bluuebunny/crossref_metadata_2025 huggingface dataset:
107 DETECTION OF BIOTINYLATED-OVULATION-INDUCING FACTOR (OIF) IN CEREBROSPINAL FLUID AND ITS ABILITY TO INDUCE OVULATION
Authors: Berland M. , Guerra M. , Bogle O. A. , Vio K. , Adams G. P. , Ratto M.
Published: December 2012

Ovulation-inducing factor (OIF) is a protein in the seminal plasma of llamas that induces a preovulatory LH surge by acting directly or indirectly on the hypothalamic GnRH neurons (Silva et al. 2011 Reprod. Biol. Endocr. 9, 74). We hypothesize that OIF crosses the blood–brain barrier and reaches the hypothalamus via secretion into the cerebro-spinal fluid (CSF) through the choroid plexus. Two experiments were designed to determine whether biotinylation of OIF (as a tracer) alters its bioactivity in a llama model (Experiment 1) and whether it crosses the blood–brain barrier in a rabbit model (Experiment 2). In Experiment 1, llamas with a follicle ≥8 mm in diameter that had grown for 3 consecutive days were assigned randomly to 5 groups (n = 2/group) and given an IV dose of 1) 800 µg of OIF, 2) 800 µg of OIF biotinylated at the amino end; 3) 1600 µg of OIF biotinylated at the amino end, 4) 800 µg of OIF biotinylated at the carboxyl end, or 5) phosphate buffered saline (control). The ovaries were examined daily by transrectal ultrasonography on Day 3 and 8 after treatment (Day 0 = treatment) to detect ovulation and corpus luteum formation. In Experiment 2, adult female rabbits were assigned randomly to 3 groups and given an IV dose of (1) 250 µg of OIF, (2) 250 µg of OIF biotinylated at the amino end, or (3) 250 µg of OIF biotinylated at the carboxyl end. A 50-µL sample of CSF was collected from the cisterna magna under general anesthesia before (0 min) and 10, 20, 30, and 45 min after treatment. The presence of biotinylated OIF in CSF samples was determined by dot blot, using streptavidin-peroxidase and diaminobenzidine. In Experiment 1, the diameter of the follicle at the time of the treatment did not differ among groups (9.7 ± 0.2, 9.4 ± 0.0, 10.5 ± 1.0, 10.1 ± 0.2, 10.3 ± 0.4). Ovulation was detected in all llamas except one llama treated with 800 µg OIF biotinylated at the carboxyl end and both llamas given PBS. The diameter of the corpus luteum did not differ among OIF-treated groups. In Experiment 2, OIF biotinylated at both amino and carboxyl ends was detected in CSF samples at 10, 20, 30, and 45 min after IV administration. No signal was recorded before IV administration (0 min) or in samples from rabbits that were given nonbiotinylated OIF. We conclude that the biotinylation process did not affect OIF bioactivity, and OIF crosses the blood–brain barrier and reaches the CSF in rabbits.
Research supported by FONDECYT 1120518, the Natural Sciences and Engineering Research Council of Canada, and the Alpaca Research Foundation.
108 EFFECTS OF UTILIZATION OF SUBCUTANEOUS IMPLANTS OF GnRH AGONISTS (DESLORELIN) ON OVARIAN ACTIVITY AND HORMONAL PROFILE DURING THE PERIOD OF IMPLANTATION AND AFTER REMOVAL IN NELORE COWS (BOS INDICUS) WITH A TOTAL IMPLANT TREATMENT OF 70 DAYS
Authors: Marques Filho W. C. , Destro F. C. , Trevisol E. , Queiroz C. M. , Martins M. C. , Ferreira J. C. P.
Published: December 2012

The aim of the present study was to evaluate the effects of subcutaneous implants of deslorelin (GnRH agonist-Suprelorin® 4.7 mg) on follicular dynamics and plasma profile of FSH, LH, and progesterone (P4) during the last 20 days of an implantation period of 70 days and after the removal of the implant until the detection of the first ovulation in Nelore cows (Bos indicus). Seven animals had their ovulation synchronized. At the day of the detection of ovulation they received subcutaneous implant of deslorelin. In the last 20 days of the period of treatment, ultrasound scans were taken every 2 or 3 days to characterize the follicular dynamics according to diameter: I (<4 mm), II (4–6 mm), and III (>6 mm). Blood samples were collected to measure plasma concentrations of FSH, LH, and P4. Results were tested by ANOVA, and difference between means was defined using Tukey’s test with significance levels of 0.05. During the 70 days of deslorelin treatment, none of the animals ovulated, despite the implant not suppressing all follicular development. The ultrasound finding was confirmed by P4 plasma concentrations (range: 0.05–0.31 ng mL–1). The normal P4 plasma concentration expected in cycling Nelore cows is about 3.8 ng mL–1 (Satrapa et al. 2010 Reprod. Anim. Dom. 45, 881–887). During the last 20 days of treatment, 4 animals presented follicular development with max diameter ranging through 4 to 6 mm, whereas in the 3 remaining animals, the observed diameter ranged through 9 to 12 mm. The secretion of FSH and LH was not entirely suppressed during the study (range: 1.69–1.39 and 1.37–1.07 ng mL–1, respectively). After the removal of the deslorelin subcutaneous implant, the period of restoration of the ovulatory capacity was 29 days, and the mean diameter of the preovulatory follicle was 12.5 mm. The P4 plasma concentrations during this time were low (range: 0.15–0.09 ng mL–1). During the recovery period, the phenomenon of follicular codominance and double ovulation in 2 animals (2/7) was observed. From the results obtained, it can be concluded that the total implant treatment of 70 days with deslorelin in cows inhibits ovulation by not allowing LH surge, although the follicular development is not completely depressed.
The financial support of FAPESP, Fundunesp, and CNPq are acknowledged.
116 EXPRESSION OF INSULIN-LIKE GROWTH FACTOR 2 RECEPTOR (IGF2R), LEPTIN RECEPTOR (LEPR) AND SERUM- AND GLUCOCORTICOID-REGULATED KINASE 1 (SGK1) mRNA IN HUMAN AND BOVINE PREGNANCIES
Authors: Yang L. , Yu W. , Yan W. L. , Peng L. G.
Published: December 2012

The objective was to investigate the expression of insulin-like growth factor 2 receptor (IGF2R), leptin receptor (LEPR), and serum- and glucocorticoid-regulated kinase 1 (SGK1) mRNA in human decidua and bovine pregnancies. The IGF2R, LEPR, and SGK1 mRNA were measured by using quantitative real-time PCR in 25 patients with spontaneous abortion, 23 early pregnancy decidua from patients that underwent elective abortion, 8 cloned bovine pregnancies, and 2 normal bovine pregnancies. The expression of LEPR and SGK1 in the human spontaneous abortion group was lower than the early pregnancy (elective abortion) group (P < 0.05) but the expression of IGF2R was increased in the spontaneous abortion group (P < 0.05). The expression of IGF2R, LEPR, and SGK1 mRNA was decreased in the cloned bovine pregnancy group (P < 0.05). Reduced expression of LEPR and SGK1 mRNA in human pregnancies undergoing spontaneous abortion indicates the key role for these transcripts in spontaneous abortion. Low expression of IGF2R, LEPR, and SGK1 in cloned bovine pregnancies may affect endometrial receptivity and contribute to high spontaneous abortion rate in cloned cattle.
118 DEEP SEQUENCING OF THE PORCINE ENDOMETRIAL TRANSCRIPTOME ON DAYS 12 AND 14 OF PREGNANCY
Authors: Samborski A. , Kessler B. , Reichenbach M. , Reichenbach H. D. , Ulbrich S. E. , Bauersachs S.
Published: December 2012

Establishment of pregnancy in mammals requires prolongation of luteal life span for sustained progesterone (P4) production. P4 stimulates secretory functions of the endometrium required for conceptus growth and development and is essential for endometrial receptivity. The porcine conceptus undergoes rapid differentiation and expansion of its trophoblastic membranes between Day 11 and Day 12 of gestation. With trophoblast elongation production of estrogen, the porcine embryonic pregnancy recognition signal, by the conceptus increases. A complex interplay of estrogen signaling and prostaglandin (PG) metabolism in the endometrium finally results in prevention of luteolysis. Conceptus attachment to the uterine surface epithelium starts around Day 14 of pregnancy, preceded by a pronounced vascularization at the implantation zones, initiating the epitheliochorial placentation. To characterize the complex transcriptome changes in the endometrium in the course of maternal recognition of pregnancy and initial placentation, deep sequencing of endometrial RNA samples of pregnant animals (n = 4) and corresponding non-pregnant controls (n = 4) was performed on Day 12 and Day 14 after ovulation using Illumina RNA-Seq (Illumina Inc., San Diego, CA, USA). Between 21 and 36 million 76-bp single-end reads were produced per sample. Reads were mapped with TopHat to the porcine genome assembly Sscrofa10.2 and relative expression values were calculated based on the National Center for Biotechnology Information’s gene annotation for the analysis of differential gene expression. Statistical analysis revealed more than 3500 differentially expressed genes (DEG) for Day 12 and almost 2500 DEG for Day 14 of pregnancy (DESeq, FDR 1%). Differential expression of selected genes was validated by the use of quantitative real-time PCR (RT-PCR). Comparison of the RNA-Seq data from Day 12 and Day 14 of pregnancy based on a comparison of the DEGs and of overrepresented functional categories revealed distinct differences in gene regulation, reflecting the different functions of the endometrium during these stages; that is, recognition of pregnancy on Day 12 and preparation for conceptus implantation on Day 14. The data from Day 14 was also compared to the published microarray data set from Day 14 of pregnancy (Østrup et al. 2010 Biol. Reprod.) and other sets of DEG by the use of gene set enrichment analysis (GSEA). Overall, a very good agreement with the results of the previous microarray study was found. Furthermore, a significant overlap with genes upregulated in bovine endometrium on Day 18 of pregnancy was found for the genes upregulated in porcine endometrium on Day 14. More than half of these genes were known interferon-regulated genes. In conclusion, RNA-Seq revealed distinct transcriptome changes on Day 12 and Day 14 of pregnancy in porcine endometrium associated with MRP and initiation of implantation.
131 EFFECTS OF HIGH MOLECULAR WEIGHT HYALURONAN ON SHEEP EMBRYO DEVELOPMENT AND SURVIVAL AFTER CRYOPRESERVATION
Authors: Ghaffarilaleh V. , Ghafari F. , Teresa-Paramio M. , Fouladi-Nashta A.
Published: December 2012

Hyaluronan (HA), a component of extracellular matrix in mammalian tissues including that of the reproductive system, has been shown to support embryo development. HA is produced in various sizes with distinct physiological functions. Cleaved sheep embryos produced after in vitro maturation and in vitro fertilization were cultured in serum free synthetic oviduct fluid medium supplemented with increasing concentrations (0, 0.25, 0.5, and 1 mg mL–1) of large size HA (Healon; 6 × 106 Da). Development to blastocyst stage was recorded at Day 7 when a group of the embryos were fixed and stained by differential staining combined with TUNEL labelling to analyse embryo quality. The remainder of the blastocysts from each treatment/repeat were vitrified in open pulled straws and then cultured for an extra period of 48 h to analyse their survival rate and quality after cryopreservation. SPSS version 20 software (SPSS Inc., Chicago, IL, USA) was used for analyzing the data with generalized linear model. Healon did not change blastocyst (33 ± 5.7, 32 ± 6.0, 35 ± 5.5; P ≥ 0.05) or survival rates (63 ± 17.1, 83 ± 15.2, 58 ± 14.2; P ≥ 0.05) as compared to the respective controls (25 ± 5.2, 38 ± 17.1). It increased the total cell (TC) number (83.6 ± 4.6, 100.7 ± 3.8, 97.2 ± 3.7, 105.0 ± 3.9; P ≤ 0.05) and trophectoderm cells (TE) (58.4 ± 3.8, 74.2 ± 3.2, 75.6 ± 3.3, 80.1 ± 3.4; P ≤ 0.05) but had no effect on the number of inner cell mass (ICM) and apoptotic cells. The ICM : TE ratio was not affected (0.45 ± 0.04, 0.36 ± 0.03, 0.33 ± 0.03, 0.30 ± 0.03; P ≥ 0.05). Surviving embryos had higher TC (63.2 ± 3.7, 130.8 ± 3.6, 113.9 ± 5.2, 149.8 ± 5.4; P ≤ 0.05), TE (42.9 ± 3.0, 96.7 ± 3.1, 85.2 ± 4.5, 111.9 ± 4.7; P ≤ 0.05) and ICM (20.3 ± 2.2, 32.9 ± 1.8, 27.7 ± 2.6, 36.5 ± 2.7; P ≤ 0.05). The apoptotic cell numbers and ICM : TE ratio of the survived embryos after cryopreservation were not affected by HA supplementation. The results indicate that large size HA improves the embryo viability and quality, which may have implication for improving embryo transfer.
133 L-CARNITINE DURING IN VITRO CULTURE ENHANCES THE CRYOTOLERANCE OF BUFFALO (BUBALUS BUBALIS) IN VITRO-DERIVED EMBRYOS
Authors: Boccia L. , De Blasi M. , Zullo G. , Longobardi V. , Vecchio D. , Gasparrini B.
Published: December 2012

In buffalo, in vitro embryo production (IVEP) technology is the best tool to improve the genetic merit through the maternal lineage. A major limitation of IVEP technology in buffalo species is the poor cryotolerance of the embryos, likely due to their high lipid content (Gasparrini 2002 Theriogenology 57, 237–256). It was previously demonstrated that supplementing bovine culture media with L-carnitine, a cofactor of β-oxidation, improves in vitro embryo development (Sutton-McDowall et al. 2012 Theriogenology 77, 1632–1641). The aim of this work was to evaluate whether L-carnitine supplementation during in vitro culture (IVC) improves blastocyst development and cryotolerance of in vitro produced buffalo embryos. After a preliminary dose response trial, we selected the concentration of 0.25 mM for the experiment. Cumulus–oocytes complexes (n = 288, over 4 replicates), recovered from slaughtered animals, were matured and fertilized in vitro according to our standard procedures (Gasparrini et al. 2006 Theriogenology 65, 275–287). On Day 1 (Day 0 = IVF), zygotes were cultured in SOF supplemented with 8 mg mL–1 BSA, in the absence (control, n = 143) or presence of 0.25 mM L-carnitine (n = 145). In vitro culture was carried out at 38.5°C under 5% CO2, 7% O2, and 88% N2. Cleavage rate was evaluated on Day 5, when the cleaved embryos were transferred into fresh medium for further 2 days. On Day 7 after IVF, embryo outcome was assessed and all the embryos were vitrified by cryotop in 16.5% ethylene glycol, 16.5% dimethyl sulfoxide (DMSO), and 0.5 M sucrose (De Rosa et al. 2007 Ital. J. Anim. Sci. 6(Suppl 2), 747–750). The resistance to cryopreservation was evaluated by assessing the survival rate, on the basis of morphological criteria, after 24 h culture. Data were analyzed by chi-square test. No differences were found in cleavage rates between the control (81.5%) and the L-carnitine group (78.8%). The blastocyst yields (calculated in relation to the cleaved embryos) were not significantly influenced by the L-carnitine treatment (40.2 and 52.9%, in the control and the L-carnitine groups, respectively). However, buffalo embryos cultured in the presence of L-carnitine showed an increased resistance to cryopreservation, as indicated by the higher survival rates recorded after 24 h culture (78.7 and 96.4%, in the control and the L-carnitine groups, respectively; P < 0.01). In conclusion, these results demonstrated that L-carnitine supplementation of culture medium improves the resistance to cryopreservation of in vitro produced buffalo embryos. We speculate that the increased cryotolerance observed in the presence of L-carnitine may be due to a better utilization of the endogenous lipid stores, resulting in improved embryo quality.
160 CONCEPTION RATES FOLLOWING TREATMENT OF LACTATING HOLSTEIN RECIPIENTS WITH A GONADOTROPIN RELEASING HORMONE (GnRH) ANALOGUE AT THE TIME OF TRANSFER OF OVUM PICK-UP/IVF-DERIVED EMBRYOS
Authors: Pinto T. L. C. , Sales J. N. S. , Carvalho R. R. , França T. M. , Souza J. C.
Published: December 2012

The use of lactating dairy cows as ovum pick-up (OPU)/IVF-derived embryo recipients in large-scale embryo transfer programs may be limited by the effects of low progesterone concentrations on the uterine environment and subsequent embryo development. It was hypothesized that the injection of a GnRH analogue on the day of embryo transfer would induce ovulation and formation of an accessory corpus luteum, increasing progesterone concentrations and, as a result, conception rates. An experiment was designed to evaluate the effects of two different GnRH analogues injected IM on the day of embryo transfer on conception rates of lactating Holstein recipients. Oestrus was not synchronized and was detected continuously by multiple observers during the day and in the evening until midnight. Recipient days in milk varied from 45 to 150, daily milk production ranged from 20 to 45 kg, body condition scores and age ranged from 2.5 to 4.5 and 2.5 to 5.5 years, respectively. Herd pregnancy rates after the first artificial insemination ranged from 15% to 70%, considering the period the experiment was conducted. Embryos were produced by standard in vitro techniques following OPU on Holstein donors. Lactating Holstein recipients (n = 224) were allocated randomly to one of three treatment groups 7 days after detected oestrus: Control (n = 77) = vehicle; Buserelin (Sincroforte®, Ouro Fino, Sao Paulo, Brazil; n = 75) = 10 µg; and Deslorelin (Sincrorrelin®, Ouro Fino; n = 72) = 750 µg. Embryos were transferred nonsurgically 6 to 8 days after observed oestrus. Pregnancy was detected by ultrasonography between 62 and 72 days after embryo transfer. Percent pregnant data were analyzed as a binomial distribution under the GENMOD procedure of SAS (SAS Institute Inc., Cary, NC, USA). Conception rates were similar (P = 0.13) between Control (28%, 22/77), Buserelin (24%, 18/75), and Deslorelin (38%, 28/72) treatment groups. These data demonstrate that conception rates from in vitro produced embryos transferred to lactating dairy cows are not improved by treatment with a GnRH analogue on the day of transfer. Although not critically tested, results indicate that adequate conception rates may be obtained in commercial dairy operations from OPU/IVF-derived embryos, adding an important management tool for reproduction and animal breeding enhancement.
161 PROCESSING OF SEMEN WITH PYOSPERMIA ALLOWS ITS USE IN EQUINE EMBRYO TRANSFER PROGRAMS
Authors: Sancler-Silva Y. F. R. , Monteiro G. A. , Ignacio F. S. , Hartwig F. P. , Papa F. O.
Published: December 2012

Previous studies in humans and current studies in horses show that the presence of leukocytes in the semen promotes increased reactive oxygen species and proinflammatory cytokines production. These substances cause damage to membrane integrity, mitochondrial function, DNA integrity, and lead to increased expression of apoptosis markers in the spermatic cell. As a result of these effects, decreased quality and longevity of the semen that lead to fertility reduction could be highlighted. A 6-year-old stallion was admitted in the Department of Animal Reproduction and Veterinary Radiology of Veterinary Medicine and Animal Science School, UNESP, Botucatu-SP, Brazil, with a history of pyospermia due to seminal vesiculitis. Therefore, because of the high demand of the stallion semen and attempting to allow its use in equine embryo transfer programs, a process to reduce leukocyte numbers was performed. For the evaluation of the percentage of leukocytes in the sample, a 10-µL aliquot of semen was smeared and stained with Quick PanoticTM. Semen was diluted in skim milk-based extender BotuSemenTM (1 : 1; Nidacon International AB, Mölndal, Sweden) and then analyzed for sperm kinetic parameters by computer assisted semen analysis (CASA). Sequentially, seminal plasma was removed by filtration with SpermFilterTM (Nidacon International AB). The sample was resuspended with BotuSemenTM to a concentration of 300 × 106 sperm mL–1 and subjected to a single layer gradient centrifugation (EquiPureTM; Nidacon International AB) at 400g for 20 min. A proportion of 5 mL of diluted semen in 5 mL of EquiPureTM (1 : 1) was used for the centrifugation. The pellet was resuspended with BotuSemenTM at a concentration of 50 × 106 sperm mL–1. Before and after processing, sperm kinetic means were, respectively: Total motility (TM) = 46% and 89%, progressive motility (PM) = 20% and 55%, and rapid sperm cells (RAP) = 32% and 81%. The mean percentages of leukocytes in semen smears before and after processing was 80% and 5%, respectively. The processed semen was used for artificial insemination of 5 mares and none of them showed signs of post-breeding endometritis. In addition, in all cases, embryos were recovered when uterine flushes were done on Day 8 post-ovulation. In conclusion, the method used for the processing of semen with pyospermia was effective in reducing leukocyte loads and promoted an improvement of seminal kinetics parameters which allowed its use in embryo transfer programs.
The authors acknowledge FAPESP (2011/23139-9) for financial support and Botupharma for donation of BotuSêmenTM extender and SpermFilterTM.
169 QUALITATIVE ASSESSMENT OF ENDANGERED ARABIAN TAHR/IBEX (HEMITRAGUS JAYAKARI) SEMEN USING BIOXCELL® AND TRILADYL® EXTENDERS
Authors: Baqir S. , Al-Zeheimi N. , Bani Orabah A. , Al-kindi A. , Al-Shakaili Y. , Al-Rasbi K.
Published: December 2012

The Arabian tahr/ibex is a small species of goat-antelope-like animal, indigenous to the United Arab Emirates and with a larger population in Oman. Although the animal was discovered in 1894, its numbers continue to decline, and the animal has been classified on the IUCN red list as an endangered species. Little is known about the male reproduction, specifically, extending and cryopreserving semen. This provides a crucial tool for the ex situ conservation of this endangered species. The objective of this study was to evaluate sperm characteristics of the Arabian ibex after dilution in 2 commercial extenders, namely Bioxcell® and Triladyl® based on multiple qualitative parameters. Semen was collected using an electroejaculator from males (n = 4, ~4 years old) during the reproductive season (February–April). Ejaculates were divided into 2 aliquots and extended 1 : 2 in Bioxcell® or Trilidayl® at 37°C. Diluted semen was evaluated for motility with computer-assisted semen analysis (Sperm Vision®, Minitube, Verona, WI, USA). Morphologically, samples were examined with eosin and nigrosin stain under light microscopy, in addition to structural or ultrastructural evaluation with the use of scanning (SEM) or transmission (TEM) electron microscopy. Viability tests were carried out with a LIVE/DEAD® sperm viability kit (Invitrogen, Carlsbad, CA, USA); samples were examined using a fluorescent microscope (Olympus BX51) or with a flow cytometer (FACSAriaTM III, FACSDiva software, BD) with an average flow of 20 000 sperm/event. Sperm samples were liquefied (37°C/5 min), washed with 0.9% NaCl, centrifuged (2000 rpm for 3 min), and filtered using 40-µm nylon mesh (BD, USA) before FACS analysis. Our data showed that semen diluted with Triladyl® had higher motility compared with samples extended with Bioxcell® (60 v. 41%). While eosin and nigrosin staining showed very low morphological abnormalities with both extenders (less than 2%), SEM and TEM assessment demonstrated slightly high incidents of cracked tails, ruptured membrane, and broken midpeace with samples diluted with Bioxcell®. Viability test showed that samples extended with Triladyl® had higher percentages of live sperm (33%) compared with Bioxcell® (21%). Similar viability numbers were obtained with FACSAriaTM III. These results indicate that diluting Arabian ibex semen with Triladyl® would provide improved motility, morphology, and viability compared with Bioxcell®. These findings would be extremely useful for an ex situ conservation program of the Arabian tahr/ibex.
180 EXPRESSION OF TUMOR NECROSIS FACTOR-STIMULATED GENE 6 PROTEIN IN BOVINE CUMULUS–OOCYTE COMPLEXES AT DIFFERENT MATURATION STATUS
Authors: Trzeciak P. , Starzyński R. , Rąpała Ł. , Dąbrowski S. , Nałęcz-Nieniewska E. , Duszewska A. M.
Published: December 2012

The TSG-6 is a ~35-kDa protein belonging to hyaluronan binding superfamily proteins. The TSG-6 plays role in inflammation and in inflammation-like processes (i.e. ovulation). The tsg-6 expression is induced in cumulus–oocyte complex (COC) cells just before ovulation. It is involved in the migration of cumulus cells though the formation and stabilisation of the extracellular matrix. Disturbances in secretion of this protein lead to a reduction in the number of ovulated oocytes. Although studies of the prevalence and role of TSG-6 in COC were conducted in several animal models, little is known about TSG-6 in cattle. The aim of this study was to assess tsg-6 mRNA expression and protein localization in bovine cumulus cells from COC at different maturation status. Ovaries were collected from the slaughterhouse. Cumulus cells were isolated from COCs in different stages of maturity. In variant I, COC were isolated from small follicles of Φ 2 to 6 mm. In variant II, COC were also isolated from small follicles and matured in vitro in TCM199 HEPES with 10% FBS and 0.02 IU NIH-pFSH mL–1, 1 µg mL–1 β-oestradiol, 0.2 µM sodium pyruvate, 50 µg mL–1 gentamicin in 5% CO2, 38.5°C for 24 h. In variant III, COCs were isolated from large follicles of Φ >15 mm. Analysis of tsg-6 expression in cumulus cells was performed using real-time PCR. Expression of Tsg-6 was normalized to that of s18. Statistical analysis of tsg-6 mRNA level in all variants was carried out by 1-way ANOVA, and comparisons of mean values were made with the Tukey honestly significant difference test (Statgraphic 5.1 Centurion); P < 0.05 was considered to reflect the presence of statistical significance. For all variants, paraffin-embedded slices of COC were carried out to localise TSG-6 by indirect method of immunofluorescence. Immunostaining of the TSG-6 protein was performed using primary polyclonal antibody raised against bovine TSG-6. Antigen–antibody complexes were visualised after incubation with secondary IgG conjugated with fluorescein isothiocynanate. Immunolocalization of TSG-6 was performed using fluorescent microscopy. The relative expression of tsg-6 mRNA in variant II was more than 2 times higher (1.887 ± 0.797 a.u.) compared with variant III (0.760 ± 0.130 a.u.). The difference was statistically significant (P < 0.05). No tsg-6 expression in variant I was detected. The presence of TSG-6 protein in the extracellular matrix of cumulus cells from variant II as well as from variant III was detected. The present data suggest that tsg-6 gene expression and its protein presence are stimulated during COC maturation in cattle as previously demonstrated for other species.
Supported by Warsaw University of Life Sciences, Faculty of Veterinary Medicine: 505-10-02330050.
182 EFFECT OF OVULATION-INDUCING FACTOR ON DEVELOPMENT AND VASCULAR PERFUSION OF THE OVULATORY FOLLICLE AND CORPUS LUTEUM IN LLAMAS
Authors: Ulloa-Leal C. , Bogle O. , Adams G. P. , Ratto M.
Published: December 2012

The aim of the study was to determine if purified ovulation-inducing factor (OIF) from llama seminal plasma evokes changes in tissue vascular area of the preovulatory follicle and CL. Mature non-lactating, non-pregnant, female llamas (n = 20) were monitored by transrectal ultrasonography using a 7.5-MHz linear-array transducer (MyLab 5, Canadian Veterinary Imaging) to determine ovarian follicular status. Llamas with a growing follicle (for 3 consecutive days) ≥8 mm were assigned randomly to 2 groups (n = 10/group) and given an i.m. dose of (1) 50 µg of gonadorelin acetate or (2) 1 mg of purified OIF from llama seminal plasma. Llamas were examined daily by transrectal ultrasonography using B-mode and power Doppler mode from Day 0 (day of treatment) to Day 16 to detect ovulation and CL development, and to calculate the area of blood-flow signal in the preovulatory follicle at 12 h after treatment and in the CL on Days 2, 4, 6, 8, 10, 12, 14, and 16. Power Doppler images were recorded, edited, and analysed using the ImageJ software (National Institutes of Health, Bethesda, MD, USA). Data were compared between groups by Student t-test, chi-square, and ANOVA for repeated measures. No differences were detected between GnRH and OIF groups, respectively, in the diameter of the preovulatory follicle at the time of treatment (9.7 ± 0.3 v. 9.9 ± 0.4 mm), ovulation rate (10/10 v. 10/10), interval from treatment to ovulation (32.0 ± 0.6 v. 30.4 ± 1.8 h), maximum CL diameter (13.05 ± 0.4 v. 13.5 ± 0.3 mm), or the day on which the CL reached the maximum diameter (8.7 ± 0.3 v. 8.2 ± 0.2). The preovulatory follicle from llamas treated with purified OIF had a greater (P ≤ 0.0001) blood-flow signal area after treatment than that of the GnRH group. Similarly, the luteal tissue of llamas treated with purified OIF had a greater (P ≤ 0.001) blood-flow signal area than that of the GnRH group on Days 4, 6, 12, and 16 after treatment. We conclude that OIF purified from llama seminal plasma increases the vascular perfusion of the preovulatory follicle and the subsequent CL, consistent with the hypothesis that OIF is luteotrophic.
Research funded by Chilean National Science and Technology Research Council (Fondecyt Regular 1120518), the Natural Sciences and Engineering Research Council of Canada, and the Alpaca Research Foundation.
197 EXPRESSION OF MELATONIN-RELATED GENES IN RAT CUMULUS–OOCYTE COMPLEXES
Authors: Coelho L. A. , Peres R. , Amaral F. G. , Cipolla-Neto J.
Published: December 2012

Melatonin is a hormone usually associated with the modulation of circadian rhythms and the regulation of seasonal reproductive function. There is evidence that melatonin acts directly on the regulation of ovary function. The mRNA expression of melatonin membrane receptors genes was detected in mammalian and non-mammalian ovaries. In spite of melatonin receptors and Asmt (limiting enzyme in melatonin biosynthesis) genes being present in cattle cumulus–oocyte complexes (COC), no information regarding rat COC has been reported. The aim of this study was to investigate the expression of melatonin receptor (Mt2) and melatonin synthesis enzyme (Asmt) genes at different meiotic cell cycle stages in COC from 27-day-old female rats. All the procedures involving animals were approved by the Animal Care Committee of the Institute of Biomedical Sciences. To obtain germinal vesicle (GV) immature COC from ovarian follicles, rats were treated with 20 IU equine chorionic gonadotropin (eCG) for induction of follicular development and killed by euthanasia 48 h later. To obtain COC at MII oocyte stage from oviducts, rats were injected with 20 IU of eCG and 48 h later with 20 IU of human chorionic gonadotropin (hCG), and then killed after 14 to 16 h. The oocytes in COC were separated from their cumulus cells by repeated pipetting through a narrow-bore pipette in culture medium. Oocytes and cumulus cells total RNA were isolated using a Trizol reagent (Invitrogen Corp., Carlsbad, CA, USA). Pools of 80 COC per cDNA sample were used. Quantitative real-time PCR (qRT-PCR) was performed (7500 Real-Time PCR System; Applied Biosystems Inc., Foster City, CA, USA) with 25-µL reactions containing 2 µL of cDNA (10 ng µL–1), SYBR green (Power SYBR Green; Applied Biosystems Inc.), and 400 nM specific intron-spanning primers. A set of 10-fold serial dilutions of each internal standard (102 to 106 copies/2 µL) was used to generate a standard curve. Transcript numbers were determined by the system software and normalized using the geometric mean calculated from the reference genes: Actb and Rpl37a. All the results were plotted as the mean ± SEM, and 4 replicates were performed. Unpaired t-test was used to evaluate the cell type (oocyte v. cumulus cells) differences. The qRT-PCR analyses revealed the presence of transcripts of the Mt2 gene only in oocytes from immature (GV) and mature (MII) COC. At both maturational stages, the copy numbers for Asmt in oocytes were significantly higher than in cumulus cells. The results confirm the presence of the Asmt gene in rat COC and suggest the possible involvement of these cells on melatonin biosynthesis. The presence of Mt2 transcript in immature and mature oocytes also suggests the potentially important role of melatonin in regulating the rat meiotic cellular cycle.
Research supported by FAPESP.
198 PROGESTERONE, ESTROGEN (ER-α AND ER-β), AND OXYTOCIN RECEPTOR GENE EXPRESSION IN CANINE EMBRYOS
Authors: Derussi A. A. P. , Castilho A. C. S. , Souza R. W. A. , Volpato R. , Guaitolini C. R. F. , Ackermann C. L. , Taffarel M. O. , Cardoso G. S. , Silva M. Dal Pai , Lopes M. D.
Published: December 2012

The aim of this study was to compare the mRNA levels of hormone receptor for progesterone (PR), oestrogen α (ER-α), oestrogen β (ER-β), and oxytocin (OTR) in canine morulae and blastocysts. Ten healthy mature bitches were inseminated based on monitoring vaginal cytology and progesterone concentration. The first insemination was performed on Day 2 after the preovulatory LH surge (progesterone 4 ng mL–1), and the second was performed 48 h later. All females were submitted to ovariohysterectomy (OVH), and the oviduct as well the uterurs were flushed with PBS solution to obtain the embryos. The females were divided into two groups: Group A (n = 5), morulae were collected 8 days after the LH surge and Group B (n = 5), blastocysts were collected 12 days after the LH surge. The pools (n = 10) of embryos (5 embryos/pool) were stored in RNAlater® (Ambion, Life Technologies, USA) at –80°C. The samples were analysed together. The RNA later was removed used PBS calcium free and the total RNA extraction was performed using the Qiagen RNeasy micro-kit (Hildesheim, Germany). Before reverse-transcription (RT) reaction, the total RNA was treated with DNase I Amplification Grade (Invitrogen Life Technologies, Carlsbad, CA, USA). The gene expression of target genes was assessed by real-time RT-qPCR, using SuperScript III for RT and power SYBR Green PCR Master Mix (Applied Biosystems, USA) for cDNA for PCR. The primers for target genes were designed using the software Primer Express® (Applied Biosystems, USA). The gene expression of target genes was normalized by HPRT gene and the relative abundance of mRNA was determined by the ΔΔct method corrected by amplification efficiency using Pffafl’s equation. The means of mRNA relative abundance were compared by t-test. The PR mRNA expression only in blastocysts is similar to the results obtained by Hou et al. (1997) in rat embryos. It is believed that the absence of PR in the early stages of cleavage is due to the indirect action of progesterone by growth factors produced by the maternal reproductive tract (2). Apparently, ER-β action does not occur in the embryo canine phases analysed; however, the action of ER-α seems related to the deployment signal as seen by Hou et al. (1996) in rats. Similarly to findings in the literature, OTR expression decreased in canine embryonic development. This receptor was produced by blastocysts while present in the uterus, which may represent an incidental mechanism to the embryo control of endometrial receptivity, such as also to prevent the development of endometrial luteolytic mechanism. The variation in hormone receptors gene expression in canine embryos can be influencing the transition from morula to blastocyst. In addition, a hormonal influence on these structures can occur in different ways.
199 EXPRESSION OF HAPTOGLOBIN mRNA IN THE OVIDUCT DURING THE OESTRUS CYCLE OF SOWS
Authors: Ramón M. C. , Acuña O. S. , Ruano M. J. , Avilés M. , Izquierdo-Rico M. J.
Published: December 2012

The haptoglobin is an acute phase protein that has been recently related with numerous events of mammalian reproduction. The objective of this study was to determine whether haptoglobin mRNA is expressed in the porcine oviduct and to analyse its expression during the different phases of the oestrus cycle. Porcine oviducts collected from a local abattoir were classified based on follicular morphology: prepuberal (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 obtained by extracting 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 d(T) as primer. This cDNA was used as template in RT-PCR and quantitative PCR (qPCR) amplifications using specific primers (Fw: gctacgtggagcacatggtt and Rv: ggagattcttagccgtggtc for RT-PCR and Fw: ggtgatgcccatttgcctccct and Rv: cagccaccggcagcatgaca for qPCR) designed based on GenBank sequence for Sus scrofa haptoglobin (NM_214000). The amplification by RT-PCR resulted in a 312-bp amplicon. This PCR product was sequenced and a 100% of identity with porcine haptoglobin sequence deposited in GenBank database was confirmed. On the other hand, analysis by qPCR revealed that the haptoglobin mRNA expression was more elevated in luteal and post-ovulatory phases than in prepuber and preovulatory phases. In conclusion, the haptoglobin mRNA is present in porcine oviduct and could be considered as a progesterone-dependent transcript. The role played by this protein in the porcine oviduct remains to be investigated.
This study was supported by MICINN (AGL2009-12512-C02-01-02).
200 DETECTION OF mRNA ENCODING HEPARANASE IN PORCINE OVIDUCT
Authors: Moros C. , Izquierdo-Rico M. J. , Acuña O. S. , Mondejar I. , Coy P. , Avilés M.
Published: December 2012

Polyspermy is an important anomaly of fertilization and is responsible for the low efficiency of IVF in the porcine species. It was reported that the exposure of oocytes to oviducal fluid increases monospermy after IVF in pigs. This fact has been related with the binding of OVGP1 to the ZP and is affected by heparin glycosaminoglycan. The OVGP1 is removed from the ZP during the embryo transit towards the uterus; however, the mechanism responsible for this process is unknown. Taking into account the contribution of the heparin in this process, the presence of heparanase in the oviduct milieu was postulated. This enzyme could degrade the heparin and unstabilised the binding of the OVGP1 to ZP. The aim of this study was to describe the presence of mRNA codifying for heparanase in the porcine oviduct. Oviducts from 4 animals in preovulatory phase were obtained. Epithelial cells from the oviducal mucosa (ampulla) was obtained by scraping, and total RNA was isolated using the RNAqueous® kit (Ambion, Austin, TX, USA) according to the manufacturer’s instructions. Complementary DNA was synthesised with an oligo dT as primer. This cDNA was used as the template in RT-PCR amplifications using specific primers designed based on GenBank sequence for Sus scrofa heparanase (NM_001146130.2). The amplification by RT-PCR resulted in a 199-bp amplicon. This PCR product was automatically sequenced and showed a high similarity with heparanase of different mammalian species. In conclusion, the heparanase mRNA is present in the porcine oviduct. This glycosidase could participate in the regulation of heparin content in the oviducal milieu. More studies are necessary to clarify its effect in the oviducal fluid and fertilization.
This study was supported by MICINN (AGL2009-12512-C02-01-02).
220 STEROID-REGULATED mRNA EXPRESSION IN OVIDUCT EPITHELIAL CELLS IN THE MARE
Authors: Nelis H. M. , Goossens K. , Leemans B. , Peelman L. , Van Soom A.
Published: December 2012

It is well known that oviducal physiology undergoes cyclical changes under the influence of steroid hormones associated with the establishment of an optimal microenvironment for fertilization and early embryonic development. Genes associated with the preovulatory phase are likely to be involved in modulation of gamete function and sperm storage, whereas those in the post-ovulatory phase are involved in embryonic development. So far, in the horse, no genes that change activity during the oestrous cycle, which may be important in fertilization, gamete and embryo passage, and early embryonic development, have been identified. Therefore, to identify steroid-regulated genes, we compared the mRNA expression of PAI-1, uPA, TGFA, TIMP-1, MMP-1, CSF, PTGER2, and PTGER4 in oviducal explants from mares in the pre- and the post-ovulatory early luteal stages. The cycle stage of healthy warmblood mares was determined in the slaughterhouse by assessment of ovarian morphology and uterine oedema. Oviducal explants were isolated by scraping the epithelium of oviducts ipsilateral to a preovulatory follicle or a recent corpus luteum and frozen in lysis buffer at –80°C until RNA extraction. The genes were quantified by RT-qPCR and normalized against the geometric mean of 6 validated reference genes (UBB, SDHA, 18S, TUB, ACTB, HPRT) according to the MIQE-guidelines (Bustin et al., 2009). Data were analysed using the independent samples t-test or the Mann–Whitney U-test. The mRNA expression of PAI-1, MMP-1, TGFA (0.01 < P < 0.05), and uPA (P = 0.007) was downregulated in the post-ovulatory phase, whereas no significant change in TIMP-1, CSF, PTGER2, and PTGER4 expression was observed. The results show that mRNA of components of the extracellular matrix turnover, such as PAI-1, uPA, MMP-1, and TIMP-1 as well as growth factors, such as TFGA and CSF and prostaglandin E2 receptors (PTGER2 and PTGER4), are produced. Moreover, down-regulation of PAI-1, MMP-1, TGFA, and uPA in the early luteal stage indicates that mRNA expression of these genes is steroid regulated. In other species, all these components are likely to protect gametes and the early embryo against proteolytic degradation and to stimulate embryonic development. Further transcriptome and proteome analyses are necessary to unravel changes in the oviducal epithelium during the oestrous cycle and early pregnancy in the mare.
222 EFFECT OF THE PRESENCE OR ABSENCE OF PERCOLL CENTRIFUGATION; PENICILLAMINE, HYPOTAURINE, AND EPINEPHRINE; AND HEPARIN ON IN VITRO PRODUCTION OF BOVINE EMBRYOS
Authors: Hasler J. F. , Stokes J. E.
Published: December 2012

Protocols for the production of bovine embryos in vitro routinely include Percoll centrifugation of semen, usually include heparin, and often include penicillamine, hypotaurine, and epinephrine (PHE) in the fertilization media. This study examined the contribution of each of these components to the success of in vitro fertilization of bovine oocytes and subsequent blastocyst development. Bovine oocytes were aspirated from 2- to 10-mm follicles within 5 h after slaughter of cattle at a local abattoir. Groups of 30 to 40 cumulus–oocyte complexes (COC) were matured in 0.5 mL of TCM-199 with 10% FCS, 4 µg mL–1 of FSH, and 6 µg mL–1 of LH (NOBL Laboratories, Sioux Center, IA, USA) for 24 h (39°C, 4% CO2 in air). The COC were then washed and placed in 0.5 mL of modified Tyrode-lactate medium for IVF with various combinations of 2 µg mL–1 of heparin, 20 µM penicillamine, 10 µM hypotaurine, and 1 µM epinephrine. Each group of COC was inseminated with 0.25 × 106 frozen–thawed sperm from a single bull after 30 min of centrifugation with (Exp. 1) or without (Exp. 2) a 45/90% Percoll gradient with sperm TALP. Oocytes were vortexed to remove the cumulus after 18 h and placed in co-culture wells containing a monolayer of buffalo rat liver cells and 0.5 mL of Menezo’s B2 medium supplemented with 10% FCS. On the fourth day of in vitro culture, cleavage was defined as 2 cells or greater and embryos were transferred to fresh co-culture wells. There were 4 replicates in the first experiment and 6 in the second. Data were analysed by ANOVA. In the first experiment, the use of a Percoll gradient during centrifugation for separation of viable sperm from seminal plasma and cryprotectants resulted in significantly higher cleavage and Day 8 blastocyst rates than did the absence of Percoll when PHE and heparin were used together, and both cleavage and blastocyst rates were lower when only PHE or heparin was used separately compared with when both were used together (Table 1). The absence of Percoll, PHE, and heparin resulted in the lowest rates of cleavage and development. In the second experiment, the absence of either PHE or heparin resulted in lower cleavage rates, but not blastocyst rates, compared with the use of both, and the absence of both resulted in the lowest cleavage and blastocyst rates in spite of the use of Percoll.

Table 1.Effects of Percoll; penicillamine, hypotaurine, and epinephrine (PHE); and heparin on cleavage and subsequent embryo development per oocyte
226 CRYOPRESERVED EJACULATED AND EPIDIDYMAL SPERM COLLECTED FROM THE SAME HOLSTEIN BULLS USED FOR IN VITRO FERTILIZATION
Authors: Stout M. A. , Saenz J. R. , Chenevert J. F. , Gentry G. T. , Bondioli K. B. , Godke R. A.
Published: December 2012

Exposure to seminal plasma may modify the ability of sperm to survive cryopreservation, undergo capacitation, and fertilize oocytes. The present work was designed to compare embryo development after IVF of oocytes with ejaculated and epididymal bovine sperm from bulls previously tested and showing similar responses to freezing. However, we also found that this ejaculated and epididymal sperm differed in their in vitro culture dynamics (capacitation, viability, and auto-acrosome reaction) and ability to fertilize oocytes in vitro. Ejaculated and epididymal sperm were collected from the same fertile mature Holstein bulls (n = 4) by artificial vagina and post-castration retrograde caudal epididymal flush, respectively. Collection of epididymal sperm was conducted 2 weeks after the last collection of ejaculated sperm. After collection, ejaculated and epididymal sperm were cryopreserved and stored in LN until use. Before IVF, a viable sperm population was isolated by centrifugation through a Percoll density gradient. Ejaculated and epididymal sperm were then added to fertilization drops at a final concentration of 1 × 106 mL–1, and IVF was conducted with and without the capacitation agent heparin. Oocytes were washed and randomly assigned to one of four treatment groups (ejaculated ± heparin or epididymal ± heparin). Embryo development was determined at 72 and 186 h after IVF. Differences in the mean values among treatment groups were analysed by one-way ANOVA, followed by the Holm-Sidak pairwise multiple comparisons. Embryo cleavage after IVF using ejaculated sperm without heparin (45.2%) was significantly lower (P < 0.05) than in all other groups. Cleavage rates of ejaculated sperm with heparin (56.9%) and epididymal sperm with (58.1%) and without (57.5%) heparin were found to be similar. No difference was noted between ejaculated and epididymal sperm in blastocyst development, although the inclusion of heparin did significantly (P < 0.01) increase blastocyst development in both ejaculated (9.3 compared with 23.6%) and epididymal sperm (2.5 compared with 23.3%). In conclusion, cryopreserved ejaculated and epididymal sperm collected from the same bulls can be successfully used for the in vitro production of bovine blastocysts without changing the existing protocols. This may increase the efficiency when using epididymal sperm in assisted reproductive techniques.