bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of low protein diets on growth performance, carcass traits and ammonia emission of barrows and gilts
Authors: Madrid J. , Martínez S. , López C. , Orengo J. , López M. J. , Hernández F.
Published: December 2012

The aim of the present research was to determine the effect of low protein diets on growth performance, carcass traits and ammonia emission from barrows and gilts during the growing and finishing periods. For each period, three diets were formulated. A control diet (C) with 160 and 155 g crude protein (CP)/kg for the growing and finishing, respectively, a medium level diet (M) and a low level diet (L) with 10 and 20 g CP/kg less than the C, respectively. All the diets were supplemented with crystalline amino acids on an ideal protein basis. Barrows (90) and gilts (90) were sorted by bodyweight and sex and housed in three identical manually ventilated chambers. No significant effects of diet on pig performance were observed. In the finishing period, barrows were heavier (P < 0.001) and had higher average daily gain (P < 0.01) and average daily feed intake (P < 0.01) than gilts. Pigs fed the L diet had higher backfat thickness than those fed the other diets in the growing period (P < 0.05). Backfat thickness was higher (P < 0.01) in barrows than in gilts. Muscle depth decreased in pigs fed the L diet in the finishing period. Muscle depth was not affected (P > 0.05) by sex. Average ammonia emissions during the last 6 days were 117.7, 94.2 and 85.5 mg ammonia/kg pig.day for pigs fed the C, M and L diets, respectively. The results show that a reduction in dietary CP of 10 g/kg, accompanied by supplementation with crystalline amino acid reduced ammonia emission by 19.9% with no detrimental effects on growth performance and carcass characteristics, and a reduction of 20 g/kg reduced ammonia emission by 27.3% with no detrimental effects on growth performance but reduced muscle depth at slaughter.
Spawning aggregation of the lanternfish Diaphus danae (family Myctophidae) in the north-western Coral Sea and associations with tuna aggregations
Authors: Flynn Adrian J. , Paxton John R.
Published: December 2012

A spawning aggregation of the lanternfish Diaphus danae in the Coral Sea, that is fed on by spawning aggregations of yellowfin and bigeye tuna, has been sampled by mid-water trawling for the first time. Males and females in the aggregation occurred at a ratio of 23 to 1 and occupied two non-overlapping size classes (males 71.2–95.1-mm standard length (SL), females 99.0–121.4-mm SL). Hydrated oocytes with single oil droplets, which indicated imminent spawning, were in higher proportion in the first trawl (2134 hours to 2234 hours) than in the final trawl (0324 hours to 0424 hours) through the aggregation. Maximum estimated female D. danae fecundity (25 803) and gonadosomatic index (34.01) were higher than for any other lanternfish species recorded. Bigeye tuna (Thunnus obesus) individuals collected from aggregations were estimated to have 81–319 D. danae specimens in their stomachs. The annual Coral Sea D. danae aggregation is the only confirmed lanternfish spawning aggregation in Australian waters. The D. danae spawning aggregation provides a rich, transient foraging resource for spawning bigeye and yellowfin tuna in the Coral Sea, these being the only known tuna spawning aggregations in the Eastern Tuna and Billfish Fishery.
Early development of the porcine embryo: the importance of cell signalling in development of pluripotent cell lines
Authors: Hall Vanessa Jane
Published: December 2012

Understanding the cell signalling events that govern cell renewal in porcine pluripotent cells may help improve culture conditions and allow for establishment of bona fide porcine embryonic stem cells (pESC) and stable porcine induced pluripotent stem cells (piPSC). This review investigates cell signalling in the porcine preimplantation embryo containing either the inner cell mass or epiblast, with particular emphasis on fibroblast growth factor, SMAD, WNT and Janus tyrosine kinases/signal transducers and activators of transcription signalling. It is clear that key differences exist in the cell signalling events that govern pluripotency in this species compared with similar embryonic stages in mouse and human. The fact that bona fide pESC have still not been produced and that piPSC cannot survive in culture following the silencing or downregulation of the reprogramming transgenes suggest that culture conditions are not optimal. Unravelling the factor/s that regulate pluripotency in porcine embryos will pave the way for future establishment of stable pluripotent stem cell lines.
Effect of superstimulatory treatments on the expression of genes related to ovulatory capacity, oocyte competence and embryo development in cattle
Authors: Barros Ciro M. , Satrapa Rafael A. , Castilho Anthony C. S. , Fontes Patrícia K. , Razza Eduardo M. , Ereno Ronaldo L. , Nogueira Marcelo F. G.
Published: December 2012

Multiple ovulation (superovulation) and embryo transfer has been used extensively in cattle. In the past decade, superstimulatory treatment protocols that synchronise follicle growth and ovulation, allowing for improved donor management and fixed-time AI (FTAI), have been developed for zebu (Bos indicus) and European (Bos taurus) breeds of cattle. There is evidence that additional stimulus with LH (through the administration of exogenous LH or equine chorionic gonadotrophin (eCG)) on the last day of the superstimulatory treatment protocol, called the ‘P-36 protocol’ for FTAI, can increase embryo yield compared with conventional protocols that are based on the detection of oestrus. However, inconsistent results with the use of hormones that stimulate LH receptors (LHR) have prompted further studies on the roles of LH and its receptors in ovulatory capacity (acquisition of LHR in granulosa cells), oocyte competence and embryo quality in superstimulated cattle. Recent experiments have shown that superstimulation with FSH increases mRNA expression of LHR and angiotensin AT2 receptors in granulosa cells of follicles >8 mm in diameter. In addition, FSH decreases mRNA expression of growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) in oocytes, but increases the expression of both in cumulus cells, without diminishing the capacity of cumulus–oocyte complexes to generate blastocysts. Although these results indicate that superstimulation with FSH is not detrimental to oocyte competence, supplementary studies are warranted to investigate the effects of superstimulation on embryo quality and viability. In addition, experiments comparing the cellular and/or molecular effects of adding eCG to the P-36 treatment protocol are being conducted to elucidate the effects of superstimulatory protocols on the yield of viable embryos.
Dynamic regulation of sperm interactions with the zona pellucida prior to and after fertilisation
Authors: Gadella B. M.
Published: December 2012

Recent findings have refined our thinking on sperm interactions with the cumulus–oocyte complex (COC) and our understanding of how, at the molecular level, the sperm cell fertilises the oocyte. Proteomic analyses has identified a capacitation-dependent sperm surface reordering that leads to the formation of functional multiprotein complexes involved in zona–cumulus interactions in several mammalian species. During this process, multiple docking of the acrosomal membrane to the plasma membrane takes place. In contrast with the dogma that the acrosome reaction is initiated when spermatozoa bind to the zona pellucida (ZP), it has been established recently that, in mice, the fertilising spermatozoon initiates its acrosome reaction during its voyage through the cumulus before it reaches the ZP. In fact, even acrosome-reacted mouse spermatozoa collected from the perivitelline space can fertilise another ZP-intact oocyte. The oviduct appears to influence the extracellular matrix properties of the spermatozoa as well as the COC. This may influence sperm binding and penetration of the cumulus and ZP, and, in doing so, increase monospermic while decreasing polyspermic fertilisation rates. Structural analysis of the ZP has shed new light on how spermatozoa bind and penetrate this structure and how the cortical reaction blocks sperm–ZP interactions. The current understanding of sperm interactions with the cumulus and ZP layers surrounding the oocyte is reviewed with a special emphasis on the lack of comparative knowledge on this topic in humans, as well as in most farm mammals.
106 STUDIES OF NUCLEAR ARCHITECTURE IN MAMMALIAN PRE-IMPLANTATION EMBRYOS AND EMBRYONIC STEM CELLS USING SUPER-RESOLUTION FLUORESCENCE MICROSCOPY
Authors: Popken J. , Sterr M. , Markaki Y. , Cremer M. , Beck A. , Dahlhoff M. , Habermann F. A. , Fezert P. , Guengoer T. , Reichenbach M. , Wuensch A. , Wolf E. , Zakhartchenko V. , Cremer T.
Published: December 2012

Three-dimensional (3-D) super-resolution fluorescence microscopy has allowed major progress in studies of the functional nuclear organization (Markaki et al. 2010 Cold Spring Harb. Symp. Quant. Biol. 75, 475–492; Markaki et al. 2012 Bioessays 34, 412–426). We have exploited these new possibilities to explore nuclear organization at different stages of bovine pre-implantation development (4-cell, 8-cell, 16-cell, morula, and blastocyst stage). In particular, we studied the topography of RNA polymerase II and the distribution of transcriptionally competent and noncompetent chromatin using antibodies against H3K4me3 and H3K27me3, respectively. For comparison, we have started analyses of mouse pre-implantation embryos and embryonic stem cells as well. Our results support the chromosome territory-interchromatin compartment (CT-IC) model (Cremer and Cremer 2010 Cold Spring Harb. Perspect. Biol. 2, a003889; Cremer et al. 2012 In: Epigenetic Regulation and Epigenomics 451–483). In all cell types, the nuclear space is occupied by chromosome territories (CTs; Koehler et al. 2009 Exp. Cell Res. 315, 2053–2063), the interchromatin compartment (IC), and one or several nucleoli. The CTs are built up from interconnected, megabase-sized chromatin domains (CDs). These ~1-Mbp CDs may consist of a series of ~100-kbp CDs (Cremer et al. 2000 Crit. Rev. Eukaryot. Gene Expr. 10, 179–212), which globally form a compact chromatin core surrounded by a layer of decondensed chromatin, called the perichromatin region. Current evidence supports the hypothesis that the perichromatin region represents the nuclear compartment, where transcription, co-transcriptional splicing, DNA-replication, and DNA-repair take place (Rouquette et al. 2010 Int. Rev. Cell Mol. Biol. 282, 1–90). The IC provides a contiguous, crowded compartment, which starts with channels at nuclear pores and pervades the chromatin compartment both between and within CTs. Small-scale chromatin loops of the perichromatin region can protrude into the interior of IC channels allowing direct contacts between CDs in cis and trans. At other sites the IC expands to wider, chromatin-free lacunas with splicing speckles and nuclear bodies. This model is in line with a fractal higher-order chromatin arrangement at all levels from CTs, chromosome arms and bands to ~1 Mbp CDs organized as fractal globules (Mirny 2011 Chromosome Res. 19, 37–51).
This work is supported by the DFG (ZA 425/1-3, CR 59/29-2).
114 THE LIPID COMPOSITION OF THE FOLLICULAR FLUID ON DAY 6 POST-AI MAY BE ASSOCIATED WITH THE GESTATIONAL SUCCESS IN NELORE COWS
Authors: Scolari S. , D'Alexandri F. L. , Maturana M. , Binelli M.
Published: December 2012

The follicular fluid (FF) microenvironment consists of serum components and follicular cell secretions, which are essential for oocyte maturation, preparing it for fertilization. Although very important, the biochemical composition of the follicular fluid is still poorly characterised. The hypothesis is that the lipid composition of the follicular fluid on Day 6 post-artificial insemination (AI) is different between cows diagnosed as pregnant (P) or not pregnant (NP) on Day 30 post-AI. The objectives were to (1) evaluate the influence of follicle aspiration procedure performed on Day 6 post-AI on pregnancy rates and (2) characterize the lipid profile of the FF collected on Day 6 in pregnant (P) and nonpregnant (NP) cows, in order to associate the amount of molecules in the FF with the probability of conception. Eighty-two multiparous Nelore cows, between 40 and 45 days post-partum and body condition score between 4 and 6 (1 to 9 scale) were used. Fixed-time AI was performed in all the animals and 6 days post-AI the animals were divided in control group, in which animals were not manipulated (CG, n = 41), and aspiration group (AG, n = 41) from which follicular fluid was aspirated from the largest follicle present in either one of the ovaries. The FF was centrifuged and stored at –80°C. Pregnancy diagnosis was performed by ultrasonography 30 days post-AI. Results showed pregnancy rates of 53% for the AG and 56% for the CG, suggesting no detrimental effect of the follicle aspiration procedure on the success of pregnancy (Kruskal-Wallis test). A pool of the FF samples from the P and NP cows was analyzed by mass spectrometry (MS), using the high-resolution shotgun lipidomics (HRSL) approach in order to compare the lipid profile between groups. Despite the fact that HRSL is not quantitative but qualitative, the general levels of the different lipid classes can be compared between groups of study and the differences in the abundance of the ions of each lipid class can be expressed as percentage. Taking this into account, in negative mode MS, the NP group showed an increase of 300% in the levels of free fatty acids such as palmitic, oleic, and linoleic acids compared to the P group as well as an increase of 50% in the levels of phospholipids and two new species of phospholipids (phosphatidylcholine and phosphatidylserine) could be observed. Furthermore, animals from the P group showed an increase of 130% in the content of structural fatty acids such as ceramids and triacylglycerols. Our findings suggest that there is a difference between the amount of lipid classes and in the lipid species origins present in the follicular fluid of cows that were P or NP on Day 30 post-AI.
140 EFFECT OF A NOVEL BOVINE EMBRYO CULTURE MEDIUM TO IMPROVE BLASTOCYST DEVELOPMENT
Authors: Gonçalves R. F. , Figueiredo C. , Achilles M. A.
Published: December 2012

There are still immense differences in the quality of in vitro-produced embryos compared to their in vivo-generated counterparts. These differences include a higher sensitivity of in vitro-produced embryos towards cryopreservation. The quality of such embryos has been evaluated using various parameters like morphological examination, assessment of total cell numbers, or pregnancy rates after transfer. In the present study, the effects of glycine, alanine, taurine, and glutamine addition to SOF (Achilles Genetics culture medium, Achilles Genetics®, Garça, SP, Brazil) on the in vitro development (cleavage and blastocyst rates) and quality (total cell and apoptotic cell numbers) of bovine embryos were determined. Ovaries of Nelore cows were obtained from a slaughterhouse. Cumulus–oocyte complexes (COC) were collected from follicles ≥4 mm in diameter, matured in TCM-199, and fertilized with frozen–thawed Nelore bull semen (IVF = Day 0). On Day 1, presumptive zygotes were cultured in SOF supplemented with fetal bovine serum (FBS, group 1, n = 550) or in Achilles Genetics culture medium (SOF supplemented with Achilles Mixture and FBS, group 2, n = 557) at 38.5°C and 5% CO2 in air until Day 9. Embryos were evaluated during culture: at Day 3 cleavage rates, at Day 7 blastocyst rates, and on Day 9 hatching rates. Experiments were replicated 5 times, analysed using ANOVA, followed by a comparison of means by Tukey test (P ≤ 0.05). Blastocysts at Day 8 from Group 1 (n = 75) and Group 2 (n = 75) were fixed and permeabilized for TUNEL assay (DeadEndTM Florimetric TUNEL System, Promega, Madison, WI, USA), according to the manufacturer instructions. Total cell number, apoptotic cell number, and apoptotic cell index (calculated by dividing the apoptotic cell number by total cell number) were analyzed by analysis of variance and means were compared by Student Newman Keuls test. The threshold of significance was set at P ≤ 0.05. Cleavage rates were 79.2 ± 2.5 for group 1 and 91.0 ± 2.5 for group 2. Blastocyst and hatching rates (calculated on the total of zygotes) for group 2 (47.4 ± 2.8; 82.1 ± 1.5) were significantly greater than for group 1 (39.8 ± 2.8; 74.3 ± 1.5). The total cell numbers were not different (P > 0.05) between group 1 (112.7 ± 2.9) and group 2 (111.1 ± 2.7). Blastocysts from group 2 showed lower (P < 0.05) number of apoptotic cells (10.7 ± 1.2) than those from group 1 (20.9 ± 1.2). These results indicate that the addition of glycine, alanine, taurine, and glutamine to SOF (Achilles Mixture) may be an important energy source for the bovine blastocyst and could act synergistically to enhance embryo development to the hatching stage and embryo quality.
Financial support from CNPq and FAPESP.
173 USE OF CORPUS LUTEUM AREA AS A PREDICTOR OF ONGOING FUNCTIONAL LUTEOLYSIS IN DAIRY HEIFERS
Authors: Pugliesi G. , Pinaffi F. L. V. , Beg M. A. , Ginther O. J.
Published: December 2012

The beginning of functional luteolysis in cattle ranges from Days 16–19 (Day 0 = ovulation). During functional luteolysis, luteal size and blood flow decrease in heifers. The wide range of beginning of luteolysis and the low sensitivity of quick progesterone (P4) assays preclude targeting an individual for a specific period of luteolysis. The present study aimed to evaluate the changes in area and blood flow of corpus luteum (CL) during spontaneous luteolysis and test the hypothesis that a decrease in CL area (cm2) is more efficient in targeting the luteolytic period than using a specific day postovulation. Blood sampling and ultrasound scanning of CL were done every 12 h from Days 13–22 in dairy heifers (Holstein) and was used for a retrospective study (Experiment 1; n = 6 heifers). The P4 concentrations, maximum CL area, and the percentage of CL area with coloured signals of blood flow were centralized to 12-h sample when the end of luteolysis (P4 < 1 ng mL–1) was detected. The percentage decrease in CL area and blood flow between Day 13 and the 12-h sample before the end of luteolysis were calculated. In Experiment 2, a blood sampling and transrectal ultrasonography of CL were performed every 8 h starting on Day 14 to determine the beginning and end of functional luteolysis (n = 20 heifers). The hour of detection of luteolysis was defined as the scan at 8-h intervals when maximum CL area decreased by 25 or 12.5% on Day 14. Based on P4 concentrations, the preluteolytic, luteolytic, and postluteolytic periods in each heifer were identified. The area and blood flow were measured by a duplex B-mode and pulse-wave colour Doppler ultrasound instrument. In Experiment 1, the retrospective study indicated a 25% decrease in CL area and 65% decrease in blood flow 12 h before the end of functional luteolysis. In Experiment 2, the 25% and 12.5% CL area decrease from Day 14 occurred, respectively, on Days 18.1 ± 0.6 and 17.2 ± 0.5 postovulation. The hypothesis that a decrease in CL area is more efficient in targeting ongoing luteolysis than using a specific day postovulation was supported. This was indicated by the greater (P < 0.05) frequency of heifers detected in luteolytic period (12 of 20 heifers) using the criteria of a 12.5% decrease in CL area than using a specific day (Table 1). Using a day postovulation for obtaining heifers in the luteolytic phase was only 0 to 30% effective for targeting luteolysis. In conclusion, the technique of using an ultrasonic 12.5% reduction in CL area is a good tool to detect ongoing luteolysis and may be useful as an experimental design to study factors related to the luteolytic period in cattle.

Table 1.Percentage values of heifers in preluteolytic, luteolytic, or postluteolytic periods according to a decrease in CL area criteria or a day postovulation

Supported by grants from FAPESP (2012/04004-8 to GP).
194 A POTENTIAL ENDOCRINE-DISRUPTING CHEMICAL, 4-NONYLPHENOL, STIMULATED THE OVARIAN CANCER CELL GROWTH BY UPREGULATING CELL CYCLE VIA AN ESTROGEN RECEPTOR SIGNALING PATHWAY IN CELLULAR AND ANIMAL MODELS
Authors: Park M.-A. , Choi K.-C.
Published: December 2012

Nonylphenol (NP), a subset of the alkylphenols, is used as a surfactant (surface-active agent). Nonylphenol has been widely detected in waste water, which is a concern since it is toxic to many aquatic organisms. Also, NP has been classified as an endocrine-disrupting chemical because of weak ability to mimic oestrogen and in turn disrupt the natural balance of hormones in organisms. In this study, the estrogenic effect of NP was examined in ovarian cancer BG-1 cells expressing high levels of oestrogen receptors (ER) by a cell viability assay and semiquantitative reverse-transcription PCR (RT-PCR) in cellular and animal models of xenografted mice. A treatment of BG-1 cells with NP (10–8 to 10–5 M) resulted in an increase in their cell proliferation as 17-β oestradiol (E2) did. In addition, NP upregulated the expression levels of cell-cycle regulating genes (i.e. cyclin D1, which is a downstream target of ER). As a result, NP stimulated the proliferation of BG-1 cells via an increase of the cell cycle progression. In a xenografted mouse model transplanted with BG-1 ovarian cancer cells, E2 or NP treatment significantly increased the tumour proliferation compared as a vehicle (corn oil) for 10 weeks. These results were identified by the measurement of tumour volume and histological analysis on tumour masses by using H&E staining and BrdU incorporation assay. These results support that NP may have a weak estrogenic activity and increase risk of cancer proliferation in oestrogen-dependent cancers such as ovarian cancer.
This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Ministry of Education, Science and Technology (MEST) of Korea government (no. 2011-0015385).
196 EXPRESSION OF TISSUE-TYPE PLASMINOGEN ACTIVATOR IN OOCYTES AND CUMULUS CELLS FROM MOUSE, PIG, AND COW
Authors: Izquierdo-Rico M. J. , Moreno-Manrique M. , García-Vázquez F. A. , Sánchez-Calabuig M. J. , Coy P.
Published: December 2012

Tissue-type plasminogen activator (tPA) is one of the components of the plasminogen-plasmin (PLG-PLA) system, better known as fibrinolytic system for its role in the blood clot lysis. It has been demonstrated recently that the activation of plasminogen into the protease plasmin during the sperm-oocyte interaction in the pig and cow decreases the percentages of penetration and increases monospermy (Mondéjar et al. 2012). However, in the mouse species, it was showed that PLG-PLA system enhances fertilization (Huarte et al. 1993). Expression of tPA has been described in rat oocytes (Bicsak et al. 1989) and cumulus cells (Ny et al. 1987; O’Connell et al. 1987), but no clear evidence about its expression in mouse, pig, and cow oocytes or cumulus cells is available. We hypothesised that differences in the effect of PLG-PLA system on fertilization results between the species mentioned above could be related to differences in tPA expression. The aim of this study was the detection of mRNA encoding tPA in oocytes and cumulus cells in mouse, pig, and cow by molecular analysis. Total RNA was obtained from oocytes and cumulus cells and cDNA was synthesised with an oligo-dT as primer. These cDNAs were used as template in RT-PCR amplifications using specific primers designed based on the GenBank sequence for Mus musculus, Sus scrofa, and Bos taurus tPA (NM_ 008872, NM_214054, NM_174146, respectively). The results of this study showed a different expression in the 3 studied species. In mouse, amplicon encoding tPA was detected in oocytes and cumulus cells. In cow and pig, tPA transcripts were obtained only in cumulus cells. The relation between the differences in the tPA expression pattern and the role of PLG-PLA system on fertilization remains to be investigated.
This study was supported by MICINN (AGL2009-12512-C02-01-02).
202 EXPRESSION OF APOLIPOPROTEIN B mRNA IN THE PORCINE OVIDUCT
Authors: Acuña O. S. , Ramón M. C. , Ruano M. J. , Avilés M. , Izquierdo-Rico M. J.
Published: December 2012

Apolipoprotein B (APOB) has been described as the primary apolipoprotein component of low-density lipoproteins (LDL) and is absolutely required for its formation. However, other functions related with the male reproductive process may be attributed to this protein. In mouse, expression of the Apob gene has been described in the testis and epididymis. Moreover, male mice heterozygous for a targeted mutation of the Apob gene exhibit severely compromised fertility. However, the relation between this protein and the fertilization process remain to be investigated. The objective of this study was to determine whether APOB mRNA is expressed in the porcine oviduct and to analyse its expression during the different phases of the oestrus cycle. Oviducts were obtained from sows slaughtered at a local abattoir and were classified based on follicular morphology: prepuber (containing only follicles 1 to 2 mm in diameter), preovulatory (containing 6 to 12 follicles 8 to 12 mm in diameter), post-ovulatory (containing 6 to 12 hemorrhagic corpora), and luteal phase (containing 6 to 12 corpora lutea). Total RNA was extracted from scraps of isthmus-ampullar junction mucosa using RNAqueous® kit (Ambion, Austin, TX, USA) according to the manufacturer’s instructions, and cDNA was synthesised with an oligo (dT) as primer. This cDNA was used as template in RT-PCR amplifications and qPCR using specific primers designed based on GenBank sequence for Sus scrofa APOB (L11235; Fw: caacaactcaaggcccagat, Rv: ctgaattttgccgttgattc for RT-PCR and Fw: tggccagagctgtccaaggga and Rv: ccactggagctctcagcct for qPCR). The amplification by RT-PCR resulted in a 567-bp amplicon. The sequencing of this PCR product has shown a high similarity with APOB sequences of different mammalian species. On the other hand, analysis by quantitative PCR did not reveal any differences in the expression of APOB between the samples of various stages of the oestrus cycle. In conclusion, the APOB mRNA is present in the porcine oviduct and its level of expression is similar in different phases of the cycle. The role played by this protein in the oviduct remains to be clarified.
This study was supported by MICINN (AGL2009-12512-C02-01-02).
207 CIRCULATORY microRNA SIGNATURES IN FOLLICULAR FLUID IN RELATION TO THE GROWTH STATUS OF BOVINE OOCYTES
Authors: Sohel M. M. H. , Salilew-Wondim D. , Hölker M. , Rings F. , Schellander K. , Tesfaye D.
Published: December 2012

Cell-to-cell communication within the follicle involves many signalling molecules, and this process is believed to be mediated by secretion and uptake of exosomes that contained several bioactive molecules including circulatory microRNAs (miRNA). The present study was conducted to investigate the circulating miRNA expression pattern in exosome and nonexosomal portion of follicular fluid (FF) in follicles with fully grown or growing oocytes. For this, the FF and cumulus–oocyte complexes (COC) were retrieved from 5- to 8-mm individual follicles from ovaries obtained from local abattoir. Then, the oocytes were subjected to brilliant cresyl blue (BCB) staining and classified as BCB+ (fully grown oocytes) and BCB– (growing oocytes). Accordingly, the corresponding FF was classified as BCB+ and BCB– based on their oocyte source. Following this, the exosomes were trapped from each FF categories using ExoQuick™ (SBI System Bioscience). Thus, total RNA was isolated from exosomal and nonexosomal portion of the FF using miRNeasy mini kit (Qiagen) and subjected to miRNA expression studies. The human miRCURY LNA™ Universal RT miRNA PCR array system (Exiqon) was used for miRNA expression profiling. Data analysis was performed using a comparative threshold cycle (ΔCT) method. The results revealed that 26 miRNAs were found to be differentially expressed (fold change ≥2 and P < 0.05) between the exosomal portion of FF from fully grown and growing oocyte groups. Among these, 17 miRNA including miR-608, miR-654-5p, miR-640, miR-582-5p, miR-449b, miR-155 were upregulated and 9 miRNA including miR-373, miR-526b*, miR-33a*, miR-30b, miR-29a* were downregulated in exosomal portion of FF of growing oocyte groups. The ingenuity pathway analysis of genes predicted to be targeted by those miRNAs were found to be involved in WNT/β catenine, purine metabolism, protein ubiquitination, and cAMP-mediated signalling pathways. Similarly, 36 miRNA were differentially expressed between the non-exosomal portion of FF of fully grown and growing oocytes. From those, 27 miRNA including let-7i*, miR-328, miR-223, miR-19b-1*, miR-423-5p, miR-29c, miR-659 were upregulated, whereas the expression level of 9 miRNA including miR-381, miR-18a*, miR-30e*, miR-934, and miR-302c was downregulated in the nonexosomal portion of FF of growing oocyte groups. In addition, the ingenuity pathway analysis indicated that the genes predicted to be targeted by these miRNA were found to be involved in NRF2-mediated oxidative stress response, tight junction signalling, and protein ubiquitination pathways. In conclusion, this study detected the presence of exosome or non-exosome-mediated circulation of miRNA in the bovine follicular fluid and oocyte growth-dependent variation of circulatory miRNA in the follicular environment.
210 DEFINING THE MINIMAL PROMOTER REGION OF BOVINE VASA HOMOLOGUE (Bvh) BY IN SILICO ANALYSIS
Authors: Malaver-Ortega L. F. , Sumer H. , Verma P. J.
Published: December 2012

The DEAD box polypeptide 4, DDX4 or VASA, is a highly conserved gene that encodes a putative RNA helicase with the motif DEAD (Asp-Glu-Ala-Asp). Although little is known about its role in germ cell genesis, VASA is one of the earliest, specialised markers of primordial germ cell (PGC) specification. Furthermore, this process of specification has been recapitulated to some degree in vitro using embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) in mice and humans, using VASA expression as one of the criteria for differentiation and sorting of the differentiated cells. In order to establish a system for isolation and tracking of bovine iPSC undergoing germ cell specification, we analysed all regulatory elements in the 5-kb upstream region [RC 5083564–5088564 Bos taurus (Hereford) chromosome 20 genomic scaffold: NW_003104511.1] of the bovine VASA homologue (Bvh) locus, which is thought to be the putative promoter region of Bvh, and in the in vivo validated promoter regions, for the corresponding homologous genes in human and mouse. We performed the analysis using 2 different approaches: at the sequence level, by orthologous promoter alignment of transcription factor (TF) binding sites (TFBS) using DiAling®, and at the functional level, by functional unit analysis (complex model) using Frameworker® (Genomatix, Munich, Germany). The initial DiAling® analysis did not produce similarities between the 3 analysed species. In contrast, using the complex analysis of functional units, we identified 85 single elements common to all 3, and 795, 482, and 129 models composed of 2, 3, and 4 elements, respectively. The number of models was reduced to 3 [M1, M2, and M3 (P = 4.8 × 10–11)] by increasing the number of TF (each model composed of 6 different elements). As a result, members of SOX/SRY-sex/testis determining and related HMG box factor family related with germ cell specification, pluripotent-related factors such as members of the octamer binding protein family, and TFs common to numerous vertebrate genes such as homeobox transcription factors were identified (Table 1). Based on these results, we determined a region of approximate 0.6 kb upstream of the Bvh gene, which encloses a core of TFBS conserved at the functional level between species. We propose that this sequence is the best candidate for driving the expression of reporter genes under Bvh promoter control.

Table 1.Six element models
212 GENE EXPRESSION PROFILE OF PROTAMINES AND TRANSITION NUCLEAR PROTEINS IN BOVINE TESTIS
Authors: Ferraz M. A. M. M. , Simões R. , Milazzotto M. P. , Visintin J. A. , Assumpçäo M. E. O. A.
Published: December 2012

During spermiogenesis, haploid spermatids undergo complex morphological and physiological changes to differentiate into spermatozoa. These processes include chromatin remodelling mediated by the replacement of histones through transition nuclear proteins (Tnp) and protamines (PRM). These proteins have the function to compact and protect the chromatin, exerting great influence on human and mouse fertility. Several studies have demonstrated the positive relationship between unregulated production of protamines and infertility. In humans and mice, PRM1/PRM2 ratio is important to predict fertility. When the 1 : 1 ratio (ideal) is disrupted, in these species, the sperm DNA integrity is altered. Most of the infertility cases caused by protamine deficiency, in humans, are related to PRM2 (Aoki et al. 2005 Hum. Reprod. 20, 1298–1306). Expression levels of PRM2 have been correlated with the DNA damage in mice; however, its role on bull fertility is still unclear. The aim of this study was to determine the relative expression of PRM1, PRM2, PRM3, Tnp1, and Tnp2 in bovine testis. Evaluate the expression of these genes is of utmost importance to understand the role of each protamine during bull spermatogenesis. Testis from post-pubertal bulls (n = 10) were obtained from the slaughterhouse. The RNA extraction and cDNA synthesis were performed using commercial kits. The gene expression (P1, P2, P3, Tnp1, and Tnp2) was determined by real-time RT-PCR using bovine specific primers and β-actin as endogenous controls. A relative expression software tool (Pfaffl et al. 2002 Nucleic Acid Res. 30) was used to compare all samples of each group. The quantification of mRNA relative expression demonstrated a higher expression of PRM1, the relative expression of PRM2 was lower than the relative expression of PRM1 (5.008 ± 1.501 × 23.906 ± 6.174, respectively; P < 0.05). There was no difference between the relative expression of the mRNA for PRM2, Tnp1, and Tnp2 (5.008 ± 1.501, 5.023 ± 1.064, 4.266 ± 1.170, respectively; P > 0.05). The PRM3 mRNA had the lowest relative expression (2.003 ± 0.663). The PRM1/PRM2 ratio found in this study was 4.77 : 1.00. Differently from human and mice, the lower expression of PRM2 mRNA may be an evolutionary adaptation of the bull spermatogenesis, which makes the bovine sperm less susceptible to protamination changes that lead to infertility. More studies are being performed by our research group to evaluate the function of these proteins in bulls. It is fundamental to understand the biology of bovine spermiogenesis, providing knowledge to increase the fertility and be able to elucidate the evolutionary mechanisms that may have caused the possible loss of functionality of PRM2 in bulls.
This work was supported financially by FAPESP (09/17035-6 and 07/55294-8).
215 INVESTIGATION OF A PREFERENTIALLY UPREGULATED GENE CLUSTER IN DAY 7 BOVINE EMBRYOS DERIVED FROM RNA SEQUENCING DATA
Authors: Al Naib A. , Mamo S. , Lonergan P.
Published: December 2012

Successful establishment and maintenance of pregnancy requires optimum conceptus-maternal cross talk. Despite significant progress in our understanding of the temporal changes in the transcriptome of the uterine endometrium, we have only a rudimentary knowledge of the genes and pathways governing growth and development of the bovine conceptus. A recent RNA sequencing study from our group (Mamo et al. 2011 Biol. Reprod. 85, 1143–1151) described the global transcriptome profile of the bovine conceptus at 5 key stages of its pre- and peri-implantation growth (Days 7, 10, 13, 16, and 19) using RNA sequencing techniques. One cluster of genes (n = 1680 transcripts) was preferentially upregulated at Day 7 and subsequently downregulated, suggesting that these genes might be markers of blastocyst formation. The objective of this study was to characterise the pattern of expression of these genes before Day 7 (i.e. from the zygote to blastocyst stage). The list of genes was submitted to DAVID (Database for Annotation, Visualisation, and Integrated Discovery) to take advantage of available ontology information contained therein. The expression of 9 genes belonging to ontologies specifically related to embryo developmental (GINS1, TAF8, ESRRB, NCAPG2, SP1, XAB2, CDC2L1, MSX1, and AQP3) plus Na/K ATPase, a gene previously known to be involved in blastocoe formation, was studied by quantitative real-time PCR (QPCR) in 6 replicate pools of 5 embryos produced by maturation, fertilization, and embryo culture in vitro. Stages studies included immature and mature oocyte, zygote, 2- cell, 4-cell, 8-cell, 16-cell, morula, blastocyst, and hatched blastocyst. In addition, in vivo derived Day 13 and Day 16 embryos were included as controls to confirm down-regulation after Day 7. Data were analysed using the GLM procedure of SAS. The QPCR expression data supported the RNA Seq data in that expression of all transcripts was downregulated after the blastocyst stage. Expression before the blastocyst stage was characterised by 1 of 3 broad patterns: (1) the expression was of maternal origin where the expression was very high up to 8-cell stage and decreased subsequently (MSX1), (2) the expression was of embryonic origin being low up to the 8-cell stage and increasing thereafter (TAF8, ESRRB, AQP3, and Na/K ATPase), or (3) static or decreased expression from oocyte to the maternal-zygotic transition followed by increased expression from the 16-cell stage (GINS1, NCAPG2, SP1, XAB2, and CDC2L1). In conclusion, the genes identified in this cluster, despite having different patterns of expression before the blastocyst stage, may represent markers of blastocyst formation in cattle given their downregulation subsequently.
Supported by Science Foundation Ireland (07/SRC/B1156).
225 DEVELOPMENTAL COMPETENCE AND QUALITY OF PIG EMBRYOS OBTAINED FROM STANDARD IN VITRO FERTILIZATION OR INTRACYTOPLASMIC SPERM INJECTION
Authors: Grad-Mandryk I. , Kosenyuk J. , Gajda B.
Published: December 2012

In vitro production of porcine embryos is still relatively inefficient. The main reasons for this limited performance are polyspermy after IVF and the poor developmental ability of obtained zygotes. Intracytoplasmic sperm injection (ICSI) is one possible solution to eliminate polyspermy. The aim of this study was to compare the developmental competence of pig zygotes, total cell number, and DNA fragmentation of pig blastocysts derived from IVF or ICSI. Cumulus–oocyte complexes were obtained by aspiration from antral follicles of ovaries collected from slaughtered gilts. The oocytes were then cultured in modified TC-199 medium to metaphase II for 42 h. Semen for IVF was incubated in modified capacitation medium (M199) for 1 h. The sperm fraction (1 × 106 cells mL–1) was introduced into droplets containing oocytes, and then gametes were co-incubated for 4 h in modified TC-199 medium. Intracytoplasmic sperm injection was performed using a mechanical micromanipulator (Research Instruments Limited, Cornwall, UK). Micromanipulation was carried out in modified NCSU-37 medium. The tails of spermatozoa were broken, and then single spermatozoa were aspirated into the injection pipette. The oocyte was fixed by a holding pipette, and the sperm head was then introduced into the oocyte cytoplasm. Presumptive zygotes were cultured in vitro for 144 h in NCSU-23 medium. The embryo quality criteria were developmental competence (morula and blastocyst rates), total cell number per blastocyst, and degree of apoptosis assessed by TUNEL staining. Data were analysed by chi-squared test. The experiment was performed on 136 zygotes (6 replicates) obtained after IVF and 83 zygotes (4 replicates) obtained after ICSI. Percentages of embryos developed to the morula and blastocyst stages were 42.3 ± 6.1 and 28.8 ± 4.7 after IVF, respectively, and 51.7 ± 15.4 and 34.5 ± 18.9 after ICSI, respectively (no differences were observed). Significant differences were noticed in total number of cells per blastocyst between embryos after IVF and ICSI (33.7 ± 5.39 v. 22.8 ± 3.22; P < 0.01). However, there was no difference in the degree of apoptosis between IVF and ICSI embryos (5.14 ± 3.49 and 6.14 ± 4.88, respectively). Our preliminary studies demonstrated a higher proportion of cell numbers in IVF-derived embryos compared with those produced by ICSI, but the developmental competence and degree of apoptosis, as evaluated by the TUNEL method, in both groups were comparable.
This study was funded by project N N311 516140 by the NCN, Poland.
228 IN VITRO EMBRYO PRODUCTION AFTER EXPOSURE OF BOVINE OOCYTES TO DIFFERENT TRANSPORTATION MEDIA AND PERIODS: PRELIMINARY RESULTS
Authors: Rossi T. C. , Rossi J. , Miyauchi T. M. , Fernandes C. A. C. , Camargo L. S. A. , Viana J. H. M. , Palhao M. P.
Published: December 2012

This study was designed to evaluate the effect of different oocyte transportation media and time. Immature oocytes were recovered from slaughterhouse bovine ovaries. Oocytes (n = 492) of quality grades I to III were randomly allotted to one of the following transportation media based on TCM-199, either buffered with HEPES (control) or buffered with NaHCO3 and added with FSH/LH (maturing). Both media were supplemented with pyruvate, penicillin G (10 000 IU), streptomycin (0.05 mg mL–1), and 10% fetal bovine serum (FBS). In control medium, the oocytes were kept for 1 or 8 h at 37°C, and thereafter were transferred to maturing medium until the maturation period (24 h) was completed, under controlled atmosphere (5% CO2) and temperature, in an incubator. The maturing oocytes were distributed in two types of equipment developed for oocyte and embryo transportation, with and without 5% CO2, and kept for 1, 8, or 24 h before maturation or fecundation (IVF) procedures. The oocytes kept for 24 h in the transportation device were placed directly for IVF. All procedures used for in vitro maturation, IVF, and in vitro culture were the same as those adopted for commercial in vitro embryo production at Biotran LTDA (Alfenas, Minas Gerais, Brazil). The cleavage rate was evaluated on Day 3 post-insemination, and the blastocyst production was evaluated on Day 7. The statistical model included the main effects of treatment (control and maturing with or without 5% CO2), time, replicate, and the interaction of media × time. Data (3 replicates) were analysed by ANOVA and differences were identified by Tukey’s test. The time before in vitro maturation at the incubator negatively (P < 0.007) affected cleavage rates (76.4 ± 16.9 v. 58.8 ± 13.2 and 52.2 ± 18.5%, respectively, for 1, 8, and 24 h). However, treatment had no effects (P = 0.3) on cleavage (66.9 ± 15.0, 57.8 ± 19.7, and 67.7 ± 19.8% for the control and maturation with and without 5% CO2, respectively). Similarly, blastocyst production rates differed (P < 0.04) between 1 h (33.4 ± 14.7) and 24 h (19.3 ± 17.0%), whereas blastocyst production at 8 h did not show significant effects (19.3 ± 17.0). Although it was not significant (P > 0.05), only 14.1% of the oocytes kept in medium 2 with a controlled atmosphere for 24 h became embryos. This difference was probably related to the trend in treatment effect (P = 0.06) and the lower rate of embryo production (22.2 ± 13.6%) in this treatment compared with the control (33.8 ± 18.1%) and maturation without 5% CO2 (29.8 ± 17.3%). These results showed that 24 h of transportation is detrimental for the oocyte development potential and that the buffered medium used in this study with 5% CO2 did not efficiently maintain embryo production.
Supported by CNPq, CAPES, FAPEMIG, and Biotran LTDA.
248 DISTRIBUTION OF OVULATION-INDUCING FACTOR IN MALE REPRODUCTIVE TISSUES OF LLAMAS
Authors: Bogle O. A. , Singh J. , Adams G. P.
Published: December 2012

We have recently reported that the ovulation-inducing factor (OIF) in seminal plasma is the well-conserved neurotrophin, nerve growth factor. This protein has been identified in the ejaculates of a variety of species using both in vivo and in vitro bioassays. The role of OIF in the ejaculate is evident in camelids. Irrespective of whether a male is intact or vasectomised, ejaculation of this protein during copulation stimulates an increase in circulating LH in the female, which subsequently leads to ovulation. The purpose for OIF in species that are spontaneous ovulators, however, remains unclear. We hypothesise that OIF production is restricted to the secretory epithelium of accessory glands of the male, specifically the prostate gland, because of its conservation among mammals. The objective of the present study was to determine the sites of OIF secretion in the llama that contribute to high concentrations found in the ejaculate. The mucosa, submucosa, and muscularis layers of the reproductive organs (testis, epididymis, ductus deferens, and penis) and accessory sex glands (ampulla, prostate gland, and bulbourethral gland) from mature male llamas (n = 2) were probed with polyclonal rabbit anti-nerve growth factor. The secondary antibody used was horseradish peroxidase-conjugated goat-anti-rabbit immunoglobulin G. The distribution and intensity of OIF-secreting cells were identified and compared among tissues using a four-point scale. The quantity of OIF detected was highest in mucosal prostatic cells. The protein was strongly localised in the apical region of epithelial cells and frequently found within the glandular lumen. The seromucus-secreting epithelium of ampullary glands showed no reaction to OIF, whereas the bulbourethral gland epithelium showed an intermediate response. The presence of OIF was not detected in the epithelium of the penile urethra, epididymis (caput, corpus, or cauda), or seminiferous tubules. A diffuse distribution of the protein was, however, detected in the stroma of the testes, epididymides, and accessory glands. We conclude that cells that secrete OIF are distributed unequally among accessory sex glands in llamas. The bulk of OIF is produced by the prostate gland, with minimal to no contributions by the bulbourethral and ampullary glands. Future studies involve species comparisons and relating the size of the prostate gland to the concentration of OIF within an ejaculate.
Research supported by the Natural Sciences and Engineering Research Council of Canada.
249 BIOPHYSICAL AND BIOCHEMICAL CHARACTERISTICS OF ALPACA SEMEN AFTER COLLECTION BY ELECTROEJACULATION
Authors: Rodriguez J. , Huanca W. , Ramos M. , Vasquez M. , Espinoza J.
Published: December 2012

The collection of alpaca semen presents difficulties because of the characteristics of mounts and copulation time in addition to the high viscosity of semen. The biophysical and biochemical characteristics of alpaca semen were investigated. Semen samples from four adult males from 6 to 7 years of age were collected using an electroejaculation technique. The biophysical parameters measured in seminal plasma included volume, color, sperm concentration, motility, and viscosity, and the biochemical parameters included pH and glucose, calcium, triglyceride, cholesterol, high-density lipoprotein cholesterol, total protein, albumin, alanine transaminase, alkaline phosphatase, and γ-glutamyltranspeptidase concentrations. The mean time for semen collection was 10.3 ± 1.5 min; the volume varied from 1.2 to 3.8 and the colour from clear to milky, and pH was 7.4 ± 0.03. Mean concentration of spermatozoa was 69.3 ± 193 million mL–1, and motility was 52.4 ± 9.7%. Biochemical parameters were glucose (mg dL–1) 8.22 ± 0.77; cholesterol (mg dL–1) 79.78 ± 5.64; triglycerides (mg dL–1) 44.12 ± 7.38; high-density lipoprotein cholesterol (mg dL–1) 4.73 ± 0.30; total protein (g dL–1) 2.36 ± 0.15; albumin (g dL–1) 0.97 ± 0.33; calcium (mg dL–1) 11.77 ± 1.74; alanine transaminase (U L–1) 17.92 ± 9.09; alkaline phosphatase (U L–1) 288.76 ± 279.59; and γ-glutamyltranspeptidase (U L–1) 89.62 ± 39.09. These results provide information on the biophysical and biochemical characteristics of alpaca semen collected by electroejaculation, with variable results in the enzymatic components.