Bovine Morula and Blastocyst

The embryos were fixed and mounted on coverslips in such a way that the three-dimensional structure was maintained. DNA staining with DAPI is shown in white f-actin filaments !phalloidin-T"IT#$ in orange. %cale bars represent &'' (m !overviews$ or &' (m !details$. A)*+ Arrest and death of early blastomeres during the first four cleavage cycles. A)D+ ,aximum intensity --pro.ections of optical serial sections of embryos examined at day / !A 0$ or day 1 !# D$. ,any embryos show large early blastomeres that are arrested at interphase !asteris2s$ or prophase !arrow heads$ or already show clear signs of cell death+ DAPI staining reveals variably si-ed and irregularly shaped clumps of highly condensed chromatin !large arrows$. Notably fre3uent findings are remnants of mitotic chromosome structures !small arrows$. 4 *+ Two day 5 embryos that initially survived the death of large early blastomeres. "emnants of early blastomere death can be seen until blastocyst hatching. Panel 4 shows an embryo in the form of a single maximum intensity --pro.ection of the entire confocal image stac2 !left$ and as a se3uence of six pro.ections of 6' (m image sub-stac2s !right$.

Panel * presents a --pro.ection !overlay image and separate channel images$ through an entire embryo. 7)8+ #ell death in the inner cell mass of bovine I9* blastocysts examined at day 5+ In expanding and hatching blastocysts DAPI staining reveals highly condensed chromatin structures that are irregularly shaped and variable in si-e referring to different modes and stages of cell death !arrows$. 7):+ *our expanding blastocysts. 7 is a maximum intensity --pro.ection of a 6' (m image stac2 ; I : are single optical sections. Dying<dead cells are also shown at higher magnification. = =>+ ;atched blastocyst. Panel = presents a maximum intensity --pro.ection of a stac2 spanning the entire embryo !overlay image and DAPI alone$. =>+ %ingle optical section of the same blastocyst and enlarged view of the inner cell mass+ Note a dying<dead cell !arrow head$ in the interior of the inner cell mass as well as the extrusion of a dead cell into the blastocoel

Bovine Morula

A Arrest and death of early blastomeres during the first four cleavage cycles. ,aximum intensity --pro.ections of optical serial sections of embryos examined at day /. ,any embryos show large early blastomeres that are arrested at interphase !asteris2s$ or prophase !arrow heads$ or already show clear signs of cell death+ DAPI staining reveals variably si-ed and irregularly shaped clumps of highly condensed chromatin !large arrows$. Notably fre3uent findings are remnants of mitotic chromosome structures !small arrows$. The embryos were fixed and mounted on coverslips in such a way that the three-dimensional structure was maintained. DNA staining with DAPI is shown in white f-actin filaments !phalloidin-T"IT#$ in orange. %cale bars represent &'' (m !overviews$ or &' (m !details$.

Bovine Blastocyst
*+ Day 5 embryos that initially survived the death of large early blastomeres. "emnants of early blastomere death can be seen until blastocyst hatching. Panel * presents a --pro.ection !overlay image and separate channel images$ through an entire embryo. The embryos were fixed and mounted on coverslips in such a way that the three-dimensional structure was maintained. DNA staining with DAPI is shown in white f-actin filaments !phalloidin-T"IT#$ in orange. %cale bars represent &'' (m !overviews$ or &' (m !details$.

Bovine Blastocyst
7+ #ell death in the inner cell mass of bovine I9* blastocysts examined at day 5+ In expanding and hatching blastocysts DAPI staining reveals highly condensed chromatin structures that are irregularly shaped and variable in si-e referring to different modes and stages of cell death !arrows$. Image is a maximum intensity --pro.ection of a 6' (m image stac2 The embryos were fixed and mounted on coverslips in such a way that the three-dimensional structure was maintained. DNA staining with DAPI is shown in white f-actin filaments !phalloidin-T"IT#$ in orange. %cale bars represent &'' (m !overviews$ or &' (m !details$.

Bovine Development

The table below shows the general timing of early development stages in the bovine embryo as well as comparing this to other domestic species. Implantation in the uterus occurs between ?'-?1 days. 1 cell 8 cell Blastocyst Enter Uterus Length of Gestation !hours$ !days$ !days$ !days$ !days$ #attle 6/ ? @ ?.1 6@& ;orse 6/ ? A 1 ??5 %heep 6/ 6.1 5 ? &/@ %wine &/-&A 6 A 6 &&/ Species

Genital Development
The male bovine !bull$ first development of the testis at the genital ridge is triggered by %"B expression following the timeline shown below.C/D

Day 32 - (CRL 12) Genital ridges first appeared Day 37 - (CRL 18) SRY expression begins Day 39 - (CRL 20) SRY expression peaks Day 42 - (CRL 27) estis !ords disting"is#able