Integrating in vitro embryo production with embryonic microsurgery facilitates the generation of monozygotic twins. However, despite their potential benefits, these methods have not been widely adopted in commercial settings because of their substantial costs. Hence, there is a need to streamline the bisection procedure while ensuring efficient production of viable demi-embryos. In this study, we investigated the impact of different orientations of microsurgical incisions in relation to inner cell mass on embryonic development, morphology, viability, and expression of cell fate protein markers using a simplified microsurgery approach. Ovaries were transported from the slaughterhouse to the laboratory and aspirated to obtain oocytes that were selected and subjected to in vitro embryo production. The selected expanded blastocysts (n = 204) underwent microsurgery. The blastocysts were immobilized to facilitate incision using an adapted microblade, yielding demi-embryos (vertical incision) and viable embryonic fragments (transverse incision). The structures were then re-cultured for 12 h. Viability was assessed by measuring the re-expansion rate after re-culture, followed by immunofluorescence analysis of proteins (CDX2 and NANOG) and apoptosis analysis using terminal deoxynucleotyl transferase dUTP nick end-labeling (TUNEL). Microsurgically derived embryos exhibited remarkable plasticity, as evidenced by a slight reduction (P < 0.05) in the re-expansion rate (transverse 64.2 % and vertical 57.2 %) compared to that of the control group (blastocysts without microsurgery) (86.7 %). They also demonstrated the ability of morphological reconstitution after culturing. Despite the anticipated decrease (P < 0.05) in the total number of cells and embryo volume, microsurgery did not result in a significant increase (P > 0.05) in the number of apoptotic cells. Furthermore, microsurgery led to higher (P < 0.05) expression of markers associated with pluripotency, indicating its efficiency in preserving regenerative capacity. Moreover, microsurgery, whether followed by immunosurgery or not, made the isolation of embryonic cells easier. In conclusion, both transverse and vertical microsurgery incisions enabled the production of identical demi-embryos and served as tools for isolating embryonic cells without compromising the resumption of development and the apoptotic index.

In vitro-produced embryos are constantly exposed to stressful conditions that can lead to the activation of the apoptotic pathway. The nuclear Kappa B factor (NF-κB) is an inflammatory mediator that induces the expression of tumor necrosis factor (TNF-α), a pro-inflammatory cytokine, while interleukin-10 (IL-10), an anti-inflammatory cytokine, inhibits NF-κB activity. This study aimed to investigate the effects of IL-10 and TNF-α on the competence and cryosurvival of in vitro-produced bovine embryos. Embryos were produced in vitro using standard protocols, and Grade I blastocysts were vitrified using the Cryotop method. Non-vitrified and vitrified blastocysts were subjected to the TUNEL assay. In Experiment I, on day 6.5 (156 h post-insemination), the embryos were treated with PBS (control), 50 ng/mL of IL-10, or a combination of 25 ng/mL of TNF-α and 50 ng/mL of IL-10. Embryonic development and apoptotic rates were monitored. In Experiment II, the same groups were set up, with the addition of a group treated with 25 ng/mL of TNF-α alone. Grade I blastocysts were vitrified 5 h after treatment, and cryosurvival was monitored at until 48 h post-warming. The apoptosis rate and total cell number were investigated in the vitrified-hatched blastocysts. IL-10 alone did not affect developmental competence or cryosurvival (P > 0.05). The IL-10-treated embryos, when exposed in combination with TNF-α, presented a detrimental effect (P < 0.05) in the embryonic development of non-vitrified embryos. However, vitrified blastocysts had no negative effect (P > 0.05). The TNF-α treatment reduced (P < 0.05) the re-expansion rate at 6 h post-warming and increased (P < 0.05) the apoptosis rate in vitrified hatched blastocysts, whereas no effect (P > 0.05) of the treatments was detected in the hatching rate and total cell number post-warming. In conclusion, TNF-α has a detrimental effect on embryonic developmental competence and cryosurvival by compromising the development of non-vitrified embryos and apoptotic-related events of vitrified blastocysts, whereas IL-10, when in combination with TNF-α, appears to attenuate the detrimental effects of TNF-α.

A wide-ranging review study regarding the molecular characterization of the first cell lineages of the developmental embryo is lacking, especially for the primary events during earliest differentiation which leads to the determination of cellular fate. Here, a systematic review and meta-analysis were conducted according to PRISMA guidelines. MEDLINE-PubMed was searched based on an established search strategy through April 2021. Thirty-six studies fulfilling the inclusion criteria were subjected to qualitative and quantitative analysis. Among the studies, 50 % (18/36) used mice as an animal model, 22.2 % (8/36) pigs, 16.7 % (6/36) cattle, 5.5 % (2/36) humans, and 2.8 % (1/36) goats as well as 2.8 % (1/36) equine. Our results demonstrated that each of the first cell lineages of embryos requires a certain pattern of expression to establish the cellular determination of fate. Moreover, these patterns are shared by many species, particularly for those molecules that have already been identified in the literature as biomarkers. In conclusion, the present study integrated carefully chosen studies regarding embryonic development and first cellular decisions in mammalian species and summarized the information about the differential characterization of the first cell lineages and their possible relationship with specific gene expression.

In this study, we investigated the impact of incorporating Interleukin-13 (IL-13) into the embryonic culture medium and its influence on cryotolerance and cellular viability of vitrified bovine embryos. Two distinct time points for IL-13 supplementation were explored: during the final hours of culture prior to cryopreservation and during the period of recultivation following cryopreservation and warming. Cryosurvival rates, total cell count, and cell viability were assessed using the TUNEL technique to determine the apoptotic percentage. Re-expansion and hatching rates did not show differences among all groups (P > 0.05), and the total cell number was com-parable between the treated and control groups (P > 0.05). However, the group that received IL-13 before vitrification exhibited a higher apoptotic percentage (P < 0.05). This suggests that the anti-inflammatory effect of IL-13 may have impacted the embryo's defense capacity against the stress induced by cryopreservation, leading to an increased percentage of apoptosis, although it did not influence the developmental resumption capability.

Com a conscientização dos consumidores em relação às questões ambientais surgiram exigências que fizeram com que as empresas se adaptassem a essa nova realidade. Novas práticas de produção tiveram que ser implantadas com o intuito de causar o menor impacto possível ao meio ambiente. Essa pressão causada pelos consumidores pode ser considerada ao mesmo tempo uma valiosa oportunidade para as empresas atingirem um novo consumidor em potencial, o consumidor “verde” através do Marketing Verde. Dentro deste cenário questiona-se se o consumidor gabrielense leva em consideração as práticas ambientais utilizadas nas empresas e se isso afeta sua decisão de compra. Com o objetivo de responder tais questionamentos teve-se como objetivo geral, mensurar a percepção e atitude do consumidor gabrielense frente às questões ambientais no momento da compra e através dos seguintes objetivos específicos: (a) Identificar os atributos considerados mais importantes na escolha de compra; (b) Levantar, se no momento da compra, o consumidor leva em consideração se o produto é de empresas “normais” ou empresas “verdes”; (c) Investigar se o consumidor pagaria mais por produtos ambientalmente corretos. Para tanto utilizou-se uma pesquisa exploratório-descritivo com abordagem quantitativa através de uma amostra de consumidores gabrielenses. Resultados deste estudo permitem observar que as empresas verdes são consideradas muito importantes e que os consumidores estariam dispostos a pagar mais por produtos ambientalmente corretos.

Each living organism is unique because of the lipid identity of its organelles. The diverse distribution of these molecules also contributes to the role of each organelle in cellular activity. The lipid profiles of whole embryos are well documented in the literature. However, this approach can often lead to the loss of relevant information at the subcellular and consequently, metabolic levels, hindering a deeper understanding of key physiological processes during preimplantation development. Therefore, we aimed to characterize four organelles in vitro-produced bovine embryos: lipid droplets (LD), endoplasmic reticulum (ER), mitochondria (MIT), and nuclear membrane (NUC), and evaluate the contribution of the lipid species to each organelle evaluated. Expanded blastocysts were subjected to cell organelle isolation. Thereafter, lipid extraction from cell organelles and lipid analysis using the Multiple Reaction Monitoring (MRM) profiling method were performed. The LD and ER displayed a greater number of lipids (Phosphatidylcholine - PC, Ceramide - Cer, and Sphingomielin - SM) with high signal-to-noise intensities. This result is due to the high rate of biosynthesis, lipid distribution, and ability to store and recycle lipid species of these organelles. The NUC had a more distinct lipid profile than the other three organelles, with high relative intensities of PC, SM, and triacylglycerols (TG), which is consistent with its high nuclear activity. MIT had an intermediate profile that was close to that of LD and ER, which aligns with its autonomous metabolism for some classes of phospholipids (PL). Our study revealed the lipid composition of each organelle studied, and the roles of these lipids could be associated with the characteristic organellar activity. Our findings highlight the lipid species and classes that are relevant for the homeostasis and function of each associated organelle and provide tentative biomarkers for the determination of in vitro embryonic development and quality.

Although well-established and adopted by commercial laboratories, the in vitro embryo production system still requires refinements to achieve its highest efficiency. Early embryonic development is a dynamic event, demanding suitable conditions to provide a high number of embryos with quality and competence. The first step to obtaining an optimized in vitro environment is to know the embryonic metabolism and energy request throughout the different stages of development. Oxygen plays a crucial role in several key biological processes necessary to sustain and complete embryonic development. Nonetheless, there is still controversy regarding the optimal in vitro atmospheric concentrations during culture. Herein, we discuss the impact of oxygen tension on the viability of in vitro-produced embryos during early development. The importance of oxygen tension is addressed as its roles regarding essential embryonic traits, including embryo production rates, embryonic cell viability, gene expression profile, epigenetic regulation, and post-cryopreservation survival. Finally, we highlight the damage caused by in vitro unbalanced oxygen tensions and strategies to mitigate the harmful effects.

The expansion of the use of in vitro production techniques has revolutionized the bovine embryo market. In the last decade, we have seen the number of in vitro produced (IVP) embryos surpass the number of in vivo-derived (IVD) embryos obtained worldwide. Concomitantly, other biotechnologies were also improved, following the global trend. Embryo cryopreservation has received special attention, as it is one of the tools capable of disseminating in vitro production. Currently, two protocols are available: slow freezing and vitrification. Both have advantages and disadvantages regarding their application and, many aspects need to be considered before their use. In this review, we discuss in vitro production market trends, cellular and molecular features involved in embryo response to cryopreservation, and addressed cryo-storage period and embryonic developmental stage on cryosurvival. In addition, we also presented an overview of some aspects that impact the pregnancy rate following transfer of fresh and cryopreserved IVP embryos.

The differential expression of many productive and reproductive traits has already been associated with the genotype effect observed between Bos taurus indicus (Bos indicus) and Bos taurus taurus (Bos taurus) breeds. Lipids have a crucial role during oocyte development. The aim of this work was to characterize the lipid profiles of oocytes recovered from dairy breeds: Gir (Bos indicus), Holstein (Bos taurus) and their crossbred (Gir x Holstein). Cumulus-oocyte complexes were collected by ovum pick-up from Gir (n=21), Holstein (n=36), and crossbreed (n=33) cows and submitted to MALDI-MS lipid fingerprint analysis. Multi- and univariate analyses were used to characterize lipid profiles of oocytes derived from each breed. Third lipid profiles (Gir n=7; Holstein n=12; and crossbred n=11) were evaluated and allowed characterization of oocyte's lipids among breeds. The lipid profiles from Gir and Holstein were observed to be the most distinct, whereas Gir and crossbred oocytes presented greater similarities. Overall, a wide-range oocyte lipid profile characterization from distinctive bovine dairy breeds recovered by OPU were provided. We have identified two distinguishable lipid profile patterns among groups and revealed that phospholipids and triacylglycerol were the predominantly lipid assigned in Gir (Bos indicus) and Holstein (Bos taurus) oocytes, respectively.

The cryosurvival of embryos is a complex process involving dynamic and integrated morphological, functional, and molecular changes. Here, we evaluated the transcriptional profiling of bovine embryos possessing high and low cryotolerance (HC and LC, respectively) by assessing the resumption of development. Embryos were produced in vitro (N = 1137) and cryopreserved (N = 894). Blastocysts samples possessed pronounced group individualization at RNA sequencing. A total of 114 genes were differentially expressed, and 27 and 84 genes were upregulated in HC and LC, respectively. Among the over-represented biological functions, cellular growth and proliferation, cell death and survival, and organismal survival were predicted to be activated, while cellular movement and cell-to-cell signaling were predicted to be inhibited in HC embryos. Enriched canonical pathways and upstream regulators related to cellular proliferation and survival (HC), inflammatory processes, and cell death (LC) were predicted to represent two embryonic molecular profiles present during the resumption of development after cryopreservation. The marked contrast in transcriptional profiles between HC and LC strongly suggests the influence of embryonic competence after cryopreservation on its respective transcriptome and indicated that HC and LC presented two different molecular strategies to overcome cryopreservation-related stress and resume postcryopreservation development.

Microbiota e o termo utilizado para caracterizar a comunidade de microorganismos que habitam tecidos e fluidos biologicos de organismos multicelulares. Divergencias nesta composicao em varios casos podem ser relacionadas com o desenvolvimento de diversas enfermidades infecciosas, hepaticas, metabolicas, respiratorias, mentais, autoimunes e neoplasias intestinais. Esta proposta objetivou caracterizar as flutuacoes da populacao de bacterias acido laticas (BAL) cultivaveis no ambiente vaginal em resposta ao estrogeno em ovelhas de corte. Alem disso, este trabalho verificou a correlacao entre a presenca de BAL cultivaveis na vagina e o potencial oxidativo deste ambiente. Para tanto, foram realizados experimentos para caracterizacao da microbiota BAL vaginal e do potencial oxidante/antioxidante deste ambiente em resposta a estrogeno divididos em tres grupos (5 animais/grupo): (1) controles (D-2.5), (2) 12h apos injecao de benzoato de estradiol (D.0.5) e (3) 60h apos injecao de benzoato de estradiol (D.2.5). Os resultados sugerem um modelo in vivo apropriado para o estudo da microbiota vaginal e ambiente oxidante/antioxidante que foi capaz de detectar efeitos de benzoato de estradiol nas variaveis testadas. Em conclusao, estrogeno regula o ambiente vaginal microbiano pela alteracao da populacao das BAL vaginais e o balanco entre atividade oxidante e antioxidante. Finalmente, as particularidades do modelo validado in vivo merecem uma investigacao mais aprofundada para abordar questoes recentes que surgiram a partir dos dados gerados por este estudo.

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

Este trabalho baseia-se na valorizacao da bovinocultura no mercado, pois e preciso que as amostras de semen cheguem ao destino final menos danificada possivel, para que seja realizada inseminacao artificial (IA) com sucesso. Para isso, no processo de criopreservacao ha a necessidade de utilizar diluentes que protejam a celula espermatica. Este trabalho tem por objetivo geral testar diferentes extratos vegetais como agentes antioxidantes no congelamento de semen bovino. Alem de (a) avaliar os padroes seminais pos-descongelamento na presenca de diferentes antioxidantes, (b) avaliar os efeitos dos antioxidantes sobre a motilidade e o vigor das celulas espermaticas, (c) mensurar o dano de membrana nos espermatozoides atraves da medida da peroxidacao lipidica (TBARS) e (d) mensurar a atividade da enzima antioxidante catalase (CAT). O procedimento iniciou na coleta de semen dos machos e seguido do diagnostico preliminar do semen via analise por volume, aspecto, turbilhao, motilidade e vigor sob o microscopio. Em seguida as aliquotas serao separadas em seis grupos para diluicao: controle positivo (20% gema de ovo), controle negativo (meio base), tratado com antioxidantes (Anacardium microcarpum, Stryphnodendron adstringens, Croton campestris e Duguetia furfuraceae), todos com 6% de glicerol, seguido do congelamento que inclui tres etapas: 3h (4°C), 20min em vapor de nitrogenio liquido (N2) (-70°C) e finalmente a imersao em N2. Posteriormente as amostras foram descongeladas e analisadas com os mesmos criterios anteriores. Foram utilizados os ensaios bioquimicos de thiobarbitutic acid reactive substances (TBARS) e atividade da enzima antioxidante catalase (CAT) para avaliacao do dano espermatico durante o processo de congelamento do semen. Os resultados obtidos demonstram que os extratos vegetais nao tenham sido efetivos no melhoramento da congelabilidade dos espermatozoides nesses testes, mas demonstraram comportamentos bioquimicos entre si. O uso de extratos vegetais podem ser tornar uma alternativa viavel desde que haja mais estudos para identificar quais extratos podem ter um papel determinante na protecao celular durante congelamento.