Differential staining combined with TUNEL labelling to detect apoptosis in preimplantation bovine embryos.
Development of accurate laboratory methods to assess embryo quality will improve the efficiency of embryo production from in-vitro culture systems. Currently, the techniques of TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end (TUNEL) labelling for the detection of apoptosis, and differential staining for determining the ratio of inner cell mass (ICM) to trophectoderm (TE) cells, are used separately to assess embryo quality in a range of different species. This paper reports a unique, simple and fast method for the assessment of embryo quality using differential staining of TE and ICM, but combined with TUNEL labelling (DST staining). This technique was used to investigate the effect of serum supplementation on total cell number, ICM:TE ratio and apoptosis index after in-vitro production of bovine embryos. Serum supplementation increased total cell number (P < 0.01), but reduced the ratio of ICM:TE cells. No differences were observed in the number of apoptotic nuclei between treatments, or in the localization of the apoptotic nuclei. However, more apoptotic nuclei were observed in ICM than TE cells in both culture groups. In conclusion, using DST, it has been possible to carry out both a qualitative and quantitative analysis of embryos produced using the two different methods. DST provides a means of assessing the effect of culture conditions on cell number of both embryo compartments (ICM and TE), as well as providing information on the localization of apoptotic nuclei within the blastocyst.
Fouladi-Nashta
A A
AA
School of Biosciences, The University of Nottingham, LE12 5RD, UK. ali.fouladi-nashta@nottingham.ac.uk
Alberio
R
R
Kafi
M
M
Nicholas
B
B
Campbell
K Hs
KH
Webb
R
R
D000818:Animals; D017209:Apoptosis; D001755:Blastocyst / Q000166:cytology* / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D002417:Cattle / Q000196:embryology; D002452:Cell Count; D002467:Cell Nucleus / Q000648:ultrastructure; D046149:Embryo Culture Techniques; D005260:Female; D005312:Fetal Blood; D020287:In Situ Nick-End Labeling; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D013194:Staining and Labeling
D012710:Serum Albumin, Bovine