An intracytoplasmic injection of deionized bovine serum albumin immediately after somatic cell nuclear transfer enhances full-term development of cloned mouse embryos

In mouse somatic cell nuclear transfer (SCNT), polyvinylpyrrolidone (PVP) is typically included in the nuclear donor injection medium. However, the cytotoxicity of PVP, which is injected into the cytoplasm of oocytes, has recently become a cause of concern. In the present study, we determined whether bovine serum albumin deionized with an ion-exchange resin treatment (d-BSA) was applicable to the nuclear donor injection medium in SCNT as an alternative to PVP. The results obtained showed that d-BSA introduced into the cytoplasm of an enucleated oocyte together with a donor nucleus significantly enhanced the rate of in vitro development of cloned embryos to the blastocyst stage compared with that of a conventional nuclear injection with PVP in SCNT. We also defined the enhancing effects of d-BSA on the blastocyst formation rate when d-BSA was injected into the cytoplasm of oocytes reconstructed using the fusion method with a hemagglutinating virus of Japan envelope before oocyte activation. Furthermore, immunofluorescence experiments revealed that the injected d-BSA increased the acetylation levels of histone H3 lysine 9 and histone H4 lysine 12 in cloned pronuclear (PN) and 2-cell embryos. The injection of d-BSA before oocyte activation also increased the production of cloned mouse offspring. These results suggested that intracytoplasmic injection of d-BSA into SCNT oocytes before oocyte activation was beneficial for enhancing the in vitro and in vivo development of mouse cloned embryos through epigenetic modifications to nuclear reprogramming.     

Primary analysis of DNA polymorphisms in the TRIM region (MHC subregion) of the Japanese quail,Coturnix japonica

Based on sequences of two cosmid clones from Japanese quail (Coturnix japonica, Coja), we confirmed that the syntenic cluster, GNB2L1～BTN1～BTN2, is located in the quail TRIM subregion of the quail major histocompatibility complex (MHC Coja) region. These cosmids also included four CjBG loci and one CjLEC locus; therefore, the quail TRIM subregion was thought to be adjacent to the BG/LEC subregion. We then identified three polymorphic markers – CjHEP21, CjTRIM39.2 and CjBTN2 – in the TRIM subregion that may be useful for the functional analysis of the MHC‐Coja region. We examined MHC‐Coja sequences from 321 individual quails sampled from 11 inbred strains, and we found eight alleles for each of the three genes – CjHEP21, CjTRIM39.2 and CjBTN2. These polymorphisms represent the first avian DNA markers in the TRIM subregion. Additionally, we discovered a quail‐specific VNTR (variable number of long tandem repeats, 133–137 bp) in intron 7 of CjBTN2. We identified 25 haplotypes in the sample of 321 quail; these haplotypes comprised combinations of all 24 alleles of the three polymorphic genes. We suggest that there are two recombination hotspots, one between each pair of adjacent loci. All strains, except AMRP, contained multiple haplotypes; the AMRP strain contained a single, apparently fixed haplotype.     

A new method for recording activities of spinal motoneurons during fictive swimming in paralyzed teleost fish

ABSTRACT: A new method for monitoring spinal motoneuron activities during fictive swimming in teleost fish was developed. Enamel-insulated copper wire electrodes were implanted in the trunk muscle of goldfish or carp. For each freely moving fish, an electromyogram (EMG) was recorded using the electrodes. In those fish paralyzed with curare, using the same electrode set, bursts of electrical activities consisted of spikes of smaller amplitude and of shorter duration compared with those recorded by EMG. Simultaneous recording of the extracellular activity and intracellular recording from the muscle revealed that the bursts of spikes recorded in the paralyzed fish were motor nerve impulses innervating the muscle and are considered to be fictive swimming activity. The method developed in the present study provides a useful tool with which to investigate neuronal mechanisms underlying swimming activity in teleost fish.     

Allelic frequency of PCR-RFLP type of the BoLA-DRB3 in Japanese Holstein herds and the relation to mastitis

Holstein Cows ( n  = 702) from 26 dairy herds in the Tama area of Tokyo, Japan were examined for polymorphisms of the BoLA-DRB3 allele using a PCR-RFLP method. Twenty alleles were observed and allelic frequencies ranged from < 1% to 20.3%. Nine alleles ( DRB3.2 * 24, * 16, * 8, * 23, * 22, * 3, * 11 , * 10 and * 7 in order) constituted 90.0% of all alleles. Somatic cell counts (SCC) were used to classify healthy (group 1), mastitis (group 2) and suspected (group 3) cows. Frequencies of DRB3.2 * 11 and DRB3.2 * 23 were slightly higher in group 1 than in group 2, whereas, frequencies of DRB3.2 * 8 and DRB3.2 * 16 were slightly higher in group 2 than in group 1. However, none of the differences in frequencies between the two groups were statistically significant. For combinations of alleles, frequencies of DRB3.2 * 8/ * 23 ( P  < 0.1) and DRB3.2 * 16/ * 24 ( P  < 0.05) were significantly higher in group 2 than in group 1, and their odds ratios were 2.1, 2.5, respectively. However, there were no significant differences between genotypes in their effects for SCC. On the other hand, frequency of DRB3.2 * 23/ * 23 including combinations of DRB3.2 * 23 with minor alleles was significantly higher in group 1 than in group 2 ( P  < 0.01), and the odds ratio was 0.3. Therefore, it was considered that mastitis resistance or susceptibility of cows may vary with the combination of BoLA-DRB3 alleles.     

Cardiovascular and Pulmonary Effects of Sevoflurane Anesthesia in Horses

The effects of 1.0, 1.5, and 2.0 minimum alveolar concentration (MAC) of sevoflurane on hemodynamic, pulmonary and blood chemistry variables were measured during spontaneous and controlled ventilation in healthy horses. Sevoflurane was the only anesthetic drug administered to the horses. In a dose-dependent manner, sevoflurane significantly decreased ( P <.05) mean arterial blood pressure, cardiac output, and stroke volume. There was a progressive decrease in peripheral vascular resistance and an increase in heart rate as the concentration of sevoflurane was increased, but the differences were not significant. During spontaneous ventilation there was a dose-dependent decrease in respiratory rate that caused a decrease in the minute volume. As the dose of sevoflurane increased, the arterial carbon dioxide tension also increased ( P <.05). All blood chemistries remained within normal limits. Recovery from anesthesia was without incident. In conclusion, sevoflurane induces a dose-dependent decrease in hemodynamic variables and pulmonary function in horses that is not greatly different from that of other approved inhalant anesthetics.