311 EFFECT OF HEAT SHOCK DURING THE FERTILIZATION AND CULTURE PERIOD ON BOVINE EMBRYO DEVELOPMENT AND THE PROTECTIVE EFFECT OF BROWN ALGAE PHLOROTANNINS

2007 ◽  
Vol 19 (1) ◽  
pp. 271
Author(s):  
M. Sakatani ◽  
K. Nagayama ◽  
K. Kobayashi ◽  
K. Kobayashi ◽  
K. Morishita ◽  
...  

It is widely reported that heat stress adversely affects the reproductive function of cattle, such as ovarian functions, fertilization, and embryo development. In a previous study, we reported that heat shock decreases embryo development and increases intracellular reactive oxygen species (ROS). Also some antioxidants increase embryo development under conditions of heat shock by reducing the intracellular ROS. Phlorotannins extracted from brown alga are known as a strong antioxidant. However, heat shock and the antioxidative effect of phlorotannins on fertilization and embryo development has not been carefully studied. In the present study, we investigated the effect of heat shock on fertilization and early embryo development, and the protective effect of phlorotannins on embryo development under conditions of heat shock. In all experiments, bovine oocytes were collected from the local abattoir and matured with TCM-199 (Experiment 1). Bovine sperm drops prepared by BO solution were pretreated at 41�C for 4 h with or without 100 ng mL-1 of phlorotannins. After heat shock, oocytes were fertilized in drops at 38.5�C for 6 h. Putative zygotes were cultured with CR1 + 5% FCS at 38.5�C. The percentages of embryos cleaved and developed to blastocysts were evaluated on Days 2 and 8. The percentages of embryo division and development were compared with embryos fertilized with sperm pretreated at 38.5�C for 4 h (Experiment 2). Oocytes were fertilized at 41�C for 6 h with or without 100 ng mL-1 of phlorotannins. Putative zygotes were cultured with CR1 + 5% FCS at 38.5�C. On Days 2 and 8, the percentages of cleaved embryos and those developed to blastocysts were evaluated and compared with embryos fertilized at 38.5�C for 6 h (Experiment 3). Oocytes were fertilized at 38.5�C for 6 h. Putative zygotes were cultured with or without 10 ng mL-1 of phlorotannins in CR1 + 5% FCS. On Day 2, embryos were exposed to 41�C for 6 h as heat shock. After heat shock, embryos were cultured at 38.5�C to Day 8, and embryo development was evaluated. The percentages of embryo development were compared with those for embryos cultured at 38.5�C through to Day 8 without phlorotannins. Mean values were compared by Student's t-test. There were no significant differences in the percentages of embryo cleavage among all experiments. The percentages of embryo development were significantly (P < 0.05) decreased by heat shock in all experiments [Experiment 1: 45.0 vs. 29.2%; Experiment 2: 25.1 vs. 6.6%; Experiment 3: 28.6 vs. 15.3% (control vs. heat shock)]. In contrast, the addition of phlorotannins to the fertilization or culture media tended to improve the embryo development (Experiment 1: 41.9%; Experiment 2: 15.1%; Experiment 3: 22.2%). These results indicate that heat shock affects not only embryo development but also fertilization. And under conditions of heat shock, the addition of phlorotannins would be effective in improving embryo development from fertilization to development.

Author(s):  
Yulia N Cajas ◽  
Karina Cañón-Beltrán ◽  
Carolina Núñez-Puente ◽  
Alfonso Gutierrez-Adán ◽  
Encina M González ◽  
...  

Abstract During preimplantational embryo development, PI3K/AKT regulates cell proliferation and differentiation and nobiletin modulates this pathway to promote cell survival. Therefore, we aimed to establish whether, when the AKT cascade is inhibited using inhibitors III and IV, nobiletin supplementation to in vitro culture media during the minor (2 to 8-cell stage, MNEGA) or major (8 to 16-cell stage, MJEGA) phases of EGA is able to modulate the development and quality of bovine embryos. In vitro zygotes were cultured during MNEGA or MJEGA phase in SOF + 5% FCS or supplemented with: 15 μM AKT-InhIII; 10 μM AKT-InhIV; 10 μM nobiletin; nobiletin+AKT-InhIII; nobiletin+AKT-InhIV; 0.03% DMSO. Embryo development was lower in treatments with AKT inhibitors, while combination of nobiletin with AKT inhibitors was able to recover their adverse developmental effect and also increase blastocyst cell number. The mRNA abundance of GPX1, NFE2L2, and POU5F1 was partially increased in 8- and 16-cell embryos from nobiletin with AKT inhibitors. Besides, nobiletin increased the p-rpS6 level whether or not AKT inhibitors were present. In conclusion, nobiletin promotes bovine embryo development and quality and partially recovers the adverse developmental effect of AKT inhibitors which infers that nobiletin probably uses another signalling cascade that PI3K/AKT during early embryo development in bovine.


2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
A Munuer. Puigvert ◽  
V. Montalv Pallès ◽  
J Mass. Hernáez ◽  
A García-Faura ◽  
B Marquè. López-Teijón ◽  
...  

Abstract Study question Have multinucleation and reverse cleavage any effect on embryo development and clinical outcomes on IVF treatments? Summary answer Embryos capable of repairing dysmorphisms and developing up to blastocyst stage keep intact their ability to become healthy babies. What is known already Time-lapse systems allow IVF laboratories to perform in-depth analysis of embryo development using the continuous monitoring tool. Some events that are impossible to detect with conventional morphologic evaluation, such as reverse cleavage or multinucleation, can be detected using time-lapse. Even though the low scientific evidence, the presence of these events is considered a negative factor when the embryo quality assessment is performed. However, it has been described the possibility that embryos have self-repair intrinsic methods. Study design, size, duration Retrospective study including data from 3,577 cycles with 21,274 embryos cultured until blastocyst stage using one-step culture media in time-lapse incubators (Embryoscope, Vitrolife) up to day 5/6 between 2014 and 2019. Participants/materials, setting, methods Three embryo groups were considered: Control group, embryos without multinucleation or reverse cleavage (CG; n = 16,897); Multinucleation group, embryos with at least one blastomere multinucleated on D + 2/3 (MNC; n = 3,879) and Reverse Cleavage group, embryos undergoing complete fusion of two blastomeres on D + 2/3 (RC; n = 498). Single embryo transfer was performed on blastocyst stage. Clinical outcome rates were compared between groups and analyzed by Chi-square test. Main results and the role of chance As published by other groups, the 2.3% of our embryos showed at least one reverse cleavage event and we observed multinucleation in the 18.2% of the embryos. Blastocyst rate of dysmorphism groups was significantly lower (p < 0.05) than Control group (MNC=20.0%; RC = 27.7%; CG = 58.0%). Once transferred, MNC and RC evolutive embryos showed significantly lower pregnancy (MNC=47.9%; RC = 46.8%; CG = 60.8%; p < 0.05) and clinical pregnancy rates (MNC=39.4%; RC = 40.4% CG = 50.6%; p < 0.05) than the Control group (p < 0.05). However, during the post-implantational development the negative effect of dysmorphisms disappears, reaching values of live birth rate comparable to the Control group (MNC=28.3%; RC = 31.9% CG = 33.8%; p = 0.17). These results prove the importance of blastocyst culture and the inherent capability of the embryos to overcome some abnormal dynamics as multinucleation and reverse cleavage. Thus, these embryos showing the poor-prognosis events can be considered for transfer or vitrify. Limitations, reasons for caution There is a wide difference on sample size between groups despite the fact that the statistical analysis considers that into account. There are some ongoing pregnancies in all groups. Wider implications of the findings: When analyzing the development of embryos undergoing reverse cleavage and multinucleation, we hypothesize that these embryos could be showing a self-correction mechanism for some type of error detected. Embryos capable of repairing and developing up to blastocyst stage keep intact their ability to become healthy babies. Trial registration number Not applicable


2019 ◽  
Vol 20 (21) ◽  
pp. 5305 ◽  
Author(s):  
Canha-Gouveia ◽  
Paradela ◽  
Ramos-Fernández ◽  
Prieto-Sánchez ◽  
Sánchez-Ferrer ◽  
...  

The improvement of the embryo culture media is of high relevance due to its influence on successful implantation rates, pregnancy, neonatal outcomes, and potential effects in adult life. The ideal conditions for embryo development are those naturally occurring in the female reproductive tract, i.e., the oviductal and uterine fluids. To shed light on the differences between chemical and natural media, we performed the first comparative study of the low abundance proteins in plasma, uterine, and oviductal fluid collected, simultaneously, from healthy and fertile women that underwent a salpingectomy. The rationale for this design derives from the fact that high-abundant proteins in these fluids are usually those coming from blood serum and frequently mask the detection of low abundant proteins with a potentially significant role in specific processes related to the embryo–maternal interaction. The proteomic analysis by 1D-nano LC ESI-MSMS detected several proteins in higher amounts in oviductal fluid when compared to uterine and plasma samples (RL3, GSTA1, EZRI, DPYSL3, GARS, HSP90A). Such oviductal fluid proteins could be a target to improve fertilization rates and early embryo development if used in the culture media. In conclusion, this study presents a high-throughput analysis of female reproductive tract fluids and contributes to the knowledge of oviductal and uterine secretome.


2007 ◽  
Vol 19 (1) ◽  
pp. 262 ◽  
Author(s):  
I. Dimitriadis ◽  
E. A. Rekka ◽  
E. Vainas ◽  
G. S. Amiridis ◽  
C. A. Rekkas

The substrates used in in vitro embryo production (IVP) mimic the in vivo fluids in which oocytes mature, oocytes are fertilized, and the early embryos develop (follicular and oviductal fluid). It is well established that oxidative stress negatively affects in vitro culture (IVC) outcomes. Guaiazulene (G) is a component of chamomile species oil with known antioxidant properties. In the present study, all IVP media were modified by the addition of G solutions so that the former exhibited a total protection against induced lipid peroxidation (TPaLP) similar to that of the respective in vivo environment. The IVP outcomes were then compared between G-processed and control oocytes. Bovine preovulatory follicular (BF) and oviductal (BO) fluid samples were collected from 10 Holstein 4- to 5-year-old cows in estrus. TPaLP was assessed according to the samples' ability to inhibit rat hepatic microsomal lipid peroxidation, by determination of the 2-thiobarbituric acid reactive material. TPaLP (mean % � SEM) of the BF and BO were 70.63 � 10.03 and 16.33 � 4.33, respectively, whereas those of the IVP [in vitro-matured (IVM), in vitro-fertilized (IVF), and IVC] media were lower (17.94 � 1.66, -1.82 � 0.78, and 14.57 � 1.26, respectively). TPaLP of the 0.1 mM G-modified IVP medium increased to 67.2 � 5.85, 19.98 � 2.49, and 69.19 � 6.22, respectively. A total of 2041 class A oocytes were used. The proportion of cleavage, early embryo development (embryos with more than 4 cells), or both after IVP (18 h IVM–5% CO2 in air, and 18 h IVF, 48 h IVC–5% CO2, 10% O2, 85% N) in the presence of G (n = 1237) during each of the IVP phases or any possible combination of IVP phases was compared with the respective control (C, n = 804). Statistical analysis was performed by a chi-squared test; P < 0.05 was considered significant. G improved cleavage and embryo development rates when present during IVM (79.4 and 57.8% vs. 64.5 and 38.2% for C) or both IVM and IVC (78.0 and 60.7% vs. 57.8 and 36.5%, respectively). When present only during 18 h of IVF, G had no effect on embryo production. However, an increased embryo development rate resulted from the combined exposure to G during IVF and IVM (56.4 vs. 29.6%), during IVF and IVC (55.3 vs. 35.5%), or at all IVP phases (56.6 vs. 34.9%). The latter effect resembled the one obtained after G addition only to the IVC medium (62.5 vs. 39.7%, respectively). We concluded that the addition of G to IVP substrates, at concentrations that mimic the in vivo TPaLP conditions, could promote bovine IVP efficiency.


2009 ◽  
Vol 21 (1) ◽  
pp. 104
Author(s):  
J. T. Aaltonen ◽  
K. J. Mattson ◽  
N. M. Loskutoff

As described in the IETS Manual (Stringfellow and Seidel, 1995), and endorsed by the OIE, trypsin can be used (for specific pathogens and livestock) to effectively remove certain infectious agents from in vivo-derived embryos for international transport. Because of the multimillion-dollar AI industry for livestock, the OIE has encouraged more research in developing similar decontamination techniques for semen as an added safeguard to animal quarantine for the prevention of disease transmission. Most or all of the earlier studies on embryos used a porcine pancreatic-derived trypsin. Because of more stringent guidelines from international regulatory agencies on the use of animal products, several serine protease recombinants are now available. Previous experiments comparing the porcine pancreatic extract with a recombinant bovine sequence trypsin developed in corn resulted in no statistical difference in cleavage or morula/blastocyst rates. (Mattson et al. 2008 Theriogenology 69, 724–727). An additional in vivo study treating bovine sperm with a yeast-derived human-sequence trypsin resulted in significantly more transferable-quality embryos after the AI of superovulated cows as compared with sperm not treated with trypsin (Blevins et al. 2008 Reprod. Fertil. Dev. 20, 84). The goal of this experiment was to examine the in vitro development of bovine embryos produced from sperm treated with a recombinant trypsin found in a commercially available density gradient centrifugation (DGC) product (Bovipure, Nidacon, Sweden) compared with DGC without trypsin. Oocyte aspiration, maturation, fertilization, and embryo culture were performed using standard methods in 5 replications (n = 2220 oocytes). Semen was collected and pooled from 2 Bos taurus bulls and frozen in an egg-yolk cryodiluent (Biladyl, Minitube). The semen was processed using Bovipure DGC composed of 2 mL of 40% colloid of silane-coated silica particles containing either a yeast-derived human sequence recombinant trypsin containing no animal by-products (n = 1126 oocytes) or the same colloid without trypsin as the control group (n = 1094 oocytes). Both 40% concentrations were layered over 2 mL of an 80% concentration of the same colloid without any additives. The density gradients were centrifuged at 300g for 20 min, after which time the pellets were washed in 5 mL of prewarmed TL Hepes solution (Cambrex) and centrifuged at 500g for 10 min. The resulting sperm pellets were then resuspended in a volume calculated to provide 1 × 106 sperm mL–1, to be used for in vitro inseminations. Results were compared using a 2-tailed unpaired t-test. Cleavage rates for the trypsin-treated sperm (n = 969, 35.8%) and the control (n = 950, 44.3%) groups were not statistically different (P = 0.20). Although more embryos reached the morula to blastocyst stages in the control group (n = 421, 61.0%) than in the trypsinized group (n = 347, 54.7%), these differences also were not statistically significant (P = 0.85). In conclusion, trypsinized Bovipure DGC of sperm before insemination showed no detrimental effects on IVF-derived bovine embryo development.


2014 ◽  
Vol 26 (1) ◽  
pp. 157
Author(s):  
S. Demyda-Peyrás ◽  
M. Hidalgo ◽  
J. Dorado ◽  
M. Moreno-Millan

Chromosomal numerical abnormalities (CNA) were described as a major cause of developmental failures in in vitro-produced (IVP) embryos. It has been described that CNA are influenced by the post-fertilization culture environment of the embryo. Furthermore, it was demonstrated that the use of different culture media affects the CNA rates. The addition of granulosa cells during early embryo development is a well-known procedure to simplify the culture of bovine IVP and cloned embryos. This technique avoids the use of culture environments saturated with N2 (tri-gas chambers). The aim of this study was to determine the effect of the addition of granulosa cells in the chromosomal abnormalities of IVP cattle embryos. Cumulus–oocyte complexes (COC) were matured in TCM-199 medium, supplemented with glutamine, sodium pyruvate, FSH, LH, oestradiol, and gentamicin during 20 h at 38.5°C in a 5% CO2 humid atmosphere. Subsequently, matured oocytes were fertilized in IVF-TALP medium using 1 × 106 spermatozoa mL–1, selected through a Percoll gradient centrifugation. After fertilization, zygotes were divided in 2 groups and cultured in TCM-199 medium for 48 h, with (TCM-GC) or without (TCM) the addition of 1 × 106 granulosa cells. These cells were obtained by centrifuging and washing the follicular fluid remaining from searching dishes and adjusted to the working concentration. After culture, a total of 106 early embryos (72 hpi) were cytogenetically evaluated following our standard laboratory techniques. Embryos showing normal development were individually fixed onto a slide, disaggregated into blastomeres with acetic acid, and stained with Giemsa solution. Chromosomal numerical abnormalities were evaluated by direct observation at 1250× magnification in a brightfield microscope. Percentage of normal diploid embryos (D) and abnormal haploid (H), polyploid (P), or aneuploid (A) embryos were determined. Results were statistically compared between treatments using a Z test for proportions. Results were: D = 81.4%, H = 7.2%, P = 7.2%. and A = 3.6% in TCM and D = 84.3%, H = 3.9%, P = 9.8%, and A = 1.9% in TCM-GC. No significant differences (P > 0.05) were found between culture media in the chromosomal abnormality rates. According to our results, the use of somatic cells in co-culture during embryo development did not influence the appearance of abnormal complements in the produced embryos. This would allow the use of GC as a potential complement to simplify the techniques used in the culture of bovine embryos until Day 3.


1999 ◽  
Vol 1999 ◽  
pp. 2-2 ◽  
Author(s):  
M. Kuran ◽  
M.E. Staines ◽  
G.J. McCallum ◽  
A.G. Onal ◽  
T.G. McEvoy

Ovine embryos produced in synthetic oviduct fluid (SOF) medium or in coculture with granulosa cell monolayers supplemented with low (A; 120 μmol/l) and high (B; 190 μmol/l) ammonia-producing steer sera caused different degrees of fetal oversize (Carolan et al., 1998). The objective of the present study was to determine whether the effects on fetal growth induced by these sera were associated with alterations in early embryo development.A total of 911 bovine oocytes, used in 8 replicates to test the effect of three culture treatments on embryo development, were matured and fertilized in vitro (IVF= Day 0). Presumptive zygotes were allocated on Day 1 to culture in SOF supplemented with 10% v/v steer serum (SOF+A, n=308; SOF+B, n=302) or with amino acids plus 0.4% w/v crystalline BSA (SOFaaBSA, n=301). All cultures were in 20 μl droplets under oil (38.5°C; 5% CO2, 5% O2; 4 zygotes per drop) and droplets were renewed every 48 h. Cleavage rate was recorded on Day 3. On Days 7 and 8, blastocyst yields, grade 1 and 2 blastocysts, their cell numbers (by staining with Hoechst 33342) and their stage and diameter were determined.


Reproduction ◽  
2004 ◽  
Vol 128 (3) ◽  
pp. 281-291 ◽  
Author(s):  
Andrea Jurisicova ◽  
Beth M Acton

Human preimplantation embryo development is prone to high rates of early embryo wastage, particularly under currentin vitroculture conditions. There are many possible underlying causes for embryo demise, including DNA damage, poor embryo metabolism and the effect of suboptimal culture media, all of which could result in an imbalance in gene expression and the failed execution of basic embryonic decisions. In view of the complex interactions involved in embryo development, a thorough understanding of these parameters is essential to improving embryo quality. An increasing body of evidence indicates that cell fate (i.e. survival/differentiation or death) is determined by the outcome of specific intracellular interactions between pro- and anti-apoptotic proteins, many of which are expressed during oocyte and preimplantation embryo development. The recent availability of mutant mice lacking expression of various genes involved in the regulation of cell survival has enabled rapid progress towards identifying those molecules that are functionally important for normal oocyte and preimplantation embryo development. In this review we will discuss the current understanding of the regulation of cell death gene expression during preimplantation embryo development, with a focus on human embryology and a discussion of animal models where appropriate.


2004 ◽  
Vol 16 (9) ◽  
pp. 12
Author(s):  
J. G. Thompson ◽  
K. L. Kind

It has long been recognised that energy substrate supply and metabolism are key determinants of early embryo development during in vitro culture. Recently it has been revealed that exposure to suboptimal metabolic environments during early embryo development can 'programme' subsequent development, leading to perturbed fetal development. For example, amino acid uptake profiles during early cleavage have been found to predict subsequent embryo development and potentially implantation success. However, the by-product of amino acid metabolism, ammonium, has also been found to significantly alter development, possibly through perturbed methylation of imprinted genes. Our own work has focussed on the role of oxygen availability and subsequent embryo development. Somatic cells respond to changing oxygen concentration by altering intracellular REDOX state (the balance between oxidative and reductive power within a cell), which in turn can alter transcription via REDOX-sensitive transcription factor activity. Furthermore, oxygen is known to have direct effects on transcriptional activity via the hypoxia-inducible factors (HIFs), transcription factors whose stability and DNA-binding activity are directly regulated by pO2, in particular under hypoxic conditions. Using a mouse model, we have demonstrated that reducing pO2 from 50�mmHg to 15�mmHg during the compaction and blastulation periods alone significantly alters expression patterns of oxygen-sensitive genes (such as glucose transporters), without significantly altering developmental progression to the blastocyst stage. Following transfer, embryos cultured under 15�mmHg O2, despite similar implantation rates, produced fewer viable and lighter fetuses than in vivo-derived control embryos or those cultured in either atmospheric or 50�mmHg pO2. This demonstrates that mouse embryos are sensitive to changes in their metabolic state during the post compaction period and that operating through causal pathways, the environment during this period of development can significantly affect subsequent developmental potential. Ironically, bovine embryo development appears to benefit under a low O2 concentration. Furthermore, HIF protein stability appears to differ between the two species, which may be the underlying cause for the differences in gene expression and developmental competence.


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