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The objective of this study was to investigate the impact of sperm source on embryo morphokinetics and the clinical outcomes of intracytoplasmic sperm injection (ICSI) cycles by considering the clustering of data (multiple embryos per patient that share a comparable developmental timing). This matched cohort study was performed at a private university-affiliated in vitro fertilization center. Women who underwent ICSI with epididymal sperm between January 2019 and December 2020 (the percutaneous epididymal sperm aspiration group, n = 32 cycles) were matched with women who underwent ICSI with ejaculated sperm because of idiopathic male factor infertility (the male factor infertility [MFI] group, n = 32 cycles) or female infertility (the control group, n = 32 cycles). Embryos were cultured in a time-lapse imaging incubator, and morphokinetic development was recorded and compared among the groups. Significantly slower divisions were observed in embryos derived from epididymal sperm than in those derived from the MFI and control groups. Embryos derived from epididymal sperm had a significantly lower KIDScore (3.1 ± 0.2) than did those derived from ejaculated spermatozoa from the MFI (5.4 ± 0.1) and control (5.6 ± 0.2, p < 0.001) groups. Epididymal sperm-derived embryos showed a significantly greater occurrence of multinucleation (23.2%) than did those derived from ejaculated sperm from the MFI and control groups (2.8% and 3.7%, p < 0.001, respectively). Epididymal sperm-derived embryos were significantly more likely to undergo direct or reverse cleavage (11.1%) than ejaculated sperm-derived embryos in the control group (4.3%, p = 0.001). In conclusion, delayed cell cleavage and increased incidences of blastomere multinucleation and abnormal cleavage patterns are observed when epididymal-derived sperm are used for ICSI.

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OBJECTIVE: To analyze if partial premature ovulation (PPO) detection during oocyte pick-up (OPU) impairs the quality of the retrieved oocyte cohort. METHODS: The PPO concept refers to the situation when premature ovulation happens only in some of the follicles and it is detected during OPU. This study constitutes a retrospective analysis performed in an infertility clinic (Spain) during 2016-2021 with patients undergoing OPU after controlled ovarian hyperstimulation for an in vitro fertilization (IVF) treatment. Study code: 2110-VLC-091- VG, registered on December 9 2021. Data from women with PPO (n=111) were compared to a matched control sample of cycles without PPO (n=333) at a proportion of 1:3. RESULTS: Cycles were matched for age, body mass index (BMI), treatment year, embryo genetic analysis and stimulation protocol type. The mean numbers of oocytes (6.1 vs. 11.2), mature oocytes (4.7 vs. 8.8), correctly fertilized oocytes (3.6 vs. 6.6) and top-quality blastocysts (0.9 vs. 1.8) were significantly lower in the PPO group than the nonPPO group (p<0.05). However, maturation, fertilization, top-quality blastocyst and pregnancy rates were statistically comparable among groups (p>0.05). CONCLUSIONS: Cycles with PPO have fewer available oocytes and, thus, fewer available embryos for transfer, al though their quality is intact, and still offer chances of pregnancy in these cases. Hence cycle cancellation may not be worth associated money, time and morale losses once PPO is detected.

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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.

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OBJECTIVE: To determine whether human hydrosalpinx fluid might have a deleterious effect on the fertilization rate and embryonic development of the exposed mouse oocytes. METHODS: Mouse cumulus-oocyte complexes (COCs) were randomly allocated for exposure to pure hydrosalpinx fluid (100% HSF group, n=400), EBSS containing 50% of hydrosalpinx fluid (50% HSF group, n=320) and pure EBSS (control group, n=300). RESULTS: The results showed that the fertilization rate in the 100% HSF group was significantly lower than the control group (64.0% versus 73.0%, p=0.031). The blastocyst formation rate was also lower in the 100% HSF group than 50% HSF and the control group (51.5% versus 56.9% versus 56.3%, respectively), but not statistically significant (p=0.275). There was no significant difference in the mean numbers of cells in the ICM, TE, and total cell number in blastocysts from the control group and two hydrosalpinx fluid exposure groups. CONCLUSIONS: Human hydrosalpinx fluid has a negative effect on the fertilization rate of the exposed mouse oocytes. However, this effect was found only in undiluted concentration and does not affect the subsequence of embryonic development and blastocyst cell number.

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OBJECTIVE: To investigate the effect of endometriosis and its different stages over Intracytoplasmic Sperm Injection (ICSI) outcomes among infertile women without previous history of ovarian surgery. METHODS: A total of 440 women enrolled in ICSI cycles were recruited and divided into two groups: endometriosis (n=220) and control group (n=220). Endometriosis patients without previous surgical treatment and with diagnostic laparoscopy were further stratified based on disease stage. Clinical and laboratory parameters, ovarian reserve markers, the number and quality of oocytes and embryos and fertilization rate were analyzed and compared among the various severity grades of endometriosis and the control group. RESULTS: Patients with advanced endometriosis had significantly fewer retrieved oocytes with small effect size (p<0.001, η2=0.04), lower metaphase II oocytes (p<0.001, η2=0.09) and fewer total numbers of embryos (p<0.001, η2=0.11) compared with less severe disease or women with tubal factor infertility. The fertilization rate in women with severe endometriosis was similar to that of the control group and in those with minimal/mild endometriosis (p=0.187). CONCLUSIONS: Severe endometriosis negatively affects ovarian response, oocyte quality and embryos. However, fertilization rate is not different among the various stages of endometriosis.

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Background: Mexico is innovating in the livestock industry through in vitro generation of bovine embryos with technologies such as well-of-the-well (WOW) and polyester mesh (PM) single-embryo culture systems. These techniques allow to maintain embryos in separate areas of a shared culture medium. Objective: To compare the quantity and quality of bovine embryos produced in WOW and PM culture systems versus the conventional (CG) culture system. Methods: In total, 345 embryos fertilized in vitro were evaluated for blastocyst yield in the three culture systems. To count blastocyst cell numbers, 69 embryos in each system were differentially stained for trophectoderm (TE), inner cell mass (ICM), and apoptotic cells. A qPCR gene expression analysis was performed for embryos in all three systems. Results: The WOW, PM and CG systems developed similar amount of blastocysts (41, 35 and 36%, respectively; p>0.05). Blastocysts in all three systems showed adequate amounts of ICM and apoptotic cells. Blastocysts in the PM system showed a greater number of TE cells [63.7 versus 58.6% in the CG system (p0.05). The ATP5B expression was higher in WOW than in PM (p0.05). The TJP3 expression was higher in PM than in WOW and CG (p<0.05). Expression of ID2 and CLDN4 was higher in WOW than in PM and CG (p<0.05). The biplot graphic from Principal Component Analysis (PCA) revealed that CG was located near degenerated embryos, whereas PM was located near arrested embryos, larger ICM and TE, and TJP3 expression. The WOW was located toward blastocysts, morulae, and expression of CLDN4, ID2 and GNAS. Conclusion: Compared with CG, both the PM and WOW systems are good options for culturing single embryos in the bovine model. Moreover, the PCA results suggest that embryos developed in the WOW system have greater capacity for generating blastocysts with increased ability to form TE and ICM layers, which might improve implantation.

Antecedentes: México está innovando en la industria ganadera a través de la generación in vitro de embriones bovinos con tecnologías de cultivo individual como lo son Pozo dentro de Pozo (WOW) y Malla de Poliéster (PM). Estos mantienen los embriones en áreas separadas mientras comparten un mismo medio de cultivo celular. Objetivo: Comparar la cantidad y calidad de embriones bovinos producidos en los sistemas WOW y PM contra el sistema de cultivo convencional en grupo (CG). Métodos: En total se evaluaron 345 embriones fertilizados in vitro para determinar la producción de blastocistos generados en los tres sistemas. Para contar el número de células por blastocisto, 69 embriones en cada sistema se tiñeron diferencialmente para trofectodermo (TE), masa celular interna (ICM) y células apoptóticas. Se realizó un análisis de expresión génica por qPCR de los embriones obtenidos en los tres sistemas. Resultados: Los sistemas WOW, PM y CG desarrollaron similares cantidades de blastocistos (41, 35 y 36%, respectivamente; p>0,05). Los blastocistos en los tres sistemas mostraron cantidades adecuadas de ICM y células apoptóticas. Los blastocistos en el sistema PM mostraron un mayor número de células TE [63,7% versus 58,6% en el sistema CG (p0,05). La expresión de ATP5B fue mayor en WOW que en PM (p<0,05), pero similar a CG (p<0,05). La expresión de TJP3 fue mayor en PM que en WOW y CG (p<0,05). La expresión de ID2 y CLDN4 fue mayor en WOW que en PM y CG (p<0,05). El gráfico de biplot del análisis de componentes principales reveló que CG se encontró cerca de embriones degenerados, mientras que PM se encontró cerca de embriones en arresto, ICM, TE, y TJP3. El WOW se localizó hacia blastocistos, mórulas y la expresión de CLDN4, ID2 y GNAS. Conclusión: En el modelo bovino los sistemas PM y WOW son buenas opciones para cultivar embriones individuales, ya que se obtienen resultados muy similares a los obtenidos con el sistema CG. Además, los resultados de PCA sugieren que los embriones individuales desarrollados en el sistema WOW generan blastocistos con mayor capacidad de formar TE e ICM, lo que podría mejorar su éxito de implantación.

Antecedentes: O México está inovando na indústria pecuária por meio da geração in vitro de embriões bovinos com tecnologias de cultura de embriões individuais, bem como em poço (WOW) e malha de poliéster (PM). Estes mantêm os embriões em áreas separadas, enquanto compartilham o mesmo meio de cultura de células. Objetivo: Comparar a quantidade e a qualidade de embriões bovinos produzidos nos sistemas de cultura WOW e PM com o sistema convencional de cultura em grupo (CG). Métodos: No total, 345 embriões fertilizados in vitro foram avaliados para determinar a produção de blastocistos gerados nos três sistemas. O número de células por blatocisto foi contado, 69 embriões em cada sistema foram diferencialmente corados para trofectoderme (TE), massa celular interna (ICM) e células apoptóticas. Uma análise de expressão gênica qPCR foi realizada para os embriões obtidos nos três sistemas. Resultados: Os sistemas WOW, PM e CG desenvolveram quantidades semelhantes de blastocistos (41, 35 e 36%, respectivamente; p>0,05). Os blastocistos nos três sistemas mostraram quantidades adequadas de ICM e células apoptóticas. Os blastocistos no sistema PM mostraram um número maior de células TE [63,7 versus 58,6% no sistema CG (p0,05). A expressão de ATP5B foi maior no WOW do que no PM (p<0,05), mas semelhante ao GC (p<0,05). A expressão de TJP3 foi maior no PM do que no WOW e CG (p<0,05). A expressão de ID2 e CLDN4 foi maior no WOW do que no PM e CG (p<0,05). O gráfico biplot da análise de componentes principais revelou que CG foi encontrado próximo a embriões degenerados, enquanto PM foi encontrado próximo a embriões presos, ICM, TE e TJP3. WOW foi encontrado para ter blastocistos, mórulas e a expressão de CLDN4, ID2 e GNAS. Conclusão: Em comparação com o CG, os sistemas PM e WOW são boas opções para a cultura de embriões individuais no modelo bovino. Além disso, os resultados da PCA sugerem que embriões individuais desenvolvidos no sistema WOW têm maior capacidade de desenvolver blastocistos com maior capacidade de formar as camadas TE e ICM, o que poderia melhorar seu sucesso de implantação.

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The aim of this study was to evaluate the effect of age of Nellore (Bos indicus) donors on the efficiency of in vitro embryo production (IVEP) and pregnancy rate. Thirty-six donors, including 11 female calves (13 ± 0.61 months), 17 prepubertal heifers (25 ± 0.78 months) and 8 cows (83 ± 28 months), were submitted to 3 procedures of ovum pickup (OPU) on random days of the estrous cycle at intervals of 21 days. Caspase-3 and IGFBP2 were quantified in oocytes and blastocysts for the evaluation of oocyte and embryo quality. The produced embryos were vitrified (n = 445) and transferred to synchronized recipients. Cows produced a larger number of follicles (cows: 54.5 ± 6.2; calves: 20.0 ± 0.57; prepubertal heifers: 20.8 ± 0.46), total oocytes (cows: 45.97 ± 7.22; calves: 28.93 ± 6.14; prepubertal heifers: 27.21 ± 4.94) and cleaved oocytes (cows: 21.14 ± 4.22; calves: 13.09 ± 3.72; prepubertal heifers: 12.4 ± 3.19). The cleavage rate was similar between age categories; however, cows tended (p < 0.07) to produce a larger number of blastocysts (9.74 ± 2.26) per OPU than calves (5.57 ± 1.99) and prepubertal heifers tended to have a higher blastocyst yield (35.4%) than calves (27.1%) (p < .07). The expression levels of IGFBP2 and caspase-3 were higher in oocytes derived from calves compared to the other two categories. The pregnancy rate was higher in calves (43.1%) and cows (40.4%) than in prepubertal heifers (33.8%) (p = .03). Despite the larger numbers of follicles and viable oocytes in cows, the blastocyst production results and pregnancy rates obtained indicate that the use of young females as oocyte donors in IVEP is feasible and may contribute to reduce the generation interval.

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OBJECTIVE: A prolonged culture of embryos beyond day 2-3 to day 5 (blastocyst culture) after fertilization might be an alternative, simple way of selecting suitable embryos for transfer. Extending embryo culture to day 5/6 is a selection tool to choose an embryo with a greater likelihood of implantation rather than improve embryo quality. METHODS: This retrospective study analyzed 1126 fresh IVF/ICSI cycles performed between February 1, 2014 and December 30, 2018 at the University Fertility Center in Kiel, Germany, to determine the impact of blastocyst culture on pregnancy rates and the association between embryo quality and pregnancy rates. RESULTS: Clinical pregnancy was achieved in 154 cases (19.5%) after day 2/3 transfer and in 76 cases (22.7%) after day 5 transfer. Pearson's two-sided chi-squared test yielded no statistical significance (p=0.221). The analysis of clinical pregnancy rates in relation to the quality of transferred embryos yielded the following results: 49 (10.7%) pregnancies in cases of no ideal embryo(s); 122 (27.2%) in cases of at least one ideal embryo; and 59 (26.7%) for both quality groups. Pearson's two-sided Chi-squared test was statistically significant (p<0.001). CONCLUSIONS: Our data revealed no improvement of pregnancy rates after blastocyst transfer compared with day 2/3 transfers. However, we noted higher pregnancy rates when an embryo of good quality was transferred.

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Individual embryo culture is the only strategy that allows the tracking of embryos throughout the culture period. However, this procedure leads to lower embryo development. This study aimed to evaluate different alternatives to improve embryo development in a single in vitro production system. First, embryo production was compared between individual cultures on a 20 µL droplet and Cell-Tak® system. Then, various concentrations of folic acid were tested for use in combination with insulin-transferrin-selenium (ITS). To determine the concentration, embryos were analyzed not only by development but also by their methylation status. Finally, the supplementation of individual culture media with ITS and/or folic acid was evaluated. The results showed that embryos cultured in the Cell-Tak® system presented lower blastocyst rates than the microdroplets system. When the concentration of folic acid was tested, 20 µM and 500 µM presented a higher level of insulin-like growth factor (IGF2) DNA methylation pattern compared to control, suggesting that in vitro conditions alter DNA methylation pattern in that region and folic acid reestablishes the pattern. However, when it was used in an individual culture system, folic acid did not improve embryo development. Conversely, ITS which is composed of three important components, proved to be an alternative to individual embryo culture, improving embryo rates, showing similar rates to grouped culture embryos. Since Folic Acid change epigenetic profile, additional studies are needed to evaluate its use in IVP culture systems.

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OBJECTIVE: Serum anti-Müllerian hormone (AMH) presents a strong positive correlation with quantitative aspects of the ovarian reserve, while its correlation with embryo quality is unclear. This study assessed the association between serum AMH as a marker of ovarian reserve and embryo quality, in women undergoing in vitro fertilization. METHODS: This observational analytical retrospective study included patients seen between 2010 and 2018. In vitro fertilization patients with measured AMH levels were analyzed based on the following parameters: number of retrieved oocytes; number of metaphase II oocytes; embryo quality; and treatment outcome. Statistical analysis was performed using ANOVA, Mann-Whitney U test, linear regression, and Pearson and Spearman correlations. RESULTS: We found a positive correlation between AMH levels, number of retrieved oocytes and number of metaphase II oocytes (r 0.649, p=0.000). The numbers of retrieved and metaphase II oocytes were predicted in 42% (R2: 429) of the cases based on AMH levels (p=0.000). Serum AMH levels were not associated with embryo quality on Day 3 (p=0.151); an association was seen between AMH levels and embryo quality on Day 5 (p=0.006). The distribution of AMH levels was the same across patients, regardless of whether they were able to achieve pregnancy (p=0.767). CONCLUSIONS: AMH levels correlated with embryo quality on Day 5; no association was found between AMH levels and embryo quality on Day 3 or pregnancy rate. The use of AMH levels to predict embryo quality still requires further studies; therefore, AMH should be used to assess the ovarian reserve only.

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PROBLEM: Decidualized cells display an active role during embryo implantation sensing blastocyst quality, allowing the implantation of normal developed blastocysts and preventing the invasion of impaired developed ones. Here, we characterized the immune microenvironment generated by decidualized cells in response to soluble factors secreted by blastocysts that shape the receptive milieu. METHOD OF STUDY: We used an in vitro model of decidualization based on the Human Endometrial Stromal Cells line (HESC) differentiated with medroxiprogesterone and dibutyryl-cAMP, then treated with human blastocysts-conditioned media (BCM) classified according to their quality. RESULTS: Decidualized cells treated with BCM from impaired developed blastocysts increased IL-1ß production. Next, we evaluated the ability of decidualized cells to modulate other mediators associated with menstruation as chemokines. Decidualized cells responded to stimulation with BCM from impaired developed blastocysts increasing CXCL12 expression and CXCL8 secretion. The modulation of these markers was associated with the recruitment and activation of neutrophils, while regulatory T cells recruitment was restrained. These changes were not observed in the presence of BCM from normal developed blastocysts. CONCLUSION: Soluble factors released by impaired developed blastocysts induce an exacerbated inflammatory response associated with neutrophils recruitment and activation, providing new clues to understand the molecular basis of the embryo-endometrial dialogue.

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The purpose of this study was to determine effects of various sources of omega-3 and omega-6 fatty acids on ovarian response and embryo quality in Boer does when there was a superovulation treatment regimen imposed. Pluriparous does were randomly assigned to be treated with 300 g of one of four experimental supplements containing linseed oil (LO), soybean oil (SO), palm oil (PO), or a control supplement without fatty acids (CO), for 15 days. Does were fitted with a controlled internal drug release (CIDR) device containing 0.3 g progesterone for 7 days. At 48 h before CIDR withdrawal, does were treated with 80 mg follicle-stimulating hormone (FSH) administered at 12 h intervals. Embryos were collected 7 days after the last natural mating. Estrous response and interval between CIDR withdrawals to estrous onset were similar between treatments (P > 0.05). Number of ovulations was similar for does in the different groups (10.0, 9.2, 7.0, and 7.0, in LO, SO, PO, and CO, respectively; P > 0.05). There was premature luteal regression in does of the SO, PO, and CO groups, except in LO group. The LO-treated does had a larger (P < 0.05) mean number of ova/embryos recovered than does of SO, PO, and CO groups (7.2, 2.0, 0.2, 0.2, respectively) and transferable embryos (5.1, 1.4, 0.2, 0.2, respectively). These results indicate that including LO in supplements may be a feasible strategy for preventing premature luteal regression and improving embryo quality in goats treated to induce follicular super-stimulation for induction of superovulation.

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Human embryos generated in vitro have a high incidence of chromosomal abnormalities that negatively affect pregnancy rate. Embryos generated in vitro secrete extracellular vesicles (EVs) into the culture medium that could be used potentially as indicators of embryo competence. This research aimed to evaluate the concentration and size of EVs and their gDNA content as an indicator of developmental competence in human embryos. Human embryos generated by intracytoplasmic sperm injection (ICSI) were classified morphologically as of either TOP, FAIR or POOR quality. Culture medium and developmentally arrested embryos (which were not able to be used for embryo transfer) were collected. Microvesicles, exosomes (MV/Exo) and apoptotic bodies (ABs) were isolated from culture medium. Nanoparticle tracking analysis (NTA) and array comparative genomic hybridization (aCGH) analysis were performed to evaluate EVs and their gDNA content. From NTA, the diameter (mean) of MVs/Exo from TOP quality embryos was higher (112.17 nm) compared with that of FAIR (108.02) and POOR quality embryos (102.78 nm) (P < 0.05). aCGH analysis indicated that MVs/Exo and ABs carried gDNA with the presence of 23 chromosome pairs. However, when arrested embryos were compared with their respective MVs/Exo and ABs, the latter had an increased rate of chromosomal abnormalities (24.9%) compared with embryos (8.7%) (P < 0.05). In conclusion, the size of EVs from culture medium might be an alternative for evaluating competence of human embryos, however more studies are needed to validate the use of gDNA from EVs as an indicator of embryo competence.

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OBJECTIVE: The aim of the present prospective study was to evaluate which ovarian reserve marker would be more reliable as the quality of the A + B embryos (day 3 and blastocyst). METHODS: We ran a prospective study with 124 infertile women, aged 24-48 years, from 2017 to 2018. The patients were divided into 3 groups according to age and the subgroups were compared for AMH, AFC, number of A+B embryos. New division of the 3 groups was performed based on the AMH, and the subgroups were compared for age, AFC and number of A+B embryos. Finally, we divided the patients into 3 groups, based on the AFC, and we compared the subgroups for age, AMH and number of A+B embryos. P<0.05 was considered statistically significant. RESULTS: When the 124 patients were divided according to age, we found a significant fall in an A+B embryo quality (day3; blastocyst) after 35 years (p<0.038; p<0.035), and more severely after 37 years (p<0.032; p<0.027). When the 124 patients were divided according to AMH, there was a significant fall in A+B embryo quality (day 3; blastocyst), with AMH<1ng/ml (p<0.023; p<0.021). When the 124 patients were divided according to AFC, there was a significant fall in A+B embryo quality (day 3; blastocyst) with AFC<7 (p<0.025; p<0.023). These markers had significant associations with embryo quality (p<0.005). CONCLUSION: Age, AFC and AMH have significant associations with A +B embryo quality on day 3 and blastocyst.

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The objective of this study was to determine the efficiency of Ringer's lactate solution (RL) and RL + 1% fetal bovine serum (FBS) and compare them with the efficiency of Dulbecco's phosphate-buffered saline (D-PBS). Twenty-two Wagyu female cattle were subjected to superovulation and were randomly distributed to form three groups: group 1 ­ uterine flushing with RL (n = 8), group 2 ­ uterine flushing with RL + 1% FBS (n = 7), and group 3 ­ uterine flushing with D-PBS (n = 7, control group). Cows received a CIDR® device containing 1.9 g of progesterone at random stages of the estrous cycle (day 0). Progesterone withdrawal occurred on day 8 in the morning. For heifers, 160 mg of porcine follicle-stimulating hormone (FSH-P) was used and for cows, 200 mg. Prostaglandin F2α was also injected on the eighth day of FSH-P administration. On day 9, in the morning, hCG was administered. Females were superovulated and inseminated twice in a fixed time for embryo transfer. On the 16th day, females were subjected to uterine flushing for embryo collection. We collected 76 embryos from 22 females subjected to superovulation, of which 52 were transferable and 24 had degenerated. The total of embryos collected was 23, 16, and 23 for groups 1, 2, and 3, respectively. The embryo recovery rates per group were 13.86±4.23, 15.39±4.61 and 27.16±13.33%, in groups 1, 2, and 3, respectively. The means for the total structures collected per female were 2.88±0.85, 3.00±1.23, and 4.57±1.72 in groups flushed with RL, RL + 1% FBS, and D-PBS, respectively. We conclude that Ringer's lactate solution and Ringer's lactate solution + 1% of FBS and Dulbecco's phosphate-buffered saline showed no significant differences in terms of embryo quality or quantity, suggesting that Ringer's lactate solution is an alternative for collecting embryos in cattle.

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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.

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Global cattle genetic market is experiencing a change of strategy, large genetic companies, traditionally recognized in the artificial insemination field, have also begun to operate in the embryo market. Consequently, the demand for in vitro produced (IVP) embryos has grown. However, the overall efficiency of the biotechnology process remains low. Additionally, the lack of homogeneity of post-cryopreservation survival results of IVP embryos still impairing a massive dissemination of this biotechnology in the field. A great challenge for in vitro production labs is to increase the amount of embryos produced with exceptional quality after each round of in vitro fertilization. Herein, we discuss the molecular and cellular features associated with the competence and cryosurvival of IVP embryos. First, morphofunctional, cellular and molecular competence of the embryos were addressed and a relationship between embryo developmental ability and quality were established with cryosurvival and pregnancy success. Additionally, determinant factors of embryo competence and cryosurvival were discussed including the following effects: genotype, oocyte quality and follicular microenvironment, in vitro production conditions, and lipids and other determining molecules. Finally, embryo cryopreservation aspects were addressed and an embryo-focused approach to improve cryosurvival was presented.

RESUMO

The earliest stages of embryo development are deeply influenced by reactive oxygen species (ROS), byproducts of the mitochondrial oxygen metabolism that play a key role as messengers in normal cell signal transduction and cell cycling. Despite its positive roles, the imbalance caused by the excess of ROS and an inefficient antioxidant system leads to oxidative stress, with negative consequences to the cell such as DNA damage, metabolic changes, mitochondrial stress and cell death. In the present work, crocetin - a natural antioxidant - was added to the culture media of bovine embryos to evaluate the efficiency of its antioxidant capability during embryo culture. Oocytes were in vitro matured (IVM) and fertilized according to standard protocols. Embryos were cultured at 38.5 °C under humidified air with 5% CO2, 7% O2, and 90% N2 in Synthetic Oviduct Fluid (SOF) medium supplemented with amino acids and either 5% of FBS (SOFaa) (control group) or SOFaa supplemented with 1  µM crocetin (crocetin group). After 5 days from the beginning of in vitro culture (IVC) (day 5 - D5), embryos were transferred to individual drops of culture media. At day 7 (D7), embryos were assessed by means of blastocyst rates, morphophysiological analyzes (total cell number, ROS and mitochondrial activity levels), transcript quantitation of 47 genes and metabolomic evaluation of the culture media by Raman spectroscopy. In the crocetin group blastocyst rates were higher and embryos had increased total cell number and decreased intracellular levels of ROS. These embryos also had upregulation of genes related with response to stress and lipid metabolism (ATF4, BAX, FOXO3, GADD45A, GPX1, GPX4, HSF1, SOD2, ACACA, SREBF1 and SREBF2). Raman spectroscopy corroborated these results indicating more active lipid and amino acid production in this group. The absence of crocetin in the culture media resulted in higher ROS level, as well as up regulation of genes related to DNA damage, stress response and energy metabolism (MORF4L2, SOD1, TXN, PFKP, PGK1 and PPARGC1A). In conclusion, crocetin supplementation during culture protects embryos from oxidative stress and influences the adaptive response to stress conditions, leading to an increase in both blastocyst yield and quality, as well as changes in transcriptomic and metabolic profile of in vitro produced bovine embryos.

Assuntos

RESUMO

Global cattle genetic market is experiencing a change of strategy, large genetic companies, traditionally recognized in the artificial insemination field, have also begun to operate in the embryo market. Consequently, the demand for in vitro produced (IVP) embryos has grown. However, the overall efficiency of the biotechnology process remains low. Additionally, the lack of homogeneity of post-cryopreservation survival results of IVP embryos still impairing a massive dissemination of this biotechnology in the field. A great challenge for in vitro production labs is to increase the amount of embryos produced with exceptional quality after each round of in vitro fertilization. Herein, we discuss the molecular and cellular features associated with the competence and cryosurvival of IVP embryos. First, morphofunctional, cellular and molecular competence of the embryos were addressed and a relationship between embryo developmental ability and quality were established with cryosurvival and pregnancy success. Additionally, determinant factors of embryo competence and cryosurvival were discussed including the following effects: genotype, oocyte quality and follicular microenvironment, in vitro production conditions, and lipids and other determining molecules. Finally, embryo cryopreservation aspects were addressed and an embryofocused approach to improve cryosurvival was presented.(AU)

Assuntos

RESUMO

Global cattle genetic market is experiencing a change of strategy, large genetic companies, traditionally recognized in the artificial insemination field, have also begun to operate in the embryo market. Consequently, the demand for in vitro produced (IVP) embryos has grown. However, the overall efficiency of the biotechnology process remains low. Additionally, the lack of homogeneity of post-cryopreservation survival results of IVP embryos still impairing a massive dissemination of this biotechnology in the field. A great challenge for in vitro production labs is to increase the amount of embryos produced with exceptional quality after each round of in vitro fertilization. Herein, we discuss the molecular and cellular features associated with the competence and cryosurvival of IVP embryos. First, morphofunctional, cellular and molecular competence of the embryos were addressed and a relationship between embryo developmental ability and quality were established with cryosurvival and pregnancy success. Additionally, determinant factors of embryo competence and cryosurvival were discussed including the following effects: genotype, oocyte quality and follicular microenvironment, in vitro production conditions, and lipids and other determining molecules. Finally, embryo cryopreservation aspects were addressed and an embryofocused approach to improve cryosurvival was presented.

Assuntos