JOURNAL OF THE WORLD AQUACULTURE SOCIETY

Volume 36, No. 1 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 SANGNAWAIUJ AND VERAPONC 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 different extenders, 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., Staphylococcus sp., 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., Staphylococcus sp., 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 domestication of shrimp broodstocks has been recently developed to minimize the reli-

'Corresponding author

ance on wild stocks, most studies have focused on the reproductive phy 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 alternative source of reliable and high quality spermatophores.

8 Copyright by the World Aquaculture Society 2005

76

PRESERVATION OF BLACK TIGER SHIMP SPERMATOPHORES 77

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 Ringer’s 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 spermatophores using 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 ‘during chilled storage of spermatophores provides information on the status of bacterial contamina- tion of preserved spermatophores, which can be controlled by antibiotics. Development of a chilled storage technique of spermatophores could 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

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 3040% 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 spermatophore was 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, Ringer’s solution, phosphate buffer, and 0.85% sodium chloride. Mineral oil was purchased from Sigma Chemi- cal Company, USA (Lot no.21K0038). Ringer’s

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 spermatophores obtained 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-forming units' (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 spermatophores in relation to the sampling period was done in triplicate for each treatment throughout the experiments.

Evaluation of Sperm Viability Preserved spermatophores during 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 79

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 spermatophores by 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-streptomycin for 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 fiberglass tank. 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

(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 Duncan’s New Multiple Range Test and were considered to be significant at P c 0.05. Student’s 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 Ringer’s solution appeared morphologically normal and similar to those at the beginning of experiment. However, the color of Ringer’s 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 Ringer’s 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.

FIGURE I . 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.

FIGURE 2. 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.

FIGURE 3. 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 signiJicantly different ( 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 lo3 to (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 Ringer’s solution [( 136 f 24.4) x lo3 CFU/g], spermatophores preserved with mineral

FIGURE 4. Contamination of Staphylococcus sp. ( X I @ CFUIg) in black tiger shrimp spermatophores during 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 lo3 CFU/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

TABLE 1. Characteristics used to differentiate the dominant bacteria

81

~~~ ~

Characteristics Staphylococcus sp. Bacillus sp. Pseudomonas aeruginosa

Gram reaction

Cell morphology

Blood agar hemolysis

Growth on MacConke y Catalase test

Oxidase test

Coagulase test

String test

TSI (Triple sugar iron) test

Motility test

Utilization of citrate

LIA (Lysine iron agar) test

Utilization of urea

Growth in 6% NaCl

OF glucose test

Positive

Cocci (cluster)

Alpha-hemolysis

Positive

Rod

Gamma-hemolysis

Negative

Rod

Gamma-hemoly sis

+

Acid slantlacid, H,S -, gaS -

+

+

+

Alkaline slantlalkaline, H,S -, gas - +

+

+ +

+ Acid slantlacid, H,S -, gas - + +

+/-

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 Ringer’s solution, compared to other extenders (Fig. 3). Highest values of B u d - lus sp. in spermatophores preserved with Ringer’s solution (30.3 f 8.1) x 103CFU/g) were observed on day 28. The number of Staphylococcus sp. on spermatophores preserved with mineral oil was significantly higher (P c 0.05) than that found for other extender treatments, with values of (219.3 f 4.9) x lo3 CFU/g on day 42 (Fig. 4). The num- ber of l? aeruginosa present on spermatophores preserved with phosphate buffer and 0.85% NaCl

FIGURE 5 . Contamination of Pseudomonas aeruginosa (x Iff CFUIg) in black tiger shrimp spermatophores during a 42-d storage period with various extenders. Bars with different letters are signgcantly different (P < 0.05) within the same sampling date. Values of 3OOxIff CFUIg on the Y-axis represented the number of bacteria over 300 x 101 CFUIg.

82 NIMRAT ET AL.

FIGURE 6. Percentage of sperm viabiliv of black tiger shrimp spermatophores during 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.

h

u $ B x v

FIGURE 8. Contamination of Bacillus sp. (XI@ CFUIg) in black tiger shrimp spermatophores during a 42-d storage period with mineral oil supplemented with penicillin-streptomycin at 0.1%, 1 %, 2%, and 3%. Bar with similar letter is not signijkantly different (P > 0.05) within the same sampling date.

FIGURE 7. Total bacterial count (x I@ CFUIg) present 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).

Evaluation of Effect of Antibiotics on Preserving of Spermatophores

The appearance of spermatophores preserved with mineral oil containing 0.1 %, I%, 2% ,and 3% 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 treatments preserved with mineral oil containing 0.1 %, 1 %, 2%, and 3% penicillin-streptomycin, although sperm viability showed a trend of gradual decline over the course of experiment (Fig. 6).

There was no significant difference (P > 0.05) 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 lo3 and (17 f 4.7 )x lo3 CFU/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 lo3 and (9 f 3.8) x lo3 CFU/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 lo3 to (4.3 f 1.9) x lo3 CFU/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 varied between (3.3 f 0.9) x lo3 and (6.7 f 3.4) x lo3 CFU/g and decreased to undetectable levels by day 42 (Fig. 10).

Fertility and Hatching

Spermatophores preserved for up to 7-8 d were 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%).

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- fer and a light green coloration of 0.85% NaCl treatment during storage may reflect the growth of P. aeruginosa since these bacteria produces the water soluble yellow-green pigments referred to 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-

Time (d)

FIGURE 9. Contamination of Staphylococcus sp. (x 101 CFUIg) in black tiger shrimp spermatophores during a 42-d storage period with mineral oil supplemented with penicillin-streptomycin at O.l%, I %, 2%, and 3%. Bar with similar letter is not signijcantly differ- ent (P > 0.05) within the same sampling date.

Y

FIGURE 10. Contamination of Pseudomonas aeruginosa (x 1O'CFUIg) in black tiger shrimp spermatophores during a 42-d storage period with mineral oil supple- mented with penicillin-streptomycin at 0.1 %, I %, 2%, and 3%. Bar with similar letter is not signijcantly different (P > 0.05) within the same sampling date,

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 Ringer’s 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 incorporated as 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 perfluorochemical and 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 (Richardson et 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 significant differences 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 Ringer’s 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 Ringer’s 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 85

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, supplementation of penicillin-streptomycin at 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 insemination of females are well established for fish species, crustacean has received

less attention, with the research of Chow (1982) and Ishida et al. (1986) being notable exceptions. Chow (1982) fertilized eggs of M . rosenbergii with spermatophores stored in Ringer’s 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. Supplementation of 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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