MALDI‐MS Lipid Profiles of Oocytes Recovered by Ovum Pickup from Bos indicus and 1/2 indicus × taurus with High vs Low Oocyte Yields

The aim of the present study was to compare the lipid profile in oocytes of indicus and 1/2 indicus × taurus cows with high and low antral follicle count (AFC)/oocyte yields. After an OPU procedure (D0), antral follicles ≥3 mm were counted by ultrasonography (D4, 19, 34, 49, 64), and cows were assigned to groups with either high AFC (≥30 follicles; indicus, NH group; 1/2 indicus × taurus, AH group) or low AFC (≤15 antral follicles; indicus, NL group; 1/2 indicus × taurus, AL group). The lipid profiles of the oocytes were determined by MALDI‐MS. For GI, GII and GIII oocytes, the indicus samples tend to cluster separately from the 1/2 indicus × taurus samples. The lipid species [PC (P‐38:5) + H]⁺ and/or [PC (P‐36:2) + Na]⁺, [PC (38:2) + H]⁺, [PC (38:5) + Na]⁺ and [TAG (60:8) + NH⁴]⁺ were more abundant in indicus (NH and NL groups) than 1/2 indicus × taurus. The higher lipid content in the indicus oocytes likely reflects differences in the rate of lipid metabolism and may contribute to oocyte competence and embryo development.     

Immunolocalization of Melatonin and Follicle‐Stimulating Hormone Receptors in Caprine Ovaries and their Effects During in vitro Development of Isolated Pre‐Antral Follicles

The expression of melatonin type 1 (MT1) and FSH (FSHR) receptors in caprine ovaries and the effects of these hormones on the in vitro development of isolated pre‐antral follicles were evaluated. Follicles (≤200 μm) were cultured for 12 days in α‐MEM (control) or melatonin (100 or 1000 pg/ml) or sequential melatonin medium (100 pg/ml: from day 0 to day 6; 1000 pg/ml: from day 6 to day 12; experiment 1) and in control or sequential FSH (100 ng/ml from day 0 to day 6; 500 ng/ml from day 6 to day 12) or sequential melatonin or this latter plus sequential FSH (experiment 2). MT1 and FSHR expressions were observed in granulosa cells from secondary and antral follicles. The oocytes from primordial and primary follicles also express FSHR. Sequential melatonin increased the percentage of normal follicles and oocyte recovery compared with the control or melatonin (1000 pg/ml) at day 12. In experiment 2, all the treatments increased the normal follicles and growth compared with the control. In conclusion, this study demonstrated the presence of MT1 and FSHR in caprine ovaries. The addition of increased concentrations of melatonin (sequential medium) or FSH can be used to promote the in vitro development of caprine pre‐antral follicles.     

Effects of Co‐culture of Cumulus Oocyte Complexes with Denuded Oocytes During In Vitro Maturation on the Developmental Competence of Cloned Bovine Embryos

This study evaluated the effects of co‐culture of immature cumulus oocyte complexes (COCs) with denuded immature oocytes (DO) during in vitro maturation on the developmental competence and quality of cloned bovine embryos. We demonstrated that developmental competence, judged by the blastocyst formation rate, was significantly higher in the co‐cultured somatic cell nuclear transfer (SCNT+DO, 37.1 ± 1.1%) group than that in the non‐co‐cultured somatic cell nuclear transfer (SCNT‐DO, 25.1 ± 0.9%) group and was very similar to that in the control IVF (IVF, 38.8 ± 2.8%) group. Moreover, the total cell number per blastocyst in the SCNT+DO group (101.7 ± 6.2) was higher than that in the SCNT‐DO group (81.7 ± 4.3), while still less than that in the IVF group (133.3 ± 6.0). Furthermore, our data showed that mRNA levels of the methylation‐related genes DNMT1 and DNMT3a in the SCNT+DO group were similar to that in the IVF group, while they were significantly higher in the SCNT‐DO group. Similarly, while the mRNA levels of the deacetylation‐related genes HDAC2 and HDAC3 were significantly higher in the SCNT‐DO group, they were comparable between the IVF and SCNT+DO groups. However, the mRNA levels of HDAC1 and DNMT3B were significantly higher in the SCNT+DO group than in the other groups. In conclusion, the present study demonstrated that co‐culture of COCs with DO improves the in vitro developmental competence and quality of cloned embryos, as evidenced by increased total cell number.     

Cumulus cell expansion and first polar body extrusion during in vitro oocyte maturation in relation to morphological and morphometric characteristics of the dromedary camel ovary

The morphological and morphometric characteristics of the ovary are fundamental properties for in vitro oocyte maturation. Nuclear maturation, including first polar body (1PB) extrusion, cytoplasmic maturation and cumulus cell (CC) expansion are the criteria for in vitro maturation (IVM) of oocyte. This study was designed to determine the effect of morphological and morphometric features of the ovary on CC expansion and 1PB extrusion during IVM of oocyte in the adult female dromedary camel. The weight, volume and three dimensions of ovaries from slaughtered dromedary camels and oocytes inside zona diameter and zona pellucida thickness were measured. The follicles were classified in regard to the size and oocytes according to their ooplasm appearance and CC compactness. Aspirated cumulus oocyte complexes (COCs) were incubated for 48 hr (with a 6‐hr interval) in Hams‐F10, and CC expansion and 1PB extrusion were assessed. Significant differences were seen in the shape, weight, volume and three dimensions of the ovaries between ≤4‐year‐old and >4‐year‐old dromedary camel (p < .5). Approximately, 95.82% of follicles were 2–4 mm in diameter. The mean (±SD) of inside zona diameter of the oocyte and zona pellucida thickness was 132.22 ± 13.8 and 14.64 ± 2.24 μm, respectively, in >4‐year‐old dromedary camel. The CC expansion and 1PB extrusion were seen in 86% and 21.88% of COCs, respectively. Age and sexual conditions of dromedary camel influence the morphological and morphometric characteristics of the ovary. Most COCs retrieved from 2–6 mm follicles are cultivable. The most slaughterhouse‐derived COCs retrieved from 2–6 mm follicles of non‐pregnant dromedary camels are excellent and good and yielding a most favourable diameter to achieve the developmental competence for IVM in an optimal time of 24–30 hr; the optimal time for CC expansion is 24–30 hr in this species. However, the CC expansion is a prerequisite process, but not sufficient for IVM.     

Effects of melatonin on oocyte developmental competence and the role of melatonin receptor 1 in juvenile goats

Melatonin enhances in vitro embryo development in several species by improving the oocyte developmental competence during in vitro maturation (IVM). Melatonin has a wide range of actions, from scavenging reactive oxygen species (ROS) to regulating gene expression, and it can also act by way of melatonin receptors. The aim of this study was to determine the mechanism of action of melatonin during the IVM of juvenile goat oocytes and the role of the membrane receptors. Melatonin receptor 1 was immunolocalized in cumulus cells and oocytes before and after 24 hr of IVM. The effect of melatonin on oocyte developmental competence was tested in three experimental IVM groups: (a) control, (b) 10^?7 M melatonin, and (c) 10^?7 M melatonin +10^?7 M luzindole (an inhibitor of both melatonin receptors). After IVM oocytes were assessed for ROS levels, mitochondrial activity, adenosine 5′‐triphosphate (ATP) concentration and relative gene expression (ACTB, SLC1A1, SOD1, GPx1, BAX, DNMT1, GCLC and GDF9). IVM‐oocytes were in vitro fertilized and cultured under conventional conditions. Blastocyst rate and quality (differential cell count) were assessed at 8 days post‐fertilization. Melatonin decreased ROS levels, increased mitochondrial activity and ATP content and increased blastocyst quality compared to control group (55.8 vs. 30.4 inner cell mass ICM, p < 0.05). There was no effect on the relative gene expression due to treatment with melatonin. In conclusion, we have showed that melatonin improves oocyte developmental competence in juvenile goats by reducing ROS levels and improving mitochondrial activity.     

The endothelial nitric oxide synthase/nitric oxide system is involved in the defective quality of bovine oocytes from low mid-antral follicle count ovaries

In a previous survey concerning cows of reproductive age, we demonstrated that oocytes isolated from ovaries with <10 medium antral follicles of 2 to 6 mm in diameter (low ovaries; Lo) show less developmental competence than oocytes collected from ovaries with >10 medium antral follicles (high ovaries; Hi). The aim of the present study was to evaluate whether a defective endothelial nitric oxide synthase/nitric oxide (eNOS/NO) system and vasculature in healthy medium antral follicles is likely to reduce oocyte competence from Lo ovaries. Thus, experiments were conducted to 1) immunolocalize eNOS protein during folliculogenesis; 2) quantify eNOS protein/vasculature in the follicle wall; and 3) verify if NO donor, S-nitroso acetyl penicillamine (SNAP) administration during in vitro maturation affects developmental competence of oocytes isolated from Lo ovaries. Endothelial nitric oxide synthase protein was detected in granulosa and theca cells, as well as in blood vessels from primordial to antral follicles. Quantitative analysis indicated that in medium antral follicles from Lo ovaries, eNOS protein expression and vasculature were reduced (P < 0.05). The addition of SNAP improved blastocyst and hatching rates of oocytes from Lo ovaries, promoting a percentage similar to oocytes from Hi ovaries, and reduced the percentage of apoptotic nuclei in in vitro-produced blastocysts (P < 0.05). Results from our study suggest that in bovine ovaries with small mid antral follicle number, a defective eNOS/NO system is related to a reduced follicle vasculature and may affect oocyte quality, thus inducing a premature decline of fertility.     

Effect of Follicle Size on In Vitro Maturation of Pre‐Pubertal Porcine Cumulus Oocyte Complexes

Very small follicles (<3.0 mm diameter) are over‐represented on the surface of ovaries of non‐cycling pigs, and the oocytes collected from these follicles generally have reduced developmental competence in vitro. This study examined the effect of follicle size on the nuclear maturation (n = 608), the potential of parthenogenetic activation (n = 243) and the cyclic AMP (cAMP) content of pre‐pubertal porcine oocytes (n = 480). In addition, the influence of follicle size on steroid hormone synthesis was analysed. Cumulus oocyte complexes (COCs) flushed from small (2.5–4.0 mm) or large (4.5–6.0 mm) ovarian follicles were cultured for 0, 28 and 46 h. After 46 h of IVM, a greater proportion of oocytes from 4.5‐ to 6.0‐mm follicles reach metaphase II (MII) compared with those from follicles with 2.5–4.0 mm of diameter (96.1 vs 77.0%, respectively; p < 0.001). Parthenogenetic activation of oocytes from large follicles produced higher developmental rates than oocytes from large follicles (p < 0.05). At 28 h, the IVM medium with oocytes from large follicles contained significantly more 17ß‐oestradiol (E₂) than the medium with oocytes from small follicles (5.55 vs 3.45 ng/ml, respectively; p < 0.05) and at 46 h, the medium with oocytes from small follicles contained significantly more progesterone (P₄) than the medium with oocytes from large follicles (276.7 vs 108.2 ng/ml, respectively, p < 0.05). Porcine oocytes from large follicles have higher nuclear and cytoplasmic maturation capacities, but the differences did not appear to be cAMP‐mediated. Our findings also suggest that COCs from small follicles undergo more intensive luteinization than COCs from large follicles. The results show that oocytes from follicles with a diameter greater than 4.0 mm are more suitable for in vitro studies.     

Extension of the culture period for the in vitro growth of bovine oocytes in the presence of bone morphogenetic protein‐4 increases oocyte diameter,but impairs subsequent developmental competence

Bone morphogenetic protein‐4 (BMP‐4) inhibits luteinization of granulosa cells during in vitro growth (IVG) culture of bovine oocytes; however, oocytes derived from a 12 day IVG were less competent for development than in vivo‐grown oocytes. We herein investigated whether an extended IVG culture with BMP‐4 improves oocyte growth and development to blastocysts after in vitro fertilization. Oocyte‐granulosa cell complexes (OGCs) were cultured for 14 or 16 days with BMP‐4 (10 ng/mL), while a 12 day culture with BMP‐4 served as the in vitro control. OGC viability was maintained for the 16 day culture with BMP‐4 (83.2%), but was significantly lower without BMP‐4 (58.9%) than the control (83.0%). Prolong‐cultured oocytes at 16 days had statistically greater diameter (114.6 μm) than the control (111.7 μm). IVG oocytes with BMP‐4 for the 16 day culture had a similar nuclear maturation rate to the control (approximately 67%); however, blastocyst rates in BMP‐4 treated oocytes of 14 (1.8%) and 16 day (0%) IVG were statistically lower than that of 12 day IVG (9.0%). In conclusion, BMP‐4 maintained OGC viability and promoted oocyte growth in a prolonged culture, but impaired the developmental competence of oocytes. Prolonged culture may not be an appropriate strategy for enhancing the developmental competence of IVG oocytes.     

In Vitro Development of Marmoset Monkey Oocytes by Pre-antral Follicle Culture

A technique for in vitro maturation of oocytes from small ovarian follicles of marmoset monkeys (Callithrix jacchus) has been developed. We employed a two‐step culture system for primary follicles (45–85 μm) and a one‐step culture technique for secondary follicles (>85 μm). The two‐step technique started with the culture of stromal tissue fragments for 2 days. Thereafter, mechanically isolated follicles were transferred to a culture system where they attached to the culture surface and grew for up to a further 12 days. Significant growth of the small follicles and their oocytes was only achieved with gonadotrophins in the medium. Oocytes with a mean diameter of 39 μm from follicles <85 μm reached a mean diameter of 90 μm by the end of the two‐step culture. After in vitro maturation, 19% of oocytes from these follicles had progressed to the germinal vesicle breakdown (GVBD). Follicles between 85 and 170 μm in diameter were isolated from the stroma and placed directly in the culture. Oocytes from these follicles had a mean diameter of 64 μm. The maximum size the oocytes reached in culture was related to the age of the females (pre‐pubertal females: 102 ± 1.3 μm; adults: 96 ± 1.4 μm). Twenty‐seven per cent of oocytes from pre‐pubertal ovaries achieved GVBD and nearly two‐thirds of these progressed to polar body stage. From adult ovaries, only 12% progressed to GVBD and one‐third of these to polar body stage. It is possible to develop mature oocytes in vitro from marmoset secondary pre‐antral follicles (>85 μm). From primary follicles, although near full size oocytes were developed, maturation capacity was incomplete.     

Gonadal Development of the Piau Leporinus copelandii (Characiformes,Anostomidae) in a Tropical River in South‐eastern Brazil

Histological analysis of the gonadal development of Leporinus copelandii Steindachner, 1875, a rheophilic Characiformes species in the Paraiba do Sul River, South‐eastern Brazil, was described. We expect that this species adapt gonadal development to succeed in this river basin that has its longitudinal profile blocked by several impoundments. Fishes were examined by routine macroscopic and histological techniques. Stages of oocyte and spermatocyte development were described, and gonadal maturation was proposed. Mean oocyte diameter obtained from histological observations increased from the pre‐spawning (4.2–175.5 μm) to spawning (148.5–262.0 μm) phases, followed by a sharp decrease in the post‐spawning (27.0–56.7 μm) phase. Based on occurrence of different oocytes phases and oocyte size distribution, this species has group‐synchronic development of oocytes. Further studies are necessary to clarify the spawning grounds for L. copelandii in the Paraíba do Sul River basin, especially considering that several impoundments obliterate the natural river course and this could limit spawning grounds.     

Relationship between DNA fragmentation of equine granulosa cells and oocyte meiotic competence after in vitro maturation

The acquisition of equine oocyte developmental capacity is ensured by the follicular environment, such as granulosa cells, which could reflect the meiotic development potential of immature oocytes. This study evaluated the relationship between DNA fragmentation of granulosa cells, using the chromatin dispersion test, and equine oocyte meiotic development after in vitro maturation. Granulosa cells and cumulus–oocytes complexes (n = 50) were recovered from slaughterhouse‐derived ovaries. Oocytes were in vitro matured, stained and evaluated under fluorescence microscopy. Maturation rates were classified into outstanding, medium and poor levels of maturation using 25th and 75th percentiles as thresholds. For DNA assessment, each sample was processed with the Ovoselect^® kit (Halotech DNA). High, low and total DNA fragmentation percentages were compared among levels of maturation rates by ANOVA, followed by Duncan test. Results were expressed as mean ± SE. Total and high DNA fragmentation rates of granulosa cells were significantly higher (p < 0.05) in follicles whose oocytes had reached outstanding maturation level than those originating from follicles whose oocytes had reached poor maturation level. In conclusion, the DNA fragmentation analysis of equine granulosa cells can be a valuable test to identify equine oocytes showing the best meiotic competence after in vitro maturation.     

Reestablishment of transzonal projections and growth of bovine oocytes in vitro

Transzonal projections (TZPs) that maintain bidirectional communication between oocytes and granulosa cells or cumulus cells are important structures for oocyte growth. However, whether TZPs develop between TZP-free oocytes and granulosa cells, and whether reestablished TZPs support oocyte growth, is unknown. We first examined changes in TZPs after denudation of bovine oocytes collected from early antral follicles (0.5–0.7 mm). Twenty-four hours after denudation, almost all the TZPs disappeared. We also examined the reestablishment of TZPs by coculturing TZP-free denuded oocytes (DOs) with mural granulosa cells (MGCs) collected from early antral follicles. In addition, to confirm if the reestablished TZPs were functional, the reconstructed complexes (DO+MGCs) were subjected to in vitro growth culture and found that the MGCs adhered to TZP-free DOs and TZPs were reestablished. During in vitro growth culture, DO+MGCs developed and formed antrum-like structures. After culture, the number of TZPs in DO+MGCs increased, and the oocytes grew fully and acquired meiotic competence. These results suggest that reestablished TZPs are able to support oocyte growth.     

Effect of the Medium Replacement Interval on the Viability,Growth and In Vitro Maturation of Isolated Caprine and Ovine Pre‐Antral Follicles

The aim of the present study was to evaluate the effects of different medium replacement intervals on the viability, antral cavity formation, growth and in vitro maturation (IVM) of oocytes from caprine and ovine pre‐antral follicles. Pre‐antral ovarian follicles (≥150 μm) were isolated from the ovarian cortex of goats and sheep and were individually cultured for 24 days using two different medium replacement intervals [2 days (T₁) or 6 days (T₂)]. Follicle development was evaluated on the basis of antral cavity formation, increases in follicular diameter and the presence of healthy cumulus oocyte complexes and fully grown oocytes. For caprine species, results showed a higher percentage (p < 0.05) of viable follicles in T₁ than T₂ from day 6 until the end of the culture. In addition, when comparing both treatments after the same culture duration, the rate of antrum formation was significantly higher in T₁ than in T₂ from day 12 onwards. Yet, in ovines, when both treatments were compared on day 24 of the culture, there were more viable follicles in T₂ than in T₁ (p < 0.05). In the caprine species, percentages of fully grown oocytes (≥110 μm) acceptable for IVM after 24 days of culture were significantly higher in normal follicles cultured in T₁ (30.0%) than in T₂ (6.7%; p < 0.05). On the other hand, in ovines, at the end of the culture, the percentage of oocytes destined for IVM was higher in T₂ than in T₁ (23.5% vs 2.9%; p < 0.05). In conclusion, under the same conditions, the frequency of medium replacement significantly affected the in vitro development of caprine and ovine pre‐antral follicles. To improve the efficiency of the culture system, the medium must be replaced every 2 and 6 days for goat and sheep pre‐antral follicles, respectively.     

Protein Localization of Epidermal Growth Factor in Sheep Ovaries and Improvement of Follicle Survival and Antrum Formation In Vitro

The aims of this study were to characterize EGF protein expression in ovine ovaries and to verify the effect of EGF on the in vitro development of isolated pre‐antral follicles. After collection, ovarian tissue was fixed for immunohistochemical analysis. Additional pairs of ovaries were collected, and secondary follicles were cultured for 18 days in α‐MEM⁺ (control) alone or supplemented with EGF (1, 10 or 50 ng/ml). The immunostaining for EGF was observed in oocytes from pre‐antral and antral follicles, in granulosa cells of primary and secondary follicles, as well as in cumulus and mural cells of antral follicles. After 18 days, the results showed that treatment with 50 ng/ml EGF significantly increased the percentage of morphologically normal follicles compared with the control group (α‐MEM⁺) and significantly reduced the precocious extrusion of oocytes and increased the percentage of antral follicles compared with the control and 1 ng/ml EGF. All the treatments induced a progressive and significant increase of the follicular diameter throughout the period of culture. However, there were no significant differences in follicular diameter or in the daily growth rate among treatments. In conclusion, this study demonstrated the presence of EGF in ovine ovaries. Moreover, 50 ng/ml EGF increased the percentage of normal follicles and improved antrum formation in isolated ovine follicles after 18 days of in vitro culture.     

m‐carboxycinnamic acid bishydroxamide improves developmental competence,reduces apoptosis and alters epigenetic status and gene expression pattern in cloned buffalo (Bubalus bubalis) embryos

Incomplete or aberrant reprogramming of nuclear genome is one of the major problems in somatic cell nuclear transfer. In this study, we studied the effect of histone deacetylase inhibitor m‐carboxycinnamic acid bishydroxamide (CBHA) on in vitro development of buffalo embryos produced by Hand‐made cloning. Cloned embryos were treated with CBHA (0, 5, 10, 20 or 50 μM) for 10 hr from the start of reconstruction till activation. At 10 μM, but not at other concentrations examined, CBHA increased (p < .05) the blastocyst rate (63.77 ± 3.97% vs 48.63 ± 3.55%) and reduced (p < .05) the apoptotic index of the cloned blastocysts (8.91 ± 1.94 vs 4.36 ± 1.08) compared to untreated controls, to levels similar to those in IVF blastocysts (4.78 ± 0.74). CBHA treatment, at all the concentrations examined, increased (p < .05) the global level of H3K9ac in cloned blastocysts than in untreated controls to that observed in IVF blastocysts. Treatment with CBHA (10 μM) decreased (p < .05) the global level of H3K27me3 in cloned blastocysts than in untreated controls but it was still higher (p < .05) than in IVF blastocysts. CBHA (10 μM) treatment increased (p < .05) the relative expression level of pluripotency‐related genes OCT‐4 and NANOG, and anti‐apoptotic gene BCL‐XL, and decreased (p < .05) that of pro‐apoptotic gene BAX than in untreated controls but did not affect the relative expression level of apoptosis‐related genes p53 and CASPASE3 and epigenetics‐related genes DNMT1, DNMT3a and HDAC1. These results suggest that treatment of cloned embryos with 10 μM CBHA improves the blastocyst rate, reduces the level of apoptosis and alters the epigenetic status and gene expression pattern.     

Expression of gap junctional connexin proteins in ovine fetal ovaries: effects of maternal diet

Gap junctions have been implicated in the regulation of cellular metabolism and the coordination of cellular functions during growth and differentiation of organs and tissues, and gap junctions play a major role in direct cell-cell communication. Gap junctional channels and connexin (Cx) proteins have been detected in adult ovaries in several species. Furthermore, it has been shown that several environmental factors, including maternal diet, may affect fetal organ growth and function. To determine whether maternal diet affects expression of Cx26, Cx32, Cx37, and Cx43 in fetal ovaries, sheep were fed a maintenance (M) diet with adequate (A) selenium (Se) or high (H) Se levels from 21 d before breeding to day 132 of pregnancy. From day 50 to 132 of pregnancy (tissue collection day), a portion of the ewes from the ASe and HSe groups was fed a restricted (R; 60% of M) diet. Sections of fetal ovaries were immunostained for the presence of Cxs followed by image analysis. All four Cxs were detected, but the distribution pattern differed. Cx26 was immunolocalized in the oocytes from primordial, primary, secondary, and antral follicles; in granulosa and theca layers of secondary and antral follicles; stroma; and blood vessels. Cx32 was in oocytes, granulosa, and theca cells in a portion of antral follicles; Cx37 was on the borders between oocyte and granulosa/cumulus cells of primordial to antral follicles and in endothelium; and Cx43 was on cellular borders in granulosa and theca layers and between oocyte and granulosa/cumulus cells of primordial to antral follicles. Maternal diet affected Cx26 and Cx43 expression, Cx26 in granulosa layer of antral follicles was decreased (P < 0.01) by HSe in the M and R diets, and Cx43 in granulosa layer of primary and granulosa and theca of antral follicles was increased (P < 0.05) by the M diet with HSe. Thus, Cxs may be differentially involved in regulation of fetal ovarian function in sheep. These data emphasize the importance of maternal diet in fetal growth and development.     

Fibrin–alginate hydrogel supports steroidogenesis,in vitro maturation of oocytes and parthenotes production from caprine preantral follicles cultured in group

This study aimed to establish a culture system that improves the in vitro development of caprine preantral follicles. In a first experiment, follicles were encapsulated as a single unit per bead and cultured singly or in groups or with five follicles in the same alginate (ALG) bead for 18 days. In a subsequent experiment, the “five follicles per bead” design was chosen to culture in ALG, fibrin–alginate (FA) or hyaluronate (HA) for 18 days. In a third experiment, we chose the five follicles per bead in FA to culture for 30 days. The culture set‐up of five follicles per ALG bead increased antrum formation and follicle diameter compared to the other culture designs (p < .05). Moreover, under this condition, 44.44% of the oocytes from in vitro cultured preantral follicles reached meiotic resumption. A significant increase of follicle diameter occurred in attachment system and FA (p < .05), but the ALG condition reached the highest among all groups on day 18 (p < .05). Follicles encapsulated in matrix produced more estradiol and progesterone than attachment system (p < .05). The expression of MMP‐9 mRNA was higher in FA than in other groups (p < .05) and similar to antral follicles from in vivo control (p > .05). Only FA group resulted in oocytes matured. After 30 days, oocytes from preantral follicles in vitro grown in FA developed to eight‐cell parthenotes. In conclusion, a culture system using FA supported the development of caprine preantral follicles cultured in group and included in the same bead of hydrogel, improving the oocyte maturation and producing parthenotes.