bluuebunny/crossref_metadata_2025 huggingface dataset:
Cotorsion pairs and model structures on Ch(R)
Authors: Yang Gang , Liu Zhongkui
Published: August 2011

We show that if the given cotorsion pair $(\mathcal{A},\mathcal{B})$ in the category of modules is complete and hereditary, then both of the induced cotorsion pairs in the category of complexes are complete. We also give a cofibrantly generated model structure that can be regarded as a generalization of the projective model structure.
The morphology and genetic characterization of Iheringascaris goai n. sp. (Nematoda: Raphidascarididae) from the intestine of the silver whiting and spotted catfish off the central west coast of India
Authors: Malhotra A. , Jaiswal N. , Malakar A.K. , Verma M.S. , Singh H.R. , Lakra W.S. , Malhotra S.K. , Shamsi S.
Published: August 2011

In this study a new species of nematode, Iheringascaris goai n. sp., is reported from two fish hosts, including silver whiting, Sillago sihama, and spotted catfish, Arius maculatus, caught off the Central West Coast of India at Goa. The new species can be differentiated morphologically from I. inquies, the most closely related species collected from cohabiting marine fish. The distinguishing characteristics are distinct cuticular striations, a unilateral excretory system, the presence of dentigerous ridges on the inner margin of the lips and the ratio of oesophagus to body length. In males, the ratio of spicules to body length is higher and the number of pre-anal papillae is less in comparison to those in I. inquies. In addition, the tail curves ventrad in males, while in females, the vulva is post-equatorial. The sequence alignment of 18S rDNA and cytochrome c oxidase subunit I with sequences of known species selected from the same superfamily shows a significant difference. The morphological and molecular differences reported here can, therefore, be used to assign the specimen to a new species.
Religiosity and resilience in persons at high risk for major depression
Authors: Kasen S. , Wickramaratne P. , Gameroff M. J. , Weissman M. M.
Published: August 2011

Background

Few studies have examined religiosity as a protective factor using a longitudinal design to predict resilience in persons at high risk for major depressive disorder (MDD).

Method

High-risk offspring selected for having a depressed parent and control offspring of non-depressed parents were evaluated for psychiatric disorders in childhood/adolescence and at 10-year and 20-year follow-ups. Religious/spiritual importance, services attendance and negative life events (NLEs) were assessed at the 10-year follow-up. Models tested differences in relationships between religiosity/spirituality and subsequent disorders among offspring based on parent depression status, history of prior MDD and level of NLE exposure. Resilience was defined as lower odds for disorders with greater religiosity/spirituality in higher-riskversuslower-risk offspring.

Results

Increased attendance was associated with significantly reduced odds for mood disorder (by 43%) and any psychiatric disorder (by 53%) in all offspring; however, odds were significantly lower in offspring of non-depressed parents than in offspring of depressed parents. In analyses confined to offspring of depressed parents, those with high and those with average/low NLE exposure were compared: increased attendance was associated with significantly reduced odds for MDD, mood disorder and any psychiatric disorder (by 76, 69 and 64% respectively) and increased importance was associated with significantly reduced odds for mood disorder (by 74%) only in offspring of depressed parents with high NLE exposure. Moreover, those associations differed significantly between offspring of depressed parents with high NLE exposure and offspring of depressed parents with average/low NLE exposure.

Conclusions

Greater religiosity may contribute to development of resilience in certain high-risk individuals.
Dynamic task allocation in cooperative robot teams
Authors: Tsalatsanis Athanasios , Yalcin Ali , Valavanis Kimon. P.
Published: August 2011

In this paper, a dynamic task allocation and controller design methodology for cooperative robot teams is presented. Fuzzy-logic-based utility functions are derived to quantify each robot's ability to perform a task. These utility functions are used to allocate tasks in real time through a limited lookahead control methodology partially based on the basic principles of discrete event supervisory control theory. The proposed controller design methodology accommodates flexibility in task assignments, robot coordination, and tolerance to robot failures and repairs. Implementation details of the proposed methodology are demonstrated through a warehouse patrolling case study.
Pursuing the method of multiple working hypotheses for hydrological modeling
Authors: Clark Martyn P. , Kavetski Dmitri , Fenicia Fabrizio
Published: August 2011

Ambiguities in the representation of environmental processes have manifested themselves in a plethora of hydrological models, differing in almost every aspect of their conceptualization and implementation. The current overabundance of models is symptomatic of an insufficient scientific understanding of environmental dynamics at the catchment scale, which can be attributed to difficulties in measuring and representing the heterogeneity encountered in natural systems. This commentary advocates using the method of multiple working hypotheses for systematic and stringent testing of model alternatives in hydrology. We discuss how the multiple‐hypothesis approach provides the flexibility to formulate alternative representations (hypotheses) describing both individual processes and the overall system. When combined with incisive diagnostics to scrutinize multiple model representations against observed data, this provides hydrologists with a powerful and systematic approach for model development and improvement. Multiple‐hypothesis frameworks also support a broader coverage of the model hypothesis space and hence improve the quantification of predictive uncertainty arising from system and component nonidentifiabilities. As part of discussing the advantages and limitations of multiple‐hypothesis frameworks, we critically review major contemporary challenges in hydrological hypothesis‐testing, including exploiting different types of data to investigate the fidelity of alternative process representations, accounting for model structure ambiguities arising from major uncertainties in environmental data, quantifying regional differences in dominant hydrological processes, and the grander challenge of understanding the self‐organization and optimality principles that may functionally explain and describe the heterogeneities evident in most environmental systems. We assess recent progress in these research directions, and how new advances are possible using multiple‐hypothesis methodologies.
Dynamic Optical Imaging of Metabolic and NADPH Oxidase-Derived Superoxide in Live Mouse Brain Using Fluorescence Lifetime Unmixing
Authors: Hall David J , Han Sung-Ho , Chepetan Andre M , Inui Edny G , Rogers Mike , Dugan Laura L
Published: August 2011

Superoxide is the single-electron reduction product of molecular oxygen generated by mitochondria and the innate immune enzyme complex, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox), and its isoforms. Initially identified as critical to the host defense against infection, superoxide has recently emerged as an important signaling molecule and as a proposed mediator of central nervous system injury in stroke, neurodegenerative conditions, and aging itself. Complete understanding of superoxide in central nervous system disease has been hampered by lack of noninvasive imaging techniques to evaluate this highly reactive, short-lived molecule in vivo. Here we describe a novel optical imaging technique to monitor superoxide real time in intact animals using a fluorescent probe compound and fluorescence lifetime contrast-based unmixing. Specificity for superoxide was confirmed using validated mouse models with enhanced or attenuated brain superoxide production. Application of fluorescence lifetime unmixing removed autofluorescence, further enhanced sensitivity and specificity of the technique, permitted visualization of physiologically relevant levels of superoxide, and allowed superoxide in specific brain regions (e.g., hippocampus) to be mapped. Lifetime contrast-based unmixing permitted disease model-specific and brain region-specific differences in superoxide levels to be observed, suggesting this approach may provide valuable information on the role of mitochondrial and Nox-derived superoxide in both normal function and pathologic conditions in the central nervous system.
A range-based method to calibrate a magnetic spectrometer measuring the energy spectrum of the backward electron beam of a plasma focus
Authors: Ceccolini E. , Rocchi F. , Mostacci D. , Sumini M. , Tartari A.
Published: August 2011

The electron beam emitted from the back of plasma focus devices is being studied as a radiation source for intraoperative radiation therapy applications. A plasma focus device is being developed for this purpose, and there is a need for characterizing its electron beam, particularly, insofar as the energy spectrum is concerned. The instrument used is a magnetic spectrometer. To calibrate this spectrometer, a procedure relying on the energy-range relation in Mylar® has been devised and applied. By measuring the transmission through increasing thicknesses of the material, electron energies could be assessed and compared to the spectrometer readings. Thus, the original calibration of the instrument has been extended to higher energies and also to better accuracy. Methods and results are presented.
Australian And New Zealand Fur Seals At The Skerries, Victoria: Recovery Of A Breeding Colony
Authors: Littnan CL , Mitchell AT
Published: August 2011

The population size of Australian fur seals Arctocephalus pusillus doriferus and New Zealand
fur seals A. forsteri at The Skerries, Victoria was estimated in two consecutive breeding
seasons, 1999-2000 and 2000-2001 using both mark-recapture procedures and aerial surveys.
675 and 746 A. p. doriferus pups and 37 and 47 A. forsteri pups were captured and marked in
1999-2000 and 2000-2001, respectively. Resights (1999-2000 N = 3; 2000-2001 N = 6) were
conducted 2 - 3 days after marking and pup population estimates were calculated using a
modified Lincoln-Petersen estimate. The arithmetic mean for A. p. doriferus pup abundance was
1,867 in the first season and 2,237 in the second. A. forsteri abundance was 75 and 78,
respectively. The A. p. doriferus population is estimated to have increased an average of 19.7%
(r = 0.18) between 1999 and 2000. The arithmetic mean from five counts of aerial photographs
of total animals present at the colony was 1,758 in 1999-2000 and 2,965 in 2000-2001. Due to
high variation between counts, aerial surveys proved to be an inconsistent and inaccurate
method for estimating the population of fur seals at The Skerries.
Breeding age of a captive male Australian sea lion, Neophoca cinerea, at the Adelaide Zoo.
Authors: Ling J.K. , Guy M.
Published: August 2011

A captive 3 year 2 month old female sea lion (Neophoca cinerea) mated with a 3 year 3-6 month old male and she gave birth 530 days later.
Development of an allele-specific marker for Glu-B3 alleles in common wheat and establishment of a multiplex PCR assay
Authors: Sui Xinxia , Wang Linhai , Xia Xianchun , Wang Zhenlin , He Zhonghu
Published: August 2011

Low-molecular-weight glutenin subunits (LMW-GS) have significant effects on the processing quality of end-use products of common wheat. It is more efficient to discriminate LMW-GS alleles with PCR-based molecular markers than with SDS-PAGE. In the present study, we developed an allele-specific PCR marker, designated Glu-B3abefg, which can be used to discriminate protein alleles Glu-B3a, b, e, f, and g simultaneously. Based on ten previously developed allele-specific STS markers, three multiplex PCRs, viz. Glu-B3c + Glu-B3d, Glu-B3b + Glu-B3g, and Glu-B3h + Glu-B3i, were established. Six Glu-B3 alleles (b, c, d, g, h, and i) could be discriminated using the three multiplex PCRs. Results of tests on 158 wheat varieties and lines using Glu-B3abefg and the three multiplex PCRs were consistent with those using the ten STS markers separately. The new allele-specific marker and three multiplex PCRs represent an efficient way to undertake marker-assisted selection of Glu-B3 alleles.
Diffusible factors involved in early interactions of actinorhizal symbiosis are modulated by the host plant but are not enough to break the host range barrier
Authors: Gabbarini Luciano Andrés , Wall Luis Gabriel
Published: August 2011

Nodulation kinetics were analysed in two nitrogen-fixing actinorhizal symbioses that show different pathways for infection: Alnus acuminata H. B. K., which is infected by Frankia ArI3, and Discaria trinervis (Hooker et Arnot) Reiche, which is infected by Frankia BCU110501. Both pairs are incompatible in cross-inoculation experiments. The dose–response effects in nodulation were studied in A. acuminata seedlings using different concentrations of compatible and incompatible bacteria in co-inoculation experiments. Restriction fragment length polymorphism PCR analysis and plant-trapping analysis showed no co-occupation in A. acuminata nodules when plants were co-inoculated with Frankia BCU110501 and Frankia ArI3. Despite the lack of co-occupation, the noninfective BCU110501 could modify the nodulation parameters of the non-host A. acuminata when infective ArI3 was present in the inoculum. The results suggest that although BCU110501 was not able to induce nodulation in A. acuminata, its interaction with the plant could induce autoregulation as if some level of infection or partial recognition could be achieved. We explored the possibility that physiological complementation of the heterologous Frankia BCU110501 for nodulation of A. acuminata originated in the homologous Frankia ArI3 in the presence of compatible root exudates. Despite the possibility of full activation between bacteria and the host, there was no co-infection of Frankia BCU110501 in Alnus or of Frankia ArI3 in Discaria either. These negative results suggest a physical recognition barrier in actinorhizal symbiosis that operates after early interactions, involving something other than root exudates and diffusible factors of bacterial or plant origin, regardless of the infection pathway.
Actinorhizal plant defence-related genes in response to symbiotic Frankia
Authors: Ribeiro Ana , Graça Inês , Pawlowski Katharina , Santos Patrícia
Published: August 2011

Actinorhizal plants have become increasingly important as climate changes threaten to remake the global landscape over the next decades. These plants are able to grow in nutrient-poor and disturbed soils, and are important elements in plant communities worldwide. Besides that, most actinorhizal plants are capable of high rates of nitrogen fixation due to their capacity to establish root nodule symbiosis with N2-fixing Frankia strains. Nodulation is a developmental process that requires a sequence of highly coordinated events. One of these mechanisms is the induction of defence-related events, whose precise role in a symbiotic interaction remains to be elucidated. This review summarises what is known about the induction of actinorhizal defence-related genes in response to symbiotic Frankia and their putative function during symbiosis.
Antioxidant profiling of Hippophae salicifolia growing in sacred forests of Sikkim, India
Authors: Goyal Arvind K. , Basistha Bharat C. , Sen Arnab , Middha Sushil K.
Published: August 2011

Hippophae commonly known as sea-buckthorn, is a versatile plant with multipurpose uses including the control of soil erosion, biological nitrogen fixation and medicinal properties. In India, two species of sea-buckthorn; namely, Hippophae salicifolia D. Don and Hippophae rhamnoides L., are very common. H. salicifolia has a shrub-to-tree habit and is restricted to the Himalayan region, whereas H. rhamnoides is bushy, growing at higher altitude in India, and widely distributed in Europe and Asia. In this study, aqueous, methanol and acetone extracts of various parts of female and male plants of H. salicifolia were evaluated for total antioxidant activity, phenolic and flavonoid contents, and correlations were made. The total phenolic and flavonoid content in all the samples ranged from 99 ± 32 to 1459 ± 53 mg gallic acid equivalent g–1, 135 ± 12 to 707 ± 62 mg quercetin equivalent g–1 of dry extract, respectively. The highest free radical (DPPH) scavenging activity was found in methanol extracts of female bark and male leaf. Methanol extracts of male bark and male leaf showed maximum H2O2 activity at a concentration of 1 mg mL–1. Antioxidant activity (FRAP method) ranged from 0.021 to 1.041 at concentrations ranging from 20 to 200 µg mL–1 with a reducing capacity of extracts as water > methanol > acetone. Strong correlation (P < 0.05) was observed between DPPH scavenging activity and total phenolic content from all the extracts under study.
New perspectives on nodule nitrogen assimilation in actinorhizal symbioses
Authors: Berry Alison M. , Mendoza-Herrera Alberto , Guo Ying-Yi , Hayashi Jennifer , Persson Tomas , Barabote Ravi , Demchenko Kirill , Zhang Shuxiao , Pawlowski Katharina
Published: August 2011

Nitrogen-fixing root nodules are plant organs specialised for symbiotic transfer of nitrogen and carbon between microsymbiont and host. The organisation of nitrogen assimilation, storage and transport processes is partitioned at the subcellular and tissue levels, in distinctive patterns depending on the symbiotic partners. In this review, recent advances in understanding of actinorhizal nodule nitrogen assimilation are presented. New findings indicate that Frankia within nodules of Datisca glomerata (Presl.) Baill. carries out both primary nitrogen assimilation and biosynthesis of arginine, rather than exporting ammonium. Arginine is a typical storage form of nitrogen in plant tissues, but is a novel nitrogen carrier molecule in root nodule symbioses. Thus Frankia within D. glomerata nodules exhibits considerable metabolic independence. Furthermore, nitrogen reassimilation is likely to take place in the host in the uninfected nodule cortical cells of this root nodule symbiosis, before amino acid export to host sink tissues via the xylem. The role of an augmented pericycle in carbon and nitrogen exchange in root nodules deserves further attention in actinorhizal symbiosis, and further highlights the importance of a comprehensive, structure–function approach to understanding function in root nodules. Moreover, the multiple patterns of compartmentalisation in relation to nitrogen flux within root nodules demonstrate the diversity of possible functional interactions between host and microsymbiont that have evolved in the nitrogen-fixing clade.
Actinorhizal plants
Authors: Ribeiro Ana , Berry Alison M. , Pawlowski Katharina , Santos Patrícia
Published: August 2011

Actinorhizal plants are a group of taxonomically diverse angiosperms with remarkable economic and ecological significance. Most actinorhizal plants are able to thrive under extreme adverse environmental conditions as well as to fix atmospheric nitrogen due to their capacity to establish root nodule symbioses with Frankia bacteria. This special issue of Functional Plant Biology is dedicated to actinorhizal plant research, covering part of the work presented at the 16th International Meeting onFrankia and Actinorhizal Plants, held on 5–8 September 2010, in Oporto, Portugal. The papers (4 reviews and 10 original articles) give an overall picture of the status of actinorhizal plant research and the imposed challenges, covering several aspects of the symbiosis, ecology and molecular tools.
Changes to demersal fish communities in Port Phillip Bay, Australia, over two decades, 1970-91
Authors: Hobday D. K. , Officer R. A. , Parry G. D.
Published: August 2011

Changes to the structure of fish communities in Port Phillip Bay between 1970
and 1991 were analysed by trawling at 14 stations sampled regularly during
1970-75 and in 1990/91. Differences between stations were compared by
using Bray-Curtis dissimilarity indices, multidimensional scaling and analysis
of variance for the most abundant species. Differences between two sampling
periods in the 1970s appear to be the result of the use of different vessels
for trawling. A third vessel was used during 1990/91, but differences in
catches between 1972-75 and 1990/91 appear to represent real temporal
changes rather than differences between trawl efficiencies. Between 1972-75
and 1990/91, increased fishing pressure is the most likely explanation for
declines in several important commercial and recreational species. A
consequent decrease in competition may have caused an increase in the
abundance of stingarees. A decline in seagrass abundance in the western bay
probably reduced the abundance of several seagrass-dependent species. The
Japanese goby was introduced into the bay after the mid 1970s, and populations
of little rock whiting in the western bay and globefish in deep regions of
Port Phillip Bay appear to have increased because of the recent establishment
of additional exotic invertebrates.
Facilitating teamwork in general practice: moving from theory to practice
Authors: Taggart J. , Schwartz A. , Harris M. F. , Perkins D. , Davies G. Powell , Proudfoot J. , Fanaian M. , Crookes P.
Published: August 2011

The aims of this paper are to describe the development of an intervention to improve teamwork and systems in general practice that support the care of patients with diabetes, ischaemic heart disease and hypertension and to identify the challenges to implementing the intervention. Effective teamwork in general practice encompasses general practitioners (GP), clinical and non-clinical staff, each with clearly defined roles and opportunities to provide feedback and input into how the practice is run and chronic disease managed. The intervention implemented in this study provided an opportunity for key members of general practice teams to work with a facilitator on changes to improve teamwork over three practice visits over 6–12 months. Facilitators had experience in practice support and goal setting, an understanding of the Medicare Items and knowledge about teamwork and systems. The visits focussed on the specific needs and capacities of each practice, assisting the team to set manageable goals and building systems that best utilise the systemic and human resources available. Successful implementation of sustained change depended on strong leadership in the practice and cooperation between team members as well as clear and achievable goals being set. Structured facilitation of teamwork in general practice should focus on goal setting and the development of leadership and communication rather than delivery of information or resources.
113 EFFECT OF SOME FACTORS ON CONCEPTION RATE IN ALPACAS UNDER PERUVIAN HIGHLAND CONDITION
Authors: Diaz A. , Huanca W. , Ampuero A. , Huaman H. , Camacho J. , Huanca T. , Quispe D. , Diaz H.
Published: August 2011

Alpacas are a domestic species of South American camelids with a great importance to highland people because of their high-quality fibre production. However, their reproductive performance has been reported to be poor, with a birth rate of 50% under the Andean community’s conditions. Two experiments were designed to evaluate the effect of some factors on the pregnancy rate of alpacas at first service in lactating, 3- to 8-years old, without parity problems. The objective of the first experiment (n = 85) was to evaluate the effect of age (G1: 3, G2: 4 to 5, G3: ≥ 6 years old); mating time (G1: 15 min, G2: 16–24 min, G3: ≥ 25 min), and interval from parturition to mating (G1: 20 days, G2: ≥ 20 days) on first service conception rate. A second experiment (n = 174) evaluated the effect of month of calving (January, February, or March) on conception rate in females with a postpartum interval ≥20 days. Animals were mated with male of good fertility after a receptivity test and confirmation of presence of a dominant follicle ≥7 mm by ultrasonography. Pregnancy was determined by ultrasonography 25 days after mating. Proportional data were compared by Fisher’s exact test. In the first experiment, conception rates were 57.9, 66.7, and 47.2% in females of G1, G2, and G3, respectively (P ≤ 0.05); 50.0, 54.5, and 59.0% in females with mating time of 15, 16–24, or ≥ 25 min of mating; 48.0 and 59.4% in those with a postpartum interval <20 days and ≥20 days (P ≤ 0.05). In the second experiment, conception rates were 58.3, 70.7, and 82.1% in alpacas calving in January, February, and March, respectively (P ≤ 0.05). The results suggest that age of female, postpartum interval, and month of calving are factors that affect conception rates in alpacas. Therefore, a reproductive management system that includes these factors would improve the pregnancy rate in alpacas under highland Peruvian conditions.

Consejo Superior de Investigacion – UNMSM.
140 PRODUCTION OF RECONSTRUCTED IN VITRO PRODUCED BOVINE EMBRYOS BY INNER CELL MASS AND TROPHECTODERM AGGREGATION IN VITRO
Authors: Emanuelli I. P. , Razza E. , Barros C. M. , Nogueira M. F. G.
Published: August 2011

The efficiency of embryonic chimerism tends to decrease when embryos in advanced stages of development, such as morulae and blastocysts, are used. To perform the inner cell mass (ICM) transfer to a trophectoderm (TE) receptor, it is essential to use embryos at an advanced stage and blastocoel presence. This method of embryo reconstruction has been performed only by the micromanipulator microinjection method (Zheng et al. 2005 Zygote 1, 73–71; Loi et al. 2007 Trends Biotechnol. 25, 195–200; Roth et al. 1989 Biol. Reprod. 41, 675–682; and Murakami et al. 2006 Cloning Stem Cells 8, 51–60). This study aimed to validate a manual procedure to reconstruct embryos using the method of ICM and TE approximation in the presence of phytohemagglutinin. Bos indicus ovaries from the abattoir were used to obtain 230 cumulus–oocyte complexes (COC; quality I and II). The COC were matured in 90-μL drops of TCM-199 bicarbonate supplemented with 10% fetal calf serum (FCS) and incubated in vitro for 22 to 24 h. Fertilization occurred in TALP-IVF medium, and the COC were incubated for 18 h. Presumptive zygotes were transferred to SOF medium to in vitro culture. Incubation conditions were 38.5°C and 5% CO2 in air. In vitro produced (IVP) embryos 8.5 days after fertilization were used for the experiment. Ninety-three hatching or hatched blastocysts were put into 3-μL microdrops of protein-free HEPES-buffered SOF (HSOF) medium to hold the embryos on the dish bottom and to allow handmade sections of ICM and TE. The section was performed with a microblade (Ultra-Sharp Splitting Blade, Bioniche, Bogart, GA, USA) under a stereomicroscope (35× magnification). Seventy half-structures from 35 different blastocysts were obtained to form pairs (ICM+TE). Each pair was transferred to drops with 500 μg mL–1 of phytohemagglutinin-L (3 min) before the approximated pairs were transferred to SOF medium in cell aggregation well-of-the-well (WOW) micro-wells (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264) to in vitro culture (38.5°C; 5% O2 and 5% CO2). The aggregation rate was 25.7% (9/35) and all the reconstructed blastocysts by aggregation (24 h) re-expanded after 48 h of culture. The technique of handmade ICM and TE section and posterior aggregation in the presence of an agglutinating agent was feasible for the structural and functional reconstruction and re-expansion of the blastocyst produced.

Financial support: FAPESP, Brazil (06/06491-2, 07/07705-9, 09/10679-5, and 09/04888-0).
17 PREGNANCY RATE OBTAINED WITH THE REUTILIZATION OF INTRAVAGINAL DEVICE DICO AFTER 6 DAYS OF TREATMENT FOR ESTRUS SYNCHRONIZATION IN SHEEP
Authors: dos Santos-Neto P. C. , García-Pintos C. , Menchaca A.
Published: August 2011

The new intravaginal device DICO (Dispositivo Intravaginal Caprino Ovino, 0.3 mg progesterone, Syntex, Buenos Aires, Argentina) is effective in controlling serum progesterone concentrations, follicular development and time of ovulation in sheep, even in its second use after 6 days of treatment (Vilariño et al. 2010 Small Rumin. Res. 91, 219–224). The pregnancy rate obtained with DICO in its first use is similar to that obtained with CIDR-G (Pfizer, New York, NY, USA) (abstract of García-Pintos et al. in this issue). However, pregnancy rate with the reutilisation of DICO has not been evaluated yet. The aim of this study was to determine fertility rates in ewes synchronized with a reused DICO in comparison with a reused CIDR-G. The experiment was carried out on 314 Corriedale multiparous ewes during breeding season (March, 33°S, Uruguay), with a body condition score of 3.0 ± 0.1 (scale 0–5). Ewes received a short-term-protocol using DICO (n = 139) or CIDR-G (n = 175) during 6 days, previously used for 6 days. One dose of prostaglandin F2α analogue (125 μg cloprostenol, Ciclase DL, Syntex) and 300 IU eCG (Novormon, Syntex) were given at the time of device removal. Cervical insemination (200 million spermatozoa) or laparoscopic intrauterine insemination (100 million spermatozoa) was performed with fresh semen at fixed-time 48 or 54 h from device removal, respectively. Pregnancy diagnosis was done by transrectal ultrasonography (5 MHz, Well D, Beijing, China) 40 days after insemination. Pregnancy rate was analysed by logistic regression, and number of fetus/pregnant ewes and fetus/inseminated ewes was analysed by Poisson regression. No differences were found between reused DICO and CIDR-G devices (Table 1). Overall, pregnancy rate was affected by the method of insemination (P ≤ 0.05), being lower for cervical (37.0%; 57/154) than intrauterine insemination (55.0%; 88/160). In conclusion, the reutilization of DICO devices in the short-term protocol (6 days of treatment) allows similar fertility rates compared to re-used CIDR-G devices, by both cervical and intrauterine insemination.

Table 1.Fertility rate obtained with the reutilisation of DICO and CIDR in short-term protocol (6 days of treatment) in sheep
191 TRANSIENT RECEPTOR POTENTIAL SUPERFAMILY OF ION CHANNELS, TRPV6, IS CONSTITUTIVELY EXPRESSED AND REGULATED BY ESTROGEN IN THE HUMAN UTERUS DURING THE MENSTRUAL CYCLE
Authors: Kim Y.-K. , Yang H. , Jeung E.-B.
Published: August 2011

Two highly selective calcium channels at the apical sides of cells, members of the transient receptor potential (TRP) superfamily of ion channels (TRPV6 and TRPV5), are the main calcium ion entry channels. Previously, the location of TRPV6 has been described in the intestine in several species, including humans. It is located in the apical brush-border membrane of the intestinal enterocyte, where it regulates calcium entry into the cell. It is most abundant in the proximal small intestine (duodenum and jejunum), where calbindin and the calcium-pumping ATPase are also found. The TRPV6 calcium transporter is also found in the human placenta, pancreas, and prostate gland in some species. However, TRPV6 expression and its potential roles remain to be clarified in the endometrium of humans during the menstrual cycle. In this study, we used a human endometrial model to examine the expression of TRPV6 and its potential roles in the human menstrual cycle. A significant increase (1.5-fold) in the TRPV6 transcript and protein was observed in the human uterus at the proliferation phase compared with other phases. In addition, the spatial localization of TRPV6 in the human uterus was determined by immunohistochemistry. Uterine TRPV6 was abundantly localised in the cytoplasm of the endometrial and glandular epithelial cells in the menstrual phases. Overall, these results demonstrate that TRPV6 is abundantly expressed in human uterine tissue, suggesting that this protein may be involved in reproductive functions during the menstrual cycle in humans.
210 CO-CULTURE WITH AUTOLOGOUS CUMULUS CELLS SUPPORTS THE INDIVIDUAL DEVELOPMENT OF SINGLY IN VITRO-MATURED AND FERTILIZED BOVINE OOCYTES
Authors: Goovaerts I. G. F. , Leroy J. L. M. R. , Merckx E. , Andries S. , Bols P. E. J.
Published: August 2011

The ability to produce embryos singly in vitro (in vitro production, IVP) would be a useful tool for many purposes. Without the interfering effects of other developing or degenerating oocytes or embryos, such an individual IVP system is the tool of choice for studies on oocyte quality and oocyte–embryo metabolism. Unfortunately, individual IVP in most cases leads to unsatisfactorily low blastocyst rates. Earlier work showed that individual culture of zygotes on a cumulus cell (CC) monolayer resulted in comparable numbers of good-quality embryos, as obtained following regular group culture (Goovaerts et al. 2009 Theriogenology 71, 729–738). However, co-culture with somatic cells is often criticised because of the undefined culture conditions and for sanitary reasons. In the cited study, CC for monolayer production were obtained from a different batch of ovaries. Our specific aim was to use CC from the zygote itself (autologous CC). Grade I COC (n = 660) were collected from slaughterhouse ovaries and randomly assigned to 2 treatments (5 replicates): a completely individual ‘single-oocyte’ IVP protocol, or routine group IVP as a control. Individual maturation (TCM-199 + 20% serum) and fertilization were performed in 20-μL droplets under oil in 24-well plates. Subsequently, each zygote was stripped and cultured in 20 μL of medium (SOF + 5% serum, 90% N2, 5% CO2, 5% O2), to which the autologous stripped CC were added. Group maturation and fertilization were carried out per 100 COC in 500 μL, whereas group culture was performed per 25 zygotes in 50-μL droplets under oil. Cleavage, blastocyst, and hatching rates were determined 2, 8, and 10 days post-fertilization, respectively. Possible effects of the individual and group cultures were evaluated with binary logistic regression (SPSS 15.0, SPSS Inc., Chicago, IL). No interactions between replicate and treatment were found (P > 0.05). Although a blastocyst rate of 15.1% was obtained using single IVP, the general efficacy of the single-embryo production system was lower when compared with group culture (Table 1). In conclusion, although developmental competence was impaired using individual IVP, co-culture with autologous cumulus cells can be useful in specific experimental setups in which the influence of other oocytes or embryos or heterologous somatic cells is unacceptable.

Table 1.Cleavage, blastocyst, and hatching rates after individual and group in vitro production (IVP)
218 ANTIBIOTIC-RESISTANT MICROBIAL CONTAMINATION (ENTEROBACTER CLOACAE) DERIVED FROM EGG YOLK, FROZEN SEMEN EXTENDER, IN PORCINE IN VITRO-FERTILIZED EMBRYOS
Authors: Jang S. H. , Kwak S. S. , Biswas D. , Hyun S. H.
Published: August 2011

The semen storage medium and the embryo culture environment are important for the further development of pre-implantation embryos, and these environments provide an ideal habitat for the propagation of a variety of microorganisms. Even though all precautions are taken to prevent contamination, this happens frequently during IVF and in vitro culture (IVC). Therefore, the aim of the present study was to investigate the source of contamination during semen processing for in vitro uses. In the present study, frozen semen was prepared from liquid semen (liquid semen was prepared according the method of (Weitze 1990 Reprod. Dom. Anim., 231–253 suppl. 1), and purchased from the Veterinary Service Laboratory, Department of Livestock Research (Yongin city, Gyeonggi-do, Korea) in our laboratory for IVF experiments because of a lack of fresh semen. Antibiotics were added to the frozen semen extender (kanamycin and gentamicin) and IVC medium (gentamicin) to further inhibit the growth of microorganisms. Nevertheless, microorganisms were observed to proliferate in the IVC drop when culturing IVF embryos using frozen semen. Three samples were randomly taken from the liquid semen, frozen semen, and egg yolk. Contaminated IVC medium, frozen–thawed semen, liquid semen, and egg yolk were cultured in de Man, Rogosa, and Sharpe agar medium. Identical whitish colonies were detected in the contaminated IVC drop, frozen–thawed semen samples, and egg yolk, but no colonies were formed in liquid semen samples. Identical gram-negative and rod-shaped bacteria were found in both the frozen–thawed semen sample and the contaminated IVC drop and egg yolk samples. Enterobacter cloacae were confirmed by API 20E kit according to the manufacturer’s instructions, with an identification value of 94.3% and a T index of 0.88. Antibiotic susceptibility tests were done according to CLSI (Wayne, PA) by using an ampicillin, amikacin, cephalothin, gentamicin, kanamycin, tetracycline, oxytetracycline, sulfamethoxazole, trimethoprim, norfloxacin, and ciprofloxacin test. Among them, E. cloacae were resistant to ampicillin, amikacin, cephalothin, gentamicin, and kanamycin but were susceptible to tetracycline, oxytetracycline, sulfamethoxazole and trimethoprim, norfloxacin, and ciprofloxacin. We suggest, based on these findings, that the sources of contamination might be from egg yolk. Therefore, synthetic semen extenders, which are free of egg yolk, might be considered during semen preparation.

This work was supported by a grant (20070301034040) from the BioGreen 21 Program, Rural Development Administration, Republic of Korea.
224 LOCALIZATION OF NITRIC OXIDE SYNTHASE ACTIVITY IN BUFFALO (BUBALUS BUBALIS) OOCYTES AND EMBRYOS
Authors: Babu K. R. , Sharma R. , Singh K. P. , George A. , Chauhan M. S. , Singla S. K. , Manik R. S. , Palta P.
Published: August 2011

Ovarian nitric oxide (NO) and that produced within the oocytes and embryos have been reported to play important roles in oocyte meiotic maturation and embryo development. Production of NO is catalyzed by NO synthase (NOS), which exists in 3 isoforms, the constitutive endothelial (eNOS) and neuronal (nNOS) isoforms and the inducible (iNOS) isoform. We have previously shown that low concentrations of NO stimulate and high concentrations inhibit embryo development, and that endogenous NO produced by iNOS is necessary for optimal embryo development in the buffalo. The present study was aimed at localizing different isoforms of NOS and examining their relative mRNA abundance in buffalo oocytes and embryos. Oocytes from slaughterhouse ovaries were subjected to in vitro maturation in 100-μL droplets (10 to 15 oocytes/droplet) of in vitro maturation medium (TCM-199 + 10% FBS + 5 μg mL–1 of pFSH + 1 μg mL–1 of oestradiol-17β + 0.81 mM sodium pyruvate + 10% buffalo follicular fluid + 50 μg mL–1 of gentamicin) for 24 h in a CO2 incubator (5% CO2 in air) at 38.5°C. In vitro fertilization was carried out by incubating in vitro-matured oocytes with 2 to 4 million spermatozoa mL–1 for 18 h. The presumed zygotes were cultured on original beds of cumulus cells in in vitro culture medium (mCR2aa + 0.6% BSA + 10% FBS) for up to 8 days post-insemination. Immature and in vitro-matured oocytes and embryos at the 2-cell, 4-cell, 8- to 16-cell, morula, and blastocyst stages were examined for the presence of NOS isoforms by indirect immunofluorescence staining using epifluorescence microscopy and RT-PCR. Each experiment was repeated in triplicate, and data were analysed using one-way ANOVA, after arcsine transformation of percentage values. Expression of all 3 NOS isoforms was detected inside the cytoplasm, in all the stages of oocytes and embryos examined, by both immunofluorescence and RT-PCR. Abundance of the iNOS transcript was significantly higher (P ≤ 0.01) in the morula and blastocyst stages compared with that in immature and in vitro-matured oocytes and in embryos at the 2-cell, 4-cell, and 8- to 16-cell stages, indicating that its expression was up-regulated at the 8- to 16-cell stage. The expression of eNOS was significantly higher (P ≤ 0.05) in the immature and mature oocytes and in 8- to 16-cell stage embryos, morulae, and blastocysts than in the early-cleavage embryos at the 2- and 4-cell stages, indicating that it was down-regulated after fertilization and was up-regulated again at the 8- to 16-cell stage. Abundance of the nNOS transcript was not significantly different among all the stages of oocytes and embryos examined. These results demonstrate that different NOS isoforms are expressed in a dynamic manner during embryonic development in the buffalo. The role of an increase in expression of iNOS and eNOS at the 8- to 16-cell stage, at which a developmental block occurs in this species, needs to be examined.
238 ASSOCIATION BETWEEN SPERM MORPHOLOGY AND IN VITRO EMBRYO PRODUCTION IN MICE
Authors: Vilariño M. , Crispo M. , Pinczak A. , Menchaca A.
Published: August 2011

Although sperm morphology affects male fertility in several species, normally, it is not considered for in vitro fertilization (IVF) programs in mice. In order to correlate sperm morphology with IVF rates in murines, a total of 3336 oocytes were used in 11 identical IVF replicates using 8-wk-old B6D2 F1 (C57BL/6 × DBA/2J) males and 3- to 4-wk-old C57BL/6J donors females. For each replicate, 10 females were injected intraperitoneally with 5 IU of eCG (Novormon, Syntex, Buenos Aires, Argentina), and 5 IU of hCG (Ovusyn, Syntex) 48 h apart. Superovulated females were killed 14 to 16 h after hCG injection. The oviducts were isolated and cumulus–oocyte complexes were recovered and introduced into each sperm suspension drop. Previously, sperm was obtained from males killed by cervical dislocation, the cauda epididymis were recovered, minced with fine scissors in equilibrated human tubal fluid medium (HTF, EmbryoMax, Chemicon International, Phillipsburg, NJ, USA), and resuspended in a total volume of 200 μL of HTF. The sperm suspension was incubated at 0.05 or 3 × 106 spermatozoa/mL concentration in equilibrated 100-μL drops of HTF under embryo tested mineral oil (Sigma, St. Louis, MO, USA), at 37°C in 5% CO2, 95% air for 1 h before insemination. To evaluate sperm morphology, a sample of each replicate was fixed with 10% formalin and observed under phase-contrast microscopy (Olympus IX81) at 100× magnification. Sperm abnormalities regarding head and neck, tail, and cytoplasmic droplet were recorded from 200 cells. Five hours after insemination, the fertilized eggs were washed and transferred in groups of 50 to 100 into 100-μL drops of equilibrated culture medium (KSOM, EmbryoMax, Chemicon International) under the same culture conditions. The number of 2- (cleavage) and 8- (development) cell embryos was scored after 24 and 72 h in culture, respectively. Statistical analysis was performed by logistic regression taking into account the effect of the sperm abnormalities, the replicate, and the sperm dose. The effect of the sperm abnormalities is shown in Table 1. Results were not significantly influenced by the replicate or the sperm dose (P > 0.05). In conclusion, sperm morphology affects cleavage and embryo development rates in mice, and it should be taken into account as a source of variation in the success of IVF.

Table 1.Effect of total sperm abnormalities on cleavage rate and embryo development in mice
245 ANALYSIS OF CORTICAL GRANULE EXUDATE OBTAINED BY CHEMICAL OOCYTE ACTIVATION IN PIGS
Authors: Romar R. , Izquierdo-Rico M. J. , Funahashi H.
Published: August 2011

Cortical granules (CG) are clue organelles in the mammalian oocyte because once released, their content modifies the zona pellucida (ZP) and oolema, thus preventing polyspermy. However, research on putative CG proteins has progressed slowly because of the picogram amount of proteins contained in CG. Isolation and identification of CG contents in porcine oocytes would help to elucidate the molecular mechanism involved in blocking polyspermic fertilization. Our objective was to study the contents of CG from in vitro-matured (IVM) porcine oocytes, and to achieve this objective, CG exudate was collected after its release from chemically activated oocytes. Oocytes were subjected to IVM in porcine oocyte medium supplemented with 50 μM β-mercaptoethanol for 44 h. After the IVM period, the ZP was removed by protease treatment (0.5% pronase in PBS), and the ZP-free oocytes were activated with calcium ionophore A23187 (6.5 μM, 2 min) in a medium consisting of 114.06 mM NaCl, 3.20 mM KCl, 0.50 mM MgCl2·6H2O, 10.00 mM sodium lactate, 0.35 mM NaH2PO4, 5.00 mM glucose, 25.07 mM NaHCO3, and 8.00 mM calcium lactate·5H2O. After activation, oocytes were transferred to fresh medium without calcium ionophore and kept for 30 min to allow release of the CG content. After this time, medium containing the CG exudate was collected, as well as the activated oocytes, and both samples were stored at –80°C until analysis. Samples were thawed and the CG proteins were concentrated by centrifugation in 10-kDa centrifugal devices (Microcon, Millipore, Billerica, MA) following the manufacturer’s instructions. The CG exudates from activated oocytes (n = 300) and activated oocytes (n = 125) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. In brief, 4% stacking and 12% separating gel was used and run using 25 mM Tris–0.2 M glycine buffer, pH 8.6, containing 0.1% SDS for 1.5 h at 150 V and room temperature. After electrophoresis, the gel was silver stained. Thirteen strong bands were identified in the CG exudate lane, with an approximate molecular mass from approximately 45 to 105 kDa. However, the lane for activated oocytes showed faint protein bands. The presence of well-defined bands in the CG exudate lane might correspond to different CG-derived proteins. These preliminary results show a new approach for studying CG content. Further proteomic analysis of the bands will help to describe specific proteins contained in these organelles, shedding light on the role of the cortical reaction in pigs.

Supported by MEC and FEDER (AGL2009-12512-C02-01) and Okayama Universit R. R. was granted funding by JSPS (Ref. S-09210).