Long-term efficacy of encapsulated xenogeneic islet transplantation: Impact of encapsulation techniques and donor genetic traits.

AIMS/INTRODUCTION: To investigate the long-term efficacy of various encapsulated xenogeneic islet transplantation, and to explore the impact of different donor porcine genetic traits on islet transplantation outcomes. MATERIALS AND METHODS: Donor porcine islets were obtained from wild-type, α1,3-galactosyltransferase knockout (GTKO) and GTKO with overexpression of membrane cofactor protein genotype. Naked, alginate, alginate-chitosan (AC), alginate-perfluorodecalin (A-PFD) and AC-perfluorodecalin (AC-PFD) encapsulated porcine islets were transplanted into diabetic mice. RESULTS: In vitro assessments showed no differences in the viability and function of islets across encapsulation types and donor porcine islet genotypes. Xenogeneic encapsulated islet transplantation with AC-PFD capsules showed the most favorable long-term outcomes, maintaining normal blood glucose levels for 180 days. A-PFD capsules showed comparable results to AC-PFD capsules, followed by AC capsules and alginate capsules. Conversely, blood glucose levels in naked islet transplantation increased to >300 mg/dL within a week after transplantation. Naked islet transplantation outcomes showed no improvement based on donor islet genotype. However, alginate or AC capsules showed delayed increases in blood glucose levels for GTKO and GTKO with overexpression of membrane cofactor protein porcine islets compared with wild-type porcine islets. CONCLUSION: The AC-PFD capsule, designed to ameliorate both hypoxia and inflammation, showed the highest long-term efficacy in xenogeneic islet transplantation. Genetic modifications of porcine islets with GTKO or GTKO with overexpression of membrane cofactor protein did not influence naked islet transplantation outcomes, but did delay graft failure when encapsulated.

The AIRN lncRNA is imprinted and paternally expressed in pigs.

Genomic imprinting plays critical roles during the development of mammalian species and underlying epigenetic mechanisms frequently involve long non-coding RNAs (lncRNAs). The paternal transcription of the antisense Igf2r RNA noncoding (Airn) is responsible for paternal silencing of the mouse insulin-like growth factor 2 receptor (Igf2r) gene and maternal Igf2r expression. Although the corresponding maternal DNA methylation imprint is conserved in humans and pigs, the orthologous AIRN lncRNA has been identified in humans but not in pigs. Here, we aimed to examine imprinted allelic expression of the porcine AIRN lncRNA along with a corresponding differentially methylated region (DMR) and to analyze allelic expression of AIRN and IGF2R in pigs. By comparing parthenogenetic and control porcine embryos, we identified a maternally methylated DMR and a significantly higher expression of AIRN lncRNA in control embryos (P < 0.05) indicating its paternal expression. Further analyses revealed that the expression of AIRN lncRNA was enriched in the pig brain and its subregions, and it was monoallelically expressed; whereas, IGF2R was expressed biallelically suggesting an absence of allele-specific transcriptional regulation. Our findings will lead to further investigations into the role of the imprinted porcine AIRN lncRNA during pig development.

Genomic imprinting is important for the development of mammals and long non-coding RNAs are often involved in the imprinting process. In mice, Airn encodes a long non-coding RNA that is imprinted, and therefore, transcribed only from the paternal allele. This paternal transcription of Airn interferes with the adjacent Igf2r promoter, leading to maternal expression of Igf2r. In pigs, the orthologous AIRN has not been identified as well as its imprinting. In the current study, we report porcine AIRN and allelic expression of both AIRN and IGF2R using our parthenogenetic embryo models and various normal pig tissues.

Retrospective study using biosensor data of a milking Holstein cow with jejunal haemorrhage syndrome.

Jejunal haemorrhage syndrome (JHS) is a sporadic and fatal enterotoxaemic disease in dairy cows associated with acute development and poor prognosis despite treatment. A 5-year-old Holstein cow with no reported pregnancy, three calving numbers, and 303 days in milk presented with hypothermia, discomfort, and inappetence. Anaemia, dehydration, faeces with blood clots, and absence of rumen and bowel movements were observed. We identified the presence of neutrophilia, hyperglycaemia, hypoproteinaemia, azotaemia, hyperlactatemia, hypocalcaemia, hypermagnesemia, hypokalaemia, and hypochloraemia through blood analyses. Necropsy and histopathologic examination revealed a dilated bluish-purple jejunum, blood clots within the jejunum, neutrophil infiltration into the submucosa of the jejunum, and vascular necrosis. Retrospective examination revealed extraordinary patterns of rumination time, activity, rumen mobility, and rumen temperature using biosensors and decreased milk yield. The abnormalities in the affected cow were detected before recognition by farm workers. To the best of our knowledge, this is the first report to examine data from biosensors in a cow with JHS. Our findings suggest that using biometric data may help understand the development of JHS.

Imprinting at the KBTBD6 locus involves species-specific maternal methylation and monoallelic expression in livestock animals.

BACKGROUND: The primary differentially methylated regions (DMRs) which are maternally hypermethylated serve as imprinting control regions (ICRs) that drive monoallelic gene expression, and these ICRs have been investigated due to their implications in mammalian development. Although a subset of genes has been identified as imprinted, in-depth comparative approach needs to be developed for identification of species-specific imprinted genes. Here, we examined DNA methylation status and allelic expression at the KBTBD6 locus across species and tissues and explored potential mechanisms of imprinting. RESULTS: Using whole-genome bisulfite sequencing and RNA-sequencing on parthenogenetic and normal porcine embryos, we identified a maternally hypermethylated DMR between the embryos at the KBTBD6 promoter CpG island and paternal monoallelic expression of KBTBD6. Also, in analyzed domesticated mammals but not in humans, non-human primates and mice, the KBTBD6 promoter CpG islands were methylated in oocytes and/or allelically methylated in tissues, and monoallelic KBTBD6 expression was observed, indicating livestock-specific imprinting. Further analysis revealed that these CpG islands were embedded within transcripts in porcine and bovine oocytes which coexisted with an active transcription mark and DNA methylation, implying the presence of transcription-dependent imprinting. CONCLUSIONS: In this study, our comparative approach revealed an imprinted expression of the KBTBD6 gene in domesticated mammals, but not in humans, non-human primates, and mice which implicates species-specific evolution of genomic imprinting.

Effects of heat stress on conception in Holstein and Jersey cattle and oocyte maturation in vitro.

Korea, located in East Asia in the northern hemisphere, is experiencing severe climate changes. Specifically, the heat stress caused by global warming is negatively affecting the dairy sector, including milk production and reproductive performance, as the major dairy cattle Holstein-Friesian is particularly susceptible to heat stress. Here, we collected artificial insemination and pregnancy data of the Holstein and the Jersey cows from a dairy farm from 2014 to 2021 and analyzed the association between the conception rate and the temperature-humidity index, calculated using the data from the closest official weather station. As the temperature-humidity index threshold increased, the conception rate gradually decreased. However, this decrease was steeper in the Holstein breed than in the Jersey one at a temperature-humidity index threshold of 75. To evaluate the effects of heat stress on the oocyte quality, we examined the nuclear and cytoplasmic maturation of Holstein (n = 158, obtained from six animals) and Jersey oocytes (n = 123, obtained from six animals), obtained by ovum pick-up. There were no differences in the nuclear maturation between the different conditions (heat stress: 40.5°C, non- heat stress: 37.5°C) or breeds, although the Holstein oocytes seemed to have a lower metaphase II development (p = 0.0521) after in vitro maturation under heat stress conditions. However, we found that the Holstein metaphase II oocytes exposed to heat stress presented more reactive oxygen species and a peripheral distribution of the mitochondria, compared to those of the Jersey cattle. Here, we show that weather information from local meteorological stations can be used to calculate the temperature-humidity index threshold at which heat stress influences the conception rate, and that the Jersey cows are more tolerant to heat stress in terms of their conception rate at a temperature-humidity index over 75. The lower fertility of the Holstein cows is likely attributed to impaired cytoplasmic maturation induced by heat stress. Thus, the Jersey cows can be a good breed for the sustainability of dairy farms for addressing climate changes in South Korea, as they are more resistant to hyperthermia.

A novel testis-enriched gene, Samd4a, regulates spermatogenesis as a spermatid-specific factor.

Spermatogenesis is the highly orchestrated process involving expression of a series of testicular genes. Testis-enriched genes are critical for cellular processes during spermatogenesis whose disruption leads to impaired spermatogenesis and male infertility. Nevertheless, among poorly investigated testicular genes are the mouse Samd4a and human SAMD4A which were identified in the current study as novel testis-enriched genes through transcriptomic analyses. In particular, as orthologous alternative splicing isoforms, mouse Samd4a E-form and human SAMD4AC-form containing the SAM domain were specific to testes. Western blot analyses revealed that the murine SAMD4AE-form was predominantly found in the testis. Analyses on GEO2R and single-cell RNA-seq datasets revealed that the Samd4a/SAMD4A expression was enriched in spermatids among various types of cells in adult testes. To investigate in vivo functions of Samd4a, Samd4a knockout mice were generated using the CRISPR/Cas9 system. The Samd4a deficiency resulted in lower testis weight, absence of elongated spermatids, and an increased number of apoptotic cells. Profiling of gene expression in human testis samples revealed that the SAMD4A expression was comparable between obstructive azoospermia patients and normal controls, but significantly lowered in nonobstructive azoospermia (NOA) patients. Among three subgroups of NOA, pre-meiotic arrest (NOA-pre), meiotic arrest (NOA-mei), and post-meiotic arrest (NOA-post), expression level of SAMD4A was higher in the NOA-post than the NOA-mei, but there was no difference between the NOA-pre and NOA-mei. The current studies demonstrated spermatid stage-specific expression of Samd4a/SAMD4A, and impairment of the late stages of spermatogenesis by disruption of the mouse Samd4a gene. These data suggest that Samd4a/SAMD4A plays an essential role in normal spermatogenesis, and SAMD4A, as a spermatid specific marker, can be used for subcategorizing NOA patients. Further understanding the molecular role of SAMD4A will advance our knowledge on genetic regulations in male infertility.

Loss of Monoallelic Expression of IGF2 in the Adult Liver Via Alternative Promoter Usage and Chromatin Reorganization.

In mammals, genomic imprinting operates via gene silencing mechanisms. Although conservation of the imprinting mechanism at the H19/IGF2 locus has been generally described in pigs, tissue-specific imprinting at the transcript level, monoallelic-to-biallelic conversion, and spatio-temporal chromatin reorganization remain largely uninvestigated. Here, we delineate spatially regulated imprinting of IGF2 transcripts, age-dependent hepatic mono- to biallelic conversion, and reorganization of topologically associating domains at the porcine H19/IGF2 locus for better translation to human and animal research. Whole-genome bisulfite sequencing (WGBS) and RNA sequencing (RNA-seq) of normal and parthenogenetic porcine embryos revealed the paternally hypermethylated H19 differentially methylated region and paternal expression of IGF2. Using a polymorphism-based approach and omics datasets from chromatin immunoprecipitation sequencing (ChIP-seq), whole-genome sequencing (WGS), RNA-seq, and Hi-C, regulation of IGF2 during development was analyzed. Regulatory elements in the liver were distinguished from those in the muscle where the porcine IGF2 transcript was monoallelically expressed. The IGF2 transcript from the liver was biallelically expressed at later developmental stages in both pigs and humans. Chromatin interaction was less frequent in the adult liver compared to the fetal liver and skeletal muscle. The duration of genomic imprinting effects within the H19/IGF2 locus might be reduced in the liver with biallelic conversion through alternative promoter usage and chromatin remodeling. Our integrative omics analyses of genome, epigenome, and transcriptome provided a comprehensive view of imprinting status at the H19/IGF2 cluster.

Development of a Chimeric Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)-2 Vaccine Candidate Expressing Hypo-Glycosylated Glycoprotein-5 Ectodomain of Korean Lineage-1 Strain.

Vaccination is a practical method to provide protection against porcine reproductive and respiratory syndrome virus (PRRSV), but current PRRSV vaccines show limited efficacy against divergent field strains. Lineage 1 PRRSV includes virulent strains such as NADC30 and MN184 and now has become one of the most prevalent viruses in Korea. Accordingly, there is an urgent need to develop a new vaccine for Korean lineage-1 strains. In this study, a vaccine candidate against Korean lineage-1 PRRSV, vCSL1-GP5-N33D, was developed by reverse genetics technology. vCSL1-GP5-N33D was designed as a hypo-glycosylated chimeric virus containing the glycoprotein 5 ectodomain region of the Korean lineage-1 wild-type strain. An inactivated vaccine of vCSL1-GP5-N33D was applied to a PRRS-endemic farm and elicited high serum virus neutralization (SVN) antibody titers. The vaccinated group induced SVN antibody titers of 4.40 (log2) ± 2.46, which were approximately 2-fold higher than those of the negative control at 8-weeks post-vaccination. Moreover, 60% of pigs in the vaccinated group displayed SVN antibody titers of ≥5 (log2), while none of the pigs in the negative control exhibited SVN antibody titers of ≥5 (log2). The overall results of the animal experiment suggest that the vCSL1-GP5-N33D inactivated vaccine is a promising vaccine candidate.

Genomic Imprinting at the Porcine DIRAS3 Locus.

The epigenetic mechanisms underlying genomic imprinting include DNA methylation and monoallelic expression of genes in close proximity. Although genes imprinted in humans and mice have been widely characterized, there is a lack of detailed and comprehensive studies in livestock species including pigs. The purpose of this study was to investigate a detailed methylation status and parent-of-origin-specific gene expression within the genomic region containing an underexamined porcine DIRAS3 locus. Through whole-genome bisulfite sequencing (WGBS) and RNA sequencing (RNA-seq) of porcine parthenogenetic embryos and analyses of public RNA-seq data from adult pigs, DNA methylation and monoallelic expression pattern were investigated. As a result, maternal hypermethylation at the DIRAS3 locus and hypothalamus-specific and monoallelic expression of the DIRAS3 gene were found in pigs. In conclusion, the findings from this study suggest that the presence of maternal hypermethylation, or imprints, might be maintained and related to monoallelic expression of DIRAS3 during pig development.

N-acetyl-L-cysteine Improves the Developmental Competence of Bovine Oocytes and Embryos Cultured In Vitro by Attenuating Oxidative Damage and Apoptosis.

Oxidative stress has been suggested to negatively affect oocyte and embryo quality and developmental competence, resulting in failure to reach full term. In this study, we investigated the effect of N-acetyl-L-cysteine (NAC), a cell-permeating antioxidant, on developmental competence and the quality of oocytes and embryos upon supplementation (0.1-10 mM) in maturation and culture medium in vitro using slaughterhouse-derived oocytes and embryos. The results show that treating oocytes with 1.0 mM NAC for 8 h during in vitro maturation attenuated the intracellular reactive oxygen species (ROS) (p < 0.05) and upregulated intracellular glutathione levels (p < 0.01) in oocytes. Interestingly, we found that NAC affects early embryonic development, not only in a dose-dependent, but also in a stage-specific, manner. Significantly (p < 0.05) decreased cleavage rates (90.25% vs. 81.46%) were observed during the early stage (days 0-2), while significantly (p < 0.05) increased developmental rates (38.20% vs. 44.46%) were observed during the later stage (from day 3) of embryonic development. In particular, NAC supplementation decreased the proportion of apoptotic blastomeres significantly (p < 0.05), resulting in enhanced hatching capability and developmental rates during the in vitro culture of embryos. Taken together, our results suggest that NAC supplementation has beneficial effects on bovine oocytes and embryos through the prevention of apoptosis and the elimination of oxygen free radicals during maturation and culture in vitro.

Genomic Imprinting at the Porcine PLAGL1 Locus and the Orthologous Locus in the Human.

Implementation of genomic imprinting in mammals often results in cis-acting silencing of a gene cluster and monoallelic expression, which are important for mammalian growth and function. Compared with widely documented imprinting status in humans and mice, current understanding of genomic imprinting in pigs is relatively limited. The objectives of this study were to identify DNA methylation status and allelic expression of alternative spliced isoforms at the porcine PLAGL1 locus and assess the conservation of the locus compared to the orthologous human locus. DNA methylome and transcriptome were constructed using porcine parthenogenetic or biparental control embryos. Using methylome, differentially methylated regions between those embryos were identified. Alternative splicing was identified by differential splicing analysis, and monoallelic expression was examined using single nucleotide polymorphism sites. Moreover, topological boundary regions were identified by analyzing CTCF binding sites and compared with the boundary of human orthologous locus. As a result, it was revealed that the monoallelic expression of the PLAGL1 gene in porcine embryos via genomic imprinting was maintained in the adult stage. The porcine PLAGL1 locus was largely conserved in regard to maternal hypermethylation, tissue distribution of mRNA expression, monoallelic expression, and biallelic CTCF-binding, with exceptions on transcript isoforms produced by alternative splicing instead of alternative promoter usage. These findings laid the groundwork for comparative studies on the imprinted PLAGL1 gene and related regulatory mechanisms across species.

Research Note: Increased myostatin expression and decreased expression of myogenic regulatory factors in embryonic ages in a quail line with muscle hypoplasia.

Genetic selection of quail for a low body weight for more than 80 generations established a low-weight (LW) Japanese quail line that has been previously characterized to have a muscle hypoplasia phenotype. The aim of this study is to investigate the relationship of temporal expression levels of myostatin (Mstn) and myogenic regulatory factors (MRFs) with hypoplastic muscle growth in the LW line. During embryonic day (E) 13 to 15, gain of embryo weight was 2-fold lower (P < 0.001) in the LW line than that in the random bred control (CON). Gains in body weight and pectoralis muscle weight from hatch to posthatch day (P) 28 were also significantly lower (P < 0.01) in the LW line but increased by 4-fold (P < 0.05) during P42 to P75. PCR analysis showed that expression levels of Mstn were greater in the LW at embryonic stage (E12 to E14, P < 0.05), but there was no difference after hatch. In addition, expression levels of Pax7 and myogenin (MyoG) at E12 were 23-fold (P < 0.05) and 3.4-fold (P < 0.05) lesser in the LW line, respectively. At E14, expression of Pax3, Pax7, and MyoG gene was 3.5-fold (P < 0.05), 6.5-fold (P = 0.065), and 4.4-fold (P < 0.01) less than that in the CON. Taken together, high expression levels of Mstn and low expression of MRFs during embryonic stages can be associated with development of muscle hypoplasia and delayed muscle growth in the LW quail line. These data provide evidence that genetic selection for a low body weight resulting in an avian model with muscle hypoplasia has altered the expression profiles of myogenic factors.

Effect of sex-specific differences on function of induced hepatocyte-like cells generated from male and female mouse embryonic fibroblasts.

BACKGROUND: The liver is one of the vital organs involved in detoxification and metabolism. The sex-based differences between the functionality of male and female liver have been previously reported, i.e., male's liver are good in alcohol clearance and lipid metabolism, while female's liver are better in cholesterol metabolism. To date, studies on novel drug toxicity have not considered the sex-specific dimorphic nature of the liver. However, the use of hepatocyte-like cells to treat liver diseases has increased recently. METHODS: Mouse embryos were isolated from a pregnant female C57BL/6J mouse where mouse embryonic fibroblasts (MEFs) were isolated from back skin tissue of each embryo. MEFs were transduced with human transcription factors hHnf1α, hHnf4α, and hFoxa3 using the lentiviral system. The transduced MEFs were further treated with hepatocyte-conditioned media followed by its analysis through RT-qPCR, immunofluorescence, functional assays, and finally whole-transcriptome RNA sequencing analysis. For in vivo investigation, the mouse hepatocyte-like cells (miHep) were transplanted into CCl4-induced acute liver mouse model. RESULTS: In this study, we evaluated the sex-specific effect of miHep induced from male- and female-specific mouse embryonic fibroblasts (MEFs). We observed miHeps with a polygonal cytoplasm and bipolar nucleus and found that male miHeps showed higher mHnf4a, albumin secretion, and polyploidization than female miHeps. Transcriptomes from miHeps were similar to those from the liver, especially for Hnf4a of male miHeps. Male Cyps were normalized to those from females, which revealed Cyp expression differences between liver and miHeps. In both liver and miHeps, Cyp 4a12a and Cyp 4b13a/2b9 predominated in males and females, respectively. After grafting of miHeps, AST/ALT decreased, regardless of mouse sex. CONCLUSION: In conclusion, activation of endogenic Hnf4a is important for generation of successful sex-specific miHeps; furthermore, the male-derived miHep exhibits comparatively enhanced hepatic features than those of female miHep.

A chimeric porcine reproductive and respiratory syndrome virus (PRRSV)-2 vaccine is safe under international guidelines and effective both in experimental and field conditions.

Vaccination is currently the most effective strategy to control porcine reproductive and respiratory syndrome (PRRS). New-generation PRRS vaccines are required to be safe and broadly cross-protective. We have recently created the chimeric PRRS virus K418DM which proved to be a good vaccine candidate under field conditions. In the present study, we designed safety and efficacy tests under experimental and field conditions for further evaluation of K418DM1.1, a plaque-purified K418DM. In the homologous challenge study, K418DM1.1 induced high serum virus neutralization (SVN) antibody titers (i.e., 4.2 log2 ± 1.7) at 21 days post-challenge (dpc) and provided protection as demonstrated by the significantly lower levels of viremia at 3 and 7 dpc and significantly lower microscopic lung lesion scores compared to the unvaccinated group. K418DM1.1 was also protective in the heterologous challenge study, with vaccinated pigs showing significantly lower levels of viremia at 14 dpc compared to the unvaccinated pigs. A field study was performed to evaluate the efficacy of K418DM1.1 against heterologous exposure and vaccinated pigs presented significantly lower viremia than unvaccinated pigs. According to the safety test for the examination of virulence reversion, no infectivity was observed in tissue homogenate filtrate both in the vaccinated and comingled groups. Thus, the risk of virulence, as well as transmission, appeared negligible. These overall results indicate that K418DM1.1 is a good vaccine candidate based on its safety and protective efficacy.

Isolation and characterization of cultured chicken oviduct epithelial cells and in vitro validation of constructed ovalbumin promoter in these cells.

OBJECTIVE: Transgenic hens hold a great promise to produce various valuable proteins. Through virus transduction into stage X embryo, the transgene expression under the control of constructed chicken ovalbumin promoters has been successfully achieved. However, a validation system that can evaluate differently developed ovalbumin promoters in in vitro, remains to be developed. METHODS: In the present study, chicken oviduct epithelial cells (cOECs) were isolated from oviduct tissue and shortly cultured with keratinocyte complete medium supplemented with chicken serum. The isolated cells were characterized with immunofluorescence, western blot, and flow cytometry using oviduct-specific marker. Chicken mutated ovalbumin promoter (Mut-4.4-kb-pOV) was validated in these cells using luciferase reporter analysis. RESULTS: The isolated cOECs revealed that the oviduct-specific marker, ovalbumin protein, was clearly detected by immunofluorescence, western blot, and flow cytometry analysis revealed that approximately 79.40% of the cells contained this protein. Also, luciferase reporter analysis showed that the constructed Mut-4.4-kb-pOV exhibited 7.1-fold (p<0.001) higher activity in the cOECs. CONCLUSION: Collectively, these results demonstrate the efficient isolation and characterization of cOECs and validate the activity of the constructed ovalbumin promoter in the cultured cOECs. The in vitro validation of the recombinant promoter activity in cOECs can facilitate the production of efficient transgenic chickens for potential use as bioreactors.

The Landscape of Genomic Imprinting at the Porcine SGCE/PEG10 Locus from Methylome and Transcriptome of Parthenogenetic Embryos.

In mammals, imprinted genes often exist in the form of clusters in specific chromosome regions. However, in pigs, genomic imprinting of a relatively few genes and clusters has been identified, and genes within or adjacent to putative imprinted clusters need to be investigated including those at the SGCE/PEG10 locus. The objective of this study was to, using porcine parthenogenetic embryos, investigate imprinting status of genes within the genomic region spans between the COL1A2 and ASB4 genes in chromosome 9. Whole-genome bisulfite sequencing (WGBS) and RNA sequencing (RNA-seq) were conducted with normal and parthenogenetic embryos, and methylome and transcriptome were analyzed. As a result, differentially methylated regions (DMRs) between the embryos were identified, and parental allele-specific expressions of the SGCE and PEG10 genes were verified. The pig imprinted interval was limited between SGCE and PEG10, since both the COL1A2 and CASD1 genes at the centromere-proximal region and the genes between PPP1R9A and ASB4 toward the telomere were non-imprinted and biallelically expressed. Consequently, our combining analyses of methylome, transcriptome, and informative polymorphisms revealed the boundary of imprinting cluster at the SGCE/PEG10 locus in pig chromosome 9 and consolidated the landscape of genomic imprinting in pigs.

Low expression of mitofusin 1 is associated with mitochondrial dysfunction and apoptosis in porcine somatic cell nuclear transfer embryos.

Mitochondria are necessary for the transition from oocyte to embryo and for early embryonic development. Mitofusin 1 is the main mediator of mitochondrial fusion and homeostasis. We investigated Mitofusin 1 expression levels in porcine somatic cell nuclear transfer (SCNT) embryos. The rate of blastocyst formation in SCNT embryos was reduced significantly compared with that of parthenogenetic activation embryos. SCNT embryos showed significantly decreased Mitofusin 1 expression and mitochondrial membrane potential, while exhibiting increased reactive oxygen species and apoptosis. Mitochondrial functional changes were observed in the SCNT embryos and may be correlated with low levels of Mitofusin 1 to negatively affect development.

Research Note: Association of temporal expression of myostatin with hypertrophic muscle growth in different Japanese quail lines.

Myostatin (MSTN) negatively regulates in muscle growth and development. Among alternative splicing isoforms of avian MSTN, MSTN-A has antimyogenic activities and MSTN-B functions as a promyogenic factor. In this study, different lines of Japanese quail were used: a random bred control (RBC) and a heavy weight (HW) quail line with muscle hypertrophy. The objectives of the current study are to compare temporal expression of the MSTN isoforms in pectoralis major muscle (PM) between 2 quail lines and to relate MSTN expression with temporal changes in muscle growth and total amounts of DNA in PM. Gains of body weight (BW) and PM weight were greater until posthatch day (D) 28 (P < 0.001), and the fold increases in total DNA contents of PM were greater in the HW line compared with the RBC line during D7 to D28 (P < 0.05). PCR analysis showed that MSTN-A expression was greater at 14 D (E14) of embryonic age (P < 0.01), D7 (P = 0.052), and D14 (P < 0.01) in the RBC line compared with the HW line. At D28 and D75, expression of MSTN-A was greater in the HW line compared with the RBC line (P < 0.05). MSTN-B expression was barely detectable from E14 to D14 and measurable from D28 to D75 in the muscle of both lines. Ratios of the MSTN-B/-A form ranging from 0.15 to 0.29 indicate a minor expression of the B form. Taken together, the lesser expression levels of MSTN-A at E14, D7, and D14 are associated with the fast growth of PM, and greater MSTN-A expression at D28 and D75 are associated with a slowdown of PM growth in the HW line. These data indicate a negative association of MSTN expression with PM growth and provide a scientific basis for potential usage of MSTN expression as a selection marker for greater muscle growth in poultry.

The protective effect of Leucosporidium-derived ice-binding protein (LeIBP) on bovine oocytes and embryos during vitrification.

Ice-binding proteins (IBPs) facilitate organism survival under extreme conditions by inhibiting thermal hysteresis and ice recrystallization. IBPs have been widely used as cryoprotectants to cryopreserve mammalian gametes and embryos. In the present study, we evaluated the protective effects of an Arctic yeast, Leucosporidium sp. AY30 derived ice-binding protein (LeIBP), on the vitrification of bovine metaphase II (MII) oocytes and embryos. When oocytes and embryos were frozen using the two-step vitrification method, the survival rate was significantly increased in the presence of LeIBP. The LeIBP supplementation decreased the levels of intracellular reactive oxygen species (ROS) and enhanced mitochondrial functions in the vitrified-warmed oocytes. Furthermore, LeIBP improved the developmental potential and suppressed apoptosis of the embryos derived from vitrified-warmed oocytes. Collectively, these data indicate that LeIBP can be used as a promising cryoprotectant to prevent cryoinjury during vitrification in bovine oocytes.

Effects of Leucosporidium-derived ice-binding protein (LeIBP) on bull semen cryopreservation.

We examined the effect of ice-binding protein derived from Leucosporidium (LeIBP) on the cryopreservation of bull semen and compared it with that derived from previously reported Antifreeze Protein III (AFPIII). Six concentrations of LeIBP (10-1  ~ 104  µg/ml) and AFPIII (10-1  ~ 104  µg/ml) were added to the bull semen extender, respectively. Sperm kinematic parameters were measured to examine sperm toxicity and cryopreserved sperm quality. Measures of antioxidant activity such as superoxide dismutase (SOD), reduced glutathione/oxidative glutathione (GSH/GSSG), and total antioxidant capacity (TAC) were analysed to identify the effect of LeIBP on sperm quality. In addition, sperm viability was analysed using a flow cytometer and fluorescence microscope by SYBR14/PI staining. The results showed that the LeIBP groups (0.1, 1 and 10 µg/ml) were less toxic, and the quality of the sperm were dramatically improved in the extenders containing 0.1 µg/ml LeIBP among concentrations of LeIBP and AFPIII. The SOD activity of LeIBP was greater than that of AFPIII and control. In addition, sperm viability was enhanced in the LeIBP-treated group. In summary, LeIBP is a useful cryoprotective adjuvant for bull sperm cryopreservation, and the most efficient concentration of LeIBP is 0.1 µg/ml.