Effect of temperature and spawning agents on wild female asp (Leuciscus aspius) reproductive efficiency under controlled conditions.

Artificial reproduction is a bottleneck to produce stocking material for many species of freshwater fish. One of these species is the asp, Leuciscus aspius. Research in the field of artificial reproduction of this species is very scarce and often incomplete. There are no breeding protocols specifying optimal environmental conditions and hormonal stimulation for many species of rheophilic cyprinids, including asp. Since the number of natural asp populations is constantly decreasing, it is important to support natural stocks by restocking with high quality stocking material. For this reason, optimized protocols are needed to breed this species under controlled conditions to produce stocking material with high biodiversity and good health. Such an approach will make it possible to maintain the population of natural asp at a constant level. The aim of this study was to develop the protocol of asp artificial reproduction using optimized thermal conditions and appropriate hormonal stimulation. In experiment I, the influence of constant temperature (10.0, 12.0 and 14.0 °C) on the effectiveness of artificial reproduction of asp. In experiment II, the effectiveness of asp reproduction was checked after the application of spawning agents: Ovopel, Ovaprim or a combination of these two agents The obtained results indicate that for the final maturation of oocytes (FOM) and artificial reproduction of asp in controlled conditions, water temperatures of 10-12 °C are the most useful. Under these thermal conditions, the highest percentages of female's ovulation and embryo survival, as well as the percentage of hatching, were obtained. Hormone injections are necessary to perform final oocyte maturation (FOM) in female asp in captivity. All spawning agents used were especially useful for artificial reproduction of asp, however, the best values of the studied indices, such as ovulation rate and embryo survival, were obtained after the application of Ovaprim or the combination of Ovopel and Ovaprim in water temperature at a range of 10-12 °C. It was found that the pH of ovarian fluid may be a preliminary indicator of the biological quality of eggs in asps. The optimal pH value is 8.0-8.4. At pH below 7.4, no viable embryos were observed.

Effect of anti-adhesive supplement doses on sperm motility characteristics in the European whitefish (Coregonus lavaretus).

Sperm adhering to glass slides is one of the main problems during fish sperm motility analyses with CASA systems. To mitigate this, albumin is the supplement added most frequently to activating solutions. However, there is no data on the use of supplements other than albumin (in various concentrations) in analyses of European whitefish (Coregonus lavaretus) sperm motility. This issue was investigated in the presented research using three anti-adhesive supplements (albumin, casein, Pluronic F-127) that were added to Billard solution (BS: 20 mM Tris, 1 mM CaCl2, 154 mM NaCl, 30 mM glycine at pH 9.0) at different concentrations (0.0; 0.1; 0.2; 0.5; 1.0; 2.0%). It was noted that the addition of the lowest concentration (0.1%) of albumin, casein, or the pluronic to BS had a significant effect on the motility and kinetic parameters of whitefish sperm compared to pure BS. BS supplemented with 0.2-0.5% albumin was the most appropriate variant used for whitefish sperm motility activation in the present experiment. BS supplemented with the pluronic at 1.0-2.0% concentrations resulted in significantly higher values of almost all CASA parameters compared to casein at the same concentrations. Moreover, CASA parameters determined in this variant of the pluronic (1.0-2.0%) were similar to those when BS was supplemented with the same albumin concentrations. This indicated that instead of albumin, the pluronic at higher concentrations in BS might be used to analyze whitefish sperm motility.

Effect of Different Water Salinities on the Larvae of the Blue Bream Ballerus ballerus (Linnaeus, 1758) during Rearing.

The influence of water salinities of 3, 5, and 7 ppt on the growth and survival of Ballerus ballerus (L.) larvae was studied. The control group was fish reared in freshwater (0 ppt). The larvae showed high tolerance to water salinities of 5-7 ppt. The mean final weight of the larvae ranged from 48.6 to 64.1 mg, with corresponding mean total lengths from 18.9 to 22.6 mm, depending on the water salinity level. The best larval length increments were recorded in water with salinity of 3 ppt. They were only slightly lower in 0 ppt water, and there were no statistically significant differences between the breeding rates calculated for larvae reared in 3 ppt water. Depending on the salinity level of the water, the final survival rate of the blue bream larvae ranged from 83.5 to 98.6%. The blue bream larvae reared in water with salinity levels of 5 and 7 ppt were statistically smaller than the others, but the results obtained were equally satisfactory.

Influence of Water Salinity on the Growth and Survivability of Asp Larvae Leuciscus aspius (Linnaeus, 1758) under Controlled Conditions.

The effect of water with a salinity 3, 5, 7, 9 and 11 ppt on the growth and survivability of asp Leuciscus aspius (L.) larvae was investigated. A control sample consisted of asp larvae reared up in freshwater (0 ppt). Larvae were fed for 21 days with nauplii of the brine shrimp Artemia salina. Water salinity was observed to have a considerable effect on the growth and survivability of asp larvae. In addition, saline water extended the life span of Artemia salina nauplii, which resulted in their prolonged availability to asp larvae. Asp larvae showed low tolerance to the salinity of water, reaching 9-11 ppt. Depending on the degree of salinity, the mean final weight of larvae varied from 122.6 to 139.4 mg, at body lengths from 23.8 to 25.6 mm, respectively. The best body length increments were recorded among asp larvae maintained in water with a salinity of 3 ppt. Depending on the level of water salinity, the final survivability of asp larvae ranged from 16.9 to 94.5%. The highest and increasing mortality was demonstrated among the larvae reared in water of the salinity equal to 11 ppt. It is not recommended to rear asp larvae in water with a salinity above 7 ppt due to the low survivability and large differences in the body size of the larvae that managed to survive.

Unique Features of River Lamprey (Lampetra fluviatilis) Myogenesis.

The river lamprey (L. fluviatilis) is a representative of the ancestral jawless vertebrate group. We performed a histological analysis of trunk muscle fiber differentiation during embryonal, larval, and adult musculature development in this previously unstudied species. Investigation using light, transmission electron (TEM), and confocal microscopy revealed that embryonal and larval musculature differs from adult muscle mass. Here, we present the morphological analysis of L. fluviatilis myogenesis, from unsegmented mesoderm through somite formation, and their differentiation into multinucleated muscle lamellae. Our analysis also revealed the presence of myogenic factors LfPax3/7 and Myf5 in the dermomyotome. In the next stages of development, two types of muscle lamellae can be distinguished: central surrounded by parietal. This pattern is maintained until adulthood, when parietal muscle fibers surround the central muscles on both sides. The two types show different morphological characteristics. Although lampreys are phylogenetically distant from jawed vertebrates, somite morphology, especially dermomyotome function, shows similarity. Here we demonstrate that somitogenesis is a conservative process among all vertebrates. We conclude that river lamprey myogenesis shares features with both ancestral and higher vertebrates.

Comparison of different methods of wild vimba bream Vimba vimba (L.) spawning under controlled conditions.

This paper describes a study in which different types of the reproduction of wild vimba bream (Vimba vimba) under controlled conditions were compared. Reproduction was performed by using three methods: spontaneous (natural spawning- NS groups): without hormones but with nests, semi-artificial (S groups) with hormones and nests, and artificial (A groups) with hormones and manual stripping of gametes. In groups S and A, different types of hormonal stimulation were tested: common carp pituitary homogenate (CPH), human chorionic gonadotropin (hCG), and commercial pellets containing mammalian GnRHa and dopamine antagonist-metoclopramide (Ovopel). The number of spawning fish, i.e., spermiating males and ovulating females, were compared depending on the type of breeding and the stimulant used. The lowest number of finally matured fish: spermiating males (67 %) and ovulating females (0 %) were noted in the spontaneously reproduced group. The best method of reproducing wild vimba bream under controlled conditions was the semi-artificial approach with hormonal stimulation and spawning nests, where ovulation rates in hormonally treated groups were between 43 % and 71 %, vs 26-57 % in artificial spawning groups. No ovulation observe in control groups in S and A types of reproduction. The highest embryo survival to the eyed-egg-stage was noted in group A-hCG (average 88.2). A higher value of spawning effectiveness (Se) with the use of the same hormones was obtained for groups S (0.374-0.574) than for groups A (0.252-0.493). In both cases, the highest Se was recorded for Ovopel stimulated fish and the lowest in hCG stimulated groups.

Microscopic study of the primary growth ovarian follicles of the pike-perch Sander lucioperca (Linnaeus 1758) (Actinopterygii, Perciformes).

The ovaries of Sander lucioperca (Actinopterygii, Perciformes) are made up of the germinal epithelium and ovarian follicles, in which primary oocytes grow. Each follicle is composed of an oocyte surrounded by flattened follicular cells, the basal lamina, and thecal cells. The early stages of oocyte development (primary growth = previtellogenesis) are not fully explained in this species. The results of research with the use of stereoscopic, light, fluorescence, and transmission electron microscopes on ovarian follicles containing developing primary oocytes of S. lucioperca are presented. The polarization and ultrastructure of oocytes are described and discussed. The deposition of egg envelopes during the primary growth and the ultrastructure of the eggshell in maturing follicles of S. lucioperca are also presented. Nuclei in primary oocytes comprise lampbrush chromosomes, nuclear bodies, and nucleoli. Numerous additional nucleoli arise in the nucleoplasm during primary growth and locate close to the nuclear envelope. The Balbiani body in the cytoplasm of oocytes (ooplasm) is composed of nuage aggregations of nuclear origin and mitochondria, endoplasmic reticulum (ER), and Golgi apparatus. The presence of the Balbiani body was reported in oocytes of numerous species of Actinopterygii; however, its ultrastructure was investigated in a limited number of species. In primary oocytes of S. lucioperca, the Balbiani body is initially located in the perinuclear ooplasm on one side of the nucleus. Next, it surrounds the nucleus, expands toward the plasma membrane of oocytes (oolemma), and becomes fragmented. Within the Balbiani body, the granular nuage condenses in the form of threads, locates near the oolemma, at the vegetal oocyte pole, and then dissolves. Mitochondria and cisternae of the rough endoplasmic reticulum (RER) are present between the threads. During primary growth micropylar cells differentiate in the follicular epithelium. They contain cisternae and vesicles of the RER and Golgi apparatus as well as numerous dense vesicles suggesting high synthetic and secretory activity. During the final step of primary growth several follicular cells delaminate from the follicular epithelium, migrate toward the oocyte and submerge in the most external egg envelope. In the ooplasm, three regions are distinguished: perinuclear, endoplasm, and periplasm. Cortical alveoli arise in the perinuclear ooplasm and in the endoplasm as a result of the fusion of RER vesicles with Golgi ones. They are evenly distributed. Lamellar bodies in the periplasm store the plasma membrane and release it into a space between the follicular cells and the oocyte. The developing eggshell in this space is made up of two egg envelopes (the internal one and the external) that are pierced by canals formed around the microvilli of oocytes and the processes of follicular cells. In the deposition of egg envelopes the oocyte itself and follicular cells are engaged. In maturing ovarian follicles the eggshell is solid and the internal egg envelope is covered with protuberances.

Rearing of Bitterling (Rhodeus amarus) Larvae and Fry under Controlled Conditions for the Restitution of Endangered Populations.

Among the several dozen European freshwater fish species, only European bitterling (Rhodeus amarus Bloch) and Rhodeus meridionalis belong to the group of ostrakophilous fish. The embryonic and larval development of the fish in this reproductive group until the time of the yolk sac resorption takes place in the gill cavity of river mussels (Anodonta sp. or Unio sp.). This paper presents the results of the European bitterling Rhodeus amarus being reared under controlled conditions. Bitterling larvae were caught together with river mussels in the natural environment and subsequently placed in a tank for behavioural observations. Bitterling larvae were seen swimming in the water within a week of placing the bivalves under controlled conditions. The bitterling larvae were 8.6 ± 0.11 mm long when they started to swim actively. The rearing was conducted in water at 20 and 26 ± 0.5 °C and lasted for 6.5 months (200 days) in both variants. Initially, the larvae were fed with live nauplii of Artemia salina and subsequently with fodder. The bitterlings in tanks with water at 26 ± 0.5 °C were 66.2 ± 3.0 mm long and weighed 3389 ± 548 mg. For comparison, bitterlings kept in water at 20 ± 0.5 °C were 64.48 ± 3.4 mm long and weighed 3242 ± 427 mg. No larval malformities or mortality were observed during the larvae and fry rearing. The bitterlings had well-developed secondary sexual characteristics and exhibited pre-spawning behaviour at the end of the rearing. This produced suitable bitterling stocking material to be used in the conservation of small or endangered populations.

Artificial Reproduction of Blue Bream (Ballerus ballerus L.) as a Conservative Method under Controlled Conditions.

The blue bream Ballerus ballerus (L.) is one of two species of the Ballerus genus occurring in Europe. The biotechnology for its reproduction under controlled conditions needs to be developed to conserve its local populations. Therefore, a range of experiments were performed to obtain valuable gametes from blue bream following hormonal stimulation with carp pituitary homogenate (CPH), Ovopel and Ovaprim. CPH and Ovopel were injected twice and Ovaprim-once, under a pectoral fin. The spawners were inspected 12 h after the last injection, and the gametes were collected. Eggs were collected from spawning females and fertilized with sperm from three males. The smallest number of ovulating blue bream (four females) was obtained from individuals stimulated with Ovaprim. There were six to nine ovulating females in the other groups, accounting for 60-90% of the females in the respective groups. The latency period was the shortest in the spawners stimulated with Ovopel. The fish in this group ovulated 14 h after the hormonal injection. Spawning was synchronized and all females spawned simultaneously. The highest average weight of eggs per fish (75.4 g) was obtained from the females stimulated with Ovopel, whereas the individuals stimulated with Ovaprim provided the smallest average amount of eggs (49.5 g). The study showed that blue bream spawners are not very sensitive to reproduction-related handling under controlled conditions.

Dynamics of mercury content in adult sichel (Pelecus cultratus L.) tissues from the Baltic Sea before and during spawning.

This study compares the content of mercury in the muscles, liver, kidneys, gonads and gills of male and female individuals of sichel (Pelecus cultratus). Moreover, the trend of changes of mercury concentration before (March) and during (May) the spawning season was examined. Sichel brooders were caught in the Vistula Lagoon during commercial fishing. The mercury content in tissues was determined by atomic absorption using a Milestone DMA-80. The tests revealed a statistically higher mercury concentration in muscles, liver and gonads in male vs. female fish. Moreover, significantly higher mercury concentration was found in male and female fish caught during the spawning season (May) than in those caught before this season (March). Moreover, testes (0.011 ±â€¯0.007 mg kg-1 w/w) were found to contain 12 times, and ovaries (0.004 ±â€¯0.001 mg kg-1 w/w) - approx. 19 times less mercury than the muscular tissue of those same fish. This may suggest the existence of a protective barrier, defending future offspring against the transfer of toxic mercury from the parent body to gonads and gametes.

Use of ultrasonography to determine sex in sexually immature European river lamprey Lampetra fluviatilis (L.).

In this study, there was the first attempt to sex immature European river lampreys Lampetra fluviatilis, classified as Agnatha using ultrasonography. This species starts a spawning migration from seas to rivers in the autumn and reproduction is initiated in the late spring. It is recommended to collect breeders soon after the beginning of the spawning migration, however, to date no method has been developed for distinguishing the sex of individuals during this developmental period. The lampreys for the present study were caught in autumn (November) in the Vistula River (northern Poland) during the period of spawning migration and transported to the laboratory. The lampreys were anaesthetised (MS-222, dose: 0.1 g/dm3 prior to sex determinations (n = 100) using ultrasonography. The images obtained using ultrasonography were verified with post-mortem and histological examinations. The findings with this study confirmed that the sex of the European river lamprey can be effectively ascertained much earlier than can occur with assessment of external secondary sexual characteristics (e.g., sexual papilla, ovipositor, skinfold). The advantages of the method include: 100% effectiveness, survival of the fish after examination, non-invasiveness, rapid verification of the lamprey sex and the possibility of determining extent of gonadal development.

Reproduction of endangered river lamprey (Lampetra fluviatilis) in controlled conditions.

The research reported focuses on reproduction of the river lamprey Lampetra fluviatilis,(Linnaeus, 1758) in controlled conditions. There was specific emphasis on fish harvesting dates (autumn and spring), holding conditions and reproduction in a controlled environment. Attempts were also made to synchronize the time of ovulation among river lampreys, egg and sperm collections. Hormonal stimulation was conducted using carp pituitary homogenate (CPH) at a total dose of 4 mg/kg which allowed for shortening of the egg-laying period from 2 to 3 weeks to a few days while sustaining embryo survival rates and larvae quality. River lamprey males were found to not require hormonal treatment to yield good-quality sperm, as measured using the CASA system. River lamprey broodstocks adapted well to different manipulations in hatchery conditions when harvested in the autumn and spring. The results of the present study may be used to restore endangered natural populations of the river lamprey (egg and sperm collection, fertilization or gamete preservation) because ovulation and spermiation synchronization is very difficult to achieve without hormonal treatment in controlled conditions.

Out-of-season artificial reproduction of common dace (Leuciscus leuciscus L.) under controlled conditions.

This study focused on the artificial reproduction of common dace Leuciscus leuciscus (L.) outside the reproductive season. The results indicate there is the possibility of initiating the reproducion in individuals of this before the natural spawning season. There could be induction of spermiation by altering the environmental conditions. For females, hormonal injections were necessary for induction of final oocyte maturation and ovulation. Generally, there were high percentages of spermiation (100%) and ovulation (87%-100%) as well as low mortality (0%-11% and 7%-13% for males and females, respectively) among the induced spawners. The greatest embryo survival occurred when Ovaprim (84.4%) or hCG (89.6%) was administered, although the latency time using hCG was at least twice as long compared to when other spawning agents were used (84-92 hrs and 30-44 hrs). The results from present study could be applicable for optimization of breeding stock numbers in aquaculture enterprises and in providing insights for conservation of L. leuciscus endangered populations.

The effect of different ambient temperatures on river lamprey (Lampetrafluviatilis) egg and sperm production under controlled conditions.

Reproductive performance (ovulation/spermation rate and relative fecundity of females) of adult river lamprey (Lampetrafluviatilis) was compared among adults held under three different controlled thermal regimes (7, 10 and 14°C). The quantity of semen (volume of semen, sperm concentration, total sperm production and total number of sperm) and the weight of the eggs as well as the semen quality (sperm motility, seminal plasma osmolality and pH, sperm pH and total protein content) were determined. Housing temperature had no apparent effect on quality or quantity of eggs produced, but did influence time of ovulation. On the other hand, temperature had a significant effect on the quantity and quality of sperm produced; 70% of males held at 10°C and 14°C did not spermiate. The best ambient temperature for river lamprey adults held under controlled conditions was 7°C. All males kept in this temperature yielded mature semen with the highest sperm motility parameters. It was also found that among the six tested solutions, the most suitable artificial medium for river lamprey sperm activation was 20mM Tris buffer containing 40mM NaHCO3 at pH 8.5 and 100mOsmkg-1.

Histological Aspects of the Early Development of the Digestive System of Burbot Lota lota L. (Lotidae, Gadiformes).

The ontogeny of the digestive tract was studied histologically in burbot, Lota lota L., from hatching to 42 days post-hatch (dph). At hatching, the digestive tract consisted of a straight tube with discernible digestive accessory glands (the liver and the pancreas) dorsally attached to the yolk sac. Most of the yolk sac reserves were consumed during the first 12 days and were completely depleted by 17 dph. The first PAS-positive goblet cells appeared at 6 dph, dispersed within the epithelium of the oesophagus and increasing substantially in number and distribution as development progressed. At 12 dph, the first vacuoles (neutral lipids) appeared in the intestine, indicating the functional absorption of nutrients from food. Differentiation of gastric glands was first noticed at 17 dph and was extensive by 27 dph. L. lota larvae have a morphologically complete digestive tract by 32 dph. These findings on the development of the digestive system in L. lota may contribute to a better understanding of its ontogeny and can be useful for improvement of the larval rearing techniques of this promising species for freshwater aquaculture diversification.

Early Development and Post Embryonic Skeletal Morphology of the Progeny of Spined Loach Cobitis taenia L. (Teleostei, Cobitidae) and its Naturally Occurring Allotriploids.

Polyploid fishes of the genus Cobitis represent a valuable model system to study the origin and consequences of hybridization and polyploidization within vertebrates. These naturally accessible polyploids are an excellent subject to determine the advantages or disadvantages of polyploidy. We investigated the embryonic and larval development with skeletal morphology of diploid and polyploid Cobitis progeny, obtained from crosses between females and males of Cobitis taenia and between allotriploid Cobitis females and C. taenia males. Observations were made during first fourteen days post fertilization. The pattern of development of all investigated individuals was the same. However the diploids developed synchronically, achieving successive stages faster than the polyploid ones; hatching was observed at 50 and 63 hours post fertilization, respectively. Statistically significant differences in hatching success and survival rate between diploid and polyploid progeny were not observed. All newly hatched larvae were characterized by a large amount of yolk, forty myomeres, body pigmentation and four external gills. Skeletal elements of the chondrocranium in the first days post hatching consisted of the otic capsule, ethmoid plate, trabeculae cranii and Meckel's cartilage. In contrast to the diploids, the polyploid larvae were characterized by a higher number of deformities. This study gives new comparative data on the features of early development of diploid and polyploid Cobitis progeny.

Ploidy-dependent survival of progeny arising from crosses between natural allotriploid Cobitis females and diploid C. taenia males (Pisces, Cobitidae).

Crosses between 21 triploid hybrid Cobitis females and 19 C. taenia (2n = 48) males led to viable progeny; whereas no embryonic development was observed in crosses with tetraploid males (4n = 98). The ploidy status of 491 progenies randomly selected with flow cytometry (316) or chromosome analysis (175) revealed an average of 55.2 % triploids and 44.8 % tetraploids, but the ratio of 3n versus 4n fish did change during development. In the first 2 days after hatching, approximately 65.1 % of tetraploid larvae were observed. Their number decreased significantly to 30.8 and 6.2 % on average during 2-5 and 10-15 months of life, respectively. The karyotype of tetraploid progeny (4n = 98) included 3n = 74 chromosomes of the parental female and n = 24 of C. taenia male. The number of tetraploid progeny indicated indirectly that about 66 % of eggs from 3n females were fertilized with C. taenia. The rest of the eggs developed clonally via gynogenesis or hemiclonally via hybridogenesis into triploids of the same karyotype structure as parental females. We have documented for the first time that (at least under experimental conditions) tetraploids are commonly formed, but are less viable than triploids, and a ratio similar to what is found under natural conditions is finally attained. The current explanation concerning the ploidy and karyotype structure of the progeny confirms that the eggs of 3n Cobitis females are not only capable of maintaining all chromosomes but are also capable of incorporating the sperm genome, thus creating the potential to produce tetraploids.