintained up to 4 d after storage in Ringers
solution at 2 C (Chow, 1982). However, chilled
storage of crustacean spermatophores was devel-
oped by Ishida et al. (1986) who stored lobster
spermatophores in paraffin oil at 4-7 C for up
to 289 d. Despite the success of storage, chilled
storage of spermatophoresusing these approaches
could not prevent bacterial growth in the sperm
mass of preserved spermatophores. Controlling
the bacterial proliferation on spermatophores is a
prerequisite for preservation of spermatophores.
Bacteria may cause a negative effect on fertiliza-
tion rate of preserved spermatophores and on the
health of artificially inseminated female brood-
stock or seed. Assessment of bacterial propagation
duringchilled storage of spermatophoresprovides
information on the status of bacterial contamina-
tion of preserved spermatophores, which can be
controlled by antibiotics.Development of a chilled
storage technique of spermatophorescould provide
a reliable and steady supply of high quality sper-
matophores, facilitate an artificial insemination
program, improve hatchery management, and
allow easy transportation of male gamete without
physically moving male broodstock as well as
cross-breeding between different strains and spe-
cies (intra- and inter-specific crosses). Presently,
no work has been reported on the chilled storage
of black tiger shrimp spermatophores. We have
developed techniques for preservation of black
tiger shrimp spermatophores at 2-4 C in order
to evaluate their feasibility and application for
hatchery operation. The objectives of this study
were to compare extenders for preservation of
black tiger shrimp spermatophores with the oc-
currence of bacteria during storage at 2-4 C and
to evaluate the total bacterial number and identify
the predominant bacteria inside spermatophores
during chilled storage with or without antibiotic
supplementation.
Materials and Methods
Broodstock Management
Sexually mature Penaeus monodon males and
females were collected from natural spawning
77
grounds around Si Chang Island in the Gulf of
Thailand, Chonburi Province, and transported to
a hatchery within 48 h after capture. All captured
males were held for 3-4 d in aerated seawater
prior to an experiment. They were fed twice a day
with mixed fresh squid, mussels, and polychaetes
at a rate of 5 1 0 % body weight by dividing equal
portions of the daily ration for the morning and
afternoon. Broodstocks were maintained under a
natural photoperiod of approximately 12-h light
and 12-h dark, in 15-m2 rectangular tanks with
water depth of 90 cm. Shrimp were maintained
at a density of three individuals per m2. Water
exchange was about 3 0 4 0 % daily, and organic
waste from the bottom of experimental tanks
was removed each night. Shrimp were weighed
to the nearest 0.1-g body weight and measured
to the nearest 0.1-cm total body length (base of
eyestalk to tip of telson). Average weight and
total length of males (N= 144) was 64.1 f 5.3 g
*
and 18.6 0.6 cm, respectively. Average values
of water temperature, salinity and pH of holding
tankswere27.6*2.4C,30.5*1.1ppt,and8.0*
0.6, respectively.
Collection of Spermatophores
Spermatophores of P. monodon were removed
by dissecting both spermatophores from individu-
als at the base of the fifth pairs of walking legs
(pereiopods)using forceps and aseptic techniques.
Each spermatophorewas transferred into eppendorf
tubes containing different kinds of extenders using
sterile forceps. Tubes were immediately placed in
the incubator at 2-4 C and kept for variable periods
of time. Males without mature spermatophores
were not used in the experiments. Only spermato-
phores that were not melanized were selected for
preservation studies, since captive penaeid males
often develop necrotic spermatophores,referred to
as black or melanized spermatophores (Dougherty
and Dougherty 1990).
Extenders
In order to determine the appropriate extender
for the chilled storage, spermatophores were
preserved with mineral oil, Ringers solution,
phosphate buffer, and 0.85% sodium chloride.
Mineral oil was purchased from Sigma Chemi-
cal Company, USA (Lot no.21K0038). Ringers
78 NIMRAT ET AL.
solution and phosphate buffer were prepared ac-
cording to protocols developed by Chow (1982)
and Jeyalectumie and Subramoniam (1989).
respectively. The composition of Ringer's solu-
tion (per 1,000-mL distilled water) was 9.88-g
NaCl, 0.44-g CaC1,.2H,O, 0.37-g KC1, 0.04-g
NaH,P04.H2O, 0.21-g Na,HP0,.2H20, and 0.3-
g MgC1,.6 H,O. The composition of phosphate
buffer (per 1,000 mL distilled water) was 20-g
KH,PO, and 20-g Na,HPO,. 12H,O. In each
treatment, spermatophores were immersed with
1-mL solution inside 1.5-mL sterile eppendorf
tubes and kept at 2-4 C without renewing the
preserving medium during a 42-d storage period.
Once the best extender for chilled storage of
spermatophores was identified, the addition of
an antibiotic into the extender was examined.
In order to observe the effect of antibiotics on
bacteria during storage, spermatophores were
preserved with the most suitable extender (min-
eral oil) to which were added one of four concen-
tration levels of penicillin-streptomycin (0.18,
1%, 2%, and 3%, v h ) . Penicillin-streptomycin
was prepared with 10,000 units/mL penicillin G
sodium and 10,000 pg/mL streptomycin sulfate
in 0.85% saline (Gibco BRL, Cat. no. 15140-
148). Spermatophores were preserved in a 1-mL
solution containing different levels of antibiotic
inside a I .5-mL sterile eppendorf tube at 2-4 C
without renewing the preserving medium during
a 42-d storage period inside the incubator. In
both experiments, spermatophores were placed
randomly in eppendorf tubes immediately after
removal from males. Each tube received one
spermatophore. The lid of tubes was closed
during storage but opened once every 7 d for 10
min for oxygen transfer. For each treatment, 24
preserved spermatophores from 12 males were
used to evaluate sperm viability once a week for
6 wk. Therefore, four preserved spermatophores
were removed every week for evaluation of sperm
viability in each treatment. Similarly, 12 preserved
spermatophoresobtained from the other males (N
= 6) were used for evaluation of bacterial growth
and identification of bacteria once every 2 wk.
As a result, four preserved spermatophores were
removed from each treatment once every 2 wk
for evaluation of bacterial growth. Evaluation of
sperm viability and bacterial propagation in both
experiments was performed on a 42-d storage
period basis to obtain baseline data of chilled stor-
age of spermatophores for further development
of the chilled storage technique. Equipment and
solutions were sterilized by autoclaving 120 C at
15 pounds per square inch for 15 min. Changes
in the color of the extender were observed dur-
ing storage.
Enumeration and Identtjkation of Bacteria
Total bacterial count was evaluated by the
pour plate technique for variable periods of time.
Spermatophores were dissected, weighed and
homogenized before serial diluting in 500-pL
sterile distilled water. A 100-pL diluted sample
was pipetted into a petri disc prior to adding 20
mL of plate count agar (PCA) media (Difco, De-
troit, Michigan, USA) following the pour plate
technique. The plates were incubated at 35 C for
48 h. Total bacterial count as colony-formingunits'
(CFU/g) was recorded and the characteristics of the
colonies were observed. Isolated colonies on PCA
plates were purified onto new PCA and identified
to genera on the basis of cell morphology, Gram
staining technique and standard biochemical tests
according to Bergey's manual (Palleroni 1984;
Schleifer 1986; Sneath 1986). The biochemical
tests performed included Gram's reaction, cell
morphology, catalase, oxidase, motility, citrate
utilization, urease, growth in 6.5% NaCl, and
triple sugar iron (TSI) reaction. Evaluation of the
bacterial growth inside spermatophoresin relation
to the sampling period was done in triplicate for
each treatment throughout the experiments.
Evaluation of Sperm Viability
Preserved spermatophoresduring storage were
stained with the eosin-nigrosin (Jeyalectumie and
Subramoniam 1989). Spermatophores were dis-
sected and part of the sperm mass was extruded to
form a suspension (50 pL) prior to adding 50 pL
of 0.5% eosin and 100 pL of 10%nigrosin. The
smear was then mixed thoroughly, dried under
flame and examined using a light microscope at
1000-fold magnification. Dead sperm cells ap-
peared pink, whereas live sperm were unstained
against a red background of nigrosin. Percentage
of viable sperm was calculated in triplicate, each
from counting of a minimum number of 250 sperm
 PRESERVATION OF BLACK TIGER SHIMP SPERMATOPHORES
cells on a slide. Evaluation of the percentage of
viable sperm at different sampling periods was
monitored throughout the experiments.
Art$cial Insemination
After obtaining data on sperm viability and
bacterial count from the two experiments, a pre-
liminary attempt was made to determine the fertil-
ization efficiency of preserved spermatophoresby
artificial insemination. Six spermatophores were
collected from mature male P. monodon (N= 3 ) on
the same day and immediately preserved in min-
eral oil without penicillin-streptomycinfor 7-8 d at
2-4 C before being used for artificial insemination.
Female P. monodon were induced to maturity by
unilateral eyestalk ablation. Newly molted females
(N = 3) with average weight and length of 105.5
2 5.7 gm and 23.6 k 3.1 cm, respectively, were
artificially inseminated with preserved spermato-
phores. Two preserved spermatophores from the
same male were inserted into the thelycum of a
female with sterile forceps. When females were
ready to ovulate, each was individually transferred
to a 500-L fiberglasstank. Females spawned within
6-7 d after artificial insemination. In the control
group, fresh spermatophores were collected and
immediately placed into the thelycum of females
(N = 3). Fertilization rate (number of fertilized
eggs to total eggs x 100) was evaluated 7-8 h after
spawning when fertilized eggs reached the gastrula
stage, by randomly counting 300 eggs with four
replications under the light microscope. The eggs
were allowed to hatch inside the tank with con-
tinuous aeration. Hatching rate of nauplii (number
of nauplii to total eggs x 100) was determined 30
h after spawning by counting number of nauplii
in four 500-mL aliquots from the tank. Care was
taken to stir the water before sampling to ensure
uniform distribution of eggs and nauplii
Statistical Analysis
Results are shown as means f standard error
of the mean (SEM). Data on percentage of sperm
viability, bacteria counts, fertilization rate, and
hatching rate were arcsine transformed prior to sta-
tistical analysis using statistical analysis software
(SPSS). To examine treatment effects of extenders
and antibiotic supplementation over time, data
were analyzed by a two-way analysis of variance
79
(ANOVA). When differences were significant,data
from the same sampling date were subjected to a
one-way ANOVA to detect significant differences
between treatments. Means were separated using
Duncans New Multiple Range Test and were
considered to be significant at P c 0.05. Students
t-test (P c 0.05) was used to compare fertilization
rate and hatching rate of eggs artificially insemi-
nated with freshly collected spermatophores and
preserved spermatophores.
Results
Identification of the Appropriate Extender for the
Chilled Storage of Spermatophores
The characteristics of preserved spermato-
phores in all treatments at the beginning of the
experiment (day 0) did not show any significant
change in external morphology of spermatophores.
At the end of the experiment (day 42), the ex-
ternal appearance of spermatophores preserved
with mineral oil and Ringers solution appeared
morphologically normal and similar to those at the
beginning of experiment. However, the color of
Ringers solution started to become cloudy while
that of mineral oil remained unchanged. Abnormal
morphology of preserved spermatophores was
clearly observed with treatments using phosphate
buffer and 0.85% NaCl as spermatophores started
to distort and lose their structural integrity. Dete-
rioration of spermatophores preserved with phos-
phate buffer and 0.85% NaCl also occurred at the
end of the experiment and was evident from the
odor of preserved spermatophores when opening
the vials. Also, the color of the treatment using
phosphate buffer changed to light yellow by day 42
while that of treatment in 0.85% NaCl developed
light green color.
At the beginning of the experiment (day 0),
there was no significant difference (P > 0.05) in
the percentage of viable sperm among treatments
preserved with extenders, averaging from 91.7 f
2.9% to 93.3 f 2.9% (Fig. 1). In all treatments,
percentage of viable sperm decreased significantly
(P c 0.05) with time. At the end of the experiment
(day 42), spermatophores preserved with mineral
oil and Ringers solution remained alive with aver-
age values of 58.3 f 2.9%and 1 1.7 f 2.9%,respec-
tively. However, viable sperm in the treatments
80 NIMRAT ET AL.
FIGUREI . Percentage of sperm viability of black tiger
shrimp spe-tophores during a 42-d storage period
with various extenders. Bars with different letters
are significantly different (p < 0.05) within the Same
sampling date.
FIGURE3. Contamination of Bacillus sp. (x IeCFUIg)
in black tiger shrimp spermatophores during a 42-d
storage period with various extenders. Bars with
different letters are signiJicantlydifferent ( P < 0.05)
within the same sampling date.
using phosphate buffer and 0.85% NaCl were no
longer observed by day 35 and 2 1, respectively.
At the beginning of the experiment (day 0),
there was no significant difference ( P > 0.05) in
total bacteria count of spermatophores among
treatments, averaging from (7 f 1.7) x lo3to (21.3
f 8.5)x103 CFU/g (Fig. 2). However, total bacteria
count in all treatments increased significantly ( P <
0.05) during the storage period. Also, there were
significant differences ( P < 0.05) in total bacterial
count of spermatophores among the treatments.
Except for Ringers solution [( 136 f 24.4) x lo3
CFU/g], spermatophores preserved with mineral
FIGURE2. Total bacterial count ( X CFUIg) present
in black tiger shrimp sPe-tOPhores during a 42-d
storage period with various extenders. Bars with
different letters are significantly different ( P < 0.05)
within the same sampling date. Values of 300 x l @
CFUlg on the Y-axis represented the number of bac-
teria over 300 x I @ CFUIg.
FIGURE4. Contamination of Staphylococcus sp. ( X I @
CFUIg) in black tiger shrimp spermatophoresduring
a 42-d storage period with various extenders. Bars
with different letters are significantly different ( P <
0.05)within the same sampling date.
oil, phosphate buffer and 0.85% NaCl had bacterial
counts of more than 300 x lo3CFU/g by the end
of the experiment.
Identification of bacteria by biochemical tests
revealed that there were three dominant types of
bacteria present in preserved spermatophores,
Bacillus sp., Staphylococcus sp. and Pseudomonas
aeruginosa (Table 1). The appearance of isolated
Bacillus colonies was white with a rough perim-
 PRESERVATION OF BLACK TIGER SHRIMP SPERMATOPHORES 81

1. Characteristics used to differentiate the dominant bacteria

TABLE
~~~ ~

Characteristics Staphylococcus sp. Bacillus sp. Pseudomonas aeruginosa

Gram reaction Positive Positive Negative
Cell morphology Cocci (cluster) Rod Rod
Blood agar Alpha-hemolysis Gamma-hemolysis Gamma-hemolysis
hemolysis
Growth on +
MacConkey
Catalase test + + +
Oxidase test +
Coagulase test
String test +
TSI (Triple sugar Acid slantlacid, Alkaline slantlalkaline, Acid slantlacid,
iron) test H,S -,gaS - H,S -,gas - H,S -,gas -
Motility test + +
Utilization of citrate +
LIA (Lysine iron +
agar) test
Utilization of urea
Growth in 6% NaCl +
OF glucose test +/-

eter, gram positive, rod shaped with a large spore

in the vegetative cell. Staphylococcus sp. formed
characteristics of yellow colonies with a round
prominent shape and smooth perimeter on the sur-
face of culture media, gram positive, and spherical
in shape. However, P. aeruginosa appeared as
transparent colonies with a round prominent shape
and smooth surface. Cells were gram negative
and small rod-shaped. The presence of Bacillus
sp. was observed prominently in spermatophores
preserved with Ringers solution, compared to
other extenders (Fig. 3). Highest values of B u d -
lus sp. in spermatophorespreserved with Ringers
solution (30.3 f 8.1) x 103CFU/g)were observed
on day 28. The number of Staphylococcus sp. on FIGURE5 . Contamination of Pseudomonas aeruginosa
spermatophores preserved with mineral oil was ( x Iff CFUIg) in black tiger shrimp spermatophores
during a 42-d storage period with various extenders.
significantly higher (P c 0.05) than that found for
Bars with different letters are signgcantly different
other extender treatments, with values of (219.3 (P < 0.05)within the same sampling date. Values of
f 4.9) x lo3CFU/g on day 42 (Fig. 4). The num- 3OOxIff CFUIg on the Y-axisrepresented the number
ber of l? aeruginosa present on spermatophores of bacteria over 300 x 101 CFUIg.
preserved with phosphate buffer and 0.85% NaCl
82 NIMRAT ET AL.

FIGURE7. Total bacterial count (x I@ CFUIg) present

FIGURE6. Percentage of sperm viabiliv of black tiger
shrimp spermatophoresduring a 42-d storage period
with mineral oil supplemented with penicillin-strep-
tomycin at 0.1 %, I%, 2 8 , and 3%.Bar with similar
letter is not signijicantly diyerent ( P > 0.05) within
the same sampling date.
in black tiger shrimp spermatophores during a 42-d
storage period with mineral oil supplemented with
penicillin-streptomycin at 0.1 %, 1%, 2 8 , and 3%.
Bars with different letters are signijicantly different
( P < 0.05)within the same sampling date.

was significantly higher ( P < 0.05) than those of

mineral oil and Ringer's solution (Fig. 5).
h
Evaluation of Effect of Antibiotics on Preserving
of Spermatophores
$
u
The appearance of spermatophores preserved
B with mineral oil containing0.1%, I%, 2% ,and 3%
v
x
of penicillin-streptomycin at the end of experiment
(day 42) did not show any significant change,
compared to the beginning of experiment (day 0).
In all treatments, the color of the extender solution
remained unchanged throughout the study. There
was no significant difference (P >0.05) in percent-
age of viable sperm of spermatophores among
FIGURE8. Contamination of Bacillus sp. (XI@ CFUIg) treatments preserved with mineral oil containing
in black tiger shrimp spermatophores during a 42-d 0.1 %, 1%, 2%, and 3% penicillin-streptomycin,
storage period with mineral oil supplemented with although sperm viability showed a trend of gradual
penicillin-streptomycin at 0.1%, 1%, 2%, and 3%. decline over the course of experiment (Fig. 6).
Bar with similar letter is not signijkantly different There was no significant difference (P > 0.05)
(P > 0.05) within the same sampling date.
in total bacteria counts of spermatophores pre-
served with four levels of penicillin-streptomycin
(Fig. 7). Average values of total bacteria count
at the beginning of the experiment (day 0) in all
treatments ranged between (10.3 f 2.3) x lo3and
(17 f 4.7 )x lo3CFU/g. However, total bacteria
count in all treatments declined rapidly by day 14
and were undetectable on day 42.
Three bacteria, Bacillus sp., Staphylococcus
sp. and P. aeruginosa, were also present in pre-
 PRESERVATION OF BLACK TIGER SHRIMP SPERMATOPHORES 83

served spermatophores containing varying levels

of penicillin-streptomycin during the storage
period. The number of Bacillus sp. found among
treatments at the beginning of the experiment was
between (6 f 1.7) x lo3and (9 f 3.8) x lo3CFU/g
and declined to an undetectable level by day 42
(Fig. 8). Similarly, the number of Staphylococcus
sp. on spermatophores in all treatments at the be-
ginning of the experiment ranged from (1 f 0.6)
x lo3to (4.3 f 1.9) x lo3CFU/g and decreased to
undetectable levels by day 42 (Fig. 9). Average
number of P. aeruginosa on spermatophores in
all treatments at the beginning of the experiment
Time (d)
varied between (3.3 f 0.9) x lo3 and (6.7 f 3.4)
x lo3CFU/g and decreased to undetectable levels FIGURE9. Contamination of Staphylococcus sp. (x 101
by day 42 (Fig. 10). CFUIg) in black tiger shrimp spermatophores during
a 42-d storage period with mineral oil supplemented
Fertility and Hatching with penicillin-streptomycin at O.l%, I %, 2%, and
3%. Bar with similar letter is not signijcantly differ-
Spermatophorespreserved for up to 7-8 d were
ent (P > 0.05)within the same sampling date.
successful in fertilizing eggs with no significant
differences (P> 0.05) in fertilization rate between
fresh (90.1 f 1.1%) and preserved spermatophores
(88.3 f 0.9%). Hatching rate of eggs fertilized with
preserved spermatophores was 87.6 f 1.2%. not
significantly different (P> 0.05) from that of the
control group (88.4 f 1.3%). Y

Discussion
Mineral oil was the only suitable extender for
chilled storage of spermatophores producing sig-
nificant percentage of viable sperm. Degenerative
changes of preserved spermatophores in terms of
odor and morphology distortion developed rap-
idly when preserved with phosphate buffer and
0.85% NaCI. The development of a light yellow
coloration to the treatment using phosphate buf- FIGURE10.Contamination of Pseudomonas aeruginosa
fer and a light green coloration of 0.85% NaCl (x 1O'CFUIg) in black tiger shrimp spermatophores
during a 42-d storage period with mineral oil supple-
treatment during storage may reflect the growth mented withpenicillin-streptomycinat 0.1%, I %, 2%,
of P. aeruginosa since these bacteria produces the and 3%. Bar with similar letter is not signijcantly
water soluble yellow-green pigments referred to different (P > 0.05)within the same sampling date,
as pseudobactins or pyoverdins (Julich et al. 200 1;
Mureseanu et al. 2003). However, the presence
of bacterial contamination in the treatment with
mineral oil may not directly affect the viability of
sperm, although high numbers of Staphylococcus
sp. and total bacteria count on day 42 were clearly
observed. These observations may indicate that the
type and number of bacteria on spermatophores
preserved with mineral oil did not affect the vi-
84 NIMRAT ET AL.
ability of spermatozoa.Presently, it is not known if
bacterial flora present in preserved spermatophores
affects the health of female broodstocks. Bacterial
propagation on spermatophores may be largely
prevented by replacing the preserving medium
at regular intervals. Chow (1982) reported that
spermatophores of Macrobrachium rosenbergii
preserved for periods up to 9 d at 2 C resulted in
successful fertilization when preserving medium
was renewed every 2 d during chilled storage.
Although maintenance of spermatophores at low
temperatures of about 2-4 C may aid in retarding
the growth of bacteria during storage, the relatively
longer successful storage period and higher per-
centage of sperm viability in the treatment using
mineral oil than other treatments using Ringers
solution, phosphate buffer and 0.85% NaCl, also
indicated the superiority of mineral oil over the
other extenders. However, this low-temperature
storage of black tiger shrimp spermatophores
could not prevent bacterial growth in the sperm
mass, similar to lobster spermatophores reported
by Ishida et al. (1986).
Mineral oil has been incorporatedas an extender
for short-term preservation of cells. The successful
storage of spermatophores of the present study
using mineral oil may derive from the fact that it
functions as a common moisturizing ingredient
that can prevent dehydration of the cells (Richard-
son et al. 2002). Sankai et al. (2001) reported on
the successful storage of mouse epididymal sper-
matozoa with a high percentage of progressively
motile spermatozoa (78.3%)after a short-term
storage of 8 d in mineral oil at 5 C. Richardson et
al. (2002) have studied short-term preservation of
rainbow trout Oncorhynchus mykiss eyed eggs us-
ing a perfluorochemicaland found that eggs stored
in medium with mineral oil had higher hatching
rates than other treatments, after storage for 3 wk
and incubation at 10 C. However, mineral oil has
been reported to adversely affect the hatching rates
of Japanese parrotfish and sea bass eggs (Mori et
al. 1983).The discrepancy on the role of mineral
oil for short-term storage may also depend on the
storage time. As rainbow trout eyed eggs preserved
with perfluorochemical and mineral oil had poor
hatching rates after 5 wk of storage (Richardsonet
al. 2002), our study indicated that a 42-d storage
of black tiger shrimp spermatophores resulted in
the decline of sperm viability. Bray and Lawrence
(1998) initiated a comparison study on the use of
seawater and calcium-free saline solution in an
attempt to store gonad tissue of marine shrimp
Penaeus vannamei and found that there were no
significantdifferences in either sperm count or per-
cent abnormal sperm between the two treatments
after a short storage period of up to 36 h at 15 C.
In the present study, sperm viability may have
been improved by replacing the extender medium
as founded by Chow (1982) for M. rosenbergii.
Sperm quality evaluation by eosin and nigrosin
staining was based on the dye exclusion principle.
Eosin and nigrosin cannot penetrate the plasma
membrane of live cells but can enter dead cells and
stain them; thus assessing the structural integrity
of the sperm plasma membrane (Schrader et al.
1986). Further studies are necessary to clarify
the effect of storage period on sperm viability of
preserved spermatophores using mineral oil, and
to explore other extenders to prolong sperm viabil-
ity of black tiger shrimp spermatophores during
chilled storage. It is not yet known whether sperm
viability of black tiger shrimp may be affected by
the osmolality of extenders.
Although storage in Ringers solution main-
tained sperm viability for 42 d, it was not an
appropriate extender for chilled storage due to
low sperm viability. The degenerative change of
spermatophores preserved with Ringers solution
may be partially associated with the presence of
massive numbers of Bacillus sp. in samples and
moderate counts of total bacteria (136 f 24.4)
x lo3 CFU/g). Chow (1982) reported that the
propagation of bacilli inside spermatophores of
M . rosenbergii during chilled storage accelerated
the degeneration of matrices, which resulted in the
degeneration of sperm.
Bacterial contamination of spermatophores
in all treatments during the first experiment may
not be related to the collection procedure of sper-
matophores, since spermatophores were collected
from the body by dissecting prior to using sterile
forceps. Harper and Talbot (1984) suggested that
the bacterial epibionts on the lobster body surface
could be picked up by the spermatophores at the
time of manual extrusion.The increase in bacterial
growth during storage in the present study may be
explained by the sperm mass acting as an enriched
 PRESERVATION OF BLACK TIGER SHRIMP SPERMATOPHORES
food source due to lipoproteinaceous property of
spermatophores (Subramoniam 1993). Bacteria
would be capable of multiplying rapidly with ap-
propriate food sources. The presence of bacteria in
the sperm mass of preserved spermatophores has
also been reported in lobster Homarus americanus
(Ishida et al. 1986). On the contrary, Martin et al.
( 1987) reported that bacterial colonization was
present on the hardened spermatophore surface of
spiny lobster Panulirus interruptus, but was not
found to penetrate into deeper layers of spermato-
phores due to antibacterial activity of phenolic
substances inside spermatophores.
Similar values of sperm viability after 42 d in
mineral oil with and without penicillin-streptomy-
cin may suggest that incorporation of the antibiotic
in mineral oil did not affect sperm viability. How-
ever, supplementationof penicillin-streptomycinat
the range from 0.1 to 3% could effectively reduce
bacterial contamination in preserved spermato-
phores since bacterial contamination was greatly
reduced after 14 d before declining to undetectable
level after 42 d. Incorporation of penicillin-strepto-
mycin into mineral oil strongly inhibited bacteria
growth during chilled storage. Also, the numbers
of Bacillus sp., Staphylococcus sp., and P. aerugi-
nosa declined rapidly to undetectable levels at the
end of the storage period after incorporation of the
antibiotics.These findings indicate the addition of
only 0.1% penicillin-streptomycin in mineral oil
could efficiently control bacteria growth during
the chilled storage of spermatophores.
The fertilizing capacity of preserved spermato-
phores of black tiger shrimp at low temperatures
of 2-4 C requires a complete evaluation at longer
storage period. However, this is the first report on
successful fertilization of P. monodon-spermato-
phores. Similar rates of fertilization and hatching
between the preserved and fresh spermatophores
indicated that chilled storage of spermatophores
for 7-8 d did not affect fertilization and hatching
success, supporting the use of the chilled storage
technique of spermatophores for spawning of P.
monodon. It is interesting to note that these pre-
served spermatophores had been retained in the
females for an additional period of 6-7 d prior to
spawning. While the benefits of chilled storage of
sperm on artificial inseminationof females are well
established for fish species, crustacean has received
85
less attention, with the research of Chow (1982)
and Ishida et al. (1986) being notable exceptions.
Chow (1982) fertilized eggs of M . rosenbergii
with spermatophoresstored in Ringers solution at
2 C for 4 d and found no impairment in hatching
success. Ishida et al. (1986) reported that female
lobsters H . americanus artificially inseminated
with preserved spermatophores stored in paraffin
oil at 4-7 C for 108 d produced fertilized eggs that
developed to the eyespot stage. Further studies are
necessary to investigate the fertilizing capacity of
preserved spermatophores of P. monodon stored
for longer time periods with or without antibiotic
supplementation.
In conclusion, mineral oil was the best extender
for chilled storage of P. monodon spermatophores
at low temperature. Supplementationof penicillin-
streptomycin at a level as low as 0.1% in mineral
oil was sufficient to control bacterial contamina-
tion of preserved spermatophores without causing
a negative effect on sperm viability. Sperm mass
of spermatophores preserved in mineral oil for
7-8 d resulted in successful fertilization of eggs
and hatching of nauplii. These methods make
possible the long-distance transport of black tiger
shrimp spermatophores for in vitro and artificial
insemination efforts.
Acknowledgements
This research was funded in part by the National
Science Technology and Development Agency of
Thailand, under grant number BT-B-06-SG-25-
4404, to Dr. Verapong Vuthiphandchai. We thank
Dr. F. W. Beamish for critical review and correc-
tions to the manuscript.
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