Interference of fixatives and fixation period on the morphologic analysis of ovarian preantral follicles.

Ovine ovarian fragments (3 × 3 × 1 mm) were fixed in neutral buffered formalin (NBF), Carnoy's solution (CAR), Davidson's solution (DAV), or paraformaldehyde (PFA) for 12 h or 24 h. After this fixation time, each fragment was prepared for histological analysis. Although fixative and fixation period did not affect follicular and stromal cells density, the percentages of morphologically normal primordial and primary follicles was affected by the fixative type and period of fixation. Paraformaldehyde was not indicated as a fixative for ovarian fragments. Formalin was a suitable fixative only when the period of fixation was 12 h, while Carnoy was efficient after a fixation period of 24 h. In conclusion, the most indicated fixative for the morphological evaluation of ovarian preantral follicles was DAV, regardless of the fixation period, that is 12 or 24 h.

Effects of in vitro exposure of sheep ovarian tissue to zearalenone and matairesinol on preantral follicles.

The aim of this study was to evaluate the effect of 1 µmol/l zearalenone (ZEN) and 1 µmol/l matairesinol (MAT), alone or in combination, on the morphology of in vitro-cultured ovarian preantral follicles. Ovaries from four adult sheep were collected at a local slaughterhouse and fragmented, and the ovarian pieces were submitted to in vitro culture for 3 days in the presence or absence of the test compounds. The morphology of primordial and primary follicles was impaired by ZEN. The plant lignan MAT alone did not maintain the morphology of the ovarian follicles; its combination with ZEN counteracted the negative effects observed when follicles were cultured in the presence of the mycotoxin alone. However, MAT was not able to promote the in vitro development of the ovarian follicles.

Betaine-loaded CaCO3 microparticles improve survival of vitrified feline preantral follicles through higher mitochondrial activity and decreased reactive oxygen species.

Ovary fragments from six sexually mature cats were vitrified in the presence or absence of betaine or ascorbic acid, loaded (7.4 or 74µM betaine; 20 or 200µM ascorbic acid) or not (1mM betaine or 0.3mM ascorbic acid) into CaCO3 microparticles, and assessed for follicular morphology, oxidative stress and mitochondrial activity Feline ovarian tissue was successfully preserved after vitrification in the presence of 74µM betaine loaded in CaCO3 microparticles, as confirmed by morphological analysis and the density of preantral follicles and stromal cells, as well as by the increased mitochondrial activity and decreased production of reactive oxygen species.

Anethole improves blastocysts rates together with antioxidant capacity when added during bovine embryo culture rather than in the in vitro maturation medium.

We performed the exposure of bovine oocytes to anethole during in vitro maturation (0 or 300 µg/ml), during in vitro embryo production (0, 30, 300 or 2000 µg/ml), or during both periods to determine the rates of 2-4 cells embryos, blastocysts rates and cells numbers, as well as the production of reactive oxygen species (ROS). Bovine ovaries (n = 240) were collected from a local abattoir after slaughter and cumulus-oocyte complexes (COCs) with homogeneous and non-dark cytoplasm, surrounded by two or more compact layers of cumulus cells, and an intact zona pellucida were selected for in vitro maturatuion (IVM). Mature oocytes were then submitted to in vitro fertilization (IVF) and in vitro embryo production (IVP) in culture medium supplemented or not with different concentrations of anethole, as described above. Although IVM medium supplementation with 300 µg/ml anethole improved the rates of bovine blastocysts formation, we demonstrated that IVP medium supplementation with 30 µg/ml anethole, regardless of IVM medium enrichment, considerably enhanced blastocysts rates. Furthermore, ROS levels were decreased only when anethole was added to the IVP medium without previous IVM medium supplementation.

In vivo and in vitro strategies to support caprine preantral follicle development after ovarian tissue vitrification.

The aim of the present study was to compare fresh and vitrified goat ovarian tissue after autotransplantation and in vitro culture. Adult goats were completely ovariectomised and each ovarian pair was sliced and distributed among six different treatment groups: fresh control, fresh transplant, fresh culture, vitrified control, vitrified transplant and vitrified culture. Follicular morphology, development, growth, density, revascularisation and hormone production were evaluated in all groups. Three antral follicles (two in the fresh transplant and one in the vitrified transplant groups) were observed on the surface of the graft 90 days after transplantation. The percentage of morphologically normal follicles was similar in the fresh control, fresh transplant and vitrified transplant groups. The percentage of developing (transition, primary and secondary) follicles was higher after in vitro culture of fresh or vitrified tissue. Transplantation resulted in a lower follicle density. Serum oestradiol concentrations remained constant during the entire transplantation period. In contrast, progesterone production decreased significantly. Expression of CD31 mRNA was lower in fresh culture. In conclusion, restoration of goat ovarian function can be successfully achieved following transplantation of both fresh and vitrified goat ovarian tissue. However, transplantation induced higher follicle loss than in vitro culture.

Morphology and morphometry of preantral follicles, and immunolocalization of angiogenic factors in ovarian tissue from the neotropical primate Sapajus apella.

SummaryOvarian biopsies from five health adult monkeys were collected by exploratory laparotomy. Preantral follicles (primordial, primary, and secondary) were classified as normal or degenerated and submitted to morphometric analysis in which granulosa cell counts and the areas of follicles, oocytes, and oocyte nuclei were measured. Ovarian fragments were also immunolabelled for the quantitative analysis of VEGFA and CD31 protein expression in the ovarian tissue and in the preantral follicles. In total, 213 preantral follicles was examined for morphometry and morphological classification. From this total, 20 (9.4%) were follicles enclosing two or more oocytes, i.e. multi-oocyte follicles (MOFs). From the 193 follicles enclosing only one oocyte, 46.3% were classified as primordial, 24,1% as transition, 23.3% as primary, and 6.3% as secondary follicles. The mean number of granulosa cells surrounding primordial, transition, primary, and secondary follicles was 9.2, 12.1, 18.7, and 45.3, respectively. Increase in oocyte diameter was observed from primary to secondary follicles, while the oocyte nucleus increased only when follicles reached the secondary stage. The expression of CD31 was strong in vessels, corpus luteum, and in normal oocytes and granulosa cells from preantral follicles at all developmental stages. Likewise, VEGFA expression was observed in vessels and preantral follicles (granulosa cells, the oocyte and the oocyte nucleus). We characterized the morphology, and morphometry and expression of angiogenic factors in normal and atretic preantral follicles from Sapajus apella. This description can support the analysis of follicular quality and survival after procedures such as transplantation and cryopreservation.

The effects of Trolox on the quality of sperm from captive squirrel monkey during liquefaction in the extender ACP-118™.

SummaryThe aim of this study was to evaluate the effect of incubating semen for different periods (90, 270 or 450 min) with or without Trolox® (100 or 150 µM) on the quality of sperm from Saimiri collinsi. Sperm motility, vigour, and plasma membrane integrity (PMI) were evaluated in both fresh semen and semen incubated for different time periods, i.e. 90, 270 or 450 min of incubation. Supplementation of semen extender with Trolox® 100 µM improved sperm motility, vigour and PMI for up to 270 min of incubation.

Vitrification of bovine embryos followed by in vitro hatching and expansion.

The objective of this study was to assess the effects of bovine embryo vitrification by applying three different vitrification solutions containing ethylene glycol (EG) and dimethylsulphoxide (DMSO) at different concentrations (10, 20 or 25% each) combined with 1.0 M glucose or 1.0 M sucrose, on the in vitro hatching and expansion rates. Healthy oocytes were selected for in vitro maturation and fertilization from 200 bovine ovaries, and subsequently cultured up to the blastocyst stage (n = 800). Control (n = 200) and vitrified cells (n = 100 per treatment; 600 in total) were cultured for an extra 24 or 48 h to evaluate hatching and expansion, respectively. Vitrification significantly decreased embryonic re-expansion and hatching rates independently of the tested solution when compared with control embryos, but solutions with 25% EG + 25% DMSO resulted in the highest re-expansion (75%) and hatching (70%) rates, independently of the added sugar. The addition of sucrose resulted in higher rates of re-expanded and hatched embryos when compared with glucose addition. We concluded that the combination of 25% EG + 25% DMSO and 1.0 M sucrose allowed hatching and expansion of vitrified-warmed bovine embryos produced in vitro.

Therapy with mesenchymal stromal cells or conditioned medium reverse cardiac alterations in a high-fat diet-induced obesity model.

BACKGROUND: Obesity is associated with numerous cardiac complications, including arrhythmias, cardiac fibrosis, remodeling and heart failure. Here we evaluated the therapeutic potential of mesenchymal stromal cells (MSCs) and their conditioned medium (CM) to treat cardiac complications in a mouse model of high-fat diet (HFD)-induced obesity. METHODS: After obesity induction and HFD withdrawal, obese mice were treated with MSCs, CM or vehicle. Cardiac function was assessed using electrocardiography, echocardiography and treadmill test. Body weight and biochemical parameters were evaluated. Cardiac tissue was used for real time (RT)-polymerase chain reaction (PCR) and histopathologic analysis. RESULTS/DISCUSSION: Characterization of CM by protein array showed the presence of different cytokines and growth factors, including chemokines, osteopontin, cystatin C, Serpin E1 and Gas 6. HFD-fed mice presented cardiac arrhythmias, altered cardiac gene expression and fibrosis reflected in physical exercise incapacity associated with obesity and diabetes. Administration of MSCs or CM improved arrhythmias and exercise capacity. This functional improvement correlated with normalization of GATA4 gene expression in the hearts of MSC- or CM-treated mice. The gene expression of connexin 43, troponin I, adiponectin, transforming growth factor (TGF) ß, peroxisome proliferator activated receptor gamma (PPARγ), insulin-like growth factor 1 (IGF-1), matrix metalloproteinase-9 (MMP9) and tissue inhibitor of metalloproteinases 1 (TIMP1) were significantly reduced in MSCs, but not in CM-treated mice. Moreover, MSC or CM administration reduced the intensity of cardiac fibrosis. CONCLUSION: Our results suggest that MSCs and CM have a recovery effect on cardiac disturbances due to obesity and corroborate to the paracrine action of MSCs in heart disease models.

Short communication: Interaction of the isomers carvacrol and thymol with the antibiotics doxycycline and tilmicosin: In vitro effects against pathogenic bacteria commonly found in the respiratory tract of calves.

Bovine respiratory disease is the major problem faced by cattle, specially calves, leading to reduced animal performance and increased mortality, consequently causing important economic losses. Hence, calves must be submitted to antibiotic therapy to counteract this infection usually initiated by the combination of environmental stress factors and viral infection, altering the animal's defense mechanism, and thus allowing lung colonization by the opportunistic bacteria Mannheimia haemolytica and Pasteurella multocida. Essential oils appear to be candidates to replace antibiotics or to act as antibiotic adjuvants due to their antimicrobial properties. In the present study, we aimed to evaluate the 4 essential oil components carvacrol, thymol, trans-anethole, and 1,8 cineole as antibacterial agents or as adjuvants for the antibiotics doxycycline and tilmicosin against M. haemolytica and P. multocida. Bacteria were cultured according to standard protocols, followed by the determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration. A checkerboard assay was applied to detect possible interactions between components, between antibiotics, and between components and antibiotics. Doxycycline at 0.25 and 0.125 µg/mL inhibited the growth of P. multocida and M. haemolytica, respectively, whereas tilmicosin MIC values were 1.0 and 4.0 µg/mL for P. multocida and M. haemolytica, respectively. Carvacrol MIC values were 2.5 and 1.25 mM for P. multocida and M. haemolytica, respectively, whereas thymol MIC values were 1.25 and 0.625 mM for P. multocida and M. haemolytica, respectively. Trans-anethole and 1,8 cineole did not present any antibacterial effect even at 40 mM against the investigated pathogens. All minimum bactericidal concentration values were the same as MIC, except when thymol was tested against M. haemolytica, being twice the MIC data (i.e., 1.25 mM thymol). Based on fractional inhibitory concentration checkerboard assay, no interaction was observed between doxycycline and tilmicosin. Carvacrol and thymol presented an additive effect when one of them was combined with tilmicosin. Additive effect was also observed when doxycycline was combined with thymol. Synergism was observed when carvacrol was combined with doxycycline or with thymol. Although the antibacterial effects of the tested essential oil components were observed at high concentrations for in vitro conditions, the additive and synergic effects of carvacrol and thymol with antibiotics suggest the option to apply them as antibiotic adjuvants.

Modulation of aquaporins 3 and 9 after exposure of ovine ovarian tissue to cryoprotectants followed by in vitro culture.

Our aim has been to evaluate the effect of cryoprotective agents (CPAs) on the exposure, vitrification (VIT), and in vitro culture (IVC) of ovarian tissue with regard to the expression and immunolocalization of aquaporins (AQPs) 3 and 9 in ovine preantral follicles. Tissues were treated as follows: Experiment I: (1) control (without exposure to CPAs), (2) e-EG (exposure to ethylene glycol), (3) er-EG (exposure to and removal of EG), (4) e-DMSO (exposure to dimethyl sulfoxide), (5) er-DMSO (exposure to and removal of DMSO), (6) e-EG+DMSO (exposure to EG+DMSO), (7) er-EG+DMSO (exposure to and removal of EG+DMSO); Experiment II: (1) control, (2) VIT, (3) IVC, (4) VIT-IVC. In Experiment I, following er-EG or er-DMSO, tissue showed the down-regulation (P < 0.05) of AQP3 mRNA. The mRNA transcript levels were reduced (P < 0.05) for AQP9 in tissue following er-EG+DMSO. Immunolocalization was positive for both proteins (AQP3 and AQP9) on ovine preantral follicles following all treatments, except in the e-EG+DMSO group. In Experiment II, the mRNA levels of AQP3 and AQP9 following VIT treatment were similar (P > 0.05) to that of the control group. Nevertheless, VIT-IVC treatment led to the down-regulation of mRNA of AQP3 and AQP9. Thus, AQP3 and AQP9 act in a mutually dependent way, maintaining the cell homeostasis that is essential for the ovary cryopreservation process. Furthermore, the changes in the expression profiles of mRNA and protein after culture are a strong indicator that in vitro conditions have to be strictly controlled to ensure follicle viability and functionality.

Population genetic structure of Attalea vitrivir Zona (Arecaceae) in fragmented areas of southeast Brazil.

Attalea vitrivir Zona (synonym Orbignya oleifera) is one of the six species of Arecaceae known as "babassu". This species is used to make cosmetics, food, and detergents due to the high concentration of oil in the seeds. It is found only in fragmented areas of southern Bahia State and northern Minas Gerais State, southeast Brazil, and this fragmentation has affected both its ecological and genetic characteristics. We evaluated the genetic diversity and population genetic structure of A. vitrivir in six areas of two different regions at the extremes of its geographical range, in order to gain a better understanding of the factors that affect the distribution and partitioning of its diversity. Nine inter simple sequence repeat (ISSR) markers amplified 74 polymorphic bands, resulting in large diversity values (Shannon diversity index, 0.37-0.47; intrapopulation genetic diversity, 0.25-0.34). Analysis of molecular variance (AMOVA) revealed considerable differentiation between sampling sites (30.03%) and regions (12.08%), although most of the diversity was observed within sampling sites (69%). Further differentiation between sampling sites was noted more in the northern region than in the southern region, highlighting the genetic connectivity between the sampling sites within Rio Pandeiros Environmental Protection Area (southern region). The identification of two distinct genetic clusters (K = 2) corresponded to the northern and southern regions, and corroborated the AMOVA results. We suggest that the northern area, outside Rio Pandeiros Environmental Protection Area, must be included in future management plans for this species.

Vitamin E-analog Trolox prevents endoplasmic reticulum stress in frozen-thawed ovarian tissue of capuchin monkey (Sapajus apella).

Ovarian fragments were exposed to 0.5 M sucrose and 1 M ethylene glycol (freezing solution; FS) with or without selenium or Trolox. Histological and ultrastructural analyses showed that the percentages of normal follicles in control tissue and in tissue after exposure to FS + 50 µM Trolox were similar. Trolox prevented endoplasmic reticulum (ER)-related vacuolization, which is commonly observed in oocytes and stromal tissue after exposure to FS. From the evaluated stress markers, superoxide dismutase 1 (SOD1) was up-regulated in ovarian tissue exposed to FS + 10 ng/ml selenium. Ovarian fragments were subsequently frozen-thawed in the presence of FS with or without 50 µM Trolox, followed by in vitro culture (IVC). Antioxidant capacity in ovarian fragments decreased after freeze-thawing in Trolox-free FS compared with FS + 50 µM Trolox. Although freezing itself minimized the percentage of viable follicles in each solution, Trolox supplementation resulted in higher rates of viable follicles (67 %), even after IVC (61 %). Furthermore, stress markers SOD1 and ERp29 were up-regulated in ovarian tissue frozen-thawed in Trolox-free medium. Relative mRNA expression of growth factors markers was evaluated after freeze-thawing followed by IVC. BMP4, BMP5, CTGF, GDF9 and KL were down-regulated independently of the presence of Trolox in FS but down-regulation was less pronounced in the presence of Trolox. Thus, medium supplementation with 50 µM Trolox prevents ER stress and, consequently, protects ovarian tissue from ER-derived cytoplasmic vacuolization. ERp29 but not ERp60, appears to be a key marker linking stress caused by freezing-thawing and cell vacuolization.

Effect of dietary treatments on performance, oocysts shedding and lesion scores in broiler chickens experimentally challenged with Eimeria infection.

Although anticoccidials effectively control coccidiosis, a needed reduction in the reliance on antimicrobials in animal production leads to the exploration of alternative compounds. The present study aimed to test five different dietary treatments to counteract the negative impact of coccidiosis on broiler chickens' health and performance. 1-day-old male Ross 308 broilers (n = 960) were randomly assigned to one of eight treatments, with six cages per treatment (20 birds/cage). To the diet of the broiler chickens of treatments (Trt) 1-5, a synbiotic was added from d0-10. From d10-28, birds of Trt1 and Trt2 were fed synbiotics, whereas birds of Trt3 were fed diets with glutamine, and birds of Trt4 and Trt5 were fed diets with a combination of ß-glucans and betaine. From d28-35 onwards, birds of Trt1 were fed a diet with a synbiotic, whereas birds of Trt2-4 received diets with glutamine, and birds of Trt5 were fed a non-supplemented diet. Birds of the positive control group (PC; Trt6) were fed a standard diet supplemented with an anticoccidial (Decoquinate). The challenged negative control (NCchall; Trt7) and non-challenged negative control (NC) Trt8 were fed a standard diet without anticoccidial or other dietary treatment. At 7 days (d) of age, all birds were inoculated with 1 023, 115, and 512 sporulated oocysts of E. acervulina, E. maxima, and E. tenella, respectively, except for Trt8. Body weight gain (BWG), feed intake, and feed conversion ratio were assessed for each feeding phase (d0-10, d10-28 and d28-35) and overall experimental period (d0-35). Oocyst shedding, Eimeria lesion scores, cecal length, and relative weight were assessed at d13, d22, d28 and d35. Additionally, oocyst shedding was determined at d9 and d17. Litter quality was evaluated at d27 and d34, and footpad lesions at d34. During the starter (d0-10) and finisher (d28-35) periods, performance did not differ between the treatments. During the grower period (d10-28), Trt6 (PC) and Trt8 (NC) chickens had the highest BWG of all treatments (P < 0.001). Dietary treatment had no effect on litter quality and severity of footpad lesions. In the PC group (Trt6), low oocyst excretion and lesion scores were found. When comparing Trt1-5 with NCchall (Trt7), none of the treatments significantly reduced oocyst output or lesion scores. In conclusion, in this experiment, none of the dietary treatments performed similar or better compared to the PC group (Trt6) regarding performance or reducing Eimeria oocyst shedding or lesion scores.

Validation of reference genes for ovarian tissue from capuchin monkeys (Cebus apella).

There is no tradition in studies reporting the effect of exposure to cryoprotectants or simply hypoxia and hypothermia on gene expression in the ovarian tissue and there has been only one study on reference or target genes quantification, and comparisons of normoxic with hypoxic, hypothermic and toxic conditions. Our aim in the present study was to investigate the stability of three reference genes in the ovarian tissue of capuchin monkeys (Cebus apella). To this end, fresh and cryoprotectant-exposed ovarian biopsies were used. Both fresh and exposed ovarian tissues were subjected to total RNA extraction and synthesis of cDNA. cDNA was amplified by real-time polymerase chain reaction (PCR), and GeNorm, BestKeeper and NormFinder software were used to evaluate the stability of glyceraldehyde-2-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyltransferase 1 (HPRT1) and TATA-binding protein (TBP). Results demonstrated that, in the ovarian tissue from capuchin monkeys, HPRT1 and TBP were the most suitable reference genes and thus could be used as parameters to normalize data in future studies. In contrast, GAPDH appeared as the least stable gene among the tested reference genes. In conclusion, HPRT1 and TBP were the most stable reference genes in fresh and cryoprotectant-exposed ovarian tissue from capuchin monkeys.

Nutritional interventions to support broiler chickens during Eimeria infection.

Different combinations of gut health-promoting dietary interventions were tested to support broilers during different stages of Eimeria infection. One-day-old male Ross 308 broilers (n = 720) were randomly assigned to one of 6 dietary treatments, with 6 pens per treatment and 20 birds per pen, for 35 d. At 7 d of age (d7), all birds were inoculated with 1000, 100, and 500 sporulated oocysts of E. acervulina, E. maxima, and E. tenella, respectively. A 4-phase feeding schedule was provided. The dietary treatments (TRT) 1 to 4 included the basal diet supplemented with multispecies probiotics from d0 to 9 and coated butyrate and threonine from d28 to 35 but received four different combinations of prebiotics and phytochemicals from d9 to 18 and d18 to 28. The basal diet for the positive control (PC, TRT5) included diclazuril as a anticoccidial. The negative control (NC, TRT6) contained no anticoccidial. Performance was assessed for each feeding phase, and oocyst output, Eimeria lesion scores, cecal weight, litter quality, and footpad lesions were assessed at d14, d22, d28, and d35. Body weight gain (BWG) and feed intake (FI) were not affected by dietary treatment. PC broilers had the best feed conversion ratio (FCR) of all treatments from d0 to 35 (P < 0.001). None of the dietary treatments resulted in better litter quality or reduced footpad lesions compared to the PC. Moreover, the PC was most effective in reducing oocyst output and lesion scores compared to all other treatments. However, broilers that received the multispecies probiotics (d0 to 9), saponins (d9 to 18), saponins, artemisin, and curcumin (d18 to 28), and coated butyrate and threonine (d28 to 35) had the best FCR (P < 0.001) and lowest oocyst output and lesion scores compared to other dietary treatments. This study suggests that although the tested compounds did not perform as well as the anticoccidial, when applied in the proper feeding period, they may support bird resilience during coccidiosis infection.

Validation of the online PREDICT tool in a cohort of early breast cancer in Brazil.

PREDICT is a tool designed to estimate the benefits of adjuvant therapy and the overall survival of women with early breast cancer. The model uses clinical, histological, and immunohistochemical variables. This study aimed to evaluate the model's performance in a Brazilian population. We assessed the discrimination and calibration of the PREDICT model to estimate overall survival (OS) in five and ten years of follow-up in a cohort of 873 women with early breast cancer diagnosed from January 2001 to December 2016. A total of 743 patients had estrogen receptor (ER)-positive and 130 had ER-negative tumors. The area under the receiver operating characteristic (ROC) curve (AUC) for discrimination was 0.72 (95%CI: 0.66-0.78) at five years and 0.67 (95%CI: 0.61-0.72) at ten years for women with ER-positive tumors. The AUC was 0.72 (95%CI: 0.62-0.81) at five years and 0.67 (95%CI: 0.54-0.77) at ten years for women with ER-negative tumors. The predicted survival in ER-positive tumors was 91.0% (95%CI: 90.2-91.6%) at five years and 79.3% (95%CI: 77.7-81.0%) at ten years, and the observed survival 90.7% (95%CI: 88.6-92.9%) and 77.2% (95%CI: 73.4-81.4%), respectively. The predicted survival in ER-negative tumors was 84.5% (95%CI: 82.5-86.6%) at five years and 75.0% (95%CI: 71.6-78.5%) at ten years, and the observed survival 76.3% (95%CI: 69.1-84.3%) and 67.9% (95%CI: 58.6-78.6%), respectively. In conclusion, PREDICT was accurate to estimate OS in women with ER-positive tumors and overestimated the OS in women with ER-negative tumors.

Cryopreservation and in vitro culture of caprine preantral follicles.

Preantral follicles (PFs) form a far larger oocyte reservoir (~90% of the follicular population) than antral follicles. Several laboratories have focussed efforts on cryopreservation and in vitro culture (IVC) of PFs to obtain large numbers of fertilisable oocytes. This technology could be used to improve the reproductive potential of economically important animals, including goats, to preserve endangered species and breeds and improve fertility after chemotherapy in young women. Caprine PFs have been successfully cryopreserved using either vitrification or slow freezing. In addition, in vitro embryo production from oocytes enclosed in caprine PFs grown and matured in vitro was also achieved. The present paper selectively reviews the published studies on cryopreservation and IVC of caprine PFs to highlight advances, limitations and prospects.

Irreversible damage in ovine ovarian tissue after cryopreservation in propanediol: analyses after in vitro culture and xenotransplantation.

Current progress in cancer treatment has increased the incidence of long-term patient survival. Ovarian tissue cryopreservation (OT) is still the most promising fertility saving method offered to young female patients with cancer prior to the onset of radio-chemotherapy. Further follicular development of immature primordial follicles depends on transplantation or in vitro culture (IVC). Aim of this study was to evaluate the appropriateness of cryopreserved ovine OT with 1,2-propanediol (PROH) after short-term IVC and xenotransplantation (XT). Ovarian tissue fragments from young adult sheep were cryopreserved using a standard slow-freezing protocol with 1.5 M PROH. Cryopreserved OT was assessed by light- and transmission electron microscopic analyses after thawing, IVC or XT in severe immunodeficient mice. Control OT showed the presence of healthy preantral follicles (Mean: 78.8%; SE 2.9%) and normal structure of the stromal tissue. After thawing and IVC over 80% of damaged primordial follicles and poor preservation of the stromal tissue was observed. After XT, OT demonstrated deficient follicles and huge areas of vacuolization in the stromal tissue confirmed by ultrastructural assessment. In conclusion, because of the irreversible character of the follicular and stromal damage of cryopreserved ovine ovarian tissue after IVC and XT, strong improvement of the utilized protocol is needed to be suitable for the preservation of ovine ovarian tissue. The deleterious effects of PROH do not imply its exclusion as cryoprotectant, but more research is needed for the development of less toxic cryoprotectant mixtures and toxicity neutralizers with attested cryoprotectant capacity for the safe and feasible freezing of human ovarian tissue.

Adding ascorbic acid to vitrification and IVC medium influences preantral follicle morphology, but not viability.

In this study, we analysed the effect on morphology and viability of ovine primordial follicles, when ascorbic acid (AA) was added to vitrification and in vitro culture (IVC) media. For morphological analysis, ovarian tissue was vitrified using DMSO or ethylene glycol (EG), to which AA was added or omitted. After warming, the tissue was fixed for histology or 1-day cultured in the presence or absence of AA. Isolated primordial follicles from ovine ovarian tissue vitrified with DMSO or EG, both supplemented with AA were stained with trypan blue for viability analysis, or 5-day cultured with or without AA followed by a viability analysis. In this study, we report on the successful vitrification protocol developed for ovine ovarian tissue using EG. Vitrification using DMSO reduced the percentage of morphological normal primordial follicles, whereas addition of AA to the vitrification and culture media did enhance these results (p < 0.05). However, vitrification in a DMSO + AA medium followed by 5-day IVC resulted in a significant decrease in the follicular viability, independently of the presence of AA in the IVC medium.