68 Developmental competency of bovine embryos derived from oocytes with granulated ooplasm after in vitro culture

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.

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

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab83 ◽

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.

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240 QUALITY AND VIABILITY OF IVF BOVINE EMBRYOS AFTER INTRACYTOPLASMIC INJECTION OF DNA–LIPOSOME COMPLEXES

Reproduction Fertility and Development ◽

10.1071/rdv24n1ab240 ◽

2012 ◽

Vol 24 (1) ◽

pp. 232

Author(s):

L. N. Moro ◽

G. Vichera ◽

D. Salamone

Keyword(s):

In Vitro Culture ◽

Dna Fragmentation ◽

Transgene Expression ◽

Fluorescent Protein ◽

Transgenic Animals ◽

Egfp Expression ◽

Bovine Embryos ◽

Exogenous Dna ◽

Intracytoplasmic Injection

Transgenic animals have important applications in agriculture and human medicine; nevertheless the available techniques still remain inefficient and technically difficult. We have recently developed a novel method to transfect bovine embryos that consists of intracytoplasmic injection of exogenous DNA–liposome complexes (eDNA-LC) in IVF zygotes. This study was designed to evaluate the quality and viability of IVF bovine embryos, after intracytoplasmic injection of pCX-EGFP–liposome complexes (EGFP-LC) or pBCKIP2.8-liposome complexes (plasmid that codifies the human insulin gene, HI-LC). First, we evaluated embryo development and enhanced green fluorescent protein (EGFP) expression of IVF embryos injected with both plasmids separately. This treatment was analysed by Fisher's Exact test (P ≤ 0.05). Cleavage rates for EGFP-LC, HI-LC and IVF embryos injected with liposomes alone (IVF-L) and IVF control (IVF-C) were 62% (63/102), 67% (67/100), 66% (67/101) and 79% (98/124); blastocysts rates were 17% (17/102), 21% (21/100), 21% (21/101) and 23% (28/124), respectively. No statistical differences were seen among groups. The percentage of EGFP-positive embryos (EGFP+) after EGFP-LC injection was 42.9% after 3 days of culture and 41.8% at the blastocyst stage. In the second experiment, the blastocysts obtained, EGFP+ or EGFP-negative (EGFP–), were analysed by TUNEL assay at Day 6 (Bd6), 7 (Bd7) and 8 (Bd8) of in vitro culture, in order to evaluate the effect of the transgene and culture length, on DNA fragmentation. This treatment was analysed by the difference of proportions test (P ≤ 0.05) using statistical INFOSTAT software. All EGFP+ blastocysts showed TUNEL positive cells (T+). The percentage of T+ in Bd6, Bd7 and Bd8 were 91, 73.7 and 99.5%, respectively (P ≤ 0.05). EGFP– blastocysts showed lower fragmented nuclei (0, 44.6 and 85%, respectively; P ≤ 0.05). Groups IVF-L and IVF-C were also evaluated. In both groups, there was no evidence of DNA fragmentation in Bd6 and Bd7, but T+ were detected in Bd8 (66.4 and 85.8%, respectively; P ≤ 0.05). In the third experiment, bovine blastocysts obtained from the HI-LC group were individually transferred to recipient cows after 6 (n = 11), 7 (n = 5) and 8 (n = 5) days of culture post-IVF and HI-LC injection. The pregnancies obtained were from Bd6 [18.2% (2/11)] and Bd7 [40% (2/5)], although none of the recipients receiving Bd8 were diagnosed pregnant. Two pregnancies developed to term, one derived from Bd6 and the other from Bd7. Analysis by PCR determined that none of the born cows were transgenic. In summary, IVF bovine embryos could be easily transfected after the injection of eDNA-LC and the technique did not affect offspring viability. The results indicate that extended time in in vitro culture increases the percentage of fragmented nuclei in blastocysts. Moreover, this parameter increases in blastocysts with transgene expression compared with those without expression. Finally, more transfers are required in order to obtain the real efficiency of this new technique and to overcome the drawbacks generated by in vitro culture length and transgene expression.

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

Reproduction Fertility and Development ◽

10.1071/rd15238 ◽

2017 ◽

Vol 29 (3) ◽

pp. 621 ◽

Cited By ~ 28

Author(s):

Ricaurte Lopera-Vasquez ◽

Meriem Hamdi ◽

Veronica Maillo ◽

Valeriano Lloreda ◽

Pilar Coy ◽

...

Keyword(s):

In Vitro Culture ◽

Embryo Development ◽

Cell Mass ◽

Water Channel ◽

Calf Serum ◽

Cell Counting ◽

Bovine Embryos ◽

Oviductal Fluid

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

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