Lead poisoning is a stealthy threat to human physiological systems as chronic exposure can remain asymptomatic for long periods of time before symptoms manifest. We presently review the biophysical mechanisms of lead poisoning that contribute to male infertility. Environmental and occupational exposure of lead may adversely affect the hypothalamic−pituitary−testicular axis, impairing the induction of spermatogenesis. Dysfunction at the reproductive axis, namely testosterone suppression, is most susceptible and irreversible during pubertal development. Lead poisoning also appears to directly impair the process of spermatogenesis itself as well as sperm function. Spermatogenesis issues may manifest as low sperm count and stem from reproductive axis dysfunction or testicular degeneration. Generation of excessive reactive oxygen species due to lead-associated oxidative stress can potentially affect sperm viability, motility, DNA fragmentation, membrane lipid peroxidation, capacitation, hyperactivation, acrosome reaction, and chemotaxis for sperm-oocyte fusion, all of which can contribute to deter fertilization. Reproductive toxicity has been tested through cross-sectional analysis studies in humans as well as in vivo and in vitro studies in animals.

Previous investigations suggested that elevated cell-free DNA (cfDNA) can indicate non-healthy states. However, the potential association between cfDNA seminal plasma levels and fertility sperm parameters has not yet been determined. Therefore, the present study evaluated the association between seminal cfDNA levels and sperm fertility criteria to determine the use of seminal cfDNA quantification. An in vivo protocol quantified cfDNA levels of semen samples obtained from 163 male patients using fluorescent PicoGreen dye staining. To confirm if semen cfDNA quantification is realistic, an in vitro complementary test was performed using three or four semen samples. The fresh sperm samples were exposed to paraquat that generates high levels of superoxide anion causing oxidative stress and cell mortality. The results showed significant association between dsDNA levels and several sperm fertility parameters, such as low viability and alterations of motility and morphology. The in vitro analysis confirmed the association between dsDNA levels and sperm viability. Together, these results suggest that dsDNA levels could be an important biomarker to test sperm fertility.

Maternal effect genes (MEG) play a crucial role in early embryogenesis. In vitro culture conditions may affect MEG expression in porcine oocytes and embryos. We investigated whether in vitro culture medium supplementation with epidermal growth factor (EGF), IL-1β or LIF (leukemia inhibitory factor) affects the mRNA level of ZAR-1 (zygote arrest 1), NPM2 (nucleoplasmin 2) and DPPA3 (developmental associated protein 3) in porcine MII oocytes and embryos. Cumulus–oocyte complexes (COCs) were matured in NCSU-37 medium (control) or in NCSU-37 with EGF 10 ng/ml, IL-1β 10 ng/ml or LIF 50 ng/ml. After maturation for 44–46 h, MII oocytes were preserved for the analysis of MEG mRNA levels (experiment 1). In experiment 2, COCs were fertilized, and the presumptive zygotes were cultured in the same groups. Then, 2-, 4-, 8-cell embryos, morulae and blastocysts were collected for the analysis of MEG mRNA levels. LIF addition to the maturation medium increased MII oocyte numbers (P < 0.05), while EGF and IL-1β did not affect oocyte maturation. Medium supplementation with EGF resulted in lower DPPA3 mRNA levels in MII oocytes and in 2- and 4-cell embryos versus control embryos (P < 0.05). LIF treatment increased DPPA3 mRNA levels in morulae and blastocysts (P < 0.05). Culture with EGF and IL-1β decreased ZAR-1 and NPM2 mRNA levels in 2-cell embryos (P < 0.05). The inclusion of EGF or IL-1β in the porcine in vitro production system influences ZAR-1, NPM2 and DPPA3 mRNA in MII oocytes and embryos but not beyond the 4-cell stage. LIF stimulates oocyte maturation and affects DPPA3 mRNA in porcine morulae and blastocysts in vitro.

Bovine embryos produced in vivo and in vitro differ with respect to molecular profiles, including epigenetic marks and gene expression profiles. This study investigated the CpG methylation status in bovine testis satellite I (BTS) and Bos taurus alpha satellite I (BTαS) DNA sequences, and concomitantly the relative abundance of transcripts, critically involved in DNA methylation (DNMT1 and DNMT3A), growth and development (IGF2R) and pluripotency (POU5F1) in Bos indicus embryos produced in vitro or in vivo. Results revealed that methylation of BTS were higher (P < 0.05) in embryos produced in vitro compared with their in vivo produced counterparts, while the methylation status of BTαS was similar in both groups. There were no significant differences in transcript abundance for DNMT3A, IGF2R and POU5F1 between blastocysts produced in vivo and in vitro. However, a significantly lower amount of DNMT1 transcripts was found in the in vitro cultured embryos (P < 0.05) compared with their in vivo derived counterparts. In conclusion, this study reported only minor changes in the expression of developmentally important genes and satellite DNA methylation related to the in vitro embryo production system.

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.

The Zungaro jahu is an important large catfish of the order Siluriformes that is in danger of extinction due to habitat destruction. Studies on its biology are scarce and the majority relates only to nutrition or parasitology. In order to provide greater information on its morphology and aid husbandry and larviculture studies, the aim of this study was to characterize larval development in Z. jahu from hatching to total yolk absorption. Samples were collected at pre-established times, processed, stained, and analyzed under stereomicroscopy, light microscopy, and scanning electron microscopy. Total yolk absorption was observed by 60 hours post-hatching (hph) at 28.75 ± 0.59°C. The newly hatched larvae showed slightly pigmented body, the outline of the digestive tract, evident eyes, and the first swimming movements. Mouth opening took place at 12 hph and the connection between the oral cavity and the rudimentary intestine was observed at 24 hph. Were analyzed the main larval organs and systems: digestive organs, heart, gill arches, sensory system, thyroid, kidney, and swim bladder. As the larvae grew, these organs became more mature and functional. The development of the sensory and feeding structures was observed at the start of larval development, and thus before depletion of endogenous energy reserves, the strategy for this species is to increase its chances of survival in the environment.

This study attempted to investigate and validate whether epididymis cold storage could be a suitable alternative for short-term preservation of spermatozoa. Mouse cauda epididymides and spermatozoa were preserved at 4–8°C from 1 day to 6 weeks. From days 1 to 10, motility and fertility were daily examined when motility loss occurred. From week 1, spermatozoa were used for intracytoplasmic sperm injection (ICSI) at weekly intervals to test their fertility, and spermatozoa DNA integrity was determined by comet assay. We found that motility and progressive motility scores gradually decreased with storage time. In nearly all spermatozoa, DNA integrity was maintained from days 1 to 10, but the percentage of spermatozoa with damaged DNA significantly increased from week 2 to week 6. Spermatozoa retained fertility until day 6, although fertility gradually decreased after day 3. From week 1 to week 5, fertilization rates by ICSI were more than 82.69% but decreased gradually after week 3. We found that spermatozoa preserved in the epididymis at 4–8°C had progressively lower motility, fertility and proportion of undamaged DNA, but could still fertilize oocytes. However, all the parameters of cold-preserved spermatozoa were completely inferior to that from cold-preserved cauda epididymides. The results imply that cold storage of cauda epididymides could be conducive to short-term preservation of spermatozoa, and the cold-stored spermatozoa can resist DNA denaturation, which is necessary for maintaining reproductive ability.

High levels of reactive oxygen species in the semen of infertile patients or spontaneously generated during in vitro sperm handling may impair sperm quality, fertilization and embryo developmental competence. We recently reported that zinc, d-aspartate and co-enzyme Q10, contained in the dietary supplement Genadis® (Merck Serono), have protective effects on human and bull sperm motility, lipid peroxidation and DNA fragmentation in vitro; furthermore, in bovine, treated spermatozoa had an improved ability to support embryo development. However, only a few studies have investigated the protective role of antioxidants during in vitro sperm handling in the presence of an exogenous oxidative stress. Herein, to simulate such conditions in an animal model, we induced exogenous oxidative stress on spermatozoa through the xanthine–xanthine oxidase system and investigated its effects on sperm function and subsequent embryo developmental competence in the presence of zinc, d-Asp and CoQ10 protection. The main results showed that exogenous oxidative stress decreased sperm motility, increased sperm DNA fragmentation, and reduced fertilization and blastocyst rates and quality. Pre-treatment with zinc, d-aspartate and co-enzyme Q10 before exogenous oxidative stress was able to prevent these effects. Supplementation of sperm culture media with zinc, d-aspartate and co-enzyme Q10 could protect sperm from oxidative stress damage during in vitro handling in assisted reproductive technologies.

The aim of this study was to evaluate the performance of cavy (Galea spixii) epididymal sperm following addition to TES or TRIS extenders and using a thermal resistance test (TRT), as well as fluorescence analysis as a complementary method to predict the viability of these gametes. Nine testicle–epididymis complexes were used for sperm collection using a flotation method. Epididymis tails were sliced and one was immersed in 3 ml of TRIS buffer, and the other in 3 ml of TES, for 5 min. After sperm recovery, the samples were subjected to a TRT which involved incubation in a water bath at 37°C for 3 h. During incubation, sample parameters were assessed at 0, 15, 30, 60, 90, 120, 150 or 180 min intervals. Results indicated that the TRIS diluent was more efficient than TES (P < 0.05) for the maintenance of sperm parameters in Spix's yellow-toothed cavies over the whole TRT, maintaining sperm longevity for an extended time. In conclusion, we indicate the use of TRIS diluent for recovery and maintenance of longevity of epididymal sperm from cavies (G. spixii).

The purpose of this study was to investigate the possible molecular pathways through which ghrelin accelerates in vitro oocyte maturation. Bovine cumulus–oocyte complexes (COCs), after 18 or 24 h maturation in the absence or the presence of 800 pg ml–1 of acylated ghrelin were either assessed for nuclear maturation or underwent in vitro fertilization in standard media and putative zygotes were cultured in vitro for 8 days. In a subset of COCs the levels of phosphorylated Akt1 and ERK1/2 (MAPK1/3) were assessed at the 0th, 6th, 10th, 18th and 24th hours of in vitro maturation (IVM). At 18 and 24 h no difference existed in the proportion of matured oocytes in the ghrelin-treated group, while in the control group more (P < 0.05) matured oocyte were found at 24 h. Oocyte maturation for 24 h in the presence of ghrelin resulted in substantially reduced (P < 0.05) blastocyst yield(16.3%) in comparison with that obtained after 18 h (30.0%) or to both control groups (29.3% and 26.9%, for 18 and 24 h in maturation, respectively). Ghrelin-treated oocytes expressed lower Akt1 phosphorylation rate at the 10th hour of IVM, and higher ERK1/2 at the 6th and 10th hours of IVM compared with controls. In cumulus cells, at the 18th and 24th hours of IVM Akt1 phosphorylation rate was higher in ghrelin-treated oocytes. Our results imply that ghrelin acts in a different time-dependent manner on bovine oocytes and cumulus cells modulating Akt1 and ERK1/2 phosphorylation, which brings about acceleration of the oocyte maturation process.

The aim was to investigate the relationship between the morphological parameters of metaphase II (MII) oocytes with morphokinetic variables of embryos following an intra-cytoplasmic sperm injection (ICSI) procedure. Morphokinetic behaviour and abnormal cleavage patterns of 334 zygotes were analyzed using time-lapse monitoring (TLM). In addition, oocyte morphology was assessed in relation to embryo morphokinetic (absolute time point, including time to second polar body (PB) extrusion (ESPB), pronuclei (PN) appearance (PNA), PN fading (PNF), time to 2-cells (t2), 3c (t3), 4c (t4), 5c (t5), 6c (t6), 7c (t7), 8c (t8) and relative timing parameters (S1, S2, CC2 and CC3). Also, cleavage patterns (uneven blastomeres, reverse, direct and arbitrary) were assessed. The data showed that 79% of the normal fertilized oocytes had at least one abnormal morphological characteristic. Intra-cytoplasmic abnormalities were observed in 12% of the oocytes. Also, extra-cytoplasmic abnormalities were noticed in 29%, while combined intra- and extra-cytoplasmic abnormalities were responsible for the remaining 38% of the oocytes. Nearly all cleavage and interval times, except extrusion of the ESPB time (P = 0.003), were similar between normal and abnormal morphologic oocytes (P < 0.05). Moreover, there was significant relationship for oocyte morphology abnormalities and cleavage patterns, including uneven blastomere (P = 0.037), reverse cleavage (RC) (P = 0.0), direct (P = 0.001) and arbitrary cleavages (P = 0.001). Using TLM, the cleavage patterns of embryos were affected by the quality of MII oocytes in the ICSI cycles. So, evaluation of oocyte morphology with subsequent embryo morphokinetics is recommended in assisted reproductive programmes.

Pou5f1/Oct4 is a key transcription factor for the induction of pluripotency and totipotency in preimplantation mouse embryos. In mice, loss or gain of function experiments have demonstrated an important role for Oct4 in preimplantation and developmental ability. In this study, using mouse preimplantation embryos as a model for the evaluation of Oct4 function, we constructed Oct4 overexpression embryos with various mutations at the N-terminal transactivation domain. Developmental competency and molecular biological phenotypes depended on the type of mutation. The replacement of serine 106 with alanine resulted in more severe phenotypes similar to that of wild type Oct4, indicating that this alteration using alanine is negligible for Oct4 function. In contrast, we found that Oct4-specific antibodies could not recognize Oct4 protein when this residue was replaced by aspartic acid (Oct4-S106D). Oct4-S106D overexpressing embryos did not show developmental arrest and aberrant chromatin structure. Thus, these results demonstrated that the Ser-106 residue within the N-terminal transactivation domain is crucial for Oct4 function and suggested that this mutation might affect Oct4 protein conformation.

Sea urchin represents an ideal model for studies on fertilization and early development, but the achievement of egg competence and mitochondrial behaviour during oogenesis remain to be enlightened. Oocytes of echinoid, such as sea urchin, unlike other echinoderms and other systems, complete meiotic maturation before fertilization. Mitochondria, the powerhouse of eukaryotic cells, contain a multi-copy of the maternally inherited genome, and are involved directly at several levels in the reproductive processes, as their functional status influences the quality of oocytes and contributes to fertilization and embryogenesis. In the present paper, we report our latest data on mitochondrial distribution, content and activity during Paracentrotus lividus oogenesis. The analyses were carried out using confocal microscopy, in vivo incubating oocytes at different maturation stages with specific probes for mitochondria and mtDNA, and by immunodetection of Hsp56, a well known mitochondrial marker. Results show a parallel rise of mitochondrial mass and activity, and, especially in the larger oocytes, close to germinal vesicle (GV) breakdown, a considerable increase in organelle activity around the GV, undoubtedly for an energetic aim. In the mature eggs, mitochondrial activity decreases, in agreement with their basal metabolism. Further and significant information was achieved by studying the mitochondrial chaperonin Hsp56 and mtDNA. Results show a high increase of both Hsp56 and mtDNA. Taken together these results demonstrate that during oogenesis a parallel rise of different mitochondrial parameters, such as mass, activity, Hsp56 and mtDNA occurs, highlighting important tools in the establishment of developmental competence.

The aim of this work was to examine the influence of cryostorage duration of Pinzgau bull's insemination doses (IDs) on some sperm traits. The IDs were frozen by a slow freezing method and stored in liquid nitrogen for different periods: less than 8 years (group 1), 8–13 years (group 2) and 14–18 years (group 3). Motility (CASA), pathological sperm rate (Giemsa staining), apoptotic (Yo-Pro-1-positive) and necrotic (propidium iodide-positive) cell occurrence and fertilizing ability (penetration/fertilization test) of spermatozoa were evaluated post-thaw. The average post-thaw sperm motility in all examined groups was over 40%. No significant influence of storage length either on the sperm total motility or progressive movement was revealed. In each tested group the average rate of malformed spermatozoa did not exceed 20%. No effect of cryostorage length on the occurrence of apoptotic or necrotic sperm was noted. Similarly, penetrating/fertilizing ability of sperm did not differ among the groups, excepting differences in the rate of pronuclei (PN) formation. In group 1, 72.9% of eggs showed two visible PN following 20 h incubation with sperm, whilst in groups 2 and 3 only 67 and 54.5% of zygotes, respectively, had both PN at this time. These results revealed no influence of storage time on the bull spermatozoa in all parameters excepting the rate of PN formation. As high inter-male variability was observed in the susceptibility of bull sperm to cryostorage, individual differences should be taken into account when semen from individual bulls is to be stored for a long time.

This study aimed to evaluate the embryo development competence, the nuclear maturation and the viability of germinal vesicle (GV) and metaphase II (MII) oocytes vitrified by the Cryotop method. Cumulus–oocyte complexes were derived from bovine ovaries and three experiments were conducted. In Experiment 1, GV oocytes were vitrified and underwent in vitro maturation (IVM) or not and their nuclear maturation was assessed by orcein staining. In Experiment 2, GV oocytes and MII oocytes were vitrified or not and the viability was assessed by calcein/ethidium homodimer-1 staining. In Experiment 3, MII oocytes matured before or after vitrification were submitted to in vitro fertilization (IVF) and parthenogenetic activation (PA) in order to evaluate embryo development. No difference was found for the nuclear maturation rate in the GV group (50%) and the GV control group (67%; P = 0.23) and for viability rate (56%; 77%; P = 0.055, respectively). However, in the MII group (27%) viability was significantly lower than that of the MII control group (84%; P < 0.0001). The cleavage rate by IVF and PA was similar in the GV group and the MII group. In contrast, vitrified MII oocytes showed no capacity for blastocyst development after IVF or PA and vitrified GV oocytes were able to develop to blastocysts only after PA, but not after IVF. In conclusion, oocyte vitrification by the Cryotop method reduced the capacity for embryo development. Vitrification of GV oocytes, however, did not influence the capacity of meiotic nuclear maturation and they exhibited higher viability following vitrification at the MII stage.

We aimed to evaluate the effect of supplementation of ACP-118® extender with the antioxidant catalase (10 and 50 µg/ml) on Sapajus apella sperm motility, vigour, and plasma membrane integrity during the processes of seminal liquefaction, cooling, and freezing. Catalase did not affect any of the evaluated parameters after semen dilution or cooling. Cryopreserved sperm in the presence of 50 µg/ml catalase presented a plasma membrane integrity similar to that fresh sperm, however.