The synthetic estrogen 17-α-ethinylestradiol (EE2), active ingredient in many oral contraceptives, belongs to the class of endocrine disrupting chemicals (EDCs). The high concentrations of EE2 detected in the aquatic environment have been shown to be harmful to the regular functioning of the reproductive system of vertebrate and invertebrate species, causing infertility, malformations and changes in sexual behaviour. Since the effects of EE2 on the reproductive behavior of aquatic species have been extensively investigated, we decided to focus the following research on non-reproductive behavioral consequences. The animal model tested is zebrafish, among the most used in neurobehavioral research. Zebrafish larvae (Danio rerio) were treated with three different concentrations of EE2 (0.5, 5, 50 ng/L) and two different exposure periods, one of short duration (4 to 7 days post-fertilization) and one of long duration (4 to 14 days post-fertilization). In addition, the reversibility of effects over time was examined following a period of suspension from the administration of the compound. The two behavioral assays proposed, the spontaneous swimming assay and the bouncing ball assay, allowed to analyze, respectively, the natural locomotor activity and the avoidance response of larvae. EE2, at concentrations of 0.5 and 5 ng/L, induced a significant decrease of the "initiations" parameter in the spontaneous swimming assay after a short period of exposure. Regarding the bouncing ball assay, concentrations of 5 and 50 ng/L in the long duration treatment impacted negatively on the avoidance response. No prolongation of effects was found after the period of interruption from the treatment. Furthermore, exposure to the compound did not induce any evidence of toxicity to the development of larvae. In conclusion, we can state that EE2 represents a danger to the survival of zebrafish as the alteration of the behavioral parameters examined is of vital importance in the control of anti-predatory maneuvers. Further investigations are necessary to determine the effects and mechanism of action of EE2 on zebrafish model. In addition to the aforementioned studies, second research was conducted with the aim of optimizing the feeding protocols commonly used in zebrafish rearing, which have always been an obstacle in terms of costs and manpower for research laboratories. The effects of two different feeding protocols, including a mix of dry and live food (Artemia), on larval survival were examined. The results did not show a significant difference on larvae survival between the protocols used.
L’estrogeno sintetico 17-α-etinilestradiolo (EE2), ingrediente attivo nella maggior parte dei contraccettivi orali, rientra nella classe dei composti chimici che alterano il sistema endocrino (Endocrine disrupting chemicals). Le elevate concentrazioni di EE2 rilevate nell’ambiente acquatico risultano dannose per il regolare funzionamento del sistema riproduttivo di specie vertebrate ed invertebrate, causando infertilità, malformazioni e cambiamenti del comportamento sessuale. Poiché gli effetti dell’EE2 sul comportamento riproduttivo delle specie acquatiche sono stati ampiamente investigati, abbiamo deciso di focalizzare la seguente ricerca sulle conseguenze comportamentali non legate alla riproduzione. Il modello animale testato è lo zebrafish (Danio rerio), tra i più impiegati nella ricerca neurocomportamentale. Le larve zebrafish sono state trattate con tre diverse concentrazioni di EE2 (0.5, 5, 50 ng/L) e due periodi di esposizione differenti, uno di breve durata (da 4 a 7 giorni post fertilizzazione) e uno di lunga durata (da 4 a 14 giorni post fertilizzazione). Inoltre, la reversibilità degli effetti nel tempo è stata esaminata a seguito di un periodo di sospensione dalla somministrazione del composto. I due saggi comportamentali proposti, lo spontaneous swimming assay e il bouncing ball assay, hanno permesso di analizzare, rispettivamente, la naturale attività locomotoria e la risposta di evitamento delle larve. L’EE2, alle concentrazioni di 0.5 e 5 ng/L, ha indotto un significativo decremento del parametro “iniziazioni” nello spontaneous swimming assay dopo un breve periodo di esposizione. Per ciò che concerne il bouncing ball assay, solo le concentrazioni di 5 e 50 ng/L nel trattamento di lunga durata hanno dimostrato avere un impatto negativo sulla risposta di evitamento. Non è stato riscontrato nessun prolungamento degli effetti a seguito del periodo di interruzione dal trattamento. Inoltre, l’esposizione al composto non ha indotto alcuna evidenza di tossicità per lo sviluppo delle larve. In conclusione, possiamo affermare che l’EE2 rappresenta un pericolo per la sopravvivenza degli zebrafish in quanto l’alterazione dei parametri comportamentali presi in esame risulta di vitale importanza nel controllo delle manovre anti-predatorie. Investigazioni future sono ritenute necessarie per determinare più approfonditamente gli effetti ed il meccanismo d’azione dell’EE2. In aggiunta agli studi sopracitati, è stata condotta una seconda ricerca con l’obiettivo principale di ottimizzare i protocolli di alimentazione comunemente utilizzati nell’allevamento dei pesci zebrafish, da sempre un ostacolo in termini di costi e manodopera per i laboratori di ricerca. Sono stati esaminati gli effetti di due diversi protocolli alimentari, comprendenti cibi secchi e vivi (Artemia), sulla sopravvivenza delle larve. I risultati ottenuti non hanno mostrato una differenza significativa tra i protocolli usati.
Neurobehavioural effects of 17-α-Ethinylestradiol on zebrafish larvae and optimization of feeding protocols
BONINA, MORGANA
2021/2022
Abstract
The synthetic estrogen 17-α-ethinylestradiol (EE2), active ingredient in many oral contraceptives, belongs to the class of endocrine disrupting chemicals (EDCs). The high concentrations of EE2 detected in the aquatic environment have been shown to be harmful to the regular functioning of the reproductive system of vertebrate and invertebrate species, causing infertility, malformations and changes in sexual behaviour. Since the effects of EE2 on the reproductive behavior of aquatic species have been extensively investigated, we decided to focus the following research on non-reproductive behavioral consequences. The animal model tested is zebrafish, among the most used in neurobehavioral research. Zebrafish larvae (Danio rerio) were treated with three different concentrations of EE2 (0.5, 5, 50 ng/L) and two different exposure periods, one of short duration (4 to 7 days post-fertilization) and one of long duration (4 to 14 days post-fertilization). In addition, the reversibility of effects over time was examined following a period of suspension from the administration of the compound. The two behavioral assays proposed, the spontaneous swimming assay and the bouncing ball assay, allowed to analyze, respectively, the natural locomotor activity and the avoidance response of larvae. EE2, at concentrations of 0.5 and 5 ng/L, induced a significant decrease of the "initiations" parameter in the spontaneous swimming assay after a short period of exposure. Regarding the bouncing ball assay, concentrations of 5 and 50 ng/L in the long duration treatment impacted negatively on the avoidance response. No prolongation of effects was found after the period of interruption from the treatment. Furthermore, exposure to the compound did not induce any evidence of toxicity to the development of larvae. In conclusion, we can state that EE2 represents a danger to the survival of zebrafish as the alteration of the behavioral parameters examined is of vital importance in the control of anti-predatory maneuvers. Further investigations are necessary to determine the effects and mechanism of action of EE2 on zebrafish model. In addition to the aforementioned studies, second research was conducted with the aim of optimizing the feeding protocols commonly used in zebrafish rearing, which have always been an obstacle in terms of costs and manpower for research laboratories. The effects of two different feeding protocols, including a mix of dry and live food (Artemia), on larval survival were examined. The results did not show a significant difference on larvae survival between the protocols used.File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.12608/10022