Identification of the DDAH2 Protein in Pig Reproductive Tract Mucus: A Putative Oestrus Detection Marker

Precisely detecting oestrus is important for artificial insemination. The aims of this study were to identify oestrus‐specific sow mucus proteins to determine the optimal time for artificial insemination. The proestrous‐ and oestrous‐stage mucus proteins were purified and analysed with proteomic tools such as two‐dimensional gel electrophoresis and matrix‐assisted laser desorption/ionization–time‐of‐flight analyses. Among the differentially expressed proteins, the dimethylarginine dimethylaminohydrolase 2 (DDAH2) protein showed a 3.6‐fold increase during the proestrous stage compared to that during the oestrous stage. A western immunoblot study revealed that two of three sow mucus samples clearly showed negative anti‐DDAH2 antibody activity during the oestrous stage. This study demonstrated that the pig DDAH2 mucus protein exists during the proestrous stage, but not during the oestrous stage, suggesting that mucus DDAH2 could be useful as an oestrus detection marker.     

Concurrent avian malaria and avipox virus infection in translocated South Island saddlebacks (Philesturnus carunculatus carunculatus)

Abstract

CASE HISTORY: Outbreaks of mortality in South Island saddlebacks (Philesturnus carunculatus carunculatus) that had been translocated to two offshore islands in the Marlborough Sounds of New Zealand were investigated during the summer of 2002 and 2007. Both outbreaks were associated with a severe decrease in numbers of saddlebacks of up to 60% of approximately 200 birds.

CLINICAL AND PATHOLOGICAL FINDINGS: Many of the surviving birds were in poor condition, and had skin lesions on the legs and head. Necropsy showed pale liver and lungs, and a swollen spleen. Histopathology revealed schizonts resembling Plasmodium spp. within the cytoplasm of many hepatocytes and splenic histiocytes. The skin lesions consisted of epithelial proliferations containing numerous Bollinger bodies typical of avipox virus (APV) infection. Two different APV were isolated, using PCR, from two different birds exhibiting skin lesions. Each isolate had 100% sequence homology with APV members from either Clade A or Clade B. In addition, PCR analysis revealed that the Plasmodium elongatum present in infected birdsbelonged to a strain that was endemic in the population of North Island saddlebacks (Philesturnus carunculatus rufusater).

DIAGNOSIS: Concurrent infections with Plasmodium spp. haemoparasites and APV were identified as the likely cause of death in the birds examined.

CONCLUSIONS AND CLINICAL RELEVANCE: Although the Plasmodium spp. identified is thought to be endemic to saddlebacks in New Zealand, the affected birds were likely to be immunocompromised by concurrent APV infection or through lack of genetic diversity. Both the introduced mosquito Culex quinquefasicatus and the native mosquito Culex pervigilans are likely vectors for both these diseases, and the provision of water supplies less favourable to mosquito-breeding is recommended.     

Effects of Developmental Stage, Embryonic Interferon-τ Secretion and Recipient Synchrony on Pregnancy Rate after Transfer of in vitro Produced Bovine Blastocysts

Three separate trials of bovine embryo transfers were performed consisting of 32, 41 and 33 transfers, respectively, to examine the effects of (a) the developmental stage of in vitro‐derived blastocysts, (b) the amount of interferon‐τ (IFN‐τ) they secreted during culture and (c) the cyclic stage of the recipient at the time of transfer on the probability of establishment of pregnancy. One blastocyst was transferred into the ipsilateral uterine horn to the CL. At the time of transfer, blastocysts were classified into one of three developmental stages (early blastocyst, blastocyst and expanded blastocyst) and the cyclic stage of each cow was assessed (?12 h, on time, +12 h, +24 h, >24 h). Prior to the second and third trials, blastocysts were individually cultured for 24 h in 50 μl medium droplets and the IFN‐τ concentration in the droplet was determined. Logistic regression analyses revealed that expanded blastocysts had a significantly higher likelihood of establishing pregnancy (p = 0.009), and that there was a significant interaction with the cyclic stage of the recipient in this group with lower rates of pregnancy resulting from decreasing synchrony with the recipient (p = 0.033). IFN‐τ secretion during culture was significantly higher in expanded blastocysts than in the other two groups (p < 0.05). A significant effect of the pre‐transfer level of IFN‐τ secretion was found only in the ‘Blastocyst’ group where transfer of embryos with lower IFN‐τ production prior to transfer resulted in higher pregnancy rates (p = 0.047). These results demonstrate that IFN‐τ secretion may be a useful tool to predict pregnancy outcome, but only within certain developmental stages.     

Interband electronic excitation-assisted atomic-scale restructuring of metal surfaces by nanosecond pulsed laser light

Interaction of high-power laser light with materials often causes irreversible damage of the near-surface region. It is shown that copper single-crystal surfaces can be patterned by laser light. Irradiation with green light produced adatoms and vacancies, which self-organized into nanoscale pyramids. This restructuring can be removed by annealing. In contrast to green light, infrared laser irradiation at equivalent absorbed energy density did not produce any structural change. This, for metallic systems, unforeseen spectral difference in laser light action points to a concerted process as the source for structural modification, which involves long-lived primary excitation of localized d-electrons through interband transition together with phonon excitation.     

Triazole fungicides and the selection of resistance to medical triazoles in the opportunistic mould Aspergillus fumigatus

Azole resistance is an emerging problem in the opportunistic mould Aspergillus fumigatus. The triazoles are the most important agents for the management of Aspergillus diseases in humans. Selection for acquired resistance may occur in the hospital setting through exposure to high doses of azoles during azole therapy, but evidence is accumulating that A. fumigatus may become resistant to medical triazoles through environmental exposure to fungicides. The recovery of A. fumigatus isolates resistant to the medical triazoles from azole‐naive patients as well as from the environment strongly indicates an environmental route of resistance selection. Molecule alignment studies have identified five fungicides that share a very similar molecule structure with the medical triazoles, and thus may have selected for mechanisms that confer resistance to both groups of compounds. It is important to explore further the presumed fungicide‐driven route of resistance selection in order to implement effective preventive measures as the prevalence of azole resistance in A. fumigatus continues to increase and causes major challenges in the management of azole‐resistant Aspergillus diseases. Copyright © 2012 Society of Chemical Industry     

Characterization of Transgenic Pigs That Express Human Decay Accelerating Factor and Cell Membrane‐tethered Human Tissue Factor Pathway Inhibitor

The expression of human complement regulatory proteins (hCRP; hDAF, hCD59, and hMCP) in pig tissues has been suggested as one of strategies to overcome the hyperacute rejection (HAR) in pig‐to‐human transplantation. Expression of human tissue factor pathway inhibitor (hTFPI) in porcine endothelial cells has been suggested as a remedy to overcome microvascular thrombosis. To investigate the effects of these combined transgenes, we established transformed pig cells expressing human decay accelerating factor (hDAF) under the control of enhancer promoter (5′LTR‐PCMVIE), and the fusion protein (hTFPI/hCD4) consisting of the functional domains (K1 and K2) of hTFPI and membrane‐tethering domains (D3 and D4) of hCD4 under the control of PCMVIE. Transgenic pigs were generated with the transformed porcine cells through somatic cell nuclear transfer (SCNT) technology. Analysis of quantitative PCR and real‐time quantitative RT‐PCR showed that four copies of hDAF were integrated and 391 copies of hDAF mRNA expressed in the cells of the transgenic pig. The enhancing activity of 5′LTR was approximately 2 fold compared to CMVIE promoter only. The cell viability test showed that more than 80% of ear cells were viable in the presence of 50% human serum. The chromogenic substrate assay and immunocytochemical staining with tail cells showed that the TFPI activity of fusion protein was observed on the cell membrane. The membrane localization of hDAF and hTFPI proteins was observed by immunocytochemical staining, and the expression of transgenes in heart and liver tissues was also confirmed by immunohistochemistry.     

Artificial Insemination of Swine: Fecundity of Boar Semen Stored in Beltsville TS (BTS), Modified Modena (MM), or MR-A and Inseminated on One,Three and Four Days After Collection12

Contents: A field trial was conducted on 1,463 farms in The Netherlands to compare the fertilizing capacity of boar spermatozoa extended in Beltsville TS (BTS), Modified (MM), or MR-A, and inseminated in 2896 sows and gilts on the first, third or fourth day following collection. Semen was collected, extended, and stored at 18°C at six different AI centers and inseminated by inseminators on their regular routes. Sows inseminated with BTS and MR-A extended semen had higher farrowing rates than MM (79.3, 77.6 us 50.4, P <.0001), higher total pigs per litter (11.4, 11.1 vs 10.0, P <.0001) and higher total pigs born alive (10.7, 10.5 vs 9.4, P <. 0001). The farrowing rate of gilts inseminated with BTS extended semen was superior to MM (73.5 vs. 50.2%, P <.004), while MR-A gave farrowing rates greater than MM (64.1 vs 50.2%, P =.06). There was no difference in litter data for gilts. Farrowing rates for 1 and 4 day semen were superior to 3 day semen (73.2, 73.8 vs 60.3, P <.0001). The semen inseminated on day 4 contained 6 billion sperm per dose rather than the 3 billion per dose for 1 and 3 day semen. Based on the results of this study, BTS and MR-A are effective diluents for extension and storage of boar semen for use within the same week of collection. In addition, semen extended in either BTS of MR-A and stored and inseminated on the fourth day after collection can give fertility equal to first day insemination if the sperm per insemination dose is doubled. Inhalt: Künstliche Besamung beim Schwein: Fruchtbarkeitsergebnisse von Ebersamen nach Flussigkonservierung mit BTS, MR-A oder modifiziertem Modena-Verdünner (MM) und Besamung am ersten, dritten und vierten Tag nach Samengewinnung In einem in den Niederlanden durchgeführten Feldversuch wurden 3 verschiedene Verdünner-Medien für Ebersamen in vivo verglichen. Die Medien waren Beltsville TS (BTS), modifizierter Modena-Verdünner (MM) und das spanische MR-A-Medium. Insgesamt wurden 2896 Jung- und Altsauen in 1463 Herden mit 1, 3 oder 4 Tage altem Sperma besamt. Der Versuch wurde als “split-sample”-Versuch an 6 Eberstationen durchgeführt, der verdunnte Samen bei + 18°Cgelagert und von den Besamungsbeauftragten im Rahmen ihrer regulären Fahrten eingesetzt. BTS- und MR-A-konserviertes Sperma gab bei Altsauen gegenüber MM höhere Abferkelraten (79,3, 77,6 vs 50,4%, P < 0.0001), eine höhere totale Wurfzahl (11,4, 11,1 vs 10,0, P < 0.0001) und eine höhere Anzahl lebendgeborener Ferkel)10,7, 10,5 vs 9,4, P < 0.0001). Die mit BTS-uerdünntem Samen inseminierten Jungsauen wiesen gegenüber MM höhere Abferkelraten auf(73.5 vs 50,2%, P < 0.004). MR-A-Sperma lag ebenfalls hüher als MM-Samen (641 vs 50,2%, P=0.06). Die Wurfgröβen der Jungsauen reigten keine Unterschiede. Die Abferkelergebnisse nach Besamung mit 1 und 4 Tage altem Samen lagen höher als die des 3 rage alten Spermas (73,2, 73,8 vs 60,3%; P < 0.0001). Die am Tag 4 verwendeten Samenportionen enthielten 6 Milliarden Spermien gegenüber 3 Milliarden der nach 1 bzw. 3 Tagen inseminierten Portionen. Die Ergebnkse dieses Versuches zeigen, daβ BTS und MR-A effektive Verdünnermedien sind und die Lagerung von Eberfrischsperma zum Einsatz innerhalb der Entnahmewoche ermoglichen. Darüber hinaus können mit BTS oder MR-A verdünntes und am Tag 4 nach Gewinnung eingesetrtes Sperma Fruchtbarkeitsergebnisse erzielt werden, die ebenso gut sind wie nach Verwendung des 1-Tage-Spermas, wenn die Spermienzahl pro Besamungsdosis verdoppelt wird.