bluuebunny/crossref_metadata_2025 huggingface dataset:
170 PREGNANCY RATES OF NONCYCLING RECIPIENT MARES TREATED WITH A NEW FORMULATION OF LONG-ACTING PROGESTERONE
Authors: Castro Chaves M. M. B. , Schutzer C. G. , Alvarenga M. A.
Published: August 2010

The mare being a seasonal polyestrous animal is a limiting factor for maintenance of embryo transfer programs during the entire year.At the beginning and end of the breeding season, the percentage of recipients showing normal estrous cycles may be low compared to donors. Exogenous progesterone has been administered to acyclic mares to allow them to be used as recipients. Noncyclic recipient mares treated once a week with long-acting progesterone (P4) have been widely used in Brazil in equine embryo transfer (ET) programs. P4LA was developed for weekly administration of 1.5 g of P4 to acyclic recipients until approximately 120 days of pregnancy. The aim of the present experiment was to evaluate the pregnancy rates of noncyclic recipients treated with a new formulation of long-acting P4 developed in our laboratory that allows a longer interval between treatments (14 days). Noncyclic mares (n = 36) were used as recipients between August and September of 2008 (transition between winter and spring in Brazil). Noncyclic mares were treated with 5 mg of estradiol benzoate during 2 consecutive days followed by the application of 10 mL (i.m.) of P4LA containing 300 mg mL-1 of P4, for a total P4 dose of 3 g. Embryos were transferred between 6 to 12 days after P4LA injection. The same dose of P4LA was repeated on the day of ET and every 14 days until 110 days after pregnancy diagnosis. One hundred fifty-five cyclic recipients were used as a control group, with embryos transferred 4 to 8 days after ovulation. No differences were observed (P > 0.05) when comparing pregnancy rates from acyclic recipients treated with P4LA (82%, 56/68) or cyclic recipients (88%; 137/155) on Day 15. The rate of embryonic loss at 50 days was similar (P > 0.05) for noncycling (11%) and cycling recipient mares (10%). The results obtained in the present study demonstrated that the new formulation of P4LA containing 300 mg mL-1 of P4 and administered every 14 days was effective in maintaining pregnancy in noncyclic recipient mares, allowing a larger treatment interval and longer window of time to transfer embryos.
197 SEMEN COLLECTION, TESTICULAR VOLUME, AND PRELIMINARY ASSAY OF COOLED SEMEN OF CAPTIVE PERUVIAN VICUNAS (VICUGNA VICUGNA MENSALIS)
Authors: Enciso M. A. , Rodríguez C. , Huanca W. , Valdivia M.
Published: August 2010

The vicuna (Vicugna vicugna) is a wild species of South American camelid (SAC) that lives in the Andean region from Peru to Argentina. It is classified at Low Risk by the International Union for Conservation of Nature (IUCN); however, it is a threatened species and conservation-related studies are needed. Assisted reproduction techniques are potentially useful as a conservation tool (Durrant B 2009 Theriogenology 71, 113-122), and the development of a sperm-based genome resource bank for subsequent use in AI in the vicuna is a priority. Although semen cryopreservation has been successfully applied in many species, difficulties have been encountered in SAC species. The aim of the present study was to obtain semen samples, measure testicular volume, and evaluate a protocol for cooling semen of Peruvian vicuna (V. v. mensalis). Six adult males, located at the Zoo Cerrito de la Libertad (Huancayo, Peru; n = 2), and Quimsachata Research Station (Puno, Peru; n = 4) were used. For semen collection, an electroejaculation procedure was carried out under general anesthesia. Semen was collected (n = 16) using a 2-cm-diameter probe with 3 ventral electrodes. Progressive electrical stimulation from 2 V to 12 V was applied in a protocol divided in 3 series: series 1, with 10 stimuli in 2 V, 4 V, and 6 V; series 2, with 10 stimuli in 4 V, 6 V, and 8 V; and series 3, with 10 stimuli in 10 V and 12 V. Fifteen ejaculates were collected. Semen samples (n = 6) were cooled to 4°C in egg yolk-Tris-citrate-glycerol extender and evaluated at 2, 4, 8, and 24 h. Seminal values of the ejaculates were as follows (mean ± SEM): volume = 0.85 ± 0.12 mL; pH = 7.09 ± 0.16; nonprogressive sperm motility = 28.08 ± 3.56%; sperm concentration = 166.29 ± 60.92 × 104 sperm/mL; and sperm normal morphology = 62.77% ± 1.96. Testicular volume was 22.95 ± 2.28 cm3 and did not show a correlation with seminal volume and sperm concentration (r = 0.06 and r = 0.16, respectively; P < 0.05). For cooling, the extender we used was able to maintain viability for 24 h: motility = >30%, viability = >25%, and normal morphology = ≈35%. The present results demonstrate the utility of our improved electroejaculation protocol in the vicuna, and the seminal values were similar to those of a previous study (Giuliano SM et al. 2002 Theriogenology 57, 583 abst) with domestic SAC. Also, we showed that vicuna semen could be successfully cooled and stored for 24 h in an egg yolk-Tris-citrate buffered extender.

T. Huanca and M. L. Gonzáles (Quimschata Res. Stat.), G Rojas and L. Bermúdez (Huachipa Zoo), and S. Gonzáles (Zoo Cerrito de la Libertad). Reseach Supported by CONCYTEC (Fellowship to MAE).
198 EXOGENOUS CONTROL OF FOLLICULAR WAVE EMERGENCE IN WOOD BISON (BISON BISON ATHABASCAE)
Authors: McCorkell R. B. , Paziuk W. , Smart L. , Woodbury M. R. , Adams G. P.
Published: August 2010

Two methods for synchronizing ovarian follicular development in both wood bison (Bison bison athabascae) and plains bison (Bison bison bison) were tested as part of a project to conserve wood bison through the application of advanced reproductive technologies. A secondary objective was to test the effect of a long-acting neurolept tranquilizer, pipothiazine palmitate, on ovarian function in bison. Female wood bison (4 years old; n = 14) and plains bison (2-8 years old, n = 10), previously conditioned to daily examination in a chute, were divided randomly into 3 groups in which 1) ovarian follicles ≥5 mm were ablated by ultrasound-guided transvaginal follicle aspiration, 2) 5 mg of estradiol-17β in canola oil was given i.m. or 3) no treatment was given (control). The experiment was conducted in 3 replicates so that each animal rotated through each of the groups. Half of the bison, blocked by subspecies and treatment group, were given a single dose (150 mg) of pipothiazine palmitate i.m. The ovaries were examined daily by transrectal ultrasonography beginning 4 days before treatment and continuing until the dominant follicle of a new wave reached a diameter of 10 mm. No effect of treatment with pipothiazine palmitate was detected for any end point. In addition, no differences were detected between wood and plains bison for any end point. Consequently the pipothiazine palmitate and subspecies treatment groups were collapsed for further analyses. The interval and variation in the interval to new follicular wave emergence were compared by ANOVA. Wave emergence was detected on Day 1.0 ± 0.2 (mean ± SEM; Day 0 = day of treatment) in the follicle ablation group, and was earlier (P < 0.05) than in both the estradiol (Day 3.3 ± 0.3) and control (Day 4.0 ± 0.4) groups. The interval to follicle wave emergence was least variable in the follicle ablation group (P < 0.05) and tended to be less variable in the estradiol group (P = 0.09) than in the control group (residuals, 0.1 ± 0.04, 1.0 ± 0.2, and 1.6 ± 0.3 days, respectively). Ovulations occurred subsequent to estradiol administration in 10 of 23 (43%) bison. In conclusion, ovarian follicular wave emergence can be synchronized in bison during the anovulatory season. Follicular ablation consistently shortened and decreased the variability in the interval to new wave emergence. The synchronizing effect of estradiol was confounded by the induction of ovulation. Progesterone will be added in future studies to control the ovulatory effect of estradiol. Pipothiazine palmitate had no discernable effect on ovarian function and maybe useful in reducing the effects of handling stress on untrained animals.

Supported by a grants from the Advancing Canadian Agriculture and Agri-Food Fund, the Agri-Food Innovation Fund, Parks Canada, the World Wildlife Fund, and the Northwest Territories.
20 PREGNANCY RATES IN SUCKLED BEEF COWS SYNCHRONIZED WITH PROGESTERONE INTRAVAGINAL DEVICES AND RECEIVING eCG FOURTEEN DAYS AFTER BREEDING
Authors: Cutaia L. , Ramos M. , Chesta P. , Bó G. A.
Published: August 2010

An experiment was designed to test the following hypotheses: (1) the administration of 400IU of eCG (Novormón, Syntex SA, Buenos Aires, Argentina) 14 days after fixed-time artificial insemination (FTAI) increases pregnancy rates in cross-breed Zebu × Bonsmara suckled cows synchronized with progesterone releasing devices (DIB 0.5, 0.5 g of progesterone, Syntex SA); and (2) pregnancy rates in cows treated with estradiol or GnRH, combined with a DIB device, are not different. Primiparous suckled cows (n = 260), between 60 and 120 days postpartum (DPP) and with a body condition score between 2.0 and 2.5 (1 to 5 scale) were used. At the beginning of the experiment (Day 0), all cows were examined by rectal palpation (only 11% had a CL), received a DIB device, and were randomly allocated to 1 of 3 treatment groups. Cows in the E2 group received 2 mg of estradiol benzoate (EB) intramuscularly (i.m.) at DIB insertion and those in the two GnRH groups received 100 μg of gonadorelin (GDR Gonasyn, Syntex SA) at the same time. On Day 7, DIB devices were removed and all cows received 500 μg of cloprostenol (Ciclase DL, Syntex SA) plus 400 IU of eCG (i.m.). Cows in the E2 group received 0.5 mg of estradiol cypionate (ECP, Cipiosyn, Syntex SA) and underwent FTAI administered by one technician, using semen from 1 of 2 bulls between 52 and 56 h later. Cows in the GnRH groups received a dose of 100 μg of gonadorelin (full GnRH group) or 50 μg of gonadorelin (1/2 GnRH group) at the time of the FTAI (52 to 56 h after DIB removal). Fourteen days after FTAI cows in the 3 groups were further subdivided (3 × 2 factorial) to receive or not 400 IU of eCG i.m. Pregnancy was determined 30 days after FTAI using real-time ultrasonography. Data were analyzed by logistic regression, taking into account the effect of the synchronization treatment, eCG or noteCG, 14 days after FTAI and sire used. No differences in pregnancy rates were found (P = 0.16) among treatment groups (E2: 39/87, 43.7%, full GnRH: 38/92, 41.3%, and 1/2 GnRH: 25/81, 30.9%). However, there was a significant (P = 0.02) effect of eCG treatment 14 days after FTAI (eCG: 60/127, 47.2% v. no eCG: 41/133, 30.8%). Furthermore, bull A (77/155, 47.1%) resulted in higher pregnancy rate (P = 0.009) than bull B (28/107, 26.2%), but there were no significant interactions between bull and synchronization treatment or eCG administration 14 days after FTAI (P > 0.1). We conclude that the application of 400 IU of eCG 14 days after FTAI increases pregnancy rates in suckled Bos taurus × Bos indicus primiparous cows with fair to poor body condition score synchronized with progesterone releasing devices. Furthermore, the use of estradiol ester or GnRH at DIB insertion and removal did not affect pregnancy rates.
206 BIOPSY ASSESSMENT OF BOVINE OVARIAN TISSUE XENOGRAFTED UNDER RENAL CAPSULE OF SEVERE COMBINED IMMUNODEFICIENCY MICE (PRELIMINARY RESULTS)
Authors: Bezerra M. B. , Macedo M. F. , Matos M. C. , Mingoti G. Z. , Garcia J. M. , Vicente W. R. R. , Pacheco M. R.
Published: August 2010

Xenografting of ovarian tissue has been considered to be a promising technique for conservation of a wide range of endangered breeds and wild animals. However, this technique requires immunosuppressed animals as recipients. Reduction of their number is desirable according to ethical principles of animal experimentation and to the costs associated with the acquisition and maintenance of severe combined immunodeficiency (SCID) or nude mouse strains. This work aims to evaluate 4 grafts per animal biopsied under the left renal capsule of SCID mice on Days 18, 30, 45, and 60 post-grafting. Thus, 8 SCID male mice that were previously orchiectomized and xenografted with 4 pieces of bovine ovarian tissue under the renal capsule were anaesthetized intraperitoneally with 10 mL kg-1 of body weight of tribromoethanol (2.5%), shaved, and antiseptically submitted to a left sublumbar incision of the skin and muscle until the identification of the left kidney containing grafts and its exposure for a biopsy procedure. One graft per animal was gently punched with a 2-mm-diameter dermatologic needle, collected with iris forceps, and immediately immersed in Bouin’s solution for histology and staining with Masson’s trichrome. Our preliminary results indicated that animals submitted to xenografting of ovarian tissue and animals that reached the first observation day (2 animals from 8) presented small subcapsular bleeding during the biopsy procedure that was easily controlled by hemostasis with local compression using sterile gauzes. Biopsied grafts presented adequate dimensions for evaluation. Ovarian tissues interacted with the surrounding renal parenchyma, were well vascularized, and contained preantral follicles. The biopsy of 1 of 4 grafts in 2 mice is part of an ongoing study. However, the first attempts on Day 18 seemed encouraging.

This work was supported by a grant from the National Counsel of Technological and Scientific Development (CNPq-Brazil) and financial support of State of São Paulo Research Foundation. (FAPESP-07/04507-1)
211 EFFECT OF ESTRADIOL BENZOATE ON THE FIRST WAVE DOMINANT FOLLICLE IN HOLSTEIN HEIFERS
Authors: Eslami Mohsen , Niasari-Naslaji Amir , Nikjou Darab , Bolourchi Mahmoud , Mousavi Sadegh
Published: August 2010

The objective of this study was to evaluate the effect of different doses of estradiol benzoate on the first wave dominant follicle in Holstein heifers. Estrous cycle of cyclic Holstein heifers (n = 16; 13.84 ± 0.44 months of age; 335.5 ± 6.65 kg) was synchronized using CIDR (14 days; VetoCIDR®, Aburaihan, Tehran, Iran) in association with GnRH analogue (12.5 μgi.m. Vetaroline®, Aburaihan) on Day 6 and PGF2 analogue (500 μg; Vetaprost®, Aburaihan) on day 13 after CIDR insertion. On Days 5 to 7 of the ensuing cycle (Day 0 of the experiment), heifers were assigned into 4 experimental groups. Heifers in Groups 1, 2, and 3 (n = 4 in each group) received 2, 5, and 10 mg of estradiol benzoate (i.m. Vetastrol®, Aburaihan), respectively, concurrent with 100 mg of progesterone (i.m. Vetagesterone®, Aburaihan). Heifers in the control group (n = 4) received distilled water as a placebo intramuscularly. Ovarian follicles were monitored daily, from Days 0 to 16, using an ultrasound scanner (Sonovet 2000, Korea) equipped with a 7.5-MHz transrectal probe. Initiation of follicle regression was defined when there was a sudden decrease of 1 to 2 mm followed by a progressive decrease in the diameter of the ovarian follicle. Emergence of anew follicle wave was defined as the day that the dominant follicle was, retrospectively, identified at a diameter of 4 to 5 mm. Data were analyzed using Kruskal-Wallis one-way ANOVA in SAS (SAS Institute, Cary, NC). On Day 0, there was a functional CL (19.62 ± 0.57 mm) and a growing dominant follicle (11.25 ± 0.62 mm) on the ovary of all heifers. The dominant follicle regressed on Day 3.5 ± 1.04, 3.2 ± 0.85, 2.7 ± 0.75, and 8.5 ± 0.50 in Groups 1, 2, 3, and control, respectively (P < 0.05). A new follicle wave emerged on Day 7.7 ± 0.25, 8.2 ± 0.48, 9.7 ± 1.44, and 7.2 ± 1.11 in Groups 1, 2, 3, and control, respectively (P > 0.05). Estrus was detected on Day 12.0 ± 1, 11.7 ± 0.34, 13.0 ± 0.00, and 15.2 ± 1.75 in Groups 1, 2, 3, and control, respectively (P > 0.05). The interval between steroid injections (Day 0) and ovulation were 13 ± 0.41, 13 ± 0, 14.3 ± 0.33, and 16.2 ± 1.75 days for Groups 1, 2, 3, and control, respectively (P > 0.05). Interovulatory intervals were 18.7 ± 0.33, 18.7 ± 0.33, 20.7 ± 0.67, and 20.5 ± 1.55 days for Groups 1, 2, 3, and control, respectively (P > 0.05). In conclusion, estradiol benzoate induced early regression of the dominant follicle of the first wave but did not affect the interval between steroid treatment and ovulation in Holstein heifers.

This research was funded by Aburaihan Pharmaceutical Company (Tehran, Iran).
213 ANALYSIS OF STEROID HORMONE RECEPTOR GENE EXPRESSION IN THE BOVINE OVARY AFTER LASER-CAPTURE MICRODISSECTION
Authors: Kenngott R. , Palma G. , Wendl M.J. , Vermehren M. , Sinowatz F.
Published: August 2010

Developmental processes in complex organs like the ovary are difficult to study in terms of a biochemical and molecular biological analysis. Laser-assisted microdissection allows the efficient and precise capture of single cells or groups of cells of an organ within the context of time and space and permits their subsequent molecular characterization. Together with real-time PCR techniques, it is now feasible to study gene expression in defined cell populations of complex tissues, but it is essential to create standards optimized for fixation, preparation, and isolation of RNA, reverse transcription reaction, and real-time PCR protocol for every tissue of interest. The aim of our study was to develop protocols for a precise analysis of estrogen receptor alpha (ER-α) and progesterone receptor (PR) in defined compartments of the ovary (granulosa cells, theca interna cells, zona vasculosa, and zona parenchymatosa of the stroma). Additionally, the receptor proteins were localized by immunohistochemistry. A special focus was put on the question of how formalin fixation and paraffin embedding influences the quality of the isolated RNA from microdissected material, which was used for quantitative reverse transcription-PCR (qRT-PCR). Quality and quantity of total RNA extracted from formalin-fixed, paraffin-embedded (FFPE) sections and from material immersed in RNAlater® (Ambion, Foster City, CA, USA) was checked using an Experion automated electrophoresis system (Bio-Rad, Munich, Germany). The RNA quality indicator for microdissected material was between 6 and 7, and for RNAlater® material was 9 or better. Online qRT-PCR using the iCycler SYBR GreenTM protocol (Bio-Rad) was performed in a 96-well plate. Primer pairs were chosen to generate PCR products between 100 bp (ER-α) and 140 bp (PR), as RNA recovered from FFPE-laser microdissected material was expected to be considerably fragmented. Using GenEx software (BioEPS, Freisling, Germany), we showed that the expression of mRNA for PR was much stronger in the theca interna than in the 3 other compartments. Estrogen receptor alpha, on the other hand, was nearly exclusively expressed in the zona parenchymatosa and zona vasculosa of the stroma. Our results show that cells obtained after laser microdissection from FFPE ovarian material can be successfully used for subsequent real-time PCR, despite the fact the RNA quality indicator number of the isolated RNA was usually comparatively low. The data of our immunohistochemical analysis support the expression data of our RNA studies. In conclusion, laser-capture microdissection in combination with quantitative PCR is a reproducible and reliable technique for quantification of a small number of cells from FFPE material.

We gratefully acknowledge the continuous support by the DFG-Graduiertenkolleg 1029 and the BMBF (ARG 08/013).
222 CHARACTERIZATION OF CO-CULTURES OF GRANULOSA CELLS AND THECA CELLS AS EXPLANTS OF BOVINE OVARY FOLLICULAR WALLS
Authors: Salles L. P. , Vasconcelos R. B. , Oliveira e Silva I. , Gulart L. V. M. , Torres F. A. G. , de Souza D. K. , Rosa e Silva A. A. M.
Published: August 2010

Granulosa cells (GC) and theca cells (TC) luteinize in culture because of the presence of serum in the medium and, as a consequence, the production of progesterone (P4) increases and that of 17β-estradiol (E2) decreases. The follicular phase of the bovine estrous cycle is characterized by the presence of E2, as well as aromatase activity, that is turned off in the atresia or in the luteal phase of the bovine estrous cycle. We propose to characterize 2 co-culture systems utilizing slices of follicular walls (explants) containing both GC and TC and to cultivate them in nondefined medium (NDM; TCM199+serum) or defined medium (DM; α MEM+polyvinyl alcohol). To prepare the explants, 80 follicles measuring 4 to 6 mm in diameter were selected. Concentrations of P4 and E2 were evaluated by RIA after 24, 48, and 72 h of culture and values were corrected by tissue weight (10 mg). Morphological analysis of the explants was stained with hematoxylin and eosin. The total mRNA from GC-TC explants was purified and the expression of the following genes was estimated by RT-PCR: bovine 3 beta-hydroxysteroid dehydrogenase (HSD3B1), cholesterol side-chain cleavage cytochrome P450 (CYP11A1), 17|3-hydroxylase (CYP17), aromatase cytochrome P450 (CYP19), steroidogenic acute regulatory protein (StAR), follicle-stimulating hormone receptor (FSHr), and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPD) as internal control. The results were analyzed by two-way ANOVA followedby Bonferroni least-significant tests. Microscopic analyses showed that cells cultured in NDM lost their polyhedral shape and acquired a fibroblast-like form. Defined medium maintained GC morphology with low cytoplasm:nucleus ratio and GC-GC contact, similar to in vivo cells. In NDM, the concentration of P4 increased as opposed to that of E2, which decreased after 48 h of culture. In DM, the concentrations of P4 and E2 were maintained in the first 48 h of culture. The level of P4 in NDM was higher than in DM during all periods tested. Our data show that CYP11A1 expression remains absent in NDM for follicles explants that initially did not express CYP11A1, whereas in DM the expression of this gene appears after 24, 48, or 72 h of culture, evidence that indicates rescue from atresia. It is important to notice that gene expression of steroidogenic enzymes in DM were higher than in NDM. Very low levels of CYP11A1 and CYP19 were observed in the NDM group. On the other hand, in the DM group there was a progressive increase of all the steroidogenic enzymes and FSHr, which reached a peak at 48 h of culture. In conclusion, our results suggest that DM showed a stimulating effect on steroidogenic activity of GC-TC co-culture similar to that observed in vivo for functional growing follicle, whereas in NDM the explants seemed to mimic luteinizing follicles.

Grants from FAP-DF, Finatec, CNPq, and Capes.
235 EXPRESSION OF PLURIPOTENCY-ASSOCIATED GENES IN BOVINE FETAL FIBROBLAST CELLS AND ADIPOSE-DERIVED STEM CELLS
Authors: Coley L. W. , Godke R. A. , Bondioli K. R.
Published: August 2010

The extraordinary nature of embryonic stem cells (ESC) lends them the remarkable ability to give rise to all the cell types of a mammalian organism, an attribute known as pluripotency. The pluripotent state of ESC is dependent on the expression of the genes Oct-4, Nanog, and Sox-2, which have also been identified as the key transcriptional regulators of pluripotency. Until recently, Oct-4, Nanog, and Sox-2 were believed to be expressed solely in ESC; however, studies have reported the expression of these genes in some sources of adult stem cells (ASC) of both the mouse and pig. In the current study, we examined cells derived from bovine adipose tissue and fetal fibroblast for the expression of Nanog and Sox-2. Cells were isolated from the adipose tissue of an adult cow and the skin of 2 male fetuses, approximately 70 and 80 d old, and cultured through 6 passages. Total RNA was isolated from each of the 3 cell lines at passages 2, 4, and 6 using TRI Reagent®. Using the Bio-Rad iScript™ cDNA Synthesis Kit, the resulting RNA products were transcribed into cDNA 3 times for separate RT-PCR reactions. RT-PCR was performed with primers for Nanog, Sox-2, and Poly A Polymerase (PAP), as a reference gene for normalization. Primer sets for Nanog and PAP have previously been verified to amplify their respective transcripts in bovine embryos. Reaction products of PCR were subjected to electrophoresis and analyzed by Quantity One software (Bio-Rad). The number of pixels in each electrophoresis band was used to determine the relative transcript levels and was expressed as a ratio of Nanog or Sox-2 to PAP (Table 1). Both Nanog and Sox-2 were present in all cell lines at all 3 passages. Because Oct-4 is known to function synergistically with Nanog and Sox-2 to confer pluripotency, we are currently examining these samples for the presence of Oct-4. The presence of transcripts for the pluripotency-associated genes Nanog and Sox-2 in cells that are not inherently pluripotent suggests that there might be another level of regulation at the translational level for these genes.

Table 1.Transcript abundance of pluripotency-associated genes relative to PA P in bovine fetal fibroblast (BFF) and adipose-derived adult stem cells (ADAS)
243 GENE EXPRESSION PROFILING BY NEXT-GENERATION SEQUENCING OF cDNA SAMPLES FROM INNER CELL MASS AND TROPHECTODERM OF A SINGLE-DAY 12 PORCINE EMBRYO
Authors: Isom S. C. , Prather R. S.
Published: August 2010

Traditional microarray approaches to gene expression profiling often require RNA or cDNA amplification when working with extremely small or valuable tissue samples.This process is generally viewed as being undesirable because there is potential for bias to be introduced during amplification. Very recently, the so-called next-generation sequencing technologies were adapted for use in global gene expression profiling. Herein we report our efforts to apply these sequencing technologies to assess relative transcript abundances in pre-implantation-stage porcine embryos, without additional nucleic acid amplification before sequencing. As a proof-of-principle experiment, we have isolated total RNA from the embryonic disc (inner cell mass; ICM) and a small piece of trophectoderm (TE) from a Day 12 in vivo-produced embryo, which were estimated to be composed of 500 to 1000 cells each. The RNA was reverse transcribed using oligo-dT priming followed by second-strand cDNA synthesis. The double-stranded cDNA was then randomly sheared by sonication, and 10 ng of double-stranded cDNA fragments was used for sample preparation before sequencing. Prepared cDNA fragments (at 7 picomolar concentrations) were submitted for sequencing using the Illumina/Solexa platform as recommended. The millions of short (36 bp) reads generated by Illumina sequencing for each sample were then aligned to the swine UniGene database from NCBI, allowing for zero or one mismatches. Relative transcript abundances between cell types were profiled by considering the read counts for a given UniGene member as a percentage of the total number of reads generated for each cell type. It was demonstrated that approximately 11 000 and 9000 UniGene members were represented by a normalized average of 5 or more short reads per lane (0.001% of the total) in the ICM and TE samples, respectively. As expected, pluripotency factors, chromatin remodeling components, and cell-cell communication molecules were overrepresented in the ICM sample as compared with the TE sample. Conversely, epithelial determinants, ion transporters, and components of the steroid biosynthesis pathways were more abundant in the TE sample than in the ICM sample. Relative abundances of representative transcripts in these samples were verified by quantitative RT-PCR. In conclusion, we demonstrate the utility of next-generation sequencing technologies for gene expression profiling using even minute tissue samples and show that such analyses are possible even in species without a sequenced genome.
244 CHANGES IN THE TRANSCRIPTOME OF THE BOVINE PRE-IMPLANTATION EMBRYO INDUCED BY COLONY-STIMULATING FACTOR-2
Authors: Loureiro B. , Oliveira L. , Hansen P. J.
Published: August 2010

Colony-stimulating factor-2 (CSF-2) is a cytokine expressed in bovine oviduct and endometrium that has been reported to improve the proportion of embryos that become blastocysts in vitro and survive after transfer to recipients. One effect of CSF-2 that might be related to increased embryonic survival is a preferential increase in the number of cells in the inner cell mass. The objective of the current study was to determine changes in the embryo transcriptome caused by CSF-2 that promote blastocyst formation and establishment and maintenance of pregnancy after transfer. Bovine embryos were produced in vitro and cultured in KSOM-BE2 +10 ng/mL recombinant BoCSF-2 added at Day 5 after insemination. On Day 6 (24 h after treatment), embryos at the morula and early blastocyst stage were harvested and stored in groups of 50 at -80°C. A total of 4 pools of GM-colony-stimulating factor treated blastocysts and 4 control blastocysts were subjected to transcriptional profiling using the Bos taurus 2-color Agilent chip (4 × 44 K format). Before labeling, total RNA starting sample was spiked with control genes (artificial clones) of known concentration provided by Agilent. Labeling was done simultaneously with complimentary RNA (cRNA) amplification. Two rounds of linear RNA amplification were employed. Images were extracted using the Agilent Feature Extraction Software (Agilent Technologies, Santa Clara, CA, USA) and normalized within arrays by the Lowess method. Statistical analysis was performed using the JMP Genomics program (SAS Inst., Cary, NC, USA). The normalized data were log2 transformed, and the quantile normalization method was used to normalize data between arrays. Differences in gene expression were determined using PROC ANOVA (fixed false discovery rate = 0.01). Only genes with a 1.5-fold difference and P < 0.05 were considered differentially expressed. A total of 216 genes were differentially expressed between CSF-2 and control embryos. Of these, 141 could be annotated (61 genes up-regulated and 80 genes down-regulated by CSF-2). These included 13 genes involved in Wnt pathways, including 5 inhibitors of Wnt signaling (FRP, MAB21L2, PCDH24, PDE7, PPPR23A) that were up-regulated by CSF-2 and 5 genes involved in transmission of Wnt signals (WNT16, ROR2, CSNK2B, CELSR2, DTX3) that were down-regulated by CSF-2. Several other genes associated with differentiation were down-regulated by CSF-2 including CXCL12, FEZF1, PLD2, and RGS12. Expression of 1 gene that inhibits apoptosis (PRKAR2B) was increased by CSF-2, whereas expression of 6 genes involved in apoptosis pathways (DAPK1, MADD, NOD2, PIK3IP1, RIPK3, RNF7) were down-regulated. Results indicate that CSF-2 promotes pluripotency and decreases apoptosis in bovine pre-implantation embryos.

This research was supported by USDA-AFRI. B. Loureiro andL. Oliveira were supported by a CAPES (Brazil)/Fulbright Fellowship.
245 EFFECT OF BOVINE OOCYTE MATURATION SYSTEM ON RELATIVE ABUNDANCE OF mRNA
Authors: Pereira M. M. , Costa F. Q. , Oliveira A. P. , Assunção C. M. , Serapião R. V. , Viana J. H. M. , Machado M. A. , Camargo L. S. A..
Published: August 2010

The oocyte cytoplasm contains several transcripts that are important for early pre-implantation embryo development, and alterations on the amount of these stored mRNA can disturb oocyte competence. The aim of this study was to evaluate the relative abundance of specific transcripts in oocytes matured in vivo or in vitro. For in vitro maturation, immature oocytes were obtained by ovum pickup from 4 crossbred cows (group 1: G1) or from ovaries collected at a slaughterhouse (group 2: G2) and matured in TCM-199 containing 10% estrus cow serum and 2 μg FSH for 24 h under 5% CO2 in air at 38.5°C. For in vivo maturation, the same crossbred cows used in G1 received a progesterone intravaginal implant (CIDR®, Eazi-Breed CIDRO, São Paulo, Brazil) and 2 mg of estradiol benzoate (Estrogin®, Farmavet, São Paulo, Brazil) on Day 0. On Day 4, cows were superstimulated with 180 mg FSH (Folltropin®, Bioniche, Canada) injected in 6 decreasing doses every 12 h, and on Day 6, the cows received 0.53 mg of sodium cloprostenol (Ciosin®, Cooper, São Paulo, Brazil). On Day 7, CIDR® was removed and 2.5 mg of gonadorelina (Gestran-Plus®, Tecnopec, São Paulo, Brazil) was injected. Ovum pickup was performed 18 h after gonadorelina injection. Oocytes with expanded cumulus cell were then pooled and used as in vivo-matured oocytes (group 3: G3). Oocytes from all groups were denuded and frozen in liquid nitrogen. Pools of 10 oocytes for each group were subject to RNA extraction and reverse transcription. cDNA was amplified by real-time PCR using the beta-actin gene as the endogenous reference. The transcripts analyzed are encoded by TEA domain 2 (TEAD2), high mobility group N1 (HMGN), zygotic arrest 1 (ZAR1), maternal antigen that embryo requires (MATER), growth differentiation factor-9 (GDF9), and peroxiredoxin 1 (PRDX1) genes. Results were analyzed by REST software v.2 using the pair-wise, fixed reallocation randomization test. Data from G3 were used as calibrator. There was no difference (P > 0.05) on relative abundance of all transcripts between pools of oocytes matured in vitro or in vivo obtained from the same cows (G1 and G3, respectively). However, the relative abundance of GDF9 (0.22 ± 0.04-fold) was less (P < 0.05), whereas the relative abundance of TEAD2 transcripts (4.27 ± 2.14-fold) was greater (P < 0.05) for in vitro-matured oocytes obtained from slaugterhouse ovaries (G2) when compared with in vivo-matured oocytes (G3). No difference (P > 0.05) on relative abundance was found between G2 and G3 for the other genes. These data suggest that in vitro maturation does not alter the relative abundance of some transcripts stored into oocytes when compared with the ones stored in oocytes obtained from the same donors by means of multiple ovulation.

Financial support was provided by CNPq and FAPEMIG.
251 METHYLATION PATTERN OF A DIFFERENTIALLY METHYLATED REGION LOCATED IN THE LAST EXON OF THE Igf2 GENE IN OOCYTES FROM NELLORE COWS
Authors: Simarro Fagundes N. , Alice Michalczechen Lacerda V. , Siqueira Caixeta E. , Marinheiro Machado G. , de Oliveira Melo E. , Rumpf R. , Alves Nunes Dode M. , Machaim Franco M.
Published: August 2010

Assisted reproductive technologies are essential in modern cattle breeding. The relevance of in vitro embryo production (IVP) has been increasing around the world, particularly in Brazil. Acknowledging the positive effect of IVP regarding the utilization of female reproductive potential, its productivity can be improved. Methylation pattern of DNA in oocytes is a key factor for the improvement of IVP efficiency because it is related to oocyte competence. The objective of this study was to evaluate the methylation pattern of a differentially methylated region (DMR) located in the exon 10 of the imprint gene IGF2 in immature and in vitro-maturated oocytes from different follicle sizes. Small follicles (1-3 mm in diameter) and large follicles (≥8.1 mm in diameter) were dissected from slaughterhouse ovaries, and the COC were removed. Immature and in vitro-maturated oocytes, presenting the first polar body, were denuded and frozen in PBS at -80°C until DNA extraction. After extraction, DNA was treated with sodium bisulfite using EZ DNA Methylation™ Kit (Zymo Research, Orange, CA, USA). Bisulfite-treated DNA was amplified in a 2-round PCR strategy (nested-PCR), the amplicons purified with Geneclean® III Kit (MP Biomedicals, Irvine, CA, USA), cloned using pGEM®-T Easy Vector System (Promega, Madison, WI, USA), and sequenced. The number of follicles and in vitro maturation data were analyzed with chi-square test, and the methylation status of 27 CpG islands was analyzed with Student (normal distribution) or Mann-Whitney tests in the Prophet software (BBN Systems and Technologies, Cambridge, MA, USA). Cumulus-oocyte complexes from ≥8.1-mm follicles presented greater percentages of viable oocytes (40.5%) and nuclear maturation (60.6%) (P ≤ 0.05). Immature oocytes from 1- to 3-mm follicles were less methylated (33.33%) than those from ≥8.1-mm follicles (83.69%). After in vitro maturation, oocytes from ≥8.1-mm follicles were less methylated (18.51%) than the immature oocytes from ≥8.1-mm follicles (83.69%) and 1- to 3-mm maturated oocytes (49.62%). The less methylated pattern in the oocytes from ≥8.1-mm maturated follicles can be associated with greater competency of those oocytes, because this was the expected pattern. The lower level of methylation in the immature oocytes group (1- to 3-mm follicles) can be associated with the incomplete establishment of imprinting reprogramming pattern. Finally, we concluded that the methylation pattern of DMR, in the last exon of the IGF2 gene, can be used as a molecular marker for epigenetic reprogramming status in oocytes, helping the development of new IVP protocols.

Supported by Embrapa Genetic Resources and Biotechnology and CAPES.
259 SODIUM/POTASSIUM/CALCIUM EXCHANGER 3 IS REGULATED BY STEROID HORMONES, ESTROGEN AND PROGESTERONE, IN THE UTERUS OF RAT DURING ESTROUS CYCLE
Authors: Yang H. , Jeung E. B.
Published: August 2010

As a member of the family of potassium-dependent sodium/calcium exchangers, a sodium/calcium/potassium exchanger, NCKX3, plays a critical role in transport of 1 intracellular calcium and potassium ion in exchange for 4 extracellular sodium ions. Transcripts of NCKX3 were most abundant in the brain and smooth muscle, but in the uterus, aorta, and intestine, this gene expressed at lower levels. Its expression and role in the rat uterus during the estrus cycle have not been elucidated yet. In this study, we further examined the uterine expressions of NCKX3 mRNA and protein in stages during the estrous cycle of mature female Sprague-Dawley (SD) rats and in the absence or presence of sex-steroid hormones estrogen (E2) and progesterone (P4) in immature female SD rats. NCKX3 mRNA was verified by RT-PCR. Western blot analysis was applied to detect NCKX3 with an anti-NCKX3 goat polyclonal antibody. During the rat estrous cycle, uterus expressions of NCKX3 mRNA and proteins were highly expressed 2.5-fold at proestrus compared with those at estrus and diestrus. To examine the role(s) of sex steroids on the regulation of NCKX3 in the uterus of the immature female rat, hormones were prepared in ethanol and the rats were treated with E2 [40 (μg/kg of body weight (BW)], P4 (4 mg/kg of BW), or E2 plus P4 for 3 days. The expression of NCKX3 mRNA and protein increased 2-fold with E2, whereas P4 antagonized E2-stimulated NCKX3 expression, as similarly observed in the uterus. In addition, spatial expression of NCKX3 protein was detected by immunohistochemistry. Uterine NCKX3 protein was abundantly localized in the cytoplasm of the luminal and glandular epithelial cells at proestrus. Taken together, these results indicate that uterine NCKX3 is abundantly expressed in the uterus and controlled by steroid hormones E2 and P4, suggesting that uterine expression of NCKX3 might be involved in reproductive function during the cycle in female rat.
277 EFFECTS OF ERYTHROCYTE AND ERYTHROCYTE HEMOLYSATE ON BOVINE PREIMPLANTATION EMBRYO DEVELOPMENT IN VITRO UNDER REACTIVE OXYGEN SPECIES CONDITION
Authors: Ideta A. , Tsuchiya K. , Nakamura Y. , Urakawa M. , Murakami M. , Hayama K. , Aoyagi Y.
Published: August 2010

Reactive oxygen species (ROS) damage preimplantation embryos by increasing DNA fragmentation, leading to early embryonic death. Erythrocytes have been shown to protect other cells and tissues against ROS. In mice, erythrocytes were recently found to improve the early development of embryos by their antioxidant effect. The purpose of the present study was to examine the effect of erythrocytes on the in vitro development of bovine IVF embryos in medium supplemented with ROS. COCs were aspirated from ovaries collected from a local slaughterhouse and were cultured for 22 h in TCM-199 containing 5% fetal bovine serum. IVF was performed using an IVF100 (Research Institute for the Functional Peptides, Yamagata, Japan) according to the manufacturer’s instructions. In experiment 1, IVF embryos were cultured in CR1aa medium supplemented with an oxidizing agent, 0.5 mM hypoxanthine and 0.01 U mL-1 xanthine oxidase (HX/XOD), in the presence and absence of erythrocytes (5 × 104, 5× 105, 5×106, and 5 × 107 erythrocytes mL-1). In experiments 2 and 3, the development of embryos under the condition without ROS was assessed in the presence and absence of erythrocytes (5 × 106 erythrocytes mL-1) or erythrocyte hemolysate (hemoglobin concentration of 1.9 g L-1), respectively. At 7 days after in vitro culture, the development to the blastocyst stage of IVF embryos was examined using a stereomicroscope. Data were analyzed using Fisher’s PLSD test and Student’s t-test In experiment 1, the presence of HX/XOD significantly inhibited embryo development to the blastocyst stage in vitro (P < 0.05). The addition of erythrocytes to medium supplemented with HX/XOD markedly improved preimplantation development (Table 1). In experiments 2 and 3, supplementation of erythrocytes or erythrocyte hemolysate promoted the development of embryos to the blastocyst stage (experiment 2: erythrocyte 42.4 ± 3.1%, control 28.5 ± 5.7%, P < 0.1; experiment 3: erythrocyte hemolysate 39.1 ± 3.3%, control 30.2 ± 1.0%, P < 0.1). In conclusion, we suggest that the addition of erythrocytes to culture medium can counteract the negative effects of ROS on embryo development and blastocyst formation.

Table 1.Effect of HX/XOD and erythrocyte supplementation on embryo development to blastocyst stage
287 EFFECT OF FOLLICULAR WAVE SYNCHRONIZATION AND ABSENCE OF CORPUS LUTEUM ON COC RECOVERY IN Gyr (BOS TAURUS INDICUS) COWS
Authors: Miyauchi T. , Fernandes C. A. C. , Oliveira E. R. , Alves B. F. L. , Viana J. H. M.
Published: August 2010

Transvaginal guided follicle aspiration (TGFA) is the main technique used in Brazil to recover oocytes for in vitro embryo production (IVP) in bovine. Different protocols have been proposed to synchronize follicular emergence in oocyte donors, but most of them were developed for use in European breeds of cattle, which show many differences in ovarian physiology when compared with Zebu breeds. The aim of this study was to compare different protocols for preparation of Gyr (dairy zebu breed) oocyte donors. The TGFA were performed in a donor management facility located in Minas Gerais State, southeast Brazil. Pluriparous cycling Gir cows (n = 42) were used as donors. All cows underwent 3 treatments: G1 (control), no treatment before TGFA; G2, 2 mg of estradiol benzoate (EB) for follicular wave synchronization given i.m. 5 days before TGFA; and G3, norgestomet auricular implants given 9 days and 2 mg of EB plus 0.53 mg of cloprostenol given 5 days before TGFA. The interval between TGFA in the same donor was greater than 30 days. All procedures were made by the same technician, using a portable ultrasound device, disposable 19G or 20G needles, and a vacuum pressure of 80 mm Hg. The aspirated follicular fluid was collected in 50-mL Falcon tubes and sent to the laboratory for COC identification and classification under 50 × magnification. Recovered oocytes were classified according to cumulus cell layers and cytoplasm morphology. The total number of oocytes and viable COC recovered and the procedure length (min), including time spent for TGFA and laboratory manipulation, were compared. Data were evaluated by ANOVA, and means compared by Tukey’s test. A total of 126 TGFA sessions were performed, with recovery of 2,809 oocytes (20.31 ± 12.32 of COC and 14.83 ± 7.97 of viable COC per cow/session; mean ± SD). The total number of recovered oocytes and viable COC recovered were lower in G1 compared with G2 and G3 (15.18 ± 11.07 v. 21.18 ± 9.71 and 24.68 ± 9.03; and 9.53 ± 7.22 v. 16.97 ± 6.47 and 18.84 ± 8.90, respectively; P < 0.05) There was no difference (P > 0.05) between G2 and G3 on the number of oocytes or viable COCs recovered. The procedure length, however, was longer in G1 and G2 compared with G3 (49.6 ± 15.1 and 46.9 ± 13.4 v. 35.8 ± 13.1 min, respectively; P < 0.05) The shorter procedure length in G3 was probably associated with the reduced number of cows showing no corpora lutea (38/42, 90%), which resulted in reduction of bleeding and clot formation in the aspirated fluid. These results show that (1) previous follicular wave synchronization by EB can improve the number and quality of recovered oocytes; (2) the absence of corpora lutea does not increase the number of recovered COC and viable COC, but reduces the time spent in the procedure.
Intervet Schering Plough Animal Health.
288 PRELIMINARY RESULTS OF THE USE OF TUMOR CELLS OF A DOG TO CONDITION BOVINE EMBRYO IN VITRO CULTURE MEDIUM
Authors: Moreno Millán M. , Ocaña Quero J. , De Luca L. J. , Hidalgo Prieto M. , Dorado Martín J. , Demyda S. E.
Published: August 2010

The aim of this study was to analyze the response of bovine embryos fertilized in vitro to a tumor dog cell- conditioned TCM-199 culture medium used in the development stage. Two hundred ninety-five oocytes obtained from slaughterhouse ovaries were cultured in groups of 20 cells at 39°C in TCM-199 medium droplets with 20% estrous cow serum (ECS) for 24h in a humid atmosphere containing 5% CO2. After incubation, these oocytes were fertilized with a 1 × 106 sperm cell dose, previously capacitated by a 1-h incubation in SP-TALP medium under usual conditions, by incubation in small petri dishes with 1 mL of Fert-TALP medium for 24 h under the conditions described previously. Alleged fertilized oocytes were randomly separated into 2 groups. A control group (n = 144) was cultured in TCM-199 medium with 10% fetal calf serum (FCS) and the other (n = 151) was cultured in TCM-199 medium previously conditioned by incubation for 72 h with 1 × 106 cells of a complex adenocarcinoma grade 1 obtained from a 6-year-old boxer dog and sterilized by filtration with 0.2-(im membrane. After 72 h of culture, the zygotes were harvested and prepared for a quick microscope observation. Alleged zygotes were classified as “not fertilized,” “divided,” or “degenerated.” Statistical differences (P < 0.01) were found among both groups with a greater number of normal divided embryos (65.2%) in the conditioned medium group and a lower number (30.4%) in the control group. The number of normal divided embryos in the conditioned medium after 72 h of incubation was similar to that achieved by current methods. These preliminary results are consistent with results obtained by other researchers in which growth factors derived from established cell lines can be used to condition culture medium for in vitro bovine embryo development. Further studies will be necessary to clarify how these factors contribute to in vitro embryo development.
Spanish Retinta Breed Asociation, Spain.