Rabbit oocyte can be used as the recipient in interspecies somatic cell nuclear transfer (iSCNT). This work was undertaken in order to study the developmental competence of Capra ibex somatic cells reprogrammed by rabbit oocytes and the fate of mitochondria in iSCNT embryos. Metaphase II (MII) oocytes from superovulated rabbit were used as nuclear recipients. The nuclear donors were Capra ibex somatic cells with different proliferative status: population doubling time (PDL)=15±2 (group 1), 35±2 (group 2), 55±2 (group 3) and 70±2 (group 4). Oocytes reconstructed with electrical pulses (2.1 kV/cm, 10 μs, 2 times) were activated (1.4 kV, 20 μs, 2 times) and then cultured in Medium199 containing 10% fetal bovine serum at 38.5 °C, 5% CO2 in air. In groups 1, 2, 3 and 4, the fusion rates were 35.83%, 66.03%, 65.40% and 35.35%, respectively. Similar cleavage rates were observed among the four groups. However, the developmental potential to morula/blastocyst from early nuclear donor embryos (16.42%/10.45%) was significantly higher (p < 0.05) than in terminal donor embryos (9.52%/3.81%). Polymerase chain reaction analysis of the mitochondrial (mt) DNA cytb gene demonstrated that mtDNAs from ibex and rabbit could be detected at various developmental stages before implantation. In conclusion, our results provide some original information about rescuing Capra ibex using the iSCNT technique. These results indicate that: (1) enucleated rabbit oocytes make Capra ibex fibroblast nuclei reprogramme; (2) the proliferative status of donor cells affects the efficiency of iSCNT; and (3) rabbit ooplasm rescues the donor-derived mtDNAs, resulting in mtDNA heteroplasmy before implantation.

This study compared the developmental competence of somatic cell nuclear transfer (SCNT) embryos reconstructed with different donor cells and analysed gene expression in the resulting embryos. Bovine fetal/adult ear fibroblasts and cumulus cells were used as donor cells and the developmental competence of the reconstructed embryos was monitored. The cell number and allocation in blastocysts were determined by differential staining. The Bax, E-cad, IF-tau, Hsp (heat shock protein) 70, Igf2r (insulin-like growth factor 2 receptor), DNMT (DNA methyltransferase) 1 and Mash (mammalian achaete-scute homologue) 2 genes were selected for gene expression analysis. The relative abundance (ratio to GAPDH mRNA) of gene transcripts in blastocysts was measured by semiquantitative reverse transcription-polymerase chain reaction. In experiment 1, development of SCNT preimplantation embryos and the cell numbers of inner cell masses and trophoblasts were not different among SCNT embryos derived from different cell types. In experiment 2, the relative expression of GAPDH and Hsp 70 transcripts was similar in all embryos. The expression of Bax, Igf2r and Mash2 transcripts was significantly increased in SCNT embryos reconstructed with adult fibroblasts. The E-cad transcript levels were reduced in SCNT embryos reconstructed with fetal fibroblasts. Relative abundance of DNMT1 in SCNT embryos derived from fetal fibroblasts was increased, and IF-tau expression in SCNT embryos derived from cumulus cells was increased. In conclusion, depending on the type of donor cells, preimplantation SCNT embryos displayed marked differences in gene expression. This may affect the developmental competence of SCNT embryos reconstructed with different cell types after implantation or during fetal growth in vivo.

We report on the first attempts, using both light and fluorescence microscopy, to visualize the developing embryo of the butterfly Bicyclus anynana. We developed a new protocol that enabled the clear visualization of the internal egg structures in early embryogenesis (1–24 h after egg laying). Dechorionation was followed by fixation and physical dissection of the external egg structures. Observations of embryonic and extra-embryonic cells were made using a Hoechst nuclear stain that fluoresces in the blue spectrum when bound to DNA and excited with ultraviolet (UV) light under a fluorescence microscope. Preliminary data on the developmental rate of the early embryo are also presented.

The aim of this study was to compare three different culture systems for in vitro follicular growth and oocyte maturation in ovarian follicles of mice in order to assess the technique with the optimal growth and improved rate of meiotic maturation. The three systems tested were culture under oil, on a hydrophobic membrane and on agar respectively. Early preantral follicles were cultured for 12 days in α-MEM GlutaMAX medium. Follicular growth, oocyte meiotic maturation, oocyte extrusion, atresia and estradiol production were analysed. Follicular development showed two phases in the three systems, with slow growth before day 5 and subsequent acceleration. The percentage of follicles transferred into oocyte maturation medium was significantly higher after culture under oil. The proportion of oocytes that achieved nuclear maturation (metaphase II) was higher when follicles were cultured under oil or on a hydrophobic membrane than on agar. Our results support the use of culture under oil for in vitro follicular growth from the early preantral stage in order to obtain metaphase II oocytes. Fertilization ability of these oocytes and the capacity to obtain healthy mice in a reproducible manner warrants further investigation.

Plasminogen activators (PAs) are highly specific serine proteases that convert the extracellular zymogen plasminogen into the active proteinase plasmin. Plasminogen-dependent proteolytic activity was detected by zymography both in the tissue membrane fraction of oviducts and in the oviductal flushing obtained at the preovulatory (Pre-Ov), postovulatory (Post-Ov) and mid-luteal (Mid-L) stages of the estrous cycle. A main proteolytic band, with a relative mobility similar to a human melanoma cell tissue-type plasminogen activator (t-PA), was found in all samples. Two additional components were observed in Pre-Ov and Post-Ov oviductal flushing but not in the tissue membrane fraction. In the oviductal flushing the PA activity was significantly higher in the Post-Ov stage than in the Pre-Ov one. Both urokinase-type plasminogen activator (u-PA, 50 kDa) and t-PA (72 kDa) were detected by Western blot; they showed differences in their relative concentration between Post-Ov and Pre-Ov oviductal flushing. The main PA substrate, plasminogen, was detected by indirect immunofluorescence in the cumulus cell extracellular matrix (ECM) and oocyte zona pellucida (ZP). In denuded oocytes, plasminogen was also detected on the surface of the plasma membrane. It is possible that oviductal PAs may act on the plasminogen present in the cumulus cell ECM and ZP; consequently, the generated plasmin could be involved in the rebuilding or degradation of these oocyte structures during fertilization or early development.

The present experiments were designed to study the effects of Robertsonian translocations on the efficiency and kinetics of in vitro fertilization and early and advanced embryo development. Spermatozoa from bulls with rob(16;20), rob(1;29) and normal karyotype (A, B and C, respectively) were used. Oocytes were matured, fertilized and cultured by the standard protocol described previously. Twenty-four hours after fertilization, adequate numbers of oocytes were fixed, stained and examined. The development of embryos was evaluated on days 2 (D2), 7 (D7) and 8 (D8) after fertilization. The rate of normally fertilized oocytes was significantly lower (p≤0.01) for bull A than for bulls B and C. However, no significant differences in the kinetics of fertilization were found between bulls A, B and C. The D2 cleavage rate of embryos was significantly lower (p≤0.01) for bull A than for bulls B and C. Both D7 and D8 blastocyst rates for bull A or bull B were significantly lower (p≤0.01 or p≤0.01) than those for bull C. The percentages of both D7 advanced blastocysts and D8 expanded blastocysts were significantly lower (p≤0.01) for bulls A and B than for bull C. In conclusion, for rob(16;20), the efficiency of fertilization was strongly reduced; it resulted in low early and advanced embryo development. On the other hand, for the rob(1;29), neither fertilization nor early embryo development were affected and only advanced embryo development was decreased. But for both translocations, blastocyst formation was significantly delayed.

G2/M somatic nuclei were introduced into enucleated meiotically competent oocytes and subsequently cultured in TCM199 plus 10% fetal calf serum (FCS). Pseudo-first polar bodies could be extruded, but the chromosomes failed to arrange normally. Kinetochores were traced with immunofluorescent microscopy using autoimmune sera from patients with CREST (Calcinosis, Raynaud's phenomenon, Esophageal dysmotility, Sclerodactyly, Telangiectasia) scleroderma. In vitro matured oocytes arrested at second meiotic metaphase and kinetochores were detectable as paired structures aligned at the spindle equator. At meiotic anaphase, present or past the kinetochores separated and remained aligned at the distal sides of the chromosomes until telophase, when their alignment perpendicular to the spindle axis was lost. Kinetochores failed to arrange normally after transferring somatic nuclei into oocytes. Our results suggest that somatic cell nuclei are unable to proceed normally through meiosis when introduced into oocyte meiotic cytoplasm.

Prostaglandin E2 (PGE2) is shown to be essential for female reproduction. Cyclooxygenase (COX) is a rate-limiting enzyme in prostaglandin synthesis from arachidonic acid and exists in two isoforms: COX-1 and COX-2. Prostaglandin E synthase (PGES) is a terminal prostanoid synthase and can catalyse the isomerization of the COX product PGH2 to PGE2, including microsomal PGES-1 (mPGES-1), cytosolic PGES (cPGES) and mPGES-2. This study examined the protein expression of COX-1, COX-2, mPGES-1, cPGES and mPGES-2 in preimplantation mouse embryos by immunohistochemistry. Embryos at different stages collected from oviducts or uteri were transferred into a flushed oviduct of non-pregnant mice. The oviducts containing embryos were paraffin-embedded and processed for immunostaining. COX-1 immunostaining was at a basal level in zygotes and a low level at the 2-cell stage, reaching a high level from the 4-cell to blastocyst stage. COX-2 immunostaining was at a low level at the zygote stage and was maintained at a high level from the 2-cell to blastocyst stages. A low level of mPGES-1 immunostaining was observed from the zygote to 8-cell stages. The signal for mPGES-1 immunostaining became stronger at the morula stage and was strongly seen at the blastocyst stage. cPGES immunostaining was strongly observed in zygotes, 2-cell and 8-cell embryos. There was a slight decrease in cPGES immunostaining at the 4-cell, morula and blastocyst stages. mPGES-2 immunostaining was at a low level from the zygote to morula stages and at a high level at the blastocyst stage. We found that the COX-1, COX-2, mPGES-1, cPGES and mPGES-2 protein signals were all at a high level at the blastocyst stage. PGE2 produced during the preimplantation development may play roles during embryo transport and implantation.

The aim of our study was to analyse the cytoskeletal organization of prepubertal goat oocytes. Microtubule and microfilament organization during in vitro maturation of prepubertal and adult goat oocytes and presumptive zygotes of in vitro matured–in vitro fertilized (IVM-IVF) prepubertal goat oocytes were analysed. Oocytes were matured in M-199 with hormones and serum and inseminated with frozen-thawed spermatozoa. Oocytes and presumptive zygotes were treated with anti-α-tubulin antibody and fluorescein isothiocyanate (FITC)-labelled goat anti-mouse antibody to stain the microtubules. Microfilaments were localized by means of phalloidin 5 μg/ml conjugated with fluorescein isothiocyanate (FITC-phalloidin). DNA was stained with propidium iodide. Stained oocytes were observed under a confocal laser scanning microscope. At the germinal vesicle nuclear stage, microfilaments were distributed at the cortex of the oocytes. After in vitro maturation, 91.7% of metaphase II (MII) oocytes from adult goats displayed microfilaments in the cortex and within the polar body and were characterized by the presence of a microfilament thickening at the cortical region over the meiotic spindle. In prepubertal goat MII oocytes only 5.7% of oocytes displayed microfilaments at the cortex and within the polar body. After insemination, most of the zygotes displayed microfilaments distributed at the cortex. An undefined microtubular network was observed in adult and prepubertal goat oocytes at the germinal vesicle stage. After in vitro maturation, 100% of MII oocytes from adult goats displayed microtubules on the meiotic spindle and within the polar body. This pattern of distribution was observed in 71.6% of prepubertal goat oocytes. Undefined microtubule networks were present in most of the zygotes analysed. In conclusion, cytoskeletal differences were found between prepubertal and adult goat MII oocytes. Furthermore, most of the zygotes from IVM-IVF prepubertal goat oocytes displayed cytoskeletal anomalies.

Inbred BALB/c mice are one of the most difficult inbred strains to fertilize in vitro. In this study we examined the abilities of various media used for mouse in vitro fertilization (IVF) to support capacitation and sperm penetration through the zona pellucida (ZP) of inbred BALB/c spermatozoa. Media examined were TYH, M16, CZB, mWhitten medium, T6, modified Tyrode's solution (mTyrode's), mKSOM, MEM and TCM199. Modified human tubal fluid (mHTF) was used as a control medium. When sperm were capacitated and inseminated in the same medium, mHTF showed the best fertilization (approximately 80%) scored by male pronuclear formation (<26%) at 5 h post-insemination (PI). When sperm were capacitated in various media and inseminated in mHTF, sperm capacitated in TYH solution (93%) but no other media (<45%) showed a significantly higher level of sperm nuclear decondensation (SND) than mHTF at 2 h PI (approximately 65%). When sperm were capacitated in mHTF and inseminated in various media, only mTyrode's (52%) was not significantly lower than mHTF (66%) in terms of SND at 2 h PI (<49%). Sperm capacitation also was examined by chlortetracycline (CTC) staining. Sperm capacitated in TYH solution showed a significantly higher percentage of capacitation (46%) than those treated in HTF (28%) and other media (<24%). These results indicate that the best approach for IVF in the BALB/c strain is capacitation in TYH and insemination in mHTF. Poor fertilization of BALB/c may result from suboptimal conditions of sperm capacitation and insemination, and overall IVF success may differ depending on strains used.

The present study was conducted to evaluate the function of cumulus cells during bovine IVF. Oocytes within cumulus–oocyte complexes (COCs) or denuded oocytes (DOs) were inseminated in control medium, or DOs were inseminated in cumulus cell conditioned medium (CCCM). DOs exhibited reduced cleavage and blastocyst formation rates when compared with intact COCs. The reduced blastocyst formation rate of DOs resulted from reduced first cleavage but subsequent embryo development was not changed. Live-dead staining and staining for apoptotic cells revealed no differences in blastocysts from oocytes fertilized as COC or DO. Fertilization of DOs in CCCM partially restored the cleavage rate, suggesting that factors secreted by cumulus cells are important for fertilization but that physical contact between oocytes and cumulus cells is required for optimal fertilization and first cleavage. Exposure of COCs to hydrogen peroxide shortly before fertilization reduced the cleavage rate, but did not lead to enhanced death of cumulus cells or oocyte death. Exposure of DOs to hydrogen peroxide, however, resulted in oocyte death and a complete block of first cleavage, suggesting that cumulus cells protect the oocyte against oxidative stress during fertilization.

Microinjection (Mi) of gene constructs into pronuclei of fertilized eggs is a widely used method to generate transgenic animals. However, the efficiency of gene integration and expression is very low because of the low viability of reconstructed embryos resulting from cell fragmentation and cleavage arrest. As a consequence, only a few viable embryos integrate and express transgene. Since cellular fragmentation and cleavage stage arrest in embryos may be associated with apoptosis, we aimed to test the hypothesis that the low viability of Mi-derived eggs is caused by a high rate of apoptosis in embryos, as a result of the detrimental effect of Mi. Pronuclear stage eggs (19–20 hours post-coitum, hpc) were microinjected with several picolitres of DNA construct into the male pronucleus (gene-Mi); the intact eggs (non-Mi) or eggs microinjected with phosphate-buffered saline (PBS-Mi) served as controls. Epidermal growth factor (EGF; 0, 20 and 200 ng/ml) was added to the culture medium and the embryos were cultured up to 94–96 hpc. Apoptosis was detected using the TUNEL assay, and the ultrastructure was analysed using electron microscopy of Durcupan ACM thin sections of the embryo. Gene-Mi embryos had significantly lower (p<0.05) blastocyst yields and a higher percentage of cleavage-arrested embryos than those in the non-Mi group. In gene-Mi groups, approximately 40% of all cleavage-stage-arrested embryos had fragmented blastomeres. Both gene-Mi-and PBS-Mi-derived blastocysts had a significantly higher TUNEL index (p<0.001) and lower total cell number (p<0.05) than the non-Mi embryos. Comparison of the quality of gene-Mi embryos with that of PBS-Mi embryos indicated that the deleterious effect of Mi on the embryo was caused by the Mi procedure itself, rather than DNA. EGF (at 20 ng/ml) had beneficial effects on the quality of gene-Mi-derived embryos, eliminating the influence of the Mi procedure on apoptosis and embryo cell number. Ultrastructural analysis confirmed a higher occurrence of apoptotic signs (nuclear membrane blebbing, areas with electron-dense material, numerous apoptotic bodies) in Mi-derived cleavage-arrested embryos compared with untreated or Mi-derived normal-looking embryos. These findings suggest an association between embryo cleavage arrest and apoptosis in Mi-derived embryos. Inclusion of EGF in the embryo culture medium can eliminate the detrimental effect of Mi on embryo quality.

The role of the ubiquitin-proteasome pathway (UPP) in mitosis is well known. However, its role in meiotic division is still poorly documented, especially in the activation of mammalian oocytes. In this study, the role of proteasome in the spontaneous and parthenogenetic activation of rat oocytes was investigated. We found that ALLN, an inhibitor of proteasome, when applied to metaphase II oocytes, inhibited spontaneous activation, blocked extrusion of the second polar body (PB) and caused the withdrawal of the partially extruded second PB. ALLN also inhibited the parthenogenetic activation induced by cycloheximide, but had no effect on the formation of pronuclei in activated eggs. In metaphase and anaphase, ubiquitin and proteasome localized to the meiotic spindle, concentrating on both sides of the oocyte–second PB boundary during PB extrusion. This pattern of cellular distribution suggests that UPP may have a role in regulating nuclear division and cytokinesis. Ubiquitin was seen to form a ring around the pronucleus, whereas proteasome was evenly distributed in the pronuclear region. Taken together, our results indicate that (1) UPP is required for the transitions of oocytes from metaphase II to anaphase II and from anaphase II to the end of meiosis; and (2) the UPP plays a role in cytokinesis of the second meiotic division.

A 2×3 factorial designed experiment was conducted in order to examine whether freeze-dried rat spermatozoa can participate in full-term development following intracytoplasmic sperm injection (ICSI). A sperm suspension from cauda epididymides of Sprague–Dawley (SD) rats was prepared with or without ultrasonic treatment. The sonicated and non-sonicated sperm suspensions were processed for freeze-thawing (FT groups; 100μl sample was cooled in liquid nitrogen vapor, stored for 1 day at −196 °C, and thawed in a 25 °C water bath) and freeze-drying (FD groups; 100 μl sample was frozen in liquid nitrogen for 20 s, lyophilized for 6 h, stored at 4 °C for 2 days, and rehydrated with 100μl ultrapure water), or were subjected to immediate use for ICSI (fresh control groups). The sperm heads were microinjected into denuded SD oocytes using a piezo-driven micropipette 2–4μm in diameter. The presumptive zygotes were transferred into oviducts of pseudopregnant Wistar female rats. Viable rat offspring were produced from all six experimental groups. Ultrasonic treatment of rat spermatozoa was effective in increasing the offspring rate (23.3% vs 6.7% in fresh control groups, 35.0% vs 7.6% in FT groups, 9.2% vs 2.5% in FD groups). The acrosomal region appeared to be intact even after ultrasonic FT and FD treatments as well as in the fresh controls, while the lateral dorsal region of the sperm membrane was more or less damaged in the sonicated, FT and FD samples. Thus, the successful participation of freeze-dried spermatozoa in full-term development was demonstrated by applying ICSI in the rat.

In the starfish, Asterias amurensis, the cooperation of three components of the egg jelly, namely ARIS (acrosome reaction-inducing substance), Co-ARIS and asterosap, is responsible for the induction of acrosome reaction. For the induction, ARIS alone is enough in high-Ca2+ or high-pH seawater, but, besides ARIS, the addition of either Co-ARIS or asterosap is required in normal seawater. Asterosap transiently increased both the intracellular pH (pHi) and Ca2+ ([Ca2+]i), while ARIS slightly elevated the basal level of [Ca2+]i. However, a sustained elevation of [Ca2+]i and acrosome reaction occurred if sperm were simultaneously treated with ARIS and asterosap. EGTA inhibited the sustained [Ca2+]i elevation and acrosome reaction. The sustained [Ca2+]i elevation and acrosome reaction were highly susceptible to SKF96365 and Ni2+, specific blockers of the store-operated Ca2+ channel (SOC). These results suggest that sustained [Ca2+]i elevation, mediated by the SOC-like channel, is a prerequisite for the acrosome reaction. In high-pH seawater, ARIS alone induced a prominent [Ca2+]i increase and acrosome reaction, which were similarly sensitive to SKF96365. The acrosome reaction was effectively induced by ARIS alone when pHi was artificially increased to more than 7.7. Asterosap increased pHi from 7.6±0.1 to 7.7±0.1. Furthermore, the sustained [Ca2+]i elevation and acrosome reaction, induced by a combination of ARIS and asterosap, were drastically inhibited by a slight reduction in pHi. Taking these results into account, we suggest that an asterosap-induced pHi elevation is required for triggering the ARIS-induced sustained [Ca2+]i elevation and consequent acrosome reaction.

The developmental ability of reconstructed blastocysts from C57BL/6 strain mouse inner cell masses (ICMs) and Kunming strain mouse trophoblasts was assessed. The procedure of ICM replacement was as follows: C57BL/6 ICMs were separated from the blastocysts using immunosurgery. A slit was made in the zona pellucida of a Kunming blastocyst to allow its ICM to extrude. The C57BL/6 ICM was injected into the Kunming blastocoele, and the extruded Kunming ICM was cut off. The reconstructed blastocysts were able to re-expand (77%) and hatch (27.3%) in vitro. A total of 64 reconstructed blastocysts and 124 Kunming blastocysts were co-transferred into the uteri of 11 pseudopregnant Kunming mice, and an ICM replacement offspring was born. The results indicate that reconstructed embryos obtained by inter-strain ICM replacement have the ability to develop to term. This technique may provide a method to solve the pregnancy failure in interspecific cloning.

In this study, inter-strain reconstructed embryos were produced by combining the female pronucleus of Kunming mouse (white) with male pronucleus of C57BL/6 strain (black). Metaphase II (MII) oocytes of Kunming mouse were enucleated and the zona pellucida was removed. Then, the enucleated oocytes were inseminated by capacitated sperm of C57BL/6 mouse in vitro. At the same time, MII oocytes of Kunming mouse were artificially activated using strontium chloride solution, which did not contain cytochalasin B. Finally, we removed the male pronucleus derived from C57BL/6 sperm and injected it into a parthenogenetically activated one-pronucleus oocyte by micromanipulation. The reconstructed 2-cell embryos were transplanted into the oviducts of 22 foster mother mice, each receiving about 20 embryos. In the end, seven healthy and live pups were born from one recipient.

The seminal plasma PSP-I/PSP-II spermadhesin is able to preserve, in vitro, the viability of highly extended boar spermatozoa, suggesting it might be used as a suitable ameliorator for the damaging effects of sperm handling, including in vitro fertilization. However, little is known about the ligand capability of PSP-I/PSP-II as regards the zona pellucida (ZP) or its possible role in gamete interaction. The present study evaluated the effect of the presence of PSP-I/PSP-II (1.5 mg/ml) during in vitro oocyte maturation and also during co-incubation of frozen-thawed boar spermatozoa with either immature (IM) or in vitro matured (IVM) oocytes, either enclosed by cumulus cells or denuded. Exposure of the gametes to the heterodimer during in vitro gamete co-incubation showed a significant blocking effect of sperm penetration rates and a decreased number of spermatozoa per oocyte in both IM and IVM denuded oocytes. Such an effect was not present in cumulus-enclosed oocytes, suggesting the effect could be mediated by exposed ZP receptors. In addition, when PSP-I/PSP-II was added to the IVM medium, oocyte maturation rates were significantly reduced. In conclusion, the results suggest that PSP-I/PSP-II, when present in vitro, blocks sperm–ZP binding.

Robertsonian translocation rob(16;20) in the heterozygous state was discovered in a subfertile bull of the Czech Siemmental breed. A chromosomal analysis of its family has shown that this dicentric fusion is formed de novo. The present experiments were designed to detect rob(16;20) and determine its incidence for in vitro produced embryos, using fluorescence in situ hybridization (FISH) and rob(1;29) as a detection control. To characterize semen of both bulls with the rob translocations, their sperm was examined for DNA integrity by the sperm chromatin structure assay (SCSA). For in vitro fertilization of oocytes, spermatozoa from a rob(16;20) bull carrier (Czech Siemmental breed) and those from a rob(1;29) bull carrier (Charolais breed) were used. Embryos at the 6- to 8-cell stage were cultured in a vinblastine-supplemented medium for 17 h, and embryos at the blastocyst stage were cultured in a colcemide-supplemented medium for 4 h. The embryos were fixed in methanol and acetic acid with Tween-20. Painting probes for chromosomes 16 (Spectrum Green) and 20 (Spectrum Orange) and chromosomes 1 (Spectrum Orange) and 29 (Spectrum Green) were simultaneously hybridized. In the embryos derived from the rob(16;20) bull, the presence of this translocation was not detected. On the other hand, 52.5% of the embryos derived from the rob(1;29) bull were translocation carriers. There was no significant difference in the frequency of this translocation between early and advanced embryos.

Although both intracytoplasmic sperm injection (ICSI) and round spermatid injection (ROSI) are used in infertility treatments, the rate of offspring achieved with ROSI is low compared with that achieved with ICSI. The difficulty in correctly selecting round spermatids from testicular cells is one of the causes of this phenomenon. We easily selected live round spermatids from testicular cells stained with 20 nM MitoTracker, which visualizes mitochondria without killing the cell. Using this method, we divided round spermatids into three groups based on the polarization of their mitochondria, and performed ROSI. The rate of successful offspring achieved with MitoTracker-stained ROSI was the same in all groups. This indicates that changes in the polarization of mitochondria in round spermatids are not directly related to the developmental capacity of subsequently fertilized embryos. Because this staining has no harmful effects on embryo development, the selection of spermatids by MitoTracker under a fluorescence microscope should be useful in research into and the treatment of infertility.