Bovine embryos are typically cultured at reduced oxygen tension to lower the impact of oxidative stress on embryo development. However, oocyte in vitro maturation (IVM) is performed at atmospheric oxygen tension since low oxygen during maturation has a negative impact on oocyte developmental competence. Lycopene, a carotenoid, acts as a powerful antioxidant and may protect the oocyte against oxidative stress during maturation at atmospheric oxygen conditions. Here, we assessed the effect of adding 0.2 μM lycopene (antioxidant), 5 μM menadione (pro-oxidant), and their combination on the generation of reactive oxygen species (ROS) in matured oocytes and the subsequent development, quality, and transcriptome of the blastocysts in a bovine in vitro model. ROS fluorescent intensity in matured oocytes was significantly lower in the lycopene group, and the resulting embryos showed a significantly higher blastocyst rate on day 8 and a lower apoptotic cell ratio than all other groups. Transcriptomic analysis disclosed a total of 296 differentially expressed genes (Benjamini–Hochberg-adjusted p < 0.05 and ≥ 1-log2-fold change) between the lycopene and control groups, where pathways associated with cellular function, metabolism, DNA repair, and anti-apoptosis were upregulated in the lycopene group. Lycopene supplementation to serum-free maturation medium neutralized excess ROS during maturation, enhanced blastocyst development and quality, and modulated the transcriptomic landscape.

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.

In vitro embryo production has evolved rapidly in the horse over the past decade, but blastocyst rates from vitrified equine oocytes remain quite poor and further research is needed to warrant application. Oocyte vitrification is affected by several technical and biological factors. In the horse, short exposure of immature oocytes to the combination of permeating and non-permeating cryoprotective agents has been associated with the best results so far. High cooling and warming rates are also crucial and can be obtained by using minimal volumes and open cryodevices. Vitrification of in vivo-matured oocytes has yielded better results, but is less practical. The presence of the corona radiata seems to partially protect those factors that are necessary for the construction of the normal spindle and for chromosome alignment, but multiple layers of cumulus cells may impair permeation of cryoprotective agents. In addition to the spindle, the oolemma and mitochondria are also particularly sensitive to vitrification damage, which should be minimised in future vitrification procedures. This review presents promising protocols and novel strategies in equine oocyte vitrification, with a focus on blastocyst development and foal production as most reliable outcome parameters.

No abstract

Anti-Müllerian hormone (AMH) reflects the population of growing follicles and has been related to mammalian fertility. In the horse, clinical application of ovum pick-up and intracytoplasmic sperm injection (OPU-ICSI) is increasing, but results depend largely on the individuality of the mare. The aim of this study was to assess AMH as a predictor for the OPU-ICSI outcome in horses. Therefore, 103 mares with a total follicle count above 10 were included in a commercial OPU-ICSI session and serum AMH was determined using ELISA. Overall, the AMH level was significantly correlated with the number of aspirated follicles and the number of recovered oocytes (p < 0.001). Mares with a high AMH level (≥2.5 µg/L) yielded significantly greater numbers of follicles (22.9 ± 1.2), oocytes (13.5 ± 0.8), and blastocysts (2.1 ± 0.4) per OPU-ICSI session compared to mares with medium (1.5–2.5 µg/L) or low AMH levels (<1.5 µg/L), but no significant differences in blastocyst rates were observed. Yet, AMH levels were variable and 58% of the mares with low AMH also produced an embryo. In conclusion, measurement of serum AMH can be used to identify mares with higher chances of producing multiple in vitro embryos, but not as an independent predictor of successful OPU-ICSI in horses.

No abstract

No abstract

Excessive production and accumulation of reactive oxygen species (ROS) may cause embryo damage associated with oxidative stress. Lycopene, a natural antioxidant, can scavenge singlet oxygen and is one of the most effective antioxidants among carotenoids. We evaluated the effects of supplementation of lycopene (antioxidant), menadione (prooxidant), and their combination during invitro oocyte maturation on ROS generation in matured oocytes and the quality of vitrified-warmed embryos. Cumulus–oocyte complexes, collected from the slaughterhouse, were matured in groups of 60 in 500μL of TCM-199 medium+50mg mL−1 gentamycin+20ng mL−1 epidermal growth factor, for 22h at 38.5°C in 5% CO2 in air and then supplemented with (1) 0.2μM lycopene, (2) 5μM menadione, (3) 0.2μM lycopene+5μM menadione (L+M), or (4) not supplemented (control). Fertilization and embryo culture were performed similarly for all the groups. In the first experiment, ROS measurement (n=236; via fluorescent microscopy) was performed in denuded, matured oocytes incubated in 5μM CellROX® Green (ThermoFisher Scientific) for 1h. Fluorescent intensity was measured in Image-J. In the second experiment, embryos in the blastocyst stage (n=143) were vitrified as previously described by Ortiz-Escribano et al. (2017 Biol. Reprod. 96, 288-301). Vitrified blastocysts were then warmed and washed in decreasing concentrations of sucrose and incubated for 2 days in culture medium [50µL of synthetic oviductal fluid (SOF)+(5g mL−1 insulin, 5g mL−1 transferrin, 5ng mL−1 selenium)]. The quality of vitrified-warmed blastocysts was assessed using a differential staining as described by Wydooghe et al. (2011 Anal. Biochem. 416, 228–230). The effects of pro- and antioxidant supplementation on oocyte fluorescent intensity and embryo quality parameters were fitted in linear mixed-effects models, and results are expressed as least squares means and standard errors. The fluorescent intensity for ROS was lower (P&lt;0.05) in lycopene (10.06±2.92) than in menadione (16.8±2.92). No differences (P&gt;0.05) in ROS intensity values were found among the other groups [control (13.5±2.92) and L+M (13.7±2.90)]. Total cell number (TCN) was similar (P&gt;0.05) in lycopene (153±2.95), L+M (143±4.59), and control (145±3.67) but lower (P&lt;0.05) in menadione (134±6.08). Lesser numbers of apoptotic cells (AC) and AC/TCN values (P&lt;0.05) were recorded in lycopene (4.12±3.07 and 2.71±2.21) compared with control (6.18±3.82 and 4.31±2.75), L+M (6.00±4.79 and 4.22±3.45), and menadione (7.75±6.33 and 5.82±4.56). For the remaining embryo quality parameters, no differences were found (P&gt;0.05). In conclusion, lycopene supplementation during invitro oocyte maturation effectively scavenged free radicals, lowering oxidative stress and improving embryo quality post-vitrification and warming.