Cytoplasmic strings in human blastocysts: hypotheses of their role and implications for embryo selection.

STUDY QUESTION: What are the implications of the presence cytoplasmic strings (Cyt-S) and their quantity and dynamics for the pre-implantation development of human blastocysts? SUMMARY ANSWER: Cyt-S are common in human embryos and are associated with faster blastocyst development, larger expansion, and better morphological quality. WHAT IS KNOWN ALREADY: Cyt-S are dynamic cellular projections connecting inner cell mass and trophectoderm (TE) cells, that can be observed during blastocyst expansion. Their prevalence in human embryos has been estimated to be between 44% and 93%. Data relevant to their clinical implications and role in development are lacking, limited, or controversial. STUDY DESIGN, SIZE, DURATION: Retrospective study conducted at a single IVF center between May 2013 and November 2014 and involving 124 pre-implantation genetic testing for aneuploidy cycles in a time-lapse incubator with ≥1 blastocyst biopsied and vitrified (N = 370 embryos assessed). These cycles resulted in 87 vitrified-warmed single-euploid blastocyst transfers. PARTICIPANTS/MATERIALS, SETTING, METHODS: ICSI, continuous blastocyst culture (Days 5-7), TE biopsy of fully expanded blastocysts without Day 3 zona pellucida drilling, qPCR to assess uniform full-chromosome aneuploidies, and vitrification were all performed. Only vitrified-warmed euploid single-embryo-transfers were conducted. Blastocyst morphological quality was defined according to Gardner's criteria. The AI-based software CHLOE™ (Fairtility) automatically registered timings from time of starting blastulation (tSB) to biopsy (t-biopsy, i.e. blastocyst full-expansion) as hours-post-insemination (hpi), embryo area (including zona pellucida in µm2), and spontaneous blastocyst collapses. One senior embryologist manually annotated Cyt-S presence, quantity, timings, and type (thick cell-to-cell connections and/or threads). All significant associations were confirmed through regression analyses. All couples', cycles', and embryos' main features were also tested for associations with Cyt-S presence, quantity, and dynamics. MAIN RESULTS AND THE ROLE OF CHANCE: About 94.3% of the patients (N = 117/124) had ≥1 embryo with Cyt-S. Out of a total of 370 blastocysts, 55 degenerated between blastulation and full-expansion (N = 55/370, 14.9%). The degeneration rate among embryos with ≥1 Cyt-S was 10.8% (N = 33/304), significantly lower than that of embryos without Cyt-S (33.3%, N = 22/66, P < 0.01). Of the remaining 315 viable blastocysts analyzed, 86% (N = 271/315; P < 0.01) had ≥1 Cyt-S, on average 3.5 ± 2.1 per embryo ranging 1-13. The first Cyt-S per viable embryo appeared at 115.3 ± 12.5 hpi (85.7-157.7), corresponding to 10.5 ± 5.8 h (0.5-31) after tSB. Overall, we analyzed 937 Cyt-S showing a mean duration of 3.8 ± 2.7 h (0.3-20.9). Cyt-S were mostly threads (N = 508/937, 54.2%) or thick cell-to-cell connections becoming threads (N = 382/937, 40.8%) than thick bridges (N = 47/937, 5.0%). The presence and quantity of Cyt-S were significantly associated with developmentally faster (on average 6-12 h faster) and more expanded (on average 2700 µm2-larger blastocyst's area at t-biopsy) embryos. Also, the presence and duration of Cyt-S were associated with better morphology. Lastly, while euploidy rates were comparable between blastocysts with and without Cyt-S, all euploid blastocysts transferred from the latter group failed to implant (N = 10). LIMITATIONS, REASONS FOR CAUTION: Cyt-S presence and dynamics were assessed manually on seven focal planes from video frames recorded every 15 min. The patients included were mostly of advanced maternal age. Only associations could be reported, but no causations/consequences. Lastly, larger datasets are required to better assess Cyt-S associations with clinical outcomes. WIDER IMPLICATIONS OF THE FINDINGS: Cyt-S are common during human blastocyst expansion, suggesting their physiological implication in this process. Their presence, quantity and dynamics mirror embryo viability, and morphological quality, yet their role is still unknown. Future basic science studies are encouraged to finally describe Cyt-S molecular nature and biophysical properties, and Artificial Intelligence tools should aid these studies by incorporating Cyt-S assessment. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: N/A.

Bovine embryo production in vitro: evolution of culture media and commercial perspectives.

In vitro produced embryos exhibit lower viability compared to their in vivo counterparts. Mammalian preimplantation embryos have the ability to reach the blastocyst stage in diverse culture media, showcasing considerable metabolic adaptability, which complicates the identification of optimal developmental conditions. Despite embryos successfully progressing to the blastocyst stage, adaptation to suboptimal culture environments may jeopardize blastocyst viability, cryotolerance, and implantation potential. Enhancing our capacity to support preimplantation embryonic development in vitro requires a deeper understanding of fundamental embryo physiology, including preferred metabolic substrates and pathways utilized by high-quality embryos. Armed with this knowledge, it becomes achievable to optimize culture conditions to support normal, in vivo-like embryo physiology, mitigate adaptive stress, and enhance viability. The objective of this review is to summarize the evolution of culture media for bovine embryos, highlighting significant milestones and remaining challenges.

Evolution over the last 40 years of the assisted reproductive technologies in cattle - the Brazilian perspective for embryo transfer and resynchronization programs (part II).

The productivity of the beef and dairy industries depends directly on the reproductive efficiency and genetic gain of the herd, which are directly associated with the appropriate use of Assisted Reproductive Technologies (ARTs). The objective of this review is to show from a Brazilian perspective the evolution over the last 40 years of ARTs related to ovulation resynchronization programs and embryo transfer in cattle. Despite significant improvements and high fertility obtained in timed artificial insemination (TAI) protocols (Sales et al., 2024 - Part I), the improvement of the use of in vitro-produced embryos, development of resynchronization programs, and the advance in Doppler ultrasonography (Doppler-US) for reproductive assessments of bovine females were the ARTs that presented the greatest relevance on reproductive effectiveness in cattle. In the last seven years, the embryo transfer (ET) technology using in vitro-produced (IVP) embryos took over the conventional ET of in vivo produced embryos after donor's superovulation. Also, procedures and pregnancy rates after ET of IVP embryos were improved in dairy and beef operations. The Doppler-US allows the identification of non-pregnant females at an early stage based on the evaluation of blood perfusion of the corpus luteum. Recent studies in beef and dairy cows indicate satisfactory accuracy when Doppler-US is used at 20-22 days after TAI. Consequently, super-early resynchronization programs have been developed and are being implemented in commercial programs, thereby facilitating earlier conception through the use of semen from superior bulls, providing genetic and economic improvements in herds. Likewise, the assessment of luteal function by Doppler-US allows the selection of embryo recipients with greater receptivity, and consequently may increase the effectiveness of timed ET programs.

Serum progesterone measurement on the day of fresh embryo transfer and its correlation with pregnancy success rates: A prospective analysis.

Studies regarding serum Progesterone (P4) concentration and Clinical Pregnancy Rates (CPR) in fresh Embryo Transfer (ET) after Controlled Ovarian Stimulation Cycles (COS) remain inconclusive. To find a P4 cutoff point on fresh ET day associated with higher CPR, and to identify predictive factors of CPR and P4, the authors conducted a prospective cohort of 106 patients who underwent COS at a public IVF center. The luteal phase was supported with vaginal micronized progesterone (200 mg, 8/8h), beginning on oocyte retrieval day. The primary outcome was CPR beyond the 8th week of pregnancy. A ROC curve was constructed to identify the best cutoff point correlated with higher CPR. Multivariate analysis evaluated predictive variables of CPR and P4 concentration. P4 levels showed no significant differences between pregnant and non-pregnant patients (67.12 ± 31.1 ng/mL vs. 64.17 ± 61.76, p = 0.7465). The cutoff point correlated with higher CPR was P4 ≥ 28.9 ng/mL (AUC 0.5654). Women's age (OR = 0.878; 95 % CI 0.774-0.995) and top-quality embryo transfer (OR = 2.89; 95 % CI 1.148-7.316) were associated with CPR. Women's age ≥ 40 years (OR = 0.0956; 95 % CI 0.0156-0.5851), poor response to COS (OR = 0.0964; 95 % CI 0.0155-0.5966), and follicles ≥ 10 mm (OR = 1.465; 95 % CI 1.013-2.117) were associated with the cutoff point. As the ROC curve was unsatisfactory, P4 ≥ 28.9 ng/mL should not be used to infer gestational success. In fresh ET, P4 concentration may merely reflect a woman's age and individual response to COS rather than being a reliable CPR predictor.

The effect of single dose of gonadotropin-releasing hormone agonist injection in frozen-thawed embryo transfer on pregnancy outcomes: A systematic review and meta-analysis.

This systematic review and meta-analysis of randomized controlled trials aimed to evaluate the effect of a single-dose gonadotropin-releasing hormone agonist administration in the frozen-thawed embryo transfer cycle on pregnancy outcomes. A literature search was strategically conducted using PubMed, EMBASE, and the Cochrane Controlled Trials Register. The primary outcome was the clinical pregnancy rate. The secondary outcomes combined chemical pregnancy rate, implantation rate, ongoing pregnancy rate, live birth rate, miscarriage rate, and extrauterine pregnancy rate. Out of the 1594 citations that were found, only six met the criteria for being included in the meta-analysis. The clinical pregnancy rate was higher in the treatment group than in the control group (52.05% vs. 47.29%; p=0.04; RR=1.09; 95% CI=1.00-1.18). According to subgroup analysis based on the natural cycle, the clinical pregnancy rate with the agonist administration is significantly higher (43.75% vs. 27.35%; p=0.01; RR=1.6; 95% CI=1.10-2.32). However, there was no difference between the groups in terms of artificial cycles (p=0.80; 95% CI=0.96-1.20). The secondary outcomes did not show significant differences. We concluded that supplementing with a single dose of gonadotrophin-releasing hormone agonist can marginally increase the clinical pregnancy rate, particularly in the natural cycle. Other pregnancy outcomes do not improve with the treatment.

Use of Zebrafish (Danio rerio) for Biosafety Evaluation of Strontium Nanostructured Hydroxyapatite.

Despite the numerous studies on biocompatibility with nano-biomaterials, the biological effects of strontium-substituted HA nanoparticles (nSrHA) need to be better understood. So, we conducted an embryotoxicity test using zebrafish (Danio rerio) according to the OECD 236 guideline, a model that represents a viable alternative that bridges the gap between in vitro and mammalian models. Zebrafish embryos were exposed for 120 h to microspheres containing nSrHA nanoparticles with low and high crystallinity, synthesized at temperatures of 5°C (nSrHA5) and 90°C (nSrHA90). We evaluated lethality, developmental parameters, and reactive oxygen species (ROS) production. The larval behavior was assessed at 168 hpf to determine if the biomaterials affected motor responses and anxiety-like behavior. The results showed that the survival rate decreased significantly for the nSrHA5 group (low crystalline particles), and an increase in ROS was also observed in this group. However, none of the biomaterials caused morphological changes indicative of toxicity during larval development. Additionally, the behavioral tests did not reveal any alterations in all experimental groups, indicating the absence of neurotoxic effects from exposure to the tested biomaterials. These findings provide valuable insights into the biosafety of modified HA-based nanostructured biomaterials, making them a promising strategy for bone tissue repair. As the use of hydroxyapatite-based biomaterials continues to grow, it is crucial to ensure rigorous control over the quality, reliability, and traceability of these materials.

Is Artificial Intelligence (AI) currently able to provide evidence-based scientific responses on methods that can improve the outcomes of embryo transfers? No.

OBJECTIVE: The rapid development of Artificial Intelligence (AI) has raised questions about its potential uses in different sectors of everyday life. Specifically in medicine, the question arose whether chatbots could be used as tools for clinical decision-making or patients' and physicians' education. To answer this question in the context of fertility, we conducted a test to determine whether current AI platforms can provide evidence-based responses regarding methods that can improve the outcomes of embryo transfers. METHODS: We asked nine popular chatbots to write a 300-word scientific essay, outlining scientific methods that improve embryo transfer outcomes. We then gathered the responses and extracted the methods suggested by each chatbot. RESULTS: Out of a total of 43 recommendations, which could be grouped into 19 similar categories, only 3/19 (15.8%) were evidence-based practices, those being "ultrasound-guided embryo transfer" in 7/9 (77.8%) chatbots, "single embryo transfer" in 4/9 (44.4%) and "use of a soft catheter" in 2/9 (22.2%), whereas some controversial responses like "preimplantation genetic testing" appeared frequently (6/9 chatbots; 66.7%), along with other debatable recommendations like "endometrial receptivity assay", "assisted hatching" and "time-lapse incubator". CONCLUSIONS: Our results suggest that AI is not yet in a position to give evidence-based recommendations in the field of fertility, particularly concerning embryo transfer, since the vast majority of responses consisted of scientifically unsupported recommendations. As such, both patients and physicians should be wary of guiding care based on chatbot recommendations in infertility. Chatbot results might improve with time especially if trained from validated medical databases; however, this will have to be scientifically checked.

40 'wild' years: the current reality and future potential of assisted reproductive technologies in wildlife species.

Over the past 40 years, assisted reproductive technologies (ARTs) have grown significantly in scale and innovation, from the bovine embryo industry's shift from in vivo derived to in vitro produced embryos and the development of somatic cell-based approaches for embryo production. Domestic animal models have been instrumental in the development of ARTs for wildlife species in support of the One Plan Approach to species conservation that integrates in situ and ex situ population management strategies. While ARTs are not the sole solution to the biodiversity crisis, they can offer opportunities to maintain, and even improve, the genetic composition of the captive and wild gene pools over time. This review focuses on the application of sperm and embryo technologies (artificial insemination and multiple ovulation/in vitro produced embryo transfer, respectively) in wildlife species, highlighting impactful cases in which significant progress or innovation has transpired. One of the key messages following decades of efforts in this field is the importance of collaboration between researchers and practitioners from zoological, academic, governmental, and private sectors.

Pressing needs and recent advances to enhance production of embryos in vitro in cattle.

Embryo transfer in cattle is an increasingly important technique for cattle production. Full attainment of the benefits of the technology will depend on overcoming hurdles to optimal performance using embryos produced in vitro. Given its importance, embryo technology research should become a global research priority for animal reproduction science. Among the goals of that research should be developing methods to increase the proportion of oocytes becoming embryos through optimization of in vitro oocyte maturation and in vitro fertilization, producing an embryo competent to establish and maintain pregnancy after transfer, and increasing recipient fertility through selection, management and pharmacological manipulation. The embryo produced in vitro is susceptible to epigenetic reprogramming and methods should be found to minimize deleterious epigenetic change while altering the developmental program of the resultant calf to increase its health and productivity. There are widening opportunities to rethink the technological basis for much of the current practices for production and transfer of embryos because of explosive advances in fields of bioengineering such as microfluidics, three-dimensional printing of cell culture materials, organoid culture, live-cell imaging, and cryopreservation.

Extracellular vesicles: emerging paradigms in bovine embryo-maternal communication.

The oviduct and uterus provide an optimal environment for early embryo development, where effective communication between the embryo and the maternal reproductive tract is crucial for establishing and maintaining pregnancy. Oviductal and uterine-derived EVs play pivotal roles in this maternal-embryonic communication and in facilitating early embryo development. However, despite the ability of in vitro culture methods to produce viable embryos, the lack of exchange between the embryo and the mother often results in lower-quality embryos than those derived in vivo. Therefore, there is a pressing need to increase our understanding of the physiological mechanisms underlying embryo interaction with the oviduct and endometrium through EVs and to develop models capable of mimicking the in vivo environment. This review aims to provide up-to-date insights into the communication between the mother and pre-implantation bovine embryo, exploring their applications and perspectives in the field.

How pollen and ovule development underlay dioecy in Chloroluma gonocarpa (Sapotaceae).

Previous studies have determined that Chloroluma gonocarpa (Sapotaceae), is a species that has cryptic dioecy. This type of sexual system is characterized by flowers that are morphologically perfect (both sexual whorls are present) but functionally pistillate or staminate (in each type of flower one of the sexual whorls is non-functional). In C. gonocarpa the pistillate flowers present well-developed stigma, functional ovules, and staminodes, while the staminate flowers present a poorly developed stigma, collapsed ovules, and pollen-producing anthers. In angiosperms, the abortion of sexual organs can occur at different stages of development (from pre-meiosis to post-meiosis), that is why we conducted an anatomical analysis of both flower types at various developmental stages. Using light microscopy, we described the processes of sporogenesis and gametogenesis to establish when the staminate flowers lose their pistillate function. To achieve this, we collected, fixed, and processed the flowers following conventional anatomical techniques for observation under a light microscope. Our findings reveal that pollen development occurs only in staminate flowers, while ovule development begins in both types of flowers but ceases in staminate flowers due to post-meiosis abortion. In contrast, normal development continues in pistillate flowers. These results suggest that dioecy in C. gonocarpa may have arisen from a gynodioecious pathway.

Cellular responses to developmental exposure to pyriproxyfen in chicken model: Contrasting embryos with and without exencephaly.

The insecticide pyriproxyfen (PPF), commonly used in drinking water, has already been described as a potential neurotoxic agent in non-target organisms, particularly during embryonic development. Consequently, exposure to PPF can lead to congenital anomalies in the central nervous system. Therefore, understanding the impact of this insecticide on developing neural cells is a relevant concern that requires attention. Thus, this study aimed to investigate the effects of PPF on the proliferation, differentiation, migration, and cell death of neural cells by comparing embryos that develop exencephaly with normal embryos, after exposure to this insecticide. Chicken embryos, used as a study model, were exposed to concentrations of 0.01 and 10 mg/L PPF on embryonic day E1 and analyzed on embryonic day E10. Exposed embryos received 50 µL of PPF diluted in vehicle solution, and control embryos received exclusively 50 µL of vehicle solution. After exposure, embryos were categorized into control embryos, embryos with exencephaly exposed to PPF, and embryos without exencephaly exposed to PPF. The results showed that although the impact was differentiated in the forebrain and midbrain, both brain vesicles were affected by PPF exposure, and this was observed in embryos with and without exencephaly. The most evident changes observed in embryos with exencephaly were DNA damage accompanied by alterations in cell proliferation, increased apoptosis, and reduced neural differentiation and migration. Embryos without exencephaly showed DNA damage and reduced cell proliferation and migration. These cellular events directly interfered with the density and thickness of neural cell layers. Together, these results suggest that PPF exposure causes cellular damage during neurogenesis, regardless of whether embryos display or do not display external normal morphology. This nuanced understanding provides important insights into the neurotoxicity of PPF and its potential effects on inherent events in neurogenesis.

Haploid embryos and embryonic stem cells to produce offspring with predetermined parental genomes in cattle.

Selection strategies are performed post-fertilization when the random combination of paternal and maternal genomes has already occurred. It would be greatly advantageous to eliminate meiotic uncertainty by selecting genetically superior gametes before fertilization. To achieve this goal, haploid embryonic cells and embryonic stem cell lineages could be derived, genotyped, and used to substitute gametes. On the paternal side, androgenetic development can be achieved by removing the maternal chromosomes from the oocyte before or after fertilization. We have shown that once developed into an embryo, haploid cells can be removed for genotyping and, if carrying the selected genome, be used to replace sperm at fertilization. A similar strategy can be used on the maternal side by activating the oocyte parthenogenetically and using some embryonic cells for genotyping while the remaining are used to produce diploid embryos by fertilization. Placed together, both androgenetic and parthenogenetic haploid cells that have been genotyped to identify optimal genomes can be used to produce offspring with predetermined genomes. Successes and problems in developing such a breeding platform to achieve this goal are described and discussed below.

Successful transposition of sperm freezing, in vitro fertilization and artificial insemination methodologies in the Tadjik markhor species (Capra falconeri heptneri).

The objective of the present study was to transpose sperm freezing methodology from domestic goat to the Tadjik markhor (Capra falconeri heptneri) and to address the feasibility to develop IVP and artificial insemination using such frozen semen. Semen of different adult markhor males were successfully recovered by electro-ejaculation and were then frozen using caprine methodology. Frozen semen showed good survival rates at thawing and good fertility rates were assessed in heterologous in vitro fertilization system with goat oocytes. LOPU/IVF was applied for Tadjik markhor females allowing the first successful blastocyst production in vitro. In an applied program, we also transposed successfully intrauterine AI method with frozen/thawed semen to the Tadjik markhor.

From the laboratory to the field: how to mitigate pregnancy losses in embryo transfer programs?

Pregnancy losses negatively affect the cattle industry, impacting economic indices and consequently the entire production chain. Early embryonic failure has been an important challenge in the embryo industry because proper identification of embryo death at the beginning of gestation is difficult. This review aimed to provide a better understanding on reproductive failure and the relationship between early embryonic loss and different reproductive biotechniques. This review also considers insights and possible strategies for reducing early embryonic loss. The strategies addressed are as follows: i) great impact of rigorous embryo evaluation on reducing embryo losses; ii) selection of recipients at the time of transfer, taking into account health and nutritional status, and classification of the corpus luteum using ultrasound, either in area or vascularization; and iii) paternal effect as one of the factors that contribute to pregnancy losses, with a focus on embryo transfer.

Oocyte developmental potential and embryo production before puberty in cattle.

With the development of in vitro technologies, embryos can be produced using oocytes retrieved directly from the ovaries, i.e., regardless of ovulation. This has allowed the use of different animal categories as oocyte donors, including prepubertal cattle. The advantages of using this strategy to shorten the generation interval and accelerate genetic gain over time were soon recognized, and the first offspring generated using oocytes collected from calves were born in the early 1990s. Nevertheless, embryo production from calves and prepubertal heifers remains a challenge. The oocytes collected before puberty present low in vitro developmental potential, and the subsequent blastocyst rates are consistently lower than those from pubertal females. The acquisition of developmental competence by the oocytes occurs progressively throughout the prepubertal period, which can be subdivided into an early, intermediate, and late prepubertal (or peripubertal) phases, each characterized by different physiological and endocrine features. Therefore, embryo yield increases with age but will only achieve its maximum after puberty. The most common strategy to improve oocyte developmental potential before puberty is the use of gonadotrophic stimulation prior to oocyte retrieval. The results with superstimulation, however, vary among studies, depending on the source, dose, and length of FSH treatment, as well as the age and breed of the donors. The use of calves and prepubertal heifers as oocyte donors should also consider the possible impacts of the oocyte retrieval technique (LOPU or OPU) and the use of exogenous hormones on their subsequent fertility and productive life.

Solutions to the fertility equation in beef embryo recipients.

In beef cattle operations that conduct embryo transfer, the overall success depends on the pregnancy outcome that results from every pregnancy opportunity. In this review, we dissected the main components that determine if a recipient will sustain the pregnancy after embryo transfer up to calving. Specifically, we describe the effect of the uterus on its ability to provide a receptive environment for embryo development. We then discuss the capacity of the embryo to thrive after transfer, and especially the contribution of the sire to embryo fitness. Finally, we review the interaction between the uterus and the embryo as an integrated unit that defines the pregnancy.

Recent advancements to increase success in assisted reproductive technologies in cattle.

Assisted reproductive technologies (ART) are fundamental for cattle breeding and sustainable food production. Together with genomic selection, these technologies contribute to reducing the generation interval and accelerating genetic progress. In this paper, we discuss advancements in technologies used in the fertility evaluation of breeding animals, and the collection, processing, and preservation of the gametes. It is of utmost importance for the breeding industry to select dams and sires of the next generation as young as possible, as is the efficient and timely collection of gametes. There is a need for reliable and easily applicable methods to evaluate sexual maturity and fertility. Although gametes processing and preservation have been improved in recent decades, challenges are still encountered. The targeted use of sexed semen and beef semen has obliterated the production of surplus replacement heifers and bull calves from dairy breeds, markedly improving animal welfare and ethical considerations in production practices. Parallel with new technologies, many well-established technologies remain relevant, although with evolving applications. In vitro production (IVP) has become the predominant method of embryo production. Although fundamental improvements in IVP procedures have been established, the quality of IVP embryos remains inferior to their in vivo counterparts. Improvements to facilitate oocyte maturation and development of new culture systems, e.g. microfluidics, are presented in this paper. New non-invasive and objective tools are needed to select embryos for transfer. Cryopreservation of semen and embryos plays a pivotal role in the distribution of genetics, and we discuss the challenges and opportunities in this field. Finally, machine learning (ML) is gaining ground in agriculture and ART. This paper delves into the utilization of emerging technologies in ART, along with the current status, key challenges, and future prospects of ML in both research and practical applications within ART.

Impact of Heat Stress on Oocyte Developmental Competence and Pre-Implantation Embryo Viability in Cattle.

Rectal and vaginal temperatures are utilised in both in vivo and in vitro models to study the effects of heat stress on oocyte competence and embryo viability in cattle. However, uterine temperature increases by only 0.5 °C in heat-stressed cows, significantly lower than simulated increases in in vitro models. Temperature variations within oviducts and ovarian follicles during heat stress are poorly understood or unavailable, and evidence is lacking that oocytes and pre-implantation embryos experience mild (40 °C) or severe (41 °C) heat stress inside the ovarian follicle and the oviduct and uterus, respectively. Gathering detailed temperature data from the reproductive tract and follicles is crucial to accurately assess oocyte competence and embryo viability under realistic heat stress conditions. Potential harm from heat stress on oocytes and embryos may result from reduced nutrient availability (e.g., diminished blood flow to the reproductive tract) or other unidentified mechanisms affecting tissue function rather than direct thermal effects. Refining in vivo stress models in cattle is essential to accurately identify animals truly experiencing heat stress, rather than assuming heat stress exposure as done in most studies. This will improve model reliability and aid in the selection of heat-tolerant animals.

Proteomic Profiling of Cocos nucifera L. Zygotic Embryos during Maturation of Dwarf and Tall Cultivars: The Dynamics of Carbohydrate and Fatty Acid Metabolism.

There is a limited number of studies analyzing the molecular and biochemical processes regulating the metabolism of the maturation of Cocos nucifera L. zygotic embryos. Our research focused on the regulation of carbohydrate and lipid metabolic pathways occurring at three developmental stages of embryos from the Mexican Pacific tall (MPT) and the Yucatan green dwarf (YGD) cultivars. We used the TMT-synchronous precursor selection (SPS)-MS3 strategy to analyze the dynamics of proteomes from both embryos; 1044 and 540 proteins were determined for the MPT and YGD, respectively. A comparison of the differentially accumulated proteins (DAPs) revealed that the biological processes (BP) enriched in the MPT embryo included the glyoxylate and dicarboxylate metabolism along with fatty acid degradation, while in YGD, the nitrogen metabolism and pentose phosphate pathway were the most enriched BPs. Findings suggest that the MPT embryos use fatty acids to sustain a higher glycolytic/gluconeogenic metabolism than the YGD embryos. Moreover, the YGD proteome was enriched with proteins associated with biotic or abiotic stresses, e.g., peroxidase and catalase. The goal of this study was to highlight the differences in the regulation of carbohydrate and lipid metabolic pathways during the maturation of coconut YGD and MPT zygotic embryos.