Cryopreservation of caprine ovarian tissue using glycerol and ethylene glycol

Cryopreservation of ovarian tissue may be a potential alternative for the conservation of genetically superior animals, including high milk- and meat-producing goat breeds. However, until now, no information was available concerning the cryopreservation of preantral follicles (PF) enclosed in caprine ovarian tissue. The objective of the present study was to evaluate the structural and ultrastructural characteristics of caprine PF after exposure to and cryopreservation of ovarian tissue in 1.5 and 3 M glycerol (GLY) and ethylene glycol (EG). At the slaughterhouse, each ovarian pair from five adult mixed breed goats was divided into nine fragments and randomly distributed into treatment groups. One fragment was immediately fixed for histological examination and ultrastructural analysis, after slaughter (control). Four of the ovarian fragments were equilibrated at 20 °C for 20 min in 1.8 ml of MEM containing 1.5 or 3 M GLY or EG for a toxicity test and the final four fragments were slowly frozen using these cryoprotectants at the concentrations above. After toxicity testing and freezing/thawing, the ovarian fragments were fixed for histological examination. Histological analysis showed that after toxicity testing and cryopreservation of the ovarian tissue in GLY or EG at both concentrations, the percentage of normal PF was significantly lower than controls. Ultrastructural analysis of PF frozen in 1.5 and 3 M GLY, as well as 3 M EG demonstrated that these follicles remained morphologically normal. In conclusion, we demonstrated cryopreservation of caprine PF in ovarian tissue

Degeneration rate of preantral follicles in the ovaries of goats

The degeneration rate of ovarian preantral follicles in goats, and the distribution in the follicular classes (primordial, primary or secondary) was assessed. Ovaries from adult goats were collected at a local slaughterhouse. To evaluate the morphology of the caprine preantral follicles in situ, one fragment from each ovary was fixed individually in Carnoy for 12 h, sectioned serially at a thickness of 7 μm and stained with Periodic Acid Shiff-hematoxylin. Preantral follicles were then classified according to the stage of development. Preantral follicles were classified individually either as morphologically normal; as Type 1 degenerated follicles (only the oocyte was degenerated); or as Type 2 degenerated follicles (when degeneration occurred at both oocyte and granulosa cells). The total examined was 235 primordial, 195 primary and 101 secondary follicles. The distribution of degenerated follicles as primordial, primary and secondary follicles was 8.5, 14.3 and 16.8%, respectively. When Types 1 and 2 degenerated follicles were pooled, secondary follicles were significantly more degenerated than primordial and primary follicles. When degeneration Types 1 and 2 was compared in each follicular class, a higher (P<0.05) percentage of Type 1 degeneration was observed in primordial and primary follicles. Conversely, secondary follicles were significantly more affected by Type 2 degeneration. When the follicular classes were taken together, a significantly higher percentage of Type 1 degenerated preantral follicles was observed when compared with Type 2 degenerated follicles (P<0.05). In conclusion, a low percentage of degenerated preantral follicles was observed and secondary follicles were more affected by degeneration than primordial follicles. Thus, primordial follicles constitute a large and potentially valuable source of oocytes for reproductive programs after in vitro growth and maturation

Histological and ultrastructural analysis of cryopreserved sheep preantral follicles

The aim of this study was to verify the histological and ultrastructural characteristics of sheep preantral follicles after exposure of ovarian tissue to cryopreservation in glycerol (GLY), ethylene glycol (EG), propanediol (PROH) or dimethyl sulfoxide (DMSO) in order to determine the optimum method to store sheep ovarian tissue for later experimental or clinical use. Each ovarian pair from five mixed-breed ewes was divided into 17 fragments. One (control) fragment was immediately fixed for routine histological and ultrastructural studies and the remaining (test) fragments were randomly distributed in cryotubes, equilibrated at 20 °C/20 min in 1.8 mL of minimal essential medium (MEM) containing 1.5 or 3 M GLY, EG, PROH or DMSO and then either fixed for morphological studies to determine their possible toxic effect or frozen/thawed and then fixed to test the effect of cryopreservation on preantral follicles. Histological analysis showed that, compared to control fragments, all cryoprotectants at both concentrations significantly reduced the percentage of normal preantral follicles in ovarian fragments prior to or after cryopreservation. PROH 3.0 M appeared to exert a more toxic effect (P < 0.05) than the other cryoprotectants in noncryopreserved tissues. After freezing/thawing, the highest (P < 0.05) percentages of lightmicroscopical normal preantral follicles were observed in ovarian fragments cryopreserved in EG (1.5 and 3 M) or DMSO (1.5 M). However, transmission electronic microscopical (TEM) examination showed that only the DMSO-cryopreserved preantral follicles had normal ultrastructure. The data suggest that sheep preantral follicles should be cryopreserved with 1.5 M DMSO for later clinical or experimental application

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

The objective was to determine the morphological and ultrastructural features of sheep primordial follicles preserved in either 0.9% saline solution or TCM 199 at different temperatures. Soon after death, the ovarian pair of each ewe (n=5) was divided into 25 fragments. One fragment was immediately fixed for morphological evaluation (control). The other 24 fragments were randomly distributed in tubes containing 2 ml of 0.9% saline solution or TCM 199 and maintained at 4, 20 or 39 °C for 2, 4, 12, or 24 h. Based on histological assessment, storage of ovarian fragments in 0.9% saline solution at 20 °C for up to 24 h and in both solutions at 39 °C for 4, 12 or 24 h increased (P<0.01) the percentage of degenerate primordial follicles compared with controls. In contrast, preservation at 4 °C in both solutions, kept the percentage of morphologically normal primordial follicles similar to control values. Although histological integrity of primordial follicles was maintained in fragments stored at 20 °C for up to 24 h in TCM 199, these results were not confirmed by ultrastructural analysis. Based on transmission electron microscopy, only primordial follicles stored at 4 °C for up to 24 h, at 20 °C for up to 12 h and at 39 °C for up to 2 h in both solutions were ultrastructurally normal. In conclusion, sheep primordial follicles were successfully preserved at 4 °C for up to 24 h, at 20 °C for up to 12 h and at 39 °C for 2 h in 0.9% saline solution or TCM 199

Cultivo in vitro de folículos pré-antrais isolados a partir de ovários de vacas zebuínas (Bos taurus indicus)

Dentre as biotecnologias da reprodução, a manipulação de folículos pré-antrais tem sido vista como uma alternativa para a preservação de gametas femininos, uma vez que possibilita o isolamento e o cultivo de um grande número de ovócitos. Assim, o presente estudo teve por objetivo avaliar o crescimento in vitro de folículos isolados a partir de ovários de vacas zebuínas. Os folículos foram isolados mecanicamente e cultivados por 4 dias utilizando-se o meio Waymouth MB 752/1 suplementado com piruvato, l-glutamina, hipoxantina, insulina, transferrina, selênio, ácido ascórbico, soro fetal bovino e eCG. Após o período de cultivo, os folículos passaram de 61,0 ± 18,5 (D0) para 86,2 ± 29,6 mm (D4) de diâmetro. Dos 78 folículos pré-antrais analisados 83,5% apresentavam-se morfologicamente normais no último dia de cultivo, enquanto 11,5% mostravam-se degenerados e 5% rompidos. Um total de 23 folículos foi processado para análise por microscopia de luz. Pela observação de figuras mitóticas constatou-se que 47,8% dos folículos apresentavam proliferação das células da granulosa. Em conclusão, este estudo mostrou que folículos pré-antrais isolados a partir de ovários de vacas zebuínas podem crescer in vitro.Dentre as biotecnologias da reprodução, a manipulação de folículos pré-antrais tem sido vista como uma alternativa para a preservação de gametas femininos, uma vez que possibilita o isolamento e o cultivo de um grande número de ovócitos. Assim, o presente estudo teve por objetivo avaliar o crescimento in vitro de folículos isolados a partir de ovários de vacas zebuínas. Os folículos foram isolados mecanicamente e cultivados por 4 dias utilizando-se o meio Waymouth MB 752/1 suplementado com piruvato, l-glutamina, hipoxantina, insulina, transferrina, selênio, ácido ascórbico, soro fetal bovino e eCG. Após o período de cultivo, os folículos passaram de 61,0 ± 18,5 (D0) para 86,2 ± 29,6 mm (D4) de diâmetro. Dos 78 folículos pré-antrais analisados 83,5% apresentavam-se morfologicamente normais no último dia de cultivo, enquanto 11,5% mostravam-se degenerados e 5% rompidos. Um total de 23 folículos foi processado para análise por microscopia de luz. Pela observação de figuras mitóticas constatou-se que 47,8% dos folículos apresentavam proliferação das células da granulosa. Em conclusão, este estudo mostrou que folículos pré-antrais isolados a partir de ovários de vacas zebuínas podem crescer in vitro

Histopathologic evaluation of the peritoneum exposed to heat shock: experimental study in rats Avaliação histopatológica do peritônio exposto a choque térmico: estudo experimental em ratos

PURPOSE: To evaluate histopathologic alterations of the peritoneum exposed to heat shock. METHODS: Sixty rats were randomly distributed into 6 groups: Heat Shock (HS), High Temperature (HT), Body Temperature (BT), Temperature 0oC (TZ), Sham (SH) and Control (CG) with 10 animals each. The peritoneal cavity of animals from groups HS, HT, BT and TZ was irrigated with NaCl solution 0.9% at temperatures 50ºC, 0ºC, 50ºC, 37ºC and 0ºC, respectively. For animals from group SH, the procedures were simulated and those from group CG, laparotomy and biopsies were conducted. Twenty-four hours later, biopsies of the peritoneum for exams under light and electronic microscopy were performed. RESULTS: Edema was found in groups HS 80%, HT 60%, BT 30% TZ 70%, SH 40% and CG 30%. Vascular congestion was found in groups HS 20%, HT 30%, BT 10% and TZ 20%. Erythrocyte extravasation was found in groups HT 60% and SH 10%. Mesothelium destruction was found in 100% of specimens from groups HS, HT, BT, TZ, SH and CG 90%. Necrosis was found in groups HS 30%, HT 20% and BT 10%. The mean peritoneal thickness ranged from 42.26 µm (TZ) to 26.42 µm (CG). CONCLUSION: The heat shock caused no deaths, but promoted significant peritoneal edema without affecting the other histopathologic indicatives.<br>OBJETIVO: Avaliar alterações histopatológicas do peritônio exposto a choque térmico. MÉTODOS: Sessenta ratos foram distribuídos aleatoriamente em seis grupos: Choque Térmico (CT), Temperatura Elevada (TE), Temperatura 0ºC (TZ) Sham (SH) e Controle (GC) com 10 animais. A cavidade peritoneal dos animais dos grupos CT, TE, TC e TZ foi irrigada com solução de NaCl 0,9% nas temperaturas, 50ºC e 0ºC, 50ºC, 37ºC e 0ºC, respectivamente. Nos animais do grupo SH foram simulados os procedimentos e nos do GC laparotomia e biópsias. Depois de 24 horas foram realizadas biópsias do peritônio para exames sob microscopia de luz e eletrônica. RESULTADOS: Edema foi encontrado nos grupos CT 80%, TE 60%, TC 30%, TZ 70%, SH 40% e GC 30%. Congestão vascular foi encontrada nos grupos CT 20%, TE 30%, TC 10% e TZ 20%. Extravasamento de hemácias foi encontrado nos grupos TE 60% e SH 10%. Destruição de mesotélio foi encontrada em 100% dos espécimes dos grupos CT, TE, TC, TZ, SH e no grupo GC 90%. Necrose foi encontrada nos grupos CT 30%, TE 20% e TC 10%. A espessura média do peritônio variou de 42,26 µm (TZ) a 26,42 µm (GC). CONCLUSÃO: O choque térmico não causou óbitos, mas promoveu edema peritoneal significante sem alterar os demais indicadores histopatológicos

Cryopreservation of caprine ovarian tissue using dimethylsulphoxide and propanediol.

The caprine ovary is a rich source of potentially viable immature oocytes enclosed in preantral follicles (PF). Previous experiments showed that these oocytes can be successfully cryopreserved in ovarian tissue of several species. However, until now, no information about the caprine PF cryopreservation is available in the literature. The aim of the present research was to evaluate the structural and ultrastructural characteristics of caprine PF after treatment and cryopreservation of ovarian tissue with 1.5 and 3 M dimethylsulphoxide (DMSO) and propanediol (PROH). One fragment of ovarian tissue was immediately fixed for histological examination and ultrastructural analysis, after slaughter (control). Four fragments were equilibrated at 20 degrees C/20 min in 1.8 ml of minimum essential medium (MEM) containing 1.5 or 3 M DMSO or PROH for the toxicity test, and the other four fragments were slowly frozen in each cryoprotectant at the concentrations previously described. After toxicity test and freezing/thawing procedures, the ovarian fragments were fixed for histological examination. The results showed that after toxicity test and cryopreservation of ovarian tissue using both cryoprotectants, the percentage of normal PF was less (P < 0.05) as compared with the control group. The present study revealed that the percentage of normal PF after toxicity test and cryopreservation in 1.5 M DSMO was significantly greater (P < 0.05) as compared with results obtained with 3 M DMSO or 1.5 and 3 M PROH. This result was confirmed by transmission electron microscopy, which showed that the PF were preserved in a higher quality state with 1.5 M DMSO. In conclusion, the present study demonstrated that caprine PF can be cryopreserved in ovarian tissue using 1.5 M DMSO