bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of titanium in electromigrated tunnel junctions
Authors: Frimmer Martin , Puebla-Hellmann Gabriel , Wallraff Andreas , Novotny Lukas
Published: December 2014

A standard route for fabrication of nanoscopic tunnel junctions is via electromigration of lithographically prepared gold nanowires. In the lithography process, a thin adhesion layer, typically titanium, is used to promote the adhesion of the gold nanowires to the substrate. Here, we demonstrate that such an adhesion layer plays a vital role in the electrical transport behavior of electromigrated tunnel junctions. We show that junctions fabricated from gold deposited on top of a titanium adhesion layer are electrically stable at ambient conditions, in contrast to gold junctions without a titanium adhesion layer. We furthermore find that electromigrated junctions fabricated from pure titanium are electrically exceptionally stable. Based on our transport data, we provide evidence that the barrier in gold-on-titanium tunnel devices is formed by the native oxide of titanium.
Effects of crossbreeding on milk production and composition in dairy sheep under organic management
Authors: Ortega Octavio A. Castelan , Ronquillo Manuel González
Published: December 2014

The crossbreeding of local sheep breeds with dairy breeds is an option to improve dairy production parameters in organic sheep dairy systems. Weekly milk yield (WMY) was recorded and individual samples of milk for chemical analysis were taken during 17 weeks from 45 dairy ewes of the following three genotypes: 15 East Friesian (EF), 15 EF × Suffolk (EF × SF) and 15 EF × Pelibuey (EF × PL) under organic management. For analysis of the lactation curve the Wood gamma model was used. The effect of genotype on the WMY was analysed using repeated-measures. The comparison of the least square means among genotypes for total milk yield (TMY), daily milk yield, protein content, protein yield, fat content, fat yield, non-fat solids concentration, non-fat solids yield, total solids yield and acidity was analysed using a general linear model. The genetic group influenced only in the ascent phase of the lactation curve, with values of the Parameter b of model Wood higher in EF (P = 0.01). There were no differences (P > 0.05) between genotypes in relation to the WMY, TMY, protein content and acidity; however, the effects of week of lactation trial and the interaction of genotype and week of lactation trial on WMY were significant (P < 0.05). The values of daily milk yield, fat yield, protein yield and total solids yield were higher (P < 0.005) in EF and EF × SF than EF × PL. Fat content was higher in EF × PL. EF × SF had similar values of TMY than EF and better chemical composition, which places this genotype as an option of crossbreeding in dairy sheep systems under organic management with similar agro climatic characteristics to the present study.
Estimation of genetic parameters for litter traits in grasscutters (Thryonomys swinderianus)
Authors: Henry A. J. , Ibe S. N. , Asuquo B. O.
Published: December 2014

A study was conducted to estimate genetic parameters for litter traits in grasscutters. Data were obtained using 209 progenies from an unbalanced design of 18 does nested under six bucks. Heritability values of 0.56 ± 0.21 and 0.22 ± 0.22 were obtained for litter weight at birth from dam component and both sire and dam components, respectively. For litter weight at weaning, the heritability estimate was 0.66 ± 0.22 using the dam component of variance. Genetic correlation between litter weight at birth and litter weight at weaning was 0.59 revealing a positive relationship between both traits.
101 SUPPLEMENTATION WITH SUNFLOWER SEED ALTERS THE ENDOMETRIAL LIPID COMPOSITION IN BEEF COWS
Authors: Membrive C. M. B. , Cordeiro M. B. , Pazzanese D. , Pugliesi G. , Sá Filho M. F. , Binelli M.
Published: December 2014

Embryo death between 15 and 19 days of pregnancy is caused by the increase in the release of endometrial prostaglandin F2α (PGF2α) involved in the luteolysis process in cattle. Compounds rich in linoleic acid, such as sunflower seeds, provide lipid changes in the endometrium, and may be involved in the ability of PGF2α biosynthesis. Previous studies observed that the conception rate increased in Nelore cows supplemented with sunflower seed for 22 days from the timed AI (66.7% v. 46.3%; Peres et al. 2008, Acta Sci. Vet. 36, 639) and in crossbred heifers submitted to timed embryo transfer (55.66% v. 36.94%; Membrive et al. 2013 Acta Sci. Vet. 36, 603). We aimed to test the hypothesis that supplementation with sunflower seed promotes endometrial changes in lipid composition. Thus, we compared the composition of fatty acids in endometrial tissue in cows supplemented or not with sunflower seed. Nelore (n = 30) cows received an intravaginal device containing progesterone (1 g; DIB, Syntex Biochemistry & Pharmaceutical Industries SA, Buenos Aires, Argentina) associated with an im injection of oestradiol benzoate (2 mg; Benzoate HC, Hertape Calier Animal Health SA, Juatuba, MG, Brazil). The devices were removed after 8 days, when cows were treated im with cloprostenol sodium (2 mg; Sincrocio®, Ourofino Animal Health Ltd., Cravinhos, SP, Brazil), oestradiol cypionate (0.5 mg; ECP®, Zoetis Ltda., São Paulo, Brazil) and eCG (300 IU; Folligon®, Intervet Veterinary Ltda., Cotia, Brazil). Two days after removal of the device, females were assigned into 6 groups to receive 1.7 kg/animal/day of 40% soybean meal, 44% crude protein (CP) + 60% sunflower seed for 6 (n = 4), 14 (n = 5) and 22 days (n = 6), or 53% soybean meal, 44% CP + 47% corn for 6 (n = 4), 14 (n = 5) and 22 days (n = 6). Both diets were formulated with 72% total digestible nutrients and 24% CP. Females were slaughtered 24 h after the end of supplementation and endometrial tissue was isolated and stored at –196°C. The fatty acids in endometrial tissue were assessed by gas chromatography. Data were analysed by SAS Proc GLIMMIX (SAS Institute Inc., Cary, NC, USA). The fatty acid profile (54 compounds) was analysed and 43 fatty acids were present in the endometrial tissue. The lacking fatty acids in endometrial tissue were C4:0, C11:0, C12:1, C: 13:0, C13:0 iso, C13:0 anteiso, C14:0 iso, C15:1, C18:1 trans-16, C18:2 cis-12, trans-10, and C21:0. The fatty acids that showed a higher percentage compared with the Control group were C18:1 trans-10-trans-11-trans-12 and C10:1. The fatty acids that showed low percentage compared with the Control group were C15:0 iso, C20:5, C20:3n-3, C23:0, C24:0, and C22:5. In conclusion, supplementation with sunflower seed promotes changes in the endometrial lipid profile that may reduce the pregnancy loss in beef cows.
Research supported by FAPESP, FUNDUNESP, and Santa Encarnação Farm.
108 CHARACTERIZATION OF BOVINE OVIDUCTAL EXOSOMES FROM IN VIVO AND IN VITRO ORIGIN
Authors: Almiñana C. , Corbin E. , Tsikis G. , Soleilhavoup C. , Galio L. , Sandra O. , Mermillod P.
Published: December 2014

Successful pregnancy requires an appropriate communication between the mother and the embryo(s). Recent studies indicate that exosomes, small (30–200 nm) membrane vesicles of endocytotic origin, could act as intercellular vehicles in this unique communication system. Exosomes have been identified in vivo in all body fluids including follicular, uterine, and oviductal fluids and can be secreted by most cell types in vitro. Bovine oviductal epithelial cells (BOEC) have been thoroughly used to study embryo-maternal communication and to improve embryo development in vitro. Hence, our objective was to provide a morphologic and proteomic characterisation of exosomes secreted by BOEC in vivo in the oviductal fluid and in vitro in the conditioned media. Oviducts from cows were flushed to recover in vivo exosomes and then BOEC were scraped in order to derive primary cultures. In vitro exosomes were collected from conditioned media of BOEC primary cultures after reaching confluence (10 days). Isolation of exosomes from in vivo and in vitro origin was performed by ultracentrifugation. The presence of exosomes was confirmed in oviductal flushings and conditioned media by electron microscopy. Further characterisation of exosomes was carried out based on morphology (transmission electron microscopy), size (dynamic light scattering, DLS), and protein composition (protein profile analysis by SDS-PAGE and Western immunoblotting). Preliminary results by DLS revealed different size distribution profiles in exosome samples (in vivo: mean size of 93.41 nm; in vitro: 433.5 nm). Because exosomes are considered as “micromaps” of the originating cells, protein patterns expressed by in vivo exosomes and in vitro exosomes were compared with scraped and cultured BOEC, respectively. Protein profile analysis by SDS-PAGE showed quantitative and qualitative differences among the exosome samples, their cells of origin, and the milieu (conditioned media or flushing). Exosome-specific protein bands were detected and will be further characterised. In addition, exosomes from in vivo and in vitro origin exhibited distinct proteomic profiles. Western blot analysis demonstrated that (1) both exosomal protein samples were positive for HSP70, a known exosomal protein, and negative for Grp78, an endoplasmic reticulum marker detected in BOEC; (2) in vivo exosomes expressed oviductal glycoprotein (OVGP), heat shock protein A8 (HSPA8), and myosin 9 (MYH9), 3 oviductal proteins with known roles in fertilization and early pregnancy. However, only HSPA8 and MYH9 were detected in in vitro exosomes. Our results provide the first extensive characterisation of oviductal exosomes from in vivo and in vitro origin, an essential step in furthering our understanding of the early embryo-maternal cross talk.
133 NONINVASIVE ANALYSIS OF EMBRYO METABOLITES USING NUCLEAR MAGNETIC RESONANCE
Authors: Rubessa M. , Herzog K. K. , Ambrosi A. , Stewart J. W. , Polkoff K. M. , Denmark S. E. , Wheeler M. B.
Published: December 2014

Wide-spread use of IVF has significantly increased the number of multiple births (Janvier et al. 2011 J. Pediatr. 159, 409–413). A potential solution to this problem is to develop improved methods for embryo selection to permit single-embryo transfer. Identification of a noninvasive technique to assess embryo implantation potential in assisted reproduction would greatly increase success rates and lead to more efficient single-embryo transfer. The aim of this study was to assess whether there are metabolic differences among embryos produced by IVF and embryos obtained by parthenogenetic activation. 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. For the parthenogenetic group, the oocytes were activated by 5 μM ionomycin in M199 + 10% FCS for 5 min, and incubation in 2 mM 6-DMAP in M199 + 10% FCS for 4 h. 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. 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 in 50 mL of deuterium oxide. The sodium 3-(trimethylsilyl)-2,2′,3,3′-tetradeuteropropionate acted both as a chemical shift reference and as an internal standard for the purposes of quantitation. 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 (IVF or parthenogenetic 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 with standard error of the means. The results, reported in Table 1, show that there were no statistical differences between embryo metabolites with IVF or parthenogenetic activation when we evaluated lactate, formate, myo-inositol, and pyruvate. However, we can see that there are differences when we focused on acetate and citrate. Parthenogenetic embryos produced more citrate than IVF embryos. It is well known that the Krebs cycle produces one molecule of acetate for each molecule of citrate. The present results support that as well with the concentration of acetate being greater in parthenogenetic than in the IVF embryos. These results are a first step in identifying noninvasive, quantitative parameters that indicate which embryos may be the most viable before transfer.

Table 1.Results (least squares means ± s.e.)
138 DEVELOPMENTAL COMPETENCE OF BIOPSIED AND SPLIT BOVINE EMBRYOS
Authors: Reichenbach M. , Jung S. , Fries R. , Wolf E. , Gschoederer C. , Scherzer J. , Grupp T. , Reichenbach H.-D.
Published: December 2014

The aim of the present study was to develop a reliable method to simultaneously split and biopsy valuable bovine embryos for a complete genomic evaluation (gender, polledness, and hereditary abnormalities) and to estimate the breeding value of progeny for traits of economic importance immediately after embryo recovery. A total of 208 good quality embryos collected from superovulated German Simmental animals were biopsied immediately after recovery using an inverse microscope (Zeiss, Germany) at 50× magnification with a single-use steel blade mounted on a holder (Bausch & Lomb, Germany) attached to a micromanipulator (Eppendorf, Germany). Biopsy was performed either by splitting the embryo and cutting of one-third of a half [G1: morulae (M), n = 50; early blastocysts (EB), n = 24; blastocysts (B), n = 16], by just splitting in equal halves (G2: M, n = 16; B, n = 2), or by cutting of just a small biopsy of the embryo (G3: M, n = 53) or of the trophoblast (G3: EB, n = 19; B, n = 28). Biopsied cells were immediately used for DNA amplification. Biopsied embryos (E) and demi-embryos (DE) were in vitro cultured in SOF, under mineral oil, at 39°C and 5% CO2, 5% O2, 90% N2 for 24 h, after which survival was recorded. Survival rate of G1 (survival of at least 1 DE: M, 98.0%; EB, 100.0%; B, 93.8%), G2 (survival of DE: M, 75.0%; B, 100.0%), and G3 (embryo survival: M, 96.3%; EB, 100.0%; B, 96.4%) were similar, but in relation to the number of original embryo the highest ratio of DE was obtained in G1 (1.67) v. G2 (0.88) and G3 (0.97; G1:G2/G3; P < 0.01). Within G1, the highest ration to the original number of embryos was by using M (1.78), followed by EB (1.75) and B (1.19; M/EB:B; P < 0.05). To verify the viability of biopsied embryos some DE from G1 (1, the nonbiopsied DE, n = 7, or 2, the biopsied and the nonbiopsied DE per recipient, n = 21), G2 (1 DE per recipient, n = 13), and G3 (1 E per recipient, n = 8) were transferred after 24 h of culture. Overall pregnancy rate (Day 42) of G1, G2, and G3 was 64.3, 23.1, and 50.0%, respectively (G1 : G2; P < 0.05). In G1, pregnancy rates (Day 42) of biopsied embryos differed significantly if either 1 or 2 DE were transferred per recipient (28.6 v. 76.2%, respectively; P < 0.05). A twin pregnancy rate of 38.9% was observed by ultrasonography in recipients when 2 DE were transferred. The results suggest that high survival rates can be obtained with the G1 technique, and splitting during biopsy can increase productivity in programs aimed to evaluate the genomic constitution of early stage embryos.
Funded by the Bayerische Forschungsstiftung (AZ-1031-12).
141 EFFECT OF LASER-ASSISTED EMBRYO BIOPSY AND DEVELOPMENTAL STAGE ON EMBRYO SURVIVAL AT TERM IN SHEEP
Authors: Alabart J. L. , Lahoz B. , Sánchez P. , Folch J. , Calvo J. H. , Cocero M. J.
Published: December 2014

A biopsy procedure causing minimal injury to embryos is essential to exploit the possibilities of the preimplantation genetic diagnosis in sheep biotechnologies. The effect of laser-assisted embryo biopsy on embryo survival at term was studied in ovine in vivo-derived embryos recovered at 2 different developmental stages. We used 294 embryos (quality scores 1 and 2; IETS manual, 1998 Edition) at the stages of compact morula (n = 136) or blastocyst (n = 158) recovered from superovulated Rasa aragonesa ewes at Day 8 after sponge removal. Embryos from each donor ewe were either biopsied (n = 160; 76 compact morulas and 84 blastocysts) or directly incubated at 38.5°C in TCM199 and 5% CO2/air (n = 134; 60 compact morulas and 74 blastocysts) during 18 to 22 hours before transfer. During biopsy, embryos were held by a holding pipette (15 µm internal diameter) in Dulbecco's phosphate-buffered saline without bovine serum albumin (100-µL microdrops). A 40× objective equipped with an infrared laser (1480 nm; 300 mW; XYClone, Hamilthon-Thorne, Parallabs Ltd, St Albans, UK) was used to open a hole in the zona pellucida by one 1-ms laser pulse. Whenever possible, only extruded cells were aspirated through the hole (usually, 3 to 10 cells) using a rounded-end aspiration pipette (19 µm internal diameter). In embryos without sufficient observable extruded cells, the cell mass was aspirated throughout the hole and a portion of the protruded part was ablated using one or several laser pulses of up to 2.4 ms. When performed in blastocysts, cell ablation was carried out in the trophoblastic region opposite to the inner cell mass. After biopsy, embryos were incubated in the same conditions as non-biopsied embryos. Embryos reaching the expanded or hatching/hatched blastocyst stages after culture were transferred in pairs to synchronized recipient ewes. Percentages were analysed by ANOVA for categorical variables using the CATMOD procedure of SAS. The percentage of arrested embryos tended to be higher (P < 0.07) in compact morulas either biopsied (11.8%) or not (10.0%) than in blastocysts (4.8 and 5.4%, respectively). The effects of the biopsy procedure and its interaction with the developmental stage were not significant (P < 0.85 and P < 0.70, respectively). The survival rate at term of the transferred embryos was similar in biopsied and nonbiopsied embryos (P < 0.31), either at the compact morula (62.7 and 72.2%, respectively) or the blastocyst (72.5 and 74.3%) stages. The effects of the developmental stage and its interaction with biopsy were not significant (P < 0.29 and P < 0.49, respectively). The effects of biopsy procedure and its interaction with the developmental stage were not significant (P < 0.33 and P < 0.44, respectively). In conclusion, the procedures presented here allow performing embryo biopsy with minimal injury, either in the compact morula or blastocyst stages. These results highlight the usefulness of laser in embryo biopsy.
153 SEASON DOES NOT INFLUENCE EARLY CONCEPTUS GROWTH AND RECOVERY IN THE HORSE
Authors: Aurich C. , Budik S.
Published: December 2014

Although the horse is a seasonal breeding species, a considerable number of mares continues to cycle throughout autumn and winter. This allows for embryo collection outside the breeding season. However, slower equine embryo growth during the non-breeding season has been hypothesised. Because in this species, smaller embryo size (<0.3 mm diameter) is beneficial for conceptus cryopreservation, embryo collection outside the breeding season could be an interesting approach for the production of frozen horse embryos. In the present study we have therefore analysed embryo recovery rates in a herd of research mares (Haflinger breed, n = 30) that are used for embryo production throughout the year. Oestrous mares were inseminated (500 Mio progressively motile sperm) at 48-h intervals and ovulation was detected by transrectal ultrasound. Diameter of the conceptus at collection was measured with a microscope scale (day 10). Statistical analysis was done with the SPSS Statistics 21 software. The influence of collection day (7 to 14 after ovulation) and season (SPR: March – May; SUM: June – August; AUT: September – November; WIN: December – February) on conceptus size was analysed by GLM univariate analysis. Influence of season on recovery rate was compared by Chi-squared analysis. Values for conceptus diameter are given as means ± s.e.M. A total of 352 embryo collection attempts were performed. Mean recovery was 64.2%. It was not affected by season (number of embryos collected: SPR: 51/72, SUM 67/105, AUT 71/114, WIN 37/61 flushes). The conceptus diameter was evaluated in a total of 165 embryos. Conceptus diameter significantly increased (P < 0.001) with day of pregnancy (e.g. day 7: 0.5 ± 0.05, day 8: 0.76 ± 0.2, day 10: 3.8 ± 0.2, day 12: 9.8 ± 0.6, day 14: 17.5 ± 1.0 mm), but was not influenced (P = 0.957) by season (e.g. day 10: SPR 4.2 ± 0.6, SUM 3.6 ± 0.4, AUT 3.9 ± 0.4, WIN 3.8 ± 0.6 mm). These results demonstrate that embryo collection in horses can be performed successfully throughout the year if mares continue to ovulate during the non-breeding season. Because conceptus growth is similar with regard to season, production of cryopreserved equine embryos is not facilitated at a specific time of the year.
175 FOLLICULAR DEVELOPMENT AND OVARIAN BLOOD FLOW IN MARES WITH EARLY OR LATE OVULATION POSTPARTUM
Authors: Lemes K. M. , Silva L. A. , Celeghini E. C. C. , Alonso M. A. , Pugliesi G. , Carvalho H. F. , Affonso F. J. , Silva D. F. , Leite T. G. , Arruda R. P.
Published: December 2014

The postpartum period is characterised by the rapid uterine involution process and return of ovarian activity (foal heat), resulting in a fertile oestrus in most of the mares. However, the follicular development and selection processes during this period are not completely known in horses. We aimed to study the characteristics of follicular growth and vascular perfusion in the ovary during the early postpartum period in mares that demonstrated oestrous behaviour and had early (<10 days) or late (≥10 days) ovulation. Ten mares were scanned daily from the first day postpartum (Day 1) until the day of the first postpartum ovulation (Day 0). The animals were split in the early (n = 3) and late (n = 7) ovulation groups (averaged interval between parturition and ovulation: 8.0 ± 0.0 and 14.7 ± 1.2 days, respectively). For ultrasound exams a Duplex B-mode and colour Doppler instrument (M5VET®, Mindray, Shenzhen, China) was used with a multifrequency linear probe. Data were analysed for the main effects of group, day, and their interaction using the PROC MIXED procedure of SAS software (version 9.3, SAS Institute Inc., Cary, NC, USA). For the follicular growth, no difference (P > 0.05) was detected between the groups when the data were analysed for the days relative to ovulation (from Day 7 to Day 1). However, the dominant follicle was larger (P < 0.05) in the early-ovulated group (37.2 ± 1.6 v. 21.9 ± 1.1) in all days during early postpartum (Day 1 to Day 7). The number of follicles with >25 mm diameter was also greater (P < 0.05) in the early-ovulated group (1.1 ± 0.1 v. 0.1 ± 0.1) during the first 3 days postpartum. In addition, the late-ovulated mares showed greater number of follicles with 20–25 mm during Day 4 to Day 7 (2.0 ± 0.2 v. 0.7 ± 0.1). For the blood flow characteristics, no difference (P > 0.05) was detected in the coloured signals of blood flows in the follicular wall of the dominant follicle or in the ovarian pedicle ipsilateral to the largest follicle. Therefore, the characteristics of the follicle growth on the preceding days of ovulation were similar between the early- and late-ovulated mares and consistent with the follicular dynamics expected in non-pregnant and non-lactating mares. However, when the data were analysed for the days relative to parturition, a greater follicle development was present in mares that ovulate earlier during the postpartum period (<10 days). In conclusion, the results suggest that important events may occur previous to the parturition, resulting in an early follicle development, mainly in those mares that show heat signs and ovulate within 10 days postpartum.
Research was supported by FAPESP process number 2010/10692-9 and CNPq process number 135954/2011-8.
179 INTRAFOLLICULAR CONCENTRATION OF FATTY ACIDS IN GOAT ACCORDING TO FOLLICLE DIAMETER AND AGE OF DONOR
Authors: Roura M. , Catalá M. G. , Hammami S. , Rodriguez M. , Moll X. , Garcia F. , Izquierdo D. , Paramio M. T.
Published: December 2014

Oocytes from adult goats have better competence to develop up to blastocyst stage than those from prepubertal females. Previous results in our laboratory showed that oocyte developmental competence in prepubertal goats is positively related to follicle diameter, as oocytes from follicles larger than 3 mm have the same competence as adult goat ones, demonstrating that follicular environment affects oocyte competence for embryo development. Other studies concluded that fatty acids (FA) in follicular fluid (FF) play an important role in oocyte and embryo development. The aim of this study was to analyse FF composition regarding FA percentage comparing samples from adult (3 years old, forage diet) or prepubertal (1 month suckling) Murciano-Granadina goats. FF samples were recovered from follicles larger and smaller than 3 mm by laparoscopy (adult) or aspiration of ovaries collected at slaughterhouse (prepubertal). For the FA analysis, the Sukhija and Palmquist protocol was used with some modifications. Briefly, 200 μL of FF sample was vortexed for 60 s with 250 μL of toluene and 1 mL of HCL (5%) and then warmed in water bath for 1 h at 70°C. Subsequently, 1.25 mL of K2CO3 (12%) and 500 μL of toluene was added, vortexed for 30 s, and centrifuged for 5 min (3000 rpm). Finally the supernatant was recovered and dried with Na2SO4. The extracted samples were maintained at –20°C until gas chromatographic analysis (123–2362, Agilent Technologies Inc., Santa Clara, CA). The results are shown in Table 1. The analysis of FA composition in FF showed significantly higher concentrations of C15:0 (pentadecanoic), C16:0 (palmitic), C17:0 (margaric), C18:0 (stearic), linolenic acid (ALA), and EPA (eicosapentanoic) in adult goats compared to prepubertal goats, and C17:0, C18:0 linoleic acid (LA), and ALA in larger follicles of prepubertal females, showing some coincidences in follicular environment that could explain previous studies. These results are in agreement with those of Matoba et al. (2014; Rep. Fertil. Dev. 26, 337) who showed that FF associated with more competent oocytes had a significantly higher concentration in ALA than FF from incompetent oocytes in cattle.

Table 1.Concentration of FA (%) in FF in adult and prepubertal goats (top) and in FF of large and small follicles (bottom) from prepubertal goat ovaries (3 replicates)
191 IN VITRO CULTURE DURING OOCYTE MATURATION AND FERTILIZATION INFLUENCES THE TRANSCRIPTOME PROFILES OF BOVINE BLASTOCYSTS
Authors: Gad A. , Besenfelder U. , Havlicek V. , Hölker M. , Rings F. , Dufort I. , Sirard M. A. , Schellander K. , Tesfaye D.
Published: December 2014

Early embryonic development, the period from oocyte maturation until blastocyst formation, is the most critical period of mammalian development. It is well known that in vitro maturation, fertilization, and culture of bovine embryos is highly affected by culture conditions. However, the stage-specific effect of culture environment is poorly understood. Therefore, we aimed to examine the effect of in vitro culture conditions during oocyte maturation and fertilization on the transcriptome profile of the resulting blastocysts. Bovine oocytes were matured in vitro and then either directly transferred to synchronized recipients, fertilized, and cultured in vivo (Vitro_M), or transferred after in vitro fertilization (Vitro_F), or at zygote stage (Vitro_Z) and blastocysts were collected at Day 7 by uterine flushing. For in vivo or in vitro fertilization, the same frozen-thawed commercial bull semen has been used. Complete in vitro (IVP) and in vivo produced blastocysts were used as controls. Gene expression patterns between each blastocyst group and in vivo produced blastocyst group were compared using EmbryoGENE's bovine microarray (EmbryoGENE, Québec, QC, Canada) over six replicates of each group (10 blastocyst/replicate). Microarray data were statistically analysed using the Linear Models for Microarray Data Analysis (LIMMA) package under the R program (The R Project for Statistical Computing, Vienna, Austria). Results showed that, the longer the embryos spent under in vitro conditions, the higher was the number of differentially expressed genes (DEG, fold-change = 2 with adjusted P-value = 0.05) compared with in vivo control group. The Vitro_M group showed the lowest number of DEG (149); in contrast IVP group represented 841, DEG, respectively compared to in vivo control group. Ontological classification of DEG showed that lipid metabolism was the most significant function influenced by in vitro maturation conditions. More than 55% of DEG in the Vitro_M group were involved in the lipid metabolism process and most of them showed down-regulation compared to in vivo control group. On the other hand, Vitro_F and Vitro_Z groups showed nearly similar numbers of DEG (584 and 532, respectively) and the majority of these genes in both groups were involved in cell-death- and cell-cycle-related functions. Pathway analysis revealed that retinoic acid receptor activation pathways were the common ones in the Vitro_M and Vitro_F groups. However, different signalling pathways were commonly dominant in the Vitro_F and Vitro_Z groups. This study provides the transcriptome elasticity of bovine embryos exposed to different environments during maturation, fertilization, and culture periods of development.
193 PROGESTERONE CONCENTRATIONS AND MESSENGER RNA EXPRESSION OF PROGESTERONE RECEPTORS IN ENDOMETRIAL TISSUE OF BOVINE UTERINE HORN IPSILATERAL AND CONTRALATERAL TO CORPUS LUTEUM
Authors: Takahashi H. , Haneda S. , Matsui M.
Published: December 2014

Generally, conception is established in the uterine horn ipsilateral to the corpus luteum (CL) in cattle. When a bovine embryo is transferred into the uterine horn contralateral to CL, conception rate is low. Since progesterone (P4) is essential for the establishment of pregnancy in cattle, locational effects of P4 released from CL at the uterus may cause the differences in fertility. The aim of this study was to determine the endometrial tissue P4 concentrations (EndP4) and the mRNA expression of nuclear progesterone receptor (PGR), and progesterone receptor component-1 (PGRMC-1) and -2 (PGRMC-2) in the endometrial tissues from the ipsi- and contralateral horn. The uteruses of Holstein cows were obtained at a local abattoir. Endometrial tissues were collected from both horns. Based on ovarian morphology, the oestrus cycle of the cow was estimated as follows: early luteal phase (ELP, Day 5–6, Day 0 = oestrus), mid luteal phase (MLP, Day 8–12), late luteal phase (LLP, Day 15–17), and follicular phase (FP, Day 18–20). EndP4 was measured by enzyme immunoassay. Expressions of mRNA were analysed by real-time RT–PCR. Two-way factorial ANOVA and the Steel-Dwass test were applied for a multiple comparison of means. The interrelations between both parameters were expressed by Spearman correlation coefficient. At ELP and MLP, EndP4 in ipsi-horn were higher than that in contra-horn (P < 0.05, see Table 1). Higher mRNA expression of PGRMC-1 in ipsi-horn was observed at ELP compared with contra-horn (P < 0.05). Expressions of mRNA for PGR and PGRMC-2 were similar in both horns. In ipsi-horn at ELP, EndP4 was positively correlated with PGRMC-1 mRNA (r = 0.87, P < 0.05), but was negatively correlated with PGR mRNA (r = –0.76, P < 0.05). However, in contra-horn, EndP4 has no correlation to mRNA expression of P4 receptors. In conclusion, EndP4 was influenced by the location of CL and stage of oestrus cycle. Higher expression of PGRMC-1 mRNA in endometrial tissue of ipsi-horn at ELP might be up-regulated by higher EndP4. These locational effects of CL on uterus may provide an intrauterine environment suitable for embryo development.

Table 1.End P4 and expression of mRNA for P4 receptors in bovine uterine horn1
201 USING THE TRANSCRIPTOME SIGNATURE TO PREDICT PREGNANCY SUCCESS IN BEEF COWS AT 6 DAYS AFTER ARTIFICIAL INSEMINATION
Authors: Scolari S. , Pugliesi G. , Andrade S. C. S. , D'Alexandri F. , Gasparin G. , Gonella-Diaza A. M. , Coutinho L. L. , Binelli M.
Published: December 2014

Pregnancy success is critical to the profitability of cattle operations. Attempts to reduce high rates of early embryonic loss mainly focus on the critical phase of embryo recognition by maternal tissue. However, the molecular events driving the uterine tissue toward a favourable stage, facilitating the maternal receptivity, are poorly understood. This study aimed to characterise the endometrial transcriptome profiles of pregnant versus nonpregnant beef cows during early pregnancy and attempted to define a potential set of marker genes that can be valuable for predicting pregnancy outcome. Therefore, pluriparous, cyclic Nellore (Bos indicus) cows were synchronized (n = 51) and artificially inseminated (n = 36) at detected oestrus using semen from a single high-fertility bull. Six days after AI (Day 6), jugular blood samples and an endometrial biopsy from the uterine horn contralateral to the ovary containing the CL were collected. Pregnancy diagnosis was performed by ultrasonography on Days 22 and 30. Based on pregnancy outcome, samples were retrospectively allocated to the following groups: pregnant (P; n = 6) and nonpregnant (NP; n = 5). Both groups had similar plasma progesterone concentrations on Day 6 (less than 1 ng mL–1 between lowest and greatest concentrations). Endometrial biopsies were submitted to RNA-Seq analysis in an Illumina single flow cell line (Illumina Inc., San Diego, CA). The 272 685 768 million filtered reads were mapped to the Bos taurus UMD3.1 reference genome and 14 654 genes were effectively analysed for differential expression between groups. Transcriptome data showed that 216 genes are differently expressed when comparing P v. NP endometrial tissue (adjusted P < 0.1). More specifically, 36 genes showed a significantly up-regulated expression for pregnant cows and 180 are up-regulated for non-pregant cows. Functional enrichment and pathway analyses revealed enriched expression of genes associated with extracellular matrix remodelling in NP cows and nucleotide binding, microsome, and vesicular fraction in P cows. From the 40 top-ranked genes, six that were down- and three that were up-regulated in pregnant cows were further analysed by qRT–PCR in an additional 26 cows. Subsequent quantitative expression data were evaluated using multivariate statistics. Both principal component analysis (PCA; R2 = 0.82 and Q2 = 0.40) and orthogonal projections to latent structures analysis (OPLS), using pregnancy as the dependent variable (R2Y = 0.95 and Q2 = 0.86), efficiently separated P from NP animals. In conclusion, this study characterizes a unique set of genes, expressed in the endometrium as early as 6 days after AI, that indicate a receptive state leading to pregnancy success. Furthermore, expression of such genes can be used as potential markers to efficiently predict pregnancy success.
224 A GROWTH OF HUMAN BG-1 CANCER CELLS EXPRESSING ESTROGEN RECEPTORS WAS ENHANCED BY SYNTHETIC PYRETHROIDS, LAMBDA-CYHALOTHRIN AND CYPERMETHRIN, VIA AN ESTROGEN RECEPTOR-DEPENDENT SIGNALING PATHWAY
Authors: Kim C.-W. , Go R.-E. , Choi K.-C.
Published: December 2014

Synthetic pyrethroids (SP) are the most common pesticides in recent use, which are used as indoor pest control. The widespread use of SPs has resulted in extensive exposure to wildlife and human. Recently some SPs are suspected as endocrine disrupting chemicals (EDC) and have been assessed for their potential estrogenicity by various assays. In this study, we examined the estrogenic effects of lambda-cyhalothrin (LCT) and cypermethrin (CP), the most commonly used pyrethroid insecticides in Korea, using BG-1 ovarian cancer cells expressing oestrogen receptors (ER). To evaluate the estrogenic activities of two SPs, LCT and CP, we employed MTT assay and reverse-transcription polymerase chain reaction (RT–PCR). In MTT assay, LCT (10–6 M) and CP (10–5 M) significantly induced the growth of BG-1 cancer cells, 1.61 ± 0.1 and 1.45 ± 0.06 times, respectively, as 17β-oestradiol (E2, 10–9 M, 2.73 ± 0.25 times) did. LCT or CP-induced cell growth was reversed to a control level (DMSO) by addition of ICI 182 720 (10–8 M), an ER antagonist, suggesting that this effect appears to be mediated by an ER-dependent manner. Moreover, RT–PCR results showed that transcriptional level of ERα expression was significantly down-regulated by LCT and CP as in case of E2. Taken together, these results indicate that LCT and CP may possess estrogenic potentials to stimulate ovarian cancer cells expressing ERs via an ER-dependent manner, and these collective results confirm the carcinogenicity of these SP, LCT and CP, in ER-positive cells or tissues.
232 ADDITION OF VERY LOW AMOUNTS OF SERUM (ESTRUS COW SERUM) IMPROVES IN VITRO EMBRYO PRODUCTION IN DAIRY CATTLE
Authors: Mullaart E. , Dotinga F. , Ponsart C. , Knijn H. , Schouten J.
Published: December 2014

Improving the efficiency of the in vitro production (IVP) process is very important because it results in more embryos to be used in breeding programs or as commercial service. At CRV, a culture medium consisting of SOF with amino acids and BSA is used. In the past, richer culture media were used with 10% fetal calf serum combined with BRL cell co-culture. Although the efficiency of the IVP process of these media was good, these rather high serum concentrations were quite often related to large offspring syndrome (LOS). The switch to a culture system without serum resulted in a significant reduction in LOS but also in a reduction of embryo yield. The aim of the present study was to investigate the effect of adding low amounts of serum to the culture medium on efficiency of embryo production. Immature cumulus-oocyte complexes (COC) were recovered from ovaries 6 to 8 h upon slaughter. The COC were matured in vitro in TCM199/FCS/LH/FSH supplemented with cysteamine (0.1 mM). Subsequently, matured oocytes were fertilised with frozen-thawed gradient-separated semen and further cultured for 7 days in SOFaaBSA. The SOF medium contained either 0 (control), 0.1, 0.5, or 1.0% oestrus cow serum (ECS). Embryos development was scored at Day 7. Three replicates were performed and results were analysed by chi-square analyses. The results clearly show that adding ECS significantly improved embryo production (Table 1). Interestingly, already very low amounts (0.1%) of serum gave a significant increase in embryo percentage. In conclusion, addition of very low amounts of ECS (0.1%) is beneficial for embryo production, resulting in significantly higher embryo production (from 19 to 27%). In a subsequent field trial with OPU-derived embryos, the effect of addition of 0.1% ECS on birth weight (LOS) of the calves has to be investigated.

Table 1.Percentage of blastocysts at Day 7 after culture in SOF medium with different amounts of serum
241 EFFECT OF PRODUCTION EFFICIENCY IN THE LIKELIHOOD OF PREGNANCY OF IN VITRO-DERIVED BOVINE EMBRYOS
Authors: Feres L. F. , Camargo L. S. A. , Palhao M. P. , Brandao F. Z. , Viana J. H. M.
Published: December 2014

Improving in vitro culture systems to optimize embryo yield has been a major research goal. The relationship between the efficiency of embryo production systems and the pregnancy outcomes, however, remain controversial. The aim of the present study was to evaluate the likelihood of pregnancy of in vitro-produced embryos derived from batches with different relative efficiency indexes. Data of 702 ovum pick-up (OPU) and in vitro embryo production (IVEP) sessions, and of 2456 embryo transfers, recorded from 2008 to 2012, were evaluated. All donors were from the same herd, and were of the same breed (Gir, Bos indicus), as well as the semen used for IVF. The cumulus-oocycte complex (COC) recovery and IVEP were performed by the same team, in a single IVF laboratory, and using standard medium and procedures. Only data from embryos transferred as fresh were used, and records from 97 OPU/IVEP sessions in which no embryo was produced, or embryos were frozen or discharged due to lack of recipients, were discharged. The remaining 605 sessions were stratified in quartiles (I to IV, each one corresponding to 25% of total data) according to COC production of the donors, or stratified in ranges (0–25%, 26–50%, 51–75%, and 76–100%) according to COC quality (percentage of viable COC or of grade I COC) and to embryo production efficiency endpoints (cleavage rate, blastocyst rate). Pregnancy rates were compared among quartiles or ranges by the chi-square method. On average, the Gir donors produced 24.8 ± 0.6 COC per OPU, from which 14.4 ± 0.4 were classified as viable (57.8%), and 3.2 ± 0.1 as grade I (12.9%). On average 6.1 ± 0.2 embryos (morulas and blastocysts) were produced per OPU per donor, and mean pregnancy rate was 30.9%. As expected, donors with greater total COC yield (quartile I) also produced more viable oocytes (25.5 ± 0.7 v. 15.7 ± 0.3, 10.5 ± 0.2 and 5.8 ± 0.2), more COC grade I (4.8 ± 0.4 v. 3.9 ± 0.3, 2.6 ± 0.2 and 1.6 ± 0.1), and more embryos (9.0 ± 0.4 v. 6.9 ± 0.3, 5.0 ± 0.2 and 3.3 ± 0.1) than donors from quartiles II, III, or IV, respectively (P < 0.0001). Nevertheless, there was no difference (P > 0.05) in pregnancy rates for embryos produced from donors ranked in the different quartiles (30.9 v. 29.3, 31.5, and 30.5% for quartiles I to IV, respectively). Similarly, there was no difference (P > 0.05) in the pregnancy rate of embryos derived from OPU sessions in which there was a high or low percentage of viable or grade I COC. In vitro production efficiency (cleavage and blastocyst rates) also had no effect (P > 0.05) on further pregnancy rates. In conclusion, these results suggest that there is no relationship among the average number or quality of the COC recovered by OPU, the efficiency of IVEP, and the likelihood of pregnancy of in vitro-derived embryos.Research was supported by Fazendas do Basa, CNPq, and Fapemig.
261 HIGH NUTRITION DURING EARLY LIFE IMPROVES REPRODUCTIVE POTENTIAL OF HOLSTEIN BULLS
Authors: Dance A. , Thundathil J. , Wilde R. , Blondin P. , Kastelic J.
Published: December 2014

The objective was to determine effects of early-life nutrition on reproductive potential of Holstein bulls. Twenty-six bull calves were randomly allotted to 3 groups and fed ~70, 100, or 130% of National Research Council recommendations for both energy and protein from 2 to 31 wk; thereafter, all were fed a 100% diet (adequate vitamins and minerals were constantly available) until slaughter (72 wk). Growth rate, scrotal circumference, and paired testis volume were determined every 4 wk during the differential feeding period. Once scrotal circumference reached 26 cm, semen collection was attempted (to confirm puberty). Post-pubertal semen quality was monitored; once bulls were producing 70% morphologically normal sperm, semen was cryopreserved (programmable freezer). These semen samples (3 bulls per group and 3 ejaculates per bull) were used in an IVF system to determine the fertilizing ability of sperm and developmental competence of resulting embryos. In the low-, medium-, and high-nutrition groups, respectively, bulls were 369.3 ± 14.1, 327.4 ± 9.5, and 324.3 ± 11.7 days at puberty; their paired testes weights were 561.6 ± 23.1, 611.1 ± 59.1, and 727 ± 33.0 g; cleavage rates were 68.0 ± 8.7, 77.1 ± 3.5, and 68.7 ± 4.5%; and blastocyst rates were 31.5 ± 5.6, 41.4 ± 4.9, and 33.7 ± 4.6% (no significant differences among the 3 nutrition groups for rates of cleavage or blastocyst formation). We concluded that early-life supplementation of energy and protein hastened puberty (P < 0.05) and increased testicular size (P < 0.05), without compromising sperm fertilizing ability. Therefore, feeding dairy bull calves a high plane of nutrition early in life is recommended as a management strategy to improve their reproductive potential.
269 EFFECT OF DIFFERENT COLLECTION FREQUENCY INTERVALS ON THE QUALITY OF SEMEN OF FRENCH BULLDOGS
Authors: Deorce C. B. , Leite F. L. G. , Loureiro B.
Published: December 2014

Dogs produce fewer sperm than other species. Furthermore, for French Bulldogs, anatomical peculiarities, low libido, and increased sensitivity to stress could cause further reductions in sperm count. The objective was to compare effects of semen collection at 24- versus 48-h intervals on semen quality of French Bulldogs. Five purebred French Bulldogs, 19 to 48 months old, were subjected to 5 semen collections (24 h apart). After a 30-day rest, collections were repeated, but the interval between collections was 48 h. Semen was collected (all 3 fractions) by digital manipulation without female stimuli. Volume was measured in a 20-mL syringe. Sperm concentration was determined with a Neubauer counting chamber. Motility and vigor were evaluated with a coverslipped drop of semen on a slide (preheated to 37°C). Motility was expressed as a percentage of motile sperm, and vigor was classified on a scale of 1 to 5. Morphology was evaluated by the panoptic method; 100 cells were counted and results expressed as the percentage of normal or defective cells. Effects of collection interval were analysed using PROC MIXED of SAS (animal as subject and collection as a repeated measure), with collections 2 to 5 compared with collection 1 (using the DIFF option). For collection every 24 h, the third, fourth, and fifth sperm collections were lower than the first collection for volume (10.4 ± 1.1, 8.3 ± 1.1, 5.6 ± 1.1, 3.5 ± 1.1, 2.4 ± 1.1 mL), concentration (437 ± 24, 448 ± 24, 370 ± 24, 322 ± 27, 258 ± 31 ×106 sperm mL–1), vigor (4.6 ± 0.2, 4.2 ± 0.2, 3.6 ± 0.2, 3.7 ± 0.2, 3 ± 0.2), and morphologically normal sperm (82 ± 2.2, 83 ± 2.2, 72 ± 2.2, 68 ± 2.5, 66 ± 2.9%). However, when the interval was increased to 48 h, only the fourth and fifth collections were lower (P < 0.05) than the first for volume (11.8 ± 1.1, 10.2 ± 1.1, 8.8 ± 1.1, 6.6 ± 1.1, 2.5 ± 1.1 mL), concentration (447 ± 24, 410 ± 24, 407 ± 24, 322 ± 24, 241 ± 31 ×106 sperm mL–1), vigor (5 ± 0.2, 4.8 ± 0.2, 4.4 ± 0.2, 4.2 ± 0.2, 4 ± 0.2), and sperm with normal morphology (92 ± 2.2, 90 ± 2.2, 87 ± 2.2, 80 ± 2.2, 81 ± 2.9%). Motility decreased (P < 0.05) following the fourth collection at 24-h intervals and decreased (P < 0.05) after the third collection at 48-h intervals. With a 24-h interval, 4 dogs had <60% motility lower at the fifth collection, whereas only 2 dogs had motility <60% on the fifth collection at 48-h intervals. In conclusion, semen collected at 48-h intervals was of better quality than semen collected at 24-h intervals.