OOCYTE DEVELOPMENT OF BILATERAL SALMON (SALMO SALAR L., 1758) AND SEA TROUT (SALMO TRUTTA M. TRUTTA L., 1758) HYBRIDS AND PURE SPECIES IN THE FRESHWATER PERIOD OF THEIR LIFE

Lucyna Kirczuk¹, Józef Domagała^2
1 Department of General Zoology, University of Szczecin, Poland
² Department of General Zoology, University of Szczecin, Szczecin, Poland

ABSTRACT

The experiment was performed on the gonads of the following: 205 females of the hybrids ♀ Salmo trutta m. trutta × ♂ Salmo salar, 64 females of the hybrids ♀ Salmo salar × ♂ Salmo trutta m. trutta, 120 female trout and 120 female salmon, aged 0+, 1+. The fish were obtained from 8 experiments, in which a reciprocal cross was made of salmon and trout caught in their natural environment. The hybrids grew in small watercourses which do not join open waters. The watercourses are in the Bukowa Forest. The hybrids and the salmon and trout were caught in cycles. After the fish were caught, they were weighed, measured and their gonads were cut out and fixed. Histological preparations were made and the gonad stages and the degree of oocyte development and oocyte size were analysed. Results showed that the gonads of females of reciprocal hybrids aged 0+ or 1+ were in developmental stage I or II. In the second stage of gonadal development these oocytes reached 1, 2, 3^o of protoplasmatic growth, while in the gonads of salmon and sea trout, oocytes reached 4^o protoplasmatic growth. In the hybrids, ♀ trout × ♂ salmon remained at stage I till the age of 14 months, while in the reverse hybrid till the age of 9 months. This is much longer than in species in which parents at the age of 4 months, were in stage II. In addition, a distinctive feature of hybrids from parental species was that in the gonads of all female hybrids, of the two oocytes there were only 1–3^o protoplasmatic growth (second stage of development of the gonads), and in the parent species growth was more advanced. In hybrids protoplasmatic growth of oocytes in the sections of the gonad were few, with salmon and sea trout filling the entire cross-section of gonad.

Key words: hybrids, oocytes, ♀ salmon × ♂ trout ♀ trout × ♂ salmon, salmon, trout.

INTRODUCTION

Hybridisation between salmon and trout in commonly shared localities is a relatively frequent phenomenon. The exemplary levels of hybridisation between these species are 0.15% in Finland and Norway [13], 0.4% in Ireland [8], 2.3% in Spain [15], 1.0% in Great Britain [36,38,27], 13.3% in Sweden (locally even 28%) [24]. There are many  reasons for an increased level of hybridisation between these species. Some of the reasons include, first of all, the reduction of spawning site areas in the localities of their common occurrence, excessive fish catching [30], and  restitution of salmon over a limited area [24]. In addition, salmon and sea trout hybrids also occur more frequently in rivers where salmon populations have a high share of dwarf males. The number of dwarf males rises as the  population decreases [15,24,13].

These dwarf males also show aggressive reproductive behaviour [3,17]. Moreover, the presence of fertile female salmon × brown trout hybrids in small populations, can lead to cross-fertilization of pure species and consequently the loss the spawning sites of salmon and sea trout [16]. Hence, it is extremely important to do an assessment of the development of the gonads of female hybrids in ontogenesis. This assessment will determine their reproductive potential as compared to pure species. Therefore, the aim of this study was to assess the state of gonads and oocyte development of bilateral salmon and sea trout hybrids and pure species in freshwater during their lifetime.

MATERIAL AND METHODS

Reciprocal hybrids of salmon and sea trout were derived from eight experiments carried out during the artificial spawning period. The salmon spawn and milt were obtained from individuals from the Miastko Fish Farm, and in the River Wieprza, originating from the school imported from the River Dougava. The trout spawn and milt were taken from the school brought from the River Rega. Fertilization was performed in the PZW Hatchery in Goleniow, where the spawn was also incubated. The hatch was introduced into the watercourses near Szczecin. The watercourses end in the municipal sewage system which ensures that the hybrids would not get into the natural environment of the pure species. The growing fish were regularly caught with the help of an electric current producing aggregate JUP-23 (Approved by the Local Commission for Ethical Research no. 24/02 of 3.06.2002). Salmons and sea trout come from from rivers: Trawna, Chojnówka, Chotla, Czarna, Gowienica, Rudzianka. After the fish were caught, the body length of the fish was measured to the accuracy of 0.1 mm. They were weighed on an electronic scale to the accuracy of 0.1 g. Then, the gonads were prepared and fixed in Bouin fluid and weighed to the accuracy of 0.1 mg. Histological analysis was performed on 509 gonads of females. According to the standard paraffin technique, from the middle section of the gonad a histological preparation of the thickness of 5 µm was made and stained by Heidenhain ferruginous haematoxylin. One microscopic slide contained 100 snips of the gonad. The preparations were evaluated under a Nikon Eclipse 80i microscope, which has a maximum magnification of 1000x.

The stage of gonad development and the degree of oocyte development were determined. For evaluation of gonad maturity, the Persov scale was used (1966). The stage of gonad maturity was classified on the basis of the most developed oocytes [42]. The measurements were performed to the accuracy of 0.01 µm using the NIS Elements BR 3.0 system of digital image analyser. The oocyte diameter and nucleus were calculated from measurements of the longest and shortest diameters of the mid cross section of the oocyte [22]. Statistical analysis was made using the program STATISTICA (data analysis software system), version 8.0. www.statsoft.com., StatSoft, Inc. (2008). Photographs of the gonad preparations were taken with a Nikon digital camera.

RESULTS

Gonads of all females of ♀ trout × ♂ salmon hybrids were in developmental stage I or II and salmon and sea trout in stage II. In the gonads of first stage maturity, there were numerous oocytes, which came into mejotic divisions (mainly at the stages of zygotene and pachytene). The oldest female salmon × brown trout with gonads in stage I of maturity, were 14 months old, and the reverse crossroads of 9 months. The youngest brown trout × salmon hybrids with gonads in stage II of maturity of oocytes 1^o previtellogenesis were aged 4 months and 2^o, and 3^o, respectively appeared at the age of 5 and 9 months. Gonads in the second stage of the previtellogenesis oocytes (1 and 2^o previtellogenesis) in female salmon × brown trout hybrids appeared much later, up until the age of 9 months and oocytes 3^o, aged 14 months. During the hybrids freshwater period of life I have never observed the development of oocytes 4^o o protoplasmatic growth. In both interspecific hybrids in the second stage of the development of oocytes in previtellogenesis were several and occupied a small area of the gonad cross-section, [29]. Trout female gonads at the age of 4 months were in the second stage of maturity of oocytes 1^o – 3^o previtellogenesis and the largest oocytes were 2 and 3^o previtellogenesis. In one month older individuals there appeared 4^o degree protoplasmatic growth oocytes (5 – month old fish).

Analyzing the development of salmon and sea trout, it was found that the degree of 1^o previtellogenesis in salmon have already been reported in 3 – month old specimens, 2^o o the age of 4 months and 3^o at the age of 6 months. Oocytes in the previtellogenesis in pure species from 5 months old, filled the entire cross-section of gonads (Fig. 1). It follows that hybrid gonads grow more slowly in relation to salmon and sea trout gonads. The first stage of gonad development persisted longer. In addition, oocytes in the gonads in hybrid previtellogenesis were few (Fig. 2) and lasted longer following previtellogenesis degrees. Analyzing the size of oocytes and their nuclei in previtellogenesis I found that reciprocal hybrids of both salmon and sea trout aged 0 + and 1 + had smaller sized oocytes in gonads of pure species (Ryc. 3a-f). Exceptions are 1^o previtellogenesis oocytes, which in some hybrids have a larger diameter than the diameter of these cells in the parent species (Ryc. 3a). In this case, the extent of oocytes remained much longer than in the pure species (Ryc. 3d). The diameters of oocytes and their nuclei in the subsequent steps previtellogenesis, as well as gonads of reciprocal hybrids of salmon and sea trout, and in pure species are shown in Table 1. Statistical analysis (U test Mann-Whitney test, Statistica 8.0) showed that in terms of the size of oocytes in the 1–3^o previtellogenesis, significant differences (P <0.05) occurred between each of the crosses and the salmon and sea trout. These differences were not significant between the salmon and trout as well as hybrids between salmon and sea trout and salmon.

diameters of oocytes diameters of nuclei, µm

DISCUSSION

Assessment of fertility, the quality of gametes and the possibility of obtaining hybrids F2 generation hybrids or backward, is of particular importance for aquaculture [39,7,4]. Fertility reciprocal hybrids of salmon and sea trout has been the subject of numerous experiments, and results were not always clear. Hybrids derived from these gametes were incorrect, or if they were morphologically normal, the percentage of fertilization, hatching and survival were very low [44,1,32]. Reverse-crossing reciprocal hybrids of female salmon and sea trout pure species with males, and male salmon × brown trout hybrids with females of pure species, and hybrid crosses of salmon and sea trout between them, failed in the early experiments because these hybrids die during embryonic or fry stage [44]. Jones [26] also bred salmon and trout hybrids. When they reached sexual maturity the males gave active sperm, and a mature female gonad had a few pieces of ripe eggs. Further studies of the crossing of male hybrids of Salmo salar × Salmo trutta with females of pure parental species were done. They showed a total mortality over a period of several months from the start of food intake, with a few exceptions where some hybrids survived [32,43,14], and by far the greater share triploids, compared to the control salmon [44]. In the experiment of Garcia-Vazquez and others [18], the rear cross between female and male salmon × salmon brown trout hybrids gave diploid progeny (having a low degree of survival). Such a result suggests that these hybrids produce haploid gametes. However, hybrid males reverse hybrids Salmo salar × Salmo trutta fell from the female trout in the early period of development [18]. Low viability of gametes and low survival was also found in hybrid crosses of Salmo salar × Salmo trutta. This undoubtedly results from the different karyotypes of pure species and the difficulties in mating chromosomes during meiosis [32,25,37]. Cyprinidae Bakos and others [2], obtained similar results, with a reverse cross of hybrids. Their results showed that despite obtaining 5–10% fertilization, the development phase did not exceed blastodysk. Mean values of fork length and weight of most reciprocal hybrids of salmon and sea trout aged 0 + and 1 + in relation to salmon [12] and trout [28] were lower (author's unpublished data). An analyzed cross between salmon and sea trout shows smaller sizes of oocytes. Subsequent steps of previtellogenesis analyzed in relation to salmon and sea trout from Pomeranian rivers and the sea trout Murza and Kristoforow [31], where the oocytes 1^o, 2^o o and 3^o previtellogenesis have the following ranges: 1923–1940, 50–170, 180–330 mm. Also, the size of the analyzed previtellogenesis oocytes of trout are similar to that found in the data of Domagała [10]. The gonads of female salmon and sea trout from 5 months of age in previtellogenesis oocytes were numerous and occurred throughout the gonad cross-section as was described by other authors [31,10,11].

In addition, studies found that hybrids later obtained further levels of previtellogenesis oocytes and those on the cross gonads were few. Despite this, Garcia-Vazquez and others [18] found that salmon and sea trout hybrids reached maturity. However, low survival, changes during development, and poor growth of diploid hybrids mean these hybrids are of limited use in aquaculture [4,35,45]. Research has shown that the gonads of female hybrids grow more slowly as their development progresses, in relation to the gonads of female parent species. Therefore, hybrids may pose a threat to salmon and sea trout due to the deterioration of the species clean gene pool. Spawning areas are reduced as a result of the anthropogenic increase of these processes. It is therefore necessary to protect the gene pool of the pure species by protecting their environment and restoration of normal water relations so that there is no hybridization between them.

CONCLUSION

1. The gonads of females of reciprocal hybrids, salmon and sea trout aged 0+ or 1+ were in developmental stage I or II.

2. In the second stage of gonadal development the oocytes in the gonads of hybrids were only 1–3° protoplasmatic growth, of salmon and trout, in addition 4°.

3. The first stage of maturity of gonads hybrids persisted much longer than the parent species.

4. In hybrids protoplasmatic growth of oocytes in the sections of the gonad were few, with salmon and sea trout filling the entire cross-section of gonad.

5. The majority of oocytes in previtellogenesis in the hybrids gonads were smaller in size relative to the size of oocytes of the parent species.

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Accepted for print: 2.03.2011

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