Ultrastructural Characterization of Fresh and VitrifiedIn Vitro-andIn Vivo-Produced Sheep Embryos

Abstract

The lower results in cryopreservation of in vitro-produced (IVP) sheep embryos, when compared to the in vivo derived, limits its use. Four groups of blastocyst (BL) were evaluated: fresh IVP (n = 3), fresh in vivo derived (n = 3), warmed IVP cryopreserved in open pulled straws (OPS, n = 3) and warmed in vivo derived cryopreserved in OPS (n = 3). Ultrastructural observation of processed fresh embryos showed a reduced number of microvilli and mitochondria in the IVP ones, as well as a lower number of mature mitochondria, that can be associated with deficient metabolism in IVP embryos, possibly involved in the lower resistance to cryopreservation. Both in vivo-derived and IVP embryos had a large number of vesicles, with light and dense content. In embryos vitrified by OPS, major changes were observed mainly in IVP embryos with small changes in grade 2 (fair) and high changes in grade 3 (bad) semithin scoring. The main changes associated with cryopreservation included disruption of cellular membranes and poor intracellular preservation, with loss of microvilli and the presence of cellular debris. In conclusion, ultrastructural evaluation of IVP blastocysts cryopreserved in OPS was herein described for the first time, reporting more severe cellular damage in these embryos when compared to those produced in vivo. This is probably associated with a lower cryotolerance that can be related to their lipid content and metabolism.

Cryopreservation of Sheep Produced Embryos – Current and Future Perspectives

“…Several reasons have been indicated to justify this fact, mainly cellular [21,22], metabolic [141,142], and biochemical [143] changes. In an ultrastructural evaluation, IVP blastocysts exhibited less microvilli and a less extensive network of intercellular junctions, specifically an apparent lack of desmosomal junctions, a higher number of lipid droplets, and incidence of cellular debris compared to their in vivo counterparts [18]. After cryopreservation, both in vivo and IVP embryos exhibited ultrastructural damages that were much more severe in the latter [17,18].…”

“…Moreover, these problems could be extended after birth, as higher growing rates were found until weaning age in lambs born from IVP [107]. It is known that IVP embryos have altered morphology compared with in vivo derived [18,108]. On the contrary, an increase in the lipid content of IVP was observed compared to in vivo bovine morulae and blastocysts regardless of the composition of the medium of culture, although differences also depend on the culture medium used [22].…”

“…IVM is usually performed at 38-39°C in a humidified atmosphere in air and 5% CO 2 [8,18,50,112]. Tissue Culture Medium (TCM199) supplemented with bicarbonate, pyruvate, LH, FSH, and estradiol (E 2 ) and with 10% fetal calf serum [10,101] or 10% estrous sheep serum [50,122] is the widest used medium for IVM.…”

“…In the last 15 years, researchers have tried to understand the reason for the low cryotolerance of IVP embryos and keep on searching for new methods to enhance the results in this species. Both in vivo and IVP sheep cryopreserved embryos present ultrastructural damage [17,18] corresponding to the expression of damage to cells during the process. Cryopreserved embryos show less microvilli and lower mitochondria activity compared to fresh ones [17][18][19].…”

“…Both in vivo and IVP sheep cryopreserved embryos present ultrastructural damage [17,18] corresponding to the expression of damage to cells during the process. Cryopreserved embryos show less microvilli and lower mitochondria activity compared to fresh ones [17][18][19]. The lower cryotolerance of IVP blastocysts can be also related to the excessive accumulation of lipids [13,[20][21][22][23], which can be favored by their culture in serum-containing media [22,24,25].…”