70 NON-PLATED GRANULOSA AND CUMULUS CELLS AND FIRST PASSAGE FIBROBLASTS AS NUCLEUS DONOR FOR GOAT CLONING

2006 ◽  
Vol 18 (2) ◽  
pp. 143
Author(s):  
D. Salamone ◽  
M. Catala ◽  
A. Gibbons ◽  
F. Pereyra Bonnet ◽  
M. Cueto
Keyword(s):  
Granulosa Cells ◽  
Room Temperature ◽  
Uv Light ◽  
Cumulus Cells ◽  
Cleavage Rate ◽  
First Passage ◽  
Chi Square ◽  
Donor Nucleus ◽  
Chi Square Test

Different types of somatic cells have been used as nucleus donors for cloning. Most of them were previously cultured in vitro as a monolayer through several plate passages. The experiment reported here was conducted to study the potential usages of granulosa and cumulus cells for cloning without previous culture as a monolayer. A first-plate-passage fibroblast was also used. Oocytes were aspirated by laparoscopy from Criolla goats and matured in TCM-199 + 5% FCS at 39°C for 24 h. Matured oocytes were denuded by vortexing for 3 min in TL HEPES with 1 mg/mL bovine testis hyaluronidase. Metaphases were assessed and oocytes were enucleated by visualization with Hoechst 33342 (5 μg/mL) under UV light (<6 s). Granulosa and cumulus cells were also recovered by laparoscopy and maintained in maturation medium in cryotube for 20 h at room temperature or 39°C, respectively. Goat adult ear fibroblasts were cultured for 1 or 2 weeks and used 2 days after confluence. All types of donor cells were transferred to the perivitlline space of enucleated oocytes and fused by an electrical pulse. After 2 h, activation was induced by incubation in TL-HEPES with 5 µM ionomycin for 4 min and 2 mM 6-DMAP for 3 h. The oocytes were then washed with TL-HEPES and cultured in SOF medium and atmosphere of 5% CO2 + 5% O2 + 90% N2. Cleavage (Day 2) and development to blastocysts (Day 6) were recorded and analyzed by chi-square test. The cleavage rate for non-plated granulosa cells was higher than for the other treatment goups; cumulus cells had a lower rate of development to blastocysts (Table 1). These results suggest that granulosa cells collected and maintained for 24 h at room temperature could be used to produce cloned blastocysts. Table 1. Effect of non-plated granulosa and cumulus cells and first passage fibroblasts as donor nucleus oocytes in goat cloning

scholarly journals 46 Effect of the addition of α-tocopherol to in vitro maturation media of bovine oocytes

2020 ◽  
Vol 98 (Supplement_2) ◽  
pp. 2-3
Author(s):  
Theisy P Acosta Pérez
Keyword(s):  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Control Group ◽  
Chi Square ◽  
Cumulus Expansion ◽  
Minimum Concentration ◽  
Maturation Rate ◽  
Chi Square Test ◽  
System Data

Abstract α-tocopherol is known to be a powerful antioxidant, in this regard, it was added to bovine oocyte in vitro maturation media to evaluate its effect on oocyte maturation. Oocytes (n = 624) aspirated from ovaries of slaughtered cows were classified by quality and divided in four categories according to cytoplasm appearance and cumulus cells layers. Oocytes were washed in TCM-199 supplemented with fetal bovine serum (FBS) and FSH, then distributed in maturation media (TCM-199 supplemented with FBS, FSH and gentamicin). Three experimental groups of α-tocopherol (50, 100 and 200 mM) and a control group without α-tocopherol were used. Maturation was carried 22 h at 38.5°C in a 5% CO2 atmosphere. Oocytes were examined to determine cumulus expansion as categorical data (expansion or no expansion), as well as cumulus expansion Index (CEI). For CEI determination oocytes were graded 0 to 4, being 0 those with null expansion and 4 those with a noticeable cell expansion, then the number of oocytes were multiplied by the grade given and a sum of the totals was obtained, the new total was divided by the total of oocytes in the group and the result obtained corresponded to the CEI of the group. Results were analyzed with Chi Square test (for maturation rates) and an ANOVA (for the CEI) using the SAS system, data are presented as mean ± standard error. There was no statistical difference between control and α-tocopherol groups (P &gt;0.05). Numerically, the control group showed a higher maturation rate (100%) and obtained a higher CEI (2.44±0.20), followed by the 50 mM group (98.16%; 2.39±0.13), the groups 200 mM (97.40%; 2.00±0.14) and 100 mM (96.25%; 2.06±0.24) were the lowest. The addition of the minimum concentration (50 mM) of α-tocopherol to the maturation media could improve maturation rates without exposing oocytes to toxic effects.

284 FERTILIZING CAPACITY OF BUFFALO (BUBALUS BUBALIS) SPERM CO-CULTURED WITH OVIDUCT EPITHELIAL CELLS

2010 ◽  
Vol 22 (1) ◽  
pp. 299 ◽  
Author(s):  
E. Mariotti ◽  
S. Di Francesco ◽  
M. De Blasi ◽  
C. Siniscalchi ◽  
M. V. Suárez ◽  
...  
Keyword(s):  
Production Efficiency ◽  
Penetration Rate ◽  
Bubalus Bubalis ◽  
Cleavage Rate ◽  
Chi Square ◽  
Chi Square Test ◽  
Group A ◽  
Group B ◽  
Bovine Oocytes

The overall in vitro embryo production efficiency in buffalo is hampered by the poor IVF efficiency. The aim of this work was to evaluate whether the fertilizing ability of buffalo sperm is improved by the presence of bovine oviductal cells (BOEC) during IVF. Because of limited availability of buffalo oocytes, this was assessed by heterologous IVF. Bovine oviducts were obtained at a local abattoir from cows that were in the preovulatory phase of a normal estrous cycle. BOEC recovered from 5 oviducts as previously described (Gualtieri and Talevi 2000 Biol. Reprod. 62, 1754-1762) were pooled and plated in 100 μL drops of TCM-199 + 10% FCS, 100 U mL-1 penicillin, 100 μg mL-1 streptomycin and 0.25 μg mL-1 amphotericin B under mineral oil. Medium was changed every 48 h up to Day 6, when cell confluence and cilia activity were optimal. On day of IVF the medium was removed from the drops and replaced with TALP supplemented with 0.2 mM penicillamine, 0.1 mM hypotaurine, and 0.01 mM heparin (IVF medium). Frozen-thawed sperm from an IVF-tested buffalo bull, treated by Percoll gradients, were used for all IVF groups (2 × 106 sperm mL-1). In vitro-matured bovine oocytes (n = 409), over 3 replicates, were distributed in 4 fertilization groups: (A) IVF medium alone (control); (B) BOEC monolayer + IVF medium; (C) sperm preincubated for 6 h in IVF medium; and (D) sperm preincubated for 6 h with BOEC + IVF medium. After 20 h of coincubation at 38.5°C and 5% CO2 in air, putative zygotes were denuded, washed, and cultured in SOF medium. Forty-eight hours after IVF, cleavage rate was evaluated, and cleaved and uncleaved oocytes were fixed in 60% methanol and stained with DAPI for nuclei examination under fluorescence microscope. Data were analyzed by chi-square test. Although cleavage rate was not different among groups (46.2, 55.8, 50.0, and 50.0% for A, B, C, and D, respectively), the monospermic penetration rate increased (P < 0.01) in group B (79.3%) compared with group A (69.6%), with intermediate values in groups C (75.2%) and D (76.0%). Interestingly, the percentage of advanced embryos (>4 cells) was higher (P < 0.01) in groups C and D (47.9 and 37.1%, respectively) than in group A (12.1%), whereas group B (21.0%) was only different from group C. We demonstrated that the fertilizing capacity of buffalo sperm, evaluated as oocyte penetration rate after heterologous IVF, is enhanced by the presence of BOEC. This suggests that IVF of buffalo oocytes on BOEC monolayer may improve the IVF efficiency in buffalo. The higher incidence of advanced embryos in both groups with preincubated sperm may be accounted for by an earlier accomplishment of capacitation, leading to anticipated oocyte penetration. However, because the penetration rate in these groups was not improved compared with the control, we hypothesize that sperm viability may have decreased and hence that shorter incubation times should be tested in further studies.

205 EFFECT OF OSTEOPONTIN ON CLEAVAGE AND BLASTOCYST RATES IN BUFFALO SPECIES (BUBALUS BUBALIS)

2009 ◽  
Vol 21 (1) ◽  
pp. 200
Author(s):  
S. Di Francesco ◽  
E. Mariotti ◽  
M. Rubessa ◽  
G. Campanile ◽  
R. Di Palo ◽  
...  
Keyword(s):  
Embryo Development ◽  
Cumulus Cells ◽  
Bubalus Bubalis ◽  
The Other ◽  
Control Group ◽  
Single Chain ◽  
Cleavage Rate ◽  
Chi Square ◽  
Vitro Fertilization

It was previously reported that osteopontin (OPN), an acidic single-chain phosphorylated glycoprotein found in the oviductal fluid in cattle (Gabler C et al. 2003 Reproduction 126, 721–729), is able to facilitate fertilization in this species (Gasparrini B et al. 2008 Reprod. Fertil. Dev. 20(Suppl. I), 180 abst). The present study aimed to investigate whether the addition of OPN to the fertilization medium would affect both cleavage and postfertilization embryo development in the buffalo. To assess the influence of OPN on cleavage and blastocyst rates, in vitro-matured oocytes were fertilized in modified Tyrode’s albumin lactate pyruvate medium (Lu KH et al. 1987 Vet. Rec. 121, 259–260) supplemented with penicillamine, hypotaurine, and heparin, in the presence of 0.0 (n = 258), 0.1 (n = 263), 1 (n = 261), and 10 μg mL–1 (n = 264) of OPN. In vitro fertilization was carried out with frozen–thawed spermatozoa from a bull already tested for IVF. After 20 to 22 h of co-incubation at 38.5°C and 5% CO2 in air, putative zygotes were gently pipetted to remove cumulus cells, washed, and transferred, 10 per droplet, into 20 μL of SOF medium including essential and nonessential amino acids and BSA (Tervit HR et al. 1972 J. Reprod. Fertil. 30(3), 493–497), in a controlled gas atmosphere consisting of 5% CO2, 7% O2, and 88% N2, in humidified air, at 38.5°C. The culture medium was changed on Day 5 (Day 0 = day of insemination), when cleavage rate was assessed and embryos were moved into fresh medium for an additional 2 days. On Day 7, development rates into blastocysts of superior quality were recorded. Differences in the percentages of both cleavage and blastocyst rates among groups were analyzed by chi-square test. Significantly higher cleavage rates (59.3, 70.3, 71.6, and 42.4%, respectively, in the control group and in the groups with 0.1, 1, and 10 μg mL–1 of OPN; P < 0.01) were observed in the groups with 0.1 and 1 μg mL–1 of OPN compared with the other groups. Likewise, higher blastocyst rate percentages (17.4, 27.4, 29.9, and 9.5%, respectively, in the control group and in the groups with 0.1, 1, and 10 μg mL–1 of OPN; P < 0.01) were observed in the groups with 0.1 and 1 μg mL–1 of OPN compared with the other groups. In conclusion, these results showed that addition of low concentrations of OPN in the fertilization medium improved both cleavage and postfertilization embryo development in the buffalo, whereas the higher concentration resulted in impaired late-stage embryo development.

137 EFFECT OF THE ADDITION OF FOLIC ACID TO MATURATION AND CULTURE MEDIA ON DEVELOPMENT OF THE BOVINE BLASTOCYST AND ITS SURVIVAL RATE AFTER FREEZE-THAWING

2017 ◽  
Vol 29 (1) ◽  
pp. 177
Author(s):  
S. Sato ◽  
O. Dochi ◽  
K. Imai
Keyword(s):  
Folic Acid ◽  
Survival Rate ◽  
Culture Media ◽  
Cell Damage ◽  
Calf Serum ◽  
Cleavage Rate ◽  
Chi Square ◽  
Exact Test ◽  
Chi Square Test

Reactive oxygen species (ROS) are the main causes of cell damage in bovine embryos in vitro. Folic acid (FA) is an antioxidant that protects cells from ROS. We studied the effect of the addition of FA to maturation and culture media on development of bovine blastocysts and their survival rate after freeze-thawing. Cell-oocyte complexes (COC) were allowed to mature in HEPES (25 mM)-buffered TCM199 (TCM199) supplemented with 5% calf serum (CS), 0.02 AU mL−1 of FSH, and FA (0, 2.5, 25, and 50 µM) for 20 hours (20–25 COC/100-µL droplet of the medium). After 6 hours of gamete co-culture (5 × 106 sperm/mL), presumptive zygotes were cultured in CR1aa medium supplemented with 5% CS and FA (0, 2.5, 25, and 50 μM) for 9 days (day of fertilization = Day 0). Expanded blastocysts that developed from Day 7 to 9 were frozen for further study. Each embryo was frozen in Dulbecco’s PBS (D-PBS) supplemented with 20% CS, 1.5 M ethylene glycol (EG), and 0.1 M sucrose (SUC). Embryos were equilibrated with their freezing medium for 15 min and loaded individually into a 0.25-mL straw. These straws were put into the cooling chamber of a programmable freezer precooled at −7°C. After 2 min, straws were seeded and held for 13 min at −7°C. Next, straws were cooled to −30°C at −0.3°C/min before being plunged into liquid nitrogen. Frozen embryos were thawed by allowing straws to stand in air for 7 s and warming them in a 30°C water bath for 20 s. Thawed embryos were washed twice with D-PBS supplemented with 20% fetal calf serum (FCS), which was warmed to 38°C. They were immersed into the same medium at 38°C for 10 min, and each embryo was cultured in a 20-μL droplet of TCM199 supplemented with 10% FCS and 0.1 mM β-mercaptoethanol (TCM-199-βME) for 72 h. Embryo cleavage rate was observed at 55 h post-insemination. Blastocyst rates were analysed at 9 days post-insemination. Rates of embryos developing into reexpanded, hatching, and hatched blastocyst stages were determined after 72 h of thawing. All data were analysed by the chi-square test and Fisher’s exact test. Cleavage and blastocyst rates after insemination at 55 hours and 9 days, respectively, were not significantly different among media containing 0 μM (n = 278; 74.1% and 39.9%), 2.5 μM (n = 260; 74.2% and 45.8%), 25 μM (n = 258; 75.6% and 45.7%), and 50 μM (n = 253; 76.3% and 42.7%) FA. Survival and hatching rates of frozen and thawed expanded blastocysts after 72 h in culture were 62.5% and 56.3%, respectively, in 0 μM FA (n = 16); 85.2% and 74.1% in 2.5 μM FA (n = 27); 66.7% and 62.5% in 25 μM FA (n = 24); and 68.0% and 64.0% in 50 μM FA (n = 25). Blastocysts cultured in media containing 2.5 μM FA tended to have a higher survival rate than those cultured in media containing 0 μM FA, although this difference was not significant (P = 0.09). Inclusion of FA did not appear to influence development or post-thaw survival of bovine blastocysts produced in vitro.

360 EMBRYONIC DEVELOPMENT AND STEROIDOGENESIS OF BOVINE CUMULUS - OOCYTE COMPLEXES MATURED IN α-MEM MEDIUM SUPPLEMENTED WITH PVA OR PVP-40

2010 ◽  
Vol 22 (1) ◽  
pp. 337
Author(s):  
A. A. Vireque ◽  
L. S. A. Camargo ◽  
E. M. Ferreira ◽  
A. A. M. Rosa e Silva ◽  
Y. F. Watanabe ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Culture Medium ◽  
Cumulus Cells ◽  
Control Group ◽  
Aromatase Activity ◽  
Cleavage Rate ◽  
Chi Square ◽  
Chi Square Test ◽  
Sigma Chemical ◽  
Hatching Rates

The influence of the culture medium and its supplements on IVM and steroid secretion by bovine COCs was examined. Immature COCs were matured for 24 h in a-MEM supplemented with IGF-1 (10 ng mL-1), insulin (100 ng mL-1) and 0.1% polyvinyl alcohol (PVA; Sigma Chemical Co., St. Louis, MO, USA) or 0.1% polyvinylpyrrolidone-40 (PVP; molecular weight of 40 000; Sigma). Neither FSH nor LH was used in either treatment. The control group consisted of COCs matured in TCM supplemented with FSH (20 μg mL-1) and 10% estrous cow serum. After fertilization, presumptive zygotes were co-cultured with cumulus cells until 224 h post-insemination. Steroid production was measured in culture medium after IVM and cumulus cells (CC) aromatase activity was estimated by estradiol (E2) production and by the determination of the ratio of E2 to testosterone (T). Cleavage, blastocyst, and hatching rates were evaluated 168-224 h post-insemination. Hormone determination data were analyzed using the GraphPad 5.0 for Windows software (GraphPad Software, San Diego, CA, USA). The means and standard deviations were first calculated for all variables, followed by the Kolmogorov-Smirnov normality test. Since the values were not normally distributed, the treatments were compared by the Kruskal-Wallis test. Blastocyst formation, cleavage, and hatching rates were analyzed by the chi-square test. The level of significance was set at P < 0.05 in all analyses. High aromatase activity (E2:T ratio > 1.0) was detected in the culture medium of both chemically defined IVM systems. E2 concentrations were 22.86 ng mL-1 and 22.46 ng mL-1 for PVA and PVP-40, respectively, and 0.27 ng mL-1 (P < 0.001) for the control group. Progesterone secretion was lower in a-MEM + PVP-40 medium. Testosterone was not secreted by COCs matured in control medium. There was a significantly higher cleavage rate in the control group, but no differences (P > 0.05) in blastocyst (48.92%, 49.56%, and 44.21%) or hatching (38.46%, 41.96%, and 40.78%) rates between the PVA, PVP, and control groups, respectively. Our results show that CC of COCs cultured in serum-supplemented medium show decreased aromatase activity. Also, the addition of IGF-I/insulin and PVA or PVP-40 to IVM medium had no significant effect on the rates of oocyte maturation and embryonic development when compared with results obtained in medium supplemented with estrous cow serum and FSH. Supported by FAPESP/FAEPA-HCFMRP/USP.

265 INHIBITION OF CLASS III PHOSPHATIDYLINOSITOL-3-KINASE, BY 3-METHYLADENINE, REVERSIBLY ARRESTS PORCINE OOCYTES AT GERMINAL VESICLE STAGE

2011 ◽  
Vol 23 (1) ◽  
pp. 230
Author(s):  
M. R. Park ◽  
M. K. Gupta ◽  
S. J. Uhm ◽  
S. T. Shin ◽  
Y. M. Han ◽  
...  
Keyword(s):  
Specific Inhibitor ◽  
Indirect Evidence ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Class Iii ◽  
Cleavage Rate ◽  
Chi Square ◽  
Meiotic Progression ◽  
Phosphatidylinositol 3

Phosphatidylinositol-3-kinases (PI3K) play pivotal roles in the meiotic progression of oocytes from metaphase I to metaphase II stage. This study evaluated the effect of 3-methyladenine (3MA), a specific inhibitor of Class III PI3K, on the meiotic progression of porcine oocytes. Immature porcine oocytes (n = 4744) retrieved from abattoir-derived oocytes were cultured in the absence or presence (10 mM) of 3MA for 22 h and evaluated for meiotic progression by florescent Hoechst 33342 staining. Data were analysed by chi-square test or ANOVA using SPSS software, and differences at P < 0.05 were considered significant. Results showed that 3MA treatment arrested the immature porcine oocytes at germinal vesicle (GV) stage in the presence (99.2 ± 0.8 v. 54.0 ± 10.1%) or absence (96.5 ± 1.8 v. 41.0 ± 17.6%) of cumulus cells. Furthermore, a significantly high proportion (60.9 ± 13.8%) of 3MA-treated oocytes acquired a nucleolus completely surrounded by a rim of highly condensed chromatin (GV-II stage). When immature oocytes, arrested at GV stage for 22 h by 3MA, were further cultured for 22 h in the absence of 3MA, 96.1 ± 1.5% of oocytes reached metaphase II stage at 42 h of in vitro maturation and did not differ (P > 0.05) from nontreated control oocytes with respect to their ability to fertilize, cleave (74.1 ± 1.2 v. 72.7 ± 2.8%), and form blastocyst (15.4 ± 1.5 v. 12.7 ± 0.6%) upon IVF or parthenogenetic activation (cleavage rate: 89.8 ± 1.7 v. 84.6 ± 5.1%; blastocyst rate: 44.3 ± 12.4 v. 45.1 ± 7.6%). These data suggest that 3MA reversibly blocks and synchronizes the meiotic progression of porcine oocytes at GV stage without affecting their ooplasmic maturation in terms of post-fertilization/activation in vitro embryonic development. Our data also provide indirect evidence for the likely participation of Class III PI3K in meiotic maturation of porcine oocytes beyond GV stage. This work was supported by grants (Code #200901FHT010305191 and #20070401034017) from BioGreen 21 program of RDA, Republic of Korea.

54 ESTABLISHMENT OF PREGNANCY BY OVINE NUCLEAR TRANSFER EMBRYOS RECONSTRUCTED USING CAFFEINE-TREATED IN VITRO-MATURED OOCYTES AS CYTOPLAST RECIPIENTS

2006 ◽  
Vol 18 (2) ◽  
pp. 135 ◽  
Author(s):  
J.-H. Lee ◽  
M. Marfil ◽  
M. Panarace ◽  
M. Medina ◽  
K. H. S. Campbell
Keyword(s):  
Nuclear Transfer ◽  
Animal Health ◽  
Subsequent Development ◽  
Blastocyst Stage ◽  
Interferon Tau ◽  
Chi Square ◽  
Developmental Potential ◽  
Donor Nucleus ◽  
Chi Square Test

In embryos reconstructed by somatic cell nuclear transfer (SCNT), components of the oocyte cytoplasm are capable of reprogramming the somatic genome to control subsequent development. Although the mechanisms that control nuclear reprogramming are unknown, we have previously hypothesized that the occurrence of nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC) in the donor nucleus are beneficial. In previous studies we have demonstrated that treatment of ovine oocytes with caffeine (10 mM), a protein phosphatase inhibitor, increased the activities of both MPF and MAPK in enucleated oocytes (Lee and Campbell 2004 Reprod. Fertil. Dev. 16, 125) and additionally resulted in a significant increase in the occurrence of NEBD and PCC in donor nuclei. Furthermore, SCNT embryos reconstructed following caffeine treatment had significantly increased cell numbers at the blastocyst stage. More recently we have demonstrated that the use of caffeine treated ovine oocytes as cytoplast recipients can regulate the expression of several developmentally important genes in SCNT embryos, including Oct-4 and interferon-tau (Choi et al. 2006 Reprod. Fertil. Dev. 18, in press). This study was designed to establish the developmental potential of NT embryos reconstructed using caffeine treated oocytes as cytoplast recipients. Ear skin fibroblast cells established from a Merino ram were quiesced in DMEM containing 0.1% fetal bovine serum (FBS) for 3 days. Oocyte maturation and embryo reconstruction and culture were performed as previously described (Lee and Campbell 2004 Reprod. Fertil. Dev. 16, 125) with the exception that ovaries from Merino � Romney Marsh cross ewes were stimulated with FSH sponge (Folltropin�-V; Bioniche Animal Health, Beltsville, Ontario, Canada) and were collected at slaughter on Day 13 following sponging. Blastocyst stage embryos were surgically transferred to the uterine horn of synchronized Merino � Romney Marsh cross recipients (three blastocysts per recipient). Recipient ewes were scanned by ultrasonography at Days 30, 60, and 90 following embryo transfer. All data were analyzed by chi-square test. There were no differences in fusion (145/167; 86.8% vs. 174/205; 84.9%), cleavage (123/145; 84.8% vs. 135/174; 77.6%), or the development to blastocyst (33/145; 22.8% vs. 34/174; 19.5%) between control SCNT embryos and caffeine treated SCNT embryos. However, although the frequency of pregnancy between control and caffeine-treated NT groups (5/15; 33.3% vs. 7/14; 50.0%) at 30 days was not significantly different, control SCNT embryos showed significantly lower pregnancies (1/15; 6.7%) than caffeine treated SCNT embryos (4/14; 28.6%) at both 60 and 90 days. In conclusion, embryos reconstructed using caffeine-treated cytoplasts can induce pregnancy at the same frequency as untreated controls; furthermore, the results suggest that SCNT embryos produced in this way are more able to maintain pregnancy.

124 CO-CULTURE WITH BOVINE INTACT CUMULUS - OOCYTE COMPLEXES DURING IN VITRO FERTILIZATION OF BUFFALO DENUDED OOCYTES COMPLETELY RESTORES THEIR FERTILIZING AND DEVELOPMENTAL COMPETENCE

2011 ◽  
Vol 23 (1) ◽  
pp. 167
Author(s):  
M. De Blasi ◽  
M. Rubessa ◽  
L. Boccia ◽  
S. Di Francesco ◽  
M. V. Suárez Novoa ◽  
...  
Keyword(s):  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Negative Control ◽  
Developmental Competence ◽  
Control Group ◽  
Chi Square ◽  
Oocyte Vitrification ◽  
Vitro Fertilization ◽  
Chi Square Test

Removal of cumulus cells is necessary for several technologies such as vitrification, intracytoplasmic sperm injection, and nuclear transfer. However, it is known that the presence of cumulus cells during IVF of buffalo oocytes is fundamental for fertilization and embryo development (Gasparrini et al. 2007 Anim. Reprod. Sci. 98, 335–342; Nandi et al. 1998 Theriogenology 50, 1251–1262). The aim of this work was to evaluate whether co-culture with intact bovine cumulus–oocyte complexes (COC) during IVF would restore the developmental competence of denuded buffalo oocytes. Due to the scarce availability of buffalo ovaries, the somatic support was provided by bovine cumulus cells. Abattoir-derived COC were matured in vitro according to our standard procedures (Gasparrini et al. 2006, Theriogenology, 65, 275–287) and randomly distributed in 3 fertilization groups: 1) a control group of COC (n = 122), 2) a negative control of denuded oocytes (DO; n = 119), and 3) DO co-cultured with in vitro matured bovine COC (DO+COC; n = 103) in a 1:1 ratio (3 bovine COC + 3 denuded buffalo oocytes/50 μL drop). Fertilization was carried out with frozen–thawed spermatozoa from a tested bull in TALP medium supplemented by 0.2 mM penicillamine, 0.1 mM hypotaurine, and 0.01 mM heparin at 38.5°C under a controlled gas atmosphere of 5% CO2 in humidified air. After fertilization the zygotes were cultured in SOF medium including essential and nonessential amino acids and 8 mg mL–1 BSA, at 38.5°C under humidified 5% CO2, 7% O2, and 88% N2, up to the blastocyst stage. On Day 5 and on Day 7 (Day 0 = IVF) cleavage and blastocyst rates were respectively recorded. Data were analysed by chi-square test. As expected, cleavage and blastocyst rates were lower (P < 0.01) in DO (36.1 and 9.2%, respectively) compared with the control (67.2 and 27.1%, respectively). However, co-culture during IVF (DO+COC) significantly increased (P < 0.01) both parameters compared with DO, giving cleavage (70.9%) and blastocyst (27.2%) rates similar to the control. The results of this study demonstrated that co-culture with bovine intact COC during IVF of buffalo denuded oocytes completely restores their fertilizing capability and blastocyst developmental competence. We conclude that this may be a suitable strategy for preserving the developmental competence of oocytes devolved to technologies, such as oocyte vitrification, that require cumulus removal.

202 EFFECT OF OSTEOPONTIN ON IN VITRO EMBRYO DEVELOPMENT IN CATTLE

2008 ◽  
Vol 20 (1) ◽  
pp. 180 ◽  
Author(s):  
B. Gasparrini ◽  
E. Monaco ◽  
L. Boccia ◽  
A. De Rosa ◽  
L. Attanasio ◽  
...  
Keyword(s):  
Embryo Development ◽  
Bovine Milk ◽  
Cumulus Cells ◽  
Control Group ◽  
Single Chain ◽  
Chi Square ◽  
Vitro Fertilization ◽  
Chi Square Test ◽  
Oviduct Fluid

Osteopontin (OPN) is an acidic single-chain phosphorylated glycoprotein found both in the oviduct fluid (ODF) and oviductal epithelium in cattle, which is believed to facilitate fertilization. It was recently reported that addition of a rabbit polyclonal immunoglobulin G antibody against purified bovine milk OPN with sperm oocytes, bovine oocytes, or both decreased (P < 0.05) fertilization compared with the in vitro-fertilized control (Goncalves et al. 2007 Theriogenology 67, 468–74). The aim of the present work was to determine the effect of in vitro addition of OPN to the fertilization medium on both cleavage and postfertilization embryo development in cattle. In the first experiment, in vitro-matured oocytes were fertilized in modified TALP medium in the presence of 0.0, 0.1, 1.0, or 10 µg mL–1 of OPN. In a second experiment, matured oocytes were in vitro-fertilized in modified TALP medium in the presence of 0.0, 10, 20, or 40 µg mL–1 of OPN. In vitro fertilization was carried out with frozen–thawed spermatozoa from a bull previously tested for IVF. After 20 to 22 h of coincubation at 39�C, 5% in CO2 in air, presumptive zygotes were vortexed to remove cumulus cells, washed, and transferred, 30 to 50 per well, into 400 µL of SOF modified medium. Zygotes were incubated in a humidified mixture of 5% CO2, 7% O2, and 88% N2 in air at a temperature of 39�C. On Day 7 of development (Day 0 = day of insemination), cleavage and development rates into transferable embryos (TE)–tight morulae (TM) and blastocysts (Bl) of superior quality were recorded. Differences in the percentages of both cleavage and blastocyst rates among groups were analyzed by a chi-square test. In experiment 1, numerically higher percentages of TM–Bl (29.5, 29.5, 30.5, and 37.5%, respectively, in the control group and in the groups with 0.1, 1, and 10 µg mL–1 of OPN; P = 0.25) and Bl (28.6, 27.5, 29.1, and 36.7, respectively, in the control group and in the groups with 0.1, 1, and 10 µg mL–1 of OPN; P = 0.24) were observed with 10 µg mL–1 of OPN. In experiment 2, significantly more cleavage (80.0 v. 71.3%; P < 0.05) and higher percentages of TM–Bl (44.6 v. 34.5%; P < 0.05) and Bl (39.2 v. 30.6%; P = 0.06) were observed with 10 µg mL–1 of OPN v. the control. Combined analysis from both experiments showed an overall effect of 10 µg mL–1 of OPN v. the control in the percentages of TM–Bl and Bl (respectively, 41.1 v. 33.3% and 37.7 v. 30.5%; P < 0.05). These results indicate that it is possible to improve the efficiency of bovine in vitro embryo production by adding the oviductal protein OPN.

298 THE INFLUENCE OF DISTINCT CENTRAL BULL STATIONS ON THE IN VITRO EMBRYONIC DEVELOPMENT OF NELORE BULLS

2010 ◽  
Vol 22 (1) ◽  
pp. 305
Author(s):  
A. Renzi ◽  
F. P. Elias ◽  
R. A. Vila ◽  
R. B. Lôbo
Keyword(s):  
Calf Serum ◽  
Cumulus Cells ◽  
In Vitro Production ◽  
Freezing And Thawing ◽  
Chi Square ◽  
Frozen Semen ◽  
Chi Square Test ◽  
Vitro Production ◽  
In Vitro Embryonic Development

Reproductive biotechnology is growing worldwide as one of the most important tools of cattle breeding because it accelerates the process of genetic improvement. Most of the embryos produced are obtained using frozen semen from different AI centers. During freezing and thawing of semen, the sperm can be damaged by the rapid and dramatic changes in the physicochemical conditions that occur during cooling and ice formation. It has previously been described that bad management of frozen semen can result in reduced fertilization. This study investigated the influence of different central bull stations on the development of in vitro-produced bovine embryos. We compared semen of 154 Nelore bulls, used for IVF, from 8 different central bull stations (all of which used the same cryopreservation protocol) in the development of blastocysts. The in vitro production of embryos was performed as described: oocytes were collected from the slaughterhouse and matured in TCM-199 + 10% fetal calf serum (FCS) +0.5 μg mL-1 FSH + 50 μg mL-1 LH+ 1 μg mL-1 estradiol, for 24 hat 38.5°C in 5%CO2 in atmospheric air. Viable spermatozoids were obtained by centrifugation in Percoll gradient (45 and 90%), and used for IVF in a concentration of 2 million spermatozoa per mL in TALP + 10 μg mL-1 of heparin medium. After 12 h, the presumptive zygotes were transferred to a CR2+ 10% FCS medium and co-cultured with cumulus cells. After 168 h of IVF, we evaluated the number and stage of cleaved embryos produced with the semen of each bull. Statistical analyses were performed by using the chi-square test. Our results suggest that there are differences among distinct central bull stations in the proportion of embryos that developed into blastocysts and the different stages they hatched. FAPESP, CNPq, PROEX, FAEPA.

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