Resveratrol supplementation and cryopreservation of buck semen.

Resveratrol (Resv) was tested to assess its effects on buck semen freezability. Ejaculates of 4 bucks were collected, washed and diluted in a commercial extender at 30 °C. Extended semen was divided into 4 aliquots supplemented with increasing concentrations of Resv: 0 µM (control); 10 µM; 25 µM and 50 µM. Aliquots were cooled to 4 °C in 5h and frozen in LN2. Thawing was performed at 37 °C for 30 s. At the 3 stages of the experiment (30 °C, 4 °C, thawing), motility (CASA), osmotic resistance (Hos test) and integrity of cytoplasm and acrosome membranes (PI/PSA staining) were assessed. Moreover, in thawed samples, the oxidative status (MDA assay) and early apoptosis (DNA fragmentation by TUNEL assay) were evaluated. Resveratrol supplementation did not affect most of the motility parameters analysed, except for total motility, ALH (lateral head displacement) and velocity distribution (P < 0.05). Functional and morphological integrity of membranes was not affected at any stage of the experiment (P > 0.05). In thawed spermatozoa, the oxidative status was not preserved by Resv (P > 0.05) while early apoptosis, was significantly decreased in the 50 µM Resv group (P < 0.05). Resveratrol did not improve buck semen freezability; the observed effects on motility and DNA were not dose dependent and not mediated by a potential anti-oxidant activity.

Resveratrol supplementation during in vitro maturation improves embryo development of prepubertal goat oocytes selected by brilliant cresyl blue staining.

This study aimed to investigate the effect of resveratrol supplementation in maturation medium on the developmental ability and bioenergetic\oxidative status of prepubertal goat oocytes selected by brilliant cresyl blue (BCB). Oocytes collected from slaughterhouse-derived ovaries were selected by 13 µM BCB staining and classified as grown BCB+ and growing BCB- oocytes. All oocytes were matured in vitro in our conventional maturation medium and supplemented with 1 µM (BCB+R and BCB-R) and without (Control groups: BCB+C and BCB-C) resveratrol. After 24 h, IVM-oocytes were fertilized with fresh semen and presumptive zygotes were in vitro cultured for 8 days. Oocytes were assessed for blastocyst development and quality, mitochondrial activity and distribution, and levels of GSH, ROS, and ATP. BCB+R (28.3%) oocytes matured with resveratrol presented significantly higher blastocyst development than BCB+C (13.0%) and BCB- groups (BCB-R: 8.3% and BCB-C: 4.7%). Resveratrol improved blastocyst development of BCB-R oocytes at the same rate as BCB+C oocytes. No differences were observed in blastocyst quality among groups. GSH levels were significantly higher in resveratrol groups (BCB+R: 36554.6; BCB-R: 34946.7 pixels/oocyte) than in control groups (BCB+C: 27624.0; BCB-C: 27655.4 pixels/oocyte). No differences were found in mitochondrial activity, ROS level, and ATP content among the groups. Resveratrol-treated oocytes had a higher proportion of clustered active mitochondria in both BCB groups (BCB+R: 73.07%; BCB-R: 79.16%) than control groups (BCB+C: 19.35%; BCB-C: 40%). In conclusion, resveratrol increased blastocyst production from oocytes of prepubertal goats, particularly in better quality oocytes (BCB+).

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.

Effect of exposure to CeO2 nanoparticles on ram spermatozoa during storage at 4 °C for 96 hours.

BACKGROUND: Cerium oxide nanoparticles (CeO2 NPs) are able to store and release oxygen, conferring them scavenger activity against oxidative stress. However, their effects in reproductive systems are not yet well understood. The aim of the study was to investigate the effects of exposure of refrigerated ram semen to CeO2 NPs for 96 h on the main structural and kinematic parameters of spermatozoa. METHODS: The ejaculates of 5 Sarda rams were collected, pooled and diluted in a soybean lecithin extender. Samples were exposed to increasing doses of CeO2 NPs (0, 44 and 220 µg/mL) and stored at 4 °C for 96 h. Analyses of kinematic parameters (computer assisted sperm analysis, CASA), integrity of membranes (PI/PSA staining), ROS production (H2DCFDA staining) and DNA damage (sperm chromatin structure assay with acridine orange, SCSA) were performed every 24 h (0, 24, 48, 72 and 96 h of incubation). The experiment was carried out in 6 replicates. Data were analysed by repeated measures ANOVA with Bonferroni's as post hoc test. When the assumption of normality was not met (ROS), non-parametric Kruskal-Wallis rank test was carried out. RESULTS: Exposure of ram spermatozoa to increasing doses of CeO2 NPs had a beneficial effect on the main motility parameters from 48 h of incubation onward. Velocity of sperm cells was enhanced in the groups exposed to CeO2 NPs compared to the control. Incubation with NPs had beneficial effects on the integrity of plasma membranes of spermatozoa, with higher percentage of damaged cells in the control group compared to the exposed ones. Production of ROS was not affected by exposure to NPs and its levels rose at 96 h of incubation. The integrity of DNA remained stable throughout the 96 h of storage regardless of co-incubation with NPs. CONCLUSIONS: We reported beneficial effects of CeO2 NPs on kinematic and morphologic parameters of ram semen, such as motility and membrane integrity following 96 h of exposure. Furthermore, we also proved no genotoxic effects of CeO2 NPs. These effects could not be related to an antioxidant activity of CeO2 NPs, since ROS levels in exposed cells were similar to those of unexposed ones.

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.

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.

Evaluation of the impact of vitrification on the actin cytoskeleton of in vitro matured ovine oocytes by means of Raman microspectroscopy.

PURPOSE: Investigation of the changes induced by vitrification on the cortical F-actin of in vitro matured ovine oocytes by Raman microspectroscopy (RMS). METHODS: Cumulus-oocyte complexes, recovered from the ovaries of slaughtered sheep, were matured in vitro and vitrified following the Minimum Essential Volume method using cryotops. The cortical region of metaphase II (MII) oocytes (1) exposed to vitrification solutions but not cryopreserved (CPA-exp), (2) vitrified/warmed (VITRI), and (3) untreated (CTR) was analyzed by RMS. A chemical map of one quadrant of single CPA-exp, VITRI and CTR oocytes was, also, performed. In order to identify the region of Raman spectra representative of the cortical F-actin modification, a group of in vitro matured oocytes were incubated with latrunculin-A (LATA), a specific F-actin destabilizing drug, and processed for RMS analysis. Thereafter, all the oocytes were stained with rhodamine phalloidin and evaluated by fluorescence confocal microscopy. Raman spectra of the oocytes were, statistically, analyzed using Principal Component Analysis (PCA). RESULTS: The PCA score plots showed a marked discrimination between CTR oocytes and CPA-exp/ VITRI groups. The main differences, highlighted by PCA loadings, were referable to proteins (1657, 1440 and 1300 cm(-1)) and, as indicated by LATA experiments, also included the changes of the F-actin. Analysis by confocal microscopy revealed a clear alteration of the cortical F-actin of CPA-exp and VITRI oocytes confirming RMS results. CONCLUSIONS: Raman microspectroscopy may represent an alternative analytical tool for investigating the biochemical modification of the oocyte cortex, including the F-actin cytoskeleton, during vitrification of in vitro matured ovine oocytes.

Expression of maternally derived KHDC3, NLRP5, OOEP and TLE6 is associated with oocyte developmental competence in the ovine species.

BACKGROUND: The sub-cortical maternal complex (SCMC), located in the subcortex of mouse oocytes and preimplantation embryos, is composed of at least four proteins encoded by maternal effect genes: OOEP, NLRP5/MATER, TLE6 and KHDC3/FILIA. The SCMC assembles during oocyte growth and was seen to be essential for murine zygote progression beyond the first embryonic cell divisions; although roles in chromatin reprogramming and embryonic genome activation were hypothesized, the full range of functions of the complex in preimplantation development remains largely unknown. RESULTS: Here we report the expression of the SCMC genes in ovine oocytes and pre-implantation embryos, describing for the first time its expression in a large mammalian species. We report sheep-specific patterns of expression and a relationship with the oocyte developmental potential in terms of delayed degradation of maternal SCMC transcripts in pre-implantation embryos derived from developmentally incompetent oocytes. In addition, by determining OOEP full length cDNA by Rapid Amplification of cDNA Ends (RACE) we identified two different transcript variants (OOEP1 and OOEP2), both expressed in oocytes and early embryos, but with different somatic tissue distributions. In silico translation showed that 140 aminoacid peptide OOEP1 shares an identity with orthologous proteins ranging from 95% with the bovine to 45% with mouse. Conversely, OOEP2 contains a premature termination codon, thus representing an alternative noncoding transcript and supporting the existence of aberrant splicing during ovine oogenesis. CONCLUSIONS: These findings confirm the existence of the SCMC in sheep and its key role for the oocyte developmental potential, deepening our understanding on the molecular differences underlying cytoplasmic vs nuclear maturation of the oocytes. Describing differences and overlaps in transcriptome composition between model organisms advance our comprehension of the diversity/uniformity between mammalian species during early embryonic development and provide information on genes that play important regulatory roles in fertility in nonmurine models, including the human.

Use of assisted reproductive technologies (ARTs) to shorten the generational interval in ruminants: current status and perspectives.

The challenges posed by climate change and increasing world population are stimulating renewed efforts for improving the sustainability of animal production. To meet such challenges, the contribution of genomic selection approaches, in combination with assisted reproductive technologies (ARTs), to spreading and preserving animal genetics is essential. The largest increase in genetic gain can be achieved by shortening the generation interval. This review provides an overview of the current status and progress of advanced ARTs that could be applied to reduce the generation time in both female and male of domestic ruminants. In females, the use of juvenile in vitro embryo transfer (JIVET) enables to generate offspring after the transfer of in vitro produced embryos derived from oocytes of prepubertal genetically superior donors reducing the generational interval and acceleration genetic gain. The current challenge is increasing in vitro embryo production (IVEP) from prepubertal derived oocytes which is still low and variable. The two main factors limiting IVEP success are the intrinsic quality of prepubertal oocytes and the culture systems for in vitro maturation (IVM). In males, advancements in ARTs are providing new strategies to in vitro propagate spermatogonia and differentiate them into mature sperm or even to recapitulate the whole process of spermatogenesis from embryonic stem cells. Moreover, the successful use of immature cells, such as round spermatids, for intracytoplasmic injection (ROSI) and IVEP could allow to complete the entire process in few months. However, these approaches have been successfully applied to human and mouse whereas only a few studies have been published in ruminants and results are still controversial. This is also dependent on the efficiency of ROSI that is limited by the current isolation and selection protocols of round spermatids. In conclusion, the current efforts for improving these reproductive methodologies could lead toward a significant reduction of the generational interval in livestock animals that could have a considerable impact on agriculture sustainability.

Raman spectroscopy-based approach to detect aging-related oxidative damage in the mouse oocyte.

PURPOSE: Detection of chemical modifications induced by aging-related oxidative damage in mouse metaphase II (MII) oocytes by Raman microspectroscopy. METHODS: CD-1 mice at the age of 4-8 weeks (young mice) and 48-52 weeks (old mice), were superovulated and oocytes at metaphase II stage were recovered from oviducts. MII oocytes from young animals were divided into three groups: A) young oocytes, processed immediately after collection; B) in vitro aged oocytes, cultured in vitro for 10 h before processing; C) oxidative-stressed oocytes, exposed to 10 mM hydrogen peroxide for 2 min before processing. Oocytes from reproductively old mice were referred to as old oocytes (D). All the oocytes were analyzed by confocal Raman microspectroscopy. The spectra were statistically analyzed using Principal Component Analysis (PCA). RESULTS: PCA evidenced that spectra from young oocytes (A) were clearly distinguishable from those obtained from in vitro-aged, oxidative-damaged and old oocytes (B, C, D) and presented significant differences in the bands attributable to lipid components (C = C stretching, 1,659 cm⁻¹; CH2 bending, 1,450 cm⁻¹; CH3 deformation,1,345 cm⁻¹; OH bending, C-N stretching, 1,211 cm⁻¹) and protein components (amide I band,1,659 cm⁻¹; CH2 bending modes and CH3 deformation, 1,450 cm⁻¹; C-N and C-C stretching vibrations, 1,132 cm⁻¹; phenylalanine's vibration, 1,035 cm⁻¹) CONCLUSIONS: Raman spectroscopy is a valuable non-invasive tool for the identification of biochemical markers of oxidative damage and could represent a highly informative method of investigation to evaluate the oocyte quality.

Heavy Metal(loid) Accumulation in the Ovarian Tissue of Free-Ranging Queens and Bitches Inhabiting Highly Polluted Urban Environments.

There is strong scientific evidence that exposure to environmental contaminants, such as heavy metal(loid)s (HMs), can impair female reproductive function. Pets, such as cats and dogs, who share the same habitat as humans, may be particularly useful sentinel models for detecting HMs in the ovary. In the present study, we compared the concentration of essential (Ems; Cu, Fe, Mn, Se, and Zn) and non-essential metal(loid)s (NEMs; Al, As, Cd, and Pb) in the ovarian tissues of free-ranging queens and bitches of different ages living in industrialized/highly polluted (south group) and non-polluted (north group) urban areas of the island of Sardinia, Italy. The results showed that both EMs and NEMs were present at detectable concentrations in feline and canine ovaries and their levels varied according to geographical areas and animal age. Among the EMs, Cu was found elevated in older queens and bitches inhabiting the southern area. Cadmium and lead were higher in feline and canine ovaries of older animals from the south compared to those living in the north. In addition, Cd and Pb concentrations increased in individuals of both species living in the south. These findings showed new perspectives for the use of pets as early warning sentinels of environmental pollution by HMs and for the risk of human exposure within a "One Health" approach. Pets may help to study the link between exposure to metals and female reproductive disturbances in mammals.

Adaptive Response to Gillnets Bycatch in a North Sardinia Mediterranean Shag (Gulosus aristotelis desmarestii) Population.

Mediterranean Shag (Gulosus aristotelis desmarestii) is a seabird endemic to the Mediterranean and Black Seas, recently included in the IUCN list of threatened Species. Most of the reproductive colonies are hosted in Sardinia and surrounding islets. Bycatch in fishing nets is one of the most significant threats for this population. Our work aimed to assess alterations in the sex ratio caused by bycatch and to study the adaptive response of the population to a skewed adult sex ratio. The sex ratio of Mediterranean Shags found drowned in the gillnets near the colonies and that of the nestlings of the Corcelli (northeast Sardinia) colony was determined using the sex-linked polymorphism of the gene Chromobox-Helicase-DNA-binding 1. The data of the shags found drowned in gillnets evidenced a high mortality rate (83.3%; p < 0.001) and a larger size of males (35% heavier than females, p < 0.05) compared to females, supporting the theory that heavier individuals are able to forage at great depths. With 64.8% of the nestlings being male, the sex ratio of nestlings was statistically different from parity (p < 0.05). Furthermore, it was related to the brood size. In one- and two-chick broods, 73% and 70% of nestlings, respectively, were males, while in three-chick broods, only 33% were males. Our data identify the higher rate of male shags drowned in gillnets as a factor causing an alteration of the sex ratio in the Mediterranean Shag population. According to the Sex Allocation Theory, an adaptive adjustment of sex made by adult females restores the Mendelian sex ratio in the population.

Cumulus Cell Transcriptome after Cumulus-Oocyte Complex Exposure to Nanomolar Cadmium in an In Vitro Animal Model of Prepubertal and Adult Age.

Cadmium (Cd), a highly toxic pollutant, impairs oocyte fertilization, through oxidative damage on cumulus cells (CCs). This study analysed the transcriptomic profile of CCs of cumulus-oocyte complexes (COCs) from adult and prepubertal sheep, exposed to Cd nanomolar concentration during in vitro maturation. In both age-groups, CCs of matured oocytes underwent RNA-seq, data analysis and validation. Differentially expressed genes (DEGs) were identified in adult (n = 99 DEGs) and prepubertal (n = 18 DEGs) CCs upon Cd exposure. Transcriptomes of adult CCs clustered separately between Cd-exposed and control samples, whereas prepubertal ones did not as observed by Principal Component Analysis. The transcriptomic signature of Cd-induced CC toxicity was identified by gene annotation and literature search. Genes associated with previous studies on ovarian functions and/or Cd effects were confirmed and new genes were identified, thus implementing the knowledge on their involvement in such processes. Enrichment and validation analysis showed that, in adult CCs, Cd acted as endocrine disruptor on DEGs involved in hormone biosynthesis, cumulus expansion, regulation of cell signalling, growth and differentiation and oocyte maturation, whereas in prepubertal CCs, Cd affected DEGs involved in CC development and viability and CC-oocyte communications. In conclusion, these DEGs could be used as valuable non-invasive biomarkers for oocyte competence.

Raman microspectroscopy as a non-invasive tool to assess the vitrification-induced changes of ovine oocyte zona pellucida.

Cryopreservation-induced modifications of zona pellucida (ZP) have been explored to a lesser extent compared to other oocyte compartments. Different methods have been applied to identify ZP changes, but most of them are invasive and measure only few properties of ZP. Raman microspectroscopy (RMS) is a powerful technique for studying the molecular composition of cells but to date few studies have been performed on the oocytes using this method. The aim of the present study is to investigate the structural modifications of ZP of vitrified/warmed in vitro matured ovine oocytes by means of RMS. Cumulus-oocyte complexes were recovered from the ovaries of slaughtered adult sheep, matured in vitro and vitrified following the Minimum Essential Volume method using cryotops. ZPs of vitrified/warmed oocytes (VITRI), were exposed to vitrification solutions but not cryopreserved (CPA-exp) and untreated oocytes (CTR) were analyzed by RMS. We focused our analysis on the ZP protein and carbohydrate components by analyzing the 1230-1300 cm(-1) amide III region and the 1020-1140 cm(-1) spectral range in RMS spectra, respectively. The spectral profiles in the ranges of proteins and carbohydrates were comparable between CTR and CPA-exp ZPs, whereas VITRI ZPs showed a significantly altered protein secondary structure characterized by an increase in ß-sheet content and a decrease in the α-helix content. A significant modification of the carbohydrate components was also observed. This study demonstrates that vitrification of ovine oocytes induces biochemical changes of ZP related to the secondary structure of proteins and carbohydrate residues. Cryoprotectants do not strongly alter the molecular composition of ZP which is affected mainly by cooling. Raman technology offers a powerful and non-invasive tool to assess molecular modifications induced by cryopreservation in oocytes.

Protective effect of resveratrol against cadmium-induced toxicity on ovine oocyte in vitro maturation and fertilization.

BACKGROUND: Heavy metal cadmium (Cd) is a widespread environmental contaminant with a potential toxicity that might negatively affect female reproduction and fertility. It has been reported that Cd exposure impaired the quality of oocytes and led to a defective maturation and fertilization, through oxidative stress induction. Resveratrol (Res) is a natural polyphenol with strong antioxidant properties that exhibited protective role in preventing oocyte redox homeostasis disruption and quality decline. Here, we explored whether the addition of Res to in vitro maturation (IVM) medium might act as a protection against Cd-induced toxicity on ovine oocyte maturation and fertilization. Firstly, we evaluated the effect of supplementing IVM medium with two different Res concentrations (1 and 2 µmol/L) on nuclear maturation and fertilization of oocytes matured under CdCl2 (2 µmol/L) exposure. Therefore, the concentration of 1 µmol/L Res was selected to analyse the effects of this compound on intracellular ROS levels, mitochondrial (mt) distribution and activity, chromatin configuration, cytoskeleton morphology, cortical granules (CGs) distribution and mRNA expression of genes associated with cellular response to oxidative stress (i.e. SIRT1, SOD 1, GPX1, GSR, CAT) in Cd-exposed in vitro matured oocytes. RESULTS: We found that 1 µmol/L Res restored the reduced oocyte meiotic competence induced by Cd exposure as well as, Res sustained oocyte ability to be normally fertilized and decreased polyspermic fertilization at both tested concentrations. Moreover, we demonstrated that 1 µmol/L Res mitigated Cd-induced alterations of oocyte cytoplasmic maturation by reducing reactive oxygen species (ROS) accumulation, preventing mt dysfunction, maintaining the correct meiotic spindle and cortical F-actin assembly and the normal cortical granule distribution as well as up-regulating SIRT1, SOD1 and GPX1 genes. CONCLUSIONS: Taken together, our findings highlighted the beneficial influence exerted by Res in preventing Cd-induced disturbance of nuclear and cytoplasmic maturation and subsequent fertilization in ovine oocytes. Res treatment may help to establish defence strategies counteracting Cd-induced toxicity on the female gamete.

High hydrostatic pressure treatment improves the quality of in vitro-produced ovine blastocysts.

Exposure to sub-lethal hydrostatic pressure (HP) treatment is emerging as an approach to improve the general resistance of gametes and embryos to in vitro conditions. The present study was aimed to evaluate the effect of HP treatment on in vitro-produced ovine blastocysts. Experiment 1 was aimed to define optimal treatment parameters: two different HP treatments were applied to blastocysts and embryo survival was evaluated. In Experiment 2, HP parameters (40 MPa, 70 min, 38 °C) selected in Experiment 1 were used to treat blastocysts. Embryo quality was assessed and compared with untreated controls by counting total cell number, the inner cell mass (ICM) and trophectoderm (TE) cells and by evaluating nuclear picnosis. HP-treated blastocysts were processed for gene expression analysis (AQP3, ATP1A1, BAX, CDH1, HSP90ß, NANOG, OCT4 and TP53) 1, 5h after the end of HP exposure. Results showed that the hatching rate of embryos treated at 40 MPa was significantly higher than that of the 60 MPa-treated group (P<0.01) and similar to untreated embryos. Blastocysts exposed at 40 MPa showed higher ICM (P<0.05) and TE (P<0.01) cell number and a lower percentage of picnotic nuclei (P<0.05) compared with the control group. Significantly lower abundance for BAX (P<0.01) and OCT4 (P<0.05) transcripts were observed in HP embryos than in the control group. In conclusion, treatment with HP improved the quality of in vitro-produced ovine blastocysts by increasing their cell number and reducing the proportion of nuclear picnosis.

Raman spectroscopy-based approach to study the female gamete.

In the last years, an increasing interest has emerged on the development of new non-invasive methods for the assessment of oocyte quality in order to improve outcomes of assisted reproductive technologies (ARTs) either in medical or veterinary fields. Raman microspectroscopy (RMS) has been proposed as a promising tool for the examination of the mammalian female gamete and identification of markers of its developmental competence. This technique provides a unique spectral fingerprint indicative of molecular composition of the cell and allows probing subcellular compartments. Studies have been carried out analysing by RMS fixed or living oocytes derived from different animal models. RMS imaging has been successfully applied to discriminate the biochemical changes of the global molecular architecture of mouse oocytes at different stages of maturation and those occurring in different conditions of maturation and oocyte aging. RMS can also detect modifications of specific structural components, including the oocyte zona pellucida and F-actin subcortical cytoskeleton in fresh sheep oocytes and those underwent to vitrification procedures. Finally, the recent application of Coherent anti-Stokes Raman scattering (CARS) microscopy for examination of oocyte lipid component will be briefly discussed. CARS overcomes some limits of RMS providing vibrational and spectral information with higher sensitivity, spatial resolution which is ideal to study living oocytes. This review summarizes the research on RMS approaches for oocyte evaluation showing the high potential use, current limitations and new improvements.

Selection of Immature Cat Oocytes with Brilliant Cresyl Blue Stain Improves In Vitro Embryo Production during Non-Breeding Season.

In domestic cats, the maturation, fertilization, and development potential in vitro decreases during the non-breeding season. This study aims at evaluating the efficacy of Brilliant Cresyl Blue (BCB) staining in selecting developmentally competent oocytes to be used in in vitro embryo production (IVEP) programs in order to overcome the season variability in blastocyst yield. Cumulus-oocytes complexes (COCs) collected from antral follicles of domestic cat ovaries during the anestrus phase (July to November) were selected by BCB staining and classified as BCB+ (colored cytoplasm) and BCB- (colorless cytoplasm). COCs not exposed to BCB staining were used as control. Before and after in vitro maturation mitochondrial activity and reactive oxygen species (ROS) were measured. Following in vitro fertilization, blastocyst rate, hatching rate, and blastocyst cell numbers were recorded. The results show that BCB staining did not alter the mitochondrial function and ROS production in cat oocytes. BCB+ oocytes presented a higher (p < 0.05) blastocyst rate, hatching rate, and blastocyst cell number than BCB- and control oocytes. In conclusion, BCB staining does not affect the bioenergetic/oxidative status of the oocyte while being a useful tool for selecting good quality oocytes to increase IVEP in domestic cats during non-breeding season.

Hercynine, Ergothioneine and Redox State in Stallion's Seminal Plasma.

The dependence of a stallion's spermatozoa on oxidative phosphorylation for energy requirements results in an unconventional relationship between reactive oxygen species (ROS) production and fertility. In such a scenario, antioxidant activity must be finely controlled and not affect the essential functions of ROS. Some in vivo evidence suggests that the naturally occurring antioxidant ergothioneine (ERT) interferes with the critical roles of ROS/reactive nitrogen species (RNS) in pro-oxidant states but not in healthy tissues. The measurement of ERT in seminal plasma collected from 14 stallions (five Anglo-Arab, five Sella Italiano and four Thoroughbreds of which three are Arabian and one English) aged 16 ± 6 years (range 6-25 years) confirms that ERT is present at high concentrations in this biological fluid, between 16.80 and 971.48 µmol/L. Although the presence of high ERT concentrations in the seminal plasma of a stallion has long been known, its exact biological role is uncertain. This might be due to the peculiar antioxidant cycle of ERT, specifically its rapid recovery, which potentially masks concentration fluctuations and, therefore, the extent of its physiological effects. The measurement of the ERT precursor and redox metabolite hercynine (ERY) may overcome such issues, as ERY does not undergo regeneration processes. ERY was detectable and measurable in the seminal plasma of all stallions at a median concentration of 7.50 (IQR 15.26) nmol/L. The analysis of the association between the ERT and ERY, as well as with other established antioxidants such as glutathione and cysteine, suggests that ERT may play a major role in the antioxidant machinery of seminal plasma, and that ERY might serve as a new combined marker of oxidative stress and semen quality.

Reproductive Performance Following Transcervical Insemination with Frozen Thawed Semen in Ewes Submitted to Surgical Incision of Cervical Folds (SICF): Comparison with Laparoscopic Artificial Insemination.

Transcervical artificial insemination (AI) after the surgical incision of cervical folds (SICF) could represent a valid alternative to laparoscopic AI when frozen thawed semen is used. The aim of this experiment was to compare pregnancy (PR) and lambing rates (LR) of ewes submitted either to transcervical AI after SICF or to laparoscopic AI using frozen thawed semen. Pregnant at term ewes (n = 80) were allocated in two experimental groups. After lambing, one group (n = 39) was submitted to SICF. The remaining ewes that were regularly lambed were allocated to the group of laparoscopic AI (n = 40). Six months later, oestrous cycle of both experimental groups was synchronised and all ewes were artificially inseminated with frozen thawed semen. Ewes submitted to SICF underwent transcervical insemination and intrauterine deposition of semen was recorded. The remaining animals were submitted to laparoscopic AI. Pregnancy and LR were recorded. Intrauterine deposition of semen was possible in 89.7% pf ewes submitted to SICF. This group showed similar PR and LR compared to the laparoscopic group (respectively: PR, 71.8% vs. 70% and LR, 64.1% vs. 65%; p > 0.05). Transcervical AI after SICF may represent a valid alternative to laparoscopy in AI protocols requiring the use of frozen thawed semen.