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Cryopreservation of immature testicular tissues is essential for increasing the possibilities of offspring generation by testicular xenografting for agricultural or medical purposes. However, successful production of offspring from the sperm involved has never been reported previously. In the present study, therefore, using intracytoplasmic sperm injection (ICSI), we examined whether xenogeneic sperm obtained from immature pig testicular tissue after cryopreservation would have the capacity to produce live piglets. Testicular fragments from 9- to 11-day-old piglets were vitrified after 10- or 20-min immersion in vitrification solution containing ethylene glycol (EG), polyvinyl pyrrolidone (PVP) and trehalose as cryoprotectants, and then stored in liquid nitrogen for more than 140 days. Thirty nude mice were assigned to each immersion-time group. Testicular fragments were transplanted under the back skin of castrated mice immediately after warming and removal of the cryoprotectants. Blood and testicular grafts were then recovered from the recipient mice on days 60, 120, 180 and 230-350 (day 0 = grafting). Histological assessment of the testicular grafts and analyses of inhibin and testosterone production revealed no significant differences between the two immersion-time groups, indicating equal growth activity of the cryopreserved tissues. A single sperm obtained from a mouse in each group on day 230-350 was injected into an in vitro-matured porcine oocyte, and then the ICSI oocytes were transferred to the oviducts of estrus-synchronized recipient gilts. One out of 4 gilts that had received oocytes fertilized using sperm from the 10-min immersion group delivered 2 live piglets, and one of another 4 gilts from the 20-min group delivered 4 live piglets. Thus, we have successfully generated porcine offspring utilizing sperm from immature testicular tissues after cryopreservation and transplantation into nude mice. The present model using pigs will be applicable to many large animals, since pigs are phylogenetically distant from the murine recipients.

The present study investigated the effects of vitrification of porcine oocytes either at the immature Germinal Vesicle (GV) stage before in vitro maturation (GV-stage oocytes) or at the pronuclear stage after in vitro maturation and fertilization (zygotes) on DNA integrity in relevance with their subsequent embryo development. Vitrification at the GV stage but not at the pronuclear stage significantly increased the abundance of double-strand breaks (DSBs) in the DNA measured by the relative fluorescence after γH2AX immunostaining. Treatment of GV-stage oocytes with cryoprotectant agents alone had no effect on DSB levels. When oocytes were vitrified at the GV stage and subjected to in vitro maturation and fertilization (Day 0) and embryo culture, significantly increased DSB levels were detected in subsequent cleavage-stage embryos which were associated with low cell numbers on Day 2, the upregulation of the RAD51 gene at the 4-8 cell stage (measured by RT-qPCR) and reduced developmental ability to the blastocyst stage when compared with the non-vitrified control. However, total cell numbers and percentages of apoptotic cells (measured by TUNEL) in resultant blastocysts were not different from those of the non-vitrified control. On the other hand, vitrification of zygotes had no effect on DSB levels and the expression of DNA-repair genes in resultant embryos, and their development did not differ from that of the non-vitrified control. These results indicate that during vitrification GV-stage oocytes are more susceptible to DNA damages than zygotes, which affects their subsequent development to the blastocyst stage.

We report the successful piglet production from cryopreserved oocytes for the first time by using a simple, high capacity vitrification protocol for preservation and a defined system for in vitro embryo production. Immature cumulus-oocyte complexes (COCs) from prepubertal gilts were vitrified in microdrops and stored in liquid nitrogen. After warming, COCs were subjected to in vitro maturation (IVM), fertilization (IVF), and subsequent culture (IVC). Adjusting warmplate temperature to 42 °C during warming prevented temperature drops in a medium below 34.0 °C and significantly increased the percentage of oocyte survival and thus blastocyst yields obtained from total vitrified oocytes compared with that of warming at 38 °C (87.1% vs 66.9% and 4.4% vs 2.7%, respectively). Nuclear maturation and fertilization of oocytes were not affected by vitrification and warming temperature. Blastocyst development on day 7 (day 0 = IVF) of the surviving oocytes after warming at 38 °C and 42 °C was not different but lower (P

Conventionally, in vitro-fertilized (IVF) bovine embryos are morphologically evaluated at the time of embryo transfer to select those that are likely to establish a pregnancy. This method is, however, subjective and results in unreliable selection. Here we describe a novel selection system for IVF bovine blastocysts for transfer that traces the development of individual embryos with time-lapse cinematography in our developed microwell culture dish and analyzes embryonic metabolism. The system can noninvasively identify prognostic factors that reflect not only blastocyst qualities detected with histological, cytogenetic, and molecular analysis but also viability after transfer. By assessing a combination of identified prognostic factors--(i) timing of the first cleavage; (ii) number of blastomeres at the end of the first cleavage; (iii) presence or absence of multiple fragments at the end of the first cleavage; (iv) number of blastomeres at the onset of lag-phase, which results in temporary developmental arrest during the fourth or fifth cell cycle; and (v) oxygen consumption at the blastocyst stage--pregnancy success could be accurately predicted (78.9%). The conventional method or individual prognostic factors could not accurately predict pregnancy. No newborn calves showed neonatal overgrowth or death. Our results demonstrate that these five predictors and our system could provide objective and reliable selection of healthy IVF bovine embryos.