Cerium oxide nanoparticles (CeO2 NPs) improve the developmental competence of in vitro-matured prepubertal ovine oocytes.

The present study investigated whether supplementation with different doses of cerium dioxide nanoparticles (CeO2 NPs) during in vitro maturation (IVM) of prepubertal ovine oocytes influenced their embryonic development in vitro. Cumulus-oocyte complexes derived from the ovaries of slaughtered prepubertal sheep underwent IVM with CeO2NPs (0, 44, 88 or 220µg mL-1). Matured oocytes were fertilised in vitro and zygotes were cultured for 7 days. The results demonstrated that CeO2NPs were internalised in the cumulus cells and not in the oocyte. The treatment with CeO2NPs did not affect nuclear maturation or intracellular levels of reactive oxygen species of the oocytes. The percentage of oocytes with regular chromatin configuration and cytoskeleton structures when treated with 44µg mL-1 CeO2NPs was similar to oocytes matured in the absence of CeO2NPs and significantly higher than those treated with 88 or 220µg mL-1 CeO2NPs. The relative quantification of transcripts in the cumulus cells of oocytes matured with 44µg mL-1 CeO2NPs showed a statistically lower mRNA abundance of BCL2-associated X protein (BAX), B-cell CLL/lymphoma 2 (BCL2) and superoxide dismutase 1 (SOD1) compared with the 0µg mL-1 CeO2 NPs group. A concentration of 44µg mL-1 CeO2NPs significantly increased the blastocyst yield and their total, inner cell mass and trophectoderm cell numbers, compared with the 0 and 220µg mL-1 groups. A low concentration of CeO2NPs in the maturation medium enhanced in vitro embryo production of prepubertal ovine oocytes.

In vitro Developmental Competence of Adult Sheep Oocytes Treated with Roscovitine.

The efficiency of in vitro sheep embryo production is still low compared to that observed in vivo and in other species. In this context, meiotic inhibition strategies emerged as a promising alternative to improve this biotechnology. So, this study aimed to evaluate, for the first time, the effects of roscovitine on in vitro maturation of sheep oocytes and their subsequent embryo development. For this, cumulus-oocyte complexes (COCs) were cultured for 6 h in the presence (Rosco) or absence (Control) of 75 µm roscovitine and, subsequently, in vitro matured (IVM) for 18 h with gonadotropins. At 0 (Immature), 6 and 24 h of culture, the nuclear status of oocytes was evaluated by Hoechst staining. Embryo cleavage and blastocyst formation were recorded 30 h after in vitro fertilization and on day 7 of culture, respectively. Blastocyst quality was evaluated by differential staining. At 6 h, the GV rate in the Rosco treatment (93.8%) was similar to that observed in the Immature oocytes (94.9%) and significantly higher compared to Control (41.3%). After IVM for 18 h, a high and similar proportion of oocytes from Rosco (93.6%) and Control (88.4%) reached the MII stage. In both treatments, approximately 70% of oocytes cleaved and 50% of them developed up to blastocyst. The mean percentage of blastocyst cells, embryoblast, trophoblast and pyknosis did also not differ between Control and Rosco. In conclusion, roscovitine, at the studied experimental conditions, was efficient to reversibly inhibit the meiosis of adult sheep oocytes without detrimental effect on development and quality of the in vitro produced embryos.

A novel technique for in vitro maturation of sheep oocytes in a liquid marble microbioreactor.

PURPOSE: The aim of this work was to develop a microbioreactor using liquid marble (LM) as a novel system for oocyte in vitro maturation (IVM) in small volumes. METHODS: Cumulus-oocyte complexes (COCs) obtained from slaughterhouse sheep ovaries were in vitro matured in a LM system prepared by placing a drop (30 µl containing 10 COCs) suspended in TCM 199 supplemented with 10 % (v/v) oestrus sheep serum (OSS) and 0.1 IU FSH and LH onto a polytetrafluoroethylene (PTFE) particle bed (LM group). As a control group (CTRL group), COCs were in vitro matured in standard volume and conditions (600 µl of IVM medium in a four-well dish). After 24-h culture at 38.5 °C in 5 % CO2 in air, COCs were released from LM and the following parameters were evaluated: (a) percentage of MII oocytes, (b) oocyte developmental competence following in vitro fertilization (IVF) or parthenogenetic activation (PA) and embryo culture for 8 days in synthetic oviductal fluid (SOF) medium at 38.5 °C in 5 % O2, 5 % CO2, and 90 % N2. RESULTS: The results indicated similar percentage of MII oocytes in LM and CTRL groups (88.0 vs. 92.0 %). No differences were observed in blastocyst rate after IVF (LM 47.5 % vs. CTRL 50.2 %, P=0.637) or PA (LM 44.4 % vs. CTRL 48.3 %, P=0.426). CONCLUSIONS: The results indicate that LM microbioreactor is a viable technique that provides a suitable microenvironment to induce oocyte in vitro maturation.

Differences in amniotic amino acid concentrations between pregnancies obtained with transfer of vitrified thawed in vitro-produced embryos and with natural mating in sheep.

In vitro embryo production (IVP) and cryopreservation are associated with a high incidence of pregnancy complications and fetal abnormalities that may be linked with alterations of placental development. The amniotic fluid is partly derived from the transport of water and solutes across the placenta and provides the fetus with amino acids (AAs), which are the building blocks for biomolecules involved in physiological growth and development. To better understand the anomalies associated with IVP pregnancies, the present study was conducted to test the hypothesis that amniotic concentrations of AAs differ in pregnancies derived from vitrified/thawed (V/T) IVP embryos compared with gestations obtained with natural mating (NM) in sheep. Amniotic fluid was sampled in ewes that were pregnant after transfer of V/T IVP embryos and that had conceived with NM between Days 60 and 65 (V/T, n = 6; NM, n = 11) and between Days 80 and 85 (V/T, n = 5; NM, n = 14) of gestation via ultrasound-guided amniocentesis. Concentrations of 16 AAs in the amniotic fluid were measured using high-performance liquid chromatography. From Days 60 to 65 of gestation, concentrations of cystine, phenylalanine, and isoleucine were lower in V/T compared with NM ewes. From Days 80 to 85 of pregnancy, the mean concentrations of cystine and lysine were lower in the V/T versus NM groups. The total AA concentration per ewe was similar between the groups from Days 60 to 65 and 80 to 85 of gestation and decreased by 55% from Days 60 to 65 and 80 to 85 of gestation in all ewes. The most abundant AA from Days 60 to 65 of gestation was alanine in both groups, whereas from Days 80 to 85, the most abundant AAs were alanine in NM and glycine in V/T ewes; cystine was the less abundant detectable AA in all ewes at both stages of gestation. Results report that V/T IVP embryos have decreased concentrations of individual AAs in the amniotic fluid during the second trimester of gestation possibly because of an impaired placental vasculogenesis or because of a reduced placental transport. These novel findings are relevant to unravel the mechanisms responsible for the issues of pregnancies achieved with the transfer of IVP and cryopreserved embryos.

Prooxidant effects of verbascoside, a bioactive compound from olive oil mill wastewater, on in vitro developmental potential of ovine prepubertal oocytes and bioenergetic/oxidative stress parameters of fresh and vitrified oocytes.

Verbascoside (VB) is a bioactive polyphenol from olive oil mill wastewater with known antioxidant activity. Oxidative stress is an emerging problem in assisted reproductive technology (ART). Juvenile ART is a promising topic 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, VB exerted prooxidant short-term effects, that is, catalase activity increase and uncoupled increases of mitochondria and reactive oxygen species (ROS) fluorescence signals, and long-term effects, that is, reduced blastocyst formation rate. In vitrified oocytes, VB increased ROS levels. Prooxidant VB effects in ovine prepubertal oocytes could be related to higher VB accumulation, which was found as 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 culture, may have contributed to significant increase of oocyte oxidation. Further studies are needed to identify lower concentrations and/or shorter exposure times to figure out VB antioxidant effects in juvenile ARTs.

Effect of caffeine treatment before vitrification on MPF and MAPK activity and spontaneous parthenogenetic activation of in vitro matured ovine oocytes.

BACKGROUND: Molecules that stabilize protein kinases may be useful in overcoming the deleterious effects of cryopreservation. OBJECTIVE: To evaluate the effect of caffeine treatment before vitrification of in vitro matured ovine oocytes on the activity of MPF and MAPK as well as the spontaneous parthenogenetic activation after 24 h culture. MATERIALS AND METHODS: Oocytes obtained from slaughterhouse sheep ovaries were in vitro matured for 21 h, incubated for 3 h with or without caffeine and then vitrified. After warming, oocytes were processed for the analysis of chromatin configuration and the evaluation of spontaneous parthenogenetic activation (24 h in vitro culture). Fresh in vitro matured oocytes were used as control. RESULTS: Caffeine treatment before vitrification maintained the MPF activity at a level similar to that of fresh oocytes, and reduced the spontaneous parthenogenetic activation in comparison with oocytes that were not-treated with caffeine. CONCLUSION: Caffeine treatment prolongs the meiotic arrest of vitrified MII oocytes, likely via its action of stabilizing the MPF level.

Characterization, isolation and culture of primordial germ cells in domestic animals: recent progress and insights from the ovine species.

Primordial germ cell (PGC) allocation, characterization, lineage restriction, and differentiation have been extensively studied in the mouse. Murine PGC can be easily identified using markers as alkaline phosphatase content or the expression of pluripotent markers such as Pou5f1, Nanog, Sox2, Kit, SSEA1, and SSEA4. These tools allowed us to clarify certain aspects of the complex interactions of somatic and germinal cells in the establishment of the germ cell lineage, its segregation from the neighbouring somatic tissue, and the guidance mechanisms during migration that direct most of the germ cells into the genital ridges. Few data are available from other domestic animals and here we reported our preliminary studies on the isolation, characterization, and in vitro culture of sheep PGCs. Sheep PGCs can be identified with the markers previously used in mouse, but, in some cases, these markers are not coherently expressed in the same cell depending on the grade of differentiation and on technical problems related to commercial antibodies used. Pluripotency of PGCs in culture (EGCs) from domestic animals also needs further evaluation even though the derivation of embryonic pluripotent cell lines from large mammals may be an advantage as they are more physiologically similar to the human and perhaps more relevant for clinical translation studies. Comprehensive epigenetic reprogramming of the genome in early germ cells, and derived EGCs including extensive erasure of epigenetic modifications, may be relevant for gaining insight into events that lead to reprogramming and establishment of totipotency. EGCs can differentiate in vitro in a various range of tissues, form embryonic bodies, but in many cases failed to generate tumours when transplanted into immunodeficient mice and are not able to generate germline chimeric animals after their transfer. Such incomplete information clearly indicates the urge to improve the studies on derivation of stem cells in farm animals and shows the need for a multidisciplinary investigation in order to create farm animal models to set up suitable ethical and technical systems for cell regenerative therapies in humans.

Use of a neuroleptic in assisted reproduction of the critically endangered Mohor gazelle (Gazella dama mhorr).

Stress is a limiting factor in assisted reproduction in wild animals maintained in captivity and measures to reduce it should improve reproductive success. The effect of the long-acting neuroleptic (LAN) perphenazine enanthate was assessed on ovarian stimulation for the recovery of immature oocytes from Mohor gazelle (Gazella dama mhorr) and their subsequent in vitro maturation, fertilization and embryo culture. The viability of embryos after transfer was also examined. Perphenazine enanthate decreased activity levels and facilitated handling of treated animals when compared to controls. LAN-treated animals showed a more regular pattern of respiratory and heart rates and body temperature than controls; no major differences were found in hematological and biochemical parameters between groups. Perphenazine-treated females had lower plasma cortisol levels during the days of intense handling. No significant differences were found in the number of punctured follicles and recovered oocytes between groups. The percentage of mature oocytes per female was significantly higher in the LAN-group. Fertilization and cleavage rates were not significantly different between groups. Embryos developed in culture but none reached the blastocyst stage, and those transferred to the oviduct of synchronized recipients did not develop to term. In conclusion, treatment of females with perphenazine enanthate during ovarian stimulation did not have negative effects on maturation, fertilization and embryo development in vitro. Moreover, an increase in oocyte maturation rate per female was observed. Thus, the use of LANs could be useful to alleviate the effects of handling-stress during assisted reproductive procedures in wild ungulates.

Morphological and biochemical analysis of immature ovine oocytes vitrified with or without cumulus cells.

The cryopreservation of oocytes is an open problem as a result of their structural sensitivity to the freezing process. This study examined (i) the survival and meiotic competence of ovine oocytes vitrified at the GV stage with or without cumulus cells; (ii) the viability and functional status of cumulus cells after cryopreservation; (iii) the effect of cytochalasin B treatment before vitrification; (iv) chromatin and spindle organization; (v) the maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activity of vitrified oocytes after in vitro maturation. Sheep oocytes were vitrified at different times during in vitro maturation (0, 2, and 6 h) with (COCs) or without cumulus cells (DOs). After warming and in vitro maturation, oocytes denuded at 0 h culture showed a significantly higher survival and meiotic maturation rate compared to the other groups. Hoechst 33342/propidium iodide double staining of COCs and microinjection of Lucifer Yellow revealed extensive cumulus cell membrane damage and reduced oocyte-cumulus cell communications after vitrification. Cytochalasin B treatment of COCs before vitrification exerted a negative effect on oocyte survival. After in vitro maturation, the number of vitrified oocytes with abnormal spindle and chromatin configuration was significantly higher compared to control oocytes, independently of the presence or absence of cumulus cells. The removal of cumulus cells combined with vitrification significantly decreased the MPF and MAPK levels. This study provides evidence that the removal of cumulus cells before vitrification enhances oocyte survival and meiotic competence, while impairing the activity of important proteins that could affect the developmental competence of oocytes.