PROLONGED EXPOSURE OF HUMAN BLASTOCYSTS TO HYALURONAN-ENRICHED TRANSFER MEDIA HAS NO EFFECT ON PERI-IMPLANTATION STAGE EMBRYO DEVELOPMENT DURING IN VITRO CULTURE

The in vitro developmental abilities of porcine oocytes are generally increasing steadily at a similar ratio to those of in vivo embryos. However, it has been suggested that the in vitro culture system for the development of porcine embryos is not optimal. In this study, we investigated the effect of 2 oxygen concentrations (5 and 20%) on porcine embryo development during in vitro maturation and in vitro culture and analyzed differences in gene expression of resulting blastocysts. Oocytes were recovered by aspiration of slaughterhouse ovaries and then matured in tissue culture medium (TCM) 199 supplemented with 10% porcine follicular fluid (pFF), epidermal growth factor (EGF), insulin, pyruvate, cystine, and gonadotropin. Matured oocytes were then activated parthenogenetically, cultured in PZM-3 media for 7 days. In vitro maturation (M group) of oocytes was carried out under two oxygen concentration (5 and 20%) in terms of nuclear maturation (polar body extrusion; Exp. 1). The developmental differences between 5% oxygen culture group and 20% oxygen culture group during in vitro culture (C group) of embryos after parthenogenetic activation was investigated in terms of first cleavage and blastocyst formation (Exp. 2). Relative mRNA abundance of multiple genes in blastocysts was analyzed for transcript abundance of genes related with metabolism (GLUT1, LDHA), oxidative response (MnSOD, GPX1), apoptosis (BAX, Bcl2), and developmental competence (CCNB1, IGF2R; Exp. 3). The results show there were no significant differences in maturation rate between 2 oxygen concentrations during in vitro maturation (83 v. 86%). It was thought that cumulus cells surrounding oocytes might have attenuated oxidative stress, but number of resulting blastocysts were (P < 0.05) increased in 5% IVC group when compared with 20% IVC group (18.67 v. 14.09%, respectively). Moreover, the M20C5 group (23.01%) had a beneficial effect on in vitro culture compared with M5C5 (14.32%), M5C20 (10.30%), and M20C20 (17.88%) groups. Total cell numbers were not significantly different among groups. According to mRNA abundance data of multiple genes, each group altered the expression of genes in various patterns. Therefore, it could be concluded that high oxygen tension during in vitro maturation and low oxygen tension during in vitro culture might alter the expression of multiple genes related to oocyte competence and improve (P < 0.05) embryo development, but not blastocyst quality. This study was supported by MKE (#2009-67-10033839, #2009-67-10033805), NRF (#M10625030005-508-10N25), BK21 for Veterinary Science, IPET (#109023-05-1-CG000), and Hanhwa L&C.

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134 CYSTEINE AND CATALASE SUPPLEMENTATION FOR 7 DAYS DURING IN VITRO CULTURE IMPAIRS BOVINE EMBRYO DEVELOPMENT

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab134 ◽

2013 ◽

Vol 25 (1) ◽

pp. 214

Author(s):

B. C. S. Leão ◽

N. A. S. Rocha ◽

M. F. Accorsi ◽

É. Nogueira ◽

G. Z. Mingoti

Keyword(s):

In Vitro Culture ◽

Embryo Development ◽

Cellular Proliferation ◽

Mitochondrial Respiratory Chain ◽

Organic Peroxide ◽

Redox Status ◽

Culture Period ◽

Bovine Embryo ◽

Embryo Survival

The production of reactive oxygen species (ROS), such as superoxide anion (O2–), hydroxyl radical (OH–), hydrogen peroxide (H2O2) and organic peroxide, is a normal process that occur in the cellular mitochondrial respiratory chain (Morado et al. 2009 Reprod. Fert. Dev. 21, 608–614). Supplementation with antioxidants during in vitro culture (IVC) appears to increase the resistance of bovine embryos to the oxidative stress, and consequently improve embryo development and cryotolerance (Rocha et al. 2011 Reprod. Fert. Dev. 23 157–158). This study was conducted to evaluate the effects of period of supplementation with intra (cysteine, CIST) or extracellular (catalase, CAT) antioxidants during IVC on embryo development and cryotolerance. Cumulus–oocyte complexes (n = 1132) were maturated for 24 h in B199 medium, at 38.5°C and 5% CO2 in air. After fertilization (Day 0), zygotes were IVC for 7 days in SOF medium (0.5% BSA + 2.5% FCS) in 7% O2, 5% CO2 e 88% N2 atmosphere, at 38.5°C. The antioxidant supplementation was performed during all of the culture period (from Day 1 to Day 7) or during the first 72 h (from Day 1 to Day 3), with 0.6 mM CIST, 100 UI CAT or without antioxidants (CONTR). The cleavage and blastocyst rates were evaluated, respectively, at 72 and 168 h post-insemination, when expanded blastocysts grade I were vitrified (n = 91) by Vitri-Ingá® protocol (Ingámed®, Maringá, PR, Brazil). Then, they were thawed and cultured for 24 h to evaluate re-expansion rates. The differences between groups were analyzed by ANOVA followed by Tukey’s test, and re-expansion rates by chi-square test (P ≤ 0.05). The cleavage and blastocyst rates were, respectively, 83.52 ± 4.52a/36.19 ± 3.21a (CONTR), 79.16 ± 4.52a/38.08 ± 3.21a (CIST Day 3), 77.74 ± 4.52a/42.09 ± 3.21a (CAT Day 3), 73.57 ± 4.05a/11.15 ± 2.87b (CIST Day 7), 71.83 ± 4.05a/15.07 ± 2.87b (CAT Day 7). The embryo re-expansion rates were 90.00%a (CONTR), 93.33%a (CIST Day 3), 75.00%a (CIST Day 7), 63.64%a (CAT Day 3) and 75.00%a (CAT Day 7). Supplementation with antioxidants for 7 days of IVC impaired embryo development, compared with addition up to Day 3 (P ≤ 0.05). However, it did not affect in vitro embryo cryotolerance (P ≥ 0.05). Supplementation with antioxidants throughout all the IVC significantly impaired blastocyst rate, probably by exerting a toxic effect leading to an arrest of embryonic development. It is believed that prolonged culture in the presence of antioxidants results in excessive reduction of ROS leading to an imbalance of the cellular redox status. It is known that ROS, particularly H2O2, act on signaling pathways involved in the cellular proliferation and differentiation, in gene expression and metabolism during embryo development. Supplementation with antioxidants up to Day 3 did not differ from CONTR, probably due to low O2 tension, and the presence of antioxidants in FBS and BSA. In conclusion, supplementation with cysteine and catalase during all of the culture period impaired embryo development, however this reduction did not affect embryo survival after vitrification. Financial support was provided by FAPESP (#2011/18257-2). The authors acknowledge Ingámed, Alta Genetics Brazil.

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35 EFFECTIVENESS OF MICROWELL-BASED IN VITRO CULTURE SYSTEMS FOR BOVINEZONA-FREE CLONED EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab35 ◽

2011 ◽

Vol 23 (1) ◽

pp. 124

Author(s):

C. Feltrin ◽

M. Machado ◽

L. M. V. Queiroz ◽

M. A. S. Peixer ◽

P. F. Malard ◽

...

Keyword(s):

In Vitro Culture ◽

Embryo Development ◽

Zona Pellucida ◽

Blastocyst Stage ◽

Aggregation State ◽

Developmental Potential ◽

In Vitro Development ◽

Vitro Development

In vitro embryo production by handmade cloning (HMC) usually requires individual embryo culture, because zona-free embryos cannot be grouped in standard in vitro culture (IVC) protocols. The aim of this study was to evaluate the developmental potential of bovine embryos produced by HMC (Ribeiro et al. 2009 Cloning Stem Cells 11, 377–386) after in vitro culture (IVC) in 3 microwell (WOW) systems. After in vitro maturation, oocytes were denuded and incubated in demecolcine (Ibáñez et al. 2003 Biol. Reprod. 68, 1249–1258), followed by zona pellucida removal, oocyte bisection, embryo reconstruction, electrofusion, and chemical activation. Cloned embryos were allocated to 1 of 3 IVC groups: cWOW: conventional microwells (250 μm, round; Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264); mWOW: modified microwells (130 μm, conical; Feltrin et al. 2006 Reprod. Fert. Dev. 18, 126); and WOW-PDMS: microwells in polydimethylsiloxane chips (170 μm, cylindrical with microchannels); IVF embryos were used as controls (Bertolini et al. 2004 Reproduction 128, 341–354). Cleavage (Day 2), blastocyst (Day 7), and pregnancy (Day 30) rates were analysed by the chi-square test, for P < 0.05. Results are shown in Table 1. Cleavage rates were similar between groups, but development to the blastocyst stage was higher in IVF controls than cloned embryo groups. Among cloned embryo groups, blastocyst rate was higher in the mWOW group than the conventional and the PMDS-based microchannels. Nevertheless, in vivo development to Day 30 of pregnancy was not different between cloned groups. Our results for in vitro embryo development indicated that the mWOW provided more suitable conditions for embryo development to the blastocyst stage when compared with cWOW or even WOW-PDMS. Among some possible reasons include the physical advantage of a smaller microwell that may better mimic the constraining effect of the zona pellucida on the developing embryo. That may also provide greater blastomere stability, favouring the aggregation state during the first rounds of cleavages, also aiding compaction and subsequent cavitation. The narrower microwell system appeared to have promoted better in vitro development than the conventional and the DMPS-based microwell systems, with no impact on subsequent in vivo development. However, the IVC in the WOW-PDMS system supported reasonable rates of development, in accordance with the current literature. Table 1.In vitro development of bovine IVF and cloned embryos produced after the in vitro culture in distinct IVC systems

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242 USE OF BRAIN-DERIVED NEUROTROPHIC FACTOR IN IN VITRO PREMATURATION OF BOVINE OOCYTES SUBJECTED TO PARTHENOGENETIC ACTIVATION

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab242 ◽

2008 ◽

Vol 20 (1) ◽

pp. 200

Author(s):

T. H. C. De Bem ◽

R. Rochetti ◽

P. R. L. Pires ◽

F. F. Bressan ◽

P. R. Adona ◽

...

Keyword(s):

In Vitro Culture ◽

Embryo Development ◽

Polar Body ◽

Hatching Rate ◽

Blastocyst Development ◽

Embryo Production ◽

Parthenogenetic Activation ◽

Bovine Oocytes ◽

Hatching Rates

Prematuration provides an additional time for oocyte capacitation and maturation in an attempt to improve in vitro embryo production (IVP) rates and allows media supplementation during this period for IVP. The aim of this study was to use brain-derived neurotropic factor (BDNF) in prematuration to improve maturation of bovine oocytes subjected to parthenogenetic activation and cultured with different media. Oocytes were subjected to prematuration in TCM-199 medium supplemented with 10 µm butyrolactone I, 2.0 mm pyruvate, and 10 µg mL–1 gentamicin for 24 h in the absence of BDNF (control) or in the presence of 10 ng mL–1 BDNF (BD). Oocytes were then in vitro-matured (IVM) in TCM-199 medium supplemented with 10% FCS, 0.5 µg mL–1 FSH, 5.0 µg mL–1 LH, 2.0 mm pyruvate, and 10 µg mL–1 gentamicin at 38.5�C under 5% CO2 in air. After 19 h oocytes were denuded using hyaluronidase and vortexing for 3 min for the 1st polar body (1PB) selection. Those which extruded the 1PB were maintained in IVM until 26 h, when parthenogenetic activation was performed (5 min in 5 µm ionomycin, followed by 3 h in 2 mm 6-DMAP). Activated oocytes were then transferred to in vitro culture (IVC) for embryo development evaluation. Embryos from both groups were cultured in SOF medium with 2.5% FCS, 0.05 g mL–1 BSA, 0.2 mm pyruvate, and 10 mg mL–1 gentamicin. Cleavage rates on the second day of in vitro culture (D2), embryo production at Days 7 and 8 (D7 and D8), and hatching rate at Day 8 were evaluated. Data regarding 1PB extrusion, cleavage, blastocyst development on D7 and D8, and blastocyst D8 hatching rates of three replicates were analyzed by chi-square test at 5% significance using the BIOESTATS 4.0 software. Control and BD, respectively, did not show differences (P > 0.05) regarding 1PB extrusion (n = 164, 63.81%, and n = 175, 66.79%) or cleavage (n = 117, 71.34%, and n = 138, 78.86%). However, for control and BD, respectively, blastocyst development on D7 (n = 63, 38.41%, and n = 89, 50.86%), D8 (n = 63, 38.41%, and n = 91, 52.00%), and hatching on D8 (n = 22, 34.92%, and n = 39, 43.82%) were all significantly higher for BD when compared with control (P < 0.05). In conclusion, BDNF during prematuration improved in vitro embryo development by increasing blastocyst and hatching rates of parthenogenetic embryos.

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18 EMBRYO AGGREGATION IN PIG IMPROVES CLONING EFFICIENCY AND EMBRYO QUALITY

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab18 ◽

2016 ◽

Vol 28 (2) ◽

pp. 139

Author(s):

C. Buemo ◽

A. Gambini ◽

L. Moro ◽

R. F. Y. Martin ◽

D. Salamone

Keyword(s):

In Vitro Culture ◽

Dna Fragmentation ◽

Embryo Development ◽

Embryo Quality ◽

T Test ◽

Control Group ◽

Reconstructed Embryo ◽

Student’S T ◽

Student’S T Test

In this study, we analysed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst size and cell number, DNA fragmentation levels by TUNEL assay, and the relative expression of genes associated with pluripotency, apoptosis, trophoblast markers, and DNA methylation in the porcine. Cumulus-oocyte complexes were recovered from slaughterhouse ovaries by follicular aspiration. Maturation was performed in TCM for 42 to 48 h at 39°C and 5% CO2. After denudation by treatment with hyaluronidase, mature oocytes were stripped of the zona pellucida using a protease and then enucleated by micromanipulation; staining was performed with Hoëchst 33342 to observe metaphase II. Ooplasms were placed in phytohemagglutinin to permit different membranes to adhere between each other; the ooplasm membrane was adhered to a porcine fetal fibroblast from an in vitro culture. Adhered membranes of the donor cell nucleus and enucleated oocyte cytoplasm were electrofused through the use of an electric pulse (80 V for 30 μs). All reconstituted embryos were electrically activated using an electroporator in activation medium (0.3 M mannitol, 1.0 mM CaCl2, 0.1 mM MgCl2, and 0.01% polyvinyl alcohol) by a DC pulse of 1.2 kVcm for 80 μs. Then, embryos were incubated in 2 mM 6-DMAP for 3 h. In vitro culture of zona-free embryos was achieved in a well of wells system in 100 μL of SOF medium. Two experimental groups were used, one control group with a single reconstructed embryo per microwell (1×) and the other group placing 3 reconstructed embryo per microwell (3x aggregation group). Embryos were cultivated at 39°C in 5% O2, 5% CO2 for 7 days in SOF medium with a supplement of 10% fetal bovine serum on the fifth day. At Day 7, resulting blastocysts were classified according to their morphology and diameter to determine their quality. Our results showed that aggregation of 3× embryos increased blastocyst formation rate and blastocyst size of pig cloned embryos (Fisher’s test P < 0.05 and Student’s t-test P < 0.05, respectively). The DNA fragmentation levels in 3× aggregated cloned blastocysts were significantly decreased compared to 1x blastocyst (Student’s t-test P < 0.05). Levels of Oct4, Klf4, Igf2, Bax, and Dnmt1 transcripts were significantly higher in aggregated embryos, whereas Nanog levels were not affected. Transcripts of Cdx2 and Bcl-xl were essentially nondetectable (Student’s t-test P < 0.05). Our study suggests that embryo aggregation in the porcine may be beneficial for cloned embryo development and embryo quality, through a reduction in apoptotic levels and an improvement in cell reprogramming.

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Effect of bovine oviductal fluid on development and quality of bovine embryos produced in vitro

Reproduction Fertility and Development ◽

10.1071/rd15238 ◽

2017 ◽

Vol 29 (3) ◽

pp. 621 ◽

Cited By ~ 28

Author(s):

Ricaurte Lopera-Vasquez ◽

Meriem Hamdi ◽

Veronica Maillo ◽

Valeriano Lloreda ◽

Pilar Coy ◽

...

Keyword(s):

In Vitro Culture ◽

Embryo Development ◽

Cell Mass ◽

Water Channel ◽

Calf Serum ◽

Cell Counting ◽

Bovine Embryos ◽

Oviductal Fluid

To evaluate the effect of bovine oviductal fluid (OF) supplementation during in vitro culture of bovine embryos on their development and quality, in vitro-produced zygotes were cultured in synthetic oviductal fluid (SOF; negative control; C–) supplemented with OF or 5% fetal calf serum (positive control; C+). Embryo development was recorded on Days 7–9 after insemination and blastocyst quality was assessed through cryotolerance, differential cell counting of the inner cell mass and trophectoderm, and gene expression. OF was added to the culture medium at concentrations ranging from 0.625% to 25%. The higher OF concentrations (5%, 10% and 25%) had a detrimental effect on embryo development. Lower OF concentrations (1.25% and 0.625%) supported embryo development until Day 9 (27.5%) and produced higher-quality blastocysts, as reflected by their cryotolerance (53.6% and 57.7% survival at 72 h, respectively, vs 25.9% in C+) and total cell number (mean (± s.e.m.) 165.1 ± 4.7 and 156.2 ± 4.2, respectively, vs 127.7 ± 4.9 in C– and 143.1 ± 4.9 in C+). Consistent with these data, upregulation of the water channel aquaporin 3 (AQP3) mRNA was observed in blastocysts supplemented with 1.25% OF compared with C– and C+. Serum supplementation resulted in a reduction in the expression of glucose and lipid metabolism-related genes and downregulation of the epigenetic-related genes DNA methyltransferase 3A (DNMT3A) and insulin-like growth factor 2 receptor (IGF2R). In conclusion, in vitro culture with low concentrations of OF has a positive effect on the development and quality of bovine embryos.

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An ultrastructural study of embryo and endosperm development during in vitro culture of maize ovaries (Zea mays)

Canadian Journal of Botany ◽

10.1139/b86-297 ◽

1986 ◽

Vol 64 (10) ◽

pp. 2227-2238 ◽

Cited By ~ 16

Author(s):

J. H. N. Schel ◽

H. Kieft

Keyword(s):

In Vitro Culture ◽

Embryo Development ◽

Light And Electron Microscopy ◽

Culture Method ◽

Endosperm Development ◽

Endosperm Formation ◽

Development In Vitro ◽

Maize Embryos

A culture method is described which allows the continuous supply of fresh liquid medium and which prevents the accumulation of toxic metabolites. Development of maize embryos and endosperm after various periods of in vitro ovary culture was studied by light and electron microscopy. Using this method the ultrastructural features of embryo development in vitro were similar to those of in vivo embryos. In contrast, the formation of endosperm was irregular with the absence of cellularization of the inner endosperm being frequent. In some cases, only the endosperm developed without any indication of embryo formation. In a calcium-depleted medium, embryo development was normal but again, endosperm formation was aberrant. No cells were formed in the central part of the endosperm and near the placental region degeneration took place, resulting in vacuoles with dark inclusions, clumps of rough endoplasmic reticulum membranes, and cellular breakdown. The events occurring after in vitro culture strongly resemble those taking place after intergeneric crosses or crosses between diploid and tetraploid strains. It is concluded that defective endosperm development is probably the main factor for the failure of embryo development.

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27 SOMATIC CELL NUCLEAR TRANSFER CLONING AND EMBRYO AGGREGATION IN PIGS

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab27 ◽

2014 ◽

Vol 26 (1) ◽

pp. 128

Author(s):

C. P. Buemo ◽

A. Gambini ◽

I. Hiriart ◽

D. Salamone

Keyword(s):

In Vitro Culture ◽

Somatic Cell ◽

Embryo Development ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Electric Pulse ◽

Blastocyst Stage ◽

Control Group

Somatic cell nuclear transfer (SCNT) derived blastocysts have lower cell number than IVF-derived blastocysts and their in vivo counterparts. The aim of this study was to improve the blastocyst rates and quality of SCNT blastocysts by the aggregation of genetically identical free zona pellucida (ZP) porcine clones. Cumulus–oocyte complexes were recovered from slaughterhouse ovaries by follicular aspiration. Maturation was performed in TCM for 42 to 48 h at 39°C and 5% CO2. After denudation by treatment with hyaluronidase, mature oocytes were stripped of the ZP using a protease and then enucleated by micromanipulation; staining was performed with Hoechst 33342 to observe metaphase II. Ooplasms were placed in phytohemagglutinin to permit different membranes to adhere between each other; the ooplasm membrane was adhered to a porcine fetal fibroblast from an in vitro culture. Adhered membranes of the donor cell nucleus and enucleated oocyte cytoplasm were electrofused through the use of an electric pulse (80 V for 30 μs). All reconstituted embryos (RE) were electrically activated using an electroporator in activation medium (0.3 M mannitol, 1.0 mM CaCl2, 0.1 mM MgCl2, and 0.01% PVA) by a DC pulse of 1.2 kV cm–1 for 80 μs. Then, the oocytes were incubated in 2 mM 6-DMAP for 3 h. In vitro culture of free ZP embryos was achieved in a system of well of wells in 100 μL of medium, placing 3 activated oocytes per microwell (aggregation embryo), whereas the control group was cultivated with equal drops without microwells. Embryos were cultivated at 39°C in 5% O2, 5% CO2 for 7 days in SOF medium with a supplement of 10% fetal bovine serum on the fifth day. The RE were placed in microwells. Two experimental groups were used, control group (not added 1X) and 3 RE per microwell (3X). At Day 7, resulting blastocysts were classified according to their morphology and diameter to determine their quality and evaluate if the embryo aggregation improves it. Results demonstrated that aggregation improves in vitro embryo development rates until blastocyst stage and indicated that blastocysts rates calculated over total number of oocytes do not differ between groups (Table 1). Embryo aggregation improves cleavage per oocyte and cleavage per microwell rates, presenting statistical significant differences and increasing the probabilities of higher embryo development generation until the blastocyst stage with better quality and higher diameter. Table 1.Somatic cell nuclear transfer cloning and embryo aggregation

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200 EFFECT OF EXOGENOUS PROGESTERONE DURING IN VITRO CULTURE ON EARLY EMBRYO DEVELOPMENT IN CATTLE

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab200 ◽

2008 ◽

Vol 20 (1) ◽

pp. 179

Author(s):

M. Clemente ◽

P. Lonergan ◽

C. Borque ◽

J. de La Fuente ◽

D. Rizos

Keyword(s):

Gene Expression ◽

In Vitro Culture ◽

Embryo Development ◽

Mineral Oil ◽

Blastocyst Stage ◽

Early Embryo ◽

Early Embryo Development ◽

Significant Difference ◽

The Media

Preimplatation embryos grown in vitro are sensitive to their environment, and the conditions of culture can affect developmental potential. Progesterone (P4) is the key hormone responsible for maintenance of pregnancy in mammals, and circulating levels in the early postconception period have been associated with pregnancy success. It is not clear whether P4 acts directly or indirectly on the embryo to alter gene expression and development. The aim of this study was to assess the effect of varying levels of exogenous P4 on the development of bovine zygotes to the blastocyst stage in vitro. A preliminary study was conducted to analyze the media used for culture (stock of P4, SOF, SOF + 1 × 10–7 M P4) on Days 1 (day of culture), 4, and 7 for P4 concentration in 25-μL droplets overlain with mineral oil or 500 μL in wells with or without mineral oil. P4 was measured using an ELISA kit, prepared for human serum or plasma (DE1561 Dimeditec Diagnostics GmbH, Kiel, Germany). Inter- and intra-assay coefficients of variation were 6.63 and 6.42%, respectively, and recovery was 95%. P4 concentration on Day 1 in all media was the expected (40 ng mL–1). However, on Days 4 and 7 in media under mineral oil, the level of P4 was nearly zero (0.1 to 1.6 ng mL–1) compared with the media without mineral oil, which remained unchanged (39 to 40 ng mL–1) through the 7 days of culture. Zygotes (n = 1467) were produced in 8 replicates by in vitro oocyte maturation and fertilization, and were cultured in groups of 40 to 50 in wells of 500 μL without mineral oil in (1) SOF supplemented with 5% fetal calf serum (control–), (2) SOF with ethanol (control+), (3) SOF with P4 0.1 × 10–7 M, (4) SOF with P4 1 × 10–7 M, and (5) SOF with P4 10 × 10–7 M at 39°C, 5% CO2 and 5% O2, with maximum humidity. No significant difference was found between groups in cleavage rate or blastocyst yield on Days 6, 7, and 8 (Table 1). These results indicate that the addition of P4 to the in vitro culture medium (SOF) did not enhance the development of bovine embryos to the blastocyst stage. However, further studies on the quality of these embryos in terms of gene expression are in preparation. Table 1. Effect of P4 on bovine in vitro early embryo development

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149 EFFECT OF RESVERATROL ANALOGUE ON DEVELOPMENT OF IN VITRO-FERTILIZED BOVINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv29n1ab149 ◽

2017 ◽

Vol 29 (1) ◽

pp. 183 ◽

Cited By ~ 1

Author(s):

T. A. Patrocínio ◽

C. A. C. Fernandes ◽

L. S. Amorim ◽

J. R. Ribeiro ◽

G. C. Macedo ◽

...

Keyword(s):

In Vitro Culture ◽

Embryo Development ◽

Culture Medium ◽

Synthetic Analogue ◽

Bovine Embryo ◽

High Humidity ◽

Cleavage Rate ◽

Antioxidant Agents ◽

Blastocyst Rate

Oxidative stress is one of the main effects of in vitro culture. Generation of reactive oxygen species (ROS) by embryos can be enhanced by the sub-optimal in vitro culture conditions and are associated with a delay in embryonic development. However, supplementation of culture medium with antioxidant agents can minimize the effects of ROS (Guérin et al. 2001 Hum. Reprod. Update 7, 175–189). Resveratrol is an example of a potent antioxidant, and modifications in its structure can improve its biological activity. This study evaluated the effect of AR33 (formula with patent pending), an analogue of resveratrol with high antioxidant activity, on embryo development. Bovine cumulus-oocyte complexes recovered from ovaries collected at the slaughterhouse were in vitro matured for 24 h and oocytes were in vitro fertilized for 20 h, both at 38.8°C under 5% CO2 in air and high humidity. Partially denuded presumptive zygotes were randomly distributed in 4 treatments (with 6 replicates): 0 µM (control, n = 347), 0.1 µM (n = 337), 0.5 µM (n = 277), and 2.5 µM (n = 343) of AR33. The base medium was SOFaa supplemented with 2.5% FCS and incubation conditions were 38.8°C under 5% CO2 in air and high humidity. Half of culture medium was renewed (feeding) at Day 3 and 5 post-fertilization. Cleavage was evaluated at Day 3 and blastocyst rates at Day 7 and 8 post-fertilization. Data were analysed by logistic regression considering the significance level of P < 0.05. Values are shown as mean ± SEM. Cleavage rate was higher (P < 0.05) for 2.5 µM (69.0 ± 4.4%) than for 0, 0.1, and 0.5 µM AR33 (62.1 ± 2.0%, 60.7 ± 5.9%, and 56.7 ± 5.8%, respectively). At Day 7, the blastocyst rate was similar (P > 0.05) among 0.1, 0.5, and 2.5 µM (18.1 ± 5.4%, 17.5 ± 2.9%, and 19.4 ± 3.3%, respectively) and all of them were higher (P < 0.05) than 0 µM AR33 (12.4 ± 2.5%). At Day 8, there was again no difference (P > 0.05) among 0.1, 0.5, and 2.5 µM AR33 (21.0 ± 5.0%, 18.4 ± 2.1%, and 24.6 ± 3.3%, respectively) but only 0.1 and 2.5 µM showed higher (P < 0.05) blastocyst rate than 0 µM AR33 (15.2 ± 2.5%). In conclusion, the synthetic analogue of resveratrol tested in this study can improve bovine embryo development in culture medium supplemented with 2.5% FCS under 5% CO2 in air. A concentration of 2.5 µM AR33 can be a choice for further studies. This study was supported by Fapemig, CAPES, and CNPq.

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