Optimization of parthenogenetic activation protocol in porcine

The effects of the electrical field strengths, number of pulses, and post-activation media on chromatin conformation and parthenogenetic development were studied to optimize the activation protocol for porcine nuclear transfer. In experiment 1, electrical field strengths were examined. Oocytes were subjected to square direct current pulses at output voltages of 1.2, 1.7, 2.2, and 2.7 kV/cm for 1 x 30 microsec. The voltage resulting from experiment 1 was 2.2 kV/cm, in which 50.0% of activated oocytes developed to blastocysts in vitro. In experiment 2, the influence of 1, 2, and 3 pulses on blastocyst development was tested using field strengths and post-activation medium described in experiment 1. Oocytes activated by a single 30 microsec pulse of 2.2 kV/cm DC yielded a higher blastocyst rate (56.3%) than oocytes activated by 2 or 3 pulses (<42.5%). In experiment 3 and 4, we investigated the effects of cytochalasin B (CB), cycloheximide (CH), and CB + CH on nuclear development stages and parthenogenetic development following a single 30 microsec pulse of 2.2 kV/cm DC. The percentage of activated oocytes was not different among CB (93.3%), CB + CH (98.3%), control (80.0%), and CH (80.0%) groups 12 hr after activation. Treatment with CB (57.5%) or CB + CH (53.8%) enhanced the blastocyst rate compared with other groups, CH (23.8%) treated- and control group (18.8%). The results demonstrated that a single 30 microsec pulse of 2.2 kV/cm DC followed by culturing in post-activation medium with CB for 5 hr were effective parameters for parthenogenetic activation and blastocyst formation of in vitro matured porcine oocytes which suggests that a single calcium rise is sufficient to activate pig oocytes and to achieve high rate of blastocyst development.     

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.     

Effect of sperm immobilisation and demembranation on the oocyte activation rate in the mouse.

To analyse the effect of the state of the sperm plasma membrane on oocyte activation rate following intracytoplasmic sperm injection (ICSI), three types of human and mouse spermatozoa (intact, immobilised and Triton X-100 treated) were individually injected into mouse oocytes. At 30, 60 and 120 min after injection, maternal chromosomes and sperm nuclei within oocytes were examined. Following human sperm injection, the fastest and the most efficient oocyte activation and sperm head decondensation occurred when the spermatozoa were treated with Triton X-100. Intact spermatozoa were the least effective in activating oocytes. Thus, the rate of mouse oocyte activation following human sperm injection is greatly influenced by the state of the sperm plasma membrane during injection. When mouse spermatozoa were injected into mouse oocytes, the rates of oocyte activation and sperm head decondensation within activated oocytes were the same irrespective of the type of sperm treatment prior to injection. We witnessed that live human spermatozoa injected into moue oocytes often kept moving very actively within the ooplasm for more than 60 min, whereas motile mouse spermatozoa usually became immotile within 20 min after injection into the ooplasm. In 0.002% Triton X-100 solution, mouse spermatozoa are immobilised faster than human spermatozoa. These facts seem to suggest that human sperm plasma membranes are physically and biochemically more stable than those of mouse spermatozoa. Perhaps the physical and chemical properties of the sperm plasma membrane vary from species to species. For those species whose spermatozoa have 'stable' plasma membranes, prior removal or 'damage' of sperm plasma membranes would increase the success rate of ICSI.     

Effects of fusion/activation methods on development of embryos produced by nuclear transfer of porcine fetal fibroblast

The present studies were carried out to investigate the effects of intensity of dc pulse, number of dc pulse and equilibration before fusion/activation on developmental ability of porcine embryos derived from nuclear transfer. In experiment 1, different fusion/activation intensity (two dc pulses of 0.4, 0.8, 1.2, 1.6 and 2.0 kV/cm for 30 micros, respectively) was carried out to investigate development of embryos. In experiment 2, the reconstructed oocytes were fused and activated with one, two or three dc pulses of 1.2 kV/cm for 30 micros. In experiment 3, reconstructed oocytes were equilibrated in TCM-199 medium for 0-6 h, respectively, and fused/activated with one dc pulse of 1.2 kV/cm for 30 micros. The reconstructed embryos were cultured in PZM-3 medium containing 0.3% BSA. When oocytes were fused with donor cell by two dc pulses of 0.4 kV/cm for 30 micros, the rates of cleavage and blastocyst formation were significantly lower (32.9% and 2.5%) than those of fused by 0.8 kV/cm (59.0% and 17.4%) or 1.2 kV/cm (63.3% and 18.4%), respectively. One dc pulse of 1.2 kV/cm for 30 micros was enough to fuse and activate embryos to develop to blastocyst (24.8%). Equilibration for 2-3 h in TCM-199 before fusion/activation was beneficial for improving the developmental ability of embryos produced by nuclear transfer (25.6-23.3% at blastocysts).     

Viability of ICSI oocytes after caffeine treatment and sperm membrane removal with Triton X-100 in pigs

Non-adequate decondensation of injected sperm nucleus is one the main problems of intracytoplasmic sperm injection (ICSI) in porcine. With the aim of improving pronuclear formation, the effects on activation and embryo development rates of 0.1% Triton X-100 (TX) sperm pre-treatment for membrane removal and/or 5 mM Caffeine (CAF) addition in oocyte manipulating and culture medium for 2 h after ICSI or artificial activation were studied. The effects of 4 different Ca²⁺ concentrations contained in the injection medium on embryo development after sham injection were also analysed. In Experiment 1, no significant effect on cleavage or blastocyst rate was detected independently of Ca²⁺ concentration contained in the injection medium. In Experiment 2, oocytes injected with TX pre-treated sperm showed a significant higher rate of male pronuclear formation in comparison with oocytes from control group (2PN; 54.1 vs 36.6%). However, no differences on in vitro embryo development, cleavage or blastocyst rates were observed. In Experiment 3, oocytes treated with CAF during and after micromanipulation and injected with sperm pre-treated with TX had a significantly lower oocyte activation rate than any other experimental groups (25.7 vs 56.3–66.3%). No differences were observed in cleavage rates among different experimental groups. However, the CAF group showed a higher blastocyst rate significantly different from TX+CAF group (12.0 vs 1.9%, respectively). In a second approach, the effect of electric field strengths and CAF treatments on oocyte activation was studied. In Experiment 4, oocytes submitted to 0.6 kV/cm showed significant higher activation rates than 1.2 kV/cm ones regardless of the caffeine treatment (83.7 vs 55.9% and 75.7 vs 44.3%; in control and caffeine groups, respectively). No effect of caffeine treatment was observed in any experimental group. In conclusion, TX sperm treatment before ICSI without an additional activation procedure improved male pronuclear formation, but did not improve embryo development until blastocyst stage. No significant effect of caffeine was found when sperm was not treated with TX, although in membrane absence caffeine avoided oocyte activation and embryo development. Finally, caffeine had no effect on female pronuclear formation regardless of electric field strengths applied to the parthenogenetic activation.     

Viability of ICSI oocytes after caffeine treatment and sperm membrane removal with Triton X-100 in pigs

Non-adequate decondensation of injected sperm nucleus is one the main problems of intracytoplasmic sperm injection (ICSI) in porcine. With the aim of improving pronuclear formation, the effects on activation and embryo development rates of 0.1% Triton X-100 (TX) sperm pre-treatment for membrane removal and/or 5 mM Caffeine (CAF) addition in oocyte manipulating and culture medium for 2 h after ICSI or artificial activation were studied. The effects of 4 different Ca²⁺ concentrations contained in the injection medium on embryo development after sham injection were also analysed. In Experiment 1, no significant effect on cleavage or blastocyst rate was detected independently of Ca²⁺ concentration contained in the injection medium. In Experiment 2, oocytes injected with TX pre-treated sperm showed a significant higher rate of male pronuclear formation in comparison with oocytes from control group (2PN; 54.1 vs 36.6%). However, no differences on in vitro embryo development, cleavage or blastocyst rates were observed. In Experiment 3, oocytes treated with CAF during and after micromanipulation and injected with sperm pre-treated with TX had a significantly lower oocyte activation rate than any other experimental groups (25.7 vs 56.3-66.3%). No differences were observed in cleavage rates among different experimental groups. However, the CAF group showed a higher blastocyst rate significantly different from TX+CAF group (12.0 vs 1.9%, respectively). In a second approach, the effect of electric field strengths and CAF treatments on oocyte activation was studied. In Experiment 4, oocytes submitted to 0.6 kV/cm showed significant higher activation rates than 1.2 kV/cm ones regardless of the caffeine treatment (83.7 vs 55.9% and 75.7 vs 44.3%; in control and caffeine groups, respectively). No effect of caffeine treatment was observed in any experimental group. In conclusion, TX sperm treatment before ICSI without an additional activation procedure improved male pronuclear formation, but did not improve embryo development until blastocyst stage. No significant effect of caffeine was found when sperm was not treated with TX, although in membrane absence caffeine avoided oocyte activation and embryo development. Finally, caffeine had no effect on female pronuclear formation regardless of electric field strengths applied to the parthenogenetic activation.     

精子和卵母细胞质量控制对山羊卵胞质内精子注射（ICSI）受精的影响

ABSTRACT Effects of sperm and oocyte quality control on the efficiency of ICSI of in vitro matured goat oocytes were studied in this paper. The results showed that when injected intracytoplasmically, spermatozoa from caput, corpus and cauda epididymidis resulted in similar rates of fertilization, cleavage and morulae/blastocysts, but when injected subzonally, spermatozoa from caput and corpus gave rise to significantly lower rates of fertilization and embryo development than spermatozoa from the cauda epididymidis and ejaculates. When dead spermatozoa collected from semen that had been preserved in different ways were used for ICSI, those dead from liquid storage at 20 degrees C for 24 h gave rise to the best, but those dead from liquid storage at 5 degrees C for 15 days produced the poorest fertilization and embryo development. When spermatozoa were treated with different concentrations of Triton X-100 before ICSI, significantly higher rates of fertilization, cleavage and morulae/blastocysts were obtained with 0.0005% Triton X-100 than with other concentrations and manual immobilization. Oocytes were classified as of good and poor qualities by treatment in hypertonic sucrose solution, and rates of fertilization and embryo development were significantly higher in the good than in the poor oocytes after ICSI. Post-injection activation of oocytes with either A23187 or ionomycin/6-DMAP significantly increased the rates of fertilization, cleavage and morulae/blastocysts after ICSI. It is therefore concluded that (i) epididymal maturation mainly endowed spermatozoa with the capacity to fuse with the egg plasma membrane; (ii) different methods of semen storage caused different impairment of sperm fertilizing capacity; (iii) pre-injection treatment of spermatozoa with proper concentrations of Triton X-100 might be used to replace manual immobilization for ICSI; (iv) oocyte quality was a major factor influencing the efficiency of ICSI; (v) post-injection activation treatment of oocytes improved fertilization and embryo development after ICSI.     

Birth of normal calves after intracytoplasmic sperm injection of bovine oocytes: a methodological approach

Intracytoplasmic sperm injection (ICSI) is advantageous when only very few spermatozoa are available for insemination. Bovine spermatozoa were injected individually into matured oocytes using a piezo electric actuator. Spermatozoa were "immobilized", by scoring their tails immediately before injection, or "killed", by repeated freezing and thawing. About 4 h after ICSI, the oocytes with two polar bodies (activated by sperm injection) were selected and treated 5 min with 7% ethanol before further culture. When examined 19-21 h after ICSI, nearly 90% of the oocytes were fertilized normally (two pronuclei and two polar bodies) irrespective of the sperm treatment (immobilization or killing) prior to ICSI, but subsequent preimplantation embryo development was much superior (cleavage 72%: blastocysts 20%) after ICSI with immobilized spermatozoa than by using killed spermatozoa (cleavage 28%; blastocysts 1%). Ethanol activation of bovine oocytes with two polar bodies 4 h after ICSI improved the cleavage (33% versus 72%) and blastocyst (12% versus 20%) rates markedly (P < 0.05). Five normal calves were born after transplantation of ten blastocysts to ten surrogate cows. These results show that piezo-ICSI using immobilized spermatozoa, combined with ethanol treatment of sperm-injected oocytes, is an effective method to produce bovine offspring.     

Efficient in vitro production of porcine blastocysts by handmade cloning with a combined electrical and chemical activation

The purpose of our work was to establish an efficient protocol for activation of porcine cytoplast-fibroblast constructs produced by the handmade cloning technique. Firstly, we investigated a combined electrical and chemical activation protocol for parthenogenetic development of in vitro matured zona-free oocytes. Oocytes were activated by one 80 micros pulse and subsequently cultured in cytochalasin B and cycloheximide. Developmental rates of blastocysts from activated oocytes were 49+/-1 and 40+/-2%, when using one 80 micros pulse of 0.85 or 1.25 kV/cm, respectively. The activation procedure was further confirmed by a simultaneous re-fusion and activation of bisected oocytes, resulting in a blastocyst rate of 41+/-8%. Secondly, the activation protocol was applied in the handmade cloning technique. In vitro matured zona-free porcine oocytes were bisected and halves containing no chromatin, i.e. the cytoplasts, were selected. Reconstructed embryos were produced by a two-step fusion procedure. At the first step, one cytoplast was fused to one fibroblast by one 80 micros pulse of 1.25 kV/cm. After 1h, the cytoplast-fibroblast pair and another cytoplast were fused and activated simultaneously by one 80 micros pulse of 0.85 kV/cm, and subsequently cultured in cytochalasin B and cycloheximide. The development of reconstructed embryos to the blastocyst stage was in average 21+/-4%, and total blastocyst cell counts were in average 48+/-3. Thus, the combined electrical and chemical activation procedure resulted in efficient blastocyst development in the handmade cloning technique.     

Production of piglets using intracytoplasmic sperm injection (ICSI) with flowcytometrically sorted boar semen and artificially activated oocytes

The purpose of the present study was to develop a protocol for the successful production of piglets employing intracytoplasmic sperm injection (ICSI) with flowcytometrically sexed spermatozoa and artificially activated porcine oocytes. In vitro matured oocytes were fertilized by ICSI using non-sorted frozen/thawed epididymal semen. Oocytes were either activated by CaCl(2), Ca(2+)-ionophore or electrical pulse. Activation and fertilization rates of sperm injected oocytes stimulated by CaCl(2)-injection were significantly higher than those without activation (70.4% versus 45.9%; 49.9% versus 33.2%, respectively; P<0.001). Activation rate of sham injected oocytes increased in parallel (11.2% versus 26.3%, P<0.05), parthenogenetic development remained low (2.8% versus 8%). Co-incubation in Ca(2+)-ionophore did not improve activation rates as compared to non-activated oocytes (44.8% versus 42.5%). Fertilization rate decreased as compared to non-treated sperm injected oocytes (36.8% versus 24.5%, P<0.05). Activation of oocytes with a single electrical pulse resulted in significantly higher activation rates in all groups of oocytes as compared to non-stimulated ones (sperm injected oocytes: 65.6% versus 43.1%, P<0.001; sham injected oocytes: 48.5% versus 5.6%, P<0.001; control oocytes: 50.7% versus 0.0%, P<0.001). Fertilization rates (32.3% versus 48.2%) and parthenogenetic development (0.7% versus 38.9%, 0.0% versus 30.9%, P<0.001) increased significantly in parallel. In addition, in four replicates of flowcytometrically sorted Y-chromosome bearing spermatozoa were injected into in vivo matured oocytes, activated with 1.2 pl of a 30 mM CaCl(2) solution. On average 85.3 fertilized oocytes were transferred surgically into four recipients. Pregnancies delivered a total of 13 male piglets. These are the first piglets born from ICSI with sorted spermatozoa.     

Effects of electric field strengths on fusion and in vitro development of domestic cat embryos derived by somatic cell nuclear transfer

The present study was conducted to determine the effect of electric field strength on the rate of membrane fusion between the somatic cell and cytoplast and on subsequent in vitro development of reconstructed embryos. Additionally, the in vitro developmental competence of cat oocytes artificially activated after 44 h of maturation culture was examined. An efficient fusion rate (64.2%) was obtained by applying a single pulse of 1.5 kV/cm for 50 micros, and the fusion rate remained almost constant at the higher field intensity (59.8 and 54.9% at 1.7 and 2.0 kV/cm, respectively). Although the cleavage rate of fused embryos increased with an increase of the electric field strength, there were no differences among the groups with respect to the proportion of development to the morula and blastocyst stages. In the additional experiment, oocytes at the metaphase II stage after culture for 44 h were activated by the combination of calcium ionophore (CaI) with cycloheximide (CHX). Some (11.8%) of activated oocytes developed to the blastocyst stage. Results from this study indicated that electric field strength affects the rates of fusion and cleavage but has no significant effects on the development to the blastocyst stage of reconstructed embryos. Prolonged maturation culture of cat oocytes (up to 44 h) decreased their ability to develop to the blastocyst stage.     

Equine blastocyst development after intracytoplasmic injection of sperm subjected to two freeze-thaw cycles

This study was conducted to evaluate the effects of thawing, division into aliquots and refreezing on fertilizing capacity (ability to support embryo development after intracytoplasmic sperm injection; ICSI) of frozen stallion semen. Frozen semen from a fertile stallion was thawed, diluted 1:100 with freezing extender, and refrozen (2F treatment). Control semen was frozen only once. In vitro matured equine oocytes were injected with: (1) motile control spermatozoa; (2) motile 2F spermatozoa; (3) non-motile 2F spermatozoa; or (4) non-motile 2F spermatozoa, followed by injection of sperm extract. Blastocyst development after ICSI was equivalent between control spermatozoa and motile 2F spermatozoa (27 and 23%, respectively). Blastocyst development after injection of non-motile 2F spermatozoa (13%) tended (P=0.07) to be lower than that for control spermatozoa. Injection of sperm extract into oocytes that received non-motile 2F spermatozoa resulted in a significant decrease in blastocyst development (to 2%) compared with injection of non-motile 2F spermatozoa alone. Spermatozoa from a subfertile stallion was similarly processed and used for ICSI; blastocyst development for both motile control (once frozen) spermatozoa and motile 2F spermatozoa was 9%. In conclusion, frozen stallion semen may be thawed, diluted, and refrozen without effect on the ability of motile spermatozoa to initiate embryo development after ICSI. Non-motile spermatozoa from reprocessed semen may also achieve embryo development after ICSI. To our knowledge, this is the first report evaluating the ability of refrozen spermatozoa to produce embryos by ICSI in any species.     

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.     

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.     

Mouse ICSI with frozen-thawed sperm: the impact of sperm freezing procedure and sperm donor strain

Normal mouse offspring can be obtained from oocytes injected with frozen-thawed spermatozoa without cryoprotection, however, embryo development can be affected by sperm freezing procedure and sperm donor strain. In this study we observed that direct contact of mouse spermatozoa with liquid nitrogen did not affect their ability to activate injected oocytes but severely restricted subsequent in vitro embryo development to blastocyst stage. Tris-EDTA buffer and M2 were also shown to be better sperm freezing extenders than DPBS, allowing higher developmental potential. In addition, differences in embryo development obtained by intracytoplasmic sperm injection (ICSI) with frozen-thawed spermatozoa were observed between hybrid sperm donor strains. Frozen-thawed B6D2F1 spermatozoa provided higher embryo development than sperm cells from C57CBAF1.     

Failure of male pronucleus formation is the major cause of lack of fertilization and embryo development in pig oocytes subjected to intracytoplasmic sperm injection

The objectives of this study were 1) to compare the efficiency of intracytoplasmic sperm injection (ICSI) with and without additional artificial stimulation using frozen-thawed sperm and in vitro-matured porcine oocytes and 2) to determine the nuclear anomalies of ICSI oocytes that failed to fertilize or develop. In experiments 1 and 2, we evaluated the effects of additional activation treatments, e.g., electrical stimulus, Ca ionophore (A23187), and/or cycloheximide, on fertilization and development of ICSI porcine oocytes. Significantly higher fertilization, cleavage, and blastocyst rates were obtained for oocytes treated with a combination of ICSI and electrical activation (EA) (P < 0.05) than for those treated with ICSI alone. However, different combinations of electrical and chemical activation treatments did not further improve the rates of fertilization, cleavage, and blastocyst development for ICSI embryos. To elucidate the association between sperm head decondensation and oocyte activation and to investigate the cause of embryonic development failure, in experiment 3 we evaluated the nuclear morphology of oocytes 16-20 h after ICSI. Nearly 100% of oocytes showed female pronucleus formation after ICSI regardless of activation treatment. However, failure of male pronucleus formation with intact or swelling sperm heads was observed in some ICSI embryos, suggesting that these embryos underwent cell division with the female pronucleus only. Artificial activation (EA and A23187) had a beneficial effect on embryonic development, sperm decondensation was independent of the resumption of meiosis, and the failure of formation of a male pronucleus was the major cause for fertilization failure in porcine ICSI embryos.     

Activation, pronuclear formation, and development in vitro of pig oocytes following intracytoplasmic injection of freeze-dried spermatozoa

The fertilization of pig oocytes following intracytoplasmic injection of freeze-dried spermatozoa was evaluated. Activation and male pronuclear (MPN) formation were better in oocytes injected with isolated freeze-dried sperm heads than whole freeze-dried spermatozoa, but cleaved embryos were generally difficult to develop to the morula or blastocyst stage. When spermatozoa were freeze-dried for 24 h, oocyte activation and MPN formation in activated oocytes after sperm head injection were inhibited. Embryo development to the blastocyst stage was only obtained after injecting sperm heads isolated from spermatozoa freeze-dried for 4 h and stored at 4 degrees C. The proportion of embryos that developed to the blastocyst stage was not increased by the treatment of injected oocytes with Ca ionophore (5-10 microM). Increasing the sperm storage time did not affect oocyte activation or MPN formation, but blastocyst development was observed only after 1 mo of storage. These results demonstrate that pig oocytes can be fertilized with appropriately freeze-dried spermatozoa and that the fertilized oocytes can develop to the blastocyst stage.     

Live rats resulting from injection of oocytes with spermatozoa freeze-dried and stored for one year

This study was designed to examine whether rat spermatozoa after freeze-drying and 1-year storage can participate in full-term development following intracytoplasmic sperm injection (ICSI). Cauda epididymal spermatozoa from Crlj:Wistar rats were frozen in liquid nitrogen (LN(2)), first dried for 14 hr at 0.37 hPa and then for 3 hr at 0.001 hPa. The dried spermatozoa were stored for 1 year in a desiccator at +25 degrees C, or in a refrigerator at +4 degrees C, or in LN(2) at -196 degrees C. Controls consisted of sperm that had only been frozen and stored in LN(2). After being stored, spermatozoa were sonicated to dissociate the sperm tail and were injected into oocytes from superovulated Slc:SD rats. The respective fertilization rates of oocytes injected with frozen sperm, or with freeze-dried sperm stored at +25, +4, and -196 degrees C were 79%, 75%, 70%, and 73%. However, the corresponding cleavage rates of injected oocytes were 63%, 1%, 38%, and 36%. After transfer of >80 zygotes of each group into recipients, the respective percentages of full-term normal offspring resulting from frozen sperm or from freeze-dried sperm stored at +25, +4, and -196 degrees C were 36%, 0%, 7%, and 14%. These results demonstrate that the storage temperature significantly influenced the likelihood of term development of rats produced by injection of oocytes with freeze-dried spermatozoa. Chromosomal analysis of the rat spermatozoa in the ICSI oocytes indicated that chromosomal aberration in freeze-dried spermatozoa stored at +25 degrees C (100%) occurred more frequently than in frozen control spermatozoa (41%) and freeze-dried spermatozoa stored at -196 degrees C (35%), and the frequency of chromosomal aberrations in freeze-dried spermatozoa stored at +4 degrees C (65%) was the intermediate. In conclusion, rat spermatozoa freeze-dried and stored at +4 degrees C for 1 year are capable of participating in full-term development after ICSI.     

Intracytoplasmic sperm injection of bovine oocytes with stallion spermatozoa

Five experiments were designed to study the fertilizability and development of bovine oocytes fertilized by intracytoplasmic sperm injection (ICSI) with stallion spermatozoa. Experiment 1 determined the time required for pronuclear formation after ICSI. Equine sperm head decondensation began 3 h after ICSI; 42% were decondensed 6 h after ICSI. Male pronuclei (MPN) began to form 12 h after ICSI. Female pronuclei (FPN), however, formed as early as 6 h after ICSI. In Experiment 2, ionomycin, ionomycin plus 6-dimethylaminopurine (DMAP), and thimerosal were used to activate ICSI ova. None of the ICSI ova cleaved after treatment with thimerosal. Ionomycin activation after 24 and 30 h of oocyte maturation resulted in 29 and 48% cleavage rates, respectively. Ionomycin combined with DMAP resulted in 49, 6 and 3% cleavage, morula and blastocyst rates, respectively, when oocytes were activated after 24 h maturation. In Experiment 3, rates of cleavage (45-60%) and development to morulae (4-13%) and blastocysts (1-5%) stages following ICSI were not different (P>0.05) among three stallions. Treatment of stallion spermatozoa with ionomycin did not affect cleavage or development of ova fertilized by ICSI. The chromosomal constitution of blastocysts derived from ICSI was bovine, not bovine and equine hybrids. In Experiment 4, to make male and FPN form synchronously, colchicine and DMAP were used for 4 h to inhibit oocytes at metaphase during activation; 63% of oocytes were still at metaphase 8h after ICSI when treated with colchicine, and 50% of sperm nuclei were decondensed. About 18 h after ICSI, 21 and 50% male and FPN had formed, respectively, but cleavage rates were low, and only 1% developed to morulae. In Experiment 5, to test if capacitated equine sperm could fuse with the bovine oolemma, capacitated spermatozoa were injected subzonally (SUZI). Of the 182 SUZI oocytes, 49 (27%) contained extruded second polar bodies. After activation of oocytes with second polar bodies, 44, 22 and 15% developed to 2-, 4- and 8-cell stages, respectively, but development stopped at the 8-cell stage. None of the unactivated oocytes cleaved. In conclusion, equine spermatozoa can decondense and form MPN in bovine oocytes after ICSI, but subsequent embryonic development is parthenogenetic with only bovine chromosomes being found.     

Fertilisability of ovine, bovine or minke whale (Balaenoptera acutorostrata) spermatozoa intracytoplasmically injected into bovine oocytes

This study was conducted to investigate the possibility of using bovine oocytes for a heterologous fertility test by intracytoplasmic sperm injection (ICSI) and to compare the pronuclear formation of ram, bull and minke whale spermatozoa after injection into bovine oocytes. Bovine oocytes were cultured in vitro for 24 h and those with a polar body were selected for ICSI. Frozen-thawed semen from the three species were treated with 5 mM dithiothreitol for 1 h and spermatozoa were killed by storing them in a -20 degrees C refrigerator before use. ICSI was performed using a Piezo system. Three experiments were designed. In experiment 1, a higher (p < 0.05) male pronuclear formation rate was found in the oocytes injected with ram (52.6%) or bull (53.4%) spermatozoa than with minke whale spermatozoa (39.1%). In experiment 2, sperm head decondensation was detected at 2 h after ICSI in the oocytes injected with a spermatozoon of each species. Male pronuclei were first observed at 4 h in the oocytes injected with ram or bull spermatozoa and at 6 h in oocytes injected with minke whale spermatozoa. The mean diameters of male pronuclei derived from both whale and bull spermatozoa were larger than those from ram spermatozoa (30.4 microm and 28.3 microm vs 22.4 microm, p < 0.005). The mean diameter of female pronuclei in the oocytes injected with whale spermatozoa was also larger than with ram spermatozoa (29.3 microm vs 24.7 microm, p < 0.05). The development of male and female pronuclei was synchronous. In experiment 3, ethanol-activated oocytes injected with a spermatozoon from any of the three species achieved significantly higher (p < 0.05-0.001) cleavage rates than control oocytes. Blastocyst formation was only observed when bull spermatozoa were used. The results of this study indicate that dead foreign spermatozoa can participate in fertilisation activities in bovine oocytes after ICSI.