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METTL3-mediated N6-methyladenosine (m6A) modification is critical for gametogenesis and early embryonic development. However, the function of METTL3-mediated m6A modification in the early development of somatic nuclear transfer embryos (SCNT) remains unclear. Here, we found that METTL3 mRNA and protein levels exhibit dynamic changes during the early development of porcine SCNT embryos. The levels of METTL3 mRNA and protein in SCNT embryos at specific developmental stages differ from those in parthenogenetic activation (PA) counterparts. SiRNA injection effectively reduced the levels of METTL3 mRNA and protein in 4-cell embryos and blastocysts. METTL3 knockdown significantly reduced the cleavage and blastocyst rates of SCNT embryos. METTL3 knockdown significantly reduced the number of total cells and trophectoderm (TE) cells in the resulting blastocysts and perturbed cell lineage allocation. In addition, METTL3 knockdown reduced the levels of m6A modification in 4-cell embryos and blastocysts. Importantly, METTL3 knockdown decreased the expression levels of CDX2, GATA3, NANOG and YAP, and increased the expression levels of SOX2 and OCT4. Taken together, these results demonstrate that METTL3-mediated m6A modification regulates early development and lineage differentiation of porcine SCNT embryos., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Decreased oocyte quality is a significant contributor to the decline in female fertility that accompanies aging in mammals. Oocytes rely on mRNA stores to support their survival and integrity during the protracted period of transcriptional dormancy as they await ovulation. However, the changes in mRNA levels and interactions that occur during porcine oocyte maturation and aging remain unclear. In this study, the mRNA expression profiles of porcine oocytes during the GV, MII, and aging (24 h after the MII stage) stages were explored by transcriptome sequencing to identify the key genes and pathways that affect oocyte maturation and postovulatory aging. The results showed that 10,929 genes were coexpressed in porcine oocytes during the GV stage, MII stage, and aging stage. In addition, 3037 genes were expressed only in the GV stage, 535 genes were expressed only in the MII stage, and 120 genes were expressed only in the aging stage. The correlation index between the GV and MII stages (0.535) was markedly lower than that between the MII and aging stages (0.942). A total of 3237 genes, which included 1408 upregulated and 1829 downregulated genes, were differentially expressed during porcine oocyte postovulatory aging (aging stage vs. MII stage). Key functional genes, including ATP2A1, ATP2A3, ATP2B2, NDUFS1, NDUFA2, NDUFAF3, SREBF1, CYP11A1, CYP3A29, GPx4, CCP110, STMN1, SPC25, Sirt2, SYCP3, Fascin1/2, PFN1, Cofilin, Tmod3, FLNA, LRKK2, CHEK1/2, DDB1/2, DDIT4L, and TONSL, and key molecular pathways, such as the calcium signaling pathway, MAPK signaling pathway, TGF-β signaling pathway, PI3K/Akt signaling pathway, FoxO signaling pathway, gap junctions, and thermogenesis, were found in abundance during porcine postovulatory aging. These genes are mainly involved in the regulation of many biological processes, such as oxidative stress, calcium homeostasis, mitochondrial function, and lipid peroxidation, during porcine oocyte postovulatory aging. These results contribute to a more in-depth understanding of the biological changes, key regulatory genes and related biological pathways that are involved in oocyte aging and provide a theoretical basis for improving the efficiency of porcine embryo production in vitro and in vivo., Competing Interests: Declaration of competing interest None., (Copyright © 2024. Published by Elsevier Inc.)

Tumour necrosis factor (TNF) superfamily member 11 (TNFSF11), also known as RANKL, plays a crucial role in regulating several physiological and pathological activities. Additionally, it is a vital factor in bone physiology, and the sex hormone progesterone regulates the expansion of stem cells and the proliferation of mammary epithelial cells. It is essential for animal growth and reproductive physiological processes. This study aimed to evaluate the tissue-specific expression characteristics and promoter activity of the TNFSF11 gene in pigs. As a result, the study examined the presence of TNFSF11 expression in the tissues of Xiangsu pigs at 0.6 and 12 months of age. Moreover, the core promoter region of TNFSF11 was also identified by utilizing a combination of bioinformatic prediction and dual-luciferase activity tests. Finally, the effect of transcription factors on the transcriptional activity of the core promoter region was determined using site-directed mutagenesis. TNFSF11 was uniformly expressed in all tissues; however, its expression in muscles was comparatively low. The core promoter region of TNFSF11 was located in the -555 to -1 region. The prediction of the transcription start site of TNFSF11 gene-2000 ∼ + 500bp showed that there was a CpG site in 17 ∼ + 487bp. Analysis of mutations in the transcription factor binding sites revealed that mutations in the Stat5b, Myog, Trl, and EN1 binding sites had significant effects on the transcriptional activity of the TNFSF11 gene, particularly following the EN1 binding site mutation (P < 0.001). This study provides insights into both the tissue-specific expression patterns of TNFSF11 in the tissues of Xiangsu pigs and the potential regulatory effects of transcription factors on its promoter activity. These results may be helpful for future research aimed at clarifying the expression and role of the porcine TNFSF11 gene., (Copyright © 2024 Elsevier Inc. All rights reserved.)

With the rapid development of intensive animal husbandry in the livestock industry, large quantities of manure waste containing phytate phosphorus are being generated. Phytase can effectively solve the problem of high phosphorus pollution in the feces of monogastric animals. Enviropig, which produces phytase in the salivary glands and secretes the enzyme in the saliva, were first generated in 1999. However, phytase is easily inactivated during digestion. To address this problem, cleavage-resistant phytase transgenic pigs were generated using handmade cloning in this study. Transgene construction was improved and three cell lines carrying Cafp were obtained. In total, 810 blastocysts were generated and 712 good-quality were transferred into six recipients. Fourteen piglets were born, of which six survived after weaning. Polymerase chain reaction and sequencing results showed that seven (three live and four dead) of the fourteen piglets carried Cafp. Phytase activity in the saliva of the six live cloned pigs was tested at four months of age, and only one pig had 0.155 FTU/mL enzyme activity. The other five pigs may not have been activated in the transgenic parotid gland. Among all the transgenic pigs, the highest phosphorus digestion rate was 59.2% of intake, representing a 25.4% decrease in fecal emission compared to the average of controls. Immunohistochemical results on the three Cafp-positive pigs that died after six months of age showed that the transgene was only expressed in parotid glands, confirming tissue-specific gene expression. In conclusion, cleavage-resistant phytase transgenic pigs were successfully produced through handmade cloning. The cloned pigs offer a unique biological approach to managing phosphorus nutrition and environmental pollution in animal husbandry., Competing Interests: Declaration of competing interest None of the authors have any conflict of interest to declare., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Genome editing in pigs for xenotransplantation has seen significant advances in recent years. This study compared three methodologies to generate gene-edited embryos, including co-injection of sperm together with the CRISPR-Cas9 system into oocytes, named ICSI-MGE (mediated gene editing); microinjection of CRISPR-Cas9 components into oocytes followed by in vitro fertilization (IVF), and microinjection of in vivo fertilized zygotes with the CRISPR-Cas9 system. Our goal was to knock-out (KO) porcine genes involved in the biosynthesis of xenoantigens responsible for the hyperacute rejection of interspecific xenografts, namely GGTA1, CMAH, and β4GalNT2. Additionally, we attempted to KO the growth hormone receptor (GHR) gene with the aim of limiting the growth of porcine organs to a size that is physiologically suitable for human transplantation. Embryo development, pregnancy, and gene editing rates were evaluated. We found an efficient mutation of the GGTA1 gene following ICSI-MGE, comparable to the results obtained through the microinjection of oocytes followed by IVF. ICSI-MGE also showed higher rates of biallelic mutations compared to the other techniques. Five healthy piglets were born from in vivo-derived embryos, all of them exhibiting biallelic mutations in the GGTA1 gene, with three displaying mutations in the GHR gene. No mutations were observed in the CMAH and β4GalNT2 genes. In conclusion, in vitro methodologies showed high rates of gene-edited embryos. Specifically, ICSI-MGE proved to be an efficient technique for obtaining homozygous biallelic mutated embryos. Lastly, only live births were obtained from in vivo-derived embryos showing efficient multiple gene editing for GGTA1 and GHR., (Copyright © 2024. Published by Elsevier Inc.)

The quality of sperm significantly influences the reproductive efficiency of pig herds. High-quality sperm is necessary for efficient fertilization and to maximize the litter numbers in commercial pig farming. However, the understanding of genes regulating porcine sperm motility and viability is limited. In this study, we validated porcine sperm/Sertoli-specific promoters through the luciferase reporter system and identified vital genes for sperm quality via loss-of-function means. Further, the shRNAs driven by the ACE and SP-10 promoters were used to knockdown the SPAG6 and PPP1CC genes which were provisionally important for sperm quality. We assessed the effects of SPAG6 and PPP1CC knockdown on sperm motility by using the sperm quality analyzer and flow cytometry. The results showed that the ACE promoter is active in both porcine Sertoli cells and sperms, whereas the SP-10 promoter is operating exclusively in sperm cells. Targeted interference with SPAG6 and PPP1CC expression in sperm cells decreases the motility and increases apoptosis rates in porcine sperms. These findings not only offer new genetic tools for targeting male germ cells but also highlight the crucial roles of SPAG6 and PPP1CC in porcine sperm function., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Over the last few decades, there has been a constant increase in sow litter size, the consequences of which include parturition duration extension, an increase in the percentage of stillborn and hypoxic piglets, and increased variation in piglet birth weight, which reduces their vitality. As such, it seems clear that further increasing sow fertility will generate difficulties and costs in rearing numerous litters with low birth weights. Therefore, the current study aimed to analyze the genetic background of sow hyperprolifcacy using a genome-wide association study (GWAS). The research included 144 sows in the maternal component, divided into two equal groups. The first group (control) consisted of females giving birth to the optimal number of piglets in their third and fourth litters (14-16), while the second group (cases) included those with excessive litter size (>16). The analyzed sows were genotyped using Illumina's PorcineSNP60v2 BeadChip microarray, comprising 64,232 single nucleotide polymorphisms (SNPs). Statistical analysis using R included quality control of genotyping data and GWAS analysis based on five logistic regression models (dominant, codominant, overdominant, recessive, and log-additive) with a single SNP marker as the explanatory variable. On this basis, one SNP (SIRI0000069) was identified on chromosome seven within the EFCAB11 (EF-hand calcium binding domain 11) gene that had a statistically significant effect on sow hyperprolificacy. Additionally, ten SNPs (INRA0007631, ALGA0011600, ALGA0043433, ALGA0043428, M1GA0010535 ALGA00443338, ALGA0087116, MARC0056787, ALGA0112928, and ALGA0089047) had a relationship with the analyzed feature at a level close to significance, set at 1 -5 . These SNPs appear important since they are located on chromosomes on which a large number of quantitative trait loci (QTLs) and SNPs associated with reproductive characteristics, including litter size, have been identified., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The purpose of this study was to compare the efficiency of the production of cloned transgenic Yucatan miniature pigs (YMPs) using two recipient breeds, i.e., YMPs and domestic pigs (DPs), under various embryo transfer conditions. We initially assessed the in vitro developmental competence of embryos obtained via somatic cell nuclear transfer (SCNT) from three different transgenic donor cells. No difference was observed among the three groups regarding developmental competence. Furthermore, the cloning efficiency remained consistent among the three groups after the transfer of the SCNT embryos to each surrogate mother. Subsequently, to compare the efficiency of the production of cloned transgenic YMPs between the two recipient breeds using varying parameters, including ovulation status (preovulation and postovulation), duration of in vitro culture (IVC) (incubated within 24 h and 24-48 h), and the number of transferred SCNT embryos (less than and more than 300), we assessed the pregnancy rates, delivery rates, mean offspring counts, and cloning efficiency. Regarding the ovulation status, YMPs exhibited higher pregnancy rates, delivery rates, and cloning efficiency compared with DPs in both statuses. Moreover, the pregnancy rates, delivery rates, and cloning efficiency were affected by the ovulation status in DPs, but not in YMPs. The comparison of IVC duration between groups revealed that YMPs had higher pregnancy rates vs. DPs in both conditions. SCNT embryos cultured for 24-48 h in YMPs yielded higher delivery rates and cloning efficiency compared with those cultured for less than 24 h in DPs. Finally, the analysis based on the number of transferred SCNT embryos showed that both the pregnancy and delivery rates were higher in YMPs vs. DPs. However, the highest average number of offspring was obtained when more than 300 SCNT embryos were transferred into DPs, whereas the cloning efficiency was higher in YMPs vs. DPs. Our results suggest that YMPs are more suitable recipients than are DPs under various conditions for the production of cloned transgenic YMPs., Competing Interests: Declaration of competing interest The authors have no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Genetically modified pigs play a critical role in mimicking human diseases, xenotransplantation, and the development of pigs resistant to viral diseases. The use of programmable endonucleases, including the CRISPR/Cas9 system, has revolutionized the generation of genetically modified pigs. This study evaluates the efficiency of electroporation of oocytes prior to fertilization in generating edited gene embryos for different models. For single gene editing, phospholipase C zeta (PLC ζ) and fused in sarcoma (FUS) genes were used, and the concentration of sgRNA and Cas9 complexes was optimized. The results showed that increasing the concentration resulted in higher mutation rates without affecting the blastocyst rate. Electroporation produced double knockouts for the TPC1/TPC2 genes with high efficiency (79 %). In addition, resistance to viral diseases such as PRRS and swine influenza was achieved by electroporation, allowing the generation of double knockout embryo pigs (63 %). The study also demonstrated the potential for multiple gene editing in a single step using electroporation, which is relevant for xenotransplantation. The technique resulted in the simultaneous mutation of 5 genes (GGTA1, B4GALNT2, pseudo B4GALNT2, CMAH and GHR). Overall, electroporation proved to be an efficient and versatile method to generate genetically modified embryonic pigs, offering significant advances in biomedical and agricultural research, xenotransplantation, and disease resistance. Electroporation led to the processing of numerous oocytes in a single session using less expensive equipment. We confirmed the generation of gene-edited porcine embryos for single, double, or quintuple genes simultaneously without altering embryo development to the blastocyst stage. The results provide valuable insights into the optimization of gene editing protocols for different models, opening new avenues for research and applications in this field., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Mummified piglets are among the leading causes of fertility loss and severely hamper reproductive performance in pigs. However, the contributions of genomic variation to the emergence of mummified piglets (MUM) have rarely been studied. This study aims to (1) elucidate the genetic architecture of MUM in sows of parity 1 - 3 using a single-step genome-wide association study (ssGWAS). The ssGWAS involved genotyping-by-sequencing of Large White and Landrace pig breeds. (2) Explore the biological role of the candidate genes at the cellular level. A total of 185 and 48 genome-wide significant SNPs are associated with MUM in Large White and Landrace pigs, explaining 0.01-36.52% genetic variance for different significant loci, respectively. All the significant SNPs are parity-specific, and the numerous, consecutive significant loci likely generated the nine significant peaks in different parities. Multiple candidate genes (including CYP24A1, FBXO30, and ARHGEF28) are associated with fetal congenital and maternal diseases. Collectively, CYP24A1 regulation contributes to steady-state levels of embryo development genes. CYP24A1 is involved in reproduction and, immune and gestational disorders. Thus, it is associated with known newborn death traits and MUM in Large White sows. Altogether, these results improve the current understanding of the genetic architecture of MUM and expand the knowledge on genetic variations for selecting against mummified piglets in pig breeding., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Comprehending mechanisms controlling corpus luteum (CL) angiogenesis and apoptosis in pregnant sows is essential to understand the physiological role of these processes in CL function, progesterone production and consequently in conceptus development and prenatal mortality. CL from 54 sows from two genetic groups, a commercial line (COM) and the local Piau breed (LPB), were obtained for gene expression ( n = 3 COM; n = 6 LPB), histological and protein analysis ( n = 3 COM; n = 3 LPB), divided in six gestational ages (seven, 15, 30, 45, 60 and 90 days). We observed differences between gestational ages in CL morphology, in which the average number of blood vessels/capillaries at 90-days was greater than at the seventh day by Tukey test. RT-qPCR analysis revealed that apoptotic genes ( BAX , BCL2 and CASP3 ) were differentially expressed between genetic groups and gestational ages in each group. Angiogenesis genes also presented differences between genetic groups ( ANGPT1 ) and gestational ages ( MMP9 , VEGFA and ANGPT1 ). No differences in protein abundance of steroidogenic enzymes (CYP11A1 and HSD3B1) were observed. Our findings indicate that despite the differences in gene expression, differences in corpus luteum vascularization were observed only across gestational ages, with no dissimilarities between genetic groups. [ABSTRACT FROM AUTHOR]

Historically, the conservation or preservation of mammalian genetic resources, especially farm animals, has been conducted under in situ conditions by maintaining living individuals as “livestock.” However, systems for laboratory in vitro embryo production using gametes such as spermatozoa and oocytes are now available, in addition to ex situ preservation methods for mammalian genetic resources. One of these methods is the cryopreservation of gametes, embryos, and gonadal tissues. In pigs, freezing of sperm is the most reliable and well-established method for this purpose. On the other hand, cryopreservation of female gametes (oocytes) and gonadal tissues—usually by vitrification—has been associated with very low efficacies. Recently, in our laboratory, some research themes related to this issue have been pursued. We have been focusing on advances in porcine in vitro embryo production systems, and here, we introduce recent data on the vitrification of porcine immature oocytes and gonadal tissues followed by their xenografting into host mice to produce gametes. [ABSTRACT FROM AUTHOR]

Reproductive traits, such as the number of teats and litter size, are essential for animal breeding programs due to the importance of the production chain, since they influence the maternal ability of the sow and can affect the number of weaned piglets. We aim to identify candidate genes associated with reproductive traits in pigs, using GWAS data from a systematic review combined with sequencing data, to build networks of biological processes and transcription factors (TFs) from the identified genes to highlight the most candidate genes for litter size and the number of teats. In the systematic review, only peer-reviewed articles were used, with descriptors related to the evaluated traits, and selected based on eligibility criteria. Fourteen papers were selected and classified for functional analysis of gene networks with 2077 candidate genes identified. After combining with the list of genes presenting known structural variants in the 5'UTR and/or coding region, 306 genes remained to be used to build the gene networks of biological processes and TFs, highlighting processes associated with litter size (e.g., ionotropic glutamate receptor signaling pathway and blastocyte growth) and the number of teats (e.g., growth hormone receptor, regulation of the BMP - Bone Morphogenetic Proteins signaling pathway and blood vessel proliferation). Two most candidate genes for litter size trait (GRID2 and PALB2) and six most candidate genes for the number of teats (GHR, IFT80, FSTL3, SKOR1, SMURF1, and AKT3) were prioritized. TFs associated with candidate genes were also identified for litter size (PALB2 and GRID2) and the number of teats (RIN, LTBP2, and COL6A6). Thus, it is suggested that the most candidate genes and TFs presented in this study may play an important role in the traits studied, being important for genetic studies and animal breeding., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The piRNA pathway plays an essential role in defense against transposable elements in the germline tissues of animals and contributes to post-transcriptional regulation of genes. Xiang pigs present an earlier sexual maturation compared with most European pig breeds, but the role that the piRNA pathway plays in the development of Xiang pigs is currently not understood. In this study, we sequenced and analyzed piRNAs expressed in the testes of Xiang pigs at four different ages, and identified endogenous piRNAs which were highly abundant at each time point. The lengths of the identified piRNAs ranged from 24 to 34 nucleotides (nt), with the most abundant length being 29 nt. Additionally, there was a strong bias for uracil at the first position, a slight bias for adenine at position 10 and frequent 5'-10 nt complementary sequences, suggesting that ping-pong-mediated silencing is present in the Xiang pig germline. We observed that the piRNA composition changed from TE-associated piRNAs in two- and three-month-old testes to predominantly gene-derived and intergenic piRNAs in six- and twelve-month-old testes, with a gradual increase in the expression level of piRNAs over the course of testis development. And more than half of piRNA reads mapped to just a few of 473 predicted piRNA clusters. Additionally, we found that several genes were highly enriched by piRNA reads, including CYP19A1, PRMT8, SUZ12, WWOX, SGSM1 and MIF. The functions of these genes are primarily associated with steroidogenesis and histone modification. Changes in piRNA composition and widespread expression patterns during spermatid development indicate that these small ncRNAs may be responsible not only for transposon suppression but also for post-transcriptional regulation of several protein-coding genes essential for normal spermatogenesis., (Copyright © 2022 Elsevier Inc. All rights reserved.)

We have shown that STK35 and IFT27 genes are differentially expressed in spermatozoa from boars with good and poor semen freezability (GSF and PSF, respectively). STK35 is a stress-related gene that is implicated in spermatogenesis, whereas IFT27 is a motility-related gene that is mainly involved in intracellular protein transport. In this study we hypothesized that polymorphic variants in the 5'-flanking regulatory regions of STK35 and IFT27 genes could contribute to differences in semen freezability. We also predicted the interactions of the polymorphic variants with transcription factors on the gene promoter activity, using bioinformatics. The 5'-flanking region sequences of the STK35 and IFT27 were PCR amplified and analyzed by Sanger sequencing method. Protein expression in STK35 and IFT27 was determined in pre-freeze (PF) and frozen-thawed (FT) spermatozoa, using western blotting analysis. Sanger sequencing revealed a single nucleotide polymorphism (SNP) rs327863835 (C > T) in STK35 promoter, while two SNPs (rs337563873, A > T; rs331520020, T > C) were detected in IFT27 promoter. STK35 and IFT27 promoter polymorphisms showed significant allele frequency differences between the GSF and PSF groups. Using bioinformatics approaches, we predicted that SNPs resulted in the generation of additional transcription factor binding sites for NFATC2, ELK1 and GR-β, which appeared to enhance or repress the promoter activity of STK35 or IFT27 in either freezability group. Wide variations in STK35 and IFT27 protein expression were observed among the boars, however, significantly higher protein expression was detected in IFT27 in FT spermatozoa of the GSF group. We suggest that the upstream variants, detected in STK35 and IFT27 promoters, might regulate the transcriptional activity of the genes by affecting their potential binding of transcription factors. The results indicate that the allelic variants in STK35 and IFT27 could be considered as potential genetic markers for predicting boar sperm freezability., Competing Interests: Declaration of competing interest None of the authors have any conflict of interest to declare., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Limb girdle muscular dystrophy type R1 (LGMDR1) is an autosomal recessive myopathy described in humans resulting from a deficiency of calpain-3 protein (CAPN3). This disease lacks effective treatment and an appropriate model, so the generation of KO pigs by CRISPR-Cas9 offers a way to better understand disease ethology and to develop novel therapies. Microinjection is the main method described for gene editing by CRISPR-Cas9 in porcine embryo, but electroporation, which allows handling more embryos faster and easier, has also recently been reported. The objective of the current study was to optimize porcine oocyte electroporation to maximize embryo quality and mutation rate in order to efficiently generate LGMDR1 porcine models. We found that the efficiency of generating CAPN3 KO embryos was highest with 4 electroporation pulses and double sgRNA concentration than microinjection. Direct comparison between microinjection and electroporation demonstrated similar rates of embryo development and mutation parameters. The results of our study demonstrate that oocyte electroporation, an easier and faster method than microinjection, is comparable to standard approaches, paving the way for democratization of transgenesis in pigs., Competing Interests: Declaration of competing interest None of the authors have any conflict of interest to declare., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Abstract: Advances in porcine assisted reproductive technology (ART) make it possible to use cryopreserved sperm, embryos and somatic cells in the maintenance, relocation and regeneration of swine genetics. In this review, development of key application-limiting technology is discussed in each cell type, focusing on the efficiencies, ease of storage and transportation, and minimization of pathogen transmission. Methods to regenerate swine genetics and/or models using frozen sperm, embryos and somatic cells in combination with other porcine ARTs, such as in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and somatic cell nuclear transplantation (SCNT), are also discussed. The applications of these ARTs utilizing cryopreserved cells will greatly increase the efficiency as well as biosecurity for maintenance, relocation and rederivation of swine genetics/models. [Copyright &y& Elsevier]

With continuous improvement of sow litter size, breeders are gradually paying more attention to the quality of litter traits that directly impact the production efficiency of pig companies, such as the rate of piglets born alive (RBA) and the rate of healthy births (RHB). The objectives of this study are to dissect the genetic basis of litter traits in pig and to identify valuable genes and genetic markers, especially pleiotropic, for pig breeding. Herein, 1140 Duroc pigs and 2046 reproduction records, 5 litter traits, including the number of healthy births (NHB), number of deformed fetuses (NDF), number of stillborn (NSB), RBA, and RHB, were used in this study. Subsequently, a genome-wide association study (GWAS) was performed for the five litter traits in the first two parities from two Duroc populations. A total of 76 significantly related SNPs and 10 potential candidate genes (CAV1, DAB2, FGF12, FHOD3, DYNC2H1, GRHL1, TCTN3, PYROXD2, MMP8, MMP13, and PGR) were detected, including 13 pleiotropic SNPs that affected more than one litter trait. Finally, the functional enrichment analysis of functional genes that were closest to these significant SNPs indicated that most of the significant pathways were associated with hormone secretion and embryo and organ development. This study advances our understanding of the genetic mechanisms of litter traits, especially the survival rate of piglets born, and provides an opportunity to increase the quality of litter using marker-assisted selection or genomic selection in pigs., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Single nucleotide polymorphisms (SNPs) in the 5'-flanking regulatory regions of genes could affect their expression levels. This is a follow-up study aimed to identify polymorphic variants in the 5'-flanking regulatory regions of genes expressed in boar spermatozoa, and to predict the interactions of such variants with transcription factors (TFs) on the gene promoter activity, using bioinformatics. Five and six boars were classified as having good and poor semen freezability (GSF and PSF, respectively) according to post-thaw (PT) assessment of sperm motility and membrane integrity characteristics. The 5'-flanking region sequences of the 14 genes (FOS, NFATC3, EAF2, FGF-14, BAMBI, RAB33B, CKS2, LARS2, SLC25A16, ACADM, CPT2, CCT3, DTD2 and CCDC85A) were PCR amplified and analyzed by Sanger sequencing method. A total of 32 polymorphic variants were identified in the 5'-flanking regions of the genes, including 4 insertion/deletion (indel) polymorphisms, and 8 unknown (novel) SNPs. Multiple sequence alignment analysis revealed a 26-bp indel variant in the 5'-flanking region of the LARS2 gene, which showed greater protein expression in spermatozoa from boars of the PSF group. It was found that 17 polymorphic variants, observed in the differentially expressed (DE) genes, showed significant allele frequency differences between the GSF and PSF groups. Polymorphic variants in the 5'-flanking regulatory regions of the genes contributed to the decrease or increase in the binding affinity for different testis-specific TFs, such as SMAD1, NF-1, FOXMI, RXRA, STAT4 and C/EBPβ. This study provides more insights into the mechanisms responsible for variations in transcriptional activity in promoters of genes expressed in boar spermatozoa. The allelic variants are promising genetic markers for predicting the freezability of boar spermatozoa., (Published by Elsevier Inc.)

TGF-β1, an important multi-functional cytokine of the TGF-β signaling pathway, has been reported to be crucial for ovarian granulosa cell (GC) states and female fertility. However, the molecular mechanism underlying TGF-β1 regulation of GC states remains largely unknown. Here, we provide a comprehensive transcriptomic view on TGF-β1 regulation of cell states in porcine GCs. We first confirmed that TGF-β1 can control GC states (apoptosis and proliferation) in pig ovary. RNA-seq showed that 909 differentially expressed genes (DEGs), including 890 DEmRNAs and 19 DEmiRNAs, were identified in TGF-β1-treated porcine GCs. Functional annotation showed that these DEGs were mainly involved in regulating cell states. In addition, multiple hub genes were identified by constructing the protein-protein interaction network, DEmiRNA-DEmRNAs regulatory network, and gene-pathway-function co-expression networks, which were further found to be enriched in FoxO, TGF-β, Wnt, PIK3-Akt, p53 and Ras signaling pathways that play important roles in regulating cell states, cell cycle, proliferation, stress-responses and inflammation. The current research deeply reveals the effects of TGF-β1 on porcine GCs, and also identifies potential therapeutic RNA molecules for inhibiting and rescuing female infertility., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

TGF-β1 is a ligand of the TGF-β superfamily and an important cytokine that regulates ovarian functions including follicular development, steroid production, ovulation, luteinization, and female fertility. However, little is known about the regulation of TGF-β1 expression in ovary. Here, we identified that TGF-β1 is a functional target of miR-130a in porcine ovarian granulosa cells (GCs). The 3'-UTR sequence of TGF-β1 gene (1137 bp in length) in Large White (LW) pig was isolated, and multiple RNA regulatory elements (RREs), including several binding motifs of different miRNAs, were identified in this region. Luciferase activity assay showed that miR-130a dramatically suppresses the 3'-UTR luciferase activity of TGF-β1 gene, and further inhibits the expression of TGF-β1 in porcine GCs. FACS revealed that miR-130a acts as a pro-apoptotic factor and promotes GC apoptosis by inhibiting TGF-β1. Two novel linked mutations (-573G > A and -540T  >  C) were identified in the promoter region of ssc-miR-130a, but their polymorphisms are not associated with sow reproductive traits. Importantly, combined genotype analysis with a known mutation (c.1583 A  >  G) in the 3'-UTR of porcine TGF-β1 gene showed a significant association with reproductive performance in LW sow population. Overall, our findings defined a novel regulatory axis, miR-130a/TGF-β1 axis, which is involved in regulating sow fertility., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

This study aimed to determine the effect of orexin B (OXB) on the global expression pattern and the relationships among differentially expressed genes (DE-genes) in the transcriptome of myometrial explants during the early implantation period in the pig (day 15 of pregnancy). The changes in the transcriptome profile of the porcine myometrium were investigated using the Porcine (V2) Two-colour Gene Expression Microarray, 4 × 44. An analysis of the data from the microarray experiment revealed that 1540 DE-genes were affected by OXB, of which 1135 exhibited fold changes (FC) greater than 1.2 (P < 0.05). Among these, 576 genes were up-regulated and 559 genes were down-regulated. Among the affected biological processes in the myometrial tissue, 76 were enhanced and 31 were suppressed. Furthermore, the differential expression of nine genes, related to the regulation of reproductive functions and metabolic homeostasis, was confirmed by quantitative RT-PCR. A functional analysis of the relationships between DE-genes indicated that OXB interacts with the genes involved in the processes such as the inflammatory response, the response to interleukin-6, cytokine receptor activity, the regulation of cell activation, growth factor receptor binding, lipid modification and the steroid metabolic process. An analysis of DE-genes and their functional relationships suggests that OXB could be involved in the mechanisms such as the regulation of cell proliferation and development, inhibition of contractility, regulation of programmed cell death, and the development of blood vessels, all of which facilitate implantation., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The implantation process requires precisely controlled interactions between the maternal uterine endometrium and the implanting conceptus. Conceptus-derived secretions affect endometrial cells to facilitate the adhesion and attachment of trophoblasts, and endometrial secretions support the growth and development of the conceptus. In pigs, the conceptus secretes a large amount of type II interferon, interferon-γ (IFNG), during the implantation period. However, the role of IFNG in the implantation process has not been fully understood in pigs. Thus, to determine the role of IFNG in the endometrium during early pregnancy in pigs, we treated endometrial explant tissues with increasing doses of IFNG and analyzed the transcriptome regulated by IFNG using an RNA-sequencing analysis. Data analyses identified 276 differentially regulated genes, their Gene Ontology terms, and 94 signature genes in a Gene Set Enrichment Analysis. Furthermore, we analyzed the expression of IFNG-regulated genes, including CIITA, KYNU, IDO1, WARS, and MHC class II molecules, in the endometrium throughout pregnancy and found that levels of those genes in the endometrium were highest on Day 15 of pregnancy, corresponding to the time of peak IFNG secretion by porcine conceptuses. In addition, immunohistochemical analyses revealed that CIITA, KYNU, and IDO proteins were expressed in a cell type- and pregnancy status-specific manner in the endometrium. These results show that genes overrepresented in endometrial tissues in response to IFNG were mainly related to immune responses, suggesting that conceptus-derived IFNG could play critical roles in regulating the maternal immune response for the establishment of pregnancy in pigs., (Copyright © 2020 Elsevier Inc. All rights reserved.)

We aimed to evaluate apoptosis and myogenesis related genes expression in embryos and fetuses from two divergent genetic groups of pigs: Piau breed and a commercial line. Thirty females (15 Piau and 15 commercial line) were selected at 120 days of age. Estrous cycle was observed and on the third estrus females were considered sexually mature. Gilts were inseminated with semen from males of the respective breed. Three females from each breed were slaughtered at five different gestational ages: 15, 30, 45, 60 and 90 days. Whole embryos (15 and 30 d) and samples of longissimus dorsi muscle from fetuses (45, 60 and 90 d) were collected for RNA extraction. Expression of apoptosis and myogenesis related genes (BAX, BCL2, FGF4, IHH, HHIP, SHH, SOX2, WNT1 and WNT4) were evaluated by quantitative real time PCR. There was significant effect of interaction between breeds and gestational ages for all genes evaluated (P < 0.05). The BCL2 gene expression differed throughout pregnancy in Piau group with lower expression on day 15. The IHH gene expression throughout pregnancy was lower on days 15 and 60 in Piau group and lower on day 90 in commercial line group; and the SHH gene expression throughout pregnancy was higher on day 30 and lower on day 60 in Piau group and lower on day 45 in commercial group. The WNT1 gene expression along pregnancy was lower on day 15 and higher on days 30 and 45 in Piau group, and it was higher on day 30 than days 15 and 60 in commercial group. WNT4 gene expression throughout pregnancy was lower on day 15 in Piau group, and it was lower on day 30 in commercial pigs. Piau group presented higher expression of the FGF4 gene on days 45, 60 and 90, and commercial group showed higher expression on day 15 and 90. SOX2 gene expression was lower on day 15 in Piau pigs and it was constant throughout pregnancy in commercial group. Overall, the expression of IHH, SHH, WNT1, WNT4 and FGF4 genes were higher in commercial than Piau pigs on day 15, besides expression of BCL2 gene in Piau embryos was lower on day 15; these results might indicate that the muscle precursor cells are allowed to proliferate for a longer time in commercial than in Piau embryos by the balance of proliferative and apoptotic genes. Therefore, the expression differential between breeds can stimulate proliferation and differentiation of cells in different ways, explaining the postnatal differences in the muscularity between pigs from Piau breed and a commercial line., Competing Interests: Declaration of competing interest Authors declare that there is no conflict of interest., (Copyright © 2020. Published by Elsevier Inc.)

Semen freezability is associated with genetic markers, and there is a diverse set of sperm transcripts that have been attributed to various cellular functions. RNA-Seq was performed to compare the transcript profiles of spermatozoa from boars with different semen freezability. We examined ejaculates from the Polish large white (PLW) boars that were classified as having good and poor semen freezability (GSF and PSF, respectively; n = 3 boars per group) by assessing post-thaw motility characteristics, mitochondrial membrane potential, plasma membrane and acrosome integrity. Total RNA was isolated from fresh spermatozoa from boars of the GSF and PSF groups and subjected to RNA-Seq (Illumina NextSeq 500 platform). Transcript abundance was assessed with the DESeq2, DESeq, and EdgeR Bioconductor R packages, and varying numbers of differentially expressed gene (DEG) transcripts were detected in the spermatozoa of each boar. Using RNA-Seq, we identified several genes associated with inflammation and apoptosis (FOS, NFATC3, ITGAL, EAF2 and ZDHHC14), spermatogenesis (FGF-14 and BAMBI), autophagy (RAB33B), protein phosphorylation (PTPRU and PTPN2) and energy metabolism (ND6 and ACADM) that were predominantly up-regulated in poor freezability ejaculates. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) validated the transcript expression levels detected by RNA-Seq and thus confirmed the reliability of this technique. Subsequent validation with western blotting showed that the expression of three proteins was in accordance with the transcript abundance. Overall, we demonstrated that the up-regulation of the DEG transcripts in spermatozoa was associated with poor semen freezability. We suggest that spermatozoa transcriptome profiling provides a foundation to further elucidate the relevance of sperm-related transcripts on cryo-survival. The sperm-related transcripts, namely FOS, NFATC3, EAF2, BAMBI, PTPRU, PTPN2, ND6 and ACADM, are potential markers for predicting the freezability of boar semen., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The maternal endometrium undergoes transformations during early pregnancy period to regulate the paracellular permeability across the epithelium and to enable adhesion between the trophoblast and endometrial epithelial cells. These transformations, under the influence of ovarian hormones, are associated with a partial loss in polarity of epithelial cell that is regulated by tight junctions (TJ), adherens junctions (AJ) and associated polarity protein complexes. This study examined the change in expression and distribution of proteins associated with TJs, AJs and apical partition defective (PAR) complex in porcine endometrium on Days 10, 13 and 16 of estrous cycle and pregnancy. Moreover, effect of hormones, progesterone (P4) and 17-β estradiol (E 2 ) on polar phenotype of endometrial epithelial cells was also investigated in vitro. There was pregnancy induced increase in gene and protein expression of TJ associated claudin-1 (CLDN1) on Day 13 of pregnancy as compared to corresponding day of estrous cycle and a decrease in TJ protein, zona occludens-1 (ZO-1) and PAR complex associated PAR6 expression levels on Day 16 of pregnancy (P < 0.05). Immunofluorescence studies revealed that on Days 10 and 13, TJ proteins occludin (OCLN) and ZO-1were primarily present in the apical region of lateral epithelial membrane. On Day 16 of pregnancy, whereas, OCLN redistributed into cytoplasm, ZO-1 decreased apically but was found to localize in the basal epithelium. The AJ proteins cadherin and β-catenin were located at the apical epithelium on Day 10 of estrous cycle and pregnancy and Day 13 of estrous cycle. On Days 13 and 16 of pregnancy both proteins were expressed in the lateral membrane and co-localization between these proteins was observed on Day 16. On Day 10, PAR complex proteins PAR3, cell division control protein 42 (CDC42) and atypical protein kinase C (aPKC) ζ were observed in apical epithelium and in lateral membrane and CDC42 was also present in the cytoplasm of epithelium. Pregnancy induced redistribution of aPKCζ to cytoplasm and CDC42 to apical surface of luminal epithelium was observed on Days 13 and 16. The in vitro P4 and E 2 treatment of epithelial cells mimicked in vivo results. These results indicate that P4 and E 2 regulate alterations in epithelium that may facilitate embryo implantation and given the role of cadherin, catenin and CDC42 in embryo invasion, change in distribution of these proteins may limit the invasiveness of porcine conceptuses into the stroma., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Artificial insemination (AI) has been used as a routine technology globally in the pig production industry since 1930. One of the preferable advantages of AI technology is that the semen of elite boars can be disseminated to the commercial sow population rapidly. Understanding the genetic background of semen traits may help in developing genetic improvement programs of boars by including these traits into the selection index. In this study, we utilized weighted single-step genome-wide association study (wssGWAS) to identify genetic regions and further candidate genes associated with sperm morphology abnormalities (proximal droplet, distal droplet, bent tail, coiled tail, and distal midpiece reflex) in a Duroc boar population. Several genomic regions explained 2.76%-9.22% of the genetic variances for sperm morphology abnormalities were identified. The first three detected QTL regions together explained about 7.65%-25.10% of the total genetic variances of the studied traits. Several genes were detected and considered as candidate genes for each of the traits under study: coiled tail, HOOK1, ARSA, SYCE3, SOD3, GMNN, RBPJ, STIL, and FGF1; bent tail, FGF1, ADIPOR1, ARPC5, FGFR3, PANX1, IZUMO1R, ANKRD49, and GAL; proximal droplet, NSF, WNT3, WNT9B, LYZL6, FGFR1OP, RNASET2, FYN, LRRC6, EPC1, DICER1, FNDC3A, and PFN1; distal droplet, ARSA, SYCE3, MOV10L1, CBR1, KDM6B, TP53, PTBP2, UBR7, KIF18A, ADAM15, FAAH, TEKT3, and SRD5A1; and distal midpiece reflex, OMA1, PFN1, PELP1, BMP2, GPR18, TM9SF2, and SPIN1. GO and KEGG enrichment analysis revealed the potential function of the identified candidate genes in spermatogenesis, testis functioning, and boar spermatozoa plasma membrane activating and maintenance. In conclusion, we detected candidate genes associated with the coiled tail, bent tail, proximal droplet, distal droplet, and distal midpiece reflex in a Duroc boar population using wssGWAS. Overall, these novel results reflect the polygenic genetic architecture of the studied sperm morphology abnormality traits, which may provide knowledge for conducting genomic selection on these traits. The detected genetic regions can be used in developing trait-specific marker assisted selection models by assigning higher genetic variances to these regions., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Litter size is one of the most important economic traits for pig production as it is directly related to the production efficiency. As an important litter size trait in pigs, the number of piglets born alive at birth (NBA) receives widespread interests in the pig industry. However, traits of piglets born dead, including the number of stillborn piglets (NS) and the piglets mummified at birth (NM) should be noted to explain the loss of reproduction. Herein, in the present study, a total of 803 producing sows were sampled and 2807 farrowing records for NBA, NM, and NS traits were collected in a Duroc swine population. Subsequently, a genome-wide association study (GWAS) was performed for NBA, NS and NM in parity groups 1 to 5. In total, 10 putative regions were found associated with these traits. After stepwise conditional analyses around the putative regions, eight independent signals were ultimately identified for NBA, NS, and NM, and there were seven promising candidate genes related to these traits, including ARID1A, RXRG, NFATC4, ABTB2, GRAMD1B, NDRG1, and APC. Our findings contribute to the understanding of the significant genetic causes of piglets born alive and dead, and could have a positive effect on pig production efficiency and economic profits., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Sex determining region Y-box 9 (SOX9), an important member of the SRY- type HMGbox (SOX) gene family, plays an important role in the regulation of mammalian reproduction, including sex differentiation during the embryonic development stage and spermatogenesis after birth. To explore the roles of polymorphism and expression of the SOX9 gene in the development of testes, we analyzed the indel of SOX9 in pigs and the corresponding expression level of the SOX9 gene in 7-day and 5-month-old porcine Sertoli cells. Results revealed that the DD haplotype of SOX9 gene as well as the ID genotype were significantly associated with larger testicular weight, while the II haplotype was closely related to the smaller testicular weight. More importantly, the SOX9 gene expression of ID genotyped group was significantly higher than that in II genotyped group. Our results first revealed that the indel polymorphism and expression of SOX9 were significantly associated with pig reproduction traits indicating the critical roles of SOX9 gene in testes development. The study provides a new clue for understanding the regulation of animal reproductive activities., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In livestock industries, one sex is usually preferred because of the impact on the production (e.g. milk from cows, eggs from laying hens). Furthermore, in pig production, the male-specific boar taint is a big hurdle for consumer acceptance. Consequently, a shift in the ratio towards the desired sex would be a great benefit. The most widely applied method for pre-determination of the sex is fluorescence-activated sperm sorting, which relies on the different DNA content of the X- and Y-chromosomal sperm. However, the successful practical adaption of this method depends on its ease of use. At present, sperm sexing via fluorescence-activated cell sorting (FACS) has only reached commercial application in cattle. Nevertheless, sperm sexing technology still needs to be improved with respect to efficiency and reliability, to obtain high numbers of sexed sperm and less invasive sperm treatment to avoid damage. New genome editing technologies such as Zinc finger nucleases (ZFN), Transcription-activator like endonucleases (TALENs) and the CRISPR/Cas system have emerged and offer great potential to affect determination of the sex at the genome level. The sex-determining region on the Y chromosome (SRY) serves as a main genetic switch of male gender development. It was previously shown that a knockout of the SRY gene in mice and rabbits displayed suppressed testis development in the fetal gonadal ridges resulting in a female phenotype. These new technologies hold great opportunities to pre-determine sex in pigs. However, further investigations are needed to exploit their full potential for practical application., (Copyright © 2019 Elsevier Inc. All rights reserved.)

We aimed to characterize the expression of angiogenesis-related genes in corpus luteum (CL) and cumulus cells (CC) during estrous cycle in gilts of different genetic groups, as well as to study the relation between gene expression and phenotypic data. Forty five gilts were used as follows: L1, Commercial Line 1 (Large White x Landrace x Duroc) (n = 15); L2, Commercial Line 2 (Large White x Landrace x Pietrain) (n = 15); and Piau, Piau breed gilts (n = 15). Estrus observation started from 120 days of age. After the second observed estrus females were slaughtered (n = 3) on days 3, 5, 10, 14 and 18 of estrous cycle (first day of estrous cycle as Day 0). CL sampling was performed on days 3, 5, 10 and 14 and collection of CC and follicular fluid on days 14 and 18. Follicular fluid was used for analysis of estradiol levels and CC and CL samples for analysis of angiogenesis-related genes expression, ANGPT-1/-2 and TEK in CC and MMP-2, VEGFA, VEGFR-1/-2, ANGPT-1/-2 and TEK in CL. Piau gilts showed lower ovulation rate than both L1 and L2 gilts (P < 0.05), lower number of large antral follicles (>6 mm) at 18 days than L2 gilts (P < 0.05), and smaller diameter of the largest follicles at 14 days than L1 gilts (P < 0.05). Piau and L2 gilts showed higher estradiol levels in follicular fluid on day 18. Expression of ANGPT-1 and -2 genes in CC did not differ among genetic groups neither among days of the estrous cycle, but TEK gene expression was higher in L1 than L2 gilts on day 18. Expression of VEGFA, VEGFR-2 and MMP-2 genes in CL did not differ among genetic groups and days of cycle, but VEGFR-1 expression was higher in Piau than L2 gilts on days 10 and 14, and it was higher in L1 than L2 gilts on day 14. The ANGPT-1/-2 and TEK genes expression in CL were significantly higher in Piau than L1 gilts on day 10. The ANGPT-2/ANGPT-1 gene expression ratio in CL was higher in L1 than Piau and L2 gilts at 14 days, suggesting a shorter luteal phase for L1 gilts. Results indicated differences among genetic groups for the pattern of the angiogenesis-related genes expression in CL along estrous cycles, which may be reflected in phenotypic traits such as ovulation rate, estradiol levels in follicular fluid and number and diameter of antral follicles., (Copyright © 2019. Published by Elsevier Inc.)

Lysine demethylase 6A (KDM6A, also known as UTX), which belongs a type of histone demethylase, is essential for spermatogenesis and embryo development. However, in pig, the coding sequence, mRNA expression and insertion/deletion (indel) variants of KDM6A remain unclear. Herein, the open-reading frame (ORF) of pig KDM6A gene was cloned and characterized. The entire ORF sequence was 4206 bp in length encoding a deduced protein of 1401 amino acid residues. The phylogenetic tree and signatures of selection on the pig KDM6A gene were calculated, and the results revealed that the pig KDM6A gene has been under strong positively selection. Expression analysis results showed that KDM6A gene was expressed in all tissues tested both in male piglets and adult boars. Meanwhile, with testicular development, the KDM6A expression levels in testis were significantly increased. Additionally, three intronic indels of 11-bp insertion, 17-bp deletion and 16-bp insertion were identified. Association analyses revealed that the 11-bp indel was associated with testicular short perimeter (TSP) (P < 0.01), testicular long perimeter (TLP) (P < 0.01) and testicular weight (TW) (P < 0.01) in 15-day-old Yorkshire and TLP (P < 0.01) in 40-day-old Yorkshire. The 16-bp indel was associated with TLP, TSP and TW (P < 0.05) in 15-day-old Yorkshire. All these findings would provide a foundation for the further research of KDM6A gene and the application of marker-assisted selection to pig breeding., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The EZH2 protein endows the polycomb repressive complex 2 (PRC2) with histone lysine methyltransferase activity that is associated with transcriptional repression. Recent investigations have documented crucial roles for EZH2 in mediating X-inactivation, stem cell pluripotency and cancer metastasis. However, there is little evidence demonstrating the maternal effect of EZH2 on porcine preimplantation development. Here, we took parthenogenetic activation embryos to eliminate the confounding paternal influence. We showed that the dynamic expression of EZH2 during early development was accompanied by changes in H3K27me3 levels. Depletion of EZH2 in MII oocytes by small interfering RNA not only impaired embryonic development at the blastocyst stage (P < 0.05), but also disrupted the equilibrium of H3K4me3 and H3K27me3 in the embryo. Interestingly, the expression of TET1, a member of Ten-Eleven Translocation gene family for converting 5-methylcytosine (5 mC) to 5-hydroxymethylcytosine (5hmC), was decreased after EZH2 knockdown, in contrast to the increase of the other two members, TET2 and TET3 (P < 0.05). These results indicate a correlation between histone methylation and DNA methylation, and between EZH2 and TET1. Along with the downregulation of TET1, the expression of the pluripotency gene NANOG was decreased (P < 0.05), which is consistent with a previous finding in mouse ES cells. Meanwhile, the abundance of OCT4 and SOX2 were also down-regulated. Moreover, EZH2 knockdown reduced the capacity of cells in the blastocysts to resist apoptosis. Taken together, our data suggest that EZH2 is integral to the developmental program of porcine parthenogenetic embryos and exerts its function by regulating pluripotency, differentiation and apoptosis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Atypical chemokine receptor (ACKR) 1, ACKR2, ACKR3, and ACKR4, chemokine decoy receptors that lack G-protein-mediated signaling pathways, internalize and degrade chemokines to control their availability and function. Chemokines play important roles in the endometrium during the estrous cycle and pregnancy, but the expression and regulation of ACKRs have not been determined in pigs. Therefore, we examined the expression of ACKRs in the endometrium throughout the estrous cycle and pregnancy and in conceptus tissues in pigs. ACKR1, ACKR2, ACKR3, and ACKR4 mRNA was expressed in the endometrium, with higher levels of ACKR3 on day 12 of the estrous cycle than in pregnancy and higher levels of ACKR4 on day 15 of pregnancy than in the estrous cycle. ACKR1, ACKR2, and ACKR3, but not ACKR4, mRNA was detected in conceptus and chorioallantoic tissues during pregnancy. ACKR2 and ACKR3 mRNA and ACKR4 protein were mainly localized to luminal epithelial cells and weakly to glandular epithelial cells in the endometrium. Increasing doses of progesterone increased the expression of ACKR2 and ACKR4 and decreased the expression of ACKR3 in endometrial tissues. On day 12 of pregnancy, the expression of ACKR4 mRNA was lower in the endometria of gilts with somatic cell nucleus transfer-derived conceptuses than in the endometria of gilts carrying conceptuses derived from natural mating. These results indicate that the expression of ACKRs is dynamically regulated at the maternal-conceptus interface, suggesting that ACKR proteins might play critical roles in regulating endometrial chemokines to support the establishment and maintenance of pregnancy in pigs., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The determination of specific reproductive parameters, which allow the early selection of high genetic merit boars from different lines, is critical as it may avoid the high maintenance costs of keeping unfertile males in the production system. The aim of this work was to evaluate body and testicular measurements during the pre-pubertal phase, and associate them with reproductive characteristics, such as precocity and libido, and seminal characteristics during puberty in two different lines. Two pure breeds used in the crossing of female lines (FL) and two crossbred terminal lines (TL) were evaluated through body and testicular biometrical measures and seminal characteristics. Terminal line boars presented greater body weights and testicular measures (P < 0.01) compared to FL animals throughout the pre-pubertal period. TL males had their first collection at 24.0 ± 0.3 weeks, while their FL counterparts started about one week later (25.0 ± 0.3 weeks, P < 0.05) and age of selection presented a two-week delay in FL males (29.0 ± 0.7 versus 27.0 ± 0.6 weeks). Overall, 57% of FL boars were selected up to 31 weeks of age, while 90% of TL males were selected during the same period (P < 0.05). It was observed that genotype did not affect the seminal characteristics evaluated: volume, concentration, number of total spermatozoa, number of viable spermatozoa or sperm motility and kinetics. However, TL crossbred males showed higher percentage of normal spermatozoa and lower percentage of tail and head defects (P < 0.05). Sperm concentration in the 28th week was positively correlated with body weight in the 1st (r = 0.58, P < 0.001) and 15th (r = 0.39, P < 0.05) weeks of age. Moreover, sperm concentration in the 28th week was also correlated with right testicle length (RTL) in the 3rd week (r = 0.40, P < 0.05), and right testicle width (RTW) at week 15 (r = 0.36, P < 0.05). There was a negative correlation between RTW at week 15 and age of selection (r = -0.32, P < 0.05). Therefore, body and testicular measurements in the pre-pubertal period may predict semen concentration and can be used as early classification criteria for the selection of boars from different lines, without the risk of culling high value boars., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Predicting male fertility is extremely important for artificial insemination and profitable farm management. Conventional semen assessment together with computer-assisted sperm analysis is widely used to predict male fertility under field conditions. However, the clinical validation and sensitivity of these methods remain unclear. Therefore, a new approach is needed to predict male fertility. Here, we investigated the use of a transcriptomic marker (solute carrier family 9, subfamily A, member 3, regulator 1; SLC9A3R1) together with sperm motility parameters and capacitation status to predict fertility/infertility in boars at the commercial level. Our data showed that among motility parameters and the capacitation status, hyperactivation (HYP) differed between high- and low-litter size boars. HYP showed a significant positive correlation (R = 0.468) with boar litter size. Simultaneously, the expression of SLC9A3R1, a gene important in sperm ion channel regulation, was significantly negatively correlated (R = -0.523) with boar litter size. Quality assessment revealed that both HYP and SLC9A3R1 showed considerable sensitivity (71.43 vs. 100%), specificity (100 vs. 71.43%), and overall accuracy (90%) for predicting male fertility. Interestingly, the potential of SLC9A3R1 expression to increase the average piglet number per breeding was higher (0.7 piglets) than that of HYP (0.5 piglets). Thus, measuring SLC9A3R1 expression in spermatozoa may be a more accurate marker for evaluating male fertility/infertility than conventionally used motility parameters and capacitation status., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The processes underlying maturation of mammalian oocytes are considered crucial for the oocytes ability to undergo monospermic fertilization. The same factors of influence are suggested to impact the development of sex associated characteristics, allowing sex differentiation to progress during embryonic growth. The primary aim of the study was to analyze the gene ontology groups involved in regulation of porcine oocytes' response to endogenous stimuli. The results obtained would indicate potential genes influencing sex differentiation. Additionally, they could help to determine new genetic markers, expression profile of which is substantially regulated during porcine oocytes' in vitro maturation. To achieve that, porcine oocytes were collected for analysis before and after in vitro maturation. Pigs were used as they are a readily available model that presents significant similarity to humans in terms of physiology and anatomy. Microarray analysis of oocytes, before and after in vitro maturation was performed and later validated by RT-qPCR. We have particularly detected and analyzed genes belonging to gene ontology groups associated with hormonal stimulation during maturation of the oocytes, that exhibited significant change in expression (fold change ≥ |2|; p < 0.05) namely "Female sex differentiation" (CCND2, MMP14, VEGFA, FST, INHBA, NR5A1), "Response to endogenous stimulus" (INSR, ESR1, CCND2, TXNIP, TACR3, MMP14, FOS, AR, EGR2, IGFBP7, TGFBR3, BTG2, PLD1, PHIP, UBE2B) and "Response to estrogen stimulus" (INSR, ESR1, CCND2, IHH, TXNIP, TACR3, MMP14). Some of them were characteristic for just one of the described ontologies, while some belonged into multiple ontological terms. The genes were analyzed, with their relation to the processes of interest explained. Overall, the study provides us with a range of genes that might serve as molecular markers of in vitro maturation associated processes of the oocytes. This knowledge might serve as a reference for further studies and, after further validation, as a potentially useful knowledge in assessment of the oocytes during assisted reproduction processes., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Cytochrome B is an important polypeptide of the mitochondria helpful in energy metabolism through oxidative phosphorylation. Cytochrome B plays an immense role in the reproduction of animals and due to its mutation prone nature, it can affect the basic physiology of animals. Cytochrome B affects reproductive system in males and equally plays an important role in transferring and providing energy in the development of the embryo, zygote, and oocytes precisely in females. The present study was conducted on Ghungroo pig to study their molecular and reproductive traits and the effect of the cytochrome B gene in the female reproduction of the Ghungroo pig. Although studies are available for cytochrome B gene analysis for evolutionary studies through phylogenetic analysis. This is the first report for the study of Cytochrome B gene on reproduction in pigs. Cytochrome B gene was sequenced and seven SNPs were observed out of which three were non-synonymous. INDEL mutation was detected in Variant B which had lead to Frame Shift mutation resulting in a stop codon AGA. The effect in the reproductive traits of the sow was studied due to the occurrence of nucleotide substitution. Bioinformatics analysis (I-mutant, PROVEAN, and SIFT) had revealed that the mutations were deleterious for the mutant type. Mutation leading to alterations in post-translational modification sites as phosphorylation site, leucine-rich nuclear export signal, occurrence of transmembrane helices, arginine and lysine peptide cleavage site for the mutant variant had resulted in a reduced physiological response. 3 D protein structure, (predicted through bioinformatics analysis) for cytochrome B has revealed distinct structural differences in mutated form with truncated protein by RMSD analysis through TM-Align software. Associated studies of genotype variants with reproductive traits have revealed the significant effect of variants of cytochrome B gene on reproductive traits namely litter size at first, second and third furrowing, piglet mortality, age at first furrowing and furrowing interval. Mitochondrial gene as Cytochrome B variants might be used as a marker for studying female reproduction of Ghungroo sow in future., (Copyright © 2018 Elsevier Inc. All rights reserved.)

To further understand the role of microRNA (miRNA) during testicular development, we constructed four small RNA libraries from the testes of the Chinese indigenous Xiang pig at four different ages, which were sequenced using high-throughput Solexa deep sequencing methods. It yielded over 23 million high-quality reads and 1,342,579 unique sequences. At two and three months of age, the proportion which represented miRNAs was the most abundant class of small RNAs, but it was gradually replaced by the category that represented piRNAs in adult testes. We identified 543 known and homologous conserved porcine miRNAs and 49 potential novel miRNAs. There were 306 known miRNAs which were co-expressed in four libraries. Six miRNAs and three potential novel miRNAs were validated in testes and sperms of Xiang pig by RT-qPCR method. Many clusters of mature miRNA variants were observed, in which let-7 family was the most abundant one. After comparison among libraries, 204 miRNAs were identified as being differentially expressed and likely involved in the development and spermatogenesis of pig testes. This work presented a general genome-wide expression profile of the testes-expressed small RNAs in different ages of pig testes. Our results suggested that miRNAs performed a role in the regulation of mRNAs in puberty pig testes while piRNAs likely functioned mainly in sexually mature pig testes., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The reproductive efficiency of Meishan pigs is higher than that of Duroc pigs, but the underlying molecular mechanism for this disparity remains unclear. No systematic quantitative proteomics studies, comparing global proteins in Meishan and Duroc boar spermatozoa have been reported. Therefore, we applied iTRAQ labeling coupled with mass spectrometry, and analyzed the differences in proteins between Meishan and Duroc sperm. In the present study, a total of 1597 proteins were quantified. Of these proteins, 190 showed statistically significant fold changes between Meishan and Duroc spermatozoa. Bioinformatics analysis revealed that these differentially abundant proteins were primarily involved in energy metabolism, sperm motility, capacitation and sperm-oocyte binding. Remarkably, SPAG6, ACR, LDHC, CALM, ACE and ENO1 which are positively related to high litter size, were more abundant in Meishan spermatozoa than in Duroc spermatozoa. Moreover, APOA1, NDUFS2 and RAB2A which are negatively related to farrowing rates, were less abundant in Meishan spermatozoa than in Duroc spermatozoa. Interestingly, essential enzymes in Glycolysis/Gluconeogenesis, such as HK1, ALDH2, LDHA and LDHC, were markedly up-regulated in Meishan spermatozoa compared to Duroc spermatozoa. In addition, we first demonstrated that the levels of protein phosphorylation in Meishan spermatozoa were higher than those in Duroc. Taken together, the physiologically and functionally differential proteins may be one main reason for explaining the high reproductive efficiency of Meishan boar., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Whereas stage-specific markers for spermatogonial cells have been well investigated in mouse, the specific markers of germ cells in the testis of domestic animals have not been well defined. Phosphoglycerate kinase (PGK), an enzyme that converts 1,3-bisphosphoglycerate and adenosine diphosphate to 3-phosphoglycerate and adenosine triphosphate, has two isozymes: PGK1 and PGK2. In mouse, PGK1 exists only during the early stages of spermatogenesis, and PGK2 is then expressed during the pachytene spermatocyte stage. In this study, we investigated the localization of PGK2 in the developing porcine testis, and compared the similarities and differences in its expression with that of the PGK2 in mouse. The PGK2 protein was found to be exclusively expressed in spermatids of the adult mouse testis, whereas PGK2-positive cells were observed in the prepubertal and postpubertal testes of pigs. Based on this result, we examined the expression of PGK2 in in vitro-cultured porcine undifferentiated spermatogonia and found it to be maintained in the cultured cells. To verify this result and identify the spermatogonial stem cell-like potential in recipient testes, PKH26 dye-stained PGK2-positive cells were transplanted into the testes of busulfan-treated immunodeficient mouse that had been depleted of both testicular germ cells and somatic cells. The transplanted cells colonized the recipient testis at 8 weeks post transplantation, and fluorescence microscopy identified the cells in the basement membranes of the seminiferous tubules of the injected mouse. Taken together, our results suggest that PGK2 is expressed differently in the testes of mouse and pigs according to developmental stage. This finding should contribute to the study of spermatogenesis and the production of transgenic domestic animals through in vitro spermatogonial sperm cell culture., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Recently, successful one-step genome editing by microinjection of CRISPR/Cas9-related mRNA components into the porcine zygote has been described. Given the relatively long gestational period and the high cost of housing swine, the establishment of an effective microinjection-based porcine genome editing method is urgently required. Previously, we have attempted to disrupt a gene encoding α-1,3-galactosyltransferase (GGTA1), which synthesizes the α-Gal epitope, by microinjecting CRISPR/Cas9-related nucleic acids and enhanced green fluorescent protein (EGFP) mRNA into porcine oocytes immediately after electrical activation. We found that genome editing was indeed induced, although the resulting blastocysts were mosaic and the frequency of modified cells appeared to be low (50%). To improve genome editing efficiency in porcine oocytes, cytoplasmic injection was performed 6 h after electrical activation, a stage wherein the pronucleus is formed. The developing blastocysts exhibited higher levels of EGFP. Furthermore, the T7 endonuclease 1 assay and subsequent sequencing demonstrated that these embryos exhibited increased genome editing efficiencies (69%), although a high degree of mosaicism for the induced mutation was still observed. Single blastocyst-based cytochemical staining with fluorescently labeled isolectin BS-I-B 4 also confirmed this mosaicism. Thus, the development of a technique that avoids or reduces such mosaicism would be a key factor for efficient knock out piglet production via microinjection., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The aim of this study was a detailed analysis of the boar genotypes used in AI stations with an indication of their production capacity, including age and a precise analysis of their culling time and reason. The study included 334 boars: 81 Polish Large White (PLW), 108 Polish Landrace (PL), 49 Pietrain (P), 56 Duroc × Pietrain (D × P) and 40 Hampshire × Pietrain (H × P). Semen volume, spermatozoa concentration, total number of spermatozoa, number of motile spermatozoa, and number of insemination doses were analyzed. Quadratic regression was used to illustrate the selected sperm parameters at specific ages. Among all the studied boars the lowest motilities of spermatozoa were identified in white breeds PLW and PL, and the difference between motility extremes was 3.53% (P ≤ 0.01). The highest number of insemination doses were produced from D × P crossbreed boars: about 0.7 portions more compared to PL, 1.13 to PLW, 1.18 to H × P and 1.8 to P (all differences P ≤ 0.01). It has been shown in the case of ejaculate volume that for PLW and H × P boars the culling moment was far too early in terms of production capacity and differences were, respectively, 16.35 ml for PLW and 12.61 ml for H × P. Based on the developed regression equations, the earliest maximum number of motile sperm (73.82 × 10 9 ) was obtained by H × P crossbreed boars as early as at age 24 months. The highest values for this parameter were achieved, however, by other D × P crossbreed boars: 74.30 × 10 9  at the later age of 32 months. A consequence of the high number of motile sperm in young H × P boars was that the theoretical maximum value of the number of AI doses was produced as early as the 14th month (25.59 portions). Curves of similar shape were obtained for PL and D × P boars; the difference in maximal values was 0.54 portions in favor of crossbreeds, at a later age of 7 months. It was noted that for PLW and D × P boars the highest number of cullings were due to reduced demand (24.47% and even 31.19%, respectively). Other boars - PL, P and H × P - were most frequently culled because of low semen values. Regardless of the genotype, high survival probabilities (over 0.96) were noted for the age of 12 months. The highest probabilities (still over 0.96) were noted in the longest time period (up to 18 months) for P boars. The results of our study can be used in AI stations as reference points for the exploitation and culling of boars with different genotypes., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Recent decades have seen revolutionary advances in our understanding of cancer, with the molecular mechanisms underlying many human cancers now reasonably well understood. The challenge now is to bridge the gap between laboratory and clinical oncology, so these accomplishments can be translated into practical benefits for human patients. Although genetically modified mice are powerful tools to investigate the molecular basis of many human diseases, they are less suitable for many preclinical studies. Other animals can provide important complementary resources to aid the development, validation, and application of new medicines and procedures. Powerful methods of genetic engineering have now been extended to physiologically more relevant species, particularly the pig, opening the prospect of more representative, genetically defined, cancer models at human scale. Here, we provide a brief review of the genetically modified porcine cancer models described in the scientific literature., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Genetic selection in sows has substantially increased the total number of pigs born, but there has been an associated decrease in litter birth weight. Furthermore, irrespective of the total number of pigs born, a repeatable low litter birth weight phenotype has evolved in a subpopulation of mature sows, in which a high ovulation rate drives extremes of intrauterine crowding in early gestation and limits placental development: placental development continues to be compromised in later gestation and is associated with intrauterine growth restriction and a low litter birth weight. Furthermore, although contemporary commercial sows are increasingly resilient to the challenges of lactational catabolism in terms of the traditional measures of reproductive performance, gender-specific effects on early embryonic development of the subsequent litter are apparent and are likely linked to effects of sow catabolism on folliculogenesis and oocyte maturation. Recent transcriptomic studies that have helped elucidate some of the regulatory mechanisms mediating the evolving changes in the reproductive biology of contemporary commercial sows are summarized in the present review. However, the lack of clearly differentiated reproductive phenotypes among commercial sow populations is a problem for ongoing studies. Access to an unselected "wild-type" population with which to make comparisons, or the application of more standardized experimental approaches to mechanistic studies, may help advance the field of sow reproductive biology., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Use of artificial insemination (AI) for breeding pigs has been instrumental for facilitating global improvements in fertility, genetics, labor, and herd health. The establishment of AI centers for management of boars and production of semen has allowed for selection of boars for fertility and sperm production using in vitro and in vivo measures. Today, boars can be managed for production of 20 to 40 traditional AI doses containing 2.5 to 3.0 billion motile sperm in 75 to 100 mL of extender or 40 to 60 doses with 1.5 to 2.0 billion sperm in similar or reduced volumes for use in cervical or intrauterine AI. Regardless of the sperm dose, in liquid form, extenders are designed to sustain sperm fertility for 3 to 7 days. On farm, AI is the predominant form for commercial sow breeding and relies on manual detection of estrus with sows receiving two cervical or two intrauterine inseminations of the traditional or low sperm doses on each day detected in standing estrus. New approaches for increasing rates of genetic improvement through use of AI are aimed at methods to continue to lower the number of sperm in an AI dose and reducing the number of inseminations through use of a single, fixed-time AI after ovulation induction. Both approaches allow greater selection pressure for economically important swine traits in the sires and help extend the genetic advantages through AI on to more production farms., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Ovarian follicular cysts are anovulatory follicular structures that have been identified in sows and are known to cause infertility. The pathogenesis of follicular cysts remains poorly understood. Hormones play key roles in the formation and persistence of cysts. The hormone inhibin is a member of the TGF-β superfamily and is named for its negative regulation of FSH, another hormone that controls follicular recruitment and growth. In the present study, 48 sows with follicular cysts and 60 normal sows with no cysts were screened for mutations in the inhibin-α gene to examine the association of inhibin-α gene polymorphisms with the presence of follicular cysts. The results show that the c.-42G>A and c.3222G>A polymorphisms are significantly associated with follicular cysts and that sows with c.-42GG and c.3222GG genotypes have lower risk of developing cysts. Our findings may provide novel biological biomarkers and promising gene therapy candidates for follicular cyst formation in sows, which would greatly benefit pig breeding programs., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Important factors contributing to the well-known high mortality of piglets produced by SCNT are gross malformations of vital organs. The aim of the present retrospective study was to describe malformations found in cloned piglets, transgenic or not, dying or culled before weaning on Day 28. Large White (LW) embryos were transferred to 78 LW recipients, while 72 recipients received Göttingen embryos (67 transgenic and five not transgenic) and 56 received Yucatan embryos (43 transgenic and 13 not transgenic). Overall pregnancy rate was 76%, and there were more abortions in recipients with minipig embryos than in those with LW embryos (26% and 24% vs. 6%). Piglets (n = 815) were born from 128 sows with 6.5 ± 0.4 full-born piglets per litter. The overall rate of stillborn piglets was 21% of all born with the number of stillborn piglets ranging from one to nine in a litter. The mortality of the surviving piglets during the first month was 48%. Thus, altogether 58% of the full-born piglets died before weaning. In 87 of the 128 litters (68%), one to 12 of the piglets showed major or minor malformations. Malformations were found in 232 piglets (29.5% of all born). A single malformation was registered in 152 piglets, but several piglets showed two (n = 58) or more (n = 23) malformations (7.4% and 2.8% of all born, respectively). A significantly higher malformation rate was found in transgenic Göttingen and Yucatan piglets (32% and 46% of all born, respectively) than in nontransgenic LW (17%). There was a gender difference in the transgenic minipigs because male piglets had a higher rate of malformations (49.1%) than females (29.7%). The most common defects in the cloned piglets were in the digestive (12.2%), circulatory (9.4%), reproductive (11.3%), and musculoskeletal (9.1%) systems. Malformations of the musculoskeletal system were most frequent in Göttingen (16.3% vs. approximately 5.5% in the two other breeds), whereas abnormal cardiopulmonary systems were most frequent in Yucatan piglets (26.9% vs. 2.1% in LW and 5.3% in Göttingen). In conclusion, these results show that pig cloning results in a considerable loss of piglets and that many of these can be related to various malformations that all are also seen in noncloned piglets. Because approximately half of the cloned piglets still survive, even with eventual unknown minor malformations, use of pigs as models for human diseases is still realistic. However, continued efforts are needed to further reduce the level of malformations., (Copyright © 2015 Elsevier Inc. All rights reserved.)

It is well known that puberty has a strong impact on oocyte developmental competence in vitro; however, the reason for this phenomenon at the cellular level has not been clarified yet. It is hypothesized that cytoplasmic maturation is responsible for oocyte quality and may be impaired in prepubertal gilts. Previous results on mitochondrial DNA copy number and mitochondria and cortical granule distribution showed that cytoplasmic maturation is a complex trait and should include multithreaded analysis. Therefore, the aim of the present research was to analyze the transcript abundance of developmentally important genes (BMP15, GDF9, GSTA2, ATP5A1, EEF1A1, BAX, BCL2) followed by investigation of the glutathione and apoptosis level in oocytes of prepubertal and cyclic gilts. We found differences in relative transcript abundance of BMP15 and GDF9 genes after IVM, whereas different concentrations of glutathione were noted before IVM (5.3 vs. 2.9 pmol, respectively). The glutathione level was equalized after IVM (10.3 vs. 9.1 pmol), whereas the incidence of apoptosis remained similar before (3.9% vs. 1.1%) and after IVM (4.5% vs. 1.9%) being higher in prepubertal oocytes. A potential impact of gilt puberty on oocyte quality has been therefore masked by the significant effect of IVM. Because the maternal effect genes, BMP15 and GDF9, play key roles in regulation of folliculogenesis and oocyte-cumulus interaction, their upregulation in oocytes of cyclic gilts may result in increased developmental competence. On the basis of findings from this and our previous research, we suggest that the reduced quality of oocytes from prepubertal females is a complex phenomenon and is not related to a single marker trait., (Copyright © 2015 Elsevier Inc. All rights reserved.)

DNA active demethylation is an important epigenetic phenomenon observed in porcine zygotes, yet its molecular origins are unknown. Our results show that 5-methylcytosine (5mC) converts into 5-hydroxymethylcytosine (5hmC) during the first cell cycle in porcine in vivo fertilization (IVV), IVF, and SCNT embryos, but not in parthenogenetically activated embryos. Expression of Ten-Eleven Translocation 1 (TET1) correlates with this conversion. Expression of 5mC gradually decreases until the morula stage; it is only expressed in the inner cell mass, but not trophectoderm regions of IVV and IVF blastocysts. Expression of 5mC in SCNT embryos is ectopically distinct from that observed in IVV and IVF embryos. In addition, 5hmC expression was similar to that of 5mC in IVV cleavage-stage embryos. Expression of 5hmC remained constant in IVF and SCNT embryos, and was evenly distributed among the inner cell mass and trophectoderm regions derived from IVV, IVF, and SCNT blastocysts. Ten-Eleven Translocation 3 was highly expressed in two-cell embryos, whereas TET1 and TET2 were highly expressed in blastocysts. These data suggest that TET1-catalyzed 5hmC may be involved in active DNA demethylation in porcine early embryos. In addition, 5mC, but not 5hmC, participates in the initial cell lineage specification in porcine IVV and IVF blastocysts. Last, SCNT embryos show aberrant 5mC and 5hmC expression during early porcine embryonic development., (Copyright © 2014 Elsevier Inc. All rights reserved.)