Simplified cryopreservation of porcine cloned blastocysts

Recently, a non-invasive delipation (lipid removal) method combined with ultrarapid vitrification has been used successfully for in vitro produced (IVP) porcine embryos. In the present study, this method was combined with parthenogenesis and a recent form of somatic cell nuclear transfer (SCNT) - handmade cloning (HMC) - to establish a simplified and efficient cryopreservation system for porcine cloned embryos. In Experiment 1, zonae pellucidae of oocytes were partially digested with pronase, followed by centrifugation to polarize lipid particles. Ninety percent (173/192) oocytes were successfully delipated in this way. Parthenogenetic activation (PA) after complete removal of zona resulted in similar blastocyst rates in delipated vs. control oocytes (28+/-7% vs. 28+/-5%, respectively). Subsequent vitrification of produced blastocysts with the Cryotop technique resulted in higher survival rates in the delipated group compared to the control group (85+/-6% vs. 32+/-7%, respectively; P<0.01). In Experiment 2, delipated oocytes were used for HMC with normal oocytes as control. Partial zona digestion was further applied before enucleation both in delipated and control groups, to bisect oocyte successfully. Although the blastocyst rate of reconstructed embryos was similar between groups derived from delipated vs. control oocytes (21+/-6% and 23+/-6%, respectively), after vitrification higher survival rates were achieved in the delipated groups than in controls (79+/-6% vs. 32+/-8%, respectively). Our results prove that porcine embryos produced from delipated oocytes by PA or HMC can be cryopreserved effectively by ultrarapid vitrification. Further experiments are required to assess the in vivo developmental competence of the cloned-vitrified embryos.     

Piglets born from vitrified cloned blastocysts produced with a simplified method of delipation and nuclear transfer

Successful cryopreservation of porcine embryos offers a promising perspective in the fields of agriculture, animal science, and human medical research. The objective of the present work was to establish a system facilitating the cryopreservation of porcine embryos produced by somatic cell nuclear transfer (SCNT). Several key techniques including micromanipulator-based enucleation, noninvasive delipation, zona-free fusion, and activation were combined with high efficiency. After a partial zona digestion and high-speed centrifugation, 89.8+/-2.1% (mean+/-SEM) of enucleated oocytes were successfully delipated. Delipated cytoplasts were incubated for an additional 0.5 or 2 h before fusion with somatic cells. After activation and 6 days of in vitro culture, no significant difference in the rate of blastocysts per reconstructed embryo was observed between the two groups (33.1+/-1.8% and 26.0+/-4.3% for 0.5 and 2 h recovery time, respectively). Cryopreservation of the blastocysts was performed with a Cryotop device and factory-prepared vitrification and warming solutions. One hundred fifty-five vitrified SCNT embryos were transferred surgically into two recipient sows to test their developmental capacity in vivo. One recipient became pregnant and delivered six piglets. In conclusion, our simplified delipation and SCNT procedure resulted in viable piglets after vitrification and embryo transfer at the blastocyst stage.     

Piglets born from handmade cloning, an innovative cloning method without micromanipulation

Porcine handmade cloning (HMC), a simplified alternative of micromanipulation based traditional cloning (TC) has been developed in multiple phases during the past years, but the final evidence of its biological value, births of piglets was missing. Here we report the first births of healthy piglets after transfer of blastocysts produced by HMC. As a cumulative effect of technical optimization, 64.3+/-2.3 (mean+/-S.E.M.) reconstructed embryos from 151.3+/-4.8 oocytes could be obtained after 3-4h manual work, including 1h pause between fusion and activation. About half (50.1+/-2.8%, n=16) of HMC reconstructed embryos developed to blastocysts with an average cell number of 77+/-3 (n=26) after 7 days in vitro culture (IVC). According to our knowledge, this is the highest in vitro developmental rate after porcine somatic cell nuclear transfer (SCNT). A total of 416 blastocysts from HMC, mixed with 150 blastocysts from TC using a cell line from a different breed were transferred surgically to nine synchronized recipients. Out of the four pregnancies (44.4%) two were lost, while two pregnancies went to term and litters of 3 and 10 piglets were delivered by Caesarean section, with live birth/transferred embryo efficiency of 17.2% (10/58) for HMC. Although more in vivo experiments are still needed to further stabilize the system, our data proves that porcine HMC may result in birth of healthy offspring. Future comparative examinations are required to prove the value of the new technique for large-scale application.     

Improving in vitro development of cloned bovine embryos with hybrid (Holstein-Chinese Yellow) recipient oocytes recovered by ovum pick up

In the present study, oocytes from F1 hybrid cattle, as well as their parental lines, were recovered by ovum pick up (OPU) and used as recipient cytoplasm for somatic cell nuclear transfer (SCNT). Four F1 hybrid (Holstein dam x Chinese Yellow sire), 10 Holstein and four Chinese Yellow cattle were subjected to OPU once weekly. There were no significant differences among breeds for number of recovered oocytes per session (overall average, 7.8+/-0.5; mean+/-S.E.M.), quality of the recovered oocytes, or oocyte maturation rate (72-73%). Matured oocytes were all used as recipient cytoplasm (without selection) and a single batch of cumulus cells collected from a Holstein cow were used as donor cells. Although reconstructed embryos initiated cleavage sooner when the recipient cytoplasm was from hybrid cattle versus the two parental breeds, the overall cleavage rate was indistinguishable among breeds. At Day 8, the blastocyst rate from the cleaved embryos (51% versus 37% and 27%), the total number of cells per blastocyst (135+/-4.1 versus 116+/-3.6 and 101+/-4.2), and the percentage of Grade-A (excellent quality) blastocysts (54% versus 42% and 29%) in the hybrid group were all higher than that of Holstein and Yellow groups. Furthermore, the proportion of blastocysts obtained at Day 7 (as a percentage of the total number of blastocysts) was greater in the hybrid group than in Holstein and Yellow groups (89% versus 71% and 63%). In conclusion, the use of F1 hybrid oocytes as recipient cytoplasm significantly improved in vitro development of cloned bovine embryos relative to oocytes derived from the parental lines.     

Duration of in vitro maturation of recipient oocytes affects blastocyst development of cloned porcine embryos

This study investigated the effects of different incubation periods for oocyte maturation and contact inhibition of donor cells as well as different osmolarities for storage of recipient oocytes on fusion rates, cleavage rates, and blastocyst yields of porcine somatic nuclear transfer (SCNT) derived embryos. In addition, the in vivo developmental potential of cloned embryos derived from the most promising SCNT protocol was tested by transfer to recipient gilts. Storage of in vitro-matured oocytes for 7.5 h in calcium-free TL-HEPES medium at 295 or 320 mOsmol prior to activation yielded significantly (p < 0.05) higher parthenogenetic blastocyst rates compared to storage in TL-HEPES with an osmolarity of 270 mOsmol (24.4 +/- 3.0% and 26.2 +/- 4.3% vs. 18.3 +/- 6.4%, respectively, mean +/- SD) and improved the visibility of the polar body. Electrical fusion of fibroblasts to enucleated oocytes matured for 38, 40, or 42 h resulted in similar fusion and cleavage rates (74.8-84.4%). However, nuclear transfer with oocytes matured for 40 h in vitro yielded significantly higher (p < 0.05) development to the blastocyst stage after 7 days of culture (14.7 +/- 1.7%) than with oocytes matured for 38 h (9.5 +/- 2.1%) or 42 h (5.1 +/- 2.1%). Contact inhibition for 24, 48, or 72 h significantly (p < 0.05) increased the proportion of cells at G0/G1 compared with cycling fibroblasts. However, duration of contact inhibition of the donor cells for either 24, 48, or 72 h had no effect on blastocyst rates of SCNT embryos. Four gilts received an average of 150 SCNT embryos (range 138-161) reconstructed with oocytes matured for 40 h; two of these became pregnant; one of them went to term and farrowed four piglets on day 115 of pregnancy. Microsatellite analysis confirmed that the clones were genetically identical with the donor cells. These results show that changes of the in vitro maturation protocol may affect in vitro development of reconstructed porcine embryos, while duration of the contact inhibition period plays a minor role for the success of porcine SCNT. The effects on in vivo development are yet to be determined.     

Cloning of bovine embryos by multiple nuclear transfer

The in vitro development of multiple generation bovine nuclear transferred embryos to blastocysts and their survival ability after freezing and thawing were examined. Parent donor embryos which had 20 to 50 cells were recovered from superovulated cows. Follicular oocytes matured in vitro were used as recipient oocytes. The recipient oocytes enucleated at 22 to 24 h after the onset of maturation were preactivated at 33 h. Enucleated oocytes with a donor blastomere were fused 9 h after activation by an electric stimulus and the fused oocytes were cultured in vitro (first generation). Reconstituted oocytes that had developed to the 8- to 16-cell stage 3 to 4 d after fusion were used as donor embryos for the next generation. Recloning procedures were performed twice (second and third generations). The proportion of recipient oocytes successfully fused with a blastomere increased with the cycle of nuclear transfer. Eighty to 86% of fused oocytes developed to the 2-cell stage and there was no significant difference with the generation. The proportion of reconstituted embryos receiving blastomeres derived from first generation embryos had higher developmental ability in vitro, than those derived from other generations (43 vs 31% for 8 to 16-cell stage, 37 vs 20 and 21% for blastocyst stage). The number of cloned blastocysts increased with repeated nuclear transfer (once: 6.2 +/- 4.3, twice: 19.8 +/- 9.2 and three times: 30.0 +/- 14.7) but varied greatly with each parent donor embryo. The in vitro viability of cloned blastocysts after freezing and thawing (59%) was low but not significantly different from that obtained for in vitro fertilized blastocysts (72%). After transfer of either fresh or frozen-thawed cloned blastocysts to 21 recipients, 10 of them were pregnant on Day 60. Four and 3 offspring were produced from 20 fresh and 14 frozen-thawed blastocysts,respectively.     

Developmental,qualitative, and ultrastructural differences between ovine and bovine embryos produced in vivo or in vitro

The objective of this study was to compare bovine and ovine oocytes in terms of (1) developmental rates following maturation, fertilization, and culture in vitro, (2) the quality of blastocysts produced in vitro, assessed in terms of their ability to undergo cryopreservation, and (3) the ultrastructural morphology of these blastocysts. In vitro blastocysts were produced following oocyte maturation/fertilization and culture of presumptive zygotes in synthetic oviduct fluid. In vivo blastocysts were used as a control from both species. In Experiment 1, the cleavage rate of bovine oocytes was significantly higher than that of ovine oocytes (78.3% vs. 58.0%, respectively, P < 0.001). The overall blastocyst yield was similar for both species (28.7% vs. 29.0%). However, when corrected for cleavage rate, significantly more ovine oocytes reached the blastocyst stage at all time-points (36.6% vs. 50.0% on day 8, for bovine and ovine, respectively, P < 0.001). Following vitrification, there was no difference in survival between in vivo produced bovine and ovine blastocysts (72 hr: 85.7% vs. 75.0%). However, IVP ovine blastocysts survived at significantly higher rates than IVP bovine blastocysts at all time points (72 hr: 47.4% vs. 18.1%, P < 0.001). At the ultrastructural level, compared with their in vivo counterparts, IVP blastocysts were characterized by a lack of desmosomal junctions, a reduction in the microvilli population, an increase in the average number of lipid droplets and increased debris in the perivitelline space and intercellular cavities. These differences were more marked in bovine IVP blastocysts, which also displayed electron-lucent mitochondria and large intercellular cavities. These observations may in part explain the species differences observed in terms of cryotolerance. In conclusion, the quality of ovine blastocysts was significantly higher than their bovine counterparts produced under identical in vitro conditions suggesting inherent species differences between these two groups affecting embryo quality.     

Development of porcine embryos and offspring after intracytoplasmic sperm injection with liposome transfected or non-transfected sperm into in vitro matured oocytes.

The objective of this study was to evaluate in vitro and in vivo development of porcine in vitro matured (IVM) porcine oocytes fertilised by intracytoplasmic sperm injection (ICSI) and the possibility of producing transgenic embryos and offspring with this procedure. Activated ICSI oocytes had a higher pronuclear formation than non-activated ICSI oocytes (mean 64.8+/-17.3% vs 28.5+/-3.4%, p<0.05). When the zygotes with two pronuclei were cultured to day 2, there was no difference (p<0.05) in the cleavage rate (mean 60.0+/-7.0% vs 63.3+/-12.7%) between the two groups. The blastocyst rate in the activation group was significantly higher than that in the non-activation group (mean 30.0+/-11.6% vs 4.6+/-4.2%, p<0.05). After injection of the sperm transfected with DNA/liposome complex, destabilised enhanced green fluorescent protein (d2EGFP) expression was not observed on day 2 in either cleaved or uncleaved embryos. But from day 3, some of the embryos at the 2-cell to 4-cell stage started to express d2EGFP. On day 7, about 30% of cleaved embryos, which were in the range of 2-cell to blastocyst stage, expressed d2EGFP. However, for the IVF oocytes inseminated with sperm transfected with DNA/liposome complex, and for oocytes injected with sperm transfected with DNA/liposome complex, and for oocytes injected with DNA/liposome complex following insemination with sperm not treated with DNA/liposome complex, none of the embryos expressed d2EGFP. Sixteen day 4 ICSI embryos derived from sperm not treated with DNA/liposome complex were transferred into a day 3 recipient. One recipient delivered a female piglet with normal birthweight. After transfer of the ICSI embryos derived from sperm transfected with DNA/liposome complex, none of the four recipients maintained pregnancy.     

High overall in vitro efficiency of porcine handmade cloning (HMC) combining partial zona digestion and oocyte trisection with sequential culture

We investigated the in vitro developmental competence of porcine embryos produced from in vitro matured (IVM) oocytes by improved HMC and parthenogenetic activation (PA). Embryos were cultured in a modified North Carolina State University (NCSU37) medium. Firstly, we compared the developmental competence between oocytes from sows and gilts by zona-intact (ZI) and zona-free (ZF) PA. Significantly higher (p < 0.05) blastocyst rates were obtained from sow oocytes (42 +/- 4% for ZF and 55 +/- 6% for ZI) than gilt oocytes (20 +/- 2% for ZF and 26 +/- 5% for ZI). Secondly, sow oocytes were used to establish the modified HMC that was based on a modified enucleation with partial zona digestion and trisection of porcine oocytes and the use of three cytoplasts and one somatic cell for embryo reconstruction. In vitro fertilization (IVF) and in parallel ZF PA were used as the control systems. After oocyte trisection, >90% of oocyte fragments were recovered, resulting in an average of 37 reconstructed embryos from 100 oocytes. Blastocyst rates of HMC, IVF, and ZF PA embryos were 17 +/- 4%, 30 +/- 6%, and 47 +/- 4%, respectively. Our results prove that HMC in pigs may result in high in vitro efficiency up until the blastocyst stage. In vivo developmental competence will be confirmed in embryo transfer experiments.     

Do age and extended culture affect the architecture of the zona pellucida of human oocytes and embryos?

Advanced female age and extended in vitro culture have both been implicated in zona pellucida (ZP) hardening and thickening. This study aimed to determine the influence of (i) the woman's age and (ii) prolonged in vitro culture of embryos on ZP thickness and density using non-invasive polarized light (LC-PolScope) microscopy. ZP thickness and density (measured as retardance) were determined in oocytes, embryos and blastocysts in women undergoing intracytoplasmic sperm injection (ICSI) in two age groups (older, > 38 years; younger, < or = 38 years). A total of 193 oocytes from 29 patients were studied. The younger group contained 100 oocytes and the older group 93 oocytes. The ZP was significantly thicker in metaphase II oocytes in the older group compared with the younger group (mean +/- SD: 24.1 +/- 2.5 microm vs 23.1 +/- 3.3 microm; p = 0.01) but ZP density was equal (2.8 +/- 0.7 nm). By day 2 of culture, embryos from the two groups had similar ZP thickness (22.2 +/- 2.2 microm vs 21.7 +/- 1.6 microm; p = 0.28) and density (2.9 +/- 0.7 nm vs 2.8 +/- 0.8 nm; p = 0.57). For the embryos cultured to blastocyst (older: n = 20; younger: n = 18) ZP thickness was similar in the two groups (19.2 +/- 2.7 microm vs 19.1 +/- 5.0 microm; p = 0.8) but thinner than on day 2. The older group had significantly denser ZP than the younger group (4.2 +/- 0.5 nm vs 3.3 +/- 1.0 nm, p < 0.01). Blastocysts from both groups had significantly denser ZP than their corresponding day 2 embryos (older: 4.2 +/- 0.5 nm vs 2.9 +/- 0.7 nm, p < 0.001; younger: 3.3 +/- 1.0 nm vs 2.8 +/- 0.8 nm, p = 0.013). It is concluded that there is little relationship between ZP thickness and its density as measured by polarized light microscopy. While ZP thickness decreases with extended embryo culturing, the density of the ZP increases. ZP density increases in both age groups with extended culture and, interestingly, more in embryos from older compared with younger women.     

Batchwise assessment of porcine embryos for cryotolerance

The viability or developmental ability of porcine embryos after slow-freezing and thawing differs depending on the embryonic stage or the batch, which is defined as a group of embryos obtained from one donor at one time. We froze porcine blastocysts in batches and assessed their cryotolerance by using two expanded blastocysts (EBs) as samples to predict the developmental potential of other blastocysts from the same batch at different stages. Two EBs from the same batch that had been separately frozen were thawed and cultured in vitro for 48 h to examine their in vitro ability to develop to the hatched blastocyst stage. Thereafter, each batch was assigned to Grade A, B, or C according to the viability of the two EBs, i.e., 100% viability (2/2: number of hatched blastocysts/number of cultured EBs) was Grade A; 50% (1/2) was Grade B; and 0% (0/2) was Grade C. The viability of EBs after freeze-thawing and in vitro culture varied depending on the batch and was lower (31.0+/-10.2%, mean+/-S.E.M.; P<0.01) than that of unfrozen controls (96.8+/-2.3%). The viability of frozen-thawed hatched blastocysts (HBs) did not differ among the graded batches, but the blastocyst diameter decreased (from 409 to 326 microm) as the batch grade decreased (from A to C). When both EBs and HBs from batches of the same grade were transferred to recipients (average 11.7 EBs and 16.0 HBs per recipient), the rate of pregnancy and farrowing in recipients decreased (from 77.8% to 0%) and the number of piglets obtained decreased (from 15.3 to 0) as the batch grade decreased. However, when not only frozen-thawed EBs from Grade B or C batches, but also four helper embryos at the morula to early blastocyst stage (which were expected to support the pregnancy) were transferred, the number of piglets generated was higher from EBs from Grade B batches (16.0) than from EBs from Grade C batches (0.0). When frozen-thawed HBs and helper embryos were transferred, the number of piglets generated was higher from HBs from Grade B batches (12.7) than that from HBs from Grade C batches (1.9). After slow-freezing of porcine blastocysts, their rate of survival to the piglet stage differs batchwise, and in vitro viability assessment of sample EBs after freezing and thawing may help in assessing the post-freezing and post-thawing developmental potential of other blastocysts at different stages from the same batch.     

Effect of prooxidant agents added at the morula/blastocyst stage on bovine embryo development,cell death and glutathione content

Two prooxidant agents, 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), a generator of free radicals in the culture medium, and buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, were used to reinforce from the morula stage (day 5 post-insemination, p.i.) the oxidative stress encountered by bovine embryos in culture. Exposure to increasing concentrations of both prooxidants from the morula stage did not affect blastocyst formation but some blastocysts were found degenerated on day 8 in a dose-dependent manner (0, 0.001, 0.01, 0.1 mM AAPH gave respectively 0, 10%, 32%, 48% degeneration, while 0, 0.1, 0.2, 0.4 mM BSO led respectively to 0, 14%, 30%, 41% degeneration). Hatching rates and cell numbers of surviving blastocysts were not affected. Morulae and early blastocysts exposed from day 5 to day 6 p.i. appeared more resistant than expanded blastocysts (75-80% survival vs 20-65%; p < 0.05). Treatment with BSO significantly decreased the level of reduced glutathione in day 7 blastocysts (0.02 vs 0.42 pmol per embryo in the control) while AAPH had no effect (0.38 pmol per embryo). The proportion ofcells showing membrane lesions was increased in degenerated blastocysts from day 7.5 p.i. In AAPH-treated, but not in BSO-treated embryos, cell membrane permeabilisation seems to occur before blastocyst degeneration. DNA fragmentation evaluated by the TUNEL technique was increased in day 7 blastocysts by both prooxidants (2.8 +/- 0.4 in the control group vs 4.5 +/- 0.4 and 6.0 +/- 0.4 respectively in the AAPH- and BSO-treated groups). Addition of an inhibitor of caspase-3, DEVD-CHO, partially prevented DNA fragmentation, indicating that prooxidant treatment induced a caspase-dependent pathway of apoptosis.     

Effect of maturation media and oocytes derived from sows or gilts on the development of cloned pig embryos

In order to develop a culture system and recipient cytoplasm that could improve the developmental competence of somatic cell nuclear transfer (SCNT) embryos for successful cloning of pigs, we evaluated the effect of donor oocytes and in vitro maturation (IVM) media on maturation of oocytes and developmental competence of SCNT embryos. In Experiment 1, oocytes derived from sows or gilts were matured in two IVM media (TCM-199 versus NCSU-23) and maturation of oocytes was evaluated by the status of chromatin configuration, the diameter of matured oocytes, the thickness of the zona pellucida, and the size of the perivitelline space (PVS). Sow oocytes matured in TCM-199 (S-TCM group) and NCSU-23 (S-NCSU group) showed significantly higher (P<0.05) maturation rates (S-TCM and S-NSCU, 86+/-4 and 82+/-4%, respectively) when evaluated by metaphase-II status than the gilt oocytes matured in TCM-199 (G-TCM group, 71+/-3%) and in NCSU-23 (G-NCSU-23 group, 71+/-3%). Oocyte diameter, the thickness of the zona pellucida, and the perivitelline space of sow oocytes (S-TCM and S-NCSU) were larger than those of gilt oocytes (G-TCM and G-NCSU) after IVM (P<0.05). In Experiment 2, SCNT was performed, using in vitro-matured oocytes from each group as recipient cytoplasm and porcine fetal fibroblasts as karyoplasts. The reconstructed embryos were electrically fused and activated, and cleavage and blastocyst formation were monitored under a stereomicroscope. The total cell number of flattened blastocysts stained with 5 microM bisbenzimide on day 7 were counted. In addition, in vitro matured non-enucleated oocytes were also electrically activated (parthenogenetic activation) and pronuclear formation was monitored. No difference in pronuclear formation rate after parthenogenetic activation and fusion rate after SCNT was observed among experimental groups. A significantly higher cleavage rate (P<0.05) was observed in S-TCM (69+/-4%) when compared with only G-NCSU (58+/-4%), but not with G-TCM (60+/-4%) or S-NCSU (68+/-4%). The rate of blastocyst formation was significantly higher (P<0.05) in sow oocytes (24% in S-TCM and S-NCSU), when compared to that observed in G-TCM (15%), and G-NCSU (14%). When the same source of oocytes was used, there was no significant difference in rate of blastocyst formation in the two culture media. Total cell number of blastocysts were not significantly different among experimental groups. In conclusion, the present study clearly demonstrated that sow oocytes have a greater developmental competence than gilt oocytes, regardless of the maturation medium examined.     

In vitro development to blastocysts of early porcine embryos produced in vivo or in vitro

The objective of this study was to compare the development of porcine embryos from the 2- and 4-cell stages to the blastocyst stage after in vivo or in vitro fertilization and in vivo or in vitro culture. Early-stage embryos were collected either from superovulated gilts 36 h after the second mating or after in vitro fertilization (IVF) of in vivo-matured oocytes, both followed by in vitro culture to the blastocyst stage. Blastocysts collected from superovulated donors served as controls. In the first experiment, a total of 821 2- and 4-cell embryos derived from in vivo-fertilized oocytes was cultured either in medium NCSU 23, modified Whittens' medium or modified KRB for 5 d. Significantly (P < 0.05 and P < 0.001) more embryos overcame the 4-cell block and developed to the blastocyst stage in medium NCSU 23 than in the 2 other culture media. Hatching was only observed in medium NCSU 23. In the second experiment, embryos derived from in vivo-matured oocytes fertilized in vitro were cultured in medium NCSU 23. Of 1869 mature oocytes 781 (41.8%) cleaved within 48 h after in vitro fertilization. A total of 715 embryos was cultured to the morula and blastocyst stages, and 410 (57.3%) overcame the developmental block stage, with 358 embryos (50.1%) developing to the morula and blastocyst stages. None of the embryos hatched, and the number of nuclei was significantly (P < 0.05) lower compared with that of in vivo-fertilized embryos (18.9 +/- 9.8 vs 31.2 +/- 5.8). In the third experiment, 156 blastocysts derived from in vitro fertilization and 276 blastocysts derived from in vivo fertilization and in vitro culture were transferred into synchronized recipients, while 164 blastocysts were transferred immediately after collection into 6 recipients, resulting in a pregnancy rate of 83.3%, with 35 piglets (on average 7.0) born. From the in vitro-cultured embryos, 58.3% (7/12) of the recipients remained pregnant at Day 35 after transfer, but only 33.3% maintained pregnancy to term, and 14 piglets (on average 3.5) were born. In contrast, the transfer of embryos derived from in vitro-fertilized oocytes did not result in pregnancies. It is concluded that 1) NCSU 23 is superior to modified Whittens' medium and modified KRB and 2) blastocysts derived from in vitro fertilization have reduced viability as indicated by the lower number of nuclei and failure to induce pregnancy upon transfer into recipients.     

Viable piglets generated from porcine oocytes matured in vitro and fertilized by intracytoplasmic sperm head injection

Intracytoplasmic sperm injection (ICSI) of a nonmotile cell into the ooplasm for assisted fertilization is a highly specialized procedure for producing the next generation. The production of piglets by ICSI has succeeded when in vivo-matured oocytes have been used as recipients. Our objective was to generate viable piglets by using porcine oocytes matured in vitro and fertilized by ICSI after evaluating the efficacy of using donor spermatozoa in which the acrosome had been artificially removed by treatment with calcium ionophore A23187 (Ca-I). The rate of acrosomal loss in spermatozoa was increased significantly as the duration of treatment with 10 micro M Ca-I was prolonged for 30-120 min (Ca-I treated; 55.6-78.6%), whereas the rate was not different as the duration of incubation without Ca-I was prolonged for 30-120 min (control; 45.3-58.4%). On the sixth day of in vitro culture after injection of the sperm head and subsequent stimulation with an electrical pulse, the rates of blastocyst formation were not significantly different between the two groups: the rates for oocytes injected with Ca-I-treated sperm heads (incubated for 120 min) and for those injected with control sperm heads were 8.6% and 4.0%, respectively. The mean cell numbers of the blastocysts were not significantly different between the two groups (25.6 and 22.7, respectively). Within 2 h after the stimulation, the injected oocytes were transferred to estrous-synchronized recipients. The three recipients that received oocytes injected with Ca-I-treated sperm heads (77-150 oocytes per recipient) were not pregnant, whereas two of the four recipients given oocytes injected with control sperm heads (55-100 oocytes per recipient) were pregnant. One of these farrowed three (a male and two female) healthy piglets. The results demonstrate clearly that in vitro-matured oocytes injected with sperm heads are developmentally competent and can produce viable piglets. They also suggest that removal of the acrosome from the spermatozoon before injection does not affect the development of the blastocyst in vitro. This might not also improve the production of piglets in vivo.     

Effect of genetic merit, milk yield, body condition and lactation number on in vitro oocyte development in dairy cows

The effects of milk yield, body condition score (BCS) and lactation number on the number of oocytes recovered and blastocysts formed were studied following in vitro maturation, fertilization and culture of bovine oocytes collected from 48 high and 46 medium genetic merit dairy cows in their first and third lactation. The cows were slaughtered between 125 and 229 d post partum. Ovaries were recovered, and 2- to 10-mm follicles were aspirated. Cleavage rate and number of blastocysts were determined at 44 h and 7 d after insemination, respectively. Oocytes from high genetic merit cows formed fewer blastocysts and had lower cleavage and blastocyst formation rates than those from medium genetic merit cows (0.36 +/- 0.19, 70.4 and 6.8% vs 0.85 +/- 0.22, 77.4 and 11.4%, respectively). The effect of milk production was tested by grouping cows in their third lactation into high and low groups. There was no difference in number of oocytes recovered and subsequent development into blastocysts between the cows in the high milk production group (4559 to 5114 kg, n = 20) and cows in the low yield (3162 to 3972 kg, n = 20) group (6.9 +/- 1.34 vs 8.9 +/- 1.32, respectively). The effect of BCS was tested by grouping cows in their first or third lactation into high and low groups. Cleavage and blastocyst formation rates were greater for oocytes from cows with a high BCS (3.3 to 4.0, n = 20) than a low BCS (1.5 to 2.5, n = 20) (75.7 vs 61.9% and 9.9 vs 3.0%, respectively). Cows in the first lactation yielded fewer oocytes (5.7 +/- 1.24) than cows in the third lactation (7.8 +/- 0.79). Thus, the quality of oocytes probably contributes to reduced fertility, often evident in high genetic merit dairy cows.     

Successful piglet production by IVF of oocytes matured in vitro using NCSU-37 supplemented with fetal bovine serum

Recently, piglets have been obtained from in vitro-produced blastocysts by using in vitro maturation systems in which oocytes have been matured in North Carolina State University (NCSU) solution supplemented with porcine follicular fluid (PFF). However, PFF is not available commercially. To prepare PFF from the ovaries required time and effort and there is substantial variation in quality among batches. Furthermore, PFF is considered a potential source of infectious agents. We evaluated another commercially available potential protein source, fetal bovine serum (FBS), for in vitro maturation, to produce embryos and piglets. Cumulus-oocyte complexes were matured in NCSU-37 with PFF or with one of four batches of FBS. The proportions of oocytes with expanded cumulus cells were lower in all FBS batch groups (P < 0.05, 15-41%) than that in the PFF group (74%). The proportions of oocytes that matured were also lower in all FBS batch groups (P < 0.05, 26-41%) than in the PFF group (73%), irrespective of cumulus expansion. However, the proportions of oocytes that underwent germinal vesicle breakdown were almost the same in all groups (76-96%). After in vitro fertilization, the rate of sperm penetration into matured oocytes was higher in the PFF group (P < 0.05, 63%) than in one batch of FBS (22%) and removal of the compacted cumulus cells after maturation did not affect fertilization status (21%). Subsequent in vitro embryo culture of the PFF and FBS groups for 6 day resulted in similar rates of blastocyst formation (17 and 19%, respectively) and similar numbers of cells per blastocyst (43 and 46 cells, respectively). When blastocysts obtained from oocytes matured with FBS were transferred into two recipients, one became pregnant and farrowed seven piglets. Transfer of blastocysts obtained from oocytes matured with PFF into two other recipients resulted in one pregnancy and production of four piglets. These data suggested that porcine in vitro maturation in NCSU-37 supplemented with FBS reduced the maturational ability of oocytes, but once oocytes have matured, they have the same ability to develop to term after in vitro fertilization and embryo transfer as those matured with PFF.     

Regenerated bovine fetal fibroblasts support high blastocyst development following nuclear transfer

Regenerated bovine fetal fibroblast cells were derived from a fetus cloned from an adult cow and passaged every 2-3 days. Serum starvation was performed by culturing cells in DMEM/F-12 supplemented with 0.5% FCS for 1-3 days. In vitro matured bovine oocytes were enucleated by removing the first polar body and a small portion of cytoplasm containing the metaphase II spindle. Cloned embryos were constructed by electrofusion of fetal fibroblast cells with enucleated bovine oocytes, electrically activated followed by 5 h culture in 10 microg/mL cycloheximide + 5 microg/mL cytochalasin B, and then cultured in a B2 + vero-cell co-culture system. A significantly higher proportion of fused embryos developed to blastocysts by day 7 when nuclei were exposed to oocyte cytoplasm prior to activation for 120 min (41.2%) compared to 0-30 min (28.2%, p < 0.01). Grade 1 blastocyst rates were 85.1% and 73.3%, respectively. The mean number of nuclei per grade 1 blastocyst was significantly greater for 120 min exposure (110.63 +/- 7.19) compared to 0-30 min exposure (98.67 +/- 7.94, p < 0.05). No significant differences were observed in both blastocyst development (37.4% and 30.6%) and mean number of nuclei per blastocyst (103.59 +/- 6.6 and 107.00 +/- 7.12) when serum starved or nonstarved donor cells were used for nuclear transfer (p > 0.05). Respectively, 38.7%, 29.4%, and 19.9% of the embryos reconstructed using donor cells at passage 5-10, 11-20 and 21-36 developed to the blastocyst stage. Of total blastocysts, the percentage judged to be grade 1 were 80.9%, 79.2%, and 54.1%, and mean number of nuclei per grade 1 blastocysts, were 113.18 +/- 9.06, 100.04 +/- 6.64, and 89.25 +/- 6.19, respectively. The proportion of blastocyst percentage of grade 1 blastocysts, and mean number of nuclei per grade 1 blastocyst decreased with increasing passage number of donor cells (p < 0.05). These data suggest that regenerated fetal fibroblast cells support high blastocyst development and embryo quality following nuclear transfer. Remodeling and reprogramming of the regenerated fetal fibroblast nuclei may be facilitated by the prolonged exposure of the nuclei to the enucleated oocyte cytoplasm prior to activation. Serum starvation of regenerated fetal cells is not beneficial for embryo development to blastocyst stage. Regenerated fetal fibroblast cells can be maintained up to at least passage 36 and still support development of nuclear transfer embryos to the blastocyst stage.     

Growth retardation of inner cell mass cells in polyspermic porcine embryos produced in vitro

The in vitro viability of polyspermic pig eggs was investigated. Immature oocytes were matured and fertilized in vitro. Approximately 10 h after insemination, the eggs were centrifuged at 12 000 x g for 10 min and individually classified into two (2PN)- and poly-pronuclear (PPN, 3 or 4 pronuclei) eggs. The classified eggs were cultured in vitro or in vivo. Nuclei numbers of inner cell mass (ICM) and trophectoderm (TE) were compared between 2PN- and PPN-derived blastocysts. The frequency of development in vitro of 2PN and PPN eggs to the blastocyst stage was 53.6% and 40.7%, respectively. The mean number (8.2 +/- 0.7, n = 48) of ICM nuclei of 2PN-derived blastocysts was higher than that (4.2 +/- 0.8, n = 37) of PPN-derived blastocysts (p < 0.001), whereas there was no difference (p > 0.05) in mean numbers of total (46.7 +/- 3.4 vs. 39. 9 +/- 3.9) and TE nuclei (38.5 +/- 2.9 vs. 35.7 +/- 3.3) between the two groups. Development of 2PN and PPN eggs cultured in vivo to the blastocyst stage was 33.3% and 27.4%, respectively. The numbers of ICM and TE nuclei of these embryos cultured in vivo showed a pattern similar to that for the in vitro-produced blastocysts. Additionally, fetuses were obtained on Day 21 from both the 2PN and the PPN groups. This suggests that polyspermic pig embryos develop to the blastocyst stage and beyond, although showing a smaller ICM cell number as compared to normal embryos.     

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.