Multiple aster formation is frequently observed in bovine oocytes retrieved from 1-day stored ovaries.

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 α-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.

Microtubule assembly and in vitro development of bovine oocytes with increased intracellular glutathione level prior to vitrification and in vitro fertilization.

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 ß-mercaptoethanol (ßME) 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 ßME/Cys × 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 ßME/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.

High incidence of multiple aster formation in vitrified-warmed bovine oocytes after in vitro fertilization.

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 < 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.

Vitrification of ICSI- and IVF-derived bovine blastocysts by minimum volume cooling procedure: effect of developmental stage and age.

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 < 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 < 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.

Stimulatory effect of Rho-associated coiled-coil kinase (ROCK) inhibitor on revivability of in vitro-produced bovine blastocysts after vitrification.

Inhibition of Rho-associated coiled-coil 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 improved 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 0.5M sucrose. When post-warm blastocysts were cultured in mSOF medium, survival rate (re-expansion of blastocoel at 24h of culture) was improved (P<0.05) by the addition of 10 microM 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 rate (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<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.

A combined treatment of ionomycin with ethanol improves blastocyst development of bovine oocytes harvested from stored ovaries and microinjected with spermatozoa.

Regardless of the presence of sperm-borne oocyte-activating factors, activation of bovine oocytes with exogenous activation stimuli is required for further development after intracytoplasmic sperm injection (ICSI). The current study was designed to develop a new activation regimen for improving the blastocyst yield after ICSI of bovine oocytes harvested from ovaries stored at 10 to 12 degrees C for 24h. After ICSI, oocytes were treated with 5microM ionomycin for 5 min, 7% ethanol for 5 or 10min, ionomycin followed by ethanol (5 or 10 min), ionomycin followed by 10 microg/mL cycloheximide for 5h, or ionomycin followed by 1.9 mM 6-dimethylaminopurine for 3h. Across the activation regimens, the cleavage rates of ICSI oocytes (45% to 77%) were higher than those of parthenogenetically activated oocytes (11% to 21%; P<0.05). Activating the ICSI oocytes with ionomycin plus ethanol improved the blastocyst yield (29% to 30%) compared with that of nontreated oocytes (12%; P<0.05), but the other regimens did not improve the blastocyst yield (9% to 18%; P>0.05). Higher blastocyst yields were due to increasing the proportion of ICSI oocytes that passed through the early postfertilization events until cleavage. None of the regimens have any adverse effect on the quality of the blastocysts regarding the total cell number or the proportion of the inner cell mass cells. Thus, a new activation regimen using two triggers for single calcium increase effectively improved blastocyst yield after bovine ICSI using oocytes harvested from stored ovaries.

The ability of freeze-dried bull spermatozoa to induce calcium oscillations and resumption of meiosis.

The objective was to investigate the ability of freeze-dried (FD) bull spermatozoa to induce calcium oscillations in mouse oocytes and meiosis resumption in in vitro-matured bovine oocytes after intracytoplasmic sperm injection (ICSI). Bull spermatozoa were freeze-dried and stored for 1 y at +25, +4, or -196 degrees C. In the first experiment, rehydrated sperm heads were microinseminated into hybrid mouse oocytes loaded with fluo-3/AM, and the kinetics of intracellular calcium concentration was monitored for 1h. Repetitive increases of intracellular calcium concentration were recorded in the majority of injected oocytes, with exception of a few oocytes injected with FD sperm heads stored at +4 degrees C (11%) and +25 degrees C (8%) that exhibited a single increase or no response (non-oscillated). The proportion of oocytes that oscillated with high frequency (>or=10 spikes/h) was higher in the non-dried control group (79%; P<0.05) than in the FD groups (58, 55, and 54% for storage at -196, +4, and +25 degrees C, respectively). In the second experiment, control and FD spermatozoa were microinseminated into in vitro-matured, denuded bovine oocytes. The oocytes were fixed and stained 12h after ICSI. A higher proportion of bovine oocytes injected with control spermatozoa (70%; P<0.05) resumed meiosis than those injected with +25, +4 and -196 degrees C stored FD spermatozoa (53, 48, and 57%, respectively). The proportion of ICSI oocytes that developed to the pronuclear stage (complete activation) was higher in the control group (64%; P<0.05) than those in all the FD groups (34, 27, and 28% for storage at -196, +4, and +25 degrees C, respectively). Thus, the ability of bull spermatozoa to induce frequent intracellular calcium spikes in mouse oocytes was impaired by the process of freeze-drying, without differences among storage at +25, +4 or -196 degrees C, probably resulting in a lower proportion of bovine oocytes that resumed meiosis and/or developed to the pronuclear stage.

Effect of post-IVF developmental kinetics on in vitro survival of vitrified-warmed domestic cat blastocysts.

A limited number of reports is available on cryopreservation of in vitro fertilization (IVF)-derived cat blastocysts. In the present study, IVF-derived domestic cat embryos which reached the blastocyst stage either on day 6 or day 7 were cryopreserved by vitrification using Cryotop as a cryodevice. Fresh control and post-warm surviving blastocysts were examined by differential cell staining with Hoechst 33342 and propidium iodide to determine total cell number and inner cell mass (ICM) ratio, and the post-warm survival rate was determined by re-expansion of the blastocoel during 24 h of in vitro culture. In fresh control, the mean number of total cells of day 7 blastocysts (61.4 cells) tended to be smaller than that of day 6 blastocysts (81.9 cells, p = 0.096). The post-warm survival rates of day 6 and day 7 blastocysts were not statistically different (73.8%; 31 of 42 vs 66.7%; 18 of 27). There were no significant differences in the total cell number and ICM ratio between fresh control and vitrified blastocysts, although the ICM ratio of surviving day 7 blastocysts was significantly smaller than that of fresh controls (stained at day 8, 18.9% vs 28.9%, p < 0.05). These results indicate that IVF-derived domestic cat embryos that reached the blastocyst stage earlier can survive the Cryotop vitrification without a reduction in the parameters studied.

The ability of whale haploid spermatogenic cells to induce calcium oscillations and its relevance to oocyte activation.

Interspecies microinsemination assay was applied to examine the ability of minke whale haploid spermatogenic cells to induce Ca2+ oscillations and oocyte activation. Populations of round spermatids (RS), early-stage elongating spermatids (e-ES), late-stage elongating spermatids (1-ES) and testicular spermatozoa (TS) were cryopreserved in the presence of 7.5% glycerol on board ship in the Antarctic Ocean. Repetitive increases of intracellular Ca2+ concentration occurred in 0, 65, 81 and 96% of BDF1 mouse oocytes injected with the postthaw RS, e-ES, 1-ES and TS, respectively. A normal pattern of the Ca2+ oscillations was observed in 26-47% of the responding oocytes. Most oocytes that exhibited Ca2+ oscillations, regardless of the oscillation pattern, resumed meiosis (83-94%). These results indicate that whale spermatogenic cells acquire SOAF activity, which is closely related to their Ca2+ oscillation-inducing ability at the relatively early stage of spermiogenesis.

Quality analysis of buffalo blastocysts derived from oocytes vitrified before or after enucleation and reconstructed with somatic cell nuclei.

We investigated the potential of vitrified-warmed buffalo oocytes to develop to blastocysts after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT). In vitro-matured oocytes before and after enucleation (M-II oocytes and enucleated oocytes, respectively) were put in 7.5% DMSO and 7.5% ethylene glycol (EG) for 4, 7 and 10 min, and then vitrified (Cryotop device) after 1-min equilibration in 15% DMSO, 15% EG and 0.5M sucrose. Following 4-, 7- and 10-min exposure, proportions of the post-warm oocytes with a normal vitelline membrane were similar (66-71% in M-II oocytes and 69-71% in enucleated oocytes). However, 18-20% of the normal M-II oocytes had no detectable first polar body in their perivitelline space (no potential for subsequent enucleation). When the post-warm M-II oocytes were treated for PA by 7% ethanol, 10 microg/mL cycloheximide and 1.25 microg/mL cytochalasin-D, parthenogenetic development into Day-7 blastocysts occurred in 10-13% of cultured oocytes, lower (P<0.05) than fresh (control) oocytes (24%). In the absence of the cooling and warming, blastocyst rates in the 4-min exposure group (22%), but not in the 7-min and 10-min exposure groups (14-15%), were similar to that in the fresh group (23%). The total cell number (group average 117-132 cells) and the ICM ratio (22-24%) of the PA blastocysts derived from vitrified M-II oocytes were comparable with fresh oocytes (127 cells and 25%). After SCNT (with fibroblast cells and vitrified-warmed oocytes), blastocyst rates were similar for the three exposure periods for M-II oocytes (8-10%) and enucleated oocytes (7-9%), but were lower (P<0.05) than in the fresh group (15%). The total cell number of the SCNT blastocysts derived from vitrified M-II and enucleated oocytes (80-90 and 82-101 cells) was smaller (P<0.05) than from fresh oocytes (135 cells); the ICM ratio of blastocysts derived from the M-II and enucleated oocytes after vitrification in 7- or 10-min exposure groups (20-22%) was not different (P>0.05) from fresh control oocytes (24%) or those in 4-min exposure group (M-II 23%, enucleated 24%). Thus, SCNT of swamp buffalo oocytes following vitrification before or after enucleation resulted in blastocysts with a slightly decreased cell number.

Successful vitrification of pronuclear-stage rabbit zygotes by minimum volume cooling procedure.

Rabbit zygotes at the pronuclear-stage were cryopreserved by vitrification using a gel-loading tip (GL-tip), Cryoloop or Cryotop. In GL-tip and Cryoloop methods, zygotes were first exposed to 10% ethylene glycol (EG)+10% DMSO in TCM199+20% fetal bovine serum (FBS) for 2 min, and then equilibrated for 30 s in a vitrification solution composed of 20% EG+20% DMSO+0.6 M sucrose in TCM199+20% FBS. In Cryotop method, zygotes were first exposed to 7.5% EG+7.5% DMSO+20% FBS in TCM199 for 3 min, and then equilibrated for 1 min in a vitrification solution composed of 15% EG+15% DMSO+0.5 M sucrose+20% FBS in TCM199. In vitro culture of vitrified-warmed zygotes using GL-tip and Cryoloop resulted in low cleavage rates (2 and 5%, respectively) and no development into blastocysts. In contrast, zygotes vitrified-warmed using Cryotop exhibited higher proportions of cleavage (58%) and development into blastocysts (24%). When compacted morulae or early blastocysts were vitrified by these three procedures, 80-93% of them exhibited blastocoele expansion or zona hatching during the subsequent 48 h of culture. Use of Cryotop instead of GL-tip or Cryoloop for zygote vitrification, without changing conditions of solutions and periods for exposure, equilibration and post-warm dilution, resulted in cleavage and blastocyst development rates of 88 and 45%, respectively. A longer exposure time (10 min) of zygotes to 7.5% EG+7.5% DMSO+20% FBS in TCM199 resulted in higher proportions of zygotes cleaving (94%) and developing into blastocysts (51%) after Cryotop vitrification. Proportions of post-warm zygotes (10-min exposure group) and fresh control zygotes developing into newborn offspring were 36 and 53%, respectively. Pronuclear-stage rabbit zygotes were successfully cryopreserved by vitrification using the Cryotop method.

Effects of cryopreservation of pronuclear-stage rabbit zygotes on the morphological survival, blastocyst formation, and full-term development after DNA microinjection.

The objectives of this study were to examine the freezing sensitivity of pronuclear-stage rabbit zygotes and to produce transgenic rabbits using the cryopreserved zygotes. Zygotes were cryopreserved either by one of two vitrification protocols or by one of the two conventional freezing protocols. The morphological survival rates of zygotes subjected to two-step freezing in 1.5 M ethylene glycol and 0.1 M sucrose (74%) or to vitrification in 7.2 M ethylene glycol and 1.0 M sucrose (81%) were higher than those subjected to freezing in 1.5 M DMSO (46%) or to vitrification in a mixture of 2.0 M DMSO, 1.0 M acetamide, and 3.0 M propylene glycol (41%). But the in vitro development into blastocysts of zygotes cryopreserved by vitrification (17%) or to a lesser extent by freezing (52%) was impaired, when compared to that of fresh control zygotes (89%). Next, a fusion gene composed from bovine aS1-casein promoter and a human GH structural gene (2.8 kb) was microinjected into the pronucleus of rabbit zygotes frozen-thawed in ethylene glycol and sucrose. Then, the presence of exogenous DNA in the genome of newborn offspring was determined by PCR. The post-injection survival of frozen zygotes (97%) was the same as that of fresh control zygotes (96%). However, of 18 offspring derived from 414 frozen-thawed and DNA-injected zygotes, no transgenic rabbits were produced. Of 52 offspring derived from 403 DNA-injected fresh zygotes, 3 transgenic rabbits were found. Here we report the first rabbit offspring resulting from zygotes cryopreserved at the pronuclear-stage, although the cryopreservation procedure employed must be improved if zygotes are to be used for systematic production of transgenic rabbits.

A comparative study on the integration of exogenous DNA into mouse, rat, rabbit, and pig genomes.

Transgenic mammals, from small laboratory rodents to domestic animals, have been successfully produced to date, but their production efficiency within or across species has been variable. This is probably due to the differences in the type of injected DNA and/or technical procedures employed in each laboratory, as well as the reproductive characteristics of the species. Here we report the direct comparison of the efficiencies of producing transgenic mice, rats, rabbits and pigs by one technician using a fusion gene composed of the bovine alpha S1-casein promoter and human growth hormone (hGH) gene. Before the fusion gene was injected into the zygotes, high magnitude centrifugation to visualize the pronuclei was necessary for all of the pig zygotes and one-third of the rabbit zygotes, but not for mouse and rat zygotes. Post-injection survival of the mouse zygotes (67.1%) was lower than those of the rat, rabbit and pig zygotes (89.6 to 100%). The volume change of the pronucleus following DNA injection was the lowest in mice (50% increase), moderate in rabbits (148% increase), and the most prominent in rats (238% increase). The data from only 1 pig zygote indicated a 22% increase in the pronucleus volume by DNA injection. The PCR analyses of the tail DNA of new born offspring indicated that 0.8% (4/493), 4.8% (22/463), 0.8% (3/367) and 0.9% (2/221) of the injected eggs in mice, rats, rabbits and pigs, respectively, developed into transgenic offspring. Some of the founder animals in all four species expressed the transgene in the mammary gland which was confirmed in hGH mRNA by RT-PCR and/or hGH peptide in Witch's milk with ELISA. These results suggest that the maximum volume of DNA solution injectable into the pronucleus is a possible factor explaining the species differences in the production of transgenic animals.

Effects of cooling and warming rates during vitrification on fertilization of in vitro-matured bovine oocytes.

The purpose of this study was to clarify the relationship of cooling rates (CR) and warming rates (WR) during vitrification with postwarming viability of in vitro-matured bovine oocytes. In Experiment 1, oocytes were vitrified in a solution containing 7.2 M ethylene glycol and 1.0 M sucrose by use of open-pulled glass capillaries with five different outer diameters and were warmed by placement of the capillaries into 0.25 M sucrose solution. The capillaries of 2000-, 1400-, 1000-, 630-, and 440-mm diameters provided CR of 2000, 3000, 5000, 8000, and 12,000 degrees C/min and WR of 5000, 8000, 17,000, 33,000, and 62,000 degrees C/min, respectively. In oocytes vitrified in capillaries of 1400-mm diameter (CR, 3000 degrees C/min; WR, 8000 degrees C/min), the morphological survival rate (86% of vitrified), penetration rate (79% of inseminated), and normal fertilization rate (69% of penetrated) were higher or tended to be higher than those in the other vitrification groups. In Experiment 2, oocytes cooled at 2000, 3000, or 12,000 degrees C/min were warmed at 8000 degrees C/min, and oocytes cooled at 3000 degrees C/min were warmed at 5000, 8000, or 33,000 degrees C/min. Among these CR-WR combinations, cooling of oocytes at 3000 degrees C/min regardless of the WR resulted in higher postwarming survival. These results indicate that survival of in vitro-matured bovine oocytes after vitrification and subsequent warming is improved by a slightly rapid cooling rate in open-pulled glass capillaries compared to that obtained in conventional straws.

Production of transgenic rats using young Sprague-Dawley females treated with PMSG and hCG.

The aim of this study was to examine the effects of gonadotrophin treatments on estrus synchronization and superovulation in young Sprague-Dawley (SD) rats that had not yet exhibited defined estrus cycles (5 to 7 weeks old), and to produce transgenic rats using these females as embryo donors and recipients. In Experiment 1, female rats were injected with PMSG and hCG (12.5, 25, 50 and 100 IU/kg each) and were mated with stud males. The reproductive performance of young rats were highest when PMSG and hCG at doses of 25 IU/kg each were injected (delivery rate 87.5%, nursing rate 92.9%). In Experiment 2, female rats were injected with PMSG and hCG (100, 150 and 300 IU/kg each) to induce superovulation. More eggs were recovered from the rats injected with PMSG and hCG at 150 and 300 IU/kg than from those treated with 100 IU/kg (33.4 and 41.3 vs. 13.3 eggs per female, respectively; p < 0.05). In Experiment 3, pronuclear-stage zygotes from 150 IU/kg PMSG/hCG-treated rats were used for microinjection of the fusion gene of bovine alpha S1-casein gene promoter and human growth hormone gene (2.8 kb), and the microinjected zygotes were transferred into the oviduct ampullae of the 25 IU/kg PMSG/hCG-treated rats. Seventeen transgenic rats were obtained from the 334 DNA-injected zygotes (5.1%). These results indicate that recipients and embryo donors for the production of transgenic rats can be prepared by the appropriate PMSG and hCG treatments of young SD rats, regardless of their estrus stages.

Production of transgenic rabbits using centrifuged pronuclear zygotes.

Superovulation of female rabbits was induced by subcutaneous injection(s) of porcine FSH. Zygotes were recovered 17 to 19 hr after hCG injection and were classified into two categories under a microscope equipped with Nomarski interference-contrast optics at x 200 magnification: (A) zygotes with clearly visible pronuclei, or (B) zygotes with visualized pronuclei after 10 min centrifugation at 12,000 x g. No significant difference between strains was found in the proportion of category-A zygotes (JW 72.6% vs NZW 79.3%). Pronuclei of category-A zygotes were located in the center of the cytoplasm, and the pronuclei of category-B zygotes were slightly moved by centrifugation toward the mass of cytoplasmic lipid droplets. Exogenous DNA solution (5 microg/ml of fusion gene composed of bovine alphaS1-casein promoter and human growth hormone structural gene) was microinjected into the pronucleus of the JW zygotes. The pronucleus of category-A zygotes with a mean volume of 7.4 pl swelled up to 16.6 pl (132% increase), while that of category-B zygotes with a mean volume of 6.1 pl swelled up to 15.9 pl (148% increase). Nevertheless, similar proportions of category-A and category-B zygotes developed into offspring after transfer to recipient females (11.1 and 11.2%, respectively). The efficiency to produce hGH-carrying transgenic rabbits was 0.9% (2/235) from category-A zygotes and 0.5% (1/215) from category-B zygotes (P>0.05). To date, transgenic rabbits have been produced without centrifugation of pronuclear zygotes. However approximately 25% of fertilized rabbit zygotes can be used for DNA microinjection after they have been centrifuged to visualize their pronuclei.

Nuclear transfer in cattle : effect of linoleic acid-albumin on freezing sensitivity of enucleated oocytes.

The effect of linoleic acid-albumin (LAA) supplementation to the media for IVM, enucleation, and activation on the developmental potential of bovine embryos produced by nuclear transfer (NT) into frozen-thawed cytoplasts was investigated. Blastomeres derived from morulae was placed in the perivitelline space of frozen-thawed cytoplasts, which were then fused by a DC pulse. The proportion of fused embryos was similar between groups with and without LAA (87 vs. 90%). The proportion of development to blastocysts of NT embryos derived from the media with LAA (14%) was higher than that without LAA (4%), indicating that LAA treatment of bovine oocytes during IVM, enucleation and activation can improve the ability of such cytoplasts after freezing and thawing to develop into blastocysts after NT.

Effects of timing of oocyte cryopreservation on in vitro development of nuclear-transferred bovine zygotes.

The utility of cryopreserved bovine oocytes as recipient cytoplasts for nuclear transfer (NT) was examined. In vitro-matured (IVM), metaphase-II oocytes were enucleated by mechanical suction and activated parthenogenetically. The cytoplasts were fused with blastomeres of in vitro-produced day-5 morulae by a DC electropulse, and then cultured up to 8 days (non-frozen controls; group I). Oocytes were frozen-thawed in 1.5-M ethylene glycol and 0.1-M sucrose before enucleation (group II), after enucleation (group III), after enucleation and aging culture (group IV), or after activation (group V). In group I, 91% of IVM oocytes could be used for NT and 89% of them fused successfully. Finally, 36% of the fused zygotes developed into blastocysts. The proportions of morphologically normal oocytes after thawing in groups IV and V (70 and 69%, respectively) were higher than in group III (56%), and the proportion of IVM oocytes used for NT in group IV (56%) was higher than those in groups II, III, and V (33%, 35%, and 38%, respectively). Fusion rates of the NT zygotes in groups III, IV, and V (90%, 88%, and 88%, respectively) were higher than the rate in group II (75%). Rates of development into blastocysts of the fused zygotes in groups II, III, IV, and V were 0%, 3%, 2%, and 6%, respectively (P < 0.05, group II vs. groups III, IV, and V). Developmental kinetics and cell numbers of the blastocysts were similar among the groups. It was suggested that timing of oocyte cryopreservation is among the factors influencing efficiency of production of cloned embryos in cattle.

Effect of linoleic acid-albumin in the culture medium on freezing sensitivity of in vitro-produced bovine morulae.

The objective of this study was to improve the survival of in vitro-produced bovine morulae after cry opreservation. In Experiment 1, presumptive zygotes at 20 h post-insemination (hpi) were cultured in a mixture of modified synthetic oviduct fluid (m-SOF)/0.3% BSA and m-SOF/0.3% linoleic acid-albumin from bovine serum (LAA) at 39.0 degrees C in 5% O2, 5% CO2 and 90% N2 (final LAA concentration: 0, 0.01, 0.03, 0.1 or 0.3%). Morulae harvested at 138 hpi were frozen and thawed in m-PBS/0.3% BSA containing 1.5 M ethylene glycol and were cultured for 96 h in m-SOF/10% FBS to assess further development. The post-thaw survival of morulae derived from culture in 0.1% LAA (60%, P < 0.01) and in 0.03% LAA (55%, P < 0.05) was higher than that in 0% LAA (32%). Lowering the LAA concentration below 0.1% resulted in similar rates of morula development as in m-SOF/0.3% BSA. In Experiment 2, zygotes were cultured in m-SOF/0.1% LAA from 20 to 90 hpi and/or from 90 to 138 hpi. Post-thaw survival of morulae that had been exposed to LAA from 20 to 90 hpi (39%) or from 90 to 138 hpi (56%) was higher than that of morulae cultured without LAA from 20 to 138 hpi (12%, P < 0.02). These survival rates were lower than that of morulae cultured with LAA over a period of 20 to 138 hpi (76%, P < 0.001). The results indicate that cell-free culture of IVM/IVF bovine zygotes in m-SOF supplemented with LAA produces morula-stage embryos relatively tolerant to the process of freezing and thawing.

Effect of nuclear stages during IVM on the survival of vitrified-warmed bovine oocytes.

The effect of nuclear stages during IVM on the survival of vitrified-warmed bovine oocytes was investigated. Oocytes with compact cumulus cells were cultured for 0, 6, 12 and 24 h in TCM199 supplemented with 5% fetal bovine serum (FBS) in 3% CO2 in air. The oocytes were first exposed to 20% ethylene glycol solution and were subjected to vitrification in a solution containing 40% ethylene glycol, 18% Ficoll-70 and 0.3 M sucrose. After warming in 20 degrees C water, oocytes which had been vitrified at less than 24-h of IVM were again cultured to complete the 24-h of IVM period. Oocytes were then incubated with frozen-thawed spermatozoa in Brackett and Oliphant (BO) medium containing 60 micrograms/ml heparin and 0.25% BSA for 20 h. In vitro fertilization rates of oocytes vitrified-warmed at 0, 6, 12 and 24-h IVM were 75.2, 68.0, 82.0 and 72.4%, respectively, comparable to the rates for unvitrified control oocytes (80.6%). A higher incidence of polyspermic fertilization was observed in oocytes vitrified at 24-h IVM (44.9 vs 22.6% in the control group, P < 0.05). Vitrification of oocytes at 12-h IVM seemed to be better than that of other IVM groups, since the normal fertilization rate of all treated oocytes was the highest (36.0%) among the vitrification groups. Developmental competence of the oocytes following vitrification and in vitro fertilization (12-h IVM group) was examined by cell-free culture of presumptive zygotes up to 9 d in modified synthetic oviduct fluid (mSOF) in 5% CO2, 5% O2 and 90% N2. The cleavage rate of zygotes from vitrified oocytes 48 h after insemination was 29.8%, which was lower than that of the control group (57.0%, P < 0.05). Development to blastocysts from the vitrified oocytes (4.8%) was much lower than that of the control group (27.0%, P < 0.05). These results indicate that cryopreservation of bovine oocytes by vitrification may be affected by their maturation stage in vitro, and that developmental competence to blastocysts of cleaved oocytes following vitrification may be impaired compared with unvitrified control oocytes.