Viability of zebrafish (Danio rerio) ovarian follicles after vitrification in a metal container

Cryopreservation of ovarian tissue has been studied for female germline preservation of farm animals and endangered mammalian species. However, there are relatively few reports on cryopreservation of fish ovarian tissue and especially using vitrification approach. Previous studies of our group has shown that the use of a metal container for the cryopreservation of bovine ovarian fragments results in good primordial and primary follicle morphological integrity after vitrification. The aim of this study was to assess the viability and in vitro development of zebrafish follicles after vitrification of fragmented or whole ovaries using the same metal container. In Experiment 1, we tested the follicular viability of five developmental stages following vitrification in four vitrification solutions using fluorescein diacetate and propidium iodide fluorescent probes. These results showed that the highest viability rates were obtained with immature follicles (Stage I) and VS1 (1.5 M methanol + 4.5 M propylene glycol). In Experiment 2, we used VS1 to vitrify different types of ovarian tissue (fragments or whole ovaries) in two different carriers (plastic cryotube or metal container). In this experiment, Stage I follicle survival was assessed following vitrification by vital staining after 24 h in vitro culture. Follicular morphology was analyzed by light microscopy after vitrification. Data showed that the immature follicles morphology was well preserved after cryopreservation. Follicular survival rate was higher (P < 0.05) in vitrified fragments, when compared to whole ovaries. There were no significant differences in follicular survival and growth when the two vitrification devices were compared.     

Effect of ovarian tissue vitrification method on mice preantral follicular development and gene expression

Vitrification is considered a viable method for cryopreservation of ovarian tissue and selection of methods that minimize follicular damage is important. The objective of the present study was to evaluate the effects of two vitrification methods on ovarian tissue morphology, preantral follicles survival rate during in vitro culture, and relative expression of genes associated with oocyte maturation and cumulus expansion. Ovaries from 12-day-old mice were vitrified in media containing ethylene glycol, dimethyl sulphoxide, and sucrose. Before plunging in liquid nitrogen, ovaries were first loaded into an acupuncture needle (needle immersion vitrification [NIV]) or placed on a cold steel surface for 10 to 20 seconds (solid surface vitrification [SSV]). The integrity of the ovarian tissue was well-preserved after vitrification and was similar controls. Follicle viability in the SSV group was lower (P < 0.05) than in the control group after 6 days of culture and the NIV group after 10 day of culture. Follicle viability after 12 day of culture was 92.8%, 82.1%, and 58.4% in control, NIV, and SSV groups, respectively. Bmp15, Gdf9, BmprII, Alk6, Alk5, Has2, and Ptgs2 gene expression patterns were similar among groups. However, the level of gene expression in the vitrification groups during Days 6 to 10 were higher compared with the control group. In conclusion, ovarian tissue morphologic integrity was well-preserved, regardless of the vitrification method. Vitrification using the needle immersion method resulted in greater follicular survival after 12 day of culture than the SSV method. Gene expression patterns during culture did not seem to explain the reduced survival rate observed in the solid surface group.     

Influence of vitrification techniques and solutions on the morphology and survival of preantral follicles after in vitro culture of caprine ovarian tissue

The objective was to compare the efficiency of various vitrification techniques and solutions for preserving morphology and viability of preantral caprine follicles enclosed in ovarian tissue. Fragments of ovarian cortex were cryopreserved by conventional vitrification (CV) in French straws, vitrification in macrotubes (MTV), or solid-surface vitrification (SSV). Six solutions containing 6 M ethylene glycol, with or without sucrose (SUC; 0.25 or 0.50 M) and/or 10% fetal calf serum (FCS) were tested (Experiment I). After 1 wk, samples were warmed and preantral follicles were examined histologically. To evaluate follicular viability (Experiment II), ovarian fragments were vitrified with the three techniques listed above, in a solution containing 0.25 M SUC and 10% FCS. After warming, follicles were assessed by the trypan blue dye exclusion test. In Experiment III, preantral follicles enclosed in ovarian tissue were vitrified using the protocol which yielded the highest percentage of viable preantral follicles (SSV with 0.25 M SUC and 10% SFB). After warming, the preantral follicles enclosed in ovarian tissue were cultured in vitro and then, were analyzed by histology and fluorescence microscopy (calcein-AM and ethidium homodimer-1). Every vitrification protocol significantly reduced the percentages of morphologically normal follicles relative to the control (88.0%); however, the addition of 0.25 M SUC and 10% FCS to the vitrification solution improved preservation of follicular morphology (67.4, 67.4, and 72.0% for CV, MTV, and SSV, respectively). Although follicular viability after SSV (80.7%) did not differ from that in fresh (non-vitrified) ovarian tissues (88.0%), after in vitro culture, percentages of viable follicles were significantly reduced (70.0%). Percentages of morphologically normal follicles after in vitro culture of vitrified ovarian tissue were similar (76.0%) to those in ovarian cortex fragments cultured without previous vitrification (83.2%). In conclusion, SSV using a solution containing 0.25 M SUC and 10% FCS, was the most efficient method for vitrifying caprine ovarian tissue.     

Cryopreservation of human ovarian tissue: Comparison of novel direct cover vitrification and conventional vitrification

Objective

To compare the effect of novel direct cover vitrification (DCV) and conventional vitrification (CV) for human ovarian tissue.

Study design

Ovarian biopsy specimens obtained from 12 patients were randomly allocated into five groups: Fresh, DCV1, DCV2, DCV3 and CV. Three concentrations of cryoprotectants were used in DCV group. The equilibration solution of DCV1, DCV2, DCV3 was 5% EG + 5% DMSO + DPBS, 7.5% EG + 7.5%DMSO + DPBS, 10% EG + 10% DMSO + DPBS, respectively. And the vitrification solution of DCV1, DCV2, DCV3 was 10% EG + 10% DMSO + DPBS, 15%EG+15% DMSO + DPBS, 20% EG + 20% DMSO + DPBS, respectively. The equilibration solution and the vitrification solution of CV group was same as DCV3 group. The effects of cryopreserved procedure on human ovarian tissue were studied by histology, TUNEL assay, transmission electron microscopy (TEM) and heterotopic allograft.

Results

The percentages of morphologically normal and viable follicles of DCV2 were significantly higher than DCV1, DCV3 and CV groups (P < 0.05). TUNEL assay demonstrated that the incidence of apoptotic cell in vitrification ovarian tissue was significantly higher than fresh tissue (P < 0.05), but there were no difference in various groups with cryopreservation. TEM showed that less damage was detected in DCV2 group. After grafting, the follicle density of DCV2 was greater than DCV1, DCV3 and CV groups (P < 0.05).

Conclusions

The novel cover vitrification with optimal concentration of cryoprotectants is superior to conventional vitrification. It is suitable for human ovarian tissue fragments with high efficiency and facility.     

Cryopreservation of ovarian tissues temporarily suppresses the proliferation of granulosa cells in mouse preantral follicles

Cryopreservation of ovarian tissue has been reported to delay the development of preantral follicles during in vitro culture, but the mechanism causing this impairment has not been brought to light. In order to elucidate the underlying mechanism of delayed follicular development, we evaluated the effects of cryopreservation on the proliferation of granulosa cells during culture of mouse preantral follicles, as a sufficient population of granulosa cells is critical for normal follicular development. Additionally the initial cell death of granulosa cells was estimated immediately after cryopreservation. The ovarian tissues obtained from 12-day-old female mice were cryopreservation by vitrification. The granulosa cell proliferation was evaluated by measuring the PCNA expression and the expression of cell cycle regulators such as cyclin D2, CDK4, cyclin E and CDK2 in preantral follicles isolated from fresh and cryopreserved ovarian tissues that were cultured for 48 h. The viability of granulosa cells was evaluated by measuring the proportion of necrotic areas. The granulosa cell proliferation of the cryopreserved preantral follicles was decreased significantly compared to that of the fresh controls at 0 and 24 h after culture (P < 0.05), and this was increased to the control levels after 48 h of culture. The expressions of cyclin D2, Cdk 4, cyclin E and Cdk2 were also decreased in the cryopreserved ovarian tissues at 0 and 24 h after culture (P < 0.05), but they were increased to the control levels after 48 h of culture. The proportion of the necrotic area was significantly higher in cryopreserved preantral follicles compared to that of the fresh preantral follicles (P < 0.05). This suggests that cryopreservation of ovarian tissues may delay the preantral follicle development by temporary suppressing the granulosa cell proliferation through the cell cycle regulators (cyclin D2, Cdk4, cyclin E and Cdk2) and by granulosa cell death immediately after warming.     

Slow freezing versus vitrification technique for human ovarian tissue cryopreservation: An evaluation of histological changes,WNT signaling pathway and apoptotic genes expression

This study compared slow freezing and vitrification of ovarian tissue by evaluation of histological changes, WNT signaling pathway and apoptotic genes expression. Ovarian tissue was obtained from women aging 27–38 years old. Ovarian cortex from each patient was divided into three pieces and randomly grouped as slow freezing, vitrification and control groups for investigation of WNT signaling gene expression and β-CATENIN presence as well as histological studies. The stromal structure of all ovaries were preserved. The number of secondary follicles decreased in vitrified group (P < 0.05). WNT-3, β-CATENIN, FZD-2 and GSK-3β expressions were significantly higher in slow frozen and vitrified groups, compared to control group (P < 0.05). On the contrary, AXIN1 expression in slow frozen samples were significantly lower than that of the vitrified and control group. The expression of apoptotic genes, excluding CASP3, was significantly decreased in slow-frozen samples (P < 0.05). Conversely, BAX:BCL-2 percentage significantly increased in vitrification versus slow freezing and control(P < 0.05). Follicles in slow frozen samples displayed nuclear and cytoplasmic β-CATENIN staining, while control and vitrification groups only showed β-CATENIN protein in the cytoplasm. The presented data show that slow freezing results in a better preservation regardless of the type of follicle. Therefore, it is concluded that slow freezing is still an ideal method for ovary cryopreservation.     

Generation of reactive oxygen species during cryopreservation may improve Lilium × siberia pollen viability

During biotic and abiotic stress in plants, reactive oxygen species (ROS) may play two very different roles: high ROS concentrations can exacerbate damage, whereas low concentrations can activate defense responses. The aim of this study was to investigate the relationship between ROS generation and pollen viability after cryopreservation. ROS generation was detected from ‘Siberia’ (Lilium?×?siberia) pollen using flow cytometry with 2′,7′-dichlorodihydrofluorescein diacetate as a fluorescent probe. Pollen viability was determined by 2,3,5-triphenyltetrazolium chloride staining. ROS generation was slightly increased by rapid cooling (26.13?±?4.74 vs. 15.80?±?2.30 for fresh pollen) and significantly increased by vitrification (49.74?±?1.43; P?<?0.01). Pollen viabilities after rapid cooling and vitrification were significantly increased (58.88?±?3.76% and 70.35?±?2.90%, respectively) over that of fresh pollen (46.65?±?1.61%; P?<?0.01). No significant differences in ROS generation were associated with cold acclimation at different temperatures before rapid cooling. However, sharp decreases in viability were observed with cold acclimation at 4°C and ?20°C relative to rapid cooling without acclimation (P?<?0.01). We observed nonsignificant decreases in ROS generation among vitrification treatments that omitted different steps and a significant decrease when the unloading step was omitted (P?<?0.05). Pollen viabilities were significantly reduced when the loading or dehydration steps were omitted (P?<?0.01). No significant differences were observed in ROS generation or pollen viability among the treatments when 200 U/ml catalase was added to different solutions used in the vitrification process. Comprehensive analysis of all data indicated a positive correlation between ROS generation and pollen viability (r?=?0.651, P?<?0.001). Therefore, increasing ROS generation during cryopreservation may improve the viability of ‘Siberia’ pollen.     

Dietary energy source and feeding levels during the rearing period affect ovarian follicular development and oocyte maturation in gilts

Fifty-four Landrace × Yorkshire gilts (59.0 ± 4.2 kg and 147 ± 3 d old) were used to examine the effects of dietary energy source (starch or mixed fat) at high [112.5% of energy requirements recommended by NRC (1998)], normal (100%), and low (87.5%) energy feeding levels on ovarian follicular development and oocyte maturation. Forty-seven estrus gilts were slaughtered at Day 19 after the second estrus; oocytes were recovered from follicles >4 mm in diameter, and matured in vitro for 44 h. Gilts fed high-energy diets had more follicles >4 mm (mean, 25.8 vs. 19.1, P < 0.05) and more oocytes that reached metaphase II (80.3 vs. 64.0%, P < 0.05) than those fed the low-energy diet. Furthermore, gilts fed starch-rich diets had enhanced oocyte nuclear maturation relative to those fed fat-rich diets (75.4 vs. 68.0%, P < 0.05). Compared to the lower-energy feeding groups, high-energy feeding groups had higher (P < 0.05) blood concentrations of postprandial insulin (1562.4 vs. 990.0 ng/4 h), IGF-I (321.2 vs. 256.9 ng/mL), and LH pulses (2.7 vs. 1.4 pulses/6 h). Follicular fluid concentrations of IGF-I (198.5 vs. 143.1 ng/mL) and estradiol (152.6 vs. 124.8 ng/mL) were higher (P < 0.05) in the high-energy group than in the normal group. Compared with gilts fed the high-energy diet supplemented with fat, gilts fed the high-energy diet supplemented with starch had a tendency (P < 0.10) towards increased IGF-I concentration in both blood and follicular fluid, and improved oocyte nuclear maturation during culture in vitro. We inferred that starch-rich, high-energy diets during rearing may improve ovarian follicular development and oocyte maturation in replacement gilts.     

Addition of cholesterol loaded cyclodextrin prior to GV-phase vitrification improves the quality of mature porcine oocytes in vitro

The purpose of this study was to determine whether the mitochondrial membrane potential, pro-apoptotic gene expression, and ubiquitylation status of zona pellucida proteins (ZP1, ZP2, and ZP3) of vitrified GV-stage mature oocytes could be protected by treatment with cholesterol-loaded methyl-β-cyclodextrin (CLC) prior to vitrification. Porcine GV oocytes were treated with CLC prior to the vitrification process, and the effects on the mitochondrial membrane potential and ZP ubiquitylation status were determined by JC-1 single staining and western blot assays. We found that porcine GV-stage oocytes were treated with CLC at different concentrations (0.5, 5, and 10 mg/mL) prior to vitrification improved in vitro maturation of these oocytes (P < 0.05). The mitochondrial membrane potential of matured oocyte without vitrification or treated with 5 mg/mL CLC vitrification treatment was higher than that of the 0 mg/mL CLC group and other treatment groups (vitrified) (P < 0.05). The expression of Caspase 3, Caspase 8, and Caspase 9 genes in the high concentration CLC treatment groups (5 and 10 mg/mL) was significantly lower than that in the 0 (vitrified) mg/mL CLC group (P < 0.05). ZPs protein and ZP3 protein ubiquitylation were also higher in the non-vitrified controls, 5 and 10 mg/mL CLC-treated oocytes than in the 0 (vitrified) and 0.5 mg/mL vitrified groups (P < 0.05). Whereas the sperm–oocyte binding capacity was improved in the CLC treatment groups (P < 0.05) but the embryonic development rate was not improved. In conclusion, pretreatment with CLC can improve the survival rate and maturation rate of oocytes and protect their mitochondria and zona pellucida of porcine oocytes from cryodamage during the vitrification process.     

Long-term in vitro culture of ovarian cortical tissue in goats: effects of FSH and IGF-I on preantral follicular development and FSH and IGF-I receptor mRNA expression

Long-term in vitro culture (16?days) of caprine ovarian cortical tissue was performed to test the effect of FSH and IGF-I on the viability and development of preantral follicles and mRNA expression for FSH and IGF-I receptors. Fragments were cultured in ??-MEM⁺ alone or supplemented with different combinations of FSH and IGF-I (sequential medium). The culture period was divided into two parts. Follicles were isolated and classified as normal or abnormal and primordial, primary or secondary. Viability of isolated follicles was determined by staining with Trypan Blue dye. Expression of FSHR and IGFR-1 mRNA was evaluated by qPCR. At day 8 of culture, more (P?<?0.05) follicles in treatments containing IGF-I alone or associated with FSH were normal and viable (overall mean, 81?% and 79?% respectively) than the treatments cultured with FSH or ??-MEM⁺ alone (68?% and 63?%). At day 16 of culture, treatments with FSH and/or IGF-I had more (P?<?0.05) viable follicles (69?%) than ??-MEM⁺ (38?%). The percentages of follicular development observed in the IGF-I/FSH, FSH+IGF-I/FSH+IGF-I and FSH/IGF-I treatments were similar but higher (P?<?0.05) than the other treatments. FSH and IGF-I during the entire culture period maximized (P?<?0.05) follicular and oocyte diameters and the percentage of secondary follicles (28?%). FSHR mRNA expression in the non-cultured control was similar to the treatment supplemented with FSH and IGF-I but higher (P?<?0.05) than ??-MEM⁺. IGFR-1 expression did not differ among treatments. Association of FSH and IGF-I in long-term in vitro culture promoted follicular development, maintaining FSHR mRNA expression.     

Vitrification of goat preantral follicles enclosed in ovarian tissue by using conventional and solid-surface vitrification methods

Caprine preantral follicles within ovarian fragments were exposed to or vitrified in the presence of sucrose, dimethyl sulfoxide (DMSO), ethylene glycol (EG), or various combinations thereof. The fragments were cryopreserved by using either a conventional (CV) or a solid-surface vitrification (SSV) protocol, and the cryoprotectants were removed by equilibrating vitrified ovarian fragments in “warming solution” consisting of minimum essential medium and heat-inactivated fetal calf serum (MEM⁺) followed by washes in MEM⁺ with or without sucrose. Histological analysis of follicle integrity showed that the percentages of normal follicles in ovarian fragments vitrified in sucrose mixed with EG and/or DMSO (CV method) or mixed with EG or DMSO (SSV method) followed by washes in MEM⁺ plus sucrose were similar to those of controls (ovarian fragments fixed without previous vitrification). Unlike for MEM⁺ (supplemented or unsupplemented by sucrose) and DMSO followed by washes in the absence of sucrose, the percentages of normal follicles found after exposure to cryoprotectant did not significantly differ from that found after vitrification, indicating that follicular degeneration was attributable to a toxic effect of cryoprotectants and not to the vitrification procedure. The viability of preantral follicles after the CV and SSV procedures was investigated by using calcein-AM and the ethidium-homodimer as “live” and “dead” markers, respectively. In both tested vitrification procedures, the highest percentages of viable follicles were observed when a mixture of sucrose and EG (70.3% for CV and 72.4% for SSV) was used. Preantral follicles were also vitrified (either by CV or SSV) in sucrose and EG and then cultured for 24 h, after which their viability was compared with that of cultured fresh and uncultured vitrified follicles. The viability of these follicles was maintained after SSV, but not after CV. Thus, the viability of caprine preantral follicles can be best preserved after SSV in a mixture of sucrose and EG, followed by washes in medium containing sucrose.CAPES/Brazil supported this work. Regiane Rodrigues dos Santos is a recipient of a grant from CAPES/Brazil.     

Effects of inulin and di-d-fructose dianhydride-enriched caramels on intestinal microbiota composition and performance of broiler chickens

In vitro and in vivo experiments were designed to evaluate the effectiveness of laboratory-made di-d-fructose dianhydride (DFA)-enriched caramels. The DFA-enriched caramels were obtained from d-fructose (FC), d-fructose and sucrose (FSC), or d-fructose and β-cyclodextrin (FCDC). In the in vitro experiment, raftilose and all caramels increased (P<0.05) l-lactate concentration and decreased (P<0.05) pH. Total short-chain fatty acid concentration was higher (P<0.05) than controls in tubes containing raftilose, FSC, FCDC and commercial sucrose caramel (CSC). Raftilose, and all caramels tested except FSC and FC (1%), increased (P<0.01) lactobacilli log₁₀ number of copies compared with the non-additive control. FSC, FCDC and CSC increased (P<0.01) the bifidobacteria number of copies as compared with controls. All additives, except FCDC, decreased (P<0.01) Clostridium coccoides/Eubacterium rectale log number of copies. Compared with controls, raftilose, FC and CSC led to lower (P<0.01) Escherichia–Shigella and enterobacteria. For the in vivo experiment, a total of 144 male 1-day-old broiler chickens of the Cobb strain were randomly assigned to one of the three dietary treatments for 21 days. Dietary treatments were control (commercial diet with no additive), inulin (20 g inulin/kg diet) and FC (20 g FC/kg diet). Final BW of birds fed FC diet was higher (P<0.01) than controls or inulin-fed birds, although feed: gain values were not different. Feed intake of chickens fed FC was higher (P<0.01) than that of inulin-fed birds but not statistically different from controls. Crop pH values were lower (P<0.01) in birds fed FC diet as compared with control diet, with inulin-fed chickens showing values not different from control- or FC-fed birds. Lower (P<0.05) lactobacilli number of copies was determined in the crop, ileum and caeca of birds fed the inulin diet compared with the control diet. Inulin supplementation also resulted in lower (P<0.05) C. coccoides/E. rectale, bacteroides and total bacteria in caecal contents. Addition of FC to broiler diets gave place to lower (P<0.05) enterobacteria and Escherichia–Shigella in crop and caecal contents compared with controls. The bacteroides number of copies increased (P<0.05) as compared with controls in the ileum, but decreased (P<0.05) in the caeca of chickens fed the FC diet. Energy, ADF, NDF and non-starch polysaccharides faecal digestibilities were greater (P<0.05) than controls in chickens fed diets containing inulin or FC. Fat digestibility was higher (P<0.05) in FC-fed birds compared with controls or inulin-fed chickens. In conclusion, DFA-enriched caramels tested here, particularly FC, may represent a type of new additives useful in poultry production.     

Effects of active immunization against a 13-amino acid receptor-binding epitope of FSHβ on fertility regulation in female mice

Follicle-stimulating hormone (FSH) is crucial for ovarian folliculogenesis and thus essential for female fertility. Here, we developed a novel FSH vaccine based on the tandem of a 13-amino acid receptor-binding epitope of FSHβ (FSHβ13AA-T) and used a mouse model to test its efficacy in female fertility regulation. Compared to placebo-immunized controls, FSHβ13AA-T vaccination: induced a marked (P < 0.05) antibody generation; reduced (P < 0.05) serum concentrations of FSH, inhibin B and 17β-estradiol; disrupted (P < 0.05) normal estrous cyclicity; delayed (P = 0.08) establishment of pregnancy; blocked (P < 0.05) folliculogenesis; and reduced (P < 0.05) litter size. Mechanistically, FSH vaccination reduced (P < 0.05) ovarian estrogen production by decreasing Lhcgr, Cyp19a1 and HSD3β1 expression, and suppressed ovarian follicular development by decreasing ovarian Fshr, Inhα, Foxo3a, Bmp15 and Cdh1 expression. Overall, vaccination of female mice with FSHβ13AA-T substantially disrupted FSH-dependent ovarian steroidogenesis and folliculogenesis, and caused subfertility. Therefore, vaccines based on FSHβ13AA-T have potential as anti-fertility/contraceptive agents in females.     

Effects of extracellular calcium on viability and osteogenic differentiation of bone marrow stromal cells in vitro

Bone marrow stromal cells (BMSCs) have been extensively used for tissue engineering. However, the effect of Ca²⁺ on the viability and osteogenic differentiation of BMSCs has yet to be evaluated. To determine the dose-dependent effect of Ca²⁺ on viability and osteogenesis of BMSCs in vitro, BMSCs were cultured in calcium-free DMEM medium supplemented with various concentrations of Ca²⁺ (0, 1, 2, 3, 4, and 5 mM) from calcium citrate. Cell viability was analyzed by MTT assay and osteogenic differentiation was evaluated by alkaline phosphatase (ALP) assay, Von Kossa staining, and real-time PCR. Ca²⁺ stimulated BMSCs viability in a dose-dependent manner. At slightly higher concentrations (4 and 5 mM) in the culture, Ca²⁺ significantly inhibited the activity of ALP on days 7 and 14 (P < 0.01 or P < 0.05), significantly suppressed collagen synthesis (P < 0.01 or P < 0.05), and significantly elevated calcium deposition (P < 0.01) and mRNA levels of osteocalcin (P < 0.01 or P < 0.05) and osteopontin (P < 0.01 or P < 0.05). Therefore, elevated concentrations of extracellular calcium may promote cell viability and late-stage osteogenic differentiation, but may suppress early-stage osteogenic differentiation in BMSCs.     

Three dimensional in vitro culture of preantral follicles following slow-freezing and vitrification of mouse ovarian tissue

To evaluate the effects slow-freezing and vitrification on three dimensional in vitro culture of preantral follicles, ovaries of 12–14 days old female NMRI mice were isolated and randomly assigned to fresh control, slow-freezing and vitrification groups. Slow-freezing was performed using programmable freezer. Vitrification was carried out in a medium consisting of ethylene glycol (EG) and dimethyl sulphoxide (Me₂SO) by needle immersion method. middle sized preantral follicles were mechanically isolated and cultured for 12 days in 0.7% sodium alginate gel. The follicles development and quantitative expression of oocyte specific genes (Bmp15, Gdf9, Fgf8) and the growth related genes (Igf1, Kit, Kit-l) were assessed after 1, 8 and 12 days of culture. Both cryopreserved groups showed reduction of follicular survival rates compared to the control group on days 8 and 12 of culture (P < 0.05). Antrum formation rates reduced in slow-freezing after 12 days of culture (P < 0.05). Evaluation of gene expression showed reduction of Bmp15, Gdf9, Fgf8, Kit and Kit-l during 12 days of culture (P < 0.05). Kit and Kit-l expression in slow-freezing group significantly reduced on day 8 of culture (p < 0.05). Igf1 expression was lower in slow-freezing group on 1st day of culture than vitrification and control groups (P < 0.05). Finally, intergroup comparison showed same expression pattern of genes after 12 days of culture. Thus, cryopreservation of mouse ovaries by both methods can preserve most developmental parameters and expression of maturation genes. However, vitrification is a better method for cryopreservation of mouse ovaries due to greater antrum formation and expression of growth related markers.     

In vitro survival and development of goat preantral follicles in two different oxygen tensions

The aim of the present study was to evaluate the effect of two different oxygen (O₂) concentrations on survival and development of preantral follicles of goats cultured in vitro. Preantral ovarian follicles (≥150 μm) were isolated from ovarian cortex fragments of goats and individually cultured for 30 days under two different O₂ concentrations (5% and 20% O₂). Follicle development was evaluated on the basis of antral cavity formation, increase in follicular diameter, presence of healthy cumulus oocyte complexes and fully grown oocytes. Results showed with progression of culture period from 6 to 12 days, a decrease in follicular survival was observed in both O₂ concentrations (P < 0.05). When the O₂ tensions were compared to each other in the different days of culture, 20% O₂ was more efficient in promoting an increase in follicular diameter from day 24 of culture onward than 5% O₂ (P < 0.05). However, follicles cultured with 5% O₂ had an increased percentage of antrum formation from 12 days to the end of culture, compared with 20% O₂ (P < 0.05). Moreover, there was no difference in percentage of fully developed oocytes with the different O₂ tensions. However, only oocytes (16.7%) from follicles cultured in 20% O₂ resumed meiosis. In conclusion, concentration of 20% O₂ was more efficient in promoting follicular growth and oocyte meiosis resumption from preantral follicles of goats when grown in vitro.     

The Local Effects of Ovarian Diathermy in an Ovine Model of Polycystic Ovary Syndrome

In order to develop a medical alternative to surgical ovarian diathermy (OD) in polycystic ovary syndrome (PCOS) more mechanistic information is required about OD. We therefore studied the cellular, molecular and vascular effects of diathermy on the ovary using an established ovine model of PCOS. Pregnant sheep were treated twice weekly with testosterone propionate (100 mg) from day 30–100 gestation. Their female offspring (n = 12) were studied during their second breeding season when the PCOS-like phenotype, with anovulation, is fully manifest. In one group (n = 4) one ovary underwent diathermy and it was collected and compared to the contralateral ovary after 24 hours. In another group a treatment PCOS cohort underwent diathermy (n = 4) and the ovaries were collected and compared to the control PCOS cohort (n = 4) after 5 weeks. Ovarian vascular indices were measured using contrast-enhanced ultrasound and colour Doppler before, immediately after, 24 hours and five weeks after diathermy. Antral follicles were assessed by immunohistochemistry and ovarian stromal gene expression by quantitative RT-PCR 24 hours and 5 weeks after diathermy. Diathermy increased follicular atresia (P<0.05) and reduced antral follicle numbers after 5 weeks (P<0.05). There was an increase in stromal CCL2 expression 24 hours after diathermy (P<0.01) but no alteration in inflammatory indices at 5 weeks. Immediately after diathermy there was increased microbubble transit time in the ovarian microvasculature (P = 0.05) but this was not seen at 24 hours. However 24 hours after diathermy there was a reduction in the stromal Doppler blood flow signal (P<0.05) and an increased ovarian resistance index (P<0.05) both of which persisted at 5 weeks (P<0.01; P<0.05). In the ovine model of PCOS, OD causes a sustained reduction in ovarian stromal blood flow with an increased ovarian artery resistance index associated with atresia of antral follicles.     

Evaluation of vitrification protocol of mouse ovarian tissue by effect of DNA methyltransferase-1 and paternal imprinted growth factor receptor-binding protein 10 on signaling pathways

Transplantation of cryopreserved ovarian tissue has been considered as a promising way of fertility preservation for women. however, this cryopreservation method is prone to post-resuscitation follicle proliferation and oocyte development stagnation, affecting late transplant survival. To evaluate current vitrification works, we investigated the critical pathway alternations in vitrified-warmed juvenile 10-day-old mouse ovary. We showed a significant decrease of protein kinase B (Akt) and Mitogen-activated protein kinase (Mapk) phosphorylation, during which serine/threonine kinases play central roles in coordinating follicle and oocyte development and stress response. Inhibition of Akt and Mapk activity were associated with one of the imprinted insulin pathway negative regulatory genes, Growth factor receptor-binding protein 10 (Grb10) which remarkably increased in vitrified-warmed juvenile mouse ovary than that of fresh group (p < 0.05). RNAi-induced Grb10 down-regulation reversed the decrease in Akt and Mapk phosphorylation. The increase of Grb10 expression was partially caused by the hyper-methylation of the promoter region, associated with the decrease of follicular DNA methyltransferase (Dnmt) 1 protein in different stages of vitrified-warmed group, compared to fresh group (p < 0.05). The mRNA and protein expression of Dnmt1 in ovary of vitrified-warmed juvenile mouse were remarkably lower than those in fresh group (p < 0.05). Dnmt1 overexpression dramatically reversed Grb10 up-regulation and Akt and Mapk phosphorylation reduction. Taken together, our findings suggest that Grb10 expression might be helpful in evaluation of effectiveness of vitrification, and considered as a potential target for further vitrification protocols improvement in the future.     

Consequences of rearing feeding programme on the performance of rabbit females from 1st to 2nd parturition

To evaluate how rearing programmes could affect resources allocation and reproductive performance of primiparous rabbit females, a total of 118 rabbit females were used to evaluate the effects of five rearing feeding programmes on their performance from 1st to 2nd parturition: CAL, fed ad libitum C diet (11.0 MJ digestible energy (DE), 114 g digestible protein (DP) and 358 g NDF/kg dry matter (DM) until 1st parturition; CR, fed ad libitum with C diet until 12 weeks of age and then C diet restricted (140 g/day) until 1st parturition; F, fed ad libitum with F diet (8.7 MJ DE, 88 g DP and 476 NDF/kg DM) until 1st parturition; FC, fed with F diet ad libitum until 16 weeks of age, and C diet ad libitum until 1st parturition; FCF, fed with F diet ad libitum until 16 weeks of age, then C diet ad libitum until 20 weeks and then F diet ad libitum until 1st parturition. From 1st parturition, C diet was ad libitum offered to all the experimental groups until 2nd parturition. CAL females presented lower feed intake than females of F, FC and FCF groups in the 1^st week of lactation (on av. −16.6%; P<0.05). During 1st lactation, the perirenal fat thickness change in CAL females was not different from 0 (+0.02 mm), while in the other four groups it increased (on av. +0.44 mm; P<0.05). Plasma of females fed with F diet during rearing (F, FC and FCF) had lower non-esterified fatty acids content than those exclusively fed with C diet (–0.088 and –0.072 mmol/l compared to CAL and CR, respectively; P<0.05). FCF litters had higher weight than F litters at day 21 of lactation (+247 g; P<0.05), but FCF litter had significantly lower weight than FC litters at weaning (+170 g; P<0.05). CR females had the shortest average interval between the 1st and 2nd parturition (49 days) and FCF females the longest (+ 9 days compared to CR; P<0.05). At 2nd parturition, liveborn litters of F females were larger and heavier than litters of FCF females (+2.22 kits and +138 g; P<0.05), probably due to the lower mortality at birth of F litters (–16.5 percentage points; P<0.05). In conclusion, rearing females on fibrous diets seems to increase the ability of primiparous rabbit females to obtain resources, especially at the onset of lactation.     

Ovarian follicular dynamics, follicle deviation, and oocyte yield in Gyr breed (Bos indicus) cows undergoing repeated ovum pick-up

The objective of this study was to evaluate ovarian follicular dynamics during intervals between successive ovum pick-up (OPU) and determine its effects on the number and quality of recovered cumulus-oocyte complexes (COCs) in Zebu cows (Bos indicus). Pluriparous nonlactating Gyr cows (Bos indicus; n = 10) underwent four consecutive OPU sessions at 96-h intervals. The dynamics of ovarian follicular growth between OPU sessions was monitored by twice-daily ultrasonographic examinations. A single dominant follicle (DF) or two codominant (CDF) follicles (>9 mm) were present in 63.3% (19 of 30) of intervals studied, with follicle deviation beginning when the future dominant follicle (F1) achieved a diameter of 6.2 ± 0.3 mm. The phenomenon of codominance was observed in four (13.3%) of the inter-OPU intervals. The remaining intervals (36.6%, 11 of 30) were characterized by a greater follicular population, lower rate of follicular growth, and a smaller diameter F1 (P < 0.0001). There was a tendency (P = 0.08) toward an increase in the number of recovered COCs when dominant follicles were not present (NDF). The quality of COCs was not affected by the presence of a single dominant follicle, but codominant follicles resulted in recovery of a lower proportion of viable embryos (40.0%, 62.1%, and 63.6%; P < 0.05) and higher proportions of degenerate COCs (56.0%, 30.3%, and 28.6%; P < 0.05) for CDF, NDF, and DF respectively. We concluded that, in Zebu cows, (a) repeated follicle aspirations altered ovarian follicular dynamics, perhaps by increasing follicular growth rate; (b) follicular dominance could be established in cows undergoing twice-a-week OPU; and (c) the presence of a dominant follicle during short inter-OPU intervals may not affect COC quality, except when a codominant follicle was present.