bluuebunny/crossref_metadata_2025 huggingface dataset:
3 ANALYSIS OF HOX EXPRESSION IN BOVINE OOCYTES AND EARLY EMBRYOS
Authors: Paul D. , Sonnet W. , Rezsohazy R. , Donnay I.
Published: August 2010

HOX genes encode transcription factors known to play a major role in patterning the main body axis of vertebrate embryos from the gastrulation stage onward. A few studies have provided evidence that some HOX genes might be expressed before implantation in mammalian embryos. Translation of maternally inherited transcripts is regulated by modifications of the poly(A) tail length until embryonic genome activation (EGA), occurring during the 4th cell cycle in the bovine. The objective of this work was to establish the expression pattern of various HOX genes and to study the polyadenylation of their transcripts during oocyte maturation and early embryonic development. Pools of 20 bovine oocytes before and after in vitro maturation and 20 in vitro-produced embryos at different stages of development up to the blastocyst stage were collected. Three to 12 pools were used for each stage. RNA was extracted and reverse transcribed (RT) using random hexamers. Quantitative real-time PCR (qPCR) was performed to establish expression profiles of 4 HOX genes: HOXD1, HOXA3, HOXB9, and HOXC9. Two distinct patterns of expression were observed. First, relative amounts of HOXD1, HOXA3, and HOXC9 were lower in morulae and blastocysts than in oocytes. On the other hand, relative expression of HOXB9 increased between the 5 to 8 cell stage and the morula stage (Mann-Whitney, P < 0.05). Those expression patterns were not modified when embryos were cultured in presence of α-amanitin, a RNA polymerase II inhibitor, indicating the maternal origin of the transcripts until EGA. Total amount of mRNAs, estimated by RT-qPCR with random hexamers, was stable for all studied genes during oocyte maturation. The relative amount of polyadenylated GAPDH mRNAs, estimated by RT-qPCR with poly(dT), decreased greatly in mature oocytes compared with immature oocytes indicating massive deadenylation of those transcripts. The relative amount of polyadenylated HOXC9 transcripts decreased slightly but significantly during oocyte maturation (Mann-Whitney, P < 0.05).The relative amount of polyadenylatedm RNAs corresponding to HOXD1, HOXA3, and HOXB9 was stable during oocyte maturation. This indicates that those transcripts escape the default deadenylation pathways followed by housekeeping genes. This experiment has been repeated 3 to 4 times. In conclusion, we confirmed the presence of HOXD1, HOXA3, HOXB9, and HOXC9 transcripts in bovine oocytes and early-stage embryos. Their role during oocyte maturation and the first stages of embryonic development will be investigated through loss of function studies.

This work is funded by the Fonds National de la Recherche Scientifique (Belgium).
304 EFFECT OF SYNTHETIC OVIDUCT FLUID MEDIUM (SOF) v. OPTIMIZED SIMPLE POTASSIUM MEDIUM (KSOM) ON EMBRYO DEVELOPMENT OF IN VITRO-FERTILIZED BOVINE OOCYTES
Authors: Zapata J. , Fernandez F. , Berland M. A. , Colazo M. , Peralta O. , Felmer R. , Ratto M. H.
Published: August 2010

The objective of the study was to compare the effect of 2 free serum chemically defined media, synthetic oviduct fluid medium (SOF) and optimized simple potassium medium (KSOM) on embryo development of in vitro-produced embryos. Bovine ovaries were collected from a local abattoir. Cumulus oocyte complexes (COC) were obtained by follicular aspiration of 3- to 6-mm follicles and in vitro-matured in TCM-199 for 24 h at 39°C, 5% CO2, and high humidity. After IVM, COC were co-incubated with sperm at a concentration of 1.5 × 106 spermatozoa per mL in TALP medium for 18 h at 39°C, 5% CO2, and high humidity. Presumptive zygotes were randomly assigned to SOF (n = 312) or KSOM (n = 290) media, both supplemented with essential and nonessential amino acids plus 0.6% BSA. Zygotes were cultured at 39°C, 5% CO2, 5% O2, 90% N2, and high humidity for 9 days. Cleavage rate was evaluated on Day 2 (Day 0 = day of insemination) and the number of blastocysts and hatched blastocysts were recorded on Days 8 and 9. Expanded blastocysts from Day 8 (n = 45/per culture medium) were fixed and stained with bisbenzimide (Hoechst 33242), and the total number of embryonic nuclei was counted by fluorescence microscopy (Optiphot, Nikon, Tokyo, Japan). The study was conducted in 6 replicates for embryo development assessment and 3 replicates for embryonic nuclei counting. Data were analyzed using Fisher’s exact test and Student’s t-test. The percentage of presumptive zygotes to develop into 2 to 4 cells (22.4 v. 24.2%) or > 4 cells (47.7 v. 53.4%) did not differ (P > 0.05) between SOF and KSOM on Day 2, but total embryo cleavage ≥ 2 cells was greater (P < 0.05) in KSOM (76.8%) than in SOF (68.5%). However, the percentage of blastocysts developed on Day 8 and 9 was greater (P < 0.05) for zygotes cultured in SOF (22.1 and 25.6%) than for those cultured in KSOM (15.1 and 18.9%). Furthermore, the percentages of hatched blastocysts on Day 8 and 9 were also greater (P < 0.05) for zygotes cultured in SOF (18.8 and 36.2%) than for those cultured in KSOM (4.7 and 12.5%). In addition, expanded blastocysts developed in SOF had higher total mean number of embryonic nuclei than those developed in KSOM (P < 0.05; 128.8 ± 7.5 v. 84.3 ± 8.9, respectively). In conclusion, the use of SOF resulted in significantly greater percentage of blastocysts and hatched blastocysts compared with KSOM. Additionally, expanded blastocysts developed in SOF displayed a higher number of cells.

This study was supported by Convenio Desempeño en Investigacion (2007-DGI-CDA-04), Universidad Catolica de Temuco and DiD-UAcH: S-2009-26.
305 OPTIMIZING DONOR AND RECIPIENT FACTORS IN XENOGRAFTING OF TESTIS TISSUE
Authors: Abbasi S. , Honaramooz A.
Published: August 2010

Grafting of donor mammalian testis tissue into recipient mice allows completion of spermatogenesis in the grafted tissue and therefore can serve as a new option for preservation of male germ line. For testis tissue xenografting, castrated male nude mice typically serve as recipients, each receiving 8 testis tissue fragments; however, no study has comprehensively investigated donor and recipient factors. The objective of this study was to determine the effects of strain of immunodeficient recipient mouse (nude v. SCID), gonadal status (intact v. gonadectomized), and gender (male v. female) on the outcome of testis tissue xenografting. A secondary objective was to determine the optimal number of testis tissue fragments per mouse most suitable for xenografting. Testis parenchyma from newborn piglets were cut into small fragments (5 mg each) and grafted under the back skin of different groups of immunodeficient mice. In Experiment 1, 8 groups of mice (n = 7/group) served as recipients: castrated male nude, intact male nude, ovariectomized female nude, intact female nude, castrated male SCID, intact male SCID, ovariectomized female SCID, and intact female SCID. In Experiment 2, 4 groups of mice (n = 10/group) served as recipients of 2, 4, 8, or 16 testis tissue fragments per mouse. Recipient mice were sacrificed 8 months after grafting and the weight of the grafts and vesicular glands (male mice) were compared among groups by analysis of variance. In Experiment 1, mouse gonadal status (intact v. gonadectomized) did not affect the total graft weight (P > 0.05), but both the recipient mouse strain (nude v. SCID) and gender (male v. female) affected the total graft weight (2460 ± 320.9, 1420 ± 290.0, 758 ± 156.7, and 2780 ± 297.4, mean ± SEM, P < 0.0001 for SCID, nude, female, and male mice, respectively). In Experiment 2, the total graft weight was highest in the group of mice receiving 8 testis tissue fragments (192 ± 76.1, 695 ± 96.5, 2443 ± 338.8, and 1458 ± 305.4, mean ± SEM, P < 0.0001 for 2, 4, 8, or 16 fragment groups, respectively). These results collectively indicate that male SCID mice receiving 8 testis tissue fragments provide optimized conditions for the recovery of largest grafts.

Research was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Saskatchewan Health Research Foundation (SHRF) to A. Honaramooz and scholarships from the Western College of Veterinary Medicine and the International Peace Scholarship to S. Abbasi.
31 PREGNANCY RATE OF CROSSBRED COWS SUBMITTED TO FIXED-TIME AI AND ADMINISTRATION OF GONADOTROPIN-RELEASING HORMONE ANALOGUE TWELVE DAYS AFTER AI
Authors: Moura G. S. , Nascimento V. A. , Oliveira F. A. , Dias A. R. , Dias M. , Torres C. A. A.
Published: August 2010

The importance of serum progesterone (P4) during the first weeks of pregnancy to reduce the embryo mortality in cattle herds has been demonstrated (Mann and Lamming 2001 Reproduction 121, 175-180). Studies show that a P4 peak at the beginning of pregnancy (about 5 days after the natural breeding or AI) helps development of the concept via secretion of interferon-τ (Mann 2002 XXII World Buiatrics Congress 18-23, 300-306). Studies have shown that the administration of gonadotropin-releasing hormone (GnRH) or its agonists after AI may stimulate corpus luteum function, induce the formation of an accessory corpus luteum, or increase of P4 serum concentrations and, therefore, improve embryonic survival (Bartolome et al. 2005 Theriogenology 63, 1026-1037). This work aims to evaluate the effect of GnRH injection 12 d after fixed-time AI(FTAI) on P4 serum levels and pregnancy rate of beef crossbred cows. The cows (Bos taurus indicus × Bos taurus taurus, n = 59), range in body condition (BCS) from 2.5 to 3.5 (scale of 1 to 5), were not previously pregnant. The animals were allocated to 2 treatments: TEB treatment (n = 30) consisted of Day 0 insertion of progesterone intravaginal device (ID) plus 2 mg of estradiol benzoate (EB), i.m. followed on Day 8 by removal of the ID and 300 IU of eCG and 0.15 mg of PGF2α, i.m. on Day 9, 1 mg injection of EB (i.m.) was given, followed by FTAI at 48 to 56 h after the withdrawal of ID. The TEBGnRH12 treatment (n = 29) protocol was similar to TEB, but with administration of 25 μg of GnRH analogue (Lecirelina) 12 d after the FTAI. Transrectal ultrasound was used for pregnancy diagnosis on Day 35 after AI. Blood samples for P4 serum assay were collected from 30% of animals in each treatment, at the time of AI and on Days 5, 12, and 20 after FTAI. For all statistical analysis, we used the program SAS 9.0 (2002; SAS Institute Inc., Cary, NC, USA) at 5% probability. The pregnancy rate was analyzed by logistic regression. The effects of treatment and day in P4 concentration were analyzed in a split plot, with the effect of day in plot, in mixed model, considering the error and repetition as a random effect, and comparison of means by Tukey-Kramer. Pregnancy rate and the concentration of serum P4 on different days was analyzed by Spearman correlation. The pregnancy rate for TEB cows was 53.33% (16/30) for the first service compared with 37.93% (11/29) for TEBGnRH12 cows. The protocol used did not affect the pregnancy rate of crossbred cows (P > 0.05). There were no treatment differences (P > 0.05) between the concentrations of P4 (TEB = 3.88 ng mL-1; TEBGnRH12 = 3.12 ng mL-1). In conclusion, the administration of the analogue of GnRH 12 days after FTAI does not affect the rates of pregnancy or the concentration of P4.

Supported by grants from CNPq, FAPEMIG and DZO-UFV.
312 MORPHOLOGICAL AND MORPHOMETRICS CHARACTERISTICS OF NUCLEAR SPERM IN THE EPIDIDYMAL TRANSIT OF BULLS
Authors: Fernandes C. E. , Sodré D. N. , Zart A. L. , Campos L. J. F.
Published: August 2010

During the transit through the epididymis, many morphological, physiological, and biochemical characteristics of spermatozoa are modified, as part of the maturation process. Nuclear maturation continues in the epididymis through an increase in formation of protamine disulfide. Thus, penetration through the oocyte membranes could be facilitated for elongated spermatozoa with dramatically condensed chromatin and nuclear integrity. Therefore, size, shape, and nuclear defects could be used to estimate the stage-related nuclear transformations from early spermiogenesis to the end of epididymal transit. Nellore bulls (n = 9), 30-36 months old, with high seminal quality (>80% motile and morphologically normal sperm) were submitted to orchiectomy. Impressions in slides of the caput, corpus, and caudal regions of the epididymis were prepared for evaluation of morphology (Feulgen stain, phase-contrast microscopy at 1000 ×) and morphometry of the nuclear sperm. The slides were captured in a Motic 2300 camera adapted to the microscope and digitally assessed. Nuclear morphology was considered normal (without visible alterations), head defect (variations in shape and form), and nuclear defects (abnormal chromatin condensation and presence of vacuoles). Base, width, length (μm), and area (μm2) were estimated in least 60 sperm nuclei. No difference (P > 0.05) among epididymal regions for normal nuclei (70.3 ± 3.1%), head defects (3.4 ± 0.5%), and nuclear defects (5.3 ± 1.3%) were seen. The base was higher (2.68 ± 0.5 μm, P < 0.01) in the caput than corpus (2.44 ± 0.4 μm) and caudal regions (2.41 ± 0.4μm). Normal nuclei were associated (P < 0.01) with width (r = 0.20), length (r = 0.27), and area (r = 0.44) in the caput and with width (r = 0.21), length (r = 0.40), and area (r = 0.33) in the corpus of epididymis. Epididymal transit affected (P < 0.001) the measures and nuclear status that accounted for regression analysis: normal nucleus (40.254 + 3.027, length; R2 = 0.20), head defects (0.922 + 1.097, width + 0.093 × area; R2 = 0.26), and nuclear defects (6.993-0.496, length + 0.454, base; R2 = 0.23). The results suggest that important variations occur in the nuclear status during the epididymal transit in the bovine spermatozoa. The higher measures in the sperm base of the caput suggest a narrowing probably indicating the continuity of nuclear remodeling from the final steps of spermiogenesis. The area accounts for 20% of the nuclear shape variations along the epididymal segment. These events characterize the adaptation of nuclear membranes and chromatin structure surrounded by epididymal environment and comprise a part of the maturation process. Additionally, morphometric variations are associated with defects in the nuclear structures and can be used to determine the conditions of spermatogenesis and sperm maturation based on the evaluation of ejaculated semen.

We thank CNPq/PROPP and Fundect for financial support.
315 REACTIVE OXYGEN SPECIES AND OXIDATIVE STRESS IN CANINE SEMEN FRACTIONS
Authors: Lucio C. F. , Nichi M. , Regazzi F. M. , Rück T. F. , Silva L. C. G. , Vannucchi C. I.
Published: August 2010

Reactive oxygen species (ROS) are physiologically produced by spermatozoa and leukocytes present in the seminal plasma. In low concentrations, ROS play an important role in sperm function because they are required for sperm fertilizing capacity, mainly during sperm capacitation and hyperactivation (Saleh RA and Agarwal A 2002 J. Androl. 23, 737-752). However, an imbalance between the formation of free radicals and the capacity for defense of the antioxidant mechanisms may lead to cell damage (Rover Jr L et al. 2001 Química Nova 24, 112-119). Spermatozoa are sensitive to lipid peroxidation due to its high content of polyunsaturated fatty acids and low concentrations of protective enzymes (Sharma RK and Agarwa LA 1996 Urology 48, 835-850). The aims of the present study were to compare ROS content among the 3 fractions of canine semen and to correlate these values with sperm variables. Semen samples were collected from 15 healthy dogs of distinct breeds aged 2 to 6 years. Sperm analysis was performed through motility, forward progressive velocity, morphology, and the percentage of viable sperm with the use of the eosin/nigrosin stain. The determination of thiobarbituric acid reactive substances (TBARS) was used to estimate the degree of lipid peroxidation in each sperm fraction. Values were compared using ANOVA and Tukey’s test for multiple comparisons at a significance level of 5%. Pearson correlation was used to calculate the relationship between sperm variables in the second fraction. Sperm motility, velocity, and percentage of viable sperm were within the normal range for canine semen: 84±2%, 3.4±0.1%, and 83 ± 2%, respectively. The sperm-rich fraction presented statistically higher concentrations of TBARS (1474.19 ± 245.78 ng mL-1) compared to the first and third fractions (579.41 ± 171.23 and 399.62 ± 58.08 ng mL-1, respectively; P < 0.05), indicating that spermatozoa and epididymal secretions are the main source of free radicals. No statistical correlation among TBARS and sperm motility and velocity were verified. However, a positive correlation was observed between the percentage of sperm proximal droplets and TBARS (r = 0.44, P = 0.7). This result suggests that a high incidence of sperm proximal droplets can enhance ROS formation in seminal plasma. Hence, canine sperm presenting delayed maturation in the epididymes produce higher concentrations of free radicals. In fact, sperm production of ROS occurs mainly by abnormal cells, especially the ones containing cytoplasm residues (Gomez E et al. 1998 Int. J. Androl. 21, 81-94). However, no protective effect of the sperm distal droplets was verified in canine semen as observed elsewhere for the bovine spermatozoa (Nichi M et al. 2007 Theriogenology 67, 334-340). In conclusion, spermatozoa and epididymal fluids are the primordial source of free radicals in canine seminal plasma, mainly when sperm proximal droplets are present.
316 FOURIER TRANSFORM INFRARED MICROSPECTROSCOPY: A NEW APPROACH FOR MOLECULAR PROFILE ANALYSIS OF SPERMATOZOA AND SEMINAL PLASMA
Authors: Milazzotto M. P. , Bitar R. A. , Visintin J. A. , Assumpção M. E. O. A. , Martinho H. S.
Published: August 2010

Fourier transform infrared (FT-IR) spectroscopy is a potential tool for noninvasive optical tissue diagnosis. Applications of this technique for non-clinical purposes have increased since it has been shown that it can also provide the identification of functional groups, bonding types, and molecular conformations. Moreover, the spectral bands are molecule-specific and provide direct information about the biochemical composition. However, the exploitation of FT-IR spectroscopy for reproductive studies is still limited. The aim of this study was to determine molecular profiles of spermatozoa and seminal plasma from different species to define important peaks present in the natural tissues. The study was based on fresh semen samples from 4 different species (human, bovine, ovine, and porcine). Semen samples were collected and evaluated for concentration and motility parameters. After that, samples were centrifuged and the seminal plasma was transferred to another tube. Spermatozoa were washed three times in PBS and 5 × 106 cells were resuspended in 50 μL of deionized water. Samples of 2 μL from spermatozoa and seminal plasma were placed separately on a gold slide and left to dry for 15 min in air. Spectral data were collected on a FT-IR spectrometer (Model 610; Varian Inc., Palo Alto, CA, USA). The absorbance spectra were acquired in reflectance mode at a spectral resolution of 2 cm-1 with 400 scans co-added. Apodization was performed using a triangular function. The system enabled spectra to be acquired from a sample area of approximately 200 μm2 in about 2 min. The preliminary tentative vibrational bands assignment was performed based on literature. Spectral differences among species were statistically validated with principal components analysis and clustering. The main spectral differences among spermatozoa spectral bands were observed in the DNA/RNA (A, B, and Z conformations), glycogen, lipids, fatty acids, cholesterol, and phospholipids vibrational bands. For seminal plasma, main spectral differences were found at the 1500-1700 cm-1 spectral region assigned to protein secondary structure (amide I and amide II). Increasing a-helix, fi-sheet, fi-turn bands assignments suggests the presence of fragmented protein in human, ovine, and porcine seminal plasma spectra. These results suggest compositional similarity among these 3 species compared to the bovine. In conclusion, FT-IR spectroscopy could provide relevant information related to spermatozoa and seminal plasma physiology useful in improving freezing methods and fertility monitoring. More studies will be needed to establish the relationship between spectral features and physiological processes.
32 USE OF HORMONE WITH ACTION FOLLICLE-STIMULATING IN OVULATION SYNCHRONIZATION OF CROSSBRED HOLSTEIN-GYR COWS
Authors: Nascimento V. A. , Moura G. S. , Dias M. , Oliveira F. A. , Dias A. R. , Torres C. A. A.
Published: August 2010

The objective of this study was to evaluate the administration of the follicle-stimulating hormone (FSH-p) or equine chorionic gonadotropin (eCG) in protocols of ovulation synchronization. One hundred forty-nine crossbred Holstein-Gyr cows were used in 2 properties in Minas Gerais and 3 treatments: TControl (n = 49 cows), Day 0: insert of a progesterone device (Primer®, Tecnopec, São Paulo, Brazil) + 2 mg of estradiol benzoate (RIC-BE®); Day 8: removal of Primer + 112 mg of D-cloprostenol (Prolise®, ARSA SRL, Buenos Aires, Argentina) + 1 mg of estradiol benzoate and fixed-time artificial insemination (FTAI) accomplished 44 h after the removal of Primer; TFSH (n = 50 cows), similar to TControl, but on Day 8, 15 mg of FSH-p (Folltropin®, Bioniche Animal Health) was administered; TeCG (n = 50 cows), similar to TControl, with administration on Day 8 of 300 IU of eCG (Novormon®, Syntex SA, Buenos Aires, Argentina). Cows that returned to estrus 14 to 28 days after the FTAI were inseminated once again. For all statistical analysis, we used the program SAS 9.0 (2002; SAS Institute Inc., Cary, NC, USA) at 5% probability. Quantitative variables (live weight, days postpartum, follicular average per cow) were analyzed by variance, and body condition score by Friedman test. The pregnancy rates were submitted to logistic analysis considering the effect of local experimental factors and were analyzed by Spearman correlation among the variables studied and the pregnancy rate. There was no difference (P > 0.05) between treatment for pregnancy rate in FTAI protocols (TControl = 40.82%; TFSH = 38.00%; TeCG = 26.00%), in the first insemination after FTAI (TControl = 56.25%; TFSH = 70.59%; TeCG = 68.42%), and in FTAI plus first insemination after FTAI (TControl = 59.18%; TFSH =62.00%; TeCG = 52.00%), as well as for the estrus repetition (TControl = 55.17%; TFSH = 54.84%; TeCG = 51.35%) and the correlation (P > 0.05) among the pregnancy rate and postpartum period, milk production, body weight and body condition. No effect was observed on the pregnancy rate by the addition of the hormones FSH and eCG in the protocol of ovulation synchronization in crossbred Holstein-Gyr cows; however, reinsemination in the period from 14 to 28 days after FTAI increased the pregnancy rate.

CNPq; FAPEMIG; TECNOPEC/São Paulo, Brazil.
320 SUSCEPTIBILITY OF GOAT SPERM TO DIFFERENT REACTIVE OXYGEN SPECIES
Authors: Silva R. O. C. , Perez E. G. A. , Cabral R. P. , Silva D. G. , Viana C. H. C. , Nichi M. , Goes P. A. A. , Ferreira G. H. , Teodoro A. C. , Barnabe R. C. , Barnabe V. H.
Published: August 2010

Semen quality is one of the main limiting factors for the success of artificial insemination in goats. It is well known that reactive oxygen species (ROS) lead to structural and functional damages to sperm, impairing or avoiding fecundation. The understanding of sperm oxidative mechanisms in goats may provide information on possible treatments to improve semen quality and fertility rates. The aim of the present study was to verify the resistance of goat spermatozoa to different reactive oxygen species. Sperm samples from 4 goats were collected using an artificial vagina. Sperm samples were then incubated (1 h, 37°C) with 4 ROS inducer mechanisms: xanthine/xanthine oxidase (produces superoxide anion), hydrogen peroxide (4 mM), ascorbate/ferrous sulfate (4 mM; produces hydroxyl radical), and malondialdehyde (MDA, lipid peroxidation product). Samples were analyzed for mitochondrial activity using the 3,3′ diaminobenzidine stain, for membrane integrity using the eosin/nigrosin staining, for acrosome integrity using the simple stain (fast green/Bengal rose), and for lipid peroxidation by dosing thiobarbituric acid reactive substances (TBARS). Results showed that goat sperm is more sensitive to hydrogen peroxide, when compared to superoxide anion, hydroxyl radical, and MDA, when considering acrosome integrity, membrane integrity, and mitochondrial potential (Table 1). On the other hand, TBARS production was increased in samples submitted to hydroxyl radical incubation. Strong negative correlations were found between sperm samples showing impaired mitochondrial potential and both membrane and acrosome integrity (r = -0.97, P < 0.0001 and r = -0.91, P < 0.0001, respectively). The concentration of TBARS correlated negatively with the percentage of sperm showing intact membranes (r = -0.53, P = 0.06), and the later correlated negatively with sperm showing no mitochondrial activity (r = -0.78, P = 0.0006). Results of the present experiment suggest that goat sperm are extremely susceptible to the attack of hydrogen peroxide, being resistant to other ROS. Therefore, an alternative to improve the use of goat semen in reproductive biotechnologies would be the treatment with catalase or glutathione peroxidase, important hydrogen peroxide scavengers.

Table 1.Effect of different ROS on goat sperm

The authors thank Nutricell for the media used in this experiment.
326 MICROINJECTIONS OF SMALL INTERFERING RNA AND COMPLEMENTARY RNA TO ELUCIDATE THE INVOLVEMENT OF ENDOGENOUS PHOSPHOLIPASE C ISOFORMS IN BOVINE OOCYTE ACTIVATION DURING FERTILIZATION
Authors: Sessions B. R. , Bayles A. H. , Collier J. , Perry K. , Whitaker L. S. , White K. L.
Published: August 2010

Phospholipase C (PLC) isoforms stimulate the hydrolysis of phosphatidyl inositol (4,5)-bisphosphate (PIP2) to produce diacylglycerol (DAG) and 1,4,5 inositol trisphosphate (IP3), with IP3 regulating the release of calcium (Ca2+) from the endoplasmic reticulum. This release of calcium is essential for oocyte activation, and a sperm-specific PLC isoform, PLCγ;, has been proposed as the primary agent that initiates the activation process. However, the oocyte contains many endogenous PLC isoforms (PLC β, γ, and δ) that could also be involved in regulating or initiating these calcium oscillations downstream of other initiating events. In order to better elucidate the involvement of endogenous PLC isoforms as well as the specific role of the sperm-specific form, small interfering RNA (siRNA) directed against the specific bovine PLC isoforms (PLCζ;, PLCγ1, PLCγ2, PLCδ1, PLCδ3, PLCδ4, PLCβ1, PLCβ3) were microinjected into bovine oocytes, and the subsequent effects on PLC mRNA levels and bovine fertilization were evaluated. Real-time PCR (qPCR) was used to quantify the levels of PLC message present in bovine oocytes at the time of injection (15 h post-maturation) and 6, 10, and 14 h post-injection. The qPCR results indicated a near-complete knockdown of mRNA levels in bovine oocytes 10 h post-injection for the isotypes PLCγ1, PLCγ2, PLCδ3, PLCδ4, PLCβ1, PLCβ3, but only partial knockdown of PLCS 1 mRNA. Oocytes microinjected with PLC siRNA were also fertilized and cultured in vitro according to our standard laboratory procedures (Reed et al. 1996 Theriogenology 45, 439-449). The oocytes microinjected with PLCζ;, PLCδ1, PLCδ3, PLCδ4, PLCβ1, PLCβ3 siRNA resulted in cleavage rates similar to the negative control siRNA, non-injected, and sham-injected treatment groups, whereas bovine oocytes microinjected with PLCγ1 and PLCγ2 siRNA had significantly lower cleavage rates compared with the controls. Additionally, complementary cRNA for each specific PLC isoform was microinjected into bovine oocytes to ascertain each isoform’s ability to induce parthenogenetic activation. Development was observed in oocytes microinjected with a variety of cRNAs, and the activating effects of the cRNA were negligible if the oocytes were microinjected with the corresponding siRNA before microinjection with cRNA. Interestingly, siRNA specific for PLCζ; failed to reduce cleavage when treated bovine oocytes were fertilized. These data illustrate the potential involvement of multiple endogenous PLC isoforms and not just the sperm-specific PLCζ; isoform in bovine oocyte activation during fertilization.
333 EFFECTS OF BOVINE OOCYTE QUALITY ON KINETICS OF NUCLEAR MATURATION AND EMBRYONIC DEVELOPMENT AFTER IN VITRO FERTILIZATION
Authors: Carvalhais I. , Faheem M. , Habibi A. , Geraldo A. , Agrícola R. , Chaveiro A. , Moreira da Silva F.
Published: August 2010

Many factors act together to prepare the immature oocyte for successful development to a competent embryo after fertilization. Defects in oocyte maturation and further development can possibly be caused by the oocyte quality or an inadequate nuclear maturation or even by a failure of both. In the present study, the effect of COCs quality on meiotic development and further embryo-development after in vitro fertilization was evaluated. A total of 3604 COCs was separated according to their morphological aspect and were classified as A, B, and C categories. Briefly, in class A, oocytes possessed compact layers of cumulus cells, being difficult to evaluate their number having a homogenous ooplasm with uniform color. In class B, oocytes show more or equal to five layers of cumulus cells, easily identifiable under a stereomicroscope and/or granulations in the ooplasm. In class C, some granulation was observed in oocytes with about three layers of cumulus cells. The total number of oocytes was divided into two groups (I and II) in which in the group I, COCs (n = 540) were fixed 0, 6, 12, 18, 24, and 30 h following ovarian aspiration, DNA was stained with aceto-orcein, and the nucleus were observed under a phase contrast microscope. In the Group II, COCs (n = 3064) were fertilized with frozen/thawed bull semen after 24 h of maturation, which was made in M199 medium (Sigma, St, Louis, MO, USA). The development of the embryos was evaluated on the third and seventh day after fertilization. Embryos were co-cultured with monolayers of granulosa cells in 45 μL droplets of B2 medium (CCD Laboratory, Paris, France), supplemented with 10% serum under mineral oil, at 39°C and 5% CO2 in air. It was observed that, other than the oocytes achieved metaphase II at 24 h was greater for the oocytes classified as A (65.4%), and B (61.0%) greater than C (51.2%), no statistical difference was observed between oocyte quality and capability to maturation. As far as the embryonic development is concerned, the same tendency was observed for the cleavage and for the morulae/blastocyst stage after 7 days after fertilization (P < 0.001). The percentages of cleaved oocytes classified as A, B, and C, were respectively 65.2%, 58.4%, and 48.0%. The development to the morulae/blastocyst stage of the cleaved embryos was A = 38.5%/27.4%, B = 33.6%/25.0%, and C = 30.9%/17.2% (Table 1). The results of our study clearly demonstrate that the morphology of the oocytes plays an important role on the in vitro embryonic developmental competence after fertilization.

Table 1.Development of oocytes according to COCs quality, evaluated 3 and 7 days after fertilization

The first author is supported by the Regional Foundation for Science and Technology of the Azores Government. This study was supported by the IBBA Institute grant number M2.1.2/I/022/2008 CITA-A is fully acknowledged.
334 EFFECT OF A GROWTH MEDIUM DURING IVM ON EMBRYO DEVELOPMENT OF PREPUBERTAL EWE OOCYTES
Authors: Catalá M. G. , Izquierdo D. , Romaguera R. , Roura M. , Paramio M. T.
Published: August 2010

The aim of this study was to assess the effect of an in vitro growth medium (De Wu et al. 2006 Biol. Rep. 75, 547-554) in prepubertal ewe oocytes selected by the brilliant cresyl blue (BCB) test. Prepubertal ewe oocytes were recovered by slicing ovaries of slaughtered animals and immediately exposed during 1 h to 13 μM BCB and classified according to their cytoplasm coloration (Rodriguez-Gonzalez E et al. 2002 Theri- ogenology 57(5), 1397-1409): BCB+ (blue cytoplasm, hypothetically grown oocytes) and BCB- (uncolored cytoplasm, hypothetically growing oocytes). Uncolored oocytes (BCB-) were matured using three culture media: growth medium (GM: TCM-199, 0.04 μg mL-1 FSH, 0.04 μg mL-1 LH, 0.004 μg mL-1 estradiol, 100 μg mL-1 ascorbic acid, and 5 μL mL-1 ITS: insulin transferrin selenium), conventional maturation medium (CM: TCM-199, 10 μg mL-1 FSH, 10 μg mL-1 LH and 1 μg mL-1 estradiol) and modified maturation medium (MM: CM with the addition of 100 μg mL-1 ascorbic acid and 5 μL mL-1 ITS). Oocytes were matured in GM for 12 h and then separated into 2 groups, CM (GM+CM) and MM (GM+MM) for another 12 h of maturation. Two extra groups of BCB- oocytes were directly cultured for 24 h in CM or MM media (BCB-/CM and BCB-/MM). Colored oocytes (BCB+) and a control group (oocytes not exposed to BCB) were matured for 24 h in CM. All groups were cultured at 38.5°C and 5% CO2 in humidified atmosphere. Fertilization took place in SOF medium supplemented with 10% of estrous sheep serum during 20 h with a sperm concentration of 1 × 106 spermatozoa/mL. Presumptive zygotes were cultured for 8 days in SOF with 10% FCS at 38.5°C, 5% CO2 and 5% O2. Results are shown in Table 1. The percentage of morula plus blastocyst obtained from BCB - oocytes was significantly increased in oocytes exposed to growth medium (containing ITS, ascorbic acid and low hormone concentrations; groups GM+CM and GM+MM) for the first 12 h. An increasing tendency has also been observed in blastocyst yield in these two groups. Regarding maturation rate, no differences were found in all groups (data not shown). In conclusion, as De Wu et al. (2006) showed in prepubertal gilts, we also achieved some improvements in embryo development of growing oocytes when the first 12 h of maturation took place in a growth medium. However, embryo developmental potential of BCB- oocytes is still lower compared with that of BCB+ oocytes.

Table 1.Effect of GM on embryo development of BCB- oocytes

Grant sponsor Spanish Ministry of Science and Innovation.Code: AGL2007-60227-CO2-01
341 IN VITRO MATURATION AND IN VITRO FERTILIZATION OF ALPACA (VICUGNA PACOS) OOCYTES: EFFECT OF TIME OF INCUBATION ON NUCLEAR MATURATION AND CLEAVAGE
Authors: Huanca W. , Condori R. , Cainzos J. , Chileno M. , Quintela L. , Becerra J. , Herradon P. G.
Published: August 2010

Experiments were carried out to evaluate the effect of incubation time on nuclear maturation (Experiment 1) and determine the cleavage rate of alpaca oocytes after of IVF time (Experiment 2) In Experiment 1, CCOs were collected from slaughterhouse ovaries and transported to the laboratory in a thermos flask containing a saline solution 0.9% with antibiotic antimycotic at 35°C. CCOs were aspirated from follicles >2 mm and pooled in a conical tube to sedimentation previous to evaluation under stereomicroscope and CCOs with a cytoplasm homogeneous and 2 or more layers of cumulus cells were transferred to plates with a 40-μL drop of maturation medium TCM-199 supplemented with 10% FCS (v : v) plus 0.5 μg mL-1 FSH, 10 μg mL-1 hCG, 0.2 mM sodium pyruvate, 50 μg mL-1 gentamicine, and 1 μg mL-1 Estradiol under mineral oil with 10-12 oocytes/drop. Oocytes were incubated under the following maturation times: 30, 34, and 38 h at 39°C in an atmosphere of 5% CO2 and high humidity. After each maturation time, CCOs were removed from maturation medium and washed with PBS supplemented with 10% FCS and 1 mgmL-1 of hyaluronidase and fixed in ethanol: acetic acid (3 : 1). Oocytes were placed on the slide with minimum medium and stained with 1% orcein for 5 min The slides were examined under a phase contrast microscope at × 400 to evaluate status of nuclear maturation and classified as germinal vesicle (GV); metaphase I (M-I), anaphase-telophase; metaphase II (M-II) and degenerated. Experiment 2: The same maturation method as Experiment 1 was used. Testes were collected of mature males from slaughterhouse and transported to the laboratory. Caudal epididymide was isolated. A prick was made on the convoluted tubules with a sterile hypodermic needle and the fluid, rich in spermatozoa, was aspirated in syringes containing 2 mL of Tris-fructose egg yolk extender. Motile spermatozoa were obtained by centrifugation: 700 g on a Percoll discontinuous gradient (22.5 :45.0%) for 25 min. The supernatant was removed by aspiration and pellet (containing viable spermatozoa) was resuspended in TL stock. Spermatozoa and oocytes were co-incubated for 18-20 h at 39°C with 5% CO2 and then cultivated in TCM-199 supplemented with 10% FCS (v: v), 0.2 mM sodium pyruvate, and 50 μg mL-1 gentamicine and evaluated at 48 h. Data were subjected to ANOVA. For Experiment 1, the proportions of oocytes reaching M-II stage was 18.9 ± 15.7, 42.9 ± 16.2, and 65.8 ± 8.1% for the 30, 34, and 38 h of culture, respectively, with difference to maturation time (P < 0.05). For Experiment 2, the cleavage rate was 9.5, 7.7, and 15.4% to 30, 34, and 38 h after of fertilization time 48 h culture. These results indicate that 38 or more h is required for the maturation and fertilization of alpaca oocytes.
Grant 064 FINCyT-PIBAP 2008.
342 EFFECTS OF MELATONIN ON PREIMPLANTATION DEVELOPMENT OF PORCINE PARTHENOGENETIC EMBRYOS
Authors: Kang J.-T. , Koo O.-J. , Kwon D.-K. , Park S.-J. , Atikuzzaman M. , Gomez N. , Jang G. , Lee B.-C.
Published: August 2010

In mammalian species, melatonin is a multi-functional pineal gland hormone that regulates several circadian and seasonal rhythms including reproduction. However, the melatonin study was not common as to the oocytes in the pig. Recently, we reported that exogenous melatonin has beneficial effects on nuclear and cytoplasmic maturation during porcine oocyte IVM and we also reported an existence of melatonin receptor on the cumulus cells and granulose cells (Kang JT et al. 2009 J. Pineal Res. 46(1), 22-28). In this study, as adding further experiments rather than our previous study, we investigated effect of exogeneous melatonin (10 ng mL-1) on the porcine oocytes and analyzed possible factors which can be responsible for that results. Oocytes were recovered by aspiration of slaughterhouse ovaries, and then matured in TCM-199 supplemented with EGF, insulin, pyruvate, cystine, and gonadotropin. Expression of apoptosis-related genes mRNA in oocytes cultured with melatonin were evaluated by real-time PCR (Exp 1), cumulus cell expansion on COC was assessed on the microscopes during in vitro maturation (Exp 2), and developmental effects between melatonin treatement group and non-treatment group on the in vitro culture of parthenogenetically activated oocytes was investigated (Exp 3). In results, oocytes matured with melatonin were assessed for the expression of apoptosis-related genes Bcl-xl (anti-apoptotic gene) and Bax (proapoptotic gene) by real-time quantitative PCR. Analysis of data showed that the expression of Bcl-xl was higher compared to the control while the expression of Bax was decreased relative to the control (P < 0.05). Cumulus cell expansion was evaluated under a stereomicroscope at 22 h, 44 h during IVM. Representative photomicrographs of porcine COC at the start of the IVM, after 22 h and 44 h treatment with melatonin, are shown in Figure. After 22 h of melatonin treatment, cumulus cells were visually expanded compared with non-treatment group. We analyzed significantly greater proportion of parthenogenetically activated oocytes developed to blastocyst when the IVM medium was supplemented with melatonin. Melatonin treatment in the IVM has consequently beneficial effect on the blastocyst formation rates on the development of porcine parthenogenetic embryos (15.4%) compared to non-treatment group (10.7%, P < 0.05). However, cleavage frequency was not affectedby the treatment. In conclusion, the present study demonstrated that melatonin had a beneficial effect on the development of parthenogenetically activated porcine embryos, probably through decreased apoptosis rate and increased cumulus cell expansion.
This study was supported by Korean MKE, MEST (BK21 program), and Hanhwa L&C
35 FERTILITY AFTER FIXED-TIME AI OR INSEMINATION AT DETECTED ESTRUS FOLLOWING CIDR SYNCHRONIZATION PROTOCOLS IN FRENCH DAIRY AND BEEF CATTLE
Authors: Ponsart C. , Gatien J. , Besognet B. , Fanuel P. , Humblot P.
Published: August 2010

The CIDR synchronization protocol was introduced in France since 2008, as a 7-day intravaginal progesterone treatment (1.38 g of progesterone) coupled with AI at detected estrus. The objective of this study was to compare the synchronization, ovulation, and conception rates following fixed-timed AI (FTAI) and AI on observed heat (HAI) following a CIDR synchronization program in dairy and beef cattle under on-farm conditions. A total of 466 females from Holstein, Limousin, and Charolais breeds were synchronized from October 2008 to March 2009, and inseminated by AI technicians from 4 French cooperatives (Amélis, CECNA, COOPELSO, Groupe Altitude) with the following inclusion criteria : heifers from 14 to 28 months old, cows with 1 to 5 calvings, interval from calving from 50 to 150 days, first breeding following calving without any complication. The CIDR device was left for a 7-day period and combined with a PGF2α injection 1 day before CIDR removal and an eCG injection at the time of CIDR removal. Cows within each herd were allocated to 2 insemination groups. In the FTAI group, a single insemination was performed 56 h after CIDR removal, whereas the females from the HAI group were inseminated once from 6 to 18 h after detected estrus. For each female, cyclicity was assessed before CIDR insertion with 2 progesterone measurements assayed 10 days apart. The CIDR device was lost in 25 females during treatment. In the remaining females, estrus synchronization was estimated from a progesterone measurement 56 h after CIDR removal. Ovulation rate following CIDR programs was determined by progesterone measurements 14 days after AI. Pregnancy was diagnosed by ultrasonography on Day 35 after AI in 417 females (24 missing values). From results of cycling females prior to treatment (n = 369), synchronization rates averaged 95.6% and 94.3% in HAI and FTAI groups, respectively. Ovulation rate, calculated from 399 synchronized or noncycling females, averaged 93.4% and 91.6% in HAI and FTAI groups, respectively. Conception rate at first AI averaged 51.9% (HAI: 52.6% v. FTAI: 47.4%; P > 0.05), and conception rate following induced estrus (i.e. calculated from ovulated females only) averaged 55% (HAI: 55.3% v. FTAI: 52.2%; P > 0.05). Conception rate was mainly influenced by breed and parity, ranging from 34% in Holstein cows to 58.7% in Holstein heifers (Table 1). To conclude, fixed-time insemination can be coupled to CIDR synchronization protocols in beef and dairy cattle and can be used to as a substitute for heat detection under on-farm conditions.

Table 1.Conception rates depending on breed and parity
353 CUMULUS CELLS RESPONSE TO HEAT SHOCK AND INSULIN-LIKE GROWTH FACTOR-I
Authors: Paula-Lopes F. F. , Mendes C. M. , Risolia P. H. B. , Gonçalves J. S. A. , Feitosa W. B. , Raposo M. , Assumpção M. E. O. A. , Visintin J. A.
Published: August 2010

Heat-stress induced maternal hyperthermia has been shown to compromise the series of events associated with oocyte growth and maturation reducing oocyte competence. Such events are regulated by a variety of growth factors and dynamic communication between the oocyte and its surrounding cumulus cells. The objective of the current study was to evaluate the modulatory effects of COCs quality and IGF-I on mitochondrial membrane potential (MMP) and apoptosis in cumulus cells induced by heat shock. In this study high (≥3 layers of compact cumulus cells and homogeneous cytoplasm) and low-grade COCs (<3 layers of less compact cumulus cells and irregular cytoplasm) derived from slaughterhouse ovaries were exposed to control (CTR: 39°C) or heat shock (HS: 41°C) treatments in the presence of 0 or 100 ng mL-1 IGF-I during the first 12 h of in vitro maturation (12 h-IVM). Immediately after 12 h-IVM COCs were denuded by repeated pipetting and cumulus cells evaluated for MMP (MitoProbe JC-1 assay kit. JC-1 is a cationic dye that exhibits potential-depend accumulation in the mitochondria) and apoptosis (Annexin V-FITC and propidium iodide) by flow cytometry (Guava EasyCyte Mini Flow Cytometry System, Millipore, Billerica, MA, USA). This factorial experiment was replicated 4 times using 75-100 COCs per treatment. Data were subjected to three-way analysis of variance using the General Linear Models procedure of SAS. Results are shown in Table 1. Exposure of high and low-grade COCs to HS reduced (P < 0.01) the percentage of cumulus cells carrying high MMP regardless of IGF-I. Even though HS caused cumulus cells mitochondrial membrane depolarization there was neither temperature nor COCs quality effect on cumulus cells apoptosis as indicated by the lack of phosphatidylserine (PS) translocation from the inner to the outer leaflet of the plasma membrane. On the other hand, addition of IGF-I to maturation medium reduced (P < 0.05) the percentage of cumulus cells labeled with Annexin V + PI regardless of COCs quality or temperature. There was no statistical interaction between COCs quality × IGF-I × temperature. In conclusion, exposure of COCs to HS during 12 h-IVM caused cumulus cells mitochondrial depolarization without inducing apoptosis. It is possible that a period longer than 12 h is required for most PS translocation to occur in cumulus cells. Moreover, IGF-I exerted protective effect reducing cumulus cells late apoptosis/necrosis events.

Table 1.Effect of heat-shock and IGF-I on cumulus cells mitochondrial membrane potential and apoptosis. Results are least-squares means ± SEM.