bluuebunny/crossref_metadata_2025 huggingface dataset:
Radiofrequency Ablation, Where It Stands in Interventional Radiology Today
Authors: Patel Vipulkumar , Ritchie Charles A. , Padula Carlos , McKinney J. Mark
Published: December 2019

Radiofrequency ablation (RFA) is one of the first developed minimally invasive definitive cancer therapies. The safety and efficacy of RFA is well documented and has led to its incorporation into multiple international societal guidelines. By expanding on the body of knowledge acquired during the clinical use of RFA, alternative ablative technologies have emerged and are successfully competing for locoregional therapy market share. The adaption of newer ablative technologies is leading to a rapid decline in the utilization of RFA by interventional radiologists despite the lack of proven superiority. In their 2010 article, Hong and Georgiades stated “… RFA is likely to remain the mainstay of ablations for small tumors until sufficient experience emerges for the widespread acceptance for alternative ablative modalities.” Within a decade of this publication, has this time arrived?
Sana Dialog 2019: Wertschöpfung, Wachstum, Wettbewerb
Authors: Salein Tobias
Published: December 2019

Aktuelle Fragen der Kliniklandschaft aus jeweils unterschiedlicher Perspektive zu diskutieren, das ist das Ziel der Jahresveranstaltung „Dialog“. Unter dem Veranstaltungsmotto (UM)SETZEN. MACHEN! hatte Sana Einkauf & Logistik am 30. September 2019 zum Dialog nach Neuss eingeladen.
Technologiepartnerschaften: Schulterschluss auf Zeit
Authors: Rößing Sabine
Published: December 2019

Systempartnerschaften versprechen finanzielle Planungssicherheit und schonen die Kapitaldecke von Krankenhäusern. Vor dem Hintergrund überstrapazierter Investitionsbudgets und steigender Anforderungen an die Medizintechnik steigt die Nachfrage.
Interview mit Markus Wild: Transparenz schaffen: Herausforderungen im Klinikeinkauf
Authors: Pott Ariane
Published: December 2019

Markus Wild ist CEO und Geschäftsführer der Prospitalia GmbH. Seit mehr als 10 Jahren gestaltet er maßgeblich die Entwicklung des Einkaufsdienstleisters hin zur Unternehmensgruppe. Mit seiner langjährigen Erfahrung berichtet er, wie man den Schwierigkeiten im modernen Beschaffungswesen begegnet.
Prozessoptimierung Beginnt bei Sauberen Stammdaten: So halten Kliniken ihre Stammdaten sauber
Authors: Luz Christoph
Published: December 2019

Damit das Bestellwesen in Kliniken einwandfrei funktioniert, benötigen diese neben durchgängigen elektronischen Prozessen vor allem eines: saubere Stammdaten. Wie gelingt es Kliniken, ihre Stammdaten sauber zu halten? Und wie können spezialisierte Dienstleister mit Mapping-Services dabei helfen?
Entscheidungsprozesse im Einkauf: Entscheidungsprozesse ändern sich – und damit auch das Kompetenzprofil der Einkäufer
Authors: Süme Christoph , Rong Oliver
Published: December 2019

Entscheidungsprozesse in der Beschaffung haben sich vom fachlichen Anwender auf den kaufmännischen Entscheider verschoben. Der aktuelle Trend zum Einbinden von Einkaufsgemeinschaften (EKG) führt zu einem Outsourcing von originären Produktentscheidungen. Durch den direkten Impact auf die Patientenversorgung sind hier klare Prozesse und eindeutige Kompetenzverteilungen und -ausbau essenziell.
Logistik und Bauplanung (Teil 2): Effizienter Krankenhausneubau durch ein logistisches Betriebskonzept
Authors: Raida Andrea , Moll Beate , Wibbeling Sebastian
Published: December 2019

Nachträgliche bauliche Anpassungen im laufenden Krankenhausbetrieb sind zeit- und kostenintensiv oder aus baulicher Sicht nicht mehr durchführbar. Durch eine frühzeitige Integration der Logistik in den Bauplanungsprozess in Form eines logistischen Betriebskonzepts sind solche Probleme vermeidbar.
Healthcare-Control-Center: Mit Flughafenlösungen ein Krankenhaus steuern
Authors: Emmermann Marco
Published: December 2019

Das Healthcare-Control-Center-Tool (HCC) ist ein bisher auf Flughäfen angewandtes Konzept der digitalen Planung und Steuerung von Prozessbläufen. Dessen Übertragung auf Krankenhausprozesse wurde im Rahmen der med.Logistica 2019 mit dem Leipziger Preis für Krankenhauslogistik ausgezeichnet. Das Ergebnis dieser Steuerung sind schneller versorgte Patienten, kürzere Liegezeiten sowie eine reibungslosere und transparentere Organisation des Entlassmanagements.
Planungsleistungen Rechtssicher Ausschreiben: Architekten und Fachplaner beauftragen: So holen Sie das Beste für Ihre Klinik heraus
Authors: Marx Christopher
Published: December 2019

„Die Mindest- und Höchstsätze der HOAI sind unwirksam.“ Ein Paukenschlag des Europäischen Gerichtshos (EuGH)! Bislang schrieben Kliniken ihre Architekten und Fachplaner im Wettbewerb aus und vergüteten diese nach der Honorarordnung für Architekten und Ingenieure (HOAI). Diesem Vorgehen hat der EuGH nun ein Ende gesetzt.
Compliance im Beschaffungsprozess: Prinzipien der Anti-Korruption: Der Einkäufer muss unkäuflich bleiben!
Authors: Schneider Hendrik
Published: December 2019

„Wir halten uns an die Gesetze!“, so oder ähnlich beginnen Compliance-Richtlinie vieler Unternehmen, auch im Bereich des Gesundheitswesens. Der Satz drückt Selbstverständliches aus – ist aber im Alltag des Klinikeinkäufers manchmal nicht einfach zu befolgen.
Unitary dilations of discrete-time quantum-dynamical semigroups
Authors: vom Ende Frederik , Dirr Gunther
Published: December 2019

We show that the discrete-time evolution of an open quantum system generated by a single quantum channel T can be embedded in the discrete-time evolution of an enlarged closed quantum system, i.e., we construct a unitary dilation of the discrete-time quantum-dynamical semigroup (Tn)n∈N0. In the case of a cyclic channel T, the auxiliary space may be chosen (partially) finite-dimensional. We further investigate discrete-time quantum control systems generated by finitely many commuting quantum channels and prove a similar unitary dilation result as in the case of a single channel.
Proton firehose instability: An interplay of thermal core and non-thermal halo protons
Authors: Sarfraz M. , Noureen S. , Abbas G. , Zeba I. , Jamil M. , Gillani S. S. A.
Published: December 2019

Higher levels of solar wind temperatures are reported to be constrained. Microinstabilities play a key role under dilute space plasma conditions. The present study highlights the role of proton firehose instability in defining parallel temperatures of protons. Considering reality, we chose a bi-Maxwellian model for core protons, while halo protons are best modeled with kappa distribution. Taking different sets of input parameters like temperature anisotropy, plasma beta, and kappa index into account, the growth rate levels and associated domains for an unstable firehose mode are investigated.
Structural and electronic properties of dilute-selenide gallium oxide
Authors: Liu Xiaoli , Tan Chee-Keong
Published: December 2019

First-principles density functional theory is applied to investigate the electronic and structural properties of dilute-Se β-Ga2(O1−xSex)3 alloys with the Se-content ranging from 0% to 16.67%. The findings showed that the addition of Se has significant effect on the β-Ga2O3 alloy properties. The equilibrium volume and lattice parameters of β-Ga2(O1−xSex)3 alloys are presented, showing a general expansion with increasing Se-content. Further analysis indicates the expansion rate in the c (001) direction is much larger than that in the a and b directions, in which the information provides important guidance for the manufacturing of the β-Ga2(O1−xSex)3/Ga2O3-based material. From our analysis of the band structures, the β-Ga2(O1−xSex)3 alloys exhibit indirect bandgap property with the bandgap energy decreasing dramatically from 4.868 eV to 2.759 eV. The wavelength derived from the direct bandgap energy covers a regime from 255 nm to 475 nm, implying the potential of β-Ga2(O1−xSex)3 alloys in an ultraviolet photodetector and visible light applications. In addition, electron effective masses are calculated and presented for the β-Ga2(O1−xSex)3 alloys, in which the electron effective mass reduces as the Se-content increases. As a part of a highly mismatched alloy semiconductor class, dilute-Se Ga2(O1−xSex)3 is discussed for the first time with no prior literature in our work, and our findings indicate the potential implementation of GaOSe alloys for electronic and optoelectronic device applications.
Lithium-ion battery pioneers awarded Chemistry Nobel
Authors: Miller Johanna L.
Published: December 2019

The batteries have already powered one revolution in wireless consumer electronics. Now they’re launching a new one in transportation.
Climate and weather in Physics Today
Authors: Day Charles
Published: December 2019

The magazine marks the centenary of the American Meteorological Society by looking back at how our coverage of meteorology has evolved.
The Wonderful World of Poo: The Turdome and Beyond
Authors: Nice Edouard C.
Published: December 2019

Defecate: it is something we all do, it is something we joke about, yet for many in real life it is a subject that is taboo. However, it is now being realised that faeces are a veritable scientific goldmine, have many potential uses, and may even save your life! In this article I will review the history behind the use of faecal material and look at some of its emerging playing fields, in particular its role in medical diagnosis. I will discuss faecal proteomics and other omics technologies (Proteogenomics: The Omics Pipeline), including studies on the microbiome, in order to understand, diagnose, and treat gastrointestinal tract pathologies and other diseases, and show how these technologies will play a role in the move towards personalized medicine.
100 Relationship between corpus luteum blood flow evaluated via colour Doppler ultrasound and pregnancy rate in bovine embryo transfer recipients
Authors: Sermersheim M. K. , Lindsey B. R. , Naves L. M. , Rubessa M. , Wheeler M. B.
Published: December 2019

The advent of colour Doppler ultrasound has allowed evaluation of blood flow to bodily tissues. This novel technology is being tested as a tool to improve efficiency in a variety of livestock reproduction programs. The objective of the present study was to evaluate the relationship between blood flow to the ovarian corpus luteum (CL), imaged via colour Doppler ultrasound, and pregnancy rate in crossbred dairy recipients for embryo transfer (ET). Oestrous cycles of Bos indicus×Bos taurus dairy heifers (n=90, 16-24 months of age) were synchronized for embryo transfer on Days 7-8. Immediately before ET, heifers were palpated for the presence of ovarian CL and CL papillae. Presence of CL, CL papillae, and CL lacunae were confirmed via transrectal B-mode ultrasound (Ibex EVO I, E. I. Medical Imaging). Transrectal colour Doppler ultrasound (Ibex EVO I, E. I. Medical Imaging) was used to evaluate blood flow to the CL. Invitro-produced (IVP) blastocysts (qualities I and II) were transferred to the uterine horn ipsilateral to the CL. All palpation, imaging, and embryo transfer was performed by a single technician. Pregnancy was determined via transrectal ultrasound 24-32 days post-embryo transfer (31-39 days of embryo age). Blood flow to the CL was split into three categories: high, median, and low. Presence or absence of two additional CL structures, CL papillae and lacunae, were recorded. Fisher's exact test was used to evaluate data. Statistical differences were considered significant at P<0.05. The overall pregnancy rate was determined to be 40%, 36 out of 90. Pregnancy rates were similar in high (33.9%, 20 of 59), median (59.09%, 13 of 22), and low (33.33%, 3 of 9) blood flow categories. Consistent with previous studies, absence of CL papillae and presence of CL lacunae at the time of ET did not affect pregnancy rate. Doppler blood flow, papillae, and lacunae were also evaluated together in each possible combination. Only one outlier was present; all other differences were not significant. The highest pregnancy rate (66.67%, 10 of 15) was observed in recipients with CLs with median Doppler blood flow, a palpable CL papillae, and no CL lacunae. A larger sample size is required to determine the accuracy of this measurement. In conclusion, recipients with high to low CL blood flow are suitable for IVP embryo transfer programs. Alone, colour Doppler imaging of the CL does not predict pregnancy rate.
108 Use of seminal plasma as ovulation inductor in alpacas (Vicugna pacos) embryo recipient and its effect on pregnancy rate
Authors: Huanca W. , Garcia K. , Huanca W. F. , Cordero A. , Malaga J.
Published: December 2019

Alpacas, like other camelids, are classified as induced ovulators because an external stimulus is required for the occurrence of ovulation. Recent studies have identified the β-nerve growth factor (β-NGF), a protein present in the seminal plasma (SP), as responsible for inducing ovulation in this species and having potent luteotropic function. We speculate that SP can be used in reproductive biotechnologies, such as embryo transfer (ET), to increase the number of genetically superior animals produced in breeding programs. The objective of this study was to evaluate the effect of inducing ovulation with SP or a gonadotrophin-releasing hormone (GnRH) analogue on pregnancy rate in recipients of an alpaca ET program. Semen from 5 adult male alpacas was collected with an artificial vagina and diluted 1:1 (v/v) with phosphate-buffered saline (PBS; Gibco-BRL). The diluted semen was centrifuged for 30min at 1200g, and the supernatant or SP was decanted and examined for absence of sperm. Then, the SP was centrifuged again for 20min at 1200×g. The SP was stored at −80°C until the use. Female alpacas (n=38; 6-8 years) with a body condition score of 2.5-3.5 (scale 1-5) were used for the experiment. Animals were evaluated daily by transrectal ultrasonography to determine the presence of a dominant follicle ≥7mm and randomly assigned to one of two groups: (1) GnRH (n=20), 0.04mg of Buserelin acetate IM, or (2) SP (n=18): 1.0mL of seminal plasma IM. Seven days after GnRH or SP treatment (Day 0) the recipients received a fresh embryo. Alpaca donors (n=18) were given GnRH (0.04mg IM) and treated 36h later with 700IU of equine chorionic gonadotropin (ECG). Donors were mated with fertile males 5 days after ECG (Day 0 of recipients), and embryos were recovered 7 days later. Embryos of similar quality were assigned to both groups and transferred nonsurgically to the uterine horn ipsilateral to the corpus luteum. Ultrasonography examinations were performed on Day 2 to confirm ovulation and Day 25 to determine pregnancy in all of the recipients. Data was analysed by chi-squared test. Ovulation rate was not different between groups (100% each). Pregnancy rate was 45% (9 out of 20) and 44% (8 of 18) in GnRH and SP groups, respectively (P=0.77). In conclusion, SP was effective to induce ovulation in alpacas and was able to produce a pregnancy rate similar to that of GnRH as an ovulation-inducing treatment. Thus, SP can be used as an alternative for ET programs in alpacas.
Research was funded by the project Role of Seminal Plasma in Reproductive Physiology and Application of Biotechnologies in Camelids (149-2017-CIENCIACTIVA).
113 Effect of antioxidants on motility and fertility of liquid-stored bovine sperm
Authors: Honkawa Y. , Fujikawa T. , Miura N. , Kubota C.
Published: December 2019

It is difficult to maintain sperm in liquid storage for a long time, compared with permanent frozen storage in liquid nitrogen. Antioxidants have been reported to improve the quality and fertility of liquid-stored semen. In this study, we investigated whether antioxidants can extend the motility and fertility of frozen-thawed sperm in liquid storage. Frozen-thawed semen from one Japanese black bull (one ejaculate) was diluted in Tris-citrate-fructose (TCF) diluent with 10% (v/v) egg yolk to a sperm concentration of 1×107 spermmL−1. The antioxidants β-mercaptoethanol (βMe) and glutathione (GSH) were added independently, at various concentrations (0.1, 0.5, 1, and 5mM) to sperm suspensions, and these preparations were compared with Control (no added antioxidant). Sperm suspensions were packaged in centrifuge tubes and placed at 17°C in air and monitored daily until sperm motility had stopped (up to 14 days). Sperm motility was analysed by the Sperm Motility Analysis System (SMAS; Ditect Co. Ltd), and the percentage of progressively motile sperm (straight-line velocity (VSL) of >25μm s−1; Grade A classified by WHO manual), compared with that recorded on Day 0 (100%), was determined each day. For evaluation of fertilizing ability, after incubation in liquid storage for 0, 3, 5, and 7 days, sperm were used for IVF with invitro-matured oocytes (30 oocytes per treatment, three replicates). Embryo development was recorded as the proportion of embryos that reached blastocyst by 8 days after IVF. Data for motility were analysed using one-way ANOVA with Tukey test, and embryo development using chi-squared test. A P-value<0.05 was considered statistically significant. At 7 days, the percentage of progressively motile sperm was significantly higher for 0.5, 1, and 5mM βMe than for Control (30.8%, 48.1%, and 50.3%, vs. 0%, respectively). Treatments with 1 and 5mM βMe maintained some sperm progressive motility for 14 days (9.5% and 14.5%). Treatment with GSH showed the same trend at 7 days (32.2%, 36.3%, and 13.7% for 0.5, 1, and 5mM, vs. 0% for Control); 1 and 5mM GSH maintained sperm progressive motility over 10 days (24.8% and 4.4%). In both antioxidant treatments, embryo development was achieved with sperm stored for up to 5 days (Day 0 vs. Day 5 for 0.1mM βMe: 17.6% vs. 13.8%; for 1.0mM GSH: 26.0% vs. 6.7%; for Control: 17.6% vs. 0%). In this study, antioxidants extended both motility and fertility of frozen-thawed bovine sperm in liquid storage. This result suggests the possibility of application to AI using liquid-stored bovine semen.
117 Supplementation of IVF medium with nerve growth factor improved bovine embryonic cleavage rates during summer months
Authors: Amiss E. , Stewart J. W. , Negrón-Pérez V. M. , Jones K. , Haines H. , Rhoads M. L. , Lima F. S. , Stewart J. L.
Published: December 2019

Nerve growth factor-β (NGF), a protein originally associated with regulation of neuron development, has been found to play a role in the reproductive system of mammals. Previous research showed that administration of NGF to cows resulted in enhanced conceptus development. Although these effects were speculated to be a result of improved corpus luteum function, whether NGF could act directly on the embryo remained undetermined. Therefore, the direct effects of NGF on fertilization and embryo development warrant investigation to see whether it can be used as a novel tool to improve cleavage and blastocyst rates when producing embryos via IVF during periods of suboptimal oocyte quality, such as with heat stress. The objective of this study was to explore how supplementation of NGF, purified from bull seminal plasma, during IVF may directly affect embryo development in oocytes harvested in the summer. Abattoir-derived bovine ovaries were used for recovery of cumulus-oocyte complexes (COCs) over eight replicates through May and June. On Day −1, COCs were collected and matured for 20h in oocyte maturation medium incubated at 38.5°C. On Day 0, matured oocytes were added to a solution of IVF-Tyrode's albumin lactate pyruvate (TALP) and either phosphate-buffered saline (PBS; control) or 100ngmL−1 NGF. Pooled frozen-thawed semen from two different bulls per replicate were added to the IVF solutions and incubated with COCs for 20h at 38.5°C in a humidified atmosphere of 5% CO2. On Day 1, zygotes were washed in HEPES-TALP, and cumulus cells were removed using 1% hyaluronidase. The zygotes were plated in synthetic oviductal fluid (SOF-BE2) culture medium and incubated at 38.5°C in a tri-gas chamber (5% CO2, 5% O2, and balanced N2). Cleavage rates were recorded at 24 and 48h, calculated by dividing the number of cleaved embryos by the total zygote count. Embryos were incubated until Day 8, when the rate of blastocysts was assessed. This study found that the treatment of IVF medium with NGF increased the cleavage rate of embryos after 48h (Control: 59%; NGF: 66%; P=0.04) and the hatched blastocyst percentage per oocyte on Day 8 (Control: 6.7%; NGF: 9.4%; P=0.01). The treatment did not affect the percentage of blastocysts per cleaved embryos (Control: 21%; NGF: 22%; P=0.16) or the hatched blastocyst rate at Day 8 (Control: 53%; NGF: 55%; P=0.67). These results show that NGF can act directly on the oocyte during fertilization to alter subsequent development, specifically through increased embryonic cleavage rates. Further studies are needed to assess different dosages of NGF in order to mitigate the detrimental effects of heat stress on oocyte competence for use in IVF. Follow-up studies using a whole-animal model are needed to understand the clinical relevance of these findings in the ability of embryos to promote maternal recognition of pregnancy.
121 Effect of pentoxifylline on motility of good- and poor-quality frozen-thawed equine sperm
Authors: Felix M. , Ortiz I. , Resende H. , Brom-de-Luna J. , Love C. , Hinrichs K.
Published: December 2019

Equine semen used for intracytoplasmic sperm injection (ICSI) is typically frozen-thawed and may be of poor quality. To prepare sperm for ICSI, semen is typically centrifuged to remove freezing extender. However, centrifugation can cause damage to sperm, which is especially meaningful if sperm quality is already poor. We evaluated a method for selection of sperm without centrifugation, using a “swim-over” technique, and assessed the effect of pentoxifylline, a phosphodiesterase inhibitor that increases sperm motility in other species. To mimic poor-quality semen, we thawed frozen semen (1×) and re-froze it three additional times (4×). Aliquots (0.25 µL; 50,000 sperm) of 1× or 4× semen were placed at the bottom of the right leg of an “H,” made using 15µL of medium by tracing a template placed below a Petri dish. The medium used (Hanks’ balanced salt solution with 40mg mL BSA and added lactate and pyruvate) contained different concentrations of pentoxifylline (0, 0.5, 1, 2 or 4mgmL−1). One µL of medium was removed from the tip of the left arm of the H after 15 and 30min incubation, and the number of sperm were counted. In a second study, we evaluated the effect of pentoxifylline on sperm motility parameters using computer-assisted sperm motility analysis. After thawing, 1× and 4× semen was washed to remove freezing extender and resuspended in the same medium but with 7mgmL−1 bovine serum albumin (BSA), containing the different pentoxifylline concentrations. In Study 1, the number of collected sperm did not differ significantly for 1× sperm exposed to 0 to 4mgmL−1 pentoxifylline (means of 15 to 23 sperm at 15min, and 18 to 25 sperm at 30min). Similarly, in 4× frozen semen, there was no significant difference in number of collected sperm between 0mgmL−1 and 2 or 4mgmL−1 pentoxifylline concentrations (<1 to 6 at 15 min; 5 to 6 at 30min). In Study 2, at 0min,% total motility was significantly higher in 1 and 2mgmL−1 pentoxifylline than in 0mgmL−1 for 1× sperm (47.8±1.7 and 49.3±1.9, vs. 32.1±3.9, respectively; P=0.018) and significantly higher for 1, 2, and 4mgmL−1 pentoxifylline than for 0mgmL−1 for 4× sperm (3.9±0.9, 5.7±0.4, and 8.2±0.5, vs. 1.2±0.4; P=0.0001). Similar results were found at 15 and 30min for 1×, and at 15min for 4×. Pentoxifylline at 1 to 4mgmL−1 significantly increased the percentage of progressive motility in 1× sperm at 30min (17.8±1.3, 21.8±2.7, and 20.3±1.2, vs. 10.0±0.4; P=0.002) and, at 4mgmL−1, increased the percentage of progressive motility in 4× sperm at 0min (1.43±0.1 vs. 0.2±0.1; P=0.005) and 15min (1.4±0.2 vs. 0.1±0.0; P=0.0001). Exposure of poor-quality semen to pentoxifylline at 4mgmL−1 improved total and progressive motility but did not increase the recovery of motile sperm in a swim-over collection preparation.
127 Chemokine receptor 2 (CCR2) is expressed in growing oocytes, and its deficiency affects follicular activation and long-term female fertility in mice
Authors: Santos A. G. A. , Pereira L. A. A. C. , Russo R. C. , Viana J. H. M. , Campos-Junior P. H. A.
Published: December 2019

Chemokine receptor 2 (CCR2) has important functions in several biological processes, including activation of PI3K/Akt/mTOR signalling, a key pathway in follicular activation. However, there is no report about the role of CCR2 in ovarian follicular physiology. The objectives of this study were (1) to immunolocalize CCR2 in mice ovaries and (2) to evaluate the effects of CCR2 deficiency on follicular growth during adult life and aging. A total of 74 C57Bl/6 (wild type, WT) and 68 B6.129S4-Ccr2tm1Ifc/J (CCR2−/− (knockout)) female mice were used. For objective 1, ovaries were collected from WT mice at 1.5 months old (m.o.), fixed in 4% PFA, embedded in paraffin, and used for immunoperoxidase staining with an anti-CCR2 antibody [EPR19698] (ab222496; 1:200) and using anti-rabbit IgG (Ab6721, 1:100) as a secondary antibody. Also, MII oocytes from oviducts of superovulated WT mice were processed with the same primary antibody for immunofluorescence. For objective 2, body and ovarian weight were evaluated. Follicle populations were assessed in WT and CCR2−/− mice at 1.5, 2.5, 6, 10, and 12m.o., by serial sectioning; the total follicle population was counted in every third section in the whole ovary. Additionally, ovarian total RNA isolation was performed from WT and CCR2−/−. Real-time PCR was used to evaluate differential gene expression according to standard protocols, using primers for Bax, Casp3, Bcl2, Fshr, and B-actin (endogenous control). The data were analysed using the GraphPad Prism Software, using t-test, and a P-value of 0.05 was considered as significant. Localization of CCR2 was observed exclusively on the membrane and cytoplasm of growing oocytes in primary, secondary, antral, and atretic follicles, as well as on ovulated MII oocytes membrane and cytoplasm. Body and ovarian weight were similar between WT and CCR2−/− mice. At 1.5m.o., CCR2−/− mice had more primordial follicles and fewer primary and secondary follicles compared with WT (P<0.05), whereas there was no difference in the antral follicle populations. Follicular activation (primordial to primary transition) and atresia rates were decreased in CCR2−/− (P<0.05) at 1.5m.o. A downregulation of pro-apoptotic genes (Bax and Casp3) was observed on CCR2−/− (P<0.05), while anti-apoptotic Bcl2 was upregulated (P<0.05) compared with WT in 1.5-m.o. animals. A larger ovarian follicular reserve at 1.5, 2.5, and 6m.o., but not at 10 or 12m.o., was observed in CCR2−/− mice. At 6 to 12m.o., CCR2−/− ovulated more (P<0.05) MII oocytes than WT. Altogether, these data may suggest that CCR2 plays an important role in the regulation of ovarian follicular reserve mobilization, potentially affecting reproductive lifespan.
Financial support was provided by Fapemig and CNPq.
155 Fertility of sexed semen in Holstein heifers and cows
Authors: Yamaguchi M. , Takayama M. , Nishisouzu T. , López H. , Dochi O.
Published: December 2019

Compared with conventional semen, use of sexed semen decreases the conception rate (CR); therefore, it is important to address the factors that lead to sperm damage. Recently, a novel sexed semen technology was developed for improving dairy cattle fertility (Betthauser et al. 2016 J. Dairy Sci. 99 (E-Suppl. 1), 534). However, there are few reports about the fertility of this novel sexed semen in dairy cattle in Japan. The objective of this study was to compare the CR of traditional sorting semen (S1: flow cytometry/cell sorting) and the novel sexed semen (S2: SexcelTM, ABS Global Inc.) in Holstein heifers and cows in Japan. The CR for the first insemination was obtained from 391 Holstein cows and 148 heifers (10.7-17.8 months old) from 14 dairy farms in Hokkaido from June 2017 to April 2019. Semen used for AI was collected from six bulls and packaged into 0.25-mL straws and frozen. Cows inseminated with conventional frozen semen (S3: unsexed semen in 0.5-mL straws) were used as controls. Calving data were collected from 123 cows and 69 heifers that had calved between January 2018 and May 2019. The diagnosis of pregnancy was confirmed using ultrasonography between 30 and 45 days after insemination. The CR and sex ratio were analysed using chi-square test. The average parity number was 1.9±1.1, and average days open was 84.9±20.4 days. The average interval between calving and the first service was 86.2±20.3 days. The average milk yield (at 305 days) was 12 195±1595 kg. All of the animals were inseminated after the onset of standing heat or removal of the tail chalk of natural heats and oestrus and ovulation synchronisation programmes on the farm. Conception rates of heifers of the first service of S1 (n=54) and S2 (n=94) were 68.5 and 70.2%, respectively. Conception rates of cows of the first service of S1 (n=32), S2 (n=173), and S3 (n=186) were 56.3, 57.8, and 53.8%, respectively. There were no significant differences in CR between these groups (P>0.4). The proportion of female calves produced using S1 (n=61) and S2 (n=57) was 91.8 and 93.0%, respectively, which was significantly more (P<0.01) compared with the 50.0% produced using S3 (n=74). There were no significant differences between S1 and S2 (P>0.8) in terms of the proportion of calves produced. The results of this study demonstrated that the traditional sorting and novel sexed semen (S1 and S2, respectively) did not differ in terms of the first-service CR and produced considerably more female calves than when the conventional semen (S3) was used. Furthermore, these findings indicate that the use of this novel sexed semen could achieve a sex ratio close to 90% female offspring.
194 Extracellular vesicles derived from ampullary oviductal fluid improve developmental competence of bovine oocytes
Authors: Asaadi A. , Pavani K. , Azari Dolatabadi N. , Van Damme P. , Van Soom A.
Published: December 2019

Invivo, oocytes mature in the presence of follicular fluid (FF) and ampullary oviductal fluid (AOF), starting from premature to final maturation stages (3-6h postovulation). Extracellular vesicles (EVs) have been identified as important mediators of gamete/embryo-maternal interactions. They carry regulatory molecules, such as microRNAs and mRNAs. So far, the functionality of EVs derived from FF and AOF has not yet been determined. The aim of this study was to evaluate the effect of EVs derived from FF and AOF during invitro oocyte maturation (IVM) on the development and quality of resulting bovine embryos. Follicular fluid of a preovulatory follicle was collected by ovum pickup, and AOF was collected from the oviducts of slaughtered cows in early luteal phase. Then, EVs from FF and AOF were isolated by size exclusion chromatography and confirmed by western blot. The concentration of EVs was determined by NanoDrop and Nanoparticle tracking analysis. Integrity and size of EVs were assessed by electron microscopy. Bovine oocytes (n=1331) were allocated at random to five groups. In the control group (C), oocytes (n=347) were cultured for 22.5h in maturation medium (TCM-199 and epidermal growth factor 20ngmL−1) (MM). In the negative control group (NC), oocytes (n=331) were cultured for 18h in MM, and then transferred into fresh MM for 4.5h. In the FF group, oocytes (n=162) were cultured in MM supplemented with FF EVs (12.5µgmL−1) for 18h, and then transferred to MM for 4.5h. In the AOF group, oocytes (n=328) were cultured for 18h in EV-free MM, and then transferred to MM supplemented with AOF EVs (1.7µgmL−1) for 4.5h. In the FF+AOF group, oocytes (n=163) were cultured in MM supplemented with FF EVs for 18h, and then transferred into MM supplemented with AOF EVs for 4.5h. At 22.5h post-incubation, mature oocytes were fertilized invitro. At 21h post-insemination, presumptive zygotes were denuded and cultured in synthetic oviductal fluid with insulin-transferrin-selenium for 8 days. Cleavage rate and blastocyst rate were recorded at 48h and 8 days post-insemination, respectively. Blastocyst quality was assessed by differential apoptotic staining. The data were analysed using one-way ANOVA. Cleavage rate was not affected by the treatment, whereas blastocyst yield in the AOF group (48.75%) was significantly higher than that in the C (42.91%) and NC (37.72%) groups. In addition, embryos produced in the AOF group showed the highest number of trophectoderm cells and inner cell mass cells, and lowest number of apoptotic cells (P<0.05). Trophectoderm and inner cell mass cells were higher and apoptotic cells were lower in FF and FF+AOF compared with the C and NC groups. In conclusion, adding AOF EVs to oocyte MM in the last 4.5h of IVM improves quantity and quality of blastocysts. In addition, although FF EVs showed no effect on blastocyst yield, they improved blastocyst quality by increasing cell number.
199 Strategies to improve canine oocyte invitro maturation
Authors: Cittadini C. , Duque M. , De Stefano A. , Salamone D.
Published: December 2019

Canine oocyte invitro maturation (IVM) is one of the challenges of animal reproduction because of low maturation and high degeneration rates. In the bitch, after ovulation, oocytes remain in an immature stage and acquire their competence in the intra- and extrafollicular (oviductal) environments. Oxidative stress and reactive oxygen species affect canine oocytes, which can be related to the high amount of lipids they contain. Therefore, the use of antioxidants such as insulin-transferrin-selenium (ITS) and lower oxygen tension during IVM could be beneficial for oocyte maturation and survival. The purpose of this study was to determine an optimum IVM culture medium and to evaluate the effect of ITS and lower oxygen tension in canine IVM. In experiment 1, TCM-199 and synthetic oviductal fluid (SOF) media were evaluated for their ability to promote nuclear maturation at 72 and 48h of culture. Also, two protein sources were used: 8% bovine serum albumin (BSA) and 2.5% fetal bovine serum (FBS), and media were supplemented with hormones. The results revealed that SOF with FBS and BSA had similar results to TCM-199 supplemented with FBS after 72 and 48h of IVM (MII rates of 7% and 4% for the 72-h group, and 4% and 10% for the 48-h group). Synthetic oviductal fluid supplemented with BSA but without FBS produced significantly higher degeneration rates compared with SOF with FBS and BSA (44% and 23%, respectively). Forty-eight hours of IVM decreased degeneration rates, with higher MII rates compared with 72h of IVM. In experiment 2, SOF medium supplemented with FBS and BSA was chosen. Oocytes were cultured in SOF with FBS and BSA supplemented at two concentrations of ITS (1 and 10μLmL−1 ITS). Supplementation with 1μLmL−1 ITS demonstrated a beneficial effect by improving maturation rates up to 20%, compared to control and 10μLmL−1 supplemented group (4% and 6% MII, respectively) after 72h of IVM. For experiment 3, oocytes were cultured in SOF medium with or without ITS (0 and 1μLmL−1 ITS) under two oxygen tensions (5% and 20% O2) for 48h. Results from this experiment demonstrated that the combination of low oxygen tension and ITS (5% O2 and 1μLmL−1 ITS) improved maturation rates up to 26.2%, although there were no statistically significant differences compared with high oxygen and ITS (20% O2 and 1μLmL−1 ITS) and low oxygen without ITS (5% O2 and 0μLmL−1 ITS) groups. These treatments were able to increase MII rates compared with the control group (20% O2 and 0μLmL−1 ITS). Parthenogenetic activation was performed on the low oxygen with or without ITS supplemented groups. The untreated group generated higher degeneration rates after 7 days of culture, and cleavage rates were low for both groups. Nevertheless, an oocyte at the 8-cell stage was obtained in the ITS-supplemented group. Taken together, these results indicate that ITS supplementation and low oxygen tension during IVM improve canine oocyte maturation.
206 Overexpression of germ cell genes DAZL, STRA8, and BOULE in bovine adipose tissue-derived mesenchymal stem cells for male germ cell derivation
Authors: Cordero P. , De los Reyes M. , Parraguez V. H. , Varas-Godoy M. , Torres C. , Peralta O.
Published: December 2019

Invitro gamete derivation from stem cells has potential applications as an alternative method for dissemination of elite animal genetics, production of transgenic animals, and conservation of endangered species. Mesenchymal stem cells (MSC) may be suitable candidates for invitro gamete derivation given their differentiation capacity and their potential for cell therapy. DAZL (deleted in azoospermia-like) and BOULE (also called BOLL) encode RNA-binding proteins that control differentiation of germ cells; STRA8 (stimulated by RA-8) encodes a protein required for meiosis. Considering the crucial roles of these factors, the aim of the present study was to evaluate co-overexpression of different combinations of DAZL, STRA8, and BOULE on germ cell gene expression profile in adipose tissue-derived MSC (AT-MSC). AT-MSC were harvested from abattoir-derived male bovine fetuses (n=9; 7-8 months of gestation). The optimal concentration of Lipofectamine 2000 (1, 1.5, 2, 1.5ng µL−1; Invitrogen) was analysed in AT-MSC transfected with plasmid pSIN-EF2-Puro containing DAZL, STRA8, and BOULE coding sequences (pSIN-EF2-DAZL-P2A-STRA8-T2A-BOULE-Puro). Then, AT-MSC were transfected either with plasmids containing DAZL, STRA8, and BOULE genes (pSIN-EF2-DAZL-P2A-STRA8-T2A-BOULE-Puro), DAZL and BOULE genes (pSIN-EF2-DAZL-P2A-STRA8-T2A-Puro), DAZL (pSIN-EF2-DAZL-P2A-T2A-Puro), or empty plasmid at a concentration of 1ng µL−1 of Lipofectamine. Cell samples were obtained from each plasmid treatment and analysed for expression of housekeeping genes ACTB and GAPDH and germ cell genes DAZL, PIWIl2, STRA8, and BOULE by quantitative-PCR using relative values (Quantity) through the ΔΔCT formula. AT-MSC transfected with pSIN-EF2-DAZL-P2A-STRA8-T2A-BOULE-Puro using 1ng µL−1 of Lipofectamine achieved higher (P<0.05) expression of DAZL (13.3-fold) compared with cells transfected with empty vector. Moreover, AT-MSC transfected with pSIN-EF2-DAZL-P2A-STRA8-T2A-BOULE-Puro had higher (P<0.05) levels of DAZL mRNA (3.8-fold) compared with empty vector. Messenger RNA levels of STRA8 (1.4-fold) were only detected in AT-MSC transfected with pSIN-EF2-DAZL-P2A-STRA8-T2A-Puro; PIWIl2 and BOULE were not detected in transfected or untransfected AT-MSC. In conclusion, bovine fetal AT-MSC are amenable for overexpression of germ cell markers DAZL and STRA8. Transfection with plasmid containing three germ cell genes (DAZL, STRA8, and BOULE) allowed overexpression of DAZL, whereas transfection with plasmid containing two germ cell genes (DAZL and STRA8) achieved overexpression of STRA8.
This study was supported by Fondecyt grant 1191114, Government of Chile.
217 Induction and differentiation of porcine induced pluripotent stem cells into neuronal precursor cell-like cells
Authors: Machado L. S. , Pieri N. C. G. , Botigelli R. C. , Castro R. V. G. , de Souza A. F. , de Lima M. A. , Bridi A. , Andrade A. F. C. , Meirelles F. V. , Freude K. K. , Bressan F. F.
Published: December 2019

The invitro cellular induction into pluripotency [induced pluripotent stem cell (iPSC) generation] has much evolved since its first report and may play a central role in veterinary regenerative medicine. In particular, the generation of invitro neural precursor cells (NPCs) enabled the creation of models for human genetic diseases, such as autism, schizophrenia, and Parkinson disease. The swine model is known to present many advantages over others, including the physiological similarities of pigs and humans. This project aimed to reprogram porcine embryonic fibroblasts (pEF) into iPSCs, and further differentiate them into NPCs. The iPSCs were generated through lentiviral transduction of OCT4, SOX2, c-MYC, and KLF4 genes and cultured with Dulbecco's modified Eagle's medium (DMEM)/F12 KO, 20% knockout serum replacement (KSR), nonessential amino acids, glutamine, β-mercaptoethanol, 10ngmL−1 basic fibroblast growth factor (bFGF), and antibiotics. Three clonal iPSCs lines with typical pluripotent morphology (high nuclear/cytoplasm ratio and well-defined colony borders) were alkaline phosphatase (AP) positive and were further analysed. All three lines [porcine (p)iPSCs lines 1, 2, and 3] expressed exogenous factors for at least 10 passages. Quantitative real-time (qRT)-PCR showed that line 1 expressed endogenous OCT4 and all three lines expressed NANOG. The pEFs were used as controls for 2−ΔΔCt analysis. piPSC line 1 was positive for OCT4, SOX2, NANOG, SSEA-1, TRA-1-60 through immunofluorescence; line 2 was negative for NANOG; and line 3 only presented SOX2 expression. piPSCs lines 2 and 3 differentiated into NPC-like cells. NPC induction was accomplished by 14 days of culture in commercial extracellular matrix (Geltrex) and neural induction media supplemented by SMAD inhibitors LDN193189 (0.2 μM) and SB431542 (10 μM), and then maintained at 20ngmL−1 of epidermal growth factor and basic fibroblast growth factor. They expressed NESTIN and GFAP by qRT-PCR and were positive for NESTIN, β-tubulin III and vimentin by immunofluorescence, confirming the NPC-like cell status. Interestingly, during all stages (pEF, iPSCs, and NPC-like cells), cells were positive to some degree for the neural markers tested by immunofluorescence. Flow cytometry analyses showed a 30% higher intensity of those markers at the NPC-like stage than the pEF and pluripotent stage. These results suggest preliminary evidence for neural differentiation. This will contribute to the use of the porcine model in future regenerative and translational medicine research.
This study was funded by FAPESP (grant nos. 2015/26818-5, 2017/02159-8) and CAPES.
222 Effective donor selection before superovulation treatment for Japanese Black beef cattle
Authors: Muraguchi N. , Uemura H. , Kubota C.
Published: December 2019

Japanese Black beef cattle (JBBC) are a leading Wagyu breed in Japan. Embryo transfers are being used to improve JBBC, a sturdy, fast-maturing breed prized for its marbled beef; however, relevant donor selection techniques have not been fully established. Accordingly, in this study, in order to produce embryos efficiently, we aimed to investigate donor selection techniques based on follicle counts at different time points (Experiment 1) and an associated genetic marker (Experiment 2). Statistical differences were evaluated with Student's t-test or Welch's t-test, regarding P<0.05 as significant. In Experiment 1, we initially targeted 57 JBBCs for evaluation. Their follicle counts were determined by ultrasound 3 days before the start of superovulation (pre), close to the time of follicular wave emergence, and they were grouped according to number of follicles (0-19, 20-29, 30-39, and ≥40). Each cow was administered a tapered dose of FSH-R (20 AU) for superovulation, and then subjected to AI with cryopreserved semen from bulls of proven fecundity. Embryos were collected 7 days after AI by non-surgical intrauterine reflux. Post-AI follicle counts were done before ovulation and did not differ significantly from pre-superovulation follicle count in any group (range: −1.09 to 0.61). We then targeted a further 12 JBBC undergoing superovulation and AI with the same procedures, for counting of follicles at 30 days before (Day −30) embryo recovery (ER), pre-superovulation, and AI, and counting of corpora lutea and large follicles at ER, to investigate correlations between counts. We used decision coefficient (R2), regarding R2 ≥ 0.5 as significant. We found positive correlations (R2) at Day −30 with pre-superovulation (0.91), AI (0.63), and ER (0.63), and at pre-superovulation with AI (0.69) and ER (0.74). In Experiment 2, we targeted 69 JBBCs for detection of mutations in the GRIA1 gene, which encodes inotropic glutamate receptor AMPA1 with a known association with ovulation rate, and embryo yield measurements. DNA obtained from blood was subject to microsatellite polymorphism analysis, for mutations at base 917 of GRIA1 exon 7. We identified 33 cows (48%) as bearers of the wild-type allele (GG), 29 cows (42%) as bearers of the heterozygous mutation (GA), and seven cows (10%) as bearers of the homozygous mutation (AA). Average embryo yield was highest in wild-type cows, followed by heterozygous mutants and then homozygous mutants, and was significantly lower for homozygous mutants than for the other two types. Our study results demonstrated that follicle count before superovulation and GRIA1 gene analysis have utility for selecting donors for embryo transfer in JBBC. Furthermore, follicle counts at the early time point of 30 days before ER can be a useful indicator for this selection.
225 Evaluation of superovulatory response in Brazilian native sheep by B-mode ultrasonography
Authors: Vergani G. B. , Lima M. S. D. , Silva K. M. , Monteiro A. W. U. , Ramos A. F. , Batista R. I. T. P. , Vicente W. R. R. , Oliveira M. E. F. , Fonseca J. F.
Published: December 2019

The objective of this study was to evaluate the correlation between the number of corpora lutea (CL), as determined by B-mode ultrasonography, and the total number of structures recovery by non-surgical embryo recovery (NSER) from superovulated ewes. Somali (n=18), Santa Inês (n=18), and Morada Nova (n=16) ewes received an intravaginal device with progesterone (0.33g, controlled internal drug release device, CIDR, Zoetis), which was maintained for 9 days. The superovulatory protocol started 60h before device removal, with 6 decreasing doses (25, 25, 15, 15, 10, and 10%) of p-FSH (IM 133mg, Folltropin V, Vetoquinol) injected every 12h. D-Cloprostenol (IM 37.5μg, Prolise, Agener Union) was injected 12h before and at the moment of device removal. Ewes in oestrus were mated by fertile male rams three times after the onset of oestrus with a 12-h interval. Flunixin meglumine (24.9 mg; Banamine, MSD Animal Health) was injected IM on Days 12, 13, and 15. One day before NSER, B-mode ultrasound evaluations were performed using portable equipment (Z5 Vet, Mindray) with a stiffened multifrequency linear probe to evaluate the number of structures present in the ovaries. Embryo recovery was performed 7 days after progesterone CIDR removal (Day 16). The number of CL and the number of recovered structures were determined and their association evaluated using the Pearson correlation test (P<0.05). Only 17 Somali, 16 Santa Inês, and 15 Morada Nova ewes were submitted to NSER; the other 4 ewes could not be flushed because of low cervical dilation or cervical puncture. After B-mode ultrasound evaluation, 168, 217, and 131 CL were observed, with an average of 9.9 (range: 1 to 12), 13.5 (range: 2 to 47), and 8.7 (range: 3 to 18) CL per donor for Somali, Santa Inês, and Morada Nova ewes, respectively. The recovery rates (i.e. structures recovered by the number of CL counted) were 60.1% (101/168), 96.3% (209/217), and 103.5% (135/131) for the 3 breeds, respectively. There was a positive correlation (r=0.69; P<0.01) between the number of recovered structures and number of CL. Those findings confirm results from other studies, in which the recovery rate varied from 35 to 91%. In some cases, recovery rate can be overestimated because of the difficulty in quantifying CL. In conclusion, B-mode ultrasonography can be used to estimate superovulatory response in ewes and thus as a criterion to decide whether a donor will undergo embryo flushing.
Financial support for this study was provided by Embrapa (02.13.06.026.00.04) and Fapemig (CVZ-PPM 00201-17).