Current status of the intrafollicular transfer of immature oocytes (IFIOT) in cattle: A review.

Intrafollicular Transfer of Immature Oocytes (IFIOT) has emerged as an alternative to the currently used systems for bovine embryo production. This technique associates the rapid multiplication of bovine females under a completely in vivo culture condition, eliminating the need for superstimulatory hormones in the in vivo system (IVD) and the costly laboratory setup required for in vitro embryo production (IVP). Despite being a promising technique, the results obtained to date have been unsatisfactory for commercial use. Only approximately 10 % -12 % of viable embryos are recovered from the total number of injected oocytes, which limits their use in genetic improvement programs. IFIOT problems can occur in any of the steps involved; therefore, each step must be carefully examined to identify those that have the most negative impact on the final embryo recovery. This review summarizes the different studies conducted using the IFIOT to provide a comprehensive analysis of the main factors that can influence the effectiveness of this technique.

Biochemical profiling of the follicular environment to predict oocyte competence in cattle.

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.

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.

Seasonality does not influence cortisol or testosterone production, or seminal quality of stallions located at low latitudes.

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.

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.

Protein source in maturation media affects gene expression in cumulus cells and embryo development in cattle.

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.

Using Cumulus Cell Biopsy as a Non-Invasive Tool to Access the Quality of Bovine Oocytes: How Informative Are They?

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.

The use of insulin-transferrin-selenium (ITS), and folic acid on individual in vitro embryo culture systems in cattle.

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.

Effects of Refrigeration at 5°C for Long Periods of Time on Bovine Ear Skin as a Strategy to Transport Biological Material and Isolate Fibroblasts to Use in the Nuclear Transfer.

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.

Transcriptome of D14 in vivo x in vitro bovine embryos: is there any difference?

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.

Biochemical markers for pregnancy in the spent culture medium of in vitro produced bovine embryos†.

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.

Electrospray mass spectrometry analysis of blastocoel fluid as a potential tool for bovine embryo selection.

PURPOSE: The aim of this study was to analyze the metabolic profiles of blastocoel fluid (BF) obtained from bovine embryos produced in vivo and in vitro. METHODS: Expanded blastocysts (20/group) that were in vitro and in vivo derived at day 7 were used. BF was collected and analyzed under direct infusion conditions using a microTOF-Q® mass spectrometer with electrospray ionization and a mass range of 50-650 m/z. RESULTS: The spectrometry showed an evident difference in the metabolic profiles of BF from in vivo and in vitro produced embryos. These differences were very consistent between the samples of each group suggesting that embryo fluids can be used to identify the origin of the embryo. Ions 453.15 m/z, 437.18 m/z, and 398.06 m/z were identified as biomarkers for the embryo's origin with 100% sensitivity and specificity. Although it was not possible to unveil the molecular identity of the differential ions, the resulting spectrometric profiles provide a phenotype capable of differentiating embryos and hence constitute a potential parameter for embryo selection. CONCLUSION: To the best of our knowledge, our results showed, for the first time, an evident difference between the spectrometric profiles of the BF from bovine embryos produced in vivo and in vitro.

Histone deacetylase inhibitor during in vitro maturation decreases developmental capacity of bovine oocytes.

This study aimed to evaluate the effect of scriptaid during pre-maturation (PIVM) and/or maturation (IVM) on developmental competence of bovine oocytes. Cumulus-oocyte complexes (COCs) were submitted to PIVM for 6 h in the presence or absence of scriptaid. COCs were distributed into five groups: T1-IVM for 22 h, T2-PIVM for 6 h and IVM for 22 h, T3-PIVM with scriptaid for 6 h and IVM for 22 h, T4-PIVM for 6 h and IVM with scriptaid for 22 h, and T5-PIVM with scriptaid for 6 h and IVM with scriptaid for 22 h. Nuclear maturation, gene expression, cumulus cells (CCs) expansion, and embryo development and quality were evaluated. At the end of maturation, all groups presented the majority of oocytes in MII (P>0.05). Only HAT1 gene was differentially expressed (P<0.01) in oocytes with different treatments. Regarding embryo development at D7, T4 (23%) and T5 (18%) had lower blastocyst rate (P<0.05) than the other treatments (T1 = 35%, T2 = 37% and T3 = 32%). No effect was observed when scriptaid in PIVM was used in less competent oocytes (P>0.05). In conclusion, presence of scriptaid in PIVM and/or IVM did not improve developmental competence or embryo quality.

Blastocoel fluid removal and melatonin supplementation in the culture medium improve the viability of vitrified bovine embryos.

In this study, we investigated the effects of melatonin supplementation in the culture medium and blastocoel fluid removal (BFR) before vitrification on the quality and viability of in vitro-derived bovine embryos. After fertilization, presumptive zygotes were assigned to one of the following treatments: control, in vitro standard culture (IVC) medium; IVC + M10-9, IVC medium supplemented 10-9 M melatonin; or IVC + M10-9 BFR, IVC medium supplemented with 10-9 M melatonin plus BFR on day 7 (D7) of culture. D7 blastocysts were vitrified by the Cryotop method and, after 5 mo of storage, were warmed and incubated for an additional 72 h. The re-expansion rate was evaluated after 2 and 24 h, and the hatching rate was evaluated after 24, 48, and 72 h. At 72 h, the total number of cells (TNC); number of apoptotic cells (NAC); and expression of genes related to oxidative stress (HSPA5), cell metabolism (SLC2A3), cell repair (MSH6), placentation (KRT8 and PLAC8), and implantation (FOSL1) were assessed in the blastocysts. Less than 30% of the control blastocysts re-expanded until 2 h, whereas more than 85% of the IVC + M10-9 and IVC + M10-9 BFR blastocysts re-expanded (P < 0.05). The hatching rate of IVC + M10-9 BFR blastocysts increased at all time points (P < 0.05), reaching 66.8% at 72 h of incubation. The TNC was similar among treatments (P > 0.05), regardless of vitrification/warming and re-cultivation. The NAC:TNC was smaller for melatonin-treated blastocysts (P < 0.05). BFR increased HSPA5 (P = 0.0118) expression and did not affect SLC2A3, MSH6, KRT8, and FOSL1 expression (P > 0.05). In conclusion, melatonin (10-9 M) supplementation in the culture medium and BFR on D7 of culture increased the hatching rate 24, 48, and 72 h after warming of the vitrified embryos, indicating an improvement in cryotolerance.

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

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

Effects of the addition of oocyte meiosis-inhibiting drugs on the expression of maturation-promoting factor components and organization of cytoplasmic organelles.

The present study evaluated the effects of the blockade of meiosis in bovine oocytes by the cyclin-dependent kinase inhibitors roscovitine (ROS) and butyrolactone-I (BL-I) on nuclear maturation and extracellular signal-regulated kinase 1/2 (ERK1/2), cyclin B1 and p34cdc2 protein expression and localization. We also evaluated ultrastructural changes in oocytes. Immature oocytes were obtained from slaughtered bovines and divided into: (1) control (oocytes for in vitro maturation only in tissue culture medium-199 for 24 h), (2) oocytes that were treated with 12.5µMROS for 6 h, (3) oocytes that were treated with 50µMBL-I for 6 h and (4) oocytes that were treated with 6.25 µMROS+25 µMBL-I for 6 h. Incubation with inhibitors was followed by the reversal of blockade for 18 h. Oocytes then underwent immunohistochemical analysis to visualize chromatin and assess ERK1/2, cyclin B1 and p34cdc2 localization/expression, followed by preparation of the cells for ultrastructure analysis by electron microscopy. The groups at 6 h of maturation and before IVM exhibited the lowest number of oocytes in metaphase I. ROS group had the highest number of degenerating oocytes (p < 0.05). After maturation, majority of oocytes were in metaphaseII with no differences among groups (p> 0.05). ERK1/2, cyclin B1 and p34cdc2 expression differed throughout inhibition and oocyte maturation (p < 0.05). No difference was observed in the localization of these proteins in the ooplasm. No ultrastructural changes in oocytes were observed between treatments, with the exception of treatment with drugs that augmented lipid metabolism (p < 0.05). Results indicate that the effects of CDK1 inhibitors are reversible in bovine oocytes, indicated by nuclear, cytoplasmic, and molecular maturation parameters.

Shape and size of epididymal sperm from Gir bulls using atomic force microscopy: A nanoscale characterization of epididymal sperm.

As epididymal sperm (EP) are not exposed to seminal plasma, they are physiologically different from ejaculated spermatozoa (EJ). Therefore, the aim of this study was to morphologically characterize the head of EP recovered from the epididymis tail, and to evaluate if the physiological differences between EP and EJ were also expressed in the head's shape and size. EP and EJ were recovered from seven Gir bulls and were individually assessed. Sperm cells were washed, fixed, and 20 cells from each animal were analyzed by atomic force microscopy (AFM). The images were acquired through contact mode. Then, an off-line processing software was used and the images acquired were manually segmented using digital zoom of the original images. Twenty-four structural features were assessed including one, two, and three dimensional parameters, and also shape descriptors which were calculated based on the one and two dimensional parameters. Data were compared by t-test, then, a collective analysis was performed using principal component analysis (PCA). The EP group presented higher roughness and elongation (P ≤ 0.05), and smaller form factor and circularity rate than that of the EJ group (P ≤ 0.05). For the other parameters no differences (P ≥ 0.05) were observed. In addition, in the PCA analysis no differences among EP and EJ were observed either (P ≤ 0.05). This study showed that EP and EJ collected from the same sire presented similar characteristics in nineteen of the twenty-four parameters evaluated, indicating that absence of seminal plasma does not affect the morphology of EP.

Effect of delipidant agents during in vitro culture on the development, lipid content, gene expression and cryotolerance of bovine embryos.

In vitro produced embryos are still sensitive to the freezing process which can be explained, in part, by the high-lipid accumulation that characterizes these embryos. Therefore, we aimed to evaluate the effect of delipidating agents, L-carnitine and the trans-10 cis-12 conjugated linoleic acid (CLA) isomer, on blastocyst development, lipid content, gene expression and cryotolerance when added to embryo culture media. Embryos were cultured in four different media: T1: control (n = 616), synthetic oviduct fluid (SOF) media with 5% foetal bovine serum (FBS); T2: L-carnitine (n = 648), SOF medium with 5% FBS and 0.6 mg/ml of L-carnitine; T3: CLA (n = 627), SOF medium with 5% FBS and 100 µM trans-10 cis-12 CLA; and T4: L-carnitine + CLA: (n = 597), SOF medium with 5% FBS plus 0.6 mg/ml L-carnitine and 100 µM trans-10 cis-12 CLA. Supplementation of culture medium with either or both delipidating agents reduced (p < .05) blastocyst rate on D7 (T1 = 49 ± 3.5; T2 = 39 ± 3.0; T3 = 42 ± 3.9 and T4 = 39 ± 3.9), but did not affected gene expression (p > .05). Although embryos cultured in the presence of L-carnitine contained fewer (p < .05) lipid droplets than the control embryos, they showed a lower re-expansion rate 24 hr post-thaw than those (p < .05). In conclusion, although L-carnitine reduced the amount of lipids in cultured embryos, the use of L-carnitine and CLA during in vitro culture was not able to improve the embryo production and the response to cryopreservation.

DNA Methylation of the Insulin-Like Growth Factor 2-Imprinted Gene in Trophoblast Cells of Elongated Bovine Embryo: Effects of the In Vitro Culture.

DNA methylation is an essential epigenetic mark for embryo development and can be susceptible to environment factors such as in vitro conditions. The aim of this study was to verify the effect of in vitro culture until Day (D) 14 of the development on the embryo size and DNA methylation pattern of the insulin-like growth factor 2 (IGF2)-imprinted gene. To achieve this, we produced bovine embryos completely in vivo, completely in vitro, and in vitro until D7 and then in vivo up to D14. The embryos produced in in vitro were smaller than those in other two groups (p = 0.024); no differences in embryo size were observed between genders. The in vitro embryos showed a higher level of DNA methylation in the IGF2 as compared with that in the completely in vivo-produced (IVV) embryos (p = 0.009). Furthermore, totally in vitro-produced male embryos showed higher levels of DNA methylation as compared with those observed for the totally IVV male embryos (p = 0.034). No differences were observed among genders for IGF2 DNA methylation. These results showed that the window between D7 and D14 is critical for embryo development and alterations in the environmental conditions during this period can impair DNA methylation establishment of important developmental imprinted genes. This study brings unprecedented data for bovine embryos regarding the impact of the environmental conditions during the posthatching development.

The dynamics of gene expression, lipid composition and DNA methylation reprogramming are different during in vitro maturation of pig oocytes obtained from prepubertal gilts and cycling sows.

This study aimed to characterize the gene expression, lipid composition and DNA methylation reprogramming during in vitro maturation (IVM) of pig oocytes with different developmental competencies. We used prepubertal gilts and cycling sows as a model to obtain oocytes with different levels of competency. We found that genes involved in lipid metabolism, SLC27A4, CPT2 and PLIN2, and DNA methylation, DNMT3A, TET1 and TET3, possessed altered transcript expression levels during IVM. Specifically, SLC27A4 mRNA (p = 0.05) increased in oocytes from cycling females, whereas CPT2 (p = 0.05), PLIN2 (p = 0.02) and DNMT3A (p = 0.02) increased in oocytes from prepubertal females during IVM. Additionally, TET3 mRNA increased during IVM in oocytes from prepubertal (p = 0.0005) and cycling females (p = 0.02). The TET1 transcript decreased (p = 0.05) during IVM in oocytes from cycling sows. Regarding lipid composition, mass spectrometry revealed a cluster of ions, with molecular masses higher than m/z 700, which comprises a group of complex phospholipids, was identified in all groups of oocytes, except in those from prepubertal gilts. With respect to DNA methylation reprogramming, it was noted that the less competent oocytes were not able to reprogramme the XIST gene during IVM. We conclude that the maternal mRNA store, lipid composition and epigenetic reprogramming are still being established during maturation and are related to oocyte competence. In addition, we propose that the methylation pattern of the XIST may be used as molecular marker for oocyte competence in pigs.