Microdroplet In Vitro Fertilization Can Reduce the Number of Spermatozoa Necessary for Fertilizing Oocytes

Successful in vitro fertilization (IVF) in mice has been achieved using spermatozoa at concentrations specifically optimized for the experimental conditions, such as species and source of spermatozoa. Although IVF in mice is mostly performed using about 80–500 µl drops, it is expected that the number of spermatozoa used for insemination can be reduced by decreasing the size of the IVF drops. The present study was undertaken to examine the extent to which the number of spermatozoa used for IVF could be reduced by using small droplets (1 µl). We devised the experimental parameters using frozen–thawed spermatozoa from C57BL/6 mice in anticipation of broader applications to other mouse facilities. We found that as few as 5 spermatozoa per droplet could fertilize oocytes (1 or 3 oocytes per droplet), although the fertilization rates were low (13–15%). Practical fertilization rates (> 40%) could be achieved with frozen-thawed C57BL/6J spermatozoa, which are sensitive to cryopreservation, when 20 sperm per droplet were used to inseminate 3 oocytes. Even with spermatozoa from a very poor quality suspension (10% motility), about 25% of oocytes were fertilized. Our calculations indicate that the number of inseminated spermatozoa per oocyte can be reduced to 1/96–1/240 by this method. In two separate embryo transfer experiments, 60% and 47%, respectively, of embryos developed to term. Our microdroplet IVF method may be particularly advantageous when only a limited number of motile spermatozoa are available because of inadequate freezing-thawing or genetic reasons.     

Determination phase at transition of gonocytes to spermatogonial stem cells improves establishment efficiency of spermatogonial stem cells in domestic cats

The development of germ cells has not been entirely documented in the cat especially the transition phase of the gonocyte to the spermatogonial stem cell (G/SSC). The aims of study were to examine testicular development and to identify the G/SSC transition in order to isolate and culture SSCs in vitro. Testes were divided into 3 groups according to donor age (I, < 4 months; II, 4–6 months; and III, > 6 months). In Exp. 1, we studied testicular development by histology, transmission electron microscopy and immunohistochemistry. In Exp. 2, we determined the expression of GFRα-1, DDX-4 and c-kit and performed flow cytometry. The SSCs isolated from groups II and III were characterized by RT-PCR and TEM (Exp. 3). Chronological changes in the G/SSC transition were demonstrated. The size, morphology and ultrastructure of SSCs were distinguishable from those of gonocytes. The results demonstrated that group II contained the highest numbers of SSCs per seminiferous cord/tubule (17.66 ± 2.20%) and GFRα-1⁺ cells (14.89 ± 5.66%) compared with the other groups. The findings coincided with an increased efficiency of SSC derivation in group II compared with group III (74.33 ± 2.64% vs. 23.33 ± 2.23%). The colonies expressed mRNA for GFRA1, ZBTB16, RET and POU5F1. Our study found that the G/SSC transition occurs at 4–6 months of age. This period is useful for isolation and improves the establishment efficiency of cat SSCs in vitro.     

In Vitro Fertilization and Development of Porcine Oocytes Matured in Follicular Fluid

This study was conducted to assess the fertilization and development of porcine oocytes matured in a solo follicular fluid (pFF) using different in vitro culture systems and insemination periods. Cumulus-oocyte complexes (COCs), follicular cells (FCs), and pFF were collected from the follicles of ovaries. The pFF was used as a maturation medium (MpFF) after supplementation with follicle stimulating hormone (FSH) and antibiotics. The COCs were matured in a 15 ml test tube containing 3.5 ml of MpFF with FCs (5.2 × 10⁶ cells/ml; rotating culture system) or 2 ml of MpFF without FCs in a 35-mm petri dish (static culture system) for 44 to 48 h. After maturation culture, oocytes were co-incubated with frozen-thawed spermatozoa for 5 h and then cultured for 7 days. The total mean rates of sperm penetration, normal fertilization, male pronucleus (MPN) formation, cleavage, and development to the blastocyst stage of oocytes after insemination were significantly higher (P<0.01) in the rotating culture system than in the static culture system. In conclusion, compared with the static culture system, the rotating culture system is adequate for the production of developmentally competent porcine oocytes when MpFF is used as a maturation medium.     

The effects analysis of two neonicotinoid insecticides on in vitro maturation of porcine oocytes using hanging drop monoculture method

Acetamiprid (ACE) and imidacroprid (IMI) are known neonicotinoid insecticides with strong affinities for the insect-selective nicotinic acetylcholine receptor. These provide insect control by hyperstimulating insect nerves and are used for agricultural pest management. However, it has also been reported that ACE and IMI affect mammalian reproductive function. We determined the effects of ACE and IMI on the in vitro maturation of porcine oocytes. Significant decreases in nuclear maturation rates were observed in the ACE or IMI-exposed groups. Also, in matured oocytes from the ACE or IMI-exposed groups, irregular chromosomes were observed. Our results suggest that ACE and IMI exposure was detrimental to porcine oocytes and the extent of the effects depends on the concentration of exposure.     

Effects of inactive parapoxvirus ovis on cytokine levels in rats

The aim of this study is to determine the effects of iPPOV on pro-inflammatoryand anti-inflammatory cytokine levels in rats. iPPOV (1 ml/rat) wasadministered intraperitoneal route to 49 rats, except for 7 rats (Control, 0 group). Serumsamples were collected from 7 rats at 1st, 2nd, 4th, 8th, 12th, 16th and 24th hr aftertreatments. Levels of TNF-α, IL-6, IL-12 and IL-10 were determined using ELISA.Administration of iPPOV stimulated TNF-α (16th and 24th hr) and IL-6 (12th, 16th and 24thhr) synthesis and caused fluctuations in IL-10 and IL-12 concentrations. In conclusion,increased cytokine levels could be attributed to immunomodulatory activity of iPPOV,however, detailed studies are required to fully understand effects of iPPOV on immunesystem.     

Overexpression of OCT4A ortholog elevates endogenous XIST in porcine parthenogenic blastocysts

X-chromosome inactivation (XCI) is an epigenetic process that equalizes expression of X-borne genes between male and female eutherians. This process is observed in early eutherian embryo development in a species-specific manner. Until recently, various pluripotent factors have been suggested to regulate the process of XCI by repressing XIST expression, which is the master inducer for XCI. Recent insights into the process and its regulation have been restricted in mouse species despite the evolutionary diversity of the process and molecular mechanism among the species. OCT4A is one of the represented pluripotent factors, the gate-keeper for maintaining pluripotency, and an XIST repressor. Therefore, in here, we examined the relation between OCT4A and X-linked genes in porcine preimplantation embryos. Three X-linked genes, XIST, LOC102165544, and RLIM, were selected in present study because their orthologues have been known to regulate XCI in mice. Expression levels of OCT4A were positively correlated with XIST and LOC102165544 in female blastocysts. Furthermore, overexpression of exogenous human OCT4A in cleaved parthenotes generated blastocysts with increased XIST expression levels. However, increased XIST expression was not observed when exogenous OCT4A was obtained from early blastocysts. These results suggest the possibility that OCT4A would be directly or indirectly involved in XIST expression in earlier stage porcine embryos rather than blastocysts.     

Production of Diabetic Offspring Using Cryopreserved Epididymal Sperm by In Vitro Fertilization and Intrafallopian Insemination Techniques in Transgenic Pigs

Somatic cell nuclear transfer (SCNT) is a useful technique for creating pig strains that model human diseases. However, production of numerous cloned disease model pigs by SCNT for large-scale experiments is impractical due to its complexity and inefficiency. In the present study, we aimed to establish an efficient procedure for proliferating the diabetes model pig carrying the mutant human hepatocyte nuclear factor-1α gene. A founder diabetes transgenic cloned pig was generated by SCNT and treated with insulin to allow for normal growth to maturity, at which point epididymal sperm could be collected for cryopreservation. In vitro fertilization and intrafallopian insemination using the cryopreserved epididymal sperm resulted in diabetes model transgenic offspring. These results suggest that artificial reproductive technology using cryopreserved epididymal sperm could be a practical option for proliferation of genetically modified disease model pigs.     

Rescue In Vitro Fertilization Method for Legacy Stock of Frozen Mouse Sperm

Sperm cryopreservation has been widely adopted for maintenance of the genetically engineered mouse (GEM). The cryopreserved sperm are being exchanged among many institutes worldwide. However, the recipients are not always able to obtain high fertilization rates with the frozen sperm shipped from senders. In this study, we cryopreserved mouse sperm via various methods and performed in vitro fertilization (IVF) in which the combination of methyl-beta-cyclodextrin for sperm preincubation and reduced glutathione for insemination was used (the MBCD-GSH IVF). In addition, frozen sperm sent from the Jackson Laboratory (USA) were thawed and used for IVF in the same manner. The fertilization rates of both the sperm cryopreserved via the methods applied in some countries and the cryopreserved GEM sperm improved when used with the MBCD-GSH IVF method. Therefore, we strongly believe that the MBCD-GSH IVF method brings about relatively high fertilization rates with any strain of frozen mouse sperm.     

High Prevalence of BlaCTX-M Group Genes in Aeromonas dhakensis Isolated from Aquaculture Fish Species in South Korea

The prevalence of resistant genes against β-lactams in 119 Aeromonas strains was determined. A large number (99.2%) of the present fish strains were resistant to one or more β- lactams including ceftiofur, amoxicillin-clavulanic acid, ampicillin, piperacillin and cefpodoxime. Among antibiotic resistance phenotypes, the simultaneous resistance to all β-lactams occurred in 25.2% (n=30) of all strains, which consisted of 18 strains of A. dhakensis, 8 strains of A. caviae, 2 strains of A. hydrophila and only one strain of A. veronii. For exploring genetic background of the antibiotic resistances, multiple PCR assays were subjected to detect β-lactamase-encoding genes, bla_TEM, bla_OXA-B and bla_CTX-M. In the results, the bla_TEM-1 gene was harbored in all strains, whereas only 3 strains harbored bla_OXA gene. In the case of bla_CTX-M gene, the gene was detected in 21.0% (25 out of 119) of all strains, which countered with 80% (20 out of 25) of A. dhakensis, 8% (2 out of 25) of A. caviae and 12% (3 out of 25) of A. hydrophila. In addition, most of the bla_CTX-M positive strains showed simultaneous resistance to all β-lactams (18 out of 30 strains). In sequence analysis for bla_CTX-M genes detected, they were CTX-M group 1-encoding genes including bla_CTX-M-33 from 3 eel strains of A. dhakensis. Therefore, A. dhakensis obtained from cultured fish could represent a reservoir for spreading genes encoding CTX-M group 1 enzymes and hence should be carefully monitored, especially for its potential risk to public health.     

Laser induced injury caused hyperglycemia-like effect in Drosophila larva: a possible insect model for posttraumatic diabetes

Diabetic patients need particular care in case of infection, digestive disorder or external injury, because external stress often exasperates the glucose metabolism, which is known as “sick day management”. In addition, severe trauma can be a cause of hyperglycemia with insulin resistance. In spite of critical component of the treatment, the precise mechanisms of how trauma develops posttraumatic diabetes remain unknown. Here, we ablated body wall muscles in Drosophila larvae by laser beam and found that the level of trehalose, the principal sugar circulating in the hemolymph or in the tissues of most insects, was increased. The model may provide a helpful tool to understand the relationship between trauma and sugar metabolism.     

Comparison of immune responses to intranasal and intrapulmonary vaccinations with the attenuated Mycoplasma hyopneumoniae 168 strain in pigs

The aim of this study was to evaluate the immune responses to intranasal and intrapulmonary vaccinations with the attenuated Mycoplasma hyopneumoniae (Mhp) 168 strain in the local respiratory tract in pigs. Twenty-four pigs were randomly divided into 4 groups: an intranasal immunization group, an intrapulmonary immunization group, an intramuscular immunization group and a control group. The levels of local respiratory tract cellular and humoral immune responses were investigated. The levels of interleukin (IL)-6 in the early stage of immunization (P<0.01), local specific secretory IgA (sIgA) in nasal swab samples (P<0.01); and IgA- and IgG-secreting cells in the nasal mucosa and trachea were higher after intranasal vaccination (P<0.01) than in the control group. Interestingly, intrapulmonary immunization induced much stronger immune responses than intranasal immunization. Intrapulmonary immunization also significantly increased the secretion of IL-6 and local specific sIgA and the numbers of IgA- and IgG-secreting cells. The levels of IL-10 and interferon-γ in the nasal swab samples and the numbers of CD4⁺ and CD8⁺ T lymphocytes in the lung and hilar lymph nodes were significantly increased by intrapulmonary immunization compared with those in the control group (P<0.01). These data suggest that intrapulmonary immunization with attenuated Mhp is effective in evoking local cellular and humoral immune responses in the respiratory tract. Intrapulmonary immunization with Mhp may be a promising route for defense against Mhp in pigs.     

Sequence differences in the internal transcribed spacer 1 and 5.8S ribosomal RNA among three Moniezia species isolated from ruminants in Japan

This study was designed to clarify the differences in the internal transcribed spacer (ITS) 1 and 5.8S nucleotide sequences of Moniezia expansa, M. benedeni and M. monardi isolated from ruminants in Japan and to determine their phylogenetic relationships. A 98% similarity in the 5.8S sequences was observed among the 3 Moniezia species, whereas many nucleotide indels and substitutions were observed in the ITS1 sequences among the three Moniezia species. These results suggest that the ITS1 region could serve as a potential marker for discriminating the 3 Moniezia species. In the phylogenetic tree based on the ITS1 sequences, M. monardi and M. benedeni showed genetically closer relationship to each other than to M. expansa.     

Decaying toxic wood as sodium supplement for herbivorous mammals in Gabon

African rainforest harbors herbivores at high density. However, because plants and soils typically lack in some essential minerals, rainforest is not always a suitable habitat for herbivores. How they fulfill the mineral requirements is therefore an important question to animal ecology and conservation. Although large marshes, called ‘bais’, are often mentioned as efficient mineral-resource, little information on other sodium resources has still been available. Our laboratory works and field surveys found that a peculiar item, decaying wood stumps of Anthostema aubryanum, played as a major sodium resource for herbivores in Moukalaba-Doudou National Park, Gabon. When A. aubryanum is alive, the sodium content of its bark is low and its latex is toxic. Sodium is accumulated in decaying stumps (mean=1,343 mg/kg dry matter). Eight herbivores visited stumps to ingest the dead wood. Fecal sample analysis revealed that western lowland gorillas, a species most-frequently using the stumps, consumed large amount of the dead wood as regular food. Our findings suggest that decaying A. aubryanum is critical sodium-resources and is a key species for herbivores in our study area. Importance of the A. aubryanum may be particularly large there, because it is a limited sodium-rich material that is available year round. Our study site is known as the site where the densities of several herbivores are among the highest at Central Africa. The relatively high herbivores density in our study site may partly depend on decaying A. aubryanum as sodium resources.