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BackgroundStorage conditions during transportation of explanted ovaries are a critical step in setting up fertility preservation protocols in both animal and human fields. Here, we evaluated the effects of ovary storage at 4 °C on the preservation of preantral follicles and oocytes retrieved from antral follicles using the domestic cat as model.MethodsOvaries were harvested from fifty-five healthy domestic queens during ovariectomy and stored at 4 °C for 0 (control), 24, 48, 72 and 96 h. In Experiment 1, the effects of the storage period at 4 °C on the morphology, cytoskeleton (α/β tubulin) and DNA integrity (phosphorylation of histone H2AX) of preantral follicles were investigated. In Experiment 2, oocytes recovered from antral follicles were matured and fertilized in vitro to evaluate their meiotic and developmental competence. Reactive oxygen species (ROS), glutathione (GSH) and lipid peroxidation were measured in matured oocytes.ResultsThe results showed that: a) storage up to 24 h did not affect the morphology and the DNA integrity of preantral follicles; b) extended storage times caused progressive morphological abnormalities, disassembling of microtubules and DNA damage; c) storage up to 48 h did not influence in vitro meiotic maturation of oocytes nor cleavage after in vitro fertilization. However, only oocytes stored within the ovary for 24 h produced blastocysts in a percentage similar to control oocytes; d) GSH levels of in vitro matured oocytes did not change at any time during ovary storage; a progressive increase in ROS levels was detected from 48 h associated with elevated lipid peroxidation at 72 and 96 h of storage.ConclusionsStorage of cat ovaries for up to 24 h caused minimal alteration of preantral follicles and oocytes. The extension of the storage period beyond 24 h progressively impaired the structure of follicles, and modified the oxidative status of in vitro matured oocytes and their developmental competence after in vitro fertilization. This information may help when setting up programs for fertility conservation, especially for wild feline species which die in geographic areas located far away from ARTs centers.

Background Heavy metal cadmium (Cd) is a widespread environmental contaminant with a potential toxicity that might negatively affect female reproduction and fertility. It has been reported that Cd exposure impaired the quality of oocytes and led to a defective maturation and fertilization, through oxidative stress induction. Resveratrol (Res) is a natural polyphenol with strong antioxidant properties that exhibited protective role in preventing oocyte redox homeostasis disruption and quality decline. Here, we explored whether the addition of Res to in vitro maturation (IVM) medium might act as a protection against Cd-induced toxicity on ovine oocyte maturation and fertilization. Firstly, we evaluated the effect of supplementing IVM medium with two different Res concentrations (1 and 2 μmol/L) on nuclear maturation and fertilization of oocytes matured under CdCl2 (2 μmol/L) exposure. Therefore, the concentration of 1 μmol/L Res was selected to analyse the effects of this compound on intracellular ROS levels, mitochondrial (mt) distribution and activity, chromatin configuration, cytoskeleton morphology, cortical granules (CGs) distribution and mRNA expression of genes associated with cellular response to oxidative stress (i.e. SIRT1, SOD 1, GPX1, GSR, CAT) in Cd-exposed in vitro matured oocytes. Results We found that 1 μmol/L Res restored the reduced oocyte meiotic competence induced by Cd exposure as well as, Res sustained oocyte ability to be normally fertilized and decreased polyspermic fertilization at both tested concentrations. Moreover, we demonstrated that 1 μmol/L Res mitigated Cd-induced alterations of oocyte cytoplasmic maturation by reducing reactive oxygen species (ROS) accumulation, preventing mt dysfunction, maintaining the correct meiotic spindle and cortical F-actin assembly and the normal cortical granule distribution as well as up-regulating SIRT1, SOD1 and GPX1 genes. Conclusions Taken together, our findings highlighted the beneficial influence exerted by Res in preventing Cd-induced disturbance of nuclear and cytoplasmic maturation and subsequent fertilization in ovine oocytes. Res treatment may help to establish defence strategies counteracting Cd-induced toxicity on the female gamete.

In domestic cats, the maturation, fertilization, and development potential in vitro decreases during the non-breeding season. This study aims at evaluating the efficacy of Brilliant Cresyl Blue (BCB) staining in selecting developmentally competent oocytes to be used in in vitro embryo production (IVEP) programs in order to overcome the season variability in blastocyst yield. Cumulus-oocytes complexes (COCs) collected from antral follicles of domestic cat ovaries during the anestrus phase (July to November) were selected by BCB staining and classified as BCB+ (colored cytoplasm) and BCB− (colorless cytoplasm). COCs not exposed to BCB staining were used as control. Before and after in vitro maturation mitochondrial activity and reactive oxygen species (ROS) were measured. Following in vitro fertilization, blastocyst rate, hatching rate, and blastocyst cell numbers were recorded. The results show that BCB staining did not alter the mitochondrial function and ROS production in cat oocytes. BCB+ oocytes presented a higher (p < 0.05) blastocyst rate, hatching rate, and blastocyst cell number than BCB− and control oocytes. In conclusion, BCB staining does not affect the bioenergetic/oxidative status of the oocyte while being a useful tool for selecting good quality oocytes to increase IVEP in domestic cats during non-breeding season.

Freeze-drying is a novel technique that permits the storage of semen at room temperature for long time periods, retaining their fertilizing capacity. The main objective of this work was to compare the fertilization ability of frozen-thawed (FT) and freeze-dried (FD) ram semen following intracytoplasmic sperm injection (ICSI) of invitro-matured (IVM) sheep oocytes. Oocytes were recovered by slicing the ovaries of slaughtered sheep. Selected cumulus-oocyte complexes (COCs) were IVM for 24h in tissue culture medium 199 (TCM-199) supplemented with 10% heat-treated oestrous sheep serum (ESS), 0.36mM pyruvate, FSH (1IUmL−1), and luteinising hormone (LH; 1IUmL−1) under mineral oil in a humidified atmosphere of 5% CO2, at 38.5°C. Semen was collected from fertile adult rams using an artificial vagina and processed for (1) freezing and thawing (Khalifa and Lymberopoulos, 2013 Cell Tissue Bank 14, 687-698; https://doi.org/10.1007/s10561-012-9357-6) or (2) freeze-drying and rehydration according to Arav et al. (2018 J. Assist. Reprod. Genet. 35, 1149-115; https://doi.org/10.1007/s10815-018-1145-1) protocols. For FD protocol, sperm samples were diluted in a sugar solution of trehalose and sorbitol (LyoB) and dehydrated for 24h. Later, the samples were rehydrated in a warming solution and diluted in TCM-199 before ICSI. After maturation, metaphase II (MII) oocytes with a polar body were injected with FT or FD sperm. Briefly, oocytes were transferred into groups of six in an ICSI dish containing 6-µL drops of holding medium (TCM-199 + 5% fetal bovine serum) and 3-µL drops of PVP for the sperm samples. Injection was carried out with an inverted microscope (Olympus IX73) connected to a micromanipulation system (Narishige) using ICSI pipettes with 7-µm internal diameter. Within 1h, ICSI oocytes were activated with 5 µM ionomycin for 4min and invitro cultured in modified synthetic oviductal fluid medium (Bogliolo et al. 2011 Reprod. Fertil. Dev. 23, 809-817; https://doi.org/10.1071/RD11023). After 17-21h, injected oocytes were fixed and stained in a solution of ethanol Hoechst 33342 and classified as FPN (one female pronucleus and one condensed sperm head), MPN (one male pronucleus and one MII), 2PN (two pronuclei, male and female), 3PN (three or more pronuclei), and NPN (no pronuclei). Data were analysed using analysis of variance (two-way ANOVA) followed by Tukey post hoc test with SAS software, version 9.4. The ICSI-FD group had a higher number of NPN and a lower number of 2PN than did the ICSI-FT group (P&lt;0.05). We think that more technical advances in the FD process as well as the rehydration procedure are necessary to improve the application of FD ovine semen for invitro fertilization by ICSI in sheep, but in any case these results have showed that FD could be a useful tool for the future of invitro embryo production. Table 1.Pronuclear formation at 17-21h post-injection1 Treatment n FPN MPN 2PN 3PN NPN FT 71 9.66±4.12 4.26±1.48 48.13±2.79a 5.97±4.16 31.98±6.75a FD 65 6.16±2.26 1.39±1.39 20.15±4.14b 10.57±6.59 61.73±6.89b a,bValues in the same column with different superscript letters differ significantly (P&lt;0.05). 1Data are presented as mean±s.e.m. FPN=female pronucleus, MPN=one male pronucleus and one metaphase II oocyte, 2PN=two pronuclei, male and female, 3PN=three or more pronuclei, NPN=no pronuclei. Funding was provided by Spanish MINECO Grant AGL2017-85837-R, Spanish MECD pre-doctoral grant FPU15/00773, and Spanish MECD mobility grant EST18/00472 to Irene Menéndez Blanco.