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.

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.

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.

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.

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.

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.

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.

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.

Effect of the interval of serial sections of ovarian tissue in the tissue chopper on the number of isolated caprine preantral follicles.

The present work investigated the effect of the interval of serial sections of ovarian tissue on the number of isolated preantral follicles in the goat. Goat ovaries were cut in the tissue chopper at eight different intervals. The quality of isolated follicles were evaluated by histology and transmission electron microscopy. Best results were obtained when the ovaries were cut in the tissue chopper at intervals of 75.0 microm (9664 preantral follicles per ovary). Histochemical and ultrastructural analysis showed that the follicular morphology was preserved after mechanical isolation as demonstrated by the normality of oocytes and granulosa cells as well as by preservation of basement membrane. The percentages of isolated primordial, primary and secondary follicles were 96.3%, 2.5%, and 1.2% and their average diameters were 21.5, 34.7 and 65.3 microm, respectively. It was concluded that the interval of serial sections of ovarian tissue in the tissue chopper affects the number of isolated preantral follicles, and that the follicles remained intact after mechanical isolation in goats.

Study of preantral follicle population in situ and after mechanical isolation from caprine ovaries at different reproductive stages.

The purposes of this study were to estimate the population of caprine preantral follicles, and to evaluate quantitatively and qualitatively the efficiency of a specific mechanical method for the isolation of preantral follicles from mixed breed goats at different reproductive stages. On average, 37,646+/-4277 preantral follicles were present in goat ovaries, and 13,631+/-2399 preantral follicles were obtained after isolation. The number of preantral follicles isolated or in situ was not significantly affected by the reproductive stage. The mean recovery rate per ovary ([number of isolated follicles/number of in situ follicles] x 100) of isolated follicles was 36.2%. The distribution of follicles in situ was 67.8% primordial, 25.8% primary and 6.4% secondary; the respective distribution after isolation was 93.8%, 5.2% and 1.0%. In this study, many polyovular follicles were also observed, mainly in prepubertal goat ovaries. Histological analysis showed that few preantral follicles were atretic in situ (4.83%+/-0.35) or after the isolation procedure (4.67%+/-0.65) in the three reproductive stages. The percentage of atretic follicles was not affected either by the mechanical method or by the reproductive stage. It is concluded that a large number of preantral follicles can be successfully isolated mechanically, with a high recovery rate and a low rate of follicular atresia, irrespective of the reproductive stage of the caprine female.

Effect of Braun-Collins and saline solutions at different temperatures and incubation times on the quality of goat preantral follicles preserved in situ.

The present work has investigated the efficiency of Braun-Collins and saline (0.9%) solutions in the conservation of goat preantral follicles in situ, at different temperatures and incubation times. For each animal the ovarian pair was divided into 19 fragments. One ovarian fragment was taken randomly and immediately fixed (control). The other 18 ovarian fragments were randomly distributed in tubes containing Braun-Collins or saline (0.9%) solutions at 4, 20 or 39 degrees C for 4, 12 or 24h. A total of 3385, 372 and 191 primordial, primary and secondary follicles were examined, respectively. The quality of preantral follicles was evaluated by histology and transmission electron microscopy. The storage of ovarian fragments in saline (0.9%) or Braun-Collins solutions at 4 degrees C did not reduce significantly the percentage of morphologically normal follicles when compared with the control. The histological analysis revealed a morphological integrity of goat preantral follicles stored at 4 degrees C for up to 24h in both solutions, but these results were not confirmed by ultrastructural analysis. The transmission electron microscopy revealed that only preantral follicles stored at 4 degrees C for a maximum of 12h in both solutions were ultrastructurally normal. In conclusion, this study shows for the first time that goat preantral follicles can be stored in situ successfully at 4 degrees C in saline (0.9%) or Braun-Collins solution for up to 12h.

Morphological and ultrastructural changes occurring during degeneration of goat preantral follicles preserved in vitro.

The present work has investigated the morphological and ultrastructural changes occurring during degeneration of goat preantral follicles preserved in vitro and showed quantitative data about the distribution of follicular degeneration types in the control and after preservation in coconut water solution or Braun-Collins solution at different temperatures (4, 20 or 39 degrees C) and incubation times (4, 12 or 24h). At the slaughterhouse, the pair of ovaries of each animal was divided into 19 fragments. One ovarian fragment was immediately fixed (control: Time 0). The other 18 fragments were randomly distributed in tubes containing 2ml of coconut water or Braun-Collins solution at 4, 20 or 39 degrees C and stored for 4, 12 or 24h. Normal preantral follicles exhibited a healthy oocyte surrounded by one or more well-organized layers of granulosa cells. The ooplasm contained numerous rounded or elongated mitochondria with continuous mitochondrial membranes. Golgi complexes were rare. Both smooth and rough endoplasmic reticulum were observed, either as isolated aggregations or complex associations with mitochondria and vesicles. Degenerated preantral follicles in the control tissue exhibited pycnotic nuclei of the oocyte, vacuolated ooplasm and normal granulosa cells. This kind of degeneration also predominated significantly (P<0.05) after preservation at 4 degrees C. In contrast, after preservation at 20 or 39 degrees C a significant predominance (P<0.05) of preantral follicles showing a retracted oocyte and swollen granulosa cells was observed. These follicles showed large irregularity of the oocyte and nuclear outlines. The ooplasm exhibited moderate proliferation of the endoplasmic reticulum and mitochondria showed disappearance of most of the cristae and damage to the mitochondrial membrane. Some follicles had numerous vacuoles in the ooplasm. Granulosa cells were spread and a low density of organelles was observed. The alterations in follicular structure progressed with an increase of temperature from 20 to 39 degrees C as well as with an increase of the incubation time from 4 to 12, or 24h. In conclusion, the present study shows for the first time that initial proliferation of the endoplasmic reticulum and damage to mitochondria are the first signs of degeneration in goat preantral follicles during storage in vitro.

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.

Effect of coconut water and Braun-Collins solutions at different temperatures and incubation times on the morphology of goat preantral follicles preserved in vitro.

Preservation of preantral follicles becomes very important to ensure follicle quality at the onset of cryopreservation or in vitro culture. However, for domestic animals, the ovarian donor of preantral follicles for in vitro studies is commonly encountered far away from reproduction laboratories. We investigated the effectiveness of coconut water and Braun-Collins solutions on the preservation of goat preantral follicles. At the slaughterhouse, the ovarian pair of each animal was divided into 19 fragments. One ovarian fragment was immediately fixed (Control - Time 0). The other 18 fragments were randomly distributed into tubes containing 2 mL of coconut water or Braun-Collins solution at 4 degrees, 20 degrees or 39 degrees C and then stored for 4, 12 or 24 h. Histological analysis showed that the storage of ovarian fragments in coconut water and Braun-Collins solutions at 20 degrees or 39 degrees C for 12 or 24 h significantly reduced (P < 0.05) the percentage of morphologically normal preantral follicles when compared with the control. However, storage in coconut water at 20 degrees C for 4 h and in both solutions at 4 degrees C kept the percentage at control values. Ultrastructural analysis of follicles exposed to the stated conditions confirmed the integrity of preantral follicles stored at 4 degrees C in Braun-Collins and coconut water solutions for up to 12 and 24 h, respectively. Reduced cellular metabolism at 4 degrees C may explain why the best preservation of preantral follicles was at 4 degrees C, which may suggest a useful method for ovary transport in the future.

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 3M 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 degrees C for 20 min in 1.8 ml of MEM containing 1.5 or 3M 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 3M GLY, as well as 3M EG demonstrated that these follicles remained morphologically normal. In conclusion, we demonstrated cryopreservation of caprine PF in ovarian tissue.

Quantitative and qualitative analysis of the effectiveness of a mechanical method for the isolation of preantral follicles from ovine ovaries.

The preantral follicles are the major source of oocytes and its utilization has been investigated as an important tool to store large numbers of female gametes for further utilization in reproductive programs. The aim of the present study was to perform quantitative and qualitative analyses of the efficacy of a mechanical method for isolating of preantral follicles from the ovaries of fetuses and from nonpregnant and pregnant ewes, using as reference the population of preantral follicles in situ. In the isolation method the ovaries were cut into fragments in the tissue chopper. Then, the suspension was filtered through nylon mesh filters. The number of isolated follicles per ovary was 1655, 4735 and 4770, respectively, for the fetus, nonpregnant ewe and pregnant ewe. The number of in situ preantral follicles per ovary was 32961, 16627 and 17794, respectively, for the fetus, nonpregnant ewe and pregnant ewe. The follicle recovery rate (number of isolated preantral follicles/number of in situ preantral follicles x 100) was higher in adult ewes (26 and 28%, respectively, for nonpregnant and pregnant ewes) than in fetuses (5%). Histological analysis showed that very few preantral follicles (less than 0.26% in situ and 0.46% after the isolation procedure) were degenerated. In conclusion, this study showed that a mechanical method could be used effectively to isolate a large number of intact ovine preantral follicles. In the future, with improvements in culture systems, the isolation of a great number of oocytes enclosed in preantral follicles will make a valuable contribution to the rare breeds and endangered species, agricultural efficiency and basic research in folliculogenesis.