1. TECHNOLOGY FOR ARTIFICIAL SPAWNING OF STURGEONS SPECIES Object Object The present technology is after to achieve by artificial spawning of parental species of sturgeons. The present technology is after to achieve by artificial spawning of parental species of sturgeons. Objectives pursued: Objectives pursued: Providing descendants for rehabilitating wild population of sturgeons from Danube river; Providing descendants for rehabilitating wild population of sturgeons from Danube river; Inserting sturgeons species in aquaculture; Inserting sturgeons species in aquaculture; Fries achieved from pure species of sturgeons will form the basis of genetic fond reared strictly under conditions of aquaculture which will contribute to its development and protection of sturgeon stocks from natural environment. Fries achieved from pure species of sturgeons will form the basis of genetic fond reared strictly under conditions of aquaculture which will contribute to its development and protection of sturgeon stocks from natural environment. Area of implementation Area of implementation Technology for artificial spawning of parental species of sturgeons is intended to economical agents that have the main activity fishing and aquaculture. Technology for artificial spawning of parental species of sturgeons is intended to economical agents that have the main activity fishing and aquaculture.

2. Technological system for artificial spawning of parental species of sturgeons Technological system for artificial spawning of parental species of sturgeons Technological system of artificial spawning will be apportion depending on production capacity agreed by costumer, and will ensure by its capacity the spawning in series of two families of beluga, five families of Russian sturgeon, 20 families of sevruga and 20 families of starlet. Technological system of artificial spawning will be apportion depending on production capacity agreed by costumer, and will ensure by its capacity the spawning in series of two families of beluga, five families of Russian sturgeon, 20 families of sevruga and 20 families of starlet. Correlate with the capacity for incubation, technological system will be framed by a pumping group, modules for standing and maturation of breeders, modules for incubation of embrionated eggs and modules for standing larvae whose sizing and technical characteristics are following Correlate with the capacity for incubation, technological system will be framed by a pumping group, modules for standing and maturation of breeders, modules for incubation of embrionated eggs and modules for standing larvae whose sizing and technical characteristics are following

3. No. Crt. Type of facilityNo. of unitsTechnical features 1.Pumping system2Q - 110 m 3 /h, înălţimea de pompare 36m 2.Module for standing breeders4Shape: circular; Diameter – 6 m Height – 1.2 m Capacity – 22.6 m3 Stocking capacity – 150 kg 3.Modules for maturation of breeders4Rectangular form: 2,8 x 0,61 x 0,61 4.Modules for hatching fertilized eggs40Tipe: Brates, capacity 15 l Minimum capacity of storage 300 g 5.Modules for standing larvae36Rectangular form: 2,2 x 0,42 x 0,17

4. 3. DESCRIPTION OF TECHNOLOGY 3. DESCRIPTION OF TECHNOLOGY Technology for artificial breeding and rearing of parental species of sturgeons imply the passing through of following phases: Technology for artificial breeding and rearing of parental species of sturgeons imply the passing through of following phases: breeders catch; breeders catch; evaluation of phenotypic characters; evaluation of phenotypic characters; assessment of reproductive condition; assessment of reproductive condition; transportation and standing; transportation and standing; induction of sexual cells maturation; induction of sexual cells maturation; gathering sexual products; gathering sexual products; fertilization; fertilization; incubation of sexual products in special installed enclosures; incubation of sexual products in special installed enclosures; standing larvae; standing larvae; sacking and transportation of larvae; sacking and transportation of larvae; rearing larvae up on achieving adult-like phenotypic characters; rearing larvae up on achieving adult-like phenotypic characters;

5. Capturing and standing breeders on fishing area Capturing and standing breeders on fishing area Sturgeon breeders needed for stations of spawning and rearing fries will be achieved by fishing, during spring migration, at temperature of Danube waters of 10 – 22 0C. Sturgeon breeders needed for stations of spawning and rearing fries will be achieved by fishing, during spring migration, at temperature of Danube waters of 10 – 22 0C. Catch will be achieved with decline seines having mash size of 60 – 100 mm. Catch will be achieved with decline seines having mash size of 60 – 100 mm. Assessment of phenotypic characters and reproductive condition Assessment of phenotypic characters and reproductive condition Sturgeon breeders will be examined immediately after catch to evaluate phenotypic characters and sex determination. Examination in the field for purpose to determining sex and maturation stage will be accomplished by internal palpation. Sturgeon breeders will be examined immediately after catch to evaluate phenotypic characters and sex determination. Examination in the field for purpose to determining sex and maturation stage will be accomplished by internal palpation. Transporting and standing breeders Transporting and standing breeders Will be used fiber glass tanks of different capacities, well sealed, sized depending on sturgeons species and provided with installation of aeration. Will be used fiber glass tanks of different capacities, well sealed, sized depending on sturgeons species and provided with installation of aeration. In case of a transportation tank well equipped, stocking density can be almost 150 kg/m3. In case of a transportation tank well equipped, stocking density can be almost 150 kg/m3.

6. Hormonal induction. Hormonal induction. The product NERESTIN 5A will be used for stimulation. Induction of complete ovulation and elimination of sexual products will be accomplished by dosing two injections with hormonal preparation. Time interval between initial injection, prime one and the second injection, decisive one is of 12 hours. The product NERESTIN 5A will be used for stimulation. Induction of complete ovulation and elimination of sexual products will be accomplished by dosing two injections with hormonal preparation. Time interval between initial injection, prime one and the second injection, decisive one is of 12 hours. Doses of synthetic hormones will be: Doses of synthetic hormones will be: –0.2 ml/kg of body weight on females; –0.1 ml/kg of body weight on males. Gathering sexual products Gathering sexual products Gathering sexual products on female start at the time when it starts to eliminate a consistently quantity of limy eggs. Gathering sexual products on female start at the time when it starts to eliminate a consistently quantity of limy eggs. The method for gathering eggs will consist in abdominal palpation or so called mode of “milking”. In case that female does not release eggs it will be incised and eggs will be gathering by slowly palpation. In males sperm will be collected when the females begin to eliminate sticky eggs either with a few hours before these. Gathering sperm will be accomplished with syringes fitted with tubes of 5 – 6 cm. The method for gathering eggs will consist in abdominal palpation or so called mode of “milking”. In case that female does not release eggs it will be incised and eggs will be gathering by slowly palpation. In males sperm will be collected when the females begin to eliminate sticky eggs either with a few hours before these. Gathering sperm will be accomplished with syringes fitted with tubes of 5 – 6 cm.

7. Fertilization Fertilization Once collected, the eggs from a single female will be equally spread in jars in which is to make the fertilization. Once collected, the eggs from a single female will be equally spread in jars in which is to make the fertilization. Currently 1 kg of eggs uses 10 ml of sperm with viability of 90 %. Currently 1 kg of eggs uses 10 ml of sperm with viability of 90 %. Time for fertilization will be of 3 minutes. After fertilization, eggs will be degumming. For degumming use a single process, i.e. stopping the sticky surface of fertilized eggs with mud. Time for fertilization will be of 3 minutes. After fertilization, eggs will be degumming. For degumming use a single process, i.e. stopping the sticky surface of fertilized eggs with mud. Hatching eggs Hatching eggs Hatching eggs will begin immediately after degumming, with the passage into the hatchery devices. Hatching duration vary with species and within the same species according to daily average temperature. Hatching eggs will begin immediately after degumming, with the passage into the hatchery devices. Hatching duration vary with species and within the same species according to daily average temperature. On sturgeons, optimal temperature for hatching is 13 – 14 0C. Average time duration of embryogenesis on these values of water temperature is around of 5 – 7 days. These temperatures provide good conditions for embryonic development and do not facilitate mould development. On sturgeons, optimal temperature for hatching is 13 – 14 0C. Average time duration of embryogenesis on these values of water temperature is around of 5 – 7 days. These temperatures provide good conditions for embryonic development and do not facilitate mould development. Mould control during hatching time will be accomplish by bathing eggs, directly in hatcheries, with malachite green 67 ppm for 30 seconds in two times, after 12 and 24 hours from fertilization. Mould control during hatching time will be accomplish by bathing eggs, directly in hatcheries, with malachite green 67 ppm for 30 seconds in two times, after 12 and 24 hours from fertilization.

8. Sampling and standing larvae Sampling and standing larvae Sampling hatched larvae is accomplished continuously on cushion of water, in individual tanks for gathering. Gathering tank is fitted with a nytal cage having mesh size of 0.3 – 0.5 mm to constrict the larvae. Stocking density will be of 40 – 60 ex/l. Water flow in tanks for standing of larvae will be adjusted to 5 l/min. Larvae will stay in these enclosures for 3 – 5 days. Sampling hatched larvae is accomplished continuously on cushion of water, in individual tanks for gathering. Gathering tank is fitted with a nytal cage having mesh size of 0.3 – 0.5 mm to constrict the larvae. Stocking density will be of 40 – 60 ex/l. Water flow in tanks for standing of larvae will be adjusted to 5 l/min. Larvae will stay in these enclosures for 3 – 5 days. Growth of larvae up on achieving adult-like phenotypic characters Growth of larvae up on achieving adult-like phenotypic characters Stocking rate Stocking rate Larvae achieved from each female are stoked independently on rearing enclosures. Stoking rate is 10.000 ex/m3 and is determined taking into account technological losses for rearing phase which in general are estimated on 30 %. Larvae achieved from each female are stoked independently on rearing enclosures. Stoking rate is 10.000 ex/m3 and is determined taking into account technological losses for rearing phase which in general are estimated on 30 %. Preparing of rearing modules and stoking of larvae. Preparing of rearing modules and stoking of larvae. Preparing of rearing enclosures is made with 48 hours afore stoking. Preparing of rearing enclosures is made with 48 hours afore stoking. Enclosures are rigorously cleaned with Germostop solution 10 %, this solution is keep on for an hour and then banished by repeated wash off. Enclosures are rigorously cleaned with Germostop solution 10 %, this solution is keep on for an hour and then banished by repeated wash off. The height of water column in rearing enclosures is adjusted to 20 cm. The height of water column in rearing enclosures is adjusted to 20 cm.

9. Feeding Feeding Rearing of larvae up to outlining phenotypic characters conformable with adults is achieved on account of a feeding schedule involving deliver of live food and blended yolk of egg in first 5 – 7 days of growth and then a composite diet composed of natural food and forage. Rearing of larvae up to outlining phenotypic characters conformable with adults is achieved on account of a feeding schedule involving deliver of live food and blended yolk of egg in first 5 – 7 days of growth and then a composite diet composed of natural food and forage. Combinations of natural feeding stuffs charged are the following: Combinations of natural feeding stuffs charged are the following: 90 % Tubifex sp. and 10 % zooplankton 90 % Tubifex sp. and 10 % zooplankton 50 % Tubifex sp. and 50 % fresh or saline zooplankton 50 % Tubifex sp. and 50 % fresh or saline zooplankton Feeding rate is fixed daily depending on weight of stock. Delivering rate in 24 hours is determined as percentage of 100 % from stocking weight on that day. Feeding rate is fixed daily depending on weight of stock. Delivering rate in 24 hours is determined as percentage of 100 % from stocking weight on that day. Equal parts from this rate are delivered at every 3 hours day and night. Equal parts from this rate are delivered at every 3 hours day and night. Feeding of larvae using natural live food in a composite diet with forage will imply the use of following feeding schedule that will allow gradually passing to a feeding based exclusively on forage: Feeding of larvae using natural live food in a composite diet with forage will imply the use of following feeding schedule that will allow gradually passing to a feeding based exclusively on forage: first 5 days: 5 % zooplankton, 85 % benthos, 10 % forage; first 5 days: 5 % zooplankton, 85 % benthos, 10 % forage; next 5 days: 5 % zooplankton, 60 % benthos, 35 % forage; next 5 days: 5 % zooplankton, 60 % benthos, 35 % forage; next 5 days: 5 % zooplankton, 45 % benthos, 50 % forage; next 5 days: 5 % zooplankton, 45 % benthos, 50 % forage; next 5 days: 5 % zooplankton, 10 % benthos, 85 % forage; next 5 days: 5 % zooplankton, 10 % benthos, 85 % forage; After that feeding will be assure only by forage. After that feeding will be assure only by forage.

10. Growth gain to be obtained from the stages of development up to outlining adult-like phenotypic characters are the following: VariantSpor de creştere - g Parental species of sturgeons10 zile20 zile30 zile40 zile Huso huso - beluga0,925,28 Acipenser gueldenstaedti – Russian sturgeon0,51,645 Acipenser stellatus – sevruga0,31,533 Acipenser ruthenus – starlet0,150,81,52