This study was designed to determine the effect of melatonin on the in vitro maturation (IVM) and developmental potential of bovine oocytes denuded of the cumulus oophorus (DOs). DOs were cultured alone (DOs) or with 10−9 M melatonin (DOs + MT), cumulus–oocyte complexes (COCs) were cultured without melatonin as the control. After IVM, meiosis II (MII) rates of DOs, and reactive oxygen species (ROS) levels, apoptotic rates and parthenogenetic blastocyst rates of MII oocytes were determined. The relative expression of ATP synthase F0 Subunit 6 and 8 (ATP6 and ATP8), bone morphogenetic protein 15 (BMP-15) and growth differentiation factor 9 (GDF-9) mRNA in MII oocytes and IFN-tau (IFN-τ), Na+/K+-ATPase, catenin-beta like 1 (CTNNBL1) and AQP3 mRNA in parthenogenetic blastocysts were quantified using real-time polymerase chain reaction (PCR). The results showed that: (1) melatonin significantly increased the MII rate of DOs (65.67 ± 3.59 % vs. 82.29 ± 3.92%; P < 0.05), decreased the ROS level (4.83 ± 0.42 counts per second (c.p.s) vs. 3.78 ± 0.29 c.p.s; P < 0.05) and apoptotic rate (36.99 ± 3.62 % vs. 21.88 ± 2.08 %; P < 0.05) and moderated the reduction of relative mRNA levels of ATP6, ATP8, BMP-15 and GDF-9 caused by oocyte denudation; (2) melatonin significantly increased the developmental rate (24.17 ± 3.54 % vs. 35.26 ± 4.87%; P < 0.05), and expression levels of IFN-τ, Na+/K+-ATPase, CTNNBL1 and AQP3 mRNA of blastocyst. These results indicated that melatonin significantly improved the IVM quality of DOs, leading to an increased parthenogenetic blastocyst formation rate and quality.

Studies on bovine oocytes have revealed that the activation of adenosine monophosphate activated protein kinase (AMPK) by millimolar concentrations of metformin controls nuclear maturation. Tuberous sclerosis complex 2 (TSC2) has been identified as a downstream target of AMPK. The objective of this study was to investigate the effects of addition of low concentrations of metformin (1 nM to 10 μM) on the percentage of cultured cumulus–oocyte complexes (COC) giving rise to cleavage-stage embryos and AMPK-mediated TSC2 activation. Metformin was supplemented either throughout in vitro embryo production (IVP) or only during in vitro fertilization (IVF). COC were matured in vitro, inseminated, and presumptive zygotes cultured for a further 72 h post insemination before the percentage of COC that gave rise to zygotes and early embryo development was assessed. The presence of TSC2 in bovine embryos and its possible AMPK-induced activation were assessed by immunocytochemistry. Metformin had a dose-dependent effect on the numbers of cultured COC that gave rise to embryos. Drug treatment either throughout IVP or only during IVF decreased the percentage of ≥8-cell embryos (1 μM, P < 0.05; 10 μM, P < 0.01; and 0.1 μM, 10 μM, P < 0.01, respectively) and increased the percentage of 2-cell embryos (10 μM, P < 0.01 and P < 0.05 respectively). The percentage of cultured COC that gave rise to zygotes was not affected by metformin. TSC2 is expressed in early embryos. Metformin (10 μM) either throughout IVP or during IVF only, increased AMPK-induced PhosphoS1387-TSC2 immunoreactivity (P < 0.01) and this increase corresponded to the total TSC2 protein levels expressed in cells. Our results suggest that there is a dose-dependent negative effect of metformin on the ability of oocytes to cleave following insemination, possibly mediated through an AMPK-induced activation of TSC2.

This study aims to investigate the effects of jacalin and follicle-stimulating hormone (FSH) on activation and survival of goat primordial follicles, as well as on gene expression in cultured ovarian tissue. Ovarian fragments were cultured for 6 days in minimum essential medium (MEM) supplemented with jacalin (10, 25, 50 or 100 μg/ml – Experiment 1) or in MEM supplemented with jacalin (50 μg/ml), FSH (50 ng/ml) or both (Experiment 2). Non-cultured and cultured tissues were processed for histological and ultrastructural analysis. Cultured tissues from Experiment 2 were also stored to evaluate the expression of BMP-15, KL (Kit ligand), c-kit, GDF-9 and proliferating cell nuclear antigen (PCNA) by real-time polymerase chain reaction (PCR). The results of Experiment 1 showed that, compared with tissue that was cultured in control medium, the presence of 50 μg/ml of jacalin increased both the percentages of developing follicles and viability. In Experiment 2, after 6 days, higher percentages of normal follicles were observed in tissue cultured in presence of FSH, jacalin or both, but no synergistic interaction between FSH and jacalin was observed. These substances had no significant effect on the levels of mRNA for BMP-15 and KL, but FSH increased significantly the levels of mRNA for PCNA and c-kit. On the other hand, jacalin reduced the levels of mRNA for GDF-9. In conclusion, jacalin and FSH are able to improve primordial follicle activation and survival after 6 days of culture. Furthermore, presence of FSH increases the expression of mRNA for PCNA and c-kit, but jacalin resulted in lower GDF-9 mRNA expression.

The oviduct is a dynamic organ in which final gamete maturation, fertilization and early embryo development take place. It is considered to be a sterile site; however the mechanism for sterility maintenance is still unknown. S100A7 is an anti-microbial peptide that has been reported in human reproductive tissues such as prostate, testicle, ovary, normal cervical epithelium and sperm. The current work reports the presence of S100A7 in the Fallopian tube and its localization at the apical surface of epithelial cells. For comparison, porcine S100A7 was used for antibody development and search for peptide in reproductive tissues. Although present in boar seminal vesicles and seminal plasma, S100A7 was not detected on female porcine organs. Also, in contrast with the human protein, porcine S100A7 did not show anti-microbial activity under the conditions tested. Phylogenetic analyses showed high divergence of porcine S100A7 from human, primate, bovine, ovine and equine sequences, being the murine sequence at a most distant branch. The differences in sequence homology, Escherichia coli-cidal activity, detectable presence and localization of S100A7 from human and pig, suggest that there are possible different functions in each organism.

Octamer-binding transcription factor 4 (Oct4) is a critical molecule for the self-renewal and pluripotency of embryonic stem cells. Recent reports have shown that Oct4 also controls cell-cycle progression and enhances the proliferation of various types of cells. As the high proliferation of donor fibroblasts is critical to the production of transgenic pigs, using the somatic cell nuclear transfer technique, we analysed the effect of Oct4 overexpression on the proliferation of porcine fibroblasts and embryos. Porcine endogenous Oct4 cDNA was cloned, sequenced and inserted into an expression vector. The vector was transfected into porcine fibroblasts, and a stable Oct4-overexpressed cell line was established by antibiotic selection. Oct4 expression was validated by the immunostaining of Oct4. Cell morphology was changed to sharp, and both proliferation and migration abilities were enhanced in Oct4-overexpressed cells. Real-time RT-PCR results showed that p16, Bcl2 and Myc were upregulated in Oct4-overexpressed cells. Somatic cell nuclear transfer was performed using Oct4-overexpressed cells, and the development of Oct4 embryos was compared with that of wild-type cloned embryos. The cleavage and blastocyst formation rates were improved in the Oct4 embryos. Interestingly, blastocyst formation of the Oct4 embryos was observed as early as day 5 in culture, while blastocysts were observed from day 6 in wild-type cloned embryos. In conclusion, the overexpression of Oct4 enhanced the proliferation of both porcine fibroblasts and embryos.

The effects of Percoll density gradient centrifugation on sperm quality, in vitro fertilizability and developmental capacity of frozen–thawed boar sperm were evaluated. Two-step density gradient centrifugation by Percoll enhanced significantly the motility parameters of sperm compared with a simple centrifugation procedure. Percentages of motile sperm and sperm with intact plasma and acrosome membranes after Percoll separation were significantly greater than those after simple centrifugation. The rates of penetration, cleavage and blastocyst formation after in vitro fertilization were significantly improved by Percoll separation compared with simple centrifugation and were influenced positively by the intactness of sperm head membranes, but not any sperm motility parameters. However, insemination with increased concentrations of sperm prepared by Percoll gradient centrifugation did not improve the success of fertilization and embryo development in vitro. Our results indicate that the integrity of sperm head membranes after Percoll separation is important for successful embryo development in vitro, more so than sperm motility.

Light exposure is a common stress factor during in vitro handling of oocytes and embryos that originates from both microscope and ambient light. In the current study, the effect of two types of ambient light (daylight and laboratory light) on porcine parthenogenetically activated (PA) embryos was tested in two experiments: (1) ambient light on medium subsequently used for embryo in vitro development; and (2) ambient light exposure on activated oocytes before in vitro development. The results from Experiment 1 showed that exposure of culture medium to both types of ambient light decreased the percentage of blastocysts that showed good morphology, only after 24 h exposure. The results from Experiment 2 revealed a reduction in both blastocyst formation and quality when activated oocytes were exposed to both types of ambient light. This effect was seen after only 1 h exposure and increased with time. In conclusion, exposure to ambient light can be harmful to embryo development, both when medium is exposed for a long period of time and, to a greater extent, when the embryo itself is exposed for >1 h. In practice, it is therefore recommended to protect both culture medium and porcine embryos against ambient light during in vitro handling in the laboratory.

This study was aimed at investigating zona pellucida glycoproteins (ZP) ZP2, ZP3 mRNA expression as well as ZP3, ZP4 (ZPB) protein distribution before and after in vitro maturation (IVM) in canine oocytes. The cumulus–oocyte complexes (COCs) were recovered from 27 anoestrous mongrel bitches and matured for 72 h in TCM199 medium. The canine COCs were analysed before and after IVM. Using real-time quantitative polymerase chain reaction (RQ-PCR), both groups of oocytes were analysed for detection of ZP2 and ZP3 mRNA profiles as well as using confocal microscopic analysis for observation of ZP3 and ZP4 protein distribution. In post-IVM canine oocytes an increase in transcript content of ZP2 and ZP3 genes as well as a decrease in ZP3 and ZP4 protein levels were observed when compared with pre-IVM oocytes. Moreover, the ZP4 protein before IVM was significantly distributed in the peripheral area of cytoplasm, whereas after IVM it was localized rather than in the entire cytoplasm. In contrast, the ZP3 protein was found both before and after IVM was distributed in the peripheral area of the cytoplasm. In conclusion, we suggest that the expression of ZP2 and ZP3 genes is associated with the maturation stage of canine oocytes, as higher mRNAs levels were found after IVM. However, a decreased expression of ZP3 and ZP4 proteins after IVM suggests maturation-dependent down-regulation of these protein translations, which may result in disturbed fertilization.

The efficiency of somatic cell nuclear transfer (SCNT) cloning remains low, thus limiting the applications of this technique. In this study, we used immunochemistry and confocal microscopy to detect the microtubule component, β-tubulin, in SCNT, parthenogenetic (PA), and intracytoplasmic sperm injection (ICSI) embryos before the first mitotic division. β-Tubulin is the component subunit of microtubule, which plays critical roles in regulating localization of cellular organelles, and the growth, maturation and fertilization of oocytes. Our results demonstrated similar changes of spindle patterns in PA and ICSI embryos. The second meiotic division resumed 1 h post-treatment, and the cytoplasmic asters (CAs) disappeared. After about 4–6 h of treatment, pronuclei formed with the midbodies connecting each other. Meanwhile, the CAs reappeared and a microtubule network developed in the cytoplasm. However, SCNT embryos showed abnormal multipolar spindles, and the pseudopronuclei that contained many nucleoli existed after 6 h of SrCl2 activation. Enucleated oocytes alone did not form spindle-like structures when they were artificially activated for 6 h, indicating that somatic cell chromosomes might be necessary for spindle formation in SCNT embryos. These results demonstrated abnormal changes of β-tubulin in mouse SCNT embryos, compared with PA and ICSI embryos.

The aim of this study was to determine the morphological characteristics of the older reproductive aged women's oocytes and to reveal the influence of these characteristics on intra-cytoplasmic sperm injection (ICSI) outcomes. The oocytes of women older than 35 years of age were evaluated retrospectively. Non-invasive polarization microscopy (PolScope) examinations of mature oocytes were performed by measurement of meiotic spindles’ length, area and retardance and zona pellucida thickness and retardance. Fertilization and conception competence and the correlation with the birefringent structures were assessed. Two hundred and thirteen mature oocytes from 54 women were evaluated with a PolScope. Length of the meiotic spindle was shown to be related to fertilization success of women with advanced maternal age. In conclusion, the PolScope is a useful device used to identify the oocyte quality. Quantitative measurements of meiotic spindle parameters may be valuable for the selection of high-quality oocytes that have the potential for embryo development in the in vitro fertilization (IVF) laboratory of women older than 35 years of age who are mostly poor responders.

The effects of the paternal breed on early embryo and later pre- and postnatal development are well documented. Several recent studies have suggested that such paternal effects may be mediated by the paternally induced epigenetic modifications during early embryogenesis. The objective of this study was to investigate the effects of the paternal breed on the early embryonic development and relative expression of the maternally imprinted gene, IGF-II, and the apoptosis-related genes BAK1 and BCL2-L1 in in vitro produced (IVP) bovine embryos derived from two unrelated paternal breeds (Holstein and Brown Swiss). The degree of correlation of IGF-II expression pattern with embryo developmental competence and apoptosis-related genes was also investigated. The relative abundance of IGF-II, BCL2-L1 and BAK1 transcripts in day 8 embryos was measured by quantitative reverse-transcription polymerase chain reaction using the comparative Cp method. Our data revealed that the paternal breed did not influence cleavage rate, blastocyst rate and relative abundance of IGF-II, BAK1 and BCL2-L1 in day 8 blastocysts (P > 0.05). Nevertheless, IGF-II expression levels were highly correlated with embryonic developmental competence (r = 0.66, P < 0.1), relative expression of BCL2-L1 (r = 0.72, P < 0.05) and ratio of BCL2-L1/BAK1 (r = 0.78, P < 0.05). In conclusion, our data show that IGF-II, BCL2-L1 and BAK1 expression is not related to the chosen combination of paternal breed, but that IGF-II expression is correlated with embryonic viability and apoptosis-related gene expression.

Exposure to lead may cause changes in the male reproductive system. We evaluated the effect of lead chloride (PbCl2) in vitro on semen quality from 31 individuals. Samples were incubated at room temperature for two exposure times (4 h and 8 h) and with two concentrations of PbCl2 (15 μg/ml or 30 μg/ml). Results showed that PbCl2 significantly inhibited rapid progressive motility and caused an increase in the percentage of tail anomalies in both times and concentrations assessed, as well as a decrease in vitality in the group exposed to 30 μg/ml PbCl2. A significant increase in immotile sperm was also observed between the group control and the groups submitted to lead. Total motility and DNA fragmentation also showed a significant decrease and increase, respectively, after 4 h of incubation in the group exposed to 30 μg/ml and in both groups after 8 h of incubation. In conclusion, PbCl2 affected sperm parameters and DNA integrity, which are essential for male fertility.

Eukaryotic species commonly contain a diploid complement of chromosomes. The diploid state appears to be advantageous for mammals because it enables sexual reproduction and facilitates genetic recombination. Nonetheless, the effects of DNA ploidy on mammalian ontogeny have yet to be understood. The present study shows phenotypic features and expression patterns of imprinted genes in tripronucleate diandric and digynic triploid (DAT and DGT) mouse fetuses on embryonic day 10.5 (E10.5). Measurement of crown–rump length revealed that the length of DGT fetuses (1.87 ± 0.13 mm; mean ± standard error of the mean) was much smaller than that of diploid fetuses (4.81 ± 0.05 mm). However, no significant difference was observed in the crown–rump length between diploid and DAT fetuses (3.86 ± 0.43 mm). In DGT fetuses, the expression level of paternally expressed genes, Igf2, Dlk1, Ndn, and Peg3, remained significantly reduced and that of maternally expressed genes, Igf2r and Grb10, increased. Additionally, in DAT fetuses, the Igf2 mRNA expression level was approximately twice that in diploid fetuses, as expected. These results provide the first demonstration that imprinted genes in mouse triploid fetuses show distinctive expression patterns independent of the number of parental-origin haploid sets. These data suggest that both DNA ploidy and asymmetrical functions of parental genomes separately influence mammalian ontogeny.

Oscar (Astronotus ocellatus) is an important fish from the Amazon Basin that has great potential for fish farming, human consumption, sport fishing and fish keeping. This study aimed to evaluate the effect of two hormonal treatments on the induction of artificial reproduction in broodstock and to describe the histological development of embryos and larvae. Broodstocks were selected and induced using two different hormones: (i) extract of carp pituitary (ECP); and (ii) synthetic human chorionic gonadotropin (hCG). Spawnings were transferred to hatcheries, collected at pre-established times, processed and analysed by histology. Astronotus ocellatus did not respond well to induced reproduction. From 16 couples of breeding fish, only five out of the eight females released oocytes after the hormonal action time, three with hCG and two with ECP; just one male responded positively to hCG. Oscar eggs were oval, and semi-adhesive, the yolk contained granules, and egg diameter was approximately 1.65 ± 0.057 to 1.98 ± 0.038 mm. Development from the initial collection (IC) point until the total absorption of the yolk lasted 315 h, at an average temperature of 27.45 ± 2.13°C. Several events marked embryonic and larval development, including the formation of the optic cup, forebrain, otic vesicle and cephalic divisions. The newly hatched larvae had non-pigmented eyes, and a closed mouth and anus, as well as the presence of adhesive glands on the head. Larval development was characterized by formation of the heart, liver, gaseous bladder, gills, pronephros, brain, fins and also the digestive tract. These results provide important information for the rearing and reproduction of A. ocellatus.

Betta splendens is a very important ornamental species. The current paper describes the embryonic and larval development of B. splendens under stereomicroscopy and scanning electron microscopy. Eggs and larvae from natural spawning were collected at different developmental stages at previously established intervals and analysed. The eggs of B. splendens are yellowish, clear, spherical, demersal, translucent and telolecithal with a large amount of yolk. Between 0–2 h post-initial collection (hpIC), the eggs were at the egg cell, first cleavage and morula stages. The blastula stage was identified at 2–3 hpIC and the early gastrula phase was observed at 3–4 hpIC with 20% epiboly, which was finalized after 13–18 hpIC. When the pre-larvae were ready to hatch, the appearance of somites and the free tail were observed, at 23–25 hpIC. At 29 hpIC, the majority of larvae had already hatched at an average temperature of 28.4 ± 0.2°C. The newly hatched larvae measured 2.47 ± 0.044 mm total length. The mouth opened at 23 h post-hatching (hPH) and the yolk sac was totally absorbed at 73 hPH. After 156 hPH, the heart was pumping blood throughout the entire larval body. The caudal fin, operculum and eyes were well developed at 264 hPH. When metamorphosis was complete at 768 hPH, the larvae became juveniles. The current study presents the first results about early development of B. splendens and provides relevant information for its reproduction, rearing and biology.

The order Caudata includes about 660 species and displays a variety of important developmental traits such as cleavage pattern and egg size. However, the cleavage process of tailed amphibians has never been analyzed within a phylogenetic framework. We use published data on the embryos of 36 species concerning the character of the third cleavage furrow (latitudinal, longitudinal or variable) and the magnitude of synchronous cleavage period (up to 3–4 synchronous cell divisions in the animal hemisphere or a considerably longer series of synchronous divisions followed by midblastula transition). Several species from basal caudate families Cryptobranchidae (Andrias davidianus and Cryptobranchus alleganiensis) and Hynobiidae (Onychodactylus japonicus) as well as several representatives from derived families Plethodontidae (Desmognathus fuscus and Ensatina eschscholtzii) and Proteidae (Necturus maculosus) are characterized by longitudinal furrows of the third cleavage and the loss of synchrony as early as the 8-cell stage. By contrast, many representatives of derived families Ambystomatidae and Salamandridae have latitudinal furrows of the third cleavage and extensive period of synchronous divisions. Our analysis of these ontogenetic characters mapped onto a phylogenetic tree shows that the cleavage pattern of large, yolky eggs with short series of synchronous divisions is an ancestral trait for the tailed amphibians, while the data on the orientation of third cleavage furrows seem to be ambiguous with respect to phylogeny. Nevertheless, the midblastula transition, which is characteristic of the model species Ambystoma mexicanum (Caudata) and Xenopus laevis (Anura), might have evolved convergently in these two amphibian orders.

We measured the ATP concentrations in the porcine follicular fluid derived from three sizes of follicles (small: <3 mm, medium: 3–6 mm, large: >6 mm in diameter). Then, the effects of pre-treatment (100 μM each for 30 min before maturation) with antagonists for extracellular ATP receptor P2X or P2Y on the nuclear maturation rate of cumulus-cell-enclosed (COs) or -denuded oocytes (DOs) up to the preovulatory stage in the presence or absence of 20 nM ATP (a similar concentration to that of medium-sized follicle fluid) were investigated. The antagonists used were pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) or reactive blue 2 (RB2), for extracellular ATP receptor P2X and P2Y, respectively. In addition, the embryonic development rates of COs pre-treated with RB2 were also evaluated. It was found that when the follicular sizes increased, the ATP concentrations significantly decreased (P < 0.05). No differences were observed in the nuclear maturation rates among all COs, regardless of pre-treatment with (+) or without (–) PPADS and in the presence (+) or absence (–) of ATP during maturation. In contrast, the nuclear maturation rate of the COs, but not DOs, in the ATP(–) RB2(+) group was significantly lower (P < 0.05) than that of the ATP(–) RB2(–) and ATP(+)RB2(–) groups. The pronuclear formation and blastocyst formation rates by parthenogenetic activation in the ATP(–) RB2(+) and ATP(+) RB2(+) groups were significantly lower (P < 0.05) than those in the ATP(–) RB2(–) group. In conclusion, it is suggested that the nuclear maturation of porcine oocytes may be influenced by the ATP receptor P2Y present in the cumulus cells.

The expression of nine serotonin (5-HT) receptor transcripts was studied using reverse transcription polymerase chain reaction (RT-PCR) in germ cells, cleavage and gastrulation stages of Japanese quail, and qPCR for 5-HT3 and 5-HT4 receptors in oocytes and embryos. We show the presence/absence of nine serotonin transcripts known in birds for receptors 5-HT1A, 5-HT1F, 5-HT2B, 5-HT2C, 5-HT3, 5-HT4, 5-HT5A, 5-HT6 and 5-HT7A in avian germ cells and early embryos. The absence of 5-HT3 and 5-HT5A in primordial germ cells and of 5-HT3 and 5-HT7A in sperm is characteristic. All transcripts appeared in oocytes at all stages (except for 5-HT3 and 5-HT5A transcripts) and all were present in cleaving embryos and at gastrulation, except for 5-HT3, which was permanently observed as late as in stage 4. Interestingly, 5-HT3 and 5-HT5A receptors accumulated in 3-mm and F1 oocytes but were degraded at ovulation and started to be re-transcribed in cleavage stage II embryos and beyond. The selective appearance of 5-HT receptors in germ cells and early embryos supports the hypothesis that serotonin may act as a signalling molecule at early stages of germ line and embryo differentiation via individual receptors present during different stages, when specialized communication systems are not yet developed.

Natural resistance-associated macrophage protein 1 (Nramp1) plays an important role in restraining the growth of intracellular pathogens within macrophages. In this study, Nramp1 cDNA was cloned from Qinchuan cattle and its anti-bacterial activity was demonstrated as being able to significantly inhibit the growth of Salmonella abortusovis and Brucella abortus in macrophages. Calf fibroblasts stably transfected with pSP–NRAMP1–HA vector were used to reconstruct bovine embryos by somatic cell nuclear transfer (SCNT). Reconstructed embryos were maturated in vitro and the blastocyst formation rate (14.0%) was similar to that of control embryos (14.5%). Transgenic blastocysts were transplanted into 43 recipient cattle, of which 14 recipients became pregnant as evidenced by non-return estrus and by rectal palpation. One fetus was aborted after 6½ months of pregnancy and transgene integration was confirmed by semi-quantitative polymerase chain reaction. Together, this study showed that bovine Nramp1 retains biological function against the growth of intracellular bacteria and can be used to reconstruct embryos and produce Nramp1 transgenic cattle, which may benefit the animal and enhance their ability to prevent attack by intracellular pathogens.

The impact of sperm DNA fragmentation on assisted reproductive technology (ART) successes, in terms of outcome, is now established. High levels of DNA strand breaks severely affect the probability of pregnancy. The importance of sperm nucleus condensation in early embryogenesis and, subsequently, on the quality of the conceptus is now emerging. In this article we have compared in situ analyses with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling (TUNEL) (for DNA fragmentation) with aniline blue (AB) (for nucleus decondensation), versus flow cytometry (FC) after acridine orange staining, in a double-blinded analysis. In our hands, TUNEL and acridine orange give perfectly comparable results. For decondensation the results are also comparable, but the double-stranded green fluorescence obtained with acridine orange seems to slightly underestimate the decondensation status obtained with AB.