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.

175 EFFECT OF HEAT STRESS ON GENE EXPRESSION OF IN VITRO-PRODUCED BOVINE EMBRYOS (BOS TAURUS VS. BOS INDICUS)

2012 ◽  
Vol 24 (1) ◽  
pp. 199
Author(s):  
C. F. Silva ◽  
A. C. S. Castilho ◽  
R. A. Satrapa ◽  
R. Z. Puelker ◽  
E. M. Razza ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heat Stress ◽  
Mrna Expression ◽  
Reverse Transcription ◽  
Target Genes ◽  
Bos Taurus ◽  
Bos Indicus ◽  
Mrna Levels ◽  
Bovine Embryos

Heat stress (HS) reduces the production of bovine embryos, especially taurine embryos, which are not adapted to heat. However, little is known about the competence of embryos produced under HS in breeds adapted or not adapted to heat. The aim of this study was to compare the gene expression of PLAC8, HSF1, COX2 and CDX2, related to competence and implantation, in bovine in vitro-produced embryos (Bos taurus vs Bos indicus), submitted or not submitted to HS. Oocytes from Nelore (zebu) and Jersey (taurine) cows were aspirated by ovum pickup, in vitro-matured in TCM-199 medium with bicarbonate containing 10% FCS, 2 μg mL–1 of pyruvate, 75 μg mL–1 of gentamicin, 20 μg mL–1 of FSH and 10 IU mL–1 of LH for 22 h at 38.5°C in 5% CO2 in air. Matured oocytes were fertilized with semen from Nelore (n = 6) and Jersey (n = 6) bulls, respectively, at 38.5°C in 5% CO2 in air. The fertilization medium was TALP-IVF supplemented with 6 mg mL–1 of fatty acid-free BSA, 2 μL mL–1 of pyruvate, 75 μg mL–1 of gentamicin, 11 μg mL–1 of heparin and 44 μL mL–1 of penicillamine, hypotaurine and epinephrine. The day of fertilization was considered Day 0. Twelve hours post-insemination, presumptive zygotes were denuded and randomly divided into 2 groups, nonstressed or stressed and both were in vitro cultured at 38.5°C in 90% N2, 5% CO2 and 5% O2 in SOFaaci medium supplemented with 5% FCS, 5% BSA and 0,2% sodium pyruvate. In the stressed group, 96-h post-insemination embryos were subjected to HS of 41°C for 6 consecutive hours and then returned to 38.5°C. On Day 7, pools with 5 blastocysts [Nelore (n = 9); Nelore HS (n = 7); Jersey (n = 5); Jersey HS (n = 5)] were subjected to RNA extraction (RNeasy, Qiagen Inc., Valencia, CA, USA). The expression of target genes was analysed by real-time reverse transcription PCR with oligo-dT in reverse transcription and bovine specific-primers in PCR. The expression of cyclophilin A was used as an internal control. The mean mRNA levels of target genes among groups were compared by parametric ANOVA, followed by orthogonal contrast. Heat stress reduced (P < 0.05) mRNA expression of CDX2 and PLAC8 in both breeds; additionally, the expression of these genes was higher in the zebu breed when compared with the taurine breed. Messenger RNA expression of COX2 did not differ between groups, under HS or not, in both the Jersey and Nelore breeds. Moreover, HS reduced the mRNA expression of HSF1 (P < 0.05) in Nelore groups, but not in Jersey groups. The highest levels of PLAC8 and CDX2 in nonstressed Nelore embryos indicate better competence and a higher capacity of implantation of these embryos when compared with Jersey and HS embryos in both breeds. Moreover, low HSF1 levels in stressed Nelore embryos indicate the thermotolerance ability of this breed. In conclusion, the data indicate that HS alters the pattern of gene expression in Nelore and Jersey in vitro-produced bovine embryos. This research was supported by FAPESP.

296 INFLUENCE OF SHORT-TERM HEAT STRESS ON THE BLASTOCYST RATE OF BOVINE EMBRYOS (INDICUS VS. TAURUS) FROM OOCYTES OBTAINED BY OVUM PICKUP

2006 ◽  
Vol 18 (2) ◽  
pp. 255
Author(s):  
E. Sartorelli e Sartorelli ◽  
A. C. Z. Barcelos ◽  
R. A. Satrapa ◽  
D. F. Martins ◽  
M. F. G. Nogueira ◽  
...  
Keyword(s):  
Heat Stress ◽  
High Temperatures ◽  
Bos Indicus ◽  
Bovine Embryos ◽  
Short Term ◽  
Cleavage Rate ◽  
Blastocyst Rate ◽  
The Difference ◽  
Resistance To Heat

There is evidence that the deleterious effects of heat stress (HS) on fertility are less pronounced in Bos indicus than in B. taurus breeds, due primarily to differences in their thermoregulatory capacity. In the present work, the resistance to heat stress of Nelore embryos (B. indicus) was compared to either a breed not adapted (Angus; B. taurus) or adapted to high temperatures (Bonsmara; 5/8 B. indicus × 3/8 B. taurus). In Experiments (Exp.) 1 (Nelore vs. Angus) and 2 (Nelore vs. Bonsmara), oocytes obtained by ovum pickup OPU (during autumn) were matured (TCM-199), fertilized, and cultured (SOFaaci) in vitro. Ninety-six hours post-insemination (hpi), embryos with more than 16 cells were randomly allocated in two main groups: Group Control (embryos were maintained at 39°C all of the time) and Group HS (embryos were maintained at 41°C during 12 h and afterwards returned to 39°C). Blastocyst rates were determined on the 7th day of culture. In Exp. 1, 294 oocytes from Nelore and 144 from Angus cows had a cleavage rate of 67.9 and 59.4%, respectively. Ninety-six-hpi embryos (>16 cells) were distributed in four groups: Nelore Control (n = 97), Nelore HS (n = 95), Angus Control (n = 34) and Angus HS (n = 25). The blastocyst rates were 39/97 (40.2%), 23/95 (24.2%), 19/34 (55.9%), and e 8/25 (32.0%), respectively. The difference in rate of blatocyst formation caused by heat stress on Nelore (16.0%) and Angus (23.9%) was not significantly different (P < 0.05), and suggests, from oocytes obtained by OPU, that Nelore embryos may be more tolerant to HS than Angus embryos. However, it is necessary to increase the number of blastocysts per group in order to better characterize the effects of heat stress on these embryos. In Exp. 2, 294 oocytes from Nelore and 101 from Bonsmara cows had a cleavage rate of 41.2 and 51.2%, respectively. Ninety-six-hpi embryos (>16 cells) were distributed in four groups: Nelore Control (n = 44), Nelore HS (n = 49), Bonsmara Control (n = 22), and Bonsmara HS (n = 22). The blastocyst rates were 35/44 (79.5%), 30/49 (61.2%), 10/22 (45.5%), and 6/22 (27.3%), respectively. In spite of the fact that Bonsmara embryos had a lower blastocyst rate as compared to Nelore, the decline on blastocyst rate caused by HS was very similar in Nelore (18.3%) and Bonsmara embryos (18.2%). Additional OPU are underway to test the hypothesis that thermotolerance of Nelore embryos is similar to that in embryos from a breed adapted to high temperatures (Bonsmara), and superior to embryos from a non adapted breed (Angus). E. S. S., R. A. S., and M. F. G. N. were supported by a fellowship from FAPESP, and A. C. Z. B. by a fellowship from CAPES of Brazil.

scholarly journals Effects of heat stress on development, quality and survival of Bos indicus and Bos taurus embryos produced in vitro

2013 ◽  
Vol 79 (2) ◽  
pp. 351-357 ◽  
Author(s):  
C.F. Silva ◽  
E.S. Sartorelli ◽  
A.C.S. Castilho ◽  
R.A. Satrapa ◽  
R.Z. Puelker ◽  
...  
Keyword(s):  
Heat Stress ◽  
Bos Taurus ◽  
Bos Indicus

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&apos;s t-test and chi-square test. The cleavage and blastocyst rates were significantly higher (P &lt; 0.05) after activation with VitE (78.1&percnt; and 16.3&percnt;, n &equals; 80) than without VitE (66.7&percnt; and 11.0&percnt;, n &equals; 60). Total cell numbers were also significantly higher (P &lt; 0.05) in blastocysts after activation with VitE (143.0 &plusmn; 34.02, n &equals; 21) than in those without VitE (127.63 &plusmn; 40.25, n &equals; 20). However, the percentage of TUNEL-positive (apoptotic) cells was similar between blastocysts activated with VitE (5.38 &plusmn; 2.22) and those without VitE (6.76 &plusmn; 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.

31 DEVELOPMENT AND APOPTOSIS IN BOVINE CLONED EMBRYOS RECONSTRUCTED WITH OOCYTES COLLECTED BY REPEATED OVUM PICKUP SESSIONS OR FROM SLAUGHTERED COW OVARIES

2011 ◽  
Vol 23 (1) ◽  
pp. 122
Author(s):  
L. S. A. Camargo ◽  
M. M. Pereira ◽  
C. C. R. Quintao ◽  
J. N. S. Sales ◽  
L. T. Iguma ◽  
...  
Keyword(s):  
Apoptotic Cell ◽  
Bos Indicus ◽  
Cell Number ◽  
Total Cell ◽  
Bovine Embryos ◽  
Total Cell Number ◽  
Cell Index ◽  
Sh Groups ◽  
Sh Group

The oocyte has important components for nuclear reprogramming and its cytoplasmic background may influence the somatic cell nuclear transfer success. The current study attempted to evaluate the competence of cytoplasm from oocytes recovered by repeated ovum pickup (OPU) in living cows (OPU group) or obtained from ovaries collected at slaughterhouse from unknown source crossbred cows (SH group) to produce nuclear-transferred bovine embryos. For the OPU group, oocytes were recovered from 4 Bos indicus × Bos taurus crossbred cows in 4 repeated OPU sessions. Oocytes of OPU and SH groups were matured in vitro for 17 to 18 h, denuded and exposed to Hoechst 33342 (Sigma, St. Louis, MO, USA) and cytochalasin (Sigma) before enucleation. Embryos of OPU (n = 100) and SH (n = 105) groups were reconstructed with somatic cells from adult Gyr (Bos indicus) cow, fused with double electric pulse of 2.4 kV cm–1 for 30 μs and activated with ionomycin (Sigma) and 6-DMAP (Sigma). Embryos were cultured in CR2aa medium supplemented with 2.5% fetal calf serum (Nutricell, Campinas, Brazil) under 5% CO2, 5% O2, and 90% N2 at 38.5°C. Cleavage and blastocyst rates were evaluated at 72 h and 168 h post-activation, respectively. Blastocysts at 168 h post-activation were fixed and permeabilized for TUNEL assay (DeadEnd™ Fluorimetric TUNEL System, Promega, Madison, WI, USA), according to the manufacturer instructions. IVF bovine blastocysts (IVF group; n = 245) obtained with oocytes of slaughtered cows were used as control group. Fusion, cleavage, and blastocyst rates were analysed by chi-square test and total cell number, apoptotic cell number, and apoptotic cell index (calculated by dividing the apoptotic cell number by total cell number) were analysed by ANOVA. There were no differences (P > 0.05) in fusion (71.0% and 61.0%), cleavage (74.6% and 78.1%) or blastocyst (32.3% and 31.2%) rates between OPU and SH groups, respectively, but both groups presented greater (P < 0.05) blastocyst rates than the IVF group (15.1%). Total cell number (80.66 ± 5.36 and 82.10 ± 4.79), apoptotic cell number (12.66 ± 3.20 and 15.60 ± 3.04), and apoptotic cell index (0.15 ± 0.03 and 0.20 ± 0.04) were also similar (P > 0.05) between OPU and SH groups, respectively. However, apoptotic cell number (7.40 ± 0.93) and apoptotic cell index (0.07 ± 0.01) were lower (P < 0.05) in the IVF group than the SH group and similar (P > 0.05) to the OPU group. In conclusion, oocytes cytoplasm from both groups (OPU and SH) have the same potential to produce nuclear-transferred bovine embryos but only blastocysts from the OPU group present apoptosis levels similar to its in vitro-fertilized counterpart. Financial support: Fapemig and CNPq.

214 INFLUENCE OF THE BREED OF BULL (GIR v. HOLSTEIN) ON THE RESISTANCE OF BOVINE EMBRYOS TO HEAT SHOCK AT EARLY STAGES OF IN VITRO DEVELOPMENT

2008 ◽  
Vol 20 (1) ◽  
pp. 186 ◽  
Author(s):  
D. Y. Saito ◽  
R. A. Satrapa ◽  
R. M. Romão ◽  
T. Nabhan ◽  
R. A. L. Simões ◽  
...  
Keyword(s):  
Heat Shock ◽  
Bos Taurus ◽  
Bos Indicus ◽  
Holstein Cows ◽  
Bovine Embryos ◽  
In Vitro Development ◽  
Early Stages ◽  
Holstein Bulls ◽  
Vitro Development

Heat shock (HS) has negative effects on pregnancy rates in lactating dairy cows. There are genetic differences in tolerance to HS, with Bos indicus cattle and embryos being more resistant to elevated temperatures than Bos taurus. Recently, Pegorer et al. (2007 Theriogenology 67, 692–697) observed that Gir semen (Bos indicus), when compared with Holstein semen (Bos taurus), increased pregnancy rates of lactating Holstein cows during the Brazilian summer. The objective of the present study was to evaluate the influence of breed of bull (Gir or Holstein) on the resistance of embryos to HS during the early stages of in vitro development. Oocytes from Holstein ovaries obtained from a local abattoir were matured in TCM 199 medium, fertilized with semen from 1 of 12 bulls (6 Gir and 6 Holstein), and cultured in synthetic oviduct fluid to the blastocyst stage. Ninety-six hours after insemination (96 hpi), embryos with less than 16 cells were discarded; half of the embryos (e16 cells) that were fertilized by each bull were submitted to HS (41�C for 12 h) and the other half (e16 cells) were maintained at 39�C (control group). After the HS period, embryos were maintained at 39�C until the end of the experiment. Six semen straws from different Gir bulls (Bos indicus) and 6 straws from Holstein bulls (Bos taurus) were used, in a total of 6 assays, to minimize the bull effect. Each breed of bull (Gir or Holstein) was used to fertilize a total of at least 300 Holstein oocytes per treatment (control v. HS). Cleavage (48 hpi), blastocyst (144 hpi), and hatched blastocyst (216 hpi) rates of embryos submitted or not submitted to HS were analyzed by PROC GLM of SAS (SAS Institute Inc., Cary, NC, USA) to adjust the model of ANOVA, with each combination of assays used as a block. The proportion data were arcsine transformed to study the effects of breed, treatment, and their interaction. There was no difference in cleavage rate when Gir semen was used to fertilize oocytes from Holstein cows (Gir � Holstein; 76.6%) as compared with Holstein � Holstein (75.5%). However, HS significantly decreased (P < 0.01) blastocyst rates (in relation to embryos e16 cells) from both Gir (74 and 62.6%, control and HS, respectively) and Holstein bulls (69 and 55%, respectively). The same was observed for hatched blastocysts from Gir (42 and 28.3%) and Holstein bulls (37 and 25%). Nevertheless, there was no interaction of breed of bull � treatment (HS) for blastocysts (P > 0.09) and hatched blastocysts (P > 0.1). It was concluded that the breed of bull used in the present experiment (Gir v. Holstein) did not increase the resistance of bovine embryos to HS at early stages of in vitro development.

Expression of candidate genes for residual feed intake in tropically adapted Bos taurus and Bos indicus bulls under thermoneutral and heat stress environmental conditions

2021 ◽  
pp. 102998
Author(s):  
Bianca Vilela Pires ◽  
Nedenia Bonvino Stafuzza ◽  
Luara Afonso de Freitas ◽  
Maria Eugênia Zerlotti Mercadante ◽  
Ester Silveira Ramos ◽  
...  
Keyword(s):  
Heat Stress ◽  
Candidate Genes ◽  
Feed Intake ◽  
Environmental Conditions ◽  
Bos Taurus ◽  
Bos Indicus ◽  
Residual Feed Intake

scholarly journals In vivo culture of bovine embryos and quality assessment of in vivo vs. in vitro produced embryos

2012 ◽  
Vol 50 (No. 4) ◽  
pp. 149-158 ◽  
Author(s):  
V. Havlicek ◽  
M. Lopatarova ◽  
S. Cech ◽  
R. Dolezel ◽  
T. Huber ◽  
...  
Keyword(s):  
Quality Assessment ◽  
Recovery Rate ◽  
Bovine Embryos ◽  
In Vivo Culture ◽  
Blastocyst Rate ◽  
Culture Procedure ◽  
Recovery Methods

Routine access to the bovine oviduct for in vivo culture accomplishes various demands on embryo production for scientific as well as commercial purposes. The experiments conducted in the present study focused on the efficiency of recovery methods after temporary in vivo culture of bovine embryos in oviducts of the homologous species using transvaginal endoscopy (Experiment I) and on the quality assessment of recovered blastocysts (Experiment II). In Experiment I in vitro matured oocytes were fertilized, cultured for 1 to 3 days and transferred unilaterally into the ipsilateral oviducts of 54 heifers by the means of transvaginal endoscopy. After 4 to 6 days of in vivo culture embryos were re-collected either by non-surgical flushing of uterine horns (U-group) or by combined flushing of the oviducts and uterine horns (OU-group). In total the recovery rate was 38.4% (780/2029). After flushing at day seven, 106 blastocysts (blastocyst rate: 13.6% ) were found. The additional 24 h of in vitro culture (day eight) resulted in 153 blastocysts (blastocyst rate: 19.6% ). The recovery rate in the OU-group was twice as efficient as in the U-group (390/1358 vs. 390/671, P &lt; 0.01). The recovery rates among the different stages of transferred embryos did not differ significantly; likewise cross-effects among the stages and the recovery methods were non-significant. The recovery methods (P &lt; 0.001) and the interaction between the recovery methods and the stages of transferred embryos (P &lt; 0.01) had an influence on blastocyst yields on day seven (U-group 37/1358 vs. OU-group 69/671) and day eight (U-group 48/1358 vs. OU-group 105/671). In Experiment II embryo quality was assessed by the survival rate of blastocysts after freezing in ethylene glycol. Day seven embryos were produced in vitro (in vitro group D7) or by IVM/IVF followed by a combined culture procedure (2 to 3 days in vitro prior to 4&nbsp;to 5 days in vivo) (in vivo group D7) or after superovulation and collection at day seven (superovulation group). Embryos from in vitro group D7 re-expanded only for 6 h after thawing, embryos from in vivo group D7 and superovulation group were alive for 24 h and 72 h of culture, respectively. Only embryos derived by superovulation showed hatching activity. Blastocysts from the in vitro group D7 and the in vivo group D7 that were held in culture medium for additional 24 h (day eight) showed an analogous post-thawing culture behaviour. In conclusion, the results of the present study demonstrated that some embryos transferred for in vivo culture remain in the oviduct even at day seven. Hence, combined flushing of oviducts and uterine horns after in vivo culture in the bovine oviduct is necessary for effective embryo re-collection. The quality of recovered embryos after temporary in vivo culture assessed by cryotolerance was in-between those produced in vitro or recovered after superovulation.

286 INFLUENCE OF REVERSIBLE MEIOSIS INHIBITION ON SWINE EMBRYOS PRODUCED BY IN VITRO FERTILIZATION AND PATHENOGENETIC ACTIVATION

2006 ◽  
Vol 18 (2) ◽  
pp. 250
Author(s):  
M. G. Marques ◽  
A. B. Nascimento ◽  
V. P. Oliveira ◽  
A. R. S. Coutinho ◽  
M. E. O. A. Assumpção ◽  
...  
Keyword(s):  
Culture Medium ◽  
Cell Number ◽  
Control Group ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Electric Pulses ◽  
Cell Numbers ◽  
Blastocyst Rate ◽  
And Control

The present work evaluated the reversible meiosis inhibition effect on the development of swine embryos produced by in vitro fertilization (IVF) or parthenogenetic activation (PA). The efficiency of PZM3 and NCSU23 embryo culture media was also evaluated. Oocytes from ovaries collected at a slaughterhouse were subjected to IVM in two different groups: CHX (cycloheximide 5 µM for 10 h) and control, both with TCM-199 + 3.05 mM glucose + 0.91 mM sodium pyruvate + 10% porcine follicular fluid (pFF) + 0.57 mM cystein + 10 ng epidermal growth factor (EGF)/mL + 10 IU eCG/mL + 10 IU hCG/mL for the initial 22 h. In the remaining period (20 h for CHX and 22 h for control), medium without hormones was utilized. After IVM, oocytes were denuded and fertilized for 6 h (IFV) or the matured oocytes were submitted to activation by electric pulses (PA) (2 DC of 1.5 kV/cm for 30 µs), incubated for 1 h in culture medium with 10 μM of CHX, and again submitted to the same electric pulses for 60 µs. Embryo development was evaluated by cleavage rate on Day 3 and blastocyst rate and blastocyst cell number on Day 7 of culture. Cleavage and blastocyst rates were analyzed by the equality-of-two-ratios test and cell number by the Kruskal-Wallis and Mann-Whitney tests (P < 0.05). In relation to IVF, the PZM3 medium was more efficient than NCSU23 for cleavage rate in the CHX group (PZM3: 68.4%, NCSU23: 44.4%) and had a better blastocyst rate in the control group (PZM3: 13.4%, NCSU23: 5.6%). With reference to PA, NCSU23 presented better cleavage and blastocyst rates than PZM3 in the CHX group (NCSU23: 89.5%, PZM3: 78.5% and NCSU23: 20.4%, PZM3: 13.0%, respectively). In the control group, only the NCSU23 blastocyst rate was higher than that for PZM3 (NCSU23: 22.5%, PZM3: 10.8%). No culture medium effect on cell number mean of IVF and PA blastocysts was observed. Maturation block improved cleavage rates in IVF groups cultured with PZM3 (68.4% and 50.6%, respectively, for CHX and control) and in PA groups cultured with NCSU23 (89.5% and 80.3%, respectively, for CHX and control), but no improvement of blastocyst rates in both groups (IVF and PA) was verified. Table 1 below shows that maturation block decreased the IVF and increased the PA blastocyst cell numbers. As older oocytes are more effectively activated, oocytes blocked with CHX achieved the maturation stage faster than the control group, therefore resulting in high-quality PA blastocysts. In conclusion, PZM3 was more efficient for IVF embryo production in contrast to NCSU23, whereas NCSU23 can be indicated for PA embryo production. Moreover, maturation blockage with CHX influenced blastocyst cell number, decreasing in IVF embryos and increasing in PA embryos. Table 1. Mean (±SD) of blastocyst cell numbers for IVF or PA groups after in vitro maturation without (control) or with cycloheximide (CHX) and cultured in NCSU23 or PZM3 medium This work was supported by FAPESP 02/10747–1.

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