Definition of protocols for cryopreservation and three-dimensional in vitro culture of prepubertal goat testicular tissue after histomorphological, ultrastructural, and functional analysis.

This study aims to define the best method (slow freezing or vitrification) and fragment size (1, 5, or 9 mm³) for prepubertal goat testis cryopreservation, as well as to evaluate testicular morphological integrity after cryopreservation and in vitro culture (IVC). Initially (experiment I), 1, 5, or 9 mm³ testis fragments were cryopreserved by slow freezing using a Mr. Frosty container with 20% Dimethylsulfoxide (DMSO) or vitrified using the Ovarian Tissue Cryosystem (OTC) device, (Equilibration solution - ES: 10% DMSO and 10% ethylene glycol - EG; Vitrification solution - VS: 20% DMSO and 20% EG) and then subjected to morphological analysis, type I and III collagen quantification and gene expression (Oct4, C-kit, Bax, and Bcl-2). Subsequently, (experiment II), fresh or cryopreserved by slow freezing testis fragments were cultured in vitro and submitted to morphological analysis by scanning electron microscopy. The data from the experiment I revealed fewer morphological alterations in 1 and 5 mm³ fragments after vitrification and slow freezing, respectively. The percentage of type I collagen fibers in 5 and 9 mm³ frozen was higher than in fresh or vitrified fragments. For type III collagen, fresh or frozen fragments of 1 and 5 mm3 showed a higher percentage than fragments of 9 mm3. Gene expression for Oct4 and C-kit after slow freezing or vitrification in the 5 mm3 fragments was lower than that observed in the fresh fragments. The Bax:Bcl-2 ratio in the 1 and 9 mm³ fragments was lower than in the 5 mm³ fragments for fresh fragments or after freezing. In experiment II, fragments cultured in vitro, previously frozen or not, showed more morphological alterations than fresh or frozen fragments. We concluded that slow freezing of 5 mm³ fragments was the best protocol for cryopreserving prepubertal goat testis and although the results of IVC are encouraging, it still needs improvement to restore testicular function after cryopreservation.

Bovine somatic cell nuclear transfer using mitomycin C-mediated chemical oocyte enucleation.

SummaryChemical oocyte enucleation holds the potential to ease somatic cell nuclear transfer (SCNT), although high enucleation rates remain limited to micromanipulation-based approaches. Therefore, this study aimed to test mitomycin C (MMC) for use in bovine functional chemical oocyte enucleation. Incubation of denuded eggs in 10 µg ml-1 MMC for different periods did not affect most maturation rates (0.5 h: 85.78%A, 1.0 h: 72.77%B, 1.5 h: 83.87%A, and 2.0 h: 82.05%A) in comparison with non-treated controls (CTL; 85.77%A). Parthenogenetic development arrest by MMC was efficient at cleavage (CTL: 72.93%A, 0.5 h: 64.92%A,B, 1.0 h: 60.39%B,C, 1.5 h: 66.35%A,B, and 2.0 h: 53.84%C) and blastocyst stages (CTL: 33.94%A, 0.5 h: 7.58%B, 1.0 h: 2.47%C, 1.5 h: 0.46%C, and 2.0 h: 0.51%C). Blastocysts were obtained after nuclear transfer (NT) using MMC enucleation [NT(MMC): 4.54%B] but at lower rates than for the SCNT control [NT(CTL): 26.31%A]. The removal of the meiotic spindle after MMC incubation fully restored SCNT blastocyst development [NT(MMC+SR): 24.74%A]. Early pregnancies were obtained by the transfer of NT(MMC) and NT(MMC+SR) blastocysts to synchronized recipients. In conclusion, MMC leads to functional chemical oocyte enucleation during SCNT and further suggests its potential for application towards technical improvements.

Cryopreservation and characterization of canine preantral follicles.

The aim of this study was to define the population, morphological and ultrastructural characteristics of bitch preantral follicles (PAFs) and to compare the effects on the morphology of PAF of two cryopreservation techniques - slow freezing (SF) and vitrification (V) - of bitches' ovarian tissue. The average population (number per ovary) of PAFs was 48,541 ±â€¯18,366, where 94.25% were primordial (45,145 ±â€¯16,076). The average diameter of the primordial follicles was 27.5 ±â€¯4.2 µm. The overall percentage of morphologically normal PAFs was 93.66 ±â€¯6.81% for the control group, 86.16 ±â€¯11.05% after SF and 68.14 ±â€¯12.75% after V. The percentage of normal primordial follicles was 96.69 ±â€¯4.72% in control, 89.51 ±â€¯10.39% in SF and 75.32 ±â€¯9.23% in V. There was no significant difference in the overall percentage of normal PAFs among SF and the control. However, slow frozen follicles presented ultrastructural damage, while vitrified primordial and primary follicles were well preserved. In conclusion, although slow freezing seemed to be a good preserving method, vitrification was more effective than slow freezing in preserving the ultrastructure of primordial and primary follicles of bitches.

Further insights into the impact of mouse follicle stage on graft outcome in an artificial ovary environment.

STUDY QUESTION: Are mouse preantral follicles differently affected by isolation, encapsulation and/or grafting procedures according to stage? SUMMARY ANSWER: Isolated secondary follicles showed superior ability to survive and grow after transplantation, which was not related to a particular effect of the isolation and/or grafting procedure, but rather to their own ability to induce neoangiogenesis. WHAT IS KNOWN ALREADY: Isolated and encapsulated mouse preantral follicles can survive (6-27%) and grow (80-100%) in a fibrin matrix with a low concentration of fibrinogen and thrombin (F12.5/T1) after short-term transplantation. STUDY DESIGN, SIZE, DURATION: An in vivo experimental model using 20 donor Naval Medical Research Institute (NMRI) mice (6-25 weeks of age) and 14 recipient severe combined immunodeficient (SCID) mice (11-39 weeks of age) was applied. Each NMRI mouse underwent mechanical disruption of both ovaries and isolation of primordial-primary and secondary follicles with ovarian stromal cells, in order to encapsulate them in an F12.5/T1 matrix. Twelve out of 40 fibrin clots were immediately fixed as controls (D0) (10 for histology and 2 for scanning electron microscopy [SEM]) and the others (n = 28) were grafted to the inner part of the peritoneum for 2 (16 fibrin clots) or 7 (12 fibrin clots) days (D2 and D7). PARTICIPANTS/MATERIALS, SETTING, METHODS: This study involved the participation of the Gynecology Research Unit (Universitè Catholique de Louvain) and the Physiological Sciences Department (University of Brasília). Specific techniques were used to analyze the follicle recovery rate (hematoxylin-eosin staining), vascularization (CD34) and follicle ultrastructure (transmission electron microscopy [TEM] and SEM). MAIN RESULTS AND THE ROLE OF CHANCE: After follicle isolation and encapsulation, a statistically higher percentage of normal follicles was observed in the secondary group (62%) than in the primordial-primary group (47%). Follicle recovery rates were 34% and 62% for primordial-primary and secondary follicles on D2, respectively, and 12% and 42% on D7, confirming that secondary follicles survive better than primordial-primary follicles after grafting. Concerning vascularization, both follicle stages exhibited similar vascularization to that seen in control mouse ovary on D7, but a significantly higher number of vessels and greater vessel surface area were detected in the secondary follicle group. Despite structural differences in fiber density between fibrin clots and ovarian tissue observed by SEM and TEM, preantral follicles appeared to be well encapsulated in the matrix, also showing a normal ultrastructure after grafting. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: As demonstrated by our results during the isolation procedure, we encapsulated a significantly higher number of round structures in the primordial-primary group than in the secondary group, which could partially explain the lower recovery rate of early-stage follicles in our previous study. However, it is not excluded that the physical and mechanical properties of the fibrin matrix may also play a role in follicle survival and growth, so further investigations are needed. WIDER IMPLICATIONS OF THE FINDINGS: This research represents one more key step in the creation of the artificial ovary. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS) to C.A. Amorim as a research associate at FRS-FNRS and (grant 5/4/150/5 awarded to M.M. Dolmans), Fonds Spéciaux de Recherche, Fondation St Luc, Foundation Against Cancer, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES-Brazil) (grant #013/14 CAPES/WBI awarded to C.M. Lucci, with F. Paulini receiving a post-doctoral fellowship), and Wallonie-Bruxelles International, and donations from the Ferrero family. None of the authors have any competing interests to declare in relation to the topic.

Culture of domestic cat ovarian tissue in vitro and in the chick embryo chorioallantoic membrane.

In vitro culture and transplantation procedures are essential protocols employed in the evaluation of ovarian follicle survival and development. Culture in the chorioallantoic membrane (CAM) of chick embryos is an intermediate method that provides important follicle development information and has not been tested for cat ovaries to date. The aim of this study was to investigate if in vitro and CAM culture could be used as short-term systems to study cat ovarian tissue development. The ovaries of eight cats were dissected into 3-mm(3) cubes, cultured in vitro and in CAM for up to 5 days, and stained with hematoxylin-eosin and Gomori trichrome. Cell proliferation was analyzed using anti-Ki67. Possible differences among groups were investigated by analysis of variance or the Kruskal-Wallis test followed by Bonferroni correction. The T-test or Wilcoxon test was used to verify differences between the CAM and IVC. Results revealed that 87.5% of all follicles were primordial during culture. The percentage of primordial follicles in the morphologically normal follicles (MNF) pool was always higher than 80%, with the exception of Day 3 of CAM culture, but the number of MNF reduced significantly from Day 0 (600 out of 777 follicles) to Day 5 in the CAM (91 out of 171) and IVC (296 out of 686). The number of primordial follicles in 1 mm(3) in Days 2, 3, and 5 in the CAM was significantly lower than that in the control (Day 0). No cellular proliferation was observed in culture. Vascularization occurred in the CAM culture, but with no association to follicular viability. In addition, both methods showed an increase in connective tissue during culture. Although no significant differences were observed in the percentage of MNF, there was a reduction in the total number of follicles, both for IVC and CAM-cultured ovarian tissue. Furthermore, anti-Ki67 did not stain any follicle after Day 0 in IVC or in CAM culture. Neither system was capable of promoting follicle growth and/or development. The results show that the CAM is not a suitable system for feline ovarian tissue and highlight the necessity to improve IVC systems in cats.

Post-hatching development of in vitro bovine embryos from day 7 to 14 in vivo versus in vitro.

This study evaluates the post-hatching development of in vitro-produced (IVP) embryos until Day 14. On Day 7, IVP embryos were either transferred to recipient uteruses or placed in a post-hatching development (PHD) system. As a control group, in vivo-produced (IVV), Day-7 embryos were also transferred to recipient uteruses. All groups were collected on Day 14 and were morphologically evaluated. Day-7 and Day-14 IVV and IVP embryos were used for quantification of eight genes (PLAC8, CD9, SLC2A1, SLC2A3, KRT8, SOD2, HSP1A1, and IFNT2) by reverse transcriptase qPCR. Day-14 embryos from the PHD system were smaller (2.92 ± 0.45 mm) and had a lower embryonic disk diameter (0.14 ± 0.00 mm) than those produced by IVV (24.18 ± 3.71; 0.29 ± 0.03 mm, respectively) or IVP (19.06 ± 2.43; 0.28 ± 0.01 mm) culture and transferred to the uterus (P > 0.05). Day-7 IVP embryos had a higher expression of the HSP1A1, SCL2A1, and SCL2A3 genes than IVV embryos. When these embryos were cultured in the uterus, no differences in gene expression were observed on Day 14. Conversely, Day-14 IVP embryos cultured in the PHD system showed a higher expression of PLAC8, SOD2, and SLC2A3 genes. It is concluded that Day-7 IVP embryos are different from IVV embryos in regards to gene expression, although exposure to the uterine environment during the elongation period allowed the IVP embryos to overcome this difference. In contrast, IVP embryos cultured in the PHD system were morphologically and molecularly different, being of poorer quality than those cultured in the uterus.

Limb immobilization alters functional electrophysiological parameters of sciatic nerve.

Immobilization, used in clinical practice to treat traumatologic problems, causes changes in muscle, but it is not known whether changes also occur in nerves. We investigated the effects of immobilization on excitability and compound action potential (CAP) and the ultrastructure of the rat sciatic nerve. Fourteen days after immobilization of the right leg of adult male Wistar rats (n=34), animals were killed and the right sciatic nerve was dissected and mounted in a moist chamber. Nerves were stimulated at a baseline frequency of 0.2 Hz and tested for 2 min at 20, 50, and 100 Hz. Immobilization altered nerve excitability. Rheobase and chronaxy changed from 3.13 ± 0.05 V and 52.31 ± 1.95 µs (control group, n=13) to 2.84 ± 0.06 V and 59.71 ± 2.79 µs (immobilized group, n=15), respectively. Immobilization altered the amplitude of CAP waves and decreased the conduction velocity of the first CAP wave (from 93.63 ± 7.49 to 79.14 ± 5.59 m/s) but not of the second wave. Transmission electron microscopy showed fragmentation of the myelin sheath of the sciatic nerve of immobilized limbs and degeneration of the axon. In conclusion, we demonstrated that long-lasting leg immobilization can induce alterations in nerve function.

Limb immobilization alters functional electrophysiological parameters of sciatic nerve

Immobilization, used in clinical practice to treat traumatologic problems, causes changes in muscle, but it is not known whether changes also occur in nerves. We investigated the effects of immobilization on excitability and compound action potential (CAP) and the ultrastructure of the rat sciatic nerve. Fourteen days after immobilization of the right leg of adult male Wistar rats (n=34), animals were killed and the right sciatic nerve was dissected and mounted in a moist chamber. Nerves were stimulated at a baseline frequency of 0.2 Hz and tested for 2 min at 20, 50, and 100 Hz. Immobilization altered nerve excitability. Rheobase and chronaxy changed from 3.13±0.05 V and 52.31±1.95 µs (control group, n=13) to 2.84±0.06 V and 59.71±2.79 µs (immobilized group, n=15), respectively. Immobilization altered the amplitude of CAP waves and decreased the conduction velocity of the first CAP wave (from 93.63±7.49 to 79.14±5.59 m/s) but not of the second wave. Transmission electron microscopy showed fragmentation of the myelin sheath of the sciatic nerve of immobilized limbs and degeneration of the axon. In conclusion, we demonstrated that long-lasting leg immobilization can induce alterations in nerve function.

Morphometry, estimation and ultrastructure of ovarian preantral follicle population in queens.

The aim of the present study was to estimate the population and morphometrically and ultrastructurally characterize preantral follicles from queen ovaries. Ovaries from 5 queens were collected and processed for light and electron microscopy. A total of 190 preantral follicles (100 primordial, 60 primary and 30 secondary) were analyzed by light microscopy. The diameters of the follicle, oocyte and oocyte nucleus were taken and the number of granulosa cells was counted using a computer program. Queen ovaries presented 37,853 +/- 6,118 preantral follicles on average, with 87% primordial, 10.4% primary and 2.3% secondary follicles. Significant differences were observed in the 3 follicular classes in regard to follicular, oocyte and oocyte nucleus diameters and granulosa cell number (p < 0.05). In regard to ultrastructure, queen preantral follicles presented many unique characteristics, such as early zona pellucida formation in primary follicles and the organization of mitochondria and other organelles in conglomerates and cortical granules aligned at the peripheral zone in secondary follicles. In conclusion, this study described the morphometry and ultrastructure of queen preantral follicles and the preantral follicle population in the ovaries, establishing a pattern for the species and consequently allowing comparisons with other species.

Glycerol, methyl-formamide and dimethyl-formamide in canine semen cryopreservation.

The aim of the present work was to compare the efficiency of methyl-formamide (MF), dimethyl-formamide (DF) and glycerol (GL) as cryoprotectants in canine semen cryopreservation. For the experiment, pooled semen was submitted to one of the three cryoprotectants, with a final concentration of 3% in egg yolk-TRIS extender. Semen was subjectively evaluated for total and progressive motility, vigour and morphology. Sperm membrane functional integrity was assessed by hypo-osmotic swelling test (HOST), and longevity was assessed using the thermoresistance test (TRT). Fresh semen showed normal physical and morphological characteristics. After thawing, differences were observed between semen frozen using GL and DF, regarding total and progressive motility and vigour (p < 0.05), but not between MF and GL or MF and DF. Means for total motility, progressive motility, vigour and morphologically normal spermatozoa were, respectively, 69.0 +/- 5.4%, 61.0 +/- 7.4%, 2.9 +/- 0.5 and 57.1 +/- 5.0% for GL; 59.0 +/- 8.9%, 50.0 +/- 10.0%, 2.5 +/- 0.7 and 66.9 +/- 7.7% for MF; and 44.0 +/- 21.0%, 37.0 +/- 19.8%, 2.1 +/- 0.6 and 61.1 +/- 5.5% for DF. On HOST, GL was superior (p < 0.05) to MF and DF (57.8 +/- 12.4%, 35.8 +/- 18.4% and 34.4 +/- 9.4%, respectively). During the TRT, both GL and MF were superior to DF, with no differences between GL and MF. In conclusion, the use of MF as cryoprotectant showed results similar to GL, and can be considered as an alternative in canine semen cryopreservation. Further studies testing different concentrations of MF may improve its effects on cryopreservation of canine semen.

Impact of pituitary FSH purification on in vitro early folliculogenesis in goats.

Porcine pituitary follicle stimulating hormone (pFSH) is known to regulate the production of growth factors that have an essential role in early foliculogenesis. However, the effects of different preparations of pFSH on the survival and development of caprine follicles are not yet known. The aim of this study was to evaluate the effects of different pFSH (Stimufol and Folltropin) on the in vitro survival and growth of caprine preantral follicles. Pieces of caprine ovarian tissues were cultured for either one or seven days in a supplemented Minimum Essential Medium, alone or containing either Stimufol (50 ng/mL) or Folltropin (10, 50, 100 and 1000 ng/mL). Fresh control ovarian tissues as well as cultured tissued were processed for histological and ultrastructural studies. The results showed that after seven days, only Stimufol maintained follicular morphology similar to control. Moreover, follicular degeneration was higher in medium alone or with Folltropin at 50, 100 and 1000 ng/mL. However, at day seven, the percentage of growing follicles was higher in 100 ng/mL of Folltropin than Stimufol. In conclusion, FSH preparations affect differently the performance of in vitro culture of caprine preantral follicles. Stimufol was better to preserve follicular morphology while Folltropin was more efficient to promote follicular growth.

Impact of pituitary FSH purification on in vitro early folliculogenesis in goats

Porcine pituitary follicle stimulating hormone (pFSH) is known to regulate the production of growth factors that have an essential role in early foliculogenesis. However, the effects of different preparations of pFSH on the survival and development of caprine follicles are not yet known. The aim of this study was to evaluate the effects of different pFSH (Stimufol and Folltropin) on the in vitro survival and growth of caprine preantral follicles. Pieces of caprine ovarian tissues were cultured for either one or seven days in a supplemented Minimum Essential Medium, alone or containing either Stimufol (50 ng/mL) or Folltropin (10, 50, 100 and 1000 ng/mL). Fresh control ovarian tissues as well as cultured tissued were processed for histological and ultrastructural studies. The results showed that after seven days, o nly Stimufol maintained follicular morphology similar to control. Moreover, follicular degeneration was higher in medium alone or with Folltropin at 50, 100 and 1000 ng/mL. However, at day seven, the percentage of growing follicles was higher in 100 ng/mL of Folltropin than Stimufol. In conclusion, FSH preparations affect differently the performance of in vitro culture of caprine preantral follicles. Stimufol was better to preserve follicular morphology while Folltropin was more efficient to promote follicular growth.(AU)

Impact of pituitary FSH purification on in vitro early folliculogenesis in goats

Porcine pituitary follicle stimulating hormone (pFSH) is known to regulate the production of growth factors that have an essential role in early foliculogenesis. However, the effects of different preparations of pFSH on the survival and development of caprine follicles are not yet known. The aim of this study was to evaluate the effects of different pFSH (Stimufol and Folltropin) on the in vitro survival and growth of caprine preantral follicles. Pieces of caprine ovarian tissues were cultured for either one or seven days in a supplemented Minimum Essential Medium, alone or containing either Stimufol (50 ng/mL) or Folltropin (10, 50, 100 and 1000 ng/mL). Fresh control ovarian tissues as well as cultured tissued were processed for histological and ultrastructural studies. The results showed that after seven days, o nly Stimufol maintained follicular morphology similar to control. Moreover, follicular degeneration was higher in medium alone or with Folltropin at 50, 100 and 1000 ng/mL. However, at day seven, the percentage of growing follicles was higher in 100 ng/mL of Folltropin than Stimufol. In conclusion, FSH preparations affect differently the performance of in vitro culture of caprine preantral follicles. Stimufol was better to preserve follicular morphology while Folltropin was more efficient to promote follicular growth.

Cryopreservation of swine ovarian tissue: effect of different cryoprotectants on the structural preservation of preantral follicle oocytes.

The present study aimed to test different cryoprotectants on cryopreservation of pig ovarian tissue. Pig ovaries (n=3) were collected at a local slaughterhouse. From each ovary, ten cortex samples were taken. One was immediately fixed (control) and another placed in short-term tissue incubation (STTI control). The other 8 samples were cryopreserved, in pairs, using 4 different cryoprotectants: dimethyl sulphoxide (Me2SO - 1.5M), ethylene glycol (EG - 1.5M), propanediol (PROH - 1.5M) and glycerol (GLY - 10%), all with 0.4% sucrose. Samples were slow cooled and stored in liquid nitrogen for 7 days. After thawing and cryoprotectant removal, one sample from each treatment was immediately fixed and the other was placed in short-term tissue incubation (STTI) for 2h and then fixed. Samples were processed for histology and transmission electron microscopy. The percentages of morphologically normal follicles (MNF) in cryopreserved tissue using Me2SO (67.0+/-4.9), EG (81.8+/-1.4) and PROH (55.9+/-9.9) were significantly lower (P<0.05) than observed in fresh control tissue (97.7+/-1.2). When ovarian tissue was cryopreserved with GLY, no morphologically normal follicles could be found (0%). After STTI, PROH showed a significantly lower percentage of MNF when compared with all other treatments and the control. After ultrastructural analysis, follicles cryopreserved with Me2SO and EG showed some small alterations, but no signs of advanced degeneration. Overall, these were similar to follicles from the control group. In conclusion, it is possible to cryopreserve preantral follicles from pig ovarian tissue using Me2SO or EG.

Growth and differentiation factor-9 stimulates activation of goat primordial follicles in vitro and their progression to secondary follicles.

The aim of the present study was to investigate the effects of growth and differentiation factor-9 (GDF-9) on the survival and activation of preantral follicles, as well as their subsequent progression to secondary follicles, using goat ovarian cortical culture in vitro. Pieces of ovarian cortex were cultured for 1 and 7 days in minimum essential medium (MEM) with or without different concentrations of GDF-9 (1-200 ng mL(-1)). On Day 0 and after 1 and 7 days of culture, cortical pieces were fixed for histological and transmission electron microscopy evaluation. Preantral follicles were classified according to their development stage (primordial, intermediate, primary and secondary) and on the basis of morphological features (normal or degenerated). In addition, follicular and oocyte diameters were determined before and after culture. The results showed that, compared with non-cultured cortical tissue (Day 0), the culture of ovarian tissue significantly reduced (P < 0.05) the percentage of normal follicles in all media tested, except for tissue cultured in the presence of 200 ng mL(-1) GDF-9. Furthermore, in all media tested, the percentage of primordial follicles was significantly reduced (P < 0.05), with a concomitant increase in the percentage of developing follicles. The highest percentage of secondary follicles was observed after 7 days of culture in MEM plus 200 ng mL(-1) GDF-9. At all concentrations of GDF-9 tested, follicular diameter increased significantly after 7 days of culture compared with non-cultured cortical tissue. In conclusion, the results of the present study indicate that 200 ng mL(-1) GDF-9 maintains the survival of preantral follicles and promotes activation of primordial follicles. Furthermore, GDF-9 stimulates the transition from primary to secondary follicles, maintaining ultrastructural integrity of the follicles.

Effects of fibroblast growth factor-2 on the in vitro culture of caprine preantral follicles.

The aims of the present study were to evaluate the effects of fibroblast growth factor-2 (FGF-2) on survival, activation and growth of caprine early-staged (preantral) follicles using histological and ultrastructural studies. Fragments of caprine ovarian cortex were cultured for 1 or 5 days in an enriched minimum essential medium, supplemented or not with different concentrations of FGF-2 (10, 50 or 100 ng/ml). Fragments from non-cultured ovarian tissue (control) and from tissues cultured for 1 or 5 days in a specific medium were processed for transmission electron microscopy (TEM) or classical histology to evaluate the morphological quality of caprine preantral follicles and to calculate the percentages of normal follicles. Additionally, effects of FGF-2 on oocyte and follicle diameter of cultured preantral follicles were investigated. Our results showed that, although the percentages of histologically normal follicles were lower in cultured than in non-cultured ovarian tissue fragments, there were no differences in this regard among treatments, neither on day 1 nor on day 5 of culture. After 1 and 5 days of culture, a significantly higher percentage of growing follicles was observed in the medium supplemented with 50 ng/ml of FGF-2. This FGF-2 treatment furthermore resulted in an increase in diameter of both oocytes and follicles that were cultured for 5 days. TEM showed that the ultrastructural integrity of caprine preantral follicles was maintained during their 5-day culture in the presence of 50 ng/ml FGF-2. In conclusion, this study demonstrated that at a concentration of 50 ng/ml FGF-2 not only maintains the morphological integrity of caprine preantral follicles cultured for 5 days, but also stimulates the activation of primordial follicles and the growth of activated follicles.

Essential role of follicle stimulating hormone in the maintenance of caprine preantral follicle viability in vitro.

The aims of the present study were to investigate the effects of follicle-stimulating hormone (FSH) on survival, activation and growth of caprine primordial follicles using histological and ultrastructural studies. Pieces of caprine ovarian cortex were cultured for 1 or 7 days in minimum essential medium (MEM - control medium) supplemented with different concentrations of FSH (0, 10, 50 or 100 ng/ml). Small fragments from non-cultured ovarian tissue and from those cultured for 1 or 7 days in a specific medium were processed for classical histology and transmission electron microscopy (TEM). Additionally, effects of FSH on oocyte and follicle diameter of cultured follicles were evaluated. The results showed that the lowest percentage of normal follicles was observed after 7 days of culture in control medium. After 1 day of culture, a higher percentage of growing follicles was observed in the medium supplemented with 50 ng/ml of FSH. In the presence of 10 and 50 ng/ml of FSH, an increase in diameter of both oocyte and follicle on day 7 of culture was observed. TEM showed ultrastructural integrity of follicles after 1 day of culture in MEM and after 7 days in MEM plus 50 ng/ml FSH, but did not confirm the integrity of those follicles cultured for 7 days in MEM. In conclusion, this study demonstrated that FSH at concentration of 50 ng/ml not only maintains the morphological integrity of 7 days cultured caprine preantral follicles, but also stimulate the activation of primordial follicles and the growth of activated follicles.

Cryopreservation of sheep primordial follicles.

The aim of this study was to evaluate the efficiency of 1 M dimethylsulphoxide (DMSO), ethylene glycol (EG), propylene glycol (PROH) and glycerol (GLY) to cryopreserve primordial follicles. The first evaluation was performed soon after cryopreservation and the second evaluation after 4 days of in vitro culture, using the cryoprotectants that allowed the higher results (higher follicular survival rate) after cryopreservation. The results after follicular isolation (control) and cryopreservation using 1 M DMSO, EG, PROH and GLY showed that the mean number (+/- SEM) of live follicles per millilitre was 3204 (100%) +/- 319.27, 2798 (87%) +/- 239.14, 2492 (78%) +/- 345.8, 448 (14%) +/- 46.3 and 208 (7%) +/- 75.26, respectively. Higher follicular survival was reported when DMSO and EG were used. Control follicles and follicles cryopreserved with these two cryoprotectants were cultured and the percentage of follicular survival was 55% (control), 42% (EG) and 34% (DMSO). Similar results were found between control and follicles cryopreserved with EG. In conclusion, 1 M EG is the most effective cryoprotectant to preserve primordial follicles isolated from ovaries of sheep.

Effects of storing pig ovaries at 4 or 20 degrees C for different periods of time on the morphology and viability of pre-antral follicles.

The purpose of this study was to evaluate the effect of cooling ovarian tissue on pig pre-antral follicles. Ovaries were maintained in saline solution (0.9%) at 4 or 20 degrees C for 6, 12 or 18 h. After storage, pre-antral follicles were morphologically evaluated. While primordial follicles were not affected by the storage, the percentage of morphologically normal growing follicles was significantly reduced in ovarian tissue stored at 20 degrees C for 12 or 18 h. To test the viability of stored follicles, growing follicles isolated from ovaries stored at 4 degrees C for 18 h and at 20 degrees C for 6 h were cultured for 3 days. Follicles stored in either condition presented the same growth pattern in vitro as fresh follicles. We conclude that storage of pig ovaries at 4 degrees C for up to 18 h or at 20 degrees C for up to 6 h does not affect the morphology of growing follicles or their ability to grow in vitro.

Light microscopical and ultrastructural characterization of black howler monkey (Alouatta caraya) ovarian follicles.

The present study describes the morphological characteristics of black howler monkey (Alouatta caraya) ovarian follicles. One ovary of an adult healthy black howler monkey was collected and processed for light and electron microscopy. Primordial, primary, secondary, tertiary and pre-ovulatory follicles were evaluated for their morphometrical aspects. The ovary of black howler monkey presented a distinct conformation with a uniform distribution of the follicles mostly in the peripheric cortex. This black howler monkey ovary presented a total of 59 921 ovarian follicles. From this amount, 71.1% were classified as primordial, 18.9% as primary, 8.1% as secondary, 1.4% as tertiary and 0.5% as pre-ovulatory follicles. From all these developmental stages, the mean diameters of follicles, oocytes, oocytes nuclei and the mean number of granulosa cells are described. Moreover, primordial, primary and secondary follicles have been observed by electron microscopy.