Cystic endometrial hyperplasia-pyometra syndrome impairs the preantral follicle reserve in domestic bitches (Canis familiaris).

Cystic endometrial hyperplasia (CEH)-pyometra syndrome is the most common uterine pathological condition reported in breeding bitches, however, their described effects on fertility are limited to uterine disorders and conception rates. As the preantral follicle population represents the available reserve of gametes recruited during the lifespan, the aim of this study was to evaluate the effects of CEH-pyometra syndrome on the: (i) preantral follicle morphology, (ii) developing follicle rates, and (iii) preantral follicle and stromal cell densities. Ovarian fragments from bitches subjected to elective or therapeutic ovariohysterectomy were allocated according to uterine diagnosis as follows: control (n = 7, clinically healthy), CEH-mucometra (n = 8, uterine lumen filled with a sterile mucus), and pyometra (n = 17, presence of a purulent mucus) groups. Overall, the control group had 3.4 and 4.1-fold higher probability (P < 0.0001) of the presence of normal preantral follicles compared with CEH-mucometra and pyometra groups, respectively. Moreover, ovarian fragments from the pyometra group showed an increase in the percentage of developing follicles (P < 0.05) compared to the control. Both CEH-mucometra and pyometra groups showed lower (P < 0.05) preantral follicle and stromal cell densities (P < 0.05) compared to the control. In summary, the CEH-pyometra syndrome decreased the percentage of morphologically normal follicles and enhanced the developing follicle rates. Additionally, a reduction of preantral follicle and stromal cell densities suggests that the inappropriate uterine environment induced by CEH-pyometra syndrome can lead to premature depletion of ovarian reserve.

Trimethylation profile of histones H3 lysine 4 and 9 in late preantral and early antral caprine follicles grown in vivo versus in vitro in the presence of anethole.

This study assessed the histones methylation profile (H3K4me3 and H3K9me3) in late preantral (PA) and early antral (EA) caprine follicles grown in vivo and in vitro, and the anethole effect during in vitro culture of PA follicles. Uncultured in vivo-grown follicles (PA, n = 64; EA, n = 73) were used as controls to assess the methylation profile and genes' expression related to apoptosis cascade (BAX, proapoptotic; BCL2, antiapoptotic), steroidogenesis (CYP17, CYP19A1), and demethylation (KDM1AX1, KDM1AX2, KDM3A). The isolated PA follicles (n = 174) were cultured in vitro for 6 days in α-MEM+ in either absence (control) or presence of anethole. After culture, EA follicles were evaluated for methylation, mRNA abundance, and morphometry. Follicle diameter increased after culture, regardless of treatment. The methylation profile and the mRNA abundance were similar between in vivo-grown PA and EA follicles. Anethole treatment led to higher H3K4me3 fluorescence intensity in EA follicles. The mRNA abundances of BAX, CYP17, and CYP19A1 were higher, and BCL2 and KDM3A were lower in in vitro-grown EA follicles than in vivo-grown follicles. In conclusion, in vitro follicle culture affected H3K4me3 fluorescence intensity, mRNA abundance of apoptotic genes, and steroidogenic and demethylase enzymes compared with in vivo-grown follicles.

Effects of different subcutaneous sites on heterotopic autotransplantation of canine ovarian tissue.

Ovarian tissue transplantation makes it possible to restore fertility; however, the success of this technique depends on the transplant region used. Therefore, this study aimed to evaluate the effect of two subcutaneous regions on canine ovarian transplantation, pinna (Pi) and neck (Ne), for 7 and 15 days. Ovaries collected by ovariosalpingohysterectomy were fragmented using a punch device. Fresh fragments were fixed, and the others were immediately grafted onto the animal itself in the Pi and Ne regions for 7 and 15 days. Recovered fragments were evaluated for histology (morphology, development and stromal density), picrosirius (collagen fibers), and immunohistochemistry (fibrosis and cell proliferation). The results showed that follicular normality rates were lower in Pi-7 (78%) vs. control (90%) and Pi-15 (86%), similar in Ne-7 (92%) and superior in Ne-15 (97%) compared to the control, with the effect of the region Ne (94%) superior (P < 0.05) to Pi (82%). Stromal density reduced in both regions vs. control but was similar within 15 days. Fragments from both regions showed higher fibronectin labeling and deposition of type I and lower type III collagen fibers (P < 0.05) vs. control. Proliferation rates in Ne-7 were higher (P < 0.05) than in control, and Pi-15 was higher (P < 0.05) than Ne-15. In conclusion, the pinna may be a region with greater potential than the neck after a 15-day autotransplantation of canine ovarian tissue.

Characterization of preantral follicle clustering and neighborhood patterns in the equine ovary.

Understanding the transition from quiescent primordial follicles to activated primary follicles is vital for characterizing ovarian folliculogenesis and improving assisted reproductive techniques. To date, no study has investigated preantral follicle crowding in the ovaries of livestock or characterized these crowds according to follicular morphology and ovarian location (portions and regions) in any species. Therefore, the present study aimed to assess the crowding (clustering and neighborhood) patterns of preantral follicles in the equine ovary according to mare age, follicular morphology and developmental stage, and spatial location in the ovary. Ovaries from mares (n = 8) were collected at an abattoir and processed histologically for evaluation of follicular clustering using the Morisita Index and follicular neighborhoods in ovarian sections. Young mares were found to have a large number of preantral follicles with neighbors (n = 2,626), while old mares had a small number (n = 305). Moreover, young mares had a higher number of neighbors per follicle (2.6 ± 0.0) than old mares (1.2 ± 0.1). Follicle clustering was shown to be present in all areas of the ovary, with young mares having more clustering overall than old mares and a tendency for higher clustering in the ventral region when ages were combined. Furthermore, follicles with neighbors were more likely to be morphologically normal (76.5 ± 6.5%) than abnormal (23.5 ± 6.5%). Additionally, morphologically normal activated follicles had increased odds of having neighbors than normal resting follicles, and these normal activated follicles had more neighbors (2.6 ± 0.1) than normal resting follicles (2.3 ± 0.1 neighbors). In the present study, it was demonstrated that preantral follicles do crowd in the mare ovary and that clustering/neighborhood patterns are dynamic and differ depending on mare age, follicular morphology, and follicular developmental stage.

Preantral follicle population and distribution in the horse ovary.

Characterization of the ovarian preantral follicle population is a necessary step to improve understanding of folliculogenesis and ovarian physiology. Therefore, in the present study, the preantral follicle population in the equine ovary in young and old mares was investigated according to follicular morphology, follicular class, distance from the geometric center using ovarian maps, and follicular density within ovarian portions (lateral vs intermediary) and regions (dorsal vs ventral). Ovaries were collected from an abattoir and histologically processed for evaluation, and the follicle population was calculated. Overall, in the current detailed study, a higher preantral follicle population per mare ovary (mean: 82,206 ± 50,022; range: 1477 to 773,091) than originally reported was identified. Additionally, a mare age effect was observed in the follicle population (young: 152,664 vs old: 11,750) and the spatial distribution of morphologically normal and abnormal follicles and the density and population of follicular classes. These results demonstrate that, in addition to the preantral follicle population in the mare ovary being comparable to that of other species, the location and spatial distribution of these follicles is dynamic and varies depending on mare age and follicle status (i.e. morphology and developmental stage). The characterization of the distribution and population of preantral follicles in the mare ovary provided by this study can potentially aid in improving reproductive studies and assisted reproductive techniques and may expand the understanding of mechanisms involving ovarian plasticity and follicular migration. Lay summary: Knowledge of the distribution and population of immature eggs within follicles (preantral follicles) in the ovaries of mares can improve approaches to assisted reproductive techniques and fertility preservation. As the existing research on horse preantral follicle population was focused solely on large follicles, the present study provides an updated investigation of small and large preantral follicles in the mare, showing that the population is similar to those in other species. This study also shows that the way these follicles are distributed in the ovary varies depending on age and follicle characteristics. Results from this study may help to highlight which areas of the mare ovary should be looked at to find samples of good-quality follicles.

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.

Stx1 and Stx2 subtyping and antimicrobial resistance in Shiga toxin-producing Escherichia coli (STEC) isolates from cattle and sheep feces in the Southeastern region of the State of Goiás, Brazil

ABSTRACT: The present study was aimed at subtyping of Stx1 and Stx2 genes and characterization of antimicrobial resistance in 106 Shiga toxin-producing Escherichia coli (STEC) strains isolated from cattle and sheep feces. PCR was used to determine the subtypes, and the disk-diffusion method was used to evaluate the antimicrobial resistance. Ten antibiotics from five different classes were tested. Among the isolates of bovine origin, two subtypes of Stx1 (Stx1a and Stx1c), and four subtypes of Stx2 (Stx2a, Stx2b, Stx2c, and Stx2d) were identified. In isolates of sheep origin, two subtypes of Stx1 (Stx1a and Stx1c), and four subtypes of Stx2 (Stx2a, Stx2b, Stx2c, and Stx2 g) were identified. The results obtained suggest the presence of high diversity in Stx1 and Stx2 genes. Further, 96.6% (57/59) of bovine fecal strains and 89.4% (42/47) of sheep fecal strains showed resistance to at least one tested antibiotic. In both animal species, most strains were multidrug-resistant (MDR) (67.8% in cattle and 59.6% in sheep), with no significant difference between host animals. Adult animals were eight times more likely to have STEC with greater pathogenic potential. STEC with the highest pathogenic potential were three times more likely to be multidrug-resistant than STEC with the lowest pathogenic potential. The data reported in this study suggests the occurrence of strains with high potential pathogenicity in the region studied. Therefore, the ruminants of this region are carriers of strains that can cause infections in humans.

RESUMO: O presente estudo teve como objetivo subtipar os genes Stx1 e Stx2 e caracterizar a resistência antimicrobiana em 106 isolados de Escherichia coli produtoras de toxinas Shiga (STEC) isoladas de fezes de bovinos e ovinos. A PCR foi utilizada para determinar os subtipos e o método de difusão em disco foi utilizado para avaliar a resistência antimicrobiana. Dez antibióticos de cinco classes diferentes foram testados. Entre os isolados de origem bovina, foram identificados dois subtipos de Stx1 (Stx1a e Stx1c) e quatro subtipos de Stx2 (Stx2a, Stx2b, Stx2c e Stx2d). Nos isolados de origem ovina, foram identificados dois subtipos de Stx1 (Stx1a e Stx1c) e quatro subtipos de Stx2 (Stx2a, Stx2b, Stx2c e Stx2g). Os resultados obtidos sugerem a presença de alta variabilidade nos genes Stx1 e Stx2. Além disso, 96,6% (57/59) dos isolados fecais de bovinos e 89,4% (42/47) dos isolados de ovinos mostraram resistência a pelo menos um antibiótico testado. Em ambas as espécies animais, a maioria das cepas foi multirresistente (MDR) (67,8% em bovinos e 59,6% em ovinos), sem diferença significativa entre as espécies animais do reservatório. Os animais adultos tiveram oito vezes mais chances de apresentar STEC com maior potencial patogênico. STEC com o maior potencial patogênico teve três vezes mais chances de ser multirresistente do que o STEC com o menor potencial patogênico. Os dados relatados neste estudo sugerem a ocorrência de cepas com alto potencial de patogenicidade na região estudada. Portanto, os ruminantes dessa região são hospedeiros de isolados que podem causar infecções em humanos.

Stx1 and Stx2 subtyping and antimicrobial resistance in Shiga toxin-producing Escherichia coli (STEC) isolates from cattle and sheep feces in the Southeastern region of the State of Goiás, Brazil / Subtipagem de Stx1 e Stx2 e resistência antimicrobiana em isolados de Escherichia coli produtoras de toxina Shiga (STEC) de fezes de bovinos e ovinos na região sudeste de Goiás, Brasil

The present study was aimed at subtyping of Stx1 and Stx2 genes and characterization of antimicrobial resistance in 106 Shiga toxin-producing Escherichia coli (STEC) strains isolated from cattle and sheep feces. PCR was used to determine the subtypes, and the disk-diffusion method was used to evaluate the antimicrobial resistance. Ten antibiotics from five different classes were tested. Among the isolates of bovine origin, two subtypes of Stx1 (Stx1a and Stx1c), and four subtypes of Stx2 (Stx2a, Stx2b, Stx2c, and Stx2d) were identified. In isolates of sheep origin, two subtypes of Stx1 (Stx1a and Stx1c), and four subtypes of Stx2 (Stx2a, Stx2b, Stx2c, and Stx2 g) were identified. The results obtained suggest the presence of high diversity in Stx1 and Stx2 genes. Further, 96.6% (57/59) of bovine fecal strains and 89.4% (42/47) of sheep fecal strains showed resistance to at least one tested antibiotic. In both animal species, most strains were multidrug-resistant (MDR) (67.8% in cattle and 59.6% in sheep), with no significant difference between host animals. Adult animals were eight times more likely to have STEC with greater pathogenic potential. STEC with the highest pathogenic potential were three times more likely to be multidrug-resistant than STEC with the lowest pathogenic potential. The data reported in this study suggests the occurrence of strains with high potential pathogenicity in the region studied. Therefore, the ruminants of this region are carriers of strains that can cause infections in humans.(AU)

O presente estudo teve como objetivo subtipar os genes Stx1 e Stx2 e caracterizar a resistência antimicrobiana em 106 isolados de Escherichia coli produtoras de toxinas Shiga (STEC) isoladas de fezes de bovinos e ovinos. A PCR foi utilizada para determinar os subtipos e o método de difusão em disco foi utilizado para avaliar a resistência antimicrobiana. Dez antibióticos de cinco classes diferentes foram testados. Entre os isolados de origem bovina, foram identificados dois subtipos de Stx1 (Stx1a e Stx1c) e quatro subtipos de Stx2 (Stx2a, Stx2b, Stx2c e Stx2d). Nos isolados de origem ovina, foram identificados dois subtipos de Stx1 (Stx1a e Stx1c) e quatro subtipos de Stx2 (Stx2a, Stx2b, Stx2c e Stx2g). Os resultados obtidos sugerem a presença de alta variabilidade nos genes Stx1 e Stx2. Além disso, 96,6% (57/59) dos isolados fecais de bovinos e 89,4% (42/47) dos isolados de ovinos mostraram resistência a pelo menos um antibiótico testado. Em ambas as espécies animais, a maioria das cepas foi multirresistente (MDR) (67,8% em bovinos e 59,6% em ovinos), sem diferença significativa entre as espécies animais do reservatório. Os animais adultos tiveram oito vezes mais chances de apresentar STEC com maior potencial patogênico. STEC com o maior potencial patogênico teve três vezes mais chances de ser multirresistente do que o STEC com o menor potencial patogênico. Os dados relatados neste estudo sugerem a ocorrência de cepas com alto potencial de patogenicidade na região estudada. Portanto, os ruminantes dessa região são hospedeiros de isolados que podem causar infecções em humanos.(AU)

Heterotopic autotransplantation of ovarian tissue in a large animal model: Effects of cooling and VEGF.

Heterotopic and orthotopic ovarian tissue autotransplantation techniques, currently used in humans, will become promising alternative methods for fertility preservation in domestic and wild animals. Thus, this study describes for the first time the efficiency of a heterotopic ovarian tissue autotransplantation technique in a large livestock species (i.e., horses) after ovarian fragments were exposed or not to a cooling process (4°C/24 h) and/or VEGF before grafting. Ovarian fragments were collected in vivo via an ultrasound-guided biopsy pick-up method and surgically autografted in a subcutaneous site in both sides of the neck in each mare. The blood flow perfusion at the transplantation site was monitored at days 2, 4, 6, and 7 post-grafting using color-Doppler ultrasonography. Ovarian grafts were recovered 7 days post-transplantation and subjected to histological analyses. The exposure of the ovarian fragments to VEGF before grafting was not beneficial to the quality of the tissue; however, the cooling process of the fragments reduced the acute hyperemia post-grafting. Cooled grafts compared with non-cooled grafts contained similar values for normal and developing preantral follicles, vessel density, and stromal cell apoptosis; lower collagen type III fibers and follicular density; and higher stromal cell density, AgNOR, and collagen type I fibers. In conclusion, VEGF exposure before autotransplantation did not improve the quality of grafted tissues. However, cooling ovarian tissue for at least 24 h before grafting can be beneficial because satisfactory rates of follicle survival and development, stromal cell survival and proliferation, as well as vessel density, were obtained.

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.

Pituitary porcine FSH, and recombinant bovine and human FSH differentially affect growth and relative abundances of mRNA transcripts of preantral and early developing antral follicles in goats.

Three different sources of FSH (porcine pituitary, pFSH; recombinant bovine, rbFSH; and recombinant human, rhFSH) were compared during in vitro culture of preantral and early antral follicles of goats for 18 days. Treatments were: base medium supplemented with no FSH (control), 10, 50, or 100 mIU/mL pFSH (pFSH10, pFSH50, and pFSH100, respectively), 100 ng/mL rbFSH (rbFSH), and 50 mIU/mL rhFSH (rhFSH). There were evaluations of follicle morphology, antrum formation, growth rate, estradiol production, oocyte viability and chromatin configuration, and follicle wall relative abundance of mRNA transcript for MMP-9, TIMP-2, CYP17, CYP19A1, FSHR, Insulin-R, and BAX/BCL-2 ratio. Follicle degeneration rates were similar among all treatment groups at the end of culturing. When there were treatments with pFSH, however, there was a lesser (P < 0.05) percentage of intact follicles and estradiol production, and greater (P < 0.05) extrusion rates. Furthermore, with only pFSH10 (antral follicles) and pFSH100 (preantral and antral follicles) treatments, there was a lesser (P < 0.05) follicle growth. For preantral follicles, when there was addition of pFSH10, pFSH100, and rhFSH there was lesser (P < 0.05) oocyte meiotic resumption compared to control and rbFSH treatments. For antral follicles, when there were treatments with rhFSH and pFSH10 there was greater (P = 0.08 - P < 0.05) oocyte maturation. In conclusion, the source of FSH differentially affected gene expression, as indicated by mRNA abundances, and follicular dynamics of preantral and antral follicles in vitro. Addition of FSH during the in vitro culture improved the developmental outcomes only for antral follicles.

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.

Heterotopic ovarian allotransplantation in goats: Preantral follicle viability and tissue remodeling.

An appropriate implantation site favors angiogenesis and avoids ovarian tissue damage after tissue grafting. The objective of this study was to evaluate the effects of intramuscular (IM) and subcutaneous (SC) sites for ovarian grafts in goats by evaluating follicular morphology and activation, preantral follicle and stromal cell densities, tissue DNA fragmentation, collagen types I and III depositions, and graft revascularizations. Ovarian cortical tissue was transplanted in IM or SC sites and recovered 7 or 15 days post-transplantation. There was a greater percentage of developing follicles and lesser follicular and stromal cell densities in all grafted tissues as compared to ovarian tissues of the control group. The stromal cell density and percentage of normal follicles were positively associated. At 15 days post-transplantation, tissues at the SC and IM sites had similar amounts of DNA fragmentation and type III collagen content. In contrast, tissues at the SC, as compared with IM site, had greater abundances of collagen type I. Furthermore, there was a positive association between collagen type I and percentage of morphologically normal follicles post-transplantation. In addition to a marked decrease in follicular density 15 days post-transplantation in ovarian grafts at the SC and IM sites, low percentages of normal follicles and follicular activation were observed similarly in both transplantation sites. There were also positive associations of stromal cell density and abundance of type I collagen fibers with the percentage of intact follicles in grafted ovarian tissues.

Activation of goat primordial follicles in vitro: Influence of alginate and ovarian tissue.

The present study aimed to evaluate the effect of three culture systems on caprine primordial follicle activation in vitro: follicles cultured either in the isolated form within alginate (Isolated follicles + Alginate treatment), or enclosed in ovarian tissue (in situ), with or without alginate (Fragment + Alginate, and Fragment alone treatments, respectively). After culture, the Isolated follicles + Alginate treatment presented a percentage of morphologically normal follicles (MNF) similar to both the non-cultured control and the Fragment Alone treatments. Nevertheless, Fragment + Alginate treatment showed a significant reduction in the number of MNF when compared to the other treatments. Regarding follicle development, our results showed that regardless of the alginate, the presence of ovarian tissue limited primordial follicle activation during in vitro culture. Remarkably, the Isolated primordial follicle + Alginate treatment was the only one that significantly promoted follicle activation and increased both follicle and oocyte diameters during IVFC, pointing out a higher cell proliferation. In conclusion, the presence of ovarian tissue with or without alginate limited follicle development (activation) after culture. Nevertheless, when primordial follicles were isolated and encapsulated in alginate they presented suitable survival rates, higher rates of follicle activation and continued to grow throughout the culture period.

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 culture of isolated preantral and antral follicles of goats using human recombinant FSH: Concentration-dependent and stage-specific effect.

The present study aimed to investigate a concentration-response curve of human recombinant FSH (hrFSH) for in vitro culture of isolated preantral and early antral follicles of goats. Isolated follicles were cultured for 18 days using the following treatments: basic culture medium (control); or control medium supplemented with 10, 50, and 100 mIU/mL of hrFSH. At the end of the culture, cumulus-oocyte complexes were recovered and subjected to in vitro maturation. The following endpoints were evaluated: follicle morphology, growth rate and antrum formation, oocyte viability and meiotic stage, and estradiol production, as well as relative expression of FSH receptor (FSHR), and steroidogenic enzyme (3ß-HSD, CYP17, and CYP19A1) genes. In antral follicles, the FSH addition at 50 mIU/mL increased follicular diameter and growth rate, percentage of fully developed oocytes, and oocyte diameter (P < 0.05), and tended to increase the percentage of MII oocytes when compared to the control (P = 0.07). With preantral follicles, FSH addition at 100 mIU/mL increased relative abundance of mRNA for CYP19A1 when compared to the control (P < 0.05). At the same FSH concentrations of 100 and 50 mIU/mL, there was a greater relatively abundance of mRNA for 3ß-HSD and CYP17 in preantral than in antral follicles (P < 0.05). For preantral and antral follicle comparisons when the same treatments were imposed, there were greater concentrations of estradiol for antral follicles (P < 0.05). In conclusion, hrFSH enhanced in a concentration-dependent manner the in vitro development of caprine antral follicles; however, there was no positive effect in the culture of preantral follicles.

Spatial distribution of preantral follicles in the equine ovary.

Comprehensive studies on spatial distribution of preantral follicles in the ovary are scarce. Considering that preantral follicles represent the main ovarian reserve, harvesting of these follicles is crucial for the development/use of assisted reproductive techniques. Therefore, knowledge on follicle spatial distribution can be helpful for targeting areas with richer number of preantral follicles through biopsy procedures. The aim of this study was to assess the distribution and localization of equine preantral follicles according to: (i) age, (ii) ovarian portion (lateral and intermediary) and region (dorsal and ventral), (iii) distance from the geometric center, and (iv) follicular class. Ovaries from young and old mares (n = 8) were harvested in a slaughterhouse and submitted to histological processing for further evaluation. For data analyses, a novel methodology was developed according to the geometric center of each histological section for a precise determination of preantral follicle distribution. Results indicated that (i) equine preantral follicles are clustered and located near to the ovarian geometric center, and that aging induced their dispersion through the ovarian cortex; (ii) the distance from the geometric center was shorter for developing follicles than primordial; and (iii) secondary follicles were more distant from the geometric center but closer to the ovulation fossa. In conclusion, the spatial distribution of preantral follicles was successfully determined in the equine ovary and was affected by age, region, and portion.

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.

Laparoscopic ovarian biopsy pick-up method for goats.

Biopsy pick-up (BPU) has been considered a safe method to harvest ovarian fragments from live animals. However, no studies have been reported on the use of BPU to collect in vivo ovarian tissue in goats. The goals of this study were: (i) to test different biopsy needle sizes to collect ovarian tissue in situ using the BPU method (Experiment 1), and (ii) to study ovarian tissue features such as preantral follicle density, morphology, class distribution, and stromal cell density in ovarian fragments obtained in vivo through a laparoscopic BPU method (Experiment 2). In Experiment 1, goat ovaries (n = 20) were collected in a slaughterhouse and subjected to in situ BPU. Three needles (16, 18, and 20G) were tested. In Experiment 2, the most efficient biopsy needle from Experiment 1 was used to perform laparoscopic BPU in goats (n = 8). In Experiment 1, the recovery rate was greater (P < 0.05; range 50-62%) with 16G and 18G needles than the 20G (17%) needle. The mean weight of ovarian fragments collected by the 16G needle was greater (P < 0.05) than the 18G and the 20G needle. In Experiment 2, 62 biopsy attempts were performed and 52 ovarian fragments were collected (90% success rate). Overall, 2054 preantral follicles were recorded in 5882 histological sections analyzed. Mean preantral follicular density was 28.4 ± 1.3 follicles per cm2. The follicular density differed (P < 0.05) among animals and ovarian fragments within the same animal. The mean stromal cell density in the ovarian fragments was 37.1 ± 0.5 cells per 2500 µm2, and differed (P < 0.05) among animals. Moreover, preantral follicle density and stromal cell density were associated (P < 0.001). The percentage of morphologically normal follicles was 70.1 ± 1.2, and differed (P < 0.05) among animals. The majority (79%) of the morphologically normal follicles was classified as primordial follicles, and differed (P < 0.05) among animals and between ovaries. In summary, a laparoscopic BPU method has been developed to harvest ovarian tissue in vivo with a satisfactory success rate in goats. Furthermore, this study described for the first time that goat ovarian biopsy fragments have a high heterogeneity in follicular density, morphology, class distribution, and stromal cell density.

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.