bluuebunny/crossref_metadata_2025 huggingface dataset:
Previously undiagnosed aortic stenosis revealed by auscultation in the hip fracture population – echocardiographic findings, management and outcome
Authors: McBrien M. E. , Heyburn G. , Stevenson M. , McDonald S , Johnston N. J. , Elliott J. R. M. , Beringer T. R. O.
Published: July 2009

The 2001 Report of the National Confidential Enquiry into Perioperative Deaths recommended that an echocardiogram should be performed on patients with aortic stenosis prior to anaesthesia. In this study we present the patient details, management and outcome of the 272 hip fracture patients with a previously undiagnosed murmur and echocardiographically proven aortic stenosis admitted from 2001–2005 in our hospital. The patients with aortic stenosis were significantly older, and had significantly lower Abbreviated Mental Test Scores, than the control group of 3698 hip fracture patients without aortic stenosis. There were significant trends toward general anaesthesia over spinal anaesthesia, and use of invasive monitoring of blood pressure, as the severity of the aortic stenosis increased. There were no significant trends towards higher 30‐day or 1‐year mortality rates as the severity of the aortic stenosis increased. Resources for rapid pre‐operative echocardiograms should be made available for hip fracture patients as the results have significant implications for their subsequent anaesthetic management.
Use of the McGrath video laryngoscope in awake patients
Authors: McGuire B. E.
Published: July 2009

Reported are three cases of successful tracheal intubation using the McGrath Video Laryngoscope in awake patients. All three patients had predicted difficult direct laryngoscopy and signs of upper airway obstruction.
Cardiopulmonary exercise testing as a risk assessment method in non cardio‐pulmonary surgery: a systematic review
Authors: Smith T. B. , Stonell C. , Purkayastha S. , Paraskevas P.
Published: July 2009

This study reviews the predictive value of maximum oxygen consumption () and anaerobic threshold, obtained through cardiopulmonary exercise testing, in calculating peri‐operative morbidity and mortality in non‐cardiopulmonary thoraco‐abdominal surgery. A literature review provided nine studies that investigated either one or both of these two variables across a wide range of surgical procedures. Six of the seven studies that reported sufficiently detailed results on peak oxygen consumption and four of the six studies that reported sufficiently detailed results on anaerobic threshold found them to be significant predictors. We conclude that peak oxygen consumption and possibly anaerobic threshold are valid predictors of peri‐operative morbidity and mortality in non‐cardiopulmonary thoraco‐abdominal surgery. These indicators could potentially provide a means of allocating increased care to high‐risk patients.
Sedimentology and sequence stratigraphy of the middle Jurassic Tarbert Formation, Oseberg South area (northern North Sea)
Authors: Løseth Tore M. , Ryseth Alf E. , Young Mike
Published: July 2009

This paper describes the development of a regressive‐to‐transgressive shoreline wedge within the Middle Jurassic Tarbert Formation in the Oseberg South area (northern North Sea), as interpreted from core and log data from more than hundred wells. The wedge is described in terms of four facies associations (FA1–FA4). The lower, regressive portion of the wedge (FA1–FA2) contains both coarsening upward wave/storm‐dominated shoreline deposits as well as coal‐bearing paralic deposits, and was deposited during ascending regression. The upper, transgressive portion of the wedge (FA3–FA4) is characterised by wave‐dominated estuarine deposits, exhibiting an upward change from inner to central to outer estuarine deposits. In contrast to some earlier studies, it is argued that this part was deposited during accretionary transgression. The present study documents an estuarine system that developed without any preceding fall of relative sea level and valley incision. It is argued that differential fault‐induced subsidence created a broad gentle sag wherein one or several estuarine systems developed as the depositional system became transgressive. The subtle fault‐induced subsidence is related to the tectonic evolution in the North Sea Basin.
The anti‐histaminic cyproheptadine synergizes the antineoplastic activity of bortezomib in mantle cell lymphoma through its effects as a histone deacetylase inhibitor
Authors: Paoluzzi Luca , Scotto Luigi , Marchi Enrica , Seshan Venkatraman E. , O’Connor Owen A.
Published: July 2009

Cyproheptadine, an inhibitor of the H1 histamine receptors, has recently shown activity in models of leukaemia and myeloma, presumably through inhibition of cyclin‐D expression. Mantle cell lymphoma (MCL) is an aggressive subtype of non‐Hodgkin lymphoma characterized by overexpression of cyclin‐D1. We investigated the effect of cyproheptadine alone and in combination with the proteasome inhibitor bortezomib in models of MCL. The combination of these drugs was mathematically synergistic, producing significant reductions in the mitochondrial membrane potential leading to apoptosis. In a severe combined immunodeficient beige mouse model, cyproheptadine plus bortezomib demonstrated a statistically significant advantage compared to either agent alone.
Spatiotemporal expression of chemokines and chemokine receptors in experimental anti-myeloperoxidase antibody-mediated glomerulonephritis
Authors: Van Der Veen B S , Petersen A H , Belperio J A , Satchell S C , Mathieson P W , Molema G , Heeringa P
Published: July 2009

Myeloperoxidase (MPO)-anti-neutrophil cytoplasmic autoantibody (ANCA)-associated necrotizing crescentic glomerulonephritis (NCGN) is characterized by abundant leucocyte infiltration. Chemokines are chemotactic cytokines involved in receptor-mediated recruitment of leucocytes. Our objective was to analyse spatiotemporal gene expression of chemokines and chemokine receptors in anti-MPO-mediated NCGN, to find potential targets for intervening with leucocyte influx. NCGN was induced in mice by co-administration of anti-MPO immunoglobulin (Ig)G and lipopolysaccharide. mRNA expression levels of chemokines and chemokine receptors were analysed in whole kidney lysates as well as in laser microdissected glomeruli and tubulo-interstitial tissue 1 and 7 day(s) after NCGN induction. Several chemokines and chemokine receptors were induced or up-regulated in anti-MPO-mediated NCGN, both on day 1 (chemokines CCL3, 5; CXCL2, 5, 13; receptor CXCR2) and on day 7 (chemokines CCL2, 5, 7, 8, 17, 20; CXCL1, 2, 5, 10; CX3CL1; receptors CCR2, 8; CX3CR1). The expression levels of most chemokines and receptors were higher in glomeruli than in the tubulo-interstitium. Because of the temporal induction of CXCR2 on day 1, we hypothesized CXCR2 as a potential target for treatment in anti-MPO-induced NCGN. Inhibition of CXCR2 using a goat-anti-CXCR2 serum prior to NCGN induction increased glomerular neutrophil influx but did not affect crescent formation and albuminuria. In conclusion, expression levels of various chemokines and chemokine receptors were increased in anti-MPO NCGN, and expressed particularly in glomeruli. These chemokines and receptors may serve as potential targets for treatment. Inhibition of a single target, CXCR2, did not attenuate anti-MPO NCGN. Combinatorial interventions may be necessary to avoid redundancy.
PPE and PE_PGRS proteins of Mycobacterium marinum are transported via the type VII secretion system ESX‐5
Authors: Abdallah Abdallah M. , Verboom Theo , Weerdenburg Eveline M. , Gey van Pittius Nicolaas C. , Mahasha Phetole W. , Jiménez Connie , Parra Marcela , Cadieux Nathalie , Brennan Michael J. , Appelmelk Ben J. , Bitter Wilbert
Published: July 2009

ESX‐5 is one of the five type VII secretion systems found in mycobacteria. These secretion systems are also known as ESAT‐6‐like secretion systems. Here, we have determined the secretome of ESX‐5 by a proteomic approach in two different strains of Mycobacterium marinum. Comparison of the secretion profile of wild‐type strains and their ESX‐5 mutants showed that a number of PE_PGRS and PPE‐MPTR proteins are dependent on ESX‐5 for transport. The PE and PPE protein families are unique to mycobacteria, are highly expanded in several pathogenic species, such as Mycobacterium tuberculosis and M. marinum, and certain family members are cell surface antigens associated with virulence. Using a monoclonal antibody directed against the PGRS domain we showed that nearly all PE_PGRS proteins that are recognized by this antibody are missing in the supernatant of ESX‐5 mutants. In addition to PE_PGRS and PPE proteins, the ESX‐5 secretion system is responsible for the secretion of a ESAT‐6‐like proteins. Together, these data show that ESX‐5 is probably a major secretion pathway for mycobacteria and that this system is responsible for the secretion of recently evolved PE_PGRS and PPE proteins.
Dynamics of cell wall components of Magnaporthe grisea during infectious structure development
Authors: Fujikawa Takashi , Kuga Yukari , Yano Shigekazu , Yoshimi Akira , Tachiki Takashi , Abe Keietsu , Nishimura Marie
Published: July 2009

Oligosaccharides derived from cell wall of fungal pathogens induce host primary immune responses. To understand fungal strategies circumventing the host plant immune responses, cell wall polysaccharide localization was investigated using fluorescent labels during infectious structure differentiation in the rice blast fungus Magnaporthe grisea. α‐1,3‐glucan was labelled only on appressoria developing on plastic surfaces, whereas it was detected on both germ tubes and appressoria on plant surfaces. Chitin, chitosan and β‐1,3‐glucan were detected on germ tubes and appressoria regardless of the substrate. Major polysaccharides labelled at accessible surface of infectious hyphae were α‐1,3‐glucan and chitosan, but after enzymatic digestion of α‐1,3‐glucan, β‐1,3‐glucan and chitin became detectable. Immunoelectron microscopic analysis showed α‐1,3‐glucan and β‐1,3‐glucan intermixed in the cell wall of infectious hyphae; however, α‐1,3‐glucan tended to be distributed farther from the fungal cell membrane. The fungal cell wall became more tolerant to chitinase digestion upon accumulation of α‐1,3‐glucan. Accumulation of α‐1,3‐glucan was dependent on the Mps1 MAP kinase pathway, which was activated by a plant wax derivative, 1,16‐hexadecanediol. Taken together, α‐1,3‐glucan spatially and functionally masks β‐1,3‐glucan and chitin in the cell wall of infectious hyphae. Thus, a dynamic change of composition of cell wall polysaccharides occurs during plant infection in M. grisea.
Host legume‐exuded antimetabolites optimize the symbiotic rhizosphere
Authors: Cai Tao , Cai Wentong , Zhang Jiang , Zheng Huiming , Tsou Amy M. , Xiao Lin , Zhong Zengtao , Zhu Jun
Published: July 2009

Rhizobia form symbiotic nodules on host legumes and fix nitrogen for their hosts in exchange for nutrients. In order to establish this mutually beneficial relationship, rhizobia must compete with other soil bacteria in the host legume rhizosphere to colonize plant roots efficiently. A promoter‐trap transposon screen in Mesorhizobium tianshanense, a Rhizobium that forms nodules on licorice (Glycyrrhiza uralensis) plants revealed that the expression of msiA, which encodes a putative exporter protein belonging to the LysE family of translocators, is activated by both legume exudates and MsiR, a LysR family transcriptional regulator. Chemical analysis suggests that the msiA‐inducing signal in exudates is canavanine, an anti‐metabolite present in the seeds and exudates of a variety of legume plants. We show that MsiA serves as a canavanine exporter that is indispensable for canavanine resistance in M. tianshanense. We also show that the expression of MsiA homologues in other rhizobial species is induced by canavanine and is critical for canavanine resistance. Furthermore, rhizobial canavanine resistance is important for root hair adherence as well as for survival in a canavanine‐producing legume rhizosphere. Together, these data suggest that host legumes may exude specific antimetabolites into their surroundings to optimize the bacterial population in order to have successful symbiotic events with rhizobia.
CzcP is a novel efflux system contributing to transition metal resistance inCupriavidus metalliduransCH34
Authors: Scherer Judith , Nies Dietrich H.
Published: July 2009

Cupriavidus metalliduransCH34 possesses a multitude of metal efflux systems. Here, the function of the novel PIB4‐type ATPase CzcP is characterized, which belongs to the plasmid pMOL30‐mediated cobalt‐zinc‐cadmium (Czc) resistance system. Contribution of CzcP to transition metal resistance inC. metalliduranswas compared with that of three PIB2‐type ATPases (CadA, ZntA, PrbA) and to other efflux proteins by construction and characterization of multiple deletion mutants. These data also yielded additional evidence for an export of metal cations from the periplasm to the outside of the cell rather than from the cytoplasm to the outside. Moreover, metal‐sensitiveEscherichia colistrains were functionally substitutedin transwith CzcP and the three PIB2‐type ATPases. Metal transport kinetics performed with inside‐out vesicles identified the main substrates for these four exporters, the Kmvalues and apparent turn‐over numbers. In combination with the mutant data, transport kinetics indicated that CzcP functions as ‘resistance enhancer’: this PIB4‐type ATPase exports transition metals Zn2+, Cd2+and Co2+much more rapidly than the three PIB2‐type proteins. However, a basic resistance level has to be provided by the PIB2‐type efflux pumps because CzcP may not be able to reach all different speciations of these metals in the cytoplasm.
A probiotic dairy product containing L. gasseri CECT5714 and L. coryniformis CECT5711 induces immunological changes in children suffering from allergy
Authors: Martínez‐Cañavate Ana , Sierra Saleta , Lara‐Villoslada Federico , Romero Julio , Maldonado José , Boza Julio , Xaus Jordi , Olivares Mónica
Published: July 2009

The increase in the prevalence of allergic diseases in children has been attributed to an unbalanced immune response probably due to environmental factors. The immunoregulatory properties of probiotic bacteria could balance the disequilibrium in the immune response causing the allergic response. The aim of this study was to evaluate the immunological effects of the consumption of a dairy product containing two probiotic strains in children suffering from allergy. A double‐blinded, randomized, control comparative study was performed with 44 allergic children. Children were randomly distributed in two groups, a control Yogurt and a Probiotic group. Both groups daily consumed 200 ml of a dairy fermented product for 3 months. The Yogurt group consumed a conventional yogurt, whereas the Probiotic group consumed a similar dairy product where Lactobacillus bulgaricus was substituted by a mixture of Lactobacillus gasseri CECT5714 and Lactobacillus coryniformis CECT5711 (at least 106 cfu/g each strain). Intestinal and immunological parameters were measured in fecal and blood samples. The consumption of the probiotic product induced a significant decrease in the level of IgE in plasma (p = 0.03) and an increase in CD4+/CD25+ T regulatory cells (p = 0.01). The decrease in IgE was accompanied by a significant increase in mucosal IgA (p = 0.01). However, changes in other effector cells potentially involved in allergic reactions such as eosinophiles, basophiles or other IgE+ cells were not detected. The consumption of the probiotic product also induced significant changes in innate response as a significant increase in natural killer cells was detected (p = 0.03). The daily consumption of a probiotic product containing L. gasseri CECT5714 and L. coryniformis CECT5711 for 3 months induces, in allergic children, beneficial effects on immune parameters involved in the allergic response such as a reduction of IgE in plasma and an increase in regulatory T cells. The probiotic product also enhanced innate and specific immune parameters that may improve the general health status of children.
Interscalene block using ultrasound guidance: impact of experience on resident performance
Authors: OREBAUGH S. L. , WILLIAMS B. A. , KENTOR M. L. , BOLLAND M. A. , MOSIER S. K. , NOWAK T. P.
Published: July 2009

Background: We evaluated the weekly progress of anesthesiology residents performing an interscalene block with ultrasound guidance (UG) for block success rates and for the specific time intervals: (i) time to image the brachial plexus and (ii) time from insertion of the block needle until motor stimulation occurred. Our primary objective was to characterize the influence of experience over the course of the regional anesthesia rotation on the performance of a UG interscalene block by anesthesiology residents.

Methods: Residents conducted an interscalene block with UG under the supervision of attending anesthesiologists experienced in this technique. Block efficacy, time intervals required to perform the block, and acute complications were recorded. We compared success rates over the course of the rotation, and analyzed process time data with respect to trainee level of experience, week of the trainee rotation, and patient body habitus.

Results: Twenty‐one trainees conducted 222 blocks over a consecutive 7‐month period. Block success rate was 97.3%, and did not change significantly over the course of the 4‐week rotation. Total block time and imaging time significantly decreased over the 4‐week rotation, while the needle insertion‐to‐stimulation time did not change. Slower imaging time was predicted by obesity.

Conclusion: The success rates for a UG interscalene block provided by supervised residents were initially high, and remained so throughout the 4‐week rotation. Trainees required less time to image the nerves and to perform the block over the course of the rotation.
Advances in Reproductive Science for Wild Carnivore Conservation
Authors: Comizzoli P , Crosier AE , Songsasen N , Gunther M Szykman , Howard JG , Wildt DE
Published: July 2009

Contents

Knowledge about reproduction is critical for predicting the viability of wildlife populations in nature and for managing breeding programmes in captivity. Intensive species‐based studies are the priority, because reproductive mechanisms are extraordinarily diverse, even within the same taxonomic family. Carnivores deserve more attention as such species are highly vulnerable to environmental change and human persecution. The present review provides contemporary illustrations of how reproductive science is contributing to understand unique reproductive mechanisms that are both of fundamental and applied interest. In the case of the endangered African wild dog (Lycaon pictus) free‐living in South Africa, non‐invasive faecal corticosteroid assessments have yielded new insights about the impact of animal relocation and reintroduction on adaptive responses, reproductive fitness and survival. For the maned wolf (Chrysocyon brachyurus), advances have been made in characterizing and comparing reproductive traits in free‐ranging vs captive individuals. For the cheetah (Acinonyx jubatus), recent studies have focused on the cryosensitivity of sperm and the ability to develop a field‐friendly sperm cryo‐method. The by‐product has been a large‐scale frozen repository of sperm from wild‐caught cheetahs useful for infusing new genes intoex situpopulations. Finally, rigorous, multi‐disciplinary and cross‐institutional reproductive studies of the black‐footed ferret (Mustela nigripes), including the use of artificial insemination, have contributed to the remarkable recovery and restoration of this species, once on the brink of extinction. In summary, advances in reproductive science are not necessarily related to ‘assisted breeding’. However, understanding the unique ways of carnivore reproduction greatly contributes to species management and conservation.
Western Blot Analysis of Proacrosin/Acrosin in Frozen Dog Sperm During In Vitro Capacitation
Authors: De los Reyes M , Medina G , Palomino J
Published: July 2009

Contents

Acrosin is an acrosomal protease synthesized as an inactive precursor, proacrosin, which is processed via autoproteolysis into active forms alpha‐ and beta‐acrosin. In this paper, a comparative study on the immunoreactivity of acrosin during in vitro capacitation of frozen and fresh (control) canine sperm using Western blot analysis is reported. Semen samples were obtained by digital stimulation and ejaculates processed as fresh and frozen samples and then capacitated for 0, 30, 60 and 90 min. At each time period, samples were analyzed with monoclonal antibody C5F10 by Western blot. The antibody specifically recognized, in fresh and frozen/thawed spermatozoa, a 40‐, 32‐ and 27‐kDa bands corresponding to proacrosin, alpha‐ and beta‐acrosin, respectively, during capacitation. Western immunoblots showed that the beta‐acrosin reactivity in fresh sperm was directly proportional to the time of capacitation, whereas a decreased reactivity of active form of acrosin was observed with frozen‐thawed sperm (p < 0.05). These results suggest that proacrosin is activated to beta‐acrosin earlier in frozen/thawed dog spermatozoa than in fresh dog spermatozoa.
Recrudescence of Spermatogenesis in the Dog Following Downregulation Using a Slow Release GnRH Agonist Implant
Authors: Goericke‐Pesch S , Spang A , Schulz M , Özalp G , Bergmann M , Ludwig C , Hoffmann B
Published: July 2009

Contents

The present study examined the degree to which downregulation with a GnRH agonist impaired spermatogenesis and the time course of morphological and hormonal changes that occurred during recrudescence of spermatogenesis. Using a control group (group 1, n = 5) of dogs, the effect of a removable slow release GnRH‐agonist implant was investigated in beagle dogs (group 2, n = 30). The implant was removed after 5 months (week 0) and three to four dogs were castrated at weeks 0, 3, 6, 9, 12, 15, 18, 21 and 24. The degree of downregulation and recrudescence of spermatogenesis was assessed by evaluation of 200 tubular cross‐sections, resulting in an assigning of dogs of group 2 to testis developmental groups (DG) according to the most developed germ cell observed: DG A, spermatocytes; DG B, round spermatids; DG C, elongating spermatids and DG D, elongated spermatids. Downregulation led to an arrest of spermatogenesis at the level of spermatogonia/primary spermatocytes. The time course of recrudescence showed high individual variations and the number of dogs falling into DG A, B, C and D was 4, 3, 6 and 17 respectively. Spermatogenesis in group 2, DG D was not different from group 1 (control). In DG A, mean area of Leydig‐cell nuclei was lower (p < 0.001) than in the other DG and group 1 and resembled that of juvenile dogs (group 3, n = 3); nuclei of Sertoli cells had changed from more flat/polygonal (group 1, group 2, DG C and D) to round/ovoid and had moved to a more luminal position. As indicated by basal testosterone (T), luteinizing hormone (LH) and follicle stimulating hormone (FSH) concentrations at implant removal, full downregulation had been obtained. Testosterone, LH and FSH concentrations [x̄g (DF), ng/ml] increased (p < 0.05) from implant removal to DG B [T: 0.1 (1.24) vs 2.12 (2.31); LH: 0.2 (2.15) vs 1.11 (1.7); FSH: 0.37 (3.50) vs 6.37 (1.68)] and were more or less constant thereafter indicating that onset of spermatogenesis was related to an increase of plasma T occurring in a very narrow time window. Following GnRH implantation, the size of the testes and the prostate decreased by approximately 55% (p < 0.001), they increased to sizes similar to pre‐treatment values following implant removal.
Comparison of the Effect of the Aromatase Inhibitor, Anastrazole, to the Antioestrogen, Tamoxifen Citrate, on Canine Prostate and Semen
Authors: Gonzalez G , Guendulain C , Maffrand C , Gobello C
Published: July 2009

Contents

This study compared the efficiency of the aromatase inhibitor, anastrazole, with the antioestrogenic receptor blocker, tamoxifen, on normal (NRL) and hyperplastic prostate glands. Forty healthy dogs were classified as NRL (n = 18) or abnormal (ABN) with benign prostate hyperplasia (n = 22). The dogs were randomly assigned to one of the following six groups, treated for 60 days; oral placebo for normal (NRL‐PLC; n = 6) and abnormal (ABN‐PLC; n = 6), oral anastrazole 0.25–1 mg/day, for normal (NRL‐ANZ, n = 6) and abnormal (ABN‐ANZ, n = 8) and oral tamoxifen citrate 2.5–10 mg/day for normal (NRL‐TMX; n = 6) and abnormal (ABN‐TMX; n = 8) dogs. The dogs were evaluated before treatment and then monthly for 4 months. At the end of the treatment, the prostatic volume decreased by 28.5 ± 4.3%, 21.6 ± 6.3% and 0.7 ± 1.0% in the ABN‐TMX, ABN‐ANZ and ABN‐PLC (p < 0.01), respectively. From then on, prostatic volume began to increase without reaching pre‐treatment values at the end of the study. In the ABN animals, there were no differences for this parameter between ANZ and TMX treatment (p > 0.1), whereas in the NRL animals ANZ produced a less pronounced decrease (p < 0.05), libido, testicular consistency and scrotal diameter decreased during treatment in the TMX group (p > 0.05). These parameters and sperm volume, count, motility and morphological abnormalities remained unaltered throughout the study in the ANZ and PLC groups (p > 0.05). There were no haematological nor biochemical side effects. Anastrazole might offer a safe and effective alternative for the medical management of dogs with benign prostatic hyperplasia.
Influence of Progesterone Withdrawal on Pregnancy‐Related Parameters During Post‐Implantation Early Pregnancy Loss
Authors: Günzel‐Apel AR , Höftmann T , Nottorf S , Politt E , Meyer‐Lindenberg A , Hoppen HO , Einspanier A , Knijn HM , Mischke R
Published: July 2009

Contents

Pharmacologically‐induced luteolysis or treatment with an antiprogestin in early post‐implantation pregnancy in dogs results in asynchronous death and resorption of conceptuses, indicating variable rates of response of individual conceptuses towards progesterone deficiency. This variability also seems to occur in bitches showing pregnancy failure in response to spontaneous luteal deficiency. In a total of 10 beagle pregnancies (two consecutive pregnancies of five bitches), abortifacient treatments beginning on day 24 after ovulation (ov) involved either administration of a progestin antagonist (total of six pregnancies, in three bitches) or a luteolytic regimen of prostaglandin F2α‐analogue together with a dopamine agonist (total of four pregnancies, in two bitches). The outcomes were evaluated in relation to four control pregnancies in two bitches by assay of serum progesterone, prolactin and relaxin at selected time points or within selected time periods, by ultrasound of conceptuses including measurement of uterine blood flow, and parameters of the blood fibrinolytic system including plasma fibrinogen and plasminogen. The process of embryonic death and conceptus resorption was variable in onset and duration both in bitches that received the progesterone antagonist aglepristone (AGLE) and in those under the luteolytic treatment (cloprostenol combined with cabergoline). Pregnancy termination (death of all embryos or foetuses, respectively) occurred as early as day 29 and as late as day 41 after ov in AGLE‐treated bitches, and not earlier than day 37 after ov in luteolytic‐treatment bitches. Impending embryonic death was not predicted by changes in relaxin concentration, parameters of the fibrinolytic system, or in the perfusion of small uteroplacental vessels.
Expression of ABC‐Transport Proteins in Canine Mammary Cancer: Consequences for Chemotherapy
Authors: Honscha KU , Schirmer A , Reischauer A , Schoon H‐A , Einspanier A , Gäbel G
Published: July 2009

Contents

Intrinsic or acquired drug resistance is a major barrier for chemotherapy of cancer. Importantly, the presence of ATP‐binding cassette, ABC‐transport proteins in tumour cells circumvents an intracellular accumulation of chemotherapeutic drugs. In this study, 103 canine mammary tumour probes were investigated for mRNA expression of seven ABC‐transporters by RT‐PCR. All tumour samples expressed multidrug resistance‐associated protein 1 (MRP1) and breast cancer resistance protein (BCRP). MRP7 was detected in 97.1% of tumour probes, MRP3 in 96.1%, Pgp in 92.2%, MRP5 in 85.4% and MRP6 in 64.1%. More of the half of tumour samples (56.1%) expressed all of the examined ABC‐transport proteins. Approximately one‐third of the tumour samples (32.7%) were lacking in one transporter and only 11.2% possessed from three to five transporters. The canine transporter cBCRP was functionally analysed in stable transfected Madin‐Darby canine kidney‐II cells using an MTT viability test. cBCRP transfected cells showed a 5.4‐fold resistance to 10 μm doxorubicin. Cell survival in the presence of methotrexate was not affected by cBCRP. In conclusion, absence of efficiency of chemotherapy of canine mammary cancer can be caused by expression of seven various ABC‐transport proteins. Because cBCRP is expressed in all examined tumour probes and induces resistance to doxorubicin, the application of doxorubicin for treatment of canine mammary is inappropriate.
Investigation of the Local Expression of the Relaxin System in Canine Mammary Tumours
Authors: Lamp O , Honscha KU , Jakob J , Lamp J , Schweizer S , Reischauer A , Gottschalk J , Hahn A , Ebert M , Rothemund S , Blaschzik S , Einspanier A
Published: July 2009

Contents

While mammary tumours are the main reasons of death in bitches, early detection of tumours and metastases is crucial for survival of affected dogs. Invasiveness and angiogenesis, which are important processes of tumour growth and spreading, require connective tissue remodelling. This process is dominantly mediated by matrix metalloproteinases (MMP), which are well known to be positively regulated by relaxin (RLX) in various tissues, including human breast cancer. So far, the presence of RLX and its receptor RXFP‐1 as well as their linkage with MMP in canine mammary tumours (CMT) is completely unknown. In the first part of the present study, concentrations of RLX, oestradiol and progesterone from plasma samples of bitches with CMT were compared with clinical and survival data to investigate the predictive value of these hormones. In the second part, the expressions of RLX, RXFP‐1 and MMP‐2, ‐9 and ‐13 were examined by real‐time reverse transcriptase polymerase chain reaction (RT‐PCR) in 31 CMT samples. Finally, relationships of systemic plasma RLX or locally expressed RLX with expression of MMP in CMT were analyzed for the first time. Comparison of hormone concentrations in 93 bitches in terms of benign or malignant nature of the CMT, lung metastases, recidivation and 12‐month survival discovered no significances. The expressions of RLX, RXFP‐1 and MMP were independent from plasma RLX, but expressions of local RLX and RXFP‐1 showed a strong correlation (p = 0.00004,r = 0.671) as well as RXFP‐1 and MMP‐2 (p = 0.009,r = 0.463), indicating a possible significant role of the locally produced RLX in CMT pathogenesis as an inducer of connective tissue remodelling.
Elective Caesarean Operation in the Bitch Using Aglepristone Before the Pre‐Partum Decline in Peripheral Progesterone Concentration
Authors: Levy X , Fontaine E , Segalini V , Fontbonne A
Published: July 2009

Contents

The aim of this study was to investigate caesarean operation (CO) undertaken before the pre‐partum decrease of progesterone but following administration of a progesterone receptor antagonist and to evaluate the innocuity of this procedure for the dam and pups. Thirty seven bitches of 15 different breeds, received an injection of 15 mg/kg aglepristone 59 or 60 days after the estimated day of ovulation, determined by progesterone quantitative assays, and caesarean section (CS) was performed between 20 and 24 h after administration. Progesterone remained above 6 nmol/l at the time of CS (mean = 15.75, SD = 3.84). No post‐operative clinical complications were reported in any of the bitches. All bitches were able to nurse and feed their puppies in the first 24 h following surgery. No pups showed any signs of prematurity and 5 out of 188 pups (2.6) died in the first 2 weeks after delivery. This small study demonstrates that a CS may be safely and successfully performed an average of 2 days before the expected date of parturition following the administration of aglepristone, without any harmful consequence for the dam and her neonates.
Ultrastructural Characteristics of Non‐matured and In Vitro Matured Oocytes Collected from Pre‐pubertal and Adult Domestic Cat Ovaries
Authors: Martins LR , Fernandes CB , Minto BW , Landim‐Alvarenga FC , Lopes MD
Published: July 2009

Contents

The present study describes the ultrastructural characteristics of cat oocytes before maturation and after 12‐ and 24‐h in vitro maturation (IVM). Oocytes were recovered from pre‐pubertal and adult queen ovaries after ovariohysterectomy and a proportion were stored in glutaraldehyde at 4°C until examination by transmission electronic microscopy (TEM). Those selected for maturation were cultured before TEM in DMEM for 12 and 24 h at 38°C in a humidified environment of 5% O2, 5% CO2 and 90% N2. Specimens were divided into six groups: non‐matured oocytes from pre‐pubertal queens (PP0), non‐matured oocytes from adult queens (A0), 12‐h in vitro matured oocytes from pre‐pubertal queens (PP12), 12‐h in vitro matured oocytes from adult queens (A12), 24‐h in vitro matured oocytes from pre‐pubertal queens (PP24) and 24‐h in vitro matured oocytes from adult queens (A24). Across the treatment groups, it was possible to observe differences in the thickness of the perivitelline space, the penetration of cumulus cell projections forming a junctional complex, distribution and density of small vesicles, lipid droplets, microvilli, mitochondria and cortical granules and variable degrees of development of Golgi complexes. These findings demonstrated that ultrastructural analysis of oocytes matured in vitro is a valuable tool to evaluate oocyte cytoplasmic maturation and that this IVM protocol was efficient in inducing gradual morphological changes necessary for cytoplasmic maturation of pre‐pubertal and adult cat oocytes.
Uterine Progesterone Receptor and Leukaemia Inhibitory Factor mRNA Expression in Canine Pregnancy
Authors: Schäfer‐Somi S , Klein D , Beceriklisoy HB , Sabitzer S , Ay SS , Agaoglu AR , Kücükaslan I , Kaya D , Aksoy OA , Aslan S
Published: July 2009

Contents

The study investigated the expression of genes for progesterone receptor (PR) and for the cytokine leukaemia inhibitory factor (LIF) in the uterine tube and uterine horn tissues from pregnant and non‐pregnant bitches. The aim was to study whether a relation existed between the likely biological effectiveness of progesterone (P4) and the change in the uterine expression of LIF mRNA during pregnancy, as has been described in primates. For this purpose, 20 pregnant bitches were ovariohysterectomized after being allotted to three groups according to gestational age (pre‐implantation: days 10 to 12, n = 7; peri‐implantation: days 18 to 25, n = 7; post‐placentation: days 28 to 45, n = 7). Tissue samples were obtained from the uterine tubes, one uterine horn (including placentation sites and interplacental sites in bitches that had already implanted) and the corpus uteri, stored at −80°C, and then analysed by qualitative and quantitative PCR for PR and LIF mRNA expression. From the pre‐implantation to the placentation stage, a decrease in the relative expression of PR mRNA in uterine tissue was obvious and significant when expressed relative to β‐actin (11.2 ± 6.8 vs 2.7 ± 1.9; p < 0.05). However, over the same period, the relative expression of LIF mRNA increased (10.1 ± 16.1 vs 50.0 ± 32.3; p < 0.05). In addition, PR mRNA went from being detectable to no longer detectable in the uterine tube, and no longer detectable in interplacental‐site uterine tissue. We conclude that LIF is important for the establishment of canine pregnancy; that decreased uterine PR mRNA expression may contribute to the increase in uterine LIF mRNA; and, that the ability of the embryo to preserve PR mRNA expression at implantation and placentation sites while expression is lost in the remainder of the uterus represent an effect important to the establishment and maintenance of pregnancy. We additionally propose that canine embryo secretory proteins have a regulatory effect on both PR and LIF before as well as at and after implantation.
The Influence of Oxygen Tension on Cumulus Cell Viability of Canine COCs Matured in High‐Glucose Medium
Authors: Silva AEF , Rodriguez P , Cavalcante LF , Rodrigues BA , Rodrigues JL
Published: July 2009

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High in vitro oxygen (O2) tensions are associated with enhanced levels of reactive oxygen species and cumulus oocyte complex (COC) apoptosis. The objective of this study was to determine the influence of O2 tension on cumulus cell (CC) viability from canine oocytes. Cumulus oocyte complexes were distributed into three groups (CG, T20 and T5) and two O2 tension levels (20% and 5%). The control group (CG) was matured in vitro in a humidified atmosphere with 5% CO2 in air in TCM199 with 26.19 mm sodium bicarbonate, 10% (v/v) foetal calf serum (FCS), 0.10 mm gentamicin, 0.20 mm pyruvic acid, 20 μg/ml oestradiol, 0.5 μg/ml follicle‐stimulating hormone, 0.03 IU/ml human chorionic gonadotropin, and 1.0 μg/ml human somatotropin. Groups T20 and T5 were matured under 20% or 5% O2 tensions respectively in a high‐glucose medium, without FCS. T20 and T5 were as CG, and supplemented with 0.1% Polyvinyl Alcohol, and 5.5 mm glucose. After 48 h of IVM, CCs from COCs were stained with propidium iodide (1.50 mm). The results showed that viability of CCs (cytoplasmic features and nuclear morphological integrity) was different for the three groups. Rates of apoptosis were at 57.9% (521/900) for CG, 54.4% (490/900) for T20 and 38.9% (350/900) for T5 (p < 0.001). Predominant features in apoptotic cells (n = 1361) were DNA nuclear fragments (94.0%). It was concluded that CCs of canine COCs cultured in high‐glucose medium showed significantly less apoptosis than those cultured in medium with FCS. Low O2 tension was efficient in reducing apoptosis in canine CCs.
Tris‐Lecithin Extender Supplemented With Antioxidant Catalase for Chilling of Canine Semen
Authors: Beccaglia M , Anastasi P , Chigioni S , Luvoni GC
Published: July 2009

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The aims were to evaluate the suitability of a non‐commercial TRIS‐lecithin (LC) extender and the effect of different concentrations of catalase (CAT) on motility, capacitation status (Chlortetracycline‐assay) and zona pellucida (ZP) binding capacity of canine spermatozoa stored at +5°C for 4 days. The sperm‐rich fractions of the ejaculates of four stud dogs were divided into four aliquots. After centrifugation, sperm pellets were diluted (200 × 106 sperm/ml) in TRIS buffer, citric acid, fructose, antibiotics, supplemented with 20% egg yolk (TRIS‐EY) or 0.04% soybean lecithin (TRIS‐LC) with CAT (150 or 450 UI/ml) or without CAT, and then preserved at 5°C for 4 days. The results showed that LC is a valid alternative to EY for chilling canine semen, as similar rates of motility, number of uncapacitated spermatozoa and of spermatozoa binding the oocyte ZP were obtained in semen chilled in TRIS‐LC or TRIS‐EY. Different concentrations of CAT in a TRIS‐LC based extender did not improve the quality of semen after chilling. However, a concentration of 150 UI/ml CAT resulted in an increased number of spermatozoa bound to the oocyte ZP, after 4 days of chilling when compared to semen chilled with TRIS‐EY and TRIS‐LC. In conclusion, an animal protein‐free extender with soybean LC, as a replacement of EY, is suitable for 4 days chilling of canine spermatozoa, but the addition of CAT does not improve general semen quality except for a slight effect on sperm‐ZP binding.
Follicular Dynamics, Ovulation and Conception Rates in Bitches
Authors: England GCW , Russo M , Freeman SL
Published: July 2009

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Real‐time ultrasound imaging was used in a clinical study to estimate the number of follicles of different sizes, ovulation and conception rates, and to study follicle dynamics following oestrus‐induction of bitches.

Follicles were identified during late anoestrus (between 100 and 60 days prior to the pre‐ovulatory LH surge) and there appeared to be a shift in the population from small follicles (1–3 mm in diameter) to large follicles (>4 mm diameter) approximately 2 days prior to the LH surge. Corpora lutea could be reliably identified although the majority were cavitated. High ovulation rates (97–100%) and pregnant rates (86–100%) were detected, and although the conception rate was approximately 70% it varied between 8 and 92%. Within the narrow range of the clinical population studied there were trends relating age to reproduction performance. Oestrus induction with a gonadotrophin regime appeared to result in large numbers of small follicles that did not ovulate, whilst when using cabergoline the number of small and large follicels and the number of copora lutea were similar to those of control cycles.
Cryopreservation of Canine Semen – New Challenges
Authors: Farstad W
Published: July 2009

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Egg yolk (EY) protects cell membranes against cold shock, and it prevents or restores the loss of phospholipids from the membrane. EY has been widely used in semen extenders. It has been added to Tris–Glucose buffer and has been widely used for cooling and cryopreservation of canine semen. EY is not a defined entity, but a complex biological compound containing proteins, vitamins, phospholipids, glucose and antioxidants which are all potentially useful for cell membrane integrity. Unfortunately, it also is a biologically hazardous compound. Hence, whole EY needs to be replaced by other chemically defined components for semen processing in dogs. Freezing poor semen does not improve its quality, so attention must be focused on how to cope with dogs whose semen does not freeze well, and on designing individual freezing extenders for semen from such males. Furthermore, differences have been found among canid species in the ability of their spermatozoa to withstand freezing. There are differences in sperm membrane fatty acid composition among species, which may explain part of these differences. If the presence of long‐chained polyunsaturated fatty acids contributes to increased membrane fluidity, this relationship may be biphasic, i.e. either too much membrane fluidity, or too little, could compromise successful sperm cryopreservation. An increase in fluidity of the outer leaflet of the plasma membrane has been shown in frozen thawed dog spermatozoa. The protective effect of exogenous lipids may lie in close association with the membrane rather than in modification or rearrangement of the membrane. This also points at lipids as an important, if not entirely new group of substances, which may substitute standard EY‐based diluents in preserving sperm survival during freezing. EY‐derived phospholipids or lecithin could be used to replace whole EY. Vegetable lecithin is currently investigated to avoid using substances of animal origin. EY also contains antioxidants which prevent cells from oxidative damage due to the generation of reactive oxygen species. An increasing number of publications now recognize the significance of protecting sperm from this damage during processing by using dietary or diluent supplemented antioxidants. This paper aims at looking at some of the new challenges in freezing of dog semen.
Comparison of Selected Endocrine Parameters During Luteal Phase and Pregnancy in German Shepherd Dogs and Beagles
Authors: Günzel‐Apel AR , Beste N , Nottorf S , Eschricht F , Hoppen HO , Dieleman S , Einspanier A
Published: July 2009

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Concentrations of progesterone, prolactin and relaxin in serum at predetermined intervals after ovulation (day 0) in non‐pregnant and pregnant normocyclic Beagles were assayed and results compared with those observed in German Shepherd dogs (GSD) in a previous study. The goal was to determine possible reproductive hormone specificities related to the GSD breed. Furthermore, the effects of medroxyprogesterone acetate (MPA)‐treatment in non‐pregnant Beagles and of progesterone supplementation in pregnant Beagles on the hormone concentrations were examined. Mean concentrations of progesterone and prolactin were not different in the non‐pregnant Beagles compared with those seen in non‐pregnant GSD, except at days 50–60, when progesterone concentrations were found to be higher in Beagles (p < 0.05). Mean progesterone concentrations in pregnant Beagles at days 50–60 after ovulation (day 0) were higher (p < 0.05) than in GSD at that time, but not at earlier time periods. Prolactin concentrations were higher (p < 0.05) in Beagles throughout pregnancy compared with those in the GSD. Mean relaxin concentrations were numerically but not significantly lower in GSD than in Beagles throughout pregnancy. A 10‐day oral MPA treatment did not affect progesterone or prolactin secretion in normocyclic non‐pregnant Beagles. Medroxyprogesterone acetate serum concentrations were approximately 3.9 ng/ml during treatment and decreased to 0.42 and 0.021 ng/ml within 5 and 15 days after end of treatment, respectively. Intramuscular progesterone supplementation from days 30 to 40 in pregnant Beagles resulted in higher concentrations of progesterone in the 36‐ to 45‐day time periods; prolactin and relaxin concentrations were not significantly affected during or after treatment compared with administration of placebo. The results suggest a tendency towards deficient luteal function in the short‐cycle GSD bitches previously studied, which in pregnancy may reflect the observed decreased prolactin concentrations; the possibility that GSD relaxin secretion is deficiency required needs further study. As oral treatment with MPA did not affect progesterone and prolactin release, it may be useful for studying luteal function in pregnant bitches with suspected hypoluteoidism.
Follicular Morphology, Oocyte Diameter and Localisation of Fibroblast Growth Factors in the Domestic Dog Ovary
Authors: Songsasen N , Fickes A , Pukazhenthi BS , Wildt DE
Published: July 2009

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Remarkably little is known about folliculogenesis in the dog. Objectives were to characterise (1) changes in follicle/oocyte diameter and granulosa cell number and (2) localisation of fibroblast growth factor (FGF)‐2 and FGF‐7 during dog ovarian follicle development. Fourteen ovarian pairs were excised and processed for histological evaluation. Two to four serial sections/bitch were stained with hematoxylin, and follicle/oocyte diameters and granulosa cell number were determined at each developmental stage. Mean follicle and oocyte size were compared among stages by one‐way analysis of variance. Relationships between follicle and oocyte size and granulosa cell number were determined using correlation and regression analysis, respectively. Another eight serial sections/bitch were processed for immunostaining to determine FGF‐2 and FGF‐7 localisation. Primordial and primary follicles were similar in size, but smaller than the progressively increasing (p < 0.05) diameter of the later stages. Oocyte diameter gradually increased (p < 0.05) among oocytes derived from primordial, primary, secondary and early antral follicles with no difference (p > 0.05) thereafter. Oocyte size and granulosa cell number increased (p < 0.01) with follicular diameter. Except during anoestrus, FGF‐2 occurred in oocytes and granulosa cells of primordial to secondary follicles. In adult bitches, FGF‐7 was localised in granulosa cells of primary and secondary follicles and also occurred in the theca layer of antral follicles during prooestrus and oestrus. In summary, folliculogenesis in the domestic dog occurs in two phases: pre‐antral phase characterised by increasing follicle size in association with oocyte growth and granulosa cell proliferation and antral phase linked with marked granulosa cell proliferation and accumulation of antral cavity fluid. Finally, the temporal localisation pattern of FGF‐2 implies its role in follicular activation, whereas FGF‐7 activities appear related to later folliculogenesis.