bluuebunny/crossref_metadata_2025 huggingface dataset:
173 ESTRADIOL-17β CONCENTRATIONS IN BLOOD AND MILK DURING SUPEROVULATORY TREATMENT IN DAIRY COWS
Authors: Dupras R. , Dupras J. , Chorfi Y.
Published: August 2010

In cows, estradiol-17β is usually used to synchronize follicular wave emergence during superovulatory treatment. This approach, however, raises some concerns about the presence of estrogens in bovine products and their possible association with some human estrogen-sensitive cancers. The objective of this study was to determine estradiol-17β concentrations in blood and milk of dairy cows after i.m. injection of estradiol-17β and to compare these concentrations to those obtained during standard superovulation protocols. Six cows were used for this experiment. On Day 0, corresponding to Day 7 of their ensuing cycle, cows received 4.5 mg of estradiol-17β (Gentes et Bolduc, St-Hyacinthe, Québec, Canada) via i.m. injection and a progesterone-releasing vaginal insert (1.9 g of progesterone, CIDR, Pfizer Animal Health, Kirkland, Québec, Canada). Blood and milk samples were taken at 0, 24, 48, and 72 h after injection. From Day 4 evening to Day 8 evening, the cows received a total of 380 mg of NIH-FSH-P1 (Folltropin-V, Bioniche Animal Health, Belleville, Ontario, Canada) administered i.m. through 9 injections of decreasing dose (from 70 to 20 mg) at 12-h intervals. On Day 7, the cows received 2 injections consisting of 500 μg of cloprostenol (prostaglandin F2 α analogue, Estrumate, Shering-Plough, Pointe-Claire, Québec, Canada) given approximately 12 h apart and vaginal inserts were removed 12 h after the last injection. Artificial insemination was performed on Day 9 and 10 after treatment with 100 μg of GnRH i.m. (Cystorelin, Merial Canada Inc., Baie Urfe, Québec, Canada). A second batch of blood and milk samples was taken at Day 8, 9, 10, and 11. Measurement of estradiol-17β was performed with an IMMULITE chemiluminescent counter using an IMMULITE Estradiol Kit (Siemens Diagnostic Products Corporation, Los Angeles, CA, USA). Concentrations of estradiol-17β in blood (37.1 ± 15.6 pg mL-1 at 24 h, 19.1 ± 14.2 pg mL-1 at 48 h) and milk (38.4 ± 29.5 pg mL-1 at 24 h, 9.3 ± 4.9 pg mL-1 at 48 h) were significantly higher after i.m. injection of 4.5 mg of estradiol-17β. In comparison, superovulation heat (Day 9 to 11) increased estradiol-17β concentrations in blood (20 ± 13.6 pg mL-1 at 24 h, 32.5 ± 16.3 pg mL-1 at 48 h) but not in milk.
179 COMPARISON OF OOCYTE AND EMBRYO PRODUCTION AMONG BOS TAURUS, BOS INDICUS, AND INDICUS-TAURUS DONOR COWS
Authors: Pontes J. H. F. , Silva K. C. F. , Basso A. C. , Ferreira C. R. , Santos G. M. G. , Sanches B. V. , Porcionato J. P. F. , Vieira P. H. S. , Sterza F. A. M. , Seneda M. M.
Published: August 2010

In recent years, Brazil has become the leading country in the world for the number of embryos produced in vitro (Thibier M 2009 IETS Embryo Transfer Newsletter 22, 12-19). This is partly due to the large numbers of Bos indicus animals in Brazil, making up about 80% of the total cattle. The mean oocyte production per ultrasound-guided follicular aspiration from Bos indicus is higher than those for European breeds (Pontes JHF et al. 2009 Theriogenology 71, 690-697). In the present study, we analyzed 5407 ovum pick ups (OPU) and compared the average production of total (n = 90,086) and viable (n = 64,826) oocytes and the number of embryos produced in vitro from Gir (Bos taurus indicus), Holstein (Bos taurus taurus), 1/4 Holstein × 3/4 Gir, and 1/2 Holstein-Gir crossbreed cows. To obtain oocytes, OPU was repeated from 4 to 7 times (mean = 5.7 ± 2.4) in each donor cow aged from 3 to 7 years (mean = 5.0 ± 2.3) during a 12-mo period. COCs (n = 90,086) obtained were classified according to the presence of cumulus cells and the oocyte cytoplasm aspect (homogeneous or heterogeneous/fragmented). The viable oocytes (n = 64,826) were in vitro matured for 24 h at 38.8°C in an atmosphere of 5% CO2 in air. Since this was a commercial programm, frozen sexed semen (2 × 106 mL-1) from Gir (n = 8) or Holstein (n = 7) sires previously tested for high efficiency was used for IVF. Fertilization was carried out (18-20 h) and the presumed embryos were cultured for 7 days in the same conditions as were used for IVM. Data were analyzed by ANOVA. On average, 16.7 ± 6.2 oocytes were obtained per OPU/IVF procedure and 71.96% were considered viable. The mean numbers of total oocytes per OPU/IVF procedure were 17.1 ± 4.4 for Gir cows (n = 617), 11.4 ± 3.9 for Holstein cows (n = 180), 20.4 ± 5.8 for 1/4 Holstein × 3/4 Gir (n = 44), and 31.4 ± 5.6 for 1/2 Holstein-Gir crossbreed females (n = 37, P < 0.01). The mean numbers of viable oocytes per OPU/IVF procedure were 12.1 ± 3.8 for Gir cows, 8.0 ± 2.6 for Holstein cows, 16.8, ± 5.0 for 1/4 Holstein × 3/4 Gir, and 24.3 ± 4.7 for 1/2 Holstein-Gir crossbreed females (P < 0.01). The average number of embryos produced by OPU/IVF were 3.2 (n = 12,243/3378) for Gir cows, 2.2 (n = 2426/1138) for Holstein cows, 3.9 (n = 1033/267) for 1/4 Holstein × 3/4 Gir, and 5.5 (n = 1222/224) for 1/2 Holstein-Gir. The average number of embryos produced per IVF session from 1/2 taurus × indicus donor cows was greater (P < 0.01) than from Bos indicus cows. The number of recoverable and viable oocytes and the number of embryos produced in vitro from Bos indicus donors were higher than from Bos taurus females. Therefore, the highest oocyte yield and the greatest embryo production were obtained from 1/2 taurus × indicus females.

This work was supported by In Vitro Brasil.
181 CHANGES IN THE BODY CONDITION STATUS OF DONOR MARES THROUGHOUT THE BREEDING SEASON AND THEIR RELATIONSHIP WITH REPRODUCTION TRAITS
Authors: Rodrigues P. G. , Raymundo C. M. , Miranda M. C. M. G. , Bastos J. B. , DeSouza J. C.
Published: August 2010

The objective was to investigate the relationship between changes in body condition on reproductive traits of donor mares in an embryo collection program. Fifty-six non-pregnant mares, 3 to 18 years old, were studied from August 2008 through April 2009. Body condition score (BC; 1-9 scale, thin to obese) was estimated monthly. Body weight and tail and rib s.c. fat depths (SF) were measured monthly. SF was determined by ultrasound. Dominant follicle diameter (DF) was measured daily during the follicular phase. Mares were artificially inseminated and embryo transfers were conducted 6 d after ovulation. Analyses were performed with SAS® (SAS Institute Inc., Cary, NC, USA). Age classes were as follows: age 1, 3 to 6.9 (n = 17); age 2, 7 to 12.9 (n = 25), and age 3, ≥13 years (n = 14). Months in experiment were T1 to T6. BC classes were as follows: B1, <6.5; B2, 6.5 to 6.9; B3, 7.0 to 7.5; and B4, >7.5. Embryo recovery rate was compared by chi square. Effects of month and age on BC, weight, and SF were analyzed by a mixed model. Age, BC, and month fixed effects on DF diameter were analyzed by ANOVA with mare within class as the error term. Means were compared by contrasts. BC was higher (P = 0.02) in older mares (6.78 ± 0.2 and 6.93 ± 0.2 in age 1 and age 2 v. 7.25 ± 0.2 in age 3) and increased (P = 0.009) with time (from 6.79 ± 0.20 to 7.42 ± 0.26 for T1 to T6, respectively). Weight (kg) was not associated with age but increased (P = 0.03) throughout the experiment. Tail fat increased with age (P = 0.04) and decreased with increasing months on experiment (P = 0.02). DF diameter increased daily through the follicular phase (P < 0.0001) and was smaller (P < 0.05) in mares with lower body condition (33.80 ± 0.31 in BC1 v. 34.17 ± 0.31 mm in BC4). DF diameter decreased (P < 0.01) with month on study. Embryo recovery rate was not affected by age or BC but was lower (P = 0.003) in mares that ovulated follicles smaller than 40 mm compared to mares ovulating follicles greater than 40 mm. BC above 6.5 was associated with higher embryo recovery rate, which was, in turn, correlated positively with DF diameter. Thus, our results support the use of BC scoring to predict performance in equine embryo transfer programs. Body condition scoring was not a good indicator of SF under the current experimental conditions because SF decreased during the breeding season. BC scoring may indicate lean tissue gain, which may be a better indicator of reproductive efficiency in mares.

FAPEMIG, CAPES, Haras El Far.
190 RELATIONSHIP BETWEEN BLUETONGUE VIRUS INFECTION AND FETAL MORTALITY IN CATTLE
Authors: Ponsart C. , Gatien J. , Pozzi N. , Humblot P. , Guérin B.
Published: August 2010

Infection by Bluetongue virus (BTV) may affect reproductive performance but little is known about its real impact on fertility and occurrence of abortions. The aim of this study was to describe the effects of BTV infection on fetal mortality in 53 herds located in the east of France that were infected by the virus between August and November 2007. In 2008, 2 visits were performed 50 days apart by local veterinary surgeons and pregnancy was checked by ultrasonography. The first visit aimed to record data on pregnancy and infection status and to register clinical signs. A total of 1939 cows were classified within 3 groups: nonpregnant (n = 262), early pregnant (<3 months, n = 1041), and late pregnant (>3 months, n = 636). For each cow, a blood sample was collected approximately 8 days after the first visit and BTV status was determined by ELISA. The rates of clinical symptoms and fetal losses were compared in seronegative and seropositive cows using chi-square tests. Bluetongue status was found positive in 69.5% of the cows (80% of beef v. 67% of dairy cows). Prevalence was highly variable between herds: 19% of herds had 100% of cows with positive serological results and 10 herds presented at least 70% of seronegative results. Clinical signs were reported by farmers in 40% (457/1141) of seropositive cows compared to 20% (90/460) of seronegative cows (P < 0.001). As a consequence, clinical signs should not be considered as a reliable tool for diagnosing BTV infection with enough accuracy. At first visit, ultrasonography was performed in cows that were supposed to be pregnant. At second visit, a positive pregnancy status was confirmed in 78% of the cows, but this proportion was higher in seronegative cows when compared with seropositive cows (81 v. 76%; P < 0.02). This proportion was associated with the presence of clinical signs. Out of 1311 pregnant cows at first visit, only 37 presented a fetal mortality at second pregnancy check (2.8%).The rate of fetal mortality was related to the BTV-seropositive status of cows as well as stage of pregnancy (P < 0.05; Table 1). Calving interval did not differ between seropositive and seronegative females (411 ± 77 days v. 405 ± 82 days; P > 0.10); however, number of AI per conception was significantly higher in seropositive than in seronegative females (2.0 ± 1.2 v. 1.8 ± 1.1; P < 0.001). These results show that a seropositive BTV status assessed by serological test (ELISA) is associated with an increased frequency of fetal mortality in beef and dairy cattle, and with an increased number of AI per conception.

Table 1.Rate of fetal mortality observed between 2 pregnancy diagnosis 50 days apart (negative pregnancy check during the 2nd visit in cows which were pregnant at first visit)

The authors thank the farmers and veterinarians who participated to this trial, as well as the GDS (Groupements de Defense Sanitaire) organization from Moselle and Meuse departments for collecting the data.
21 GENETIC APPROACH TO IMPROVE FERTILITY IN CATTLE
Authors: Di Croce F. A. , Saxton A. M. , Casanova D. , Schrick F. N.
Published: August 2010

In spite of the economic importance of fertility and increased knowledge of reproductive biology in the cow, efficiency of reproductive performance has continued to decline in dairy cattle. Improvements in fertility through genetic selection may be a possible approach to increase reproductive efficiency in dairy cattle. Although progress may be slow because of low heritabilities, ignoring fertility in genetic improvement programs has contributed to the current fertility problems. The objectives of this study were to (1) estimate genetic parameters and breeding values for fertility traits based on data from milk recording programs on a Holstein population and (2) develop a multi-trait selection index (dollar fertility value; $F) based on estimated breeding values. Data representing 3,282,843 lactations and 1,622,088 animals (cows and bulls from 1936 to 2007) were used for the analysis and obtained from official records maintained by the Argentinean Holstein Association (ACHA). Data were collected from official milk records, and lactations were standardized to 305 DIM for milk, protein, and fat. Gestation period was considered to be 282 days and restrictions were applied to ensure the quality of data (days open <40 and >350 days; calving interval <300 and >600 days; and contemporary groups with <25 lactations were eliminated). Days open (DO), calving interval (CI), age to first calving (AFC), and daughter pregnancy rate (DPR) were traits chosen for genetic parameter estimation. Daughter pregnancy rate was calculated from DO as PR = 0.25 × (233 DO); Kuhn et al. 2004 J. Dairy Sci. 87, 2277-2284. Animal models by lactation for each fertility trait included contemporary group (dairy herd and calving year), calving month, animal effect, milk production as a covariate and error term. Multiple-trait derivative-free restricted maximum likelihood (MTDFREML) was used to estimate covariance components (Boldman et al. 1995 ARS, USDA). Solutions for fixed effects, breeding values, and sampling variances (accuracies) were obtained for each trait. The proposed selection index ($F) included AFC and CI, with economic weights from -0.28 and -4.9 $/year per cow for AFC and CI, respectively (Gonzalez-Recio et al. 2004 J. Dairy Sci. 87, 3053-3061). Heritabilities for DO and DPR ranged from 2 to 7%; CI ranged from 3 to 8%; and AFC was 16%. Predicted transmitting ability values across different lactations ranged from -18.5 to 15.8 days, -23.15 to 16.0 days, -4.0 to 4.6%, and -120.3 to 76.2 days for DO, CI, DPR, and AFC, respectively. Values for $F ranged from -$89.16 to $147.12 in this Holstein population. Results indicate substantial variation in fertility traits, suggesting that genetic selection may be effective in improving declines in fertility.

Asociacion Criadores Holando Argentino (ACHA).
212 EXPRESSION OF FIBROBLAST GROWTH FACTOR 18 (FGF18) AND COGNATE RECEPTORS (FGFR3C AND FGFR4) DURING LUTEAL DEVELOPMENT AND INDUCED LUTEOLYSIS IN CATTLE
Authors: Guerra D. M. , Castilho A. C. S. , Machado M.F. , Berisha B. , Schams D. , Price C. A. , Amorim R. L. , Buratini Jr J.
Published: August 2010

Fibroblast growth factor receptor 2 (FGFR2) has been shown to induce luteinization in granulosa cells, luteal angiogenesis, and luteal growth. Alternative splicing of 4 genes give rise to 7 subtypes of fibroblast growth factor receptors (FGFR) with varying affinity for different fibroblast growth factors (FGF). Fibroblast growth factor receptor 2 and FGF18 efficiently activate FGFR3C and FGFR4 and may act in cooperation in tissues expressing these receptors. We aimed to determine mRNA expression patterns for FGF18, FGFR3C, and FGFR4 during bovine luteal development and following induced luteolysis. In addition, we assessed FGF18 localization in the bovine CL. Bovine CL were obtained from abattoir ovaries and classed into 4 stages of development: stage 1 =corpus hemorragicum; stage 2 = developing CL; stage 3 = mature or early functional luteolysis CL; and stage 4 = structural luteolysis. To assess FGF18 and FGFR mRNA expression during induced luteolysis, adult cows (Bos taurus Holstein-Friesians) were injected with the PGF2 analogue cloprostenol (500 mg i.m. Intervet, Unterschleissheim, Germany) during the mid-luteal phase of the cycle (Days 8-12). Corpus luteum were collected by transvaginal ovariectomy at 0, 0.5, 2, 4, 12, 24, 48, and 64 hr (n = 5/time point) after PGF2 injection. Tissue samples were submitted to total RNA extraction. Expression of FGF18, FGFR3C, and 4 mRNA during the bovine CL lifespan and induced luteolysis were measured by real-time RT-PCR with oligo-dT in the RT and bovine-specific primers in the PCR. Expression of cyclophilin was used as internal control. The effect of developmental stage and time post-PGF2 on gene expression was tested by ANOVA, followed by Tukey- Kramer HSD test. Immunohistochemical analysis was performed with a commercial human antibody (anti-FGF18; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Fibroblast growth factor 18, FGFR3C, and FGFR4 mRNA was detected in all 4 developmental stages; FGF18 mRNA abundance was higher in stage 3 (2.89 ± 0.05; mean ± SEM) compared with stages 1 (0.3 ± 0.27), 2 (0.56 ± 1.27), and 4 (0.99 ± 0.32). Fibroblast growth factor 18 and FGFR4 mRNA expression did not significantly change during induced luteolysis. Fibroblast growth factor receptor 3C mRNA abundance peaked 4 h after PGF2 injection and significantly decreased at 24 h post-treatment in comparison with peak levels. Immunohistochemical analysis revealed the presence of FGF18 in small and large luteal cells and in blood vessels. In conclusion, the mRNA expression patterns of FGF18 and its receptors suggest their participation in the control of luteal differentiation, particularly during functional luteolysis. The localization of FGF18 protein to blood vessels suggests it may play a role in the control of angiogenesis in the bovine CL.

Supported by CAPES/FAPESP.
214 PREANTRAL FOLLICLE BANKING AND ULTRASTRUCTURAL DEFORMITY INDUCED BY DIFFERENT CRYOPRESERVATION PROTOCOLS
Authors: Kim G. A. , Kim H. Y. , Kim J. W. , Lee G. , Lee E. S. , Park J. H. , Lim J. M.
Published: August 2010

Establishment of cryopreserved follicle banks would provide a valuable source of immature oocytes for subsequent follicular culture and embryo pro- duction. In order to develop an optimal cryopreservation protocol, a comparison of ovarian and follicular cyropreservation procedures was performed. Whole ovaries or isolated follicles of B6CBAF1 mice were either frozen slowly or vitrified, and post-thaw follicle growth and oocyte maturation, parthenogenetic activation and embryo development after IVF, and follicle ultrastructure were subsequently monitored. In ovary cryopreservation, intrafollicular oocytes (n = 67-80) can mature (n = 20-21; 25-31%) and develop into blastocysts (n = 8-12; 12-16%) after parthenogenesis. When comparing optimal methods, slow freezing using 1.5 M of DMSO with a 0.5-mL straw resulted in more cleavage than did vitrification using 7.5% ethylene glycol (EG) + DMSO with an EM grid (P < 0.05) after IVF. In follicle cryopreservation, slow freezing (n = 79) yielded only mature oocytes (n = 19; 24%), but none were parthenogenetically activated. Major cryoinjury after ovary cryopreservation was intracytoplasmic vacuole formation and mitochondrial deformity. In follicular cells, vitrification induced more damage in mitochondria than did slow freezing, whereas both induced mitochondrial damages and vacuole formation in ooplasm. Slow freezing did not damage zona pellucida. In conclusion, banking of the whole ovary yields better outcome of follicle culture than follicle banking and, compared with vitrification, slow freezing efficiently supports post-thaw survival and ultrastructure normality in ovary cryopreservation. However, each protocol induces cell-specific damage.

This research was supported by a grant (Code 200504676) from BioGreen 21 Program, Rural Development Administration, Republic of Korea. The authors also acknowledge a world-class university program supported by the Korean Ministry of Education, Science, and Technology.
216 DINOPROST ADMINISTRATION, PERIOD OF OVULATION, AND OVULATORY FOLLICULAR SIZE ON DAYS FOUR TO SIX OF ESTROUS CYCLE IN EWES
Authors: Nascimento P. M. P. , Oliveira A. P. , Brandao F. Z. , Maia A. L. R. S. , Pinto P. H. N. , Oliveira M. E. F. , Bruschi J. H. , Viana J. H. M. , Fonseca J. F.
Published: August 2010

The aim of this study was to evaluate the effects of 2 doses of dinoprost at the time of ovulation on Days 4 to 6 of the estrous cycle in ewes. The experiment was conducted in the city of Piau (Minas Gerais, Brazil) from July to August 2008 during the local no-breeding season. Twenty-four ewes with body condition score 3.0 ± 0.75 were used for this experiment. The estrous cycle was synchronized with intravaginal releasing devices containing 0.33 g of progesterone for 6 days (Eazi-Breed CIDR®, Pfizer Animal Health, São Paulo, Brazil), and 12.5 mg of dinoprost (Lutalyse®, Pfizer do Brasil Saúde Animal, São Paulo, Brazil) laterovulvar plus 300 IU of eCG 24 h before device removal. Animals were monitored by transrectal ultrasonography every 8 h and subjected to 5 or 10 mg of dinoprost on Days 4, 5, or 6 of the cycle. Results were submitted to ANOVA and Tukey test, using all tests at the 95% confidence interval (SAEG program, Funarbe, Viçosa, Brazil). Data were reported as percentage or mean ± SD. The period from device removal to ovulation was 48.0 ± 7.07 h. The 5-mg dinoprost dose evoked similar (P > 0.05) results for Days 4, 5, and 6 regarding the time of ovulation after luteal gland regression induction [71.3 ± 28.6, 71.46 ± 30.4, and 68.2 ± 29.2 h for Days 4 (n = 4), 5 (n = 4), or 6 (n = 4), respectively] and follicle size (6.48 ± 0.34, 6.41 ± 0.35, and 6.5 ± 0.32 mm for Days 4, 5, and 6, respectively) at the time of ovulation. In animals that received 10 mg of dinoprost, the time of ovulation was affected (P ≤ 0.05) by Day of treatment [71.3 ± 27.6, 68.3 ± 30.6, and 64 ± 29.2 h for Days 4 (n = 4), 5 (n = 4), and 6 (n = 4), respectively]. However, there was no effect of the 10 mg on follicle size at ovulation (6.48 ± 0.34, 6.41 ± 0.35, and 6.3 ± 0.33 mm for Days 4, 5, and 6, respectively). There was no difference (P ≥ 0.05) on the size of ovulatory follicle(s) among animals with 1 (n = 4, 4, and 1 for Days 4, 5, and 6, respectively), 2 (n = 3, 3, and 4 for Days 4, 5, and 6, respectively), and 3 (n = 1, 1, and 3 for Days 4, 5, and 6, respectively) ovulations. The difference in the interval from luteal gland regression to ovulation in animals that received 10 mg of dinoprost on Day 6 of the estrous cycle suggests a higher sensitivity of corpora lutea at that moment. Results showed that both doses of dinoprost were capable of inducing early luteal gland regression; this fact supports the use of short-term protocols for the induction of estrus in ewes.

Pfizer Brazil, CNPq, FAPEMIG.
219 FOLLICLE AND HORMONE DYNAMICS IN SINGLE- v. DOUBLE-OVULATING HEIFERS
Authors: Palhao M. P. , Beg M. A. , Rodrigues M. T. , Araújo R. R. , Viana J. H. M. , Borges A. M. , Ginther O. J.
Published: August 2010

The present experiment used the Day 4 ablation model for increasing the incidence of double ovulations in heifers. The objective was to compare follicle growth and plasma hormone concentrations associated with single v. double ovulations. Follicles ≥5 mm were ablated at 4 days post-ovulation to induce a prominent FSH surge and a new follicular wave, and 2 injections of PGF2 (12 h apart) were given 2 days later (Day 6) to favor ovulation. Beginning on Day 5, the 3 largest follicles of the induced wave were scanned twice a day until 36 h after the next ovulation. Blood samples were taken at 6-h intervals starting when the largest follicle reached ≥8.0 mm (expected deviation at 8.5 mm; Ginther et al. 1996) and continued until 36 h after the next ovulation. Concentrations of LH and FSH were measured by validated RIA for cattle (LH measured by Ginther et al. 1999; FSH measured by Adams et al. 1992) and concentrations of oestradiol measured by a commercially available RIA kit (Siddiqui et al. 2009). From a total of 31 heifers, 16 (52%) or 15 (48%) developed a single or more than 1 dominant (≥ 10 mm) follicle in the follicular wave after ablation, respectively. For heifers with 2 dominant follicles, the second-largest follicle ovulated in 9 (60%) heifers and the overall double ovulation rate was 29% (9/31). Follicle diameters and plasma hormone concentrations were compared between single ovulators (n = 12) and double ovulators (n = 8). Diameter of the preovulatory follicles did not increase between the LH peak and ovulation in either the single or double ovulations. In double ovulators, the interval from follicle deviation to the peak of the preovulatory LH surge was shorter (1.9 ± 0.2 days v. 2.5 ± 0.2 days; P < 0.02) and the diameter of the largest preovulatory follicle was smaller (12.2 ± 0.5 mm v. 13.3 ± 0.3 mm; P < 0.02) than in single ovulators, respectively. The LH concentrations of the preovulatory surge did not differ between single and double ovulators for 24 h on each side of the peak (main effect of hour only; P < 0.0001). When data were normalized to the LH peak, the peak of the preovulatory FSH and estradiol surges occurred in synchrony with the peak of LH surge for both groups. A group effect (P < 0.0001) for FSH resulted from a lower concentration averaged over hours in double ovulators. Estradiol showed a group by hour interaction (P < 0.008), reflecting greater concentrations in the double ovulators before and at peak. In conclusion, an increased

Supported by the Eutheria Foundation, Cross Plains, WI, USA. Submission supported by FAPEMIG.
226 EFFECT OF AMMONIA IN BOVINE FOLLICULAR FLUID AND OOCYTE MORPHOLOGY DERIVED FROM ABATTOIR OVARIES
Authors: Urakawa M. , Ideta A. , Tsuchiya K. , Aoyagi Y.
Published: August 2010

In cattle, high intake of crude protein elevates rumen ammonia and blood urea nitrogen and has been associated with decreased fertility. The aim of this study was to analyze the concentration of ammonia in bovine follicular fluid derived from abattoir ovaries and to examine the relation between ammonium concentration and morphology of oocytes from antral follicles. In the first experiment, follicular fluid was collected within 1 h of slaughter from 3919 small (2-5 mm in diameter), 366 medium (6-9 mm), and 159 large (10-20 mm) follicles from 203 ovaries. Ammonium concentration was analyzed in 6 batches pooled from follicular fluids of each size (derived from 15, 39, 7, 36, 81, and 25 ovaries, respectively). Ammonium concentration was measured by the microdiffusion method using an Ami Check Meter kit (Kyoto Daiichi Kagaku Co., Ltd., Japan). In the second experiment, follicular fluids were aspirated from small (2-5 mm) follicles of 38 cows to evaluate the relationship between ammonium concentration and oocyte morphology. Oocytes were classified into 4 morphological categories according to the surrounding cumulus cells (Konishi M et al. 1996 Theriogenology 45, 573-581): grade 1 (≥5 layers); grade 2 (3-4 layers); grade 3 (1-2 layers); and grade 4 (denuded, degenerated cumulus cell mass or ooplasm). The oocyte morphology for the low-ammonium group (<330 μmol, 239 oocytes from 19 cows) and high-ammonia group (≥330 μmol, 255 oocytes from 19 cows) was compared. Statistical significance was tested by chi-square test or t-test Ammonium con- centrations of small (281.8 ± 20.0 μM), medium (175.4 ± 15.8 μM), and large (96.3 ± 9.3 μM) follicles were significantly different (P < 0.0001). Ammonium concentrations in follicular fluid of the 38 cows ranged from 180.6 to 802.4 μM. In a comparison of oocyte morphology between the low- (263.8 ± 41.6 μM) and high-ammonium (423.0 ± 108.9 μM) groups, the number of grade 1, 2, 3, and 4 oocytes in the former (80, 68, 18, and 73, respectively) were not significantly different from the numbers in the latter (96, 61, 27, and 71, respectively; P = 0.30-0.99). These results indicate that, in follicles of different sizes derived from abattoir ovaries, the ammonium concentration was higher in small follicles than in the other sizes of follicles. However, there was no evidence that in vivo exposure of bovine oocytes to ammonium badly influenced oocyte morphology. Further studies are needed to examine the relationship between ammonium concentration and the developmental potential of oocytes.
227 EFFECTS OF A SINGLE DOSE OF A NONSTEROIDAL AROMATASE INHIBITOR ON OVARIAN FUNCTION IN CATTLE
Authors: Yapura M. J. , Mapletoft R. J. , Singh J. , Pierson R. A. , Adams G. P.
Published: August 2010

Many countries have banned the use of estrogenic products in farm animals. Nonsteroidal aromatase inhibitors prevent the body from producing its own estrogen. The effects on the ovary are thought to be a result of suppression of estrogen-producing follicles and a rebound in endogenous levels of FSH through the removal of the negative feedback effect of estradiol. An experiment was designed to determine the effects of a nonsteroidal aromatase inhibitor, letrozole, on ovarian function in cattle. The specific objective was to test the hypothesis that letrozole will arrest growth of the dominant follicle, resulting in emergence of a new follicular wave at a predictable interval post-treatment. Beef heifers were assigned randomly to 4 groups and given phosphate-buffered saline [control; (n = 10)] or letrozole at a dose of 500 (n = 9), 250 (n = 10), or 125 (n = 10) μgkg-1 i.v. 4 days after follicular ablation (≈ 2.5 days after wave emergence, at the time dominant follicle selection is manifest). Blood samples were collected and ovarian structures were monitored daily by transrectal ultrasonography. Analysis of variance for repeated measures, one-way ANOVA, paired t-test, and 2-sample t-test were applied to the analysis of the data. The diameter profile of the dominant follicle was larger in heifers treated with letrozole than in control heifers (P < 0.05). The intervals from treatment to new wave emergence and from treatment to onset of regression of the extant dominant follicle were longer (P < 0.05) in heifers treated with letrozole than in controls, although variances in the intervals were not different. A small but significant reduction in circulating estradiol concentration was observed, and plasma LH concentrations were higher (P < 0.05) in letrozole-treated heifers than in controls. Lower plasma concentrations of FSH in letrozole-treated heifers than in controls (P < 0.03) was interpreted as an indirect effect resulting from prolonged follicular dominance. In summary, a single dose of letrozole did not induce regression of the extant dominant follicle, nor did it directly affect FSH release. Conversely, letrozole extended the lifespan of the dominant follicle, in association with increased endogenous levels of LH, thereby delaying the next FSH surge and subsequent follicular wave emergence. Results suggest that letrozole has potential as a nonsteroidal method of controlling ovarian function in cattle but further studies are needed to clarify dosage and timing of treatment.

Research supported by the Natural Sciences and Engineering Research Council of Canada and Bioniche Life Science Inc.
229 EXPRESSION OF mRNA ENCODING GLYCOLYTIC ENZYMES IN BOVINE CUMULUS CELLS DURING IN VITRO MATURATION: EFFECTS OF TIME AND FSH
Authors: Caixeta E. S. , Ripamonte P. , Machado M. F. , da Silva R. B. , Price C. , Barros C. M. , Buratini Jr J.
Published: August 2010

Mammalian oocytes require pyruvate as an energy source for growth and resumption of meiosis. Because oocytes are not competent to carry out glycolysis, cumulus cells (CC) are responsible for metabolizing glucose into pyruvate and providing it to the oocyte through gap junctions. The understanding of the energetic metabolism of CC in culture conditions might provide basis for the improvement of COC in vitro maturation. The aim of this study was to determine the temporal patterns of mRNA expression of glycolytic enzymes [phosphofructokinase (PFKP), aldolase (ALDOA), triosephosphate isomerase (TPI), enolase (ENO1), pyruvate kinase (PKM2), and lactate dehydrogenase (LDHA)] in bovine CC during COC in vitro maturation with or without FSH. Immature COC (grades 1 and 2) were obtained from 2- to 8-mm follicles from abattoir ovaries (predominantly Bos indicus). Cumulus cells were separated from COC and frozen before (immature group) or after COC culture for 4, 8, 12, 16, and 20 hours with (10 ng/mL) or without FSH. Total RNA was extracted using RNeasy® (Qiagen, Valencia, CA, USA), and 100 ng of RNA was reverse transcribed using oligo dT primers and Omniscript® (Qiagen). Relative expression of target genes was assessed by real-time PCR using bovine-specific primers and Power SYBR green master mix in an ABI Prism® 7300. To select the most stable housekeeping gene for expression normalization, cyclophilin-A (CYC-A), GAPDH, and histone H2AFZ amplification profiles were compared using the geNorm applet for Microsoft Excel (Vandesompele J et al. 2002 Genome Biol. 3, 1-11); the most stable housekeeping gene was CYC-A. Relative expression values were calculated using the AACt method with efficiency correction (Pfaffl MW 2001 Nucleic Acids Res. 29, 2002-2007). Effects of time in culture and of FSH treatment were tested by ANOVA, and groups were compared by Tukey-Kramer Honestly Significant Difference test. Nonparametric analysis was used when data were not normally distributed. Abundance of mRNA of all glycolytic enzymes decreased during in vitro maturation with or without FSH. Expression of PFKP, ALDOA, TPI1, ENO1, and LDHA genes was decreased to around half of the initial value (time 0) by 4 to 8 h of culture (P < 0.05) and did not increase thereafter. A similar expression pattern was observed for PKM2, although mRNA abundance was reduced later in comparison with other enzymes; levels were decreased by 16 (without FSH) to 20 h (with FSH) of culture. The presence of FSH did not alter the overall temporal pattern of gene expression but decreased mRNA abundance for PFKP, ALDOA, and TPI1 at 20, 16 and 16 h of culture, respectively. In conclusion, gene expression of glycolytic enzymes decreased with time during COC in vitro maturation in cattle, and FSH did not have a major influence on this expression pattern.

This study was supported by CAPES and FAPESP.
24 EFFECT OF GnRH ON FIXED-TIMED ARTIFICIAL INSEMINATION PREGNANCY RATES OF WHITE-TAILED DEER
Authors: Lambe J. , Forbes W. , Olcott B. M. , Sanders D. E. , Godke R. A. , Gentry G. T.
Published: August 2010

During the fall 2008 breeding season in Louisiana, 2 synchronization protocols for fixed-timed artificial insemination (FTAI) in White-tailed deer were assessed. The objective was to determine if GnRH at FTAI improved pregnancy rates in White-tailed deer. White-tailed does (n = 35) with a mean body weight of 65.8 kg (range: 54.0 to 79.8 kg) and average age of 5.9 years (range: 1.5 to 9.5 years) were stratified by weight, age, and last fawning date into 2 groups. Treatment and control synchronization protocols were randomly assigned to each group. Does were synchronized with a CIDR-G device for 14 d and were then subjected to either FTAI 60 hpost-CIDR removal (control, n = 17) or 100 μg (i.m) injection of GnRH at FTAI 60 h post-CIDR removal (treated, n = 18). At insemination (AI), insemination pipette placement score (IP; 0 = at cervical os to 3 = within uterine body), mucous scores (clear/cloudy, viscous/nonviscous, or no secretions), vulva assessments (hyperemic/nonhyperemic and swollen/not swollen), and sperm progressive motility were recorded. Does were inseminated with frozen-thawed sperm (5 × 107 progressively motile cells pre-freeze) from 2 fertile bucks stratified across each treatment group. Starting 28 d following AI, intact bucks (ITB) were introduced into both groups for natural mating. Pregnancy was determined via transrectal ultrasonography 73 or 80 d postinsemination. Does, confirmed pregnant via ultrasonography, had fawns within the reported gestation range of 187 to 222 days. Placentomes were visualized and measured in AI pregnancies (range: 32.7 to 56.2 mm in length), whereas pregnancies derived from ITB presented no identifiable placentomes. However, crown-rump measurements were obtainable from ITB pregnancies (range: 13.4 to 21.7 mm). Five does were not included in the final analyses as they were either lost to predators or removed because of illness. IP (1.4 ± 0.24 v. 1.3 ± 0.30; P = 0.671), mucous classifications (3.0 ± 0.35 v. 2.5 ± 0.39; P = 0.311), vulva assessments (2.1 ± 0.29, 1.7 ± 0.23; P = 0.223), and sperm motility (1.6 ± 0.16, 1.7 ± 0.22; P = 0.829) were not different for pregnant and nonpregnant AI does, respectively. Treatment did not affect AI pregnancy rates (53 v. 27%) or fecundity rates (1.6 v. 1.3 offspring/doe) for the GnRH treated and control groups, respectively. Addition of GnRH to a 14-day estrus synchronization protocol did not result in significantly higher pregnancy rates compared with controls. More studies are needed to determine the effect of GnRH on White-tailed pregnancies following FTAI protocols. We have demonstrated that differentiating pregnancies derived from AI and ITB could be accomplished by utilizing transrectal ultrasonography as early as 73 d postinsemination in White-tailed does.
240 DIFFERENCES IN GLOBAL TRANSCRIPTOME PROFILE OF BOVINE BLASTOCYSTS DERIVED FROM SUPEROVULATED OR SYNCHRONIZED CYCLIC HEIFERS
Authors: Gad A. , Hoelker M. , Rings F. , Ghanem N. , Salilew-Wondim D. , Tesfaye D. , Phatsara C. , Schellander K. , Havlicek V. , Besenfelder U.
Published: August 2010

Estrus synchronization and superovulation are the most widely used procedures in embryo transfer technology. However, changes in the oviduct and uterine environment due to these procedures and the subsequent influence on embryos have not yet been investigated. This study was con- ducted to investigate the effect of oviduct environment of only synchronized or superovulated cyclic heifers on the gene expression profile of blastocysts. Bovine Affymetrix array analysis was performed using 2 groups of blastocysts. The first group was bovine blastocysts produced after superovulation of Simmental heifers (n = 9) using 8 consecutive FSH injections over 4 days in decreasing doses (in total, 300-400 mg of FSH equivalent according to body weight) and flushed at Day 7 by nonsurgical endoscopic method. The second group was bovine blastocysts derived from synchronized Simmental heifers (n = 4) after transfer of 2-cell stage embryos from superovulated donor Simmental heifers (n = 9) by nonsurgical transvaginal endoscopy tubal transfer method. Total RNA was extracted from 3 pools of embryos from each experimental group (6 embryos per pool). A total of 6 biotin-labeled cRNA samples were hybridized on 6 bovine Affymetrix arrays. Data analysis was performed using LIMMA written on R package, which maintained the Bioconductor. Array data analysis revealed a total of 454 transcripts to be differen- tially expressed (P < 0.05, fold change >2) between the 2 groups. Of these, 429 and 25 were up- and down-regulated, respectively, in blastocysts derived from superovulated heifers compared with those derived from synchronized animals. Genes involved in response to stress (HSPA14 and HSPE1), cellular and metabolic processes (CPSF3, ATPIF1, POMP, and MDH2), translation (RPS17, EEF1B2, and EIF4E), and cell commu- nication (FN1, KRT18, and DSG2) were found to be enriched in blastocysts derived from superovulated animals. On the other hand, protein metabolic processes related genes (CLGN) were found to be enriched in blastocysts derived from the synchronized group. The KEGG analysis of the differentially expressed genes showed that the ribosome and oxidative phosphorylation pathways are the dominant pathways and genes involved in these pathways are greatly abundant in the blastocysts derived from superovulated animals. Quantitative real-time PCR has confirmed the transcript abundance of 7 out of 8 genes selected for validation. In conclusion, blastocysts cultured in synchronized animals post 2-cell stage showed significant differences in transcriptome profile compared with their counterparts that remained in superovulated heifers until Day 7. Further functional analysis of some selected candidate genes could give new insights into mechanisms regulating the ability of embryos to survive after transfer.
262 REPORTS OF IN VITRO PRODUCTION AND PREGNANCY RATES OF BOVINE EMBRYOS PRODUCED BY POST-THAWING SEXED SEMEN
Authors: Avelino K. B. , Rossetto M. R. , Maraia A. C. , Lima M. R. , Mansano A. T. R. , Garcia J. M.
Published: August 2010

Births of predetermined-sex calves illustrates the importance of sperm sexing technology for in vitro production of bovine embryos (Johnson, 2000). Flow cytometry techniques for sperm sexing are usually performed on fresh semen. However, many bulls presenting high genetic value died or became unproductive before development of sperm sexing technologies. The technique of fresh semen sexing and semen reverse sorting method is based on separation of sperm according to differences in DNA content (X or Y). The objective of this study is to examine the blastocysts and pregnancy rates of bovine embryos produced by fertilization with sperm sexed with the post-thawing procedure, called reverse sorting. Oocytes were obtained by ovum pick-up from Dairy Gir, Nelore, and Guzera cows. The COC were transported to the laboratory and matured at 38.5°C and 5% of CO2 in air, TCM-199 medium with FCS (10% vol/vol), FSH (1.0 μg mL-1), hCG (50 μg mL-1), estradiol (1.0 μg mL-1), sodium pyruvate (0.20 mM), and amicacin (83.4 μg mL-1). IVF was performed 24 to 26 h after the onset of maturation. Doses of commercialized semen (3 to 5) from the specified breeds were sent to Goyaike Brazil Agropecuária Ltda. The semen sexing process was performed by flow cytometry and semen was returned to the IVF laboratory at 18°C. In the Tecgene laboratory, semen was centrifugated using Percoll gradient and evaluated. After 18 h, the presumptive zygotes were transferred to IVC in SOF medium at 38.5°C and 5% CO2 in air. The cleavage rate was evaluated 48 h after IVF and embryos were transferred at Day 7. A total of 5213 viable oocytes were obtained from 266 donors, fertilized with 7 different reverse sorted semen samples (1 Dairy Gir, 2 Nelore, and 4 Guzera) resulting in 1333 embryos. From these, 1084 transferred embryos resulted in 260 pregnancies to this time, with 226 females and 34 males. Each bull was evaluated separately according to cleavage rate, production of blastocysts, pregnancy rate, and percentage of females, respectively. Bull 1 presented 100%, 38%, 25%, and 90%; bull 2: 98%, 38%, 21%, and 90%; bull 3: 84%, 23%, 21%, and 84%; bull 4: 87%, 15%, 29%, and 86%; bull 5: 92%, 53%, 70%, and 71%; bull 6: 78%, 15%, 34%, and 71%, and bull 7: 55%, 27%, 50%, and 78%. The average of in vitro blastocysts (26%), the pregnancy rate (24%), and the percentage of females (87%) are similar to rates obtained by commercial fresh semen sexed by Goyaike Brazil Agropecuária Ltda. In conclusion, semen reverse sorting is an alternative method for selecting sex of in vitro production of bovine embryos using thawed semen collected from extinct or unproductive bulls.
264 EFFECT OF FROZEN SEMEN BATCH ON THE IN VITRO PRODUCTION OF BOVINE EMBRYOS
Authors: Fernandes M. B. , Torregrossa T. L. G. , Prado R. B. , Vila R. A. , Elias F. P. , Netto H. A.
Published: August 2010

Within an in vitro production controlled system, bulls differ with respect to their semen potential in generating embryos when the variables of maternal effect are minimized (Marquant-le-Guienne and Humblot 1992 Ann. Zootech. 41, 361-370). We have tested the hypothesis that even with this variation among bulls, there is also an intra-bull variation, according to the frozen semen batch used in the in vitro fertilization, identified with the date of ejaculate and its freezing. In an embryo commercial production system, over 12 months, 10 619 viable oocytes were obtained by ultrasound-guided follicular aspiration from 642 Nelore cows (Bos indicus). The oocytes were matured in vitro for 24 h in TCM-199 supplemented with 0.5 μg mL-1 FSH, 50 μg mL-1 LH, and 10% fetal bovine serum. They were then inseminated for 18 hours in IVF-TALP medium, using the semen from 4 bulls (A to D) subdivided into 4 frozen batches (I to IV) and selected by 45/90% Percoll gradient. Putative zygotes surrounded in cumulus cells were transferred in CR2aa medium drops (Rosenkrans and First 1994 J. Anim. Sci. 72, 434-437) for 163 h at 39°C in a humidified atmosphere of 5% CO2 in air. The oocyte distribution, the total number of blastocysts, and the embryo development rate by each bull and respective batch are described in Table 1. The chi-square test was measured with a significance level of P < 0.05 and showed that there is a difference between the used batches of each bull regarding the development rate of blastocysts 163 h after IVF Therefore, there is intra-bull variation in the ability to develop in vitro embryos according to the batch of frozen semen.

Table 1.Viable oocytes (VO), total blastocysts (TB), and embryo development rate (%E) by bull and batch used in IVF
268 IN VITRO EVALUATION OF FRESH SPERM QUALITY IN TOMCATS: A COMPARISON OF COLLECTION TECHNIQUES
Authors: Filliers M. , Rijsselaere T. , Bossaert P. , Anastasi P. , Hoogewijs M. , Van Soom A.
Published: August 2010

Semen can be collected from tomcats by urethral catheterization (CT) after medetomidine administration, offering a novel approach to obtain sperm for in vitro fertilization. This study was designed to determine motion characteristics of CT sperm samples by means of computer-assisted sperm analysis (CASA) and to compare sperm quality parameters and in vitro fertilizing capacity of CT spermatozoa with those of spermatozoa retrieved after epididymal slicing (EP). Semen was collected in 17 adult cats by urethral catheterization as reported by Zambelli et al. (2008 Theriogenology 69, 485-490), after which the cat was orchidectomized. Motility (subjective and objective by means of CASA), morphology [eosin/nigrosin (E/N)], plasma membrane integrity (E/N and SYBR14-PI staining), and acrosomal status (FITC-PSA staining) of fresh CT and EP samples were evaluated and compared between both methods with a paired t-test or the Wilcoxon Rank test, dependent on normality of the data. In vitro maturation (24 h), fertilization (18 h), and culture (6h) of grade I to III oocytes were carried out as described by Pope et al. (2009 Theriogenology 71, 864-871). Twenty-four hours after in vitro insemination, fertilization rates were assessed for group 1 (CT; n = 148) and group 2 (EP; n = 159) presumptive zygotes. The distribution of presumptive zygotes between CT and EP over the different developmental stages was compared using Pearson chi-square test. Results showed that total and progressive motility as well as the percentage of normal spermatozoa were higher for EP sperm compared with CT sperm (P < 0.01). Epididymal sperm had a lower percentage of spermatozoa with an intact acrosome (P < 0.01), whereas CT sperm contained more spermatozoa with tail abnormalities (P < 0.01). Other sperm parameters did not differ (P > 0.05) between collection techniques. In group 1, 84% of in vitro-matured oocytes (metaphase II) were penetrated (40.2% cleaved, 24.4% with 2 pronuclei, 12.2% with >2 pronuclei, 7.3% with expanded sperm head), whereas in group 2, penetration rate was 88.5% (42.7% cleaved, 21.8% with 2 pronuclei, 16.7% with >2 pronuclei, 7.3% with expanded sperm head). No difference (P > 0.05) in in vitro fertilizing capacity between spermatozoa collected by means of the 2 methods was found. In conclusion, semen collection by means of CT yields fertilization results similar to epididymal slicing, despite the fact that several sperm variables were different. Because CT is repeatable and easy to perform and does not require a trained male/queen in heat, it may be preferable for routine IVF experiments with fresh spermatozoa.

The first author is a research fellow of the Fund for Scientific Research-Flanders (Belgium).