In vitro Growth and Maturation of Caprine Oocytes

This study was conducted to culture in vitro caprine pre-antral follicles for determining the competence of growth and maturation of oocytes and establishing a suitable culture system for oocyte maturation from pre-antral follicles. Two different culture methods (microdrop and agar gel clot) were employed to culture caprine pre-antral follicles. The pre-antral follicles were isolated from prepubertal goat ovaries by treatment with collagenase and DNase. The isolated pre-antral follicles were cultured in basic culture medium for 9 days (for growth). And oocytes were cultured in maturation culture medium for another 2 days for maturation. The result demonstrated that the growth rate of oocytes cultured in microdrops was significantly (p < 0.05) higher than that in agar gel clots, whereas the viability of oocytes in microdrops was considerably (p < 0.05) lower than that in agar gel clots. The oocytes grew over 150 microm in diameter, and two of 151 oocytes cultured in microdrops yielded morphologically abnormal first polar bodies. However, the size of oocytes cultured in agar gel approached to 120 microm in diameter and no polar body was produced.     

Impact of Growth Factors on In Vitro Development of Caprine Oocytes at Pre-antral Stage

The objective of this study was to examine the effect of various growth factors such as the epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) either individually or in association, in the presence of follicle-stimulating hormone (FSH) on the in vitro growth and viability of caprine oocytes at pre-antral stage. Pre-antral follicles were disassociated enzymatically and mechanically from pre-pubertal caprine ovaries after the animals were anaesthetically ovariectomized. Caprine pre-antral follicles in group 1, 2, 3 and 4 were cultured in growth culture medium, growth culture medium + EGF, growth culture medium + IGF-I and growth culture medium + IGF-I + EGF, respectively, for 9 days. The results indicated that EGF (50 mg/l) increased the survival rate of oocytes, but decreased the growth rate of oocytes; IGF-I (100 mg/l) effectively maintained the survival of oocytes and stimulated their growth; IGF-I (100 mg/l) and EGF (50 mg/l) in combination produced a higher effect on both the survival and the growth rate of oocytes than IGF-I or EGF alone. Conclusively, the growth factors can effectively maintain the survival of caprine oocytes from pre-antral follicles and regulate their growth in culture. EGF and IGF-I in association could synergically meliorate the culture system of caprine pre-antral follicles.     

The effect of LIF in the absence or presence of FSH on the in vitro development of isolated caprine preantral follicles

We investigated the effect of the leukaemia inhibitory factor (LIF) alone or in association with FSH on the in vitro culture (IVC) of caprine preantral follicles. Preantral follicles >200 μm in size were isolated and cultured for 18 days in basic medium either alone (control) or supplemented with LIF (10 or 50 ng/ml) in the absence or presence of FSH. Every 6 days, follicular survival, growth and antrum formation were evaluated. At the end of the culture period, the oocytes underwent in vitro maturation (IVM), and their viability and chromatin configuration were assessed. Follicles of the control group and those cultured in 10 ng/ml LIF maintained the structural integrity (particularly the preservation of the basement membrane) when compared to the oocytes cultured in 50 ng/ml LIF, regardless the presence of FSH. In the absence of FSH, the percentage of antrum formation after 18 days of culture in the 50 ng/ml LIF group was significantly lower than in either the control group or the 10 ng/ml LIF group. However, this effect was not observed in the presence of FSH. The rate of resumption of meiosis was significantly higher in the 50 ng/ml LIF group in the absence of FSH in comparison with the control and 10 ng/ml LIF groups. Metaphase II was observed only when follicles were cultured in a combination of FSH and 50 ng/ml LIF. In conclusion, LIF alone does not interfere with antral formation and oocyte growth, but at concentration of 50 ng/ml and combined with FSH, it promotes oocyte maturation.     

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.     

Expression levels of mRNA-encoding PDGF receptors in goat ovaries and the influence of PDGF on the in vitro development of caprine pre-antral follicles

The aims of this study were to investigate the expression levels of mRNA for platelet-derived growth factor (PDGF) receptors (PDGFR-α and -β) in caprine follicles at different developmental stages and to evaluate the influence of PDGF on the in vitro development of pre-antral follicles. For this, goat primordial, primary and secondary follicles, as well as small (1-3 mm) and large (3-6 mm) antral follicles, were obtained, and PDGFR-α and -β mRNA levels were quantified by real-time PCR. Furthermore, pre-antral follicles (≥ 200 μm) were isolated from goat ovaries and cultured for 18 days in α- minimum essential medium supplemented with PDGF at 50 or 100 ng/ml, containing or not FSH. Real-time PCR showed highest PDGFR-α mRNA levels in secondary follicles, while PDGFR-β mRNA levels were highest in primary follicles onwards. Both receptors showed higher mRNA levels in granulosa/theca cells from small and large antral follicles than in their corresponding cumulus-oocyte complexes. In culture, the percentage of antrum formation was significantly higher in 100 ng/ml PDGF compared with the same PDGF concentration associated with FSH. After 18 days, PDGF in both concentrations associated with FSH promoted follicular growth significantly higher than the control. Moreover, the addition of FSH to 50 ng/ml PDGF positively influenced the follicular growth when compared with the same PDGF concentration in the absence of FSH. In conclusion, PDGF is important for early goat folliculogenesis, because the presence of PDGFR-α and -β mRNA was detected in all follicular categories, and PDGF associated with FSH stimulated the growth of goat pre-antral follicles isolated and cultured in vitro.     

Bovine oocytes in secondary follicles grow in medium containing bovine plasma after vitrification

There has been no culture system that supports the growth of bovine oocytes for more than 2 weeks. In the present study, bovine secondary follicles were cultured for 4 weeks, and the effects of supplemented protein components and FSH in the culture medium on the growth of the oocytes were examined. The effect of vitrification of secondary follicles on the subsequent oocyte growth was also examined. Secondary follicles (150 to 200 μm in diameter) containing growing oocytes (approximately 60 μm in diameter) were dissected from ovaries and cultured in a medium supplemented with FSH (0, 25 or 50 ng/ml) and one of the following four kinds of protein components: bovine serum albumin (BSA), bovine plasma (BPL), fetal calf serum (FCS) and bovine follicular fluid (BFF). In BSA- and BPL-supplemented media with 0 or 25 ng/ml FSH, more than 50% of follicles showed no degenerative signs during culture, and oocytes significantly increased in size after 4 weeks (P<0.05). Higher percentages of granulosa cell-enclosed oocytes were recovered from the follicles cultured in BPL-supplemented media with 0 and 25 ng/ml FSH, and the oocytes grew to 90 μm or more in diameter. In FCS- and BFF-supplemented media, FSH increased the numbers of degenerating follicles. Next, vitrified-warmed secondary follicles were cultured in BPL-supplemented medium. One third of the follicles showed no degenerative signs, and the oocytes increased in diameter to 88.8 ± 3.1 μm after 4 weeks of culture. These results suggest that a BPL-supplemented medium supports oocyte growth in bovine secondary follicles for 4 weeks, even after vitrification and warming of the follicles.     

In vitro development of primordial follicles after long-term culture of goat ovarian tissue

This study aims to investigate the effects of follicle stimulating hormone (FSH) and fibroblast growth factor-2 (FGF-2) on the survival and growth of caprine preantral follicles. Ovarian tissues were cultured for 1, 7, 14, 21 or 28 days in medium supplemented with FSH (FSH-2d or FSH-7d, i.e., with replacement of the culture medium every 2 or 7 days, respectively) or FSH + FGF-2 (replacement of the medium every 2 days). Non-cultured (control) and cultured ovarian fragments were processed for histological and ultrastructural analysis. After 28 days of culture, the media supplemented with FSH-2d was the most effective in maintaining the percentage of normal follicles and in promoting follicular growth. Furthermore, both treatments with FSH increased the percentage of the primary follicles. However, ultrastructural studies did not confirm follicular integrity from 14 days of culture onward. In conclusion, culturing tissue for up to 7 days in medium containing FSH alone or combined with FGF-2 maintains caprine preantral follicle integrity and promotes their growth in vitro.     

Ca2+ Cascade and Meiotic Resumption of the Caprine Primary Oocyte

In this study, we investigated the fluctuations of concentration of intracytoplasmic free Ca(2+) during in vitro maturation of caprine primary oocytes and its role in meiotic resumption. Oocytes that were extracted from caprine ovaries were cultured and allowed to mature in vitro to determine their developmental stages including germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase of the first meiotic division (MI) and metaphase of the second meiotic division (MII). Intracytoplasmic free Ca(2+) turnovers of caprine oocytes at these different developmental stages were measured using the calcium fluorescent probe Fura-2/AM (C(44)H(47)N(3)O(24)) to investigate the dynamics of cytosolic free Ca(2+) during in vitro maturation of oocytes and the role of Ca(2+) in inducing the initiation of meiotic resumption of oocytes. Moreover, the oocytes were cultured in Ca(2+) culture medium and Ca(2+)-free culture medium to examine the effect of extracellular Ca(2+) on the oocyte maturation. The results indicated that Ca(2+) concentrations at GV, GVBD, MI and MII stages were 78.06, 147.41, 126.97 and 97.73 nmol/l, respectively, and that 86.30% of oocytes remained at the GV stage and no oocyte developed to MII in Ca(2+)-free culture medium, and 1.1% of oocytes stayed at the GV stage and 83.5% of oocytes developed to MII in Ca(2+) culture medium. These results suggest that the occurrence of GVBD and cell cycle progression to MI and MII stages are closely related to Ca(2+), and that extracellular Ca(2+) performs a specific function for the initiation of meiotic resumption in caprine oocytes.     

Meiosis Resumption of Canine Oocytes Cultured in the Isolated Oviduct

The aim of this study was to investigate the effects of culture in isolated oviducts relative to meiotic maturation, the time required to resume meiosis and the viability of the canine oocytes. For this purpose, cumulus–oocyte complexes and isthmus–ampullar tracts of the oviducts were collected from bitches undergoing ovariohysterectomies and destined to two experiments of culture. In experiment 1, the oocytes were cultured for 24 or 30 h: (1) in 100 μl drops under oil; (2) on the mucosal epithelium of the open oviducts; (3) in the ligated oviducts. In experiment 2, oocytes were cultured in the ligated oviduct for 24, 30 and 48 h. A group of control oocytes was not cultured (0 h). The results showed that within 30 h of culture, a higher proportion of oocytes (p < 0.001) resumed meiosis in the ligated oviduct (63.8%) than in drop (20.4%) or in the open oviduct (27.1%). Moreover, 24 and 30 h of culture assured higher proportions of meiosis resumption than 48 h (69.2 and 59.1% vs 35.8%, p < 0.005). Oocyte resumption of meiosis was mainly determined by oocytes at meiotic stages preceding metaphase I, while stages between metaphase I and II in the ligated oviduct ranged between 12.5 and 31.9%. The extension of the culture time up to 48 h in the oviduct increased oocyte degeneration significantly (59.3%, p < 0.0001) compared with 24 and 30 h (18.7 and 27.3%, respectively) and the oviductal epithelium showed nuclear picnosis and degeneration following culture. The present study suggests that the close physical interaction between the canine oocytes and the oviductal tract positively affects oocyte maturation, and meiosis is resumed within 30 h of culture. Moreover, the oocyte survival is better preserved within 30 h in the ligated oviduct compared with the conventional culture in drop or to the culture in the open oviduct, but the ligated oviduct does not assure viability of the oocytes up to 48 h of culture.     

The effect of ovarian status and follicular diameter on maturational ability of domestic cat oocytes

The objective of this study was to clarify the effect of ovarian status and follicular size on morphological normality and maturational ability of cat oocytes. Ovarian status was classified into inactive, follicular, luteal and prepubertal, and follicles were classified into three groups according to their diameter (400-800, 800-1200 and 1200-2000 μm). In each ovarian status, the number of follicles decreased but the percentage of morphologically normal oocytes increased with the growth of follicles (p<0.05). Only a single follicle that was 1200-2000 μm in diameter was observed in two of the five prepubertal cats. In follicles that were 800-1200 μm in diameter, the percentage of normal oocytes and maturation rate were higher in prepubertal cats than in mature cats (p<0.05). Oocyte diameter tended to increase with the growth of follicles. After oocytes were cultured individually in droplets of maturation medium, the oocyte maturation rate increased with the growth of follicles in each ovarian status (p<0.05). In conclusion, oocytes collected from larger follicles possess higher maturational ability in vitro in sexually mature cats. In prepubertal cats, a higher maturation rate can be obtained from oocytes derived from small follicles compared with in mature cats.     

Follicle formation in the canine ovary after autografting to a peripheral site

This study reports about follicular development on the surface of canine ovarian tissue after autografting under the fascia of the thoracolumbar muscle and about meiotic resumption of follicle-derived oocyte after maturation culture. After ovarian excision from a bitch, each ovary of the pairs was cut approximately into half. The hemi-ovaries were transplanted into the bitch of origin at three different body sites (under the fascia of the quadriceps femoris muscle and the thoracolumbar muscle, and in the deltoid muscle in the scapular region). All grafted ovaries were recovered from the bitch at 35 days post-transplantation. A visible antral follicle was observed on the surface of the ovary grafted under the thoracolumbar fascia. Histological examination revealed viable follicles at different stages of development irrespective of graft site. Most granulosa cells in the follicles at different stages of development expressed proliferating cell nuclear antigen (PCNA). A total of three oocytes were collected from an ovary grafted under the fascia of the thoracolumbar muscle, wherein an oocyte reached metaphase I after maturation culture. This is the first report to demonstrate follicular development and meiotic resumption of oocytes recovered from autografted canine ovarian tissues.     

Mitochondrial Patterns in Bovine Oocytes with Different Meiotic Competence Related to Their in vitro Maturation

This study was designed to specify chromatin and mitochondrial patterns in bovine oocytes with different meiotic competence in relation to maturation progress, resumption of meiosis, MII onset and completion of maturation. Oocytes with greater or lesser meiotic competence, recovered separately from medium (MF) and small follicles (SF), were categorized according to morphology. Four oocyte categories, healthy and light‐atretic MF and healthy and light‐atretic SF oocytes were matured and collected at 0, 3, 7, 16 and 24 h of maturation. Specific differences in terms of chromatin and mitochondrial patterns were found among the maturing oocyte categories. Resumption of meiosis was accelerated in light‐atretic oocytes, as compared with healthy oocytes, regardless of their meiotic competence. More competent oocytes activated mitochondria twice during maturation, before resumption of meiosis and before completion of maturation, while less competent oocytes did it only once, before completion of maturation. Changes in mitochondrial activity differed in light‐atretic compared with healthy in both more and less competent oocytes. Healthy meiotically more competent oocytes formed clusters and produced ATP for the whole time of maturation until its completion, while light‐atretic more competent oocytes and healthy less competent oocytes reduced these activities earlier, at MII onset. Contrary to these oocyte categories, light‐atretic less competent oocytes increased cluster formation significantly before resumption of meiosis. It can be concluded that bovine oocytes with different meiotic competence and health differed in the kinetics of mitochondrial patterns during maturation.     

Regulation of mitogen-activated protein kinase 3/1 activity during meiosis resumption in mammals

In vivo, resumption of oocyte meiosis occurs in large ovarian follicles after the preovulatory surge of luteinizing hormone (LH). The LH surge leads to the activation of a broad signaling network in mural granulosa cells equipped with LH receptors. The signals generated in the mural granulosa cells are further augmented by locally produced peptides or steroids and transferred to the cumulus cell compartment and the oocyte itself. Over the last decade, essential progress has been made in the identification of molecular events associated with the final maturation and ovulation of mammalian oocytes. All new evidence argues for a multiple roles of mitogen-activated protein kinase 3/1 (MAPK3/1) in the gonadotropin-induced ovulation processes. However, the knowledge of gonadotropin-induced signaling pathways leading to MAPK3/1 activation in follicular cells seems limited. To date, only the LH-induced transactivation of the epidermal growth factor receptor/MAPK3/1 pathway has been described in granulosa/cumulus cells even though other mechanisms of MAPK3/1 activation have been detected in other types of cells. In this review, we aimed to summarize recent advances in the elucidation of gonadotropin-induced mechanisms leading to the activation of MAPK3/1 in preovulatory follicles and cultured cumulus-oocyte complexes and to point out a specific role of this kinase in the processes accompanying final maturation of the mammalian oocyte.     

Cumulus and oocyte maturation and in vitro and in vivo fertilization of oocytes in relation to follicular steroids,prolactin, and glycosaminoglycans throughout the estrous period in superovulated heifers with a normal LH surge,no detectable LH surge,and progestin inhibition of LH surge

Crossbred heifers (n = 103) were synchronized to estrus with prostaglandin (PGF_2α) and superovulated with follicle stimulating hormone (FSH-P). Animals were ovariectomized every 12 hr after the PGF_2α injection (n = 7 to 9/time) up to 108 hr to monitor the follicular, hormonal, and oocyte changes associated with follicular development and ovulation. Twenty-eight animals were implanted with Norgestomet implants 12 hr before PGF_2α and ovariectomized at 72, 84, 96, and 108 hr post PGF_2α injection to monitor effects of progesterone and suppression of the luteinizing hormone (LH) surge on oocyte maturation and quality. Follicular fluid was collected and analyzed for progesterone, estradiol, prolactin, and glycosaminoglycan content in conjunction with cumulus maturation and nuclear stage of oocyte maturation. Analysis of in vivo matured oocytes by in vitro fertilization was carried out at 60, 72, 84, and 96 hr post PGF_2α and in vitro matured oocytes at 12 to 108 hr post PGF_2α. No developmental changes in cumulus cells surrounding the oocyte of small follicles was noted (≤ 4 mm dia) indicating a static population. Medium (> 4 ≤ 8 mm) and large size (> 8 mm) follicles developed to the corona radiata and loose cumulus stages in animals in which an LH surge was detected but cumulus status remained primarily in the tight cumulus stage for animals without an LH surge. The estradiol-to-progesterone ratio for tight cumulus (TC), corona radiata (CR), and loose cumulus (LC) stages was 1.8 ± .1, 1.0 ± .1, and .4 ± .2, respectively (P < .01). Nuclear maturation of oocytes in small follicles from animals without a detectable LH surge seem to indicate early maturation (48 to 72 hr post PGF_2α) in conjunction with a high percent of degenerate oocytes not seen in animals exhibiting an LH surge. Oocytes from medium size follicles matured to germinal vesicle breakdown (GVBD) and early meiosis (metaphase I; MI) stages of development in all treatments. Most oocytes were degenerate in Norgestomet-implanted animals. Oocytes from large follicles (> 8 mm dia) from animals exhibiting an LH surge were in MI and metaphase II (MII) stages (48 to 84 hr post PGF_2α) in preparation of ovulation whereas oocytes from animals not exhibiting an LH surge had oocytes that early matured to MII (48 to 72 hr post PGF_2α), later regressing to degenerate oocytes (84 to 108 hr). Follicular progesterone, estradiol, and prolactin increased with oocyte maturation, particularly in medium and large follicles. In vivo matured oocytes for fertilization (60, 72, 84, and 96 hr post PGF_2α) were nude (from the oviduct) and primarily CR from follicles. Tubal oocytes (37%) were fertilized more frequently by a single sperm than follicular oocytes (14.3%; P < .01) and single sperm penetration peaked at 72 hr post PGF_2α. Follicular hormone concentrations were not related to sperm penetration. Oocytes (n = 101) matured in vivo had lower fertilization potential from ovaries producing < 14 or > 50 follicles (39.3%) as compared to 21 to 45 aspirated follicles (68.2%; P < .05), with a peak penetration at 32 follicles (86.7% penetration). No treatment differences (LH surge or no detectable LH surge) were noted in relation to in vivo matured oocytes. Oocytes with single sperm penetration had the lowest estradiol/progesterone ratio of 2.2 vs polyspermic penetration of 13.7.     

Vitrification with DAP 213 and Cryotop of Ex Situ and In Situ Feline Cumulus–Oocyte Complexes

This study was undertaken to compare cryotolerance, in terms of viability and resumption of meiosis after warming and culture (24 and 48 h), of ex situ (isolated) and in situ (enclosed in the ovarian tissue) feline cumulus–oocyte complexes (COCs) vitrified with DAP 213 (2 m DMSO, 1 m acetamide, 3 m propylene glycol) in cryotubes or Cryotop method. Ovaries were harvested from 49 pubertal queens. Of each pair of ovaries, one was dissected to release COCs randomly divided into three groups: fresh COCs (control), ex situ COCs vitrified with DAP 213 and Cryotop. The cortex of the other ovary was sectioned into small fragments (approximately 1.5 mm³) and randomly assigned to be vitrified by DAP 213 or Cryotop. After warming, ex situ and in situ (retrieved form vitrified ovarian tissue) COCs were matured in vitro. Viability of oocytes was highly preserved after warming and culture in all treatments. Proportions of oocytes surrounded by complete layers of viable cumulus cells were remarkably decreased (p < 0.00001) in both vitrification procedures compared to fresh oocytes. Resumption of meiosis occurred in all treatments. After 24 h of culture, results were similar in ex situ and in situ vitrified oocytes regardless of the vitrification protocol used (range 29–40%), albeit lower (p < 0.05) than those of fresh oocytes (65.8%). After 48 h of culture, ex situ oocytes vitrified with Cryotop achieved the rates of meiosis resumption similar to fresh oocytes (53.8% vs 67.5%; p > 0.05) and ex situ and in situ oocytes vitrified with DAP 213 showed similar rates of resumption of meiosis. These findings demonstrated that DAP 213 and Cryotop preserve the viability of ex situ and in situ oocytes, but cumulus cells are highly susceptible to vitrification. However, the capability to resume meiosis evidences that feline immature oocytes vitrified as isolated or enclosed in the ovarian cortex have comparable cryotolerance.     

Follicular development after ovum pick-up and fertilizability of retrieved oocytes in postpartum dairy cattle

This study aimed to evaluate gonadotropin secretion and the developmental competence of follicular oocytes in dairy cattle during the early postpartum (PP) period. The number of follicles developed after transvaginal ultrasound-guided ovum pick-up (OPU) and fertilizability of retrieved oocytes were compared between cows in which the first dominant follicle (DF) ovulated (ovulated group, n=4) and did not ovulate (non-ovulated group, n=3), and between early PP (early PP group, n=2) and after the resumption of the estrous cycle (cyclic group, n=2). Follicular ablation was performed 2-4 days after the detection of DF in the second follicular wave PP. OPU was repeated 3-5 times at 3 or 4-day intervals from 3-4 days after the follicular ablation. At OPU, the follicles were enumerated and all those > or = 5 mm in diameter were aspirated. Recovered oocytes were subjected to in vitro maturation and fertilization. Both criteria were similar between ovulated and non-ovulated groups, and between early PP and cyclic groups. These results suggest that FSH/LH secretions required for follicle recruitment and subsequent follicular growth during the early PP period are similar to those after resumption of the estrous cycle. They also indicate that follicular oocytes during the early PP period have developmental competence.     

In vitro Development of Buffalo Oocytes in Media-containing Fluids from Different Size Class Follicles

Studies were conducted to investigate the effect of supplementation of fluid from different sized class [small (SFF, < 3 mm), medium (MFF, 3-8 mm) and large (LFF, > 8 mm)] of normal and cystic (CFF) ovarian follicles in oocyte culture media on oocyte maturation rate and embryo development in vitro and to test the efficacy of follicular fluid (FF) from different size classes as a whole oocyte maturation medium. Results suggested that FF were capable of developing buffalo oocytes to embryonic stage in vitro although its efficacy was lower than that of serum. Regardless of high maturation rates after in vitro maturation (IVM) in media containing FF or IVM in whole FF, low blastocyst rates were obtained after in vitro fertilization (IVF) and culture of embryos. Follicular fluid from small follicles had significantly (p < 0.05) higher potential of developing buffalo oocytes to embryonic stage in vitro than that from medium and large follicles. Cystic FF was not capable of supporting development of buffalo oocytes in vitro.     

Vitrification of Immature Feline Oocytes with a Commercial Kit for Bovine Embryo Vitrification

The aim of this study was to evaluate the suitability of a commercial kit for bovine embryo vitrification for cryopreserving cat oocytes and to evaluate comparatively the effects of its use with slow freezing procedure on cryotolerance in terms of morphology and oocyte resumption of meiosis. Germinal vesicle stage oocytes isolated from cat ovaries were either vitrified (n = 72) using a vitrification kit for bovine embryo or slow frozen (n = 69) by exposing oocyte to ethylene glycol solution before being transferred to a programmable embryo freezer. After thawing and warming, oocytes were cultured for 48 h and then were examined for meiosis resumption using bisbenzimide fluorescent staining (Hoechst 33342). Fresh immature oocytes (n = 92) were used as the control group. The proportion of oocytes recovered in a morphologically normal state after thawing/warming was significantly higher in frozen oocytes (94.5%) than in the vitrified ones (75%, p < 0.01). Morphological integrity after culture was similar in vitrified (73.6%) and slow frozen oocytes (76.8%); however, only 37.5% of the morphologically normal oocytes resumed meiosis after vitrification compared to 60.9% of those submitted to slow freezing procedure (p < 0.01). Fresh oocytes showed higher morphological integrity (91.3%) and meiosis resumption rates (82.6%, p < 0.002) than cryopreserved oocytes, irrespective of the procedure used. These results suggest that immature cat oocytes vitrified with a kit for bovine embryos retain their capacity to resume meiosis after warming and culture, albeit at lower rates than slow frozen oocytes. Vitrification and slow freezing methods show similar proportions of oocytes with normal morphology after culture, which demonstrate that thawed and warmed oocytes that resist to cryodamage have the same chances to maintain their integrity after 48 h of culture.     

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.     

Interaction between ascorbic acid and follicle-stimulating hormone maintains follicular viability after long-term in vitro culture of caprine preantral follicles

This study evaluates the effects of ascorbic acid and its interaction with follicle-stimulating hormone (FSH) on the morphology, activation, and in vitro growth of caprine preantral follicles. Ovarian fragments were cultured for 1, 7, or 14 d in minimum essential medium (MEM) containing ascorbic acid (50 or 100 μg/mL), FSH (50 ng/mL), or both of these substances. Ovarian tissue that was either fresh (control) or cultured for 1, 7, or 14 d was processed for histological and ultrastructural evaluation. The results showed that after 14 d of culture, medium supplemented with 50 μg/mL of ascorbic acid alone or combined with FSH showed higher rates of follicular survival compared with MEM. After 7 d of culture, FSH, ascorbic acid at 50 μg/mL with or without FSH, and ascorbic acid at 100 μg/mL increased the percentage of follicular activation compared to fresh control. In addition, FSH alone significantly increased the percentage of growing follicles after 14 d. The combination of 50 μg/mL of ascorbic acid and FSH promoted a significant increase in oocyte and follicular diameter after 7 d of culture. Ultrastructural and fluorescent analysis confirmed the integrity of follicles cultured with 50 μg/mL of ascorbic acid and FSH after 14 d. In conclusion, the combination of 50 μg/mL of ascorbic acid and FSH maintained follicular integrity and promoted follicular activation and growth after long-term in vitro culture of caprine preantral follicles.