EMBRYONIC DEVELOPMENT OF ASPIUS ASPIUS L. (ACTINOPTERYGII: CYPRINIFORMES: CYPRINIDAE)

Agata Korzelecka-Orkisz¹, Małgorzata Bonisławska², Adam Tański¹, Izabela Smaruj¹, Joanna Szulc¹, Krzysztof Formicki^3
1 Division of Hydrobiology, Ichthyology and Biotechnology of Biotechnology of Reproduction, West Pomeranian University of Technology in Szczecin, Szczecin, Poland
² Department of Aquatic Sozology, West Pomeranian University of Technology in Szczecin, Szczecin, Poland
³ Division of Hydrobiology, Ichthyology and Biotechnology of Biotechnology of Reproduction,
West Pomeranian University of Technology in Szczecin, Szczecin, Poland

ABSTRACT

Fertilized eggs asp (Aspius aspius L.) were incubated in water under constant temperature of 10 ±0.2°C. Egg membranes were observed under a scanning electron microscope. Images of the eggs and newly hatched larvae were recorded, measured and analyzed. Asp eggs are surrounded by 12.1 µm thick egg membrane. On the outside, the membrane comprises of club-shaped formations connected together in a network, they are weakly joint with the deeper layer of the membrane, hence when the egg is detached from the substrate this layer becomes detached from the parent structures.

During embryogenesis, which lasted 20 days, it was observed that: diameter of hydrated egg is 2.03 mm, while the diameter of the yolk sphere is 1.37 mm, volume of the egg is 4.36 ±0.19 mm³ and yolk sphere volume 1.33 ±0.14 mm³. The blastodisc, and later the embryos, is situated in side position in the egg; the first single movements of the trunk appeared before 40% of the period of embryonic development had elapsed. The greatest embryo activity was observed after ½ of the embryogenesis. Start of operation of the heart primordia, and later heart caused a drop in somatic activity of the embryo. Newly hatched asps measured 7.43 ±0.27 mm and were equipped with double-chamber yolk sac of volume 0.72 ±0.13 mm³. Pigment of hatched individuals could be seen in the eyes (intense) and on the body.

Key words: fisheries, asp (Aspius aspius L.), embryonic development, egg membranes.

INTRODUCTION

One of the most interesting among the carp family fish, in addition to quite unusual feeding habits, is the asp (Aspius aspius L.), which up to the end of the eighteenth century were fished in large quantities. Today, this species has no significant economic importance. However, due to its predatory lifestyle it plays an important regulatory role in biocenoses. It is also an interesting fishing trophy. Continual degradation of the asp natural spawning habitat is limiting its population.  On the other hand, because of the rapid multiplication of this species the  asp reproduction and breeding cycle has became a target for research studies in Poland and many European countries [2, 15, 17, 27, 28]. This in turn promotes efforts to rebuild and increase its population, which is of significance in maintaining biodiversity [21].

In the category of endangered species, in accordance with the IUCN/WCU classification, Witkowski et al. [33] qualified asp species to the NT/LC (near threatened/least concern) category. It has to be emphasized that this species has been qualified to a group of animals whose conservation requires designation of special conservation habitats.

Asp is found in Europe, to the east of the Rhine, in the catchment areas of the Northern, Black and Caspian Seas. The eastern limits of its distribution is bound by the rivers: Ural, Emba, Sagiz, Bolshoi and Malyj Uzeń [18, 23]. It inhabits fresh water, lower reaches of large rivers, sometimes standing water – backwaters and oxbow lakes as well as lakes and reservoirs.

The absolute fecundity of this species ranges from 80 000 to 400 000 eggs [10]. These fish spawn from March to May, when the water level in most European rivers is high [1, 18, 23]. Average water temperature during the spawning season ranges from 5 to 12°C. Asp spawn, like many other fish of the carp family, has a sticky coating, which allows it to attach itself to the substrate, hence the embryo develops under relatively comfortable aerobic conditions, away from the bottom sediments, where there might be oxygen deficit [19]. The incubation period of asp eggs varies with water temperature and ranges between 6 (underwater temperature of 15–16°C) and 21 days [15]. Asp egg diameter is in the range 1.3–1.7 mm. Asp embryogenesis runs normally in a fairly wide temperature range, from 7.0 to 17.2°C, and even 19.7–24.3°C [19, 16]. The survival rate of embryos at 12.8°C is the highest, it was 86.3% and the duration of embryonic development was 179 D° [15].

Literature on the biology of asp is extensive, but much less data on various aspects of embryonic development of asp is available, and yet a more and more better understanding of the biotechnology of reproduction of this valuable and interesting species would help to effectively protect and restore its population This is the reason why attention has been focused on the structure of egg membranes while morpho-physiological changes occur during embryogenesis, from the moment of activation to hatching, under the environmental conditions considered optimal. In addition, features of developing larvae were analyzed and presented.

MATERIALS AND METHODS

The asp spawners used for the experiments came from a pond farm of the Polish Anglers Association Stocking and Breeding Centre in Goleniów. After transfer of males and females to the hatchery, they were kept in 1000-liter, constantly aerated, water tanks at a temperature of 6 ±0.5°C. The females were twice, in 24-hour interval, subjected to hormonal stimulation using the Ovopel (D-Ala6, Pro9-Net-mGnRH; Unic-trade, Hungary) preparation and at the same time water temperature was raised by 2ºC within 12 hours. Eggs were collected from the females 24 hours after the last dose. The sperm collected from the males did not require any additional treatment.

Standard fertilization, “dry” method, was carried out in the Department of Hydrobiology, Ichthyology and Biotechnology of Reproduction laboratory. After hydration the eggs were placed in crystallizers with water, equipped with aerators for adequate water aeration and analyzed for water quality parameters (Table 1).

Table 1. Selected physical and chemical incubation water parameters

Parameter	Tap water
Temperature [°C]	9,8–10,2
pH	7.03–7.60
Dissolved oxygen [mgO2dm-3]	9.80–10.55
Electrolytic conductivity. [μScm-1]	325–435
Alkalinity [mg CaCO3dm-3]	164–185
General hardness [mg CO3∙dm-3]	210–215
Chlorides Cl- [mg∙dm-3]	51.8–56.8
Ammonium nitrogen N- NH4+ [mg∙dm-3]	0.009–0.010
Nitrite nitrogen N-NO2- [mgN∙dm-3]	0.005–0.008

Egg membranes
Egg membranes were collected three minutes and five hours after fertilization, they were next preserved in 4% formaldehyde, rinsed in distilled water, dehydrated in a series of alcohol solutions of increasing concentrations (70% and 96%), and then further dehydrated using acetone. After spraying with palladium and gold alloy, samples were observed under a scanning electron microscope (FEI Quanta 200), photographs were taken and documented.

Embryogenesis
Live registration of asp embryonic development was performed using sets of equipment consisting of a light microscope (Nikon–ECLIPSE TE–2000S) and stereomicroscope (SMZ 1500) with microprocessor controller (Trol–8100/9100) and color digital camera (Nikon DS. Fi-1.), monitor (LG), VCR (JVC-HR-S7700) and computer (PC). Duration of asp embryogenesis was, during the early stages of development, given in degree-hours (H° – calculated as the sum of water temperature at hourly intervals during the development period), then next in degree-days (D° – calculated as the sum of water temperature each days during the development period).The respective stages of embryo development were determined when about 60% of the subjects had attained particular stage of development. In the course of embryonic development the percentage of fertilized eggs (at the stage of closing blastopore, by analyzing 500 eggs), the survival rate (number of larvae hatched from fertilized group, n=500) was determined.

Upon completion of the process of hydration of the eggs (n=30), their pictures were taken using NIS Elements, and then analyzed: two diameters measured [3]. Next, their volume and area were calculated using the formulae:

V = 4/3·πr³ mm³, S = 4·πr² mm²

where r is sphere radius [mm]

After obtaining the calculation results, the ratio S/V of  was determined.

In addition, a live documentation of 5 embryos that left the membranes, which were naturally torn, in the form of films and photographs was obtained, and this allowed a capture of progressive morphological changes taking place during embryogenesis of the asp, but is difficult to observe due to restricted transparency of the egg membrane.

Embryonic motor skills – the motor activity of asp embryos (n=30) was analyzed from the moment of the first movement (heart and body) to the moment of hatching. All types of movements in the developing embryos were recorded (for 5 minutes each). The number of contractions per minute of the heart primordia, and later of the heart of fully developed embryo as well as somatic movements were counted. Over the same time range (1 minute) the number of somatic movements of the body was determined.

Larvae
After hatching, measurements were done of total body length (longitudo totalis –l.t.) of larvae (n=30), and length (l) and height of their double-chamber yolk sacs. The yolk sac volume was calculated using the elongated ellipsoid volume formula (the front part of the bag) and a cylinder (rear part of the sac) [5, 8]:

V = (π/6·l₁·h₁²) + [π·(h₂/2)²·l₂]  [mm³]

where:  
l₁ – length of first part of yolk sac (ellipsoid) [mm];
h₁ – height of first part of yolk sac (ellipsoid) [mm];
l₂ – length of second part of yolk sac (cylinder) [mm];
h₂ – height of second part of yolk sac (cylinder) [mm]

The results obtained were processed using Excel 7.0.

The experiment was performed twice.

RESULTS AND DISCUSSION

Egg membranes
SEM pictures of the membranes, taken 3 minutes after activation, revealed that the micropyles are quite large – inlet measures about 80 μm, membrane thickness 12.1 µm (Fig. 1). The membrane is a structure with sub-layers of different thickness, externally equipped with club-shaped pads, between which numerous small pores can be distinguished. There are regularly spaced vertical cavities in the middle layer. The slope of the cavities becomes more inclined at the outer layer (Fig. 2).

Fig. 1. Micropyle (magnification 800 ×), 3 minutes after activation. SEM, scale bar 100 µm.

Fig. 2. Cross-section through egg membrane (magnification 15000 ×) - Pictures in the SEM, 5 hours after activation. SEM, scale bar 5 µm.

Spherical formations connected together (responsible for the adhesiveness of eggs) are found on the outer surface of the membrane (Fig. 3, 4). The club-shaped formations connect together in a network. While the egg detaches from the substrate, this layer becomes detached from the parent structures (Fig. 5).

Fig. 3. The outer surface of asp egg membrane – numerous cavities and adhesive discs (magnification 10000 ×), 5 hours after activation SEM, scale bar 10 µm.

Fig. 4. Magnified club-shaped formations (magnification 40000 ×), 5 hours after activation. . SEM, scale bar 3 µm.

Fig. 5. Excerpt of egg membrane with damaged sticky layer (magnification 5000 ×) 5 hours after activation. .SEM, scale bar 20 µm.

Eggs and the yolk sphere
Hydrated egg diameter is 2.03 ±0.03 mm and the yolk sphere diameter – 1.37 ±0.04 mm, which translates into an average volume of eggs of 4.36 ±0.19 mm3 and yolk spheres – 1.33 ±0.14 mm3 (Table 2). Eggs do not vary much in overall size, there is only about 20% difference between the smallest and largest. However, they vary more in terms of yolk sphere size, the difference is up to 40%. The space around the yolk that emerges within 30 minutes of activation in the asp eggs on average constitute up to about 70% of entire volume of the whole egg (Table 3). The ratio of egg surface area to its volume – the S/V coefficient, an indicator of the breathing efficiency of the developing embryo, is up to 2.96, for the yolk sphere this it reaches 4.41 (Table 2).

Table 2. Asp eggs features (±SD) – measurements taken in 8 Dº

Eggs parameters	±SD	Minimum	Maximum
Eggs diameter [mm]	2.03 ±0.03	1.94	2.06
Yolk sphere diameter [mm]	1.37 ±0.04	1.30	1.46
Eggs volume [mm3]	4.36 ±0.19	3.82	4.59
Yolk sphere volume [mm3]	1.33 ±0.14	1.16	1.63
Eggs S/V ratio	2.96 ±0.05	2.91	3.09
Yolk sphere S/V ratio	4.41 ±0.15	4.11	4.60
Perivitelline space [%]	69 ±3.5	63	74

Table 3. Process and period of asp embryogenesis (D°)

Stages of embryonic development	Development time (D°)
End of process of swelling of the egg (Fig. 6)	0.17
Emergence of fertilization cone	0.33
2 blastomeres	1.44
8 blastomeres	5.83
High-molecular-weight blastula (Fig. 7)	8.33
Epibole 1/3	23.02
Epibole 3/4	31.00
Development of the cephalic part (Fig. 8)	47.00
Emergence of 5-7 myomeres in the trunk section, start of division of yolk sac	63.00
First somatic movements of embryo trunk-tail section (Fig. 9)	71.10
Formation of lens in the eyes (Fig. 10)	79.00
Pigment in the eyes	111.10
Commencement of hatching (Fig.11)	169.00
50% hatch	200.00
End of hatching	245.00

Embryonic development
Shortly after fertilization ectoplasm elevation emerges inside the egg on the upper pole of the yolk sphere, giving rise to a blastodisc, due to the lack of a fat raft, and "slips" on to the side. This lateral position is maintained at later stages of embryogenesis.

The process of embryonic development, with separation of the different stages, from activation of the egg to the moment of larvae hatch, is shown in Table 2, while selected stages are shown in photographs (Fig. 6–11). A fertilization percentage of 80% was attained.

Fig. 6. Egg activation, emergence of perivitelline space and formation of fertilization cone. . Scale bar 1 mm.

Fig. 7. Early stages of asp embryonic development – high-molecular-weight morula. Scale bar 1 mm.

Fig. 8. Early organogenesis. Scale bar 1 mm.

Fig. 9. Division of yolk sac into two chambers. Scale bar 1 mm.

Fig. 10. Progressive resorption of rear section of the yolk sac

Fig. 11. Embryo preparing to leave the egg membranes. Scale bar 1 mm.

Cleavage
The process of division of blastodisc into blastomeres begins shortly after fertilization. The emergence of the first groove (2 blastomeres) is registered 3.5 hours after fertilization of the eggs (1.44 D°). The next groove dividing each of the blastomeres into two parts appears 7.5 hours after fertilization (3.13 D°), as a result four blastomeres are formed, the third groove (8 blastomeres) emerges 14 hours after fertilization (5.83 Dº). Finally, blastula emerges  after 20 hours from the moment of fertilization (8.23 D°) (Table 2).

Gastrulation
Gastrulation begins a day after fertilization, and after 23 Dº the eggs are at the stage of 1/3 epibole. Embryo body outline emerges 100 hours after fertilization (41.67 Dº)

Organogenesis
As the embryo develops, the head area begins to increase and become more and more distinct on the surface of the yolk sac (Fig. 8). The first myomers, emerging from the trunk region after 126 hours after fertilization (52.50 D°), begin to develop on the body of the embryo (Table 3).

Embryonic motility
The first movements appeared at 70.1 D° of embryonic development, they are isolated contractions of the trunk, of limited reach and low regularity. Over time, the number of movements increased to 4–5 movements/minute, and their reach also expands (whole-body movements). The greatest activity of the embryos were observed in the second half of embryogenesis. Start of operation of the heart leads to a slowdown in embryo’s somatic motility. Heart primordia in asp embryos started work after 120 D° of embryonic development had elapsed (ca), while the initially low frequency of contractions increased almost twice just before hatching (Fig. 12).

Fig. 12. Embryonic motility during embryogenesis.

Larvae
Individuals hatched with both head (Fig. 13) and tail. Newly hatched asp individuals measured 7.43 ±0.27 mm and were equipped with a double-chamber, 0.72 ±0.13 mm³ in volume, yolk sac. Pigment of hatched individuals could be seen in the eyes (intense) (Fig. 14, 15) and on the body (delicate). The head still adhered to the yolk sac, and the occluded mouth opening was at the bottom of the head.

Fig. 13. Embryo during hatching. Scale bar 1 mm.

Fig. 14. Intensive pigment in the eye, visible otoliths (arrow) as well as heart and network of blood vessels in the yolk sac. Scale bar 1 mm.

Fig. 15. Front section of the larvae with emerging brain Scale bar 100 µm.

DISCUSSION

The results obtained in this study, after a detailed analysis, allowed us to obtain a complete picture of the morpho-physiological changes taking place during early ontogenetic development of asp (Aspius aspius L.).

Asp spawn, like most of the fish of the carp family [6, 11, 12], does not contain the so-called structural fat in the form of droplets in the yolk sphere. This fact results in lateral alignment of the blastodisc, and later the embryo itself, because the very swollen ectoplasm accumulating at the vitellus (which later is transformed into blastodisc), in the absence of structural fat in the form of raft that could have lifted it towards the top, slides to the side and this allows  emergence of very expansive perivitellar space.

The asp eggs laid between March and May are larger in comparison with eggs of other Cyprinidae, that lay their eggs at a later period: May – June, such as gold fish Carassius auratus gibelio (Bloch, 1783) – Ø 1.29 ±0.05 mm, sunbleak, Leucaspius delineatus (Heckel, 1843) Ø 1.25 ±0.04 mm, rudd, Scardinius erythrophthalmus (L.) – Ø 1.41 ±0.04 mm; common bream Abramis brama (L.)–Ø 1.64 ±0.04 mm [7], tench Tinca tinca (L.) – Ø1.21 ±0.04 mm [13], when water temperatures are already much higher. This is reflected in the ratio of the surface area of the egg to its volume – the S/V coefficient, indicating the rate of metabolism processes in the tissues dictating the rate of embryogenesis, and thus the longer embryonic development [7]. Asp eggs are characterized by a less favorable S/V ratio (value = 2.96 ±0.05) – than eggs of the specified species of the carp family: gold fish S/V = 4.64 ±0.18, sunbleak S/V = 4.80 ±0.16, rudd S/V = 5.01 ±0.65; common bream S/V = 3.65 ±0.10 [7], tench S/V = 4.95 ±0.18 [13]. This is connected with the rate of gas exchange, which is directly proportional to the surface area through which the gas diffuses. This in turn can be explained by the longer period of embryonic development (200 D°) compared with the development of goldfish 90 D°, sunbleak 122 D°, common bream 122 D° [7] or tench 66 D° [13].

A more expansive perivitelline space (as in most species  of the carp family) provides the embryo with favorable respiratory conditions, allowing for free movement of the developing embryo. This causes mixing of perivitelline fluid, and increases the availability of oxygen to the embryo epithelium through which gas diffusion takes place.

The pattern of changes in asp embryonic movement [6], unlike other fish of other Cyprinidae was most probably influenced by lower incubation temperature, hence automatically higher amount of dissolved oxygen. The observed phenomenon of reduction, in the course of development, in the number of somatic contractions of asp embryo as movement of heart muscles increases may be explained by the fact that before the formation of embryo circulation system, the somatic movements that cause mixing of the perivitelline fluid caused supply of oxygen through the epithelium to the interior of the embryo. Later on, owing to the more and more efficient and faster heart action in the expanding circulatory system, oxygen was more efficiently transported in the bloodstream. Continued maintenance of somatic motility at a higher level would expose the developing embryo to energy loss in situation (as the structures develop) of enhanced demand for oxygen.

Division of the yolk sac into two chambers is a characteristic for the carp family [32]. Just as in other fish species of the carp family (bleak, rudd, sunbleak, common bream), this division takes place after about 30% of the duration of embryogenesis, from the moment of egg fertilization [32]. In the later stage of embryonic development the rear section of the yolk sac expands with rapidly growing tail section, but after resorption of yolk contained in it, it transforms into a caudal section of larvae body cavity.

The club-shaped formations, which stick eggs to the ground – more often underwater vegetation, present at the egg membranes also occur in other fish of the carp family [9, 20, 22, 24, 25]. These outgrowths, just like those found in silver bream or vimba egg membranes [26], play a very important role, since, despite close contact of the egg with the ground, they allow flow of water between it and the membranes. Meandering between the outgrowths, this water washes the whole surface of the egg and thus increases the surface through which the process of gas exchange takes place, and as a result of which an adequate amount of oxygen delivered to the embryo is guaranteed. Significant adhesiveness of the membrane is of great importance because it makes it possible for the eggs to stick to the vegetation, at a given distance above the bottom, which often is covered with a layer of sediments where there could be oxygen deficiency, and sufficient amount of oxygen is a prerequisite for proper development of the embryo. Downfall of oxygen amount delivered to the embryo leads to slowdown of embryonic development.

The size of hatching asp larvae in this experiment was very similar to data reported by Mitrofanov et al. [23]. In comparison with other species of the carp family, they are comparable to those of blue bream – 7.7 mm [29], developing under similar temperature conditions. However, in fish of the carp family developing at much higher temperatures, such as tench, the sizes of hatched individuals are about half the size – 3.49 mm [13] or rudd 4.7 mm.

The low survival rate of embryos may have been caused by the use of the hormone Ovopel which, as it turned out after experiments, gives worse results than the new hormonal agent Ovaprim used in asp aquaculture. Recent studies have shown that use of Ovopel leads to a lower embryo survival rate to the stage than use of Ovaprim [30, 31, 34]. Moreover, the use of Ovaprim produced gametes of better biological quality and featured greater percentage of ovulating females [34].

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Accepted for print: 26.08.2013

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Agata Korzelecka-Orkisz
Division of Hydrobiology, Ichthyology and Biotechnology of Biotechnology of Reproduction,
West Pomeranian University of Technology in Szczecin, Szczecin, Poland
K. Królewicza 4
71-550 Szczecin
Poland
email: Agata.Korzelecka-Orkisz@zut.edu.pl

Małgorzata Bonisławska
Department of Aquatic Sozology, West Pomeranian University of Technology in Szczecin, Szczecin, Poland
Kazimierza Królewicza 4B
71-550 Szczecin
Poland
email: Malgorzata.Bonislawska@zut.edu.pl

Adam Tański
Division of Hydrobiology, Ichthyology and Biotechnology of Biotechnology of Reproduction,
West Pomeranian University of Technology in Szczecin, Szczecin, Poland
K. Królewicza 4
71-550 Szczecin
Poland
email: adam.tanski@zut.edu.pl

Izabela Smaruj
Division of Hydrobiology, Ichthyology and Biotechnology of Biotechnology of Reproduction,
West Pomeranian University of Technology in Szczecin, Szczecin, Poland
K. Królewicza 4
71-550 Szczecin
Poland

Joanna Szulc
Division of Hydrobiology, Ichthyology and Biotechnology of Biotechnology of Reproduction,
West Pomeranian University of Technology in Szczecin, Szczecin, Poland
K. Królewicza 4
71-550 Szczecin
Poland

Krzysztof Formicki
Division of Hydrobiology, Ichthyology and Biotechnology of Biotechnology of Reproduction,

West Pomeranian University of Technology in Szczecin, Szczecin, Poland
K. Królewicza 4
71-550 Szczecin
Poland
email: Krzysztof.Formicki@zut.edu.pl

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Responses to this article, comments are invited and should be submitted within three months of the publication of the article. If accepted for publication, they will be published in the chapter headed 'Discussions' and hyperlinked to the article.

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