Modern Sterlet Acipenser ruthenus Broodstock Management in Southern Russia

Modern Sterlet Acipenser ruthenus Broodstock Management in Southern Russia E.N. Ponomareva and M.M. Belaya The maintenance of optimal heterogeneity and decrease of probable inbreeding during broodstock collection is achieved by fully renewing broodstock every 4 years (8 reproductive cycles) with an annual change of 30 percent of breeders with different reproductive origins: 10 percent from hatchery broodstock, 10 percent from the wild and 10 percent from females and cryopreserved sperm. In an attempt to counter the catastrophic decrease in populations of sturgeon and the accompanying pressure on breeders, artificial reproduction in hatcheries has an important place in Russia. Fish hatcheries supplying mature breeders is a faster way to obtain sturgeon broodstock, using new biotechnological methods. When establishing broodstock of sturgeon for fish hatcheries, it is important to conserve the integrity of the gene pool and carefully plan the release of these fishes into natural water bodies (Popova et al. 2001). More than 20 broodstock lines have been established across several fish hatcheries in Russia. The main problems are estimation of optimal conditions for breeding, joint breeding of different sturgeon species and age groups, and a search for effective methods of selective breeding for the development of highly productive broodstock for breeding and commercial purposes (Burtsev et al. 1989, Vasil eva 2000). Development of productive broodstock is accomplished in two ways: domestication of wild breeders and rearing from eggs to eggs. Domestication of wild sturgeon is realized by obtaining sexual products from them over their lifetimes. The basis of successful domestication is training fish to accept formulated feeds and acclimate to unusual environments. Formation of broodstock on the principle of eggs to eggs is realized by obtaining offspring from breeders and rearing them in controlled conditions to maturation. The use of females captured from natural water bodies, with eggs in the second stage of maturation, is a common practice. Females are then bred in hatcheries until the end of gametogenesis. Formation of broodstock in this manner has a spontaneous quality; there can be no selection of fish specific to sturgeon breeding (Podushka 1997, Barannikova et al. 2000). One advantage for fish hatcheries in using mature breeders is faster maturation of males and females, using different combinations of breeding methods, manipulation of environmental factors and other opportunities (Samoilenko 2011). With development of modern biotechnology, the most sure and effective method of faster maturation of sturgeon is the use of industrial breeding, particularly in installations with closed water supplies. The main purpose of our investigations is to develop new concepts of productive sturgeon broodstock formation and improve existing methods of broodstock exploitation in installations with closed water supplies. The use of recirculation systems with complex regulation of basic environmental factors (temperature, light, feeding regime, mineral content of water and water use) allows juveniles and breeders to achieve maturation quickly, approximately twice as fast as in nature. In conditions of intensive breeding, the maturation of males is faster than that of females. Sterlet mature at 2 years, sturgeon of Lena River at 2-3 years, Russian sturgeon at 3-4 years and beluga at 4-5 years. The method of early maturation of females requires further improvement (Samoilenko 2011). The object of this investigation was sterlet a freshwater, early-maturing species of sturgeon. Two subspecies of sterlet were used: from populations in the Volga and Don rivers. In formation and exploitation of fish broodstock, decreased heterogeneity of offspring is a problem because the supply of breeders used for spawning is often limited. Inbreeding leads to deterioration of physiological status with a decrease in immunity and vitality of offspring. To avoid the negative consequences of inbreeding, the quantity of breeders must be increased. This is very difficult because of a current lack of sturgeon breeders. To increase the population heterogeneity, it is reasonable to exchange the sexual products (milt) between fish hatcheries. However, broodstock in different hatcheries do not always mature at the same time. Furthermore, the quality of milt can be unsatisfactory and quality can decrease during transportation beyond usability, posing a threat to the entire spawning campaign. Maintenance of an optimal level of genetic diversity in hatchery populations is a primary and simultaneously difficult undertaking because the decrease of genetic heterogeneity results in deterioration of fish quality. In nature, there is adaptive flexibility and the opportunity for populations to adapt to different environmental conditions. In hatcheries, the reduction of genetic diversity results in the loss of unique properties and characteristics of these engineered fish populations. Maintenance of balance among traits is the basis for extended exploitation of hatchery populations. We have developed the following criteria for broodstock formation based on this principle. The effective number of broodstock (N e ) should be at least 100-200 breeders of different ages and the quantity of females and males should be equal to decrease the risks of inbreeding. Other criteria for broodstock development provides for decreased inbreeding and genetic drift. This can be achieved by collection of broodstock from different areas. Our broodstock population was formed from mature and nearly mature fish that were caught in natural waters at different (CONTINUED ON PAGE 56) WWW.WAS.ORG WORLD AQUACULTURE JUNE 2013 55

FIGURE 1. Recirculation system for broodstock formation. FIGURE 2. Obtaining sterlet eggs. FIGURE 3. Workplace of scientist at SSC RAS. FIGURE 4B. Samples of milt in cryofreezer. FIGURE 4A. Samples of milt in cryofreezer. FIGURE 5. Cryostorage of milt samples in liquid nitrogen. 56 JUNE 2013 WORLD AQUACULTURE WWW.WAS.ORG

times followed by a final selection. The maintenance of optimal heterogeneity and decrease of probable inbreeding during broodstock collection was achieved by fully renewing broodstock every 4 years (8 reproductive cycles) with an annual change of 25-30 percent of breeders with different reproductive origins: 10 percent from hatchery broodstock, 10 percent from the wild and 10 percent from females and cryopreserved sperm. At the Southern Scientific Center of the Russian Academy of Science (SSC RAS), an experimental fish breeding complex allows year-round investigations, independent of weather. The complex consists of 9-m 3 tanks for breeders, 2-m 3 tanks for recuperation groups (i.e. juveniles chosen to relieve spent broodstock), 1-m 3 tanks for fry, tanks for artificial wintering, cooling systems, and filtration systems for maintenance of optimal hydrochemical and hydrological regimes. There is a place for long-term cryostorage of frozen sperm and other special equipment (Figs. 1-5). We have developed a method of cryopreservation of cells using electrostimulation, which gives a high proportion (90-95 percent) of live sperm after thawing. A sperm cryobank is used in the formation of several sterlet broodstocks. In this facility, the development of sterlet broodstock was maintained in two lines: from imported breeders of Volga and Don River populations and from the eggs of those fish. The starlet broodstock population is increased every year with up to 10 percent of new adult fish and up to 10 percent of imported fry (5-10 g). Since 2010, in the process of broodstock formation, a line of sperm was stored in liquid nitrogen. It also amounted to 10 percent. The scheme of sterlet broodstock formation is shown in Figure 6. We have formed lines of broodstock and now can make forecasts of estimated spawning dates. The structure of different sturgeon species broodstock populations was formed to avoid inbreeding. Therefore, groups with different origins were formed. They were imported from different areas of southern Russia (Astrakhan, Volgograd and Rostov regions). Spawning interval decreases with age. Sterlet from the Volga River have the shortest spawning interval (5.5-6.0 months; Table 1). Specialists of SSC RAS have developed a software program for sturgeon broodstock registration that allows creation of a database that includes information on individual biological, biometric, physiological and reproductive parameters of all tagged fish that are maintained as broodstock. In this database program, information about species and population of each individual, age, date of importation from other hatcheries and tank number is shown. There is also the opportunity to trace individual fish growth. To accommodate the rapid selection of tagged individuals, the search string capability is provided. In the process of ultrasonic scanning, the sex and stage of sexual maturity (I V) are recorded in the program. During the spawning season, the date of each spawn and the quantity and quality of obtained eggs of sturgeon breeders is recorded. The program allows tracing of the physiological state of individual broodstock on the basis of the following hematological parameters: hemoglobin concentration erythrocyte sedimentation rate hematocrit value (CONTINUED ON PAGE 58) OM Farming our waters - Agrifood Innovations Cultiver nos eaux : Innovation en agroalimentaire Co-Hosts / Hôtes conjoints General Information/ Renseignements généraux Conference coordinator Joanne Burry Tel : 709-437-7203 Email : jmburry@nl.rogers.com www.aquacultureassociation.ca WWW.WAS.ORG WORLD AQUACULTURE JUNE 2013 57

TABLE 1. Duration of spawning intervals of sterlet broodstock that was formed in recirculation system. Sex First spawning interval (month) Second spawning interval (month) Third spawning interval (month) 1 2 1 2 1 2 STERLET OF VOLGA RIVER 11 12 9 9.5 7 7.5 10 11 8 9 6.5 6 STERLET OF DON RIVER 10 11 8 9 6 6.5 9 10 9 10 5.5 6 TABLE 2. Characteristic of females of River Don sterlet broodstock that are formed from fry (generation of 2005 year). Age Weight, kg Length, cm Work fecundity Relative fecundity Spawning interval (thousand pieces) (thousand pieces/ kg of weight) (months) 3+ 0.97±0.08 60.2±1.6 15.5±1.5 15.9±1.6 9.0 4+ 1.09±0.16 62.3±1.7 22.7±1.9 20.8±1.6 8.0 5+ 1.38±0.20 65.1±1.7 36.3±1.5 26.3±2.2 7.5 TABLE 3. Characteristic of females of River Don sterlet broodstock that are formed from breeders (generation of 2006 year). Age Weight, kg Length, cm Work fecundity Relative fecundity Spawning interval (thousand pieces) (thousand pieces/ kg of weight) (months) 4+ 1.25±0.11 62.8±1.7 24.9±1.6 19.9±1.9 8.0 5+ 1.46±0.13 66.2±1.3 30.5±2.1 20.9±1.7 7.8 6+ 1.65±0.16 68.1±1.3 35.4±1.8 21.5±1.7 7.5 TABLE 4. Characteristic of males of River Don sterlet broodstock that are formed from fry (generation of 2005 year). TABLE 5. Characteristic of males of River Don sterlet broodstock that are formed from breeders (generation of 2006 year). Age Weight, kg Length, cm Ejaculate Relative ejaculate volume, ml volume ml/kg of weight 3+ 0.90±0.07 59.3±1.6 20.2±2.1 22.4±1.5 4+ 1.06±0.14 60.2±1.7 22.2±1.5 21.0±1.5 5+ 1.36±0.21 61.4±1.6 28.4±2.2 20.9±2.0 Age Weight, Length, Ejaculate Relative ejaculate kg cm volume, ml v ml/kg of weight 4+ 1.26±0.13 62.0±1.7 26.4±2.2 21.0±1.5 5+ 1.35±0.20 63.2±1.9 33.0±1.5 24.5±1.8 6+ 1.52±0.24 65.1±1.9 42.2±1.8 24.8±1.9 TABLE 6. Quality of cryopreserved sperm. Characteristics Native sperm Cryopreserved sperm Spermatozoa activity (%) 100 70 Fertilization rate (%) 90 80 Amount of fingerlings (%) 75 60 58 JUNE 2013 WORLD AQUACULTURE WWW.WAS.ORG

FIGURE 6. Scheme of sterlet broodstock formation. FIGURE 7. Page of a fish electronic passport that was made with a computer program. serum protein level serum lipid concentration serum cholesterol concentration leukocytic formula: white blood cells, eosinophils, stab neutrophils, segmental neutrophils, basophiles, monocytes, lymphocytes and blasts. There is also an opportunity to record the results of histological investigations of liver and gonads, for example, in descriptive form. Such a program allows matching, with high accuracy, individual breeders using their electronic passports and eliminating the probability of mistakes in crossing different lines. Each broodfish has an electronic passport that shows its origin, physiological status and all control research (Fig. 7). The characteristics of sterlet broodstock from the River Don are shown in the Tables 2 and 3. At present, it consists of individuals 3-6 years old. Stable hydrological and hydrochemical conditions of the water environment and effective feeding allow high growth rates of female broodstock. Multiple generations of fry and broodstock have high reproductive characteristics; average fecundity ranged from 15.5 to 36.3 thousand eggs, confirmation of the good quality of sterlet females. Males of the River Don population also have high reproductive indicators. Reproductive characteristics of different generations of males did not differ practically (Table 4, 5). This is the result of the stable regime of environmental factors in the recirculation system. Comparative analysis of formed lines of broodstock of Volga River sterlet indicates high biological performance of females and males of different ages. This was also connected to stable hydrological and hydrochemical conditions of the water environment and optimal feeding of breeders at different stages of ontogenesis. Sterlet eggs were artificially inseminated with thawed, cryopreserved sperm that had been stored in liquid nitrogen for 2 years. This reproductive material was 10 percent of the total number of broodstock that took part in spawning. Before fertilization, the quality of thawed sperm was good, resulting in a fertilization rate of 80 percent, compared to 90 percent in controls. Survival of fry after incubation for 6 days was 75 percent in the control group and 60 percent in the treatment group (Table 6). Morphometric measurements of the larvae of investigated groups were not significantly different. The same results were obtained in trials of fingerlings and fry. Development of starlet broodstock by several methods, including sperm cryopreservation, is reasonable in recirculation systems. It reduces inbreeding and decreases the area required to contain broodstock. Development of sturgeon culture broodstocks and sperm cryobanks allows conservation of the gene pool and decreases the risks of anthropogenic influence on productivity. Notes E.N. Ponomareva and M.M. Belaya, 41 Chekhov Avenue, Rostovon-Don, Russia, 344006 344006, Россия, Ростов-на-Дону, проспект Чехова 41 References Barannikova, I.A., S.I. Nikanorov and A.N. Belousov. 2000. The problem of sturgeon fishes conservation in Russia today. Pages 7-8 In: Sturgeon Fishes at the Turn of the XXI Century. Astrakhan, Russia. Burtzev, I.A. and A.I. Nikolaev. 1999. Methods of formation and exploitation of sturgeon broodstocks in conditions of sturgeon fish hatcheries to purposes of pasturable mariculture. Pages 20-21 In: Resource-Saving Technologies in Aquaculture. Krasnodar. Russia. Podushka, S.B. 1997. Formation and exploitation of sturgeon fish s broodstocks for the purposes of obtain the material to let it out in natural water bodies. Page 293 In: First Congress of Ichthyologists in Russia. Moscow, Russia. Popova, A.A., V.N. Shevchenko, L.V. Piskunova, P.V. Chernova and G.P. Marinova. 2001. Results of experimental-industrial work in creation of sturgeon fishes broodstocks. Pages 58-61 In: Problems of Present Commercial Sturgeon Breeding. Astrakhan, Russia. Samoilenko, D.A. 2011. About the factors of faster sexual maturation of sturgeon fishes. Pages 176-179 In: Sturgeon Fishes and their Future. Berdyansk, Ukraine. Vasil eva, L.M. 2000. Biological and technological peculiarities of commercial aquaculture of sturgeon fishes in conditions of Lower Volga Region. Astrakhan, Russia. WWW.WAS.ORG WORLD AQUACULTURE JUNE 2013 59