scholarly journals Pengaruh Penambahan Chorionic Gonadotrophin pada Medium Maturasi terhadap Kemampuan Maturasi, Fertilisasi, dan Perkembangan Embrio secara In Vitro Kambing Peranakan Ettawa (The Effect of Chorionic Gonadotrophin Addition Into Maturation Medium on The Abili

2012 ◽  
Vol 34 (1) ◽  
pp. 8
Author(s):  
Nurvina Septi Adifa ◽  
Pudji Astuti ◽  
Diah Tri Widayati
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
Chorionic Gonadotrophin ◽  
Fertilization In Vitro ◽  
Cleavage Rate ◽  
Group I ◽  
Maturation Medium ◽  
Frozen Semen ◽  
Significant Difference

<p>This research was conducted to investigate the effect of chorionic gonadotrophin addition into maturation medium on oocyte maturation, fertilization, and embryo development in vitro of Ettawa crossbred. Oocytes were divided into 3 groups, group I: maturation medium without addition of chorionic gonadotrophin (0), group II: 10 μl/10 ml chorionic gonadotrophin was added into maturation medium (1), group III: 20 μl/10 ml chorionic gonadotrophin was added into maturation medium (2). Oocytes were transferred into 50 μl maturation medium, then covered by mineral oil. Oocyte was incubated at 39oC, 5% CO2, 95% humidity for 24 hours for maturation. Matured oocytes were inseminated with frozen semen–thawed concentration 12.5 x 106/ml. Process of fertilization were carried out on incubator 39oC, 5% CO2, 95% humidity for 5 hours. The fertilized oocytes were transferred into 50 μl drop G–1, then incubated at 39oC, 5% CO2, 95% humidity. Embryo development was monitored every 24 hours. Culture medium was changed every 48 hours. G–2 medium used second day after culture. The variables measured involved oocyte maturation, fertilization, and in vitro cleavage rate. The data were analyzed by chi–square, using SPSS 15.0 program. The result showed no significant difference on the percentage of mature oocytes and fertilization rate were 78.0%, 72.8%, 75.0% and 76.6%, 74.5%, 77.8% respectively. But cleavage rate showed significant difference (P≤0.05) with<br />the values of 40.8%, 11.4%, and 12.2% respectively. Based on the result it could be concluded that chorionic gonadotrophin addition into maturation medium had not increased ettawa crossbred oocytes maturation, fertilization, and in vitro cleavage rate. The best maturation, fertilization, and in vitro cleavage rate were found using maturation medium without any addition of chorionic gonadotrophin.</p><p>(Key words: Does oocyte, Chorionic gonadotrophin, In vitro maturation, In vitro fertilization, In vitro embryo development)<br /><br /></p>

150 DEVELOPMENT OF ZONA-FREE AND WITH ZONA PARTHENOGENETIC GOAT EMBRYOS IN DIFFERENT MEDIA

2010 ◽  
Vol 22 (1) ◽  
pp. 233
Author(s):  
M. K. Jena ◽  
D. Malakar ◽  
A. K. De ◽  
S. Garg ◽  
Y. S. Akshey
Keyword(s):  
Embryo Development ◽  
Chemical Activation ◽  
Formation Rate ◽  
Electrical Activation ◽  
Cleavage Rate ◽  
Maturation Medium ◽  
Parthenogenetic Embryo ◽  
Oviductal Fluid ◽  
Phosphate Buffered Saline

The present study was carried out to see the developmental efficiency of zona-free and with zona parthenogenetic goat embryos cultured in Research Vitro Cleave from Cook Australia (RVCL), Embryo Development Media (EDM), modified synthetic oviductal fluid (mSOF), and modified Charles Rosenkrans media (mCR2a). Zona-free embryos were cultured in 4 media, whereas with zona embryos were cultured in 3 media except mCR2a. Ovaries were collected from slaughterhouse and oocytes were isolated by puncturing the follicles in medium containing Dulbecco’s phosphate-buffered saline, 3% BSA, and 50 μg mL-1 gentamicin. Oocytes were matured in maturation medium containing TCM-199 (HEPES modified), 0.05 mg mL-1 Na pyruvate, 0.003 mg mL-1 L-glutamine, 5.5 mg mL-1 glucose, 3 mg mL-1 BSA, 5 μg mL-1 FSH, 10 μg mL-1 LH, 1 μg mL-1 estradiol-17β, 50 μg mL-1 gentamicin, and 10% FBS in 5% CO2 in air at 38.5°C. The COC (15 to 20 oocytes) were placed in 100-μL droplets of maturation medium and incubated in a CO2 incubator (5% CO2 in air) with maximum humidity at 38.5°C for 27 h. Matured oocytes were made cumulus free by treatment with hyaluronidase (0.5 mg mL-1) and zona-free by pronase (2 mg mL-1) in zona-free parthenogenesis. Then the oocytes were activated by 5 μM Ca ionophore for 5 min in a CO2 incubator and then treated with 2 mM 6-DMAP for 4 h. Activation was also done by electrical activation with DC 1.78 kV cm-1, 20 μs, and 2 pulses. Then the zona-free oocytes were kept for in vitro culture in 4 types of media such as RVCL, EDM, mSOF, andm CR2a for 7 days in 5% CO2 in air at 38.5°C. The cleavage rate andmorulae formation were observed in RVCL 40.95%, 13.95%, in EDM 46.92%, 14.75%, in mCR2a 56.66%, 5.88%, and in mSOF 48.23%, 14.63%, respectively. The cleavage rate and morulae formation were also found 55.9%, 14.63% during chemical activation and 32%, 12.5% in electrical activation. Hence, better result was found in chemical activation than electrical activation. For with zona parthenogenesis, the matured oocytes were chemically activated by 5 μM Ca ionophore for 5 min and 2 mM 6-DMAP for 4 h. Then the oocytes were cultured in RVCL, EDM, and mSOF in 100-μL micro-drops media for 7 days. The cleavage, morulae, and early blastocyst production rate were as follows: cleavage rate 75.68%, 72.03%, and 57.11%; morulae 44.61%, 30.29%, and 40.22%; and early blastocyst 17.49%, 11.88%, and 25.01% in RVCL, EDM, and mSOF, respectively. Hatched blastocyst formation rate was 6.75%, 5.48%, and 1.15% in RVCL, EDM, and mSOF, respectively. It could be concluded that zona-free parthenogenetic embryos were produced better in EDM medium and with chemical activation. With zona parthenogenetic embryo development was significantly (P < 0.05) higher in RVCL and EDM media.

34 EFFECTS OF L-CARNITINE AND trans-10,cis-12 CONJUGATED LINOLEIC ACID SUPPLEMENTATION DURING MATURATION ON DEVELOPMENT AND CRYOTOLERANCE OF BOVINE EMBRYOS PRODUCED IN VITRO

2016 ◽  
Vol 28 (2) ◽  
pp. 147
Author(s):  
J. Block ◽  
A. M. Zolini ◽  
E. Carrascal-Triana ◽  
A. Ruiz ◽  
P. J. Hansen ◽  
...  
Keyword(s):  
Linoleic Acid ◽  
Conjugated Linoleic Acid ◽  
Embryo Development ◽  
Blastocyst Stage ◽  
Cleavage Rate ◽  
Sodium Pyruvate ◽  
Maturation Medium ◽  
Fetal Bovine ◽  
Hatching Rates

The objective of the present study was to determine the effect of supplementation of maturation media with L-carnitine and trans-10,cis-12 conjugated linoleic acid (CLA) on embryo development and survival following cryopreservation. Immature bovine cumulus-oocyte complexes (n = 1796) were harvested from abattoir-derived ovaries and randomly assigned in a 2 × 2 factorial design to be matured in maturation medium [TCM-199 with Earle salts supplemented with 10% (vol/vol) bovine steer serum, 2 μg mL–1 oestradiol 17-β, 20 μg mL–1 bovine FSH, 22 μg mL–1 sodium pyruvate, 50 μg mL–1 gentamicin sulfate, and 1 mM glutamax®] supplemented with or without 100 mM CLA and with or without 3.03 mM L-carnitine for 22 to 24 h at 38.5°C in a humidified atmosphere of 5% CO2. The proportion of oocytes that cleaved was determined on Day 3 after insemination, and the proportion of oocytes developing to the blastocyst and advanced blastocysts stages (expanded, hatching, and hatched) was assessed on Day 7. Blastocyst and expanded blastocyst stage embryos (n = 270) were harvested on Day 7 and subjected to controlled-rate freezing following equilibration in 1.5 M ethylene glycol. Embryos were thawed and then cultured for 72 h in SOF-BE1 (Fields et al. 2011) supplemented with 10% (vol/vol) fetal bovine serum and 50 μM dithiothreitol. Post-thaw re-expansion and hatching rates were determined at 24, 48, and 72 h. The experiment was replicated 5 times. There was no effect of supplementation of maturation medium with either CLA or L-carnitine on the proportion of oocytes that cleaved at Day 3 or that developed to the blastocyst and advanced blastocyst stages at Day 7 after insemination. There was no interaction between CLA and L-carnitine affecting cleavage rate or embryo development. Supplementation of maturation medium with L-carnitine did not affect post-thaw re-expansion or hatching rates. In contrast, treatment with CLA during maturation reduced (P < 0.05) post-thaw re-expansion (24 h: 75.2 ± 3.8% v. 60.3 ± 4.1%; 48 h: 82.0 ± 3.4% v. 64.9 ± 4.0%; 72 h: 78.9 ± 3.6% v. 65.9 ± 4.0%, respectively) and hatching (24 h: 33.7 ± 4.2% v. 23.5 ± 3.6%; 48 h: 61.1 ± 4.3% v. 44.0 ± 4.2%; 72 h: 62.6 ± 4.3% v. 50.2 ± 4.2%, respectively) rates at all time points. There was no interaction between CLA and L-carnitine affecting post-thaw viability. In conclusion, supplementation of maturation medium with L-carnitine did not affect embryo development or post-thaw viability. Although addition of CLA during maturation did not affect embryo development, post-thaw cryotolerance was reduced following CLA supplementation. There was no beneficial effect of supplementing maturation medium with both CLA and L-carnitine on embryo development or post-thaw cryosurvival.

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).

260 BREED INFLUENCES OUTCOME OF IN VITRO PRODUCTION OF EMBRYOS IN CATTLE

2010 ◽  
Vol 22 (1) ◽  
pp. 287 ◽  
Author(s):  
M. C. Abraham ◽  
A. Ruete ◽  
Y. C. B. Brandt
Keyword(s):  
Body Fat ◽  
Age Distribution ◽  
Time Of Day ◽  
Developmental Competence ◽  
Carcass Weight ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Frozen Semen ◽  
Significant Difference

Fertility among cattle breeds can vary. The Swedish Red and White dairy breed (SRB) has been systematically bred for good reproductive traits since 1970 and might therefore have retained a better oocyte quality than other dairy breeds. The aim of this study was to determine if the breed of oocyte donor affects the development of embryos using IVM, IVF, and IVC. Oocyte developmental competence in vitro was compared between the SRB (n = 77 animals), the Swedish Holstein breed (SLB, n = 49), and beef breeds (mixed breeds, n = 97). The oocytes (n = 1380, 18 batches) were aspirated from abattoir-derived ovaries from healthy animals with known identity. Statistical analyses were performed using Student’s t-tests and generalized linear mixed models with random effects. The time of collection in relation to slaughter and time of day, as well as aspiration and the following in vitro procedures, were consistent throughout the experiment. The oocytes were matured, fertilized (frozen semen), and cultured according to conventional protocols without serum. Data are presented as mean ± SEM. The SRB and SLB groups were comparable in age [SRB: 66% cows (over 3 years of age), 27% young cows (calved at least once but not over 3 years of age), and 7% heifers; SLB: 63% cows, 20% young cows, and 17% heifers], carcass classification (scale 1-15, where 15 = highest amount of muscle; SRB: 3.8 ± 0.2, SLB 3.5 ± 0.3), body fat (scale 1-15, where 15 =highest amount of fat; SRB: 8.4 ± 0.4, SLB 8.8 ± 0.5) and kilograms of carcass weight (SRB: 297.3±7.4, SLB: 311.6 ± 9.0). The beef group had a significantly higher mean carcass classification (6.2 ± 0.2) and a different age distribution with a higher proportion of heifers (38% cows, 12% young cows, and 50% heifers), but was comparable in body fat content (8.5 ± 0.4) and kilograms of carcass weight (310.9 ± 7.9). Cleavage rate, number of embryos developed beyond the 2-cell stage by 44 h post-fertilization, and the number of blastocysts developed by Days 7 and 8 were noted. All blastocysts were graded and stained with Hoechst 33 342 and the number of nuclei was determined. Cleavage rate was not different among the breeds (SRB: 71.9 ± 0.03%, SLB: 72.5 ± 0.02%, beef: 73.9 ± 0.03%). The percentage of embryos developed beyond 2-cells (from cleaved) did not differ between the beef and SRB (beef: 65.1 ± 6.1%; SRB: 70.4 ± 4.9%) but SLB was significantly greater than than the other breeds (75.4 ± 4.5%). The percentage of blastocysts developed by Day 8 was significantly higher in the beef (21.1 ± 2.7%) and SRB (23.3 ± 3.5%) breeds compared with the SLB (12.5 ± 2.4%). There was no significant difference in blastocyst grades among breeds (scale 1-4, where 1 = highest grade; SRB: 2.4 ± 0.1, SLB: 2.4 ± 0.2, beef: 2.1 ± 0.2), but the number of nuclei in Day 8 blastocysts was significantly lower in the SLB (SRB: 98.9 ± 7.7, SLB: 79.2 ± 8.7, beef: 101.4 ± 6.9). In conclusion, the breed of origin of the oocytes is an important factors affecting the development during in vitro embryo production in cattle. Funded by Formas.

344 EFFECTS OF BMP4 AND ITS INHIBITOR, NOGGIN, ON OOCYTE MATURATION AND DEVELOPMENT OF BOVINE PREIMPLANTING EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 328
Author(s):  
I. La Rosa ◽  
R. Fernandez y Martín ◽  
D. A. Paz ◽  
D. F. Salamone
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Cell Number ◽  
Bmp Signaling ◽  
Control Group ◽  
Cleavage Rate ◽  
Exact Test ◽  
Student’S T

BMP4 regulates different events during development in all vertebrates and Noggin is one of its powerful inhibitors that blocks BMP4 interaction with its receptors (Groppe et al. 2002). In this work, the effect of these factors on bovine oocyte maturation and subsequent embryo development has been investigated. COCs were aspirated from abattoir ovaries and in vitro-matured for 22 h or 24 h in a 5% CO2 humidified atmosphere at 39°C in TCM containing 0.6% BSA, 2 mM FSH, 10 mM cysteamine, 1% antibiotic and 1% pyruvate, control group (C), plus 100 ng mL-1 of BMP4 (B), or 100 ngmL-1 of Noggin (NOG). Oocytes were stained with Hoechst 33342 and classified by their nuclear stage. Effects on embryo development were investigated for embryos produced by parthenogenic activation (PA) and IVF For PA, denuded oocytes were chemically activated in 5 μM ionomycine for 4 min, and immediately incubated in 1.9 mM of 6-dimethilaminopurine for 3 h. For IVF, frozen-thawed semen was centrifuged and resuspended in Bracket and Oliphant (BO) solution and incubated with 22 h matured COCs for 5 h. Embryos were cultured in CR2 medium free of serum and co-culture. Cleavage and blastocyst formation were registered at Day 2 and 9 respectively. Fischer’s exact test was used and P ≤ 0.05 was considered significant. Nuclear progression was not affected by maturation treatments [% of MII: 79.4(C, n = 102), 72.4 (B, n = 98), 80.9 (NOG, n = 89)]. For PA, both factors significantly increased cleavage rates [%: 51.7 (C, n = 284), 65 (B, n = 186), 62.1 (NOG, n = 198)] while blastocyst rates were not affected [%: 8.8 (C), 7.5 (B), and 8.6 (NOG)]. On the other hand, for IVF, cleavage rate was statistically lower for Noggin group [%: 70.7 (C, n = 140), 71.3 (B, n = 157), 64 (NOG, n = 159)] while blastocyst rates were similar between groups [%: 15.7 (C), 13.4 (B), 14.5 (NOG)]. Any of the added factors affected cell number of the embryos at Day 2. Blastocysts did not differ in the number of cells at Day 9 (Student’s t-test was used) neither for PA [mean ± SD: 100 ± 33 (C, n = 9), 88 ± 14 (B, n = 3) and 68 ± 8,(NOG, n = 3)] nor for IVF [mean ± SD: 90 ± 24 (C, n = 9), 132 ± 18 (B, n =4) and 99 ± 8 (NOG, n = 3)]. It is noticeable that addition of these factors during in vitro maturation showed different effects on subsequent embryo development depending on whether the embryos were PA or IVF. Probably, these responses represent differences in the BMP signaling system between these embryos which could be associated with different imprinting pattern. Further experiments are needed to elucidate clearly the mechanisms implicated. To our knowledge, this is the first work to study BMP4 inhibition during bovine in vitro maturation. To “Merlo” and “Nueva Escocia” Slaughterhouses

PSX-A-23 Late-Breaking: Supplementation of Nerve Growth Factor (β-NGF) in the maturation medium and efficiency of in vitro embryo production in cattle

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 365-365
Author(s):  
Lucas Gonçalves ◽  
Muller C Martins ◽  
Natalia Arle ◽  
Rafaela T Torres ◽  
Luisa Migilo ◽  
...  
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Oocyte Maturation ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Nerve Growth ◽  
Maturation Medium ◽  
In Vitro Embryo Production ◽  
Blastocyst Rate

Abstract The aim of this study was to evaluate the supplementation of Nerve Growth Factor (β-NGF) in the maturation medium in in vitro embryo production routines. Antral follicles were aspirated from ovaries of cows obtained from slaughterhouses and then oocytes were selected for quality (grades I and II) for in vitro maturation and subjected to 4 successive in vitro embryo production routines (IVEP). Supplementation of 100 ng of β-NGF was performed in the oocyte maturation medium 22 hours before in vitro fertilization. 48 hours after fertilization of the oocytes, an analysis was made of their cleavage rate by counting blastomeres with the aid of a stereoscopic microscope (cleavage rate = number of embryos / number of initial oocytes). Seven days after fertilization, the blastocyst rate was determined through the relation to the number of oocytes that started cleavage and reached this stage of development (blastocyst rate = number of blastocyst / number of oocytes that started cleavage). To verify the existence of a difference between the supplemented and the non-supplemented groups, the paired T test was applied, using the Excel / Action software (Microsoft). In vitro embryo production routines supplemented with β-NGF in the maturation medium had, on average, a higher cleavage rate (P = 0.0072) and a higher blastocyst rate (P = 0.0033) compared to non-supplemented routines with β-NGF. In this study was demonstrated that Nerve Growth Factor supplementation in the maturation medium improves the efficiency of in vitro embryo production in cattle, and this protein has a probable action in the oocyte maturation process.

Effect of butafosfan supplementation during oocyte maturation on bovine embryo development

Zygote ◽  
2019 ◽  
Vol 27 (05) ◽  
pp. 321-328
Author(s):  
Lucas Teixeira Hax ◽  
Joao Alveiro Alvarado Rincón ◽  
Augusto Schneider ◽  
Lígia Margareth Cantarelli Pegoraro ◽  
Letícia Franco Collares ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryo Development ◽  
Oocyte Maturation ◽  
Cumulus Cells ◽  
Oocyte Quality ◽  
Bovine Embryo ◽  
Cleavage Rate ◽  
Nuclear Maturation

SummaryAround 60–80% of oocytes maturated in vivo reached competence, while the proportion of maturation in vitro is rarely higher than 40%. In this sense, butafosfan has been used in vivo to improve metabolic condition of postpartum cows, and can represent an alternative to increase reproductive efficiency in cows. The aim of this study was to evaluate the addition of increasing doses of butafosfan during oocyte maturation in vitro on the initial embryo development in cattle. In total, 1400 cumulus–oocyte complexes (COCs) were distributed in four groups and maturated according to supplementation with increasing concentrations of butafosfan (0 mg/ml, 0.05 mg/ml, 0.1 mg/ml and 0.2 mg/ml). Then, 20 oocytes per group were collected to evaluate nuclear maturation and gene expression on cumulus cells and oocytes and the remaining oocytes were inseminated and cultured until day 7, when blastocysts were collected for gene expression analysis. A dose-dependent effect of butafosfan was observed, with decrease of cleavage rate and embryo development with higher doses. No difference between groups was observed in maturation rate and expression of genes related to oocyte quality. Our results suggest that butafosfan is prejudicial for oocytes, compromising cleavage and embryo development.

scholarly journals Effect of Rolipram on in vitro maturation, gene expression and embryonic development in bovines

2019 ◽  
Vol 71 (5) ◽  
pp. 1433-1444 ◽  
Author(s):  
B.B. Santana ◽  
G.G. Sobral ◽  
E.T. Gomes ◽  
A.M. Batista ◽  
L.P.R. Teixeira ◽  
...  
Keyword(s):  
Gene Expression ◽  
Control Group ◽  
Cleavage Rate ◽  
Time Control ◽  
Follicular Aspiration ◽  
Maturation Medium ◽  
Significant Difference ◽  
Group A ◽  
Bovine Oocytes

ABSTRACT The aim of this work was to evaluate the effect of the Rolipram during the maturation of bovine oocytes and gene expression of embryos produced in vitro. Bovine ovaries were collected in slaughterhouse. The COCs were selected and divided into 5 groups: Control 0 time; Control: IVM for 24 hours; Rolipram treatments with IVM blocking for 24 hours in maturation medium containing (100, 150 and 200µM). After 24 hours all groups were reseated in IVM for another 24 hours. Subsequently COCs were subjected to the same IVM system and fertilized, being checked for cleavage post fertilization and for blastocyst. In addition, performed expression of the following genes: Mater, BMP15 and Bax. No difference was found in gene expression. Of oocytes evaluated shortly after follicular aspiration, 79.00% were in GV, GVBD, MI, while 13.40%, were in MII and 7.60%, D/NI. Significant difference was observed in different concentrations (T100, T200 and T150µM) in oocytes that have reached the MII phase compared to control treatments (P= 0.003). Differences were observed in cleavage rate (P< 0.05) between T150 and T200 when compared to the C/24 Group. A high difference was observed on blastocyst rate (P< 0.001) among treatments compared to the control group.

336 SHEEP OOCYTES MATURED IN A SIMPLEX MEDIUM (HECM-1), AN ANSWER TO IN VITRO FERTILIZATION

2006 ◽  
Vol 18 (2) ◽  
pp. 275
Author(s):  
C. Navarro-Maldonado ◽  
Y. Ducolomb-Ramirez ◽  
A. Galindo-Rodriguez ◽  
A. Rosado-Garcia
Keyword(s):  
In Vitro Fertilization ◽  
Embryo Development ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Culture Media ◽  
Sperm Quality ◽  
Complete Control ◽  
Vitro Fertilization ◽  
Significant Difference

In vitro maturation and in vitro fertilization (IVM and IVF) of mammalian oocytes still show unsatisfactory results when applied to the study of embryo development. This is probably due to inadequate information about the use of media components and supplements for oocyte maturation and to a discrepancy between results obtained by focusing strictly on oocyte maturation and those that are focused on IVF. A conventional medium that provides adequate results in studies of oocyte maturation (TCM-199) contains hypoxanthine, phosphate ions, and glucose, all known to inhibit embryo development in vitro in some species. In contrast, it has been shown that a simpler medium (HECM-9) increases embryo development in bovine although its use for oocyte maturation has not been defined. This medium contains taurine (an amino acid that reduces intracellular peroxide content) and is supplemented with polyvinyl alcohol (PVA) instead of using protein components, making it a simple defined medium that reduces variability in embryo development. Adding sodium panthothenate to media also confers cell protection against reactive oxygen species. Finally, supplements such as epidermal growth factor (EGF) increase the number of oocytes that complete maturation (Metaphase II, MII) and facilitate embryo development. An adequate combination of our knowledge about in vitro maturation and fertilization of oocytes, together with the requirements for embryo development, is important for the preparation of culture media to study regulatory mechanisms for embryo development and to increase the number of viable and normal term individuals. In this study we compared the effects of HECM-9 (containing panthothenate) vs. TCM-199 (both media supplemented with PVA, EGF, and FSH/LH) on the integrated processes involving IVM and IVF. No significant differences were found between the results obtained with these media in relation to oocyte maturation (65% MII for HECM-9 vs. 71% for TCM-199); however, those oocytes matured in HECM-9 showed a highly significant difference in in vitro fertilization using a conventional IVF medium (SOFm) (25% in HECM-9 vs. 6% in TCM-199). Maturation results were relatively low but in accordance with those reported by other groups, whereas IVF results are lower than those reported in the literature, perhaps because we have been using frozen and thawed samples and do not have complete control over the sperm quality. At present, we are extending our investigation using fresh semen samples.

191 SUPPLEMENTATION OF MATURATION MEDIUM WITH FOLLICULAR FLUID, EPIDERMAL GROWTH FACTOR AND NEUREGULIN AFFECTS MITOCHONDRIAL DNA REPLICATION, OOCYTE MATURATION AND EMBRYO DEVELOPMENT IN PIGS

2012 ◽  
Vol 24 (1) ◽  
pp. 208
Author(s):  
J. Mao ◽  
K. M. Whitworth ◽  
L. D. Spate ◽  
E. M. Walters ◽  
J. Zhao ◽  
...  
Keyword(s):  
Growth Factor ◽  
Embryo Development ◽  
Oocyte Maturation ◽  
Follicular Fluid ◽  
Copy Number ◽  
In Vitro Maturation ◽  
Meiotic Maturation ◽  
Mtdna Copy Number ◽  
Maturation Medium

Mitochondria supply the majority of ATP in a cell. Mitochondrial DNA (mtDNA) copy number in oocytes might be used as a marker of viability and might be a key determinant of pre-implantation embryo development. However, little is known about mtDNA copy number changes during porcine oocyte maturation and its regulation by extracellular growth factors. The objectives of the current study were to determine the effects of supplementation of in vitro maturation medium with porcine follicular fluid (pFF; 0, 10, 20 and 30%), epidermal growth factor (EGF; 10 ng mL–1), neuregulin 1 (NRG; 20 ng mL–1) and NRG + IGF1 (insulin-like growth factor-1; 100 ng mL–1 + NRG, 20 ng mL–1) during in vitro maturation on mtDNA copy number, oocyte meiotic maturation and subsequent embryo development after parthenogenic activation. Follicular fluid used for the pFF supplementation experiment was prepared from medium-sized (3–6 mm in diameter) healthy follicles. Cumulus–oocyte complexes (COCs) were collected from antral follicles (3–6 mm in diameter), cultured in LH- and FSH-containing maturation medium for 22 h at 38.5°C, transferred into basic maturation medium without FSH and LH and cultured for another 22 h. The basic maturation medium was TCM-199 supplemented with 0.1% polyvinylalcohol (w/v), 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 10 μg mL–1 of gentamicin, 0.57 mM cysteine and without or with different growth factors depending on the experimental design. In total, 177 germinal vesicle (GV) oocytes and 3837 MII oocytes were used for this study. All data were analyzed by the general linear model (GLM) procedure of SAS software (V9.2). The mtDNA copy number in oocytes increased (P < 0.05) from GV to MII stage oocytes (MII oocytes from all treatment groups pooled). Supplementation of IVM media with 10% pFF decreased mtDNA copy number (P < 0.05), whereas 20 and 30% pFF had no major effect on mtDNA copy number, resulting in a quadratic correlation between percentage of pFF and mtDNA copy number. There was a negative linear correlation between percentage of pFF and oocyte meiotic maturation, with a higher percentage of pFF inhibiting meiotic maturation (73.2 ± 5.2, 71.9 ± 4.8, 64.1 ± 8.5 and 65.8 ± 6.4% for 0, 10, 20 and 30% pFF groups, respectively). The mtDNA copy numbers in EGF and NRG-treated MII oocytes were significantly higher than those in GV oocytes, whereas the control was not different (EGF, 237 042.6 ± 22 198.2; NRG, 281 293.4 ± 22 893.5; and control, 231 856.8 ± 21 883.5 in MII oocytes vs 192 288.7 ± 21 675.4 in GV oocytes). The EGF, NRG and NRG+IGF1 treatments enhanced oocyte maturation as well. There was no difference in Day-7 blastocyst formation between EGF, NRG+IGF1 and the control, whereas the NRG treatment enhanced blastocyst formation as compared to the control (23.8 ± 2.4 vs 15.1 ± 2.1%; P < 0.05). This study demonstrated that there was an increase in mtDNA copy number during in vitro maturation. The EGF and NRG treatments stimulated mitochondria biogenesis, which may provide new means to increase oocyte quality and enhance embryonic development.

Sign in / Sign up

Export Citation Format

Share Document