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To identify markers of oocyte competence, we compared the biochemical characteristics of fluid and cells from follicles containing oocytes with different capacities to form an embryo. Follicles (5-6 mm) were dissected, and follicular fluid (FF), granulosa cells (GC), cumulus cells (CC) from immature and mature cumulus-oocyte-complexes (COC) were individually collected. The oocytes were matured, fertilized, and cultured individually until day 8 (D8) of development. On D8, the samples were grouped according to embryo production into those that gave rise to blastocysts (EMB) and those that did not reach the blastocyst stage (NEMB). In CCs from immature and mature COCs and GCs, expression of CASP3, SERPINE2, VCAN, LUM, FSHR, EGFR, PGR, and GHR genes was quantified. Cell-free DNA (cfDNA), progesterone, and estradiol concentrations in the FF were determined. Data were analyzed by Mann-Whitney U test (GraphPad Prism 9). GHR was highly expressed in immature CCs from the EMB group, whereas CASP3 was highly expressed in mature CCs from the NEMB group (P<0.05). During maturation, the expression of CASP3 and GHR genes increased only in the NEMB group. ART2 cfDNA was highly detected in FF of the NEMB compared to the EMB group. Progesterone concentration was similar between the groups, whereas estradiol concentration was higher (P<0.05) in the EMB than in the NEMB group. It was concluded that a higher level of GHR transcripts in immature CCs, lower CASP3 expression in CCs from matured COCs, lower levels of ART2, and higher estradiol concentrations in FF may indicate oocytes with greater potential for development.

Assuntos

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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.

Assuntos

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The effects of seasonality on the reproduction of stallions vary based on the latitude. Although previous studies have shown the influence of seasonality in raw semen quality in south-eastern Brazil, data regarding the influence of seasonality in cooled and frozen stored semen in Brazil is limited. Therefore, in this study, we have analysed if seasonality influences the hormone production (i.e., cortisol and testosterone), spermatogenesis, and quality of fresh, cooled, and frozen semen of stallions in central Brazil, and established the season most suitable for semen cryopreservation in a latitude of 15°S. Ten stallions were followed-up for one year, which was divided into two seasons, namely, drought, and rainy. Fresh, cooled, and frozen-thawed semen samples were assessed using CASA and flow cytometry. Additionally, the temperature and humidity index (THI) was calculated to determine the thermal stress. Although the THI varied between the two seasons, no thermal stress was observed throughout the year, nor were there differences in the physiological parameters of the stallions or plasma cortisol or testosterone levels. Furthermore, differences were not detected in total and progressive motility, sperm capacitation, and sperm membrane integrity, as well as in the number of live sperm with intact acrosomes and high mitochondrial membrane potential, between the two seasons in the fresh and frozen-thawed semen. Our data suggest that semen can be effectively collected and cryopreserved throughout the year within central regions of Brazil.

Assuntos

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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.

Assuntos

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We aimed to evaluate if protein source (PS) alterations during IVM affect embryo sex/development and gene expression profile in cumulus cells (CCs). Bovine oocytes were matured and cultured in the presence of FBS or BSA. Then, the PS effect during IVM on gene expression (GPC4, VCAN, GHR, PTGS2, and ALCAM) was determined. CC biopsy was removed before and after IVM treatments. After fertilization and cultured, CCs were grouped according to their fate into CCs from immature COCs, CCs from COCs that did or did not result in embryos (according to PS). Results showed that when the culture was performed in FBS presence, blastocyst rate was higher (p < 0.05) than BSA. However, when embryos were cultured with BSA, no effect (p > 0.05) of PS during IVM was observed. PS used during IVM did not affect embryos sex (p > 0.05) but changed VCAN, GHR, PTGS2, and ALCAM genes expression. No differences (p > 0.05) were observed between immature and mature CCs groups in gene expression, regardless of their fate. Only the GHR gene was related to embryo production but just with FBS on IVM. In conclusion, PS can affect embryo development when using the serum on IVM and IVC, influences CCs gene expression, and has to be considered when studying oocyte quality markers.

Assuntos

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The present study aimed to determine whether cumulus cells (CC) biopsy, acquired before or after in vitro maturation (IVM), presents similar gene expression pattern and if would compromises oocyte quality. First, immature cumulus oocyte complexes (COCs) were distributed: (1) maturated in groups (control); (2) individually maturated, but not biopsied; (3) subjected to CC biopsy before maturation and individually matured; (4) individually matured and submitted to CC biopsy after maturation; (5) individually matured and CC biopsied before and after maturation. Secondly, candidate genes, described as potential markers of COCs quality, were quantified by RT-qPCR in CCs before and after IVM. After in vitro fertilization (IVF), zygotes were tracked and sorted regarding their developmental potential: fully developed to embryo, cleaved and arrested, and not-cleaved. The COC's biopsy negatively affects embryo development (p < 0.05), blastocyst cell number (p < 0.05), and apoptotic cell ratio (p < 0.05), both before and after IVM. The PTGS2, LUM, ALCAM, FSHR, PGR, SERPINE2, HAS2, and PDRX3 genes were differentially expressed (p < 0.05) on matured CCs. Only PGR gene (p = 0.04) was under-expressed on matured CCs on Not-Cleaved group. The SERPINE2 gene was overexpressed (p = 0.01) in the Cleaved group on immature CCs. In summary, none of the selected gene studies can accurately predict COC's fate after fertilization.

RESUMO

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.

Assuntos

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Animal cloning is an important technique used to produce clones from valuable farm animals, to rescue animals in risk of extinction, and for producing transgenic animals. The objective of this work was to evaluate the effects of refrigeration on bovine ear skin as a strategy to transport biological material for long periods of time to isolate viable fibroblasts. Ears from eight cows were collected after death and stored for 30 days at 5°C. On days 0, 2, 4, 7, 14, 21, and 30, skin biopsies were cultured in vitro for fibroblast isolation. The time for first fibroblast outgrowth, time to reach 100% confluence. and cell concentration before freezing were observed for each period. In addition, plasma membrane integrity, cell apoptosis, and necrosis in cells were evaluated through fluorescent colorant combination in a flow cytometer from all periods after thawing. Fibroblasts obtained after 30 days of storage, considered a critical period, were tested for embryo production using nuclear transfer (NT) with micromanipulators. All time points allowed for cell culture. The time of cell growth onset was longer in samples refrigerated for 14, 21, and 30 days. The time to reach confluence also increased with longer refrigeration periods. Cells from day 0 reached confluence in 24 ± 2 days, while day 30 cells took 31 ± 0 days. Cell concentration and viability dropped with increased storage time and freezing/thawing, respectively. It was found that a long period of sample storage results in cell damage, making cultivation more difficult and decreasing cell viability post-thawing and cell concentration. However, when cells from day 30 were used as nuclei donors in NT, a 26.05% blastocyst rate after 7 days in culture was obtained. In conclusion, refrigeration at 5°C was shown to be efficient in maintaining viable tissue for up to 30 days, and fibroblasts isolated can be used for cloned embryo production.

Assuntos

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It is well-established that in vitro culture affects quality, gene expression, and epigenetic processes in bovine embryos and that trophectoderm cells are the most susceptible to abnormalities. These changes have been reported as the main factors responsible for losses observed after transfer of in vitro-produced embryos. The present study aimed to investigate the effect of an in vitro system on bovine embryo transcriptional profiles on D14 of development. Two groups were used-one with embryos produced in vitro until D7 (day 7; VT group) and another with embryos produced in vivo by hormonal stimulation, with embryos collected on D7 (VV group). D7 embryos at similar developmental stages from both treatments were transferred to recipient uteri and recollected on D14. From D14 embryos of both treatments, trophoblast samples were removed by biopsy for sexing and transcriptome analyses. Embryos were sexed by polymerase chain reaction (PCR), and only males were used for RNA sequencing. In total, 29,005 transcripts were expressed, from which 900 were differentially expressed, but only 29 genes were significantly differentially expressed. In addition, 20 genes were found uniquely for VV and 27 for VT. These findings suggested that although the uterine environment minimized transcriptional differences, it was not able to make trophoblasts from the in vitro embryos similar to the in vivo ones. The few genes exhibiting differences are in control of important events that may be responsible for embryonic losses occurring during the first period of gestation.

Assuntos

RESUMO

The present study aimed to identify biomarkers to assess the quality of in vitro produced (IVP) bovine embryos in the culture media. IVP embryos on Day (D) 5 of development were transferred to individual drops, where they were maintained for the last 48 h of culture. Thereafter, the medium was collected and the embryos were transferred to the recipients. After pregnancy diagnosis, the media were grouped into the pregnant and nonpregnant groups. The metabolic profiles of the media were analyzed via electrospray ionization mass spectrometry, and the concentrations of pyruvate, lactate, and glutamate were assessed using fluorimetry. The spectrometric profile revealed that the media from embryos from the pregnant group presented a higher signal intensity compared to that of the nonpregnant group; the ions 156.13 Da [M + H]+, 444.33 Da [M + H]+, and 305.97 Da [M + H]+ were identified as biomarkers. Spent culture medium from expanded blastocysts (Bx) that established pregnancy had a greater concentration of pyruvate (p = 0.0174) and lesser concentration of lactate (p = 0.042) than spent culture medium from Bx that did not establish pregnancy. Moreover, pyruvate in the culture media of Bx can predict pregnancy with 90.9% sensitivity and 75% specificity. In conclusion, we identified markers in the culture media that helped in assessing the most viable IVP embryos with a greater potential to establish pregnancy.

Assuntos