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Reproduction, Fertility and Development Reproduction, Fertility and Development Society
Vertebrate reproductive science and technology
RESEARCH ARTICLE

Effect of dietary administration of letrozole and tamoxifen on gonadal development, sex differentiation and biochemical changes in common carp (Cyprinus carpio L.)

Atul K. Singh A C , P. P. Srivastava B , Rita Verma A , Sharad C. Srivastava A , Dinesh Kumar A and Abubakar Ansari A
+ Author Affiliations
- Author Affiliations

A Exotic Germplasm Section of Fish Health Management Division, National Bureau of Fish Genetic Resources, Canal Ring Road, P.O. Dilkusha, Lucknow-226002 (Uttar Pradesh), India.

B Biochemistry and Genomics Laboratory of Molecular Biology and Biotechnology Division, National Bureau of Fish Genetic Resources, Canal Ring Road, P.O. Dilkusha, Lucknow-226002 (Uttar Pradesh), India.

C Corresponding author. Email: aksingh56@rediffmail.com

Reproduction, Fertility and Development 27(3) 449-457 https://doi.org/10.1071/RD13234
Submitted: 29 July 2013  Accepted: 23 November 2013   Published: 13 January 2014

Abstract

The effect of letrozole and tamoxifen on the specific growth rate (SGR; % day–1), gonado–somatic index (GSI), total haemoglobin (g%), gonadal and serum protein as well as lipid, sex differentiation and 17β-oestradiol levels were studied in sexually undifferentiated Cyprinus carpio fingerlings 30 days post fertilisation (30 dpf) for 60 days. Results showed decreased GSI with tamoxifen treatment whereas letrozole increased it. There were reduced protein, lipid, triglyceride and cholesterol levels after treatment with tamoxifen and letrozole during gonadal development. Tamoxifen (200 mg kg–1 feed) induced 82.5% masculinisation, whereas letrozole in the same dose produced 98.5% males. Gonadal 17β-oestradiol significantly declined from 86.0 ± 1.41 pg per 100 mg (control) to 45.5 ± 1.94 pg per 100 mg with tamoxifen and 36.0 ± 0.72 pg per 100 mg with letrozole treatment. Similarly, serum 17β-oestradiol levels also decreased after tamoxifen and letrozole treatments. Testicular development in 37.8% of fish treated with tamoxifen and letrozole was found to be more advanced (spermatocytes) than in the control (spermatogonium); however, there was reduced ovarian growth and increased atresia. It was concluded that letrozole and tamoxifen both significantly affect sex differentiation and gonadal maturity in C. carpio leading to the production of sex-reversed males, yet the effect of letrozole was more potent.

Additional keywords: aromatase, 17β-oestradiol.


References

Andersen, L., Kinnberg, K., Holbech, H., Korsgaard, B., and Bjerregaard, P. (2004). Evaluation of a 40-day assay for testing endocrine disrupters: effects of an anti-oestrogen and an aromatase inhibitor on sex ratio and vitellogenin concentrations in juvenile zebrafish (Danio rerio). Fish Physiol. Biochem. 30, 257–266.
Evaluation of a 40-day assay for testing endocrine disrupters: effects of an anti-oestrogen and an aromatase inhibitor on sex ratio and vitellogenin concentrations in juvenile zebrafish (Danio rerio).Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXht1OqtrvM&md5=704e50d383e50fe26ce2854b9a538291CAS |

Ankley, G. T., Jensen, K. M., Durhan, E. J., Makynen, E. A., Butterworth, B. C., Kahl, M. D., Villeneuve, D. L., Linnum, A., Gray, L. E., Cardon, M., and Wilson, V. S. (2005). Effects of two fungicides with multiple modes of action on reproductive endocrine function in the fathead minnow (Pimephales promelas). Toxicol. Sci. 86, 300–308.
Effects of two fungicides with multiple modes of action on reproductive endocrine function in the fathead minnow (Pimephales promelas).Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXlvF2htrY%3D&md5=fc901161e58f5397582e10c0fcbde6fcCAS | 15901916PubMed |

Barber, B. J., and Blake, N. J. (2006) Reproductive physiology. In ‘Scallops: Biology, Ecology and Aquaculture’, 2nd edn. (Eds S. E. Shumway and G. J. Parsons.) pp. 357–406. (Elsevier: Amsterdam.)

Barney, M. L., Patil, J. G., Gunaskera, R. M., and Carter, C. G. (2008). Distinct cytochrome P450 aromatase isoforms in the common carp (Cyprinus carpio): sexual dimorphism and onset of ontogenic expression. Gen. Comp. Endocrinol. 156, 499–508.
Distinct cytochrome P450 aromatase isoforms in the common carp (Cyprinus carpio): sexual dimorphism and onset of ontogenic expression.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXlsF2qsL4%3D&md5=96913c1f4dbf3b2dd637ea2cdddfecefCAS | 18430424PubMed |

Baumann, L., Holbech, H., Keiter, S., Kinnberg, K. L., Knörr, S., Nagel, T., and Braunbeck, T. (2013). The maturity index as a tool to facilitate the interpretation of changes in vitellogenin production and sex ratio in the fish sexual development test. Aquatic Toxicology 128–129, 34–42.
The maturity index as a tool to facilitate the interpretation of changes in vitellogenin production and sex ratio in the fish sexual development test.Crossref | GoogleScholarGoogle Scholar | 23261669PubMed |

Bhandari, R.K., Komuro, H., Higa, M., and Nakamura, M. (2004). Sex inversion of sexually immature honeycomb grouper Epinephelus merra by aromatase inhibitor. Zoolog. Sci. 21, 305–310.
Sex inversion of sexually immature honeycomb grouper Epinephelus merra by aromatase inhibitor.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXltV2lur8%3D&md5=9b76f0e67f7fa00a80562b244a66e705CAS | 15056925PubMed |

Blaxhall, P. C., and Daisley, K. W. (1973). Routine haematological methods for use with fish blood. J. Fish Biol. 5, 771–781.
Routine haematological methods for use with fish blood.Crossref | GoogleScholarGoogle Scholar |

Bligh, E. G., and Dyer, W. J. (1959). A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917.
A rapid method of total lipid extraction and purification.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaG1MXhtVSgt70%3D&md5=29e471d45a73b03917193fb2d374b94dCAS | 13671378PubMed |

Cheshenko, K., Pakdel, F., Segner, H., Kah, O., and Eggen, R. I. L. (2008). Interference of endocrine-disrupting chemicals with aromatase cyp19 expression or activity, and consequences for reproduction of teleost fish. Gen. Comp. Endocrinol. 155, 31–62.
Interference of endocrine-disrupting chemicals with aromatase cyp19 expression or activity, and consequences for reproduction of teleost fish.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXhtlGiu77J&md5=6e305bd875964d4da0c93021b5ed6c4dCAS | 17459383PubMed |

Das, R., Rather, M. A., Basavaraja, N., Sharma, R., and Udit, U. K. (2012). Effect of nonsteroidal aromatase inhibitor on sex reversal of Oreochromis mossambicus (Peters, 1852). Israeli J. Aquaculture – Bamigdeh 64, 1–9.

De Silva, S. S., Nguyen, T. T., Turchini, G. M., Amarasinghe, U. S., and Abery, N. W. (2009). Alien species in aquaculture and biodiversity: a paradox in food production. Ambio 38, 24–28.
Alien species in aquaculture and biodiversity: a paradox in food production.Crossref | GoogleScholarGoogle Scholar | 19260343PubMed |

Devlin, R. H., and Nagahama, Y. (2002). Sex determination and sex differentiation in fish: an overview of genetic, physiological and environmental influences. Aquaculture 208, 191–364.
Sex determination and sex differentiation in fish: an overview of genetic, physiological and environmental influences.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38XktFKjsbY%3D&md5=b091c7d71fb4d48e33967c639b521ff4CAS |

Diotel, N., Le Page, Y., Mouriec, K., Tong, S.-K., Pellegrini, E., Vaillant, C., Anglade, I., Brion, F., Pakdel, F., Bon-chu, C., and Kah, O. (2010). Aromatase in the brain of teleost fish: expression, regulation and putative functions. Front. Neuroendocrinol. 31, 172–192.
Aromatase in the brain of teleost fish: expression, regulation and putative functions.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXktlCisrw%3D&md5=991e6ddaab510eca6fa96bcd7ac9cf18CAS | 20116395PubMed |

Fenske, M., and Segner, H. (2004). Aromatase modulators alter gonadal differentiation in developing zebrafish (Danio rerio). Aquat. Toxicol. 67, 105–126.
Aromatase modulators alter gonadal differentiation in developing zebrafish (Danio rerio).Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXhs12htro%3D&md5=502610003fda81991187e8930e35a1bfCAS | 15003697PubMed |

Gao, Z. X., Wang, H. P., Wallat, G., Yao, H., Rapp, D., O’Bryant, P., MacDonald, R., and Wang, W. M. (2010). Effects of a nonsteroidal aromatase inhibitor on gonadal differentiation of bluegill sunfish Lepomis macrochirus. Aquacult. Res. 41, 1282–1289.
Effects of a nonsteroidal aromatase inhibitor on gonadal differentiation of bluegill sunfish Lepomis macrochirus.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXhtlOqsLnP&md5=3e0c51dac5627bdec033e9eefa21154bCAS |

Gomelsky, B. (2003). Chromosome set manipulation and sex control in common carp: a review. Aquat. Living Resour. 16, 408–415.
Chromosome set manipulation and sex control in common carp: a review.Crossref | GoogleScholarGoogle Scholar |

Guerrero, R. D., and Shelton, W. I. (1974). An acetocarmine squash method of sexing juvenile fishes. Prog. Fish-Cult. 36, 56–58.
An acetocarmine squash method of sexing juvenile fishes.Crossref | GoogleScholarGoogle Scholar |

Guiguen, Y., Fostier, A., Piferrer, F., and Chang, C.-F. (2010). Ovarian aromatase and oestrogen: pivotal role for gonadal sex differentiation and sex change in fish. Gen. Comp. Endocrinol. 165, 352–366.
Ovarian aromatase and oestrogen: pivotal role for gonadal sex differentiation and sex change in fish.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXktVegtQ%3D%3D&md5=338c149d0a1921347ecf554229a59b54CAS | 19289125PubMed |

Hudson, Q. J., Smith, C. A., and Sinclair, A. H. (2005). Aromatase inhibition reduces expression of FOXL2 in the embryonic chicken ovary. Dev. Dyn. 233, 1052–1055.
Aromatase inhibition reduces expression of FOXL2 in the embryonic chicken ovary.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXmtFKgsbc%3D&md5=2c26f94a8b3ddcefbfb2db836c943de0CAS | 15830351PubMed |

Huffman, L. S., Lauren, A. O. C., and Hofmann, H. A. (2013). Aromatase regulates aggression in the African cichlid fish Astatotilapia burtoni. Physiol. Behav. 112–113, 77–83.
Aromatase regulates aggression in the African cichlid fish Astatotilapia burtoni.Crossref | GoogleScholarGoogle Scholar | 23438371PubMed |

Hulak, M., Psenicka, M., Gela, D., Rodina, M., and Linhart, O. (2010). Morphological sex change upon treatment by endocrine modulators in meiogynogenetic tench (Tinca tinca L). Aquacult. Res. 41, 233–239.
Morphological sex change upon treatment by endocrine modulators in meiogynogenetic tench (Tinca tinca L).Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXitF2itLg%3D&md5=3f8d13243a62b2cac45fe6c5b5f59508CAS |

Ito, L. S., Yamashita, M., Takashima, F., and Strussman, C. A. (2005). Dynamics and histological characteristics of gonadal sex differentiation in pejerrey (Odontesthes bonariensis) at feminising and masculinising temperatures. J. Exp. Zool. Part A (Comparative and Experimental Biology) 303, 504–514.

Kazeto, Y., Tosaka, R., Mastubara, H., Shigeho, I., and Adachi, S. (2011). Ovarian steroidogenesis and the role of sex steroid hormones on ovarian growth and maturation of the Japanese eel J. Steroid Biochem. Mol. Biol. 127, 149–154.
Ovarian steroidogenesis and the role of sex steroid hormones on ovarian growth and maturation of the Japanese eelCrossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXhsFSksrnO&md5=a57d1ce3506e6c301257a9bc320c5220CAS | 21414407PubMed |

Kinnberg, K., Holbech, H., Petersen, G. I., and Bjerregaard, P. (2007). Effects of the fungicide prochloraz on the sexual development of zebrafish (Danio rerio). Comp. Biochem. Physiol. C Toxicol. Pharmacol. 145, 165–170.
Effects of the fungicide prochloraz on the sexual development of zebrafish (Danio rerio).Crossref | GoogleScholarGoogle Scholar | 17229592PubMed |

Kobayashi, Y., Nagahama, Y., and Nakamura, M. (2013). Diversity and plasticity of sex determination and differentiation in fishes. Sex Dev. 7, 115–125.
Diversity and plasticity of sex determination and differentiation in fishes.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC38XhvVClsbvN&md5=eac4d0689a0772afd5fb2c2ff440c517CAS | 22948719PubMed |

Komatsu, T., Nakamura, S., and Nakamura, M. (2006). Masculinisation of female golden rabbitfish Sigomus guttatus using an aromatase inhibitor treatment during sex differentiation. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 143, 402–409.
Masculinisation of female golden rabbitfish Sigomus guttatus using an aromatase inhibitor treatment during sex differentiation.Crossref | GoogleScholarGoogle Scholar | 16807123PubMed |

Kwon, J. Y., Haghpanah, V., Kogson-Hurtado, L. M., McAndrew, B. J., and Penman, D. J. (2000). Masculinisation of genetic female Nile tilapia (Oreochromis niloticus) by dietary administration of an aromatase inhibitor during sexual differentiation. J. Exp. Zool. 287, 46–53.
Masculinisation of genetic female Nile tilapia (Oreochromis niloticus) by dietary administration of an aromatase inhibitor during sexual differentiation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXktlKhtb0%3D&md5=22d5172e0a3ba9c2a2010704517b2b7dCAS | 10861549PubMed |

Lawson, C., Gieske, M., Murdoch, B., Ye, P., Li, Y., Hassold, T., and Hunt, P. A. (2011). Gene expression in the fetal mouse ovary is altered by exposure to low doses of bisphenol A. Biol. Reprod. 84, 79–86.
Gene expression in the fetal mouse ovary is altered by exposure to low doses of bisphenol A.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXlvVegur4%3D&md5=9add1e4ff5c1540e5df73e6497d166dbCAS | 20739668PubMed |

Le Page, Y., Diotel, N., Vaillant, C., Pellegrini, E., Anglade, I., Mérot, Y., and Kah, O. (2010). Aromatase, brain sexualisation and plasticity: the fish paradigm. Eur. J. Neurosci. 32, 2105–2115.
Aromatase, brain sexualisation and plasticity: the fish paradigm.Crossref | GoogleScholarGoogle Scholar | 21143665PubMed |

Li, G. L., Liu, X. C., and Lin, H. R. (2005). Aromatase inhibitor letrozole induces sex inversion in the protogynous red-spotted grouper (Epinephelus akaara). Acta Physiologica Sinica 57, 473–479.
| 1:CAS:528:DC%2BD28XhtFejurzJ&md5=188bf9e234408d74edc90a530852d495CAS | 16094495PubMed |

Li, G. L., Liu, X. C., Zhang, Y., and Lin, H. R. (2006). Gonadal development, aromatase activity and P450 aromatase gene expression during sex inversion of protogynous red-spotted grouper Epinephelus akaara (Temminck and Schlegel) after implantation of the aromatase inhibitor, fadrozole. Aquacult. Res. 37, 484–491.
Gonadal development, aromatase activity and P450 aromatase gene expression during sex inversion of protogynous red-spotted grouper Epinephelus akaara (Temminck and Schlegel) after implantation of the aromatase inhibitor, fadrozole.Crossref | GoogleScholarGoogle Scholar |

Lowry, O. H., Rosenbrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265–275.
| 1:CAS:528:DyaG38XhsVyrsw%3D%3D&md5=8394b58439cc3617994b5b970205f81fCAS | 14907713PubMed |

Mikolajczyk, T., Sokosmallerska-Mikolajczyk, M., Chyb, J., Szczerbik, P., Socha, M., Foks, M., Duc, M., and Epler, P. (2007). LH secretion and 17β-oestradiol concentration in the blood plasma and hypothalamus of goldfish (Carassius auratus gibelio B.) and common carp (Cyprinus carpio L.) treated with fadrozole (aromatase inhibitor) and GnRH analogues. Czech J. Anim. Sci. 52, 354–362.
| 1:CAS:528:DC%2BD2sXhtl2gsLrK&md5=baff6c7bd45b27f61ec19fb013d5199eCAS |

Nakamura, M. (2013). Morphological and physiological studies on gonadal sex differentiation in teleost fish. Aqua-BioScience Monographs 6, 1–47.
Morphological and physiological studies on gonadal sex differentiation in teleost fish.Crossref | GoogleScholarGoogle Scholar |

Olmstead, A. W., Kosian, P. A., Korte, J. J., Holcombe, G. W., Woodis, K. K., and Degitz, S. J. (2009). Sex reversal of the amphibian, Xenopus tropicalis, following larval exposure to an aromatase inhibitor. Aquat. Toxicol. 91, 143–150.
Sex reversal of the amphibian, Xenopus tropicalis, following larval exposure to an aromatase inhibitor.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXntlymtg%3D%3D&md5=c2176f64d0909349e6c9d382bacde0b0CAS | 18804292PubMed |

Panter, G. H., Hutchinson, T. H., Hurd, K. S., Sherren, A., Stanley, R. D., and Tyler, C. R. (2004). Successful detection of (anti-) androgenic and aromatase inhibitors in pre-spawning adult fathead minnows (Pimephales promelas) using easily measured endpoints of sexual development. Aquat. Toxicol. 70, 11–21.
Successful detection of (anti-) androgenic and aromatase inhibitors in pre-spawning adult fathead minnows (Pimephales promelas) using easily measured endpoints of sexual development.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXnvV2jsbk%3D&md5=fd39c2adb1dc17faec9633546db557feCAS | 15451604PubMed |

Pieau, C., and Dorizzi, M. (2004). Oestrogens and temperature-dependent sex determination in reptiles: all is in the gonads. J. Endocrinol. 181, 367–377.
Oestrogens and temperature-dependent sex determination in reptiles: all is in the gonads.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXlslWqtb8%3D&md5=cf2733079c981f1ea37d93345e29be73CAS | 15171684PubMed |

Piferrer, F., and Blazquez, M. (2005). Aromatase distribution and regulation. Fish Physiol. Biochem. 31, 215–226.
Aromatase distribution and regulation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XptF2rsbo%3D&md5=d7e3aee6c177358769b76017a28ace07CAS | 20035461PubMed |

Pinto, P. I. S., Teodosio, H. R., Galay-Borgos, M., Power, D. M., and Sweeney, G. E. (2006). Identification of oestrogen-responsive genes in the testis of sea bream (Sparus auratus) using suppression subtractive hybridisation. Mol. Reprod. Dev. 73, 318–329.
Identification of oestrogen-responsive genes in the testis of sea bream (Sparus auratus) using suppression subtractive hybridisation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XhsVKht7Y%3D&md5=9b953e1116f8665ddb980456a0f9d3d5CAS |

Rothbard, S. (2006). A review of ploidy manipulations in aquaculture: the Israeli experience. The Israeli Journal of Aquaculture – Bamidgeh 58, 266–279.

Sharp, R. L., and MacLatchy, D. L. (2007). Lipid dynamics in goldfish (Carassius auratus) during a period of gonadal recrudescence: effects of β-sitosterol and 17β-estradiol exposure. Comp. Biochem. Physiol. 145, 507–517.

Shen, Z.-G., Fan, Q.-X., Yang, W., Zhang, Y.-L., Hu, P.-P., and Xie, C.-X. (2013). Effects of non-steroidal aromatase inhibitor letrozole on sex inversion and spermatogenesis in yellow catfish Pelteobagrus fulvidraco. Biol. Bull. 225, 18–23.
| 1:CAS:528:DC%2BC3sXhslGgurbP&md5=79e490948de93f18a46446e4787f9218CAS | 24088793PubMed |

Shine, R., Warner, D. A., and Radder, R. (2007). Windows of embryonic sexual lability in two lizard species with environmental sex determination. Ecology 88, 1781–1788.
Windows of embryonic sexual lability in two lizard species with environmental sex determination.Crossref | GoogleScholarGoogle Scholar | 17645024PubMed |

Singh, A. K. (2013). Introduction of modern endocrine techniques for the production of monosex population of fishes. Gen. Comp. Endocrinol. 181, 146–155.
Introduction of modern endocrine techniques for the production of monosex population of fishes.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC38XhsFymu73P&md5=dbb4af3e9778ba998b8cfeffe9b8e846CAS | 23063432PubMed |

Singh, A. K., and Kapila, R. (2007). Sex control in endangered upland golden mahseer, Tor putitora (Hamilton 1822) for reproductive management. In ‘Proceedings of the 7th Indian Fisheries Forum’. (Eds. C. Vasudevappa, Y. Basavaraju, D. Seenappa, S. Ayyappan and S. Ravichandra Reddy.) pp. 165–172. (Asian Society of Fisheries, Indian Branch: Mangalore, India.)

Singh, A. K., and Singh, R. (2013). In vivo response of melatonin, gonadal activity and biochemical changes during aromatase-inhibited sex reversal in common carp Cyprinus carpio (L). Anim. Reprod. Sci. 136, 317–325.
In vivo response of melatonin, gonadal activity and biochemical changes during aromatase-inhibited sex reversal in common carp Cyprinus carpio (L).Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC38Xhslynu77P&md5=3f7d3cefd286dd35ec3d3bb46e3dd0feCAS | 23218911PubMed |

Singh, R., Singh, A. K., and Tripathi, M. (2012). Melatonin-induced changes in specific growth rate, gonadal maturity, lipid and protein production in Nile tilapia, Oreochromis niloticus (Linnaeus 1758). Asian-Aus. J. Anim. Sci. 25, 37–43.
Melatonin-induced changes in specific growth rate, gonadal maturity, lipid and protein production in Nile tilapia, Oreochromis niloticus (Linnaeus 1758).Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC38XltlaqtLg%3D&md5=70ffe1c8c273611b4171a34dcb98ead2CAS |

Stein, E. A. (1986). Quantitative enzymatic colorimetric determination of total cholesterol in serum or plasma. In ‘Textbook of Clinical Chemistry’, (Ed. W.B. Saunders.) pp. 879–886. (Nwtietz: Philadelphia.)

Sun, L., Zha, J., Spear, P. A., and Wang, Z. (2007). Tamoxifen effects on the early life stages and reproduction of Japanese medaka (Oryzias latipes). Environ. Toxicol. Pharmacol. 24, 23–29.
Tamoxifen effects on the early life stages and reproduction of Japanese medaka (Oryzias latipes).Crossref | GoogleScholarGoogle Scholar | 21783785PubMed |

Sun, L., Shao, X., Chi, J., Hu, X., Jin, Y., and Fu, Z. (2011). Transcriptional responses in the brain, liver and gonad of Japanese ricefish (Oryzias latipes) exposed to two anti-oestrogens. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 153, 392–401.
Transcriptional responses in the brain, liver and gonad of Japanese ricefish (Oryzias latipes) exposed to two anti-oestrogens.Crossref | GoogleScholarGoogle Scholar | 21281741PubMed |

Taranger, G. L., Carrillo, M., Schulz, R. W., Fontaine, P., Zanuy, S., Felip, A., Weltzien, F.-A., Dufour, S., Karlsen, Ø., Norberg, B., Andersson, E., and Hansen, T. (2010). Control of puberty in farmed fish. Gen. Comp. Endocrinol. 165, 483–515.
Control of puberty in farmed fish.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXktVentg%3D%3D&md5=5c80cf13f56cf5fa2d82851e6d6731c5CAS | 19442666PubMed |

Thresher, R., Gurney, R., and Canning, M. (2011). Effects of lifetime chemical inhibition of aromatase on the sexual differentiation, sperm characteristics and fertility of medaka (Oryzias latipes) and zebrafish (Danio rerio). Aquat. Toxicol. 105, 355–360.
Effects of lifetime chemical inhibition of aromatase on the sexual differentiation, sperm characteristics and fertility of medaka (Oryzias latipes) and zebrafish (Danio rerio).Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXhtl2hu7%2FM&md5=a38179bb521b586b4c3700aa78791c0eCAS | 21819815PubMed |

Wu, G. C., and Chang, C. F. (2013). The switch of secondary sex determination in protandrous back porgy, Acanthopagrus schlegeli. Fish Physiol. Biochem. 39, 33–38.
The switch of secondary sex determination in protandrous back porgy, Acanthopagrus schlegeli.Crossref | GoogleScholarGoogle Scholar | 22411079PubMed |

Yang, X., Zheng, J., Na, R., Li, J., Xu, G., Qu, L., and Yang, N. (2008). Degree of sex differentiation of genetic female chickens treated with different doses of an aromatase inhibitor. Sex Dev. 2, 309–315.
Degree of sex differentiation of genetic female chickens treated with different doses of an aromatase inhibitor.Crossref | GoogleScholarGoogle Scholar | 19276633PubMed |