Vitrified before and after genome editing via electroporation.

BACKGROUND: Cryopreservation of bovine zygotes allows for a flexible schedule of genome editing via electroporation. However, vitrification-induced cell membrane damage may not only affect embryonic development but also genome mutation. OBJECTIVE: To investigate the effects of vitrification of zygotes before and after electroporation treatments on the development and genome mutation of bovine presumptive zygotes. MATERIALS AND METHODS: In vitro-derived bovine zygotes were electroporated with the CRISPR/Cas9 system immediately (Vitrified-EP) or 2 h after incubation (Vitrified-2h-EP) following vitrification and warming, or electroporated before vitrification (EP-vitrified). RESULTS: The development rates of vitrified-warmed zygotes were significantly lower (p < 0.05) than those of control zygotes that were not vitrified. Moreover, no differences were observed in the mutation rates and mutation efficiency of the blastocysts resulting from electroporated zygotes, irrespective of the timing of electroporation treatment. CONCLUSION: Our results suggest that vitrification before and after electroporation treatments does not affect the genome editing of zygotes.

Effects of Antifreeze Protein Supplementation on the Development of Porcine Morulae Stored at Hypothermic Temperatures.

BACKGROUND: Short-term storage is valuable method to reuse manipulated embryos. OBJECTIVE: The present study evaluated the effects of antifreeze protein (AFP) supplementation on the quality and development of in vitro-produced porcine morulae after short-term storage (24 h). MATERIALS AND METHODS: The morulae were stored with various concentrations of AFP type III for 24 h at 5, 15 and 25C. RESULTS: Supplementation of AFP type III (1.0 microgram per mL) improved the developmental competence of embryos stored at 25C. The proportions of DNA-fragmented nuclei in the blastocysts did not differ between the embryos stored at 25C and the control embryos without storage treatment. However, the developmental competence of embryos stored at hypothermic temperatures decreased relative to that of the control embryos. CONCLUSION: Supplementation of AFP type III (1.0 microgram per mL) maintained the quality of embryos stored at 25C, but did not have beneficial effects on the development of embryos stored at hypothermic temperatures.

EFFECTS OF ORVUS ES PASTE ON THE MOTILITY AND VIABILITY OF YAK (BOS GRUNNIENS) EPIDIDYMAL AND EJACULATED SPERMATOZOA AFTER FREEZING AND THAWING.

BACKGROUND: The addition of the detergent Orvus ES Paste (OEP) to semen freezing extenders has been observed to improve the post-thaw survival and longevity of spermatozoa from various species but has never been evaluated for yak spermatozoa. OBJECTIVE: This study evaluated the effects of OEP on the post-thaw motility and viability of epididymal and ejaculated yak spermatozoa. MATERIALS AND METHODS: Semen samples were frozen and thawed in semen freezing extender supplemented with 0 %, 0.375 %, 0.75 % or 1.5 % OEP. The motility and viability of frozen-thawed spermatozoa were evaluated before and after 3 h of incubation. RESULTS: The addition of 0.75 % OEP to the freezing extender significantly improved the mean motility and viability values of both the epididymal and ejaculated spermatozoa immediately after thawing, but the beneficial effects on motility disappeared after 3h of incubation. CONCLUSION: Our findings indicate that the addition of 0.75 % OEP is effective for the preservation of yak spermatozoa.

Melatonin Supplementation During In Vitro Maturation and Development Supports the Development of Porcine Embryos.

Melatonin has been reported to improve the in vitro development of embryos in some species. This study was conducted to investigate the effect of melatonin supplementation during in vitro maturation (IVM) and development culture on the development and quality of porcine embryos. In the first experiment, when the in vitro fertilized embryos were cultured with different concentrations of melatonin (0, 10, 25 and 50 ng/ml) for 8 days, the blastocyst formation rate of embryos cultured with 25 ng/ml melatonin (10.7%) was significantly increased (p < 0.05) compared to the control embryos cultured without melatonin (4.2%). The proportion of DNA-fragmented nuclei in blastocysts derived from embryos cultured with 50 ng/ml melatonin was significantly lower (p < 0.05) than that of embryos cultured without melatonin (2.1% vs 7.2%). In the second experiment, when oocytes were cultured in the maturation medium supplemented with different concentrations of melatonin (0, 10, 25 and 50 ng/ml), fertilized and then cultured with 25 ng/ml melatonin for 8 days, there were no significant differences in the rates of cleavage and blastocyst formation among the groups. However, the proportions (2.7-5.4%) of DNA-fragmented nuclei in blastocysts derived from oocytes matured with melatonin were significantly decreased (p < 0.05) compared to those (8.9%) from oocytes matured without melatonin, irrespective of the concentration of melatonin. Our results suggest that supplementation of the culture media with melatonin (25 ng/ml) during IVM and development has beneficial effects on the developmental competence and quality of porcine embryos.

Chelation of trace elements in preservation medium influences the quality of boar spermatozoa during liquid preservation at 5°C for 4 weeks.

BACKGROUND: The addition of a metal chelator, ethylenediaminetetraacetic acid (EDTA), to semen extender has the purpose of capturing trace element ions. OBJECTIVE: This study was conducted to evaluate the effects of EDTA on the quality and in vitro fertilisability of liquid-preserved boar spermatozoa. METHODS: In Experiment 1, semen samples were preserved in the semen extender supplemented with 0, 3, 6, or 12 mM of Na-EDTA at 5 degree C for 4 weeks. In Experiment 2, semen samples were preserved in the extender supplemented with 3 mM of Na-EDTA, Ca-EDTA, or Zn-EDTA and without chelator EDTA. RESULTS: When Na-EDTA was used as a chelating substance in the extender, 3 mM was a most suitable concentration for sperm motility and viability after cold preservation. The supplementation of 3 mM Ca-EDTA had advantages regarding sperm motility, viability and plasma membrane integrity. CONCLUSION: Our findings indicate that 3 mM Ca-EDTA is the most suitable metal-chelating substance for the liquid preservation of boar semen.

Effect of sericin supplementation during in vitro maturation on the maturation, fertilization and development of porcine oocytes.

This study aimed to examine the effects of sericin supplementation during in vitro oocyte maturation on the nuclear maturation, fertilization and development of porcine oocytes. Cumulus-oocyte complexes (COCs) were cultured in maturation medium supplemented with 0 (control), 0.1, 0.5, 1.0, 2.5 or 5.0% sericin and were then subjected to in vitro fertilization and embryo culture. More COCs matured with 1.0% sericin underwent germinal vesicle breakdown and reached metaphase II compared with the control COCs matured without sericin (p < 0.01). The proportions of oocytes with DNA-fragmented nuclei did not differ between the groups, regardless of the sericin level. The total fertilization rate of oocytes matured with 1.0% sericin was higher (p < 0.05) than that of oocytes matured with 0.1%, 2.5% and 5.0% sericin. Supplementation with more than 1.0% sericin decreased the DNA fragmentation index of the blastocysts compared with the control group (p < 0.05). However, the supplementation of the maturation medium with sericin had no beneficial effects on the cleavage, development to the blastocyst stage and the total cell number of the embryos. Our findings indicate that supplementation with 1.0% sericin during maturation culture may improve the nuclear maturation and the quality of the embryos but does not affect blastocyst formation.

In vivo tear-film thickness determination and implications for tear-film stability.

PURPOSE: Previous measurements of tear-film thickness in vivo are limited and cannot be easily applied in a clinical setting. A novel technique to measure tear-film thickness indirectly is introduced here, requiring only a slit lamp, video camera, and computer. A recent fluid mechanical theory relates tear-film thickness h, to the tear meniscus radius R, tear surface tension or, tear viscosity mu, and upper lid velocity U. This theory yields the result that h/R = 2.12 (microU/sigma)2/3. All parameters except h/R are taken as known physical constants, and R was measured for each subject, allowing the above equation to establish h. Tear-film breakup was also evaluated and correlated with tear-film thickness. METHODS: A clinical study was performed in which aqueous tear-film thickness was determined for 45 subjects, including 24 non-lens subjects, 15 hydrogel contact lens wearers, and 6 RGP lens wearers. R was measured by instilling fluorescein dye in the form of an eyedrop and videotaping the tear meniscus in profile. Tear-film breakup was videotaped through the ocular port of the slit lamp and evaluated based on a severity scale. RESULTS: Aqueous tear-film measurements are in the same range as literature values, with most measured values falling between 6 and 12 microm. Average tear-film thicknesses for non-lens, hydrogel, and RGP subjects are 10.4, 6.5, and 5.8 microm, respectively. Tear-film breakup is most severe in subjects with thin tear films, especially in contact-lens wearers. CONCLUSIONS: Tear-film thickness is an important parameter that varies among individuals. These variations correlate with differences in tear-film stability.

A novel sensitive and selective bioassay for human type I interferons.

We describe a novel MxA gene-induction assay for type I interferons (IFN-alpha and IFN-beta) based on the specific induction of the MxA gene in cultured human cells. Accumulated intracellular MxA protein is determined by immunologic measurement by a rapid method using commercially available materials. IFN activity can be measured accurately over a concentration range of 0.1-30 IU/ml. In contrast, type II IFN and other cytokines are not significantly detected. The MxA-induction assay has advantages in terms of specificity, reliability, and sensitivity over other methods for assaying type I IFN. It has also been adapted and validated for measuring the titers of anti-IFN-beta neutralizing antibodies in human sera.

A novel bioassay for the determination of neutralizing antibodies to IFN-beta1b.

We have adapted the new MxA gene-induction bioassay to measure neutralizing antibodies to interferon-beta1b (IFN-beta1b, the active ingredient in Betaseron) in sera from patients treated with Betaseron. This antibody assay has been validated to quantify neutralizing titers of 1:20 and above, with a precision of +/- 0.20 in log10. We have used this MxA gene-induction antibody assay to reinvestigate serum samples from multiple sclerosis (MS) patients treated with Betaseron. The titers measured were closely comparable to those obtained in antiviral assays. Data obtained by both methods show that neutralizing antibodies may appear and subsequently disappear over time in the sera of some patients treated with Betaseron. Sera from some patients contain binding antibodies to IFN-beta1b. It was shown that binding antibody titers do not correlate quantitatively or qualitatively with neutralizing antibody titers, and indeed, a number of patients develop high levels of binding antibodies but never form measurable levels of neutralizing antibodies.

Site-directed mutagenesis of the subunit PsaC establishes a surface-exposed domain interacting with the photosystem I core binding site.

We have postulated that the orientation of PsaC on the photosystem I core involves electrostatic interactions between charged residues on the core binding site and the subunit [Rodday, S. M., Jun, S.-S., & Biggins, J. (1993) Photosynth. Res. 36, 1-9]. We, therefore, changed eight acidic residues on PsaC to arginine and examined the efficiency of the mutant subunits in the reconstitution of P700-Fx cores in vitro. Reconstitution of the cores by the mutant subunits was determined by analysis of the kinetics of recombination reactions between P700+ and reduced acceptors as measured optically. Restoration of complete forward electron transfer, indicative of efficient subunit binding, was estimated from the ca. 30 ms decay component in the flash transients. Slightly reduced levels of reconstitution were observed for the mutants D24R, E46R/D47R. D61R, and E72R. In contrast, mutants D9R, E27R, and D32R showed significantly lower efficiencies. The presence of the iron-sulfur centers, FA and FB, in these three mutant subunits was confirmed by low-temperature EPR spectroscopy indicating that the polypeptides had folded correctly. We conclude that the introduction of positively charged side chains at positions 9, 27, and 32 seriously disrupts PsaC binding. However, when the wild-type acidic residues in these positions were changed to alanine, only mutant D9A showed a reduced level of reconstitution, suggesting that this aspartate is the most important residue participating in the electrostatic interaction with the core. The results are discussed in relation to the photosystem I crystal structure and support an orientation of PsaC on the core such that center FB is proximal to Fx.