原 弘真

We conducted two lines of genome-editing experiments of mouse hematopoietic stem cells (HSCs) with the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9). First, to evaluate the genome-editing efficiency in mouse bona fide HSCs, we knocked out integrin alpha 2b (Itga2b) with Cas9 ribonucleoprotein (Cas9/RNP) and performed serial transplantation in mice. The knockout efficiency was estimated at approximately 15%. Second, giving an example of X-linked severe combined immunodeficiency (X-SCID) as a target genetic disease, we showed a proof-of-concept of universal gene correction, allowing rescue of most of X-SCID mutations, in a completely non-viral setting. We inserted partial cDNA of interleukin-2 receptor gamma chain (Il2rg) into intron 1 of Il2rg via non-homologous end-joining (NHEJ) with Cas9/RNP and a homology-independent targeted integration (HITI)-based construct. Repaired HSCs reconstituted T lymphocytes and thymuses in SCID mice. Our results show that a non-viral genome-editing of HSCs with CRISPR/Cas9 will help cure genetic diseases.

A knock-in can generate fluorescent or Cre-reporter under the control of an endogenous promoter. It also generates knock-out or tagged-protein with fluorescent protein and short tags for tracking and purification. Recent advances in genome editing with clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) significantly increased the efficiencies of making knock-in cells. Here we describe the detailed protocols of generating knock-in mouse and human pluripotent stem cells (PSCs) by electroporation and lipofection, respectively.

We report that a sheep fetal liver provides a microenvironment for generating hematopoietic cells with long-term engrafting capacity and multilineage differentiation potential from human induced pluripotent stem cell (iPSC)-derived hemogenic endothelial cells (HEs). Despite the promise of iPSCs for making any cell types, generating hematopoietic stem and progenitor cells (HSPCs) is still a challenge. We hypothesized that the hematopoietic microenvironment, which exists in fetal liver but is lacking in vitro, turns iPSC-HEs into HSPCs. To test this, we transplanted CD45-negative iPSC-HEs into fetal sheep liver, in which HSPCs first grow. Within 2 months, the transplanted cells became CD45 positive and differentiated into multilineage blood cells in the fetal liver. Then, CD45-positive cells translocated to the bone marrow and were maintained there for 3 years with the capability of multilineage differentiation, indicating that hematopoietic cells with long-term engraftment potential were generated. Moreover, human hematopoietic cells were temporally enriched by xenogeneic donor-lymphocyte infusion into the sheep. This study could serve as a foundation to generate HSPCs from iPSCs.

Pigs with X-linked severe combined immunodeficiency (X-SCID) caused by a mutation of the interleukin-2 receptor gamma chain gene (IL2RG) are of value for a wide range of studies. However, they do not survive longer than 8 weeks because of their susceptibility to infections. To allow longer survival of X-SCID pigs, the animals must be born and reared under germ-free conditions. Here, we established an efficient system for piglet derivation by hysterectomy and used it to obtain and maintain a germ-free X-SCID pig. In four trials using pregnant wild-type pigs, 66% of piglets after hysterectomy started spontaneous breathing (range of 20–100% per litter). The resuscitation rate was found to negatively correlate with elapsed time from the uterus excision to piglet derivation (r=−0.97, P&lt 0.05). Therefore, it is critical to deliver piglets within 5 min to achieve a high resuscitation rate (82% estimated from regression analysis). In a fifth trial with an IL2RG+/− pig, four piglets were delivered within 4.2 min of uterus excision and three were alive (75%). One of the live born piglets was genotypically and phenotypically determined to be X-SCID and was reared for 12 weeks. The X-SCID piglet was free from both bacteria and fungi at all time points tested by microbial culture and grew without any abnormal signs or symptoms. This study showed successful production and rearing of germ-free pigs, enabling experiments involving long-term follow-up of X-SCID pigs.

The present study was conducted to establish haploid embryonic stem (ES) cell lines using fluorescent marker carrying rats. In the first series, 7 ES cell lines were established from 26 androgenetic haploid blastocysts. However, only 1 ES cell line (ahES-2) was found to contain haploid cells (1n = 20 + X) by fluorescence-activated cell sorting (FAGS) and karyotypic analyses. No chimeras were detected among the 10 fetuses and 41 offspring derived from blastocyst injection with the FACS-purified haploid cells. In the second series, 2 ES cell lines containing haploid cells (13% in phES-1 and 1% in phES-2) were established from 2 parthenogenetic haploid blastocysts. Only the phES-2 cell population was purified by repeated FAGS to obtain 33% haploid cells. Following blastocyst injection with the FAGS-purified haploid cells, no chimera was observed among the 11 fetuses; however, 1 chimeric male was found among the 47 offspring. Although haploid rat ES cell lines can be established from both blastocyst sources, FAGS purification may be necessary for maintenance and chimera production.

Islet transplantation is an established therapy for diabetes. We have previously shown that rat pancreata can be created from rat pluripotent stem cells (PSCs) in mice through interspecies blastocyst complementation. Although they were functional and composed of rat-derived cells, the resulting pancreata were of mouse size, rendering them insufficient for isolating the numbers of islets required to treat diabetes in a rat model. Here, by performing the reverse experiment, injecting mouse PSCs into Pdx-1-deficient rat blastocysts, we generated rat-sized pancreata composed of mouse-PSC-derived cells. Islets subsequently prepared from these mouse-rat chimaeric pancreata were transplanted into mice with streptozotocin-induced diabetes. The transplanted islets successfully normalized and maintained host blood glucose levels for over 370 days in the absence of immunosuppression (excluding the first 5 days after transplant). These data provide proof-of-principle evidence for the therapeutic potential of PSC-derived islets generated by blastocyst complementation in a xenogeneic host.

The Foxn1 gene is known as a critical factor for the differentiation of thymic and skin epithelial cells. This study was designed to examine the phenotype of Foxn1-modified rats generated by the CRISPR/Cas9 system. Guide-RNA designed for first exon of the Foxn1 and mRNA of Cas9 were co-injected into the pronucleus of Crlj:WI zygotes. Transfer of 158 injected zygotes resulted in the birth of 50 offspring (32 %), and PCR identified five (10 %) as Foxn1-edited. Genomic sequencing revealed the deletion of 44 or 60 bp from and/or insertion of 4 bp into the Foxn1 gene in a single allele. The number of T-cells in the peripheral blood lymphocytes of mutant rats decreased markedly. While homozygous deleted mutant rats had no thymus, the mutant rats were not completely hairless and showed normal performance in delivery and nursing. Splicing variants of the indel-mutation in the Foxn1 gene may cause hypomorphic allele, resulting in the phenotype of thymus deficiency and incomplete hairless. In conclusion, the mutant rats in Foxn1 gene edited by the CRISPR/Cas9 system showed the phenotype of thymus deficiency and incomplete hairless which was characterized by splicing variants.

Pronucleus-like vesicle formation following premature chromosome condensation (PCC) of the donor cell nucleus is the key event for successful generation of cloned rodents by nuclear transplantation (NT). However in rat cloning, this change is difficult to induce in enucleated recipient oocytes because of their inability to maintain maturation-promoting factor levels. In this study, intact oocytes retrieved from nuclear-visualized H2B-tdTomato knock-in rats were injected with Venus-labeled cell nuclei. Because the incidence of PCC under MG-132 treatment significantly increased with the culture period (0%, 10.8%, 36.8%, and 87.5% at 0, 0.5, 1, and 2 h postinjection, respectively), the metaphase plate of the oocyte was removed 1-2 h after the nuclear injection. The NT-derived rat zygotes (n = 748) were activated with ionomycin/cycloheximide and transferred into temporal host mothers, resulting in the harvest of three blastocysts (0.4%) with Venus fluorescence. Two blastocysts were examined for their potential to commit to NT-derived embryonic stem cells (ntESCs). One ntESC line was established successfully and found to be competent in terms of karyotype, stem cell marker expression, and pluripotency. In conclusion, time-lagged enucleation of visualized oocyte nuclei allows the PCC incidence of donor nuclei and generation of NT blastocysts, and the blastocysts can commit to germline-competent ntESCs.

We have recently reported that multiple aster formation after in vitro fertilization (IVF) was one of the factors that negatively affected the developmental competence of vitrified-warmed bovine matured oocytes, and that short-term culture of the post-warm oocytes with an inhibitor of Rho-associated coiled-coil kinase (ROCK) suppressed the multiple aster formation and improved the blastocyst yield. The present study was conducted to investigate whether increased multiple aster formation following IVF was involved in impaired developmental competence of stored ovary-derived bovine oocytes. Oocytes retrieved from 1-day stored ovaries had lower developmental potential to day 8 blastocysts when compared with those from fresh ovaries (37 versus 63%). Immunostaining of alpha-tubulin 10 h post-IVF revealed that a higher incidence of multiple aster formation occurred in oocytes retrieved from stored ovaries than from fresh ovaries (31 versus 15%). Treatment of post-in vitro maturated (post-IVM) oocytes with ROCK inhibitor for 2 h significantly suppressed the incidence of multiple aster formation (10 versus 32% in the control group). However, the suppression effect of ROCK inhibitor on multiple aster formation in IVM/IVF oocytes did not improve blastocyst yield from stored ovary-derived oocytes (41 versus 37% in the control group). These results suggested that the higher incidence of multiple aster formation by bovine ovary storage was not responsible for the decreased developmental competence of IVF oocytes.

Although vitrification is a useful technique for preservation of bovine oocytes, the yield of blastocysts derived from the vitrified oocytes is still low. We have recently reported a new type of cryoinjury, multiple aster formation, by which pronuclear migration and development of vitrified-warmed and in vitro-fertilized bovine oocytes are impaired. The aim of the present study was to investigate the effect of glutathione (GSH) content of vitrified bovine oocytes on multiple aster formation and subsequent in vitro development. Treatment of bovine cumulus-oocyte complexes with beta-mercaptoethanol (beta ME) and L-cysteine (Cys) during in vitro maturation resulted in 2.5-fold higher GSH content not only in fresh control but also in vitrified-warmed oocytes. The percentage of normally fertilized zygotes exhibiting sperm aster(s) was >95% in all four groups (with or without beta ME/Cys x fresh control or vitrified). The frequency of multiple aster formation in vitrified oocytes (three-fold higher than that in fresh control oocytes) was not affected by the increased level of intracellular GSH with beta ME/Cys. Consequently, the migration and development of pronuclei as well as the yield of blastocysts from vitrified-warmed oocytes (17 versus 41%) were not improved. In addition, there was no effect of increased GSH level on the yield of blastocysts in fresh control groups.

Under optimal freeze-drying conditions, solutions exhibit a cake-like porous structure. However, if the solution temperature is higher than the glass transition temperature of the maximally freeze-concentrated phase (T-g') during drying phase, the glassy matrix undergoes viscous flow, resulting in cake collapse. The purpose of the present study was to investigate the effect of cake collapse on the integrity of freeze-dried bull spermatozoa. In a preliminary experiment, factors affecting the T-g' of conventional EGTA buffer (consisting of Tris-HCl, EGTA and NaCl) were investigated in order to establish the main experimental protocol because EGTA buffer T-g' was too low (-45.0 degrees C) to suppress collapse. Modification of the EGTA buffer composition by complete removal of NaCl and addition of trehalose (mEGTA buffer) resulted in an increase of T-g' up to -27.7 degrees C. In the main experiment, blastocyst yields after ooplasmic injection of freeze-dried sperm preserved in collapsed cakes (drying temperature: 0 or -15 degrees C) were significantly lower than those of sperm preserved in non-collapsed cake (drying temperature: -30 degrees C). In conclusion, freeze-dried cake collapse may be undesirable for maintaining sperm functions to support embryonic development, and can be inhibited by controlling both T-g' of freeze-drying buffer and temperature during the drying phase. (C) 2014 Elsevier Inc. All rights reserved.

This study was designed to investigate whether supplementation of 2i medium with leukemia inhibitory factor (LIF) and/or forskolin would support establishment of germline-competent rat embryonic stem (ES) cell lines. Due to the higher likelihood of outgrowth rates, supplementation of forskolin with or without LIF contributed to the higher establishment efficiency of ES cell lines in the WDB strain. Germline transmission competency of the chimeric rats was not influenced by the profile of ES cell lines until their establishment. When the LIF/forskolin-supplemented 2i medium was used, the rat strain used as the blastocyst donor, such as the WI strain, was a possible factor negatively influencing the establishment efficiency of ES cell lines. Once ES cell lines were established, all lines were found to be germline-competent by a progeny test in chimeric rats. In conclusion, both LIF and forskolin are not essential but can play a beneficial role in the establishment of "genuine" rat ES cell lines. © 2014 by the Society for Reproduction and Development.

This study was undertaken to establish rat embryonic stem (ES) cells from parthenogenetically developing blastocysts. Ten blastocysts were prepared by treatment of ovulated rat oocytes with ionomycin and cycloheximide, and three alkaline phosphatase-positive ES cell lines were established using the N2B27 medium supplemented with mitogen activated protein kinase kinase inhibitor PD0325901, glycogen synthase kinase 3 inhibitor CHIR99021, rat leukemia inhibitory factor, and forskolin. Expression of stem cell marker genes (Oct-4, rNanog, Fgf-4, and Rex-1) was confirmed in all three ES cell lines by reverse transcriptase-polymerase chain reaction (RT-PCR). Combined bisulfite restriction analysis showed that the differentially methylated region locus of five imprinted genes (H19, Meg3IG, Igf2r, Peg5, and Peg10) in these ES cells remained to be demethylated or was hypomethylated, which was similar to that in control ES cells established from normal blastocysts. Characteristics of the parthenogenetic blastocyst-derived ES cells were successfully transmitted to the next generation through a chimeric rat for one of the three ES cell lines. This is the first report on germline-competent (genuine) ES cells derived from parthenogenetically developing rat blastocysts.

Cryotolerance of matured bovine oocytes is not fully practical even though a promising vitrification procedure with a ultrarapid cooling rate was applied. The present study was conducted to investigate whether recovery culture of vitrified-warmed bovine oocytes with an inhibitor (Y-27632) of Rhoassociated coiled-coil kinase (ROCK) can improve the developmental potential after in vitro fertilization (IVF) and in vitro culture. Immediately after warming, almost all oocytes appeared to be morphological normal. Treatment of the postwarming oocytes with 10 mu M Y-27632 for 2 h resulted in the significantly higher oocyte survival rate before IVF as well as higher cleavage rate and blastocyst formation rate. Quality analysis of the resultant blastocysts in terms of total cell number and apoptotic cell ratio also showed the positive effect of the Y-27632 treatment. Time-dependent change in mitochondrial activity of the vitrified-warmed oocytes was not influenced by ROCK inhibition during the period of recovery culture. However, the ability of ooplasm to support single-aster formation was improved by the ROCK inhibition. Thus, inhibition of ROCK activity in vitrified-warmed bovine oocytes during a short-term recovery culture can lead to higher developmental competence, probably due to decreased apoptosis and normalized function of the microtubule-organizing center.

We have proposed a new hypothesis for cryodamage of bovine oocytes; multiple aster formation frequently observed in vitrified-warmed and fertilized oocytes may be related to loss of ooplasmic function responsible for normal microtubule assembly from sperm-aster. The present study was conducted to investigate whether recovery culture of vitrified-warmed bovine oocytes with an inhibitor (Y-27632) of Rho-associated coiled-coil kinase (ROCK) can improve the developmental potential after IVF and IVC. Cumulus-free bovine IVM oocytes were subjected to Cryotop vitrification. Treatment of the post-warm oocytes with 10 μM Y-27632 for 2 h resulted in the significantly higher oocyte survival rate prior to the IVF (97.8 vs 90.2%), cleavage rate (72.4 vs 56.2%) and Day-8 blastocyst formation rate (21.4 vs 13.9%). Time-dependent change in mitochondrial activity of the vitrified-warmed oocytes was not influenced by the ROCK inhibition during the period of recovery culture. Also, kinetics of actin filaments until 10 hpi was not affected by the Y-27632 treatment. At the 10 hpi, proportions of oocytes formed aster(s) were comparable (80.7 and 81.8%), but the ability of ooplasm to support single-aster formation was improved by the ROCK inhibition (67.1 vs 49.4%). In conclusion, inhibition of ROCK activity in vitrified-warmed bovine oocytes during a short-term recovery culture can lead to the higher developmental competence, probably due to decreased apoptosis and normalized function of microtubule-organizing center.

【目的】精子の凍結保存に代わる保存方法として凍結乾燥が注目されているが，満足のいく発生成績が報告されている動物種は限られている。凍結乾燥を適切な条件下で行うと溶液は多孔質のスポンジ様構造 (凍結乾燥ケーキ) を形成する。しかし，溶液の最大凍結濃縮相ガラス転移温度 (Tg') よりも高い温度で乾燥を行うと，コラプスと呼ばれる凍結乾燥ケーキの構造崩壊が生じやすい。本研究では，凍結乾燥ケーキに生じたコラプスが凍結乾燥ウシ精子の発生能に及ぼす影響について調べた。【方法と結果】示差走査熱量計を用いて測定した，10 mM Tris-HCl，50 mM EGTA，0.5 M トレハロースからなる凍結乾燥バッファーのTg'は，−28℃だった。次に，同バッファーに懸濁した凍結融解ウシ精子を予備凍結したのち，−30℃または0℃に制御した凍結乾燥機中で6時間の乾燥を行った。乾燥温度が−30℃の凍結乾燥ケーキは完全な形状を保っていた一方，Tg'より高い0℃で乾燥した場合にはコラプスが生じていた。続いて，得られた凍結乾燥ケーキの乾燥程度を調べるために含水率およびガラス転移温度 (Tg) の測定を行った。0℃で乾燥した場合の含水率は−30℃の場合と比べて約5倍 (3.6 vs. 0.7%, P < 0.05) だったが，Tgの測定によりいずれも十分な乾燥状態にあることが確認できた。−20℃で一晩保存した凍結乾燥精子を顕微授精 (ICSI) に供したところ，卵割率に差は認められなかった。しかし，胚盤胞発生率については0℃で乾燥した場合の値 (1%：卵割ベース) が−30℃で乾燥したときの値 (14%) よりも有意 (P < 0.05) に低下した。現在さらに，凍結乾燥精子の微細構造やDNA損傷に対する影響を調べている。以上，凍結乾燥ケーキのコラプスはウシ精子に対する乾燥ストレスを増加させ，ICSI後の胚盤胞発生能を低下させることが明らかとなった。

In vitro-matured bovine oocytes do not tolerate vitrification as well as mature murine or human oocytes. Delayed first cleavage in vitrified and in vitro-fertilized bovine oocytes may be responsible for the decreased yield of blastocysts in vitro. Because formation of sperm-aster and the subsequent assembly of microtubule network play an important role for migration and fusion of both pronuclei, aster formation in vitrified-warmed oocytes was analyzed by confocal laser-scanning microscopy. At 10 h post-insemination (hpi), proportions of oocytes fertilized normally were comparable between the vitrified and fresh control groups (67 and 70%, respectively). Proportions of oocytes that exhibited microtubule assembly were similar between the two groups (95% each), but the proportion of oocytes with multiple asters was higher in the vitrified group when compared with the fresh control group (68 vs 29%, P &lt; 0.05). Both migration and development of two pronuclei were adversely affected by multiple aster formation. In the next experiment, multiple asters observed in 5.5 vs 8 hpi pronuclear zygotes were located near the male pronucleus, suggesting that those multiple asters were not the cytoplasmic asters of maternal origin. In conclusion, multiple aster formation frequently observed in vitrified-warmed bovine oocytes may be related to loss of ooplasmic function responsible for normal microtubule assembly from the sperm-aster. (C) 2012 Elsevier Inc. All rights reserved.

【目的】ガラス化・加温したウシ卵子では体外受精後に複数の星状体が出現する例が多発し，これが一因となって胚盤胞発生率が低下している可能性がある (Theriogenology 2012; 77: 908-15)。また，ウシ精子中心体のMTOC機能はグルタチオン合成阻害剤による卵子処理で損なわれるという報告がある。本研究ではウシ卵子の体外成熟行程において細胞内グルタチオンレベルの上昇が期待できる薬剤処理を行い，ガラス化・体外受精後の精子星状体形成ならびに胚盤胞発生との関係を調べた。【方法】屠殺の翌日まで約10℃で保存したウシ卵巣から卵丘卵母細胞複合体を回収し，&beta;-メルカプトエタノール (&beta;-ME) とL-システイン (L-Cys) を添加した成熟培地で22時間培養した。裸化後に成熟卵子率を求め，クライオトップをデバイスとしてガラス化保存した。加温卵子のグルタチオンレベルはDTNB-グルタチオン還元酵素リサイクリングアッセイにより，体外受精10時間目の星状体形成率と複数星状体の発生頻度は&alpha;チューブリンに対する免疫染色により，そして胚盤胞発生率は低酸素濃度下での体外培養により求めた。【結果】成熟卵子率に&beta;-ME / L-Cysの添加による影響はなかったが，無処理対照区よりも細胞内グルタチオンレベルは約3倍に上昇しており，このレベルはガラス化行程が加わっても下がることはなかった。精子星状体形成率は実験区に関わらず，いずれも95%以上だった。これらの星状体形成卵子のうち複数の精子星状体が観察された卵子の割合はガラス化区で新鮮対照区よりも3〜4倍多くなっており，&beta;-ME / L-Cys添加によって細胞内グルタチオンレベルが高まっている場合でも同様であった。胚盤胞発生率においても&beta;-ME / L-Cys添加の有無に関わらず，ガラス化区と新鮮対照区のあいだで2倍強の差が認められた。以上の結果から，ガラス化・加温卵子におけるグルタチオンレベルと胚盤胞発生率との因果関係の存在について否定的な見解に達した。

This study was designed to investigate whether freeze-dried (FD) bull spermatozoa maintained the function of the microtubule-organizing center (MTOC) after rehydration and intracytoplasmic sperm injection (ICSI). In a preliminary attempt, the cleavage and blastocyst formation rates in FD-ICSI zygotes (36 and 1%, respectively) were found to be considerably lower than those in control ICSI zygotes (67 and 21%, respectively) or in IVF zygotes (78 and 43%, respectively). An alkaline comet assay indicated that the DNA fragmentation index (length of comet tail x % DNA liberated) was not significantly different between fresh and FD spermatozoa. In the main experiment, formation of sperm-asters in the FD-ICSI oocytes 7 h postinsemination occurred at a similar rate when compared with the control ICSI oocytes (41 vs. 49%). Among the oocytes exhibiting sperm aster formation, the extent of microtubule network assembly was comparable between the FD-ICSI and control ICSI groups. However, the MTOC of the ICSI oocytes was not as functional as that of IVF oocytes in terms of the aster formation rate (97%) and the fluorescent intensity of the microtubule network (2.0 folds). These results suggest that the freeze-drying process per se had no adverse effect on maintaining the MTOC function in bull spermatozoa.

The objective was to investigate the effects of developmental stage (fully-expanded or expanding blastocysts) and/or age (harvested on Days 7 or 8) on post-vitrification in vitro survival of bovine blastocysts derived from intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF). Post-warming survival (re-expansion of blastocoele within 24 h) of ICSI-derived fully-expanded blastocysts (80%) was similar to that of their IVF-derived counterparts (88%). However, the ability of ICSI-derived expanding blastocysts to survive vitrification procedures (61%) was lower than that of IVF-derived blastocysts (85%; P &lt; 0.05), although the ICSI- and IVF-derived fresh blastocysts were of similar quality. The age of the blastocysts before vitrification did not affect cryotolerance for either ICSI-derived (73 and 59% for Days 7 and 8 embryos, respectively) or IVF-derived blastocysts (86% for both Days 7 and 8 embryos). At 24 h of post-warming culture, ICSI-derived blastocysts surviving vitrification contained a higher proportion of dead cells than their IVF-derived counterparts (5-13% vs. 2-4%; P &lt; 0.05), but these proportions were not different from those of fresh control embryos. There was an adverse effect of vitrification on the ability of blastocysts to hatch within 72 h of culture only in IVF-derived Day 8 blastocysts (41 and 70% in vitrified and fresh control groups, respectively). In conclusion, the proportion of blastocysts that survived vitrification procedures was similar for ICSI- and IVF-derived bovine blastocysts if the former were cultured to the fully-expanded stage prior to vitrification, with no significant difference between embryos harvested on Day 7 versus Day 8. (C) 2010 Elsevier Inc. All rights reserved.

Inhibition of Rho-associated kinase (ROCK) activity promoted recovery and growth of frozen-thawed human embryonic stem cells. The primary objective was to determine if a ROCK inhibitor (Y-27632) in post-thaw culture medium unproved revivability of vitrified IVP bovine blastocysts. Expanding or expanded blastocysts (7 d after IVF) were vitrified (minimum volume cooling procedure, using a Cryotop) in 15% ethylene glycol. 15% DMSO and 0 5 M sucrose When post-warm blastocysts were cultured in inSOF medium, survival rate (re-expansion of blastocoel at 24 h of culture) was improved (P &lt; 0 05) by the addition of 10 mu M Y-27632 (94 9 +/- 2 4%, mean +/- SEM) compared to a control (78 0 +/- 6 0%) Conversely. after 48 h of culture, there were no significant differences in hatching late (62.8 +/- 11 1 vs 59 6 +/- 9.4%) and mean total cell number (135 2 +/- 13 1 vs. 146 7 +/- 13 3) In non-vitrified IVP bovine blastocysts, the hatching rate on Day 9 was improved by Y-27632 (91 7 +/- 3 8 vs 54 7 +/- 8 9%. P &lt; 0 05). with no difference in mean total cell number of blastocysts (230 0 +/- 23 0 vs 191 2 +/- 22 2, P = 0 23). In an additional experiment, Y-27632 was added to culture medium on either Day 0, Day 2. or Day 4 (and remained present until Day 8). resulting in no improvement in blastocyst yield compared to a control group (7.5 +/- 2 1, 31 4 +/- 2 3, 36 2 +/- 3.2. and 28.6 +/- 6.9%. respectively) In conclusion, adding a ROCK inhibitor to post-thaw culture medium improved revivability of IVP bovine blastocysts after vitrification and warming (C) 2010 Elsevier Inc. All rights reserved