Morphological and ultrastructural analysis of sheep primordial follicles preserved in 0.9% saline solution and TCM 199

Matos, Maria Helena Tavares de; Andrade, Evelyn Rabelo; Lucci, Carolina Madeira; Báo, Sônia Nair; Silva, José Roberto Viana; Santos, Regiane R.; Ferreira, Marcos; Costa, Sónia; Celestino, Juliana Jales de Hollanda; Figueiredo, José Ricardo de

doi:10.1016/j.theriogenology.2003.07.025

Cited by 25 publications

(35 citation statements)

References 38 publications

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“…In this context, studies have assessed short-term preservation of preantral follicles combining different temperatures and media in several species such as caprine (Silva et al, 2000, ovine , Andrade et al, 2002a, Andrade et al, 2002band Matos et al, 2004, bovine and swine (Lucci et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

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

Lopes

Santos

Celestino

et al. 2009

Animal Reproduction Science

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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.

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Section: Introductionmentioning

confidence: 99%

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

Lopes

Santos

Celestino

et al. 2009

Animal Reproduction Science

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“…In sheep, Carnoy effectively preserved the basement membrane of the cells, leading to 80% of normal follicles (MATOS et al, 2004). However, Santos et al (2012) observed that the use of Bouin and Carnoy resulted in a better maintenance of bovine ovarian morphology.…”

Section: Discussionmentioning

confidence: 99%

“…The 10% Formaldehyde is commonly used for tissues assigned to evaluation by optical microscopy and is viable for different tissue types (FOX et al, 1985). Good results were obtained with 4% Paraformaldehyde , Bouin (ANDRADE et al, 2005) and Carnoy (MATOS et al, 2004) for fixation of ovarian tissue obtained from sheep and cattle. However, data regarding the use of these fixatives in equine ovarian tissue are scarce.…”

Section: Introductionmentioning

confidence: 99%

Efeito do tipo de fixador e tempo de fixação na morfologia de folículos pré-antrais equinos

et al. 2016

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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 µm 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. Key words: Bouin, Carnoy, equine, preantral follicles, 10% formaldehyde, ovary ResumoO 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...

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“…Studies have demonstrated that storage of ovine and caprine ovarian fragments at 4°C results in proper rates of morphologically normal follicles compared to higher temperatures such as 20 or 35 °C (ANDRADE et al, 2001;MATOS et al, 2004;COSTA et al, 2005). Chaves et al (2008) have shown that ovarian tissue transportation in Minimal Essential Medium (MEM) at 4°C for up to 4 h maintained the percentages of morphologically normal follicles similar to those observed in fresh tissues even after 7 days of in vitro culture.…”

Section: Introductionmentioning

confidence: 97%

Effect of ovarian tissue storage in Morus nigra extract on the morphology and DNA fragmentation of ovine preantral follicles

Cavalcante

Barberino

Gouveia

et al. 2017

SCA

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This study demonstrated the effect of Morus nigra leaf extract during ovine ovarian tissue transportation on the survival and apoptosis of preantral follicles in vitro. High Performance Liquid Chromatography (HPLC) was used to determine the fingerprint chromatogram of the crude ethanolic extract. Four pairs of ovaries from four sheep were collected. The ovarian cortex was fragmented and one fragment was fixed in 10% buffered formaldehyde and processed for histological and TUNEL analyses (fresh control). The other fragments were placed in Minimal Essential Medium (MEM -control medium) or M. nigra extract (0.025; 0.05 or 0.1 mg/mL) and stored (simulating transport) at 4ºC for 6, 12 or 24 h. Preserved fragments (6 h) were also destined to histological and TUNEL analyses. HPLC analysis confirmed the presence of antioxidant compounds (rutin, isoquercetin e kaempferitrin) in the extract. There was a decrease (P < 0.05) in the percentage of morphologically normal preantral follicles after preservation in all treatments compared to the fresh control. The percentage of normal preantral follicles after preservation in M. nigra at 0.05 mg/mL for 6 h was higher (P < 0.05) than in MEM or 0.025 mg/ mL M. nigra and similar (P > 0.05) to 0.1 mg/mL of the extract. Apoptosis increased (P < 0.05) after conservation for 6 h in all treatments compared to the fresh control. Moreover, TUNEL positive cells decreased (P < 0.05) after preservation in 0.05 or 0.1 mg/mL M. nigra compared to MEM or 0.025 mg/ mL M. nigra. In conclusion, 0.05 mg/mL M. nigra extract can be used as a preservation medium for ovine ovarian tissue at 4°C for up to 6 h. ResumoEste estudo demonstrou o efeito do extrato das folhas de Morus nigra durante o transporte na sobrevivência e apoptose de folículos pré-antrais in vitro. A CLAE (Cromatografia Líquida de Alta Eficiência) foi usada para determinar a impressão digital cromatográfica do extrato etanólico. Foram coletados 4 pares de ovários de 4 ovelhas. O córtex ovariano foi fragmentado e um dos fragmentos foi fixado em formaldeído tamponado a 10% e destinado às análises histológica e de TUNEL (controle fresco). Os demais fragmentos foram colocados em Meio Essencial Mínimo (MEM -meio controle) ou extrato de M. nigra (0,025, 0,05 ou 0,1 mg/mL) e conservados (simulando transporte) à 4 ºC por 6, 12 ou 24 h. Os fragmentos conservados (6 h) também foram destinados às análises histológica e de TUNEL. A análise de CLAE confirmou a presença de compostos antioxidantes (rutina, isoquercetina e canferitrina) no extrato. Houve uma redução (P < 0,05) na percentagem de folículos pré-antrais morfologicamente normais após conservação em todos os tratamentos em comparação com o controle fresco. A percentagem de folículos pré-antrais normais após conservação em M. nigra a 0,05 mg/mL por 6 h foi maior (P < 0,05) do que em MEM ou M. nigra a 0,025 mg/mL e similar (P > 0,05) ao extrato a 0,1 mg/mL. A apoptose aumentou (P < 0,05) após conservação durante 6 h em todos os tratamentos em comparação com o controle fresco. Além diss...

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Morphological and ultrastructural analysis of sheep primordial follicles preserved in 0.9% saline solution and TCM 199

Cited by 25 publications

References 38 publications

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

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

Efeito do tipo de fixador e tempo de fixação na morfologia de folículos pré-antrais equinos

Effect of ovarian tissue storage in Morus nigra extract on the morphology and DNA fragmentation of ovine preantral follicles

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