Cryotolerance Of Embryos Research Articles

Effect of Alpha-Lipoic Acid on the Development, Oxidative Stress, and Cryotolerance of Bovine Embryos Produced In Vitro

Oxidative stress (OS) induced by an imbalance in reactive oxygen species (ROS) levels in vitro impairs embryonic development. Here, we assessed the effects of alpha-lipoic acid (ALA) in in vitro production media on OS reduction, embryonic development, and cryotolerance of bovine embryos. We evaluated the effects of adding different concentrations of ALA (2.5, 5, 10, and 25 μM) to in vitro maturation (IVM) or in vitro culture (IVC) medium on embryonic development. We also determined the effects of adding ALA (25 μM) to the IVM and IVC medium in the same routine on the development and quality of embryos, ROS levels, and cryotolerance. Embryos were produced in vitro using conventional protocols for each treatment. The inclusion of ALA in the IVM and IVC media did not affect the development or quality of embryos; however, it reduced ROS levels in grade II embryos and increased hatching after 12 h on day 7 in grade I embryos and on day 8 in grade II embryos after warming. These findings prompt questions regarding the potential of ALA in improving embryo metabolism, considering the initial embryo recovery in the first few hours of embryo warming.

Expression profile of viability and stress response genes as a result of resveratrol supplementation in vitrified and in vitro produced cattle embryos.

Resveratrol, a potent antioxidant, is known to induce the up-regulation of the internal antioxidant system. Therefore, it holds promise as a method to mitigate cryopreservation-induced injuries in bovine oocytes and embryos. This study aimed to (i) assess the enhancement in the quality of in vitro produced bovine embryos following resveratrol supplementation and (ii) monitor changes in the expression of genes associated with oxidative stress (GPX4, SOD, CPT2, NFE2L2), mitochondrial function (ATP5ME), endoplasmic reticulum function (ATF6), and embryo quality (OCT4, DNMT1, CASP3, ELOVL5). Three groups of in vitro bovine embryos were cultured with varying concentrations of resveratrol (0.01, 0.001, and 0.0001 µM), with a fourth group serving as a control. Following the vitrification process, embryos were categorized as either good or poor quality. Blastocysts were then preserved at - 80°C for RNA isolation, followed by qRT-PCR analysis of selected genes. The low concentrations of resveratrol (0.001 µM, P < 0.05 and 0.0001 µM, P < 0.01) significantly improved the blastocyst rate compared to the control group. Moreover, the proportion of good quality vitrified embryos increased significantly (P < 0.05) in the groups treated with 0.001 and 0.0001 µM resveratrol compared to the control group. Analysis of gene expression showed a significant increase in OCT4 and DNMT1 transcripts in both good and poor-quality embryos treated with resveratrol compared to untreated embryos. Additionally, CASP3 expression was decreased in treated good embryos compared to control embryos. Furthermore, ELOVL5 and ATF6 transcripts were down-regulated in treated good embryos compared to the control group. Regarding antioxidant-related genes, GPX4, SOD, and CPT2 transcripts increased in the treated embryos, while NFE2L2 mRNA decreased in treated good embryos compared to the control group. Resveratrol supplementation at low concentrations effectively mitigated oxidative stress and enhanced the cryotolerance of embryos by modulating the expression of genes involved in oxidative stress response.

Impact of reducing lipid content during in vitro embryo production: A systematic review and meta-analysis

There is still no consensus regarding the role of lipid modulators during in vitro embryo production. Thus, we investigated how lipid reducers during the in vitro maturation of oocytes (IVM) or in vitro culture (IVC) of embryos impact their cryotolerance. A literature search was performed using three databases, recovering 43 articles for the systematic review, comprising 75 experiments (13 performed in IVM, 62 in IVC) and testing 13 substances. In 39 % of the experiments, an increase in oocyte and/or embryo survival after cryopreservation was reported, in contrast to 48 % exhibiting no effect, 5 % causing negative effects, and 8 % influencing in a dose-dependent manner. Of the 75 experiments extracted during IVM and IVC, 41 quantified the lipid content. Of those that reduced lipid content (n = 26), 50 % increased cryotolerance, 34 % had no effect, 8 % harmed oocyte/embryo survival, and 8 % had different results depending on the concentration used. Moreover, 28 out of the 43 studies were analyzed under a meta-analytical approach at the IVC stage in cattle. There was an improvement in the cryotolerance of bovine embryos when the lipid content was reduced. Forskolin, l-carnitine, and phenazine ethosulfate positively affected cryotolerance, while conjugated linoleic acid had no effect and impaired embryonic development. Moreover, fetal bovine serum has a positive impact on cryotolerance. SOF and CR1aa IVC media improved cryotolerance, while mSOF showed no effect. In conclusion, lipid modulators did not unanimously improve cryotolerance, especially when used in IVM, but presented positive effects on cryotolerance during IVC when reaching lipid reduction.

Blastocoel fluid aspiration improves vitrification outcomes and produces similar sexing results of in vitro-produced cattle embryos compared to microblade biopsy

The potential applications of in vitro-produced (IVP) cattle embryos are significantly enhanced when combined with genotype selection and cryopreservation techniques. While trophectoderm (TE) biopsies are frequently used for genotyping, cell-free DNA (cfDNA) found in blastocoele fluid (BF) arises as a less-invasive method. Moreover, the blastocoel collapse produced by BF aspiration could be beneficial for embryo cryotolerance. This study was conducted to test the BF as a source of cell free-DNA (cfDNA) and to compare the BF to the TE biopsy in terms of sexing efficiency/accuracy, embryo survival and gene expression after vitrification/warming. IVP day 7 expanded blastocysts were artificially collapsed by aspiration of BF (VIT-Collapsed) or biopsied (VIT-Biopsied). After sample collection, embryos were vitrified/warmed by the Cryotop method and individually cultured in vitro. Intact fresh non-vitrified and vitrified/warmed blastocysts served as Fresh Control and VIT-Control, respectively. After sex identification of BF or TE biopsies and the corresponding surviving embryos, amplification efficiency and sexing accuracy were assessed. There were no differences between the BF and TE biopsy samples in terms of sexing accuracy or efficiency. Although all vitrified groups showed lower post-warming re-expansion rates (p < 0.05), the blastocyst re-expansion rates in the VIT-Collapsed group were comparable to those in the Fresh Control group whereas biopsied blastocysts showed the lowest (p < 0.05) re-expansion rates. VIT-Collapsed blastocysts had hatching rates that were comparable to those of Fresh Control blastocysts but significantly higher than those of the other vitrification treatments. Proapoptotic gene BAX was overexpressed in VIT-Biopsied embryos, whereas BCL2 transcripts were more abundant in the VIT-Collapsed group. On the other hand, VIT-Biopsied embryos showed altered ATP1B1- and AQP3-mRNA levels. The analysis of the cfDNA present in the BF is an efficient, minimally invasive approach to sex IVP cattle embryos. Besides, the artificial collapse of blastocoel prior to vitrification resulted in higher re-expansion and hatching ability than when embryos were vitrified after being biopsied.

Embryo transfer: past, present, future - a personal perspective.

Embryo transfer is just one of a range of assisted reproductive technologies - often the last one in a sequence of others - that has revolutionised the cattle breeding industry. The number of in vitro-produced embryos transferred annually now surpasses the number derived by traditional superovulation by a factor of four. Although issues with cryotolerance of IVP embryos, embryo loss, and, in some cases, calf birth weight remain to be fully resolved, IVP embryos are likely here to stay as a tool for genetic improvement in dairy herds, offering increased flexibility in sire usage allowing multiple pregnancies from elite dam-bull combinations to be generated and the ability to produce more embryos per unit time than traditional superovulation. What follows is a short personal look back at the last 30 years; if you are looking for deep insights into the underlying biology regulating embryo development, this is not the place to look! Please refer to some of the excellent recent reviews and research papers cited herein.

Effects of stearic acid on the embryo cryopreservation in mouse

BACKGROUND: Intracellular lipids are sensitive to freezing. Lipidome modification is an important tool for studying the role of intracellular lipids in cryotolerance of mammalian oocytes and preimplantation embryos. OBJECTIVE: To study the effects of in vitro exposure of murine embryos to saturated stearic acid (SA) on the lipid content, embryo development and cryotolerance. MATERIALS AND METHODS: In vivo derived mouse embryos were cultured with 100 μM SA for 48 h up to the morula/blastocyst stage. Some of the SA-treated embryos were chosen for the evaluation of their development competence and the change in the lipidome, and other embryos were either slowly frozen or rapidly vitrified. RESULTS: Nile red staining combined with confocal laser scanning microscopy revealed a decrease in the total amount of lipids in the SA-treated embryos. Raman measurements showed that the lipid unsaturation was lower in embryos after in vitro SA culture. The addition of SA did not affect the embryo development before cryopreservation, but negatively affected the results of slow freezing cryopreservation and vitrification. CONCLUSION: In vitro SA exposure lowered the total amount of intracellular lipids and unsaturation in mouse embryos. The changes were accompanied with a significantly lower efficacy of embryo cryopreservation.

Lipid modification to improve cryotolerance of gametes, embryos and larvae and its potential application in aquaculture species: a review

Cryopreservation is a technique to maintain biological materials’ physiological and genetic stability at an ultralow temperature. For commercially important livestock or aquatic species, gamete and embryo cryopreservation could play a significant role in breeding programs and commercial production. For example, it could help overcome key problems such as asynchronous maturation and an unbalanced sex ratio. However, the physiochemical stresses imposed by cryopreservation can negatively affect gametes and embryos, leading to a poor survival rate. Recent studies on cryoinjury have demonstrated that the cryosensitivity of lipids is one of the key causes of cryodamage in mammalians, as lipid compositions in membranes of gametes and embryos are closely related to their cryoresistance. In addition, the cryotolerance of gametes and embryos in some mammalian species has been improved by lipid modification. However, studies on the role of lipids in the cryopreservation of gametes, embryos, and larvae are rare in fish and shellfish. Therefore, this review focuses on recent methodological advances to improve cryotolerance by lipid modification, including lipid application or manipulation in human and livestock sperm, oocytes, and embryos, and how these novel approaches could improve cryopreservation techniques in aquatic species, especially for oocytes and embryos.

CRIOTOLERANCIA DE EMBRIONES BOVINOS CULTIVADOS INDIVIDUALMENTE EN UN MEDIO SUPLEMENTADO CON L-CARNITINA

&lt;p&gt;&lt;strong&gt;Background:&lt;/strong&gt; L-carnitine is a lipid metabolism enhancer and a potent antioxidant that prevents oxidative damage and improves cryotolerance of bovine embryos. &lt;strong&gt;Objective:&lt;/strong&gt; To determine the effect of L-carnitine during oocyte maturation on developmental competence and cryotolerance of single bovine embryo cultured. &lt;strong&gt;Methodology:&lt;/strong&gt; Embryos were produced in vitro using abattoir-derived cumulus-oocyte complexes (COCs). In experiment 1, two individual maturation, fertilization and culture systems were used in 24-well plates with 20 µL drops of medium covered with mineral oil and 96-well plates with 30 µL drops of medium. In experiment 2, oocytes were randomly distributed into two groups and single matured in 96-well plates in medium supplemented or not with 0.6mg/mL L-carnitine. On day 7 post fertilization, blastocysts were vitrified on solid surface in Fiberplug. Non-vitrified blastocysts were used as control. Embryonic survival after devitrification was determined by blastocysts re-expansion and hatching rate at 24 and 48 hours of post-devitrification culture. Total cell number and apoptotic rate by TUNEL-DAPI staining were used as quality and cryotolerance indicator. In both cases, cleavage and blastocyst rates were evaluated at 48 hours and 7 days post fertilization, respectively. &lt;strong&gt;Results:&lt;/strong&gt; No significant differences were found for embryonic development between single culture systems. There was no effect of L-carnitine supplementation during maturation on embryo development, but embryo survival had increased (P &amp;lt; 0.05) at 24- and 48-hours post devitrification. &lt;strong&gt;Implications:&lt;/strong&gt; Treatment with L-carnitine had increased (P &amp;lt; 0.05) post-thaw re-expansion rates (86.8 ± 3.0 vs 70.0 ± 4.4) and it was similar to non-vitrified control (89.7 ± 2.6). Mean cell number and apoptotic cell index, were similar for all treatment groups. &lt;strong&gt;Conclusion:&lt;/strong&gt; L-carnitine supplementation during maturation, does not improve division rate and subsequent development of single cultured embryos, however increases cryotolerance post devitrification.&lt;/p&gt;

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 3h 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.

Novel Synthetic oviductal fluid for Conventional Freezing 1 (SCF1) culture medium improves development and cryotolerance of in vitro produced Holstein embryos.

In vitro produced (IVP) embryos hold great promise in the cattle industry; however, suboptimal in vitro culture conditions induce metabolic dysfunction, resulting in poor development and low cryotolerance of IVP embryos. This limits the use of IVP embryos in the cattle industry for embryo transfer and commercial scale-up. Previous studies have reported the use of individual metabolic regulators in culture media to improve blastocyst development rates and cryopreservation. In this study, we hypothesized that using a combination of select regulators, chosen for their unique synergistic potential, would alleviate metabolic dysfunction and improve the development of in vitro produced embryos to make them more closely resemble in vivo derived embryos. To test this, we first compared lipid content between Holstein and Jersey embryos produced in vivo and in vitro, and then systematically determined the combination of metabolic regulators that led to the greatest improvements in embryonic development, lipid content, mitochondrial polarity, and cryotolerance. We also tested different slow freezing techniques to further improve cryotolerance and finally validated our results via a clinical trial. Overall, we found that the use of multiple metabolic regulators in one culture media, which we refer to as Synthetic oviductal fluid for Conventional Freezing 1 (SCF1), and an optimized slow freezing technique resulted in improved pregnancy rates for frozen IVP embryos compared to embryos cultured in a synthetic oviductal fluid media. Additionally, there was no difference in pregnancy rate between frozen and fresh IVP embryos cultured in SCF1. This suggests that optimizing culture conditions and slow freezing technique can produce cryotolerance IVP and should allow further dissemination of this assisted reproductive technology.

Extracellular vesicles improve embryo cryotolerance by maintaining the tight junction integrity during blastocoel re-expansion.

Cryopreservation is a process in which the intact living cells, tissues, or embryos are preserved at subzero temperatures for preservation. The cryopreservation process highly impacts the survival and quality of the in vitro-produced (IVP) embryos. Some studies have highlighted the use of oviduct extracellular vesicles (EVs) to improve the cryotolerance of IVP embryos but the mechanism has not been well studied. The present study unravels the role of in vitro cultured bovine oviduct epithelial cells-derived EVs in improving the re-expansion and hatching potential of thawed blastocysts (BLs). The comparison of cryotolerance between synthetic oviduct fluid (SOF) and SOF + EVs-supplemented day-7 cryopreserved BLs revealed that the embryo’s ability to re-expand critically depends on the intact paracellular sealing which facilitates increased fluid accumulation during cavity expansion after shrinkage. Our results demonstrated that BLs cultured in the SOF + EVs group had remarkably higher re-expansion (67.5 ± 4.2%) and hatching rate (84.8 ± 1.4%) compared to the SOF group (53.4 ± 3.4% and 63.9 ± 0.9%, respectively). Interestingly, EVs-supplemented BLs exhibited greater influence on the expression of core genes involved in trophectoderm (TE) maintenance, formation of tight junction (TJ) assembly, H2O channel proteins (aquaporins), and Na+/K+ ATPase alpha 1. The EVs improved the fluid flux and allowed the transport of H2O into an actively re-expanded cavity in EVs-cultured cryo-survived BLs relative to control BLs. Our findings explored the function of EVs in restoring the TE integrity, improved the cell junctional contacts and H2O movement which helps the blastocoel re-expansion after thawing the cryopreserved BLs.

Impact of L-carnitine supplementation on the in vitro developmental competence and cryotolerance of buffalo embryos.

Background and Aim:Despite many trials, buffalo embryos have poor cryosurvivability because of their high lipid content. L-carnitine was found to be a lipid-reducing agent when added to oocyte and embryo culture media. The study aimed to determine the most effective concentration of L-carnitine to improve the oocyte developmental competence and cryotolerance of buffalo embryos.Materials and Methods:In vitro maturation and embryo culture media were supplemented with four concentrations of L-carnitine: 0 (control), 0.25, 0.5, and 1 mM. Good-quality embryos on 7 days were vitrified using mixtures of dimethyl sulfoxide and ethylene glycol at two concentrations (3.5 and 7 M).Results:The result showed that the cleavage and morula rates were significantly (p<0.05) higher in the 0.5 mM group. Blastocyst rates were significantly (p<0.05) higher at both 0.5 and 1 mM. The rates of viable embryos directly after thawing were significantly (p<0.05) increased in the 0.5 mM group. No significant difference was found in embryos cultured for 24 h after warming among all the groups.Conclusion:The addition of L-carnitine at a concentration of 0.5 mM to the culture media improves the oocyte developmental competence and cryotolerance of buffalo embryos directly after warming but not after 24 h of culture. Nevertheless, further studies must identify how L-carnitine exerts its beneficial micromechanisms.

24 Lipid composition of fresh or frozen sexed bovine blastocysts produced invivo or invitro

Currently, invitro embryo production (IVP) is successfully applied commercially in cattle. However, the high sensitivity of embryos to cryopreservation compared with invivo-derived (IVD) embryos still impairs the dissemination of this biotechnology. Reduced cryotolerance is frequently associated with lipid accumulation in the cytoplasm mainly due to invitro culture conditions. The objective of this study was to evaluate the lipid content of fresh and frozen sexed bovine grade 1 IVP or IVD embryos. The same 8 Holstein heifers were used in a Latin square design for both IVP and IVD embryo production. Zygotes were cultured in synthetic oviductal fluid (SOF) supplemented with 1% oestrus cow serum. The same bull was used for IVP and IVD. All expanded Day 7 blastocysts (n=40 IVP and 40 IVD) were biopsied and sexed. Half of the embryos (n=20 in each group) was slow frozen (1.5M ethylene glycol, 0.1m sucrose) and thawed before lipid extraction. Remaining embryos underwent lipid extraction in the fresh state. Briefly, the liposoluble fraction of the embryos was extracted according to the Bligh and Dyer method using chloroform and methanol. Liquid chromatography–high-resolution mass spectrometry (LC-HRMS) analysis was performed and operated in positive ionization mode. Lipids with variance intensities greater than 30% in quality control samples were removed as well as those identified as background noise. Partial least square discriminant analysis (PLS-DA) was used to show the relationship between variance in the data and difference among embryo origin (IVP vs. IVD), state before extraction (fresh vs. frozen), and sex of the embryos (male vs. female). The differentially lipid species groups were identified using Wilcoxon test, and considered significantly different when P&amp;lt;0.05. LC-HRMS analysis allowed us to identify 75 lipids. PLS-DA showed that embryo origin (IVP vs. IVD) and state before extraction (fresh vs. frozen) can be determined by LC-HRMS profiles by group in PLS-DA plot, despite slight overlaps. Sex of the embryos did not allow us to differentiate the lipid profile. However, 15 lipids varied significantly between male and female IVD, predominantly triglycerides (TG), whereas no lipid varied between the sexes in the IVP homologues. Moreover, 26 lipids varied significantly between IVP and IVD fresh embryos with enrichment of IVP embryos in TG, phosphatidyl choline, cholesteryl ester, and less diglyceride and lysophospholipid (LP) compared with IVD embryos. The comparison of the lipid profiles before and after freezing for IVP embryos showed that only 7 lipids varied significantly between fresh and frozen states with a decrease in LP for the frozen embryos. For the invivo counterparts, 13 lipids varied significantly, including the same LP as those identified for IVP embryos in the same way. Our results showed that the embryonic lipid profile is mainly affected by IVP and slow freezing protocols and, to a lesser extent, by sex. Further studies are needed to improve IVP protocols and optimize the cryotolerance of IVP embryos in cattle.

Ethanolic Extract of Dried Leaves from the Cerrado Biome Increases the Cryotolerance of Bovine Embryos Produced In Vitro.

In vitro embryo production (IVP) induces excessive production of reactive oxygen species (ROS), which affects blastocyst quality. Therefore, the supplementation of culture media with antioxidants is an alternative to overcome oxidative stress damage. However, there is a growing demand for the use of antioxidant compounds that are more natural and less toxic in cell cultures. The present study is aimed at evaluating the effect of ethanolic extracts from cerrado leaves on IVP. First, the antioxidant capacity and the amount of phenolic compounds of the leaves were evaluated. Then, the best ethanolic extract concentration composed of cagaita (Eugenia dysenterica) and murici (Byrsonima crassifolia) to be used during the in vitro culture of in vitro-produced embryos was determined. Afterward, we evaluated the influence of the extract of both plants on ROS and glutathione (GSH) production, while also evaluating the apoptosis and ROS metabolism gene expression. In a subsequent step, the effect of the ethanolic extracts of dried cagaita and murici leaves during embryonic cultivation on the cryotolerance of expanded blastocysts was studied. The results showed a significant reduction in the proportion of apoptotic cells from embryos cultivated with 0.01 mg/mL of the cagaita ethanolic extract, besides inducing an increase in the GPX4 and PRDX3 transcription levels. The murici ethanolic extract induced an increase in the transcription abundance of these genes but did not reduce the proportion of apoptotic cells. In addition, expanded blastocysts cultivated with extracts at a concentration of 0.01 mg/mL and cryopreserved had higher hatching rates and lower degeneration rates when compared to the frozen group previously supplemented with the extracts. Moreover, the apoptosis rate of embryos cultured for 12 h after cryopreservation was lower in groups previously exposed to extracts during in vitro cultivation. Such extracts may be used as alternatives to increase the cryotolerance of in vitro-produced embryos.

Factors Affecting Embryo Recovery Rate, Quality, and Diameter in Andalusian Donkey Jennies.

Simple SummaryEmbryo transfer has been successfully used for the conservation of equine endangered species, but a number of factors may affect the outcome of these techniques in mares. However, only a few studies have evaluated these factors in donkeys. The present study was conducted to determine which factors affect the recovery rate, morphological quality, and diameter in embryos from Andalusian donkey jennies. According to our results, the factors affecting embryo recovery rate were donor jenny, donor age, successive cycle within donor, number of flushings, and jack. Day of flushing and number of flushings had an effect on embryo diameter, whereas donor jenny and day of flushing had an effect on embryo quality. The knowledge of these factors is crucial to achieve a higher efficiency of embryo transfer in endangered donkey breeds.Embryo transfer and the vitrification of embryos could be used for the conservation and recovery of endangered donkey breeds. It is important to develop techniques that optimize recovery rates and the cryotolerance of donkey embryos. This study evaluates factors affecting the recovery rate, quality, and diameter of embryos obtained from donor jennies as a starting point for the use of vitrification and embryo transfer in the conservation of the Andalusian donkey. A total of 100 embryos were recovered out of 124 estrous cycles (80.6%). The donor jenny affected the rates of positive flushings (PFR; p = 0.040) and embryo recovery (ERR; p < 0.05) as well as embryo quality (p = 0.004). ERR was also affected by the number of flushings (p < 0.001), donor age (p < 0.05), successive cycle within donor (p < 0.001), and jacks (p < 0.05). Number of flushings (p < 0.001) and jack (p < 0.05) had a significant effect on PFR, whereas the day of flushing influenced the developmental stage (p < 0.001), embryo quality (p < 0.05), and diameter of embryos (p < 0.001). The number of flushings significantly influenced the diameter (p = 0.038) and embryo developmental stage (p = 0.001), whereas the developmental stage was statistically different between herds (p = 0.020). The factors influencing the success of this assisted reproductive technique were donor jenny, donor age, successive cycle within donor, day of flushing, number of flushings, and jack. The identification of these key points is crucial to achieve a higher efficiency of embryo transfer and vitrification processes, before considering their application in the conservation of endangered donkey breeds.

The SPOM-adapted IVM system improves in vitro production of bovine embryos

This study aimed to test the effects of an IVM SPOM adaptation (SPOM-adapted IVM) on the production, total number of cells (TNC), apoptosis, and cryotolerance (post-warming survival and cytoskeleton actin integrity) of bovine IVP embryos. Two experiments were conducted with two experimental groups based on IVM treatment: A control group (TCM 199 without FCS) and an SPOM-adapted group (TCM 199 with forskolin and IBMX in pre-IVM and IVM with cilostamide). The first experiment evaluated embryo in vitro production, TNC, and apoptosis rate on D9 of development. In the second experiment, embryos were vitrified/warmed at D7 (control fresh and vitrified; SPOM-adapted fresh and vitrified) and assessed regarding post-warming survival rates and cytoskeleton actin integrity. Statistical analysis was performed using GraphPad INSTAT software at a significance level of 5%. An increase (p < 0.05) in blastocyst production was observed in the SPOM-adapted group comparing to the control group. There was no difference (p > 0.05) in the TNC or apoptosis rate between the groups. Regarding cryopreservation, no differences were found (p > 0.05) in actin integrity or post-warming survival rates between the vitrified groups. In both vitrified groups, we observed a significantly lower uninjured pattern of actin integrity compared to the fresh groups (p < 0.05). We conclude that the SPOM-adapted IVM system is beneficial for blastocyst production and does not affect the quality and cryotolerance of the produced embryos.

Inhibiting diacylglycerol acyltransferase-1 reduces lipid biosynthesis in bovine blastocysts produced in vitro

Diacylglycerol acyltransferase-1 (DGAT1) is one of the DGAT enzymes that catalyzes the final step in the synthesis of triacylglycerol, which is a major component of the lipid droplets in embryos. Intracellular lipids accumulated in embryos produced in vitro have been associated with reduced cryotolerance and quality. The objective of the present study was to investigate the influence of DGAT1 inhibition on embryo development, quality, and post-vitrification survival, in addition to expression profiles of selected lipid metabolism-regulating and oxidative stress genes. Bovine cumulus-oocyte complexes were matured and fertilized in vitro and were cultured in synthetic oviduct fluid (SOF) supplemented with 5% fetal calf serum (FCS) alone (Control) or with 1, 5, 10 or 50 μM DGAT1 inhibitor (A922500®; D1, D5, D10, and D50, respectively) or 0.1% dimethyl sulfoxide (CDMSO: vehicle for DGAT1 inhibitor dilution) from 54 h post-insemination until Day 8 post insemination. No differences were found in blastocyst yield on days 7 and 8 in Control, CDMSO, D10, and D50 groups. Embryos cultured with 10 or 50 μM DGAT1 inhibitor had greater mitochondrial activity (P < 0.01), and increased number of cells (P < 0.05), while the cytoplasmic lipid content was reduced (P < 0.01), the latter associated with altered expression profiles of selected genes regulating lipid metabolism or genes related with oxidative stress (transcript abundance increased for SLC2A1 and SLC2A5 and decreased for DGAT1 and GPX1). Importantly, the survival rate of blastocysts produced with 10 μM DGAT1 was higher than that of Control, CDMSO and D50 groups at 72 h after vitrification and warming (73.8 vs 57.1, 55.9 and 56.1%, respectively, P < 0.001). In conclusion, inhibition of DGAT1 synthesis in bovine embryos produced in vitro abrogates the negative effect of FCS by decreasing their lipid content, increasing mitochondria activity and improving embryo cryotolerance, as well as favoring the expression of lipid metabolism regulating and oxidative stress-related transcripts.

Recent progress in bovine in vitro-derived embryo cryotolerance: Impact of in vitro culture systems, advances in cryopreservation and future considerations.

Cryopreservation of in vitro-derived bovine embryos is a crucial step for the widespread reproduction and conservation of valuable high-merit animals. Given the current popularity of bovine in vitro embryo production (IVP), there is a demand for a highly efficient ultra-low temperature storage method in order to maximize donor ovum pickup (OPU) turn-over, recipient availability/utilization and domestic/overseas commercial trading opportunities. However, IVP bovine embryos are still very sensitive to chilling and cryopreservation, and despite recent progress, a convenient (simple and robust) protocol has not yet been developed. At the moment, there are two methods for bovine IVP embryo cryopreservation: slow programmable freezing and vitrification. Both of the aforementioned techniques have pros and cons. While controlled-rate slow cooling can easily be adapted for direct transfer (DT), ice crystal formation remains an issue. On the other hand, vitrification solved this problem but the possibility of successful DT commercial incorporation remains to be determined. Moreover, simplification of the vitrification protocol (including warming) through the use of an in-straw dilution without the use of a microscope is a prerequisite for its use under farm conditions. This review summarizes the bovine IVP embryo cryopreservation achievements, strengths and limitations of both freezing systems and prospective improvements to enhance cryosurvival, as well as perspectives on future directions of this assisted reproductive technology.

Cryotolerance of somatic embryos of guinea (Petiveria alliacea) to V-cryoplate technique and histological analysis of their structural integrity

Optimized cryopreservation protocols are essential for the safe, cost-effective and long-term conservation of germplasm of great economic interest, such as Guinea (Petiveria alliacea L.), a medicinal species that synthesizes a great diversity of bioactive substances, among them polysulfides with antitumor activity. Somatic embryos (SEs) produced from in vitro roots were cryopreserved using the V-cryoplate technique. Their viability was measured using the triphenyltetrazolium test and their recovery by counting the number of secondary SEs produced per cryopreserved SE 90 days after liquid nitrogen (LN) exposure. Structural alterations were evaluated qualitatively and quantitatively during the successive stages of the protocol. SEs were dehydrated in 0.5 M sucrose, attached to cryoplates using sodium alginate solution (3%) and treated with PVS2 for different periods. After immersion in LN, SEs were rewarmed in unloading solution (1.2 M sucrose) at room temperature (25 °C) for 20 min. Viability based on the triphenyltetrazolium test was 100% after 15 min of treatment with PVS2 and LN exposure, and recovery, based on multiplication rate, was 21 somatic embryos produced per cryopreserved somatic embryo after 90 days of culture. The histological analysis of cryopreserved somatic embryos showed plasmolysis in the different cell types after treatment with PVS2, with meristematic and parenchymatic cells presenting a higher plasmolysis level. However, after rewarming, the level of plasmolysis decreased over cultivation time, reaching only 2% in meristematic cells after 30 days, indicating the good ability of Petiveria alliacea SEs to develop cryotolerance under the experimental conditions tested.

Conjugated linoleic acid as a potential bioactive molecule to modulates gamete and embryo cryotolerance

Abstract Conjugated linoleic acid (CLA) is a mixture of positional isomers of linoleic acid found in meat and dairy products from ruminants. It is a trans fat widely used by athletes as a food supplement, due to a supposed effect of maximizing the use of body fat reserves. The interest in diet and culture media supplementation with CLA is an emerging area, demanding studies in order to elucidate its benefits in the reproductive parameters, as well as in cryopreservation. Therefore, the aim of this review was to discuss the effects of CLA on the oocytes, sperm and embryos cryotolerance. Some studies have already demonstrated its use in cryopreservation of germline. Among those, it was observed that CLA supplementation during oocyte in vitro maturation can increase their viability post-freezing and developmental capacity. Regarding the use of CLA on sperm, there are few studies and their results are still inconclusive. Finally, studies about CLA supplementation on embryo culture media have shown promising results, indicating that this bioactive molecule is able to modulate lipid uptake on blastomeres. Altogether, these findings demonstrate the potential use of CLA as a bioactive molecule to improve germline and embryo cryotolerance and open new perspectives on human and animal reproduction field.