Potential of preimplantation genomic selection using the blastomere separation technique in bovine in vitro fertilized embryos.

Preimplantation genomic selection combined with an in vitro embryo production system is expected as a means of accelerating genetic improvement in cattle. While micromanipulation-based biopsy approaches are often used to collect embryonic cells for genetic testing, they require expensive equipment and sophisticated skills, hindering the adoption of this system. In the present study, to develop a simple method for preimplantation genomic selection using the blastomere separation (BS) technique in bovine in vitro fertilized embryos, we examined the accuracy of single nucleotide polymorphism (SNP) genotyping and optimal cryopreservation method in demi-blastocysts produced by the BS technique. We demonstrated reliable SNP genotyping using DNA derived from demi-blastocysts. We indicated a suitable equilibrium time in vitrification solution for demi-blastocysts and succeeded obtaining pregnancies by the transfer of vitrified demi-blastocysts. In conclusion, our findings suggest that the BS technique provides a simple method for preimplantation genomic selection in bovine in vitro fertilized embryos.

Discrimination between L-type and C-type bovine spongiform encephalopathy by the strain-specific reactions of real-time quaking-induced conversion.

Real-time quaking-induced conversion (RT-QUIC) assays using Escherichia coli-derived purified recombinant prion protein (rPrP) enable us to amplify a trace amount of the abnormal form of PrP (PrPSc) from specimens. This technique can be useful for the early diagnosis of both human and animal prion diseases and the assessment of prion contamination. In the present study, we demonstrated that there are strain-specific differences in the RT-QUIC reactions between an atypical form of bovine spongiform encephalopathy (BSE), l-BSE, and classical BSE (C-BSE). Whereas mouse rPrP (rMoPrP) was efficiently converted to amyloid fibrils in the presence of PrPSc seed derived from either l-BSE or C-BSE, hamster rPrP (rHaPrP) was converted only in l-BSE, not C-BSE. These characteristics were preserved in the second round reaction, but gradually weakened in the subsequent rounds and were completely lost by the fifth round, most likely due to the selective growth advantage of nonspecific rPrP amyloid fibrils in the RT-QUIC. Our findings further enhance the discrimination of prion strains using RT-QUIC, and further our understanding of the molecular basis of prion strains.

Expression of C-C motif chemokines and their receptors in bovine placentomes at spontaneous and induced parturition.

In bovine placentomes, the inflammatory response is considered important for the detachment of the fetal membrane from the caruncle after parturition. Glucocorticoids, a trigger of the onset of parturition, facilitate functional maturation of placentomes via prostaglandin (PG) and estrogen production in cattle. This study investigated how exogeneous glucocorticoids, which exert immunosuppressive effects, affect placental inflammation at parturition. Placentomes were collected immediately after spontaneous or induced parturition. Parturition was conventionally induced using PGF2α or dexamethasone or with a combination of triamcinolone acetonide and high-dose betamethasone (TABET treatment). Polymerase chain reaction (PCR) array analysis indicated that 9/13 C-C motif chemokine ligands (CCLs) were upregulated > two-fold in spontaneous parturition, with CCL2 and CCL8 being highly expressed. The expressions of CCL2, CCL8, C-C motif chemokine receptor 1 (CCR1), and CCR5 in caruncles were significantly higher in spontaneous parturition than in induced parturition. Although the clinical dose of dexamethasone did not influence the expression of these CCLs and CCRs, TABET treatment increased CCR1 expression. CCL8, CCR1, CCR2, and CCR5 were localized in the caruncular epithelial cells. CCR2 was also localized in the epithelial cells of the cotyledonary villi. This study is the first report to reveal the disruption in CCL and CCR expression in bovine placentomes at induced parturition. Enhanced glucocorticoid exposure for the induction of parturition may upregulate CCR1 expression in placentomes, but the treatment does not adequately promote CCL expression. Additionally, immunohistochemistry suggested that the CCL-CCR system is involved in the functional regulation of maternal and fetal epithelial cells in placentomes at parturition.

Enhanced glucocorticoid exposure facilitates the expression of genes involved in prostaglandin and estrogen syntheses in bovine placentomes at induced parturition.

The mechanism by which the fetal membrane detaches after parturition in cattle is poorly understood, but the upregulation of placentomal prostaglandin and estrogen synthesis are considered to be important. This study investigated whether enhanced glucocorticoid exposure affected the functional maturation of placentomes at induced parturition. Placentomes were collected immediately after spontaneous (beef; n = 5, dairy; n = 5) or induced parturition in beef and dairy cattle. Parturition was induced conventionally using prostaglandin F2α (beef; n = 7, dairy; n = 6) or dexamethasone (beef; n = 6) or with a combination of triamcinolone acetonide (a long-acting glucocorticoid) and a high dose of betamethasone (TABET treatment, beef; n = 6, dairy; n = 9). Gene expression levels and protein localization in placentomes were analyzed by RT-qPCR and immunohistochemistry, respectively. Compared with the conventional methods, TABET treatment resulted in upregulated PTGS2 expression in cotyledons. The expression levels of PTGS2 and PGES were positively correlated in both cotyledons and caruncles. TABET treatment also upregulated the expression of CYP17A1, but not of CYP19A1, in cotyledons. The results revealed, for the first time, that PLA2G4A was localized in microvascular endothelial cells in the cotyledonary villi and the maternal septum. PTGS2 and PGES were colocalized in mononucleated cells of the cotyledonary villi and caruncle epithelial cells adjacent to the chorionic plate. TABET treatment upregulated the expression of placentomal genes involved in PGE2 synthesis and the conversion of pregnenolone to androstenedione. Thus, enhanced glucocorticoid exposure might partially facilitate the functional maturation of placentomes at induced parturition in cattle.

Potential of preimplantation genomic selection for carcass traits in Japanese Black cattle.

Preimplantation genomic selection using genomic estimated breeding values (GEBVs) based on single nucleotide polymorphism (SNP) genotypes is expected to accelerate genetic improvement in cattle. To develop a preimplantation genomic selection system for carcass traits in Japanese Black cattle, we investigated the accuracy of genomic evaluation of carcass traits using biopsied embryonic cells (Experiment 1); we also performed an empirical evaluation for embryo transfer (ET) of vitrified GEBV-evaluated blastocysts to assess the efficiency of the preimplantation genomic selection system (Experiment 2). In Experiment 1, the mean call rate for SNP genotyping using approximately 15 biopsied cells was 98.1 ± 0.3%, whereas that for approximately 5 biopsied cells was 91.5 ± 2.4%. The mean concordance rate for called genotypes between ~15-cell biopsies and the corresponding biopsied embryos was 99.9 ± 0.02%. The GEBVs for carcass weight, ribeye area, and marbling score calculated from ~15-cell biopsies closely matched those from the corresponding calves produced by ET. In Experiment 2, a total of 208 in vivo blastocysts were biopsied (~15-cell) and the biopsied cells were processed for SNP genotyping, where 88.5% of the samples were found to be suitable for GEBV calculation. Large variations in GEBVs for carcass traits were observed among full-sib embryos and, among the embryos, some presented higher GEBVs for ribeye area and marbling score than their parents. The conception rate following ET of vitrified GEBV-evaluated blastocysts was 41.9% (13/31). These findings suggest the possible application of preimplantation genomic selection for carcass traits in Japanese Black cattle.

Effects of genetic background on responses to superovulation in Japanese Black cattle.

We investigated the effects of genetic background on the responses to superovulation in Japanese Black cattle. The genotype frequencies of GRIA1 and FSHR relating to ovulation and follicular development in each of the major bloodlines-Tajiri, Fujiyoshi, and Kedaka-were analyzed. The Tajiri line had the lowest frequency of G allele homozygosity of c.710A>G in GRIA1 among the three bloodlines, and deviation from Hardy-Weinberg equilibrium was detected. Genotype frequencies of c.337C>G, c.871A>G, and c.1973C>G in FSHR were in Hardy-Weinberg equilibrium in all bloodlines. The results of generalized linear mixed-model analyses showed that farm, levels of plasma anti-Müllerian hormone (AMH) concentration, age in months, repeated superovulation, c.337C>G in FSHR, and bloodlines had significant effects on the responses to superovulation. The number of transferable embryos in the group heterozygous for c.337C>G in FSHR was significantly higher than that in the group homozygous for the C allele. The Kedaka line showed a significantly higher number of ova/embryos, fertilized embryos, and transferable embryos than the Tajiri and Fujiyoshi lines. The concentration of circulating AMH is a useful endocrine marker for antral follicle counts. This study revealed the effects of genetic background on the responses to superovulation using levels of plasma AMH concentration as a covariate. The prominent effect of genetic background on superovulation in the Kedaka line requires additional studies to confirm the genomic regions and polymorphisms that are involved in the trait.

Expression and localization of aquaporins 3 and 7 in bull spermatozoa and their relevance to sperm motility after cryopreservation.

Artificial insemination with cryopreserved semen is a well-developed technique commonly used for controlled reproduction in cattle. However, despite current technical advances, cryopreservation continues to damage bull spermatozoa, resulting in a loss of approximately 30 to 50% of viable spermatozoa post thawing. To further improve the efficiency of cryopreservation of bull spermatozoa, understanding the molecular mechanisms underlying the cryobiological properties that affect cryoinjuries during cryopreservation process of bull spermatozoa is required. In this study, we examined the expression and localization of aquaporin (AQP) 3 and AQP7 in fresh, cooled, and frozen-thawed bull spermatozoa. Furthermore, we investigated the relevance of AQP3 and AQP7 to motility and to membrane integrity in frozen-thawed bull spermatozoa. Western blotting against AQP3 and AQP7 in bull spermatozoa revealed bands with molecular weights of approximately 42 kDa and 53 kDa, respectively. In immunocytochemistry analyses, immunostaining of AQP3 was clearly observed in the principal piece of the sperm tail. Two immunostaining patterns were observed for AQP7 -pattern 1: diffuse staining in head and entire tail, and pattern 2: diffuse staining in head and clear staining in mid-piece. Cooling and freeze-thawing did not affect the localization pattern of AQP7 and the relative abundances of AQP3 and AQP7 evaluated by Western blotting. Furthermore, we demonstrated that the relative abundances of AQP3 and AQP7 varied among ejaculates, and they were positively related to sperm motility, particularly sperm velocity, post freeze-thawing. Our findings suggest that AQP3 and AQP7 are possibly involved in the tolerance to freeze-thawing in bull spermatozoa, particularly in the sperm's tail.

Production of calves by the transfer of cryopreserved bovine elongating conceptuses and possible application for preimplantation genomic selection.

Preimplantation genomic selection based on single nucleotide polymorphism (SNP) genotypes is expected to accelerate genetic improvement in cattle. However, genome-wide genotyping at the early embryonic stage has several limitations, such as the technical difficulty of embryonic biopsy and low accuracy of genotyping resulting from a limited number of biopsied cells. After hatching from the zona pellucida, the morphology of the bovine embryo changes from spherical to filamentous, in a process known as elongation. The bovine nonsurgical elongating conceptus transfer technique was recently developed and applied for sexing without requiring specialized skills for biopsy. In order to develop a bovine preimplantation genomic selection system combined with the elongating conceptus transfer technique, we examined the accuracy of genotyping by SNP chip analysis using the DNA from elongating conceptuses (Experiment 1) and optimal cryopreservation methods for elongating conceptuses (Experiment 2). In Experiment 1, the call rates of SNP chip analysis following whole genome amplification in biopsied cells from two elongating conceptuses were 95.14% and 99.32%, which were sufficient for estimating genomic breeding value. In Experiment 2, the rates of dead cells in elongating conceptuses cryopreserved by slow freezing were comparable to those in fresh elongating conceptuses. In addition, we obtained healthy calves by the transfer of elongating conceptuses cryopreserved by slow freezing. Our findings indicate that the elongating conceptus transfer technology enables preimplantation genomic selection in cattle based on SNP chip analysis. Further studies on the optimization of cryopreservation methods for elongating conceptuses are required for practical application of the selection system.

Long-term changes in plasma anti-Müllerian hormone concentration and the relationship with superovulatory response in Japanese Black cattle.

The concentration of circulating anti-Müllerian hormone (AMH) in cattle is a useful endocrine marker for ovarian response to superovulation. Although the AMH concentration undergoes little variation throughout the estrous cycle, its long-term changes remain incompletely understood. Here, we investigated the relationship between superovulation response and plasma AMH concentration in Japanese Black cattle and the long-term changes in plasma AMH concentration of embryo donor cows and heifers. The median, 25th percentile, and 75th percentile of AMH concentrations in 222 mature animals were 0.265, 0.118, and 0.488 ng/ml, respectively. The numbers of ova/embryos, fertilized embryos, and transferable embryos in a total of 295 superovulations were significantly different among the H (AMH ≥ 0.488 ng/ml), M (AMH 0.487-0.119 ng/ml), and L (AMH ≤ 0.118 ng/ml) groups. AMH concentrations during repeated superovulation in ten donor cows were significantly decreased after the third treatment. In heifers, the highest AMH concentration was observed in individuals during 2-13 months of age, with considerable individual variability. AMH concentrations of heifers at 10 or 11 months correlated with the number of ova/embryos during superovulation at 13-18 months (r = 0.641, P < 0.05). These results suggest that the 25th and 75th percentile values of AMH concentration would give a useful rough estimate of ovarian response; however, repeated superovulation may reduce the predictive accuracy of single measurements of AMH concentration. It would be possible to evaluate AMH concentration in heifers after approximately 11 months of age.

The Necessity of OCT-4 and CDX2 for Early Development and Gene Expression Involved in Differentiation of Inner Cell Mass and Trophectoderm Lineages in Bovine Embryos.

The functions of POU class 5 transcription factor 1 (Oct-4) and caudal-type homeobox 2 (Cdx2) in the differentiation of the murine inner cell mass (ICM) and trophectoderm (TE) have been described in detail. However, little is known about the roles of OCT-4 and CDX2 in preimplantation bovine embryos. To elucidate their functions during early development in bovine embryos, we performed OCT-4 and CDX2 downregulation using RNA interference. We injected OCT-4- or CDX2-specific short interfering RNAs (siRNAs) into bovine zygotes. The rate of blastocyst development of OCT-4-downregulated embryos was lower compared with uninjected or control siRNA-injected embryos. Gene expression analysis revealed decreased CDX2 and fibroblast growth factor 4 expression in OCT-4-downregulated embryos. CDX2-downregulated embryos developed to the blastocyst stage; however, in most cases, blastocoel formation was delayed. Gene expression analysis revealed decreased GATA3 expression and elevated NANOG expression in CDX2-downregulated embryos. In conclusion, OCT-4 and CDX2 are essential for early development and gene expression involved in differentiation of ICM and TE lineages in bovine embryos.

Regulation of bovine oviductal NO synthesis by follicular steroids and prostaglandins.

Nitric oxide (NO) is a regulator of sperm motility, oocyte/embryo survival, and waves of contraction/relaxation in mammalian oviducts. As follicles control oviductal functions by two routes at least, (1) a systemic way via blood vessels before ovulation, (2) a direct way by entering of follicular fluid through fimbria at ovulation, we hypothesized that NO synthesis in the bovine oviduct is regulated by follicular steroids and prostaglandins (PGs). Quantification of mRNA expressions in the ampullary tissues showed that inducible NO synthase (NOS2) mRNA expression was highest on the day of ovulation (day 0). By contrast, NOS2 mRNA expression in the isthmus was highest on days 5-6 and lowest on days 19-21. Endothelial NOS (NOS3) mRNA expressions in either the ampulla or the isthmus did not change during the estrous cycle. PGE2 and PGF2α increased NOS2 mRNA expressions in cultured ampullary oviductal epithelial cells after 1-h incubation. These increases were suppressed by an antagonist of E-prostanoid receptor type 2, one of the PGE2 receptor. Estradiol-17ß decreased the expression of NOS2 mRNA expression in cultured isthmic epithelial cells 24h after treatment. This effect was suppressed by an antagonist of estrogen receptorα(ESR1). Expression of ESR1 was highest on days 19-21 in the isthmic tissues. The overall findings indicate region-specific difference of NO synthesis in the oviduct. PGs flowed from ruptured follicle may up-regulate NO synthesis in the oviductal epithelium, whereas circulating E2 seems to inhibit NO synthesis via ESR1 in the isthmus at the follicular stage.

Localization of TGF-ß and TGF-ß receptor in bovine term placentome and expression differences between spontaneous and induced parturition.

INTRODUCTION: Mechanisms of detachment of fetal membrane after parturition in cattle are poorly understood. Glucocorticoids trigger the initiation of parturition and may facilitate the placental maturation. We compared the disappearance of trophoblast binucleate cells (BNCs) and expression of transforming growth factor-ß (TGFB) in term placentomes between spontaneous and induced parturition to investigate the influences of glucocorticoids on the placental maturity. METHODS: Cows were delivered spontaneously (SP group) or after the administration of prostaglandin (PG) F(2)α (PG group); dexamethasone, PGF(2)α, and estriol (DEX group); and triamcinolone acetonide, PGF(2)α, and betamethasone (BET group) and placentomes were collected immediately after parturition. The number of BNCs in hematoxylin and eosin stained section was examined. Protein localization and mRNA levels of TGFB and its receptor (TGFBR) were analyzed using immunohistochemistry and qRT-PCR, respectively. RESULTS: TGFB1 is characteristically localized in the maternal septum in caruncle in contrast to TGFB2 and TGFB3, which are mainly found in cotyledonary villi and maternal epithelial cells. TGFBR1 and TGFBR2 colocalized in BNCs. The number of BNCs was lower in the SP group than in PG and DEX groups. mRNA levels of TGFB1, TGFBR1 and TGFBR2 in the SP group differed from PG and DEX groups. There was no difference between SP and BET groups in all analyses. DISCUSSION: These results indicate that parturition inductions using PGF(2)α or dexamethasone were not able to induce disappearance of BNCs and change of TGFB signaling. Results in the BET group suggest that investigation into types, dose, and dosage schedule of glucocorticoids may facilitate placental maturation.

Genetic diagnosis of band 3 deficiency using a quenching probe (QProbe)-PCR assay in bovine embryos.

The present study was conducted to develop a simple and rapid procedure to determine the genotype of band 3 deficiency in bovine embryos by a novel real-time PCR method using a fluorescent quenching-based probe (QProbe-PCR). QProbe-PCR successfully distinguished wild type and R664X mutant alleles by melting curve analysis. Minimal amounts of DNA template were required for the detection of wild type/wild type alleles, mutant/mutant alleles, and wild type/mutant alleles; their amounts were 10 pg, 25 pg, and 50 pg, respectively. When 10 blastomeres were used as a DNA sample, accuracies of genotyping by QProbe-PCR were 100% and 89% in embryos homozygous for the wild type allele and heterozygous for the wild type and mutant alleles, respectively. QProbe-PCR takes approximately 2 h for genotyping and requires lesser time than the conventional method using PCR-RFLP, which requires digestion with a restriction enzyme and electrophoresis. Our data showed that QProbe-PCR is a useful method for rapid analysis of the genetic deficiency in preimplantation embryos. Reduction in the time required for genotyping enabled the transfer of genetically selected embryos to recipient cows on the day of embryo collection. These results suggest that determination of the genotype for the genetic deficiency in embryos is useful to select animals free from the genetic disease, and it also makes it possible to produce an animal model homozygous for the mutation.

Enhancement of maternal recognition of pregnancy with parthenogenetic embryos in bovine embryo transfer.

This study was performed to elucidate the changes in IFNT messenger RNA (mRNA) levels in in vivo-fertilized and parthenogenetic bovine embryos and their interferon-τ (IFNT) secretion amounts during the elongation phase. We assessed the induction capability of maternal recognition of pregnancy by parthenogenetic embryos and attempted cotransfer of in vivo-fertilized and parthenogenetic embryos. The expression level of IFNT mRNA in in vivo-fertilized embryos peaked on Day 18 after estrus, and the highest amount of uterine IFNT was observed on Day 20. Transfer of 10 parthenogenetic embryos produced a detectable amount of uterine IFNT. Transfer of one or three parthenogenetic embryos inhibited luteolysis. An increase in ISG15 mRNA levels in peripheral granulocytes was induced by the transfer of three parthenogenetic embryos. Cotransfer of three parthenogenetic embryos significantly improved the pregnancy rate on Day 40 in code 3 in vivo-fertilized embryos compared with single transfer without parthenogenetic embryos (65% vs. 35%). However, the pregnancy rate on Day 90 (35%) in cotransfer of code 3 in vivo-fertilized embryos did not differ from that upon single transfer (29%), because the cotransfer group had a higher incidence of pregnancy loss than with single transfer (47% vs. 17%) after Day 40. Cotransfer did not affect the pregnancy rate of code 2 in vivo-fertilized embryos. The incidence of pregnancy loss was higher in cotransfer of code 2 in vivo-fertilized embryos than in single transfer (30% vs. 7%). In conclusion, parthenogenetic embryos in the elongation phase secreted IFNT, enabling induction of maternal recognition of pregnancy. The present study revealed that enhancement of the maternal recognition of pregnancy using parthenogenetic embryos promoted the viability of poor-quality embryos until Day 40 of gestation. However, the incidence of pregnancy loss increased after Day 40 in the cotransfer of parthenogenetic embryos. A technique for promoting the full-term survival of poor-quality embryos is needed.

The presence of disease-associated prion protein in skeletal muscle of cattle infected with classical bovine spongiform encephalopathy.

The aim of this study was to investigate the presence of disease-associated prion protein (PrP(Sc)) in the skeletal muscle of cattle infected with classical bovine spongiform encephalopathy (C-BSE). The study was carried out systematically in 12 different muscle samples from 43 (3 field and 40 experimental) cases of C-BSE; however, muscle spindles were not available in many of these cases. Therefore, analysis became restricted to a total of 31 muscles in 23 cattle. Even after this restriction, low levels of PrP(Sc) were detected in the muscle spindles of the masseter, intercostal, triceps brachii, psoas major, quadriceps femoris and semitendinosus muscles from 3 field and 6 experimental clinical-stage cases. The present data indicate that small amounts of PrP(Sc) are detectable by immunohistochemistry in the skeletal muscles of animals terminally affected with C-BSE.

Embryo sexing and sex chromosomal chimerism analysis by loop-mediated isothermal amplification in cattle and water buffaloes.

In domestic animals of the family Bovidae, sex preselection of offspring has been demanded for convenience of milk/beef production and animal breeding. Development of the nonsurgical embryo transfer technique and sexing methods of preimplantation embryos made it possible. Sexing based on detection of Y chromosome-specific DNA sequences is considered the most reliable method to date. PCR enables amplification of a target sequence from a small number of blastomeres. However, it requires technical skill and is time consuming. Furthermore, PCR has the risk of false positives because of DNA contamination during handling of the PCR products in duplicate PCR procedures and/or electrophoresis. Therefore, for embryo sexing to become widely used in the cattle embryo transfer industry, a simple, rapid and precise sexing method needs to be developed. Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification method, and the reaction is carried out under isothermal conditions (range, 60 to 65 C) using DNA polymerase with strand displacement activity. When the target DNA is amplified by LAMP, a white precipitate derived from magnesium pyrophosphate (a by-product of the LAMP reaction) is observed. It is noteworthy that LAMP does not need special reagents or electrophoresis to detect the amplified DNA. This review describes the development and application of an embryo sexing method using LAMP in cattle and water buffaloes.

Differences in apoptotic status in the bovine placentome between spontaneous and induced parturition.

We conducted this study to analyze apoptotic changes in the bovine placentome at spontaneous and induced parturition. Cows delivered i) after the administration of dexamethasone followed by prostaglandin F(2α) and estriol, ii) after the administration of prostaglandin F(2α) and estriol or iii) spontaneously. Prepartum changes in plasma progesterone and estradiol-17ß concentrations were similar between spontaneous and induced parturition. Messenger RNA of BCL2-related protein A1 (BCL2A1), an antiapoptotic gene, was expressed by trophoblast binucleate cells and caruncular epithelial cells. Quantitative RT-PCR showed that the expression of BCL2A1 mRNA in cotyledonary and caruncular portions was significantly lower in spontaneous parturition than induced parturition. The expression of BCL2-associated X protein (BAX) mRNA, a proapoptotic gene, was significantly higher in cotyledons at spontaneous parturition than parturition induced without dexamethasone. Caspase-3 (CASP3) mRNA and pre-activated CASP3 protein were predominantly detected in caruncular epithelial cells regardless of how parturition proceeded. Activated CASP3 protein was found in trophoblast uninucleate cells and binucleate cells rather than caruncular epithelial cells. In spontaneous parturition, intense staining of activated CASP3 was detected in caruncular epithelial cells. Spontaneous and dexamethasone-induced parturition increased apoptotic cells in the placentome compared with parturition induced without dexamethasone. The number of binucleate cells was significantly decreased in spontaneous parturition. The present results suggest that although the clinical dose of dexamethasone induces apoptosis in the placentome at term, neither dexamethasone nor prostaglandin F(2α) evoke normal physiological changes in the placentome during delivery such as a change in the balance of apoptosis-related genes and disappearance of binucleate cells.

Prediction of superovulatory response in Japanese Black cattle using ultrasound, plasma anti-Müllerian hormone concentrations and polymorphism in the ionotropic glutamate receptor AMPA1/GRIA1.

The aim of this study was to improve the reliability of predicting the superovulatory response in Japanese Black cattle. Follicle counts and plasma anti-Müllerian hormone concentrations were analyzed within four days prior to the initiation of superovulation. The single nucleotide polymorphism (guanine or adenine) of the ionotropic glutamate receptor AMPA1 was determined. The plasma anti-Müllerian hormone concentration was positively correlated (P<0.001) with the numbers of all follicles and small (<5 mm) follicles and with the numbers of ova/embryos (P<0.001), fertilized embryos (P<0.001) and transferable embryos (P=0.005). There was no significant difference in follicle counts and superovulatory responses between donor cows bearing guanine/adenine or guanine/guanine alleles of AMPA1. Donor cows with a high plasma anti-Müllerian hormone concentration and homozygous for the guanine-containing allele of AMPA1 were most responsive to superovulation. The results suggest that physiological and genetic markers of superovulation have a synergistic effect on the accuracy of predictions of responsiveness.

Neuroanatomical distribution of disease-associated prion protein in experimental bovine spongiform encephalopathy in cattle after intracerebral inoculation.

The pathologic disease-associated prion protein (PrP(Sc)) has been shown to be expressed in the central nervous system of Holstein cattle inoculated intracerebrally with 3 sources of classical bovine spongiform encephalopathy (BSE) isolates. Several regions of the brain and spinal cord were analyzed for PrP(Sc) expression by immunohistochemical and Western blotting analyses. Animals euthanized at 10 months post-inoculation (mpi) showed PrP(Sc) deposits in the brainstem and thalamus, but no vacuolation; this suggested that the BSE agent might exhibit area-dependent tropism in the brain. At 16 and 18 mpi, a small amount of vacuolation was detected in the brainstem and thalamus, but not in the cerebral cortices. At 20 to 24 mpi, when clinical symptoms were apparent, heavy PrP(Sc) deposits were evident throughout the brain and spinal cord. The mean time to the appearance of clinical symptoms was 19.7 mpi, and the mean survival time was 22.7 mpi. These findings show that PrP(Sc) accumulation was detected approximately 10 months before the clinical symptoms of BSE became apparent. In addition, the 3 sources of BSE prion induced no detectable differences in the clinical signs, incubation periods, neuroanatomical location of vacuoles, or distribution and pattern of PrP(Sc) depositions in the brain.

Prepartum maternal plasma glucose concentrations and placental glucose transporter mRNA expression in cows carrying somatic cell clone fetuses.

In this study, the plasma glucose concentrations of cows carrying a somatic cell clone fetus during late pregnancy and placental glucose transporter (GLUT) mRNA levels at parturition were examined. Parturition was induced using dexamethasone, prostaglandin F(2α) and estriol in cows bearing a clone (Clone) or a fetus fertilized in vivo as a control (DEX). Plasma glucose concentrations were measured in the cows (days 257 and 271 of pregnancy and at parturition) and newborn calves. Cotyledon and caruncle tissues removed just after parturition were used for mRNA extraction. Expression of mRNA was also analyzed in control cows that were induced to undergo parturition without dexamethasone (PG) or that spontaneously delivered (SP). The glucose concentrations of the Clone group were significantly low at all points examined, but those of the calves were normal. The increase in the maternal glucose concentration from day 257 to parturition was significantly lower in the Clone group. Glucose concentrations were negatively correlated with birth weight for clones (day 257; r=-0.584, day 271; r=-0.286, parturition; r=-0.549). There was no difference in mRNA levels in the cotyledons among the animals examined. In the caruncles, the Clone and PG groups showed significantly higher GLUT1 and GLUT3 mRNA levels than the SP group, and the GLUT3 mRNA level was significantly higher in the Clone group than in the DEX group. The glucocorticoid receptor α mRNA level was significantly lower in the SP group than in the DEX group. Although spontaneous parturition and administration of dexamethasone suppressed the placental GLUT mRNA levels, the action was not observed in clone pregnancy. These results raise the possibility of facilitation of glucose transportation through the placenta to meet increased nutritional requirements of overgrown clone fetuses.