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 voltage strength during electroporation on the development and quality of in vitro-produced porcine embryos.

This study was conducted to determine suitable conditions for an experimental method in which the CRISPR/Cas9 system is introduced into in vitro-produced porcine zygotes by electroporation. In the first experiment, when putative zygotes derived from in vitro fertilization (IVF) were electroporated by either unipolar or bipolar pulses, keeping the voltage, pulse duration and pulse number fixed at 30 V/mm, 1 msec and five repeats, respectively, the rate of blastocyst formation from zygotes electroporated by bipolar pulses decreased compared to zygotes electroporated by unipolar pulses. In the second experiment, the putative zygotes were electroporated by electroporation voltages ranging from 20 V/mm-40 V/mm with five 1-msec unipolar pulses. The rate of cleavage and blastocyst formation of zygotes electroporated at 40 V/mm was significantly lower (p < .05) than that of zygotes electroporated at less than 30 V/mm. Moreover, the apoptotic nuclei indices of blastocysts derived from zygotes electroporated by voltages greater than 30 V/mm significantly increased compared with those from zygotes electroporated by voltages less than 25 V/mm (p < .05). When zygotes were electroporated with Cas9 mRNA and single-guide RNA (sgRNA) targeting site in the FGF10 exon 3, the proportions of blastocysts with targeted genomic sequences were 7.7% (2/26) and 3.6% (1/28) in the embryos derived from zygotes electroporated at 25 V/mm and 30 V/mm, respectively. Our results indicate that electroporation at 25 V/mm may be an acceptable condition for introducing Cas9 mRNA and sgRNA into pig IVF zygotes under which the viability of the embryos is not significantly affected.

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.

Sequence polymorphisms in porcine homologs of murine coat colour-related genes.

Herein, we report the variability among 57 porcine homologs of murine coat colour-related genes. We identified single nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) within 44 expressed gene sequences by aligning eight pig complementary DNA (cDNA) samples. The sequence alignment revealed a total of 485 SNPs and 15 InDels. The polymorphisms were then validated by performing matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with reference DNA samples obtained from 384 porcine individuals. Of the 384 individuals, three parents of the experimental F(2) family were included to detect polymorphisms between them for linkage mapping. We also genotyped previously reported polymorphisms of 12 genes, and one SNP each in three genes that were detected by performing a BLAST search of the Trace database. A total of 211 SNPs and three InDels were successfully genotyped from our porcine DNA panel. We detected SNPs in 33 of the 44 genes among the parents of an experimental F(2) family and then constructed a linkage map of the 33 genes for this family. The linkage assignment of each gene to the porcine chromosomes was consistent with the location of the BAC clone in the porcine genome and the corresponding gene sequence. We confirmed complete substitutions of EDNRB and MLPH in the Jinhua and Clawn miniature breeds, respectively. Furthermore, we identified polymorphic alleles exclusive to each pig group: 13 for Jinhua, two for Duroc, three for Meishan, four for the Japanese wild boar, one for the Clawn miniature pig and four for the Potbelly pig.

Fine mapping of quantitative trait loci for meat color on Sus scrofa chromosome 6: analysis of the swine NUDT7 gene.

In the livestock industry, meat color has become important because consumer acceptance is subject to the appearance of the product in the marketplace. Our previous analyses of a whole genome QTL scan for various meat qualities using 2 F(2) families from Japanese wild boar (known as a red meat) x Large White and from Duroc x Chinese Jinhua suggested that a meat color (heme content) QTL is located on SSC6. The objective of this study was to fine-map this SSC6 meat color QTL and subsequently investigate positional candidate genes for polymorphisms that may cause changes in meat color. Therefore, we conducted interval mapping on SSC6 using an additional 9 gene markers through combined analyses of the 2 F(2) families of Japanese wild boar x Large White (353 progeny) and Duroc x Chinese Jinhua (204 progeny). Comparative analysis with humans, mice, and cattle suggested that there were 10 functional genes in the region. Among these genes, we suggested that a novel pig gene encoding a nudix (nucleoside diphosphate linked moiety X)-type motif 7 (NUDT7, a member of the nudix hydrolases) is a strong candidate for the QTL because the mouse Nudt7 is reported to hydrolyze succinyl-CoA, a substrate of the reaction limiting the rate of heme biosynthesis. We therefore determined the pig NUDT7 gene sequence including the 5' promoter region and explored genetic polymorphisms between Japanese wild boar and Large White. We identified 116 polymorphisms within the NUDT7 CDS or in the 5' region. None of the AA substitutions were associated with the meat color QTL; however, 3 polymorphisms were found in putative transcription factor recognition sites. We then investigated the differential expression of NUDT7 in Japanese wild boar and Large White by allele-specific quantitative real-time PCR. The expression level of the Large White type allele was greater than that of the Japanese wild-boar-type allele. Consequently, we speculated that the difference in meat color between Japanese wild boar and Large White is caused partly by differential expression of this candidate gene. Upregulation of NUDT7 expression in muscle may reduce succinyl-CoA content and thus reduce the level of heme biosynthesis.

Quantitative trait loci mapping for fatty acid composition traits in perirenal and back fat using a Japanese wild boar x Large White intercross.

Here, we analysed quantitative trait loci (QTL) for fatty acid composition, one of the factors affecting fat quality, in a Japanese wild boar x Large White cross. We found 25 significant effects for 17 traits at 13 positions at the 5% genome-wise level, of which 16 effects for 12 traits at 10 positions were significant at the 1% level. QTL for saturated fatty acids (SFA) in back fat were mapped to swine (Sus scrofa) chromosomes (SSC) 1p, 9 and 15. QTL for unsaturated fatty acids in back fat were mapped to SSC1p, 1q, 4, 5, 9, 15 and 17. Using a regression model that fits back fat thickness as a covariate, two of the QTL for linoleic acid content on SSC4 and SSC17 were not significant, but one QTL for total SFA composition was detected on SSC5 with correction for back fat thickness. Wild boar alleles at six of seven QTL tended to increase SFAs and to decrease unsaturated fatty acids. QTL for fatty acid composition in perirenal fat were mapped on SSC2, 3, 4, 5, 6, 14, 16 and X. QTL for melting point (in back fat samples) were mapped on SSC1, 2 and 15. Wild boar alleles in QTL on SSC1 and SSC15 were associated with elevated melting points whereas those on SSC2 were associated with lower melting point measurements.

Assessment of fetal atrioventricular time intervals by tissue Doppler and pulse Doppler echocardiography: normal values and correlation with fetal electrocardiography.

OBJECTIVE: To establish gestational age-specific reference values of normal fetal atrioventricular (AV) time interval by spectral tissue Doppler imaging (TDI) and pulse-wave Doppler (PD) methods, and to assess their correlation with signal-averaged fetal PR intervals (ECG). DESIGN: Cohort study. SETTING: Tertiary centre for fetal cardiology. PATIENTS AND MEASURES: 131 pregnant women between 14 and 42 weeks' gestation underwent 196 fetal echocardiograms and 158 fetal ECG studies. TDI-derived AV intervals were measured as the intervals from atrial contraction (Aa) to isovolumic contraction (IV) and from Aa to ventricular systole (Sa) at the right ventricular free wall. PD-derived AV intervals were measured from simultaneous left ventricular inflow/outflow (in/out) and superior vena cava/aorta (V/AO) recordings. RESULTS: Measurements were possible by ECG in 61%, by TDI in 100%, by in/out in 100% and by V/AO in 97% of examinations. Aa-IV correlated significantly better with PR intervals (y = 0.67x + 38.29, R(2) = 0.15, p < 0.0001, mean bias 8.0 ms) than did in/out (R(2) = 0.10, p = 0.002, bias 18.7 ms) and V/AO (R(2) = 0.06, p = 0.02, bias 12.4 ms). Gestational age and AV intervals were positively correlated with all imaging modalities (R(2) = 0.19-0.31, p < 0.0001). CONCLUSION: This study showed the feasibility of fetal AV interval measurements by TDI, and established gestational age-specific reference data. TDI-derived Aa-IV intervals track ECG PR intervals more closely than PD-derived AV intervals and thus should be used as the ultrasound method of choice in assessing fetal AV conduction.

Analysis of DNA fragmentation of porcine embryos exposed to cryoprotectants.

The chemical toxicity of cryoprotectants to porcine embryos was examined by the evaluation of survival and DNA damage after exposure to cryoprotectants. Porcine blastocysts were exposed to 10% of ethylene glycol (EG), 1,2-propanediol (PD) or glycerol (GLY) for 1 h at room temperature (23-25 degrees C) and then cultured in vitro for 24 h. The survival rates of blastocysts exposed to PD and GLY were significantly lower than those of control blastocysts in which the embryos were exposed to carrier solution without cryoprotectants. Significantly more DNA-fragmented nuclei occurred in the cryoprotectant-exposed blastocysts, compared with the control blastocysts. Moreover, the indices of DNA-fragmented nuclei in the blastocysts without blastocoele re-formation after culture were significantly higher than those with blastocoele re-formation, irrespective of the exposure treatment. These results indicate that the exposure of porcine blastocysts to cryoprotectant decreases the survival rates and increases the DNA-fragmented nuclei in embryos.

Two quantitative trait loci on Sus scrofa chromosomes 1 and 7 affecting the number of vertebrae.

The objective of the research was to identify QTL affecting the number of vertebrae in swine, one of the major determining factors of growth and body composition. Previously, we reported a QTL for the number of vertebrae located on SSC1qter (terminal band of the q arm of SSC 1) in an F2 family produced by crossing a Göttingen miniature male with two Meishan females. Eight other swine families were subsequently produced by crosses between different breeds of European, Asian, and miniature pigs. In these families, the QTL on SSC1qter for the number of vertebrae was detected. Unlike the Asian alleles, all European alleles in this study had the effect of increasing the number of vertebrae by 0.44 to 0.69 and acted additively without dominance. The Göttingen miniature sire, for which we previously reported a smaller additive effect, seemed to be heterozygous at the QTL. In the present study, another QTL was found for the number of vertebrae on SSC7. This QTL was not fixed in the European pigs used as parents in our experimental families, and some of the European alleles increased the number of vertebrae. A half-sib analysis confirmed that this QTL was segregating in a commercial Large White population. Analysis in an F2 family in which the parental pigs were fixed for alternative alleles revealed that the effects of the QTL on SSC1 and on SSC7 were additive and similar in size. The two QTL acted independently without epistatic effects and explained an increase of more than two vertebrae.

Effect of the removal of cumulus cells on the nuclear maturation, fertilization and development of porcine oocytes.

The present study was conducted to investigate the effects of attachment of cumulus cells to porcine oocytes during the process of maturation and fertilization on the nuclear maturation, fertilization and subsequent development after in vitro fertilization (IVF). In the first experiment, the cumulus cells were removed from cumulus-oocyte complexes (COCs) at 0, 24 and 42 h after the onset of maturation culture and were then cultured until reaching 42 h of cultivation. In the second experiment, COCs were denuded as described in the first experiment, then fertilized and cultured for 7 days. As a control, cumulus cells were allowed to maintain attachment to the oocytes until the end of IVF. The proportion of oocytes reaching metaphase II significantly increased with the delay in the removal treatment of cumulus cells. The proportion of normal fertilization gradually increased with delay in the removal treatment of cumulus cells from COCs until the end of IVF. However, no significant difference in the proportion of normal fertilization was found between the 42-h and control groups. The removal treatment of cumulus cells in the 0- and 24-h group significantly (p < 0.05) decreased the proportion of cleaved embryos when compared with the control, and none of them developed to the blastocyst stage. The proportion of development to the blastocyst stage was significantly higher (p < 0.05) in the control group than in the 42-h group (18.1% vs 12.4%; p < 0.05). The present study indicates that the attachment of cumulus cells to the oocyte during maturation and fertilization is important to support oocyte nuclear maturation, fertilization and subsequent embryo development. Particularly, the attachment of cumulus cells to the oocyte during IVF promotes embryonic development.

Quantitative trait loci mapping for meat quality and muscle fiber traits in a Japanese wild boar x Large White intercross.

Three generations of a swine family produced by crossing a Japanese wild boar and three Large White female pigs were used to map QTL for various production traits. Here we report the results of QTL analyses for skeletal muscle fiber composition and meat quality traits based on phenotypic data of 353 F(2) animals and genotypic data of 225 markers covering almost the entire pig genome for all of the F(2) animals as well as their F(1) parents and F(0) grandparents. The results of a genome scan using least squares regression interval mapping provided evidence that QTL (<1% genome-wise error rate) affected the proportion of the number of type IIA muscle fibers on SSC2, the number of type IIB on SSC14, the relative area (RA) of type I on SSCX, the RA of type IIA on SSC6, the RA of type IIB on SSC6 and SSC14, the Minolta a* values of loin on SSC4 and SSC6, the Minolta b* value of loin on SSC15, and the hematin content of the LM on SSC6. Quantitative trait loci (<5% genome-wise error rate) were found for the number of type I on SSC1, SSC14, and SSCX, for the number of type IIA on SSC14, for the number of type IIB on SSC2, for the RA of type IIA on SSC2, for the Minolta b* value of loin on SSC3, for the pH of loin on SSC15, and for the i.m. fat content on SSC15. Twenty-four QTL were detected for 11 traits at the 5% genome-wise level. Some traits were associated with each other, so the 24 QTL were located on 11 genomic regions. In five QTL located on SSC2, SSC6, and SSC14, each wild boar allele had the effect of increasing types I and IIA muscle fibers and decreasing type IIB muscle fibers. These effects are expected to improve meat quality.

Pulsed wave Doppler tissue echocardiography assessment of the long axis function of the right and left ventricles during the early neonatal period.

OBJECTIVE: To assess the long axis function of both ventricles during the early neonatal period by using pulsed wave Doppler tissue (PWDT) echocardiography. DESIGN: PWDT echocardiography was recorded from the lateral sites of the mitral and tricuspid annuluses and the tip of interventricular septum in 130 neonates within 24 hours after birth (day 0 group), in 135 neonates 1-7 days after birth (day 1-7 group), and in 131 healthy children (children group). RESULTS: Peak systolic motion velocity (Sw) of the three ventricular walls positively correlated with the number of days after birth (p < 0.005). Compared with the children group, in neonates Sw in the right ventricle and peak early diastolic motion velocity (Ew) and peak atrial systolic motion velocity in the interventricular septum were lower than in the remaining two walls (p < 0.0005, p < 0.0001, and p< 0.0001, respectively). Although peak mitral and tricuspid flow velocities during early diastole (E) correlated with the number of hours after birth in the day 0 group, there was no significant change in the Ew of either ventricle. The E:Ew ratio of both ventricles was significantly higher in both neonate groups than in the children group (p < 0.001). The E:Ew ratio of the left ventricle was higher in the day 0 group than in the day 1-7 group (p < 0.005). CONCLUSIONS: The two ventricles differ in their normal PWDT echocardiographic values and in the parameter change after birth during the early neonatal period, which may reflect differences in ventricular adaptation after birth.

Effects of cooling ovaries before oocyte aspiration on meiotic competence of porcine oocytes and of exposing in vitro matured oocytes to ambient temperature on in vitro fertilization and development of the oocytes.

Experiments were conducted to evaluate the effects of cooling porcine ovaries to low temperature (4 degrees C, 15 degrees C, 20 degrees C, 25 degrees C or 30 degrees C) for 1 h on the meiotic competence of their oocytes. Moreover, it was determined whether or not the exposure of in vitro matured oocytes to ambient temperature (20 degrees C, 25 degrees C or 30 degrees C) for 1 h affects the fertilization and developmental competence of the oocytes. There was no difference between the proportions of oocytes that underwent maturation to metaphase II when isolated from control ovaries held at 35 degrees C and ovaries exposed to 30 degrees C. However, the percentages of oocytes from ovaries exposed to 25 degrees C or less were significantly lower than those of oocytes from ovaries exposed to 30 degrees C and control ovaries. The proportions of total and normal fertilization of oocytes that had been exposed to 20 degrees C before in vitro fertilization (IVF) were significantly lower than those of control oocytes maintained at 38.5 degrees C. However, cooling in vitro matured oocytes had no effects on their cleavage and development to blastocysts after IVF. These data suggest that exposing porcine ovaries to a low temperature of 25 degrees C or less before aspiration of oocytes may adversely affect their subsequent in vitro maturation. It may be necessary to maintain the oocytes at a temperature of more than 25 degrees C during manipulation of oocytes for retaining the fertilizability of the oocytes.

Plasma levels of natriuretic peptide and echocardiographic parameters in patients with Duchenne's progressive muscular dystrophy.

We investigated the relationship between plasma atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) levels and systolic and diastolic cardiac function, determined by echocardiography, in 63 patients with Duchenne's progressive muscular dystrophy (DMD) (age range 8-21 years). The relationship between shortening fraction of the left ventricle and ANP and BNP levels was curvilinear rather than linear: When the shortening fraction was >15%, increases in ANP and BNP levels were minimal. However, if the shortening fraction was <15%, both natriuretic peptide levels increased dramatically. Stepwise regression analysis revealed that only the deceleration time of the early diastolic filling wave predicted plasma BNP concentration among various diastolic echocardiographic parameters determined by mitral flow. Three patients died of cardiac dysfunction during a 2-year follow-up period. These patients had a severely decreased deceleration time (<65% of normal) in association with increases in both natriuretic peptide levels. In conclusion, plasma ANP and BNP levels are not sensitive markers for the early detection of cardiac systolic dysfunction in patients with DMD. However, in patients with systolic dysfunction, an increase in the concentrations of these peptides, associated with a decrease in the deceleration time of early diastolic filling, suggests poor prognosis.

The usefulness of brain perfusion SPECT in an infant with focal convulsions associated with hemophilus influenzae central nervous system infection.

Left hemiconvulsions occurred in a 13-month-old girl with Hemophilus influenzae (H. influenzae) central nervous system (CNS) infection. Tc-99m HMPAO brain SPECT showed a focal hyperperfusion area in the right frontal lobe. The patient recovered without complications, and follow-up SPECT revealed markedly improved findings. Tc-99m HMPAO brain SPECT is useful for evaluating focal convulsions associated with H. influenzae CNS infection.

Role of hypertension, dyslipidemia and diabetes mellitus in the development of coronary atherosclerosis in Japan.

The present study evaluated the effect of hypertension (HT), dyslipidemia and diabetes mellitus (DM) on the development of coronary atherosclerosis in the Japanese population, using a cross-sectional study of 433 patients (254 men and 179 women) aged 30 years or older who underwent coronary angiography for suspected or known coronary heart disease angina at 5 cardiology departments in the Fukuoka area between September 1996 and August 1997. Patients with a disease duration of 6 months or more were excluded. The main outcome measure was angiographically defined coronary artery stenosis and was found to a significant degree in 146 patients (33.7%). HT, DM, low levels of high-density lipoprotein cholesterol (HDL-C) and hypertriglyceridemia remained as significant coronary artery disease (CAD) risk factors even after controlling for age, sex, hospital, smoking, alcohol use, body mass index and leisure time physical activity. However, hypercholesterolemia was not a significant risk factor after adjusting for these variables. After controlling for these variables, DM, low HDL-C and hypertriglyceridemia were significant CAD risk factors for men, but only DM was a significant CAD risk factor in women. These results indicate that in Japan DM, low HDL-C and hypertriglyceridemia may be more important CAD risk factors than hypercholesterolemia.

Left ventricular wall motion velocities in healthy children measured by pulsed wave Doppler tissue echocardiography: normal values and relation to age and heart rate.

Left ventricular wall motion velocities were measured by pulsed wave Doppler tissue (PWDT) echocardiography in 131 healthy children (mean age 7.5 +/- 5.5 years) at the interventricular septum and the posterior wall in the left ventricular short-axis view, and at the interventricular septum and the lateral wall in the 4-chamber view. The systolic wave (Sw) consisted of 2 components, and the difference between the 2 components was greater in the lateral wall than in the other walls. The peak early diastolic wave (Ew) velocity was also highest in the lateral wall. Most variables during systole correlated with age. The ratio of peak atrial systolic wave (Aw) velocity to peak Ew velocity (Aw/Ew) correlated with heart rate. The Aw/Ew in each wall correlated with the ratio of late (A) to early (E) peak mitral flow, although regression slopes differed among different wall segments. In younger children with increased heart rates, the Aw/Ew ratio increased because the Ew velocity decreased, although the A/E ratio increased because of an increased A velocity. Normal values for the PWDT variables change with heart rate and age in the pediatric population. The data reported in this study can be used as normal values for left ventricular function for PWDT echocardiography.