bluuebunny/crossref_metadata_2025 huggingface dataset:
11 CALF REMOVAL IMPROVES CO-SYNCH + CONTROLLED INTERNAL DRUG-RELEASE PREGNANCY RATES IN BEEF COWS
Authors: Kesler D. J. , Faulkner D. B. , Ireland F. A. , Dahlquist J. , El-Zarkouny S. Z. , Ferguson C. E.
Published: December 2008

Improving AI pregnancy rates in beef cattle will enhance the profitability of beef herds during these current times of increasing cost inputs by enhancing the quality of the calf crop. This experiment was aimed at determining the effect of calf removal (from CIDR removal to timed AI) during estrous cycle synchronization on AI pregnancy rates. This experiment was conduced at the Dixon Springs Research Station (Simpson, IL) during the fall 2007 breeding season. The beef cows used for this research project were crossbred Angus cows between 2 and 8 years of age that had calved in August to October of 2007. Cows were at least 45 days postpartum at the timed AI (November 2007). All cows were randomly divided into 1 of 2 treatment groups: cows in treatment group 1 (control group, n = 284) were allowed to keep their calves for the entire estrous cycle synchronization and AI program (except during brief times of treatment administration), whereas cows allotted to treatment group 2 (n = 258) had their calves removed at the time of CIDR implant removal/PGF2α treatment and returned following AI. The calves were maintained on creep feed out of sight and sound of their dams. All cows were synchronized by using the CO-Synch + CIDR protocol [CIDR (1.38 g) + GnRH (100 μg) on Day 0 and CIDR removal on Day 7 + PGF2α (25 mg)] and were inseminated at a predetermined time of 60 to 66 h post PGF2α with semen from proven bulls. At AI, cows also received an injection of GnRH (100 μg). All cows were pregnancy checked 50 to 65 days after timed AI via ultrasonography. Statistical comparisons were performed by using the chi-square test of SAS. The pregnancy rate for cows in the control treatment group was 47% (132 of 284) and was 57% (147 of 258) for the calf-removal treatment group. There was a significant increase in pregnancy rate in the treatment group (P < 0.02) that resulted in 10 more calves per 100 synchronized AI breedings. Although excellent facilities are needed to maintain calves away from their dams during this 60 to 66 h and there are additional costs for the feedings required for the calves, there is a savings in time (one calf separation from the cow is eliminated). In conclusion, removing the calves at the onset of estrous cycle synchronization can improve pregnancy rates to synchronized timed AI and can increase the ease of cattle handling during this process by reducing the number of calf removals during routine cow workings.

Table 1.Pregnancy rates to single synchronized timed AI for control and calf removal treatment groups
110 DEVELOPMENTAL CHANGES IN ACTIONS OF INSULIN-LIKE GROWTH FACTOR-I IN THE PREIMPLANTATION BOVINE EMBRYO-RECEPTOR EXPRESSION AND THERMOTOLERANCE
Authors: Bonilla A. Q. , Hansen P. J.
Published: December 2008

Insulin-like growth factor-I (IGF-I) can affect function of the preimplantation bovine embryo by increasing the proportion of cultured embryos that become blastocysts, reducing effects of heat shock on development and apoptosis, and enhancing survival of embryos transferred into heat-stressed recipients. At Day 5 postinsemination (pi), the embryo is sensitive to IGF-I as determined by activation of the phosphatidylinositol 3-kinase/Akt pathway and activation of thermoprotective mechanisms. It is not known how early in development IGF-I can affect embryo physiology. The overall objective of the present study was to determine whether IGF-I protects two-cell embryos from heat shock. In the first experiment, the presence of the IGF-I receptor (IGF-IR) was evaluated by immunofluorescence using a rabbit polyclonal antibody against a synthetic peptide of the human IGF-IRβ subunit. Specific labeling for IGF-IR was observed for two-cell embryos (n = 20) and embryos ≥16 cells collected at Day 5 pi (n = 17). In the second experiment, it was tested whether IGF-I would protect two-cell embryos from heat shock. Two-cell embryos were collected at 28 hpi and cultured ±100 ng mL–1 recombinant human IGF-I. After 1 h, embryos were heat-shocked (41°C for 15 h and 38.5°C for 9 h) or maintained at 38.5°C for 24 h. Embryos were then washed to remove IGF-I and cultured in KSOM-BE2 until Day 8 pi. The percent of embryos that became blastocysts at Day 8 was reduced by heat shock (P < 0.005) but was not affected by IGF-I or IGF-I v. heat shock. The least-squares means for percent blastocyst was 38.1% (control) v. 19.3% (heat shock) for embryos without IGF-I and 32.8% (control) v. 20.8% (heat shock) for embryos cultured with IGF-I (n = 11 replicates, n = 169–174 embryos/group; SEM = 2.0%). The third experiment was performed to verify that IGF-I protects Day 5 embryos from heat shock. Embryos ≥16 cells were collected at Day 5 pi and cultured ±100 ng mL–1 IGF-I. After 1 h, embryos were heat-shocked (42°C for 15 h and 38.5°C for 9 h) or maintained at 38.5°C for 24 h. Embryos were washed and cultured in KSOM-BE2 until Day 8 pi. The percent of embryos that became blastocysts was reduced by heat shock (P < 0.001) and increased by IGF-I (P < 0.05). The least-squares means for percent blastocyst at Day 8 pi was 86.9% (control) v. 47.7% (heat shock) for embryos without IGF-I and 88.7% (control) v. 66.3% (heat shock) for embryos cultured with IGF-I (n = 4 replicates, n = 59–60 embryos/group; SEM = 5.6%). In conclusion, IGF-I does not induce thermotolerance in two-cell embryos despite the presence of IGF-IR.

Support: USDA NRI 2007-35203-18070 and BARD US-3986-07.
128 N-METHYL-D-ASPARTIC ACID CAN BE USED TO PARTIALLY REPLACE BOVINE SERUM ALBUMIN IN CULTURE MEDIUM
Authors: Spate L. D. , Bauer B. K. , Murphy C. N. , Prather R. S.
Published: December 2008

One major obstacle in mammalian embryo culture has been unidentifiable biological contaminants in the media due to the inclusion of Bovine Serum Albumin (BSA) or Fetal Bovine Serum. The goal of this study was to remove BSA from culture media and develop chemically defined media based off the embryo’s biological and physiologic makeup. We evaluated the presence of message in various stages of porcine embryos and found that the message for the ionic glutamate receptor, N-Methyl-D-aspartic acid (NMDA) increased about 3-fold from oocyte to blastocyst. Thus, this study was conducted to determine if the addition of NMDA (0.5 mm) would improve development of embryos in an already chemically defined medium. Slaughterhouse derived ovaries were aspirated, cumulus–oocyte complexes were identified and then matured for 42 h in M199 base medium supplemented with EGF, FSH, and LH. Metaphase II oocytes were selected and fertilized in modified Tris buffered medium with 0.25 × 106 mL–1 frozen–thawed porcine semen for 5 h. Presumptive zygotes were then transferred to Porcine Zygote Medium with 0.3% BSA (PZM3) or 0.1% PVA (PZM4). After 28 h, cleaved embryos were selected and embryos were placed into treatment groups: (1) PZM3, (2) PZM4, or (3) PZM4 + 0.5 mm NMDA. Embryos were cultured in 5% CO2, 5%O2, 90% N2 until Day 7. For this experiment the number of cleaved embryos cultured in each treatment group were 260 for group 1, 220 for group 2 and 300 for group 3. Percentage of development to blastocyst was determined and analyzed with SAS Proc GENMOD Procedure (a,b P < 0.05). The percentage developed to blastocyst was (1) 47.5% a, (2) 29.6% b, and (3) 36.1% a,b, respectively. Total cell number of the blastocysts was determined by using Hoechst nuclear stain and statistically analyzed by SAS Proc GENMOD Procedure. The average cell number for the treatment groups was (1) 25.8 a, (2) 19.6 b, and (3) 22.9 a,b, respectively. Culture without BSA significantly reduced development to blastocyst and total cell number; however, with the addition of 0.5 mm NMDA there was no significant difference from media containing BSA. This indicates that NMDA can be used to partially replace BSA to form a chemically defined media.

Funded by a grant from the USDA NRI 2006-35203-17282.
144 PREGNANCY RETENTION OF BOVINE RECIPIENTS FOLLOWING TRANSFER OF EMBRYOS EXPOSED TO A PROSTAGLANDIN2ALPHA RECEPTOR ANTAGONIST DURING COLLECTION
Authors: Roper D. A. , Scenna F. N. , Saxton A. M. , Edwards J. L. , Rohrbach N. R. , Schrick F. N.
Published: December 2008

Increasing efficiency and success of MOET continues to be a goal of researchers and practitioners. Although numerous studies report success in establishing pregnancies, fewer evaluate term development and report number of live calves born. In a previous study, Scenna FN et al. (2008 Reprod. Fertil. Dev. 20, 154) exposed embryos to 3 different medium treatments while being collected from superovulated beef donors on Day 7. Medium treatments consisted of a commercially available medium plus 1 mL of DMSO (Control), commercial medium plus 100 nm of AL-8810 (AL100), or a commercial medium plus 1000 nm of AL-8810 (AL1000). Embryos were evaluated for grade and stage according to IETS guidelines. Embryos (n = 1734 at 6 locations across 13 replicates) were transferred (fresh or frozen in ethylene glycol) by 4 experienced technicians. Pregnancy rates were determined by ultrasonography 28 to 35 days after transfer and were increased in recipients receiving embryos collected in media containing AL100 (65%) and AL1000 (60%) compared with Control (50%; P < 0.05) as previously reported. As a continuum of this research interest, pregnancy retention rates (PRR) of recipient animals receiving embryos exposed to a prostaglandin F2α receptor antagonist (AL100 or AL1000) during collection or serving as nontreated controls were determined. Pregnancy retention rate was defined as the percentage of animals calving that were diagnosed pregnant at 28 to 35 days after transfer. From this pool of recipient animals, calving information was available to date on 494 confirmed pregnancies (presence of an embryo with a heartbeat). Data were obtained on 192 Control, 108 AL100, and 194 AL1000 recipients and analyzed using generalized mixed model analysis of variance (Glimmix) in SAS (SAS Institute, Cary, NC). Similar to initial pregnancy rates, PRR did not differ between AL100 and AL1000 (89 ± 0.04% and 90 ± 0.03%, respectively; P > 0.10); therefore, these groups were combined (AL) for subsequent analysis of PRR. Regardless of treatment, overall PRR were 88 ± 0.01% for fresh and 85 ± 0.03% for frozen embryos (P > 0.10). Additionally, gestation length (days) was similar between recipient animals receiving Control (282 ± 1.19), AL100 (281 ± 1.30) or AL1000 (281 ± 1.17; P > 0.10). However, addition of AL to collection medium of embryos increased PRR compared with Control (90 ± 0.03% v. 83 ± 0.03%, respectively; P = 0.06). Currently, an interaction was not noted between freezing method and media treatment (P > 0.10). Although collection of data continues, addition of a prostaglandin F2α receptor antagonist to the collection medium of embryos improves PRR of recipient animals without altering gestation length.
157 FIRST IN VITRO EMBRYO PRODUCTION IN ALPACAS (LAMA PACOS)
Authors: Gamarra G. , Huaman E. , León S. , Carpio M. , Alvarado E. , Asparrin M. , Vivanco H. W.
Published: December 2008

The objective was to produce alpaca embryos in laboratory due to its potential role for the multiplication of genetically superior animals and for conservation purposes. Ovaries were collected from an alpaca abattoir located in the Central Highlands of Peru and transported in a thermos flask with warm saline and antibiotics to the laboratory located 200 km away on the coast. Alpaca epididymal sperm to be used for fertilization was previously frozen by diluting in a TRIS-Fructose based extender with 10% glycerol and frozen as pellets in liquid nitrogen vapor. From 31 ovaries, 262 cumulus–oocyte complexes (COCs) were collected (mean of 8.5 COCs per ovary) which were matured in TCM-199 supplemented with 10% heat inactivated FCS plus epidermal growth factor (EGF), FSH, LH, estradiol, and cysteamine for 30 h incubation at 38.5°C, 5% CO2 and 90% humidity. The selected oocytes post-maturation were fertilized with the frozen/thawed sperm that was subjected post-thawing to Percoll gradient (90 and 45% Percoll), centrifugation and resuspension in TALP-IVF medium supplemented with 20 μm D-penicillamine, 10 μm hypotaurine, 1 μm epinephrine and 1.1 μg mL–1 of heparin. The oocytes were inseminated with a concentration of 10 × 106 spermatozoa per drop of 100 mL of fertilization medium containing 30 oocytes each and incubated for 24 h at 38.5°C, 5% CO2 and 90% humidity. The presumptive zygotes were transferred to 200 μL drops (30 zygotes per drop) of SOFaa media supplemented with 5% heat-inactivated FCS which was replaced by SOFaa plus 1% heat-inactivated FCS on day 5 after fertilization. The incubation period post-fertilization was up to day 7 at 38.5°C, 5% CO2 and 90% humidity, when the embryos were inspected and graded. The cleavage rate was evaluated at 72 h post-fertilization and embryo development was evaluated on day 5 and 7 post-fertilization. The cleavage rate was 27.1% (71/262) and the percentage of oocytes that reached the stage of morula and blastocyst was 8.0% (21/262). The percentage of blastocyst that hatched when incubated after day 7 was 14.28% (3/21). The in vitro embryo production in alpacas was successful and suggests the possibility for application in intensive reproduction for conservation of South American camelids and for genetic improvement.

Research was partially funded by contributions of BIONICHE and SAIS TUPAC AMARU, Junin, Peru.
160 FREEZABILITY OF ELECTROEJACULATED AND EPIDIDYMAL SPERMATOZOA FROM BLUE WILDEBEEST (CONNOCHAETES TAURINUS)
Authors: Mata-Campuzano M. , Alvarez M. , Borragan S. , Martinez-Pastor F. , Nicolas M. , Tamayo J. , Anel L. , de Paz P.
Published: December 2008

Maintenance of population viability, especially endangered species, requires preservation of as much genetic variability as possible, therefore genetic resource banks are very important. For male gametes, preservation of all available sources (ejaculates and epididymal) are useful. Information regarding sperm characteristics of most wild ruminant species is limited compared to that from domestic species. The objective of this work was to characterize the freezability of electroejaculated and epididymal spermatozoa from a wildebeest (5 year old; housed in Cabárceno Nature Park, Cantabria, Spain) that was castrated because of behavioral problems. After general anesthesia (ethorfine + xilazine, 1.8 mL + 0.5 mg kg–1) with dart, semen was collected by electroejaculation (3 V and 75 mA). Sperm concentration was 250 × 106 mL–1 (total spermatozoa: 1128.6 × 106). After castration, epididymides were disected and spermatozoa were collected by making several incisions in the caudal epididymis. Concentration was 12 441 × 106 spermatozoa mL–1 (total spermatozoa: 24 882 × 106). Samples were diluted to 200 × 106 spermatozoa mL–1 (TesT-Fructose-Egg yolk-Glycerol-Antibiotics) and chilled to 5°C during 2 h. Diluted semen was packaged in 0.25-mL straws and frozen from 5°C to –100°C (–20°C min–1) in a programmable cell freezer (Kryo 10, Planer). Straws were plunged into liquid nitrogen until analysis and thawed in a water bath (65°C, 6 s). Fresh, pre-freezing and post-thawed samples were analysed for motility (total motility TM, %; progressive motility PM, %; path velocity VAP, μm s–1; track speed VCL, μm s–1; progressive velocity VSL, μm s–1) using a CASA (ISAS, Proiser, Valencia, Spain). Viability (VIAB %) (SYBR-14 and propidium iodide) and mitochondrial membrane potential (MIT %) (JC1) were assessed by flow cytometry. Post-thawing results for electroejaculated v. epididymal samples were, respectively: TM: 87.0 v. 64.6%; PM: 68.7 v. 33.4%; VAP: 95.9 v. 49.8 μm s–1; VCL: 108.3 v. 71.6 μm s–1; VSL: 86.7 v. 40.2 μm s–1; VIAB: 57.0 v. 73.9%; MIT: 59.5 v. 77.5%. Motility parameters were higher for the electroejaculated sample; however, viability was higher for the epididymal sample. Recovery rates (post-thawed value/pre-freezing value × 100) for electroejaculated v. epididymal samples were: TM: 97.2 v. 93.4%; PM: 113.3 v. 103.2%; VAP: 88.9 v. 122.0 μm s–1; VCL: 87.5 v. 126.0 μm s–1; VSL: 93.4 v. 125.3 μm s–1; VIAB: 75.0 v. 97.7%; MIT: 69.2 v. 95.9%). These rates suggest a good freezability of electroejaculated and epididymal spermatozoa in blue wildebeest.

This work was supported in part by Cantur. 3 Supported by Juan de la Cierva program (MICINN, Spain).
174 MicroRNA EXPRESSION PROFILE IN BOVINE CUMULUS-OOCYTE COMPLEXES DURING LATE OOGENESIS
Authors: Miles J. R. , McDaneld T. G. , Wiedmann R. T. , Cushman R. A. , Echternkamp S. E. , Vallet J. L. , Smith T. P. L.
Published: December 2008

During late oogenesis, the mammalian oocyte synthesizes and stores mRNA necessary to guide the early stages of embryo development before the activation of embryonic transcription. The oocyte also contains many post-transcriptional regulatory mechanisms that coordinate mRNA stability and translation before specific activation. MicroRNAs (miRNAs) are short noncoding RNAs (17–25 nucleotides) that repress translation of target genes through sequence complementation and have recently been identified in murine oocytes. The objective of the current study was to identify and characterize the expression of miRNAs in bovine cumulus–oocyte complexes (COC) during late oogenesis as a potential mechanism for post-transcriptional regulation of mRNA in developing bovine oocytes. Ovaries from beef cattle (mixed populations) were obtained at a local abattoir. The COC were aspirated from 2- to 10-mm follicles and were pooled from each of 5 replicate collections for RNA extraction (n = 2241 total COC). Small RNA in the 16- to 27-bp range was isolated and used to construct cDNA libraries for sequencing, producing 2529 successful sequences that were clustered based on matching 14 consecutive bases to the most common member of the cluster. The consensus sequences of the clusters were screened for mitochondrial RNA, rRNA, tRNA, and snoRNA contaminants, leading to removal of 774 (31%) sequences from consideration. The remaining 1755 putative miRNA sequences were compared with known miRNA in miRBase, revealing 62 bovine COC miRNA clusters matching previously known sequences and 4 with no match. The cluster with the largest number of sequences identified in bovine COC matched the sequence of the let-7 miRNA family (657 sequences or 37% of putative miRNA). Within the let-7 family, let-7b (459 sequences or 26%) was the most abundant followed by let-7i (135 sequences or 8%). The four clusters that did not match sequences in miRBase represent putative novel miRNA. One of these four clusters had relatively high expression in bovine COCs (308 sequences or 18%), whereas the other 3 clusters had relatively low expression (total of 55 combined sequences or 3%). Expression of several putative miRNAs (let-7b, let-7i, miR-106a, and the abundant novel miRNA) in bovine COC were confirmed using TaqMan miRNA assays. These results demonstrate the presence of miRNA within bovine COC during late oogenesis, which suggests that these post-transcriptional regulatory elements may play a role in coordinating mRNA stability and translation in bovine oocytes.
18 RELATIONSHIP BETWEEN MULTIPLE OVULATIONS AND CONCEPTION IN LACTATING DAIRY COWS
Authors: Yoshioka H. , Matsuda H. , Yamanouchi Y. , Aoki Y. , Konishi K. , Imai K.
Published: December 2008

The relationship between multiple ovulations and various factors involved in conception in lactating Holstein cows was investigated in this study. A total of 53 AI cows housed in a free-stall barn were assigned to this study. They were offered a balanced TMR ad libitum during the experimental period. Cows were milked at 0830 and 1900 h. Milk yields were recorded at each milking. Estrus was detected by radiotelemetric pedometers (Yoshioka et al. 2007 Reprod. Fertil. Dev. 19, 129). The body condition score (BCS) of cows was recorded at estrus. Artificial insemination was performed according to the pedometry data. Pregnancy was diagnosed 42 days post-AI by transrectal ultrasonography. Ovulation was assessed by transrectal ultrasonography of the ovary every 3 h from the end of estrus. Ovulation time was measured from the onset of estrus to ovulation. Milk production of each cow was determined from 14 days before the day of estrus. Blood samples for estradiol-17β (E2) were collected at the peak of estrus, and for progesterone (P4), samples were collected on Days 0, 3, 6, 9, and 12 (estrus = Day 0). The E2 and P4 concentrations were measured by enzyme immunoassay. Preovulatory follicles at estrus and CL on Day 0, 3, 6, 9, and 12 were monitored by transrectal ultrasonography. The overall incidence of multiple ovulations was 28.3% (15/53). There was no difference in duration of estrus between cows with multiple ovulations and cows with single ovulations (10.2 h, n = 15 v. 9.8 h, n = 38). Ovulation time was longer for cows with multiple ovulations than that for cows with single ovulations (30.4 h, n = 10 v. 28.7 h, n = 26; P = 0.0545). In all cases of multiple ovulations, 2 follicles ovulated simultaneously. The milk production of cows with multiple ovulations was higher than that of cows with single ovulations (40.1, n = 15 v. 36.2 kg d–1, n = 38; P = 0.0515); however, BCS showed the opposite results (2.86, n = 15 v. 3.13, n = 38; P < 0.05). There was a significant inverse correlation between milk production and BCS in cows with multiple ovulations (r = –0.768; P < 0.01). The E2 concentrations showed no difference between cows with multiple and single ovulations (9.6 pg mL–1, n = 8 v. 8.2 pg mL–1, n = 23). The diameters of preovulatory dominant follicles and secondary follicles at estrus in cows with multiple ovulations were significantly smaller than those in cows with single ovulations (18.2 and 14.2 mm, n = 14 v. 19.9 mm, n = 38, respectively; P < 0.05). Cows with multiple ovulations showed a significantly higher P4 concentration and a larger CL than those with single ovulations from Day 3 to 12. The conception rate for AI was significantly higher for cows with multiple ovulations than for cows with single ovulations (73.3%, n = 15 v. 36.2%, n = 38; P < 0.05). Our results showed that high conception rates, high levels of milk production, and low BCS were associated with multiple ovulations.
181 THE POTENTIAL ROLE OF NON-GENOMIC PATHWAYS AND THEIR EFFECTS ON THE REGULATION OF CALBINDIN-D9k IN VITRO
Authors: Dang V. H. , Jeung E.-B.
Published: December 2008

Calbindin-D9k (CaBP-9k), a cytosolic protein, is one of the members of the family of vitamin D-dependent calcium-binding proteins with high affinity for calcium. The previous in vitro studies indicated that this gene is controlled by 17β-estradiol (E2), a physiological estrogen, via both genomic (through its classical nuclear receptors) and non-genomic (through different cypoplasmic signals) mechanisms. In order to provide a better understanding in molecular events by which E2 exerts its actions in the regulation of CaBP-9k, we employed GH3 cells as an in vitro model to examine the possible non-genomic effects of E2 on the induction of CaBP-9k. GH3 cells were treated dose-dependently (10–5, 10–6, 10–7, 10–8, and 10–9 m) with E2-BSA, a membrane-impermeable E2 conjugated with BSA, for 24 h. To examine the time dependency, the cells were also exposed to a high concentration (10–6 m) of E2-BSA and harvested at various time points (5 min, 15 min, 30 min, 1 h, 3 h, 6 h, 12 h, 24 h, and 48 h). Furthermore, in order to determine the potential involvement of non-genomic signaling pathways in E2-BSA-induced expression of CaBP-9k, several inhibitors also were employed, including ICI 182 780 for membrane estrogen receptor (ER) pathway, pertussis toxin (PTX) for G protein signaling, U0126 for ERK pathway, and wortmannin for Akt pathway. The non-genomic effects of E2-BSA on the induction of CaBP-9k mRNA and protein were determined by semi-quantitative RT-PCR and Western blotting, respectively. In a dose-dependent manner, administration with E2-BSA (10–6 m) induced the highest response of CaBP-9k at transcriptional (mRNA) level, whereas protein level of CaBP-9k peaked at E2-BSA concentration (10–7 m) at 24 h. In a time course, E2-BSA (10–6 m) exposure caused a significant increase in both CaBP-9k mRNA and protein expressions as early as 15 min and peaked at 24 h. Co-treatment with ICI 182 780 and PTX completely inhibited E2-BSA-induced CaBP-9k mRNA and protein expressions. Interestingly, although co-treatments with U0126 and/or wortmannin alone failed to attenuate the effects of E2-BSA, a combination of 2 inhibitors completely reversed E2-BSA-induced CaBP-9k expressions at both transcriptional (mRNA) and translational (protein) levels, suggesting their involvement in the regulation of CaBP-9k in GH3 cells. Taken together, these results demonstrate that various signaling pathways may be involved in E2-induced regulation of CaBP-9k in which membrane ER and G protein signaling pathways play a central role in non-genomic responses. Further in vitro experiments are required to elucidate additional details of the interaction of ERK and Akt pathways in the regulation of CaBP-9k in these cells, offering a new insight into the mode of E2 action in the pituitary gland of human and wildlife.
182 EXPRESSION PATTERN OF EMBRYONIC STEM CELL-SPECIFIC microRNAs IN MOUSE PREIMPLANTATION EMBRYOS
Authors: Fu T.-Y. , Tang P.-C.
Published: December 2008

The endogenous non-coding microRNAs (miRNAs) of 18–25 nucleotides (nt) have been shown to involve in a wide variety of cellular processes as the posttranscriptional regulators by repression of translation or cleavage of mRNAs. In mammals, there are approximately 250 miRNAs that have been identified, and the cluster of miRNA-290 s (miR-290 s) has been demonstrated to express dramatically from the 2-cell to the 4-cell stage in mouse embryos examined from oocytes to the 8-cell stage. The association of miR-290 to 295 with pluripotency has been reported according to their specific expression in embryonic stem (ES) cells. It is interesting to explore the roles of these ES cell-specific miRNAs during the preimplantation stages and early differentiation at the blastocyst stage. Therefore, the objective of this study was to profile the expression pattern of ES cell-specific miRNAs (miR-291-5p, miR-293-3p, and miR-294-3p) from the 4-cell, 8- to 16-cell, morula, and blastocyst stages of mouse embryos. CD-1 F1 embryos at various developmental stages were collected from superovulated and naturally mated CD-1 mice. Total miRNAs of each stage analyzed were collected from 3 embryos for every replicate. Real-time RT-PCR was performed by using the specific stem-loop primers and the embryo lysate as template, which was prepared by heating in 4 μL of PBS at 95°C. Additionally, the in situ expressions of miR-291-5p, miR-293-3p, and miR-294-3p in mouse preimplantation embryos were confirmed by LNA™ probes specific for individual miRNAs. The embryo was fixed with 4% paraformaldehyde for 2 h at room temperature, followed by 3 times wash in PBST (0.1% TritonX-100 in PBS). After hybridization with individual 5′-fluorescein-labeled LNA™ probe, the embryo was washed with 0.1 × SSC, 2 × SSC, and TN buffer (0.1 m Tris-HCl, pH 7.5, 0.15 m NaCl) subsequently. The in situ expressions of miRNAs were detected by immunocytochemical reaction. The results indicated that the expressions of miR-291-5p, miR-293-3p, and miR-294-3p were up-regulated from the 4-cell to the morula stage and then down-regulated afterwards. It was found that the signals of miR-293-5p in an expanded blastocyst were weaker than those at the early blastocyst stage. However, it showed that the intensity of expression at the morula stage was 2 to 4 folds higher compared to that at the 4-cell stage in each miRNA analyzed. Also, the result showed that the ES cell-specific miRNAs examined were expressed in all cells in a blastocyst, i.e. tropectoderm and inner cell mass. In conclusion, we have established the expression profile of ES cell-specific miRNAs during preimplantation stages in mouse embryos. The specific roles of these miRNAs would be further investigated in the short future.
185 THE PORCINE EPIBLAST AND NOT THE INNER CELL MASS HAS DEVELOPED CONVENTIONAL PATHWAYS FOR REGULATION OF PLURIPOTENCY
Authors: Hall V. J. , Christensen J. , Maddox-Hyttel P.
Published: December 2008

Pluripotency in mice and human embryonic stem cells is regulated by a number of transcription factors, notably including Oct-4, Sox-2, and Nanog. However, in the pig, previous research indicates that Oct-4 protein and mRNA is not specifically localized to the inner cell mass (ICM) of the zona-intact (ZI) blastocyst. Levels of expression of Nanog mRNA, on the other hand, appear to be low in the ZI blastocyst, and protein has not been detected. Similarly, Sox-2 expression in the ZI blastocyst is relatively low and not specific to the ICM. In this study, we investigated the mRNA expression of Oct-4, Sox-2, and Nanog in D6/D7-derived ZI porcine in vivo-derived blastocysts compared with epiblasts mechanically isolated from hatched D10/D11 in vivo-derived blastocysts. We then investigated components involved in pathways important for regulating pluripotency, including JAK/STAT (i.e. gp130, LIFr), FGF (i.e. bFGF, FGFr1, FGFr2), and BMP (bmp4, smad4) signaling pathways and their downstream targets, stat3, c-myc, c-fos, by using RT-PCR. Sows were artificially inseminated, and embryos were flushed from uteri following slaughter. Single D6/D7 blastocysts (n = 3), single mechanically isolated D10/D11 epiblasts (n = 3), endometrium, and oviduct total RNA was isolated using the RNeasy Micro Kit (Qiagen, Valencia, CA, USA). Total RNA from the blastocysts and epiblasts was then amplified to form cDNA using the QuantiTect Whole Transcriptome kit (Qiagen). Positive control tissues (oviduct and endometrium) were reverse transcribed using the RevertAid First Strand cDNA synthesis kit (Fermentas, Burlington, Ontario, Canada). Primers were designed to span introns in highly homologous sequences to human mRNA. Primers were tested in both oviduct and endometrium tissue, and products were sequenced to confirm specificity. PCR was performed at 55°C for 35 cycles. Results indicate that D6/D7 blastocysts only expressed Oct-4 and not Nanog and Sox-2. In contrast, all 3 transcripts were expressed in D10/D11 epiblasts. The D10/D11 epiblasts also expressed LIFr, bFGF, FGFr1, FGFr2, bmp4, smad4, stat3, c-myc, and c-fos. The cytokine receptor gp130 was only weakly expressed in a single epiblast. In contrast, the earlier stage D6/D7 blastocysts failed to express these messengers with the exception of weak expression of gp130 in all 3 blastocysts, and only a single blastocyst expressed LIFr, smad4, c-myc, and c-fos. In conclusion, this study indicates that the ICM of the porcine D6/D7 ZI blastocyst has not developed pluripotency signaling as observed in mice and humans at this developmental stage. Furthermore, without expression of gp130, the JAK/STAT pathway is unlikely to play a role in regulating pluripotency in the epiblast. It is likely that the later stage epiblast may be more amenable for the derivation of porcine embryonic stem cells.
195 TRANSCRIPTOME PROFILING OF BOVINE ENDOMETRIUM BASED ON THE PREGNANCY OUTCOME AFTER TRANSFER OF IN VIVO-DERIVED EMBRYOS
Authors: Salilew-Wondim D. , Ghanem N. , Grosse-Brinkhaus C. , Becker A. , Rings F. , Hoelker M. , Phatsara C. , Tholen E. , Zimmer A. , Schellander K. , Tesfaye D.
Published: December 2008

Transcriptome profiling of pre-transfer cycle endometrium in relation to its ability to sustain the upcoming pregnancy may pave the way to develop or identify molecular markers that can be utilized to detect and select receptive endometrium before embryo transfer. Here, we aim to show differential expression of genes between endometrium biopsies derived from recipients during pre-transfer cycle based on the pregnancy successes after embryo transfer. For this, endometrium biopsies were taken from 56 Simmental cyclic heifers of the same age at day 7 and 14 of the estrous cycle. On the next cycle, in vivo-produced day 7 blastocysts were transferred to all animals at day 7 of the estrous cycle. Pregnancy diagnosis was done at 28, 42, and 56 days of gestation. Thirty-two cows were returned to heat after 21 days, 7 were pregnant until day 42 but no pregnancy after that, and 15 resulted in calf delivery. Subsequently, the endometrium biopsies sampled during the pre-transfer period were categorized based on the pregnancy outcome. Those endometrial biopsies taken at days 7 and 14 during the pre-transferred period from those that resulted in successful calf delivery were named as d7CD and d14CD, respectively, and those endometrial samples collected at days 7 and 14 during pre-transferred period from those groups that resulted in no pregnancy were named as d7NP and d14NP, respectively. Total RNA was extracted from 3 pools of each experimental group in 3 replicates using RNeasy mini kit (Qiagen, Hilden, Germany). A total of 12 biotin-labeled cRNA samples were hybridized on 12 bovine Affymetrix arrays (Affymetrix, Santa Clara, CA, USA) consisting of 24128 probe sets. The microarray data normalization and background correction was performed using guanine cytosine robust multi-array analysis, and the data analysis was performed using LIMMA written on R package, which maintained the bioconductor. The results showed that 1130 transcripts were differentially expressed between d7CD and d7NP, of which 626 and 504 were up- and down-regulated, respectively, in d7CD. Genes that involve in regulation of transcription (PPARA, NR2F1, MYB, MYB, and CHF2) and the collagen families (COL1A1 and COL1A2) were enriched in d7CD. A total of 234 transcripts were differentially expressed between d14CD and d14NP, of which 94 and 140 were up- and down-regulated, respectively, in d14CD compared to d14NP. Transcripts involved in protein amino acid phosphorylation (MA2K6, GATM, AK3L1, and MAPK10) were found to be enriched in d14CD compared to d14NP. In conclusion, pre-transfer endometrium biopsies showed significant differences in transcriptome profile depending on the pregnancy outcome after transfer of in vivo-derived blastocysts and enable to identify transcripts related to pregnancy establishment.
199 EFFECT OF PRODUCTION SYSTEM AND BREED ON RELATIVE GENE EXPRESSION IN BOVINE EMBRYOS
Authors: Wohlres-Viana S. , Boite M. C. , Pereira M. M. , Sa W. F. , Viana J. H. M. , Machado M. A. , Camargo L. S. A.
Published: December 2008

Embryos produced in vivo and in vitro show morphological and developmental differences, which can be related to culture environment. Nevertheless, there are a few studies showing the effect of in vitro environment on embryos from different bovine subspecies, such as Gyr (Bos indicus) and Holstein (Bos taurus). The aim of this study was to evaluate the relative abundance of aquaporin 3 (AQP3) and ATPase-α1 (Na/K-ATPase alpha 1) transcripts in blastocysts produced in vivo or in vitro from Gyr and Holstein cattle. The production system effect (in vivo × in vitro) for Gyr cattle and the breed effect (Holstein × Gyr) for in vitro-produced embryos were evaluated. For each group, blastocysts (n = 15) distributed in 3 pools were used for RNA extraction (RNeasy MicroKit, Qiagen, Valencia, CA), followed by RNA amplification (Messageamp II amplification kit, Ambion-Applied Biosystems, Foster City, CA) and reverse transcription (SuperScript III First-Stand Synthesis Supermix, Invitrogen, Carlsbad, CA). The cDNA obtained were submitted to real-time PCR, using the H2a gene as endogenous control, and analyzed with REST software© using the pair wise fixed reallocation randomization Test. There was no difference (P > 0.05) in gene expression for AQP3 and ATPase-α1 between in vivo- and in vitro-produced Gyr embryos, although the results suggest that the APQ3 gene was down-regulated (0.81 ± 0.31) and the ATPase-α1 gene was up-regulated (1.20 ± 0.65) in embryos produced in vitro. For breed effect within in vitro production system, ATPase-α1 gene was down-regulated in Holstein (0.56 ± 0.30) when compared with Gyr embryos (P < 0.05). The same trend was observed for AQP3 (0.58 ± 0.25), but with no difference (P > 0.05). In conclusion, the data suggest that embryo production system does not interfere with the transcript amount of the genes studied for Gyr cattle; however, the in vitro production system may have different effects on gene expression according to embryo breed. Other genes should be evaluated for a better understanding of these differences.

Financial support: CNPq, Fapemig.
2 EVIDENCE FOR THE PRESENCE OF OVULATION-INDUCING FACTOR IN PORCINE AND EQUINE SEMINAL PLASMA
Authors: Bogle O. A. , Ambati D. , Davis R. P. , Adams G. P.
Published: December 2008

The presence of an ovulation-inducing factor (OIF) in the seminal plasma of llamas and alpacas (reflex ovulators) and cattle (spontaneous ovulators) has been reported previously (Ratto MH et al. 2006 Theriogenology 66, 1102–1106). The presence of this protein in unrelated species supports the hypothesis that OIF is a conserved factor among species. The objectives of this study were to determine if OIF was present in equine and porcine seminal plasma, and whether the proportion of test animals (llamas) that ovulated in response to treatment with seminal plasma was related to dose. In Experiment 1, female llamas were assigned randomly to four groups (n = 8 or 9 per group) and treated intramuscularly with 1 mL llama seminal plasma (positive control), 3 mL equine seminal plasma, 3 mL porcine seminal plasma, or 2 mL saline (negative control). Ovulation and maximum corpus luteum diameter were compared using ultrasonography and confirmed with blood samples taken on Day 7 (Day 0 = day of treatment) to determine plasma progesterone concentration. The diameter of the preovulatory follicle at the time of treatment did not differ among groups. Equine seminal plasma induced ovulations in 3/8 (38%) llamas compared to 0/8 (0%) llamas treated with saline or porcine seminal plasma (P = 0.1). The proportion of females that ovulated was lower in the equine group (P < 0.01) compared with those animals treated with llama seminal plasma (9/9; 100%). Of the animals that ovulated, maximum CL diameter did not differ between llama and equine seminal plasma-treated groups (mean ± SEM; 11.1 ± 1.1, 11.5 ± 1.5, respectively). Similarly, progesterone concentrations were not different among llamas treated with llama seminal plasma or equine seminal plasma (mean ± SEM; 3.1 ± 0.4, 3.7 ± 1.2, respectively). The design of Experiment 2 was the same, but the dose of equine and porcine seminal plasma was increased to 8 mL and 10 mL, respectively. The proportion of females that ovulated was less (P < 0.05) in equine (2/9) and porcine (3/9) seminal plasma groups compared with the group treated with llama seminal plasma (9/9). There were no ovulations detected in llamas treated with saline (0/8). Although differences between equine, porcine, and negative control groups did not reach significance, results provide some evidence for the presence of OIF in equine and porcine seminal plasma. The effect of dose of equine and porcine seminal plasma is equivocal, suggesting that the concentration of OIF in the seminal plasma of these species may be very low and the optimal dose for inducing ovulation in test animals had not been reached.

Research supported by the Natural Sciences and Engineering Council of Canada.
20 IMMUNOCYTOCHEMICAL CHARACTERIZATION OF DAYS 17 AND 19 OVINE IN VIVO AND SOMATIC CELL NUCLEAR TRANSFER EMBRYOS
Authors: Alexopoulos N. I. , Schauser K. , Holland M. K. , Peura T. T. , Hartwich K. M. , Walker S. K. , D'Cruz N. T. , French A. J. , Maddox-Hyttel P.
Published: December 2008

The present study aimed to characterize development of in vivo and somatic cell nuclear transfer (SCNT) Merino ovine embryos on Days 17 and 19 using the mesoderm marker vimentin, the neuroectoderm marker β-tubulin III, and the pluripotency marker OCT4 for primordial germ cells. In vivo embryos were obtained by transferring 10 to 20 zygotes to each of 20 intermediate recipient ewes. On Day 6, 4 final recipients were used to obtain embryos for recovery at Day 17 (n = 2) and at Day 19 (n = 2). SCNT embryos were constructed from in vitro-matured oocytes and adult granulosa cells. On Day 6, 9 embryos (with intact inner cell masses) were transferred to each recipient for collection at Day 17 (n = 2) and Day 19 (n = 2). Ewes were euthanazed with phenobarbitone and excised reproductive tracts flushed with saline. A total of 24 embryos at Day 17 (14 in vivo and 10 SCNT) and 23 embryos at Day 19 (11 in vivo and 12 SCNT) were collected and processed for immunohistochemistry. On Day 17, length of embryo proper for in vivo and SCNT embryos was 6.2 and 5.6 mm, width of allantois was 9.3 and 3.8 mm, and number of somites was 19 and 13, respectively. On Day 19, length of embryo proper for in vivo and SCNT embryos was 11.5 and 7.3 mm, width of allantois was 70.6 and 13.7 mm, and number of somites was 28 and 22, respectively. On both Day 17 and Day 19, in vivo embryos had a much larger allantois, more somites, and were longer in length. Vimentin staining was observed in all in vivo embryos at Day 17 and Day 19; however, in 75% of Day 17 SCNT embryos, differentiation of the somites into dermatome with underlying sclerotome was either delayed or absent. Similarly, at both Day 17 and Day 19, a larger proportion of embryos did not stain for β-tubulin III, and of those that did, only a small amount was found in the neural tube. The intensity of both stains was much weaker in the SCNT embryos compared to in vivo embryos. Oct-4 was initially found in the splanchnic mesoderm lining the endoderm in the wall of the yolk sac and later in the dorsal mesentery and medial aspect of the mesonephros with in vivo embryos having double the number of positively stained cells than SCNT embryos. The delay in mesoderm compartments of SCNT embryos could imply future problems in the musculoskeletal system. However, lack of any neuroectoderm staining in more than half SCNT embryos could point to neurological problems being more of an issue in terms of survival and well-being. Equally, the smaller allantois could induce later placental abnormalities.

The current project was funded by the Co-operative Research Centre for Innovative Dairy Products (CRC-IDP), Australia.
209 A COMPARISON OF THE EFFECTS OF DEFINED, SEMI-DEFINED, AND UNDEFINED MATURATION MEDIA ON CLEAVAGE AND SUBSEQUENT EMBRYO DEVELOPMENT OF SHEEP OOCYTES
Authors: Karami Shabankareh H. , Zandi M.
Published: December 2008

The objectives were to determine the effects of addition of growth factor(s) and antioxidant to defined (DMM), semi-defined (SDMM), or undefined (UDMM) sheep oocyte maturation media on cleavage and subsequent embryo development. Exogenous epidermal growth factor (EGF, 10 ng mL–1), insulin-like growth factor-I (IGF-I, 50 ng mL–1), or cysteamine (Cys, 100 m) was added to DMM, SDMM, or UDMM. In the experiments, 967 oocytes were collected from 1160 ovaries (Experiment 1), 770 oocytes were collected from 831 ovaries (Experiment 2), and 778 oocytes were collected from 845 ovaries (Experiment 3). After in vitro fertilization with fresh ram semen in SOF medium, groups of 10 oocytes were stripped free of cumulus cells and transferred into 50 μL of a 2-step SOF medium. The incubations were conducted under paraffin oil at 38.5°C in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2. In all experiments, data were analyzed by ANOVA. Duncan’s multiple range test was used to determine the difference between 2 means after ANOVA. Results were expressed as mean ± SEM, and statistical significance was accepted from P < 0.05. In Experiment 1, the effects of IGF-I and EGF in DMM (TCM-199 supplemented with FSH, LH, 17β-estradiol, Na pyruvate, gentamycin sulfate, and polyvinyl alcohol) were evaluated in 4 treatment groups (DMM as a control, DMM + EGF, DMM + IGF-I, and DMM + EGF + IGF-I). Cleavage, morula, and blastocyst production rates were higher in the DMM + EGF + IGF-I (83.1 ± 0.5, 47.7 ± 0.5, and 30.8 ± 1.2) than in the DMM (72.9 ± 1.3, 37.0 ± 1.1, and 20.2 ± 1.2), DMM + EGF (77.2 ± 1.0, 43.2 ± 2.1, and 26.1 ± 1.8), or DMM + IGF-I (79.9 ± 0.6, 43.3 ± 1.2, and 25.7 ± 1.3) groups (P < 0.05). In Experiment 2, the effects of Cys in DMM were evaluated. The addition of Cys to DMM + EGF + IGF-I resulted in a mean blastocyst rate of 35.0 ± 0.9% compared with 31.5 ± 1.3% in DMM + EGF + IGF-I alone (P < 0.05); however, mean cleavage rates (84.5 ± 1.3 v. 82.8 ± 1.0) did not differ (P = 0.32). In Experiment 3, the effects of DMM + EGF + IGF-I + Cys, SDMM (TCM-199 supplemented with FSH, LH, 17β-estradiol, Na pyruvate, gentamycin sulfate, and BSA) + EGF + IGF-I + Cys, and UDMM (TCM-199 supplemented with FSH, LH, 17β-estradiol Na pyruvate, gentamycin sulfate, and FBS) + EGF + IGF-I + Cys on cleavage and embryo developmental were compared. The UDMM supplemented with EGF, IGF-I, and Cys resulted in higher proportions of cleavage, morula yields, and blastocyst yields (P < 0.05) than the DMM or SDMM supplemented with EGF + IGF-I + Cys. There was no significant difference between DMM and SDMM in the proportions of oocytes reaching the morula and blastocyst stages. In conclusion, an efficient system for in vitro production of sheep blastocysts was developed by using a defined oocyte maturation system combined with growth factors, hormones, and an antioxidant, but the UDMM resulted in higher cleavage, morula yields, and blastocyst yields than the DMM or SDMM.