bluuebunny/crossref_metadata_2025 huggingface dataset:
152 STUDY OF THREE CONSECUTIVE ELECTROEJACULATIONS IN BROWN BEAR (URSUS ARCTOS)
Authors: Anel L. , Borragan S. , Alvarez M. , Martinez-Pastor F. , Mata-Campuzano M. , Gomes-Alves S. , Anel E. , de Paz P.
Published: December 2008

The preservation of threatened species, such as the cantabric brown bear, requires the establishment of genetic resource banks. In these species it is important to increase the efficiency of the electroejaculation techniques so as to collect as many gametes as possible from each collection, and to decrease risks of anesthesia and immobilization. Our objective was to study several characteristics of brown bear semen quality obtained in three consecutive electroejaculations (a, b, and c) in the same anesthetic session. Ejaculates were collected from 11 adult males living in the Cabárceno Nature Park during the breeding season (May–July). Animals were anesthetized by administration of tiletamine + zolazepan (Zoletil 100®) and ketamine (Imalgene 1000®) before being subjected to electroejaculation (6 to 10 V; 250 to 300 mA). From each ejaculate we assessed motility (CASA: SCA, Microptic, Barcelona, Spain), osmolality (mOsm k–1), and viability (VIAB: % viable spermatozoa (spz), SYBR-14 and propidium iodide) and mitochondrial membrane potential (MIT: % spz, JC-1) by flow cytometry. Our results show that total spz (×106 spz) varies widely among individuals depending on the number of electroejaculation. In five males we observe a decreasing pattern (a: 454.50; b: 341.7; c: 138.0); in other two males it is observed an increasing pattern (a: 24.9; b: 70.3; c: 334.3) while in the remaining four males we see a varied pattern, with the sperm production peak in the second electroejaculation (a: 53.4; b: 270.6; c: 107.5). Motility parameters do not show differences among the three electroejaculations, showing a reduction of the progressive motility in the males with increasing pattern with respect to the other patterns. Also, spermatozoa physiology indicators show a relation with the sperm production patterns. For viability (%) it is shown a rising tendency in the increasing pattern (a: 64.0, b: 80.0, c: 79.5) and a reduction tendency in the decreasing pattern (a: 68.7, b: 61.0, c: 58.7). The same is observed in the case of mitochondrial membrane potential (%) (increasing pattern [a: 77.0, b: 89.0, c: 87.0]; decreasing pattern [a: 80.0, b: 76.3, c: 55.7]). Ejaculates of the varied pattern show irregular data for these parameters. On the other hand, osmolality changes depending on the number of electroejaculation (increasing pattern [a: 324.0, b: 289.0, c: 298.3]; decreasing pattern [a: 333.0, b: 297.0, c: 283.0]; varied pattern [a: 264.0, b: 294.6, c: 318.54]) which would determine a change in the spz microenvironment that regulates their physiological activity. Although the high individual variability observed does not lead to solid conclusions, our results indicate that consecutive electroejaculations can be useful for increasing the technique yield in brown bear.

Supported by CICYT (CGL2007-63788/BOS) and Cantur SA. 3Supported by Juan de la Cierva program (MICINN, Spain).
17 THE EFFECT OF EXOGENOUS PROGESTERONE ON CONCEPTION RATE OF COOLED COWS DURING THE SUMMER AND AUTUMN
Authors: Wolfenson D. , Friedman E. , Lavon Y. , Roth Z.
Published: December 2008

Dairy cows exhibit low concentrations of plasma progesterone during the summer. The effect of exogenous progesterone supplementation postinsemination (AI) in the summer on conception is equivocal. Moreover, the variability among studies in thermal stress severity contributes to the variable effect of exogenous progesterone on summer fertility. This study examined the effect of supplementing exogenous progesterone post-AI during the summer and autumn on conception rate of lactating cows that were efficiently cooled during the hot season. The study was conducted in Israel on 2 commercial farms of high-milk-producing cows, yielding, on average, 41.3 kg of milk day–1 around AI (124 days postpartum). These farms were equipped with efficient cooling systems along the feeding line and in the holding area. Mean body temperatures during the afternoon and evening hours in summer (air temperature and relative humidity of 30 to 32°C and 41 to 57%) were 39.1 ± 0.1°C, with a peak value of 39.5°C. Cows at first, second, or third AI that were inseminated after estrus (Day 0; detected by visual observation and computerized pedometric system; Afimilk, Israel) from July to August (summer) or September to October (fall) were included in the study (n = 377 cows). Treated cows (n = 181 cows) were fitted with an intravaginal device containing progesterone (CIDR) on Day 5 ± 1 after AI for 12 days; the device was removed on Day 17. Administration of the CIDR increased progesterone concentration by 2.3 ng mL–1 (P < 0.05). Conception rates were analyzed by a logistic regression procedure. Overall, the CIDR treatment increased conception rate by 6% compared with controls (39 v. 33%; NS). Day of CIDR insertion tended to affect conception rate (43, 39, and 34% for Days 4, 5, and 6, respectively; NS). The CIDR treatment significantly increased, compared with the control (+23%, P < 0.05), the conception rate of cows with a low body condition score at peak lactation (49.3 v. 26.6%). Similarly, the CIDR significantly increased (+22%, P < 0.05) the conception rate of cows that exhibited a uterine disorder at parturition (46.3 v. 24.6% in untreated control cows). The CIDR treatment also increased conception rate (NS) in the fall (+13%) in mature cows (+8%) and in the first AI (+12%). Similarly, cows treated with a CIDR before Day 120 postpartum, or cows yielding less than 40 kg of milk day–1 exhibited a numerically higher (+8%; NS) conception rate. Results indicated that exogenous progesterone administered post-AI increases conception during the summer and autumn in efficiently cooled cows during the hot months. The most beneficial effect was documented in cows that had been diagnosed as having a uterine disease postpartum, and in those that exhibited a low body condition score.
172 IDENTIFICATION OF MICRORNAS IN BOVINE OVARY
Authors: Hossain M. M. , Hoelker M. , Phatsara C. , Tholen E. , Schellander K. , Tesfaye D.
Published: December 2008

Tightly regulated expression and interaction of a multitude of genes for ovarian folliculogenesis leading to successful oocyte development could be regulated by recently identified new class of small RNAs of ~22 nt (i.e. microRNAs), which are already proved as one of the vital transcriptional regulators in different biological processes including development. But their presence and expression in bovine ovary has not yet been determined. Here, we have attempted to identify miRNAs in bovine ovary by small RNA-cDNA library construction through 5 ligation independent cloning. For this purpose, total RNA enriched with small RNA was isolated from ovary and size fractionated (18 to 24 nt) by denaturing PAGE. Extracted RNA was first 3′ linkered and after template switching by RT, the second 3′ linkering of the first strand cDNA was performed. These linkered small RNA-cDNAs were then amplified with linker-specific primers consisting of BAN I restriction sites, concatemerized by serial ligation, cloned into TOPO TA vector, and transformed into TOP 10 chemically competent cells. After screening, colonies were picked and sequenced. Bioinformatic analysis was done according to the published criteria for the small RNAs. From 233 clones a total of 479 reads were identified. Frequency of sequence length found in the library was 26.8% for ≤18 nt, 55.1% for 19 to 22 nt, and 18.1% for ≥23 nt. The total 479 sequences identified in the library represent 35% miRNAs, 12% mRNA, 12.1% rRNA, 5.6% tRNA, 4.2% repeat associated siRNA, 3.8% non-repeat-associated siRNA, 4% tiny noncoding RNA, 1% small nuclear RNA, and 16% sequences not matched to bovine genome. All 171 miR sequences comprised 79 distinct miRNAs, of which 45 miRNAs already annotated in miRBase for bovine and the other 34 miRNAs are new discoveries. Of the 34 newly identified miRNAs, 12 are described in other species but not yet in bovine. Most of the miRNAs cloned into multiple times, where let-7a cloned for 10, let-7b for 28, let-7c for 13, miR-21 for 4, miR-23b for 11, miR-24 for 7, miR-27a for 6, miR-126 for 4, and miR-143 for 11 times. Based on best hit score, P-value and free energy by online target prediction, some of the bta-miR identified in the library (let-7b, 15b, 18a, 23b, 101, 125b, 126, 140, 145, 199a) are found to target hundreds of genes related to follicular development, ovulation and hormonal regulation. Further functional characterization of some selected miRNAs including expression profiling and in situ localization in follicles of different size and cycles may supplement the results of this study and will enable us to gain insight into their relation to female fertility.
173 THE EFFECT OF EXERCISE ON FOLLICULOGENESIS IN MARES
Authors: Kelley D. E. , Gibbons J. R. , Pratt S. E. , Smith R. L. , Mortensen C. J.
Published: December 2008

The effects of exercise on follicular development in mares have not been well established; however, a combination of exercise and heat stress can negatively affect embryo recovery rate in mares. The purpose of this research was to examine the effects of exercise, independent of heat stress, on follicular development in mares. One thoroughbred and 12 quarter horse mares, aged 3 to 15 years, were used in this study. Mares were assigned to the control group or exercised at 0700 h, 6 days a week, as follows: warmed up 5 min at a walk for 3 m s–1 and then exercised 30 min, 9 m s–1 for 10 min, 13 m s–1 for 5 min, 9 m s–1 for 5 min, 13 m s–1 for 5 min, and 9 m s–1 for 5 min. Exercised mares were given a 1-month conditioning period. Rectal temperatures were recorded immediately before and after exercise. The mean rectal temperature of mares increased by 0.85°C during exercise, whereas control mares under the same environmental conditions did not experience temperature increases. Individual mares underwent a daily transrectal ultrasound examination with a 7.5-MHz linear rectal probe to monitor follicular development. Estrous cycles were normalized to 17 days for linear follicular growth analysis. Exercise had a significant impact on follicular growth at deviation, with a greater diameter of the largest (P < 0.05) and second largest (P < 0.01) follicles in exercised v. control mares (Table 1). Additionally, growth rate of the second largest follicle was greater in the exercised group (P < 0.01). Deviation was observed in 13 of 14 cycles in control mares, whereas in exercised mares, it was clear in only 11 of 16 cycles (χ2, P = 0.086). Time of deviation was defined as the day when a difference in the growth rate between the dominant and largest subordinate follicle of a wave was first observed. The largest subordinate follicle either declined in growth rate or regressed. At Day –5 (relative to ovulation), exercised mares had significantly greater follicle diameters in both the largest and second largest follicle (P < 0.05). Exercised mares had double ovulations in 4 of 16 cycles when compared with control mares, which had double ovulations in 1 of 14 cycles. No differences were found between groups in the length of the estrous cycle or ovulatory follicular diameter. Exercised mares exhibited larger diameter follicles and an increased growth rate of the largest subordinate follicle, suggesting that exercise delayed deviation. Further research is needed to assess the impact of exercise, independent of heat stress, on fertility.

Table 1.Follicle dynamics in exercised v. non-exercised mares
175 FOLLICULAR DIAMETER AND OVULATION RATE IN NELLORE COWS TREATED WITH LH
Authors: Simões R. A. L. , Rosa F. S. , Piagentini M. , Satrapa R. A. , Ereno R. L. , Nabhan T. , Trinca L. A. , Nogueira M. F. G. , Barros C. M.
Published: December 2008

The aim of the present experiment was to verify the correlation between follicular diameter and ovulation rate in Nellore cows submitted to LH treatment to add information to the limited literature about this subject. A preliminary test (experiment 1) was performed to determine the lowest dose of LH necessary to induce ovulation in follicles with diameter ≥10 mm. At a random stage of the estrous cycle (D0), Nellore cows (n = 20) received a progesterone intravaginal device (1.0 g, Primer®, Tecnopec, Sao Paulo, Brazil) and 2.5 mg of estradiol benzoate (EB, i.m., Estrogin®; Farmavet, Sao Paulo, Brazil). Eight days later (D8) PGF2α was administered (150 μg of D-cloprostenol, i.m., Prolise® ARSA S.R.L., Buenos Aires, Argentina) and the device was removed. Twenty-four hours after device removal, cows were treated with EB (1.0 mg, i.m.) and 48 h afterward; ovulation was determined by ultrasonography (US, Aloka 900, Tokyo, Japan). Three days after ovulation, the follicular growth was daily observed by US, and when the follicles reached ≥10 mm diameter, the cows were randomly allocated in 4 groups and treated with different doses of LH (i.m., Lutropin-V®, Bioniche, Ontario, Canada): Groups 1.56; 3.12; 6.25, and 12.5 mg. The data were analyzed by Fisher’s Exact Test (PROC FREQ, SAS). The ovulation rates were 0% (0/5), 80% (4/5), 80% (4/5), and 80% (4/5) respectively, and it was lower in group 1.56 when compared with the others (P < 0.02). Taking in account the results above, the dose of 6.25 mg to induce ovulation in Nellore cows (n = 58, experiment 2) submitted to the same protocol used in experiment 1 and randomly allocated in 5 groups according to follicular diameter at the time of LH administration: G1 (7.0–7.5 mm), G2 (7.6–8.0 mm), G3 (8.1–8.5 mm), G4 (8.6–9.0 mm), and G5 (9.1–10.0 mm). The results were analyzed by Logistic Regression (PROC GENMOD, SAS). The ovulation rates were 0% (0/11), 20% (2/10), 36% (4/11), 36% (4/11), and 90% (9/10), respectively, for G1, G2, G3, G4, and G5. When the data from 2 groups (except G5) were combined, G1 + G2 (7.0–8.0 mm), G3 + G4 (8.1–9.0 mm), and G5 (9.1–10.0) ovulation rates were, respectively, 9% (2/21), 36% (8/22), and 90% (9/10). There were significant differences when comparing G1 + G2 v. G5 (P < 0.01), G3 + G4 v. G5 (P < 0.02) and G1 + G2 v. G3 + G4 (P < 0.03). After LH application, the smallest follicle that ovulated had a diameter of 7.65 mm. In summary, although LH administration induced ovulation in one follicle of 7.65 mm, high ovulation rates were observed only when the follicles reached <9.0 mm. These data will be used to study the correlation between ovulation rate and expression of isoforms of LH mRNA receptors in theca and granulosa cells.

Fellowship from *FAPESP (Sao Paulo, Brazil) and **CAPES (Brasilia, Brazil).
176 GENE EXPRESSION OF LUTEINIZING HORMONE RECEPTOR (LHR) ISOFORMS IN GRANULOSA CELLS OF FOLLICLES FROM NELLORE HEIFERS BEFORE, DURING, AND AFTER FOLLICULAR DEVIATION
Authors: Barros C. M. , Ereno R. L. , Machado M. F. , Buratini J. , Pegorer M. F. , Simões R. A. L. , Satrapa R. A.
Published: December 2008

During bovine follicular development, there is a phase known as follicular deviation in which the future dominant follicle grows faster than the other follicles and acquires LH receptors (LHR). In Nellore breed, deviation occurs 2.5 days after ovulation, and at this time, the dominant follicle has in average a diameter of 6.0 mm. Some authors believe that LHRs are present in the future dominant follicle before deviation and are essential for this process. However, others are convinced that LHRs are present only during or after follicular deviation. The aim of the present experiment was to evaluate the expression of 4 LHR isoforms (M1 to M4) in granulosa cells of follicles from Nellore heifers before, during, and after follicular deviation. At a random stage of the estrous cycle (D0), Nellore heifers (n = 21) received a progesterone intravaginal device (1.0 g, Primer®, Tecnopec, Sao Paulo, Brazil) and 2.5 mg of estradiol benzoate (EB, i.m., Estrogin®, Farmavet, Sao Paulo, Brazil). Eight days later (D8) PGF2α was administered (150 μg d-cloprostenol, i.m., Prolise®, ARSA S.R.L., Buenos Aires, Argentina), and the device was removed. Twenty-four hours after device removal, cows were treated with EB (1.0 mg, i.m.), and from this point in time, the growth of the dominant follicle growth was observed by ultrasonography (US, Aloka 900, Tokyo, Japan) every 12 h. The animals were allocated in 3 groups: Group 2 (G2, 2 days after ovulation, n = 7), Group 2.5 (G2.5, 2.5 days after ovulation, n = 7), and Group 3 (G3, 3 days after ovulation, n = 7), and were slaughtered 2, 2.5, and 3 days after ovulation, respectively, in order to remove the ovaries. The granulosa cells, obtained from ovarian follicles, were separated for total RNA extraction, and the gene expression of LHR isoforms was measured by semiquantitative RT-PCR. Since LHR expression was not detected in Group 2 (follicles with 4.5 to 6.7 mm), comparisons were performed between groups G2.5 and G3 by ANOVA. The LHR expression was detected only in 2 samples of Group G2 (7.0-mm follicles) and was significantly higher in Group G3 (63.6%; follicles from 8 to 14 mm, P < 0.05). In all samples that expressed LHR, the 4 isoforms were present. It is concluded that LHR expression is present in granulosa cells of follicles from Nellore heifers after follicular deviation.

Support and fellowship from FAPESP (Sao Paulo, Brazil).We are grateful to Tecnopec (Sao Paulo, Brazil) for providing intravaginal devices used in the experiment.
19 OPTIMIZATION OF ENUCLEATION TIME AFTER IVM FOR SOMATIC CELL NUCLEAR TRANSFER IN GOAT
Authors: Ajithkumar G. S. , Krishnamohan B. , Sarkhel B. C.
Published: December 2008

For cloning by somatic cell nuclear transfer (SCNT) in goat, there are conflicting reports about the proper time of enucleation after IVM of oocytes, which varied from 20 to 27 h (Das SK et al. 2003 Small Rumin. Res. 48, 217–225; Keefer CL et al. 2002 Biol. Reprod. 66, 199–203; Daniel SM et al. 2007 Small Rumin. Res. 77, 45–50). The present investigation has been undertaken to standardize the optimum time of enucleation after IVM of oocytes. The hypothesis behind the study was that enucleation performed during early stages of maturation maintains the MII plate and polar body (PB) in a closer position and therefore makes it easy to enucleate. To test this hypothesis, caprine COCs were aspirated from slaughterhouse ovaries of goats and good quality oocytes were matured in TCM 199 containing 7.5% FBS supplemented with FSH, LH, and estradiol. Enucleation was performed in four different interval groups after IVM (20–23 h, 23–26 h, 26–29 h and 29–32 h). The enucleation of oocytes was conducted as per method described by (Du F et al. 2006 Theriogenology 65, 642–665). Briefly, IVM oocytes were enucleated by squashing and compressing out the first PB along with 10 to 15% of its surrounding cytoplasm with an enucleation needle through a slit made on the zona pellucida. Successful enucleation was confirmed by fluorescent microscopy of removed ooplasm after staining with Hoechst 33342. The enucleation percentage values after arcsine transformation was analyzed by completely randomized design ANOVA. The result of enucleation at different intervals has been summarized in Table 1. There was no significant difference (P > 0.05) in number of PB observed among the four enucleation groups, however the enucleation percentage decreased significantly (P < 0.05) with increase in enucleation time (70.29% and 70.51% in G1 and G2 v. 59.52% and 55.61% in G3 and G4 respectively). With increase in time of enucleation after maturation the size of perivitelline space increases, causing deviation of PB from spindle, thus the success rate of enucleation is reduced (Song K et al. 2007 Repro. Fertil. Dev. 19, 293–294). In G1 and G2 groups the PB and MII chromosomes are located close together with stronger spindle force that requires minimum ooplasm to be removed with higher percentage of successful enucleation. Hence, it was concluded that G1 and G2 groups may be considered as most efficient for enucleation but the developmental potential of reconstructed oocytes after nuclear transfer in each group needs to be tested (study under progress).

Table 1.Enucleation results at different intervals
196 EMBRYO BIOPSY TRANSCRIPTOMICS: A POTENTIAL TOOL TO IDENTIFY TRANSCRIPTS DIRECTLY RELATED TO THE ABILITY OF THE EMBRYO TO INDUCE PREGNANCY AFTER TRANSFER
Authors: Tesfaye D. , Ghanem N. , Rings F. , Tholen E. , Phatsara C. , Schellander K. , Hoelker M.
Published: December 2008

The incidence of pregnancy loss due to embryonic mortality in cattle is one of the major causes of reproductive failure. The early embryonic loss can be due to problems with the embryo itself, the uterine environment, or interactions between the embryo and the uterus. So, this study was conducted to investigate the gene expression profile of bovine embryo biopsies produced in vivo and in vitro that resulted in different pregnancy outcomes. For this, biopsies representing 30 to 40% of the intact in vitro and in vivo blastocysts were taken, and 60 to 70% part was allowed to re-expand prior to transfer to recipients. Based on the pregnancy outcome after transfer, biopsies (n = 10 per pool) were grouped into 3 distinct phenotypes: those that resulted in no pregnancy, those that resulted in resorption, and those that resulted in successful pregnancy and subsequent calf delivery. A bovine cDNA microarray with 2000 clones was used to analyze the gene expression profiles of 3 replicates from each embryo biopsy group. Array data analysis revealed a total of 50 and 52 genes to be differentially expressed between biopsies derived from in vivo blastocysts that resulted in no pregnancy v. calf delivery and resorption v. calf delivery, respectively. Similarly, a total of 52 and 58 transcripts were differentially expressed between biopsies derived from in vitro-produced blastocysts that resulted in no pregnancy v. calf delivery and resorption v. calf delivery, respectively. Quantitative real-time PCR has confirmed the expression profile of 6 selected candidate genes. A distinct set of genes were found to be commonly expressed between in vitro- and in vivo-derived blastocyst biopsies, which ended up with the same pregnancy outcome. Biopsies, which ended up with calf delivery, were found to be enriched with transcripts involved in nucleosome assembly (KRT8), translation (RPLPO), electron transport (COX-2), and placenta specific (PLAC8). On the other hand, transcripts regulating immune response (TNFa), response to stress (HSPD1), and cell adhesion (CD9) were up-regulated in embryos that resulted in no pregnancy or resorption. Differences in transcript abundance of some genes have been seen between biopsies derived from in vitro and in vivo blastocysts. Biopsies from in vivo-derived blastocysts and that ended up with resorption were found to be enriched with transcripts regulating calcium-binding protein (S100A10, S100A14). Transcription factor-related transcripts (CDX2, HOXB7) were up-regulated in vitro-derived blastocyst biopsies that resulted in no pregnancy. In conclusion, the results evidenced that embryos derived from either in vitro or in vivo have more similarities than differences in their transcript abundance with respect to the ability in initiating pregnancy.
201 SODIUM/POTASSIUM/CALCIUM EXCHANGER 3, NCKX3, IS REGULATED BY ESTROGEN AND PROGESTERONE IN THE UTERUS OF MICE DURING ESTROUS CYCLE
Authors: Yang H. , Choi K.-C. , Jeung E.-B.
Published: December 2008

As a member of family of potassium-dependent sodium/calcium exchangers, a sodium/potassium/calcium exchanger NCKX3 (slc24a3), plays a critical role in transport of 1 intracellular calcium and potassium ion in exchange for 4 extracellular sodium ions. Transcripts of NCKX3 were most abundant in the brain, but many other tissues have been shown to express NCKX3 at lower levels, especially uterus, aorta, and intestine, which are rich in smooth muscle. Thus, we further investigated uterine NCKX3 mRNA expression during the estrous cycle in the absence or presence of sex-steroid hormones estrogen (E2) and progesterone (P4) in mature (>14 wk old) female ICR mice (total n = 15). NCKX3 was expressed in the uterus of female reproductive system and regulated by E2 or P4. During estrous cycle of the mice, uterine NCKX3 mRNA level fluctuated, and its mRNA level was down-regulated when E2 and P4 levels were high (proestus and estrus), whereas its level disappeared at metestrus and diestrus. In addition, NCKX3 protein expression was identified in the uterus of mice by immunohistochemistry. To examine the roles of sex steroids, the mice were treated with E2 [40 μg kg–1 body weight (BW)] or P4 (4 mg kg–1 of BW) and E2 plus P4 for 3 days. The expression level of NCKX3 mRNA was significantly decreased by E2 or P4 in the uterus of mice. However, co-administration of E2 and P4 caused a significant decline at the transcriptional level of this gene when compared with vehicle or P4 treatment alone, suggesting an inhibition role of E2 in response to P4. Taken together, these results indicate that uterine NCKX3 expression is regulated during the estrous cycle, and its expression is mainly controlled by E2 and P4, suggesting that uterine NCKX3 in female mice may be involved in the function of female reproductive system.
210 CLEAVAGE ASSESSMENT AT DAY 2 TO PREDICT BLASTOCYST DEVELOPMENTAL POTENTIAL IN PORCINE IN VITRO-FERTILIZED EMBRYOS
Authors: Kim Y. J. , Jeon Y. P. , Hyun S. H.
Published: December 2008

Porcine embryos could be a valuable tool to study preimplantation development, implantation, and pregnancy, but to do this it is necessary to establish an efficient in vitro embryo production system. Because the cause of high mortality in embryos during preimplantation development is not clear, a noninvasive method of determining the developmental potential of cleavage-stage embryos is needed. The objective was to evaluate the developmental potential of Day 2 embryos in a porcine in vitro fertilization (IVF) system. Specifically, this study was conducted to examine the relationship between embryo morphology 48 h after IVF on rates of blastocyst formation 5 days later. To prepare in vitro maturation (IVM) of porcine oocytes, cumulus–oocyte complexes were obtained from slaughterhouse-derived ovaries and matured in M-199 medium supplemented with 10% pig follicular fluid and 0.57 mm cysteine for 44 h and then freed from cumulus cells. After IVM, cumulus-free oocytes were coincubated with frozen–thawed sperm (2 × 106 cells mL–1) and 2 mm caffeine for 6 h. Inseminated embryos were cultured in NCSU-23 medium that was supplemented with 0.5 mm pyruvate and 0.5 mm lactate. Data were analyzed by ANOVA and Duncan’s test (P < 0.05). Morphology data on a total of 919 embryos were analyzed retrospectively. Forty-eight hours after insemination, embryos were classified into the following 5 groups based on the cleavage state: 1 cell, 2 cells, 4 cells, 5 to 8 cells, and fragmentation. These groups were cultured another 120 h and then evaluated for blastocyst formation. Blastocyst formation rates were significantly higher in the 4-cell (38.07%) and 5- to 8-cell (40.65%) cleaving groups than in the other groups (P < 0.05). In contrast, the 2-cell and fragmentation groups produced 7.5 and 2.9% blastocysts, respectively. Data suggest that embryos reaching 4 cells and 5 to 8 cells by 48 h after insemination have high developmental competence, and this parameter may be useful to predict the development of preimplantation embryos and their ability to establish pregnancy.

This work was supported by a grant (No. 20070301034040) from the BioGreen 21 program, Rural Development Administration, Republic of Korea.
216 COMPARISON BETWEEN A TRI-GAS THERMO INCUBATOR AND A MODULAR CHAMBER WITH PREMIXED GAS ON THE DEVELOPMENT OF BOVINE EMBRYOS IN VITRO
Authors: Raito M. B. , Mphaphathi M. L. , Schwalbach L. M. , Greyling J. P. C. , Nedambale T. L.
Published: December 2008

In an attempt to optimize germplasm and reproduction biotechnology IVF laboratory conditions in South Africa, we compared the effects of 2 triple-gas incubation systems, a tri-gas thermo incubator and a modular chamber with premixed gas, on the development of bovine embryos in vitro. After aspirating ovaries collected from a local abattoir, 778 oocytes were matured for 24 h in M-199 supplemented with 10% FBS, and 1 μg mL–1 of FSH and LH at 39°C in 5% CO2. Oocytes were then fertilized in vitro in Brackett and Oliphant (BO) medium at 39°C in 5% CO2. Presumptive zygotes were randomly allocated to the tri-gas thermo incubator or the modular chamber with premixed gas and cultured in synthetic oviductal fluid (SOF) medium at 39°C in 5% CO2, O2, and 90% N2. Total cleavage (Day 2), 8-cell (Day 2), morula (Day 6), and blastocyst (Day 7) rates were recorded postfertilization. Data were analyzed by ANOVA. There was no statistical difference in total cleavage rate between the 2 incubation systems. However, the 8-cell, morula, and blastocyst rates were significantly higher for the modular chamber group compared with the tri-gas incubator group (Table 1). In summary, this study suggests that the modular chamber with premixed gas was a better system for culturing zygotes of South African domestic animals to the blastocyst stage.

Table 1.Effect of modular chamber and tri-gas incubator on embryo development in vitro

This work was funded by the South African National Department of Agriculture, DST-PDP, and the National Research Foundation (NRF, Grant Nos. RT21 and 24000).
218 GENE AND PROTEIN EXPRESSION OF ANTIOXIDANT ENZYMES IN BOVINE EMBRYOS EXPOSED TO HEAT SHOCK
Authors: Sakatani M. , Yamanaka K. , Takahashi M.
Published: December 2008

In a previous study, we reported that 8-cell-stage embryos exposed to a temperature of 41°C for 6 h had significantly increased embryonic mortality and intracellular reactive oxygen species (ROS). There have been some reports that ROS regulates the expression of genes encoding antioxidant enzymes in culture cells. In this study, we investigated the gene and protein expression of antioxidant enzymes in bovine 8-cell-stage embryos exposed to heat shock. In vitro-produced bovine embryos were used for the experiment. Embryos were cultured with CR1aa + 5% FCS at 38.5°C in 5% CO2 and 5% O2. On Day 2 after fertilization, 8-cell-stage embryos were exposed to heat shock at 41°C in 5% CO2 and 5% O2 for 6 h (HS). Eight-cell-stage embryos cultured at 38.5°C in 5% CO2 and 5% O2 were sampled at the same collection time as controls. After HS, 20 embryos were immediately collected for gene expression analysis. Expression of heat shock protein 70 (HSP70), CuZn-containing superoxide dismutase (SOD), catalase (CAT), and glutathione peroxide (GPx) genes was examined by real-time polymerase chain reaction. Twenty embryos were also collected after 3 h of HS (3 h) and at 18 h after HS (18 h) to evaluate the expression of proteins. Expression of HSP70, SOD, and CAT proteins was examined by Western blotting. Both the gene and protein expression levels of HS groups were normalized to those of the controls to obtain the relative expression levels. All results were analyzed by Student’s t-test. Expression of the HSP70 gene significantly increased in HS embryos (P < 0.05). Expression of the SOD and CAT genes tended to increase in HS embryos (P < 0.07), but there were no significant differences in expression of the GPx gene. There was no significant difference in protein expression in all the antioxidant enzymes in 3-h-sampled embryos. Expression of the HSP70 protein increased significantly in heat-shocked embryos sampled at 18 h (P < 0.05). These results indicate that expression of antioxidant enzymes was not greatly affected in 8-cell-stage embryos exposed to HS. Thus, these results suggest the possibility that the early-stage embryos were stressed and damaged from heat shock because of their poor antioxidative potency.

Table 1.Gene and protein expression of embryos

This work was supported by KAKENHI [16780209, Grant-in-Aid for Young Scientists (B)].
222 THE USE OF IN VITRO FERTILIZATION TO STUDY BOVINE IDIOPATHIC INFERTILITY
Authors: Siqueira L. G. B. , Palmer C. W. , Lessard C.
Published: December 2008

A 4-year-old beef bull underwent a breeding soundness evaluation at the Western College of Veterinary Medicine (Veterinary Hospital, University of Saskatchewan); no apparent abnormalities were observed after conventional semen evaluations. However, the clinical history of this bull indicated that no pregnancies resulted from natural service of 52 cycling cows over a period of 2 years. Completed services were observed. The objective of this study was to use in vitro fertilization (IVF) technology to evaluate whether the sperm from this infertile bull could successfully fertilize oocytes in vitro. Fresh semen was collected with an electroejaculator and frozen in a computer-controlled rate freezer. Concentration and motility parameters were assessed by using computer-assisted semen analyses. Sperm morphology was evaluated on eosin-nigrosin-stained slides, and Coomassie blue staining was used to evaluate the presence of intact acrosomes. Within each evaluation technique, frozen–thawed semen from bulls (n = 2) with proven fertility was used as a positive control and samples of dead sperm (produced by repeated frozen–thawed cycles until reaching no sperm motility) were used as a negative control. Frozen semen from the infertile bull was used for IVF assay. Data were analyzed by ANOVA (sperm motility and the presence of acrosomes) or the chi-square test (cleavage and blastocyst rates), with a P-value of 0.05. Our infertile bull showed an average motile sperm percentage of 91.7 ± 2.2%, with 78.6 ± 3.5% progressive motility. After cryopreservation procedures, frozen–thawed semen had an acceptable general and progressive percentage of motility of 57.8 ± 6.7% and 43.9 ± 9.2%, respectively. Sperm stained with Coomassie blue showed a greater proportion of intact acrosomes in the fresh semen (63.6 ± 4.3% v. 40.4 ± 3.7%; P < 0.05); however, frozen–thawed semen from both the fertile bull and the control were similar (40.4 ± 3.7b% v. 45.5 ± 2.2b%, P < 0.05). In vitro fertilization results revealed a low cleavage rate at 48 h postfertilization (19.8 ± 6.3%) and blastocyst rate (2.4 ± 2.8%) when using frozen–thawed semen from the infertile bull, compared with the control (56.7 ± 8.2% and 26.3 ± 4.5%, respectively; P < 0.001). Moreover, cleavage and blastocyst rates obtained by using the negative control (21.1 ± 3.2% and 1.1 ± 1.9%, respectively) were similar to those of the infertile bull (P > 0.10). It was noted that ova fertilized with either frozen–thawed semen from the infertile bull or the negative control did not produce blastocysts before Days 8 and 9 of embryo culture, which is a characteristic of parthenogenesis. The results from this IVF study suggest that in this bull, there was a missing or defective factor blocking one of the steps in the fertilization process. Further investigations to identify this factor will increase our knowledge of male fertility, and could lead to new methods of evaluating and regulating fertilizing ability.
229 INFLUENCE OF SPERM TREATMENT ON SPERM HEAD DECONDENSATION FERTILIZING WITH EJACULATED AND EPIDIDYMAL PORCINE SPERM BY ICSI
Authors: Garcia-Vazquez F. A. , Aviles-Lopez K. , Matas C.
Published: December 2008

Intracytoplasmic sperm injection (ICSI) provides a possible remedy to produce high numbers of monospermic zygotes. However, the efficiency of ICSI in pigs is low, mainly due to a failure of oocyte activation and low incidence of sperm head decondensation. The persistence of an intact sperm acrosome, plasma membrane, and perinuclear theca, which are removed during the process of sperm penetration in natural fertilization, are considered major reasons for low male pronuclear formation after ICSI in the oocytes, which are capable of supporting male pronuclear formation after IVF. Failure of male pronucleus formation is the major fertilization defect causing embryo development failure in pig oocytes subjected to ICSI (Lee JW et al. 2003 Theriogenology 59, 305). The main objective of this experiment was to determine the effect of sperm treatment on sperm head decondensation for ejaculated and epididymal spermatozoa used with ICSI. We divided ejaculated (EJ) and epididymal (EP) sperm into 3 treatment groups: 1) sperm without any treatment (control = C), 2) spermatozoa washed through a Percoll® gradient (P), and 3) spermatozoa washed through Percoll® followed by 30 min of incubation in porcine oviductal fluid (POF). Oocytes were matured in vitro 44 h in NCSU-37 and microinjected with sperm from the different treatment. Four hours after injection, the putative zygotes were stained with Hoescht and classificated as: (i) intact, (ii) low level of decondensation, and (iii) high level of decondensation and male pronuclei. A total of 461 oocytes were injected in 6 replicates. Sperm head decondensation (categorical data) was modeled using a binomial model of parameters and analyzed by ANOVA. The EP-C treatment showed a higher level of intact sperm (21.95 ± 3.4) than the other treatments (EP-P: 16 ± 3.6; EP-POF: 6.58 ± 3.6; EJ-C: 12.33 ± 3.7; EJ-P 5 ± 3.5; EJ-POF: 5.3 ± 3.6). Ejaculated sperm showed lower decondensation levels (C: 49.32 ± 5.7; P: 38.75 ± 5.4; POF: 32.00 ± 5.6) compared to epididymal sperm (C: 67.07 ± 5.4; P: 64 ± 5.6; POF: 61.84 ± 5.6). Sperm decondensation and male pronuclear formation were higher in EJ-P (56.25 ± 5.0) and EJ-POF (62.67 ± 5.2) compared to other groups (EP-C: 10.89 ± 4.9; EP-P: 20.00 ± 5.2; EP-POF: 31.58 ± 5.1; EJ-C: 38.36 ± 5.2). In conclusion, the EJ sperm exhibited higher levels of head sperm decondensation and pronuclei formation than EP sperm; the modification of sperm membrane mediated by washing sperm through Percoll® or incubating with POF induced faster decondensation than sperm without any treatment.

Supported by MEC (AGL2006-03495).
232 OIL SOURCES AND VITAMIN E IN THE DIET ON THE BOAR SEMEN QUALITY PRESERVED AT 5°C
Authors: Torres C. A. A. , Amorim E. A. M. , Amorim L. S. , Graham J. K. , Guimarães J. D. , Murgas L. D. S. , Costa E. P. , Carvalho G. R. , Romualdo P. L.
Published: December 2008

Spermatozoa experience physical and chemical stresses during cooling and freezing as a result of ice formation and osmotic changes in the medium. Sperm cryosurvival appears to depend on intrinsic properties of the sperm plasma membrane, such as biochemical composition, thermal behavior, osmotic resistance, and the physical stresses determined by the protocol. The objective of this study was to evaluate the addition of oil sources and dietary supplementation of the vitamin E on boar sperm characteristics cooled at 5°C. Twenty-four mature Dalboard 85 boars, of proven fertility and in routine semen production for artificial insemination, were randomly divided, in factorial arrangement 2 × 3, with 2 oil sources (35 g kg–1 of soybean or salmon oil were added to the basal diet as a supplement) and 3 levels of antioxidants (150, 300, and 450 vitamin E mg kg–1). The antioxidant used in this study was vitamin E provided as DL-α-tocopheryl acetate. At the start of the experiment, the age of the boars ranged from 1 to 2 years old. Sperm samples were collected after 0, 1a, 4a, 7a, and 10a weeks of feeding the experimental diets, and total motility, vigor, hypoosmotic swelling (HOST), sperm morphology, and the fatty acid composition of the sperm were determined. Ejaculates from each of 24 boars were diluted to 3 × 109 sperm to 80 mL in a Beltsville thawing solution diluent and cooled to 5°C in a bottle (3 bottles/boar were cooled). After 24, 48, and 72 h, sperm samples from 1 bottle/boar were analyzed. Treatment differences for sperm parameters were determined using ANOVA. Dietary supplementation with salmon oil reduced (P < 0.05) the proportion of ω6 fatty acid in fresh sperm, specifically of docosapentaenoic acid (22:5ω6), and it increased (P < 0.05) the proportion of docosahexanoic acid (22:6ω3). The motility and HOST of the animals sperm treated with salmon oil at 5°C was superior (P < 0.05) than animals treated with soybean oil, after 24, 48, and 72 h. Feeding salmon oil also increased (P < 0.05) sperm vigor after 24 and 48 h. The motility, vigor, and HOST of sperm at 5°C differed (P < 0.05) over the time they were preserved, with values at 24 h of storage being superior to sperm stored for 48 h and both being superior to sperm stored for 72 h. The percentages of sperm with abnormal morphology increased (P < 0.05) in the semen from animals treated with soybean oil. The percentages of sperm with abnormal morphology were higher (P < 0.05) in the semen at 5°C after 24, 48, and 72 h. Feeding boars salmon oil improved the sperm characteristics of boar semen cooled at 5°C during the 24 and 48 h of preservation time without using a specific diluent designed for this temperature.

Supported by grants from: CNPq, FAPEMIG, Perdigao S/A., MINITUB, and Lagoa da Serra.
250 EFFECT OF LOW OXYGEN TENSION ATMOSPHERE AND MATURATION MEDIA ON IN VITRO-MATURED SWINE OOCYTES
Authors: Marques M. G. , de Barros F. R. O. , Goissis M. D. , Cavalcanti P. V. , Nicacio A. C. , Feitosa W. B. , Assumpção M. E. O. A. , Visintin J. A.
Published: December 2008

The aim of this study was to evaluate the efficiency of a low oxygen tension atmosphere (5% CO2, 5% O2, and 90% N2) on swine oocyte maturation in chemically defined media or when supplemented with porcine follicular fluid (PFF). Briefly, oocytes were in vitro matured for 44 h in TCM-199 with 10% PFF or 0.1% PVA added, under a low oxygen tension atmosphere or a normal oxygen tension atmosphere (5% CO2 in air, approximate 20% O2). At 0 and 44 h of maturation, cumulus oophorus cells were removed. To evaluate the migration of cortical granules, oocytes were fixed, permeabilized, and incubated in 100 μg of FITC-PNA mL–1 for 30 min. Oocytes were then incubated in 10 μg mL–1 of Rnase for 30 min and in 10 μg mL–1 of propidium iodide for 10 min to verify nuclear maturation by confocal microscopy (Zeiss LSM 510 Meta). Heat shock protein 70 (HSP70) content was assessed as described in Kawarsky and King (2001 Zygote 9(3), 39–50) to verify oxidative stress. Data were analyzed by the SAS System for Windows (2000). The nonparametric ANOVA NPAR1WAY procedure was applied to evaluate nuclear maturation rate by comparing groups in pairs. Migration of cortical granules and HSP70 content were analyzed using PROC GLM (LSD test of means). The effects of treatment and manipulation were verified in all analyses. The significance level was 5%, and data were presented as means ± SEM. Results indicated that the percentage of metaphase II oocytes did not differ among groups after 44 h of maturation [PFF 5% O2 (89.16 ± 3.73a), PFF 20% O2 (86.59 ± 6a), PVA 5% O2 (79.62 ± 8.22a), and PVA 20% O2 (93 ± 5.17a)]. However, these groups were different from the 0-h group (0 ± 0b). Results for the percentage of cortical granule migration showed that 0-h oocytes (38.92 ± 2.75a) had lower migration rates compared with other groups. After 44 h of maturation, migration of the cortical granule rate of the PFF-supplemented group under a 5% O2 atmosphere (61.66 ± 3.21b) was different when compared with the PVA 20% O2 group (50.97 ± 3.48c). The other groups showed intermediate results, but without statistical differences [PFF 20% O2 (58.51 ± 2.5bc) and PVA 5% O2 (53.75 ± 3.14bc)] for the migration of cortical granules. Moreover, no difference at pixel quantification of HSP70 was observed among groups after 44 h of maturation [PFF 5% O2 (116.45 ± 40.94a), PFF 20% O2 (44.44 ± 12.66a), PVA 5% O2 (29.95 ± 7.95a), and PVA 20% O2 (58.49 ± 22.2a)], although these groups were different from the 0-h group (247.41 ± 38.59b). Although the HSP content decreased throughout in vitro maturation of swine oocytes under the low and high oxygen tension atmospheres, it can be concluded that a low oxygen tension atmosphere did not affect nuclear maturation and rates of cortical granule migration regardless of maturation media supplementation.

Financial support: FAPESP (grant no. 05/01420-7).
251 EVALUATION OF mRNA POLYADENYLATION STATUS OF CELL CYCLE-RELATED GENES IN BOVINE OOCYTES DURING MATURATION: PARTIAL RESULTS
Authors: Milazzotto M. P. , Paula-Lopes F. F. , Feitosa W. B. , Simoes R. , Nicacio A. C. , Giassetti M. I. , Visintin J. A. , Assumpção M. E. O. A.
Published: December 2008

During oocyte growth, transcription activity results in the production of RNA and proteins, which are immediately used or stored. At this time, gene expression is, in part, controlled at the post-transcriptional level through deadenylation and polyadenylation processes. After fertilization, early embryonic cell cycles are mainly supported by maternal mRNA and proteins until the maternal embryonic transition. The extent of the poly A tail of mRNA is known as an important element to determine their stability and can be considered a key marker in embryonic development. Because cell cycle-related genes are implicated in the maturation process and early embryonic cell cycle, study of the poly A tail in oocytes during maturation and early embryonic development may be an useful marker of differential developmental competence. Thus, the aim of the present work was to determine the polyadenylation status of the cell cycle-related genes cdc2, CDK2, CDK4, and Cyclin A, B, D, and E during bovine oocyte in vitro maturation (IVM). A PCR-based technique previously developed by Salles and Strickland (1995 PCR Meth. Appl. 4, 317–321) was used, with some modifications. Cumulus–oocyte complexes obtained from 2- to 8-mm follicles were immediately frozen in liquid nitrogen or IVM for 24 h in TCM-199 supplemented with fetal bovine serum and hormones (LH, FSH, and estradiol). In both cases, cumulus cells were removed by pipetting after a 10-min 0.2% hyaluronidase incubation. The RNA was extracted from pools of 10 immature or IVM oocytes and incubated with phosphorylated oligo-dT and DNA ligase. Thereafter, an anchor adapter [poly(dT) anchor] was targeted to the 5 end of the oligo-dT and incubated with Superscript II (Invitrogen) for reverse transcription. Specific primers were designed at the end of a full known sequence of the genes described above, and PCR was conducted for each gene using a specific primer and the anchor primer. The PCR products were digested with restriction enzymes to verify their specificity and were submitted to polyacrylamide gel electrophoresis. All sequences were amplified in immature and matured oocytes from 2 replicates by using a very small sample. After the restriction enzyme treatment, the amplified products were digested as expected. Although differences in amplicon sizes could be observed between the immature and matured oocytes, these are preliminary results and a higher number of replicates are necessary to determine the changes in the polyadenylation status of these cell cycle-related genes and their biological function. In conclusion, the polyadenylation status of the cell cycle-related genes cdc2, CDK2, CDK4, and Cyclin A, B, D, and E could be effectively performed in small samples, and this may be a powerful tool for studying the differential developmental competence of bovine oocytes and early cleavage embryos.

FAPESP.