Aquaculture 195 Ž2001.

331–352
www.elsevier.nlrlocateraqua-online

Bio-markers for egg quality determination in

cyprinid fish
F. Lahnsteiner a,) , B. Urbanyi b, A. Horvath b, T. Weismann c
a
Institute for Zoology, UniÕersity of Salzburg, Hellbrunnerstrasse 34, A-5020 Salzburg, Austria
b
¨ ¨ ¨ UniÕersity of Agricultural Sciences, Institute of Animal Husbandry, Laboratory of Fish Culture,
Godollo
¨ ¨ ¨ Pater K. St. 1., Hungary
H-2103, Godollo,
c
Bundesamt fur ¨ Wasserwirtschaft, Institut fur
¨ Gewasserokologie,
¨ ¨ Fischereibiologie und Seenkunde,
Scharfling 18, A-5310 Mondsee, Austria
Received 22 December 1998; received in revised form 30 September 2000; accepted 8 October 2000

Abstract

The present study investigated potential bio-markers Žovarian fluid parameters, egg weight and
weight increase during water hardening, biochemical egg composition, egg enzyme activities. for
egg quality determination in the common carp Ž Cyprinus carpio ., the silver carp Ž Hypophthal-
michthys molitrix ., the grass carp Ž Ctenopharyngodon idella. and the bleak Ž Chalcalburnus
chalcoides ..
For all investigated species, the study revealed highly significant correlations between the egg
fertilization rate and the weight of water-hardened eggs, the percent weight increase during water
hardening and the ovarian fluid pH, protein concentration, and aspartate aminotransferase activity.
The fertilization rate was further correlated with the activities of malate dehydrogenase and
pyruvate kinase of the eggs, and in C. carpio and Cha. chalcoides with the respiration activity,
too. Investigated biochemical parameters of the eggs Žprotein, peptides, fructose, galactose,
glucose, non-esterified fatty acids, esterified fatty acids, total DNA and RNA. were not correlated
with the fertilization rate.
The possible use of the analysed parameters for prediction of egg quality during short-term
storage was also investigated in C. carpio and Cte. idella: during short-term storage for 4 h at
48C, the fertilization rate significantly decreased. Also, the weight of the eggs after water
hardening, the percent weight increase due to water hardening and the ovarian fluid pH and
ovarian fluid aspartate aminotransferase changed in comparison to fresh samples and were highly
significantly correlated with the fertilization rate. In contrast, the biochemical composition of the
eggs Žprotein, peptides, fructose, galactose, glucose, non-esterified fatty acids, esterified fatty

)
Corresponding author.
E-mail address: Franz.Lahnsteiner@sbg.ac.at ŽF. Lahnsteiner..

0044-8486r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 0 4 4 - 8 4 8 6 Ž 0 0 . 0 0 5 5 0 - 0
332 F. Lahnsteiner et al.r Aquaculture 195 (2001) 331–352

acids, total DNA and RNA. and egg enzyme activities Žphosphofructokinase, pyruvate kinase,
protease, lipase, NAD-dependent malate dehydrogenase, respiration rate, NADP-dependent isoci-
trate dehydrogenase, aspartate aminotransferase. remained constant. q 2001 Elsevier Science B.V.
All rights reserved.

Keywords: Chalcalburnus chalcoides; Ctenopharyngodon idella; Cyprinus carpio; Hypophthalmichthys

molitrix; Egg quality; Ovarian fluid; Physiology; Metabolism; Bio-marker; Short-term storage

1. Introduction

Today, there exist several parameters for determination of egg quality ŽKjørsvik et
al., 1990; Bromage and Roberts, 1994.: Ž1. for the unfertilized eggs, the transparency
and the homogenous distribution of lipid droplets are described as viability criteria in the
Salmonidae ŽCraik and Harvey, 1984; Lahnsteiner et al., 1999a., for the fertilized eggs
blastomere morphology is a predictor for the quality as its structure correlates with the
embryo viability in the wolffish Ž Anarhichas lupus ., the cod Ž Gadus morhua. and the
Atlantic halibut Ž Hippoglossus hippoglossus. ŽPavlov and Moksness, 1994; Kjørsvik,
1994; Shields et al., 1997.. Ž2. Intensity of the cortical reaction, egg osmolality and
mechanical strength of the chorion affect egg viability in the cod ŽKjørsvik and Lønning,
1983.; the increase in egg wet weight due to water influx during water hardening is
correlated with the number of eyed stage embryos in the lake trout, Salmo trutta f.
lacustris ŽLahnsteiner et al., 1999a.. Ž3. Ovarian fluid pH and protein levels correlate
with the egg viability in the turbot, Scophthalmus maximus ŽFauvel et al., 1993. and in
the lake trout Ž S. trutta f. lacustris . ŽLahnsteiner et al., 1999a.; also the activity of
ovarian fluid aspartate aminotransferase activity in the lake trout. Ž4. Biochemical
parameters such as yolk composition, egg enzyme activities, and metabolite levels are
correlated with the egg viability in rainbow trout and lake trout, too ŽCraik and Harvey,
1984; Lahnsteiner et al., 1999a..
Although cyprinid fish are of great commercial importance, there exists no data
useful for determination of their egg quality. Therefore, the present study investigated
correlations between the egg fertilization rate and selected egg parameters in freshly
collected individual samples of varying quality and compared these with short-term
stored samples.
The following species were studied: Ž1. the common carp Ž Cyprinus carpio ., as it is
the traditionally cultured European cyprinid species, Ž2. two Southeast Asiatic species,
the silvercarp Ž Hypophthalmichthys molitrix . and the grass carp Ž Ctenopharyngodon
idella., as these are frequently introduced in carp hatcheries because of their beneficial
dietary behavior, Ž3. the bleak Ž Chalcalburnus chalcoides. as a model for closely related
endangered species Že.g. Alburnoides bipunctatus, Leucaspius delineatus., which are
involved in restocking and breeding programs.
The following parameters were investigated for correlation with the fertilization rate.
Ž1. Ovarian fluid pH, protein levels and aspartate aminotransferase activities as these
parameters were found to correlate with egg viability in other species ŽFauvel et al.,
1993; Lahnsteiner et al., 1999a..
 F. Lahnsteiner et al.r Aquaculture 195 (2001) 331–352 333

Ž2. The water uptake of the eggs during water hardening, which is an easy assay for
determining the intensity of cortical reaction, as water uptake depends on osmolytes
released from the cortical vesicles ŽAlderdice, 1988..
Ž3. The main biochemical composition of the eggs, which gives information about
energy reserves Žprotein, peptides, fructose, galactose, glucose, non-esterified fatty acids,
esterified fatty acids. and about biosynthetic outfit Žtotal DNA and RNA..
Ž4. Selected enzyme activities of the eggs which are important for energy metabolism
Žphosphofructokinase, pyruvate kinase, protease, lipase, NAD-dependent malate dehy-
drogenase, respiration rate., and for biosynthetic processes ŽNADP-dependent isocitrate
dehydrogenase.. Aspartate aminotransferase was selected to monitor the integrity of the
oolemma as this enzyme is highly soluble in the cytoplasm and it leaks out of the cell
when the membrane is damaged.

2. Material and methods

2.1. Fish

Mature C. carpio Žmales—age: q4 years, TL Žtotal length.: 30–35 cm, BW Žbody

weight.: 3–3.5 kg; females—age: 5 years, TL: 40–50 cm, BW: 4–5 kg. were obtained
from the commercial hatchery of the National Federation of Hungarian Fish Producers at
Dinnyes´ near Szekesfehervar
´ ´ ´ ŽHungary. at the end of May. They were kept in ponds
which had a water temperature of 27.0 " 0.58C during spawning. About 150 fish were
stocked per hectare and fed natural zooplankton supplemented with a diet of cereal
grains Žbarley, wheat, corn. at a rate of 2–5% of their body weight per day.
Mature Cte. idella Žmales—age: 5 years, TL: 60–65 cm, BW: 5.0–5.5 kg; females—
age: 7 years, TL: 60–65 cm, BW: 7.0–7.5 kg. and H. molitrix Žmales—age: 5 years,
TL: 50–60 cm, BW: 5.0–6.0 kg; females—age: q6 years, TL: 60–65 cm, BW: 7.0–8.0
kg. were obtained from the Warm Water Fish Hatchery ŽTEHAG. Szazhalombatta,
Hungary at the end of May. There they were kept in ponds at a density of 100–150
spawnersrha and at a water temperature of 28.0 " 0.58C. Food requirements of Cte.
idella were grass, water-weeds and legumes in amounts of about 15% of their body
weight per day. For H. molitrix, the food consisted of unicellular algae and plants,
bacteria, and organic detritus. During the growing season Žbefore reproduction., the
ponds were fertilized with nitrogen fertilizer at doses of 50–60 kgrha for sufficient
algae production.
Cha. chalcoides ŽTL: 10–15 cm, BW: 15–50 g. were electro-fished from Austrian
wild populations spawning in the Zeller Ache near Mondsee in May and June. Water
temperature was 15 " 18C in the spawning grounds. Mature fish being in the running
stage were transported to the Bundesamt fur ¨ Wasserwirtschaft in Scharfling in contain-
ers supplied with oxygen, where the gametes were collected within 6 h. In Cha.
chalcoides kept under these conditions, no significant changes in egg quality due to
overripening were observed during a time span of 6 h but changes became very
significant after 24–36 h Ždecrease in fertilization rate for ) 50%. ŽLahnsteiner and
Weismann, unpublished investigations..
334 F. Lahnsteiner et al.r Aquaculture 195 (2001) 331–352

2.2. Collection and processing of gametes

In Cte. idella, H. molitrix and C. carpio, spermiation and ovulation were induced by
a gonadotropin releasing hormone analogue ŽGnRH-a. ŽD-ala6 ,pro 9 NEt. ŽMagyary et
al., 1996.. One pellet of GnRH-a consisted of 10–15 mg GnRH-a and 2.5–3.0 mg of
water soluble dopamine antagonist meta-chloramide. Before hormone induction, the fish
were anaesthetized in a diluted solution of quinaldin Ž1:10,000 in water.. Females
received two intramuscularly injected doses of GnRH-a. As the first dose, one pellet
GnRH-a Ždissolved in 0.7% NaCl. per 5 kg of body weight was injected, and as the
second dose, one pellet GnRH-a per kilogram of body weight 14 h later. Ovulation
occurred 24 h after the first injection. The genital opening of the females was sewed up
with two ligatures after the second injection to prevent the deposition of eggs into the
tanks. Males received one dose of GnRH-a Žone pelletr5 kg body weight. when females
were injected the second time. As Cha. chalcoides were collected from spawning wild
populations, the individuals were in a running stage.
For collection of gametes, Cte. idella, H. molitrix and C. carpio were anaesthetized
in quinaldin. Because of small body size, there was no necessity to anaesthetize Cha.
chalcoides. Semen of all investigated species was collected by pressure on their
abdomen and sampled into centrifuge tubes. Sperm density was counted in a Makler
counting chamber after 1000-fold dilution in sperm motility inhibiting saline solution
and was Ž1.17 " 0.47. = 10 10 spermatozoarml in Cha. chalcoides, Ž2.37 " 0.70. = 10 11
spermatozoarml in C. carpio, and Ž1.42 " 0.80. = 10 11 spermatozoarml in Cte. idella
and in H. molitrix. Immediately after collection, semen was diluted in 48C cold buffered
sperm motility inhibiting saline solution Ž75 mmolrl NaCl, 70 mmolrl KCl, 2 mmolrl
CaCl 2 , 1 mmolrl MgSO4 , and 20 mmolrl Tris, pH 8. ŽMorisawa, 1983. at a ratio of
1:5 Žsemenrsalt solution. to stabilize semen quality, and to retard aging processes.
Then, the semen quality was estimated by evaluation of the motility rate under a light
microscope Žpredilution: 1:10 in sperm motility inhibiting saline solution, activation:
1:100 in 50 mmolrl NaCl, 20 mmolrl Tris, pH 9.. Thereafter, in each species, four
semen samples with motility rates of ) 70% were pooled in equal ratios. The prediluted
semen pools were stored in centrifuge tubes at 48C maximally for 1 h for insemination
of the individual freshly collected egg batches and for 4 h for insemination of the
short-term stored egg batches. Semen storage conditions derived from earlier experi-
ments ŽLahnsteiner et al., 1999b..
Eggs were collected from 27 Cha. chalcoides, 18 Cte. idella, eight H. molitrix, and
32 C. carpio. From individuals of Cte. idella, H. molitrix, and C. carpio, approximately
10-g portions of eggs were stripped into petri dishes. The total amount was about
500–1000 g eggsrfemale in C. carpio and Cte. idella, and 300–800 g eggsrfemale in
H. molitrix. The remaining eggs were pooled in each species and used for commercial
hatching. In Cha. chalcoides, the eggs were completely stripped out and about 5–10 g
eggs were obtained per female. Egg samples were processed within 30 min after
collection and were kept at 48C for this time period. The following experiments were
performed: Ž1. fertilization assays, Ž2. determination of the egg weight and of the weight
increase during water hardening, Ž3. collection and analysis of the ovarian fluid, Ž4.
extraction and determination of egg enzymes and metabolites.
 F. Lahnsteiner et al.r Aquaculture 195 (2001) 331–352 335

2.3. Storage of eggs of Cte. Idella and C. Carpio

From the 32 individual egg batches of C. carpio, 14 batches were randomly selected
from the 18 individual egg batches of Cte. idella eight batches. From these selected egg
batches, subsamples of approximately 1 g were taken and stored in a refrigerator at 48C
for 4 h in petri dishes. Samples were stored at 48C to decrease the egg metabolism and
avoid the spontaneous activation of eggs. Subsamples were not separated from the
ovarian fluid for storage. After the storage was terminated, the egg batches were
processed in a similar way as the freshly collected, unstored egg batches.

2.4. Fertilization assays

In Cte. idella, H. molitrix and C. carpio, fertilization assays were performed at 258C
and exactly 300 mg of eggs were used. In Cha. chalcoides, fertilization assays were
performed at 158C and exactly 400 mg of eggs were used. There were 200 " 30 eggs in
all species. In all cases, the ovarian fluid was removed before weighing. The eggs were
fertilized in petri dishes using the wet technique. Three hundred microliters Ž Cte. idella,
H. molitrix, C. carpio . or 400 ml Ž Cha. chalcoides. sperm motility activating saline
solution adjusted to the respective fertilization temperature was pipetted onto the eggs
and immediately thereafter 50 ml prediluted semen was added and mixed with the eggs
with a fine glass rod. The resulting sperm to egg ratio was 1:2 = 10 7 Žeggrspermatozoa.
for all species. For insemination of the short-term stored egg batches, semen of Cte.
idella and C. carpio was stored for 4 h. Over this period, the motility rates decreased by
20–40% as found by re-investigation of the motility rates and therefore the two-fold
amount of semen was used to compensate for the decrease in semen quality.
In Cte. idella, H. molitrix and C. carpio, the sperm motility activating saline solution
was drained after 5 min, and the eggs gently spread out in the petri dishes in a single
layer. Then, the petri dishes were filled with about 50 ml of hatchery water and the eggs
were incubated for 4 h at 258C whereby the water was renewed every 30 min. The
fertilization rate was evaluated as the number of eggs reaching the morula stage in
relation to the total number of eggs using a stereomicroscope at 10-fold magnification.
As the eggs of Cha. chalcoides had longer development periods, they were trans-
ferred into small flow through incubators Ž15 = 15 cm. 5 min after fertilization and
spread out there in one layer. For water supply, a circulating system was used and water
temperature was 158C. After 72–84 h incubation, the number of eyed embryos in
relation to the total number of eggs was determined.

2.5. Determination of egg weight and of weight increase during water hardening

The weights of the fresh unhardened and the water-hardened eggs were determined
with an analytical balance Žaccuracy 0.1 mg.. Special care was taken to avoid damage of
eggs. All measurements were performed in glass vessels with a flat bottom Ždiameter 10
mm, volume 5 ml.. The dry and empty vessel was weighed, approximately 30 mg of
eggs Ž Cte. idella, H. molitrix, C. carpio . or 40 mg of eggs Ž Cha. chalcoides. were
placed in the tube with a spatula and the vessel was re-weighed. Then, 4 ml hatchery

Table 1
Methodology for enzymatic assay
Enzyme, assay
Aspartate aminotransferase, UV-spectrophotometric ŽBergmeyer, 1985.
Isocitrate dehydrogenase, UV-spectrophotometric ŽBergmeyer, 1985.
Malate dehydrogenase, UV-spectrophotometric ŽBergmeyer, 1985.
Pyruvate kinase, UV-spectrophotometric ŽBergmeyer, 1985.
Phosphofructokinase, UV-spectrophotometric ŽLoewenstein, 1983.
Lipase, turbidimetric assay ŽBergmeyer, 1985.
Respiration, colourimetric assay ŽClark and Switzer, 1977.
Protease, fixed time, UV-spectrophotometric assay ŽBergmeyer, 1985.
336
F. Lahnsteiner et al.r Aquaculture 195 (2001) 331–352
Assay conditions
substrates: 240 mmolrl aspartic acid, 12 mmolrl 2-oxoglutarate;
co-substrates: 0.20 mmolrl NADH, 0.10 mmolrl pyridoxal phosphate; pH: 7.8
substrate: 3 mmolrl isocitrate; co-substrate: 0.42 mmolrl NADP;
ions: 1.50 mmolrl MnCl 2 ; pH: 7.4
substrate: 2.5 mmolrl malate; co-substrate: 0.20 mmolrl NAD;
ions: 0.05 mmolrl MgCl 2 ; pH: 9.4
substrate: 2.5 mmolrl phospoenolpyruvate; co-substrate:
0.20 mmolrl NADH; ions: 10 mmolrl MgCl 2 , 100 mmolrl KCl; pH: 9.4
substrate: 1.5 mmolrl fructose-6-phosphate; co-substrates: 0.20 mmolr
l NADH; 1 mmolrl ATP; ions: 5 mmolrl MgSO4 , 0.05 mmolrl KCl; pH: 8.0
substrate: 0.14 mmolrl triolein; ions: 35 mmolrl NaCl,
1 mmolrl CaCl 2 ; incubation period: 5 min; pH: 9.0
substrate: 5 mmolrl succinate; co-substrate: 0.40 mmolrl NAD;
incubation period: 5 min; pH: 7.4; colour reagent: 2.5 mmolr
l dichlorophenol–indophenol; determination:
dichlorophenol–indophenol reduction at 500 nm
substrate: 0.25% gelatine; ions: 0.05 mmolrl CaCl 2 ;
pH: 7.4; incubation period: 30 min; determination: liberated peptides at 280 nm
 F. Lahnsteiner et al.r Aquaculture 195 (2001) 331–352 337

water was added and the eggs were spread out to find space for water hardening. After
the desired time periods, the water was removed. This was easy in C. carpio and Cha.
chalcoides since the eggs stuck to the vessel and the water could be poured off. In Cte.
idella and H. molitrix whose eggs were floating, the water was carefully pipetted out of
the tube. Remaining droplets of ovarian fluid and water were removed with absorbent
paper. Then, the vessel was re-weighed for determination of the weight of the water-
hardened eggs. Finally, the exact number of eggs was counted for each subsample.
To standardize the assay monitoring the egg weight increase during water hardening,
additional experiments with six egg batches were performed in each species. From each
egg batch, two subsamples were used. The wet weight of non-water-hardened eggs was
determined. Then, in one subsample, eggs were fertilized as described whereby the
amount of sperm motility activating saline solution and of semen was adjusted. After 5
min, the eggs were rinsed and finally 4 ml hatchery water was added. For the other
subsample, the unfertilized eggs were immersed in 4 ml water. The wet weight of the
water-hardened eggs was determined at intervals of 30 min for 120 min. At the desired
time intervals, water was removed as described, the eggs were weighed and then
re-incubated.
Since there was no difference in the weight increase of fertilized and unfertilized eggs
and since the water hardening was completed within 60 min, for routine analysis
unfertilized eggs and incubation periods of 60 min were used.

2.6. Collection and analysis of oÕarian fluid

From the individual egg samples, the ovarian fluid was collected with a fine pipette.
In Cte. idella, H. molitrix and C. carpio, 100–200 ml per 10 mg eggs were collected. In
H. molitrix only in four of eight egg batches ovarian fluid could be obtained. Due to the
low sample number, the samples were not included into the statistical analysis. Gener-
ally, for those three species, 200–400 ml ovarian fluid are obtained per female. In Cha.

Table 2
Changes in the weight of eggs of Cte. idella, H. molitrix, C. carpio and Cha. chalcoides during water
hardening
0 min 30 min 60 min 90 min
Egg wet weight (mg r egg)
Cte. idella, H. molitrix 1.7"0.2 a 8.4"1.1b 21.6"3.9 c 21.6"3.9 c
C. carpio 1.4"0.1a 3.1"0.2 b 3.3"0.2 c 3.3"0.2 c
Cha. chalcoides 2.1"0.2 a 5.5"0.5 b 6.4"0.4 c 6.4"0.4 c

Weight increase during hardening (%)

Cte. idella, H. molitrix 0"0 a 445"51b 1342"350 c 1295"210 c
C. carpio 0"0 a 110"9 b 120"9 c 121"5c
Cha. chalcoides 0"0 a 166"1b 206"17 c 206"17 c

Values represent mean"standard deviation from the fertilized egg batches and are similar for unfertilized egg
batches. Data were combined for Cte. idella and H. molitrix. Values within a row superscripted with the same
letter are not significantly different, one-way multifactorial analysis of variance with subsequent Tukey’s
b-test, P ) 0.001.
338 F. Lahnsteiner et al.r Aquaculture 195 (2001) 331–352

chalcoides, 100–500 ml ovarian fluid were obtained per female. Ovarian fluid of all
species was frozen in liquid nitrogen until analyses.

Table 3
Fertilization rates, ovarian fluid composition and egg parameters investigated in Cte. idella, H. molitrix, C.
carpio and Cha. chalcoides
Parameter Unit Cte. idella, C. carpio Cha.
H. molitrix Ž ns 32. chalcoides
Ž ns 26. Ž ns 27.
Eggs
Fertilization rate % 39.5"44.4 a,b 86.3"17.7 c 56.9"34.8 b,c
Weight of unhardened eggs mgregg 1.4"0.2 a 1.4"0.3 a 2.1"0.4 b
Weight of hardened eggs mgregg 8.9"2.7 a 3.1"0.6 b 5.8"1.1c
Weight increase after 60 min % 535q240 a 106"56 b 185"62 c
Protein mgrmg egg 0.20"0.06 a 0.07"0.04 b 0.6"0.3 c
mgregg 0.40"0.36 a 0.10"0.06 b 1.16"0.70 c

Metabolites (units)
DNA mgrmg egg 1.1"0.4 a 0.9"0.2 a 7.2"2.9 b
mgregg 0.9"0.3 a 1.2"0.4 a 14.5"0.8 b
RNA mgrmg egg 20.9"14.7 a 39.2"24.2 a 18.8"19.5a
mgregg 35.6"20.4 a 55.1"30.1a 38.3"39.1a
Esterified fatty acids mgrmg egg 229.3"74.5a 189.3"47.3 a 466.6"254.5 b
mgregg 310.0"118.6 a 273.1"65.4 a 942.7"551.8 b
Non-esterified fatty acids mgrmg egg 5.1"1.6 a 3.5"0.7 b 125.7"146.1c
mgregg 6.8"1.7 a 5.2"1.2 b 254.4"296.9 c
Galactose nMrmg egg 0.1"0.2 a 0.01"0.01b 0.3"0.5a
mgregg 0.2"0.2 a 0.01"0.05 b 0.6"1.1a
Glucose nMrmg egg 3.6"2.5 a 7.1"4.1a 17.3"12.2 b
mgregg 4.0"3.6 a 9.7"4.7 b 36.8"27.7 b
Fructose nMrmg egg 3.6"2.3 a 2.3"1.8 b 9.5"8.0 c
nMregg 4.7"2.8 a 3.1"2.4 a 20.3"19.7 b
Peptides nMrmg egg 0.18"0.05a 32.10"19.26 b 0.65"0.46 c
nMregg 0.25"0.09 a 45.13"24.0 b 1.32"0.78 c

Enzymes
Aspartate aminotransferase nMrminrmg protein 5.5"2.4 a 5.2"1.9 a 14.6"11.0 b
nMrminregg 1.6"0.8 a 0.4"0.1b 12.6"9.3 c
Isocitrate dehydrogenase nMrminrmg protein 1.2"0.3 a 0.3"0.5 b 0.3"0.3 b
nMrminregg 0.3"0.1a 0.02"0.04 b 0.2"0.2 a
Lipase nMrminrmg protein 0.1"0.1a 0.1"0.1a 0.5"1.0 b
nMrminregg 0.02"0.03 a 0.01"0.01a 0.3"0.6 b
Malate dehydrogenase nMrminrmg protein 8.0"5.4 a 27.3"14.4 b 11.4"7.9 a
nMrminregg 2.5"1.4 a 2.4"1.3 a 9.9"5.2 b
Phosphofructokinase nMrminrmg protein 0.9"0.7 a 2.7"2.0 b 1.3"1.5a
nMrminregg 0.3"0.2 a 0.2"0.2 a 1.1"1.3 a
Protease nMrminrmg protein 5.4"5.9 a,b 6.4"5.4 a 2.3"2.7 b
nMrminregg 1.7"1.8 a 0.6"0.5 b 2.0"2.3 a
Pyruvate kinase nMrminrmg protein 4.2"6.9 a 19.4"15.4 b 1.5"1.7 a
nMrminregg 1.2"1.8 a 1.5"0.7 a 1.3"1.2 a
Respiration rate nMrminrmg protein 2.7"1.8 a 21.7"15.3 b 4.4"3.8 a
nMrminregg 0.8"0.7 a 1.1"0.9 a 4.7"3.9 b
 F. Lahnsteiner et al.r Aquaculture 195 (2001) 331–352 339

Table 3 Ž continued .
Parameter Unit Cte. idella, C. carpio Cha.
H. molitrix Ž ns 32. chalcoides
Ž ns 26. Ž ns 27.
OÕarian fluid parameters
pH 9.07"0.20 a 8.89"0.26 a 8.83"0.39 a
Protein mgr100ml 711"287 a 1154"611b 313"194 c
Aspartate aminotransferase mMrminrl 23.0"21.8 a 50.8"30.1b 5.4"4.8 c

Values are mean"standard deviation. Data were combined for Cte. idella and H. molitrix. Values super-
scripted by the same letter are not significantly different, one-way multifactorial analysis of variance with
subsequent Tukey’s b-test, P ) 0.001.

The ovarian fluid pH value was determined with a micro-electrode. Protein concen-
tration and aspartate aminotransferase activity were assayed as described below.

2.7. Extraction of enzymes and metabolites from the eggs

From each egg batch, five portions of approximately 30 eggs were randomly taken
and placed in eppendorf tubes of 1.5 ml volume, weighed and the number of eggs
determined by computation. Samples were taken only from unfertilized eggs. Live and
dead eggs were not distinguished by visual inspection during sampling as egg viability
was not related to morphological parameters. Each subsample was rinsed in a 0.2 molrl
Tris buffer ŽpH 8.2. of 48C. Then, 0.5 ml of 48C appropriate extraction solution was
added and the subsample was homogenized manually on ice using a conically shaped
glass rod. After homogenisation, 0.5 ml more extraction solution was added, the
homogenate was frozen in liquid nitrogen and thawed for additional destruction of cell
structure. The sample was centrifuged for 15 min at 1000 = g, the supernatant was
collected, diluted with extraction solution in a ratio of 1:1 and stored in liquid nitrogen
until analysis.
Extraction of enzymes and metabolites followed the methodology described by
Lahnsteiner et al. Ž1999a. for salmonid eggs. For preparation of crude enzyme extracts
100 mmolrl Tris buffer ŽpH 7.5. and Triton X-100 Ž0.1%. were used. After homoge-
nization and centrifugation, a 50-ml subsample was taken for determination of prote-
olytic activity. To the remaining enzyme extract, EDTA was added to a final concentra-
tion of 2 mmolrl to inhibit proteolytic activity. Acid-stable metabolites ŽDNA, fructose,
galactose, glucose, glycogen, NAD, peptides, RNA. were extracted in 3 molrl perchlo-
ric acid and the supernatant was neutralized in 2 molrl KOH. Esterified fatty acids were
extracted in isopropanol, and non-esterified fatty acids in chloroform, heptane, methanol
Ž2.5:2.5:1..

2.8. Analysis of metabolites and determination of protein content

Photometric measurements were carried out with a Shimadzu-UV spectrophotometer.

Fructose, galactose, and the total amount of glucose after enzymatic degradation of
 340
Table 4
Simple and multiple regression models for the prediction of the egg fertilization rate using the weight of water-hardened eggs, the weight increase during hardening,
and parameters of egg composition as independent variables
Independent variable Equation R2 F-value

F. Lahnsteiner et al.r Aquaculture 195 (2001) 331–352

Cte. idella, H. molitrix
Weight of hardened eggs y s9.46 x y24.31 0.711 47.11
x 1 —weight of hardened eggs, y s 5.54 x 1 q7.59 x 2 q0.20 x 3 y51.70; 0.911 Ž0.750. 47.95 Ž8.98.
x 2 —fructose, x 3 —peptides Ž y s 4.31 x 1 q9.557x 2 q0.003 x 3 y38.29.
Weight increase during hardening y s 0.127x y14.55 0.753 63.68
x 1 —weight increase during hardening, y s 0.080 x 1 q6.17x 2 q0.24 x 3 y49.34; 0.895 Ž0.735. 42.82 Ž11.08.
x 2 —fructose, x 3 —peptides Ž y s 0.068 x 1 q9.00 x 2 q0.03 x 3 y46.16.

C. carpio
Weight of hardened eggs y s 30.39 x y22.31 0.632 56.80
x 1 —weight of hardened eggs, y s 45.78 x 1 y3.46 x 2 y0.00059 x 3 y41.53; 0.664 Ž0.653. 11.85 Ž18.20.
x 2 —fructose, x 3 —peptides Ž y s 30.89 x 1 y0.39 x 2 y0.00013 x 3 y16.01.
Percent weight increase y s 0.48 x y35.26 0.403 22.23
during hardening

Cha. chalcoides
Weight of hardened eggs y s133.05 x y9.96 x 2 y365.80 0.562 14.09
Weight increase during hardening y s 0.684 x y131.10 0.532 25.02
x 1 —weight increase during hardening, y s 0.78 x 1 y0.014 x 2 y148.95; 0.560 Ž0.547. 11.47 Ž12.96.
x 2 —peptides Ž y s 0.73 x 1 y0.01 x 2 y130.62.

Regressions for egg metabolites were calculated in units per milligram egg and in units per egg Žparenthesis.. P F 0.001 for all regression models.
 F. Lahnsteiner et al.r Aquaculture 195 (2001) 331–352 341

glycogen were measured by routine spectrophotometric assays described in Bergmeyer

Ž1985.. Degradation of glycogen was performed by amyloglucosidase digestion at pH
4.5 for 3 h at 378C ŽBergmeyer, 1985.. Non-esterified and esterified fatty acids were
determined by colorimetric assays described in Chaudhry Ž1989., the RNA levels were
determined with orcinol, and DNA levels with diphenylamine ŽHoltzhauer, 1988..
Protein was determined by the Lowry procedure ŽLowry et al., 1951. in the crude
enzyme extract and in the ovarian fluid. Peptides were determined in the deproteinized
and neutralized perchloric acid extract with the Folin–Ciocalteu phenol reagent
ŽBergmeyer, 1985..

2.9. Determination of enzyme actiÕities

Routine enzymatic assays were used which had been optimized for fish tissue for
substrate and co-enzyme saturation, pH and temperature by determining conditions

Fig. 1. Relation between the weight of hardened eggs, the percent weight increase during hardening and the
fertilization rate in C. carpio.
 342
Table 5
Simple and multiple regression models for the prediction of the egg fertilization rate using egg enzymes as independent variables
Independent variable Equation R2 F-value

F. Lahnsteiner et al.r Aquaculture 195 (2001) 331–352

Cha. chalcoides
Malate dehydrogenase y s 4.17x y0.115 x 2 q40.46; Ž y s13.16 x y0.57x 2 y0.40. 0.392 Ž0.621. 7.08 Ž18.10.
Pyruvate kinase y s 47.80 x y11.80 x 2 q26.25; Ž y s 41.80 x y11.01 x 2 q37.84. 0.390 Ž0.203. 4.75 Ž2.08.
Respiration rate y s13.79 x y0.63 x 2 q24.22; Ž y s 20.68 x y1.20 x 2 q19.84. 0.517 Ž0.640. 11.25 Ž19.54.
x 1 —respiration rate, x 2 —malate y s8.69 x 1 y0.84 x 12 q2.61 x 2 y0.08 x 22 q30.29 x 3 y9.17x 32 q 0.748 Ž0.944. 6.35 Ž50.14.
dehydrogenase, x 3 —pyruvate kinase 0.91 x 1 x 2 q10.10; Ž y sy0.13 x 1 q0.09 x 12 q12.52 x 2 y
0.44 x 22 y3.35 x 3 qy0.08 x 32 q0.89.
C. carpio
Malate dehydrogenase y s 5.28 x y0.074 x 2 y8.79; Ž y s 29.83 x y2.07x 2 y27.69. 0.449 Ž0.458. 13.05 Ž13.96.
Pyruvate kinase y s6.93 x y0.14 x 2 y6.06; Ž y s 77.81 x y13.19 x 2 y36.55. 0.366 Ž0.557. 9.22 Ž20.56.
Respiration rate y s 2.89 x y0.03 x 2 q27.43; Ž y s 72.05 x y18.94 x 2 q17.33. 0.684 Ž0.556. 30.42 Ž20.15.
x 1 —respiration rate, x 2 —malate y s 3.80 x 1 y0.06 x 12 q1.49 x 2 y0.02 x 22 q3.96 x 3 y0.09 x 32 y 0.808 Ž0.761. 18.91 Ž14.87.
dehydrogenase, x 3 —pyruvate kinase 0.05 x 1 x 3 y20.52; Ž y s14.96 x 1 y3.24 x 12 q14.99 x 2 y1.03 x 12 q
52.08 x 3 y8.56 x 32 y60.50.
H. molitrix, Cte. idella
Malate dehydrogenase y s17.18 x y0.87x 2 y7.15; Ž y s 47.21 x y4.89 x 2 y40.25. 0.383 Ž0.534. 6.20 Ž13.15.
Pyruvate kinase y s12.11 x y0.65 x 2 q19.04; Ž y s 44.12 x y7.18 x 2 q5.15. 0.412 Ž0.522. 8.04 Ž12.54.
x 1 —malate dehydrogenase, y s6.89 x 1 y0.39 x 12 q5.41 x 2 y0.52 x 22 q0.30 x 1 x 2 q36.71; 0.648 Ž0.681. 6.24 Ž8.54.
x 2 —pyruvate kinase Ž y s 44.96 x 1 y4.74 x 12 q9.53 x 2 y2.38 x 22 y0.51 x 1 x 2 qy30.85.

P F 0.001 for all regression models.

 F. Lahnsteiner et al.r Aquaculture 195 (2001) 331–352 343

Fig. 2. Relations between enzyme activities of eggs Žmalate dehydrogenase, pyruvate kinase, respiration. and
the fertilization rate in C. carpio.
344 F. Lahnsteiner et al.r Aquaculture 195 (2001) 331–352

yielding the highest reaction velocity. The standardisation was done in preliminary
investigations ŽLahnsteiner, unpublished., the resulting assay conditions are shown in
Table 1. The reaction temperature was 208C.

2.10. Statistical analysis

Protein concentration and enzyme activity of the ovarian fluid was expressed in units
per volume ovarian fluid. Data on egg compounds were expressed in two data sets. Ž1.
To define substance concentration independent of the egg size, protein and metabolite,
values were expressed as units per milligram unhardened egg, and enzyme activities as
units per milligram protein concentration. Ž2. Since the physiological and biochemical
processes during storage could affect the weight and the protein content of the eggs
analytes, values were also expressed in units per egg. For Cte. idella and H. molitrix,
the investigated parameters revealed no statistically significant differences and therefore
they were combined as a common data set.
Scatterplots and the bivariate correlation coefficient of Pearson ŽLorenz, 1984. were
used to detect relations between analyzed parameters and egg viability. The relations
were described by simple and stepwise multiple regression models Žtest procedure:
linearity of relation x; curvature of relation x 2 , x 3 ; interaction of relation beyond their
independent effects x 1 x 2 . using non-transformed and transformed data sets Žlogarithmic
transformation for metrical data, angular transformation for relative abundances.. Since
the statistical relations were similar for non-transformed and transformed data, the

Table 6
Simple and multiple regression models for the prediction of the egg fertilization rate using ovarian fluid
parameters as independent variables
Independent variable Equation R2 F-value
Cha. chalcoides
pH y s1208.42 x y70.05 x 2 y5127.68 0.574 14.12
Aspartate aminotransferase y sy1.68 x q79.10 0.548 21.82
Protein y sy0.01 x y0.0002 x 2 q83.66 0.700 23.29
x 1 —pH, x 2 —protein y s 2212.64 x 1 y132.04 x 12 q0.01 x 2 – 0.925 46.70
0.00008 x 22 y9181.87

C. carpio
pH y s 2604.10 x y150.97x 2 y11133.42 0.585 9.16
Aspartate aminotransferase y sy0.84 x q91.76 0.744 32.04
Protein y sy0.01 x y0.00005 x 2 q102.10 0.651 12.10
x 1 —pH, x 2 —protein y s1875.62 x 1 y110.52 x 12 y0.42 x 2 y 0.893 9.98
0.000003 x 22 q0.04 x 1 x 2 y7832.03

Cte. idella
pH y sy174.60 x q1620.15 0.615 14.39
Aspartate aminotransferase y sy1.85 x q92.88 0.494 9.75
Protein y sy0.098 x q147.39 0.605 19.66
x 1 —pH, x 2 —protein y sy179.27 x 1 q0.0005 x 2 q1666.24 0.685 7.62

P F 0.001 for all regression models.

 F. Lahnsteiner et al.r Aquaculture 195 (2001) 331–352 345

Fig. 3. Relations between the ovarian fluid pH, protein levels and aspartate aminotransferase activity and the
fertilization rate in C. carpio.
346 F. Lahnsteiner et al.r Aquaculture 195 (2001) 331–352

results are shown for the non-transformed data set to ascertain clarity and simplicity
especially under aspects of practical application.
A minimal fertilization rate of 70% was considered as tolerable for efficiently
working hatcheries and therefore egg batches with G 70% fertilized eggs were arbitrar-
ily classified to be of high quality, while egg batches with - 70% fertilized eggs were
classified to be of low quality. This classification was done before the analysis of data.
Optimal analyte values characterizing high quality egg batches were calculated from the
simple regression models.
To define differences in the analytes values between the species, one-way multifacto-
rial analysis of variance with subsequent Tukey’s b-test was used. To define the changes
occurring during storage of eggs, unstored samples Ž0 h. were compared with the stored
samples Ž4 h. using Student’s t-test.

3. Results
3.1. Egg parameters and oÕarian fluid composition
In all species, the weight increase during water hardening was similar for unfertilized
and fertilized egg batches, and was completed during the first 60 min of water
incubation ŽTable 2.. In Cte. idella and H. molitrix, there were no differences in egg
weights at any time. For the egg batches of Cte. idella, H. molitrix, C. carpio and Cha.
chalcoides, the analysed parameters are shown in Table 3.
3.2. Regression models for the prediction of the fertilization rate
The weight of the unhardened eggs did not correlate with the weight of the hardened
eggs in any of the species. The weight of the hardened eggs and the percent weight

Table 7
Optimal analyte values for characterization of high quality egg batches Ž G 70% fertilization rate. of
Cyprinidae as calculated from simple regression models
Parameter Unit Cte. idella, C. carpio Cha.
H. molitrix chalcoides
Weight of hardened eggs mgregg G10.0 G 3.0 G6.0
Weight increase during % G665.0 G 210.0 G 290.0
hardening
Malate dehydrogenase nMrminrmg protein 10.0–23.0 21.2–50.0 9.6–26.9
nMrminregg 4.0–5.6 5.1–9.3 7.0–20.1
Pyruvate kinase nMrminrmg protein 6.4–12.9 16.4–33.0 1.4–2.6
nMrminregg 2.6–3.5 2.3–3.8 1.2–2.6
Respiration rate nMrminrmg protein – G18.0 G 2.8
nMrminregg – G1.2 G 5.5
Ovarian fluid pH F8.88 a 8.35–8.85 8.43–8.80
Ovarian fluid protein mgr100ml F870 a F1250 F 300
Ovarian fluid aspartate mMrminrl F12.4 a F 25.9 F 5.4
aminotransferase
a
Data only for Cte. idella.
 F. Lahnsteiner et al.r Aquaculture 195 (2001) 331–352 347

increase during hardening correlated significantly Ž P F 0.01. with the fertilization rate
in Cte. idella, H. molitrix, C. carpio and Cha. chalcoides ŽTable 4.. Further, the weight
of the hardened eggs and the percent weight increase during water hardening correlated
significantly and positively Ž P F 0.01. with the levels of peptides and of fructose. The
following correlation coefficients were calculated Ždata in units per milligram egg.:
weight of hardened eggs—peptides: Cte. idella: 0.492, H. molitrix: 0.615, C. carpio:
0.371; weight of hardened eggs—fructose: Cte. idella: 0.709, H. molitrix: 0.668, C.
carpio: no significant correlation; percent weight increase during water hardening—
peptides: Cte. idella: 0.744, H. molitrix: 0.508, C. carpio: 0.539; percent weight
increase during water hardening-fructose: Cte. idella: 0.729, H. molitrix: 0.686, C.
carpio: 0.533. Simple and multiple regression models which use weight of the hardened
eggs, the percent weight increase during water hardening and the levels of peptides and
of fructose to predict the fertilization rate are shown in Table 4 Žscatterplots for simple
regression models in Fig. 1..
The biochemical composition of the eggs did not significantly Ž P ) 0.01. correlate
with the fertilization rate in any of the species. From the analyzed enzymes, the
activities of malate dehydrogenase and of pyruvate kinase revealed significant Ž P F 0.01.
relations to the fertilization rate in all investigated species, in Cha. chalcoides and C.
carpio the respiration rate, too. The regression models are shown in Table 5, scatterplots
in Fig. 2.
Also, the ovarian fluid pH, protein concentration and activities of aspartate amino-
transferase significantly Ž P F 0.01. correlated with the fertilization rate in all three
species. The regression models are shown in Table 6 and relevant scatterplots in Fig. 3.

Table 8
Changes in egg parameters and ovarian fluid composition of Cte. idella and C. carpio during storage together
with the ovarian fluid for 4 h at 48C
Parameter Fresh Stored r
Cte. idella
Fertilization rate Ž%. 91.6"10.5a 2.8"3.4 b
Wet weight of hardened eggs Žmgregg. 11.1"2.1a 6.54"1.4 b 0.812
Weight increase during hardening Ž%. 849.6"177.7 a 434.6"187.9 b 0.843
Ovarian fluid ŽpH. 8.85"0.09 a 9.19"0.10 b 0.603
Ovarian fluid aspartate aminotransferase 13.0"12.5a 35.4"16.0 b y0.640
ŽmMrminrL.

C. carpio
Fertilization rate Ž%. 84.7"12.1a 20.7"17.3 b
Wet weight of hardened eggs Žmgregg. 3.1"0.3 a 2.4"0.6 b 0.510
Weight increase during hardening Ž%. 123.7"30.4 a 76.0"26.8 b 0.506
Ovarian fluid ŽpH. 8.85"0.19 a 8.97"0.53 b 0.511
Ovarian fluid aspartate aminotransferase 39.8"24.3 a 113.8"121.7 b y0.490
ŽmMrminrL.

Fresh and stored samples were compared with the Student’s t-test and values superscripted with the same letter
are not significantly different Ž P ) 0.001.. Rs bivariate correlation coefficient of Pearson for the correlations
between fertilization rate and the listed parameters. All correlation coefficients have a probability of
P F 0.001.
348 F. Lahnsteiner et al.r Aquaculture 195 (2001) 331–352

Optimal analyte values characterizing high quality egg batches G 70% fertilization rate
are shown in Table 7 and were calculated from the simple regression models.

3.3. Changes in egg parameters and oÕarian fluid composition during short-term
storage

When eggs of Cte. idella and C. carpio were stored for 4 h in ovarian fluid, the
fertilization rate, wet weight of water-hardened eggs and the percent weight increase due
to hardening significantly decreased in comparison to fresh samples, but the ovarian
fluid pH and aspartate aminotransferase activity significantly increased ŽTable 8.. Also,
under storage conditions, the wet weight of water-hardened eggs, the percent weight
increase due to water hardening, and the ovarian fluid pH, and aspartate aminotrans-
ferase activity correlated highly significantly with the fertilization rate ŽTable 8..

4. Discussion

To detect correlations between egg parameters and egg viability, it is necessary to

analyze samples exhibiting a wide range of quality. Therefore, all available egg batches
were included into the experiments. In individuals from wild populations, egg composi-
tion and quality are probably influenced mainly by external, environmental factors,
whereas in individuals in culture, the characteristics are likely to be more affected by the
response of individual fish to hormone treatment or by their tolerance to stress during
handling.
Preliminary experiments on Cha. chalcalburnus ŽLahnsteiner, unpublished., C. car-
pio ŽMagyary et al., 1996., and Cte. idella ŽHorvath, unpublished. revealed that the rate
of morula stage embryos and the rate of hatched embryos were highly significantly
correlated with each other. Therefore, egg viability was determined at the morula stage
in Cte. idella, H. molitrix, and C. carpio. The fact that the fertilization rate correlated
with numerous and similar biochemical and physiological parameters in Cte. idella, H.
molitrix, and C. carpio also indicated that the survival rates of morula stage embryos
and hatched embryos were closely related with each other and with egg viability.
During the 4-h short-term storage of eggs of Cte. idella and C. carpio in ovarian
fluid, the egg fertilization rates decreased by more than 50%. Unpublished data
demonstrated that the egg viability decreased to a similar extent when the ovarian fluid
was removed from the eggs before storage. Therefore, the viability of cyprinid eggs is
unstable during short-term storage which is in accordance with earlier data ŽWithler,
1980; Billard, 1988.. By contrast, in the Salmonidae egg viability is maintained constant
for up to 1 month when the ovarian fluid is drained off and the eggs are stored dry or in
artificial saline solutions at 0–38C ŽRosenberg, 1983; Munkittrick et al., 1992..
For statistical analysis, the data were expressed as units per egg and as units per
milligram egg. As similar statistical relationships were obtained for the two data sets,
both were suitable for the determination of egg quality. The described regression models
not only described the relations between the analytes and the fertilization rate but also
distinguished high and low quality eggs using 70% fertilization rates as the bordering
line between. In comparison to other statistical procedures Že.g. ANOVA models., the
 F. Lahnsteiner et al.r Aquaculture 195 (2001) 331–352 349

regression models have advantages as they are continuous mathematical functions which
allow infinitely variable classifications.
4.1. Egg weight, egg weight increase during hardening and the correlation with the
fertilization rate
In Cte. idella, H. molitrix, C. carpio and Cha. chalcoides, the weight of the fresh,
unhardened eggs gave no indication of egg viability defined as fertilization rate. During
water hardening, the egg weight increased due to water influx. The weight increase was
significantly higher for the pelagic eggs of Cte. idella and H. molitrix, than for the
demersal eggs of C. carpio and Cha. chalcoides which indicates that the eggs of Cte.
idella and H. molitrix became buoyant. This was confirmed by the fact that only
water-hardened eggs floated. Generally, two different mechanisms are responsible for
water influx during water hardening: Ž1. water flows into the perivitelline space
following the osmotic gradient originating from non-permeable osmolytes such as the
polysaccharides and muco-polysaccharides released from the cortical vesicles ŽJaffe,
1985; Alderdice, 1988.. The cortical reaction itself is induced after depolarization of the
egg membrane which is triggered by a contacting sperm cell ŽAlderdice, 1988.. Since in
the investigated species, the weight increase during hardening was similar for unfertil-
ized and fertilized egg batches, the cortical reaction was also induced by membrane
depolarization due to changes in the environment which was established by histological
investigations in Cha. chalcoides ŽLahnsteiner, unpublished data.. Ž2. Water flows also
into the egg as the membrane of the fresh, unfertilized egg is highly permeable
ŽAlderdice, 1988.. In the investigated species, the occurrence of this mechanism was
established by the correlation between the weight of water-hardened eggs and the weight
increase during water hardening and the levels of peptides and fructose, two osmotically
active compounds of the egg.
In Cte. idella, H. molitrix, C. carpio and Cha. chalcoides, fertilization rate was
positively correlated with the weight of the water-hardened eggs and with the rate of
water uptake during hardening. Therefore, reduced water uptake possibly indicated
insufficient cortical reaction and impaired water flow into the egg due to low levels of
osmolytes. In short-term stored egg batches of Cte. idella and C. carpio, the weight of
the hardened eggs and the weight increase due to water hardening were significantly
lower than in fresh samples while the levels of the osmotically active compounds
fructose and peptides remained constant. Probably the potential and permeability of the
oolemma changed during storage and cortical reaction and water flows into the egg were
reduced or inhibited.
The present data are in accordance to earlier ones on the cod, G. morhua. In this
species, the cortical reaction is rapid and homogenous, and is followed by significant
increases in osmolality and egg diameter in high quality eggs. However, it is incomplete
and takes twice as long in low quality eggs ŽKjørsvik and Lønning, 1983; Kjørsvik et al.,
1984..
4.2. OÕarian fluid parameters and fertilization rate
Ovarian fluid pH significantly correlated with the fertilization rate in the form of a
quadratic function in C. carpio and Cha. chalcoides but negatively and linearly in Cte.
350 F. Lahnsteiner et al.r Aquaculture 195 (2001) 331–352

idella, possibly, as the low sample numbers did not allow the full range of fluctuations
to be monitored. Ovarian fluid pH may have changed due to Hq diffusion or transport
between ovarian fluid and eggs or epithelium of the ovarian cavity or due to leakage of
metabolites, e.g. lactic acid, amino acids or proteins. In the stored samples also the
lability of the ovarian fluid carbonic acid-bicarbonate buffer to changes in environmental
CO 2 partial pressures ŽBell et al., 1968. could have contributed to pH changes. As
physiological consequence pH may have changed the potential and conductivity of the
oolemma ŽGilkey, 1983; Jaffe, 1985..
Increased levels of ovarian fluid proteins and of aspartate aminotransferase could
have originated from ovarian synthetic activities, from cell leakage, or lysis of eggs in
the ovaries. Aspartate aminotransferase is generally an enzyme which is highly soluble
in the cytoplasm and catalyses the reversible transamination of aspartate into ketoglu-
tarate ŽRawn, 1983.. The fact that the ovarian fluid aspartate aminotransferase activity
significantly increased during short-term storage conditions is an indication of its
leakage from the eggs.
In the turbot, Sco. maximus ŽFauvel et al., 1993. ovarian fluid pH and protein
correlated with fertilization rate and were indicative for overripe eggs. In S. trutta f.
lacustris ŽLahnsteiner et al., 1999a. ovarian fluid pH, protein levels and activities of
aspartate aminotransferase are markers for the egg quality.

4.3. Egg metabolism and fertilization rate

In Cte. idella, H. molitrix, C. carpio and Cha. chalcoides, the egg fertilization rate
correlated with the activities of malate dehydrogenase and pyruvate kinase, and in C.
carpio and Cha. chalcoides with respiration activity, too. The reasons why the respira-
tion activity did not correlate with the fertilization rate in Cte. idella and H. molitrix is
uncertain, possibly it may depend on lower sample numbers. In summary, these enzymes
are involved in the cell energy metabolism whereby the NAD-dependent malate
dehydrogenase reduces NAD to NADH which is mainly used as co-substrate in the
respiration chain, and pyruvate kinase catalyzes the transformation of phosphoenolpyru-
vate to pyruvate whereby ADP is phosphorylized to ATP ŽRawn, 1983.. The regression
models for fertilization rate and malate dehydrogenase and fertilization rate and pyruvate
kinase were quadratic functions, with both high and low activities correlating with
reduced egg viability and alterations in egg metabolism. By contrast, respiration rate was
positively correlated with the fertilization rate and a high activity was indicative of good
egg quality. Generally, glycolysis is considered as necessary for energy supply of
unfertilized eggs ŽMommsen and Walsh, 1988. and aerobic energy metabolism for
embryogenesis ŽRombough, 1988; Finn et al., 1995..
Parameters which describe the egg energy reserves Žprotein, peptides, fructose,
galactose, glucose, non-esterified and esterified fatty acids. were not correlated with the
fertilization rate. The yolk energy reserves are most important for the larvae before they
start exogenous food uptake ŽMommsen and Walsh, 1988.. Neither the levels of nucleic
acids nor the ratio of DNA to RNA levels were correlated with egg viability although
these are helpful in estimating the growth rates of embryos and larvae ŽClemmesen,
1988.. In comparison to fresh samples, the ovarian fluid protein levels and the egg
 F. Lahnsteiner et al.r Aquaculture 195 (2001) 331–352 351

enzyme activities in the stored samples were not correlated with fertilization rates. This
indicates that mechanisms influencing the egg viability in stored samples differ from
those in fresh samples; however, the distinct metabolic processes are still unknown.

Acknowledgements

¨
Supported by AStiftung Aktion Osterreich-UngarnB ¨ ., Austrian Bun-
Žproject 34OU7
¨ Land-und Forstwirtschaft, Austrian AFond zur Forderung
desministerium fur ¨ der wis-
senschaftlichen ForschungB Žproject P10577-BIO., Hungarian Scientific Fund ŽOTKA
´
no. F025306., Orszagos ˆ
Muszaki ´ Bizottsag
Fejlesztesi ´ and Bundesministerium fur ¨
¨
auswartige Angelegenheiten ŽTET A-32r98.. The authors wish to express their gratitude
to Dinnyes fish farm and TEHAG fish farm for providing animal stock and to Mr. Szolt
¨
Mullner, ´ Sule
Mr. Peter ¨ and Wallis Motor LDT for supply with equipment.

References

Alderdice, D.F., 1988. Osmotic and ionic regulation in teleost eggs and larvae. In: Hoar, W.S., Randall, D.J.
ŽEds.., Fish Physiology. The Physiology of Developing Fish. Eggs and Larvae, vol. 11. Academic Press,
London, pp. 163–251, part A.
Bell, G.H., Davidson, J.N., Scarborough, H., 1968. Textbook of Physiology and Biochemistry. Livingstone,
London.
Bergmeyer, H.U. ŽEd.. 1985. Methods of Enzymatic Analysis. VCH Verlagsgesellschaft, Weinheim.
Billard, R., 1988. Artificial insemination and gamete management in fish. Mar. Behav. Physiol. 14, 3–21.
Bromage, N.R., Roberts, R.J., 1994. Broodstock Management and Egg and Larval Quality. Blackwell, Oxford.
Chaudhry, K., 1989. Biochemical Techniques. Medical Publishers, New Delhi.
Clark, J.M., Switzer, R.L., 1977. Experimental Biochemistry. Freeman, San Francisco.
Clemmesen, C.M., 1988. A RNA and DANN fluorescence technique to evaluate the nutritional stage of
individual fish larvae. Meeresforschung 32, 134–143.
Craik, J.C.A., Harvey, S.M., 1984. Egg quality in rainbow trout: the relation between egg viability, selected
aspects of egg composition, and time of stripping. Aquaculture 40, 115–134.
Fauvel, I.C., Omnes,` M.H., Suquet, M., Normant, Y., 1993. Reliable assessment of overripening in turbot
Ž Scophthalmus maximus . by a simple pH measurement. Aquaculture 117, 107–113.
Finn, R.N., Rønnestad, I., Fyhn, H.J., 1995. Respiration, nitrogen and energy metabolism of developing
yolk-sac larvae of Atlantic halibut Ž Hippoglossus hippoglossus L... Comp. Biochem. Physiol. 111A,
647–671.
Gilkey, J.C., 1983. The role of calcium and pH in activation of eggs of the medaka fish, Oryzias latipes. J.
Cell. Biol. 76, 448–466.
Holtzhauer, M., 1988. Biochemische Arbeitsmethoden. Springer–Verlag, Berlin.
Jaffe, L.F., 1985. The role of calcium explosions, waves, and pulses in activating eggs. In: Metz, C., Monroy,
B. ŽEds.., Biology of Fertilization, vol. 3. Academic Press, Orlando, FL, pp. 127–165.
Kjørsvik, E., 1994. Egg quality in wild and broodstock cod Gadus morhua L. J. World Aquacult. Soc. 25,
22–29.
Kjørsvik, E., Lønning, S., 1983. Effects of egg quality on normal fertilization and early development of the
cod, Gadus morhua L. J. Fish Biol. 23, 1–12.
Kjørsvik, E., Stene, A., Lønning, S., 1984. Morphological, physiological and genetical studies of egg quality
in cod Ž Gadus morhua L... In: Dahl, E., Danielssen, D.S., Moksness, E., Solemdal, P. ŽEds.., The
Propagation of Cod, Gadus morhua L. Flødevigen rapportser, vol. 1, pp. 67–86.
352 F. Lahnsteiner et al.r Aquaculture 195 (2001) 331–352

Kjørsvik, E., Mangor Jensen, A., Holmefjord, T., 1990. Egg quality in fishes. Adv. Mar. Biol. 26, 71–113.
Lahnsteiner, F., Weismann, T., Patzner, R.A., 1999a. Physiological and biochemical parameters for egg
quality determination in lake trout, Salmo trutta lacustris. Fish Physiol. Biochem. 20, 375–388.
Lahnsteiner, F., Berger, B., Weismann, T., 1999b. Sperm metabolism of the teleost fishes Oncorhynchus
mykiss and Chalcalburnus chalcoides and its relation to motility and viability. J. Exp. Zool. 284, 454–465.
Loewenstein, J.M., 1983. Methods in Enzymology: Carbohydrate Metabolism. Academic Press, New York.
Lorenz, R.J., 1984. Biometrie. Gustav Fischer Verlag, Stuttgart.
Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol
reagent. J. Biol. Chem. 193, 265–275.
´
Magyary, I., Urbanyi, ´ L., 1996. Cryopreservation of common carp Ž C. carpio L.. sperm. II:
B., Horvath,
Optimal conditions for fertilization. J. Appl. Ichthyol. 12, 117–119.
Mommsen, T.P., Walsh, P.J., 1988. Vitellogenesis and oocyte assembly. In: Hoar, W.S., Randall, D.J. ŽEds..,
Fish Physiology. The Physiology of Developing Fish. Eggs and Larvae, vol. 11. Academic Press, London,
pp. 347–406, part A.
Morisawa, M., 1983. Effects of osmolality and potassium on motility of spermatozoa from freshwater cyprinid
fishes. J. Exp. Zool. 107, 95–103.
Munkittrick, K.R., McGeachy, S.M., Burke, M.G., Flett, P.A., 1992. Effect of delay in water addition or
rinsing on fertilization rates of Chinook salmon, Coho salmon, Atlantic salmon and rainbow trout eggs.
Prog. Fish-Cult. 54, 14–20.
Pavlov, D.A., Moksness, E., 1994. Production and quality of eggs obtained from wolffish Ž Anarhichas lupus
L.. reared in captivity. Aquaculture 122, 295–312.
Rawn, J.D., 1983. Biochemistry. Harper and Row, New York.
Rombough, P.J., 1988. Respiration, gas exchange, aerobic metabolism, and effects of hypoxia during early
life. In: Hoar, W.S., Randall, D.J. ŽEds.., Fish Physiology. The Physiology of Developing Fish. Eggs and
Larvae, vol. 11. Academic Press, London, pp. 59–161, part A.
Rosenberg, D.L., 1983. Fertilization success of coho salmon gametes: effects of storage under various
atmospheric conditions, temperature and acclimation, and temperature variations. Prog. Fish Cult. 45,
84–87.
Shields, R.J., Brown, N.P., Bromage, N.R., 1997. Blastomere morphology as a predictive measure of fish egg
viability. Aquaculture 155, 1–12.
Withler, F.C., 1980. Chilled and cryogenic storage of gametes of Thai carps and catfishes. Can. Tech. Rep.
Fish. Aquat. Sci. 948, Ottawa.