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OBJECTIVE: To evaluate the effect of dynamized follicle-stimulating hormone (FSH) on the survival, activation and growth of ovine preantral follicles (PFs) in vitro. METHODS: Ovarian fragments were cultured for 1 or 7 days in alpha minimum essential medium (α-MEM(+)) control in the absence or presence of alcohol (Al control) or FSH (6cH, 12cH and 30cH) added at intervals of 24 or 48 h. The ovarian fragments were processed, coded and analyzed by a blinded observer by classical histology (CH), fluorescence microscopy (FM) and transmission electron microscopy (TEM). RESULTS: After 7 days of culture, the group which to which FSH 6cH was added at 24 h intervals showed better rates of follicle survival and activation compared to α-MEM(+) control or Al control (p < 0.05). This group also showed higher follicle and oocyte growth than α-MEM(+) control (p < 0.05). FM and TEM techniques confirmed that FSH 6cH promoted viability and ultrastructural integrity of follicles after 7 days of culture. CONCLUSIONS: FSH 6cH (24 h) treatment maintained the viability, and promoted the activation and in vitro growth of ovine PFs.

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The objective was to develop an efficient protocol for cryopreservation of agouti (Dasyprocta aguti) ovarian tissue. Agouti ovarian fragments were placed, for 10 min, in a solution containing MEM and fetal bovine serum plus 1.5 M dimethyl sulfoxide (DMSO), ethylene glycol (EG) or propanediol (PROH); some of those fragments were subsequently cryopreserved in a programmable freezer. After exposure and/or thawing, all samples were fixed in Carnoy prior to histological analysis. To evaluate ultrastructure, follicles from the control and all cryopreserved treatments were fixed in Karnovsky and processed for transmission electron microscopy. After exposure and freezing, there was a significant decrease in the percentage of morphologically normal preantral follicles in all treatments when compared to the control (92.67 ± 2.79, mean ± SD). However, there were no significant difference when the exposure and freezing procedures were compared using the same cryoprotectant. Moreover, there was no significant difference among cryoprotectants at the time of exposure (DMSO: 64.7 ± 3.8; EG: 70.7 ± 11.2, PROH: 63.3 ± 8.5) or after freezing (DMSO: 60.6 ± 3.6, EG: 64.0 ± 11.9; PROH: 62.0 ± 6.9). However, only follicles frozen with PROH had normal ultrastructure. In conclusion, preantral follicles enclosed in agouti ovarian tissue were successfully cryopreserved using 1.5 M PROH, with satisfactory maintenance of follicle morphology and ultrastructure.

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The aim of this study was to evaluate the influence of the number of follicles per drop (one or three) and antral follicles on in vitro development of isolated goat preantral follicles. Preantral follicles were isolated through microdissection and distributed individually (control) or in groups of three follicles (treatment) in microdroplets of α-MEM with or without 1000 ng/ml follicle stimulating hormone (FSH) for Experiments 1 and 2, respectively. Experiment 3 was divided into four treatments according to the presence of one or three preantral follicles, associated or not with antral follicles. After culture, oocytes were retrieved from morphologically normal follicles and submitted to in vitro maturation (IVM) and live/dead fluorescent labelling. Results of Experiment 1 (basic medium without FSH) showed that culture of preantral follicles in groups enhances viability, growth and antrum formation after 12 days. However, in the presence of FSH (Experiment 2), only the recovery rate of fully grown oocytes for IVM was significantly affected by grouping of follicles. In Experiment 3, in general, co-culture of preantral follicles with an early antral follicle had a detrimental effect on viability, antrum formation and production of oocytes for IVM. In conclusion, the performance of in vitro culture of goat preantral follicles is affected by the number of follicles per drop, the presence of an antral follicle and FSH.

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Este trabalho teve como objetivo avaliar o efeito do hormônio folículo estimulante recombinante (FSHr) sobre o desenvolvimento in vitro de folículos pré-antrais (FOPA) caprinos e ovinos isolados. Folículos pré-antrais (≥ 150 µ m) foram isolados de fragmentos do córtex ovariano de cabras e ovelhas e cultivados individualmente por 24 dias em α-MEM suplementado com 10% de soro fetal bovino (SFB), 1% de insulina-transferrina-selênio (ITS), 1% de antibióticos e 50µg/mL de ácido ascórbico, na ausência (controle) ou presença de FSHr (100 ou 1000 ng/mL), a 39 °C com 5% de CO2 em ar. Durante o cultivo avaliou-se a morfologia, o crescimento folicular e a formação de antro. Foi observado que, na espécie caprina, o cultivo com FSHr apresentou percentuais de folículos morfologicamente normais semelhantes ao controle até o 12º dia de cultivo (P>0,05), ocorrendo uma redução deste parâmetro no 18º dia (P<0,05) somente no tratamento com 1000 ng/mL de FSRr. Na espécie ovina, o percentual de folículos morfologicamente normais foi semelhante em todos os tratamentos, observando-se um decréscimo a partir do 12º dia (P<0,05). Com relação ao desenvolvimento folicular, em caprinos, a adição de FSHr aumentou a taxa de crescimento em relação ao controle (P<0,05). Entretanto, em ovinos, isso só foi observado no tratamento com 1000 ng/mL de FSHr. No que se refere à formação de antro, em ambas as espécies, não houve diferença significativa entre os tratamentos com FSHr e o controle. Quanto à análise da configuração da cromatina, todos os oócitos permaneceram em estádio de vesícula germinativa. Assim, pôde-se concluir que FOPA caprinos e ovinos são capazes de manter a sobrevivência e o crescimento in vitro até o estádio antral na ausência de FSH. Contudo, este hormônio teve um papel importante na taxa de crescimento folicular em ambas as espécies.

The aim of this work was to evaluate the effects of recombinant follicle stimulating hormone (rFSH) on the in vitro development of isolated caprine and ovine preantral follicles. Preantral follicles (≥ 150 micron) were isolated from fragments of goats and sheeps ovarian cortex and individually cultured for 24 days in α-MEM supplemented with 10% fetal calf serum (FCS), 1% insulin-transferrin -selenium (ITS), 1% antibiotics and 50 mg / mL ascorbic acid, in the absence (control) or presence of FSHR (100 or 1000 ng / mL) at 39 ° C with 5% CO2 in air. Follicular morphology, growth and antral cavity formation were assessed throughout the culture. The results showed that addition of rFSH to the medium had no effect on the percentages of caprine morphologically normal follicles until day 12 as compared with the control, but a decrease was observed on day 18 with the use of the hormone (p<0.05) only rFSH (1000 ng/mL). Regarding ovine follicles, percentages of normal follicles were significantly reduced from day 12 onwards in all treatments, which led to similar results. Growth rates were increased in caprine follicles by use of rFSH (100 and 1000 ng/ml) in comparison to the control (p<0,05). In ovine follicles, such effect was observed only when rFSH was used at 1000 ng/ml. With respect to antral cavity formation, no diference among treatments was observed for both species. The analysis of chromatin configuration revealed that all oocytes remained at the germinal vesicle stage. In conclusion, caprine and ovine preantral follicles are capable to survive and develop to the antral stage in vitro without FSH. However, this hormone is important for increasing follicular growth rates in both species.

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The objective was to evaluate the effects of various concentrations of exogenous FSH during in vitro culture of isolated canine preantral follicles. Preantral secondary follicles (>200 microm) were isolated by microdissection and cultured for 18 d in supplemented alpha-Minimum Essential Medium (alpha-MEM). There were three treatment groups: 1) absence of FSH (control medium); 2) FSH100 (fixed concentration of 100 ng/mL throughout the entire culture period); and 3) sequential FSH (FSHSeq - 100, 500, and 1,000 ng/mL were added sequentially). Following culture, all follicles from all treatments were still viable (marked green by calcein-AM). The initial (D0) average follicle diameter for the control, FSH100, and FSHSeq was (mean +/- SEM) 298.96 +/- 7.02, 286.00 +/- 5.87, and 275.39 +/- 174 6.55 microm, respectively (P > 0.05). Mean diameter of follicles treated with FSHSeq on Day 18 (D18-439.80 +/- 14.08 microm) was greater than those of the other treatments (P < 0.05). Daily follicular growth rate (microm/d) of follicles in the FSHSeq treatment (6.47 +/- 0.55) was significantly faster than for both the control (3.67 +/- 0.32) and FSH100 (4.47 +/- 0.38) treatments. Furthermore, FSH100 and FSHSeq treatments had a significantly higher rate of antrum formation than the control group on D12 of culture, whereas after D12, FSH100 had a significantly higher rate of extrusion compared to the control (P < 0.05). In conclusion, the sequential addition of FSH to the culture medium maintained the survival of isolated canine preantral follicles and promoted an increased rate of follicular growth and antrum formation.

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Este trabalho teve como objetivo avaliar o efeito do hormônio folículo estimulante recombinante (FSHr) sobre o desenvolvimento in vitro de folículos pré-antrais (FOPA) caprinos e ovinos isolados. Folículos pré-antrais ( 150 µm) foram isolados de fragmentos do córtex ovariano de cabras e ovelhas e cultivados individualmente por 24 dias em -MEM suplementado com 10% de soro fetal bovino (SFB), 1% de insulina-transferrina-selênio (ITS), 1% de antibióticos e 50 µg/mL de ácido ascórbico, na ausência (controle) ou presença de FSHr (100 ou 1000 ng/mL), a 39 °C com 5% de CO2 em ar. Durante o cultivo avaliou-se a morfologia, o crescimento folicular e a formação de antro. Foi observado que, na espécie caprina, o cultivo com FSHr apresentou percentuais de folículos morfologicamente normais semelhantes ao controle até o 12º dia de cultivo (P>0,05), ocorrendo uma redução deste parâmetro no 18º dia (P<0,05) somente no tratamento com 1000 ng/mL de FSRr. Na espécie ovina, o percentual de folículos morfologicamente normais foi semelhante em todos os tratamentos, observando-se um decréscimo a partir do 12º dia (P<0,05). Com relação ao desenvolvimento folicular, em caprinos, a adição de FSHr aumentou a taxa de crescimento em relação ao controle (P<0,05). Entretanto, em ovinos, isso só foi observado no tratamento com 1000 ng/mL de FSHr. No que se refere à formação de antro, em ambas as espécies, não houve diferença significativa entre os tratamentos com FSHr e o controle. Quanto à análise da configuração da cromatina, todos os oócitos permaneceram em estádio de vesícula germinativa. Assim, pôde-se concluir que FOPA caprinos e ovinos são capazes de manter a sobrevivência e o crescimento in vitro até o estádio antral na ausência de FSH. Contudo, este hormônio teve um papel importante na taxa de crescimento folicular em ambas as espécies.(AU)

The aim of this work was to evaluate the effects of recombinant follicle stimulating hormone (rFSH) on the in vitro development of isolated caprine and ovine preantral follicles. Preantral follicles ( 150 micron) were isolated from fragments of goats and sheeps ovarian cortex and individually cultured for 24 days in-MEM supplemented with 10% fetal calf serum (FCS), 1% insulin-transferrin -selenium (ITS), 1% antibiotics and 50 mg / mL ascorbic acid, in the absence (control) or presence of FSHR (100 or 1000 ng / mL) at 39 ° C with 5% CO2 in air. Follicular morphology, growth and antral cavity formation were assessed throughout the culture. The results showed that addition of rFSH to the medium had no effect on the percentages of caprine morphologically normal follicles until day 12 as compared with the control, but a decrease was observed on day 18 with the use of the hormone (p<0.05) only rFSH (1000 ng/mL). Regarding ovine follicles, percentages of normal follicles were significantly reduced from day 12 onwards in all treatments, which led to similar results. Growth rates were increased in caprine follicles by use of rFSH (100 and 1000 ng/ml) in comparison to the control (p<0,05). In ovine follicles, such effect was observed only when rFSH was used at 1000 ng/ml. With respect to antral cavity formation, no diference among treatments was observed for both species. The analysis of chromatin configuration revealed that all oocytes remained at the germinal vesicle stage. In conclusion, caprine and ovine preantral follicles are capable to survive and develop to the antral stage in vitro without FSH. However, this hormone is important for increasing follicular growth rates in both species.(AU)

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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 degrees 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 degrees C for up to 6h and at 38 degrees C for 2 h. Using MEM, such preservation was possible for 12h at 4 or 20 degrees C, and for 6h at 38 degrees C. These results indicate that preservation of canine preantral follicles might be better accomplished through hypothermic (4 or 20 degrees C) storage in MEM, which ensures maintenance of morphology and viability for up to 12h.

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Bovine preantral follicles within ovarian fragments were exposed and cryopreserved in absence or presence of 1.5M glycerol (GLY), ethylene glycol (EG), propanediol (PROH) or dimethyl sulfoxide (DMSO), undergoing a previous cooling at 20 degrees C for 1h (protocol 1) or at 4 degrees C for 24h (protocol 2) in 0.9% saline solution. At the end of each treatment, preantral follicles were classified as non-viable/viable when they were stained/not stained with trypan blue, respectively. To confirm viability staining, ultra-structure of the follicles was evaluated by transmission electronic microscopy (TEM). Data were compared by Chi-square test (P<0.05). The storage of the ovaries at 20 degrees C for 1h (78%) and 4 degrees C for 24h (80%) did not reduce significantly the percentage of viable preantral follicles when compared to the control (75%). Similar results were obtained when ovarian fragments, respectively, for protocols 1 and 2, were exposed to MEM (78 and 77%), 1.5M EG (78 and 71%), as well as frozen in 1.5M EG (74 and 77%). Percentages of viable follicles in control were similar to those observed after exposure (75%) and freezing (76%) in presence of 1.5M DMSO only when protocol 1 was used. The increase of the concentration from 1.5 to 3.0M, for all cryoprotectants, reduced significantly the percentage of viable preantral follicles after freezing. Ultra-structural analysis has confirmed trypan blue results, showing that not only basement membrane, but also organelles, were intact in viable preantral follicles. In conclusion, ovarian tissue cooling at 4 degrees C for 24h before cryopreservation (protocol 2) does not affect the viability of bovine preantral follicles when 1.5M EG is present in the cryopreservation medium.

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