bluuebunny/crossref_metadata_2025 huggingface dataset:
298 THE INFLUENCE OF DISTINCT CENTRAL BULL STATIONS ON THE IN VITRO EMBRYONIC DEVELOPMENT OF NELORE BULLS
Authors: Renzi A. , Elias F. P. , Vila R. A. , Lôbo R. B.
Published: August 2010

Reproductive biotechnology is growing worldwide as one of the most important tools of cattle breeding because it accelerates the process of genetic improvement. Most of the embryos produced are obtained using frozen semen from different AI centers. During freezing and thawing of semen, the sperm can be damaged by the rapid and dramatic changes in the physicochemical conditions that occur during cooling and ice formation. It has previously been described that bad management of frozen semen can result in reduced fertilization. This study investigated the influence of different central bull stations on the development of in vitro-produced bovine embryos. We compared semen of 154 Nelore bulls, used for IVF, from 8 different central bull stations (all of which used the same cryopreservation protocol) in the development of blastocysts. The in vitro production of embryos was performed as described: oocytes were collected from the slaughterhouse and matured in TCM-199 + 10% fetal calf serum (FCS) +0.5 μg mL-1 FSH + 50 μg mL-1 LH+ 1 μg mL-1 estradiol, for 24 hat 38.5°C in 5%CO2 in atmospheric air. Viable spermatozoids were obtained by centrifugation in Percoll gradient (45 and 90%), and used for IVF in a concentration of 2 million spermatozoa per mL in TALP + 10 μg mL-1 of heparin medium. After 12 h, the presumptive zygotes were transferred to a CR2+ 10% FCS medium and co-cultured with cumulus cells. After 168 h of IVF, we evaluated the number and stage of cleaved embryos produced with the semen of each bull. Statistical analyses were performed by using the chi-square test. Our results suggest that there are differences among distinct central bull stations in the proportion of embryos that developed into blastocysts and the different stages they hatched.
FAPESP, CNPq, PROEX, FAEPA.
311 MELATONIN PROMOTES IN VITRO SPERM CAPACITATION IN BUFFALO (BUBALUS BUBALIS)
Authors: Di Francesco S. , Mariotti E. , Tsantarliotou M. , Sattar A. , Venditto I. , Rubessa M. , Zicarelli L. , Gasparrini B.
Published: August 2010

Melatonin, the main hormone secreted by the pineal gland, plays many roles in reproduction. In ram spermatozoa, melatonin administration increases plasminogen activator activity (Tsantarliotou MP et al. 2007 Theriogenology 69, 458-465), known to be involved in sperm capacitation and acrosome reaction (Taitzoglou IA et al. 1996 Mol. Androl. 8, 187-197). The aim of this study was to evaluate the efficiency of melatonin to induce buffalo in vitro sperm capacitation, indirectly assessed by estimating the capability of spermatozoa to acrosome-react. Frozen-thawed semen from 4 different bulls was pooled and treated by swim-up in order to select only the motile population. Spermatozoa (n = 829) were assessed immediately after swim- up separation, to evaluate the incidence of acrosomal loss in non-treated cells (time 0). The remaining spermatozoa were incubated in the absence of capacitating agents (negative control; n = 513), in the presence of 0.01 mM heparin (positive control; n = 775), 10 μM melatonin (n = 684), 100 μM melatonin (n = 751), and 1 mM melatonin (n = 650), for 2 h. Sperm were then exposed for 15 min to 60 μg mL-1 of lysophosphatidylcholine, an agent known to induce acrosome reaction (AR) only on capacitated spermatozoa. Trypan blue was used first to differentiate live from dead spermatozoa and the dried smears were then fixed in 37% formaldehyde and stained with Giemsa for acrosome evaluation by microscopic examination. The percentage of acrosome-reacted spermatozoa in each group was used to assess the efficiency of capacitation under different incubation conditions. The experiment was repeated 4 times. Differences among groups were analyzed by chi-square test. Acrosomal loss was observed only in 2.1% of the sperm population at time 0. Interestingly, sperm treatment with both heparin and the different concentrations of melatonin resulted in a significantly higher incidence of AR compared to the negative control (24.4, 20.5, 20.0, 23.6 v. 8.0% for the positive control, 10 μM melatonin, 100 μM melatonin, 1 mM melatonin, and the negative control, respectively; P < 0.01). These results demonstrated that melatonin determines capacitation of buffalo spermatozoa in vitro. Furthermore, the effect of melatonin was comparable to that of heparin, that is, the capacitating agent currently used in the IVF system. The capacitating effect was observed at all the tested concentrations, and viability was not affected. This suggests to extend the range of concentrations to test in future studies, in order to identify the optimal dose. Moreover, considering the seasonality of the species and the great differences in fertility attitude of buffalo bulls, it would be interesting to investigate the capacitation effect of melatonin in relation to both the season and the bull.
321 CO-CULTURE WITH OVIDUCT EPITHELIAL CELLS PROMOTES CAPACITATION OF BUFFALO (BUBALUS BUBALIS) SPERM
Authors: Siniscalchi C. , Mariotti E. , Boccia L. , Albero G. , Di Francesco S. , Suárez M. V. , Di Palo R. , Gasparrini B.
Published: August 2010

The process of capacitation, necessary for spermatozoa to acquire fertilizing ability, normally takes place in the oviduct. It has been demonstrated that oviduct fluid improves capacitation in bovine spermatozoa (McNutt TL and Killian GJ 1991 J. Androl. 12, 244-252). The aim of this study was to evaluate whether co-culturing buffalo sperm with bovine oviduct epithelial cells (BOEC) promotes in vitro capacitation of buffalo spermatozoa. Bovine oviducts were obtained at a local abattoir from cows that were in the preovulatory phase of a normal estrous cycle. BOEC recovered as previously described (Gualtieri R and Talevi R 2000 Biol. Reprod. 62, 1754-1762) from 5 oviducts were pooled and plated in dishes of TCM-199 + 10% FCS, 100 U mL-1 penicillin, 100 μg mL-1 streptomycin, and 0.25 μg mL-1 amphotericin B. Medium was changed every 48 h up to Day 6, when cell confluence and cilia activity were optimal. On day of trial, the medium was removed from the BOEC dishes and replaced with modified TALP containing 30 μM penicillamine, 15 μM hypotaurin, 0.15 μM epinephrine, and 1% bovine serum (mTALP). Buffalo frozen- thawed sperm from 4 bulls, pooled and treated by Percoll gradients, were incubated for 3 h (1 million sperm/mL): in mTALP with 30 μg mL-1 of heparin (Group A), on the BOEC monolayer (Group B), on the BOEC monolayer with 30 μg mL-1 of heparin (Group C), and in medium lacking capacitating agents, as negative control (Group D). After 3 h, sperm were recovered by centrifugation and capacitation was indirectly evaluated by assessing their ability to undergo acrosome reaction (AR) after 10 min of exposure to 10 μM calcium ionophore A23187. The AR was determined by the double-staining technique with trypan blue-Giemsa (Kovács A andFoote RH 1992 Biotec. Histochem. 67, 119-124). Spermatozoa (n = 294, 204, 225, and 222 for Groups A, B, C, and D, respectively) were then examined by microscopic evaluation. Differences in the percentages of AR among groups were analyzed by chi-square test. After Percoll separation, sperm viability was 95.8% and acrosomal loss was observed only in 5% of the sperm population. After 3 h of incubation, sperm viability decreased (P < 0.01) in all groups compared to the post-Percoll time, whereas no differences were found among groups (73, 80.4, 80.0, and 76.2%, respectively, for Groups A, B, C, and D). Sperm co-incubation with both BOEC and heparin for 3 h improved capacitation, as indicated by the significant increase (P < 0.01) of AR compared to the negative control (29.9, 30.5, 30.6, and 14%, respectively, for Groups A, B, C, and D). Furthermore, both BOEC groups gave percentages of AR similar to those observed after incubation with heparin, that is, the most widely used capacitating agent. In conclusion, it was demonstrated that co-culturing sperm with BOEC, both in the presence or absence of heparin, enhances buffalo sperm capacitation in vitro.
325 VIABILITY AND GROWTH OF CATTLE PREANTRAL FOLLICLES AFTER IN VITRO CULTURE OF OVARIAN FRAGMENTS IN α-TOCOPHEROL
Authors: Lisboa L. A. , Andrade E. R. , Hertel M. F. , Melo-Sterza F. A. , Moreno K. , Bracarense A. P. F. R. L. , Alfieri A. A. , Seneda M. M.
Published: August 2010

The development of culture systems to support the initiation of growth of primordial follicles is important to the study of the factors that control the earliest stages of folliculogenesis. The aims of the present study were to investigate the effects of α-tocopherol on survival, activation, and growth of cattle preantral follicles using histological and immunohistochemistry proliferating cell nuclear antigen (PCNA) studies. The ovarian cortex was divided into small fragments; one fragment was immediately fixed in Bouin (control). The other fragments were cultured for 2, 4, 6, or 8 d in culture plates with Minimum Essential Medium supplemented with insulin-transferrin-selenium (ITS), pyruvate, glutamine, hypoxanthine, BSA, and antibiotics (MEM+); and MEM+ plus α-tocopherol (5, 25, 50, 100, or 200 ng mL-1). Preantral follicles were classified according to their developmental stage (primordial, intermediate, primary, or secondary) and on the basis of morphological features (normal or degenerated). Pair-wise comparisons were done using Tukey’s procedure. Chi-square test was used to compare the percentage of follicles with PCNA-positive granulosa cells. All analyses were done with the SAS software (SAS Institute, Cary, NC, USA), and P < 0.05 was considered significant. The results showed that, compared with non-cultured cortical tissue (Day 0), the culture of ovarian tissue significantly reduced (P < 0.05) the percentage of normal follicles in all media tested, except for tissue cultured in the presence of 200 ng mL-1 of a-tocopherol. Furthermore, in all media tested, the percentage of primordial follicles was significantly reduced (P < 0.05), with a concomitant increase in the percentage of developing follicles. The highest percentage of secondary follicles was observed after 6 days of culture in MEM plus 200 ng mL-1 of a-tocopherol. The PCNA analysis confirmed the viability of follicles cultured with 200 ng mL-1 of a-tocopherol after 6 d. After 8 days of in vitro culture, we observed severe follicular degeneration in all media tested, suggesting that other supplements are recommended for longer periods of culture. In conclusion, the results of the present study indicate that 200 ng mL-1 of a-tocopherol maintains the survival of cattle preantral follicles and promotes activation of primordial follicles after 6 days of in vitro culture.
Financial support: L. A. Lisboa is a recipient of CAPES support; E. R. Andrade and A. A. Alfieri are recipients of PRODOC/CAPES fellowships.
330 ROLE OF STAT3 PATHWAY IN THE LEPTIN-INDUCED SIGNALING DURING OOCYTE IN VITRO MATURATION AND STEROIDOGENIC RESPONSE IN RABBIT MODEL
Authors: Arias-Alvarez M. , Garcia-Garcia R. M. , Torres-Rovira L. , Gonzalez-Bulnes A. , Rebollar P. G. , Lorenzo P. L.
Published: August 2010

The effect of leptin on oocyte maturation and steroidogenic response could be mediated by activation of specific transcription factors. The aim of this work was to establish if the Signal Transducer and Activator of Transcription 3 (STAT3) pathway mediates leptin’s influence on oocyte maturation and steroid secretion of COC by using a rabbit oocyte model. A total of 522 COC from follicles >1 mm were IVM for 16 h (38°C, 5% CO2) in TCM-199 serum free medium plus: 0 or 10 ng mL-1 leptin. Each group was further treated with 10 and 100 μM of specific inhibitor for STAT3 pathway (AG490, Sigma, St. Louis, MO, USA). COC were treated progressively with hyaluronidase (2 mM), 0.5% pronase, 4% paraformaldehyde, 0.02% Triton X-100, and 7.5% BSA after IVM. Oocytes examined under a confocal laser-scanning microscope were previously incubated with 100 mg mL-1 fluorescein isothiocyanate of Lens culinaris (FITC-LCA) and then with 10 mg mL-1 propidium iodide, for cortical granule (CG) and nuclear staining, respectively. Spent maturation media were analyzed for oestradiol and progesterone secretion using Enzimoinmunoassay. Statistical analysis was performed by Chi-square and ANOVA tests. COC supplemented with 10 ng mL-1 leptin had significantly higher metaphase IIrate than those IVM without leptin (P < 0.05); but it did not enhance the percentage of oocytes with peripheral migration of CG (Table 1). Leptin-stimulated nuclear oocyte maturation was significantly impaired when the STAT3-inhibitor was added to the media (P < 0.005). CG migration index was not modified when inhibitors were included in IVM media compared to control groups. Estradiol and progesterone secretion by COC were similar in all groups tested; these concentrations were not affected when STAT3-pathway inhibitor was added. The results confirmed that 10 ng mL-1 leptin enhances meiotic oocyte maturation but not CG migration, through the STAT3 pathway. In the present rabbit model, this effect is not mediated by an increase of steroid secretion by COC.

Table 1.Metaphase II, CG migration rate and steroid production in rabbit COC after use of different concentrations of STAT3-inhibitor

We acknowledge CM, FSE, and MICINN for funding.
331 EFFECT OF SUCESSIVE LAPAROSCOPIC OOCYTE RECOVERY AFTER HORMONAL TREATMENT IN CROSSBRED GOATS
Authors: Avelar S. R. G. , Almeida K. C. , Pereira A. F. , Sousa F. C. , Moura R. R. , Teles Filho A. C. A. , Melo C. H. S. , Albuquerque E. S. , Pereira F. B. , Melo L. M. , Teixeira D. I. A. , Freitas V. J. F.
Published: August 2010

Laparoscopic oocyte recovery (LOR) is a valuable tool for obtaining oocytes for in vitro embryo production. When preceded by a treatment of ovarian stimulation, this technique produces an increase in the amount of oocytes recovered. However, a little information has been found to respect the effect of successive hormonal treatments on both oocyte quantity and quality. Therefore, the objectives of this study were to evaluate the ovarian response and quantitative and qualitative COC production. Five adult crossbred goats were hormonally treated with intravaginal sponges containing 60 mg of medroxyprogesterone acetate (MAP, Progespon, Syntex, Buenos Aires, Argentina) for 11 days. In the 8th day of progestagen treatment, 50 μg of prostaglandin F2α analogue (Ciosin, Coopers, São Paulo, Brazil) was administered by i.m. injection. At this time, ovarian stimulation was initiated by the administration of 120 mg pFSH (Folltropin-V, Vetrepharm, Canada) distributed in five decreasing doses (30/30, 20/20, 20 mg), at 12-h intervals. The animals were fasted for 24 h before the laparoscopic procedure, which was performed just after the sponge removal. A laparoscopic system connected to a 22-gauge needle (WTA, Watanabe, Brazil) and a vacuum pump (Biovacuum, Biocom, Brazil) providing 30 mm Hg was used. All follicles with a size larger than 2 mm present in both ovaries were counted and aspirated. The collection medium was TCM-199 supplemented with HEPES (10 mM), heparin (20 IU mL-1), and gentamicin (40 μg mL-1). The COCs were graded based on presence of cumulus cells and cytoplasm homogeneity (I to IV). The hormonal treatment and LOR procedures were repeated three times at 14-day intervals. Data were expressed in percentage or mean ± SEM. The differences were analyzed using ANOVA and Tukey’s or Fischer’s exact test when appropriate, with P < 0.05. No statistical differences were found (P > 0.05) for the number of follicles obtained in each LOR session: 17.0 ± 3.91, 18.75 ± 2.59, and 18.0 ± 4.73, respectively. Repeated LOR procedures also did not affect (P > 0.05) the number of aspirated follicle (15.0 ± 3.92, 15.5 ± 2.33, and 16.0 ± 4.36), resulting from the three sessions, respectively. Average recovery rates were not statistically different (P > 0.05), resulting in 74.7%, 62.9%, and 64.6% between sessions. With respect to the percentage of viable COCs (GI and GII) were not observed statistical differences (P > 0.05), as verified the follow values at 1st to 3rd sessions: 76.79%, 84.62%, and 74.19%. In conclusion, three successive hormonal stimulation LOR procedures did not cause detrimental effects on quantitative and qualitative oocyte production, suggesting that this protocol can be used for programs of in vitro goat embryo production.
This study was supported the following Brazilian agencies: FINEP, CNPq, FUNCAP, and CAPES.
344 EFFECTS OF BMP4 AND ITS INHIBITOR, NOGGIN, ON OOCYTE MATURATION AND DEVELOPMENT OF BOVINE PREIMPLANTING EMBRYOS
Authors: La Rosa I. , Fernandez y Martín R. , Paz D. A. , Salamone D. F.
Published: August 2010

BMP4 regulates different events during development in all vertebrates and Noggin is one of its powerful inhibitors that blocks BMP4 interaction with its receptors (Groppe et al. 2002). In this work, the effect of these factors on bovine oocyte maturation and subsequent embryo development has been investigated. COCs were aspirated from abattoir ovaries and in vitro-matured for 22 h or 24 h in a 5% CO2 humidified atmosphere at 39°C in TCM containing 0.6% BSA, 2 mM FSH, 10 mM cysteamine, 1% antibiotic and 1% pyruvate, control group (C), plus 100 ng mL-1 of BMP4 (B), or 100 ngmL-1 of Noggin (NOG). Oocytes were stained with Hoechst 33342 and classified by their nuclear stage. Effects on embryo development were investigated for embryos produced by parthenogenic activation (PA) and IVF For PA, denuded oocytes were chemically activated in 5 μM ionomycine for 4 min, and immediately incubated in 1.9 mM of 6-dimethilaminopurine for 3 h. For IVF, frozen-thawed semen was centrifuged and resuspended in Bracket and Oliphant (BO) solution and incubated with 22 h matured COCs for 5 h. Embryos were cultured in CR2 medium free of serum and co-culture. Cleavage and blastocyst formation were registered at Day 2 and 9 respectively. Fischer’s exact test was used and P ≤ 0.05 was considered significant. Nuclear progression was not affected by maturation treatments [% of MII: 79.4(C, n = 102), 72.4 (B, n = 98), 80.9 (NOG, n = 89)]. For PA, both factors significantly increased cleavage rates [%: 51.7 (C, n = 284), 65 (B, n = 186), 62.1 (NOG, n = 198)] while blastocyst rates were not affected [%: 8.8 (C), 7.5 (B), and 8.6 (NOG)]. On the other hand, for IVF, cleavage rate was statistically lower for Noggin group [%: 70.7 (C, n = 140), 71.3 (B, n = 157), 64 (NOG, n = 159)] while blastocyst rates were similar between groups [%: 15.7 (C), 13.4 (B), 14.5 (NOG)]. Any of the added factors affected cell number of the embryos at Day 2. Blastocysts did not differ in the number of cells at Day 9 (Student’s t-test was used) neither for PA [mean ± SD: 100 ± 33 (C, n = 9), 88 ± 14 (B, n = 3) and 68 ± 8,(NOG, n = 3)] nor for IVF [mean ± SD: 90 ± 24 (C, n = 9), 132 ± 18 (B, n =4) and 99 ± 8 (NOG, n = 3)]. It is noticeable that addition of these factors during in vitro maturation showed different effects on subsequent embryo development depending on whether the embryos were PA or IVF. Probably, these responses represent differences in the BMP signaling system between these embryos which could be associated with different imprinting pattern. Further experiments are needed to elucidate clearly the mechanisms implicated. To our knowledge, this is the first work to study BMP4 inhibition during bovine in vitro maturation.
To “Merlo” and “Nueva Escocia” Slaughterhouses
345 GENE EXPRESSION IN OOCYTES AFTER MEIOTIC INHIBITION WITH THE CYCLIN-DEPENDENT KINASE INHIBITOR BUTYROLACTONE I
Authors: Leal C. L. V. , Mamo S. , Fair T. , Lonergan P.
Published: August 2010

Once removed from the follicle, mammalian oocytes resume meiosis spontaneously and progress through breakdown of the germinal vesicle to the matured state at metaphase II. The ability to reversibly inhibit such meiotic resumption has been reported and is a potentially useful method for studying developmental competence acquisition in oocytes as well as in some cases allowing flexibility in an IVF system where oocytes are collected from distant locations or on different days. The aim of the present study was to determine the effect of temporary inhibition of meiotic resumption using the cyclin-dependent kinase inhibitor butyrolactone I (BLI) on gene expression in bovine oocytes. Immature bovine oocytes were recovered from the ovaries of slaughtered heifers at a commercial abattoir and assigned to 1 of 4 groups: (1) Control: immature oocytes were collected either immediately or (2) after IVM for 24 h in TCM-199 containing 10 ng mL-1 EGF and 10% (v/v) FCS, (3) Inhibited oocytes collected either 24 h after incubation in the presence of 100 μM BLI in TCM-199 with 3 mg mL-1 BSA or (4) after meiotic inhibition for 24 h followed by in vitro maturation. All cultures were carried out at 38.5°C under 5% CO2 in air and maximum humidity. For mRNA relative abundance analysis, cumulus cells were removed and pools of 10 denuded oocytes were snap frozen in liquid nitrogen and stored at -80°C until use. A total of 42 transcripts, previously reported to be related to cell cycle regulation and/or oocyte competence were evaluated by quantitative real time PCR. Differences in relative abundance were analyzed by ANOVA and Student’s t-test. The majority of transcripts were downregulated (P < 0.05) after IVM in control oocytes (23 out of 42) and the same pattern was observed in inhibited oocytes that were allowed to mature. Twelve transcripts remained stable (P > 0.05) after IVM in control oocytes; of these, only two (PTTG1 and INHBA) did not show the same pattern in inhibited and matured oocytes. Few genes (7) were upregulated after IVM in control oocytes (P < 0.05) and of these, three (PLAT1, RBP1, and INHBB) were not upregulated in inhibited oocytes after IVM. Inhibited oocytes showed similar levels of expression (P > 0.05) as immature control oocytes, except for two genes (LUM and INHBB), which were increased in these oocytes (P < 0.05). The expression profiles of cell cycle genes were mostly unaffected by the BLI treatment. The few genes affected were previously reported as competence-related and could be useful markers of oocyte competence following pretreatment. In conclusion, the changes occurring in transcript abundance during oocyte maturation in vitro were to a large extent mirrored following inhibition of meiotic resumption prior to IVM and subsequent release from inhibition and maturation.
CLV Leal was supported by CNPq, Brazil (PDE 201487/2007-1); Supported by Science Foundation Ireland (07/SRC/B1156).
350 EFFECT OF PROGESTERONE SUPPLEMENTATION OF MATURATION MEDIUM ON DEVELOPMENT OF IVM-IVF-IVC BOVINE EMBRYOS
Authors: Miyata K. , Koyama H. , Lessard C. , Singh J. , Dochi O.
Published: August 2010

Follicular fluid from small and large bovine follicles contains large amounts of progesterone, and during preovulatory period progesterone concen- tration increase markedly by 18 h after LH surge. Furthermore, cumulus cells express membrane progestin receptor beta (Liu et al. 2008 Steroids 73, 1416-1423). For these reasons, we hypothesized that progesterone supports maturation of preovulatory bovine oocytes to MII stage. The object of this study was to investigate the effect of progesterone supplementation of in vitro maturation medium on competence of bovine oocyte to develop into blas- tocysts in vitro. COCs were collected by the aspiration of 2-6 mm follicles from ovaries within 6 h of slaughter. The COCs were divided into 5 groups: (1) a control group, TCM-199 supplemented with 5% calf serum (CS) as IVM medium, and (2 to 5) progesterone (P4) supplementation groups, TCM- 199 supplemented with 5% CS and 1, 3, 5, and 10 μg mL-1 of P4. Groups of 10 COCs were incubated in 50-μL drops of IVM media at 38.5°C under an atmosphere of 5% CO2 in air for 20 h. The matured COCs were inseminated with 3 × 106 sperm mL-1. After 18 h of gamete co-culture, the pre- sumptive zygotes were cultured in CR1aa media supplemented with 5% CS for 9 days at 38.5°C under an atmosphere of 5% CO2, 5%O2, and 90% N2. Embryonic development was evaluated at 48 h after IVF (total cleavage rates) and on Days 7 to 9 (blastocyst rate). Data was analyzedby chi-square test. The results are presented in Table 1. There were no significant differences in the cleavage rates between treatments. However, the blastocyst formation rate of 5 μg mL-1 P4 supplementation group was significantly higher than that of 10 μg mL-1 P4 supplementation group (P < 0.05). In addition, the blastocyst formation rates of 10 μg mL-1 P4 supplementation group was lower than the other groups. These results suggest that progesterone supple- mentation of in vitro, maturation medium affects the competence of the oocytes to develop into blastocysts in vitro, and 5 μg mL-1 P4 supplementation may be effective in increasing embryo production. Furthermore, 10 μg mL-1 P4 supplementation has negative effect on the oocyte competence.

Table 1.Effect of progesterone supplementation on development of IVF bovine embryos
355 INFLUENCE OF NITRIC OXIDE AND CYCLIC GMP SIGNALING PATHWAY ON THE IN VITRO MATURATION OF BOVINE OOCYTES: PRELIMINARY RESULTS
Authors: Schwarz K. R. L. , de Bem T. H. C. , Pires P. R. L. , Mesquita L. G. , Remy L. , Leal C. L. V.
Published: August 2010

Nitric oxide (NO) is a chemical messenger generated by the activity of the nitric oxide synthase enzyme (NOS) and has been shown to be involved in oocyte maturation. NO is known to act through the guanylate cyclase (GC) signaling pathway, stimulating the production of cyclic guanosine monophosphate (cGMP), which in turn activates protein kinase G (PKG). The objective of the present study was to investigate the involvement of NO and GC/cGMP/PKG pathway on the IVM of bovine oocytes. Slaughterhouse ovaries were transported to the laboratory and oocytes were aspirated from 2 to 8 mm follicles. Oocytes were submitted to IVM (TCM-199+10% fetal calf serum + hormones) for 24 h (38.5°C and 5% CO2 in air) and were assessed for nuclear maturation by acetic-orcein (1%) staining. Maturation rates were analyzed by ANOVA. Five replicates were performed with 20 oocytes per group per replicate. When the oocytes were matured with the NO donor [(0, 10-9, 10-8 and 10-7M S-nitroso-N-acteyl-D,L- penicillamine (SNAP)] germinal vesicle break down (GVBD) rates after 7 h in IVM were 36, 31, 42, and 24%, respectively (P > 0.05). Maturation rates after 24 h IVM ranged from 80 to 85% (P > 0.05). The inhibition of GC [(0, 0.1, 10, and 100 μM 1, H-[1, 2, 4]oxadiazole[4, 3-a]quinoxalon-1-one (ODQ)] and PKG (0, 1, 10, and 100 μM KT5823) did not affect (P > 0.05) the ability of oocytes to form the first polar body (average of 83 and 88%, respectively). When the cGMP-analogue (0, 1, 2, and 4 mM 8-Bromo-cGMP) and the GC-stimulator (0, 5, 10, and 50 μM Protoporphyrin IX) were used during IVM, maturation rates were over 85% in all groups (P > 0.05). To confirm the lack of effect of the inhibitors, another evaluation with higher concentrations of inhibitors in semi-defined IVM medium (TCM-199 + 0.04% BSA) was carried out. Maturation rates were 70 to 75% (P > 0.05) with ODQ and 57 to 76% (P > 0.05) with KT5823. The evaluation with the GC stimulator and the cGMP analogue in semi-defined medium is currently underway. In conclusion, under the conditions studied, the GC/cGMP/PKG signaling pathway is not involved in the nuclear maturation of bovine oocytes.

Supported by FAPESP, Brazil.
389 CULTURE OF EQUINE INNER CELL MASS USING EQUINE MESENCHYMAL CELLS AS A FEEDER LAYER
Authors: Lima-Neto J. F. , Araujo G. H. M. , Alvarenga M. L. , Moya-Araujo C. F. , Blanco I. D. P. , Rascado T. S. , Macedo C. C. , Wodewotzky T. I. , Alvarenga M. A. , Landim-Alvarenga F. C.
Published: August 2010

Stem cells can originate from embryo, fetus, and adults tissues. Adult stem cells are present in almost all organs and are responsible for tissue regeneration and repair. The mesenchymal stem cells (MSC) are multipotent cells present in the bone marrow. The plasticity of these cells allows them to be used in cell therapy because they have the potential to differentiate into several tissues with mesenchymal origin. On the other hand, embryonic stem cells (ESC) have the ability to differentiate into the 3 embryonic tissues (endoderm, mesoderm, and ectoderm) generating all kinds of tissue in the living organism. Embryonic stem cells can be maintained in their undifferentiated state when cultured in vitro in the presence of the leukemia inhibitory factor (LIF) and over a feeder layer composed normally by mouse fibroblasts. Because of the small number of articles studying equine ESC, the current experiment aimed to compare the utilization of equine fibroblasts and equine MSC as feeders for inner cell mass (ICM) culture. Eighteen blastocysts were collected on Day 7 after ovulation from 5 fertile mares that had been artificially inseminated. The embryos were placed in a petri dish with Dulbecco’s phosphate buffered saline + 10% FCS, and the ICM was mechanically removed using 2 insulin needles. The ICM was then transferred to one well of 24 well plates containing either an equine fibroblast monolayer (9 embryos) or a MSC monolayer (9 embryos). Both feeders layers were inactivated with 10 μg mL-1 mitomycine C, and the cell concentration was 140 000 cells/well. The culture media utilized was the DMEM/F12 with 20% FCS, 1% essential amino acids, 1% nonessential amino acids, 0.1 mM β-mercaptoetanol, 10 ng mL-1 LIF, penicillin, streptomycin, and amphotericin B. The ICM was cultured during 5 days for the adherence to the plate, and the media was change every 2 days. Data was analyzed by ANOVA. Results indicated no significant differences between the use of equine fibroblasts or MSC as feeder layer to support equine ICM in culture. The expansion of the ICM cells was observed after 10 to 12 days in culture in 44.4% (4/9) of ICM cultured in MSC monolayer and 55.5% (5/9) of the ICM cultured over a fibroblast layer. The formation of embryoid body-like structures were observed after the second passage (5-7 days in culture) in 50% (2/4) of embryos using MSC feeders and in 40% of embryos (2/5) for which fibroblasts were used. Cultures were then positively marked with the Oct 4 antibody for characterization of the undifferentiated lineage. The results of the current experiment showed that although further patronization of the culture system is still needed, both fibroblasts and MSC are suitable as feeders for the culture of equine ICM when aiming to establish ESC lineage in vitro.

Financial support was provided by FAPESP and CNPq.
405 LEVELS OF HORMONES AND NITRIC OXIDE PRESENT IN FOLLICULAR FLUID UNDER OR NOT UNDER SUPEROVULATION IN MARES
Authors: Carmo M. T. , Losinno L. , Aguilar J. , Rose J. , Araujo G. H. M. , Alvarenga M. A.
Published: August 2010

The aim of the present study was to compare the levels of 17β-estradiol, testosterone, progesterone, inibin, and nitric oxide in follicular fluid between superovulated mares (G1) using equine pituitary extract (EPE) and nonsuperovulated mares (G2) used as control. Two estrus cycles from 24 mares (G1 = 12; G2 = 12) were monitored. In G1, the first cycle was used to determine the ovulation day (Day 0), followed by the administration of PGF2 (250 μg daily of cloprostenol; Sincrocio™, Ouro Fino, São Paulo, Brazil) on the first and second day of treatment with EPE. During the second cycle, EPE (25 mg i.m.) was administered twice daily from Day 7 to the ultrasound detection of a majority of follicles at least 35 mm in diameter. At this time, 2500 IU of hCG IV (Vetecor™, Hertape Calier, Minas Gerais, Brazil) was given. The same protocol was used for G2 as was used for G1 except that EPE administration was replaced by saline injections. For follicular aspiration, the mares were sedated with xilazine (0.5 mg kg-1; Sedazine™, Fort Dodge, São Paulo, Brazil) and acepromazine (0.05 mg kg-1; Acepram 1%™, Univet, Milton, Ontario Canada); hioscin bromide (Buscofin™, Agener União, São Paulo, Brazil) was also administered to reduce intestinal motility and to make ovary manipulation by way of the rectum easier. All follicles larger than 35 mm were aspirated using a Cook® double lumen needle (12 GA) in a transvaginal aspiration probe guided by ultrasonography. The number of aspirated preovulatory follicles was 4.75 ± 2.7 and 1.0 ± 0.0 for G1 and G2, respectively. The follicular fluid collected was centrifuged at 600 × g for 10 min in order to separate the fluid from the cells. The fluid was stored at -20°C and sent to the University of California (Davis, USA) for hormonal assay. The nitric oxide dosage was determined at São Paulo State University (Botucatu, Brazil) using the luminescent reaction of Griess. The comparison between groups of hormonal levels and nitric oxide was performed using the t-test at 5% significance. The results of the present study showed that the EPE treatment (G1), under the experimental conditions, did not lead to a significant change in the mean concentrations of the evaluated hormones or of nitric oxide in the follicular fluid when compared with the levels in G2. However, differences were found among the individuals only in G1 in all hormones (P < 0.05), with exception of progesterone (P > 0.05).

Table 1.Mean and standard error of the follicular fluid hormones and nitric oxide levels of groups G1 and G2

Supported by FAPESP, Brazil.
409 SUPERSTIMULATED BOVINE DONORS INSEMINATED WITH SEMEN WITH DIFFERENT LINEARITY EVALUATED BY COMPUTER-ASSISTED SPERM ANALYSIS
Authors: Garcia Guerra A. , Villareal J. , Larraburu G. , Brogliatti G. M.
Published: August 2010

There is a highly significant relationship between semen quality and the percentage of fertilized ova and transferable embryos in superovulated donors (Stroud B and Hasler JF 2006 Theriogenology 65, 65-76). Computer-assisted sperm analysis (CASA) provides an objective and highly repeatable system for sperm analysis. It has been observed that linearity (LIN) measured by CASA has a high correlation with fertility (Foote RH et al. 1998 Theriogenology 49, 871-879). In the present report, retrospective analysis was done to determine the effect of sperm linearity as assessed by CASA on the number of ova and viable embryos recovered from superstimulated cows. This research was carried out using different breeds of donors (n = 150, 80% Angus) during the last 5 years in an embryo transfer center in Buenos Aires province, Argentina. Donors with a CL received an intravaginal progesterone device (DIB®, Syntex, Buenos Aires, Argentina), 2 mg of 17? estradiol, and 50 mg of progesterone (Rio de Janeiro, Brazil), i.m. on the same day. On Day 4 after DIB® insertion, superstimulatory treatment was initiated as a decreasing dose regimen of FSH i.m. (Pluset®, Callier, Spain, or Folltropin®, Bioniche Animal Health, Inc., Belleville, Ontario, Canada) every 12 h during 4 days. On Day 6, DIB® devices were removed and cows were administered cloprostenol (2 mL) twice at 12-h intervals. When estrus was detected, donors received GnRH (2 mL i.m. Dalmarelin®, Von Franken, Buenos Aires, Argentina) and were inseminated 12 and 24 h thereafter. Semen was thawed in a water bath at 37°C for 1 min. Before insemination, every semen dose was evaluated for motility characteristics using the IVOS Sperm Analyzer (Hamilton Thorne Biosciences, Beverly, MA, USA). Two chambers of 20-(im depth and 5 fields per chamber were analyzed (30 frames/0.5 s for each field). According to CASA results, 3 groups were defined based on LIN: Group 1 = <50%; Group 2 =≥50% and <60%; and Group 3 =≥60%. Seven days later, embryo collection was performed and ovarian response was evaluated by transrectal ultrasonography to assess number of CL + anovulatory follicles. Ova/embryos were evaluated and classified according to the IETS manual. Kruskal-Wallis test was used to compare variables among groups, and results are shown in Table 1. There was a significant difference between Groups 1 and 3 for total number and percentage of viable embryos. More embryos were recovered from donors inseminated with higher LIN semen. Also, there was a significant difference between Group 1 and both Groups 2 and 3 in the number of total fertilized ova, percentage of fertilized ova, and percentage of unfertilized ova. The present results suggest that insemination of donors with semen of high LIN results in higher fertilization rates and number of viable embryos.

Table 1.Number of fertilized ova and viable embryos of donor cows inseminated with semen with different linearity
410 ENHANCED OVARIAN RESPONSE TO LOW-DOSE TREATMENT WITH EQUINE PITUITARY EXTRACT IN MARES
Authors: Gimenes A. M. , Ignacio F. S. , Boff A. L. N. , Bergfelt D. R. , Meira C.
Published: August 2010

The present study aimed to evaluate the ovarian response to relatively low doses of equine pituitary extract (EPE) involving 40 estrous cycles in 15 horse mares. The mares were Mangalarga Marchador and ranged in age from 5 to 24 years. Transrectal ultrasonography was used at 24-h intervals to monitor follicular development and to detect ovulations (Day 0). On Day 8, all mares received 7.5 mg i.m. of dinoprost tromethamine (Lutalyse®, Pfizer, Paulínia, Brasil) and were randomly assigned to treatment and control groups as follows: Groups 1 and 2 (n = 8 cycles/group) received 6 mg of EPE i.m. twice daily, beginning when the largest follicle was 20 to 23 mm (Group 1) or regardless of follicle size on Day 8 (Group 2); Groups 3 and 4 (n = 8 cycles/group) received 8 mg of EPE i.m. twice daily, beginning when the largest follicle was 20 to 23 mm (Group 3) or regardless of follicle size on Day 8 (Group 4); Group 5 (control; n = 8 cycles) received saline i.m. starting on Day 8. Treatments with EPE or saline continued until the largest follicle reached ≥35 mm, at which time a single dose of hCG (2500 IU i.v. Vetecor®, Hertape Calier Saúde Animal S/A, Juatuba, Brasil) was given. Groups were compared using ANOVA and mean differences among groups were located with ScottKnott test. There was an effect of group (P < 0.05) for number of follicles >30 mm, ovulations, and days of treatment as indicated in the table. In conclusion, starting EPE treatment with 8 mg (16 mg day-1) when the largest follicle reached 20 to 23 mm (Group 3) was the most effective for enhancing the number of >30 mm follicles and ovulations with minimal days of treatment.

Table 1.

FAPESP and JM Farm for funding the project.
411 EFFICIENCY OF DAY SEVEN COLLECTION OF BOVINE EMBRYOS AFTER SUPEROVULATION BY FLUSHING THE OVIDUCTS AND THE UTERINE HORNS
Authors: Havlicek V. , Kuzmany A. , Beckers J. F. , Remy B. , Brem G. , Besenfelder U.
Published: August 2010

Embryo collection on Day 7 following superovulation is characterized by wide variation in the number of ova/embryos recovered. The differences between the numbers of corpora lutea and recovered ova/embryos provide evidence for incomplete recovery. The aim of this study was to examine the effect of flushing only the uterine horns v. endoscopical flushing of both the oviducts and the uterine horns on embryo recovery rates. A total of 12 superovulated Simmental heifers, aged 18 to 24 months were presynchronized by i.m. administration of PGF2 (500 μg of cloprostenol; Estrumate®, Essex Tierarznei, Munich, Germany) twice within 11 days. Two days after each of the PGF2 treatments, the animals received 0.02 μg of GnRH (Receptal®, Intervet, Boxmeer, the Netherlands). Between Days 9 and 11 of the estrous cycle, the animals got the first of 8 consecutive, twice-daily FSH injections in decreasing doses (in total 300-400 mg of FSH equivalent according to body weight; Stimufol®, ULg FMV PhR, Tilman, Belgium). Two PGF2 treatments were administered 60 and 72 h after the initial FSH treatment. Finally, 48 h after the first PGF2 application, ovulation was induced by injecting 0.02 μg of GnRH simultaneously with the AI. The AI was performed by using 1 straw of semen from a Simmental bull of proven fertility. Seven days after estrus, the heifers were restrained and an epidural anesthesia was performed. The uterine horns of the heifers were first flushed nonsurgically (300 mL of PBS + 1% FCS). Additionally, the oviducts were flushed by a transvaginal endoscopic technique (50 mL of PBS+1% FCS) followed by repeated flushing (250 mL of PBS + 1% FCS) of the uterine horns (combined flushing). In total, 12 animals delivered 132 ova/embryos (mean = 11 ± 6.5; recovery rate = 83%), with 83 developed to blastocysts/morulae (mean = 6.9 ± 6.4). The first flush of the uterine horns gave 3.8 ova/embryos on average with 2.8 being blastocysts/morulae (developmental rate = 75.6%). The following combined flush gave 7.3 ova/embryos with 4.1 developed to blastocysts/morulae (developmental rate = 56.3%). No differences were found between flushing the left and right sides. In conclusion, the present study shows that the additional step involving flushing of oviducts plus uterine horns of superovulated heifers provided recovery of a higher number of ova/embryos. It is assumed that these extra numbers might be caused by the flushing of (1) the uterine horns repeatedly, (2) the region of the utero-tubal junctions, and (3) ova/embryos from the oviducts, probably as a result of incomplete migration.
415 INDUCTION OF FOLLICULAR WAVE EMERGENCE IN WOOD BISON BY FOLLICULAR ABLATION OR TREATMENT WITH ESTRADIOL AND PROGESTERONE
Authors: Palomino J. M. , McCorkell R. B. , Balog B. , Ambati D. , Woodbury M. , Adams G. P.
Published: August 2010

Reclamation of Canada’s threatened wood bison (Bison bison athabascae) herd is complicated by endemic disease. As part of an overall goal to conserve bison genetics, the specific objective of this study was to develop a protocol to control follicular wave emergence in bison for the purposes of ovarian superstimulation and collection of disease-free oocytes. In an initial study, the synchronizing effect of follicular ablation and estradiol (E2) treatment was examined. Ablation was effective, but the effects of E2 (5 mg) were confounded by treatment-induced ovulation in some animals. Two experiments were done to determine the effect of a reduced dose of E2 or the addition of progesterone (P4) in comparison with follicular ablation. Bison cows (n = 19), ≥3 years old, were scanned for 14 days to determine the mean and variance in the interval to follicular wave emergence (control phase). In Experiment 1, bison were assigned randomly to 2 groups: follicular ablation (n = 9) or 2 mg of E2i.m. (n = 10). In Experiment 2, the same bison were randomly assigned to 2 groups: follicular ablation (n = 9) or 2 mg of E2+ 100 mg of P4 i.m. (n = 10). Ablation involved transvaginal ultrasound-guided aspiration of all follicles ≥5 mm. Wave emergence was determined retrospectively by identification of the follicle destined to become dominant at an initial diameter of 4 to 5 mm, with a concurrent increase in the number of follicles ≥4 mm. The interval and variation in the interval to emergence of a new follicular wave was compared among the control phase and treatment groups by ANOVA. In Experiment 1, the interval to new wave emergence (mean ± SEM) was 4.9 ± 0.66, 1.1 ± 0.11, and 2.4 ± 0.47 days from the start of the control phase, follicular ablation, and E2 treatment, respectively (P < 0.001). The degree of synchrony (residuals) was 2.4 ± 0.36, 0.2 ± 0.09, and 1.2 ± 0.24 days for the control phase, follicular ablation, and E2 treatment, respectively (P < 0.001). Ovulation was detected in 1 bison treated with E2. In Experiment 2, the interval to new wave emergence (mean ± SEM) was 4.9 ± 0.66, 1.2 ± 0.15, and 2.9 ± 0.31 days from the start of the control phase, follicular ablation, and E2 + P4 treatment, respectively (P < 0.001). The degree of synchrony was 2.4 ± 0.36, 0.2 ± 0.08, and 0.7 ± 0.20 days for the control phase, follicular ablation, and E2 + P4 treatment, respectively (P < 0.001), and no ovulations were detected. In conclusion, follicular ablation, E2, and E2 + P4 treatments all shortened and decreased the variability in the interval to new wave emergence in bison, but follicular ablation consistently produced a quicker and more synchronous response.

Supported by grants from the Advancing Canadian Agriculture and Agri-Food Fund (ACAAF), the Agri-Food Innovation Fund, Parks Canada, the World Wildlife Fund, and the Northwest Territories.
419 OVARIAN ULTRASONOGRAPHY TO ANALYZE FOLLICULAR DYNAMICS DURING SUPEROVULATORY TREATMENT IN RED DEER (CERVUS ELAPHUS) HINDS
Authors: Soler J. P. , Mucci N.
Published: August 2010

Inconsistency of the superovulatory responses of donor hinds has been a general feature of all red deer MOET programs (Asher GW et al. 2000 Anim. Reprod. Sci. 59, 61-70). The development of the techniques has been by trial and error as there is usually a lack of basic information on which to base MOET protocols (Fennessy PF et al. 1994 Theriogenology 41, 133-138). The objective of this study was to understand follicle development during a superovulatory treatment in order to improve ovulation rates and quantity of transferable embryos produced. During the breeding season, 10 mature (3-5 years old) red deer hinds were synchronized receiving an intravaginal sponge containing 100 mg of medroxiprogesterone acetate for 13 days, with device replacement on Day 11. Four days prior to the beginning of the FSH treatment, 0.5 mg of estradiol benzoate (Syntex SA, Buenos Aires, Argentina) was given i.m. to synchronize the follicular wave. Superovulation was conducted with a total dose of 120 mg of NIH-FSH-P1 (Folltropin®-V, Bioniche Animal Health, Belleville, Ontario, Canada) given i.m. in 4 equal doses of 30 mg every 24 h, from Day 11 to 14. Forty-eight hours after sponge withdrawal, 0.84 mg of buserelin acetate (Receptal®, Intervet, Boxmeer, the Netherlands) was injected i.m. to stimulate and synchronize ovulations. Ovarian scanning was performed by transrectal ultrasonography using a multifrequency linear transducer (Tringa Linear, Esaote Pie Medical, Genoa, Italy) on Days -1, 0, 1, 2, 3, 4, 5, and 6, Day 0 being the day of sponge withdrawal. The diameters of all follicles ≥3 mm were measured and their 3-dimensional position recorded to determine growth and ovulation. The average ovulation rate was 10.8 ± 1.6. The distribution of ovulations was 9.3, 31.5, 24.1, 22.2, 9.2, and 3.7% at 24, 48, 72, 96, 120, and 144 h after sponge withdrawal, respectively. The proportion of follicles that did not ovulate during the period of this study was 16.9%. The proportion of ovulated follicles according to their diameter was 9.3, 68.5, and 22.2% for 3 mm, 4 to 5 mm, and ≥6 mm, respectively, and were during the 24- to 96-h period for the first 2 follicles categories and after 96 h for the last category. This study showed a great variability of ovulations in the superovulatory protocols routinely used in red deer donor hinds. Improvement of the hormone treatment to induce a greater degree of ovulation synchrony (within 72 h after progesterone device withdrawal) would increase the fertilization rates and the quantity of transferable embryos produced in red deer MOET programs.

Table 1.Mean (±SEM) ovulation rate and number of follicles recorded at each ultrasonography day before and after sponge withdrawal