Expression and Characterization of Constitutive Heat Shock Protein 70.1 (HSPA‐1A) Gene in In Vitro Produced and In Vivo‐Derived Buffalo (Bubalus bubalis) Embryos

Cells are blessed with a group of stress protector molecules known as heat shock proteins (HSPs), amongst them HSP70, encoded by HSPA‐1A gene, is most abundant and highly conserved protein. Variety of stresses hampers the developmental competence of embryos under in vivo and in vitro conditions. Present work was designed to study the quantitative expression of HSPA‐1A mRNA in immature oocytes (IMO), matured oocytes (MO), in vitro produced (IVP) and in vivo‐derived (IVD) buffalo embryos to assess the level of stress to which embryos are exposed under in vivo and in vitro culture conditions. Further, HSPA‐1A gene sequence was analysed to determine its homology with other mammalian sequences. The mRNA expression analysis was carried out on 72 oocytes (40 IMO; 32 MO), 76 IVP and 55 IVD buffalo embryos. Expression of HSPA‐1A was found in oocytes and throughout the developmental stages of embryos examined irrespective of the embryo source; however, higher (p < 0.05) expression was observed in 8–16 cell, morula and blastocyst stages of IVP embryos as compared to IVD embryos. Phylogenetic analysis of bubaline HSPA‐1A revealed that it shares 91–98% identity with other mammalian sequences. It can be concluded that higher level of HSPA‐1A mRNA in IVP embryos in comparison with in vivo‐derived embryos is an indicator of cellular stress in IVP system. This study suggests need for further optimization of in vitro culture system in which HSPA‐1A gene could be used as a stress biomarker during pre‐implantation development.     

Two Methods of Vitrification Followed by In Vitro Culture of the Ovine Ovary: Evaluation of the Follicular Development and Ovarian Extracellular Matrix

The aim of this study was to evaluate the influence of two vitrification techniques on the extra cellular matrix (ECM) and ovarian follicular development. The ovarian cortex was fragmented (9 mm³) and divided into six groups, viz. fresh control, cultured control, vitrified by the Ovarian Tissue Cryosystem (OTC) method, conventional solid surface vitrification (SSV) method, OTC/cultured and SSV/cultured. Follicles from all the fragments were analysed for morphology, development and viability. The ECM was evaluated based on the condition of collagen and reticular fibres and the immunolocalization of type I collagen and fibronectin. After 7 days of culture, the tissue vitrified by OTC revealed a higher percentage (p < 0.05) of morphologically normal (30.66%) and viable (60.00%) follicles when compared with those vitrified using the SSV technique (21.33% and 23.00%). In all the fragments cultured, regardless of the vitrification method, a significantly higher percentage of developing follicles was observed when compared with the non‐cultured tissue. Analysis of the type I collagen showed increased immunostaining after the in vitro culture in the vitrified fragments. In conclusion, the OTC is better for preserving the follicular viability and morphology and maintaining the integrity of the extracellular matrix components of the ovine ovary.     

Comparative IFN-τ Secretion after Hatching by Bovine Blastocysts Derived Ex Vivo and Completely Produced In Vitro

The interferon-tau (IFN-tau) secretion levels after hatching by bovine blastocysts derived from in vitro maturated oocytes (Group A) and from in vivo (Group B) were investigated considering embryo quality. Only very homogeneous blastocysts of excellent or good quality were considered from day 7 of culture (Group A) and day 7 after artificial insemination with frozen-thawed from the same bull used for in vitro fertilization (Group B). All embryos were individually cultured into a 50 microl droplet of synthetic oviduct fluid medium with 10% fetal calf serum. After 24-h culture both Group A (n=44) and B (n=40) secreted <54 pm IFN-tau. After 48-, 72-, 96- and 120-h culture, Group A daily secreted 143 +/- 24 pm IFN-tau (n=19) vs 85 +/- 12 pm IFN-tau (n=21) for Group B (p < 0.01), 491 +/- 128 pm IFN-tau (n=29) vs 216 +/- 37 pm IFN-tau (n=23) (NS), 499 +/- 135 pm IFN-tau (n=26) vs 353 +/- 93 pm IFN-tau (n=21) (NS), 559 +/- 136 pm IFN-tau (n=22) vs 333 +/- 75 pm IFN-tau (n=20) (NS), respectively. Taken all together during 5 days, Group A produced per embryo 1690 +/- 290 pm IFN-tau (n=22) vs 982 +/- 182 pm IFN-tau (n=20) for Group B (p < 0.05). For all culture time there were sizable percentages of embryos that did not produce concentrations of IFN-tau above a certain cut-off level, and as such were not used to compute the means. In respect of the embryo quality whatever the groups after days 7-12 of culture, IFN-tau secretions were 1815 +/- 453 pm (n=10) for the embryos of excellent quality vs 1356 +/- 200 pm (n=28) for those of good quality (NS) and 360 +/- 188 pm (n=4) (p < 0.05) for embryos of fair quality. A positive relationship between IFN-tau production and in vitro development of quality I embryos was observed, whatever the embryos origins and, the embryos completely produced in vitro secreted more IFN-tau than the embryos produced in vivo.     

Differential Expression of IGF Family Members in Heat‐Stressed Embryos Produced In Vitro from OPU‐Derived Oocytes of Nelore (Bos indicus) and Holstein (Bos taurus) Cows

The IGF system is related to embryo quality. We aim to determine the effect of the heat stress on the mRNA expression of IGF1 and IGF2, IGFR1 and IGFR2, IGFBP2 and IGFBP4, and PAPPA in in vitro production (IVP) blastocysts from Nelore and Holstein after ovum pick up (OPU) to better understand the differences between these breeds. Oocytes from four Nelore and seven Holstein were collected in six OPU sessions. Following in vitro maturation and fertilization using six Nelore or Holstein sires, embryos were divided into control (cultured at 39°C) and heat stress (HS; exposed to 41°C for 9 h). Blastocysts were submitted to RNA extraction. The IGF1 expression was higher in blastocysts under HS in both breeds, and the expression of IGFBP2 and IGFBP4 was higher in Holstein blastocysts under HS. The high PAPPA expression and the low expression of IGFBP2 and IGFBP4 are associated with a more efficient degradation of IGFBPs, which results in greater IGF bioavailability in Nelore blastocysts and may contribute to the superior HS tolerance in Nelore, when compared to Holstein.     

Some In Vitro Invasion Inhibition of Red Cells by In Vivo Non‐protective Anti‐LDH Antibodies of Plasmodium berghei

In Plasmodium berghei, sephadex G‐200 purified lactate dehydrogenase (LDH) fraction immunized mice did not exhibit protection when challenged with 1 × 10⁶P. berghei‐parasitized erythrocytes. However, LDH immunized mice seroconverted and showed an antibody titre of 1:2048 by indirect haemagglutination (IHA) and 1:160 by indirect fluorescent antibody (IFA) assays. Fluorescence was distributed evenly on P. berghei‐parasitized red cells showing no specific location of parasite LDH. Anti‐LDH antibodies supplemented in 19 h in vitro culture of P. berghei exhibited 9.2% invasion inhibition into the fresh red cells.     

Apoptosis‐Like Events and In Vitro Fertilization Capacity of Sex‐Sorted Bovine Sperm

This study utilized three staining assays (Annexin V, mitochondrial membrane potential (JC‐1) and TUNEL) for flow cytometric analysis of apoptosis in sex‐sorted sperm from four different bulls (A, B, C and D). Correlations between sperm quality and IVF efficiency were then assessed to determine which assay provided the best prediction of IVF efficiency. The results of the Annexin V assays, as well as measures of viable sperm, early apoptosis, necrotic sperm and mitochondrial membrane potential (?ψm) showed that the sex‐sorted sperm collected from bull A significantly differed from those of the other three bulls (p < 0.05). In addition, the levels of DNA fragmentation in sex‐sorted sperm from bull A were significantly lower than those from bulls B and C (p < 0.05). The percentage of cells reaching the cleavage and blastocyst stages in sex‐sorted sperm from bull A were significantly greater than those from the other bulls (p < 0.05). A significant positive correlation was observed between viable sperm and the percentage of cells at the cleavage or blastocyst stages (p < 0.05). In contrast, a negative correlation was found between early apoptotic sperm and the percentage of cells at the cleavage or blastocyst stages (p < 0.05). In conclusion, these results indicated that the Annexin V assay was the most reliable technique for the prediction of the IVF success of sex‐sorted bovine sperm.     

细胞外基质蛋白mindin的原核表达及对日本血吸虫Sjc23抗原的免疫增强作用研究

根据GenBank中公布的鼠源spon2基因序列,合成全长基因片段,用BamH Ⅰ和Hin dⅢ双酶切后将目的片段插入表达载体pET-22b(+)中,构建pET-22b-mindin重组质粒,并转入大肠埃希菌BL21(DE3)后用IPTG进行诱导表达,产物经SDS-PAGE、Western blot进行检测.将表达蛋白...     

In Vitro Elution and Antibacterial Activity of Clindamycin,Amikacin, and Vancomycin from R‐gel Polymer

Objective: To characterize the in vitro elution and bioactivity of 2 formulations of antibiotics in a novel, dissolvable, cross‐linked dextran polymer matrix: Formulation 1—amikacin and clindamycin (AC); Formulation 2—amikacin, clindamycin, and vancomycin (ACV). Study Design: Prospective, in vitro, experimental study. Methods: Aliquots of the antibiotic impregnated polymer were incubated in PBS buffer for 10 days. PBS was changed every 24 hours and concentrations of the antibiotics eluted into saline were quantified. Antimicrobial activity of the eluent from each sampling period was tested for growth inhibition of Staphylococcus aureus. Results: Both formulations of R‐gel^? had a rapid initial release of antibiotics within the first 24 hours and then the concentrations decreased gradually over 10 days. The concentration of amikacin, clindamycin, and vancomycin remained above the breakpoint minimum inhibitory concentration of each drug for a minimum of 9 days. No significant difference (P=.9938, P=.9843) was present in the elution pattern or total amount of antibiotic eluted from clindamycin or amikacin, respectively. Eluent from both groups demonstrated bioactivity against S. aureus for the entire 10‐day study period. Conclusions: Amikacin and clindamycin together, or in combination with vancomycin, elute from R‐gel^? effectively and at gradually decreasing concentrations for at least 10 days. The antibiotics maintained their bioactivity following polymerization and elution from the R‐gel^?.     

Effects of N‐acetyl‐cysteine and N‐acetyl‐cysteine‐amide Supplementation on In Vitro Matured Porcine Oocytes

This study was conducted to evaluate the effects of different concentrations of the antioxidant N‐acetyl‐cysteine (NAC) supplemented to the maturation medium on porcine embryo development. Concentrations of NAC and its synthetic derivative, NAC‐amide (NACA) were evaluated for effects on nuclear maturation, fertilization success and embryo development. Concentrations of NAC (0, 0.5, 1.0, 1.5, 2.0, 2.5 and 5.0 mm ) were supplemented to maturing oocytes, and embryo development was analysed at 48 and 144 h post‐fertilization. There were no differences among cleavage rates for any of the treatment groups. Blastocyst formation for 1.5 mm NAC (56.5 ± 9.2%) was higher (p < 0.05) than all other supplementations. There were no differences in nuclear maturation or fertilization or in cleavage rates when comparing 1.5 mm NAC and 1.5 mm NACA supplementation to the control. Blastocyst formation for 1.5 mm NAC (44.4 ± 4.7%) and 1.5 mm NACA (46.2 ± 3.4%) supplementation were higher (p < 0.05) than the control (32.1 ± 6.2%) oocytes. These results indicate that supplementing 1.5 mm of NAC or NACA to the oocyte maturation medium increased the percentage of viable embryos reaching the blastocyst stage of development.     

Expression of Matrix Metalloproteinases (MMP‐2, MMP‐9) and their Inhibitors (TIMP‐1, TIMP‐2) in Canine Testis,Epididymis and Semen

This study aims to investigate the role of matrix metalloproteinases (MMPs) in determining semen quality and to evaluate the expression and cellular localization of MMP‐2, MMP‐9, tissue inhibitor of metalloproteinase‐1 (TIMP‐1) and TIMP‐2 in the testes, epididymis and ejaculated spermatozoa. Gelatinase activities between normal (n = 21) and abnormal (n = 25) semen samples showed a significant, sixfold increase in proMMP‐2 and MMP‐2 activity in high than low sperm concentration samples (p < 0.001). ProMMP‐9 and MMP‐9 levels were significantly elevated in samples with low sperm counts compared to those with high sperm density (p < 0.001). High levels of proMMP‐2 and MMP‐2 were associated with high sperm motility (≥70%, p < 0.001). Sperm‐rich fraction showed significantly (eight‐fold) higher proMMP‐9 enzymatic activity compared with prostatic fraction. The mRNA expressions of MMP‐2, MMP‐9, TIMP‐1 and TIMP‐2 were confirmed in testicular and epididymal tissues. Immunohistochemical staining illustrated the MMP‐2‐specific strong immunoreactivity in the head of mature spermatids during spermatogenesis, whereas MMP‐9, TIMP‐1 and TIMP‐2 were absent in these cells. Matrix metalloproteinase‐9 immunoreactivity was observed in the spermatocyte and round spermatid, whereas TIMP‐1 was only exhibited in the residual bodies. Immunolabeling of epididymal and ejaculated sperm demonstrated MMP‐2 localization along acrosomal region of sperm, while MMP‐9, TIMP‐1 and TIMP‐2 localization was merely limited to the flagella. In conclusion, spermatozoa initially acquire MMP‐2 during their formation at testicular level, and the presence of this protein persists through the epididymal transit and up to ejaculate. The enzymatic activity of MMP‐2 and MMP‐9 may serve as an alternative biomarker in determining semen quality.     

Exposure Time‐Dependent Bactericidal Activities of Amoxicillin Against Actinobacillus pleuropneumoniae; an In Vitro and In Vivo Pharmacodynamic Model

The pharmacodynamic effects of amoxicillin against Actinobacillus pleuropneumoniae at exposure concentration above and below minimum inhibitory concentration (MIC) were evaluated in both in vitro and in vivo. In vitro, the growth and morphological change of A. pleuropneumoniae in culture medium was observed. In vivo, the efficacy of amoxicillin on experimentally induced A. pleuropneumoniae infection in disease‐free pigs was evaluated. Fifteen pigs were divided into three groups (n = 5 per group). After the onset of clinical respiratory disease symptoms, 6 h post‐infection, amoxicillin sustained‐release injectable formulation was injected intramuscularly at 7.5 mg/kg/day (group I) and 15 mg/kg/day (group II). Then the serum concentration of amoxicillin was measured. An untreated infected group served as controls. In each amoxicillin administration group, if symptoms were not absent after 48 h, the pig was injected with the amoxicillin sustained‐release injectable formulation again using the same dosage. In vitro, the growth of A. pleuropneumoniae inhibited by amoxicillin exposure at the concentration above the MIC (1.28 × MIC), and the inhibition time was in directly proportion to the time of amoxicillin exposure. Moreover, all the cells were lysed. Whereas the bacterial growth inhibition at the amoxicillin exposure concentration below the MIC (0.25 × MIC) was not done, and the shape of cells were normal or long filamentous. In vivo, the group I clinical and pathological score was higher than the group II, and the group I weight gain was significantly less than the group II. Performance with respect to weight gain corresponded with clinical signs. The infected control group was severely affected with an 80% (4/5) mortality rate 24–96 h post‐challenge. The duration of time above MIC (T > MIC) of serum amoxicillin concentration in the group I was less than group II. The present studies suggest that amoxicillin has exposure time‐dependent bactericidal activity against A. pleuropneumoniae.