Short-term preservation of canine preantral follicles: Effects of temperature, medium and time

Abstract

The use of the large pool of preantral follicles is a promising alternative to provide high numbers of fertilizable oocytes to reproductive biotechnology. This issue is particularly important to canids, since current rates of success of in vitro techniques using oocytes are very limited, and many species within this family are threatened by extinction. The aim of this study was to evaluate effects of temperature, medium and time on morphology and viability of canine preantral follicles during short-term preservation. Canine ovaries were cut into fragments which were incubated in 0.9% NaCl solution or in minimum essential medium (MEM) at 4, 20 or 38 °C for 2, 6, 12 or 24 h. Afterwards, preantral follicles were analyzed by histology, transmission electron microscopy and viability testing using trypan blue, calcein-AM and ethidium homodimer-1. Percentages of morphological normal and viable follicles were maintained similar to control (time 0 h) after incubation in 0.9% NaCl at 4 or 20 °C for up to 6 h and at 38 °C for 2 h. Using MEM, such preservation was possible for 12 h at 4 or 20 °C, and for 6 h at 38 °C. These results indicate that preservation of canine preantral follicles might be better accomplished through hypothermic (4 or 20 °C) storage in MEM, which ensures maintenance of morphology and viability for up to 12 h.

In vitro development of primordial follicles after long-term culture of goat ovarian tissue

“…Therefore, despite being a time-consuming and expensive technique, TEM is an important tool for detecting early morphological changes after follicular culturing in vitro. In addition, according to Lopes et al (2009), follicular atresia changes occur first at the ultrastructural level before being able to be identified by light microscopy.…”

Efeito Do Intervalo De Secção Sobre O Isolamento Mecânico De Folículos Pré-Antrais De Cadelas

“…A presença da membrana basal em folículos isolados pode afetar a eficiência do cultivo in vitro oferecendo uma série de vantagens, como a preservação da morfologia celular e manutenção da adesão aos componentes extracelulares Machado et al, 2002). Vários corantes como o azul de trypan, calceína-AM e etídio homodímero podem ser associados ao corante Hoechst 33342 para testar a viabilidade, número de células da granulosa e integridade de folículos pré-antrais caninos em diferentes meios de preservação (Lopes et al, 2009).…”

“…As biotécnicas reprodutivas em pequenos animais têm sido pouco empregadas, sendo que um dos fatores limitantes em cães são a anatomia e a fisiologia peculiares desta espécie (Lopes, 2001). Diferente das demais espécies, pouco sucesso tem sido obtido com a transferência de embriões, bem como com a indução da superovulação em cadelas (Tsutsui et al, 1989).…”

Estimation, morphometry and ultrastructure of ovarian preantral follicle population in agouti (Dasyprocta leporina)

“…PFs were classified and counted as morphologically normal, when the oocyte nucleus and an intact oocyte was present surrounded by granulosa cells that were well organized in one or more layers , and organized layers of granulosa cells, or atretic, when the oocyte exhibited pycnotic nucleus and/or ooplasma shrinkage, and occasionally granulosa cell layers became disorganized, and/or included enlarged cells. To avoid evaluating and counting a follicle more than once, preantral follicles were analyzed only in the sections where oocyte nucleus were observed (Lopes et al 2009). …”