Aquaculture 198 Ž2001.

209–218
www.elsevier.nlrlocateraqua-online

Effect of temperature on sperm quality of captive

Litopenaeus Õannamei broodstock
Martin Perez-Velazquez a,) , William A. Bray a ,
Addison L. Lawrence a , Delbert M. Gatlin III b,
Mayra L. Gonzalez-Felix a
a
TAES Shrimp Mariculture Project, Texas A&M UniÕersity System, 1300 Port Street, Port Aransas,
TX 78373, USA
b
Department of Wildlife and Fisheries Sciences, Texas A&M UniÕersity, College Station, TX 77843, USA

Received 22 June 2000; accepted 19 December 2000

Abstract

Effect of temperature on reproductive quality of captive male Litopenaeus Õannamei brood-

stock was investigated by measuring sperm count and percentage of abnormal sperm per
compound spermatophore. Variability in these responses among and within experimental units
also was evaluated. Male shrimp with an initial average weight of 48.0 g were maintained in
3.7-m diameter circular tanks with recirculating seawater, and exposed to constant temperatures of
268C, 298C, and 328C for 42 days. Significantly higher mean sperm count Ž18.6 million cells. and
lower percentage of sperm abnormalities Ž36.7%. were observed in shrimp held at 268C, as
compared to broodstock at 298C Žmean sperm count s 0.1 million cells; percentage of sperm
abnormalitiess 99.7%. or 328C Žno sperm cells. Ž P - 0.05.. Results indicate that adequate sperm
count and percentage of abnormal sperm of L. Õannamei broodstock can be maintained at water
temperature of 268C, but not at 298C or 328C, at least within the time span of 42 days. The
importance of treatment replication and sampling sufficient numbers of shrimp from culture tanks
in experiments concerning male reproductive quality is discussed. q 2001 Elsevier Science B.V.
All rights reserved.

Keywords: Litopenaeus; Broodstock; Spermatophore; Reproductive quality; Variability; Sperm

)
Corresponding author. Tel.: q1-361-749-4625; fax: q1-361-749-5756.
E-mail address: mperezvelazquez@hotmail.com ŽM. Perez-Velazquez..

0044-8486r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 0 4 4 - 8 4 8 6 Ž 0 1 . 0 0 5 1 0 - 5
210 M. Perez-Velazquez et al.r Aquaculture 198 (2001) 209–218

1. Introduction
Deterioration of the reproductive tract is a recurrent problem in captive male shrimp.
It is characterized by a gradual loss of viable sperm and is accompanied by melanization
of the reproductive tract and spermatophores ŽChamberlain et al., 1983., and has been
referred to as male reproductive tract degenerative syndrome ŽMRTDS. ŽTalbot et al.,
1989.. The presence of deteriorating spermatophores in the terminal ampulla of male
Atlantic white shrimp Litopenaeus setiferus was documented by Brown et al. Ž1979..
Additional incidences were subsequently reported for this species, which appeared to be
especially susceptible to the disease ŽBray et al., 1985; Leung-Trujillo and Lawrence,
1987; Talbot et al., 1989; Alfaro, 1990; Alfaro et al., 1993.. Other studies revealed that
MRTDS also affects other penaeid shrimp of economic importance, including L.
stylirostris ŽChamberlain et al., 1983; Chamberlain and Gervais, 1984. and L. Õannamei
ŽAcuacop, 1983; Alfaro and Lozano, 1993.. The black pigment associated with severely
degenerated spermatophores was initially believed to be melanin ŽChamberlain et al.,
1983.. Histochemical tests and ultrastructural examination confirmed this presumption
ŽDougherty and Dougherty, 1989, 1990.. Causes of MRTDS have not been elucidated at
this time. Factors suggested as possibly causative include repetitive mechanical or
electrical ejaculation of spermatophores, bacterial infection, and nutritional factors
ŽChamberlain et al., 1983; Sandifer et al., 1984; Dougherty and Dougherty, 1990..
However, melanization also occurs spontaneously in spermatophores of animals showing
no bacterial infection and not subjected to manipulation of spermatophores ŽDougherty
and Dougherty, 1990.. As to nutritional factors, shrimp receiving diets presumed to be
nutritionally complete have developed deterioration of spermatophores ŽBrown et al.,
1979.. Interestingly, temperature also seems to be associated with the condition. Bray et
al. Ž1985. pointed out that water temperatures used in commercial hatcheries are
generally higher than those found in the natural environment where mating and
spawning take place. They showed that deterioration of the reproductive tract could be
delayed in male L. setiferus by maintaining a reduced water temperature of 25–268C,
but under those experimental conditions, deterioration still occurred. Also working with
L. setiferus, Pascual et al. Ž1998. found a similar trend. Temperature between 268C and
278C was necessary to prevent deterioration of the reproductive tract and sper-
matophores for at least 30 days, and deteriorated sperm quality was observed at high
temperature Ž308C.. However, the effect of temperature on the Pacific white shrimp L.
Õannamei, the most widely cultured species in the Western hemisphere, is not known.
This study describes the effect of temperature on sperm quality of captive L. Õannamei
broodstock and evaluates variability in male quality parameters.

2. Materials and methods

The study was conducted at the Texas Agricultural Experiment Station ŽTAES.,
Shrimp Mariculture Project, Texas A & M University System, Port Aransas, TX, USA,
using three indoor semi-closed, recirculating seawater systems with each system contain-
ing 3.7-m diameter circular tanks. Male L. Õannamei, originating from a domesticated
strain ŽOceanic Institute, HI, U.S. Marine Shrimp Farming Program., were reared in
 M. Perez-Velazquez et al.r Aquaculture 198 (2001) 209–218 211

ponds at low-salinity Ž1.7–2.0 ppt. ground water at Wood Brothers Shrimp Farm, Gila
Bend, AZ, until they attained a size of approximately 29.7 " 2.2 g Žmean " standard
deviation Žs.d.... Water temperature during this period ranged from 248C to 338C. They
were then transported to the TAES Shrimp Mariculture Project, acclimated to seawater,
and maintained for 5 months for further growth under laboratory conditions. Salinity
acclimation was carried out at 268C at a rate of 1 pptrday until equilibrium with
seawater was achieved. Three experimental temperatures of 268C, 298C, and 328C were
tested over a 42-day study period. Temperatures were maintained using an Accu-Therm
heat exchanger ŽPaul Mueller, Springfield, MO, USA. operated in connection with a
78.4-ton Flotronic Plus Reciprocating Liquid Chiller ŽCarrier, Syracuse, NY, USA. and
a 1.5-MBTUrh Boiler ŽRaypack, Westlake Village, CA, USA.. Experimental treatments
consisted of 13–14 shrimp randomly stocked into each tank Žf 1 shrimprm2 ., with
three replicate tanks per treatment. Seawater recirculated at a rate of 62 lrmin per tank
and fresh filtered seawater was exchanged daily at a rate of 13 lrmin per tank for 30
min Žapproximately 5%.. Shrimp were fed a diet composed of 75% standard dry
commercial feed with a protein content of 40% ŽRangen, Buhl, ID, USA., and 25%
fresh frozen squid at a rate of 3.5% of body weight on a dry weight basis. Temperature,
salinity, and dissolved oxygen were monitored daily. Ammonia, nitrite, nitrate, and pH
were monitored weekly. Concentration of nitrogenous wastes was calculated following
procedures adapted from Spotte Ž1979. and Solarzano Ž1969.. Manual ejaculation of
spermatophores was performed on all males at initiation and termination to estimate
sperm quality parameters, i.e., sperm counts and percentage of abnormal sperm per
compound spermatophore, according to methods previously described ŽLeung-Trujillo
and Lawrence, 1987.. The following criteria were used to determine spermatophore
condition. Healthy: white spermatophores with normal morphology; early deterioration:
spermatophores develop a tan color and may present melanization at anterior end;
advanced deterioration: spermatophores become dark brown in color, melanization
spreads to more areas. Erosion of peripheral areas Žflaps, flanges, outer margins. may
occur; severe deterioration: the entire surface of spermatophores is usually melanized
Žblack spermatophores.. Extensive erosion of structures Žgeminate body, glutinous
material, flaps, flanges, etc.. is evident. The degree of variability in sperm quality
parameters among and within experimental units was also evaluated.
Two-level nested analysis of variance ŽANOVA. was employed to evaluate treatment
differences using a significance level of P F 0.05. Differences among treatments were
identified by Tukey’s Honest Significant Difference ŽHSD. method. Transformed sperm
count Žsquare root Y q 3r8. and percentage of abnormal sperm Žarcsine. data were
employed for statistical analysis. Untransformed values are presented. All data analyses
were performed using Statistical Analysis System software ŽSAS Institute, 1989–1995..

3. Results
3.1. Water quality
Stable water temperatures were recorded throughout the experiment. Mean treatment
temperatures" s.d. were 26.0 " 0.18C Žrange s 25.8–26.28C., 29.0 " 0.18C Žrange s
212 M. Perez-Velazquez et al.r Aquaculture 198 (2001) 209–218

28.8–29.28C., and 32.0 " 0.18C Žrange s 31.6–32.38C., respectively. Dissolved oxygen
was maintained at a concentration above 5.9 mgrl; whereas nitrogenous wastes were
maintained at low concentrations in all three systems, not exceeding 0.02 mgrl of
NH 3 –N, 0.08 mgrl of NO 2 –N, and 1.20 mgrl of NO 3 –N. Overall mean salinity and
pH were 36.1 Ž"0.7. ppt and 8.2, respectively.

3.2. Shrimp growth, surÕiÕal, spermatophore condition, and sperm quality

Mean final weight and percent survival did not differ significantly among treatments
ŽTable 1..
Healthy white spermatophores were observed in all shrimp at the onset of the
experiment, although some males with spermatophores showing early signs of deteriora-
tion Žsee spermatophore condition criteria in Section 2. were culled from the population
at initiation. Observations on final spermatophore condition are shown in Table 1. Most
of the shrimp Ž70%. kept at 268C maintained healthy spermatophores. After 42 days of
exposure to experimental temperatures, only early signs of spermatophore deterioration,
consisting of tan coloration, were observed in 11 individuals Ž30%. maintained at 268C.
In contrast, advanced and severe deterioration, and no healthy spermatophores were
recorded for shrimp exposed to 298C and 328C.
In the initial sample of males, sperm count and percentage of abnormal sperm per
compound spermatophore were similar for all treatments, with no significant differences
among them ŽFigs. 1 and 2.. After 42 days of exposure to experimental treatments,
temperature was found to significantly affect sperm quality. Mean sperm count"
standard error Žs.e.m.. per compound spermatophore in shrimp maintained at 268C
Ž18.6 " 1.75 million cells. was significantly higher Ž P - 0.05. than estimates from
animals kept at 298C and 328C. At 298C, most of the spermatophores contained pieces of
disrupted sperm cells, and in only a few cases complete cells could be counted, yielding

Table 1
Initial weight, final weight, survival Žtreatment means"s.d.., and spermatophore conditiona of male L.
Õannamei broodstock held at three temperatures
Water temperature Ž8C.
26 29 32
Initial weight Žg. 48.3"2.6 Ž ns 42. 47.9"4.2 Ž ns 40. 47.7"3.5 Ž ns 39.
Final weight Žg. 49.2"2.5 Ž ns 38. 48.6"3.9 Ž ns 32. 47.9"3.6 Ž ns 34.
Survival Ž%. 95"4 87"9 90"10
Spermatophore condition
Žpercent of shrimp.
Healthy 70 0 0
Early deterioration 30 27 3
Advanced deterioration 0 27 41
Severe deterioration 0 46 56
a
Spermatophore condition criteria described in Section 2.
 M. Perez-Velazquez et al.r Aquaculture 198 (2001) 209–218 213

a mean sperm count of 0.1 " 0.05 million cells. At 328C, only sperm cell pieces were
found Ž0.0 " 0.00 million cells. ŽFig. 1.. Consequently, high percentages of abnormal
sperm Žmostly representing cell pieces. were recorded at both of these temperatures
Ž99.7 " 0.23% and 100 " 0.00% at 298C and 328C, respectively., whereas a signifi-
cantly lower value Ž36.7 " 3.32%. was found at 268C ŽFig. 2..

3.3. Variability in male quality parameters

Identified sources of variation in final sperm count data and their contribution
towards total variability were estimated. Significant differences were found for treatment
effects of this parameter Ž P s 0.0001.. The corresponding variance component ac-
counted for 88.8% of the total variation. Variation among experimental tanks was not
significant Ž P s 0.9998., and there was not an added variance component. At the level
of variation within experimental tanks Žerror., the contribution of the variance compo-
nent to total variability was 11.2%.
A similar pattern, in terms of variance attributable to different sources of variation,
also was observed for percentage of abnormal sperm values. The variance component of
the treatment effects was significant Ž P s 0.0001. and explained 91.8% of the total
variation. The variation among experimental tanks was not significant Ž P s 0.61190.,

Fig. 1. Sperm count per compound spermatophore Žmeans"s.e.m.. of male L. Õannamei held at three
temperatures. Initial sperm counts shown as reference, but not incorporated in statistical analysis of final
values. Final sperm count values with similar superscripts are not significantly different Ž P ) 0.05..
214 M. Perez-Velazquez et al.r Aquaculture 198 (2001) 209–218

Fig. 2. Percentage of abnormal sperm per compound spermatophore Žmeans"s.e.m.. of male L. Õannamei
held at three temperatures. Initial percentage of abnormal sperm values shown as reference, but not
incorporated in statistical analysis of final values. Final percentage of abnormal sperm values with similar
superscripts are not significantly different Ž P ) 0.05..

and the variance component did not add to total variability. Added variance within
experimental tanks accounted for 8.2% of the total variation.

4. Discussion

The present study demonstrated for the first time a strong sensitivity to temperature
of male L. Õannamei. Under our experimental conditions, not only was high water
temperature found to be an important factor in male gamete viability but effects were
observed at surprisingly moderate temperatures. The data indicate a critical high
temperature for L. Õannamei males between 268C and 298C. These results have
implications for maintaining optimal breeding conditions and perhaps more importantly,
for culture of shrimp broodstock from sub-adulthood onwards.
In hindsight, it is possible to review the literature and find examples of deterioration
of spermatophores possibly related to high temperature, which at the time were not
correlated with this factor. For wild-caught L. setiferus, spermatophore deterioration
was observed in males after being transferred into a recirculating system at 29.4 " 0.88C
ŽTalbot et al., 1989.. At day 35 in that study all shrimp Ž n s 45. were infertile.
 M. Perez-Velazquez et al.r Aquaculture 198 (2001) 209–218 215

Likewise, Leung-Trujillo and Lawrence Ž1987. reported greatly reduced fertility in

wild-caught male L. setiferus after 5 weeks of captivity at 27–298C, and complete
absence of sperm after 6 weeks. Earlier reports suggested an apparently slower process
of spermatophore deterioration in L. Õannamei. Employing subadult L. Õannamei reared
in captivity, Alfaro and Lozano Ž1993. observed varying degrees of spermatophore
deterioration, after 4 months of culture, in a population of males held at 28 " 18C. The
authors showed that spermatophores of some males deteriorated in 2–3 months, while
others maintained normal white spermatophores. Our study indicates that L. Õannamei
can be susceptible to rapid deterioration of sperm quality and that water temperature
should be monitored carefully, especially when maintaining male-only populations.
Objective comparison of our results and those of Alfaro and Lozano Ž1993. is difficult
because they did not maintain a consistently stable temperature and spermatophores of
their group of animals were not subjected to microscopic evaluation. Additionally,
shrimp of different size Žmean final weight. were employed: 48.6 g Žthis study. vs. 24.2
g ŽAlfaro and Lozano, 1993.. Deterioration of spermatophores seems to be less prevalent
in young, developing males than in fully-grown, mature animals Žpersonal observations..
If the process of spermatophore deterioration is associated with changes related to age of
shrimp, as suggested by Dougherty and Dougherty Ž1989, 1990., the larger shrimp of
our study might have been more susceptible to declining sperm quality at the experimen-
tal temperatures imposed.
Optimal temperature for most penaeid species reproduced in captivity is considered to
range between 278C and 298C ŽBray and Lawrence, 1992.. Although stability of water
temperature is desirable in reproduction tanks holding L. Õannamei ŽBray and Lawrence,
1992., some degree of variation is normally observed, reaching values of 298C or higher
in some instances ŽNaessens et al., 1997.. In this respect, the severe deterioration of
sperm observed at 298C ŽFigs. 1 and 2. was unexpected. Nevertheless, important
differences can be identified between our experimental shrimp and broodstock com-
monly employed by reproduction systems at commercial scale. In this study, domesti-
cated male shrimp, raised in extremely low-salinity water, were kept in recirculating
systems; whereas, marine shrimp hatcheries rely primarily on wild-caught broodstock
ŽBrowdy, 1998. that are most commonly maintained in open or flow-through systems
ŽBray and Lawrence, 1992.. In addition, natural variations in penaeid shrimp popula-
tions, based upon geographical origin, have been demonstrated ŽBenzie, 1995; Daud et
al., 1996.. The population of shrimp described in the present study may possess a
particular susceptibility to sperm deterioration at elevated temperatures, as compared to
other shrimp populations, in terms of geographical origin, source of animals Žwild vs.
captive., prior rearing history, and breeding culture system Žrecirculating vs. flow-
through.. These aspects remain to be investigated.
Among the different stages of spermatophore deterioration, early signs of deteriora-
tion were present in spermatophores of shrimp held at all three temperatures ŽTable 1..
However, sperm of contrasting characteristics was observed: commonly found propor-
tions of morphologically normal and abnormal sperm in shrimp maintained at 268C, and
deteriorating sperm in shrimp at 298C and 328C. This suggests that assessment of
spermatophore condition is not a sensitive indicator of quality of sperm, at least during
the early stages of spermatophore deterioration. Sampling of animals for microscopic
216 M. Perez-Velazquez et al.r Aquaculture 198 (2001) 209–218

examination of sperm is therefore necessary in order to determine suitability for

reproduction.
Sperm counts of shrimp held at 268C increased from an initial value of 7.7 to 18.6
million cells per compound spermatophore ŽFig. 1.. Taking into account that growth of
shrimp was limited Žinitial and final mean weights were 48.3 and 49.2 g, respectively.,
and that prior to the experiment shrimp were fed the dry commercial feed but no fresh
frozen squid supplement, the higher final numbers of sperm cells might reflect a
nutritionally more adequate dietary regime.
Very significant differences were found for both final sperm count and percentage of
abnormal sperm values, with most of the variation clearly accounted for by the treatment
effects. This is the first study, to our knowledge, that reports variability of male quality
data among experimental units Žtanks stocked with a determined number of shrimp.. In
previous studies, males have generally been stocked into a single tank, considering each
individual shrimp as an experimental unit ŽAlfaro, 1993; Wang et al., 1995; Pascual et
al., 1998.. Variation in male quality parameters, as a result of individual measurements,
was observed in our study, e.g., 18.6 " 1.75 Žmean " s.e.m.. million cells Ž n s 36. for
shrimp held at 268C ŽFig. 1.; whereas Pascual et al. Ž1998. found 12.1 " 2.78 Žmean "
s.e.m.. million cells Ž n s 8. after maintaining 35 g male L. setiferus at the same
temperature for 30 days. According to our data, variation within experimental units
represented 11.2% and 8.2% of the total variation for sperm counts and percentage of
sperm abnormalities, respectively. In view of the considerable variation exhibited by
these variables in this and other studies, the importance of sampling sufficient numbers
of shrimp from more than one experimental tank is emphasized.
It is concluded that adequate sperm count and percentage of abnormal sperm of L.
Õannamei broodstock can be maintained at water temperature of 268C, but not at 298C
or 328C, at least within the time span of 42 days. Early stages of deterioration in
spermatophores are not necessarily concomitant with the process of degeneration of
sperm cells. Male sperm quality, as estimated by sperm count and percentage of
abnormal sperm, is a more precise method in the assessment of male broodstock
reproductive quality. Variability in sperm count and percentage of abnormal sperm data
was mainly accounted for by treatment effects, and to some degree, by observations
within experimental units, indicating that sampling large enough numbers of shrimp
from experimental tanks is essential. Variation among experimental units was not
significant. In summary, this study suggests that high water temperature is important in
male viability. However, other factors, such as nutrition, may be influencing
spermrspermatophore quality of shrimp in captivity. Further research is needed to
investigate this and other aspects.

Acknowledgements

This research was funded in part by Project H-8158 of the Texas Agricultural
Experiment Station, United States Department of Commerce Marine Shrimp Farming
Program CSREES Grant No. 95-38808-1424, to Dr. Addison Lee Lawrence, Principal
Investigator.
 M. Perez-Velazquez et al.r Aquaculture 198 (2001) 209–218 217

References

Acuacop, Mayra L., 1983. Constitution of broodstock, maturation, spawning, and hatching systems for penaeid
shrimp in the Centre Oceanologique du Pacifique. In: McVey, J.P., Moore, J.R. ŽEds.., CRC Handbook of
Mariculture. Crustacean Aquaculture, vol. 1, CRC Press, Boca Raton, FL, USA, pp. 105–121.
Alfaro, J., 1990. A contribution to the understanding and control of the male reproductive system melanization
disease of broodstock. Master’s thesis. Texas A&M University, College Station, TX, USA.
Alfaro, J., 1993. Reproductive quality evaluation of male Penaeus stylirostris from a grow-out pond. J. World
Aquacult. Soc. 24, 6–11.
Alfaro, J., Lozano, X., 1993. Development and deterioration of spermatophores in pond-reared Penaeus
Õannamei. J. World Aquacult. Soc. 24, 522–529.
Alfaro, J., Lawrence, A.L., Lewis, D., 1993. Interaction of bacteria and male reproductive system blackening
disease of captive Penaeus setiferus. Aquaculture 117, 1–8.
Benzie, J.A.H., 1995. Genetics in the domestication of the giant tiger prawn Penaeus monodon in Australia.
Book of Abstracts, Aquaculture ’95. World Aquaculture Society, Baton Rouge, LA, USA, p. 32.
Bray, W.A., Lawrence, A.L., 1992. Reproduction of Penaeus species in captivity. In: Fast, A.W., Lester, L.J.
ŽEds.., Marine Shrimp Culture: Principles and Practices. Elsevier, Amsterdam, The Netherlands, pp.
93–170.
Bray, W.A., Leung-Trujillo, J.R., Lawrence, A.L., Robertson, S.M., 1985. Preliminary investigation of the
effects of temperature, bacterial inoculation, and EDTA on sperm quality in captive Penaeus setiferus. J.
World Maric. Soc. 16, 250–257.
Browdy, C., 1998. Recent developments in penaeid broodstock and seed production technologies: improving
the outlook for superior captive stocks. Aquaculture 164, 3–21.
Brown Jr., A., McVey, J.P., Middleditch, B.S., Lawrence, A.L., 1979. Maturation of white shrimp Penaeus
setiferus in captivity. Proc. World Maric. Soc. 10, 435–444.
Chamberlain, G.W., Gervais, N.F., 1984. Comparison of unilateral eyestalk ablation with environmental
control for ovarian maturation of Penaeus stylirostris. J. World Maric. Soc. 15, 29–30.
Chamberlain, G.W., Johnson, S.K., Lewis, D.L., 1983. Swelling and melanization of the male reproductive
system of captive adult penaeid shrimp. J. World Maric. Soc. 14, 135–136.
Daud, S.K., McAndrew, B.J., Penman, D., 1996. Genetic population subdivision in Malaysian P. monodon
Fabricius and its relation to hatchery stock management. In: Creswell, R.L. ŽEd.., Book of Abstracts,
World Aquaculture ’96. World Aquaculture Society, Baton Rouge, LA, USA, p. 98.
Dougherty, W.J., Dougherty, M.M., 1989. Electron microscopical and histochemical observations on melanized
sperm and spermatophores of pond-cultured shrimp, Penaeus Õannamei. J. Invertebr. Pathol. 54, 331–343.
Dougherty, W.J., Dougherty, M.M., 1990. Ultrastructural observations on melanized sperm in developing and
fully formed spermatophores of male shrimp, Penaeus Õannamei. In: Jenkins, J.O., Cheng, T.C. ŽEds..,
Pathology in Marine Science. Academic Press, Inc., London, pp. 387–394.
Leung-Trujillo, J.R., Lawrence, A.L., 1987. Observations on the decline in sperm quality of Penaeus setiferus
under laboratory conditions. Aquaculture 65, 363–370.
Naessens, E., Lavens, P., Gomez, L., Browdy, C.L., McGovern-Hopkins, K., Spencer, A.W., Kawahigashi, D.,
Sorgeloos, P., 1997. Maturation performance of Penaeus Õannamei co-fed Artemia biomass preparations.
Aquaculture 155, 87–101.
Pascual, C., Valera, E., Re-Regis, C., Gaxiola, G., Sanchez, A., Ramos, L., Soto, L.A., Rosas, C., 1998. Effect
of water temperature on reproductive tract condition of Penaeus setiferus adult males. J. World Aquacult.
Soc. 29, 477–484.
Sandifer, P.A., Lawrence, A.L., Harris, S.G., Chamberlain, A.D., 1984. Electrical stimulation of sper-
matophore expulsion in marine shrimp, Penaeus spp. Aquaculture 41, 181–187.
SAS Institute, 1989–1995. SAS System for Windows 6.11. Cary, NC, USA, 27513.
Solarzano, L., 1969. Determination of ammonia in natural waters by the phenolhypochlorite method. Limnol.
Oceanogr. 14, 799–801.
Spotte, S., 1979. Fish and Invertebrate Culture: Water Management in Closed Systems. 2nd edn. Wiley, New
York, NY, USA.
218 M. Perez-Velazquez et al.r Aquaculture 198 (2001) 209–218

Talbot, P., Howard, D., Leung-Trujillo, J., Lee, T.W., Li, W.Y., Ro, H., Lawrence, A.L., 1989. Characteriza-
tion of male reproductive tract degenerative syndrome in captive penaeid shrimp Ž Penaeus setiferus ..
Aquaculture 78, 365–377.
Wang, Q., Misamore, M., Jiang, C.Q., Browdy, C.L., 1995. Egg water induced reaction and biostain assay of
sperm from marine shrimp Penaeus Õannamei: dietary effects on sperm quality. J. World Aquacult. Soc.
26 Ž3., 261–271.