212 INSEMINATION OF OVUM PICKUP-DERIVED DAIRY COWS RESULTS IN OFFSPRING WITH NORMAL BIRTH WEIGHT

2008 ◽  
Vol 20 (1) ◽  
pp. 185
Author(s):  
E. Mullaart ◽  
B. Landman ◽  
J. S. Merton
Keyword(s):  
Birth Weight ◽  
First Generation ◽  
Second Generation ◽  
Calf Serum ◽  
Control Group ◽  
High Birth Weight ◽  
Gestation Length ◽  
Aberrant Methylation ◽  
Epigenetic Changes

Animals derived by ovum pickup-in vitro production (OPU-IVP) have a higher birth weight compared with animals derived by AI (Wagtendonk et al. 2000 Theriogenology 53, 575–597). It has been suggested that this higher birth weight is the result of epigenetic changes such as aberrant methylation and gene expression pattern, which are caused by the presence of serum in the culture medium (Wrenzycki et al. 2004 Anim. Reprod. Sci. 82–83, 593–603). The present study aimed to investigate whether the higher birth weight, possibly caused by epigenetic changes, is a permanent characteristic that is transmitted to the offspring. We therefore monitored the birth weight of calves born after insemination of OPU-IVP-derived animals. Ovum pickup-IVP was performed according to routine procedures. Immature COC were recovered by OPU. The COC were matured in vitro in TCM-199 supplemented with fetal calf serum (FCS)/LH/FSH. Subsequently, matured oocytes were fertilized with frozen–thawed gradient-separated semen and further cultured for 7 days in TCM-199/10% FCS on a BRL monolayer (CoCul group) or in SOFaaBSA (SOF group). First-generation OPU-IVP animals were produced from oocytes collected by OPU of AI-derived animals. The second generation was produced by inseminating OPU-IVP animals. Calves generated by inseminating AI animals were used as a control group. Birth weights of control AI, first-generation, and second-generation calves were analyzed by using restricted maximum likelihood (Genstat 9.1). Model Birth Weight: *Fixed: Parity Recipient + Sex + Gestation Length + Year + Embryo Type (AI, first, or second generation) + Culture System (CoCul or SOF). *Random: Sire + Barn. The results (Table 1) clearly show that the first-generation (OPU-IVP) calves had, on average, a 3.4-kg greater birth weight than the AI calves. The second-generation calves, however, had approximately the same birth weight as the calves in the AI control group. Our results indicated that the high birth weight of OPU-IVP-derived calves is not a permanent characteristic that is transmitted to their offspring. Previous studies have demonstrated that the fertility of OPU-IVP-derived animals is in the normal range (Wagtendonk et al. 2000 Theriogenology 53, 575–597). Table 1. Birth weight (least squares means ± SE) of AI calves (control), first generation OPU-IVP-derived calves, and second generation AI derived calves from OPU-IVP mothers

79RELATIONSHIPS BETWEEN SURVIVAL RATE AND BIRTH WEIGHT IN CLONED KOREAN NATIVE CALVES

2004 ◽  
Vol 16 (2) ◽  
pp. 161
Author(s):  
B.C. Yang ◽  
G.S. Im ◽  
D.H. Kim ◽  
S.K. Lee ◽  
H.S. Park ◽  
...  
Keyword(s):  
Birth Weight ◽  
Survival Rate ◽  
Caesarean Section ◽  
Survival Rates ◽  
Blastocyst Stage ◽  
High Birth Weight ◽  
Gestation Length ◽  
Average Birth Weight ◽  
Fetal Bovine

Cloning of somatic cells has been investigated actively in cattle, but the cloned calves have been characterized by high birth weight and low survival rate. The present study was conducted to investigate the relationships between survival rate and birth weight in cloned and AI calves. The ear skin fibroblasts were obtained from 2- to 3-year-old Korean native cows (Hanwoo) and the cells were cultured in Dulbeccos Modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 38.5°C, 5% CO2 in air. Bovine oocytes collected from ovaries obtained from a nearby slaughterhouse were cultured in vitro and then enucleated, injected with donor cells and fused, and cultured to produce cloned embryos at the blastocyst stage. Somatic cell cloning and in vitro culture of embryos were performed by the procedures described previously (Im et al., 2001 AJAS 14, 759–764, and Im et al., 2001 AJAS 14, 1260–1266). A total of 580 cloned embryos at blastocyst stage were transferred to 293 recipient cows; 32 female calves (5.5%) were born (2 of them were born dead). Thirty-four (15 female and 19 male ) calves (57.6%) were born from 59 artificially inseminated Korean native cows as control. Fifteen of the 32 cloned calves were delivered by caesarean section. However, all the artificially inseminated cows delivered naturally. Birth weights of 30 live cloned calves averaged 31.08kg (>15kg:3, 20kg:2, 25kg:2, 30kg:5, 35kg:9, 40kg:6, <45kg:3), while those of female AI calves averaged 23.67kg (>15kg:0, 20kg:3, 25kg:6, 30kg:6, 35kg:0, 40kg:0, <45kg:0). After calving, 11 of 30 cloned calves survived for more than 365 days (birth weight of these calves averaged 28.25kg), but 19 of 30 calves died within 175 days and their average birth weight was 32.80kg (650kg). Gestation length of cows that received cloned embryos was 287 (279–295) days on average (excluding the data of calves delivered by caesarean section) and that of cows artificially inseminated was 287 (255–293) days. In conclusion, the birth weight was significantly correlated (P<0.05) with survival rate of cloned calves, and survival rates of calves with extremely high or low birth weights were significantly low. However, there was no relationship between gestation length and survival rate.

scholarly journals 240 GESTATION LENGTH AND BIRTH WEIGHT OF IN VITRO PRODUCED EMBRYOS FROM ZEBU DAIRY CATTLE

2005 ◽  
Vol 17 (2) ◽  
pp. 270 ◽  
Author(s):  
L.S.A. Camargo ◽  
J.H.M. Viana ◽  
A.A. Ramos ◽  
W.F. de Sa ◽  
A.M. Ferreira ◽  
...  
Keyword(s):  
Birth Weight ◽  
Sex Ratio ◽  
Sao Paulo ◽  
Control Group ◽  
Gestation Length ◽  
São Paulo ◽  
Embryo Production ◽  
In Vitro Embryo Production ◽  
And Control

Gir cattle are a popular zebu dairy breed in tropical and subtropical regions because of their tolerance of heat stress and resistance to tick-borne disease. The use of in vitro embryo production (IVP) technology may help accelerate genetic improvement of this breed. However, in general, IVP systems have been implicated in the production of large offspring and a greater proportion of male calves. Natural breeding results in newborn Gir calves weighing around 25 kg despite the fact that dams may weigh over 500 kg. It is unknown whether in vitro-produced Gir embryos also result in large offspring. The aim of this work was to evaluate the effect of in vitro embryo production on gestation length, birth weight, and sex ratio in Gir cattle. COCs were harvested by oocyte pickup from mature non-lactating Gir cows and in vitro-matured in TCM 199 medium (Gibco, Sao Paulo, Brazil) with 10% inactivated estrous cow serum for 24 h under 5% CO2 at 38.5°C in air. Gir spermatozoa were obtained through the swim-up method and co-incubated with oocytes in Fert-TALP media (Parrish JJ et al. 1988 Biol. Reprod. 38, 1171–1180) with 10 μg/mL heparin (Sigma, Sao Paulo, Brazil) and 6 mg/mL fatty acid-free bovine albumin (Sigma) for 18 h in 5% CO2 at 38.5°C in air. Presumptive zygotes were co-cultured with their own cumulus cells in CR2aa medium (Wilkinson RF et al. 1996 Theriogenology 45, 41–49) with 10% fetal calf serum in humid atmosphere of 5% CO2 at 38.5°C in air. Fresh Day 7 blastocysts were transferred to synchronized B. indicus × B. taurus crossbred recipients. Data of gestation length, birth weight, and gender ratio from 26 IVP calves (IVP group) were recorded and compared to data obtained from Gir calves produced by artificial insemination or natural mating (n = 24; control group) using ANOVA or chi-square analysis. There was no difference (P > 0.05) in gestation length between pregnancies of the IVP and control groups (mean ± SEM, 285.4 ± 1.5 vs. 284.4 ± 1.1 days, respectively). IVP and control calves showed similar (P > 0.05) weight at calving (29.6 ± 0.9 vs. 26.9 ± 1.2 kg for IVP and control male calves, and 27.0 ± 2.5 vs. 25.2 ± 0.5 kg for IVP and control female calves, respectively). The percentage of male calves was greater (P < 0.05) in the IVP group than in the control group (76.9% vs. 43.4%, respectively). IVP calves did not show abnormalities associated with Large Offspring Syndrome, such as breathing difficulty and perinatal death. These data suggest that in vitro production may affect the development of Gir embryos, biasing the sex ratio in a manner similar to previously reported for in vitro-produced embryos from Bos taurus breeds. This work was supported by FAPEMIG and CNPq.

scholarly journals Identification of Novel Mutations Responsible for Resistance to MK-2048, a Second-Generation HIV-1 Integrase Inhibitor

2010 ◽  
Vol 84 (18) ◽  
pp. 9210-9216 ◽  
Author(s):  
Tamara Bar-Magen ◽  
Richard D. Sloan ◽  
Daniel A. Donahue ◽  
Björn D. Kuhl ◽  
Alexandra Zabeida ◽  
...  
Keyword(s):  
Viral Replication ◽  
First Generation ◽  
Second Generation ◽  
Integrase Inhibitor ◽  
Replication Capacity ◽  
Strand Transfer ◽  
Viral Replication Capacity ◽  
Transfer Inhibitor ◽  
Potential Basis

ABSTRACT MK-2048 represents a prototype second-generation integrase strand transfer inhibitor (INSTI) developed with the goal of retaining activity against viruses containing mutations associated with resistance to first-generation INSTIs, raltegravir (RAL) and elvitegravir (EVG). Here, we report the identification of mutations (G118R and E138K) which confer resistance to MK-2048 and not to RAL or EVG. These mutations were selected in vitro and confirmed by site-specific mutagenesis. G118R, which appeared first in cell culture, conferred low levels of resistance to MK-2048. G118R also reduced viral replication capacity to approximately 1% that of the isogenic wild-type (wt) virus. The subsequent selection of E138K partially restored replication capacity to ≈13% of wt levels and increased resistance to MK-2048 to ≈8-fold. Viruses containing G118R and E138K remained largely susceptible to both RAL and EVG, suggesting a unique interaction between this second-generation INSTI and the enzyme may be defined by these residues as a potential basis for the increased intrinsic affinity and longer “off” rate of MK-2048. In silico structural analysis suggests that the introduction of a positively charged arginine at position 118, near the catalytic amino acid 116, might decrease Mg2+ binding, compromising enzyme function and thus leading to the significant reduction in both integration and viral replication capacity observed with these mutations.

294 EFFECTS OF FOLLICULAR FLUID OBTAINED FROM REPEAT BREEDER DAIRY HEIFER ON MATURATION OF BOVINE OOCYTES IN VITRO

2015 ◽  
Vol 27 (1) ◽  
pp. 236
Author(s):  
M. Kafi ◽  
M. R. Divar ◽  
S. Gharib-Zadeh
Keyword(s):  
Follicular Fluid ◽  
Present Experiment ◽  
Calf Serum ◽  
Low Fertility ◽  
Control Group ◽  
Normal Fertility ◽  
Maturation Rate ◽  
Bovine Oocytes ◽  
Repeat Breeder

The cause of repeat breeding syndrome is often difficult to explain in dairy heifers with no clinical abnormalities. The aim of the present experiment was to determine the effect of follicular fluid obtained from the preovulatory follicle of repeat breeder heifers on maturation of bovine oocytes in vitro. Holstein virgin heifers either with normal fertility (VH, n = 5) or repeat breeder syndrome (RB, n = 5) were used in the present experiment. The RB heifers had a history of at least 5 unsuccessful consequent artificial breeding. The reason for using such RB heifers was to exclude the possibility of the presence of usual causes of infertility in heifers. Oestrus cycles of all heifers were synchronized using 2 injections of PGF2a 11 days apart. Six to 12 h after oestrus detection, clear follicular fluid samples from the ovulatory follicles were collected transrectally using a long fine-needle covered by a hard plastic tube. Follicular fluid samples were pooled, centrifuged, and frozen until used in the maturation medium. A total of 483 good or excellent quality bovine cumulus-oocytes complexes (COC) were obtained from 2 to 6 mm follicles in diameter from slaughterhouse ovaries and randomly allocated in 3 groups; in group 1 (control, n = 180), oocytes were cultured in TCM-199 supplemented with 10% heat-treated fetal calf serum and hormones (5 IU mL–1 of hCG plus 0.1 IU mL–1 of rFSH); in group 2 (n = 126), oocytes were cultured in TCM-199 supplemented with 10% filtered follicular fluid of VH without hormones; in group 3 (n = 177), oocytes were cultured in TCM-199 supplemented with 10% filtered follicular fluid of RB heifers without hormones. All oocytes were cultured for 24 h at 39°C in an atmosphere of 5% CO2 under 90% humidity. At the end of maturation, the degree of cumulus expansion was evaluated and scored under a stereomicroscope. Then, oocytes were mechanically denuded using 3% sodium citrate and repeated pipetting and were fixed in ethanol/acetic acid (3 : 1) for 24 h. The oocytes were subsequently stained with 1% aceto-orcein and evaluated for meiotic resumption. Proportions were statistically analysed using a Chi-squared test (significant at P < 0.05; SPSS program, 11.5). The percentages of fully expanded COC differed among groups (P < 0.001). The maturation rate (MII stage) was 83% (150/180) in oocytes that were cultured in the presence of FCS as the control group. However, a reduction in the maturation rate was observed when oocytes were cultured either in VH follicular fluid (71.4%, 90/126; P < 0.01) or RB follicular fluid (59.3%, 105/177; P < 0.001) compared to the control group. The percentages of matured oocytes were also different between VH and RB follicular fluid (71.4 v. 59.3%; P < 0.01, respectively). In conclusion, the quality of follicular fluid of the preovulatory follicles of repeat breeder heifers is lower than that of the virgin heifers with normal fertility. This may explain the cause of the low fertility in some repeat breeder Holstein heifers.

165 FETAL CALF SERUM INFLUENCES CYCLIC GMP PATHWAY, WHICH IN TURN AFFECTS THE LIPID CONTENT IN IN VITRO-MATURED BOVINE OOCYTES

2014 ◽  
Vol 26 (1) ◽  
pp. 196
Author(s):  
K. R. L. Schwarz ◽  
R. C. Botigelli ◽  
F. C. Castro ◽  
M. R. Chiaratti ◽  
C. L. V. Leal
Keyword(s):  
Lipid Content ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Control Group ◽  
Maturation Medium ◽  
Cgmp Pathway ◽  
Bovine Oocytes ◽  
Synthesis And Degradation

The sensitivity of IVP embryos to cryopreservation is often associated with lipid accumulation in the cytoplasm induced by the presence of fetal calf serum (FCS) during culture. Intracellular levels of cyclic (c)AMP and cGMP are involved in the regulation of lipolysis in adipocytes; high levels stimulate lipolysis whereas low levels lead to lipogenesis. Both nucleotides are present in bovine oocytes, together with the enzymes for their synthesis and degradation. The aim of this study was to analysis the effect of FCS on the cGMP pathway and the influence of cGMP on cytoplasmic lipids in bovine oocytes. In experiments 1 and 2, cumulus–oocyte complexes (COC) were cultured for 24 h in maturation medium with different proportions of FCS (2 and 10%) and a control group was matured with 0.4% BSA. After this period, transcripts for cGMP pathway were assessed by real-time PCR (GUCY1B3 and PDE5, cGMP synthesis and degradation enzymes, respectively; experiment 1) in oocytes and cumulus cells, and cGMP levels were measured in COC using commercial enzyme immunoassay kits (EIA; experiment 2). In experiments 3 and 4, COC were matured for 24 h with 0.4% BSA and different concentrations of the phosphodiesterase (PDE)5 inhibitor (0, 10–7, and 10–5 M sildenafil) to inhibit cGMP degradation and a control group was matured with 0.4% BSA. The nucleotide levels were measured in COC (experiment 3) and the oocytes were stained with Nile Red (1 μg mL–1) for evaluation of lipid content (experiment 4). Statistical analyses were performed by ANOVA followed by Tukey post hoc test using SAS software (SAS Institute Inc., Cary, NC, USA). Data for gene expression from 5 replicates and for cGMP measurements and lipid content from 3 replicates were log10-transformed into before analyses. The level of significance was 5%. The presence of FCS reduced GUCY1B3 expression in both cells and increased PDE5A in cumulus cells (P < 0.05). In experiment 2, the groups treated with 2 (0.64 fmol/COC) and 10% FCS (1.04 fmol/COC) showed decreased cGMP levels compared with control (9.46 fmol/COC; P < 0.05). In experiment 3, inhibition of PDE5A increased cGMP levels in the treated groups (36 and 56 fmol/COC for 10–7 and 10–5 M sildenafil, respectively) compared with control (9.5 fmol/COC; P < 0.05). Therefore, sildenafil showed inverse effects compared with FCS (experiment 2). In experiment 4, oocytes treated with 10–7 and 10–5 M sildenafil showed a reduced lipid content compared with controls (11.6 ± 9.4 v. 13.9 μm2 fluorescence intensity, respectively; P < 0.05). The results suggest that FCS in maturation medium affects the cGMP pathway, interfering with the transcription of genes that control its levels, which in turn results in nucleotide reduction. Inhibition of PDE5 increases cGMP levels and reduces the lipid content of oocytes, indicating that changes in this pathway caused by FCS may affect lipid metabolism of oocytes. More studies are underway to better understand this mechanism. The authors acknowledge FAPESP 2012/00170-0 for financial support.

354 IN VITRO DEVELOPMENT OF BOVINE EMBRYOS AFTER NITRIC OXIDE INHIBITION

2007 ◽  
Vol 19 (1) ◽  
pp. 292
Author(s):  
K. R. L. Schwarz ◽  
T. H. C. de Bem ◽  
T. T. Zampieri ◽  
P. R. Adona ◽  
C. L. V. Leal
Keyword(s):  
Nitric Oxide ◽  
Cell Death ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Control Group ◽  
Sperm Cells ◽  
Blastocyst Development ◽  
Nuclear Maturation ◽  
Cytoplasmic Maturation

Nitric oxide (NO) is a chemical messenger detected in several cell types such as endothelial cells, neurons, and macrophages, exerting varied functions including vasodilatation, neurotransmission, and cell death induction. NO is generated by the activity of the enzyme nitric oxide synthase (NOS), which has been detected in several organs and tissues including the reproductive system. The aim of the present study was to assess the dose-response effect of N-omega-nitro-l-arginine-methyl ester (l-NAME), an NOS inhibitor, on in vitro nuclear and cytoplasmic maturation of bovine oocytes. Slaughterhouse ovaries were collected and their follicles (2–6 mm) were aspirated to obtain cumulus–oocyte complexes (COCs). Increasing l-NAME concentrations (0, 10-7, 10-5, 10-4, and 10-3 M) were added to IVM medium (TCM-199, supplemented with 10% fetal calf serum, 0.5 �g mL-1 FSH, 5.0 �g mL-1 LH, 0.2 mM pyruvate, and 10 mg mL-1 gentamicin); oocytes were cultured for 22 h. Nuclear maturation was assessed by propidium iodide staining (10 �g mL-1). For IVF, frozen–thawed semen prepared by Percoll gradient was used. Sperm cells were co-cultured with the oocytes at a final concentration of 2 � 106 sperm cells mL-1 in TALP-IVF medium supplemented with 2 �M penicillamine, 1 �M hypotaurine, 250 �M epinephrine, and 20 �g mL-1 heparin. After 20 h, presumptive zygotes were partially denuded and transferred to IVC medium (TCM-199 supplemented with 10% fetal calf serum, 2.0 mM pyruvate, and 10 mg mL-1 gentamicin). All cultures were at 38.5�C under 5% CO2 in air and maximum humidity. Cytoplasmic maturation was assessed by blastocyst development rates on Day 7. DNA fragmentation was assessed on Day 8 embryos by TUNEL (In Situ–Cell Death Detection kit, fluorescein; Roche Diagnostica Brasil, Sao Paulo, Brazil). Data were analyzed by ANOVA using the GLM procedure (SAS Institute, Inc., Cary, NC, USA), and means were compared by Duncan test at a 5% level. After IVM, the control group (0 M l-NAME) showed a greater number of oocytes in metaphase II (MII: 95.8 � 3.7%; P &lt; 0.05), whereas the groups cultured with l-NAME had lower MII rates (78–82%; P &lt; 0.05), irrespective of concentration (P &gt; 0.05). Many oocytes remained in metaphase I (MI: 18–22%). Cleavage rates at 48 h IVC was not affected (77–88%; P &gt; 0.05). Blastocyst rates (34.0 � 7.2% to 41.5 � 4.8%; P &gt; 0.05) and total cell numbers (151 to 174) were also unaffected by NO inhibition by l-NAME. However, the number of TUNEL-positive cells was lower in the control group (1.4 � 4.7; P &lt; 0.05) than in the treated groups (2.7 � 4.8 to 4.4 � 6.4; P &gt; 0.05). In conclusion, NO synthesis inhibition in oocytes during IVM reduces nuclear maturation, particularly during MI–MII transition, and increases apoptosis in blastocysts, suggesting that NO may be involved in oocyte maturation and apoptosis protection.

Gestation length, birth weight and offspring gender ratio of in vitro-produced Gyr (Bos indicus) cattle embryos

2010 ◽  
Vol 120 (1-4) ◽  
pp. 10-15 ◽  
Author(s):  
Luiz Sergio Almeida Camargo ◽  
Celio Freitas ◽  
Wanderlei Ferreira de Sa ◽  
Ademir de Moraes Ferreira ◽  
Raquel Varela Serapiao ◽  
...  
Keyword(s):  
Birth Weight ◽  
Bos Indicus ◽  
Gestation Length ◽  
Gender Ratio ◽  
Offspring Gender

scholarly journals High Birth Weight Increases the Chance of Mortality in Girolando Calves 2 Conceived Through in Vitro Fertilization

2021 ◽  
Author(s):  
Maria Amélia Agnes Weiller ◽  
Evandro Schmoeller ◽  
Antônio Amaral Barbosa ◽  
Adriane Dalla Costa de Matos ◽  
Marcio Nunes Correa ◽  
...  
Keyword(s):  
Birth Weight ◽  
In Vitro Fertilization ◽  
Mortality Rate ◽  
Dairy Farm ◽  
High Birth Weight ◽  
Gamma Glutamyl Transferase ◽  
Vitro Fertilization ◽  
Clinical Complications ◽  
Glutamyl Transferase

Abstract The aim of this study was to determine zootechnical and health performance of Girolando calves born with high or low birth weight, and compare metabolic parameters between groups. The study was carried out on a commercial dairy farm located in Passos, Minas Gerais, Brazil. In this sense, a hundred Girolando calves were divided into 2 groups: Control, which consisted of calves that were born weighing ≤ 35 kg; and HBW, calves that were born weighing > 35 kg. Calves were monitored for zootechnical parameters; epidemiological indexes such as morbidity, mortality, recurrence of diarrhea, pneumonia; as well as serum concentrations of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma-glutamyl transferase, cholesterol, triglycerides, paraoxonase1, albumin, urea and globulin). Calves from the HBW group had a higher mortality rate as well as a tendency to more cases of pneumonia, but no effect on zootechnical performance was seen. The reasons for the differences in mortality need to be clarified since our study found no changes in biochemical parameters between the groups. The results allow us to conclude that Girolando calves from in vitro fertilization that are born heavier have a greater chance of clinical complications and a higher mortality rate, but the birth weight does not influence the zootechnical performance.

scholarly journals Effects of Current Provisional Restoration Materials on the Viability of Fibroblasts

2009 ◽  
Vol 03 (02) ◽  
pp. 114-119 ◽  
Author(s):  
Mustafa Ulker ◽  
H. Esra Ulker ◽  
Mustafa Zortuk ◽  
Mehmet Bulbul ◽  
Ali Riza Tuncdemir ◽  
...  
Keyword(s):  
Calf Serum ◽  
Basal Medium ◽  
In Vitro Study ◽  
Fibroblast Cell ◽  
Control Group ◽  
Mitochondrial Activity ◽  
Fibroblast Cells ◽  
Cytotoxic Effects ◽  
Provisional Restoration

ABSTRACTObjectives: The aim of the present study was to evaluate the cytotoxic effects of three different provisional restoration materials on fibroblasts. Two bis-acrylic based [Tempofit Duomix (Detax), Protemp 3 Garant (3M ESPE)] and one urethan dimethacrylate [Revotek LC (GC Corporation)] based provisional restoration materials used.Methods: Materials were prepared according to the manufacturers’ instructions in standard teflon disks (2×5mm) and four samples were extracted in 7 ml of Basal Medium Eagle with 10% new born calf serum and 100 mg/ml penicillin/streptomycin for 24 hours. The L929 fibroblast cells were plate (25.000 cells/ml) in well plates, and maintained in a CO2 incubator at 37°C for 24h. After 24 hours, the incubation medium was replaced by the immersed medium in which the samples were stored and the L929 fibroblasts were incubated in contact with eluates for 24 hours at 37°C for 24h. The fibroblast cell viability was analyzed by measuring the mitochondrial activity with the methyltetrazolium test (MTT). Twelve well used for each specimen and experiment repeated for two times. The data was statistically analyzed by Mann-Whitney U tests.Results: The results showed that, Revotek LC and Protemp 3 Garant were not cytotoxic for fibroblast cells when compared to control group (P>.05). However, Tempofit duomix was cytotoxic for L929 fibroblasts when compared to control group and other tested materials (P%.05).Conclusions: Taking into consideration the limitations of an in vitro study, our study indicate that provisional restoration materials might have cytotoxic effects on fibroblasts and should be selected carefully for clinical applications. (Eur J Dent 2009;3:114-119)

68 EFFECT OF CO-CULTURE DURING FERTILIZATION OF BUFFALO (BUBALUS BUBALIS) IN VITRO-MATURED DENUDED OOCYTES VITRIFIED BY CRYOTOP

2008 ◽  
Vol 20 (1) ◽  
pp. 115
Author(s):  
L. Attanasio ◽  
A. De Rosa ◽  
L. Boccia ◽  
R. Di Palo ◽  
G. Campanile ◽  
...  
Keyword(s):  
Survival Rate ◽  
In Vitro Fertilization ◽  
Survival Rates ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Control Group ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Group B

Although removal of cumulus cells improves the efficiency of vitrification of buffalo (Bubalus bubalus) in vitro-matured (IVM) oocytes (Gasparrini et al. 2007 Anim. Reprod. Sci. 98, 335–342), the lack of cells impairs the fertilization process. Therefore, the aim of the present work was to evaluate the influence of a somatic support during in vitro fertilization (IVF) of buffalo vitrified denuded matured oocytes. Since IVF on a cumulus cells monolayer was inefficient, we verified the effects of co-culture with cumulus-enclosed oocytes (COCs). IVM buffalo oocytes (n = 316) were vitrified by the Cryotop� method (Kuwayama and Kato 2000, J. Assist. Reprod. Genet. 17, 477 abst) that was recently proven suitable for buffalo oocyte cryopreservation (Attanasio et al. 2006 Reprod. Domest. Anim. 41, 302–310). Denuded buffalo oocytes were equilibrated in 10% ethylene glycol (EG) and 10% dimethyl sulfoxide (DMSO) for 3 min, transferred into 20% EG and 20% of DMSO in TCM199 with 20% fetal calf serum (FCS) + 0.5 m sucrose, loaded on Cryotops, and plunged into liquid nitrogen within 25 s. For warming, oocytes were exposed for 1 min to 1.2 m sucrose and then to decreasing concentrations of the sugar (0.6, 0.4, 0.3 m for 30 s) in TCM199 + 20% FCS. Oocytes were rinsed and allocated to IVM drops for 1.5 h. Survival rate was evaluated at this point and the oocytes that had survived (292/316 = 92.4%) were split into 2 fertilization groups: (A) approximately 5 buffalo oocytes per 50-µL drop of IVF medium, and (B) approximately 3 buffalo oocytes + 3 bovine fresh COCs per 50-µL drop of IVF medium. Since buffalo COCs easily lose their cells following IVF, for better identification we used bovine COCs that have a brighter and more compact cumulus mass. In vitro fertilization and culture were carried out as previously described (Gasparrini et al. 2007). As control, buffalo oocytes (n = 104) were in vitro-matured, fertilized, and cultured up to the blastocyst stage. On Day 1, survival rate was evaluated in the two vitrification groups; cleavage and blastocyst rates were recorded on Days 5 and 7, respectively, in all groups. The experiment was repeated 4 times. Differences in the percentages of survival, cleavage, and blastocyst formation among treatments were analyzed by chi-square test. Within vitrification groups, despite similar survival rates on Day 1 (90.6% v. 93.3%, respectively, in Groups A and B), cleavage rate was significantly improved in Group B compared to Group A (59.2% v. 45.4%, respectively; P < 0.01). Interestingly, the cleavage rate in Group B was not significantly different from that recorded in the control group (71.0%). Although blastocysts were produced in both vitrification groups (3.6% v. 4.1%, respectively, in Groups A and B), the yield was significantly lower than that of the control group (29.0%, P < 0.01). In conclusion, co-culture with bovine COC during fertilization improves the capability of buffalo denuded vitrified oocytes to cleave.

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