Alpha lipoic acid controls degeneration and ensures follicular development in ovine ovarian tissue cultured in vitro.

This study aims to evaluate the effects of adding alpha lipoic acid (ALA) to the in vitro ovarian tissue culture medium, either fresh or after vitrification/warming. For this purpose, 10 ovaries from five adult sheep were used. Each pair of ovaries gave rise to 16 fragments and were randomly distributed into two groups: fresh (n = 8) and vitrified (n = 8). Two fresh fragments were fixed immediately and considered the control, while another six were cultured in vitro for 14 days in the absence; presence of a constant (100 µM/0-14 day) or dynamic (50 µM/day 0-7 and 100 µM/day 8-14) concentration of ALA. As for the vitrified fragments, two were fixed and the other six were cultured in vitro under the same conditions described for the fresh group. All the fragments were subjected to morphological evaluation, follicular development and stromal density (classical histology), DNA fragmentation (TUNEL), senescence (Sudan Black), fibrosis (Masson's Trichome), and endoplasmic reticulum stress (immunofluorescence). Measurements of the antioxidant capacity against the free radicals 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) and estradiol (E2) levels in the culture medium was performed. The results showed that in the absence of ALA, in vitro culture of vitrified ovarian fragments showed a significant reduction (P < 0.05) in follicular morphology and increased the presence of senescence and tissue fibrosis (P < 0.05). Dynamic ALA maintained E2 levels unchanged (P > 0.05) until the end of vitrified ovarian tissue culture and controlled the levels of ABTS and DPPH radicals in fresh or vitrified cultures. Therefore, it is concluded that ALA should be added to the vitrified ovarian tissue in vitro culture medium to reduce the damage that leads to loss of ovarian function. To ensure steroidogenesis during in vitro culture, ALA should be added dynamically (different concentrations throughout culture).

Anethole supplementation during in vitro maturation increases in vitro goat embryo production in a concentration-dependent manner.

During in vitro maturation (IVM) cumulus-oocyte complexes (COCs) are exposed to conditions that can trigger oxidative stress, thus, reducing oocyte maturation and viability. Aiming to mitigate these detrimental conditions, the effects of IVM medium supplementation with anethole have been tested. Anethole, also known as trans-anethole (1-methoxy-4 [1-propenyl]-benzene), is a naturally occurring phenylpropanoid with various pharmacological properties, including antioxidant effects. However, no study has examined anethole effect on goat COCs during IVM. Thus, the aim of this study was to evaluate the effects of different anethole concentrations on oocyte maturation, oxidative stress, and in vitro development of caprine embryos after parthenogenetic activation. Goat COCs were selected and randomly distributed into the following treatments: TCM-199+ medium (control), or TCM-199+ medium supplemented with 30 µg/mL (AN30); 300 µg/mL (AN300) or 2000 µg/mL (AN2000) of anethole. After IVM, part of the COCs was chosen for oocyte viability and chromatin configuration, intracellular reactive oxygen species levels, and mitochondrial membrane potential assessment. Another part of COCs was parthenogenetically activated, and presumptive zygotes were cultured for 7 days. Results demonstrated that anethole at 30 µg/mL increased oocyte maturation and cleavage rates when compared to the other treatments (P < 0.05), as well as oocyte viability and in vitro embryo production when compared to the control treatment (P < 0.05). Additionally, treatment with anethole at 2000 µg/mL decreased oocyte nuclear maturation and cleavage rates when compared to other treatments (P < 0.05) and embryo production if compared to control and AN30 treatments (P < 0.05). Moreover, anethole at 2000 µg/mL increased mitochondrial membrane potential when compared to the other treatments (P < 0.05). In conclusion, anethole exerts a concentration-dependent effect during goat COCs IVM. For a more desirable outcome of oocyte viability and maturation, and in vitro embryo production, the use of anethole at 30 µg/mL is recommended.

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.

In vitro- and in vivo-derived early antral follicles have comparable in vitro follicular growth and oocyte maturation rates in goats.

Recent in vitro follicle culture (IVFC) studies in caprine have yielded lower maturation rates using late preantral follicles compared to early antral follicles. Thus, research focusing on developing stage-specific customized culture systems able to improve the efficiency of IVFC for late preantral follicles are warranted. This study aimed to compare the morphometric features, estradiol production, and gene expression between early antral caprine follicles produced in vitro and in vivo. In vitro-derived early antral follicles were produced after a 6-day in vitro culture of late preantral follicles, while in vivo-derived early antral follicles were yielded immediately after isolation from the ovaries; antral follicles were, thereafter, cultured for 18 days. In vitro-derived antral follicles were cultured either in a medium developed for preantral follicles (PF medium) or in a medium developed for antral follicles (AF medium). In vivo-derived early antral follicles, on the other hand, were cultured in AF medium (Control treatment). Results demonstrated that in vitro-derived antral follicles cultured in PF medium produced higher estradiol concentration, and m-RNA expression for matrix metalloproteinase-9 (MMP-9), and insulin receptor when compared to both in vitro- and in vivo-derived antral follicles cultured in AF medium. Remarkably, in vitro-derived antral follicles cultured in PF medium had similar MII and oocytes ≥110 µm rates compared with in vivo-derived antral follicles (Control treatment). In conclusion, when cultured in a single and appropriate medium (i.e., PF medium), in vitro-derived early antral follicles had comparable oocyte maturation rates to the in vivo-derived early antral follicles.

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.

Transcriptional downregulation of ABC transporters is related to follicular degeneration after vitrification and in vitro culture of ovine ovarian tissue.

ATP-binding cassette (ABC) transporters perform multiple functions in reproductive tissues. During ovarian tissue vitrification, the plasma membrane has important functions in the influx or efflux of water, and substances such as cryoprotectants and channel proteins that are required in this process. Thus, the present study aimed to verify the relative abundance of mRNA transcript of ABC transporters ABCB1, ABCG2, and MRP2 after vitrification and in vitro culture (IVC) of ovine ovarian tissue. For this study, the ovarian cortex fragments were proportioned into four groups as fresh control, vitrified control, fresh culture, and vitrified culture groups. After vitrification and in vitro culture, the ovarian tissue was evaluated using morphological procedures. Further, relative abundance of ABCB1, ABCG2, and MRP2 transporter mRNA transcripts in the ovarian cortex subjected to aforementioned treatment conditions were evaluated using qPCR. Our results showed a negative association between degenerated follicles and mRNA transcript abundances of ABCB1 and ABCG2. In addition, the percentage of growing follicles in the ovine ovarian cortex after vitrification was similar to that of the fresh control tissue without in vitro culture. The in vitro culture of fresh and vitrified tissue however, showed a significant decrease in the percentage of growing follicles. To the best of our knowledge, we believe that our data for the first time has studied the relative abundances of ABCB1 and ABCG2 mRNA transcripts in the ovine ovarian cortex. In addition, alterations of these protein channels may be indicative of a deleterious effect of osmotic stress on follicular survival during vitrification. Furthermore, these effects were detectable only after the IVC of the ovarian tissues. Nonetheless, further studies are required to investigate the functions of ABC transporters in ovine folliculogenesis, especially after in vitro culture of ovarian tissue.

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.

In vitro exposure of sheep ovarian tissue to the xenoestrogens zearalenone and enterolactone: Effects on preantral follicles.

The aim of this study was to evaluate the effect of 1 µmol/L zearalenone (ZEN) and 1 µmol/L enterolactone (ENL), alone or in combination, on the survival and morphology of in vitro cultured ovarian preantral follicles. Ovaries from 10 sheep were collected at a local abattoir 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, whereas that of cultured secondary follicles was improved by ENL. However, the combination of ENL with ZEN impaired the quality of primary and secondary follicles. Both ZEN and ENL induced apoptosis, but only ZEN was responsible for oocyte autophagy. None of these xenoestrogens affected endoplasmic reticulum stress as observed by the unaltered expression of ERP29. Differently from ZEN, ENL increased the expression of the efflux transporter ABCG2. In conclusion, although ENL can counteract the negative effects of ZEN on primordial and primary follicles, this positive effect is not similar to that observed in ovarian tissue cultures in the presence of ENL alone.

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.

The subtle balance of insulin and thyroxine on survival and development of in vitro cultured caprine preantral follicles enclosed in ovarian tissue.

Numerous studies have reported the importance of thyroid hormones on the development of later preantral and antral follicles, but their interactions with other hormones and effects in regulating early preantral follicle growth remain unclear. Here we investigated the in vitro effects of thyroxine combined with insulin on caprine preantral follicle survival and development. Sliced ovarian tissues were cultured for 1 or 7 days using 10 ng/mL (low) or 10 µg/mL (high) insulin in the presence of thyroxine at 0, 0.5, 1 or 2 µg/mL. Post-culture, we evaluated the follicular survival and development, assessed the expression of apoptotic-related genes (Bcl2/Bax) and receptors of insulin and thyroid hormones, and quantified the estradiol and reactive oxygen species (ROS) production levels. Follicular survival in low-insulin culture conditions was enhanced by the presence of 0.5 µg/mL thyroxine (P < 0.05) as compared to the thyroxine-free medium but remained similar to non-cultured control in the presence of 2 µg/mL (P > 0.05). Significantly higher ROS production was measured from Day 1 to Day 7 in low-insulin culture media containing 0.5 or 2 µg/mL thyroxine (P < 0.05). When compared to high insulin level, the presence of thyroxine in low insulin culture conditions yielded higher stromal cell density (P < 0.05), increased estradiol production on Day 1, and higher Bcl2/Bax ratio on Day 7. Cultures with high levels of both insulin and thyroxine led to follicles and oocytes with larger diameters (P < 0.05). The RNA transcript levels of insulin and thyroid receptors were reduced in the presence of high insulin cultures when compared to controls (non-cultured). In conclusion, the combination of low concentrations of insulin and thyroxine better maintained follicle survival, while high levels ensured better follicular development.

Effect of different extracts and fractions of Senecio biafrae (Oliv. &Hiern) J. Moore on in vivo and in vitro parameters of folliculogenesis in experimental animals.

BACKGROUND: Senecio biafrae is a medicinal plant widely used in traditional medicine to cure female infertility. Some effects have been pharmacologically demonstrated on immature female rats but in vivo and in vitro investigations are still necessary for determining its mechanism of action. The aim of the present study was to evaluate the estrogenic and FSH-like effects of the plant extracts and fractions on some fertility parameters in immature female rats and on in vitro survival and growth of swine preantral follicles. METHODS: 21-23 days old female Wistar rats orally received extracts and fractions of S. biafrae at 0, 8 and 64 mg/kg doses over 20 days. The LH, FSH, estradiol and progesterone serum levels were evaluated as well as the ovarian cholesterol, uterus and ovaries masses and proteins. The numbers of follicles at different developmental stages were recorded in ovarian cortexes after histology. Slices of swine ovarian cortexes were cultured along 1 or 7 days in alpha-minimum essential medium (α-MEM) and fixed for morphological analysis of preantral follicles. The fresh control, cultured control (CIV control) and different Senecio biafrae-treated ovarian fragments were analyzed for preantral follicles development. Treatments that showed the best follicle growth in culture were submitted to AgNOR test. The aqueous and MeOH/CH2Cl2 extracts as well as the ethyl acetate and hexane fractions of S. biafrae were submitted to the HPLC for analysis of polyphenolic secondary metabolites. RESULTS: Ovarian and uterine proteins were significantly high (p < 0.01) in animals treated with the two dosages of ethyl acetate and n-butanol fractions. The same result was recorded with uterine proteins in animals treated with the hexane fraction. The FSH level significantly dropped with all ethanolic extract doses and with the 64 mg/kg dosage of the methanol/methylene chloride (MeOH/CH2Cl2) extract while LH was reduced (p < 0.01) in almost all the treated groups. Estradiol level was significantly increased (p < 0.001) in the three groups receiving the extracts, but reduced (p < 0.001) in the three groups receiving the fractions of the plant. The progesterone level increased with almost all the treated groups. Primary and secondary follicles augmented (p < 0.01) in MeOH/CH2Cl2 extract and n-butanol fraction while tertiary follicles increased with the same extract and the ethyl acetate fraction (p < 0.05). Treatments with aqueous and ethanolic extracts as well as ethyl acetate fraction led to a significant increase (p < 0.05) in the number of morphologically normal follicles after 7 days of culture as compared to the CIV control. The number of AgNOR dots per follicle was significantly low (p < 0.05) in all cultured groups as compared to the fresh control, except the ethyl acetate 2.8 ng/ml dosage. The same observation was done with AgNOR dots per cell in the 2.8 ng/ml dosage aqueous extract-treated fragments. The phenolic compounds mainly encountered in the plant, independently of the extract or fraction are apigenin, eugenol and rutin. CONCLUSION: Extracts and fractions of S. biafrae have an important FSH-like effect which induces follicular survival and growth.

Linfoma leucemizado em felino coinfectado com os vírus da imunodeficiência felina e da leucemia felina: relato de caso / Lymphoma in leukemic phase in feline coinfected with feline immunodeficiency virus and feline leukemia virus: case report

O presente trabalho teve por objetivo relatar um caso de linfoma leucemizado em um felino coinfectado com os vírus da imunodeficiência felina (FIV) e o da leucemia felina (FeLV). Foram realizados exames de hemograma, contagem de reticulócitos, mielograma, bioquímica, teste de imunocromatografia para FIV e FeLV, imunofluorescência indireta (IFA) para FeLV, radiografia torácica e citologia renal. Esse último exame revelou um linfoma extranodal. Foi determinante para a conclusão diagnóstica a associação dos sinais clínicos corroborados com a infiltração de elevada quantidade de células linfoblásticas na medula óssea, exibindo critérios citomorfológicos de malignidade, como mitoses atípicas, relacionadas à presença de corpúsculos linfoglandulares e material hematopoiético inter-relacionado. O linfoma é uma neoplasia relativamente comum em felinos, entretanto, a apresentação leucemizada é rara, podendo representar um desafio diagnóstico clínico, o que torna fundamental a inclusão da citologia medular na prática clínica dessa espécie.(AU)

The present study aimed to report a case of lymphoma in leukemic phase in feline coinfected with feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV). Blood counts, reticulocyte counts, bone marrow avaluation, biochemistry, immunochromatography assay for FIV and FeLV, indirect immunofluorescence (IFA) for FeLV, thoracic radiography and renal citology were performed. This last examination revealed extranodal lymphoma. The association of the clinical signs with the infiltration of a high number of lymphoblastic cells in the bone marrow with cytomorphological criteria of malignancy, atypical mitoses, lymphoglandular corpuscles and hematopoietic material were determinant for the diagnostic conclusion. Lymphoma is a relatively common neoplasm in felines, however the leukemic phase is rare and may represent a clinical diagnostic challenge, making it essential to include bone marrow cytology in the clinical practice of this species.(AU)

Cilostamide affects in a concentration and exposure time-dependent manner the viability and the kinetics of in vitro maturation of caprine and bovine oocytes.

This study investigated: 1) the kinetics of oocyte chromatin configuration during in vitro maturation (IVM) of caprine and bovine oocytes; and 2) the effect of in vitro pre-maturation (IVPM) with cilostamide with or without association of the follicular wall (FW) on the same parameters. In experiment I, cumulus-oocyte complexes (COCs) were cultured in vitro in a standard maturation medium for 6, 12, 18 or 30 h. For experiment II, the COCs were cultured for 30 h, either in a standard IVM medium or in IVPM containing cilostamide (10 or 20 µM) and FW alone or in combination, for 6 or 12 h before the onset of maturation. The MII rate was similar (P > .05) between 18 and 30 h of maturation, both of which were higher (P < .05) than 6 and 12 h IVM in both species (Experiment I). Contrary to caprine, all IVPM treatments presented a higher (P < .05) percentage of bovine oocytes arrested at the GV stage than the control treatment after 6 h of culture. The percentage of MII oocytes after 30 h (IVPM+IVM) of culture in bovine oocytes treated with 10 µM cilostamide associated with FW and FW alone cultured for 6 h presented MII percentages similar to the control. However, in caprine, these treatments significantly reduced the percentages of MII in relation to the control treatment (Experiment II). In conclusion, the combination of concentration-exposure time to cilostamide during IVPM delayed meiotic progression in bovine after 6 and 12 h of culture. However, overall the culture period (IVPM+IVM) influenced the oocyte chromatin configuration and kinetics in both species.

Cryopreservation and in vitro culture of white-tailed deer ovarian tissue.

The aims of this study were to evaluate (1) the survivability of white-tailed deer ovarian tissue after cryopreservation by slow-freezing (SF) and vitrification (VIT) techniques and in vitro culture (IVC) for up to 7 days, and (2) the effects of cryopreservation techniques on protein expression of proliferative and apoptotic markers of ovarian tissue pre- and post-in vitro culture. Ovaries (n = 14) of seven white-tailed deer fawns (<1.5 years old) were used. Ovarian cortexes were cut into fragments (2 × 2 × 0.5 mm) and split into nine treatment groups: (1) fresh noncultured control, (2) fresh-IVC 1 day, (3) fresh-IVC 7 days, (4) SF noncultured, (5) SF-IVC 1 day, (6) SF-IVC 7 days, (7) VIT noncultured, (8) VIT-IVC 1 day, and (9) VIT-IVC 7 days. Preantral follicle morphology, class distribution, and density; stromal cell density; EGFR, Ki-67, Bax, and Bcl-2 protein expression; and DNA fragmentation were assessed. Results showed that: (i) white-tailed deer fresh ovarian tissue can be cultured for up to 7 days, preserving the tissue integrity and 50% of morphologically normal preantral follicles; (ii) cryopreservation of white-tailed deer ovarian tissue by either slow-freezing or vitrification does not disrupt markers of proliferation and apoptosis after thawing; (iii) ovarian fragments cryopreserved by the vitrification method had greater follicle viability during in vitro culture than the slow-freezing method; and (iv) fragments cryopreserved by slow-freezing suffered apoptosis earlier than those preserved by vitrification. The findings herein reported advance knowledge towards development of adequate cryopreservation protocols for long-term banking programs for Cervidae species.

Cryopreservation of domestic cat (Felis catus) ovarian tissue: Comparison of two vitrification methods.

We aimed to evaluate the effect of two vitrification methods on the morphology and functionality of vitrified feline preantral follicles. Feline ovarian tissue was vitrified with EG + trehalose combined or not with dimethyl sulphoxide (DMSO), using two different techniques (open or closed systems). Morphology, developmental capacity and mRNA expression of markers for follicle survival and quality were assessed before and after in vitro culture (IVC). Both vitrification and culture media were serum-free. Vitrification of feline ovarian tissue from five adult domestic cats was performed with EG + trehalose combined or not with DMSO. Two systems were used: the open system solid-surface vitrification (SSV) and the closed system ovarian tissue cryosystem (OTC). Histological analysis of follicle integrity showed that the percentages of normal follicles in previously vitrified ovarian fragments decreased after 7 days of in vitro culture (IVC), independently of the protocol used. Although follicular activation was observed by Ki-67 labelling, this was accompanied by extensive follicular degeneration as detected by a 3-4-fold decrease in follicular density. Remarkable follicle activation was observed in the ovarian tissue vitrified using OTC and subjected to IVC, probably due to a higher rate of degeneration of developing follicles. Even with such follicular loss, the results are promising for the combination of EG + DMSO + trehalose in a serum-free medium when applying the SSV method, with this approach resulting in the highest rates of normal developing follicles (19%) after 7 days IVC, together with granulosa cells proliferating at the same rate observed in fresh tissue.

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.

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.

Relationship between follicular dynamics and oocyte maturation during in vitro culture as a non-invasive sign of caprine oocyte meiotic competence.

The search for non-invasive signs of oocyte meiotic competence is very important for the development of in vitro follicle culture (IVFC) systems. The aims of the present study were: (1) to investigate the effect of in vitro maturation (IVM) of in vivo grown goat COCs, in group or individually, on oocyte chromatin configuration (Experiment 1), and (2) the influence of IVFC period (12 vs. 18 days) on the ability of the oocyte to resume meiosis immediately after IVFC (before in vitro maturation; IVM), or after IVM (Experiment 2). In experiment 1, in vivo grown cumulus-oocyte complexes (COCs) were submitted to IVM in groups (10 COCs/100 µL-drop) or individually (1 COC/10 µL-drop), and chromatin configuration was assessed. In experiment 2, isolated follicles were individually cultured for 12 or 18 days, and submitted to individual IVM afterwards. The following end points were evaluated: follicular growth and morphology, oocyte diameter, viability and chromatin configuration, as well as individual follicular estradiol production. Similar maturation rates were obtained between in vivo grown COCs matured individually and in groups (66.7% vs. 63.6%, respectively) (Experiment 1). Only after 18 days of IVFC, oocytes were able to grow during IVM, reaching a mean oocyte diameter of 119 µm. Also, this treatment produced the highest rate of metaphase II oocytes (46.2% out of the total number of cultured follicles). Finally, it was observed that follicles with a daily growth rate >7.1 µm/day (fast-growing) and that reached at least 600 µm in diameter, were more likely (P < 0.05) to produce oocytes capable of attaining MII. In conclusion, caprine oocytes can be individually matured in vitro, as efficiently as in groups. This result was essential to pair in vitro follicle development and in vitro oocyte maturation with specific individual follicles. Using this approach, it was possible to establish non-invasive signs for the efficiency of IVFC based on follicle daily growth rate and diameter, and oocyte diameter: follicle daily growth >7 µm, follicle diameter of at least 600 µm, and oocyte diameter ≥120 µm. In addition, 18 days seems to be the most suitable culture time for caprine early antral follicles.

Refining insulin concentrations in culture medium containing growth factors BMP15 and GDF9: An in vitro study of the effects on follicle development of goats.

The aim of the present study was to investigate the influence of two insulin concentrations (10ng/mL and 10µg/mL) combined or in the absence of BMP15 and/or GDF9, on the in vitro survival and development of preantral follicles of goat ovarian tissue. Ovarian slices from the same goat ovary pair were randomly assigned to a non-cultured control treatment or to be in vitro cultured for 1 or 7days in α-MEM containing 10ng/mL (Low) or 10µg/mL (High) of insulin in the absence or presence of BMP15 and/or GDF9. With the low insulin treatment, there was a greater percentage of normal follicles than with the high insulin treatment. The addition of BMP15 alone or in association with GDF9 to the medium containing low insulin resulted in a lesser percentage of normal follicles (P<0.05). The addition of BMP15 and GDF9 separately or in combination with the high insulin concentration enhanced the percentage of normal follicles. On day 7 of culture, the use of medium containing low insulin alone or high insulin supplemented with BMP15 and BMP15+GDF9 resulted in a greater percentage of secondary follicles than the non-cultured control, although follicles cultured with low insulin were smaller than those from the control group and had greater rates of oxidative stress. In conclusion, in the presence of physiological concentrations of insulin (10ng/mL), medium supplementation with GDF9 and BMP15 alone or in combination is unnecessary for normal follicle development in vitro.

Expectations and limitations of ovarian tissue transplantation.

Constant progress in the diagnosis and treatment of cancer disease has increased the number and prognosis of cancer survivors. However, the toxic effects of chemotherapy and radiotherapy on ovarian function have resulted in premature ovarian failure. Patients are, therefore, still expecting methods to be developed to preserve their fertility successfully. Several potential options are available to preserve fertility in patients who face premature ovarian failure, including immature or mature oocyte and embryo cryopreservation. However, for children or prepubertal women needing immediate chemotherapy, cryopreservation of ovarian tissue is the only alternative. The ultimate aim of this strategy is to implant ovarian tissue into the pelvic cavity (orthotopic site) or in a heterotopic site once oncological treatment is completed and the patient is disease free. Transplantation of ovarian tissue with sufficiently large numbers of follicles could potentially restore endocrine function and allow multiple cycles for conception. However, the success of ovarian tissue transplantation still has multiple challenges, such as the low number of follicles in the graft that may affect their longevity as well as the survival of the tissue during ex vivo processing and subsequent transplantation. Therefore, this review aims to summarize the achievements of ovary grafting and the potential techniques that have been developed to improve ovarian graft survival.