Lycopene supplementation to serum-free embryo culture medium and its effect on development and quality of bovine blastocysts produced in vitro.

Selenium is commonly used as an antioxidant in a serum-free culture medium setting. However, lycopene has emerged as a potent antioxidant being twice as efficient as ß-carotene and 10 times as efficient as α-tocopherol with beneficial effects when supplemented in a serum-free maturation medium. Here, we aimed to evaluate the effect of lycopene supplementation in a serum-free culture medium on blastocyst development and quality. After in vitro maturation and fertilization, presumed zygotes were cultured in groups of 25 in 50 µl droplets of synthetic oviductal fluid. Culture medium supplementation was done using four experimental groups: insulin, transferrin, selenium (ITS, control); ITS + DMSO (diluent control); ITS + DMSO-lycopene 0.1 µM (ITSL); and IT + DMSO-lycopene 0.1 µM (ITL). DMSO was used as a diluent for lycopene. Blastocyst development among experimental groups was fitted in mixed-effects models, and blastocyst quality parameters (assessed via differential apoptotic staining) were evaluated in mixed linear regression models. The cleavage (85.3 ± 2.4, 82.6 ± 2.7, 86 ± 2.3 and 86.4 ± 2.3% for control, diluent control, ITSL and ITL, respectively) and day 8 blastocyst rates (37.4 ± 3.3, 36.9 ± 3.4, 39.7 ± 3.3 and 46.2 ± 3.4% for control, diluent control, ITSL and ITL, respectively) were not different (p > .1) among experimental groups. Embryos produced in the ITL group resulted in blastocysts with higher total cell numbers (TCN; 141 ± 19.2), inner cell mass (ICM; 65.3 ± 11.6) and trophectoderm cells (TE; 75.2 ± 8.8) compared with the control (129 ± 19.2, 56.3 ± 11.6 and 72.7 ± 8.8, for TCN, ICM and TE; p < .01, respectively). Lycopene-supplemented groups (ITSL and ITL) resulted in blastocysts with similar TCN, ICM and TE (p > .2). The number of apoptotic cells was not different among experimental groups (p > .1). Lycopene supplementation to the culture medium only produced a numerical increase in the blastocyst rate but replacing selenium with lycopene in a serum-free culture medium resulted in blastocysts with more cells.

Embryo morphokinetics derived from fresh and vitrified bovine oocytes predict blastocyst development and nuclear abnormalities.

Embryo development is a dynamic process and critical stages may go unnoticed with the use of traditional morphologic assessments, especially the timing of embryonic divisions and aberrant zygotic cleavage patterns. Bovine embryo development is impaired after oocyte vitrification, but little is known about the underlying morphokinetic behavior. Here, bovine zygotes from fresh (n = 708) and vitrified oocytes (n = 182) were monitored by time-lapse imaging and the timing and nature of early blastomere divisions were modeled to find associations with blastocyst development at day 8. The predictive potential of morphokinetic parameters was analyzed by logistic regression and receiver operating characteristic curve analysis to determine optimal cut-off values. Lag-phase was highly correlated with embryo development. Remarkably, 100% of zygotes that reached the blastocyst stage showed a lag-phase. Fast first cleavage increased the chance of blastocyst development to 30% with a cut-off of 32 h and 22 min. Aberrant zygotic cleavage events, including multipolar division, unequal blastomere sizes, and membrane ruffling resulted in decreased blastocyst development. Multipolar division leads to uneven blastomeres, which was associated with anuclear and multinuclear blastomeres, indicating genome segregation errors. Moreover, we described for the first time morphokinetics of embryos derived from vitrified bovine oocytes. Vitrification severely affected blastocyst development, although lower cryoprotectant concentration in equilibration solutions seems to be less detrimental for embryo yield. Impaired development was linked to slow cleavages, lower lag-phase incidence, and increased early embryonic arrest. Typically, less than 15% of the embryos produced from vitrified oocytes reached more than eight cells. Interestingly, the rate of abnormal first cleavage events was not affected by oocyte vitrification. In conclusion, time to first cleavage, the presence of a lag-phase, and the absence of aberrant zygotic cleavage were the best predictors of bovine blastocyst development for both fresh and vitrified oocytes.

Alternative Culture Systems for Bovine Oocyte In Vitro Maturation: Liquid Marbles and Differentially Shaped 96-Well Plates.

In vivo-matured oocytes exhibit higher developmental competence than those matured in vitro but mimicking the in vivo environment by in vitro conditions has been challenging. Until now, conventional two-dimensional (2D) systems have been used for in vitro maturation of bovine cumulus-oocytes-complexes (COCs). However, using such systems present certain limitations. Therefore, alternative low-cost methodologies may help to optimize oocyte in vitro maturation. Here, we used two different systems to culture COCs and evaluate their potential influence on embryo development and quality. In the first system, we used treated fumed silica particles to create a 3D microenvironment (liquid marbles; LM) to mature COCs. In the second system, we cultured COCs in 96-well plates with different dimensions (flat, ultra-low attachment round-bottom, and v-shaped 96-well plates). In both systems, the nuclear maturation rate remained similar to the control in 2D, showing that most oocytes reached metaphase II. However, the subsequent blastocyst rate remained lower in the liquid marble system compared with the 96-well plates and control 2D systems. Interestingly, a lower total cell number was found in the resulting embryos from both systems (LM and 96-well plates) compared with the control. In conclusion, oocytes matured in liquid marbles or 96-well plates showed no remarkable change in terms of meiotic resumption. None of the surface geometries influenced embryo development while oocyte maturation in liquid marbles led to reduced embryo development. These findings show that different geometry during maturation did not have a large impact on oocyte and embryo development. Lower embryo production after in vitro maturation in liquid marbles was probably detected because in vitro maturation was performed in serum-free medium, which makes oocytes more sensitive to possible toxic effects from the environment.

Oocyte developmental capacity is influenced by intrinsic ovarian factors in a bovine model for individual embryo production.

The ovary and its hormones may have major effects on the in vitro developmental capacity of the oocytes it contains. We related intrinsic ovarian factors namely the presence of corpus luteum (CL) and/or dominant follicle (>8 mm) and the follicular count to cumulus expansion (CE), embryo development, and blastocyst quality in a bovine model. Cumulus-oocyte-complexes (COCs) were aspirated from follicles between 4 and 8 mm in diameter. In vitro embryo production was performed in a fully individual production system. The follicular fluid from which COCs were collected was pooled (per ovary) to evaluate the estrogen, progesterone, and insulin-like growth factor-1 (IGF-1) concentrations. Cumulus oocyte complexes collected from ovaries without a CL presented a greater CE than COCs derived from ovaries bearing CL. The absence of ovarian structures increased the blastocyst rate when compared to oocytes derived from ovaries with a CL, a dominant follicle, or both. Blastocysts derived from ovaries without a dominant follicle presented higher total cell numbers and a lower proportion of apoptosis than blastocysts derived from ovaries containing a dominant follicle. Cumulus oocyte complexes collected from ovaries with high follicular count resulted in higher cleavage than from ovaries with low follicular count, but the blastocyst rate was similar between groups. Ovaries bearing a CL had greater progesterone and IGF-1 follicular fluid concentrations in neighboring follicles than ovaries without a CL. Selection for bovine ovaries without CL or dominant follicle can have positive effects on CE, embryo development, and blastocyst quality in an individual embryo production system set-up.

New Alternative Mixtures of Cryoprotectants for Equine Immature Oocyte Vitrification.

Equine oocyte vitrification would benefit the growing in vitro embryo production programs, but further optimization of the protocol is necessary to reach clinical efficiency. Therefore, we aimed to perform a direct comparison of non-permeating and permeating cryoprotective agents (CPAs) during the vitrification and warming of equine immature oocytes. In the first experiment, cumulus oocytes complexes (COCs) were vitrified comparing sucrose, trehalose, and galactose in combination with ethylene glycol (EG) and dimethyl sulfoxide (DMSO). In the second experiment, the COCs were vitrified using three mixtures of permeating CPAs in a 50:50 volume ratio (ethylene glycol-dimethyl sulfoxide (ED), propylene glycol-ethylene glycol (PE), and propylene glycol-dimethyl sulfoxide (PD)) with galactose and warmed in different galactose concentrations (0.3 or 0.5 mol/L). Overall, all the treatments supported blastocyst formation, but the developmental rates were lower for all the vitrified groups in the first (4.3 to 7.6%) and the second (3.5 to 9.4%) experiment compared to the control (26.5 and 34.2%, respectively; p < 0.01). In the first experiment, the maturation was not affected by vitrification. The sucrose exhibited lower cleavage than the control (p = 0.02). Although the galactose tended to have lower maturation than trehalose (p = 0.060) and control (p = 0.069), the highest numerical cleavage and blastocyst rates were obtained with this CPA. In the second experiment, the maturation, cleavage, and blastocyst rates were similar between the treatments. Compared to the control, only the ED reached similar maturation (p = 0.02) and PE similar cleavage (p = 0.1). The galactose concentration during warming did not affect the maturation, cleavage, or blastocyst rates (p > 0.1), but the PE-0.3 exhibited the highest blastocyst rate (15.1%) among the treatments, being the only one comparable to the control (34.2%). As such, PE-galactose provides a valuable option for equine immature oocyte vitrification and should be considered for the future optimization of the protocol.