Aquaculture International 12: 3–10, 2004.© 2004 Kluwer Academic Publishers. Printed in the Netherlands.

The first attempt to artificially reproducethe endangered cyprinid lake minnowEupallasella perenurus (Pallas)

RAFAŁ KAMINSKI1,∗, JAN KUSZNIERZ2, LESZEK MYSZKOWSKI1

and JACEK WOLNICKI1

1Pond Fishery Department, The Stanisław Sakowicz Inland Fisheries Institute in Olsztyn,Zabieniec, 05-500 Piaseczno, Poland; 2Zoological Institute, University of Wrocław,Sienkiewicza 21, 50-335 Wrocław, Poland; ∗Author for correspondence(e-mail: erka@infish.com.pl)

Received 10 November 2002; accepted 9 July 2003

Abstract. The standard artificial reproduction technique with the use of Ovopel (GnRH ana-logue) was evaluated for the critically endangered cyprinid species in Poland, the lake minnowEupallasella perenurus. Wild spawners were angled at the beginning of the spawning season.Only females with a condition coefficient value higher than the mean were included in theexperiment and then stimulated with Ovopel. Group A (n = 20) was given standard treatment(0.2 pellet kg−1 and 1.0 pellet kg−1 after 12 h), whereas group B (n = 8) was treated witha single dose of 2.0 pellets kg−1. After fertilisation with the dry method, the glutinous eggsattached to the bottom of flow-through aquaria were incubated at 17.1–18.4◦C. Throughoutincubation (85D◦), dead eggs were removed and counted. The same was done with deadlarvae, those with deformed bodies or those with empty alimentary tracts after 2 days of ex-ternal feeding. Two injections of Ovopel resulted in a significantly (P ≤ 0.05) higher ovulationrate in comparison with the single dose (70 and 25% in groups A and B, respectively). Theindividual hatching rates were very high (98.4–100%), as was the share of good quality larvae(91.1 and 96.5% of stripped eggs in groups A and B, respectively). These results indicate thatthe standard propagation method used with commercially important cyprinid species can alsobe used to successfully breed E. perenurus.

Key words: Eupallasella perenurus, Fecundity, Larval quality, Ovopel stimulation,Reproduction

Introduction

The lake minnow Eupallasella perenurus (Pallas), formerly classified in thegenus Phoxinus (Kottelat, 1997), is a member of the Cyprinidae family. Thisfish is widely distributed throughout Eurasia. Its geographical range extendsfrom central Poland in the west to northern Japan and the Korean Peninsulain the east (Witkowski, 1992; Kusznierz, 1998). This short-living (up to 6

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years) fish only reaches a maximum total length of merely 13 cm, and it hasno commercial value anywhere (Kusznierz, 1998). It is a phytophilous speciesthat inhabits extremely vulnerable to destruction, tiny and shallow still waterbodies with dense submerged vegetation, mostly natural dystrophic pools orman-made peat-hags and clay-pits. It can occur solely but its common cohab-itants are the dwarf form of the crucian carp Carassius carassius and/or thetench Tinca tinca (Kusznierz, 2001).

In Europe, E. perenurus is considered an endangered species (Maitland,1995). In Poland, many habitats of this fish have disappeared over the pastfew decades due to anthropogenic factors such as swamp draining, ameliora-tion, deforestation and the filling in of field water bodies (Witkowski, 1992).As a consequence, this species is presently listed as being in critical dangerof extinction in the Red Data Book of Polish Animals (Kusznierz, 2001),and is protected by law (Witkowski, 1992). Today the total number of itspopulations in Poland can be estimated at about 25 (Kusznierz et al., 2002);however, those of the least size count only a few hundred specimens.

It is widely recognised that E. perenurus urgently requires active pro-tection based on the captive breeding and stocking of hatchery-reared ju-venile individuals in an attempt to restore or maintain its wild populations(Kusznierz, 1998; Kusznierz et al., 2002). This is the case with many fresh-water fish species (Brown and Laland, 2001). The present work describes thefirst ever attempt to artificially reproduce wild E. perenurus under controlledconditions. The purpose of the study was to determine the effectiveness of thestandard propagation technique used with commercially important cyprinids,based on the application of a synthetic GnRH analogue in two doses (Horváthet al., 1997) for the breeding of E. perenurus. Additionally, the effectivenessof a single dose of the same hormone was evaluated using a smaller fishsample.

Material and methods

Fish

With the permission of The Ministry of the Environment, 91 wild E. peren-urus spawners were angled in a small dystrophic pool located near Warsaw,Poland, at the end of April, that is, at the beginning of the spawning sea-son when the diurnal water temperature range was 13–16◦C. All the fishwere transferred to a laboratory and held together in a shadowed tank (V =1000 L) fed continuously (0.3 L min−1) with well-aerated water of 13◦C. Thefish were kept for a natural photoperiod and not fed. On the next day, theirindividual total length and body weight were determined, and then their con-

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Table 1. Total length, body weight and condition of E. perenurus females stimulated with two(group A) or one (group B) Ovopel injections

Group n Total length (mm) Body weight (g) Condition coefficient (K)

A 20 79.3 ± 3.6a 5.82 ± 0.85a 1.16 ± 0.07a

B 8 76.5 ± 2.2b 5.19 ± 0.61a 1.15 ± 0.05a

Data are means ± SD.In columns, data with the same superscripts are not significantly different (Student’s t-test;P ≤ 0.05).

dition coefficient was calculated. The males producing the largest amounts ofdense milt (24 individuals) were placed in an aerated flow-through aquariumwith a water volume of 40 L. Their total length, body weight and conditioncoefficient were 76.1 ± 6.4 mm, 3.75 ± 1.00 g and 0.83 ± 0.05 (mean ± SD),respectively. For all females, the condition coefficient value was 1.08 ± 0.08(mean ± SD). Only 28 females with well-rounded bellies and condition coef-ficients higher than 1.08 were assumed to be ripe and were used in the presentexperiment (Table 1). The remaining fish were considered immature or notripe or already spawned in the pool.

Experimental procedures

All the selected females were stimulated with intraperitoneally injected GnRHanalogue, Ovopel, which is obtainable as pellets weighing approximately25 mg (Horváth et al., 1997). Each pellet contains 18–20 µg of D-Ala6,Pro9NEt-mGnRH and 8–10 mg of dopamine antagonist, metoclopramide. Inexperimental group A, Ovopel was injected in two doses of 0.2 pellet kg−1

and 1.0 pellet kg−1 after 12 h. In group B, Ovopel was injected in a singledose of 2.0 pellets kg−1. The males were not stimulated.

The female groups were kept separately in aerated flow-through aquaria(V = 40 L). The fish were checked for ovulation 12, 24, 28, 36 and 48 hafter the first hormone injection. Only females that released more than 0.1 gof eggs were considered ovulated. All spawners were anaesthetised in 2-phenoxyethanol (0.45 g L−1) prior to any manipulation.

Eggs, brownish and opaque, were stripped from each female onto a sepa-rate, small plastic spoon and weighed. Dry eggs were stirred with sperm takenfrom at least 4 males, then fertilised in a small amount of water for 15 s.

Fertilised eggs were incubated in separate 10 L flow-through aquariawithout removing their natural strong stickiness. The eggs were dispersedon the aquarium bottom (540 cm2) to which they attached in a single layer. Inaddition, to determine egg diameter, a sample of 25 swollen eggs was takenfrom one average-sized female.

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Eight females were chosen at random from the two experimental groups(six from group A and two from group B) in order to determine the fecundityand hatching rates individually. The dead eggs were removed and countedthree times daily. Mass hatching took place in all the aquaria after 120 h(85D◦).

The larvae that hatched from eggs taken from eight females mentionedabove remained in their aquaria and were used to evaluate biological quality.From day 4 post-hatch, when the swim bladder inflated, the larvae were fedfor 2 days with freshly hatched, live Artemia nauplii (INVE Aquaculture)ad libitum five times a day. During feeding time (08:00–21:00), the aquariawere illuminated with fluorescent tubes, which provided a light intensity ofabout 1000 lx at the water surface. Dead larvae and larvae with visible bodydeformities were removed and counted three times a day. On day 6 post-hatch, 30 min after feeding, the final counting of all live larvae was performed.Only larvae with food in the alimentary tract were considered to be of goodbiological quality.

The stripping fecundity was calculated as a sum of all dead eggs foundwhile incubating and all the hatched larvae.

Water properties

All aquaria used in the experiment were fed water originating from the re-circulating system with diatomite bed biofilter. Water flow in the aquariawas set at 0.2 L min−1 before and during hormonal treatment and in the in-cubation and rearing periods. The water temperature and dissolved oxygencontent were measured twice a day, whereas nitrites, ammonia and pH weremeasured at the beginning and end of the experiment. Water temperaturethroughout the experiment was kept between 17.1 and 18.4◦C. It is very sim-ilar to the temperature conditions in the E. perenurus habitat in the middleof the spawning season (16–19◦C; Kusznierz, 1998). Oxygen saturation al-ways exceeded 90%. The nitrite concentration was below 0.02 mg L−1, theammonia concentration was lower than 0.1 mg L−1 and pH was 7.8.

Measurements, calculations and data analysis

The fish were measured to the nearest 0.1 mm. The fish and stripped eggswere weighed to the nearest 0.01 g. The eggs were measured using a micro-scope to the nearest 0.01 mm.

The condition coefficient (K) was calculated following the formula: K =105 BW TL−3, where BW is body weight in g, and TL is total length in mm.

The average weight of an egg was calculated dividing weight of all strippedeggs by their number.

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The mean total length, mean body weight and mean condition coefficientof females were compared using Student’s t-test.

Ovulation rates were normalised using√

arcsine transformation (Sokaland Rohlf, 1969).

Results

When used in two doses, Ovopel induced ovulation in 70% of 20 females ingroup A (Table 2). In group B, this figure was 25% of 8 individuals. Thesevalues differed significantly (P ≤ 0.05).

Table 2. Ovulation rate and fecundity in E. perenurus stimulated with two (group A) or one(group B) Ovopel injections

Group Ovulation Relative weight of Stripping fecundity Relative stripping

rate (%) stripped eggs (egg number) fecundity (egg number

(% of BW) per 1 g BW)

A 70 13.7 ± 0.02∗ 771 ± 142∗∗ 141 ± 21∗∗B 25 13.0 ± 0.01∗∗∗ 806 ± 3∗∗∗ 135 ± 13∗∗∗

Data are means ± SD.∗n= 14; ∗∗n= 6; ∗∗∗n= 2; percent of ovulated females differ significantly at P ≤ 0.05.

In group A, 14, 36 and 50% of the ovulated females gave eggs after 24,28 and 36 h following the first injection, respectively, and in group B, 50% offemales gave eggs after 28 and the remaining after 36 h.

The weight of stripped eggs was dependent on the fish body weight (y =0.14 × BW −0.023, r2 = 0.52, P ≤ 0.01, where y is the weight of the strippedeggs) in all the ovulated females (both groups pooled). The relative weight ofthe stripped eggs ranged from 10% to 16% of the fish body weight. The meanabsolute stripping fecundities and mean relative fecundities were similar in

Table 3. Hatching rate and percent of E. perenurus feeding larvae in female group A (twoOvopel injections) and female group B (single Ovopel injection)

Group Hatching rate (%) Feeding larvae (% of

stripped egg number)

A 99.3 ± 8.5 91.1 ± 21.2

B 99.5 ± 1.4 96.5 ± 13.0

Data are means ± SD.n= 6 for group A, n= 2 for group B.

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groups A and B (Table 2). The unswollen egg wet weight was 1.00 ± 0.05 mg(mean ± SD), the swollen egg diameter was 1.43 ± 0.04 mm (mean ± SD).

The individual hatching rates ranged from 98.4% to 100%. The mean val-ues for groups A and B exceeded 99% (Table 3). The mean share of feedinglarvae exceeded 90% of stripped eggs in groups A and B.

Discussion

In the present experiment, the ovulation rate induced by Ovopel in two in-jections was high (70%), and similar to the values of 60–100% recorded incommon carp Cyprinus carpio, white amur Ctenopharyngodon idella andsilver carp Hypophthalmichthys molitrix, which were treated in the samemanner (Horváth et al., 1997). The single dose of Ovopel given to E. peren-urus was considerably less efficient (Table 2), as has also been observed inbarbel Barbus barbus (Wolnicki J., unpublished data). The lower efficiencyof a single hormone dose was also recorded in C. carpio treated with carppituitary (Tamás and Horváth, 1984).

In the present study, the fish were bred at a temperature that was within therange of temperatures that occur during their natural spawning (Kusznierz,1998). The latency time recorded in the present experiment was 24–36 h fromthe first Ovopel injection. These values are similar to those observed in othercyprinid species stimulated with GnRH analogues at the same temperaturesas those during spawning in the wild (Herzig and Winkler, 1985), includingB. barbus (Ciesla et al., 2000), T. tinca (Barth et al., 1997), vimba Vimbavimba (Sliwinski et al., 2000) and C. carassius (Wolnicki J., unpublisheddata).

According to the extensive literature survey by Kusznierz (1998), the ab-solute fecundity of E. perenurus of a size similar to fish used in the presentstudy ranges from around 400 to 1200 eggs per female. The individual strip-ping fecundities recorded in our experiment were within this range, whereasthe relative weight of stripped eggs ranged from 10% to 16%. These figuresdo not differ much from those found for such cyprinids as asp Aspius aspius(Sliwinski, 2000), bream Abramis brama (Adámek et al., 2002), C. carpio(Brzuska, 1997), and T. tinca (Barth et al., 1997). Since the gonadosomaticindex in E. perenurus females at the beginning of the spawning season is closeto 18% (Kusznierz, 1998), one can conclude that ovulation in the presentexperiment was complete.

It should then be stressed, in this experiment, the hatching rates were ex-tremely high, in some cases reaching even 100%. It is also noteworthy that, inmost cases, the feeding larvae constituted more than 90% of the egg numberstripped from a female (Table 3). This is considerably better than 40–60%

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usually recorded for larvae before the first feeding in the mass production ofcyprinids such as C. carpio, C. idella or T. tinca (Okoniewski Z.J., personalcommunication).

In contrast with the exceptionally high hatching rates obtained in thiswork, the E. perenurus ovulation rate of 70%, though satisfactory, couldbe improved considerably. Hormonal stimulation can induce ovulation onlywhen the ovary is at an appropriate stage of maturation (Bieniarz and Epler,1991). Therefore, differences in ovary maturation might have been the reasonthat some of the E. perenurus females did not ovulate. A more effectivemethod of female ripeness evaluation, based not only on fish condition coef-ficients and body shape but also on the gonad maturation assessment, seemsnecessary. However, the standard method of oocyte sampling by means ofa probe inserted into the genital opening seems to be difficult to apply anddangerous for the females due to their small size.

Acknowledgement

This study was supported by a Grant from the State Committee for ScientificResearch No. 6 P04G 055 21.

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