The results showed that the matology parameters, packed cell volume, haemoglo-bin, erythrocyte sedimentation rate, red blood cell,white blood cell, total serum protein, Mg21, Ca21, hae-C
Trang 2Low-density culture of red abalone juveniles, Haliotis
system and flow-through system
Miroslava Vivanco-Aranda1, Cristian Jorge Gallardo-EscaŁrate2& Miguel AŁngel del R|¤o-Portilla1
1 Laboratorio de Gene¤tica, Departamento de Acuicultura, Centro de Investigacio¤n Cient|¤¢ca y de Ecuacio¤n Superior de Ensenada Ensenada, BC, Me¤xico
2 Departamento de Oceanograf|¤a, Centro de Biotecnolog|¤a, Universidad de Concepcio¤n, Barrio Universitario s/n Casilla 160-C, Concepcio¤n, Chile
Correspondence: M A del R|¤o-Portilla, CICESE, Acuicultura, PO Box 434844, San Diego, CA 92143-4844, USA E-mail: mdelrio@ cicese.mx
Abstract
Commercial abalone culture is carried out using
£ow-through systems with a high water volume
ex-change in Baja California, Mexico The objective of
this work was to compare the growth rate and
survi-val of red abalone cultured in two systems Flow
through (daily water exchange rate of 800%) and
re-circulating systems consisted of a 250 L ¢breglass
tank and constant aeration, but bio¢ltration in the
re-circulating system was provided with a 28 L (1ft3)
bubble-washed bead ¢lter.Water variables were
mea-sured either daily (dissolved oxygen, temperature, pH
and salinity) or three times a week (total ammonia
nitrogen, nitrate-nitrogen, nitrite-nitrogen and
alkali-nity) Shell length was measured every 2 weeks for 18
weeks Only the alkalinity and pH were signi¢cantly
di¡erent due to the addition of sodium bicarbonate to
the recirculating system Abalone growth rate was
26.1 15.96 mm day 1in the recirculating systems
and 22.21 18.69 mm day^1in the £ow-through
sys-tems The ¢nal survival was 78.74% in the
recirculat-ing systems and 71.82% in the £ow-through systems
Signi¢cant di¡erences in the ¢nal size and survival of
the abalones were found between systems (Po0.05)
Therefore, recirculating aquaculture systems is a
fea-sible alternative for juvenile red abalone culture
Keywords: red abalone, Haliotis rufescens,
recircu-lating systems, growth, closed system
Introduction
Abalones, Haliotis, are marine gastropod molluscs,
with a high commercial value There is a high
inter-est in abalone culture for commercial and stock hancement In Baja California, abalone culturestarted in 1984 with the red abalone, Haliotis rufes-cens Swainson 1822 (Mazo¤n-SuaŁstegui, Mucino-D|¤az
en-& Bazu¤a-Sicre 1996), although blue, Haliotis fulgensPhilippi 1845, and yellow, Haliotis corrugata Wood
1828, abalone culture are also of interest The through system is commonly used both commer-cially and for stock enhancement In this system,water is constantly introduced to provide good waterquality to the organisms with an 800% daily waterexchange This practice requires large amounts ofwater to be pumped and, thus, yields a high cost.Recently, the abalone industry has implementedimportant technical changes to reduce costs and toimprove the production procedures, and recirculat-ing systems are considered to be a feasible option toreduce the pumping cost, but without reducing aba-lone growth Commercial recirculating systems havebeen used for nearly three decades (Masser, Rakocy &Losordo 1992) This type of system allows high envir-onmental control and feasible cultivation conditions
£ow-as well £ow-as a reduction of water and land (M£ow-asser et al.1992)
Abalone requires a good water quality to obtainhigh growth rates (Basuyaux & Mathieu 1999) thisinvolves the control and monitoring of di¡erent phy-sical^chemical variables Among the most importantare the temperature, dissolved oxygen, pH, salinity,concentrations of total ammonia nitrogen (TAN), ni-trite-nitrogen (NO2-N), nitrate-nitrogen (NO3-N) andalkalinity In general, control of these variableswill provide good water quality for abalone growthand development (Huchette, Koh & Day 2003) TheAquaculture Research, 2011, 42, 161^168 doi:10.1111/j.1365-2109.2010.02545.x
r 2010 CICESE
Trang 3objective of this work was to evaluate and compare
the growth rate and survival of red abalone cultured
in a recirculating and in a £ow-through system
Materials and methods
Juvenile abalone
Seven-month-old red abalone (batch 200D, produced
by spawning 33 males and 73 females), were obtained
from the commercial farm ‘Abulones Cultivados S.A
de C.V.’ located in Ejido Ere¤ndira, Baja California,
Me¤x-ico (3111603300N,11612205300W) Juvenile abalones were
separated from substrata using CO2and were
trans-ported on a wet sponge in plastic bags with oxygen at
the Aquaculture Department (Departamento de
Acui-cultura) at the Centre for Scienti¢c Research and
Higher Education in Ensenada (Centro de
Investiga-cio¤n Cient|¤¢ca y de EducaInvestiga-cio¤n Superior de Ensenda,
CICESE) After arriving at our facilities, individual
se-paration was not possible; thus, abalone distribution
was based on the weight of several abalones, which
were then transferred to culture systems In each tank
50 g of abalones were placed, which corresponds to a
low stocking density of 0.070 kg m 2570 g cm 2
(2265 organisms m 2)
Culture systems
One £ow-through system and one recirculating
sys-tem were used in the growth trial Three individual
£ow-through systems, each one with individual
water supply plus three individual recirculating
tems, were used Each one of the recirculating
sys-tems consisted of a cylindrical £at-bottom ¢breglass
tank (250 L capacity) connected to a magdrive 500
pump (Danner Manufacturing, Islandia, NY, USA)
and a 28.31 L (1ft3) bubble wash bead ¢lter
(Aquacul-ture System Technologies, New Orleans, LA, USA) as
a bioclari¢er (Malone & Beecher 2000), with a
back-wash frequency of two to three times a week The
bead ¢lters were acclimated for 60 days at 20 1C
be-fore the experiment as recommended by Malone and
Beecher (2000) In the recirculating systems, the £ow
rate was 30 L min 1 The three individual
£ow-through systems consisted of similar tanks (250 L
ca-pacity) mentioned above A £ow rate of 1.39 L min l
was set in the three tanks for a daily water exchange
rate of 800% The water used was pumped from the
ocean and ¢ltered to 70mm before reaching the
tanks Constant aeration was provided throughout
the experiment to both systems The experiment
lasted 4 months, beginning on 9 October 2003 andconcluding on 13 February 2004 Fifty grams of or-ganisms were randomly allocated to each tank Theinitial mean shell length was 5.88 0.04 mm forabalones maintained in the recirculating system and6.21 0.01mm in the £ow-through system All dataare average standard error Abalones were fed at aratio of 70% of abalone weight per week with Macro-cystis pyrifera according to the alimentation of theabalones in the commercial farms (N Garc|¤a, pers.comm.) Every week, macroalgae were removed andreplaced with fresh ones
Abalone and environmental variablemeasurements
Every 2 weeks, the shell length of 50 randomly lected animals per tank was measured using an elec-tronic vernier calliper (precision to 0.01mm) modelS225 (Fowler Company, Newton, MA, USA) Everyday, temperature and dissolved oxygen were mea-sured using an oxymeter model YSI 55 (YSI, YellowSprings, OH, USA) On 24 November, in both systems,
se-a hese-ater (precision of 0.55 1C) model Pro Heat II
150 W (Won Brother’s, Fredericksburg, VA, USA) wasplaced to maintain the temperature above 16 1C Sali-nity was measured using a temperature-compen-sated refractometer (conventional refractometer)and pH was measured using a Hanna Hi98127 meter(Hanna Instruments,Woonsocket, RI, USA) Alkalinitywas measured two or three times a week with 1.6 N
H2SO4and a bromcresol green-methyl red indicator(Hach Company, Loveland, CO, USA) Because an alka-linity concentration between 80 and 200 mg L 1ofCaCO3is optimal for bacterial survival in the bio¢lters(Loyless & Malone 1997), when alkalinity values de-clined below 100 mg L 1of CaCO3, alkalinity was ad-justed to 150 mg L 1of CaCO3with the addition ofcommercial sodium bicarbonate, Iris quality (Smart
& Final, Tijuana, BCN, Mexico), to the recirculatingsystems according to the method described by Loylessand Malone (1997) The concentrations of TAN, NO2-Nand NO3-N were determined two or three times perweek using a saltwater master liquid test kit (Aqua-rium Pharmaceuticals, Chalfont, PA, USA); for colora-tion of each nutrient, the absorbance was read using aspectrophotometer Shimadzu UV-1201 (ShimadzuScienti¢c Instruments, Columbia, MD, USA)
Statistical analyses
At the beginning of the experiment, abalone shelllengths were compared among tanks with anANOVA
Trang 4after assumptions of normality and homogeneity of
variances were met At the end of the experiment,
the abalone shell lengths and the survival between
the two culture systems were compared using a
cov-ariance analysis (ANCOVA) to determine the di¡erences
among the slope of the two culture systems against
time Di¡erences between treatment means were
considered to be signi¢cant at Po0.05 Growth rates
[(shell length at a given time shell length at the
time of the previous measurement)/shell length at
the time of the previous measurement 100] were
calculated for every period of measurement Survival
was estimated by counting empty shells at each
mea-surement time For the di¡erent physical^chemical
variables (temperature, dissolved oxygen, pH,
sali-nity,TAN, NO2-N, NO3-N and alkalinity), paired t-tests
were carried out to determine whether there was a
tendency for the data to have a higher value between
culture systems using [recirculating datum]
[£ow-through datum] to calculate the t-value in the t-tests
(paired comparisons, Sokal & Rohlf 1995) Statistical
analyses were carried out usingMINITABforWINDOWS
release 10.2 (Minitab, State Collage, PA, USA)
Results
The initial mean shell length (10 October) was
5.88 0.04 mm for abalones maintained in the
recir-culating system and 6.21 0.01mm in the
£ow-through system (Fig 1) Signi¢cant di¡erences in the
initial abalone size were found between systems
(F(1,298)5 12.25; Po0.01) The ¢nal mean shell length
was 9.17 0.03 mm for organisms in the
recirculat-ing system and 9.01 0.02 mm for organisms in the
£ow-through system (Fig 1) Marginally signi¢cant
di¡erences were found between systems in the
aba-lone growth rate (F(1,2996)5 4.16; P 5 0.04)
There-fore, throughout the experiment, abalones that were
in the recirculating system grew more in comparisonwith the £ow-through system Abalone growthrates ranged from 6.43 to 58.64 (total mean26.1)mm day 1in the recirculating system and from2.3 to 65.87 (total mean 22.21)mm day 1in the £ow-through system (Fig 2) Juvenile survivals (at day126)were 78.74% for organisms in the recirculation sys-tem and 71.82% for organisms in the £ow-throughsystem (Fig 3) No signi¢cant di¡erence was found
in abalone survival (F(1,4)5 3.78, P 5 0.12)
Temperature, salinity and dissolved oxygen did notshow a signi¢cant di¡erence between treatments(Table 1) The values of dissolved oxygen found in thiswork were below the saturation point (72.54% for therecirculating system and 71.62% for the £ow-through system), but never below 5 mg L 1.Although in the £ow-through system, there was
a tendency (paired samples test) to have a highertemperature (n 5128, t 5 13.89, Po0.001), lower
NO2-N concentration (n 5 45, t 5 2.53, P 5 0.015),
Figure 1 Shell length (mm) of juvenile Haliotis rufescens
in two culture systems Mean SE Three £ow-through
system (n 5 50150150 at each time point) Three
recircu-lating systems (n 5 50150150 at each time point)
Figure 2 Growth rate of juvenile Haliotis rufescens intwo culture systems Mean SE Three £ow-through sys-tem (n 5 50150150 at each time point) Three recirculat-ing systems (n 5 50150150 at each time point)
Figure 3 Survival (%) of juvenile red abalones Haliotisrufescens in two culture systems Mean SE Three
£ow-through system (n 5 50150150 at each time point).Three recirculating systems (n 5 50150150 at each timepoint)
Aquaculture Research, 2011, 42, 161^168 Recirculating and £ow through red abalone culture M.Vivanco-Aranda et al.
r 2010 CICESE
Trang 5lower dissolved oxygen concentration (n 5128,
t 513.51, Po0.001) and higher salinity (n 5128,
t 5 2.45, P 5 0.016)
Alkalinity and pH were signi¢cantly di¡erent
be-tween systems (Table 1) due to the addition of sodium
bicarbonate in the recirculating system, where
alkali-nity and pH were higher (n 5 45, t 511.2, Po0.001;
Abalone growth is in£uenced by environmental
con-ditions, water quality (Leitman 1992; Hoshikawa,
Sa-kai & Kijima 1998; Harris, Maguire, Edwards & Johns
1999), diet type (Viana, Cervantes-Trujano &
Solana-Sansores 1994; Capinpin Jr & Corre 1996; Viana,
Cervantes-Trujano & Solana-Sansores 1996; Haaker,
Parker, Barsky & Chun 1998; Lopez, Tyler & Viana
1998; Bautista-Teruel & Millamena 1999; Capinpin Jr,
Toledo, Encena & Doi 1999), culture density (Day &
Fleming 1992; Mgaya & Mercer 1995; Mgaya, Gosling,
Mercer & Donlon 1995; Clarke & Creese 1998;
Valdes-Urriolagoitia 2000) and abalone size at the beginning
of the experiment (Corazani & Illanes 1998;
Trevel-yan, Mendoza & Buckley 1998; Steinarsson &
Ims-land 2003) Abalone size is a principal factor
a¡ecting the feeding rates of gastropods Generally,
feeding rates per biomass unit are higher in smaller
and faster growing juveniles than in larger abalone
(Marsden & Williams, 1996)
H rufescens fed with arti¢cial diets and di¡erent
types of macroalgae produce similar growth rates
as macroalgae (Table 2) It can be observed that red
abalone growth rate/initial shell length (GR/ISL)
obtained in this study were similar to other GR/ISLvalues Furthermore,Table 3 shows a comparison be-tween studies carried out with arti¢cial diets againstmacroalgae with other abalone species The highestgrowth rates in most of these works were achieved
by feeding abalones with an arti¢cial diet This may
be explained by the fact that both the arti¢cial dietshad higher protein and fat contents and producedthe best growth rates in terms of the total weightand shell length (Capinpin & Corre 1996), which sug-gests that diet type has a direct e¡ect on abalonegrowth rate
In contrast to this study, for other abalone species ofsimilar sizes growth rates of the100mm day 1growthrate of similar abalone size were obtained with aba-lone juveniles of Haliotis tuberculata (Linnaeus, 1758)fed with an arti¢cial diet (Lopez et al 1998) and withJapanese abalone Haliotis discus hannai Ino,1953 (Hos-hikawa et al 1998) fed with diatoms The highestgrowth rates have been reported in juveniles between
10 and 40 mm shell length in other abalone species:Haliotis asinina Linnaeus, 1758 (Capinpin & Corre,1996; Fermin 2002), black abalone Haliotis cracherodiiLeach, 1814 (Leighton & Boolootian 1963), paua aba-lone Haliotis iris Gmelin, 1791 (Clarke & Creese 1998),
H fulgens (Leighton, Byhower, Kelly, Hooker & Morse1981), greenlip abalone Haliotis laevigata Donovan,
1808 (Gilroy & Edwards 1998) and H tuberculata pez et al 1998), all fed with an arti¢cial diet
(Lo-An other aspect that a¡ects the growth rate is theculture density For abalone cultured in £ow-throughsystems, growth is inversely related to density(Mgaya & Mercer1995; Capinpin et al.1999;Valde¤s-Ur-riolagoitia 2000) Culture density has an inverse ef-fect on abalone survival and may a¡ect abalonegrowth directly through competition for foodand space However, in this study, density was not acritical factor for the growth rate because a low
Table 1 Water quality parameters measured in two systems of culture of juvenile red abalones Haliotis rufescens
Water parameters
Recirculating system Flow-through system
Temperature ( 1C) 16.07 0.98 10.40–20.80 16.77 0.88 12.90–20.90 Dissolved oxygen (mg L 1) 7.08 0.51 5.34–8.50 6.89 0.55 5.23–8.28 Salinity (g L 1) 35.35 0.38 35.00–38.00 35.23 0.29 34.00–37.00
pH 8.13 0.04 7.90–8.30 8.01 0.03 7.80–8.20 Alkalinity (mg L 1 ) of CaCO3 139.00 7.99 100.00–165.00 115.11 4.76 95.00–140.00 TAN (mg L 1 ) of CaCO 3 0.11 0.04 0.00–0.36 0.12 0.05 0.01–0.49
NO 2 -N (mg L 1 ) of CaCO 3 0.04 0.05 0.00–0.65 0.01 0.01 0.00–0.05
NO 3 -N (mg L 1 ) of CaCO 3 1.08 1.21 0.02–17.29 0.83 0.33 0.01–4.61 Temperature, dissolved oxygen, salinity and pH (n 5128) Alkalinity, TAN, nitrite-nitrogen and nitrates-nitrogen (n 5 45).
Trang 6stocking density was used for both culture systems to
eliminate potential complications due to a high
stock-ing rate The low stockstock-ing density used in this study
can explain the highest survival rate obtained
com-pared with the survival rates reported in other
stu-dies (Nie, Ji & Yan 1996, Park, Rho & Song 1995)
Also, the di¡erences may be due to the size of the ganisms and the duration of the experiment There-fore, it is possible to obtain high growth rates of H.rufescens even on feeding with macroalgae if the phy-sical^chemical variables are within the ranges ofgood water quality and low stocking density
or-Table 3 Data of di¡erent relating experimental studies for the growth juvenile of diverse species
Species
Initial shell length (mm)
Growth rate
Haliotis fulgens 10.00 2.71 0.271 Egregia laevigata and
Macrocystis pyrifera
Leighton et al (1981) 25.00 1.74 0.070
42.00 0.71 0.017 Haliotis tuberculata 15.30 1.75 0.114 Palmaria palmata Mgaya and Mercer (1995)
15.20 1.07 0.070 19.60 1.61 0.082 23.80 1.67 0.070 16.80 1.81 0.108 Haliotis asinina 15.80 4.07 0.258 Gracilaria bailinae Bautista-Teruel and Millamena (1999)
15.20 6.67 0.439 Artificial diet 15.80 7.33 0.464 Artificial diet 15.90 7.43 0.467 Artificial diet Haliotis asinina 19.00 4.20 0.221 Gracilaria heteroclada Capinpin et al (1999)
Haliotis rubra 33.91 1.30 0.038 Artificial diet Huchette et al (2003)
34.23 0.90
Present study Haliotis rufescens 5.88 0.78 0.133 Macrocystis pyrifera Recirculating system
6.21 0.67 0.108 Flow through system Initial shell length (ISL) is the average length of the abalone’s shell in the beginning of the experiment and growth rate (GR) is the monthly growth of the abalones shell (evaluated in millimetres).
Table 2 Data from di¡erent studies on the growth juvenile red abalones Haliotis rufescens
Aquaculture Research, 2011, 42, 161^168 Recirculating and £ow through red abalone culture M.Vivanco-Aranda et al.
r 2010 CICESE
Trang 7Mortality not only depends on the culture density
but also on inadequate handling (stress) of food
transfer (Searcy-Bernal, Salas-Garza &
Flores-Agui-lar 1992) The mortality rate is also associated with
the organism’s size and stage of development
(Ma-zo¤n-SuaŁstegui et al.1996).Water quality variables like
salinity, temperature, dissolved oxygen, pH and
ni-trogen wastes are also important factors governing
the growth of abalones (Harris, Maguire, Edwards &
Hindrum 1998) The results obtained in the present
work for all physical^chemical variables are within
the ranges of good water quality
Special attention should be paid to the pH and
al-kalinity variables, because they were the only two
quality parameters that di¡ered considerably
be-tween the £ow-through and the recirculating system
due to the addition of sodium bicarbonate to the
re-circulating system However, the pH range observed
in the recirculating system was similar to those
shown in other studies with di¡erent abalone species
(Nie et al 1996; Harris, Maguire, Edwards & Hindrum
1997; Harris et al 1998; Basuyaux & Mathieu 1999;
Bautista-Teruel & Millamena 1999) The pH interval
obtained in this study is within the pH values that
promoted the activities of nitrifying bacteria and
pre-vents ammonia toxicity (Loyless & Malone 1997,
Mal-one & Beecher 2000) To our knowledge, there are no
studies on the in£uence of alkalinity on abalone
me-tabolism; however, the alkalinity values in the
recir-culating system (100^165 mg L 1 CaCO3) were
within the ranges recommended for bacterial
survi-val in bio¢lters (Masser et al 1992; Loyless & Malone
1997; Malone & Beecher 2000) Further studies on
the e¡ect of alkalinity on abalone growth could be
carried out; however, it is considered that low
alkali-nity and pH values may have a higher e¡ect on
aba-lone growth than alkalinity values between 100 and
165 L 1CaCO3, because these values were close to
the alkalinity values found in the £ow-through
sys-tem and in nature
In the case of the waste nitrogen compounds, the
TAN, NO2-N and NO3-N concentrations presented in
this study are below the toxic levels reported for
aba-lone (Harris et al 1997; Harris et al 1998) Basuyaux
and Mathieu (1999) found that H laevigata mortality
and growth were a¡ected by a concentration of
1mg TAN L 1; they also found that a concentration
of 1^5 mg NO2-N L 1 does not in£uence abalone
growth, and in contrast, a concentration of
2 mg NO2-N L 1stimulates the growth in H
tubercu-lata On the other hand, they report that toxic levels
are from 8.5 to 15.4 NO2-N L 1 For the NO3-N,
Basuyaux and Mathieu (1999) reported that H culata supports (without a¡ecting growth) a concen-tration range of 100^250 mg of NO3-N L 1 The lowconcentration of the waste nitrogen compounds(TAN, NO2-N and NO3-N) presented in this study wasprobably due to the low density of abalones used andhad no e¡ect on abalone growth
tuber-In the £ow-through system, nearly 15^30% of theproduction costs are associated with the mainte-nance of a high rate of exchange One way to de-crease the cost of production associated withconstantly pumping water through the abalonegrow-out tanks can be with the use of recirculatingsystems (Badillo, Segovia & Searcy-Bernal 2007) For
a recirculating system to be considered a closed tem, it should haveo10% of water exchange per day
sys-of the total volume sys-of the system (Masser et al 1992;Loyless & Malone 1997) In our experiment, the waterexchange was 4.8% of the total volume per day andthe only water replaced was that lost by evaporationand bio¢lter back£ushes; thus, it can be considered as
a closed system
Table 4 shows a cost analysis of the two culturesystems used in this study In the recirculating sys-tem, the high initial costs are due to the equipmentpurchased (bead ¢lter) However, with the saving ofthe rate of water exchange in the recirculating sys-tems, it is possible to recover the initial investment
in a short period of time In this case, the investmentrecovery time is close to 8 months
An economic analysis to the commercial level isnecessary and will be important for the choice of theoptimum stocking density to maximize the produc-
Table 4 Cost analysis of the two systems of culture of nile red abalones Haliotis rufescens
juve-Cost (US$) Recirculating system
Flow-through system
Equipment cost Bubble wash bead filter 571.67 Fibreglass tank 154.30 154.30 PVC pipes 30.86 30.86 Magdrive 500 pump 51.02
Electric energy Pumping cost (per day) 0.09 1.76 Pumping total cost (127 days) 11.43 223.52 Total 819.28 408.68 Difference between systems 410.60 Costs calculated by tank in the experiment time The cost of water pumped from the ocean and the equipment associated was not considered.
Trang 8tion of H rufescens in recirculation culture systems.
With the present work, it is possible to state that
recir-culating systems are a feasible alternative for the
culture of juvenile red abalone at a low density
Acknowledgments
The CONACYT supported the M.Sc studies of M.V.-A
with a scholarship This study was partially ¢nanced
by means of the CONACYT project ‘Genetic markers
of abalone, Haliotis spp.’ (33018 B) and by CICESE
pro-ject number 655 The authors are particularly
grate-ful to the commercial farm ‘Abulones Cultivados S.A
de C.V.’for providing the abalone used in this research
The authors also thank Marisela Aguilar-JuaŁrez for
the support provided and Oscar B Del R|¤o Zaragoza
for the technical assistance
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Trang 10Assay performance during validation of freezing
infected with a Gram-negative bacterium
Julie Bebak1, Craig Shoemaker1, Covadonga Arias2& Phillip Klesius1
1 USDA ARS AAHRU, Auburn, AL, USA
2 Department of Fisheries and Allied Aquaculture, Auburn University, Auburn, AL, USA
Correspondence: J Bebak, USDA ARS AAHRU, 990 Wire Rd., Auburn, AL 36832, USA E-mail: julie.bebak@ars.usda.gov
Abstract
Recovery of bacteria from infected ¢sh during
popula-tion sampling can be a¡ected by factors including the
type of assay, method of specimen preservation and
concentration of bacteria present Consequently, before
use in ¢eld sampling, methods should be validated The
three objectives of this study were, ¢rst, to determine
whether a channel cat¢sh Ictalurus punctatus
(Ra¢n-esque) ¢ngerling classi¢ed as positive for Gram-negative
Edwardsiella ictaluri infection according to bacterial
culture before freezing was also classi¢ed as positive
after freezing, second, to determine how direct culture
from the kidney (DIRECT), culture of homogenate
(HOMOG) and standard PCR (PCR) agree with bacterial
culture in terms of classifying ¢sh as positive or
nega-tive and third, to estimate diagnostic sensitivity (dSe)
and diagnostic speci¢city (dSp) for DIRECT, HOMOG
and PCR In fresh and frozen ¢sh, as bacterial
concen-tration decreased, the ability of each assay to detect
po-sitive ¢sh also decreased, especially when there were
o104colony-forming units per gram (CFU g 1) tissue
HOMOG proved to be the most reliable at correctly
clas-sifying cat¢sh, whether they were subclinically or
clini-cally infected PCR assay was the least reliable Overall,
values for this study population for dSe were 0.66, 0.92
and 0.43, and for dSp were 0.86, 0.91 and 0.95, for
DI-RECT, HOMOG and PCR respectively
Keywords: validation, freezing, Edwardsiella, cat¢sh,
PCR, bacterial culture
Introduction
Studies conducted on ¢eld populations of ¢sh may
require sampling large numbers of individuals
to achieve su⁄cient statistical power For example,
studies that estimate prevalence, incidence or otherepidemiological measures generally require sam-pling hundreds, or even thousands, of ¢sh Assuming100% diagnostic sensitivity (dSe) and speci¢city(dSp), for a prevalence estimate in a population of
100 000 animals, an error of 5%, and con¢dence of95%, the sample size for simple random samplingwill be 138 animals if the expected prevalence is 10%and 384 animals if the expected prevalence is 50%(Thrus¢eld 1995) If the animals sampled are warm-water pond-cultured ¢sh species, then sampling willmost likely occur in an environment with daytimetemperatures that range from an average of about
27 1C to 40 1C, processing ¢sh will proceed slowlyand the distance from laboratory facilities would pre-clude transporting large numbers of dead ¢sh for la-boratory analysis while still maintaining sampleintegrity The transport of live ¢sh back to the labora-tory may be considered, but would likely be con-strained by the need to keep groups of ¢sh separatedand the lack of adequate culture facilities at the desti-nation One possible solution to this problem would
be to place dead ¢sh on dry ice (i.e at least
78.2 1C), and then transport them to the laboratoryfor storage at 80 1C and processing at a later date.Freezing can result in the inactivation of bacteriaand a loss of infectious units (Sheridan 1997; Archer2004) Suomalainen, Reunanen, Ijas, Valtonen andTiirola (2006) froze enriched Flavobacterium colum-nare from rainbow trout skin mucus at 20 1C for
an unspeci¢ed length of time and then thawed it at
23 1C and found that bacterial DNA could not bedetected with PCR after freezing Electron micro-scopy revealed that frozen then thawed bacterialcells had disintegrated They hypothesized thatbacterial Dnases, lyases and proteases present in
F columnare were responsible for the destruction ofAquaculture Research, 2011, 42, 169^176 doi:10.1111/j.1365-2109.2010.02551.x
r 2010 Blackwell Munksgaard
Trang 11bacterial cell walls and of the PCR-ampli¢able DNA.
Therefore, before freezing is incorporated as a
sam-pling strategy for pond-based samsam-pling, the method
should be validated
In addition to Suomalainen et al (2006), some
other studies have examined the e¡ect of freezing at
20 1C and/or at 70 1C on positive or negative
re-sults for ¢sh pathogens isolated from various organs
after injecting ¢sh with a known concentration of
bacteria (Brady & Vinitnantharat 1990; Evans,
Wie-denmayer, Klesius & Shoemaker 2004) However, to
our knowledge, no studies have quanti¢ed the
survi-val of bacteria pre- and post freezing in the same ¢sh
in the same organ Nor have any studies linked the
concentration of bacteria in the organ with the
re-sults of molecular assay
The three main objectives of this laboratory study
were, ¢rst, to determine whether a channel cat¢sh
¢ngerling classi¢ed as positive for Edwardsiella
icta-luri (Enterobacteriaceae, Gram-negative rod)
infec-tion according to bacterial culture before freezing
was also classi¢ed as positive after freezing at
80 1C for 58^60 days, second, to determine how
di-rect culture from the kidney, culture of homogenate
and standard PCR agree with bacterial culture
[col-ony-forming units per gram (CFU g 1) kidney tissue]
in terms of classifying ¢sh as positive or negative and
third, to estimate dSe and dSp for direct culture,
cul-ture of homogenate and standard PCR
Methods
Speci¢c pathogen-free channel cat¢sh were obtained
from an Alabama farm as sac fry They were kept in
£ow-through dechlorinated fresh water and fed one
to four times per day, depending on age and water
temperature Fish were 120.6 2.1g [mean
weight standard error (SE)] and from 6 to 14
months old when they were used for the study
Before injection with E ictaluri, ¢sh were stocked
individually into 57 L £ow-through tanks and
accli-mated for at least 48 h Water temperature was
29 2 1C.Water £ow rate was about 500 mL min 1
Photoperiod was 12 h light:12 h dark
Edwardsiella ictaluri (AL-93-75), isolated previously
from a naturally occurring outbreak of ESC in
Alaba-ma industry cat¢sh, was the isolate used for the study
(Bader, Shoemaker & Klesius 1998; Shoemaker,
Kle-sius & Bricker 1999) The stock culture was stored in
brain heart infusion (BHI) broth at 80 1C Before
use, the stock culture was thawed, 100mL was added
to 25 mL BHI and grown overnight at 28 1C After22^26 h growth, a 10-fold serial dilution was madeand ¢sh were injected intracoelomically with 100mL
of the dilution needed to infect the ¢sh This dilutionvaried according to whether ¢sh were to be infectedwith low numbers or high numbers of bacteria Therange in numbers of bacteria injected was 102 to
107CFU E ictaluri Numbers were estimated by ing and counting the 10-fold serial dilution in tripli-cate
plat-Fish were injected and processed in groups of 20^
40 animals Fish that died before the sampling daywere placed in 0 1C and processed within 24^48 h.Surviving ¢sh were sampled at 6^9 days post injec-tion to allow the infection to establish itself as clinical
or subclinical, and to allow time to prepare BHI brothand tubes, Edwardsiella isolation medium (EIM)plates and other supplies needed for sampling Onsample day, ¢sh were euthanized by immersion in atleast 250 mg L 1tricaine methanesulphonate (MS-222; Argent Chemical, Redmond,WA, USA) Each ¢shwas weighed, then the anterior and posterior kidneywere exposed A 1mL culture loop was inserted via astabbing technique into the left anterior and then theleft posterior kidney and streaked onto an EIM (selec-tive for E ictaluri; Shotts & Waltman 1990) plate (DI-RECT) These DIRECT samples were collected induplicate on a divided culture plate The left anteriorand left posterior kidney were then removed andplaced in the same preweighed sterile 5 mL tube.The body cavity of the ¢sh was then closed and theremainder of the ¢sh was placed in a plastic bagand frozen at 80 1C The kidney tissue was homo-genized (TissueTearor, Biospec Products, Bartlesville,
OK, USA), and15^25 mg was immediately transferred
to a 1.5 mL microcentrifuge tube for DNA extractionthat same day (PCR) The remaining homogenate wasthen streaked in triplicate onto a divided EIM plate,using a 1mL culture loop each time (HOMOG) Thehomogenate and tube were weighed, and the weightwas recorded Two millilitres of BHI broth was added
to the homogenate and three 10-fold dilution series(i.e in triplicate) were made using 500mL of thehomogenate plus BHI for each dilution series Onehundred microlitres of each of the target dilutionswere plated with a plate spreader onto EIM plates Re-sults were recorded after plates were incubated atroom temperature for 48^72 h, which we had deter-mined previously to be su⁄cient time for colonies to
be visualized, if present For plates that were streaked
in duplicate directly from the kidney or that werestreaked in triplicate from the kidney homogenate,
Trang 12results were recorded as positive or negative For
plates spread in triplicate from serial dilutions, the
number of bacterial colonies were counted and
recorded and the kidney was classi¢ed as positive or
negative for E ictaluri infection This result was used
as the reference test (gold standard) for comparison
with DIRECT, HOMOG and PCR samples The mean
( SE) analytical sensitivity of plating the dilution
series was 123 ( 23) CFU g 1tissue
The ¢sh were frozen at 80 1C, which was chosen
because it is a typical storage temperature for the
pre-servation of stocks of bacteria To allow for the
amount of time that it may take to process large
num-bers of samples (e.g hundreds), ¢sh were removed
from the freezer after 59^63 days After removal, they
were thawed within about 30 min at room
tempera-ture The right anterior and posterior kidneys were
then assayed as described above
To test the assumption that the left and right sides
of the kidney contain the same log concentration of
bacteria in each ¢sh, 40 ¢sh were injected with
vary-ing concentrations of E ictaluri, then sampled 7^8
days later The left and right kidneys were removed
from each ¢sh, homogenized separately and 2 mL
BHI was added Appropriate dilutions were made
and plated on an EIM plate in triplicate using a plate
spreader Results were recorded after plates were
in-cubated at room temperature for 48^72 h
DNA extraction and ampli¢cation
DNA from the homogenized tissue samples was
extracted and puri¢ed using the animal tissue
spin-column protocol and the Qiagen Dneasy Blood and
Tissue Kit (QIAGEN, Germantown, MD, USA) with
one minor modi¢cation in that after adding Bu¡er
ATL to the sample it was ground with a sterile pestle
Nucleic acid concentration was quanti¢ed using a
nucleic acid spectrophotometer (ND-1000
Spectro-photometer, Nano-drop Technologies, Wilmington,
DE, USA) The ampli¢cation pro¢le for this standard
PCR was the same as used by the Bilodeau,
Waldbie-ser, Terhune, Wise and Wolters (2003) real-time PCR
assay with minor modi¢cations The thermal cycler
(MyCycler Thermal Cycler, Bio-rad Laboratories,
Hercules, CA, USA) was hot-started at 94 1C before
samples were loaded and the ampli¢cation cycle was
started at 94 1C for 5 min instead of 10 min One
posi-tive (75 ngmL 1E ictaluri DNA) and two negative
controls (75 ngmL 1¢sh DNA; master mix with no
template) were included in each batch Each
ampli¢-cation reaction mixture (50mL) contained a DNA
sample (1mL, approximately 75 ng of bacterial and
cat-¢sh genomic DNA); 25mL of 2 Epicentre Bu¡er D;
15 pmol Eict forward primer (ACTTATCGCCCTCGCAACTC) and 15 pmol Eict reverse primer (CCTCTGATAAGTGGTTCTCG) (Bilodeau et al 2003); and 1.0 UTaq polymerase (5 UmL 1) Ampli¢cation was veri¢ed
by electrophoresis of 12mL of the PCR product through1.0% agarose gels Gels were interpreted by an observerwho was blinded to the results of the bacterial culture.For each 1mL template, the analytical sensitivity of thisprocedure for bacterial DNA only was 500 fg to 5 pgand for bacterial DNA plus ¢sh DNA it was 500 fg bac-terial DNA 100 ng ¢sh DNA 1 Consequently, at leastabout100 bacterial genomes must be present for detec-tion by this PCR method
Data analysisThe number of CFU of E ictaluri per gram of kidneytissue was estimated as
CFU g1tissue¼ CFU
inoculumðmLÞ
1dilution
ðg tissue mL1BHIÞwhere CFU is the average colony-forming units perplate, inoculum the volume (ml) of sample inoculatedonto plate, dilution is that which corresponds to platecount, g tissue ml 1BHI the weight of the kidney tis-sue in 2 mL of BHI
For comparison of CFU E ictaluri g 1kidney in theleft and right side of the kidney to test the assumptionthat both sides contain the same log concentration,bacterial numbers were expressed as 10x, where x isthe number of logs for the sample The agreement be-tween the left and right sides of the kidney, based onlog number, was examined by plotting the di¡erence
in log concentration over the average log tion in the left and right side (Altman & Bland 1983;Bland & Altman 1986) To obtain the values betweenwhich 95% of the results should lie, two times thestandard deviation of the di¡erence in the value be-tween the left and right side was estimated and sub-tracted from the mean di¡erence If, for post-freezingsamples, the change in concentration was outsidethis range, then freezing was assumed to have af-fected the concentration of E ictaluri
concentra-Thek statistic was used to estimate the agreementbetween sample sets (Thrus¢eld 1995; StataCorp2005) The interpretation of k values followed thecategorization proposed by Everitt (1989), whereinAquaculture Research, 2011, 42, 169^176 Assay performance during validation of freezing J Bebak et al.
r 2010 Blackwell Munksgaard
Trang 13k values 40.81 represent an almost perfect agreement,
values from 0.61 to 0.80 represent substantial
agree-ment, values from 0.41 to 0.60 represent moderate
agreement, values from 0.21 to 0.40 represent fair
agreement, values from 0 to 0.20 represent a slight
agreement, and a value of 0 represents poor agreement
Diagnostic sensitivity (proportion of true positives
detected by a test) and dSp (proportion of true
nega-tive detected by a test) (Saah & Hoover 1997) with
95% con¢dence intervals (95% CI) were estimated
for DIRECT, HOMOG and PCR for pre-freezing
com-bined with post-freezing samples The results from
bacterial culture (CFU g 1tissue) of the serial
dilu-tions were used as the reference test to classify ¢sh
as positive or negative
Results
Comparison of right and left kidney
The mean SE body weight of the 40 ¢sh used to
test the assumption that the right and left sides of
the kidney would contain the same log concentration
of E ictaluri was132.7 7.5 g.When bacterial
concen-trations for the left and right side of the kidney were
compared, 24/40 kidneys were within the same log
concentration (i.e 10x, x is the log number of bacteria
present), 15/40 were 1 log di¡erent and one was 2 logs
di¡erent These results indicate that the left and right
kidney contained unequal log concentrations of
bac-teria Therefore, the pre-freezing vs post-freezing
com-parisons that could be made were limited However,
analysis of agreement indicated that if, post freezing,
the right kidney was greater than 101.5CFU g 1tissue
below the pre-freezing results, then the sample could
be classi¢ed as a¡ected by freezing
E¡ect of freezing on log bacterial
concentration in kidney
Thirty of the ¢sh died before the day of sampling
and were placed at 0 1C These ¢sh were processed
within 24^48 h of death Mean weight SE was
120.6 2.1g for the 162 ¢sh sampled pre- and post
freezing Results for the culture of serial dilutions
ranged from 0 to 108CFU E ictaluri g 1 kidney
tissue After freezing for 58^60 days, the proportion
of ¢sh that were still classi¢ed as positive according
to the culture of serial dilutions depended on how
high the log concentration was in the kidney before
Ten out of 57 of the ¢sh with 0 CFU g 1tissue freezing were positive post freezing, that is, they had
pre-an increase in bacterial titre, as did 2/7 of the ¢shthat were 101CFU g 1tissue pre-freezing None ofthe rest of the ¢sh in any other log category had ahigher post-freezing log concentration
Bacterial concentration compared withDIRECT, HOMOG and PCR
Pre- and post-freezing samples were combined for acomparison between bacterial concentration resultsand DIRECT, HOMOG and PCR samples For each as-say type, the per cent correctly classi¢ed as positiveincreased as the log number concentration increased(Table 2) The best performing assay was HOMOG,which correctly classi¢ed490% of the positive sam-ples as positive for all concentrations 101
CFU
E ictaluri g 1tissue The worst performer was PCR,which correctly classi¢ed o65% of the samplesbelow the 105
CFU g 1category When tration was 0 CFU g 1, DIRECT, HOMOG and PCRmisclassi¢ed 14%, 8% and 5% of the samples aspositive respectively
concen-Table 1 Pre-freezing (Pre FC) and post-freezing (Post FC) concentration (CFU E ictaluri g 1 kidney tissue) for indivi- dual channel cat¢sh ¢ngerlings (n 5162)
Number
of fish
Pre FC
Post FC
% Pos
Trang 14Overall k values for the three assays compared
with the reference test were 0.49 0.05, 0.75 0.05
and 0.34 0.04 for DIRECT, HOMOG and PCR
respectively (test prevalence 5 61%; Table 3) When
kidneys contained o104CFU g 1 tissue (test
pre-valence 5 38%),k was 0.21 0.06, 0.62 0.06 and
0.12 0.05 for DIRECT, HOMOG and PCR
respec-tively When there was 104CFU g 1 tissue
present, most comparison categories were 100% in
agreement (Table 2)
Diagnostic sensitivity varied widely depending on
the assay and whether the samples were pooled or
the cutpoint was set at 104CFU E ictaluri g 1tissue
(Table 4) PCR had the lowest dSe (and the highest
false-negative fractions) for all categories, and was
as low as 0.15 (95% CI 5 0.07, 0.23) for samples with
o104
CFU g 1tissue The highest dSe for PCR was
0.62 for samples 104CFU g 1tissue The HOMOG
assay had consistently the highest dSe values of 0.92
(95% CI 5 0.89, 0.96), 0.80 (95% CI 5 0.71, 0.89)
and 1.00 for all samples combined, o104
and
104CFU g 1 respectively There was much less
variability in dSp for the three assays The lowest
dSp was 0.86 (95% CI 5 0.80, 0.92) for DIRECT and
the highest was 0.95 (95% CI 5 0.92, 0.95) for PCR
Discussion
Under the conditions evaluated in this study, the
low-er the concentration of E ictaluri in the kidney, the
greater the chance that ¢sh would be misclassi¢ed
post freezing This chance was at least 59% wheno104
CFU g 1tissue was present For studies that tempt to measure population parameters in a cat¢shpopulation that includes subclinically infected ¢sh,freezing may signi¢cantly bias the result towards aninaccurate, low estimate of prevalence There wasalso some likely misclassi¢cation error due to e¡ects
at-of freezing when concentrations were higher, inthe range of 104to 105CFU g 1tissue Edwardsiellaictaluri is a Gram-negative bacterium In general,because of di¡erences in cell wall structure, Gram-positive bacteria are more resistant to freezing thanGram-negative bacteria (Lund, Baird-Parker & Gould2000) Therefore, freezing ¢sh for analysis of a Gram-positive bacterium, such as S iniae, may result in lessloss of bacterial cells
A few ¢sh had an ‘increase’ in bacterial tion while they were in the freezer, even though theywere speci¢c pathogen free and even when the nor-mal variation between left and right sides of the kid-ney was considered Error is inherent in evaluation ofdiagnostic tests and should be allowed to be ex-pressed Therefore, all ¢sh were included in the re-sults This di¡erence may be explained by samplingerror in that the sampling technique did not capturebacteria present at low concentrations while takingthe pre-freezing sample These results also demon-strate the well-known concept that reference tests areoften imperfect, and this one likely is as well (Enoe,Georgiadis & Johnson 2000; Cameron 2002) It is alsopossible that viable bacteria were clustered in macro-phages (Blazer 1991), which could account for this re-sult especially at the very low log concentrations
concentra-If the two sides of the kidney had been the same interms of concentration of E ictaluri, it would be valid
Table 2 Per cent of channel cat¢sh ¢ngerlings with 0^
108CFU g 1kidney tissue that were also positive (% Pos)
for direct culture of Edwardsiella ictaluri from kidney tissue
(DIRECT), culture of homogenate (HOMOG) or PCR (PCR)
Results for samples taken before freezing were combined with
samples taken after freezing.
n, total number of samples included in each group; CFU,
colony-forming unit.
Table 3 k statistics standard error (SE) for the tion of channel cat¢sh ¢ngerling kidney samples as positive
classi¢ca-or negative by culture (CFU g 1kidney tissue of
Edwardsiel-la ictaluri) compared with direct (DIRECT) culture from the kidney, culture of homogenate (HOMOG) or PCR
j SE All samples o10 4
CFU g 1 tissue.
Aquaculture Research, 2011, 42, 169^176 Assay performance during validation of freezing J Bebak et al.
r 2010 Blackwell Munksgaard
Trang 15to compare post-freezing results for DIRECT, HOMOG
and PCR to pre-freezing estimates of E ictaluri
concentration However, as can be seen from Table 2,
a 1 log change in concentration greatly a¡ected the
ability of DIRECT and PCR assays to detect positive
samples Therefore, comparing pre-freezing
con-centration with post-freezing results for DIRECT,
HOMOG and PCR is not valid
Microbiological assay and standard PCR assay
pro-vided less than perfect estimates of infection status
of channel cat¢sh infected with E ictaluri As the
bacterial concentration decreased, the ability of each
assay to detect positive ¢sh also decreased Out of
the three assays tested, only HOMOG consistently
demonstrated an agreement with the results in
CFU g 1tissue Neither DIRECT nor PCR were able
to detect480% of the positive ¢sh until the
concen-tration in the kidney was 104
or 105CFU g 1respectively
Analytical sensitivity is the smallest amount of
substance in a sample that can accurately be
mea-sured by an assay Diagnostic sensitivity is the
pro-portion of individuals who have a given condition
that are identi¢ed as positive for the condition High
analytical sensitivity does not guarantee that dSe
will also be high (Saah & Hoover 1997) This
relation-ship can be observed in the results for both DIRECT
and PCR methods The target bacterium may have
been missing from the sample taken by the culture
loop or from the 15 to 25 mg tissue sample removed
for PCR analysis The dSe of the PCR method used
de-clined abruptly at about 104^105CFU E ictaluri g 1
kidney tissue At these concentrations, even if
bacter-ia were evenly distributed in kidney tissue, there
would be only about 60^125 E ictaluri bacterial cells
present in the 15^25 mg sample used for PCR As
described previously, the analytical sensitivity of theassay was about 100 bacterial genomes Therefore,the results for dSe make sense given the analyticalsensitivity of the PCR assay
DIRECT, HOMOG and PCR also classi¢ed some ¢sh
as positive when, according to culture in CFU g 1sue, they were actually negative As discussed pre-viously, this result could have been due to animperfect reference test and/or sampling error and/
tis-or the presence of bacteria in the phagocytic phages
macro-Thek results corresponded with the other ods used in this study to determine how likely ¢shwould be to be misclassi¢ed.k, the level of agreementbeyond chance for two observations, ranged fromslight to substantial agreement, and was the lowestfor PCR assay and the highest for HOMOG.k esti-mates are a¡ected by the underlying prevalence ofthe trait in question (Sargeant & Martin 1998) Inthis study, the underlying prevalence did not changefor the comparisons being made as long ask valuesfor assays are compared within a cut-o¡ group.The use ofk was appropriate as long as k values for
meth-‘all samples’ are not compared with k values foro104
CFU g 1.Bayesian modelling may also be used to estimatedSe and dSp when the reference test is imperfect orthe true disease state is unknown (Enoe et al 2000;Branscum, Gardner & Johnson 2005) This approachcan provide a useful way to evaluate the diagnostictests in a future work
Sample sizes for the estimation of population meters such as prevalence and incidence should beadjusted to account for the less than perfect dSe anddSp of bacterial culture and PCR Estimates of dSeand dSp can be used to determine how sample sizes
para-Table 4 Estimates of diagnostic sensitivity (dSe) and diagnostic speci¢city (dSp) 95% con¢dence intervals (95% CI) for culture of Edwardsiella ictaluri from serial dilutions of channel cat¢sh kidney tissue compared with direct culture from the kidney (DIRECT), culture from kidney homogenate (HOMOG) and PCR (PCR) results
Concentration in
kidney (CFU g 1) Test prevalence (%) Type dSe 95% CI (lower, upper) dSp 95% CI (lower, upper)
All 61 DIRECT 0.66 0.60, 0.73 0.86 0.80, 0.92
HOMOG 0.92 0.89, 0.96 0.91 0.86, 0.96 PCR 0.43 0.36, 0.50 0.95 0.92, 0.99 o10 4 38 DIRECT 0.32 0.22, 0.43 0.86 0.80, 0.92
HOMOG 0.80 0.71, 0.89 0.91 0.86, 0.96 PCR 0.15 0.07, 0.23 0.95 0.92, 0.99
All samples combined (All), results for o10 4
CFU g 1tissue and results for 10 4
CFU g 1tissue were estimated.
Trang 16should be adjusted for estimates of prevalence or
in-cidence Additionally, if the dSe and dSp of the test are
known or can be estimated, it is possible to correct for
mistakes, and convert the apparent prevalence to the
true prevalence (Greiner & Gardner 2000; Cameron
2002)
Transferring estimates of dSe and dSp from one
po-pulation to another should be carefully considered
However, the estimates provided by this laboratory
study provide a starting point for sample size
esti-mates for ¢eld studies, if a test prevalence of 61% is
acceptable If the infection status of a population is
unknown, and standard PCR is going to be used as
the assay, then a dSe 5 0.43 (95% CI 5 0.36, 0.50)
and a dSp 5 0.95 (95% CI 5 0.92, 0.99) provide a good
starting point If the population is composed of ¢sh
that are all subclinically infected, then dSe 5 0.15
(95% CI 5 0.07, 0.23) and dSp 5 0.95 (95% CI 5 0.92,
0.99) may be used
If the infection status is to be estimated with the
direct culture of the kidney, then a dSe and dSp of
0.66 (95% CI 5 0.60, 0.73) and 0.86 (95% CI 5 0.80,
0.92), respectively, will be informative for a
popula-tion with unknown status and dSe 5 0.32 (95%
CI 5 0.22, 0.43) and dSp 5 0.86 (95% CI 5 0.80, 0.92)
for a subclinically infected population For assay
using homogenized kidney samples, an overall
dSe 5 0.92 (95% CI 5 0.89, 0.96) and dSp 5 0.91 (95%
CI 5 0.86, 0.96) will provide a starting point For a
subclinically infected population, dSe 5 0.80 (95%
CI 5 0.71, 0.89) and dSp 5 0.91 (95% CI 5 0.86, 0.96)
may be considered
This work demonstrated that, especially at lower
log concentrations, E ictaluri bacterial cell count
may decrease during freezing Brady and
Vinit-nantharat (1990) injected individual cat¢sh
¢nger-lings averaging 4.7 g with 1.8 107
cells of E
ictaluri and found that they could isolate bacteria
from the kidney and liver with an inoculating loop
for no longer than 30 days after freezing at 20 1C
It is possible that bacteria were not viable after 30
days, but it is also possible that as CFU g 1tissue
de-clined in the organs, isolation with an inoculating
loop became a less accurate way of isolating E
icta-luri and the possibility of false negative results
in-creased
As mentioned previously, the results of this study
should be extended to other techniques, ¢sh species
and bacterial species with caution Evans et al (2004)
injected Nile tilapia (Oreochromis niloticus) with
5.5 102
or 4.5 106
CFU Streptococcus agalactiaeper ¢sh, and placed them in the freezer 24 h later
After 180 days at 70 1C, all ¢sh were positive for
S agalactiae infection using kidney culture with asterile swab and overnight culture in enrichmentbroth The concentration in the ¢sh tissue whenfrozen was unknown, but ¢sh injected with 102CFU
S agalactiae probably had a concentration ofo104
CFU g 1tissue when they were frozen The ference in recovery results could have been due to theuse of overnight culture in enrichment broth or thefact that S agalactiae is a Gram-positive bacterium
dif-In conclusion, ¢ngerling cat¢sh kidneys lost a ni¢cant number of E ictaluri cells when they wereplaced in a 80 1C freezer for 60 days Comparedwith direct culture or PCR, culture of homogenizedkidney tissue proved to be the most reliable at cor-rectly classifying ¢sh, whether they were clinically
sig-or sub-clinically infected When estimating ¢shpopulation parameters for E ictaluri infection,sample size estimates should be adjusted to accountfor the error when using these less than perfectassays This study provides some suggested valuesfor dSe and dSp
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Bader J.A., Shoemaker C.A & Klesius P.H (1998) Genomic subtyping of Edwardsiella ictaluri isolated from diseased channel cat¢sh by arbitrarily primed polymerase chain reaction Journal of Aquatic Animal Health 10, 22^27 Aquaculture Research, 2011, 42, 169^176 Assay performance during validation of freezing J Bebak et al.
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Wolters W.R (2003) A real-time polymerase chain
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assessing agreement between two methods of clinical
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and disease prevalence when the true disease state is
un-known PreventiveVeterinary Medicine 45, 61^81.
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(2004) Survival of Streptococcus agalactiae from frozen ¢sh
following natural and experimental infections
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Investiga-tions Oxford University Press/Edward Arnold, New York,
NY, USA/London, UK.
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on attribute prevalence when assessing the repeatability
of questionnaire data Preventive Veterinary Medicine 34, 115^123.
Sheridan J.J (1997) The e¡ect of freezing on the survival of pathogens in di¡erent meat types and the e¡ect of varying lean fat ratios The Society of Food Hygiene and Technology Hygiene Review 1–8 Available at http:// www.sofht.co.uk/isfht/irish_97_freezing.htm.
Shoemaker C.A., Klesius P.H & Bricker J.M (1999) E⁄cacy of
a modi¢ed live Edwardsiella ictaluri vaccine in channel cat¢sh as young as seven days post hatch Aquaculture
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selec-StataCorp (2005) Stata Statistical Software: Release 9 Corp LP, College Station,TX, USA.
Stata-Suomalainen L.R., Reunanen H., Ijas R.,Valtonen E.T & ola M (2006) Freezing induces biased results in the mole- cular detection of Flavobacterium columnare Applied and Environmental Microbiology 72, 1702–1704.
Tiir-Thrus¢eld M (1995) Veterinary Epidemiology, 2nd edn well Science Ltd, Oxford, UK, 483pp.
Trang 18Black-Sperm capacitation of the shrimp Litopenaeus
vannamei
Sirinda Aungsuchawan1, Craig L Browdy2& Boonsirm Withyachumnarnkul3
1
Department of Anatomy, Faculty of Medicine, Chiang Mai University, Chiang Mai,Thailand
2 South Carolina Department of Natural Resources,Waddell Mariculture Center, Charleston, SC, USA
3 Department of Anatomy, Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp), Faculty of Science, Mahidol University, Bangkok,Thailand
Correspondence: B Withyachumnarnkul, Department of Anatomy, Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp), Faculty of Science, Mahidol University, Rama 6 Rd., Bangkok 10400,Thailand E-mail address: boonsirm@yahoo.com
Abstract
Litopenaeus vannamei is one of the most important
spe-cies of farmed shrimp The females have
an‘open’thely-cum Mating is accomplished by attaching the male
spermatophore onto the surface of the thelycum 4^
6 h before spawning During this period, sperm may
have to undergo morphological changes associated
with a capacitation process that has been described
for other shrimp species The objective of this research
was to extend research on sperm capacitation in L
vannamei by ultrastructural and biochemical means
The sperm of L vannamei were divided into those
freshly prepared from the spermatophore (S-sperm),
extracted from the male gonopores, and those
ex-tracted from the female thelycum (T-sperm) Under
transmission electron microscopy, ultrastructural
dif-ferences were detected between the S- and theT-sperm
in the nuclear material, the ¢lamentous meshwork and
the cytoplasmic particles Under scanning electron
mi-croscopy, the di¡erence was observed in the cap and
spike regions Immuno£uorescence using confocal
mi-croscopy to detect tyrosine phosphorylated proteins
revealed di¡erent distribution patterns between
S-and T-sperm The location of phosphorylation activity
changed from the spike in S-sperm, to the ¢lamentous
meshwork in the T-sperm These morphological and
biochemical changes con¢rm that capacitation of L
vannamei sperm takes place following mating
Keywords: Litopenaeus vannamei, sperm,
sperma-tophore, thelycum, capacitation, tyrosine
phos-phorylation
Introduction
In many mammalian species, sperm must undergo acapacitation process in order to be able to fertilizeeggs The process is usually accomplished in thefemale reproductive organ, with complex morphologi-cal and biochemical changes in the sperm (Yanagi-machi 1994; Cooper 1998) The capacitation process
in invertebrates has not been as thoroughly gated as in mammals, and some investigatorshave suggested that invertebrate sperm did not re-quire capacitation before fertilization (Vacquire1998) Nevertheless, a capacitation process hasbeen observed in tick, nematode and shrimp (Oliver
investi-Jr & Shephard 1981; Clark investi-Jr & Gri⁄n 1988; nayake, Uhlinger, Gri⁄n & Clark Jr 1992; Vani-chviriyakit, Kruevaisayawan, Weerachatyanukul,Tawipreeda, Withyachumnarnkul, Pratoomchat,Chavadej & Sobhon 2004) In shrimp, the phenomen-
Wikrama-on has been described in SicyWikrama-onia ingentis (Clark Jr &Gri⁄n 1988; Wikramanayake et al 1992) and Penaeusmonodon (Vanichviriyakit et al 2004), both of whichare close-thelycum species In P monodon, it wasfound that the nuclear material of the sperm takenfrom the female thelycum (T-sperm) is less condensedthan that of the sperm taken from the male terminalampoule of the vas deferens (S-sperm) In addition,the protein pro¢le of the plasma membrane of S- andT-sperm is also di¡erent Certain proteins wereabsorbed while others were removed from theperipheral and integral membrane Tyrosinephosphorylation, an index of sperm capacitation(Visconti, Bailey, Moore, Pan, Olds-Clarke & KopfAquaculture Research, 2011, 42, 188^195 doi:10.1111/j.1365-2109.2010.02579.x
r 2010 The Authors
Trang 191995), was also increased in T-sperm, compared with
that of S-sperm These morphological and
biochem-ical modi¢cations were accomplished within 3 days
after the sperm sac was inserted into the female
the-lycum The modi¢cation was accompanied by an
in-crease in the acrosome reaction, a necessary step
before fertilization (Kruevaisayawan,Vanichviriyakit,
Weerachatyanuku, Iamsaard, Withyachumnarnkul,
Basak, Tanphaichitr & Sobhon 2008) Morphological
changes in sperm have also been observed in
S ingentis (Talbot & Chanmanon 1980; Lynn & Clark
1983; Arsenault 1984; Gri⁄n, Shigekawa & Clark Jr
1988; Dougherty 1990)
In the open-thelycum species, capacitation was
also observed in Litopenaeus occidentalis and possibly
in Litopenaeus vannamei (Alfaro, Ulate & Vargas
2007) In L occidentalis, the acrosome reaction was
observed in T-sperm but not in S-sperm In L
vanna-mei, the ultrastructure of the S- and T-sperm is
simi-lar, except for some changes in the ¢lamentous
network that lies between the nucleus and the
hemi-spherical cap On the other hand, in vitro fertilization
with the production of viable nauplii using S-sperm
has been reported, although the fertilization rate is
low (Misamore & Browdy1997) Thus, it is still a
ques-tion whether capacitaques-tion is required for L vannamei
In L vannamei, mating is accomplished by attaching
male spermatophores or sperm sacs onto the surface
of the thelycum and the spermatophores remain
at-tached to the female thelycum about 4^6 h from
mat-ing to spawnmat-ing (Misamore & Browdy 1996) The
purpose of this study was therefore to explore the
morphological changes in sperm of L vannamei at
4 h after mating using transmission electron
micro-scopy (TEM), scanning electron micromicro-scopy (SEM), as
well as the distribution of tyrosine phosphorylated
protein using confocal laser scanning microscopy
The results suggested that sperm capacitation is also
required in this economically important species
Materials and methods
Animals
Male and female broodstock of L vannamei were
ob-tained from commercial hatcheries They were
main-tained under a 12 h:12 h (L:D) photoperiod and given
commercial pellets and fresh feed Water qualities
(temperature, 28 1C; dissolved oxygen, 5 ppm;
sali-nity, 34 ppt; pH, 7.5; alkalisali-nity, 150 ppm; total
ammo-nia nitrogen,o0.1ppm; and total nitrite, o0.1ppm)
were monitored daily to assure an optimal ment for shrimp health
environ-TEMSpermatophores were collected manually from theterminal ampoule of the vas deferens of male brood-stock and from the thelycum of female broodstock at
4 h after mating The spermatophores were cut openusing a sharp knife to obtain S- and T-sperm.The spermwere ¢xed in 2.5% glutaraldehyde in arti¢cial seawater
at pH 8.0 overnight The rest of the spermatophore wascut into small pieces, approximately 1 1mm, andwashed with 0.2 M cacodylate bu¡er, at 4 1C, threetimes They were post-¢xed in 1% OsO4in 0.2 M caco-dylate bu¡er for 30 min, rinsed three times with bu¡erand dehydrated in ethanol series The samples weresubsequently washed in propylene oxide (PO) for
10 min and then 20 min After that, the tissues wereembedded in a series of mixed resin:PO (1:2,1:1 and 2:1)and pure Epon 812 for 1h each, followed by pure Epon
812 overnight On the following day, they were placed
in embedding moulds in fresh resin at 60 1C overnight.The tissue blocks were sectioned using a Reichert-JungUltracut E Ultra-microtome (Capovani Brothers, Scotia,
NY, USA) Thin sections were doubly stained with asaturated solution of uranyl acetate in methanol, fol-lowed by Reynold’s lead citrate and viewed under FEITecnai-20 TEM (Eindhoven, the Netherlands)
SEMS- and T-sperm were processed in the same manner
as in the TEM procedure from the beginning to thedehydration step From that step, they were critical-point dried and mounted on stubs The samples werecoated with 25 nm of platinum and paradium andviewed under a Hitachi S-2500 SEM (Tokyo, Japan)
In addition to the free sperm, S-sperm in the phore were also processed and visualized under SEM
spermato-Confocal laser scanning microscopyThis procedure was applied to localize tyrosinephophorylation, which is one of the indices of spermcapacitation in mammalian species (Visconti et al.1995) and in shrimp (Vanichviriyakit et al 2004).The S- and T-sperm were ¢xed with 4% paraformal-dehyde, washed twice with 0.1 M phosphate-bu¡eredsaline (PBS) and smeared on histoGrip adhesiveslides (Zymed, San Francisco, CA, USA) The smeared
Trang 20slides were dried at room temperature To
permeabi-lize the plasma membrane of the sperm,Triton X-100
solution (0.1%) was gently dropped onto the slides to
cover the whole smear and incubated for 5 min To
prevent non-speci¢c binding, the slides were
incu-bated with 10% normal bovine serum for 30 min
Subsequently, they were incubated overnight with
monoclonal anti-tyrosine phosphate
immunoglobu-lin G (IgG) (clone P-Try-102: Cell Signaimmunoglobu-ling, Beverly,
MA, USA), followed by a gentle wash with PBS For
£uorescent visualization, the slides were incubated
with Alexa Flours546 goat anti-mouse IgG (H1L)
(Molecular Probe, Invitrogen, CA, USA), 1:100 dilution,
for 30 min and gently washed with PBS to remove
un-bounded antibody Further, the slides were secondarily
¢xed with 4% formaldehyde in PBS for 15 min,
coun-terstained with To-Pro-3 (1:500) for 1h, mounted with
antifade and observed under an Olympus FV-1000
confocal laser scanning microscope (Tokyo, Japan)
Results
Sperm of L vannamei were non-£agellated and thus
non-motile The basic ultrastructures of both S- and
T-sperm were similar, except for details of certaincomponents The features were the same as those de-scribed in Alfaro et al (2007), and their terminologieshave been used here The sperm can be divided intothe spike (S), the hemispherical cap (C), a ¢lamentousmeshwork (FM) and a hemispherical rim of cytoplas-mic particles (CP) (Fig 1a) The hemispherical capcontained acrosome (ACr) and there was a clear zone(CZ) between the ¢lamentous meshwork and the ac-rosome The cap covered about half of the area of thesperm and thus, at both peripheral ends of the cap, adiameter of 4mm could be drawn The nucleus con-tained ¢brillar chromatins, without a nucleolus and
a nuclear membrane The spike, about 3^4mm inlength, contained abundant micro¢laments, align-ing in a parallel fashion (Fig 1b), and on the base ofthe spike, two electron-dense triangular dots wereobserved (Fig 1c)
The most distinctive di¡erence between the structures of the S- and T-sperm was in the nuclearmaterial Chromatin ¢bres in the nucleus of theT-sperm were more de-condensed than that of theS-sperm (Fig 2a vs b) This made the nucleus ofthe T-sperm more vesicular than that of the S-sperm
ultra-In most cases, the ¢lamentous meshwork of the
Figure 1 Transmission electron microscopy of Litopenaeus vannamei sperm taken from the spermatophore (S-sperm) (a)The sperm is composed of an anterior spike (S), a cap region (C) and a posterior main body (MB) The cap region containsthe acrosome (ACr) and the subacrosomal region, which is composed of a clear zone (CZ) and the ¢lamentous meshwork(FM) The posterior main body contains the nucleus (N) and cytoplasmic particles (CP) (b) The spike contains abundantmicro¢laments (c) Two prominent electron-dense triangular dots (arrows) at the base of the spike
Sperm capacitation of L vannamei S Aungsuchawan et al Aquaculture Research, 2011, 42, 188–195
r 2010 The Authors
Trang 21T-sperm was larger and denser than those of the
S-sperm (Fig 2c vs d), and in a few cases, it was
di⁄-cult to distinguish the di¡erence; however, it has
never been observed that the ¢lamentous meshwork
of the S-sperm is larger than that of the T-sperm
In the hemispherical rim of cytoplasmic particles
containing several small and large electron-dense
granules, the T-sperm occasionally contained largevesicles (V), which were not found in the S-sperm(Fig 2e vs f)
Under SEM, a spiral line on the external surface ofthe spike and a basal circular line on the external sur-face of the cap region of S-sperm, especially thosethat were embedded in the spermatophoric matrix,
Figure 2 Transmission electron microscopy of S- and T-sperm of Litopenaeus vannamei showing ultrastructural ences All pictures use the same scale bar (a, b) The nucleus (N): The chromatin ¢bres of the T-sperm are more de-con-densed than those of the S-sperm (c, d) The ¢lamentous meshwork (FM): The granules in the T-sperm were larger thanthose of the S-sperm (e, f) The cytoplasmic particles: Electron-lucent vesicles (V) were occasionally found in the T- spermbut not in the S-sperm
Trang 22di¡er-were observed (Fig 3a) The two lines di¡er-were less
pro-minent in T-sperm (Fig 3b), compared with those of
the S-sperm
The immuno£uorescent localization of tyrosine
phosphorylated proteins on intact free S- and
T-sperm revealed a di¡erence in the area staining
positive S-sperm showed positive £uorescent signals
on their plasma membrane and spike, but T-sperm
was positive on the plasma membrane and
¢lamen-tous meshwork, and on the base of spikes of very few
sperm (Fig 4) A bright spot of red £uorescence was
present at a central part of the ¢lamentous meshwork
of many T-sperm, but not in the S-sperm The positive
signal of tyrosine phosphorylated proteins at the
spike was decreased from S- to T-sperm and it
disap-peared completely when the bright red spot apdisap-peared
on the ¢lamentous meshwork
Discussion
The basic ultrastructure of L vannamei sperm was
similar to that described by Alfaro et al (2007)
Simi-lar to the spike of Penaeus aztecus and S ingentis
sperm, abundant micro¢laments run along its length
Figure 3 (a) Scanning electron microscopic of S-spermembedded in the matrix of spermatophore; (b) T-spermisolated from the thelycum of Litopenaeus vannamei Thebasal line (BL) surrounding the base of the spike and thespiral line (SL) on the spike are less prominent in the T-sperm Both pictures use the same scale bar
Figure 4 Immuno£uorescent localization of tyrosine phosphorylation (P-tyr) on S- and T-sperm of Litopenaeus mei Four pictures in (a), as well as in (b), were taken from the same sperm but di¡ered in the staining selection: the red
vanna-£uorescence signal represents P-tyr, green vanna-£uorescence represents the sperm nucleus or DNA, no-colour picture showsthe phase-contrast one and the fourth picture shows the overlapping of P-tyr and DNA stains The P-tyr signal was found
on the sperm membrane surrounding the cap and the main body of both sperm types; however, in S-sperm, it was tionally found on the spike (arrows, a&c), and in T-sperm, on the ¢lamentous meshwork (FM) A few T-sperm had the P-tyrsignal on the base of the spike (d, arrow), which disappeared when a bright red spot of the P-tyr signal appeared at thecentre of FM (d, arrowhead) All pictures use the same scale bar
addi-Sperm capacitation of L vannamei S Aungsuchawan et al Aquaculture Research, 2011, 42, 188–195
r 2010 The Authors
Trang 23without microtubules or centrioles, and unlike
Macrobrachium rosenbergii sperm, which possessed a
pair of centrioles in closely associated with the base
of the spike (Lynn & Clark 1983) Interestingly, a
pro-minent pair of triangular electron dense spots was
present at the base of spike in both S- and T-sperm
This structure, which was also observed in the TEM
picture by Alfaro et al (2007), may act as an anchor
to ¢x the cap with the spike or may be the centre of
the origin of micro¢laments that run into the length
of the spike, or may control the retraction of spike
during an acrosome reaction Similar to the previous
¢nding (Alfaro et al 2007), the nucleus of L vannamei
has no nuclear membrane, but instead, is surrounded
by an amorphous cytoplasm; this lack of nuclear
en-velop is also shared by S ingentis sperm (Kleve,Yudin
& Clark 1980)
In this study, three basic di¡erences in the
ultra-structure were detected between the S- and the
T-sperm: the nucleus, the ¢lamentous meshwork and
the cytoplasmic particles Alfaro et al (2007)
des-cribed the di¡erence in the ¢lamentous meshwork in
both L vannamei and Litopenaeus stylirostris, which
was larger in T-sperm, and suggested that the change
signi¢ed its role in the acrosome reaction This
di¡er-ence was also detected in our study in that the
¢la-mentous meshwork was not only larger but also
denser in the T-sperm This change was also clearly
observed in the capacitation process in S ingentis
(Wikramanayake et al 1992), where the ¢lamentous
meshwork was termed the granular region and the
increase in this area was termed an extended saucer
In L stylirostris (Alfaro et al 2007) and P monodon
(Pongtippatee, Vanichviriyakit, Chavadej, Plodpai,
Pratoomchart, Sobhon & Withyachumnarnkul
2007), the ¢lamentous meshwork was not observed
in S-sperm, but it appeared in sperm that entered the
early stage of the acrosome reaction in P monodon
(Pongtippatee et al 2007), where it was described as
polymerization of the anterior granulation
The signi¢cant di¡erence between the S- and
T-sperm is in the nucleus T-T-sperm had more
deconden-sation than S-sperm, which was probably a part of
the capacitation process similar to that observed in
P monodon (Vanichviriyakit et al 2004) However, this
di¡erence was not observed by Alfaro et al (2007)
The di¡erence was not distinctive and might become
overlooked, perhaps requiring densitometry to
quan-tify the density of the nuclear material In our TEM
studies, this di¡erence was observable and
consis-tent In S ingentis, it was shown that the nuclear
ma-terial was devoid of basic histones and protamines
and was composed of naked DNA (Kleve et al 1980)
If L vannamei sperm lacks one or both of these cleoproteins, then the more decondensed appearance
nu-in the T-sperm may be due to uncoilnu-ing of its DNA orwater in£ux into the nucleus
At present, we cannot o¡er any explanation for thedi¡erences in cytoplasmic particles, the spiral lineand the basal line between the S- and the T-spermfound in this study Interestingly, the spiral line wasalso observed in S ingentis S-sperm, and was also ab-sent in its T-sperm (Wikramanayake et al 1992).The immuno£uorescent analyses to localize tyro-sine phosphorylation protein revealed striking di¡er-ences between S- and T-sperm The reaction wentfrom the spike to the ¢lamentous meshwork The redspot at the ¢lamentous meshwork could indicate anearly acrosome reaction event similar to the spheri-cal mass formation as shown in P monodon sperm(Pongtippatee et al 2007) Because the spike was stillpresent in the T-sperm (Figs 3 and 4), the absence ofimmuno£uorescence from the spike and its presence
as a red spot on the ¢lamentous meshwork was fore a purely biochemical phenomenon, i.e., tyrosinephosphorylation
there-In rat, di¡erences in tyrosine phosphorylationhave been shown between caput and caudal epididy-mal sperm (Lewis & Aiken 2001) Tyrosine phosphor-ylation was localized in the acrosomal region of thecaput sperm, whereas a majority of caudal sperm ex-hibited a narrow band of tyrosine phosphorylation
at the posterior acrosomal margin These di¡erences
in tyrosine phosphorylation protein expressionbetween caput and caudal epididymal spermatozoaappeared to re£ect the competence of the sperm Inmouse sperm, increased tyrosine phosphorylation inthe £agellum during sperm maturation was detected(Urner, Leppens-Luisier & Sakkas 2001) In the blacktiger shrimp P monodon, tyrosine phosphorelationwas found at a low level in S-sperm A stepwiseincrease in tyrosine phosphorylation markedlyappeared in T-sperm from day 1 to day 2 and its levelwas relatively constant in day 3 (Vanichviriyakit et al.2004) It was not shown, however, where thetyrosine phosphorylation took place, and whetherits location changed within the sperm cell
It is clear in this study that sperm of L vannameishows morphological and biochemical changes afterbeing transferred from the male to the female thely-cum; it is likely that the changes suggest a capacita-tion process of the sperm The questions are howthis process occurs, within 4^6 h, and whetherthe female has any in£uence on these changes
Trang 24In P monodon, it takes at least 3 days for the
capacita-tion process to be complete, and it is conceivable that
the female may play a signi¢cant role in inducing
these changes because the sperm sacs have to be
in-serted into the female thelycum and surrounded by
components within the thelycal £uid But in L
vanna-mei, the sperm sacs are completely outside the female
body as the shrimp has the open-type thelycum
Therefore, the female may not play any part in the
in-duction of the capacitation process, unless there are
minute pores or channel connecting the sperm sacs
with the inner part of the female Another possibility
is that the sperm of L vannamei may have a
sponta-neous capactation after being released from the male
gonopore, i.e., just being exposed to seawater It is
also possible that the capacitation may be inhibited
by certain factor(s) inside the terminal part of the
vas deferens and disinhibition occurs as the sperm
sac is released out from the male gonopore The latter
would explain the potential for in vitro fertilization
using S-sperm observed by Misamore and Browdy
(1997) These two possibilities are related and are
un-der further investigation In light of the ease of sperm
capacitation of L vannamei, it can be stated that
sperm of this shrimp species are quite di¡erent from
those of P monodon, and this phenomenon may be
one of the basic di¡erences between the closed- and
the open-type thelycum species regarding the
phylo-genetic development of the reproductive function of
the shrimp
Acknowledgments
We would like to thank Eleanor F Shepard and
Laur-inda L Smith for their kind support and technical
help The study was funded by The Commission on
Higher Education and Mahidol University grant in
2007
References
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Clark W.H Jr & Gri⁄n F.J (1988) The morphology and
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dec-apod, Sicyonia ingentis Development, Growth and Di¡erentiation 30, 451^462.
Cooper T.G (1998) Interactions between epididymal tions and spermatozoa Journal of Reproduction and Ferti- lity Supplement 53, 119^136.
secre-Dougherty W.J (1990) Ultrastructural observation on nized sperm in developing and fully formed spermato- phores of male shrimp, Penaeus vannamei In: Pathology
mela-in Marmela-ine Science (ed by JO Jenkmela-ins & TC Cheng), pp 387^
394 Academic Press, London, UK.
Gri⁄n F.J., Shigekawa K & Clark W.H Jr (1988) Formation and structure of the acrosomal ¢lament in the sperm of Sicyonia ingentis Journal of Experimental Zoology 246, 94^102.
Kleve M.G., Yudin A.I & Clark W.H (1980) Fine structure of the unistellate sperm of the shrimp, Sicyonia ingentis (Na- tantia) Tissue and Cell 12, 29^45.
Kruevaisayawan H., Vanichviriyakit R., Weerachatyanuku W., Iamsaard S., Withyachumnarnkul B., Basak A., Tan- phaichitr N & Sobhon P (2008) Induction of the acro- some reaction in black tiger shrimp (Penaeus monodon) requires sperm trypsin-like enzyme activity Biology of Re- production 79, 134^141.
Lewis B & Aiken R.J (2001) Impact of epididymal tion on the tyrosine phosphorylation patterns exhibited
matura-by rat spermatozoa Biology of Reproduction 64, 1545^ 1556.
Lynn J.W & Clark W.H Jr (1983) Morphology of sperm-egg interaction in the eggs of the freshwater prawn Macrobra- chium rosenbergii Biological Bulletin 164, 446^458 Misamore M & Browdy C.L (1996) Mating behavior in the white shrimp Penaeus setiferus and Penaeus vannamei: a generalized model for Penaeus mating Journal of Crusta- cean Biology 16, 61^70.
Misamore M & Browdy C.L (1997) Evaluating hybridization potential between Penaeus setiferus and Penaeus vannamei through natural mating, arti¢cial insemination and in vi- tro fertilization Aquaculture 150, 1^10.
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activa-Pongtippatee P., Vanichviriyakit R., Chavadej J., Plodpai P., Pratoomchart B., Sobhon P & Withyachumnarnkul B (2007) Acrosome reaction in the sperm of the black tiger shrimp Penaeus monodon (Decapoda, Penaeidae) Aquacul- ture Research 38, 1635^1644.
Talbot P & Chanmanon P (1980) The structure of sperm from the lobster, Homarus americanus Journal of Ultra- structure Research 70, 275^286.
Urner F., Leppens-Luisier G & Sakkas D (2001) Protein osine phosphorelation in sperm during gamete interac- tion in the mouse: the in£uence of glucose Biology of Reproduction 64, 1350^1357.
tyr-Vacquire V.D (1998) Evolution of gamete recognition teins Science 281, 1995^1998.
pro-Sperm capacitation of L vannamei S Aungsuchawan et al Aquaculture Research, 2011, 42, 188–195
r 2010 The Authors
Trang 25Vanichviriyakit R., Kruevaisayawan H., Weerachatyanukul
W., Tawipreeda P., Withyachumnarnkul B., Pratoomchat
B., Chavadej J & Sobhon P (2004) Molecular modi¢cation
of Penaeus monodon sperm in female thelycum and its
consequent responses Molecular Reproduction and
Devel-opment 69, 356^363.
Visconti P.E., Bailey J.L., Moore G.D., Pan D., Olds-Clarke P &
Kopf G.S (1995) Correlation between the capacitation
state and protein tyrosine phosphorylation Development
121, 1129^1137.
Wikramanayake A.H., Uhlinger K.R., Gri⁄n F.J & ClarkW.H.
Jr (1992) Sperm of the shrimp Sicyonia ingentis undergo a bi-phasic capacitation accompanied by morphological changes Development, Growth and Di¡erentiation 34, 347^355.
Yanagimachi R (1994) Mammalian fertilization The siology of Reproduction (ed by E Knobil & JD Neill), pp 189^317 Raven Press, NewYork, NY, USA.
Trang 26Phy-Evaluating the use of Lactobacillus acidophilus as
a biocontrol agent against common pathogenic
bacteria and the effects on the haematology
parameters and histopathology in African catfish
Mohammed Abdullah Al-Dohail1,2, Roshada Hashim1& Mohammed Aliyu-Paiko1
1 Laboratory of Aquafeed and Feeding Development, Aquaculture Research Group, School of Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia
2 Faculty of Environmental Sciences and Marine Biology, Hadhramout University of Science and Technology, Mukalla,Yemen
Correspondence: M A Al-Dohail, Laboratory of Aquafeed and Feeding Development, Aquaculture Research Group, School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia E-mail: aldohial@yahoo.com
Abstract
This study was carried out to evaluate the use of
Lac-tobacillus acidophilus as a biocontrol agent against
some common ¢sh pathogenic bacteria
(Staphylococ-cus xylosus, Aeromonas hydrophila gr.2 and
Streptococ-cus agalactiae) in African cat¢sh, Clarias gariepinus
Eight treatments were designed inclusive of 10 C
gar-iepinus juveniles (mean weight 190 g) per tank, each
in triplicate Four groups of ¢sh were fed a diet
supple-mented with L acidophilus, comprising about 3.01
107colony-forming units per gram of diet (the
probio-tics diet), while the other four groups were fed a diet
not supplemented with probiotics (the non-probiotics
diet) In the ¢rst group, ¢sh were injected with 1mL
physiological saline and fed the non-probiotic
diet (non-probiotic control); in the second, third
and fourth groups, ¢sh were injected with 1mL each
of S xylosus, A hydrophila gr.2 and S agalactiae,
respectively, and were all fed the non-probiotic diet
(designated as non-probiotic treatments; NPsx, NPah
and NPsa respectively) In the ¢fth group, ¢sh were
injected with 1mL physiological saline but fed the
probiotic diet (probiotic control), while ¢sh in the
sixth, seventh and eighth groups were each injected
with 1mL of S xylosus, A hydrophila gr.2 and S
aga-lactiae, respectively, and were all fed the probiotic diet
(and designated as probiotic treatments; Psx, Pah and
Psa respectively) Blood samples were collected for
haematology analysis, while samples of the liver and
kidney were examined for pathohistology after 7 and
21 days of infection The results showed that the matology parameters, packed cell volume, haemoglo-bin, erythrocyte sedimentation rate, red blood cell,white blood cell, total serum protein, Mg21, Ca21,
hae-Cl, glucose, cholesterol and total immunoglobulinconcentrations and the pathohistology of the liverand kidney were better in the challenged ¢sh (in-fected) maintained on the probiotic diet than those
in the groups fed the non-probiotic diet It is cluded, based on these results, that L acidophilus isuseful as a probiotic agent in C gariepinus againstthese pathogenic bacteria (S xylosus, A hydrophilagr.2 and S agalactiae)
con-Keywords: Probiotic, Lactobacillus acidophilus,pathogenic bacteria, blood, histopathology, Clariasgariepinus, ¢sh diseases
IntroductionFish diseases are widely distributed worldwide andare considered to be serious problems in aquaculture(Lim & Webster 2001) The advancement in intensiveaquaculture is usually accompanied by several dis-ease problems, often due to opportunistic pathogens,
as evident from general aquaculture (Abraham, dal & Babu 2008) This is because at higher stockingdensities, high feed inputs and other organic loadsAquaculture Research, 2011, 42, 196^209 doi:10.1111/j.1365-2109.2010.02606.x
Mon-r 2010 Universiti Sains Malaysia
Trang 27stimulate the selective proliferation of opportunistic
bacteria, leading to ¢sh diseases (Austin, Stuckey,
Ro-bertson, E¡endi & Gri⁄th 1995) In freshwater ¢sh
culture, disease outbreaks due to pathogenic bacteria
are very common, especially in the production of
cat¢sh (Lim & Webster 2001) Among the common
bacteria pathogens, Staphylococcus xylosus,
Aeromo-nas hydrophila gr.2 and Streptococcus agalactiae are
known to seriously infect ¢sh in culture systems,
sometimes causing heavy mortalities (Schperclaus,
Kulow & Schreckenbach 1992)
Blood parameters of ¢sh provide accurate
indica-tions of any changes occurring in the organism as a
result of injuries to organs or tissues related to
infec-tious diseases, similar to those of warm-blooded
ani-mals (Adeyemo 2007) Ranzani-paiva, Ishikawa, Das
Eiras and Felizardo (2000) also reported that the
blood parameters of ¢sh are important tools useful
for detecting abnormalities related to temperature
and dissolved oxygen variations, diseases and other
factors Thrall (2004) and Pimpao, Zampronio and
Silva de Assis (2007) reported that the
haematologi-cal and biochemihaematologi-cal parameters of ¢sh can be used
to evaluate the health condition of the organism
Furthermore, haematological variations are useful
for the clinical diagnosis of ¢sh physiology, to
deter-mine the e¡ects of external stressors and toxic
sub-stances (Cech Jr, Bartholow, Young & Hopkins 1996;
Bonga 1997) For these reasons, many authors have
used haematological parameters to report the e¡ects
of environmental pollutants and pathogenic diseases
causing changes in blood characteristics of ¢sh such
as Clarias gariepinus (Onusiriuka & Ufodike 2000;
Ezeri 2001; Gabriel, Alagoa & Allison 2001)
Probiotics can be de¢ned as living microbial cells
that exert bene¢cial e¡ects on the health and
well-being of their host (Salminen, Ouwehand, Benno &
Lee 1999) The use of probiotic bacteria in
aquacul-ture to enhance the growth performance and to also
improve the quality of water in which ¢sh are
cultured has received considerable attention
(Verschuere, Rombaut, Sorgeloos & Verstraete 2000)
Generally, probiotics are associated with bacteria,
which produce bacteriocins and other compounds
that may inhibit the growth of pathogenic bacteria
(Klaenhammer & Russell 2000) Lactic acid bacteria
have been identi¢ed to possess probiotic properties
and some bene¢cial advantages, as it enhances the
digestive process in its host and could therefore be
useful due to its bene¢cial e¡ects on the health of its
consumers (Schillinger 1999) Strm and Ring
(1993), Stosik and Szenfeld (1996), Rengpipat,
Ruk-pratanporn, Piyatiratitivorakul and Menasaveta(2000) and Vine (2004) all concluded that probioticshave the ability to improve ¢sh health and preventbacterial diseases in ¢sh Consequently, the use ofprobiotics as a new technique to confer protection
in the host ¢sh against pathogenic bacteria in themost economic and environment-friendly manner iscertainly worth evaluating in aquaculture
Information regarding the e¡ects of probiotics ondiseases of C gariepinus is limited; therefore, thisstudy was conducted to evaluate the use of Lactoba-cillus acidophilus as a biocontrol agent against somecommon pathogenic bacteria (S xylosus, A hydrophi-
la gr.2 and S agalactiae) and its e¡ects on the tological parameters and histopathology of Africancat¢sh C gariepinus juveniles
haema-Materials and methods
L acidophilusLactobacillus acidophilus was isolated from a knownsource (Vitagenryoghurt drink, produced by Malay-sia Milk Sdn Bhd Penang, Malaysia), identi¢ed usingthe Biolog plate technique (Choi & Dobbs 1999) andsub-cultured on de Man, Rogosa and Sharpe (MRS)media in the microbiology laboratory, UniversitiSains Malaysia One millilitre of Vitagen yoghurtdrink was added to 99 mL of MRS media broth andincubated at 37 1C for 36 h About 1.0 mL of this mix-ture was serially diluted in 10-fold (10 1^10 10)physiological saline as a diluent to determine the vi-able cell count of L acidophilus in a 10-fold dilution ofthe solution (Koch 1994) Subsequently, 0.1mL ofeach dilution (10 1^10 10) was taken and evenlyspread on MRS agar plates and incubated at 37 1Cfor 48 h to produce L acidophilus colonies The num-bers of colonies produced on the plates were countedmanually
Pathogenic bacteriaThree common pathogenic bacteria of fresh water
¢sh, S xylosus, A hydrophila gr.2 and S agalactiae,were obtained from the National Fish Health Re-search Center Penang, Malaysia The bacteria werecultivated at 30 1C for 24 h in the Microbiology La-boratory, Universiti Sains Malaysia, and maintained
on nutrient agar slants kept in a refrigerator at 4 1Cuntil used The viable cell count of pathogenic bacter-
ia on the plates was also performed manually
Trang 28Experimental ¢sh and husbandry conditions
This experiment was conducted in the Diseases
La-boratory at the Aquaculture Research Complex of
Universiti Sains Malaysia, in May 2008 Two groups
of juvenile African cat¢sh, C gariepinus, were reared
for12 weeks on a probiotic diet containing 3.01 107
colony-forming units (CFU) per gram and a
non-pro-biotic supplemented diet respectively (refer to
Al-Do-hail, Hashim & Aliyu-Paiko 2009) From these, 120
homogenous juveniles (of mean weight 190 5 g)
were collected from each group and used for the
pre-sent trial Fish were acclimated to laboratory
condi-tions for 2 weeks in a £ow-through water system
before the commencement of this study, feeding on
the same diets as those in the previous experiment
Ten juveniles were randomly distributed into each of
24 glass aquariums (measuring 60 35 30 cm,
L W H) Three replicate groups of ¢sh were
maintained on each assigned diet and ¢sh were
reared under a natural photoperiod of approximately
12/12 h light/dark cycles Each glass aquarium was
connected to the £ow-through water system, which
was continuously supplied with de-chlorinated tap
water, with the £ow rate set at 1.5 L min 1and also
aerated continuously Water quality parameters
were monitored weekly, and the mean values
re-corded were as follows: ammonia (mg L 1) 0.9
0.10, nitrite (mg L 1) 0.05 0.02, nitrate (mg L 1)
3.07 0.29, temperature ( 1C) 29.47 0.50 and pH
6.52 0.11 At the end of the ¢rst and third weeks of
the trial, three ¢sh from each aquarium were
ran-domly removed for haematology and patho-histology
analysis
Preparation of experimental diets
Two practical diets were formulated to contain 35%
crude protein each, using soybean and ¢sh meals
as the protein sources The ¢rst diet was
supplemen-ted with L acidophilus (probiotic diet), containing
3.01 107CFU g 1, while the second diet was
not supplemented with probiotic bacteria
(non-pro-biotic diet) The lipid level in the two diets was 10%,
comprising a 1:1 ratio of ¢sh oil to vegetable oil Under
sterile conditions, the probiotic diet (supplemented
with L acidophilus) was prepared by slowly spraying
250 mL of media broth containing about 3.7 109
CFU mL 1of live L acidophilus (grown in MRS media
and1% w/v of skimmed milk as a cryoprotective
solu-tion) into a clean plate containing the dry pellets
while slowly mixing for 3^5 min to prepare1kg of
ex-perimental feedstu¡ The pellets were dried in anoven at 30 1C for 18 h, packed and stored in a freezer
at 20 until used The ¢nal concentration of L ophilus in the probiotic diet was 3.01 107
acid-g 1,whereas the formulation of diets used in the feedingtrial is as described previously in Al-Dohail et al.(2009)
Feeding trialThe feeding trial was performed for eight treatments
in a completely randomized design, each in triplicate.Juvenile C gariepinus were hand fed the assigneddiets to visual satiation three times daily at 08:00,14:00 and 18:00 hours respectively The feeding trialand treatments used for this study are as presented inTable 1 All groups were maintained on the assigneddiets for 3 weeks after infection with the designatedpathogenic bacteria
Challenging ¢sh with pathogensThree di¡erent pathogenic bacteria, S xylosus, A hy-drophila gr.2 and S agalactiae, were used in the pre-sent study Under sterile conditions, the ¢rst and
¢fth groups of ¢sh were injected with physiologicalsaline to serve as the controls However, the secondand sixth groups were infected with S xylosus, thethird and seventh groups were infected with A hy-drophila gr.2 and the fourth and eighth groups wereinfected with S agalactiae
Fish in all groups (except the ¢rst and the ¢fth) wereinjected intraperitoneally with1mL of the pathogenicbacteria, containing 2 106
CFU mL 1 pended in physiological saline Fish in the ¢rst and
sus-¢fth groups were injected intraperitoneally with1mL of physiological saline, using 1mL capacity syr-inges (TERUMOssyringes and needles, Somerset, NJ,USA) The amount of 2 106CFU mL 1of patho-genic bacteria injected into C gariepinus juvenilewas determined to be the infectious dose, in a sepa-rate preliminary study before the current feedingtrial (Al-Dohail, Hashim & Aliyu-Paiko 2008)
In another preliminary study, the in vitro inhibition
of the three pathogenic bacteria by L acidophilus wasassessed and found to be 15.50 mm for A hydrophilagr.2 and 20.33 mm for S xylosus, while the inhibitionzone for S agalactiae was 21.17 mm (Al-Dohail et al.2008)
L acidophilus as a probiotic in African cat¢sh M A Al-Dohail et al Aquaculture Research, 2011, 42, 196–209
r 2010 Universiti Sains Malaysia
Trang 29Haematological parameters
For the determination of each of the following
hae-matology parameters, blood was collected from the
caudal vein of three randomly selected ¢sh per tank
All the procedures used are as described previously
(Al-Dohail et al 2009)
Haematocrit
Haematocrit was measured according to the
proce-dures of Schperclaus et al (1992) as described
pre-viously The haematocrit value was calculated based
on the following formula:
PCV¼ Height of packed red cells ðmmÞ=
Height of packed red cells and plasmaðmmÞ
100
where PCV is the packed cell volume
Haemoglobin (Hb) concentration
The Hb content was determined according to the
Cyan-methaemoglobin method (Blaxhall & Daisley
1973) as described previously The Hb concentration
was determined as g dL 1of blood from a standard
graph, using the following equations:
Haemoglobin concentration (g dL 1) 5 Absorbance
of sample/Absorbance of standard concentration of
standard
Mean corpuscular haemoglobin concentration(MCHC) g dL 15Haemoglobin g %/Haematocrit
volume % 100Mean corpuscular haemoglobin (MCH)
pg cell 15Haemoglobin g %/erythrocyte
(millions mm 3) 10Mean corpuscular volume (MCV)mm 35 Haema-tocrit volume/erythrocyte (millions per mm 3) 10
Erythrocyte sedimentation rate (ESR)The ESR was calculated according to the method ofWestergren (Britton1963) as described previously Er-ythrocyte sedimentation rates were expressed as thedistance moved in mm per hour of corpuscles in theblood
Total red blood cell count (erythrocyte or RBC
106mm 3)The total RBC counts were carried out based on theprocedure of EWOS Technology Center (2000) andJohnson, Timmons and Hall (2002) as described pre-viously The RBC count was recorded as millions ofcells per cubic millimetre Total RBC was calculatedaccording to the following formula:
RBCðmm3Þ ¼ ðN 5 10 200Þwhere N is the number of cells in ¢ve squares,5 repre-sents multiplication factor to yield the number ofcells in 1mm2, 10 represents multiplication factor tobring the depth of the chamber from 0.1 to 1mm and
200 represents the dilution factor
Total white blood cells (WBCs)
A leucocyte count (LC) was performed according toStoskopf (1993) and Johnson et al (2002) as describedpreviously The LC was expressed as thousands ofcells in mm3
Leucocyteðmm3Þ ¼ LC 500where LC represents number of cells in 4 mm2squares and 500 represents dilution and volume cor-rection factor
Serum mineral content (Mg21, Ca21and Cl)Serum minerals (magnesium, calcium and chloride)were measured as described previously (Al-Dohail
et al 2009) The serum mineral values were lated and recorded as (mg dL 1) or (mmole L 1)according to the following formula:
calcu-Table 1 De¢nition of ¢sh groups and treatments used for
the feeding trial
Group Treatment Feeding trial descriptions
1 NPc Fish injected with 1 mL of physiological saline
and fed the non-probiotic diet (control)
2 NPsx Fish injected with 1 mL of Staphylococcus
xylosus and fed the non-probiotic diet
3 NPah Fish infected with 1 mL of Aeromonas
hydrophila gr.2 and fed the non-probiotic diet
4 NPsa Fish infected with 1 mL of Streptocossus
agalactiae and fed the non-probiotic diet
5 Pc Fish injected with 1 mL of physiological saline
and fed the probiotic diet (control)
6 Psx Fish infected with 1 mL of S xylosus and fed
the probiotic diet
7 Pah Fish infected with 1 mL of A hydrophila gr.2
and fed the probiotic diet
8 Psa Fish infected with 1 mL of S agalactiae and
fed the probiotic diet
Fish in all the treatments were injected with pathogenic
bacter-ia or physiological saline after a 3-month rearing period (initbacter-ial
time of starting the experiment).
Trang 30Serum minerals (mg dL 1) or (mmole L 1) 5
(Absorbance of sample/Absorbance of standard)
concentration of standard
Cholesterol concentration
The blood cholesterol level was calculated as
described previously (Al-Dohail et al 2009)
Cholesterol (mg dL 1) 5 Absorbance of sample/
Absorbance of standard) concentration of standard
(mg dL 1)
Glucose concentration
Blood glucose was measured as described previously
(Al-Dohail et al 2009) and calculated according to
the following formula:
Glucoseðmg dL1Þ ¼ ðDA sample=DA standardÞ
concentration of standard ðmg dL1Þ
Total protein content
Total serum protein was measured as already
de-scribed (Al-Dohail et al 2009) The total protein value
was expressed as (g dL 1), calculated according to
the following formula:
Proteinðg dL1Þ ¼ ðD Absorbance sample=
D Absorbance standardÞ
concentration of standard ðg dL1Þ
Total immunoglobulin (Ig) concentration
The total Ig concentration was measured according
to the method of Siwicki and Anderson (1993) and
Amar, Kiron, Satoh, Okamoto and Watanabe (2000)
as also described previously The total Ig value was
expressed as (mg mL 1), calculated according to the
following formula:
Total Igðmg mL1Þ ¼ total protein in serum sample
total protein treated with PEG
Histopathology assessment
Samples of the liver and kidney were collected for a
pathohistological examination and ¢xed in 10%
for-malin Afterwards, the samples were dehydrated in a
series of ethanol solutions: 50%,70%, 80%, 90%, 95%
and 100% for 1, 1, 2, 2, 1.5 and 16 hours respectively
Each of the samples was then transferred into a
xy-lene solution for another 30 min and then embedded
in a solution of xylene wax for 1h A 5-mm-thick
sec-tion was cut using a rotary microtome (A.O Spencer,model Reicheit-Jurg 820, Leica, Germany) and quicklytransferred onto a slide and kept in an oven at 40^
50 1C for 24 h Each sample was observed under acompound light microscope attached with a camera
Statistical analysisThe data for haematology parameters and Ig concen-tration were analysed using one-way analysis of var-iance (ANOVA), and the di¡erences in the meansbetween groups were tested for signi¢cance at the95% con¢dence level P valueso0.05 were consid-ered to be signi¢cant, using Duncan’s multiple rangetest All statistical analyses were performed using the
SPSSsoftware package, version 11.5
ResultsSeven days post infection with pathogenicbacteria
Fish survival rate in the controls and infected groupsfed the probiotic diet was100%, whereas in the groupsinfected with S xylosus, A hydrophila gr.2 and S aga-lactiae fed the non-probiotic diet, ¢sh survival re-corded was 83.3%, 76.6% and 80.0% respectively Theresults for the monitored haematology parameters of
C gariepinus juveniles fed probiotic or non-probioticdiets after 1 week of infection with the three patho-genic bacteria are presented in Table 2 Signi¢cant(Po0.05) di¡erences were observed in the haematol-ogy parameters between the infected groups fed theprobiotic diet and those maintained on the non-pro-biotic diet Overall, the haematocrit (PCV), Hb, RBC,cholesterol, total serum protein, calcium (Ca12), mag-nesium (Mg12), chloride (Cl) and total Ig values fol-lowed a similar pattern; the values were signi¢cantlyhigher in the infected ¢sh maintained on the probioticdiet than in those fed the non-probiotic diet Conver-sely, the ESR, WBC and serum glucose concentrationwere signi¢cantly higher in the infected ¢sh fed thenon-probiotic diet than in those infected with patho-genic bacteria and maintained on the probiotic dietand the two controls respectively
The ESR values in the ¢sh groups infected with S.agalactiae and S xylosus and maintained on the pro-biotic diet did not signi¢cantly di¡er from both theprobiotic and the non-probiotic controls, whereasthe non-probiotic treatment di¡ered signi¢cantlyfrom both controls in their ESR values
L acidophilus as a probiotic in African cat¢sh M A Al-Dohail et al Aquaculture Research, 2011, 42, 196–209
r 2010 Universiti Sains Malaysia
Trang 31The Hb and PCV values of the probiotic treatments
infected with S xylosus and A hydrophila gr.2 were
similar to those of the non-probiotic control Fish
in-fected with S agalactiae and fed the probiotic diet, on
the other hand, showed Hb and PCV values similar to
those of the probiotic control
The RBC values of the ¢sh infected with S
agalac-tiae and fed the probiotic diet did not di¡er
signi¢-cantly from the non-probiotic control, whereas all
the ¢sh infected with all pathogenic bacteria and
maintained on the non-probiotic diet showed
signi¢-cant di¡erences in RBC between both probiotic and
non-probiotic controls respectively
For the RBC indices, MCHC, MCH and MCV in the
infected groups of ¢sh maintained on the probiotic
diet were signi¢cantly higher than the groups fed the
non-probiotic diet, even though both controls were
si-milar However, MCH and MCV in ¢sh fed either the
probiotic or the non-probiotic diets were signi¢cantlyhigher than both the controls respectively
The WBC level was signi¢cantly higher in theblood of all the infected ¢sh fed both probiotic andnon-probiotic diets than in both the controls, respec-tively, whereas the levels in all the infected ¢sh fed thenon-probiotic diet were signi¢cantly and consistentlyhigher than those in their counterpart groups main-tained on the probiotic diet respectively
The serum total Ig concentration was signi¢cantlyhigher (Po0.05) in the infected ¢sh fed the probioticdiet than in the counterpart groups maintained onthe non-probiotic diet, although infected ¢sh fed theprobiotic diet showed no signi¢cant di¡erences fromthose recorded for the probiotic control group, norwas there any noticeable di¡erence in the Ig levels be-tween the non-probiotic treatments and the non-pro-biotic control
Table 2 Haematological parameters of Clarias gariepinus at 7 days post infection and fed non-probiotic and probiotic diets
Results are expressed as mean SD.
Mean values in the same row with di¡erent superscripts show signi¢cant di¡erence (P o0.05).
NPc, non-probiotic control; NPsx, ¢sh infected with Staphylococcus xylosus and fed the non-probiotic diet; Npah, ¢sh infected with Aeromonas hydrophila gr.2 and fed the non-probiotic diet; Npsa, ¢sh infected with Streptocossus agalactiae and fed the non-probiotic diet;
Pc, probiotic control; Psx, ¢sh infected with Staphylococcus xylosus and fed the probiotic diet; Pah, ¢sh infected with Aeromonas phila gr.2 and fed the probiotic diet; Psa, ¢sh infected with Streptocossus agalactiae and fed the probiotic diet; WBC, white blood cell, RBC, red blood cell; MCHC, mean corpuscular haemoglobin concentration; MCH, mean corpuscular haemoglobin; MCV, mean corpuscular volume.
Trang 32hydro-The serum blood protein, cholesterol, Mg12and
Cllevels in all the infected ¢sh groups fed the
pro-biotic diet were signi¢cantly di¡erent from those of
their counterparts maintained on the non-probiotic
diet respectively The serum Ca12concentration was
signi¢cantly lower in infected ¢sh groups fed the
non-probiotic diet than in the other groups, with no
signi¢cant di¡erences recorded between the infected
¢sh groups maintained on the probiotic diet and the
non-probiotic control group
The serum glucose level was signi¢cantly higher in
infected groups fed the non-probiotic diet than in the
groups fed the probiotic diet and both the control
groups respectively
Twenty-one days post infection with
pathogenic bacteria
The results of the haematology parameters
moni-tored for C gariepinus ¢ngerlings fed probiotic or
non-probiotic diets on the third week after infecting
with three di¡erent pathogenic bacteria are
pre-sented in Table 3 At 21 days post infection withpathogens, the levels of Hb, PCV, RBC, total serumprotein, Ca12, Mg12, Clserum glucose and Ig con-centrations in all treatments followed a pattern simi-lar to that recorded at 7 days post infection, althoughthe di¡erences in these parameters between the in-fected groups maintained on probiotic diets com-pared with the groups fed the non-probiotic dietswere clearer and more pronounced after 21 days
HistopathologyMonitoring of the histopathologic e¡ects in the inter-nal organs (liver and kidney) of C gariepinus revealedsevere damage to these organs in the infected groupsmaintained on the non-probiotic diet when the speci-mens were collected at 7 and 21 days post infectionwith the three pathogenic bacteria (Fig 1; images7
NPsx,7NPah and7NPsa and Fig 2; images21NPsx,21
NPah and21NPsa), compared with the very mild fects observed in the infected ¢sh fed the probioticdiet during the same period (Fig 1; images 7Psx,
ef-Table 3 Haematological parameters of clarias gariepinus at 21 days post infection and fed non-probiotic and probiotic diets
Results are expressed as mean SD.
Mean values in the same row with di¡erent superscripts show signi¢cant di¡erence (P o0.05).
See Table 1 for details of abbreviations.
WBC, white blood cell, RBC, red blood cell; MCHC, mean corpuscular haemoglobin concentration; MCH, mean corpuscular bin; MCV, mean corpuscular volume.
haemoglo-L acidophilus as a probiotic in African cat¢sh M A Al-Dohail et al Aquaculture Research, 2011, 42, 196–209
r 2010 Universiti Sains Malaysia
Trang 33B A
Figure 1 (a) Livers were stained with haematoxylin and eosin, and shown at 40., bar 200 mm; sampling collection at
7 days post infection with pathogenic bacteria of images:7NPc,7NPsx,7NPah,7NPsa,7Pc,7Psx,7Pah and7Psa Liver ture of non-probiotic control (7NPc) Note (A) blood vessel and (B) hepatocyte NPsx: ¢sh infected with Staphylococcusxylosus and fed the non-probiotic diet Note the acute hepatitis area (arrowheads).7NPah: ¢sh infected with Aeromonoshydrophila and fed the non-probiotic diet Note the acute necrosis area (arrowheads).7NPsa: ¢sh infected with Streptococ-cus agalactiae and fed the non-probiotic diet Note acute hepatitis with the necrosis area (arrowheads).7Pc: probiotic con-trol: (A) blood vessel and (B) hepatocyte.7Psx: ¢sh infected with S xylosus and fed the probiotic diet Note the mild hepatitisarea (arrowheads).7Pah: ¢sh infected with A hydrophila and fed the probiotic diet Note the mild necrosis area (arrow-heads).7Psa: ¢sh infected with S agalactiae and fed the probiotic diet Note mild hepatitis with the necrosis area (arrow-heads) (b) Livers were stained with haematoxylin and eosin, and shown at 40, bar 200 mm; sampling collection at 21days post infection with pathogenic bacteria of images:21NPc,21NPsx,21NPah,21NPsa,21Pc,21Psx,21Pah and21Psa Liverstructure of non-probiotic control (21NPc) Note (A) blood vessel and (B) hepatocyte.21NPsx: ¢sh infected with Staphylococ-cus xylosus and fed the non-probiotic diet Note the acute hepatitis area (arrowheads).21NPah: ¢sh infected with Aeromo-nos hydrophila and fed the non-probiotic diet Note very acute necrosis, swelling, vacuolation and the degeneration area(arrowheads).21NPsa: ¢sh infected with Streptococcus agalactiae and fed the non-probiotic diet Note very acute hepatitis,swelling and the degeneration area (arrowheads).21Pc: probiotic control (A) blood vessel and (B) hepatocyte.21Psx: ¢shinfected with S xylosus and fed the probiotic diet Note the mild hepatitis area (arrowheads).21Pah: ¢sh infected with A.hydrophila and fed the probiotic diet Note the mild necrosis area (arrowheads).21Psa: ¢sh infected with S agalactiae andfed the probiotic diet showed a normal area in hepatocyte (arrowheads)
Trang 34Haematological characteristics have been studied in
many ¢sh species to determine the normal range,
and any variations from these ranges were indicative
of pathophysiology (Ranzani-paiva et al 2000) In the
present study, at 7 days post infection, all the blood
parameters, Ig, Mg21, Ca21, Cl, total serum protein
and cholesterol levels in the infected ¢sh fed the
pro-biotic diet were signi¢cantly higher (Po0.05) than
those in the infected groups maintained on the
non-probiotic diet, whereas ESR, WBC and glucose were
signi¢cantly higher in the infected ¢sh fed the
non-probiotic diet than in the other groups These
obser-vations, in general, could be because infected ¢sh fed
probiotic diets had a better capacity (and also lowerstressor levels) to resist infection compared with theother infected ¢sh fed the non-probiotic diets Thehigher ESR level noted in the present study could berelated to damage to RBCs, due to the poison released
by pathogenic bacteria during infection This tion is supported by Haney, Hursh, Mix & Winton(1992), Harikrishnan, Rani and Balasundaram(2003) and Scott and Rogers (1981), who all claimedthat the RBC swells during bacterial infection Itcould also be that the probiotic treatments weremuch better at resisting infection than the othergroups, due to induced immune response when Lac-tobacillus was used as a probiotic, as reported by Pa-nigrahia, Kirona, Puangkaewa, Kobayashib, Satohaand Sugitac (2005) and Nikoskelainena, Ouwehanda,Bylundb, Salminena and Lilius (2003) Results simi-lar to those reported in the current study have beenobtained with numerous other ¢sh species, where
L acidophilus as a probiotic in African cat¢sh M A Al-Dohail et al Aquaculture Research, 2011, 42, 196–209
r 2010 Universiti Sains Malaysia
Trang 35the ESR values were shown to increase with bacterial
infection or toxins (Gabriel, Ezeri and Opabunmi
(2004)
For RBC indices, the mean range reported for
nor-mal MCHC is 32^36 g dL 1, 27^31pg cell 1for MCH
and 80^100mm 3 for MCV (Johnson et al 2002;
George & Parker 2003) The results obtained in the
present study were higher than these values and
were better in the probiotic treatments infected with
all the three pathogenic bacteria than those fed the
non-probiotic diet, possibly due to lower swelling
and hence reduced RBC damage (Scott & Rogers
1981; Haney et al 1992; Harikrishnan et al 2003) in
the probiotic-treated groups
Moreover, serum protein, cholesterol, Mg12, Ca12
and Clwere signi¢cantly decreased in infected ¢sh
fed either probiotics or non-probiotic diets in
compar-ison with both the probiotic and the non probiotic
controls In many ¢sh species, the blood chemistry
parameters were altered throughout the period of
se-vere bacterial infection as reported when rainbow
trout was infected with a combination of Aeromonas
and Streptococcus (Barham, Smith & Schoonbee
1980) and in tilapia infected with Edwardsiella tarda(Benli & Yildiz 2004) In addition, decreased choles-terol, Mg12, Ca12and Clcould be related to the bio-chemical requirements of organic and inorganiccompounds for growth and reproduction or loss ofelectrolytes (Benli & Yildiz 2004) due to the perme-ability of renal tubules (Barham et al 1980)
WBC in the infected ¢sh fed the non-probiotic dietwas signi¢cantly higher than that in the infected ¢shmaintained on the probiotic diet possibly due tothe induction of the non-speci¢c defence system(Panigrahia et al 2005) and/or increased phagocyto-sis and cytotoxic activity (Salinas, Cuesta, Esteban &Meseguer 2005; Picchietti, Mazzini, Taddei, Renna,Fausto, Mulero, Carnevali, Cresci & Abelli 2007) inthe infected ¢sh maintained on the probiotic diet Ser-
um glucose was signi¢cantly the highest in thegroups of ¢sh fed the non-probiotic diet than the othergroups as reported when tilapia was infected with Ed-wardsiella tarda (Benli & Yildiz 2004) andVibrio vulni-
¢cus or Streptococcus iniae (Chen, Wooster & Bowser2004) as well as in Anguilla anguilla infected with A.hydrophilus (Yildiz, Bekans, Benli & Akan 2005)
Figure 2 (a) Kidneys were stained with haematoxylin and eosin, and shown at 200, scale bar 50 mm; sampling tion at 7 days post infection with pathogenic bacteria of images:7NPc,7NPsx,7NPah,7NPsa,7Pc,7Psx,7Pah and7Psa.Kidney structure of non-probiotic control (7NPc) Note (A) glomerulus, (B) proximal convoluted tubules and (C) Bowman’scapsule.7NPsx: ¢sh infected with Staphylococcus xylosus and fed the non-probiotic diet Note glomerular capillaries areextremely dilated and Bowman’s capsule space is blocked (arrowheads).7NPah: ¢sh infected with Aeromonos hydrophilaand fed the non-probiotic diet Note glomerular capillaries are extremely dilated and Bowman’s capsule space is blocked(arrowheads) and glomerular capillaries are degenerated (star).7NPsa: ¢sh infected with Streptococcus agalactiae and fedthe non-probiotic diet Note glomerular capillaries are extremely dilated and Bowman’s capsule space is blocked (arrow-heads); necrosis in the epithelia cell of proximal convoluted tubules (star).7Pc: probiotic control Note (a) glomerulus, (b)rental tubules and (c) Bowman’s capsule.7Psx: ¢sh infected with S xylosus and fed the probiotic diet Note that the glo-merular capillaries are dilated and Bowman’s capsule space is blocked in some areas (arrowheads).7Pah: ¢sh infected with
collec-A hydrophila and fed the probiotic diet Note that the glomerular capillaries are dilated and Bowman’s capsule space isblocked at one part (arrowheads), and slight mesangial cell proliferation (star).7Psa: ¢sh infected with S agalactiae and fedthe probiotic diet Note that the glomerular capillaries are dilated and Bowman’s capsule space is blocked at one side (ar-rowheads) (b) Kidneys were stained with haematoxylin and eosin, and shown at 200., scale bar 50 mm; samplingcollection 21 days post infection with pathogenic bacteria of images:21NPc,21NPsx,21NPah,21NPsa,21Pc,21Psx,21Pah and
21Psa Kidney structure of non-probiotic control (21NPc) Note (A) glomerulus, (B) proximal convoluted tubules and (C)Bowman’s capsule.21NPsx: ¢sh infected with S xylosus and fed the non-probiotic diet Note glomerular capillaries areextremely dilated and Bowman’s capsule space is blocked (arrowheads), and mesangial cell proliferation and degenerated(star).21NPah: ¢sh infected with A hydrophila and fed the non-probiotic diet Note very acute necrosis and the degenera-tion area of architecture kidney structure (circle).21NPsa: ¢sh infected with S agalactiae and fed the non-probiotic diet.Note the expansion of space inside the Bowman’s capsule, shrinking of convoluted tubules (arrowheads) and contraction
of the glomerulus (star).21Pc: probiotic control Note (A) glomerulus, (B) rental tubules and (C) Bowman’s capsule.21Psx:
¢sh infected with S xylosus and fed the probiotic diet Note glomerulusis appears mostly normal (arrowheads), and withsome necrosis in the epithelia cell of proximal convoluted tubules (star).21Pah: ¢sh infected with A hydrophila and fed theprobiotic diet Note the expansion of space inside the Bowman’s capsule with contraction of the glomerulus (arrowheads),thickening of Bowman’s capsule basement membrane (arrowheads-A) and degenerated glomerulus (star).21Psa: ¢sh in-fected with S agalactiae and fed the probiotic diet Note that the glomerulus capillaries are very slight dilated and Bow-man’s capsule space is not blocked (arrowheads)
Trang 36The increased glucose level in the non-probiotic
treatments observed in the present study could be
be-cause ¢sh require more energy to support all
meta-bolic processes under abnormal conditions
However, during emergencies such as the
stimula-tion of the immune defence system during bacterial
infection, the ¢sh could have also converted several
lipids into glucose, and this may explain the
de-creased cholesterol level observed in this study The
increase in blood glucose may also be attributed to a
slow metabolism or an increase in stress (Smith,
Hat-tingh & Burger 1976; Connors, Schneider, Genoway &
Barraclough 1978; Best, Eddy & Codd 2001)
The observation in the present study that total Ig
was signi¢cantly higher in infected ¢sh fed the
pro-biotic diet was expected and agrees with reports for
trout, Oncorhynchus mykiss, maintained on a
probio-tic-based diet containing Lactobacillus rhamnosus
strains (Nikoskelainena et al 2003; Panigrahia et al
2005)
At 21 days post infection, however, the WBC levels
in infected groups fed probiotic diets were decreased
and became similar to those in the probiotic and
non-probiotic control groups, whereas the level in ¢sh fed
the non-probiotic diet remained higher than in their
probiotic counterparts and both controls These
ob-servations could indicate that the immune system
was successful in controlling the bacterial infection
in the probiotic treatments, returning the WBC count
back to the normal condition This suggestion is also
supported by the similar pattern observed in the
other haematology parameters, which increased or
decreased from 7 days post infection and became
si-milar to the levels of the controls (normal) after 21
days Reports by many authors (Van Vuren, van der
Merwe & Du Preez 1994; Ranzani-paiva et al 2000;
Ezeri 2001; O’Neal & Weirich 2001; Omoregie &
Oye-banji 2002; Gabriel et al 2004) support this opinion,
whereby haematological deviations were indicative
of pathophysiology and health status
In addition to haematology parameters,
histo-pathology of the liver, the main organ for processing
and removal of toxic substances or transforming
them into harmless materials for excretion (Dutta,
Adhikari, Singh & Munshi 1993), was assessed The
liver also has the ability to accumulate xenobiotics,
and bio-transforms and transports them to the bile
for removal (Metcalfe 1998) In the present study, the
histopathology of the liver at 7 days post infection
with the three pathogenic bacteria revealed acute
he-patitis, with large necrosis areas appearing in the
non-probiotic treatments (Fig 1a) and subsequent
worsening conditions at 21 days post infection Thesechanges and damage to the hepatic cells could haveoccurred due to the poisons released by the patho-genic bacteria during infection and the long-term ac-tion of poisonous compounds and their metabolites
on the liver cells This suggestion concurs with theobservations of Bowser, Wooster, Chen and Mo(2004), who reported that hybrid striped bass had ne-crotic foci in the liver when it was infected with Aero-monas salmonicida, Mycobacterium sp and Vibrioanguillarum Stoskopf (1993) also reported thatvacuolar degeneration in the cytoplasm of liver cellswas observed when Atlantic salmon was infectedwith vibirios
In the kidney, capillaries and mesangial cells form important functions in the normal ¢ltration ofblood In the present study, histopathology examina-tion of the kidney at 7 days post infection revealedconditions such as extreme dilatation of the glomer-ular capillaries, blocked Bowman’s capsule space andnecrosis in epithelial cells of the proximal convolutedtubules, when ¢sh were raised on the non-probioticdiet The situation worsened at 21 days post infection,evident by very acute necrosis and degeneration ofareas of the kidney architectural structure, expan-sion of spaces inside the Bowman’s capsule andshrinking of the convoluted tubules (refer to plates
per-in Fig 2b) Kidney cell damage due to pathogenic terial infection such as multiple necrotic foci in hy-brid striped bass, Morone chrysops Morone saxatilis(Bowser et al 2004), congested kidneys in silver pom-fret Pampus argenteus, Euphrasen (Duremdez,Al-marzouk, Qasem, Al-harbi & Gharabally 2004),haemorrhaging and necrosis in turbot Scophthalmusmaximus (Bj˛rnsdo¤ttir, Gudmundsdo¤ttir, Bambir &Gudmundsdo¤ttir 2005; Padr, Zarza, Dopazo, Cuadra-
bac-do & Crespo 2006), lesions, enlarged kidneys andnecrosis in rainbow trout (Hirvel-Koski, Pohjanvir-
ta, Koski & Sukura 2006; Altinok, Balta, Capkin &Kayis 2007) and embolism in capillary and lymphaticvessels in Seriola dumerili (Hagiwara, Takano, Nogu-chi & Narita 2009) have also been reported pre-viously
In conclusion, the results of the present studyclearly demonstrate that the infected groups of ¢shmaintained on probiotic diets yielded signi¢cantlybetter haematology parameters and histopathologythan the infected groups fed with the non-probioticdiets This supports the bene¢cial e¡ects of probioticbacteria (L acidophilus) as a bio-control agent in as-sisting ¢sh (C gariepinus in this case) to better with-stand bacterial infections (especially S agalactiae,
L acidophilus as a probiotic in African cat¢sh M A Al-Dohail et al Aquaculture Research, 2011, 42, 196–209
r 2010 Universiti Sains Malaysia
Trang 37S xylosus and A hydrophila gr.2) respectively It is
known that the e¡ect of a bacterial toxin depends on
the pathogenic bacteria species from which it is
de-rived Therefore, the results of this study showed that
S agalactiae was the highest inhibited by probiotic
bacteria (L acidophilus), whereas A hydrophila gr.2
was the lowest inhibited in both the in vitro and the
in vivo challenge L acidophilus is therefore useful as a
bio-control agent against bacterial infections in C
gariepinus
Although the market value of cat¢sh is relatively
low per kilo, compared with other species, the
con-sumption, on the other hand, is quite high in Asian
markets Therefore, the use of single probiotic
bacter-ia such as L acidophilus rather than a cocktail of
bac-teria (as has been suggested by other authors) for the
treatment of cat¢sh diseases is more a¡ordable,
trans-lating into higher pro¢ts in the aquaculture industry
Acknowledgments
This study was funded by the Malaysian Government
Probiotic Project number 305/PBIOLOGI/613514 and
supported by the USM fellowship scheme The ¢rst
author was ¢nancially supported by the
Hadhram-out University of Science and Technology (Yemen)
The authors will like to express their gratitude to the
National Fish Health Research Center Penang,
Ma-laysia, for the supply of the pathogenic bacteria The
authors also express their gratitude to Professor
Abdlhakim Alrawi of Baghdad University, who was
a visiting Professor to USM, for his technical
assis-tance during pathohistology analysis and Dr Saeed
Alfadly of Ministry of health, Hadhramount, Yemen,
for his technical assistance during the haematology/
immunology analysis
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Trang 40Microsatellite–centromere mapping in walking catfish
gynogenetic diploids
Supawadee Poompuang1& Chantapim Sukkorntong2
1 Department of Aquaculture, Faculty of Fisheries, Kasetsart University, Bangkok,Thailand
2 Center for Agricultural Biotechnology, Kasetsart University, Nakorn Pathom,Thailand
Correspondence: S Poompuang, Department of Aquaculture, Faculty of Fisheries, Kasetsart University, 50 Paholyothin Road, Bangkok 10900,Thailand E-mail: supawadee.p@ku.ac.th
Abstract
Thirty new microsatellite loci were isolated from a
mi-crosatellite-enriched genomic library of walking
cat-¢sh Clarias macrocephalus The CA motif was the most
abundant, although several other motifs were also
isolated Two gynogenetic diploid families, each
con-sisting of a female and 50 o¡spring, were genotyped
at 56 microsatellite loci, including 30 loci developed
in the present study and 26 loci reported in the
previous study Overall, 33 anonymous microsatellites
derived from genomic library and11 EST-linked
micro-satellites were mapped to their centromeres High
levels of chiasma interference were apparent in the
walking cat¢sh genome as indicated by an average
fre-quency of second-division segregation (y) of 0.643
0.248 Twenty-six loci (59%) showed a high
microsa-tellite^centromere recombination, with a frequency
40.67, and three loci had recombination frequencies
40.9 This study demonstrated that gene^centromere
mapping provided a rapid method for the expansion of
the initial linkage map of walking cat¢sh
Keywords: Clarias macrocephalus, centromere, gene
mapping, gynogenetics, microsatellite
Introduction
Gene^centromere mapping is the technique used to
determine the position of a centromere on the genetic
linkage map In species with a small number of
avail-able genetic markers, it is di⁄cult to construct a
ge-netic map, but the distances of these markers from
their centromere can be estimated (Thorgaard,
Allen-dorf & Knudsen 1983; AllenAllen-dorf, Seeb, Knudsen,Thorgaard & Leary 1986) The principle of gene^cen-tromere mapping is based on half-tetrad analysis, forwhich two products of second meiotic division can berecovered by inhibiting the release of the secondpolar body (Zhao & Speed 1998) In ¢sh and shell¢sh,two products of second meiotic division ^ includingthe second polar bodies and the secondary oocytes ^can be recovered using gynogenesis techniques(Thorgaard et al 1983; Streisinger, Singer, Walker,Knauber & Dower 1986)
Walking cat¢sh (Clarias macrocephalus) is an portant food ¢sh species in Thailand.Walking cat¢shhas superior £esh quality and commands high retailprices Although culture of walking cat¢sh has beenwell established in Thailand for over 30 years, the po-tential of the industry is limited by the species’ slowgrowth rate and disease susceptibility (Na-Nakorn,Rangsin & Witchasunkul 1993) A molecular ap-proach could be applied for implementing geneticmarker-assisted selective breeding of walking cat¢sh.The application of this method, however, requires in-formation on its genome including a high-resolutiongenetic map A ¢rst generation of genetic map based
im-on AFLP markers has been generated for walkingcat¢sh, but the marker density was low (Poompuang
& Na-Nakorn 2004) Because the localization ofquantitative trait loci (QTLs) is essential for the appli-cation of marker-assisted selection, QTL mappingstrategies would bene¢t from a higher gene density
on the map (Poompuang & Hallerman 1997) over, the genetic map consisted of 31 linkage groups,whereas walking cat¢sh has 27 chromosomes, indi-cating that there are at least four gaps in the map ToAquaculture Research, 2011, 42, 210^220 doi:10.1111/j.1365-2109.2010.02613.x
More-r 2010 The Authors