Introduction
Under in vitro conditions, isolated fish ovarian follicles retain the ability to resume meiosis and progress through the last stages of oogenesis when exposed to maturation-promoting stimuli. Assessing the dynamic response of follicles during in vitro maturation (IVM) can help estimate the reproductive state of the broodstock and gamete quality. To maintain viability and induce follicle development in culture, it is necessary to consider both effective hormonal stimulation and optimal media composition. In fish, gonadotropins and maturation-inducing hormones, such as 17α,20β-dihydroxy-4-pregnen-3-one (DHP), are commonly used to induce maturation of isolated ovarian follicles. Furthermore, both amino-acid rich culture media and additional incorporation of protein and lipid sources—such as serums and bovine serum albumin (BSA)—were found beneficial for maintaining follicular cell and oocyte viability, as well as promoting maturation and ovulation.
In pikeperch (Sander lucioperca), IVM was used to predict the maturational competence and latency time of female breeders (Ljubobratović et al., 2023). The aim of this study was to further optimize the established IVM protocol for immature, postvitellogenic ovarian follicles of this species. This was done by testing different culture media types and supplementation, as well as the effect of maturation-promoting hormones in a range of concentrations.
Materials and methods
Adult pikeperch females (TL: 37 ± 2 cm; W: 424 ± 63 g; GSI: 12 ± 1%) were sampled during the chilling period of the photo-thermal spawning induction (6 °C, 8 h light/16 h dark cycle at sampling moment), prior to any hormonal stimulation. Intact fully grown ovarian follicles were isolated manually and subsequently placed in culture media consisting either of 90% Leibovitz L-15 medium or full strength Cortland’s medium. Both media types were supplemented with antibiotics and 15 mM HEPES, with pH adjusted to 7.5. Furthermore, the effect of additional culture media supplementation in the form of BSA (0-1%) or fetal bovine serum (FBS; 0-20%) was tested. Maturation was induced by adding either DHP (1-1000 ng/ml), human chorionic gonadotropin (hCG; 1-20 IU/ml) or a combination of the two, after which the follicles were incubated at 12 °C, under gentle agitation for 6 days. Resumption of meiosis and maturation progress was evaluated regularly, by scoring the percentage of follicles that underwent germinal vesicle breakdown (GVBD), ooplasm clearing and lipid droplet coalescence, compared to the control group with no hormonal stimulation. In addition, ovulation was monitored by checking the integrity of the follicular layer under light and fluorescent stereo microscope, after staining the follicular cells with SYBR Green.
Results and discussion
At the time of sampling, most of the fish contained follicles in postvitellogenic stage of development that have attained maturational competence (Ø 898 ± 15 µm). Both media types successfully maintained the viability and responsiveness of follicles throughout treatments; however, the rate of GVBD a 4-day stimulation with 100 ng/ml DHP was higher in Leibovitz L-15 medium (83 ± 11%), compared to ones in Cortland’s medium (67 ± 9). The rate and speed of maturation were dependent on the concentration of DHP used. Furthermore, a high percentage of maturing follicles had issues with lipid droplet fragmentation. Treatment with only hCG resulted in comparable final GVBD rates (85 ± 13%); however, the length of incubation notably increased (up to 6 days), regardless of hormone concentration. Slower response during hCG stimulation enabled gradual and synchronized nuclear and cytoplasmic events, resulting in proper lipid droplet coalescence in all mature follicles, as well as evident hydration – increase in diameter was 33 ± 10 µm at the time of GVBD, as opposed to 14 ± 8 µm in the DHP-groups. The rate of ovulation was low after both DHP- (10 ± 8%) and hCG-induced maturation (9 ± 8%), with the majority of non-ovulated mature oocytes showing signs of activation and cell cleavage with longer incubation times. In hCG-groups, higher ovulation rates were achieved by subsequently including DHP (10-100 ng/ml) to the ongoing treatment, shortly before the follicles reached GVBD. Incorporation of supplements in the form of BSA (0.1-0.5%) and FBS (2-10%) to the 90% L-15 medium improved the hormone-induced GVBD of pikeperch follicles in culture. Moreover, it was noted that FBS in concentrations higher than 10% promotes maturation (>32 ± 6%), even with no additional stimulation with DHP or hCG. This outcome might be due to the complex biomolecular composition of FBS, which can include hormones and growth factors that can influence follicle development and induce oocyte maturation. However, although favorable for nuclear maturation, high concentrations of both BSA (≥1%) and FBS (≥15%) seemingly affected the lipid metabolism and cytoplasmic maturation of the oocyte, most often leading to disruption in the lipid droplet formation.
Conclusions
An in vitro maturation protocol was successfully developed for fully grown, postvitellogenic pikeperch ovarian follicles. The culture media consisting of 90% Leibovitz L-15 media (pH 7.5) with the addition of 0.5% BSA or 5% FBS maintains viability and responsiveness of follicles to hormones. The highest quality and rate of maturation was obtained after treatment with 2-5 IU/ml hCG after 6 days of incubation at 12 °C. Subsequent addition of DHP (10 ng/ml) to the culture media during an ongoing hCG treatment improves ovulation rates of matured oocytes, without notable changes in the GVBD rate and lipid droplet morphology. The protocol detailed here presents a useful tool for refining pikeperch spawning practices in the future, as it enables an in vitro assessment of the maturational competence of individual fish, as well as the prospective outcome of hormone-induced ovulation during artificial spawning.
Acknowledgements
This research was supported by the Ministry of Innovation and Technology within the framework of the Thematic Excellence Programme 2020, National Challenges Subprogramme (TKP2020-NKA-16) and National Research, Development and Innovation Office of Hungary (K138425, and PD-139053).
References
Ljubobratović, U., Kitanović, N., Milla, S., Marinović, Z., Fazekas, G., Stanivuk, J., Nagy, Z. and Horváth, Á., 2023. Predicting population’s oocyte maturation competence and evaluating individual’s latency time using in vitro oocyte maturation in pikeperch (Sander lucioperca). Aquaculture, 562, p.738851.