Intracytoplasmic sperm injection (ICSI) has been successfully achieved in mice and rats using a piezo-driven injection pipette. More than 30% of ICSI oocytes are capable of developing to full-term when the isolated sperm heads are microinjected. The ICSI technique has been applied not only to rescue infertile male strains, but also to produce transgenic rodents. ICSI-mediated DNA transfer, which mixes sperm heads and exogenous DNA solution and co-injects them into ooplasm, was as effective as conventional pronuclear DNA microinjection. The production efficiency of transgenic founders by ICSI-mediated DNA transfer was comparable between mice and rats, while the optimal DNA concentration for 1-min exposure was lower in rats than in mice. The production efficiency was improved when the membrane structure of sperm heads was partially disrupted by detergent or ultrasonic treatment before exposure to the exogenous DNA solution. Exogenous DNAs with various chain lengths have been stably integrated into rodent genomes of various genetic backgrounds using this method. ICSI-mediated DNA transfer in which preparation of pronuclear-stage fertilized zygotes is not required would be alternative to conventional pronuclear DNA microinjection.

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Attempts to freeze-dry spermatozoa are not new. Successful conception and full-term development using freeze-dried spermatozoa were reported in the cow and rabbit prior to 1960. However, these preliminary results have not been confirmed. Spermatozoa become defective in motility after freeze-drying and are unable to fertilize eggs both in vivo and in vitro. However, in 1998, freeze-drying of mammalian spermatozoa was demonstrated without loss of genetic or reproductive potential. Freeze-dried spermatozoa support normal development when injected directly into oocytes by means of an intracytoplasmic sperm injection (ICSI) technique. Recent investigations of freeze-dried sperm have focused on the factors affecting freeze-drying in spermatozoa in an effort to make it possible to store the male gamete at ambient temperature. The pressure level at primary dryied of freeze-drying spermatozoa, in addition to the components of the suspending solution, appears to be an important factor for long-term preservation of freeze-dried spermatozoa.

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In vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are highly specialized procedures used for artificial production of successive generations of domestic animals. However, in pigs, the efficacy of these procedures in generating viable embryos is still poor. We focus here on both IVF and ICSI in pigs in relation to the physiology of fertilization (morphological changes and nuclear remodeling). This knowledge should contribute to the development of advanced technologies for in vitro production of zygotes that have the potential for full-term development to the offspring stage after transfer to recipients.

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In general, in vitro-matured oocytes and commercially available frozen semen are used for bovine intracytoplasmic sperm injection (ICSI), unlike ICSI in humans and experimental animals, which uses in vivo-matured oocytes and fresh semen. Bovine ICSI is also characterized by difficulties in pronuclear formation. Therefore, increasing in vitro development of produced ICSI embryos is considered to necessitate use of an artificial activation treatment after injection of motile sperm. However, because parthenogenetic embryos are found amongst produced ICSI embryos, it is necessary to establish a bovine ICSI protocol that leads to normal karyomorphism.

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The cytogenetic normality and developmental competence of mouse oocytes derived from prepubertal females were investigated to determine ooplasmic maturity after in vitro fertilization (IVF) and to examine the ability of the resultant blastocysts to develop to term. To estimate the effect of body weight and age on ovulation, prepubertal female (BALB/c × C57BL/6J) F₁ mice, 20-30 days of age, were classified into two groups according to body weight as follows: a light group (L) of 8.5-12 g and a heavy (H) group of 13-16 g. The IVF blastocysts were fixed as chromosome samples, and some of the blastocysts were transferred to pseudopregnant recipients. The implantation rates and number of fetuses were subsequently evaluated at 20 days post-coitus. The average number of ovulated oocytes differed significantly, with 46.2, 18.1, and 37.1 in the H, L, and control groups, respectively. All fertilization rates were high, and there were no significant differences. However, 16.8% of zygotes from the L group were arrested at the 1-cell stage, with mostly male premature condensed chromosomes (PCC). The rate of development to the blastocyst stage was significantly low in the L group (36.6%). The rates of implantation and development to newborns were significantly lower in the H group than in the pubertal mice.

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This study was conducted to compare the quantity of total protein in miniature pig sperm extract (SE) extracted by two methods [sonication and a method using Mammalian Protein Extraction Reagent (M-PER)]. Furthermore, the effects of injection with SE extracted by M-PER and fractionated SE on porcine oocyte activation were examined. The total amount of protein extracted from miniature pig spermatozoa (1 × 10⁹ sperm) by M-PER was significantly (P<0.05) larger than that extracted by sonication. When the porcine oocyte activation rate after injection with SE extracted by M-PER at different protein concentrations was examined, the highest activation (31.4%) was induced by injection with 0.08 mg/ml SE, and the value was significantly (P<0.05) higher than the control (buffer injection). When the effect of injection with fractionated SE (>100 kDa and <100 kDa) was examined, the activation rates of the control (non-fraction), >100 kDa, and <100 kDa groups were 63.6, 48.7, and 46.4%, respectively. Significant differences (P<0.05) were observed between the control and <100 kDa groups. These results indicate that SE extracted by M-PER from miniature pig sperm is effective for porcine oocyte activation. Oocyte activation could be induced efficiently by injection with SE using M-PER. Both <100 kDa SE and >100 kDa SE contain effective materials for porcine oocyte activation.

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To clarify the causes of low viability of hamster embryos following in vitro culture, the present study compared the distributions of mitochondria and the cytoskeleton in embryos grown in vivo and in vitro. Hamster 2- and 4-cell embryos were characterized by perinuclear clustering of mitochondria, the degree of which was almost the same for in vivo and in vitro embryos. In the cell cortex and cell-to-cell contact region, however, microfilaments were located less densely in in vitro embryos than in in vivo ones. The nucleus moved towards the apex of the blastomere at the late 8-cell stage, when embryos begin the process of compaction. The density of mitochondria seemed to increase in the cell-to-cell contact region during this cellular rearrangement. Mitochondria were concentrated at the perinuclear region of in-vivo 8-cell embryos, whereas they were diffused into the subcortical region of in-vitro 8-cell embryos. Such a diffusion pattern of mitochondrial distribution was also noted in the morulae and blastocysts grown in vitro. These results show that both mitochondrial translocation and cytoskeletal reorganization did not proceed normally in the hamster embryos cultured in vitro, probably resulting in decreased viability of these embryos.

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We predicted the success or failure of fertilization by the presence or absence of a second polar body 6 hours after insemination, and we performed intracytoplasmic sperm injection (ICSI) for oocytes that did not produce a second polar body to investigate the efficacy of such rescue ICSI. Cases in which ICSI was performed as scheduled after oocyte collection were classified together as the ICSI group. Those in which the second polar body was confirmed in at least half of the oocytes 6 hours after insemination by in vitro fertilization (IVF) were classified as the IVF group, and those in which the second polar body was not confirmed in at least half of the oocytes 6 hours after insemination and on which ICSI was thus performed were classified as the rescue ICSI group. The fertilization rates in the ICSI group, IVF group and rescue ICSI group were 73.8%, 75.4% and 69.0% without significant differences. Pregnancy rates were 41.0%, 41.0% and 29.8%, in the respective groups without significant differences. Our results strongly suggest that cancellation of embryo transfer is avoided by rescue ICSI when IVF results in all unfertilized oocytes.

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