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Increasing evidence suggests that environmental exposures can modify epigenetic marks in the germline, leading to the transmission of abnormal post-fertilization sperm epigenetic indicators and affecting embryonic development. Given the pivotal role of sperm cells in determining embryo quality, there is growing interest in understanding the potential effects of sperm sex sorting on embryo quality. This study aimed to investigate the impact of bovine sperm sexing on in vitro embryo production (IVP) and to associate molecular aspects of embryos analysis. Frozen semen samples from five Nellore bulls were used, with each bull contributing unsexed sperm (conventional semen - CV treatment) and female and male sexed sperm pooled after thawing (SX treatment). First, semen quality was assessed, including motility, morphology, acrosome integrity, and chromatin integrity to denaturation. Then, IVP was carried out, focusing on embryonic production and developmental kinetics. In the third experiment, embryo quality was evaluated by examining the gene expression of key markers (OCT4, NANOG, DNMT3A, TET1, and Fematrin-1) and the methylation pattern of the Satellite-1 and α-Satellite genes in blastocysts. Differences between CV and SX semen were only observed in motility, which was lower in SX compared with CV (P < 0.05). Although cleavage was similar, the SX groups showed lower blastocyst production than CV (P < 0.05). Of the genes evaluated, only NANOG showed high expression in the CV blastocysts compared with the SX blastocysts, but the methylation pattern revealed no differences. In conclusion, sex sorting markedly affects sperm motility and in vitro embryo production but showed no significant impact on embryo quality., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest I, Margot Alves Nunes Dode, author responsible for submitting the manuscript entitled “Current status of the intrafollicular transfer of immature oocytes (IFIOT) in cattle: a review” declare that I am not subject to any type of conflict of interest with the participants or any other collaborator, direct or indirect., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Purpose: This study aimed to identify a marker for freezability and in vitro fertility of sperm samples before freezing., Methods: Semen was collected from nine Nelore bulls; half of the ejaculate was used for seminal plasma cell-free DNA (cfDNA) quantification, and the other half was cryopreserved. Evaluation of sperm movement using computer-assisted semen analysis and plasma membrane integrity and stability, acrosomal integrity, apoptosis, and mitochondrial potential using flow cytometry were performed on fresh and frozen/thawed semen at 0, 3, 6, and 12 h after thawing. Frozen/thawed sperm was also used for in vitro embryo production. cfDNA was extracted from each bull, and the total DNA and number of cell-free mitochondrial DNA (cfmtDNA) copies were quantified. Semen from each animal was used for IVF, and cleavage, blastocyst formation, and cell counts were evaluated., Results: Two groups were formed and compared based on the concentrations of cfDNA and cfmDNA present: low-cfDNA and high-cfDNA and low-cfmtDNA and high-cfmtDNA. Up to 12 h post-thawing, there were no differences between the groups in the majority of the sperm parameters evaluated. Cleavage, day 6 and 7 blastocyst rates, and the number of cells were higher in the high cfDNA group than in the low cfDNA group. Similar results were observed for cfmtDNA, except for the number of cells, which was similar between the groups., Conclusion: The concentration of cfDNA and the relative number of copies of cfmtDNA in seminal plasma cannot predict the freezability of semen but can be used to predict in vitro embryo production., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Scrotal surface thermography is a non-invasive method for assessing testicular thermoregulation in stallions; however, few studies have explored the application of this technique concerning the thermal physiology of equine reproductive systems. This study aimed to evaluate the consistency of testicular thermoregulation in stallions over a year using thermography to measure the scrotal surface temperature (SST). Moreover, we assessed the best region for measuring the surface body temperature compared with the SST. Ten light-breed stallions were used in the experiment. Thermographic images of the scrotal and body surfaces (neck and abdomen) were captured. Fresh, cooled and frozen-thawed semen samples were evaluated to verify the impact of thermoregulation on semen quality. Testicular thermoregulation was maintained throughout the year in stallions amidst changes in the external temperature, as evidenced by the weak correlation between the SST and ambient temperature. A lower correlation was observed between the environmental temperature and body surface temperature (BTS) obtained from the abdomen (BTS-A; R = .4772; p < .0001) than with that obtained from the neck (BTS-N; R = .7259; p < .0001). Moreover, both BTS-A and SST were simultaneously captured in a single image. The consistent quality of the fresh, cooled and frozen semen suggests efficient thermoregulation in stallions throughout the year., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Kussano et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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.

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.

This study aimed to evaluate the effects of donor age on lipid metabolism during in vitro maturation (IVM) of pigs cumulus-oocyte complexes (COCs). We evaluated transcript levels of genes, the percentage of ooplasm occupied by lipid droplets (LD) and evaluated DNA methylation in COCs from sows and prepubertal gilts. Transcript levels of six genes (ACACA, ACSS2, FASN, FABP3, SLC27A4, PLIN2), which were analyzed in cumulus cells (CCs), increased after 44 h of IVM in the sow group. In the gilt group, only FASN expression increased, while NR3C1 expression decreased after IVM. The measurement of LD in oocytes showed an accumulation of lipids in sow oocytes during IVM, while gilt oocytes showed a decrease in LD. FABP3 and NR3C1 methylation patterns exhibited a demethylation pattern in CCs and oocytes from gilts and sows and showed statistical differences between groups. CCs from sows had a better capacity to change transcription levels of the major genes involved in lipid metabolism during IVM than CCs from gilts. This difference may be involved in accumulation of lipids, acquisition of competence, and maturation of enclosed oocytes. Our results contribute to a better understanding of mechanisms involved in lipid metabolism and acquisition of competence in porcine COCs., (© 2023 Wiley Periodicals LLC.)

Despite many studies in humans and mice using genome transfer (GT), there are few reports using this technique in oocytes of wild or domestic animals. Therefore, we aimed to establish a GT technique in bovine oocytes using the metaphase plate (MP) and polar body (PB) as the sources of genetic material. In the first experiment, GT was established using MP (GT-MP), and a sperm concentration of 1 × 10 6 or 0.5 × 10 6 spermatozoa/ml gave similar fertilization rates. The cleavage rate (50%) and blastocyst rate (13.6%) in the GT-MP group was lower than that of the in vitro production control group (80.2% and 32.6%, respectively). The second experiment evaluated the same parameters using PB instead of MP; the GT-PB group had lower fertilization (82.3% vs. 96.2%) and blastocyst (7.7% vs. 36.8%) rates than the control group. No differences in the amount of mitochondrial DNA (mtDNA) were observed between groups. Finally, GT-MP was performed using vitrified oocytes (GT-MPV) as a source of genetic material. The cleavage rate of the GT-MPV group (68.4%) was similar to that of the vitrified oocytes (VIT) control group (70.0%) and to that of the control IVP group (81.25%, P < 0.05). The blastocyst rate of GT-MPV (15.7) did not differ neither from the VIT control group (5.0%) nor from the IVP control group (35.7%). The results suggested that the structures reconstructed by the GT-MPV and GT-PB technique develop in embryos even if vitrified oocytes are used., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Somatic cell nuclear transfer (SCNT) is commercially used despite incomplete nuclear reprogramming of the somatic cell nucleus by the enucleated oocyte compromising its efficiency. Oocyte selection is a key factor in increasing this efficiency as its cytoplasm reprograms the differentiated cell. In this study, we adapted a methodology to characterize epialleles in potential epigenetic markers in single in vitro matured oocytes. Characterization of the regions that control the expression of imprinted genes, X-chromosome inactivation, and satellite I DNA (IGF2, ICR-H19, XIST, RepA, and SAT1) showed methylated and unmethylated alleles in the imprinted genes IGF2 and ICR-H19 while XIST-DMR1 and RepA showed hypermethylated alleles. There was great variation in methylation patterns for candidate regions which may be related to oocyte quality. Moreover, the identification of different epialleles in the same oocyte suggests that, at least for those loci, the epigenome of the metaphase plate and polar body is different. The single-cell bisulfite polymerase chain reaction technique can be used to improve the precision of selecting the best oocytes for SCNT procedures, thereby increasing its efficiency., (© 2023 Wiley Periodicals LLC.)

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., Competing Interests: Declaration of interest None of the authors of this manuscript has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of this article., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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., (© 2022 Wiley-VCH GmbH.)

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.

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.

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., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Maternal nutrition is critical in mammalian development, influencing the epigenetic reprogramming of gametes, embryos, and fetal programming. We evaluated the effects of different levels of sulfur (S) and cobalt (Co) in the maternal diet throughout the pre- and periconceptional periods on the biochemical and reproductive parameters of the donors and the DNA methylome of the progeny in Bos indicus cattle. The low-S/Co group differed from the control with respect to homocysteine, folic acid, B12, insulin growth factor 1, and glucose. The oocyte yield was lower in heifers from the low S/Co group than that in the control heifers. Embryos from the low-S/Co group exhibited 2320 differentially methylated regions (DMRs) across the genome compared with the control embryos. We also characterized candidate DMRs linked to the DNMT1 and DNMT3B genes in the blood and sperm cells of the adult progeny. A DMR located in DNMT1 that was identified in embryos remained differentially methylated in the sperm of the progeny from the low-S/Co group. Therefore, we associated changes in specific compounds in the maternal diet with DNA methylation modifications in the progeny. Our results help to elucidate the impact of maternal nutrition on epigenetic reprogramming in livestock, opening new avenues of research to study the effect of disturbed epigenetic patterns in early life on health and fertility in adulthood. Considering that cattle are physiologically similar to humans with respect to gestational length, our study may serve as a model for studies related to the developmental origin of health and disease in humans.

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2021. The Society for In Vitro Biology.)

To evaluate the addition of antioxidants in extenders on post-thaw bovine semen quality and in vitro embryo production efficiency. Six semen samples were collected from five Holstein bulls. In the experiment I, the samples were diluted with AndroMed® and Bovimix® and added antioxidants glutathione (1.5 and 2.5 mM) and melatonin (0.5 and 1.0 mM). In the experiment II, the best treatments obtained in experiment I were used for in vitro fecundation. Glutathione did not improve sperm viability. Melatonin had a negative effect on semen characteristics. Andromed® showed better results in sperm kinetics parameters. Bovimix® was more efficient in maintaining cell integrity parameters. Significant correlation was found between sperm kinetics parameters and between cell integrity parameters. For in vitro embryo production, after oocyte selection, maturation, fertilization and cultivation were performed using the four treatments previously evaluated. Andromed® was more efficient in the cleavage rate, no effect of the addition of glutathione. However, the addition of 2.5 mM glutathione in the Bovimix® improved the cleavage rate. There was a significant moderate correlation between cleavage rate and sperm kinetic characteristics. Glutathione did not improve sperm viability. Melatonin reduced the maintenance of sperm characteristics. Andromed® was more efficient in in vitro embryo production and no effect of glutathione was found in this extender. Addition of 2.5 mM glutathione in the Bovimix® extender provided a higher cleavage rate.

The maturation kinetics and in vitro fertilization of immature bovine oocytes injected by the intra-follicular oocyte injection (IFOT) technique into pre-ovulatory follicles of previously synchronized cows were evaluated. In Experiment 1, grade I, II and III cumulus-oocyte complexes (COCs) were randomly distributed to one of three Groups: Matvitro22 (COCs matured in vitro for 22 h), MatFol20 and MatFol28 (COCs matured in vivo after being injected into a pre-ovulatory follicle of previously synchronized cows for 19.8 ± 0.1 h and 28.3 ± 0.1 h, respectively). Cows received 12.5 mg of LH (Lutropin, Bioniche, Canada) at the time of IFOT in the MatFol20 Group or 10 h after IFOT in the MatFol28 Group. MatFol20 and MatFol28 COCs were aspirated approximately 20 h after the LH injection for nuclear maturation kinetics and recovery rate assessment. In Experiment 2, grade I, II, and III COCs were randomly distributed into two Groups: Matvitro22 Group, COCs were matured and fertilized in vitro, and MatFol20 Group, COCs were matured as in the MatFol20 Group in Experiment 1, but COCs were fertilized in vitro. Putative zygotes were classified as fertilized, unfertilized or polyspermic. In Experiment 1, the recovery rate was lower (P < 0.001) in the MatFol20 Group (52.9%, 91/172) compared with MatFol28 (72.9%, 113/155). Rate of oocytes in germinal vesicle stage, metaphase I, anaphase I and telophase I were similar among Groups. However, oocytes matured in vivo for 28.3 h had lower rate of metaphase II (P = 0.001) and greater rates of degenerated (P = 0.001) and parthenogenetically activated (P = 0.001) oocytes. In experiment 2, the rates of polyspermy and degenerated were similar between Groups. However, the rate of fertilized oocytes was greater (P = 0.05) in oocytes in the MatFol20 Group. It is concluded that oocyte in vivo maturation for 19.8 h after IFOT does not compromise the nuclear maturation kinetics and increases in vitro fertilization rates. However, the extra 10 h of intra-follicular incubation time decreased oocyte viability., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist.

This study evaluated the effects of three maturation systems, namely invitro (MatV) and invivo (MatS) systems, as well as intrafollicular transfer of immature oocytes (IFIOT; MatT), on the accumulation of lipid droplets in bovine oocytes. Lipids were evaluated using confocal microscopy and transmission electron microscopy. The expression of genes related to lipid metabolism, namely acyl-CoA synthetase short chain family member 2 (ACSS2), ELOVL fatty acid elongase 1 (ELOVL1) and fatty acid binding protein 3 (FABP3), was quantified by quantitative polymerase chain reaction. The mean (±s.d.) area occupied by lipids in immature oocytes (13±2%) was similar to those matured invivo (MatS, 16±2%; MatT, 12±2%). However, there was a significant increase in lipids in oocytes in the MatV group (24±2%) compared with all other groups (P<0.001). In the ultrastructural evaluations, MatV oocytes also showed the highest lipid content. The expression of ELOVL1 and FABP3 was similar in the MatS and IFIOT groups. However, transcript levels of ACSS2 were lower in IFIOT than MatV oocytes. These results indicate, for the first time, that oocytes matured by IFIOT are similar to those matured invivo with regard to lipid accumulation, which indicates better quality than those matured invitro.

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., (Copyright © 2020. Published by Elsevier Inc.)

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., Competing Interests: The authors report no conflicts of interest., (Copyright © 2020 Andrei Antonioni Guedes Fidelis et al.)

We aimed to elucidate the effects of PGE2 and PGF2α on the in vitro maturation (IVM) of bovine oocytes. First, cumulus-oocyte complexes were matured in the media supplemented with or without PGE2, PGF2α, or PGE2 plus PGF2α for the final 24, 12, or 6 h of culture. Then, the cumulus-oocyte complexes were matured in the absence or presence of a PG endoperoxide synthase 2 (PTGS2) enzyme inhibitor (NS398) supplemented with PGE2, PGF2α, or PGE2 plus PGF2α. Finally, the expression of genes associated with PGs activity in cumulus cells (PTGS2, PG E-synthase-1 [PTGES1], and aldo-keto reductase 1 [AKR1B1]) or oocytes (receptors for PGE2 [PTGER2] and PGF2α [PTGFR]) of different competencies was quantified. Supplementation of the IVM medium with PGs did not improve in vitro embryo production or embryo quality (P > 0.05). During maturation, the relative abundance of PTGS2 transcripts increased (P < 0.05) only in the less-competent group, whereas those of PTGES1 increased in the less-competent and in the more-competent groups. Conversely, AKR1B1 expression decreased only in the less-competent group (P < 0.05). Receptors for the PGE2 and PGF2α genes were very low or undetectable in oocytes. In conclusion, PGE2 and PGF2α are not recommended for media supplementation during maturation because they have no effect on embryo development. Although genes related to PGs activity are differentially expressed in cumulus cells of cumulus-oocyte complexes of different competence during maturation, the expression of PGE2 and PGF2α receptor genes was either not detectable or was detected at low levels in oocytes., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In the present study, four experimental groups were used: fresh embryos, cultured during in vitro maturation and in vitro culture in media supplemented with bovine serum albumin (BSA) (fresh BSA) or fetal bovine serum (FBS) (fresh FBS); and two groups of cryopreserved and thawed embryos, produced under the same conditions (frozen BSA and frozen FBS). Experiment 1 evaluated the protein source effect on embryo development and response to cryopreservation. At day 7, half of the expanded blastocysts (Bx) from each group were cryopreserved and warmed and the other half were used as controls. After warming, embryos were incubated under the same conditions for 48 hours, and the hatching rate was measured at 24 and 48 hours. The total and the apoptotic cell numbers were measured in a subset of Bx after 24 hours. Experiment 2 used the Bx of experiment 1 to compare the expression of KRT8 , PLAC8 , FOSL1 , HSP1A1 , and HSPA5 genes in hatched blastocysts at 24 and 48 hours for all groups. The FBS group showed a higher percentage ( p  < 0.05) of embryos (42.8% vs. 27.9%) and higher rates of Bx (75.0% vs. 63.8%) on day 7, compared with the BSA group. At 24 hours postwarming, the fresh FBS group showed the highest hatching rate ( p  < 0.05) in comparison with other treatments. However, at 48 hours, the hatching rate was similar ( p  > 0.05) among groups: fresh FBS (68.1% ± 23.3%), fresh BSA (70.0% ± 31.0%), frozen FBS (39.2 ± 27.1), and frozen BSA (38.2 ± 23.9). After 24 hours, frozen BSA showed a higher number of cells compared with frozen FBS ( p  < 0.05). The expression of the PLAC8 gene was higher ( p  < 0.05) in fresh BSA embryos compared with frozen FBS embryos at 24 hours. In the present study, BSA replacement reduced embryo development, but did not affect the response to cryopreservation. However, upregulation of the PLAC8 gene suggests that embryos cultured in BSA might have better quality to support further development.

Valuable female cattle are continuously subject to follicular puncture (ovum pick-up - OPU). This technique is commonly used for in-vitro embryo production, but may result in ovarian lesion. Mesenchymal stem cells (MSC) ameliorate the function of injured tissues, but their use to treat ovarian lesions in cattle has not been established. We investigated whether a local injection of MSC would reduce the negative effects of repeated OPU under acute and chronic scenarios in bovines. First, we performed four OPU sessions and injected 2.5 × 10 6 MSCs immediately after the 4th OPU procedure (n = 5). The treated organs (right ovary) were compared to their saline-treated counterparts (left), and presented superior production of oocytes and embryos in the three following OPU sessions (P < 0.05). Then, cows with progressive fertility loss went through three OPU sessions. Animals received MSC, saline, or MSC + FSH in both ovaries after the first OPU. In the two following OPU sessions, the MSC and MSC + FSH - treated groups failed to present any significant alteration in the number of oocytes and embryos compared to saline-treated animals. Thus, MSC have beneficial effects on the fertility of OPU-lesioned cows, but not in cows with cystic ovarian disease and chronic ovarian lesions.

We aimed to analyse the histone acetylation status and expression profile of genes involved in histone acetylation (histone acetyltransferase 1 (HAT1), lysine acetyltransferase 2A (KAT2A), histone deacetylase 1(HDAC1), HDAC2 and HDAC3) in bovine oocytes of different competences during invitro maturation (IVM). Cumulus-oocyte complexes were recovered from two groups of follicles: minor follicles (1.0-3.0mm in diameter), classified as low competence (LC) and large follicles (6.0-8.0mm in diameter) classified as high competence (HC). Oocytes were submitted to IVM for 0, 8 and 24h and stored for analysis. Acetylation status of histone H4 on lysine K5, K6, K12 and K16 was assessed by immunohistochemistry. For gene expression, mRNA levels were determined by real-time quantitative polymerase chain reaction. All oocytes, regardless of their competence, showed a gradual decrease (P<0.05) in acetylation signals during IVM. From 0 to 8h of maturation, an increase (P<0.05) in the relative abundance of HAT1 mRNA was observed only in the HC oocytes. In this group, higher (P<0.05) mRNA levels of HDAC1 at 8h of maturation were also observed. In conclusion, in the present study, LC oocytes were shown to have adequate acetylation levels for the resumption and progression of meiosis; however, these oocytes do not have the capacity to synthesise RNA during IVM as the HC oocytes do.

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 p34 cdc2 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 p34 cdc2 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 p34 cdc2 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., Competing Interests: Declaration of Competing Interest Authors have no conflict of interest with this study., (Copyright © 2020 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The author affirm that this study don’t have conflict of interest., (Copyright © 2019 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

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., (© 2019 Blackwell Verlag GmbH.)

Male and female embryos are known to be different in developmental kinetics, metabolism, gene expression, and epigenetic patterns. Therefore, the objective of this study was to clarify whether the morphological criteria used to select embryos for cryopreservation lead to a deviation in the male:female ratio, and whether vitrification effects vary according to embryo sex. Initially, five sires were tested to evaluate the effect of the bull on embryo development, sex ratio, speed of development, and response to cryopreservation. Results showed that bulls affected (P < 0.05) embryo production, response to cryopreservation, and sex ratio. Then, one bull was selected, and used to produce embryos in vitro to characterize the responses of male and female embryos to vitrification. Results suggested that male and female embryos have the same morphological responses to vitrification, as no differences (P > 0.05) were observed between the two sexes in post-warming survival and re-expansion rates. However, their molecular responses as evaluated by gene expression (FOSL1, HSPB1, CASP3, CASP8, HSPA5, HSPA1A, G6PD, and PGK1) analysis indicated an effect of sex on vitrification; vitrified female embryos exhibited higher mRNA levels of HSPA1A, CASP3, and G6PD compared to their male counterparts. In conclusion, bulls affected embryo production, speed of development, sex ratio, and response to cryopreservation. Male and female embryos differed in their molecular responses to vitrification; and also, deviations in the male:female ratio when selecting embryos for cryopreservation were confirmed., (Copyright © 2019. Published by Elsevier Inc.)

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.

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., (© 2019 Blackwell Verlag GmbH.)

XIST, in association with the shorter ncRNA RepA, are essential for the initiation of X chromosome inactivation (XCI) in mice. The molecular mechanisms controlling XIST and RepA expression are well characterized in that specie. However, little is known in livestock. We aimed to characterize the DNA methylation status along the 5' portion of XIST and to characterize its transcriptional profile during early development in cattle. Three genomic regions of XIST named here as promoter, RepA and DMR1 had their DNA methylation status characterized in gametes and embryos. Expression profile of XIST was evaluated, including sense and antisense transcription. Oocytes showed higher levels of methylation than spermatozoa that was demethylated. DMR1 was hypermethylated throughout oogenesis. At the 8-16-cell embryo stage DMR1 was completed demethylated. Interestingly, RepA gain methylation during oocyte maturation and was demethylated at the blastocyst stage, later than DMR1. These results suggest that DMR1 and RepA are transient differentially methylated regions in cattle. XIST RNA was detected in matured oocytes and in single cells from the 2-cell to the morula stage, confirming the presence of maternal and embryonic transcripts. Sense and antisense transcripts were detected along the XIST in blastocyst. In silico analysis identified 63 novel transcript candidates at bovine XIST locus from both the plus and minus strands. Taking together these results improve our understanding of the molecular mechanisms involved in XCI initiation in cattle. This information may be useful for the improvement of assisted reproductive technologies in livestock considering that in vitro conditions may impair epigenetic reprogramming.

The hG-CSF (human Granulocyte Colony-Stimulating Factor) is a growth and stimulation factor capable of inducing the proliferation of bone marrow cells, several types of leukocytes, among other hematopoietic tissue cells. hG-CSF is used in used to treat anomalies that reder a small number of circulating white blood cells, which may compromise the immune defenses of the affected person. For these reasons, the production of hG-CSF in a bioreactor system using the mammary gland of genetic modified animals is a possibility of adding value to the bovine genetic material and reducing the costs of hG-CSF production in pharmaceutical industry. In this study, we aimed the production of transgenic hG-CSF bovine through the lipofection of bovine primary fibroblasts with an hG-CSF expression cassette and cloning these fibroblasts by the somatic cell nuclear transfer (SCNT) technique. The bovine fibroblasts transfected with the hG-CSF cassette presented a stable insertion of this construct into their genome and were efficiently synchronized to G0/G1 cell cycle stage. The transgenic fibroblasts were cloned by SCNT and produced 103 transferred embryos and 2 pregnancies, one of which reached 7 months of gestation., Competing Interests: No potential conflict of interest relevant to this article was reported.

This study aimed to establish a protocol for in vitro embryo production using epididymal sperm (EP). Samples were obtained from ejaculated sperm (EJ) and the epididymis of 7 Gir bulls. First, the effect of heparin (+) on the viability, longevity (Experiment 1) and fertilization rates (Experiment 2) of the EP was evaluated. In experiment 2, a pool of EP and EJ sperm (n = 7) was coincubated with cumulus-oocyte complexes (COCs) for 0, 3, 6, 12 and 18 h, and the fertilization rate (FR) was evaluated. A third experiment was performed to test sperm treatments for IVP using the Percoll (P) or PureSperm (PS) gradients or a spTALP wash for sperm selection. Cleavage, blastocyst rate (BR) and embryo sex were evaluated. In experiment 4, embryos were produced using 6, 12, and 18 h of sperm-oocyte coincubation. The cleavage, BR, and total number and percentage of apoptotic cells were determined. Heparin affected EP viability, longevity and FR. After 6 h, 82% of the oocytes were fertilized in the EP+ group, a higher value (P<0.05) than that in the EJ (19%) and EP- (42%) groups. At 12 and 18 h, FR remained higher in the EP+ group, and a gradual increase in polyspermy was observed. The use of a P or PS gradient yielded a similar BR on D7 (54% and 52%), which was higher than the rate obtained using the washing method (37%). The embryos produced by EP and selected in a P or PS gradient resulted in a sex deviation in favor of male embryos (P>0.05). No differences (P>0.05) were observed among the groups that were coincubated for 6, 12 and 18 h with respect to embryo production, kinetics of development, total cell number and percentage of apoptotic cells. In conclusion, IVF time can be reduced to 6 h without affecting embryo production and quality. In addition, EP sperm selection can be performed by either a PS or P gradient., Competing Interests: The authors have declared that no competing interests exist.

Effects of adding different concentrations of melatonin (10 -7 , 10 -9 and 10 -11  M) to maturation (Experiment 1; Control, IVM + 10 -7 , IVM + 10 -9 , IVM + 10 -11 ) and culture media (Experiment 2; Control, IVC + 10 -7 , IVC + 10 -9 , IVC + 10 -11 ) were evaluated on in vitro bovine embryonic development. The optimal concentration of melatonin (10 -9  M) from Experiments 1-2 was tested in both maturation and/or culture media of Experiment 3 (Control, IVM + 10 -9 , IVC + 10 -9 , IVM/IVC + 10 -9 ). In Experiment 1, maturated oocytes from Control and IVM + 10 -9 treatments showed increased glutathione content, mitochondrial membrane potential and percentage of Grade I blastocysts (40.6% and 43%, respectively). In Experiment 2, an increase in the percentage of Grade I blastocysts was detected in IVC + 10 -7 (43.5%; 56.7%) and IVC + 10 -9 (47.4%; 57.4%). Moreover, a lower number and percentage of apoptotic cells in blastocysts were observed in the IVC + 10 -9 group compared to Control (3.8 ± 0.6; 3.6% versus 6.1 ± 0.6; 5.3%). In Experiment 3, the IVC + 10 -9 treatment increased percentage of Grade I blastocysts with a lower number of apoptotic cells compared to IVM/IVC + 10 -9 group (52.6%; 3.0 ± 0.5 versus 46.0%; 5.4 ± 1.0). The IVC + 10 -9 treatment also had a higher mRNA expression of antioxidant gene (SOD2) compared to the Control, as well as the heat shock protein (HSPB1) compared to the IVM + 10 -9 . Reactive oxygen species production was greater in the IVM/IVC + 10 -9 treatment group. In conclusion, the 10 -9  M concentration of melatonin and the in vitro production phase in which it is used directly affected embryonic development and quality., (© 2017 Blackwell Verlag GmbH.)

In an attempt to improve in vitro embryo production, we investigated the effect of FGF10 and 0.1 mM of cilostamide, cAMP modulator, during in vitro prematuration (PIVM) and in vitro maturation (IVM) on the developmental capacity of bovine oocytes. Five treatments (T) were used as follows: T1) IVM (control): COCs were matured for 22 h; T2) PIVM/IVM: COCs were submitted to 22 h of PIVM and 22 h of IVM; T3) PIVM + FGF10/IVM: COCs were submitted to PIVM for 22 h in the presence of FGF10 and matured for 22 h; T4) PIVM/IVM + FGF10: COCs were submitted to PIVM for 22 h and matured for 22 h in the presence of FGF10; and T5) PIVM + FGF10/IVM + FGF10: COCs were submitted to 22 h of PIVM and 22 h of IVM, both in the presence of FGF10. COCs were evaluated for CC expansion and nucleus configuration at 0 h, 22 h of PIVM and 22 h after IVM. At those time points, CCs were used for expression analysis of PTGS2, TNFAIP6, PTX3, HAS2, CASP8, CASP3, SLC2A1, SLC2A3 and FGFR2 genes. Then, embryo production was evaluated on D2 for cleavage and at D6, D7 and D8 for embryo development. Embryo quality was measured by the speed of development and by expression of KRT8, PLAC8, CD9, PAG2, PAG2, HSPB1, MSH6 genes. The percentage of COC that remained at GV after 22 h PIVM was lower (P < 0.05) in the treated group than in the control group, regardless of the addition of FGF10. Nevertheless at 22 h of maturation, none of the treatments affected the final rate of MII oocytes. No expansion of CCs was observed during the PIVM period. After 22 h of maturation expansion was similar (P > 0.05) for all groups but they all had lower expansion (P < 0.05) than control group. Except for PTGS2 and SLC2A1 which were unchanged during PIVM, all other genes changed their expression during PIVM. When we evaluated the changes in gene expression during IVM on the different groups, we noted a change in the expression of four genes (PTGS2, CASP8, PTX3 and TNFAIP6). No differences (P > 0.05) in the cleavage and blastocyst rates at D6 were observed among treatment groups. However, the blastocyst rates at D7 were lower (P < 0.05) than the control groups in which FGF10 was present either during PIVM or during IVM. When the speed of embryo development was evaluated on D7, embryos from groups PIVM-IVM and PIVM - IVM + FGF10 developed faster than the other groups. However, at D8, the hatching rate was similar (P > 0.05) for all groups. Of all the analysed genes, only PLAC8 was shown to be overexpressed in PIVM/IVM + FGF10, and MSH6 was less expressed (P < 0.05) in PIVM + FGF10/IVM + FGF10 group when compared with the control. According to these findings, the PIVM of COCs in the presence of FGF10 affected the molecular profile and the expansion of CCs without affecting the nuclear maturation and embryo production rates. Although the presence of FGF10 changed the expression of genes related to embryo quality it did not show a great impact when added either or both during PIVM and IVM., (Copyright © 2017. Published by Elsevier Inc.)

Assisted reproductive techniques have significantly contributed to animal breeding programs. Similarly, genomics has provided important information and tools to improve the accuracy of selection. However, the greatest benefits of those tools can only be expected when they are combined, allowing animals to be selected accurately early in life. Therefore, obtaining DNA samples from embryos without compromising their viability is essential for the consolidation of preimplantation genomic selection. We aimed to evaluate the effect on the gestation rate of conducting a biopsy of in vivo (VV) and in vitro-produced (IVP) bovine embryos. The VV and IVP embryos were distributed into two groups: VV-B (biopsied embryos; n = 380) and VV-C (intact embryos-controls; n = 229) and IVP-B (biopsied embryos; n = 91) and IVP-C (intact embryos-controls; n = 227), respectively. After biopsy, embryos from both groups VV-B and IVP-B were cultured for an additional 3 hours before being transferred to synchronized recipients. To evaluate the quality of the DNA obtained in the biopsies, this was used to determine the sex of embryos by polymerase chain reaction. No effect (P > 0.05) of the biopsy was observed for any of the treatments, the pregnancy rate at D 60 post-transfer being similar for VV-B: 206/380 (54.21%) and VV-C: 128/229 (55.89%) and for IVP-B: 24/91 (26.37%) and IVP-C: 45/227 (19.82%). Also, no effect (P > 0.05) of the embryo's stage of development was detected on percentage of pregnant recipients when in vitro embryos were transferred. From the biopsies analyzed, about 90% had the sex determined, confirming that DNA was there and it was efficiently amplified. The results indicated that biopsy does not affect the viability of IVV and IVP bovine embryos and can be used in commercial programs to associate assisted reproductive technologies with genomic selection., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In this study we examined quality, longevity and ability of epididymal sperm (EP) to bind to oviduct explants (OE) after cooling and cryopreservation. Ejaculated (EJ) and EP sperm from seven bulls were evaluated before, during and after cryopreservation for total (TM), progressive motility (PM), sperm morphology, plasma membrane integrity (PMI) and acrosome integrity (ACI). For longevity, cryopreserved EP, EJ and a third group of cells in which EP spermatozoa were incubated with seminal plasm (SP) for 10 min after thawing (EPP group), were compared, and the groups were analyzed at 0, 3, 6, and 24 h for all parameters. Sperm from each group were co-incubated with OE for 30 min, 6 h, and 24 h for binding evaluation. Data were analyzed by the generalized linear models SAS 9.1 (P < 0.05). After cooling, EP displayed higher TM, higher PMI, and higher ACI (P < 0.05) than EJ. No differences were noted in the percentage of spermatozoa with PMI and AI between EJ and EP for fresh, cooled or cryopreserved sperm. However, a reduction in motility occurred in the EJ sperm after cooling, while in EP group such reduction occurred only after cryopreservation. At 6 h of incubation EP and EPP had higher PMI and ACI than EJ (P < 0.05). The number of spermatozoa bound to OE was similar P > 0.05) for all groups either at 30 min or 24 h. We conclude that EP are more resistant to cooling than EJ, and can bind to OE similarly to EJ., (Copyright © 2016 Elsevier Inc. All rights reserved.)