Association analysis of antibiotic and disinfectant resistome in human and foodborne E. coli in Beijing, China.

The widespread use of chemical disinfectants and antibiotics poses a major threat to food safety and human health. However, the mechanisms of co-transmission of antimicrobial resistance genes (ARGs) and biocides and metal resistance genes (BMRGs) of foodborne pathogens in the food chain is still unclear. This study isolated 343 E. coli strains from animal-derived foods in Beijing and incorporated online data of human-derived E. coli strains from NCBI. Our results demonstrated a relatively uniform distribution of strains from various regions in Beijing, indicating a lack of region-specific clustering. Additionally, predominant sequence types varied between food- and human-derived strains, suggesting a preference for different hosts and environments. Phenotypic association analysis showed that the chlorine disinfectants peroxides had a significant positive correlation with tetracyclines. Many more ARGs and BMRGs were enriched in human-associated E. coli compared with those in chicken- and pork-origin. The quaternary ammonium compounds (QACs) resistance gene qacEΔ1 had a strong correlation with aminoglycoside resistance gene aadA5, folate pathway antagonist resistance gene dfrA17, sul1 and macrolide resistance gene mph(A). The correlation results indicated a significant association between the copper resistance gene cluster pco and the silver resistance gene sil. Coexistence of many resistance genes was observed within the qacEΔ1 gene structure, with qacEΔ1-sul1 being the most common combination. Our findings demonstrated that the epidemiological spread of resistance is affected by a combination of heavy metals, disinfectants and antibiotic use, suggesting that the prevention and control strategies of antimicrobial resistance need to be multifaceted and comprehensive.

[Determination of trace anions in battery-grade lithium carbonate by double-inhibition on-line matrix-removal ion chromatography].

A method was developed for the determination of trace anions in battery-grade lithium carbonate. In this method, lithium carbonate was dissolved in ultrapure water with ultrasound assistance, and its matrix was removed using an on-line matrix-removal method. In the matrix-removal process, the sample was first passed through an ADRS600(4 mm) suppressor (suppressor current, 150 mA; external water flow rate, 2 mL/min). Hydrogen and lithium ions were then completely exchanged via the ion-exchange membrane in the suppressor, converting the lithium carbonate into carbonic acid. The carbonic acid entered the waste-liquid channel in the form of carbon dioxide through a CRD 200(4 mm) carbonate removal device to remove the lithium carbonate matrix. Finally, the target anions were automatically enriched on an IonPac UTAC-LP2 concentration column (35 mm×3 mm) and automatically transferred to a chromatographic system using valve-switching technology. The chromatographic system featured an IonPac AG18 column (50 mm×2 mm) as the protection column and an IonPac AS18 column (250 mm×2 mm) as the analytical column. The column temperature was 30 ℃, gradient elution was performed using KOH solution as the eluent, and the pump flow rate was 0.30 mL/min. An ADRS600(2 mm) suppressor, suppressor current of 25 mA, injection volume of 250 µL, and conductance detector were also used. The results showed good linear relationships (r≥ 0.999) for F-, Cl-, [Formula: see text] in their respective concentration ranges. The limits of detection (LODs) and quantification (LOQs) were 0.05-0.88 and 0.15-2.92 µg/L, respectively. Lithium carbonate samples were tested six consecutive times, and the relative standard deviations (RSDs) of the peak areas of each ion were less than 0.73%. The same lithium carbonate samples were injected after 0, 2, 4, 8, 12, 18, and 24 h, and the RSD of the peak areas of each ion was less than 0.96%. The average recoveries ranged from 93.3% to 99.3%, and the RSDs (n=6) of samples spiked at three levels were in the range of 0.97%-3.45%. The proposed method has a low method limit of quantification of only 0.5 mg/kg for each ion analyzed and is capable of the simultaneous analysis of multiple ions. Thus, it is suitable for the detection of trace anions in battery-grade lithium carbonate.

Transcriptome analysis of porcine embryos derived from oocytes vitrified at the germinal vesicle stage.

Vitrification of porcine immature oocytes at the germinal vesicle (GV) stage reduces subsequent embryo yield and changes at the molecular level may occur during embryonic development. Therefore, the present study used porcine parthenogenetic embryos as a model to investigate the effect of GV oocyte vitrification on the transcriptional profiles of the resultant embryos at the 4-cell and blastocyst stages using the Smart-seq2 RNA-seq technique. We identified 743 (420 up-regulated and 323 down-regulated) and 994 (554 up-regulated and 440 down-regulated) differentially expressed genes (DEGs) from 4-cell embryos and blastocysts derived from vitrified GV oocytes, respectively. Functional enrichment analysis of DEGs in 4-cell embryos showed that vitrification of GV oocytes influenced regulatory mechanisms related to transcription regulation, apoptotic process, metabolism and key pathways such as the MAPK signaling pathway. Moreover, DEGs in blastocysts produced from vitrified GV oocytes were enriched in critical biological functions including cell adhesion, cell migration, AMPK signaling pathway, GnRH signaling pathway and so on. In addition, the transcriptomic analysis and quantitative real-time PCR results were consistent. In summary, the present study revealed that the vitrification of porcine GV oocytes could alter gene expression patterns during subsequent embryonic developmental stages, potentially affecting their developmental competence.

Decoding the influence of semen collection processes on goat sperm quality from a perspective of seminal plasma proteomics.

This study aims to assess the impact of semen collection methods on goat semen quality and seminal plasma (SP) proteomes. Semen was collected by artificial vagina (AV) or electro-ejaculator (EE) and semen parameters were evaluated. Tandem mass tag coupled with liquid chromatography-tandem mass spectrometry was used to screen SP differentially abundant proteins (DAPs) between EE and AV. PRM was used to confirm the reliability of the data. In contrast to EE, a lower volume, higher progressive motility and concentration were observed in AV. No differences were found in total motility, membrane integrity, acrosome integrity, and ROS production between EE and AV. In total, 1692 proteins were identified in SP, including 210 DAPs. Among them, 120 and 90 proteins were down-regulated and up-regulated in AV compared to EE, respectively. The GO annotation showed that DAPs are mainly localized in the membrane, involved in deference responses to bacterium, RNA processing, and related to oxidoreductase activity. KEGG demonstrated tight associations of DAPs with specific amino acids, carbon metabolism, citrate cycle, and propanoate metabolism. In conclusion, this study provides valuable insights into the effects of semen collection on goat semen quality and explores the potential action mechanism based on the modification of SP proteomes. SIGNIFICANCE OF THE STUDY: The quality of fresh semen directly influences the results of artificial insemination and semen cryopreservation in livestock. This study represents the first attempt to evaluate the impact of semen collection methods including electroejaculation and artificial vagina on sperm quality and seminal plasma proteomes in goat. The results of this study demonstrated that semen collection methods directly impacted the quality of goat semen. Then, the proteomic strategy was used to explore the potential action mechanism of semen collection methods on sperm. Some differentially abundant proteins that potentially influence semen quality were identified. Furthermore, this study suggests the possibility of utilizing specific proteins as predictive markers for goat semen quality.

The Proteomic Modification of Buck Ejaculated Sperm Induced by the Cryopreservation Process.

Using two-dimensional electrophoresis along with mass spectroscopy, we have investigated how the cryopreservation process affected the protein profile of goat ejaculated sperm. In this study, five bucks were used for semen collection. After removal of seminal plasma, the Tris-based extender containing glycerol and egg yolk was used to freeze semen. The results indicated that the post-thaw sperm quality showed a significant reduction compared with fresh sperm. The numbers of protein spots acquired in fresh and post-thaw sperm were 2926 ± 57 and 3061 ± 81, respectively. Twenty-two different abundant proteins (DAPs) were identified between fresh sperm and frozen-thawed sperm (≥3.0-folds, p < 0.05). The abundances of 19 proteins were significantly higher in the fresh sperm than the post-thaw sperm. The results of the gene ontology annotation showed the primary location of the DAPs on sperm cytoskeleton, protein complex, cytoplasm, and mitochondria. In addition, these proteins were mainly involved in ion binding, small molecular metabolic processes, structure molecule activity, guanosine triphosphatase activity, oxidoreductase activity, and protein complex assembly. The interaction networks among these DAPs demonstrated that they may play roles in oxidoreductase activity, structure, acrosomal function, and motility of sperm. Collectively, the proteome of goat sperm was altered during the cryopreservation process, demonstrating that protein modification induced by cryopreservation may be associated with the reduced quality of goat sperm after thawing.

Fertility results after exocervical insemination using goat semen cryopreserved with extenders based on egg yolk, skim milk, or soybean lecithin.

To evaluate the effects of four extenders on the post-thaw quality and fertility of goat semen, six Yunshang Black bucks' semen was collected, pooled, diluted with Andromed® (Andr®), Optidyl® (Opt®), P3644 Sigma l-phosphatidylcholine (l-α SL), and skim milk-based (Milk) extenders, and then cryopreserved. The sperm motilities, abnormalities, membrane and acrosome integrity, mitochondrial activity, apoptosis, and reactive oxygen species (ROS) production were evaluated after thawing. After exocervical insemination with the thawed semen, the pregnancy, lambing, and twinning rates were recorded and compared. The results showed that sperm motilities, membrane integrity, acrosome integrity, mitochondrial activity, and viable spermatozoa were significantly higher in the Andr® and Opt® groups than those in the l-α SL and Milk groups (p < .05). Furthermore, there was no difference between Andr® and Opt® (p > .05). The sperm abnormality was lower in semen frozen with the Andr® or Opt® extenders, as compared to the l-α SL or Milk extender (p < .05). Regarding, the viable cells with low ROS production, the optimal results were obtained in the semen frozen with Andr® and Opt® extenders. Following exocervical insemination, the pregnancy and lambing rates in the Milk group were significantly lower than those in the other groups (p < .05). No difference was found in the pregnancy and lambing rates between Andr®, Opt®, and l-α SL (p > .05). Furthermore, the twinning rates were similar between these four groups (p > .05). In conclusion, egg yolk or skim milk can be substituted by soybean lecithin during cryopreservation of goat semen.

The establishment of goat semen protein profile using a tandem mass tag-based proteomics approach.

In this study, the complete proteome of goat ejaculated semen including spermatozoa and seminal plasma was established, applying a tandem mass tag (TMT) labeling together with liquid chromatography-tandem mass spectrometry (LC-MS/MS). In seminal plasma, 2299 proteins were identified and 2098 proteins were quantified. The GO analysis demonstrated that 32% proteins were involved in metabolic activities. 46% proteins are located at intracellular region, intracellular organelle, and membrane-bounded organelle. Regarding molecular function, 40% proteins are engaged on protein binding, hydrolase activity, and ion binding. The KEGG analysis indicated a primary involvement of the identified proteins in protein processing in endoplasmic reticulum, lysosome, and proteome. In spermatozoa, 2491 proteins were identified and quantified. 39% proteins are involved in metabolic activities. 48% proteins are located at intracellular region, intracellular organelle, and membrane-bounded organelle. 38% proteins are engaged on protein binding, hydrolase activity, and ion binding. The KEGG analysis demonstrated their roles derived from the identified proteins in proteasome, glycolysis, pyruvate metabolism, and citrate cycle. Additionally, 1312 proteins were simultaneously presented in spermatozoa and seminal plasma. The involvement of 42% proteins in metabolic activities were observed. 47% proteins are located at intracellular region, intracellular organelle, and membrane-bounded organelle. The common proteins are mainly engaged on protein processing in endoplasmic reticulum, proteome, glycolysis, lysosome, and citrate cycle. Collectively, this study established the protein database of goat semen. More studies should be used to elucidate functionality of these identified proteins.

Mito-Q promotes porcine oocytes maturation by maintaining mitochondrial thermogenesis via UCP2 downregulation.

Mitochondrial thermogenesis is an adaptive response of cells to their surrounding stress. Oxidative stress is one of the common stresses during in vitro maturation (IVM) of oocytes, which leads to mitochondrial dysfunction. This study aimed to probe the effects of the mitochondria-targeted antioxidant Mito-Q on oocyte development and unravel the role of Mito-Q in mitochondrial ATP production and thermogenesis regulation. Our results showed that Mito-Q had a positive effect on porcine oocytes maturation and subsequent embryo development. During oocytes IVM, Mito-Q could reduce ATP levels and ROS, increase lipid droplets accumulation, induce autophagy, and maintain mitochondrial temperature stability. Moreover, in metaphase II (MII) oocytes, Mito-Q would induce mitochondrial uncoupling manifested by decreased ATP, attenuated mitochondrial membrane potential (MMP), and increased mitochondrial thermogenesis. Notably, the expression of mitochondrial uncoupling protein (UCP2) was significantly reduced in oocytes treated with Mito-Q. Further study indicated that specific depletion of UCP2 in oocytes also resulted in increased thermogenesis, decreased ATP and declined MMP, suggesting that UCP2 downregulation may participate in Mito-Q-induced mitochondrial uncoupling. In summary, our data demonstrate that Mito-Q promotes oocyte maturation in vitro and maintains the stability of mitochondrial thermogenesis by inhibiting UCP2 expression.

Astaxanthin Supplementation Improves the Subsequent Developmental Competence of Vitrified Porcine Zygotes.

Cryopreservation of embryos has been confirmed to cause oxidative stress as a factor responsible for impaired developmental competence. Currently, astaxanthin (Ax) raises considerable interest as a strong exogenous antioxidant and for its potential in reproductive biology. The present study aimed to investigate the beneficial effects of Ax supplementation during in vitro culture of vitrified porcine zygotes and the possible underlying mechanisms. First, the parthenogenetic zygotes were submitted to vitrification and then cultured in the medium added with various concentrations of Ax (0, 0.5, 1.5, and 2.5 µM). Supplementation of 1.5 µM Ax achieved the highest blastocyst yield and was considered as the optimal concentration. This concentration also improved the blastocyst formation rate of vitrified cloned zygotes. Moreover, the vitrified parthenogenetic zygotes cultured with Ax exhibited significantly increased mRNA expression of CDX2, SOD2, and GPX4 in their blastocysts. We further analyzed oxidative stress, mitochondrial and lysosomal function in the 4-cell embryos and blastocysts derived from parthenogenetic zygotes. For the 4-cell embryos, vitrification disturbed the levels of reactive oxygen species (ROS) and glutathione (GSH), and the activities of mitochondria, lysosome and cathepsin B, and Ax supplementation could fully or partially rescue these values. The blastocysts obtained from vitrified zygotes showed significantly reduced ATP content and elevated cathepsin B activity, which also was recovered by Ax supplementation. There were no significant differences in other parameters mentioned above for the resultant blastocysts. Furthermore, the addition of Ax significantly enhanced mitochondrial activity and reduced lysosomal activity in resultant blastocysts. In conclusion, these findings revealed that Ax supplementation during the culture period improved subsequent embryonic development and quality of porcine zygotes after vitrification and might be used to ameliorate the recovery culture condition for vitrified embryos.

Ultrastructural Modification of Ram Sperm Frozen with Cyclohexanediol and Trehalose.

In this study, the effects of trehalose and 1, 3-cyclohexanediol (1, 3-CHD) on the ultrastructure of frozen-thawed ram sperm were assessed and compared. In the control group, sperm were frozen without trehalose and 1, 3-CHD. In the trehalose group, 100 mM trehalose was used for sperm cryopreservation. In the cyclohexanediol group, the freezing extender contained 100 mM 1, 3-CHD. The transmission electron microscope (TEM) was used to observe the ultrastructural alterations of sperm. For verification of the TEM results, the plasma membrane and acrosome integrity of ram frozen sperm was assessed. Three fertility-proven rams were used in this study. Semen collection was repeated 6 times. The collected semen was pooled to preclude the individual difference each time. The sperm collected from a representative ram were used for ultrastructural observation. The TEM results indicated extensive and severe cryoinjuries on the main organelles of ram frozen sperm. Some alterations in plasma membrane, including detachment, rupture, dilation, or loss, appeared in post-thaw sperm. The bending shape and leakage of genetic materials were also observed in the nucleus. In addition, the outer acrosome membrane in some frozen sperm was broken or partly lost. Further, leakage of the inner contents of acrosomes also occurred. Sperm mitochondria was negatively influenced by cryopreservation. With 1, 3-CHD or trehalose, the percentage of sperm with normal acrosomes was 62% or 64%, and it was significantly higher than that of the control (41.51%, p < 0.05). However, different from trehalose, 1, 3-CHD cannot efficiently protect the post-thaw integrity of the plasma membrane (48.09% vs. 26.92%, p < 0.05). The TEM results were consistent with the quality assessment of frozen-thawed sperm. Collectively, trehalose and 1, 3-CHD can mitigate cryoinjuries on sperm ultrastructure. The cryoprotective effects of trehalose on sperm plasma membrane are superior to 1, 3-CHD. Sperm plasma membrane is the most sensitive to cryoinjuries, followed by acrosomes, mitochondria, and nuclei.

Identification and validation of ram sperm proteins associated with cryoinjuries caused by the cryopreservation process.

The change of sperm protein profile after the cryopreservation process may influence fertilization and early embryonic development. The purpose of the present study was to identify ram sperm proteomic modifications induced by the cryopreservation process using the isobaric tags for relative and absolute quantification labeling technology (iTRAQ) coupled with the parallel reaction monitoring (PRM) technology. Semen samples were collected from five Yunnan semi-fine wool rams using an electroejaculator. Sperm motility (CASA), plasma membrane (HOST test), and acrosome integrity (FITC-PSA) were evaluated after freeze-thawing. The total proteins of fresh and frozen-thawed sperm were extracted and purified, followed by identifying ram sperm proteomic modifications using the isobaric tags for relative and absolute quantification labeling technique (iTRAQ) coupled with the parallel reaction monitoring (PRM) technology. The results showed a significant reduction (P < 0.05) in all sperm parameters after thawing. 126 differentially abundant proteins (DAPs) were identified through comparison of the proteomes between fresh and frozen-thawed sperm. Among them, 90 proteins were down-regulated after the cryopreservation process. The remaining 36 proteins were up-regulated in frozen-thawed sperm. The results of functional annotation demonstrated the potential relationship of 10 DAPs with oxidoreductase activity. 18 and 15 DAPs may be involved in the stress and carbohydrate metabolic process, respectively. Furthermore, 8 DAPs may be functionally associated with reproduction. The Kyoto Encyclopedia of Genes and Genomes (KEGG) results demonstrated the primary enrichment of these identified DAPs in metabolic activities, disease, and oxidative phosphorylation. In order to confirm the reliability of the iTRAQ results, the changing trends of 10 proteins analyzed by PRM were similar to those of the corresponding proteins identified by iTRAQ. In conclusion, the cryopreservation process modifies the proteome of ram sperm, possibly leading to compromised fertility of post-thaw sperm. Additionally, the identified DAPs in this study may function as potential biomarkers for assessing the post-thaw quality of ram semen.

Sequencing Reveals Population Structure and Selection Signatures for Reproductive Traits in Yunnan Semi-Fine Wool Sheep (Ovis aries).

Yunnan semi-fine wool sheep are among the most important cultivated sheep breeds in China. However, their population structure, genetic characteristics and traits of interest are poorly studied. In this study, we systematically studied the population characteristics and selection signatures of 40 Yunnan semi-fine wool sheep using SNPs obtained from whole-genome resequencing data. A total of 1393 Gb of clean data were acquired. The mapping rate against the reference genome was 91.23% on average (86.01%-92.26%), and the average sequence depth was 9.51X. After filtering, 28,593,198 SNPs and 4,725,259 indels with high quality were obtained. The heterozygosity rate, inbreeding coefficient and effective population size of the sheep were calculated to preliminarily explore their genetic characteristics. The average heterozygosity rate was 0.264, the average inbreeding coefficient was 0.0099, and the effective population size estimated from the heterozygote excess (HE) was 242.9. Based on the Tajima's D and integrated haplotype score (iHS) approaches, 562 windows and 11,356 core SNPs showed selection signatures in the Yunnan semi-fine wool sheep population. After genome annotation and gene enrichment analysis, we found traces of early domestication in sensory organs, behavioural activity and the nervous system as well as adaptive changes in reproductive and wool traits under selection in this population. Some selected genes related to litter size, including FSHR, BMPR1B and OXT, were identified as being under selection. Specific missense mutations of the FSHR gene that differed from the reference genome were also identified in the population, and we found some SNP variations that may affect litter size. Our findings provide a theoretical basis for the conservation and utilization of Yunnan semi-fine wool sheep. Furthermore, our results reveal some changes common to sheep after domestication and provide a new opportunity to investigate the genetic variation influencing fecundity within a population evolving under artificial selection.

The Assessment of Various Factors Affecting the Postwarming Viability and Developmental Capability of Goat Metaphase II Oocytes Vitrified by Cryotop.

The effects of the equilibration time, the vitrification procedure, and the warming procedure on the quality of goat oocytes vitrified by Cryotop were assessed. In the first part of the study, oocytes were exposed to 10% dimethyl sulfoxide (DMSO) and 10% ethylene glycol (EG) for 1, 3, 5, or 10 minutes, respectively, followed by vitrification. In the second part, after equilibration in 7.5% DMSO +7.5% EG for 3 minutes, 10% DMSO +10% EG for 3 minutes, or 4% EG for 10 minutes, oocytes were equilibrated in 15% DMSO +15% EG, 20% DMSO +20% EG, or 35% EG for 30 seconds before vitrification. The vitrification procedures were designated as first vitrification procedure (VPI), second vitrification procedure (VPII), and third vitrification procedure (VPIII), respectively. In the third part, oocytes vitrified using VPIII were warmed by the three procedures (first warming procedure [TPI], second warming procedure [TPII], or third warming procedure [TPIII]) containing different concentrations of trehalose. The results showed that after equilibration for 1 or 3 minutes in 10% DMSO and 10% EG, the viability and developmental capability of vitrified oocytes were significantly superior to the groups after equilibration for over 5 minutes (p < 0.05). With the VPIII procedure, the frequencies with normal morphology, cleavage, and blastocyst formation of vitrified oocytes were 91.87% ± 4.14%, 76.51% ± 4.37%, and 39.84% ± 2.91%, respectively, demonstrating a significant increase compared to the VPI or VPII group (p < 0.05). The rates of vitrified oocytes with normal morphology and cleavage in the TPI group were higher than the TPII or TPIII group (p < 0.05). In conclusion, equilibration in 10% DMSO and 10% EG for <3 minutes benefits the viability of vitrified oocytes. EG may be more efficient for vitrification of goat oocytes compared to DMSO. Higher concentrations (more than 1 M) of trehalose enhance cryosurvival of goat oocytes when warming.

The characteristics of proteome and metabolome associated with contrasting sperm motility in goat seminal plasma.

Sperm motility is an index tightly associated with male fertility. A close relationship between seminal plasma and sperm motility has been confirmed. This study was to assess the protein and metabolite profiles of seminal plasma obtained from adult goats with high or low sperm motility using the proteomic and metabolomic strategies. In total, 2098 proteins were found. 449 differentially abundant proteins (DAPs) were identified, and 175 DAPs were enriched in the high motility group. The obtained DAPs primarily exist in cytoplasma and extra-cellular portion. The Gene Ontology enrichment analysis demonstrated the main functional roles of these DAPs in regulating biological process, metabolic process of organic substances, cellular-metabolic process, primary-metabolic process, metabolic process of nitrogen compounds, etc. Additionally, the Kyoto-Encyclopedia of Genes and Genomes (KEGG) analysis revealed that these DAPs were primarily involved in phosphatidylinositol signaling system, salivary secretion, proteasome, apoptosis, mitophagy-animal, etc. Aided by the parallel reaction monitoring technology, the abundance changing pattern of 19 selected DAPs was consistent with that of the corresponding proteins obtained by TMT. A total of 4603 metabolites were identified in seminal plasma. 1857 differential metabolites were found between the high motility group and the low motility group, and 999 metabolites were up-regulated in the high motility group. The KEGG analysis demonstrated the primary involvement of the differential metabolites in metabolic and synthetic activities. In conclusion, we first established the proteome and metabolome databank of goat seminal plasma, detecting some proteins and metabolites which may affect sperm motility. This study will be valuable for understanding mechanisms leading to poor sperm motility.

Trehalose modifies the protein profile of ram spermatozoa during cryopreservation.

As a magical oligosaccharide, trehalose has been revealed to enhance the post-thaw quality of stock semen. However, information regarding the cryoprotective mechanism of trehalose during cryopreservation has not yet been determined. This study was designed to observe the effects of trehalose on the proteome of ram frozen spermatozoa by applying the isobaric tag for relative and absolute quantification (iTRAQ) strategy combined with parallel reaction monitoring (PRM). A total of 1269 proteins were identified. Among them, there were 21 differentially expressed proteins (DEPs), with 9 up-regulated proteins and 11 down-regulated proteins in spermatozoa frozen with trehalose. These DEPs were primarily located in nucleus, cytoplasm, and extracellular region. The Gene Ontology (GO) enrichment analysis demonstrated the involvement of the DEPs in signal transduction, ion binding, oxidoreductase activity, response to stress, and catabolic processes. Based on the STRING analysis, tight functional correlations were observed between 6-phosphogluconate dehydrogenase, fructose-bisphosphate aldolase A isoform 1, 14-3-3 protein epsilon, tyrosine-protein kinase Fer, and beta-hexosaminidase subunit alpha precursor. Furthermore, 10 DEPs were verified using PRM, confirming the accuracy of the iTRAQ data acquired in this study. In conclusion, trehalose can modify the protein profile of ram spermatozoa during cryopreservation, which may be associated with its cryoprotective effects. Additionally, trehalose may function on frozen spermatozoa through antioxidation, involvement in glycolysis, and increment of spermatozoa tolerance to various stresses.

Role of astaxanthin as an efficient antioxidant on the in vitro maturation and vitrification of porcine oocytes.

As one of the most powerful natural antioxidants, astaxanthin (Ax) has begun to be applied to the field of reproductive biology. Here we used porcine oocyte as a model to explore how Ax improves the oocyte potential during in vitro maturation (IVM), and we also investigated the cytoprotective effects of Ax on the vitrified oocytes. Ax supplementation (final concentration of 2.5 µM) was subjected for immature oocytes during vitrification and subsequent IVM; fresh oocytes were also matured in vitro in the presence or absence of 2.5 µM Ax. Our results showed that Ax significantly increased the survival rate of vitrified oocytes, and promoted the blastocyst yield of both fresh and vitrified oocytes after parthenogenetic activation and somatic cell nuclear transfer. The oocytes treated with Ax displayed significantly lower reactive oxygen species generation and higher glutathione level. Vitrification of oocytes had no impact on caspase-3, cathepsin B and autophagic activities; Ax significantly decreased the cathepsin B activity in both fresh and vitrified oocytes. Moreover, the relative fluorescence intensity of lysosomes was significantly increased in vitrified oocytes, which was recovered by Ax treatment. The mitochondrial activity did not differ between fresh and vitrified oocytes, and was significantly enhanced in Ax-treated oocytes. Furthermore, Ax significantly restored the decreased expression of BMP15, ZAR1, POU5F1, GPX4 and LAMP2 genes in vitrified oocytes. Both fresh and vitrified oocytes treated with Ax showed significantly higher mRNA levels of GDF9, POU5F1, SOD2, NRF2 and ATG5. Taken together, this study provides new perspectives in understanding the mechanisms by which Ax improves the developmental competence of both fresh and vitrified porcine oocytes.

The Effects of Antifreeze Protein III Supplementation on the Cryosurvival of Goat Spermatozoa During Cryopreservation.

Antifreeze protein (AFP) has been shown to have beneficial effects on frozen mammalian spermatozoa. However, rare reports have been published regarding the use of AFPs in storage of goat spermatozoa. The aim of this study was to investigate the effects of AFPIII on the quality of goat semen during cryopreservation. Ejaculates were collected from six Yunshang black goats through an artificial vagina. The collected semen was pooled, divided into five aliquots, and diluted with the commercial bull semen extender containing: no AFPIII (AFP-0, control), 1 µg/mL AFPIII (AFP-1), 10 µg/mL AFPIII (AFP-10), 50 µg/mL AFPIII (AFP-50), and 100 µg/mL AFPIII (AFP-100), respectively. Spermatozoa motility, membrane integrity, acrosome integrity, mitochondrial function, distribution of phosphatidylserine, and formation of reactive oxygen species (ROS) were measured after the freezing and thawing process. The results showed that the spermatozoa motility, membrane integrity, acrosome integrity, and mitochondrial function were significantly higher in frozen spermatozoa using the extender containing 1 µg/mL AFPIII as compared with the other groups (p < 0.05). Furthermore, the extender supplemented with 1 µg/mL of AFPIII resulted in higher viable and lower nonviable spermatozoa compared with the other treated groups (p < 0.05), after staining using Annexin V-fluoresceine isothiocyanate (Annexin V-FITC) and Propidium Iodide. No significant differences were found between these groups in relation to viable cells with lower ROS production. In conclusion, the addition of AFPIII to the freezing extender improved the post-thaw quality of goat semen. The optimal concentration used in this study was 1 µg/mL. However, excessively high concentrations of AFPIII were unable to exhibit their cryoprotective effects on goat spermatozoa. However, the presence of AFPIII cannot mitigate oxidative stress caused by the freezing and thawing process. In addition, in vitro fertilization or artificial insemination can further evaluate the effects of AFPIII on frozen-thawed goat spermatozoa.

Transcriptome Analysis of mRNAs and Long Non-Coding RNAs During Subsequent Embryo Development of Porcine Cloned Zygotes After Vitrification.

Cryopreservation of porcine cloned zygotes has important implications for biotechnology and biomedicine research; however, lower embryo developmental potential remains an urgent problem to be resolved. For exploring the sublethal cryodamages during embryo development, this study was designed to acquire the mRNA and long non-coding RNA (lncRNA) profiles of 2-cells, 4-cells and blastocysts derived from vitrified porcine cloned zygotes using transcriptome sequencing. We identified 167 differentially expressed (DE) mRNAs and 516 DE lncRNAs in 2-cell stage, 469 DE mRNAs and 565 lncRNAs in 4-cell stage, and 389 DE mRNAs and 816 DE lncRNAs in blastocyst stage. Functional enrichment analysis revealed that the DE mRNAs during embryo development were involved in many regulatory mechanisms related to cell cycle, cell proliferation, apoptosis, metabolism and others. Moreover, the target genes of DE lncRNAs in the three embryonic stages were also enriched in many key GO terms or pathways such as "defense response", "linoleic acid metabolic process", "embryonic axis specification", "negative regulation of protein neddylation", etc., In conclusion, the present study provided comprehensive transcriptomic data about mRNAs and lncRNAs for the vitrified porcine cloned zygotes during different developmental stages, which contributed to further understand the potential cryodamage mechanisms responsible for impaired embryo development.

Proteomic Exploration of Porcine Oocytes During Meiotic Maturation in vitro Using an Accurate TMT-Based Quantitative Approach.

The dynamic changes in protein expression are well known to be required for oocyte meiotic maturation. Although proteomic analysis has been performed in porcine oocytes during in vitro maturation, there is still no full data because of the technical limitations at that time. Here, a novel tandem mass tag (TMT)-based quantitative approach was used to compare the proteomic profiles of porcine immature and in vitro mature oocytes. The results of our study showed that there were 763 proteins considered with significant difference-450 over-expressed and 313 under-expressed proteins. The GO and KEGG analyses revealed multiple regulatory mechanisms of oocyte nuclear and cytoplasmic maturation such as spindle and chromosome configurations, cytoskeletal reconstruction, epigenetic modifications, energy metabolism, signal transduction and others. In addition, 12 proteins identified with high-confidence peptide and related to oocyte maturation were quantified by a parallel reaction monitoring technique to validate the reliability of TMT results. In conclusion, we provided a detailed proteomics dataset to enrich the understanding of molecular characteristics underlying porcine oocyte maturation in vitro.

Inhibitory effects of astaxanthin on postovulatory porcine oocyte aging in vitro.

Mammalian oocytes represent impaired quality after undergoing a process of postovulatory aging, which can be alleviated through various effective ways such as reagent treatment. Accumulating evidences have revealed the beneficial effects of astaxanthin (Ax) as a potential antioxidant on reproductive biology. Here, porcine matured oocytes were used as a model to explore whether Ax supplement can protect against oocyte aging in vitro and the underlying mechanism, and therefore they were cultured with or without 2.5 µM Ax for an additional 24 h. Aged oocytes treated with Ax showed improved yield and quality of blastocysts as well as recovered expression of maternal genes. Importantly, oxidative stress in aged oocytes was relieved through Ax treatment, based on reduced reactive oxygen species and enhanced glutathione and antioxidant gene expression. Moreover, inhibition in apoptosis and autophagy of aged oocyte by Ax was confirmed through decreased caspase-3, cathepsin B and autophagic activities. Ax could also maintain spindle organization and actin expression, and rescue functional status of organelles including mitochondria, endoplasmic reticulum, Golgi apparatus and lysosomes according to restored fluorescence intensity. In conclusion, Ax might provide an alternative for ameliorating the oocyte quality following aging in vitro, through the mechanisms mediated by its antioxidant properties.