bluuebunny/crossref_metadata_2025 huggingface dataset:
175 REPRODUCTIVE PERFORMANCE OF MANGALARGA MARCHADOR MARES AFTER INDUCTION OF OVULATION WITH hCG OR DESLORELIN
Authors: Faria D. R. , Boffe A. L. N. , Grossi S. F. , Gradela A.
Published: August 2010

The use of biotechnology, such as AI, embryo transfer, and hormonal protocols, have accelerated the genetic improvement in the equine specie, and inducers of ovulation, such as hCG, are important tools in implementing them. However, the repeated use of ovulation inducers can promote the development of antibodies and undermine their purpose. Thus, a synthetic GnRH, deslorelin, has been used. In this study, we compared the reproductive performance of 44 Mangalarga Marchador donor mares, aged between 5 and 21 years and with sound reproductive history after treatment with hCG or deslorelin. During estrus, mares were examined by transrectal ultrasonography every other day until a follicle of 30 mm in diameter was detected and then daily until detection of a >35-mm follicle and moderate uterine edema. At this time, mares were treated with either 2 mL of saline, i.m. as controls (CON = 16); 2500IU of hCG, i.v. (Chorulon, Intervet Inc., Millsboro, DE, USA; hCG = 14); or 2 mg mL-1 deslorelin in BRT vehicle (BET Pharm, Lexington, KY, USA), i.m. (DES = 14). After treatments, mares were examined at 12-h intervals until ovulation and were inseminated with fresh semen 12 h after ovulation. On Day 8, embryos were recovered and evaluated. All mares were ovulated, and follicle diameters were not different (P > 0.05) at the time of treatment (38 ± 0.029 v. 39 ± 0.029 v. 38 ± 0.029 mm). Embryo recovery was similar among groups (CON, 37.5%; hCG, 42.8%; DES, 50.0%, respectively). The interval from treatment to ovulation did not differ (P > 0.05) between hCG (1.43 +0.56 days) and DES mares (1.14 + 0.36 days), and both were less (P > 0.05) than for controls (CON, 2.94 + 0.77 days). The number of mares that ovulated per ovulation interval were as follows: <24 h = CON: 0%, hCG: 7.14%, and DES: 14.29%, respectively; from 24 to 48 h = 6.25%, 64.29%, and 78.57%; from 49 to 72 h = 37.5%, 28.57%, and 7.14%; >72 h = 56.25%, 0%, and 0%. Time to ovulation was negatively correlated (P < 0.05) with follicle diameter in CON (r = -0.50, P < 0.05) but not for hCG (r = 0.05) or DES (r = 0.022) and was positively correlated with embryo recovery for DES (r = 0.46, P < 0.10), but not hCG (r = 0.23) or CON (r = 0.09). Based on the results, we concluded that hCG and DES effectively synchronize the time of ovulation. Follicle size did not affect the time of ovulation in hCG- and DES-treated mares.

We thank the Farm Water supply for the animals and drugs.
176 PREGNANCY AND PREGNANCY LOSS RATES OF ACYCLIC RECIPIENT MARES TREATED WITH TWO DIFFERENT LONG-ACTING PROGESTERONE CONCENTRATIONS
Authors: Greco G. M. , Dell'aqua Jr J. A. , Alvarenga M. A.
Published: August 2010

Long-acting progesterone compounds containing concentrations of 150 mg mL-1 (P4LA150) have been routinely used in Brazil, especially at the beginning and end of the breeding season when the percentage of recipient mares showing normal estrous cycles is low compared to donors (Greco GM et al. 2008 Proceedings 7th International Symposium on Equine Embryo Transfer, 59-60). However, recipients treated with P4LA150 must receive weekly administrations until pregnancies achieve 100 days. The present study aimed to evaluate pregnancy and pregnancy loss rates from acyclic mares treated at 7-day intervals with P4LA150 and 14-day intervals with a long-acting progesterone formulation containing 300 mg mL-1 (P4LA300). Acyclic Campolina breed mares (n = 80) ranging from 4 to 13 years old showing considerable endometrial edema 2 days after estradiol benzoate injection (5 mg, i.m.) were split in 2 groups. Group 1 mares (n = 40) received 10 mL of P4LA150 (1500 mg) i.m. every 7 days, whereas Group 2 mares (n = 40) were treated with 10 mL of P4LA300 (3000 mg) i.m. every 14 days. Steroid hormone formulations were produced in our research laboratory, at São Paulo State University, Botucatu, Brazil. Embryos 7 to 8 days of age were transferred into the uteri of recipients between 4 and 7 days after the first progesterone administration. Following a positive pregnancy diagnosis at 14 to 15 days, mares were examined at Days 50 and 100, when progesterone treatment was discontinued. These evaluations were conducted in order to determine pregnancy loss rates during both periods. Results were analyzed through the chi-square test and significance was set at P < 0.05. Pregnancy rates and pregnancy loss rates at Day 50 from mares treated with P4LA150 (Group 1) and P4LA300 (Group 2) did not differ. Pregnancy rates were 60.0% (24/40) and 75.0% (30/40) at Day 50 for mares treated with P4LA150 (Group 1) and P4LA300 (Group 2), respectively (P > 0.10). Pregnancy loss rates at Day 50 were 8.3% (2/24) and 10.0% (3/30) for mares treated with P4LA150 and P4LA300, respectively (P > 0.10). Pregnancy losses were not identified at Day 100. Based on the results from the present study, we concluded that acyclic recipient mares treated with P4LA150 or P4LA300 are able to obtain satisfactory pregnancy rates with low pregnancy losses. Moreover, the use of 3000 mg of long-acting progesterone every 14 days reduces overall progesterone administrations, minimizes stress in horse handling, and makes the choice for acyclic recipient mares more appealing in commercial embryo transfer programs.

Authors would like to thank FAPESP for financial support.
184 EFFECTS OF X-RAY ELECTROMAGNETIC RADIATION ON BOVINE OOCYTE DEVELOPMENT
Authors: Snyder W. E. , Aaltonen J. T. , Sigal H. D. , Loskutoff N. M.
Published: August 2010

This study examined the effects of X-rays on bovine oocytes used for in vitro embryo production. In recent years, airport security has become more stringent and the use of X-ray screening may be required for all items, which could be problematic when transporting oocytes. Ionizing radiation such as X-rays are high-energy particles that can penetrate into the nucleus of a cell and cause single and double-strand breaks in the DNA chain. Oocytes exposed to X-ray radiation may have DNA damage affecting maturation, fertilization, and embryonic development. Bovine ovaries obtained from a local abattoir were used to collect immature oocytes by follicular aspiration. The oocytes were divided into a control group and an experimental group then placed in maturation medium. The oocytes in the experimental group were placed in a hospital X-ray machine with a single exposure of 100 mA, 120 kVp for 0.3 s. This exposure challenged the oocytes with a comparable, calculated amount of mrems equivalent to a single screening from an airport X-ray machine (30 mrems). After X-ray exposure, the experimental group was cultured with the control at 37°C and 5% CO2. After 18 h, cryopreserved bovine sperm were thawed and processed using BoviPure (Nidacon International, Mölndal, Sweden) density gradient centrifugation and the oocytes were inseminated with 1 × 106 sperm in fertilization medium at 37°C and 5% CO2. After 24 h, both groups were transferred to 50-μL droplets of G1 medium (Vitrolife, Göteborg, Sweden) at 37°C and 6% CO2 and both were transferred into G2 medium after 72 h. Development of the oocytes was scored after an additional 72 h. Results were based on 2 criteria: the percent cleaved (presumptive fertilization) and the percent cleaved that developed to morula and blastocyst stages. The results indicated no significant difference in the percentages of oocytes cleaving after insemination for the control and experimental groups (P = 0.403, chi-square test). However, there was a significant increase (P = 0.037, chi-square test) in the percentages of morulae and blastocysts developing in the control (non-irradiated) v. experimental (X-rayed) groups. The outcome of this study cautions on the potential damage caused to bovine oocytes used for embryo production in vitro when exposed to X-ray electromagnetic radiation during routine air transport screening operations.

Table 1.
185 NONSURGICAL EMBRYO TRANSFER FOR PRODUCTION OF PIGS BY IN VITRO FERTILIZATION AND SOMATIC CELL NUCLEAR TRANSFER
Authors: Yoo J.-G. , Park M.-R. , Kim H.-N. , Ko Y.-G. , Lee J.-Y. , Hyung N.-W. , Yang B.-S. , Park S.-B. , Woo J.-S.
Published: August 2010

Instead of surgical embryo transfer (ET) in the pig, nonsurgical ET is a hopeful method to increase the efficiency of biotechnology applications such as cloning and transgenesis. In this study, we conducted surgical and nonsurgical ET methods after somatic cell nuclear transfer (SCNT) with MHC miniature pig cells to find out the best condition for production of cloned miniature pigs. Ovaries were obtained from prepubertal crossbred gilts at a local slaughterhouse. Oocytes were matured for 40 to 44 h at 38.5°C under 5% CO2 in air. As donor cells, fibroblast cells were cultured from ear skin tissue of 8-month-old MHC inbred miniature pigs. Fibroblast cells were cultured, passaged (3 to 8 passages), and used as donor cells for NT. After the enucleation and injection process, eggs were held in TCM-199. For fusion, 2 DC pulses of 1.2 kV cm-1 were applied for 30 μs. Both IVF and SCNT embryos were cultured in PZM-3 medium. After IVF, 84.9% (411/484) of embryos cleaved and 27.3% (132/484) of embryos reached the blastocyst stage. In the SCNT group, 80.8% (231/286) of eggs fused and 25.9% (60/286) of embryos developed to blastocysts. For surgical ET, approximately 200 SCNT embryos were transferred into oviducts of each synchronized recipient. For nonsurgical ET, embryos were cultured in PZM-3 for 6 days after SCNT and IVF, and then good quality blastocyst stage embryos were selected for ET. The pregnancy status of recipients at Day 30 was determined by ultrasound scanning. Using Day 30 of gestation as an endpoint, the nonsurgical ET method (47.3%, 9/19) had a similar pregnancy rate as the surgical ET method (56.5%, 13/23). Further study is needed to optimize the nonsurgical ET method especially for SCNT eggs.

This work received grant support from the Agenda Program (no. 200901FHT010305535), Rural Development Administration, Republic of Korea.
194 ESTROUS SYNCHRONIZATION AND FIXED-TIME AI IN WOOD BISON (BISON BISON ATHABASCAE)
Authors: Adams G. P. , McCorkell R. B. , Jurgielewicz V. C. , Ambati D. , Woodbury M. R.
Published: August 2010

As part of a project involving reproductive biotechnology as a method of preserving Canada’s threatened wood bison population, an experiment was designed to test the effectiveness of steroid-induced ovarian synchronization and fixed-timed AI. The experiment was performed in the early ovulatory season (September) with female wood bison (4 years old, n = 13) and plains bison (2 years old, n = 4, and 8 years old, n = 3). Based on results of a preceding study, progesterone was combined with estradiol as a synchronization treatment to mitigate against untimely ovulation. The bison were blocked by subspecies and assigned randomly to a control group (no treatment; n = 10) or given estradiol 17β (2.5 mg) + progesterone (50 mg) in canola oil i.m. and a progesterone-releasing intravaginal device (Cue-mate™, Bioniche, Belleville, Ontario, Canada) on Day 0 (n = 10). On Day 8, the Cue-mate™ device was removed and PGF (500 mg Estrumate®, Mallinckrodt Vet GmbH, Friesoythe, Germany) was given to induce luteolysis. On Day 10, all bison were given 5 mg of LH (Lutropin®-V, Bioniche) and artificially inseminated 12 h later with semen collected and frozen previously from wood bison of the same herd. The ovaries were examined daily by transrectal ultrasonography beginning 5 days before treatment and thereafter until the first post-treatment ovulation. Ultrasonographic pregnancy diagnosis was done 30 days post-insemination. No differences were detected between wood and plains bison for any end point, and data were combined for further statistical analyses. Ovarian follicular wave emergence occurred on Day 4.1 ± 0.8 (mean ± SEM) and 4.1 ± 0.3 in the control and treatment groups, respectively (P = 99.3). The interval to new wave emergence was less variable in bison treated with estradiol + progesterone than in untreated controls (residuals, 0.7 ± 0.2 and 1.9 ± 0.5 days, respectively; P < 0.05). The interval from LH administration to ovulation was 2.7 ± 0.6 and 5.4 ± 1.9 days for the treatment and control groups, respectively, and was less variable in the treatment group than in controls (residuals, 1.2 ± 0.4 and 5.3 ± 0.8 days, respectively; P < 0.05). The diameter of the preovulatory follicle was not different between groups and was, on average, 15.2 ± 1.1 mm. Pregnancy was diagnosed in 3 bison in the treatment group and 2 in the control group. In conclusion, treatment with estradiol and progesterone effectively synchronized the interval to wave emergence, and subsequent LH treatment resulted in a synchronous ovulatory response necessary for fixed-time AI in bison. Although the pregnancy rate was modest, perhaps because of issues with semen quality, timing of AI, or quality of the ovulated oocyte, this represents the first report of pregnancy in bison from fixed-time insemination.

Supported by grants from the Advancing Canadian Agriculture and Agri-Food Fund, the Agri-Food Innovation Fund, Parks Canada, the World Wildlife Fund, and the Northwest Territories.
195 EVIDENCE OF SEASONALITY IN SEMEN CHARACTERISTICS OF CAPTIVE ANDEAN HAIRY ARMADILLO (CHAETOPHRACTUS NATIONI)
Authors: Bermúdez L. , Enciso M. A. , Rojas G. , Alvis R. , Valdivia M.
Published: August 2010

Chaetophractus nationi is an armadillo species that inhabits in the high Andes of Peru, Chile, and Bolivia at altitudes of over 3000 m. The main threats to this species are habitat loss and over-hunting; in the last 10 years it has been estimated that the population has declined by 30%. The species is categorized as Vulnerable (VU, A2d) by International Union for Conservation of Nature (IUCN) and Peruvian legislation (DS-034-2004-AG) and is listed in appendix II of Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). Chaetophractus nationi is 1 of 7 species of armadillos in which further studies on their reproductive biology and physiology are considered to be urgently needed. The aim of the present study was to determine the semen ejaculate values of C. nationi males in 2 opposite seasons. Six adult males of C. nationi belonging to the Huachipa Zoological Park (Lima, Peru) collection under the same captive conditions were used. Semen samples were collected coinciding with the Peruvian warm season (February) and cold season (June). The animals were chemically immobilized using ketamine (15 mg kg-1), xylazine (1 mg kg-1), and midazolam (0.4 mg kg-1) i.m. Semen was collected by an electroejaculation technique using commercial equipment (Eletrojet®, Eletrovet, São Paulo, Brazil) and a rectal probe specifically designed in Peru for the species. Progressive electrical stimulation from 2 to 6 V was applied in a protocol of 30 stimuli divided into 3 series: series 1, with 10 stimuli of 3 sec at 2 V; series 2, with 10 stimuli of 3 sec at 4 V; and series 3, with 10 stimuli of 3 sec at 6 V The results (mean ± SEM) for the warm season (26-28°C) were n = 6; volume = 80 ± 10.34 μL; pH = 8.2 ± 0.3; progressive motility = 77% ± 0.06; and sperm concentration/mL = 46.2 ± 2.9 × 106. The results (mean ± SEM) for the cold season (13-18°C) were n = 6; volume = 8.6 ± 2.27 μL; pH = 8.7 ± 0.18; and progressive motility = 80%. Sperm concentration could not be determined on the cold season samples because of the low volume collected. In summary, our results are suggestive of a seasonal pattern in semen characteristics of the Andean hairy armadillo that may be related to its reproductive activity in the wild.

J. Pino and B. Shiga (UNMSM), for help with the rectal probe elaboration.
205 CUMULUS CELL MORPHOLOGY BEFORE AND AFTER IN VITRO MATURATION AS AN INDICATOR OF PORCINE OOCYTE DEVELOPMENTAL COMPETENCE
Authors: Bertoldo M. , Holyoake P. K. , Evans G. , Grupen C. G.
Published: August 2010

Effective in vitro maturation (IVM) is essential for successful in vitro embryo production. The morphology of the cumulus investment before and after IVM may be a useful noninvasive indicator of oocyte quality. In pigs, oocyte developmental competence is reduced during the summer months. The aim of this study was to determine whether the morphology of cumulus-oocyte complexes (COC) before and after IVM are associated with oocyte quality, using COC collected from small and large follicles in summer and winter as models of poor and good oocyte quality. Ovaries were collected from sows slaughtered 4 days after weaning. The COC recovered from small (3-4 mm) and large (5-8 mm) antral follicles were morphologically graded and parthenogenetically activated following IVM during winter (n = 1419; 10 replicates) and summer (n = 2803; 10 replicates). Grade 1 and 2 COC had >2 layers of compact cumulus cells and a homogenous cytoplasm. Grade 3 COC were either partially or fully denuded, had a heterogeneous cytoplasm, or were vacuolated or dark in color. Grade 4 COC had expanded cumulus cells. Cumulus expansion was also assessed subsequent to IVM. The COC recorded as having a cumulus expansion index (CEI) of 1 had the poorest expansion with no detectable response to IVM, whereas those with a CEI of 4 had the greatest amount of expansion, including that of the corona radiata. Data were analyzed using a generalized linear mixed model in GenStat® (release 10, VSN International, Hemel Hempstead, UK). There was an effect of follicle size for Grade 1 COC, with COC from large follicles in both seasons yielding better quality COC (P < 0.05). The proportion of COC in Grade 2 was higher in small follicles during winter compared with large follicles, but there were no differences between follicle sizes during summer (P < 0.05). The proportion of COC with CEI 1 was highest in COC from small follicles during summer (P < 0.05). The proportion of COC from large follicles with CEI 2 was higher during summer compared with winter (P < 0.05). There were no seasonal or follicle size effects on COC with CEI 3 or 4 (P > 0.05). The proportion of oocytes that developed to blastocysts was greater in winter than in summer (39.06% ± 5.67 v. 22.27% ± 4.01; P < 0.05). Oocytes derived from large follicles had a greater ability to form blastocysts compared with those from small follicles (37.13% ± 5.65 v. 23.32% ± 4.56; P < 0.06). Morphological assessment of cumulus cells before and after IVM may be a useful tool to evaluate the effects of follicle size on oocyte developmental competence. However, the results of the present study indicate that cumulus cell morphology is not a good indicator of the effect of season on oocyte developmental competence.
208 OVARIAN FOLLICULAR DYNAMICS IN SOUTH AMERICAN CAMELIDS: EFFECT OF PLANE OF NUTRITION AND SPECIES
Authors: Cervantes M. P. , Orban T. , Adams G. P.
Published: August 2010

Controversy exists regarding characteristics of follicular waves in llamas and alpacas. Lactational status has been shown to influence follicular dynamics, but the effects of species and nutrition have not been critically examined. A 2 × 2 experimental design was used to determine the effects of species (llama v. alpaca) and nutritional status (high-plane v. low-plane) on ovarian follicular wave dynamics. Adult female llamas (n = 16) and alpacas (n = 19), ≥ 3 years old, were assigned randomly to either a high or low plane of nutrition. Nutritional planes were defined by the grazing condition of the native pasture. The respective nutritional conditions were imposed 2 weeks before the start of the observational period. Body condition was estimated at the start of the observational period using a subjective scoring system (1 = very thin, 10 = very fat) and ovarian dynamics were monitored daily by transrectal ultrasonography for 38 days. Data were analyzed by two-way ANOVA and are expressed as mean ± SEM. Body condition scores were not different among groups (6.9 ± 0.35 and 6.6 ± 0.19 for llamas on high and low planes of nutrition, respectively, and 7.2 ± 0.25 and 6.8 ± 0.18 for alpacas on high and low planes of nutrition, respectively). The growing phase of the dominant follicle tended (P = 0.1) to be longer in llamas than in alpacas (9.8 ± 0.47 v. 8.8 ± 0.45 days) and in animals on a high plane of nutrition than in animals on a low plane (9.6 ± 0.50 v. 8.6 ± 0.42 days). Accordingly, the maximum diameter of the dominant follicle tended to be larger in llamas than in alpacas (10.1 ± 0.37 v. 9.1 ± 0.30 mm; P = 0.06) and in animals on a high plane of nutrition than in animals on a low plane (9.9 ± 0.39 v. 9.1 ± 0.27 mm; P = 0.14). The interwave interval was similar between llamas and alpacas (16.5 ± 0.66 v. 15.6 ± 0.42 days; P = 0.29), but was longer (P < 0.01) in animals on a high plane of nutrition than in animals on a low plane (16.9 ± 0.54 v. 15.0 ± 0.44 days); there was no interaction between main effects (P = 0.31). The total lifespan (duration of detection) of the dominant follicle was similar in both llamas and alpacas (22.9 ± 0.75 v. 21.9 ± 0.73 days; P = 0.38) and in animals on a high plane of nutrition than in animals on a low plane (22.7 ± 0.78 v. 22.0 ± 0.70 days; P = 0.53). There was no interaction between main effects (P = 0.21). All females (n = 35/35, 100%) had a follicle ≥ 7 mm (ovulatory size) from Days 7 to 12 after wave emergence. In conclusion, a low plane of nutrition had a suppressive effect on dominant follicle growth, resulting in a shortened interwave interval in llamas and alpacas. The interwave interval was not significantly longer in llamas than in alpacas despite a tendency for a longer growing phase and a larger dominant follicle.

Research supported by Mitchell Group’s Mallkini Alpaca Breeding and Genetic Centre and the Natural Sciences and Engineering Research Council of Canada.
209 SEASONAL CHANGES IN CHARACTERISTICS OF THE ESTROUS CYCLE IN BOS TAURUS INDICUS (NELORE) HEIFERS
Authors: Corte Jr A. O. , Navarrete B. J. F. , Nogueira G. P.
Published: August 2010

To evaluate the possible seasonal variation in estrous cycle characteristics, 8 heifers were synchronized with 2 prostaglandin injections and their estrous cycles were followed during different seasons of the year (autumn, n = 413; winter, n = 8; spring, n = 9; and summer, n = 9) with daily ultrasonographic exams to count and measure follicles ≥3 mm. Blood was collected every 12 h for luteinizing hormone (LH) and progesterone (P4), and every 3 h from estrus until ovulation to characterize the LH peak. Five ovariectomized heifers were injected with 17β-estradiol (2 μg/kg of liveweight) every season and blood samples were collected thereafter at 3 h intervals for LH quantification. The monthly body weight percentile difference (Δ%) did not vary among seasons. Average P4 concentration for the cycle was higher (P = 0.001) and follicle numbers lower during autumn (2.5 ± 0.2 ng mL-1; 7.8 ± 0.1) and summer (2.9 ± 0.3 ng mL-1; 6.8 ± 0.2; P = 0.001) compared with winter (1.4 ± 0.2 ng mL-1; 9.6 ± 0.3) and spring (1.6 ± 0.2 ng mL-1; 9.7 ± 0.3). During winter, there were more estrous cycles with 3 follicle waves (5/8) and during summer only there were cycles with 2 follicular waves (P = 0.009). Because LH secretion did not vary despite seasonal variation in P4 concentration and because there was a negative correlation between higher P4 values and daily percentile variation of photoperiod (A%; P = 0.0038; r = -0.4533), seasonal variation in the luteal cell sensitivity to LH needs to be evaluated. In the ovariectomized Nelore heifers, the LH circannual concentration without estradiol stimulus was significant (P = 0.02). The LH response to estradiol treatment was lower in summer (0.8 ± 0.2 ng mL-1 v. 1.3 ± 0.5 ng mL-1). We hypothesize that seasonal variation exists in hypothalamic sensitivity to estradiol. The present experiment provides evidence that seasonality in reproductive responses remains in Nelore heifers.

FAPESP (07-53539-3), UNESP, and Devani Mariano Pinheiro.
210 EXPRESSION OF mRNA ENCODING STEROIDOGENIC ENZYMES IN THE DEVELOPING BOVINE OVARY
Authors: da Silva R. B. , Caixeta E. S. , Ripamonte P. , Castilho A. C. S. , Price C. , Buratini Jr J.
Published: August 2010

Recent findings suggest a role for estradiol in the regulation of early folliculogenesis. Estradiol production is greatest in the fetal ovary during early gestation in cattle, and both estradiol and progesterone inhibit primordial follicle activation (Yang MY and Fortune JE 2008 Biol. Reprod. 78 (Suppl 6), 1153-1161). Aromatase expression is detected in early stages of bovine pregnancy (Garverick HA et al. 2009 Anim. Reprod. Sci. in press). The mechanisms controlling steroidogenesis in the bovine fetal ovary remain to be fully elucidated. The objective of this study was to assess mRNA expression patterns of enzymes involved in steroid production [steroidogenic acute regulatory protein (STAR), side-chain cleavage P450 (CYP11), cytochrome P450 17 alpha-hydroxylase (CYP17A1), 3 beta-hydroxysteroid dehydrogenase (3fi-HSD), aromatase cytochrome P450 (CYP19), and 17 beta-hydroxysteroid dehydrogenase (17fi-HSD)] in bovine fetal ovaries during gestation. Bovine fetal ovaries were obtained in a local slaughterhouse, fetal age was estimated by the crown-rump length, and samples were grouped according with days of gestation as follows: 60 (n = 5), 75 (n = 8), 90 (n = 6), 120 (n = 7), 150 (n = 7), and 210 (n = 6). Expression of mRNA encoding steroidogenic enzymes was determined by semiquantitative real-time RT-PCR using bovine-specific primers and cyclophilin A as endogenous control. Reverse transcription was performed with SuperScriptIII® (Invitrogen, Carlsbad, CA, USA) and PCR with Power SYBR green master mix (Applied Biosystems, Foster City, CA, USA) in an ABI Prism® 7500 (Applied Biosystems). Gene expression values were determined by the Pfaffl equation and effect of day of gestation on gene expression was analyzed with Fisher’s protected test, except when data were not normally distributed and nonparametric analysis was performed. Expression of mRNA encoding all steroidogenic enzymes was detected throughout gestation. The mRNA abundance of CYP17A1 and CYP19 was highest at 60 days of gestation and decreased thereafter (P < 0.05). Expression of all other genes did not significantly vary with time of gestation. In conclusion, all major enzymes required for steroidogenesis were expressed in the bovine fetal ovary. Expression of CYP17A1 and CYP19 was suppressed after 60 days of gestation, suggesting that these enzymes may be involved in the mechanisms controlling estradiol production and follicle formation in the bovine fetal ovary.

Supported by CAPES and FAPESP.
228 microRNA REGULATION OF GENES IN BOVINE OOCYTES AND EMBRYOS
Authors: Barfield J. P. , Bouma G. J. , Seidel Jr G. E.
Published: August 2010

Little is known about expression of microRNA (miRNA) in bovine oocytes and pre-implantation embryos. These molecules likely have an important role in regulating development. For example, differences in quality of oocytes matured in vivo v. in vitro might be due, in part, to altered miRNA expression. In Experiment 1, in vivo-matured COC were collected by transvaginal aspiration of 7 superstimulated cows 21 to 23 h after GnRH injection, given 48 h after prostaglandin F2α and the last of 6 FSH injections given b.i.d. Oocytes aspirated from abattoir ovaries were matured in vitro for 23 h in a chemically defined medium. After vortexing, maturation of both groups of oocytes was confirmed by visualization of the first polar body, and oocytes were snap frozen in mirVana lysis buffer (Applied Biosciences, Foster City, CA, USA). In Experiment 2, in vitro-matured oocytes were generated as described. Subsets were fertilized in vitro or activated parthenogenetically by incubation in 5-μM ionomycin for 5 min followed by 10 μg mL-1 cycloheximide plus 5 μg mL-1 cytochalasin B for 5 h. After 18 h and 12 h, respectively, fertilized and activated oocytes were centrifuged at 10 000 × g for 10 min to enable visualization of pronuclei. Zygotes with 2 polar bodies and 2 pronuclei and parthenotes with 2 pronuclei were snap frozen in mirVana lysis buffer. Total RNA was extracted from 30 pooled oocytes for each replicate using the mirVana MiRNA Isolation Kit (Ambion, Inc., Austin, TX, USA). Reverse transcription of RNA was performed using the QuantiMir RT kit (System Biosciences, Mountain View, CA, USA), and miRNA expression was evaluated by real-time PCR using the Mouse miRNome Profiler plate, which contains primers for 384 miRNA (System Biosciences). Three plates were analyzed for each group (30 oocytes per plate). Changes in relative expression levels were analyzed with a t-test of values normalized to miR-181a, which was consistently expressed in all samples. In Experiment 1, compared with in vitro-matured oocytes, in vivo-matured oocytes had 11-fold higher (P = 0.02) expression of miR-375, which targets numerous genes involved in electron transport chain and oxidative phosphorylation pathways according to the bioinformatic database mirGator. MiR-291a-5p, miR-494, miR-539, and miR-547 were expressed in in vivo-matured oocytes only; the converse was found for miR-575-5p. Results from Experiment 2 are in the table. Major pathways associated with potential targets of the detected miRNA include TGF-beta signaling, Wnt signaling, tight junction formation, DNA replication reactome, steroid biosynthesis, mRNA processing binding reactome, and glutamate metabolism. Several of these candidate miRNA might be important for regulation of bovine oocyte maturation and embryo development.

Table 1.Experiment 2: Fold change expression of miRNA
231 SELECTION OF UBIQUITOUS AND ENDOTHELIUM-SPECIFIC PROMOTERS FOR EXPRESSING TRANSGENES IN PIG
Authors: Chen Y. , Mir B.
Published: August 2010

Several promoters of different origins have been used to drive expression of transgenes in pig with variable results. However, there has not been a systemic search to identify the most reliable, ubiquitous, and tissue-specific promoters, such as endothelium-specific promoters, to drive expression of transgenes with high predictability. In this study we tested 9 ubiquitous mammalian promoters, generated by assembling enhancers, minimal promoters, and 5′untranslated region of different origins (Table 1) and 5 endothelial-specific promoters: hFlt-1 (fms-like tyrosine kinase), hICAM-2 (intercellular adhesion molecule-2), andhEndoglin promoters from human; mouse Tie-2 promoter (Schlaeger et al. 1997 PNAS 94, 3058-3063); and pig ICAM-2 promoter (Godwin et al. 2006 Xenotransplantation 13, 514-521). These promoters drive the expression of either GFP or LacZ reporter genes and were electroporated into 9 different pig cell types: endothelium-derived 2A2 and PEDSV15 cells (Seebach et al. 2001 Xenotransplantation 8, 48-61), nasal fibroblasts, kidney epithelial cells, and lung macrophages (ATCC, CRL-2528, 2842, and 2843), pig fetal fibroblasts, liver stem cells, fat-derived stem cells, and immortalized liver endothelial cells (generated in the lab). The expression was graded as week, moderate, or strong depending on the brightness of GFP signal or intensity of LacZ staining. Out of 9 ubiquitous promoters, 7 showed either low expression in all cell types or different levels of expression in some of the cell types. However, 2 promoters, hCMV-hCMV-HTLV and SV40-hCMV-HTLV, showed strong and stable expression across all 9 cell types. Among endothelial-specific promoters, pig ICAM-2 promoter showed moderate and specific expression in all 3 endothelial cell types (strong expression with the enhancer) and no expression in non-endothelial cells. Human EC-specific promoter hFlt-1 showed moderate expression in all 3 endothelial cell types, as well as in some of the non-endothelial cells such as liver stem cells, fat-derived stem cells, and lung macrophages. The other endothelial-specific human and mouse promoters did not show any expression in both endothelial and non-endothelial cells. In summary, we identified hCMV-hCMV-HTLV and SV40-hCMV-HTLV as 2 ubiquitous promoters and pICAM2 as an endothelial-specific promoter for expression of transgenes in pig. In the future, we will test these 3 promoters for in vivo expression studies.

Table 1.Ubiquitous promoter characteristics
234 EXPRESSION OF NUCLEAR AND MEMBRANE MELATONIN RECEPTORS GENES AND THE CLOCK GENES IN RAT OOCYTES: PRELIMINARY RESULTS
Authors: Coelho L. A. , Peres R. , Cipolla-Neto J.
Published: August 2010

There is evidence that melatonin acts directly on the regulation of ovary function. This action is probably attributed in part to melatonin receptors, which are known to be present in granulosa and cumulus cells (Kang J-T et al. 2009 J. Pineal Res. 46, 2228). Melatonin is also known to be associated with the modulation of circadian rhythms and the regulation of seasonal reproductive function (Arendt J 1998 Rev. Reprod. 3, 1322). Circadian rhythms and clock genes appear to be involved in reproductive processes (Dolatshad H et al. 2009 Repro. Fertil. Dev. 21, 19). However, the presence of melatonin receptor genes and the clock genes has not been so widely studied or has never been reported to exist in mammalian oocytes.The aim of this study was to investigate the presence of nuclear (Rorα) and membrane (Mt1 and Mt2) melatonin receptors genes and the clock genes (Clock, Bmal1, Cry1, Cry21, Per1, Per2) in rat oocytes by reverse RT-PCR. Twenty-seven-day-old Wistar female rats were treated with 20 UI of pregnant mares serum gonadotropin for induction of follicular development and slaughtered 48 h later. All the procedures involving animals were approved by the Animal Care Committee of the Institute of Biomedical Sciences. The ovaries were removed and placed in TCM-199 supplemented with 100 UI mL-1 penicillin, 100 μg mL-1 streptomycin, and 0.1% polyvinyl alcohol (H-199 medium). Germinal vesicle-intact oocytes, isolated from the ovarian follicles, were denuded from cumulus cells by vortexing for 5 to 8 min. The denuded oocytes were incubated for 5 min in H-199 medium with 0.1% pronase for removal of the zona pellucida. Pools of 80 oocytes per cDNA sample were used.As an internal control for the sample integrity, additional primers for RPL37a were included in each PCR reaction. All the 3 control PCR replicates showed a repeatable amplification. Polymerase chain reaction amplifications of cDNA yielded Rorα, Clock, Bmal1, and Cry1 products in 2 of 3 replicates. No expression of the MT1 and MT2 mRNA was observed. The preliminary results suggest the presence of a nuclear melatonin receptor gene and some clock genes in rat oocytes. However, additional studies are necessary to confirm this hypothesis.

This research was supported by FAPESP.
238 RNA BINDING PROTEIN EXPRESSION IN CULTURED EARLY BOVINE EMBRYOS
Authors: Favetta L. A. , Van de Laar E. , King W. A. , LaMarre J.
Published: August 2010

The control of gene expression in the early embryo requires a highly regulated turnover of specific mRNA, particularly those of maternal origin, as the embryo becomes transcriptionally autonomous. In cattle, the period during which maternal transcripts persist can last 72 to 96 h or longer, suggesting a dynamic, regulated interplay between factors that protect transcripts before this point and those that subsequently facilitate decay. Some decay pathways for specific embryonic transcripts are now known, but many are not. In somatic cells, mRNA decay is often mediated by interactions between defined sequence elements (ARE) in the 3′ untranslated region of important target genes and specific RNA-binding proteins (AUBP) that promote or inhibit decay of the associated transcript. These have not been extensively characterized in embryos. We hypothesized that changes in the pattern of expression of one or several AUBP in the developing bovine embryo would support a role for these proteins in mRNA turnover and the control of gene expression. We, therefore, evaluated the expression of different AUBP (HuR, AUF1, TTP) in bovine oocytes and early embryos in vitro. Bovine oocytes obtained at slaughter were matured, fertilized, and cultured using standard protocols. Oocytes and embryos from different stages were either placed in Trizol for subsequent RNA isolation and RT-PCR analysis or fixed in 4% paraformaldehyde and subsequently processed for immunohistochemical detection of AUBP. Analysis by RT-PCR revealed that AUF1, an mRNA destabilizing protein, was expressed at all stages examined (immature oocyte, mature oocyte, 2 to 4 cells, 8 to 16 cells, morulae, and blastocyst) except in morulae. Another mRNA destabilizing protein, TTP, was expressed at the morula stage only. An mRNA stabilizing factor, HuR, was expressed at all stages except the morula. Immunohistochemical analysis revealed that the pattern of protein expression for AUF1 and TTP essentially mirrored that observed at the RNA level as detected by RT-PCR. Together, these results show that AUBP expression in the early bovine embryo is dynamic, with RNA-binding proteins present at all times during development and changes in expression evident at the morula stage. This suggests that modification of presynthesized (i.e. maternal) AUBP is likely to control mRNA decay during the maternal to embryonic transition (8-cell stage) and that the expression of TTP at the morula stage might mark the onset of embryonic control of mRNA stability.

Research was supported by NSERC, OMAFRA, and the Canada Research Chairs Program.
242 POTENTIAL ESTROGENIC EFFECT(S) OF PARABENS AT THE NEONATAL STAGE OF AN IMMATURE FEMALE RAT MODEL
Authors: Hyun S. H. , Jeung E. B.
Published: August 2010

In this study, to examine the estrogenic activity effects of parabens on hormonal responsiveness and on change in the morphology of reproductive target tissues during a critical development stage in female rats, analyses for parabens including methyl-, ethyl-, propyl-, isopropyl-, butyl-, and isobutylparaben were performed in an immature female Sprague-Dawley rat model. Two hundred female immature rats (n = 10/group) were orally treated with these parabens from postnatal day 21 to 40 in a dose-dependent manner based on our previous study [62.5, 250, and 1000 mg/kg of body weight (BW) per day]. 17α-ethinylestradiol (EE;1 mg/kg of BWper day) was used as a positive control and corn oil as a vehicle.A high doseofmethyl- and isopropylparaben (1000 mg/kg of BW per day) resulted in a significant delay in the date of vaginal opening and a decrease in length of the estrous cycle (P < 0.05). In measurements of organ weight and body weight, we observed significant weight changes in ovaries, adrenal glands, thyroid glands, liver, and kidneys(P < 0.05); conversely, body weight was not altered following paraben treatment. In all groups exposedto paraben treatment, histological analysis of the ovaries from the immature rats revealed interstitial cell disorders, a lack of corpora lutea, an increase in the number of cystic follicles, and thinning of the follicular epithelium, which occurred in a dose-dependent manner. In addition, morphological studies of the uterus revealed the myometrial dysplasia suchas myometrial hyperplasia inthe high-doseofpropyl- and isopropylparaben (1000 mg/kgof BWper day) group and in all dose of butyl- and isobutylparabens groups. We also observed a significant decrease in serum estradiol and T4 concentrations in methyl-, ethyl-, propyl-, isopropyl-, and isobutylparaben-treated groups (P < 0.01 and 0.05).A receptor-binding assay indicated that the relative binding affini- ties of parabens to estrogen receptors occurred in the order: isobutylparaben > butylparaben > isopropylparaben = propylparaben > ethylparaben. These values were much less than the binding affinity for 17?-estradiol. Taken together, long-term exposure to parabens, which show less estrogenic activity than EEl, can produce suppressive effects on hormonal responsiveness and can disrupt the morphology of reproductive target tissues during this critical stage of development in immature female rats.
247 ASSOCIATIONS BETWEEN HEAT SHOCK PROTEIN 70 GENETIC POLYMORPHISMS AND CALVING RATES OF BRAHMAN-INFLUENCED COWS
Authors: Rosenkrans Jr C. , Banks A. , Reiter S. , Starkey L. , Looper M.
Published: August 2010

Stress proteins and their genetic polymorphisms have been associated with decreased male and female fertility. Our objectives were to 1) identify single nucleotide polymorphisms (SNP) located in the promoter region of the bovine heat shock protein 70 (Hsp70) gene and 2) evaluate associations between Hsp70 SNP and calving rates of multiparous Brahman-influenced cows (n = 99). Genomic DNA was extracted from the buffy coats of EDTA- treated whole blood. Primers HSP-Pro749F (GCCAGGAAACCAGAGACAGA) and HSP-Pro1268R (CCTACGCAGGAGTAGGTGGT) were used for PCR amplification of a 539-base segment of the bovine Hsp70 promoter (GenBank accession number M98823). Eleven single nucleotide polymorphisms were detected: 8 transitions (G1013A, n = 2; G1045A, n = 8; C1069T, n = 4; A1096G, n = 14; G1117A, n = 12; T1134C, n = 7; C1154G, n = 11; andT1204C, n = 56), 2 transversions (A1125C, n = 53; and G1128T, n = 51), and 1 deletion at base position 895 (n = 37). Within an SNP, calving percentages were compared by chi-square analysis. Concentrations of Hsp70 and Julian date were analyzed by ANOVA, with each SNP represented as the main effect in the model. Cows that were homozygous for the minor allele at both transversion (A1125C and G1128T) sites had lower (P < 0.05) calving rates when compared with cows that were homozygous for the primary allele (48 v. 75%). Homozygous and heterozygous deletion of cytosine at base 895 resulted in lower (P < 0.05) calving percentages than homozygous cytosine cows (8, 50, 82%; respectively). In addition, DD cows had the latest (P < 0.05) Julian calving date. Eighteen Hsp70 promoter haplotypes were deduced, and 7 of those haplotypes (n = 37) included the deletion at base 895. Thirty-two cows had the haplotype consistent with the sequence deposited at GenBank, and the remaining 30 cows had an SNP other than the deletion. Cows with the deletion haplotypes had greater (P < 0.05) serum Hsp70 concentrations and lower (P < 0.05) calving rates (5.1, 4.7, and 3.5 MSE 0.5 ng mL-1; and 35, 78, and 87%; respectively, for Deletion, No, and Yes). Furthermore, cows with the deletion haplotypes had the latest (P < 0.05) Julian calving date (85, 77, and 73 d, respectively, for Deletion, No, and Yes). Our results suggest that the promoter region of the bovine Hsp70 gene is polymorphic and might be useful in selecting cows with greater fertility.
283 INFLUENCE OF THE MOMENT OF EMBRYO TRANSFER IN PREGNANCY RATES IN FEMALE BOVINE INSEMINATED WITH IVP EMBRYOS OFTHE NELLORE BREED
Authors: Mariani A. C. B. , Souza J. F. , Lopes F. B. , Souza J. A. T. , Ferreira J. L. , Cavalcante T. V.
Published: August 2010

The in vitro production (IVP) of bovine embryos is a biotechnique that has created new perspectives in animal reproduction and improvement. However, one of the main issues in the diffusion of this technology concerns the rate of pregnancy of the recipient cows. The objective was to verify the relationship between the fertilization in vitro, the day the recipient female was in estrus, and the pregnancy rates. Two hundred sixty crossbred recipient cows were selected from July to November 2008, synchronized with 2 different protocols at a random moment of the estrous cycle. One of the groups (G1) composed of 110 females that received 2.0 mL of D-cloprostenol, i.m. in only dose. The other group (G2), with 150 females, received a norgestomet auricular implant and 2.0 mg of estradiol benzoate i.m. On the fifth day, the females received, i.m. 200 IU of equine serum gonadotrophin and 150 μg of D-cloprostenol. The implant was removed on the eighth day, and 24 h later all animals received 1.0 mg of estradiol benzoate. The recipients entered in estrus 1 day before, 1 day after, and on the day of IVF. The embryos were transferred on Day 7 after fertilization, trancervically in the ipsilateral uterine horn that contained corpus luteum. The gestation diagnosis was made by ultrasound scan 30 days after the transfer. Data were analyzed using SAS software (version 9.0, SAS Institute, Cary, NC, USA). Initially, data were analyzed by normality test using Proc Univariate; however, a normal distribution was not observed. Data were then analyzed by PROC FREQ with nonparametric method of chi-square test and a significance level of 5%. Among the recipients, 40 were observed in estrus 1 day before IVF (D - 1), 137 on the IVF day (D0) and 83 were observed in estrus 1 day after (D + 1), resulting in a total rate of pregnancy of 38.8%. For each day the resulting pregnancy rates were 42.50% in D - 1, 41.61% in D - 0, and 30.12% in D + 1. These results suggest that there is no relationship between the day of the recipient’s estrus, the day of IVF, and pregnancy rates. There was no influence of the day of estrus in relation to the day of IVF in the gestation rates of the recipients of IVP embryos. However, more studies concerning the recipients are necessary, because they assume an important role in the success of the IVP.

The authors thank Brio Genetic and Technology.
296 EFFECT OF GROWTH HORMONE ON EMBRYO DEVELOPMENT AND ULTRASTRUCTURE OF BOS TAURUS IVP BOVINE EMBRYOS
Authors: Pegoraro L. M. C. , Dode M. N. , Weissheimer C. F. , Leivas F. G. , Vieira A. , Mozzaquatro F. , Rocha N. E. M. , Castro L. A. S. , Vianna L. L. , Pradieé J. , Anghonni L. B.
Published: August 2010

Bovine in vitro production systems are one of the most used assisted reproductive technique. However, this technique has some limitations especially in Bos taurus breeds, because of the low percentage of viable blastocysts produced (around 40% of oocytes inseminated) and higher cryosensitivity due to higher lipid content. Growth hormone (GH) can be a promising additive to increase in vitro embryo production. The aim of this study was to evaluate the embryo developmental rates (blastocyst/oocytes cleaved and blastocyst/oocytes inseminated) and ultrastructural features in Bos taurus embryos produced in SOFaa medium with or without GH. Cumulus oocyte complexes (COC) were recovered from slaughterhouse-derived ovaries (Angus Red crosses) and by ovum pick-up (OPU) from Jersey donors. After IVM and IVF, the presumptive zygotes were allocated in the SOFaa medium without (control) or with addition of GH (100 ng mL-1), for culture at 39°C in an atmosphere of 5% CO2. The cleavage and viable blastocyst rates were recorded 2 and 8 days after initiation of IVF, respectively. The results were compared by chi-square analysis. Similar (P > 0.05) cleavage rates were found in different culture medium and bovine breeds (61 v. 63% for Jersey control and Jersey GH; 71 v. 72% for cross-breed control and cross-breed GH). The development rates (blastocyst/oocytes cleaved and blastocyst/oocytes inseminated) did not differ in culture medium with or without GH within breeds (35 v. 30% for Jersey control and GH; 52 v. 56% for cross-breed control and GH; 21 v. 20% for Jersey control and GH; 36 v. 41% for cross-breed control and GH, respectively; P > 0.05). However, when breeds were compared, higher development rates were observed in cross-breed obtained from slaughterhouses than Jersey donors obtained by OPU (35 v. 52% for Jersey v. cross breed control; 30 v. 56% for Jersey v. cross-breed GH; 21 v. 36% for Jersey v. cross-breed control; 20 v. 41% for Jersey v. cross-breed GH. P < 0.001). The analyses of ultrastructure demonstrated no difference in the lipid proportion and organelle distribution of embryos produced with or without GH. We concluded that GH addition to SOFaa medium did not increase developmental rates for cross-breed or Jersey IVP embryos.