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Background: In recent times, various algorithms have been developed to assist in the selection of embryos fortransfer based on artificial intelligence (AI). Nevertheless, the majority of AI models employed in this context werecharacterized by a lack of transparency. To address these concerns, we aim to design an interpretable tool to automatehuman embryo evaluation by combining artificial neural networks (ANNs) and genetic algorithms (GA).Materials and Methods: This retrospective cohort study included 223 human blastocyst time-lapse (TL) imagestaken at 110 hours post-injection. All the images were evaluated by five embryologists from different clinics in termsof blastocyst expansion (BE), quality of the inner cell mass (ICM), and trophectoderm (TE). The embryo databasewas used to develop an AI system (70% training, 15% validation, and 15% test) for automate blastocyst assessment.The entire set of images underwent a standardization process, followed by processing and segmentation using Matlabsoftware. The resulting quantified variables were utilized in AI techniques (ANN and GA). Finally, the accuracy andperformance of the automation tool was assessed with the area under the receiver operating characteristic (ROC)curve (AUC). Then, the level of agreement among embryologists and between embryologists and the AI system wascompared with Kappa Index.Results: The overall agreement among embryologists was low (Kappa: 0.4 for BE; and 0.3 for TE and ICM). The AItool achieved higher consistency (Kappa 0.7 for BE and ICM; and 0.4 for TE). The AI exhibited high accuracy in classifyingBE (test 81.5%), ICM (test 78.8%), and TE (test 78.3%) and better performance for BE (AUC 0.888-0.956)than for ICM (AUC 0.605-0.854) and TE (AUC 0.726-0.769) assessment.Conclusion: Our AI tool highlighted the superior consistency of AI compared to human operators in grading blastocystmorphology. This research represents an important step towards fully automating objective embryo evaluation.

The aim of this study was to evaluate the effect of multilamellar vesicles (MLVs) of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in co-culture with in vitro-produced bovine embryos (IVPEs). The stability of five concentrations of MLVs (1.0, 1.25, 1.5, 1.75, and 2.0 mM) produced using ultrapure water or embryonic culture medium with 24 or 48 h of incubation at 38.5 °C with 5% CO2 was assessed. In addition, the toxicity of MLVs and their modulation of the lipid profile of the plasma membrane of IVPEs were evaluated after 48 h of co-culture. Both media allowed the production of MLVs. Incubation (24 and 48 h) did not impair the MLV structure but affected the average diameter. The rate of blastocyst production was not reduced, demonstrating the nontoxicity of the MLVs even at 2.0 mmol/L. The lipid profile of the embryos was different depending on the MLV concentration. In comparison with control embryos, embryos cultured with MLVs at 2.0 mmol/L had a higher relative abundance of six lipid ions (m/z 720.6, 754.9, 759.0, 779.1, 781.2, and 797.3). This study sheds light on a new culture system in which the MLV concentration could change the lipid profile of the embryonic cell membrane in a dose-dependent manner.

Thoroughly analyzing the sperm and exploring the information obtained using artificial intelligence (AI) could be the key to improving fertility estimation. Artificial neural networks have already been applied to calculate zootechnical indices in animals and predict fertility in humans. This method of estimating the results of reproductive biotechnologies, such as in vitro embryo production (IVEP) in cattle, could be valuable for livestock production. This study was developed to model IVEP estimates in Senepol animals based on various sperm attributes, through retrospective data from 290 IVEP routines performed using 38 commercial doses of semen from Senepol bulls. All sperm samples that had undergone the same procedure during sperm selection for in vitro fertilization were evaluated using a computer-assisted sperm analysis (CASA) system to define sperm subpopulations. Sperm morphology was also analyzed in a wet preparation, and the integrity of the plasma and acrosomal membranes, mitochondrial potential, oxidative status, and chromatin resistance were evaluated using flow cytometry. A previous study identified three sperm subpopulations in such samples and the information used in tandem with other sperm quality variables to perform an AI analysis. AI analysis generated models that estimated IVEP based on the season, donor, percentage of viable oocytes, and 18 other sperm predictor variables. The accuracy of the results obtained for the three best AI models for predicting the IVEP was 90.7, 75.3, and 79.6%, respectively. Therefore, applying this AI technique would enable the estimation of high or low embryo production for individual bulls based on the sperm analysis information.

Abstract Giant unilamellar vesicles (GUVs) are composed of lipophilic layers and are sensitive to the action of reactive oxygen species (ROS). The use of GUVs as microcarriers of biological macromolecules is particularly interesting since ROS produced by gametes or embryos during in vitro culture can induce the opening of pores in the membrane of these vesicles and cause the release of their content. This study investigated the behavior of GUVs [composed of 2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)] in co-culture with in vitro produced bovine embryos, as well as their embryotoxicity and effectiveness as cysteine carriers in culture medium. Embryonic developmental rates were unaffected, demonstrating the absence of toxicity of GUVs co-cultured with the embryos. No increase of intracellular ROS levels was observed in the embryos co-cultured with GUVs, indicating that the higher lipid content of the culture environment resulting from the lipid composition of the GUV membrane itself did not increase oxidative stress. Variations in the diameter and number of GUVs demonstrated their sensitivity to ROS produced by embryos cultured under conditions that generate oxidative stress. Encapsulation of cysteine in GUVs was found to be more effective in controlling the production of ROS in embryonic cells than direct dilution of this antioxidant in the medium. In conclusion, the use of GUVs in in vitro culture was found to be safe since these vesicles did not promote toxic effects nor did they increase intracellular ROS concentrations in the embryos. GUVs were sensitive to oxidative stress, which resulted in structural changes in response to the action of ROS. The possible slow release of cysteine into the culture medium by GUV rupture would therefore favor the gradual supply of cysteine, prolonging its presence in the medium. Thus, the main implication of the use of GUVs as cysteine microcarriers is the greater effectiveness in preventing the intracytoplasmic increase of ROS in in vitro produced bovine embryos.

Several opportunities for embryo development, stem cell maintenance, cell fate, and differentiation have emerged using induced pluripotent stem cells (iPSCs). However, the difficulty in comparing bovine iPSCs (biPSCs) with embryonic stem cells (ESCs) was a challenge for many years. Here, we reprogrammed fetal fibroblasts by transient expression of the four transcription factors (Oct4, Sox2, Klf4, and c-Myc, collectively termed “OSKM” factors) and cultured in iPSC medium, supplemented with bFGF, bFGF2i, leukemia inhibitory factor (LIF), or LIF2i, and then compared these biPSC lines with bESC to evaluate the pluripotent state. biPSC lines were generated in all experimental groups. Particularly, reprogrammed cells treated with bFGF were more efficient in promoting the acquisition of pluripotency. However, LIF2i treatment did not promote continuous self-renewal. biPSCs (line 2) labeled with GFP were injected into early embryos (day 4.5) to assess the potential to contribute to chimeric blastocysts. The biPSC lines show a pluripotency state and are differentiated into three embryonic layers. Moreover, biPSCs and bESCs labeled with GFP were able to contribute to chimeric blastocysts. Additionally, biPSCs have shown promising potential for contributing to chimeric blastocysts and for future studies.

In several species, oocyte and embryo competence are improved by the addition of endoplasmic reticulum (ER) stress inhibitors to in vitro maturation (IVM) medium and/or in vitro culture (IVC) medium. This study aimed to evaluate the effects of three concentrations of tauroursodeoxycholic acid (TUDCA; 50, 200, and 1,000 μM), a chemical chaperone for relieving ER stress, during IVM of bovine cumulus–oocyte complexes (COCs) for 24 h. Treated oocytes were analyzed for nuclear maturation, reactive oxygen species (ROS) production, mitochondrial activity, and abundance of target transcripts. In addition, the number of pronuclei in oocytes was evaluated after 18–20 h of insemination, and the rates of blastocyst and hatched blastocyst formation were evaluated after 7 and 8/9 days of culture, respectively. We further evaluated the transcript abundance of embryonic quality markers. Our findings showed that supplementation of IVM medium with 200 μM of TUDCA decreased ROS production and increased abundance of transcripts related to antioxidant activity in oocytes (CAT, GPX1, and HMOX1) and embryos (GPX1 and PRDX3). Interestingly, high concentration of TUDCA (1,000 μM) was toxic to oocytes, reducing the nuclear maturation rate, decreasing mitochondrial activity, and increasing the abundance of ER stress (HSPA5) and cellular apoptosis (CASP3 and CD40) related transcripts. The results of this study suggest that treatment with 200 μM of TUDCA is associated with a greater resistance to oxidative stress and indirectly with ER stress relief in bovine oocytes.

Three-dimensional in vitro maturation (3D IVM) is a promising approach to improve IVM efficiency as it could prevent cumulus-oocyte complex (COC) flattening and preserve its structural and functional integrity. Methods reported to date have low reproducibility and validation studies are limited. In this study, a bioprinting based production process for generating microbeads containing a COC (COC-microbeads) was optimized and its validity tested in a large animal model (sheep). Alginate microbeads were produced and characterized for size, shape and stability under culture conditions. COC encapsulation had high efficiency and reproducibility and cumulus integrity was preserved. COC-microbeads underwent IVM, with COCs cultured in standard 2D IVM as controls. After IVM, oocytes were analyzed for nuclear chromatin configuration, bioenergetic/oxidative status and transcriptional activity of genes biomarker of mitochondrial activity (TFAM, ATP6, ATP8) and oocyte developmental competence (KHDC3, NLRP5, OOEP and TLE6). The 3D system supported oocyte nuclear maturation more efficiently than the 2D control (P

Despite the use of new techniques on embryo selection and the presence of equipment on the market, such as EmbryoScope® and Geri®, which help in the evaluation of embryo quality, there is still a subjectivity between the embryologist’s classifications, which are subjected to inter- and intra-observer variability, therefore compromising the successful implantation of the embryo. Nonetheless, with the acquisition of images through the time-lapse system, it is possible to perform digital processing of these images, providing a better analysis of the embryo, in addition to enabling the automatic analysis of a large volume of information. An image processing protocol was developed using well-established techniques to segment the image of blastocysts and extract variables of interest. A total of 33 variables were automatically generated by digital image processing, each one representing a different aspect of the embryo and describing a different characteristic of the blastocyst. These variables can be categorized into texture, gray-level average, gray-level standard deviation, modal value, relations, and light level. The automated and directed steps of the proposed processing protocol exclude spurious results, except when image quality (e.g., focus) prevents correct segmentation. The image processing protocol can segment human blastocyst images and automatically extract 33 variables that describe quantitative aspects of the blastocyst’s regions, with potential utility in embryo selection for assisted reproductive technology (ART).

Abstract Background The timing of the first cell divisions may predict the developmental potential of an embryo, including its ability to establish pregnancy. Besides differences related to metabolism, stress, and survival, embryos with different speeds of development present distinct patterns of gene expression, mainly related to energy and lipid metabolism. As gene expression is regulated by epigenetic factors, and that includes DNA methylation patterns, in this study we compared the global DNA methylation profile of embryos with different kinetics of development in order to identify general pathways and regions that are most influenced by this phenotype. For this purpose, bovine embryos were in vitro produced using sexed semen (female), classified as fast (four or more cells) or slow (two cells) at 40 hpi and cultured until blastocyst stage, when they were analyzed. Results Genome-wide DNA methylation analysis identified 11,584 differently methylated regions (DMRs) (7976 hypermethylated regions in fast and 3608 hypermethylated regions in slow embryos). Fast embryos presented more regions classified as hypermethylated distributed throughout the genome, as in introns, exons, promoters, and repeat elements while in slow embryos, hypermethylated regions were more present in CpG islands. DMRs were clustered by means of biological processes, and the most affected pathways were related to cell survival/differentiation and energy/lipid metabolism. Transcripts profiles from DM genes connected with these pathways were also assessed, and the most part disclosed changes in relative quantitation. Conclusion The kinetics of the first cleavages influences the DNA methylation and expression profiles of genes related to metabolism and differentiation pathways and may affect embryo viability.

Abstract Morphological analysis is the standard method of assessing embryo quality; however, its inherent subjectivity tends to generate discrepancies among evaluators. Using genetic algorithms and artificial neural networks (ANNs), we developed a new method for embryo analysis that is more robust and reliable than standard methods. Bovine blastocysts produced in vitro were classified as grade 1 (excellent or good), 2 (fair), or 3 (poor) by three experienced embryologists according to the International Embryo Technology Society (IETS) standard. The images (n = 482) were subjected to automatic feature extraction, and the results were used as input for a supervised learning process. One part of the dataset (15%) was used for a blind test posterior to the fitting, for which the system had an accuracy of 76.4%. Interestingly, when the same embryologists evaluated a sub-sample (10%) of the dataset, there was only 54.0% agreement with the standard (mode for grades). However, when using the ANN to assess this sub-sample, there was 87.5% agreement with the modal values obtained by the evaluators. The presented methodology is covered by National Institute of Industrial Property (INPI) and World Intellectual Property Organization (WIPO) patents and is currently undergoing a commercial evaluation of its feasibility.

In this study, we evaluated the modulation effect of long-chain Acyl-CoA synthetase during early embryo development. Bovine embryos were cultured in four groups: positive modulation (ACS+) with GW3965 hydrochloride, negative modulation (ACS-) with Triacsin C, association of both modulators (ACS±), and control. Embryo development rates were not altered (P>0.05) by treatments. Embryonic cytoplasmic lipid content increased in ACS+ but reduced in ACS- compared to the control (P < 0.05), whereas the membrane phospholipids profile was not altered by treatments. The total number of blastomeres did not differ (P > 0.05) between groups; however, an increased apoptotic cells percentage was found in ACS- compared to control. Twenty-four hours after warming, ACS+ and control grade I embryos presented the best hatching rates, whereas the ACS+ group equaled the hatching rates between their embryos of grades I, II and III 48 hours after warming. The relative abundance of transcripts for genes associated with lipid metabolism (ACSL3, ACSL6, ACAT1, SCD, and AUH), heatshock (HSP90AA1 and HSF1), oxidative stress (GPX4), and angiogenesis (VEGF), among other important genes for embryo development were affected by at least one of the treatments. The treatments were effective in modulating the level of transcripts for ACSL3 and the cytoplasmic lipid content. The ACS- was not effective in increasing embryonic cryosurvival, whereas ACS+ restored survival rates after vitrification of embryos with low quality, making them equivalent to embryos of excellent quality.

The event of cellular reprogramming into pluripotency is influenced by several factors, such as in vitro culture conditions (e.g., culture medium and oxygen concentration). Herein, bovine iPSCs (biPSCs) were generated in different levels of oxygen tension (5% or 20% of oxygen) and supplementation (bFGF or bFGF + LIF + 2i—bFL2i) to evaluate the efficiency of pluripotency induction and maintenance in vitro. Initial reprogramming was observed in all groups and bFL2i supplementation initially resulted in a superior number of colonies. However, bFL2i supplementation in low oxygen led to a loss of self-renewal and pluripotency maintenance. All clonal lines were positive for alkaline phosphatase; they expressed endogenous pluripotency-related genes SOX2, OCT4 and STELLA. However, expression was decreased throughout the passages without the influence of oxygen tension. GLUT1 and GLUT3 were upregulated by low oxygen. The biPSCs were immunofluorescence-positive stained for OCT4 and SOX2 and they formed embryoid bodies which differentiated in ectoderm and mesoderm (all groups), as well as endoderm (one line from bFL2i in high oxygen). Our study is the first to compare high and low oxygen environments during and after induced reprogramming in cattle. In our conditions, a low oxygen environment did not favor the pluripotency maintenance of biPSCs.

Reproductive efficiency plays a major role in the long-term sustainability of livestock industries and can be improved through genetic and genomic selection. This study aimed to estimate genetic parameters (heritability and genetic correlation) and identify genomic regions and candidate genes associated with anti-Müllerian hormone levels (AMH) and antral follicle populations measured after estrous synchronization (AFP) in Nellore cattle. The datasets included phenotypic records for 1099 and 289 Nellore females for AFP and AMH, respectively, high-density single nucleotide polymorphism (SNP) genotypes for 944 animals, and 4129 individuals in the pedigree. The heritability estimates for AMH and AFP were 0.28 ± 0.07 and 0.30 ± 0.09, and the traits were highly and positively genetically correlated (rG = 0.81 ± 0.02). These findings indicated that these traits can be improved through selective breeding, and substantial indirect genetic gains are expected by selecting for only one of the two traits. A total of 31 genomic regions were shown to be associated with AMH or AFP, and two genomic regions located on BTA1 (64.9–65.0 Mb and 109.1–109.2 Mb) overlapped between the traits. Various candidate genes were identified to be potentially linked to important biological processes such as ovulation, tissue remodeling, and the immune system. Our findings support the use of AMH and AFP as indicator traits to genetically improve fertility rates in Nellore cattle and identify better oocyte donors.

The bovine pre-implantation embryo secretes bioactive molecules from early development stages, but effects on endometrial function are reported to start only after elongation. Here, we interrogated spatially defined regions of the endometrium transcriptome for responses to a day 7 embryo in vivo. We hypothesize that exposure to an embryo changes the abundance of specific transcripts in the cranial region of the pregnant uterine horn. Endometrium was collected from the uterotubal junction (UTJ), anterior (IA), medial (IM) and posterior (IP) regions of the uterine horn ipsilateral to the CL 7 days after estrus from sham-inseminated (Con) or artificially inseminated, confirmed pregnant (Preg) cows. Abundance of 86 transcripts was evaluated by qPCR using a microfluidic platform. Abundance of 12 transcripts was modulated in the Preg endometrium, including classical interferon-stimulated genes (ISG15, MX1, MX2 and OAS1Y), prostaglandin biosynthesis genes (PTGES, HPGD and AKR1C4), water channel (AQP4) and a solute transporter (SLC1A4) and this was in the UTJ and IA mainly. Additionally, for 71 transcripts, abundance varied according to region of the reproductive tract. Regulation included downregulation of genes associated with proliferation (IGF1, IGF2, IGF1R and IGF2R) and extracellular matrix remodeling (MMP14, MMP19 and MMP2) and upregulation of anti-adhesive genes (MUC1) in the cranial regions of uterine horn. Physical proximity to the embryo provides paracrine regulation of endometrial function. Embryo-independent regulation of the endometrial transcriptome may support subsequent stages of embryo development, such as elongation and implantation. We speculate that successful early embryo-dependent and -independent programming fine-tune endometrial functions that are important for maintenance of pregnancy in cattle.

In this study, we developed an online graphical and intuitive interface connected to a server aiming to facilitate professional access worldwide to those facing problems with bovine blastocysts classification. The interface Blasto3Q, where 3Q refers to the three qualities of the blastocyst grading, contains a description of 24 variables that were extracted from the image of the blastocyst and analyzed by three Artificial Neural Networks (ANNs) that classify the same loaded image. The same embryo (i.e., the biological specimen) was submitted to digital image capture by the control group (inverted microscope with 40× magnification) and the experimental group (stereomicroscope with maximum of magnification plus 4× zoom from the cell phone camera). The images obtained from the control and experimental groups were uploaded on Blasto3Q. Each image from both sources was evaluated for segmentation and submitted (only if it could be properly or partially segmented) for automatic quality grade classification by the three ANNs of the Blasto3Q program. Adjustments on the software program through the use of scaling algorithm software were performed to ensure the proper search and segmentation of the embryo in the raw images when they were captured by the smartphone, since this source produced small embryo images compared with those from the inverted microscope. With this new program, 77.8% of the images from smartphones were successfully segmented and from those, 85.7% were evaluated by the Blasto3Q in agreement with the control group.

Mouse embryo production by superstimulation is a multifactorial process. To minimize the number of sacrificed animals and to maximize the results of the superstimulatory treatment, it should be possible to predict the risk of do not get embryos from such a treated animal. This work aimed to evaluate if the variables - hormone concentration and the timing of its administration, the copulatory plug presence and individual male used to mating – could be predictive factors on the mouse embryo production. Females were distributed in four groups (cross-over design) related to scheduled superstimulation treatment (1300h or 1700h) and eCG/hCG administered concentration (5 or 10IU). After the hCG treatment, females were put to mate. On the next morning it was verified the presence of a copulatory plug (D0.5). Embryo recovery was performed from D2.5 to D4.5 by flushing the oviducts and uterine horns. Total structures recovered (TSR) and the viable embryos (VE) were classified by its morphology. Viability rate (VR) was calculated with VE in relation to TSR (x100). Group comparison was analyzed with 5% of significance. There were no significant differences among groups, even when only main effects were analyzed (hormone concentration and timing of its administration). There was significant difference in VR from animals with or without plug and from the worst and best males used. It was concluded that neither the hormone concentration nor timing of its administration - or their association – was significant as predictive factors for the embryo production. However, the plug presence was related to higher VR.

A superestimulação visando a produção embrionária, em camundongos, é um procedimento multifatorial. Para minimizar o número de animais sacrificados e maximizar o resultado do tratamento superestimulatório, é necessário que haja uma predição do risco de não obter embriões de determinado animal. Este trabalho objetivou avaliar se as variáveis - concentração e horário da administração hormonal, presença de “plug” copulatório e macho utilizado – seriam fatores preditivos na produção embrionária. Camundongas foram distribuídas em quatro grupos (fatorial 2x2), com o tratamento superovulatório iniciado às 13h ou às 17h e os hormônios (eCG e hCG) administrados com 5 ou 10UI. Após o hCG, os animais foram colocados com machos para a cópula. Na manhã seguinte foi verificada a presença de “plug” (D0,5). A recuperação dos embriões foi realizada entre D2,5 e D4,5, mediante lavagem dos ovidutos e cornos uterinos. As estruturas recuperadas (ETO) e os embriões viáveis (EVI) foram morfologicamente classificados. A porcentagem de EVI, em relação às ETO, foi considerada taxa de viabilidade embrionária (TVE). Os grupos foram comparados com a significância sendo considerada quando p