bluuebunny/crossref_metadata_2025 huggingface dataset:
162 FERTILITY OF BEEF RECIPIENTS FOLLOWING A FIXED-TIME EMBRYO TRANSFER PROTOCOL WHICH INCLUDED FOLLICLE STIMULATING HORMONE DILUTED IN HYALURONAN
Authors: Thorne J. W. , Looney C. R. , Hasler J. F. , Hockley D. K. , Forrest D. W.
Published: December 2012

This study was performed to test the viability of administering Folltropin-V® (FSH, Bioniche Animal Health) diluted in hyaluronan (MAP-5 50 mg, sodium hyaluronate, Bioniche Animal Health) to beef cows enrolled in a recipient synchronization protocol to evaluate its effect on recipient fertility. All recipients were administered an estradiol 17β (2.5 mg, IM) and progesterone (50 mg, IM) combination injection on Day 0, a CIDR® (progesterone 1.34 g, Pfizer Animal Health, Groton, CT, USA) was inserted for 7 days. Lutalyse® (dinoprost tromethamine, Pfizer Animal Health, 25 mg, IM) was administered at the time of CIDR removal on Day 7, and estradiol 17β (1 mg, IM) was administered on Day 8. On Day 16, the presence of at least one corpus luteum, detected via ultrasound, resulted in the recipient receiving an embryo (both fresh and frozen–thawed embryos were used). Embryos were not transferred into cows that did not show ultrasonic evidence of a CL. Dependent variables for which data were collected included circulating progesterone levels at the time of transfer and CL diameter, area, and circumference; measured in millimeters. The total study (n = 274) consisted of both wet (n = 85) and dry (n = 189) cows and included both Bos indicus (Brahman-influenced) crossbred (n = 93) and Bos taurus (Angus-based) cows (n = 181). The experiment consisted of cows being placed in either the treated or control groups, with treated cows receiving a single 40 mg (1 mL) IM injection of Folltropin-V in hyaluronan on Day 5 and control cows receiving no additional injections. Results are shown in Table 1. Transfer rate, conception rate, and pregnancy rate were tested for significance with chi-square analysis and remaining statistics were analyzed with a t-test: two-sample assuming equal variances. There were no significant differences found between the treated and control groups for transfer rate, conception rate, or pregnancy rate. Corpus luteum diameter was shown to be larger in control cows (P < 0.05); however, CL area and circumference did not differ significantly. Folltropin-V given with hyaluronan at a 40-mg dose on Day 5 did not improve fertility, induce a larger CL, or increase circulating progesterone levels in synchronized beef recipients as hypothesized. Further work is needed with Folltropin-V in hyaluronan to determine if an alternative dose or timing of administration would be more appropriate for improving fertility in recipients.

Table 1.Fertility data in beef recipients following synchronization for fixed-time embryo transfer with a protocol that included (Treated) or did not include (Control) FSH in hyaluronan
168 A NONINVASIVE, TRANSDERMAL ABSORPTION APPROACH FOR EXOGENOUS HORMONE INDUCTION OF SPAWNING IN THE NORTHERN CRICKET FROG, ACRIS CREPITANS: A MODEL FOR SMALL ENDANGERED AMPHIBIANS
Authors: Snyder W. E. , Trudeau V. L. , Loskutoff N. M.
Published: December 2012

The World Conservation Union (IUCN) currently recognises Eleutherodactylus locustus from Puerto Rico and Plectrohyla acanthodes from Mexico and Guatemala as critically endangered, and they are recommended for ex situ breeding by several amphibian conservation organizations (e.g. AZA, ATAG, RCP, and AARK). Because of their very small and delicate bodies, conventional injectable methods developed for hormonally stimulating spawning in larger frogs and toads are undesirable, because this can result in trauma and injury. Recently, a protocol was developed for inducing spawning in frogs by co-injection of a gonadotropin-releasing hormone agonist (GnRHa) with metoclopramide-HCl (MET), a dopamine antagonist (Amphiplex; Trudeau et al. 2010 Rep. Biol. Endo. 8, 36). The goal of this study, therefore, was to determine the effectiveness of Amphiplex using 3 routes of exogenous treatments: (1) direct IP injection (0.4 µg of GnRHa + 10 µg of MET per gram of body weight), (2) injection into prey (1 µg of GnRHa + 25 µg of MET per insect) before feeding, and (3) a 60-min immersion in 10 mL of pond water containing 0.17 µg of GnRHa + 0.42 µg of MET per microliter. The northern cricket frog (Acris crepitans), a very common local species, was used as a model because it is also small, delicate, and difficult to handle. A total of 110 cricket frogs were caught locally either late or after their normal breeding season, sexed as males or females, and segregated into a control group that was housed similarly ex situ but not exposed to Amphiplex and the 3 Amphiplex experimental groups. The frogs were monitored daily for egg production and behavioural amplexus (potential spermiation). The results are summarised as follows (Table 1): of the frogs exposed to the Amphiplex-supplemented pond water, 84% of 38 females deposited eggs, and 6 pairs were observed in amplexus (indicating spermiation). Approximately 27% of more than 2242 eggs were found to develop cleavage divisions, and tadpoles were first observed in vitro after 4 days. In conclusion, to our knowledge this is the first report indicating that transdermal absorption of a reproductive hormone cocktail (e.g. Amphiplex) may serve in the application of assisted reproductive technology to increase the populations of critically endangered amphibians such as frogs and toads that may be too small or delicate for conventional methods of exogenous hormone treatments that require handling and injections.

Table 1.Treated northern cricket frogs
17 MANIPULATION OF PROESTRUS PERIOD BY EXOGENOUS GONADOTROPIN AND ESTRADIOL DURING TIMED ARTIFICIAL INSEMINATION PROTOCOL IN SUCKLED BEEF COWS
Authors: Pitaluga P. C. S. F. , Sá Filho M. F. , Sales J. N. S. , Baruselli P. S. , Vincenti L.
Published: December 2012

The objective of this study was to evaluate the effect of eCG or estradiol cypionate (ECP) during the proestrus period on estrus occurrence, ovarian responses, and pregnancy per AI (P/AI) in suckled Bos indicus beef cows subjected to timed AI (TAI) protocols. A total of 393 cows in random stages of estrus (Day 0), received an intravaginal progesterone (P4) device (1.9 g of P4; CIDR®, Pfizer Animal Health, New York, NY, USA) and 2.0 mg of estradiol benzoate IM (EB; Gonadiol®, MDS Animal Health). Eight days later, the P4 devices were removed, and cows were given 0.15 mg of d-cloprostenol (Prostaglandina Tortuga®, Tortuga Companhia Zootécnica Agrária, São Paulo, Brazil). At this time, cows were randomly assigned to 1 of 3 treatments: 300 IU of eCG (Group eCG; n = 138), 300 IU of eCG plus 1 mg of ECP IM (Group eCG+ECP; n = 124), or no additional treatment (control; n = 131). Cows were timed inseminated 48 h after P4 device removal and were administrated simultaneously 100 µg of gonadorelin IM (Profertil®, Tortuga Companhia Zootécnica Agrária). A subset of cows (n = 98) were evaluated according to the occurrence of estrus between the P4 device removal and TAI and their ovarian follicles were evaluated by ultrasound at P4 device removal and corpus luteum (5 days after TAI). The data were analyzed using the GLIMMIX procedure of SAS (SAS Institute Inc., Cary, NC, USA) and differences with P < 0.05 were considered statistically significant. A greater pregnancy rate (P/AI; P = 0.04) was observed in cows receiving the eCG treatment at P4 device removal [eCG = 42% (58/138) and eCG+ECP = 50.8% (63/124)] than cows from the control group [29.8% (39/131)]. There was no additive effect of ECP supplementation on P/AI. Cows that received ECP [eCG+ECP = 56.3% (18/32)] displayed more estrus (P = 0.002) compared with those receiving eCG [eCG = 23.5% (8/34)] or only GnRH at TAI [control = 15.6% (5/32)]. In addition, regardless of the ECP supplementation, cows receiving the eCG treatment at P4 device removal [eCG = 85.3% (29/34) and eCG+ECP = 90.1% (29/32)] presented a greater ovulation rate than cows from the control group [65.6% (21/32)]. In conclusion, exogenous estradiol administrated at device removal increased the proportion of suckled Bos indicus cows that displayed estrus. Cows receiving eCG treatment (with or without estradiol supplementation) had greater ovulatory and pregnancy responses after the estradiol/P4-based synchronization protocol.
185 DEVELOPMENT CAPACITY OF PRE- AND POSTPUBERTAL PIG OOCYTES EVALUATED BY SOMATIC CELL NUCLEAR TRANSFER AND PARTHENOGENETIC ACTIVATION
Authors: Pedersen H. S. , Li R. , Liu Y. , Løvendahl P. , Holm P. , Hyttel P. , Callesen H.
Published: December 2012

Most of the porcine oocytes used for in vitro studies are collected from gilts. Our aims were to study development capacity of gilt v. sow oocytes (pre- and postpubertal respectively) using 2 techniques illustrating development competence [parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT)], and to describe a simple method to select the most competent oocytes. Inside-ZP diameter of in vitro-matured gilt oocytes was measured (µm; small ≤110; medium >110; large ≥120). Gilt and sow oocytes were morphologically grouped as good (even cytoplasm, smooth cell membrane, visible perivitelline space) or bad before used for PA (good and bad) or SCNT (good). The PA and SCNT were performed as before with minor modifications (Cryobiol. 64, 60; Cell. Reprogr. 13, 521) before culture for 6 days in a standard or timelapse incubator. Rates of cleavage (CL%, Day 2), blastocyst (BL%, Day 6), and blastocyst cell number (Hoechst 33342) were recorded. For PA embryos in a timelapse incubator (26 oocytes/group; 2 replicates), the first appearance of 2-cell stage was recorded. Between groups, CL% and BL% were analysed by chi-square and cell number by t-test. Results are presented in the table for the development of good oocytes after PA. The results show a low CL% of small-gilts compared with the other groups. The BL% increased with gilt-oocyte-diameter; however, sow oocytes reached the highest BL%. Total cell number was higher in sow than in gilt blastocysts. The SCNT experiments showed no differences in CL% (90–96) and blastocyst cell number (51–59) between groups. The BL% was higher in medium gilts and sows (41; 45) compared with large gilts (21). The BL% of bad oocytes was 1% from all 4 groups (176 oocytes, 25 replicates). Time interval for appearance of 2-cell stage for embryos developing into blastocysts showed no differences between groups (19–20 h). Within groups, this time interval showed a larger standard deviation for embryos not developing v. embryos developing into blastocysts. It is concluded that (a) sow oocytes have higher developmental capacity compared to gilts, (b) small gilt oocytes are not developmentally competent, (c) measurement of inside-ZP diameter, combined with morphological selection, is useful to remove non-competent oocytes. Further studies are needed to dissect the developmental capacity of medium and large gilt oocytes. Also, further timelapse studies may reveal a time interval in which the first cleavage of embryos with high developmental capacity takes place.

Table 1.Rates of cleavage (CL%), blastocyst (BL%), and total no. of cells (mean ± SEM) in blastocysts of PA embryos from gilts and sows1
186 EFFECT OF POLYVINYLPYRROLIDONE SUPPLEMENTATION IN CULTURE MEDIUM ON THE GROWTH OF MOUSE OOCYTES
Authors: Mizumachi S. , Sasaki K. , Matsubara K. , Hirao Y.
Published: December 2012

A high volume of polyvinylpyrrolidone (PVP) supplementation in culture medium has a significant impact on the growth of bovine oocytes. The objective of the present study was to determine whether or not PVP affects oocyte growth in the mouse. Oocyte–granulosa cell complexes were isolated from 11- or 12-day-old mice (ICR) by mechanical isolation of follicles, followed by a collagenase treatment (0.1%; 10 min). Twenty complexes were placed on each insert fit in the 24-well culture plate and cultured for 10 days in an atmosphere of 5% CO2 in air at 37°C. The culture medium was a modified α-MEM supplemented with 5% fetal bovine serum and 1 ng mL–1 FSH. The concentration of PVP (molecular weight of 360 000) was 0%, 1%, 2%, or 3% (w/v). During the first 2 days, only medium with 0% PVP was used. The oocytes recovered on Day 10 were subjected to in vitro maturation, IVF, and embryo culture. In 12 replications, the total numbers of oocytes cultured in medium with 0%, 1%, 2%, and 3% PVP were 235, 233, 233, and 231, respectively. In some additional experiments, oocytes were fixed on Day 10 and processed for transmission electron microscopy (TEM). The oocytes in medium with 0% PVP became located within an enlarged dome-like structure. In medium with 2% PVP and 3% PVP, no such domes were formed, and the oocytes within several granulosa cell layers were exposed to medium; however, the cumulus cell mass specifically became larger than that in medium with 0% PVP. The viabilities of oocytes recovered from medium with 0%, 1%, 2%, and 3% PVP were 83%, 81%, 91%, and 93%, respectively. The survival rate was significantly higher in medium with 3% PVP than in medium with 0% PVP or 1% PVP (P < 0.05). The mean oocyte diameter increased from 59 µm (Day 0) to 72, 71, 71, and 72 µm in medium with 0, 1, 2, and 3% PVP, respectively, but they continued to be smaller than in vivo grown oocytes (81.0 µm; P < 0.01). When maturation was induced, cumulus cell mucification occurred irrespective of PVP concentration during the growth. No significant differences were found between the groups in the percentage of polar body extrusion (ranging from 78 to 88%). Developmental outcomes based on oocytes used for in vitro fertilization were the following: cleavage rates were 67, 78, 74, and 76%; and blastocyst rates were 37, 44, 47, and 36% of oocytes that had been grown in medium with 0, 1, 2, and 3% PVP, respectively. The numbers of oocytes included were 60, 59, 68, and 66, respectively. The TEM observation suggests that more intimate contacts were maintained between the oocyte and cumulus cells in medium with 2% PVP than in medium with 0% PVP. Taken together, PVP supplementation in medium has a considerable influence on the morphology of mouse oocyte–granulosa cell complexes and close contacts within the complexes in the long-term culture, as having been observed with bovine oocytes.
193 BISPHENOL A AND PHTHALATE ENHANCED THE GROWTH OF PROSTATE CANCER CELLS AND ALTERED TGF-β SIGNALING MOLECULES VIA AN ESTROGEN RECEPTOR OR ANDROGEN RECEPTOR-DEPENDENT PATHWAY IN IN VITRO AND IN VIVO MODELS
Authors: Lee H.-R. , Hyun S.-H. , Jeung E.-B. , Choi K.-C.
Published: December 2012

Endocrine-disrupting chemicals (EDC) can bind to the hormone receptor and induce an unexpected hormone response to activate oestrogen receptor (ER)- and androgen receptor (AR)-mediated signalling pathways. Among EDC, bisphenol A (BPA) has a detrimental effect on the endocrine system and is suspected to promote human breast and ovarian cancers. Recent studies have reported that phthalate can disrupt the endocrine system and has a weak estrogenic activity with binding to ER. In this study, we demonstrated whether BPA and dibutyl phthalate (DBP) stimulate the proliferation of prostate cancer cells, LNCaP cells, which have both ER and AR. We evaluated the proliferative rate of LNCaP cells following BPA and DBP treatment using a cell viability assay compared with EtOH treatment as a negative control. Further, we examined the alteration of cell cycle-related gene expressions and TGF-β signalling molecules by semiquantitative RT-PCR. Both BPA and DBP increased LNCaP cell growth more than 2-fold. Moreover, these EDC altered transcriptional expressions of cell cycle-related genes, cyclin D1 and p21, at 6 h in LNCaP cells after exposure of BPA and DBP. Like 17β-oestradiol (E2) and dihydrotestosterone (DHP), treatments of BPA and DBP lead to an increase of the transcriptional levels of c-myc and c-fos in LNCaP cells from 30 min to 6 h. In addition, BPA and DBP decreased the protein level of not only p-smad but also total smad, suggesting that these EDC can affect the molecules of the TGF-β signalling pathway. It was of interest that these effects of EDC were reversed by an antagonist of ER or AR signalling pathways in these prostate cancer cells. These results suggest that BPA and phthalate can alter various gene expressions in TGF-β signalling molecules and stimulate cell growth in prostate cancer cells in vitro. In addition, the growth of prostate cancer cells was stimulated following the exposure of E2, DHT, and DBP in vivo. Taken together, these results indicate the potential of BPA and phthalate in the carcinogenesis of prostate cancer by the oestrogen or androgen-dependent signalling pathway.
This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Ministry of Education, Science and Technology (MEST) of Korea government (no. 2011-0015385).
209 9-cis RETINOIC ACID INHIBITS EXPRESSION OF AKR1B1 TRANSCRIPT IN THE BOVINE OOCYTES AND PRE-IMPLANTATION EMBRYOS
Authors: Deb G. K. , Dey S. R. , Huque K. S. , Fokruzzaman M. , Lee K. L. , Bang J. I. , Kong I. K.
Published: December 2012

Quantitative real-time PCR has enabled quality evaluation of oocyte and pre-implantation embryo through monitoring expression of several molecular markers that are involved in metabolic activity, stress response, reprogramming, and other biological events. The aldo-keto reductase family 1 member B1 (AKR1B1) transcript is potentially involved in pregnancy failure through metabolism of progesterone and synthesis of prostaglandin F2α in the bovine uterine endometrium. High expression of the transcript in blastocysts correlates inhibition of embryo implantation and/or embryo resorption. Maturation of immature oocyte in presence of 9-cis retinoic acid (9-cis RA) increases in vitro bovine embryo development rates and embryo quality. These beneficial effects of 9-cis RA are mediated through multiple mechanisms, including FSH/LH receptor expression, polyadenylation, growth factor signalling, oxidative-stress protection, or decreasing oocyte TNFα gene expression and inhibiting cumulus cell apoptosis during maturation. The present study aimed to evaluate the effect of 9-cis RA on expression pattern of AKR1B1 transcript in the oocyte matured in vitro and embryos (8-cell and Day 8 blastocyst) produced from in vitro matured oocytes in presence or absence of 9-cis RA. Bovine cumulus–oocyte complexes, isolated from ovaries collected at the abattoir, were matured in vitro in the presence of zero (control) or 5 nM 9-cis RA in the maturation medium (TCM199 + 10% fetal bovine serum + 1 µg mL–1 β-oestradiol + 10 µg mL–1 follicle stimulating hormone + 0.6 mM cystein and 0.2 mM Na-pyruvate). After maturation, the oocytes were subjected to standardized in vitro embryo production protocol or oocyte samples were collected for gene expression analysis. The expression of AKR1B1 transcript was quantified in zona-free oocytes, 8-cell embryos, and Day 8 blastocysts by real-time PCR using SYBER green. Not less than 4 biological replicates (oocytes: 50 to 60 per replicate and 8-cell embryos/day-8 blastocyst: 3 to 5 per replicate) were done for each group. The expression was normalized against a minimum of 2 out of 4 reference transcripts (18S rRNA, β-actin, glyceraldehyde-3-phosphate dehydrogenase and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide) analysed each time with AKR1B1. The best combination of reference genes was automatically calculated by the CFX manager V1.1 program (Bio-Rad) based on M-value. The differences in gene expression levels were tested by Student’s t-test. Results indicated that 9-cis RA decreased expression of AKR1B1 transcript in the oocyte (1.0- v. 2.0-fold; P < 0.05), 8-cell-embryos (1.0- v. 10.1-fold; P < 0.03), and blastocyst (1.0- v. 2.1-fold; P < 0.03) compared with control. In conclusion, the present study indicates that 9-cis RA inhibits AKR1B1 transcript expression in oocytes and pre-implantation embryos.
219 EXPRESSION PATTERN OF THE SUB-CORTICAL MATERNAL COMPLEX IN OVINE OOCYTES AND PRE-IMPLANTATION EMBRYOS
Authors: Bebbere D. , Bogliolo L. , Ariu F. , Murrone O. , Strina A. , Nieddu S. , Rosati I. , Ledda S.
Published: December 2012

The sub-cortical maternal complex (SCMC) is a multi-protein complex located in the sub-cortex of the oocyte. In mouse, it assembles during oocyte growth and is essential for zygotes to progress beyond the first embryonic cell divisions (Li et al., 2008). At least 4 proteins contribute to the complex: oocyte expressed protein (OOEP), maternal antigen that embryo requires (MATER), transducin-like enhancer of split 6 (TLE6), and ES cell associated transcript 1 (ECAT1), all encoded by maternal effect genes. In mouse, the relative transcripts are degraded during meiotic maturation and ovulation, whereas the SCMC proteins persist in the early embryo. Whereas MATER expression has been studied in several species, the existence of the genes encoding the other components has been assessed in few mammalian species and their pattern of expression during pre-implantation development has been analysed only in mouse (Li et al. 2008 Dev. Cell 15, 416–425). In a previous work (Bebbere et al. 2008 Reprod. Fertil. Dev. 20, 908–915), we assessed MATER existence and pattern of expression in the ovine species. The aim of the present work was to assess the existence of OOEP, TLE6, and FILIA in the ovine species and to analyse the expression pattern of the 4 genes in the oocytes and during pre-implantation embryo development. Total RNA was isolated and reverse transcribed from pools of immature (GV) and in vitro matured (IVM) metaphase II (MII) oocytes, from in vitro fertilized and cultured (IVFC) embryos at the 2-, 4-, 8-, and 16-cell stage and from blastocysts. Three pools of 10 oocyte/embryos were analysed for each class. Primers were designed on the basis of the sequences conserved among orthologs and amplify intron-spanning regions. The PCR products were sequenced, and the alignment, performed with BLASTn, confirmed the homology with the orthologous genes present in public databases. Real-time PCR analysis revealed that all 4 transcripts are present at its highest level in the GV oocyte but decrease during embryo pre-implantation development with a gene-specific pattern. Conversely to the pattern of expression observed in mouse, all 4 transcripts persisted until the 8-cell stage embryo, disappearing only at the 16-cell stage. No transcripts were detected at the blastocyst stage. This study confirms the existence of transcripts related to SCMC also in the ovine species, but highlights species-specific patterns of expression in the 2 species, possibly related to the different time of activation of the embryo genome in mouse and in sheep. The observed expression patterns suggest an involvement of the protein complex in oocyte maturation and in the very first phases of life, possibly in the transition from the maternal to embryonic program of development.
22 EFFECT OF CHOLESTEROL ADDITION TO EQUINE SPERM MEMBRANE ON FERTILITY
Authors: Hartwig F. P. , Lisboa F. P. , Monteiro G. A. , Maziero R. R. D. , Alvarenga M. A. , Papa F. O. , Dell'aqua J. A.
Published: December 2012

Artificial insemination with cooled-shipped semen has been widely used in horse breeding. However, some stallions, referred to as poor coolers, present abrupt fertility decrease when their semen is processed, cooled, and transported. Cholesterol incorporation into sperm membranes improves the quality of cryopreserved semen by increasing the sperm membrane stability and fluidity at low temperatures. Despite the beneficial effect of cholesterol addition on sperm quality, studies demonstrate that the presence of large amounts of cholesterol in the plasma membrane interferes with the physiological process of sperm capacitation and is detrimental to frozen equine sperm fertility. The aim of this study was to assess the fertility of cooled semen from good-cooler and poor-cooler stallions after adding cholesterol to sperm membranes. Two stallions were used and classified as good cooler (n = 1) and poor cooler (n = 1) based on the ability to maintain sperm progressive motility after 24 h of cooling at 5°C. For classification of the stallions, the fertility history was also taken into account through the results of pregnancy per cycle using inseminations with cooled semen (<50% for poor cooler and >70% for good cooler). Ejaculates of these stallions were subjected to 2 treatments: control (CON) and cholesterol (CLC). In the CON group, the semen was extended to 30 × 106 sperms mL–1 with skim milk-based extender (BotuSemen™). In the CLC group, the semen was extended as in the CON group plus 0.25 µL/1 × 106 sperms of 6.1 mM cholesterol-loaded cyclodextrin was added. Afterwards, both treatments were cooled at 5°C for 24 h. To test the fertility of poor-cooler and good-cooler stallions, 2 cycles from 25 mares and 2 cycles from 10 mares were respectively used. For both stallions, randomly for each mare, the inseminations were performed by alternating both treatments. If the mare was first inseminated with the CLC treatment, in the next cycle the CON treatment was used and vice versa. After 24 h of ovulation induction, the inseminations were done in the uterine body with 1 × 109 viable cells. Statistical analyses were performed using the Fisher exact test and significance was set at P < 0.05. For the poor cooler, the CON treatment presented 44% pregnancy/cycle compared to 76% for the CLC treatment (11/25a v. 19/25b). For the good cooler, both treatments presented 80% (8/10) pregnancy/cycle. The results suggest that the fertility capability of stallions is not affected by incorporation of cholesterol-loaded cyclodextrin to the sperm plasma membrane. Additionally, the utilization of cholesterol-loaded cyclodextrin may be an option to enable the utilization of cooled-shipped semen from poor cooler stallions for AI programs.
23 RELATIONSHIP BETWEEN CONCENTRATION OF SUPEROXIDE DISMUTASE WITH TIME AND TIMED ARTIFICIAL INSEMINATION PREGNANCY RATE IN COWS WITH HIGH MILK PRODUCTION
Authors: Maturana Filho M. , Santin T. , Nishimura T. K. , Nunes Barbosa V. , Naves J. R. , Arantes Fonseca G. , Sucupira Araripe M. C. , Hoffmann Madureira E.
Published: December 2012

Disorders related to oxidative metabolism, which occur in the production cycle of high-producing dairy cows (HPC) are more frequent during the transition period and early lactation. Blood concentrations of the enzyme superoxide dismutase (SD) are closely related to the activity of stabilizing conditions of oxidative stress (OS). It is very important to know the main mechanisms involved in regulation of metabolic events, to develop new strategies to increase the productive and reproductive performance of HPC. The objective of this study was to evaluate differences in pregnancy rates and pregnancy losses of HPC with different concentrations of SD with fixed-time AI (FTAI). The experiment was conducted on a commercial farm with 140 HPC (multiparous pregnant Holsteins). The evaluations of concentrations of SD were done from 21 days before birth to the day of the first-service FTAI (75 days in milk), with intervals of 14 days. The animals were retrospectively divided into 2 groups (G1 and G2) in accordance with the average output from all cows in peak lactation (4–8 weeks). Data were analyzed using logistic regression to determine the pregnancy rates between groups. There was a time effect and group effect for the concentrations of SD (P < 0.05) only on the first day of FTAI (3054.4a ± 37.4 and 3662.4 ± 42.1b) for G1 and G2, respectively. But this was not reflected in differences in pregnancy rates between the experimental groups (G1 and G2) and 30 (26.7 v. 31.4) and 60 (20 v. 19.6) days. Although the dismutation activity was high, it seems that the plasma concentration of this enzyme alone does not explain the effects of oxidative stress on fertility in HPC.
230 EVALUATION OF APOPTOSIS IN IN VITRO-PRODUCED BOVINE EMBRYOS MATURED WITH FORSKOLIN
Authors: Paschoal D. M. , Sudano M. J. , Maziero R. R. D. , Guastali M. D. , Crocomo L. F. , Vergara L. E. , Magalhães L. C. O. , Lima-Neto J. F. , Rascado T. S. , Martins A. , Landim F. C.
Published: December 2012

The maintenance of oocytes in the germinal vesicle stage for a few hours could result in more competent oocytes for use in biotechnology. Related to this, forskolin (Sigma-Aldrich, St. Louis, MO, USA) is an efficient inhibitor of nuclear maturation because of its ability to increase the levels of intracellular cyclic adenosine monophosphate. This study aimed to show whether the use of forskolin would be able to inhibit maturation in bovine oocytes, producing a higher rate of in vitro embryos. Nellore oocytes from a slaughterhouse (n = 960) were matured in TCM-199 with Earle’s salt + 10% FCS, FSH, and LH, in a 5% CO2 atmosphere. To delay meiosis, the oocytes were maintained for 6 h in medium with forskolin at 3 different concentrations, 0. 1 mM (n = 240), 0.05 mM (n = 240), and 0.025 mM (n = 240), whereas untreated oocytes acted as controls (n = 240). The oocytes were then cultured for 18 h in agent-free medium to resume meiosis, completing 24 h of maturation. After 24 h (Day 0) of maturation, oocytes were fertilized in human tubal fluid (HTF, Irvine, New Zealand) under the same conditions as described above. Semen was selected through Percoll gradient, and the concentration was adjusted to 2 × 106 sperm mL–1. The presumed zygotes were cultured in 90-µL droplets of SOFaa + 0.6% BSA + 2.5% FCS in a 5% CO2, 5% O2, and 90% N2 atmosphere until Day 7, when blastocysts were evaluated. Apoptosis in blastocysts was accessed through TUNEL reaction. Data were analysed by ANOVA, followed by Tukey’s test using the general linear models procedure (PROC GLM) of SAS (SAS Institute Inc., Cary, NC, USA). The level of significance adopted was 5%. No statistical differences were observed in blastocyst production rate (n = 297): control (n = 88): 36.7% ± 3.7; 0.1 mM forskolin (n = 61): 25.1% ± 3.7; 0.05 mM forskolin (n = 70): 29.2% ± 3.7; 0.025 mM forskolin (n = 78): 32.6% ± 3.7 (P > 0.05). However, when we analysed the apoptosis rates, differences were found among groups: control: 6.0% ± 6.3a; 0.1 mM forskolin: 33.4% ± 6.3b; 0.05 mM forskolin: 27.2% ± 6.3ab; 0.025 mM forskolin: 10.0% ± 6.3ab (P < 0.05). Although there was no difference in blastocyst rate, the TUNEL technique allowed us to identify that a high dose of forskolin was detrimental for in vitro-produced bovine embryos.
FAPESP: 10/50410-2.
239 INFLUENCE OF BREED PREDOMINANCE ON MATURATION COMPETENCE, FERTILIZATION, AND DEVELOPMENT IN VITRO OF BOVINE OOCYTES
Authors: Pelaez J. , Hernandez-Fonseca H. , Pirela A. , Baez F. , Villamediana P. , Urribarri Y.
Published: December 2012

The purpose of this research was to compare the competence of bovine oocytes of different breed predominance (Bos taurus v. Bos indicus) to mature and to be fertilized. This was done through the collection, selection, maturation, and fertilization of oocytes from slaughtered cows, predominantly either B. taurus or B. indicus. Only cows that were at least 5/8 B. taurus or 5/8 B. indicus, according to a series of phenotypic characteristics, such as the presence of a hump, dewlap, length of the ears, and others, were selected. To obtain cumulus–oocyte complexes, ovarian follicles (3 to 10 mm in diameter) were aspirated, and only oocytes with 2 or more layers of cumulus cells, an intact zona pellucida, and a homogeneous granular cytoplasm were selected. After selection, oocyte maturation [in vitro maturation (IVM)] and IVF were done. Frozen–thawed semen was used from one Brahman bull (B. indicus). For the evaluation of IVM as for IVF, oocytes were fixed for approximately 24 h at 4°C in a solution of ethanol : acetic acid (3 : 1). They were then stained with 1% acetic orcein. A chi-squared test was performed for all reported rates. The rate of maturation of oocytes from cows with a predominant B. indicus phenotype was 66.93%, whereas cows with a B. taurus phenotype reached 43.10% (P < 0.001). Regarding the fertilization rate, predominantly B. indicus females had 43.68% of oocytes normally fertilized and 41.74% of oocytes were abnormally penetrated. This category included polyspermic and asynchronic (abnormally developed pronucleus) oocytes. In cows with B. taurus predominance, 31.96% of oocytes were normally penetrated and 46.39% were abnormally penetrated by spermatozoa (no significant differences were found). The rate of non-fertilized oocytes was significantly different (P < 0.05) among B. indicus and B. taurus oocytes (6.79 and 17.52%, respectively). A small and nonsignificant proportion of degenerated oocytes resulted in both groups (7.79% for B. indicus and 4.14% for B. taurus). The cleavage rate was not different among phenotypic groups (36.12 and 32.30%, respectively, for B. indicus and B. taurus). In conclusion, the present results indicate that oocytes from predominantly B. indicus cows were more competent than oocytes from cows with a predominance of the B. taurus breed. Nonetheless, this superiority was not evident in terms of cleavage rates. Semen from other B. indicus and B. taurus breeds must be tested to clarify any breed interactions.
243 IMPACT OF MATERNAL LOW-DOSE ESTRADIOL-17β EXPOSURE DURING PREGNANCY ON THE REPRODUCTIVE ORGANS OF MALE PIG PROGENY
Authors: Fürst R. W. , Pistek V. L. , Bauersachs S. , Waberski D. , Meyer H. D. D. , Ulbrich S. E.
Published: December 2012

In male developing piglets, low endogenous oestrogenic hormone levels prevail. Sensitive tissues expressing high levels of oestrogen receptors, such as the epididymis, might thus be of specific relevance for early disturbances by estrogenic endocrine disruptors. We have previously shown that distinct concentrations of the natural oestrogen oestradiol-17β (E2) orally applied during the whole period of pregnancy in sows lead to a distinct shift in body composition. Therefore, we aimed at investigating whether prenatal E2 exposure had the potential to program male reproductive organs toward altered semen quality. The E2 treatment groups (n = 4 sows/group) were designed to represent doses of pharmacological relevance (PR; 1000 µg kg–1 of body weight per day), oral no-effect level (10 µg kg–1 of body weight per day), and acceptable daily intake level (0.05 µg kg–1 of body weight per day). Total RNA was extracted from the epididymis of 8-week-old piglet twins (n = 2 per sow). The RNA of littermates was pooled and subjected to microarray analysis (MA) using Agilent Porcine Genome Arrays (Agilent Technologies Inc., Santa Clara, CA, USA). Ejaculates of adult male pigs from the control group and the pharmacological group (n = 4) were collected and analysed for quantitative and qualitative parameters by using computer-assisted sperm analysis and flow cytometry. In total, 105 transcripts exhibited a significant difference of more than 1.5-fold for at least one of the treatment groups compared with the control group (P < 0.05; FDR 5%). Several transcripts revealed gradually decreasing amounts subject to the dose of E2 exposure. Eight transcripts showed significant differences in all 3 treatment groups compared with the control group. Thirty-two transcripts had reduced abundance in the PR group and the oral no-effect level group, whereas 20 transcripts were downregulated in the PR group only. Semen parameters of adult males did not differ regarding volume and semen concentration, morphology, and membrane integrity. Computer-assisted sperm analysis evaluations did not show differences in motility and other kinematic parameters. Eight weeks after E2 exposure, male prepubertal piglets exhibited significant differences in epididymal messenger RNA transcript abundance subject to an in utero E2 treatment. This points toward possible epigenetic programming of the epididymis. Although the differential transcript abundance indicates a perturbation, normal semen quality was found after adolescence. Either these transcriptional changes are of minor functional importance or flexible mechanisms account for the developmental adaptations. Considering that identical transcripts were regulated in offspring exposed to both pharmacological and very low doses, further investigations of set points obviating long-term adverse consequences are needed.
251 DEVELOPMENTAL AND BIOENERGY/OXIDATIVE CHARACTERIZATION OF PREPUBERTAL OVINE OOCYTES MATURED IN VITRO
Authors: Martino N. A. , Russo R. , Filioli Uranio M. , Bogliolo L. , Amati F. , Sardanelli A. M. , Lacalandra G. M. , Dell'Aquila M. E.
Published: December 2012

Juvenile in vitro embryo transfer in farm animals reduces the generation interval and increases the rate of genetic gain. In human reproductive medicine, it enables the preservation of female fertility of young patients affected by cancer or by premature ovarian failure. The developmental competence of in vitro-produced juvenile embryos is strictly related to oocyte quality. The aim of the present study was to analyse the developmental potential and the mitochondrial/oxidative status of ovine prepubertal oocytes matured in vitro to clarify their suitability in juvenile in vitro embryo transfer programs. Oocytes from the ovaries of slaughtered prepubertal lambs (less than 6 months of age) were analysed after in vitro maturation. After cumulus cell removal, oocytes at the metaphase II stage (MII) underwent either IVF plus in vitro embryo culture (Experiment 1; n = 200; Bogliolo et al. 2011 Reprod. Fert. Dev. 23, 809–817) or confocal analysis of mitochondria (mt) and reactive oxygen species (ROS) fluorescence distribution, intensity, and colocalization (Experiment 2; n = 30; Martino et al. 2012 Fertil. Steril. 97, 720–728) or scavenger enzyme [superoxide dismutase (Ambruosi et al. 2011 PLoS ONE 6, e27452) and catalase (Beers and Sizer 1952 J. Biol. Chem. 195, 133–140)] activity analyses in cell lysates of individual oocytes (Experiment 3; n = 7). In Experiment 1, 150 of 200 MII oocytes (75%) cleaved after 30 h of in vitro embryo culture, and 36 of 150 2- to 4-cell-stage embryos (24%) reached the blastocyst stage at Day 8. In Experiment 2, 60 of 111 (54%) oocytes selected for in vitro maturation culture reached the MII stage, and 30 of them (50%) with a regular size (>150 µm in diameter) and morphology were analysed for bioenergy/redox parameters. Fourteen of 30 oocytes (47%) showed a heterogeneous (perinuclear, pericortical, or both) mt distribution pattern, and the remaining 16 of 30 oocytes (53%) showed a homogeneous distribution of small mt aggregates. Intracellular ROS were uniformly distributed, thus not corresponding to the mt distribution pattern. Fluorescent intensity of mt and ROS labelling, expressed as arbitrary densitometric units, were 821.4 ± 274.7 and 737.6 ± 226.5 in oocytes with a heterogeneous pattern and 723.7 ± 371.6 and 831.7 ± 263.7 in oocytes with a homogeneous pattern, respectively (not significant). The mt/ROS colocalization (Pearson correlation coefficient) did not differ between heterogeneous (0.47 ± 0.2) and homogeneous (0.51 ± 0.09; not significant) oocytes. In Experiment 3, superoxide dismutase (n = 4) and catalase activity (n = 3) values were 1.09 ± 0.03 and 10.63 ± 1.96 IU mg–1 of protein, respectively. This study provides basal values of bioenergy/redox parameters in prepubertal lamb MII oocytes as related to their developmental potential and may increase the knowledge of prepubertal oocyte physiology compared with their young adult counterparts.
263 DESCRIPTION OF CORTICAL GRANULE DISTRIBUTION IN OVINE MATURED OOCYTES: PRELIMINARY RESULTS
Authors: Padilha L. C. , Saraiva N. Z. , Dell Collado M. , Teixeira P. P. M. , Pires-Buttler E. A. , Apparício M. , Vicente W. R. R.
Published: December 2012

In mammalian oocytes, the migration of cortical granules (CG) is an important step in cytoplasmic maturation and has been used as a criterion in the assessment of maturity and organelle organisation. With the use of fluorescent lectins, it is possible to identify specific sugars originating from the CG and to localise them to the oolemma, within the perivitelline space, or both. To our knowledge, no reports are available on fluorescence assessment of CG organisation in ovine oocytes. Therefore, the aim of this study was to describe the CG distribution of ovine oocytes after in vitro maturation (IVM). One hundred twenty-five oocytes from adults ewes were submitted to IVM in TCM-199 medium supplemented with 10% FBS for 24 h. The denuded oocytes were immersed in 0.1% pronase for 5 min to dissolve the zona pellucidae, fixed in 3% paraformaldehyde for 30 min, and incubated overnight in blocking solution at 4°C. Oocytes were placed into 0.1% Triton X-100 for 5 min for permeabilization and incubated in 10 µg mL–1 of fluorescent Lens culinaris agglutinin–fluorescein complex for 15 min at 37°C before being rinsed and mounted onto histological slides. The distribution of CG was evaluated with an Olympus fluorescence microscope (wavelength: 420 to 490 nm; Olympus, Tokyo, Japan), and oocytes were classified into three types according to the observed distributional pattern of the CG: (I) aggregates of CG distributed over the entire cytoplasm, (II) CG distributed in the cortex and forming a fluorescent halo around the plasma membrane, and (III) CG distributed uniformly over the entire cytoplasm without the presence of aggregates. Proportions of oocyte types were analysed by chi-square or, when appropriate, by Fisher’s exact test, using SAS version 8.2 (SAS Institute Inc., Cary, NC, USA). The proportion of type III oocytes (98/125, 78.4%) was significantly higher (P < 0.05) than those of types I (10/125, 8%) and II (17/125, 13.6%). No significant difference was observed between the proportions of type I and II oocytes. Analogous with the results described in the bovine, the type I distribution of CG in ovine oocytes has been considered a sign of a lack of maturation, even if the aggregates of CG are not as evident as in the bovine; types II and III were considered mature oocytes based on the cortical migration of CG. The cytoplasmic maturation is an essential process in directing and supporting the events of fertilization and early embryonic development. Therefore, describing the species-specific pattern of CG distribution is important for the assessment of oocyte maturation. More studies in the ovine are necessary to confirm the correlation between CG distribution and maturation status, as in bovine species.
270 HIGH GASEOUS PRESSURE PRETREATMENT IN IN VITRO MATURATION OF CANINE OOCYTES
Authors: Rodrigues B. A. , Rodrigues C. A. , Salviano M. B. , Willhelm B. R. , Collares F. J. F. , Rodrigues J. L.
Published: December 2012

Stress processes, such as hydrostatic pressure treatment of oocytes in different species, have been reported to increased embryo rate following in vitro maturation (IVM). However, studies on high gaseous pressure (HGP) pretreatment in IVM of oocytes from domestic animals are lacking in the literature. This experiment aimed to test HGP pretreatment of canine oocytes to increase meiosis achievement (metaphase II) after IVM. A total of 502 canine oocytes (6 replicates) were used in this study. Ovaries from 15 bitches were obtained from local shelters or rescue organizations after ovariohysterectomy. Sample collection was blind as to reproductive stage and dog age. The ovaries were transported to the laboratory in 0.9% NaCl and were processed within 3 h of collection. The ovarian cortex was sliced and washed in PBS with 1% FCS to release cumulus–oocyte complexes. Grade 1 and 2 cumulus–oocyte complexes were selected for IVM and randomly distributed into 3 treatment groups: HGP (oocytes placed in PBS and subjected to pressure chamber; 206 oocytes), ambient control (oocytes maintained in TCM-HEPES at room temperature for 60 min; 130 oocytes), and laboratory protocol (oocytes IVM after morphologic selection; 166 oocytes). The average pressure, initial and final temperature, and duration of oocytes in the HGP pretreatment were 76.19 atm (±0.92), 32.20°C (±5.17) and 27.71°C (±3.17), and 60 min, respectively. In vitro maturation was carried out for 72 h at 37°C in a high-glucose medium, consisting of TCM-199 with 2.2 mg mL–1 of sodium bicarbonate (11150, Gibco, Grand Island, NY, USA), and supplemented with 0.1% polyvinyl alcohol (P-8136, Sigma, St. Louis, MO, USA), 0.991 mg mL–1 of glucose (108337, Merck, Darmstadt, Germany), 50 µg mL–1 of gentamicin, 22 µg mL–1 of pyruvic acid, 20 µg mL–1 of oestradiol (E-8875, Sigma), 0.5 µg mL–1 of FSH (Folltropin-V, Vetepharm Inca), 0.03 IU mL–1 of hCG (Chorulon®, Intervet, Kenilworth, NJ, USA), under 20% oxygen tension. The number of oocytes at each stage (prophase to metaphase II) was recorded according to the morphology of nuclear content after staining with Hoechst 33342. For comparison purposes of nuclear maturation in oocytes, data were analysed by Fisher’s exact test. Differences at a P-value ≤0.05 were considered significant. Oocytes from the HGP, ambient control, and laboratory protocol groups had similar meiotic progression to the metaphase II stage (metaphase I–anaphase I–telophase I–metaphase II), and were 35.4% (73/206), 30.8% (40/130), and 34.9% (58/166), respectively (P ≥ 0.05). The proportion of oocytes without chromatin or having an irregular organisation was not different among groups. In conclusion, results indicate that HGP pretreatment as used in this experiment did not improve meiosis rates in IVM canine oocytes. Further investigations to understand the significance of HGP pretreatment in IVM and in vitro production of canine embryos are ongoing in our laboratory.
278 Jak/Stat3 SIGNALING PROMOTES SOMATIC CELL REPROGRAMMING BY EPIGENETIC REGULATION
Authors: Tang Y. , Luo Y. , Jiang Z. , Carter M. , Tian X. (Cindy)
Published: December 2012

Although leukemia inhibitory factor (LIF) maintains the ground state pluripotency of mouse embryonic stem cells and induced pluripotent stem cells (iPSC) by activating the Janus kinase/signal transducer and activator of transcription 3 (Jak/Stat3) pathway, the mechanism remains unclear. Stat3 has only been shown to promote complete reprogramming of epiblast and neural stem cells, and the partially reprogrammed cells (pre-iPSC). We investigated if and how Jak/Stat3 activation promotes reprogramming of terminally differentiated mouse embryonic fibroblasts (MEF). We demonstrated that activated Stat3 not only promotes but is essential for the pluripotency establishment in MEF cell reprogramming. We further demonstrated that, during reprogramming, inhibiting Jak/Stat3 activity blocks demethylation of Oct4 and Nanog regulatory DNA sequences in induced cells, which are marked by suppressed endogenous pluripotent gene expression. These are correlated with significant upregulation of DNA methyltransferase (Dnmt) 1 and histone deacetylases (HDAC) expression, as well as the increased expression of lysine-specific histone demethylase 2 and methyl-CpG binding protein 2. Inhibiting Jak/Stat3 also blocks the expression of Dnmt3L, which is correlated with the failure of retroviral transgene silencing. Furthermore, Dnmt or HDAC inhibitor but not overexpression of Nanog rescues the reprogramming arrested by Jak/Stat3 inhibition or LIF deprivation. Finally, we demonstrated that LIF/Stat3 signal also represents the prerequisite for complete reprogramming of pre-iPSC. We conclude that Jak/Stat3 activity plays a fundamental role in promoting the establishment of pluripotency at the epigenetic level, by facilitating DNA demethylation/de novo methylation and open-chromatin formation during late stage reprogramming.