Our aim was to optimize the cryoprotectant treatment for the preservation of immature porcine cumulus-oocyte
complexes (COCs) by solid surface vitrification. In each experiment, the vitrification solution consisted of
50 mg/ml polyvinyl pyrrolidone, 0.3 M of the actual sugar and in total 35% (v/v) of the actual permeating
cryoprotectant (pCPA) combination. After warming, the COCs were subjected to in vitro
maturation, fertilization and embryo culture. In Experiment 1, trehalose and sucrose were equally effective
during vitrification and warming in terms of facilitating oocyte survival and subsequent embryo development.
In Experiment 2, when equilibration was performed at 38.5 C in a total of 4% (v/v) pCPA for 15 min, the
combination of ethylene glycol and propylene glycol (EG + PG = 1:1) was superior to EG and dimethyl sulfoxide
(EG + DMSO = 1:1) in terms of oocyte survival after vitrification and the quality of resultant blastocysts. In
Experiment 3, equilibration in 4% (v/v) pCPA for 15 min before vitrification was superior to that in 15% (v/v)
CPA for 5 min for achievement of high survival rates irrespective of the pCPA combination used. In Experiment
4, when equilibration was performed in 4% EG + PG for 5 min, 15 min or 25 min, there was no difference in
oocyte survival and subsequent embryo development after vitrification and warming; however, the developmental
competence of cleaved embryos was tendentiously reduced when equilibration was performed for 25 min. In
conclusion, trehalose and sucrose were equally effective in facilitating vitrification, and the optimum pCPA
treatment was 5–15 min equilibration in 4% (v/v) of EG + PG followed by vitrification in 35% (v/v) EG +
PG.
Testicular xenografting, combined with cryopreservation can assist conservation of the genetic diversity of indigenous pigs by salvaging germ cells from their neonatal testes. Using Meishan male piglets as an example, we examined whether testicular tissue would acquire the ability to produce sperm after cryopreservation and grafting into nude mice (MS group). For comparison, testicular tissue from neonatal Western crossbreed male piglets was used (WC group). Sixty days after xenografting (day 0 = grafting), MS grafts had already developed seminiferous tubules containing sperm, whereas in the WC grafts, sperm first appeared on day 120. The proportion of tubules containing spermatids and sperm was higher in the MS group than in the WC group between days 90 and 120. Moreover, in vitro‐matured porcine oocytes injected with a single sperm obtained from the MS group on day 180 developed to the blastocyst stage. The blastocyst formation rate after injection of the xenogeneic sperm was 14.6%, whereas the ratio in the absence of such injection (attributable to parthenogenesis) was 6.7%. Thus, cryopreserved Meishan testicular tissue acquired spermatogenic activity in host mice 60 days earlier than Western crossbreed tissue. Such xenogeneic sperm are likely capable of generating blastocysts in vitro.
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