Effects of oocyte activation and sperm preparation on the development of porcine embryos derived from in vitro-matured oocytes and intracytoplasmic sperm injection

The objective was to determine the effects of various methods of oocyte activation and sperm pretreatment on development of porcine embryos derived from in vitro-matured oocytes and intracytoplasmic sperm injection (ICSI). The second polar body was extruded in the majority (>78.4%) of in vitro-matured (IVM) oocytes 4h after electrical pulse activation. In embryos generated by ICSI and sham-ICSI, a combination of an electrical pulse, with various chemical activators 4 h later, improved (P < 0.05) blastocyst formation rate compared to activation only with a pulse. Treatment with 6-dimethylaminopurine (DMAP) after electrical activation significantly increased the oocyte activation rate. The effects of exposure of sperm to repeated freeze-thaw cycles (without cryoprotectant) on oocyte activation and the effects of sperm pre-incubated with dithiothreitol (DTT) or Triton X-100 on early embryo development were also examined. Blastocyst formation rates after ICSI did not differ between motile sperm and those rendered immotile by one-time freezing and thawing without cryoprotectant. However, sperm rendered immotile by three cycles of freezing/thawing without cryoprotectant had a significantly lower blastocyst formation rate. Although oocytes injected with sperm pre-incubated with Triton X-100 had a higher normal fertilization rate than those pre-incubated with DTT or one-time frozen/thawed sperm, rates of blastocyst formation and cell numbers were similar among the three groups. In conclusion, various methods of oocyte activation and sperm preparation significantly affected the developmental capacity of early porcine embryos derived from IVM and ICSI.     

Normal reproductive development of offspring derived by intracytoplasmic injection of porcine sperm grown in host mice

For establishment of gonadal xenografting, it is essential to clarify whether offspring derived from gametes grown in host mice harboring xenografts have normal reproductive development. This study examined the secretory profiles of gonadal hormones in relation to sexual maturation or ovarian cyclicity in pigs generated by intracytoplasmic sperm injection using xenogeneic sperm (Xeno-ICSI pigs, four males and one female). We also assessed the developmental activity of gametes obtained from these pigs using in vitro culture systems, or by mating with conventionally produced (conventional) pigs. During the growth of male Xeno-ICSI pigs, serum inhibin and testosterone concentrations were generally within ranges for those hormones in conventional pigs. Histologically, there were no differences in the growth and differentiation of seminiferous tubules between Xeno-ICSI and conventional pigs. Parameters of semen quality, including volume, pH, sperm concentration, and the percentage of motile sperm were not different from those in conventional pigs. Among the Xeno-ICSI pigs, individual differences were noted in the ability of sperm to penetrate oocytes and to produce blastocysts. However, oocytes after in vitro fertilization using these sperm developed into blastocysts containing more than 31 cells. One conventional sow delivered 12 piglets after being mated with a male Xeno-ICSI pig. During growth of the female Xeno-ICSI pig, serum progesterone concentrations had a sudden increase at 41 wk of age, suggesting CL formation. After puberty, this animal showed cyclic changes in the serum concentrations of progesterone and inhibin, and delivered 10 piglets after AI using fresh sperm obtained from a conventional boar. In conclusion, these findings demonstrated that both male and female Xeno-ICSI pigs had normal reproductive abilities.     

Non-surgical deep intra-uterine transfer of in vitro produced porcine embryos derived from sex-sorted frozen-thawed boar sperm

Embryos and offspring of a pre-determined sex have been produced in pigs using AI and IVF with unfrozen sperm, and after surgical insemination with sex-sorted frozen-thawed sperm. The aims of this study were to demonstrate that sex-sorted frozen-thawed boar sperm could be incorporated into pig IVF for the production of embryos of a pre-determined sex and that these embryos could be successfully non-surgically transferred. Oocytes were matured in vitro, fertilised with either unsorted or sex-sorted frozen-thawed sperm and cultured until the eight-cell stage. These embryos were then transferred to recipients (n = 7) non-surgically (n = 70 embryos per sow). Oocyte cleavage was similar between sex-sorted (1538/5044; 30.5%) and unsorted (216/756; 28.6%) frozen-thawed sperm, and PCR sex-determination of the embryos confirmed that they were of the predicted sex (n = 16). Delayed return to oestrus (>23 days) was observed in five recipient sows (71.4%). Fetal sacs were observed by transcutaneous ultrasound on Day 18 in one of these sows. Pre-sexed porcine IVP embryos can be successfully produced using sex-sorted frozen-thawed boar sperm, and these embryos are capable of initiating pregnancies when transferred to recipients. However, further refinement of porcine ET protocols are required to enable development to term.     

Cryopreservation of porcine embryos derived from in vitro-matured oocytes

This study describes a cryopreservation method for porcine in vitro-produced (IVP) embryos using as a model parthenogenetic embryos derived from in vitro-matured (IVM) oocytes. IVP embryos at the expanded blastocyst stage were cryopreserved by vitrification using the minimum volume cooling (MVC) method and exhibited an embryo survival rate of 41.2%. Survival was then significantly improved (83.3%, P < 0.05) by decreasing the amount of cytoplasmic lipid droplets (delipation) prior to vitrification. IVP embryos at the 4-cell stage also survived cryopreservation when vitrified after delipation (survival rate, 36.0%), whereas post-thaw survival of nondelipated embryos was quite low (9.7%). Furthermore, it was demonstrated that porcine IVP morulae can be cryopreserved by vitrification following delipation by a noninvasive method (survival rate, 82.5%). These results clearly confirm that porcine embryos derived from IVM oocytes can be effectively cryopreserved with high embryo survival using the MVC method in conjunction with delipation.     

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.     

Births of calves derived from embryos produced by intracytoplasmic sperm injection without exogenous oocyte activation

Tail-cut bovine spermatozoa were microinjected into ooplasmic lipid polarised, in vitro matured bovine oocytes using a piezomicropipette-driving system. No exogenous oocyte activation treatment was used. Of the sperm-injected oocytes, 86.3% were activated, 71.8% cleaved and 22.7% developed to the blastocyst stage. The average cell count of the blastocysts was 122.5 +/- 15 and a majority (81.8%) of the blastocysts were cytologically normal (diploid). When transferred to recipient cows, 5 of 8 blastocysts developed to fetuses and 4 of 7 recipients became pregnant. Normal offspring were born.     

Intracytoplasmic sperm injection in bovine: effects of oocyte activation, sperm pretreatment and injection technique

Intracytoplasmic sperm injection (ICSI) is a very important technique for treating male subfertility and for basic research. The efficiency of ICSI in bovine is very limited because of the necessity for additional oocyte activation before or after the ICSI procedure. In this study, we compared the effects of seven different protocols on activation and fertilization rates of bovine oocytes after ICSI and on their subsequent development under in vitro conditions. The protocols include 1) different chemical activation of oocytes, 2) pretreated or nonpretreated sperm, and 3) conventional or Piezo-driven injection techniques. In all three groups, ICSI, sham-injected, and noninjected, the highest activation rates were obtained after treatment of oocytes with ionomycin followed by 6-dimethylaminopurine (6-DMAP). Using this treatment for oocyte activation, 59% of oocytes were activated and 31% of oocytes were fertilized using dithiothreitol (DTT) pretreated spermatozoa and Piezo-driven injection. Using the protocols with the same oocyte activation or activation with calcium ionophore (Ca-I) and cycloheximide (CHX), nonpretreated sperm, and conventional injection technique, early cleavage rate (79.6% and 77.6%, respectively) were significantly (P <0.01) higher when compared with all other protocols. The latter protocol resulted in 8% blastocyst and 90% of the obtained blastocysts were found to be diploid. Our results demonstrate that activation of oocytes, sperm treatment, and injection technique separately or together could improve the success of bovine ICSI.     

Effects of dithiothreitol and boar on pronuclear formation and embryonic development following intracytoplasmic sperm injection in pigs

The objective of this study was to improve normal fertilization, male pronuclear formation and embryonic development following intracytoplasmic injection of dithiothreitol (DTT)-treated boar spermatozoa. To determine the effect of DTT treatment, frozen-thawed boar spermatozoa were treated with DTT for 0, 10, 30, and 60 min, and injected into porcine oocytes. The effects of DTT and male difference on normal fertilization and embryonic development were investigated. The mean normal fertilization rate in the groups treated with DTT for 30 min (73.8%) and 60 min (74.9%) was higher (P < 0.05) than that in the control group (49.3%). The mean blastocyst formation rate in the group treated with DTT for 30 min (23.2%) was higher (P < 0.05) than that in the other groups (8.7-10.9%). Among boars there was no difference in normal fertilization, but there was a significant difference between the non-treated and the DTT-treated groups. The mean rate of blastocyst formation was different (P < 0.05) among boars, and between the non-treated and DTT-treated groups. The mean number of cells in blastocysts was similar among the boars and between the non-treated and the DTT-treated groups. In conclusion, DTT treatment for 30 min increased the rate of normal fertilization and embryonic development to the blastocyst stage. Furthermore, the rate of blastocyst formation of oocytes injected with spermatozoa differed among boars.     

Chordin is required for neural but not axial development in sea urchin embryos

The oral-aboral (OA) axis in the sea urchin is specified by the TGFβ family members Nodal and BMP2/4. Nodal promotes oral specification, whereas BMP2/4, despite being expressed in the oral territory, is required for aboral specification. This study explores the role of Chordin (Chd) during sea urchin embryogenesis. Chd is a secreted BMP inhibitor that plays an important role in axial and neural specification and patterning in Drosophila and vertebrate embryos. In Lytechinus variegatus embryos, Chd and BMP2/4 are functionally antagonistic. Both are expressed in overlapping domains in the oral territory prior to and during gastrulation. Perturbation shows that, surprisingly, Chd is not involved in OA axis specification. Instead, Chd is required both for normal patterning of the ciliary band at the OA boundary and for development of synaptotagmin B-positive (synB) neurons in a manner that is reciprocal with BMP2/4. Chd expression and synB-positive neural development are both downstream from p38 MAPK and Nodal, but not Goosecoid. These data are summarized in a model for synB neural development.     

Cleavage, development and competence of sheep embryos fertilized by intracytoplasmic sperm injection and in vitro fertilization

More abnormal fertilization has been found in sheep oocytes after intracytoplasmic sperm injection (ICSI) than after in vitro fertilization (IVF). Although the birth of a normal lamb has been reported, the efficiency of blastocyst production is low. We therefore evaluated the cleavage, development and viability of sheep embryos obtained from ICSI, IVF and sham injection. In vitro matured oocytes either injected or inseminated with spermatozoa were assessed for cleavage 1 and 4 d after injection or insemination, and for development to blastocyst after 7 d of culture. A total of 699 oocytes was injected (ICSI); 198 (30.6%) were activated and 55 (8.5%) developed to the blastocyst stage. Of the 17 recipient ewes with 1, 2, 3 or 4 embryos, 15 (88.2%) were pregnant on Day 18; of these 17 recipients, 7 (41.1%) and 6 (35.2%) ewes remained pregnant on Days 45 and 110, respectively. Two normal lambs were born, one ewe died on Day 110 with 2 normal male fetuses, another ewe aborted on Day 90 and 4 pregnancies were maintained. A total of 517 oocytes was inseminated (IVF); 296 (62%) were activated and 90 (18.8%) reached the blastocyst stage. A total of 19 ewes received 1, 2, 3 or 4 embryos; of these, 13 (68.4%) were pregnant on Day 18, 8 (42.1%) ewes remained pregnant on each of Days 45 and 110. Three ewes delivered 5 lambs. Five pregnancies were maintained. A total of 156 oocytes was sham injected, 38 (24.3%) were activated and no blatocysts were obtained after culture. The results of this study showed that blastocysts obtained after ICSI are potentially viable and are not a result of parthenogenesis.     

Effects of oocyte age, cumulus cells and injection methods on in vitro development of intracytoplasmic sperm injection rabbit embryos

This study assessed the effects of oocyte age, cumulus cells and injection methods on in vitro development of intracytoplasmic sperm injection (ICSI) rabbit embryos. Oocytes were recovered from female rabbits superovulated with PMSG and hCG, and epididymal sperm were collected from a fertile male rabbit. The oocyte was positioned with the first polar body at 12 o'clock position, and a microinjection needle containing a sperm was inserted into the oocyte at 3 o'clock. Oolemma breakage was achieved by aspirating ooplasm, and the aspirated ooplasm and sperm were re-injected into the oocyte. The injected oocytes were cultured in M199 medium containing 10% fetal calf serum at 38 degrees C with 5% CO2 in air. The results showed that oocytes injected at 1 h post-collection produced a higher (p < 0.05) fertilization rate than those injected at 4 or 7 h post-collection. Blastocyst rate in the 1 h group was higher (p < 0.05) than in the 7 h group. Denuded oocytes (group A) and oocytes with cumulus cells (group B) were injected, respectively. Rates of fertilization and development of ICSI embryos were not significantly different (p > 0.05) between the two groups. Four ICSI methods were applied in this experiment. In methods 1 and 2, the needle tip was pushed across half the diameter of the oocyte, and oolemma breakage was achieved by either a single aspiration (method 1) or repeated aspiration and expulsion (method 2) of ooplasm. In methods 3 and 4, the needle tip was pushed to the oocyte periphery opposite the puncture site, and oolemma breakage was achieved by either a single aspiration (method 3) or repeated aspiration and expulsion (method 4) of ooplasm. Fertilization rate in method 2 was significantly higher (p < 0.05) than in methods 1 and 3. Blastocyst rates were not significantly different (p > 0.05) among methods 1, 3 and 4, but method 2 produced a higher (p < 0.05) blastocyst rate than method 3.     

Germinal vesicle materials are not required for the activation of MAP kinase in porcine oocyte maturation

The requirement of the germinal vesicle (GV) for the normal kinetics of mitogen-activated protein (MAP) kinase activity during porcine oocyte maturation was investigated. Porcine follicular oocytes were enucleated, and the locations of their extracellular signal-regulated kinases 1 and 2 (ERK1/2), major MAP kinases in maturating porcine oocytes, were detected by indirect immunofluorescent microscopy. The MAP kinase activity was assayed as myelin basic protein (MBP) kinase activity, and the phosphorylation states of ERK1/2 were detected by immunoblotting analyses. Translocation of MAP kinase into the GV and association with the spindle were observed in intact oocytes, while MAP kinase in enucleated oocytes was distributed almost uniformly in cytoplasm throughout the culturing period. The phosphorylation and the activation of MAP kinase were induced, and the activity was comparable with that of control denuded oocytes. The high level of activity was maintained through maturation, even in the absence of spindle formation. These results indicate that the presence of nuclear material and translocation into the GV are dispensable for the activation of MAP kinase and that associating with the spindle is not required for maintenance of its activity though porcine oocyte maturation.     

Cyclin E/Cdk2 is required for sperm maturation, but not DNA replication, in early sea urchin embryos

The cell cycle is driven by the activity of cyclin/cdk complexes. In somatic cells, cyclin E/cdk2 oscillates throughout the cell cycle and has been shown to promote S-phase entry and initiation of DNA replication. In contrast, cyclin E/cdk2 activity remains constant throughout the early embryonic development of the sea urchin and localizes to the sperm nucleus following fertilization. We now show that cyclin E localization to the sperm nucleus following fertilization is not unique to the sea urchin, but also occurs in the surf clam, and inhibition of cyclin E/cdk2 activity by roscovitine inhibits the morphological changes indicative of male pronuclear maturation in sea urchin zygotes. Finally, we show that inhibition of cyclin E/cdk2 activity does not block DNA replication in the early cleavage cycles of the sea urchin. We conclude that cyclin E/cdk2 activity is required for male pronuclear maturation, but not for initiation of DNA replication in early sea urchin development.     

Effects of chemically defined medium on early development of porcine embryos derived from parthenogenetic activation and cloning

The present study was to investigate if a completely chemically defined medium (PZM-4) could support the early development of porcine embryos derived from parthenogenetic activation (PA) and cloning (somatic cell nuclear transfer, SCNT), and to lay the foundation for determining the physiological roles of certain supplements in this medium. Porcine embryos derived from PA and SCNT were cultured in media: PZM-3 (a chemically semi-defined medium), PZM-4 (a fully defined medium), and PZM-5 (an undefined medium). Early embryo development was observed. We found that the three medium groups (PZM-3, PZM-4 and PZM-5) exhibited no significant differences in cleavage rates of PA embryos (p > 0.05), while the blastocyst rate in PZM-3 was significantly higher than in PZM-4 and PZM-5 (78.9% vs. 36.0% and 52.3%) (p < 0.05). Moreover, total cell number per blastocyst in PZM-3 was clearly higher than in PZM-5 but similar to that in PZM-4. As for SCNT embryos, no significant differences were observed for the cleavage rates or the blastocyst rates among the three groups (p > 0.05). However, total cell number per blastocyst in PZM-3 was notably higher than in PZM-5, but was similar to that in PZM-4. In conclusion, our results suggested that the completely chemically defined medium PZM-4 can be used to efficiently support the early development of porcine PA and SCNT embryos.     

Neurotrophins are not required for normal embryonic development of olfactory neurons

Neurons of the vertebrate olfactory epithelium (OE) regenerate continuously throughout life. The capacity of these neurons to regenerate and make new and precise synaptic connections in the olfactory bulb provides a useful model to study factors that may control or mediate neuronal regeneration. Expression and in vitro studies have suggested potential roles for the neurotrophins in the olfactory system. To directly examine whether neurotrophins are required for olfactory neuron development, we characterized in vivo the role of the neurotrophins in the primary olfactory system. For this, we generated mutant mice for TrkA, TrkB, TrkC, and also for BDNF and NT3 together with P2-IRES-tau-LacZ trangenic mice. Histochemical staining for beta-galactosidase at birth allowed in vivo analysis of the P2 subpopulation of olfactory neurons as well as their projections to the olfactory bulb. Our data indicate that Trk signaling is not required for normal embryonic development of the olfactory system.     

Fragmentation and development of preimplantation porcine embryos derived by parthenogenetic activation and nuclear transfer

Fragmentation occurs during early developmental stages of electrically activated oocytes and nuclear transfer (NT) embryos. It might contribute to the low developmental rate of porcine NT embryos. The present study was conducted to investigate whether the addition of sugars such as sorbitol or sucrose suppresses fragmentation and supports the development of electrically activated oocytes and NT embryos. The activated oocytes were cultured in Porcine Zygote Medium-3 (PZM-3) supplemented with sorbitol or sucrose for 2 days after electric activation, and then cultured in the PZM-3 for the remaining 4 days. The osmolarities of PZM-3, PZM-3 supplemented with 0.05 or 0.1 M sorbitol, and PZM-3 with 0.05 M sucrose were 269 +/- 6.31, 316 +/- 3.13, 362 +/- 4.37, and 315 +/- 5.03 mOsm, respectively. When parthenogentically activated oocytes were cultured in PZM-3 supplemented with 0.05 M sorbitol or sucrose for the first 2 days and then cultured in PZM-3 without sugar, a significantly higher (P < 0.05) cleavage rate and blastocyst rate were observed. Interestingly, addition of sugar to PZM-3 for 2 days reduced the fragmentation rate compared to PZM-3 without sugar. In NT embryos, sugar addition into PZM-3 increased the fusion rate (84.2% +/- 6.07 vs. 95.1% +/- 2.52), cleavage rate (67.6% +/- 5.80 vs. 77.3% +/- 3.03), and developmental rate to the blastocyst stage (10.2% +/- 0.79 vs. 19.4% +/- 1.77). There was no significant difference between treatments for the number of the blastocysts. In addition the fragmentation rate was reduced compared to PZM-3 without sorbitol (26.1 +/- 4.30 vs. 14.5 +/- 1.74). In conclusion, increasing the osmolarity of PZM-3 through addition of either sorbitol or sucrose for 48 hr increased the cleavage and developmental rate to the blastocyst stage by reducing the fragmentation rate through increasing osmolarity.     

Sperm transfer during mating, movement of sperm in the female reproductive tract, and sperm precedence in the common cutworm Spodoptera litura

Abstract. Mating behaviour, sperm transfer and sperm precedence were studied in the moth Spodoptera litura (Fabr.) (Lepidoptera: Noctuidae). There existed a rhythmic, diel pattern of mating behaviour of this moth during the scotophase, presumably set with respect to an endogenous activity rhythm. Approximately 30 min after copulation had started, the formation of the corpus of the spermatophore began in the bursa copulatrix of the female moth, but full inflation of the corpus was not completed until 45–60 min after mating had started. The mature spermatophore contained about 350 eupyrene sperm bundles and a large number of individual (loose) apyrene spermatozoa. The mating status and the age of the male insect influenced the number of sperm transferred to the female within the spermatophore, and also affected the consequent fertility. There was no evidence of sperm reflux within the male tract. Within the female, dissociation of eupyrene sperm bundles was evident within the spermatophore less than 15 min after the completion of mating. Spermatozoa began to move from the bursa (in which the spermatophore is lodged) into the spermatheca 30–45 min after the end of the copulation, and the quantity of sperm in the spermatheca reached a plateau at 90 min after mating. Apyrene sperm reached the spermatheca first, followed by eupyrene sperm. Examination of total (apyrene plus eupyrene) sperm in the female tract showed that 86% of mated females received an apparently normal amount of total sperm from the male. Examination of eupyrene sperm alone showed that 81% of matings resulted in an apparently normal transfer of eupyrene sperm. A small proportion (approximately 8%) of the matings, however, were identified as transferring a clearly subnormal quantity of eupyrene sperm to the spermatheca. The eggs produced as a result of such pairings displayed much reduced fertility (about 43%) compared to those from matings confirmed to have transferred normal quantities of sperm, which showed about 92% fertility. This shows that the availability of eupyrene sperm in the spermatheca may be an important constraint on fertility in normal populations of insects. In the laboratory, S. litura females exhibited multiple matings. Of the females, 93% mated, and the mean frequency of mating was 1.69. Mating with a fertile male led to the oviposition of an increased number of eggs. This effect continued even when the female subsequently mated with an infertile male. Displacement of sperm from previous matings is known to be an important factor in the evolution of multiple mating strategies. Our results on sperm utilization by S. litura indicated that after a second mating, the sperm utilized for subsequent fertilization were almost exclusively from the last mating with little mixing. The proportion of eggs fertilized by sperm from the second mating (P₂) was calculated as 0.95, indicating almost complete sperm precedence from the last mating.     

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.