In Vitro Activation and Development of Goat Preantral Follicles Enclosed in Ovarian Tissue Co-cultured with Mesenchymal Stem Cells.

The development of culture systems capable of maintaining follicular growth since the preantral stage has been the target of investigations. Mesenchymal stem cells (MSC) present potential for use in a wide range of applications, including research aimed at preserving fertility. Therefore, this study investigated the use of caprine Wharton's Jelly Mesenchymal Stem Cells (WJMSC) on the survival and in vitro development of goat preantral follicles enclosed in ovarian fragments cultured for 1 or 7 days. Fragments of the ovarian cortex were immediately fixed (non-cultured control) or distributed in four treatments: ovarian tissue cultured in control medium (α-MEM+); ovarian tissue cultured in α-MEM+ supplemented with FBS (α-MEM+ + FBS); ovarian tissue co-cultured with stem cells in α-MEM+ (α-MEM+ + SC); and ovarian tissue co-cultured with stem cell in α-MEM+ + FBS (α-MEM+ + SC + FBS). The rates of cell proliferation, follicular survival, and activation, as well as follicular diameter, were evaluated. After 7 days, the treatment co-cultured with stem cells showed a higher (P < 0.05) percentage of morphologically normal preantral follicles compared to the other treatments, as well as a higher (P < 0.05) activation rate compared to cultured control. Moreover, the follicular diameter was higher (P < 0.05) in the treatment co-cultured with stem cells compared to co-cultured with stem cells plus FBS. This study demonstrates for the first time that in vitro co-culture of caprine WJMSC with preantral follicles enclosed in goat ovarian tissue improves activation and early follicular development.

First pregnancy after in vitro culture of early antral follicles in goats: Positive effects of anethole on follicle development and steroidogenesis.

This study aimed to evaluate the role of anethole during the in vitro culture of caprine early antral follicles. Early antral follicles were isolated from caprine ovaries and cultured for 18 days without (control) or with anethole (300 µg/ml). After culture, the cumulus-oocyte complexes were subjected to in vitro maturation, followed by parthenogenetic activation or in vitro fertilization (IVF) and embryo culture. Follicular walls were used for the quantification of messenger RNA (mRNA) of CYP19A1, CYP17, MMP-9, TIMP-2, Bax, and Bcl-2 genes, and culture medium was used for evaluation of ferric reducing antioxidant power (FRAP) and estradiol levels. After in vitro follicle culture (IVFC), anethole induced higher total antioxidant capacity, that is, it produced higher FRAP levels, reduced the Bax/Bcl-2 ratio, and increased the levels of mRNA for CYP19A1 and CYP17, which was associated with a greater estradiol production (p < .05). Also, anethole improved the ability of oocytes to resume meiosis and reach metaphase II stage, as well as yielded higher (p < .05) embryo production (e.g., morulas and blastocysts) in both parthenogenetic activation and IVF techniques. One pregnancy (Day 30) was obtained from IVFC with anethole. In conclusion, anethole promoted in vitro growth and maturation of goat early antral follicles and oocytes and enabled embryo production. Furthermore, this study reports, for the first time in goats, a pregnancy after IVF using oocytes originated from early antral follicles grown in vitro.

Anethole Supplementation During Oocyte Maturation Improves In Vitro Production of Bovine Embryos.

Oxidative stress is one of the most detrimental factors that affect oocyte developmental competence and embryo development in vitro. The impact of anethole supplementation to in vitro maturation (IVM) media on oocyte maturation and further bovine in vitro embryo production was investigated. Oocytes of slaughterhouse-derived bovine ovaries were placed in IVM with anethole at different concentrations of 30 (AN30), 300 (AN300), and 2000 µg/mL (AN2000), or without (control treatment). The oocytes were assessed for maturation rates, and for reactive oxygen species (ROS) and ferric reducing antioxidant power (FRAP) levels, and mitochondrial membrane potential. Embryo development was assessed by cleavage and blastocyst rates, and embryo cell number. The percentage of metaphase II oocytes were similar among the treatments (range, 77%-96%). Anethole at 300 µg/mL was the only treatment that yielded higher cleavage and embryo development (morula and blastocyst) rates compared to the control treatment. The ROS production in the oocytes after maturation did not differ among treatments. However, oocytes treated with anethole at 300 µg/mL had higher (P < .05) FRAP and mitochondrial membrane potential compared to the control treatment. Furthermore, AN300 treatment increased (P < .05) the average number of total cells in blastocysts compared to the control and AN30 treatments. The use of anethole at 300 µg/mL during IVM is suggested to improve the quantity and quality of bovine embryos produced in vitro. The beneficial effects of anethole on embryonic developmental competence in vitro seems to be related to its capacity to regulate the redox balance and improve mitochondrial function in oocytes and embryos.

In Vitro Probiotic and Antioxidant Potential of Lactococcus lactis subsp. cremoris LL95 and Its Effect in Mice Behaviour.

The composition of intestinal microbiota is widely believed to not only affect gut health but also influence behaviour. This study aimed to evaluate the probiotic characteristics, antioxidant activity, and antidepressant- and anxiolytic-like activities of Lactococcus lactis subsp. cremoris LL95. This strain showed probiotic properties such as resistance in a simulated gastric tract model and survival at different concentrations of NaCl and bile salts. Moreover, antioxidant activity of LL95 was demonstrated through DPPH radical scavenging activity, scavenging of ABTS•+ radical and ferric ion reducing antioxidant power (FRAP) assays. Female C57BL/6 mice received LL95 orally at a dose of 109 UFC/day for 28 days. LL95 improved depressive- and anxiety-like behaviour, demonstrated by decreased immobility time in the tail suspension test and forced swim test and increased per cent of time spent in the open arms on the elevated plus maze. These findings indicate the potential antioxidant activity of LL95 and its role in behaviour, suggesting that probiotic may have therapeutic applications.

Anethole Supplementation During Oocyte Maturation Improves In Vitro Production of Bovine Embryos.

Oxidative stress is one of the most detrimental factors that affect oocyte developmental competence and embryo development in vitro. The impact of anethole supplementation to in vitro maturation (IVM) media on oocyte maturation and further bovine in vitro embryo production was investigated. Oocytes of slaughterhouse-derived bovine ovaries were placed in IVM with anethole at different concentrations of 30 (AN30), 300 (AN300), and 2000 µg/mL (AN2000), or without (control treatment). The oocytes were assessed for maturation rates, and for reactive oxygen species (ROS) and ferric reducing antioxidant power (FRAP) levels, and mitochondrial membrane potential. Embryo development was assessed by cleavage and blastocyst rates, and embryo cell number. The percentage of metaphase II oocytes were similar among the treatments (range, 77%-96%). Anethole at 300 µg/mL was the only treatment that yielded higher cleavage and embryo development (morula and blastocyst) rates compared to the control treatment. The ROS production in the oocytes after maturation did not differ among treatments. However, oocytes treated with anethole at 300 µg/mL had higher ( P < .05) FRAP and mitochondrial membrane potential compared to the control treatment. Furthermore, AN300 treatment increased ( P < .05) the average number of total cells in blastocysts compared to the control and AN30 treatments. The use of anethole at 300 µg/mL during IVM is suggested to improve the quantity and quality of bovine embryos produced in vitro. The beneficial effects of anethole on embryonic developmental competence in vitro seems to be related to its capacity to regulate the redox balance and improve mitochondrial function in oocytes and embryos.

Influence of follicle size on bovine oocyte lipid composition, follicular metabolic and stress markers, embryo development and blastocyst lipid content.

This study assessed the lipid composition of oocytes from different follicle sizes and compared the expression of lipid-related genes and follicular fluid (FF) molecules between groups. We also investigated the functional consequences of differences on embryo development and blastocyst lipid deposits. Oocytes and FF were recovered from different follicle sizes. Oocytes from small (≤5mm) and large (≥6mm) bovine follicles were used to produce Day 7 expanded blastocysts (Day7Ex) and blastocysts that only became expanded at Day 8 (Day8Ex) after insemination. Oocytes from >8mm follicles had the highest lipid content. Few oocyte phospholipid variations were identified between groups. Very long chain fatty acid elongase 6 (ELOVL6) mRNA abundance was reduced in larger follicle-derived oocytes compared with the ≤2mm group. Increased levels of glucose, reactive oxygen species, glutathione and superoxide dismutase activity were also identified in FF from larger follicles. Large follicle-derived embryo development and lipid content of Day7Ex were greater than those derived from small follicles. Day8Ex had greater lipid deposition than Day7Ex. Oocytes and blastocysts exhibited follicle size-specific lipids. Large-follicle oocytes had increased lipid content and became Day7Ex with greater lipid deposition whereas delayed blastocoel expansion associated with a prolonged period of culture determined the lipid accumulation of Day8Ex. The FF microenvironment of large follicles seems to favour embryo development.

Effect of Catalase or Alpha Lipoic Acid Supplementation in the Vitrification Solution of Ovine Ovarian Tissue.

AIM: The present study evaluates the effect of different concentrations of antioxidants (catalase - CAT and alpha lipoic acid - ALA) on the follicular activation and morphology, DNA damage, ROS production, and mitochondrial activity in vitrified sheep ovarian tissue. METHODS: This experiment was divided into two steps. First, ovarian fragments were distributed into the following treatments: fresh tissue or control (CTR), incubation (INC), vitrification without antioxidant (VWA), with CAT (10, 20, or 40 IU mL-1) or ALA (25, 50, or 100 µM mL-1). After vitrification/warming, the fragments were additionally incubated for 24 hours and evaluated for morphology and follicular activation, as well as reactive oxygen species (ROS) levels in the culture medium. For the second step, other ovarian fragments were submitted to CTR, VWA, CAT40, and ALA100. After vitrification/warming, the fragments were incubated for 24 hours and evaluated by cell density of ovarian stroma, DNA damage, and mitochondrial and intracellular ROS levels. RESULTS: The percentage of morphologically normal follicles in vitrified ovarian tissue in the presence of ALA in all concentrations did not differ (p > 0.05) from fresh tissue or CTRs. The percentage of activated follicles was higher in ALA100 µM mL-1 than those observed for the treatments INC, CAT (40 IU mL-1), or ALA (25 or 50 µM mL-1). The use of CAT affected (p < 0.05) the density of stromal cells (40 IU mL-1), ROS levels (10 and 20 IU mL-1), as well as DNA damage revealed by ©H2AX (40 IU mL-1). CONCLUSIONS: Although 100 µM/mL of ALA did not alter intracellular ROS, this concentration reduced the levels of ROS in the culture medium, preserved both the follicular morphology, as well as the mitochondrial activity, promoted follicle activation, and protected the follicles from DNA damage.

In vitro growth and development of isolated secondary follicles from vitrified caprine ovarian cortex.

The aim of this study was to evaluate the viability, antrum formation and in vitro development of isolated secondary follicles from vitrified caprine ovarian cortex in a medium previously established for fresh isolated secondary follicles, in the absence (α-minimum essential medium (α-MEM+) alone) or presence of FSH and vascular endothelial growth factor (VEGF; α-MEM++FSH+VEGF). Ovarian fragments were distributed among five treatments (T1 to T5): fresh follicles were fixed immediately (T1), follicles from fresh tissue were cultured in vitro in α-MEM+ (T2) or α-MEM++FSH+VEGF (T3) and follicles from vitrified tissue were cultured in vitro in α-MEM+ (T4) or α-MEM++FSH+VEGF (T5). After 6 days of culture, treated follicles (T2, T3, T4 and T5) were evaluated for morphology, viability and follicular development (growth, antrum formation and proliferation of granulosa cells by Ki67 and argyrophilic nucleolar organiser region (AgNOR) staining). The levels of reactive oxygen species (ROS) in the culture media were also assessed. Overall, morphology of vitrified follicles was altered (P<0.05) compared with the fresh follicles. Follicular viability, antrum formation and ROS were similar between treatments (P>0.05). The average overall and daily follicular growth was highest (P<0.05) in T3. Granulosa cells in all treatments (T1, T2, T3, T4 and T5) stained positive for Ki67. However, fresh follicles from T3 had significantly higher AgNOR staining (P<0.05) compared with follicles of T1, T2, T4 and T5. In conclusion, secondary follicles can be isolated from vitrified and warmed ovarian cortex and survive and form an antrum when growing in an in vitro culture for 6 days.