Histological Evaluation of the Effects of Cryopreservation in Bovine Ovarian Tissue

Abstract

The aim of this study was to show the effects of freezing and thawing in bovine ovarian tissue by histological analysis. Ten cortical slices (2-4 mm in diameter) were obtained from each ovary by tru-cut biopsy and randomly divided into two groups: five fragments were immediately processed as a fresh tissue control group, while the remaining 5 fragments were slowly frozen using DMSO plus sucrose as cryoprotectors, then stored for two weeks and quickly thawed. Histological examination of all cryopreserved ovarian fragments showed no damage in the structure of the organ. Furthermore, there was no difference in the average number of primordial and primary follicles between the two groups of ovarian tissue. These data suggest that the bovine ovary can be used as a suitable model to test new freezing and thawing procedures in search for a standard protocol of human ovary cryopreservation.

Use of trypan blue staining to assess the quality of ovarian cryopreservation

“…These results are consistent with the results of a comparative study on fresh and frozen-thawed ovarian tissue using DMSO or 1,2-propanediol (PROH) as cryoprotectants in human (34,35) and in bovine ovarian tissue (36). The complementary results of the study show that the freezing combined with thawing induce an increase in the detection of apoptotic cells.…”

“…The complementary results of the study show that the freezing combined with thawing induce an increase in the detection of apoptotic cells. The studies on the sheep did not find more fragmentation of DNA after freezing (36,37), but the interpretation of results and the study model were different from ours. However, results of these investigators regarding the percentage and distribution of morphologic abnormalities showed tissue injury by cryopreservation.…”

Effects of neutral red assisted viability assessment on the cryotolerance of isolated bovine preantral follicles

“…To our knowledge, we are the first to report on successfully vitrified isolated bovine follicles, in combination with repeated NR viability assessment prior and following cryopreservation and subsequently cultured for a short-term period. In line with earlier work on follicles embedded in cortical tissue strips [56,57], we showed that cryopreservation, as compared with non-cryopreserved follicles, has an overall negative effect on the viability of isolated (bovine) PAFs, as noticed immediately after warming and at 4 days of culture. We have used NR as an indicator or build-in sentinel for easy and routine applicable assessment of PAF's metabolic activity.…”

Morfometria, células da granulosa e cromatina nuclear de folículos préantrais em associação ou não com a vitrificação de tecido ovariano bovino

“…Gandolfi et al (2006) compararam duas técnicas de vitrificação com a técnica de congelação lenta usando fragmentos ovarianos de humanos, suínos e bovinos, e concluíram que a vitrificação, independentemente de espécie, classe folicular e técnica, não foi adequada para a preservação de folículos pré-antrais quando comparada com a técnica de congelação lenta. Têm-se obtido excelentes resultados com a congelação lenta de tecido ovariano bovino, em determinados estudos, mais de 70% de preservação da normalidade folicular (Paynter et al, 1999;Lucci et al, 2004;Lamaita et al, 2005;Celestino et al, 2007), levando a assumir que a técnica de congela- Tabela 2. Diâmetro do folículo (mm) e ovócito (mm) e número de células da granulosa (média ± desvio padrão) de folículos primários no T 0 (controle), T I (teste de toxicidade) e T II (vitrificação)* ção lenta é a mais apropriada para criopreservação de tecido ovariano bovino.…”