Irma Rosati

Influence of cadmium exposure on in vitro ovine gamete dysfunction

This study was conducted to determine the in vitro effects of three different cadmium concentrations (0, 2, and 20 microM CdCl(2)) on oocyte maturation, fertilisation, and acrosome integrity and sperm viability in sheep. Cumulus-oocyte complexes were recovered from ovaries of slaughtered sheep and sperm were collected by artificial vagina from adult rams. The oocyte maturation rate was significantly affected (P < 0.001) by Cd at both concentrations, with a metaphase II (MII) rate of 96.8, 63.8, and 32.0% for 0, 2, and 20 microM cadmium, respectively. In the second experiment, the presence of Cd significantly decreased (P < 0.01) the rate of oocytes resting in MII after 24-h postmaturation culture, compared with the control group (93.8 versus 29.0 and 19.8%, respectively, for 0, 2, and 20 microM Cd). Oocytes cultured with Cd 2 microM showed a higher activation rate (59.5%, P < 0.001) with one or two pronucleus than with 0 and 20 microM Cd (6.2 and 22.9%, respectively). During fertilisation the presence of fertilised oocytes was decreased in both culture systems with Cd compared with the control (76.1, 25.9, and 4.7% for 0, 2, and 20 microM Cd, respectively; P < 0.001) while polyspermy was increased in the 2 microM Cd group (23.5 for 2 microM versus 6.7 and 0%, respectively, for 0 and 20 microM groups). In both experiments Cd significantly increased (P < 0.001) the rates of oocyte degeneration. In the third experiment, Cd 20 microM significantly decreased (P < 0.01) the viability rate (35.6%) of spermatozoa compared with 2 microM (57.6%) and 0 microM (54.4%) while Cd 2 microM increased (P < 0.01) acrosome-reacted spermatozoa (45.2%) compared with 20 microM (32.5%) and control (31.9%). The results suggest that in vitro cadmium at the lowest dose tested affects the physiological function of both ovine gametes but at higher dose tested can compromise cell viability.

Defined media for vitrification, warming, and rehydration: effects on post-thaw protein synthesis and viability of in vitro derived ovine embryos

The purpose of this study was to assess the viability (rates of re-expanding and hatching in vitro), of in vitro derived ovine blastocysts using vitrification and warming/rehydration media containing fetal calf serum (20% FCS) or polyvinyl alcohol (0.1% PVA), and the incorporation of labelled methionine in protein synthesised during the first 4h after cryopreservation. In experiment 1, after 60 h culture in TCM-199 supplemented with 10% FCS, the hatching rates of blastocysts that had been vitrified, warmed, and rehydrated in media containing only PVA (p/p) were significantly (P<0.05) lower than those vitrified in medium containing PVA with warming and rehydration in medium containing FCS (p/s). Blastocysts that were vitrified in medium containing FCS and warmed and rehydrated in medium with PVA (s/p) had hatching rates that were significantly lower (P<0.01) than those vitrified, warmed, and rehydrated in media with only FCS (s/s). After warming, the number of dead cells in the p/p group was significantly (P<0.05) lower than in all other groups. In experiment 2, the [35S]methionine uptake by embryonic cells of the s/p group was significantly (P<0.01) higher than in other groups. The incorporation of labelled methionine into newly synthesised proteins was significantly lower in the p/p group (P<0.01) than in all other groups. No differences in the newly synthesised proteins were observed between groups. In conclusion, these results suggest that it is possible to replace serum with defined macromolecules in vitrification and warming/rehydration media for in vitro derived ovine blastocysts but this leads to a decrease in viability and a reduction in protein synthesis after warming.

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

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

Epidemiological study of Toxoplasma gondii infection in ovine breeding.

An outbreak of toxoplasmosis occurring in a typical farm of 524 ovines was monitored for 1 year after the occurrence of 31 abortions. Abortion events involved 7.2% of 430 pregnant sheep. Presence of antibodies to Toxoplasma gondii in sheep sera was investigated by the indirect fluorescence antibody test (IFAT). A total of 422 ewes were bled four times during the year, and an epidemiological analysis was performed on all serology data collected in this subgroup. The prevalence of IgG positives ranged from 31.52% (133/422) at the first sampling to 62.56% (264/422) at the fourth sampling. Incidence of IgG antibodies was 38.75% at the second sampling, 14.92% at the third and 29.28% at the fourth sampling. At the beginning of the study, prevalence was 70.7% in primiparous sheep and 20.9% in sheep older than 5 years; at the third sampling, prevalence was stable at 70% in pluriparous sheep. The mean prevalence of IgM antibodies was 14.87%. A total of 147 out of all 524 ovines of the flock tested positive for IgM in more than one sampling. After an initial positivity, 60 sheep tested negative for IgG at the following serological controls (4 between the first and the second sampling, 30 between the second and the third and 28 between the third and the fourth sampling). One stray cat was positive for IgG, with a titre of 1 : 320. Moreover, one of the farmers was also positive, with a titre of 1 : 160 for IgG. A positive PCR result for T. gondii DNA was also observed in aliquots of grain and pellets taken from feed stocks amassed inside the sheds without protection, suggesting that an adequate management of the farm might be useful, if not essential, for controlling T. gondii outbreaks in ovine flocks.

The effect of co-culture on the development of in vitro matured equine oocytes after intracytoplastic sperm injection.

It is clear that, in the horse, there are many weak links in the process of in vitro embryo production; an optimal culture system for equine oocytes does not exist, and related data are conflicting. Therefore, the ability of 3 different culture systems to support embryonic development of ICSI horse oocytes was examined. Oocytes (n = 261) suitable for culture were collected from 55 ovaries and divided, according to cumulus morphology, into 2 categories: expanded cumulus and compacted cumulus. Oocytes with expanded and compacted cumulus were cultured for in vitro maturation in TCM 199 + 10% FCS + 0.1 iu/ml FSH/LH at 38.5 degrees C under 5% CO2 in air for 24 and 40 h, respectively. Oocytes (n = 149) reached metaphase II and were subjected to ICSI with frozen semen and then incubated in 3 different culture systems: A) TCM 199 + 10% FCS alone or B) on granulosa cell monolayer, C) SOF + MEM amino acids + 0.8% BSA. Cultural conditions were 39 degrees C and 5% CO2 in air for A and B, while a gas mixture (5% CO2, 5% O2, 90% N2) was used for C. The fertilisation rate was 32%. The cleavage rate in Group A was 74.4% (32/43); 18 embryos reached 2-6 cell stage, eight 8-16 cell, four 16-32 cell and two >32 cell. In Group B, the cleavage rate was 73.5% (36/49) with better results in embryonic development; 14 reached 2-6 cell stage, eighteen 8-16 cell, twelve 16-32 cell and five >32 cell. In Group C, the cleavage rate was significantly lower then in A and B; only 15 of 47 ICSI oocytes (39.1%) cleaved with maximum development to 2-6 cell stage. The remaining oocytes (68.1%) degenerated during culture. In conclusion, IVM horse oocytes can be fertilised in vitro with high efficiency with ICSI and co-culture systems showed to be superior in supporting in vitro embryo culture compared to simple ones. The identification of the factors beneficial to in vitro embryo development provided by the somatic cells could be important to optimise the embryo culture systems for equine embryos.

The effect of okadaic acid on meiotic maturation of canine oocytes of different size

The present study was conducted to determine the effect of okadic acid (OA), a potent inhibitor of seronine/treonine 1 and 2A phosphatase, on meiotic resumption and progression in canine oocytes with different diameters. Cumulus-oocyte complexes were collected from ovaries of bitches at different oestrous phases. In Experiment 1, to determine the optimal concentration of OA (0.5 or 2 μM), the oocytes were pre-incubated for 1, 3, and 20 h in TCM 199 supplemented with 20% SCE and thereafter cultured in the same medium without OA. In Experiment 2, the selected oocytes were divided into three groups according to their diameter: <110 μm, 110-120 μm, >120 μm, and pre-incubated in OA 0.5 μM for 1 h. Oocytes were cultured in vitro as previously described. After 72 h of IVM, in Experiment 1, significantly more oocytes reached MII stage with 0.5 μM for 1 h (30.8% P<0.001%) for oocytes cultured in other OA condition and in control group. In Experiment 2, OA induced a significantly higher incidence of MII oocytes in the 110-120 μm and >120 μm groups (P<0.001) compared to control group, but a significantly higher proportion of the oocytes>120 μm pre-incubated with OA progressed to MII (51.3% P<0.001). In contrast, smaller oocytes (<110) did not develop to MII stage with or without OA. In conclusion, treatment of canine oocytes with 0.5 μM for 1 h, improves meiotic maturation. The culture of fully grown (>120 μm) oocytes with OA at the onset of in vitro maturation can result in a higher frequency of meiotic maturation.

Clinical and cytogenetic studies in intersex ewes

Abstract Nine Sarda x Lacaune ewes with intersexual characteristics and an infertility condition at the reproductive anamnesis were analysed. In order to make a diagnosis, we have evaluated their behaviour and performed clinical and laparoscopic examination of the reproductive tract, as well as cytogenetic analysis. The ewes showed basically a female phenotype but a clinical examination revealed a different degree of masculinization in the morphology of external genital organs. A shorter vagina was observed in female-like ewes and a hypertrophic clitoris in male-like ewes. Laparoscopic analysis evidenced the presence of testis in seven individuals and, for two of them, the gonadal position was subcutaneous. Different male characteristics in the nine subjects, were also observed in their behaviour with a different degree of masculinization. Their blood samples were used for determining the percentage of male cells on lymphocytes chromosome spreads by using the C-banding technique. The haematopoietic chimeras (XX/XY) found in the lymphocytes confirmed the diagnosis of freemartinism for seven out of the nine subjects.

Safety in Assisted Reproductive Technologies: Insights from Gene Expression Studies During Preimplantation Development

The mammalian oocyte and embryo display considerable plasticity. Even in sub-optimal conditions, as in vitro environment may be certainly considered, the oocyte is able to mature, face fertilization and develop first into an embryo and finally to a live offspring. Such ability has encouraged, over the decades, the development of numerous in vitro assisted reproductive technologies (ART) in several species, included human. Nowadays, more than 30 years after its inception, human ART are routinely and successfully applied to solve fertility problems, which affect ~ 15% of reproductive age couples and have significant medical, social and financial implications.

321 Oocyte diameter influences the meiotic resumption and progression induced by okadaic acid in dog.

The acquisition of meiotic competence, in the bitch as in many other mammalian species, is related to the oocyte diameter. This study was designed to determine the effect of okadaic acid (OA), a potent inhibitor of seronine/threonine 1 and 2A phosphatases, on meiotic resumption and progression in canine oocytes with different diameters. In two experiments, healthy cumulus-oocytes complexes were collected from ovaries of bitches at various stages of the estrous cycle and divided, by diameters, into three treatment groups for in vitro maturation: 120 ¼m. In Experiment 1, oocytes were pre-incubated for 1 h in TCM-199 + 20% estrous canine serum (SCE) + cysteamine + OA (0.5 ¼M). Then, oocytes were cultured for 48 h in the same medium without OA at 38.5°C, 5% CO2 in air. As a control group, oocytes were matured in vitro under the same conditions but without pre-incubation with OA. In Experiment 2, to determine if the effect of OA is mediated by cumulus cells, >120 ¼m oocytes were denuded from cumulus cells, incubated with or without OA, and cultured in vitro as previously described. At 48 h, all oocytes were stained and fixed with glycerol-Hoechst 33342 to assess the stage of meiotic maturation. In Experiment 1, OA induced a significantly higher incidence of meiotic resumption in oocytes 120 ¼m OA group (64/78, 82.0%) was similar to the >120 ¼m control group (56/72, 77.8%), but a significantly higher proportion of the oocytes pre-incubated with OA progressed to MII than did the control oocytes (40/78, 51.3% vs. 12/72, 16.7%, respectively; P 120-¼m oocytes with (7/63, 11.1%) or without OA (7/55, 12.7%) and none of them progressed to MII. In conclusion, the results of the present study indicate that treatment of fully grown (>120 ¼m) oocytes with okadaic acid at the onset of in vitro maturation can result in a higher frequency of meiotic maturation than previously reported. Also, we determined that the beneficial effect of okadaic acid was mediated by cumulus cells.