361 EFFECT OF HORMONES, EGF AND β-MERCAPTOETHANOL ON IN VITRO MATURATION OF CAPRINE OOCYTES

2010 ◽  
Vol 22 (1) ◽  
pp. 337 ◽  
Author(s):  
P. Yadav ◽  
S. D. Kharche ◽  
A. K. Goel ◽  
S. K. Jindal ◽  
M. C. Sharma
Keyword(s):  
In Vitro Maturation ◽  
Calf Serum ◽  
Basic Medium ◽  
Control Group ◽  
Vitro Fertilization ◽  
Base Medium ◽  
Maturation Medium ◽  
Maturation Rate ◽  
Group 5

In vitro maturation of oocytes is an integral part of in vitro culture system. In almost all studies of mammalian in vitro maturation, the basic medium is supplemented with serum and hormones. The maturation medium and selection of protein supplements, growth factors, antioxidants and hormones for IVM play an important role in subsequent in vitro fertilization and in vitro embryo development. The objective of the present experiment was to study the effect of exogenous addition of hormones, epidermal growth factor, insulin and β-mercaptoethanol to the maturation medium for in vitro maturation of caprine oocytes. A total of 1540 oocytes were collected from slaughtered goat of 1.5 to 2.5 year of age and randomly divided in to treatment groups. Group 1; COCs were matured in TCM-199 medium containing 10% calf serum (NCS) and 3 mg mL-1 BSA (used as a base medium) for control, Group 2; COCs were matured in a base medium supplemented with hormones (5 μg mL-1 FSH, 5 μg mL-1 LH and 1 μg mL-1 estradiol-17β), Group 3β COCs were matured in a base medium supplemented with 50 ng mL-1 insulin, Group 4β COCs were matured in a base medium supplemented with hormones and 10 ng mL-1 EGF, Group 5; COCs were matured in a base medium supplemented with hormones and 50 mM β-mercaptoethanol and Group 6; COCs were matured in a base medium supplemented with hormones and 50 ng mL-1 insulin. These COCs were matured at 38.5°C in 5% CO2 in air for 27 h. After the maturation, oocytes were separated from cumulus and corona cells by treatment with 0.1% hyaluronidase and by passing through a fine pipette. They are them fixed in 2.5% glutaraldehyde, stained with DAPI and observed under fluorescent microscope for evidence of nuclear maturation. The maturation rates in groups 1 to 6 were 33.6%, 38.0%, 39.7%, 60.0%, 37.4%, and 44%, respectively. Statistical analysis (ANOVA) after arcsin transformation revealed that the maturation rate in group 4 was statistically significant (P < 0.05) as compared to those in groups 1, 2, 3, 5, and 6. The results suggest that the supplementation of EGF in maturation medium significantly enhances the in vitro maturation rate of caprine oocytes.

scholarly journals LÍQUIDO FOLICULAR EQÜINO NA MATURAÇÃO IN VITRO DE OÓCITOS BOVINOS

2004 ◽  
Vol 9 (1) ◽  
Author(s):  
M.G.L. PINTO ◽  
M.I.B. RUBIN ◽  
C.A.M. SILVA ◽  
T.F. HILGERT ◽  
M.F. SÁ FILHO ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Mineral Oil ◽  
Control Group ◽  
Sodium Pyruvate ◽  
Vitro Fertilization ◽  
Treatment Groups ◽  
Maturation Medium ◽  
Bovine Oocytes

O desenvolvimento embrionário de oócitos bovinos maturados in vitro (MIV) foi avaliado em meio suplementado com líquido folicular eqüino (Lfe). Foram distribuídos 1045 oócitos em 11 repetições formando três grupos tratamentos (T1, T2, T3) e um controle (C). O meio de maturação utilizado foi o TCM-199 acrescido de piruvato de sódio, hormônio folículo estimulante recombinante (rFSHh) e hormônio luteinizante equino (LHe). Suplementou-se esse meio com 10% de soro de égua em estro para o grupo controle e para T1, T2 e T3, o meio foi suplementado com 5, 10, e 20% de LFe, respectivamente. Os oócitos foram maturados in vitro (MIV) por 24h. A fecundação in vitro (FIV) foi realizada em meio Talp-Fert. A MIV e a FIV foram realizadas em estufa a 39ºC com 5% de CO2 em ar e umidade saturada. Os zigotos foram cultivados em meio SOFaaci, sob óleo mineral no interior de bolsas plásticas gaseificadas. As taxas de clivagem e de blastocistos foram observadas diariamente (D), e em D7, foram superiores (P0,05) às do grupo controle. Em D9, a taxa de blastocistos do T2 foi superior (P0,05). O LFe, na concentração de 10% pode ser utilizado, em substituição ao soro de égua em estro para suplementar o meio de MIV de oócitos bovinos. Equine follicular fluid on in vitro maturation of bovine oocytes Abstract Embryo development of bovine oocytes was evaluated using maturation medium supplemented with equine follicular fluid (eFF). One thousand and forty five (1045) oocytes were distributed in 11 replications forming three treatment groups (T1, T2 e T3) and one Control (C). TCM-199 added with sodium pyruvate, rFSHh and LHe was used as maturation medium. This medium was supplemented with 10% estrous mare serum for Control group, and 5, 10, and 20% eFF, respectively, for T1, T2 e T3 groups. In vitro maturation (IVM) of all groups was performed during 24h. In vitro fertilization (IVF) was performed in TALP-FERT medium. IVM and IVF were carried out in an incubator at 39ºC with 5% CO2 in air and saturated humidity. Zygotes were cultured in SOFaaci medium, under mineral oil in gasified bags. Cleavage and blastocyst rates were daily observed (D), and at D7, were higher (P0.05) for those from control group. At D9, blastocyst rate of T2 was higher (P0.05). The eFF, at a 10% concentration, can replace the use of estrous mare serum to supplement the IVM medium of bovine oocytes.

scholarly journals Use of Melatonin in the In Vitro Production of Bovine Embryos

2020 ◽  
Vol 21 ◽  
Author(s):  
Alan da Silva LIRA ◽  
Ricardo de Macedo CHAVES ◽  
Felipe de Jesus MORAES JUNIOR ◽  
Sergio Henrique COSTA JUNIOR ◽  
Brenda Karine Lima do AMARAL ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Control Group ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Maturation Medium ◽  
Significant Difference ◽  
Maturation Rate ◽  
Vitro Production

ABSTRACT We aimed to assess the effects of melatonin in the in vitro production of bovine embryos. Our experiment was conducted at the Laboratório de Reprodução Animal of the Universidade Estadual do Maranhão. The cumulus-oocyte complexes (COCs) were distributed among treatments at concentrations of 0, 10-1, 10-3 and 10-5 µMol/L melatonin. Our experiment was further divided into two: the first was to assess the effect of different concentrations of melatonin (treatments) on the maturation rate of COCs, and the second was to assess the effects of melatonin treatments on the in vitro production of bovine embryos. The results from the first experiment demonstrated no significant difference between the in vitro maturation rate of the cultivated COCs in treatments with melatonin. In the second experiment, however, melatonin treatments yielded statistically higher cleavage, morula and blastocyst rates in the 10-5 µM group (52.9%, 52.9%, and 35.3%, respectively), and lower rates in the 10-1 µM group (19.5%, 19.5% and 7.8%, respectively), compared to the others. The control group (no melatonin) and the 10-3 µM group showed similar results. We concluded that supplementation of melatonin in the in vitro maturation medium resulted in no improvement in the oocyte maturation rate, but in the in vitro production of embryos at different concentrations, the 10-5 µM group displayed better results, but with no improvement in the variables (P < 0.05).

286 EFFECTS OF PORCINE FOLLICULAR FLUID DURING IN VITRO MATURATION OF OOCYTES ON IN VITRO FERTILIZATION AND EMBRYONIC DEVELOPMENT

2007 ◽  
Vol 19 (1) ◽  
pp. 258
Author(s):  
B. Agung ◽  
T. Otoi ◽  
D. Fuchimoto ◽  
S. Senbon ◽  
A. Onishi ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Embryonic Development ◽  
Follicular Fluid ◽  
Culture System ◽  
In Vitro Maturation ◽  
Control Group ◽  
Rotating System ◽  
Vitro Fertilization ◽  
Maturation Medium

When used as a solo maturation medium for oocytes, porcine follicular fluid (pFF) promoted male pronucleus formation (MPF) of oocytes after in vitro maturation (IVM), using a static system, and in vitro fertilization (IVF) in pigs (Naito et al. 1988 Gamete Res. 21, 289–295). However, the developmental competence of oocytes matured in pFF after IVM/IVF has not been reported. This study was conducted to assess the ability of pFF as a maturation medium to support IVM/IVF of porcine oocytes and their subsequent in vitro development. pFF, including cumulus–oocyte complexes (COCs), was aspirated from follicles (2–5 mm in diameter) of prepubertal crossbred gilt ovaries, and large clusters of follicular cells (FC) were removed from pFF by filtration through 212 �m of mesh. All of the COCs in filtered pFF were collected, and COCs with compact cumulus cells were selected for IVM. Also, small clusters of FC were collected by centrifugation of the filtered pFF, and pFF without any cells was prepared by centrifugation and used as a maturation medium (MpFF) after supplementation with FSH and antibiotics. COCs were transferred to 3.5 mL (in a 15-mL test tube) of MpFF with FC (5.2 � 106 cells mL-1) and cultured for 44–48 h at 38.5�C in 5% O2 and 5% CO2 using the rotating culture system. As a control group, COCs were cultured in 2 mL of MpFF without FC in a 35-mm Petri dish by the standard static culture system. After maturation, culture oocytes were co-incubated (IVF) for 5 h with frozen–thawed sperm in vitro, as reported previously (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041), and then some of them were fixed 10 h after IVF to assess the fertilization status; the rest of them were cultured in PZM (Yoshioka et al. 2002 Biol. Reprod. 60, 112–119) for 7 days to assess their early embryonic development. All of the data were analyzed by ANOVA. Oocytes cultured with FC in the rotating system (R group) showed significantly higher sperm penetration (71.0%), MPF formation (70.5%), and normal fertilization (monospermic fertilization with female and male pronuclei; 31.5%) rates than those in the control group (56.0%, 56.9%, and 17.6%, respectively; P &lt; 0.05). Also, the R group showed significantly higher rates of 8-cell embryos at 2 days after IVF and blastocyst formation at 7 days after IVF than those of the control group (17.2% vs. 8.3% and 10.9% vs. 4.5%, respectively; P &lt; 0.05). These results indicate that porcine oocytes matured in pFF supplemented with FC using the rotating system have the ability to be penetrated by sperm and form MPF, and to develop to the blastocyst stage at higher rates, than oocytes cultured in the standard static maturation culture system. In conclusion, the pFF can be a sole and simple maturation culture medium useful for the in vitro production of blastocysts in pigs.

scholarly journals Evaluation of the sperm separability of blanc-blue-belge bull by swim-up method and in vitro embryo production with hybrid Zebu bovine oocyte in Vietnam

2020 ◽  
Vol 18 (1) ◽  
pp. 59-66
Author(s):  
Nguyen Huu Duc ◽  
Pham Thu Giang ◽  
Tran Thi Binh Nguyen ◽  
Bui Dai Phong
Keyword(s):  
In Vitro Fertilization ◽  
Calf Serum ◽  
Basic Medium ◽  
Embryo Production ◽  
Vitro Fertilization ◽  
Bovine Sperm ◽  
Maturation Medium ◽  
In Vitro Embryo Production ◽  
The Right

The objective of this study was to determine the right conditions for the separation of Blanc-Blue-Belge bovine sperm (BBB) by swim-up mothed; determine the maturity of hybrid Zebu bovine eggs; and culture of embryos after in vitro fertilization. After 60-80 minutes of swim-up in CAP-05, BBB bovine sperms were healthy, straight movement and separated with a concentration of 106 sperm/ml. Hybrid Zebu bovine eggs developed and matured in the maturation medium with the basic medium TCM-199 supplemented with 10% calf serum, FSH (0.75 µg / ml), LH (0.15 µg / ml) and Estradiol (2.5 µg / ml), the results showed that the IVM-08 medium had significantly higher maturation rates than IVM-03, the proportion of mature eggs reached 71,11% compared to 51.69%, respectively (P <0.01). In vitro fertilization of hybrid Zebu bovine egg in IVF-08 medium. In vitro fertilized embryos (BBB x hybrid Zebu) developed from bovine sperms separated by the swim-up method achieved a better rate of morula-blastocysts in IVC-09 than IVC-06 medium, 21.68% compared to 8.56%, respectively (P <0.01). The conclusion was that the suitable conditions for BBB bovine sperm separation and in vitro embryo production (BBB x hybrid Zebu) were determined. This is the premise to create bovine semen, BBB bovine embryos with defined gender.

350 EFFECT OF PROGESTERONE SUPPLEMENTATION OF MATURATION MEDIUM ON DEVELOPMENT OF IVM-IVF-IVC BOVINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 331
Author(s):  
K. Miyata ◽  
H. Koyama ◽  
C. Lessard ◽  
J. Singh ◽  
O. Dochi
Keyword(s):  
In Vitro Maturation ◽  
Formation Rate ◽  
Calf Serum ◽  
Control Group ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Oocyte Competence ◽  
Maturation Medium ◽  
Progesterone Supplementation

Follicular fluid from small and large bovine follicles contains large amounts of progesterone, and during preovulatory period progesterone concen- tration increase markedly by 18 h after LH surge. Furthermore, cumulus cells express membrane progestin receptor beta (Liu et al. 2008 Steroids 73, 1416-1423). For these reasons, we hypothesized that progesterone supports maturation of preovulatory bovine oocytes to MII stage. The object of this study was to investigate the effect of progesterone supplementation of in vitro maturation medium on competence of bovine oocyte to develop into blas- tocysts in vitro. COCs were collected by the aspiration of 2-6 mm follicles from ovaries within 6 h of slaughter. The COCs were divided into 5 groups: (1) a control group, TCM-199 supplemented with 5% calf serum (CS) as IVM medium, and (2 to 5) progesterone (P4) supplementation groups, TCM- 199 supplemented with 5% CS and 1, 3, 5, and 10 μg mL-1 of P4. Groups of 10 COCs were incubated in 50-μL drops of IVM media at 38.5°C under an atmosphere of 5% CO2 in air for 20 h. The matured COCs were inseminated with 3 × 106 sperm mL-1. After 18 h of gamete co-culture, the pre- sumptive zygotes were cultured in CR1aa media supplemented with 5% CS for 9 days at 38.5°C under an atmosphere of 5% CO2, 5%O2, and 90% N2. Embryonic development was evaluated at 48 h after IVF (total cleavage rates) and on Days 7 to 9 (blastocyst rate). Data was analyzedby chi-square test. The results are presented in Table 1. There were no significant differences in the cleavage rates between treatments. However, the blastocyst formation rate of 5 μg mL-1 P4 supplementation group was significantly higher than that of 10 μg mL-1 P4 supplementation group (P < 0.05). In addition, the blastocyst formation rates of 10 μg mL-1 P4 supplementation group was lower than the other groups. These results suggest that progesterone supple- mentation of in vitro, maturation medium affects the competence of the oocytes to develop into blastocysts in vitro, and 5 μg mL-1 P4 supplementation may be effective in increasing embryo production. Furthermore, 10 μg mL-1 P4 supplementation has negative effect on the oocyte competence. Table 1.Effect of progesterone supplementation on development of IVF bovine embryos

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.

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.

293 EFFECT OF DIFFERENT CONCENTRATIONS OF LH, FSH, AND E2 ON THE MATURATIONAL RATE OF INDIGENOUS SOUTH AFRICAN CATTLE OOCYTES SELECTED BY BRILLIANT CRESYL BLUE STAINING

2015 ◽  
Vol 27 (1) ◽  
pp. 235
Author(s):  
K. P. M. Lekola ◽  
J. W. Ng'ambi ◽  
N. Nkadimeng ◽  
M. L. Mphaphathi ◽  
T. L. Nedambale
Keyword(s):  
South African ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Blue Staining ◽  
Brilliant Cresyl Blue ◽  
Cresyl Blue ◽  
African Cattle ◽  
Maturation Medium ◽  
Maturation Rate

In vitro maturation of indigenous African cattle oocytes is a major challenge even though different maturation protocols work successfully in other breeds. The objective of this study was to determine the maturation rate of indigenous South African cattle oocytes following in vitro maturation in media supplemented with different concentrations of hormones and selected using brilliant cresyl blue (BCB) staining. Indigenous cattle ovaries were collected from the slaughterhouse and then oocytes were retrieved by aspiration method. A total of 966 oocytes were exposed to 26 µM BCB stain and 700 oocytes were not exposed to the BCB stain. Thereafter, oocytes exposed to the BCB stain were grouped according to the colour of their cytoplasm BCB+ (oocytes with blue cytoplasm, low G6PDH) and BCB– (unstained oocytes, increased G6PDH). The BCB exposed (BCB+ and BCB–) and the oocytes not exposed to BCB were then randomly allocated into tissue culture medium (TCM199) + 10% (vol/vol) fetal bovine serum (FBS) supplemented with 3 different concentrations of hormones as treatments (T). The T1 group was matured in the presence of 0.5 µg mL–1 of FSH, 5 mg mL–1 of LH, and 2 µg mL–1 of E2; the T2 group was matured in the presence of 1 µg mL–1 of FSH, 6 mg mL–1 of LH, and 2.5 µg mL–1 of E2; and the T3 group was matured in the presence of 1.5 µg mL–1 of FSH, 7 mg mL–1 of LH, and 4.5 µg mL–1 of E2. For IVM, 20 to 25 COC were placed in 50-µL droplets of IVM medium containing the 3 different levels of hormones. Maturation rate of oocytes was determined by the extrusion of the first polar body after 24 h of incubation in maturation medium. Data was analysed by ANOVA using SAS with 4 replicates per treatment. Treatment 2 yielded higher maturation rate for both BCB+ (65.6%) and not exposed to BCB (60.3%) oocytes compared to T1 (22, 3.03, and 16% for BCB+, BCB–, and not exposed to BCB, respectively) and T3 (48, 2.2, and 48% for BCB+, BCB–, and not exposed to BCB respectively). However, BCB– oocytes had lower polar body extrusion for T1, T2, and T3 (3.03, 8.1, and 2.2%, respectively) compared to BCB+ oocytes (22, 65.6, and 48% for T1, T2, and T3, respectively). In conclusion, immature oocytes that were cultured into TCM199 supplemented with 10% FBS, 1 µg mL–1 of FSH, 6 mg mL–1 of LH, and 2.5 µg mL–1 of E2 showed maturation rate for BCB+ oocytes and those not exposed to BCB. Oocytes selection using BCB staining was a useful test to classify good quality cattle oocytes. Therefore, it is suggested that treatment 2 is a suitable in vitro-maturation medium to mature indigenous South African cattle oocytes.

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.

Sign in / Sign up

Export Citation Format

Share Document