The aim of this study was to establish a functional freezing–thawing protocol for epididymal sperm of collared peccaries (Pecari tajacu L., 1758) by comparing different extenders. The epididymal sperm from 12 sexually mature males was recovered by retrograde flushing using Tris-based or coconut water-based (ACP®-116c) extenders. After initial evaluation, samples were diluted and frozen with the same extenders to which 20% egg yolk and 6% glycerol were added. After 2 weeks, thawing was performed at 37°C/60 s and sperm motility, vigour, morphology, functional membrane integrity, sperm viability, sperm plasma membrane integrity, and a computer-assisted semen analysis (CASA) were assessed. In addition, to evaluate the survival of frozen–thawed sperm, a thermal resistance test (TRT) was executed. Samples preserved using Tris were in better condition compared with those preserved using ACP®, showing higher values for most assessments performed, including CASA and the TRT (P<0.05). After determining Tris to be the better of the two extenders, additional samples were thawed using different thawing rates (37°C/60 s, 55°C/7 s, 70°C/8 s). Sperm thawed at 37°C/60 s had the greatest preservation (P<0.05) of viability (54.1 ± 5.9%) and functional membrane integrity (43.2 ± 5.4%), and had higher values for various CASA parameters. In conclusion, we suggest the use of a Tris-based extender added to egg yolk and glycerol for the cryopreservation of epididymal sperm obtained from collared peccaries. In order to achieve better post-thawing sperm quality, we suggest that samples should be thawed at 37°C/60 s.

This study aimed to understand how germ cell development occurs in females of Devario aequipinnatus, by morphologically describing oogenesis and the reproductive phases. Sexually mature females of D. aequipinnatus (n = 70) were obtained from commercial fisheries and delivered to the Laboratório de Ictiologia Neotropical, UNESP, Ilha Solteira, SP, Brazil. The ovaries were removed, fragmented and fixed following the usual techniques for light microscopy. The stages of ovarian development in D. aequipinnatus begin with the oogonia, which proliferate into new cells or differentiate into prophasic oocytes that, at the end of this process, form the ovarian follicle and end folliculogenesis. In the previtellogenic stage, the oocytes were characterized mainly by the gradual loss of basophilia and an increase in oocyte diameter. Vitellogenesis was marked mainly by the incorporation of yolk granules. Mature oocytes were defined by their migration from the nucleus to the micropyle. Postovulatory follicles and atresic oocytes were also observed. The reproductive phases were classified as: immature, early and final developing, spawning capable, regressing and regenerating. Therefore, the development of an understanding of cell modifications that occurs up to oogenesis is a basic step that is essential for the description of the reproductive biology of D. aequipinnatus, given the lack of information about the reproductive aspects of this species.

The present study examines the role of RNA polymerase I (RPI)-mediated transcription, maternally inherited rRNA and nucleolar proteins in the resumption of fibrillogranular nucleoli during embryonic genome activation (EGA) in porcine embryos. Late 4-cell embryos were incubated in the absence (control) or presence of actinomycin D (AD) (0.2 μg/ml for inhibition of RPI; 2.0 μg/ml for inhibition of total transcription) and late 2-cell embryos were cultured to the late 4-cell stage with 0.2 μg/ml AD to block EGA. Embryos were then processed for reverse-transcriptase polymerase chain reaction (RT-PCR), and for autoradiography (ARG), transmission electron microscopy (TEM), fluorescence in situ hybridization (FISH), silver staining and immunofluorescence (for RPI). Embryos in the control group displayed extranucleolar and intranucleolar ARG labelling, and exhibited de novo synthesis of rRNA and reticulated functional nucleoli. Nucleolar proteins were located in large foci. After RPI inhibition, nucleolar precursors transformed into segregated fibrillogranular structures, however no fibrillar centres were observed. The localization of rDNA and clusters of rRNA were detected in 57.1% immunoprecipitated (IP) analyzed nucleoli and dispersed RPI; 30.5% of nuclei showed large deposits of nucleolar proteins. Embryos from the AD-2.0 group did not display any transcriptional activity. Nucleolar formation was completely blocked, however 39.4% of nuclei showed rRNA clusters; 85.7% of nuclei were co-localized with nucleolar proteins. Long-term transcriptional inhibition resulted in the lack of ARG and RPI labelling; 40% of analyzed nuclei displayed the accumulation of rRNA molecules into large foci. In conclusion, maternally inherited rRNA co-localized with rDNA and nucleolar proteins can initiate a partial nucleolar assembly, resulting in the formation of fibrilogranular structures independently on activation of RPI-mediated transcription.

This study investigated and analysed survival, growth and macro- and microscopic damage during the development of zebrafish embryos up to the adult stage after undergoing cooling. The embryos at 50% epiboly stage were selected, submerged in cryoprotectant solution of methanol and sucrose, cooled gradually to 0 ± 2°C temperature, and divided into two groups with different storage times (6 and 18 h). Subsequently, the embryos were reheated, rehydrated and incubated normally. The experiment lasted 5 months and, from hatching onward, the larvae were examined, collected and processed at pre-established time intervals. The hatching rate was significantly higher for the larvae stored for 18 h compared with the 6-h group. However, embryos from this group gave rise to a larger number of malformations, and these were much more severe compared with those in the 6 h group, which led to a higher mortality in the long term. Regarding larval length, the animals of the 6 h group had higher mean total length compared with the 18 h group, but both treatments were inferior to the control. Numerous macro- and microscopic malformations were observed and, in both treatments, only the morphologically normal individuals were able to develop to the adult stage, with organ development similar to the control, except for the gonads that were still undifferentiated in treated animals.

To investigate the effects of physiologically relevant heat shock during oocyte maturation, buffalo cumulus–oocyte complexes (COCs) were cultured at 38.5°C (control) or were exposed to 39.5°C (T1) or 40.5°C (T2) for the first 6 h of in vitro maturation (IVM), followed by 38.5°C through the next 18 h/IVM and early embryonic development up to the blastocyst stage. Gene expression analysis was performed on selected target genes (HSF-1, HSF-2, HSP-70, HSP-90, BAX, p53, SOD1, COX1, MAPK14) in denuded oocytes and their isolated cumulus cells resulting from control COCs as well as from COCs exposed to a temperature of 39.5°C (T1). The results indicated that heat shock significantly (P < 0.01) decreased the maturation rate in T1 and T2 cells compared with the control. After in vitro fertilization (IVF), cleavage rate was lower (P < 0.01) for oocytes exposed to heat stress, and the percentage of oocytes arrested at the 2- or 4-cell stage was higher (P < 0.01) than that of the control. The percentage of oocytes that developed to the 8-cell, 16-cell or blastocyst stage was lower (P < 0.01) in both T1 and T2 groups compared with the control group. mRNA expression levels for the studied genes were decreased (P < 0.05) in treated oocytes (T1) except for HSP-90 and HSF-1, which were increased. In cumulus cells isolated from COCs (T1), the expression for the target genes was upregulated except for BAX, which was downregulated. The results of this study demonstrated that exposure of buffalo oocytes to elevated temperatures for 6 h severely compromised their developmental competence and gene expression.

The negative effects of cryopreservation on sperm parameters are well documented but little information is known about molecular basis of the process. The aim of the present study was to investigate the possible effects of sperm cryopreservation on main apoptotic signs including DNA fragmentation and caspase-3 activation and to determine if these effects vary according to sperm parameters. Sperm samples of 72 patients were cryopreserved. The patients were sub-grouped as normozoospermic or non-normozoospermic patients according to their semen parameters. DNA fragmentation rates and caspase-3 activation levels were analyzed before and after cryopreservation in both groups. Mean DNA fragmentation rate was increased significantly from 23.98% in neat semen samples to 27.34% after cryopreservation (P = 0.03). DNA fragmentation rates were slightly higher in non-normozoospermic patients compared with the normozoospermic patients in both the neat semen and after cryopreservation (23.25 and 24.71% vs. 26.32 and 28.36%, respectively) although the difference obtained were not statistically significant. An increasing trend for caspase-3 activations (0.093 vs. 0.116) was observed after cryopreservation but the differences were not statistically significant. Caspase-3 activation was found to be slightly higher in non-normozoospermic patients both in the neat semen and after cryopreservation compared with the normozoospermic patients but the differences were not statistically significant. Caspase-3 expression was also shown using immunocytochemistry in both fresh ejaculated sperm and thawed sperm after cryopreservation but at different localizations. The cryopreservation process had detrimental effects on sperm quality but the quality of the sperm samples was not adversely effective for the apoptotic markers including DNA fragmentation and caspase-3 activation patterns. In fact, it was the cryopreservation process itself that adversely effected the above apoptotic markers and apoptosis. It was concluded therefore that sperm cell cryopreservation triggers apoptosis after thawing and this process adversely affects semen parameters.

This study aimed to compare the efficiency of different incubation systems for in vitro embryo production in bovine. Oocytes/embryos were cultured in three incubators: conventional – CONV, mini bench – MINI and portable – PORT. After in vitro maturation (IVM), oocytes were verified for maturation rate. The remaining structures were submitted to in vitro fertilization and culture to verify cleavage (day 2) and blastocyst (day 7) rates. Reactive oxygen species (ROS) were evaluated in post-IVM oocytes and embryos (days 2 and 7) using arbitrary fluorescence units (AFUs). No significant difference (P>0.05) was observed for maturation rate. The CONV system (74.0%) produced the highest cleavage rate (P<0.05) when compared with PORT (59.5%), but similar (P>0.05) to MINI (65.0%). The same pattern and differences were observed for blastocyst rate: CONV (33.3%), MINI (32.3%) and PORT (21.9%). ROS levels were not different (P>0.05) in post-IVM oocytes: CONV (35.6±4.5), MINI (29.4±4.0) and PORT (35.6±4.5). For day-2 embryos, ROS levels were higher (P<0.05) in MINI (44.2±3.1) in comparison with CONV (27.7±3.7) and PORT (33.3±3.2). No significant difference (P>0.05) was observed in blastocysts. In conclusion, although it produced high ROS levels at day 2 of culture, the MINI system was as efficient as the CONV system for blastocyst production. This option may be an interesting and economical for the in vitro embryo industry.

One of the methods to generate transgenic animals is called sperm-mediated gene transfer (SMGT). Mature sperm cells can take up exogenous DNA molecules intrinsically and transfer them into the oocyte during fertilization. This study assessed the effect of dimethyl sulfoxide (DMSO) and electrolyte-free medium (EFM) on DNA uptake (EGFP–N1plasmid) in mouse sperm. Sperms cells cultured in human tubular fluid (HTF) without any treatment were considered as the control group. Sperms cells that were incubated in EFM and HTF with DNA/DMSO at 4°C were classified into EFM and HTF groups. Sperm motility and viability were assessed following treatment. In vitro fertilization (IVF) with sperm in all groups was performed. Fertilization, embryo development and GFP-positive blastocyst rates were analyzed and compared. The result showed that sperm motility and viability in EFM were better than those in the HTF group. The rate of development to reach the blastocyst stage and GFP-positive blastocysts was significantly higher in the EFM group compared with the HTF group (P<0.05). Our data demonstrate that sperm stored in the EFM group can improve the efficiency of SMGT for the generation of GFP-positive blastocysts.

In fish, many factors can affect reproduction during in vitro fertilization, therefore determination of the factors that affect affecting gamete quality is needed. However, few studies have focused on gamete quality and the ploidy status. This study was conducted to elucidate whether oocyte storage can affect ploidy status, survival, and embryo viability in the characid species Astyanax altiparanae. Oocytes were stored in Dulbecco's phosphate-buffered saline (PBS) at 26°C, then aliquots were fertilized immediately after extrusion (control) and also after 60, 120, 180, and 240 min of storage. Fertilization and hatching rates were measured, and the developmental stages were analyzed at each stage before describing the main abnormalities. Ploidy status was analyzed by flow cytometry and blood smear. In the control group, 100% of the samples were diploid. After treatment for 60 min, 95.56 ± 4.44% samples were diploid and 4.44 ± 4.44% were triploid. After 120 min, 94.44 ± 9.62% of the samples was diploid and 5.56 ± 5.56% were triploid; 100% of the samples were diploid after 180 min and, after 240 min, there was no survival. In other treatments, the highest percentage of hatching was after 60 min (88.93 ± 5.15%; P = 0.015), and treatment with 180 min storage resulted in the highest percentage of abnormal larvae (95.76 ± 12.67%; P = 0.012). These results show that oocyte storage can affect ploidy status and may be an interesting parameter for analysis in studies on chromosome set manipulation and micromanipulation.

Digyny, the presence of a third pronucleus due to the failure of second polar body extrusion, is problematic after intracytoplasmic sperm injection (ICSI) practices. Mitochondria have critical roles such as production of adenosine triphosphate (ATP) and regulation of Ca2+ homeostasis during oocyte maturation, fertilization and the following development, while the regulation of meiotic spindle formation, chromosome segregation, pronuclear apposition and cytokinesis is closely associated with the cytoskeleton. In this study, mitochondrial membrane potential, distribution of F-actin and γ-tubulin, and the ultrastructure of three pronuclear (3PN) oocytes were investigated. 3PN oocytes after ICSI procedure were taken from patients who were enrolled in assisted reproduction programmes. For mitochondrial membrane potential analysis, fresh oocytes stained with the mitochondrial membrane potential probe JC-1, were evaluated under fluorescence microscopy. The mitochondrial membrane potential of three pronuclear oocytes showed similar results to normal zygotes. γ-Tubulin was stained strongly at the subplasmalemmal domain and microfilaments were localized at the cortical, but not the perinuclear, area. Cytoplasmic halos were moderately or not detected by electron microscopy; lipofuscin granules, degenerated mitochondria, and multilamellated bodies were seen in the ooplasm. Immunohistochemistry and electron microscopic findings suggested that mitochondrial membrane potential has no direct effect on second polar body extrusion. This abnormality can be associated with an altered cytoskeleton due to poor oocyte quality.

The objective of this study was to assess the effects of bovine embryo vitrification by applying three different vitrification solutions containing ethylene glycol (EG) and dimethylsulphoxide (DMSO) at different concentrations (10, 20 or 25% each) combined with 1.0 M glucose or 1.0 M sucrose, on the in vitro hatching and expansion rates. Healthy oocytes were selected for in vitro maturation and fertilization from 200 bovine ovaries, and subsequently cultured up to the blastocyst stage (n = 800). Control (n = 200) and vitrified cells (n = 100 per treatment; 600 in total) were cultured for an extra 24 or 48 h to evaluate hatching and expansion, respectively. Vitrification significantly decreased embryonic re-expansion and hatching rates independently of the tested solution when compared with control embryos, but solutions with 25% EG + 25% DMSO resulted in the highest re-expansion (75%) and hatching (70%) rates, independently of the added sugar. The addition of sucrose resulted in higher rates of re-expanded and hatched embryos when compared with glucose addition. We concluded that the combination of 25% EG + 25% DMSO and 1.0 M sucrose allowed hatching and expansion of vitrified-warmed bovine embryos produced in vitro.

Polyspermy was initiated by microinjecting a multiple number of sperm into the activated and dechorionated eggs of dojo loach Misgurnus anguillicaudatus (Teleostei: Cobitidae). A 10-nl sperm suspension from an albino (recessive trait) male (105, 106, 107 or 108 sperm ml −1) was microinjected into eggs from a wild-type female. Although the rates of embryos developing into the blastula stage in the injection group at the highest sperm concentration were similar to that of the control group, the hatching rates of the injection group were much lower. A large proportion of embryos that developed from the injected eggs was haploid and were mosaics containing haploid cells. Most of the haploid and mosaic embryos inherited only paternally derived alleles in the microsatellite markers (i.e. androgenesis was initiated by injecting multiple sperm). In contrast, some haploid embryos contained both paternal and maternal alleles despite haploidy, suggesting that they were mosaics consisting of cells with either paternal or maternal inheritance. The injected eggs displayed diploid, hypotriploid and triploid cells, all of which included both maternally and paternally derived alleles. One albino tetraploid with only paternal alleles was also observed from the injected eggs. These results suggested that part of the sperm microinjected into the ooplasm should form a male pronucleus/pronuclei, which could develop by androgenesis or could fuse with the female pronucleus/pronuclei. Therefore, microinjection of multiple sperm should be considered a potential technique to induce androgenesis and polyploidy.

This study investigated the in vitro culture of bovine follicles included in ovarian tissue for 2 or 6 days (D2 or D6), with the addition of different concentrations of follicle-stimulating hormone (FSH) (0, 10, 50, 100 or 200 ng/ml). Data were compared for follicular development, morphological integrity and diameter of follicles and oocytes. Ovaries (n = 10) from Nelore cows (n = 5) were divided into fragments (n = 11 per ovary) and were immediately fixed in Bouin’s solution (D0) or were individually cultured for 2 or 6 days in one of the described concentrations of FSH and then processed for histology. Compared with the rates of follicular development at D2 for minimal essential medium (MEM) (75.0%) and 50 ng/ml of FSH (71.1%), the best rates of follicular development at D2 were obtained with 10 (84.7%), 100 (87.5%) and 200 ng/ml of FSH (85.0%; P<0.05). After 6 days of cultivation, there were no differences among treatments regarding follicular growth. The morphological integrity of preantral follicles was better maintained by 100 ng/ml FSH for 2 and 6 days of cultivation (51.2 and 40.4%, respectively; P<0.05) than that for MEM (D2: 30.9%, D6: 20.8%), 10 (D2: 39.2%, D6: 22.8%), 50 (D2: 30.4%, D6: 28.8%) and 200 ng/ml FSH (D2: 45.2%, D6: 36.8%). FSH at 100 ng/ml provided the highest mean diameter averages: 34.5±10.8 µm at D2 and 33.2±12.5 µm at D6 (P<0.05). We concluded that the medium supplemented with 100 ng/ml FSH during in vitro culture provided appropriate conditions for the development and morphological integrity of preantral follicles in cattle.

Oxidative stress is a major cause of defective embryo development during in vitro culture. Retinoids are recognized as non-enzymatic antioxidants and may have an important role in the regulation of cell differentiation and vertebrate development. However, there are not enough reports discussing the antioxidant and developmental capacity of retinoids, including retinol (RT), on the in vitro development of embryos recovered from livestock animals, particularly in rabbit species. Therefore, morula embryos obtained from nulliparous Red Baladi rabbit does were cultured for 48 h in TCM199 medium in the absence of RT (control group) or in the presence of RT at concentrations of 10, 100 and 1000 nM. The developmental capacity to the hatched blastocyst stage, the antioxidant biomarker assay and the expression of several selected genes were analyzed in each RT group. The data show that RT significantly (P<0.001) promoted the embryo hatchability rate at the concentration of 1000 nM to 69.44% versus 29.71% for the control. The activity of malondialdehyde (MDA) level was significantly (P<0.05) lower in the RT groups than in the control group, while the total antioxidant capacity (TAC), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities were significantly (P<0.05) higher following treatment with RT. Furthermore, RT treatment considerably upregulated the relative expression of gap junction protein alpha 1 (GJA1), POU class 5 homeobox 1 (POU5F1) and superoxide dismutase 1 (SOD1) genes compared with the control group. The current study highlights the potential effects of RT as antioxidant in the culture medium on the in vitro development of rabbit embryos.

Sperm-mediated gene transfer (SMGT) is a simple, fast, and economical biotechnological tool for producing transgenic animals. However, transgene expression with this technique in bovine embryos is still inefficient due to low uptake and binding of exogenous DNA in spermatozoa. The present study evaluated the effects of sperm membrane destabilization on the binding capacity, location and quantity of bound exogenous DNA in cryopreserved bovine spermatozoa using Triton X-100 (TX-100), lysolecithin (LL) and sodium hydroxide (NaOH). Effects of these treatments were also evaluated by intracytoplasmic sperm injection (ICSI)-SMGT. Results showed that all treatments bound exogenous DNA to spermatozoa including the control. Spermatozoa treated with different membrane destabilizing agents bound the exogenous DNA throughout the head and tail of spermatozoa, compared with the control, in which binding occurred mainly in the post-acrosomal region and tail. The amount of exogenous DNA bound to spermatozoa was much higher for the different sperm treatments than the control (P < 0.05), most likely due to the damage induced by these treatments to the plasma and acrosomal membranes. Exogenous gene expression in embryos was also improved by these treatments. These results demonstrated that sperm membrane destabilization could be a novel strategy in bovine SMGT protocols for the generation of transgenic embryos by ICSI.

The aim of this study was to evaluate the effect of incubating semen for different periods (90, 270 or 450 min) with or without Trolox® (100 or 150 µM) on the quality of sperm from Saimiri collinsi. Sperm motility, vigour, and plasma membrane integrity (PMI) were evaluated in both fresh semen and semen incubated for different time periods, i.e. 90, 270 or 450 min of incubation. Supplementation of semen extender with Trolox® 100 µM improved sperm motility, vigour and PMI for up to 270 min of incubation.

Ovarian biopsies from five health adult monkeys were collected by exploratory laparotomy. Preantral follicles (primordial, primary, and secondary) were classified as normal or degenerated and submitted to morphometric analysis in which granulosa cell counts and the areas of follicles, oocytes, and oocyte nuclei were measured. Ovarian fragments were also immunolabelled for the quantitative analysis of VEGFA and CD31 protein expression in the ovarian tissue and in the preantral follicles. In total, 213 preantral follicles was examined for morphometry and morphological classification. From this total, 20 (9.4%) were follicles enclosing two or more oocytes, i.e. multi-oocyte follicles (MOFs). From the 193 follicles enclosing only one oocyte, 46.3% were classified as primordial, 24,1% as transition, 23.3% as primary, and 6.3% as secondary follicles. The mean number of granulosa cells surrounding primordial, transition, primary, and secondary follicles was 9.2, 12.1, 18.7, and 45.3, respectively. Increase in oocyte diameter was observed from primary to secondary follicles, while the oocyte nucleus increased only when follicles reached the secondary stage. The expression of CD31 was strong in vessels, corpus luteum, and in normal oocytes and granulosa cells from preantral follicles at all developmental stages. Likewise, VEGFA expression was observed in vessels and preantral follicles (granulosa cells, the oocyte and the oocyte nucleus). We characterized the morphology, and morphometry and expression of angiogenic factors in normal and atretic preantral follicles from Sapajus apella. This description can support the analysis of follicular quality and survival after procedures such as transplantation and cryopreservation.

Currently, rescue in vitro maturation (IVM) is not a routine method in assisted reproductive treatment (ART) programmes but is a promising procedure for ART to improve IVM. The aim of this study was to compare embryo morphokinetics of germinal vesicles (GV) with metaphase II (MII) oocytes from controlled ovarian hyperstimulation (COH) cycles by time-lapse photography monitoring (TLM). Morphokinetics of the same number of embryos derived from the in vivo (group I) and rescue of in vitro matured oocytes (group II) from 310 patients were analyzed and compared retrospectively. The time to form second PB extrusion (tPB2), time of pronuclei appearance (tPNa), time of pronuclei fading (tPNf) and time of two to eight discrete cells (t2–t8) were assessed. Abnormal cleavage patterns such as uneven blastomeres at the two-cell stage, cell fusion (Fu), trichotomous mitoses (TM), and the rates of embryo arrest were assessed. These data showed that tPB2, tPNa, tPNf, t2, t3 and t4 stages took place later in group II compared with group I (P<0.001, P=0.017, P<0.001, P<0.001, P<0.001, P<0.001, respectively). The rates of uneven blastomeres, Fu, TM, and embryo arrest were increased significantly in group II compared with group I (P=0.001, P<0.001, P=0.003, P<0.001, respectively). Based on the exact annotation of timing parameters and cleavage patterns, the present data agreed with the concept that rescue IVM of oocytes negatively influences embryo morphokinetics. Therefore, cautious use of embryos derived from rescue IVM of GV oocytes should be made.

Previous studies have established a model of atresia in preovulatory follicles after stimulation of immature rats with equine chorionic gonadotropin (eCG). This gonadotropin recruits a follicular pool and the deprivation of preovulatory luteinizing hormone (LH) surge induces the atresia in preovulatory follicles. The present study investigated the occurrence of ovulation and provided some morphological features of granulosa cell (GC) apoptosis of atretic follicles at 0, 48, 72 and 120 h after eCG stimulation. Histological sections of ovaries from untreated animals (0 h) showed primordial, primary, secondary and early antral follicles. After 48 h ovaries showed large antral follicles. Preovulatory follicles were observed at 72 h, and two out of five rats displayed cumulus–oocyte complexes (COCs) in the oviducts. All animals exhibited corpora lutea after 120 h. We observed increased estradiol (E2) levels 48 h after eCG treatment that might trigger an endogenous preovulatory gonadotropin surge. Higher progesterone (P4) level, which is the hallmark of a functional corpus luteum, was observed at 120 h. Atresia in secondary and antral follicles was observed by pyknotic granulosa cell nuclei in histology and positive immunolabelling for cleaved caspase 3. We also observed macrophages in secondary and antral follicles in atresia. Transmission electron microscopy revealed GCs with compacted chromatin against the nuclear envelope, nuclear fragmentation, cell shrinkage and fragmentation. No preovulatory follicles showed apoptosis of GCs. In conclusion, our results suggested the occurrence of an endogenous gonadotropin surge, promoting ovulation and preventing atresia of preovulatory follicles.