Use of time-lapse imaging to evaluate morphokinetics of in vitro equine blastocyst development after oocyte holding for two days at 15°C versus room temperature before intracytoplasmic sperm injection.

Time-lapse imaging was used to establish the morphokinetics of equine embryo development to the blastocyst stage after invitro oocyte maturation (IVM), intracytoplasmic sperm injection (ICSI) and embryo culture, in oocytes held overnight at room temperature (22-27°C; standard conditions) before IVM. Embryos that developed to the blastocyst stage underwent precleavage cytoplasmic extrusion and cleavage to the 2-, 3- and 4-cell stages significantly earlier than did embryos that arrested in development. We then determined the rate of blastocyst formation after ICSI in oocytes held for 2 days at either 15°C or room temperature before IVM (15-2d and RT-2d treatment groups respectively). The blastocyst development rate was significantly higher in the 15-2d than in the RT-2d group (13% vs 0% respectively). The failure of blastocyst development in the RT-2d group precluded comparison of morphokinetics of blastocyst development between treatments. In any condition examined, development to the blastocyst stage was characterised by earlier cytoplasmic extrusion before cleavage, earlier cleavage to 2- and 4-cell stages and reduced duration at the 2-cell stage compared with non-competent embryos. In conclusion, this study presents morphokinetic parameters predictive of embryo development invitro to the blastocyst stage after ICSI in the horse. We conclude that time-lapse imaging allows increased precision for evaluating effects of different treatments on equine embryo development.

Exposure to cadmium during in vitro maturation at environmental nanomolar levels impairs oocyte fertilization through oxidative damage: A large animal model study.

Cadmium is a highly toxic heavy metal with negative effects on oocyte fertilization. The aim of this study was to analyse whether cadmium-induced impairment of fertilization is caused by mitochondria dysfunction and oxidative stress in the cumulus-oocyte complex (COC). Preliminarily, 19 trace element levels were measured in ovaries from juvenile and adult ewes and age-related cadmium ovarian bioaccumulation at nanomolar concentrations was found. COCs from juvenile and adult ewes, exposed during in vitro maturation to 1nM or 100nM CdCl2, and subjected to in vitro fertilization showed significantly lower fertilization rates in exposed COCs compared with controls. In vitro matured exposed and control COCs underwent confocal microscopy analysis of mitochondria activity and reactive oxygen species (ROS) levels and lipid peroxidation (LPO) assay at cumulus cell and oocyte level. In both age groups, cadmium at nanomolar concentrations induced cumulus-oocyte mitochondria over-activity and oxidative damage which were related to impaired oocyte fertilization.

Morphological, ultrastructural and functional imaging of frozen/thawed and vitrified/warmed human ovarian tissue retrieved from oncological patients.

STUDY QUESTION: Which is the best method for human ovarian tissue cryopreservation: slow freezing/rapid thawing (SF/RT) or vitrification/warming (V/W)? SUMMARY ANSWER: The conventional SF/RT protocol used in this study seems to better preserve the morpho-functional status of human cryopreserved ovarian tissue than the used open carrier V/W protocol. WHAT IS KNOWN ALREADY: Cryopreservation of human ovarian tissue is generally performed using the SF/RT method. However, reduction in the follicular pool and stroma damage are often observed. An emerging alternative procedure is represented by V/W which seems to allow the maintenance of the morphological integrity of the stroma. STUDY DESIGN, SIZE, DURATION: This is a retrospective cohort study including six patients affected by oncological diseases and enrolled from January to December 2014. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian tissue was laparoscopically harvested from the right and left ovaries and was cryopreserved using a routinary SF/RT protocol or a V/W method, involving tissue incubation in two solutions (containing propylene glycol, ethylene glycol and sucrose at different concentrations) and vitrification in an open system. For each patient, three pieces from each ovary were collected at the time of laparoscopy (fresh tissue) and after storage (SF/RT or V/W) and processed for light microscopy (LM) and transmission electron microscopy (TEM), to assess the morphological and ultrastructural features of follicles and stroma, and for laser scanning confocal microscopy (LSCM), to determine the functional energetic/redox stroma status. The preservation status of SF/RT and V/W ovarian tissues was compared with that of fresh ones, as well as between them. MAIN RESULTS AND THE ROLE OF CHANCE: By LM and TEM, SF/RT and V/W samples showed cryodamage of small entity. Interstitial oedema and increased stromal cell vacuolization and chromatin clumping were observed in SF/RT samples; in contrast, V/W samples showed oocyte nuclei with slightly thickened chromatin and irregular shapes. The functional imaging analysis by LSCM revealed that the mitochondrial activity and intracellular reactive oxygen species levels were reduced both in SF/RT and in V/W samples compared with fresh samples. The study also showed progressive dysfunction of the mitochondrial activity going from the outer to the inner serial section of the ovarian cortex. The reduction of mitochondrial activity of V/W samples compared with fresh samples was significantly higher in the inner section than in the outer section. LIMITATIONS, REASONS FOR CAUTION: The results report the bioenergetic and oxidative status assessment of fresh and cryopreserved human ovarian tissue by LSCM, a technique recently applied to tissue samples. The use of LSCM on human ovarian tissues after SF/RT or V/W is a new application that requires validation. The procedures for mitochondrial staining with functional probes and fixing are not yet standardized. Xenografting of the cryopreserved ovarian tissue in severe combined immunodeficient mice and in vitro culture have not yet been performed. WIDER IMPLICATIONS OF THE FINDINGS: The identification of a cryopreservation method able to maintain the morpho-functional integrity of the ovarian tissue and a number of follicles comparable with those observed in fresh tissue might optimize results in clinical practice, in terms of recovery, duration of ovarian function and increased delivery outcomes after replanting. The SF/RT protocol allowed better morpho-functional tissue integrity than the V/W procedure. STUDY FUNDING/COMPETING INTERESTS: Funding was provided by Fondazione del Monte di Bologna e Ravenna, Italy. Dr N.A.M. was granted by the project ONEV MIUR PONa3 00134-n.254/R&C 18 5 2011 and the project GR-2011-02351396 (Ministry of Health, Young Researchers Grant 2011/2012). There are no competing interests. TRIAL REGISTRATION NUMBER: Clinical trial 74/2001/0 (approved:13 2 2002): 'Pilot study on cryopreservation of human ovarian tissue: morphological and immunohistochemical analysis before and after cryopreservation'.

Effects of kisspeptin-10 on in vitro proliferation and kisspeptin receptor expression in primary epithelial cell cultures isolated from bovine placental cotyledons of fetuses at the first trimester of pregnancy.

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.

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.

Assessment of different functional parameters of frozen-thawed buffalo spermatozoa by using cytofluorimetric determinations.

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 (AR) by FITC-PNA staining. Semen samples from five 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 (Δψ(high)), with no significant differences between subjects. Sperm chromatin structure assay differed significantly between the five 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 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 Ca(2+) ionophore A23187 for 3 h, AR significantly differed between subjects and was characterized by an increase in both ARL (10.8%) and ARD population (22.0%). This 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 in sperm functional parameters sensitive enough for the diagnosis of frozen-thawed semen fertilizing potential.

Isolation, proliferation, cytogenetic, and molecular characterization and in vitro differentiation potency of canine stem cells from foetal adnexa: a comparative study of amniotic fluid, amnion, and umbilical cord matrix.

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.

Expression of maternal transcripts during bovine oocyte in vitro maturation is affected by donor age.

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.

Effects of in vitro exposure to natural levels of zearalenone and its derivatives on chromatin structure stability in equine spermatozoa.

The purpose of this study was to assess the natural exposure of male 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 the DNA 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, alpha-zearalenol, and beta-zearalenol proved to be more toxic compounds for sperm chromatin stability than other tested derivatives. A nongenomic mechanism of action can be hypothesized.

Expression of the micro-opioid receptor on Malassezia pachydermatis and its effect in modulating phospholipase production.

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.

Lectin-binding sites in isolated equine cumulus-oocyte complexes: differential expression of glycosidic residues in complexes recovered with compact or expanded cumulus.

Equine cumulus-oocyte complexes (COCs) were analyzed by means of 13 lectins to evaluate their glycoconjugate patterns and to verify differences between COCs recovered with compact (Cp) and expanded (Exp) cumulus. Cumulus cells showed a similar staining pattern in both Cp and Exp COCs with all lectins used, except for a higher reactivity with SNA and GSA II in Cp COCs and SBA in Exp COCs. The zona pellucida (ZP) showed (1) uniform staining with MAL II, RCA(120), and SBA in both Cp and Exp COCs, (2) trilaminar binding pattern with WGA as well as higher Con A reactivity in the outer region of both types of COCs, (3) uniform staining with PNA only in Exp COCs, (4) uniform and trilaminar binding pattern with SNA in Cp and Exp COCs, respectively, and (5) major reactivity with GSA II in Exp COCs. Ooplasm showed similar staining intensity with Con A, HPA, GSA I-B(4), and WGA in both Cp and Exp COCs, with stronger reactivity to GSA II in Exp COCs, whereas SNA, UEA I, and LTA binding sites were present only in Cp COCs. Oocyte cortical granules of both Cp and Exp COCs reacted with Con A and WGA. These results suggest that, in the mare, viable (Cp) and atretic (Exp) COCs display different glycoconjugate staining pattern, which may account for the different maturation and developmental competence of COCs.

Cytoplasmic lipid droplets and mitochondrial distribution in equine oocytes: Implications on oocyte maturation, fertilization and developmental competence after ICSI.

Lipid droplets (LDs) and mitochondria in the ooplasm are essential for energy production required for maturation, fertilization and embryo development. This study investigates the correlations between cytoplasmic LDs polar aggregation and: (1) nuclear maturation (Experiment 1); (2) mitochondrial (mt) distribution pattern and localization (Experiment 2); (3) fertilization and embryonic development after intracytoplasmic sperm injection (ICSI; Experiment 3) in equine oocytes recovered from slaughtered mares and matured in vitro. Morphologically normal oocytes were selected after culture and categorized as having polar (P) aggregation or uniform (U) distribution of LDs. In Experiment 1, the maturation rate was significantly higher in P compared with U oocytes (69%, 40/58 vs. 32%, 13/41; P<0.001). In Experiment 2, it was observed that P and U oocytes showed heterogeneous mt distribution at comparable rates (68%, 25/37 vs. 50%, 2/4 for P and U respectively; NS). Moreover, only in 8/25 (32%) of P oocytes, LDs overlapped with mt aggregates in the area containing meiotic spindle. In Experiment 3, normal fertilization (51%, 19/37 vs. 60%, 6/10, for P and U) and cleavage rates (83%, 20/24 vs. 67%, 4/6, for P and U) did not differ between groups, also in oocytes with LDs located nearby the polar body. Overall, P aggregation of LDs was related to cumulus expansion at collection. In conclusion, in equine matured oocytes, P aggregation of LDs is related with cumulus expansion and nuclear maturation. However, it is not related with heterogeneous mt distribution and cannot be considered a predictive indicator for normal fertilization and embryo development.

In vitro equine oocyte maturation in pure follicular fluid plus interleukin-1 and fertilization following ICSI.

The interleukin-1 (IL-1) system is thought to be involved in periovulatory events in the mare. Previous in vivo studies have demonstrated that IL-1beta induces oocyte maturation, but depresses the pregnancy rate 14 days after ovulation. To better understand the role of IL-1 in oocyte maturation and fertilization, the effects of IL-1 on the in vitro maturation rate of equine oocytes in pure follicular fluid were evaluated and fertilization rate assessed following intracytoplasmic sperm injection (ICSI). Oocytes collected from slaughterhouse ovaries were cultured in four different media for 30 h prior to fertilization. Two experiments were performed, each using three maturation media as the experimental treatments. Medium 1 was pure follicular fluid from subordinate follicles. Medium 2 was medium 1 plus 50 ng/ml recombinant human IL-1beta. Medium 3 was pure follicular fluid collected from mares administered crude equine gonadotropin (CEG). Medium 4 was medium 2 plus 50 ng/ml of recombinant human IL-1 receptor antagonist. Media 1, 2 and 3 were compared in experiment 1. In experiment 2, media 1, 2 and 4 were compared. After maturation, metaphase II oocytes were submitted to microinjection and assessed for signs of fertilization. In experiment 1, 101 oocytes were evaluated. The rate of polar body extrusion was 66, 51 and 68% and the proportions of normally fertilized oocytes after ICSI were 40, 18 and 38% for media 1, 2 and 3, respectively. In experiment 2, 122 oocytes were evaluated. The rate of polar body extrusion was 55, 48 and 42% and the proportions showing normal fertilization after ICSI were 14, 25 and 29% for media 1, 2 and 4, respectively. There was no positive effect of IL-1beta on maturation in both experiments, but the fertilization rate and percentage of embryos reaching four-cell were low in the presence of IL-1beta, indicating that this cytokine may interfere with fertilization and early embryo development.

Role of beta-endorphin on phospholipase production in Malassezia pachydermatis in dogs: new insights into the pathogenesis of this yeast.

Malassezia spp. are lipophilic yeasts that are part of the normal cutaneous microflora and sometimes act as pathogens causing dermatitis. This study investigated the interactions occurring between beta-endorphin and phospholipase activity in isolates of M. pachydermatis in dogs presenting cutaneous lesions. Phospholipase production was evaluated and quantified on 144 isolates suspended in Dixon broth to which different beta-endorphin concentrations (from 600 to 0.6 pM) were added. The isolates were divided into three groups: group A comprised isolates from lesional skin of dogs with dermatitis confined to one site, group B consisted of isolates from the healthy skin of the same dogs with localized lesions, and group C was made up of isolates from assorted skin sites of healthy dogs. A statistically higher phospholipase activity than that of the controls was recorded in group B at all tested beta-endorphin concentrations. In groups A (Pz=0.62) and C (Pz=0.62) phospholipase activity was statistically higher than the controls only at a concentration of 600 pM. This study suggests that beta-endorphin plays an important role in the production of phospholipase in M. pachydermatis isolates and provides evidence that beta-endorphin concentrations affect the number but not the Pz value of phospholipase-producing isolates. B-endorphin concentrations may play a relevant role in inducing M. pachydermatis cell differentiation towards the production or non-production of phospholipase.

Effects of beta-endorphin and Naloxone on in vitro maturation of bovine oocytes.

Bovine cumulus-oocyte complexes (COCs) and mural granulosa cells express the mRNA coding for the micro-opioid receptor. The addition of beta-endorphin (beta-end) to oocytes cultured in hormonally-supplemented in vitro maturation (IVM) medium had no effect on the rates of oocytes reaching the metaphase II (MII) stage, but significantly decreased the maturation rate (P < 0.05) and arrested oocytes at metaphase I (MI) after culture in hormone-free medium (P < 0.001). Naloxone (Nx) reverted this inhibitory effect of beta-end. Moreover, Nx "per se" showed a dose-dependent dual effect. When added at high concentration (10 x (-3) M), it significantly reduced the rate of oocytes in MII (P < 0.001), thus increasing the rate of oocytes arrested in MI. However, Nx added at low concentration (10 x (-8) M) significantly increased oocyte maturation (P < 0.001). High concentration of Nx induced an increase in both intracellular calcium concentration ([Ca(2+)](i)) and in the activity of the mitogen-activated protein kinase (MAPK) also called extracellular-regulated kinase (ERK) in cumulus cells of bovine COCs. Blocking the rise in [Ca(2+)](i) with the calcium chelator acetoxymethylester-derived form of bis (o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM) reversed the Nx-dependent inhibition of meiotic maturation observed at high Nx concentrations. Whereas blocking ERK with the MAPK/ERK kinase (MEK) inhibitor, PD98059, had no effect on this process. Therefore, we concluded that the mocro-opioid receptor, by inducing [Ca(2+)](i) increase, participates in the cumulus-oocyte coupled signaling associated with oocyte maturation.

Influence of oocyte collection technique on initial chromatin configuration, meiotic competence, and male pronucleus formation after intracytoplasmic sperm injection (ICSI) of equine oocytes.

There is a great variability in the success of horse oocyte maturation and fertilization among laboratories. This study was conducted to determine if the meiotic and developmental competence of horse oocytes could be dependent on the method of oocyte collection, i.e., aspiration of follicular fluid with a vacuum apparatus, or opening follicles and scraping the granulosa layer. Horse oocytes were recovered from abattoir ovaries by aspiration or scraping and classified as having compact (Cp), expanded (Ex), or partial (P) cumuli. In Experiment 1 (Part A in May and Part B in October), oocytes were fixed immediately after collection to assess whether the collection method influenced the initial chromatin configuration of oocytes. In Experiment 2, in vitro maturation rates of oocytes recovered by aspiration or scraping were compared. In Experiment 3, oocytes were matured in vitro and submitted to intracytoplasmic sperm injection (ICSI). Initial chromatin configuration differed according to collection method in that there was a significantly higher prevalence of diffuse chromatin within the germinal vesicle in oocytes recovered by scraping than in oocytes recovered by aspiration (29/87, 33% and 28/166, 17%, respectively; P < 0.01). Maturation of oocytes to metaphase II did not significantly differ between scraped and aspirated oocytes (56/101, 55.4 % vs. 65/106, 61.4%, respectively). The overall pronucleus formation rate after ICSI of oocytes recovered by scraping was not significantly different than that of oocytes recovered by aspiration (50/99, 52.6% vs. 50/85, 68.5 %, respectively); however, the rate of abnormal fertilization was significantly higher for oocytes collected by aspiration (14/73, 19% vs. 6/94, 6%, respectively; P <0.05). These results demonstrate that the collection method affects the population of recovered oocytes and may contribute to differences in results observed among laboratories working with horse oocytes.

Toxic effects of the mycotoxin zearalenone and its derivatives on in vitro maturation of bovine oocytes and 17 beta-estradiol levels in mural granulosa cell cultures.

Moulds parasites of livestock foodstuffs alter the quality of grains by synthesizing mycotoxins. Zearalenone (ZEA) and its derivatives (alpha- and beta-zearalenol, zeranol, taleranol and zearalanone) are produced by fungi of the genus Fusarium and, after ingestion via contaminated cereals, may lead to fertility disturbances and other reproductive pathologies. Zearalenone, alpha-zearalenol and zearalanone were tested, at levels ranging from 0.3 to 30 microg/ml, in order to evaluate the effect on the in vitro maturation (IVM) rate of bovine oocytes and on the formation of 17 beta-estradiol in supernatants of mural granulosa cells (GC) cultures. These compounds induced dose-dependent oocyte maturation delay and chromatin abnormalities. Maturation of oocytes to metaphase II (M II) was inhibited in oocytes cultured in the presence of 30 microg/ml ZEA, alpha-zearalenol or zearalanone, with a significant increase in chromatin abnormalities occurring in the presence of ZEA (P<0.05) and alpha-zearalenol (P< 0.001). In preliminary trials on 17 beta-estradiol formation, at the same testing concentration, higher levels of 17 beta-estradiol were found in the presence of alpha-zearalenol (mean value 1.6 ng/ml) with respect to ZEA and zearalanone (mean estradiol concentrations of 0.06 and 0.5 ng/ml, respectively). These data demonstrate a negative effect of ZEA and its derivatives on meiotic progression of bovine oocytes, possibly attributable to a toxic mechanism not related to the binding affinity of these compounds to estrogen receptor sites, and support previous observations that alpha-zearalenol acts as a stronger estrogenic inducer than the original molecule (ZEA).

Effects of naloxone on calcium turnover in cows affected by milk fever.

Milk fever is a metabolic disorder of calcium homeostasis that affects about 2 to 6% of postpartum cows. Current therapy is based on the administration of calcium gluconate. On the basis of the clinical signs, and given that endorphins increase at parturition, we supposed that endogenous opioid peptides (EOP) could be responsible for this pathology. In this study, cows with milk fever were administered the opiate antagonist, Naloxone (Nx; experiment 1) or Nx with calcium salts (experiment 2). In experiment 1, Nx induced the recovery of affected cows. The effects of Nx therapy, expressed in terms of proportion of recovered cows, of cows recovering in less than 30 min and cows requiring repeated treatments, were not statistically different than those obtained by means of calcium administration (17/17, 100%; 10/17, 59% and 7/17, 41% vs. 33/35, 94%; 22/35, 63% and 11/35, 31%, respectively; NS). In experiment 2, a significantly higher ratio of cows recovered in less than 30 min in the group of animals treated with Nx in association with calcium salts, compared with the group of cows treated with the calcium traditional therapy (106/118, 90% for calcium-Nx treated cows vs. 34/62, 55% for calcium-treated cows). Moreover, in the group of cows treated with calcium-Nx, the number of cows requiring repeated treatments was significantly reduced and no unrecovered cows were observed. The results support the idea that high EOP levels interfere with inward movement of calcium through the cell membrane and with calcium activity. The association of calcium and Nx at low dosage is a safe method to treat milk fever in cows and reduces muscular complications.

Effects of fetuin on zona pellucida hardening and fertilizability of equine oocytes matured in vitro.

In vitro fertilization (IVF) has had poor success in the horse, a situation related to low rates of sperm penetration through the zona pellucida (ZP). Zona pellucida hardening (ZPH) is seen in mouse and rat oocytes cultured in serum-free medium. The hardened ZP is refractory to sperm penetration. Fetuin, a component of fetal calf serum, inhibits ZPH and allows normal fertilization rates in oocytes cultured in the absence of serum. We evaluated whether fetuin is present in horse serum and follicular fluid (FF) and whether fetuin could inhibit ZPH in equine oocytes matured in vitro, thus increasing sperm penetration during IVF. The presence of fetuin in equine serum and FF was confirmed by immunoblotting. Oocytes submitted to in vitro maturation (IVM) in medium containing fetuin were used for ZPH assay or IVF. Intracytoplasmic sperm injection (ICSI) was carried out as a control procedure. The presence of fetuin during IVM did not affect the rate of maturation to metaphase II. Maturation of oocytes in the presence of fetuin reduced ZPH in a dose-dependent manner. After both IVF and ICSI, there was no significant difference in oocyte fertilization between fetuin-treated and untreated oocytes. The fertilization rate was significantly higher after ICSI than after IVF, both in fetuin-treated and in untreated oocytes. In conclusion, fetuin reduced ZPH in equine oocytes but did not improve sperm penetration during IVF. This implies that, in the horse, "spontaneous" ZPH is unlikely to be the major factor responsible for inhibiting sperm penetration in vitro.

Intracytoplasmic sperm injection (ICSI) versus conventional IVF on abattoir-derived and in vitro-matured equine oocytes.

Conventional IVF as well as several assisted microfertilization techniques have shown limited success in the horse. After recent positive results achieved with intracytoplasmic injection of a single spermatozoon (ICSI) in human IVF, we chose to try the method in the horse. We compared conventional IVF to ICSI by fertilization rates of oocytes with compact and expanded cumuli and by developmental potential of the resulting embryos. Cumulus-oocyte complexes (COCs) were obtained by aspirating the follicular fluid from the ovaries of slaughtered mares. Complexes showing complete cumulus investment, either compact or expanded, were randomly assigned to IVF or ICSI trials and separately cultured for IVM. Frozen-thawed stallion spermatozoa were prepared for IVF with a swim-up procedure conducted in Talp-Hepes with heparin or for ICSI in Earle's balanced salt solution (EBSS) supplemented with human serum albumin (HSA). Oocytes for IVF were partially decumulated by pipetting, whereas those for ICSI were totally denuded with 80 UI/ml hyaluronidase. Oocytes were fixed, stained and examined for signs of fertilization the day after IVF or ICSI. The percentage of normally fertilized oocytes showing 2 pronuclei or cleavage was significantly higher with ICSI than IVF (29.8%, 17/57 vs 8.7%, 9/103 ; P < 0.01). Significantly higher fertilization rates were observed in oocytes retrieved with an expanded cumulus when submitted to ICSI procedure as compared with IVF (52.2%, 12/23 vs 17.1%, 6 35 ; P < 0.01), whereas in oocytes recovered with a compact cumulus, fertilization rates were low (14.7%, 5/34 with ICSI and 4.4%, 3 68 with IVF; NS). Embryonal development did not occur after culture following IVF, as indicated by absence of cleavage in any of the 93 inseminated oocytes. Following ICSI, 7 of 55 injected oocytes cleaved, 5 of which had shown expanded cumuli; of the 5, 2 were at the 16-cell stage and one each at the 8-, 3- and 2-cell stage, respectively. The other 2 fertilized oocytes, originating from compact cumuli, reached 4- and 8- cell stages, respectively. These results indicate that ICSI can be applied successfully to in-vitro matured equine oocytes to increase the fertilization rates. In addition, it seems that in vitro cytoplasmic maturation of oocytes issuing from a compact cumulus may not be complete enough to lead to a successful fertilization and that ICSI may be a tool to evaluate ooplasmic maturation.