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.

Identification of Biomarkers Affecting Cryopreservation Recovery Ratio in Ram Spermatozoa Using Tandem Mass Tags (TMT)-Based Quantitative Proteomics Approach.

Sperm proteins play vital roles in improving sperm freezing resilience in domestic animals. However, it remains poorly defined which proteins regulate the freezing resilience of spermatozoa in rams (Ovis aries). Here, we compared the proteome of ram sperm with a high cryopreservation recovery ratio (HCR) with that of ram sperm with a low cryopreservation recovery ratio (LCR) using a tandem mass tag-based quantitative proteomics approach. Bioinformatic analysis was performed to evaluate differentially expressed proteins (DEPs). A total of 2464 proteins were identified, and 184 DEPs were screened. Seventy-two proteins were higher in the LCR group. One hundred and twelve proteins were more abundant in the HCR group, and they were mainly involved in the regulation of oxidative phosphorylation and thermogenesis pathways. Proteins in high abundance in the HCR group included the S100A family, such as S100A8, S100A9, S100A14, and S100A16, effectively controlling for CA2+ and maintaining flagella structure; HYOU1 and PRDX1, which participate in antioxidant protection and anti-apoptosis to prevent cell death; and HSP90B1, which maintains cell activity and immune response. Our results could help illuminate the molecular mechanisms underlying cryopreservation of ram semen and expand the potential direction of cryopreservation of high-quality semen.

Microbiome-Metabolome Reveals the Contribution of the Gut-Testis Axis to Sperm Motility in Sheep (Ovis aries).

A close association exists among testicular function, gut microbiota regulation, and organismal metabolism. In this study, serum and seminal plasma metabolomes, and the rumen microbiome of sheep with significant differences in sperm viability, were explored. Serum and seminal plasma metabolomes differed significantly between high-motility (HM) and low-motility (LM) groups of sheep, and 39 differential metabolites closely related to sperm motility in sheep were found in seminal plasma metabolomes, while 35 were found in serum samples. A 16S rRNA sequence analysis showed that the relative abundance of HM and LM rumen microorganisms, such as Ruminococcus and Quinella, was significantly higher in the HM group, whereas genera such as Rikenellaceae_RC9_gut_group and Lactobacillus were enriched in the mid-LM group. Serum hormone assays revealed that serum follicle-stimulating hormone (FSH) and MT levels were significantly lower in the LM group than in the HM group, whereas serum glucocorticoid (GC) levels were higher in the LM group than in the HM group, and they all affected sperm motility in sheep. Ruminococcus and other rumen microorganisms were positively correlated with sperm motility, whereas Lactobacillus was negatively correlated with FSH and GCs levels. Our findings suggest that rumen microbial activity can influence the host metabolism and hormone levels associated with fertility in sheep.

Changes on proteomic and metabolomic profiling of cryopreserved sperm effected by melatonin.

Cryopreservation may reduce sperm fertility due to cryodamage including physical-chemical and oxidative stress damages. As a powerful antioxidant, melatonin has been reported to improve cryoprotective effect of sperm. However, the molecular mechanism of melatonin on cryopreserved ram sperm hasn't been fully understand. Give this, this study aimed to investigate the postthaw motility parameters, antioxidative enzyme activities and lipid peroxidation, as well as proteomic, metabolomic changes of Huang-huai ram spermatozoa with freezing medium supplemented with melatonin. Melatonin was firstly replenished to the medium to yield five different final concentrations: 0.1, 0.5, 1.0, 1.5, and 2.0 mM. A control (NC) group without melatonin replenishment was included. Protective effects of melatonin as evidenced by postthaw motility, activities of T-AOC, T-SOD, GSH-Px, CAT, contents of MDA, 4-HNE, as well as acrosome integrity, plasma membrane integrity, with 0.5 mM being the most effective concentration (MC group). Furthermore, 29 differentially abundant proteins involving in sperm functions were screened among Fresh, NC and MC groups of samples (n = 5) based on the 4D-LFQ, with 7 of them upregulated in Fresh and MC groups. 26 differentially abundant metabolites were obtained involving in sperm metabolism among the three groups of samples (n = 8) based on the UHPLC-QE-MS, with 18 of them upregulated in Fresh and MC groups. According to the bioinformatic analysis, melatonin may have positive effects on frozen ram spermatozoa by regulating the abundance changes of vital proteins and metabolites related to sperm function. Particularly, several proteins such as PRCP, NDUFB8, NDUFB9, SDHC, DCTN1, TUBB6, TUBA3E, SSNA1, as well as metabolites like L-histidine, L-targinine, ursolic acid, xanthine may be potential novel biomarkers for evaluating the postthaw quality of ram spermatozoa. In conclusion, a dose-dependent replenishment of melatonin to freezing medium protected ram spermatozoa during cryopreservation, which can improve motility, antioxidant enzyme activities, reduce levels of lipid peroxidation products, modify the proteomic and metabolomic profiling of cryopreserved ram spermatozoa through reduction of oxidative stress, maintenance of OXPHOS and microtubule structure. SIGNIFICANCE: Melatonin, a powerful antioxidant protects ram spermatozoa from cryopreservation injuries in a dose-dependent manner, with 0.5 mM being the most effective concentration. Furthermore, sequencing results based on the 4D-LFQ combined with the UHPLC-QE-MS indicated that melatonin modifies proteomic and metabolomic profiling of ram sperm during cryopreservation. According to the bioinformatic analysis, melatonin may have positive effects on frozen ram spermatozoa by regulating the expression changes of vital proteins and metabolites related to sperm metabolism and function. Particularly, several potential novel biomarkers for evaluating the postthaw quality of ram spermatozoa were acquired, proteins such as PRCP, NDUFB8, NDUFB9, SDHC, DCTN1, TUBB6, TUBA3E, SSNA1, as well as metabolites like L-histidine, L-targinine, ursolic acid, xanthine.

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.

Proteomic analysis of physiological function response to hot summer in liver from lactating dairy cows.

Lactation performance of dairy cattle is susceptible to heat stress. The liver is one of the most crucial organs affected by high temperature in dairy cows. However, the physiological adaption by the liver to hot summer conditions has not been well elucidated in lactating dairy cows. In the present study, proteomic analysis of the liver in dairy cows in spring and hot summer was performed using a label-free method. In total, 127 differentially expressed proteins were identified; most of the upregulated proteins were involved in protein metabolic processes and responses to stimuli, whereas most of the downregulated proteins were related to oxidation-reduction. Pathway analysis indicated that 3 upregulated heat stress proteins (HSP90α, HSP90ß, and endoplasmin) were enriched in the NOD-like receptor signaling pathway, whereas several downregulated NADH dehydrogenase proteins were involved in the oxidative phosphorylation pathway. The protein-protein interaction network indicated that several upregulated HSPs (HSP90α, HSP90ß, and GRP78) were involved in more interactions than other proteins and were thus considered as central hub nodes. Our findings provide novel insights into the physiological adaption of liver function in lactating dairy cows to natural high temperature.