TMT-based quantitative proteomics reveals the targets of andrographolide on LPS-induced liver injury.

BACKGROUND: Andrographolide (Andro) is a diterpenoid derived from Andrographis paniculate, which has anti-inflammatory, antibacterial, antiviral and hepatoprotective activities. Gram-negative bacterial infections can cause varying degrees of liver injury in chickens, although Andro has been shown to have a protective effect on the liver, its underlying mechanism of action and effects on liver proteins are not known. METHODS: The toxicity of Andro on the viability of leghorn male hepatoma (LMH) cells at different concentrations and times was analyzed by CCK-8 assays. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in the culture supernatants were measured using an automatic biochemical analyzer to evaluate the protective effect of androscopolide on LPS-induced injury of LMH cells. Subsequently, TMT proteomics analysis were performed on the negative control group (NC group), LPS, and LPS-Andro groups, and bioinformatics analysis was performed on the differentially expressed proteins (DEPs). RESULTS: It was found that Andro reduced ALT and AST levels in the cell supernatant and alleviated LPS-induced injury in LMH cells. Proteomic analysis identified 50 and 166 differentially expressed proteins in the LPS vs. NC group and LPS-Andro vs. LPS group, respectively. Andro may be involved in steroid metabolic processes, negative regulation of MAPK cascade, oxidative stress, and other processes to protect against LPS-induced liver injury. CONCLUSIONS: Andro protects against LPS-induced liver injury, HMGCS1, HMGCR, FDPS, PBK, CAV1, PRDX1, PRDX4, and PRDX6, which were identified by differential proteomics, may be the targets of Andro. Our study may provide new theoretical support for Andro protection against liver injury.

Assessment of semen quality and anti-oxidative enzyme activity between bovine sex-sorted and non-sex-sorted frozen-thawed semen.

In the current study, the difference between the sex-sorted and non-sex-sorted frozen semen of Holstein Friesian breed cattle was investigated. Significant variation (p < .05) was found in the semen quality parameters such as motility; vitality; acrosome integrity rate; the anti-oxidative enzyme activity including GSH (glutathione); SOD (superoxide dismutase); CAT (catalase); GSH-Px (glutathione peroxidase) and the rate of fertilization. The results showed that the sperm acrosome integrity and motility of the non-sorted sperm were higher compared to sex-sorted sperm (p < .05). The linearity index and mean coefficient analysis revealed that the percentage of 'grade a' in sex-sorted sperm were significantly (p < .05) lower than non-sorted sperm. Interestingly, low SOD level and high CAT level was found in the non-sexed semen than in the sexed semen (p < .05). Furthermore, the GSH and GSH-Px activity in the sexed semen was found lower than the non-sexed semen (p < .05). In conclusion, sperm motility characteristics were lower in sex-sorted semen than in non-sex-sorted semen. This might be related to the complex process of sexed semen production, which could reduce sperm motility and movement characteristics, acrosomal integrity, CAT, SOD, GSH and GSH-Px, and finally lead to the decline in the fertilization rate.

Metabolomic profiling of Dezhou donkey seminal plasma related to freezability.

Donkeys are indispensable livestock in China because they have transport function and medicinal value. With the popularization of artificial insemination on donkeys, semen cryopreservation technology has gradually become a research hotspot. Seminal plasma is a necessary medium for transporting sperm and provides energy and nutrition for sperm. Seminal plasma metabolites play an important role in the process of sperm freezing, and also have an important impact on sperm motility and fertilization rate after freezing and thawing. In this study, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was used to compare the metabolic characteristics of seminal plasma of high freezability (HF) and low freezability (LF) male donkeys. We identified 672 metabolites from donkey seminal plasma, of which 33 metabolites were significantly different between the two groups. Metabolites were identified and categorized according to their major chemical classes, including homogeneous non-metal compounds, nucleosides, nucleotides, and analogues, organosulphur compounds, phenylpropanoids and polyketide, organoheterocyclic compounds, organic oxygen compounds, benzenoids, organic acids and derivatives, lipids and lipid-like molecules, organooxygen compounds, alkaloids and derivatives, organic nitrogen compounds. The results showed that the contents of phosphatidylcholine, piceatannol and enkephalin in donkey semen of HF group were significantly higher than those of LF group (p < .05), while the contents of taurocholic and lysophosphatidic acid were significantly lower than those of LF group (p < .05). The different metabolites were mainly related to sperm biological pathway response and oxidative stress. These metabolites may be considered as candidate biomarkers for different fertility in jacks.

Changes in sperm metabolites of Dezhou donkey after cryopreservation.

Sperm cryopreservation technology has laid the foundation for promoting the popularity of artificial insemination in donkey reproduction, but the freeze-thaw process can cause sperm damage, and the viability of frozen sperm is greatly reduced, resulting in low insemination ability. Sperm metabolites play an important role in the freezing process of spermatozoa and have a major influence on the freezability of spermatozoa. The aim of this study was to explore the differential metabolites in donkey spermatozoa before and after cryopreservation by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We analysed ejaculate samples from male donkeys obtained before and after freezing and identified 1323 metabolites. Compared with fresh sperm (F), the metabolites of cryopreserved sperm (CRY) were significantly changed, and 570 metabolites were significantly different between the two groups (p < .05). Among them, 277 metabolites were higher in frozen sperm, while the opposite was true for 293 metabolites. These metabolites mainly include phospholipids, lysophospholipids and amino acids., most of which are associated with oxidative stress and sperm capacitation. We describe significantly different metabolites before and after freezing that are significantly associated with decreased sperm motility post-freezing and can be used as biomarkers of decreased sperm motility post-freezing.

Effects of long-distance transportation on blood constituents and composition of the nasal microbiota in healthy donkeys.

BACKGROUND: This study aims to determine the effects of transportation on the nasal microbiota of healthy donkeys using 16S rRNA sequencing. RESULTS: Deep nasal swabs and blood were sampled from 14 donkeys before and after 21 hours' long-distance transportation. The values of the plasma hormone (cortisol (Cor), adrenocorticotrophic hormone (ACTH)), biochemical indicators (total protein (TP), albumin (ALB), creatinine (CREA), lactic dehydrogenase (LDH), aspartate transaminase (AST), creatine kinase (CK), blood urea (UREA), plasma glucose (GLU)) and blood routine indices (white blood cell (WBC), lymphocyte (LYM), neutrophil (NEU), red blood cell (RBC), hemoglobin (HGB)) were measured. 16S rRNA sequencing was used to assess the nasal microbiota, including alpha diversity, beta diversity, and phylogenetic structures. Results showed that levels of Cor, ACTH, and heat-shock protein 90 (HSP90) were significantly increased (p < 0.05) after long-distance transportation. Several biochemical indicators (AST, CK) and blood routine indices (Neu, RBC, and HGB) increased markedly (p < 0.05), but the LYM decreased significantly (p < 0.05). Nine families and eight genera had a mean relative abundance over 1%. The predominant phyla in nasal microbiota after and before transportation were Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. Transportation stress induced significant changes in terms of nasal microbiota structure compared with those before transportation based on principal coordinate analysis (PCoA) coupled with analysis of similarities (ANOSIM) (p < 0.05). Among these changes, a notably gain in Proteobacteria and loss in Firmicutes at the phylum level was observed. CONCLUSIONS: These results suggest transportation can cause stress to donkeys and change the richness and diversity of nasal microbiota. Further studies are required to understand the potential effect of these microbiota changes on the development of donkey respiratory diseases.

Label-free based comparative proteomic analysis of whey proteins between different milk yields of Dezhou donkey.

Donkey milk, similar to human milk in compositions, has been suggested as the best potential hypoallergenic replacement diet for babies suffering from cow milk protein allergens and a promising nutraceutical for aged people. In this study, label-free mass spectrometry analysis was conducted to quantitatively identify the whey proteins differentially expressed in high-milk-yield samples compared with low-milk-yield samples. A total of 216 whey proteins were identified, and 19 of them showed significant differences in high-milk-yield samples. Of these proteins, 16 were upregulated and 3 were downregulated. Differentially expressed proteins (DEPs) were subjected to intensive bioinformatic analysis. Results revealed that the majority of DEPs participated in protein processing in endoplasmic reticulum, estrogen signaling pathway, progesterone-mediated oocyte maturation, and PI3K-Akt signaling pathway. Functional protein analysis suggested that proteins functioned in binding, catalytic activity, molecular function regulation, structural molecule activity, and transporter activity. Our study was the first to analyze the whey protein profile of different samples of donkey milk and to identify candidate proteins that could be used to explore the molecular mechanism related to the yield traits of Dezhou donkey milk. This study provided the biomarkers for the selection of high-milk-yielding donkey and obtained valuable information for future studies.

Exosomes Mediate Intercellular Transmission of Porcine Reproductive and Respiratory Syndrome Virus.

Exosomes are small membrane-enclosed vesicles produced by various cells and actively released into the extracellular space. They participate in intercellular communication and transfer of biologically active proteins, lipids, and nucleic acids. Accumulating evidence suggests that exosomes derived from cells infected by some viruses selectively encapsulate viral proteins, genetic materials, or even virions to mediate cell-to-cell communication and/or virus transmission. Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has been devastating the global swine industry since the late 1980s. Recent studies have shown that major proteins secreted from PRRSV-infected cells are exosomal proteins and that the serum-derived exosomes from PRRSV-infected pigs contain viral proteins. However, the role of exosomes in PRRSV infection remains unclear. In this study, purified exosomes isolated from PRRSV-infected cells were shown with reverse transcription-PCR and mass spectrometry to contain viral genomic RNA and partial viral proteins. Furthermore, exosomes from PRRSV-infected cells established productive infection in both PRRSV-susceptible and -nonsusceptible cells. More importantly, exosome-mediated infection was not completely blocked by PRRSV-specific neutralizing antibodies. In summary, this study demonstrated that exosomes can mediate PRRSV transmission and are even resistant to antibody neutralization, identifying a potential immune evasion mechanism utilized by PRRSV.IMPORTANCE Exosomes have recently been characterized as bioactive vesicles that function to promote intercellular communication. The exosomes from virally infected cells containing altered compositions confer numerous novel functionalities. A study of the secretome of cells infected with PRRSV indicated that the exosomal pathway is strongly activated by PRRSV infection. Here, we demonstrate that PRRSV can utilize host exosomes to infect naive healthy cells. Furthermore, exosome-mediated viral transmission is largely resistant to PRRSV-specific neutralizing antibodies. Our study provides novel insights into an alternative mechanism of PRRSV transmission that can compromise the host's anti-PRRSV immune response.

SILAC-based quantitative proteomic analysis of secretome of Marc-145 cells infected with porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of PRRS, which causes severe reproductive failure in sows, respiratory disease in young and growing pigs, and enormous economic losses to the global swine industry. In this study, SILAC combined with MS/MS was used to quantitatively identify the secretory proteins differentially expressed in PRRSV-infected Marc-145 cells compared with mock-infected controls. In total, we identified 204 secretory proteins showing significant differences in infected cells (163 upregulated, 41 downregulated). Intensive bioinformatic analysis of secretome data revealed that PRRSV infection strongly activated nonclassical protein secretion, especially vesicle-mediated release of exosomal proteins, including different danger-associated molecular pattern molecules and the majority of secreted proteins involved in protein binding and transport, regulation of response to stimulus, metabolic processes, and immune responses. According to the functional proteins analysis, we speculate that proteins functioning in binding, transport, and the immune response are exploited by PRRSV to facilitate virus replication and immune evasion. Our study for the first time analyzes the secretory protein profile of PRRSV-infected Marc-145 cells and provides valuable insight into the host response to PRRSV infection.

Porcine bocavirus NP1 protein suppresses type I IFN production by interfering with IRF3 DNA-binding activity.

Type I interferon (IFN) and the IFN-induced cellular antiviral responses are the primary defense mechanisms against viral infection; however, viruses always evolve various mechanisms to antagonize this host's IFN responses. Porcine bocavirus (PBoV) is a newly identified porcine parvovirus. In this study, we found that the nonstructural protein NP1 of PBoV inhibits Sendai virus-induced IFN-ß production and the subsequent expression of IFN-stimulating genes (ISGs). Ectopic expression of NP1 significantly impairs IRF3-mediated IFN-ß production; however, it does not affect the expression, phosphorylation, and nuclear translocation of IRF3, the most important transcription factor for IFN synthesis. Coimmunoprecipitation and Chromatin immunoprecipitation assays suggested that NP1 interacts with the DNA-binding domain of IRF3, which in turn blocks the association of IRF3 with IFN-ß promoter. Together, our findings demonstrated that PBoV encodes an antagonist inhibiting type I IFN production, providing a better understanding of the PBoV immune evasion strategy.

The complete chloroplast genome of Eurya rubiginosa var. attenuata H. T. Chang (Pentaphylacaceae).

Eurya rubiginosa var. attenuata is a valuable multiuse tree with a long history of use in China. It has great economic and ecological importance and is used for landscape and urban planting, soil improvement, and raw materials for food production. However, genomic studies of E. rubiginosa var. attenuata are limited. Meanwhile, the classification of this taxon is controversial. In this study, the complete plastome of E. rubiginosa var. attenuata was successfully sequenced and assembled. The chloroplast genome is 157,215 bp in length with a 37.3% GC content. The chloroplast genome structure includes a quadripartite structure comprising a pair of inverted repeat (IR) sequences of 25,872 bp, a small single-copy (SSC) region of 18,216 bp, and a large single-copy (LSC) region of 87,255 bp. The genome contains 128 genes, including 83 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Phylogenetic inference based on complete plastome analysis showed that E. rubiginosa var. attenuata is closely related to E. alata and belongs to the family Pentaphylacaceae, which differs from the results of the traditional Engler system. The chloroplast genome sequence assembly and phylogenetic analysis enrich the genetic resources of Pentaphylacaceae and provide a molecular basis for further studies on the phylogeny of the family.

TMT-based comparative proteomic analysis of Dezhou donkey spermatozoa related to freezability.

The freezability difference between donkey ejaculates is a limiting factor of sperm cryopreservation. Our recent study shows that the freezability of donkey semen is related to the seminal plasma proteome. In this study, we aimed to identify the different abundance sperm proteins in good freezability ejaculates (GFEs) and poor freezability ejaculates (PFEs) using a Tandem Mass Tag (TMT) peptide labeling coupled with LC-MS/MS approach. A total of 2682 proteins were identified, among which 58 were significantly up-regulated in GFEs and 16 were down-regulated compared with PFEs. Bioinformatic analysis results revealed that the majority of different abundance proteins (DAPs) participated in copper and calcium binding, regulation of RNA biosynthetic process, positive regulation of innate immune response, and negative regulation of programmed cell death. KEGG pathway enrichment analysis showed the up-regulated proteins in GF group were mainly involved in N-Glycan biosynthesis and protein processing in endoplasmic reticulum. Our study was the first to analyze the proteome of sperm from donkey ejaculates with different freezabilities. The identified candidate proteins might be used to explore the molecular mechanism related to donkey sperm cryotolerance and might improve the screening of jacks with good sperm freezability. SIGNIFICANCE: Cryopreserved semen has been widely used in assisted reproductive technology. However, semen cryopreservation is a damaging process, which can cause oxidative stress, reduce sperm motility and motility. There are differences in sperm freezability reported to exist between or within breeds, and even between fractions coming from the same ejaculate. The freezability difference between donkey ejaculates is a limiting factor of sperm cryopreservation. The mechanisms that affect the freezing difference in sperm quality remain to be investigated, and freezability differences was found to be related to protein composition of spermatozoa. Some protein markers that can indicate good freezability or poor freezability semen have been identified in mammals. Until now, there is no information about the relationship between donkey spermatozoa proteome and freezability. Additional novel biomarkers of semen freezability in donkey spermatozoa are also needed. The identified candidate proteins might be used to explore the molecular mechanism related to donkey sperm cryotolerance and might improve the screening of jacks with good sperm freezability.

Bioactive isoquinoline alkaloids from Corydalis saxicola.

Twelve isoquinoline alkaloids including two new nitro-containing tetrahydroprotoberberines, (-)-2,9-dihydroxyl-3,11-dimethoxy-1,10-dinitrotetrahydroprotoberberine (1) and (+)-4-nitroisoapocavidine (2), were isolated from the whole plant of Corydalis saxicola Bunting. The structures of the new compounds were established by spectroscopic analysis and chemical evidence. The inhibitory activity of these isolates against cholinesterase and canine parvovirus were evaluated. Compounds 1 and 1A, (+)-1-nitroapocavidine (5), berberine (8), palmatine (9), dehydrocavidine (10), and sanguinarine (11) showed potent inhibitory activity against acetylcholinesterase with IC(50) values of less than 10 µM, while only compound 1 possessed weak activity against canine parvovirus. Structure-activity studies demonstrated that the nitro substituents at ring A in the tetrahydroprotoberberines led to an increase in the anti-acetylcholinesterase activity.

Metabolomic profiling of Dezhou donkey sperm associated with freezability.

The cryopreservation technology of sperm has promoted the popularization of artificial insemination in the reproductive process of donkeys to a certain extent, but the freezing-thawing process would bring damage to the sperm, and the vitality of the sperm would be greatly reduced after freezing. Sperm metabolites play an important role in the process of sperm freezing, and also have an important impact on the vitality and fertilization rate of sperm after freezing-thawing. In this study, the LC-MS/MS analysis method was used to compare the metabolic profiles of high freezability (HF) and low freezability (LF) male donkey sperm after freezing-thawing. We identified 1323 metabolites in total, of which 17 metabolites are significantly different between the two groups. Most of these metabolites belong to fatty acids and phospholipids, including phosphatidylcholine, stearic acid and so on. These different metabolites are mostly related to the plasma membrane fatty acids of sperm and oxidative stress. Our results illustrate several metabolites related to sperm freezability and provide corresponding biomarkers.

Comparative Whey Proteome Profiling of Donkey Milk With Human and Cow Milk.

Donkey milk (DM), similar to human milk (HM) in chemical composition, has been suggested as the best potential hypoallergenic replacement diet for babies suffering from Cow milk (CM) protein allergy. In order to better understand DM protein, many studies based on proteomic have been performed. In this study, the label-free quantitative proteomic approach was conducted to quantitatively identify the differentially expressed whey proteins (DEPs) in DM vs. HM group and DM vs. CM group. In total, 241 and 365 DEPs were found in these two groups, respectively. Bioinformatics analysis of DEPs showed that the majority of DEPs participated in the lipoprotein metabolic process, regulation of cytokine production, chemical homeostasis, and catabolic process. The Kyoto Encyclopedia of Gene and Genomes (KEGG) pathways analysis found that these DEPs mainly participated in an antigen processing, complement, and coagulation cascades. These results may provide valuable information in the composition of milk whey proteins in DM, HM, and CM, especially for low abundant components, and expand our knowledge of different biological functions between DM and HM or CM.

Proteomic analysis of donkey sperm reveals changes in acrosome enzymes and redox regulation during cryopreservation.

Sperm cryoinjuries caused by cryopreservation restrict the application of donkey frozen semen in artificial insemination (AI). Identification of differentially represented proteins in fresh and frozen-thawed spermatozoa is of great significance to optimize the cryopreservation process and modify the component of cryopreservation extender. In this study, protein samples prepared from fresh (F) and frozen-thawed (FT) donkey spermatozoa were compared. 2682 proteins were quantitatively identified by tandem mass spectrometry (TMT) polypeptide labeling technique and LC-MS/MS method, of which 28 were more abundant in thawed samples and 147 in fresh spermatozoa. The differential abundant proteins (DAPs) were analyzed by bioinformatics. Most of the DAPs in intensive bioinformatic analysis were involved in the process of regulation of biological process and metabolism. Functional protein analysis showed that DAPs process mainly protein hydrolase activity and oxidoreductase activity. Cellular Component analysis showed that DAPs were related to vesicle transport and membrane system. This is the first analysis and study on differential proteomics of donkey sperm proteins before and after cryopreservation, which has a certain guiding significance for studying the mechanism of sperm damage caused by cryopreservation and improving the freezing and thawing procedure. SIGNIFICANCE: In recent years, the commercial value of donkey products has been discovered. Improving the breeding efficiency of donkeys can save the stock of donkeys which is decreasing rapidly, and allow people to continuously benefit from the nutritional value brought by donkey milk. Sperm cryopreservation technology has laid the foundation for encouraging the spread of artificial insemination in donkey reproduction, but the freezing and thawing process causes damage to sperm, which dramatically reducing the viability of frozen sperm and leading to low fertility. At present, the mechanism of damage to donkey sperm caused by cryopreservation is still unclear, and studying this mechanism can provide a direction for improving the quality of frozen semen. Protein is a potential key factor affecting sperm cryopreservation activity. Studying changes in the sperm proteome during cryopreservation can provide promising evidence for revealing sperm cryopreservation damage, which is of great significance for optimizing the cryopreservation process, improving the composition of cryopreservation extender, and seeking directions for improving the quality of frozen semen.

Cytotoxic amide alkaloids from Piper boehmeriaefolium.

Eight new amide alkaloids (1-8) and 19 known ones were isolated from the whole plant of Piper boehmeriaefolium. Their structures were determined through spectroscopic data analyses. Cytotoxic activity of these amides against human cervical carcinoma HeLa cells was evaluated, and 1-[(9E)-10-(3,4-methylenedioxyphenyl)-9-decenoyl]pyrrolidine (9) exhibited significant inhibitory activity with an IC(50) value of 2.7 µg/mL.

Quantitative Label-Free Proteomic Analysis of Milk Fat Globule Membrane in Donkey and Human Milk.

Previous studies have found donkey milk (DM) has the similar compositions with human milk (HM) and could be used as a potential hypoallergenic replacement diet for babies suffering from cow's milk allergy. Milk fat globule membrane (MFGM) proteins are involved in many biological functions, behaving as important indicators of the nutritional quality of milk. In this study, we used label-free proteomics to quantify the differentially expressed MFGM proteins (DEP) between DM (in 4-5 months of lactation) and HM (in 6-8 months of lactation). In total, 293 DEP were found in these two groups. Gene Ontology (GO) enrichment analysis revealed that the majority of DEP participated in regulation of immune system process, membrane invagination and lymphocyte activation. Several significant Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were determined for the DEP, such as lysosome, galactose metabolism and peroxisome proliferator-activated receptor (PPAR) signaling pathway. Our study may provide valuable information in the composition of MFGM proteins in DM and HM, and expand our knowledge of different biological functions between DM and HM.

An ethnobotanical study of forage plants in Zhuxi County in the Qinba mountainous area of central China.

In the Qinba mountainous area of Central China, pig farming has a significant impact on the growth of the rural economy and has substantially increased farmer incomes. Traditional knowledge plays an important role in the selection of forage plant species for pig farming by local people. This study aimed to identify the forage plants used for pig feeding and to catalog indigenous knowledge regarding their use. During 2016 and 2017, ethnobotanical surveys and inventories were conducted in Zhuxi County, Hubei Province, China. Data were collected using semi-structured interviews, key informant reports, free listings, guided field walks, and participatory observations with 77 households in 16 villages in 13 towns/townships. The obtained data were analyzed using a relative frequency citation (RFC) index. Overall, 145 wild forage plants from 91 genera and 31 families were recorded. The most cited families were Asteraceae, Polygonaceae, Urticaceae, Amaranthaceae, Fabaceae, Cruciferae, Caryophyllaceae, and Lamiaceae. Whole plants (75.9%) and tender leaves (12.4%) were the most frequently used parts of the plants. Most of the forage plants were herbaceous (88.9%). Almost all forage plants could be collected throughout the year (62.7%). Raw and cooked were the two main preparation methods. The most frequently cited species were Taraxacum mongolicum, Bidens pilosa, Sonchus oleraceus, Pilea verrucosa, and Pilea pumila var. obtusifolia. A total of 14 species were identified as the top forage plants in Zhuxi County based on their RFC values (RFC value greater than 0.5). Local people possess rich traditional knowledge about the utilization and management of forage plants for pig feeding. However, the maintenance of this traditional knowledge may be seriously threatened by changes in pig feeding modes and the lack of successors. Appropriate strategies and action plans have been suggested for the conservation of traditional knowledge associated with biodiversity and the sustainable use of forage species resources. These include 1) taking targeted measures to protect forage resources and associated traditional knowledge; 2) strengthening research on the forage plants with the highest RFC values for nutritional value, digestibility, other functions, and ecological status; and 3) enhancing the identification of poisonous forage plants.

Transport stress affects the fecal microbiota in healthy donkeys.

BACKGROUND: With the development of large-scale donkey farming in China, long-distance transportation has become common practice, and the incidence of intestinal diseases after transportation has increased. The intestinal microbiota is important in health and disease, and whether or not transportation disturbs the intestinal microbiota in donkeys has not been investigated. OBJECTIVES: To determine the effects of transportation on the fecal microbiota of healthy donkeys using 16S rRNA sequencing. ANIMALS: Fecal and blood samples were collected from 12 Dezhou donkeys before and after transportation. METHODS: Prospective controlled study. Cortisol, ACTH, and heat-shock protein 90 (HSP90) concentrations were measured. Sequencing of 16S rRNA was used to assess the microbial composition. Alpha diversity and beta diversity were assessed. RESULTS: Results showed significant (P < .05) increases in cortisol (58.1 ± 14.6 to 71.1 ± 9.60 ng/mL), ACTH (163.8 ± 31.9 to 315.8 ± 27.9 pg/mL), and HSP90 (10.8 ± 1.67 to 14.6 ± 1.75 ng/mL) on the day of arrival. A significantly lower (P = .04) level of bacterial richness was found in fecal samples after transportation, compared with that before transportation without distinct changes in diversity. Most notably, donkeys had significant decreases in Atopostipes, Eubacterium, Streptococcus, and Coriobacteriaceae. CONCLUSIONS AND CLINICAL IMPORTANCE: Transportation can induce stress in healthy donkeys and have some effect on the composition of the in fecal microbiota. Additional studies are required to understand the potential effect of these microbiota changes, especially significantly decreased bacteria, on the development intestinal diseases in donkeys during recovery from transportation.

Daily Sperm Output, Spermatogenic Efficiency, and Sexual Behavior of Dezhou Donkey Jacks Mounting Jennies in Estrus.

This study aimed to assess the sexual behavior of jacks mounting jennies in estrus and determine the daily sperm output (DSO) and spermatogenic efficiency using two equations to calculate testicular volume (TV). Eight sexually rested mature jacks, aging 5 to 10 years old, had semen collected once a day for 10 consecutive days using jennies in good standing estrus for mounting. Sexual behavior and semen parameters were assessed during each collection. Testicular measurements of height, width, and length were taken immediately before the first semen collection, and these measurements were used to calculate TV. After that, the TV was used to predict the DSO. The average total sperm number (TSN) obtained on days 8 to 10 was deemed the actual DSO. Differences in the predicted vs. the actual DSO were used to calculate the spermatogenic efficiency. In addition, the actual DSO was also used to calculate the number of inseminating doses a jack could produce for both on- and off-site breeding. Jack's sexual behavior and sperm motility did not vary across collection days. Sperm concentration and TSN reduced over time (P < .05). The actual DSO was 9.1 ± 4.1 billion, and the predicted DSO varied from 4.7 to 18 billion. Spermatogenic efficiency varied from 50 to 150% based on jack and the equation used to calculate TV. The number of inseminating doses ranged from 15 to 47 at 300-500 million progressively motile sperm (pms)/dose for on-site breeding. In contrast, the number of breeding doses with cooled-shipped semen (1 billion pms/dose) varied from 4 to 14 doses across donkeys. In conclusion, sexual behavior was not affected by daily semen collections. Sexual rest did not affect sperm motility. The predicted DSO varied with the equation used to determine TV. Clinically normal donkeys have high spermatogenic efficiency, which confirms previous histology reports.