Changes in acetylation of lysine 5 on histone H4 in canine oocytes following in vitro maturation.

Post-translational modifications of histones, such as acetylation, are involved in regulating chromatin remodelling and gene expression. Proper in vitro maturation (IVM) of canine oocytes, for many reasons, is up to now inefficient. This study aimed to evaluate the post-translational histone H4 acetylation at lysine 5 (H4K5) in immature and post-IVM canine oocytes. Oocyte nuclear stage was assessed using Hoechst 33342 staining. Acetylation patterns were determined by indirect immunofluorescence staining of immature and post-IVM oocytes, using an antibody against the acetylated lysine 5 residue on histone 4 (H4K5ac). The experiment was repeated four times, with a total of 7-17 oocytes evaluated per stage. Immunofluorescence signal was quantified using the NIHimagej software. Data were expressed as a percentage of the average fluorescence intensity of the specific antibody over the intensity of DNA, as determined by Hoescht staining. H4K5ac displayed a significantly higher acetylated pattern in immature oocytes (0.97 ± 0.08) when compared to post-IVM oocytes at different nuclear stages. There was a decrease in the fluorescence level of the matured oocytes with the progression of meiosis (GVBD: 0.47 ± 0.06 and MI/MII: 0.35 ± 0.04). Similarly to other domestic species, we hypothesized that post-translational modification of histone acetylation takes place during meiosis of in vitro matured canine oocytes. However, it remains to be investigated whether these changes occur during in vivo maturation.

Matrix-assisted laser desorption/ionization imaging mass spectrometry for the spatial location of feline oviductal proteins.

With 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 ß10, thymosin ß4 and S100, respectively. Keratin and thymosins are involved in the biological response to tissue damage. S100 proteins are calcium-modulated 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 vitro maturation of oocytes from Santa Ines ewes subjected to consecutive sessions of follicular aspiration by laparoscopy.

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.

In vitro survival of follicles collected from domestic cats' ovaries at different stages of oestrous cycle and cultured with IGF-1.

Optimal 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 µm, 66.7%, respectively) than that of follicles collected at the luteal phase and cultured without IGF-1 (26.7 ± 14.4 µm, 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 µm, 63.6%, respectively) than that cultured without IGF-1 (26.7 ± 14.4 µm, 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.

Aspectos atuais das biotecnologias da reprodução em carnívoros / Current aspects of biotechnology of reproduction in carnivores

O desenvolvimento de técnicas de reprodução assistida no cão e no gato desperta grande interesse, pois poderiam ser utilizadas para a conservação de espécies de carnívoros selvagens ameaçadas de extinção. Nos últimos anos houve um grande avanço das biotecnologias da reprodução aplicáveis aos felinos domésticos. No entanto, o seu desenvolvimento nos caninos domésticos tem sido dificultado pela baixa eficiência. Um dos maiores problemas reside na fisiologia única dos gametas caninos, que exigem meios complexos para a maturação e desenvolvimento, o que dificulta a implementação de protocolos e técnicas que têm sido eficazes em outras espécies. O objetivo desta revisão é descrever os avanços nas biotecnologias da reprodução que se aplicam aos cães e gatos, com enfoque sobre os aspectos que podem ser importantes para o uso em espécies protegidas de carnívoros.

The development of assisted reproductive techniques (ART) in dogs and cats is an area of increasing interest because ART can be potentially applied to non-domestic carnivores threatened with extinction. Much progress has been achieved in ART of the feline species. However, advances on assisted reproduction in dogs are hampered by its low efficiency. A major obstacle lies on the particular characteristics of canine gamete physiology, for instance, the highly complex requirements for oocyte maturation and development, which makes difficult to apply protocols/techniques that have been successful in other species. This review aims to depict the advances made on assisted reproductive techniques in dogs and cats, focusing on those features that may be relevant for applying these techniques to wild endangered carnivores.

CURRENT ASPECTS OF BIOTECHNOLOGY OF REPRODUCTION IN CARNIVORES. / Aspectos atuais das biotecnologias da reprodução em carnívoros

O desenvolvimento de técnicas de reprodução assistida no cão e no gato desperta grande interesse, pois poderiam ser utilizadas para a conservação de espécies de carnívoros selvagens ameaçadas de extinção. Nos últimos anos houve um grande avanço das biotecnologias da reprodução aplicáveis aos felinos domésticos. No entanto, o seu desenvolvimento nos caninos domésticos tem sido dificultado pela baixa eficiência. Um dos maiores problemas reside na fisiologia única dos gametas caninos, que exigem meios complexos para a maturação e desenvolvimento, o que dificulta a implementação de protocolos e técnicas que têm sido eficazes em outras espécies. O objetivo desta revisão é descrever os avanços nas biotecnologias da reprodução que se aplicam aos cães e gatos, com enfoque sobre os aspectos que podem ser importantes para o uso em espécies protegidas de carnívoros. SUMMARY   The development of assisted reproductive techniques (ART) in dogs and cats is an area of increasing interest because ART

Aspectos atuais das biotecnologias da reprodução em carnívoros / Current aspects of biotechnology of reproduction in carnivores

O desenvolvimento de técnicas de reprodução assistida no cão e no gato desperta grande interesse, pois poderiam ser utilizadas para a conservação de espécies de carnívoros selvagens ameaçadas de extinção. Nos últimos anos houve um grande avanço das biotecnologias da reprodução aplicáveis aos felinos domésticos. No entanto, o seu desenvolvimento nos caninos domésticos tem sido dificultado pela baixa eficiência. Um dos maiores problemas reside na fisiologia única dos gametas caninos, que exigem meios complexos para a maturação e desenvolvimento, o que dificulta a implementação de protocolos e técnicas que têm sido eficazes em outras espécies. O objetivo desta revisão é descrever os avanços nas biotecnologias da reprodução que se aplicam aos cães e gatos, com enfoque sobre os aspectos que podem ser importantes para o uso em espécies protegidas de carnívoros.(AU)

The development of assisted reproductive techniques (ART) in dogs and cats is an area of increasing interest because ART can be potentially applied to non-domestic carnivores threatened with extinction. Much progress has been achieved in ART of the feline species. However, advances on assisted reproduction in dogs are hampered by its low efficiency. A major obstacle lies on the particular characteristics of canine gamete physiology, for instance, the highly complex requirements for oocyte maturation and development, which makes difficult to apply protocols/techniques that have been successful in other species. This review aims to depict the advances made on assisted reproductive techniques in dogs and cats, focusing on those features that may be relevant for applying these techniques to wild endangered carnivores.(AU)

CANINE TRANSMISSIBLE VENEREAL TUMOR (TVT) IN THE UTERUS: CASE REPORT / TUMOR VENÉREO TRANSMISSÍVEL (TVT) CANINO NO ÚTERO: RELATO DE CASO.

The canine transmissible venereal tumor (TVT) is a sexually transmissible neoplasm found in the external genitalia of dogs. It is clinically characterized by the growth of friable and hemorrhagic tissue in the genital tract, with serosanguineous and fetid vaginal discharge. The diagnosis is based on anamnesis and on clinical signs, however, cyto or histopathological evaluation are also necessary. Several treatments have been used for the management of the tumor, which may include surgical removal, radiotherapy, immunotherapy, biotherapy and chemotherapy. The purpose of this study is to describe the clinical-surgical aspect of a female dog with transmissible venereal tumor located in the external genitalia, with an atypical metastasis in the uterus, and clinical urinary signs as dysuria and hematuria. The surgical excision of the tumor with cauterization of the remaining tissue presented good results and few complications. KEYWORDS: Transmissible venereal tumor. TVT. Uterus. Bitch.

CURRENT ASPECTS OF BIOTECHNOLOGY OF REPRODUCTION IN CARNIVORES. / Aspectos atuais das biotecnologias da reprodução em carnívoros.

O desenvolvimento de técnicas de reprodução assistida no cão e no gato desperta grande interesse, pois poderiam ser utilizadas para a conservação de espécies de carnívoros selvagens ameaçadas de extinção. Nos últimos anos houve um grande avanço das biotecnologias da reprodução aplicáveis aos felinos domésticos. No entanto, o seu desenvolvimento nos caninos domésticos tem sido dificultado pela baixa eficiência. Um dos maiores problemas reside na fisiologia única dos gametas caninos, que exigem meios complexos para a maturação e desenvolvimento, o que dificulta a implementação de protocolos e técnicas que têm sido eficazes em outras espécies. O objetivo desta revisão é descrever os avanços nas biotecnologias da reprodução que se aplicam aos cães e gatos, com enfoque sobre os aspectos que podem ser importantes para o uso em espécies protegidas de carnívoros. SUMMARY   The development of assisted reproductive techniques (ART) in dogs and cats is an area of increasing interest because ART