Beruflich Dokumente
Kultur Dokumente
1
WORLD AQUACULTURE SOCIETY March 2005
SUBUNTITH
NIMRAT
Department of Microbiology and Environmental Science Program, 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- ance on wild stocks, most studies have focused on
nomically important crustacean species in Thai- the reproductivephy siofogy of females. Neverthe-
land, generating foreign income of about US$1 less, the nonsynchronous final sexual maturation
billion (89,230 million baht) with the production between male and female stocks in a hatchery often
of 304,987 tons in the year 2000 (Department of creates a problem regarding the management of
Fisheries 2000). One of the major obstacles in the male broodstock, since fertilization requires mat-
development of the black tiger shrimp industry is ing to augment seed production. Mating in penaeid
the inadequate and inconsistent supply of good shrimp involves the transfer of spermatophores
quality seed. Currently, production of shrimp seed from males to the thelycum of females. Moreover,
is still dependent on wild stocks collected from problems sometimes arise with the quality of males
estuaries, which is unpredictable and seasonal. in that they may not have mature spermatophores
Although the domesticationof shrimp broodstocks or their sperm may have deteriorated. Access of
has been recently developed to minimize the reli- good quality sperm via spermatophore preserva-
tion would provide an alternativesource of reliable
'Correspondingauthor and high quality spermatophores.
76
PRESERVATION OF BLACK TIGER SHIMP SPERMATOPHORES 77
Successful preservation of crustacean sper- grounds around Si Chang Island in the Gulf of
matophores was first reported in freshwater Thailand, Chonburi Province, and transported to
shrimp Macrobrachiurn rosenbergii, in which a hatchery within 48 h after capture. All captured
sperm viability and successful fertilization were males were held for 3-4 d in aerated seawater
maintained up to 4 d after storage in Ringers prior to an experiment. They were fed twice a day
solution at 2 C (Chow, 1982). However, chilled with mixed fresh squid, mussels, and polychaetes
storage of crustacean spermatophores was devel- at a rate of 5 1 0 % body weight by dividing equal
oped by Ishida et al. (1986) who stored lobster portions of the daily ration for the morning and
spermatophores in paraffin oil at 4-7 C for up afternoon. Broodstocks were maintained under a
to 289 d. Despite the success of storage, chilled natural photoperiod of approximately 12-h light
storage of spermatophoresusing these approaches and 12-h dark, in 15-m2 rectangular tanks with
could not prevent bacterial growth in the sperm water depth of 90 cm. Shrimp were maintained
mass of preserved spermatophores. Controlling at a density of three individuals per m2. Water
the bacterial proliferation on spermatophores is a exchange was about 3 0 4 0 % daily, and organic
prerequisite for preservation of spermatophores. waste from the bottom of experimental tanks
Bacteria may cause a negative effect on fertiliza- was removed each night. Shrimp were weighed
tion rate of preserved spermatophores and on the to the nearest 0.1-g body weight and measured
health of artificially inseminated female brood- to the nearest 0.1-cm total body length (base of
stock or seed. Assessment of bacterial propagation eyestalk to tip of telson). Average weight and
duringchilled storage of spermatophoresprovides total length of males (N= 144) was 64.1 f 5.3 g
information on the status of bacterial contamina- *
and 18.6 0.6 cm, respectively. Average values
tion of preserved spermatophores, which can be of water temperature, salinity and pH of holding
controlled by antibiotics.Development of a chilled tankswere27.6*2.4C,30.5*1.1ppt,and8.0*
storage technique of spermatophorescould provide 0.6, respectively.
a reliable and steady supply of high quality sper-
Collection of Spermatophores
matophores, facilitate an artificial insemination
program, improve hatchery management, and Spermatophores of P. monodon were removed
allow easy transportation of male gamete without by dissecting both spermatophores from individu-
physically moving male broodstock as well as als at the base of the fifth pairs of walking legs
cross-breeding between different strains and spe- (pereiopods)using forceps and aseptic techniques.
cies (intra- and inter-specific crosses). Presently, Each spermatophorewas transferred into eppendorf
no work has been reported on the chilled storage tubes containing different kinds of extenders using
of black tiger shrimp spermatophores. We have sterile forceps. Tubes were immediately placed in
developed techniques for preservation of black the incubator at 2-4 C and kept for variable periods
tiger shrimp spermatophores at 2-4 C in order of time. Males without mature spermatophores
to evaluate their feasibility and application for were not used in the experiments. Only spermato-
hatchery operation. The objectives of this study phores that were not melanized were selected for
were to compare extenders for preservation of preservation studies, since captive penaeid males
black tiger shrimp spermatophores with the oc- often develop necrotic spermatophores,referred to
currence of bacteria during storage at 2-4 C and as black or melanized spermatophores (Dougherty
to evaluate the total bacterial number and identify and Dougherty 1990).
the predominant bacteria inside spermatophores Extenders
during chilled storage with or without antibiotic
supplementation. In order to determine the appropriate extender
for the chilled storage, spermatophores were
Materials and Methods preserved with mineral oil, Ringers solution,
Broodstock Management phosphate buffer, and 0.85% sodium chloride.
Mineral oil was purchased from Sigma Chemi-
Sexually mature Penaeus monodon males and
cal Company, USA (Lot no.21K0038). Ringers
females were collected from natural spawning
78 NIMRAT ET AL.
solution and phosphate buffer were prepared ac- experiments was performed on a 42-d storage
cording to protocols developed by Chow (1982) period basis to obtain baseline data of chilled stor-
and Jeyalectumie and Subramoniam (1989). age of spermatophores for further development
respectively. The composition of Ringer's solu- of the chilled storage technique. Equipment and
tion (per 1,000-mL distilled water) was 9.88-g solutions were sterilized by autoclaving 120 C at
NaCl, 0.44-g CaC1,.2H,O, 0.37-g KC1, 0.04-g 15 pounds per square inch for 15 min. Changes
NaH,P04.H2O, 0.21-g Na,HP0,.2H20, and 0.3- in the color of the extender were observed dur-
g MgC1,.6 H,O. The composition of phosphate ing storage.
buffer (per 1,000 mL distilled water) was 20-g
Enumeration and Identtjkation of Bacteria
KH,PO, and 20-g Na,HPO,. 12H,O. In each
treatment, spermatophores were immersed with Total bacterial count was evaluated by the
1-mL solution inside 1.5-mL sterile eppendorf pour plate technique for variable periods of time.
tubes and kept at 2-4 C without renewing the Spermatophores were dissected, weighed and
preserving medium during a 42-d storage period. homogenized before serial diluting in 500-pL
Once the best extender for chilled storage of sterile distilled water. A 100-pL diluted sample
spermatophores was identified, the addition of was pipetted into a petri disc prior to adding 20
an antibiotic into the extender was examined. mL of plate count agar (PCA) media (Difco, De-
In order to observe the effect of antibiotics on troit, Michigan, USA) following the pour plate
bacteria during storage, spermatophores were technique. The plates were incubated at 35 C for
preserved with the most suitable extender (min- 48 h. Total bacterial count as colony-formingunits'
eral oil) to which were added one of four concen- (CFU/g) was recorded and the characteristics of the
tration levels of penicillin-streptomycin (0.18, colonies were observed. Isolated colonies on PCA
1%, 2%, and 3%, v h ) . Penicillin-streptomycin plates were purified onto new PCA and identified
was prepared with 10,000 units/mL penicillin G to genera on the basis of cell morphology, Gram
sodium and 10,000 pg/mL streptomycin sulfate staining technique and standard biochemical tests
in 0.85% saline (Gibco BRL, Cat. no. 15140- according to Bergey's manual (Palleroni 1984;
148). Spermatophores were preserved in a 1-mL Schleifer 1986; Sneath 1986). The biochemical
solution containing different levels of antibiotic tests performed included Gram's reaction, cell
inside a I .5-mL sterile eppendorf tube at 2-4 C morphology, catalase, oxidase, motility, citrate
without renewing the preserving medium during utilization, urease, growth in 6.5% NaCl, and
a 42-d storage period inside the incubator. In triple sugar iron (TSI) reaction. Evaluation of the
both experiments, spermatophores were placed bacterial growth inside spermatophoresin relation
randomly in eppendorf tubes immediately after to the sampling period was done in triplicate for
removal from males. Each tube received one each treatment throughout the experiments.
spermatophore. The lid of tubes was closed Evaluation of Sperm Viability
during storage but opened once every 7 d for 10
min for oxygen transfer. For each treatment, 24 Preserved spermatophoresduring storage were
preserved spermatophores from 12 males were stained with the eosin-nigrosin (Jeyalectumie and
used to evaluate sperm viability once a week for Subramoniam 1989). Spermatophores were dis-
6 wk. Therefore, four preserved spermatophores sected and part of the sperm mass was extruded to
were removed every week for evaluation of sperm form a suspension (50 pL) prior to adding 50 pL
viability in each treatment. Similarly, 12 preserved of 0.5% eosin and 100 pL of 10%nigrosin. The
spermatophoresobtained from the other males (N smear was then mixed thoroughly, dried under
= 6) were used for evaluation of bacterial growth flame and examined using a light microscope at
and identification of bacteria once every 2 wk. 1000-fold magnification. Dead sperm cells ap-
As a result, four preserved spermatophores were peared pink, whereas live sperm were unstained
removed from each treatment once every 2 wk against a red background of nigrosin. Percentage
for evaluation of bacterial growth. Evaluation of of viable sperm was calculated in triplicate, each
sperm viability and bacterial propagation in both from counting of a minimum number of 250 sperm
PRESERVATION OF BLACK TIGER SHIMP SPERMATOPHORES 79
cells on a slide. Evaluation of the percentage of (ANOVA). When differences were significant,data
viable sperm at different sampling periods was from the same sampling date were subjected to a
monitored throughout the experiments. one-way ANOVA to detect significant differences
between treatments. Means were separated using
Art$cial Insemination
Duncans New Multiple Range Test and were
After obtaining data on sperm viability and considered to be significant at P c 0.05. Students
bacterial count from the two experiments, a pre- t-test (P c 0.05) was used to compare fertilization
liminary attempt was made to determine the fertil- rate and hatching rate of eggs artificially insemi-
ization efficiency of preserved spermatophoresby nated with freshly collected spermatophores and
artificial insemination. Six spermatophores were preserved spermatophores.
collected from mature male P. monodon (N= 3 ) on
the same day and immediately preserved in min- Results
eral oil without penicillin-streptomycinfor 7-8 d at
2-4 C before being used for artificial insemination. Identification of the Appropriate Extender for the
Female P. monodon were induced to maturity by Chilled Storage of Spermatophores
unilateral eyestalk ablation. Newly molted females The characteristics of preserved spermato-
(N = 3) with average weight and length of 105.5 phores in all treatments at the beginning of the
2 5.7 gm and 23.6 k 3.1 cm, respectively, were experiment (day 0) did not show any significant
artificially inseminated with preserved spermato- change in external morphology of spermatophores.
phores. Two preserved spermatophores from the At the end of the experiment (day 42), the ex-
same male were inserted into the thelycum of a ternal appearance of spermatophores preserved
female with sterile forceps. When females were with mineral oil and Ringers solution appeared
ready to ovulate, each was individually transferred morphologically normal and similar to those at the
to a 500-L fiberglasstank. Females spawned within beginning of experiment. However, the color of
6-7 d after artificial insemination. In the control Ringers solution started to become cloudy while
group, fresh spermatophores were collected and that of mineral oil remained unchanged. Abnormal
immediately placed into the thelycum of females morphology of preserved spermatophores was
(N = 3). Fertilization rate (number of fertilized clearly observed with treatments using phosphate
eggs to total eggs x 100) was evaluated 7-8 h after buffer and 0.85% NaCl as spermatophores started
spawning when fertilized eggs reached the gastrula to distort and lose their structural integrity. Dete-
stage, by randomly counting 300 eggs with four rioration of spermatophores preserved with phos-
replications under the light microscope. The eggs phate buffer and 0.85% NaCl also occurred at the
were allowed to hatch inside the tank with con- end of the experiment and was evident from the
tinuous aeration. Hatching rate of nauplii (number odor of preserved spermatophores when opening
of nauplii to total eggs x 100) was determined 30 the vials. Also, the color of the treatment using
h after spawning by counting number of nauplii phosphate buffer changed to light yellow by day 42
in four 500-mL aliquots from the tank. Care was while that of treatment in 0.85% NaCl developed
taken to stir the water before sampling to ensure light green color.
uniform distribution of eggs and nauplii At the beginning of the experiment (day 0),
there was no significant difference (P > 0.05) in
Statistical Analysis
the percentage of viable sperm among treatments
Results are shown as means f standard error preserved with extenders, averaging from 91.7 f
of the mean (SEM). Data on percentage of sperm 2.9% to 93.3 f 2.9% (Fig. 1). In all treatments,
viability, bacteria counts, fertilization rate, and percentage of viable sperm decreased significantly
hatching rate were arcsine transformed prior to sta- (P c 0.05) with time. At the end of the experiment
tistical analysis using statistical analysis software (day 42), spermatophores preserved with mineral
(SPSS). To examine treatment effects of extenders oil and Ringers solution remained alive with aver-
and antibiotic supplementation over time, data age values of 58.3 f 2.9%and 1 1.7 f 2.9%,respec-
were analyzed by a two-way analysis of variance tively. However, viable sperm in the treatments
80 NIMRAT ET AL.
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 the decline of sperm viability. Bray and Lawrence
bacterial flora present in preserved spermatophores (1998) initiated a comparison study on the use of
affects the health of female broodstocks. Bacterial seawater and calcium-free saline solution in an
propagation on spermatophores may be largely attempt to store gonad tissue of marine shrimp
prevented by replacing the preserving medium Penaeus vannamei and found that there were no
at regular intervals. Chow (1982) reported that significantdifferences in either sperm count or per-
spermatophores of Macrobrachium rosenbergii cent abnormal sperm between the two treatments
preserved for periods up to 9 d at 2 C resulted in after a short storage period of up to 36 h at 15 C.
successful fertilization when preserving medium In the present study, sperm viability may have
was renewed every 2 d during chilled storage. been improved by replacing the extender medium
Although maintenance of spermatophores at low as founded by Chow (1982) for M. rosenbergii.
temperatures of about 2-4 C may aid in retarding Sperm quality evaluation by eosin and nigrosin
the growth of bacteria during storage, the relatively staining was based on the dye exclusion principle.
longer successful storage period and higher per- Eosin and nigrosin cannot penetrate the plasma
centage of sperm viability in the treatment using membrane of live cells but can enter dead cells and
mineral oil than other treatments using Ringers stain them; thus assessing the structural integrity
solution, phosphate buffer and 0.85% NaCl, also of the sperm plasma membrane (Schrader et al.
indicated the superiority of mineral oil over the 1986). Further studies are necessary to clarify
other extenders. However, this low-temperature the effect of storage period on sperm viability of
storage of black tiger shrimp spermatophores preserved spermatophores using mineral oil, and
could not prevent bacterial growth in the sperm to explore other extenders to prolong sperm viabil-
mass, similar to lobster spermatophores reported ity of black tiger shrimp spermatophores during
by Ishida et al. (1986). chilled storage. It is not yet known whether sperm
Mineral oil has been incorporatedas an extender viability of black tiger shrimp may be affected by
for short-term preservation of cells. The successful the osmolality of extenders.
storage of spermatophores of the present study Although storage in Ringers solution main-
using mineral oil may derive from the fact that it tained sperm viability for 42 d, it was not an
functions as a common moisturizing ingredient appropriate extender for chilled storage due to
that can prevent dehydration of the cells (Richard- low sperm viability. The degenerative change of
son et al. 2002). Sankai et al. (2001) reported on spermatophores preserved with Ringers solution
the successful storage of mouse epididymal sper- may be partially associated with the presence of
matozoa with a high percentage of progressively massive numbers of Bacillus sp. in samples and
motile spermatozoa (78.3%)after a short-term moderate counts of total bacteria (136 f 24.4)
storage of 8 d in mineral oil at 5 C. Richardson et x lo3 CFU/g). Chow (1982) reported that the
al. (2002) have studied short-term preservation of propagation of bacilli inside spermatophores of
rainbow trout Oncorhynchus mykiss eyed eggs us- M . rosenbergii during chilled storage accelerated
ing a perfluorochemicaland found that eggs stored the degeneration of matrices, which resulted in the
in medium with mineral oil had higher hatching degeneration of sperm.
rates than other treatments, after storage for 3 wk Bacterial contamination of spermatophores
and incubation at 10 C. However, mineral oil has in all treatments during the first experiment may
been reported to adversely affect the hatching rates not be related to the collection procedure of sper-
of Japanese parrotfish and sea bass eggs (Mori et matophores, since spermatophores were collected
al. 1983).The discrepancy on the role of mineral from the body by dissecting prior to using sterile
oil for short-term storage may also depend on the forceps. Harper and Talbot (1984) suggested that
storage time. As rainbow trout eyed eggs preserved the bacterial epibionts on the lobster body surface
with perfluorochemical and mineral oil had poor could be picked up by the spermatophores at the
hatching rates after 5 wk of storage (Richardsonet time of manual extrusion.The increase in bacterial
al. 2002), our study indicated that a 42-d storage growth during storage in the present study may be
of black tiger shrimp spermatophores resulted in explained by the sperm mass acting as an enriched
PRESERVATION OF BLACK TIGER SHRIMP SPERMATOPHORES 85
food source due to lipoproteinaceous property of less attention, with the research of Chow (1982)
spermatophores (Subramoniam 1993). Bacteria and Ishida et al. (1986) being notable exceptions.
would be capable of multiplying rapidly with ap- Chow (1982) fertilized eggs of M . rosenbergii
propriate food sources. The presence of bacteria in with spermatophoresstored in Ringers solution at
the sperm mass of preserved spermatophores has 2 C for 4 d and found no impairment in hatching
also been reported in lobster Homarus americanus success. Ishida et al. (1986) reported that female
(Ishida et al. 1986). On the contrary, Martin et al. lobsters H . americanus artificially inseminated
( 1987) reported that bacterial colonization was with preserved spermatophores stored in paraffin
present on the hardened spermatophore surface of oil at 4-7 C for 108 d produced fertilized eggs that
spiny lobster Panulirus interruptus, but was not developed to the eyespot stage. Further studies are
found to penetrate into deeper layers of spermato- necessary to investigate the fertilizing capacity of
phores due to antibacterial activity of phenolic preserved spermatophores of P. monodon stored
substances inside spermatophores. for longer time periods with or without antibiotic
Similar values of sperm viability after 42 d in supplementation.
mineral oil with and without penicillin-streptomy- In conclusion, mineral oil was the best extender
cin may suggest that incorporation of the antibiotic for chilled storage of P. monodon spermatophores
in mineral oil did not affect sperm viability. How- at low temperature. Supplementationof penicillin-
ever, supplementationof penicillin-streptomycinat streptomycin at a level as low as 0.1% in mineral
the range from 0.1 to 3% could effectively reduce oil was sufficient to control bacterial contamina-
bacterial contamination in preserved spermato- tion of preserved spermatophores without causing
phores since bacterial contamination was greatly a negative effect on sperm viability. Sperm mass
reduced after 14 d before declining to undetectable of spermatophores preserved in mineral oil for
level after 42 d. Incorporation of penicillin-strepto- 7-8 d resulted in successful fertilization of eggs
mycin into mineral oil strongly inhibited bacteria and hatching of nauplii. These methods make
growth during chilled storage. Also, the numbers possible the long-distance transport of black tiger
of Bacillus sp., Staphylococcus sp., and P. aerugi- shrimp spermatophores for in vitro and artificial
nosa declined rapidly to undetectable levels at the insemination efforts.
end of the storage period after incorporation of the
antibiotics.These findings indicate the addition of Acknowledgements
only 0.1% penicillin-streptomycin in mineral oil This research was funded in part by the National
could efficiently control bacteria growth during Science Technology and Development Agency of
the chilled storage of spermatophores. Thailand, under grant number BT-B-06-SG-25-
The fertilizing capacity of preserved spermato- 4404, to Dr. Verapong Vuthiphandchai. We thank
phores of black tiger shrimp at low temperatures Dr. F. W. Beamish for critical review and correc-
of 2-4 C requires a complete evaluation at longer tions to the manuscript.
storage period. However, this is the first report on
successful fertilization of P. monodon-spermato- Literature Cited
phores. Similar rates of fertilization and hatching Bray, W. A. and A. L. Lawrence. 1998. Male viability
between the preserved and fresh spermatophores determinations in Penaeus vannamei:evaluation of
indicated that chilled storage of spermatophores short-term storage of spermatophores up to 36 hand
comparison of Ca-free saline and seawater as sperm
for 7-8 d did not affect fertilization and hatching
homogenate media. Aquaculture 160:6347.
success, supporting the use of the chilled storage Chow, S. 1982. Artificial insemination using preserved
technique of spermatophores for spawning of P. spermatophores in the palaemonid shrimp Mac-
monodon. It is interesting to note that these pre- robrachium rosenbergii. Bulletin of the Japanese
served spermatophores had been retained in the Society of Scientific Fisheries 48: 1693-1695.
females for an additional period of 6-7 d prior to Department of Fisheries. 2000. Statistics of shrimp
spawning. While the benefits of chilled storage of culture 2000. Publication no. 2/2003. Fisheries
sperm on artificial inseminationof females are well Statistics Analysis and Research Group. Ministry of
established for fish species, crustacean has received Agriculture and Cooperatives, Thailand. 50 pp.
86 NIMRAT ET AL.