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.     

Long-term effects of in vitro growth of mouse oocytes on their maturation and development

Since very few oocytes grow completely in vivo, in vitro growth (IVG) of ovarian oocytes may provide a new source of functional oocytes. The long-term effects of in vitro maturation (IVM) of oocytes and in vitro culture of fertilized eggs have been reported; however, the effects of IVG of oocytes are unknown. Here in, we report the long-term effects of IVG of oocytes. Ovaries from 1-day-old mice containing non-growing oocytes were cultured for 10 days; the isolated follicles were then cultured for 11 days. Secondary follicles from 10-day-old mice were also cultured for 11 days. The nuclei of oocytes collected from the IVG and Graafiais follicles of adult mice were transferred to enucleated oocytes grown in vivo, respectively. Developmental competence was examined following IVM of the reconstituted oocytes. Chronologically, oocytes of 1-day-old, 10-day-old and adult mice were cultured for 22, 12 and 1 day(s). The result showed that the reconstituted eggs developed into pups at high rates after nuclear transfer and in vitro fertilization (IVF) in all the experimental groups (29-45%). However, the pups from reconstituted eggs containing the nuclei of 22-day cultured oocytes were heavier than the control pups (P<0.05). We concluded that long-term culture of oocytes did not affect their nuclear ability to develop to term; however, fetal growth was affected by the culture duration or culture conditions during the initial phase of follicular growth.     

In vitro growth of mouse ovarian preantral follicles and the capacity of their oocytes to develop to the blastocyst stage.

Two groups of mouse preantral follicles with diameters of 125-150 and 151-175 microm were cultured individually for 6 days in a medium supplemented with FSH and fetal calf serum to determine their in vitro growth characteristics. Their oocyte capacity for maturation and development to the blastocyst stage following in vitro fertilization was also assessed. Antral formation rate at the end of culture was higher in the follicles of 151-175 microm (89%) than 125-150 microm (76%). The timing of antrum formation was different between the two follicle categories: most 151-175 microm follicles formed antra earlier than 125-150 microm follicles (days 4 and 5 vs. 5 and 6). However, follicle diameters at the time of antrum formation were the same regardless of the initial size and the culture period. Maturation rates of the oocytes derived from both categories of in vitro grown follicles (70 and 62%) were not different from those of oocytes from in vivo grown follicles (74%). The in vitro derived oocytes, however, showed less cleavage (30 and 35%) than the in vivo derived oocytes (89%). Although the oocytes from both follicle categories developed to the morula stage after in vitro fertilization, blastocysts were only obtained from oocytes derived from the 151-175 microm category. These results demonstrate that an individual follicle culture system using a medium with FSH and fetal calf serum supports in vitro growth of mouse preantral follicles with diameters of 151-175 microm to the preovulatory stage, and that their oocytes have the capability to develop to the blastocyst stage.     

山羊卵巢无腔卵泡卵母细胞的体外生长

山羊卵巢无腔卵泡卵母细胞在以ＤＭＥＭ为基础,添加ＨＥＰＥＳ（２０ｍｍｏｌ／Ｌ）、ＦＣＳ（１０％）、ＦＳＨ（４０ｍｇ／Ｌ）、次黄嘌呤（２ｍｍｏｌ／Ｌ）、异双丁酰环腺苷酸（２ｍｍｏｌ／Ｌ）、ＩＧＦ－Ｉ（５０μｇ／Ｌ）、氢化可的松（４０μｇ／Ｌ）和ＩＴＳ（５０μｇ／Ｌ）的培养液中得以存活并生长。在二维培养体系中,卵泡在体外的生长模式和体内有很大差别。最显著的特征是卵泡不像在体内那样保持完整的立体结构一直到结束。绝大部分卵泡不同程度地发生基膜溶解和破裂,颗粒细胞向四周扩展并贴壁,形成单层。由于卵泡原有三维立体结构的破坏,易于导致卵母细胞的迁移。在生长方式上以数个卵泡聚集生长对其卵母细胞的生长似乎较为有利。卵泡卵母细胞在体外培养９ｄ,其直径可达１５０μｍ以上,达到了成熟时体积,存活率为５３％。实验证明,山羊无腔卵泡卵母细胞在合适的培养体系中,能在体外存活并生长,并能发育到成熟时的大小。     

Growing porcine oocyte-granulosa cell complexes acquired meiotic competence during in vitro culture

The aim of this study was to establish a culture system to improve the meiotic competence of porcine oocyte-granulosa cell complexes (OGCs) obtained from preantral or early antral follicles. Porcine OGCs were recovered from follicles with diameters of 230-300 (preantral follicles), 300-500, and 500-700 mum (early antral follicles) using scalpels. The OGCs were cultured for 2 weeks in culture medium. We examined the effects of the sizes of the follicles from which OGCs were recovered, the concentrations of polyvinylpyrrolidone (PVP, 0-8%) in the culture medium, and 2 types of culture dish (Falcon 3002 vs 1007) on formation of the antrum of OGCs. After culture, the oocytes were matured for 44 h to assess their meiotic competence. OGCs recovered from small follicles (230-500 microm) required longer (P<0.05) than larger follicles to form the antrum structure. The percentage of OGCs forming the antrum structure that were cultured in 2% PVP (31%) was higher (P<0.05) than for those cultured in other PVP concentrations (0-11%). The percentages of antrum-structure formation for OGCs cultured on Falcon 3002 (83% for 2% PVP and 60% for 4% PVP) were higher (P<0.05) than those cultured on Falcon 1007 (47% for 2% PVP and 9% for 4% PVP). Furthermore, all of the intact oocytes that were obtained from culture of OGCs and that formed an antrum were in the GV stage (n=28). When these immature oocytes were cultured for 44 h, the percentage of oocytes that reached the metaphase II stage (25%, n=68) was higher (P<0.0001) than that of oocytes matured without culture (0.7%, n=137). The results of the present study show that porcine OGCs obtained from preantral or early antral follicles acquire meiotic competence in vitro.     

JSAR Outstanding Research Award. In vitro growth of mammalian oocytes

The mammalian ovary contains a huge number of small follicles of various sizes, and each follicle encloses a small oocyte. Only a small number of non-growing oocytes (30 microm in the pig and cow) grow to their final size (120 microm), mature, and are ovulated. In vitro growth (IVG) culturing of small oocytes will provide a new source of mature oocytes for livestock production. Using the IVG culture system, non-growing mouse oocytes in primordial follicles grow to their final size and acquire full developmental competence. Among large animals, babies were produced from ovarian oocytes by IVG culture only in the cow. However, the oocytes used were not non-growing ones but at the mid-growth stage (90-99 microm in diameter) in early antral follicles. Xenotransplantation of the follicles at an early stage to immuno-deficient mice is a substitute for an effective long-term IVG culture of much smaller oocytes. IVG and xenotransplantation of small oocytes at a specific size will provide a new understanding of the mechanisms regulating oogenesis and folliculogenesis in the complex mammalian ovary.     

Regulation of in vitro growth of preantral follicles by growth factors in goats

Goat preantral follicles were cultured to investigate the effects of insulin-like growth factor-I (IGF-I), epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on the in vitro growth and viability of oocytes. Preantral follicles were isolated mechanically and enzymatically (using collagenase and DNase) from prepuberal goat ovaries. The working medium was composed of Defined Eagle's Minimum Essential Medium (DMEM) supplemented with HEPES (20 mM), 10% fetal calf serum (FCS), hypoxanthine (2 mM), dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP) (2 mM), penicillin (75 ng/ml) and streptomycin (50 ng/ml). The culture medium consisted of the working medium with follicle stimulating hormone (FSH) (100 ng/ml) and hydrocortisone (40 ng/ml) added. In the experiment, goat preantral follicles were cultured for 9 days in the culture medium and in the culture medium supplemented with either IGF-I (100 ng/ml), EGF (50 ng/ml), bFGF (50 ng/ml) or IGF-I (100 ng/ml)+EGF (50 ng/ml). The results indicated that IGF-I (100 ng/ml) effectively maintained the survival of oocytes and promoted their growth; EGF (50 ng/ml) enhanced the survival rate of oocytes but had a negative effect on oocyte growth; bFGF (50 ng/ml) stimulated oocyte survival but had no obvious effect on their growth while IGF-I (100 ng/ml) and EGF (50 ng/ml) in combination had a greater effect on both survival and growth rate of oocytes than IGF-I or EGF alone. The supplementation of IGF-1 and EGF to the culture medium is recommended in the culture of goat preantral follicles.     

Attempt at intracytoplasmic sperm injection of in vitro matured oocytes in common minke whales (Balaenoptera acutorostrata) captured during the Kushiro Coast Survey

The present study was conducted during the Kushiro Coast Survey in an attempt to produce common minke whale embryos. In Experiment 1, we attempted to determine the appropriate culture duration (30 or 40 h) for in vitro maturation (IVM) of immature oocytes using the Well of the Well method. In Experiment 2, and intracytoplasmic sperm injection (ICSI) was applied to matured oocytes from prepubertal and adult common minke whales after IVM culture (40 or 48 h), and then their embryonic development was assessed. In Experiment 1, the maturation rate of oocytes cultured for 40 h (30.4%) was significantly higher than that of oocytes cultured for 30 h (6.8%; P<0.01). In Experiment 2, a total of 35 and 46 immature oocytes derived from adult (n=2) and prepubertal (n=6) minke whales, respectively, were cultured for 40 or 48 h. The maturation rate in the oocytes from the adult whales (34.2%) tended to be higher than that of the oocytes from the prepubertal whales (19.6%), but there was no significant difference. Following ICSI, 3 out of the 10 inseminated and cultured oocytes from the adult whales cleaved (2-, 8-, and 16-cell stages); all of these oocytes had been matured for 40 in culture. However, these oocytes did not develop to further stages. Only one of the 6 oocytes derived from the prepubertal whales, IVM cultured for 40 h and inseminated, developed to the 4-cell stage. The present results indicate that a 40 h IVM culture produces significantly higher rates of in vitro maturation than a 30 h IVM culture for common minke whale oocytes. Following ICSI, some oocytes cleaved to the 16-cell stage, but no further development was observed.     

In vitro culture conditions using chemically defined media for in vitro matured and intracytoplasmically inseminated porcine oocytes

The present study investigated in vitro culture methods [droplet and Well of the Well (WOW)] using semi-defined and defined media [modified porcine zygote medium (mPZM)] and the additional effects of insulin on in vitro matured and intracytoplasmically inseminated porcine oocytes. In Experiment 1, in vitro matured and intracytoplasmically inseminated porcine oocytes were cultured for 6 days in the following four groups: 1) mPZM-3 (containing bovine serum albumin) + droplet (30 mul), 2) mPZM-3 + WOW, 3) mPZM-4 (containing polyvinyl alcohol) + droplet, and 4) mPZM-4+ WOW. The culture media (mPZM-3 and mPZM-4) and methods (droplet and WOW) did not significantly affect the cleavage rate, but the blastocyst rate of the oocytes cultured in mPZM-3 was significantly (P<0.01) higher than that of mPZM-4 (20.1 and 9.4%, respectively). The blastocyst rates as percentages of the cleaved oocytes (51.8 and 16.9%) and the hatched blastocyst rate as percentages of the number of blastocysts (12.3 and 2.2%) were also significantly (P<0.01) higher in mPZM-3 compared with those in mPZM-4. There was significant interaction (P<0.05) between the two main factors; the effects of the culture media and methods on the rate of hatched blasyocysts as percentages of the blastocysts produced and, the hatched blastocyst rate (20.3%) as percentages of the number of blastocysts produced in mPZM-3 were significantly (P<0.05) higher than in the other groups. In Experiment 2, the additional effects of insulin (100 ng/ml) in mPZM-3 and mPZM-4 media was investigated in the WOW culture system. Insulin addition did not improve cleavage, blastocyst formation, or the number of cells in blastocysts. However, as in Experiment 1, mPZM-3 resulted in a significantly higher blastocyst rate as percentages of the cleaved oocytes than mPZM-4 (33.9 and 18.4%). These results indicate that a chemically defined medium (mPZM-4) needs to be improved to provide more suitable culture conditions for in vitro development of in vitro matured and intracytoplasmically inseminated porcine oocytes. However, the WOW system may be a useful IVC method for blastocyst development of in vitro matured porcine oocytes following ICSI when a semi-defined medium (mPZM-3) is used.     

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 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.     

Effect of different mechanical isolation techniques on developmental competence and survival of buffalo ovarian preantral follicles

The present study aimed to analyze different methods of mechanical isolation of buffalo preantral follicles (Experiment I), in vitro culture of isolated follicles in different groups of culture medium over collagen gel matrix (Experiment II) and subsequent in vitro development and survival of isolated preantral follicles (PFs) (Experiment III). In Experiment I, ovarian cortical pieces were separated and PFs isolated by different mechanical methods. In Experiment II, isolated follicles were divided into three groups and cultured in TCM-199 having 10% FBS, 1% ITS, and 20 ng/ml EGF (Group A, control), addition of 0.5 μg/ml FSH (Group B) or FSH + 100 ng/ml IGF-I (Group C). Follicles were incubated at 38.5 °C in 5% CO₂ with maximum humidity. In Experiment III, based on the outcome of Experiment I and II, PFs were cultured from those isolation method and treatment group, showing better growth and developmental pattern to analyze the impact of growth factors on in vitro growth of follicles in long term culture. It was found that micro-dissected PFs showed higher survival rate and growth after 15 days of culture compared to PFs isolated by other methods. Follicles cultured with FSH + IGF-1 on collagen gel matrix, showed significantly (P < 0.05) higher survival rate and mean diameter of follicles on day 15 of culture compared to control. In summary, it has been shown that isolation of follicles by micro-dissection has advantages over other methods, being relatively simple, inexpensive and less harmful to follicles. Micro-dissected buffalo PFs maintained the architecture, showed antrum formation in presence of FSH and IGF-I over the collagen gel matrix.     

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.     

Changes in the amount of proteins, glycogen and lipids in porcine oocytes during in vitro meiotic maturation

Changes in the cytoplasmic inclusions during meiotic maturation were histochemically examined in cultured porcine oocytes. The oocytes contained a small amount of protein and glycogen granules throughout the maturation culture, as well as Sudanophilic lipids composed of small, medium and large droplets. Soon after collection, the amount of Sudanophilic lipid droplets of small and medium size was small and there were 167 ± 11.2 large droplets. After being cultured for 22 h, the number of large lipid droplets decreased remarkably, while the number of small and medium ones increased. There were no differences in the number of Sudanophilic lipid droplets of different sizes between ovulated oocytes and the oocytes cultured for 44 h. The oocytes always contained a large amount of neutral fats and lipoids, but not cholesterols. In the oocytes cultured for 22 h with olomoucine, both the resumption of nuclear maturation and the decrease in the size of the Sudanophilic lipid droplets were inhibited. From the present findings, it appears that the change in the size of the Sudanophilic lipid droplets in the cytoplasm of porcine oocytes is closely related to nuclear maturation.     

Collection and in Vitro Maturation of Equine Oocytes from Estrus,Diestrus and Pregnant Mares

Two experiments were conducted to evaluate oocyte collection rates and in vitro nuclear maturation rates of equine oocytes obtained during diestrus and pregnancy, and to compare these rates with maturation rates in oocytes derived from preovulatory follicles. In Experiment I, transvaginal ultrasound-guided aspiration of follicle was performed during estrus and diestrus in 14 mares over four consecutive cycles. Follicular aspirations during estrus were performed 24 to 27 hours after injection of 2500 IU of hCG given when the largest follicle reached 35 mm in diameter. Oocyte recovery rate from preovulatory follicles was 51% (33/65) in 49 aspiration sessions. Cumulus-oocyte complexes from preovulatory follicles were cultured for 12-15 hours in TCM199 + 10% (NCS) at 38.5°C in 5% CO₂ in air, and 22/33 (67%) were in metaphase II. During diestrus, mares were treated (Group I) or not treated (Group II) daily with equine pituitary extract (EPE) during alternate cycles from days 1 to 14 after the preovulatory aspiration. Diestrous follicles were aspirated when four or more follicles greater than 12 mm in diameter were present. EPE had no effect on the number of follicles that developed during estrus or diestrus (p>0.05). Oocytes were recovered from 119 of 383 diestrous follicles (31 %)in 75 sessions. There was no difference in recovery rates between Groups I and II (p>0.05). Maturation rates for oocytes collected during diestrus, after 42 hours of culture in TCM 199 + 1 μg/ml of Estradiol and lnl/ml of EPE, were not significantly different (p>0.05) between Groups I and II (49% vs. 53%). In Experiment II, mares between 50 and 85 days of pregnancy were used as oocyte donors. The oocyte recovery rate was 53% (66/125). After in vitro maturation for 40 hours (compact COC) or 15 hours (expanded COC), 22% (7/32) and 22% (7/32), respectively, of the oocytes were in metaphase II. It was concluded that: 1) Preovulatory follicles yield a higher percentage of oocytes with a higher rate of maturation to metaphase II than follicles of diestrus and pregnant mares. 2) Diestrous follicles yielded fewer oocytes than follicles of pregnant mares but with a higher percentage of oocyte maturation. Further studies are necessary to determine if oocytes recovered from diestrous follicles and matured in vitro can be fertilized successfully.     

不同收集方法及添加半胱氨酸、肝素钠对黄牛卵母细胞体外成熟及发育的影响

试验旨在研究不同卵母细胞收集方法及添加半胱氨酸、肝素钠对黄牛卵母细胞体外成熟及体外受精的影响。以黄牛为研究对象,采用两种方法（抽卵法和割卵法）抽取卵泡中的卵母细胞,比较两种方法获取的卵母细胞成熟率,结果发现,抽卵法获得的卵母细胞成熟率显著高于割卵法（P<0.05）。将获取的卵母细胞分为4组：A组（对照组,只添加基础成熟培养液）、B组（基础成熟培养液+200 μmol/L半胱氨酸）、C组（基础成熟培养液+20 μg/mL肝素钠）、D组（基础成熟培养液+200 μmol/L半胱氨酸+20 μg/mL肝素钠）,结果发现,D组的卵母细胞成熟率显著高于A、B、C组（P<0.05）,B、C组间卵母细胞成熟率无显著差异（P>0.05）,但两组卵母细胞成熟率均显著高于A组（P<0.05）;A组卵母细胞卵裂率均显著低于B、C、D组（P<0.05）,B、C、D组间卵母细胞卵裂率无显著差异（P>0.05）;D组囊胚率显著高于其他3组（P<0.05）。结果表明,抽卵法获得卵母细胞效率显著高于割卵法,肝素钠及半胱氨酸对黄牛卵母细胞体外成熟和体外受精都有促进作用,且同时添加两种物质对体外成熟的效果更佳。     

Acceleration of follicular development by administration of vascular endothelial growth factor in cycling female rats

To address the role of follicular angiogenesis in the determination of ovulatory follicles and the effects of different vascular endothelial growth factor (VEGF) isoforms on follicular angiogenesis and development, mature female rats were treated with an angiogenic inhibitor (TNP-470), and also with VEGF 120 or 164 at different dosages (0.4, 0.8, 4.0 or 8.0 microg/kg body weight) for 3 days during the estrous cycle. Ovarian follicular angiogenesis, the population of large follicles and ovulation were examined. VEGF 120 (0.8 microg/kg) and 164 (8.0 microg/kg) treatments stimulated follicular angiogenesis in the theca interna layer, while TNP-470 treatment showed severe depression of follicular angiogenesis, and completely inhibited ovulation. After administration of VEGF 120 or 164, the number of healthy preovulatory follicles and ovulated oocytes increased significantly, concomitantly with a decrease in the number of atretic preovulatory follicles. The oocytes ovulated had normal fertilizability and developed to term with the same litter size as in the control rats. Our findings suggest that follicular angiogenesis may be a determinant of follicular development during the periovulatory phase, and that VEGF isoforms may play different important roles in regulating follicular angiogenesis.     

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.     

Effects of Follicular Fluid on the Transport of Porcine Oocytes into the Oviduct at Ovulation

Contents Three experiments were conducted to determine whether follicular fluid (FF) enters the oviduct and plays any role in the transport of oocytes into the oviduct. Experiment 1: Oestrus and ovulation were synchronized in cycling gilts (n = 21) over a 15 day period of feeding Regumate^® and injections of 1000 IU pregnant mare’s serum gonadotrophin (PMSG) 24 h after the last Regumate^® feed and 500 IU human chorionic gonadotrophin (hCG) 80 h after PMSG. Ipsi-lateral aspiration of FF and salpingectomy (group 1, n = 7), aspiration of FF without salpingectomy (group 2, n = 7) or ligation of the oviduct between the ampulla and infundibulum (group 3, n = 7) was performed endoscopically prior to ovulation (34–36 h after hCG). Ipsi-lateral (group 2 and 3) and contra-lateral salpingectomy was carried out in all gilts post ovulation, 42–44 h after hCG. The oviducts were flushed with 1 ml saline and the samples as well as the aspirated FF were analysed for progesterone and estradiol by RIA methods. In group 1 both progesterone and estradiol concentrations did not differ before and after ovulation. Withdrawal of FF from the ipsi-lateral ovary by aspiration (group 2) or ligation of the oviduct (group 3) did not influence the steroid content within the oviducts. Similarly low progesterone concentrations were measured in ipsi- and contra-lateral oviducts after ovulation (group 2: 0.29 ± 0.17 versus 0.24 ± 0.35 ng/ml and group 3: 0.22 ± 0.19 versus 0.21 ± 0.22 ng/ml). The high content of progesterone of FF (269.7 ± 67.9 and 389.6 ± 226.5 ng/ml in group 1 and 2, respectively) was not reflected in the oviductal fluid. Experiment 2: In five gilts 0.06 ml ³H-progesterone (30 000 dpm) were applied via a fine 27 G injection needle into the largest three follicles of the ipsi-lateral ovary prior to ovulation (34–36 h after hCG). The oviducts were flushed following ovario-salpingectomy 42–44 h after hCG. All follicles had ovulated. The oviductal flushings and oviductal and ovarian tissue were analysed for labelled progesterone. No differences were measured in the content of ³H-progesterone of oviductal flushings and of both oviductal and ovarian tissues between the ipsi-lateral injection and contra-lateral control sides. The main part of the counts detected was within the range of background dpm values. Only 2.4% of the initial counts were recovered from fluid and tissue samples. Experiment 3: In a subsequent study FF was cautiously aspirated by endoscopy from follicles of the ipsi-lateral ovary 34–36 h after hCG (n = 12 gilts). Postovulatory (58 h after hCG), both oviducts were flushed and the oocytes were recovered. To test the influence of follicle puncture alone on the process of ovulation (n = 8 gilts), the aspiration needle alone was pricked into the follicles of the ipsi-lateral ovary, without any fluid aspiration. Despite the cautious aspiration of FF from 89 follicles, 26 oocytes were recovered together with the FF. Eighty-six postovulatory follicles were observed on the ipsi-lateral ovary. Out of 57 oocytes able to reach the oviduct, 29 oocytes were flushed from the oviduct (50.4 ± 28.1%). From the contra-lateral control oviduct 71 oocytes out of 91 ovulations (69.0 ± 33.9%) were recaptured. Puncture of follicles without aspiration did not influence ovulation compared with the control (recovery rate 68.2 and 79.6%, respectively). Results indicate (1) on the basis of the low progesterone level within the oviductal fluid that only a small amount of FF seems to reach the oviduct at ovulation, and (2) FF does not appear to be a compulsory carrier of the porcine oocyte at ovulation.