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This study evaluated the efficiency of in vitro culture of preantral follicles (PAF) in a commonly used medium for mesenchymal stem cell (MSC) culture. Parameters assessed included follicle survival, growth, stromal cell density, levels of reduced thiols and reactive oxygen species, epigenetic changes, cell apoptosis, and mRNA abundance. Caprine ovarian tissues were cultured for 1 or 7 days in either PAF or MSC-common media, with uncultured tissues serving as controls. The MSC medium exhibited increased follicular survival and growth and remodeled stromal density potentially through the regulation of oxidative stress and epigenetic changes compared to the PAF medium. In conclusion, our results highlight the importance of the MSC medium in enhancing follicular survival and growth, changing the stromal cell density, as well as in regulating the medium oxidative stress and epigenetic changes during the in vitro culture of caprine PAF.
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Vitrification is essential for successful tissue cryopreservation and biobanking in wild cats. This study aimed to compare different methods of vitrification (Ovarian Tissue Cryosystem-OTC, Straws-STW, and Solid Surface vitrification-SSV) for testicular fragment vitrification in tom cats. Testicular fragments were recovered from five adult tom cats and subjected to equilibrium vitrification using different cryovials and methods under the same conditions of vitrification solutions and cryoprotectants. The efficiencies of the methods were evaluated using histological analysis of spermatogonia and Sertoli cell nuclei, seminiferous tubular basement membrane detachment, and the gonadal epithelium shrinkage score scale. Cell viability was assessed using Hoechst PI and Terminal deoxynucleotidyl transferase nick end labeling (TUNEL) assay. The results showed that OTC is an effective vitrification method for maintaining the distinction between spermatogonia and Sertoli cells. OTC was similar to the control for basal membrane detachment parameters (p = 0.05). Epithelial shrinkage was low in the SSV group, which showed the highest percentage of viable cells among the vitrified groups (p = 0.0023). The OTC and SSV vitrification methods were statistically similar in terms of the percentage of TUNEL-positive cells (p = 0.05). Therefore, OTC and SSV provide favorable conditions for maintaining viable cat testicular tissue cells after vitrification.
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This study assessed the histones methylation profile (H3K4me3 and H3K9me3) in late preantral (PA) and early antral (EA) caprine follicles grown in vivo and in vitro, and the anethole effect during in vitro culture of PA follicles. Uncultured in vivo-grown follicles (PA, n = 64; EA, n = 73) were used as controls to assess the methylation profile and genes' expression related to apoptosis cascade (BAX, proapoptotic; BCL2, antiapoptotic), steroidogenesis (CYP17, CYP19A1), and demethylation (KDM1AX1, KDM1AX2, KDM3A). The isolated PA follicles (n = 174) were cultured in vitro for 6 days in α-MEM+ in either absence (control) or presence of anethole. After culture, EA follicles were evaluated for methylation, mRNA abundance, and morphometry. Follicle diameter increased after culture, regardless of treatment. The methylation profile and the mRNA abundance were similar between in vivo-grown PA and EA follicles. Anethole treatment led to higher H3K4me3 fluorescence intensity in EA follicles. The mRNA abundances of BAX, CYP17, and CYP19A1 were higher, and BCL2 and KDM3A were lower in in vitro-grown EA follicles than in vivo-grown follicles. In conclusion, in vitro follicle culture affected H3K4me3 fluorescence intensity, mRNA abundance of apoptotic genes, and steroidogenic and demethylase enzymes compared with in vivo-grown follicles.
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Cabras , Lisina , Animales , Proteína X Asociada a bcl-2/metabolismo , Cabras/metabolismo , Histonas , Esteroide 17-alfa-Hidroxilasa/metabolismo , ARN Mensajero/genética , Oocitos/metabolismoRESUMEN
This study investigated the effects of cyclic adenosine monophosphate modulating during cumulus-oocyte complexes (COCs) pre-maturation and the role of melatonin on in vitro maturation (IVM) of bovine COCs. In experiment one, COCs were pre-matured for 8 h in control medium or with 3-isobutyl-1-methylxanthine (IBMX) and forskolin, IBMX and C-type natriuretic peptide, c-type natriuretic peptide and forskolin or IBMX, forskolin and c-type natriuretic peptide. Then, meiotic progression was evaluated. In experiment two, COCs were pre-matured, followed by IVM in control medium alone or with 10-6, 10-7 or 10-8 M melatonin. After IVM, chromatin configuration, transzonal projections (TZPs), reactive oxygen species, mitochondrial distribution, ultrastructure and mRNA expression for antioxidant enzymes were evaluated. In experiment 1, COCs pre-matured with both C-type natriuretic peptide and forskolin or C-type natriuretic peptide, forskolin and IBMX had lower meiotic resumption rate when compared to control. Considering that IBMX had not an additional effect to potentiate inhibition of meiotic resumption, a combination of C-type natriuretic peptide and forskolin was chosen. In experiment 2, COCs matured with 10-8 M melatonin had greater rates of meiotic resumption when compared to the other treatments (P < 0.05). The COCs matured with 10-7 or 10-8 M melatonin had greater mitochondrial activity (P < 0.05), while those matured with 10-6 or 10-8 M of melatonin had greater levels of TZPs. Ultrastructure of oocyte and cumulus cells after IVM with melatonin was relatively well preserved. COCs matured with 10-8 M melatonin increased mRNA expression for superoxide dismutase (SOD) and catalase (CAT) (P < 0.05), when compared to non-cultured and pre-matured COCs, respectively. In conclusion, bovine COC pre-maturation with C-type natriuretic peptide and forskolin, followed by IVM with 10-8 M melatonin improves meiotic resumption rates, TZPs, mitochondrial distribution and mRNA expression for SOD and CAT.
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Melatonina , Animales , Bovinos , Femenino , Melatonina/farmacología , Melatonina/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Péptido Natriurético Tipo-C/farmacología , Colforsina/farmacología , Colforsina/metabolismo , Oocitos/fisiología , AMP Cíclico/metabolismo , ARN Mensajero/metabolismo , Superóxido Dismutasa/metabolismo , Adenosina Monofosfato/metabolismo , Adenosina Monofosfato/farmacología , Células del CúmuloRESUMEN
This study aims to evaluate the effects of N-acetylcysteine (NAC) on bovine oocyte maturation, mitochondrial activity and transzonal projections (TZP), as well as on the levels of reactive oxygen species (ROS) and messenger RNA (mRNA) for catalase (CAT) superoxide dismutase (SOD), periredoxin-6 (Prdx6), glutathione peroxidase (GPx), growth and differentiation factor-9 (GDF9), histone H1Foo, cyclin B1 (CCNB1) and c-Mos. Bovine cumulus-oocyte complexes (COC) of medium-sized antral follicles (3.0-6.0 mm) were prematured in TCM-199 for 8 h at 38.5°C in 5% CO2. After prematuration in the presence of forskolin and C-type natriuretic peptide, COCs were matured in TCM-199 alone or with 0.1, 0.5 or 2.5 mM NAC. Then, oocytes were classified according to the stage of chromatin. Furthermore, mitochondrial activity and intracellular levels of ROS and TZP were also evaluated. The levels of mRNAs for CAT, SOD, Prdx6, GPx, GDF9, H1Foo, CCNB1 and c-Mos were evaluated using real-time polymerase chain reaction (RT-PCR). The results showed that NAC significantly increased the percentages of oocytes with resumption of meiosis when compared with those oocytes matured in control medium. Oocytes had homogeneous mitochondrial distribution, and those cultured with 0.1 and 0.5 mM NAC had lower levels of ROS when compared with the control. In addition, 0.5 mM NAC reduced TZP and the levels of mRNA for CCNB1. In contrast, NAC did not influence the expression of CAT, GPx, Prdx6, SOD, GDF9, H1Foo, and c-Mos. In conclusion, 0.5 mM NAC reduced the levels of ROS, TZP and mRNA for CCNB1, and improved in vitro resumption of meiosis in oocytes from medium-sized bovine antral follicles.
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Acetilcisteína , Técnicas de Maduración In Vitro de los Oocitos , Bovinos , Animales , Técnicas de Maduración In Vitro de los Oocitos/métodos , Especies Reactivas de Oxígeno/metabolismo , Acetilcisteína/farmacología , Acetilcisteína/metabolismo , Oocitos , Meiosis , Superóxido Dismutasa/metabolismo , Glutatión Peroxidasa/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Heterotopic and orthotopic ovarian tissue autotransplantation techniques, currently used in humans, will become promising alternative methods for fertility preservation in domestic and wild animals. Thus, this study describes for the first time the efficiency of a heterotopic ovarian tissue autotransplantation technique in a large livestock species (i.e., horses) after ovarian fragments were exposed or not to a cooling process (4°C/24 h) and/or VEGF before grafting. Ovarian fragments were collected in vivo via an ultrasound-guided biopsy pick-up method and surgically autografted in a subcutaneous site in both sides of the neck in each mare. The blood flow perfusion at the transplantation site was monitored at days 2, 4, 6, and 7 post-grafting using color-Doppler ultrasonography. Ovarian grafts were recovered 7 days post-transplantation and subjected to histological analyses. The exposure of the ovarian fragments to VEGF before grafting was not beneficial to the quality of the tissue; however, the cooling process of the fragments reduced the acute hyperemia post-grafting. Cooled grafts compared with non-cooled grafts contained similar values for normal and developing preantral follicles, vessel density, and stromal cell apoptosis; lower collagen type III fibers and follicular density; and higher stromal cell density, AgNOR, and collagen type I fibers. In conclusion, VEGF exposure before autotransplantation did not improve the quality of grafted tissues. However, cooling ovarian tissue for at least 24 h before grafting can be beneficial because satisfactory rates of follicle survival and development, stromal cell survival and proliferation, as well as vessel density, were obtained.
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Frío , Folículo Ovárico/trasplante , Trasplante Heterotópico , Factor A de Crecimiento Endotelial Vascular/farmacología , Animales , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Recuento de Células , Proliferación Celular/efectos de los fármacos , Femenino , Fibrosis , Caballos , Modelos Animales , Folículo Ovárico/irrigación sanguínea , Folículo Ovárico/efectos de los fármacos , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Flujo Sanguíneo Regional/efectos de los fármacos , Células del Estroma/citología , Células del Estroma/efectos de los fármacos , Trasplante AutólogoRESUMEN
This study aimed to evaluate the role of anethole during the in vitro culture of caprine early antral follicles. Early antral follicles were isolated from caprine ovaries and cultured for 18 days without (control) or with anethole (300 µg/ml). After culture, the cumulus-oocyte complexes were subjected to in vitro maturation, followed by parthenogenetic activation or in vitro fertilization (IVF) and embryo culture. Follicular walls were used for the quantification of messenger RNA (mRNA) of CYP19A1, CYP17, MMP-9, TIMP-2, Bax, and Bcl-2 genes, and culture medium was used for evaluation of ferric reducing antioxidant power (FRAP) and estradiol levels. After in vitro follicle culture (IVFC), anethole induced higher total antioxidant capacity, that is, it produced higher FRAP levels, reduced the Bax/Bcl-2 ratio, and increased the levels of mRNA for CYP19A1 and CYP17, which was associated with a greater estradiol production (p < .05). Also, anethole improved the ability of oocytes to resume meiosis and reach metaphase II stage, as well as yielded higher (p < .05) embryo production (e.g., morulas and blastocysts) in both parthenogenetic activation and IVF techniques. One pregnancy (Day 30) was obtained from IVFC with anethole. In conclusion, anethole promoted in vitro growth and maturation of goat early antral follicles and oocytes and enabled embryo production. Furthermore, this study reports, for the first time in goats, a pregnancy after IVF using oocytes originated from early antral follicles grown in vitro.
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Derivados de Alilbenceno/farmacología , Anisoles/farmacología , Cabras/fisiología , Hormonas Esteroides Gonadales/biosíntesis , Técnicas de Maduración In Vitro de los Oocitos , Folículo Ovárico , Preñez , Animales , Células Cultivadas , Medios de Cultivo/farmacología , Femenino , Fertilización In Vitro/métodos , Fertilización In Vitro/veterinaria , Técnicas de Maduración In Vitro de los Oocitos/métodos , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Redes y Vías Metabólicas/efectos de los fármacos , Oocitos/citología , Oocitos/efectos de los fármacos , Oocitos/fisiología , Oogénesis/efectos de los fármacos , Oogénesis/fisiología , Folículo Ovárico/citología , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/fisiología , EmbarazoRESUMEN
The aim of this study was to evaluate the viability, antrum formation and in vitro development of isolated secondary follicles from vitrified caprine ovarian cortex in a medium previously established for fresh isolated secondary follicles, in the absence (α-minimum essential medium (α-MEM+) alone) or presence of FSH and vascular endothelial growth factor (VEGF; α-MEM++FSH+VEGF). Ovarian fragments were distributed among five treatments (T1 to T5): fresh follicles were fixed immediately (T1), follicles from fresh tissue were cultured in vitro in α-MEM+ (T2) or α-MEM++FSH+VEGF (T3) and follicles from vitrified tissue were cultured in vitro in α-MEM+ (T4) or α-MEM++FSH+VEGF (T5). After 6 days of culture, treated follicles (T2, T3, T4 and T5) were evaluated for morphology, viability and follicular development (growth, antrum formation and proliferation of granulosa cells by Ki67 and argyrophilic nucleolar organiser region (AgNOR) staining). The levels of reactive oxygen species (ROS) in the culture media were also assessed. Overall, morphology of vitrified follicles was altered (P<0.05) compared with the fresh follicles. Follicular viability, antrum formation and ROS were similar between treatments (P>0.05). The average overall and daily follicular growth was highest (P<0.05) in T3. Granulosa cells in all treatments (T1, T2, T3, T4 and T5) stained positive for Ki67. However, fresh follicles from T3 had significantly higher AgNOR staining (P<0.05) compared with follicles of T1, T2, T4 and T5. In conclusion, secondary follicles can be isolated from vitrified and warmed ovarian cortex and survive and form an antrum when growing in an in vitro culture for 6 days.
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Criopreservación/veterinaria , Cabras/embriología , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Folículo Ovárico/fisiología , Ovario/citología , Animales , Antígenos Nucleares/metabolismo , Proliferación Celular , Forma de la Célula , Supervivencia Celular , Células Cultivadas , Femenino , Fármacos para la Fertilidad Femenina/farmacología , Hormona Folículo Estimulante/farmacología , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factores de Tiempo , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
The aim of the present study was to evaluate the development of fresh and vitrified agouti ovarian tissue after xenografting to C57Bl/6 severe combined immunodeficiency (SCID) female mice. Ovaries were obtained from five female agoutis and divided into 16 fragments. Five fragments were transplanted immediately to ovariectomised SCID mice and the others were vitrified, stored for 2 weeks and transplanted only after rewarming. Tissue fragments were transplanted under the kidney capsule in recipients. The return of ovarian activity in recipients was monitored by the observation of external signs of oestrus and vaginal cytology over a period of 40 days after transplantation, after which the grafts were removed and evaluated for morphology, cell proliferation and the occurrence of DNA fragmentation. Ovarian activity returned in four of five mice that received fresh ovarian tissue from agoutis and in one of six mice that had received vitrified tissue a mean (±s.e.m.) 20.6±8.6 days after xenotransplantation. After graft removal, a predominance of primordial and primary follicles was observed in all grafts. Vitrification reduced cell proliferation and increased the occurrence of DNA fragmentation in grafted agouti ovarian tissue. In conclusion, the present study demonstrates that xenografted agouti ovarian tissue, fresh or vitrified, is able to promote the return of ovarian activity in ovariectomised SCID C57B1/6 mice. However, improvements to vitrification protocols for agouti ovarian tissue are necessary.
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Criopreservación , Preservación de la Fertilidad/métodos , Ovariectomía , Ovario/trasplante , Animales , Proliferación Celular , Fragmentación del ADN , Ciclo Estral , Femenino , Supervivencia de Injerto , Xenoinjertos , Ratones Endogámicos C57BL , Ratones SCID , Ovario/metabolismo , Ovario/patología , Embarazo , Recuperación de la Función , Factores de Tiempo , VitrificaciónRESUMEN
The aim of the present study was to characterise the ovarian preantral follicle (PF) population and to establish a solid surface vitrification (SSV) process using dimethyl sulfoxide (DMSO) as a cryoprotectant for preservation of ovarian tissue from yellow-toothed cavies (Galea spixii). Ovaries were fixed for PF population analysis or were subjected to the SSV process. The mean (± s.e.m.) PF population per ovarian pair was estimated to be 416.0±342.8. There were 140.0±56.0 (63.4%) and 125.0±58.0 (64.0%) primary follicles on the right and left ovaries, respectively. The proportion of this follicle category was significantly greater than that of other follicle categories (P<0.05). The diameter of follicles (123.7±18.3µm), oocytes (50.1±5.0µm) and nuclei (14.27±2.01µm) was larger for secondary ones when compared with other PFs categories. Most PFs were morphologically normal (94.6%), with light microscopy identifying only a few atretic follicles (5.4%). After SSV, there was a reduction in the proportion of morphologically normal PFs compared with the non-vitrified group (69.5% vs 91.2%, respectively). Transmission electron microscopy revealed preservation of oocytes and granulosa cell membranes and the morphological aspect of follicles; the primary change observed in some vitrified PFs was the presence of vacuoles in the oocytes and granulosa cells cytoplasm and turgid mitochondria. In conclusion, the present study provides an estimative and characterization for the PF population in ovaries of G. spixii. Moreover, we report its PFs cryopreservation using an SSV process.
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Criopreservación , Folículo Ovárico/anatomía & histología , Ovario/anatomía & histología , Vitrificación , Animales , Femenino , Microscopía Electrónica de Transmisión , Folículo Ovárico/ultraestructura , Ovario/ultraestructura , RoedoresRESUMEN
Ovarian cortical fragments (3 x 3 x 1 mm) were exposed to dimethyl sulfoxide (DMSO) in different concentrations for further analysis of cryoprotectant perfusion by applying high-performance liquid chromatography (HPLC) and conventional cryopreservation. This simple perfusion test can predict the efficiency of the cryopreservation procedure.
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Cromatografía Líquida de Alta Presión/métodos , Criopreservación/métodos , Crioprotectores/farmacología , Dimetilsulfóxido/farmacología , Folículo Ovárico/efectos de los fármacos , Ovario/efectos de los fármacos , Supervivencia Tisular/efectos de los fármacos , Animales , Crioprotectores/administración & dosificación , Dimetilsulfóxido/administración & dosificación , Relación Dosis-Respuesta a Droga , Femenino , Cabras , Modelos Animales , Folículo Ovárico/fisiología , Ovario/fisiología , Perfusión/métodos , Supervivencia Tisular/fisiologíaRESUMEN
The aim of the present study was to determine the amount of dimethyl sulfoxide (DMSO) present in sheep ovarian tissue after exposure to cryoprotectant at different times (5, 10, 20, or 30 min) and at different concentrations (1.0, 1.5, or 2.0 M). To quantify the levels of DMSO in the ovarian tissue, the high-performance liquid chromatography (HPLC) method was applied. In addition, viability of preantral follicles after toxicity test and cryopreservation of ovarian tissue using the above mentioned concentrations of DMSO and exposure times was evaluated. We have observed that the presence of â¼0.6 mg of DMSO into the ovarian tissue may be deleterious to the sheep preantral follicles. In addition, the application of a short exposure time (5 min at 1.5 or 2.0 M DMSO) or low concentration (1.0 M for 10 min) of DMSO successfully preserves sheep preantral follicles following cryopreservation.
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Isolated or cortical tissue-enclosed (in situ) sheep early-stage follicles were exposed to 1.5 M dimethyl sulfoxide (DMSO), ethylene glycol (EG) or unexposed, or frozen/thawed in the presence of these cryoprotectants and then cultured for 5 days in enriched minimal essential medium (MEM) or not cultured. Cultured and uncultured follicles were classified as non-viable/viable when they were stained/not stained with trypan blue, respectively. Follicular diameter was measured and the percentages of primordial and developing follicles calculated. Exposure of isolated or in situ follicles to DMSO or EG led to a marked decrease in the percentage of viable follicles. The percentage of viable isolated and in situ follicles further decreased when they were in vitro-cultured for 5 days, EG-exposed follicles generally showing a more damaging effect than DMSO-exposed follicles. Cultured follicles, both isolated and in situ, which were exposed to EG and DMSO, as well as in situ follicles, which had been frozen/thawed in the presence of one of these cryoprotectants, showed similar growth rates as cultured, untreated follicles, while in these groups significantly lower percentages of primordial follicles and higher percentages of more advanced follicular stages were observed. Among the treated groups, the highest percentage (71-75%) of developing follicles was observed after culturing cryoprotectant-exposed isolated follicles. In contrast, when cryopreserved, isolated follicles were cultured, they did not increase in diameter and did not develop into more advanced stages. In conclusion, exposure to or cryopreservation in the presence of EG and DMSO, as well as their further in vitro culture, negatively affected the viability of ovine isolated and in situ early-stage follicles. In vitro growth of early-stage follicles and activation of primordial follicles were better maintained when follicles had been frozen/thawed and cultured in situ.
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Criopreservación/veterinaria , Folículo Ovárico/fisiología , Ovinos/fisiología , Animales , Crioprotectores/toxicidad , Dimetilsulfóxido/toxicidad , Glicol de Etileno/toxicidad , Femenino , Folículo Ovárico/citología , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/crecimiento & desarrollo , Factores de Tiempo , Azul de Tripano/metabolismoRESUMEN
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 degrees 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.