35 Ethanolic extracts of Cerrado plants in cryotolerance of invitro-produced bovine embryos

Invitro embryo culture induces excessive production of reactive oxygen species (ROS) that impair the quality of produced blastocysts, making them less cryotolerant. Supplementation of culture media with antioxidants would be an alternative to minimise these effects. The present study evaluated the effect of ethanolic extracts obtained from Brazilian Cerrado plants on the cryotolerance of invitro-produced embryos. Bovine ovaries from a slaughterhouse were used to obtain grade I and II oocytes, which were submitted to maturation, fertilization (Day 0), and invitro culture. After fertilization, zygotes were distributed into five groups: one fresh control group (GCont), cultured in high O2 tension (~21%), and four groups cryopreserved at Day 7: a control group subject to high O2 tension without extract supplementation (GDT); a group (G5%) cultured in low O2 tension (5%) without extract supplementation, and two groups cultured under high O2 tension, one supplemented with 0.01mgmL−1 of the ethanolic cagaita extract (Eugenia dysenterica; GCag) and another with 0.01mgmL−1 of murici extract (Byrsonima crassifolia; GMur). Only the expanded blastocysts (EB) of each group were submitted to the direct-transfer cryopreservation system, with slow freezing with ethylene glycol (Sanches et al. 2016 Theriogenology 85, 1147-1151). A total of 163 thawed embryos were evaluated 12, 24, and 36h post-thawing for re-expansion, hatching, and embryonic degeneration rate. In addition, after 12h of post-thaw culture, only the EB embryos were subjected to ROS measurement, quantified in confocal microscopy using 2′,7′-dichlorodihydrofluorescein diacetate, and the rate of apoptosis was obtained by the terminal deoxynucleotidyl transferase dUTP nick end labelling method. Data were analysed by analysis of variance, and the means were compared by TUKEY. A total of 133 EB were used (GCont: 20; GDT: 22; GCag: 35; GMur: 36; G5%: 20). Results are expressed as means±standard deviation. No differences were observed on re-expansion among treatments and time of culture. However, 24h post-thaw, hatching and degeneration rates differed between groups. The group GCont had a higher (P<0.05) hatching rate (47.5±10.3) than the GDT (14.4±6.7) and G5% (10.5±5.8) groups but was similar (P>0.05) to groups supplemented with extracts GCag (22.2±17.1) and GMur (17.5±17.5). Degeneration rate was higher (P<0.05) at 12h for the direct-transfer group (30.2±11.3) and similar among the others (GCont: 0; GCag: 16.8±12; GMur: 10.8±10.8; G5%: 11±11). Nevertheless, GCag had lower embryonic degeneration rates (20±16.7) than the others cryopreserved groups 24h after thawing (GDT: 37.7±5.5; GMur: 38.9±8.2; G5%: 41.3±14.4). Although ROS levels were similar among all groups (P>0.05), apoptosis rate was higher (P<0.05) in the GDT group (23.4±1.9) than in the GCont groups (10.6±1.3): GCag (10.8±1.1), GMur (10.1±1.1), and G5% (7.8±1.2). Therefore, supplementation of ethanolic extracts (0.01mgmL−1) of cagaita and murici improved the embryo quality by reducing the degeneration and apoptosis rates of cryopreserved embryos when compared with the group without supplementation (GDT). Such extracts may be an alternative to increase the cryotolerance of invitro-produced bovine embryos.

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Resveratrol-supplemented holding or re-culture media improves viability of fresh or vitrified-warmed in vitro-derived bovine embryos

Research Society and Development ◽

10.33448/rsd-v10i14.22097 ◽

2021 ◽

Vol 10 (14) ◽

pp. e367101422097

Author(s):

Arianny Rafaela Neto Silva ◽

Thaisa Campos Marques ◽

Elisa Caroline Silva Santos ◽

Tiago Omar Diesel ◽

Isabelle Matos Macedo ◽

...

Keyword(s):

Culture Media ◽

Cell Number ◽

Time Dependent ◽

Expansion Rate ◽

Ros Generation ◽

Hatching Rate ◽

Protective Effects ◽

Bovine Embryos ◽

In Vitro Production

The effect of resveratrol supplementation on fresh (E1) or vitrified/warmed (E2) in vitro produced bovine embryos was investigated by evaluating the time-dependent response. After in vitro production, resveratrol (0.5 µM) was added to the incubation media and after two incubation periods with or without resveratrol, blastocysts were re-cultured for 24h. The rates of re-expansion, hatching, total cell number (TCN), apoptotic cells (ACN), reactive oxygen species (ROS) and intracellular glutathione (GSH) content were evaluated. For E1, the re-expansion rate differed at 6 and 10h within and between treatments (P<0.05), as did the re-expansion rate after 24h (P<0.01). The hatching rate increased after 10h with resveratrol (P<0.01) with differences within (P<0.05), but not between treatments after 24h of re-cultivation. At E2, hatching rate differed between treatments at 24h (P<0.01), with higher TCN in resveratrol-treated blastocysts after 10h (P<0.01). Resveratrol supplementation reduced ROS generation in E1 and E2 after 10h of incubation and increased GSH content (P<0.01). These results indicate that supplementation of holding re-cultivation medium with resveratrol for treatment of fresh or vitrified/warmed in vitro produced bovine embryos has a positive and time-dependent effect. The reduction of ROS content, the increase of GSH and the anti-apoptotic ability of resveratrol are responsible for its protective effects, allowing an extension of embryo storage time before transfer to recipients.

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239 USE OF CYCLIC ADENOSINE MONOPHOSPHATE MODULATORS IN IN VITRO PRODUCTION OF BOVINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab239 ◽

2015 ◽

Vol 27 (1) ◽

pp. 209

Author(s):

T. Fanti ◽

N. M. Ortega ◽

R. Garaguso ◽

M. J. Franco ◽

C. Herrera ◽

...

Keyword(s):

Phosphodiesterase Inhibitor ◽

Basal Medium ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Control Group ◽

Hatching Rate ◽

Bovine Embryos ◽

Oocyte Competence

In vitro embryo production systems (IVP) try to emulate and enhance molecular events that occur in in vivo reproductive systems in order to increase, not only the number of embryos generated, but also their quality. Despite advances, IVP processes are still inefficient compared with in vivo systems. Several studies have attributed this deficiency to a lack of oocyte competence due to spontaneous premature resumption of meiotic maturation in the oocyte following the removal from its follicular environment. Therefore, our objective was to increase oocyte competence avoiding premature resumption of meiosis by using cyclic adenosine monophosphate modulators. Cumulus-oocyte complexes (COC) were obtained from ovaries of slaughterhouses, washed, and randomly allocated in 2 culture systems. Oocytes in the control group (IVM) were cultured for a period of 24 h in basal medium TCM-199 with EGF (1 µg mL–1) supplemented with rhFSH (25 mIU mL–1). Oocytes in the biphasic in vitro maturation (b-IVM) group were cultured for 2 h in a basal medium supplemented with a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX, 500 µM), and an activator of adenylate cyclase (forskolin, 100 µM). Subsequently, COC were washed and cultured in basal medium supplemented with cilostamide (20 µM) and rhFSH (25 mIU mL–1) for 24 h. Maturation rates were analysed and IVF was performed with a dose of 1 × 106 sperm cells mL–1 in IVF-SOF medium. The presumptive zygotes were cultured in continuous-single-culture medium (Irvine) supplemented with 8 mg mL–1 of BSA until they reached the blastocyst stage. No significant differences in maturation, cleavage, and cryotolerance were observed between b-IVM and IVM groups (P > 0.05; Table 1). This study showed that b-IVM produced a significant increase in IVP compared with the control (IVM) at Days 7 and 8 (P < 0.01). Blastocyst hatching rate was significant (P < 0.05) for both treatment and day of analysis. The b-IVM group yielded an increase of 10 and 7.5% at Days 7 and 8, respectively, of IVP. The biphasic maturation showed an improvement in quality regarding the control group, in the timing analysis of production, and hatching percentages, and these results show that the use of cyclic adenosine monophosphate modulators in the oocyte maturation process enhances oocyte competence, which is reflected in increased productivity and embryo quality. We propose this treatment as an alternative to the standard protocols currently used in IVP of bovine embryos. Table 1.Effect of treatment on maturation, cleavage, and cryotolerance

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Effects of different cryopreservation methods on post-thaw culture conditions of in vitro produced bovine embryos

Zygote ◽

10.1017/s0967199410000717 ◽

2011 ◽

Vol 20 (2) ◽

pp. 117-122 ◽

Cited By ~ 8

Author(s):

Alessandra Corallo Nicacio ◽

Renata Simões ◽

Fabiola Freitas de Paula-Lopes ◽

Flavia Regina Oliveira de Barros ◽

Maria Angelica Peres ◽

...

Keyword(s):

Culture Conditions ◽

Slow Freezing ◽

Control Group ◽

Hatching Rate ◽

Rapid Freezing ◽

Bovine Embryos ◽

Group 10 ◽

Controlled Freezing ◽

Hatching Rates

SummaryThe aim of this work was to evaluate the effect of cryopreservation protocols on subsequent development of in vitro produced bovine embryos under different culture conditions. Expanded in vitro produced blastocysts (n = 600) harvested on days 7–9 were submitted to controlled freezing [slow freezing group: 10% ethylene glycol (EG) for 10 min and 1.2°C/min cryopreservation]; quick-freezing [rapid freezing group: 10% EG for 10 min, 20% EG + 20% glycerol (Gly) for 30 s]; or vitrification [vitrification group: 10% EG for 10 min, 25% EG + 25% Gly for 30 s] protocols. Control group embryos were not exposed to cryoprotectant or cryopreservation protocols and the hatching rate was evaluated on day 12 post-insemination. In order to evaluate development, frozen–thawed embryos were subjected to granulosa cell co-culture in TCM199 or SOFaa for 4 days. Data were analyzed by PROC MIXED model using SAS Systems for Windows®. Values were significant at p < 0.05. The hatching rate of the control group was 46.09%. In embryos cultured in TCM199, slow freezing and vitrification group hatching rates were 44.65 ± 5.94% and 9.43 ± 6.77%, respectively. In embryos cultured in SOFaa, slow freezing and vitrification groups showed hatching rates of 11.65 ± 3.37 and 8.67 ± 4.47%, respectively. In contrast, the rapid freezing group embryos did not hatch, regardless of culture medium. The slow freezing group showed higher hatching rates than other cryopreservation groups. Under such conditions, controlled freezing (1.2°C/min) can be an alternative to cryopreservation of in vitro produced bovine embryos.

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133 EFFECTS OF ACTIVINA ON mRNA EXPRESSION IN IN VITRO FERTILIZED BOVINE EMBRYOS CULTURED FROM CHEMICALLY DEFINED TWO-STEP CULTURE MEDIUM

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab133 ◽

2008 ◽

Vol 20 (1) ◽

pp. 147

Author(s):

J. E. Park ◽

G. Jang ◽

H. J. Oh ◽

S. G. Hong ◽

I. S. Yang ◽

...

Keyword(s):

Embryo Development ◽

Culture Medium ◽

Early Stage ◽

Activin A ◽

Developmental Competence ◽

Control Group ◽

Hatching Rate ◽

Bovine Embryos ◽

Defined Culture

During the preimplantation stage, embryo development occurs in a maternal environment within the oviducts and uterine horns. It has been speculated that both the embryo itself and the maternal reproductive tract provide paracrine factors that influence embryo development (Jones et al. 2006 Reproduction 132(5), 799–810). Activins are known for FSH releasers, and several previous studies have reported that activin subunits and activin receptors mRNA were expressed in oocytes, zygotes, and oviduct (Yoshioka et al. 1998 Reprod. Fertil. Dev. 10(3), 293–298; Gandolfi et al. 1995 Mol. Reprod. Dev. 40(3), 286–291). The purposes of the present study were Experiment 1) to evaluate the effects of activin A on developmental competence of bovine embryos derived from two-step defined culture medium (Lim et al. 2007 Theriogenology 67(2), 293–302) and Experiment 2) to analyze the effects of activin A on transcriptional level of the genes in IVF embryos. Cumulus–oocyte complexs were harvested from ovaries obtained from a local slaughter house, matured, and fertilized in vitro. In vitro fertilized zygotes cultured in media supplemented with activin A in the early stage at the concentrations of 0, 10, or 100 ng mL–1 or in the later stage medium at the concentrations of 0, 10, or 100 ng mL–1. Data were analyzed using the Statistical Analysis System (SAS) program. In Exp. 1, although the development competence of embryos that cultured with activin A in the early stage medium was not significantly different, development to blastocysts on day 8 in the later stage medium with 100 ng mL–1 activin A was significantly higher than the control group [22.4% (54/264) v. 34.7% (76/233); P < 0.05]. Hatching rate of blastocyst on day 8 was significantly higher in the presence of 100 ng mL–1 activin A in the later stage culture medium compared with the control group [9.3% (5/54) v. 22.4% (17/76); P < 0.05]. In Exp. 2, the relative expression of 3 genes (Na/KATPase, E-cad, Glut-1) related to blastocyst hatching and implantation was analyzed. The relative abundance (ratio to GAPDH mRNA) of gene transcripts in blastocysts was measured by conventional semi-quantitative reverse transcription-polymerase chain reaction. The expression level of the Na/K ATPase, E-cad, and Glut-1 gene were higher in the presence of activin A in the culture medium compared with the control group. In conclusion, this study suggests that activin A during the later stage of in vitro bovine embryo development can enhance the developmental competence of preimplantation embryos, increase the hatching rate, and affect expression level of genes related to hatching and implantation in defined culture medium. This study was financially supported by KOSEF (grant ? M10625030005-07N250300510) and the Korean MOE, through the BK21 program for Veterinary Science.

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Effect of cysteamine supplementation during in vitro culture of early stage bovine embryos on blastocyst rate and quality

Acta Veterinaria Brno ◽

10.2754/avb201281030229 ◽

2012 ◽

Vol 81 (3) ◽

pp. 229-234 ◽

Cited By ~ 4

Author(s):

Martina Lojkic ◽

Iva Getz ◽

Marko Samardžija ◽

Mario Matkovic ◽

Goran Bacic ◽

...

Keyword(s):

Inner Cell Mass ◽

Culture Media ◽

Cell Mass ◽

Cell Number ◽

Total Cell ◽

Hatching Rate ◽

Bovine Embryos ◽

Total Cell Number ◽

Oviductal Fluid

The aim of this study was to evaluate whether the addition of cysteamine to the in vitro culture media enhances the yield, hatching rate, total cell number and inner cell mass/total cell number ratio of bovine embryos. A total of 933 bovine oocytes collected from ovaries of 60 slaughtered donors were subjected to in vitro maturation and in vitro fertilization. Following fertilization, embryos were cultured in synthetic oviductal fluid without glucose. After 24 h embryos were transferred into synthetic oviductal fluid with 1.5 mM glucose and 0 (control), 50, 100 and 200 µM of cysteamine. After 48 h, the embryos were transferred into synthetic oviductal fluid with glucose but without cysteamine and cultured until Day 9. The number of cleaved embryos on Day 2, the total number of blastocysts on Day 7 and the number of hatched blastocysts on Day 9 were calculated. Differential staining of inner cell mass and trophectoderm cells of blastocysts were performed on Day 7 and Day 9 of in vitro culture. Supplementation of in vitro culture media with 100 µM cysteamine increased the blastocyst yield (P < 0.05) without affecting the hatching rate. Furthermore, the embryos cultured in the presence of 100 µM cysteamine had significantly higher number of inner cell mass cells (P < 0.05) and the proportion of inner cell mass cells (P < 0.05) compared with the controls. The results of the present study demonstrated that the addition of 100 µM cysteamine to the in vitro culture media improved blastocyst production rate and enhance embryo quality, which could lead to the improvement of the in vitro culture system for bovine embryos.

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106 EFFECT OF GLYCEROL AND ETHYLENE GLYCOL ON THE DEVELOPMENT OF IN VITRO BOVINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab106 ◽

2006 ◽

Vol 18 (2) ◽

pp. 161

Author(s):

A. C. Nicacio ◽

R. Simões ◽

M. A. Peres ◽

J. S. A. Gonçalves ◽

M. E. O. D'Ávila Assumpção ◽

...

Keyword(s):

Ethylene Glycol ◽

Cell Monolayer ◽

Control Group ◽

Hatching Rate ◽

Bovine Embryos ◽

Embryo Viability ◽

Chi Square ◽

Chi Square Test ◽

Hatching Rates

The aim of this study was to evaluate the viability of in vitro-produced bovine embryos after exposure to different cryoprotectant solutions and cryopreservation. Bovine ovaries were collected at slaughterhouse and oocytes were matured, fertilized, and cultured in vitro. The embryos were co-cultured on a granulosa cell monolayer in SOF + 5% FCS and nonessential amino acids. In Experiment 1, expanded blastocysts were exposed to 10% ethylene glycol (EG) solution for 10 min (Group EG) or to 10% EG solution for 10 min and to 20% EG + 20% glycerol (Gly) solution for 30 s (Group EG/Gly). Cryoprotectants were diluted with PBS + 0.2% BSA + 0.3 M sucrose and PBS + 0.2% BSA solutions, both for 3 min, and the hatching rate was evaluated after culture. In Experiment 2, after exposure, EG Group was cryopreserved by slow freezing procedure (1.2�C/min) and EG/Gly Group was vitrified on nitrogen vapor for 2 min. After thawing, cryoprotectants were diluted using PBS + 0.2% BSA + 0.3 M sucrose and PBS + 0.2% BSA solutions, both for 3 min; hatching rate was evaluated after culture. As a control group for both experiments, non exposed embryos were cultured and evaluated for hatching rate. In Experiment 1, the hatching rates were 59.72% (43/72) for control, 62.38% (63/101) for EG, and 69.00% (69/100) for EG/Gly groups. In Experiment 2, hatching rates were 59.72% (43/72) for control, 15.22% (7/46) for EG, and 0.00% (0/46) for EG/Gly groups. Results were analyzed by chi-square test. In Experiment 1, no differences were observed among groups (P > 0.05) and in Experiment 2, differences were observed among control, EG, and EG/Gly groups (P < 0.05). In conclusion, the cryoprotectants were not deleterious to the development of in vitro bovine embryos until hatching, but the cryopreservation procedures decreased embryo viability. This work was supported by FAPESP 04/05335-1.

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237 CHROMOSOMAL ABNORMALITIES IN IN VITRO-PRODUCED EARLY BOVINE EMBRYOS: USE OF HOMOLOGOUS FOLLICULAR FLUID SUPPLEMENTATION IN THE OOCYTE MATURATION MEDIA

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab237 ◽

2013 ◽

Vol 25 (1) ◽

pp. 266

Author(s):

S. Demyda-Peyrás ◽

M. Hidalgo ◽

J. Dorado ◽

L. De Luca ◽

E. Genero ◽

...

Keyword(s):

Oocyte Maturation ◽

Follicular Fluid ◽

Chromosomal Abnormalities ◽

Culture Media ◽

Control Group ◽

Bovine Embryos ◽

Humid Atmosphere ◽

Serum Replacement ◽

Laboratory Techniques

Chromosomal abnormalities were described as a possible cause of embryo failures in cattle, even more so when they are in vitro produced. It has been widely demonstrated that the post-fertilization culture environment affects the frequency of blastomeric aneuploidies. However, the literature concerning the effect of the oocyte maturation techniques on in vitro-produced embryos is scarce. The aim of this study was to determine the effect of homologous bovine follicular fluid (BFF) as a possible replacement for commercial sera in the appearance of chromosomal abnormalities on early IVF-produced embryos. Cumulus–oocyte complexes obtained from ovarian puncture were maturated in modified bicarbonate-buffered TCM-199 media, supplemented with glutamine, sodium pyruvate, FSH, LH, oestradiol, and gentamicin in three different groups. Two treatments were performed: 1) base media supplemented with BFF, obtained aseptically from follicles between 4 and 10 mm in diameter (10 and 20%), and 2) a control group, with base media supplemented with 10% FCS without BFF. After 20 h of culture at 38.5°C in a 5% CO2 humid atmosphere, cumulus–oocyte complexes from both treatments were fertilized in IVF media and then cultured for 72 h in SOF media, according to our laboratory techniques. A total of 152 early embryos were cytogenetically evaluated following our standard laboratory techniques. Developed embryos were individually fixed onto a slide, disaggregated into blastomeres with acetic acid, and stained with Giemsa solution. Chromosomal numerical abnormalities were evaluated in each embryo by direct observation at 1250× magnification using a bright field microscope. Results were statistically compared among treatments by the expected proportion test. No significant differences (P > 0.05) were found between different culture media on the percentages of normal diploid embryos or each kind of numerical abnormality. According to our results (Table 1), the use of homologous follicular fluid as a supplement on the oocyte maturation media did not influence the appearance of abnormal complements in the embryos produced compared with the use of FCS. In conclusion, homologous follicular fluid may be considered a valid serum replacement in the maturation media on IVF-produced bovine embryos. Table 1.Analysis of chromosomal complements of Day 3 in vitro-produced bovine embryos derived from oocytes maturated in culture media with different serum supplementation1

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149 Uptake of C18:0 from culture media during invitro culture decreases cryosurvival rates of bovine embryos

Reproduction Fertility and Development ◽

10.1071/rdv33n2ab149 ◽

2021 ◽

Vol 33 (2) ◽

pp. 183

Author(s):

I. Bertijn ◽

B. M. Gadella ◽

H. T. A. van Tol ◽

A. Rijneveld ◽

P. L. A. M. Vos ◽

...

Keyword(s):

Embryo Culture ◽

Negative Binomial ◽

Culture Media ◽

Negative Binomial Regression ◽

Terminal Deoxynucleotidyl Transferase ◽

Bovine Embryos ◽

Multiple Ovulation ◽

Binomial Regression ◽

Post Hoc ◽

And Control

Cryosurvival of invitro-produced bovine embryos is lower than that of invivo-produced embryos, limiting their usability in the field. Previous work showed that the embryo’s lipid composition relates to its quality and cryosurvival. The present study aimed to investigate the effects of free fatty acid (FA) additions to embryo culture media during the oviduct phase of embryonic development on the improvement of cryosurvival of invitro-produced blastocysts. Bovine cumulus–oocyte complexes (n=1675, 3 replicates) were harvested from slaughterhouse ovaries, invitro matured (23h), and subsequently fertilized (18–20h). Embryos were cultured until Day 5 post-fertilization in synthetic oviductal fluid (SOF) with (1) bovine serum albumin (BSA; control, n=253); (2) delipidified BSA (>96% FA free, n=460); (3), delipidified BSA complexed with 25µM unsaturated oleic acid (C18:1, n=455); or (4) with saturated stearic acid (C18:0, n=507) with a stoichiometry of 5:1. At Day 5, SOF was refreshed and embryos were cultured without supplementation. At Days 7 and 8, blastocyst rates were determined. Blastocysts were LD540 stained for lipid droplets (LD), and the LD number and size were analysed by ANOVA. Cryosurvival%, defined by re-expansion of the blastocoel, was analysed by logistic regression. Additionally, fresh and frozen–thawed blastocysts were stained for apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labelling, TUNEL), necrosis (EthD-1), and DNA (Hoechst 33342) and analysed using negative binomial regression. Group differences were tested using a post hoc Tukey test. Statistical analysis was performed in R Studio (version 3.4.2), and P-values <0.05 were considered significant. FA-free culture delayed and decreased blastocyst rates to 19% compared with any FA supplementation: 35%, 27%, and 29% for control, C18:1, and C18:0, respectively (P<0.04). Cryosurvival doubled with culture in FA-free SOF (58%) and C18:1 (63%) compared with C18:0 (23% P=0.01 and P<0.01, respectively) and control (29%; P=0.15 and P<0.02, respectively), approaching cryosurvival rates of donated multiple ovulation embryo transfer (MOET) embryos (CRV Company; 67%). C18:0 exposure also resulted in elevated necrosis levels after cryopreservation (5–8% of cells), compared with all groups (2–4%; P<0.016). The LD size increased in blastocysts cultured with C18:1 compared with all groups (3.1µm2 vs. 2.4–2.7µm2; P<0.016). C18:0 addition to SOF during embryo culture invitro, as well as a mixture of FA in control SOF (including C18:0), caused a reduction of ∼50% in blastocyst cryosurvival compared with MOET blastocysts. Interestingly, either C18:1 addition or the complete omission of FA in SOF during embryo culture invitro restored the cryosurvival of blastocysts to the level of MOET blastocysts. Currently, we are investigating whether the free FA conditions in the oviduct endorse the distinct quality between invivo- and invitro-produced embryos.

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

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab70 ◽

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>0.05) and the production of blastocysts was lower in the DHA group (P<0.05). The number of cells per embryo was higher (P<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>0.05) compared with the control group. Phosphatidylcholine [phosphatidylcholine (32: 0)+H] was more abundant (P<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.

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Effect of the cryopreservation method used, the embryonic stage and the use of conjugated linoleic acid isomers on the cryotolerance of in vitro-produced bovine embryos

Semina Ciências Agrárias ◽

10.5433/1679-0359.2015v36n6supl2p4297 ◽

2015 ◽

Vol 36 (6Supl2) ◽

pp. 4297

Author(s):

Luciana Simões Rafagnin Marinho ◽

Lain Uriel Ohlweiler ◽

Marcos Henrique Barreta ◽

Paulo Bayard Dias Gonçalves ◽

Joana Claudia Mezzalira ◽

...

Keyword(s):

Linoleic Acid ◽

Embryonic Development ◽

Mrna Expression ◽

Cell Number ◽

Development Rate ◽

Control Group ◽

Hatching Rate ◽

Bovine Embryos ◽

Blastocyst Development

Conjugated linoleic acid (CLA) might be able to improve the cryotolerance of in vitro-produced (IVP) embryos. The effect of two CLA isomers on the cryotolerance of bovine IVP embryos, as well as that of the stage of embryonic development and the method used for cryopreservation was evaluated by three experiments. In Experiment 1, oocytes (n = 3,917) were fertilized in vitro and cultured with 0, 50, 100, or 200 ?M trans-10, cis-12 (t10, c12 CLA). In Experiment 2, fertilized oocytes (n = 2,131) were cultured with 100 ?M t10, c12 or cis-9, trans-11 (c9, t11 CLA), or a combination of both isomers. The embryos were vitrified at the blastocyst (BL) or the expanded blastocyst (EB) stage. In Experiment 3, oocytes (n = 1,720) were fertilized and cultured with or without 100 ?M t10, c12 CLA, and the blastocysts were vitrified or frozen. Blastocyst development rate as well as the rates of re-expansion and hatching after thawing was recorded. Moreover, the mean cell number and mRNA expression of acetyl-CoA carboxylase (ACC1) and stearoyl-CoA desaturase (SCD1) as well as fatty acid synthase (FASN) multienzyme complex were determined. In Experiment 1, the highest concentration of t10, c12 CLA that did not reduce blastocyst development rate was 100 ?M. In Experiment 2, the rates of re-expansion and hatching among the EBs obtained through IVP after supplementation with t10, c12 CLA (73.1% and 57.7%), with c9, t11 CLA (80.0% and 68.6%), with the combination (78.3% and 52.2%), and with the control group (85.4% and 58.3%) were similar. At the BL stage, the rates of re-expansion and hatching were lower than those at the EB stage, and CLA combination allowed a hatching rate (8.0%) lower than that observed in the control group (40.0%). In Experiment 3, the hatching rates for vitrified EBs (vitrified control; 67.4%) and vitrified CLA EBs (65.8%) were higher than those obtained for frozen EBs, exposed (13.3%) or not exposed (28.6%) to CLA. In addition, in Experiment 3, the hatching rate was higher at the EB stage in vitrified groups, while the rates of BL and EB were similar in frozen groups, thus proving that vitrification was more efficient than freezing for IVP bovine embryos. In Experiment 3, CLA isomer t10, C12 did not influence the embryonic cell number or mRNA expression of ACC1 and SCD1 enzymes, but decreased the mRNA expression of FASN. In conclusion, 100 ?M CLA did not affect subsequent embryonic development. However, neither CLA isomer improved the cryotolerance of IVP bovine embryos.

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