bluuebunny/crossref_metadata_2025 huggingface dataset:
111 EXPRESSION OF PHOSPHOLIPASE A2 ISOFORMS IN EQUINE ENDOMETRIUM DURING THE ESTROUS CYCLE AND EARLY PREGNANCY
Authors: Ozel C. , Guzeloglu A. , Hitit M. , Atli M. O. , Kurar E. , Kayis S. A.
Published: December 2014

Phospholipase A2 (PLA2) is involved in the synthesis of prostaglandins (PG) as it releases arachidonic acid from the membrane phospholipids to be a precursor for cyclooxygenase enzymes. Therefore, it is critically important during luteolysis and at the time of maternal recognition of pregnancy. The embryo must attenuate endometrial PGF2α production, whereas PGE2 is considered to be luteoprotective. Furthermore, implantation also requires the action of PGs. A balance should be maintained in PG synthesis in the endometrium. The objective of this study was to evaluate the expression of isoforms of PLA2 (cytosolic [cPLA2], secretory [PLA2], and calcium independent [iPLA2]) in equine endometrium during the oestrous cycle and early pregnancy. Biopsies were obtained from mares on the day of ovulation (d0, n = 4), late diestrus (LD, n = 4, high progesterone [P4]), and after luteolysis in the beginning of oestrus phase (AL, n = 4, <1 ng mL–1 P4) of the cycle. Biopsies were also taken on days 14 (P14, n = 4), 18 (P18, n = 4), and 22 (P22; n = 4) of pregnancy. Relative mRNA expression levels of genes were quantified using real-time quantitative RT-qPCR. Data were analysed using one-way ANOVA and l.s.d. test. Compared with the day of oestrus (d0), steady-state levels of cPLA2 and sPLA2 were down-regulated at LD, where P4 was high, and expression of both were up-regulated again at AL. In contrast, iPLA2 expression was higher at LD and then decreased again at AL. Pregnancy decreased expression of cPLA2 and sPLA2 mRNA at P14 and P18 compared with their respective cycle days. Late diestrus elevation in the expression of iPLA2 was suppressed by pregnancy at P14; however, it was up-regulated later in pregnancy at P22. The results suggest that expression of cPLA2 and sPLA2 is negatively correlated with circulating progesterone concentrations. Pregnancy further inhibits their expression in the equine endometrium. However, iPLA2 expression seems to be positively correlated by progesterone presence and its expression increased as equine pregnancy advanced.
This project was partially funded by TUBITAK 107O035 and SUBAP to AG.
114 ASYNCHRONOUS EMBRYO TRANSFER AFFECTS THE EXPRESSION OF IMPRINTED GENES IN EQUINE TROPHECTODERM
Authors: Gibson C. , de Ruijter Villani M. , Stout T. A.
Published: December 2014

Gene imprinting is a form of epigenetic modification that results in parent-of-origin specific monoallelic expression. Imprinted genes play important roles during fetal-placental growth with paternally imprinted genes generally promoting and maternally imprinted genes suppressing fetal growth. Imprinted genes are therefore believed to have important effects on trophoblast differentiation and placental development, and in adjusting fetal nutrition to maternal supply. The horse is an interesting model of early placental development because of its unusually long pre-implantation period (40 days), during which the conceptus is dependent on uterine secretions for nutrient provision. Moreover, horse embryos tolerate a wide range of uterine asynchrony following embryo transfer (ET), offering a unique tool to study maternal influences on conceptus development. This study examined the effect of asynchronous ET on the expression of imprinted genes in equine trophectoderm. Twenty Day 8 embryos were transferred to recipient mares that either ovulated on the same day (synchronous; n = 10) or 5 days after (asynchronous; n = 10) the donor mare. The conceptuses were recovered 6 or 11 days after ET (Day 14 or 19 of conceptus development; n = 5 per group). Bilaminar trophectoderm was isolated and mRNA expression for a range of genes known to be imprinted in equine trophectoderm (H19, PHLDA2, IGF2R, IGF2, PEG3, PEG10, SNRPN, INSR, and INS) was investigated by RT-qPCR. The effects of asynchronous ET and stage of pregnancy on gene expression were analysed by two-way ANOVA followed by independent-samples t-tests. IGF2, PEG3, PEG10, INSR, H19, and PHLDA2 all showed a significant up-regulation in gene expression between Days 14 and 19 of pregnancy; however, expression was higher in synchronous than asynchronous pregnancies at Day 19 (P < 0.05). IGF2R expression increased significantly from Day 14 to 19 in the synchronous pregnancies (P < 0.05), but did not differ between treatments at Day 19. SNRPN expression increased from Day 14 to 19, and was unaffected by asynchrony. INS mRNA was not detectable in trophectoderm. In conclusion, asynchronous ET had a significant effect on gene expression at Day 19 of gestation that was not evident at Day 14. This may be either a contributor to the delayed development that is observed in asynchronous pregnancies or a result/response; in either case, it may affect subsequent development.
This study was founded by EpiHealthNet (Project number 317146).
126 EXPRESSION OF MYO-INOSITOL OXYGENASE IN BOVINE PRE-IMPLANTATION EMBRYOS AND ITS REGULATION BY ANTI-OXIDATIVE VITAMINS
Authors: Ikeda S. , Sugimoto M. , Kume S.
Published: December 2014

Myo-inositol (MI) added into in vitro culture media stimulates blastocyst development of mammals, including cattle (Holm et al. 1999 Theriogenology 52, 683–700), and these stimulatory effects are considered to be exerted via the incorporation of MI into phosphatidylinositol (Hynes et al. 2000 Mol. Reprod. Dev. 55, 265–269). Myo-inositol is catabolized by MI oxygenase (MIOX), which may affect bioavailability of MI for phosphatidylinositol pathway. In the present study, we investigated the expression pattern of MIOX transcripts in bovine pre-implantation embryos and the effects of anti-oxidative vitamins, which promote blastocyst hatching (Ikeda 2014 Reprod. Fertil. Dev. 26, 157), on the expression levels of MIOX in bovine blastocysts in vitro. Cumulus-enclosed oocytes obtained from slaughterhouse bovine ovaries were in vitro-matured for 22 h in modified SOF (mSOF) supplemented with 10% v/v FCS and 0.2 IU mL–1 FSH. After in vitro maturation, the oocytes were subjected to IVF with Percoll gradient-selected sperm in an mSOF-based medium for 20 h. After IVF, presumptive zygotes were freed from cumulus cells and cultured in mSOF up to Day 8 (IVF = Day 0). All cultures were performed at 38.5°C under 5% CO2, 5% O2, and 90% N2. Total RNA was extracted from embryos at the 1-cell, 2-cell, 8-cell, morula, and blastocyst stages (n = 15 × duplicates for each stage) and reverse transcribed to cDNA using oligo (dT) primer in a 31.5 μL reaction volume. Transcripts for MIOX were examined by quantitative reverse transcription-PCR (qRT-PCR) using 1 μL of the cDNA solution and succinate dehydrogenase as a reference gene. Moreover, IVF-derived 8- to 16-cell embryos on Day 3 were cultured in mSOF supplemented with or without a vitamin mix (11 μM a-tocopherol and 9 nM β-carotene) up to Day 8, and blastocysts (n = 10 × 3 replicates) were collected from each treatment. The expression levels of MIOX transcripts in the blastocysts were compared between the treatments by using qRT-PCR. The data were statistically analysed by Mann–Whitney U test. The MIOX transcripts were undetectable at the 1-cell and 2-cell stages and then expressed from the 8-cell stage onward. The vitamin treatment significantly (P < 0.05) decreased the expression level of MIOX in blastocysts. These results suggest that MIOX is one of embryonic-activated genes in bovine pre-implantation embryos, and the level of its expression is regulated by anti-oxidative vitamins. The stimulatory effects of anti-oxidative vitamins on blastocyst development might be in part via the modulation of MI bioavailability.
130 METABOLIC PROFILING OF MALE AND FEMALE BOVINE EMBRYOS USING NUCLEAR MAGNETIC RESONANCE (NMR) IMAGING
Authors: Rubessa M. , Ambrosi A. , Polkoff K. M. , Stewart J. W. , Herzog K. K. , Denmark S. E. , Wheeler M. B.
Published: December 2014

It has been previously shown that during the pre-implantation phase of embryo development, the pentose phosphate energy pathway is 4 times more active in female embryos when compared with male embryos (Tiffin et al. 1991 J. Reprod. Fertil. 93, 125–132). The different metabolic and growth rates can be attributed to the different expression of X-linked genes between the sexes during the early stages of pre-implantation development, in which both X chromosomes are still active in female embryos (Okamoto et al. 2004 Science 303, 644–649). The aim of this study was to evaluate, by proton magnetic resonance (H1-NMR) imaging, the different behaviour of female and male embryos. In this study we evaluated only energetic substrates or Krebs cycle's intermediates. Matured bovine cumulus-oocyte complexes were fertilized in vitro according to our standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347–1355). Presumptive zygotes were placed in individual drops of 50 μL of SOF. Zygotes were incubated in a humidified mixture of 5% CO2, 6% O2, and 88% N2 in air at 39°C. After 48 h, the zygotes were placed into WOW culture, and the drops collected in tubes. The embryos were scored for quality on the basis of morphological criteria. In this experiment, we evaluated 10 embryos for each sex, at stage of tight morula, early blastocyst, blastocyst, and expanded; data were obtained from 2 replicates. The embryos had their sex determined according to our standard protocol (Alomar et al. 2008 Anim. Reprod. Sci. 107, 48–61). Samples of media (40 μL) were added to 660 μL of a stock solution prepared by dissolving 5.0 mg of sodium 3-(trimethylsilyl)-2,2′,3,3′-tetradeuteropropionate (TSP) in 50 mL of deuterium oxide. The TSP acted both as a chemical shift reference and as an internal standard for the purposes of quantitation. The resulting diluted samples were transferred to a 5-mm NMR tube. Samples were analysed on a Varian VNS-750 NB (750 MHz) spectrometer (Agilent Technologies, Santa Clara, CA, USA). Data were statistically analysed with ANOVA using the Generalized Linear Model (GLM) procedure (SAS, version 9, 1999, SAS Institute Inc., Cary, NC, USA), where the independent variable was the sample (female, male embryos and control media without embryos). Tukey's post-hoc test was used to perform multiple comparisons. The P-level was set at 0.05. All data were expressed as quadratic means and with standard error of the means. The results, reported in Table 1, indicate that there are no statistical differences between the sexes after 48 h of embryo culture. In conclusion, these results confirm that in the first phase of development, the embryos derive their energetic substrates from substrates contained within the embryos themselves.

Table 1.Results, least squares mean (standard error)
Corrigendum to: 131 CHROMOSOMAL ABNORMALITIES IN IN VITRO-PRODUCED 4-DAY-OLD CATTLE EMBRYOS: INFLUENCE OF THE OOCYTE MATURATION MEDIA
Authors: Demyda Peyrás S. , Moreno Millán M.
Published: December 2014

Chromosomal abnormalities were related to embryo developmental failures in cattle, being highly increased in IVF-produced compared with multiple-ovulation embryo transfer-produced embryos. The origin of this difference remains unclear, but they were related to the suboptimal environmental culture laboratory conditions. We studied the influence of 3 different maturation media in the appearance of chromosomal abnormalities in early in vitro-produced embryos. A total of 562 oocytes classified as Class A were collected by follicular aspiration in slaughterhouse ovaries and matured by 20 h in 3 different culture media supplemented with 10% FCS and antibiotics as follows: TCM-199 (T, 193 oocytes); DME (D, 178 oocytes); and RPMI-1640 (R, 191 oocytes). Matured oocytes were fertilized by 16 h with 1 × 106 spz mL–1 in IVF-TALP media and cultured by 72 h in SOF media. Later, 48 h after fertilization, 562 presumptive embryos were evaluated showing a normal cleavage rate of 64.7, 55.2, and 54.9% in T, D, and R groups, respectively. After culture, only 181 early embryos (90 hpi) showing normal development were correctly karyotyped following our standard laboratory techniques. Individual blastomeres were stained with Giemsa and assessed by direct observation at ×1250 magnification in a brightfield microscope. The percentage of normal diploid embryos (D) and abnormal haploid (H), polyploid (P), or aneuploid (A) embryos were determined. Only embryos showing almost 2 different blastomeres correctly karyotyped were included in this study. Cleavage rates were statistically higher (P < 0.05) in the oocytes matured in T media in comparison with the oocytes matured in D or R media. The percentage of diploid embryos were almost equals in the 3 groups evaluated (Table 1). There was some variation when each kind of chromosomal abnormality was assessed individually, but no statistical differences were observed. These results are in consonance with our previous studies in which we demonstrated the maturation time and morphological quality are the 2 main oocyte-derived factors influencing the ploidy of early embryos. It was also demonstrated that chromosomal complements were affected, in a much lesser extent, by the maturation media supplementation. However, in the present study, the maturation media did not statistically affected embryo ploidy. However, the higher percentage of cleaved embryos using TCM-199 observed agree with fact that this maturation media is one of the most widely used in IVF procedures in cattle. Based on our data, we suggest that oocyte maturation, a well established technique in cattle, could be performed using different maturation media without expecting major differences in the embryo ploidy, and therefore, the differences observed in cleavage rate must be originated in other physiological factors.
Table 1.Results
132 VITRIFICATION SYSTEM (OPEN AND CLOSED) IN NEW INCUBATOR WITH REDUCED OXYGEN
Authors: Watanabe O. , Schmitt E. , Meirelles F. , Oliveira A. , Bos-Mikich A. , Meirelles F. , Watanabe Y.
Published: December 2014

Most IVF laboratories uses high oxygen tension (20%) during embryonic culture. However, it is known that under physiological conditions, oxygen tension in the female reproductive tract ranges between 2 and 8%. Therefore, the aim of this study was to evaluate survival and hatching rate after in vitro culture of vitrified/thawed bovine in vitro-produced blastocysts cultured under different oxygen concentrations. The experiment consisted of comparing 2 culture systems using different concentrations of oxygen: conventional incubator (20% O2, Thermo Scientific, model 3130) and a new incubator (5% O2, WTA Watanabe Tecnologia Aplicada, model Eve). Only Day 7 expanded blastocysts grade 1 were used. Embryos were produced according to conventional IVF protocols. Briefly, cumulus-oocyte complexes were aspirated from postmortem ovarian follicles, matured in TCM199 + 10% FCS + 0.5 mg FSH mL–1 + 50 mg LH mL–1 + 1 mg oestradiol mL–1, for 24 h at 38.5°C, 5% CO2 in air. Live spermatozoa from Nellore bull were obtained by centrifugation in Percoll gradients (45 and 90%) and cultured with cumulus-oocyte complexes at 1 million of sperm mL–1 in TALP medium + 10 mg of heparin mL–1. After 20 h incubation, zygotes were transferred to CR2 + 2.5% FCS + 4 mg of BSA mL–1 and granulosa monolayer for 7 days. Expanded blastocysts were randomly allocated to 2 treatments for vitrification (open system – cryotop and closed system – HSV Kit, IMV-Technologies) using the same vitrification media and protocol (VS1: 10% ethylene glycol + 10% dimethyl sulfoxide and VS2: 20% ethylene glycol + 20% dimethyl sulfoxide for 8 min and 50 s, respectively). After exposure to the vitrification solutions, 2 embryos were loaded/straw, and the straws were plunged into LN. The warming procedure consisted of, immediately after removal from LN2, transferring the embryos in 2 successive warming solutions with decreased concentrations of sucrose (1 M and 0.50 M for 5 min each). The vitrified/rewarmed embryos were transferred to in vitro culture. There were no differences in survival rates (P < 0.05) between the open and closed vitrification system for blastocysts produced in reduced oxygen in the Eve incubator – 5% O2 (96% – 109/114 and 98% – 158/161, respectively) compared with embryos produced in the high oxygen environment in the Thermo incubator – 20% O2 (93% – 214/230 and 92% – 94/102, respectively). Hatching rates were increased for blastocysts cultured in the lower oxygen environment (EVE treatment: 95 and 98%, respectively, for open and closed vitrification protocols) when compared with the high oxygen environment (Thermo treatment: 86 and 87%, respectively, for open and closed systems); P < 0.05. In vitro culture in a reduced-oxygen environment improves blastocysts competence after vitrification.
Financial support was received from CNPq-RHAE.
15 SPERM STORAGE IN FEMALE REPRODUCTIVE TRACT: STUDY OF MOLECULES INVOLVED
Authors: Riou C. , Gargaros A. , Harichaux G. , Brionne A. , Gautron J. , Druart X. , Labas V. , Gerard N.
Published: December 2014

Because of prolonged sperm storage in their oviduct, domestic hens can produce fertile eggs for up to 3 weeks following a single AI. The oviduct secretions may have an effect on sperm survival, but its composition during fertilization is unknown. In the present study, we compared the proteomic content of uterine fluid collected from two lines of hens divergent by their duration of fertility period (DFP), which defined sperm-storage duration. The first line displays a shorter period of sperm storage (10 days, line DFP–), whereas the second displays a longer period of sperm storage (21 days, DFP+). The aim was to identify proteins or peptides that may be involved in spermatozoa survival. Uterine fluid was collected 10 h after oviposition either before (n = 5/line) or 24 h after (n = 5/line) AI. Samples were pooled by condition: DFP+ before AI, DFP+ after AI, DFP– before AI, and DFP– after AI. Bottom-up approach using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nano LC-MS/MS was performed (3 replicates). Data were matched against the NCBInr database (2014) using Mascot, and identifications were validated by the peptide and protein Prophet algorithm using Scaffold software. To determine the differences in protein expression, spectral counting and XIC quantitative methods were employed using Scaffold Q+ (P < 0.05, ratio > 2). Two proteins were up-regulated and one was down-regulated in oviductal secretion of both lines in response to AI. However, AI induced a significantly different abundance between protein content of DFP– and DFP+ fluids. A panel of 8 proteins, included one DFP+-specific protein, was more abundant in DFP+ line than in DFP–. Only one protein was less abundant in DFP+ line than in DFP–. In conclusion, the presence of sperm in the genital tract induced quantitative differences of the protein content of the uterine fluid in DFP– and DFP+ hen lines. These differences imply proteins that are known as male proteins (sperm, seminal plasma, testis). Analysis of sperm protein modifications after storage will help us to understand the functional implication of these candidates.
152 FACTORS AFFECTING FERTILITY ON AN EQUINE EMBRYO TRANSFER PROGRAM
Authors: Souza J. C. , Portilho R. V. , Pinto T. L. C. , Neto F. D. M. , Filgueiras E. P. , Silva M. G. C. M. , Moura R. S.
Published: December 2014

Assisted reproduction through embryo transfer (ET) increases the chances, within a limited group of genetically superior mares, producing a greater number of desirable offspring. Several variables, however, are involved in the process and may impact or even limit the potential of ET to increase a single mare´s foal production within a given breeding season. The objective was to analyse information on variables related to an ET operation carried over 10 years by a single veterinarian in different reproduction centers or directly from the database of all breeders. General management and reproductive data from 150 embryo donor mares, 362 recipient mares and 73 stallions were submitted to statistical analysis by the GENMOD procedure using SAS® (SAS Institute Inc., Cary, NC, USA). The proportion of positive recoveries (in which one or more embryos were actually recovered) and of pregnant recipients were dependent variables and donor breed (Campolina, Mangalarga, Thoroughbred, Haflinger and Brasileiro de Hipismo), donor (3–18 years old) and stallion age (3–20 years old) type of semen, hormonal ovulation induction (1000–2500 IU of hCG when dominant follicles reached at least 30 mm in diameter), diameter of the dominant follicles at the time of induction in donors and recipients as independent variables. Gestation rate was greater (P < 0.05) for embryos produced by donors of the Campolina breed compared to all others (74.0 v. 57.6%). The positive flush rates of donor age classes ≤7 (50.2%), >7 and <12 (60.0%) and ≥12 (53.2%) years old were similar (P = 0.45). Gestation rates were similar (P = 0.43) between stallions <6 (70.3%) and ≥6 (75.8%) years-old. The proportion of positive flushes was greater (P = 0.01) for fresh semen (59.9%) compared to those of natural service, cooled and frozen-thawed semen (54.8, 51.5 and 28.6%, respectively). Pregnancy rates were similar (P > 0.05) between recipient mares hCG-induced (70.6%) and not induced (73.3%) to ovulate. In conclusion, we confirmed that donor breed and type of semen are influential over pregnancy and positive flush rates in the present study. Stallion or donor mare age did not influence fertility traits in this study, which indicates that animals were well managed and used within appropriate age limits. More relevant data may be gathered from the field work of veterinarians in order to enhance the knowledge that supports managing decisions in assisted reproduction techniques.
158 BOVINE HERPES VIRUS 4 (BoHV4) INHIBITS BOVINE ENDOMETRIAL EPITHELIAL CELL (bEEC) PROLIFERATION
Authors: Chanrot M. , Guo Y. Z. , Blomqvist G. , Juremalm M. , Reinaud P. , Charpigny G. , Sandra O. , Chantaraprateep P. , Båge R. , Donofrio G. , Valarcher J. F. , Humblot P.
Published: December 2014

BoHV4 is a double-stranded DNA virus which has been associated to endometritis, metritis, and abortions in dairy cow. The objective of this study was to characterise its cytopathic effects on bovine endometrial epithelial cells (bEEC). Bovine uteri were collected from slaughter house and bEEC separated and cultivated as previously described (Guo et al. 2014 Reprod. Fertil. Dev. 26, 165–166). In Experiment 1 (Exp 1), bEEC (passage 5) from 3 cows were cultivated for 6 days without virus or following exposure to serial dilutions (10–4, 10–3, 10–2) of virus. Living cells were counted for each group at start of the experiment and by Day 6. Proliferation or inhibition of proliferation was calculated by (Number of cells Day 6 – Number of cells Day 0)/Number of cells Day 0. In Experiment 2 (Exp 2) cells were challenged with a single dosage of virus (MOI 0.01; 1 virus: 100 cells) and culture performed during 1, 2, 3, 4, 5, 6, or 7 days. Cells were counted at Day 0 and each day, proliferation of cells was calculated as (number of cells by Day X – number of cells Day 0)/number of cells Day 0. The effects of the dilution of virus, cow and their interaction (Exp 1) or effects of time, cow, viral exposure, and second-order interactions (Exp 2) on cow cell proliferation were analysed by ANOVA (SAS 9.2, proc GLM; SAS Institute, Inc., Cary, NC, USA). In Exp 1, the amount of living cells by Day 6 was very significantly increased in controls when compared to Day 0 (+172.6 ± 24%; P < 0.0001). A linear inhibition of proliferation was observed with increasing dilutions of virus. The number of living cells for the highest concentration of virus is not different from Day 0 numbers (–26.7 ± 24.6%). Pattern of proliferation differed between cows as evidenced by a significant interaction between cow and virus dilution (P < 0.001). In Exp 2, we observed a very strong increase of proliferation from Day 0 to Day 7 in controls (+1000 ± 87%; P < 0.0001). From Day 1 to 4, the increase in number of cells was very similar for cells exposed to BoHV4 and in controls. However, after Day 4, cells exposed to virus had a limited proliferation or expressed cell death as the number of living cells by Day 7 were not different from these observed by Day 0 (50 ± 87%; NS). These results show that both time and dose of BoHV4 affect the proliferation of bovine EEC. These results will be used to investigate further the molecular mechanisms by which BoHV4 induces cell death and their sequence.
Research was partly funded by RMUTSV.
159 CHANGES IN PROTEIN EXPRESSION PROFILES IN BOVINE ENDOMETRIAL EPITHELIAL CELLS (bEEC) FOLLOWING E. COLI LIPOPOLYSACCHARIDE CHALLENGE
Authors: Guo Y. Z. , Piras C. , Soggiu A. , Chanrot M. , Båge R. , Andersson G. , Reinaud P. , Charpigny G. , Sandra O. , Valarcher J. F. , Roncada P. , Humblot P.
Published: December 2014

E. coli is one of the most frequent bacteria involved in uterine diseases. Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria involved in the pathogenic processes leading to postpartum metritis and endometritis in cattle. It also causes inflammation of the endometrium. Increase of cell proliferation by LPS is part of the inflammatory process and has been reported in human epithelial and immune cells (Martin et al. 2000 J. Immunol. 165, 139–147) and from bovine endometrial epithelial cells (bEEC) (Guo et al. 2014 Reprod. Fertil. Dev. 26, 165–166). The aim of this study was to investigate possible changes in protein expression in relation with the proliferative response of bEEC after challenge with E. coli-LPS. In vitro culture of bEEC was performed from 3 cows. On passage 5, bEEC from each individual were exposed to 0, 8, and 16 µg mL–1 LPS for 72 h. At time 0 and 72 h later, attached cells were counted and for each time and LPS dosage, cells were frozen for proteomic analyses. The variation of cells number over time was analysed by ANOVA (SAS 9.1, proc GLM; SAS Institute, Inc., Cary, NC, USA). All samples were analysed (every sample run in triplicate) by 2-D gel electrophoresis coupled to matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF)/time-of-flight (TOF) mass spectrometry (MS) and shotgun nLC-MS/MS analysis. As reported before, a significant increase in cell number was observed for cells treated with 8 µg mL–1 LPS (P ≤ 0.001), whereas changes in cell number were highly variable and nonsignificant for 16 µg mL–1 LPS. From each sample, ~800 proteins were visualised. Results from 2-D gel coupled to MALDI-TOF/TOF were very reproducible (same responses between individual cows) and revealed changes in protein profiles very much related (from P < 0.05 to P < 0.01) to proliferative phenotypes for seven proteins. From shotgun analysis, 27 proteins were found significantly differentially expressed (P < 0.05 to P < 0.01) following exposure to LPS (21 up-regulated and 6 down-regulated). Among the 21 found as up-regulated, 20 were differentially expressed both for the 8 and 16 µg mL–1 LPS, whereas 5 out of 6 were down-regulated for both dosages. Differentially expressed proteins were associated to cell proliferation, apoptosis, oxidative stress, regulation of histones, allergy, and general cell metabolism pathways. Candidate proteins need to be confirmed from larger series of individuals and relevant pathways further studied.
Research was partially funded by RMUSTV.
164 EMBRYO SURVIVAL TO CALVING ACCORDING TO TYPE OF EMBRYO AND EMBRYO TRANSFER METHOD IN ALPACAS
Authors: Vivanco-Mackie H. W. , Salazar M. D. P. , Miguel M. , Youngs C. , Asparrin M.
Published: December 2014

The objective of the study was to determine the embryo survival up to calving of fresh and cryopreserved (frozen and vitrified) alpaca embryos transferred into alpaca recipients by nonsurgical transcervical embryo transfer and by surgical laparoscopically aided embryo transfer. For this report we have compiled the information from 127 embryo transfers in alpacas done by our group at Mallkini, Puno, Peru, at 4200 m elevation. The embryos have been collected from superovulated donor alpacas flushed at 6.5 days post mating, some were transferred as fresh and some were cryopreserved; the recipients (3 to 7 years old) were selected based on presence of functional corpora lutea at ecosonographic examination and subjected to ovarian cycle synchronization and ovulation induction as per Vivanco (2013). From a total of 133 alpacas selected, 127 were used, from which 82 received fresh, 32 frozen, and 13 vitrified embryos. All embryos were classed as A-class expanded blastocysts at time of transfer. By nonsurgical transcervical embryo transfer, 33 embryos were transferred fresh, 22 were frozen/thawed embryos, and 13 were vitrified/warmed embryos. By surgical laparoscopically aided method, 49 embryos were transferred fresh and 10 embryos were frozen/thawed; no vitrified embryos were transferred by this method. Results are detailed in Table 1. Pregnancy losses occured at up to 9 weeks (63 days) of gestation, the heaviest loss occurs in the first 3 weeks. After 9 weeks of gestation, no losses were registered. In average, 22% of fresh embryos transferred were represented as crias born. None of the cryopreserved embryos survived up to 11 weeks post-transfer. There is no difference in percentage of crias born between nonsurgical transcervical embryo transfers and surgical laparoscopically aided embryo transfers.The heavy embryo losses could be related to nutrition and high-altitude limitations; however, it is difficult to make comparisons with others because reports to date lack information on the actual crias born from embryo transfers in alpacas; most of the reports are based on pregnancy reports up to 30 to 60 days post-transfers. To date, no births from cryopreserved alpaca embryos have been reported. Furher studies on causes of embryo/fetal losses are necessary.

Table 1.Results of embryo transfer
This study was financed by the Peruvian Fund for Innovation, Science and Technology (FINCYT).
178 IS PROGESTERONE THE DETERMINING REGULATORY FACTOR BEHIND OVULATION RATE IN EWES?
Authors: Sohal J. , Paravinja V. , Baby T. , Murawski M. , Schwarz T. , Zieba D. A. , Keisler D. H. , Bartlewski P. M.
Published: December 2014

Ovarian antral follicles in the ewe grow in an orderly succession, producing 3–4 waves per oestrous cycle. In prolific sheep, some large antral follicles from the second-last wave of the oestrous cycle are added to the ovulatory follicles emerging just before oestrus to give a higher ovulation rate; it is feasible that regression of these follicles is prevented by an increase in serum concentrations of FSH and/or LH pulsatility at pro-oestrus. Prolific sheep tend to have a shorter luteal phase than non-prolific breeds and there is a great deal of evidence that luteal progesterone (P4), in addition to regulating LH release, may govern the secretion of FSH heralding the emergence of follicular waves. The specific purpose of the present experiments was to determine whether or not extending the duration of the luteal phase would alter the ovulation rate in prolific sheep. In both studies, exogenous P4 (7.5 mg ewe–1 IM) was administered on Days 11 and 12 (Day 0 = ovulation) in moderately prolific Rideau Arcott x Polled Dorset (Exp. 1, n = 8) and highly prolific Olkuska ewes (Exp. 2, n = 7), while the equinumerous groups of animals served as controls (CTR). Transrectal ovarian ultrasonography was performed daily and jugular blood samples were drawn twice a day from Day 9 until ovulation. All single-time point observations were compared between groups by Student t-test. Progesterone injections resulted in uniform increments in serum P4 levels in all animals allocated to the treatment (TRT) groups. However, the mean duration of the interovulatory interval did not differ (P > 0.05) between TRT and CTR groups of ewes in both experiments. The mean (± s.e.m.) ovulation rate was 1.6 ± 0.2 v. 3.2 ± 0.4 (Exp. 1; P < 0.001) and 3.2 ± 0.8 v. 4.0 ± 1.0 (Exp. 2; P < 0.05) in TRT v. CTR ewes, respectively. There were no differences in terms of the timing of penultimate and final wave emergence between the two subsets of animals studied in either experiment. The number/percentage of ovulating follicles from the penultimate wave of the interovulatory interval studied was 0.25 ± 0.16 v. 1.75 ± 0.45 (P < 0.01)/25.0 ± 16.4% v. 75.0 ± 16.4% (P < 0.05) in Exp. 1 and 0.50 ± 0.30 v. 1.60 ± 0.40 (P < 0.05)/13.8 ± 9.0% v. 53.4 ± 16.7% (P < 0.05) in Exp. 2, in TRT v. CTR animals, respectively. In summary, administration of P4 at the end of diestrus reduced the incidence of ovulations from the penultimate wave of the oestrous cycle in moderately and highly prolific strains of sheep. Therefore, progesterone appears to be a key endocrine signal governing the ovulation rate in cyclic sheep, presumably by acting directly at the level of the ovary. The present results may pave a way to devising a simple and inexpensive method of controlling lamb productivity in commercial flocks of sheep and fertility in other polyovulatory species.
183 ULTRASONOGRAPHIC MONITORING OF CANINE OVARIES CLAMPED AT SUBCUTANEOUS SITE AFTER FOLLICLE-STIMULATING HORMONE TREATMENT
Authors: Terazono T. , Luu V. V. , Do L. T. K. , Taniguchi M. , Takagi M. , Otoi T.
Published: December 2014

Follicle-stimulating hormone (FSH) alone can induce oestrus in bitches, but few reports describe oestrous induction by FSH because pregnant mare serum gonadotrophin (PMSG) has been more successful than FSH for oestrus induction. Real-time ultrasonography can show canine ovarian follicle development, but no method can determine or predict ovulation accurately. Moreover, the ovary location and size complicate imaging. Using ultrasonography, we investigated FSH treatment stimulation of canine ovary follicles, with clamping of the ovaries at a subcutaneous site. Bilateral malacotomy of four 5-year-old Beagle bitches (mean weight 10.3 ± 2.0 kg) with normal oestrous cycles was done using a ventral flank abdominal approach with routine techniques and materials. Each ovary that maintained blood circulation from the suspensory ligament was clamped at a subcutaneous site through muscles of the abdomen. After about six months of bilateral malacotomy, four bitches at the anestrous (two bitches) and diestrous (two bitches) stages of the oestrous cycle were given 0.5 Armour units of FSH twice daily for 5 days. Examinations with ovarian ultrasonography with 7.5 MHz sector transducer, vaginal cytology, and serum concentrations of progesterone and oestradiol were performed daily from the day before the start of FSH treatment through 7 days after FSH treatment. After 15 days of ovarian examination, each bitch received the same FSH treatment twice continually at 15-day intervals. No serosanguineous vaginal discharge was observed during the ovarian examination. The concentrations of progesterone (<0.045–9.6 ng mL–1) and oestradiol (<9.7–81.4 pg mL–1) varied through all treatments. Comparison of the concentrations of progesterone (<0.045–7.6 ng mL–1) and oestradiol (<9.7–30.3 pg mL–1) at the start of FSH administration in each trial revealed that elevated concentrations of both progesterone and oestradiol were observed in the first treatment in 3 bitches. Regarding the second and third treatments, no elevation of concentration was found for progesterone or oestradiol. A new follicular growth was observed in 1 animal after the third FSH treatment, but no follicular growth was found for the other animals. No correlation was found between follicular development and the profile of either progesterone or oestradiol. Ultrasonography proved that FSH stimulation alone cannot induce follicular growth by a single treatment, but it might increase the levels of progesterone and oestradiol, which are not correlated with follicular development and oestrous cycles at the start of FSH treatment.
19 GENOME-WIDE ANALYSIS OF DNA METHYLATION IN CLONES AND NONCLONES OF TWO DIFFERENT BREEDS: HOLSTEIN AND JAPANESE BLACK
Authors: Kiefer H. , Kaneda M. , Jouneau L. , Campion E. , Balzergue S. , Martin-Magniette M.-L. , Renard J.-P. , Nagai T. , Jammes H.
Published: December 2014

Epigenetic marks, and especially DNA methylation, are at the interplay of both environmental and genetic factors. By facilitating the metabolic adaptation of highly selected rent animals to their environment, DNA methylation could contribute to the phenotypic differences observed between breeds. The aim of this study was to assess to which extent the methylome of 2 specialised cattle breeds – the dairy breed Holstein and the beef breed Japanese Black – could show some variability. We focused on the liver, which has a central role in metabolism and is therefore most susceptible to be affected by genetic and environmental variations. For each breed, both cloned and noncloned animals were included in the study. We used 9 adult Holstein cows aged from 5 to 15 years (5 healthy clones generated from ear skin fibroblasts of 4 genotypes, 2 cell donors obtained by AI and 2 other AI controls of unrelated genotypes, and 11 Japanese Black cows aged from 4 to 10 years (5 healthy clones generated from cumulus cells of one genotype and 6 AI controls of unrelated genotypes). The Holstein breed and Japanese Black breed were therefore represented by 6 and 7 genotypes, respectively. Liver samples were snap-frozen after slaughtering, and genomic DNA was extracted. To identify methylated regions, we used immunoprecipitation of methylated DNA followed by hybridization on a bovine promoter microarray (MeDIP-chip). The microarray targets the upstream region (–2000 to +1360 bp) of 21 416 genes (UMD3.1 assembly). After normalization of the data, enriched probes were identified using ChIPmix (Martin-Magniette et al. 2008). Results of exploratory analysis, including correlation clustering and principal component analysis, show a clear separation between the two breeds. A statistical test based on differences in the proportion of the enriched probes was used to identify differentially methylated regions (DMR) related to cloning and breed (Spatstat R package; http://www.spatstat.org/spatstat/). Only a restricted number of cloning-related DMR could be found (240). Interestingly, most of these DMR showed no overlap between Holstein and Japanese Black animals, maybe reflecting the different origin of the somatic cells used for cloning (fibroblasts v. cumulus cells). In contrast, we identified an important number of breed-related DMR (3642). These DMR were significantly enriched in genes involved in placental development and lactation, suggesting an adaptation of the two breeds to the different metabolic demand during gestation. Whether these epigenetic differences rely on environmental variations or genetic polymorphism remains to be elucidated.Research was supported by grants ANR-09-GENM-012-01 and Egide-JSPS Sakura 2012.
200 STAGE-SPECIFIC EXPRESSION PATTERNS OF THE INSULIN-LIKE GROWTH FACTOR 1 RECEPTOR DURING BOVINE PRE-IMPLANTATION EMBRYONIC DEVELOPMENT
Authors: Poppicht F. , Stinshoff H. , Wrenzycki C.
Published: December 2014

Insulin-like growth factor 1 (IGF1) is a key regulator in early embryonic development, influencing physiological processes and stimulating growth and development (Fowden et al. 2003). Supplementing IGF1 during in vitro culture of bovine embryos improved cleavage and developmental rates while it reduced apoptosis (Byrne et al. 2002). The signal transduction of IGF1 is performed by its binding to the insulin-like growth factor 1 receptor (IGF1R). At the mRNA level, IGF1R is expressed throughout pre-implantation embryonic development and was identified as a potential marker of good quality embryos (Yaseen et al. 2001). However, information on protein level is rare. Therefore, protein expression of the IGF1R during early embryonic development in vitro was analysed in the present study by immunofluorescence staining. Furthermore, the mRNA expression of the IGF1R was investigated by RT-qPCR. In vitro derived embryos of different stages (2-cell, 4-cell, 8-cell, 16-cell stage, morula, blastocyst, and expanded blastocyst) were either directly subjected to immunofluorescence staining or frozen at –80°C for use in RT-qPCR. Staining was performed with a peptide antibody against two peptide sequences of the bovine IGF1R α unit, which was specifically produced. Pixel intensity of immunofluorescence was measured and a mean grey value was calculated using the cellsens® software (Olympus, Hamburg, Germany). Data were analysed by one-way ANOVA followed by a Tukey's test using SigmaStat 3.5 Software (Systat Software GmbH, Erkrath, Germany). The detection of the IGF1R mRNA and protein was possible in all stages of embryonic development beginning at the 2-cell stage up to the expanded blastocyst. The maximal mRNA expression could be observed in 2- and 4-cell embryos. It significantly decreased to the 8-cell stage, followed by an increase up to the expanded blastocyst. The IGF1R protein was mainly localised in the plasma membrane of single blastomeres and also weakly in the cytoplasm. Mean grey values are highest in the 2-cell stage, showing a significant decline up to the 16-cell stage and an increase again until the expanded blastocyst. The mRNA and protein expression showed similar patterns during early embryonic development. IGF1R expression started to increase at the 8-cell stage (mRNA) and 16-cell stage (protein) indicating a link to the maternal-embryonic transition. For the first time, these results show that in bovine embryos, the IGF1R expression is related to the activation of the embryonic genome.
We gratefully acknowledge the financial support of the H. Wilhelm Schaumann Foundation (Hamburg, Germany).
203 PI3-K PATHWAY IS ONE OF THE KEY REGULATORS OF STEROIDOGENESIS OF CUMULUS CELLS OF BOVINE CUMULUS–OOCYTE COMPLEXES MATURATED IN DEFINED MEDIUM IN THE PRESENCE OF FSH
Authors: Kaiser de Souza D. , Salles L. P. , Camargo R. , Dolabela de Lima B. , Torres F. A. G. , Rosa e Silva A. A. M.
Published: December 2014

The aim of the present study was to access the function of PI3-K pathway tested by the use of its inhibitor, LY294002, in maturation medium of bovine cumulus-oocyte complexes (COC) in steroid concentration in the medium, key enzymes of steroidogenic pathway and gonadotrophin receptors of cumulus cells. This study was performed in defined medium without serum and albumin (MIV B) in absence or presence of 10 ng mL–1 of FSH. Androstenedione 10–7 M was used as a precursor of steroidogenesis. Bovine COC (n = 35–40/well) collected from ovaries obtained at abattoirs of Brasilia (DF, Brazil) were cultivated in 400 μL of medium MIV B; MIV B + 100 μmol mL–1 of LY294002; MIV B added to 10 ng mL–1 of FSH; or MIV B added to 10 ng mL–1 of FSH + 100 μmol mL–1 of LY294002 for 22 to 24 h. After culture, COC were mechanically denuded and cumulus cells from 20 COC were isolated by centrifugation to determine the gene expression of LHR, FSHR, CYP11A1, CYP19A1, and HSD17B1 by PCR real time. Cumulus cells of immature COC were also collected and analysed as the calibrator group. Student–Newman–Keuls was performed as statistical test. The culture medium was collected after culture to determine progesterone and 17β-oestradiol concentration by quimioluminescence method and to calculate E2/P4 ratio. Two-way ANOVA, followed by Bonferroni test, and t-test were performed to determine the statistical significance. The MIVB enhanced LHR, FSHR, CYP11A1, CYP19A1, and HSD17B1 and LY294002 inhibited the expression of all genes (P < 0.05). MIVB + FSH decreased the expression of all genes (P < 0.05) except CYP11A1. LY294002 in the presence of 10 ng mL–1 of FSH did not affect the gene expression of FSHR, CYP11A1, and HSD17B1; however, it increased expression of LHR and CYP19A1. Oestradiol and progesterone production was increased by supplementation of FSH in MIVB (P < 0.05), but the E2/P4 ratio did not differ between treatments. LY294002 decreased oestradiol and E2/P4 ratio in absence or presence of FSH (P < 0.05), but did not alter progesterone concentration in MIVB+FSH. The inhibitor of PI3-K decreased the expression of steroidogenic proteins and steroid production in absence of FSH. The supplementation of FSH increased steroid production and decreased gene expression of steroidogenic enzymes, except CYP11A1. The inhibition of PI3-K in presence of FSH increases LHR and CYP19A1 expression. This fact suggests a strong role of the PI3-K pathway in the regulation of LHR and CYP19A1 expression.The authors thank FAP-DF (193.000.577/2009), CNPq, CAPES, Sabin, and Ponte Alta abattoir, Brasilia.
229 SHORT-TERM EXPOSURE OF MATURE OOCYTES TO A NITRIC OXIDE DONOR FOR INDUCING OXIDATIVE STRESS RESISTANCE ON IN VITRO-PRODUCED BOVINE EMBRYOS
Authors: Cheuquemán C. , Loren P. , Arias M. , Risopatrón J. , Felmer R. , Alvarez J. , Mogas T. , Sánchez R.
Published: December 2014

Recent studies have shown that short-term exposure of oocytes to stressors such as hydrostatic pressure, osmotic stress, and oxidative stress might induce stress tolerance in embryos. In this research we studied the effect of short-term exposure of bovine in vitro-matured cumulus-oocyte complexes (COC) with a nitric oxide donor (SNP) on IVF, embryo development, embryo quality, and relative gene expression related to cell redox state regulation. The COC were selected and matured in TCM 199 supplemented with 10% inactivated FBS, 6 mg mL–1 of LH, 6 mg mL–1 of FSH, 1 mg mL–1 of oestradiol, and 0.2 mmol of pyruvate and then incubated for 22 to 24 h at 38.5°C in 5% CO2 in a humidified atmosphere (n = 12). Before IVF, mature COC were incubated during 1 h with different concentration of sodium nitroprusside, SNP (control without SNP, 10–6 M, 10–5 M, and 10–4 M SNP) in maturation media at 38.5°C and 5% CO2 in a humidified atmosphere. For IVF procedure, oocytes of each treatment and sperm of one bull were co-incubated for 18 to 20 h at 38.5°C and 5% CO2. Presumptive zygotes were separately cultured until Day 7 under mineral oil at 38.5°C and 5% CO2, 5%O2, and 90% N2 in a humidified atmosphere. Embryo quality was analysed by staining with CDX2 antibody for trophectoderm cells and compared with total embryo cells stained with Hoechst 33342. Relative gene expression for each treatment were evaluated after RNA extraction and cDNA synthesis in Stratagene MX 3000P real-time equipment with Agilent qPCR software MX pro 4.1 version. Differences between experimental groups (n = 12) were measured using a one-way ANOVA test in the STATGRAPHICS plus 5.1 version software. P < 0.05 was considered statistically significant. Cleavage percentage at 72 h post-insemination was significantly different between the control and 10–4 M SNP group (82 ± 8.4% v. 77 ± 7.1%, respectively) and between 10–5 M and 10–4 M SNP group (84.9 ± 4.1% v. 77 ± 7.1%, respectively). Blastocyst percentage at 7 days of culture was significantly different between control and 10–4 M SNP group (34.1 ± 7.8% v. 26.2 ± 4.9%, respectively). Embryo development between control group and treatments was similar within early, expanded, and hatched blastocyst percentage. Embryo quality of expanded blastocyst was similar between control group and treatments (ICM: TE). No significant differences in gene expression after SNP exposure was observed (iNOS, eNOS, nNOS, PRDX5, HSP70, HSP90, HIF1A, BCL2A). Oocytes incubated with a high concentration of SNP showed lower cleavage and blastocyst rates, showing that this treatment was deleterious for in vitro embryo production in bovine. However, there were no significant differences on embryo quality assessed by ICM : TE ratio and/or in gene expression pattern of 7-day cultured expanded blastocysts.