JOURNALOFTHE WORLD AQUACULTURE SOCIETY

Vol. 29, No. 4 December, 1998

Effect of Water Temperature on Reproductive Tract Condition of Penaeus senifems Adult Males

CRISTINA PASCUAL AND EVANGELINA VALERA Laboratorio de Ecofsiologia, Departmento de Biologia, Facultad de Ciencias, Universidad Nacional Autdnoma Mdxico, Apartado Postal 69, Ciudad del Carmen, Campeche, Mdxico

CRISTINA RE-REGIS Centro Regional de Investigaciones Pesqueras de Lnma, Campeche, INP, SEMARNAP,

Mdxico

GABRIELA GAXIOLA AND ADOLFO SANCHEZ Laboratorio de Ecofsiologia, Departmento de Biologia, Facultad de Ciencias, Universidad Nacional Auto'noma Mdxico, Apartado Postal 69, Ciudad del Carmen, Campeche, Mdxico

LAIDA RAMOS

Centro de Investigaciones Marinas, Avenida la , No. 2808, Miramar, La Habana, Cuba

LUIS A. SOTO

Laboratorio de Ecologia del Bentos, Instituto de Ciencias del Mar y Limnologia, Universidad Nacional Autdnoma Mexico, Mexico 04510, D. F. Mdxico

CARLOS ROSAS' Laboratorio de Ecofsiologia, Departmento de Biologia, Facultad de Ciencias, Universidad Nacional Auto'noma Mdxico, Apartado Postal 69, Ciudad del Carmen, Campeche, Mdxico

Abstract. -This study was conducted to determine the effect of water temperature on male reproductive tract degenerative syndrome (MRTDS) and male re- productive system melanization (MRSM) in Penaeus setiferus, and to evaluate the effect of water tempera- ture on spermatophore regeneration time and sperm quality in electrically ejaculated adult males. Variation in sperm quality, gonadosomatic index, and histologi- cal changes in the reproductive system were used to assess reproductive tract changes in the first experi- ment. Sperm quality and regeneration time were mea- sured before and after successive regenerations in the second experiment. Sperm quality was stable when shrimp were held at 26 C for 30 d but was reduced when shrimp were held at 30 C. Reproductive tissues of 20% of the shrimp held at 30 C were melanized to some degree but tissue melanization was found in only 2.5% of the shrimp held at 26 C. Spennatophore re- generation time was also affected by temperature. Av- erage times for first spermatophore regeneration were 192 h at 25 C, 152 h at 30 C, and 144 h at 33 C. Sperm quality of regenerated shrimp was not affected at 25 C but was reduced for regenerated animals held at 33 C. Based on these results, we recommend a water temperature between 25 and 27 C to obtain adequate

I Corresponding author.

spermatophore regeneration and to maintain healthy male P. setijerus broodstock for at least 30 d.

Male reproductive tract degenerative syndrome (MRTDS) of Penaeus setifems has been the subject of considerable re- search to determine the causes of the dete- rioration of spermatophore terminal am- pule, wings, and flank. The syndrome was first noted by Brown et al. (1979) and de- fined by Chamberlain et al. (1983). This syndrome and male reproductive system melanization (MRSM) are characterized by progressive reduction in the number of sperm cells and an increase in the percent- age of abnormal and dead cells. In the past, it was believed that the two syndromes were associated with the same disease pro- cess (Chamberlain et d. 1983; Bray et al. 1985; Talbot et al. 1989). Recently, how- ever, Alfaro (1990) and Alfaro et al. (1 993) have shown that MRSM and MRTDS are two distinct phenomena. Male reproductive

8 Copyright by the World Aquaculture Society 1998

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478 PASCUAL ET AL.

system melanization is caused by melanin production by the shrimp immune system after chitinolitic bacterial infection (Alfaro et al. 1993). Although the process that trig- gers male reproductive tract degenerative syndrome remains poorly understood, it has been confirmed that electrostimulation-in- duced stress can cause spermatophore de- generation (Chamberlain et al. 1983; San- difer et al. 1984; Rosas et al. 1993). Ac- cording to Alfaro et al. (1993) and Alfaro (1993b), the appearence of MRTDS could be of endocrine nature since it has been demonstrated that a single injection of 1701- methyltestosterone at a concentration of 0.01 and 0.1 pg/g body weight improved sperm count, spermatophore weight, and re- duced sperm abnormalities of adult males of Penaeus vannamei.

Shrimp producers routinely use artificial insemination for nauplii production, even though many males must be discarded be- cause of spermatophore damage after elec- troejaculation or manual ejaculation (Rosas et al. 1993). Since the duration of male spermatophore viability is short (2-3 wk), hatcheries must continually replace male broodstock, thus increasing production costs. Production of large numbers of P. se- tijerus nauplii by natural mating has not been reported. Misamore and Browdy (1996) reported that only 27% of all pos- sible matings were successful in achieving spermatophore attachment when gravid fe- males were placed in tanks containing only males (a female to male ratio of 1 to 7). The main reason postulated for the low rate of spermatophore attachment is male ster- ilization due to MRTDS (Brown et al. 1979; Bray et al. 1985; Leung-Trujillo and Lawrence 1991).

According to Bray et al. (1985), water temperatures in broodstock culture systems are usually higher than those experienced by animals in nature. Although the recom- mended temperature range for broodstock maturation systems is generally between 28-30 C (Robertson et al. 1993). it has been shown that lower temperatures delay sper-

matophore degeneration in P. setiferus (Bray et al. 1985). Therefore, the purpose of this study was to investigate the effects of water temperature and time of exposure on MRTDS and MRSM, and to study the effect of water temperature on spermato- phore regeneration time and sperm quality in electrically stimulated P. setijerus adult males.

Material and Methods P. setijerus adult males were captured

between May and July 1994, in the coastal area of Laguna de TCrminos, Campeche, MCxico. Shrimp were transferred to the lab- oratory in plastic bags with an oxygen at- mosphere and a water temperature of 25 C. At the laboratory, the animals were placed in a 3.65-m diameter round tank filled with filtered (to 5km) sea water. Shrimp were exposed to a 14 h 1ight:lO h dark photo- period. Shrimp were fed squid Loligo sp. and oligochaetes Pontodn'lus bemudensis three times a day (0800, 1400, and 2000 h) at 20% of shrimp body weight daily.

The first experiment was designed to as- sess the effect of water temperature and time of exposure on MRTDS and MRSM at 26 and 30 C. The low temperature was maintained with a titanium chiller and the high temperature was ambient seawater temperature. The experiment at 30 C was run twice. Experiments were carried out in a 3.69-m diameter round tank with constant sea water flow at the experimental temper- ature. Water was exchanged at a rate of 160% per d. Water depth in the tank was kept at 40 cm, with pH of 7.4 2 0.1, salin- ity of 36 2 1 ppt, and the dissolved oxygen concentration was maintained above 5 mg/ L. Forty sexually mature male shrimp (35 2 2 g mean wet weight) were used in each experiment. Samples of eight randomly se- lected shrimp were collected at 0, 10, 15, 20, and 25 d after stocking to assess sper- matophore degeneration. Variation in sperm quality, gonadosomatic index (GI), and his- tological changes in the reproductive sys- tem were used to assess degeneration. The

WATER TEMPERATURE AND MRTDS IN PENAEUS SETIFERUS 479

TABLE 1. Effect of water temperature on wet weight and gonadosomatic index (%GI) of Penaeus setiferus males. The trial at 30 C was repeated twice (a and b). Values are expressed as means _f SEM. Means in the same column that are followed by a same letter do not differ at the 5% level of probability as determined by Duncan’s multiple range test (Zar I974). Gonadosomatic indices were arcsine-transformed prior to statistical analysis.

Water temperature

26 C 30 C (a) 30 C (b)

Day Weight (g) %GI Weight (g) %GI Weight (g) %GI

0 38.7 t I.la 2.38 2 0.10a 33.6 2 0.4a 1.80 2 0.04a 30.7 2 1.4a 2.10 2 0.70a 10 37.4 t 1.2a 2.15 2 0.09a 37.4 2 0.2a 2.11 2 0.03a 35.6 2 0.8b 2.21 2 0.03a 15 39.1 2 0.9a 2.01 t 0.08a 32.2 2 0.7b 2.32 t 0.Wa 33.1 ? 0.7b 2.28 t 0.05a 20 37.9 t 0.9a 2.01 t 0.05a 29.2 t O.lb 2.09 t 0.15a 33.3 2 0 . 7 ~ 1.98 rC_ 0.04a 25 38.2 t l . la 1.81 2 0.09b 29.6 2 0.6b 2.27 2 0.05a 28.6 2 1 . 2 ~ 2.25 2 0.03a 30 37.9 t 1.9a 2.04 2 0.13a - - - -

spermatophore was manually expelled to test for sperm count and sperm quality. Sperm quality was assessed by trypan blue biostain reaction and by gross morphology. The trypan blue assay was conducted as de- scribed by Ramos et al. (1994). The per- centage of morphologically abnormal cells was determined by recording the numbers of sperm with head malformations or miss- ing spike (Leung-Trujillo and Lawrence 1987). Gonadosomatic index (GI) was cal- culated as GI = 100 [gonad weight/(shrimp weight - gonad weight)]. Changes in re- productive tract tissue structure were eval- uated histologically. The animal’s reproduc- tive system was divided into three sections: testicles, vas deferens, and terminal ampule. Each section was processed with the tech- niques proposed by Bell and Lightner (1988) and stained with hematoxylin-eosin. The characteristics proposed by Chamber- lain et al. (1983) were used to classify the degree of melanization. Stages of MRSM were classified according to external obser- vations: healthy shrimp-white ampules; early infection-light brown in some areas of the ampule; advanced infection in the terminal ampule-brown areas and gradual darkening of the edges towards the center, developing to totally dark tissue; and severe infection--complete reproductive tract mel- anization, including testes.

The effect of water temperature on sper-

matophore regeneration was studied in elec- troejaculated animals. In this experiment, 15 randomly selected, sexually mature shrimp (33 2 3 g mean wet weight) were placed in three 500-L tanks (5 animals/ tank) at 25 2 0.8 C, 30 2 1.2 C, and 33 2 1.3 C. A chiller maintained the temperature at 25 C and immersion heaters were used to maintain a temperature of 33 C. All an- imals were electroejaculated and marked at the beginning of the experiment, following the method described by Rosas et al. (1993). After ejaculation, erythromycin was locally applied. The spermatophores were placed in a calcium-free solution and the shrimp were returned to their corresponding temperature. Sperm quality in these sper- matophores was termed “initial.” Shrimp were checked daily to verify regeneration based on the following criteria (bung-Tru- jillo and Lawrence 1991): 1) without sper- matophore-no organization or presence of sperm detected; 2) with spermatophore- small, thin pieces of hardened material of variable form; 3) with spermatophore- spermatophore unhardened, thin, soft, pa- pery, and white in color; and 4) with mature spermatophore-spermatophore completely hardened with a white-yellow color. Regen- erated shrimp were re-ejaculated and re- turned to their corresponding tank. Differ- ent responses to electrostimulation were classified according to the following criteria

480 PASCUAL ET AL.

TABLE 2. Effect of water temperature on total sperm counts, abnormal sperm, and dead sperm in spermato- phore of Penaeus setiferus adult males. The trial at 30 C was repeated twice (a and b). Total sperm counts are expressed as means 2 SEM in millions of cells/spermatophore. Means in the same column that are followed by a same letter do not differ at the 5% level of probability as determined b y Duncan's multiple range test (Zar 1974).

Water temperature

26 C 30 C (a)

Abnormal Dead Abnormal Dead sperm sperm sperm sperm

Day Total sperm (%) (%) Total sperm (%)

0 8.841 2 1.160a 1.6 0 8.917 2 2.030a 7.8 0.3 10 8.005 2 1.450a 10.0 0 9.337 ? 1.200a 14.2 0 15 15.964 2 2.690b 5.6 0 3.545 2 1.5OOb 19.4 0.1 20 13.125 2 1.700b 12.8 0 14.742 t 0 . 5 8 0 ~ 2.0 2.9 25 13.180 2 2.530b 5.4 0 6.464 * 1.09a 16.1 1.2 30 12.175 2 2.780b 10.1 0

(Rosas et al. 1993): total ejection, partial ejection, and no ejection of one or both am- pules.

Results Effect of Temperature and Time of Exposure on MRTDS and MRSM

Shrimp held at 26 C did not lose weight (P > 0.05) whereas those held in the two trials at 30 C lost 12 and 7% of their initial weights (P < 0.05; Table 1). Mean gona- dosomatic index on day 25 for shrimp held at 26 C was significantly lower (P > 0.05) than that measured on day 0 for shrimp held at 26 C, whereas mean gonadosomatic in- dex for shrimp held at 30 C did not change over the same time period (Table 1; P > 0.05).

Total sperm cell count for shrimp held at 26 C ranged between 8-15 million cells/ spermatophore, with highest levels 15 d af- ter the beginning of the experiment (P C 0.05; Table 2). Total sperm count did not increase after day 15. Normal sperm cells accounted for 98% of the total sperm cells on day 0; after 30 d at 26 C, 89.9% of the cells were normal. No dead sperm were ob- served. Sperm quality in shrimp held at 30 C decreased with time in both trials (Table 2). In trial (a) at 30 C, the percentage of abnormal cells increased from 7.8% on day 0 to 16.1% after 25 d. There was also an

increase in the percentage of dead cells (Ta- ble 2). In trial (b) at 30 C, the percentage of abnormal cells increased from 6.1% on day 0 to 71.1% on day 25. Likewise, the percentage of dead sperm increased from 0 to 12.6% in 25 d (Table 2).

Melanization was also affected by tem- perature (Table 3). Only one animal showed signs of melanin production after 25-30 d at 26 C, whereas melanization was evident in 8 (20%) of the shrimp in trial a and 4 (10%) of the shrimp in trial b at 30 C.

Effect of Temperature on Spemtophore Regeneration Time and Sperm Quality

At the beginning of the experiment, elec- trostimulation resulted in total spermato- phore expulsion by 6.6% of the shrimp and partial expelling of the contents of one or both ampulae by 70% of the shrimp. All five animals held at 25 C regenerated sper- matophores at 192 h. One of the five shrimp held at 25 C regenerated a second time after 336 h. Spermatophore regeneration at 30 C was quicker than at 25 C, and at 30 C there was even a third regeneration. At 152 h, four of five shrimp had regenerated, one of five regenerated a second time at 216 h, and one shrimp regenerated a third time at 264 h. At 33 C, four of five shrimp regenerated spermatophores after 144 h.

Total numbers of sperm per spermato-

WATER TEMPERATURE AND MRTDS IN PENAEUS SETIFERUS 48 1

TABLE 2. Extended.

Water temperature

30 C (b)

Abnormal Dead sperm sperm

Total sperm (%) (%)

2.860 5 0.580a 6.1 0 9.452 -+ 2 . 0 9 0 ~ 6.2 0 4.427 t 0.540b 2.1 0 6.994 2 0.570b 50.6 12.4 1.470 2 0.083d 71.1 12.6

phore increased with successive spermato- phore regenerations for shrimp held at 25 C (Table 4). The highest percentage of ab- normal cells (56%) was seen after the first regeneration, and the lowest percentage in the initial samples. In the one shrimp that regenerated twice, total number of sperm cells increased to 23.1 million cells/sper- matophore, of which only 6% were abnor- mal.

In shrimp held at 30 C, total numbers of sperm increased with successive regenera- tions with values ranging from an average of 1.59 million celldspermatophore in the initial sample to 49.9 million cellshper- matophore for the one shrimp that regen- erated a third time (Table 4). The largest percentage of abnormal cells was present in the samples after the first regeneration, rep- resenting 70% of the total number of sperm cells (Table 4). Abnormal sperm in single sample after the third regeneration consti- tuted only 6% of the total.

In shrimp held at 33 C, total sperm counts decreased from an average of 4.83 million cells/spermatophore in the initial sample to an average of 2.06 million cells/ spermatophore in the samples obtained after the first regeneration (Table 4). Abnormal cells increased from 20% of the total (1 million cells/spermatophore) in the initial sample to 78% of the total (1.6 million

TABLE 3. Numbers of adult male Penaeus setiferus showing various degrees of male reproductive tract melanization (MRTM) when held for 25-30 d at 26 or 30 C. The trial at 30 C was repeated mice (a and b). Each treatment group consisted of a total of 40 shrimp.

Number of shrimp showing melanization

Stage of melanization 26 C 30 C (a) 30 C (b)

Early 1 3 3 Advanced 0 4 1 Severe 0 1 0

Total 1 8 4

cells/spermatophore) in the samples after regeneration.

Sperm quality was highly variable among individuals throughout the experi- ment. For example, the percentage abnor- mal sperm following the first regeneration at 25 C ranged from 11.6 to 94.2%. In shrimp held at 30 C, the percentage of ab- normal sperm in the initial samples ranged from 11.5% to 50%, and from 13.3% to 83.5% after the fist regeneration. One of the two shrimp regenerated a second time had 1% abnormal sperm; the other had 61.2% abnormal sperm.

TABLE 4. Effect of water temperature on sperm qual- ity in successive spennatophore regenerations for adult male Penaeus setiferus afer spermatophores were ejaculated by electrical stimulation. Each treatment group consisted of jive shrimp. N = num- ber of animals with regenerated spennatophores. Total sperm counts are expressed as means 2 SEM in millions of cellslspermatophore.

Spermato- phore Abnormal

Temp- regener- sperm erature ation N Total sperm (%)

25 C Initial 5 2.47 t 0.26 8 1 5 8.82 2 0.86 56 2 1 23.1 1 6

30 C Initial 5 1.59 2 0.08 15 1 4 4.38 -+ 0.24 70 2 1 6.13 26 3 1 49.9 6

33 C Initial 5 4.83 t 0.06 20 1 4 2.06 2 0.52 78

482 PASCUAL ET AL.

Discussion

Results of this study indicate that holding shrimp at water temperatures of 24-27 C delays spermatophore degeneration and melanization in P. setifems male adults. These results are similar to those of Bray et al. (1985) who observed that holding P. setifems males at 25-26 C prevented de- creased sperm quality during the 30 d of exposure, and provided adequate mainte- nance of sperm quality for 60 d in 33% of the shrimp.

Several authors have reported that both MRSM and MRTDS are a result of captiv- ity-induced stress (Alfaro 1990; Leung-Tru- jillo and Lawrence 1991; Alfaro and Loz- anno 1993). In MRSM, melanin is pro- duced after bacterial infection. On the other hand, MRTDS has been related to an en- docrine-based problem, since this system is particularly sensitive to captivity-induced stress (Alfaro 1990). We have shown in this study that temperature is also a factor af- fecting reproductive performance in P. se- tifems, with higher temperatures stimulat- ing melanization and spermatophore degen- eration.

Melanized shrimp in this study had in- termediate and advanced symptoms, with the higher rate of melanization in shrimp held at 30 C (Table 3). The presence of mel- anized structures indicates that the darken- ing process in severely damaged animals is confined to surface tissues in the terminal ampule and testicular lobes. These obser- vations agree with those made by Alfaro (1990) and Chamberlain et al. (1983). In addition, Alfaro (1990) and Alfaro et al. (1993) have shown that infection by oppor- tunistic bacteria (Vibrio alginolyticus, Pseu- domonas putrefaciens and an unidentified species) is associated with MRSM.

The results from this study reveal a close association between MRSM and water tem- perature. Since immunological response in shrimp is related to MRSM, it may be that temperatures over 27 C decrease the im- munocompetence against infectious agents,

possibly by modifying phagocytic cell pro- duction mechanisms (Fontaine and Lightner 1974) or other antibacterial factors such as non-specific agglutinins (Lewis and Lawrence 1983).

Male reproductive tract degeneration syndrome is characterized by a dramatic de- crease in sperm quality. Although the cause of this syndrome is not yet clear, it has been shown that the decrease in sperm quality is associated with the decrease or total disap- pearance of glandular secretions in the am- pules. If these secretions are responsible for keeping spermatomids alive (Talbot et al. 1989), it could be presumed that these glands, or the hormonal mechanisms re- sponsible for their function, are particularly sensitive to temperatures higher than 27 C because the syndrome has been delayed by using temperatures under 27 C in this and other studies (Bray et al. 1985). Thus, a temperature range of 25-27 C may be rec- ommended for the maintenance of healthy males in shrimp maturation facilities for pe- riods up to 30 d. Many laboratories include periodic broodstock exchange in their man- agement practices (Robertson et al. 1993; Browdy 1992) and a substitution of 70% of the males is recommended in the case of P. setifens. This would allow the maintenance of consistent sperm quality for nauplii pro- duction.

Large-scale nauplii production in P. se- tifems by means of natural matings has not been reported. Misamore and Browdy (1996) reported that only 27% of all pos- sible matings were successful in achieving spermatophore attachment when 3 gravid females were placed in a tank containing 20 males. If temperatures lower than 27 C pre- serve sperm quality, it might be reasonable to expect natural copulation resulting in im- proved nauplii production at lower temper- atures. Natural copulation among P. setifer- us broodstock has been observed under ex- perimental conditions in our laboratory, and has also been reported by Leung-Trujillo and Lawrence (1991) and Misamore and Browdy (1996). In ow experimental facili-

WATER TEMPERATURE AND MRTDS IN PENAEUS SETIFERUS 483

ty. natural matings have taken place at tem- peratures ranging between 26-31 C, with improved nauplii production at the lower temperatures. We therefore recommend that water in tanks used to hold males be main- tained at 25-27 C and water in tanks used for maturation of females be maintained at 28-30 C. Gravid females could then be transferred to the tanks used to hold males for mating.

Electroejaculation has been used as an al- ternative to natural mating for insemination of mature females. This approach has been successfully used in nauplii production for P. setiferus and other open thelycum spe- cies (Ramos et al. 1994). In our study, sper- matophore regeneration in P. setiferus male adults after electrostimulation was in- creased at water temperatures of 25-30 C, and sperm quality was similar at both tem- peratures. As in the present study, Alfaro (1993a) also reported a high rate of abnor- mal sperm in regenerated male P. styliros- tris after manual ejaculation. Alfaro (1993a) observed that regeneration is a highly var- iable process which does not follow a set pattern: small animals can regenerate a sig- nificant amount of sperm cells with high percentages of abnormal cells and large an- imals can regenerate fewer cells with low abnormality rates. In Alfaro's study, MRTDS and MRTDS were not observed in P. stylirostris, which was interpreted as a result of species-specific defense response and the degree of physiological adaptation to captivity or to ejaculatory procedures.

The high variability found in P. setiferus reproductive quality could be evidence of the effects produced by the electric shock. Electric shock might affect testicular func- tion, possibly by affecting spermatophore synthesis in the ampulla, or the spermatid maturation process in others. The low per- centages of normal sperm observed in the second and third regeneration in each of the treatments may suggest that the shock strongly affects testicular function and, therefore, spermatid formation.

In a previous study, Rosas et al. (1993)

reported a decrease in sperm quality as the possible result of bacterial infection the am- pulla after electrostimulation. We used lo- cally applied powdered erythromycin to prevent infection, as recommended by Al- far0 (1990). Our results indicate that this procedure may prevent infection by oppor- tunistic bacteria, thus allowing spermato- phore regeneration. Absence of MRSM in regenerated animals suggests that this treat- ment is effective.

As expected, holding shrimp at 33 C sig- nificantly affected sperm quality. Although time of regeneration was shorter at this tem- perature, total number of viable sperm was almost 10-fold lower than that for recently captured animals.

Following spermatophore regeneration in individual shrimp showed that sperm qual- ity varied greatly among individuals, re- gardless of temperature. This was also ob- served by Alfaro (1993a) in P. stylirostris, and shows a strong individual component in sperm quality that is difficult to identify when selecting males from a broodstock batch. Therefore, analysis of sperm quality in the male population, prior to their intro- duction into laboratories, may be of value in assessing their reproductive ability.

Acknowledgments This project was developed with funds

provided by the Direcci6n General de Aten- ci6n a1 Personal Acadtmico of the UNAM for the project IN 200994 given to Luis A. Soto and Carlos Rosas. Assistance was also received from Instituto Nacional de la Pesca through the collaboration agreement with the Facultad de Ciencias, UNAM.

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