Peroxisome proliferator-activated receptor delta-PPARδ agonist (L-165041) enhances bovine embryo survival and post vitrification viability.

The effect of L-165041 (PPARδ-agonist) on decreasing apoptosis and intracellular lipid content was assessed in fresh and vitrified-warmed in vitro -produced bovine embryos. It was hypothesised that the addition of L-165041 to the culture medium enhances development and cryopreservation. Oocytes were allocated to one of two treatments: control-standard culture medium, or L-165041 added to the medium on day1 with no media change. Ultrastructure, cleavage, and blastocyst rates were evaluated in fresh, and in post-vitrification cultured embryos by optical and electronic microscopy. A subset of fresh embryos were fixed for TUNEL assay and for Sudan-Black-B histochemical staining. Vitrified-warmed embryos were assessed using MALDI-MS technique. Cleavage and blastocyst rates (control 49.4±5.2, L-165041 51.8±4.3) were not influenced by L-165041. The proportion of inner cell mass cells (ICM) was higher in fresh embryos, and the rate of total and ICM apoptosis was lower in L-165041. In warmed-embryos, total and ICM apoptosis was lower in L-165041. The overall hatching rate was higher in L-165041 (66.62±2.83% vs 53.19±2.90%). There was less lipid accumulation in fresh L-165041-embryos. In conclusion, the use of L-165041 is recommended to improve the viability of in vitro -derived bovine embryos.

ELOVL5 Participates in Embryonic Lipid Determination of Cellular Membranes and Cytoplasmic Droplets.

Embryonic lipids are crucial for the formation of cellular membranes and dynamically participate in metabolic pathways. Cells can synthesize simple fatty acids, and the elongation of fatty acids facilitates the formation of complex lipids. The aim of this work was to investigate the involvement of the elongation of very long chain fatty acid enzyme 5 (ELOVL5) in embryonic development and lipid determination. Bovine embryos were produced in vitro using a standard protocol and randomly divided to receive one of three treatments at Day 4: morpholino (Mo) gene expression knockdown assay for ELOVL5 (ELOVL5-Mo), Mo antisense oligonucleotides for the thalassemic ß-globulin human mRNA (technical control Mo), and placebo (biological control). The phenotypes of embryonic development, cell number, ELOVL5 protein abundance, lipid droplet deposits, and lipid fingerprint were investigated. No detrimental effects (p > 0.05) were observed on embryo development in terms of cleavage (59.4 ± 3.5%, 63.6 ± 4.1%, and 65.4 ± 2.2%), blastocyst production (31.3 ± 4.2%, 28.1 ± 4.9%, and 36.1 ± 2.1%), and blastocyst cell number (99.6 ± 7.7, 100.2 ± 6.2, 86.8 ± 5.6), respectively, for biological control, technical control Mo, and ELOVL5-Mo. ELOVL5 protein abundance and cytoplasmic lipid droplet deposition were increased (p < 0.05) in ELOVL5-Mo-derived blastocysts compared with the controls. However, seven lipid species, including phosphatidylcholines, phosphatidylethanolamines, and triacylglycerol, were downregulated in the ELOVL5-Mo-derived blastocysts compared with the biological control. Therefore, ELOVL5 is involved in the determination of embryonic lipid content and composition. Transient translational blockage of ELOVL5 reduced the expression of specific lipid species and promoted increased cytoplasmic lipid droplet deposition, but with no apparent deleterious effect on embryonic development and blastocyst cell number.

Lipid characterization of in vitro-produced bovine embryos with distinct kinetics of development.

Human embryo studies have proposed the use of additional morphological evaluations related to the moment of the first cell divisions as relevant to embryo viability. Nevertheless, there are still not enough data available related to morphokinetic analysis and its relationship with lipid composition in embryos. Therefore, the aim of this study was to address the lipid profile of bovine embryos with different developmental kinetics: fast (four or more cells) and slow (two or three cells) at 40 h post-insemination (hpi), at three time points of in vitro culture (40, 112 and 186 hpi) and compare these to profiles of in vivo embryos. The lipid profiles of embryos were analyzed by matrix-assisted laser desorption ionization mass spectrometry, which mainly detected pools of membrane lipids such as phosphatidylcholine and sphingomyelin. In addition to their structural function, these lipid classes have an important role in cell signalling, particularly regarding events such as stress and pregnancy. Different patterns of lipids in the fast and slow groups were revealed in all the analyzed stages. Also, differences between in vitro embryos were more pronounced at 112 hpi, a critical moment due to embryonic genome activation. At the blastocyst stage, in vitro-produced embryos, despite the kinetics, had a closer lipid profile when compared with in vivo blastocysts. In conclusion, the kinetics of development had a greater effect on the membrane lipid profiles throughout the embryo culture, especially at the 8-16-cell stage. The in vitro environment affects lipid composition and may compromise cell signalling and function in blastocysts.

The normal electrocardiograms in the conscious newborn lambs in neonatal period and its progression.

BACKGROUND: Veterinary cardiology, especially electrocardiography, has shown major advancements for all animal species. Consequently, the number of ovine species used as experimental animals has increased to date. Few studies have been published on ovine systematic electrocardiography, particularly with respect to lamb physiology and neonatology. This study aimed to standardize the values of normal waves, complexes, and intervals of the electrocardiogram (ECG) in clinically Bergamasca healthy neonatal lambs, used as experimental animals. Serial computerized electrocardiography was performed in 10 male and 12 female neonates on the 1st, 7th, 14th, 21st, 28th, and 35th days of age. The following parameters were analyzed: heart rate and rhythm, duration and amplitude of waves, duration of intervals, and heart electrical axis. RESULTS: During the first 35 days of life, (1) the sinusal heart rhythm was predominant, (2) there was a progressive decrease in the heart rate and R and T wave amplitude, and (3) a progressive increase in the PR, QT, and RR intervals. Finally, we confirmed that various components of neonatal evolution were more discernible in the augmented unipolar leads (aVF), which we recommend should be preferentially used in future studies. No significant statistical alterations were observed between males and females in relation to the analyzed parameters. CONCLUSIONS: The information assimilated in this study is anticipated to enhance the diagnosis of multiple congenital heart defects in Bergamasca lambs and could be implemented in studies that use ovine species as experimental models.

Time course of the meiotic arrest in sheep cumulus-oocyte complexes treated with roscovitine.

Temporary meiosis arrest with cyclin-dependent kinases inhibitors has been proposed in order to improve the quality of in vitro matured oocytes. In sheep, however, this phenomenon has been rarely investigated. Therefore, the present study aimed to evaluate the effect of different incubation times with roscovitine on nuclear maturation and cumulus cell expansion of sheep cumulus-oocyte complexes (COCs). For this, COCs were cultured for 0, 6, 12 or 20 h in basic maturation medium (Control) containing 75 µM roscovitine (Rosco). After, they were in vitro matured (IVM) for 18 h in the presence of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). At the end of each treatment, cumulus cell expansion and nuclear maturation were assessed under a stereomicroscope and by Hoechst 33342 staining, respectively. In the Control and Rosco groups, the absence of cumulus cell expansion prevailed at 0, 6, 12 and 20 h. After IVM for 18 h, total cumulus cell expansion in the Rosco treatments was dependent on the exposure time to roscovitine. A significantly high percentage of oocytes treated with roscovitine for 6 h (87%), 12 h or 20 h (65%) were arrested at the germinal vesicle (GV) stage. In contrast, 23% GVBD, 54% metaphase I (MI) and 61% MII oocytes were observed in the Control groups at 6, 12 and 20 h, respectively. In all treatments, a significant percentage of oocytes reached MII after IVM for 18 h. Therefore, roscovitine reversibly arrested the meiosis of sheep oocytes during different culture times with the maximal efficiency of meiotic inhibition reached at 6 h. In addition, reversibility of its inhibitory action on cumulus cells was exposure-time dependent.

In vitro embryos production after oocytes treatment with forskolin.

The inhibition of nuclear maturation allows time for the oocyte to accumulate molecules that are important for embryonic development. Thus, the objective of this work was to evaluate the effect of blocking oocyte meiosis with the addition of forskolin, an efficient inhibitor of nuclear maturation, in in vitro maturation (IVM) medium. Forskolin was added to the IVM medium for 6 h at concentrations of 0.1 mM, 0.05 mM or 0.025 mM, then the oocytes were allowed to mature in drug-free medium for 18 h. The oocytes were assessed for the stage of nuclear maturation, the activity and distribution of mitochondria, oocyte ultrastructure, the number of viable cells and the apoptosis rate. After forskolin treatment, the oocytes were fertilized in vitro and cultured for 7 days. On day 7, the blastocyst rate, the ultrastructure, the number of intact cells and the apoptosis rate of the blastocysts were measured. No differences were observed for the stage of nuclear maturation of the oocyte, the mitochondrial activity and distribution, the blastocyst rate or total number of intact cells. However, a higher rate of apoptosis was observed in the blastocysts produced from oocytes blocked for 6 h with the higher concentration of forskolin (P < 0.05). We conclude that all the experimental groups reached the MII stage after the addition of forskolin and that the highest concentration of forskolin caused cellular degeneration without harming embryo production on the 7th day.

Short and long-term repercussions of the experimental diabetes in embryofetal development.

BACKGROUND: Diabetic pregnancy have increased rates of congenital malformation and neonatal mortality. In vitro studies suggest hyperglycemia associated with diabetes impair embryogenesis but in vivo investigations on maternal hyperglycemic insult and early embryo development are scarce. We evaluated the embryofetal development on experimental diabetes models to assess whether hyperglycemia at preimplantation period impairs the progression of pregnancy. METHODS: Different hyperglycemic intensities were obtained by two experimental diabetes models. Female Sprague Dawley rats received streptozotocin at birth (mild diabetes) or at day 90 of life (severe diabetes). For both diabetic groups hyperglycemia was confirmed 5 days after diabetes induction and the mating was performed around 100 day of life. For preimplantation analysis, embryos were recovered at D4 of pregnancy. Another group of animals was submitted to laparotomy at D21 to assess contents of the uterus and fetal viability. RESULTS: Mild (i) and Severe (ii) diabetes modified the early development. Degenerating embryos percentage was higher compared to control (11%) (i) 30.7%, (ii) 37.3%. Cell number mean dropped according to hyperglycemic intensity (control 30.57, (i) 21.42, (ii) 13.42). Pre and post-implantation loss rates were higher in diabetic groups. The fetal viability also decreased from 96% in the control group to (i) 78.7% and (ii) 80.6%. CONCLUSION: Our results show that during diabetic pregnancy, preimplantation embryos present decreased cell number due to higher apoptosis rates, which are dependent of the hyperglycemic intensity. Moreover, fetal viability was also decreased suggesting that the quality of these embryos at long-term may be questioned.

Crucial surviving aspects for vitrified in vitro-produced bovine embryos.

The objective of the present study was to correlate some parameters (cleavage, blastocyst production, quality degree score, total cell number, fresh apoptosis and lipid content) with embryo survival after cryopreservation. A total of 1727 in vitro-produced bovine blastocysts were used to establish the parameters (mean ± standard error of the mean (SEM)) for cleavage (85.6 ± 0.8), blastocyst production (39.9 ± 1.4), quality degree score (1.6 ± 0.1), total cell number (140.1 ± 2.9), fresh apoptosis (20.8 ± 1.1) and lipid content (21.3 ± 0.8 droplets). On the same way 1316 blastocysts were vitrified for the determination of post-cryopreservation embryo survival (49.4 ± 1.9). Fresh apoptosis rate and total lipid droplets value were correlated (P < 0.05) with embryo survival after cryopreservation (r = 0.91 and r = 0.59; respectively). However, cleavage, blastocyst production, quality degree score and total cell number were not correlated (P > 0.05) with embryo cryotolerance (r = 0.23, r = 0.38, r = 0.22 and r = 0.28; respectively). Therefore, the increased lipid content was moderately correlated with apoptosis in vitrified blastocysts. On the other hand, increased apoptosis in fresh blastocysts was strongly correlated with apoptosis in vitrified blastocysts, which indicated that the apoptosis rate in fresh embryos was a better parameter than the lipid content to predict post-vitrification embryo survival.

Forskolin effect on the cryosurvival of in vitro-produced bovine embryos in the presence or absence of fetal calf serum.

The objective of this study was to assess the viability and cryotolerance of zebu embryos produced in vitro with or without the addition of fetal calf serum (FCS) and forskolin (F). Embryos produced in vivo were used as a control. Presumptive zygotes were cultured in modified synthetic oviductal fluid supplemented with amino acids (SOFaa), bovine serum albumin (BSA) and with (2.5%) or without (0%) FCS. On day 6 of growth, the embryos from each group were divided into treatments with or without 10 µM F to induce embryonic lipolysis, comprising a total of four experimental groups: 2.5% FCS, 0% FCS, 2.5% + F and 0% + F. For vitrification, embryos were exposed to vitrification solution 1 (5 M EG (ethylene glycol)) for 3 min and then transferred to vitrification solution 2 (7 M EG, 0.5 M galactose solution and 18% (w/v) Ficoll 70) before being introduced to liquid nitrogen. The presence of FCS in the culture medium resulted in the production of embryos with a similar rate of damaged cells compared with in vivo-produced embryos. After vitrification, the 2.5% FCS group had a significantly higher rate of damaged cells when compared with the other groups (P < 0.05). The results of this experiment indicated that the omission of FCS and the addition of forskolin do not have deleterious effect on embryo production rates. In addition, embryos produced in the presence of FCS had greater sensitivity to cryopreservation, but this effect was reversed when forskolin was added to the medium, which improved embryo survival without affecting embryo development and quality after vitrification.

Can the addition of Interleukin-13 affect the cryosurvival of bovine embryos?

In this study, we investigated the impact of incorporating Interleukin-13 (IL-13) into the embryonic culture medium and its influence on cryotolerance and cellular viability of vitrified bovine embryos. Two distinct time points for IL-13 supplementation were explored: during the final hours of culture prior to cryopreservation and during the period of recultivation following cryopreservation and warming. Cryosurvival rates, total cell count, and cell viability were assessed using the TUNEL technique to determine the apoptotic percentage. Re-expansion and hatching rates did not show differences among all groups (P > 0.05), and the total cell number was comparable between the treated and control groups (P > 0.05). However, the group that received IL-13 before vitrification exhibited a higher apoptotic percentage (P < 0.05). This suggests that the anti-inflammatory effect of IL-13 may have impacted the embryo's defense capacity against the stress induced by cryopreservation, leading to an increased percentage of apoptosis, although it did not influence the developmental resumption capability.

Effect of IL-10 and TNF-α on the competence and cryosurvival of in vitro produced Bos indicus embryos.

In vitro-produced embryos are constantly exposed to stressful conditions that can lead to the activation of the apoptotic pathway. The nuclear Kappa B factor (NF-κB) is an inflammatory mediator that induces the expression of tumor necrosis factor (TNF-α), a pro-inflammatory cytokine, while interleukin-10 (IL-10), an anti-inflammatory cytokine, inhibits NF-κB activity. This study aimed to investigate the effects of IL-10 and TNF-α on the competence and cryosurvival of in vitro-produced bovine embryos. Embryos were produced in vitro using standard protocols, and Grade I blastocysts were vitrified using the Cryotop method. Non-vitrified and vitrified blastocysts were subjected to the TUNEL assay. In Experiment I, on day 6.5 (156 h post-insemination), the embryos were treated with PBS (control), 50 ng/mL of IL-10, or a combination of 25 ng/mL of TNF-α and 50 ng/mL of IL-10. Embryonic development and apoptotic rates were monitored. In Experiment II, the same groups were set up, with the addition of a group treated with 25 ng/mL of TNF-α alone. Grade I blastocysts were vitrified 5 h after treatment, and cryosurvival was monitored at until 48 h post-warming. The apoptosis rate and total cell number were investigated in the vitrified-hatched blastocysts. IL-10 alone did not affect developmental competence or cryosurvival (P > 0.05). The IL-10-treated embryos, when exposed in combination with TNF-α, presented a detrimental effect (P < 0.05) in the embryonic development of non-vitrified embryos. However, vitrified blastocysts had no negative effect (P > 0.05). The TNF-α treatment reduced (P < 0.05) the re-expansion rate at 6 h post-warming and increased (P < 0.05) the apoptosis rate in vitrified hatched blastocysts, whereas no effect (P > 0.05) of the treatments was detected in the hatching rate and total cell number post-warming. In conclusion, TNF-α has a detrimental effect on embryonic developmental competence and cryosurvival by compromising the development of non-vitrified embryos and apoptotic-related events of vitrified blastocysts, whereas IL-10, when in combination with TNF-α, appears to attenuate the detrimental effects of TNF-α.

Mercury concentrations in South Atlantic swordfish, Xiphias gladius, caught off the coast of Brazil.

This study evaluated the concentrations of mercury in fillets (anterior, middle, and end regions) from the swordfish, Xiphias gladius, and the relationships between mercury concentration and fish weight, as well as the region of collection. Of a total of 697 swordfish analyzed, 11 had mercury concentrations above 1 mg/kg, 421 were between 0.5 and 1.0 mg/kg, and 265 were below 0.5 mg/kg. The anterior and posterior regions had greater concentrations of mercury than the middle region, and fish caught off the northern coast of Brazil had a higher concentration than those caught off the southern coast.

Species-specific molecular differentiation of embryonic inner cell mass and trophectoderm: A systematic review.

A wide-ranging review study regarding the molecular characterization of the first cell lineages of the developmental embryo is lacking, especially for the primary events during earliest differentiation which leads to the determination of cellular fate. Here, a systematic review and meta-analysis were conducted according to PRISMA guidelines. MEDLINE-PubMed was searched based on an established search strategy through April 2021. Thirty-six studies fulfilling the inclusion criteria were subjected to qualitative and quantitative analysis. Among the studies, 50 % (18/36) used mice as an animal model, 22.2 % (8/36) pigs, 16.7 % (6/36) cattle, 5.5 % (2/36) humans, and 2.8 % (1/36) goats as well as 2.8 % (1/36) equine. Our results demonstrated that each of the first cell lineages of embryos requires a certain pattern of expression to establish the cellular determination of fate. Moreover, these patterns are shared by many species, particularly for those molecules that have already been identified in the literature as biomarkers. In conclusion, the present study integrated carefully chosen studies regarding embryonic development and first cellular decisions in mammalian species and summarized the information about the differential characterization of the first cell lineages and their possible relationship with specific gene expression.

Unraveling the Consequences of Oxygen Imbalance on Early Embryo Development: Exploring Mitigation Strategies.

Although well-established and adopted by commercial laboratories, the in vitro embryo production system still requires refinements to achieve its highest efficiency. Early embryonic development is a dynamic event, demanding suitable conditions to provide a high number of embryos with quality and competence. The first step to obtaining an optimized in vitro environment is to know the embryonic metabolism and energy request throughout the different stages of development. Oxygen plays a crucial role in several key biological processes necessary to sustain and complete embryonic development. Nonetheless, there is still controversy regarding the optimal in vitro atmospheric concentrations during culture. Herein, we discuss the impact of oxygen tension on the viability of in vitro-produced embryos during early development. The importance of oxygen tension is addressed as its roles regarding essential embryonic traits, including embryo production rates, embryonic cell viability, gene expression profile, epigenetic regulation, and post-cryopreservation survival. Finally, we highlight the damage caused by in vitro unbalanced oxygen tensions and strategies to mitigate the harmful effects.

Molecular phenotypes of bovine blastocyst derived from in vitro-matured oocyte supplemented with PAPP-A.

Insulin-like growth factor-1 (IGF-1) regulates cellular lipid content, whereas pregnancy-associated plasma protein-A (PAPP-A) increases IGF-1 bioavailability. Using in vitro-matured cumulus-oocyte complexes, we aimed to evaluate the impact of PAPP-A on the blastocyst lipid content, embryo cryotolerance and embryonic transcriptional profile. We determined that PAPP-A did not affect the lipid content of oocytes, blastocysts, or blastocyst yield (P > 0.05). However, PAPP-A modulated the embryo transcriptional profiles by downregulating PPARGC1A and AKR1B1, which are related to lipid metabolism; CASP9, a pro-apoptotic gene; and IFN-τ, a marker of embryo quality (P < 0.05). Furthermore, the use of PAPP-A improved blastocyst re-expansion in the first 3 h of culture after vitrification (P < 0.05). Although PAPP-A did not affect the blastocyst lipid content or embryo production, we suggest that embryonic transcriptional modulation could contribute to maintain the balance in embryo lipid metabolism. Furthermore, PAPP-A's approach seems to control key intracellular pathways that improve post-cryopreservation development of blastocysts.

Insulin-like growth factor-1 (IGF-1) selectively modulates the metabolic and lipid profile of bovine embryos according to their kinetics of development.

Supplementation of culture media with IGF-1 during in vitro culture of embryos has had controversial results over the years. In the present study, we show that differences previously observed in response to IGF addition might be related to intrinsic heterogeneity of the embryos. In other words, the effects exerted by IGF-1 are dependent on the characteristics of the embryos and their ability to modulate metabolism and overcome stressful conditions, such as the ones found in a non-optimized in vitro culture system. To test this hypothesis, in vitro produced bovine embryos with distinct morphokinetics (fast- and slow-cleavage) were submitted to treatment with IGF-1 and then evaluated for embryo production rates, total cell number, gene expression and lipid profile. Our results show that remarkable differences were found when fast and slow embryos treated with IGF-1 were compared. Fast embryos respond by upregulating genes related to mitochondrial function, stress response, and lipid metabolism, whereas slow embryos presented lower mitochondrial efficiency and lipid accumulation. We conclude that indeed the treatment with IGF-1 selectively affects embryonic metabolism according to early morphokinetics phenotypes, and this information is relevant for decision-making in the design of more appropriate in vitro culture systems.

Contribution of lipids to the organelle differential profile of in vitro-produced bovine embryos.

Each living organism is unique because of the lipid identity of its organelles. The diverse distribution of these molecules also contributes to the role of each organelle in cellular activity. The lipid profiles of whole embryos are well documented in the literature. However, this approach can often lead to the loss of relevant information at the subcellular and consequently, metabolic levels, hindering a deeper understanding of key physiological processes during preimplantation development. Therefore, we aimed to characterize four organelles in vitro-produced bovine embryos: lipid droplets (LD), endoplasmic reticulum (ER), mitochondria (MIT), and nuclear membrane (NUC), and evaluate the contribution of the lipid species to each organelle evaluated. Expanded blastocysts were subjected to cell organelle isolation. Thereafter, lipid extraction from cell organelles and lipid analysis using the Multiple Reaction Monitoring (MRM) profiling method were performed. The LD and ER displayed a greater number of lipids (Phosphatidylcholine - PC, Ceramide - Cer, and Sphingomielin - SM) with high signal-to-noise intensities. This result is due to the high rate of biosynthesis, lipid distribution, and ability to store and recycle lipid species of these organelles. The NUC had a more distinct lipid profile than the other three organelles, with high relative intensities of PC, SM, and triacylglycerols (TG), which is consistent with its high nuclear activity. MIT had an intermediate profile that was close to that of LD and ER, which aligns with its autonomous metabolism for some classes of phospholipids (PL). Our study revealed the lipid composition of each organelle studied, and the roles of these lipids could be associated with the characteristic organellar activity. Our findings highlight the lipid species and classes that are relevant for the homeostasis and function of each associated organelle and provide tentative biomarkers for the determination of in vitro embryonic development and quality.

Global gene expression of the inner cell mass and trophectoderm of the bovine blastocyst.

BACKGROUND: The first distinct differentiation event in mammals occurs at the blastocyst stage when totipotent blastomeres differentiate into either pluripotent inner cell mass (ICM) or multipotent trophectoderm (TE). Here we determined, for the first time, global gene expression patterns in the ICM and TE isolated from bovine blastocysts. The ICM and TE were isolated from blastocysts harvested at day 8 after insemination by magnetic activated cell sorting, and cDNA sequenced using the SOLiD 4.0 system. RESULTS: A total of 870 genes were differentially expressed between ICM and TE. Several genes characteristic of ICM (for example, NANOG, SOX2, and STAT3) and TE (ELF5, GATA3, and KRT18) in mouse and human showed similar patterns in bovine. Other genes, however, showed differences in expression between ICM and TE that deviates from the expected based on mouse and human. CONCLUSION: Analysis of gene expression indicated that differentiation of blastomeres of the morula-stage embryo into the ICM and TE of the blastocyst is accompanied by differences between the two cell lineages in expression of genes controlling metabolic processes, endocytosis, hatching from the zona pellucida, paracrine and endocrine signaling with the mother, and genes supporting the changes in cellular architecture, stemness, and hematopoiesis necessary for development of the trophoblast.

Basic and applied features in the cryopreservation progress of bovine embryos.

The expansion of the use of in vitro production techniques has revolutionized the bovine embryo market. In the last decade, we have seen the number of in vitro produced (IVP) embryos surpass the number of in vivo-derived (IVD) embryos obtained worldwide. Concomitantly, other biotechnologies were also improved, following the global trend. Embryo cryopreservation has received special attention, as it is one of the tools capable of disseminating in vitro production. Currently, two protocols are available: slow freezing and vitrification. Both have advantages and disadvantages regarding their application and, many aspects need to be considered before their use. In this review, we discuss in vitro production market trends, cellular and molecular features involved in embryo response to cryopreservation, and addressed cryo-storage period and embryonic developmental stage on cryosurvival. In addition, we also presented an overview of some aspects that impact the pregnancy rate following transfer of fresh and cryopreserved IVP embryos.

Cellular and apoptotic status monitoring according to the ability and speed to resume post-cryopreservation embryonic development.

Embryonic morphofunctional competence features regulating post-cryopreservation resumption of development are still poorly understood. In this study, we investigated the correlation between embryonic viability and the speed and ability to resume post-cryopreservation development. Thus, in vitro produced blastocysts were vitrified by the Cryotop method using standard protocols. Subsequently, the embryos were warmed, re-cultured, and classified into groups according to their speed and ability to resume post-cryopreservation development: embryos not re-expanded at 12h (NE12); embryos re-expanded at 12h and hatched at 24h (E12H24); embryos re-expanded at 12h and hatched at 48h (E12H48); embryos re-expanded at 12h and not hatched at 48h (E12NH48). Subsequently, the embryos were subjected to monitoring of total cell number and apoptosis. We identified that the blastocoel's ability to re-expand was negatively affected by the significant higher percentage of apoptotic cells observed in the NE12 group than in the other groups. A greater (P < 0.05) number of total cells, found in groups E12H24 and E12H48, seems to have a positive influence on the hatching capacity of blastocysts after cryopreservation. In conclusion, the total number of cells and apoptotic index correlated with the speed and ability to resume post-cryopreservation development. Apoptosis was a determinant for embryonic re-expansion, and the total cell number was crucial for blastocyst hatching.