Maria Teresa Zedda

Intracytoplasmic sperm injection of in vitro matured oocytes of domestic cats with frozen-thawed epididymal spermatozoa.

The ability to mature and fertilize oocytes of endangered species may allow us to sustain genetic and global biodiversity. The first objective of this study was to compare the effect of two different culture media and two different incubation times on in vitro maturation (IVM) of domestic cat oocytes. The second objective was to determine the developmental competence of in vitro matured cat oocytes after intracytoplasmic sperm injection (ICSI) with cat spermatozoa. Oocytes recovered from ovaries of ovariectomized cats were cultured either in TCM 199 medium or in synthetic oviductal fluid (SOF), both of which were supplemented with cysteamine, BSA, FSH, LH. Nuclear maturation was assessed after 24 h and 40 h of incubation. Results of IVM showed that the percentage of oocytes reaching MII after 24 h and 40 h of incubation were significantly higher (P<0.001) after culture with SOF (88/110, 80% and 159/192, 82.8%) than TCM 199 (86/129, 66.7% and 58/90, 64.4%). Oocytes (n = 231) matured in vitro in SOF for 24 h were fertilized by ICSI with frozen-thawed epididymal cat spermatozoa. After ICSI, one group of oocytes (n = 129) was activated with ethanol, and a second group (n = 102) was not activated. The developmental competence of all ICSI oocytes was examined after 7 days of in vitro culture. After 28 h of culture, the cleavage frequency of ICSI-activated oocytes was significantly higher (P<0.001) than that of IC

M-phase promoting factor (MPF) and mitogen activated protein kinases (MAPK) activities of domestic cat oocytes matured in vitro and in vivo

This work was undertaken in order to examine M-phase promoting factor (MPF) and mitogen-activated protein kinases (MAPK) activities during meiotic progression of cat oocytes cultured in two different media for two different incubation times and preovulatory cat oocytes that reached MII in vivo. Oocytes recovered from ovaries of ovariectomized cats were cultured either in TCM 199 or SOF for 24 h and 40 h. In vivo matured oocytes were recovered by follicular aspiration from ovaries of domestic cats ovariectomized 24 h to 26 h after hormonal treatment. Results showed that the kinetic of MPF and MAPK activity was similar during meiotic progression of cat oocytes matured in TCM 199 and SOF. After 24 h of incubation, MII oocytes had significantly (p < 0.001) higher MPF and MAPK levels than MII oocytes cultured for 40 h in both culture media. MPF and MAPK activity was significantly (p < 0.01) lower in the oocytes matured in vitro than in those matured in vivo. This study provides evidence that the two different maturation media did not determine differences in MPF and MAPK fluctuations and levels during meiotic progression of cat oocytes and that the time of maturation influenced the level of the two kinases. Moreover, it shows that MPF and MPK activity is higher in in vivo matured oocytes than in in vitro matured oocytes, suggesting a possible incomplete cytoplasmic maturation after culture.

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.

Evaluations of Testicular Biopsy by Tru-cut in the Stallion

Testicular biopsy is one of the supplementary examinations performed in the course of andrological testing. In veterinary medicine, it is seldom used in low-fertility or sterility testing, or in the investigation of suspected cancer. Biopsies are discouraged in horses in particular, since they may result in testicular sclerosis and atrophy (Galina, 1971; Smith, 1974; Marusi and Corradi, 1989; Varner, 1991; Del Vento et al ., 1992; Threlfall and Lopate, 1993; Roser, 2000). In humans (Foresta and Varotto, 1992; Foresta et al ., 1992; Kessaris et al ., 1995; Harrington et al., 1996) good results are currently being reported with the use of tru-cut needles (needle assemblies with an inner recess that cuts and traps the tissue sample). These can be used to remove a fragment of parenchymal tissue with minimal invasiveness. Our aim was to study the use of this instrument in stallions; to assess the adequacy of the biopsy sample in relation to the size of the needle and sample retrieval mechanism used (either springloaded or manual); and to analyse the suitability for histology of the specimen. We were also interested in the short- and long-term outcomes of the procedure.

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.

Host cell tropism, genome characterization, and evolutionary features of OaPV4, a novel Deltapapillomavirus identified in sheep fibropapilloma

Investigating papillomavirus (PV) diversity is crucial to fully comprehend pathogenicity, genetic features, and evolution of taxa hosted by domestic and wild animal species. This study reports the identification of OaPV4, a novel ovine PV type within Deltapapillomaviruses 3. The study of OaPV4 genomic features combined to in situ hybridization and immunohistochemistry investigations allowed extrapolating several general biological features of ovine PVs, such as their cellular tropism, pathogenicity, and evolutionary history. Based on results, ovine PVs can be grouped into a polyphyletic ancient group of viruses, which splits in two main subgroups having peculiar cellular tropism and pathogenicity. Results add up to animal PV diversity and are crucial to future studies aimed to investigate the correlation between animal PV and cutaneous benign and malign proliferations.

Unveiling mRNA Changes During Meiotic Progression and Pre-Implantation Development: Help from Large Animal Models

Assisted reproductive technologies (ART) are successfully applied in several mammals, including humans, thanks to the ability of oocytes and embryos to face maturation, fertilization and first development in vitro. However, efficiency and safety of ART represent main issues. Mammalian oocytes and early embryos are transcriptionally inactive, and rely exclusively on maternal RNAs and proteins, deposited during oocyte growth, until embryonic genome activation (EGA). Such transcriptional quiescence needs complex post-transcriptional and post-translational mechanisms to coordinate meiotic maturation, fertilization, and reprogramming of the nascent genome. These events are the final outcome of complex, hormonally regulated biological processes that translate into specific molecular mechanisms, which are still far from being fully understood. A deep knowledge of these early phases of development is crucial to understand the core mechanisms of life onset, and to optimize the safety and efficiency of in vitro reproductive technologies. This work focuses on meiotic progression and pre-implantation development in mammals, underlining the importance of fundamental molecules stored during oocyte growth and selectively used during early embryogenic stages. Taking into account the species-specific behaviour of these pivotal molecules, this review describes the advantages of using large domestic animals for research in the reproductive field and proposes large domestic animals as models to improve human ART.

Lipid droplet distribution of immature canine oocytes in relation to their size and the reproductive stage

This study investigated the distribution of lipid droplets (LD) in immature canine oocytes in relation to their size and the reproductive stage. Oocytes were collected from the ovaries of bitches at different estrous stages, divided according to their size (110-120 µm; >120 µm), and stained with Nile Red to detect lipid droplet distribution. At the follicular phase most of the oocytes displayed a diffuse pattern of LD distribution, whereas at anestrus and luteal phase oocytes showed LD mainly in a peripheral/ perinuclear LD distribution. A significantly higher intensity of LD has been recorded in the oocytes > 120 µm compared to those of smaller size (110 - 120 µm) at all stages of the estrous cycle. At follicular phase, oocytes > 120 µm displayed LD intensity similar to that of oocytes > 120 µm at luteal phase and higher compared to the oocytes of the other groups.

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.