The effects of cumulus cells on the fertility and developmental ability of vitrified C57BL/6J mouse oocytes after IVF.

<p>IVF was carried out using frozen-thawed BDF1 mouse sperm. Data are means±S.E.M. ^a-b,c-d,e-f Different superscripts denote significant difference (<i>P</i><0.05). In each treatment group, more than 100 oocytes were examined.</p

The effects of cumulus cells on the fertility and developmental ability of C57BL/6J mouse oocytes after IVF.

<p>Data are means±S.E.M. ^a-b,c-d,e-f Different superscripts denote a significant difference (<i>P</i><0.05). In each treatment group, more than 100 oocytes were examined. PN, pronuclear stage; COC, cumulus oocyte complexes; DO, denuded oocytes.</p

In vivo development of vitrified COCs from C57BL/6J mouse after in vitro fertilization.

<p>In vivo development of vitrified COCs from C57BL/6J mouse after in vitro fertilization.</p

Generation of offspring after ICSI using sperm from cryopreserved porcine testicular xenografts.

<p>Two male and 3 female piglets were born after transfer of fertilized oocytes produced by ICSI using sperm recovered from a mouse on day 254 in the 20-min group. Tissue was stored in LN₂ for 188 days. Photographed on the fifth day after birth.</p

<i>In vitro</i> embryo development after IVM and IVF of control and vitrified oocytes.

<p>Five replications were performed. Data are presented as means ± SEM.</p>a,b<p>Percentages with different letters differ significantly (<i>P</i><0.05) in the same column by one-way ANOVA followed by Tukey’s multiple comparison test.</p><p>IVM = in vitro maturation, IVF = in vitro fertilization, Day 0 = the day of IVF.</p>§<p>Live oocytes at 44 h IVM.</p

Generation of Live Piglets from Cryopreserved Oocytes for the First Time Using a Defined System for <i>In Vitro</i> Embryo Production

<div><p>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 <i>in vitro</i> 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 <i>in vitro</i> 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 (<i>P</i><0.05) than those of non-vitrified control oocytes (4.6%, 5.2% and 17.9%, respectively). However, blastocyst cell numbers in the control and vitrified groups were similar irrespective of warming temperature. Omitting porcine follicular fluid (pFF) from IVM medium (POM) did not affect maturation, fertilization and embryo development of vitrified-warmed oocytes. Transfer of blastocysts obtained on day 5 from vitrified oocytes matured either with or without pFF into 4 recipients (2 for each group) resulted in 4 pregnancies and the delivery of a total of 18 piglets. In conclusion, optimization of warming temperature was a key factor for achieving high survival rates, and surviving oocytes could be utilized <i>in vitro</i> using defined media. Using these modifications, live piglets could be obtained from cryopreserved oocytes for the first time.</p></div

Weights of cryopreserved porcine testicular tissue in mice.

<p>All visible grafts were recovered from the recipient mice, and values are means of total weights per mouse (± SEM) for 4 to 7 mice in each group.</p

The first piglets obtained by the transfer of IVP blastocysts obtained from vitrified oocytes.

<p>The photograph was taken 5 days after delivery.</p

The effect of COOH-PLL concentration on the number of cells in a blastocyst.

<p>The effect of COOH-PLL concentration on the number of cells in a blastocyst.</p

Changes of medium temperature during consecutive insertion of 6 vitrified microdrops (denoted with arrows) in a common 35 mm petri dish containing 2.5 ml of warming medium.

<p>A) a typical pattern of temperature changes on a 38°C warmplate; B) a typical pattern of temperature changes on a 42°C warmplate; C) mean ± SEM values of maximum and minimum temperatures recorded on 38°C and 42°C warmplates. Values with different superscripts (a, b) between treatment groups differ significantly (<i>P</i><0.05) by student’s t-test.</p