bluuebunny/crossref_metadata_2025 huggingface dataset:
103 TRANSDUCTION PATHWAYS RELATED TO GLUCOSE METABOLISM IN MALE AND FEMALE BOVINE EMBRYOS PRODUCED IN VITRO
Authors: Garcia-Herreros M. , Aparicio I. M. , Rath D. , Fair T. , Lonergan P.
Published: August 2011

Glucose metabolism plays an important role in energy balance control in mammalian cells and has been widely used as an indicator of embryo developmental competence. Previous studies have shown that developmental kinetic rates following IVF are lower in female than in male blastocysts, which may be related to differences in glucose consumption and metabolism. In addition, we have demonstrated that inhibition of phosphatidylinositol 3-kinase (PI3-K) with a structurally unrelated inhibitor, LY294002, suggests a negative role for PI3-K in the regulation of bovine embryo development (Aparicio et al. 2010 Reproduction 140, 83–92). The aim of this study was to determine whether PI3-K has a role in the regulation of glucose metabolism in Day 7 bovine blastocysts and to study the possible differential protein expression involved in glucose metabolism [hexokinase-I (HK-I), phosphofructokinase-1 (PFK-1), pyruvate kinase1/2 (PMK1/2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenase A/C (LDHA/C), glucose transporter-1 (GLUT-1) and glycogen synthase kinase-3 (GSK-3A/B)] between in vitro produced male and female embryos derived from IVF with either X- or Y-sorted semen. Day 7 blastocysts derived from unsorted semen (n = 25 blastocysts per group) were incubated up to 12 h in SOF culture medium in the presence or absence of LY294002 (10 μM) and stored. Similarly, male and female Day 7 blastocysts derived from sorted semen were collected apart and stored at –80°C until proteomic analysis (Western blot analysis of proteins separated by sodium dodecyl sulfate-polyacylamide gel electrophoresis). Inhibition of PI3K significantly decreased HK-I (P < 0.01), PFK-1 (P < 0.001), GAPDH (P < 0.05), GSK-3A/B (P < 0.001), and GLUT-1 (P < 0.01) protein levels. Interestingly, protein expression of HK-1 (P < 0.001), PFK-1 (P < 0.01), PMK1/2 (P < 0.05), GAPDH (P < 0.01), and GLUT-1 (P < 0.001) was significantly higher in male compared with female blastocysts. The significant increase in the phosphorylated forms (Ser21 and Ser9) of both isoforms (GSK-3A/B) in male compared with female embryos is indicative of a higher inactivation of GSK-3A/B in males (P < 0.001). The presence of LDHA/C activity was not detected in any blastocyst group, irrespective of the gender or treatment studied. In conclusion, our data suggest that PI3K plays a major role in the regulation of glucose metabolism in bovine embryos, because pretreatment with LY294002 significantly modified the protein expression of HK-I, PFK-1, GAPDH, GSK- 3A/B, and GLUT-1, and underline the possibility of modulating glucose metabolism via the PI3K cellular pathway. The differential glycolytic metabolism between male and female blastocysts might explain the higher developmental kinetic rates in males described by other authors, because the expression of proteins involved in glycolysis and glycogenogenesis was significantly higher in male than in female in vitro-produced bovine embryos.
104 COMPARATIVE ANALYSIS OF THE YOLK SAC OF BOVINE EMBRYOS AT 30 DAYS OF PREGNANCY DERIVED FROM ARTIFICIAL INSEMINATION OR IN VITRO FERTILIZATION
Authors: Oliveira C. M. , Oliveira A. , Fonseca E. , Miglino M. A.
Published: August 2011

The yolk sac has importance related to hematopoiesis, because it carries blood cells, and is part of the circulatory system in early embryos. The yolk sac is related to proper embryonic development because the organ is responsible for transmitting metabolic, nutrition, protection, and training of key organs, such as the intestine, in early embryos before formation of the placenta. This study attempted to compare the yolk sac of bovine embryos derived from fixed-time artificial insemination (TAI) and in vitro fertilization (IVF), relating them to their stage of development and growth at 30 days of gestation. Morphological, histological and immunological diseases were also compared. Yolk sacs from TAI or IVF bovine embryos at 30 days of gestation were collected (n = 2/group). The yolk sacs were immersed in fixative solution of 10% formaldehyde and embedded in Paraplast for light microscopy and immunohistochemical techniques. Sample cuts of 5 μm thickness were stained with hematoxylin and eosin (HE) and exposed to primary (anti-Ki-67; 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and secondary (LSAB + Peroxidase Kit – K0690, DakoCytomation, Glostrup, Denmark) antibodies. To reveal immunoreactivity, diaminobenzidine (DAB, Dako) was used and the background was counter-stained with Harris hematoxylin. On Day 30 of gestation, the yolk sac of embryos from either TAI or IVF were quite developed, yellowish coloured, and presented signs of early regression. Moreover, they showed a central compact area that was clearly visible next to the embryo. The free ends were elongated and accompanied by umbilical vessels towards the ends of the gestational sac. There were no signs of fusion between the chorion and allantois, but the juxtaposition was evident with the allantois. The yolk sac is composed of three layers: the endoderm, mesothelium (simple mesothelial), and an intermediate layer (vascular mesenchymal). The layer corresponding to the mesenchyme presented thin and elongated cells with characteristics of synthesising cells. The KI-67 is a marker of cell proliferation, present in all active forms of the cell cycle and absent during quiescence. This expression reflects proliferative activity; however, in all samples subjected to immunohistochemical reaction, there was no nuclear staining of the antibody. We conclude that on Day 30 of gestation, the yolk sac shows no differences in morphological and microscopic characteristics between TAI and IVF groups. It also appears that the process of regression had not occurred as indicated by staining patterns of the Ki-67 antibody.

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Sociedade Agropecuária Imaculada Conceição Ltda. (SAIC).
106 EFFECTS OF BONE MORPHOGENETIC PROTEIN 4 (BMP4) AND ITS INHIBITOR NOGGIN ON BOVINE IN VITRO EMBRYO DEVELOPMENT
Authors: La Rosa I. , Fernandez-Martin R. , Paz D. A. , Salamone D. F.
Published: August 2011

Bone morphogenetic protein 4 (BMP4) is a member of the BMP family of conserved morphogenes in charge of many events of differentiation (Chen et al. 2004 Growth Factors 22, 233–241) BMP4 is involved in regulation of pluripotency in humans and mice though the role in bovine early embryo development is still undefined. Noggin is a BMP4 inhibitor (Groppe et al. 2002 Nature 420, 636–642) that does not have a specific receptor but functions by directly binding BMP ligands. The objective of this work was to study the effects of BMP4 and Noggin on early bovine embryo development. Cumulus–oocyte complexes (COC) were aspirated from abattoir ovaries and in vitro matured in TCM containing 10% fetal bovine serum (FBS), 2 mM FSH, 20 mM cysteamine, 1% antibiotic- antimycotic (15240, GIBCO, Grand Island, NY, USA) and 0.1 mM sodium pyruvate. Incubation conditions were a 6.5% CO2 humidified atmosphere at 39°C. After 22 h, in vitro fertilization was performed. Briefly, frozen–thawed semen was centrifuged twice at 490 × g and resuspended in B.O. solution to a final concentration of 20 × 106 mL–1 and incubation with COC was performed for 5 h. Presumptive zygotes were randomly cultured in CR2 with 0.3% BSA, free of serum and co-culture (control, n = 217) or supplemented with 100 ng mL–1 of either BMP4 (n = 218) or Noggin (n = 205). Cleavage and blastocyst rates were evaluated at Days 2 and 9 of culture. Blastocysts cell numbers were analysed by nuclear staining with Hoechst 33342. The expression pattern of the transcription factor Oct-4 was studied by immunocytochemistry and confocal microscope analysis in blastocysts. Chi-square tests were applied for cleavage, blastocyst, and hatching rates. One-way ANOVA was used to compare blastocyst cell number and a proportion test was used for Oct-4 expression. For all, P < 0.05 was considered significant. Cleavage rate was significantly lower in the Noggin group compared to control (51.2% v. 62.3%) whereas the BMP group (61.3%) did not differ from control or Noggin groups. Blastocyst rates for the BMP and Noggin groups were statistically lower than control (9.24% and 11.7% v. 20.6%, respectively). Hatching rate for the control group was significantly higher than both BMP and Noggin groups (4.6% v. 1.4% and 0.49%, respectively). Blastocyst cell number did not differ between groups (130, 117, and 128 for control, BMP4, and Noggin groups, respectively). Oct-4 expressing cells over total cell number was lower in BMP (72%; n = 3) and Noggin (72%; n = 3) groups compared to control (83%; n = 3). In our conditions, BMP inhibition with Noggin or addition of exogenous BMP4 negatively affected developmental rates and altered the proportion of pluripotent (Oct-4 positive) cells. Our results demonstrate the importance of a correct balance within the BMP signalling system for proper bovine in vitro embryo development.
107 DIFFERENCES IN THE INNER MITOCHONDRIAL MEMBRANE POTENTIAL BETWEEN NON-CULTURED AND CULTURED PORCINE EMBRYOS
Authors: Romek M. , Gajda B. , Rolka M. , Smorag Z.
Published: August 2011

In comparison to in vivo derived pig embryos, in vitro culture conditions produce embryos with altered metabolic rates of carbohydrates and fatty acids (Romek M et al. 2010 Theriogenology 74, 265–276), which may compromise embryo viability. Because various energy substrates are metabolized via several aerobic pathways leading to generation of the inner mitochondrial membrane potential (ΔΨm), value of ΔΨm is a key indicator of embryo metabolic activity, closely related to oxygen consumption and cellular energy needs. Therefore, the aim of this study was to compare ΔΨm between non-cultured and cultured pig embryos during early development. The non-cultured embryos were obtained from 6-month-old gilts, whereas those derived in vitro were cultured from zygotes to the appropriate stage in North Carolina State University 23 (NCSU-23) medium supplemented with 4 mg mL–1 of bovine serum albumin. The ΔΨm measurements were carried out on both non-cultured and cultured 4 to 8 cell embryos, morulae, blastocysts and late blastocysts. For this, embryos were labelled with 0.5 μM Mito Tracker Orange CMTMRos (MtOR) for 30 min at 39°C and then with 0.5 μM Mito Tracker Deep Red (MtDR) for 30 min at 10°C. Using a LSM 510 Meta Zeiss confocal microscope, we measured the amounts of fluorescence (IMtOR and IMtDR) emitted from embryos and values of ΔΨm were estimated as the IMtOR/IMtDR ratios. The results were analysed by ANOVA and Tukey's test. From the zygote to morula stages, ΔΨm remained unchanged and did not differ between developmentally matched non-cultured and cultured embryos (P < 0.001). The value of ΔΨm increased significantly (P < 0.05) from 0.90 ± 0.26 arbitrary units (a.u.) for morulae to 3.92 ± 0.63 and 2.06 ± 0.38 a.u. for non-cultured and cultured early blastocysts, respectively. Whereas the mean value of ΔΨm was almost 2 times higher in non-cultured than in cultured early blastocysts, the mitochondrial membrane potential was statistically similar (P < 0.05) in the in vivo derived (2.10 ± 0.37 a.u.) compared to cultured (1.87 ± 0.30 a.u.) blastocysts. The lower ΔΨm in cultured early blastocysts may be explained by several-fold higher glucose concentration in NCSU-23 medium than in the oviductal fluid. It was reported that high levels of glucose decreases the Krebs cycle metabolism of pyruvate, glutamine, and glucose, and reduces oxidation rates of fatty acids in cultured pig embryos in comparison with in vivo counterparts. Hence, this impaired metabolism reflected by decreased ΔΨm may be responsible for insufficient energy production and reduced developmental competence of cultured early blastocysts. Therefore, because embryo-cavitation is a critical event in pig development, further effort should be focused on proper blastocyst culture.

Research was partially supported by Grant NR 12 0036 06 from NCBiR, Poland.
11 PREGNANCY RATES IN ESTONIAN HOLSTEIN HEIFERS AFTER INSEMINATION WITH SEXED SPERM
Authors: Kurykin J. , Jalakas M. , Majas L. , Kaart T. , Jaakma Ü.
Published: August 2011

We analysed the results of insemination (AI) with 2.2 million X-chromosome-bearing frozen–thawed sperm in 2283 Estonian Holstein (EHF) heifers on 7 dairy herds. The heifers of 11 to 18 months of age were inseminated with sexed sperm or unsexed control semen doses (15 × 106 sperm) from 10 different bulls either 1) at fixed time following synchronization of oestrus by 2 injections of PGF2α, 2) at visually detected spontaneous oestrus or 3) at oestrus displayed after a single injection of PGF2α. At AI, the presence and intensity of estrous signs (vulvar edema, hyperemia, discharge of mucus and an ease to pass through the cervix by catheter) were recorded. Pregnancy status of heifers was diagnosed by rectal palpation of the uterus 45–60 days after AI. Statistical analyses were performed using the SAS package (1999; SAS Institute Inc., Cary, NC, USA). The pregnancy rate of heifers after fixed-time AI at synchronized oestrus was 42.7%, which is about 80% of control unsexed semen doses (53.0%, P < 0.05). The pregnancy rate after intracornual deposition of sexed sperm (44.1%) did not differ (P > 0.05) from conventional insemination into the uterine body (41.9%). The pregnancy rates after AI of heifers with sexed sperm at spontaneous oestrus (55.9%) or oestrus displayed following a single PGF2α treatment (50.8%) did not differ between each other, but were higher than at fixed-time AI (42.7%; P < 0.05). The pregnancy rates after AI with sexed sperm at spontaneous oestrus and induced oestrus were about 90% and 85% of that of unsexed semen doses, respectively (P < 0.05). The pregnancy rates varied among the farms and among the bulls. For some farms and bulls the pregnancy rates with sexed sperm doses were similar to the pregnancy rates with unsexed regular semen doses. The pregnancy rates did not differ between heifers housed in tie-stalls or free-stalls. Pooled across sperm deposition sites, heifers that showed strong estrous signs at fixed-time AI with sexed sperm had 2.7 times higher pregnancy rate than heifers with weak signs of oestrus. The difference in pregnancy rates was 1.2 times higher in heifers with strong estrous signs when unsexed semen was used at fixed-time AI (P < 0.05) and when the heifers were inseminated with sexed or unsexed sperm doses at visually detected spontaneous or induced oestrus. The mean age and body weight did not differ between the heifers that conceived and those that failed irrespective of AI treatment. In conclusion, insemination of heifers with sexed sperm at spontaneous oestrus or oestrus induced by PGF2α treatment resulted in higher pregnancy rates than insemination at fixed time after oestrus synchronization. Intracornual deposition of semen did not improve the pregnancy rate. However, good reproductive status of a herd along with the selection of heifers according to the intensity of oestrus expression improves the efficiency of using sexed sperm for the insemination of dairy heifers.

The study was supported by the ESF grant 7814.
111 SINGLE EQUINE EMBRYO LIPID FINGERPRINTING BY MASS SPECTROMETRY
Authors: Gonçalves R. F. , Ferreira C. R. , Orlandi C. M. B. , Sartori V. C. , Ferreira H. N. , Gozzo F. C. , Saraiva S. A. , Pilau E. J. , Eberlin M. N.
Published: August 2011

Matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS) allows the lipid profile study of individual mammal embryos. The data collection is rapid, highly sensitive, can tolerate some level of impurities, and is easy to interpret. The aim of this study was to report the lipid profile obtained from a single equine embryo by MALDI-MS. Follicles ≥30 mm in diameter were monitored daily until ovulation (Day 0). The insemination was performed close to ovulation with fresh diluted semen, and the embryo recovery was performed on Day 9 (D9) post ovulation. The equine embryo was placed in 50/50 (v/v) methanol/phosphate buffer solution and transported at 4°C to the laboratory. MALDI-MS spectra were acquired in the positive ion mode using MALDI Synapt HDMS mass spectrometer (Waters, Manchester, UK) m/z 700–950 range. The sample was coated with a 1.2 μL matrix of 2,5-dihydroxybenzoic acid (DHB) 1.0 mL L–1 in methanol. Due to the equine embryo volume, it was possible to divide it and get two mass spectra, which were identical. Spectra processing was performed using the MassLynx 4.0 software (Waters, Manchester, UK). It was observed the presence protonated and sodiated species of sphingomyelins (SM), phosphatidylcholines (PC), and triacylglycerols (TAG). The most intense ions assigned by comparison with data obtained from bovine embryos were m/z 723.5 [PC (34:1) and loss of N(CH3)3]+, 725.5. [SM (16:0) + Na]+, 754.6 [PC (32:1) + Na]+, 778.6 [PC (36:1) + Na]+, 780.6 [PC (34:2) + Na]+ or [PC (36:5) + H]+, 782.6 [PC (36:4) + H]+ or [PC (34:1) + Na]+, 788.6 [PC (36:1) + H]+, 806.6 [PC (38:6) + H]+ or [PC (36:3) + Na]+, 808.6 [PC (38:5) + H]+ or [PC (36:2) + Na]+, 810.6 [PC (38:4) + H]+ or [PC (36:1) + Na]+, 907.7 [TAG (54:3) + Na]+ and 909.7 [TAG (54:2) + Na] +. Regarding the lipid profile by MALDI-TOF previously reported for oocytes and embryos of several species (Ferreira et al. 2010, J. Lipid Res., 51, 1218–1227), it detected similar lipid species, but with different relative intensities. Because of the single equine embryo volume and MALDI-MS technique sensitivity, we intend to observe if there will be differences between the lipid profile of the inner cell mass and trophoblast in the future. The analysis of a greater number of embryos as well as different development periods and MS/MS experiments will contribute to building a database of lipid profiles that allows a better understanding of the lipid profile physiology in equine embryos and the meaning of differences among other mammalian embryos.

FAPESP (São Paulo Research Foundation).
115 EXPRESSION OF RADICAL S-ADENOSYL METHIONINE DOMAIN CONTAINING-2 (RSAD2) AND TRANSFORMING GROWTH FACTOR β (TGF-β) IN BOVINE PERIPHERAL BLOOD MONONUCLEAR CELLS DURING PREGNANCY: IDENTIFICATION OF POTENTIAL BIOMARKERS TO DETERMINE PREGNANCY STATUS
Authors: Mansouri-Attia N. , Oliveira L. J. , Carter F. , Forde N. , Lonergan P. , Fair T.
Published: August 2011

During the bovine peri-implantation period, immune cells are exposed to endocrine and paracrine signals, which support feto-placental development. Recent studies in cattle have shown that the antiviral factor Mx2, known to be up-regulated by conceptus-derived interferon-τ in pregnancy, is detected in peripheral blood mononuclear cells (PBMC) and can be used for pregnancy detection on Day 18. The objectives of the current study were to correlate mRNA expression levels of pregnancy associated factors and immune modulators RSAD2 (radical S-adenosyl methionine domain containing-2) and TGF-β (transforming growth factor β) in circulating PBMCs, with pregnancy status in dairy cows and heifers. Holstein-Friesian heifers (n = 12) and lactating dairy cows (n = 17) were synchronised to estrus and artificially inseminated (bred) or not (non bred, n = 4). Blood samples were collected on Day 0 (day of AI or estrus), 5, 7, 13, 16, 20, and 25 for PBMC isolation. Pregnancy was diagnosed retrospectively by transrectal ultrasonography at Days 30 and 60 after breeding. The relative quantification of RSAD2 and TGF-β transcript abundance, analysed by qPCR, was normalised against PPIA and calculated according to the relative standard curve method. Data from duplicates were pooled, and a mixed model was run on the pooled Cq values for each stage. Genes were considered as differentially expressed if the mixed model P value was <0.05. RSAD2 mRNA abundance was 2-fold higher in pregnant heifers compared with non-bred heifers on Day 20 (P < 0.01) and Day 25 (P < 0.03) post AI. Likewise, RSAD2 mRNA expression was greater (2.25 fold) in pregnant heifers compared with heifers that were inseminated but not pregnant (P < 0.007). In contrast, RSAD2 expression was not significantly different in pregnant cows compared to non-bred or bred but non-pregnant cows. TGF-β mRNA abundance was significantly lower in both pregnant cows (3.5-fold, P < 0.04) and pregnant heifers (4.5-fold, P < 0.01) compared to non-bred animals as early as Day 13. This suppression of expression was maintained until Day 20 in heifers (3.9-fold, P < 0.02). In addition, PBMC TGF-β mRNA expression was lower in pregnant cows compared with bred non-pregnant cows (1.65-fold, P < 0.06). In conclusion, Day 20 PBMC RSAD2 expression may be a useful predictor of pregnancy maintenance in dairy heifers. However, decreased TGF-β expression as early as Day 13 appears to be a more powerful indicator of the presence of a viable embryo in both dairy heifers and cows and may play a role in the establishment and maintenance of pregnancy through the restriction of monocyte recruitment or activation during the time of maternal recognition of pregnancy.

Research supported by Science Foundation Ireland (PICA-B813)
117 SERUM PROGESTERONE CONCENTRATION AND EMBRYO DIAMETER ON DAY 7 POST-OVULATION IN MARES
Authors: Scott B. R. , Carwell D. B. , Hill R. A. , Bondioli K. R. , Godke R. A. , Gentry G. T.
Published: August 2011

Monitoring serum progesterone (P4) concentrations has been a routine practice for determining phases of the oestrous cycle in mares. The use of maternal serum P4 concentration to estimate embryo size would be beneficial in determining the proper collection time for equine embryos destined for cryopreservation and transfer. Therefore, the objective of this study was to determine if embryo diameter was correlated with maternal serum P4 concentration on Day 7 post-ovulation. Light horse mares of various breeds (n = 40) were used in this study. The mares were in good body condition and ranged in age from 3 to 18 yrs. Oestrus detection was performed first with a stallion followed by reproductive tract examinations via transrectal ultrasonography to predict ovulation. Blood samples were collected via jugular venipuncture before artificial insemination (AI) (Day 0) and again on Day 7 post-ovulation. Blood samples were allowed to clot, then centrifuged for 10 min at 2600 × g and serum stored at –20°C until assayed. Each donor mare was inseminated with extended fresh semen every other day until ovulation. On Day 7 post-ovulation, a nonsurgical embryo collection was performed on inseminated mares by a single technician using a standard equine uterine flushing procedure. Only blood samples taken from mares in which an embryo was collected were analysed (n = 18). Blood samples were collected from similar noninseminated mares (n = 12) from the same research herd on the day of expected AI (Day 0) and again on Day 7 post-ovulation. Serum progesterone was determined using a P4 fluorometric enzyme immunoassay (FEIA; Tosoh Bioscience, Inc., South San Francisco, CA). Embryo diameter was measured including the zona pellucida using Micron Software (Westover Scientific). Mean P4 concentrations (±s.e.) for all inseminated donors producing an embryo were 0.49 ± 0.1 ng mL–1 at Day 0 and 12.9 ± 1.2 ng mL–1 on Day 7 post-ovulation. Mean P4 concentration of donors on Day 7 post-ovulation with embryo diameters of <400 μm (n = 7) was not different from donors producing embryos >400 μm (n = 10) and were 11.2 ± 1.7 ng mL–1 and 14.1 ± 1.7 ng mL–1, respectively. One mare produced viable twin embryos (284 and 373 μm in diameter) and had a P4 level of 35 ng mL–1 on Day 7 post-ovulation, and was not included in the data analysis. Mean P4 concentrations in the noninseminated mares were 0 0.68 ± 0.2 ng mL–1 on Day 0 and 12.3 ± 1.6 ng mL–1 on Day 7 post-ovulation and were not different from embryo-producing mares regardless of embryo size. From this preliminary study, that there is no evidence that embryo diameter is correlated with maternal progesterone concentrations on Day 7 post-ovulation.
118 A SINGLE INTRAUTERINE INFUSION OF SUSTAINED RELEASE RECOMBINANT BOVINE INTERFERON τ ON DAY 13 EXTENDS CORPUS LUTEUM LIFESPAN IN CYCLIC COWS
Authors: Takahashi H. , Hashiyada Y. , Inaba Y. , Yasuda T. , Hirako M. , Geshi M.
Published: August 2011

Bovine interferon (bIFN) τ has been implicated as a mediator of maternal recognition of pregnancy in cattle. (Geshi et al. 2001 Theriogenology 55, 325) reported that daily intrauterine infusion of recombinant (r) bIFNτ from Day 13 to 24 extended interestrous intervals in heifers. The objective of this study was to determine whether a single infusion of sustained release bIFNτ into the uterine horn would extend corpus luteum lifespan in cyclic cows. RbIFNτ was prepared from the Silkwarm-Baculovirus gene expression system (1 mg mL–1, 1 × 108 IU mg–1, Nagaya et al. 2004 J. Vet. Med. Sci. 66, 1395–1401). Two sustained release carriers, liposome and aluminum hydroxide (Al) gel were tested. Liposome encapsulated (lipo) BSA (control) and rbIFNτ were prepared from a lipid mixture solution contained 2 mg of each protein by the Bangham method including lyophilization and rehydration. The same amount of BSA or rbIFNτ was adsorbed to Al-gel containing 2.5 mg Al. Adsorption was allowed to proceed for 5 min at room temperature and unbound proteins were removed by centrifugation. Subsequently, lipo- or Al-gel adsorbed BSA and rbIFNτ were adjusted to 0.5 mL with saline and loaded into 0.5-mL AI straws. BIFNτ free from liposome or unbound to Al-gel was measured by RIA. Eight Japanese Black cows were used in this study. Their signs of oestrus were monitored twice a day. Cows were randomly assigned to four groups, receiving a single infusion of 1) lipo-BSA (n = 3); 2) lipo-rbIFN τ (n = 3); 3) BSA with Al-gel (n = 4); or 4) rbIFNτ with Al-gel (n = 4) on Day 13 (oestrus = Day 0). The BSA or rbIFNτ solution was introduced into the uterine horn ipsilateral to the corpus luteum by the cervical route. Blood samples were collected and rectal temperatures were recorded immediately preceding the infusion (0 h), thereafter every 3 h until 12 h, and 24 h after the infusion. Total white blood cells (WBC) were counted. Data were analysed by ANOVA using the STATVIEW program. Unbound bIFNτ in liposomal encapsulation and adsorption to Al-gel were estimated to be ∼90% and 2%, respectively. Corpus luteum lifespan was normal in controls (lipo-BSA; 21.6 ± 0.33 and BSA in Al-gel; 21.2 ± 0.25 days) but was extended in cows receiving rbIFNτ with Al-gel (27.0 ± 0.40 days; P < 0.01), whereas lipo-rbIFNτ was less effective. Rectal temperatures increased following rbIFNτ treatment with a peak at 6 h after infusion (P < 0.05). Correspondingly, total WBC was decreased following rbIFNτ treatment with a minimum at 9 h. Those changes were larger in liposomal encapsulation than in adsorbed to Al-gel. In conclusion, a single infusion of bIFNτ adsorbed to Al-gel can extend corpus luteum lifespan in cyclic cows.
119 FGF2 AND FGF10 STIMULATES BOVINE AND OVINE TROPHOBLAST CELL MIGRATION
Authors: Yang Q. E. , Zhang K. , Giassetti M. I. , Ozawa M. , Johnson S. E. , Ealy A. D.
Published: August 2011

Following hatching, bovine and ovine conceptuses undergo a phase of massive development and remodelling that causes elongation and filamentation. Proper trophoblast cell development and interaction with the uterus is critical for the establishment and maintenance of pregnancy. Various growth factors, including several fibroblast growth factors (FGFs), are produced by the uterus, and at least two of these, FGF2 and FGF10, are released into uterus lumen during early pregnancy. Microarray analysis found that gene products associated with migration and invasion were altered in bovine blastocysts exposed to FGF2 or 10. The objective of this work was to determine if FGF2 and FGF10 impact bovine and ovine trophoblast cell migration. The ability of FGF2 and FGF10 to influence migratory ability of trophoblast cells was examined by using an in vitro transwell migration assay. The bovine trophoblast line, CT1, was used in the first study. After serum starvation, CT1 cells were seeded on the top of each transwell membrane (50 000/transwell) in the presence of vehicle, 0.5, 5, or 50 ng mL–1 bovine recombinant FGF2 or human recombinant FGF10. After 12 h, the transwell was fixed and stained with Hoechst 33342 (0.5 μg mL–1). Migrated cells were counted on five non-overlapping areas of each filter using epifluorescence microscopy. Supplementation with 0.5 ng mL–1 FGF2 increased the number of migrated CT1 cells when compared with controls (268.3 ± 58.3 v. 167.3 ± 47.7; P < 0.01). Supplementation with 5 or 50 ng mL–1 FGF2 further increased the number of migrated CT1 cells (297.0 ± 51.4 and 429.4 ± 98.3, respectively; P < 0.001). Adding 0.5 ng mL–1 FGF10 did not affect CT1 migration but providing 5 or 50 ng mL–1 FGF10 increased CT1 migration (399.8 ± 29.7 and 392.7 ± 58.6 v. 194.2 ± 40.3 for controls; P < 0.005). A subsequent study utilised the ovine trophoblast line, oTR1 in the migration assay (30 000 cells/transwell; 8 h migration assay). Adding 0.5 ng mL–1 FGF2 or FGF10 did not affect oTR1 migration number but exposure to holdout 5 or 50 ng mL–1 FGF2 or FGF10 increased oTR1 migrated cell numbers v. controls (P < 0.05). In a subsequent study, p38 mitogen-activated protein kinase (MAPK), ERK1/2 and JNK signalling cascades utilised by FGF2 and FGF10 in oTR1 cells were investigated. Western blot analysis indicated that both FGF2 and FGF10 induced ERK1/2 and p38 MAPK phosphorylation status. Interestingly FGF10 activated JNK but not p38 MAPK. Taken together, FGF2 and FGF10 stimulate trophoblast cell migration. This response could be mediated by an ERK1/2- or p38 MAPK-dependent system.

This project was supported by NRICGP number 2008-35203-19106 from the USDA-NIFA.
12 INDUCTION OF OVARIAN ACTIVITY IN THE MANED WOLF (CHRYSOCYON BRACHYURUS) USING A GnRH-AGONIST
Authors: Johnson A. E. , Padilla L. R. , Hope K. , Wildt D. E. , Songsasen N.
Published: August 2011

Induction of ovarian activity and ovulation is a valuable tool for the genetic management of ex situ populations of wildlife. Deslorelin, a gonadotropin releasing hormone (GnRH)-agonist, has been used earlier to induce oestrus in the grey wolf (Canis lupus). The objective of the present study was to determine the efficacy of Deslorelin (2.1 mg, Ovuplant®, Peptech Animal Health, Australia) for manipulating ovarian activity of the maned wolf, a species speculated to be an induced ovulator. Eight female (4–12 years old) maned wolves were (i) paired with a male (n = 3) or (ii) housed alone (n = 5). Of the 8 females, 1 (1 in a pair and 1 singleton) were implanted with Deslorelin under the vulvar mucosa for 12 days. The remaining 6 received implants in the subcutaneous layer of the ear for 7 days. Fresh fecal samples were collected 5 to 7 days/week for 1 month before Deslorelin treatment through 6 weeks after implant withdrawal. Reproductive steroid metabolites were extracted from the fecal samples and quantified using a validated enzyme immunoassay. Baseline progestagen concentrations for each individual were calculated by an iterative process, whereby high values exceeding the mean plus one standard deviation were excluded. Comparisons of oestrogen and progestagen concentrations among pre-, peri-, and post-Deslorelin implant periods were performed using analysis of variance. Site and duration of the GnRH agonist treatment had no effect (P > 0.05) on subsequent ovarian responses. In paired females, oestrogen metabolites reached the highest (P < 0.05) concentration during Deslorelin (i.e. peri-) treatment (441.7 ± 20.2 ng g–1 of feces) compared to pre- (174.9 ± 16.7) and post- (177.8 ± 9.1) treatment. Progesterone metabolites rose (P < 0.05) above the baseline (indicative of ovulation) starting on Day 10 after the onset of Deslorelin implantation and remained elevated throughout the study (pre-, 11 645 ± 4798; peri-, 31 521 ± 6444; post-, 55 843 ± 2924 ng g–1 of feces). In singleton females, oestrogen metabolites increased (P < 0.05) immediately after implantation (from pre-, 184.2 ± 45.3 to peri-, 334.2 ± 29.8 ng g–1 of feces) and then declined (post-, 192.3 ± 12.4). Progestagen metabolites exhibited a similar pattern with a rise (P < 0.05) during Deslorelin treatment (from pre-, 3870 ± 1336 to peri-, 10 546 ± 880 ng g–1 of feces) followed by a decline after implant withdrawal (post-, 6171 ± 366), indicating that ovulation did not occur. These results suggest that Deslorelin can induce ovarian activity in the maned wolf. However, administration of an ovulatory agent after Deslorelin treatment may be required in females managed in the absence of a male, further supporting the assertion that this species is an induced ovulator.

Funded by the Morris Animal Foundation.
124 CO-CULTURE WITH BOVINE INTACT CUMULUS - OOCYTE COMPLEXES DURING IN VITRO FERTILIZATION OF BUFFALO DENUDED OOCYTES COMPLETELY RESTORES THEIR FERTILIZING AND DEVELOPMENTAL COMPETENCE
Authors: De Blasi M. , Rubessa M. , Boccia L. , Di Francesco S. , Suárez Novoa M. V. , Longobardi V. , Gasparrini B.
Published: August 2011

Removal of cumulus cells is necessary for several technologies such as vitrification, intracytoplasmic sperm injection, and nuclear transfer. However, it is known that the presence of cumulus cells during IVF of buffalo oocytes is fundamental for fertilization and embryo development (Gasparrini et al. 2007 Anim. Reprod. Sci. 98, 335–342; Nandi et al. 1998 Theriogenology 50, 1251–1262). The aim of this work was to evaluate whether co-culture with intact bovine cumulus–oocyte complexes (COC) during IVF would restore the developmental competence of denuded buffalo oocytes. Due to the scarce availability of buffalo ovaries, the somatic support was provided by bovine cumulus cells. Abattoir-derived COC were matured in vitro according to our standard procedures (Gasparrini et al. 2006, Theriogenology, 65, 275–287) and randomly distributed in 3 fertilization groups: 1) a control group of COC (n = 122), 2) a negative control of denuded oocytes (DO; n = 119), and 3) DO co-cultured with in vitro matured bovine COC (DO+COC; n = 103) in a 1:1 ratio (3 bovine COC + 3 denuded buffalo oocytes/50 μL drop). Fertilization was carried out with frozen–thawed spermatozoa from a tested bull in TALP medium supplemented by 0.2 mM penicillamine, 0.1 mM hypotaurine, and 0.01 mM heparin at 38.5°C under a controlled gas atmosphere of 5% CO2 in humidified air. After fertilization the zygotes were cultured in SOF medium including essential and nonessential amino acids and 8 mg mL–1 BSA, at 38.5°C under humidified 5% CO2, 7% O2, and 88% N2, up to the blastocyst stage. On Day 5 and on Day 7 (Day 0 = IVF) cleavage and blastocyst rates were respectively recorded. Data were analysed by chi-square test. As expected, cleavage and blastocyst rates were lower (P < 0.01) in DO (36.1 and 9.2%, respectively) compared with the control (67.2 and 27.1%, respectively). However, co-culture during IVF (DO+COC) significantly increased (P < 0.01) both parameters compared with DO, giving cleavage (70.9%) and blastocyst (27.2%) rates similar to the control. The results of this study demonstrated that co-culture with bovine intact COC during IVF of buffalo denuded oocytes completely restores their fertilizing capability and blastocyst developmental competence. We conclude that this may be a suitable strategy for preserving the developmental competence of oocytes devolved to technologies, such as oocyte vitrification, that require cumulus removal.
128 MULTIPLICATION OF 8-CELL EMBRYOS BY AGGREGATION OF A SINGLE ENHANCED GREEN FLUORESCENT PROTEIN-LABELED BLASTOMERE WITH PUTATIVE TETRAPLOID EMBRYOS
Authors: Hiriart M. I. , Bevacqua R. J. , Fernandez-Martin R. , Salamone D. F.
Published: August 2011

Isolated blastomeres from 2- and 4-cell embryos are able to generate live offspring. However, the development of each cell of an 8-cell embryo is limited. Tetraploid embryos are used for aggregation with other embryos, embryonic stem cells, and iPS cells, and they are selected against during development of the fetal tissues, but persist in extraembryonic membranes. The objective of this work was to generate a new and simple method for cloning 8-cell bovine embryos and also to explore more efficient methods to multiply transgenic embryos by aggregation of each blastomere from a day-3 embryo with putative tetraploid embryos. To this aim, bovine cumulus–oocyte complexes were in vitro matured in standard conditions and subjected to IVF (day 0) according to Bracket and Oliphant (1975). After IVF, a group of presumptive zygotes was injected with ooplasmic vesicles incubated with 50 ng mL–1 of linearized pCX–egfp. Other group was cultured for 25 additional hours (day 1). At that time 2-cell embryos were electrofused twice at 40V for 25 μs at 100-ms intervals to generate putative tetraploid embryos, visualised as a single blastomere 1 h after the fusion pulse (fused embryos, F). Two aggregation groups were included. A synchronic group (S): IVF for the production of both transgenic embryos and fused embryos was done on the same day; and an asynchronic group (AS): IVF for transgenic embryos took place 1 day before IVF for fused embryos production, so embryos from the A group were younger. Controls consisted of the same S and AS groups, but no fusion was included (NF). On day 3, the enhanced green fluorescent protein [EGFP(+)] blastomeres were selected. Using the well of well system, 1 or 2 embryos of each fusion group (S or AS and F or NF) were removed of their ZP and aggregated in a microwell with one EGFP(+) blastomere from a 5- to 8-cell stage embryo (day 3). In vitro development of the aggregates and green fluorescent protein expression localization of blastocysts were analysed. Blastocysts were obtained for all groups; however, the 2A-F and 2A-NF groups showed the highest rates (44%, P < 0.05) compared with one embryo aggregation. The highest aggregation rates of the EGFP(+) blastomere were observed for 2A-F (67%) and 2A-NF (44%) groups, too. A very poor integration was noted in the 2S-NF (100%), 2S-F (94%), 1A-NF (89%), and 1S-NF (80%) groups. Localised EGFP distribution was also high in the 2A-F group (42%). In all cases, EGFP expression seemed to localise by the inner cell mass. We demonstrated that it is possible to multiply 8-cell embryos of genetic value and also transgenic embryos, in theory reducing mosaicism rates in future offspring. Moreover, our results give rise to the possibility of using EGFP like a reporter gene that could be used to evaluate aggregation efficiency by a fluorescence microscope.
130 THE SYNERGIC EFFECT OF NERVE GROWTH FACTOR AND VASCULAR ENDOTHELIAL GROWTH FACTOR ON IN VITRO MATURATION AND DEVELOPMENTAL COMPETENCE IN BOVINE OOCYTES
Authors: Kim B. , Saadeldin I. M. , Lee B. , Jang G.
Published: August 2011

Nerve growth factor (NGF) has been reported to increase the mRNA expression of vascular endothelial growth factor (VEGF) in granulose cells of human, rat via TrkA signaling; VEGF has been shown to exert beneficial effects during bovine in vitro maturation (IVM) as well as early embryonic development. The aims of this study were 1) to investigate not only the direct effect of NGF but also the collaborative effect of NGF and VEGF during bovine in vitro maturation (IVM), in vitro culture (IVC), or both; and 2) to validate the correlation among transcript abundance of 7 genes (VEGF164, VEGF120, Flt-1, Flk-1, TrkA, PTGS2, and CYP11A1) in bovine cumulus cells and the results of IVM or IVC among the differently treated groups. In Experiment 1, concentrations of 0, 10, and 100 ng mL–1 NGF were added to our established IVM medium without serum, and in Experiment 2, control and treatment groups (concentration of 0, 10, and 100 ng mL–1 NGF with VEGF 100 ng mL–1) were added into chemically defined media. The oocytes of each group in Experiments 1 and 2 were determined by the proportion of MII oocytes after 24 h, and embryos were assessed after parthenogenetic activation. Cumulus cells from the differently treated matured cumulus cell–oocyte complexes (COC) were separated and synthesised into cDNA for RT-PCR and real-time PCR in order to measure relative abundance of 7 genes in a dose-dependent manner or a time-dependent manner. In Experiment 1, the concentration of 10 ng mL–1 (57.40%) and 100 ng mL–1 (62.75%) NGF treatment groups did not significantly increase the proportion of MII oocytes compared with the control group (55.06%). In Experiment 2, both the NGF 10 ng mL–1 with VEGF 100 ng mL–1 treated group (67.69%; P ≤ 0.01) and the NGF 100 ng mL–1 with VEGF 100 ng mL–1 treated group (72.24%; P ≤ 0.001) had a significantly higher percentage of polar body extrusion than control group (51.77%) and the group which was treated with VEGF 100 ng mL–1 (56.39%). The NGF treatment group with VEGF increased transcriptional level of VEGF164 and VEGF120 compared with the control group and only NGF- or VEGF-treated groups. In addition, either the NGF-treated group or NGF plus VEGF showed significantly increased mRNA abundance in VEGF164, VEGF120, Flt-1, Flk-1, and TrkA genes, whereas the NGF plus VEGF-treated group indicated the up-regulation of VEGF164, VEGF120, CYP11A1, and PTGS2 genes. In conclusion, NGF and exogenous VEGF have a synergic effect during bovine IVM and the early stage of embryo development; the elevated VEGF mRNA abundance in cumulus cells might contribute to the viability of bovine oocytes and early embryonic development.

This study was supported by grants from IPET (#109023-05-1-CG000), NRF (#M10625030005-10N250300510), MKE (#2009-67-10033839, #2009-67-10033805), and BK21 program.
132 EFFECTS OF OXYGEN TENSION, MEDIUM, AND WELL OF THE WELL ON IN VITRO DEVELOPMENT OF THE MOUSE EMBRYO
Authors: Li Y. S. , Cao Z. B. , Liu Y. , Cao H. G. , Tao Y. , Zhang X. R. , Zhang Y. H.
Published: August 2011

The objectives of the present research were to investigate whether embryo culture media have preferences for oxygen tension, to explore the feasibility of using physical lung air to support the in vitro development of mouse embryos, and to evaluate the effect of well of the well (WOW) culture on in vitro preimplantational development of mouse embryos. The results are as follows. First, cleavage rate and blastocyst rate were not significantly different between medium CZB and mKSOM regardless of using 3 gas phases: 4% CO2 + 16% O2 + 78% N2 + 2% H2O (lung air), 5% CO2 + 5% O2 + 90% N2 (5% O2, low oxygen), and 5% CO2 + 95% air (20% O2, high oxygen; P > 0.05), but mean total cell numbers per blastocyst cultured in CZB medium were higher than those in mKSOM when the lung air was used (P < 0.05). Second, when mKSOM was used as the basic medium, the blastocyst rate (22.6%) in the 5% O2 gas phase was notably higher than that in other 2 gas phases (P < 0.05). Third, for the CZB medium, the blastocyst rate was not different significantly among 3 gas environments (P > 0.05). Fourth, both the blastocyst rate (74.6 ± 5.1%) and the mean total cell numbers per blastocyst (76 ± 2) cultured in the WOW system were obviously higher than those in the group culture system (38.2 ± 6.6% and 58 ± 4). Taken together, our results indicate that mKSOM and CZB have preferences for oxygen tension during in vitro culture of mouse embryos, the lung air was reaffirmed to be able to effectively support in vitro preimplantation development of mouse embryos, and the WOW culture system can apparently enhance in vitro developmental competence and blastocyst quality of mouse embryos.

L. YS, C. ZB: equal contribution; supported by NSFC (30700574), 863 (2008AA101003).
133 EVALUATION OF OOCYTE DONOR SOURCE AND CULTURE MEDIUM ON DOMESTIC CAT EMBRYO DEVELOPMENT RATES
Authors: Pearks Wilkerson A. J. , Landry R. D. , Long C. R.
Published: August 2011

The use of assisted reproductive technology (ART), including in vitro maturation (IVM) and embryo culture, is well established in several species, including canine and feline culture systems. Embryo production conditions tend to be specific for each species and prepared in unique formulations by laboratory. However, the increasing numbers of commercially available media allows for new comparisons in companion animal systems. Therefore, a goal of this study was to compare the development rates of feline parthenotes cultured in a commercially available bovine embryo culture medium with those cultured in a published 3-step domestic cat-specific system. In addition, the source of ovaries utilised for oocyte retrieval was evaluated as a factor in development rates. Ovaries from 2 locations (L1 and L2) were collected on the same day, and harvested oocytes were held in meiotic arrest medium containing 25 μM roscovitine for 14 to 18 h. Oocytes were incubated in maturation medium for 24 h before cumulus cell removal with vigorous pipetting in 0.4% hyaluronidase, and a subset of each group was fixed and stained to determine meiotic maturation rates (n = 76 and 55 for L1 and L2, respectively). Following activation (day 0) by a single course of three 50-μs electric pulses at 1.2 kV cm–1 in 0.3 M mannitol, 0.1 mM CaCl2, and 0.1 mM MgSO4, parthenotes from each source were randomly divided to culture medium treatment of Bovine Evolve medium (Zenith Biotech, Guilford, CT, USA) with 4 mg mL–1 BSA (n = 209) or IVC-1 medium n = 269; (Pope et al. 2009 Theriogenology 71, 864–871), each containing 10 μg mL–1 cycloheximide and 7.5 μg mL–1 cytochalasin B. After a 4-h activation treatment, parthenotes were moved to culture media without cycloheximide and cytochalasin B for embryo development. All parthenotes in IVC-1 medium were moved to IVC-1a medium on day 2. On day 5, both sets of parthenotes were moved to culture media containing 10% heat-inactivated FBS instead of BSA. On day 7, all parthenotes were fixed and stained with Hoechst to determine cell number. No differences were seen in maturation rates between L1 and L2 (56.3 ± 9.5 v. 54.7 ± 9.5, respectively). However, cleavage rates tended to differ, and proportion of embryos greater than 64 cells was different (60.7 ± 5.8 v. 78.3 ± 5.8, P = 0.056 and 3.0 ± 3.1 v. 19.7 ± 3.1, P < 0.005; respectively). We hypothesised that the physical condition of the ovary donors may have affected development rates because cats from L1 tended to be feral animals, whereas cats from L2 were mostly privately owned. Bovine Evolve was similar to IVC-1 medium for cleavage, 32-cell, and 64-cell development rates (74.2 ± 6.7 v. 64.8 ± 6.7; 24.0 ± 7.5 v. 31.8 ± 7.5; 10.7 ± 4.8 v. 12.0 ± 4.8, respectively; P > 0.05). These results indicate that commercially available culture medium can support in vitro development, even if the commercial medium is developed for a different species, but that source of cat ovaries should be considered in feline ART.