Immature Vitrified Oocytes Research Articles

Ovary cold storage and shipment affect oocyte yield and cleavage rate of cat immature vitrified oocytes

In feline species, cooled transport of ovaries can be employed without detrimental effects to retrieve immature oocytes intended for in vitro embryo production purposes. Indeed, this is the most common way to collect gametes from gonads of wild, valuable animals after they die or are castrated far from specialized laboratories. However, fresh retrieved gametes are generally used, and their cryosensitivity is not known. This study employed ovariectomy-derived domestic cat gonads as a model for wild felids, and aimed to compare the yield and developmental competence of Cryotop-vitrified oocytes (VOs) collected and cryopreserved right after ovary excision (In loco-VOs) or after 24 h cooled transport of ovaries (Shipped-VOs). The number of collected oocytes was higher in In loco-VOs than in Shipped-VOs (mean ± SD: 8 ± 3.36 vs 5.6 ± 3.1, p = 0.05). In vitro embryo production resulted in similar maturation (35% for both vitrified groups, p = 1) and fertilization rates (In loco-VOs: 29.1%; Shipped-VOs: 22.2%; p = 0.295), but showed a difference in cleavage (In loco-VOs: 25.6%; Shipped-VOs: 14.5%; p = 0.0495). No differences were found in further embryo development. Taken together, results suggested that delayed oocyte vitrification after cooled transport of organs was feasible and allowed embryo development. However, the number of collected oocytes and the cleavage rate of matured oocytes were higher when oocyte vitrification was performed without delay after ovary excision, and this should be considered in gamete conservation programs for endangered felids.

Inhibition of Apoptotic Pathways Improves DNA Integrity but Not Developmental Competence of Domestic Cat Immature Vitrified Oocytes.

Being a model for endangered wild felids, cryopreservation protocols for domestic cat oocytes are under continuous development. Immature vitrified oocytes (VOs) are a valuable resource for fertility preservation programs, but they often degenerate after warming and their in vitro development is poor. Since the exact mechanisms are not clear, this study assessed whether vitrification might trigger two apoptotic markers (DNA fragmentation and caspase activity, Experiment I) and the effects of a chemical inhibitor (i.e., the pan-caspase inhibitor Z-VAD-FMK) on the same markers (Experiment II) and on VOs in vitro development (Experiment III). The overarching aim was to check whether apoptosis inhibition might be a strategy to improve cat oocytes cryotolerance. In Experiment I, vitrification induced DNA fragmentation and increased caspase activity in VOs incubated for 24 h after warming (DNA fragmentation: 59.38%; caspase activity: 414.6 ± 326.8) compared to a fresh control (9.68%; 199.6 ± 178.3; p = 0.02). In Experiment II, the addition of Z-VAD-FMK to vitrification-warming and incubation media decreased DNA fragmentation and caspase activity (8.82%; 243.7 ± 106.9) compared to control (untreated) VOs (69.44%; 434.5 ± 248.3; p < 0.001). In Experiment III, Z-VAD-FMK brought maturation rates of treated VOs close to those of fresh oocytes (53.13 and 65.38%, respectively, p = 0.057), but there were no differences in VOs embryo development (cleavage rates; Z-VAD-FMK-treated VOs: 34.38%; control VOs: 31.78%; p = 0.69). In summary, vitrification increased apoptotic markers in cat VOs, and while Z-VAD-FMK was able to hinder DNA damage and caspase activity, its addition was not determinant for embryo development. To make the best use of VOs, other oocyte in vitro maturation and embryo culture strategies, such as the addition of other inhibitors or their prolonged use, should be investigated.

Developmental Competence of Domestic Cat Vitrified Oocytes in 3D Enriched Culture Conditions.

Simple SummaryOocyte vitrification is a cryopreservation method that guarantees the long-term conservation of genetic material and fertility potential in humans and wild or domestic animals. However, in the domestic cat the in vitro embryo development of immature vitrified oocytes is not yet satisfactory. In this study, a three-dimensional (3D) culture system was used for the in vitro embryo production of vitrified oocytes to provide conditions more similar to those of the in vivo microenvironment, and for comparison, control vitrified oocytes were cultured in two-dimensional (2D) conditions. Embryos were cultured for seven days and their development was assessed. The results showed that the 3D enriched culture system was able to sustain the in vitro maturation and the subsequent embryo development of vitrified oocytes, but no differences were found with the 2D system and improvements to enhance the development of vitrified oocytes are still needed.Cryoinjuries severely affect the competence of vitrified oocytes (VOs) to develop into embryos after warming. The use of culture conditions that provide physical and chemical support and resemble the in vivo microenvironment in which oocytes develop, such as 3D scaffolds and coculture systems, might be useful to improve VOs outcomes. In this study, an enriched culture system of 3D barium alginate microcapsules was employed for the in vitro embryo production of domestic cat VOs. Cryotop vitrified-warmed oocytes were in vitro matured for 24 h in the 3D system with or without fresh cumulus-oocyte complexes (COCs) in coculture, whereas a control group of VOs was cultured in traditional 2D microdrops of medium. After in vitro fertilization, presumptive embryos were cultured in 3D or 2D systems according to the maturation conditions. Vitrified oocytes were able to mature and develop into embryos in 3D microcapsules (17.42 ± 11.83%) as well as in 2D microdrops (14.96 ± 8.80%), but the coculture with companion COCs in 3D resulted in similar proportions of VOs embryo development (18.39 ± 16.67%; p = 1.00), although COCs presence allowed for blastocyst formation (0.95 ± 2.52%). In conclusion, embryos until late developmental stages were obtained from cat VOs, and 3D microcapsules were comparable to 2D microdrops, but improvements in post-warming conditions are still needed.

39 THE EFFECTS OF RESVERATROL DURING IN VITRO MATURATION ON THE DEVELOPMENTAL COMPETENCE OF PORCINE OOCYTES VITRIFIED AT THE IMMATURE STAGE

Previously, live offspring have been produced from porcine oocytes vitrified at the immature stage (Somfai et al. 2014 PLoS One 9, e97731); however, their embryo developmental rates remain low. The aim of our current research was to test the effects of resveratrol, an antioxidant and anti-apoptotic agent on the developmental competence of immature vitrified oocytes during in vitro maturation (IVM) after warming. Follicular porcine cumulus-oocyte complexes (COC) were vitrified on Cryotop® sheets (Kitazato Corp. Shizuoka, Japan) using the cryoprotectant treatment and warming method of Somfai et al. (2015 J. Reprod. Dev. 61, 571–579). After warming, the oocytes were subjected to IVM for 46 h in a chemically defined porcine oocyte medium (POM) enriched with 10 ng mL−1 epidermal growth factor, 10 IU mL−1 eCG, and 10 IU mL−1 hCG. During the first 22 h of IVM, the medium was supplemented with 1 mM dibutyryl cAMP. The following 24 h of IVM was performed in POM without dibutyryl cAMP. Vitrified/warmed COC (vitrified group) and freshly collected COC (control group) were matured either in the absence or presence of 2 µM resveratrol (RES− and RES+, respectively) throughout the entire IVM. At the end of IVM, oocytes were denuded and their survival was evaluated. Then, those with 1 polar body (PB1+) were selected for parthenogenetic activation (Day 0). Activated oocytes were cultured for 7 days in PZM-3. Survival, nuclear maturation, cleavage, and blastocyst rates were assessed. The experiment was replicated 5 times. Results were analysed by one-way ANOVA and Tukey’s multiple comparison test. Vitrification reduced the percentage of live oocytes after IVM both in RES− and RES+ groups in a similar manner (47.9 and 51.8%, respectively) compared with control RES− and RES+ groups (99.4 and 100%, respectively; P &lt; 0.05) There was no statistical difference among groups in the percentage of PB1+ oocytes (ranging between 76.1 and 90.2%). On Day 2, the cleavage rate in vitrified RES− group was lower than those in control RES− and RES+ groups (55.9 v. 78.5% and 79.2%, respectively) whereas the vitrified RES+ group did not differ from the others (72.1%). The blastocyst developmental rate calculated from total cultured oocytes on Day 7 in vitrified RES+ group was significantly higher (P &lt; 0.05) than that in the vitrified RES− group (26.2% v. 6.9%, respectively) and did not differ significantly from those of control RES− and RES+ groups (32.1 and 36.0%, respectively). Blastocyst rates in control RES− and RES+ groups were significantly higher (P &lt; 0.05) than that in vitrified RES− group but did not differ from one another. In conclusion, supplementation of IVM medium with resveratrol improved the developmental competence of vitrified, but not freshly collected oocytes. This work was supported by JSPS KAKENHI (Grant Number: 26870839) and JST/JICA SATREPS. E.C.S. Santos was supported by a CNPq-Brasil fellowship.

Vitrification of immature and matured bovine oocytes: effect of brilliant cresyl blue selection and hyaluronan addition

The aim of the present study was to evaluate the effect of brilliant cresyl blue (BCB) selection, the type of oocyte (immature or matured) and the use of hyaluronan in the vitrification solution on further embryo developmental competence. Oocytes (n = 1308) obtained from abattoir ovaries were classified by BCB stain. Control oocytes were maintained in holding media for 90 min and then subdivided to be placed into maturation media without any treatment or were vitrified. Immature or matured oocytes were vitrified by the solid-surface technique using two different vitrification solutions. VS1: composed of 10% ethylene glycol (EG) for 10 min followed by 20% EG + 0.2M trehalose for 30 sec and finally into 30% EG + 0.5M trehalose for 30 sec, or VS2 composed by 10% EG for 10 min, followed by 20% EG + 0.2M trehalose for 30 sec, and finally into 30% EG+ 0.5M trehalose + 0.1 g/ml hyaluronan for 30 sec. Oocytes were then loaded into Fyberplugs™ and vitrified. After one week, Fyberplugs™ were open and placed directly into (37°C) 0.5M sucrose solution for 5 min, then into 0.25M of sucrose for another 5 min and finally placed into maturation medium for in vitro production. Cleavage and development rates were examined on days 2 and 7 after fertilization, respectively. The blastocyst rate of vitrified oocytes selected as BCB + (5.5 ± 0.6%) were higher than those selected as BCB - (1.0 ± 0.4%) and those that were not selected by BCB (2.0 ± 1.1%; P < 0.001). Furthermore, immature vitrified oocytes had greater (P < 0.05) cleavage and blastocyst rates (44.8 ± 1.9% and 4.0 ± 0.6%) than matured vitrified oocytes (38.3 ± 2.8% and 2.5 ± 0.6%). Finally, the addition of hyaluronan to the vitrification solution had no significant effect on development rates. In conclusion, the selection of oocytes by BCB and the use of immature oocytes increase the development rates of vitrified-warmed oocytes.

36 EFFECT OF cAMP MODULATORS DURING OOCYTE IN VITRO MATURATION ON GAP JUNCTIONAL ACTIVITY OF VITRIFIED BOVINE OOCYTES

Oocyte cryopreservation is a strategic tool for in vitro embryo production, but low rates of cryosurvival are reported for bovine oocytes. Simulated physiological oocyte maturation system (Albuz et al. 2010 Hum. Reprod. 25, 12) uses cAMP modulators to increase oocyte competence by the extension of meiosis block and gap junctional communications activity. The aim of this study was to investigate the effect of simulated physiological oocyte maturation system on gap junctional activity of vitrified bovine oocytes. Oocytes from slaughterhouse ovaries were divided into 4 groups: C (control: fresh immature oocytes); V (vitrified immature oocytes); PM-V (vitrified oocytes after a 2-h pre-in vitro maturation phase – in the presence of AMPc modulators, 100 μM Forskolin, and 500 μM IBMX); and PM (fresh immature oocytes subjected to pre-in vitro maturation). Viable oocytes (n = 404 obtained from 4 replicates) were stained with Calcein-AM using the protocol of Thomas et al. (2004 Biol. Reprod. 71(4), 1142–1149) in order to measure gap junctions activity. Images were captured in fluorescence microscope, and fluorescence intensity was analysed with ImageJ software. Mean fluorescence intensity of each group was normalized to control group to obtain relative intensity value. Means were compared by Kruskal-Wallis test and Dunn post-test. A second analysis was performed considering the percentage of each staining pattern (low, middle, and high intensity) for each group. Results were analysed using Fisher exact test. All statistical analysis were performed in GraphPad Instat program with 5% significance level. Results demonstrated that all treatments induced an increase (P &lt; 0.05) in fluorescence intensity (V: 1.76 ± 1.13; PM-V: 1.58 ± 0.98; PM: 1.38 ± 0.94) compared with control (C: 1.00 ± 0.48). Regarding the staining patterns analyses, immature vitrified oocytes (V group) differed from control group in middle and low patterns (G1, calibrator – high: 11.2%ab, middle: 43.8%a, low: 44.9%a; G2 – high: 8.2%ab, middle: 63.9%b, low: 27.9%b; G3 – high: 16.3%a, middle: 42.3%a, low: 41.3%a; G4 – high: 6.7%b, middle: 53.9%ab, low: 39.3a). In conclusion, unexpectedly, vitrification also increased gap junctional activity, as was found for pre-in vitro maturation group. However, staining pattern analysis results showed only vitrified group was different from control, suggesting vitrified and pre-in vitro maturation groups could have gap activity affected by different ways. This research was supported by FAPERJ (E26/111.61/2013) and CAPES.

Calves Born after Direct Transfer of Vitrified Bovine In Vitro‐produced Blastocysts Derived from Vitrified Immature Oocytes

Vitrification has been the method of choice for the cryopreservation of bovine oocytes, as rapid cooling decreases chilling sensitivity. The aim of this study was to determine the in vitro and in vivo survival and the viability of immature oocytes vitrified using super-cooled liquid nitrogen. Immature oocytes were randomly allocated to three groups: (i) non-vitrified control group, (ii) vitrified in normal (-196 degrees C) liquid nitrogen (LN(2)) and (iii) vitrified in super-cooled LN(2) (< or =-200 degrees C). Open-pulled glass micropipettes were used as vitrification containers. Immature oocytes were in vitro-matured, fertilized and cultured to the blastocyst stage. In vitro viability was assessed by cleavage and blastocyst rates on days 2 and 7 of culture respectively. Vitrified blastocysts derived from the immature vitrified oocytes were directly transferred to synchronous recipients. The in vitro embryo development of vitrified immature oocytes was not influenced by the LN(2) state. After direct transfer (one embryo per recipient) of 16 embryos obtained from immature vitrified oocytes (eight from each vitrified group), two healthy calves were born in each group. These results indicated that vitrification of immature bovine oocytes using glass micropipettes under normal or super-cooled LN(2), resulted in viable blastocysts and live calves following in vitro embryo production.