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.     

In vitro production of llama (Lama glama) embryos by intracytoplasmic sperm injection: effect of chemical activation treatments and culture conditions

Assisted reproductive technologies in the llama (Lama glama) are needed to provide alternative methods for the propagation, selection and genetic improvement; however, recovery of adequate quantity and quality of spermatozoa for conventional IVF is problematic. Therefore, an effort was made to adapt the intracytoplasmic sperm injection (ICSI) procedure for the in vitro production of llama embryos. The specific objectives of this study were: (1) to determine in vitro maturation rates of oocytes recovered by transvaginal ultrasound-guided oocyte aspiration (TUGA) or flank laparotomy; (2) to evaluate the effects of activation treatments following ICSI; (3) to evaluate the development of llama ICSI embryos in CR1aa medium or in an oviduct cell co-culture system. Llamas were superstimulated by double dominant follicle reduction followed by oFSH administered in daily descending doses over a 3-day interval. Oocytes were harvested by flank laparotomy or TUGA and matured in vitro for 30 h. Mature oocytes were subjected to ICSI followed by no chemical activation (Treatment A), ionomycin only (Treatment B) or ionomycin/DMAP activation (Treatment C). More oocytes were recovered by flank laparotomy procedure compared with TUGA (94% versus 61%, P<0.05) and a greater number of oocytes harvested by flank laparotomy reached the metaphase-II stage (77% versus 44%, P<0.05). After ICSI, the proportion of cleaved and 4-8-cell stages embryos was significantly greater when injected oocytes were activated with ionomycin/DMAP combination (63% and 38%, respectively, P<0.05). The co-culture of ICSI embryos with llama oviduct epithelial cells resulted in progression to morula (25%) and blastocyst (12%) stages; whereas, all embryos cultured in CR1aa medium arrested at the 8-16-cell developmental stage.     

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.     

Oocyte activation and parthenogenetic development of bovine oocytes following intracytoplasmic sperm injection.

Development of bovine oocytes after intracytoplasmic sperm injection (ICSI) was investigated. Oocytes were matured for 24-26 h in vitro and injected with isolated sperm heads. When treated with 7% ethanol (v/v) for 5 min, 71.7% of ICSI oocytes were activated as shown by the resumption of meiosis and the formation of female pronuclei. However, 41.5% of injected sperm heads remained condensed at 18-20 h after injection into the ooplasm. The incidence of decondensing sperm and that of male pronuclei at this stage were 15.1% and 26.4%, respectively. A total of 55.5% of oocytes reached the 2-cell stage following sperm head injection and 54.7% after sham-ICSI; these percentages were not significantly different from those following in vitro fertilisation (IVF) (73.1%). The percentage of 2-cell embryos reaching the 8-cell stage following ICSI was 37.5%, and 27.6% after sham-ICSI, which were significantly lower (p < 0.01) than the equivalent percentage following IVF (62.4%). The percentages of parthenogenetic embryos reaching the 2-cell, 4-cell and 8-cell stages following ICSI were 56.4%, 48.9% and 30.0%, respectively. These results indicate that the low rate of normal embryonic development of bovine oocytes following ICSI is largely due to the parthenogenetic activation of the oocytes.     

In vitro developmental competence of ICSI-derived activated ovine embryos

The present study was conducted to determine the necessity for activation after intracytoplasmic sperm injection (ICSI) in sheep. The effect of chemical stimulation with either 5 μM ionomycin (I) for 5 min or ionomycin + 2 mM 6-dimethylaminopurine (6-DMAP) for 3 h on the efficiency of ICSI, was compared in six experimental groups: (1) ICSI, (2) ICSI + I, (3) ICSI + I + 6DMAP, (4) Sham, (5) Sham + I, and (6) parthenogenetics (Sham and parthenogenetic groups were used as controls). In the present study, ovine oocytes needed additional chemical stimulation, after conventional ICSI, to activate (female pronucleous formation) and to form zygotes with male and female pronuclei (2PN). The percentage of cleaved embryos obtained and developed to blastocyst stage was higher (P < 0.001) for ICSI-derived zygotes, followed by activation (I and I + 6DMAP; 18.2 and 22.5%, respectively) than ICSI and Sham injection without activation (3.0 and 0.0%, respectively). There was, however, no significant difference between activation protocols I or I + 6DMAP. Furthermore, there was no significant difference among chemically activated, ICSI-derived zygotes in term of hatchability rate; however, the percentage was significantly higher in parthenogenetic and IVF groups than ICSI and Sham injection. In conclusion, neither sperm alone nor mechanical activation was sufficient for ovine oocyte activation and pronuclei formation. Therefore, in our study conditions for in vitro embryo development, chemical activation of oocytes must be considered an essential part of the ICSI procedure in sheep.     

Fertilization of mouse oocytes from in vitro-matured preantral follicles using classical in vitro fertilization or intracytoplasmic sperm injection

Early preantral mouse follicles with a diameter of 110-160 microm were cultured in vitro for 10 or 12 days. Mature oocytes were retrieved following hCG, and fertilization was attempted either by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Two-cell and blastocyst formation rates and blastocyst cell numbers were compared between 10-day and 12-day in vitro-matured oocytes versus in vivo-matured oocytes. Uncleaved IVF oocytes were subjected to chromosome analysis. The 2-cell formation rate was significantly improved by ICSI compared with IVF both in 10-day (72.1% versus 56.1%; P = 0.03) and 12-day cultures (74.1% versus 54.5%; P = 0.028). Cytogenetic analysis of uncleaved MII oocytes following IVF showed that about 30% of MII oocytes showed no sign of sperm penetration. The blastocyst formation rate was significantly lower in 12-day versus 10-day cultures, whether fertilization was by IVF (40.7% versus 62.4%, P = 0.016) or by ICSI (32.5% versus 57.1%, P = 0.035). Blastocyst cell numbers from IVF and ICSI 10-day groups were similar and both significantly higher (P < 0.001) than from IVF 12-day cultures. All above expressed values were significantly higher for in vivo-matured oocytes. In conclusion, fertilization of oocytes from in vitro-matured mouse preantral follicles can be optimized with ICSI, giving significantly higher 2-cell formation rates than IVF. Blastocyst formation rate was not influenced by the technique of fertilization but rather by the extent of the in vitro culture period. Best results on preimplantation development of oocytes for in vitro-matured preantral follicles were obtained with ICSI on oocytes from 10-day in vitro cultures.     

Development of sheep androgenetic embryos is boosted following transfer of male pronuclei into androgenetic hemizygotes

Androgenetic embryos are useful model for investigating the contribution of the paternal genome to embryonic development. Little work has been done with androgenetic embryo production in domestic animals. The aim of this study was the production of diploid androgenetic sheep embryos. In vitro matured sheep oocytes were enucleated and fertilized in vitro; parthenogenetic and normally fertilized embryos were also produced as a control. Fifteen hours after in vitro fertilization (IVF), presumptive zygotes were centrifuged and scored for the number of pronucleus. IVF, parthenogenetic, and androgenetic embryos (haploid, diploid, and triploid) were cultured in SOFaa medium with bovine serum albumin (BSA). The proportion of oocytes with polyspermic fertilization increased linearly with increasing sperm concentration. After IVF, there was no significant difference in early cleavage and morula formation rates between the groups, while there was a significant difference on blastocyst development between IVF, parthenogenetic, and androgenetic embryos, the last ones displaying poor developmental potential (IVF, parthenogenetic, and haploid, diploid, and triploid androgenetic embryos: 43%, 38%, 0%, 2%, and 2%, respectively). In order to boost androgenetic embryonic development, we produced diploid androgenetic embryos through pronuclear transfer. Single pronuclei were aspirated with a bevelled pipette from haploid or diploid embryos and transferred into the perivitelline space of other haploid embryos, and the zygotes were reconstructed by electrofusion. Fusion rates approached 100%. Pronuclear transfer significantly increased blastocyst development (IVF, parthenogenetic, androgenetic: Diploid into Haploid, and Haploid into Haploid: 42%, 42%, 19%, and 3%, respectively); intriguingly, the Haploid + Diploid group showed the highest development to blastocyst stage. The main findings of our study are: (1) sheep androgenetic embryos display poor developmental ability compared with IVF and parthenogenetic embryos; (2) diploid androgenetic embryos produced by pronuclear exchange developed in higher proportion to blastocyst stage, particularly in the Diploid-Haploid group. In conclusion, pronuclear transfer is an effective method to produce sheep androgenetic blastocysts.     

Developmental potential of oocytes fertilized by conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) after cryopreservation and mesometrial autotransplantation of rabbit ovarian tissue

Objective: To evaluate mesometrial transplantation of frozen-thawed ovarian tissue in rabbit and to choose the optimized fertilization method for oocytes retrieved from grafts by investigating the capability of oocyte fertilization and further development. Forty rabbits were divided into three groups randomly: control group, fresh tissues transplantation group and frozen-thawed tissues transplantation group. Three months after the transplantation, rabbits were stimulated with FSH and oocytes were retrieved 13 h after human chorionic gonadotropin (HCG) injection. Oocytes matured in vivo or in vitro were then fertilized by conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), followed by observation and evaluation of fertilization rate and blastocyst formation rate. Blastocytes embryos were transferred to pseudopregnancy rabbits to observe pregnancy rate and birth rate. There were no significant differences in the percentage of oocytes matured either in vivo or in vitro among the three groups. The fertilization rate, cleavage rate and blastocyst formation rate of in vivo-matured oocytes had no difference among the three groups, whether they were fertilized by IVF or ICSI. Significantly higher fertilization rates of in vitro-matured oocytes were observed with ICSI compared with IVF in each group. The blastocyst formation rate of in vitro-matured oocytes was significantly lower than that of in vivo-matured oocytes in each group. The birth rate of in vivo-matured oocytes was significantly higher than that of in vitro-matured oocytes, although the pregnancy rate was similar between them. Mesometrial transplantation of frozen-thawed ovarian tissue may provide favorable conditions for follicle development. Oocytes retrieved from mesometrial grafts can develop to the blastocyst stage and produce live offspring. ICSI can optimize the fertilization rate of in vitro-matured oocytes retrieved from grafts.     

Embryo development of prepubertal goat oocytes fertilised by intracytoplasmic sperm injection (ICSI) according to oocyte diameter

The aim of this study was to evaluate embryo development of prepubertal goat oocytes fertilised by ICSI according to their diameter. Three experiments were carried out to achieve this objective. In all experiments, oocytes were matured in TCM199 supplemented with hormones, cysteamine and serum for 27 h at 38.5 degrees C. In Experiment 1, we studied the nuclear stage of goat zygotes produced by conventional ICSI and IVF using 20 nM ionomycin plus 10 microM heparin as sperm treatment. A group of Sham-injected oocytes was used as control. Results showed differences in the percentage of 2 PN (zygotes with male and female pronuclei) between ICSI, IVF and Sham (40.9, 26.6 and 3.0%, respectively; P<0.05). In Experiment 2, we evaluated the embryo development of prepubertal goat oocytes produced by ICSI and IVF after 192 h of culture in SOF medium. The percentage of morulae plus blastocysts obtained was higher in the ICSI than in the IVF group (13.4 and 5.1%, respectively; P<0.05). In Experiment 3, IVM-oocytes were classified in four groups depending on their diameter (Group A: <110 microm; Group B: 110-125 microm; Group C: 125-135 microm; Group D: >135 microm), fertilised by ICSI and cultured for 192 h. Results showed a positive correlation between oocyte diameter and embryo development (morulae+blastocysts: Group A: 0%; Group B: 6.2%; Group C: 46.4% and Group D: 33.3%). In conclusion, sperm treatment with ionomycin plus heparin using the conventional ICSI protocol improved fertilisation rates in comparison to IVF. Oocytes smaller than 125 microm were unable to develop up to blastocyst stage.