RU2548104C1 - Method of growing of heterotic larvas of giant oyster crassostrea gigas (th) - Google Patents

Abstract

FIELD: agriculture.

SUBSTANCE: proposed process relates to mariculture and can be used for industrial cultivation of grassostrea gigas in Black Sea nursery. Nursery of the Institute of southern seas biology obtained in 2006 the heterotic hybrids of Crassostrea gigas. Sires represented three-year old mussels of two isolated colonies: Black Sea (inbred line) and Atlantic. Black sea mussels feature more flat shape of the shell: Shell shape index (IF) makes about 2.15 and 2.50. Inbread line was raised by sibs mating and at return mating. In fifth generation, "inbred minimum" was reached which can be decided on at comparison with survival rate of the fourth generation larvas.

EFFECT: higher rate of growth and survival rate compared with Atlantic mussels and inbred line larvas.

Description

The present invention relates to mariculture and is intended for industrial cultivation of a giant oyster in the Black Sea in a nursery.

The giant oyster Crassostrea gigas did not form natural settlements in the Black Sea due to the impossibility of reaching the required number of producers without mass rearing of juveniles in nurseries. Currently, the species C. gigas consists of many populations that are geographically fragmented and do not interbreed. In naturally breeding populations, the genetic variants that are most adapted to specific conditions remain the effect of natural selection. In the artificial breeding of oysters, the broodstock is a small number of individuals compared to the number of individuals of the species. In this case, some genetic variants are either completely absent or present with a frequency atypical for the species. Replenishment of the broodstock with individuals selected from natural populations avoids closely related crosses, since inbreeding can be directed against productivity. The use of inbreeding when breeding clean lines is a common selection technique for improving breeds.

A known method of cultivation (see JP A 2000354434, 12.26.2000, Cultivation of pearl oysters), where the larvae were obtained by crossing pearl oysters from natural populations of China and Japan. The producer is selected from the natural populations of China and Japan, which are geographically isolated and do not interbreed with each other. As a result of prolonged natural selection, genotypes that are most adapted to local conditions remain in each population, since the concentration of alleles in the population responsible for fitness increases with each generation and, accordingly, the rate of response of genotypes to changing living conditions decreases. Each population also retained a high level of heterozygosity as a result of free crosses.

When the pearl oyster Crassostrea nippona was artificially crossed from populations of Japan and China, in the hybrids, the genetic differences that existed between the populations were aligned. The disadvantages of this method:

- Oysters for crossing were selected from natural populations of Japan and China;

- Each population is adapted to local environmental conditions;

- Among the descendants there is a splitting into fit and poorly adapted to new conditions;

- Survival is reduced due to poorly adapted individuals.

Known (see McGoldrick DJ, Hedgecock, D. Hybrid vigor in Pacific oysters: An experimental approach using crosses among inbred lines // Journal of Shellfish Research. -1995. - Vol. 14, No. 1. - P. 229 .; Hedgecock D. Hybrid vigor is pervasive in crosses among inbred lines of Pacific oysters // Journal of Shellfish Research. - 1996. - Vol. 15, No 2. - P. 511) analogue of the proposed Method for growing heterotic giant oyster larvae. Larvae are obtained by selecting parental pairs for crossing. Moreover, each inbred male is crossed with each inbred female; those. in the presence of 5 males and 5 females, 25 crosses are made. Heterosis was observed only in 1 out of 5 such crosses. Inbred lines are removed during self-fertilization with cryopreserved sperm. Frozen semen is stored for a year, and the viability of the oysters from which it was obtained is also maintained, since sex inversion in some individuals of C. gigas occurs between two spawning grounds, i.e. a change in the sex of an individual can be fixed in a year. The disadvantages of this method:

- Inbred lines were bred as a result of self-fertilization after sex inversion by cryopreserved sperm;

- Biotechnological difficulty in producing inbred lines led to clogging of the brood stock by 60%, which is confirmed by the use of allozyme marker genes;

- When selecting parental pairs, heterotic descendants were obtained only in 1 out of 5 crosses;

- In some cases, the size of hybrids at the larval and juvenile stages is less than inbred.

The closest in technical essence are the principles of selection in the nursery SATMAR, which are described in the report of Kuczer J.- C. Rappor de Stage.

C.F.P.P.A. de Coutances session BP AM. - 1993 - 1994. - 79 p. For 40 years, since the introduction of the giant oyster onto the Atlantic coast of France, it has been propagated under natural conditions under the control of natural selection. For the broodstock, oysters are selected from various places on the coast of France. Conduct artificial selection of manufacturers according to the shape and appearance of the sinks. Group crossing is carried out between 15-20 copies. manufacturers age 2-4 years. The general principle of fertilization is the cross between the broodstock of the nursery and oysters, which are selected from natural settlements every year. Larvae are grown at a planting density of 5 and 2 thousand lic./l, respectively, at the stages of veligra and velikonha. The disadvantage of this method is that conducted outbred crosses do not provide a high level of survival and growth at a specified density of larvae.

The basis of the invention: A method for growing heterotic oyster larvae Crassostrea gigas when cultivated in a nursery, the task of obtaining hybrid offspring of high survival rate and growth rate at a high density of their cultivation.

The problem is achieved in that in the proposed method for obtaining heterotic hybrid offspring, group outbred oysters with an inbred line are grouped, while the outbred oysters can be from any geographically isolated population, then the obtained larvae are cultivated for 21 days at a planting density at the veliger stage of 30 thousand lich. / l and at the stages of the Great-Velkha and pediveliger - 15 thousand lich. / l.

A significant advantage of offspring in growth and survival compared to parents is called heterosis, and heterosis occurs in hybrids when parents have genotypic differences. Hybrid descendants are heterozygous at most loci. One of the characteristic features of heterosis is its strongest manifestation in first-generation hybrids.

To obtain heterotic descendants, genetically differentiated oysters were used as producers: the Atlantic and Black Sea cohorts. The Atlantic oyster cohort was obtained by crossing the broodstock of the nursery and oysters selected from natural settlements according to the criterion of the shape and appearance of the shells. The Black Sea cohort of inbred oysters was

obtained as a result of many years of artificial selection and closely related crosses for five years. The inbreeding coefficient [Dubinin, 1985] was at least 0.5. The inhibitory effect of inbreeding was revealed in a decrease in survival at the larval stages, especially at the stage of the early veligra, as well as during the transition of the velikonha to the pediveliger stage. An abnormal development of larvae was noted during the formation of the shell (stage D - veligra). A positive effect in closely related crosses was manifested in the elimination of lethal genotypes. Since the departure of inbred oysters mainly occurred at the larval stages, poorly adapted individuals were not involved in subsequent crosses. Survival results of the larvae in 2003 and 2006 (25 and 21.4%, respectively) indicated that the inbred minimum was reached and a further decrease in survival would not occur in subsequent inbred generations.

The invention is illustrated by illustrations. FIG. 1 - Producers of oysters Crassostrea gigas: Atlantic (above) and Black Sea (bottom) cohorts. FIG. 2 - Heterosis larvae collected on a sieve, growing density of 15 thousand individuals / l. FIG. 3 - Growth of giant oyster larvae obtained by inbred (No. 1), outbred (No. 2) crosses and heterosis (No. 3). FIG. 4 - Heterotic larvae at the late Velikonha stage: minimal size variability and maximum survival rate. FIG. 5 - Distribution of pediveligers by the H / L value of the sink.

Example.

Oysters were cultivated in the oyster nursery InBYUM NAS of Ukraine in 1999-2006.

In 1998, 25 copies were transferred from the Karadag branch of the InBYUM. three-year-old S. gigas as producers, which were approximately the 10th generation of oysters acclimatized in the Black Sea. As a result of group crosses (10♀♀ × 1♂ in 1999, the offspring of a giant oyster were obtained in the nursery. Every year, from 2000 to 2003, the larvae were obtained from crosses of the descendants of the same parents (siblings) and (or) during crossbreeding ( parents with descendants) Up to 50 specimens of oysters were used as broodstock, and about 30 specimens of ages from 1 to 4 years were selected for crossing.

In March 2004, C. gigas spat with a shell height of about 15 mm was obtained from the SATMAR nursery (France). Until maturity, he was reared in a growing cage on a mussel-oyster farm in the quarantine bay (Sevastopol). In 2006, there were three types of crosses:

No. 1 - between the oysters of the Black Sea cohort (inbreeding); No. 2 - between the oysters of the Atlantic cohort (outbreeding); No. 3 - crossbreeding carried out according to the scheme:

черноморская когорта

атлантическая когорта;

Black Sea cohort

Atlantic cohort;

атлантическая когорта

черноморская когорта.

atlantic cohort

Black Sea cohort.

Conditioning of the producers and stimulation of their spawning was carried out according to P. No. 76680 UA. Spawning of each producer took place in a separate vessel with filtered seawater and aeration. For crossing, the same number of eggs from each female and sperm from males was selected. For example, if it is necessary to obtain 1 million eggs from 20 females, 50 thousand were taken from each female. Fertilization was carried out in a 10 liter vessel, adding a suspension of sperm to the eggs. After 15 minutes after fertilization, the eggs were collected on a mill sieve with a mesh diameter of 32 μm, washed with filtered (through 1 μm) sea water and transferred to a 100 L container with filtered sea water and aeration. The entire process of growing larvae was carried out according to P. No. 76680 UA. For two days from the moment of fertilization, larvae from crosses No. 1 and No. 2 were grown at a density of 50 thousand l / l; at the veliger's stage - at 10 thousand l / l; at the stages of the great-pediveliger - 5 thousand person / liter. In crossbreeding No. 3, the density of planting of larvae at the veliger's stage was 30 thousand li / l, and at the stages of the Velikonha pediveliger - 15 thousand li / l.

The larvae obtained by crossing the Black Sea cohort of inbred oysters with the Atlantic oysters, in terms of growth rate and especially survival at the larval stages, significantly exceeded the larvae from crossing Atlantic or Black Sea ones. The duration of growing larvae, from the moment of fertilization to settling on the substrate, in crossings of the Black Sea (No. 1) and Atlantic oysters (No. 2) was 25 days, and in the cross (No. 3) - 21 days (Fig. 3). The average daily growth of hybrid larvae during the growing period was 1.2 times higher than the growth of larvae from two other crosses and amounted to 13 μm / day. At the veliger stage (age 2–10 days), the growth rate of larvae in all crosses was similar. At the stages of the late Velikonha and pediveliger, hybrid larvae exceeded in growth by 4.7 μm / day not only inbred, but also outbred larvae. In the same period, in all crosses, the maximum variation in their sizes was noted. So, the coefficient of variation of hybrid larvae was 11.5%; in crosses No. 1 and No. 2, respectively, 2.2 and 1.5 times higher.

The ratio of the height of the shell to the length H / L in pediveligers characterizes the shape of the shell. In larvae ready for sedimentation, it varied from 1.0 to 1.4. At values close to 1, the larvae resembled a circle in shape; with values close to 1.4 - an ellipse. According to this indicator, the larvae in crossing No. 3 occupied an intermediate position (Fig. 5). In crossing No. 1, 70% of the larvae had H / L values between 1.16 and 1.30; in crossbreeding No. 2, 68% ranged from 1.06 -1.15, while in 76% of hybrid offspring this indicator varied from 1.11 to 1.30. Throughout the growing period, the survival of hybrid larvae was maximum. If in crosses No. 1 and No. 2 the maximum withdrawal (30 and 16.6%, respectively) was noted within 7 days from the moment of fertilization, and on 14 · days of cultivation in inbred crossing, 52.6% remained; in outbred - 72.6%, then in crossing No. 3, there were 93.8% of the larvae of the initial amount. Three weeks later, the proportion of larvae in crosses No. 1 and No. 2 was 32.3%, respectively. and 56.3%, and in the third cross, 86.4%. During this period, all hybrid larvae were ready for subsidence. In two other crosses, the larvae were reared for another 4 days before settling.

Thus, the effectiveness of the proposed method for growing heterotic larvae of the giant oyster Crassostrea gigas in the nursery exceeds by 6.7 - 8.2 times the effectiveness of the known methods. Efficiency in this case is the total value of the indicators of the superiority of heterotic larvae in terms of growth rate (1.2 times), survival (2.5 and 4 times) with a planting density of larvae three times higher than the optimal values at which larvae were grown from crosses.

Claims (1)

A method of growing heterotic larvae of the giant oyster Crassostrea gigas when cultivated in a nursery, which includes spawning of oysters, crossbreeding, obtaining larvae and their cultivation in the nursery, characterized in that in order to obtain heterotic hybrid offspring, outbred oysters with inbred line are grouped, while outbred oysters can be from any geographically isolated population, then the obtained larvae are cultured for 21 days at a planting density at the veliger stage of 30 thousand l / l and per stage veliconcha and pediveliger - 15 tys.lich / l..

Images