Interval in the replacement of in vitro culture medium affects the integrity and development of equine preantral follicles

ABSTRACT: The efficiency of a culture system is related to the elaboration and replacement of a medium with conditions suitable for follicular development. Recent investigations suggested that in vitro culture medium should be replaced after specific time periods in various species. However, the suitable interval for the exchange of in vitro culture medium has not yet been established in equine species. The objective of this investigation was to evaluate the effect of medium exchange intervals of 24 hours (T24) or 48 hours (T48) for in vitro culture of preantral follicles at 2 or 6 days. At the end of the culture period, the fragments were processed using classical histology. Equine preantral follicles were classified according to morphological integrity and developmental stage. Data analysis was performed using Fisher’s test with a significance level of p0.05). Compared to the medium replacement at six days of culture, there was a statistically significant difference for T24 (68.9%, p<0.05). Therefore, we suggest changing the medium for equine species at 48 hours after the start of culture followed by subsequent daily replacements

Effect of fixative type and fixation time on the morphology of equine preantral ovarian follicles

O objetivo deste estudo foi investigar a eficácia dos fixadores teciduais Bouin, Carnoy ou Formol 10% em fragmentos ovarianos equinos. Ovários (n=4) de éguas, sem raça definida, foram obtidos de abatedouro local e transportados em recipiente térmico a 20 ºC. Imediatamente após a coleta, os ovários foram lavados com solução de PBS modificado (Cultilab®, Campinas-SP, Brasil), e divididos em nove fragmentos com aproximadamente 5x5x1 mm, retirados do parênquima de cada ovário. Em seguida, os fragmentos ovarianos foram imersos em um dos três diferentes fixadores, Bouin (B), Carnoy (C) ou Formol 10% (F), por 6, 12 ou 24 horas. Cada fragmento foi acondicionado individualmente em um frasco contendo aproximadamente 20 vezes o volume da solução fixadora. Após este período, foram mantidos em álcool 70% por 24 horas. Para cada fixador e tempo foram realizadas quatro réplicas. No processamento histológico, os fragmentos foram desidratados em concentrações crescentes de álcool, diafanizados em xilol e incluídos em parafina. Em seguida, foram feitos cortes seriados de 5 ?m em micrótomo rotativo (Leica®, Wetzlar-Alemanha), seguidos da montagem de lâminas e coloração com ácido periódico de Schiff (PAS) e hematoxilina. Foram avaliadas 540 lâminas com 1.620 cortes histológicos, contendo 465 folículos pré-antrais que foram classificados como íntegros ou degenerados. A degeneração foi detectada pela presença de pelo menos um dos seguintes aspectos: retração do citoplasma, núcleo picnótico, vacúolos citoplasmáticos, deslocamento das células da granulosa e/ou rompimento da membrana basal. Um teste de regressão logística foi utilizado para a análise estatística, e as diferenças foram consideradas significativas quando P<0,05. O fixador Carnoy utilizado por 24 horas proporcionou as melhores condições de integridade morfológica (53,3%; 32/60) em relação aos demais, sendo Boiun por 24 h o tratamento menos eficaz (19,1%; 9/47). Os demais tratamentos apresentaram os resultados a seguir: C12h 50% (30/60), C6h 40% (24/60), F24h 37,8% (17/45), F12h 35,1% (13/37), F6h 32% (16/50), B12h 30,5% (18/59) e B6h 24,4% (11/45). Portanto, sugerimos que a fixação de tecido ovariano equino com Carnoy por 24 horas é o mais indicado para preservação morfológica de folículos pré-antrais.The aim of this study was to investigate the efficacy of the tissue fixatives Bouin, Carnoy and 10% Formaldehyde in equine ovarian fragments. Ovaries (n=4) from mares of mixed breeds were obtained at a local slaughterhouse and transported at 20 ºC in a thermo container. Immediately after collection, the ovaries were washed with a modified PBS solution (Cultilab®, Campinas-SP, Brazil) and divided into nine fragments with approximately 5x5x1 mm, removed from the parenchyma of each ovary. The ovarian fragments were then immersed in three different fixatives, Bouin (B) Carnoy (C) or 10% Formaldehyde (F) for 6, 12 or 24 hours. Each fragment was individually immersed in a 20 mL tube containing 20 times the volume of fixative solution. After this period, the fragments were held in 70% ethanol for 24 hours. Each procedure was performed in four replicates. For histological analysis, the specimens were dehydrated in increasing concentrations of alcohol, submitted to diaphanization in xylol and embedded in paraffin. Serial sections of 5 ?m were made with the use of a rotating microtome (Leica® type, Wetzlar, Germany), followed by slide mounting and staining with periodic acid-Schiff (PAS) and hematoxylin. A total of 540 slides with 1,620 sections were evaluated, which contained 465 preantral follicles that were classified as normal or degenerated. Follicles were considered as degenerated when presented at least one of the following aspects: cytoplasm retraction, pyknotic nucleus, cytoplasmic vacuoles, displacement of granulosa cells and/or disruption of the basal membrane. A logistic regression test was used for statistical analysis, and differences were considered significant when P<0.05. The Carnoy fixative, when used for 24 hours, provided the best conditions of morphological integrity (53.3%; 32/60) compared to all others, and the use of Boiun for 24 hours was considered the worst treatment (19.1%; 9/47). The other treatments lead to the following results: C12h 50% (30/60), C6 H 40% (24/60), F24h 37.8% (17/45), F12h 35.1% (13/37), F6h 32% (16/50), B12h 30.5% (18/59) and B6h 24.4% (11/45). Therefore, we suggest that fixation of equine ovarian tissue with Carnoy for 24 hours is the most suitable protocol for morphological preservation of pre-antral follicles

Recovery of equine oocytes by scraping of the follicular wall with different specifications of needles and morphological analysis of cumulus oophorus

In follicular aspiration, physical aspects are of high significance for the technique to succeed, such as vacuum pressure, caliber of the needle and the way the follicular wall curettage is performed. The aim of this study was to investigate the recovery rate of equine oocytes aspirated by scraping of the follicular wall, testing different calibers of disposable needles, as well as the morphological evaluation of the cumulus oophorus complexes (COCs). Mares ovaries (n=447) obtained at a local slaughterhouse were transported to the laboratory in a thermal container (20 °C) and had the tunica albuginea and connective tissues dissected. The aspirated follicles had 10 to 25 mm in diameter, and 30x8 (21G 1 ¼) or 40x12 (18G 1 ½) needles were used for the aspiration, forming group A (G-A) and group B (G-B), respectively. In G-A and G-B, 480 and 548 follicles were aspirated, respectively. Under the stereomicroscope, the oocytes were evaluated according to the quality of the ooplasm and characteristics of the cumulus cells (grade I, II, III and denuded). The statistical analysis was performed using the Student’s t-test, logistic regression and test of proportions, and differences were considered significant when P&lt;0.05. There was no difference between recovery rates of groups G-A (66.5%; 330/496) and G-B (65.5%; 359/548). In the G-A group, grade II oocytes were related to higher recovery rates (46.9%; 145/330) than grade I (23.6%; 72/330), grade III (20.6%; 59/330) and denuded oocytes (8.5%; 24/330; P&lt;0.05). However, in G-B, there was no statistical difference regarding the quality of the recovered oocytes: grade I (23.4%; 77/359), grade II (43.2%; 145/359), grade III (22.5%; 73/359) and denuded (11.1%; 32/359). The 30x8 (21G 1 ¼) needle provided a higher proportion of grades I and II oocytes than the 40x12 (18G 1 ½) needle, with 72.4% (239/330) and 65% (233/359; P&lt;0.05), respectively. Both calibers of needles tested in this study provide efficient oocyte recovery rates. Aspiration with 30x8 (21G 1 ¼) needles resulted in a higher proportion of morphologically good equine oocytes for use in reproductive biotechnologies. </p