Effects of In Vitro Production on Horse Embryo Morphology, Cytoskeletal Characteristics, and Blastocyst Capsule Formation

Abstract

Blastocyst formation rates during horse embryo in vitro production (IVP) are disappointing, and embryos that blastulate in culture fail to produce the characteristic and vital glycoprotein capsule. The aim of this study was to evaluate the impact of IVP on horse embryo development and capsule formation. IVP embryos were produced by intracytoplasmic sperm injection of in vitro matured oocytes and either culture in synthetic oviduct fluid (SOF) or temporary transfer to the oviduct of a ewe. Control embryos were flushed from the uterus of mares 6-9 days after ovulation. Embryo morphology was evaluated with light microscopy, and multiphoton scanning confocal microscopy was used to examine the distribution of microfilaments (AlexaFluor-Phalloidin stained) and the rate of apoptosis (cells with fragmented or terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling-positive nuclei). To examine the influence of culture on capsule formation, conceptuses were stained with a monoclonal antibody specific for capsular glycoproteins (OC-1). The blastocyst rate was higher for zygotes transferred to a sheep's oviduct (16%) than for those cultured in SOF (6.3%). Day 7 IVP embryos were small and compact with relatively few cells, little or no blastocoele, and an indistinct inner cell mass. IVP embryos had high percentages of apoptotic cells (10% versus 0.3% for in vivo embryos) and irregularly distributed microfilaments. Although they secreted capsular glycoproteins, the latter did not form a normal capsule but instead permeated into the zona pellucida or remained in patches on the trophectodermal surface. These results demonstrate that the initial layer of capsule is composed of OC-1-reactive glycoproteins and that embryo development ex vivo is retarded and aberrant, with capsule formation failing as a result of failed glycoprotein aggregation.

Maternal age and in vitro culture affect mitochondrial number and function in equine oocytes and embryos

“…Future studies examining ROS-scavenging activity during oocyte maturation should therefore encompass both the oocyte and its cumulus investment. The trend to reduced GPX3 gene expression in IVP equine embryos may have similar implications for the prevention of ROS accumulation, and could therefore contribute to the elevated levels of apoptosis and DNA damage seen in equine IVP compared with in vivo embryos (Tremoleda et al 2003;Pomar et al 2005).…”

“…Embryos were washed 10 times with LRS to remove maternal cells and residual FCS before assessment under a dissecting microscope (SZ60; Olympus, Zoeterwoude, The Netherlands). Embryos were classified according to developmental stage (morula, early blastocyst or expanded blastocyst) and quality (1-4, good to degenerate), as described previously (Tremoleda et al 2003), and their diameter was measured using a calibrated eyepiece micrometer. The embryos were then transferred with 10 mL LRS into 0.5-mL Eppendorf tubes, snap frozen in liquid nitrogen and stored at À808C until DNA and RNA extraction.…”

Obtaining farm animal embryos <i>in vitro</i>

“…The capsule is a structure located between trophoblast and zona pellucida, which is generated by the embryo. Its role is to protect the embryo and it is found in embryos obtained both in vitro and in vivo (Tremoleda et al, 2003).…”

Foreword: A perspective on the role of emerging technologies for the propagation of companion animals, non-domestic and endangered species

“…the reproductive tract in elephants; Brown 2000), or embryo physiology (e.g. failed formation of vital glycoprotein-rich membranes in equine embryos in vitro (Tremoleda et al 2003) or formation of shells around marsupialia early embryos (Pukazhenthi and Wildt 2004)). However, the lack of basic reproductive knowledge remains the primary dilemma for each new target species.…”