Sperm morphometry can be applied to identify different animal groups and species and to evaluate sperm quality. Furthermore, knowledge on species-specific differences will help to enhance biological information, as well as to develop efficient reproductive technologies. The aims in the present study were to describe sperm morphometry from the recently characterized species S. collinsi and S. vanzolinii, to verify if the morphometric sperm patterns are similar or different between both species, and to determine if the sperm morphometry is affected by the levels of sperm defects using the S. collinsi as a model. Semen was collected from S. collinsi (n = 10) and S. vanzolinii (n = 2) monkeys, and sperm was submitted to morphological analysis. From the 10 samples from S. collinsi, five presented sperm of poor quality and two subgroups were formed for this species, i.e. high and poor quality sperm. Data on sperm motility and vigour were analysed, as well morphometric parameters on sperm head and tail. It was observed the normal morphometry was correlated with high quality sperm. Poor quality sperm presented smaller and 7% more ellipticity in their head, when compared with high quality sperm. Sperm from S. vanzolinii presented larger head than those from S. collinsi, but tail lengths were similar. Sperm morphometry can be used as a complementary tool to predict sperm motility and vigour for the S. collinsi species, and S. collinsi appear as a suitable model for S. vanzolinii.

In response to gonadotropins and androgens, testicular cells produce various molecules that control proper proliferation and differentiation of spermatogenic cells through their paracrine and autocrine actions. However, molecules functioning downstream of the hormonal stimulation are poorly understood. Leukaemia inhibitory factor (Lif) is known to maintain the pluripotency of stem cells including embryonic stem cells and primordial germ cells at least in vitro, but its actual roles in vivo remain to be elucidated. To clarify the function of Lif in teleost (medaka) testes, we examined the effects of Lif on spermatogenesis in a newly established cell culture system using a cell line (named Mtp1) derived from medaka testicular somatic cells as feeder cells. We found that addition of baculovirus-produced recombinant medaka Lif to the culture medium or co-culture with Lif-overexpressing Mtp1 cells increased the number of spermatogonia. In situ hybridization and immunohistochemical analyses of the medaka testes showed that mRNAs and proteins of Lif are expressed in spermatogonia and the surrounding Sertoli cells, with higher expression levels in type A (undifferentiated) spermatogonia than in type B (differentiated) spermatogonia. Our findings suggest that Lif regulates spermatogonial cell proliferation in the medaka.

The present study aimed to determine the relationship among changes in the number of preantral follicles and concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), progesterone (P4), androstenedione (A) and estradiol-17β (E2) in the fetal heart, umbilical cord and maternal blood. Primordial follicles had already appeared in a 20 cm fetus and primary follicles were observed in a 50 cm fetus. In a 70 cm fetus, the number of primordial and primary follicles increased rapidly and secondary follicles were present. The concentrations of LH and FSH did not change between 20 cm and 160 cm in fetal length. When the fetal length became >70 cm, serum levels in the fetus, umbilical cord and mothers, and E2 levels in umbilical cord increased synchronously (p<0.05). These results showed increases in the number of preantral follicles in the Antarctic minke whale fetal ovary along with fetal growth during the early gestation period. These findings suggest that the change in preantral follicles was associated with changes in the concentration of steroids in early gestation periods. The changes in steroid concentrations in the fetal and umbilical cord blood and the increased number of preantral follicles were coincident at around 70 cm in fetal length, whereas the growth and differentiation of primordial and primary follicles appeared to be independent of FSH and LH.

This study evaluated the effects of oocyte meiosis inhibitors roscovitine (ROS) and butyrolactone I (BL-I) on in vitro production of bovine embryos. Bovine oocytes were maintained in pre in vitro maturation (pre-IVM) with 25 µM ROS or 100 µM BL-I for 24 h to delay meiosis and for 24 h in in vitro maturation (IVM). Following this treatment, the nuclear maturation index was evaluated. All embryos degenerated following this procedure. In the second set of experiments, oocytes were maintained for 6 or 12 h in pre-IVM with the following three treatments: ROS (25 µM or 12.5 µM), BL-I (100 µM or 50 µM) or a combination of both drugs (6.25 µM ROS and 12.5 µM BL-I). Oocytes were cultivated for 18 or 12 h in IVM. When a meiosis-inducing agent was used during pre-IVM for 24 h, more degenerated oocytes were observed at the end of the IVM period. This effect decreased when the meiotic blocking period was reduced to 6 or 12 h. No significant differences were observed in the blastocyst production rate of oocytes in pre-IVM for 6 h with ROS, BL-I, or ROS + BL-I compared with that of the control group (P > 0.05). However, inhibition of oocytes for 12 h resulted in decreased embryo production compared with that in the controls (P < 0.05). There was no difference in the post-vitrification embryo re-expansion rate between the study groups, showing that the meiotic inhibition for 6 or 12 h did not alter the embryo cryopreservation process.

Stem cells are an immortal cell population capable of self-renewal; they are essential for human development and ageing and are a major focus of research in regenerative medicine. Despite considerable progress in differentiation of stem cells in vitro, culture conditions require further optimization to maximize the potential for multicellular differentiation during expansion. The aim of this study was to develop a feeder-free, serum-free culture method for human embryonic stem cells (hESCs), to establish optimal conditions for hESC proliferation, and to determine the biological characteristics of the resulting hESCs. The H9 hESC line was cultured using a homemade serum-free, feeder-free culture system, and growth was observed. The expression of pluripotency proteins (OCT4, NANOG, SOX2, LIN28, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) in hESCs was determined by immunofluorescence and western blotting. The mRNA expression levels of genes encoding nestin, brachyury and α-fetoprotein in differentiated H9 cells were determined by RT-PCR. The newly developed culture system resulted in classical hESC colonies that were round or elliptical in shape, with clear and neat boundaries. The expression of pluripotency proteins was increased, and the genes encoding nestin, brachyury, and α-fetoprotein were expressed in H9 cells, suggesting that the cells maintained in vitro differentiation capacity. Our culture system containing a unique set of components, with animal-derived substances, maintained the self-renewal potential and pluripotency of H9 cells for eight passages. Further optimization of this system may expand the clinical application of hESCs.

The large and optically clear embryos of the zebrafish provide an excellent model system in which to study the dynamic assembly of the essential contractile band components, actin and myosin, via double fluorescent labelling in combination with confocal microscopy. We report the rapid appearance (i.e. within <2 min) of a restricted arc of F-actin patches along the prospective furrow plane in a central, apical region of the blastodisc cortex. These patches then fused with each other end-to-end forming multiple actin cables, which were subsequently bundled together forming an F-actin band. During this initial assembly phase, the F-actin-based structure did not elongate laterally, but was still restricted to an arc extending ~15° either side of the blastodisc apex. This initial assembly phase was then followed by an extension phase, where additional F-actin patches were added to each end of the original arc, thus extending it out to the edges of the blastodisc. The dynamics of phosphorylated myosin light chain 2 (MLC2) recruitment to this F-actin scaffold also reflect the two-phase nature of the contractile apparatus assembly. MLC2 was not associated with the initial F-actin arc, but MLC2 clusters were recruited and assembled into the extending ends of the band. We propose that the MLC2-free central region of the contractile apparatus acts to position and then extend the cleavage furrow in the correct plane, while the actomyosin ends alone generate the force required for furrow ingression. This biphasic assembly strategy may be required to successfully divide the early cells of large embryos.

Spermatozoa from the mollusc Ostrea edulis are capable of fusing to and entering de-chorionated ascidian eggs. During interaction they generate activation currents, comparable to the fertilisation currents induced by homologous spermatozoa. Activation currents are inward at − 80 mV, with a mean initial slope of 111 ± 124 pA/s for Ciona intestinalis eggs and 47 ± 25 pA/s for Phallusia mammillata eggs, while the mean peak currents are 2782 ± 1132 pA and 1523 ± 1668 pA, respectively. The fertilisation and activation currents reverse at a holding potential of 0 mV to + 20 mV, suggesting that oyster sperm and ascidian sperm gate the same channel precursor, a non-specific, large conductance channel described previously (Dale & DeFelice, 1984). In contrast to homologous fertilisation, the activation current is not followed by a polarised contraction of the egg surface, nor other signs of egg activation. Staining eggs with Hoechst 33342 after insemination shows the female nucleus and a single oyster sperm nucleus at the antipode. This suggests a specialised predetermined site at the vegetal pole for sperm entry. Homologous and heterologous spermatozoa delivered, in a large pipette, to localised areas of the egg surface generate fast inward currents of 200–2000 pA, but do not induce contraction of the egg surface. This shows that although channel precursors are located globally over the egg surface, channel activation does not necessarily trigger the contraction wave. Subsequent induction of both a fertilisation current and a contraction by homologous sperm added to the bath, implies a regionalised activation site with an accumulation of channel precursors and a ‘pacemaker’ for the initiation of the contraction wave.

Calcium signalling is involved in important events in oocytes, such as meiotic competence acquisition. We have previously demonstrated the positive influence of animal age and gonadotropin stimulation in vivo regarding the ability of oocytes recovered from preantral follicles to exhibit calcium spikes. In the present work we determined whether preantral follicle development in vitro also allows oocytes to acquire calcium signalling activity. We also aimed to verify the influence of animal age, FSH + LH and/or insulin on oocyte calcium spike acquisition during preantral follicle culture. Early preantral follicles were isolated from 12-day-old and 1- to 3-month-old F1 hybrid mice and cultured individually for either 2 or 6 days. At the end of the culture period the oocytes were processed for calcium imaging by confocal microscopy. We show that oocytes recovered from cultured preantral follicles exhibit variable calcium spike activity rates, depending on animal age, culture duration and hormonal supplementation. Oocytes recovered from adult animals continue to exhibit calcium spikes, and those recovered from juveniles acquire that activity after culture. Insulin and gonadotropins in combination account for an early and maintained inhibitory effect on calcium signalling acquisition by oocytes. Insulin alone also leads to an early inhibitory effect, which, however, disappears with longer culture periods. Contrary to the complex in vivo situation, the acquisition of calcium signalling by oocytes in a controlled in vitro environment does not seem to be dependent on gonadotropins alone.

The presence of intra-ovarian nerves was reported more than a century ago and successive investigations have demonstrated that the mammalian ovary is innervated by both sympathetic and sensory fibres distributed to the different compartments of the gland, such as blood vessels, ovarian stroma and follicle wall. Despite the extensive ovarian innervation and the experimental evidence indicating that neuromediators can influence sex steroid production, the role of the nervous system in the control of ovarian activity is still largely unknown.

In a previous study, we have identified a set of conserved spermatogenic genes whose expression is restricted to testis and ovary and that are developmentally regulated. One of these genes, the transcription factor Mael, has been reported to play an essential role in mouse spermatogenesis. Nevertheless, the role of Mael in mouse oogenesis has not been defined. In order to analyse the role of Mael in mouse oogenesis, the expression of this gene was blocked during early oogenesis in mouse in vitro using RNAi technology. In addition, the role of Mael during differentiation of embryonic stem cells (ESC) into germ cells in vitro was analysed. Results show that downregulation of Mael by a specific short interfering RNA disrupted fetal oocyte growth and differentiation in fetal ovary explants in culture and the expression of several germ-cell markers in ESC during their differentiation. These results suggest that there is an important role for Mael in early oogenesis and during germ-cell differentiation from embryonic stem cells in mouse in vitro.

The aims of the present study were: (1) to evaluate the influence of sperm concentration (ranging from 0.5 × 106 to 4 × 106 spermatozoa/ml) and length of the gamete co-incubation time (2, 4, 6, 8, 10, 12, 16, 20, 24 or 28 h) on in vitro fertilization (IVF), assessing the sperm penetration rate; (2) to investigate the kinetics of different semen parameters as motility, viability and acrosome status during the co-culture period; and (3) to analyse the effect of the presence of cumulus–oocytes complexes (COCs) on these parameters. To achieve these objectives, several experiments were carried out using in vitro matured oocytes from prepubertal goats. The main findings of this work are that: (1) in our conditions, the optimum sperm concentration is 4 × 106 sperm/ml, as this sperm:oocyte ratio (approximately 28,000) allowed us to obtain the highest penetration rate, without increasing polyspermy incidence; (2) the highest percentage of viable acrosome-reacted spermatozoa is observed between 8–12 h of gamete co-culture, while the penetration rate is maximum at 12 h of co-incubation; and (3) the presence of COCs seems to favour the acrosome reaction of free spermatozoa on IVF medium, but not significantly. In conclusion, we suggest that a gamete co-incubation for 12–14 h, with a concentration of 4 × 106 sperm/ml, would be sufficient to obtain the highest rate of penetration, reducing the exposure of oocytes to high levels of reactive oxygen species produced by spermatozoa, especially when a high sperm concentration is used to increase the caprine IVF outcome.

This study examined the effect of a cryoprotectant with and without pentoxifylline supplementation on the motility and viability of human testicular sperm, both before and after freezing. Testicular samples were obtained from 68 patients with azoospermia who came to the Andrology Service of West China Second University Hospital, Sichuan University, for testicular biopsies from December 2019 to April 2020. All patients were assigned randomly to two groups: experimental, whose testicular sperm were added to the cryoprotectant with pentoxifylline, and the control, whose testicular sperm were added to the cryoprotectant without pentoxifylline. Both groups used the same freezing and thawing methods. Testicular sperm motility in the experimental group was significantly higher than that of the control group, both before and after cryopreservation. The recovery rate of sperm motility in the experimental group was significantly higher than that of the control group. The percentage of samples with motile testicular sperm in the experimental group was significantly higher than that of the control group after thawing. Sperm viability was unchanged between the experimental and control groups, both before and after freezing. Overall, a pentoxifylline-supplemented cryoprotectant can significantly improve the motility of testicular sperm before and after cryopreservation.

This study evaluated the effects of reversible meiotic inhibition and different culture media (PZM3 or NCSU23) on production of porcine embryos by either in vitro fertilization (IVF) or parthenogenetic activation (PA). Oocytes from abattoir-derived ovaries were allocated into two groups for maturation: CHX (5 μg/ml cycloheximide for 10 h) or Control (no CHX). The percentage of metaphase II (MII) oocytes was determined at 36, 40 or 44 h of in vitro maturation. For IVF and PA, denuded oocytes were fertilized with purified sperm for 6 h or activated by electric stimuli. Zygotes were then subdivided into two culture groups: NCSU23 or PZM3. No effect of treatment with CHX and culture media was observed on cleavage (D3) and blastocyst (D7) rates in IVF and PA groups. There are no differences of quality or development rates between IVF-derived embryos cultured in NCSU23 or PZM3. However, we observed high quality PA embryos in PZM3 compared with NCSU23. Maturation arrest with CHX decreased the average blastocyst cell number in IVF while it was increased in PA embryos. As older oocytes are more effectively activated, CHX– blocked oocytes reached the mature stage faster than the control group. In conclusion, the CHX treatment for 10 h, followed by oocyte maturation for 40 h, is an efficient protocol to produce high quality parthenote embryos, especially when they are cultured in PZM3. However, this protocol is not satisfactory for IVF embryos production. In this case, a shorter maturation period could provide better embryo quality.

Microtubule-severing protein (MTSP) is critical for the survival of both mitotic and postmitotic cells. However, the study of MTSP during meiosis of mammalian oocytes has not been reported. We found that spastin, a member of the MTSP family, was highly expressed in oocytes and aggregated in spindle microtubules. After knocking down spastin by specific siRNA, the spindle microtubule density of meiotic oocytes decreased significantly. When the oocytes were cultured in vitro, the oocytes lacking spastin showed an obvious maturation disorder. Considering the microtubule-severing activity of spastin, we speculate that spastin on spindles may increase the number of microtubule broken ends by severing the microtubules, therefore playing a nucleating role, promoting spindle assembly and ensuring normal meiosis. In addition, we found the colocalization and interaction of collapsin response mediator protein 5 (CRMP5) and spastin in oocytes. CRMP5 can provide structural support and promote microtubule aggregation, creating transportation routes, and can interact with spastin in the microtubule activity of nerve cells (30). Knocking down CRMP5 may lead to spindle abnormalities and developmental disorders in oocytes. Overexpression of spastin may reverse the abnormal phenotype caused by the deletion of CRMP5. In summary, our data support a model in which the interaction between spastin and CRMP5 promotes the assembly of spindle microtubules in oocytes by controlling microtubule dynamics, therefore ensuring normal meiosis.

Congenital domestic absence of vas deferens (CBAVD) is a common factor in male infertility, and percutaneous epididymal sperm aspiration (PESA) combined with intracytoplasmic sperm injection (ICSI) is a primary clinical treatment, but the effect of the sperm obtained on pregnancy outcome remains to be explored. This study aimed to investigate the relationship between sperm motility with clinical outcome of PESA–ICSI in infertile males with CBAVD. A cohort of 110 couples was enrolled. In total, 76 infertile males were included in the high motility group, while the remaining 34 males were placed in the low motility group. Clinical pregnancy, embryo implantation rate and live birth rate were included as the primary outcome. After all follow-ups, we found that the high motility group achieved higher normal fertilization rates, cleavage rates, transplantable embryo rates and high-quality embryo rates than those in low motility group (normal fertilization rate, 78.2 ± 11.7% vs. 70.5 ± 10.2%, P = 0.003; cleavage rate, 97.1 ± 2.9% vs. 92.3 ± 3.0%, P = 0.000; transplantable embryo rate, 66.8 ± 14.9% vs. 58.6 ± 12.6%, P = 0.009 and high-quality embryo rate, 49.9 ± 10.5% vs. 40.5 ± 11.2%, P = 0.000). Additionally, compared with the low motility group, the clinical pregnancy rates, embryo implantation rates, and live birth rates in the high motility group were significantly increased (pregnancy rate, 61.8% vs. 26.5%, P = 0.009; embryo implantation rate, 36.5% vs. 18.0%, P = 0.044; live birth rate, 55.3% vs. 17.6%, P = 0.000). We concluded that the motility of sperm obtained by PESA affected the clinical outcome of ICSI in infertile males with CBAVD.

The aim of the present study was to characterize biopsied and stripped oocytes from the Brazilian endangered teleost pirapitinga (Brycon nattereri) using histochemical and morphological analyses. Biopsied oocytes had a mean diameter of 2.225 mm (modal diameter: 2.312 mm), complete vitellogenesis and a central or slightly eccentric nucleus. Neutral polysaccharides were detected in the follicular cells, zona radiata and yolk globules, while acidic polysaccharides were detected in the follicular cells and cortical alveoli. Ten out of the 19 females treated with two doses of carp pituitary extract (cPE) released oocytes, which were also analysed. Stripping occurred 292 ± 39 degree-hours after the second dose of cPE and led to a mean spawning weight of 36.2 g, 10% spawning index, 241 oocytes/g of ova, 8222 oocytes/female and 23 oocytes/g of body weight. Stripped oocytes had a mean diameter of 2.33 mm and a mode at 2.375 mm, were weakly adhesive and coloration ranged from wine to brown. Under scanning electron microscopy, stripped oocytes exhibited a single funnel-shaped micropyle located at the animal pole and a zona radiata that measured 7.7 μm in thickness with eight pore canals/μm2. Oocyte morphology in Brycon nattereri is similar to that found in other species of the genus, except for the larger size and weaker adhesiveness. These findings provide essential information for a better understanding of the reproductive biology of B. nattereri and the establishment of conservation measures for this threatened species.

Staining with brilliant cresyl blue (BCB) may be used for oocyte selection, but BCB staining itself and the most commonly used selection medium (DMPBS) may compromise the development of porcine oocytes in vitro. This study evaluated DNA fragmentation, nuclear maturation, the area of migration of cortical granules (CG) and embryo development for stained (BCB+) and unstained (BCB−) oocytes incubated in DMPBS and in a modified medium (ReproPel) tested for the first time. Unexposed (UN), BCB+ and BCB− oocytes were incubated composing six groups: DMPBS/UN; DMPBS/BCB+; DMPBS/BCB−; ReproPel/UN; ReproPel/BCB+; and ReproPel/BCB−. There were more BCB+ oocytes in ReproPel than in DMPBS (P < 0.05). The DNA fragmentation was evaluated for oocytes in DMPBS/BCB+, DMPBS/BCB−, ReproPel/BCB+, ReproPel/BCB− and in porcine follicular fluid (control). The frequency of oocytes with no DNA fragmentation was greatest (64.6%) in DMPBS/BCB+ and lowest in ReproPel/BCB+ and ReproPel/BCB− (26.8 and 34.1%, respectively) (P < 0.05). Nuclear maturation rates were greater (P < 0.05) for DMPBS/BCB+ (63.1%), ReproPel/UN (55.1%) and ReproPel/BCB+ (50.2%) than for DMPBS/UN (40.8%) and ReproPel/BCB− (35.5%). The area of CG was greater (P < 0.05) for ReproPel/BCB− (80.7%) and DMPBS/UN (77.6%) than for ReproPel/UN (34.7%). Cleavage rates for DMPBS/BCB+ and ReproPel/BCB+ were greater than for DMPBS/UN (P < 0.05). Blastocyst development rates were greatest (P < 0.05) for ReproPel/UN and ReproPel/BCB+. In both media, BCB staining was apparently unable to select competent oocytes, which likely occurred due to toxicity. Despite the similar nuclear maturation and area of CG compared with DMPBS, oocytes selected in ReproPel presented impaired DNA integrity.

The objectives of this study were firstly to determine whether the stimulatory function of equine growth hormone (eGH) on equine oocyte maturation in vitro is mediated via cyclic adenosine monophosphate (cAMP); and secondly if the addition of eGH in vitro influences oocyte nuclear maturation and if this effect is removed when GH inhibitors are added to the culture. Cumulus–oocyte complexes (COCs) were recovered from follicles <25 mm in diameter and randomly allocated as follows: (i) control (no additives); and (ii) 400 ng/ml of eGH. A specific inhibitor against cyclic AMP-dependent protein kinase (H-89; 10−9, 10−11 or 10−15 M concentration) and a specific adenylate cyclase inhibitor, 2′,3′-dideoxyadenosine (DDA; 10−8, 10−10 or 10−14 M concentration) were used to observe whether they could block the eGH effect. After 30 h of in vitro maturation at 38.5°C with 5% CO2 in air, oocytes were stained with 10 μg/ml of Hoechst to evaluate nuclear status. More mature oocytes (P < 0.05) were detected when COCs were incubated with eGH (29 of 84; 34.5%) than in the control group (18 of 82; 21.9%). The H-89 inhibitor used at a concentration of 10−9 M (4 of 29; 13.8%) decreased (P < 0.05) the number of oocytes reaching nuclear maturation when compared with eGH (11 of 29; 38%). The DDA inhibitor at a concentration of 10−8 M (2 of 27; 7.4%) also reduced (P < 0.05) the number of oocytes reaching maturity when compared with the eGH group (9 of 30; 30%). Results from the present study show that H-89 and DDA can be used in vitro to block the eGH effect on equine oocyte maturation.

The polar bodies are derived from meiotic divisions during oogenesis and are contained together with the oocyte within the zona pellucida. Fertilisation triggers the second meiotic division, at which time the second polar body (PB2) is formed (Hogan et al., 1986; Schatten et al., 1988; Johnson & Everitt, 1995) There is no clear evidence on the fate of the polar bodies in any mammal including the mouse, which is the commonly used research model. However, the polar bodies are generally considered as waste material, and therefore not essential to embryo development. In recent years the polar bodies have gained prominence as they have been used in humans for pre-implantation genetic diagnostic purposes (PGD), of single gene disorders, such as determining whether an embryo may have inherited the cystic fibrosis allele from its mother (Munne et al., 1995; Strom et al., 1998; Rechitsky et al., 2000). PB2 also has a potential use in cloning, for the harvesting of stem cells. Wakayama et al. (1997) have shown that PB2 has the same genetic potential as the female pronuclei and can be used for the production of normal offspring in mice. The successful use of PB2 for these purposes is dependent on its age, for its longevity, rate and nature of degeneration has yet to be determined. While there is little doubt that the first polar body (PB1) experiences a necrotic fate, the same cannot be said for PB2, which may experience an apoptotic fate. Furthermore if PB2 experiences an apoptotic fate rather than a necrotic one, it would not only be the earliest evidence of apoptosis in a mammal but also provide an excellent research model for the study of apoptosis.

The efficiency of in vitro embryo production is highly variable amongst individual sires in cattle. To eliminate that this variability is not caused by sperm chromatin damage caused by separation or capacitacion, chromatin integrity was evaluated. Seventeen of AI bulls with good NRRs but variable embryo production efficiency were used. For each bull, motile spermatozoa were separated on a Percoll gradient, resuspended in IVF–TALP medium and capacitated with or incubated without heparin for 6 h. Samples before and after separation and after 3-h and 6-h capacitacion or incubation were evaluated by the Sperm Chromatin Structure Assay (SCSA) and the proportion of sperm with intact chromatin structure was calculated. Based on changes in the non-DFI-sperm proportion, the sires were categorized as DNA-unstable (DNA-us), DNA-stable (DNA-s) and DNA-most stable (DNA-ms) bulls (n = 3, n = 5 and n = 9, respectively). In DNA-us bulls, separation produced a significant increase of the mean non-DFI-sperm proportion (p ≤ 0.01), as compared with the value before separation. Capacitacion produced a significant decrease in the mean non-DFI-sperm proportion in H+ sperm (p ≤ 0.01). In DNA-s bulls, separation significantly increased the mean non-DFI-sperm proportion (p ≤ 0.01) but during capacitacion, the mean non-DFI-sperm proportion remained almost unchanged. In DNA-ms bulls, neither separation nor capacitacion had any effect on the mean non-DFI-sperm proportion. It can be concluded that, although separation and capacitacion may produce some changes in sperm chromatin integrity, these are not associated with different in vitro fertility of the bulls involved.