bluuebunny/crossref_metadata_2025 huggingface dataset:
139 INTRACELLULAR GLUTATHIONE CONCENTRATION AND IN VITRO DEVELOPMENT OF BOVINE OOCYTES SELECTED BY BRILLIANT CRESYL BLUE
Authors: Lee H. S. , Koo D. B. , Lee K. K. , Yin X. J. , Kong I. K.
Published: January 2008

During in vitro development of bovine embryos, a large proportion of embryos fail to develop to the blastocyst stage. Brilliant cresyl blue (BCB) stain determines the activity of glucose-6-phosphate dehydrogenase, an enzyme synthesized in growing oocytes. It was previously reported that BCB positive oocytes are better developed to the blastocyst stage than BCB-negative oocytes for IVF (Alm et al. 2005 Theriogenology 63, 2194–2205). The objective of this study was to compare the intracellular glutathione (GSH) level and the developmental competence of bovine oocytes selected by BCB. Bovine ovaries were obtained at a local slaughterhouse and cumulus–oocytes complexes (COCs) were collected from follicles. In this study, we only used COCs with a compact cumulus investment. The concentration of BCB was reported in a previous study (Rodriguez-Gonzalez et al. 2002 Theriogenology 57, 1397–1409). The COCs were exposed to 26 µm of BCB for 90 min at 38.5�C in humidified air. Treated oocytes were divided into BCB-positive (colored cytoplasm) and BCB-negative (colorless cytoplasm). The selected COCs (BCB-positive and negative COCs) were matured in TCM-199 supplemented with 10% FBS, and then inseminated using frozen semen (1 � 106 cells mL–1). Subsequently, presumptive zygotes were cultured in vitro in CR1-aa medium supplemented with 0.3% BSA. After 3 days of IVC, cleaved embryos were transferred to CR1-aa medium supplemented with 10% FBS and cultured for an additional 4 days at 38.5�C, 5% CO2 in air. Before (geminal vesicle (GV) stage) and after IVM (metaphase II (MII) stage), the COCs were denuded mechanically to prepare the samples for GSH assay, and then stored at –70�C until used. In the GV- and MII-stage oocytes, intracellular concentration of GSH was measured according to Mertens et al. (2005 Reprod. Domest. Anim. 40, 126–130). This experiment was replicated at least three times. GSH concentrations were analyzed by Student's t-test and the blastocyst formation rates were analyzed by chi-square analysis using the SAS 8.01 program (SAS Institute, Inc., Cary, NC, USA). Differences among treatment effects were considered significant at P < 0.05. The GSH concentration of BCB-negative oocytes (4.5 � 0.7 pmol/oocyte; n = 105) was significantly (P < 0.05) lower than that of BCB-positive oocytes (7.3 � 0.7 pmol/oocyte; n = 78) before IVM. After 24 h IVM, no differences in the GSH concentration were observed between the BCB-positive group (13.5 � 0.5 pmol/oocyte; n = 85) and the BCB-negative group (12.6 � 2.6 pmol/oocyte; n = 110). However, the percentage of embryos developed to the blastocyst stage in the BCB-positive group was significantly higher compared with that of the BCB-negative group (23.5 � 3.6 v. 9.8 � 2.1%, respectively; P < 0.05). Here, we concluded that intracellular GSH plays an important role in embryonic development to the blastocyst stage in vitro.
14 MORPHOLOGICAL AND FUNCTIONAL PARAMETERS OF ENDANGERED BERMEYA GOAT BREED SEMEN
Authors: Hidalgo C. O. , Rodríguez A. , Díez C. , Martín D. , Carbajo M. , Martínez A. , de la Fuente J. , Palasz A. T. , Benito J. M. , Tamargo C.
Published: January 2008

The Bermeya goats are an endangered autochthonous breed distributed in the north of Spain. To ensure their genetic diversity and long-term survival, morphological and functional parameters of the semen must be known in order to preserve the current genetic stock in a germplasm bank. The aim of this work was to establish basic characteristics and post-thaw survival of Bermeya goat's semen obtained by electro-ejaculation, that is not well described in the literature. The semen was collected by electro-ejaculation from 7 bucks, 1 to 7 years old, twice per week, for 9 weeks (n = 83). Fresh semen was evaluated for volume (V), concentration (C), motility, morphology, functional integrity of the sperm (spz) membranes (hypoosmotic swelling test; HOST), and acrosome integrity rate (NAR). Individual and progressive sperm motility were analyzed by means of a computer-assisted sperm analysis system (CASA: SCA 2002�, Microptic, Barcelona, Spain) immediately after dilution with the extender at 37�C, and after cooling to 4�C; five fields per sample (diluted to 204 � 106 spz mL–1) were evaluated under a phase contrast microscope (100�). The NAR and morphological abnormalities of sperm head, midpiece, tail, and cytoplasmic droplets were determined by counting 100 spz under 1000�. For freezing, ejaculates with at least 80% motile spz were diluted at 32�C with Krebs-Ringer solution containing 20% egg yolk and 14% glycerol to a final concentration of 400 � 106 spz mL–1, cooled to 4�C for 90 min, aspirated into 0.25-mL plastic straws (IMV�, L'Aigle, France), frozen at 7 cm above liquid nitrogen (LN2) phase for 10 min, and then plunged into the LN2. Straws were thawed in a water bath at 39�C for 30 s for post-thaw survival analysis. Data were analyzed by the GLM and FREQ procedures (SAS; SAS Institute, Inc., Cary, NC, USA) and expressed as means � standard error. Fresh semen characteristics were: V = 1.7 � 0.1 mL; C = 2619 � 106 � 153 spz mL–1; total and progressive motility were 89.0 � 2.1% and 66.9 � 2.1%, respectively. Percentages of head abnormalities were 4.8 � 0.5; midpiece: 3.8 � 0.7; tail: 4.7 � 1.0; cytoplasmic droplets: 8.3 � 0.7; intact acrosome: 91.8 � 0.6; and membrane integrity: 49.2 � 2.1. At 4�C, the % of total motile spz was 62.6 � 1.6, and the post-thaw survival rate was 46.3 � 1.5. There were only individual differences (P < 0.001) between bucks on sperm concentration, head abnormalities, and cytoplasmic droplets. In conclusion, our results indicate that semen quality is related to each individual animal and that electro-ejaculation allows collection of semen of satisfactory quality to use as fresh and for cryopreservation. However, the validity of our results for possible future sperm banking of endangered Bermeya goats semen must be confirmed by field trials.
150 EXAMINATION OF ESTRUS SYNCHRONIZATION USING A PRID® + PROSTAGLANDIN F2α AND PREGNANCY RATE AFTER ARTIFICIAL INSEMINATION FOLLOWING TRANSFER OF IN VITRO-PRODUCED EMBRYOS IN REPEAT-BREEDING COWS
Authors: Urakawa M. , Ideta A. , Hayama K. , Tsuchiya K. , Aoyagi Y.
Published: January 2008

Repeat-breeding Holstein cows (RBCs), i.e. cows that have undergone three or more AIs, have reduced pregnancy rates. The present study aimed to determine whether application of a combination of a PRID� (ASKA Pharmaceutical Co., Inc., Tokyo, Japan) + prostaglandin F2α (PGF2α) for estrus synchronization and AI + embryo transfer (ET) would improve pregnancy rates in RBCs. All RBCs selected showed a normal interval between the previous insemination and estrus, and were free of clinical disorders. The RBCs (n = 107) received a PRID for 9 days with a capsule containing 10 mg of estradiol benzoate, 0.5 mg IM of cloprostenol (PGF2α, Resipron�-C; ASKA, Japan) at 2 days before PRID removal. The cows were inseminated with frozen–thawed semen from a bull at 0 to 12 h after onset of estrus. Seven days after AI, single in vitro-produced embryos were transferred to the same RBCs. Pregnancy was diagnosed by palpation per rectum at e60 days post-insemination. Exp. 1 estimated the response to estrus synchronization treatment (n = 107). The factors examined were lactation number (mean � SD: 2.3 � 1.5, range: 1–6), insemination number (5.2 � 2.2, range: 3–13), and the interval from calving to next AI (275.9 � 97.6 days, range: 93–650 days). Exp. 2 compared the pregnancy rate in control RBCs (n = 161) and PRID-treated RBCs (n = 100). The factors examined included lactation number (2.5 � 1.6, range: 1–12), insemination number (6.0 � 2.5, range: 3–19), the interval from calving to next AI (288.0 � 102.1 days, range: 102–797 days), and whether the ET was into the uterine horn ipsilateral or contralateral to the CL in a total of 261 RBCs. Data were analyzed using logistic regression. The results for Exp. 1: 103 cows (96.3%) were confirmed to be in estrus (2.0 � 0.3 days after PRID removal). Although the frequency of estrus was affected by the lactation number (P = 0.014) and the interval from calving to the first AI (P = 0.005), the AI number did not have an affect (P = 0.624). Exp. 2: PRID synchronization had no effect on pregnancy rate (control: 37.3%, 60/161; and PRID-treated: 40.0%, 40/100). For the RBCs, the pregnancy rate was not affected by lactation number (P = 0.093), AI number (P = 0.694), or the interval from calving to next AI (P = 0.762). No significant difference (P = 0.428) in pregnancy rate was observed in cows when the embryos were transferred into the uterine horn contralateral to the CL (43.5%, 20/46), compared to those embryos transferred into the uterus horn ipsilateral to the CL (37.2%, 80/215). In conclusion, our findings suggest that the pregnancy rate may be improved by considering lactation number and the interval from calving to the next AI of RBCs in estrus synchronization using a combination of PRID + PGF2α and AI + ET.
152 TRANSMISSION OF BOVINE VIRAL DIARRHEA VIRUS BY IN VITRO-FERTILIZED EMBRYOS UNDER EXPERIMENTAL CONDITIONS
Authors: Bielanski A. , Algire J. , Lalonde A.
Published: January 2008

Bovine viral diarrhea virus (BVDV) infection affects cattle throughout the world. It causes significant economic losses in the cattle industry. The potential for transmission of a cytopathic biotype of BVDV by in vivo-derived embryos has been thought to be negligible. However, there is no study to prove non-transmission of the most common field isolate of noncytopathic biotype (NCPB) of BVDV by IVF embryos. Here we report on the preliminary outcome of embryo transfer (ET) of IVF embryos exposed in vitro to type-1 (NY-1) and type-2 (P-131) genotypes of NCPB of BVDV. For this experiment, IVF embryos were generated using standard methods which briefly involve: maturation of cumulus–oocyte complexes in TCM medium, fertilization of oocytes with BVDV-free semen, and culture of zygotes to the blastocyst stage in SOF medium without somatic cells. Day 7 blastocysts were exposed for 1 h to NY-1 or P-131 (103–107 TCID50 mL–1) BVDV strains before being washed (without trypsin) as recommended by IETS. Two embryos were transferred on each occasion. Embryo recipients were virus-free and anti-BVDV antibody-free prior to ET. The recipients remained individually in isolation premises after ET. In total, 126 ET procedures were performed resulting in 57 pregnancies and 34 calves born free of the infectious virus and BVDV antibodies (5 pregnancies are still pending). In total, 23 pregnancies were lost after 30 days. Exposure of embryos to type-2 BVDV resulted in a loss of 46% (17/37) of pregnancies after 30 days post-ET and 20 recipients seroconverted to BVDV. Within seroconverted and pregnant animals (n = 14), only 2 recipients maintained pregnancy and delivered uninfected calves at term. In contrast, exposure of embryos to type-1 caused 30% (6/20) of the pregnancy losses after 30 days and did not cause any seroconversion in ET recipients. After washing, 33% (3/9) and 38% (17/44) single embryos from the infected pool of IVF embryos tested positive for the BVDV. In conclusion, under these experimental conditions, a proportion of recipients was apparently infected after receipt of BVDV-exposed embryos. However, all of the calves that survived to term were BVDV-free and anti-BVDV antibody free.
154 THE QUANTITY OF BOVINE VIRAL DIARRHEA VIRUS ASSOCIATED WITH SINGLE ZONA PELLUCIDA-INTACT IN VITRO-PRODUCED BOVINE EMBRYOS FOLLOWING ARTIFICIAL EXPOSURE
Authors: Gard J. A. , Givens M. D. , Galik P. K. , Riddell K. P. , Marley M. S. D. , Stringfellow D. A. , Edmondson M. A. , Whitlock B. K.
Published: January 2008

The primary objective of this study was to determine the percentage of individual, preimplantation, in vitro-produced bovine embryos which maintained association with virus despite washing following artificial exposure to a high affinity strain of bovine viral diarrhea virus (BVDV). Another objective of this study was to determine the quantity of virus associated with these embryos. A total of eighty-seven zona pellucida-intact, Day 7, in vitro-produced bovine embryos were exposed for 1 h to 2 � 106 cell culture infected doses per mL to the 50 percent endpoint (CCID50 mL–1) of a type 1 noncytopathic strain of BVDV (SD-1). Following exposure, the embryos were washed according to International Embryo Transfer Society standards for in vitro-produced bovine embryos; they then underwent sonication, RNA extraction, and freezing at –80�C until assayed for virus. A real-time quantitative polymerase chain reaction (QPCR) was run in duplicate on each of the 87 embryos. Forty-two percent (39/87) of the embryos assayed were determined to be positive for virus. The quantity of virus associated with the embryos averaged 0.55 viral copies per 5 µL (SD = 0.89 copies/5 µL, SEM = 0.14 copies/5 µL). Assessment of data using tolerance intervals (P = 0.05) indicates that 90% of contaminated embryos were associated with ≤2.40 viral copies per 5 µL while 99% of contaminated embryos were associated with ≤3.44 viral copies per 5 µL. These findings show that there is a low level of virus associated with in vitro-produced embryos but virus is associated with a significant number of exposed embryos. In conclusion, this study indicates that the potential for transmission of BVDV via embryo transfer of in vitro-produced embryos is small given the amount of virus that was found to associate with individual embryos.
156 INVESTIGATION OF BOVINE HERPESVIRUS IN CUMULUS - OOCYTE COMPLEXES AND FOLLICULAR FLUID
Authors: Oliveira A. P. , Leite R. C. , Heinmam M. B. , Siqueira L. G. B. , Maciel A. , Viana J. H. M.
Published: January 2008

The world market for bovine embryos has increased in the past few years. However, sanitary problems such as foot and mouth disease in Brazil, vesicular stomatitis in South America, and bovine spongiform encephalopathy (BSE) in North America and Europe have increased concerns regarding the risk of introducing exotic diseases and/or more virulent serotypes of endemic diseases by embryo transfer. Many countries are trying to develop and/or improve new techniques for infectious disease detection, with the scientific basis to support the import and export of animal germplasm. Therefore, the epidemiology of the diseases and the interaction between pathogens and cumulus–oocyte complexes (COCs), embryos, and semen must be investigated. Despite the many studies that have been carried out to evaluate the possibility of transmission of infectious agents by the embryo, few data are available regarding COC susceptibility (Tsuboi et al. 1992 J. Vet. Med. Sci. 54, 1179–1181). The aim of this study was to evaluate the presence of bovine herpes virus serotype 1 (BHV-1) in COCs and follicular fluid (FF) collected from naturally infected animals in a low stress condition. Blood samples of non-lactating Gyr breed (Bos indicus) cows were collected and evaluated for BHV-1 antibodies by the serum neutralization microplate test, performed as described in the Manual for Standards for Diagnostic Tests and Vaccines (OIE, 1992). The cows were diagnosed as serologically positive (n = 38) or serologically negative (n = 8), and kept under grazing in Brachiaria decumbens pasture with mineral supplementation. The cows considered as positive showed titers greater than 1/4. COCs and follicular fluid (FF) were obtained by ovum pick-up (OPU) using sterile and disposable materials for each animal. Virus detection was performed by the PCR technique. PCR sensitivity was made using COCs and FF recovered from eight BHV-1 serologically negative animals. These samples were either artificially infected on plates with 106.5 TCID in 50 µL of IBR Colorado 1 reference serotype (ATCC, VR-864) or used as a negative control. The PCR analitical sensitivity was 100.5 TCID. The presence of BHV-1 in COCs and FF was not detected in any of the animals, despite the high sensitivity of the PCR technique. In the present in vivo model, results show that COCs collected from serologically BHV-1 positive cows presenting no clinical signs of the illness and managed in a low stress condition could be used as donors for in vitro fertilization procedures with minimal sanitary risks. Also, the absence of the virus in COCs and FF cannot be used as a predictor of BHV-1 infection status in bovine herds.
164 EFFECT OF OVULATION-INDUCING FACTOR (OIF) ON OVARIAN FUNCTION IN CATTLE
Authors: Van Steelandt M. D. , Tanco V. M. , Ratto M. H. , Adams G. P.
Published: January 2008

Systemic administration of ovulation-inducing factor (OIF), discovered recently in seminal plasma of llamas, alpacas (induced ovulators), and cattle (spontaneous ovulators), stimulated ovulation in >90% of female llamas and alpacas. The objective of the present study was to test the hypothesis that purified OIF from llama seminal plasma would induce ovulation in cattle. Peripubertal heifers, weighing 323 � 27 kg, were used to minimize the confounding effect of spontaneous ovulation. Heifers (n = 11/group) were treated intramuscularly with 1.0 mg/100 kg of purified OIF, 100 µg of GnRH (positive control), or 2.5 mL of phosphate-buffered saline (negative control). Ovarian dynamics were monitored daily by transrectal ultrasonography for 10 days post-treatment. Blood samples were collected at 0.5- to 1-h intervals for 8 h, beginning at the time of treatment. Ovulation occurred in 9/11 (82%) of GnRH-treated heifers and in 1/11 (9%) heifers in each of the OIF- and saline-treated groups (P < 0.05). A surge in plasma LH concentration was detected within 30 min of treatment in the GnRH group (2.2 � 0.1 ng mL–1; P < 0.05), but remained at the basal level in the OIF- and saline-treated groups (0.3 � 0.1 and 0.2 � 0.1 ng mL–1, respectively). The onset of regression of the dominant follicle present at the time of treatment was earlier (P < 0.05) in OIF- v. saline-treated heifers (3.1 � 0.6 days v. 6.0 � 0.7 days). The interval from treatment to follicular wave emergence was shorter (P < 0.05) in GnRH- and OIF-treated heifers than in those treated with saline (1.1 � 0.4 days, 1.5 � 0.3 days, and 3.1 � 0.3 days, respectively). A similar pattern was observed for emergence of the second follicular wave (5.1 � 0.7 days, 4.6 � 0.5 days, and 6.6 � 0.4 days, respectively). Purified OIF did not induce ovulation in heifers but hastened both the regression of the extant dominant follicle and follicular wave emergence. Results provide a rationale for the hypothesis that OIF from seminal plasma is involved in controlling follicular wave dynamics in spontaneously ovulating species (e.g., Bos taurus) through a suppressive effect on the dominant follicle. The mechanism of action on ovarian follicular wave dynamics, as well as species specificity, remains to be elucidated.
170 EXPRESSION PATTERN OF THE Sox2 GENE IN BOVINE OOCYTES AND IN VITRO-DERIVED EMBRYOS
Authors: Brevini T. A. L. , Antonini S. , Cillo F. , Pennarossa G. , Colleoni S. , Lazzari G. , Galli C. , Gandolfi F.
Published: January 2008

Sox2 is a member of the Sox (SRY-related HMGbox) family. It acts to maintain developmental potential and marks the pluripotent lineage of the early mouse embryo; in particular, as in the case of Oct-4 and Nanog, Sox2 is expressed specifically in the inner cell mass (ICM) and in the epiblast of this species. Moreover, it plays an important role in the transcription network that maintains stem cell pluripotency, interacting with other factors such as Oct-4 and Nanog. Little information is available on this gene in bovine; therefore aims of the present study were: a) to identify and characterize the Sox2 expression profile in bovine oocytes and preimplantation embryos; and b) to investigate its expression pattern in ICM and trophectoderm (TE). Bovine oocytes and embryos were obtained by in vitro maturation and fertilization; blastocysts at Day 7 post-insemination underwent microsurgery to separate TE from ICM. mRNA was isolated from 3 pools, each consisting of 5 MII oocytes, 2-, 4-, 8-, and 16-cell embryos, morulae, blastocysts, ICMs, and TEs. Semi-quantitative analysis of Sox2 expression was performed in the exponential phase of PCR amplification using rabbit globin as exogenous control. Data were analyzed with one-way ANOVA, followed by multiple pairwise comparisons with Tukey test (SigmaStat 2.03, SPSS, Inc., Chicago, IL, USA). Values are presented as mean � SEM and differences of P ≤ 0.05 are considered significant. In order to rule out false negative results, PCR amplifications of isolated ICMs and TEs were extended to the plateau phase. Fragment identity was confirmed by sequencing. Comparison of bovine Sox2 cDNA sequence (EMBL AM774325) with databases revealed a 98%, 93%, and 87% homology with sheep, human, and mouse, respectively. Sox2 mRNA was detectable in oocytes as well as in embryos at the different developmental stages analyzed. Semi-quantitative expression studies revealed that Sox2 was present as both maternal and embryonic transcript; in particular, a statistically significant increase from the 8-cell stage, concomitant with embryo genome activation, was observed. Differently from the mouse, Sox2 was expressed in both bovine ICM and TE, resembling the profile previously shown for Oct-4 (van Eijk et al. 1999 Biol. Reprod. 60, 1093–1103), and suggesting that Sox2 expression might be regulated by Oct-4 also in bovine, as described in mouse and human. These findings also suggest that its expression may become restricted to the ICM only at the expanded hatched stage, as previously described for Oct-4 in pig embryos (Vejlsted et al. 2006 Mol. Reprod. Dev. 73, 709–718).
This work was supported by PRIN 2006, FIRST 2005, TECLA-MIUR, and EUROSTELLS-ESF.
182 OSTEOPONTIN GENE EXPRESSION IN IMMATURE AND MATURE SWINE CUMULUS CELLS AND OOCYTES
Authors: Monaco E. , Lima A. , Wilson S. , Kim D. , Bionaz M. , Hurley W. L. , Wheeler M. B.
Published: January 2008

Osteopontin (OPN) is an acidic single-chain phosphorylated glycoprotein found in both the female and the male reproductive tract that is believed to facilitate reproduction. It was recently reported in swine that OPN improves in vitro fertilization (Hao et al. 2006 Biol. Reprod. 75, 726–733). In bovine, OPN improves the efficiency of in vitro embryo production, and it has been detected on the zona pellucida (ZP) of immature and mature oocytes (Monaco et al. 2007 J. Anim. Sci. 85, 529 abst). This study was designed to evaluate the gene expression of OPN (SPP1) in immature and mature swine cumulus cells and oocytes. Ovaries from a local slaughterhouse were collected on three different days. Half of the immature cumulus–oocyte complexes (COCs) from each day were vortexed in 1 mL TCM-199 HEPES containing 10 mg mL–1 hyaluronidase to separate immature cumulus cells, while the other half of the COCs were maturated for 40-42 h and then the cumulus cells were separated. RNA from cumulus cells and oocytes was extracted and genomic DNA was removed by DNase treatment. cDNA was synthesized using 100 ng of RNA and diluted 50% with DNase–RNase-free water. The RT-PCR product from cumulus cells and oocytes was run in a 2% agarose gel stained with ethidium bromide to verify the presence of SPP1. Relative mRNA abundance between immature and mature cumulus cells was assessed by SYBR green real-time RT–PCR run in triplicate using a 6-point twofold dilution standard curve. GAPDH was used as internal control. Primers for SPP1 and GAPDH were designed spanning an exon/exon junction. The presence of a single and specific PCR product was assessed by gel electrophoresis (a single band was expected at 100 and 90 bp, respectively, for SPP1 and GAPDH), dissociation curve, and sequencing. A t-test was used to assess differences between immature (n = 3) and mature (n = 3) cumulus cells. SPP1 was detected in agarose gel in both immature and mature oocytes and cumulus cells. Cumulus cells presented a Ct for SPP1 ranging from 20 (immature) to 29 (mature). Immature cumulus cells showed a 32-fold larger mRNA abundance compared to the mature cumulus cells (P < 0.05) when data were transformed using a standard curve. Results showed expression of SPP1 in porcine oocytes and cumulus cells. However, maturation significantly decreased the expression of SPP1 in cumulus cells. The presence of SPP1 mRNA in oocytes and cumulus cells and the larger mRNA abundance before maturation may suggest a role of this protein prior to maturation of oocytes. Additional studies will be required to determine the specific role of SPP1 in oocyte maturation in the pig.
189 microRNA EXPRESSION PROFILING DURING BOVINE OOCYTE MATURATION
Authors: Tesfaye D. , Worku D. , Salilew-Wondim D. , Hoelker M. , Rings F. , Un C. , Schellander K.
Published: January 2008

MicroRNAs (miRNAs) are members of noncoding small RNAs (~21–25 nucleotides long) that inhibit translation and/or induce degradation of protein-coding mRNAs that contain complementary sequences to miRNAs by interacting with the 32 untranslated region. They are known to influence the expression of hundreds of genes in numerous biological processes including development. In order to get insight into the regulation of miRNAs during bovine early development, we applied a heterologous approach to identify and predict bovine miRNAs differentially regulated during oocyte maturation. For this purpose, the miRCURYTM locked nucleic acids (LNA) array (Exiqon, Vedbaek, Denmark), consisting of 454 capture probes for human, mouse, and rat miRNAs, as registered and annotated in the miRBase release 8.0 at TheWellcomeTrust Sanger Institute, was used to compare the miRNA expression profile between bovine immature and matured oocytes. Total RNAs with miRNAs were isolated from three independent pools (each containing 200 oocytes) of immature and in vitro-matured oocytes using an miRNeasy mini kit (Qiagen, Hilden, Germany). The isolated miRNAs were amplified using a Global MicroRNA amplification kit (System Bioscience LLC, Mountain View, CA, USA) to obtain enough targets for hybridization. The RNA samples from immature and matured oocytes were labelled with either Hye3 or Hye5 dyes and hybridized on three independent slides. After 16 h of incubation at 60�C, slides were sequentially washed and scanned using an Axon GenePix 4000B scanner (Axon Instruments, Foster City, CA, USA). Species-specific GenePix� Array Lists (GAL; Molecular Devices Corp., Sunnyvale, CA, USA) files of human, mouse, and rat were used independently. Following normalization using GProcessor 2.0a, probes with an average value of ≥1.5-fold change were considered to be differentially regulated. From the total miRNA probes (454) spotted on the array, about 400 were detected in bovine oocytes. Analysis of the array data using a human, mouse, and rat files indicated that a total of 33, 46, and 76 clones were differentially regulated during oocyte maturation, respectively. Of these, 11 miRNAs were found to come out as differentially regulated in all three array analyses. Accordingly, immature oocytes were found to be enriched with miR-208, miR-125a, miR-130b, miR-25, miR-375, and miR-382, while mature oocytes were enriched with miR-122a, miR-101, miR-206, miR-515-5P, and miR-101. The candidate miRNAs were predicted to target mRNA products of genes related to growth regulating proteins, transcription, and tumor necrosis factors. Investigation of the corresponding target mRNAs and protein products during oocyte maturation will enable insight into miRNA-controlled regulation of maternal gene expression.
193 HUMAN CYTOMEGALOVIRUS PROTEIN US11 DOWN-REGULATES THE EXPRESSION OF SWINE LEUKOCYTE ANTIGEN-I IN MINIPIG CELLS
Authors: Yoo J. Y. , Choi K. M. , Hong S. P. , Han G. S. , Kim E. J. , Kim S. H. , Park Y. C. , Seol J. G. , Park K. W.
Published: January 2008

The CD8+ cytotoxic T lymphocyte (CTL)-mediated immune response is important in porcine xenotransplantation, and pigs could be used as a good model for organ donor if these immunological barriers are overcome. Human cytomegalovirus (HCMV) encodes unique short (US) 11 gene, which interferes with cellular immune responses by inducing rapid degradation of newly synthesized heavy chains of major histocompatibility (MHC) class I. The destruction of heavy chains by US11 helps the virus to hide from recognition by cytotoxic T lymphocytes at early stage. In this study we demonstrated the inhibitory effect of US11 on the cytotoxicity of CTL cells by down-regulation of swine leukocyte antigen (SLA)-I expression. We established five US11 clonal cell lines by transfection into minipig fetal fibroblasts and confirmed the integration of US11 gene by PCR and Southern blot assay. The reduction of SLA-I, which was expressed on the cell surface, was also detected by flow cytometry assay. The level (14.6% to 21.2%) of SLA-1 expression in US11 clonal cell lines was decreased relative to that in the control. In the CTL assay, the rate of CD8+ T cell-mediated cytotoxicity was significantly reduced to 31.9 � 11.3%, compared to that of the control (81.4 � 5.3%; P < 0.01; n = 4). These results indicate that HCMV viral protein US11 can effectively suppress the presentation of SLA-I in pig fetal fibroblast cells.
This work was supported by a grant (Code # 20070101034005) from the BioGreen 21 Program, Rural Development Administration, Republic of Korea.
201 LIVER AND KIDNEY FUNCTION IN BOVINE FETUSES FROM EMBRYOS PRODUCED IN VIVO OR IN VITRO
Authors: Farin P. W. , Malarkey D. E. , Alexander J. E. , Farin C. E.
Published: January 2008

Abnormal offspring syndrome can occur in fetuses and calves resulting from embryos produced in vitro or by nuclear transfer procedures. This study was conducted to determine the effects of in vitro embryo culture on fetal biochemistry profiles and histology of the liver and kidneys during late gestation. Embryos were produced in vivo by using superovulated cows (In Vivo) or in vitro by using a serum-containing culture system (In Vitro) as previously described (Miles et al. 2004 Biol. Reprod. 71, 1919–1926). Single blastocysts from each embryo production system were transferred nonsurgically into heifers. On Day 222 of gestation, fetuses from the In Vivo group (n = 12) and the In Vitro group (n = 12) were recovered in utero. Samples of fetal serum were collected for biochemical analysis. Samples of liver and kidney were prepared for histological evaluation. Stereological methods were used to determine the volume density of hepatocytes as well as kidney glomeruli and kidney tubules. Fetuses from the In Vitro group were heavier (P = 0.03) than those from the InVivo group (17.3 � 1.0 kg and 20.7 � 1.0 kg for InVivo and InVitro, respectively; least squares means � SEM). Liver and paired kidney weights per kilogram of body weight did not differ (P ≥ 0.10) with treatment (26.4 � 0.6 g kg–1 v. 27.6 � 0.6 g kg–1 and 7.8 � 0.5 g kg–1 v. 9.1 � 0.5 g kg–1 for liver and kidney, respectively). In addition, there was no effect of treatment on the volume densities of hepatocytes, kidney glomeruli, and kidney tubules. However, compared with the In Vivo group, fetuses from the In Vitro group had increased (P ≤ 0.02) concentrations of blood urea nitrogen (BUN; 13.8 � 1.8 mg dL–1 v. 19.8 � 1.8 mg dL–1) and BUN:creatinine ratio (4.6 � 0.8 v. 7.9 � 0.8). No differences were observed between the In Vivo and In Vitro groups for serum levels of creatinine, total bilirubin, total protein, albumin, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, glucose, insulin, and insulin-like growth factor-I. In summary, compared with bovine fetuses from in vivo-produced embryos, fetuses from in vitro-produced embryos had increased body weight, normal liver and kidney morphology, and increased concentrations of BUN during late gestation.
Supported by the State of North Carolina.
210 COMPARISON OF A RECOMBINANT TRYPSIN v. THE PIG PANCREATIC EXTRACT ON IN VITRO-PRODUCED BOVINE EMBRYOS
Authors: Mattson K. J. , Devlin B. R. , Loskutoff N. M.
Published: January 2008

According to the Manual of the International Embryo Transfer Society, trypsin can be used to remove certain pathogenic agents from in vivo-derived embryos. Research is currently in progress to determine whether trypsin can also remove pathogenic agents from semen. The original research on embryos involved the use of trypsin from pig pancreatic extracts. Because of stricter guidelines from international regulatory agencies on the use of animal products, several recombinant serine protease products are now becoming available. TrypZean (Sigma, St. Louis, MO, USA) is a recombinant developed from corn and is the first bovine sequence recombinant trypsin to contain no animal by-products. As part of our ongoing research on the effects of trypsin on sperm, the goal of this investigation was to examine the development of bovine embryos produced from sperm treated with the recombinant TrypZean compared with pig pancreas trypsin (Sigma) and a control (no trypsin). Oocyte aspiration, maturation, fertilization, and embryo culture were performed using standard methods in 9 replications. Semen was collected and pooled from Bos taurus and frozen in an egg-yolk cryodiluent (Biladyl, Minitube, Verona, WI). The semen was processed using density gradient centrifugation composed of 1 mL of 30% Percoll (Sigma), layered over 2 mL of 45% Percoll containing either 0.25% TrypZean (n = 972 oocytes), 0.25% trypsin (n = 1040 oocytes), or no trypsin for the control group (n = 1024 oocytes). The bottom layer for the 2 treatments and control was 2 mL of 90% Percoll containing 10 µg mL–1 of soy-based protease inhibitor (Sigma). The density gradients were centrifuged at 700g for 30 min, after which time the pellets were washed in 5 mL of prewarmed TL HEPES Solution (Cambrex) and centrifuged at 300g for 10 min. The resulting sperm pellets were then resuspended in a volume calculated to provide 1 � 106 sperm mL–1 for in vitro insemination. The results were compared using one-way ANOVA. There were no statistically significant differences (P > 0.05) between any of the measures of embryonic development for the control and either of the treatment groups. Cleavage rates were measured for TrypZean (n = 689, 70.9%), trypsin (n = 729, 70.1%), and the control (n = 757, 73.9%) groups. More embryos reached the morula to blastocyst stages with the TrypZean (n = 367, 53.3%) and trypsin (n = 389, 53.4%) groups than the control (n = 369, 48.7%) group; however, these differences also were not statistically significant (P = 0.91) because of the large variation within the groups. In conclusion, the TrypZean and pig pancreas trypsin treatments of sperm prior to insemination showed no detrimental effects on IVF-derived bovine embryo development.
216 IN VITRO CAPACITATION OF FROZEN/THAWED STALLION SPERM
Authors: Spizziri B. E. , Graham J. K. , Squires E. L. , Hudson M. R.
Published: January 2008

The inability of stallion sperm to capacitate in an in vitro setting has limited many equine assisted reproductive technologies such as gamete intrafallopian transfer (GIFT) and IVF. These experiments were conducted to develop methods for artificially capacitating frozen/thawed stallion sperm, using dilauroylphosphatidylcholine (PC-12), calcium ionophore A23187 (IONO), or methyl-β-cyclodextrins (MBC) so that sperm could be used in equine assisted reproductive technologies. Assisted reproductive technologies need to be established to maximize the number of offspring produced with cryopreserved spermfrom stallions with low cryosurvival rates or that have a very limited number of frozen sperm. Low concentrations of lysophosphatidylcholine (LPC) induce the acrosome reaction in capacitated sperm and, therefore, were used to detect fully capacitated sperm treated with PC-12, IONO, or MBC. Ejaculates from 8 stallions were diluted in a modified Tyrode's medium and centrifuged (1000g for 11 min). The sperm pellets were suspended to 400 million sperm mL–1 in EZ-Freezin LE, packaged into 0.5-mL straws, and frozen in liquid nitrogen vapor. Straws were thawed in a 37�C water bath for 30 s and washed through 35% Percoll (400g for 4.5 min) to remove egg yolk particles and seminal plasma. The sperm pellets were suspended in Medium 199 (0.6% BSA and 5 mm calcium chloride) to 50 million sperm mL–1, and stained with propidium iodide and FITC-peanut agglutinin (PNA) (1 mg mL–1 each; viability and acrosome reaction detection, respectively). Sperm were treated with PC-12 (13, 17, 20, 30, 40 µm), IONO (0.5, 1, 1.5, 2 µm), or MBC (0.4, 0.6, 0.8, 1 µm) and incubated in a 37�C water bath (20, 15, 20 min) to artificially capacitate sperm. Sperm were then challenged with LPC (2 or 5 µg), for 10 additional min, and analyzed by flow cytometry to determine the percentages of acrosome-reacted sperm. Data were analyzed by ANOVA and treatments were separated by Student-Newman-Keul's test. The PC-12 (20, 30, 40 µm), IONO (1.5, 2 µm), and MBC (0.4, 0.6, 0.8, 1 µm) artificially capacitated sperm equally well (59 to 70%, 52 to 59%, and 48 to 55%, respectively) and were higher than control sperm (13%, SEM � 9; P < 0.05). Sperm viability was similar at all IONO or MBC concentrations; however, PC-12-treated sperm (17 to 40 µm) had significantly lower sperm viability when compared with control (24 to 28% and 35%, respectively, SEM � 3; P < 0.05). In conclusion, frozen/thawed stallion sperm can be artificially capacitated by treating with PC-12, IONO, or MBC, and this may lead to practical applications for in vitro equine assisted reproductive technologies.
224 A COMPARATIVE STUDY BETWEEN WOOD AND PLAINS BISON
Authors: Lessard C. , Danielson J. , Thundatil J. , Woodbury M. , McCorkell R.
Published: January 2008

In Canada, brucellosis and tuberculosis threaten an estimated 4500 wood bison (Bison bison athabascae), a species considered at risk by the Committee on the Status of Endangered Wildlife In Canada (COSEWIC). To help rescue this species, our Wood Bison Reproductive Research group proposes to employ advanced reproductive technologies. Unfortunately, little is known about the reproductive physiology of the wood bison, which hinders the application of these reproductive technologies. In order to modify advanced reproductive techniques developed in cattle for use in wood bison, the large amounts of semen, embryos, and oocytes from wood bison required are not available. The purpose of this study was to compare semen collected from the more abundant and closely related plains bison (Bison bison bison) with that of wood bison. Semen from 3 wood and 4 plains bison were collected by electro-ejaculation during the summer of 2007. Andrological parameters of morphology and motility were recorded on fresh semen, extended semen, and post-thawed semen samples. A Student's t-test was used to compare the results of these two groups. Semen was cryopreserved using two commercially available cryopreservation media (Andromed and Triladyl, Minitube Canada, Ingersoll, Ontario, Canada). Sperm morphology and motility were not different between electro-ejaculated samples from plains and wood bison (P > 0.05). Also, no difference was found in the survival rate of sperm from the electro-ejaculated samples between plains and wood bison after freezing and then thawing using an egg-yolk based extender (Triladyl) or an extender containing no products of animal origin (Andromed). A difference between cryopreservation media was found; post-thaw motility of Triladyl-treated sperm was higher (29%) than that of the Andromed-treated sperm (12%). Due to lack of previous success with preserving electro-ejaculated semen in media free of animal-origin products, motility assays were performed to evaluate if spermatozoa retrieved from epididymides of plains bison can be cryopreserved in Andromed. Interestingly, cyropreserved epididymal spermatozoa had a higher motility than cryopreserved electro-ejaculated sperm after freezing-thawing procedures using a medium containing no products of source animal (respectively, 30% v. 7%). This result suggests that there may be a factor secreted by the reproductive accessory glands that interferes with the post-thaw survivability of bison sperm. In conclusion, this study supports the hypothesis that semen from plains bison behaves similarly to that of wood bison semen during cryopreservation and therefore could be used to establish protocols for advanced reproductive technologies in wood bison.
This project was supported by Canadian Adaptation and Rural Development in Saskatchewan.
225 DOES THE SUPPLEMENTATION OF THE BOAR SEMEN EXTENDER WITH CARNOSINE, L-HISTIDINE, AND TAURINE PRESERVE SPERM FUNCTION DURING STORAGE?
Authors: Matás C. , Gardón J. C. , Garcia-Vazquez F. A. , Pacchini S. , Ducci M.
Published: January 2008

High levels of reactive oxygen species (ROS: superoxide, hydroxyl, hydrogen peroxide, nitric oxide, peroxynitrile) endanger sperm motility, viability, and function by interaction with membrane lipids, proteins, and nuclear and mitochondrial DNA (Sikka 2004 J. Androl. 25, 5–18). ROS generation has a significant negative effect on the fertilization rate after IVF, and so measurement of ROS levels in semen specimens before IVF may be useful in predicting the IVF outcome (Agarwal et al. 2005 Fertil. Steril. 84, 228–231). Several compounds of the antioxidant systems have been identified in the epididymal environment, spermatozoa, and seminal plasma. The antioxidants carnosine, L-histidine (Ducci et al. 2006 Pol. J. Vet. Sci. 9, 159–163), and taurine (Van der Horst and Grooten 1966 Biochim. Biophys. Acta. 117, 495–497) have been detected in boar semen and added to the extender in freezing procedures in several species. The main objective of this study was to evaluate the effect of carnosine, L-histidine, and taurine supplementation of the extender on boar sperm functionality as measured by sperm motility during computer-assisted semen analysis (CASA) and by IVF ability using mature oocytes, as previously described (Selles et al. 2003 Reprod. Domest. Anim. 38, 66–72). The sperm-rich fraction from mature fertile boars was diluted with isothermal Beltsville thawing solution (BTS) extender. Diluted semen was placed at 15�C and centrifuged at 800g for 10 min. The semen pellet was resuspended with BTS supplemented by 5 mm of carnosine, L-histidine, or taurine or not supplemented (control) to provide 75 � 106 spermatozoa mL–1 and stored at 15�C for 24 h (IVF assay), or 48 or 120 h (for CASA assay). We observed that the motility parameters were affected by storage time and that the addition of taurine increased the motility at 48 h of storage. Alternately, the addition of L-histidine to the extender reduced significantly the motility parameters after 120 h. The results showed that the addition of L-histidine induced a significant (P ≤ 0.01) decrease of the penetration rate (L-histidine 75.8% v. control 89.9%) and the number of sperm per oocyte penetrated (L-histidine 3.1 v. control 4.1). The rate of male pronuclear formation was not affected by the addition of antioxidants to the extender (over 85% in all cases). The addition of carnosine and taurine had no effect on the IVF parameters. In conclusion the antioxidants carnosine, taurine, and L-histidine affect sperm functionality differently, and further studies are necessary to elucidate what changes in sperm function take place during storage and the mechanisms by which these antioxidants exert their effects.
This work was supported by Italian-Spanish research project HI2005-0165 and AGL2006-03495.
226 DAY LENGTH DOES NOT AFFECT LIVE SPERM NUCLEAR SHAPE IN THE STALLION
Authors: Parrish J. J. , Mueller C. , Ludwig E. , Susko-Parrish J. L.
Published: January 2008

Fourier harmonic analysis (FHA) of sperm nuclei is a precise and objective method to evaluate shape of the sperm head, with the calculated harmonic amplitudes highly related to male fertility. The FHA approach has been developed for use in the bull and the boar but has not yet been applied to the stallion. Direct utilization of the previous fluorescent approaches to identify and image live sperm nuclei in the bull cannot be used in the stallion due to the increased thickness of the post-nuclear region and thin anterior region of the sperm head. An alternative approach was developed in which live and motile sperm were isolated after filtration of an ejaculate through a Sephadex G-15 column. The resulting live sperm were sonicated briefly to separate tails and heads. The heads were isolated on a 45–90 discontinous Percoll gradient, fixed with paraformaldehyde (0.2%), centrifuged onto glass slides, and dried. The slides were then stained with eosin (1%), cleared with water, and dried again; Permount was added, followed by a coverslip. Slides were imaged with phase contrast microscopy; digital images were acquired and evaluated with custom software to identify perimeter coordinates of sperm nuclei. The perimeter coordinates were next converted to Fourier harmonic amplitudes 0–5 (HA0–HA5) using trigonomic regression at 1 degree equally spaced angles. Fertility of bulls were previously reported to be most related to changes in HA0 and HA2 but no information is available on stallions. As fertility data on stallions is limited, to evaluate FHA in the equine, the day length (period of light) was increased in January from the ambient 9–10 h to 16. Semen samples from 5 light horse stallions were collected at weekly intervals for 8 weeks following the increase in light. It was hypothesized that fertility would increase for each stallion over the course of the experiment, as testosterone increases and spermatogenesis improves with increasing day length, as previously shown. Each week semen samples were evaluated for FHA live sperm nuclei, as described above. All parameter means and variations were recorded on 100 randomly selected sperm nuclei per semen sample evaluated. There was no difference relative to week 0 in the least squares mean or SD of HA0, HA1, HA2, HA3, HA4, or HA5 over the 8 weeks (P > 0.05). However stallions were consistently different for all HAs (P < 0.05). The overall mean HA0–HA5 � SEM were 1.973 � 0.38, 0.087 � 0.002, 0.721 � 0.021, 0.051 � 0.004, 0.208 � 0.014, and 0.031 � 0.002, respectively. Even though libido increased during the experiment, confirming the effect of light on the stallions, no affect on sperm nuclear shape or its variation was detected using FHA. Based on work in other species involving numbers of sperm inseminated, if large numbers of sperm from these stallions were inseminated in mares, we would predict no change in fertility due to season. Further research is needed to confirm this prediction.