Calf Oocytes Research Articles

Exopolysaccharide ID1 Improves Post-Warming Outcomes after Vitrification of In Vitro-Produced Bovine Embryos.

This study aimed to assess the cryoprotectant role of exopolysaccharide (EPS) ID1, produced by Antarctic Pseudomonas sp., in the vitrification of in vitro-produced (IVP) bovine embryos. IVP day 7 (D7) and day 8 (D8) expanded blastocysts derived from cow or calf oocytes were vitrified without supplementation (EPS0) or supplemented with 10 µg/mL (EPS10) or 100 µg/mL (EPS100) EPS ID1. The effect of EPS ID1 was assessed in post-warming re-expansion and hatching rates, differential cell count, apoptosis rate, and gene expression. EPS100 re-expansion rates were significantly higher than those observed for the EPS0 and EPS10 treatments, regardless of culture length or oocyte source. EPS100 hatching rate was similar to the one of the fresh blastocysts except for those D7 blastocysts derived from calf oocytes. No differences were observed among EPS ID1 treatments when the inner cell mass, trophectoderm, and total cell number were assessed. Although apoptosis rates were higher (p ≤ 0.05) in vitrified groups compared to fresh embryos, EPS100 blastocysts had a lower number (p ≤ 0.05) of apoptotic nuclei than the EPS0 or EPS10 groups. No differences in the expression of BCL2, AQP3, CX43, and SOD1 genes between treatments were observed. Vitrification without EPS ID1 supplementation produced blastocysts with significantly higher BAX gene expression, whereas treatment with 100 µg/mL EPS ID1 returned BAX levels to those observed in non-vitrified blastocysts. Our results suggest that 100 µg/mL EPS ID1 added to the vitrification media is beneficial for embryo cryopreservation because it results in higher re-expansion and hatching ability and it positively modulates apoptosis.

The effect of age and length of gonadotropin stimulation on the in vitro embryo development of Holstein calf oocytes

The use of oocytes recovered from prepubertal donors for in vitro embryo production has great potential for accelerating the rate of genetic gain in the dairy industry. However, these oocytes are known to be less developmentally competent than those from adult donors. In this study, we investigated the effect of age and gonadotropin stimulation in Holstein heifers subjected to oocyte collection every two weeks between 2 and 6 months of age. In order to assess the effect of gonadotropin stimulation, animals were subjected to one of three treatments, namely Short (ST; 36–42 h), Long (LT; ≥72 h) and No Treatment (NT) prior to laparoscopic ovum pick-up (LOPU). Our results show that the LT significantly improved the proportion of large follicles (>5 mm diameter) present in the ovary (LT 34.0% vs. ST 11.2% vs. NT 2.4%, P < 0.05), as well as the percentage of good-quality cumulus oocyte complexes (COCs) recovered (LT 95.3 ± 18% vs. ST 85.4 ± 22% vs. NT 82.2 ± 14%, P < 0.05) and blastocyst rate (LT 36.7 ± 26% vs. ST 18.3 ± 15% vs. NT 16.7 ± 9%, P < 0.05). Recovery rate was affected by treatment (LT 70.4 ± 25 vs. ST 85.4 ± 29 vs. NT 72.7 ± 23, P < 0.05). To assess the impact of age, data was grouped into <100 days (A), 100–130 days (B) and >130 days (C) of age at LOPU. We found that as animals got older, although the average number of COCs per donor per LOPU declined (A: 17.5 ± 11 vs. B: 14.7 ± 7 vs. C: 11.9 ± 8), the blastocyst rate increased (A: 12.8 ± 20% vs. B: 17.1 ± 21% vs. C: 21.8 ± 25%, P < 0.05). We also evaluated the incidence of polyspermy and confirmed it is a critical limitation for IVF in calf oocytes. The incidence of polyspermy was unaffected by gonadotropin treatment, but significantly decreased with age. The capacity for full development to term of in vitro produced embryos from calf oocytes was tested by embryo transfer into 21 synchronized adult recipients, which resulted in 13 pregnancies (62%), full development to term and healthy calves born. Finally, the study allowed evaluating the safety of the procedure since, on average, each animal was subjected to 8 LOPU procedures over a period of 4 months. Our results showed that the procedure is safe (no incidents during laparoscopy), and was not harmful for the reproductive future of the animals, as those that were bred became pregnant after reaching sexual maturity.

Assessment of the effect of adding L-carnitine and/or resveratrol to maturation medium before vitrification on in vitro-matured calf oocytes

Cryopreservation may lead bovine oocytes to undergo morphological changes and functional damage due to the high-lipid content in the cytoplasm and the formation of reactive oxygen species. Against this background, the present study aimed to improve the cryotolerance and developmental competence of prepubertal calf oocytes by adding L-carnitine (LC) and/or resveratrol (R) to the IVM medium, as the former is involved in lipid metabolism and both are able to scavenge reactive oxygen species. With this purpose, different quality and functional oocyte parameters, such as spindle and chromosome configuration, DNA integrity, caspase activity, and the profile of genes involved in lipid metabolism and oxidative stress were evaluated in IVM bovine oocytes before or after vitrification/warming. Oocytes were matured in the absence (control) or presence of LC (3.03 mM) and/or R (1 μM) and then vitrified/warmed before IVF and embryo culture. All treatment groups (control, LC, R, and LC + R) of nonvitrified IVM oocytes showed similar rates (P > 0.05) of a normal spindle and chromosome configuration to oocytes vitrified/warmed after maturation in the presence of LC + R. When oocytes in all treatment groups were compared before and after vitrification, no significant differences were detected in DNA fragmentation as measured using the TUNEL method. However, the proportion of early apoptotic oocytes increased after vitrification/warming, except when previously matured with R. Vitrified/warmed oocytes matured in the presence of LC did not differ with nonvitrified oocytes in terms of the expression of ACACA, SLC2A1, PLIN2, HSPA1A, GPX1, and SOD1 genes. Similarly, expression of ACACA, SLC2A1, PLIN2, HSPA1A, and SOD1 genes in vitrified/warmed oocytes was similar to that of their fresh counterparts when matured in the presence of R. Finally, while the addition of LC and/or R to IVM medium had no effect on both cleavage and blastocyst rates either in fresh or vitrified oocytes. To conclude, the results of the present study report that the addition of LC and/or R to the IVM medium used for prepubertal bovine oocytes did not increase the embryo development potential of both fresh and vitrified oocytes. However, LC + R supplementation before vitrification decreased spindle damage, R addition–modulated apoptosis, and LC or R addition before vitrification positively affected the gene expression of vitrified/warmed oocytes.

Effects of Administration of Estradiol and Progesterone on Ovarian Stimulation Response, and In Vitro Developmental Competence of Oocytes in CIDR-Treated Calves

The present study was conducted to investigate the effects of administering estradiol (estradiol-17β, 2.5 mg) plus progesterone (P4, 50 mg) as a part of a controlled internal drug release (CIDR)-based superovulation protocol on ovarian follicles development, and oocyte recovery as well as on in vitro development in prepubertal calves after follicle stimulating hormone (FSH) stimulation. Calves in the treatment group exhibited significantly decreased (P < 0.05) numbers of both small and large antral follicles compared to the control group A and B (<3 mm in diameter [small], 3.4 versus 12.8, 11.6; > 7 mm in diameter [large], 0 versus 7.6, 7.2). The treated animals also exhibited an increased (P < 0.05) proportion of usable oocytes from the total oocytes recovered compared with those of the control group A and B (77% versus 50.2%, 47.8%). Moreover, the rate of cleavage and the percentage of blastocysts displayed an increased trend in the treatment group compared to controls A and B (59.2% versus 50.5%, 50.8%; 15% versus 12.4%, 11.6%, respectively). In conclusion, the application of estradiol-17β plus P4 at CIDR insertion prior to FSH stimulation affected ovarian follicles development and exerted a beneficial effect on the in vitro development of calf oocytes.

207 EFFECT OF REPEAT HORMONE STIMULATION ON THE YIELD AND IN VITRO DEVELOPMENT OF JUVENILE CALF OOCYTES

Juvenile in vitro embryo transfer can markedly reduce animal generation intervals. The purpose of this study was to investigate the ovarian response of juvenile calves and in vitro oocyte developmental capacity after superstimulation. Experiments on calves were performed in accordance with the Animal Welfare Regulations. A total of 36 donor juvenile calves on standard nutrition and in a disease-free environment, were selected from the breeding farm of the Beijing Dairy Cattle Center. At 60 days of age, calves were randomly assigned into three groups of four calves each, replicated three times. On day 1, Group 1 received a progesterone vaginal insert (CIDR, 300 mg per device); Group 2 received a CIDR and 0.5 mg oestrogen benzoate (China); Group 3 received a CIDR, 0.5 mg oestrogen benzoate, and 50 mg progesterone (China). Then, calves were injected with FSH (Folltropin-V, Bioniche Animal Health, Belleville, ON, Canada) twice daily on days 5 (40 mg/40 mg) and 6 (30 mg/30 mg) at 12 h intervals. Cumulus–oocyte complexes (COCs) were recovered from the superstimulated calves 12 to 14 h after the final FSH treatment. COCs were considered usable unless they were damaged or had expanded cumulus layers. Usable COCs were matured in vitro for 24 h in maturation medium consisting of TCM199, 10% FBS, 10 μg mL–1 FSH, 1 μg mL–1 LH, 1 μg mL–1 E2–17β, 100 IU mL–1 penicillin, 100 μg mL–1 streptomycin, with (+Cys) or without (–Cys) 100 μM Cysteamine. Each calf oocyte was cultured in one well. The final concentration added to each fertilization drop was 5 × 106 sperm mL–1. Sperm and oocytes were co-cultured in IVF-100 medium (BO liquid+10 μg mL–1 heparin, Japan) at 38.5°C, 5% CO2 and a saturated humidity for 6 to 8 h. Blastocyst production rates were determined after 7 and 8 d of in vitro culture in CR1aa medium without the addition of cysteamine. Differences among treatments in each experiment were determined by one-way ANOVA and a multiple range test. Superstimulatory results indicated that more follicles were aspirated (63.2 per calf) and more usable oocytes were recovered (48.0 per calf) in Group 1 than in the other two groups (Group 2–45.2 and 31.8, respectively; Group 3–35.4 and 28.3, respectively; P &lt; 0.05). No difference was observed between Groups 2 and 3. Superstimulation of calves twice at 30 day intervals in Group 2 (n = 12) did not affect the number of follicles or usable oocytes (overall, 44.2 and 28.0 per calf). Maturation rates (86.5% v. 85.0%, respectively) and cleavage rates (84.4% v. 80.0%, respectively) did not differ whether cysteamine was not (–Cys; n = 318) or was (+Cys; n = 330) added to the maturation medium. However, the blastocyst rate differed significantly (12.9% v. 35.2%, respectively; P &lt; 0.01). This study established a protocol for the superstimulation of juvenile calves with an average of 48 oocytes obtained per calf. Superstimulation and surgical oocyte recovery twice at an interval of 30 days had no adverse effect on follicle development or oocyte recovery. The novelty of this research is that the blastocyst production rate of calf oocytes (35.2%) in maturation medium supplemented with cysteamine was similar to that reported in the cow.

Leptin Enhances Maturation and Development of Calf Oocytes In vitro

We investigated the effects of leptin on the in vitro maturation (IVM) and development of calf oocytes. Cumulus-oocyte complexes were matured in IVM medium containing 0-100 ng/ml leptin. Experiment 1 showed that exposure of calf oocytes to IVM medium containing 1 or 10 ng/ml leptin significantly increased rates of development to the metaphase II stage compared with the control (81.7 ± 3.0% and 83.3 ± 2.1% for 1 and 10 ng/ml leptin, respectively, vs 64.1 ± 5.1% for control; p < 0.05). Experiment 2 showed that 1 or 10 ng/ml leptin significantly improved cleavage rates after in vitro fertilization when compared to control (58.6 ± 3.3% and 59.3 ± 2.9% for 1 and 10 ng/ml leptin, respectively, vs 48.5 ± 2.6% for control; p < 0.05); in addition, when compared to control medium, the addition of 10 ng/ml leptin to the IVM medium resulted in more presumptive zygotes reaching the 4- to 8-cell stage after 48 h of in vitro culture (30.3 ± 2.3% vs 20.1 ± 2.3%; p < 0.05) and developing into blastocysts after 8 days of culture (20.4 ± 1.6% vs 11.7 ± 1.7%; p < 0.05). Experiment 3 showed that the addition of 1 or 10 ng/ml leptin significantly increased the total number of blastocyst cells on day 8 of culture (114.6 ± 7.8 and 117.4 ± 5.9 for 1 and 10 ng/ml leptin, respectively, vs 92.7 ± 8.3 for control; p < 0.05) and trophectoderm (TE) cells (88.5 ± 5.5 and 90.6 ± 3.7 for 1 and 10 ng/ml leptin, respectively, vs 70.1 ± 5.9 for control; p < 0.05). In summary, these results indicate that the addition of leptin to IVM medium enhances meiotic maturation and embryo development from calf oocytes and improves the quality of embryos derived from these oocytes.

Effect of leptin during in vitro maturation of prepubertal calf oocytes: Embryonic development and relative mRNA abundances of genes involved in apoptosis and oocyte competence

During the in vitro maturation of adult bovine oocytes, leptin has beneficial effects on blastocyst development, apoptosis and transcription levels of developmentally important genes. The present study analyzes the differential effects of leptin on prepubertal bovine oocytes and cumulus cells. Effects were determined of leptin treatment during oocyte maturation on their developmental capacity after fertilization (Exp. 1), incidence of apoptosis in cumulus oocyte complexes (COCs) (Exp. 2) or on relative mRNA abundances of genes in cumulus cells and oocytes (Exp. 3). COCs were matured in serum-free medium containing 1 mg/mL polyvinyl alcohol and 0, 10, 100, or 1000 ng/mL leptin (L0, L10, L100, and L1000, respectively), or in medium supplemented with 10% fetal calf serum (FCS) as a positive control. Addition of leptin during oocyte maturation had no effect on cleavage rates after fertilization (FCS, 68.6%; L0, 62.9%; L10, 66.9%; L100, 63.4%; L1000, 60.9%). Similarly, no significant differences in blastocyst rates were observed when oocytes were matured in the presence of L0 (8.4%), L10 (9.3%), L100 (6.7%), L1000 (8.2%), compared to control FCS (9.4%). In Experiment 2, maturation in the presence of 1000 ng/mL of leptin increased the proportion of TUNEL-positive cumulus cell (6.9%) with respect to those matured in the presence of FCS (4.96%), but not at the lower leptin doses. When relative mRNA abundances were examined for seven genes by qRT-PCR, five (TP53, BAX, DNMT3A, PGTS2 and LEPR) showed differences among groups. LEPR expression was significantly higher in the oocytes matured with FCS compared with the other groups and in those matured with PVA (L0) without leptin compared with the three groups of oocytes matured in the presence of leptin. In conclusion, the addition of leptin to the in vitro maturation medium used for prepubertal bovine oocytes does not increase the development potential of the oocytes or reduce the percentage of apoptosis in cumulus cells. Leptin blocks transcription of the leptin receptor (LEPR) probably reflecting selective, differential degradation by doses of leptin.

Quantitative analysis of bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) gene expression in calf and adult bovine ovaries

BackgroundIt has been reported that calf oocytes are less developmentally competent than oocytes obtained from adult cows. Bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) play critical roles in folliculogenesis, follicular development and ovulation in mammalian ovaries. In the present study, we attempted to compare the expression patterns of BMP15 and GDF9 in the cells of calf and cow ovaries to determine a relationship between the level of these genes and the low developmental competence of calf oocytes.MethodsBovine tissues were collected from 9-11 months-old calves and from 4-6 years-old cows. We characterized the gene expression of BMP15 and GDF9 in calf and adult bovine oocytes and cumulus cells using quantitative real-time reverse transcriptase polymerase chain reaction (QPCR) and in situ hybridization. Immunohistochemical analysis was also performed.ResultsThe expression of BMP15 and GDF9 in cumulus cells of adult ovaries was significantly higher than that in calf ovaries, as revealed by QPCR. GDF9 expression in the oocytes of calf ovaries was significantly higher than in those of the adult ovaries. In contrast, BMP15 expression in the oocytes of calf and adult ovaries was not significantly different. The localization of gene expression and protein were ascertained by histochemistry.ConclusionsOur result showed for the first time BMP15 and GDF9 expression in bovine cumulus cells. BMP15 and GDF9 mRNA expression in oocytes and cumulus cells was different in calves and cows.

187 DIFFERENTIAL EXPRESSIONS OF GROWTH AND DIFFERENTIATION FACTOR 9 AND BONE MORPHOGENETIC PROTEIN 15 GENES IN OVARIES OF THE CALF AND COW

It has been reported that prepubertal calf oocytes are less developmentally competent than those obtained from cows. The bone morphogenetic protein (BMP) family of proteins regulate folliculogenesis and the ovulation rate in mammals. Of the members of the BMP family, growth and differentiation factor 9 (GDF9) and BMP15 are oocyte-derived proteins that play critical roles in granulosa cell proliferation and differentiation. In the present study, we characterised the gene expression of bovine GDF9 and BMP15 in calf and adult cow ovaries. The ovaries obtained from 4 calves at 9 to 11 months old and 4 cows at 4 to 6 years old. For quantitative real-time RT-PCR (qPCR), cumulus–oocyte complexes (COC) and mural granulosa cells were collected by aspiration of follicles 2 to 5 mm in diameter from an ovary from each animal. Ovaries from the other side were used for in situ hybridization. The COC and mural granulosa cells were separated and cultured for 22 h according to the protocol for oocyte maturation. Total RNA was isolated from denuded oocytes, cumulus cells, and mural granulosa cells. Bovine glyceraldehyde-3-phosphate dehydrogenase was used to normalise qPCR efficiency. For in situ hybridization, the collected ovaries were immediately fixed with 4% formaldehyde-PBS and embedded in a paraffin block. In situ hybridization was carried out with digoxigenin-labelled RNA probes. We confirmed there was no contamination of oocytes in the collected cumulus and mural granulosa cells by determining the mRNA expression of germ cell–oocyte markers (ZAR1 and VASA). Two, 16, 7, and 10 COC were collected from the ovary on one side of each calf, and 14, 22, 29, and 33 COC were collected from an ovary of each adult cow. Two COC from the calves could not be used for qPCR analysis. Both GDF9 and BMP15 mRNA were detected in oocytes and cumulus cells at the end of maturation culture, whereas only GDF9 mRNA was detected in mural granulosa cells. Quantitative PCR detection revealed that BMP15 and GDF9 mRNA expression of the cumulus cells from adult ovaries was significantly greater than that from calf ovaries. The expression of GDF9 mRNA was significantly greater in calf oocytes than in oocytes from cows. However, BMP15 mRNA expression in the oocytes of calf and adult ovaries was not significantly different. In mural granulosa cells, the intensities of GDF9 mRNA expression were not significantly different between calves and cows. These qPCR results were also ascertained by in situ hybridization. In conclusion, we clarified that the characteristics of bovine GDF9 and BMP15 mRNA expression in oocytes and cumulus cells were different between calves and cows. Our results indicate the possibility that the calf oocytes were less developmentally competent because of excess GDF9 on the oocytes, deficiencies of GDF9 and BMP15 on the cumulus cells, or both.

Effect of the addition of insulin-transferrin-selenium and/or L-ascorbic acid to the in vitro maturation of prepubertal bovine oocytes on cytoplasmic maturation and embryo development

This study examines the effects of adding insulin-transferrin-selenium (ITS) and/or L-ascorbic acid (ASC) to a conventional medium for maturing prepubertal calf oocytes on chromosome organization, cortical granule (CG) distribution, and embryo development to the blastocyst stage. Cumulus-oocyte complexes (COCs) were matured in medium TCM 199 containing PVA and EGF (control), and supplemented with ITS and/or ASC for 12 or 24 h at 38.5 °C in a 5% CO 2 atmosphere. Calf oocytes matured with ITS + ASC or ASC for 12 h showed significantly higher percentages of peripherally distributed CG (83.3% and 86.2% respectively) than control oocytes (71.4%) or those matured with ITS alone (71.4%). No effects on chromosome organization were detected. Conversely, 24 h of supplementation did not affect CG distribution patterns, while the addition of ASC gave rise to significantly higher percentages of oocytes showing a normal alignment of their chromosomes (72.9%) compared to controls (58.7%). At 48 hpi, similar cleavage rates were observed among treatments regardless of the treatment time. However, the presence of ITS + ASC for 12 h rendered significantly higher blastocyst rates than those recorded in the remaining groups. Supplementation for 24 h with ITS or ITS + ASC had no significant effects on the percentage of blastocysts obtained, while the presence of ASC significantly reduced the proportions of embryos developing to the blastocyst stage. Our data suggest that ITS plus L-ascorbic acid supplementation during the first 12 h of in vitro maturation improves cytoplasm maturation and the developmental competence of embryos produced from prepubertal calf oocytes.

Reconstruction of calf oocytes by germinal vesicle transfer in mature bovine oocytes: preliminary results

Reconstruction of calf oocytes by germinal vesicle transfer in mature bovine oocytes: preliminary results A. Lange Consiglio & A. Bignotti & A. M. Pecile & F. Cremonesi Published online: 14 July 2009 # Springer Science + Business Media B.V. 2009

Heterologous in vitro fertilization is a good procedure to assess the fertility of thawed ram spermatozoa

A heterologous in vitro fertilization (IVF) test using calf oocytes with zona pellucida was employed to assess the fertility of thawed ram sperm samples. Six males with significant differences in fertility ( P = 0.003) were used. The males were classified as having high fertility (≥42%) and low fertility (≤41%). Male fertility was not influenced by number of inseminated ewes ( P = 0.584), insemination technician ( P = 0.156), insemination date ( P = 0.323) or farm ( P = 0.207). Thawed sperm samples were employed to assess several sperm parameters for each male: motility, acrosomal integrity, viability, membrane stability, membrane phospholipid disorder, mitochondrial membrane potential and chromatin stability. These samples were used to carry out a heterologous in vitro fertilization. In vitro-matured calf oocytes ( n = 716) were inseminated with thawed ram semen and in vitro cultured for 40 h. Overall, at thawing, variability among males respect to sperm quality was high. Despite this variability, there were not differences ( P < 0.05) between fertility groups. Yield of hybrid embryos ranged from 31 to 59% between males. There were not differences between males ( P = 0.340). However, there were differences between fertility groups (high fertility: 55%; low fertility: 39%; P = 0.020). Multiple regression analysis showed that the heterologous in vitro fertility was the only predictive parameter for in vivo male fertility. Correlation between both parameters was fair ( r 2 = 0.760; P = 0.025). These results indicate that heterologous in vitro fertilization tests can be useful to predict the fertility of ram spermatozoa using calf oocytes with intact-zona pellucida.

Embryo development and structural analysis of in vitro matured bovine oocytes vitrified in flexipet denuding pipettes

The aim of this study was to compare the effectiveness of two different vitrification carrier systems for oocyte cryopreservation. In vitro matured (IVM) bovine oocytes were vitrified in open pulled straws (OPS) or flexipet denuding pipettes (FDP), and the effects of cryopreservation determined on the cytoskeletal components and developmental capacity of the oocytes. Three experimental groups were established according to whether the oocytes were vitrified in OPS (OPS group), FDP (FDP group) or left untreated (CTR group). Twenty two hours after the onset of maturation, sub-groups of 2–4 oocytes were pre-equilibrated in 1 mL of Hepes-TCM 199 with 20% fetal calf serum (FCS) (HM), 10% dimethyl sulfoxide (DMSO) and 10% ethylene glycol (EG) for 30 s. The oocytes were then transferred to a 20-μL drop of HM containing 20% DMSO, 20% EG and 0.5 M of sucrose, which was used to load the OPS or FDP before their immersion in liquid nitrogen (LN 2). Oocytes were thawed by plunging the OPS or FDP into 0.25 M sucrose in HM, and then placed for 5 min each in 0.15 and 0 M sucrose in HM. After warming, spindle configuration, chromosome distribution and embryo development were assessed. Frozen–thawed semen was used for fertilization. Zygotes were denuded at 22 h post-insemination, and cultured in SOF medium for 9 days at 38.5 °C in a 5% CO 2, 5% O 2 and 90% N 2 atmosphere. All experiments were performed using both cow and calf oocytes to establish sensitivity differences. After in vitro fertilization and culture, oocytes in the FDP group showed a lower cleavage rate than those in the OPS or control groups ( P < 0.05), while blastocysts were only obtained in the OPS group, at a lower rate than controls. After warming, double fluorescent staining revealed higher rates of spindle and chromosome abnormalities in the FDP group compared to the OPS group ( P < 0.05). No differences between cow and calf oocytes were observed in the different experiments. Our results indicate that the carrier system affects the capacity of IVM oocytes to survive cryopreservation. Unexpectedly, the flexipet denuding pipette failed to improve results and high rates of clustered chromatin and abnormal spindles were observed in calf and cow oocytes vitrified by the FDP method. In conclusion, the use of the flexipet denuding pipette modifies the cytoskeletal components and compromises the developmental capacity of in vitro matured calf and cow oocytes.

Cryotops versus open-pulled straws (OPS) as carriers for the cryopreservation of bovine oocytes: Effects on spindle and chromosome configuration and embryo development

Two experiments were designed to assess the effectiveness of cryopreserving bovine MII oocytes using cryotops as the carrier system for vitrification. In the first experiment, we examined the developmental competence of oocytes after: (i) vitrification in open-pulled straws (OPS method); or (ii) vitrification in <0.1 μl medium droplet on the surface of a specially constructed fine polypropylene strip attached to a plastic handle (Cryotop method). In the second experiment, warmed oocytes that had been vitrified in OPS or cryotops were fixed to analyze spindle and chromosome configuration. In all experiments both cow and calf oocytes were used. Significantly different fertilization rates were observed between the vitrification groups: 31.5% and 20.2% for the cow and calf oocytes vitrified in OPS, respectively, versus 46.1% and 46.4% for the oocytes vitrified using cryotops. After in vitro fertilization, 3.8% of the calf oocytes and 5.3% of the cow oocytes developed to the blastocyst stage. All blastocysts from vitrified oocytes resulted from the Cryotop method. A significantly lower percentage of the OPS-vitrified calf oocytes showed a normal spindle configuration (37.8%) compared to control fresh oocytes (69.9%), while normal spindle and chromosome configurations were observed in a significantly higher proportion of the cryotop-vitrified calf oocytes (60.2%). For the cow oocytes, 60.6% in the OPS group and 60.3% in the Cryotop group exhibited a normal morphology after warming. These findings suggest the cryotop system is a more efficient carrier for vitrification than OPS for the cryopreservation of bovine oocytes.

Ultrastructure of bovine oocytes exposed to Taxol prior to OPS vitrification

Our objective was to document potential subcellular consequences of treatment with the microtubule stabilizer Taxol with or without subsequent vitrification of cow and calf oocytes by the open pulled straw (OPS) method. Oocytes were divided into four experimental groups for cows and four groups for calves: (1) a control group fixed immediately after maturation; (2) an OPS group cryopreserved by conventional OPS; (3) a Taxol/CPA group exposed to 1 microM Taxol and cryoprotective agents (CPAs); and (4) a Taxol/OPS group vitrified by OPS including 1 microM Taxol to the vitrification solution. All oocytes were processed for light and transmission electron microscopy. The main injuries were observed on the metaphase plate and the spindle. In control oocytes, the metaphase appeared as condensed chromosomes arranged in a well-organized metaphase plate and the spindle showed well organized microtubules in both cow and calf oocytes. However, in cow OPS oocytes, the metaphase plate was disorganized into scattered chromosomes or the chromosomes were condensed into a single block of chromatin. In addition, microtubules were not organized as typical spindles. In contrast, cow Taxol/OPS oocytes as well as both cow and calf Taxol/CPAs oocytes showed well-organized metaphase plates and normal spindle morphology. All calf OPS and calf Taxol/OPS oocytes displayed a single block of chromatin and no microtubules could be observed around the chromosomes. In conclusion, treatment with 1 microM Taxol before and during vitrification did not induce adverse changes in the oocyte cytoplasm or metaphase spindles in adult bovine oocytes, but stabilized the metaphase and spindle morphology.

124 TAXOL™ COULD PROMOTE EMBRYO DEVELOPMENT OF BOVINE OOCYTES VITRIFIED BY OPS

Stabilizing the cytoskeleton system during vitrification could be beneficial for improving post-thawed survival and subsequent development of vitrified oocytes. Taxol™, paclitaxel, is a microtubule stabilizer that has been found to improve development competence of vitrified mouse and human oocytes. The objective of this work was to study the effect of a Taxol pretreatment before OPS vitrification on the post-thaw cow and calf oocyte development. Oocytes were aspirated from slaughterhouse-derived ovaries and matured in TCM-199. Oocytes were randomly assigned to one of 3 experimental groups: (1) control oocytes matured in vitro for 24 h, (2) oocytes matured for 22 h and vitrified by the OPS method (Vajta et al. 1998 Mol. Reprod. Dev. 51, 53–58), and (3) oocytes matured for 22 h and vitrified by OPS method with 1 µM Taxol. OPS and Taxol–OPS oocytes were transferred back into the maturation dishes and matured for 2 additional h before being subjected to fertilization. Fertilization was performed using frozen–thawed Percoll-selected sperm. At 22 h after insemination, presumptive zygotes were pipetted and then cultured in drops of 25 µL SOF medium and 5% fetal calf serum under paraffin oil at 38.5°C in 5% CO2, 5% O2, 90% N2, and maximum humidity. The Taxol–OPS group provided a significantly higher cleavage rate than the OPS group in cows (41.9% and 34.0%, respectively) or in calves (33.7% and 23.5%, respectively). However, cleavage rate in the experimental groups was significantly lower than in the control group (78.3% and 69.7% for cow and calf control groups, respectively). Blastocyst yield was also higher for the Taxol–OPS group (3.2%) than the OPS group (0%) in cow oocytes. There was no blastocyst development when calf oocytes were vitrified with or without Taxol pretreatment. As expected, cow and calf vitrification groups triggered a significantly lower blastocyst yield when compared with their control (26.7% and 14.9% for cow and calf control groups, respectively). In conclusion, this study showed that supplementation of 1 µM Taxol could promote embryo development after thawing. Further research is indicated to clarify the function of Taxol and its optimal concentration in order to improve the rate of embryo development. Table 1. Effect of Taxol pretreatment on development of cow and calf oocytes vitrified by OPS

316 DEVELOPMENTAL COMPETENCE OF OOCYTES SELECTED BY THE BRILLIANT CRESYL BLUE STAINING IN PREPUBERTAL AND ADULT CATTLE

Transvaginal ultrasound guided ovum pickup (OPU) can be carried out repeatedly not only in adult cows but also in prepubertal calves. Moreover, recently the brilliant cresyl blue (BCB) staining has been used successfully to select oocytes homologous stained with BCB as an indirect measure of oocyte growth for in vitro fertilization (IVF) in several farm animals. The aim of this study was to evaluate the effectiveness of BCB staining on the selection of developmentally competent oocytes, collected from cows and calves by OPU, before in vitro maturation by assessing the embryonic development to the blastocyst stage after IVM/IVF. OPU was performed once weekly for a period of 7 weeks on Japanese Black prepubertal calves (9 months old, n = 4) and adult cows (n = 4) without gonadotropin stimulation. Calf and cow oocytes with homogeneous cytoplasm were exposed to 26 �M BCB for 90 min and classified according to their cytoplasmic coloration: blue coloration (BCB+) or colorless (BCB-). Classified oocytes were then matured for 20 h in TCM-199 supplemented with 5% calf serum (CS). Matured oocytes were inseminated with Percoll-separated spermatozoa (3 � 106 mL) for 6 h in BO solution supplemented with 5 mM hypotaurine and 2 U mL-1 heparin. Presumptive zygotes were cultured in CR1aa supplemented with 5% CS for 8 days. Data were analyzed by Student&amp;apos;s t-test. The mean (&amp;plusmn; SEM) percentage of oocytes classified as BCB&amp;plus; in calves was significantly lower than that in cows (34.4 &amp;plusmn; 2.9&amp;percnt; and 69.2 &amp;plusmn; 2.1&amp;percnt;, respectively; P &amp;lt; 0.01). In cows, BCB&amp;plus; oocytes showed significantly higher cleavage and blastocyst formation percentages (72.5&amp;percnt; and 42.4&amp;percnt;, respectively) than those of BCB&amp;minus; oocytes (47.0&amp;percnt; and 13.0&amp;percnt;, respectively). In contrast, in calves there were no significant differences in cleavage and blastocyst formation percentages between BCB&amp;plus; oocytes (56.9&amp;percnt; and 25.3&amp;percnt;, respectively) and BCB&amp;minus; oocytes (65.4&amp;percnt; and 22.4&amp;percnt;, respectively). The mean (&amp;plusmn; SEM) numbers of usable oocytes and blastocysts obtained per calf (19.0 &amp;plusmn; 1.5 and 4.5 &amp;plusmn; 0.6, respectively) were similar to those obtained per cow (16.4 &amp;plusmn; 1.1 and 5.2 &amp;plusmn; 0.6, respectively). No significant difference was observed in the numbers between calves and cows. These results indicate that the selection of developmentally competent oocytes before IVM/IVF, using the BCB staining, was effective for cow oocytes but not for calf oocytes, and that blastocysts could be produced by OPU-IVF of oocytes from 9-month-old prepubertal calves at an efficiency equivalent to that achieved from adult cows.

Effects of roscovitine on the nuclear and cytoskeletal components of calf oocytes and their subsequent development

Roscovitine, a potent inhibitor of M-phase promoting factor kinase activity, was used to maintain calf oocytes at the germinal vesicle stage for a 24 h culture period. Cumulus–oocyte complexes were first prematured for 24 h in the presence of different levels of roscovitine (12.5, 25, 50 and 100 μM). Roscovitine was shown to block germinal vesicle breakdown in calf oocytes in a concentration dependent manner. Significantly greater inhibitory effect was observed at 50 and 100 μM with 64.6% and 63.2% oocytes being blocked in the germinal vesicle stage when compared to the control (0.0%) and the 12.5 μM (2.9%) and 25 μM (18.8%) groups. However, this inhibitory effect of roscovitine was fully reversible since a substantial number of the oocytes resumed meiosis and reached the metaphase II stage after a further 24 h of culture in a permissive medium. Cleavage rates and blastocyst yields were not significantly different for oocytes cultured under 50 μM roscovitine inhibition compared to oocytes not subjected to prematuration culture (rates of 76.7% cleavage and 8.7% blastocysts for control oocytes compared to 69.8% and 6.3%, respectively, for oocytes pretreated with 50 μM roscovitine). The morphology of the meiotic spindle was typical of metaphase II in 75.8% and 82.1% of the oocytes reaching the metaphase II stage after pretreatment with 50 μM roscovitine compared to control, respectively. A normal distribution of actin filaments was observed in 97.0% and 98.2% of oocytes exposed to 50 μM roscovitine compared to control, respectively. These results demonstrate the feasibility of maintaining calf oocytes in artificial meiotic arrest without compromising their subsequent developmental competence.

340 IN VITRO MATURATION OF PREPUBERTAL BOVINE OOCYTES ON A GRANULOSA CELL MONOLAYER FROM ADULT ANIMALS IMPROVES THEIR DEVELOPMENTAL COMPETENCE

Oocytes from prepubertal calves have a decreased developmental competence compared with oocytes from adult animals. The goal of this study was to improve the developmental competence of juvenile oocytes by maturation on granulosa cell (GC) monolayers from adult animals. Oocytes were recovered by ovum pickup (OPU) from 48 Holstein Friesian calves at 7-8 months of age and 18 adult cows. Animals received intramuscular injections of 60 mg FSH 48 h prior to each OPU session. Follicles were punctured twice per week in six consecutive OPU sessions. Cumulus oocyte complexes (COCs) recovered from calves were divided into three quality groups (classes I-III) and were then randomly distributed into three maturation groups: COCs were matured for 24 h on either GC or fibroblasts or without co-culture. Cow oocytes were matured without co-culture. TCM-199 supplemented with BSA (0.1%), hCG (5 IU/mL), and eCG (10 IU/mL) served as the medium in all groups. After maturation, all COCs were fertilized in vitro; after 18 h, presumptive zygotes were cultured in SOF+BSA for 8 days (37�C, 5% CO2). On Day 3, cleavage rates and, on Day 8, blastocyst rates were determined. The relative mRNA abundance of the following transcripts, critically involved in early embryonic development was determined: growth differentiation factor-9 (GDF-9), heat shock protein 70 (Hsp-70), and glucose transporter-3 (Glut-3). Single immature and matured oocytes (for GDF-9 and Hsp-70) and 8-16-cell embryos and expanded blastocysts (for Hsp-70 and Glut-3) from calves and cows were examined by semiquantitative RT-PCR. Cleavage and blastocyst rates were similar in oocytes derived from cows and calves matured on GC (74.3% vs. 70.0% and 22.3% vs. 22.3%, respectively), but were significantly higher (P &lt; 0.05; one way ANOVA, Student-Newman-Keuls Method) than in the group without co-culture on fibroblasts (55.2% vs. 53.6% and 11.7% vs. 5.5%, respectively). GDF-9 expression was similar in immature calf and cow oocytes. After maturation, a significant decrease in GDF-9 expression was observed in calf oocytes. Matured cow oocytes showed a significantly higher mRNA abundance of GDF-9 than matured calf oocytes. The relative abundance of Hsp-70 was decreased in matured oocytes of all groups. Expanded blastocysts derived from adult oocytes expressed Hsp-70 significantly higher than blastocysts derived from oocytes of the control calves. The relative abundance of Glut-3 mRNA was similar in 8-16 cell embryos and expanded blastocysts in all groups. Overall, mRNA expression pattern for Hsp-70 and Glut-3 in blastocysts from GC matured oocytes were similar to that of cow blastocysts. Results indicate that maturation of juvenile calf oocytes on granulosa cells from adult animals improves their developmental competence. These findings provide clues toward identification of factors critically involved in acquiring full developmental capacity at puberty.

Vitrification of calf oocytes: Effects of maturation stage and prematuration treatment on the nuclear and cytoskeletal components of oocytes and their subsequent development

This study was designed to establish the effects of the meiotic stage of bovine oocytes and of a prematuration treatment with roscovitine (ROS) on their resistance to cryopreservation. Oocytes from prepubertal calves at the stages of germinal vesicle breakdown (GVBD) or at metaphase II (MII) were vitrified by the open pulled straw (OPS) method. In another experiment, oocytes were kept under meiotic arrest with 50 microM ROS for 24 hr and vitrified at the GVBD stage. After warming, some oocyte samples were fixed, stained using specific fluorescent probes and examined under a confocal microscope. The remaining oocytes were fertilized, and cleavage and blastocyst rates recorded. Significantly lower cleavage rates were obtained for the vitrified GVBD and MII oocytes (9.9% and 12.6%, respectively) compared to control oocytes (73.9%). Significantly worse results in terms of cleavage rates were obtained when GVBD calf oocytes were exposed to cryoprotectants (CPAs: ethylene glycol plus dimethyl sulfoxide, DMSO) (13.1%) or vitrified (1.6%) after a prematuration treatment with ROS, when compared to untreated control oocytes (68.7%) or ROS-control oocytes (56.6%). None of the vitrification procedures yielded blastocysts, irrespective of the initial meiotic stage or previous prematuration treatment. Compared to the control oocytes, significantly fewer oocytes exhibited normal spindle configuration after being exposed to CPAs or after vitrification of either GVBD or MII calf oocytes. These results indicate that the vitrification protocol has a deleterious effect on the meiotic spindle organization of calf oocytes cryopreserved at both the GVBD and MII stage, which impairs the capacity for further development of the embryos derived from these vitrified oocytes. Prematuration treatment with ROS has no beneficial effect on the outcome of vitrification by the OPS method.