Zinc improves the developmental ability of bovine in vitro fertilization embryos through its antioxidative action.

Zinc, an essential trace mineral, exerts a pivotal influence in various biological processes. Through zinc concentration analysis, we found that the zinc concentration in the bovine embryo in vitro culture (IVC) medium was significantly lower than that in bovine follicular fluid. Therefore, this study explored the impact of zinc sulfate on IVC bovine embryo development and investigated the underlying mechanism. The results revealed a significant decline in zygote cleavage and blastocyst development rates when zinc deficiency was induced using zinc chelator N, N, N', N'-Tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) in culture medium during embryo in vitro culture. The influence of zinc-deficiency was time-dependent. Conversely, supplementing 0.8 µg/mL zinc sulfate to culture medium (CM) increased the cleavage and blastocyst formation rate significantly. Moreover, this supplementation reduced reactive oxygen species (ROS) levels, elevated the glutathione (GSH) levels in blastocysts, upregulated the mRNA expression of antioxidase-related genes, and activated the Nrf2-Keap1-ARE signaling pathways. Furthermore, 0.8 µg/mL zinc sulfate enhanced mitochondrial membrane potential, maintained DNA stability, and enhanced the quality of bovine (in vitro fertilization) IVF blastocysts. In conclusion, the addition of 0.8 µg/mL zinc sulfate to CM could enhance the antioxidant capacity, activates the Nrf2-Keap1-ARE signaling pathways, augment mitochondrial membrane potential, and stabilizes DNA, ultimately improving blastocyst quality and in vitro bovine embryo development.

Meta-analysis to determine efficacy of colony-stimulating factor 2 for improving pregnancy success after embryo transfer in cattle.

Results have been inconsistent as to whether addition of colony stimulating factor 2 (CSF2) to culture medium improves embryo competence for establishment of pregnancy in cattle and humans. The purpose of the current study was to use all available experiments in cattle concerning effects of CSF2 on pregnancy success after transfer into recipient cattle. The approach was to perform a meta-analysis of all published data sets as well as data from an unpublished experiment described for the first time here. Meta-analysis failed to support the hypothesis that addition of CSF2 to embryo culture medium improves competence of bovine blastocysts to increase pregnancy or calving rates after transfer into recipient females. Thus, its general use as a culture medium additive to increase pregnancy success after embryo transfer is not recommended.

Effect of food supplementation on in vitro embryo production and growth performance in prepubertal Nelore heifers.

In vitro embryos production from prepubertal heifers can help contribute to breeding programs; however, strategies are necessary to increase their embryo production. The aim of this study was to investigate the effects of two nutritional plans on oocyte recovery, embryo production and growth performance of prepubertal Nelore heifers. Thirty-four Nelore heifers with age of 6.5 months were divided into two feeding treatments (NP1 and NP2). The NP1 diets served as the control and NP2 diets were formulated to contain an average of 1.22-fold more energy than NP1. After 3 months of supplementation, the animals underwent follicular aspiration (ovum pick-up, OPU) every 21 d for 3 months and embryos were produced in vitro. Wither height, chest depth, body weight and subcutaneous fat of animals were measured. The number of retrieved and viable oocytes per OPU were 1.49-fold and 1.42-fold greater in NP2 heifers (p = 0.018 and p = 0.049, respectively) than those in NP1 heifers. Heifers administered NP2 produced 29.7% blastocysts, a percentage higher than NP1 animals that produced 24.40% embryos (p < 0.05). Consequently, females in the NP2 treatment showed improved body development. These results indicate a positive effect of a higher energy diet on assisted reproduction and body development in prepubertal heifers.

Effects of niacin supplementation during in vitro culture on the developmental competence of porcine embryos.

Niacin is a water-soluble vitamin belonging to the vitamin B complex. It has been found to possess various biological activities, including antioxidant and lipid modification capacities. This study aimed to elucidate the effects of niacin treatment in porcine in vitro culture (IVC) medium on embryo developmental competence after parthenogenetic activation. IVC medium was supplemented with different concentrations of niacin (0 [control], 300, 600 and 900 µM). The results showed that embryos cultured in an IVC medium supplemented with 300 and 600 µM niacin had an increased cleavage rate (p < .05). In addition, 300 µM niacin treatment resulted in a higher blastocyst formation rate than the control and other niacin-treated groups. However, the total cell number did not differ significantly among the experimental groups. Niacin supplementation at 600 µM decreased reactive oxygen species, whereas treatment with 300, 600 and 900 µM increased glutathione levels in day two embryos. On day seven, 300 µM niacin exhibited improved fatty acid levels and fewer lipid droplets than the control group. Furthermore, gene expression at the mRNA level was performed on day two and day seven embryos, treated with or without 300 µM niacin. The expression of anti-apoptotic BCL2 and lipid metabolism PLIN2-related genes were upregulated, whereas the pro-apoptotic BAX and CASPASE3 were downregulated with niacin supplementation compared with the control group. However, SIRT1, a gene related to energy and the oxidative state, was up-regulated in niacin-treated day two embryos (p < .05). Overall, the results indicate that niacin has a beneficial effect on pre-implantation embryo development by modulating lipid metabolism and reducing oxidative stress and apoptosis. The expression patterns of PLIN2 and SIRT1 reported here suggest that these transcripts may be involved in the mechanism by which niacin affects the developmental capacity of IVC embryos.

Atypical initial cleavage patterns minimally impact rhesus macaque in vitro embryo morphokinetics and embryo outgrowth development†.

Embryo morphokinetic analysis through time-lapse embryo imaging is envisioned as a method to improve selection of developmentally competent embryos. Morphokinetic analysis could be utilized to evaluate the effects of experimental manipulation on pre-implantation embryo development. The objectives of this study were to establish a normative morphokinetic database for in vitro fertilized rhesus macaque embryos and to assess the impact of atypical initial cleavage patterns on subsequent embryo development and formation of embryo outgrowths. The cleavage pattern and the timing of embryo developmental events were annotated retrospectively for unmanipulated in vitro fertilized rhesus macaque blastocysts produced over four breeding seasons. Approximately 50% of the blastocysts analyzed had an abnormal early cleavage event. The time to the initiation of embryo compaction and the time to completion of hatching was significantly delayed in blastocysts with an abnormal early cleavage event compared to blastocysts that had cleaved normally. Embryo hatching, attachment to an extracellular matrix, and growth during the implantation stage in vitro was not impacted by the initial cleavage pattern. These data establish normative morphokinetic parameters for in vitro fertilized rhesus macaque embryos and suggest that cleavage anomalies may not impact embryo implantation rates following embryo transfer.

Outstanding Gir oocyte donors: How does individual factor affect in vitro embryo production efficiency?

The oocyte donor plays a pivotal role in bovine in vitro embryo production (IVP) success. The individual factor affects blastocyst/oocyte ratio and determine the existence of outstanding performing animals. The aim of this study was to assess the extent of individual factor effect to IVP efficiency, in a population of Gir oocyte donors. Extreme (high or low IVP efficiency based on blastocyst/oocyte ratio) animals were selected out of a population of 250 oocyte donors (1,734 observations) to form high (>0.48, n = 40), average (0.17-0.48, n = 168), and low (<0.17, n = 42) efficiency donor groups. Cumulus-oocyte complex indicators (total number, IVF-grade number, and IVF-grade/total ratio) were lower (p < 0.05) in high efficiency donors. The number of blastocysts per OPU was analyzed for highest performing bull, and an increase (p < 0.05) in high efficiency donors and a decrease (p < 0.05) in low efficiency donors were noticed, compared to average efficiency donors. The number of pregnancies obtained per OPU was affected (p = 0.017) by donor's efficiency (low: 0.60 ± 0.09 $$ 0.60\pm 0.09 $$ , average: 1.17 ± 0.07 $$ 1.17\pm 0.07 $$ , high: 2.57 ± 0.26 $$ 2.57\pm 0.26 $$ ), being 4.3-fold higher in high than in low efficiency donors. We conclude that producing embryos from high efficiency blastocyst/oocyte ratio donors increases blastocyst and pregnancy numbers by OPU, being an important indicator for donor selection in IVP programs.

Effect of arachidonic acid on pre- and post-hatching in vitro bovine embryo development.

CONTEXT: Arachidonic acid (AA) is the precursor of prostaglandins, which may play autocrine roles during early embryo development. AIMS: To test the developmental effects of addition of AA to pre- and post-hatching culture media on in vitro -produced bovine embryos. METHODS: Pre-hatching effects of AA were tested by culturing bovine zygotes in synthetic oviductal fluid (SOF) supplemented with 100 or 333µM AA. Post-hatching effects of AA were tested by culturing Day 7 blastocysts in N2B27 supplemented with 5, 10, 20 or 100µM AA up to Day 12. KEY RESULTS: Pre-hatching development to blastocyst was completely abrogated at 333µM AA, whereas blastocyst rates and cell numbers were not altered at 100µM AA. Impaired post-hatching development was observed at 100µM AA, whereas no effect on survival rates was noted at 5, 10 and 20µM AA. However, a significant reduction in Day 12 embryo size was observed at 10 and 20µM AA. Hypoblast migration, epiblast survival and formation of embryonic-disc-like structures were unaffected at 5-10µM AA. AA exposure downregulated the genes PTGIS , PPARG , LDHA and SCD in Day 12 embryos. CONCLUSIONS: Pre-hatching embryos are mostly irresponsive to AA, whereas AA was observed to have negative effects during early post-hatching development. IMPLICATIONS: AA does not improve in vitro bovine embryo development and is not required up to early post-hatching stages.

A comparison of culture and cooling for the short term preservation of in vivo derived dromedary camel embryos of varying morphological quality.

Despite recent advancements in the cryopreservation of dromedary camel embryos, widespread application of the technique is still limited by the need for specialised vitrification equipment and supplies. Temporary, liquid-phase embryo storage methods provide a useful tool for short-term preservation of camel embryos. In the current study, we compared the use of in vitro embryo culture with cold liquid storage in order to maintain both high- (Grade 1- Excellent and 2-Good) and low- (Grade 3- Moderate and 4-Poor) morphological grade Day-7 dromedary camel embryos in vitro for up to 3 days. Embryos were either cooled and placed in Hams-F10 medium supplemented with HEPES and 10% FBS and then kept at 4 °C; or placed in Hams-F10 supplemented with sodium bicarbonate and 10% FBS and then cultured in a humidified atmosphere of 6% CO2 at 37 °C before being assessed for viability at 24 h. In high-morphological grade embryos, both cold storage and culture supported 100% viability (maintenance of normal morphology) over this period (Cooled n = 22, Cultured n = 20). In low-morphological grade embryos, culture supported higher viability (16/18, 88.9%) than did cooling (4/18, 22.2%). We then evaluated the effect of up to 3 days of cold storage or culture on embryo morphological grade, diameter, and developmental competence following embryo transfer. High-grade embryos were divided between culture and cold storage; low-grade embryos were evaluated only after culture. Over 3 days of culture, both high- and low-grade embryos tended to either maintain or improve upon their initial morphological score (P < 0.05) and increased in diameter (P < 0.001). Embryos subjected to cooling tended to have reduced morphological scores by 48 h of storage and decreased in diameter by 72 h (P < 0.05). No significant influence of storage method (cooling vs. culture), duration (24-72 h), or embryo grade (high vs low) was observed on pregnancy establishment at Day-60 (22.2%-57.2% pregnancy rates for all treatments). Overall, rates of pregnancy establishment were similar for transferred cultured (n = 45) and cooled (n = 45) embryos (pregnancy rates at Day 18, 48% vs 51.1%; at Day 60, 37.7% vs 37.7%). Rates of embryonic loss also were similar (22.7% vs 26%). In conclusion, whilst similar rates of pregnancy and pregnancy loss were observed following the transfer of both cooled and cultured embryos held in vitro for up to 3 days, amongst the two methods, only embryo culture appears to provide a means of effectively preserving Day- 7 dromedary camel embryos with reduced morphological values in vitro. Considering these embryos appear to show poor tolerance to the cooling procedure and are unlikely candidates for vitrification, embryo culture may provide an effective method for deriving pregnancies from low-morphological grade embryos when immediate transfer is not possible on the day of flushing.

Zinc oxide nanoparticles functionalized with curcumin supplementation during in vitro embryo culture impaired in a concentration-dependent-manner blastocyst production in cattle.

This work investigated the effect of zinc oxide nanoparticles functionalized with curcumin (ZnO(np) + CUR) supplementation during the in vitro embryo culture (IVC) on the bovine in vitro embryo production, and the cellular antioxidant response. The cumulus-oocyte complexes (COCs) were matured, fertilized and then the presumptive zygotes were cultured in the medium in the absence (0 µM-control) or presence of different concentrations of ZnO(np) + CUR (3, 6 or 12 µM). After IVC, the embryos were destined either to assay intracellular ROS levels and mitochondrial membrane potential. The results demonstrated that only the addition of 12 µM ZnO(np) + CUR during IVC decreased intracellular ROS production and the rate of blastocyst production when compared to the control (p < .05). In conclusion, ZnO(np) + CUR addition during the IVC impaired in concentration-dependent-manner bovine in vitro embryo production.

Pre-hatching exposure to N2B27 medium improves post-hatching development of bovine embryos in vitro.

Ungulate embryos undergo critical cell differentiation and proliferation events around and after blastocyst hatching. Failures in these processes lead to early pregnancy losses, which generate an important economic impact on farming. Conventional embryo culture media, such as SOF, are unable to support embryo development beyond hatching. In contrast, N2B27 medium supports early post-hatching development, evidencing a swift in embryonic nutritional requirements during this developmental window. Here, we investigate if earlier exposure to N2B27 could improve embryo development after hatching. Embryo culture in N2B27 from day (D) 5, 6 or 7 significantly enhanced complete hypoblast migration (>45 vs. ∼24%) and epiblast development into an embryonic disc (ED)-like structure at D12 (>40 vs. 23%), compared to embryos cultured in SOF up to D9. Culture in N2B27 from D5 significantly increased epiblast and hypoblast cell number in D8 blastocysts, but post-hatching embryos cultured in N2B27 from D5 or 6 frequently showed a disorganized distribution of epiblast cells. In conclusion, bovine embryo culture in N2B27 from D7 onwards improves subsequent post-hatching development. This improved fully in vitro system will be very useful to functionally explore cell differentiation mechanisms and the bases of early pregnancy failures without requiring animal experimentation.

Time-lapse monitoring technologies for the selection of bovine in vitro fertilized embryos with high implantation potential.

Over the years, the utilization of in vitro fertilization (IVF) in bovine embryo production has increased globally to accelerate the selection of cows with high genetic values. The selection of embryos with high implantation potential is a critical factor in establishing pregnancy. Time-lapse monitoring (TLM) has emerged as a new technique that allows frequent and non-invasive imaging of developing embryos. TLM is considered to have several advantages over the conventional morphological evaluation of embryos, which has been widely used in bovine embryo production. Establishing a novel embryo selection algorithm specifically for bovine IVF embryos is a critical challenge, but information on the association between morphokinetic data obtained using TLM and the implantation potential of embryos is still limited. This review outlines the potential application of TLM technology to improve the fertility of bovine IVF embryos, focusing on the results of human and bovine TLM studies that can be applied to select bovine embryos with high implantation potential. First, the progress of the TLM technology in bovine embryo production is summarized. The association between kinetic and morphological parameters and the developmental and implantation potential of human and bovine embryos is outlined. Finally, the benefits of evaluating blastocyst collapse and re-expansion as indicators of bovine embryo viability and the possible application of TLM to detect chromosomal abnormalities and determine embryo sex will be discussed.

Metabolic signature of spent culture media shows lipid metabolism as a determinant of pregnancy outcomes.

In the present study, we investigated the spent culture media of in vitro produced (IVP) bovine embryos which did (group Pregnant) or did not (group Non-pregnant) establish pregnancy after transfer. For that purpose, IVP embryos on D5 were transferred to individual droplets for the last 48 h of culture. Embryos at the blastocyst stage were then transferred to synchronized recipients, while respective culture media drops were collected and evaluated individually. The list of metabolites present in spent culture media was obtained by electrospray ionization mass spectrometry (ESI-MS) and analysed with Metaboanalyst® to characterize the metabolic profile of each group. The spectrometric analysis showed that pathways related to lipid metabolism, particularly fatty acids degradation via beta-oxidation, were more present in the Pregnant group whereas no significant pathway was identified in the group Non-pregnant. By using this method, we were able to identify a metabolic signature in culture media that allows for a better comprehension of preferential metabolic routes taken by the most viable embryos. These findings offer great insights into the biochemistry of embryo development and reveal a potential target for the development of better-quality IVP systems, as well as tools to identify bovine embryos with greater chances to establish and maintain pregnancy.

Alpha-lipoic acid improves bovine preimplantation blastocyst quality and cryotolerance.

In vitro embryo production has grown in recent decades due to its great potential for cattle production. However, the quality of in vitro-produced embryos is lower compared with those produced in vivo. The postfertilization culture environment has a major influence on bovine embryo quality. We hypothesize that the inclusion of the inclusion of alpha-lipoic acid (ALA) in the in vitro culture (IVC) medium during the first 24 h would have positive effects on embryo development in vitro and cryotolerance. The aims of this study were to evaluate the antioxidant effect of ALA in IVC medium for 24 h on bovine zygotes (21 h post in vitro fertilization, IVF), day 2 cleaved embryos (46 h post-IVF), and to assess embryo quality, developmental competence, and cryotolerance after vitrification. In all experiments, IVC medium was the Control, and 2.5 µM ALA was the treatment implemented. Viability and reactive oxygen species (ROS) levels in zygotes and day 2 embryos did not differ from the Control (P > 0.05). Supplementation with ALA increased total blastocyst and hatching rates (P < 0.05). It also improved embryo quality, evidenced by the increased blastocyst total cell number and the percentage of excellent-quality embryos observed (P < 0.05). In embryos cultured with ALA and then vitrified, ALA reduced intracellular ROS levels in warmed blastocysts (P < 0.05). In conclusion, ALA supplementation to IVC medium during 24 h is a new advantage in improving embryo quality for assisted bovine reproduction.

Post-thaw viability, developmental and molecular deviations in in vitro produced bovine embryos cultured with l-carnitine at different levels of fetal calf serum.

l-carnitine is a well-known an antioxidant that enhanced lipid metabolism. Therefore, this study investigated the influence of supplementing l-carnitine (LC) to in vitro culture medium on preimplantation development, quality, cryotolerance and transcription profile of candidate genes. Following in vitro fertilization, embryos at zygote stage were cultured with medium supplemented with LC at 1.5 mM and fetal calf serum (FCS) at 0, 2.5, 5, 7.5 and 10% of the CR1-aa culture media. Intracellular quality of produced embryos was measured using different fluorescent stains that measured reactive oxygen species (ROS), lipid and mitochondria intensities. In addition, total cell number and total apoptotic cells were counted per embryo. Quantitative expression of candidate genes was conducted to find out molecular response of embryos after treatment. Moreover, vitrification was done at day 8 of preimplantation development to evaluate post-thaw embryo viability. The results indicated improved blastocyst formation rate at day 8 of preimplantation development (day zero = day of IVF) when embryos cultured with LC supplementation at low FCS at levels of 2.5% (35.3%) and 5% (34.7%) compared to control (25.9%), LC + FCS 7.5% (26.5%) and LC + FCS 10% (28.1%) groups. The total number of blastocyst cells that were cultured with LC + FCS 2.5% and LC + FCS 5% was increased and the number of dead cells (apoptotic) was decreased compared to control counterparts. Intracellular mitochondria activity was enhanced and resulted in reduction of cytoplasmic lipid in embryos treated with LC + FCS 2.5% and LC + FCS 5% compared with other experimental embryo groups. In addition, intracellular reactive oxygen species level was reduced in LC + FCS 2.5%, LC + FCS 5% and LC + FCS 7.5% compared to control and LC + FCS 10% groups. The expression profile of genes regulating embryo quality (BCL2), metabolic activity (GLUT1, CPT2 and TFAM), lipolysis (LIPE, AMPKa1 and ACCα), resistance to stress (SOD2) and ability to induce pregnancy (IFNt) was up-regulated under low FCS (2.5% and 5%) combined with LC supplementation. On the other hand, genes regulating lipogenesis were down-regulated (ACSL3 and S1PR). It can be concluded that LC is an efficient culture media supplement when added with FCS at 2.5 and 5% which improved blastocyst development rate and quality. These improvements are due to enhanced utilization of intracellular embryo lipid that subsequently increased cryotolerance through orchestrating genes involved in various activities of bovine embryos.

Generation of viable calves derived from developmentally mature blastocysts produced by on-gel culture.

Conventional culture systems for bovine embryos are unable to support sustained embryonic development until the developmentally mature blastocyst stage. Although we have previously developed an on-gel culture system that enables bovine blastocysts to complete cell segregation events at day (D) 10 following in vitro culture, the development of D10 blastocysts to term has yet to be achieved. In this study, we attained full-term development of D10 mature blastocysts produced using an on-gel culture system. Two calves derived from on-gel-cultured embryos were vaginally born, showing normal birth and placental weights and no obvious morphological abnormalities. Moreover, we detected no abnormalities in blood metabolic profile analyses. Our findings indicate that on-gel culturing can be used to facilitate the development of developmentally mature blastocysts to term, and produce healthy viable calves. This culture system could make a valuable contribution to cattle production and would enable a range of analyses for characterizing bovine-specific pre-implantation development.

Oroxin A reduces oxidative stress, apoptosis, and autophagy and improves the developmental competence of porcine embryos in vitro.

Oroxin A (OA) is a flavonoid isolated from Oroxylum indicum (L.) Kurz that has various biological activities, including antioxidant activities. This study aimed to examine the viability of using OA in an in vitro culture (IVC) medium for its antioxidant effects and related molecular mechanisms on porcine blastocyst development. In this study, we investigated the effects of OA on early porcine embryo development via terminal deoxynucleotidyl transferase dUTP nick-end labeling, 5-ethynyl-2'-deoxyuridine labeling, quantitative reverse transcription PCR, and immunocytochemistry. Embryos cultured in the IVC medium supplemented with 2.5 µM of OA had an increased blastocyst formation rate, total cell number, and proliferation capacity, along with a low apoptosis rate. OA supplementation decreased reactive oxygen species levels while increasing glutathione levels. OA-treated embryos exhibited an improved intracellular mitochondrial membrane potential and reduced autophagy. Moreover, levels of pluripotency- and antioxidant-related genes were upregulated, whereas those of apoptosis- and autophagy-related genes were downregulated by OA addition. In conclusion, OA improves preimplantation embryonic development by reducing oxidative stress and enhancing mitochondrial function.

The metabolic profile of bovine blastocysts is affected by in vitro culture system and the pattern of first zygotic cleavage.

Time lapse monitoring (TLM) is a commercial system of individual embryo culture based on the well-of-the-well system. It allows for collecting a panel of intrinsic parameters of special importance to non-invasive quality assessment. Besides, a combined analysis of TLM and metabolome data provides a deeper insight into the embryo quality. Two questions have been addressed by this study: (i) whether embryo culture in the Primo Vision affects embryo development and quality compared to the classical system and (ii) whether the first zygotic cleavage (FZC) dysmorphisms affect metabolomic profile of blastocysts. Presumptive zygotes from IVM/IVF oocytes were cultured either in classical drops (pools of 30 embryos, control group) or on Primo Vision plates (16 embryos in separate wells, sharing culture medium, Primo Vision group). The two categories of blastocysts were analysed for relative transcript abundance of five genes (real time PCR; FASN, Hsp70, PLIN2, Bax, Slc2a1) and for metabolite profile (mass spectrometry). Besides, basing on retrospective analysis of the TLM files, the Primo Vision blastocysts were allocated into one of two groups depending on the FZC pattern (normal NC or direct cleavage DC) and analysed for metabolite profiles. The Primo Vision embryos showed higher cleavage rate (p < 0.01) but reduced blastocyst rate (p < 0.05) when compared to the control group. Although the culture system (Primo Vision vs classical) did not affect the relative transcript abundance, the metabolomic analysis revealed different activity of five pathways. Two of them, related to beta-oxidation of fatty acids were up-regulated, whereas the remaining three corresponding to ubiquinone synthesis, metabolism of phenyloalanine and tyrosine as well as vitamin B6 metabolism were down-regulated. The metabolite profiles were however particularly affected by the FZC pattern. The NC and DC blastocysts differed significantly in the activity of 16 metabolic pathways which mainly involved pyruvic acid. The DC embryos displayed a higher level of pyruvic acid (p < 0.05) which may imply disturbances in the switch from lipid to glucose metabolism. Besides, in our opinion, embryos cultured in separate wells in the Primo Vision may face increased ROS level which reduces their developmental potential. Summarizing we underline application of metabolome analysis to embryo quality assessment due to providing a wider insight into embryo response to environmental factors.

Lycopene supplementation to serum-free embryo culture medium and its effect on development and quality of bovine blastocysts produced in vitro.

Selenium is commonly used as an antioxidant in a serum-free culture medium setting. However, lycopene has emerged as a potent antioxidant being twice as efficient as ß-carotene and 10 times as efficient as α-tocopherol with beneficial effects when supplemented in a serum-free maturation medium. Here, we aimed to evaluate the effect of lycopene supplementation in a serum-free culture medium on blastocyst development and quality. After in vitro maturation and fertilization, presumed zygotes were cultured in groups of 25 in 50 µl droplets of synthetic oviductal fluid. Culture medium supplementation was done using four experimental groups: insulin, transferrin, selenium (ITS, control); ITS + DMSO (diluent control); ITS + DMSO-lycopene 0.1 µM (ITSL); and IT + DMSO-lycopene 0.1 µM (ITL). DMSO was used as a diluent for lycopene. Blastocyst development among experimental groups was fitted in mixed-effects models, and blastocyst quality parameters (assessed via differential apoptotic staining) were evaluated in mixed linear regression models. The cleavage (85.3 ± 2.4, 82.6 ± 2.7, 86 ± 2.3 and 86.4 ± 2.3% for control, diluent control, ITSL and ITL, respectively) and day 8 blastocyst rates (37.4 ± 3.3, 36.9 ± 3.4, 39.7 ± 3.3 and 46.2 ± 3.4% for control, diluent control, ITSL and ITL, respectively) were not different (p > .1) among experimental groups. Embryos produced in the ITL group resulted in blastocysts with higher total cell numbers (TCN; 141 ± 19.2), inner cell mass (ICM; 65.3 ± 11.6) and trophectoderm cells (TE; 75.2 ± 8.8) compared with the control (129 ± 19.2, 56.3 ± 11.6 and 72.7 ± 8.8, for TCN, ICM and TE; p < .01, respectively). Lycopene-supplemented groups (ITSL and ITL) resulted in blastocysts with similar TCN, ICM and TE (p > .2). The number of apoptotic cells was not different among experimental groups (p > .1). Lycopene supplementation to the culture medium only produced a numerical increase in the blastocyst rate but replacing selenium with lycopene in a serum-free culture medium resulted in blastocysts with more cells.

Supplementation with kaempferol relieves oxidative stress and enhances development of early bovine embryos in vitro.

Oxidative stress (OS) has been considered the principle cause of developmental failure of early embryos cultured in vitro; therefore, the addition of antioxidants is very important for improving in vitro culture (IVC) systems. Various antioxidants have been tested for IVC systems, but most have exhibited some side effects. Kaempferol (3,5,7-trihydroxy-2-[4-hydroxyphenyl]-4 h-1-benzopyran-4-one, KAE) is a flavonoid with strong antioxidant activity and no obvious side effects. This study explored the effect of KAE on antioxidant capacity and developmental competence of bovine embryos after fertilization. KAE was added to bovine IVC medium and significantly reduced reactive oxygen species (ROS) in 2-, 4- and 8-cell stage embryos and increased blastocyst formation. In addition, the level of H3K9ac was increased, the apoptotic index was reduced and total cell numbers and trophectoderm cell numbers in day 7 blastocysts were increased significantly in KAE-treated embryos compared to control. Expression of the apoptotic gene, Bcl-2, was higher in blastocysts after KAE treatment, while expression of the endoplasmic reticulum (ER) stress genes, Bip and HDAC1, and the pro-apoptotic gene, Bax, were significantly lower in the KAE group. Thus, KAE significantly reduced ROS damage and improved development of IVC bovine embryos.

Effect of DHA on the quality of In vitro produced bovine embryos.

Docosahexaenoic acid (DHA) is an n-3 polyunsaturated fatty acid (PUFA) that improves fertility by increasing membrane fluidity. Moreover, embryos produced by donor females supplied with n-3 PUFA did not show any difference in terms of the lipid profile after 7 days of culture. The present study aimed to investigate the effects of DHA (20 and 100 µM) coupled with carnosine (5 mg/mL), an antioxidant, during oocyte maturation and embryo development on the developmental and cryosurvival rates and the number of pluripotent cells. Free fatty acid receptor-4 (FFAR4), which is able to bind DHA, was visualised by immunostaining. The addition of DHA in the in vitro development (IVD) medium decreased the percentage of pluripotent SOX2 positive cells compared with the control (8.4% vs. 10.9%) without affecting the number of cells (196.7 vs. 191.6 cells) or the developmental (20.9% vs. 23.9% blastocysts rate on D7) and cryosurvival rates (86.3% vs 86.2%). Such a decrease in pluripotent cells, relevant to the differentiation of the first lineage within the inner cell mass, represents an improvement in the embryo quality. On the contrary, embryos without any pluripotent SOX2-positive cells would not be able to achieve gestation. Future studies should follow up these results by carrying out embryo transfers to assess the beneficial effects of DHA supplementation.