We examined the association between season and expression of genes involved in early embryonic development with an emphasis on cleavage rate and timing of the first embryonic cleavage. In Exp. 1, oocytes were aspirated during the cold (Dec-Apr) and hot (May-Nov) seasons. Matured oocytes were chemically activated and cultured in vitro. The developmental peak to the two-and four-cell stages occurred earlier, with a higher proportion of first-cleaved embryos, during the cold season relative to the hot season (P!0.01). In Exp. 2, a time-lapse system was employed to characterize the delayed cleavage noted for the hot season. Cleavage to the two-cell stage occurred in two distinct waves: early cleavage occurred between 18 and 25 h post activation, and late cleavage occurred between 27 and 40 h post activation. In Exp. 3, oocytes were aspirated during the cold and hot seasons, matured in vitro, fertilized, and cultured for 8 days. In each season, early-and late-cleaved two-cell stage embryos were collected. Total RNA was isolated, and semi-quantitative and real-time PCRs were carried out with primers for GDF9, POU5F1, and GAPDH using 18S rRNA as the reference gene. In both seasons, the expression of all examined genes was higher (P!0.05) in early-versus late-cleaved embryos. POU5F1 expression was higher (P!0.05) in early-cleaved embryos developed in the cold season versus the hot season counterparts. The findings suggest a deleterious seasonal effect on oocyte developmental competence with delayed cleavage and variation in gene expression.

No abstract

This method enables maintenance of high cooling rates as well as reduction of cryoprotectant concentration, despite the use of a sealed container that helps to reduce the potential risk of contamination.

Selecting an embryo with the highest probability of achieving a pregnancy is a major challenge. Early-cleavage embryos are considered to be of good quality; however, the exact developmental stage that predicts further development has not been defined. The aim of the study was to characterize cleavage rate and distribution of various stages of mouse preimplantation embryos using a time-lapse system. Mated mice were killed 20 h after human chorionic gonadotrophin administration and putative zygotes were recovered and cultured in an incubator-enclosed time-lapse imaging system. The 'shortest half' analysis was used to establish the period in which at least 50% of the embryonic population cleaved within the shortest time. Analysis indicated that through embryonic development, cleavage timing becomes less uniform and the 'shortest half' becomes longer with intervals of 2, 2.5, 3.5 and 5 h for 2-, 4-, 8-cell embryo and blastocyst stages, respectively. The 'shortest half' for the first cleavage was closely synchronized, with 80% of embryos developing to the blastocyst stage. Moreover, slow-cleaving embryos approaching the 2-cell stage expressed inferior developmental potential in comparison to those cleaving within the 'shortest half'. Thus, embryonic cleavage rate seems to be a biological indicator of developmental potential and may be useful for embryo selection.

No abstract

Heat stress is a major contributing factor to low fertility among dairy cattle, as reflected by the dramatic reduction in conception rate during the hot months. The effects of thermal stress on oocyte competence and embryonic development have been well documented. However, timing of embryonic cleavage, which may be considered a parameter for the identification of good-quality embryos, and its association with elevated temperatures have not been studied. Two experiments were performed to examine and characterize seasonal effects (i.e. thermal stress) on cleavage timing of bovine parthenogenetic embryos. Oocytes were aspirated from ovaries collected at the local abattoir in 2 seasons: cold (Dec–Apr) and hot (May–Nov). Matured oocytes were chemically activated (ionomycin followed by 6-DMAP) and cultured in vitro; cleavage timing to the 2- and 4-cell stages was observed and documented. The one-way ANOVA procedure was used for statistical analysis. In the first experiment (n = 5416 oocytes), cleavage was documented at specific time points during development post-activation. The peak in embryonic development to the 2-cell stage was earlier (22 to 27 vs. 27 to 40 h after activation) and the cleavage rate higher (39 vs. 21%; P < 0.0001) during the cold season relative to the hot season, respectively. Similarly, the peak in 4-cell-stage development was also observed earlier (46–52 vs. 52–70 h after activation) and corresponded with a higher proportion of developing embryos (33 vs. 21%; P < 0.0001) during the cold season as compared to the hot season, respectively. These results indicate that embryonic development is delayed and a lower proportion of embryos cleaved during the hot season. To better understand the delay in cleavage timing, a second experiment (n = 308 oocytes) was performed through two consecutive hot seasons. A time-lapse system (EmbryoGuard; IMT, Ltd., Ness-Ziona, Israel) was employed to collect accurate data on the first cleavage division, known to be indicative of embryo quality. The time-lapse system was pre-programmed to take photos at 1-h intervals such that culture dishes did not need to be removed from the incubator. Similar to the pattern noted for the hot season in the first experiment, a wide distribution of cleavage timing (18-40 h after activation) was observed. Further analysis revealed that embryos cleaved in 2 distinct waves: cleavage timing of the first wave (18 to 25 h after activation) was characterized by a time frame similar to that in the cold season, suggesting good-quality embryos; however, the second wave, from 27 to 40 h after activation, presented a delay in cleavage timing, suggesting that these late-cleaving embryos are of inferior quality. Taken together, the results of the 2 experiments lead to the assumption that oocytes harvested from lactating cows during the hot season are of reduced developmental potential, which may be explained, in part, by the pattern of 2 cleavage waves. Furthermore, cleavage timing appears to be a good indicator of embryo potential and may increase the chances of selecting better in vitro-derived embryos during the hot season for embryo transfer.

Hyperthermia-induced oxidative stress is one of the suggested mechanisms underlying the loss of developmental competence in heat-stressed embryos. The objective of the present study was to determine whether pretreatment with the antioxidant epigallocatechin gallate (EGCG) would counteract the negative effects of maternal hyperthermia on oocyte competence and improve subsequent embryonic development. Exp. 1 examined the effect of pretreatment with EGCG on ex vivo embryonic development under normal culture conditions (KSOM, 37°C, 5% CO2, 95% RH in air). Female mice (CB6F1) were synchronized (PMSG + hCG) and injected with 0.4 mL EGCG (100 mg/kg body weight) or with saline. Both EGCG- and saline-treated females were paired with stud males overnight. Mated mice were sacrificed and putative zygotes recovered and cultured in vitro. Cleavage and blastocyst formation rates were recorded on Days 1 and 5 post-fertilization, respectively. The percentage of putative zygotes that cleaved into the two-cell stage did not differ between the groups; however, blastocyst formation rate was higher (P < 0.05) in the EGCG group (85 ± 2%) than in the saline group (75 ± 2%). In Exp. 2 (a 2 × 2 factorial study), both EGCG- and saline-treated mice were exposed to normo-thermal conditions (NT; 22°C, 45% RH) or heat stress (HS; 40°C, 70% RH) for 1.5 to 2 h, the latter to induce a rise of 2°C in body temperature. Synchronized mice were paired with stud males overnight and mated mice were then sacrificed. Putative zygotes were recovered and cultured in vitro as described above. The number of putative zygotes recovered, cleavage and blastocyst formation rates and percentage of hatched blastocysts are presented in Table 1. Blastocyst formation rate was higher (P < 0.05) in the HS-EGCG group than in the HS-saline group. In addition, HS-EGCG embryos exhibited developmental competence similar to that of embryos from both NT groups. In summary, pretreatment with EGCG improved developmental competence under the described culture conditions. Furthermore, pretreatment with the antioxidant EGCG counteracted the negative effects of maternal hyperthermia. Further studies are warranted to evaluate the effect of pretreatment with EGCG on embryo quality. Table 1. Effect of pretreatment with EGCG on developmental competence of heat stressed oocytes

Embryonic development is a dynamic process in which embryo morphology may change immensely within several hours. Therefore, identifying and selecting embryos with the highest probability of developing and achieving a pregnancy is a major challenge. The timing of embryonic cleavage may serve as an additional indicator for the identification of quality embryos. The aim of this study was to characterize the cleavage timing of mouse embryos and to identify the stage that is most indicative of blastocyst formation. Mated mice (CB6F1) were sacrificed 20 h after hCG administration; putative zygotes were recovered and cultured (50 embryos in each 20-µL drop of M16) in a time-lapse system (EmbryoGuard; IMT, Ltd., Ness-Ziona, Israel) inside the incubator. The time-lapse system was programmed to take photos at half-hour intervals such that culture dishes were not removed from the incubator. The ‘shortest half’ statistical procedure of JMPIN (SAS Institute, Inc., Cary, NC, USA) was utilized to evaluate the period during which at least 50% of the embryonic population cleaves within the shortest time frame. Captured images made it possible to search along the time axis for the densest 50% of cleavage observations. Developing embryos were categorized into 3 groups according to the time of cleavage after hCG administration: before, during, and after the ‘shortest half’ for each developmental stage. Two hundred thirty putative zygotes cleaved and created 2-cell-stage embryos, of which 55 arrested at various stages and 175 progressed to the blastocyst stage. During embryonic development, cleavage timing appeared to become less uniform and the ‘shortest half’ became longer for each successive cell division: Whereas the shortest period in which 50% of the 2-cell-stage embryos cleaved was a 2-h interval, cleavage into the 4-cell, 8-cell, and blastocyst stages took 2.5, 3.5, and 5 h, respectively. The ‘short half’ for the first cleavage appears to be a predictive time frame for subsequent embryonic development, because cleavage was closely synchronized with 80% of the embryos developing to the blastocyst stage. Note that only a small number of embryos were actually cleaving early, while the ‘shortest half’ consisted of 50% of the embryonic population. Moreover, late-cleaving embryos in the 2-cell stage expressed inferior developmental potential relative to those that cleaved within the ‘shortest half’ (see Table 1). In summary, 2-cell-stage embryos that cleaved within the ‘shortest half’ seemed to be better synchronized and consequently more competent than the rest of the embryonic population. Embryonic cleavage timing using the ‘shortest half’ parameter can be considered a biological indicator of embryo potential. It may be useful as an additional tool for selecting embryos for transfer and cryopreservation. Table 1. Cleavage timing distribution into the 2-cell stage according to the shortest half