The latter was induced by incubating 5x106 sperm cells with 20 [micro]L of perivitelline
layer (PVL) and kept for 40 min at 37 [degrees]C (Ahammad et al., 2011b).
The oocyte maturation was classified as metaphase 2, intermediate, and germinal vesicles in terms of cumulus/corona morphology, cytoplasmic clarity, zona thickness, and extent of perivitelline
The outermost membrane, the chorion, overlays the viscous fluid-filled perivitelline
space, the vitelline membrane, and the blastoderm (or embryo) and yolk (Jones et al., 1978; Rawson et al., 2000).
In particular, the AG synthesizes and secretes perivitelline
fluid (PVF), which provides the embryos the nutrients and defenses for development (Heras et al., 1998: Dreon et al., 2013, 2014), while the CG stores large amounts of calcium that later form the eggshell (Runham, 1988).
Zygote to cleavage phase began from 0-1 h to 4 h after post fertilization and one day after post fertilization, it characterized with cytoplasmic growth, formation of blastodisc at the end of animal pole, presence of different perivitelline
gape and cleavage phase categorized with a sequence of meiotic partitions that resulted in several blastomeres.
On the other hand, if a polar body is present in the perivitelline
space, the oocytes are classified as MII.
The presence of two pronuclei in the absence of sperm head within the oocyte or perivitelline
space was an indication of the presence of both female and male pronuclei.
Three EFS cases (case 1, 5 and 6) did not have embryos for transfer as a result of poor quality embryos due to poor quality oocytes [cytoplasmic (dense granular cytoplasm and aggregation of smooth endoplasmic reticulum) and extra-cytoplasmic abnormalities (large perivitelline
space with debris and fragmented polar bodies)].
Consisely, oocytes enucleation was performed under UV light, and a single fibroblast cell was injected into the perivitelline
space of an enucleated oocyte in [M.sub.2] (Sigma, USA) medium supplemented with 10%FCS(HyClone, Beijing, China), 2 [micro]g/mL Hoechst 33342 (Sigma, USA) and 7.5 [micro]g/mL cytochalasin B (Sigma, USA).
In teleosts, the cortical alveoli content is released into the perivitelline
space, at the moment of fertilization, blocking polyspermy and contributing to chorion hardening (Hart, 1990).
The trehalose content of perivitelline
fluid influences hatching and is important for the survival of dormant, unhatched juveniles (Perry, 1989).
A fibroblast was placed in the perivitelline
space in close contact with the oocyte membrane to form a couplet.