The effects of ambient temperature and feeding regimens on cecum bacteria composition and circadian rhythm in growing rabbits.

Seasonal environmental shifts and improper eating habits are the important causes of diarrhea in children and growing animals. Whether adjusting feeding time at varying temperatures can modify cecal bacterial structure and improve diarrhea remains unknown. Three batches growing rabbits with two groups per batch were raised under different feeding regimens (fed at daytime vs. nighttime) in spring, summer and winter separately, and contents were collected at six time points in 1 day and used 16S rRNA sequencing to investigate the effects of feeding regimens and season on the composition and circadian rhythms of cecum bacteria. Randomized forest regression screened 12 genera that were significantly associated with seasonal ambient temperature changes. Nighttime feeding reduced the abundance of the conditionally pathogenic bacteria Desulfovibrio and Alistipes in summer and Campylobacter in winter. And also increases the circadian rhythmic Amplicon Sequence Variants in the cecum, enhancing the rhythm of bacterial metabolic activity. This rhythmic metabolic profile of cecum bacteria may be conducive to the digestion and absorption of nutrients in the host cecum. In addition, this study has identified 9 genera that were affected by the combination of seasons and feeding time. In general, we found that seasons and feeding time and their combinations affect cecum composition and circadian rhythms, and that daytime feeding during summer and winter disrupts the balance of cecum bacteria of growing rabbits, which may adversely affect cecum health and induce diarrhea risk.

Optimizing Feeding Strategies for Growing Rabbits: Impact of Timing and Amount on Health and Circadian Rhythms.

Mammals exhibit circadian rhythms in their behavior and physiological activities to adapt to the diurnal changes of the environment. Improper feeding methods can disrupt the natural habits of animals and harm animal health. This study investigated the effects of feeding amount and feeding time on growing rabbits in northern China during spring. A total of 432 healthy 35-day-old weaned rabbits with similar body weight were randomly assigned to four groups: whole day diet-unrestricted feeding (WUF), whole day diet-restricted feeding (WRF), nighttime diet-unrestricted feeding (NUF), and nighttime diet-restricted feeding (NRF). The results showed that nighttime diet-unrestricted feeding improved performance, circadian rhythm of behavior, and body temperature, while reducing the risk of diarrhea and death. WRF group increased daytime body temperature but had no significant difference in feed conversion rate. The study suggests that nighttime diet-unrestricted feeding in spring can improve the growth and welfare of rabbits in northern China. Our study underscores the pivotal role of feeding timing in enhancing animal health. Future investigations should delve into the underlying mechanisms and expand the application of this strategy across seasons and regions to improve rabbit husbandry practices.

Nonlinear vibration feature extraction based on power spectrum envelope adaptive empirical Fourier decomposition.

Analyzing the vibration features of a shipboard stabilized platform (SSP) is significant to the design or vibration compensation of a marine gravimetric survey vibration isolation system. Empirical Fourier decomposition (EFD) is a recently developed method for nonlinear and non-stationary signal decomposition. However, the spectral segmentation boundary needs to be set in advance according to sophisticated experience, and it is easy to be disturbed by noise, and the decomposition result is inaccurate. In order to accurately extract the nonlinear vibration characteristics of SSP, this paper proposes a new method called power spectrum envelope adaptive empirical Fourier decomposition (PSEEFD). Firstly, the number of selected modal decomposition is determined based on the mutual information to realize adaptive segmentation. Then, the improved power spectrum envelope segmentation method is adopted to effectively diminish the interference of noise since the segmentation boundary is formed by the minimum of the adjacent extreme points enveloped by the maximum value of the power spectrum. The spectrum segments obtained from segmentation contain less interference. Finally, the component signal in each frequency band is reconstructed by inverse fast Fourier transform, and the instantaneous frequency signal component with physical significance is obtained. Through the analysis of vibration simulation signals and measured data of SSP, the proposed method is compared with EMD, AFVMD, EWT and EFD. The results show that PSEEFD has a well suppression of noise interference and can effectively extract the characteristics of nonlinear vibration signals.

Decomposition-Based Multi-Step Forecasting Model for the Environmental Variables of Rabbit Houses.

To improve prediction accuracy and provide sufficient time to control decision-making, a decomposition-based multi-step forecasting model for rabbit house environmental variables is proposed. Traditional forecasting methods for rabbit house environmental parameters perform poorly because the coupling relationship between sequences is ignored. Using the STL algorithm, the proposed model first decomposes the non-stationary time series into trend, seasonal, and residual components and then predicts separately based on the characteristics of each component. LSTM and Informer are used to predict the trend and residual components, respectively. The aforementioned two predicted values are added together with the seasonal component to obtain the final predicted value. The most important environmental variables in a rabbit house are temperature, humidity, and carbon dioxide concentration. The experimental results show that the encoder and decoder input sequence lengths in the Informer model have a significant impact on the model's performance. The rabbit house environment's multivariate correlation time series can be effectively predicted in a multi-input and single-output mode. The temperature and humidity prediction improved significantly, but the carbon dioxide concentration did not. Because of the effective extraction of the coupling relationship among the correlated time series, the proposed model can perfectly perform multivariate multi-step prediction of non-stationary time series.

A game-based approach for designing a collaborative evolution mechanism for unmanned swarms on community networks.

Intelligent and coordinated unmanned aerial vehicle (UAV) swarm combat will be the main mode of warfare in the future, and mechanistic design of autonomous cooperation within swarms is the key to enhancing combat effectiveness. Exploration of the essential features and patterns of autonomous collaboration in unmanned swarms has become the focus of scientific research and technological applications, in keeping with the evolving conceptions of the military theatre. However, given the unique attributes of the military and the novelty of the warfare mode of unmanned swarms, few achievements have been reported in the existing research. In this study, we analysed the military requirements of unmanned swarm operations and proposed an analytic framework for autonomous collaboration. Then, a literature review addressing swarm evolution dynamics, game-based swarm collaboration, and collaborative evolution on complex networks was conducted. Next, on the basis of the above work, we designed a community network for unmanned swarm cooperation and constructed a collaborative evolution model based on the multiplayer public goods game (PGG). Furthermore, according to the "network" and "model", the dynamic evolution process of swarm collaboration was formally deduced. Finally, a simulation was conducted to analyse the influence of relevant parameters (i.e., swarm size, degree distribution, cost, multiplication factor) on the collaborative behaviour of unmanned swarms. According to the simulation results, some reasonable suggestions for collaborative management and control in swarm operation are given, which can provide theoretical reference and decision-making support for the design of coordination mechanisms and improved combat effectiveness in unmanned swarm operation.

The Relationship between Animal Welfare and Farm Profitability in Cage and Free-Range Housing Systems for Laying Hens in China.

Several countries and regions have regulations in place to provide standards for the welfare of production animals, which have implications for breeding, management and trade. In the chicken egg production industry, the welfare impacts of this are not well understood. In the past decades, free-range systems were widely used for local chicken breeds in poultry industry in China, but their use has gradually declined due to the lower competitiveness compared to commercial cage systems. However, the practices of free-range systems for hens raising have gradually increased again over the past decade, as consumer individualized demand for higher food quality and animal welfare has increased. We recruited 14 free-range farms and 45 cage farms from Beijing, Shandong, Hebei, Anhui, Yunnan, Gansu and Jiangsu provinces in China, for an evaluation of hen welfare, production and economic outcomes from farm operations. This study provides data for the welfare outcomes of laying hens in China and preliminarily explored the relationship between welfare level and economic income within farming system types. The researchers visited the farms and used Welfare Quality measures to investigate the welfare, and farm self-reported profits. Nonparametric Mann−Whitney U tests were used to compare the welfare scores between cage and free-range rearing farms. Correlation and regression are used for the analysis of the animal welfare scores, economic data, and production metrics. The general income from free-range farms was linearly correlated with red mite score and stocking density (p < 0.001 and p < 0.05, respectively). The results showed less centimeters of feeder and drinker space per animal in the free-range system than in cage systems (p < 0.05 and p < 0.01, respectively). Welfare scores for both the stocking density and beak condition were significantly better in the free-range systems than the cage systems (p < 0.001), as were qualitative behavior assessment scores (p < 0.05). The total egg production and peak egg production in cage farms were much higher than in free-range farms (p < 0.001), and egg loss rate was significantly lower (p < 0.001). While the production efficiency of free-range farms was lower than that of cage farms, general income per 10,000 hens was actually higher. Our results provide some evidence that some welfare indicators and general income (per 10,000 hens) in free-range farms in China were better than those of cage farms. The results indicate that better parasite control and lower stocking densities may result in improved hen welfare on free-range farms and potentially improve profitability. The level of welfare and economic benefits of free-range farms vary widely, and there was potential room for improvement in feeding space, drinking water space and human−animal relationship.

Botrytis cinerea mediated cell wall degradation accelerates spike stalk browning in Munage grape.

Munage grape (Vitis vinifera L. cv. Munage.) is a unique cultivar in southern Xinjiang, China. Spike stalk browning in this species has becomes more common in recent years, negatively impacting the shelf life, and causing severe economic losses during storage. This study investigated the changes in metabolisms of cell wall by Botrytis cinerea infection in association with spike stalk browning. Morphological and physiological observations showed that preharvest B. cinerea infection accelerates the spike stalk browning during storage in Munage grapes by promoting cell wall degradation. Accordingly, the cell structures in infected spike stalk showed severe collapse, while the cell structures in uninfected spike stalk remained relatively complete. Furthermore, the contents of CDTA-soluble pectin (CSP), Na2 CO3 -soluble pectin (NSP), cellulose, and hemicellulose were reduced, while the water-soluble pectin (WSP) content was increased during infection. In addition, the activities of polygalacturonase (PG), pectin methylesterase (PME), beta-galactosidase (ß-Gal), and cellulase (Cx) were highly promoted by B. cinerea. Correspondingly, the expression levels of VvPG were markedly upregulated after inoculation and played a major role in cell wall degradation. Additionally, the spike stalk inoculated by B. cinerea showed higher activities of PPO and POD, and content of total phenolics. These results contribute to elucidating the relationship between cell wall degradation induced by B. cinerea during spike stalk browning and provide a basis for future research on improving the ability of the host cell wall to resist degrading enzymes. PRACTICAL APPLICATIONS: Botrytis cinerea is the main fungal pathogen causing the gray mold of grapes. It usually enters the tissue early in crop development, has a long incubation period, and rapidly infects the tissue when the environment is favorable and the host physiology changes. Gray mold has been reported as one of the major postharvest diseases of grapes. However, there are relatively few reports on the pathways through which B. cinerea causes the browning of grape stalks. Controlling browning caused by B. cinerea may require clarification of the physiological and molecular mechanisms by which browning occurs. The elucidation of the role of B. cinerea in causing browning of grape stalks through the cell wall degradation pathway will help to provide scientific basis for further controlling browning, maintaining freshness of stalks, developing biological agents to prevent browning, improving grape quality, and extending storage period.

Loss of diurnal behavioral rhythms and impaired lipid metabolism in growing pigs with mistimed feeding.

The misalignment of eating time and the endogenous circadian rhythm impairs the body's ability to maintain homeostasis. Although it is well established that children and growing animals differ from adults in their energy metabolism and behavioral patterns, little is known about how mistimed feeding disturbs the diurnal rhythms of behavior and metabolism in children and growing diurnal animals. In this study, growing pigs (diurnal animal) were randomly assigned to the daytime-restricted feeding (DRF) and nighttime-restricted feeding (NRF) groups for 5 weeks. Compared with observations in the DRF group, NRF disrupted the diurnal rhythm of behavior and clock genes and lowered the serum ghrelin, dopamine, and serotonin levels during the daytime and nighttime. Microbiome analysis results suggested that NRF altered the diurnal rhythm and composition of the gut microbiota, and increased log-ratios of Catenibacterium:Butyrivibrio and Streptococcus:Butyrivibrio. Based on the serum proteome, the results further revealed that rhythmic and upregulated proteins in NRF were mainly involved in oxidative stress, lipid metabolism, immunity, and cancer biological pathways. Serum physiological indicators further confirmed that NRF decreased the concentration of melatonin and fibroblast growth factor 21 during the daytime and nighttime, increased the diurnal amplitude and concentrations of very-low-density lipoprotein cholesterol, triglyceride, and total cholesterol, and increased the apolipoprotein B/ApoA1 ratio, which is a marker of metabolic syndrome. Taken together, this study is the first to reveal that mistimed feeding disrupts the behavioral rhythms of growing pigs, reprograms gut microbiota composition, reduces the serum levels of hormones associated with fighting depression and anxiety, and increases the risk of lipid metabolic dysregulation.

Prolonged melatonin treatment promote testicular recovery by enhancing RAC1-mediated apoptotic cell clearance and cell junction-dependent spermatogensis after heat stress.

INTRODUCTION: A decline in semen quality caused by global warming and torrid working conditions is a major cause of human male infertility, and heat stress-induced decreases in male reproductive ability results in economic losses in livestock husbandry. Increasing evidence suggests that melatonin exerts protective effects on stress-induced DNA damage and apoptosis in germ cells. However, few studies have assessed the effects of melatonin on testicular recovery during post-heat stress and the underlying mechanisms. METHODS AND RESULTS: In vivo studies using 8-week-old male CD-1 mice revealed that melatonin pretreatment (50 mg/kg for 5 days) did not alleviate heat stress-induced germ cell loss and disrupted testicular histomorphology, however, long-term melatonin administration after heat stress accelerated germ cell apoptosis, spermatogenic cell regeneration, and testicular weight recovery. In vitro studies demonstrated that melatonin enhanced RAC1 activity, resulting in increased phagocytosis of apoptotic germ cells by Sertoli cells. In addition, melatonin restored gap junctions and tight junctions after heat stress, thereby promoting hollow seminiferous tubule filling. DISCUSSION: Long-term melatonin administration accelerated testicular recovery after heat stress by enhancing the phagocytotic activity of Sertoli cells and the regeneration of spermatogenic cells. This finding suggests that melatonin is a potential therapeutic for heat stress-induced male infertility.

Night-restricted feeding improves locomotor activity rhythm and modulates nutrient utilization to accelerate growth in rabbits.

An unfavorable lifestyle disrupts the circadian rhythm, leading to metabolic dysfunction in adult humans and animals. Increasing evidence suggests that night-restricted feeding (NRF) can effectively prevent ectopic fat deposition caused by circadian rhythm disruption, and reduce the risk of metabolic diseases. However, previous studies have mainly focused on the prevention of obesity in adults by regulating dietary patterns, whereas limited attention has been paid to the effect of NRF on metabolism during growth and development. Here, we used weaning rabbits as models and found that NRF increased body weight gain without increasing feed intake, and promoted insulin-mediated protein synthesis through the mTOR/S6K pathway and muscle formation by upregulating MYOG. NRF improved the circadian clock, promoted PDH-regulated glycolysis and CPT1B-regulated fatty-acid ß-oxidation, and reduced fat content in the serum and muscles. In addition, NRF-induced body temperature oscillation might be partly responsible for the improvement in the circadian clock and insulin sensitivity. Time-restricted feeding could be used as a nondrug intervention to prevent obesity and accelerate growth in adolescents.

Night-Restricted Feeding Improves Gut Health by Synchronizing Microbe-Driven Serotonin Rhythm and Eating Activity-Driven Body Temperature Oscillations in Growing Rabbits.

The circadian misalignment of the gut microbiota caused by unusual eating times in adult animals is related to disease development. However, whether the composition and diurnal rhythm of gut microbiota can be optimized by synchronizing the window period of eating with natural eating habits to reduce the risk of diarrhea remains unclear, especially in growing animals. In this study, 108 5-week-old weaned rabbits (nocturnal animals) were randomly subjected to daytime feeding (DF) and night-restricted feeding (NRF). At age 12 weeks, six rabbits were selected from each group, and caecum and cecal contents, as well as serum samples were collected at 4-h intervals during 24 h. Overall, NRF was found to reduce the risk of diarrhea in growing rabbits, improved the diurnal rhythm and abundance of beneficial microorganisms, along with the production of beneficial metabolites, whereas reduced the abundance of potential pathogens (Synergistes, Desulfovibrio, and Alistipes). Moreover, NRF improved diurnal rhythm of tryptophan hydroxylase isoform 1 and serotonin. Furthermore, NRF strengthened the diurnal amplitude of body core temperature, and promoted the diurnal expression of intestinal clock genes (BMAL1, CLOCK, REV-ERBα, and PER1), and genes related to the regulation of the intestinal barrier (CLAUDIN-1), and intestinal epithelial cell self-proliferation and renewal (BMI1). In vitro simulation experiments further revealed that synchronization of microbial-driven serotonin rhythm and eating activity-driven body temperature oscillations, which are important zeitgebers, could promote the diurnal expression of clock genes and CLAUDIN-1 in rabbit intestinal epithelial cells (RIEC), and enhance RIEC proliferation. This is the first study to reveal that NRF reprograms the diurnal rhythm of the gut microbiome, promotes the diurnal expression of clock genes and tight junction genes via synchronization of microbial-driven serotonin rhythm and eating activity-driven body temperature oscillations, thereby improving intestinal health and reducing the risk of diarrhea in growing rabbits. Collectively, these results provide a new perspective for the healthy feeding and management of growing animals.

Drinking Warm Water Improves Growth Performance and Optimizes the Gut Microbiota in Early Postweaning Rabbits during Winter.

Accumulating evidence indicates that cold exposure changes the composition of the gut microbiota and reduces intestinal immunity in early postweaning livestock. However, little is known about the effects of drinking warm water (WW) on gut microbiota during winter. In this study, we investigated the effects of drinking WW in winter on the growth performance and gut microbiota structure of rabbits raised in poorly insulated housing from the early postweaning period (day 46) to the subadult period (day 82). The average daily gain and feed conversion ratio in rabbits drinking WW were significantly improved compared to those of the rabbits drinking cold water (CW) during 47-58 days. In addition, rabbits drinking WW had a significantly decreased the risk of diarrhea during 71-82 days. 16S rRNA sequence analysis revealed that the alpha diversity of the cecal microbiota was not significantly different between the WW and CW groups, but significantly increased with age. The relative abundance of cecal microorganisms, such as Coprococcus spp. was considerably increased at day 70 in the group drinking WW. Correlation analysis indicated that Coprococcus spp. was negatively associated with pro-inflammatory factors. In conclusion, our results suggest that drinking WW has a positive effect on growth performance and gut microbiota in rabbits during the early postweaning stage in winter.

Melatonin protects in vitro matured porcine oocytes from toxicity of Aflatoxin B1.

Aflatoxin B1 (AFB1) is a major food and feed contaminant that threaten public health. Previous studies indicate that AFB1 exposure disrupted oocyte maturation. However, an effective and feasible method is unavailable for protecting oocytes against toxicity of AFB1. In the present study, using in vitro matured porcine oocytes and parthenogenetic embryos as model, we confirmed that AFB1 exposure during in vitro oocyte maturation (IVM) significantly impaired both nuclear and cytoplasmic maturation in a dose- and time-dependent manner. The different concentrations of melatonin were also tested for their protective effects on oocytes against the AFB1-induced toxicity. Our results showed that supplementation of a relative high concentration of melatonin (10-3 mol/L) during IVM efficiently reversed the impaired development rate and blastocyst quality, to the levels comparable to those of the control group. Further analysis indicated that melatonin application efficiently alleviated reactive oxygen species accumulation and initiation of apoptosis induced by AFB1 exposure. In addition, disrupted GSH/GPX system, as well as inhibited mitochondrial DNA (mtDNA) replication and mitochondrial biogenesis in AFB1-treated oocytes, can be notably reversed by melatonin application. Furthermore, cumulus cells may be important in mediating the toxicity of AFB1 to oocytes, and the metabolism of AFB1 in cumulus cells can be depressed by melatonin. To the best of our knowledge, this is the first report to confirm that melatonin application can efficiently protect oocytes from AFB1-induced toxicity. Our study provides a promising and practical strategy for alleviating or reversing AFB1-induced female reproductive toxicity in both clinical treatment and domestic reproductive management.

Natriuretic peptide receptor 2 (NPR2) localized in bovine oocyte underlies a unique mechanism for C-type natriuretic peptide (CNP)-induced meiotic arrest.

Meiosis is of prime importance for successful gametogenesis, and insufficient maintenance of oocyte meiotic arrest compromises oocyte developmental competence. Recent studies have demonstrated that the C-type natriuretic peptide (CNP)-Natriuretic peptide receptor 2 (NPR2) pathway can inhibit mammalian oocyte meiotic resumption. In mouse and porcine, the inhibitory effect of mural granulosa cell (MGC)-derived CNP on oocyte meiotic resumption is mediated by NPR2 localized in cumulus cells (CCs) surrounding the oocytes. However, in the present study, we identified a novel mechanism for CNP-induced meiotic arrest that appears to be unique to bovine oocytes. Unlike mouse and porcine, bovine NPR2 not only localizes in CCs, but also in oocyte membranes. We also showed that CNP can directly activate intra-oocyte cGMP production via NPR2 localized in oocyte membranes, in parallel with the CC-mediated pathway. Furthermore, we demonstrated that Npr2 expression in bovine CCs and oocytes were synergistically regulated by estradiol and oocyte-derived growth factors. Finally, based on the profound inhibitory effect of CNP on meiotic resumption, we established a natural factor synchronized in vitro oocyte maturation (NFSOM) system, which can significantly improve the developmental competence of matured oocytes, thereby resulting in higher in vitro embryo production efficiency. Taken together, our study not only provides new insight into understanding the crosstalk between oocytes and follicular somatic cells in mammals, but also presents a promising strategy for improving the in vitro oocyte maturation systems of assisted reproductive technology (ART).

High-resolution profiles of gene expression and DNA methylation highlight mitochondrial modifications during early embryonic development.

Well-organized mitochondrial functions and dynamics are critical for early embryonic development and are operated via a large number of mitochondria-related genes (MtGs) encoded by both the nuclear and the mitochondrial genome. However, the mechanisms underlying mitochondrial modifications during the critical window between blastocyst implantation and postimplantation organogenesis are poorly understood. Herein, we performed high-resolution dynamic profiling of MtGs to acquire a more detailed understanding of mitochondrial modifications during early development. Our data suggest that the resumption of mitochondrial mass growth is not only facilitated by increased mitochondrial biogenesis and mitochondrial DNA (mtDNA) replication, but also by the appropriate balance between mitochondrial fission and fusion. In addition, increased levels of reactive oxygen species (ROS) resulting from enhanced mitochondrial functions may be the critical inducer for activating the glutathione (GSH)-based stress response system in early embryos. The appropriate balance between the mitochondrial stress response and apoptosis appears to be significant for cell differentiation and early organogenesis. Furthermore, we found that most MtGs undergo de novo promoter methylation, which may have functional consequences on mitochondrial functions and dynamics during early development. We also report that mtDNA methylation can be observed as early as soon after implantation. DNMT1, the predominant enzyme for maintaining DNA methylation, localized to the mitochondria and bound to mtDNA by the implantation stage. Our study provides a new insight into the involvement of mitochondria in early mammalian embryogenesis. We also propose that the epigenetic modifications during early development are significant for modulating mitochondrial functions and dynamics.

Efficient biallelic mutation in porcine parthenotes using a CRISPR-Cas9 system.

The parthenotes represent ideal models mimicking the embryonic development and characterizing the function of maternal genomes as well as an alternative source of pluripotent cell lines. Besides, parthenogenetically activated (PA) embryos serve as a rapid assay system to maximize the efficiency of generating genetically modified pig CRISPR/Cas9 system, an efficient and multiplex gene editing tool, has been utilized to modify the genome of porcine parthenotes. However, lower biallelic mutation rate and high mosaicism frequency were observed. Here, we aimed to enhance the biallelic mutation rate with reduced mosaicism by optimization of the concentration and injection time of the Cas9/sgRNA mixture in porcine parthenotes. The results showed that the efficient biallelic mutation (93%) and low mosaicism (33%) could be achieved in porcine parthenotes by cytoplasmic injection of Cas9 mRNA/sgRNA (125/12.5 ng/µl) after 8 h of parthenogenetical activation. Thus, our study provides an effective strategy for increasing the biallelic mutation rate and population homogeneity of genetically modified parthenotes, which will strengthen the role of parthenotes in uncovering early embryonic development and assessing the mutation efficiency due to the simplicity and adaptability of CRISPR/Cas9.

Impaired imprinted X chromosome inactivation is responsible for the skewed sex ratio following in vitro fertilization.

Dynamic epigenetic reprogramming occurs during normal embryonic development at the preimplantation stage. Erroneous epigenetic modifications due to environmental perturbations such as manipulation and culture of embryos during in vitro fertilization (IVF) are linked to various short- or long-term consequences. Among these, the skewed sex ratio, an indicator of reproductive hazards, was reported in bovine and porcine embryos and even human IVF newborns. However, since the first case of sex skewing reported in 1991, the underlying mechanisms remain unclear. We reported herein that sex ratio is skewed in mouse IVF offspring, and this was a result of female-biased peri-implantation developmental defects that were originated from impaired imprinted X chromosome inactivation (iXCI) through reduced ring finger protein 12 (Rnf12)/X-inactive specific transcript (Xist) expression. Compensation of impaired iXCI by overexpression of Rnf12 to up-regulate Xist significantly rescued female-biased developmental defects and corrected sex ratio in IVF offspring. Moreover, supplementation of an epigenetic modulator retinoic acid in embryo culture medium up-regulated Rnf12/Xist expression, improved iXCI, and successfully redeemed the skewed sex ratio to nearly 50% in mouse IVF offspring. Thus, our data show that iXCI is one of the major epigenetic barriers for the developmental competence of female embryos during preimplantation stage, and targeting erroneous epigenetic modifications may provide a potential approach for preventing IVF-associated complications.

Dynamic comparisons of high-resolution expression profiles highlighting mitochondria-related genes between in vivo and in vitro fertilized early mouse embryos.

STUDY QUESTION: Does in vitro fertilization (IVF) induce comprehensive and consistent changes in gene expression associated with mitochondrial biogenesis and function in mouse embryos from the pre- to post-implantation stage? SUMMARY ANSWER: IVF-induced consistent mitochondrial dysfunction in early mouse embryos by altering the expression of a number of mitochondria-related genes. WHAT IS KNOWN ALREADY: Although IVF is generally safe and successful for the treatment of human infertility, there is increasing evidence that those conceived by IVF suffer increased health risks. The mitochondrion is a multifunctional organelle that plays a crucial role in early development. We hypothesized that mitochondrial dysfunction is associated with increased IVF-induced embryonic defects and risks in offspring. STUDY DESIGN, SIZE, DURATION: After either IVF and development (IVO groups as control) or IVF and culture (IVF groups), blastocysts were collected and transferred to pseudo-pregnant recipient mice. Both IVO and IVF embryos were sampled at E3.5, E7.5 and E10.5, and the expression profiles of mitochondria-related genes from the pre- to post-implantation stage were compared. PARTICIPANTS/MATERIALS, SETTING, METHODS: ICR mice (5- to 6-week-old males and 8- to 9-week-old females) were used to generate IVO and IVF blastocysts. Embryo day (E) 3.5 blastocysts were transferred to pseudo-pregnant recipient mice. Both IVO and IVF embryos were sampled at E3.5, E7.5 and E10.5 for generating transcriptome data. Mitochondria-related genes were filtered for dynamic functional profiling. Mitochondrial dysfunctions indicated by bioinformatic analysis were further validated using cytological and molecular detection, morphometric and phenotypic analysis and integrated analysis with other high-throughput data. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 806, 795 and 753 mitochondria-related genes were significantly (P < 0.05) dysregulated in IVF embryos at E3.5, E7.5 and E10.5, respectively. Dynamic functional profiling, together with cytological and molecular investigations, indicated that IVF-induced mitochondrial dysfunctions mainly included: (i) inhibited mitochondrial biogenesis and impaired maintenance of DNA methylation of mitochondria-related genes during the post-implantation stage; (ii) dysregulated glutathione/glutathione peroxidase (GSH/Gpx) system and increased mitochondria-mediated apoptosis; (iii) disturbed mitochondrial ß-oxidation, oxidative phosphorylation and amino acid metabolism; and (iv) disrupted mitochondrial transmembrane transport and membrane organization. We also demonstrated that some mitochondrial dysfunctions in IVF embryos, including impaired mitochondrial biogenesis, dysregulated GSH homeostasis and reactive oxygen species-induced apoptosis, can be rescued by treatment with melatonin, a mitochondria-targeted antioxidant, during in vitro culture. LIMITATIONS, REASONS FOR CAUTION: Findings in mouse embryos and fetuses may not be fully transferable to humans. Further studies are needed to confirm these findings and to determine their clinical significance better. WIDER IMPLICATIONS OF THE FINDINGS: The present study provides a new insight in understanding the mechanism of IVF-induced aberrations during embryonic development and the increased health risks in the offspring. In addition, we highlighted the possibility of improving existing IVF systems by modulating mitochondrial functions.

Actin Disorganization Plays a Vital Role in Impaired Embryonic Development of In Vitro-Produced Mouse Preimplantation Embryos.

Assisted reproductive technology (ART) is being increasingly applied to overcome infertility. However, the in vitro production process, the main procedure of ART, can lead to aberrant embryonic development and health-related problems in offspring. Understanding the mechanisms underlying the ART-induced side effects is important to improve the ART process. In this study, we carried out comparative transcriptome profiling between in vivo- (IVO) and in vitro- produced (IVP) mouse blastocysts. Our results suggested that aberrant actin organization might be a major factor contributing to the impaired development of IVP embryos. To test this, we examined the effect of actin disorganization on the development of IVP preimplantation embryos. Specific disruption of actin organization by cytochalasin B (CB) indicated that well-organized actin is essential for in vitro embryonic development. Supplementing the culture medium with 10(-9) M melatonin, a cytoskeletal modulator in adult somatic cells, significantly reversed the disrupted expression patterns of genes related to actin organization, including Arhgef2, Bcl2, Coro2b, Flnc, and Palld. Immunofluorescence analysis showed that melatonin treatment of IVP embryos significantly improved the distribution and organization of actin filaments (F-actin) from the 8-cell stage onwards. More importantly, we found that melatonin alleviated the CB-mediated aberrant F-actin distribution and organization and rescued CB-induced impaired embryonic development. This is the first study to indicate that actin disorganization is implicated in impaired development of IVP embryos during the preimplantation stage. We also demonstrated that improving actin organization is a promising strategy to optimize existing IVP systems.

Comparative analysis between endometrial proteomes of pregnant and non-pregnant ewes during the peri-implantation period.

BACKGROUND: Early pregnancy failure has a profound impact on both human reproductive health and animal production. 2/3 pregnancy failures occur during the peri-implantation period; however, the underlying mechanism(s) remains unclear. Well-organized modification of the endometrium to a receptive state is critical to establish pregnancy. Aberrant endometrial modification during implantation is thought to be largely responsible for early pregnancy loss. RESULT: In this study, using well-managed recipient ewes that received embryo transfer as model, we compared the endometrial proteome between pregnant and non-pregnant ewes during implantation period. After embryo transfer, recipients were assigned as pregnant or non-pregnant ewes according to the presence or absence of an elongated conceptus at Day 17 of pregnancy. By comparing the endometrial proteomic profiles between pregnant and non-pregnant ewes, we identified 94 and 257 differentially expressed proteins (DEPs) in the endometrial caruncular and intercaruncular areas, respectively. Functional analysis showed that the DEPs were mainly associated with immune response, nutrient transport and utilization, as well as proteasome-mediated proteolysis. CONCLUSION: These analysis imply that dysfunction of these biological processes or pathways of DEP in the endometrium is highly associated with early pregnancy loss. In addition, many proteins that are essential for the establishment of pregnancy showed dysregulation in the endometrium of non-pregnant ewes. These proteins, as potential candidates, may contribute to early pregnancy loss.