Role of media, protein and energy supplements on maintenance of morphology and DNA-synthesis of small preantral domestic cat follicles during short-term culture

Small preantral follicles (40 to 90 microns in diameter) from domestic cats were cultured for 10 d using different media (M199 and Dulbecco's MEM) and protein (FCS and BSA) supplements. Culture efficacy was determined by Hoechst 33258 staining and estimation of Brom-desoxyuridine (BrdU)-incorporation into oocytes and granulosa cells. Culture in M199 + FCS and in DMEM + FCS resulted in 21.6% and 38.1%, respectively, of morphologically intact preantral follicles. Adding BSA increased the rate of normal follicles to 51.7% in M199 and to 58.6% in DMEM. Oocytes were found in 40% of the follicles, when DMEM and/or BSA supplementation was used, while M199 with FCS induced acute loss of oocytes in 85% of the follicles. About 10% of the oocytes contained degenerating chromatin. Measurement of BrdU-incorporation during culture allows for quick and effective assessment of follicle viability in vitro. Comparison of M199 and Dulbecco's MEM, both with FCS or BSA and DMEM with or without pyruvate/lactate, indicated that Dulbecco's MEM + BSA without pyruvate and lactate is the best medium for culture of cat follicles. However, further research of suitable medium supplements is needed.     

In vitro culture of bovine preantral follicles

Bovine preantral follicles (40-100 microm diameter at collection) were collected from ovaries of slaughtered cows and cultured in vitro with one of the four treatments: follicle stimulating hormone (FSH; 100 ng/ml) alone; FSH plus epidermal growth factor (EGF; 100 ng/ml); FSH plus insulin-transferrin-selenium (ITS; +1%) or FSH plus hypoxanthine (4 mM) in tissue culture medium (TCM 199) supplemented with 10% fetal calf serum (FCS), 0.1 mg/ml sodium pyruvate, 100 IU/ml of penicillin and 100 microg/ml streptomycin. The control culture medium was TCM 199 with supplements without any treatments. Follicles of each size were cultured separately in groups of one to three in 24-well multidishes each containing 500 microl of the appropriate culture medium. Culture commenced at follicle recovery (day 1) and continued for 10 days (harvested on day 11). In each case, half the medium was removed and replaced by fresh medium every third day. Follicle diameters were recorded on days 1, 5 and 11 of the experiment. At the end of the 10-day culture period, half of the follicles were stained with trypan blue to assess their potential viability and half were stained with bisbenzimide plus propidium iodine to estimate various morphological features of the follicles. Follicles of all initial sizes, on all culture treatments, increased in diameter during in vitro cultures with the greatest increases, both in absolute and proportional size, occurring between days 1 and 5 of culture. All of the culture medium supplements caused greater increases in follicle diameters than control medium at both days 5 and 11 of culture for all initial sizes of follicles (p<0.01). The most effective culture supplements for follicles of 40-, 60- and 80-microm initial diameter were FSH alone and FSH+EGF. The size of these follicles at both days 5 and 11 of culture on both the treatments was significantly larger (p<0.01) than follicles cultured in the presence of the other two supplementary treatments. The growth of follicles of 100-microm initial diameter did not differ between culture medium supplements. None of the culture media caused follicle size to increase to the initial diameters of the next larger size category during the 10 days of culture although follicles of 100-microm diameter achieved a diameter of 120 microm, after 4 days of culture.The overall follicular viability and morphology were better with treatments than the controls in all cases; however, there was no significant difference (p>0.05) among them.From this experiment, FSH and FSH plus EGF may be recommended for in vitro culture of smaller (40, 60 and 80 microm) follicles.     

Effects of culture medium,serum type,and various concentrations of follicle-stimulating hormone on porcine preantral follicular development and antrum formation in vitro

Developing a culture system for preantral follicles has important biotechnological implications due to the potential to produce large number of oocytes for embryo production and transfer. As an initial step toward accomplishing this long-term goal, a study was conducted to determine the effects of culture medium, serum type, and different concentrations of FSH on preantral follicular development in vitro. Specific endpoints included follicular growth rate, antrum formation, recovery rate of cumulus cell-oocyte complexes (COCs) from follicles, and oocyte meiotic competence. Compared with the North Carolina State University medium 23 (NCSU23), preantral follicles cultured in TCM199 medium for 4 days grew faster (P < 0.02). However, more follicles cultured in NCSU23 differentiated to form an antrum than in TCM199 (P < 0.01). For this reason, NCSU23 was chosen to investigate the role of FSH and serum type in regulating preantral follicular growth. Compared with the 0 mIU/ml FSH control, addition of 2 mIU/ml FSH to the medium stimulated follicular growth and antrum formation and suppressed apoptosis of granulosa cells (P < 0.05), supporting the essential role of FSH in preantral follicular growth and development. Another experiment compared fetal calf serum (FCS) with prepubertal gilt serum (PGS) and studied different concentrations of FSH in the culture medium (0.5, 1, and 2 mIU/ml). The best follicular growth rate was obtained with 2 mIU/ml compared with 0.5 or 1 mIU/ml FSH. Compared with PGS, FCS supplementation increased the cumulative percentage of antral follicles and COC recovery rate (P < 0.04). None of the oocytes recovered from any of these experiments reached metaphase II stage after maturation in vitro. In summary, culture medium, serum type, and FSH concentration in the medium interacted to affect follicular growth and antrum formation in vitro. These results suggest that a longer term culture of preantral follicles (>4 days) may be needed to produce oocytes capable of undergoing meiosis in vitro.     

Effect of follicle size and quality on the ability of follicular fluid to support cytoplasmic maturation of bovine oocytes

Recent studies have shown that the developmental capacity of in vitro-matured oocytes is affected by the origin of follicular fluid (FF) supplemented to the maturation medium. The aims of this study were (1) to determine if follicle size and quality would influence the capacity of FF to support bovine oocyte maturation and (2) to determine if fetal calf serum (FCS) and FF had an additive effect when added together to the maturation medium. Follicular fluid collected from 108 follicles was classified according to size (<6, 6–8, >8 mm in diameter) and quality (healthy, early atretic, and atretic). Quality, first determined by mitosis/pycnosis ratios in granulosa cell smears, was subsequently confirmed by insulin-like growth factor binding protein (IGFBP) patterns and estradiol concentrations. While most small- or medium-sized follicles showed some atresia (88% and 67%, respectively), fewer of the large follicles were atretic (30%). In experiment 1 bovine oocytes (n = 2,152) were matured either in TCM199 alone, with 10% FCS, or with 10% FF from the following follicle types: small healthy (SH); small early atretic (SEA); small atretic (SA); medium healthy (MH); medium early atretic (MEA); medium atretic (MA); large healthy (LH); large early atretic (LEA); and large atretic (LA). Following IVM, oocytes were fertilized and subsequently cultured in synthetic oviduct fluid (SOF). Day 8 blastocyst yields were 23% in TCM199 alone; 37% in TCM199 plus FCS; and, in medium supplemented with FF, SH, 36%; MH, 32%; LH, 30%; SEA, 21%; MEA, 26%; LEA, 28%; SA, 32%; MA, 33%; and LA, 38%. All FF from healthy or atretic follicles resulted in significantly improved blastocyst yields compared to M199 alone (P < 0.05) However, FF from early atretic follicles irrespective of size did not yield a significant improvement. In experiment 2 we examined the effect of addition of FF-LH and serum together to the maturation medium. In terms of blastocyst yield, no additional benefit was observed when TCM199 was supplemented with 10% FCS + 10% FF (33%) compared to 10% FCS or FF alone (35% and 30%, respectively). The efficacy of FF as a supplement to the maturation medium to improve cytoplasmic maturation appears to vary with follicle quality but not size. However, in general, the addition of 10% FF or FCS to the maturation media resulted in a similar blastocyst yield with no additional improvement when media was supplemented with both FCS and FF. © 1996 Wiley-Liss, Inc.     

Hormone-controlled culture of secondary follicles of domestic cats

Ovaries were obtained from domestic cats during ovariohysterectomy. Large, preantral follicles were freed by dissection and mechanical crushing, and were cultured in TCM 199 + 10% FCS medium in the presence or absence of hormones (FSH, hydrocortisone and Insulin-Transferrin-Selenite) as well as in hypoxanthine. A decline in growth potential along with increasing follicle size were observed after one week, with no FSH added. Hormone-supplemented medium was found to induce growth to 2 or 3 times the original volume in more than 80% of follicles of all sizes. Oocyte diameters were continuously increasing, depending on follicle size, and reached 90 mum (80 %) at the point of antrum formation. Nuclear configuration of oocytes from follicles which had been cultured without addition of hormones up to the antral stage indicated a high rate of degeneration which, however, could be reduced by gonadotrophic stimulation. Meiosis at the germinal vesicle stage was found to be inhibited by hypoxanthine. For some oocytes, evidence was provided to meiotic maturation up to extrusion of the first polar body.     

Nuclear configuration of bovine oocytes derived from fresh and in vitro-cultured preantral and early antral ovarian follicles

The aim of this experiment was to characterize the growth and nuclear configuration of oocytes isolated from late preantral and early antral bovine ovarian follicles immediately after recovery and after the in vitro culture. Individual follicles were isolated by microdissection from slices of the ovarian cortex. Follicles were sorted by diameter into 175 to 224, 225 to 274 and 275 to 325 microm-size classes. The follicles selected for in vitro culture were placed singly into 40 microL droplets of medium (TCM 199 enriched with FCS, insulin, transferrin, sodium selenite, sodium pyruvate, 1-glutamine, hypoxanthine, FSH and estradiol-17beta) and cultured for 6, 8, 11, 14 or 17 d. The sizes of follicles and oocytes were related to the duration of culture and gradually increased as culture duration was prolonged. The analysis of the relationship between mean diameters of oocytes at the time of recovery and after the in vitro culture, has shown significant differences after culture lasting 8 d (76.9+/-9.9 vs. 86.1+/-11.1 microm; P < 0.05), 11 d (77.0+/-9.9 vs. 91.9+/-17.5 microm; P < 0.01), 14 d (80.0+/-9.5 vs. 97.9+/-16.5 microm; P < 0.01) and 17 d (82.6+/-6.6 vs. 97.2+/-11.5 microm; P< 0.01). No statistical differences were shown among oocytes in the 5 pre-culture groups (79.5+/-8.8; 76.9+/-9.9; 77.1+/-9.9; 80.1+/-9.5 and 82.6+/-6.6 microm). Meiotic arrest was preserved in 71.9% of oocytes in our culture system up to 14 d. Frequency of the germinal vesicle (GV) stage did not significantly differ among oocytes evaluated "fresh" or cultured for 6, 8, 11 or 14 d. No relationship was observed between the size class of follicles and the frequency of the GV-stage. Prolonging the culture period to 17 d drastically decreased the percentage of oocytes in the GV-stage (18.7%) and increased the percentage of oocytes having premature initiation of meiosis (GVBD; 46.3%) and degeneration (25.0%). These results suggest that out of all culture periods used in our experiment, Day 14 was found to be the longest culture time allowing for both oocyte growth and maintenance of nuclear configuration at the GV-stage.     

In vitro culture of buffalo (Bubalus bubalis) preantral follicles

Growth of buffalo preantral follicles in culture was studied to investigate the effect of size of preantral follicles, individual or group culture, long-term culture of preantral follicles for (40 days), addition of human follicle stimulating hormone (FSH), insulin-transferrin-selenium (ITS), growth factors (epidermal growth factor (EGF), fibroblast growth factor (FGF), vaso active intestinal polypeptide (VIP) in culture media, and substitution of pregnant mare serum gonadotrophin (PMSG) for FSH as gonadotrophin source in culture media. Preantral follicles were isolated mechanically from ovaries of matured, nonpregnant slaughtered buffaloes and cultured in droplets of culture media under mineral oil in a 35 mm petri dish in a CO2 incubator (38-39 degrees C, 5% CO2 in air, 90-95% relative humidity) for 15 days. Preantral follicle isolation and washing medium consisted of Minimum Essential Medium (MEM) supplemented with steer serum (10%), glutamine (2 mM), sodium pyruvate (0.23 mM), hypoxanthine (2 mM) and gentamycin (50 microg/ml), respectively. In Experiment 1, we placed isolated preantral follicles individually or in groups of 2-4 preantral follicles in 30 or 50 microl droplets, respectively, using two culture media: washing media and washing media + ITS (1%) + FSH (0.05 IU/ml), respectively. In Experiment 2, we grouped isolated preantral follicles were grouped into six different size classes: < or = 36, 37-54, 55-72, 73-90, 90-108 and > or = 109 microm. We cultured groups of 2-4 preantral follicles in washing media + ITS (1A) + FSH (0.05 IU/ml) in a CO2 incubator for 15 days. In Experiment 3, we allocated groups of 2-4 preantral follicles to 10 treatments: (1) only washing media, (2) washing media + FSH (0.05 IU/ml), (3) washing media + ITS (17%), (4) washing media + ITS (1%) + FSH (50 IU/ml), (5) washing media + ITS (1%) + EGF (50 ng/ml), (6) washing media + ITS (1%) + FSH (0.05 IU/ml) + EGF (50 ng/ml), (7) washing media + ITS (1%) + FGF (50 ng/ml), (8) washing media + ITS (1%) + FSH (0.05 IU/ml) + FGF (50 ng/ml), (9) washing media + ITS (1%) + VIP (50 ng/ml), and (10) washing media + ITS (1%) + FSH (0.05 IU/ml) + VIP (50 ng/ml). In Experiment 4, based on the results of Experiment 3, we incubated preantral follicles from those treatments showing significantly (P < 0.05) higher growth up to 40 days. In Experiment 5, we allocated groups of 2-4 preantral follicles to two treatments: (1) washing media + PMSG (50 IU/ml), and (2) washing media + ITS (1%) + PMSG (50 IU/ml) and cultured in a CO2 incubator for 15 days. The results indicated that the preantral follicles cultured in groups had a higher growth rate (P < 0.05) than those cultured as individuals. ITS, FSH, PMSG and growth factors significantly (P < 0.05) promoted the growth of the preantral follicles. Following 40 days of culture, follicular architecture was preserved in nearly 17% of the follicles though there was no antrum formation. The growth rate of preantral follicles was lower in buffalo than in cattle.     

In vitro development of caprine ovarian preantral follicles

The in vitro growth and developmental pattern of caprine preantral follicles cultured in agar gel was observed. Preantral follicles 50 to 150 microm in diameter were isolated from prepuberal goat ovaries by treatment with collagenase and DNase. The isolated preantral follicles were cultured in agar gel for up to 14 days. A group of 10 follicles in different developmental stages was cultured in a culture well coated with 0.6% agar gel and filled with DMEM medium supplemented with FCS (10%), hypoxanthine (2 mmol/mL), dbcAMP (2 mmol/mL), FSH (100 ng/mL), insulin-transferrin-selenium (ITS) (50 ng/mL), IGF-1 (50 ng/mL), hydrocortisone (40 ng/mL) and antibiotics. Follicle viability was determined under an inverted phase-contrast microscope according to morphological and histological criteria, and follicle growth was assessed by their size and appearance. The results showed that the three-dimensional structures and forms of follicles were basically maintained intact during culture. Primary follicles developed into secondary follicles and a few of them into antral follicles. A large portion of secondary follicles entered the antral stage, and oocytes also acquired growth. The formation of theca lamina and zona pellucida was observed. The survival capacity of secondary follicles was greater than primary follicles. The survival rates for primary and secondary follicles were 11.36% (5/44) and 71.16% (53/74), respectively. During in vitro development the follicles demonstrated dominance. This experiment revealed the preliminary characteristics of the in vitro development of caprine preantral follicles.     

Development of a combined new mechanical and enzymatic method for the isolation of intact preantral follicles from fetal, calf and adult bovine ovaries

The isolation of preantral follicles from the ovaries of bovine fetuses, calves and adult cows was performed using a simple, rapid mechanical and enzyme method. The ovaries were cut into small pieces with a tissue chopper. Then, the suspension was filtered successively through 500 and 100 mum nylon mesh filters. This simple mechanical procedure resulted in large numbers of isolated preantral follicles: 2,142 +/- 254; 512 +/- 92 and 298 +/- 54 from the ovaries of bovine fetuses, calves and cows, respectively. In addition, the ovarian fragments between 100 and 500 mum were suspended in 10 ml of M199 Hepes medium plus 5% FCS and divided into 2 equal parts: one portion was used for collagenase treatment (200 U/ml) for 20 minutes, while the other served as a control. Collagenase treatment resulted in 841 +/- 161; 216 +/- 51 and 52 +/- 17 preantral follicles from fetuses, calves and cows, respectively, compared with 312 +/- 86; 52 +/- 15 and 10 +/- 2 in the control group. The use of collagenase with ovarian fragments selected by filtration as a method for increasing the rate of recovery of preantral follicles is described here.     

Effects of storage time and temperature on atresia of goat ovarian preantral follicles held in M199 with or without indole-3-acetic acid supplementation

Maintenance of follicular quality after removal and during transport of ovaries is necessary for studies on development of preantral follicles in vitro. The present work investigated the effectiveness of M199 and M199IAA for preservation of goat preantral follicles in ovarian tissue. At the slaughterhouse, the ovarian pair of each animal was divided into 19 fragments. One ovarian fragment was immediately fixed (control--Time 0). The other 18 fragments were randomly distributed in M199 or M1991AA at 4, 20 or 39 degrees C and stored for 4, 12 or 24 h. Histological analysis showed that storage of ovarian fragments in either solution at 20 or 39 degrees C significantly reduced the percentage of normal preantral follicles when compared with the control, in all cases except after preservation in M199IAA at 20 degrees C for 4 h. In contrast, preservation at 4 degrees C, in either solution, kept the percentage of normal preantral follicles at control values. Reduced cellular metabolism may explain why the best preservation of preantral follicles was at 4 degrees C. The addition of IAA to the TCM 199 was effective for goat preantral follicle preservation at 20 degrees C for 4 h.     

Effects of fetal bovine serum, FSH and 17beta-estradiol on the culture of bovine preantral follicles

We describe a 7-d culture in droplets of collagen gel of isolated small bovine preantral follicles in medium with or without 10% fetal bovine serum (FBS). In addition, the effect of human recombinant FSH and 17beta-estradiol on the morphology and growth of the preantral follicles was investigated in medium without FBS. After culture in medium with 10% FBS, the increase in follicle diameter was 13.1 +/- 8.4 microm, the percentage of BrdU-labeled cells was 49.9 +/- 11.3 and the number of cells per area granulosa was 11.1 +/- 1.8. Omission of serum from the culture medium had no effect on the percentage of labeled cells, but the diameter increase was lower and the cells were smaller. Apparently, serum affects the size of the granulosa cells from small preantral follicles rather than the stimulation of cell proliferation. Addition of human recombinant FSH and/or 17beta-estradiol to serum-free medium resulted in a larger diameter increase during culture compared with that of the control. With FSH, this was due to an increase in cell proliferation, while with estradiol this was caused by an increase in granulosa cell size. The effects of simultaneous treatment with FSH and estradiol was simply the combination of their individual effects. In conclusion, small bovine preantral follicles can be cultured for 7 d in the absence of serum and hormones. The follicles increase in diameter and react to FSH with enhanced cell proliferation and to estradiol with an increase in cell size.     

Effects of follicular fluids on the growth of porcine preantral follicle and oocyte

We examined the effect of supplementing the culture medium with follicular fluid (FF) on the growth of porcine preantral follicles and oocytes. Firstly, preantral follicles were retrieved from ovaries and then FF was collected from all antral follicles that were 2-7 mm in diameter (AFF), which included large follicles of 4-7 mm in diameter (LFF) and small follicles of 2-3 mm in diameter (SFF). When preantral follicles with a diameter of 250 mum were cultured in medium containing AFF, the growth of follicles and oocytes was greater than when follicles were cultured in medium containing fetal calf serum (FCS). When this growth-promoting effect in AFF was compared for LFF and SFF, the LFF were shown to be significantly more effective than SFF. This LFF effect was lost, however, when the concentration of LFF in the medium was decreased from 5% to 0.5% or when LFF were heat treated (60 degrees C for 30 min) or trypsin was added. In contrast, a decrease in SFF concentration from 5% to 0.5% and heat treatment of the SFF enhanced preantral follicle growth. Furthermore, proteins obtained from LFF that had molecular weights greater than 10 kDa (LFF > 10 kDa) had similar, but relatively reduced, growth-promoting properties. The remaining three LFF protein fractions (<10 kDa or <100 kDa or >100 kDa), however, did not have these growth-promoting properties. In conclusion, the supplementation of medium with LFF, rather than serum, enhanced preantral follicle and oocyte growth. Factors that enhanced follicle development in LFF and factors that suppressed follicle development in SFF were proteins and these LFF factors ranged in size from 10 kDa to over 100 kDa.     

Expression of follicle-stimulating hormone receptor (FSHR) in goat ovarian follicles and the impact of sequential culture medium on in vitro development of caprine preantral follicles

This study evaluated the expression of FSH receptors (FSHR) in the different stages of goat follicle development and investigated whether the addition of increasing concentrations of FSH throughout the culture period influences the survival, growth and antral formation of in vitro-cultured caprine preantral follicles. The expression of FSHR was analysed before and after culturing follicles using real-time RT-PCR. For the culture, preantral follicles (≥150 μm) were isolated from ovarian fragments and cultured for 18 days in α-MEM+ alone or associated with recombinant FSH (rFSH: 100 or 1000 ng/ml), or in α-MEM+ supplemented with increasing concentrations of FSH throughout culture periods as follows: (a) sequential medium 1: FSH 100 ng/ml (from day 0 to 6), FSH 500 ng/ml (from day 6 to 12) and FSH 1000 ng/ml (from day 12 to 18); and (b) sequential medium 2: FSH 500 ng/ml (from day 0 to 9) and 1000 ng/ml (from day 9 to 18). Follicle development was evaluated on the basis of antral cavity formation, follicular and oocyte growth, and cumulus-oocyte complex health. The expression of FSHR in isolated caprine follicles increased from the preantral to antral phase. Regarding the culture, after 18 days, sequential medium 1 promoted follicular survival, antrum formation and a reduction in oocyte extrusion. Both sequential media promoted a higher rate of meiotic resumption compared with the other treatments. In conclusion, the addition of increased concentrations of FSH (sequential medium) has a significant impact on the in vitro development of caprine preantral follicles.     

Factors influencing the rate of preantral and antral growth of mouse ovarian follicles in vitro.

The development of a culture system for individual mouse ovarian follicles using a low concentration of homologous serum, human follicle-stimulating hormone (hFSH) and a simple combination of growth factors is reported. Preantral follicles, 150 microns in diameter, with thecal cells attached were isolated mechanically. After 6-7 days on a Millicell membrane, a high proportion of the preantral follicles cultured individually with hFSH grew to morphologically normal large antral follicles (400-500 microns in diameter) with high oestradiol secretion. Without hFSH, the follicles grew to approximately 275 microns diameter in 6 days, but did not form antra or secrete oestradiol. The growth trajectory (overall pattern of growth formed by daily measurements of diameter) of each follicle was recorded and used as a measurement of response to experimental variation of culture conditions. The rapidly growing follicles were morphologically normal, but those that grew more slowly showed some abnormality or atresia and secreted less oestradiol. Follicles cultured in groups without being in direct contact with each other showed much poorer growth than those grown individually, but the inhibition was not uniform and some follicles grew larger than others in the group. Follicles that contacted each other directly in culture tended to fuse into one mass and their growth was substantially inhibited. Even under these conditions, one follicle often continued to grow slowly while the others degenerated. Such alteration of growth patterns suggests interfollicular paracrine control and may be a means of three-dimensional spacing of follicle growth within the ovary, as well as part of the mechanism of follicle selection. The dose-response curve based on the mean growth trajectory of follicles cultured individually, produced increasing rates of growth with 12.5-100 miu hFSH ml-1. Higher concentrations of hFSH did not increase growth rate further, but oestradiol secretion continued to increase with increasing hFSH up to the maximum used (2000 miu ml-1).     

Effect of hormones and growth factors on in vitro development of sheep preantral follicles

The present investigation attempts to improve the frequency of in vitro maturation of oocytes by culturing small (150–250 μm) and large (>250–400 μm) preantral follicles (PFs) of sheep for 6 days in various combinations/sequences of thyroxin (T₄), FSH, LH, transforming growth factor alpha (TGF-), epidermal growth factor (EGF) and heat-treated foetal calf serum (FCS). Bicarbonate-buffered tissue culture medium 199, supplemented with 50 μg ml⁻¹ gentamicin sulphate, served as the control medium. In vitro development was initially assessed by the proportion of PFs exhibiting an increase in size, mean increase in diameter and antrum formation. Nuclear maturation to the metaphase II stage of the oocytes isolated from cultured PFs, after an additional 24-h in vitro maturation, indicated success. A total of 15% of oocytes from small PFs and 55% from large PFs, cultured in T₄ + FSH, matured to metaphase II. Culture of PFs in other combinations/sequences of hormones and growth factors, including the control medium, supported a significantly lower proportion of oocytes maturing to metaphase II stage. It is concluded that 6-day in vitro culture of sheep PFs in thyroxin and FSH greatly improves the frequency of oocyte maturation to metaphase II stage.     

Interferon stimulated gene 15 (ISG15): Molecular characterization and expression profile in endometrium of buffalo (Bubalus bubalis)

A sequential medium was evaluated on the survival, activation and growth rates of caprine preantral follicles submitted to a long-term culture period, aiming to establish an ideal in vitro culture system. Ovarian fragments were cultured for 16 days in α-MEM(+) alone or supplemented with hormones (GH and/or FSH) added sequentially on different days of culture. Ovarian fragments were cultured in the first (days 0-8) and second (days 8-16) halves of the culture period, generating 10 treatments: α-MEM(+)/α-MEM(+), FSH/FSH, FSH/GH, FSH/FSH+GH, GH/GH, GH/FSH, GH/FSH+GH, FSH+GH/FSH+GH, FSH+GH/FSH and FSH+GH/GH. Follicle morphology, viability and ultrastructure were analyzed. After day 1 of culture, FSH treatments maintained the percentage of normal follicles similar to the fresh control. At day 16 of culture, the treatment FSH/GH showed the highest (P<0.05) percentage of normal follicles. The ultrastructure of follicles was preserved in the fresh control and FSH/GH treatment. Follicles cultured with FSH/GH had a higher (P<0.05) viability than α-MEM(+); however the viability was lower (P<0.05) when compared to the fresh control. The FSH/GH treatment showed the highest (P<0.05) percentage of follicular activation and secondary follicle formation and produced the largest (P<0.05) mean follicular diameter after 16 days of culture. In conclusion, a sequential medium supplemented with FSH followed by GH during a long-term culture maintains the survival, viability and ultrastructure of goat preantral follicles, and promotes activation and secondary follicles.     

Influence of vitrification techniques and solutions on the morphology and survival of preantral follicles after in vitro culture of caprine ovarian tissue

The objective was to compare the efficiency of various vitrification techniques and solutions for preserving morphology and viability of preantral caprine follicles enclosed in ovarian tissue. Fragments of ovarian cortex were cryopreserved by conventional vitrification (CV) in French straws, vitrification in macrotubes (MTV), or solid-surface vitrification (SSV). Six solutions containing 6 M ethylene glycol, with or without sucrose (SUC; 0.25 or 0.50 M) and/or 10% fetal calf serum (FCS) were tested (Experiment I). After 1 wk, samples were warmed and preantral follicles were examined histologically. To evaluate follicular viability (Experiment II), ovarian fragments were vitrified with the three techniques listed above, in a solution containing 0.25 M SUC and 10% FCS. After warming, follicles were assessed by the trypan blue dye exclusion test. In Experiment III, preantral follicles enclosed in ovarian tissue were vitrified using the protocol which yielded the highest percentage of viable preantral follicles (SSV with 0.25 M SUC and 10% SFB). After warming, the preantral follicles enclosed in ovarian tissue were cultured in vitro and then, were analyzed by histology and fluorescence microscopy (calcein-AM and ethidium homodimer-1). Every vitrification protocol significantly reduced the percentages of morphologically normal follicles relative to the control (88.0%); however, the addition of 0.25 M SUC and 10% FCS to the vitrification solution improved preservation of follicular morphology (67.4, 67.4, and 72.0% for CV, MTV, and SSV, respectively). Although follicular viability after SSV (80.7%) did not differ from that in fresh (non-vitrified) ovarian tissues (88.0%), after in vitro culture, percentages of viable follicles were significantly reduced (70.0%). Percentages of morphologically normal follicles after in vitro culture of vitrified ovarian tissue were similar (76.0%) to those in ovarian cortex fragments cultured without previous vitrification (83.2%). In conclusion, SSV using a solution containing 0.25 M SUC and 10% FCS, was the most efficient method for vitrifying caprine ovarian tissue.     

The stage-dependent inhibitory effect of porcine follicular cells on the development of preantral follicles

The objective of this study was to examine the effects of follicular cells on the in vitro development of porcine preantral follicles. In Experiment 1, one preantral follicle alone (Trt 1) was cocultured with a follicle of the same size with oocytes (Trt 2) or without oocytes (Trt 3). Preantral follicles cultured alone in vitro for 12 days had greater follicle diameters (1017 +/- 96 microm versus 706 +/- 69 or 793 +/- 72 microm, P < 0.05), growth rates (201 +/- 0.3 versus 103 +/- 0.2 or 128 +/- 0.2, P < 0.05) and oocyte survival rates (73% versus 48, or 25%, P < 0.05) than other groups. The inhibitory effects of follicle cells on the growth of preantral follicles and oocyte survival rates were not enhanced by the addition of oocytectomized preantral follicles (Experiment 2). Follicles were cocultured with different sources of follicular cells in other experiments. Coculture with cumulus cells enhanced oocyte survival compared to the control (without coculture) and mural follicular cell groups (Experiment 3). The growth and survival rates of oocytes collected from the group of follicles cocultured with cumulus cells from large antral follicles (>3 mm) were greater (P < 0.05) than those from small antral follicles (<3 mm), or than the control group (without cumulus cells, experiment 4). No significant differences in the follicular diameters (674 +/- 30 microm versus 638 +/- 33 and 655 +/- 28 microm) and growth rate (105% versus 94 and 105%) were observed among the preantral follicles of the different treatments (P > 0.05). Taken together, coculture with the cells from large antral follicles (>3 mm) exerted a significant positive effect on oocyte survival. The growth and oocyte survival of preantral follicle cocultured with the same size of follicles (with or without oocyte) were inhibited. Growth and survival rates of preantral follicles and oocytes are improved by coculturing them with the cumulus cells derived from larger antral follicles.     

Production of buffalo embryos using oocytes from in vitro grown preantral follicles

The present study examines the use of buffalo preantral follicles as a source of oocytes for in vitro embryo production. Preantral follicles were isolated from abattoir-derived buffalo ovaries and were grown for 100 days in five different culture systems: (1) minimum essential medium (MEM); (2) coconut water; (3) MEM + ovarian mesenchymal cell (OMC) co-culture; (4) MEM + granulosa cell (GC) co-culture; or (5) MEM + cumulus cell (CC) co-culture. Low growth rates for the preantral follicles were observed when follicles were cultured in MEM or coconut water medium. Moderate growth rates were seen for OMC and GC co-cultures, and high rates of growth were observed when follicles were grown in CC co-culture. The survival of preantral follicles was low in the MEM culture (<25%), but was over 75% in the other culture systems. Oocytes were not recovered from the MEM group, while an oocyte recovery rate of 80-100% was observed when the follicles were cultured with coconut water/somatic cells. Transferable embryos could be produced only with the oocytes obtained from preantral follicles grown in the OMC and CC co-culture systems. This study demonstrates, for the first time, that it is possible to produce buffalo embryos by in vitro fertilization of oocytes derived from in vitro grown preantral follicles.