Fund Project: Preliminary Report on artificial breeding technology of naked-bellied sturgeon
Fund Project: Agricultural Finance Special Project "Fishery Resources and Environment Survey in Key Waters of Northwest China"; Ili Kazakh Autonomous Prefecture Science and Technology Program Project "Research on Artificial Breeding Technology of Naked-bellied Sturgeon in Rare and Endangered Fish species in the Ili River".
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Acipenser nudiventris, commonly known as the blue sturgeon, is a large sturgeon, good egg quality, high price of caviar produced, is an ancient species with high ecological value and economic value, can be called "living fossils in the water". It was once widely distributed in the Black Sea, the Sea of Azov and Central Asia in Europe, the Caspian Sea and the Aral Sea, as well as some rivers in the above areas, and has important value for maintaining the ecological balance of habitat waters. The caviar produced by naked-bellied sturgeon is of good quality and high price, and has always been a valuable ingredient in Europe. Influenced by years of overfishing and environmental change and wading projects, the naked-bellied sturgeon has long been classified as critically en-dangered (CR) on the International Union for Conservation of Nature (IUCN) Red List of Threatened Species (www.iucnredlist.org), as well as the International Union for Conservation of Nature (IUCN) Protected species in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). Naked-bellied sturgeon have important scientific value in many areas, including zoogeography, paleoecology, fish systems and climate change. At the same time, as an important native sturgeon germplasm resource in China, the naked-bellied sturgeon has great market development potential in aquaculture. However, among the 8 species of native sturgeon in China, except for the Yangtze River white sturgeon (Psephurus gladius), which has been declared extinct, only the Ili River naked-bellied sturgeon has not yet achieved artificial reproduction, and the situation of species protection is imminent.
Naked-bellied sturgeon were transplanted from the Syr Darya River by the Soviet Union from the Syr Darya River in 1933-1934 to the then Sino-Soviet cross-border river Ili River-Lake Balkhash water system, forming a large fishing group 16 years later. Due to the existence of suitable spawning grounds in the main tributaries of Xinjiang in the upper reaches of the Ili River, mature broodstock often breed from Kazakhstan to the Xinjiang section of China, and in the 1970s, stable economic catch was formed in the Xinjiang section of the Ili River in China, with an annual yield of about 40 t. In the 1990s, before and after the collapse of the Soviet Union, kazakhstan's fishery management order was chaotic, affected by water conservancy projects, environmental pollution, cool fishing and other factors, and wild naked-bellied sturgeon was on the verge of extinction in the Ili River-Balkhash Lake water system in Kazakhstan. At present, the population of naked-bellied sturgeon in the Ili River in Xinjiang, China, is very small, and it has been found occasionally, and it has been impossible to form a catch production. It is listed as a first-level protected wild animal at the autonomous region level in the Xinjiang Uygur Autonomous Region and a wild animal under national second-level protection. For many years, all sectors of society in Xinjiang have been paying close attention to the protection of naked-bellied sturgeon species. Therefore, the rescue and protection of wild naked-bellied sturgeon also has high social welfare value.
In the Ili River Basin, the males of wild naked-bellied sturgeon are generally 6-9 years old. Females are mostly 12 to 14 years old [6]. According to the recent fishery resources survey and monitoring, the main tributaries of the Ili River have not found sexually mature wild naked-bellied sturgeon for many years, and the live naked-bellied sturgeon caught in the wild is extremely rare and mainly young-aged individuals. The rearing cycle required for wild juvenile fish to achieve sexual maturity under artificial culture conditions is extremely long, and the economic and time cost of feeding management is huge. In addition, breeding deaths or gonadal growth retardation caused by incompatibility with the breeding environment also occur from time to time. Over the years, key technologies for the protection of naked-bellied sturgeon species in the Ili River, such as artificial breeding and seed domestication, have not yet made substantial breakthroughs.
In this study, the wild individuals of naked-bellied sturgeon were collected in the Xinjiang section of the Ili River in China for many years, domesticated in artificial environment, and continuously monitored their gonadal development for many years, and the temperature change control of the simulated field environment was implemented before birth, and the synchronous development of female and male gonads in some sexually mature individuals was realized, and on this basis, artificial reproduction was successfully achieved and broodstock recovered well. This paper is the first successful report on the artificial breeding and seed breeding technology of the critically endangered species naked-bellied sturgeon in the Ili River, and through the systematic sorting out of the technical parameters of artificial breeding, it is hoped to provide reference for the subsequent full artificial breeding and large-scale breeding of naked-bellied sturgeon, and provide seedlings and expanded populations for the restoration and utilization of naked-bellied sturgeon fishery resources in the Ili River Basin.
1 Materials and methods
1.1 Experimental materials
1.1.1 External morphological characteristics of the naked-bellied sturgeon The appearance and morphology of the adult fish in the abdomen are shown in Figure 1, the dorsal view is gray-green, the posture is similar to a long spindle, the body side color is lighter, the abdomen is generally yellowish-white, and the fin color is mostly gray. The number of rows of body inflamement is 5, of which the first row is the largest bone plate, which is at a blunt angle to the top of the head, which is obviously different from other sturgeon. There are no small bone plates between the bone plates on the surface of the body, of which there are 11 to 17 bone plates on the back, 49 to 74 on the sides, and 11 to 17 on the ventral surface. The snout is smooth, slightly rounded, almost completely conical (fig. 2), ventral orifice, larger, and not separated in the center of the lower lip. There are 4 tentacles, and the whisker length is 7.1%~17.6%; The number of gill rakers is 24 to 45; Dorsal fin strips do not branch, 39~57 strips; The fins also do not branch, and the number is 23 to 37.
Fig. 1 Adult morphological characteristics of the naked-bellied sturgeon
Fig. 2 Head morphology of an adult naked-bellied sturgeon (ventral surface)
1.1.2 Parental source and feeding management All the individuals in this study were collected from wild individuals in the Ili River Basin, which have been collected continuously by the research team since 2013, and preserved in the Gongliu Scientific Experimental Base of Gongliu Science Experimental Base jointly established by Xinjiang Yihe Sturgeon Technology Co., Ltd. and the Heilongjiang Fishery Research Institute of the Chinese Academy of Fishery Sciences. The temperature range of cultured water bodies is 0 to 22 °C. The daily feeding amount of Tianbang sturgeon granules is 0.5% to 1.0%. 13 native adult species of wild naked-bellied sturgeon have been preserved as the basis for artificial breeding. Minimally invasive surgery, ultrasound, and dynamics of hematological indicators are used to detect gonadal development in adult individuals.
1.2 Artificial induction and incubation management
In May 2020, the gonadal development of broodstock populations was monitored by minimally invasive surgical method and PI (polariza-tion index) index method, and the naked-bellied broodstock whose gonads developed to stage IV were transferred to the pond to be fertilized, and the female broodstock with oocyte polarization index PI<0.1 was selected for artificial induction experiments with male broodstock containing a small number of sperm under the microscope. The inducing drug is LHRH-A2 (Ningbo Third Pharmaceutical Factory), in which the female broodstock use the two-needle injection method and the male broodstock use the one-needle injection method. The first injection dose of female broodstock is 0.8 to 1.0 μg/kg (body weight), after 10 to 12 h, the second injection of female broodstock and male broodstock is 2~3 μg/kg (body weight), and the injection dose of male broodstock is 1/2 of the total dose of female broodstock injection. The induction temperature is 15~16 °C. After the effect period, the semen was collected in batches by abdominal compression, and stored in a refrigerator at 2~4 °C after oxygenation. Eggs are collected at one time using abdominal compression and minimally invasive surgery, semi-dry artificial insemination, and semen quality is measured before insemination.
The fertilized eggs were debonded from 15% to 20% talc solution for 45 min, debonded and placed in a bottle incubator (Figure 3), the incubation water temperature was 16.5 to 18.0 °C, dissolved oxygen ≥ 6.0 mg/L, and the incubation water temperature was determined every 30 minutes by RC-T601A intelligent portable temperature logger. During the incubation period, the whole process of embryo development of naked sturgeon was continuously observed under the dissectoscope, observed every 0.5 h in the pre-development period, observed once every 1.5-3 h after reaching the blastocyst stage, and recorded the time of different stages of embryonic development. After hatching, take 15 fish per day to observe their changes until they are open for feeding.
Fig. 3 Bottle hatching of fertilized eggs of naked-bellied sturgeon
1.3 Artificial domestication methods and daily management of fry
Hatching naked-bellied sturgeon are cultured in circular glass tanks with a radius of 0.6 m × 0.5 m deep, with a cluster phenomenon (fig. 4). The water temperature of the culture is 17~18 °C, after the culture is 7~8 d, the intestinal embolus is started, and the water earthworm section is fed (the water earthworm is broken into segments by beating the pulper), and the daily feeding is 6 to 8 times, and the feeding amount is 20% of the body weight. After 2 weeks of feeding, the water earthworm section plus artificial particles and powder (the powder is the nordic horse company imported piglet fish opening material), the artificial pellet feed domestication began, the domestication period was 30 to 40 days, the number of daily feedings was 6 times, and the feeding amount was 10% of the body weight. The feeding time is 20 to 30 minutes, and the residual bait and feces are removed in time after each feeding.
The temperature is 17~18 °C, after breeding for 7~8 d, the intestinal embolus is started, and the water earthworm section (the water earthworm is broken into segments by beating the pulper), fed 6 to 8 times a day, and the feeding amount is 20% of the body weight. After 2 weeks of feeding, the water earthworm section plus artificial particles and powder (the powder is the nordic horse company imported piglet fish opening material), the artificial pellet feed domestication began, the domestication period was 30 to 40 days, the number of daily feedings was 6 times, and the feeding amount was 10% of the body weight. The feeding time is 20 to 30 minutes, and the residual bait and feces are removed in time after each feeding.
Fig. 4 Cluster phenomenon of naked-bellied sturgeon hatching fish
1.4 Measurement methods
For the determination of egg diameter, egg quality, semen quality and other indicators, refer to Liu Jianyi et al. Among them, the egg diameter measurement is calculated by the length and egg quality, and the egg diameter and quality are extracted three times before and after the egg is excreted, and 50 eggs are randomly drawn each time, and the length and quality are measured. Estimation of spawning by volume method; After the embryos reach the primary stage of development, the fertilization rate is counted, and 200 embryos are drawn per batch to calculate the fertilization rate. During incubation, the whole process of embryonic development of naked-bellied sturgeon was observed under an autopsyctomy, observed once every 0.5 h in the pre-development period, and observed once every 1.5 to 3 h after the blastocyst stage.
2 Results and analysis
2.1 Identification of gonadal development of naked-bellied sturgeon under artificial breeding conditions
A total of 13 adults of naked-bellied sturgeon were domesticated, aged 12 to 16 years, and 5 females, 7 males, and 1 unrecognizable were identified. In September 2019, two females were identified as having reached stage IV with an average weight of 32.65 kg. The feeding status of sturgeon entering stage III of gonadal development has not changed significantly, while broodstock feeding after entering stage IV is significantly weakened (table 1). 2 to 3 months before induction of labor, the female brooding sturgeon whose gonads have developed to stage IV are transferred to the ecological control pool, and the PI value is calculated after egg collection by the minimally invasive abdominal method, and the PI index is about 0.02 when entering the pool, and the PI index reaches about 0.1, and it is transferred to indoor waiting for delivery. Male broodstock whose nests develop to the end of IV are transferred to the ecological control pool 1 month before delivery until the full gonads mature, and there is a large amount of semen spillage in the abdomen under light pressure.
Table 1 Biological indicators of naked-bellied sturgeon in stage IV of gonadal development
2.2 Reproductive performance and effect analysis of artificial induced reproduction of naked-bellied sturgeon
After synchronous ecological regulation of gonadal development, a total of 2 females and 3 males achieved synchronous gonadal maturation and artificial induced reproduction. Female broodstock begin to ovulate 15 to 18 hours after the second injection of LHRH-A2, and collect eggs at one time using abdominal compressions and minimally invasive surgery. The average prenatal weight of female parent sturgeon is 35.65 kg, and the average weight after spawning drops to about 30 kg, and there are some fish eggs in the abdominal cavity, which are gradually emptied later. The total spawning volume of the two females was 211 000 (55,000 and 156,000, respectively), with an egg diameter distribution range of 2.95-3.44 mm, an average egg diameter (3.21±0.25) mm, an average single egg mass (0.042±0.011) g, and a total spawning mass of 5.04 kg, accounting for about 14.2% of the body weight. The males collected semen in 5 batches after LHRH-A2 injection for 10 h, with a total sperm collection volume of 2 850 mL. Male sturgeon weigh 27.35 kg before childbirth and 20 kg after childbirth. The sperm collected had an average duration of vigorous exercise (46.12±6.47) s, an average lifespan of (260.18±55.62) s, an average fertilization rate of 55%, and a hatching rate of 42.5% (Table 2).
Table 2 Reproductive performance and artificial induction effect of naked-bellied sturgeon
2.3 Observation of embryonic development and emergence of fry
The developmental stages of the naked-bellied sturgeon generation of the Ili River were observed and the developmental accumulation temperature was calculated. The initial fertilization rate can be judged to begin at the second ruse, develop to 4-cell stage 3.5 h after fertilization, reach blastocyst stage at 12.5 h, reach mid-intestinal stage at 26 h, close the neural tube at 48 h, beat the heart at 62 h, reach the heart at 83 h, and emerge in large numbers at about 130 h (Table 3). At incubation water temperature of 16.5~18.0 °C, it takes 123~145 h from fertilization to hatching of naked-bellied sturgeon, and the total accumulated temperature is 2030~2755 °C·h. About 25,200 hatchlings were successfully ingested, and the body length of the hatchlings was 9.56~11.96 mm.
Table 3 Accumulated temperature at each stage of development of the naked-bellied sturgeon generation of the Ili River
3 Discussion
3.1 Conservation and utilization of sturgeon germplasm resources in China
Historically, the natural waters of China are rich in fishery resources, with a total of 8 species of sturgeon, belonging to 2 families and 3 genera, including Acipenser sinensis, Acipenser sinensis, Siberian sturgeon (Acipenser baerii), small body sturgeon (Acipenser ruthenus), naked-bellied sturgeon and Acipenser schrenkii. Husodauricus of the genus Sturgeonidae and Yangtze River Sturgeon of the genus Sturgeon of the family Sturgeonidae. Among them, 3 species of sturgeon in the Yangtze River Basin are listed as national first-level protected wild animals, and the other 5 species of sturgeon are all national second-level protected wild animals. In the early 21st century, Chinese fishery workers successively broke through the full artificial breeding technology of sturgeon such as Shi's sturgeon, Siberian sturgeon, and Chinese sturgeon. Through strategies and methods such as in situ conservation, ex situ conservation and native habitat enhancement and stocking, most of the sturgeon species in China have avoided extinction in the sense of species, except for the Yangtze River white sturgeon. However, the amount of wild sturgeon resources has still shown a sharp downward trend in recent years. In order to reduce the damage to wild sturgeon resources due to the consumption of sturgeon and caviar, all existing species of wild sturgeon in the world have been listed as protected species in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). Since the 1990s, the naked-bellied sturgeon in the Ili River Basin has not broken through artificial breeding technology for nearly 30 years, and the wild naked-bellied sturgeon in the Ili River-Balkhash Lake water system has been almost completely extinct, and through more than ten years of efforts, the research team has collected a total of 13 large individual wild naked-bellied sturgeon, which are domesticated in captive breeding environments. Up to now, only the Gongliu Scientific Experimental Base of Heilongjiang Fisheries Research Institute has preserved 5 live wild Ili River naked-bellied sturgeon to sexual maturity, which provides parental materials for the development of this artificial breeding experiment.
3.2 Suitable breeding conditions and nutritional requirements for broodstock of naked-bellied sturgeon
Factors such as water temperature changes, photoperiods and feed nutrition are the main influencing factors of sturgeon gonadal development and maturation, and play an important role in inducing and activating the "hypothalamus-pituitary-gonad axis" of the fish endocrine system. Generally speaking, the conventional pond culture environment is often not conducive to the wild sturgeon larvae to initiate reproductive endocrine regulation pathways related to gonadal development, the scientific experiment base based on this study is located in the Ili River Valley in Gongliu County, Ili Kazakh Autonomous Prefecture, Ili River and caspian Sea are at the same latitude, and the climate is relatively similar. The species protection and domestication and breeding of naked-bellied sturgeon here have unique advantages in water source, geography, climate and environment. In terms of feed nutrition, the feeding compound feed method used in this study can achieve the final maturity of the gonads of some naked-bellied sturgeon. There are also studies that show that the addition of live fish or chilled fish is more conducive to the initiation of sturgeon gonadal development, possibly due to the fact that the nutrients in fresh or fresh fish or shrimp have a higher absorption and conversion efficiency in the body, which is more conducive to the accumulation of yolk nutrients in female fish, which is more conducive to gonadal development.
3.3 Artificial reproduction of naked-bellied sturgeon under artificial domestication conditions
The naked-bellied sturgeon of the Ili River system originally originated from the Syr Darya River in the Aral Sea system, and the population of the brackish sea system naked-bellied sturgeon is almost extinct. According to historical records, in the Aral Sea, the average egg diameter of naked-bellied sturgeon eggs is 1.55 to 3 mm, and the average egg mass is 0.0114 g. The egg size distribution range of the naked-bellied sturgeon in the Ili River obtained in this study ranged from 2.95 to 3.44 mm, the mean egg diameter was 3.21 mm, and the average single egg mass was 0.042 g. The egg quality and size are significantly better than those of wild naked-bellied sturgeon in the brackish sea, and it is speculated that the reason may be that in the pond rearing environment, broodstock can obtain better feed and nutritional conditions, save safety and defense, breeding courtship and other behaviors to consume energy, thereby improving reproductive performance. The naked-bellied sturgeon, as a larger of the sturgeon, also does not have it