Fetal Growth and Osteogenesis Dynamics during Early Development in the Ovine Species.

Increased knowledge of the developmental processes during gestation could provide valuable information on potential alterations in embryonic/fetal development. We examined the development of ovine conceptus between the 20th and 70th day of gestation with three convergent analyses: (1) uterus ultrasound examination and measurement (eco) of crown-rump length (CRL) and biparietal diameter (BPD) of the conceptus; (2) direct measurement (vivo) of CRL and BPD of the conceptus outside the uterus (3) osteo-cartilage dynamics during development by differential staining. No significant differences were observed between eco and vivo measurements for CRL and BPD in all examined concepti. CRL and BPD, instead, showed a significant positive linear correlation with gestational age. The study of osteogenesis dynamics has demonstrated a completely cartilaginous ovine fetus at up to 35 days of gestation. The ossification begins in the skull (40th day) and is almost complete between the 65th and the 70th of pregnancy. Our study highlighted that CRL and BPD are accurate parameters for gestational age estimation in the first part of sheep pregnancy and provides an overview of osteochondral temporal dynamics. Furthermore, tibia ossification is a valid parameter to estimate fetal age by ultrasound.

New Challenges in Cryopreservation: A Reproductive Perspective.

Cryopreservation is a fundamental procedure to preserve the structure and function of cells and tissues by storing them at low temperatures for long periods [...].

Microtubular Assessment of C6 Rat Glioma Cell Spheroids Developed in Transparent Liquid Marbles or Hanging Drops.

Glioblastoma is a brain tumour frequently used as an experimental model to exploit innovative therapeutic approaches due to its high lethality and refractoriness to therapies. Part of these innovative anticancer therapies address cytoskeletal microtubules (MTs) since specific tubulin post-translational modifications (PTMs) are considered markers of tumour plasticity. In vitro studies, which traditionally employ two-dimensional (2D) culture systems, are now being replaced by three-dimensional (3D) systems that more closely mimic in vivo physiological conditions and allow a better understanding of the signalling between cells. In this work, we compared 2 liquid base 3D methods for the generation of spheroids from C6 rat glioma cells (RGCs) using 30 µL of liquid marble (LM) or the hanging drops (HDs), which contained 2 different cell numbers (5000 or 15,000). After 24 or 48 h of in vitro culture (IVC), the morphology of the spheroids was observed and the behaviour of the two main tubulin PTMs, tyrosinated α-tubulin (Tyr-T) and acetylated α-tubulin (Ac-T), was evaluated by fluorescence and Western blot (WB). RGCs spontaneously formed spherical agglomerates more rapidly in the LM than in the HD system. Cell density influenced the size of the spheroids, which reached a larger size (> of 300 µm Ø), with 15,000 cells compared to 5000 cells (150 µm Ø). Moreover, an increase in Tyr-T and Ac-T was observed in both the HD and LM system from 24 to 48 h, with the highest values shown in the 48 h/LM spheroids of 5000 cells (p < 0.05). In conclusion, by comparing the morphology and microtubular architecture of spheroids from C6 rat glioma cells developed by LM or HD methodology, our findings demonstrate that the use of a fumed silica microbioreactor boosts the induction and maintenance of a high plasticity state in glioma cells. RGCs cultured in LM express levels of tubulin PTMs that can be used to evaluate the efficacy of new anticancer therapies.

Oocyte aging: looking beyond chromosome segregation errors.

The age-associated decline in female fertility is largely ascribable to a decrease in oocyte quality. This phenomenon is multifaceted and influenced by numerous interconnected maternal and environmental factors. An increase in the rate of meiotic errors is the major cause of the decline in oocyte developmental competence. However, abnormalities in the ooplasm accumulating with age - including altered metabolism, organelle dysfunction, and aberrant gene regulation - progressively undermine oocyte quality. Stockpiling of maternal macromolecules during folliculogenesis is crucial, as oocyte competence to achieve maturation, fertilization, and the earliest phases of embryo development occur in absence of transcription. At the same time, crucial remodeling of oocyte epigenetics during oogenesis is potentially exposed to interfering factors, such as assisted reproduction technologies (ARTs) or environmental changes, whose impact may be enhanced by reproductive aging. As the effects of maternal aging on molecular mechanisms governing the function of the human oocyte remain poorly understood, studies in animal models are essential to deepen current understanding, with translational implications for human ARTs. The present mini review aims at offering an updated and consistent view of cytoplasmic alterations occurring in oocytes during aging, focusing particularly on gene and epigenetic regulation. Appreciation of these mechanisms could inspire solutions to mitigate/control the phenomenon, and thus benefit modern ARTs.

Molecular and Histological Evaluation of Sheep Ovarian Tissue Subjected to Lyophilization.

Cryopreservation is routinely used to preserve cells and tissues; however, long time storage brings many inconveniences including the use of liquid nitrogen. Freeze-drying could enable higher shelf-life stability at ambient temperatures and facilitate transport and storage. Currently, the possibility to freeze-dry reproductive tissues maintaining vitality and functions is still under optimization. Here, we lyophilized sheep ovarian tissue with a novel device named Darya and a new vitrification and drying protocol and assessed effects on tissue integrity and gene expression. The evaluation was performed immediately after lyophilization (Lio), after rehydration (LR0h) or after two hours of in vitro culture (IVC; LR2h). The tissue survived lyophilization procedures and maintained its general structure, including intact follicles at different stages of development, however morphological and cytoplasmic modifications were noticed. Lyophilization, rehydration and further IVC increasingly affected RNA integrity and caused progressive morphological alterations. Nevertheless, analysis of a panel of eight genes showed tissue survival and reaction to the different procedures by regulation of specific gene expression. Results show that sheep ovarian tissue can tolerate the applied vitrification and drying protocol and constitute a valid basis for further improvements of the procedures, with the ultimate goal of optimizing tissue viability after rehydration.

3D Liquid Marble Microbioreactors Support In Vitro Maturation of Prepubertal Ovine Oocytes and Affect Expression of Oocyte-Specific Factors.

In vitro oocyte maturation (IVM) is a well-established technique. Despite the high IVM rates obtained in most mammalian species, the developmental competence of IVM oocytes is suboptimal. The aim of this work was to evaluate the potential beneficial effects of a liquid marble microbioreactor (LM) as a 3D culture system to mature in vitro prepubertal ovine oocytes, as models of oocytes with intrinsic low competence. Cumulus-oocyte complexes of prepubertal sheep ovaries were in vitro matured in a LM system with hydrophobic fumed-silica-nanoparticles (LM group) or in standard conditions (4W control group). We evaluated: (a) maturation and (b) developmental rates following in vitro fertilization (IVF) and embryo culture; (c) expression of a panel of genes. LM and 4W groups showed similar IVM and IVF rates, while in vitro development to blastocyst stage approached significance (4W: 14.1% vs. LM: 28.3%; p = 0.066). The expression of GDF9, of enzymes involved in DNA methylation reprogramming and of the subcortical maternal complex was affected by the IVM system, while no difference was observed in terms of cell-stress-response. LM microbioreactors provide a suitable microenvironment to induce prepubertal sheep oocyte IVM and should be considered to enhance the developmental competence of oocytes with reduced potential also in other species, including humans.

Anthropogenic Drivers Leading to Population Decline and Genetic Preservation of the Eurasian Griffon Vulture (Gyps fulvus).

Human activities are having increasingly devastating effects on the health of marine and terrestrial ecosystems. Studying the adaptive responses of animal species to changes in their habitat can be useful in mitigating this impact. Vultures represent one of the most virtuous examples of adaptation to human-induced environmental changes. Once dependent on wild ungulate populations, these birds have adapted to the epochal change resulting from the birth of agriculture and livestock domestication, maintaining their essential role as ecological scavengers. In this review, we retrace the main splitting events characterising the vultures' evolution, with particular emphasis on the Eurasian griffon Gyps fulvus. We summarise the main ecological and behavioural traits of this species, highlighting its vulnerability to elements introduced into the habitat by humans. We collected the genetic information available to date, underlining their importance for improving the management of this species, as an essential tool to support restocking practices and to protect the genetic integrity of G. fulvus. Finally, we examine the difficulties in implementing a coordination system that allows genetic information to be effectively transferred into management programs. Until a linking network is established between scientific research and management practices, the risk of losing important wildlife resources remains high.

Mitochondrial DNA Depletion in Granulosa Cell Derived Nuclear Transfer Tissues.

Somatic cell nuclear transfer (SCNT) is a key technology with broad applications that range from production of cloned farm animals to derivation of patient-matched stem cells or production of humanized animal organs for xenotransplantation. However, effects of aberrant epigenetic reprogramming on gene expression compromise cell and organ phenotype, resulting in low success rate of SCNT. Standard SCNT procedures include enucleation of recipient oocytes before the nuclear donor cell is introduced. Enucleation removes not only the spindle apparatus and chromosomes of the oocyte but also the perinuclear, mitochondria rich, ooplasm. Here, we use a Bos taurus SCNT model with in vitro fertilized (IVF) and in vivo conceived controls to demonstrate a ∼50% reduction in mitochondrial DNA (mtDNA) in the liver and skeletal muscle, but not the brain, of SCNT fetuses at day 80 of gestation. In the muscle, we also observed significantly reduced transcript abundances of mtDNA-encoded subunits of the respiratory chain. Importantly, mtDNA content and mtDNA transcript abundances correlate with hepatomegaly and muscle hypertrophy of SCNT fetuses. Expression of selected nuclear-encoded genes pivotal for mtDNA replication was similar to controls, arguing against an indirect epigenetic nuclear reprogramming effect on mtDNA amount. We conclude that mtDNA depletion is a major signature of perturbations after SCNT. We further propose that mitochondrial perturbation in interaction with incomplete nuclear reprogramming drives abnormal epigenetic features and correlated phenotypes, a concept supported by previously reported effects of mtDNA depletion on the epigenome and the pleiotropic phenotypic effects of mtDNA depletion in humans. This provides a novel perspective on the reprogramming process and opens new avenues to improve SCNT protocols for healthy embryo and tissue development.

The subcortical maternal complex: emerging roles and novel perspectives.

Since its recent discovery, the subcortical maternal complex (SCMC) is emerging as a maternally inherited and crucial biological structure for the initial stages of embryogenesis in mammals. Uniquely expressed in oocytes and preimplantation embryos, where it localizes to the cell subcortex, this multiprotein complex is essential for early embryo development in the mouse and is functionally conserved across mammalian species, including humans. The complex has been linked to key processes leading the transition from oocyte to embryo, including meiotic spindle formation and positioning, regulation of translation, organelle redistribution, and epigenetic reprogramming. Yet, the underlying molecular mechanisms for these diverse functions are just beginning to be understood, hindered by unresolved interplay of SCMC components and variations in early lethal phenotypes. Here we review recent advances confirming involvement of the SCMC in human infertility, revealing an unexpected relationship with offspring health. Moreover, SCMC organization is being further revealed in terms of novel components and interactions with additional cell constituents. Collectively, this evidence prompts new avenues of investigation into possible roles during the process of oogenesis and the regulation of maternal transcript turnover during the oocyte to embryo transition.

High in vitro survival rate of sheep in vitro produced blastocysts vitrified with a new method and device.

BACKGROUND: To advance the use of embryo vitrification in veterinary practice, we developed a system in which embryo vitrification, warming and dilution can be performed within a straw. Ovine in vitro produced embryos (IVEP) were vitrified at either early (EBs: n = 74) or fully expanded blastocyst stage (FEBs: n = 195), using a new device named "E.Vit", composed by a 0.25-mL straw with a 50-µm pore polycarbonate grid at one end. Embryos at each stage (EBs and FEBs) were vitrified by either Two-step (TS) or Multi-step (MS; 6 different concentrations of vitrification solutions) protocol. Non-vitrified embryos (n = 102) were maintained in in vitro culture as a control. Warming consisted of placing the straws directly into 1.5 mL tubes containing a TCM-199 solution with three decreasing concentrations of sucrose. Blastocyst re-expansion, embryo survival and hatching rate were evaluated at 2, 24 and 48 h post warming. The number of apoptotic cells was determined by TUNEL assay. RESULTS: Blastocyst re-expansion (2 h) after warming was higher (P < 0.05) in FEBs group, vitrified with the MS and TS methods (77.90% and 71.25%, respectively) compared with the EBs group (MS: 59.38% and TS: 48.50%, respectively). Survival rates of vitrified FEBs after 24 h IVC were higher (P < 0.001) in both methods (MS and TS) than vitrified EBs (MS: 56.25%; TS: 42.42%) and was higher (P < 0.05) in the MS method (94.19%) compared with those in TS (83.75%). After 48 h of culture the hatching rate for FEBs vitrified in MS system (91.86%) was similar to control (91.89%), but higher than FEB TS (77.5%) and EBs vitrified in MS (37.5%) and TS (33.33%). Number of apoptotic cells were higher in EBs, irrespective of the system used, compared to FEBs. The number of apoptotic cells in FEBs vitrified with MS was comparable to the control. CONCLUSIONS: A high survival rate of IVP embryos can be achieved by the new "E.Vit" device with hatching rates in vitro comparable with control fresh embryos. This method has the potential for use in direct embryo transfer in field conditions.

Gene expression analysis of ovine prepubertal testicular tissue vitrified with a novel cryodevice (E.Vit).

PURPOSE: Testicular tissue cryopreservation prior to gonadotoxic therapies is a method to preserve fertility in children. However, the technique still requires development, especially when the tissue is immature and rather susceptible to stress derived from in vitro manipulation. This study aimed to investigate the effects of vitrification with a new cryodevice (E.Vit) on cell membrane integrity and gene expression of prepubertal testicular tissue in the ovine model. METHODS: Pieces of immature testicular tissue (1 mm3) were inserted into "E.Vit" devices and vitrified with a two-step protocol. After warming, tissues were cultured in vitro and cell membrane integrity was assessed after 0, 2, and 24 h by trypan blue exclusion test. Controls consisted of non-vitrified tissue analyzed after 0, 2, and 24 h in vitro culture (IVC). Expression of genes involved in transcriptional stress response (BAX, SOD1, CIRBP, HSP90AB1), cell proliferation (KIF11), and germ- (ZBDB16, TERT, POU5F1, KIT) and somatic- (AR, FSHR, STAR) cell specific markers was evaluated 2 and 24 h after warming. RESULTS: Post-warming trypan blue staining showed the survival of most cells, although membrane integrity immediately after warming (66.00% ± 4.73) or after 2 h IVC (59.67% ± 4.18) was significantly lower than controls (C0h 89.67% ± 1.45). Extended post-warming IVC (24 h) caused an additional decrease to 31% ± 3.46 (P < 0.05). Germ- and somatic-cell specific markers showed the survival of both cell types after cryopreservation and IVC. All genes were affected by cryopreservation and/or IVC, and moderate stress conditions were indicated by transcriptional stress response. CONCLUSIONS: Vitrification with the cryodevice E.Vit is a promising strategy to cryopreserve prepubertal testicular tissue.

Correction to: High in vitro survival rate of sheep in vitro produced blastocysts vitrified with a new method and device.

[This corrects the article DOI: 10.1186/s40104-019-0390-1.].

Structure of preantral follicles, oxidative status and developmental competence of in vitro matured oocytes after ovary storage at 4 °C in the domestic cat model.

BACKGROUND: Storage conditions during transportation of explanted ovaries are a critical step in setting up fertility preservation protocols in both animal and human fields. Here, we evaluated the effects of ovary storage at 4 °C on the preservation of preantral follicles and oocytes retrieved from antral follicles using the domestic cat as model. METHODS: Ovaries were harvested from fifty-five healthy domestic queens during ovariectomy and stored at 4 °C for 0 (control), 24, 48, 72 and 96 h. In Experiment 1, the effects of the storage period at 4 °C on the morphology, cytoskeleton (α/ß tubulin) and DNA integrity (phosphorylation of histone H2AX) of preantral follicles were investigated. In Experiment 2, oocytes recovered from antral follicles were matured and fertilized in vitro to evaluate their meiotic and developmental competence. Reactive oxygen species (ROS), glutathione (GSH) and lipid peroxidation were measured in matured oocytes. RESULTS: The results showed that: a) storage up to 24 h did not affect the morphology and the DNA integrity of preantral follicles; b) extended storage times caused progressive morphological abnormalities, disassembling of microtubules and DNA damage; c) storage up to 48 h did not influence in vitro meiotic maturation of oocytes nor cleavage after in vitro fertilization. However, only oocytes stored within the ovary for 24 h produced blastocysts in a percentage similar to control oocytes; d) GSH levels of in vitro matured oocytes did not change at any time during ovary storage; a progressive increase in ROS levels was detected from 48 h associated with elevated lipid peroxidation at 72 and 96 h of storage. CONCLUSIONS: Storage of cat ovaries for up to 24 h caused minimal alteration of preantral follicles and oocytes. The extension of the storage period beyond 24 h progressively impaired the structure of follicles, and modified the oxidative status of in vitro matured oocytes and their developmental competence after in vitro fertilization. This information may help when setting up programs for fertility conservation, especially for wild feline species which die in geographic areas located far away from ARTs centers.

Testicular development in male lambs prenatally exposed to a high-starch diet.

Maternal nutrition during critical gestation periods impacts on offspring in later life; effects of high-starch maternal diet on testicular development in lambs were addressed. Dairy ewes were fed diets providing either 27% (Starch, S) or 11% (Fiber, F) of starch from mating to lambing (∼147 days; S147, F147) or for the last 75 days of gestation (S75, F75). Testes of single male lambs were measured and then sampled for histological and gene expression analyses at selected ages. Testicular dimensions and weight were similar among groups, but the total area of seminiferous tubules increased with age and tended to be higher (p = 0.057) in lambs from starch- than fiber-fed ewes. Sertoli and germ cells number increased with age, but was not influenced by maternal diet. Transcript abundances of androgen receptor (AR), insulin-like growth factor 1 (IGF1), and hydroxysteroid (17-beta) dehydrogenase 3 (HSD17B3) was similar between S147 and F147 lambs (p > 0.1). Abundance of luteinizing hormone/choriogonadotropin receptor (LHCGR) and steroidogenic acute regulatory protein (STAR) was higher in young vs older lambs, whereas insulin-like growth factor 2 (IGF2) levels increased with age. The expression of vascular endothelial growth factor A (VEGFA), Anti-Müllerian hormone (AMH), IGF1, follicle stimulating hormone receptor (FSHR), and insulin-like growth factor 2 receptor (IGF2R) was not influenced by maternal diet or lamb age (p > 0.1). In conclusion, a high-starch maternal diet did not influence gene expression, but may have affected testicular structure in infant offspring, as seen by an increase in the total area of seminiferous tubules.

Delay in maternal transcript degradation in ovine embryos derived from low competence oocytes.

Oocytes from prepubertal animals have a reduced ability to undergo embryo development and produce viable offspring. The present work used an ovine model consisting of oocytes derived from adult and prepubertal donors to assess the molecular status of oocytes and preimplantation embryos with different developmental competence. The lower potential of oocytes of young donors was confirmed in terms of in vitro developmental capabilities and kinetics. A panel of genes including maternal effect (DPPA3, GDF9, NMP2, ZAR1) and housekeeping genes (ACTB, RPL19, SDHA, YWHAZ, ATP1A1), genes involved in DNA methylation (DNMT1, DNMT3A, DNMT3B), genomic imprinting (IGF2R), pluripotency (NANOG, POU5F1) and cell cycle regulation (CCNB1, CDK1, MELK) was relatively quantified. Temporal analysis during oocyte maturation and preimplantation embryo development evidenced patterns associated with donor age. With a few gene-specific exceptions, the differential model showed a reduced transcript abundance in immature prepubertal oocytes that completely reversed trend after fertilization, when higher mRNA levels were consistently observed in early embryos, indicating a delay in maternal transcript degradation. We propose that the molecular shortage in the prepubertal oocyte may affect its developmental potential and impair the early pathways of maternal mRNA clearance in the embryo. While confirming the different potential of oocytes derived from adult and prepubertal donors, our work showed for the first time a consistent delay in maternal transcript degradation in embryos derived from low competence oocytes that interestingly recalls the delayed developmental kinetics. Such abnormal transcript persistence may hinder further development and represents a novel perspective on the complexity of developmental competence.

Methylation dynamics during folliculogenesis and early embryo development in sheep.

Genome-wide DNA methylation reprogramming occurs during mammalian gametogenesis and early embryogenesis. Post-fertilization demethylation of paternal and maternal genomes is considered to occur by an active and passive mechanism respectively, in most mammals but sheep; in this species no loss of methylation was observed in either pronucleus. Post-fertilization reprogramming relies on methylating and demethylating enzymes and co-factors that are stored during oocyte growth, concurrently with the re-methylation of the oocyte itself. The crucial remodelling of the oocyte epigenetic baggage often overlaps with potential interfering events such as exposure to assisted reproduction technologies or environmental changes. Here, we report a temporal analysis of methylation dynamics during folliculogenesis and early embryo development in sheep. We characterized global DNA methylation and hydroxymethylation by immunofluorescence and relatively quantified the expression of the enzymes and co-factors mainly responsible for their remodelling (DNA methyltransferases (DNMTs), ten-eleven translocation (TET) proteins and methyl-CpG-binding domain (MBD) proteins). Our results illustrate for the first time the patterns of hydroxymethylation during oocyte growth. We observed different patterns of methylation and hydroxymethylation between the two parental pronuclei, suggesting that male pronucleus undergoes active demethylation also in sheep. Finally, we describe gene-specific accumulation dynamics for methylating and demethylating enzymes during oocyte growth and observe patterns of expression associated with developmental competence in a differential model of oocyte potential. Our work contributes to the understanding of the methylation dynamics during folliculogenesis and early embryo development and improves the overall picture of early rearrangements that will originate the embryo epigenome.

Insights on Cryopreserved Sheep Fibroblasts by Cryomicroscopy and Gene Expression Analysis.

Cryopreservation includes a set of techniques aimed at storing biological samples and preserving their biochemical and functional features without any significant alterations. This study set out to investigate the effects induced by cryopreservation on cultured sheepskin fibroblasts (CSSF) through cryomicroscopy and gene expression analysis after subsequent in vitro culture. CSSF cells were cryopreserved in a cryomicroscope (CM) or in a straw programmable freezer (SPF) using a similar thermal profile (cooling rate -5°C/min to -120°C, then -150°C/min to -196°C). CSSF volume and intracellular ice formation (IIF) were monitored by a CM, while gene expression levels were investigated by real-time polymerase chain reaction in SPF-cryopreserved cells immediately after thawing (T0) and after 24 or 48 hours (T24, T48) of post-thaw in vitro culture. No significant difference in cell viability was observed at T0 between CM and SPF samples, while both CM and SPF groups showed lower viability (p < 0.05) compared to the untreated control group. Gene expression analysis of cryopreserved CSSF 24 and 48 hours post-thawing showed a significant upregulation of the genes involved in protein folding and antioxidant mechanisms (HPS90b and SOD1), while a transient increase (p < 0.05) in the expression levels of OCT4, BCL2, and GAPDH was detected 24 hours post-thawing. Overall, our data suggest that cryostored CSSF need at least 24 hours to activate specific networks to promote cell readaptation.

Supplementation with nanomolar concentrations of verbascoside during in vitro maturation improves embryo development by protecting the oocyte against oxidative stress: a large animal model study.

The effects of verbascoside (VB), added at nanomolar concentrations during in vitro maturation (IVM) of juvenile sheep oocytes, on in vitro embryo development and its mechanisms of action at the oocyte level were analyzed. Developmental rates, after IVM in the presence/absence of VB (1nM for 24h; 1nM for 2h; 10nM for 2h), were evaluated. The bioenergetic/oxidative status of oocytes matured after IVM in the presence/absence of 1nM VB for 24h was assessed by confocal analysis of mitochondria and reactive oxygen species (ROS), lipid peroxidation (LPO) assay, and quantitative PCR of bioenergy/redox-related genes. The addition of 1nM VB during 24h IVM significantly increased blastocyst formation and quality. Verbascoside reduced oocyte ROS and LPO and increased mitochondria/ROS colocalization while keeping mitochondria activity and gene expression unchanged. In conclusion, supplementation with nanomolar concentrations of VB during IVM, in the juvenile sheep model, promotes embryo development by protecting the oocyte against oxidative stress.

Lipid droplet distribution of immature canine oocytes in relation to their size and the reproductive stage.

This study investigated the distribution of lipid droplets (LD) in immature canine oocytes in relation to their size and the reproductive stage. Oocytes were collected from the ovaries of bitches at different estrous stages, divided according to their size (110-120 µm; >120 µm), and stained with Nile Red to detect lipid droplet distribution. At the follicular phase most of the oocytes displayed a diffuse pattern of LD distribution, whereas at anestrus and luteal phase oocytes showed LD mainly in a peripheral/ perinuclear LD distribution. A significantly higher intensity of LD has been recorded in the oocytes > 120 µm compared to those of smaller size (110 - 120 µm) at all stages of the estrous cycle. At follicular phase, oocytes > 120 µm displayed LD intensity similar to that of oocytes > 120 µm at luteal phase and higher compared to the oocytes of the other groups.