Antioxidative Effects of Astaxanthin against Nitric Oxide‐Induced Oxidative Stress on Cell Viability and Gene Expression in Bovine Oviduct Epithelial Cell and the Developmental Competence of Bovine IVM/IVF Embryos

The aim of the present study was to elucidate the fundamental mechanism of bovine oviduct epithelial cell (BOEC) co‐culture on developmental capacity of bovine in vitro oocyte maturation/in vitro fertilization (IVM/IVF) embryos. We examined the effects of astaxanthin against nitric oxide‐induced oxidative stress on cell viability by MTT assay, lipid peroxidation (LPO) by using thiobarbituric acid (TBA) reaction for malondialdehyde (MDA) and the expression of antioxidant genes (CuZnSOD, MnSOD and Catalase) or apoptosis genes (Bcl‐2, Caspase‐3 and Bax) by RT‐PCR in BOEC. We also evaluated the developmental rates of bovine IVM/IVF embryos co‐cultured with BOEC pre‐treated with astaxanthin (500 μm ) in the presence or absence of sodium nitroprusside (SNP, 1000 μm ) for 24 h. Cell viability in BOEC treated with SNP (50–2000 μm ) lowered, while astaxanthin addition (50–500 μm ) increased it in a dose‐dependent manner. Cell viability in astaxanthin plus SNP (1000 μm ) gradually recovered according to the increase in astaxanthin additions (100–500 mm ). The LPO in astaxanthin group (50–500 μM) gradually decreased in a dose dependent manner and among SNP or astaxanthin plus SNP group, SNP alone and astaxanthin (50 μM) plus SNP shown a significant increase than other groups (p < 0.05). Expression of apoptosis or antioxidant genes was detected by RT‐PCR. Bcl‐2 and antioxidant genes were detected in astaxanthin or astaxanthin plus SNP group, and Caspase‐3 and Bax genes were only found in SNP group. When bovine IVM/IVF embryos were cultured for 6–7 days under co‐culture system such as BOEC treated with astaxanthin in the presence or absence of SNP, the developmental ability to blastocysts in 500 μm astaxanthin group was the highest of all groups. These results suggest that astaxanthin has a antioxidative effect on cell viability and LPO of BOEC, and development of bovine IVM/IVF embryos due to the induction of antioxidant genes and suppression of apoptosis genes.     

Exogenous DNA Uptake of Boar Spermatozoa by a Magnetic Nanoparticle Vector System

The sperm‐mediated gene transfer method is applicable to transgenesis in many species that use spermatozoa for reproduction recently, which has been shown various results. In the current study, we show that transgenic porcine embryos can be efficiently produced by employing a simple transfection method that uses magnetic nanoparticles (MNPs). The complexes formed between plasmid DNA and MNPs were bounded on ejaculated boar spermatozoa at a higher efficiency compared to methods using DNA alone or lipofection. Using confocal microscopy, rhodamine fluorophore‐labelled MNPs were detected on external surfaces of the spermatozoa membrane, which were bounded on zona pellucida of in vitro maturated oocyte during in vitro fertilization. Electron microscopy revealed that clusters of MNPs were detected in inside of plasma membrane and nucleus of the spermatozoa head. Additionally, we found that magnetofected boar spermatozoa could be fertilized with oocytes in vitro and that the resulting gene of green fluorescent protein was detected in fertilized eggs by genomic PCR analysis. Taken together, these results suggest that MNPs can be used to efficiently introduce a transgene into embryo via spermatozoa.     

Leukotrienes Affect Secretory Function of Ovarian Cells In Vitro*

The aim of this study was to determine which cells are the source of production and target for leukotriene (LTs) action within the bovine ovary. Luteal (CL, days 14–16 of the oestrous cycle), steroidogenic cells (LSC) and endothelial cells (LEC) of the bovine corpus luteum (CL), and granulosa cells (GC) were isolated enzymatically, cultured in a monolayer and incubated with LTC₄, LTB₄, Azelastine (an antagonist of LTC₄) or Dapsone (an antagonist of LTB₄). Then cells were collected for determination of mRNA expression for LT receptors (LTRs) and 5‐lipoxygenase (5‐LO) by real time RT‐PCR, and media were collected for determination of prostaglandin (PG)E₂, F_2α, progesterone (P4; LSC only), endothelin‐1 (ET‐1; LEC only) and 17‐β oestradiol (E2; GC only). The greatest mRNA expression for LTR‐II and 5‐LO were found in LEC, whereas LTR‐I mRNA expression did not differ among cell types. The level of PGE₂ increased after LTs treatment in each type of ovarian cell, excluding LTC₄ treatment in LEC. The secretion of PGF_2α was also increased by LTs, but decreased after LTB₄ treatment of LSC. In GC cultures, both LTs stimulated E2 secretion; in LEC cultures, LTB₄ stimulated whereas LTC₄ inhibited P4 secretion; in LEC cultures, LTC₄ stimulated but LTB₄ inhibited ET‐1 secretion. The results show that LTs are produced locally and are involved in PGs production/secretion in all examined cells (LSC, LEC and GC) of bovine ovary. Leukotriene treatment modulate secretion of E2, by GC, P4 by LSC and ET‐1 by LEC, which indicates that LTs are involved in regulation of ovarian secretory functions.     

Cytoarchitectural differences of myoepithelial cells among goat major salivary glands

Previously, the distribution of myoepithelial cells (mecs) in the salivary glands was studied by both immunohistochemistry, and transmission electron microscopy; however, little was elucidated concerning their morphological features, especially in goats. This study was performed to investigate the correlation between the cytoarchitecture of the mecs in goat major salivary glands (parotid, mandibular, and sublingual glands) and the nature of the saliva secretion. The cytoarchitectural features of the mecs were examined by scanning and transmission electron microscopy as well as immunohistochemically. The secretory endpieces in the parotid gland are of the pure serous type, but in both the mandibular and sublingual glands they are of the mixed type. In all studied glands, the intercalated ducts were covered by mecs which, unlike the large stellate cells that surrounded the secretory endpieces, were spindle-shaped with few cytoplasmic processes. Interestingly, the mecs were found to bulge on the basal surfaces of the serous acini and intercalated ducts in all glands and to be in close contact to the seromucous tubules surface in the mandibular and sublingual glands forming a continuous network around it. In conclusion, the differences in the degree of development of the mecs as well as the number of their cytoplasmic processes may be correlated with the nature of the secretion and the number of the secretory granules. Thus these observations may have some relevance in the diagnosis of atrophy and pathogenic conditions of these glands.     

Protective potential of an attenuated Pasteurella multocida,which expresses only the N-terminal truncated fragment of P. multocida toxin

Pasteurella multocida serogroup D causes progressive atrophic rhinitis in pigs and produces a potent, intracellular, mitogenic toxin known as P. multocida toxin (PMT), which is encoded by the toxA gene. Highly toxic to cells, PMT is a poor antigen and becomes more immunogenic after its native structure has been destroyed. Previously, we found that the N-terminal fragment of PMT (N-PMT) can induce a strong immune response that is protective against wild-type challenge. Here, an attenuated P. multocida mutant expressing only N-PMT was developed and its protective effect was evaluated. The mutant provides protective immune responses against bacterial and toxin challenges, and so is a good live vaccine candidate.     

饲粮不同赖氨酸水平对早期断奶仔猪生产性能的影响

试验旨在研究饲粮不同赖氨酸水平对早期断奶仔猪生产性能的影响。选择28日龄断奶、平均初始体重(6.74±0.03)kg的杜长大三元杂交仔猪150头,随机分为3组,每组2个重复(圈),每个重复25头猪,分别饲喂总赖氨酸水平为1.35%、1.42%、1.60%的3种饲粮,饲养试验时间为20d。结果表明,各项生产性能指标均随饲粮总赖氨酸水平提高呈二次曲线变化。以处理Ⅱ(总赖氨酸水平1.42%)为最佳,随着赖氨酸水平的降低或升高,生产性能均下降。当总赖氨酸水平提高到1.60%(处理Ⅲ)时,其日增重、日采食量和每克赖氨酸增重均显著低于处理Ⅱ(P0.05)。     

The optimal size and placement of transdermal electrodes are critical for the efficacy of a transcutaneous pacemaker in dogs

Transcutaneous cardiac pacing (TCP) can be used in dogs with a high risk for bradyarrhythmias prior to anesthesia, either in an emergency room or intensive care unit setting. Furthermore, TCP can also be used on patients diagnosed with bradyarrhythmias that require temporary pacing at the induction of anesthesia for the implantation of a permanent pacemaker. Despite the importance of TCP in emergency medicine, no studies have evaluated the optimal size and placement of the transdermal electrodes crucial for the efficacy of TCP in dogs.This study evaluated four different sizes of electrodes (10.5, 20, 30 and 40 cm²), and four different anatomical sites (anterior–posterior, left–right, apex–base, modified left–right) in order to optimize the efficacy of TCP in dogs. Electrodes with a surface area of 20 cm² and a modified left–right placement minimized the pacing current and involuntary skeletal muscular contraction (muscular twitching) and so achieved the most optimal effect of TCP in dogs.