bluuebunny/crossref_metadata_2025 huggingface dataset:
188 DETECTION BY POLYMERASE CHAIN REACTION OF NEOSPORA CANINUM FROM OVUM PICKUP AND UTERINE FLUSHING FLUIDS FROM DAIRY CATTLE IN MEXICO
Authors: Marques A. F. , Ortiz C. G. , Lima M. R. , Zanella E. L. , Rangel L. , Morales E. , Gutierrez C. G.
Published: August 2010

Neospora caninum, an intracellular protozoon, causes encephalomyelitis in dogs (Bjerkas I et al. 1984 Zentralblat fur Parasitenkunde 70, 271-274). For the past decade, neosporosis has been a main cause of abortion in dairy cattle worldwide (Anderson M et al. 2000 Anim. Reprod. Sci. 60-61, 417-431; Dubey JP 2003 Korean J. Parasitology 41, 1-16). Vertical transmission has been indicated as an important way of spreading neosporosis (Hall CA et al. 2005 Vet. Parasitology 31, 231-41); thus, we investigated whether the protozoon could be transferred by embryo production techniques. Blood samples were collected from 92 dairy cows with history of reproductive failure and abortion within the previous 90 days at 7 dairy farms in Tizayuca, Mexico. For serology evaluation, a commercial indirect ELISA kit (Civtest Bovis Neospora, Laboratories Hipra S.A, Girona, Spain), yielded 46.74% (43/92) positive results, 46.74% (43/92) negative results, and 6.52% (6/92) suspicious to N. caninum infection. Thirteen positive cows were chosen for uterine flush (UF), ovum pickup (OPU), and a blood sample collection. Lymphocytes from blood and cells within the UF and OPU collection fluids were collected after centrifugation and DNA was extracted. All samples were tested for the presence of N. caninum by PCR, using primers and protocols that amplified a 275-bp fragment of the genomic region (5-GGGTGAACCGAGGGAGTTG-3 and 5-CCTCCCAATGCGAACGAAA-3). The N. caninum vaccine (Bovilis® NeoGuard, Intervet, Santiago Tianguistenco, Mexico) was used as a positive control and water as a negative control. Uterine flush could not be obtained from 1 cow. From 13 cows seropositive to N. caninum, only 38% were positive to PCR from blood lymphocytes. In contrast, PCR amplification was obtained from OPU cell sediment in 92.31% (12/13) and in 33.33% (4/12) of UF. Of these 12 OPU- and 4 UF-positive samples, only 5 and 3 of their corresponding blood lymphocytes were positive. Our results using uterine and follicular fluid were contradictory to those published by Moskwa et al. (2008 Vet. Parasitology 158, 370-375) where oocytes and embryos were evaluated. These results indicate that N. caninum is present in the ovary and uterine lumen of the cows, suggesting a possible risk of neospora transmission during oocyte and embryo collection and transfer techniques.

UNAM and UPF.
192 SUSCEPTIBILITY OF BOVINE-HATCHED BLASTOCYSTS TO BLUETONGUE VIRUS SEROTYPE 8 INFECTION
Authors: Vandaele L. , Wesselingh W. , De Clercq K. , Nauwynck H. , Van Soom A.
Published: August 2010

In 2006 and 2007, Bluetongue virus serotype 8 (BTV-8) caused devastating outbreaks in Northern Europe; the outbreaks were controlled in 2008 and 2009 by an international vaccination policy. Remarkably, BTV-8 differs from other serotypes in that it spread transplacentally (De Clercq K et al. 2008 Transboundary and Emerging Diseases 55, 352-359). Apart from the transplacental spreading, a significant increase in the incidence of abortions was reported in Belgium (Meroc E et al. 2009 Transboundary and Emerging Diseases 56, 39-48). The aim of the present study was to investigate the susceptibility of bovine-hatched, in vitro-produced blastocysts to BTV-8. A total of 1390 immature bovine oocytes were matured and fertilized in vitro. Presumed zygotes (n = 1148) were denuded 24 h post-insemination and cultured in 50-μL droplets of modified synthetic oviduct fluid (SOF) medium with 10% fetal calf serum (tested negative for BTV antibodies) at 39.0°C in 5% CO2, 5% O2, and 90% N2. At 7 days post-insemination (dpi), blastocysts were grouped to enhance hatching. For virus incubation, BTV-8 Bel 2006/2 from Veterinary and Agrochemical Research Centre (VAR, Brussels, Belgium) was used. At 8.5 dpi, hatched embryos were placed in 800μL of minimum essential medium (MEM) containing 103.8 50% tissue culture infectious doses (TCID50) of BTV-8 and incubated for 1 h at 39°C in an atmosphere of 5% CO2 in air. At the same time, 2 groups of hatched control embryos were incubated under the same circumstances in 800 μL of SOF and 800 μL of MEM, respectively. After infection, all embryos were washed according to IETS guidelines with the exception that they were not zona pellucida intact and cultured in new SOF. At 48, 60, 72, and 96 h post-infection (hpi), one-fourth of the embryos of each group were fixed in 4% paraformaldehyde for 12 to 24 h and subsequently stained for BTV-8 with double immunofluorescent staining using a BTV-8 monoclonal antibody (8A3B.6, ID-Vet, Montpellier, France). All control embryos (CTRL and MEM) were negative for BTV-8 virus antigen at all time points. At 48 hpi, only 1 out of 7 infected embryos was positive for virus antigen (in all its cells). At 60 hpi, all remaining embryos (n = 6) were negative, whereas at 72 hpi and 96 hpi all embryos had 25% to 100% BTV-8-positive cells (n = 6 at 72 hpi and n = 7 at 96 hpi). Furthermore, 1 embryo at 72 hpi and 2 embryos at 96 hpi showed morphological signs of degeneration. This study has showed for the first time that hatched in vitro-produced blastocysts are susceptible for BTV-8 virus infection and replication in vitro. The relatively long time between virus infection and detection of viral antigen is in accordance with the slow replication cycle of the virus. Further research is ongoing to investigate the importance of BTV-8 infection in early embryonic death.

The first author is supported by Research Foundation-Flanders.
196 NONINVASIVE HORMONE MEASUREMENTS FOR GENDER IDENTIFICATION IN MONOMORPHIC ANURANS
Authors: Blevins B. A. , Loskutoff N. M.
Published: August 2010

Noninvasive techniques have been successful in distinguishing gender in dimorphic amphibian models (Szymanski DC et al. 2006 Zoo Biol. 25, 35-46). The aim of this study was to assess gender-specific fecal hormone metabolite concentrations in 2 dimorphic and 2 monomorphic, critically endangered species of amphibians to assist in ex situ breeding efforts. Fecal samples from 2 dimorphic anurans, the Puerto Rican Crested Toad (Bufo lemur) and the Wyoming Toad (Bufo baxteri) and 2 monomorphic species, the Mountain Chicken Frog (Leptodactylus fallax) and the Dusky Gopher Frog (Lithobates sevosa) were measured by enzyme immunoassay for sex-specific hormone differences (Student’s t-test; P < 0.05) among 6 variables (concentrations of estradiol (E2), progesterone (P), and testosterone (T), and ratios of P:E2, T:E2, and P:T). Values are presented as means ± SEM. Mountain Chicken Frog samples were obtained from individuals of known sex. Dusky Gopher Frog samples were from confirmed females but males were presumed based on subtle physical characteristics. The E2 concentrations (ng g-1) were significantly higher in females than males (P < 0.05) in all species except for the Dusky Gopher Frog. However, males in all 4 species, including the presumptive Dusky Gopher Frog males, exhibited significantly higher P:E2 and T:E2 ratios than females. Additionally, Wyoming Toad and Mountain Chicken Frog females had significantly higher P:T ratios than males (0.19 ± 0.05 v. 0.06 ± 0.00 and 0.34 ± 0.03 v. 0.20 ± 0.02, respectively). Similar to the American Toad (Szymanski et al. 2006), presumptive Dusky Gopher Frog males measured significantly higher T concentrations compared with females (376.4 ± 118.5 ng g-1 v. 174.0 ± 23.1 ngg-1, respectively). Although species-specific differences are apparent, fecal hormone measurements can be used to identify sex in monomorphic species and offer a potential tool for the captive breeding of endangered anurans.

Table 1.
2 IDENTIFICATION OF FIVE GENES EXPRESSED PREFERENTIALLY FROM THE PATERNAL X CHROMOSOME
Authors: Bermejo-Álvarez P. , Rizos D. , Lonergan P. , Gutiérrez-Adán A.
Published: August 2010

Imprinting mechanisms have been proposed to evolve from mechanisms to defend the genome against transposable elements. Compared with autosomal chromosomes, the X chromosome has generated a disproportionately high number of functional retroposed genes in mammalian species. However, few paternally expressed X-linked genes have been reported (http://igc.otago.ac.nz/home.html). In a previous microarray study that analyzed transcriptional differences between male and female bovine blastocysts, almost half of the upregulated genes in female embryos were present on the X-chromosome. The higher expression of X-linked genes in female compared with male embryos may be caused by preferential paternal allele expression or by double allele expression. In the first case, a lower expression for male and parthenogenetic embryos compared with their female counterparts is expected, as both lack a paternal X-chromosome; in the second case, a lower expression in male embryos compared with female and parthenogenetic embryos would be obtained, as they only have one X-chromosome. To discover putative imprinted genes on the X-chromosome, we analyzed the relative mRNA abundance of 8 X-linked transcripts (BEX1, CAPN6, FMR1NB, SAT1, BEX2, X24112, SRPX2, and UBE2A) in male, female, and parthenogenetic bovine blastocysts. Male and female embryos were produced by conventional IVF with unsorted semen, sexed by PCR, and pooled in groups of 10 to perform the reverse transcription (RT) reaction. Parthenogenetic embryos were obtained by activation in ionomycin + 6-DMAP. mRNA was extracted using the Dynabeads mRNA Direct Extraction Kit (Dynal Biotech, Oslo, Norway), RT was performed with Moloney murine leukemia virus reverse transcriptase, and relative abundance was analyzed by quantitative PCR using H2AFZ as a housekeeping gene. The expression level of 5 genes (BEX1, CAPN6, BEX2, SRPX2, and UBE2A) differed significantly between parthenogenetic and female blastocysts, suggesting that they are preferentially expressed from the paternal allele. These results suggest that an imprinting mechanism may play a role in preimplantation sexual dimorphism and provide a valuable tool to uncover X-linked paternally expressed imprinted genes.
22 IN VITRO FERTILITY ESTIMATES USING SPERM KINETICS VARIABLES AND CONCEPTION RATES IN BEEF HEIFERS
Authors: Ferraz Jr M. V. C. , Macedo R. S. , Barreto Filho J. B. , Silva T. F. , Braga Jr R. A. , Rabelo G. F. , Previdelli R.
Published: August 2010

Sperm motility is a physical parameter evaluated in semen samples of the bull and is thought to be related to the fertility in the male. Despite being a characteristic of simple and fast evaluation, motility estimates involve subjective components when analyzed by light microscopy (LM) that might restrain their evaluation in some conditions. Moreover, in some species, poor correlations were observed between spermatozoa motility and semen fertility. The incidence of coherent light in a semen sample generates a phenomenon called biospeckle (BSL) that is capable of measuring the kinetics activity of the spermatozoa. In this work the relationship among sperm kinetics variables evaluated by LM and BSL and conception rates in beef heifers was investigated, with the purpose to predict frozen semen fertility when laser light is used. Sixty semen samples of 6 mature AI donor bulls (Bos taurus indicus) herein named A, B, C, D, E, and F were thawed at 37°C for 30 s in a water bath and evaluated by LM and BSL. In LM evaluation, an index (IND) was proposed to group together, in a single mean estimate, the spermatozoa motility (M: % scale) and velocity (V: 1 to 5 scale) according to the equation IND = [V × 20 + M]/2. In the BSL evaluation, each sample was illuminated (n = 10 per bull) by a laser beam (He-Ne, 632 nm and 10 MW) for 40 s and a mean inertial moment (IM) was obtained for each donor bull. These semen samples were used in an AI program of beef heifers (n = 166) clinically examined for reproductive health, body condition, and weight. Pregnancy diagnosis was done by ultrasound images (Falco 100, 6 MHz, Pie Medical, Crawley, UK) 28 days after insemination. Fertility estimates were done by the generalized linear model using logistic regression (stepwise methodology), generating an equation to predict the conception rate of the semen, the variables of which were IM and IND. Results of the predicted conception rates (pCR) using IM and IND and the observed conception rates (oCR) were A (0.5490; 0.57), B (0.6483; 0.68), C (0.7108; 0.71), D (0.4552; 0.28), E (0.4797; 0.54), F (0.3825; 0.47), respectively. Positive correlations (P < 0.05) were observed between pCR and oCR (r = 0.79) showing a similar behavior between in vitro and in vivo estimates. Results of this work showed that there is a high correlation between spermatozoa kinetics and semen fertility in the bull and that BSL motility analysis could be used as an approach to evaluate the fertility of semen samples.

Financial support: FAPEMIG grant EDT 94/07, CNPq.
230 EXPRESSION OF mRNA ENCODING FGF10 AND COGNATE RECEPTORS (FGFR1B AND FGFR2B) AROUND FOLLICLE DEVIATION IN NELORE HEIFERS
Authors: Castilho A. C. S. , Machado M. F. , Guerra D. M. , Ereno R. , Barros C. M. , Price C. A. , Buratini Jr J.
Published: August 2010

A member of the FGF7 subfamily, FGF10 acts via FGFR2B and FGFR1B. In bovine antral follicles, FGF-10 was detected in oocytes and theca cells (TC). Levels of mRNA were negatively correlated with intrafollicular concentrations of estradiol, and FGF10 inhibited estradiol production from granulosa cells (GC). In Nellore (Bos indicus), morphological divergence occurs on average 2.5 days after ovulation, when dominant follicle diameter is around 6.0 mm. To gain insight into the involvement of the FGF10 system in the control of follicle selection, we assessed mRNA expression of FGF10 in TC and of FGFR1B and FGFR2B in GC from dominant and subordinate follicles around deviation in Nellore heifers. Thirteen Nellore heifers were hormonally synchronized, and ovulation was detected by ultrasound monitoring every 12 h. Heifers were slaughtered 2 (n =4), 2.5 (n = 5), and 3 (n = 4) days after ovulation. Granulosa cells and TC were separated from the 2 largest follicles and submitted to total RNA extraction. mRNA abundance of CYP19 (aromatase), FGF10, FGFR1B, and FGFR2B was measured by real-time RT-PCR and normalized by the expression of cyclophilin A (CYCA) and GAPDH, for TC and GC, respectively. Dominant and subordinate follicles were considered those expressing the greatest and second-greatest abundance of CYP19 mRNA in GC within each heifer. Effects of follicle status and day on CYP19, FGF10, FGFR2B, and FGFR1B mRNA abundance were tested by ANOVA. On Day 2, FGFR2B mRNA abundance was greater in GC of subordinate follicles compared with dominant follicles (P = 0.006), and that of FGF10 in TC tended to exhibit the same pattern (P = 0.06). Follicle diameter was not different between dominant and subordinate follicles on Day 2 (5.5 ± 0 v. 5.12 ± 0.3 cm). On Day 2.5, FGF10 expression was greater in TC from subordinate follicles (P = 0.01), and FGFR2B expression in GC was no longer different between dominant and subordinate follicles. Follicle diameter was greater in dominant follicles on Day 2.5 (6.7 ± 0.2 v. 5.8 ± 0.3 cm; P = 0.04). On Day 3, no differences were observed between dominant and subordinate follicles for any of the genes assessed. mRNA expression of FGFR1B in GC did not change with follicle status or day. In conclusion, expression of FGF10 and FGFR2B was decreased in dominant follicles around morphological divergence, suggesting their involvement in the mechanisms controlling dominant follicle selection. As FGF10 inhibits estradiol production of GC, we propose that FGF10 and FGFR2B are suppressed in the dominant follicle to allow acquisition of full steroidogenic capacity.

This research was supported by FAPESP.
232 DIFFERENTIAL EXPRESSION OF UTERINE CALCIUM-RELATED PROTEINS (NCKX3, NCX1, PMCA1, TRPV6, AND CABINDIN-D9k AND -D28k) DURING HUMAN MENSTRUAL CYCLE
Authors: Choi K. C. , Jeung E. B.
Published: August 2010

The endometrium is hostile to embryo implantation except during the window of receptivity. A change in endometrial gene expression is required for the development of receptivity. The uterine calcium balance is crucial for physiological functioning, including smooth muscle contraction and embryo implantation. The location of cytoplasmic calcium-related proteins (CRP) include the calcium transporters 1 (CaT1), calbindin-D9k/-D28k (CaBP- 9k/28k), plasma membrane Ca2+-ATPase 1b (PMCA1b), sodium/calcium exchangers (NCX1), and potassium-dependent Na+/Ca2+ exchanger (NCKX3). The expressions of these CRP and their potential roles in the uterus of human during the menstrual cycle remain to be clarified. Thus, in this current study, the expression patterns of CRP were examined for their roles in the human uterus during the menstrual cycle. Human endometrial tissues were collected by curettage from women undergoing hysteroscopy for investigation of tubal patency or tubal ligation. Approval was given by the Human Ethics Committee at SCH Medical Center, Bucheon, and signed consent was obtained in every case. Human uterus (total n = 51) were divided into 3 groups: menstrual, proliferative, and secretory phase. Reverse-transcription PCR and Western blot analysis were applied to measure the level of CRP mRNA and protein, respectively. During the menstrual cycle of human, the expression levels of CaT1 mRNA and protein were increased 5-fold at proliferative phase (Days 6 to 13) compared with secretory phase in the endometrium of uterus. The expression of CaBP-28k mRNA and protein was less 2-fold during the proliferative phase (Days 6 to 13) than during the secretory phase (Days 16 to 28). However, the expressions of NCX1, NCKX3, and PMCA1b mRNA and protein were not altered during cycle, whereas the expression of CaBP-9k was not observed in the uterus of human. In addition, spatial expression of CRP was detected by immunohistochemistry Uterine CRP was abundantly localized in the cytoplasm of the luminal and glandular epithelial cells during menstrual cycle. Taken together, these results indicate that uterine CRP is abundantly expressed in the uterus, suggesting that uterine expression of CRP might be involved in reproductive function during the menstrual cycle in human.
239 ALLELE-SPECIFIC EXPRESSION OF X CHROMOSOME-LINKED GENE MAO-A DURING PRE-IMPLANTATION DEVELOPMENT IN BOVINE EMBRYO
Authors: Ferreira A. R. , Machado G. M. , Diesel T. O. , Carvalho J. O. , Rumpf R. , Melo E. O. , Dode M. A. N. , Franco M. M.
Published: August 2010

The in vitro embryo culture might affect epigenetic mechanisms, which are involved in controlling the expression of genes related to embryonic development and inactivation of X chromosome. Female mammals have 2 X chromosomes, and males have only 1. This has led to a particular mechanism of evolution of dosage compensation, called X-chromosome inactivation, an important epigenetic event that must occur in all mammalian female embryos. During embryogenesis, at the late blastocyst development (Xue F et al. 2002 Nature Genet. 31, 216220), 1 of the 2 X chromosomes is randomly inactivated in each cell of the inner cell mass and preferentially X paternal in trophoblast. The aim of this study was to characterize the allele-specific expression of the X chromosome-linked gene monoamine oxidase type A (MAO-A) during in vitro pre-implantation embryo development in bovine. For phenotyping of the MAO-A gene, the RT-PCR restriction fragment length polymorphism technique was used. Primers were designed flanking a single nucleotide polymorphism and the sequence of forward inner primer creating a site of restriction to the RsaI enzyme, thus allowing the detection of allele-specific expression (Bos taurus Taurus × Bos taurus indicus). Oocytes were aspirated from 9 Nelore heifers homozygous for theA allele previously genotyped. The oocytes were selected, matured in vitro, and inseminated with X-sorted sperm from a Holstein bull homozygous for the G allele. Two pools of 10 heterozygous in vitro embryos of each developmental stage, 4-cell [44 h post-insemination (p.i.)], 8- to 16-cell (72 h p.i.), morula (144 h p.i.), blastocyst (156 p.i.), and expanded blastocyst (168 h p.i.), were produced and frozen until RNA extraction. Total RNA was extracted using Invisorb® Spin Cell RNA Mini Kit (Invitek, Berlin, Germany) according to the manufacturer’s protocol, and residual genomic DNA was removed with DNase I treatment. cDNA was done using Oligo dT primers (Invitrogen) and superscript III reverse transcriptase (Invitrogen). Nested PCR for each pool was performed and then the amplicons were digested with 10 U of RsaI enzyme (Promega, Madison, WI, USA). The products were separated by electrophoresis on a 3% agarose gel stained with ethidium bromide. The results showed that both alleles were expressionally represented in the 4-cell, 8- to 16-cell, and expanded blastocyst stages, with the X paternal allele disappearing in morula and blastocyst. We can conclude that both, maternal and paternal X chromosomes, are activated in the 2 earliest stages, inactivated in the morula and blastocyst stages, and reactivated in the expanded blastocyst stage.

This research was supported by Embrapa Genetic Resources and Biotechnology and the Brazilian National Council for Scientific and Technological Development (CNPq).
249 PROFILE OF MEMBERS OF IGF SYSTEM GENE EXPRESSION IN BOVINE IMMATURE OOCYTES: COMPARISON BETWEEN NELLORE (BOS INDICUS) AND HOLSTEIN (BOS TAURUS)
Authors: Satrapa R. A. , Simões R. A. L. , Castilho A. C. S. , Nabhan T. , Silva C. F. , Razza E. M. , Pegorer M. F. , Puelker R. Z. , Buratini Jr J. , Barros C. M.
Published: August 2010

There is evidence that IGF system can be involved in the oocyte competence and, consequently, in the embryo development. To better understand possible differences between Bos taurus and Bos indicus in in vitro embryo development, the current work aimed to assess the expression of IGF ligands (IGF-1 and 2), types 1 and 2 IGF receptors (IGFR1 and 2), IGF-binding proteins 2 and 4 (IGFBP-2 and 4), and type A pregnancy-associated plasmatic protein (PAPP-A) mRNA in bovine immature oocytes fromBos Taurus and Bos indicus. Nellore and Holstein cows were submitted to ovum pickup, and the oocytes with grade 1, 2, and 3 were selected. To remove cumulus cells and pellucida zone, respectively, the oocytes were submitted to vortex (900 × g for 3 min) and protease treatment (Protease® from Streptomyces griseus, Sigma-Aldrich Corporation, St. Louis, MO, USA). Pools of 20 oocytes obtained from Nellore (n = 8) and Holstein (n = 4) cows were submitted to total RNA extraction (RNeasy, Qiagen, Valencia, CA, USA). The gene expression of target genes was measured by real-time RT-PCR with oligo-dT in the RT and bovine-specific primers in the PCR. Expression of cyclophlin A (CYC-A) was used as internal control. The means of mRNA levels of target genes between the breeds were compared using t-test and Mann-Whitney test when the data had normal or not normal distribution, respectively. The means values of mRNA expression of IGF-1, IGF-2, IGF receptors (1 and 2), and IGFBP 2 and 4 were greater in Holstein (0.96 ± 0.21, 0.74 ± 0.27, 1.08 ± 0.04, 1.19 ± 0.5, 1.21 ± 0.23, 0.53 ± 0.15, respectively) compared with Nellore (0.48 ± 0.10, 0.07 ± 0.02, 0.04 ± 0.03, 0.06 ± 0.02, 0.05 ± 0.01, 0.03 ± 0.15, respectively; P < 0.01). Never- theless, mRNA expression of PAPP-A was much greater in Nellore (28.10 ± 18.96) than in Holstein (1.32 ± 0.17; P < 0.05). These results suggest that high expression of PAPP-A and low expression of IGFBP-2 and 4 could allow more efficiency on the degradation of IGFBP and increase the IGF bioavailability in the oocytes from Nellore as compared with Holstein cows.

Satrapa, Simões, and Castilho received a fellowship and funding from FAPESP (São Paulo, Brazil).
25 ADDITION OF FSH, IN CONTRAST TO eCG, DOES NOT INCREASE PREGNANCY RATES IN ANESTROUS NELLORE (BOS INDICUS) COWS TREATED WITH FIXED-TIME AI PROTOCOL
Authors: Lima L. A. , Pinheiro V. G. , Cury J. R. , Barros C. M.
Published: August 2010

There are reports in the literature indicating that use of eCG improves pregnancy rates in Bos indicus anestrous cows treated with a progesterone-based fixed-time AI (FTAI) protocol. More recently, replacement of eCG by FSH in FTAI protocols was reported to have beneficial effects (Bos indicus) or no effect (Bos taurus) on pregnancy rates. In the present experiment the effects of eCG and FSH on pregnancy rates were compared in an FTAI protocol. Primiparous lactating Nellore cows (40 to 80 days postpartum, n = 421) with a body condition score of 2.5 to 3.0 (on a 1 to 5 scale) were randomly allocated in 3 groups: Control (CTR), eCG, and FSH. In the control group, all animals received a progesterone (P4)-releasing intravaginal device (1.55 g, PRID®, Ceva Sante Animale S.A., Libourne, France) and 2.5 mg of estradiol benzoate (EB, i.m. Estrogin®, Farmavet, São Paulo, Brazil), on Day 0 (D0). Eight days later (D8), at the time of the intravaginal device withdrawal, PGF2α (150 μg, D-cloprostenol, i.m. Prolise®, ARSA S.L.R., Buenos Aires, Argentina) was administered. Twenty-four hours after PRID removal, cows were treated with EB (1.0 mg, i.m.), and FTAI was done 30 to 36 h later. In the eCG and FSH groups, the cows were treated with 20 mg of FSH (Folltropin-V®, i.m. Bioniche, Belleville, Canada) or400IU of eCG (Novormon®, i.m. Syntex, Buenos Aires, Argentina), respectively, at the time of PGF2α administration. Ovarian ultrasonography (Aloka SSD 500, 7.5-MHz probe, Aloka, Tokyo, Japan) was performed 10 days prior to and at the beginning of FTAI protocol to select the animals in postpartum anestrous (absence of CL in both examinations). The pregnancy diagnosis was performed by ultrasonography 30 days after FTAI. Data were analyzed by logistic regression (SAS Institute, Cary, NC, USA). Cows treated with eCG had a higher pregnancy rate (41.5%, 95/229; P < 0.05) than those treated with FSH (22.7%, 22/97) or in the control group (26.3%, 25/95). Pregnancy rates of animals treated with FSH did not differ (P > 0.05) from those in the control group. These results indicated that, in lactating primiparous anestrous Nellore cows, the use of eCG in a FTAI protocol improves the pregnancy rate, whereas FSH has no beneficial effect.

V. G. Pinheiro received a fellowship from FAPESP (São Paulo, Brazil). The authors are grateful to Ceva Animal Health for providing the intravaginal devices (PRID®) used in this experiment.
255 THE ESTROGENIC EVALUATION OF PARABENS USING RAT UTERINE CALBINDIN-D9k
Authors: Vo T. T. B. , Jeung E. B.
Published: August 2010

In the current study, calbindin-D9k (CaBP-9k), a potent biomarker for screening estrogen-like environmental chemicals in vivo and in vitro, was adopted to examine the potential estrogen-like property of the following parabens: propyl-, isopropyl-, butyl-, and isobutyl-paraben. Immature female rats were administered for 3 days from postnatal day 14 to 16 with 17?-ethinylestradiol (EE, 1 mg/kg of body weight (BW) per day) or parabens (62.5, 250, and 1000 mg/kg of BW per day). In uterotrophic assays, significantly increased uterus weights were detected in the EE-treated group and in the groups treated with the greatest dose of isopropyl-, butyl- and isobutyl-paraben. In addition, these parabens induced uterine CaBP-9k mRNA and protein levels, whereas co-treatment of parabens and fulvestrant (Faslodex, formerly known as ICI 182, 780), a pure estrogen receptor (ER) antagonist, completely reversed the paraben-induced gene expression and increased uterine weights. To investigate the ER-mediated mechanism(s) by which parabens exert their effects, the expression level of ERÎ± and progesterone receptor (PR) was analyzed. Exposure to EE or parabens caused a dramatic decrease in expression of both ER? mRNA and protein levels, whereas co-treatment with fulvestrant reversed these effects. These data showed the difference of CaBP-9k and ER? expression, suggesting that CaBP-9k might not express via ER? pathway. In the effect of parabens on CaBP-9k expression through PR mediation, a significantly increased expression of uterine PR gene, a well-known ER regulating gene, at both transcriptional and translational levels was indicated in the greatest dose of isopropyl- and butyl-paraben. These parabens induced PR gene expression that was completely blocked by fulvestrant. This result indicates that CaBP-9k expression might involve PR mediates in the estrogenic effect of paraben in immature rat uteri. Taken together, parabens exhibited an estrogen-like property in vivo, which might be mediated by a PR and/or ER? signaling pathway. In addition, our results expanded the current understanding of the potential adverse effects of parabens associated with their estrogen-like activities. Further investigation is needed to elucidate in greater detail the adverse effects of parabens in humans and wildlife.
26 SPERM MOTILITY DECREASING AND SEMEN FERTILITY IN THE BULL EVALUATED BY BIOSPECKLE
Authors: Macedo R. S. , Barreto Filho J. B. , Braga Jr R. A. , Rabelo G. F.
Published: August 2010

Sperm motility decreasing in a semen sample over time is an indirect approach to assess spermatozoa viability and should be related to the ejaculate fertility. The biospeckle (BSL) is an interacting phenomenon between laser light and biological specimens that allows measurement of sperm kinetic activity by means of an index called the inertial moment (IM). The aim of this work was to evaluate sperm motility diminishing in frozen bovine semen with BSL and analyze semen fertility in relation to the motility decreasing behavior patterns showed by different semen samples. Fertility was assessed by beef heifer conception rates after AI. Semen of 6 mature IA donor bulls (Bos taurus indicus) was previously divided in 2 groups (group I: motility ≥50%; group II: motility <50%), each group comprised 3 animals. Semen was thawed at 37°C for 30 sin a water bath. One aliquot of 10 μL was placed in a warmed slide, covered with a slip, and evaluated by light microscopy and BSL just after thawing. Then, samples were kept at room temperature to induce a decrease in sperm motility. Each sample was illuminated 6 times by laser light at intervals of 2 min (n = 36) and IM values were obtained. The semen samples were used in an AI program of beef heifers (n = 166) under the same nutrition and management conditions. All inseminations were done by the same technician, and donor bulls were used consecutively throughout the breeding season. Pregnancy diagnosis was done by ultrasound images (Falco 100, 6 MHz, Pie Medical, Crawley, UK) 28 days after insemination. The SISVAR software was used in variance analysis and comparison of means by the Tukey test. Group I showed greater (P < 0.05) IM mean value (153.5 ± 27.48) compared with group II (107.94 ± 30.61), which means that this group had higher sperm motility during the time of evaluation. In addition, conception rates (0.63 ± 0.49) for group I were also higher (P < 0.05) compared with group II. Apparently semen fertility is related to the sperm kinetics measured by the IM. Mean IM values (134.0 ± 22.65 and 96 ± 22.34, groups I and II, respectively) obtained in the last illumination for each group did not differ (P > 0.05), but a tendency to differ was observed when a greater number of illuminations was done. Our data suggest that the BSL was capable of identifying spermatozoa surviving between high and low motility groups, and the sperm motility decreasing measured by the IM might be an objective approach to evaluate semen fertilization potential.

Financial support: FAPEMIG, grant EDT 94/07, CNPq.
269 THE USE OF A NEW EXTENDER FOR STORING FRESH BOVINE SEMEN FOR LONG PERIODS
Authors: Frers L. G. , Hepburn J. , Hogan K. , Parminter C. , Mc Gowan L. , Vishwanath R. , Beaumont S. , McDonald R.
Published: August 2010

Processing bovine semen in fresh long life extender for use over 3 to 4 days after collection is a widely used technique in New Zealand (Shannon and Vishwanath 1995 Anim. Reprod. Sci. 39, 1-10). Advantages include greater use of valuable sires, transport without liquid nitrogen, and the possibility of more efficient use of sexed sorted semen. The new extender (Ext. A) also has the advantage of containing no egg yolk. This study compares this new long-life extender (Ext. A) with an existing product (Ext. B) and frozen/thawed semen. Semen from 12 different bulls was diluted to a concentration of 8 × 106 mL-1 and gradually cooled to 16°C. All samples were held at ambient temperature in the dark and motility was evaluated over a storage period of 4 days comparing the extenders. In this part of the trial Ext. A maintained motility better than Ext. B (P = 0.001) during the 4-day storage period (24 h: 90 v. 70%; 96 h: 85 v. 50%). The second part of the trial compared the conception rates (CR) in cows from the use of fresh long-term-extended semen and frozen/thawed semen. On 19 farms, 8546 cows were inseminated with fresh semen stored for 1 to 3 days and 7280 cows were inseminated with frozen semen. The overall CR at 7 to 8 weeks for the 19 farms was 73.7%. On 18 farms within the same farming group, 8498 cows received frozen semen and the CR was 71.1%. Pregnancy results were 2.6% (P = 0.001) higher CR at scanning in herds where fresh semen was used compared with the farms where only frozen/thawed semen was used (73.7 v. 71.1%). In the third part of the trial, semen from 4 different bulls were extended to 1 × 106 mL-1 in Ext. A and held at ambient temperature for 6 days prior to use for IVF. Our lab standard frozen/thawed bull semen was used as a control. Table 1 shows that semen held at ambient temperature in Ext. A for 6 days produced a similar percentage of transferable quality embryos to our IVP control frozen/thawed semen (26.9 v. 25.7%). We conclude that preserved bovine semen in fresh long-life extender for several days offers some advantages in AI and IVP programs compared with frozen semen.

Table 1.Fresh semen extender (Ext.A) compared with frozen semen in IVP

We appreciate the assistance of Liberty Genetics Ltd.
275 EVIDENCE FOR α5 AND αV INTEGRINS ON SPERM PLASMA MEMBRANES AND THEIR ROLE IN BOVINE FERTILIZATION
Authors: Gonçalves R. F. , Bertolla R. P. , Barnabe V. H.
Published: August 2010

Sperm-egg interaction is a complex molecular process leading to gamete fusion mediated by a series of molecular interactions. Some integrin subunits, which are adhesion molecules, are expressed on human and mouse sperm, but major questions about the roles of integrins in sperm-oocyte fusion remain unsolved. This study was conducted to determine the presence of α5 and αV integrins on cattle (Bos indicus and Bos taurus) sperm, and whether fertilization might be affected by treating sperm with antibodies to these integrin subunits. To determine if integrin subunits were present on sperm, sperm plasma membrane proteins were subjected to 1-dimensional SDS-PAGE and Western blot analysis. Frozen-thawed sperm, donated by ABS Pecplan, were centrifuged at 700 × g for 10 min, washed twice with warm PBS (Nutricell®, Campinas, Sao Paulo, Brazil), and resuspended in Jones buffer (0.4% deoxyclolic acid, 8.9 M sucrose, 0.1 M Tris, pH 8.5) for 60 min at 4°C to solubilize sperm plasma membranes. Plasma membrane proteins were then separated by SDS-PAGE and transferred to nitrocellulose. The resulting blots were probed with αV integrin antibody (Calbiochem®, San Diego, CA, USA) or α5 integrin antibody (Calbiochem) and developed using ECL. Frozen-thawed spermatozoa were washed by a 45/90% layered Percoll gradient centrifugation and incubated for 1 h in fertilization medium (FM; 1), FM with anti-integrin αV IgG (2), and FM with anti-integrin a5 IgG (3). In vitro-matured cattle oocytes were incubated (39°C, 5% CO2 in air) with 1 × 105 washed, pretreated spermatozoa per 25 oocytes for 18 h. The oocytes were fixed in acid alcohol, stained with 1% acetate-orcein, and observed to determine the presence of pronuclei. Each experiment was repeated 4 times and data from each experiment were pooled. Approximately 80 to 90 oocytes per treatment for fertilization were evaluated in each replicate. Weighted least squares means were used to analyze fertilization data (SAS software, SAS Institute, Cary, NC, USA). The significance level for all tests was P < 0.05. Both antibodies for α5 (35 kDa) and αV (34 kDa) integrins showed immunoreactivity on Western blots of sperm membrane proteins. Addition of anti-integrin αV, and anti-integrin α5 decreased fertilization (P < 0.05) compared with the control: (1) 94.1 ± 1.0%; (2) 18.2 ± 1.0%; (3) 12.2 ± 1.0%. These findings show that αV and α5 integrins are expressed by cattle spermatozoa and may be involved in sperm-oocyte fusion and fertilization.

This study was supported by FAPESP grants (2007/00363-5 and 2006/06008-0, Brazil). We acknowledge Nutricell and ABS Pecplan for their generous contribution.
276 CORTICAL GRANULE MIGRATION AND PRONUCLEAR FORMATION IN BOVINE OOCYTES TREATED WITH BUTYROLACTONE I DURING TRANSPORT
Authors: Gottardi F. , ã B. , Mingoti G.
Published: August 2010

Butyrolactone I (BLI) can be used to maintain meiosis blocked during transport of oocytes to the laboratory, aiming to maintain their quality and competence. This work evaluated the effect of BLI during transport of bovine oocytes on the cortical granules (CG) distribution, and fertilization. Meiotic block culture was in basic medium (TCM-199 with 0.2 mM pyruvate, 25 mM sodium bicarbonate, and 75 mg mL-1 gentamicin) with 10 μLM BLI (B10) or 100 (AM BLI + 0.3% BSA (B100). During meiotic block, oocytes were cultured in cryogenic vials (15/vial) containing 60 μLL of medium recovered with mineral oil, packed in a portable incubator (Thawing Unit MT 35/42, Minitub) at 38.5°C during the first 5 h, and then transferred to a incubator at 38.5°C and 5% CO2 in air for the remaining 19 h. Then, they were matured at 38.5°C and 5% CO2 in basic medium supplemented with 0.6% BSA and hormones (maturation medium, MM) for 20 h. As controls, a group of oocytes was matured in MM during 24 h, without prior meiotic block. Control oocytes were cultured at 38.5°C and 5% CO2 (control 1, C1) or in the same portable incubator conditions in which the blocked oocytes were cultured (C2). A group of oocytes was fixed immediately after follicular aspiration (C0). At the end of cultures, a group of oocytes (n = 853) were stained with 1 μg mL-1 Lens culinaris (FITC-LCA) and assessed for the distribution of CG (arrangement in clusters = immature; peripheral = mature). The remaining oocytes (n = 563) were fertilized for 18 h, and zygotes were stained with 10 μg mL-1 Hoechst 33342 and assessed for sperm penetration and pronuclear formation. Oocytes with 2 pronuclei (2PN) were considered to present normal fertilization, and oocytes with >2PN were considered to be polyspermic. The data were analyzed by ANOVA and Tukey’s test, and correlation between CG positioning and pronuclear formation was analyzed with the Pearson correlation test. After meiotic block, the proportion of immature CG were higher (P < 0.05) in B100 (90.7%) and C0 (88.2%) than in B10 (69.0%). After maturation, mature CG rates in C2 (61.7%) were similar to B10 (52.3%) and B100 (72.9%), but only B100 was similar to C1 (83.6%). Irrespective of the presence or absence of BLI, culture of oocytes in the portable incubator adversely affected the normal pronuclear formation rates, which were higher (P < 0.05) in C1 (78.9%) than in C2 (56.8%), B10 (56.0%), and B100 (51.8%). The incidence of >2PN was higher (P < 0.05) in B10 (5.9%) than in C1, C2, and B100 (0.0, 1.4, and 0.0%, respectively). There was a fine correlation between mature CG and normal pronuclear formation (R = 0.5; P < 0.05). We conclude that (1) CG migration is reversibly blocked by BLI at a concentration of 100 μLM during transport; (2) oocyte transport in the portable incubator, but not BLI itself, adversely affects the normal pronuclear formation; and (3) CG maturation is essential for normal pronuclear formation.
Financial support: FAPESP.
289 EFFECTS OF YEAR PERIOD, TECHNICAL TEAM, AND BREED ON THE PREGNANCY RATES AFTER TRANSFER OF IVF BOVINE EMBRYOS IN THE SOUTHERN REGION OF RIO DE JANEIRO
Authors: Moura B. G. , Almeida J. , Lima F. L. , Balbi G. , Calmerani R. , Resende O. A. , Silveira D. L.
Published: August 2010

The aim of the work was to study the effects of year period, technical team, breed, beef cattle and dairy cattle on the pregnancy rates in fresh embryos used in bovine transfer of IVF programs. The study was carried out at the fertilization laboratory In Vitro Nyltta Britto de Carvalho, in partnership with In Vitro Brazil, located at the Boa Vista farm, Barra do Pirai, during August 2007 to September 2008, seeking subsidies to improve the use of the technique in the field. During that period, aspirations and inovulations in 3 different periods I (August to December), II (January to April), and III (May to September) were carried out. The jobs were accomplished by 9 technical teams (A, B, C, D, E, F, G, H, and I) rendering services to the laboratory, by working with 2 beef breeds (Brahman and Nelore) and 3 dairy breeds (Gir, Girolando, and Holstein). The different breed receivers were synchronized, and in general, from 6 to 8 days after heat, they received embryo transfer, the cervical way, under low epidural anesthesia, where each female received 1 fresh embryo of IVF. All cows were submitted to gestation diagnosis by rectal palpation and ultrasonography, in general, 42 days after embryo transfer. The numbers of embryo transferred and pregnancy rates were submitted to the chi-square test, which presented significant differences (P < 0.05). There were pregnancy rates of 36.25%a (n = 960), 39.83%a (n = 1180), and 32.59%b (n = 919) in the I, II, and III periods, respectively. Among the 9 technical teams, there were verified pregnancy rates (%) of 33.51d (n = 1313), 30.30d (n = 330), 35.00cd (n = 405), 39.24cd (n = 1060), 59.25a (n = 7), 33.33d (n = 24), 53.57bc (n = 28), 43.31c (n = 157), and 58.33ab (n = 12) for A, B, C, D, E, F, G, H, and I teams, respectively. Among breeds there were rates (%) of 36.89ab (n = 412), 34.68b (n = 1286), 35.13ab (n = 74), 38.94a (n = 1140), and 37.80ab (n = 82) for Brahman, Nelore, Gir, Girolando, and Holstein, respectively. In the study, pregnancy rates (%) of 35.21b (n = 1698) in beef cattle and 38.65a (n = 1296) in dairy cattle were observed. The differences in pregnancy rates with respect to the evaluated factors, may be explained by individual, breed, and nutritional variations of the animals. There are few data in the literature with results on the embryo transfer use of IVF bovine under field conditions.
295 ASSESSMENT OF CYTOTOXICITY AND INTERFERENCE OF ATELEIA GLAZIOVIANA IN BOVINE HERPESVIRUS TYPE 1 (BoHV-1) INTERACTION WITH IN VITRO-MATURED BOVINE OOCYTES
Authors: Pavão D. L. , Piccolomini M. M. , Góes A. C. , Harakava R. , Haraguchi M. , Dangelo M.
Published: August 2010

In vitro embryo production (IVP), as well as having a biotechnical importance, is a valuable tool for studies of gamete and/or embryo interaction with pathogens and xenobiotics. In consequence, it has become an excellent model not only for investigations about sanitary aspects, but also for aspects related to toxic processes. The aim of this study was to evaluate the effect of cytotoxic aqueous extract of Ateleia glazioviana and its interference on the interaction of bovine herpesvirus type 1 (BoHV-1) with bovine oocytes during the In vitro maturation (IVM) period. The statistical analysis of the experiments was made according to Student’s t-test (P < 0.05). The parameters used for this experiment were based on the morphological, physiological, and clastogenic action analysis of the bovine oocytes. The oocytes were collected from ovaries from slaughterhouse and divided into control group (G1, n = 214), a group infected with BoHV-1 (Los Angeles sample 105.5 TCID50 mL-1(G2, n = 210), a group exposed to the extract of A. glazioviana, 0.24 g mL-1; G3, n = 228), and a group simultaneously exposed to the virus and to the extract (G4, n = 210). For IVM, the oocytes were kept in TCM-199 supplemented with hormones and incubated at 38°C, 5%CO2, and 95% humidity for 24 h. The oocytes in G1 showed high expansion of the cumulus cells and ooplasm uniform in appearance; oocytes in G2 showed uniform but moderate expansion of cumulus cells and retraction of ooplasm; the G3 group showed low and irregular expansion with degeneration of cumulus cells and retraction of ooplasm with a granular aspect; and oocytes in G4 showed degeneration of cumulus cells, retracted and granular ooplasm. We observed maturation rates of 81.3% in G1, 31.0% in G2, 5.7% in G3, and 1.4% in G4. As for the clastogenic action analysis, an additional group of oocytes, named in natura (n = 210), was evaluated and presented 41.9% of comets class 0 (zero), 34.8% class I, 12.4% class II, 7.1% class III, and 3.8% class IV G1 (n = 211) presented 6.1% of comets class 0, 47.8% class I, 31.3% class II, 11.0% class III, and 3.8% class IV Oocytes belonging to G3 (217) presented 0.5% of comets class 0, 19.8% class I, 28.1% class II, 34.1% class III, and 17.5% class IV G2 (n = 229) presented 4.4% of comets class 0, 61.2% class I, 26.6% class II, 4.8% class III, and 3.0% class IV Oocytes in G4 (n = 206) presented 3.9% of comets class 0, 26.2% class I, and similar amounts of comets level II (23.8%), III (22.8%), and IV (23.3%). The statistical analysis presented a significant difference in the final results. Such results show the cytotoxic effect of A. glazioviana in bovine oocytes. The simultaneous exposure to the virus and the extract aggravated the effect of the virus, suggesting an increase of the pathogen within the gametic cell.
Vitrocel/Embriolife.