Animal Reproduction (AR)
https://www.animal-reproduction.org/article/doi/10.1590/1984-3143-AR2025-0003
Animal Reproduction (AR)
ORIGINAL ARTICLE

Standardization of techniques and conditions for in vitro ovulation induction in Astyanax altiparanae

Cristiane Fernanda Benevente; Mariana Roza de Abreu; Queila Carla Ameno Alves; Laíza Maria de Jesus Silva; Roosevelt Passos Barbosa; Samyra Maria dos Santos Nassif Lacerda; Sergio Ricardo Batlouni

http://dx.doi.org/10.1590/1984-3143-AR2025-0003 Anim Reprod, vol.23, n1, e20250003, 2026
PDF
Downloads: 0
Views: 14

Abstract

This study aimed to establish in vitro culture conditions for inducing ovulation in lambari (Astyanax altiparanae). All fish received a priming dose of either 0.6 mg/kg or 100 IU hCG/kg (human chorionic gonadotropin) in vivo. In experiment 1, 0.6 mg and 5.4 mg of carp pituitary extract (CPE)/ kg of ovarian fragment were tested as resolving doses. In experiment 2, prostaglandin F2α (PGF2α) analogue at 100 ng/mL was added to 5.4 mg CPE/kg as the resolving dose. Since ovulation did not occur, in experiment 3 and 4, we compared diverse forms of obtaining follicles, comparing manual follicle dissociation and collagenase at 100, 200, and 400 CDU/mL. Additionally, in experiment 4, the resolving dose of CPE and hCG was replaced by 1 µg/mL 17α,20β-dihydroxy-4-pregnen-3-one (DHP). Ovulation was successful only in Experiment 4, using mechanically dissociated follicles with a priming dose of either CPE or hCG and 1 µg/mL DHP as the resolving dose. Key findings include that 5% CO2 is unnecessary, mechanical dissociation of ovarian fragments is optimal, a priming dose of CPE or hCG is required, and DHP at 1 µg/mL is effective. These results establish a standardized protocol for in vitro ovulation induction in A. altiparanae, offering a valuable tool to study ovarian function and spawning failure in tropical species.

Supplementary Material

Supplementary material accompanies this paper.
Supplementary Figure 1 - Experiment 3. Survival rate of follicles analyzed using Trypan blue.

Keywords

fish ovulation, spawning induction, DHP, PGF2alpha, pituitary extracts

References

Ariki DGF, de Abreu MR, de Jesus Silva LM, Sato RT, Batlouni SR. Attempts for increasing Astyanax altiparanae spawning rates and percentage of responsive oocytes. Anim Reprod Sci. 2023;254:107262. https://doi.org/10.1016/j.anireprosci.2023.107262. PMid:37295049.

Batlouni SR, Corrêa Filho RAC, Kuradomi RY, Spica LN, Abreu MR, Brasileiro LO, et al. Maintenance and management of breeders of Neotropical South American species in laboratories. In: Streit DP Jr, Zhang T, Paredes E, editors. Cryobiology for South American Neotropical Fish Species. Boca Raton: CRC Press; 2024. p. 142-70. https://doi.org/10.1201/9781003269441-7.

Bobe J, Montfort J, Nguyen T, Fostier A. Identification of new participants in the rainbow trout (Oncorhynchus mykiss) oocyte maturation and ovulation processes using cDNA microarrays. Reprod Biol Endocrinol. 2006;4:39. https://doi.org/10.1186/1477-7827-4-39. PMid:16872517.

Criscuolo-Urbinati E, Kuradomi RY, Urbinati EC, Batlouni SR. The administration of exogenous prostaglandin may improve ovulation in pacu (Piaractus mesopotamicus). Theriogenology. 2012;78(9):2087-94. https://doi.org/10.1016/j.theriogenology.2012.08.001. PMid:23043951.

El Mohajer L, Bulteau R, Chevalier C, Selmi S, Fontaine P, Milla S. In vitro follicle culture shows that progestagens are the maturation-inducing hormones (MIH) and possible regulators of the ovulation-mediating hormone PGE2 in female Eurasian perch Perca fluviatilis. Fish Physiol Biochem. 2021;47(4):881-94. https://doi.org/10.1007/s10695-021-00946-5. PMid:33774768.

Fujimori C, Ogiwara K, Hagiwara A, Rajapakse S, Kimura A, Takahashi T. Expression of cyclooxygenase-2 and prostaglandin receptor EP4b mRNA in the ovary of the medaka fish, Oryzias latipes: possible involvement in ovulation. Mol Cell Endocrinol. 2011;332(1-2):67-77. https://doi.org/10.1016/j.mce.2010.09.015. PMid:20932877.

Goetz FW, Nagahama Y. Effects of pH on in vitro ovulation of goldfish (Carassius auratus) oocytes. J Exp Zool. 1985;235(1):81-5. https://doi.org/10.1002/jez.1402350111. PMid:3863880.

Goetz FW, Theofan G. In vitro stimulation of germinal vesicle breakdown and ovulation of Yellow Perch (Perca flavescens) oocytes. Effects of 17α-hydroxy-20β-digydroprogesterone and prostaglandins. Gen Comp Endocrinol. 1979;37(3):273-85. https://doi.org/10.1016/0016-6480(79)90001-7. PMid:456868.

Goncharov BF, Williot P, Le Menn F. Comparison of the effects of gonadotropic preparations of the carp and Stellate Sturgeon Pituitaries on in vivo and in vitro oocyte maturation in the Siberian Sturgeon Acipenser baeri Brandt. Ontogenez. 2001;32(5):384-92. https://doi.org/10.1023/A:1012364702687. PMid:11605413.

Guo Q, Lyu L, Wen H, Li Y, Zuo C, Wang X, Jing X, Jiang T, Yang J, Qi X. Black rockfish (Sebastes schlegelii) nuclear progesterone receptor: Cloning, tissue distribution and potential role in ovoviviparity teleost pregnancy and parturition. Aquacult Rep. 2024;36:102068. https://doi.org/10.1016/j.aqrep.2024.102068.

Hagiwara A, Ogiwara K, Katsu Y, Takahashi T. Luteinizing hormone-induced expression of Ptger4b, a prostaglandin E2 receptor indispensable for ovulation of the medaka Oryzias latipes, is regulated by a genomic mechanism involving nuclear progestin receptor. Biol Reprod. 2014;90(6):126-1. https://doi.org/10.1095/biolreprod.113.115485. PMid:24790162.

Hagiwara A, Ogiwara K, Sugama N, Yamashita M, Takahashi T. Inhibition of medaka ovulation by gap junction blockers due to its disrupting effect on the transcriptional process of LH-induced Mmp15 expression. Gen Comp Endocrinol. 2020;288:113373. https://doi.org/10.1016/j.ygcen.2019.113373. PMid:31874135.

Hainfellner P, Souza TG, Moreira RG, Nakaghi LSO, Batlouni SR. Gonadal steroids levels and vitellogenesis in the formation of oocytes in Prochilodus lineatus (Valenciennes) (Teleostei: characiformes). Neotrop Ichthyol. 2012;10(3):601-12. https://doi.org/10.1590/S1679-62252012005000021.

Hartshorne GM. In vitro culture of ovarian follicle. Rev Reprod. 1997;2(2):94-104. https://doi.org/10.1530/ror.0.0020094. PMid:9414471.

Jalabert B, Szöllösi D. In vitro ovulation of trout oocytes: effect of prostaglandin on smooth muscle-like cells of the theca. Prostaglandins. 1975;9(5):765-78. https://doi.org/10.1016/0090-6980(75)90113-6. PMid:1162086.

Jalabert B, Breton B, Brzuska E, Fostier A, Wieniawski J. A new tool for induced spawning: the use of 17α-hydroxy-20β-dihydroprogesterone to spawn carp at low temperature. Aquaculture. 1977;10(4):353-64. https://doi.org/10.1016/0044-8486(77)90126-0.

Jalabert B. Particularities of reproduction and oogenesis in teleost fish compared to mammals. Reprod Nutr Dev. 2005;45(3):261-79. https://doi.org/10.1051/rnd:2005019. PMid:15982453.

Jesus LWO, Bogerd J, Vieceli FM, Branco GS, Camargo MP, Cassel M, et al. Gonadotropin subunits of the characiform Astyanax altiparanae: molecular characterization, spatiotemporal expression and their possible role on female reproductive dysfunction in captivity. Gen Comp Endocrinol. 2017;246:150-63. https://doi.org/10.1016/j.ygcen.2016.12.004. PMid:27940043.

Joy KP, Singh V. Functional interactions between vasotocin and prostaglandins during final oocyte maturation and ovulation in the catfish Heteropneustes fossilis. Gen Comp Endocrinol. 2013;186:126-35. https://doi.org/10.1016/j.ygcen.2013.02.043. PMid:23510856.

Kagawa H, Fujie N, Imaizumi H, Masuda Y, Oda K, Adachi J, Nishi A, Hashimoto H, Teruya K, Kaji S. Using osmotic pumps to deliver hormones to induce sexual maturation of female Japanese eels, Anguilla japonica. Aquaculture. 2013;388-91:30-4. https://doi.org/10.1016/j.aquaculture.2013.01.025.

Kagawa H, Tanaka H, Unuma T, Ohta H, Gen K, Okuzawa K. Role of prostaglandin in the control of ovulation in the Japanese eel Anguilla japonica. Fish Sci. 2003;69(2):234-41. https://doi.org/10.1046/j.1444-2906.2003.00613.x.

Keeley TP, Mann GE. Defining physiological normoxia for improved translation of cell physiology to animal models and humans. Physiol Rev. 2019;99(1):161-234. https://doi.org/10.1152/physrev.00041.2017. PMid:30354965.

Kim DG, Hwang IJ, Baek HJ. Potent influence of exogenous melatonin on in vitro oocyte maturation in the Longchin Goby, Chaeno gobius annularis. Dev Reprod. 2023;27(3):127-35. https://doi.org/10.12717/DR.2023.27.3.127. PMid:38074459.

Kitanović N, Marinović Z, Quyến NN, Kovács B, Müller T, Urbányi B, Horváth Á. In vitro maturation of African catfish (Clarias gariepinus, Burchell, 1822) oocytes results in viable larvae. Aquaculture. 2024;583:740617. https://doi.org/10.1016/j.aquaculture.2024.740617.

Knight OM, Van Der Kraak G. The role of eicosanoids in 17α,20β-dihydroxy-4-pregnen-3-one-induced ovulation and spawning in Danio rerio. Gen Comp Endocrinol. 2015;213:50-8. https://doi.org/10.1016/j.ygcen.2014.12.014. PMid:25573385.

Kuradomi RY, Batlouni SR. PGF 2α and gonadal steroid plasma levels of successful and unsuccessful spawning Piaractus mesopotamicus (Teleostei, Characiformes) females. Aquacult Int. 2018;26(4):1083-94. https://doi.org/10.1007/s10499-018-0269-8.

Lacerda SMSN, Batlouni SR, Silva SBG, Homem CSP, França LR. Germ cells transplantation in fish: the Nile-tilapia model. Anim Reprod (AR). 2018;3(2):146-59.

Levavi-Zermonsky B, Yaron Z. Changes in gonadotropin and ovarian steroids associated with oocyte maturation during spawning induction in the carp. Gen Comp Endocrinol. 1986;62(1):89-98. https://doi.org/10.1016/0016-6480(86)90097-3. PMid:3781219.

Lira LVG, Kuradomi RY, Souza TG, Hainfellner P, Batlouni SR. Astyanax altiparanae ovarian maturation after spawning in water recycling systems. Bol Inst Pesca. 2018;44:1-10. https://doi.org/10.20950/1678-2305.2018.44.4.207.

Lister AL, Van Der Kraak GJ. Regulation of prostaglandin synthesis in ovaries of sexually mature zebrafish (Danio rerio). Mol Reprod Dev. 2009;76(11):1064-75. https://doi.org/10.1002/mrd.21072. PMid:19551897.

Mello F, Alonso DJ, Faria NPVM, Marques VH, Oliveira EF, de Mello PH, et al. Alterations in gene expression and the fatty acid profile impact but do not compromise the in vitro maturation of zebrafish (Danio rerio) stage III ovarian follicles after cryopreservation. Animals (Basel). 2023;13(22):3563. https://doi.org/10.3390/ani13223563. PMid:38003179.

Mylonas CC, Fostier A, Zanuy S. Broodstock management and hormonal manipulations of fish reproduction. Gen Comp Endocrinol. 2010;165(3):516-34. https://doi.org/10.1016/j.ygcen.2009.03.007. PMid:19318108.

Patiño R, Thomas P. Induction of maturation of Atlantic croaker oocytes by 17α,20β,21-trihydroxy-4-pregnen-3-one in vitro: consideration of some biological and experimental variables. J Exp Zool. 1990;255(1):97-109. https://doi.org/10.1002/jez.1402550113. PMid:2391470.

Pereira TSB, Boscolo CNP, Moreira RG, Batlouni SR. Leporinus elongatus induced spawning using carp pituitary extract or mammalian GnRH analogue combined with dopamine receptor antagonists. Anim Reprod. 2018;15(1):64-70. https://doi.org/10.21451/1984-3143-2017-AR983. PMid:33365097.

Roza de Abreu M, de Jesus Silva LM, Benevente CF, Barbosa RP, Mechaly AS, Batlouni SR. Hormonal regulation of oocyte maturation and ovulation in lambari (Astyanax altiparanae) induced by different pituitary extracts. Aquaculture. 2025;609:742843. https://doi.org/10.1016/j.aquaculture.2025.742843.

Roza de Abreu M, Silva LMJ, Figueiredo-Ariki DG, Sato RT, Kuradomi RY, Batlouni SR. Reproductive performance of lambari (Astyanax altiparanae) in a seminatural system using different protocols. Aquacult Res. 2021;52(2):471-83. https://doi.org/10.1111/are.14905.

Roza de Abreu M, Silva LMJ, Figueiredo-Ariki DG, Sato RT, Kuradomi RY, Batlouni SR. The effect of LHRHa with and without dopamine antagonist on reproductive performance in lambari Astyanax altiparanae. Aquaculture. 2022;550:737883. https://doi.org/10.1016/j.aquaculture.2021.737883.

Samokhin P, Gardner GL, Moffatt C, Stuart JA. An inexpensive incubator for mammalian cell culture capable of regulating O2, CO2, and temperature. Oxygen (Basel). 2022;2(1):22-30. https://doi.org/10.3390/oxygen2010003.

Santos FBP, Affonso EG, Godoy LCD. Trypan blue staining is not efficient in determining oocyte viability in Colossoma macropomum and Brycon amazonicus. Bol Inst Pesca. 2017;44:121-9. https://doi.org/10.20950/1678-2305.2017.121.129.

Sato RT, Abreu MR, Kuradomi RY, Silva LMJ, Batlouni SR. Effects of the time of administration and the concentration of exogenous prostaglandin F on ovulation in pacu (Piaractus mesopotamicus). Aquacult Int. 2023;1:1-10. https://doi.org/10.1007/s10499-023-01332-z.

Sato RT, Kuradomi RY, Calil MC, Silva LMJ, Roza de Abreu MR, Figueiredo-Ariki DG, Freitas GA, Batlouni SR. Resumption and progression of meiosis and circulating levels of steroids and prostaglandin F2α of Piaractus mesopotamicus induced by hypophysation with prostaglandin F2α. Aquacult Res. 2021;52(3):1026-7. https://doi.org/10.1111/are.14957.

Sorbera LA, Asturiano JF, Carrillo M, Cerda J, Kime DE, Zanuy S. In vitro oocyte maturation in the sea bass: effects of hCG, pituitary extract and steroids. J Fish Biol. 1999;55(1):9-25. https://doi.org/10.1111/j.1095-8649.1999.tb00652.x.

Souza TG, Kuradomi RY, Rodrigues SM, Batlouni SR. Wild Leporinus friderici induced spawning with different doses of mGnRHa and metoclopramide or carp pituitary extract. Anim Reprod. 2020;17(1):e20190078. https://doi.org/10.21451/1984-3143-AR2019-0078. PMid:32399066.

Takahashi T, Fujimori C, Hagiwara A, Ogiwara K. Recent advances in the understanding of teleost medaka ovulation: the roles of proteases and prostaglandins. Zool Sci. 2013;30(4):239-47. https://doi.org/10.2108/zsj.30.239. PMid:23537233.

Takahashi T, Hagiwara A, Ogiwara K. Follicle rupture during ovulation with an emphasis on recent progress in fish models. Reproduction. 2019;157(1):R1-13. https://doi.org/10.1530/REP-18-0251. PMid:30394703.

Tang H, Liu Y, Li J, Li G, Chen Y, Yin Y, Guo Y, Cheng CH, Liu X, Lin H. LH signaling induced ptgs2α expression is required for ovulation in zebrafish. Mol Cell Endocrinol. 2017;447:125-33. https://doi.org/10.1016/j.mce.2017.02.042. PMid:28254490.

Tang H, Liu Y, Li J, Yin Y, Li G, Chen Y, Li S, Zhang Y, Lin H, Liu X, Cheng CH. Gene knockout of nuclear progesterone receptor provides insights into the regulation of ovulation by LH signaling in zebrafish. Sci Rep. 2016;6(1):28545. https://doi.org/10.1038/srep28545. PMid:27333837.

Welch EL, Eno CC, Nair S, Lindeman RE, Pelegri F. Functional manipulation of maternal gene products using in vitro oocyte maturation in zebrafish. J Vis Exp. 2017;(122):55213. https://doi.org/10.3791/55213. PMid:28518112.
 

Submitted date:
01/21/2025

Accepted date:
10/28/2025

698b9411a953950fc157f9a6 animreprod Articles
Links & Downloads
1984-3143 (Electronic) 1806-9614 (Printed)
Anim Reprod ©2026 All rights reserved.

Anim Reprod

Share this page
Page Sections