Vitro-produced Bovine Embryos Research Articles

Small extracellular vesicles derived from the crosstalk between early embryos and the endometrium potentially mediate corpus luteum function.

The first interactions among the embryo, endometrium, and corpus luteum (CL) are essential for pregnancy success. Small extracellular vesicles (sEVs) are part of these interactions. We previously demonstrated that sEVs from in vivo- or in vitro-produced bovine embryos contain different miRNA cargos. Herein we show: 1) the presence and origin (in vivo or in vitro) of the blastocyst differentially reprograms endometrial transcriptional profiles; 2) the endometrial explant (EE) cultured with in vivo or in vitro embryos release sEVs with different miRNA contents, and; 3) the luteal explant (CLE) exposed to these sEVs have distinct mRNA and miRNA profiles. To elucidate this, the EE were cultured in the presence or absence of a single Day-7 in vivo (EE-AI) or in vitro (EE-IVF) embryo. After of culture we found, in the EE, 45 and 211 differentially expressed genes (DEGs) associated with embryo presence and origin, respectively. SEVs were recovered from the conditioned media (CM) in which EE and embryos were co-cultured. Four miRNAs were differentially expressed between sEVs from CM-EE-AI and CM-EE-IVF. Luteal explants exposed in culture to these sEVs showed 1360 transcripts, and fifteen miRNAs differentially expressed. The DEGs associated with embryo presence and origin, modulating cells' proliferation, and survival. These results demonstrate that in vivo- or in vitro-produced bovine embryos induce molecular alterations in the endometrium; and that the embryo and endometrium release sEVs capable of modifying the mRNA and miRNA profile in the CL. Therefore, the sEVs-mediated embryo-endometrium-CL interactions possibly regulate the CL viability to ensure pregnancy success.

Comparison of Gene Expression for Preimplantation Genes in Bovine Embryos Fertilized in vitro

Background: In vitro fertilization (IVF) the technique is useful for understanding early mammalian development and maximizing and speeding up genetic improvement in livestock. Genes that function as genetic markers and are involved in the pre-implantation development of embryos may be used to evaluate the quality of an embryo and the expression of these genes is correlated with the timing of embryonic genomic activation. Methods: Maturing the oocytes of bovine, then fertilizing these matured oocytes and finally culturing the fertilized oocytes to develop into embryos. Determined the gene expression of different certain genes HSP70, OCT4, GLUT1, BAX and DNMT1 in these different cell stages of embryos that were produced. Result: The mRNA expression of these genes was estimated after collecting the embryos listed above that were produced by the in vitro technique. The results showed statistically significant differences in the relative abundance between some of the genes mentioned above in each embryo stage. The timing of the zygotic genome activation process (ZGA) can result in differences in gene expression levels between the early embryonic genes in each developmental stage of in vitro-produced bovine embryos.

Preimplantation development of in vitro-produced bovine embryos treated with hydroxychloroquine

Hydroxychloroquine (HCQ) is a safe antimalarial drug but its overdosage or inappropriate use, such as during the pandemic, may cause adverse effects once this drug is considered a potent inhibitor of autophagy. Information about HCQ's effects on the reproductive field, including gametes and initial embryos, is limited. In this study, we evaluated the effect of HCQ (1, 6, 12, and 24 μM) on pre-implantation embryo development, autophagy, and apoptosis of bovine embryos produced in vitro. A dose-response experiment showed a reduction (p < 0.05) in cleavage only at the highest concentration. Blastocyst rate was gradually reduced (p < 0.05) with the increase of HCQ dosage starting at 6 μM, with no embryo formation occurring at 24 μM. Further analysis showed that embryos treated with 12 μM of HCQ had a higher (p < 0.05) accumulation of acidic autophagic vesicles on Days 5 and 7 of development and a higher (p < 0.01) apoptotic index on Day 7. To our knowledge, this is the first study to evaluate the effects of HCQ on embryo pre-implantation development in mammals. The results contribute with more information related to the study of autophagy in embryology as well as add some discussion on HCQ toxicology and its effects on reproductive cells.

In silico-designed vitrification protocols: an approach to improve survival of in vitro produced-bovine embryos.

In silico predictions validated in this study demonstrate the potential for designing shorter equilibration protocols that improve post-warming re-expansion and hatching rates of D7 and D8 in vitro-produced bovine embryos. Our results benefit the livestock industry by providing a refined and reproducible approach to cryopreserving bovine embryos, which, in addition, could be useful for other mammalian species. The cryopreservation of in vitro-produced (IVP) embryos is vital in the cattle industry for genetic selection and crossbreeding programs. Despite its importance, there is no standardized protocol yielding pregnancy rates comparable to fresh embryos. Current approaches often neglect the osmotic tolerance responses to cryoprotectants based on temperature and time. Hereby, we propose improved vitrification methods using shorter dehydration-based protocols. Blastocysts cultured for 7 (D7) or 8 days (D8) were exposed to standard equilibration solution (ES) at 25ºC and 38.5ºC. Optimized exposure times for each temperature and their impact on post-warming re-expansion, hatching rates, cell counts, and apoptosis rate were determined. In silico predictions aligned with in vitro observations, showing original volume recovery within 8 min 30 s at 25ºC or 3 min 40 s at 38.5ºC (D7 blastocysts) and 4 min 25 s at 25ºC and 3 min 15 s at 38.5ºC (D8 blastocysts) after exposure to ES. Vitrification at 38.5ºC resulted in D7 blastocysts re-expansion and hatching rates (93.1% and 38.1%, respectively) comparable to fresh embryos (100.0% and 32.4%, respectively), outperforming the 25ºC protocol (86.2% and 24.4%, respectively; P < 0.05). No differences were observed between D7 and D8 blastocysts using the 38.5ºC protocol. Total cell number was maintained for D7 and D8 blastocysts vitrified at 38.5ºC but decreased at 25ºC (P < 0.05). Apoptosis rates increased post-warming (P < 0.05), except for D8 blastocysts vitrified at 38.5ºC, resembling fresh controls. In conclusion, based on biophysical permeability data, new ES incubation times of 3 min 40 s for D7 blastocysts and 3 min 15 s for D8 blastocysts at 38.5ºC were validated for optimizing vitrification/warming methods for bovine IVP blastocysts.

134 Effectiveness of the timing of trypsin washing to remove pathogens from in vitro-produced bovine embryos at different stages of development

134 Effectiveness of the timing of trypsin washing to remove pathogens from in vitro-produced bovine embryos at different stages of development

4 Exploring the actions of FLI on in vitro-produced bovine embryo development and viability

4 Exploring the actions of FLI on in vitro-produced bovine embryo development and viability

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.

Effects of antifreeze protein from Lolium perenne L. (LpAFP) in the vitrification of in vitro-produced bovine embryos.

In the present study, the cryoprotective effects of Lolium perenne antifreeze protein (LpAFP) on the vitrification of bovine embryos were evaluated. In vitro-produced blastocysts were divided into two groups: the control group (CG) without the addition of LpAFP and the treatment group (TG) with the addition of 500 ng/ml of LpAFP in the equilibrium and vitrification solution. Vitrification was carried out by transferring the blastocysts to the equilibrium solution [7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO)] for 2 min and then to the vitrification solution (15% EG, 15% DMSO and 0.5M sucrose). The blastocysts were deposited on a cryotop device and submerged in liquid nitrogen. Warming was carried out in three steps in solutions with different sucrose concentrations (1.0, 0.5, and 0.0 M, respectively). Embryos were evaluated for re-expansion/hatching, the total cell count, and ultrastructural analysis. There was no significant difference in the re-expansion rate 24 h after warming; however, there was variation (P < 0.05) in the hatching rate in the TG and the total number of cells 24 h after warming was higher in the TG (114.87 ± 7.24) when compared with the CG (91.81 ± 4.94). The ultrastructural analysis showed changes in organelles related to the vitrification process but, in the TG, there was less damage to mitochondria and rough endoplasmic reticulum compared with the CG. In conclusion, the addition of 500 ng/ml of LpAFP during the vitrification of in vitro-produced bovine embryos improved the hatching rate and total cell number of blastocysts after warming and mitigated intracellular damage.

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.

Biomarker metabolite mating of viable frozen-thawed in vitro-produced bovine embryos with pregnancy-competent recipients leads to improved birth rates

Selection of competent recipients before embryo transfer (ET) is indispensable for improving pregnancy and birth rates in cattle. However, pregnancy prediction can fail when the competence of the embryo is ignored. We hypothesized that the pregnancy potential of biomarkers could improve with information on embryonic competence. In vitro-produced embryos cultured singly for 24 h (from d 6 to 7) were transferred to d 7 synchronized recipients as fresh or after freezing and thawing. Recipient blood was collected on d 0 (estrus; n = 108) and d 7 (4-6 h before ET; n = 107) and plasma was analyzed by nuclear magnetic resonance (1H+NMR). Spent embryo culture medium (CM) was collected and analyzed by ultra-high-performance liquid chromatography tandem mass spectrometry in a subset of n = 70 samples. Concentrations of metabolites quantified in plasma (n = 35) were statistically analyzed as a function of pregnancy diagnosed on d 40, d 62 and birth. Univariate analysis with plasma metabolites consisted of a block study with controllable fixed factors (i.e., embryo cryopreservation, recipient breed, and day of blood collection; Wilcoxon test and t-test). Metabolite concentrations in recipients and embryos were independently analyzed by iterations that reclassified embryos or recipients using the support vector machine. Iterations identified some competent embryos, but mostly competent recipients that had a pregnancy incompetent partner embryo. Misclassified recipients that could be classified as competent were reanalyzed in a new iteration to improve the predictive model. After subsequent iterations, the predictive potential of recipient biomarkers was recalculated. On d 0, creatine, acetone and l-phenylalanine were the most relevant biomarkers at d 40, d 62, and birth, and on d 7, l-glutamine, l-lysine, and ornithine. Creatine was the most representative biomarker within blocks (n = 20), with a uniform distribution over pregnancy endpoints and type of embryos. Biomarkers showed higher abundance on d 7 than d 0, were more predictive for d 40 and d 62 than at birth, and the pregnancy predictive ability was lower with frozen-thawed (F-T) embryos. Six metabolic pathways differed between d 40 pregnant recipients for fresh and F-T embryos. Within F-T embryos, more recipients were misclassified, probably due to pregnancy losses, but were accurately identified when combined with embryonic metabolite signals. After recalculation, 12 biomarkers increased receiver operator characteristic-area under the curve (>0.65) at birth, highlighting creatine (receiver operator characteristic-area under the curve = 0.851), and 5 new biomarkers were identified. Combining metabolic information of recipient and embryos improves the confidence and accuracy of single biomarkers.

Use of Antifreeze Protein from Tenebrio molitor (TmAFP) in Vitrification of In Vitro-Produced Bovine Embryos: An Ultrastructural Study.

The objective of this study was to evaluate the effects of different concentrations of antifreeze protein (AFP) extracted from the larva of the beetle, Tenebrio molitor (TmAFP), on vitrification of in vitro-produced bovine embryos. In vitro-produced blastocysts were divided into three experimental groups and vitrified using a cryotop. TmAFP was added to the equilibrium solution (ES) and vitrification solution (VS) at a concentration of 0 ng/mL (control), 500 ng/mL (500TmAFP), or 1000 ng/mL (1000TmAFP). Vitrification was carried out by first placing the blastocysts in ES for 2 minutes (7.5% ethylene glycol [EG] and 7.5% dimethyl sulfoxide [DMSO]). The blastocysts were then transferred to VS (15% EG and 15% DMSO) and promptly deposited on a cryotop stem and submerged in liquid nitrogen. Warming was carried out in three steps with decreasing sucrose concentrations. After warming, the blast cells were cultured for 24 hours for subsequent survival analysis and ultrastructural evaluation. There was a significant difference in the survival rate and expansion in the 500TmAFP group compared with the other groups. The ultrastructural analysis revealed intracellular lesions in all vitrified embryos; however, the embryos of the 500TmAFP and 1000TmAFP groups showed fewer cytoplasmic lesions compared with the control group. Taken together, addition of TmAFP can mitigate cellular changes that involve organelles and cellular components essential for proper functioning and improve the viability of warmed and vitrified in vitro-produced bovine embryos.

Effects of fetal bovine serum on trophectoderm and primitive endoderm cell allocation of in vitro-produced bovine embryos.

Supplementing embryonic culture medium with fetal bovine serum (FBS) renders this medium undefined. Glucose and growth factors present in FBS may affect the results of cell differentiation studies. This study tested the hypothesis that FBS supplementation during in vitro culture (IVC) alters cell differentiation in early bovine embryo development. Bovine embryos were produced in vitro and randomly distributed into three experimental groups at 90 h post insemination (90 hpi): the KSOM-FBS group, which consisted of a 5% (v/v) FBS supplementation; the KSOM33 group, with the renewal of 33% of medium volume; and the KSOM-Zero group, without FBS supplementation nor renewal of the culture medium. The results showed that the blastocyst rate (blastocyst/oocytes) at 210 hpi in the KSOM-FBS group was higher than in the KSOM-Zero group but not different from the KSOM33 group. There were no significant changes in metabolism-related aspects, such as fluorescence intensities of CellROX Green and MitoTracker Red or reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD+). Immunofluorescence analysis of CDX2 revealed that the lack of FBS or medium supplementation reduced the number of trophectoderm (TE) cells and total cells. Immunofluorescence analysis revealed a reduction of SOX17-positive cell numbers after FBS supplementation compared with the KSOM33 group. Therefore, we concluded that FBS absence reduced blastocyst rates; however, no reduction occurred when there was a 33% volume renewal of the medium at 90 hpi. We also concluded that FBS supplementation altered TE and primitive endoderm cell allocation during early bovine embryo development.

Exopolysaccharide ID1 Improves Post-Warming Outcomes after Vitrification of In Vitro-Produced Bovine Embryos.

This study aimed to assess the cryoprotectant role of exopolysaccharide (EPS) ID1, produced by Antarctic Pseudomonas sp., in the vitrification of in vitro-produced (IVP) bovine embryos. IVP day 7 (D7) and day 8 (D8) expanded blastocysts derived from cow or calf oocytes were vitrified without supplementation (EPS0) or supplemented with 10 µg/mL (EPS10) or 100 µg/mL (EPS100) EPS ID1. The effect of EPS ID1 was assessed in post-warming re-expansion and hatching rates, differential cell count, apoptosis rate, and gene expression. EPS100 re-expansion rates were significantly higher than those observed for the EPS0 and EPS10 treatments, regardless of culture length or oocyte source. EPS100 hatching rate was similar to the one of the fresh blastocysts except for those D7 blastocysts derived from calf oocytes. No differences were observed among EPS ID1 treatments when the inner cell mass, trophectoderm, and total cell number were assessed. Although apoptosis rates were higher (p ≤ 0.05) in vitrified groups compared to fresh embryos, EPS100 blastocysts had a lower number (p ≤ 0.05) of apoptotic nuclei than the EPS0 or EPS10 groups. No differences in the expression of BCL2, AQP3, CX43, and SOD1 genes between treatments were observed. Vitrification without EPS ID1 supplementation produced blastocysts with significantly higher BAX gene expression, whereas treatment with 100 µg/mL EPS ID1 returned BAX levels to those observed in non-vitrified blastocysts. Our results suggest that 100 µg/mL EPS ID1 added to the vitrification media is beneficial for embryo cryopreservation because it results in higher re-expansion and hatching ability and it positively modulates apoptosis.

Modulating the lipid profile of blastocyst cell membrane with DPPC multilamellar vesicles

The aim of this study was to evaluate the effect of multilamellar vesicles (MLVs) of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in co-culture with in vitro-produced bovine embryos (IVPEs). The stability of five concentrations of MLVs (1.0, 1.25, 1.5, 1.75, and 2.0 mM) produced using ultrapure water or embryonic culture medium with 24 or 48 h of incubation at 38.5 °C with 5% CO2 was assessed. In addition, the toxicity of MLVs and their modulation of the lipid profile of the plasma membrane of IVPEs were evaluated after 48 h of co-culture. Both media allowed the production of MLVs. Incubation (24 and 48 h) did not impair the MLV structure but affected the average diameter. The rate of blastocyst production was not reduced, demonstrating the nontoxicity of the MLVs even at 2.0 mmol/L. The lipid profile of the embryos was different depending on the MLV concentration. In comparison with control embryos, embryos cultured with MLVs at 2.0 mmol/L had a higher relative abundance of six lipid ions (m/z 720.6, 754.9, 759.0, 779.1, 781.2, and 797.3). This study sheds light on a new culture system in which the MLV concentration could change the lipid profile of the embryonic cell membrane in a dose-dependent manner.

Peroxisome proliferator-activated receptor delta-PPAR&#x3b4; 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.

Silencing mark H3K27me3 is differently reprogrammed in bovine embryos with distinct kinetics of development.

The kinetics of the first cleavages is a predictor of blastocyst development and implantation. For bovine embryos, this attribute was previously related to distinct metabolic, molecular and epigenetic profiles, including DNA and histone modifications. In the present work, we described the dynamics of trimethylation of lysine 27 on histone H3 (H3K27me3) in fast and slow developing embryos and verified if this epigenetic mark was also influenced by the speed of the first cleavages. In vitro-produced bovine embryos were classified as fast (4 or more cells) or slow (2 cells) at 40hr post fertilization (hpf) and either collected or cultured until 96hpf or 186hpf. Immunofluorescence analysis was performed in these three time points and showed that although both groups presented the same levels of H3K27me3 at 40hpf, slow embryos presented a pronounced increase in this mark at 186hpf when compared to fast embryos, resulting in blastocysts with remarkable differences in H3K27me3 levels. In conclusion, the increased levels of this repressive histone post-translation modification (PTM) might be an attempt of slow embryos to promote gene expression control and chromatin integrity, since it was already reported that these embryos present reduced levels of other epigenetic repressive marks as DNA methylation and trimethylation of lysine 9 on histone H3 (H3K9me3).

89&#x2003;Influence of sperm preincubation on development and sex ratio of in vitro-produced bovine embryos.

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Small extracellular vesicles derived from in vivo- or in vitro-produced bovine blastocysts have different miRNAs profiles-Implications for embryo-maternal recognition.

In vivo- and in vitro-produced bovine embryos have different metabolic profiles and differences in gene transcription patterns. These embryos also have a distinct ability to establish and sustain early pregnancies. Small extracellular vesicles (sEVs) are secreted by embryos and carry bioactive molecules, such as miRNAs. We hypothesize that in vivo or in vitro-produced bovine hatched blastocysts on Day 9 and the sEVs secreted by them have different miRNA profiles. To address this hypothesis, embryos of both groups were placed in in vitro culture on Day 7. After 48 h, hatched embryos and hatched embryo-conditioned media (eCM) of both groups were collected. A total of 210 miRNAs were detected in embryos of both groups, of these 6 miRNAs were downregulated, while 7 miRNAs were upregulated in vitro group when compared to in vivo group. sEVs were isolated from eCM to determine miRNA profile. A total of 106 miRNAs were detected in both groups, including 14 miRNAs upregulated in sEVs from in vivo-eCM, and 2 miRNAs upregulated in sEVs from in vitro-eCM. These miRNAs express in embryos and sEVs secreted by them regulate early embryonic developmental and endometrial pathways, which can modify embryo-maternal communication during early pregnancy and consequently affect pregnancy establishment.

A Shorter Equilibration Period Improves Post-Warming Outcomes after Vitrification and in Straw Dilution of In Vitro-Produced Bovine Embryos.

Simple SummaryFor more productive and sustainable livestock activity, various reproductive biotechnologies are being incorporated into breeding programs to accelerate genetic improvement. Among these strategies, embryo cryopreservation is a key technique for the conservation and dissemination of genetic resources while also optimizing animal production and biosafety. Though vitrification techniques are rapidly gaining acceptance due to their speed, simplicity, and feasibility, their practical applications in veterinary reproduction are limited because there is no standard protocol that facilitates warming in field conditions. Moreover, working time increases when a large number of embryos has to be cryopreserved. In-straw warming/dilution methods allow for the vitrification of embryos and their direct transfer to the uterus of recipients. In order to increase vitrification efficiency by reducing the working time and simplifying warming in field conditions, in vitro-derived cattle embryos at the expanded blastocyst stage were vitrified by using two different protocols (short equilibration vitrification and long equilibration vitrification) and in straw diluted/warmed. The short equilibration protocol improved vitrification outcomes in terms of embryo survival and hatching ability, and it improved embryo quality in terms of higher total cell number and lower apoptosis rate. A gene expression analysis of surviving embryos also indicated that the short equilibration treatment could lead to the production of more high-quality blastocysts.This study was designed to the optimize vitrification and in-straw warming protocol of in vitro-produced bovine embryos by comparing two different equilibration periods, short equilibrium (SE: 3 min) and long equilibrium (LE: 12 min). Outcomes recorded in vitrified day seven (D7) and day eight (D8) expanded blastocysts were survival and hatching rates, cell counts, apoptosis rate, and gene expression. While survival rates at 3 and 24 h post-warming were reduced (p < 0.05) after vitrification, the hatching rates of D7 embryos vitrified after SE were similar to the rates recorded in fresh non-vitrified blastocysts. The hatching rates of vitrified D8 blastocysts were lower (p < 0.05) than of fresh controls regardless of treatment. Total cell count, and inner cell mass and trophectoderm cell counts were similar in hatched D7 blastocysts vitrified after SE and fresh blastocysts, while vitrified D8 blastocysts yielded lower values regardless of treatment. The apoptosis rate was significantly higher in both treatment groups compared to fresh controls, although rates were lower for SE than LE. No differences emerged in BAX, AQP3, CX43, and IFNτ gene expression between the treatments, whereas a significantly greater abundance of BCL2L1 and SOD1 transcripts was observed in blastocysts vitrified after SE. A shorter equilibration vitrification protocol was found to improve post-warming outcomes and time efficiency after in-straw warming/dilution.

Nanoparticles from culture media are internalized by in vitro-produced bovine embryos and its depletion affect expression of pluripotency genes.

Extracellular vesicles are nanoparticles secreted by cell and have been proposed as suitable markers to identify competent embryos produced in vitro. Characterizing EVs secreted by individual embryos is challenging because culture medium itself contributes to the pool of nanoparticles that are co-isolated. To avoid this, culture medium must be depleted of nanoparticles that are present in natural protein source. The aim of this study was to evaluate if the culture medium subjected to nanoparticle depletion can support the proper in vitro development of bovine embryos. Zygotes were cultured in groups on depleted or control medium for 8 days. Nanoparticles from the medium were characterized by their morphology, size and expression of EVs surface markers. Isolated nanoparticles were labelled and added to depleted medium containing embryos at different developmental stages and evaluated after 24 hours at 2, 8-16 cells, morula and blastocyst stages. There were no statistical differences on blastocyst rate at day 7 and 8, total cell count neither blastocyst diameter between groups. However, morphological quality was better in blastocysts cultured in non-depleted medium and the expression of SOX2 was significantly lower whereas NANOG expression was significantly higher. Few nanoparticles from medium had a typical morphology of EVs but were positive to specific surface markers. Punctuated green fluorescence near the nuclei of embryonic cells was observed in embryos from all developmental stages. In summary, nanoparticles from culture medium are internalized by in vitro cultured bovine embryos and their depletion affects the capacity of medium to support the proper embryo development.