An Optimized Experimental Zebrafish Breeding Scheme for Significantly Enhancing Reproductive Efficiency and Service Life

doi:10.12300/j.issn.1674-5817.2023.004

Abstract

Abstract:

Objective To solve the problems of delayed growth and development and insufficient spawning of experimental Zebrafish, so as to improve the reproductive efficiency and service life of experimental Zebrafish. Methods The zebrafish at the age of 2 months after fertilization were divided into two groups. The experimental group was fed with dry commercial diets specifically designed for ornamental fish or frozen adult brine shrimp, while control group was fed with live laval brine shrimp. Within a period of 70 days, the growth performance of the zebrafish was evaluated by measuring body length and weight, and the reproductive performance was assessed by measuring the fecundity and spawning rate. Zebrafish with apparent goiter disease were fed with dry commercial diets, and the inhibitory effect of the pellets on this disease was evaluated by measuring the diameter of the thyroid enlargement lesion. The three feeding methods were combined, and the feeding plan was optimized. The actual effects of the plan on zebrafish rearing were validated through reproductive performance tests. Results Starting from 60 days post-fertilization (dpf) until 111 dpf, the body length and weight of the dry commercial diets feed group gradually surpassed those of control group (all P<0.000 1). From 60 dpf to 96 dpf, the growth trend in body length of the adult brine shrimp group was similar to that of control group, but the female fish in the adult brine shrimp group had significantly higher body weight than the female fish in control group at 75-82 dpf (P<0.000 1). Compared to control group, there was a significant difference in body color between males and females in the adult brine shrimp group, and at 75 dpf, gender could be accurately distinguished by body color differences. Furthermore, the spawning rate of the zebrafish in the adult brine shrimp group at 3 months of age was significantly higher than that of control group (94.44% vs. 27.78%, P<0.05). Additionally, after feeding with the dry commercial diets for 130 days, all thyroid enlargement lesions in the experimental zebrafish disappeared. Based on the above results, the three feeding methods were combined and the feeding plan for zebrafish older than 2 months of age was optimized as follows: feed live brine shrimp in the morning, and alternate between dry commercial diets and adult brine shrimp in the afternoon. This feeding plan lasts until the age of 12 months. The spawning rate of Zebrafish can maintan 70%, and the spawning amount can reach (233.6±3.95) eggs. The fertilization rate and hatching rate were 97.47% and 90.24%, respectively, both significantly higher than those of control group (P<0.001, or P=0.01). Conclusion Compared to live brine shrimp feed, the dry commercial diets feed significantly improves the growth performance of zebrafish and has a therapeutic effect on thyroid enlargement disease. On the other hand, adult brine shrimp feed significantly enhances the early reproductive performance of zebrafish. The optimized feeding plan successfully improves the spawning efficiency of laboratory zebrafish, prolonging their reproductive lifespan and better supporting relevant scientific research.

Key words: Zebrafish, Breeding scheme, Reproduction, Growth

CLC Number:

Q95-33

Shirong JIN, Ye HUA, Huaxing ZI, Xufei DU, Jiwen BU. An Optimized Experimental Zebrafish Breeding Scheme for Significantly Enhancing Reproductive Efficiency and Service Life[J]. Laboratory Animal and Comparative Medicine, 2023, 43(3): 297-306.

Figures/Tables 5

Table 1 Nutrition ingredients of different diets

营养成分 Nutrition ingredients	需求量 Requirement	幼体卤虫(活) Larva brine shrimp (live)	成体卤虫(冻) Adult brme shrimp (frozen)	颗粒饲料1 Dry commercial diets-1	颗粒饲料2 Dry commercial diets-2
钙Calcium/%	0.9～1.6 (斑马鱼)^[5]	未检出	未检出	2.31	2.62
磷Phosphorus/%	0.8～1.4 (斑马鱼)^[5]	0.12	0.07	1.92	2.22
镁Magnesium/(mg·kg^-1)	600 mg/kg (鲤鱼)^[14]	186	102	6 110	5 360
铁Iron/(mg·kg^-1)	150 mg/kg (鲤鱼)^[15]	未检出	58.5	575	940
硒Selenium/(mg·kg^-1)	0.12 mg/kg (鲤鱼)^[16]	0.15	0.27	3.22	4.55
锌Zinc/(mg·kg^-1)	15～30 mg/kg (鲤鱼)^[15]	20	5.3	160	300
维生素B3 Vitamin B3/(mg·kg^-1)	30～40 mg/kg (鲤鱼)^[17]	未检出	133	未检出	未检出
维生素C Vitamin C/(mg·kg^-1)	30～50 mg/kg (鲤鱼)^[17]	50.5	102	1 000	3 960
维生素B2 Vitamin B2/(mg·kg^-1)	7～10 mg/kg (鲤鱼)^[17]	未检出	未检出	38	84.8

Figure 1 Effects of dry commercial diets and adult brine shrimp on growth of zebrafishNote： Data are presented as means±SEM （n=9）. A - B are the body length （A） and body weight （B） changes of zebrafish in dry commercial diets group and larva brine shrimp group （compared with larva brine shrimp group， **P<0.01， *P<0.05）. C-D are the changes in body length （C） and body weight （D） of male and female zebrafish in adult brine shrimp group and larva brine shrimp group （compared with male and female zebrafish in larval brine shrimp group， ****P<0.000 1， ***P<0.001， **P<0.01， *P<0.05）. dpf， age after fertilization.

Figure 2 Effects of different diets on reproductive characteristics of zebrafishNote： A, the typical appearance of zebrafish (♂ male, ♀ female) in dry commercial diets group, adult brine shrimp group and larva brine shrimp group. B is the comparison of spawning rate, spawning amount, fertilization rate and hatching rate of 3-month-old and 5-month-old zebrafish between adult brine shrimp group and larva brine shrimp group （9 zebrafish in each group. Spawning amount, fertilization rate and hatching rate are presented as means±SEM, **P<0.01).

Figure 3 Effect of dry commercial diets on goiter disease of zebrafishNote: A, Appearance of Zebrafish with goiter; B, Changes in thyroid diameter of Zebrafish after dry commercial diets feeding [Data are presented as means ±SEM (n=15), ?P<0.01, ????P<0.000 1, ns indicated no statistical difference].

Figure 4 New breeding scheme Du-Lab Zeb's Recipe 2.0

References 29

1	李阔宇, 潘鲁湲, 孙永华. 斑马鱼鱼房和养殖系统建设标准[J]. 中国比较医学杂志, 2020, 30(6):121-127. DOI: 10.3969/j.issn.1671-7856.2020.06.018 .
	LI K Y, PAN L Y, SUN Y H. Standards for constructing zebrafish houses and breeding systems[J]. Chin J Comp Med, 2020, 30(6):121-127. DOI: 10.3969/j.issn.1671-7856.2020.06.018 .
2	国家市场监督管理总局, 国家标准化管理委员会. 实验动物实验鱼质量控制: [S]. 北京: 中国标准出版社, 2020.
	State Administration for Market Regulation, China National Standardization Commission. Laboratory animal-Quality control of laboratory fish: [S]. Beijing: China Standards Press, 2020.
3	欧阳娟, 薛松磊, 徐妍, 等. 环境因素对丰年虾孵化的影响及孵化条件优化[J]. 中国饲料, 2018(18): 83-86. DOI: 10.15906/j.cnki.cn11-2975/s.20181817 .
	OUYANG J, XUE S L, XU Y, et al. The effect of environmental factors on the hatching of Artemia salina and the optimization of hatching conditions[J]. China Feed, 2018(18): 83-86. DOI: 10.15906/j.cnki.cn11-2975/s.20181817 .
4	LAWRENCE C. The husbandry of zebrafish (Danio rerio): a review[J]. Aquaculture, 2007, 269(1-4):1-20. DOI: 10.1016/j.aquaculture.2007.04.077 .
5	祝梅香, 王天奇, 张长勇, 等. 实验用斑马鱼剑尾鱼营养需求及饲料现状分析[J]. 中国比较医学杂志, 2009, 19(12): 61-65. DOI: 10.3969/j.issn.1671-7856.2009.12.012 .
	ZHU M X, WANG T Q, ZHANG C Y, et al. Nutritional requirements and current status of diets of laboratory zebrafish and swordtails[J]. Chin J Comp Med, 2009, 19(12): 61-65. DOI: 10.3969/j.issn.1671-7856.2009.12.012 .
6	SPENCE R, FATEMA M K, ELLIS S, et al. Diet, growth and recruitment of wild zebrafish in Bangladesh[J]. J Fish Biol, 2007, 71(1):304-309. DOI: 10.1111/j.1095-8649.2007.01492.x .
7	何嘉玲, 刘静, 王天奇, 等. 斑马鱼的质量标准化[J]. 中国实验动物学报, 2014, 22(6): 99-102. DOI: 10.3969/j.issn.1005-4847.2014.06.018 .
	HE J L, LIU J, WANG T Q, et al. Research of zebrafish quality standardization[J]. Acta Lab Animalis Sci Sin, 2014, 22(6): 99-102. DOI: 10.3969/j.issn.1005-4847.2014.06.018 .
8	CAHU C, INFANTE J Z. Substitution of live food by formulated diets in marine fish larvae[J]. Aquaculture, 2001, 200(1-2):161-180. DOI: 10.1016/S0044-8486(01)00699-8 .
9	Baert P, Bosteels T, Sorgeloos P. Manual on the Production and Use of Live Food for Aquaculture [J]. Fao Fisheries Technical Paper, 1996. DOI: http://tailieu.vn/doc/manual-on-the-production-and-use-of-live-f .
10	李爱杰. 水产动物营养与饲料学[M]. 北京: 中国农业出版社, 1996.
	LI A J. Aquatic animal nutrition and Feed science [M]. Beijing: China Agriculture Press, 1996.
11	WATTS S A, POWELL M, D'ABRAMO L R. Fundamental approaches to the study of zebrafi sh nutrition[J]. Ilar J, 2012, 53(2):144-160. DOI: 10.1093/ilar.53.2.144 .
12	张蓉, 王晓雯, 朱华. 观赏鱼营养需要研究与饲料配制[J]. 中国饲料, 2017(17): 5-9, 17. DOI: 10.15906/j.cnki.cn11-2975/s.20171701 .
	ZHANG R, WANG X W, ZHU H. Study on nutritional needs of ornamental fish and feed preparation[J]. China Feed, 2017(17): 5-9, 17. DOI: 10.15906/j.cnki.cn11-2975/s.20171701 .
13	王强. 浅谈观赏鱼的配合颗粒饲料[J]. 中国园林, 1992, 8(1):57.
	WANG Q. 浅谈观赏鱼的配合颗粒饲料[J]. J Chin Landsc Archit, 1992, 8(1):57.
14	汪福保, 罗莉, 王朝明, 等. 鱼类镁营养研究进展[J]. 动物营养学报, 2011, 23(6):930-936. DOI: 10.3969/j.issn.1006-267X.2011.06.007 .
	WANG F B, LUO L, WANG C M, et al. Recent advances in magnesium nutrition of fish[J]. Acta Zoonutrimenta Sin, 2011, 23(6):930-936. DOI: 10.3969/j.issn.1006-267X.2011.06.007 .
15	梁德海. 鱼类对矿物质的营养需要及其缺乏症[J]. 饲料工业, 1998,019(10):25-26. DOI: CNKI:SUN:FEED.0.1998-10-012 .
	LIANG D H. Nutritional requirement of fish for minerals and its deficiency[J]. Feed Ind, 1998(10):25-26. DOI: CNKI:SUN:FEED.0.1998-10-012 .
16	吴云发, 方春林, 王庆萍. 鱼类矿物质营养研究进展[J]. 江西水产科技, 2007(4): 19-22. DOI: 10.3969/j.issn.1006-3188.2007.04.007 .
	WU Y F, FANG C L, WANG Q P. Research progress of mineral nutrition in fish[J]. Jiangxi Fish Sci Technol, 2007(4): 19-22. DOI: 10.3969/j.issn.1006-3188.2007.04.007 .
17	韩如政. 维生素对鱼类的营养作用及在饲料中的添加[J]. 饲料工业, 1989(9):34-36.
	HAN R. 维生素对鱼类的营养作用及在饲料中的添加[J]. Feed Ind, 1989(9):34-36.
18	曹洪泽, 胡旭辉, 杨树娥. 卤虫的生活习性及其养殖技术[J]. 河北渔业, 2003(4): 45-46. DOI: 10.3969/j.issn.1004-6755.2003.04.027 .
	CAO H Z, HU X H, YANG S E. Life habits and culture techniques of Artemia[J]. Hebei Fish, 2003(4): 45-46. DOI: 10.3969/j.issn.1004-6755.2003.04.027 .
19	张宏, 陈燕琴. 卤虫的饵料价值与选用[J]. 青海师范大学学报(自然科学版), 2009, 25(1):67-71. DOI: 10.3969/j.issn.1001-7542.2009.01.019 .
	ZHANG H, CHEN Y Q. Feed value and select application of Artemia[J]. J Qinghai Norm Univ Nat Sci Ed, 2009, 25(1):67-71.DOI: 10.3969/j.issn.1001-7542.2009.01.019 .
20	郑新春, 刘莉, 戴文聪, 等. 过度喂养建立斑马鱼幼鱼肥胖模型[J]. 南方医科大学学报, 2016, 36(1):20-25. DOI: 10.3969/j.issn.1673-4254.2016.01.04 .
	ZHENG X C, LIU L, DAI W C, et al. Establishment of a diet-induced obesity model in zebrafish larvae[J]. J South Med Univ, 2016, 36(1):20-25. DOI: 10.3969/j.issn.1673-4254.2016.01.04 .
21	IZQUIERDO M S, FERNÁNDEZ-PALACIOS H, TACON A G J. Effect of broodstock nutrition on reproductive performance of fish[M] // Reproductive Biotechnology in Finfish Aquaculture. Amsterdam: Elsevier, 2001:25-42. DOI: 10.1016/b978-0-444-50913-0.50006-0 .
22	王梅, 张永勤, 黄靖, 等. 红斑马鱼体色观察及敲除mitfa基因对其体色发育的影响[J]. 激光生物学报, 2022, 31(1):19-26. DOI: 10.3969/j.issn.1007-7146.2022.01.004 .
	WANG M, ZHANG Y Q, HUANG J, et al. Observation of red zebrafish body color and the effects of knocking out mitfa gene on its body color development[J]. Acta Laser Biol Sin, 2022, 31(1):19-26. DOI: 10.3969/j.issn.1007-7146.2022.01.004 .
23	LECLERCQ E, TAYLOR J F, MIGAUD H. Morphological skin colour changes in teleosts[J]. Fish Fish, 2009, 11(2):159-193. DOI: 10.1111/j.1467-2979.2009.00346.x .
24	GOODWIN T. The biochemistry of the carotenoids: volume Ⅱanimals[M/OL]. 2nd ed. New York: Chapman and Hall, 1984. .
25	曹天义, 景晓娜. 人工投喂饵料提高卤虫养殖单产的探究[J]. 水产养殖, 2020, 41(12):40-41, 44.
	CAO T Y, JING X. 人工投喂饵料提高卤虫养殖单产的探究[J]. J Aquac, 2020, 41(12):40-41, 44.
26	林小涵, 金超凡, 高晨, 等. MiR-430对斑马鱼原始生殖细胞迁移和性腺发育影响的初步研究[J]. 中国海洋大学学报(自然科学版), 2022, 52(3):72-79. DOI: 10.16441/j.cnki.hdxb.20210144 .
	LIN X H, JIN C F, GAO C, et al. Preliminary functional analysis of miR-430 in regulating zebrafish (Danio rerio) primordial germ cell migration and gonad development[J]. Period Ocean Univ China, 2022, 52(3):72-79. DOI: 10.16441/j.cnki.hdxb.20210144 .
27	郭盼, 周继术, 吉红, 等. 不同链长脂肪酸对斑马鱼的性腺脂肪酸组成、繁殖力与仔鱼成活率的影响[J]. 水生生物学报, 2017, 41(4):766-773. DOI: 10.7541/2017.95 .
	GUO P, ZHOU J S, JI H, et al. Influence of fatty acids with different chain length on fatty acid composition of ovaries, fecundity and survival rate of larvae in zebrafish (Danio rerio)[J]. Acta Hydrobiol Sin, 2017, 41(4):766-773. DOI: 10.7541/2017.95 .
28	MURRAY K N, WOLF J C, SPAGNOLI S T, et al. Reversibility of proliferative thyroid lesions induced by iodine deficiency in a laboratory zebrafish colony[J]. Zebrafish, 2018, 15(6):558-565. DOI: 10.1089/zeb.2018.1603 .
29	D'ABRAMO L. Challenges in developing successful formulated feed for culture of larval fish and crustaceans[J]. Avances en Nutrición Acuícola Ⅵ, 2002, 143–151.

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