Time-Lapse Videomicrographic Observations of Blastocyst Hatching in Cattle

Abstract

Abstract. Morphological changes of cultured bovine blastocysts during hatching were observed using time-lapse videomicrography in order to investigate the patterns of the hatching process that occurred in the blastocysts and to determine whether the hatching patterns differed between blastocysts developed from fresh and cryopreserved embryos. Compacted morulae (CMs) were collected from superovulation-treated Japanese Black and Holstein dairy cattle and cultured in a medium in a CO2 culture chamber equipped with an inverted microscope at 38.5 C. Images of resultant blastocysts during the period from blastocoel formation to completion of hatching were taken at 4-sec intervals by a CCD color camera connected to an inverted microscope and recorded by a time-lapse video cassette recorder. In blastocysts developed from fresh CMs, hatching was found to begin with protrusion of trophectoderm cells from zonae pellucidae at the expanded stage. Protrusion of the cells occurred in any site of the trophectoderm. After protrusion, a large or small slit was formed in the zona pellucida in all blastocysts as a result of blastocyst expansion or enlargement of the protrusion. Then, blastocysts completely escaped from the zona pellucida through the slit in the state of expansion. From these findings, the hatching patterns of cattle blastocysts could be classified into 5 types. In blastocysts developed from frozen-thawed CMs, the hatching pattern and length of time needed for hatching are similar to those in blastocysts developed from fresh CMs. In addition, the pregnancy rate of recipients following transfer of frozen-thawed CMs (52.4%) did not differ from that of recipients following transfer with fresh CMs (58.3%). These results suggested that the quality of frozen-thawed cattle embryos is comparable to that of fresh embryos and that there could be a relationship between the hatching pattern of blastocysts and the viability of embryos after transfer. [13][14][15]. The process of blastocyst hatching has been extensively studied in mice [16][17][18][19][20][21][22][23] and rats [23][24][25][26], and it was reported that the process of blastocyst hatching could be classified into 6 types in the mouse and 5 types in the rat, according to the site of protrusion of trophectoderm cells from the zona pellucida, the mode of slitting in the zona pellucida and the state of blastocyst contraction at the time of escape from the zona pellucida [23].In cattle blastocysts, the morphological changes during hatching have also been investigated by scanning electron microscopy [27] and time-lapse microcinematography [28]. Fléchon and Renard [27] reported that 31 out of 47 cultured blastocysts started hatching by protrusion of trophectoderm cells from zonae pellucidae after 24 h of culture and then formed a slit in the zona pellucida by blastocyst expansion. Massip et al. [28] reported that 22 out of 27 cattle blastocysts started hatching by protrusion of trophectoderm cells from zonae pellucidae at any site of the trophectoderm. Of these 22 cat...

Prediction of blastocyst development and implantation potential <i>in utero</i> based on the third cleavage and compaction times in mouse pre-implantation embryos

“…On the other hand, embryos with good morphology, which are chromosomally normal, may fail to differentiate into blastocysts [12]. To achieve a high implantation rate, transferable embryos are selected by considering various parameters beyond morphokinetics, such as the duration of oocyte activation [13], first cell division [14,15,16,17], pronuclear (PN) scoring [18], presence of evenly cleaved blastomeres [19], degree of fragmentation [20, 21], cleavage pattern [9], contraction pattern, and time to hatching [22, 23]. …”

Intraoviductal concentrations of steroid hormones during in vitro culture changed phospholipid profiles and cryotolerance of bovine embryos

“…This is globally in accord with previous reports that used comparable culture systems without cells (20–31%, Shirazi et al, ; 0–29%, Schmaltz‐Panneau et al, ; 41 ± 4.5%, Lopera‐Vasquez et al, ; 48 ± 9%, Lopera‐Vasquez et al, ). However, embryo re‐expansion may be difficult to evaluate because, over time after warming, blastocysts often show repeated collapse and re‐expansion (Maezawa et al, ; Niimura et al, ). Moreover, the duration of the re‐expansion process, if any, is highly variable among blastocysts (Ebner et al, ; Niimura et al, ).…”

“…T A B L E 2 Developmental rates of bovine blastocysts exposed in vitro to to intraoviductal concentrations of progesterone, estradiol, cortisol, or a combination of all at Day 8 (Maezawa et al, 2014;Niimura et al, 2010). Moreover, the duration of the re-expansion process, if any, is highly variable among blastocysts (Ebner et al, 2017;Niimura et al, 2010).…”

“…T A B L E 2 Developmental rates of bovine blastocysts exposed in vitro to to intraoviductal concentrations of progesterone, estradiol, cortisol, or a combination of all at Day 8 (Maezawa et al, 2014;Niimura et al, 2010). Moreover, the duration of the re-expansion process, if any, is highly variable among blastocysts (Ebner et al, 2017;Niimura et al, 2010). Several studies have reported accurate and reproducible evaluation of cell viability after staining with the fluorescent probes calcein-AM and ethidium homodimer-1 in a variety of frozen-thawed tissues and cells, including mammalian follicles and oocytes (Eroglu, Toner, & Toth, 2002;Sanfilippo et al, 2011).…”

Time‐lapse observations to analyze the effects of assisted hatching

“…Niimura et al [25] showed that there is a relationship between the hatching patterns of blastocysts and the development of embryos after transfer, and that a large slit in the zona pellucida contributes to hatching in cattle blastocysts. This could be the theoretical basis for the clinical use of AH.…”