ContentsOptimal conditions for in vitro culture of feline ovarian follicles have not yet been defined. Follicular development is regulated by intraovarian growth factors, as insulin-like growth factor (IGF-1), and during the different stages of the oestrous cycle, follicles are exposed to specific hormonal environments. The aim of this study was to investigate the effect of IGF-1 on in vitro growth and granulosa cell (GC) viability of preantral follicles collected from domestic cats at follicular and luteal phases of the oestrous cycle. Oestrus and ovulation were induced in 12 cats. A total of 39 and 32 follicles collected at the follicular and luteal phases, respectively, were individually cultured in vitro for 6 days in minimum essential medium media supplemented with or without IGF-1 (100 ng/ ml). Follicles collected during the follicular phase and cultured without IGF-1 displayed a significant increase in size and higher GC viability (46.5 ± 22.1 lm, 66.7%, respectively) than that of follicles collected at the luteal phase and cultured without IGF-1 (26.7 ± 14.4 lm, 50%, respectively; p < 0.05). In contrast, when IGF-1 was added to the culture medium, no differences were observed in size or GC viability between follicles collected at the two phases of the cycle. Nonetheless, follicles collected at the luteal phase and cultured with IGF-1 had a significant increase in their diameter and GC viability (31.9 ± 15.9 lm, 63.6%, respectively) than that cultured without IGF-1 (26.7 ± 14.4 lm, 50%, respectively; p < 0.05). These data suggest that in vitro growth and GC survival of feline preantral follicles are affected by the oestrous cycle phase, and the IGF-1 exerts a positive effect on follicles collected at the luteal phase.
The success of embryo production in vitro depends upon the use of an efficient oocyte retrieval technique, and the best results have been obtained by laparoscopic aspiration. The aim of this study was to evaluate the effect of consecutive sessions of follicular aspiration on the quantity, quality and in vitro maturation competence of oocytes obtained from ewes subjected to hormonal stimulation. Six Santa Ines ewes underwent nine sessions of follicular aspiration by laparoscopy with a 7-day interval between sessions, totalling 56 aspirations. After 24 h of culture, oocytes were stained and classified according to the stage of nuclear and cytoplasmic maturation. Oocyte retrieval rate was 61.4 ± 2%, resulting in a total of 249 oocytes. No significant variation was observed between sessions (p > 0.05). The average number of oocytes retrieved from each ewe was 6.4 ± 2 per session and 42 ± 4 in total. No significant difference was observed between the frequencies of the different stages of nuclear maturation: 32.72% mature, 40.74% immature and 26.54% degenerated/indeterminate oocytes; however, a significant difference was observed between the frequencies of the different stages of cytoplasmic maturation: 10.7% mature, 73.25% immature and 16.05% degenerated/indeterminate oocytes. No significant difference was observed in nuclear or cytoplasmic maturation between the weeks of procedure. We conclude that after nine consecutive sessions of follicular aspiration, the quantity and quality of retrieved oocytes remained unchanged as well as the levels of nuclear and cytoplasmic maturation obtained, demonstrating the viability of this technique for repetitive follicular aspirations on the same donor.
The aims of this study were to investigate: 1) if the addition of α-tocopherol (vitamin E) in three concentrations (0.3, 0.6 and 0.9 mM) is able to preserve spermatozoa integrity after thawing and 2) the effect of α-tocopherol supplementation on lipid peroxidation. Fifty four domestic cats were used in this study constituting 18 pools (3 cats per pool). Each pool was submitted at four experimental groups: group 0 (control)-epididymal sperm were frozen with a commercial Botucrio ® extender; group 0.3, group 0.6 and group 0.9-the extender was supplemented with 0.3, 0.6 and 0.9 mM of α-tocopherol, respectively. Each semen sample was evaluated for motility, progressive forward motility, morphology, sperm viability (plasma membrane integrity-PMI), hypo-osmotic swelling test (HOST), before and after thawing. The evaluation of lipid peroxidation reaction by Thiobarbituric Acid Reactive Substances (TBARS) test was performed on thawed semen only. Results demonstrated that there was no significant difference between control and the three α-tocopherol groups with regards to motility and progressive motility after thawing (P > 0.05). As expected, in fresh samples viability was significantly higher than in all the cryopreserved groups in which there was no positive influence of any of the αtocopherol concentration used. Lipid peroxidation was higher in the supplemented groups 0.6 and 0.9 mM of α-tocopherol than in control and in 0.3 mM group. In conclusion, the addition of α-tocopherol to the commercial extender had no positive influence on reduction of lipid peroxidation. This topic deserves further investigations to better understand the effect of cryopreservation procedures on epididymal spermatozoa and to establish adequate strategies to counteract sperm cryodamages.
ContentsWith the purpose of identifying factors involved in early stages of embryo development in the domestic cat, matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) was used for the first time to describe the spatial localization of proteins in the oviducts of queens. Oviducts were obtained from two 2 and 4 years old cross-bred queens, divided into three segments, snap-frozen in liquid nitrogen and then stored at −80°C until use. Next, they were sectioned in a cryostat, fixed on ITO (indium tin oxide) conductive glass slides for MALDI-IMS and serial sections were collected on microscope slides for histology. As confirmed by histology, MALDI-IMS was able to show contrasting protein distributions in the oviductal infundibulum, ampulla and isthmus. Mass spectra were characterized by abundant ions of m/z 1,259, 4,939, 4,960 and 10,626, which have been tentatively attributed to keratin, thymosin β10, thymosin β4 and S100, respectively. Keratin and thymosins are involved in the biological response to tissue damage. S100 proteins are calciummodulated proteins implicated in a variety of cellular activities, including cell differentiation and regulation of cell motility. These results suggest that protein composition differs between segments of the cat oviduct, which corresponds to morphological changes within these sections. Further functional studies could elucidate the effects of these proteins on feline reproductive physiology.
In mammalian oocytes, the migration of cortical granules (CG) is an important step in cytoplasmic maturation and has been used as a criterion in the assessment of maturity and organelle organisation. With the use of fluorescent lectins, it is possible to identify specific sugars originating from the CG and to localise them to the oolemma, within the perivitelline space, or both. To our knowledge, no reports are available on fluorescence assessment of CG organisation in ovine oocytes. Therefore, the aim of this study was to describe the CG distribution of ovine oocytes after in vitro maturation (IVM). One hundred twenty-five oocytes from adults ewes were submitted to IVM in TCM-199 medium supplemented with 10% FBS for 24 h. The denuded oocytes were immersed in 0.1% pronase for 5 min to dissolve the zona pellucidae, fixed in 3% paraformaldehyde for 30 min, and incubated overnight in blocking solution at 4°C. Oocytes were placed into 0.1% Triton X-100 for 5 min for permeabilization and incubated in 10 µg mL–1 of fluorescent Lens culinaris agglutinin–fluorescein complex for 15 min at 37°C before being rinsed and mounted onto histological slides. The distribution of CG was evaluated with an Olympus fluorescence microscope (wavelength: 420 to 490 nm; Olympus, Tokyo, Japan), and oocytes were classified into three types according to the observed distributional pattern of the CG: (I) aggregates of CG distributed over the entire cytoplasm, (II) CG distributed in the cortex and forming a fluorescent halo around the plasma membrane, and (III) CG distributed uniformly over the entire cytoplasm without the presence of aggregates. Proportions of oocyte types were analysed by chi-square or, when appropriate, by Fisher’s exact test, using SAS version 8.2 (SAS Institute Inc., Cary, NC, USA). The proportion of type III oocytes (98/125, 78.4%) was significantly higher (P < 0.05) than those of types I (10/125, 8%) and II (17/125, 13.6%). No significant difference was observed between the proportions of type I and II oocytes. Analogous with the results described in the bovine, the type I distribution of CG in ovine oocytes has been considered a sign of a lack of maturation, even if the aggregates of CG are not as evident as in the bovine; types II and III were considered mature oocytes based on the cortical migration of CG. The cytoplasmic maturation is an essential process in directing and supporting the events of fertilization and early embryonic development. Therefore, describing the species-specific pattern of CG distribution is important for the assessment of oocyte maturation. More studies in the ovine are necessary to confirm the correlation between CG distribution and maturation status, as in bovine species.
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