Fertilizability and developmental capacity of individually cultured bovine oocytes

Culture of single oocytes throughout in vitro maturation (IVM), fertilization (IVF) and culture (IVC) provides detailed information on maturity, fertilizability and developmental capacity of individual bovine oocytes and embryos. In the present study, effects of sperm concentration (Experiment 1), microdrop size (Experiment 2), and the addition of hypotaurine (HT) or glutathione (GSH; Experiment 3) during IVF were investigated. In Experiment 4, in vitro maturity and developmental capacity of bovine oocytes cultured for IVM in a medium supplemented with fetal calf serum (FCS), bovine serum albumin (BSA) or polyvinyl alcohol (PVA) during IVM were investigated. In Experiments 1 to 3, the percentages of normal (2 pronuclei with a spermtail) and polyspermic fertilization in singly cultured oocytes were similar to those of group IVF culture (5 oocytes/drop). The addition of GSH during single oocyte IVF significantly increased the proportion of normal fertilization and decreased the polyspermic fertilization compared with addition of HT or of the control. The rates of mature oocytes (62.4 and 67.7%) and blastocyst development (12.9 and 15.2%) for single oocyte IVM cultures (Experiment 4) were also similar compared with the group culture; PVA supplementation significantly increased the matured oocyte rate, but decreased blastocyst development significantly (7.1%) as compared with FCS (19.5%) or BSA (15.6%). These results indicate that a single oocyte culture system throughout in vitro production of bovine embryos provides similar maturity, fertilizability and developmental capacity to oocytes cultured in groups.     

Bovine oocyte development following different oocyte maturation and sperm capacitation procedures

Various procedures have been reported for successful in vitro maturation and in vitro fertilization (IVM/IVF) of bovine follicular oocytes. Direct comparisons of these different recommended procedures have been rare. In this research, involving a total of 5,128 oocytes, a series of experiments were conducted to compare oocyte maturation, fertilization, and development in vitro with 2 maturation systems (with or without added hormones) and 3 types of sperm treatment procedures. Oocytes were collected from ovarian antral follicles (2–7 mm in diameter) within 3 hr after slaughter of cows or heifers. Those with intact or at least 4 layers of cumulus cells were selected for IVM/IVF. Oocytes were incubated for 22 hr in either Medium 199 with 7.5% fetal calf serum (M199 + FCS) alone or M199 + FCS with added hormones (M199 + FCS + H; oFSH 0.5 μg/ml, oLH 5.0 μg/ml, and E₂ 1.0 μg/ml) at 39°C in 5% CO₂ and 95% air. For IVF, frozen-thawed sperm were treated with either 0.1 μM calcium ionophore A23187 (A23187) for 1 min, or 10 or 100 μg/ml heparin (H10 or H100) for 15 min. Our results demonstrated the following: (1) both M199 + FCS and M199 + FCS + H supported maturation development to the metaphase II stage (90–95%, P > 0.05); (2) when oocytes were matured in M199 + FCS without added hormones, A23187 sperm treatment was superior to H10 or H100 treatment for fertilization and blastocyst development of the inseminated oocytes (P < 0.05); (3) when oocytes were matured in M199 + FCS + H, A23187 treated sperm again produced a higher fertilization rate than the H10 group (P < 0.05), but the development to the blastocyst stage was similar among all 3 sperm treatment groups (P > 0.05); (4) direct comparison of the 2 maturation systems with A23187 treated sperm resulted in no difference in all criteria measured; however, (5) when compared retrospectively, beneficial effects of added hormones are evident for blastocyst development (but not for fertilization) when sperm were treated with heparin procedures. © 1993 Wiley-Liss, Inc.     

小鼠卵母细胞体外成熟、体外受精的效果观察

目的　研究不同培养条件对小鼠卵母细胞体外成熟及体外受精率的影响。方法　小鼠卵母细胞分别在含有FSH、BSA和胰岛素的培养液中体外成熟,在Whitten 氏液中体外受精,比较体外成熟率、体外受精率。结果　1- 裸卵(DO) 的体外成熟率、体外受精率(81-4% ,31-0 % ) 均高于卵丘卵母细胞复合体(COC)(48-6 % ,27-1% ) 。2- 在培养液中添加FSH、胰岛素和BSA,卵母细胞的体外成熟率为77-9 % ,82-3% 、60-7% ;体外受精率为77-2 % 、72-6 % 、26-7% ;2 - 细胞率为49-2 % 、34-2 % 、10-0% 。胰岛素组的卵母细胞IVM 率最高,但IVF率、2 - 细胞率低于FSH 组。3- 添加BSA的两组的体外受精率只有26-7 % 、25-8 % ,显著低于其他组,其体外成熟率也较添加FSH 和胰岛素的组成。4- 排出第一极体(PbI) 的卵母细胞的体外受精率和2 - 细胞率(85-9 % ,22-4% ) 均高于GV期卵母细胞(71-1 % ,12-9 % ) 。结论　1- 卵丘卵母细胞(COC) 较裸卵(DO) 的体外成熟率、体外受精率都低,差异显著(P成熟< 0-01;P受精< 0-05) 。2-FSH 和胰岛素均能提高小鼠卵母细胞的体外成熟率、体外受精率。3-BSA可以降低小鼠卵母细胞体外受精率,差异极显著。4-GV 期卵母细胞的体外受精率显著低于体外培养的排出第一极体的卵母细胞(P2 - cell < 0-05,P受精<0-05)     

Influence of oocyte quality, culture media and gonadotropins on cleavage rate and development of in vitro fertilized buffalo embryos

The present study was designed to examine the influence of oocyte quality, culture media and gonadotropins on cleavage rate and development of in vitro fertilized buffalo embryos. Three experiments were conducted. In experiment 1, oocytes were classified by number of cumulus cell layers and morphology of the ooplasm as good, fair or poor. Oocytes were cultured for IVM, IVF and IVC in CR1aa medium. In experiment 2, good quality oocytes were cultured for maturation in: (1) CR1aa; (2) CR2aa; (3) TCM-199; (4) MEM or (5) RPMI-1640, and then fertilized using frozen thawed buffalo spermatozoa in CR1aa. The oocytes were cultured in the same medium used for maturation after fertilization. In experiment 3, oocytes were classified into three groups: group (1) was without gonadotropin and serve as a control; group (2) in which IVM medium was supplemented with 10microg/ml FSH and group (3) in which IVM medium was supplemented with 10IUml(-1) eCG. In all experiments, oocytes were kept at 38.5 degrees C under 5% CO(2) for IVM, IVF, IVC and examined for cleavage and embryo development rates on days 3 and 8, respectively. Good and fair quality oocytes produced a higher cleavage rate (P<0.01) than poor quality oocytes. Morula production rate was also higher (P<0.01) for good as compared with fair quality oocytes. Embryo development with poor quality oocytes was arrested at the two to sixteen cell stage. In experiment 2, the cleavage rate was higher (P<0.05) in CR1aa than CR2aa, and higher (P<0.01) than TCM-199, MEM and RPMI-1640. The numbers of morulae and blastocysts were higher (P<0.01) for oocytes cultured in CR1aa and CR2aa media than TCM-199 or MEM. In experiment 3, the addition of FSH or eCG to the maturation medium increased (P<0.01) cleavage and developmental rates of buffalo embryo compared with control medium. In conclusion, the IVM of good quality buffalo oocytes in CR1aa or CR2aa medium and the addition of FSH or eCG in maturation medium produced higher cleavage and developmental rates of IVF buffalo embryos.     

Effects of fetuin on zona pellucida hardening,fertilization and embryo development in cattle

Mammalian oocytes can undergo spontaneous meiotic maturation when they are liberated from their follicles and cultured in vitro; however, the zona pellucida (ZP) becomes resistant to chymotrypsin digestion, or hardens, when spontaneous maturation occurs in serum-free medium. Schroeder et al. [Biol. Reprod. 43 (1990) 891] described that fetuin, a component of fetal calf serum (FCS), inhibits ZP hardening during oocyte maturation. The aim of this experiment was to study the effect of the presence of cumulus cells and addition of hormones to maturation media on bovine zona hardening and embryo development in medium with and without fetuin. In Experiment I, different concentrations of fetuin were added to the maturation medium. The time necessary for digestion of 50% of the ZP (d50) was not different when oocytes were matured in presence of 10% FCS, 1mg/ml polyvinyl alcohol (PVA), or 4, 1 and 0.25mg/ml of fetuin; cleavage rates were also similar. However, significantly more blastocysts (P<0.05) were formed when FCS was used compared to PVA and 0.25mg/ml of fetuin. In Experiment II, we examined the influence of the presence of cumulus cells and hormones during the maturation of oocytes in media with PVA, BSA, FCS and fetuin. The d50 was significantly higher (P<0.05) when oocytes were matured in presence of cumulus cells. The cleavage rate of cumulus-intact oocytes was similar for all groups. However, when oocytes were partially stripped before maturation, the cleavage rate was significantly higher (P<0.05) when FCS or fetuin was used. In both stripped and non-stripped groups, significantly more blastocysts (P<0.05) were formed when oocytes were matured with FCS compared to BSA and PVA. These results indicate that zona hardening, as described for mouse and human oocytes, does not have a large effect on bovine cumulus-intact oocytes. Apparently fetuin can be used as a substitute for FCS during bovine oocyte maturation, since it leads to similar developmental rates as FCS in intact and partially stripped oocytes.     

Luteinizing hormone-enhanced in vitro maturation of bovine oocytes with and without protein supplementation

Luteinizing hormone was shown to enhance maturation of immature oocytes obtained from slaughtered cattle as reflected by elevated proportions of oocytes that fertilized and reached blastocyst stages in vitro after in vitro fertilization (IVF). Higher proportions of ova were fertilized in vitro after in vitro maturation (IVM) in modified TCM-199 (TCM-199 + BSA + LH [USDA-bLH-B-5, 100 micrograms/ml]) than in TCM-199 alone (p less than 0.01). Further improvement in IVF (p less than 0.005) followed IVM when 20% proestrous (Day 20) bovine serum replaced the BSA, but similar proportions of inseminated ova (22.2% and 22.6%) developed into blastocysts. The positive LH effect was verified in defined conditions for IVM. Exposure of oocytes to the purified LH preparation (without any other added protein or biological substances) during IVM improved IVF (39.7% in TCM-199 vs. 73.5% in TCM-199 + LH; p less than 0.001) and blastocyst development (7.9% vs. 28.2%; p less than 0.005), respectively. Efforts to better define effective concentrations of LH revealed no difference in viability after IVM with 50 micrograms LH/ml vs. 100 micrograms LH/ml (27.0% vs. 28.3%, respectively); 10 micrograms LH/ml did not enhance viability when compared to TCM-199 alone (10.8% vs. 9.9%). Results demonstrate potential utility of this approach for investigation of factors influencing mammalian development by specific effects initiated during the interval of oocyte maturation.     

Equine oocyte in vitro maturation: influences of sera, time, and hormones.

Objectives of the present research were to determine the influences of types of media, sera, time and hormones on equine oocyte in vitro maturation (IVM). The following types of media and sera were evaluated: Menezo's B2 medium (B2), modified Tissue Culture Medium 199 (TCM), Defined Medium (DM), fetal calf serum (FCS), mare serum collected on the first day of estrus (MS), and mare serum collected on the day of ovulation (MSO). Resultant oocyte maturation was compared with the control: DM with bovine serum albumin (BSA). Effect of culture time (0, 15, and 32 hr) and the following hormones on oocyte IVM were evaluated: none, bovine luteinizing hormone (bLH; 1, 10, 100 micrograms/ml), equine luteinizing hormone (eLH; 100 micrograms/ml), bovine follicle-stimulating hormone (FSH; 5 micrograms/ml), and equine chorionic gonadotropin (eCG; 1 and 100 IU/ml). Cumulus expansion in the media and sera experiments was 50% (DM with BSA), 80% (TCM, B2, and DM with MS or MSO), and 100% (FCS with any medium). The proportion of metaphase II (MII) oocytes was significantly (P less than 0.05) increased the percentage of MII oocytes as compared with 0 hr of culture. Cumulus expansion in the hormone experiments was 80% (none, bLH, and eLH), and 100% (eCG and FSH). Freshly prepared bLH significantly (P less than 0.05) inhibited nuclear maturation of equine oocytes. In summary, 15 hr of culture was sufficient time for equine oocyte IVM and all combinations of medium, serum, and hormone addition were equally effective in achieving IVM except fresh bLH and DM with BSA.     

The effects of macromolecular and serum supplements and oxygen tension during bovine in vitro procedures on kinetics of oocyte maturation and embryo development

Aiming to standardize in vitro production of bovine embryos and to obtain supplements to replace serum in culture media, this study evaluated the nuclear maturation kinetics and embryonic development in bovine after in vitro maturation (IVM) and culture (IVC) with several macromolecules (animal origin: bovine serum albumin (BSA), fetal calf serum (FCS); synthetic: polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), Ficoll, and Knockout) at two oxygen tensions (20% and 5% O(2)). Regarding nuclear kinetics, neither the presence of the expected stage (metaphase I, transition anaphase to telophase, and metaphase II) at each evaluation moment (6, 18, and 24?h after IVM, respectively) nor the accelerated polar body emission (at 18?h after IVM) related developmental competence to blastocyst stage when different supplements were compared. Independently of supplement, cleavage rates at 20% O(2) (61.6-79.2%) were higher than at 5% O(2) (38.9-58.7%). At 20% O(2), higher blastocyst and hatching rates, respectively, were obtained in treatments BSA, FCS, Knockout, and control group (IVM with FCS and IVC with BSA + FCS, 14.0-23.5% and 6.8-15.4%) in comparison to PVA, PVP, and Ficoll (0%). The same was observed at 5% O(2) for blastocyst rates with BSA, FCS, Knockout, and control (5.4-16.8%) and for hatching rates with BSA, FCS, and control (2.0-11.1%). We can conclude that producing bovine embryos at 20% O(2) during the entire IVP process resulted in higher developmental rates than at 5% O(2). In addition, while defined macromolecules PVA, PVP, and Ficoll were not suitable for embryonic development, the synthetic serum Knockout was able to replace serum and albumin for IVP in bovine at 20% O(2).     

Influence of insulin-like growth factor-I and its interaction with gonadotropins, estradiol, and fetal calf serum on in vitro maturation and parthenogenic development in equine oocytes

The effects of insulin-like growth factor-I (IGF-I) and its interaction with gonadotropins, estradiol, and fetal calf serum (FCS) on in vitro maturation (IVM) of equine oocytes were investigated in this study. We also examined the role of IGF-I in the presence or absence of gonadotropins, estradiol, and FCS in parthenogenic cleavage after oocyte activation with calcium ionophore combined with 6-dimethylaminopurine (6-DMAP), using cleavage rate as a measure of cytoplasmic maturation. Only equine cumulus-oocyte complexes with compact cumulus and homogenous ooplasm (n = 817) were used. In experiment 1, oocytes were cultured in TCM-199 supplemented with BSA, antibiotics, and IGF-I at 0 (control), 50, 100, 200 ng/ml, at 39 degrees C in air with 5% CO(2), 95% humidity for 36 or 48 h. In experiment 2, oocytes were cultured with FSH, LH, estradiol, and FCS with IGF-I at the concentration that promoted the highest nuclear maturation rate in experiment 1. In experiment 3, oocytes from the three experimental groups (IGF-I; hormones; and IGF-I + hormones) were chemically activated by exposure to calcium ionophore followed by culture in 6-DMAP. In experiment 1, IGF-I stimulated equine oocyte maturation in a dose-dependent manner with the highest nuclear maturation rate at a concentration of 200 ng/ml. No significant effect of IGF-I on nuclear maturation was observed in experiment 2. In experiment 3, a significant difference in cleavage rate was observed between the hormone + IGF-I group (15 of 33; 45.4%) compared with IGF-I (10 of 36; 27.8%) and hormone (4 of 31; 12.9%) alone (P < 0.05). These results demonstrated that IGF-I has a positive effect on nuclear maturation rate of equine oocytes in vitro. The addition of IGF-I to an IVM medium containing hormones and FCS did not increase nuclear maturation, but resulted in a positive effect on cytoplasmic maturation of equine oocytes measured by parthenogenic cleavage.     

Effect of macromolecules in solutions for vitrification of mature bovine oocytes

This study was designed to evaluate vitrification procedures for in vitro matured bovine oocytes for efficient blastocyst production after warming, IVF and culture. A second goal was to replace serum as the macromolecular component of the vitrification solution, without compromising efficacy. The first experiment compared two containers, open pulled straws (OPS) versus cryoloops, and two vitrification protocols: short equilibration (H-TCM-199+10% EG+10% DMSO+20% FCS for 30s, followed by H-TCM-199+20% EG+20% DMSO+20% FCS+0.48M galactose for 20s) versus long equilibration (H-TCM-199+3% EG+20% FCS for 10min, followed by H-TCM-199+31% EG+20% FCS+1M galactose for 20s). Subsequent experiments used only cryoloops and the short equilibration protocol to evaluate the effect of replacing FCS with defined macromolecules (BSA, Ficoll, PVP, and PVA) in vitrification solutions. Cryoloops were superior to OPS for vitrification of oocytes as determined by blastocyst production (P<0.05). The short and long vitrification protocols gave similar results. The presence of macromolecules in vitrification solutions for bovine oocytes was necessary for acceptable post-warming developmental capacity; 20% FCS, 1% and 2% BSA, 6% and 18% Ficoll, 6% and 20% PVP, 1% PVA, and the combinations of 18% Ficoll+1% BSA, and 6% PVP+1% BSA provided similar protection during vitrification of oocytes; development ranged from 14.8% to 23.0% blastocysts/oocyte, which was not different (P>0.05) from non-vitrified controls (26.9-34.0% blastocysts/oocyte). Too much (6%) and too little (0.3%) BSA, and 0.3% PVA for vitrification resulted in lower blastocyst production (P<0.05) relative to unvitrified oocytes.     

In vitro maturation and fertilization of buffalo oocytes (Bubalus bubalis ): Effects of media, hormones and sera

Media (TCM-199 and Ham's F-10); sera (fetal calf serum, FCS, and buffalo estrous serum, BES); and hormones (FSH, 0.5 ug/ml, LH, 5 ug/ml and estradiol 1 ug/ml) were tested to determine the efficiency of in vitro maturation and fertilization of buffalo follicular oocytes. Immature good quality cumulus-oocyte complexes (COCs) were randomly assigned to 1 of 4 experiments. Each experiment consisted of 6 treatment groups. Oocytes cultured for 24 hours in medium (TCM-199 or Ham's F-10) containing 10% FCS or BES had a significantly higher maturation rate than those in medium alone (P < 0.05). However, the maturation rate was higher in medium supplemented with 10% FCS than with 10% BES. Addition of hormones alone or in combination with sera further improved the maturation rate, but no significant difference was observed in the maturation rate among the 3 hormone-treated groups. Immature oocytes matured in the various cultures were fertilized with frozen-thawed buffalo spermatozoa. Our findings show that hormone and/or serum supplementation of TCM-199 did not improve the fertilization rate. Supplementation of Ham's F-10 with LH alone or in combination with LH + FSH + E(2) and with FCS significantly improved the fertilization rate of oocytes while medium with FSH, E(2) or no hormones did not (P < 0.05); same media supplemented with BES resulted in lower fertilization rates both in the presence or absence of hormones. The results indicate that the culture medium has a marked effect on the fertilization rate of buffalo oocytes. Ham's F-10 + LH + FSH + E(2) supplemented with FCS was the most efficacious culture system of those studied for the in vitro maturation of buffalo oocytes.     

Effects of ovarian cortex cell co-culture during in vitro maturation on porcine oocytes maturation, fertilization and embryo development

The objective of the experiments was to evaluate the effects of porcine ovarian cortex cells (pOCCs) during in vitro maturation (IVM) of porcine oocytes on IVM of porcine oocytes, in vitro fertilization (IVF) parameters and subsequent embryo development. The pOCCs was cultured in the 500 microl TCM199 without hormone until the confluence, and then cultured in 500 microl TCM199 supplemented with hormone for 12 h before the oocytes added. Porcine oocytes were co-cultured with the pOCCs monolayers in the co-culture system for 44 h, following fertilized in the mTBM for 6 h. Finally, the presumptive zygotes were cultured for 144 h in the NCSU-23 supplemented with 0.4% BSA. The results showed that matured M II oocytes in the co-culture group were higher than that in the control group (P<0.05). Although penetration did not differ between the co-culture and control groups (P=0.481), polyspermy declined in the co-culture group (P<0.05), whereas male pronucleus (MPN) formation was improved in the co-culture group compared with the control group (P<0.05). More blastocysts developed in the co-culture group than that in the control group (P<0.05); however, the cleavage rates and the mean number cells per blastocyst showed no significant difference between the treated group and the control group (P=0.560 and 0.873, respectively). In conclusion, the presence of the pOCCs monolayers during IVM enhanced the maturation quality of the porcine oocytes, reduced the polyspermy, increased the percentages of MPN formation and blastocyst, but the blastocyst quality was not improved.     

Influence of serum and hormones on bovine oocyte maturation and fertilization in vitro

Three approaches were investigated for improvement of in vitro maturation (IVM), in vitro fertilization (IVF), and early embryonic development in cattle. These were: 1) Selection of oocytes, 2) medium supplementation with fetal calf serum (FCS) and cow sera (DO, Dl, D10, and D20 to correspond with estrus, metestrus, diestrus, and proestrus, respectively), and 3) addition of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol-17β (E₂)during maturation. Greater proportions (percentage) of oocytes initially selected for their compact cumulus cells completed IVM and IVF when compared to unselected oocytes (P < .05). Proportions (percentage) of selected oocytes that matured and cleaved after in vitro insemination according to serum type used for IVM were: FCS: 110/175 (62.9%) and 37/110 (33.6%) and DO: 130/145 (89.7%) and 52/130 (40.0%); D1 127/130 (97.7%) and 41/127 (32.3%); D10 95/98 (96.9%) and 35/95 (36.8%); D20:113/116 (97.4%) and 49/113 (43.4%). A higher proportion (P < .05) of embryos resulting from the D20 group reached four- and eight-cell stages. In FCS-supplemented maturation media with no hormones added during maturation (control), results of IVM and IVF were 157/265 (59.2%) and 39/157 (24.8%), respectively. With E₂ (1 μg/ml) and FSH (5 μg/ml), comparable results were 189/215 (87.9%) and 71/189 (37.6%); with E₂ (1 μg/ml) plus LH (10 μml), 280/327 (85.6%) and 111/280 (39.6%). Added hormones improved IVM results (P < .05) and, when FSH or LH was added with E₂, in vitro development to four- and eight-cell stages was markedly enhanced (P < .05). Selection of oocytes, D20 serum, and added E₂ and FSH or LH for IVM improved in vitro development of bovine embryos after IVF.     

Effect of macromolecule supplementation during in vitro maturation of goat oocytes on developmental potential

In vitro maturation (IVM) of goat oocytes with serum-supplemented media results in oocytes with reduced developmental potential. The objective of this study was to develop a defined medium for IVM of goat oocytes that better supports subsequent embryonic development. Cumulus oocyte complexes (COC) were matured for 18-20 hr in: Experiment (1), tissue culture medium 199 (TCM199) with 10% (v/v) goat serum or modified synthetic oviduct fluid maturation medium (mSOFmat) with 2.5, 8.0, or 20.0 mg/ml bovine serum albumin (BSA); Experiment (2), mSOFmat with 4.0, 8.0, 12.0, or 16.0 mg/ml BSA; or Experiment (3), 1.0 mg/ml polyvinyl alcohol (PVA; control), 4.0 mg/ml BSA, 0.5 mg/ml hyaluronate plus 0.5 mM citrate, or hyaluronate, citrate, and BSA. Mature COC were coincubated for 20-22 hr with 12-15 x 10(6) sperm/ml in modified Brackett and Oliphant (mBO) medium. Embryos were cultured for a total of 7 days in G1/2, and evaluated for cleavage, and blastocyst development, hatching, and total cell numbers. In the first experiment, more (P < 0.05) blastocysts developed per cleaved embryo following maturation in mSOFmat with 2.5 or 8.0 mg/ml BSA than with 20.0 mg/ml BSA or TCM199 with 10% goat serum. The various concentrations of BSA used in the second experiment did not affect (P > 0.05) any of the developmental endpoints examined. In the third experiment, developmental potential of oocytes matured with PVA or hyaluronate with citrate was not different (P > 0.05) from oocytes matured in the presence of BSA. These results demonstrate that developmentally competent goat oocytes can be matured under defined conditions.     

Development of in vitro fertilized oocytes from pregnant and nonpregnant cows in oviductal epithelial and cumulus cell co-culture systems

The objectives of this study were 1) to measure cleavage, blastocyst formation, and blastocyst hatching after in vitro maturation (IVM), fertilization (IVF) and culture (IVC) of oocytes aspirated from pregnant versus nonpregnant cows, and 2) to compare embryo development in co-culture with bovine oviductal epithelial cells versus cumulus cells. No differences in cleavage (38 versus 40%), blastocyst formation (13 versus 13%), or blastocyst hatching (53 versus 51%) were observed for in vitro-matured, fertilized, and cultured oocytes from pregnant versus nonpregnant cows, respectively (P > 0.05), indicating that nonpregnant and early-pregnant cows are equally acceptable donors of oocytes for IVM/IVF/IVC procedures. Cleavage (36 versus 40%), blastocyst formation (11 versus 12%), and blastocyst hatching (50 versus 55%) were not different for embryos co-cultured with oviductal epithelial cells versus cumulus cells (P > 0.05). Thus, equivalent embryo development can be obtained with co-culture systems commonly used for in vitro-derived bovine embryos. These results help to define variables that affect comparison of results across laboratories and that are relevant to the practical application of IVM/IVF/IVC procedures to cattle.     

Effect of sheep and human follicular fluid on the maturation of sheep oocytes in vitro

This study examines the effect of sheep and human follicular fluid on the in vitro maturation (IVM) of sheep follicular oocytes. Oocyte cumulus complexes recovered post mortem were matured for 24 to 26 h at 38.6 degrees C, 5% CO(2) in air, in TCM-199 bicarbonate medium supplemented with 20% fetal calf serum (FCS) and, where stated, with maturation hormones, including FSH (5.0 ug/ml), LH (5.0 ug/ml) and estradiol (1 ug/ml), or with sheep follicular fluid recovered from large (>5mm) or small (2 to 5mm) ovarian follicles post mortem, or with human periovular follicular fluid obtained during routine IVF procedures. The matured oocytes were then denuded, and their maturation stage and developmental capacity were assessed by in vitro fertilization (IVF) and culture (IVC). It was found that inclusion of sheep or human follicular fluid or hormone supplements in the IVM media more than doubled the number of oocytes completing maturation (FCS alone 33%, compared with 76.2% for maturation hormones, 84.2% for fluid from large and 69.6% for fluid from small sheep follicles and 82.6% for human follicular fluid), and significantly increased fertilization rates (FCS alone 51.6%, compared with 71.9% for maturation hormones, 78.4% for fluid from the large and 75.7% for fluid from small sheep follicles and 73.1% for human follicular fluid) without discernible adverse effects on the development of the cleaving embryos to the morula or blastocyst stage in culture. Omission of FCS and supplements from the IVM medium resulted in a marked reduction (56%) in the number of oocytes maturing. This reduction could be offset to a large part, but not completely, by inclusion of human follicular fluid or human follicular fluid plus LH (5 ug/ml) in the medium. The results of this study show that addition of sheep or human follicular fluid to maturation medium can enhance rather than inhibit the maturation and fertilizability of sheep follicular oocytes in vitro.     

Isolation, identification and characterization of secretory proteins of IVMFC embryos and blood circulation of estrus and early pregnant goat

The aim of the present study was to isolate, identify and characterize the secretory proteins of IVM oocytes and IVMFC embryos to evaluate its immunogenecity. and identify of such proteins if any, in blood circulation of estrus and early pregnant goats. Oocytes were matured in TCM-199 with 1 microg/ml, estradiol-17beta; 0.5 microg/ml, FSH; 100 IU/ml, LH and 10% FCS on granulosa cell monolayer. After 18 hr of maturation, oocytes were further cultured in maturation medium containing 3 mg/ml polyvinyl alcohol (PVA) without serum and BSA for 12 hr and medium was collected. The IVF embryos of 4-8 cell stage were cultured in medium containing PVA without serum and BSA. Embryo culture medium was collected after 24 hr of culture and was pooled. The proteins were analyzed on SDS-PAGE (12.5%). Four secretory proteins of oocytes with approximately molecular weight of 45, 55, 65 and 95 kDa and three secretory proteins of embryos 45, 55 and 65 kDa were obtained on SDS-PAGE in silver staining. The protein profile of midluteal, estrus and early pregnant goat serum was similar and no variation was observed among the proteins on SDS-PAGE. Two secretory proteins of 55 and 65 kDa of both IVM oocytes and IVMFC embryos were observed on Western analysis. None of such proteins was observed in midluteal, estrus and early pregnant goat serum on western blotting. It can be concluded that IVM oocytes and IVMFC embryos secrete proteins in medium and two of them can develop antibody. The proteins secreted from embryos till morula stage was similar to that of oocytes. None of these oocyte/embryo released proteins were observed in blood circulation of estrus and early pregnant goats.     

In vitro embryo production in goats: Slaughterhouse and laparoscopic ovum pick up–derived oocytes have different kinetics and requirements regarding maturation media

A total of 3427 goat oocytes were used in this study to identify possible differences during in vitro embryo production from slaughterhouse or laparoscopic ovum pick up (LOPU) oocytes. In experiment 1, one complex, one semi-defined, and one simplified IVM media were compared using slaughterhouse oocytes. In experiment 2, we checked the effect of oocyte origin (slaughterhouse or LOPU) on the kinetics of maturation (18 vs. 22 vs. 26 hours) when submitted to semi-defined or simplified media. In experiment 3, we determined the differences in embryo development between slaughterhouse and LOPU oocytes when submitted to both media and then to IVF or parthenogenetic activation (PA). Embryos from all groups were vitrified, and their viability evaluated in vitro after thawing. In experiment 1, no difference (P > 0.05) was detected among treatments for maturation rate (metaphase II [MII]; 88% on average), cleavage (72%), blastocyst from the initial number of cumulus oocyte complexes (46%) or from the cleaved ones (63%), hatching rate (69%), and the total number of blastomeres (187). In experiment 2, there was no difference of MII rate between slaughterhouse oocytes cultured for 18 or 22 hours, whereas the MII rate increased significantly (P < 0.05) between 18 and 22 hours for LOPU oocytes in the simplified medium. Moreover, slaughterhouse oocytes cultured in simplified medium matured significantly faster than LOPU oocytes at 18 and 22 hours (P < 0.05). In experiment 3, cleavage rate was significantly greater (P < 0.001) in all four groups of embryos produced by PA than IVF. Interestingly, PA reached similar rates for slaughterhouse oocytes cultured in both media, but improved (P < 0.05) the cleavage rate of LOPU oocytes. Slaughterhouse oocytes had acceptable cleavage rate after IVF (∼67%), whereas LOPU oocytes displayed a lower one (∼38%), in contrast to cleavage after PA. The percentage of blastocysts in relation to cleaved embryos was not affected by the origin of the oocytes (P > 0.05). Therefore, slaughterhouse oocytes developed a greater proportion of blastocysts than LOPU ones, expressed as the percentage of total cumulus oocyte complexes entering to IVM. Vitrified-thawed blastocysts presented similar survival and hatching rates between the oocyte origin, media, or method of activation. In conclusion, slaughterhouse and LOPU derived oocytes may have different IVM kinetics and require different IVM and IVF conditions. Although the IVM and IVF systems still need improvements to enhance embryo yield, the in vitro development step is able to generate good quality embryos from LOPU-derived oocytes.     

A combination of EGF and IGF-I accelerates the progression of meiosis in bovine follicular oocytes in vitro and fetal calf serum neutralizes the acceleration effect

The effects of a combination of EGF and IGF-I (GFs) on the progress of meiosis and on their developmental competence were examined in cumulus-enclosed bovine oocytes. Exposure to GFs in serum-free, 0.3% PVP-containing maturation medium significantly (P<0.05) increased the frequency of oocytes with the first polar body (PB) at 16 h of culture and decreased those with PB at 20 h. The cleavage rates of PB-extruded oocytes after fertilization were not affected by treatment of GFs during maturation culture, and blastocyst yield was not improved by GFs treatment. Although replacement of PVP from GFs-containing medium with fatty acid-free BSA did not affect the timing of PB extrusion, replacement with 10% FCS neutralized the acceleration effects of GFs. Replacement for macromolecule in maturation medium did not improve blastocyst yield of PB-extruded oocytes after fertilization. These results indicate that the progression of meiosis in bovine oocytes with cumulus cells is accelerated by exposure to GFs in serum-free maturation medium but their developmental competence is not improved, and that the acceleration effects on the progress of meiosis is neutralized by the presence of FCS in maturation medium with no improvement of developmental competence after in vitro fertilization.     

Enrichment of in vitro maturation medium for buffalo (Bubalus bubalis) oocytes with thiol compounds: effects of cystine on glutathione synthesis and embryo development

The purpose of this study was to evaluate whether enriching the oocyte in vitro maturation medium with cystine, in the presence of cysteamine, would improve the in vitro embryo production efficiency in buffalo by further increasing the GSH reservoir created by the oocyte during maturation. Cumulus-oocytes complexes were matured in vitro in TCM 199 + 10% FCS, 0.5 microg/ml FSH, 5 microg/ml LH and 1 microg/ml 17beta-estradiol in the absence or presence of cysteamine (50 microM), with or without 0.3mM cystine. In Experiment 1, glutathione content was measured by high-performance liquid chromatography and fluorimetric analysis in representative samples of oocytes matured in the four different experimental conditions. In Experiment 2, oocytes were fixed and stained to assess nuclear maturation and normal pronuclear development following IVM and IVF respectively. In Experiment 3, mature oocytes were in vitro fertilized and cultured to assess development to blastocysts. In all supplemented groups the intracytoplasmic GSH concentration was significantly higher than the control, with the highest GSH levels in oocytes matured in the presence of both thiol compounds (3.6, 4.7, 5.4 and 6.9 picomol/oocyte in the control, cysteamine, cystine and cystine+cysteamine groups, respectively; P < 0.05). Cystine supplementation of IVM medium, both in the presence or absence of cysteamine, significantly increased the proportion of oocytes showing two normal synchronous pronuclei following fertilization. In all supplemented groups, cleavage rate was significantly improved compared to the control (55, 66.1, 73.5 and 78.4% in the control, cysteamine, cystine and cystine+cysteamine groups, respectively; P < 0.05). Similarly, blastocyst yield was also increased in the three enriched groups compared to the control (17.1, 23.8, 29.3, 30.9% in the control, cysteamine, cystine and cystine+cysteamine groups, respectively; P < 0.05). Overall, the addition of cystine to a cysteamine-enriched medium resulted in a significant increase of cleavage rate and transferable embryo yield compared to the medium supplemented with only cysteamine.