bluuebunny/crossref_metadata_2025 huggingface dataset:
293 FACTORS ASSOCIATED WITH PREGNANCY OUTCOMES OF IN VITRO-PRODUCED EMBRYOS IN ZEBU CATTLE
Authors: Oyuela L. A. , Olaya J. , Zambrano-Varón J. , Jiménez C.
Published: August 2010

Few studies have addressed the factors that can affect pregnancy rates of in vitro-produced embryos (IVPE) and fewer still when referring to zebu IVPE. This study aimed to estimate some of these factors and the proportion of pregnancy loss. A total of 1227 pure-breed IVPE (Brahman, Red Brahman, and Gyr) were transferred (ET) to an equal number of crossbred Bos indicus × Bos taurus heifers. Recipients averaged 420 kg, were grass fed, and kept in three different ET Centrals located in northern, central, and eastern regions of Colombia. IVPE (Embriogen, Bogotá, Colombia) were nonsurgically transferred into synchronized recipients by using the following protocol: Day 0 s.c. implant of norgestomet 3 mg, plus an i.m. injection of estradiol valerate 5 mg and norgestomet 3 mg; Day 7 D-cloprostenol 0.15 mg i.m. Day 9 implant removal plus 200IU of eCGi.m. Pregnancy was recorded on Days 30 and 60 by ultrasound with an 8-m Hz linear rectal probe. Data were collected for 1 year beginning in September 2007. Binary logistic regression with forward conditional elimination was performed to evaluate factors associated with pregnancy. The strength of the association between the studied variables and pregnancy rate was estimated by the odds ratio (OR) (95% CI); a P-value < 0.05 indicated statistical significance. Interaction terms were considered (SPSS Inc., Chicago, IL, USA). Variables included in the logistic model were CL diameter at the time of ET, number of previous transfers to a recipient before achieving pregnancy, difficulty during the ET, embryo quality and stage, technician and level of training, number of transferred embryos by technician per day, uterine horn where the embryo was delivered, time between laboratory release and ET, ET Central, period of the year when the ET was performed, sire and breed of the embryo. Total pregnancy rate was 38% on Day 60 (462/1227), and the proportion of pregnancy loss was 8% between Days 30 and 90. Factors associated with pregnancy were as follows: 1) excellent embryos were 2.3 times more likely to achieve pregnancy (39%) than good embryos (21%), OR = 2.3, 95%CI = 1.4-3.8, P = 0.001; 2) easy transferred embryos were 5.1 times more likely to result in pregnancy (49%) compared with difficult transferred embryos (11%), OR = 5.1, 95% CI = 2.5-10.3, P < 0.05; and 3) transferred embryos <5 h after being released from the laboratory resulted in higher pregnancy rate (42%) than those transferred >5 h (33%). No interactions were found in the final logistic model. In conclusion, pregnancy outcome of IVPE Bos indicus was strongly associated with quality of the embryo, level of difficulty at the moment of the ET, and the time between the production of the embryo and the ET. In addition, the observed low pregnancy loss in this study suggests careful selection and management of the recipients, adequate handling of the IVPE, and proper training of the ET technicians.
Embriogen S.A. Bogotá, Colombia. Cenatte, Pedroleopoldo, MG, Brazil.
300 CASE REPORT: MORE THAN ONE CALF PER WEEK PRODUCED BY NELORE DONOR
Authors: Santos G. L. , Yamazaki L. T. S. , Benzi E. C. D. , Corneglian D. P. , Romano M. , Guidorizzi F. J. , Netto A. C. , Landim Junior L. P. , Yamazaki W.
Published: August 2010

According to the animal reproduction biotechnologies, in vitro fertilization is considered one of the most important tools to improve genetic gain as well as reduce bovine herd generations. Brazil is recognized as having the greatest commercial bovine herd in the world as well as by its use of IVP; however, this biotechnology needs more studies to improve not only the oocyte-embryo conversion rate but also the competent oocyte recovery from donor cows. Recently, one Nelore donor cow in 3 sessions was able to convert 233 (53.81%) embryos from 433 oocytes recovered, which resulted in 156 pregnancies (55.95%), which means one pregnancy per 2.7 oocytes or 3.05 pregnancies per week (when 51 weeks are admitted in one year). The COC obtained from this donor were matured (TCM-199, supplemented with FCS, LH, FSH, E2, pyruvate, and antibiotic) for 24 h and fertilized (Fert-TALP supplemented with BSA, PHE, and heparin) for 18 to 22 h (Day 0) in vitro. On Day 1, presumptive zygotes were transferred to development media (SOFaa supplemented with BSA and FCS) and on Day 7 the blastocyst production rate was evaluated. Following embryo transfer, the pregnancy diagnosis was performed at 30 days and confirmed at 60 days after ET. The possibility of identification of factors related to high performance and its selection could improve the formation of a homogeneous bovine herd, highly correlated with embryo production, which numerically could promote more than fifty calves per year (more than one calf per week). This case report refers to an animal highly superior to the average and therefore needs more investigation, mainly with molecular markers able to identify and select this class of high genetic animal.

Table 1.Embryo production performance
306 THE EFFECT OF BoviPURE GRADIENT ON BISON SPERM CRYOPRESERVATION
Authors: Bogle O. A. , Lessard C. , McCorkell R. B. , Grafton T. , Adams G. P.
Published: August 2010

Endemic tuberculosis and brucellosis threaten Canada’s wood bison population. For the purposes of developing a procedure to harvest pathogen-free sperm from bison, a sperm separation and purification product, BoviPure, was tested to determine its effect on bison sperm viability before and after cryopreservation. BoviPure (NidaCon International AB, Mölndal, Sweden) is a density centrifugation gradient system that contains trypsin and is designed to separate motile from non-motile sperm and remove infectious pathogens. Spermatozoa were expelled from the caudal epididymis of bison testicles collected at slaughter and diluted in 3 to 5 mL of TCM-199 medium (Gibco, Burlington, Ontario, Canada). After 1 h of incubation at 37°C, 1 to 1.5 mL of diluted sample (approximately 300 × 106 sperm) was placed in (1) a BoviPure gradient containing trypsin (n = 18 samples), (2) a BoviPure gradient without trypsin (n = 10 samples), or 3) left untreated (control; n = 20). Gradients were centrifuged at 300g for 20 min. The sperm pellet was resuspended in 5 mL of TCM-199 medium and recentrifuged at 500 g for 10 min. Total and progressive motility were estimated using computer-assisted sperm analysis (CASA). Gradient-treated and untreated sperm samples were diluted in an egg yolk extender (Triladyl; Minitube Canada, Ingersoll, Ontario, Canada), placed in 0.5-mL straws (50-200 × 106 spermatozoa per straw), frozen, and stored in liquid nitrogen for a minimum of 24 h. Semen straws were thawed by plunging in a 37°C water bath for 30 s, and motility measurements were taken immediately by CASA. Total and progressive motility were compared among groups by analysis of variance. Before cryopreservation, total motility and progressive motility were not affected by gradient processing. Total motility was 85±5.1%, 80±4.8%, and 77 ± 3.7% for untreated, BoviPure with trypsin, and BoviPure without trypsin, respectively (mean ± SEM), and progressive motility was 74 ± 2.7%, 77 ± 3.8%, and 68 ± 4.7%, respectively. After freezing and thawing, total motility decreased compared to unfrozen samples (P < 0.0001) but was not different among groups (37 ± 3.6%, 33 ± 3.7%, and 29 ± 4.8% for untreated, BoviPure with trypsin, and BoviPure without trypsin, respectively). Similarly, progressive motility decreased compared to unfrozen samples (P < 0.0001) but was not different among groups (26 ± 3.6%, 21 ± 3.7%, and 14.5 ± 4.9% for untreated, BoviPure with trypsin, and BoviPure without trypsin, respectively). We conclude that the BoviPure gradient with or without trypsin does not influence total motility or progressive motility of bison sperm either before or after cryopreservation and has potential as a method of harvesting pathogen-free sperm from wild animals of unknown disease status.

This study was supported by Canadian Adaptation and Rural Development in Saskatchewan.
307 TESTICULAR ULTRASONOGRAPHIC CHARACTERISTICS OF GUZERAT (BOS TAURUS INDICUS) BULLS DURING THE PERIPUBERTAL PERIOD
Authors: Carmo A. S. , Osorio J. P. , Mendonça L. F. , Lucio A. C. , Perini A. P. , Miguel M. C. V. , Oliveira L. Z. , Hossepian de Lima V. F. M. , Henry M.
Published: August 2010

Recently, ultrasonography has been used to study reproductive tract and testes development in the bull. The testicular ultrasound allows identification of subtle changes in the echotexture of the testicular parenchyma in the different stages of reproductive development. The aim of the present study was to characterize the pixel intensity (echotexture) of testicular ultrasonograms of Guzerat bulls in the peripubertal period to attempt to raise efficiency of sire selection programs and puberty identification. Seventy-five animals from 9 to 30 months of age were evaluated monthly for 6 consecutive months. The testes were examined using a 5-MHz linear array transducer connected to a B-mode ultrasound scanner (Fukuda®, Tokyo, Japan). Images were frozen on the monitor of the ultrasound scanner and recorded on VHS tape. The images were digitalized and the average pixel intensity (PI) of the testes was determined by the J1.58 (National Institutes of Health, Bethesda, MD, USA) software image package. Additionally, all of the bulls were submitted to a complete andrological examination and seminal collection was attempted when males had a scrotal perimeter > 20 cm. Onset of puberty was considered to have occurred when at least one motile sperm cell was detected in the ejaculate. Statistical analysis was performed using the SAS statistical package (SAS Institute Inc., Cary, NC, USA; Student-Newman-Keuls test and Pearson correlation). The PI in pubertal animals (95.0 ± 39.01 cm3) was higher (P < 0.05) than non-pubertal animals (56.5 ± 43.1 cm3). Changes in patterns of testicular echotexture during the peripubertal stage reflect the gradual and significant increase (P < 0.05) of the intensity of pixels observed in animals from 9 months (36.7 ± 22.2 cm3) up to 30 months of age (127.4 ± 46.2 cm3). The correlation between age and intensity of pixels was significant (P < 0.05; r = 0.534). The increase of PI is an important indicator of puberty in bovine males. Nevertheless, it was not possible to establish a pattern of testicular echotexture that characterized the precise time of onset of puberty.

Thanks to Fazenda do Rosário, CAPES, and CNPq.
308 GUANACO SPERM CHROMATIN EVALUATION USING TOLUIDINE BLUE
Authors: Carretero M. I. , Giuliano S. , Agüero A. , Pinto M. , Miragaya M. , Trasorras V. , Egey J. , von Thungen J. , Neild D.
Published: August 2010

Guanacos, a wild species of South American camelids, have a high-quality fiber with great economic potential. To evaluate reproductive aptitude in guanacos, our laboratory has developed a reliable semen collection technique using electroejaculation and has applied various methods for evaluating semen characteristics. Studies for evaluating the state of sperm chromatin have also been initiated. Toluidine blue (TB) is a cationic stain that unites with the phosphate groups in the DNA, thus permitting differentiation between sperm heads according to the degree of chromatin decondensation. The objectives of this study were to determine the TB staining patterns of guanaco sperm chromatin, establish a positive control for the stain, and evaluate the effect of collagenase on sperm chromatin condensation. Semen was collected from 4 guanacos, between 6 and 9 years old, using electroejaculation. In Experiment 1, to establish a positive control for the stain, equal quantities of 1% dithiothreitol (DTT) and raw semen were incubated at room temperature for 30 s, 1.5 min, and 3 min. After incubation, smears were made and then dried, to avoid continuing the reaction, and finally were stained with 0.02% TB. A split-plot design was used with time as the splitting factor and considering the males as a block. In Experiment 2, raw semen was divided into 2 aliquots, one diluted 4 : 1 in 0.1% collagenase in HEPES-TALP-BSA medium and the other left without enzyme. Both aliquots were incubated 4 min at 37°C and, after centrifugation to remove the enzyme, smears were made and stained with TB. Spermatozoa were classified according to the degree of chromatin decondensation. Analysis of variance was performed using the males as a blocking factor and the treatment as a fixed factor. According to the degree of chromatin decondensation, three patterns of staining with TB were observed: light blue (negative, without alteration of chromatin condensation), light violet (intermediate, some degree of decondensation), and dark violet (positive, high degree of decondensation). A significant increase (P < 0.05) of sperm with highly decondensed chromatin was observed in semen incubated for 3 min with DTT when compared to 30 s of incubation. Therefore, 3 min of incubation with DTT was chosen as the positive control for Experiment 2. No significant differences in any of the 3 patterns of TB staining were observed between semen incubated with or without 0.1% collagenase. In conclusion, it is possible to use TB to evaluate the degree of chromatin decondensation in guanaco spermatozoa and to use DTT as a positive control for the stain. Treatment of guanaco semen with 0.1% collagenase did not affect sperm chromatin condensation; therefore, this enzyme can be used to decrease semen viscosity and aid handling in the laboratory.
336 MEIOTIC DEVELOPMENT AND CORTICAL GRANULES DISTRIBUTION IN CANINE OOCYTES DURING IN VITRO MATURATION
Authors: De los Reyes M. , Luna D. , Palomino J.
Published: August 2010

Low development of IVM canine oocytes could be in part attributed to an impaired cytoplasmic maturation. In mammalian oocytes, migration and the redistribution of cortical granules (CGs) around the periphery of the oocyte contribute to the inhibition of polyspermy and it is an important criterion to evaluate cytoplasmic maturation. The state of nuclear maturation and the distribution of CGs were evaluated in canine oocytes cultured for different periods in order to compare the synchrony of nuclear and cytoplasmic maturation during in vitro maturation. Bitch ovaries at different stages of the estrous cycle were obtained following ovariectomy. COCs with compact cumulus cells showing a homogeneous cytoplasm were selected for experiments. Thirty-six COCs were processed at immature stage, placed in PBS medium until evaluation. A total of 275 COCs were matured in vitro for 48, 72, and 96 h in TCM-199 with Earle’s salt supplemented with 25 mM Hepes, 10% FCS, 0.25 mM pyruvate, 10 IU mL-1 of hCG, 300 IU mL-1 penicillin, and 20 mg mL-1 streptomycin, at 38.5°C and 5% CO2. At each culture period, the oocytes were stained with Lens culinaris agglutinin (LCA), labeled with fluorescein isothiocyanate, and the CGs distributions were examined under a fluorescent microscope. The nuclear status of the denuded oocytes was determined by DAPI staining under a fluorescence microscope. For each treatment, at least four replicates were performed and the data was analyzed by ANOVA using Tukey’s test to determine the differences P < 0.05. Three types of CGs distribution were distinguished during canine oocyte maturation: (1) homogeneous distribution throughout the cytoplasm including the cortex; (2) heterogeneous (clusters) within the cytoplasm and (3) densely distributed beneath the oolemma. Nuclear stages were classified as immature or germinal vesicle (GV) stage; resumption of meiosis or germinal vesicle break down (GVBD); metaphase I to telophase I (MI toTel I); and mature or second metaphase (MII). The distribution patterns of GCs were different (P < 0.05) among oocytes cultured for different periods and the nuclear maturation status also differed between oocytes cultured for different intervals (P < 0.05). Most (>84%) of the immature oocytes at GV showed a uniform distribution of CGs throughout the cytoplasm. At 48 h of culture, CGs distribution was mainly Type 2 (25%) and 3 (61%) and the oocytes were at GVBD (33%) and MI-Tel I (33%) stages. Most nuclei of the type 3 oocytes were in the MI (40%) and MII (11%) stages, corresponding to those oocytes matured for 72 (88%) or 96 h (71%). These results indicate that canine oocytes migrate to the cortex during IVM and this process is not finished before 72 h of culture. In addition, although the re-distribution of the CGs occurred in parallel with nuclear maturation, the oocytes cannot always proceed to the MII stage; however, in such oocytes the CGs are distributed beneath the oolemma.
Supported by Grant FONDECYT 1080618.
347 TRANSCRIPTIONAL REGULATION DURING IN VITRO BOVINE OOCYTE MATURATION: FACT OR ARTIFACT?
Authors: Mamo S. , Leal C. L. V. , Al Naib A. , Lonergan P. , Fair T.
Published: August 2010

Understanding the molecular dynamics of oocyte maturation may assist in improvements to maturation protocols and outcomes of in vitro embryo production. There are conflicting results on the occurrence of transcriptional changes during meiotic maturation. A previous microarray study from our group (Carter et al. 2007 Reprod. Fertil. Dev. 19 248) indicated upregulation of a number of transcripts during oocyte maturation in vitro. The aim of this study was to attempt to ascertain whether such changes are real or artifacts of methods used for analysis. COCs were aspirated from follicles of bovine ovaries collected at the local abattoir. Immature COC were immediately denuded and half of them were snap frozen in pools of 12 oocytes in liquid nitrogen. The remaining oocytes were matured in vitro for 24 h at 39°C under an atmosphere of 5% CO2 in air with maximum humidity in TCM-199 supplemented with 10% (v/v) fetal calf serum and 10 ng mL-1 epidermal growth factor. They were subsequently denuded and snap frozen in pools of 12 as above. Samples were stored at -80°C until analysis. Six different pools per stage were collected from four separate collections. Prior to RNA isolation, samples were spiked with luciferase mRNA and total RNA was isolated using RNeasy Micro Kit (Qiagen, Hilden, Germany). The RNA was then divided into two equal parts for priming either with Oligo (dT) or random primers during cDNA synthesis.The expression of eight genes (LUM, PLAT, SERPINE1, STC1, PLAU, ANXA1, TGFB2, and MAOA), previously reportedtobe up regulated (P < 0.05) during bovine oocyte maturation and luciferase was examined. Quantitative real-time PCR was used to compare transcript abundance and data were analyzed using the relative standard curve method. The expression level of these genes was normalized to the average luciferase levels and means were compared by the student t-test. With the exception of MAOA, the expression of all transcripts up regulated (P < 0.05) in the previous microarray study showed similar trend in this analysis. However, the relative expression levels varied with the type of primer used during cDNA synthesis. Oligo (dT) primed samples had higher relative expression ratios compared to samples with random primers, indicating the preferential amplification of polyadenylated transcripts by oligo primers in matured oocytes. Despite the variations in the ratio, the expression patterns remained the same, indicating the existence of transcription during the in vitro bovine oocyte maturation. Future studies will involve in vivo-matured oocytes and identify whether the observed transcription in the in vitro samples is a normal phenomenon or a consequence of suboptimal in vitro culture environment.
Supported by Science Foundation Ireland (07/SRC/B1156).
362 STORAGE OF BOVINE SPERM FOR 20 H BETWEEN SEMEN COLLECTION AND SEXING
Authors: Anema J. L. , Graham J. K. , Lenz R. W. , Seidel G. E.
Published: August 2010

The objective of this study was to optimize bovine sperm storage for up to 20 h between semen collection and sex sorting followedby cryopreservation. Two successive ejaculates were obtained from mature dairy bulls (Holstein, n = 5; Jersey, n = 3) via artificial vagina. Treatments were then applied to the neat semen to which antibiotics were added as recommended by Certified Semen Services (Columbia, MO). Nothing further was added to the control samples until staining with Hoechst 33342 for sorting. For Treatment 1, semen was diluted 9:1 with a MOPS solution resulting in 24 mM MOPS and similarly, Treatment 2 resulted in 24 mM MOPS +2% egg yolk. A subsample of each treatment and control was sorted by flow cytometry shortly after collection, and sperm then were frozen following standard processing procedures. The other subsample was stored at 15-18°C and sorted 20 h after collection followed by cryopreservation. pH measurements were made before staining samples for sorting. Samples were evaluated post-thaw for subjective progressive and total sperm motility, by computer-assisted sperm analysis (CASA, Berkeley, CA, USA), and by flow cytometry for sperm viability using propidium iodide and SYBR-14. Treatment 1 performed better than the control (Table 1), while results for Treatment 2 were similar to the control. Second ejaculates were superior to first ejaculates. pH measurements showed that addition of MOPS kept the pH about 0.2 units higher than the control, but pH declined similarly over time in all groups. While responses for the 20 h sort were numerically lower than the 0 h sort (P > 0.1), the majority of responses were acceptable for most, but not all bulls. In conclusion, storing sperm in 24 mM MOPS was beneficial. Surprisingly, 2% egg yolk negated the beneficial effect of MOPS, possibly due to increasing osmolarity by ∼15mOsM/kg due to pH adjustment. Addition of MOPS provided better results than the control for both the 0 h and 20 h sorts.

Table 1.Main effect means of semen characteristics
364 QUANTIFICATION OF DEVELOPMENTALLY IMPORTANT GENE TRANSCRIPTS EXPRESSED IN EMBRYOS PRODUCED IN VIVO AND IN VITRO USING SEX-SORTED AND NON-SORTED RAM SPERMATOZOA
Authors: Beilby K. H. , Wilkening S. , de Graaf S. P. , Wrenzycki C. , Grupen C. G.
Published: August 2010

The AI of sex-sorted spermatozoa results in decreased levels of fertility in most species. This is not the case in sheep, where low-doseAI of sex-sorted ram spermatozoa produces similar, if not superior levels of fertility to non-sorted controls (Beilby et al. 2009 Theriogenology, 71, 829-835). In an effort to provide insight into the molecular basis for this difference in fertility between species, the aim of the present study was to examine the impact of sex-sorting technology on ovine embryo gene expression. After semen collection, ejaculates (n = 8) were split and either sex-sorted by flow cytometry and frozen, or diluted and frozen (non-sorted control). Embryos were produced in vivo by inseminating superovulated ewes with either X- or Y-chromosome enriched spermatozoa, or non-sorted control spermatozoa, and collected by flushing uterine horns on Day 6 after AI. Embryos were produced in vitro by using established oocyte in vitro maturation and in vitro fertilization procedures (using X, Y or non-sorted spermatozoa), and cultured in vitro for 6 days. The relative abundance of Glut-3, G6PD, SUV39H1, DnMT3a, and HSP70 was measured in high grade blastocysts (in vivo Day 6: n = 23; in vitro Day 6: n = 21) using quantitative, real-time PCR (iCycler5TM, BioRad, Hercules, CA, USA). Blastocyst cell numbers were quantified to ensure embryos were at a similar stage of development. The sex of all embryos was identified by PCR to allow comparison between treatments. Fold differences in gene expression, acquired through the A ACt method, were calculated and compared by an ANOVA. The expression of HSP70 was up-regulated in in vitro embryos derived from sex-sorted spermatozoa compared to those produced in vivo (P < 0.05). For all other genes examined, there was no effect of sex or sperm treatment on gene expression. Glut-3, G6PD, SUV39H1, andDnMT3a were all up-regulated in in vitro embryos compared with in vivo embryos (P < 0.05). These results suggest that fertilization with sex-sorted ram spermatozoa does not result in aberrant patterns of gene expression in embryos produced in vivo. The data from the present study provide further evidence that in vitro culture induces epigenetic modification within the embryonic genome, when compared to the in vivo physiological standard. It would be of interest to conduct a similar in vivo study in cattle, where sex-sorting technology has not been as biologically successful. The altered expression of HSP70, which is associated with cellular stress, may demonstrate a cumulative impact of in vitro reproductive technologies on the preimplantation embryo.

Research supported by XY, Inc.
365 QUALITY ASSESSMENT OF BOVINE CRYOPRESERVED SPERM AFTER SEXING BY FLOW CYTOMETRY AND ITS USE FOR IN VITRO EMBRYO PRODUCTION
Authors: Carvalho J. O. , Sartori R. , Machado G. M. , Mourão G. B. , Dode M. A. N.
Published: August 2010

Several studies using sex-sorted sperm by flow cytometry have shown that its fertility is reduced. Therefore, this study evaluated structural and functional characteristics of sperm sexed by flow cytometry. In addition, in vitro embryo production (IVP) and development was assessed when frozen-thawed unsorted and sex-sorted sperm from 4 Nellore bulls. Each ejaculate was separated into three fractions: non-sexed (NS), sexed for X-sperm (SX), and sexed for Y-sperm (SY). After thawing, each sample was analyzed for sperm motility by computer-assisted semen analysis (CASA, Berkeley, CA), sperm head agglutination, sperm morphology, membrane integrity by propidium iodide (PI) and 6-carboxy-fluorescein diacetate (CFDA) staining, acrosome integrity by peanut agglutinin (PNA), capacitation by chlortetracycline (CTC), and chromatin integrity by acridine orange staining. Then, the samples were placed in 45 : 90% (NS90) or 45 : 60% (NS60, SX, and SY) Percoll™ gradients. After Percoll™ centrifugation, sperm pellets were analyzed or used for IVP. All analyses were replicated independently three times. For IVP, 2,271 in vitro matured oocytes were used. To assess fertilization rate, presumptive zygotes were fixed and stained with lacmoid at 18 h post-insemination (hpi). Cleavage was evaluated at Day 2 (48 hpi) and blastocyst development at Days 6, 7, 8, and 9 of culture. Data were analyzed using generalized linear models. No differences (P > 0.05) were observed between SX and SY groups for e sperm variables evaluated either before or after Percoll™. However, non-sexed sperm had higher sperm motility, greater percentage of sperm with intact membranes, and greater percentage of live sperm with intact acrosomes than sexed sperm (P < 0.05). An effect of Percoll™ was observed in the non-sexed samples, with those submitted to 45 : 90% gradient having higher motility, greater percentage of cells with intact membrane, and lower recovery rate than those submitted to a 45 : 60% gradient. No differences among groups were observed for fertilization rate, being 74.0 ± 5.7, 63.2 ± 5.1, 67.2 ± 5.7, and 55.4 ± 5.9% for NS90, NS60, SX, and SY, respectively. Group NS90 showed a greater cleavage rate than did the SY group, while groups NS60 and SX had similar rates to the others. Blastocyst development rates on Day 6 to Day 9 were greater for group NS90. For example, on Day 8 the blastocyst rate was 34.9 ± 3.6, 22.2 ± 3.2, 18.1 ± 3.3, and 14.8 ± 2.9% forNS90, NS60, SX, and SY groups, respectively. All groups showed similar embryonic developmental stages on Day 6 to Day 9. Although sex-sorting affected sperm characteristics, it did not cause a decrease with in vitro fertility. However, differences in blastocyst rates between groups NS60 and NS90 indicated that the sperm selection protocol affected embryo production.

Financial support: Embrapa Genetic Resources and Biotechnology.
368 CONCEPTION RATE AT FIRST ARTIFICIAL INSEMINATION WITH SEXED SEMEN OR CONVENTIONAL SEMEN IN CROSSBRED DAIRY COWS
Authors: Gerhardt B. T. , Sinedino L. D. P. , Dourado A. P. , Alves P. A. M. , Nogueira L. A. G.
Published: August 2010

The use of sexed semen in dairy cattle almost eliminates the birth of unwanted male calves and can lead to increased milk production by means of genetic selection exerted on the females. However, AI with sexed semen still presents several obstacles to overcome. Holstein-Zebu cows are responsible for 80% of the milk produced in Brazil. These crossbred animals are used extensively throughout the country because their physiologic and morphologic characteristics are compatible with milk production in the tropics which results in an economic performance advantage. The objective of this study is to evaluate the conception rates with sexed semen, comparing it to the use of non-sorted semen in crossbred dairy cows. In addition, two insemination times had been studied to determine the best timing for AI with the sexed semen. Both studies were carried out in the Experimental Station Seropedica of Pesagro-RJ, located in the state of Rio de Janeiro, Brazil. The experiment was done in 2008/2009 (January to April). In the first Trial 56 Holstein-Zebu females, composed of 1/2 to 7/8 Holstein genetics, were inseminated 12 h after estrus detection with non-sorted semen (control group n = 29, 14 heifers and 15 cows) or with sexed semen (experimental group n = 27, 13 heifers and 14 cows). The semen utilized was from the same bull and thawing was performed intravaginally. In the second trial, 55 females were inseminated only with sexed semen from the same bull, with thawing in water at 35°C, at two different times: 12 h after estrus observation (control group n = 27, 12 heifers, 4 primiparous, and 11 multiparous) and 15 h after estrus observation (experimental group n = 28, 12 heifers, 5 primiparous, and 11 multiparous). The pregnancy diagnoses were done by rectal palpation and transrectal CAT scan at Day 45. Statistical analyses had been done using Chi-square. The conception rate in the first trial was of 17.2% (5/29) for sexed semen and 53.8% (14/26) for non-sorted semen (P < 0.05). In the second trial, the conception rate was of 37.0% (10/27) for insemination with sexed semen, 12 h after estrus observation and 50.0% (14/28) for the females inseminated 15 h after estrus observation (P > 0.05). It is possible that one of the causes of fertility reduction with sexed semen is that sexed-sperm remain viable for a shorter time after the insemination. Inseminations with sexed semen done 15 h after estrus detection, which can be more synchronized with the time of ovulation, and when accompanied by closely controlling the thawing process, show results similar to those obtained with conventional semen.
37 USE OF THERMO-RESISTANCE TEST (TRT) TO EVALUATE EQUINE COOLED SEMEN STORED AT 5°C
Authors: Pugliesi G. , Silva Filho J. M. , Torres C. A. A. , Rates D. M. , Ker P. G. , Lourenço G. G. , Matta M. P. , Oliveira R. R. , Carvalho G. R.
Published: August 2010

Evaluation of seminal characteristics is an important step to predict the reproductive potential of equine semen in natural or AI programs. Thermo-resistance test (TRT) has wide acceptance among tests in the bovine species, mainly because of its high correlation with fertility field. However, the TRT for stallion semen has not been widely studied. The objective of this study was to evaluate the effective use of TRT for equine cooled semen diluted with different extenders. Three stallions of Mangalarga Marchador breed aged between 8 and 14 years were used. Five semen samples per stallion were obtained, collected 3 times a week, with the aid of an artificial vagina (adapted Hannover model) using mares in natural estrus as dummy. The semen was diluted in 2 extenders: skim dried milk-glucose (E1) and glycine-egg yolk (E2), packaged in samples containing 12 mL of diluted semen to reach a final concentration of 30 million viable spermatozoa mL-1 and then stored at 5°C in an Equitainer® for 24 h. The cooled semen was warmed at 37°C in a water-bath. Spermatozoal progressive motility and vigor of semen were evaluated at 0 (TRT0), 30 (TRT30), 60 (TRT60), and 90 (TRT90) min after the start of warming. Treatment differences for sperm parameters were determined using ANOVA. The average values of sperm motility during TRT0, TRT30, TRT60, and TRT90 in E1 and E2 were, respectively, (E1) 37.0, 31.3, 23.7, and 19.7 and (E2) 30.3, 23.7, 18.3, and 15.7. The average values of vigor during TRT0, TRT30, TRT60, and TRT90 in E1 and E2 were, respectively, (E1) 2.4, 2.03, 1.53, and 1.43 and (E2) 1.97, 1.53, 1.33, and 1.17. During the test, the progressive motility obtained with E1 was higher (P < 0.05) than that with E2, and is within the patterns of motility considered acceptable only at 0 and 30 min of TRT. The E2 extender gave the worst result of the test, which was below the standards recommended for cooled semen. The seminal characteristics decreased in a very short time of TRT (30 min). This test is for use in insemination program. Thus, this demonstrates that changes in interpretation of the test need to be made in equine semen evaluation. A marked reduction of progressive motility at 30 min of test can be caused by loss of intracellular components or lesions in sperm movement structures. Possibly, availability of cyclic nucleotides involved in oxidative phosphorylation and motility are insufficient, although the mitochondria have the ability to produce energy. The TTR time of 90 min is long for equine cooled semen, and a duration for TTR of 30 min may be more appropriate in this species.

Supported by grants from CNPq and CAPES.
372 X-CHROMOSOME-BEARING SPERM SEPARATION BY Percoll DISCONTINUOUS DENSITY GRADIENT AND SEX RATIO DEVIATION IN IN VITRO PRODUCED BOVINE EMBRYOS
Authors: Perini A. P. , Lucio A. C. , Carmo A. S. , Miguel M. C. V. , Oliveira L. Z. , Miguel J. A. , Dernowsek-Meirelles A. P. , Moreira-Filho C. A. , Hossepian de Lima V. F. M.
Published: August 2010

The aims of this study were to separate X-chromosome-bearing bovine sperm by discontinuous Percoll ™ (GE Healthcare Bio-Science AB, Uppsala, Sweden) density gradients, validate the sexing of resultant IVF embryos by PCR, replace the bovine fetal serum (BFS) with BSA in the culture medium, to decrease male development advantage, and verify whether the gradient can be used in an IVF laboratory routine. The gradient was prepared by mixing Dubelcco’s Modified Eagle’s Medium (DMEM; Sigma Aldrich, St. Louis, MO, USA) with Percoll™ isotonic solution with 0.3% BSA, for different densities obtained ranging from 1.110 to 1.123 g mL-1, disposed in 3 layers into 15-mL conical tubes. For sexing, 40 million thawed sperm were overlaid on density gradients. The tubes were centrifuged at 500 g, for 15 min, at 22°C. After centrifugation, sperm sediment was used for IVF. For the control group, a Percoll™ 45, 90% gradient was used. The oocytes were selected from ovaries from slaughterhouse and maturated for 24 h in TCM-199 medium. After fertilization, oocytes and sperm were incubated for 20 h in 5% CO2, in humidified air at 38.5°C. Presumptive zygotes were denuded of cumulus cells, and washed in modified SOF medium and then transferred to 500 μL SOF in four well dishes. Embryo culture was carried out under mineral oil in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 at 38.5°C, and the cleavage assessed at 46 h and development to the blastocyst stage at Day 7. To obtain embryonic cell DNA for sex determination by PCR, 115 embryos of the sexed and 82 of the control group were used. Two pairs of primers of Y-specific sequences were split in two distinct samples. The first pair detected a sequence of 210 bp, and the second one 196 bp of the bovine Y-chromosome. A third one detected an autosomal sequence of 280 bp, indicating the presence of bovine genomic DNA. PCR multiplex was carried out in the same tube with first and third primers and the PCR of the second one was carried out in another tube. The results were analyzed by X2. Of the sexed group, from a total of 373 oocytes, the cleavage rate was 58.2% (n = 217); 35.6% (n = 133) produced embryos; 36.5% (n = 42) were male embryos and the female embryo rate was 63.5% (n = 73). From a total of 268 control oocytes, the cleavage rate was 63.8% (n = 171); produced embryos 37.3% (n = 100); 57.3% (n = 47) were male embryos and the female rate was 42.7% (n = 35). The Percoll™ density gradient for sperm sexing altered the proportion of IVF embryos toward more females. Because of fast and easy preparation, the gradient can be used routinely in an IVF laboratory and also, BSA can replace FBS for the IVF.

FAPESP process number 59357-9 and CAPES
379 TREATMENT OF 293T CELLS WITH XENOPUS EGG EXTRACT INDUCES NOT-STRESS-RELATED EPIGENETIC REMODELING OF RIBOSOMAL GENES
Authors: Østrup O. , Hyttel P. , Klœrke D. , Collas P.
Published: August 2010

Undefined mechanisms involved in reprogramming of somatic cells by Xenopus leavis egg extract have, to date, prevented clinical applications for cell replacement therapy. The aim of this study was to evaluate the immediate response of somatic cells to exposure to Xenopus leavis egg extract using ribosomal genes (rDNA) as a sensitive marker for stress and/or chromatin remodeling. Human epithelial 293T cells in standard culture (control), starved in glucose-free medium (stress-control), or treated for 1 h with egg extract were fixed at 6 and 24 h after treatment and analyzed for pre-rRNA synthetic activity by quantitative RT-PCR; level of H3K9me3 by immunofluorescence; and for occupancy of rDNA promoter by markers of normal activity (UBF), stress silencing (SIRT1, SUV39H1), and remodeling (SNF2H) using chromatin immunoprecipitation. Relative levels of pre-rRNA decreased in all treated groups to 20 to 35% of control levels. Chromatin immunoprecipitation did not reveal any significant effect of starvation or extract treatment on UBF and SIRT1 binding to the rDNA promoter, presumably because of the pleiotropic property of these proteins. However, starvation specifically led to enhanced SUV39H1 binding 6 h after treatment, indicating early cell response to stress. No differences were found in SUV39H1 binding between extract-treated cells and control. In contrast, binding of SNF2H at 6 h increased in extract-treated cells but decreased in starved cells. The relative level of H3K9me3 increased first after 24 h of starvation equally in all cells, suggesting later involvement of this histone modification in rDNA silencing. Extract treatment, however, led to a decrease in H3K9me3 level at 6 h, and after 24 h, 2 main cell populations were observed: one (85.4% of cells) that retained decreased H3K9me3 and one (12.4% of cells) with significantly enhanced levels. Moreover, H3K9me3 foci in the last group were associated with the periphery of presumptive nucleoli. Thus, extract treatment apparently does not initiate a stress-induced silencing of the rDNA genes; rather, it activates SNF2H-dependent chromatin remodeling, resulting in a long-term decrease in pre-rRNA synthetic activity.

The research was supported by Carlsberg Foundation 2008-01-0105.
380 ISOLATION AND DIFFERENTIATION OF MESENCHYMAL STEM CELLS DERIVED FROM CANINE AMNIOTIC FLUID
Authors: Choi S. A. , Lee J. H. , Kim K. J. , Kim E. Y. , Li X. X. , Lashgari A. , Kim M. K
Published: August 2010

The dog is biologically comparable with humans with respect to stem cell kinetics, haematopoietic demand, and responsiveness to cytokines. The availability of canine mesenchymal stem cells allows for the establishment of the dog as a large animal model for testing the safety and efficacy of mesenchymal stem cells replacement therapy. Large animal models, such as the dog, are invaluable for working out the practicalities of a therapeutic regimen in a complex system and for verifying established mechanistic theories. Therefore, canine stem cells present the potential for unique and exciting biological opportunities. Recent observations also indicate that stem cells derived from second-trimester amniocentesis are pluripotent, capable of differentiating into multiple lineages, including representatives of all 3 embryonic germ layers. Compared with embryonic stem cells, amniotic fluid stem cells can be obtained without destroying embryos, thus avoiding much ethical controversy. The aim of the current study was to investigate adipogenic, osteogenic, and chondrogenic in vitro differentiation potential of canine amniotic fluid-derived mesenchymal stem cells by biological characterization. We successfully isolated and characterized canine amniotic fluid-derived mesenchymal stem cells (cAFS). Expression of stem cell-specific marker OCT3/4, SOX2, and NANOG was confirmed by RT-PCR. Flow cytometric analysis showed that cAFS were positive for CD44, CD29, and CD90 but negative for CD34. Immunocytochemical analysis also showed the expression of alkaline phosphatase, SOX2, SSEA-1, and SSEA-4. Following incubation with specific adipogenic, osteogenic, and chondrogenic agents, cAFS stained positive by Oil Red O and Alizarin Red S, respectively. In conclusion, according to the preview studies on other mammalians, cAFS is an appropriate source of pluripotent stem cells. Here, we demonstrated that cAFS has a high adipogenic, osteogenic, and chondrogenic differentiation potential in vitro. Therefore, amniotic fluid might be a suitable alternative source of stem cells.

This study was financially supported by KOSEF (grant #R01-2008-000-21076-0), research fund of Chungnam National University, and the Korean MEST, through the BK21 program for creative research in animal biotechnology.
398 DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS INTO CARDIOMYOCYTES BY USING SLOW TURNING LATERAL VESSEL (STLV/BIOREACTOR)
Authors: Rungarunlert S. , Tar K. , Muenthaisong S. , Techakumphu M. , Pirity M. , Dinnyes A.
Published: August 2010

Cardiomyocytes derived from embryonic stem (ES) cells are anticipated to be valuable for cardiovascular drug testing and disease therapies. The overall efficiency and quantity of cardiomyocytes obtained by differentiation of ES cells is still low. To enable a large-scale culture of ES-derived cells, we have tested a scalable bioprocess that allows direct embryoid body (EB) formation in a fully controlled, bioreactor/STLV (slow turning lateral vessel, Synthecon, Inc., Houston, TX, USA) following inoculation with a single cell suspension of mouse ES cells. Technical parameters for optimal cell expansion and efficient ES cell differentiation were compared, such as ES cell seeding density (3 × 105 and 5 × 105 cells mL-1) into the bioreactor and day of transfer and plating of EB on gelatinated petri dishes (Day 2, Day 3, Day 4, and Day 5). The quantity and quality of EB production including the yield and size of EB, as well as viability and apoptosis of cells, were analyzed. Furthermore, after cultivation, well-developed contracting EB with functional cardiac muscle were obtained in which the percentage of EB beating/well and several specific cardiac genes [cardiac Troponin T (cTnT) and α-actinin] expression were also determined. Data are expressed as mean ± SEM of at least 3 independent experiments. Statistical analyses included one-way ANOVA and Student’s t-test Statistical significance was set at P < 0.05. The results showed that 5 × 105 ES cells mL-1 seeded into the STLV significantly improved the homogeneity of size of EB formed compared with 3 × 105 ES cells mL-1. The EB derived from Days 2 or 3 culturing in STLV had less necrotic cells than Days 4 and 5 groups. Furthermore, plating these EB on Days 2 and 3 resulted in significantly more EB beating/well than that of Days 4 and 5 groups. For cardiac differentiation, the group with 5 × 105 ES cells mL-1 seeded into STLV and transferred and plated on Day 3 expressed more cardiac markers than other groups. In conclusion, the optimized rotary suspension culture method can produce a highly uniform population of efficiently differentiating EB in large quantities in a manner that can be easily implemented by basic research laboratories. This method provides a technological platform for the controlled large-scale generation of ES cell-derived cells for clinical and industrial applications.

This work was financed by The Thailand Commission on Higher Education (CHE-PhD-SW-2005-100), EUFP6 CLONET (MRTN-CT-2006-035468), NKFP_07_1-ES2HEART-HU (OM-00202-2007), and EUFP7 (PartnErS, PIAP-GA-2008-218205).
403 CONTROL OF OVULATION IN SOWS IN THE TROPICAL ENVIRONMENT USING HUMAN CHORIONIC GONADOTROPHIN IN COMPARISON WITH GONADOTROPIN-RELEASING HORMONE ANALOG
Authors: Buranaamnuay K. , Wongkaweewit K. , Raksasub R. , Prommachart P. , Tummaruk P. , Techakumphu M.
Published: August 2010

The reproductive performance of sows is influenced by numerous factors, including environment. The time of ovulation in response to hormonal treatment might vary depending on climate. Because control of ovulation in pigs using hormonal treatments has not been studied in the tropical climate, the effect of hCG or GnRH analog (buserelin) on the time of ovulation in weaned sows was investigated. Thirty-three multiparous Landrace (L; n = 14) and Yorkshire (Y; n = 19) sows housed in the mating and gestation unit on a commercial swine farm in Thailand were used. The average of maximum and minimum daily temperature and daily humidity during experiment was 35°C and 25°C and 31%, respectively. Estrus detection by back pressure test and presence of a mature boar was performed every 6 h. Only sows with weaning to estrus interval (WOI) of ≤7 days were studied. Estrous sows were randomly allocated to 3 groups: a control group (4 Y and 6 L), which was given no treatment; a group (5 Y and 5 L) given 750 IU of hCG i.m. at the beginning of estrus (hCG group); and a group (5 Y and 8 L) given 10 μg of GnRH analog i.m. at the beginning of estrus (GnRH group). The mean time of ovulation was monitored by transrectal ultrasonography every 6 h from the onset of estrus. Interval from onset of estrus to mean time of ovulation (EOI) was analyzed using general linear model procedures of SAS (version 9.0; SAS Institute, Cary, NC, USA). Least squares means and standard deviations of EOI were compared using ANOVA. The proportion of sows ovulating within 45 h after onset of estrus was compared using Fisher’s exact test. Differences with P < 0.05 were considered significant. All of the sows in the control and hCG groups ovulated within 5 days after onset of estrus, but 3 out of 13 (23%) sows in the GnRH group developed cystic follicles; these 3 sows were excluded from the analyses. Overall, WOI was 3.8 ± 0.9 days and did not differ among the groups (P ± 0.05). The breed of sow had no effect on the EOI (P ± 0.05). Although the EOI did not differ among the control (43.0 ± 19.2 h), hCG (40.2 ± 5.5 h), and GnRH (37.5 ± 10.3 h) groups (P ± 0.05), variation was less (P = 0.001) after administration of hCG or GnRH. In addition, the proportions of sows that ovulated within 45 h after onset of estrus in the hCG (9/10 sows) and GnRH (8/10 sows) groups tended to be higher than in the control group (5/10 sows; P = 0.10). In conclusion, results indicate that both hCG and GnRH are efficacious in inducing ovulation at a predictable time in weaned, spontaneously estrous sows. The occurrence of follicular cysts in GnRH-treated sows requires further investigation.