Genomic structural variation is an important and abundant source of genetic and phenotypic variation. Here, we describe the first systematic and genome-wide analysis of copy number variations (CNVs) in modern domesticated cattle using array comparative genomic hybridization (array CGH), quantitative PCR (qPCR), and fluorescent in situ hybridization (FISH). The array CGH panel included 90 animals from 11 Bos taurus, three Bos indicus, and three composite breeds for beef, dairy, or dual purpose. We identified over 200 candidate CNV regions (CNVRs) in total and 177 within known chromosomes, which harbor or are adjacent to gains or losses. These 177 high-confidence CNVRs cover 28.1 megabases or ;1.07% of the genome. Over 50% of the CNVRs (89/177) were found in multiple animals or breeds and analysis revealed breed-specific frequency differences and reflected aspects of the known ancestry of these cattle breeds. Selected CNVs were further validated by independent methods using qPCR and FISH. Approximately 67% of the CNVRs (119/177) completely or partially span cattle genes and 61% of the CNVRs (108/177) directly overlap with segmental duplications. The CNVRs span about 400 annotated cattle genes that are significantly enriched for specific biological functions, such as immunity, lactation, reproduction, and rumination. Multiple gene families, including ULBP, have gone through ruminant lineage-specific gene amplification. We detected and confirmed marked differences in their CNV frequencies across diverse breeds, indicating that some cattle CNVs are likely to arise independently in breeds and contribute to breed differences. Our results provide a valuable resource beyond microsatellites and single nucleotide polymorphisms to explore the full dimension of genetic variability for future cattle genomic research.

Flow cytometry is a useful tool that provides an accurate, objective and rapid evaluation of semen quality. The use of this technique could significantly improve the quality of buffalo semen samples used in artificial insemination. This study was carried out to evaluate, by flow cytometry, frozen-thawed buffalo spermatozoa quality parameters such as: sperm viability by SYBR-14/propidium iodide staining; mitochondrial function by JC-1 potentiometric probe; sperm chromatin stability (SCSA) by acridine orange and acrosome reaction by FITC-PNA staining. Semen samples from 5 Italian Mediterranean buffalo bulls were used. Sperm viability was not different between bulls and ranged from 33.4 to 43.6%. A consistent rate (55.1±10.8%) of sperm cells showed high mitochondrial membrane potential (Δψhigh), with no significant differences between subjects. SCSA differed significantly between the 5 buffalo bulls, moreover data showed high stability within each buffalo. DNA fragmentation indexes (DFI), such as %-DFI, -DFI, SD-DFI, were 11.2±8.6, 153.3±24.6 and 81.6±21.2, respectively. Regarding acrosome reaction (AR), the percentage of acrosome-reacted live (ARL) and acrosome-reacted dead (ARD) spermatozoa was 0.3±0.2 and 15.3±5.5 respectively. This functional parameter differed significantly between buffalo bulls and showed high stability. Following to Ca2+ ionophore A23187 for 3 hrs, AR significantly differed between subjects and was characterized by an increase of both ARL (10.8%) and ARD population (22.0%). The present study indicates that flow cytometry could be a useful tool for a quick multiparametric evaluation of sperm quality in buffalo. In particular, SCSA and AR resulted sperm functional parameters sensitive enough for the diagnosis of frozen-thawed semen fertilizing potential.

Methodologies, such as flow cytometry and computer assisted sperm analysis (CASA), provide objective, reproducible, rapid and multi-parametric evaluation of semen quality. In this study, semen samples collected from six stallions were analysed for viability (by propidium iodide), chromatin stability by sperm chromatin structure assay (SCSA) and mitochondrial membrane potential by JC-1 using flow cytometry. Total and progressive motility, average path velocity (VAP), curvilinear velocity (VCL) and straight-line velocity (VSL) were determined by CASA system. The cytofluorimetric analysis provided results with low intra-assay variability respect to motility analysis by CASA system. The data on viability and mitochondrial assessment were rather uniform between stallions. The SCSA was able to distinguish potential fertility levels between stallions. In fact statistical differences were found between stallions especially for %-DFI and SD-DFI parameter. The %-DFI parameter was negatively correlated with VCL parameter. The higher repeatability of %-DFI parameter respect to those of other SCSA parameters confirms the importance of this parameter notoriously related to fertility. In conclusion, the simultaneous assessment of different functional sperm parameters, by flow cytometry and CASA, may be allow to obtain detailed and repeatable evaluations of sperm quality in the stallion, usually not considered in breeding selection programs.

The developmental competence of in vitro-produced embryos is strictly related to oocyte quality. Analyses of energy and redox status parameters are emerging technologies useful for further oocyte quality characterisation. Mitochondrial (mt) activity is a necessary feature involved in cytoplasmic maturation, and the primary function of mitochondria is adenosine triphosphate (ATP) production. Mitochondria distribution pattern and ATP content are important parameters in the evaluation of oocyte metabolic activity, particularly activities driving microtubules dynamics leading to chromosomes segregation. Superoxide dismutase (SOD), a first-line antioxidant enzyme, has also been hypothesised as being associated to oocyte quality. The aim of the present study was to analyse ATP content and SOD activity in single equine oocytes examined before and after in vitro maturation. Cumulus–oocyte complexes surrounded by a compact cumulus oophorus were recovered from the ovaries of slaughtered mares and analysed before or after in vitro maturation (Ambruosi et al. 2009 Theriogenology 71, 1093–1104). After cumulus cell removal, all oocytes underwent evaluation of signs of meiotic maturation, and only those oocytes showing cumulus expansion, regular ooplasmic size (4160 mm in diameter) and morphology, and 1st polar body extrusion were selected for analysis. Adenosine triphosphate intracellular levels were analysed by luciferin-luciferase bioluminescent reaction (ATPlite, PerkinElmer, Monza, Italy). Quantification of SOD activity was performed by spectro- photometrical assay with WST1 and by polyacrylamide native gel and nitro blue tetrazolium reduction method. Intracellular ATP levels were influenced by meiotic stage in that oocytes at the germinal vesicle stage (GV, n 1⁄4 15) showed 1.25 ` 0.8 pmol cell1, whereas metaphase II (MII) oocytes (n1⁄415) showed significantly higher levels (2.29`1.69 pmolcell1; Po0.05). This is in line with our previous observations on mt distribution pattern analysed by Mitotracker Orange CMTM Ros staining and confocal microscopy (Ambruosi et al. 2009). In vitro-matured MII oocytes showed significantly higher rates of perinuclear mt distribution pattern, indicating mt aggregation around meiotic metaphase spindle, compared with GV oocytes (3/12, 25% v. 0/13, 0% in GV oocytes; Po0.05). Superoxide dismutase spectrophotometrical activity was 0.72 ` 0.55 U mg1 prot in GV oocytes (n 1⁄4 4) and 2.33 ` 0.33 U mg1 prot in MII oocytes (n 1⁄4 2; P o 0.001). In native gel SOD activity was 16 285.05 arbitrary densitometric units (ADU) in a GV oocyte and 22 501.35 ADU in a MII oocyte. To our knowledge, this is the first study reporting intracellular SOD activity in single oocytes in mammals. Moreover, this is the first study reporting ATP content in single equine oocytes. Observed quantitative differences seem to be related to meiotic stage.

Background: Umbilical cord matrix mesenchymal stem cells (UCM-MSCs) present a wide range of potential therapeutical applications. The extracellular calcium-sensing receptor (CaSR) regulates physiological and pathological processes. We investigated, in a large animal model, the involvement of CaSR in triggering osteogenic and neurogenic differentiation of two size-sieved UCM-MSC lines, by using AMG641, a novel potent research calcimimetic acting as CaSR agonist. Methodology/Principal Findings: Large (.8mm in diameter) and small (,8mm) equine UCM-MSC lines were cultured in medium with high calcium (Ca2+) concentration ([Ca2+]o; 2.87 mM) and dose-response effects of AMG641 (0.01 to 3mM) on cell proliferation were evaluated. Both cell lines were then cultured in osteogenic or neurogenic differentiation medium containing: 1) low [Ca2+]o (0.37 mM); 2) high [Ca2+]o (2.87 mM); 3) AMG641 (0.05, 0.1 or 1 mM) with high [Ca2+]o and 4) the CaSR antagonist NPS2390 (10 mM for 30 min) followed by incubation with AMG641 in high [Ca2+]o. Expression of osteogenic or neurogenic differentiation biomarkers was compared among groups. In both cell lines, AMG641 dose- dependently increased cell proliferation (up to P,0.001). Osteogenic molecular markers expression was differentially regulated by AMG641, with stimulatory (OPN up-regulation) in large or inhibitory (RUNX2 and OPN down-regulation) effects in small cells, respectively. AMG641 significantly increased alkaline phosphatase activity and calcium phosphate deposition in both cell lines. Following treatment with AMG641 during osteogenic differentiation, in both cell lines CaSR expression was inversely related to that of osteogenic markers and inhibition of CaSR by NPS2390 blocked AMG641-dependent responses. Early-stage neurogenic differentiation was promoted/triggered by AMG641 in both cell lines, as Nestin and CaSR mRNA transcription up-regulation were observed. Conclusions/Significance: Calcium- and AMG641-induced CaSR stimulation promoted in vitro proliferation and osteogenic and early-stage neurogenic differentiation of UCM-MSCs. CaSR activation may play a fundamental role in selecting specific differentiation checkpoints of these two differentiation routes, as related to cell commitment status.

Fetal adnexa are a noncontroversial source of mesenchymal stem cells (MSCs) with high plasticity, proliferation rate and ability to differentiate towards multiple lineages. Mesenchymal SCs have been characterized for both their stemness and their differentiation abilities. Recently, the scientific debate has focused on MSCs selection and on establishing predictable elements to discriminate the cells with most promising potential in regenerative medicine. In this study, we characterized and followed in vitro proliferation and differentiation potency of canine amniotic membrane MSCs (AM-MSCs) and umbilical cord matrix MSCs (UCM-MSCs) isolated from fetuses at early (35-40 days) and late (45-55 days) gestational ages. We found that cells from both fetal gestational ages showed similar features. In all examined cell lines, the morphology of proliferating cells typically appeared fibroblast-like and the population doubling of cells, cultured up to passage 10, significantly increased with passage number. In both cell types, cell viability and chromosomal number and structure were not affected by gestational age. In AM- and UCM-MSCs of both gestational phases, the expression of embryonic (Oct-4) and mesenchymal stemness (CD29, CD44) markers was observed. Hematopoietic and histocompatibility markers were never found in any sample. Cells of the two cell types at P3 showed multipotent abilities and differentiated to neurocytes and osteocytes, as demonstrated by specific stains and molecular analysis. These results indicated that MSCs retrieved from UCM and AM in early and late fetal phase of gestation could be used in regenerative medicine approaches in the dog.

OBJECTIVE: To evaluate the effectiveness of a bioenergy/oxidative stress assessment based on confocal laser scanning microscopy (CLSM) in association with morphology and ultrastructure analyses based on light microscopy (LM) and transmission electron microscopy (TEM), to monitor the preservation status of cryopreserved human ovarian tissue from cancer patients. DESIGN: Prospective study. SETTING: University hospital. PATIENT(S): Fourteen young cancer patients. INTERVENTION(S): Human ovarian tissue biopsy, slow freezing/rapid thawing, LM, TEM, CLSM assessment of mitochondrial distribution and activity, and intracellular reactive oxygen species (ROS) localization and levels. MAIN OUTCOME MEASURE(S): In tissue examined before and after slow freezing/rapid thawing, follicular and stromal LM-based score of morphologic damage, ultrastructure, mitochondrial distribution pattern, reactive oxygen species (ROS) localization; mean ± standard deviation of stromal mitochondrial activity and ROS levels. RESULT(S): Severe (n = 6 patients), slight (n = 6 patients), or no (n = 2 patients) LM/TEM-based damage was found in fresh tissue. After freezing/thawing, no further morphologic/ultrastructural alterations were found; however, statistically significant reductions, increases, or no changes in mitochondrial activity and ROS levels were found in severely, slightly, and undamaged tissue, respectively. CONCLUSION(S): Bioenergy/oxidative functional damage was found in tissue with severe LM/TEM-assessed damage. In tissue with slight LM/TEM-assessed damage, the CLSM-based bioenergy/oxidative stress assessment was the only test that allowed discrimination between tissue that had been better (low/no difference) or worse preserved (significant differences).

Background: Reproductive biotechnologies in dromedary camel (Camelus dromedarius) are less developed than in other livestock species. The in vitro maturation (IVM) technology is a fundamental step for in vitro embryo production (IVP), and its optimization could represent a way to increase the success rate of IVP. The aim of the present study was to investigate the bioenergy/oxidative status of dromedary camel oocytes before and after IVM by confocal microscopy 3D imaging. Methods: Oocytes were retrieved by slicing ovaries collected at local slaughterhouses. Recovered oocytes were examined before and after IVM culture for nuclear chromatin configuration and bioenergy/oxidative status, expressed as mitochondria (mt) distribution and activity, intracellular Reactive Oxygen Species (ROS) levels and distribution and mt/ROS colocalization. Results: The mean recovery rate was 6 oocytes/ovary. After IVM, 61% of oocytes resumed meiosis and 36% reached the Metaphase II stage (MII). Oocyte bioenergy/redox confocal characterization revealed changes upon meiosis progression. Immature oocytes at the germinal vesicle (GV) stage were characterised by prevailing homogeneous mt distribution in small aggregates while MI and MII oocytes showed significantly higher rates of pericortical mt distribution organized in tubular networks (P<0.05). Increased mt activity in MI (P<0.001) and MII (P<0.01) oocytes compared to GV stage oocytes was also observed. At any meiotic stage, homogeneous distribution of intracellular ROS was observed. Intracellular ROS levels also increased in MI (P<0.01) and MII (P<0.05) oocytes compared to GV stage oocytes. The mt/ROS colocalization signal increased in MI oocytes (P<0.05). Conclusions: This study provides indications that qualitative and quantitative indicators of bioenergy and oxidative status in dromedary camel oocytes are modified in relation with oocyte meiotic stage. These data may increase the knowledge of camel oocyte physiology, in order to enhance the efficiency of IVP procedures.

ABSTRACT - The aim of the study was to compare the effects of slow freezing and vitrification on blastomere/chromatin integrity and energy/oxidative stress parameters of mouse preimplantation embryos. Mouse (4-cell to blastocyst stage) embryos were analyzed as fresh (controls) or after vitrification or slow freezing. Embryo collapsing was found after both slow freezing (P<0.001) and vitrification (P<0.05). Significantly higher rate of blastomere cytofragmentation was found after slow freezing (P<0.001) but it was not observed after vitrification. Chromatin damage and altered mitochondrial (mt) distribution pattern were observed, both after slow freezing (P<0.05) and vitrification (P<0.05). In embryos at the morula stage, mt activity was reduced by slow freezing (P<0.05) but it did not change by vitrification. In embryos at the blastocyst stage, mt activity was reduced by both slow freezing (P<0.05) and vitrification (P<0.05). Intracellular reactive oxygen species (ROS) level was significantly lower in slow-frozen (P<0.05) but higher in vitrified embryos (P<0.05) compared with controls. Mitochondria/ROS colocalization was significantly reduced after slow freezing (P<0.05) but it was not affected by vitrification. In conclusion, this study demonstrates that vitrification is a suitable method to preserve embryo bioenergy/redox parameters.

In: Reproduction, Fertility and Development, Vol 25(1), 2013 Juvenile in vitro embryo transfer in farm animals reduces the generation interval and increases the rate of genetic gain. In human reproductive medicine, it enables the preservation of female fertility of young patients affected by cancer or by premature ovarian failure. The developmental competence of in vitro-produced juvenile embryos is strictly related to oocyte quality. The aim of the present study was to analyse the developmental potential and the mitochondrial/oxidative status of ovine prepubertal oocytes matured in vitro to clarify their suitability in juvenile in vitro embryo transfer programs. Oocytes from the ovaries of slaughtered prepubertal lambs (less than 6 months of age) were analysed after in vitro maturation. After cumulus cell removal, oocytes at the metaphase II stage (MII) underwent either IVF plus in vitro embryo culture (Experiment 1; n 1⁄4 200; Bogliolo et al. 2011 Reprod. Fert. Dev. 23, 809–817) or confocal analysis of mitochondria (mt) and reactive oxygen species (ROS) fluorescence distribution, intensity, and colocalization (Experiment 2; n 1⁄4 30; Martino et al. 2012 Fertil. Steril. 97, 720–728) or scavenger enzyme [superoxide dismutase (Ambruosi et al. 2011 PLoS ONE 6, e27452) and catalase (Beers and Sizer 1952 J. Biol. Chem. 195, 133–140)] activity analyses in cell lysates of individual oocytes (Experiment 3; n 1⁄4 7). In Experiment 1, 150 of 200 MII oocytes (75%) cleaved after 30 h of in vitro embryo culture, and 36 of 150 2- to 4-cell-stage embryos (24%) reached the blastocyst stage at Day 8. In Experiment 2, 60 of 111 (54%) oocytes selected for in vitro maturation culture reached the MII stage, and 30 of them (50%) with a regular size (.150 mm in diameter) and morphology were analysed for bioenergy/redox parameters. Fourteen of 30 oocytes (47%) showed a heterogeneous (perinuclear, pericortical, or both) mt distribution pattern, and the remaining 16 of 30 oocytes (53%) showed a homogeneous distribution of small mt aggregates. Intracellular ROS were uniformly distributed, thus not corresponding to the mt distribution pattern. Fluorescent intensity of mt and ROS labelling, expressed as arbitrary densitometric units, were 821.4 ` 274.7 and 737.6 ` 226.5 in oocytes with a heterogeneous pattern and 723.7 ` 371.6 and 831.7 ` 263.7 in oocytes with a homogeneous pattern, respectively (not significant). The mt/ROS colocalization (Pearson correlation coefficient) did not differ between heterogeneous (0.47`0.2) and homogeneous (0.51`0.09; not significant) oocytes. In Experiment 3, superoxide dismutase (n 1⁄4 4) and catalase activity (n 1⁄4 3) values were 1.09 ` 0.03 and 10.63 ` 1.96 IU mg1 of protein, respectively. This study provides basal values of bioenergy/redox parameters in prepubertal lamb MII oocytes as related to their developmental potential and may increase the knowledge of prepubertal oocyte physiology compared with their young adult counterparts.

Glycoprotein oligosaccharides play major roles during reproduction, yet their function in gamete interactions is not fully elucidated. Identification and comparison of the glycan pattern in cumulus-oocyte complexes (COCs) from species with different efficiencies of in vitro spermatozoa penetration through the zona pellucida (ZP) could help clarify how oligosaccharides affect gamete interactions. We compared the expression and localization of 12 glycosidic residues in equine and porcine in vitro-matured (IVM) and preovulatory COCs by means of lectin histochemistry. The COCs glycan pattern differed between animals and COC source (IVM versus preovulatory). Among the 12 carbohydrate residues investigated, the IVM COCs from these two species shared: (a) sialo- and bN-acetylgalactosamine (GalNAc)-termi- nating glycans in the ZP; (b) sialylated and fucosylated glycans in cumulus cells; and (c) GalNAc and N-acetylglucosamine (GlcNAc) glycans in the ooplasm. Differences in the preovulatory COCs of the two species included: (a) sialoglycans and GlcNAc terminating glycans in the equine ZP versus terminal GalNAc and internal GlcNAc in the porcine ZP; (b) terminal galactosides in equine cumulus cells versus terminal GlcNAc and fucose in porcine cohorts; and (c) fucose in the mare ooplasm versus lactosamine and internal GlcNAc in porcine oocyte cytoplasm. Furthermore, equine and porcine cumulus cells and oocytes contributed differently to the synthesis of ZP glycoproteins. These results could be attributed to the different in vitro fertilization efficiencies between these two divergent, large-animal models.

Background: The aim of this study was to investigate the in vitro effects of the Fusarium fungus-derived mycotoxin, zearalenone and its derivatives alpha-zearalenol and beta-zearalenol on motility parameters and the acrosome reaction of stallion sperm. Since the toxic effects of zearalenone and its derivatives are thought to result from their structural similarity to 17beta-estradiol, 17beta-estradiol was used as a positive control for ‘estrogen-like’ effects. Methods: Stallion spermatozoa were exposed in vitro to zearalenone, alpha-zearalenol, beta-zearalenol or 17betaestradiol at concentrations ranging from 1 pM - 0.1 mM. After 2 hours exposure, motility parameters were evaluated by computer-assisted analysis, and acrosome integrity was examined by flow cytometry after staining with fluoroscein-conjugated peanut agglutinin. Results: Mycotoxins affected sperm parameters only at the highest concentration tested (0.1 mM) after 2 hours exposure. In this respect, all of the compounds reduced the average path velocity, but only alpha-zearalenol reduced percentages of motile and progressively motile sperm. Induction of motility patterns consistent with hyperactivation was stimulated according to the following rank of potency: alpha-zearalenol >17beta-estradiol > zearalenone = beta-zearalenol. The hyperactivity-associated changes observed included reductions in straight-line velocity and linearity of movement, and an increase in the amplitude of lateral head displacement, while curvilinear velocity was unchanged. In addition, whereas alpha- and beta- zearalenol increased the percentages of live acrosome-reacted sperm, zearalenone and 17beta-estradiol had no apparent effect on acrosome status. In short, alpha-zearalenol inhibited normal sperm motility, but stimulated hyperactive motility in the remaining motile cells and simultaneously induced the acrosome reaction. Beta-zearalenol induced the acrosome reaction without altering motility. Conversely, zearalenone and 17beta-estradiol did not induce the acrosome reaction but induced hyperactive motility albeit to a different extent. Conclusions: Apparently, the mycotoxin zearalenone has 17beta-estradiol-like estrogenic activity that enables it to induce hyperactivated motility of equine sperm cells, whereas the zearalenol derivatives induce premature completion of the acrosome reaction and thereby adversely affect stallion sperm physiology. The alpha form of zearalenol still possessed the estrogenic ability to induce hyperactivated motility, whereas its beta stereo-isomere had lost this property.

Background: Evaluation of mitochondrial function offers an alternative to evaluate embryo development for assessment of oocyte viability, but little information is available on the relationship between mitochondrial and chromatin status in equine oocytes. We evaluated these parameters in immature equine oocytes either fixed immediately (IMM) or held overnight in an Earle’s/Hank’s’ M199-based medium in the absence of meiotic inhibitors (EH treatment), and in mature oocytes. We hypothesized that EH holding may affect mitochondrial function and that holding temperature may affect the efficiency of meiotic suppression. Methods: Experiment 1 - Equine oocytes processed immediately or held in EH at uncontrolled temperature (22 to 27°C) were evaluated for initial chromatin configuration, in vitro maturation (IVM) rates and mitochondrial energy/redox potential. Experiment 2 - We then investigated the effect of holding temperature (25°C, 30°C, 38°C) on initial chromatin status of held oocytes, and subsequently repeated mitochondrial energy/redox assessment of oocytes held at 25°C vs. immediately-evaluated controls. Results: EH holding at uncontrolled temperature was associated with advancement of germinal vesicle (GV) chromatin condensation and with meiotic resumption, as well as a lower maturation rate after IVM. Holding did not have a significant effect on mitochondrial distribution within chromatin configurations. Independent of treatment, oocytes having condensed chromatin had a significantly higher proportion of perinuclear/pericortical mitochondrial distribution than did other GV configurations. Holding did not detrimentally affect oocyte energy/redox parameters in viable GV-stage oocytes. There were no significant differences in chromatin configuration between oocytes held at 25°C and controls, whereas holding at higher temperature was associated with meiosis resumption and loss of oocytes having the condensed chromatin GV configuration. Holding at 25°C was not associated with progression of mitochondrial distribution pattern and there were no significant differences in oocyte energy/redox parameters between these oocytes and controls. Conclusions: Mitochondrial distribution in equine GV-stage oocytes is correlated with chromatin configuration within the GV. Progression of chromatin configuration and mitochondrial status during holding are dependent on temperature. EH holding at 25°C maintains meiotic arrest, viability and mitochondrial potential of equine oocytes. This is the first report on the effects of EH treatment on oocyte mitochondrial energy/redox potential.

ABSTRACT - The aim of the present study was to analyze the effects of in vitro exposure to Di-(2-ethylhexyl) phthalate (DEHP), an industrial plasticizer, on cumulus-oocyte maturation and energy/oxidative status in the horse. After in vitro maturation (IVM) in presence of 0.12, 12 and 1200 μM DEHP, cumulus cells (CCs) were removed and evaluated for apoptosis and intracellular reactive oxygen species (ROS) levels. Oocytes were evaluated for nuclear chromatin configuration. Matured (Metaphase-II stage; MII) oocytes were further evaluated for cytoplasmic energy/oxidative parameters. DEHP significantly inhibited oocyte maturation when added at low doses (0.12 μM; P<0.05). This effect was related to increased CC apoptosis (P<0.001) and reduced ROS levels (P<0.0001). At higher doses (12 and 1200 μM), DEHP induced apoptosis (P<0.0001) and ROS increase (P<0.0001) in CCs without affecting oocyte maturation. In DEHP-exposed MII oocytes, mitochondrial (mt) distribution patterns, apparent energy status, intracellular ROS localization and levels, mt/ROS colocalization and total SOD activity did not vary, whereas increased ATP content (P<0.05), possibly of glycolytic origin, was found. Co-treatment with the antioxidant N-Acetyl-Cysteine reversed apoptosis and efficiently scavenged excessive ROS in DEHP-treated CCs without improving oocyte maturation. In conclusion, in vitro exposure to DEHP inhibits equine oocyte maturation without altering ooplasmic energy/oxidative parameters in matured oocytes.

ABSTRACT - Amniotic membrane (AM) and umbilical cord matrix (UCM) mesenchymal stem cells (MSCs) have been isolated and characterized in humans and large animal models. In order to distinguish which cells retain the best features for different purposes, the effects of gestational age on proliferation and differentiation potency of canine AM-MSCs and UCM-MSCs was analyzed. Samples were recovered after elective ovariohysterectomy from bitches in early (35 to 40 days) and late (45 to 55 days) fetal stage of pregnancy. The proliferation study and the molecular analysis of embryonic, mesenchymal and hematopoietic markers were performed. Cell neurogenic and osteogenic differentiation were followed. No differences were noticed when comparing data obtained from cells isolated at different gestational ages. Doubling times, cell viability and Oct-4, CD29 and CD44 stemness markers expression were similar in cell isolated from bitches in early or late pregnancy. In both gestational ages, morphological features of neuronal and osteogenic differentiation were observed which need to be confirmed by molecular analysis. In conclusion, our data indicate the possibility to isolate MSCs from canine fetuses at early and late gestational ages with the same proliferative and differentiative capabilities.

Opioid receptors (ORs) are G protein-coupled receptors. Other than antinociception, they have been recently shown to be involved in the crucial switch phase between cell proliferation and differentiation, from which the stem cell fate depend. We detected mu-OR subtype 1 (MOR-1) and kappa-OR subtype 1 (KOR-1) expression in canine umbilical cord matrix (UCM) mesenchymal stem cells (MSCs). MOR expression decreased with passage numbers, whereas KOR was expressed at constant levels throughout passages. Both ORs type were functionally active, since DAMGO and U69593, MOR- and KOR- selective agonists respectively, and CTAP and nor-BNI, MOR- and KOR- selective antagonists respectively, were significantly able to modulate cell proliferation. Both opioid agonists, when used at the concentration of 1μM, inhibited cell proliferation of canine UCM-MSCs. Inhibitory effect on cell proliferation was also observed after CTAP treatment, whereas no effect was noticed after nor-BNI treatment. By specific stain and morphology analysis, no differences were observed in the neurogenic differentiation potency of UCM-MSCs in both treatments and control conditions. Collectively our data suggest that, in canine UCM-MSC, opioids modulate cell proliferation, but further studies are needed to evaluate whether opioid modulation may play a role in directing these cells to neurogenic lineages.

The purpose of this studywas to assess the natural exposure ofmale horses (Equus caballus) to the mycotoxin zearalenone (ZEA) by using the ELISA test and to evaluate the effects of in vitro exposure of sperm cells to mycotoxin-containing urine extracts on sperm chromatin structure stability. Because of their occurrence in urine samples, ZEA and its derivatives were tested by sperm chromatin structure assay (SCSA) at natural levels detected by ELISA. Thirty-eight urine extracts of Italian (n = 11) and northeastern European (n = 27) horses were tested on frozen-thawed spermatozoa to evaluate the toxic effect of mycotoxin on their chromatin structure by flow cytometry. Different parameters of theDNA fragmentation index (DFI), such as the mean (¯X DFI), the percentage (%-DFI), and the standard deviation (SD-DFI), were analyzed. Urine samples showed a mean level of 32.3 ng/mL ZEA with significantly higher concentrations in northeastern European samples than in Italian samples, probably in relation to climatic and feeding differences. The toxic effects of ZEA-containing urine samples on SCSA parameters were found at low ZEA concentrations and were mainly observed in Italian samples. By using mycotoxin standards, ZEA, a-zearalenol, and b-zearalenol proved to be more toxic compounds for sperm chromatin stability than other tested derivatives. A nongenomic mechanism of action can be hypothesized. # 2010 Elsevier Inc. All rights reserved.

Kisspeptin (Kp) and Kiss-1 receptor (Kiss-1R) expressions have been reported to be in the placenta, and a possible involvement of the Kiss-1R/Kps system in regulating trophoblast invasion and proliferation has been hypothesized. The aim of the present study was to investigate whether Kiss-1R activation by kisspeptin-10 (Kp-10) could modulate in vitro proliferation and progesterone (P4) secretion of bovine primary placental cell lines isolated from cotyledons of fetuses in the first trimester of pregnancy. The involvement of Kiss-1R in the cell responses observed was also analyzed. Uteri from cows at the first trimester of pregnancy were obtained from local abattoirs. Fetal cotyledon fragments were digested with collagenase in low glucose Dulbecco's Modified Eagle's Medium and cell lines were isolated. After being characterized for epithelial polygonal morphology, the presence of binucleate cells, male gender, and the expression of cytokeratin and zona occludens 2, cell lines were cultured in a low glucose Dulbecco's Modified Eagle's Medium–based expansion medium in the presence of 0.01, 0.1, 1, and 10 μM Kp-10. Control cells were cultured in the absence of Kp-10. Cell population doubling time was evaluated for each culture passage (P) from P1 to P10. Cells were tested for Kiss-1R mRNA expression analysis by real-time reverse transcription–polymerase chain reaction, and culture media were analyzed for P4 concentration by radioimmunoassay. Kisspeptin-10 modulated in vitro proliferation of epithelial cell lines isolated from cotyledons recovered from bovine fetuses in the first trimester of pregnancy. Inhibitory (line A) or stimulatory (line B) effects of Kp-10 on cell proliferation were found in different cell lines and observed cell responses were found to be related to Kiss-1R mRNA levels. Inhibition of cell proliferation matched with not significant variation of Kiss-1R expression, whereas stimulation of cell proliferation was found to be related to Kiss-1R upregulation. In both cell lines, no effect of Kp-10 on P4 secretion was found at any tested concentration. These results lead to the conclusion that the Kiss-1R/Kps system is involved in the regulation of cell proliferation of bovine placental cotyledon cell lines isolated at the first trimester of pregnancy but, at this gestational stage, it may not be involved in modulating placental P4 secretion.

Kisspeptin (Kp) and Kiss-1 receptor (Kiss-1R) expressions have been reported to be in the placenta, and a possible involvement of the Kiss-1R/Kps system in regulating trophoblast invasion and proliferation has been hypothesized. The aim of the present study was to investigate whether Kiss-1R activation by kisspeptin-10 (Kp-10) could modulate in vitro proliferation and progesterone (P4) secretion of bovine primary placental cell lines isolated from cotyledons of fetuses in the first trimester of pregnancy. The involvement of Kiss-1R in the cell responses observed was also analyzed. Uteri from cows at the first trimester of pregnancy were obtained from local abattoirs. Fetal cotyledon fragments were digested with collagenase in low glucose Dulbecco's Modified Eagle's Medium and cell lines were isolated. After being characterized for epithelial polygonal morphology, the presence of binucleate cells, male gender, and the expression of cytokeratin and zona occludens 2, cell lines were cultured in a low glucose Dulbecco's Modified Eagle's Medium-based expansion medium in the presence of 0.01, 0.1, 1, and 10 μM Kp-10. Control cells were cultured in the absence of Kp-10. Cell population doubling time was evaluated for each culture passage (P) from P1 to P10. Cells were tested for Kiss-1R mRNA expression analysis by real-time reverse transcription-polymerase chain reaction, and culture media were analyzed for P4 concentration by radioimmunoassay. Kisspeptin-10 modulated in vitro proliferation of epithelial cell lines isolated from cotyledons recovered from bovine fetuses in the first trimester of pregnancy. Inhibitory (line A) or stimulatory (line B) effects of Kp-10 on cell proliferation were found in different cell lines and observed cell responses were found to be related to Kiss-1R mRNA levels. Inhibition of cell proliferation matched with not significant variation of Kiss-1R expression, whereas stimulation of cell proliferation was found to be related to Kiss-1R upregulation. In both cell lines, no effect of Kp-10 on P4 secretion was found at any tested concentration. These results lead to the conclusion that the Kiss-1R/Kps system is involved in the regulation of cell proliferation of bovine placental cotyledon cell lines isolated at the first trimester of pregnancy but, at this gestational stage, it may not be involved in modulating placental P4 secretion.

Juvenile in vitro embryo production from oocytes of prepubertal subjects is a promising assisted reproductive technology (ART). In farm animals, it reduces the generation gap and in human reproductive medicine, it helps to overcome premature ovarian failure. Oxidative stress in germ cell in vitro culture and cryopreservation procedures is an emerging problem in ART and studies aimed to evaluate the potentially antioxidant activity of natural bioactive compounds, such as phenolic compounds and polyphenols, present in plant-derived by-products would be beneficial to improve cryopreservation and in vitro culture protocols of prepubertal oocytes. Verbascoside (VB) or acteoside is a phenolic bioactive compound with known antioxidant activity, which is present in a good amount in olive oil mill wastewater (1). The aim of this study was to test the effects of VB on the developmental competence of ovine prepubertal oocytes and the bioenergetic/oxidative stress status of fresh and vitrified oocytes. Previously published methods were used for: VB extraction, purification, HPLC analysis and uptake by the cumulus-oocyte complex (1); in vitro oocyte maturation (IVM) and assessment of bioenergy redox biomarkers, such as mitochondrial distribution pattern and activity; intracellular reactive oxygen species (ROS) levels; mitochondria/ROS colocalization; catalase and total superoxide dismutase activities (2); oocyte vitrification (3); in vitro fertilization and embryo development (4). VB effects were tested at microM concentrations (1.03, 2.06 and 4.11). In fresh oocytes, 4.11 microM VB exerted pro-oxidant short-term effects, i.e. catalase activity increase and uncoupled increases of ooplasmic mitochondria and reactive oxygen species (ROS) specific fluorescence signals. It also induced pro-oxidant long-term effects, i.e reduced blastocyst formation rate, thus indicating an affected developmental competence of exposed oocytes. In vitrified oocytes, 1.03 microM VB increased ROS levels. Pro-oxidant VB effects in ovine prepubertal oocytes could be related to higher VB accumulation, which was found as being almost one thousand times higher than that reported in other cell systems in previous studies. Also, long exposure times of oocytes to VB, throughout the duration of IVM culture, may have contributed to significantly increase oocyte oxidation. In conclusion, VB, added at microM concentrations in a continuative 24 hours IVM exposure protocol, acts as a pro-oxidant molecule by impairing bioenergetic potential, oxidative status and embryo developmental competence of prepubertal lamb oocytes. Further studies are ongoing aimed to identify suitable conditions, lower concentrations and/or shorter exposure times, to figure out VB antioxidant effects in juvenile ARTs.

Malassezia spp. may act as opportunistic skin pathogens in humans and animals. Malassezia pachydermatis proliferation and phospholipase production may play a pathogenic role in the occurrence of skin lesions in dogs. This study investigates the presence of mu-opioid receptor (MOR) in M. pachydermatis strains isolated from healthy dogs and dogs with skin lesions and its effects on phospholipase activity (p.a.). P.a. of 64 M. pachydermatis isolates was evaluated using different concentrations of naloxone (Nx), a MOR antagonist. Isolates were divided into Group A (i.e., 40 isolates from 26 dogs with dermatitis) and Group B (i.e., 24 isolates from 12 healthy dogs). The MOR expression was analyzed by Western blot and immunofluorescence. A statistically higher p.a. than that of the controls was found with isolates in Group A at a Nx concentration of 10(-6) M (P<0.05). No isolate in Group B displayed p.a. in either control samples or in the presence of any Nx concentration. Immunoblotting revealed two positive MOR immunoreactive bands of approximately 65 and 98 kDa. MOR expression and localization was also demonstrated by immunofluorescence in isolates from Groups A and B. This study provides the first evidence of MOR expression on M. pachydermatis cell membranes pointing to its possible role in modulating p.a. production in isolates from dogs with skin lesions

The primary objective of this study was to compare expression of maternal transcripts in bovine oocyte populations with differential developmental competence: oocytes from prepubertal and pubertal animals; and oocytes from small (3–4 mm) and large (6–10 mm) follicles from pubertal animals. All transcripts were examined in oocytes prior to and after in vitro maturation (IVM). Genes were selected based on their known maternal effect in mouse (ZAR1, STELLA, HSF1, MATER⁄ NLRP5 and its paralogue NLRP9), or their identification as markers of oocyte maturation, either involved in redox metabolism (PRDX1, PRDX2) or meiotic progression (AURKA). Total or polyadenylated forms of the transcripts were followed by reverse transcription coupled to real-time PCR. Six polyadenylated transcripts were found significantly reduced after maturation irrespective of donor age or follicle diameter (p < 0.05). Within these six polyadenylated transcripts, ZAR1, NLRP9, HSF1, PRDX1 and PRDX2 were significantly reduced in oocytes from prepubertal animals compared to adult animals (p < 0.05). A younger age was also associated with lower abundance (total form) of PRDX2⁄ PRDX1 irrespective of maturation. Total HSF1, PRDX1 and polyadenylated NLRP9 showed a tendency (p values from 0.053 to 0.08) for a higher detection in oocytes from small follicles, thus encouraging further investigation of the follicle diameter model. However, at the present time, follicle size did not significantly affect expression of transcripts examined. In conclusion, this study demonstrates differences in the maternal store of RNA and its regulation during IVM which is dependent on donor age.

There is no published information about follicular fluid leptin concentrations or presence of 25 leptin and leptin-receptor in the equine ovary or oocyte. Three groups of mares: adult draft 26 mares, draft fillies and adult Standardbred mares were included in the study. Leptin and 27 leptin-receptor were detected in all immature oocytes by immunofluorescence with 28 intensity that was higher in oocytes from draft mares compared to draft fillies and 29 Standardbred mares. After in vitro maturation a higher proportion of oocytes reached 30 metaphase II in draft mares than in draft fillies and Standardbred mares, and in all groups 31 both leptin and leptin-receptor became localized in the oocyte cortex but with higher 32 immunopositivity in draft mares compared to draft fillies and Standardbred mares. These 33 intensities were confirmed by expression profile of leptin and leptin-receptor mRNA. 34 Moreover, leptin was detected in ovarian blood vessels in all kind of animals and within the 35 corpora lutea in adult mares. Serum and follicular fluid concentrations of leptin were 36 similar in draft and Standardbred mares but higher in draft mares than in draft fillies. This 37 study supports the hypothesis that expression of leptin and leptin-receptor mRNA and the 38 rate of maturation can be related either to adiposity or to puberty.

Background: The present study investigates the effects of high external calcium concentration ([Ca2+]o) and the calcimimetic NPS R-467, a known calcium-sensing receptor (CaSR) agonist, on growth/proliferation of two equine size-sieved umbilical cord matrix mesenchymal stem cell (eUCM-MSC) lines. The involvement of CaSR on observed cell response was analyzed at both the mRNA and protein level. Methodology/Principal Findings: A large (.8 mm in diameter) and a small (,8 mm) cell line were cultured in medium containing: 1) low [Ca2+]o (0.37 mM); 2) high [Ca2+]o (2.87 mM); 3) NPS R-467 (3 mM) in presence of high [Ca2+]o and 4) the CaSR antagonist NPS 2390 (10 mM for 30 min.) followed by incubation in presence of NPS R-467 in medium with high [Ca2+]o. Growth/proliferation rates were compared between groups. In large cells, the addition of NPS R-467 significantly increased cell growth whereas increasing [Ca2+]o was not effective in this cell line. In small cells, both higher [Ca2+]o and NPS R-467 increased cell growth. In both cell lines, preincubation with the CaSR antagonist NPS 2390 significantly inhibited the agonistic effect of NPS R-467. In both cell lines, increased [Ca2+]o and/or NPS R-467 reduced doubling time values. Treatment with NPS R-467 down-regulated CaSR mRNA expression in both cell lines. In large cells, NPS R-467 reduced CaSR labeling in the cytosol and increased it at cortical level. Conclusions/Significance: In conclusion, calcium and the calcimimetic NPS R-467 reduce CaSR mRNA expression and stimulate cell growth/proliferation in eUCM-MSC. Their use as components of media for eUCM-MSC culture could be beneficial to obtain enough cells for down-stream purposes.

The oviduct isthmus is considered to be a sperm reservoir prior to ovulation in the reproductive tract of mammals. Ovarian steroids regulate the synthesis and secretion of specific molecules such as glycoproteins that are involved in the interactions between germ cells or embryos and oviductal epithelial cells. The objective of the present study was to investigate the effects on histmus glycoprotein pattern from hormonally stimulated ewes by means of lectin histochemistry. Isthmus fragments were separated from oviducts immediately after laparatomy, and, after fixed in 4% (w/v) neutral formalin, they were embedded in paraffin wax. Then, the sections were stained with a panel of lectins which revealed: 1) an increase of reactivity with MAL II, SNA, RCA120, SBA, GSA I-B4, GSA II, UEA I and LTA in the whole cytoplasm of ciliated and non-ciliated cells of hormonally treated females, 2) a reduction of DBA affinity in the luminal surface, 3) no staining pattern modification with PNA, KOH-sialidase (s)- PNA, HPA, Con A and KOH-s-WGA. These results indicate that exogenous gonadotropin administration for superovulation may alter the oligosaccharidic composition of glycoproteins produced in the ovine isthmus.

Phthalates are ubiquitous environmental contaminants because of their use in plastics and other common consumer products. Di-(2-ethylhexyl) phthalate (DEHP) is the most abundant phthalate and it impairs fertility by acting as an endocrine disruptor. The aim of the present study was to analyze the effects of in vitro acute exposure to DEHP on oocyte maturation, energy and oxidative status in the horse, a large animal model. Cumulus cell (CC) apoptosis and oxidative status were also investigated. Cumulus-oocyte complexes from the ovaries of slaughtered mares were cultured in vitro in presence of 0.12, 12 and 1200 µM DEHP. After in vitro maturation (IVM), CCs were removed and evaluated for apoptosis (cytological assessment and TUNEL) and intracellular reactive oxygen species (ROS) levels. Oocytes were evaluated for nuclear chromatin configuration. Matured (Metaphase II stage; MII) oocytes were further evaluated for cytoplasmic energy and oxidative parameters. DEHP significantly inhibited oocyte maturation when added at low doses (0.12 µM; P<0.05). This effect was related to increased CC apoptosis (P<0.001) and reduced ROS levels (P<0.0001). At higher doses (12 and 1200 µM), DEHP induced apoptosis (P<0.0001) and ROS increase (P<0.0001) in CCs without affecting oocyte maturation. In DEHP-exposed MII oocytes, mitochondrial distribution patterns, apparent energy status (MitoTracker fluorescence intensity), intracellular ROS localization and levels, mt/ROS colocalization and total SOD activity did not vary, whereas increased ATP content (P<0.05), possibly of glycolytic origin, was found. Co-treatment with N-Acetyl-Cysteine reversed apoptosis and efficiently scavenged excessive ROS in DEHP-treated CCs without enhancing oocyte maturation. In conclusion, acute in vitro exposure to DEHP inhibits equine oocyte maturation without altering ooplasmic energy and oxidative stress parameters in matured oocytes which retain the potential to be fertilized and develop into embryos even though further studies are necessary to confirm this possibility.

In vitro matured oocytes (IVM) suffer some inadequacies when compared with in vivo matured ones1. Inadequate IVM can yield under- or overmature oocytes, which will not undergo normal fertilization and embryo development2. Glycoconjugates play a key role in oocyte maturation, and in oocyte-sperm interactions leading to fertilization 3,4, thus the knowledge of oligosaccharide pattern of equine COCs could provide useful information about the comparison between immature and matured COCs. Cumulus enclosed oocytes from abbattoir ovaries were fixed in Bouin’s solution and embedded in paraffin wax either before or after IVM. Sections were stained with 13 lectin (MAL II, SNA, PNA, DBA, RCA120, SBA, HPA, Con A, WGA, GSA I-B4, GSA II, UEA I, LTA). The radiata zone of immature COCs reacted with all used lectins, whereas matured COCs stained with MAL II, SNA, HPA, SBA, and Con A. The zona pellucida of both COCs types bound MAL II, SNA, SBA, and Con A, whereas immature COCs reacted also with RCA120, WGA, and matured ones stained with UEA I. The ooplasm of both types of COCs reacted with HPA, Con A, GSA II, UEA I and LTA, whereas immature oocytes bound also SNA, SBA, WGA, GSA I-B4. These results indicate that IVM modifies glycoprotein pattern of equine COCs and prompted us to undergo further studies to investigate the role of the modified oligosaccharides in oocyte viability, capacity to undergo fertilization and normal embryonic development. References 1. Deleuze S et al Theriogenology. 2009, 72:203-9. 2. Hinrichs K & Di Giorgio LM J Reprod Fertil Suppl 1991, 44:369–74. 3. Dell A et al Biochim Biophys Acta 1999, 1473:196-205. 4. Clark GF & Dell A. J Biol Chem 2006, 281:13853-6.

The calcium sensing receptor (CaSR) plays a key role in cells involved in calcium (Ca2+) homeostasis by directly sensing changes in extracellular Ca2+ concentration ([Ca2+]o), and external Ca2+ is a potent mediator of cell proliferation. The present study investigated the effects of high [Ca2+]o and of the CaSR agonist NPS R-467 on growth and proliferation of equine size-sieved umbilical cord matrix mesenchymal stem cells (UCM-MSC). The involvement of CaSR on observed cell response was analysed at the mRNA and protein level. Two subpopulations of UCM-MSC, isolated using multi-dishes with transwell inserts of 8-μm pores and expressing MSC markers (CD105, CD44, CD29; Corradetti et al. 2010 Reprod. Fertil. Dev. 22, 347–348), were analysed. Cells were cultured in medium containing: (A) low [Ca2+]o (0.37 mM), (B) high [Ca2+]o (2.87 mM), (C) NPS R-467 (3 μm) in the presence of high [Ca2+]o, and (D) the CaSR antagonist NPS 2390 (10 μm for 30′) followed by NPS R-467 in the presence of high [Ca2+]o. Growth and proliferation rates were compared among treatments (Student’s t-test). The CaSR expression and subcellular localization were investigated by real-time quantitative RT-PCR, immunofluorescence, and confocal microscopy. In the &gt;8-μm cell line, the addition of NPS R-467, in the presence of [Ca2+]o, significantly increased cell growth after day 7 of culture (C v. A and B; P &lt; 0.001). Increasing [Ca2+]o was not effective in this cell line (B v. A; not significant). In the &lt;8-μm cell line, NPS R-467 increased cell growth, even at a lower extent (C v. A; P &lt; 0.05), as observed on day 9 of culture. In this cell line, an increased proliferation rate was observed upon [Ca2+]o increase (B v. A; P &lt; 0.05). In both cell lines, preincubation with NPS 2390 significantly inhibited the agonistic effect of NPS R-467. In both cell lines, a stimulatory effect of additional calcium and NPS R-467 on cell proliferation, in terms of reduced DT values, was observed. In the 2 cell lines, CaSR expression was down-regulated in the presence of high calcium and in NPS R-467-treated cells compared with controls (B and C v. A cells; P &lt; 0.001). Treatment with high calcium or NPS R-467 reduced CaSR labelling in the cytosol and increased it at the cortical level. We found that CaSR is expressed at mRNA and protein levels in equine UCM-MSC, and it is functionally active because the selective CaSR agonist NPS R-467 induced a stimulatory effect on cell growth and proliferation, which was reversed by the CaSR antagonist NPS 2390. The different responses to treatments between the 2 UCM-MSC subpopulations suggest that CaSR could be differentially activated in these cell lines. The calcimimetic NPS R-467 might be useful as an adjunctive component of media for UCM-MSC culture to obtain enough cells for down-stream purposes.

The calcium sensing receptor (CaSR) plays a key role in cells involved in calcium (Ca2+) homeostasis by directly sensing changes in extracellular Ca2+ concentration ([Ca2+]o), and external Ca2+ is a potent mediator of cell proliferation. The present study investigated the effects of high [Ca2+]o and of the CaSR agonist NPS R-467 on growth and proliferation of equine size-sieved umbilical cord matrix mesenchymal stem cells (UCM-MSC). The involvement of CaSR on observed cell response was analysed at the mRNA and protein level. Two subpopulations of UCM-MSC, isolated using multi-dishes with transwell inserts of 8-μm pores and expressing MSC markers (CD105, CD44, CD29; Corradetti et al. 2010 Reprod. Fertil. Dev. 22, 347–348), were analysed. Cells were cultured in medium containing: (A) low [Ca2+]o (0.37 mM), (B) high [Ca2+]o (2.87 mM), (C) NPS R-467 (3 μm) in the presence of high [Ca2+]o, and (D) the CaSR antagonist NPS 2390 (10 μm for 30′) followed by NPS R-467 in the presence of high [Ca2+]o. Growth and proliferation rates were compared among treatments (Student’s t-test). The CaSR expression and subcellular localization were investigated by real-time quantitative RT-PCR, immunofluorescence, and confocal microscopy. In the &gt;8-μm cell line, the addition of NPS R-467, in the presence of [Ca2+]o, significantly increased cell growth after day 7 of culture (C v. A and B; P &lt; 0.001). Increasing [Ca2+]o was not effective in this cell line (B v. A; not significant). In the &lt;8-μm cell line, NPS R-467 increased cell growth, even at a lower extent (C v. A; P &lt; 0.05), as observed on day 9 of culture. In this cell line, an increased proliferation rate was observed upon [Ca2+]o increase (B v. A; P &lt; 0.05). In both cell lines, preincubation with NPS 2390 significantly inhibited the agonistic effect of NPS R-467. In both cell lines, a stimulatory effect of additional calcium and NPS R-467 on cell proliferation, in terms of reduced DT values, was observed. In the 2 cell lines, CaSR expression was down-regulated in the presence of high calcium and in NPS R-467-treated cells compared with controls (B and C v. A cells; P &lt; 0.001). Treatment with high calcium or NPS R-467 reduced CaSR labelling in the cytosol and increased it at the cortical level. We found that CaSR is expressed at mRNA and protein levels in equine UCM-MSC, and it is functionally active because the selective CaSR agonist NPS R-467 induced a stimulatory effect on cell growth and proliferation, which was reversed by the CaSR antagonist NPS 2390. The different responses to treatments between the 2 UCM-MSC subpopulations suggest that CaSR could be differentially activated in these cell lines. The calcimimetic NPS R-467 might be useful as an adjunctive component of media for UCM-MSC culture to obtain enough cells for down-stream purposes.

The possibility to isolate canine mesenchymal stem cells (MSCs) from foetal adnexa is interesting since several canine genetic disorders are reported to resemble similar dysfunctions in humans. In this study, we successfully isolated, cytogenetically and molecularly characterized, and followed the differentiation potency of canineMSCs from foetal adnexa, such as amniotic fluid (AF), amniotic membrane (AM), and umbilical cord matrix (UCM). In the three types of cell lines, the morphology of proliferating cells typically appeared fibroblastlike, and the population doubling time (DT) significantly increased with passage number. For AF- and AM-MSCs, cell viability did not change with passages. In UCM-MSCs, cell viability remained at approximately constant levels up to P6 and significantly decreased from P7 (P<0.05).Amnion andUCM-MSCs expressed embryonic and MSC markers, such as Oct- 4 CD44, CD184, and CD29, whereas AF-MSCs expressed Oct-4, CD44. Expression of the hematopoietic markers CD34 and CD45 was not found. Dog leucocyte antigens (DLADRA1 and DLA-79) were expressed only in AF-MSCs at P1. Isolated cells of the three cell lines at P3 showed multipotent capacity, and differentiated in vitro into neurocyte, adipocyte, osteocyte, and chondrocyte, as demonstrated by specific stains and expression of molecular markers. Cells at P4 showed normal chromosomal number, structure, and telomerase activity. These results demonstrate that, in dog, MSCs can be successfully isolated from foetal adnexa and grown in vitro. Their proven stemness and chromosomal stability indicated that MSCs could be used as a model to study stem cell biology and have an application in therapeutic programs.

The possibility to isolate canine mesenchymal stem cells (MSCs) from foetal adnexa is interesting since several canine genetic disorders are reported to resemble similar dysfunctions in humans. In this study, we successfully isolated, cytogenetically and molecularly characterized, and followed the differentiation potency of canine MSCs from foetal adnexa, such as amniotic fluid (AF), amniotic membrane (AM), and umbilical cord matrix (UCM). In the three types of cell lines, the morphology of proliferating cells typically appeared fibroblast-like, and the population doubling time (DT) significantly increased with passage number. For AF- and AM-MSCs, cell viability did not change with passages. In UCM-MSCs, cell viability remained at approximately constant levels up to P6 and significantly decreased from P7 (P < 0.05). Amnion and UCM-MSCs expressed embryonic and MSC markers, such as Oct-4, CD44, CD184, and CD29, whereas AF-MSCs expressed Oct-4, CD44. Expression of the hematopoietic markers CD34 and CD45 was not found. Dog leucocyte antigens (DLA-DRA1 and DLA-79) were expressed only in AF-MSCs at P1. Isolated cells of the three cell lines at P3 showed multipotent capacity, and differentiated in vitro into neurocyte, adipocyte, osteocyte, and chondrocyte, as demonstrated by specific stains and expression of molecular markers. Cells at P4 showed normal chromosomal number, structure, and telomerase activity. These results demonstrate that, in dog, MSCs can be successfully isolated from foetal adnexa and grown in vitro. Their proven stemness and chromosomal stability indicated that MSCs could be used as a model to study stem cell biology and have an application in therapeutic programs.

BACKGROUND: Infertility affects ~10-15% of couples trying to have children, in which the rate of male fertility problems is approximately at 30-50%. Copy number variations (CNVs) are DNA sequences greater than or equal to 1 kb in length sharing a high level of similarity, and present at a variable number of copies in the genome; in our study, we used the canine species as an animal model to detect CNVs responsible for male infertility. We aim to identify CNVs associated with male infertility in the dog genome with a two-pronged approach: we performed a sperm analysis using the CASA system and a cytogenetic-targeted analysis on genes involved in male gonad development and spermatogenesis with fluorescence in situ hybridization (FISH), using dog-specific clones. This analysis was carried out to evaluate possible correlations between CNVs on targeted genes and spermatogenesis impairments or infertility factors. RESULTS: We identified two genomic regions hybridized by BACs CH82-321J09 and CH82-509B23 showing duplication patterns in all samples except for an azoospermic dog. These two regions harbor two important genes for spermatogenesis: DNM2 and TEKT1. The genomic region encompassed by the BAC clone CH82-324I01 showed a single-copy pattern in all samples except for one dog, assessed with low-quality sperm, displaying a marked duplication pattern. This genomic region harbors SOX8, a key gene for testis development. CONCLUSION: We present the first study involving functional and genetic analyses in male infertility. We set up an extremely reliable analysis on dog sperm cells with a highly consistent statistical significance, and we succeeded in conducting FISH experiments on sperm cells using BAC clones as probes. We found copy number differences in infertile compared with fertile dogs for genomic regions encompassing TEKT1, DNM2, and SOX8, suggesting those genes could have a role if deleted or duplicated with respect to the reference copy number in fertility biology. This method is of particular interest in the dog due to the recognized role of this species as an animal model for the study of human genetic diseases and could be useful for other species of economic interest and for endangered animal species.

Analyses of energy and redox status parameters are emerging technologies to improve oocyte quality assessment. Mitochondria (mt) play a vital role in the oocyte to support maturation, fertilization, and pre-implantation development. They are the major source of reactive oxygen species (ROS) produced during oxidative phosphorylation, which are not only by-products of cell metabolism but also important molecules for regulation of intracellular cell signaling. The aim of the present study was to test for mt/ROS colocalization in oocytes recovered from superovulated adult ewes and examined after in vivo or in vitro maturation (IVM). Cumulus–oocyte complexes of 8 superovulated (fluorogestone acetate þ D-cloprostenol for oestrus synchronization, pFSH/pLH and eCG for superovulation) adult (2 to 8 years of age) ewes were recovered (ovariohysterectomy by midventral laparotomy performed 54 h after vaginal sponge removal) either from flushing oviducts (oviducal oocytes) or from ovarian growing follicles (1–5 mm in diameter; follicular oocytes). Follicular oocytes were analysed after IVM (Ambruosi et al. 2009 Theriogenology 71, 1093–1104). After cumulus cell removal, all oocytes underwent nuclear chromatin, mt, and ROS evaluation. Hoechst 33258 and Mitotracker Orange CMTM Ros were used to label nuclear chromatin and mt (Ambruosi et al. 2009) and 20,70-dichloro-dihydro-fluorescein diacetate was used for ROS labelling (Hashimoto et al. 2000 Mol. Reprod. Dev. 57, 353–360). Oocytes at the metaphase II (MII) stage showing regular ooplasmic size (4130 mm in diameter) and morphology were selected for confocal analysis of mt/ROS fluorescence distribution, intensity, and colocalization. Forty oviducal MII oocytes recovered from 8 ewes were analysed. Thirty-two oocytes recovered from the ovaries of 4 ewes underwent IVM, and 23 out of 32 (72%) reached nuclear maturation and were analysed. The rate of oocytes showing perinuclear mt distribution pattern did not differ between oviducal and IVM oocytes (33%, 13/40 v. 43%, 10/23; not significant). In these oocytes, fluorescent intensity of mt labelling and intracellular ROS levels did not differ between oviducal and IVM ooocytes (996.27 ` 363.57 v. 798.13 ` 275.91; not significant; and 1808.11 ` 442.78 v. 1473.29 ` 662.49, for mt and ROS, respectively; not significant), whereas mt/ROS colocalization was significantly higher in ovulated oocytes than in IVM oocytes (Pearson coefficient 0.67 ` 0.11 v. 0.39 ` 0.19, respectively; P o 0.001). In conclusion, in oocytes of adult ewes, mt aggregation, apparent energy status, and intracellular ROS levels do not differ between ovulated and IVM oocytes, but mt/ROS colocalization differs between the 2 groups. As it was reported for other cell systems that such a difference can be indicative of healthy status of ovulated oocytes, we suggest that mt/ROS colocalization could be considered as a suitable marker of oocyte quality.

This study investigates the mitochondrial (mt) distribution in canine ovarian oocytes examined at recovery time, as related to the reproductive cycle stage, and in oviductal oocytes. Ovarian Germinal Vesicle (GV) stage oocytes were recovered from bitches in anestrous (A, n = 2), follicular phase (F, n = 4), ovulation (0, n = 2), early luteal (EL, n = 7) and mid/late luteal phase (MILL, n = 2). Oviductal GV, metaphase I (MI) or MII stage oocytes were recovered from six bitches between 56 and 110 h after ovulation. Mitochondria were revealed by using MitoTracker Orange CMTM Ros and confocal microscopy. In ovarian oocytes, three mt distribution patterns were found: (1) small aggregates diffused throughout the cytoplasm; (11) diffused tubular networks; (III) pericortical tubular networks. Significantly higher rates of oocytes showing heterogeneous mt patterns (II + III) were obtained from bitches in F (75%) and in O (96%) compared with bitches in A (31%; F vs. A: P<0.05; O vs. A: P<0.001), in EL (61%; O vs. EL P<0.01), or in MLL (0%; F vs. MLL: P<0.05; O vs. MLL: P<0.001). Fluorescence intensity did not vary according to mt distribution pattern except that it was lower in oocytes recovered in EL phase and showing small mt aggregations (P<0.001). The majority of ovulated MII stage oocytes (79%) showed diffused tubular mt network. We conclude that mt distribution pattern of canine ovarian immature oocytes changes in relation to reproductive cycle stage and that patterns observed in oocytes recovered from bitches in periovulatory phases are heterogeneous and similar to those of in vivo matured oocytes. (C) 2009 Elsevier B.V. All rights reserved.

Metastatic cells are highly plastic for differential expression of tumor phenotype hallmarks and metastatic organotropism. The signaling proteins orchestrating the shift of one cell phenotype and organ pattern to another are little known. Na(+)/H(+) exchanger regulatory factor (NHERF1) is a molecular pathway organizer, PDZ-domain protein that recruits membrane, cytoplasmic, and cytoskeletal signaling proteins into functional complexes. To gain insight into the role of NHERF1 in metastatic progression, we stably transfected a metastatic breast cell line, MDA-MB-231, with an empty vector, with wild-type NHERF1, or with NHERF1 mutated in either the PDZ1- or PDZ2-binding domains to block their binding activities. We observed that NHERF1 differentially regulates the expression of two phenotypic programs through its PDZ domains, and these programs form the mechanistic basis for metastatic organotropism. The PDZ2 domain promotes visceral metastases via increased invadopodia-dependent invasion and anchorage-independent growth, as well as by inhibition of apoptosis, whereas the PDZ1 domain promotes bone metastases by stimulating podosome nucleation, motility, neoangiogenesis, vasculogenic mimicry, and osteoclastogenesis in the absence of increased growth or invasion. Collectively, these findings identify NHERF1 as an important signaling nexus for coordinating cell structure with metastatic behavior and identifies the "mesenchymal-to-vasculogenic" phenotypic transition as an essential step in metastatic progression.

Extracellular matrix (ECM) degradation is a critical process in tumor cell invasion and requires membrane and released proteases focalized at membrane structures called invadopodia. While extracellular acidification is important in driving tumor invasion, the structure/function mechanisms underlying this regulation are still unknown. Invadopodia are similar in structure and function to osteoclast podosomes responsible for bone degradation, and extracellular acidification is central to podosome action, suggesting that it could also be for invadopodial function. Here, utilizing a novel system for in situ zymography in native matrices, we show that the Na(+)/H(+) exchanger (NHE1) and NHE1-generated extracellular acidification are localized at and necessary for invadopodial-dependent ECM degradation, thereby promoting tumor invasion. Stimulation with EGF increased both NHE1-dependent proton secretion and ECM degradation. Manipulation of the NHE1 expression by RNA interference or activity via either transport-deficient mutation or the specific inhibitor cariporide confirmed that NHE1 expression and activity are required for invadopodia-mediated ECM degradation. Taken together, our data show a concordance among NHE1 localization, the generation of a well-defined acidic extracellular pH in the nanospace surrounding invadopodia, and matrix-degrading activity at invadopodia of human malignant breast carcinoma cells, providing a structural basis for the role of NHE1 in invasion and identifying NHE1 as a strategic target for therapeutic intervention.-Busco, G., Cardone, R. A., Greco, M. R., Bellizzi, A., Colella, M., Antelmi, E., Mancini, M. T., Dell'Aquila, M. E., Casavola, V., Paradiso, A., Reshkin, S. J. NHE1 promotes invadopodial ECM proteolysis through acidification of the peri-invadopodial space

Ochratoxin A (OTA) exposure during pregnancy in laboratory animals induces delayed/abnormal embryo development. Foetal adnexa-derived mesenchymal stem cells (MSCs) could help evaluate the developmental risk of exposure to chemicals in advanced gestational age. We tested the effects of OTA at concentrations ranging from 2.5×10-4 to 25nM on growth parameters of canine umbilical cord matrix (UCM)-derived MSCs. The hypothesis that oxidative chromatin and DNA damage could underlie OTA-mediated cell toxicity was also investigated. After in vitro exposure, OTA significantly decreased cell density and increased doubling time in a passage- and concentration-dependent manner and no exposed cells survived beyond passage 5. Significantly higher rates of cells showed condensed and fragmented chromatin and oxidized DNA, as assessed by OxyDNA assay. These findings showed that in vitro exposure to OTA, at picomolar levels, perturbs UCM-MSC growth parameters through oxidative chromatin and DNA damage, suggesting possible consequences on canine foetal development.

A crucial component of Assisted Reproductive Technologies (ART) is the assessment of oocyte developmental potential, to allow selection of those oocytes most likely to result in fertilization and pregnancy. Currently, oocyte quality assessment is largely based on the morphological appearance of the cumulus-oocyte complex, however the accuracy of morphological methods, as predictive of oocyte competence, is still suboptimal. Therefore, the development of objective, accurate, fast and reliable tests for assessing oocyte developmental potential remains an important aim of human and veterinary reproductive medicine. (9pt) The process of oocyte meiotic maturation, which is central to the developmental competence of the oocyte, is regulated by numerous genes (Matzuk & Lamb, 2008; Fauser et al., 2011) and protein pathways (Kubiak, 2011) and is accompanied by significant changes within the oocyte at many levels. Better understanding of oocyte meiotic maturation would allow better support of this process to increase the success of reproductive biotechnologies, and thus overcome some forms of infertility. (9pt) Recently, global assessment strategies, namely OMICS, investigating genomic, transcriptomic, proteomic, lipidomic, and glycomic profiles of oocytes, cumulus or granulosa cells have become increasingly applied to the study of oocyte physiology and pathology. Also being investigated is the oocyte-cumulus metabolome, via measurements of metabolites in biological fluids, such as follicular or tubal fluid, or in culture media. The establishment of these technologies, which are in their initial stages of application to reproductive biology, can require large sample numbers; only animal models can meet this requirement. Because of their wide availability and the body of existing knowledge regarding their biology, oocytes of large animals provide useful models for investigating the relationship between oocyte developmental competence and OMICS biomarkers. This review summarizes recent literature on the application of OMICS strategies to evaluating developmental competence of human oocytes and oocytes of large animals. (9pt) Among the available animal models, the mare is uniquely applicable to investigation of oocyte developmental competence. Horses represent the most economically valuable domestic animal, with progeny from specific mares worth hundreds of thousands of euros. Thus, there is obvious practical interest in the use of assisted reproduction in this species. In addition, similarities between mare and human follicle growth and oocyte maturation make the mare a particularly valuable model for topics at the interface between animal breeding and biomedical research, such as age-related and obesity-related oocyte dysfunction and the effects of exposure to environmental toxicants, as well as for fundamental research on factors involved in meiotic maturation. For these reasons, particular attention will be dedicated in this review to recent OMICS results obtained in the equine species and to discussion of the potential application of this animal model in future investigations.

OBJECTIVE: To analyze within-/between-subject, in vivo versus in vitro maturation (IVM), and age-related variations of mitochondrial (mt) bioenergy potential and oxidative status of metaphase II (MII) oocytes recovered from hormonally stimulated sheep. DESIGN: Prospective study. SETTING: Academic basic research laboratory. SUBJECT(S): Ten adult ewes. INTERVENTION(S):Estrus synchronization, controlled ovarian hyperstimulation (COH), ovariohysterectomy; follicular and oviductal oocyte retrieval; IVM of follicular oocytes. MAIN OUTCOME MEASURE(S): Mean ± SD, within-subject (CV(w)) and between-subject (CV(b)) variation coefficients of mt activity, intracellular reactive oxygen species (ROS) levels, and mt/ROS colocalization in sheep oocytes from young and aged donors and matured in vivo (in vivo MIIs) or in vitro (IVM MIIs). RESULT(S): Within- and between-subject, in vivo versus IVM, and age-related variations of mt activity were observed in MII oocytes from hormonally stimulated donor sheep. ROS levels increased significantly in oocytes from aged donors. Mt-ROS colocalization was consistently higher in in vivo MIIs compared with IVM MIIs. Oviductal energy/antioxidant ability is influenced by COH. CONCLUSION(S): Oocyte energy/oxidative status is affected by within-/between-subject, in vivo versus IVM, and age-related variations. Mt/ROS colocalization is a reliable marker of in vivo MII oocytes.

Verbascoside (VB) is a bioactive compound from olive oil mill wastewater with known antioxidant activity. Oxidative stress in germ cell in vitro culture and cryopreservation procedures is an emerging problem in assisted reproductive technology (ART). Juvenile in vitro embryo production from oocytes of prepubertal subjects is a promising ART because, in farm animals, it reduces the generation gap and in human reproductive medicine, it helps to overcome premature ovarian failure. The aim of this study was to test the effects of VB on the developmental competence of ovine prepubertal oocytes and the bioenergetic/oxidative stress status of fresh and vitrified oocytes. In fresh oocytes, when used at high concentration, VB exerted pro-oxidant short-term effects, i.e. catalase activity increase and uncoupled increases of ooplasmic mitochondria and reactive oxygen species (ROS) specific fluorescence signals and long-term effects, i.e reduced blastocyst formation rate. In vitrified oocytes, it increased ROS levels. Pro-oxidant VB effects in ovine prpubertal oocytes could be related to higher VB accumulation, which was found as being almost one thousand times higher than that reported in other cell systems in previous studies. Also, long exposure times of oocytes to VB, throughout the duration of in vitro maturation (IVM) culture, may have contributed to significantly increase oocyte oxidation. Further studies are needed to identify suitable conditions, lower concentrations and/or shorter exposure times, to figure out VB antioxidant effects in juvenile ARTs.

Immature equine oocytes may be held overnight in an Earle's/Hanks' M199-based medium in the absence of meiotic inhibitors (EH medium) to schedule the onset of in vitro maturation. Holding in EH has been shown not to affect meiotic or developmental competence of equine oocytes (Choi et al. 2006 Theriogenology 66, 955-963). However, no studies have been performed to identify the mode by which this medium suppresses meiosis. We hypothesised that holding temperature may affect oocyte meiotic arrest. The effect of 3 holding temperatures (25, 30, 38°C) on chromatin status was investigated after Hoechst 33258 staining (Hinrichs et al. 2005 Biol. Reprod. 72, 1142-1150). Oocytes were recovered by scraping of follicles from slaughterhouse-derived ovaries. Data were analysed by Chi-squared test and one-way ANOVA followed by Dunn's or Holm-Sidak Multiple Comparison methods. A level of P<0.05 was considered significant. There were no significant differences in chromatin configuration between oocytes held overnight at 25°C (25°C-held) and controls (immediately-fixed oocytes); the proportion of oocytes showing meiotic resumption was 1/27, 4% and 0/26, 0%, respectively (not significant, NS). In contrast, holding at higher temperature significantly increased meiosis resumption (14/38, 37% and 14/28, 50%, at 30 and 38°C, respectively; P<0.01) and reduced the proportion of oocytes showing the most meiotically-competent germinal-vesicle (GV) configuration (condensed chromatin, CC; 24 to 29% v. 65 to 70% for control and 25°C-held, respectively; P<0.05). Based on these results, a subsequent experiment was performed in which oocyte meiotic stage and mitochondrial (mt) potential of 25°C-held (n=29) and control (n=36) oocytes was evaluated. Nuclear chromatin, mt activity (MitoTracker orange), intracellular reactive oxygen species (ROS) levels (2',7'-dichlorodihydrofluorescein diacetate, DCDHFDA), and mt/ROS colocalization (Pearson's coefficient) were analysed by epifluoscence and confocal microscopy (Martino et al. 2012 Fertil. Steril. 97, 720-728). Meiotic arrest after EH treatment at 25°C was confirmed (0/29, 0% v. 5/36, 14% for meiotic resumption in 25°C-held and controls, respectively; NS). At any GV stage, 25°C-held treatment had no effect on mt activity, ROS levels, or mt/ROS colocalization. For example, in CC oocytes, values for control and 25°C-held, respectively, were: MitoTracker, 547.8±499.5 v. 722.9±390.3; DCF fluorescence intensity, 278.5±179.3 v. 378±185, and mt/ROS colocalization, 0.5±0.1 v. 0.5±0.2; these were not significantly different (NS). In conclusion, EH holding at 25°C maintains meiotic arrest, viability, and mt potential of equine oocytes.

Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium species that exerts its toxic effect through interference with the sphingolipid pathway by inhibiting ceramide synthase. A FB1-dependent sperm toxicity was reported in boars. No information on FB1-related reproductive toxicity in stallions, the most sensitive animal species, has been reported. The aim of the present study was to assess the in vitro toxicity of FB1 on fresh and frozen-thawed equine spermatozoa by analyzing sperm viability, chromatin stability (SCSA) and reactive oxygen species (ROS) production by flow cytometry and sperm motility by CASA system. Fumonisin B1 did not affect viability of fresh spermatozoa after 2h exposure up to 25 μM. Damage on sperm chromatin structure was observed only in one frozen sample after exposure up to 2.5 x 10⁻⁵ μM FB1 without associated increase of ROS. Increase of ROS, at FB1 levels up to 2.5 x 10⁻⁴ μM, was found on another frozen-thawed sperm sample, may be as a consequence of seminal plasma removal. At 7.5 and 15 μM, FB1 induced reduction of total and progressive motility.

Verbascoside (VB), a bioactive polyphenol from olive mill wastewater with known antioxidant activity, was shown to act as a pro-oxidant molecule, by impairing energy/redox status and embryo developmental competence of prepubertal ovine oocytes when added at micromolar concentrations in a continuative 24-h in vitro maturation (IVM) exposure protocol (1). The aim of the present study was to determine whether a lower (nanomolar) VB concentration and a shorter exposure time (2 v. 24h) during IVM may improve the maturation rates of prepubertal ovine oocytes and their subsequent embryonic development in vitro. Cumulus-oocyte complexes derived from the ovaries of slaughtered 1-mo-old prepubertal sheep oocytes underwent IVM in TCM 199 with 10% oestrus sheep serum, 0.1IUmL(-1) of FSH/LH, and 100µM cysteamine, in 5% CO2 in air at 38.5°C for 24h. Based on our previous results (Dell'Aquila et al. 2014 Biomed. Res. Int. 2014, 878062), VB was added in the IVM medium at 1.03nM, and 2 incubation times (24 and 2h) were tested. In the 2-h exposure group, after 2h of exposure to VB, oocytes were washed and cultured up to 24h without VB. A group of oocytes were cultured in absence of VB, as controls. Matured oocytes were fertilized with frozen-thawed ram semen in SOF medium for 22h and zygotes were cultured in vitro for 8 days. Metaphase II (MII) cleavage and blastocyst rates were analysed by Chi-squared test. Embryo quality was evaluated by staining and total cell count of the blastocyst and analysis of variance (ANOVA) was applied. Differences were considered to be significant when P<0.05. Compared to controls, VB treatment at the concentration of 1.03nM and 24h of exposure had no effect on MII rates (196/268, 73% v. 226/323, 70% MII/cultured oocytes; P>0.05). However, this treatment allowed to obtain significantly higher rates of cleaved embryos/MII oocytes (156/196, 80% v. 165/226, 73%; respectively; P<0.05), blastocyst yield/cleaved embryos (59/156, 38% v. 45/165, 27%, respectively; P<0.05), and total blastocyst cell numbers (108.62±19.87 v. 89.61±26.32, respectively; P<0.05) compared to control oocytes. The VB treatment at the same concentration but for 2h induced only significantly higher cleavage rate (196/210, 93% v. 165/226, 73%; P<0.05). In conclusion, our results showed that VB treatment at 1.03nM during 24h of IVM exerted a positive effect on in vitro embryo development of prepubertal ovine oocytes by increasing the blastocyst yield and their quality. The hypothesis that VB at nanomolar concentrations may improve cumulus-oocyte energy/redox status is under investigation.

BACKGROUND: The aim of this study was to evaluate the effects of vitrification on morpho-functional parameters (blastomere/chromatin integrity and bioenergy/oxidative potential) of mouse preimplantation embryos. METHODS: In vivo produced mouse (4/16-cell, morulae and blastocyst-stage) embryos were randomly divided into vitrification and control groups. For vitrification, embryos were exposed to a 2-step loading of ethylene glycol and propylene glycol, before being placed in a small nylon loop and submerged into liquid nitrogen. After warming, the cryoprotectants were diluted by a 3-step procedure. Embryo morphology, chromatin integrity and energy/oxidative status were compared between groups. RESULTS: Vitrification induced low grade blastomere cytofragmentation (P < 0.05) and low chromatin damage only in embryos at the morula stage (P < 0.001). Mitochondrial (mt) distribution pattern was affected by vitrification only in early embryos (P < 0.001). Mitochondrial activity did not change upon vitrification in morula-stage embryos but it was reduced in blastocyst-stage embryos (P < 0.05). Intracellular ROS levels significantly increased in embryos at the morula and blastocyst stages (P < 0.001). Colocalization of active mitochondria and ROS increased only in vitrified blastocysts. CONCLUSIONS: In conclusion, this study elucidates the developmentally-related and mild effects of vitrification on morphology, nuclear and bioenergy/oxidative parameters of mouse embryos and demonstrates that vitrification is a suitable method for preserving predictive parameters of embryo ability to induce a full-term pregnancy.