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1.
Studies were conducted to examine the effects of the cyclicity and the presence of a dominant follicle (DF) in ovary on the recovery and in vitro growth of pre-antral follicles (PFs) in sheep and buffalo. Small pre-antral follicles (SPFs, 100–250 μm) and large pre-antral follicles (LPFs, 250–450 μm) were isolated from slaughterhouse ovaries in the breeding seasons by a mechanical and enzymatic method. The sheep and buffalo PFs were cultured in vitro for 6 and 15 days, respectively, and examined for their growth, survival and antrum formation rates and growth rates of oocytes in cultured pre-antral follicles. The follicles of the sheep and buffalo were recovered and cultured simultaneously within replicates. The recovery rates (number per ovary) of both SPFs and LPFs were significantly (p < 0.05) higher in cyclic ewes (SPFs: 22.0 ± 3.3 vs 12.1 ± 2.6 and LPFs: 16.0 ± 3.6 vs 9.2 ± 1.8) and buffaloes (SPFs: 9.2 ± 1.3 vs 4.1 ± 1.0 and LPFs: 10.3 ± 2.7 vs 5.4 ± 0.7) compared with those recovered from acyclic ones. Presence of a DF in ovary significantly (p < 0.05) reduced the recovery rates of LPFs in ewes (9.06 ± 2.7 vs 16.4 ± 3.8) but had no effect in buffalo. Cyclicity of animals or follicular dominance had no effects on in vitro growth, survival and antrum formation rates and growth rates of oocytes in cultured PFs of SPFs and LPFs in both sheep and buffalo. The in vitro growth, survival and antrum formation rates of LPFs and growth rates of oocytes in cultured LPFs were significantly (p < 0.05) higher than those observed in SPFs in both sheep and buffalo. The overall recovery and growth rates of the PFs were lower in buffaloes compared with ewes.  相似文献   

2.
This study was designed to describe, both quantitatively (morphometry) and qualitatively (histological differentiation), follicle and oocyte growth in the feline ovary. The ovaries of 43 cats were collected and processed for histology. The diameters of 832 follicle/oocyte pairs were measured, with and without zona pellucida (ZP), and a special emphasis was placed on the study of early folliculogenesis. Primordial, primary, secondary, pre-antral and early antral follicles were measured at 44.3, 86.2, 126.0, 155.6 and 223.8 μm in diameter respectively. A biphasic pattern of follicle and oocyte growth was observed. Before antrum formation, follicle ( x ) and oocyte ( y ) size were positively and linearly correlated ( y  = 0.500 x  + 20.01, r 2 = 0.89). Antrum formation occurred when the follicle reached 160–200 μm in diameter (when oocyte was at 102 μm). After antrum formation, a decoupling was observed, a minimal increase in oocyte size contrasting with a significant follicle development ( y  = 0.001 x  + 114.39, r 2 = 0.01). The pre-ovulatory follicle diameter was approximately 3500 μm and the maximal oocyte diameter was 115 μm. The ZP, absent in primordial and primary follicles, appeared at the secondary stage and reached almost 6 μm at the pre-ovulatory stage. These results suggest that (i) in feline ovary, follicle and oocyte growth pattern is similar to that observed in other mammals; (ii) the antrum forms in 160–200 μm follicles, which represents 5% of the pre-ovulatory diameter and (iii) the oocyte had achieved more than 90% of its maximal growth at the stage of antrum formation.  相似文献   

3.
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.  相似文献   

4.
Vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF‐2) play a paramount role in the regulation of normal and pathologic angiogenesis in the ovary of mammals. Very little is known on the expression of these two growth factors in the avian ovary. The aim of this study was to determine for the first time the localization of VEGF and FGF‐2 in the ovary of the ostrich using immunohistochemical techniques to investigate the vascularization of the rapidly growing huge ostrich oocyte. At the oocyte periphery, distinct VEGF‐positive granules are visible. In our opinion, the expression of VEGF in the growing oocytes, which does not occur in mammals such as bovines, does not significantly contribute to angiogenesis in the theca interna and externa, where all the original and developing vessels are located, but may contribute to the mitoses and survival of granulosa cells during folliculogenesis. A different immunostaining can be demonstrated for FGF‐2: from late pre‐vitellogenic follicles, FGF‐2 immunopositivity can be observed at the inner perivitelline layer area. In the stroma, the smooth muscle cells of small arteries and the endothelial cells of venules and veins are positively stained for FGF‐2. Another interesting finding of this study is the occurrence of a significant number of VEGF‐ and FGF‐2 positive heterophilic granulocytes within the ovarian stroma, which migrate from the periphery of the ovary towards the growing follicles. We assume that the growth factors of the heterophilic granulocytes contribute significantly to the angiogenesis seen in both theca layers.  相似文献   

5.
Effect of sodium nitroprusside (SNP), a nitric oxide (NO) donor, on in vitro survival, growth, steroidogenesis, and apoptosis of buffalo preantral follicles (PFs) was investigated. PFs (200~250 µm) were isolated by micro-dissection and cultured in 0 (control), 10-3, 10-5, 10-7, and 10-9 M SNP. To examine the reversible effect of SNP, PFs were cultured with 10-5 M SNP + 1 mM Nω-nitro-L-arginine methyl ester (L-NAME) or 1.0 µg hemoglobin (Hb). The results showed that greater concentrations of SNP (10-3, 10-5, 10-7 M) inhibited (p < 0.05) FSH-induced survival, growth, antrum formation, estradiol production, and oocyte apoptosis in a dose-dependent manner. However, a lower dose of SNP (10-9 M) significantly stimulated (p < 0.05) the survival, growth, antrum formation, follicular oocyte maturation, and stimulated progesterone secretion compared to the control. A combination of SNP + L-NAME promoted the inhibitor effect of SNP while a SNP + Hb combination reversed this effect. Nitrate and nitrite concentrations in the culture medium increased (p < 0.05) in a dose-dependent manner according to SNP concentration in the culture medium. At higher concentrations, SNP had a cytotoxic effect leading to follicular oocyte apoptosis whereas lower concentrations have stimulatory effects. In conclusion, NO exerts a dual effect on its development of buffalo PFs depending on the concentration in the culture medium.  相似文献   

6.
The effect of co‐culture of buffalo preantral follicles (PFs) with different somatic cells, i.e, cumulus, granulosa, ovarian mesenchymal and oviductal epithelial cells was studied. Large PFs (250–450 μm) were isolated by microdissecting the trypsin (1%) digested ovarian cortical slices. Cumulus cells were isolated by repeated pipetting of oocytes, granulosa cells were isolated by aspirating from punctured PFs and ovarian mesenchymal cells were isolated from ovarian cortex by scraping the cortical slices and passing through 20 μm filter. Preantral follicles were cultured in standard culture medium without somatic cells or co‐cultured with cumulus cells, granulosa cells, ovarian mesenchymal cells and oviductal epithelial cells for 80 days. The growth rate (μm/day) of the PFs was monitored by measuring follicular diameter on day 0, 30, 60 and 80 days of culture. The viability of PFs was evaluated by trypan blue staining. The results indicated that PFs co‐cultured with cumulus, granulosa and ovarian mesenchymal cells had a better development and survivality compared with control and those co‐culture with oviductal epithelial cells. Maximum growth and survivality of PFs were achieved when cultured with cumulus cells. It is concluded that inclusion of somatic cells in PF culture media had beneficial effect on the growth of PFs and cumulus cells supported maximum growth and survivality of PFs in vitro of all somatic cells tested.  相似文献   

7.
The aim of this study was to identify the occurrence of polyovular follicles in porcine ovaries. We investigated the presence of such follicles in relation to age, and compared the intrafollicular concentrations of steroid hormones between poly- and uniovular follicles. Then we measured the size, viability and the in vitro fertilizing ability of the oocytes from polyovular follicles. Histological examinations documented the occurrence of polyovular follicles in pigs at various stages of follicular growth. Within antral follicles, the number of polyovular follicles was higher in the ovaries of gilts than in sows ( P  < 0.01). We noticed differences in the viability and size of oocytes recovered from the same follicles. We noted a higher concentration of oestradiol-17β and a lower concentration of progesterone in polyovular follicles as compared with uniovular follicles ( P  < 0.01). The amount of embryos after in vitro -fertilization of oocytes from polyovular follicles was significantly lower than that from uniovular ones. Nevertheless, we found that some oocytes from polyovular follicles also have the capacity to be fertilized in vitro and be developed to the blastocyst stage.  相似文献   

8.
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9.
The aim of this research was to characterize the preantral ovarian follicular population in collared peccaries (Tayassu tajacu) using light and electron microscopy. Ovaries from six mature females were collected and further fixed for histological and ultrastructural analysis. A total of 33273.45 ± 5789.99 preantral follicles (PFs) were estimated for the population in each ovary. Most preantral follicles were primordial (91.56%), followed by primary (6.29%) and secondary (2.15%) ones. Most PFs were morphologically normal (94.4%), and only a few were atretic (5.6%). At histology assessment, amounts of lipid droplets were observed into the oocyte cytoplasm, which was confirmed through ultrastructural analysis. This work characterizes for the first time the ovarian population of preantral follicles, total and per category, in collared peccaries (Tayassu tajacu). The general follicles featured at primordial, primary and secondary categories are very similar to those described for other species.  相似文献   

10.
The development of efficient ovarian preantral follicle (PF) isolation and culture systems provide a large number of oocytes for the manipulation and embryo production. It also helps for understanding the mechanisms of follicle and oocyte development. Isolation and culture protocols for PFs were developed for many domestic species like cattle, buffalo, sheep, goat, pig, horse, camel, dog and cats; however, embryo production from oocytes derived from in vitro grown PFs was reported only in pigs, buffalo, sheep and goat. The rate of oocyte maturation from PFs grown in vitro is low and requires considerable research. This paper presents an overview of isolation and culture systems of PFs that have been developed for domestic species (cattle, buffalo, sheep, goat, pigs, horse, camel, dog and cat) along with the current status of progress achieved in the direction of producing embryos using PFs as the source of oocyte in these species.  相似文献   

11.
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.  相似文献   

12.
The main objectives of the present study were to determine the ultrastructural modifications occurring in the oocyte during late folliculogenesis and to estimate pre-antral follicle population in buffalo. Half the collected ovaries were fixed and prepared for optic microscopy; the antral follicles from the other ovaries were measured and individually punctured. The cumulus–oocyte complexes (COCs) were processed for transmission electron microscopy. The number of pre-antral follicles in buffalo ovaries was estimated at 19 819 structures. Cumulus–oocyte complexes derived from 1-mm antral follicle had an eccentrical nucleus and compact corona radiata , ooplasm vilosities were fully embedded in zona pellucida (ZP) and a well-defined junction could be observed. Mitochondria were predominantly round and well distributed in ooplasm, as were small lipid vacuoles. In COCs derived from 2-mm antral follicles, the initial formation of perivitelline space was observed. The nucleus was peripherally located and the number of pleomorphic mitochondria increased. Cortical granules were clustered at oocyte periphery and lipid vacuoles increased in number and size. In COCs derived from 6-mm antral follicles, the organelles were located mainly in the perinuclear region. Golgi complexes and smooth endoplasmic reticulum (SER) were more developed. Mitochondria migrated to the cortical region and lipid vacuoles migrated to the medullar region. In COCs derived from 10-mm antral follicles, the lipid vacuoles coalesced and occupied the medullar region of the oocyte, together with a well-developed SER. Mitochondria were pleomorphic and located at the oocyte periphery. In conclusion, the morphological differences described in this paper could be responsible for some functional differences observed in in vitro embryo production and follicular dynamics for buffalo, when compared with cattle.  相似文献   

13.
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.  相似文献   

14.
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15.
Contents
This study was conducted to characterize the degree of proliferation and atresia in mid-luteal and follicular phase bovine follicles of different size containing morphologically good quality cumulus oocyte complexes. Follicular cell proliferation was estimated by enzyme immunoassay for tissue polypeptide specific antigen and atresia by an enzyme immunoassay for apoptosis. The relationship between follicular apoptosis and proliferation was inverse in the mid-luteal stage ovaries, whereas in follicular stage ovaries the tissue polypeptide specific antigen was increased in follicles with the highest degree of apoptosis. Follicular stage follicles showed a higher degree of apoptosis in comparison with the mid-luteal stage. This was caused by an increased apoptosis in follicles with less than 3 mm and greater than 5 mm diameter. In conclusion, the data indicate a sophisticated interplay between follicular growth and atresia in dependence of oestrous stage and follicular size.  相似文献   

16.
Polyphenols (PFs) extracted from green tea, known to be potent anti‐oxidants, have been reported to be effective in increasing the motility and viability of mammalian sperm, preserved in a liquid form. Therefore, we tested whether PFs might also be effective for maintaining the integrity of frozen‐thawed boar spermatozoa. Ejaculates, collected from Clawn miniature pigs, were diluted in a semen extender containing various amounts of PFs (0, 0.01, 0.05, 0.1 and 0.2% w/v) and then stored at 15°C overnight. The semen samples were processed, using the straw freezing procedure, and then frozen in liquid nitrogen. After rapid thawing at 40°C, the spermatozoa were subjected to several assays to evaluate semen quality. Spermatozoa frozen in a medium containing 0.01% w/v PFs exhibited significantly (P < 0.05) higher degrees of post‐thawed viability and acrosomal integrity than those stored in the absence of PFs. However, no change in the mitochondrial activity was noted between the two groups. The inclusion of 0.01% PFs in the semen extender was significantly (P < 0.05) effective in increasing both the rates of monospermic oocyte formation and of blastocyst formation. These findings indicate that preincubation with the semen extender, containing 0.01% PFs prior to freezing, exerts a protective effect on boar sperm by preventing injuries associated with freezing‐thawing.  相似文献   

17.
The aim of the present study was to investigate the effects of fibroblast growth factor-10 (FGF-10) on the survival, activation (transition from primordial to primary follicles), and growth of goat preantral follicles cultured in vitro. Pieces of ovarian cortex were cultured for 1 and 7 d in the absence or presence of FGF-10 (0, 1, 10, 50, 100, and 200 ng/mL). Noncultured and cultured tissues were processed and analyzed by histology, transmission electron microscopy, and viability testing. Results showed that after 7 d, a greater percentage (79.9%) of morphologically normal follicles (containing an oocyte with regular shape and uniform cytoplasm, and organized layers of granulosa cells without a pyknotic nucleus) was observed when cultured with 50 ng/mL of FGF-10 when compared with other concentrations of FGF-10 (0 ng/mL, 67.3%; 1 ng/mL, 68.2%; 10 ng/mL, 63.3%; 100 ng/mL, 64.4%; 200 ng/mL, 52.7%). Ultrastructural analyses and viability testing using fluorescent markers confirmed the follicular integrity of FGF-10 (50 ng/mL)-treated fragments after 7 d of culture. After 7 d, all FGF-10 concentrations reduced the percentage of primordial follicles and increased the percentage of developing follicles. In the presence of 50 ng/mL of FGF-10, follicles increased in diameter after 7 d of culture when compared with other concentrations tested. In conclusion, this study demonstrates that FGF-10 maintains the morphological integrity of goat preantral follicles and stimulates the growth of activated follicles in culture. The culture conditions identified here contribute to the understanding of the factors involved in goat early follicular development.  相似文献   

18.
In this study, the expressions of VEGF in dog follicles were detected by immunohistochemistry and the effects of VEGF treatment on the primordial to primary follicle transition and on subsequent follicle progression were examined using a dog ovary organ culture system. The frozen‐thawed canine ovarian follicles within slices of ovarian cortical tissue were cultured for 7 and 14 days in presence or absence of VEGF. After culture, the ovaries were fixed, sectioned, stained and counted for morphologic analysis. The results showed that VEGF was expressed in the theca cells of antral follicles and in the granulosa cells nearest the oocyte in preantral follicle but not in granulosa cells of primordial and primary follicles; however, the VEGF protein was expressed in CL. After in vitro culture, VEGF caused a decrease in the number of primordial follicles and concomitant increase in the number of primary follicles that showed growth initiation and reached the secondary and preantral stages of development after 7 and 14 days. Follicular viability was also improved in the presence of VEGF after 7 and 14 days in culture. In conclusion, treatment with VEGF was found to promote the activation of primordial follicle development that could provide an alternative approach to stimulate early follicle development in dogs.  相似文献   

19.
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.  相似文献   

20.
Experiments were designed to investigate the size distribution of queen steroidogenic luteal cells throughout pseudopregnancy. Corpora lutea were obtained from the queens following ovariohysterectomy on days 7, 15 or 25 of pseudopregnancy. Luteal cells were isolated from the ovary by collagenase digestion. Steriodogenic cells were identified by staining of cells for 3β-HSD activity. Cell diameters were measured using a microscope. Luteal cells having steroidogenic capacity covered a wide spectrum of sizes ranging from 3 to 35 μm in diameter. There was a significant increase in mean cell diameters (p < 0.01) as pseudopregnancy progressed. Mean diameter of 3β-HSD positive cells increased from 10.41 ± 0.7 μm, on day 7 of pseudopregnancy, to 19.72 ± 1.3 μm on day 25 of pseudopregnancy. The ratio of large (>20 μm in diameter) to small (3–20 μm in diameter) luteal cells was 0.08 : 1.0 on day 7 of pseudopregnancy, with the 7.5–10 μm cell size class predominant. By day 25 of pseudopregnancy, the ratio of large-to-small cells was increased to 0.87 : 1.0, and 20–25 μm cell sizes become predominant. In conclusion, this study has demonstrated that the cells of the corpus luteum undergo continuous differentiation during pseudopregnancy in queen. This study also demonstrates that luteal cells dissociated from pseudopregnant queen can be used as a model to study the physiology of corpus luteum in pregnant cats.  相似文献   

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