bluuebunny/crossref_metadata_2025 huggingface dataset:
Mirror‐mode‐like structures in Venus' induced magnetosphere
Authors: Volwerk M. , Zhang T. L. , Delva M. , Vörös Z. , Baumjohann W. , Glassmeier K.‐H.
Published: December 2008

The solar wind interaction with Venus creates an induced magnetosphere around the planet. It is shown that within the space bound by Venus' bow shock and ionopause, there is a rich occurrence of mirror‐mode‐like structures in the magnetic field data. The dayside magnetosheath and nightside magnetosheath/wake regions are investigated separately. It is shown that the probability to observe mirror mode structures is much higher at the dayside, where it is also strongly dependent on the angle between the solar wind magnetic field and the bow shock normal. In Venus' wake the chance to observe these structures is low, most likely because of the fully developed turbulence in this region, which will decrease temperature anisotropies. The results stand in contrast to the very low occurrence rate claimed from data taken by the Pioneer Venus Orbiter mission.
Evaluation of ensemble‐based distributed hydrologic model response with disaggregated precipitation products
Authors: Forman Barton A. , Vivoni Enrique R. , Margulis Steven A.
Published: December 2008

The disaggregation of coarse precipitation products is desirable for spatially distributed hydrological applications where high‐resolution rainfall fields are required. In this study, we generate an ensemble of satellite‐based precipitation estimates using a spatiotemporal disaggregation framework for use in a spatially explicit hydrologic model. The framework effectively reproduced small, localized storm events using coarse precipitation estimates while simultaneously including known error characteristics into the precipitation fields. Ensemble precipitation inputs were propagated through a distributed hydrologic model to evaluate the impact on simulated hydrologic states and fluxes, such as streamflow, soil moisture content, and evapotranspiration. The disaggregation framework adds utility to the coarse satellite precipitation measurements by inducing a realistic hydrologic response in a manner similar to high‐resolution ground‐based radar observations. Errors introduced in the precipitation ensembles, namely, the overestimation of high‐intensity, short‐duration storm events, were tracked through the hydrological system, and physical mechanisms for the soil moisture and streamflow uncertainty are discussed. It was found that increases in streamflow magnitude and decreases in catchment area resulted in increased ensemble streamflow dispersion. In addition, precipitation uncertainty affected soil moisture predictions with more pronounced ensemble dispersion near the surface. Finally, spatial ensemble statistics were used to assess the precipitation and topographic control over soil moisture statistics in the study catchment.
Developing and applying uncertain global climate change projections for regional water management planning
Authors: Groves David G. , Yates David , Tebaldi Claudia
Published: December 2008

Climate change may impact water resources management conditions in difficult‐to‐predict ways. A key challenge for water managers is how to incorporate highly uncertain information about potential climate change from global models into local‐ and regional‐scale water management models and tools to support local planning. This paper presents a new method for developing large ensembles of local daily weather that reflect a wide range of plausible future climate change scenarios while preserving many statistical properties of local historical weather patterns. This method is demonstrated by evaluating the possible impact of climate change on the Inland Empire Utilities Agency service area in southern California. The analysis shows that climate change could impact the region, increasing outdoor water demand by up to 10% by 2040, decreasing local water supply by up to 40% by 2040, and decreasing sustainable groundwater yields by up to 15% by 2040. The range of plausible climate projections suggests the need for the region to augment its long‐range water management plans to reduce its vulnerability to climate change.
Remediation of heterogeneous aquifers based on multiobjective optimization and adaptive determination of critical realizations
Authors: Kourakos G. , Mantoglou A.
Published: December 2008

A method for optimal remediation of heterogeneous aquifers based on stochastic simulation with adaptive determination of critical realizations is developed. Multiple hydraulic conductivity realizations are generated using the turning bands method while Monte Carlo simulation is incorporated into a multiobjective genetic optimization algorithm. In order to reduce the computational burden of Monte Carlo simulations, an adaptive procedure is developed for identifying “critical realizations” which are the ones that have an effect on the optimal solution depending on desired reliability level. The adaptive procedure is embedded into the genetic optimization algorithm where noncritical realizations are eliminated step by step during the advancement of optimization. When the number of remaining realizations reaches a minimum, some new realizations are added. The specific realizations which are removed or added in the critical set are selected by an automatic procedure controlled by a similarity criterion based on rankings of realizations. This is a significant improvement over the nonadaptive methodology for identification of critical realizations and does not require using a number of initial designs and a safety threshold. The methodology is applied in a remediation problem with two objectives (reduction of contaminant mass and cleanup cost). The applications indicate significant savings in computer time without loss of performance.
Effects of Age, Gender, Bolus Condition, Viscosity, and Volume on Pharyngeal and Upper Esophageal Sphincter Pressure and Temporal Measurements During Swallowing
Authors: Butler Susan G. , Stuart Andrew , Castell Donald , Russell Gregory B. , Koch Kenneth , Kemp Shannon
Published: December 2008

Purpose

The purpose of this study was to determine the effects of trial (i.e., Trial 1 vs. Trial 2); viscosity (i.e., saliva, thin, nectar-thick, honey-thick, and pudding-thick water); volume (i.e., 5 mL vs. 10 mL); age (i.e., young vs. older adults); and gender on pharyngeal (i.e., upper and lower) and upper esophageal sphincter (UES) pressures, durations, and onsets (i.e., onset of upper pharyngeal pressures relative to onsets of UES relaxations and onset of lower relative to upper pharyngeal pressures).

Method

Twenty-three young adults (M= 30 years) and 21 older healthy adults (M= 75 years) participated. Measurements were acquired with a 2.1-mm catheter during simultaneous manometric and endoscopic swallowing assessment. Participants contributed 18 swallows, affording a study total of 792 swallows for analyses.

Results

There was no significant effect of trial on any measurement of pressure, duration, and onset (ps = .63, .39, and .71, respectively). It was found that viscosity, volume, age, and gender affected pressure, duration, and onset measurements (e.g., onset of upper pharyngeal pressures relative to onsets of UES relaxations) but in varying degrees relative to the location in the pharynx or UES and the type of measurement (e.g., pressure, onset).

Conclusions

Manometric measurements vary with respect to age, gender, and bolus variables and interactions of each. Consideration of these variables is paramount in understanding normal and pathological swallowing if manometry is to develop as a quantitative adjunct to videofluoroscopic and endoscopic swallowing tools.
Comorbidity of Auditory Processing, Language, and Reading Disorders
Authors: Sharma Mridula , Purdy Suzanne C. , Kelly Andrea S.
Published: December 2008

Purpose

The authors assessed comorbidity of auditory processing disorder (APD), language impairment (LI), and reading disorder (RD) in school-age children.

Method

Children (N= 68) with suspected APD and nonverbal IQ standard scores of 80 or more were assessed using auditory, language, reading, attention, and memory measures. Auditory processing tests included the Frequency Pattern Test (FPT; F. E. Musiek, 1994; D. Noffsinger, R. H. Wilson, & F. E. Musiek, 1994); the Dichotic Digit Test Version 2 (DDT; F. E. Musiek, 1983); the Random Gap Detection Test (R. W. Keith, 2000); the 500-Hz tone Masking Level Difference (V. Aithal, A. Yonovitz, & S. Aithal, 2006); and a monaural low-redundancy speech test (compressed and reverberant words; A. Boothroyd & S. Nittrouer, 1988). The Clinical Evaluation of Language Fundamentals, Fourth Edition (E. Semel, E. Wiig, & W. Secord, 2003) was used to assess language abilities (including auditory memory). Reading accuracy and fluency and phonological awareness abilities were assessed using the Wheldall Assessment of Reading Passages (A. Madelaine & K. Wheldall, 2002) and the Queensland University Inventory of Literacy (B. Dodd, A. Holm, M. Orelemans, & M. McCormick, 1996). Attention was measured using the Integrated Visual and Auditory Continuous Performance Test (J. A. Sandford & A. Turner, 1995).

Results

Of the children, 72% had APD on the basis of these test results. Most of these children (25%) had difficulty with the FPT bilaterally. A further 22% had difficulty with the FPT bilaterally and had right ear deficits for the DDT. About half of the children (47%) had problems in all 3 areas (APD, LI, and RD); these children had the poorest FPT scores. More had APD–RD, or APD–LI, than APD, RD, or LI alone. There were modest correlations between FPT scores and attention and memory, and between DDT scores and memory.

Conclusions

LI and RD commonly co-occur with APD. Attention and memory are linked to performance on some auditory processing tasks but only explain a small amount of the variance in scores. Comprehensive assessment across a range of areas is required to characterize the difficulties experienced by children with APD.
Message and Talker Identification in Older Adults: Effects of Task, Distinctiveness of the Talkers' Voices, and Meaningfulness of the Competing Message
Authors: Rossi-Katz Jessica , Arehart Kathryn Hoberg
Published: December 2008

Purpose

In this study, the authors investigated (a) the effects of task, vocal distinctiveness of the competing talkers, and meaningfulness of the competitor on older listeners' identification of a target in the presence of competition and (b) the factors that are most predictive of the variability in target identification observed among older listeners.

Method

Seventeen older and 5 younger adults identified a target in the presence of a competing message. Identification was measured for 2 target types (message; talker), 3 vocal-distinctiveness levels (same talker; different talkers–same sex; different talkers–different sex), and 2 competitors (meaningful speech; nonmeaningful time-reversed speech). Multiple regression analyses were used to identify variables that were predictive of intersubject variability.

Results

Significant age-related differences were found in the benefit obtained from increasing vocal distinctiveness. Older listeners showed reduced target identification when (a) the competitor was normal speech compared with time-reversed speech and (b) the target was message identification compared with talker identification. Variability among listeners in the older group was partially explained by auditory and cognitive factors.

Conclusion

Age-related declines in multitalker environments are not solely due to lower-level deficits in perceptual organization but are also a consequence of an interaction between lower-level and higher-level processes.
1 EFFECT OF OXYGEN TENSION DURING IN VITRO MATURATION AND FERTILIZATION ON BOVINE EMBRYO PRODUCTION AND CUMULUS-OOCYTE-COMPLEX mRNA ABUNDANCE
Authors: Bermejo-Alvarez P. , Rizos D. , Lonergan P. , Gutierrez-Adan A.
Published: December 2008

Despite physiological oxygen tension being lower than in atmospheric gas, both in vitro maturation and fertilization are generally conducted under atmospheric oxygen tension (20%). The objective of this study was to evaluate the effect of two different oxygen concentrations (20 and 5%) during in vitro maturation (M) and fertilization (F) on bovine embryo production and to analyze differences in gene expression between cumulus–oocyte-complexes (COC) matured at 5% (M5) or 20% (M20) oxygen tension. A total of 1179 COC were matured, fertilized and cultured in vitro in 5 replicates divided in 4 groups according to the oxygen tension used (M5F5, M5F20, M20F5 and M20F20). Cleavage was assessed every 4 h from 24 to 48 h post-insemination (pi) and blastocyst yield was recorded from Day 6 to Day 8. For gene expression analysis, 5 pools of 10 COC (not denuded) of each group were snap frozen after maturation and stored at –80°C. After RNA extraction and RTPCR, qPCR was used to quantify relative abundance of 7 genes: the M-phase promoting factor subunit Cyclin B1, insulin-growth factor 2 (IGFR2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenase A (LDHA), glucose-6-phosphate dehydrogenase (G6PD), glutathione peroxidase (GPX1), and the apoptosis related gene BAX. Histone 2A was used as housekeeping gene. Statistical analysis was performed analyzing two factors (M5 v. M20 and F5 v. F20) by two-way ANOVA (P ≤ 0.05) for embryo production and by one-way ANOVA (P ≤ 0.05) for gene expression. At every time point analyzed, maturation under low oxygen tension significantly improved both cleavage rate and blastocyst yield. However, the use of low oxygen tension at fertilization has a significant negative effect on developmental rates (48 hpi: M5F5: 71.6 ± 5.5; M5F20: 76.2 ± 2.7; M20F5: 40.1 ± 5.8; M20F20: 69.9 ± 0.8). (Day 6: M5F5: 12.6 ± 3; M5F20: 17.2 ± 2.4; M20F5: 2.5 ± 1.2; M20F20: 13.3 ± 4.) (Day 7: M5F5: 32.6 ± 4.9; M5F20: 38.8 ± 3.2; M20F5: 12.2 ± 1.2; M20F20: 34.2 ± 3.9.) (Day 8: M5F5: 35.5 ± 5; M5F20: 43.1 ± 5.1; M20F5: 15.6 ± 1.4; M20F20: 37.1 ± 4.2.) (Group: %Mean ± SEM). Four genes (Cyclin B1, GAPDH, IGFR2 and LDHA) were significantly upregulated in M5 COC, and three genes (GPX1, G6PD and BAX) did not show significant differences. In conclusion, low oxygen tension during maturation exerts a beneficial effect upon embryo development, whereas the opposite situation is observed during fertilization. Furthermore, the expression of a growth factor, a meiotic promoting factor and two enzymes of anaerobic glycolysis were significantly upregulated in COCs matured under low oxygen tension, which may be linked to the improvement in developmental rates.
126 THE EFFECT OF FORSKOLIN ON IN VITRO PRODUCED BRAHMAN-SIRED BOVINE EMBRYOS
Authors: Pryor J. H. , Trant J. A. , Ponchirolli-Schneider C. B. , Looney C. R. , Long C. R. , Forrest D. W.
Published: December 2008

Forskolin (Colforsin) is a lipolytic agent that can be used to reduce intracellular lipids within in vitro produced (IVP) porcine embryos (Men et al. 2006 Theriogenology 66, 2008–2016). Lowering lipids within bovine embryos has shown to decrease sensitivity to cryopreservation. The objective of this preliminary experiment was to determine developmental differences and lipolytic effect on bovine IVP embryos exposed to 10 or 40 μm forskolin. Bovine oocytes (obtained from an abattoir) were fertilized with frozen/thawed Brahman semen (Day 0) and cultured in G1.5/G2.5 medium supplemented with 8 mg mL–1 BSA (Vitrolife, Englewood, CO, USA). On Day 6 post-fertilization, a total of 352 viable embryos were randomly submitted to 3 treatment groups; Control (no treatment, n = 116), 10 μm (n = 122) and 40 μm (n = 114) forskolin (Sigma, St. Louis, MO, USA) in G2.5 medium for 24 h. On Day 7, embryos were washed in holding medium (Vigro Holding Plus, Bioniche, Pullman, WA, USA). From each treatment group, a randomized half was cultured 72 h in G2.5 and assessed for hatched blastocyst rates (development), with the balance stained on Day 7 in Hoechst 33342 (2.5 μg mL–1) under UV light for total cell counts before being fixed/stained in Nile red (1 μg mL–1). Nile red stained embryos were mounted on glass slides with cover slips gently applied over each embryo. Embryos were photographed under UV light using the proper excitation filters. The area measurements of compact morula and blastocysts did not include the zona pellucida. Nile red units (NRU) were measured with computer software Image J (www.rbsweb.nih.gov) from 0 to 255 shades/pixel (0 = no lipids; 255 = highest lipid accrual). Each image was adjusted for variation in embryo size. Student’s t-test was used for statistical analysis. There was no difference (P > 0.05) between development rates for control, 10 μm and 40 μm forskolin (62%, 67% and 65%, respectively). Mean NRU by total cell numbers were calculated per embryo for each treatment group: control, 10 μm and 40 μm with the resulting means being 72.4 ± 5.0, 63.1 ± 5.1 and 66.6 ± 4.8 respectively. With treatment groups combined by embryo stage, compact morula tended to exhibit a higher mean NRU than blastocysts (78.6 ± 6.8 to 64.7 ± 3.1, respectively, P = 0.07). In conclusion, forskolin did not significantly affect embryo development rates or lipid content as measured by Nile Red staining. Compact morula tended to yield a higher NRU mean than blastocysts indicating an elevation in intracellular lipids. Unlike pig IVP embryos exposed to forskolin, forskolin failed to show a significant decrease in intracellular lipids within bovine IVP embryos. However, further research is in progress to test forskolin’s lipolytic effect on cryopreservation of in vitro and in vivo produced bovine embryos.

The authors acknowledge support from the American Brahman Breeders Association and Dr. Moisés Barceló Fimbres.
139 NEW APPROACH FOR BOVINE EMBRYO RECOVERY
Authors: Dupras R. , Chorfi Y.
Published: December 2008

The objective of this study was to evaluate the use of a second flush for bovine embryo recovery. A total of 319 clinically healthy Holstein cows (247 lactating, 53 dry, 19 nulliparous) with an average age of 5.5 ± 2.5 years were used for this experiment. Superovulation was performed according to a modified method of Baracaldo et al. (2000). On Day 0 (beginning of the experiment), each cow received 3 mg of estradiol-17β intramuscularly (i.m.) and a progesterone-releasing vaginal insert (1.9 g of progesterone, CIDR, Pfizer Animal Health, Kirkland, QC, Canada) at random stages of the estrous cycle. From Day 4 evening to Day 8 evening, the cows received a total of 380 mg of NIH-FSH-P1 (FolltropinV, Bioniche Animal Health) administered im through 9 injections of decreasing dose (from 70 to 20 mg) at 12-h intervals. On Day 7, the cows received 2 injections consisting of 500 μg of cloprostenol (prostaglandin F2α analogue Estrumate, Schering-Plough, Pointe-Claire, QC, Canada) given approximately 12 h apart and vaginal inserts were removed 12 h after the last injection. Artificial insemination was performed on Day 10 after treatment with 100 μg, GnRH im (Cystorelin, Merial Canada Inc, Baie d’Urfe, QC, Canada). Embryos were flushed from the uterus of donor cows 6 days after AI. The method consisted of using simultaneously 1 catheter (18Fr Silicone 2-way, Bioniche Animal Health) per uterine horn. Catheters were maintained in place to perform 2 flushes 1 h apart. A total of 1000 mL of flushing media (Complete flush, Bioniche Animal Health) were used, 700 mL and 300 mL for the first and the second flush, respectively. Embryos were assessed for viability immediately after collection using IETS classification. Data were analyzed using the SAS MIXED procedure (SAS Institue, Cary, NC). The mean (±SD) number of embryos collected at the first flushing was 5.87 ± 5.1, 0.92 ± 2.2 and 2.9 ± 4.4 for transferable, degenerate and unfertilized oocytes, respectively. The second flushing yielded 2.32 ± 2.6 transferable embryos, 0.28 ± 0.83 dead embryos and 1.2 ± 2.2 unfertilized oocytes. There was no significant effect of age, day in milk, or stage of lactation on transferable or degenerate embryos or nonfertilized oocytes in each flushing. The embryo recovery method used in this experiment could be used to recover more transferable embryos.

The authors want to thank Dr Vincent Girard for his help in statistics.
146 RELOCATION OF SWINE GENETICS USING EMBRYO TRANSFER
Authors: Terlouw S. L. , Bierman C. D. , Kohler D. L. , Didion B. A. , Dobrinsky J. R.
Published: December 2008

Swine production requires a stable health status that can be compromised by introduction of live animals for genetic change. Our objective was to use embryo transfer to avoid disease transmission during genetic relocation. Forty genotype-specific (GS) donor females were scheduled for 3 sessions of embryo recovery at 6-week intervals using Altrenogest (Matrix®, Intervet, Millsboro, DE), 1250 IU of equine chorionic gonadotropin (eCG/PMSG; Sigma, St. Louis, MO) and 750 IU of human chorionic gonadotropin (hCG; Chorulon®, Intervet). Single-sire GS matings were made 34 h after Chorulon® injection. To accomplish single-sire transfers, color specific (CS) supplemental embryos were used to assist in maintenance of recipient pregnancy. The CS embryo donors and GS embryo recipients were synchronized with Matrix®, P.G. 600® (200 IU hCG, 400 IU PMSG, Intervet) and Chorulon®. Embryos from GS donors were surgically recovered on Day 5 post-insemination, washed per IETS recommendations using a zwitterion-buffered culture medium (PorcPro E-Blast, Minitube of America, Verona, WI) and transported in a portable incubator (Minitube of America) 2.5 h to the recipient herd. Embryos were surgically transferred into –24-h asynchronous recipients within 6 to 14 h after recovery. A total of 620 embryos were recovered from 65.2% (60/92) of GS matings, and 587 (59.4%) GS and 402 (40.6%) CS embryos were transferred into 63 recipients. On average, 9.3 GS and 6.4 CS embryos were transferred per recipient (15.7). A total of 33 GS embryos were discarded before transfer. To achieve a target of 17 embryos per transfer, 59 embryo transfers required CS embryos and 4 embryo transfers were only GS embryos. Fifty-three (84.1%) recipients were confirmed pregnant by ultrasound at 35 days of gestation. Of the 40 GS donors, one was culled for genetic reasons, 6 did not give transferable embryos, and 1 gave transferable embryos but the corresponding recipient returned to estrus for a total genetic transfer rate of 80% (32/40). After 3 sessions of embryo transfer, 32/33 (97%) GS donors that produced embryos for transfer were represented by a minimum of 1 pregnant recipient at 35 days of gestation; 17/32 GS donors were represented by a single pregnancy and 15/32 by multiple pregnancies. Sera from GS donors were evaluated for porcine reproductive and respiratory syndrome (PRRSV) and porcine circovirus type 2 (PCV2) before each embryo recovery session. Serology results were negative for PRRS (0/98)) and positive for PCV2 in 27.5% (27/98) of GS donors. Embryo wash media from the last 2 washes from PCV2 positive GS donors producing embryos for transfer were pooled and evaluated for PCV2 after transfer; all samples (0/18) were negative for PCV2. In summary, zona pellucida-intact embryos were successfully used to relocate swine genetics from a donor herd into a recipient herd with no apparent health status change in the recipient herd.
148 RISK OF TRANSMISSION OF BOVINE HERPESVIRUS (BHV-1) BY INFECTED SEMEN TO RECIPIENTS AND EMBRYOS
Authors: Bielanski A. , Lalonde A. , Algire J.
Published: December 2008

Bovine herpesvirus-1 (BHV-1) causes a variety of economically important respiratory and reproductive problems, the latter including vulvovaginitis, endometritis and infertility. For that reason, several countries have eradicated the disease and others have schemes in progress to achieve freedom. Although there is a considerable amount of information about the risk of BHV-1 transmission through contaminated semen used for artificial insemination, there is no available evidence to indicate whether the resulting embryos, when used for embryo transter (ET), can lead to the transmission of BHV-1 to recipients and offspring. For this experiment, bull semen contaminated in vitro with BHV-1 at 102 TCID50 mL–1 (Colorado strain) and then cryopreserved was used for insemination (2 times at estrus) of BHV-1 seronegative, superovulated heifers (N = 18). Embryos were collected postmortem 7 days post-insemination and were washed according to the IETS recommendations (however without trypsin treatment) or left unwashed. On 4 occasions, washed embryos were transferred to BHV-1 seronegative recipients. The remaining embryos and other samples collected from the reproductive tract were tested for BHV-1 presence using the standard virus isolation test. In total, out of 144 unfertilized oocytes and embryos collected, 9 were ET quality. Most of the embryos were degenerated (N = 79) or unfertilized (N = 56). The 4 heifers, which each received a single morula-stage embryo, maintained seronegative status, but did not become pregnant. BHV-1 was detected in 43% (23/53) unwashed and 0% (0/57) of washed embryos, 78% (14/18) of follicular fluid samples, 89% (16/18) of oviductal epithelial cells, 78% (14/18) of endometrium, and 89% (16/18) of corpora lutea tissues. Results herein suggest that BHV-1 can be transmitted by infected semen to embryo donors. The resulting unwashed embryos may remain infectious. However, whether BHV-1 uninfected offspring can be produced by ET of BHV-1 contaminated embryos that are washed according to the IETS guidelines, remains to be determined.
163 DOMESTIC CAT (FELIS CATUS) EMBRYO CRYOPRESERVATION: SLOW-COOLING VERSUS VITRIFICATION
Authors: Pedersen M. J. , Watson C. A. , Blevins B. A. , Loskutoff N. M.
Published: December 2008

A variety of small domestic or nondomestic felid species have been produced from cryopreserved embryos using slow-cooling (controlled rate) cryopreservation methods (Pope et al. 2006 Theriogenology 66, 59–71; 1518–1524). However, in our laboratory these methods have not been as successful as vitrification in freezing in vitro-derived embryos of larger felid species such as tigers, Panthera tigris spp. (Crichton et al. 2000 Theriogenology 53, 238). The objective of this study was to examine the effects of slow-cooling cryopreservation v. vitrification methods on the developmental competence of in vitro-produced 2- to 4-celled domestic cat embryos as compared to non-frozen controls. Mature tomcat testicles and queen ovaries were collected from local veterinary clinics. Epididymal sperm were processed using one of three methods according to availability and time of use: 1) freshly washed if used the same day for IVF, 2) stored in cooled, non-electrolyte medium (Pope et al. 2000 ICAR Proc. 2, 191) if used within one week, or 3) frozen according to Tebet et al. (2006 Theriogenology 66, 1629–1632). Batches of oocytes were inseminated with the same pool and type of processed sperm per replicate. All in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro culture (IVC) procedures followed those previously reported (Pope et al. 2000 Theriogenology 53, 163–174). After 48 h in IVC, 2- to 4-celled embryos were equally divided into the three groups: 1) control, non-frozen; 2) slow-cooling cryopreservation using propanediol and sucrose; and 3) vitrification using DMSO, ethylene glycol and sucrose. The cryopreservation/thawing procedure followed that of Gomez et al. (2003) and the vitrification/warming procedure followed that of Vajta et al. (2000 Anim. Reprod. Sci. 60, 357–364). Results were recorded as the percentages of blastocyst formations at Day 8 of IVC and were compared between groups using chi-square analysis. In March 2000 to 2001, 806 feline oocytes were recovered from 101 ovaries and 286 blastocysts were obtained on Day 8 IVC in seven replicates. There were no significant differences in the numbers of degenerated embryos (2- to 8-celled) or morulae in all three groups (P > 0.05). There were more blastocysts that developed in the non-frozen control group (45/97; 46.4%), but this was not statistically different (P > 0.05) from the cryopreserved group (37/91; 40.7%). However, there were significantly more blastocysts (P < 0.01) produced in the non-frozen control group than the vitrified group (28/98, 28.6%). There were no differences between the cryopreserved and vitrified groups (P > 0.05). In conclusion, this study verifies previous reports that slow, or controlled-rate cryopreservation is effective for domestic cat embryos. Vitrification is less effective, as compared to non-frozen control embryos, but this may be improved with modified protocols.
166 POTENTIAL OF URINARY RELAXIN AS A USEFUL INDICATOR OF PREGNANCY IN THE MANED WOLF
Authors: Santymire R. C. , Steinetz B. , Santymire R. M. , Brown J. L. , Songsasen N.
Published: December 2008

The maned wolf (Chrysocyon brachyurus), a neotropical canid, lives in habitats severely compromised by agricultural development. Therefore, maintaining a healthy and viable zoo population is crucial, because it serves as hedge against extinction. However, the North American ex situ population of maned wolves is far from self-sustaining, mainly due to low pregnancy success and high neonatal mortality. Currently, there is no non-invasive way to determine pregnancy in this species. Therefore, the goal is to determine whether urinary relaxin can be used as an indicator of pregnancy in this species. Objectives were to (1) determine the relationship between serum and urinary relaxin in canids, using pregnant domestic bitches as a model, and (2) evaluate the patterns of urinary relaxin in pregnant v. non-pregnant female maned wolves. Serum and urine samples were obtained weekly from six pregnant domestic bitches starting from the day of natural breeding until 3 weeks before the estimated whelping dates. For the maned wolf, urine and feces were obtained weekly during a breeding season (October to February) from six females, three of which were pregnant and gave birth. Concentrations of serum and urinary relaxin were measured using the double antibody canine relaxin radioimmunoassay (Steinetz et al. 1996 Biol. Reprod. 54, 1252–1260). Fecal progestagen were determined using an enzyme immunoassay (Songsasen et al. 2006 Theriogenology 66, 1743–1750). The relationship between serum and urinary relaxin concentrations was determined using the Pearson Product Moment Correlation Analysis. The relationship between urinary relaxin concentrations and days before whelping (for pregnant females) or time after the initial rise of progestagen for each individual were determined using a linear regression analysis. Serial dilutions of pooled dog urine produced displacement curves parallel to those of the appropriate synthetic canine relaxin standards, while wolf samples showed limited parallelism with low concentration of immunoactive antigen. Concentrations of urinary relaxin were significantly correlated with the serum counterpart in pregnant dogs (P < 0.01, r2 0.58). In pregnant maned wolves, relaxin level increased two- to six-fold as gestation progressed (P < 0.05, r2 ranged from 0.15 to 0.56). In contrast, urinary relaxin remained at the baseline in non-pregnant females throughout the diestrus period (P > 0.05). In conclusion, our findings suggest that urinary relaxin mirrors serum relaxin during pregnancy in domestic bitches and may have potential as a tool to diagnose pregnancy in the maned wolf. Further studies are required to improve the specificity and sensitivity of this non-invasive assay.
191 MATERNAL AGE ALTERS FETAL AND PLACENTAL DEVELOPMENT AND EXPRESSION OF METHYLATED GENES
Authors: Paczkowski M. , Fleming-Waddell J. , Bidwell C. A. , Krisher R. L.
Published: December 2008

The objective of this experiment was to assess fetal and placental development and to determine the abundance of imprinted gene transcripts in fetal, placental, ovarian, and brain tissue, from aged (15 months old) and young (26 days old) B6D2F1 mice. Fetal and placental weight was measured, and transcript abundance of 14 imprinted genes and 3 nutrient transport genes was analyzed by quantitative PCR. The percentage of females pregnant at day 15.5 of gestation and the average number of normal implantation sites were significantly lower in aged mice, while the average number of degenerating implantation sites was significantly increased (P < 0.05). Fetal growth restriction and placental overgrowth were associated with maternal age, suggesting the balance between maternally and paternally expressed genes may be impaired. Peg3 and Grb10 tended to have decreased abundance in placental tissue collected from pregnancies in aged mice compared to young mice (P < 0.10). Abundance of Igf2, Igf2r, Cdkn1c, Slc2a3, Slc38a4, Peg3, and Phlda2 was significantly increased (P < 0.05) in ovarian tissue from aged mice. Brain tissue harvested from aged females displayed increased abundance of Igf2r, Snrpn, Slc2a3, Mest, Gtl2, Peg3, Grb10, and Lpl (P < 0.05). Aberrant transcript abundance of imprinted genes in ovarian and brain tissue harvested from aged mice suggests that hypothalamic regulation of oocyte growth, as well as oocyte imprinting, may be altered in older females. Abnormal fetal and placental development and differential expression of imprinted genes in the ovary and brain of aged mice suggests that epigenetic regulation of oocyte and fetal development is impaired with advanced maternal age.
197 DIFFERENTIAL EFFECTS OF FLUTAMIDE AND DI-(2-ETHYLHEXYL) PHTHALATE ON MALE REPRODUCTIVE ORGANS IN A RAT MODEL
Authors: Vo T. T. B. , Jung E.-M. , Back M.-G. , Dang V. H. , Choi K.-C. , Jeung E.-B.
Published: December 2008

Endocrine disruptors (EDs) with androgenic and anti-androgenic effects may alter reproductive function by binding to androgenic receptors (AR) and inducing or modulating AR-dependent responses in the male reproductive system. However, the molecular mechanism(s) underlying these events remains unclear. Thus, in the present study, we elucidated the prenatal effects of maternal testosterone propionate (TP), flutamide (Flu), and di-(2-ethylhexyl) phthalate (DEHP) on male reproductive organs of newborn rats. Pregnant Sprague-Dawley (n = 32 in total, n = 8/each group) rats were treated with these compounds at gestation days 11 to 21, and newborn males (n = 154 in total) were euthanized at post-neonatal day (PND) 63. Interestingly, maternal exposure to Flu or DEHP caused fluctuations in the neonatal levels of serum testosterone (T) and luteinizing hormone (LH). Serum T and LH were up-regulated by Flu, but these hormones were down-regulated by DEHP. The anogenital distances (AGD) of male newborns were detected at PND 1, 21, and 63. Male rats treated prenatally with DEHP (100 mg kg–1 mother’s body weight) or Flu showed an AGD shorter than that of control rats. At PND 63, the sperm concentration, viability, and mobility were reduced in the maternal DEHP and Flu-treated groups. The numbers of seminiferous tubules were reduced in the Flu- and DEHP-treated offspring when compared with vehicle- and TP-treated groups, and the tubules of the testes at PND 63 were disrupted by a high dose of Flu. In addition, we examined differential gene expression patterns in the testes by microarray analysis following ED exposure, particularly in sex determination-related genes. Significantly distinct expressions of sex determination-related genes were observed in the testes by microarray analysis following treatments with different types of EDs in this study. Although Flu and DEHP are considered to be identical with regard to their anti-androgenic effects, their effects on developing male reproductive organs were distinct, suggesting that Flu competes with endogenous T, while DEHP influences a different step in androgenesis.
200 REPROGRAMMING OF Oct-4 FOLLOWING EQUINE SOMATIC CELL NUCLEAR TRANSFER
Authors: Xiang T. , Walker S. , Gregg K. , Zhou W. , Farrar V. , Sadeghieh S. , Hwang E. , Findeisen B. , Arenivas F. , Polejaeva I.
Published: December 2008

Oct-4, a POU domain-containing transcription factor encoded by Pou5f1, is selectively expressed in pre-implantation embryos and pluripotent stem cells, but not in somatic cells. Because of such a unique expression feature, Oct-4 can serve as a useful reprogramming indicator in somatic cell nuclear transfer (SCNT). Compared with data of Oct-4 expression in mouse and bovine cloned embryos, little is known about this gene in equine nuclear transfer. In the present study, we investigated Oct-4 expression in donor cells, oocytes, and SCNT embryos to evaluate reprogramming of equine somatic cells following nuclear transfer. Horse ovaries were obtained from a local slaughterhouse and the oocytes collected from the ovaries were matured in vitro in an M199-based medium (Galli et al. 2003 Nature 424, 635) for 24 h. Donor cells were derived from biopsy tissue samples of adult horses and cultured for 1 to 5 passages. Standard nuclear transfer procedures (Zhou et al. 2008 Mol. Reprod. Dev. 75, 744–758) were performed to produce cloned embryos derived from equine adult somatic cells. Cloned blastocysts were obtained after 7 days of in vitro culture of reconstructed embryos. Total RNA were extracted using Absolutely RNA Miniprep/Nanoprep kits (Stratagen, La Jolla, CA) from oocytes (n = 200), donor cells, and embryos (n = 5). DNase I treatment was included in the procedure to prevent DNA contamination. Semiquantitative RT-PCR was performed with optimized cycling parameters to analyze Oct-4, GDF9, and β-actin in equine donor cells, oocytes, and cloned blastocysts. The RT-PCR products were sequenced to verify identity of the genes tested. The relative expression abundance was calculated by normalizing the band intensity of Oct-4 to that of β-actin in each analysis. No transcript of Oct-4 was detected in equine somatic cells used as donor nuclei, consistent with its expression patterns in other animal species, whereas Oct-4 was abundantly expressed in equine SCNT blastocysts derived from the same donor cell line. Oct-4 transcripts were also detected in equine oocytes and whether any maternally inherited Oct-4 mRNA persisted up to the blastocyst stage was unclear in this study. We selected GDF9 to address this question; GDF9 was abundantly detected in equine oocytes, consistent with its expression pattern in mouse and bovine, but not detected in donor cells and cloned blastocysts, suggesting that the GDF9 mRNA from the oocyte was degraded at least by the blastocyst stage. The results from this study imply occurrence of Oct-4 reprogramming in equine SCNT blastocysts, and future analysis for more developmentally important genes is needed to better understand reprogramming at molecular levels in this species.
202 INDUCTION OF CALBINDIN-D9k EXPRESSION BY MELATONIN APPEARS TO BE INVOLVED IN HYDROGEN PEROXIDE-INDUCED CELL DEATH IN RAT PITUITARY GH3 CELLS
Authors: Yoo Y.-M. , Jeung E.-B.
Published: December 2008

Calcium-binding protein-D9k (CaBP-9k) is a 9-kDa polypeptide, with 2 calcium binding sites, found in mammalian intestine, placenta, uterus, kidney, pituitary gland, and growth cartilage. However, the regulation of CaBP-9k by endocrine factors (i.e. melatonin) in pituitary gland is not known yet. Melatonin, the major secretory product of the pineal gland, has been known to interact with the neuroendocrine axis, circadian rhythms, and apoptotic pathways in various cell types. Thus, in this study, we investigated whether CaBP-9k gene is regulated by melatonin in association with hydrogen peroxide (H2O2)-induced cell death or apoptosis in rat pituitary GH3 cells. We examined the expression levels of CaBP-9k mRNA by RT-PCR and immunoblot analysis, and further measured protein levels of p53, Bax, and Bcl-2 by immunoblot analysis, and kit-8 cell survival marker by cell counting following mRNA or protein isolation by Trizol reagent or Proprep solution, respectively. CaBP-9k expression was increased by melatonin in a dose-dependent (0, 0.1, 0.25, 0.5, and 1 mm) and time-dependent (1 to 48 h) manner, indicating that CaBP-9k expression may be related to melatonin receptor-mediated signal transduction in these cells. Cell death increased about 10% in H2O2 (0.25 mm) plus melatonin (1 mm) compared with treatment with only H2O2 (0.25 mm) or H2O2 (0.25 mm) plus melatonin (0.5 mm), and CaBP-9k expression was augmented by this treatment with H2O2 (0.25 mm) plus melatonin (1 mm). This suggests that the mechanism between the increase in cell death and melatonin-induced CaBP-9k expression may be involved in apoptosis by hydrogen peroxide. Bax as a cell death marker was detected in cell death condition of H2O2 (0.25 mm) or H2O2 (0.25 mm) plus melatonin (0.5 or 1 mm), whereas Bcl-2 as a survival marker was identified in the culture condition with melatonin (0.5 or 1 mm) or H2O2 (0.25 mm) plus melatonin (1 mm). p53 as a stress marker mediated with calcium change was decreased in the melatonin only treatment or H2O2 plus melatonin. This result is in agreement with CaBP-9k expression level. Knockdown of CaBP-9k using its siRNA (5′GAGCG CTAAG AAATC TCCCG AAGAA3′, Invitrogen Life Technologies) showed that the reduction of H2O2-induced cell death, suggesting that induction of CaBP-9k expression caused by melatonin is associated with H2O2-induced cell death in rat pituitary GH3 cells.
205 EFFECT OF OSTEOPONTIN ON CLEAVAGE AND BLASTOCYST RATES IN BUFFALO SPECIES (BUBALUS BUBALIS)
Authors: Di Francesco S. , Mariotti E. , Rubessa M. , Campanile G. , Di Palo R. , Zicarelli L. , Gasparrini B.
Published: December 2008

It was previously reported that osteopontin (OPN), an acidic single-chain phosphorylated glycoprotein found in the oviductal fluid in cattle (Gabler C et al. 2003 Reproduction 126, 721–729), is able to facilitate fertilization in this species (Gasparrini B et al. 2008 Reprod. Fertil. Dev. 20(Suppl. I), 180 abst). The present study aimed to investigate whether the addition of OPN to the fertilization medium would affect both cleavage and postfertilization embryo development in the buffalo. To assess the influence of OPN on cleavage and blastocyst rates, in vitro-matured oocytes were fertilized in modified Tyrode’s albumin lactate pyruvate medium (Lu KH et al. 1987 Vet. Rec. 121, 259–260) supplemented with penicillamine, hypotaurine, and heparin, in the presence of 0.0 (n = 258), 0.1 (n = 263), 1 (n = 261), and 10 μg mL–1 (n = 264) of OPN. In vitro fertilization was carried out with frozen–thawed spermatozoa from a bull already tested for IVF. After 20 to 22 h of co-incubation at 38.5°C and 5% CO2 in air, putative zygotes were gently pipetted to remove cumulus cells, washed, and transferred, 10 per droplet, into 20 μL of SOF medium including essential and nonessential amino acids and BSA (Tervit HR et al. 1972 J. Reprod. Fertil. 30(3), 493–497), in a controlled gas atmosphere consisting of 5% CO2, 7% O2, and 88% N2, in humidified air, at 38.5°C. The culture medium was changed on Day 5 (Day 0 = day of insemination), when cleavage rate was assessed and embryos were moved into fresh medium for an additional 2 days. On Day 7, development rates into blastocysts of superior quality were recorded. Differences in the percentages of both cleavage and blastocyst rates among groups were analyzed by chi-square test. Significantly higher cleavage rates (59.3, 70.3, 71.6, and 42.4%, respectively, in the control group and in the groups with 0.1, 1, and 10 μg mL–1 of OPN; P < 0.01) were observed in the groups with 0.1 and 1 μg mL–1 of OPN compared with the other groups. Likewise, higher blastocyst rate percentages (17.4, 27.4, 29.9, and 9.5%, respectively, in the control group and in the groups with 0.1, 1, and 10 μg mL–1 of OPN; P < 0.01) were observed in the groups with 0.1 and 1 μg mL–1 of OPN compared with the other groups. In conclusion, these results showed that addition of low concentrations of OPN in the fertilization medium improved both cleavage and postfertilization embryo development in the buffalo, whereas the higher concentration resulted in impaired late-stage embryo development.
219 APPLICATION OF A MICROFLUIDIC SPERM SORTER TO THE IN VITRO PRODUCTION OF PORCINE EMBRYOS
Authors: Sano H. , Matsuura K. , Naruse K. , Funahashi H.
Published: December 2008

In porcine in vitro fertilization (IVF), polyspermy is a persistent obstacle to the efficient production of normal embryos. A microfluidic sperm sorter (MFSS; Strex Inc., Osaka, Japan) was developed to isolate motile human spermatozoa (from diluted semen by two laminar flows in the microchannel). The motile spermatozoa can gradually accumulate in a chamber of the MFSS. We previously reported that the monospermy rate was higher when oocytes were co-cultured with isolated spermatozoa in an MFSS for 5 min than when spermatozoa were co-cultured traditionally in drops for 8 h (P < 0.05; Reprod. Fertil. Dev. 20, 187 abst). The present study was undertaken to compare the effect of oocyte location within the MFSS chamber on early development after IVF in the MFSS. A sperm-rich fraction from Berkshire boars was diluted at 1 × 108 cells mL–1 with modified Modena solution containing 20% seminal fluid. In the first experiment, a diluted semen sample was flowed with modified TCM-199 containing 5 mm caffeine (mm199-caf) for 5 min at room temperature. Before flowing, porcine IVM oocytes were positioned in a part of the MFSS chamber where motile spermatozoa would accumulate with mm199-caf. After flowing for 5 min, those oocytes were cultured in caffeine-free mm199 for 8 h and then in a chemically defined medium (PZM-5) for 7 days. In the second experiment, denuded oocytes were co-cultured with isolated spermatozoa at several locations in the MFSS chamber, and the penetration and monospermy rates were estimated. The concentration of motile spermatozoa was also measured at each place in the MFSS chamber after isolation for 5 min. Statistical analyses of results based on 4 to 5 replicates were carried out by ANOVA and Fisher’s PLSD post hoc test (significance, P < 0.05). When IVM oocytes were co-cultured with spermatozoa gradually accumulated in the chamber of the MFSS for 5 min, the cleavage rate (83.7 ± 6.3% of 121 oocytes) was not different from that of control oocytes co-cultured with spermatozoa (5.7 × 105 cells mL–1) in 100-μL drops for 5 min (84.6 ± 6.6% of 126 oocytes). However, the blastocyst formation rate (38.2 ± 3.3%) was higher than for the controls (20.6 ± 6.8%; P < 0.05). After flowing for 5 min, the distance from the inflow opening of the MFSS chamber to the location of the oocytes did not affect the sperm penetration rate, but did affect the monospermy rate (14.0 ± 4.0% of 48 oocytes at the nearest position to 50.0 ± 5.6% of 43 oocytes at the furthest position; P < 0.05). After flowing for 5 min, the concentration of motile spermatozoa was also different at each location (57.5 ± 5.6 × 104 cells mL–1 at the nearest position to 0.8 ± 0.5 × 104 cells mL–1 at the furthest position; P < 0.05). These observations demonstrate that co-culturing oocytes with spermatozoa that gradually accumulated in the MFSS chamber improved the efficiency of blastocyst formation in the pig, whereas efficiency was affected by the position where oocytes were located in the chamber.