335 EFFECT OF VITAMIN E ON THE DEVELOPMENT OF BOVINE OOCYTES AFTER CHEMICAL ACTIVATION

2007 ◽  
Vol 19 (1) ◽  
pp. 283
Author(s):  
J. I. Park ◽  
Y. Jang ◽  
E. S. Lee
Keyword(s):  
Vitamin E ◽  
Apoptotic Cell ◽  
Chemical Activation ◽  
Calf Serum ◽  
Cell Number ◽  
Total Cell ◽  
Chi Square ◽  
Cell Numbers ◽  
Humidified Air

Oxidative stress is known to induce apoptotic cell death by reactive oxygen species (ROS) generated from in vitro culture systems. This study was conducted to evaluate the effect of Vitamin E (VitE), as antioxidant, on development of bovine embryos activated in vitro. Bovine ovaries were collected from slaughtered cows at a local abattoir. Oocytes were aspirated from follicles 3-8 mm in diameter and transferred to maturation medium: tissue culture medium (TCM)-199 supplemented with 10% (v/v) fetal calf serum, 100 mg/mL-1 l-cysteine, 20 mg/mL-1 sodium pyruvate, gonadotropins (250 IU each of eCG and hCG/mL), 10 mg/mL-1 epidermal growth factor, and 100 �M VitE. Oocytes were cultured at 38.9�C in 5% CO2 in humidified air. After 22 hours of culture, oocytes with polar bodies were selected and subjected to activation treatments. Oocytes were exposed to calcium ionomycin (5 �M for 5 min), followed by incubation with 6-DMAP (2 mM) for 3.5 hours in medium supplemented with or without VitE (100 �M). After activation, oocytes were cultured in mSOF medium containing 0.8% BSA at 38.9�C in 5% CO2, 5% O2 in humidified air for 7–8 days. Cell numbers were counted by the number of nuclei of blastocysts stained with Hoechst 33342, and apoptosis was detected by TUNEL assay using a MK500 kit (Takara Bio, Inc., Otsu, Shiga, Japan). Total cell and apoptotic cell number were determined under a fluorescence microscope. Data were analyzed using Student's t-test and chi-square test. The cleavage and blastocyst rates were significantly higher (P < 0.05) after activation with VitE (78.1% and 16.3%, n = 80) than without VitE (66.7% and 11.0%, n = 60). Total cell numbers were also significantly higher (P < 0.05) in blastocysts after activation with VitE (143.0 ± 34.02, n = 21) than in those without VitE (127.63 ± 40.25, n = 20). However, the percentage of TUNEL-positive (apoptotic) cells was similar between blastocysts activated with VitE (5.38 ± 2.22) and those without VitE (6.76 ± 1.98). The results of the present study demonstrate that vitamin E added to activation medium promoted further development of activated embryos, although its role in the alleviation of apoptosis remains unclear.

140 EFFECT OF A NOVEL BOVINE EMBRYO CULTURE MEDIUM TO IMPROVE BLASTOCYST DEVELOPMENT

2013 ◽  
Vol 25 (1) ◽  
pp. 217
Author(s):  
R. F. Gonçalves ◽  
C. Figueiredo ◽  
M. A. Achilles
Keyword(s):  
Culture Medium ◽  
Apoptotic Cell ◽  
Cell Number ◽  
Total Cell ◽  
Cell Numbers ◽  
Group 2 ◽  
Group 1 ◽  
Hatching Rates

There are still immense differences in the quality of in vitro-produced embryos compared to their in vivo-generated counterparts. These differences include a higher sensitivity of in vitro-produced embryos towards cryopreservation. The quality of such embryos has been evaluated using various parameters like morphological examination, assessment of total cell numbers, or pregnancy rates after transfer. In the present study, the effects of glycine, alanine, taurine, and glutamine addition to SOF (Achilles Genetics culture medium, Achilles Genetics®, Garça, SP, Brazil) on the in vitro development (cleavage and blastocyst rates) and quality (total cell and apoptotic cell numbers) of bovine embryos were determined. Ovaries of Nelore cows were obtained from a slaughterhouse. Cumulus–oocyte complexes (COC) were collected from follicles ≥4 mm in diameter, matured in TCM-199, and fertilized with frozen–thawed Nelore bull semen (IVF = Day 0). On Day 1, presumptive zygotes were cultured in SOF supplemented with fetal bovine serum (FBS, group 1, n = 550) or in Achilles Genetics culture medium (SOF supplemented with Achilles Mixture and FBS, group 2, n = 557) at 38.5°C and 5% CO2 in air until Day 9. Embryos were evaluated during culture: at Day 3 cleavage rates, at Day 7 blastocyst rates, and on Day 9 hatching rates. Experiments were replicated 5 times, analysed using ANOVA, followed by a comparison of means by Tukey test (P ≤ 0.05). Blastocysts at Day 8 from Group 1 (n = 75) and Group 2 (n = 75) were fixed and permeabilized for TUNEL assay (DeadEndTM Florimetric TUNEL System, Promega, Madison, WI, USA), according to the manufacturer instructions. Total cell number, apoptotic cell number, and apoptotic cell index (calculated by dividing the apoptotic cell number by total cell number) were analyzed by analysis of variance and means were compared by Student Newman Keuls test. The threshold of significance was set at P ≤ 0.05. Cleavage rates were 79.2 ± 2.5 for group 1 and 91.0 ± 2.5 for group 2. Blastocyst and hatching rates (calculated on the total of zygotes) for group 2 (47.4 ± 2.8; 82.1 ± 1.5) were significantly greater than for group 1 (39.8 ± 2.8; 74.3 ± 1.5). The total cell numbers were not different (P > 0.05) between group 1 (112.7 ± 2.9) and group 2 (111.1 ± 2.7). Blastocysts from group 2 showed lower (P < 0.05) number of apoptotic cells (10.7 ± 1.2) than those from group 1 (20.9 ± 1.2). These results indicate that the addition of glycine, alanine, taurine, and glutamine to SOF (Achilles Mixture) may be an important energy source for the bovine blastocyst and could act synergistically to enhance embryo development to the hatching stage and embryo quality. Financial support from CNPq and FAPESP.

317 EFFECT OF REACTIVE OXYGEN SPECIES (ROS) ON ACTIVATION OF BOVINE OOCYTES MATURED IN VITRO

2006 ◽  
Vol 18 (2) ◽  
pp. 266 ◽  
Author(s):  
J. I. Park ◽  
Y. Jang ◽  
E. S. Lee
Keyword(s):  
Reactive Oxygen Species ◽  
Vitamin E ◽  
Chemical Activation ◽  
Calf Serum ◽  
Reactive Oxygen ◽  
Control Treatment ◽  
Oxygen Species ◽  
Cell Stage ◽  
Humidified Air

Vitamin E is an antioxidant that protects embryos from oxidative stress and supports embryonic development in vitro. This study was carried out to assess the effect of reactive oxygen species (ROS) by measuring the effects of hydrogen peroxide (H2O2) and vitamin E (VitE) on chemical activation of in vitro-matured oocytes. Bovine ovaries were collected from slaughtered cows at a local abattoir. Oocytes were aspirated from follicles 3-8 mm in diameter and transferred to maturation medium: tissue culture medium (TCM)-199 supplemented with 10% (v/v) fetal calf serum, 100 mg/L-cysteinr, 44 mg/L sodium pyruvate, gonadotropins (each 250 IU of eCG and hCG/mL), and 10 ng/mL epidermal growth factor. Oocytes were cultured at 38.9�C in 5% CO2 in humidified air. After 22 h of culture, oocytes with polar bodies were selected and submitted to activation treatments. Oocytes were exposed to calcium ionomycin (5 �M for 5 min) followed by incubation with 6-DMAP (2 mM) for 3.5 h in medium supplemented with or without VitE (100 �m). Routine in vitro fertilization (IVF) was also performed for 18 h as control treatment with or without VitE. After activation, oocytes were cultured in mSOF medium containing 0.8% BSA at 38.9�C in 5% CO2, 5% O2 in humidified air for 16-18 h. H2O2 content was measured and rate of development was monitored. For detection of H2O2, individual oocytes were stained with 22,72-dichlorofluorescein diacetate (DCFDA) and observed under fluorescence microscope (Hashimoto et al. 2000 Mol. Reprod. Dev. 56, 520-526). Levels of H2O2 were analyzed by counting the number of black-white pixels of TIFF images (ImageJ ver. 1.32; http://rsb.info.nih.gov/ij) after conversion of fluorescence image. Data were analyzed using Student's t-test and chi-square test. The H2O2 contents were significantly higher (P < 0.05) in the activation group without VitE (218 pixels, n = 49) than IVF without VitE (210 pixels, n = 50). Levels of H2O2 in oocytes activated with VitE were also higher (P < 0.05) than in the IVF group with VitE (216 pixels, n = 50 vs. 210 pixels, n = 51). Between groups with or without VitE, the H2O2 contents were not different in both activation and IVF. The cleavage rate to the 2-cell stage and development rate to blastocysts after activation with VitE (77.8% and 15.1%, n = 72) were significantly higher (P < 0.05) than in those without VitE (63.3% and 10.2%, n = 60). The results of the present study demonstrate that the chemical activation increased accumulation of H2O2 in oocytes compared to the IVF condition. Although ROS level did not affect chemical activation, vitamin E added in the activation medium promoted further development of activated embryos. This work was supported by a grant from the BioGreen 21 Program (20050301-034-443-026-03-00), Rural Development Administration, Korea.

197 EFFECTS OF HYALURONAN ON OXYGEN CONSUMPTION AND ATP CONTENT IN PIG BLASTOCYSTS PRODUCED IN VITRO

2007 ◽  
Vol 19 (1) ◽  
pp. 215
Author(s):  
K. Yoshioka ◽  
M. Yokoo ◽  
T. Ozawa ◽  
C. Suzuki ◽  
H. Abe ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Metabolic Activity ◽  
Formation Rate ◽  
Defined Medium ◽  
Cell Number ◽  
Total Cell ◽  
Chemically Defined Medium ◽  
Atp Content ◽  
Cell Numbers

Hyaluronan (HA), a glycosaminoglycan present in follicular and oviductal fluids, has been related to sperm capacitation, fertilization, and embryo development. We have found that exogenous HA improves cell proliferation of porcine embryos cultured in a chemically defined medium (Yoshioka et al. 2004 Reprod. Fertil. Dev. 16, 264–265). Moreover, mitochondrial maturation was clearly more advanced in blastocysts cultured with HA compared to those cultured without HA, as seen by transmission electron microscopy. In the present study, the effects of HA on oxygen consumption and ATP content of blastocysts, produced in a defined system which reflects metabolic activity, were investigated. Porcine immature oocytes were matured for 44 h in porcine oocyte medium (POM) and subsequently fertilized with frozen–thawed ejaculated semen in porcine gamete medium supplemented with theophylline, adenosine, and cysteine (PGMtac4). Both POM and PGMtac4 were chemically defined media modified from porcine zygote medium (PZM)-5. After IVF, presumptive zygotes were cultured in PZM-5 containing HA (from the microorganism, Nacalai tesque, Kyoto, Japan) at concentrations of 0 [control], 10 [HA10], or 100 [HA100] �g mL-1 until 5 days after IVF. Blastocyst formation rate and total cell numbers/blastocyst at Day 5 were assessed. In addition, oxygen consumption and ATP content of single Day 5 blastocysts were measured. Blastocyst oxygen consumption was quantified using scanning electrochemical microscopy (HV-403; Research Institute for the Functional Peptides, Yamagata, Japan), and embryonic ATP content was determined using a commercial assay based on the luciferin-luciferase reaction (ATPlite; PerkinElmer, Groningen, The Netherlands). Data were statistically analyzed by ANOVA and Fisher&apos;s PLSD test. While the percentage of embryos that developed to the blastocyst stage [30.5% (63/206) to 31.7% (65/206)] did not differ among treatments, blastocyst cell number in the HA100 group [57.9 cells (n = 64)] was greater (P &lt; 0.05) compared to those in the control [48.6 cells (n = 63)] or HA10 [50.0 cells (n = 65)] groups. Blastocyst oxygen consumption rate in the HA100 group [0.629 � 10-14 mol s-1 (n = 15)] was significantly higher than in the control [0.500 � 1-14 mol s-1 (n = 16)] or HA10 [0.464 � 10-14 mol s-1 (n = 14)] groups. ATP content/blastocyst did not differ among treatments [control: 0.645 pmol (n = 38), HA10: 0.727 pmol (n = 42), and HA100: 0.704 pmol (n = 43)]. It is concluded that HA affects the metabolic activity of pig blastocysts developed in a chemically defined medium, enhancing oxygen consumption and their total cell numbers, thus improving the quality of IVP blastocysts.

50 EFFECT OF AGGREGATION OR FUSION ON DEVELOPMENT AND CELL NUMBER OF BOVINE EMBRYOS PRODUCED BY HANDMADE CLONING AND PARTHENOGENESIS

2008 ◽  
Vol 20 (1) ◽  
pp. 105
Author(s):  
E. S. Ribeiro ◽  
R. P. C. Gerger ◽  
L. U. Ohlweiler ◽  
I. Ortigari Jr ◽  
F. Forell ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Chemical Activation ◽  
Primary Cultures ◽  
Cell Number ◽  
Total Cell ◽  
Bovine Embryos ◽  
In Vitro Development ◽  
Vitro Development ◽  
Handmade Cloning

Cloning by somatic cell nuclear transfer has been associated with developmental abnormalities, with the level of heteroplasmy imposed by cell fusion being one of many potential determining factors. As the cytoplast exerts a key role in nuclear reprogramming, embryo aggregation is an alternative to minimize such negative effects during cloning. The aim of this study was to determine the effect of fusion of hemi-cytoplasts or aggregation of hemi-embryos on in vitro development and cell number of clone and parthenote embryos. Bovine cumulus–oocyte complexes (COCs) from slaughterhouse ovaries, after 17 h of IVM, were used for the production of parthenotes by chemical activation, and clone embryos by handmade cloning (HMC) (Vajta et al. 2003 Biol. Reprod. 68, 571–578). Following cumulus and zona removal, oocytes were manually bisected, followed by segregation of nucleated and enucleated hemi-cytoplasts by fluorescence using Hoechst stain. One or two enucleated hemi-cytoplasts were paired with an adult skin somatic cell from primary cultures (>90% confluence) and fused using a 25V AC pre-pulse, followed by a single 1.2 kV cm–1 DC pulse for 10 μs. Reconstructed clone structures and groups of zona-intact oocytes and nucleated hemi-cytoplasts were chemically activated in ionomycin and 6-DMAP. Clone and parthenote structures were in vitro-cultured in the WOW system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264) for 7 days, as follows: (G1) clone embryos reconstructed by aggregation of two hemi-embryos per WOW; or (G2) one embryo (two hemi-cytoplasts + cell) perWOW; and parthenote embryos composed of (G3) zona-intact oocytes cultured in wells; or aggregation of one (G4), two (G5), three (G6), or four (G7) nucleated hemi-cytoplasts per WOW. Fusion, cleavage (Day 2), and blastocyst (Day 7) rates, evaluated on a per WOW basis, were compared by the chi-square test (8 replications). Total cell number estimated by fluorescence (Hoechst stain) in blastocysts was analyzed by the Student t-test. Fusion rates of one hemi-cytoplast + cell (G1; 275/592, 46.5%) were lower than for two hemi-cytoplasts + cell (G2; 264/337, 78.3%). Cleavage rates were lower in G1 and G4 and higher in G6 and G7 than G2 and G3. A significant linear increase in blastocyst rates was observed in G5, G6, and G7. Total cell numbers were lower in parthenotes than in clones, except in G6 and G7. The lower fusion and cleavage rates after the aggregation of two clone hemi-embryos (G1) caused nearly a 50% reduction in the overall cloning efficiency. In addition, the aggregation of parthenogenetic hemi-embryos increased cleavage and blastocyst rates and cell number. However, aggregation of hemi structures did not improve blastocyst yield or cell number on a hemi-cytoplast basis. Table 1. In vitro development of parthenote or clone bovine embryos This work was supported by funding from CAPES/Brazil.

scholarly journals 271 ASSESSMENT OF NUCLEAR STATUS OF ACTIVATED BOVINE OOCYTES MATURED IN DIFFERENT MATURATION CONDITIONS IN VITRO

2005 ◽  
Vol 17 (2) ◽  
pp. 285
Author(s):  
J.I. Park ◽  
Y. Jang
Keyword(s):  
Oocyte Maturation ◽  
Polar Body ◽  
Calf Serum ◽  
Chi Square ◽  
Cell Stage ◽  
Maturation Medium ◽  
Second Polar Body ◽  
Humidified Air ◽  
Bovine Oocytes

This study was carried out to assess the nuclear status after parthenogenetic activation in in vitro matured oocytes under different conditions. Bovine ovaries were collected from slaughtered cows at a local abattoir. Oocytes were aspirated from follicles of 3–8 mm in diameter and transferred to maturation medium: tissue culture medium (TCM)-199 supplemented with 10% (v/v) fetal calf serum, 100 mg/mL l-cysteine, 20 mg/mL sodium pyruvate, gonadotropins (each 250 IU of eCG and hCG/mL), and 10 mg/mL epidermal growth factor, with or without 5 mM hypotaurine and taurine. Oocytes were cultured at 38.9°C in 5% CO2 in humidified air. After 24 h of culture, oocytes with polar body were selected and submitted to activation treatments. Oocytes were exposed to calcium ionomycin (5 μM for 5 min) followed by incubation with 6-DMAP (2 mM), roscovitine (50 μM), or 6-DMAP + roscovitine for 3.5 h. After activation, oocytes were cultured in mSOF medium containing 0.8% BSA at 38.9°C in 5% CO2, 5% O2 in humidified air for 16 h and stained with Hoechst 33342 or aceto-orcein for assessment of nuclear status. Nuclear status was recorded as follows: 1PB (polar body) + 1PN (pronucleus), 2PB + 1PN and others. Data were analyzed using chi-square test. The maturation rate of bovine oocytes cultured in maturation medium containing hypotaurine/taurine (89.3%, n = 84) was higher (P < 0.05) than those cultured without hypotaurine/taurine (72%, n = 93). In the oocytes matured with hypotaurine/taurine, the rates of diploid activation (1PB + 1PN) were 84% (n = 50) in oocytes treated with 6-DMAP + roscovitine, 78.6% (n = 56) with 6-DMAP, and 52% (n = 50) with roscovitine. In the oocytes matured without hypotaurine/taurine, the rates of diploid activation were 80% (n = 60) in oocytes treated with 6-DMAP + roscovitine, 72% (n = 50) with 6-DMAP, and 54% (n = 50) with roscovitine. The rates of diploid activation were not different in oocytes matured with or without hypotaurine/taurine and among activation treatments. The oocytes treated with roscovitine showed a lower rate (P < 0.05) of diploid activation and higher rate (39.3–40%) of second polar body extrusion (1PN + 2PB) than the other activation groups in both maturation conditions. Cleavage rates to 2-cell stage were 40–45% in all groups. Development rate of blastocysts were 7–10% in all the groups treated with 6-DMAP and 6-DMAP + roscovitine and no blastocysts were obtained from the groups treated with roscovitine alone. Hypotaurine/taurine are known to be stable and potent antioxidants, and have shown the properties of supporting oocyte maturation and further embryonic development (Guerin and Menezo 1995 Zygote 3, 333–43; Mizushima and Fukui 2001 Theriogenology 55, 1432–45). In this study, although the effectiveness of hypotaurine/taurine on promoting oocyte maturation was observed, there were no significant improvements in the rate of diploid activation in oocytes matured with hypotaurine/taurine. These results suggest that the nuclear status of activated oocytes may not have a direct relationship with the enhanced maturation condition. This work was supported by BioGreen 21 Program(#1000520030100000-1), Republic of Korea.

235 A COMPARISON OF PARTHENOGENETIC DEVELOPMENT OF PORCINE OOCYTES SYNCHRONISED DURING MATURATION BY CYCLOHEXIMIDE OR cAMP

2009 ◽  
Vol 21 (1) ◽  
pp. 215
Author(s):  
W. C. Chen ◽  
J. Zhu ◽  
P. Fisher ◽  
D. Amarnath ◽  
K. H. S. Campbell
Keyword(s):  
Time Window ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Blastocyst Formation ◽  
Cleavage Rate ◽  
Chi Square ◽  
Parthenogenetic Development ◽  
High Level

In vitro maturation of porcine oocytes is characterized by a high level of asynchrony between oocytes. Previous studies reported that cycloheximide (CHX) and 3′, 5′-cyclic AMP (cAMP) synchronize porcine oocytes and improve development to blastocyst stage following IVF or have been used for somatic cell nuclear transfer (SCNT) (Ye et al. 2005 Biol. Reprod. 72(2), 399–406; Betthauser et al. 2000 Nat. Biotechnol. 18(10), 1055–1059). We previously reported that cAMP was more effective than CHX in synchronizing porcine oocyte maturation, producing MII oocytes in a shorter time window and providing a more homogenous population for future SCNT studies (Chen et al. 2008 SRF conference, 2008 abst, p34). Here we compared parthenogenetic development of porcine oocytes synchronized by these two treatments. Selected cumulus–oocyte complexes (COC) obtained from slaughtered gilts were randomly divided into three groups and cultured at 39°C, 5% CO2 in air in modified NCSU-23 medium (with 1 μm glutathione, 1 mm cysteine, 5 mg L–1 insulin, 10 ng mL–1 epidermal growth factor, 10% (v/v) porcine follicular fluid, 1% essential and 0.5% nonessential amino acids) ± hormones (10 IU mL–1 PMSG and 10 IU mL–1 hCG): (1) with hormones for the first 22 h and then without hormones until 44 h; (2) with hormones and 5 μg mL–1 CHX for 12 h, and then with hormones but no CHX until 44 h; (3) with hormones and 1 mm cAMP for 22 h, and then without hormones and cAMP until 44 h. Parthenogenetic development of cycloheximide and cAMP treated oocytes was compared by cleavage rate at 48 h postactivation (hpa) and blastocyst formation at 168 hpa. No significant differences were observed in the frequency of cleavage (96.7 ± 2.1% v. 81.4 ± 11.6% v. 84.5 ± 5.7%), development to blastocyst (28.3 ± 11.4% v. 27.1 ± 5.7% v. 32.8 ± 5.3%) between control, CHX or cAMP treated oocytes, respectively (chi-square test, P > 0.05). However, total cell number was significantly higher in the CHX group than cAMP group (42.7 ± 4.1 v. 31.8 ± 2.0, respectively; t-test, P < 0.05). The results demonstrate that synchronization of porcine oocytes by treatment with CHX or cAMP does not affect subsequent parthenogenetic development if judged by the blastocyst formation, although the meaning of the difference of total cell numbers between CHX and cAMP treatments is still unclear.

209 EFFECT OF HEAT STRESS ON DEVELOPMENT OF IN VITRO-FERTILIZED AND PARTHENOGENETIC BOVINE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 203
Author(s):  
F. Paludo ◽  
M. M. Pereira ◽  
C. C. R. Quintao ◽  
L. T. Iguma ◽  
M. M. Gioso ◽  
...  
Keyword(s):  
Heat Stress ◽  
Bos Taurus ◽  
Apoptotic Cell ◽  
Chemical Activation ◽  
Bos Indicus ◽  
Bovine Embryos ◽  
Cell Numbers ◽  
Bovine Reproduction ◽  
Blastocyst Rate

Heat stress has been a challenge for bovine reproduction in tropical and subtropical environments. Although the role of the oocyte in thermotolerance has been studied, little attention has been paid to the contributions of sperm to embryo resistance to heat shock. The current study aimed to evaluate the development of fertilized and nonfertilized (parthenogenetic) bovine embryos undergoing heat stress during the pre-implantation stage. Cumulus–oocyte complexes obtained from ovaries collected from Bos indicus × Bos taurus crossbred cows at slaughter were in vitro matured with TCM-199 supplemented with 20 μg mL–1 of FSH, under 5% CO2 at 38.5°C for 24 h. Afterward, oocytes were randomly allocated into 2 groups: 1) IVF and 2) PART (chemical activation for parthenogenesis induction). In vitro-fertilized oocytes were cultured with 2.0 × 106 Holstein sperm mL–1 in Fert-TALP medium supplemented with heparin, for 20 h. For chemical activation, oocytes were activated with calcium ionomycin for 4 min, followed by 6-DMAP for 4 h, both in CR2aa medium supplemented with 0.1% BSA. Presumptive IVF (n = 1 262) or PART (n = 1 206) zygotes were denuded by vortexing and cultured in CR2aa medium with 2.5% of FCS under 5% CO2, 5% O2, and 90% N2 at 38.5°C. At 44 h post-insemination or chemical activation, embryos were exposed to 38.5 or 41°C for 12 h in an atmosphere of 5% CO2, 5% O2, and 90% N2. After that, embryos were cultured at 38.5°C under 5% CO2, 5% O2, and 90% N2 until Day 8 post-insemination. Blastocyst rates were evaluated at Day 7 and Day 8 post-insemination and were calculated based on the total number of presumptive zygotes. Blastocysts at 192 h post-insemination or activation were fixed and permeabilized for TUNEL assay (DeadEndTM Florimetric TUNEL System, Promega, Madison, WI) according to the manufacturer’s instructions. The effect of heat stress was compared within groups (IVF or PART) and the data were analysed by ANOVA. As expected, heat stress reduced the blastocyst rate of IVF embryos at Day 7 (24.3 ± 2.0% and 17.4 ± 2.2% for nonstressed and stressed IVF embryos; P < 0.05) and at Day 8 (32.4 ± 1.9% and 23.0 ± 2.1% for nonstressed and stressed IVF embryos; P < 0.01). However, the effect of heat stress on blastocyst rate of PART embryos was observed only at Day 8 post-insemination (30.0 ± 1.7% and 22.6 ± 2.0% for nonstressed and stressed PART embryos; P < 0.05), with no difference in blastocyst rate at Day 7 (21.6 ± 1.5% and 18.2 ± 1.8% for nonstressed and stressed PART embryos; P > 0.05). There was no difference in total cell numbers between nonstressed and stressed IVF or PART embryos. Apoptosis cell numbers and the apoptotic cell index were higher (P < 0.05) for stressed IVF (18.45 ± 1.24 and 0.16 ± 0.00) and PART (16.40 ± 5.20 and 0.17 ± 0.00) embryos than for nonstressed IVF (13.70 ± 0.75 and 0.13 ± 0.00) and PART (14.15 ± 0.86 and 0.13 ± 0.00) embryos. In conclusion, heat stress can induce apoptosis in both IVF and PART embryos, but its effect on pre-implantation development may occur at earlier stages in IVF embryos when compared with PART embryos. Financial support from Fapemig and CNPq.

292 OOCYTES FROM FOLLICLES OF DIFFERENT DIAMETERS: EMBRYO IN VITRO DEVELOPMENTAL POTENTIAL AND QUALITY

2007 ◽  
Vol 19 (1) ◽  
pp. 261
Author(s):  
T. H. C. de Bem ◽  
K. R. L. Schwarz ◽  
L. G. Mesquita ◽  
P. R. Adona ◽  
C. L. V. Leal
Keyword(s):  
Apoptotic Cell ◽  
Total Cell ◽  
Sperm Cells ◽  
Hatching Rate ◽  
Blastocyst Development ◽  
Visual Evaluation ◽  
Developmental Potential ◽  
Cell Numbers ◽  
Follicle Diameter

The present study aimed to assess the developmental potential and quality of embryos produced from oocytes originating from follicles of different sizes. Ovaries were collected at a slaughterhouse and follicles of &lt;3, 3 to 6, and &gt;6 mm were aspirated. Follicle diameters were estimated based on the size of their exposed surface on the ovarian cortex using a ruler as reference for the first aspirations and were then based on visual evaluation. Aspirated oocytes, separated by their follicular origin, were matured in vitro in TCM-199 supplemented with 10% FCS, 5.0 �g mL-1 of LH, 0.5 �g mL-1 of FSH, 0.2 mM pyruvate, and 10 �g mL-1 of gentamicin for 22 h. After in vitro maturation, oocytes were in vitro-fertilized (IVF) using frozen–thawed semen prepared by Percoll gradient. 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 of heparin. After 20 h, presumptive zygotes were partially denuded and transferred to in vitro culture (IVC) medium (TCM-199 supplemented with 10% FCS, 2.0 mM pyruvate, and 10 mg mL-1 of gentamicin). All cultures were incubated at 38.5�C under 5% CO2 in air and maximum humidity. The cleavage rate was assessed after 48 h of IVC, and blastocyst development was assessed on Day 7 (D7). On Day 9 (D9), the hatching rate was assessed and the hatched embryos were fixed for 1 h (3% paraformaldehyde in PBS), permeabilized for another h (0.5% Triton-X, 0.1% sodium citrate, and 0.1% plyvinyl alcohol in PBS), and evaluated by the TUNEL technique (in situ cell death detection kit). Total cell number and TUNEL-positive cells in embryos were counted under an epifluorescence microcope. Data of 4 replicates were analyzed by ANOVA and comparisons among groups were made by the Tukey test. The level of significance used was 5%. From the oocytes used (&lt;3 mm = 100; 3–6 mm = 99; &gt;6 mm = 88), cleavage rates increased with increasing follicular diameter. Oocytes originating from &lt;3-, 3- to 6-, and &gt;6-mm follicles resulted in 75, 93.6, and 95.5% cleavage rates, respectively (P &gt; 0.05). For blastocyst rates on D7, &lt;3-mm follicles showed 21.5% blastocyt development, which was lower (P &lt; 0.05) than for the other follicle diameter groups (3–6 mm = 35.4% and &gt;6 mm = 41.1%; P &gt; 0.05). Regarding the hatching rate, total cell numbers, and TUNEL-positive cells on D9, &lt;3 mm (20.4%, 268, and 0.37%, respectively), 3–6 mm (29.1%, 248, and 0.32%, respectively), and &gt;6 mm (32.3%, 237, and 0.23%, respectively) were not different (P &gt; 0.05). The results suggest that oocytes from larger follicles are more competent for cleavage and blastocyst development on D7; however, when oocytes reach the blastocyst stage, hatching, total cell numbers, and apoptotic cell numbers are not influenced by follicle diameter. Financial suport for this work was provided by the FAPESP, Brazil.

147 THE DEVELOPMENTAL CHARACTERISTICS OF IN VITRO-PRODUCED CATTLE-WISENT (BOS TAURUS-BISON BONASUS) HYBRID EMBRYOS

2017 ◽  
Vol 29 (1) ◽  
pp. 182
Author(s):  
E. N. Shedova ◽  
G. N. Singina ◽  
V. A. Bagirov ◽  
N. A. Zinovieva
Keyword(s):  
Bos Taurus ◽  
Apoptotic Cell ◽  
Cell Number ◽  
Total Cell ◽  
European Bison ◽  
Bison Bonasus ◽  
Epididymal Sperm ◽  
Total Cell Number ◽  
Cell Ratio

Interspecies hybrids are important resources for research and agriculture. Therefore, the aim of this study was to evaluate development, quality, and viability of embryos produced in vitro using cattle (Bos taurus) oocytes and European bison (Bison bonasus) epididymal sperm. The epididymes were obtained following a forced slaughter of one bull aged 7 years. The sperm was collected by scraping the inner surface of the epididymes, diluted with the cryopreservation medium, and equilibrated for 4 h at 4°C. Thereafter, sperm aliquots (0.2 mL) were frozen in liquid nitrogen vapor for 5 min and then plunged into liquid nitrogen for storage. Prior to fertilization, frozen semen was thawed in pre-warmed medium for 1 min at 37°C and prepared by the swim-up method. The frozen-thawed ejaculated sperm from the Russian Black Pied bulls was used as a positive control. Slaughterhouse-derived cumulus-oocyte complexes were matured for 24 h in TCM-199 supplemented with 10% FCS, 0.2 mM sodium pyruvate, 10 μg mL−1 porcine FSH, and 10 μg mL−1 ovine LH. Matured oocytes (35–40 oocytes per group) were co-incubated for 18 h with homologous (n = 266 oocytes) or heterologous (n = 292 oocytes) sperm (spermatozoa/mL) in 500 µL of TALP containing 10 μg mL−1 heparin, 20 μM penicillamine, 10 μM hypotaurine, 1 μM epinephrine, and 0.1% minimal essential medium nonessential amino acids. After IVF, the oocytes were cultured in CR1aa medium (Rosenkrans 1994 J. Anim. Sci. 72, 434–437) to the blastocyst stage. All the cultures were performed at 38.5°C and 5% CO2 in humidified air. At Days 2 and 7 after insemination, the cleavage and blastocyst rates were determined. In addition, a part of obtained blastocysts was fixed with 4% paraformaldehyde, and the total cell number and apoptotic cell ratio were determined by 4’,6-diamidino-2-phenylindole and TUNEL staining. The remaining blastocysts were cultured up to Day 10, and the hatching rates were assessed. The data (3–5 replicates) were analysed by ANOVA. The cleavage rates did not differ among both male species (72.4 and 77.1%). Furthermore, no significant effects of interspecies fertilization on the blastocyst rate or total cell number per blastocyst were found (27.4 ± 1.6% and 77.0 ± 5.7 for cattle embryos and 26.2 ± 1.9% and 83.1 ± 8.9 for cattle-wisent hybrid embryos). On the other hand, the significant differences between homologous and heterologous fertilization were detected in the rate of hatched blastocysts (60.3 ± 5.1 v. 38 ± 2.9, P < 0.05) and apoptotic cell ratio 7.3 ± 0.8 v. 11.6 ± 1.04, P < 0.05). Our findings demonstrate that hybrid embryos produced by IVF of bovine oocytes with the epididymal sperm of European bison can be developed up to advanced blastocyst stages. However, the hybrid embryos have a lower quality and viability than cattle embryos. Research was supported by the Program of Presidium of the Russian Academy of Science, project no. IV.13.3.

362 DIFFERENT IN VITRO DEVELOPMENT AFTER AGGREGATION OF BOVINE AND FELINE PARTHENOGENETIC EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 269
Author(s):  
A. De Stefano ◽  
A. Gambini ◽  
D. Salamone
Keyword(s):  
Cumulus Cells ◽  
Cell Number ◽  
Total Cell ◽  
In Vitro Development ◽  
Cell Numbers ◽  
Blastocyst Quality ◽  
Vitro Development ◽  
Parthenogenetic Embryos

Embryo aggregation has been shown to improve embryo development in several species. However, the effects seem to be different among species. Thus, the aim of this study was to compare the effect of embryo aggregation over in vitro development and blastocyst quality of bovine and feline parthenogenetic (PA) embryos. To this aim, bovine cumulus-oocyte complexes (COC) were collected from slaughterhouse ovaries, whereas cat ovaries were obtained from ovariectomized animals. The COC were in vitro matured in TCM199 supplemented following standard protocols for each species. After 24 h, cumulus cells and zona pellucidae were removed. Matured oocytes were selected and activated by 5 µM ionomycin treatment for 4 min followed by incubation in 1.9 mM 6-DMAP. Bovine and feline PA embryos were cultured in SOF medium in the well of well system in two different groups: only one PA embryo per microwell (1X); and three PA embryos per microwell (3X, aggregated embryos). Cleavage and blastocyst rates from all groups were assessed at Days 2 and 7, respectively. Size of blastocysts was measured at Day 7 using a millimetre eyepiece, and total cell number was determined by Hoechst 33342 staining. Blastocyst rates and embryo size were analysed by Fisher's test (P < 0.05) and total cell numbers by Kruskal–Wallis test with Dunn's correction (P < 0.05). Statistical differences were found in PA blastocyst rates between experimental groups (1X: 15/104, 24.6% v. 3X: 27/37, 62.2% for feline; and 1X: 21/113, 19.4% v. 3X: 20/32, 62.5% for bovine), but no differences were found between species. In addition, there was no statistical difference in the number of blastocysts obtained per oocyte used in any of the experimental groups. Bovine aggregated PA blastocysts were significantly larger than non-aggregated embryos (>200 microns, 1X: 2/20, 10% v. 3X: 9/19, 47.4%), but no differences were found in cell number. On the other hand, cat aggregated PA blastocysts had significantly higher cell numbers (1X: 122.4 ± 79.66 cells v. 3X: 259.8 ± 137.1 cells), but no differences were found in blastocyst size. This observation can contribute in the understanding of embryo physiology, suggesting that benefits of embryo aggregation in parthenogenic embryos vary among these species.

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