[Key Points of Examination after Breast Augmentation and Our Clinic's Algorithm of Immediate Reconstruction Using Silicone Implant for Breast Cancer Patient after Breast Augmentation].

Recently, the cases of breast augmentation for cosmetic purposes are rapidly increasing, there are more opportunities to examine for patient with breast augmentation history than before. In some cases, breast cancer screening is difficult due to the effects of breast augmentation. At our clinic, even in cases diagnosed with breast cancer after breast augmentation, we actively perform immediate breast reconstruction using silicone implant. However, it is necessary to consider the condition and type of breast augmentation at the time of diagnosis and also treatment. We will share our algorithm for immediate breast reconstruction using silicone implant for breast cancer after augmentation mammaplasty.

Epidermoid Cyst of Orbit Requiring Cranialization of Frontal Sinus.

How often do intracranial epidermoid cysts occur?Is a coronary incision necessary?What are the steps of the procedure, difficulties encountered, and process for circumventing those difficulties?What is the follow-up protocol and outcome?

Usefulness of customized titanium plates for midface contouring surgery.

INTRODUCTION AND IMPORTANCE: In our department, we have been performing bone reconstructions on a case-by-case basis using vascularized free tissue transfers and custom-made artificial bones (HA). While these procedures have specific advantages, they are also limited in terms of the invasiveness as well as the stability and strength of implants. In the present study, we describe the use of a CTP to achieve minimally invasive midface plastic surgery with the superior moldability of a 3D structure and reliable stability compared to the use of autologous tissue. CASE PRESENTATION: A total of three patients were included in the study. The patients (all female, ages: 66, 18, and 35 years) had bone malformation or hemifacial microsomia following surgery for maxillary cancer or multiple facial fractures. Based on DICOM data from preoperative CT, 3D models were created on a computer using CAD/CAM techniques. The models were compared in simulations to determine the optimal structure. These 3D models were used in additive manufacturing systems to create custom-made titanium alloy plates for facial reconstruction. CLINICAL DISCUSSION: Although the amount of soft tissue was insufficient in some cases, all patients were able to maintain the desired morphology without developing any complications such as infections, significant soft tissue atrophy, or implant failure. CONCLUSION: Our CTP model created by CAD/CAM was effective in contouring surgery of the midface as it had the superior stability and biocompatibility of titanium. Changes to the soft tissue should also be considered in order to further improve the procedure.

What is the most anticipated change induced by treatment using gender-affirming hormones in individuals with gender incongruence?

OBJECTIVES: To identify the most eagerly anticipated change resulting from hormone therapy using gender-affirming hormones for patients with gender incongruence undergoing a clinical trial. METHODS: Patients diagnosed with gender identity disorders based on the International Classification of Diseases 10th revision classification at three institutions in Japan for whom hormone therapy using gender-affirming hormones was initiated were analyzed. They were asked what the most anticipated change was due to gender-affirming hormone that they had thought of between giving informed consent and the first administration of the drug. RESULTS: The responders were 336 transgender men who were administered androgens and 48 transgender women who received estrogens. The median age at commencement of hormone therapy was 24 years for transgender men and 28 years for transgender women. For transgender men, the most frequent answer was cessation of menses (52.7%) followed by a deepened voice (32.4%). For transgender women, breast development (35.4%) was the most anticipated change, followed by gynoid fat deposition (29.2%). CONCLUSIONS: Cessation of menses in transgender men and breast development/gynoid fat deposition in transgender women might represent primary end-points in clinical trials evaluating the efficacy of hormonal treatment in these patients.

The optimal mechanical condition in stem cell-to-tenocyte differentiation determined with the homogeneous strain distributions and the cellular orientation control.

In tendon tissue engineering, mechanical stimulus-induced differentiation is one of the most attractive techniques for stem cell-to-tenocyte differentiation in terms of cost, safety and simplicity. However, the most effective strain amplitude for differentiation using cyclic stretching remains unknown. Existing studies have not constrained cell reorientation behavior during cyclic stretching, resulting in uncertainty regarding the loads experienced by cells. In addition, strain distribution homogeneity of the culture membrane is important. Here, we improved the strain distribution uniformity of the membrane and employed a microgrooved membrane to suppress cell reorientation. Then we evaluated the most effective strain amplitude (0, 2, 4, 5, 6, or 8%) for the differentiation of mesenchymal stem cells into tenocytes by measuring mRNA expression levels. The maximum expression of all tenogenic markers was observed at a 5% strain. These results contribute to tendon tissue engineering by clarifying the most effective strain amplitude during tenogenic differentiation induction using cyclic stretching.

Develop to term rat oocytes injected with heat-dried sperm heads.

This study investigated the development of rat oocytes in vitro and in vivo following intracytoplasmic injection of heads from spermatozoa heat-dried at 50°C for 8 h and stored at 4°C in different gas phases. Sperm membrane and chromosome are damaged by the process of heat-drying. Oocyte activation and cleavage of oocytes were worse in oocytes injected with spermatozoa heat-dried and stored for 1 week than unheated, fresh spermatozoa, but in heat-dried spermatozoa, there were no differences in these abilities of oocytes between the samples stored in nitrogen gas and in air. The oocytes injected with heat-dried spermatozoa stored for 1 week could develop to the morula and blastocyst stages without difference between the samples stored in nitrogen gas and in air after artificial stimulation. Cleavage of oocytes and development of cleaved embryos were higher when heat-dried spermatozoa were stored for 3 and 6 months in nitrogen gas than in air. However, the ability of injected oocytes to develop to the morula and blastocyst stages was not inhibited even when heat-dried spermatozoa stored in both atmosphere conditions for as long as 6 months were used. When 2-cell embryos derived from oocytes injected with heads from spermatozoa heat-dried and stored for 1 week and 1 month were transferred, each 1 of 4 recipients was conceived, and the conceived recipients delivered 1 live young each. These results demonstrate that rat oocytes can be fertilized with heat-dried spermatozoa and that the fertilized oocytes can develop to term.

Efficacy and safety of Macrolane(™) for breast enhancement: a 12-month follow-up study in Asian women.

The demand for breast enhancement has risen substantially over recent years. Stabilised hyaluronic acid of non-animal origin manufactured using NASHA(™) technology (Q-Med, Uppsala, Sweden) is an injectable gel, which has increasingly been used as a minimally invasive, non-permanent option for breast enhancement. The aim of this study was to investigate the 12-month efficacy and safety of NASHA gel, when used for breast enhancement in Asian women. Non-pregnant, non-breastfeeding women with small breasts (aged 20-50 years) were recruited into this open, prospective, non-comparative, single-centre study. Subjects received sub-glandular injections of NASHA gel. Efficacy and safety assessments were carried out at follow-up visits (1, 6, and 12 months). Physician and subject assessment of breast improvement was recorded using the Global Esthetic Improvement Scale (GEIS). Ninety-eight subjects of Asian ethnicity were enrolled; 65 subjects completed the 12-month follow-up period. Overall, a median volume of 200 mL (range 80-300 mL) NASHA gel was injected per subject. Following GEIS assessment, 79% of breasts were subject-assessed as improved, much improved, or very much improved 6 months after treatment; 48% of breasts were still considered improved after 12 months. Sub-glandular NASHA gel injection was well tolerated, eliciting no serious adverse events judged to be treatment-related. High rates of aesthetic improvement were observed for at least 6 months after NASHA gel breast enhancement. The minimally invasive injection of NASHA gel provided a treatment option, which was an attractive alternative to permanent breast implants for many women.

Effect of polyvinylpyrrolidone on bovine oocyte maturation in vitro and subsequent fertilization and embryonic development.

The exact role of polyvinylpyrrolidone (PVP) in culture medium for oocyte maturation is still largely unknown. Bovine cumulus-oocyte complexes (COC) were cultured in in-vitro maturation (IVM) medium supplemented with 10% fetal bovine serum (FBS), 0.3% PVP (K-90) or 10% serum substitute supplement (SSS) respectively. The rates of oocyte maturation, fertilization and early embryonic development were evaluated. In addition, the status of DNA fragmentation in the oocytes was determined by comet assay, and the ratio of trophectoderm (TE) cells and inner cell mass (ICM) in blastocysts was determined by differential staining. Furthermore, the percentage of apoptotic cells in the blastocysts was examined by TUNEL assay. The results indicated that the effect of PVP in IVM medium was similar to FBS in terms of oocyte maturation and subsequent embryonic development. However, the addition of SSS in IVM medium retarded further embryonic development and resulted in more oocyte DNA fragmentation and a higher ratio of TE cells and ICM in the blastocysts. However, the number of apoptotic cells in blastocysts was similar among the three groups. These results suggest for the first time that the addition of PVP in oocyte maturation medium is not only a suitable substitute for serum but is also beneficial to in-vitro oocyte maturation.

Fertilization and development in vitro of bovine oocytes following intracytoplasmic injection of heat-dried sperm heads.

This study investigated the development of bovine oocytes following intracytoplasmic injection of sperm heads from spermatozoa dried by heating. When sperm suspension was heated in a dry oven at 50, 56, 90, and 120 degrees C, the mean amounts of residual water were about 0.3 g water/g dry weight within 8 h, 6 h, 1.5 h, and 20 min of heating, respectively. Oocyte activation, cleavage of oocytes, and development of cleaved embryos to the morula stage were better in oocytes injected with spermatozoa stored at 25 degrees C for 7-10 days following drying at 50 and 56 degrees C than at 90 and 120 degrees C; however, only a small proportion of oocytes developed to the blastocyst stage. When spermatozoa were dried at 50 degrees C for 16 h, activation, male pronucleus (MPN) formation, cleavage, and development to the morula stage were less good than when spermatozoa were dried for 8 and 10 h and no blastocysts were obtained. The development of oocytes was significantly better when spermatozoa were stored for 7-10 days at 4 degrees C than 25 degrees C after drying at 50 degrees C for 8 h. Longer storage (7 days-12 mo) of heat-dried spermatozoa at 4 degrees C did not affect MPN formation in activated oocytes, but blastocyst development was significantly lower when spermatozoa were stored for 3 mo or more. These results demonstrate that bovine oocytes can be fertilized with heat-dried spermatozoa and that the fertilized oocytes can develop at least to the blastocyst stage.

Effects of partial removal of cytoplasmic lipid on survival of vitrified germinal vesicle stage pig oocytes.

This study was designed to investigate whether the partial removal of cytoplasmic lipid from immature pig oocytes prior to vitrification had any positive effects on subsequent maturation, fertilization and early development. Oocytes at the germinal vesicle stage were partially freed from cumulus cells and centrifuged, and then polarized cytoplasmic lipid was removed by micromanipulation. When cultured for 44-48 h, significantly fewer of the centrifuged oocytes reached metaphase II (M-II) than did the non-centrifuged oocytes (approximately 53% vs approximately 68%, respectively); however, no further reduction in the M-II rate was observed when centrifuged oocytes were then delipated prior to culture (approximately 47%). To evaluate their sensitivity to the equilibration and vitrification solutions containing ethylene glycol, non-centrifuged, centrifuged, and delipated oocytes were cultured continuously for several minutes in those solutions, then washed and cultured further; no significant differences in the M-II rates (approximately 20-27%) were observed among the three treatment groups. When oocytes were vitrified and then warmed, significantly more delipated oocytes reached M-II in culture (approximately 15%) than did the non-delipated oocytes, whether centrifuged or not (approximately 4% in each group). When delipated, vitrified and matured oocytes were microsurgically injected with frozen-thawed spermatozoa, approximately 39% were activated and male pronucleus formation was observed in approximately 40% of activated oocytes; none developed beyond the 4-cell stage. These results show that maturation in vitro of vitrified pig oocytes can be promoted by partial removal of cytoplasmic lipid prior to vitrification and that the vitrified oocytes can be fertilized, although the embryonic development obtained in this study was limited.

Factors required during preculture of rat oocytes soon after sperm penetration for promoting their further development in a chemically defined medium.

Blastocyst formation in a chemically defined medium (mR1ECM) of rat oocytes soon after sperm penetration is less frequent than in those undergoing male pronuclear formation. This inhibition is released by preculturing the oocytes for a few hours in modified Krebs-Ringer bicarbonate solution (mKRB). The present study examined the effects of phosphate (Pi), bovine serum albumin (BSA) and osmolarity during preculture of sperm penetrated rat oocytes on their development to blastocysts in mR1ECM in vitro. These are the major factors that differ between mR1ECM and mKRB. When oocytes collected at 0730-0800 h on the day following mating and freed from cumulus cells were precultured for 5 h in mKRB or Pi-free mKRB and then cultured for 127 h in mR1ECM, about 73-74% of oocytes developed to blastocysts. In both media, replacement of BSA with polyvinylalcohol (PVA) or osmolarity of 246 mOsM reduced blastocyst formation compared with media containing BSA or with osmolarity of 304 mOsM; blastocyst formation was greatly inhibited when oocytes were precultured in media with PVA and osmolarity of 246 mOsM. On the other hand, when precultured in mR1ECM or mR1ECM with osmolarity of 304 mOsM or BSA instead of PVA, fewer oocytes developed to blastocysts than those precultured in Pi-free mKRB and mR1ECM with osmolarity of 304 mOsM and BSA. These results indicate that both BSA and osmolarity, but not Pi, are essential factors during preculture of rat oocytes soon after sperm penetration for promoting their further development to blastocysts in a chemically defined medium.

Effects of 17beta-estradiol on in vitro maturation of pig oocytes in protein-free medium.

The present study examined the effects of 17 beta-estradiol (E(2)) on in vitro maturation and subsequent in vitro fertilization of pig oocytes matured with or without cumulus cells. When E(2) (10 ng/ml) was added to the protein-free maturation medium, the proportions of cumulus-enclosed oocytes that underwent germinal vesicle breakdown and reached metaphase II were significantly reduced (P<0.05), and cumulus expansion was also significantly inhibited (P<0.05) compared with the control (no E(2) added). Although oocytes matured in the presence of E(2) were penetrated by sperm in vitro at the same level as the control, the incidences of male pronuclear (MPN) formation and activated oocytes were significantly lower (P<0.05) than the control. These inhibitory effects of E(2) were prevented when the medium was supplemented with E(2) together with its antagonist, ICI 182,780 (1 microg/ml), although the presence of the antagonist alone in the medium had no effect on the maturation and fertilization in vitro of oocytes. In cumulus-free oocytes, E(2) had no effect on nuclear maturation and penetration in vitro, but low MPN formation was observed in oocytes matured in the presence and absence of E(2). When cumulus-enclosed oocytes were cultured in the presence of progesterone (P(4); 600 ng/ml) alone or together with E(2), no significant differences in nuclear maturation, cumulus expansion or penetration in vitro were observed compared with control oocytes. The concentration of P(4) in maturation medium was significantly (P<0.01) lower when cumulus-enclosed oocytes were cultured for 44 h in the medium with E(2) than in medium without E(2). These results indicate that E(2) inhibits both nuclear and cytoplasmic maturation of cumulus-enclosed pig oocytes, and that this inhibition can be prevented by an E(2) antagonist or P(4). This E(2) inhibition may occur indirectly via the cumulus cells and inhibition of P(4) synthesis.

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.

Activation and penetration in vitro of pig oocytes treated with calcium ionophore.

The present study examined the effects of pre-treatment of pig oocytes with different concentrations (0-50 microM) of calcium ionophore A23187 (CaA) on their activation, development and penetration in vitro. Although untreated oocytes were not activated and did not cleave in culture, high proportions of treated oocytes did so and 20% of oocytes developed to the blastocyst stage when treated with 6.25 microM CaA for 2 min. However, these proportions were reduced in a concentration-dependent manner. When inseminated in vitro with 1 x 10(6) spermatozoa/ml, the penetration rate of oocytes treated with 6.25 microM CaA was similar to that of untreated oocytes. However, fewer oocytes treated with 12.5 and 50 microM CaA were penetrated than untreated oocytes. On the other hand, the proportion of monospermy of oocytes treated with 6.25 microM CaA was higher than the values in oocytes not treated or treated with 50 microM CaA. The time required for zona dissolution of oocytes treated with 6.25 and 12.5 microM CaA was not different from that in untreated oocytes, but oocytes treated with 50 microM CaA required a longer time than untreated oocytes, indicating that zona solubility by protease does not reflect penetrability of oocytes in vitro. When oocytes were inseminated with different concentrations (1-10 x 10(6) cells/ml) of spermatozoa, the highest penetration rate was observed at 1 x 10(6) cells/ml in untreated oocytes and a similar result was obtained in oocytes treated with 6.25 microM CaA. There was no difference in the rate of monospermy in untreated oocytes among different concentrations of spermatozoa, but in treated oocytes, higher proportions of monospermy were observed at 0.5-5 x 10(6) than 10 x 10(6) cells/ml. At 1 x 10(6) cells/ml, the proportion of monospermy was higher in treated than untreated oocytes. These results suggest that pre-treatment of pig oocytes with 6.25 microM CaA, an appropriate concentration, inhibits polyspermic penetration in vitro when insemination occurs with spermatozoa at a concentration of 1 x 10(6) cells/ml.

In vivo development of vitrified rat embryos: effects of timing and sites of transfer to recipient females.

In cryopreserved rat embryos, survival rates obtained in vitro are not always consistent with the rates obtained in vivo. To determine the optimal conditions for in vivo development to term, rat embryos at the 4-cell, 8-cell, and morula stages were vitrified in EFS40 by a one-step method and transferred into oviducts or uterine horns of recipients at various times during pseudopregnancy. Vitrified and fresh 4-cell embryos only developed after transfer into oviducts of asynchronous recipients on Days -1 to -2 of synchrony (i.e., at a point in pseudopregnancy 1-2 days earlier than the embryos). Approximately half the vitrified embryos transferred into oviducts on Day -1 developed to term, but only a minority of embryos, whether vitrified (10%-34%) or fresh (24%-33%), transferred at later times did so, suggesting that this may not be the most suitable stage for cryopreservation. Very few 8-cell embryos, either vitrified or fresh, developed when transferred into oviducts on Day 0 to -0.5. However, when transferred into uterine horns, high proportions of vitrified 8-cell embryos ( approximately 63%) developed to term in reasonably synchronous recipients (Day 0 to -0.5) but not in more asynchronous ones (6%; Day -1). A majority of vitrified morulae also developed to term (52%-68%) in a wider range of recipients (Days 0 to -1), the greatest success occurring in recipients on Day -0.5. Similar proportions of vitrified and fresh 4-cell embryos, 8-cell embryos, and morulae developed to term when appropriate synchronization existed between embryo and recipient. Thus, vitrification of preimplantation-stage rat embryos does not appear to impair their developmental potential in vivo.

Two-phase chemically defined culture system for preimplantation rat embryos.

A limiting factor in the development of new technologies and transport of rats worldwide has been the inability to robustly culture preimplantation embryos. Previously, culture in vitro to the blastocyst stage from one-cell embryos was successful only if the one-cell embryos were isolated near the time of the first cleavage and from only a few strains. Here we report the use of commonly available, chemically defined culture media to overcome these limitations. In vitro culture of young one-cell embryos using common embryo media (KSOM, BMOC, or HTF) for 18-22 h followed by culture in mR1ECM medium allows the successful in vitro development of blastocysts from one-cell embryos after 5 days from both outbred (SD) and inbred strains of rat (WF, LEW, F344, and PVG). This system allows the parthenogenetic development of chemically activated, unfertilized oocytes to the blastocyst stage. Embryos cultured in this system develop to term and are live-born following transfer to surrogate mothers.

Effects of BSA and fetal bovine serum in culture medium on development of rat embryos.

Rat 1-cell embryos, recovered from naturally mated females, were cultured in a chemically defined medium (mR1ECM) or in mR1ECM supplemented with BSA (4 mg/ml; mR1ECM-BSA) or fetal bovine serum (FBS; 10%, v:v; mR1ECM-FBS) instead of polyvinylalcohol. There was no difference in percentages of embryos that developed to the 2-cell to blastocyst stages between mR1ECM and mR1ECM-BSA, but in mR1ECM-FBS, no development beyond the 2-cell stage was observed. When embryos were transferred to mR1ECM-FBS from mR1ECM after 24 to 64 h of culture, development of embryos to and beyond the 4-cell stage was inhibited. However, when transferred after 80 h of culture, more embryos developed to blastocysts and hatching or hatched blastocysts than in embryos cultured in mR1ECM. When 8-cell embryos and early morulae obtained after 72 and 80 h of culture in mR1ECM, respectively, were cultured in mR1ECM-FBS, a higher proportion of early morulae developed to the blastocyst stage than did 8-cell embryos. When morulae obtained after culture in mR1ECM or mR1ECM-BSA were transferred to recipient females, there was no difference in proportions of fetuses obtained. However, a higher proportion of blastocysts cultured in mR1ECM-FBS developed to fetuses compared with those obtained in mR1ECM. These results indicate that BSA has neither deleterious nor beneficial effects on development of rat 1-cell embryos. In contrast, FBS has deleterious effects on early cleavage of embryos but it promotes more rapid development of morulae to blastocysts, resulting in better quality blastocysts.

Localisation of the hyaluronan receptor CD44 in porcine cumulus cells during in vivo and in vitro maturation.

Polyspermy is fairly common during porcine in vitro fertilisation (IVF), perhaps due to incomplete in vitro oocyte maturation (IVM). Porcine cumulus cells (CCs) layered around the oocyte produce large amounts of extracellular hyaluronan (HA) when forming an expanding cell cloud during the last phase of oocyte maturation. The specific actions of HA are mediated via HA-binding proteins (HABPs), such as CD44, which act as receptors. In this study using immunocytochemistry and western blotting we investigated the localisation of CD44 in CCs obtained from in vivo-matured pig cumulus-oocyte complexes (COCs) and compared it with that in CCs from immature COCs and of COCs subjected to IVM and IVF procedures. Immunolabelling of CD44 was absent or very weak in CCs from immature COCs but strongly present on the surface of the CCs obtained from in vivo, displaying a similar localisation in the in vitro-matured COCs. In the latter, the labelling decreased but did not disappear in CCs 4 h after sperm co-incubation during IVF. Immunoblotting detected bands of between 73 and 88 kDa, corresponding to CD44, in the protein extract from in vivo CCs collected immediately prior to, or following spontaneous ovulation. The in vitro-matured CCs, however, presented bands ranging from 81 kDa to 88 kDa. Also, the bands found in the in vivo-matured CCs showed a larger variation of intensity and migration among animals than did the batches of in vitro-matured CCs. No CD44 band was detected on aliquots of the frozen-thawed boar spermatozoa used for IVF. The results clearly demonstrate that the specific HA receptor CD44 is present in expanding CCs of in vivo-matured pig COCs, in relation to increasing amounts of inter-CC HA. The subtle differences in molecular weight and migration ability observed between in vivo and in vitro samples may relate to differences in glycosylation and thus explain differences in HA-binding ability, of consequence for optimising in vitro culture conditions.

Penetration in vitro of zona-free pig oocytes by homologous and heterologous spermatozoa.

We examined the penetrability of pig, rat and bull spermatozoa into zona-free pig oocytes. Frozen-thawed boar spermatozoa penetrated into both zona-intact and zona-free oocytes with similar efficacy in a modified Tris-buffered medium (mTBM) supplemented with BSA and caffeine, but not in medium without caffeine. Rat epididymal spermatozoa did not readily penetrate into zona-free pig oocytes in mTBM with BSA. However, when a modified Krebs-Ringer bicarbonate solution was used, penetration rate varied with sperm concentrations at insemination: 79% of the oocytes were penetrated at 1.0 x 10(6) cells/ml, but very few at 0.1 x 10(6) and 10.0 x 10(6) cells/ml. In all oocytes penetrated, no activation was observed and the sperm nucleus was fully decondensed but did not transform into a male pronucleus. Frozen-thawed bull spermatozoa were also found to penetrate into zona-free pig oocytes in mTBM with BSA, caffeine and heparin: higher penetration rates were obtained with 1.0 x 106 and 10.0 x 10(6) spermatozoa/ml compared with 0.1 x 10(6) spermatozoa/ml. The penetration rate with 1.0 x 10(6) spermatozoa/ml was stable in five different bulls. All oocytes penetrated were activated and male pronuclear formation was observed in 57-79% of the penetrated oocytes. These results suggest that capacitation or the acrosome reaction is required for boar, rat, and possibly, bull spermatozoa to penetrate into zona-free pig oocytes. Bull spermatozoa can easily induce activation of pig oocytes and form male pronuclei, but rat spermatozoa cannot do so, indicating species differences in the ability of spermatozoa to activate pig oocytes and to transform to male pronuclei in the ooplasm.