Timing of the first cleavage post‐insemination affects cryosurvival of in vitro–produced bovine blastocysts

Abstract

The time of the first cleavage of bovine zygotes during in vitro culture can affect the rate of development and cell number of the blastocysts. The aim of this work was to study the effect of the timing of first cleavage on the cryosurvival of the resulting blastocysts. Following standard IVM and IVF, zygotes were cultured in modified synthetic oviduct fluid (SOF), with 10% fetal calf serum (FCS) added 48 hr post insemination, in a humidified atmosphere of 5% CO 2, 5% O 2 and 90% N 2. Embryos which cleaved by 24, 27 30, 33, or 36 hr after insemination (IVF) were harvested and further cultured to the blastocyst stage (day 7 or day 8 post IVF). All developing blastocysts on days 7 and 8 were classified into three groups and were cryopre-served by vitrification. Group A consisted of blastocysts (150 m, small blastocysts); group B consisted of expanded or hatching blastocysts (150 m, large blastocysts); and group C consisted of morphologically poor quality blastocysts. The vitrification solution consisted of 6.5 M glycerol and 6% bovine serum albumin in PBS (VS3a). Thawed embryos were cultured further and survival was defined as the re-expansion and maintenance of the blastocoel over 24, 48, and 72 hr, respectively. Overall survival and hatching at 72 hr post-thawing was higher in blastocysts formed by day 7 than those formed by day 8 (60% vs. 40% survival; 63% vs. 45% hatching). Large blastocysts from day-7 and day-8 groups survived significantly better than small or poor quality blastocysts (76% vs. 63% and 31%; 72% vs. 30% and 26%, respectively; P 0.05). Day-7 blasto-cysts from the 27-and 30-hr cleavage groups survived significantly better than those from the 36-hr group (63% and 66% vs. 25%, P 0.05). Day-8 blastocysts from later cleaved (30 hr) zygotes had a higher survival than the 27-hr cleavage groups (52% vs. 26%, P 0.05). These results indicate that the day of blastocyst appearance, developmental stage, and timing of the first cleavage post-insemination can influence the cryo-survival of bovine blastocysts following vitrification.

A new simple and reliable vitrification device based on Hollow Fiber Vitrification (HFV) method evaluated using IVP bovine embryos

“…Obtained results are in agreement with data shown in literature (Han et al, 1994;Hasler et al, 1997;Dinnyes et al, 1999;Rios et al, 2010), as it is generally expected for morphologically normal and more developmentally advanced embryos on the given day to be of higher quality and have higher viability after cryopreservation. It was also shown that day 7 blastocysts and expanded blastocysts survived cryopreservation better than their slower developing day 8 and 9 counterparts (Han et al, 1994;Saha et al, 1996;Dinnyes et al, 1999;Gomez et al, 2008).…”

Temporal detection of caspase-3 and -7 in bovine in vitro produced embryos of different developmental capacity

“…Fast cleaving bovine embryos have an altered gene expression (Lonergan et al 2000, Ward et al 2001, Gutiérrez-Adán et al 2004, Dode et al 2006 or altered polyadenylation status of several developmentally important gene transcripts (Pocar et al 2001, Brevini-Gandolfi et al 2002 and a higher chance of reaching advanced developmental stages in comparison with late cleaving embryos (Van Soom et al 1992, Dinnyés et al 1999, Lequarré et al 2003, Favetta et al 2004, Gutiérrez-Adán et al 2004. Studies investigating the effect of developmental kinetics on the incidence of apoptosis during preimplantation embryo development in bovine are scarce.…”

“…1). The time points and criteria for the selection of fast or slow developing embryos were determined on the basis of previous studies in similar culture conditions (Dinnyés et al 1999, Yoshioka et al 2000, Lequarré et al 2003 and verified in a preliminary experiment (data not shown).…”

High incidence of multiple aster formation in vitrified-warmed bovine oocytes after in vitro fertilization

“…Incidence of delayed cleavage and overall lower cleavage rate in vitrified-warmed oocytes after IVF were also reported in sheep [16]. Higher potential of oocytes cleaving earlier, compared to those cleaving later, to reach blastocysts [24,25], to survive cryopreservation [26] and to achieve pregnancy [24] have been reported in cattle. Although genetic factor [30], bull individuals for sperm [25], chromosomal normality [31], embryonic sex [24,31,32], and culture conditions [33] are involved in the developmental kinetics of embryos, the principle factor that controls the timing of first cleavage remains unclear.…”

“…Hence, the oocytes use their own MTOC dispersed in the cytoplasm for aster formation; that is called cytoplasmic aster [22,23]. Timing of first cleavage in IVF-derived bovine oocytes is important for yield and quality of blastocysts, as oocytes cleaving earlier are more likely to become blastocysts [24,25], and the resulting blastocysts have higher cryosurvival potential [26] and higher pregnancy rates [24] than those cleaving later. Thus, developmental kinetics can be used as a proxy of embryo quality.…”