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Mesangial cell-glomerular basement membrane (GBM) connections play an important role in maintaining the structural integrity of the glomerular tuft. However, information remains scarce about how GBM maturation affects the formation of these connections during glomerular development. Here, we show that abnormal GBM maturation due to tensin 2 deficiency affects mesangial cell differentiation by impairing mesangial cell-GBM connections during postnatal glomerulogenesis.

Tensin2 (Tns2), an integrin-linked protein, is enriched in podocytes within the glomerulus. Previous studies have revealed that Tns2-deficient mice exhibit defects of the glomerular basement membrane (GBM) soon after birth in a strain-dependent manner. However, the mechanisms for the onset of defects caused by Tns2 deficiency remains unidentified. Here, we aimed to determine the role of Tns2 using newborn Tns2-deficient mice and murine primary podocytes. Ultrastructural analysis revealed that developing glomeruli during postnatal nephrogenesis exhibited abnormal GBM processing due to ectopic laminin-α2accumulation followed by GBM thickening. In addition, analysis of primary podocytes revealed that Tns2 deficiency led to impaired podocyte-GBM interaction and massive expression of laminin-α2in podocytes. Our study suggests that weakened podocyte-GBM interaction due to Tns2 deficiency causes increased mechanical stress on podocytes by continuous daily filtration after birth, resulting in stressed podocytes ectopically producing laminin-α2, which interrupts GBM processing. We conclude that Tns2 plays important roles in the podocyte-GBM interaction and maintenance of the glomerular filtration barrier.

In female mammals, luteal cells rapidly proliferate and form corpora lutea (CLs) after ovulation. The corpus luteum (CL) plays crucial roles in establishing and maintaining pregnancy. To gain further insights into the role of cellular FLICE-like inhibitory protein (cFLIP), an anti-apoptosis factor that is structurally similar to procaspase-8 but lacks proteolytic enzyme activity, we examined the expression in CLs of Thai swamp buffalos (Bubalus bubalis) during the early, mid, and late stage of the estrous cycle and pregnancy. cFLIP short form and long form (cFLIP S and cFLIP L , respectively) mRNA and protein levels were assessed by reverse transcription-polymerase chain reaction and western blotting, respectively. cFLIP S mRNA levels were low in the mid and late stages of the estrous cycle and increased during pregnancy (P L mRNA was highly expressed in CLs during pregnancy and was lower in the mid and late stages of the estrous cycle. The level of cFLIP S protein was high in CLs during pregnancy and low levels were noted in the mid stage of the estrous cycle (P L protein were demonstrated in CLs during pregnancy and lower levels were found in CLs during the early stage of the estrous cycle. Strong positive immunohistochemical staining for cFLIP S/L proteins was observed in luteal cells during pregnancy. The present findings revealed that cFLIP was at the highest level in CLs during pregnancy, and this may act as a dominant survival anti-apoptotic factor by inhibiting intracellular apoptosis signal transduction in luteal cells of CLs during pregnancy.

C1s deficiency is strongly associated with the development of human systemic lupus erythematosus (SLE); however, the mechanisms by which C1s deficiency contributes to the development of SLE have not yet been elucidated in detail. Using ICR-derived-glomerulonephritis (ICGN) mouse strain that develops SLE and very weakly expresses C1s in the liver, we investigated the protective roles of C1s against SLE. A genetic sequence analysis revealed complete deletion of the C1s1 gene, a mouse homolog of the human C1s gene, with partial deletion of the C1ra and C1rb genes in the ICGN strain. This deletion led to the absence of C1r/C1s and a low level of C1q in the circulation. In order to investigate whether the C1r/C1s deficiency induces SLE, we produced a congenic mouse strain by introducing the deletion region of ICGN into the C57BL/6 strain. Congenic mice exhibited no C1r/C1s and a low level of C1q in the circulation, but did not have any autoimmune defects. These results suggest that C1r/C1s deficiency is not sufficient to drive murine SLE and also that other predisposing genes exist in ICGN mice.

Due to the Fukushima Daiichi nuclear power plant accident, a tremendous amount of organic waste (e.g., baled grass silage) contaminated with radioactivity was generated in Tohoku region, northeastern Japan. To establish a safe and efficient way to treat cesium contaminated silage, we investigated the use of aerobic, high temperature composting. Radiocesium (137Cs and 134Cs) contaminated silage (2000 kg, approximately 2700 Bq/kg), water (4000 kg) and matured compost soil (as inoculum, 16,000 kg) were mixed by a wheel loader, and then the mixture was piled up. Air was supplied from the bottom of a compost pile continuously, and the fermentation continued for 7 weeks. The temperature at 100 cm below the surface reached approximately 100 °C. The water content decreased to less than 30% after 7 weeks. The level of radioactive cesium in the final product (18,000 kg) was 265 Bq/kg, which was below the tolerance value for fertilizer (400 Bq/kg) suggested by the Japanese government. The radioactive cesium within silage remained in the final products. We cultivated tomato (fruit), soybean (seed), carrot (root), Italian ryegrass (leaf feed for livestock), Swiss chard (leaf), cosmos (flower) and field mustard (seed) in an experimental farm fertilized with the matured compost made from the radiocesium contaminated silage, for 3 months. Radiocesium levels of edible parts and non-edible parts of each crop were lower than 20 Bq/kg, which was less than one-fifth of the Japanese government value for food (100 Bq/kg). This research demonstrated that the final product can be used safely as an organic fertilizer.

The antioxidant effect of N, N-dimethylglycine (DMG) on in vitro-produced (IVP) bovine embryos was examined. After in vitro fertilization, presumptive zygotes were cultured with or without 0.1 μM DMG under different oxygen tensions. The percentage of embryos developing to the blastocyst stage was lowest under a 20% oxygen concentration without DMG, and it was significantly increased (P < 0.05) by applying a 5% oxygen concentration. Under the 20% oxygen concentration, supplementation of the medium with DMG significantly improved blastocyst development, which was nearly equal to that achieved under 5% oxygen without DMG. Furthermore, a tendentious increase (P = 0.06) in blastocyst cell numbers was observed when DMG was applied. In the second experiment, addition of H2O2 (0.5 mM) to the culture medium significantly (P < 0.01) reduced the percentage of embryos developing to the blastocyst stage. However, DMG supplementation prevented this reduction. In conclusion, DMG enhanced the in vitro development of IVP bovine embryos by acting as an antioxidant.

The antioxidant effect of N, N-dimethylglycine (DMG) on in vitro-produced (IVP) bovine embryos was examined. After in vitro fertilization, presumptive zygotes were cultured with or without 0.1 μM DMG under different oxygen tensions. The percentage of embryos developing to the blastocyst stage was lowest under a 20% oxygen concentration without DMG, and it was significantly increased (P < 0.05) by applying a 5% oxygen concentration. Under the 20% oxygen concentration, supplementation of the medium with DMG significantly improved blastocyst development, which was nearly equal to that achieved under 5% oxygen without DMG. Furthermore, a tendentious increase (P = 0.06) in blastocyst cell numbers was observed when DMG was applied. In the second experiment, addition of H2O2 (0.5 mM) to the culture medium significantly (P < 0.01) reduced the percentage of embryos developing to the blastocyst stage. However, DMG supplementation prevented this reduction. In conclusion, DMG enhanced the invitro development of IVP bovine embryos by acting as an antioxidant.

Tensin2 (Tns2) is an essential component for the maintenance of glomerular basement membrane (GBM) structures. Tns2-deficient mice were previously shown to develop mild glomerular injury on a DBA/2 background, but not on a C57BL/6J or a 129/SvJ background, suggesting that glomerular injury by the deletion of Tns2 was strongly dependent on the genetic background. To further understand the mechanisms for the onset and the progression of glomerular injury by the deletion of Tns2, we generated Tns2-deficient mice on an FVB/N (FVB) strain, which is highly sensitive to glomerular disease. Tns2-deficient mice on FVB (FVBGN) developed severe nephrotic syndrome, and female FVBGN mice died within 8 weeks. Ultrastructural analysis revealed that FVBGN mice exhibited severe glomerular defects with mesangial process invasion of glomerular capillary tufts, lamination and thickening of the GBM and subsequent podocyte foot process effacement soon after birth. Aberrant laminin components containing α1, α2 and β1 chains, which are normally expressed in the mesangium, accumulated in the GBM of FVBGN, suggesting that these components originated from mesangial cells that invaded glomerular capillary tufts. Compared to Tns2-deficient mice on the other backgrounds in previous reports, FVBGN mice developed earlier onset of glomerular defects and rapid progression of renal failure. Thus, this study further extended our understanding of the possible genetic background effect on the deterioration of nephrotic syndrome by Tns2 deficiency.

Cooperation of the Hungarian Research Institute for Animal Breeding and Nutrition (ATK) with the German Leibniz Institute for Farm Animal Biology (FBN) goes back many decades. In 1988 the two departments of reproductive biology began a joint project concerning on ovulation investigation in Landrace pigs. This joint project laid the foundation for further joint projects and has existed for almost 30 years. Over the years, the main focus has always been on the events of the female reproductive tract in pigs, i.e., follicular growth, ovulation, transport of gametes in the oviduct, fertilization and early embryonic development. Nearly all studies were done under in vivo circumstances and using different clinical and endocrinological methods, enabling us to obtain more profound knowledge of the dynamics of reproductive processes. Even results considered to be basic scientific achievements were available for utilization in the daily practice of porcine reproductive management. Since the end of the 1990s, the common projects have been gradually shifted to the physiology of the Hungarian indigenous pig breed Mangalica. Research partners were convinced that modern utilization of indigenous pig breeds would open new doors for premium pork production. In addition to the Mangalica breed, this principal was broadened to non-European fatty-type native pig breeds as well and resulted in long-term, intercontinental scientific cooperation.

In Japan, a criterion value of histamine residue in food is not clearly defined and there is no official test method. We examined histamine in fish and fish products according to the food sanitation test guideline (fluorescence derivatization of histamine with dansyl chloride and quantification by LC-FL: the LC-FL method). Positive samples were confirmed by determining dansylated histamine using our developed LC-MS/MS procedure (the LC-MS/MS method) when histamine was detected. Validation was earried out according to the validation test guideline using fresh fish. Recovery tests of histamine from fresh fish spiked at the level of 20 ppm were carried out. The limit of quantification was 5 ppm. The results confirmed that our LC-MS/MS method is applicable for the inspection of fish and fish products. This LC-MS/MS method has a lower false-positive ratio and a higher selectivity than the LC-FL method.

Collagen in an eggshell membrane is important for egg preservation, medical burn treatment and manufacturing of cosmetics. Because collagen in the membrane is little, it is a need to improve the accumulation in the membrane to develop these applications. Wood charcoal powder with vinegar (WCV) is a natural substance that improves poultry production. In hen fed with WCV, total collagen in the eggshell membrane increased with an increase in dietary WCV and significantly increased in the 1.0% WCV group (p < 0.05). Scanning and light microscopic images revealed that this group had thicker eggshell membranes and a fine mesh structure composed of finer and more densely distributed fibres than in the control. Eggs from WCV group showed slow Haugh unit decrease during egg storage and the decrease correlated with total collagen in eggshell membrane. In intact chicken, type I and type III collagens were found in different specific locations in the oviduct but not in the membrane. The finding that collagen accumulates in the eggshell membrane under WCV feeding suggests that feeding chicken with WCV will permit long-term storage of eggs in poultry production, and the increased volume of total collagen will facilitate its application in medicine and cosmetics.

Background/Aims: Tenc1 (also known as tensin2) is an integrin-associated focal adhesion molecule that is broadly expressed in mouse tissues including the liver, muscle, heart and kidney. A mouse strain carrying mutated Tenc1, the ICR-derived glomerulonephritis (ICGN) strain, develops severe nephrotic syndrome. Methods: To elucidate the function of Tenc1 in the kidney, Tenc1ICGN was introduced into 2 genetic backgrounds, i.e. DBA/2J (D2) and C57BL/6J (B6), strains that are respectively susceptible and resistant to chronic kidney disease. Results: Biochemical and histological analysis revealed that homozygous Tenc1ICGN mice develop nephrotic syndrome on the D2 background (D2GN) but not on the B6 background (B6GN). Initially, abnormal assembly and maturation of glomerular basement membrane (GBM) were observed, and subsequently effacement of podocyte foot processes was noted in the kidneys of D2GN but not B6GN mice. These defects are likely to be involved in the integrin signaling pathway. Conclusion: This study suggests that Tenc1 contributes to the maintenance of GBM structures and that the genetic background influences the severity of nephrotic syndrome.

Background: ICR-derived glomerulonephritis (ICGN) strain is a novel inbred strain of mice with a hereditary nephrotic syndrome. Deletion mutation of tensin 2 (Tns2), a focal adhesion molecule, has been suggested to be responsible for nephrotic syndrome in ICGN mice; however, the existence of other associative factors has been suggested. Methods and Results: To identify additional associative factors and to better understand the onset mechanism of nephrotic syndrome in ICGN mice, we conducted a comprehensive gene expression analysis using DNA microarray. Immune-related pathways were markedly altered in ICGN mice kidney as compared with ICR mice. Furthermore, the gene expression level of complement component 1, s subcomponent (C1s), whose human homologue has been reported to associate with lupus nephritis, was markedly low in ICGN mouse kidney. Real-time quantitative reverse transcription-polymerase chain reaction confirmed a low expression level of C1s in ICGN mouse liver where the C1s protein is mainly synthesized. A high serum level of anti-dsDNA antibody and deposits of immune complexes were also detected in ICGN mice by enzyme-linked immunosorbent assay and immunohistochemical analyses, respectively. Conclusion: Our results suggest that the immune system, especially the complement system, is associated with nephrotic syndrome in ICGN mice. We identified a low expression level of C1s gene as an additional associative factor for nephrotic syndrome in ICGN mice. Further studies are needed to elucidate the role of the complement system in the onset of nephrotic syndrome in ICGN mice.

The present study aimed to clarify changes of oxidative stress and antioxidative functions in treadmill-exercised Thoroughbred horses (n=5, 3 to 7 years old), using recently developed techniques for measurement of serum d-ROMs for oxidative stress, and BAP for antioxidative markers. Also, the effect of nasogastric administration of hydrogen-rich water (HW) or placebo water preceding the treadmill exercise on these parameters was examined. Each horse was subjected to a maximum level of treadmill exercise in which the horses were exhausted at an average speed of 13.2 ± 0.84 m/sec. Blood samples were taken 4 times, immediately before the intake of HW or placebo water at 30 min preceding the treadmill exercise, immediately before the exercise (pre-exercise), immediately after the exercise (post-exercise) and at 30 min following the exercise. In all horses, both d-ROMs and BAP values significantly increased at post-exercise. The increase in d-ROMs tended to be lower in the HW trial, as compared to the placebo trial at pre-exercise. The increase in BAP was considerable at approximately 150% of the pre-exercise values in both the HW and placebo treatment trials. The BAP/d-ROMs ratio was significantly elevated at post-exercise in both treatment trials, while a significant elevation was also observed at pre-exercise in the HW trial. BAP, d-ROM, and the BAP/d-ROM ratio tended to decline at 30 min after the exercise, except BAP and BAP/d-ROMs in the placebo trial. These results demonstrate that the marked elevation of oxidative stress and anitioxidative functions occurred simultaneously in the intensively exercised horses, and suggest a possibility that HW has some antioxidative efficacy.

Mitochondria are reported to be critical in in vitro maturation of oocytes and subsequent embryo development after fertilization, but their contribution for fertilization has not been investigated in detail. In the present study, we investigate the contribution of mitochondria to fertilization using reconstructed porcine oocytes by fusion of ooplasmic fragments produced by serial centrifugations (centri-fusion). Firstly, we evaluated the characteristics of ooplasmic fragments. Three types of fragments were obtained by centrifugation of porcine oocytes matured in vitro for 46 h: brownish (B), transparent (T) and large (L) fragments containing both B and T parts in a fragment. The production efficiencies of these types of fragments were 71.7, 91.0 and 17.8 fragments/100 oocytes, respectively. In experiments, L fragments were excluded because they contained both brownish and transparent components that were apparently intermediate between B and T fragments. Observations by confocal microscopy after staining with MitoTracker Red CMXRos® and transmission electron microscopy revealed highly condensed active mitochondria in B fragments in contrast to T fragments that contained only sparse organelles. We reconstructed oocytes by fusion of a karyoplast and two cytoplasts from B and T fragments (B and T oocytes, respectively). The B oocytes showed higher sperm penetration (95.8%) and male pronuclear formation rates (94.2%) by in vitro fertilization than T oocytes (66.7% and 50.0%, respectively). These results suggest that the active mitochondria in oocytes may be related to their ability for fertilization.

Selective breeding by humans has resulted in various breeds of domestic animals. Great diversity between different breeds has been created not only in body size and coat color, but also in behavioral traits and personality. For example, dogs of different breeds differ greatly in social behavioral traits [9, 15]. Differences between breeds in personality, as measured by questionnaire surveys, are also reported in horses [11]. Breed differences in behaviors are especially important in companion animals such as dogs or horses. Scientific evaluation of behavioral differences between breeds should be useful for the development of methods of management or training better suited to each breed. Variability of behavioral traits between breeds may be partly affected by genes. Recent studies have shown that genetic polymorphisms in genes related to neurotransmitters or hormones influence individual differences in personality [7]. The dopamine receptor D4 gene (DRD4) encodes one of the subtypes of dopamine receptor, and is one of the candidate genes underlying individual differences in personality in many species including humans. In human DRD4 there is a variable number of tandem repeats (VNTR), which is reported to be associated with a “novelty-seeking” trait [1, 2]. In dogs, breed differences in the allele frequency of DRD4 have been reported. For example, a VNTR in the canine DRD4 exon3 region is reported to be associated with aggressiveness and activity/impulsivity [5]. The allele frequency of this polymorphism is different among breeds [8], particularly between Asian breeds and European breeds [10]. In horses, DRD4 is located on chromosome 12 [13], and two types of polymorphisms have been found. They are VNTR consisting of 18 base pairs (6 amino acids) and some single nucleotide polymorphisms (SNPs) in the exon3 region [3]. One of these SNPs, G292A, was reported to be associated with horse personality scores measured by a questionnaire survey. The A allele in G292A is associated with low curiosity and high vigilance in Thoroughbred horses [12]. If there are breed differences in personality related genes such as DRD4, it may be possible to predict the behavioral traits of breeds based on the genotype. A previous study reported that one Kiso horse, a native Japanese horse breed, had shorter repeats in the VNTR region than Thoroughbred horses [3]. However, whether the allele frequency of this polymorphism differs between breeds has not yet been verified. Samples from more breeds are needed to validate the breed differences of DRD4 in horses. In this study, we investigated breed differences in equine DRD4 using samples from more breeds than previously studied. We collected samples from two breeds of European origin (Thoroughbred and Selle Francais), one South American breed (Criollo), three native Japanese breeds (Hokkaido, Taishu and Yonaguni) and one native Korean breed. We explored the polymorphisms in DRD4 and compared its allele frequency among breeds.

Contamination of food and animal products by radioactive cesium represents an important potential route of exposure in the human food chain. Therefore, following the Fukushima Daiichi nuclear power plant accident, the development of solutions for radiocesium contamination is a serious social issue in Japan. Most farm animals are kept in closed barns in Japan; this reduced the initial contamination of animal products by radioactive nuclides in the early phase of the accident. Furthermore, pigs and chickens were given imported feed that was not contaminated. However, more than 10 million tons of grass feed were needed for herbivorous livestock in Japan. We report the effects of “clean feeding” management on livestock products contaminated with radioactive cesium due to the nuclear accident. The present results concerning products from herbivores (horses, sheep, and cattle) revealed that radiocesium levels were undetectable in the meat or milk of herbivores that fed on non-contaminated feed after an initial period of consuming radiocesium-contaminated feed. Thus, we conclude that “clean feeding” management can play a crucial role in solving the social problem of food contamination.

Appropriate treatment is urgently needed for farm and ranch waste that has been contaminated with radioactive cesium from the Fukushima Daiichi nuclear power plant accident. We previously developed an aerobic ultra-high temperature fermentation (more than 115 °C) method to inhibit intestinal infectious diseases. Fermented waste (compost), in which pathogens were sterilized, was useful as a fertilizer to grow crops. In the present study, we examined the kinetics of radioactive cesium in farming fields to promote sustainable circular agriculture including livestock in farms and pasture fields in wide areas of Tohoku and Kanto, in an approximately 200 km radius from the nuclear power plant. The compost produced at the experimental ranch of the Animal Resource Science Center, the University of Tokyo, was contaminated with radioactive cesium (approximately 900 Bq/kg). Some crops (soybean, sweet corn, eggplant, bitter gourd, potato, cabbage, and ginger) were cultivated in cubic holes (approximately 1 × 1 × 1 m) that were filled with contaminated compost in the field of the experimental ranch. Each crop was planted in a hole and cultivated in an appropriate manner for a suitable time period. Radiocesium levels in the roots, stems, leaves, and fruits of each crop at harvest were lower than 20 Bq/kg, which was below the new reference/regulation value (100 Bq/kg) for food. In conclusion, when crops were planted using compost contaminated with radioactive cesium (900 Bq/kg; more than twice the new tolerance value of 400 Bq/kg for fertilizer and compost), the radiocesium levels in the crops were as low as one-fifth of the new reference/regulation value.

To clarify the effect of lactation period on ovarian follicular activity and associated hormonal levels in goats, six goats were monitored daily by ultrasonographic examination with blood sampling during early (Days 5 to 25; Day 0 was the day of kidding) and late (Days 40 to 60) lactation. While the presence of a corpus luteum of pregnancy retarded follicular growth in the ipsilateral ovary until Days 11-13 postpartum, the total follicular number (TFN) and area (TFA) increased during late lactation due to the significant increase in the number of medium- and large-sized follicles and decrease in the number of small follicles. Four goats showed a similar pattern of follicular development during the period studied characterized by the emergence of five and six waves during the early and late lactation, respectively. The largest follicle diameter of the first three waves monitored during early lactation was significantly smaller as compared with the diameter of those existing during late lactation. TFN showed a positive correlation with FSH but showed a negative correlation with immunoreactive (ir-) inhibin and estradiol during the postpartum period. TFA was positively correlated with ir-inhibin, estradiol and PRL and negatively correlated with FSH during the monitored periods. The plasma levels of ir-inhibin and progesterone were significantly higher during late lactation compared with the levels recorded during early lactation. Ir-inhibin levels showed a significant positive correlation with LH and estradiol during early and late lactation but showed a negative correlation with FSH during the whole lactation period. LH was positively correlated with estradiol and PRL during early and late lactation, respectively. These results suggest that the lactation period has a detrimental effect on ovarian activity during the early postpartum period in goats.

The mammalian ovary is an extremely dynamic organ in which a large majority of follicles are effectively eliminated throughout their reproductive life. Due to the numerous efforts of researchers, mechanisms regulating follicular growth and atresia in mammalian ovaries have been clarified, not only their systemic regulation by hormones (gonadotropins) but also their intraovarian regulation by gonadal steroids, growth factors, cytokines and intracellular proteins. Granulosa cells in particular have been demonstrated to play a major role in deciding the fate of follicles, serving molecules that are essential for follicular growth and maintenance as well as killing themselves by an apoptotic process that results in follicular atresia. In this review, we discuss the factors that govern follicular growth and atresia, with a special focus on their regulation by granulosa cells. First, ovarian folliculogenesis in adult life is outlined. Then, we explain about the regulation of follicular growth and atresia by granulosa cells, in which hormones, growth factors and cytokines, death ligand-receptor system and B cell lymphoma/leukemia 2 (BCL2) family members (mitochondria-mediated apoptosis) are further discussed.

Pig production continues to be an important livelihood activity in the Lao People's Democratic Republic (Lao PDR) which base on traditional methods. However, it is increasingly affected by social and market pressures. More than 80 % of pig herds are native breeds and belong to smallholders with combined keeping systems. Based on a national field survey, the native pigs have been characterized and classified in four phenotypes. The native pig types and their local distribution are described. Most of them have short ears, black colour and potbelly. Their production performance, i.e. growth rates and daily weight gain is lower and the carcass fat considerably higher than that of exotic (i.e. in Lao terms »modern, intensive«) breeds. However, they require lower inputs and have excellent adaptation traits.

Contents Despite normal eucrasia, mating desire and semen quality, sire bulls sometimes have spermatozoa with poor freezing tolerance. This study assessed effects of the addition of linoleic acid albumin (LAA) and long-term (LT) equilibrium to frozen semen on their sperm freezing tolerance. Immediately after collection using an artificial vagina and a breeding mount, semen was diluted with yolk citrate buffer; then, it was cooled slowly to 4°C during more than 5 h. Equilibrium treatment at 4°C was applied using the same extender supplemented with glycerol. Semen of bull A, with low sperm freezing tolerance, was treated with 1 mg/ml of LAA added to the first extender. The equilibrium treatment at 4°C was prolonged to 30 h. Significantly higher motility rates were obtained for the LT + LAA-treated sperm before and after freezing–thawing. However, for semen of bulls B and C with normal sperm freezing tolerance, the LT + LAA treatment barely exhibited a small effect on the motility rate. Almost no difference was found among bulls A, B and C in the motility rates of LT + LAA-treated sperm after freezing–thawing. No difference of fertility was apparent on LT + LAA-treated frozen sperm in comparison with normal sperm in embryonic collection and in vitro fertilization. It was not an aberration of fertility in vivo or in vitro. In addition, the conception rate of artificial insemination did not have a difference, and a normal calf was obtained. Results show that addition of LAA to an extender for frozen bovine spermatozoa and 30 h of low-temperature equilibrium might improve the motility of freezing–thawing spermatozoa with poor freezability. Sperm exhibited normal fertilization capability and ontogenic capability.

We have shown in pigs that oocytes denuded of cumulus cells at 24 h of in vitro maturation culture and subsequently matured for a total of 46 h (DO24 oocytes) have lower cytoplasmic maturity than those matured with cumulus cells for 46 h and then denuded (DO46 oocytes). In the present study, DO24 zona-free oocytes were fused with one (1C) or two (2C) cytoplasmic fragments produced by serial centrifugation ("centri-fusion") of DO46 oocytes (DO24+1C and DO24+2C oocytes, respectively). Groups of (1) DO46 (a control), (2) DO24, (3) DO24+1C and (4) DO24+2C oocytes were partheno-activated by an electrical pulse or fertilized in vitro and subsequently cultured for 6 days. In the fused groups, female pronucleus (FPN) formation rates were higher than that in the DO24 group after parthenogenetic activation (PA); however, the blastocyst rates were intermediate between those of the control and DO24 groups. After in vitro fertilization, the male pronucleus (MPN) formation rates in the fused groups were similar to that in the control group and higher than that in the DO24 group; the normal fertilization rate in the DO24+2C group was higher than that in the DO24 group and similar to that in the control group, resulting in significantly higher blastocyst rates in the DO24+2C and control groups than that in the DO24 group. These results suggest that centri-fusion using ooplasm from fully matured DO46 oocytes can offer a potentially novel approach for restoration of cytoplasmic maturity to oocytes with low developmental ability and subsequent improvement of fertilization and developmental competence.

More than 99% of follicles undergo "atresia" during follicular development and growth. Follicular atresia is predominantly regulated by granulosa cell apoptosis. However, the intracellular signaling pathway of apoptosis in granulosa cells has not been revealed. In the present study, we examined changes in the expression of BH3-interacting domain death agonist (Bid) and Bcl-2-associated X protein (Bax), which are considered to promote the cell death ligand/receptor-mediated process in mitochondrion-dependent type II apoptosis, in porcine granulosa cells during atresia. Levels of mRNA and protein of Bid and Bax were determined by the reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting techniques, respectively. Levels of Bid and Bax mRNA and protein were markedly increased in granulosa cells of early atretic follicles compared with those of healthy follicles. In situ hybridization and immunohistochemical staining revealed that mRNA and protein of Bid and Bax were present in the granulosa cells, though only traces were found in healthy follicles; however, strong staining was noted in atretic follicles. These results indicate that Bid and Bax appear to be signal transduction factors in granulosa cells during follicular atresia and appear to play proapoptotic roles and confirm that the porcine granulosa cell is a mitochondrion-dependent type II apoptotic cell.

福島第一原子力発電所事故の3週後(4月8日)及び12週後(6月3日)に東京大学大学院農学生命科学研究科附属牧場(原発から直線距離で約130km南方の茨城県笠間市に位置する)において採取した土壌，牧草，乳牛飲料用井戸水，及び牛乳中の131I，134Cs及び137Csの放射能濃度を測定したので報告する。測定の結果，飼料中の放射性核種は速やかに牛乳中に移行することがわかった。しかし，いずれの測定でも牛乳中の131I，134Cs及び137Csの濃度は国の暫定許容値(放射性ヨウ素：300Bq/kg，放射性セシウム：200Bq/kg)を下回る値であった。

In mammalian ovaries, most follicles are lost by atresia before ovulation. It has become apparent that the apoptosis of granulosa cells induces follicular atresia. Forkhead box O3 (FOXO3), also called FKHRL1 (forkhead in rhabdomyosarcoma-like 1), is a proapoptotic molecule that belongs to the FOXO subfamily of forkhead transcription factors. Foxo3-deficient female mice were reported to be infertile because of abnormal ovarian follicular development, but the precise influences of FOXO3 on follicular atresia of mature ovary have not been determined. Therefore, we examined the expression and function of FOXO3 in porcine ovarian follicles and granulosa-derived cells. FOXO3 mRNA levels in granulosa cells of porcine ovaries increased during atresia, while FOXO3 protein was abundant in granulosa cells of early atretic follicles. By immunohistochemistry, the inner surface area of the granulosa layer in early atretic follicles was strongly stained with anti-FOXO3 antibody. The granulosa cells expressing FOXO3 coincided with apoptotic cells, indicating a role of FOXO3 as a proapoptotic factor in granulosa cells of porcine ovaries. In porcine (JC-410) and human (KGN) granulosa-derived cells, cell death was induced by transfection of FOXO3 expression vectors. Expression of the proapoptotic factors Fas ligand (FASLG) and BCL2-like 11 (BCL2L11) was upregulated by FOXO3 in KGN cells. In conclusion, FOXO3 is expressed in porcine ovarian follicles and induces apoptosis in granulosa cells, suggesting that it is a candidate for the initiator of follicular atresia.

To clarify the toxic effects and fate of zearalenone (ZEA) in ruminants, we studied histopathological changes and toxicokinetic profiles in goats administered with a single intravenous (iv) injection of ZEA at doses of 2.4 mg/kg bw and 1.2 mg/kg bw, respectively. The expression of the mRNA of estrogen receptor (ER) alpha and beta in tissues was also investigated. The histopathological study revealed that ZEA caused hepatocellular swelling and lymphocytic infiltration in the liver, kidney, and uterus. The expression of ERalpha mRNA was enhanced by ZEA in association with the histopathological changes, indicating the possible involvement of ERalpha in the toxic effects of ZEA. For toxicokinetic profiles, blood plasma, urine, and feces were collected consecutively after iv injection of ZEA and analyzed for ZEA and its metabolites with high performance liquid chromatography (HPLC). alpha-Zearalenol (ZOL) and beta-ZOL were detected with ZEA, but alpha-zearalanol (ZAL), beta-ZAL, and zearalanone were below the detection limits. The distribution half-life (t(1/2alpha)) and elimination half-life (t(1/2beta)) of ZEA were 3.15 and 28.58h, respectively. ZEA, alpha-ZOL, and beta-ZOL were excreted in urine and feces, with beta-ZOL being the predominant metabolite. The ZEA and ZOL in urine were largely in their glucuronide and/or sulphate conjugated forms, while those in feces were largely in their free forms. This study showed the toxic effect of zearalenone and its metabolites, and their pharmacokinetic characteristics in goats.

The current study was performed to follow up the circulating hormonal changes and to correlate the findings with the physiological activity of the corpus luteum (CL) and placenta during pregnancy in goats. Blood samples were collected weekly from five goats during pregnancy for measuring steroid and protein hormones. A gradual increase was observed in immunoreactive (ir-) inhibin, with maximal levels at the 17th week. The plasma concentrations of estradiol and prolactin (PRL) showed nearly similar patterns during pregnancy, where they declined to basal levels during the first 4 weeks post-breeding and then increased significantly, with the maximal concentration during late pregnancy. The plasma FSH and LH concentrations were maintained at basal levels throughout the gestation period. The plasma progesterone concentration abruptly increased in the first week post-breeding and remained at high values throughout the pregnancy period. Immunohistochemical localization of inhibin alpha, beta(A), beta(B) and steroidogenic enzymes cytochrome P450 aromatase, 3alpha-hydroxysteroid dehydrogenase (3betaHSD), cytochrome 17alpha-hydroxylase P450 and cholesterol side-chain cleavage cytochrome P450 in the cyclic and pregnant goat CL revealed positive immunoreactivity without affinity differences between the luteal and pregnancy stages. The placental syncytiotrophoblasts also showed positive staining, except for inhibin beta(A) and 3betaHSD. The giant binucleate cells of the placenta showed positive immunoreactions to PRL. These results suggest that the high concentrations of ir-inhibin, estradiol and PRL during late pregnancy are of placental origin and that the placenta may have a vital role in the maintenance of pregnancy, regulation of mammary growth and preparation for kidding and lactation in goats.

The present study was conducted to characterize follicular development and its hormonal control during early pregnancy in goats. The ovaries of goats (n=8) were scanned daily for follicles (> or = 2 mm in diameter) and corpora lutea by transrectal ultrasound with blood sampling from the jugular vein for monitoring the hormonal changes during the first thirty-five days after mating. During early pregnancy, three (37.5%), four (50%) and one (12.5%) goat showed nine, eight and seven waves of follicular development, respectively. The corpora lutea were detected as early as Day 3.61 ± 0.45 (7.47 ± 0.43 mm) of pregnancy (Day 0=day of mating) and attained their maximal cross-sectional diameter (10.64 ± 0.37 mm) on Day 25.7 ± 0.8 of pregnancy, respectively. A transient rise in FSH levels was temporally associated with the day of follicular wave emergence (up to three days prior to wave emergence). The plasma LH and estradiol levels were negatively correlated with the progesterone concentration. The rise in plasma immunoreactive (ir) inhibin levels was negatively correlated with the FSH concentration and positively correlated with the number of large-sized follicles. Alternatively, the mean plasma ir-inhibin levels showed a noticeable decline with the progression of pregnancy. The present results demonstrated that follicular development during early pregnancy shows a wave-like pattern, with seven to nine waves developing until Day 35 after breeding, and that the number of follicular waves can be predicted by the number of FSH peaks. The current study also demonstrated that the role of inhibin as an FSH regulator is maintained throughout early pregnancy.

Background/Aims: Growth hormone (GH) is secreted in pulsatile fashion, but the involvement of neuropeptides in the generation of GH pulses is not fully understood. The present study was conducted to elucidate the relationship between GH pulses and neuropeptide levels in the cerebrospinal fluid (CSF) of the third ventricle in ovariectomized goats. Methods: CSF and plasma samples were collected every 15 min. Levels of plasma GH and profiles of GH-releasing hormone (GHRH), somatostatin (SRIH) and neuropeptide Y (NPY) in the CSF were measured by radioimmunoassay. Pulse/trough characteristics and correlations were assessed by the ULTRA algorithm, cross-correlation analysis and approximate entropy test. Results: The periodicity of GH pulses was 2.20 h. Although most GH pulses were associated with GHRH peaks, there was no correlation between GH and GHRH or GH and SRIH. NPY levels in the CSF fluctuated episodically at 2.03-hour intervals. GH pulse peaks occurred 0–30 min after NPY troughs, and there was a significant negative cross-correlation and negative synchronicity between GH and NPY profiles. In addition, intracerobroventricular infusion of NPY suppressed GH secretagogue (KP102)-induced GH release. Conclusion: The periodic decrease in NPY may be involved in the generation of GH pulsatility in goats.

Deproteinization has been recognized as a prerequisite for amino acid analysis of plasma samples. For plasma stored at room temperature, delaying deproteinization for 30, 60 or 120 minutes did not result in significant changes in the mean CV (coefficient of variation), which ranged from 4.4 to 5.6%. However the mean CV of aspartic acid, α-aminoadipic acid, alanine and lysine was about 10%. When the plasma was stored frozen at -20°C, the CV was increased at 0 and 120 minutes after thawing, to 12.4% (range, 4.1 to 35.3%) and 8.0% (2.5 to 30.7%), respectively. The concentrations in plasma during storage at room temperature of all the amino acids analyzed showed significant changes. In plasma stored for 30 minutes at room temperature, 17 amino acids increased in concentrations and two decreased. Extending this period to 60 or 120 minutes increased the instability as compare to the reference group. Storing plasma at -20°C for 2 weeks resulted in significantly greater changes in the amino acid concentrations than at room temperature. On extending the storage time at room temperature, after thawing, to 30, 60, and 120 minutes, 21, 20, and all 22 amino acids respectively changed significantly (p

ICR-derived glomerulonephritis (ICGN) mice are a known inbred strain with hereditary nephrotic syndrome and are considered a good animal model of human idiopathic nephrotic syndrome. ICGN mice show proteinuria at a young age, and hypoalbuminemia, hyperlipidemia, anemia and edema accompanies their symptoms with aging. In addition, ICGN mice develop severe anemia with the progression of renal fibrosis similar to human chronic kidney disease (CKD). Recently, tissue transglutaminase (tTG) has been shown to be related to the renal fibrosis in several animal models and CKD patients. In the present study, we investigated the relationship between the progression of renal fibrosis and the localization of tTG in the kidneys using histochemistry and image analysis. Male ICGN mice aged 26-43 weeks were used. They were divided into two groups of early and terminal stages of renal fibrosis, based on plasma levels of blood urea nitrogen (BUN). Normal ICR males aged 11 weeks were used as a control group. tTG was localized to the interstitium in the normal ICR mice. In the early stage of renal fibrosis, the localization of tTG increased in renal tubules showing luminal dilation, as well as in the interstitium; however, the amount of tubular and interstitial tTG decreased in the late stage. In the glomeruli, tTG-immunoreactivity decreased in the late stage of renal fibrosis, despite the progression of glomerular sclerosis. The results suggest that epsilon(gamma-glutamyl) lysine cross-linking is not directly related to the progression of renal fibrosis in ICGN mice.

It is suggested that pathogenic prion protein (PrP(sc) may accumulate in peripheral nervous tissues in addition to the specified-risk materials. To assess the risk of PrP(sc) accumulation in peripheral tissues, the present study was designed to study the ratio of peripheral nervous tissues in tongues, skeletal muscles and intestines in 2-week- and 59-month-old cows. The nervous tissues could be detected by immunohistochemistry using antibody Neurofilament 150. After immunostaining with Neurofilament 150, the photographs taken from each section were applied for Scion Image software. Then, the ratio of nervous tissues in tongues, skeletal muscles and intestines were calculated. The highest ratio of peripheral nervous tissues was observed in intestines, the second highest ratio was found in tongues, and the lowest ratio was recognized in skeletal muscles in both 2-week- and 59-month-old cows. The ratio of nervous tissues in intestines was approximately 2.5 fold and 10 fold, compared to that in skeletal muscles in 2-week- and 59-month-old cows, respectively. These results suggest that intestines and tongues may have a higher risk of PrP(sc) accumulation, compared to skeletal muscles.

More than 99% of follicles in mammalian ovaries undergo atresia, but the mechanisms regulating the strict selection process are still unclear. Granulosa cell apoptosis is considered the trigger of follicular atresia, which occurs in advance of the death of an oocyte. Cellular FLICE-like inhibitory protein (cFLIP), a homologue of procaspase-8 (also called FLICE), is an intracellular anti-apoptotic protein. It is expressed in granulosa cells of porcine ovaries, where its levels decreases during follicular atresia. We hypothesized that cFLIP regulates granulosa cell apoptosis by acting as a pro-survival factor. In the present study, to further reveal the function of cFLIP in granulosa cells, we examined the anti-apoptotic mechanism of cFLIP using KGN, a human granulosa tumor cell line. Fas-mediated apoptosis was induced by co-treatment with anti-Fas antibody (CH-11), which acts as an agonist of Fas-ligand, and cycloheximide (CHX). When cFLIP was stably expressed in KGN cells following transfection of an expression vector, the Fas-mediated apoptosis was inhibited. Suppression of cFLIP by small interfering RNA (siRNA) spontaneously induced cell death. Silencing of cFLIP promoted cleavage of procaspase-8, and the cell death caused by cFLIP siRNA was completely blocked by a caspase-8 inhibitor (Z-IETD-FMK), indicating that cFLIP regulates apoptosis in KGN cells by inhibiting cleavage of procaspase-8. In conclusion, cFLIP is an essential pro-survival factor for granulosa cells, and it prevents granulosa cell apoptosis by inhibiting procaspase-8 activation.

The apoptosis inhibitory ligand (Netrin-1) and its receptor (p53-regulated receptor for death and life: p53RDL1) play an important role in the regulation of selective apoptosis. When Netrin-1 binds to p53RDL1, p53-dependent apoptosis is inhibited. We identified porcine (Sus scrofa) cDNAs encoding Netrin-1 [pNetrin-1; 1,803 base pairs (bp) and 600 amino acids (aa)] and p53RDL1 (pp53RDL1; 2,838 bp and 945 aa). Porcine p53RDL1 (pp53RDL1) contains a death domain (DD), a tandem specific amino acid region, in its C-terminal, suggesting that it mediates death signaling by binding with other pro-apoptotic factors via the DD. Porcine Netrin-1 (pNetrin-1), pp53RDL1 and the DD in pp53RDL1 showed high levels of identity in aa sequence with human and murine Netrin-1 (98 and 97%, respectively), p53RDL1 (94 and 91%, respectively) and the DD in p53RDL1 (96 and 95%, respectively). Reverse transcription-polymerase chain reaction (RT-PCR) revealed that the levels of pNetrin-1 and pp53RDL1 mRNAs were moderate in granulosa cells compared with their expression in other tissues and that their levels during follicular atresia were stable. The Netrin-1 and p53RDL1 system may regulate the induction of apoptosis in porcine granulosa cells.

Several hundred thousand primordial follicles are present in the mammalian ovary, however, only 1% develop to the preovulatory stage and finally ovulate. The remainder will be eliminated via a degenerative process called ‘atresia’. The endocrinological regulatory mechanisms involved in follicular development and atresia have largely been characterized but the precise temporal and molecular mechanisms involved in the regulation of these events remain unknown. Many recent studies suggest that apoptosis in ovarian granulosa cells plays a crucial role in follicular atresia. Notably, death ligand-receptor interaction and subsequent intracellular signaling have been demonstrated to be the key mechanisms regulating granulosa cell apoptosis. In this review we provide an overview of granulosa cell apoptosis regulated by death ligand-receptor signaling. The roles of death ligands and receptors [Fas ligand (FasL)]-Fas, tumor necrosis factor α (TNFα)-TNF receptor and TNFα-related apoptosis-inducing ligand (TRAIL)-TRAIL receptor (TRAILR)] and intracellular death-signal mediating molecules (Fas-associated death domain protein), TNF receptor 1-associated death domain protein, caspases, apoptotic protease-activating factor 1, TNFR-associated factor 2 and cellular FLICE-like inhibitory protein in granulosa cells are discussed.

To clarify whether enzymes involved in aflatoxin B1 (AFB1) metabolism in pigs respond to antioxidant agents, the effect of feeding piglets with diets containing green tea extracts (Sunphenon) and coumarin on in vitro AFB1 metabolism by their liver and intestinal tissues was studied. The results showed that coumarin reduced AFB1-DNA adduct formation by both liver and intestinal microsomes, while Sunphenon did not have any effects. Both coumarin and Sunphenon enhanced the glutathione S-transferase (GST) activity to conjugate AFB1 to glutathione GSH in the intestine, although no effects were noted in the liver. Changes of the expression of mRNA of GSTA2 and GSTO1 were not in parallel with the observed changes of GST activity, suggesting that other GST subtypes are involved in the GST activity toward AFB1. As for lipophilic-free AFB1 metabolites, coumarin reduced the liver microsomal conversion of AFB1 to aflatoxin M1 (AFM1) and aflatoxin Q1 (AFQ1), but Sunphenon exerted no effects. Both coumarin and Sunphenon enhanced the conversion of AFB1 to aflatoxicol in the liver. All the results suggest that feeding with a diet containing coumarin affects AFB1 metabolism to enhance AFB1 detoxification through the suppression of P450 enzyme activity in the liver and the enhancement of GST activity in the intestine. Feeding with a diet containing Sunphenon enhances AFB1 detoxification, but the effects are noted mainly in the intestine.

More than 99% of follicles undergo a degenerative process known as "atresia" in mammalian ovaries, and only a few follicles ovulate during follicular growth and development. Follicular selection predominantly depends on granulosa cell apoptosis. To reveal the molecular mechanisms of selective follicular atresia, we examined the changes in the levels of interleukin-6 (IL-6) receptors expressed in the granulosa cells of pig ovaries. The levels of IL-6 receptor (IL-6R)-alpha mRNA and protein in granulosa cells were quantified by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively. IL-6R alpha mRNA and protein were highly expressed in the granulosa cells of progressed atretic follicles. Enzyme-linked immunosorbent assay showed that the expression of IL-6 soluble receptor (IL-6sR) protein in follicular fluid decreased during atresia. Moreover, we isolated porcine cDNA encoding an IL-6 signal transducer, gp130. Porcine gp130 (2,754 bp and 917 amino acids) was identified from a cDNA library prepared using follicular granulosa cells of pig ovaries. Porcine gp130 was highly homologous with human and murine gp130. RT-PCR analysis revealed that the level of gp130 mRNA also decreased during atresia. We presume that IL-6sR and gp130, but not IL-6R alpha, play important roles in regulation of granulosa cell survival.

Tumor necrosis factor (TNF) alpha can induce both cell death and proliferation by binding to either TNF receptor (TNFR) 1 or 2. In the granulosa cells of porcine ovaries, TNFalpha is considered to act as an anti-apoptotic/survival factor during follicular atresia. As a first step toward elucidating the function of TNFR2 in regulating follicular development/growth and atresia in porcine ovaries, we isolated the porcine (Sus scrofa) cDNA encoding TNFR2, which was identified from a cDNA library prepared from the follicular granulosa cells of pig ovaries. Porcine TNFR2 (1,125 bp, 375 amino acid residues), which contains specific amino acid region of transmembrane, indicated high identities with human and murine TNFR2 (78 and 69% at mRNA level, respectively; 73 and 61% at protein level, respectively), suggesting that the function of porcine TNFR2 is similar to that of human and murine homologues. Understanding the expression patterns of porcine TNFR2 mRNA in various organs, which we confirmed by reverse transcription polymerase chain reaction analysis, would help to elucidate the physiological role of TNFR2 in the regulation of apoptosis in porcine organs.

The mRNA expression of GPRC5B, an orphan G protein-coupled receptor, is induced by retinoic acid (RA). Because RA plays critical roles in embryonic development, reproductive functions, metabolism and homeostasis, GPRC5B is also considered crucial in these physiological events. We investigated the changes in expression of GPRC5B and RA receptor (RAR) alpha mRNAs and immunohistochemical localization of their proteins in the murine placenta and yolk sac at 13.5, 15.5 and 17.5 days post coitus. Stable levels of GPRC5B and RARalpha mRNAs were detected in the placenta and yolk sac. In the placenta, GPRC5B was present in maternal and fetal vascular endothelial cells, stromal cells, fibroblast-like cells and glycogen cells. A strong reaction to RARalpha was detected in maternal and fetal vascular endothelial cells and stromal cells. The levels of GPRC5B and RARalpha proteins in maternal and fetal vascular endothelial cells decreased with gestation. In the yolk sac, GPRC5B and RARalpha proteins were detected in vascular endothelial cells, but their levels did not change during the gestation period. These findings indicate that GPRC5B is involved in RA-dependent morphogenesis/angiogenesis and regulation of extracellular matrix synthesis in the murine placenta and yolk sac.

Two major apoptotic signaling pathways have been defined in mammals, the extrinsic pathway, initiated by ligation of death receptors, and the intrinsic pathway, triggered by cytochrome c release from mitochondria. Here, we identified and characterized the Xenopus homologs of caspase-10 (xCaspase-10beta), a novel initiator caspase, and Bid (xBid), a BH3-only molecule of the Bcl-2 family involved in both the extrinsic and intrinsic pathways. Exogenous expression of these molecules induced apoptosis of mammalian cells. By biochemical and cytological analyses, we clarified that xCaspase-10beta and xBid exhibit structural and functional similarities to their mammalian orthologues. We also detected xCaspase-10beta and xBid transcripts during embryogenesis by whole-mount in situ hybridization and RT-PCR analysis. Microinjection of mRNA encoding a protease-defect xCaspase-10beta mutant into embryos resulted in irregular development. Enforced expression of active xBid induced cell death in developing embryos. Using transgenic frogs established to allow monitoring of caspase activation in vivo, we confirmed that this form of cell death is caspase-dependent apoptosis. Thus, we demonstrated that the machinery governing the extrinsic and intrinsic apoptotic pathways are already established in Xenopus embryos. Additionally, we propose that the functions of the initiator caspase and BH3-only molecule are evolutionarily conserved in vertebrates, functioning during embryonic development.

The Wuzhishan miniature pig originates from Hainan Island, P. R. China, and is considered useful for medical and veterinary research due to its small size; a mature adult weights less than 25 kg. To reveal the genetic characteristics of this breed, the genetic polymorphism in 4 inbred strains was estimated using 30 microsatellite genes, multiplex polymerase chain reaction, and gene scanning. The average number of alleles at the 30 marker loci was 8.300, 4.533, 6.700, and 7.433, respectively. The polymorphism information content was 0.632, 0.507, 0.653, and 0.691, respectively. The average homozygosity in these strains was 0.537, 0.546, 0.549, and 0.520, respectively. These results indicate a relatively high degree of heterozygosity, perhaps becaquse these strains were inbred for 3 generations. Therefore, a long-term inbred breeding program should be conducted for Wuzhishan pigs to establish an experimental model.

In the pig, a temporal relationship is suggested between sperm release from the sperm reservoir (SR) and ovulation, but the mechanism(s) is still under discussion. In two experiments, the influence of transferred ova on the release of SR-spermatozoa at ovulation and the effect of supplementation with non-sulfated glycosaminoglycan hyaluronan (HA) on embryo development and the number of accessory spermatozoa, respectively, were examined. PMSG/hCG primed ovectomized gilts that had previously received endoscopic low-dose insemination into the cranial uterine horn were used as an experimental model. After salpingectomy, tubal segments (ampulla, cranial, and caudal isthmus) were flushed and sperm numbers or respective accessory spermatozoa were counted. In Experiment 1, the distribution of the sperm population was altered in the presence of cumulus-oocyte-complexes (COCs). A higher proportion of spermatozoa was found after transfer of COCs into one oviduct in the ampulla and cranial isthmus segments compared with the controls (17.5 vs. 4.9%, p

Intravascular accumulation of blood cells after brain ischemia-reperfusion can cause obstruction of cerebral blood flow and tissue hypoxia/ischemia as a consequence. In the present study, we examined temporal and topographic changes of tissue hypoxia/ischemia after occlusion of the middle cerebral artery (MCA) for 60 min in rats with immunohistochemical staining for hypoxia (2-nitroimidazole hypoxia marker: hypoxyprobe-1 adducts). Our results showed that tissue hypoxia expressed as positive staining for hypoxyprobe-1 adducts preceded neuronal degeneration. Platelets and granulocytes were detected close to the hypoxyprobe-1 adducts positive area. These results suggested that the hypoxic environment could persist even after reperfusion of MCA, because of vascular obstruction with accumulation of platelets and granulocytes.