83 VITAMIN K2 SUPPLEMENTATION IMPROVES BLASTOCYST RATE BY RECOVERY OF MITOCHONDRIA IN IN VITRO-CULTURED BOVINE EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 155
Author(s):  
L. Baldoceda ◽  
C. Vigneault ◽  
P. Blondin ◽  
C. Robert
Keyword(s):  
In Vitro Culture ◽  
Fluorescent Microscopy ◽  
Vitamin K2 ◽  
Control Group ◽  
Mitochondrial Activity ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Blastocyst Rate ◽  
Mitotracker Red

Mitochondria play an important role during early mammalian embryo development through their diverse cellular functions, in particular creating balance between production of ATP by electron transport chain and oxidative stress. Embryonic mitochondria are inherited maternally and independently of the nuclear genome. They show limited activity during the early developmental stages before embryonic genome activation. It has been shown that in vitro culture (IVC) has an adverse effect on mitochondrial function in embryos. So far several attempts have been performed to improve and rescue the impaired mitochondria. It has been shown that vitamin K2 (a membrane-bound electron carrier, similar to ubiquinone) was used to rescue mitochondrial dysfunction and resulted in more efficient ATP production in eukaryotic cells (Vos et al. 2012 Science 336, 1306–1310). Therefore, the aim of the present study was to investigate the effects of supplementation of vitamin K2 on mitochondrial activity and blastocyst rate. Cumulus–oocytes complexes (n = 687) recovered from slaughtered animals, were matured and fertilized in vitro according to our standard procedures. After fertilization, zygotes were cultured in SOF media supplemented with 10 mg mL–1 BSA. At 96 h post-fertilization, vitamin K2 was added to the culture media (n = 448 oocytes). On Day 7, treatment embryos were compared with untreated controls (n = 239 oocytes). In vitro culture was carried out at 38.5°C under 5% CO2, 7% O2, and 88% N2. Differences among groups in blastocyst yield were analysed by ANOVA. Mitochondrial activity data was analysed by unpaired 2-tailed t-tests. Results show that the vitamin K2-treated group had a significantly (P < 0.05) higher blastocyst rate (+8.6%), expanded blastocyst rate (+7.8%), as well as better morphological quality compared with the control group. Furthermore, to evaluate mitochondria activity, pools of embryos of each treatment were labelled with a specific dye for active mitochondria (Mitotracker Red). A significantly higher intensity of Mitotracker Red (P < 0.05) was observed in the vitamin K2 treatment versus control group, as measured by fluorescent microscopy. In conclusion, for the first time, our data prove that supplementation of vitamin K2 during IVC of bovine embryos increases blastocyst rates and embryo quality. Future studies will focus on gene expression to identify targets implicated in impaired mitochondrial activity in in vitro bovine embryo production.

scholarly journals l-Carnitine Supplementation during In Vitro Maturation and In Vitro Culture Does not Affect the Survival Rates after Vitrification and Warming but Alters Inf-T and ptgs2 Gene Expression

2020 ◽  
Vol 21 (16) ◽  
pp. 5601
Author(s):  
Diego F. Carrillo-González ◽  
Nélida Rodríguez-Osorio ◽  
Charles R. Long ◽  
Neil A. Vásquez-Araque ◽  
Juan G. Maldonado-Estrada
Keyword(s):  
Gene Expression ◽  
In Vitro Culture ◽  
In Vitro Maturation ◽  
Survival Rates ◽  
Cell Number ◽  
Control Group ◽  
Mitochondrial Activity ◽  
Bovine Embryo ◽  
Carnitine Supplementation

l-carnitine is a potent antioxidant used for in vitro culture systems. Controversial results have been reported using l-carnitine in culture medium at different stages of in vitro bovine embryo production. Cumulus-oocyte complexes (n = 843) were in vitro-fertilized and cultured and added (treatment group) or not added (control group) with l-carnitine. At day three of culture, each group was subdivided into two subgroups receiving no l-carnitine (group 1), 3.8 mM l-carnitine added during in vitro maturation (group 2), 1.5 mM added during the in vitro culture (group 3), and 3.8 mM and 1.5 mM added during the maturation and culture, respectively (group 4). At day 8, blastocyst embryos were examined for mitochondrial activity, the presence of lipid droplets, total cell number, gene expression, and cryotolerance by vitrification. The data were analyzed with a one-way analysis of variance. l-carnitine added in the late in vitro culture significantly reduced mitochondrial activity and lipid content, and upregulated ifn-τ and ptgs2 gene expression compared to controls (p < 0.05). l-carnitine supplementation did not significantly affect the embryo rate production or survival rate after vitrification and warming (p > 0.05). l-carnitine supplementation significantly improved embryo potential to develop viable pregnancies in agreement with a study reporting improved pregnancy rates.

scholarly journals Effect of insulin–transferrin–selenium (ITS) and l-ascorbic acid (AA) during in vitro maturation on in vitro bovine embryo development

Zygote ◽  
2016 ◽  
Vol 24 (6) ◽  
pp. 890-899 ◽  
Author(s):  
A.L.S. Guimarães ◽  
S.A. Pereira ◽  
M. N. Diógenes ◽  
M.A.N. Dode
Keyword(s):  
Ascorbic Acid ◽  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Bovine Embryo ◽  
Total Cell Number ◽  
Embryo Production ◽  
Embryo Size ◽  
Blastocyst Rate

SummaryThe aim of this study was to evaluate the effect of adding a combination of insulin, transferrin and selenium (ITS) and l-ascorbic acid (AA) during in vitro maturation (IVM) and in vitro culture (IVC) on in vitro embryo production. To verify the effect of the supplements, cleavage and blastocyst rates, embryo size and total cell number were performed. Embryonic development data, embryo size categorization and kinetics of maturation were analyzed by chi-squared test, while the total cell number was analyzed by a Kruskal–Wallis test (P < 0.05). When ITS was present during IVM, IVC or the entire culture, all treatments had a cleavage and blastocyst rates and embryo quality, similar to those of the control group (P < 0.05). Supplementation of IVM medium with ITS and AA for 12 h or 24 h showed that the last 12 h increased embryo production (51.6%; n = 220) on D7 compared with the control (39.5%; n = 213). However, no improvement was observed in blastocyst rate when less competent oocytes, obtained from 1–3 mm follicles, were exposed to ITS + AA for the last 12 h of IVM, with a blastocyst rate of 14.9% (n = 47) compared with 61.0% (n = 141) in the control group. The results suggest that the addition of ITS alone did not affect embryo production; however, when combined with AA in the last 12 h of maturation, there was improvement in the quantity and quality of embryos produced. Furthermore, the use of ITS and AA during IVM did not improve the competence of oocytes obtained from small follicles.

scholarly journals 136 EVIDENCE OF A DIRECT EFFECT OF P4 ON IVF-DERIVED BOVINE 8-CELL EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 219 ◽  
Author(s):  
C.E. Ferguson ◽  
T.R. Davidson ◽  
M.R.B. Mello ◽  
A.S. Lima ◽  
D.J. Kesler ◽  
...  
Keyword(s):  
Embryo Development ◽  
Direct Effect ◽  
Blastocyst Stage ◽  
Control Group ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Direct Role ◽  
Treatment Groups ◽  
And Control

There has been much debate over a direct role for progesterone (P4) in early bovine embryo development. While previous attempts to supplement bovine embryos in vitro with P4 produced results that vary and are often contradictory, this may be a response of administering P4 at inappropriate times. Therefore, the objective of these experiments was to determine if P4 could exert a direct effect on developing IVF-derived bovine embryos when administered at an appropriate time of embryo development. In Exp. I, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 168); (2) vehicle, CR1aa + ETOH (0.01%) (n = 170); and (3) P4, CR1aa + ETOH + P4 (20 ng/mL in 50-μL droplet) (n = 173). In Exp. II, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 160); (2) vehicle, CR1aa + DMSO (0.01%) (n = 180); and (3) P4, CR1aa + DMSO (0.01%) + P4 (20 ng/mL in 50-μL droplet) (n = 170). All embryos were evaluated on Days 6 to 9 post-insemination and rates calculated from 8-cell embryos. In Exp. I, ETOH tended to have a detrimental effect with significantly fewer (P < 0.05) embryos (53%) developing to the blastocyst stage on Day 7 compared with the control (62%) and P4 (71%) groups. At Day 7, significantly more embryos cultured in P4 (71%) developed to the blastocyst stage compared with the control group (62%). P4 treatment significantly increased the number of Grade 1 blastocysts (25%) on Day 7 compared with vehicle (15%) and control (17%) groups. At the end of culture, there were also significantly more Day 9 hatched blastocysts in the P4 group (33%) compared with vehicle (22%) and control (21%) groups. Supplementing P4 in the culture medium increased the rate of development, resulting in significantly more blastocysts (8%) on Day 6 and hatched blastocysts (21%) on Day 8 compared with vehicle (3% and 12%) and control (0% and 8%) groups, respectively. In Exp. II, there were no significant differences between treatment groups for Day 7 blastocysts (control 54%, DMSO 61%, P4 57%) and Day 9 hatched blastocysts (control 46%, DMSO 51%, P4 46%). However, there were significantly more Grade 1 blastocysts in the P4 group (22% and 36%) on Days 6 and 8 compared with vehicle (11% and 23%) and control (13% and 23%) groups, respectively. The lack of improvement in Day 7 blastocysts and Day 9 hatched blastocysts rates leads to further uncertainty in understanding the P4 vehicle interactions. In conclusion, the results of these two experiments indicate that P4 can exert a direct effect on the developing IVF-derived bovine embryo; however, due to P4 vehicle interactions; other inert vehicles need to be explored to further evaluate the direct effects of P4 on the developing bovine embryo.

302 REPLACEMENT OF FETAL BOVINE SERUM WITH KNOCKOUT SERUM REPLACER AND STEM CELLS CONDITIONED MEDIUM DURING IN VITRO CULTURE OF BOVINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 307
Author(s):  
M. M. Souza ◽  
N. Z. Saraiva ◽  
C. S. Oliveira ◽  
T. A. D. Tetzner-Nanzeri ◽  
R. Vantini ◽  
...  
Keyword(s):  
Stem Cells ◽  
In Vitro Culture ◽  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Protein Supplementation ◽  
Control Group ◽  
Bovine Embryos ◽  
Fetal Bovine

The use of fetal bovine serum (FBS) as protein supplementation in IVP of bovine embryos has presented difficulties because it can introduce a number of pathogenic components in culture systems, can be related to the birth of calf with abnormal growth and development, and precludes the establishment of the actual nutritional needs of the embryo, because it contains an unlimited variety of substances. This study evaluated the replacement of the FBS in the medium of in vitro culture (IVC) of bovine embryos, using the knockout serum replacer (KSR) as protein supplementation and culture medium conditioned with stem cells. Therefore, bovine oocytes from ovaries of slaughterhouse were selected and matured in vitro in TCM-199 medium supplemented with 10% FBS (Crypion), 1.0 μg mL-1 FSH (Pluset®, Calier, Barcelona, Spain), 50 μg mL-1 hCG (Profasi®, Serono, Geneva, Switzerland), 1.0 μg mL-1 estradiol (Sigma E-2758, Sigma Chemical, St. Louis, MO, USA), 0.2 mM sodium pyruvate, and 83.4 μg mL-1 amikacin for 24 h. After that, 1144 oocytes were fertilized in IVF-TALP medium containing 6 mg mL-1 of BSA. After 18 to 22 h, the zygotes were cultured in SOF + 5% FBS (group 2); SOF + 5% KSR (group 3); SOF (5% FBS) + 10% SOF (5% FBS) conditioned by stem cells (group 4); or SOF (5% KSR) + 10% SOF (5% KSR) conditioned by stem cells (group 5), in an atmosphere of 5% O2 at 38.5°C for 8 days. A control group outside the controlled atmosphere was added, supplemented with 5% FBS (group 1). The SOF medium supplemented with 5% FBS or KSR was conditioned by stem cells and added to SOF medium for the culture of embryo at a concentration of 10%. The rates of cleavage and production of blastocysts were assessed 48 hours and 7 days after IVF, respectively, and analyzed by chi-square test, with a significance level of 5% in the statistical program Minitab® (release 14.1, Minitab, State College, PA, USA). On the eighth day, the TUNEL test for determination of the percentage of apoptosis and the differential staining technique for determination of inner cell mass (ICM) and trophoblast (TF) were performed. The results were submitted to ANOVA, followed by comparing the means by Tukey’s test using the program GraphPad Prism (GraphPad, San Diego, CA, USA). The treatments did not differ in the production of embryos, being similar to the control group: G1 = 31.75% (74/233), G2 = 35.26% (79/224), G3 = 32.70% (74/226), G4 = 28.76% (63/219), and G5 = 26.85% (65/242). With regard to the assessment of embryonic quality, the treatments showed similar results to the control groups. No differences were observed among groups both in color and ICM/TF ratio (G1 = 0.60, G2 = 0.62, G3 =0.65, G4 = 0.60, and G5 = 0.60). Furthermore, the TUNEL showed no significant difference in the percentage of apoptosis among groups (G1 = 7.10%, G2 = 3.76%, G3 = 5.58%, G4 = 4.50%, and G5 = 4.11%). The data obtained so far indicate that it is possible to produce embryos in vitro by replacing the FBS in the culture, achieving results similar to those obtained with serum. Financial support: FAPESP 2007/58506-6.

scholarly journals Genetic influence on the reduction in bovine embryo lipid content by L-carnitine

2016 ◽  
Vol 28 (8) ◽  
pp. 1172 ◽  
Author(s):  
Luis Baldoceda ◽  
Dominic Gagné ◽  
Christina Ramires Ferreira ◽  
Claude Robert
Keyword(s):  
Lipid Content ◽  
Culture Medium ◽  
Cell Damage ◽  
Mitochondrial Activity ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Intracellular Lipid ◽  
Embryo Culture Medium

The decreased rate of pregnancy obtained in cattle using frozen in vitro embryos compared with in vivo embryos has been associated with over-accumulation of intracellular lipid, which causes cell damage during cryopreservation. It is believed that the higher lipid content of blastomeres of bovine embryos produced in vitro results in darker-coloured cytoplasm, which could be a consequence of impaired mitochondrial function. In this study, l-carnitine was used as a treatment to reduce embryonic lipid content by increasing metabolism in cultured bovine embryos. We have observed previously that in vivo embryos of different dairy breeds collected from cows housed and fed under the same conditions differed in lipid content and metabolism. As such, breed effects between Holstein and Jersey were also examined in terms of general appearance, lipid composition, mitochondrial activity and gene expression. Adding l-carnitine to the embryo culture medium reduced the lipid content in both breeds due to increased mitochondrial activity. The response to l-carnitine was weaker in Jersey than in Holstein embryos. Our results thus show that genetics influence the response of bovine embryos to stimulation of mitochondrial metabolism.

141 BIOPSY TECHNIQUE IN BOVINE EMBRYOS PRODUCED IN VITRO AT EARLY STAGES OF DEVELOPMENT: EVALUATION OF QUALITY AND DEVELOPMENT CAPACITY

2008 ◽  
Vol 20 (1) ◽  
pp. 151
Author(s):  
J. Polisseni ◽  
M. O. Guerra ◽  
R. V. Serapião ◽  
M. M. Pereira ◽  
I. M. Folhadella ◽  
...  
Keyword(s):  
Preimplantation Genetic Diagnosis ◽  
Embryo Quality ◽  
Genetic Diagnosis ◽  
Blastocyst Stage ◽  
Chromosome Abnormalities ◽  
Control Group ◽  
Bovine Embryos ◽  
Blastocyst Rate ◽  
Number Of Cells

One of the causes of embryo mortality is chromosome abnormalities that occur during gametogenesis, fertilization, and embryo early development. Thus, a combination of morphological standards and techniques of molecular analyses could identify abnormal embryos. Preimplantation genetic diagnosis (PGD) is an emergent technology for use with farm animal embryos. With this procedure, blastomeres are removed by the biopsy of embryos at the 8- to 16-cell stage to provide cells for analyses of chromosome abnormalities prior to transfer. The aim of this study was to evaluate the effect of biopsy in bovine 8- to 16-cell embryos fertilized in vitro on embryo quality and subsequent development in vitro. A group of 706 oocytes were obtained from slaughterhouse ovaries, matured, and fertilized in vitro at 38.8�C with 95% humidified air and 5% CO2. The zygotes were semi-denuded and cultured in CR2aa medium under the same conditions as for in vitro fertilization. The rate of cleavage was 78.20%. Three days after fertilization, part of the 8- to 16-cell (298/706) embryos were distributed randomly across two groups: control (n = 103) and biopsy (n = 92) of blastomeres, and then returned to in vitro embryo culture to evaluate development until the blastocyst stage and the capacity to hatch. The amount of cells removed was one-fourth of the embryo. The blastocyst rate was evaluated on Day 8 after fertilization and the hatching rate on Day 10. Embryo morphology and quality were evaluated as previously described in the International Embryo Transfer Society manual (1998). To evaluate overall quality, embryos were stained on the 10th day of culture and the blastomeres were counted with the imaging software AxioVision 3.1 (Carl Zeiss, Feldbach, Switzerland). The blastocyst rate was analyzed by treatment groups with the chi-square test and the number of cells/embryo was analyzed by ANOVA with SAS (SAS Institute, Inc., Cary, NC, USA). The percentage of 8- to 16-cell embryos that developed to the blastocyst stage was similar (P > 0.05) between the control (66.0%, 68/103) and the biopsied (53.3%, 49/92) groups. Furthermore, no difference was noted in the hatching rates between the control group and the biopsied group (42.6%, 29/42 v. 44.9%, 22/49, respectively). Overall, no impact was detected on embryo quality from embryo biopsy with no difference in mean (�SE) blastocyst cell number between the control group (blastocysts: 67.1 � 3.1; expanded blastocysts: 100.7 � 6.9; hatched blastocysts: 189.9 � 16.1) and the biopsied group (blastocysts: 61.1 � 5.5; expanded blastocysts: 121.87 � 10.6; hatched blastocysts: 187.3 � 18.5). In conclusion, the biopsy used on 8- to 16-cell bovine IVF-derived bovine embryos does not affect the subsequent embryo development and number of cells/embryo or blastocyst, showing that it can be used to provide genetic material for preimplantation genetic diagnosis without affecting embryo quality. This work was supported financially by FAPEMIG.

29 SCRIPTAID IMPROVES SOMATIC NUCLEAR TRANSFER EFFICIENCY DURING IN VITRO CULTURE OF PORCINE EMBRYOS DERIVED FROM INBRED MINIATURE PIG FETAL FIBROBLASTS

2015 ◽  
Vol 27 (1) ◽  
pp. 107
Author(s):  
R. Koppang ◽  
N. R. Mtango ◽  
M. Barcelo-Fimbres ◽  
J. P. Verstegen
Keyword(s):  
In Vitro Culture ◽  
Nuclear Transfer ◽  
Fibroblast Cell ◽  
Treated Group ◽  
Culture Conditions ◽  
Control Group ◽  
Miniature Pig ◽  
Fetal Fibroblasts ◽  
Blastocyst Rate

Porcine somatic cell nuclear transfer (SCNT) is limited to the same or next day surgical embryo transfer due to poor culture conditions in vitro. In this study, we aimed to overcome this problem by treating SCNT embryos with scriptaid, an inhibitor of histone deacetylase (HDACi) that helps with epigenetic reprogramming of the somatic nuclei. Scriptaid was chosen over other HDACi because it has been shown to improve histone acetylation in the same pattern as that of IVF embryos as well as its low toxicity characteristic (Zhao et al. 2009 Biol. Reprod. 81, 525–530; Zhao et al. 2010 Cell Reprogram. 12, 75–78). An inbred miniature pig fetal fibroblast cell line that is known to give low blastocyst rate in culture was used as a source of donor cells transferred into enucleated oocytes. Traditional SCNT was performed; embryos were fused and chemically activated in 10 µM ionomycin for 5 min and 2 mM DMAP for 3 to 4 h before being transferred into scriptaid. Embryos were treated with 500 nM scriptaid (Zhao et al. 2010) for 18 h and the untreated group was used as control. A total of 806 oocytes were used in 8 replicates. The constructed embryos were cultured in modified porcine zygote medium 5 (mPZM-5) for 7 days at 39°C in 5% O2, 5% CO2, 90% N2 atmosphere. Cleavage rates were assessed at 2.5 days and blastocyst rates at Day 7 after activation. Data were analysed by ANOVA using GLM, and percentages were transformed using arcsin square root using Statistix 10 software (Tallahassee, FL, USA). There were no differences in cleavage rates for control group v. scriptaid (55.3 v. 49.9%; P > 0.1; Table 1). The blastocyst rate per construct showed remarkable increase in the scriptaid treated group compared with the control group (12.8 v. 2.2%; P < 0.01; Table 1). Similarly, a significant effect was observed for blastocyst per embryos cleaved where scriptaid had higher rates compared with control (25.8 v. 5.8%; P < 0.01). These results indicated that improving nuclear reprogramming of miniature porcine SCNT clones by scriptaid treatment enhanced blastocyst production during the in vitro culture of porcine embryos. Table 1.Mean (± s.e.m.) measures of embryonic development of SCNT embryos

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

2017 ◽  
Vol 29 (1) ◽  
pp. 183 ◽  
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.

131 Effect of Oviductal Fluid During In Vitro Culture on Bovine Embryo Development and Quality

2018 ◽  
Vol 30 (1) ◽  
pp. 205 ◽  
Author(s):  
R. Emmerstorfer ◽  
K. Radefeld ◽  
V. Havlicek ◽  
U. Besenfelder ◽  
H. Yu ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Specific Effect ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Control Group ◽  
Hatching Rate ◽  
Bovine Embryo ◽  
Cleavage Rate ◽  
Significant Difference

The aim of this work was to establish an in vitro culture approach using bovine oviducal fluid (OF) to improve embryo quality and to provide an in vitro system to study oviduct function. Bovine oviducts ipsilateral to ovulation were collected at the slaughterhouse, 1 to 4 days after ovulation. The OF was collected by flushing the oviducts with 1 mL of Charles Rosenkrans 1 medium (CR1). Samples from 21 oviducts were pooled and proteins were concentrated using centrifugal filter devices. Aliquots of 3 different protein concentrations, determined by Bradford assay, were prepared and stored at –20°C. Abattoir-retrieved cumulus–oocyte complexes were used for standard in vitro maturation (IVM) and IVF (Day 0). On Day 1, presumptive zygotes (n = 1498) were randomly allocated to 4 different culture groups and cultured up to Day 9. The presumptive zygotes of the control group (n = 364) were cultured in CR1 with 5% oestrous cow serum (OCS) supplemented with 1 mg mL−1 hyaluronan. In the experimental groups, OCS was replaced by OF, resulting in 3 groups with final protein concentrations of 0.1 mg mL−1 (n = 380), 0.5 mg mL−1 (n = 380) or 1 mg mL−1 (n = 374). Cleavage rate was recorded on Day 2 and blastocyst yield on Days 7, 8, and 9 after fertilization. On Day 7, blastocysts were removed and either stained (Hoechst 33342) for cell number or subjected to a slow freezing protocol using 1.5 M ethylene glycol. After thawing, the re-expansion and hatching rate of blastocysts were determined at 24, 48 and 72 h. Eight replicates were carried out and data were analysed by ANOVA. Cleavage rate increased with increasing protein concentration (0.1 mg mL−1: 80.9 ± 4.2%; P > 0.05; 0.5 mg mL−1: 83.4 ± 2.5%; P < 0.1) and was significantly higher in the 1 mg mL−1 group (84.5 ± 4.4%; P < 0.05) compared with the control group (79.7 ± 3.4%). The cumulative blastocyst rate on Day 9 was significantly lower (P < 0.05) in all experimental groups (0.1 mg mL−1: 15.8 ± 8.9%; 0.5 mg mL−1: 18.7 ± 12.0%; 1 mg mL−1: 17.0 ± 11.2%) compared with the control group (34.1 ± 5.4%). The total number of cells was not affected by OF (P > 0.05). There was no significant difference (P > 0.05) in the post-thaw re-expansion rate between the experimental groups (0.1 mg mL−1: n = 26 thawed blastocysts; 0.5 mg mL−1: n = 27; 1 mg mL−1: n = 23) and the control group (n = 58). The post-thaw hatching rate was significantly higher at 24 and 72 h, respectively, in the 0.5 mg mL−1 group (44.4% and 74.1%; P < 0.05) and the 1 mg mL−1 group (47.8%; P < 0.05; and 82.6%; P < 0.01) compared with the control group (18.9% and 44.8%). The replacement of serum with OF during in vitro culture of bovine embryos had a stage specific effect, resulting in higher cleavage rates but lower blastocyst rates. To address this issue, OF will be collected at different stages and applied in the matching in vitro culture phases in future studies. Interestingly, the post-thaw hatching rate was up to twice as high in the experimental groups, indicating better quality of those embryos developing to blastocyst stage.

78 SUPPLEMENTATION WITH CARNOSINE DURING IN VITRO CULTURE IMPROVES THE QUALITY OF IN VITRO-PRODUCED BOVINE EMBRYOS

2017 ◽  
Vol 29 (1) ◽  
pp. 146
Author(s):  
D. Le Bourhis ◽  
M. Verachten ◽  
P. Salvetti ◽  
M. Hochet ◽  
L. Schibler
Keyword(s):  
Reactive Oxygen Species ◽  
Reactive Oxygen ◽  
Control Group ◽  
Hatching Rate ◽  
Bovine Embryo ◽  
Oxygen Species ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Blastocyst Rate

The objective of the present study was to determine the effect of supplementation of culture medium with carnosine (β-alanyl-l-histidine; Sigma, St-Quentin Fallavier, France), a reactive oxygen species scavenger, on in vitro bovine embryo development and survival following cryopreservation. Abattoir-derived bovine oocytes (4 replicates) were in vitro matured and fertilized with frozen-thawed semen of one bull, according to our standard procedures. In Experiment 1, 20 h after IVF, groups of presumptive zygotes were cultured in 30 μL of SOF BSAaa + 1% oestrus cow serum with 0 (control; n = 205) or 5 μg mL−1 of carnosine (n = 209) under humidified air with 5% CO2, 5% O2, and 88% N2. Cleavage rates were determined on Day 2, and the blastocyst rates and grade were assessed on Day 7 according to IETS classification. Day 7 grade 1 expanded blastocysts (n = 25 control and n = 27 carnosine) were frozen in 1.5 M ethylene glycol + 0.1 M sucrose. Embryos were thawed and then cultured for 72 h in SOF-BSAaa + 1% oestrus cow serum for re-expansion and hatching rate assessments at +24 h, +48 h, and +72 h post-thawing. In Experiment 2, presumed zygotes were cultured in SOF BSAaa + 1% oestrus cow serum with 0 (control; n = 48) or 5 μg mL−1 of carnosine (n = 48) in a WOW dish and observed with Time Laps Cinematography (Primo Vision®, VitroLife, Göteborg, Sweden). Images were recorded every 15 min for up to 168 h post-insemination. For embryos that reached the blastocyst stage, mean timing of the first cleavage (C1; 2-cell stage), second cleavage (C2; 4-cell stage), second cleavage to compaction (C3), and blastocoel cavity appearance (B4) were recorded. Chi-square test for Experiment 1 and Student’s t-test for Experiment 2 were used, and differences were considered significant at P < 0.05. In Experiment 1, no differences were observed in cleavage rate, blastocyst rate on Day 7, and grade 1 blastocyst rate between both control and carnosine groups (84.0 ± 4.2 v.85.2 ± 3.8, P = 0.7; 46.9 ± 7.1 v. 45.0 ± 7.5, P = 0.7; 24.1 ± 2.0 v. 24.0 ± 6.5, P = 0.6; respectively). After thawing, the re-expansion at +24 h was not different between groups (74.1 v. 48.0% for carnosine and control groups, respectively; P = 0.06). However, at +48 h and +72 h, the survival rate of carnosine treated blastocysts was significantly higher than that of blastocysts in the control group: 70.4 ± 4.5% v. 40.0 ± 3.8% and 59.3 ± 3.8% v. 24.0 ± 3.6%, respectively. Results from Experiment 2 indicated no difference between control and carnosine groups for C1 (32.1 ± 3.9 v. 33.8 ± 6.1; P = 0.3), C2 (8.2 ± 8.9 v. 8.9 ± 0.9; P = 0.07), and B4 (147.0 ± 9.5 v. 145.4 ± 11.6; P = 0.6), whereas C3 was significantly different within groups: 59.9 ± 9.6 v. 51.8 ± 6.7 (P = 0.008). In conclusion, bovine blastocysts derived from zygotes cultured in the presence of 5 μg mL−1 carnosine possess a significantly faster kinetic from 4-cell stage to compaction and show a higher post-thawing viability. However, further analyses are still needed to clarify the relationship between the reactive oxygen species intracellular levels after carnosine treatment and in vitro bovine embryo quality. This work was supported by FECUND European project (grant agreement number 312097).

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