216 EFFECT OF SPERM PRETREATMENT WITH LYSOLECITHIN AND Triton X-100 ON PRONUCLEAR FORMATION AND QUALITY OF BOVINE EMBRYOS PRODUCED BY INTRACYTOPLASMIC SPERM INJECTION

2016 ◽  
Vol 28 (2) ◽  
pp. 239
Author(s):  
F. Zambrano ◽  
M. E. Arias ◽  
T. Vargas ◽  
L. Aguila ◽  
R. Felmer
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Cell Mass ◽  
Harmful Effect ◽  
Hydrolytic Enzymes ◽  
Control Group ◽  
Cleavage Rate ◽  
Blastocyst Rate ◽  
Triton X ◽  
Pronuclear Formation

In cattle, intracytoplasmic sperm injection (ICSI) has low efficiency and has application for sexed semen, semen of poor quality of high genetic value, or semen of endangered breeds. The acrosome content has a harmful effect within the oocyte because of the presence of hydrolytic enzymes. With the aim of removing the acrosome and destabilising the membranes, cryopreserved bovine spermatozoa were treated with lysolecithin (LL) and Triton X-100 (TX). Frozen straws of bull semen were thawed and the spermatozoa were selected by Percoll gradient. The selected spermatozoa were incubated at concentrations of 0.05% LL (ICSI-LL), 0.05% TX (ICSI-TX), and a control group (without treatment) for 1 min with vortex, and were then immediately washed by centrifugation at 400 × g in Sp-TALP medium. Following ICSI, oocytes were activated with 5 mM ionomycin and 5 µg mL–1 cycloheximide. Embryos were cultured in KSOM medium at 38.5°C, 5% CO2, 5% O2, and 90% N2, to Day 8 to record blastocyst rate. Pronuclear formation was evaluated at 19 h after activation, and the presumptive zygotes were fixed in methanol-glacial acetic acid (3 : 1), stained with Hoechst, and analysed with a fluorescence microscope. The results were evaluated by a chi-squared test with Bonferroni’s correction. Significance was set at P < 0.05. The quality of embryos was evaluated by counting the total cell numbers (T), inner cell mass cells (ICM), and trophectoderm cells (TE) after staining with Hoechst and propidium iodide. We injected 345 oocytes in the control group, 335 in ICSI-LL, 304 in ICSI-TX, and 351 were parthenogenetically activated (12 replicates for each group). A higher cleavage rate was observed in ICSI-TX (66%; 231/351) and ICSI-LL (62%; 207/335) compared the control group (53%; 183/345). In addition, a significantly higher blastocyst rate was observed in both treatments: 27% (82/304) and 27.5% (92 of 335) for ICSI-TX and ICSI-LL groups, respectively, compared with the control group (22.6%; 78/345). In the parthenogenetic activation group without ICSI, the cleavage rate was 41.9% (147/351) and blastocyst rate was 15.4% (54/351). Differences among treatments were analysed using one-way ANOVA after arcsine transformation, and significance was set at P < 0.05. No differences were observed in pronuclear formation (with male and female pronuclei and 2 polar bodies) for ICSI-LL (77%; 48/53), ICSI-TX (76.4%; 39/51), and the control group (71.6%; 38/53) with 3 replicates for each group. No significant differences were observed in the quality of the embryos, ICM cell number (control: 35.7 ± 2.8, ICSI-LL: 38 ± 4.5, and ICSI-TX: 40.8 ± 8.2), TE (control: 139 ± 31.5, ICSI-LL: 124 ± 10.4, and ICSI-TX: 129 ± 18.5), T (control: 170 ± 25.6, ICSI-LL: 156 ± 14.3, and ICSI-TX: 176 30.3), with 8 replicates for each group. In conclusion, this study proposed treatments able to improve the rate of development without affecting the quality of embryos produced by ICSI.

60 IMPROVEMENT IN EFFICIENCY AND QUALITY OF CLONED AND PARTHENOGENETIC PORCINE EMBRYOS BY AMINO ACIDS

2007 ◽  
Vol 19 (1) ◽  
pp. 147
Author(s):  
H. T. Lee ◽  
J. M. Jang ◽  
S. H. Lee ◽  
M. K. Gupta
Keyword(s):  
Amino Acids ◽  
Culture Media ◽  
Cell Number ◽  
Control Group ◽  
In Vitro Production ◽  
Cleavage Rate ◽  
Fetal Fibroblasts ◽  
Blastocyst Rate

In vitro production of cloned porcine embryos by somatic cell nuclear transfer (SCNT) has become routine in several laboratories but the efficiency and quality of the resultant blastocysts remains sub-optimal. Cloned porcine blastocysts show low cell number, high fragmentation rate, and apoptosis which results in lower pregnancy rates upon embryo transfer. Earlier we reported that supplementation of culture media with amino acids benefit pre-implantation embryo development of in vivo- as well as in vitro-fertilized porcine embryos (Koo et al. 1997 Theriogenology 48, 791–802). This study evaluated how exogenous amino acids could affect pre-implantation development and quality of cloned or parthenogenetic porcine embryos. The effects of commercially available amino acids, referred to as Eagle&apos;s non-essential amino acids (NEAA), added or not added (control) to NCSU23 medium containing fatty acid-free BSA were studied. Oocytes recovered from abattoir-derived prepubertal porcine ovaries were matured in vitro and parthenogenetically activated (PA) or nuclear-transferred with fetal fibroblasts (SCNT), as described earlier (Uhm et al. 2000 Mol. Reprod. Dev. 57, 331–337). At 168 h post-activation, blastocysts were harvested for assessment of embryo quality by TUNEL labeling, Hoechst 33342 staining, and gene expression analysis. Results showed that, in the PA group, the cleavage rate was not affected by the supplementation of NEAA. However, the blastocyst rate was significantly improved when NEAA was present in the medium compared to that of the control group (38.9 &plusmn; 0.3 vs. 27.5 &plusmn; 0.3&percnt;, respectively) throughout the culture period. The supplementation during the pre-compaction period alone gave better results than during the post-compaction period alone (59.5 &plusmn; 0.9 vs. 33.4 &plusmn; 0.3&percnt;, respectively). In the SCNT group, however, both cleavage (73.6 &plusmn; 0.2 vs. 64.2 &plusmn; 0.4&percnt;) and blastocyst rate (18.7 &plusmn; 0.2 vs. 13.8 &plusmn; 0.3&percnt;) were improved by NEAA supplementation. Furthermore, these blastocysts had higher hatching ability (30.0 &plusmn; 1.8 vs. 14.6 &plusmn; 4.9&percnt;) than those of control group (P &lt; 0.05). Supplementation of NEAA also increased the mean nuclei number of PA-derived (76.1 &plusmn; 4.9 vs. 66.5 &plusmn; 3.3) as well as SCNT-derived (43.1 &plusmn; 2.6 vs. 31.8 &plusmn; 1.9) blastocysts and reduced the time during which blastocysts formed. TUNEL assay revealed that incidence of nuclear fragmentation and apotosis was reduced by NEAA. Real-time qRT-PCR for Bax and Bcl-XL transcripts revealed that the relative abundance of Bax was reduced while that of Bcl-XL was increased. These effects were more pronounced when NEAA was present during the pre-compaction period alone. Thus, our data suggest that NEAA improves the yield and quality of cloned porcine embryos by enhancing blastocyst expansion and positively modulating the total cell number and apoptosis. These data may have implications for understanding the nutritional needs of cloned porcine embryos produced in vitro and for optimizing the composition of culture media to support their development. This work was supported by the Research Project on the Production of Bio-Organs (No. 200503030201), Ministry of Agriculture and Forestry, Republic of Korea.

194 Extracellular vesicles derived from ampullary oviductal fluid improve developmental competence of bovine oocytes

2020 ◽  
Vol 32 (2) ◽  
pp. 225
Author(s):  
A. Asaadi ◽  
K. Pavani ◽  
N. Azari Dolatabadi ◽  
P. Van Damme ◽  
A. Van Soom
Keyword(s):  
Extracellular Vesicles ◽  
Follicular Fluid ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Control Group ◽  
Cleavage Rate ◽  
Blastocyst Quality ◽  
Oviductal Fluid ◽  
Bovine Oocytes

Invivo, oocytes mature in the presence of follicular fluid (FF) and ampullary oviductal fluid (AOF), starting from premature to final maturation stages (3-6h postovulation). Extracellular vesicles (EVs) have been identified as important mediators of gamete/embryo-maternal interactions. They carry regulatory molecules, such as microRNAs and mRNAs. So far, the functionality of EVs derived from FF and AOF has not yet been determined. The aim of this study was to evaluate the effect of EVs derived from FF and AOF during invitro oocyte maturation (IVM) on the development and quality of resulting bovine embryos. Follicular fluid of a preovulatory follicle was collected by ovum pickup, and AOF was collected from the oviducts of slaughtered cows in early luteal phase. Then, EVs from FF and AOF were isolated by size exclusion chromatography and confirmed by western blot. The concentration of EVs was determined by NanoDrop and Nanoparticle tracking analysis. Integrity and size of EVs were assessed by electron microscopy. Bovine oocytes (n=1331) were allocated at random to five groups. In the control group (C), oocytes (n=347) were cultured for 22.5h in maturation medium (TCM-199 and epidermal growth factor 20ngmL−1) (MM). In the negative control group (NC), oocytes (n=331) were cultured for 18h in MM, and then transferred into fresh MM for 4.5h. In the FF group, oocytes (n=162) were cultured in MM supplemented with FF EVs (12.5µgmL−1) for 18h, and then transferred to MM for 4.5h. In the AOF group, oocytes (n=328) were cultured for 18h in EV-free MM, and then transferred to MM supplemented with AOF EVs (1.7µgmL−1) for 4.5h. In the FF+AOF group, oocytes (n=163) were cultured in MM supplemented with FF EVs for 18h, and then transferred into MM supplemented with AOF EVs for 4.5h. At 22.5h post-incubation, mature oocytes were fertilized invitro. At 21h post-insemination, presumptive zygotes were denuded and cultured in synthetic oviductal fluid with insulin-transferrin-selenium for 8 days. Cleavage rate and blastocyst rate were recorded at 48h and 8 days post-insemination, respectively. Blastocyst quality was assessed by differential apoptotic staining. The data were analysed using one-way ANOVA. Cleavage rate was not affected by the treatment, whereas blastocyst yield in the AOF group (48.75%) was significantly higher than that in the C (42.91%) and NC (37.72%) groups. In addition, embryos produced in the AOF group showed the highest number of trophectoderm cells and inner cell mass cells, and lowest number of apoptotic cells (P&lt;0.05). Trophectoderm and inner cell mass cells were higher and apoptotic cells were lower in FF and FF+AOF compared with the C and NC groups. In conclusion, adding AOF EVs to oocyte MM in the last 4.5h of IVM improves quantity and quality of blastocysts. In addition, although FF EVs showed no effect on blastocyst yield, they improved blastocyst quality by increasing cell number.

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.

77 Development and quality of in vitro bovine hemi embryos produced by blastomere separation and embryo bisection

2019 ◽  
Vol 31 (1) ◽  
pp. 164
Author(s):  
A. E. Ynsaurralde Rivolta ◽  
M. Suvá ◽  
V. Alberio ◽  
C. Vazquez Echegaray ◽  
A. Guberman ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Monozygotic Twin ◽  
Cell Number ◽  
Control Group ◽  
Cell Stage ◽  
Exact Test ◽  
Development Rates

Bovine monozygotic production of twins became popular in the 1980s as a technique to multiply high value genetics. Moreover, it also became a powerful model for research. Different techniques have been used on bovine embryos obtained by superovulation. In this work, we compared the development rates and quality of monozygotic twin embryos produced by blastomere separation (BS) and embryo bisection (EB) of IVF embryos. To this aim, cumulus-oocytes complexes collected from slaughterhouse ovaries were in vitro matured in TCM 199 containing 10% fetal bovine serum, 10µg mL−1 FSH, 0.3mM sodium pyruvate, 100mM cysteamine, and 2% antibiotic-antimycotic for 24h, at 6.5% CO2 in humidified air and 38.5°C. The IVF was performed with 16×106 spermatozoa per mL for 5h. Afterward, presumptive zygotes were cultured in SOF medium for 7 days at 38.5°C and 5% O2. After 24h of culture, blastomeres of 2-cell stage embryos (N=114) were separated and each one was cultured individually in a microwell for 7 days. Embryo bisection (N=179) was performed manually on Day-7 blastocysts previously depleted of their zonae pellucidae, under stereoscopic microscope. Hemi embryos were cultured for 24h and then twins and single blastocyst rates were calculated. For quality assessment, diameter, total and inner cell mass (ICM) cell number of hemi embryos (BS: 6 couples; ES: 10 couples) and the control group (C: 11) were evaluated. The ICM cell number was measured by immunofluorescence staining using SOX2 antibody and the percentage of ICM and trophectoderm (TE) cells was calculated. The results were analysed using Fisher’s exact test and ANOVA with mean comparison using Tukey’s test (P=0.05). No statistical differences were found in blastocyst rates of twins and single hemi embryos produced by BS (28 and 25%) or EB (23 and 32%). Blastocyst diameter was similar between groups and control. Hemi embryos exhibited lower total and ICM cell number than control (BS: 43±18, EB: 57±14v. C: 93±35 and BS: 16±7, EB: 12±8v. C: 34±19). However, BS hemi embryos had higher ICM and lower TE percentage (40/60%) compared with the EB group (20/80%). The control group did not differ with hemi embryo treatments for ICM and TE (30/70%). Our preliminary results have indicated that although the development rates of hemi embryos produced in vitro were similar between both techniques, blastomere separation generates better quality embryos than blastocyst bisection.

Effect of sperm pretreatment with sodium hydroxide and dithiothreitol on the efficiency of bovine intracytoplasmic sperm injection

2014 ◽  
Vol 26 (6) ◽  
pp. 847 ◽  
Author(s):  
M. E. Arias ◽  
R. Sánchez ◽  
J. Risopatrón ◽  
L. Pérez ◽  
R. Felmer
Keyword(s):  
Embryonic Development ◽  
Intracytoplasmic Sperm Injection ◽  
Membrane Integrity ◽  
Control Group ◽  
Developmental Potential ◽  
Bovine Spermatozoa ◽  
Pronuclear Formation ◽  
First Time ◽  
In Vitro Embryonic Development

The efficiency of intracytoplasmic sperm injection (ICSI) in bovines is lower than in other species due, in part, to a lack of optimal conditions for its implementation; this has hindered the achievement of high rates of embryonic development and the birth of live offspring. The aim of the present study was to evaluate the effects of pretreatment of bovine spermatozoa with NaOH and dithiothreitol (DTT) on the viability, plasma membrane integrity, DNA fragmentation and in vitro developmental potential of embryos generated by ICSI. Following pretreatment of spermatozoa with 5 mM DTT for 20 min and a low concentration of NaOH (1 mM for 60 min), there were fewer live and acrosome reacted spermatozoa (44% and 34%, respectively) than in the control group without treatment (82%). Spermatozoa subjected to higher alkali concentrations (10–50 mM) were mostly dead and reacted. However, pronuclear formation, cleavage, blastocyst rate and embryo quality did not differ between these pretreatment groups and the untreated control group. In conclusion, we have described, for the first time, the effects of NaOH treatment on bovine spermatozoa and subsequent in vitro embryonic development after ICSI, and have demonstrated that pretreatment of bovine spermatozoa with NaOH or DTT is not necessary for an appropriate in vitro embryo development in this species.

A combination of calcium ionophore and puromycin effectively produces human parthenogenones with one haploid pronucleus

Zygote ◽  
2001 ◽  
Vol 9 (1) ◽  
pp. 83-88 ◽  
Author(s):  
Koji Nakagawa ◽  
Shuji Yamano ◽  
Hisayo Nakasaka ◽  
Kenji Hinokio ◽  
Midori Yoshizawa ◽  
...  
Keyword(s):  
Polar Body ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Control Group ◽  
Ionophore A23187 ◽  
Cleavage Rate ◽  
Parthenogenetic Activation ◽  
Second Polar Body ◽  
Activation Rates ◽  
Pronuclear Formation

Parthenogenetic activation with various combinations of the calcium ionophore A23187 and protein synthesis or phosphorylation inhibitors was investigated as a means of producing human parthenogenones with one haploid pronucleus. Unfertilised human aged oocytes exposed to 5 μM A23187 for 5 min were treated with 10 μg/ml puromycin (puromycin group, 46 oocytes) or 2 mM 6-dimethylaminopurine (DMAP group, 42 oocytes) for 5 h. Oocytes treated only with A23187 served as a control (control group, 40 oocytes). After washing the oocytes, they were incubated for up to 37 h. Evidence of activation (pronuclear formation) and cleavage was observed 18 h and 42 h after A23187 treatment, respectively. Activation rates in the puromycin and DMAP groups were significantly higher than in the control group (91% (42/46) and 77% (34/44) vs 20% (8/40), p < 0.05, respectively). In the puromycin group, 81% (34/42) of the activated oocytes showed one pronucleus with the second polar body (2ndPB), whereas none (0/34) of the activated oocytes in the DMAP group extruded the 2ndPB. The cleavage rate in the puromycin group was significantly lower than in the DMAP group (38% vs 68%, p < 0.05). The activated oocytes which had one pronucleus with the 2ndPB in the puromycin group showed a haploid set of chromosomes (10/13). In conclusion, the combination of A23187 and puromycin is effective for producing human parthenogenones with one haploid pronucleus.

scholarly journals Effect of deslorelin acetate treatment in oocyte recovery and in vitro embryo production in domestic cats

2016 ◽  
Vol 19 (10) ◽  
pp. 1091-1095
Author(s):  
Camila Louise Ackermann ◽  
Eduardo Trevisol ◽  
Leticia Ferrari Crocomo ◽  
Tatiana da Silva Rascado ◽  
Rodrigo Volpato ◽  
...  
Keyword(s):  
Treated Group ◽  
Control Group ◽  
Domestic Cats ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Oocyte Recovery ◽  
Significant Difference ◽  
In Vitro Embryo Production ◽  
Blastocyst Rate

Objectives The present study investigated the effect of contraceptive treatment with deslorelin acetate on in vitro embryo production and oocyte recovery in domestic queens. Methods Twenty-one mature domestic cats were used. Eleven queens (treated group) and one tom were kept in an experimental cattery, and 10 queens were privately owned (control group). When in interestrus or diestrus (day 0) a deslorelin acetate implant (Suprelorin, 4.7 mg/animal) was inserted into the subcutaneous tissue of the interscapular region in all queens in the treated group. After 6 months of treatment, all animals were ovariohysterectomized, and the ovaries were used for in vitro embryo production. Percentage of cleavage was determined 18 h after oocyte insemination and blastocyst formation was assessed on the eighth day of culture. The rate of cumulus-oocyte complexes (COCs) recovery was analyzed by an unpaired t-test. The cleavage and blastocyst rates were expressed as percentages and analyzed by Fisher’s exact test. All analyses were performed using GraphPad Prism v5.0, with P <0.05 set as the level of significance. Results In the treated group, we recovered 8.3 ± 1.15 grade I COCs per queen; the cleavage rate was 60% and the blastocyst rate was 36%. In the control group, we recovered 18.4 ± 3.21 grade I COCs per queen; the cleavage rate was 55.97% and the blastocyst rate was 34%. Forty percent of treated females did not produce any blastocysts. In the treated group, we observed a significant decrease in COC recovery. Although there was no significant difference in cleavage and blastocyst rates between groups, 40% of treated females did not produce any blastocysts. Conclusions Recovery of grade I COCs is negatively affected by deslorelin treatment in domestic cats. Regarding embryo production, new studies are still necessary to evaluate the success of this technique owing to the individual effect caused by deslorelin acetate.

High hydrostatic pressure treatment improves the quality of in vitro-produced ovine blastocysts

2011 ◽  
Vol 23 (6) ◽  
pp. 809 ◽  
Author(s):  
Luisa Bogliolo ◽  
Federica Ariu ◽  
Giovanni Leoni ◽  
Stefania Uccheddu ◽  
Daniela Bebbere
Keyword(s):  
Hydrostatic Pressure ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Lower Percentage ◽  
Control Group ◽  
Hatching Rate ◽  
Embryo Survival

Exposure to sub-lethal hydrostatic pressure (HP) treatment is emerging as an approach to improve the general resistance of gametes and embryos to in vitro conditions. The present study was aimed to evaluate the effect of HP treatment on in vitro-produced ovine blastocysts. Experiment 1 was aimed to define optimal treatment parameters: two different HP treatments were applied to blastocysts and embryo survival was evaluated. In Experiment 2, HP parameters (40 MPa, 70 min, 38°C) selected in Experiment 1 were used to treat blastocysts. Embryo quality was assessed and compared with untreated controls by counting total cell number, the inner cell mass (ICM) and trophectoderm (TE) cells and by evaluating nuclear picnosis. HP-treated blastocysts were processed for gene expression analysis (AQP3, ATP1A1, BAX, CDH1, HSP90β, NANOG, OCT4 and TP53) 1, 5 h after the end of HP exposure. Results showed that the hatching rate of embryos treated at 40 MPa was significantly higher than that of the 60 MPa-treated group (P < 0.01) and similar to untreated embryos. Blastocysts exposed at 40 MPa showed higher ICM (P < 0.05) and TE (P < 0.01) cell number and a lower percentage of picnotic nuclei (P < 0.05) compared with the control group. Significantly lower abundance for BAX (P < 0.01) and OCT4 (P < 0.05) transcripts were observed in HP embryos than in the control group. In conclusion, treatment with HP improved the quality of in vitro-produced ovine blastocysts by increasing their cell number and reducing the proportion of nuclear picnosis.

70 Does the addition of docosahexaenoic acid to in vitro systems during culture improve the quality of bovine embryos?

2019 ◽  
Vol 31 (1) ◽  
pp. 160
Author(s):  
J. A. Sánchez Viafara ◽  
G. Lopez de Vasconcelos ◽  
R. Maculan ◽  
N. Gomes Alves ◽  
J. Camisão de Souza
Keyword(s):  
Docosahexaenoic Acid ◽  
Cell Mass ◽  
Control Group ◽  
Hatching Rate ◽  
Link Function ◽  
Bovine Embryos ◽  
Cleavage Rate ◽  
Internal Cell ◽  
Fresh Embryos

The aims of this study were to decrease the apoptotic index and increase cryotolerance of bovine embryos produced in vitro with the addition of 1 µM docosahexaenoic acid (DHA). On Day 1, presumed zygotes were cultivated with 1µM DHA (Sigma, St. Louis, MO, USA; n=437), and without the agonist (control group; n=450). The cleavage rate (%) was evaluated on Day 2 and the development of blastocysts on Day 7. Embryos before and after vitrification were fixed for the TUNEL trial. After vitrification, the embryos were heated and re-cultivated to evaluate the hatching rate at 12, 24, 36, 48, 60, and 72h. A sample of re-cultivated embryos at 12h of DHA (n=5), and without the agonist (control group; n=6), was frozen for mass spectrometry (MALDI-MS). Statistical analyses of deviance were carried out considering generalized mixed linear models, and the effect of the collection day (block) was considered as random. For the count variables, the Poisson distribution and the log link function were considered. In the cases of variables represented by rates, binomial distribution and the logit link function were used. In the study of cryotolerance, ANOVA of the hatching rate for each one of the times evaluated was carried out. In cases of significance of the effect of treatments, the Dunnett test was applied to compare treatments. Multivariate and univariate statistical models were used for analysis of MALDI-MS. All analyses were made using the GLIMMIX procedure of the SAS software (SAS Institute Inc., Cary, NC, USA). The cleavage rate was not different between the groups (P&gt;0.05) and the production of blastocysts was lower in the DHA group (P&lt;0.05). The number of cells per embryo was higher (P&lt;0.05) by the addition of 1μM DHA in blastocysts pre- and post-vitrification. The rate of total and internal cell mass apoptosis in fresh embryos (11.73 and 15.98%) increased compared with the control group (9.62% and 11.03%, respectively). The proportion of internal cell mass in fresh embryos decreased in the DHA group (39.93%) compared with the control group (57.00%). Hatching rates at 48, 60, and 72h after devitrification in the group treated with 1μM DHA were not different (P&gt;0.05) compared with the control group. Phosphatidylcholine [phosphatidylcholine (32: 0)+H] was more abundant (P&lt;0.05) in embryos cultured with DHA, and thus was considered as a negative apoptosis biomarker. In conclusion, the use of 1μM DHA in vitrification of bovine embryos impairs embryonic quality and development under the conditions of the present study. Research was supported by CAPES, FAPEMIG, PPGCV/UFLA, EVUFMG, CENATTE EMBRIÕES.

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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