Phenylbutyrate and Dichloroacetate Enhance the Liquid-Stored Boar Sperm Quality via PDK1 and PDK3.

Artificial insemination (AI) with liquid-stored semen is the most prevalent and efficient assisted reproduction technique in the modern pork industry. Pyruvate dehydrogenase complex component X (PDHX) was demonstrated to be associated with sperm metabolism and affected the boar sperm viability, motility, and fertility. Pyruvate Dehydrogenase Kinases (PDKs) are the key metabolic enzymes that regulate pyruvate dehydrogenase complex (PDHC) activity and also the conversion from glycolysis to oxidative phosphorylation. In the present study, two PDK inhibitors, Dichloroacetate (DCA) and Phenylbutyrate (4-PBA), were added to an extender and investigated to determine their regulatory roles in liquid-stored boar sperm at 17 °C. The results indicated that PDK1 and PDK3 were predominantly located at the head and flagella of the boar sperm. The addition of 2 mM DCA and 0.5 mM 4-PBA significantly enhanced the sperm motility, plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), and ATP content. In addition, DCA and 4-PBA exerted their effects by inhibiting PDK1 and PDK3, respectively. In conclusion, DCA and 4-PBA were found to regulate the boar sperm metabolic activities via PDK1 and PDK3. These both can improve the quality parameters of liquid-stored boar sperm, which will help to improve and optimize liquid-stored boar semen after their addition in the extender.

Cryopreservation Induces Acetylation of Metabolism-Related Proteins in Boar Sperm.

Cryodamage affects the normal physiological functions and survivability of boar sperm during cryopreservation. Lysine acetylation is thought to be an important regulatory mechanism in sperm functions. However, little is known about protein acetylation and its effects on cryotolerance or cryodamage in boar sperm. In this study, the characterization and protein acetylation dynamics of boar sperm during cryopreservation were determined using liquid chromatography-mass spectrometry (LC-MS). A total of 1440 proteins were identified out of 4705 modified proteins, and 2764 quantifiable sites were elucidated. Among the differentially modified sites, 1252 were found to be upregulated compared to 172 downregulated sites in fresh and frozen sperms. Gene ontology indicated that these differentially modified proteins are involved in metabolic processes and catalytic and antioxidant activities, which are involved in pyruvate metabolism, phosphorylation and lysine degradation. In addition, the present study demonstrated that the mRNA and protein expressions of SIRT5, IDH2, MDH2 and LDHC, associated with sperm quality parameters, are downregulated after cryopreservation. In conclusion, cryopreservation induces the acetylation and deacetylation of energy metabolism-related proteins, which may contribute to the post-thawed boar sperm quality parameters.

Odorant Receptor OR2C1 Is an Essential Modulator of Boar Sperm Capacitation by Binding with Heparin.

Heparin, a class of glycosaminoglycans (GAGs), is widely used to induce sperm capacitation and fertilization. How heparin induces sperm capacitation remains unclear. Olfactory receptors (ORs) which are G protein-coupled receptors, have been proposed to be involved in sperm capacitation. However, the interaction between ORs and odor molecules and the molecular mechanism of ORs mediating sperm capacitation are still unclear. The present study aimed to explore the underlying interaction and mechanism between heparin and ORs in carrying out the boar sperm capacitation. The results showed that olfactory receptor 2C1 (OR2C1) is a compulsory unit which regulates the sperm capacitation by recognizing and binding with heparin, as determined by Dual-Glo Luciferase Assay and molecular docking. In addition, molecular dynamics (MD) simulation indicated that OR2C1 binds with heparin via a hydrophobic cavity comprises of Arg3, Ala6, Thr7, Asn171, Arg172, Arg173, and Pro287. Furthermore, we demonstrated that knocking down OR2C1 significantly inhibits sperm capacitation. In conclusion, we highlighted a novel olfactory receptor, OR2C1, in boar sperm and disclosed the potential binding of heparin to Pro287, a conserved residue in the transmembrane helices region 7 (TMH7). Our findings will benefit the further understanding of ORs involved in sperm capacitation and fertilization.

Genistein Promotes Skeletal Muscle Regeneration by Regulating miR-221/222.

Genistein (GEN), a phytoestrogen, has been reported to regulate skeletal muscle endocrine factor expression and muscle fiber type switching, but its role in skeletal muscle regeneration is poorly understood. As a class of epigenetic regulators widely involved in skeletal muscle development, microRNAs (miRNAs) have the potential to treat skeletal muscle injury. In this study, we identified miR-221 and miR-222 and their target genes MyoG and Tnnc1 as key regulators during skeletal muscle regeneration, and both were regulated by GEN. C2C12 myoblasts and C2C12 myotubes were then used to simulate the proliferation and differentiation of muscle satellite cells during skeletal muscle regeneration. The results showed that GEN could inhibit the proliferation of satellite cells and promote the differentiation of satellite cells by inhibiting the expression of miR-221/222. Subsequent in vitro and in vivo experiments showed that GEN improved skeletal muscle regeneration mainly by promoting satellite cell differentiation in the middle and late stages, by regulating miR-221/222 expression. These results suggest that miR-221/222 and their natural regulator GEN have potential applications in skeletal muscle regeneration.

Transcriptome-wide m6A profiling reveals mRNA post-transcriptional modification of boar sperm during cryopreservation.

BACKGROUND: Cryopreservation induces transcriptomic and epigenetic modifications that strongly impairs sperm quality and function, and thus decrease reproductive performance. N6-methyladenosine (m6A) RNA methylation varies in response to stress and has been implicated in multiple important biological processes, including post-transcriptional fate of mRNA, metabolism, and apoptosis. This study aimed to explore whether cryopreservation induces m6A modification of mRNAs associated with sperm energy metabolism, cryoinjuries, and freezability. RESULTS: The mRNA and protein expression of m6A modification enzymes were significantly dysregulated in sperm after cryopreservation. Furthermore, m6A peaks were mainly enriched in coding regions and near stop codons with classical RRACH motifs. The mRNAs containing highly methylated m6A peaks (fts vs. fs) were significantly associated with metabolism and gene expression, while the genes with less methylated m6A peaks were primarily involved in processes regulating RNA metabolism and transcription. Furthermore, the joint analysis of DMMGs and differentially expressed genes indicated that both of these play a vital role in sperm energy metabolism and apoptosis. CONCLUSIONS: Our study is the first to reveal the dynamic m6A modification of mRNAs in boar sperm during cryopreservation. These epigenetic modifications may affect mRNA expression and are closely related to sperm motility, apoptosis, and metabolism, which will provide novel insights into understanding of the cryoinjuries or freezability of boar sperm during cryopreservation.

Complete mtDNA genome of Otus sunia (Aves, Strigidae) and the relaxation of selective constrains on Strigiformes mtDNA following evolution.

Most of owls are nocturnal raptor and usually use their soft and fluffy feathers to flight silently to catch prey while other diurnal raptors prefer fierce attack and swift flight. For energy cost of these different hunting strategies can be greatly different, we speculate that mitochondrial gene of owls may undergo a different evolution pattern following raptors evolution. To test our hypothesis, we sequenced the mtDNA genome of Otus sunia and calculated the ratio of nonsynonymous to synonymous nucleotide substitutions (ω, Ka/Ks, dN/dS) of raptors. The mtDNA genome of O. sunia was 17,609 bp in length, containing 13 PCGs, 2 ribosomal RNAs, 22 transfer RNAs and a control region. Secondly structure of tRNAs and rRNAs were predicted and conserved sequence blocks (CSBs) on control region were identified. The Bayesian inference tree and maximum likelihood tree based on 13 PCGs and 2 rRNAs suggested the owls were related to other raptors. Finally, calculation of ω-values of each owls and other raptors mtDNA PCGs indicated that owls accumulated more nonsynonymous nucleotide substitutions relative to synonymous substitutions compared to other raptors. For mtDNA PCGs associated with energy metabolism, this finding may reveal the degeneration of flight abilities of owls.

Exploration of miRNA and mRNA Profiles in Fresh and Frozen-Thawed Boar Sperm by Transcriptome and Small RNA Sequencing.

Due to lower farrowing rate and reduced litter size with frozen-thawed semen, over 90% of artificial insemination (AI) is conducted using liquid stored boar semen. Although substantial progress has been made towards optimizing the cryopreservation protocols for boar sperm, the influencing factors and underlying mechanisms related to cryoinjury and freeze tolerance of boar sperm remain largely unknown. In this study, we report the differential expression of mRNAs and miRNAs between fresh and frozen-thawed boar sperm using high-throughput RNA sequencing. Our results showed that 567 mRNAs and 135 miRNAs were differentially expressed (DE) in fresh and frozen-thawed boar sperm. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the majority of DE mRNAs were enriched in environmental information processing such as cytokine-cytokine receptor interactions, PI3K-Akt signaling, cell adhesion, MAPK, and calcium signaling pathways. Moreover, the targets of DE miRNAs were enriched in significant GO terms such as cell process, protein binding, and response to stimuli. In conclusion, we speculate that DE mRNAs and miRNAs are heavily involved in boar sperm response to environment stimuli, apoptosis, and metabolic activities. The differences in expression also reflect the various structural and functional changes in sperm during cryopreservation.

High throughput small RNA and transcriptome sequencing reveal capacitation-related microRNAs and mRNA in boar sperm.

BACKGROUND: Capacitation, a prerequisite for oocyte fertilization, is a complex process involving series of structural and functional changes in sperms such as membrane modifications, modulation of enzyme activities, and protein phosphorylation. In order to penetrate and fertilize an oocyte, mammalian sperms must undergo capacitation. Nevertheless, the process of sperm capacitation remains poorly understood and requires further elucidation. In the current study, via high throughput sequencing, we identified and explored the differentially expressed microRNAs (miRNAs) and mRNAs involved in boar sperm capacitation. RESULTS: We identified a total of 5342 mRNAs and 204 miRNAs that were differentially expressed in fresh and capacitated boar sperms. From these, 12 miRNAs (8 known and 4 newly identified miRNAs) and their differentially expressed target mRNAs were found to be involved in sperm capacitation-related PI3K-Akt, MAPK, cAMP-PKA and Ca2+signaling pathways. CONCLUSIONS: Our study is first to provide the complete miRNA and transcriptome profiles of boar sperm. Our findings provide important insights for the understanding of the RNA profile in boar sperm and future elucidation of the underlying molecular mechanism relevant to mammalian sperm capacitation.

Expression of CD9 and CD81 in bovine germinal vesicle oocytes after vitrification followed by in vitro maturation.

The present study aimed to investigate the effect of vitrification on the expression of fertilization related genes (CD9 and CD81) and DNA methyl transferases (DNMT1 and DNMT3b) in bovine germinal vesicle (GV) oocytes and their resulting metaphase Ⅱ (MⅡ) stages after in vitro maturation culture. GV oocytes were vitrified using the open-pulled straw method; after warming, they were cultured in vitro. The vitrified-warmed GV oocytes and more developed MII oocytes were used to calculate the maturation rates (first polar body extrusion under a stereomicroscopy), and to detect mRNA expression (qRT-PCR). Fresh GV oocytes and their in vitro-derived MII oocytes served as controls. The results showed that both the maturation rate (54.23% vs. 42.93%) and the relative abundance of CD9 mRNA decreased significantly (p < 0.05) in bovine GV oocytes after vitrification, but the expression of CD81 and DNMT3b increased significantly. After in vitro maturation of vitrified GV oocytes, the resulting MII oocytes showed lower (p < 0.05) mRNA expression of genes (CD9, CD81, DNMT1 and DNMT3b) when compared to the control group (MII oocytes). Altogether, vitrification decreased the maturation rate of bovine GV oocytes and changed the expression of fertilization related genes and DNA methyl transferases during in vitro maturation.

Transcriptome Sequencing Reveals the Differentially Expressed lncRNAs and mRNAs Involved in Cryoinjuries in Frozen-Thawed Giant Panda (Ailuropoda melanoleuca) Sperm.

Sperm cryopreservation and artificial insemination are important methods for giant panda breeding and preservation of extant genetic diversity. Lower conception rates limit the use of artificial insemination with frozen-thawed giant panda sperm, due to the lack of understanding of the cryodamaging or cryoinjuring mechanisms in cryopreservation. Long non-coding RNAs (lncRNAs) are involved in regulating spermatogenesis. However, their roles during cryopreservation remain largely unexplored. Therefore, this study aimed to identify differentially expressed lncRNAs and mRNAs associated with cryodamage or freeze tolerance in frozen-thawed sperm through high throughput sequencing. A total of 61.05 Gb clean reads and 22,774 lncRNA transcripts were obtained. From the sequencing results, 1477 significantly up-regulated and 1,396 significantly down-regulated lncRNA transcripts from fresh and frozen-thawed sperm of giant panda were identified. GO and KEGG showed that the significantly dysregulated lncRNAs and mRNAs were mainly involved in regulating responses to cold stress and apoptosis, such as the integral component of membrane, calcium transport, and various signaling pathways including PI3K-Akt, p53 and cAMP. Our work is the first systematic profiling of lncRNA and mRNA in fresh and frozen-thawed giant panda sperm, and provides valuableinsights into the potential mechanism of cryodamage in sperm.

Selenium, Selenoproteins, and Female Reproduction: A Review.

Selenium (Se) is an essential micronutrient that has several important functions in animal and human health. The biological functions of Se are carried out by selenoproteins (encoded by twenty-five genes in human and twenty-four in mice), which are reportedly present in all three domains of life. As a component of selenoproteins, Se has structural and enzymatic functions; in the latter context it is best recognized for its catalytic and antioxidant activities. In this review, we highlight the biological functions of Se and selenoproteins followed by an elaborated review of the relationship between Se and female reproductive function. Data pertaining to Se status and female fertility and reproduction are sparse, with most such studies focusing on the role of Se in pregnancy. Only recently has some light been shed on its potential role in ovarian physiology. The exact underlying molecular and biochemical mechanisms through which Se or selenoproteins modulate female reproduction are largely unknown; their role in human pregnancy and related complications is not yet sufficiently understood. Properly powered, randomized, controlled trials (intervention vs. control) in populations of relatively low Se status will be essential to clarify their role. In the meantime, studies elucidating the potential effect of Se supplementation and selenoproteins (i.e., GPX1, SELENOP, and SELENOS) in ovarian function and overall female reproductive efficiency would be of great value.

Female Reproductive Performance in the Mouse: Effect of Oral Melatonin.

Although melatonin has some of the broadest ranges of actions on the physiology of vertebrates, especially on their reproductive processes, the mechanism by which melatonin regulates animal reproduction is still incompletely understood. This study was designed to determine the effect of oral melatonin on the reproductive performance of female mice. Female ICR mice (7 weeks old) were given melatonin-containing water (3, 30 and 300 µg/mL; melatonin) or water only (control) until 10 weeks of age. Then, some of the mice were successfully mated (confirmed by vaginal plugs), and the number of live births and their weights were recorded. Some mice were used for a histological analysis of the number of follicles in the ovaries. Others were used for oocyte collection after superovulation, and in vitro fertilization (IVF) was performed. The mRNA expression of the apopotosis-related genes (BAX, BCL2) in the IVF embryos were analyzed. After melatonin administration, the mice showed similar serum melatonin levels to that of the control. The number of antral follicles per mm² unit area in the 30 µg/mL melatonin-treated group (14.60) was significantly higher than that of the control (7.78), which was lower than that of the 3 µg/mL melatonin-treated group (12.29). The litter size was significantly higher in the 3 µg/mL melatonin-treated group (15.5) than in the control (14.3). After IVF, the hatched blastocyst formation rate in the 30 µg/mL melatonin-treated group (85.70%) was significantly higher than that of the control (72.10%), and it was the same for the BCL2/BAX expression ratio. Although oral melatonin did not appear to have an effect on the serum melatonin rhythm in the mouse, melatonin did increase litter size at the 3 µg/mL dose level, and improved the developmental competency of IVF embryos at the 30 µg/mL level.

Cryopreservation of boar sperm induces differential microRNAs expression.

Lower conception rates and litter sizes limit the wide use of artificial insemination with frozen-thawed boar sperm, due to a lack of understanding of the mechanisms that cause cryodamage and cryoinjury to sperm during cryopreservation. CryoMiRs, a family of freeze-related microRNAs (miRNAs), are associated with freeze tolerance, and regulate metabolism in mammalian hibernators and insects. Thus, we speculate that miRNAs maybe involved in the regulation of the freeze-thaw process and may affect boar sperm function. In this study, we studied the differential expression of 46 miRNAs that have roles in spermatogenesis, sperm maturation, and sperm quality in response to cryopreservation (with or without 3% glycerol). The results indicated that, in response to cryopreservation with 3% glycerol, 14 miRNAs were significantly up-regulated, but only two miRNAs (miR-22 and miR-450b-5p) were significantly down-regulated, relative to fresh sperm. Preservation with 3% glycerol caused up-regulation of 17 miRNAs, but only caused down-regulation of one miRNA (miR-24), relative to sperm cryopreserved without glycerol. Functional annotations of these differentially expressed miRNAs indicated that these miRNAs and their targets are mainly associated with metabolic and cellular processes. Therefore, our findings show that cryopreservation results in changes in miRNA expression, and suggest that the anti-freeze mechanisms of boar sperm need to be studied further.

Improved development by melatonin treatment after vitrification of mouse metaphase II oocytes.

The study was aimed to investigate the effect of melatonin on the development potential of mouse MII oocytes after cryopreservation. Mouse MII oocytes were subjected first to vitrification/warming and 2 h of in vitro culture (phase 1), then to parthenogenetic activation (PA) followed by in vitro culture of parthenogenetic embryos (phase 2). Different concentrations of melatonin (0, 10-9, 10-6 mol/L) were added to the medium during either phase 1, phase 2 or both phases. The fresh oocytes were used as control. When melatonin was used during both phases, 10-9 mol/L melatonin-treated group showed similar rates of cleavage and 4-cell embryo development compared with control, which were significantly higher than those of melatonin-free group, while the rates in either 10-6 mol/L melatonin-treated or melatonin-free groups were significantly lower than that in control. When 10-9 mol/L melatonin was added during either phase 1 or phase 2, both cleavage and 4-cell embryo development rates of either group were significantly lower than those of control. After oocyte vitrification/warming and PA, the ROS levels increased significantly and maternal-to-zygotic transition (MZT) related genes (Dcp1a, Dcp2, Hspa1a, Eif1ax, Pou5f1, Sox2) expression were disorganized. However, after 10-9 mol/L melatonin supplementation, the ROS levels decreased significantly compared with melatonin-free group, and the gene expressions were almost recovered to normal level of control group. These results demonstrated that 10-9 mol/L melatonin supplementation could increase the developmental potential of vitrified-warmed mouse MII oocytes, which may result from ROS scavenging activities and recovery of normal levels of the expressions of MZT-related genes.

Vitrification transiently alters Oct-4, Bcl2 and P53 expression in mouse morulae but does not affect embryo development in vitro.

This study was conducted to determine the impact of vitrification on the expression of genes regulating pluripotency and apoptosis in mouse morulae. The morulae were randomly allocated into three groups: (1) untreated (control), (2) exposed to vitrification solution without freezing (toxicity), or (3) vitrified by open-pulled straw method (vitrification). In vitro development was evaluated by morphology and assessed by the blastocyst rate and the blastocyst total cell number. Gene expression in morulae and blastocysts was assessed by quantitative Real Time-PCR (qRT-PCR) and western blot. The results showed that at morulae stage, the POU class 5 homeobox1 (Oct-4) and B-cell lymphoma2 (Bcl2) mRNA levels of vitrification group were significantly lower (P < 0.05) than those of control. Strikingly, the p53 mRNA level was significantly higher in vitrification group. However, the Oct-4, Bcl2 and p53 mRNA levels in mouse blastocysts were not statistically different. Furthermore, western blot results showed that there was no significant difference in Oct-4, Bcl2 and p53 expression at protein level in mouse morulae among three groups. Additionally, the blastocyst rate (96.67%-100.00%) and the average cell number of blastocysts (89.67-92.33) were similar between all groups. The data demonstrate that vitrification transiently changes the mRNA expression of several key genes in mouse morulae regulating early embryo development but does not affect embryo developmental potential in vitro.

A preliminary study on epigenetic changes during boar spermatozoa cryopreservation.

Artificial insemination (AI) with cryopreserved boar semen is limited to no more than 1% of the total number of inseminations due to low conception rates and litter sizes. Cryopreservation causes a dramatic decrease in the viability, motility and fertility of spermatozoa, but the underlying mechanism remains unknown. In this study, mRNA expression and protein levels of epigenetic-related genes (Dnmt3a, Dnmt3b, Jhdm2a, Kat8, Prm1, Prm2 and IGF2) in fresh and cryopreserved boar spermatozoa were evaluated using qRT-PCR and ELISA. The results showed that cryopreservation or freezing, which drastically alter the environmental stimuli, can induce epigenetic changes of boar spermatozoa. Dramatic changes of mRNA expression of epigenetic-related genes were observed before and after cryopreservation, and low protein levels of multiple genes were mainly found in program freezing groups with or without LEY. The addition of different cryoprotective agents to the freezing extender can provide better protective effects for boar spermatozoa to avoid freezing or cryopreservation-induced expression changes of epigenetic-related genes.

Effects of glycerol on apoptotic signaling pathways during boar spermatozoa cryopreservation.

Artificial insemination (AI) with post-thawed boar spermatozoa results in low farrowing rates and reduced litter sizes mainly due to cryoinjury or damages to spermatozoa during cryopreservation. Low viability and motility of post-thawed boar spermatozoa are highly associated with apoptosis during cryopreservation. Although glycerol is widely used a cryoprotectant (CPA) for boar spermatozoa cryopreservation, the mechanism and relationship between glycerol and apoptosis-related gene expression needs to be clarified. In this study, we treated boar spermatozoa with different concentrations of glycerol in lactose egg yolk (LEY) extender to evaluate the apoptosis-related gene expression and protease activities of caspases. These results show that: (1) low concentrations of glycerol (2% and 3%) were more suitable for boar spermatozoa cryopreservation; (2) apoptosis-related genes involved in intrinsic mitochondrial and extrinsic death receptor apoptotic signaling pathways were widely expressed in different concentrations of glycerol treated boar spermatozoa; (3) there was a significant positive correlation (r=0.840, P=0.037) between the percentage of Annexin V(+)/PI(+) staining spermatozoa and caspase-6/9 protease activity. In conclusion, 2% and 3% glycerol have the best anti-apoptotic effects, and the expression of Fas/FasL and Bcl-2/Bax have a strong correlation with spermatozoa parameters.

Selection of optimal reference genes for quantitative RT-PCR studies of boar spermatozoa cryopreservation.

Reference genes can be used to normalize mRNA levels across different samples for the exact comparison of the mRNA expression level. It is important to select reference genes with high quality for the accurate interpretation of qRT-PCR data. Although several studies have attempted to validate reference genes in pigs, no validation studies have been performed on spermatozoa samples frozen with different cryoprotectants. In this study, 11 commonly used reference genes (ACTB, B2M, GAPDH, HPRT1, RPL4, SDHA, YWHAZ, PPIA, PGK1, S18, and BLM) were investigated in boar spermatozoa frozen with six different cryoprotectants using qRT-PCR. The expression stability of these reference genes in different samples was evaluated using geNorm (qbase(plus) software), NormFinder, and BestKeeper. The geNorm results revealed that PGK1, ACTB, and RPL4 exhibit high expression stability in all of the samples, and the NormFinder results indicated that GAPDH is the most stable gene. Furthermore, the BestKeeper results indicated that the three most stable genes are PPIA, GAPDH, and RPL4 and that S18, B2M and BLM are the three least stable genes. There are a number of differences in the ranking order of the reference genes obtained using the different algorithms. In conclusion, GAPDH, RPL4, and PPIA were the three most stable genes in frozen boar spermatozoa, as determined based on the cycle threshold coefficient of variation (Ct CV%) and the comprehensive ranking order, and this finding is consistent with the BestKeeper results.

Ram sperm cryopreservation disrupts metabolism of unsaturated fatty acids.

In ram sperm, metabolites are important components of the plasma membrane, energy metabolism cycle, and precursors for other membrane lipids, and they may have important roles in maintaining plasma membrane integrity, energy metabolism, and regulation of cryotolerance. In this study, the ejaculates from 6 Dorper rams were pooled and sperm were systematically investigated by metabolomics at various steps of cryopreservation (37 °C, fresh [F]; from 37 to 4 °C, cooling [C]; and from 4 to -196 to 37 °C, frozen-thawed [FT]) to identify differential metabolites (DM). There were 310 metabolites identified, of which 86 were considered DMs. Regarding the DMs, there were 23 (0 up and 23 down), 25 (12 up and 13 down), and 38 (7 up and 31 down) identified during cooling (C vs F), freezing (FT vs C), and cryopreservation (FT vs F), respectively. Furthermore, some key polyunsaturated fatty acids (FAs), particularly, linoleic acid (LA), docosahexaenoic acid (DHA), and arachidonic acid (AA) were down-regulated during cooling and cryopreservation. Significant DMs were enriched in several metabolic pathways including biosynthesis of unsaturated FAs, LA metabolism, mammalian target of rapamycin (mTOR), forkhead box transcription factors (FoxO), adenosine monophosphate-activated protein kinase (AMPK), phosphatidylinositol 3-kinase/protein kinase B (PI3K-Akt) signaling pathways, regulation of lipolysis in adipocytes, and FA biosynthesis. This was apparently the first report to compare metabolomics profiles of ram sperm during cryopreservation and provided new knowledge to improve this process.

Poly(I:C) exposure during in vitro fertilization disrupts first cleavage of mouse embryos and subsequent blastocyst development.

The reproductive system can be infected by a variety of double-stranded RNA viruses, which disrupt ovary function and pregnancy. However, whether viral infection directly affects early embryonic development remains unknown. Here we show that Poly(I:C), which mimics a double-stranded RNA virus, significantly impaired mouse early embryonic development in vitro, and up-regulated TLR3 and IFNα at the two cells embryo stage. Further studies indicated that Poly(I:C)-treatment caused DNA damage and abnormal spindle morphology at the first cleavage. Moreover, CDX2 and SOX2 expression was decreased while blastocyst cell apoptosis was increased. Altogether, Poly(I:C) decreased the rate of successful in vitro fertilization via DNA damage and abnormal spindle morphology at the first cleavage and inhibited early embryonic development by inducing immune response and promoting blastocyst cell apoptosis. This study provides an implication for exploring the causes of reproductive disorders in mammals and humans caused by infection of double-stranded RNA virus.