bluuebunny/crossref_metadata_2025 huggingface dataset:
272 OOCYTE SPONTANEOUS ACTIVATION IN DIFFERENT RAT STRAINS
Authors: Ross P. , Yabuuchi A. , Cibelli J.
Published: December 2004

Oocyte spontaneous activation (OSA) has been reported to occur during in vitro culture of ovulated rat eggs. Approximately 1.5 h after isolation and culture, unfertilized oocytes extrude the second polar body and enter a metaphase arrest, and by 3 h individual chromatids separate and scatter throughout the egg's cytoplasm. The objective of this study was to compare the proportion of OSA and the level of maturation promoting factor (MPF) activity in oocytes from different strains. Twelve strains were selected from two commercial sources based on litter size. Mature female rats (9 to 12 weeks old) were superovulated using 20 IU eCG s.c. followed by 30 IU eCG s.c. 5 h later. After 48 h, a dose of 50 IU hCG was administered intraperitonealy and females were mated with vasectomized males. Oocytes were collected 17 h after hCG injection and cumulus cells were removed by transfer to M2 medium containing hyaluronidase (1 mg mL−1). Denuded oocytes were cultured in 50 μÂ­L drops of M16 medium under oil at 37°C and 5% CO2 in air. In order to assess OSA, oocytes were mounted in glycerol containing Hoechst 33342 (10 μg mL−1) on a glass slide after 6 h of in vitro culture. The proportion of activated oocytes was determined by epifluorescence microscopy. Oocytes were considered to be in metaphase II if they presented a compact group of chromosomes at the metaphase plate, and were considered activated when chromosome dispersion at different degrees was observed. Data were analyzed by ANOVA, considering each animal as an experimental unit. Significant differences were observed between strains (P < 0.01, Table). In order to determine MPF activity of each strain, 10 oocytes were removed from culture at 0, 1.5, and 3 h after oocyte collection and immediately stored in kinase buffer in LN2 for posterior analysis using an ELISA based kit (MESACUP cdc2 Kinase Assay Kit, MBL, Nagoya, Japan). The log ratio of the MPF activity at 1.5 and 3 h relative to 0 h for each animal (5 per strain) was analyzed by ANOVA. There were no MPF differences between or within strains (P > 0.3 and P > 0.05, respectively). We did not observe the expected decrease in MPF activity that allows for the exit of metaphase II arrest. This could imply that OSA is not associated with a decrease in MPF, or that MPF decreased rapidly and returned to metaphase levels by 1.5 h after culture. In conclusion, different levels of OSA were observed between strains, however, no differences in MPF activity were detected at the analyzed time points.
281 MITOGEN-ACTIVATED PROTEIN KINASE IN PORCINE CUMULUS CELLS
Authors: Ebeling S. , Boesebeck C. , Meinecke B.
Published: December 2004

Mitogen-activated protein kinase (MAPK) is involved in many signal processes within eukaryotic organisms. Its active form is phosphorylated. For meiotic resumption in oocytes the MAPK cascade plays a central role, because it participates in the transfer of the extracellular gonadotropin signal into the nucleus. In pigs it could be shown that for a gonadotropin-induced germinal vesicle breakdown (GVBD), an activation of MAPK in oocytes is not essential, but in the surrounding cumulus cells the MAPK has to be phosphorylated (Ohashi et al. 2003 Biol. Reprod. 68, 604–609). Because cumulus cells are very important for signal transfer, the present investigation dealt with the relevance of porcine cumulus cells and the phosphorylation of MAPK for the resumption of meiosis. Oocytes of slaughtered pigs were collected and cultured (medium: TCM 199, insulin, l-glutamine, gentamycin, 20% (v/v) FCS, and with or without 2.5 μg/mL FSH and 5.0 μg/mL LH). The proteins of isolated cumulus cells and oocytes were separated by gel electrophoresis (cumulus cells of 10 cumulus-oocyte complexes and 40 oocytes per lane, respectively) followed by an immunoblot with antibodies against MAPK and p90rsk (ERK 1 (sc-94) and Rsk-1 (sc-231), respectively; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Alternatively the nuclear maturation was determined by orceine staining. The following results were achieved: The phosphorylation of MAPK in cumulus cells began very early during the in vitro maturation period. This was demonstrated already after 0.5 h unlike in oocytes where phosphorylation of MAPK does not occur until 18 h. The phosphorylation in cumulus cells occurs both in the presence and in the absence of FSH/LH, but without FSH/LH almost no GVBD occurs (after 26 h IVM: 86.9% GV oocytes, n = 59). The phosphorylation in the absence of gonadotropins could be caused by components of FCS, but with an exchange against polyvinylpyrrolidone (0.3%), the phosphorylation without FSH/LH still existed. The specificity was examined with the MAPK kinase inhibitor U0126. A concentration of 10 μM U0126 prevented GVBD and phosphorylation of MAPK in oocytes. However, in cumulus cells the phosphorylation of MAPK was reduced only minimally. In the presence of 50 μM U0126, a distinct decrease was observed during the first hours of maturation. But after 26 h phosphorylated MAPK appeared in cumulus cells despite the high concentration of U0126. The p90rsk is an important substrate of MAPK, which is phosphorylated by activated MAPK in oocytes. In our investigations we could detect only unphosphorylated forms of p90rsk in the cumulus cells. It seems that there are different ways for phosphorylation of MAPK to occur in cumulus cells, but they do not have the same consequences. The phosphorylation of MAPK in cumulus cells is necessary for a gonadotropin induced meiotic resumption, but phosphorylation does not always lead to GVBD. Furthermore, the p90rsk appears not to have the same importance as a substrate of MAPK in cumulus cells as in oocytes.
286 THE EFFECT OF IN VITRO MATURATION MEDIUM ON CRYOSURVIVAL, CELL NUMBERS AND APOPTOTIC INDEXES OF BOVINE EMBRYOS
Authors: Korhonen K. , Matomäki J. , Ketoja E. , Kananen-Anttila K. , Halmekytö M. , Räty M. , Peippo J.
Published: December 2004

The success of in vitro maturation (IVM) has a significant impact on the oocytes ability to develop to blastocyst stage. The quality of the produced blastocysts can be evaluated using staining techniques. The aims of this study were (a) to compare the effect of different IVM media on embryo production rates, and (b) to utilize differential (DF) and TUNEL staining to evaluate the quality and cryosurvival of the produced blastocysts. Abattoir-derived oocytes were randomly divided into the IVM groups: (1) M199 IVM (n = 2305): TCM-199 with glutamax-I (GIBCO, Paisley, UK), 0.25 mM Na-pyruvate, 100 IU mL−1 penicillin, and 100 μg mL−1 streptomycin; (2) FBS IVM (n = 2484): M199 IVM medium with hormones (10 μg mL−1 LH, 2 μg mL−1 FSH, and 100 μg mL−1 β-estradiol), and 10% FBS (GIBCO, New Zealand); and (3) FAFBSA IVM (n = 2411): as group (2), but FBS was replaced with 4 mg mL−1 fatty acid free albumin. Fertilized oocytes were denuded and cultured in modified SOFaaci + 4 mg mL−1 FAFBSA in 5% O2 (Holm P et al. 1999 Theriogenology 52, 683–700). Fresh, Grade I Day 7 blastocysts were stained with TUNEL (n = 114) or with DF (n = 149). In addition, 184 Grade I Day 7 blastocysts were frozen in AG Freeze (AB Technology, Pullman, WA, USA), thawed, and cultured for 24 h. The re-expanded embryos were stained with TUNEL (n = 96) or with DF (n = 88). Modified TUNEL protocol (Upstate, Lake Placid, NY, USA; Makarevich A and Markkula M 2002 Biol. Reprod. 66, 386–392) and DF staining protocol (Thouas G.A. et al. 2001 Reprod Biomed Online 3, 25–29) were used. The results of embryo cleavage and D7 embryo development are based on logistic regression models, and the results of proportion of ICM and apoptotic index on general linear mixed models.
After FBS IVM, 83.6% of the fertilized oocytes were estimated to cleave and 25.5% to develop to the blastocyst stage by Day 7. The estimations for embryo cleavage and Day 7 development rates were significantly lower in FAFBSA IVM and M199 IVM groups (P < 0.0001): 74.0% and 15.0% for the FAFBSA, and 76.1% and 8.8% for the M199, respectively. The re-expansion rates (%) after thawing were 86.5, 90.6, and 73.3 for the FBS, FAFBSA, and M199 IVM groups, respectively. Freezing reduced the ICM proportions and elevated the apoptotic indexes (P < 0.001). The rate of ICM reduction after freezing was not influenced by the IVM medium. There was a significant interaction between the apoptotic index and the IVM group (P = 0.04). The increase of the apoptotic index was smallest in FAFBSA IVM and greatest in M199 IVM. The results indicate that exclusion of serum from IVM medium results in lower embryo cleavage and development rates. Freezing reduced ICM and increased apoptotic index of Day 7 embryos in every IVM group studied. FAFBSA IVM seemed to produce embryos of better quality as evidenced by the smallest increase in the apoptotic index after freezing.
287 INHIBITION OF THE PENTOSE PHOSPHATE PATHWAY RESULTS IN MEIOTIC ARREST IN PORCINE OOCYTES THAT CAN BE OVERCOME BY THE ADDITION OF PATHWAY COFACTORS AND END PRODUCTS
Authors: Krisher R.
Published: December 2004

Glucose metabolism is an indicator of oocyte developmental competence, and is also correlated with meiotic maturation. In vitro maturation of porcine oocytes with the pentose phosphate pathway (PPP) inhibitor diphenyleneiodonium (DPI) blocks meiotic progression to metaphase II. The objectives of this study were (1) to examine the reversibility of meiotic arrest induced by DPI; and (2) to overcome metabolically induced meiotic arrest by the addition of PPP end products and cofactors downstream of DPI inhibition. Oocytes were matured for 40 h in standard (defined) maturation media containing 0, 25, 50, or 100 nM DPI. At that time half the oocytes in each treatment (TRT) were fixed, and half were moved into standard maturation medium with no DPI for an additional 40 h, at which time all remaining oocytes were fixed. Two oocytes were matured for 40 h in one of 11 media: standard (defined) maturation medium (STND), standard with 50 nM DPI (DPI), standard with 50 nM DPI and 0.25, 2.5, or 5 mM phosphoribose diphosphate (PRPP), nicotinamide adenine dinucleotide phosphate (NADP), or ribose-5-phosphate (R5P). Additionally, 10 mM R5P and 12.5 mM PRPP were examined. All oocytes were fixed. Oocytes were assigned a meiotic score; germinal vesicle (GV) = 1, GV breakdown (GVBD) = 2, condensed chromatin (CC) = 3, metaphase I (MI) = 4, anaphase (A) = 5, telophase (T) = 6, and metaphase II (MII) = 7. Immature oocytes were classified as those at GV or GVBD stages, and mature oocytes as those at A, T or MII stages. Data were analyzed by ANOVA and are presented as mean ± SEM. After 40 h of arrest (n = 79–87/TRT), increasing concentrations of DPI significantly increased the % of immature oocytes (0, 7.2 ± 2.9; 25, 26.4 ± 4.8; 50, 53.2 ± 5.7; 100, 75.9 ± 4.8) and decreased the % of mature oocytes (0, 73.5 ± 4.9; 25, 52.9 ± 5.4; 50, 20.3 ± 4.6; 100, 0). After an additional 40 hours in standard maturation medium (n = 89–93/TRT), there was no difference in the % of immature oocytes between treatments (0, 7.5 ± 2.8; 25, 14.4 ± 3.7; 50, 13.0 ± 3.5; 100, 9.0 ± 3.0) although the % of mature oocytes significantly decreased with increasing DPI concentration (0, 90.3 ± 3.1; 25, 68.9 ± 4.9; 50, 35.9 ± 5.0; 100, 10.1 ± 3.2). Data from experiment 2 are presented below. Meiotic maturation is significantly inhibited by DPI in a dose-dependent manner. Ability of the oocyte to reach MII following 40 h of arrest is also concentration-dependent, although all treatments resulted in GVBD following removal from DPI. Metabolic arrest can be overcome, resulting in numbers of mature oocytes equal to standard controls, by NADP and PRPP but only moderately by R5P. These data demonstrate that glucose metabolism via the PPP is a critical control mechanism of meiotic maturation in porcine oocytes.
Table 1.
Effect of PPP cofactors and end products on overcoming metabolically induced meiotic arrest in porcine oocytes
305 FIRST REPORT OF THE USE OF SEXED SEMEN BY FLOW CYTOMETRY IN BOS INDICUS
Authors: Cattaneo L. , Galassi A. , Franco R. , Duarte A. , Mello K. , Jáuregui G. , Lagioia J. , Basualdo M.
Published: December 2004

The only method of sexing sperm that works in any practical sense is measuring DNA content of individual sperm by flow cytometry, and sorting them based on that information (Schenk and Seidel 2002 J. Anim. Sci. 80 (Suppl. 1), 188). This is, also, the first reference of the use of sexed semen in Brazil. The objective of this experiment was to compare the fertility of sexed and non-sexed conventional semen in Bos indicus purebreeds. Ejaculates from eight Nelore bulls were collected by artificial vagina, diluted, and stained with Hoeschst 33342 (Schenk et al. 1999 Theriogenology 52, 1375–1391). A high-speed flow cytometer (SX-MOFLO®, DakoCytomation, Inc., Fort Collins, CO, USA) was used for sorting X sperm. Sexed semen was then packaged at 3 million total sperm in 0.25-mL straws and frozen. The average estimated purity obtained by resort analysis of one straw per batch was 91.5%. An aliquot of each ejaculate was frozen unsexed at a concentration of 50 × 106 sperm/mL in 0.5-mL straws (control). A total of 283 Nelore heifers were synchronized with two doses of PGF2α 12 days apart and inseminated in the body of the uterus 12 h after heat detection. Field trials were carried out in four different states in Brazil: Minas Gerais (MG), Mato Grosso (MT), Mato grosso do Sul (MS), Rondônia (RO). Pregnancy diagnosis and fetal sex determination were performed by ultrasound 30 and 60 days post-AI, respectively. Data were analyzed by chi-square and Monte Carlo methods. There was no significant difference (P < 0.05) in pregnancy rate between sexed and non-sexed semen (see Table 1). At the time of abstract submission, of 41 pregnant heifers from sexed semen confirmed by ultrasound, 40 had a female fetus (97%). These results indicate that sexing semen by flow cytometry could be a very important tool to accelerate the genetic gain in Bos indicus cattle. Brazil has the largest commercial herd in the world and, therefore, a great potential to incorporate this technology that is now available in the country.

Table 1.
309 SEX SORTED BOAR SPERMATOZOA: TIME COURSE AND PROFILE OF THEIR IN VITRO PENETRATION ABILITY AFTER STORAGE IN THE PRESENCE OF HOMOLOGOUS SEMINAL PLASMA
Authors: Parrilla I. , Vazquez J.M. , Gil M.A. , Caballero I. , Almiñana C. , Roca J. , Martinez E.A.
Published: December 2004

Addition of seminal plasma (SP) to the collection medium has been shown to be beneficial for motility and viability of sex-sorted and stored spermatozoa. However, SP could not only delay but also decrease the in vitro fertilization rates of IVM pig oocytes. In the present study, the time-course of IVM pig oocyte penetration of sex sorted boar spermatozoa stored in the presence or absence of SP was evaluated. Spermatozoa were sex-sorted following the Beltsville sperm sexing technology (Johnson and Welch 1999 Theriogenology 52, 1323–1342) and collected in TEST-egg yolk buffer (2%) with (10%) or without (control) SP. Sex-sorted spermatozoa were stored at 20°C during 0, 2, 5, and 10 h after sorting. Oocytes were matured in vitro in NCSU23 (Peters and Wells 1663 J. Reprod. Fertil. 48, 61–73) for 44 h in 5% CO2 in air at 39°C. The in vitro penetration time-course was determined by co-incubating the sex-sorted and stored spermatozoa with IVM oocytes during 3, 6, and 18 h in modified TRIS-buffered medium (mTBM) (Abeydeera and Day 1997 Theriogenology 48, 537–544) at 39°C in an atmosphere of 5% CO2 in air. Penetration rates and number of spermatozoa per oocyte were assessed after fixation and staining of the oocytes. Statistical analyses were conducted by ANOVA. Presence of SP did not delay the onset of the oocyte penetration. Moreover, at 3 h of co-incubation, SP increased (P < 0.05) both penetration rates and mean number of spermatozoa per oocyte in sorted and stored boar spermatozoa when compared with control (45 vs. 20, 50 vs. 32, 38 vs. 23, 15 vs. 8, at 0, 2, 5, and 10 h of storage with SP and control, respectively). High penetration rates were reached after 6 h of co-incubation (82 vs. 51, 96 vs. 76, 83 vs. 48, 31 vs. 24, at 0, 2, 5, and 10 h of storage with SP and control, respectively) in sorted and stored samples, with no further increase at 18 h (70 vs. 63, 92 vs. 79, 87 vs. 53, 55 vs. 40, at 0, 2, 5, and 10 h of storage with SP and control, respectively). Spermatozoa stored 2 h in the presence of SP showed the best penetration rate and highest mean number of spermatozoa per oocyte. The mean number of spermatozoa per oocyte increased as the co-incubation time increased (ranging from 2.1 to 5.8 for sorted spermatozoa stored 2 h in the presence of SP at 3 h and 18 h of co-incubation, respectively). In conclusion, the presence of SP during the storage of sex-sorted spermatozoa improves their in vitro fertilizing ability without affecting the onset of the oocyte penetration time.
This work was supported by DGICYT (AGL 2001-0471), Fundación Seneca (PB174/FS/02) and CTIC (2103SIU0040).
313 EFFECTS OF ETHANOL TREATMENT AFTER INTRACYTOPLASMIC SPERM INJECTION (ICSI) ON SPERM AFTER FORMATION AND THE MICROTUBULE ORGANIZATION OF BOVINE OOCYTES
Authors: Fujinami N. , Hosoi Y. , Kato H. , Mitani T. , Matsumoto K. , Saeki K. , Iritani A.
Published: December 2004

The cleavage rate of bovine embryos is very low without activation of oocytes after intracytoplasmic sperm injection (ICSI), although both male and female pronuclei are formed. We previously reported that the stimulus due to the injected sperm alone was sufficient to lower the MPF activity of bovine oocytes after ICSI, and the activation treatment of oocytes with ethanol at 4 h after ICSI served to maintain the low levels of MPF activity until the next cell cycle started (Fujinami et al. 2004 J. Reprod. Dev. 50, 171–178). These results suggested that activation treatment is necessary to improve the embryonic development after bovine ICSI. In bovine fertilization, the sperm introduces the centrosome into the oocyte. The centrosome acts as the microtubule-organizing center and microtubules are organized within the oocyte. It is reported that the sperm aster is important for the normal fertilization process. Therefore, failure of sperm aster formation possibly causes the failure of cleavage following fertilization. To investigate the reason of the low cleavage rate after bovine ICSI without artificial activation treatment, we examined sperm aster formation and the microtubule organization in bovine oocytes with or without activation treatment after ICSI. Bull spermatozoa immobilized by piezopulse was injected into bovine oocytes matured in vitro. At 4 h after ICSI, oocytes were treated with 7% ethanol in TCM199 for 5 min for activation. Oocytes were fixed at 6 and 12 h after ICSI, and the microtubule organization was examined by using specific antibodies and immunofluorescence microscopy. The cleavage rate (51% vs. 15%) and the developmental rate to the blastocyst stage (13% vs. 3%) were increased by ethanol treatment after ICSI (with or without ethanol treatment, respectively, P < 0.05). In oocytes activated with ethanol after ICSI, both the sperm aster formation rate at 6 h and the microtubule organization rate at 12 h after ICSI were significantly higher than in oocytes without activation treatment (58%, 80% vs. 12%, 26%, P < 0.05). It was reported that the sperm aster has an important role for the pronuclear movement to make the male and female pronuclei come into close apposition. From these results, it was concluded that oocyte activation after bovine ICSI promoted sperm aster formation and microtubule organization, and was effective to improve embryonic development.
This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan MEXT, and by a grant from the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence of the JST.
326 OPTIMIZING FREQUENCY OF FSH APPLICATION FOR SUPEROVULATORY TREATMENT IN CATTLE
Authors: Martens G. , Nohner H.P. , Leiding C. , Schneider F. , Becker F. , Nuernberg G. , Kanitz W.
Published: December 2004

The aim of the study was to examine the influence of the application frequency of Pluset® (Calier, Spain) and Folltropin® (Vetrepharm, London, Ontario, Canada) on the results of embryo recovery in Simmental cattle. Moreover, pulsatility of LH concentrations in plasma should be studied before and after application of Pluset. FSH was given in a treatment of eight or four injections on four consecutive days starting between Days 9 and 13 of the estrous cycle. Luteolysis was induced by application of 500 μg Estrumate® (Essex Tierarznei, Munich, Germany), applied 72 and 84 h after the first FSH injection. Embryo recovery was performed non-surgically on Day 7 of the following estrous cycle. Heifers received either a total amount of 500 IU Pluset according to the International Standard for human urine FSH and LH (P1: 8 injections, n = 20; P2: 4 injections, n = 54) or 200 mg Folltropin according to NIH bFSH-B1 (F1: 8 injections, n = 117; F2: 4 injections, n = 60). Cows received either 550 IU Pluset (P3: 8 injections, n = 54; P4: 4 injections, n = 183) or 260 mg Folltropin (F3: 8 injections, n = 36; F4: 4 injections, n = 159). Altogether 8 heifers were bled throughout 8 h every 10 min on Days 9 and 12 (before and during stimulation with FSH). The samples were analyzed for LH by ECLIA (intra-assay coefficient of variation (VK) 6.4%, inter-assay VK 8.9%). The evaluation of the data was carried out with the procedure ‘GLM’ of the statistics software package SAS (SAS Institute, Inc., Cary, NC, USA) and PULSAR analysis. As a post hoc test Student's t-test was used. Significance was set at P = 0.05. Results of embryo recovery are shown in the table. Number of LH pulses in 8 h (LSM ± SE) was 2.3 ± 0.4 on Day 9 and 0.6 ± 0.4 on Day 12 (P < 0.05). We conclude that four injections of both FSH products can lead to results comparable with those reached with eight injections in a 12 hours interval. Therefore, the effort for superovulatory treatments can be reduced. However, it has to be considered that the portion of transferable embryos was reduced, partly significantly, after four FSH injections. This result was independent of the drug used.

Table 1.
Results of embryo recovery
327 CHARACTERIZATION OF FETAL AND ADULT FIBROBLASTS FROM NELORE BOVINE FOR NUCLEAR TRANSFER
Authors: Caetano H.V.A. , Milazzotto M.P. , Goissis M.D. , Yamada C. , Marques M.G. , Assumpcao M.E.O.A. , Visintin J.A.
Published: December 2004

Cloning by nuclear transfer is a technology that has provided major advances in reproductive sciences. It is an efficient method: (1) to produce transgenic embryos, fetuses, and animals; (2) to multiply genetically superior adult animals; and (3) to generate proteins, tissues, and organs for xenotransplants of human interest. Basic studies on nuclear transfer have contributed to understanding how genomic activation and cell cycle synchrony affect nuclear reprogramming and cloning efficiencies. The aim of this study was to establish a stable lineage of Nelore adult and fetal fibroblasts for embryo reconstruction. Cultures were characterized by morphological aspects, and by immunocytochemical and ultrastructural analysis. Cultures of fetal and adult fibroblasts at passage 2 stained positively for cytokeratin, a typical protein from the intermediate filament of epithelial cells, in some cells but staining was not found in subsequent passages. A dense and organized network occurred in adult and fetal fibroblast cultures presenting positive immunostainig for vimentin in pass 5, 10, and 15 subcultures, but did not stain for cytokeratin (negative control). The results demonstrated that fibroblasts continued expressing vimentin and maintained their morphological aspects in culture. The ultrastructural analysis showed the presence of organelles involved in protein synthesis including dilated rough endoplasmatic reticulum, Golgi cisternae, and polyribosomes. Elongate mitochondria were distributed around the Golgi complex, suggesting high metabolic activity. These results demonstrated that adult and fetal fibroblasts continued to develop biological activities and maintained specific characteristics under these culture conditions. In conclusion, adults and fetal fibroblasts can be successfully used as donor cells for nuclear transfer.
This work was supported by FAPESP 01/11931-8 and 01/13944-0.
4 EFFECT OF THE TIMING OF ARTIFICIAL INSEMINATION ON THE NUMBER OF SPERMATOZOA DISCOVERED IN THE UTERINE CRYPTS OF THE BITCH
Authors: Rijsselaere T. , Van Soom A. , Van Cruchten S. , Coryn M. , Gortz K. , Maes D. , de Kruif A.
Published: December 2004

Canine spermatozoa may be stored for several days within the genital tract of the bitch since natural matings 8 to 9 days before ovulation may result in litters. Several studies have suggested that the sperm reservoir in the dog is located in the uterine crypts and the uterotubal junction (UTJ). In the present study, we investigated the effect of the timing of artificial insemination (AI) in relation to ovulation on the sperm distribution in the genital tract of the bitch. Ten beagle dogs were inseminated intravaginally with 500 × 106 spermatozoa. Based on progesterone concentration, three dogs were inseminated 1–2 days before ovulation, four dogs during ovulation, and three dogs 2–3 days after ovulation. Ovariohysterectomy was performed 24 h after AI. The genital tract was divided into eight segments (i.e. corpus uteri; caudal, middle, and cranial parts of the uterine horn; UTJ; isthmus; ampulla; and infundibulum) which were processed for histology. From each segment, 30 histological sections were evaluated. For the UTJ and the different segments of the oviduct, the total number of spermatozoa was determined. For the different parts of the uterus, on each of these 30 sections, 100 uterine crypts of comparable size were evaluated for the presence of spermatozoa. The crypts were divided into crypts without spermatozoa, crypts with 1 sperm cell, crypts with 2 to 5 spermatozoa, and crypts with either more than 5 spermatozoa or in which the spermatozoa were clustered. The data were analyzed using univariate analysis of variance. Histology revealed that the spermatozoa were located mainly in the uterine crypts and at the UTJ, while very few spermatozoa were detected in the different parts of the oviduct. Insemination during ovulation resulted in higher percentages of crypts with spermatozoa in the different parts of the uterus (P < 0.05). Moreover, for the ovulatory group, 54.7% of the uterine crypts with spermatozoa contained more than 5 spermatozoa (or clusters) compared to 19.9% and 28.2% for the pre- and post-ovulatory groups, respectively (P < 0.05). In the pre-ovulatory group, 59.6% of the uterine crypts with spermatozoa contained only 1 sperm cell whereas in the post-ovulatory group, frequently 1 (34.0%) or 2 to 5 spermatozoa (37.9%) were found per crypt. In conclusion, sperm transport in the genital tract of the bitch is affected by the time of AI in relation to ovulation. Insemination during the ovulation period resulted in higher percentages of uterine crypts with spermatozoa, and most of these crypts contained 5 or more spermatozoa. Further research should determine whether the number of sperm binding sites expressed on the epithelium of the canine uterine crypts is influenced by the ovulation event.
This research was supported by the UGent Special Research Fund, Grant numbers 011 B8698 and 011 B8301.
49 EFFECT OF REPEATED CELL FREEZINGS ON PREGNANCY RATE OF BOVINE NUCLEAR TRANSFER DERIVED EMBRYOS
Authors: Marfil M. , Révora M. , Gutierrez J. , Sosa S. , Lagioia J.J. , Panarace M. , Medina M.
Published: December 2004

Cell line cryopreservation is nowadays one of the most useful tools in somatic cell nuclear transfer. Although this technique guarantees the genetic storage for an unlimited period of time, many studies have shown that it produces different kinds of cellular damage such as DNA fragmentation (Men et al. 2003 Mol. Reprod. Dev. 64, 245–250) and ultrastructural cell anomalies (Taddei et al. 2001 Cryobiology 42, 244–555; Nardid et al. 1997 Cryobiology 34, 107–113). The aim of the present study was to evaluate how repeated cell freezing/thawing processes could affect the pregnancy rate of bovine nuclear transfer-derived embryos. Two adult fibroblast cell lines from different animals were separated into two groups according to the number of freezing/thawing processes they went through (1 vs. 3). For both groups, the first freezing process was performed with cells from passage 1. Cells from passages 3 and 4 were used for the second and third freezings, respectively. The time interval between thawing and next freezing was 20 days. Cells were harvested at 80% confluence using trypsin, and cryopreservation was performed in D-MEM with 35% FCS and 10% DMSO. Enucleation and nuclear transfer (NT) were performed as described by Cibelli et al. (1998 Science 280, 1256–1258) with modifications. For both groups, cells from the same number of passages were used for the NT assays (between passages 2 and 7). Cytoplasts were activated using 5 μM ionomycin for 4 min and the couplets were subsequently fused. The fused units were cultured in 10 μg/mL cycloheximide and 5 μg/mL cytochalasin B for 6 h. Embryo culture was performed at 38.5°C in a 5% O2, 5% CO2, 90% N2 atmosphere, in 50-μL drops of KSOM. On Day 3 of culture, the KSOM was supplemented with 2% FCS and 0.2 mM glucose. After 6–7 days, the embryos were non surgically transferred to synchronized recipients. Pregnancy at 30 and 60 days was recorded by ultrasonography using an Aloka 500® scanner (Aloka Co., Tokyo, Japan). Data were analyzed by ANOVA (InfoStat, Austin, TX, USA) (Table 1). The results show an association between the number of cell freezing/thawing processes and a higher pregnancy loss at 60 days. This could be related to the cellular damages caused by multiple cryopreservation procedures, which could lead to chromosomal abnormalities in the donor cells and thus in the nuclear transfer (NT) embryos and pregnancies derived from them. Further studies should be done in order to evaluate the chromosomal status of the cell lines used in this work.

Table 1.
Numbers of cell freezings/thawings and their effect on pregnancy rate of bovine NT-derived embryos
7 HETEROSPERMIC INSEMINATION AT TWO SPERM CONCENTRATIONS IN TIMED AI: CASA SEMEN PARAMETERS AND PREGNANCY RATES
Authors: Albrecht A. , Cavia R. , Larraburu G. , Garcia Migliaro F. , Brogliatti G.
Published: December 2004

The latest entry in the field of semen evaluation is computer assisted semen analysis (CASA). Heterospermic insemination has been used to increase pregnancy rates from low fertile bulls. The objective of this study was to evaluate, with the aid of CASA, heterospermic semen characteristics and pregnancy rates using different concentrations of bull semen in a timed artificial insemination protocol. Semen was collected from two bulls of known fertility by artificial vagina and all CASA motility parameters were evaluated individually and combined. Straws were filled using a semi-defined semen extender (Andromed, Minitüb, Tiefenbach, Germany) as follows: single bull A and single bull B (12 × 106 of progressive motile cells after thawing); Mixed bull semen: A + B (12 × 106 of progressive motile cells after thawing) and Supermix bull semen: A + B (28 × 106 of progressive motile cells after thawing). All cows received a P4 intravaginal device (DIB, Syntex, Argentina) and 2 mg of EB i.m. (Syntex) on Day 0, 500 mg cloprostenol (Estroplan, Syntex) at the time of DIB removal (Day 8), and 1 mg EB i.m. on Day 9. Fixed-time insemination (FTAI) was performed at 52 to 56 h after DIB removal. A total of 249 cows were randomly allocated to be inseminated with bulls A and B (n = 76), with Mixed A + B (n = 87), and with Supermixed A+B at a high concentration (n = 86) by a single inseminator. Pregnancy rates were evaluated at 38 days after insemination by transrectal ultrasonography. Means and standard deviations or each characteristic were calculated, compared, and statistically analyzed. The following sperm motility parameters were determined with the Ceros 12.1 sperm analyzer (Hamilton Thorne Biosciences, Inc., Beverly, MA, USA) on at least 1000 spermatozoa: velocity average path (VAP), velocity straight line (VSL), curvilinear velocity (VCL), amplitude lateral head (ALH), beat cross frequency (BCF), straightness (STR), linearity (LIN), and percentage of rapid or static cells. There were no significant differences (P > 0.05) in VAP, VSL, VCL, ALH, STR, or LIN. There was a numerically higher percentage of rapid cells in the Supermix semen. Pregnancy rate from bulls A and B was 61% and from Mixed A + B 60%, while that from Supermixed A + B was 69%. Results from the analysis indicate that semen concentration is an important element to be considered in a timed artificial insemination program. Numerically higher pregnancy rates were obtained with double semen concentration in the straw. More research is required to evaluate the interaction between different breeds within a timed artificial insemination program.
This research was supported by Centro Genético Bovino de EOLIA sa and Syntex sa Argentina.
77 FREEZING OF MOUSE SPERM BY THREE DIFFERENT CRYOPROTECTANTS
Authors: An C.S. , Uhm S.J. , Ko D.H. , Chung H.M. , Cho S.-G. , Chung K.S. , Lee H.T.
Published: December 2004

The cryopreservation of sperm has contributed greatly to animal breeding and reproduction. This study was designed to examine the effect of raffinose, sucrose, and trehalose as cryoprotectants for freezing of mouse sperm. The cryoprotectant solution (CPA) consisting of 3% skim milk (Skim Milk dehydrated, Bacto, Difco, Seoul, Korea) as buffer or extender was prepared and supplemented with 0.3 M raffinose (D[+]raffinose pentahydrate, Sigma) or sucrose or trehalose as non-permeating cryoprotectants. Sperm samples for cryopreservation were collected from caudae epididymides and vas deferens of males of four mouse strains (ICR, FVB, C57BL/6, and CBA). Sperm samples from individual males were aliquoted in cryotubes, placed immediately in the vapor phase of a liquid nitrogen storage container for 10 min, and then plunged directly in liquid nitrogen. For thawing, frozen cryotubes were removed from liquid nitrogen and placed directly into a water bath kept at 37°C for approximately 2 min until the ice melted. Survival of mouse sperm was measured by vital staining. Survival rates of spermatozoa frozen and thawed in freezing solution supplemented with raffinose were higher (ICR: 51.0%, FVB: 27.6%, C57BL/6: 25.7%, and CBA: 23.3%) than those supplemented with sucrose (35.8, 19.6, 12.3, and 19.7%) or trehalose (16.0, 25.4, 25.3, and 24.7%), all respectively. Furthermore, in vitro fertilization of mouse oocytes with sperm frozen in raffinose gave cleavage rates of 61.1 (FVB: 217/355 eggs), 59.4 (C57BL/6: 165/278 eggs), and 57.1% (CBA: 144/252 eggs), respectively. These cleaved embryos (FVB: 163, C57BL/6: 137, and CBA: 134) were transferred to 7, 5, and 6 pseudopregnant females, respectively. A total of 4, 3, and 4 females in the respective strains became pregnant and delivered 17 (10.4%), 17 (12.4%), and 18 (13.4%) offspring, respectively. Our results suggest that raffinose is a good cryoprotectant for freezing of sperm for production of inbred mice.
81 EFFECT OF SUCROSE CONCENTRATION IN WARMING MEDIUM ON THE DEVELOPMENT POTENTIAL OF VITRIFIED BOVINE OOCYTES
Authors: Chang W.C. , Xu J. , Jiang S. , Tian X.C. , Yang X. , Du F.L.
Published: December 2004

The aim of this experiment was to determine the effect of the sucrose concentration (0 to 0.33 M) in the dilution medium on the viability, fertilizability, and development of vitrified bovine oocytes. Bovine oocyte-cumulus complexes were collected from slaughterhouse ovaries and in vitro-matured as reported previously. After 24-h maturation in TCM199-based medium under 5% CO2 humidified air at 39°C, these were exposed to hyaluronidase and carefully pipetted to remove all except the 3–5 innermost layers of cumulus. Oocytes were put into the pre-equilibration medium for 3 min and then into vitrification solution containing HEPES-buffered TCM199 supplemented with 20% FBS, ethylene glycol, and dimethylsulphoxide for 25–30 s; they were then vitrified by modified solid surface vitrification (Dinnyes et al. 2000 Biol. Reprod. 63, 513–518).The oocytes were warmed at 39°C by placing them in holding medium with 0, 0.08, 0.17, 0.25, or 0.33 M sucrose. Non-vitrified oocytes were used as controls. Oocytes were inseminated 30 min after warming, and the presumptive zygotes were cultured in CR1-aa medium supplemented with 6 mg/mL BSA at 39°C in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 for eight days. Data were analyzed by one-way ANOVA. As shown in Table 1, there was no significant difference in survival rate (P > 0.05) of the vitrified oocytes that were placed in dilution solution containing 0.17, 0.25, or 0.33 M sucrose and the non-treated controls. On Day 2 (fertilized on Day 0), cleavage to the 8-cell stage was similar for the 0.17, 0.25, and 0.33 M dilution groups, but the rates for all three were significantly lower (P < 0.05) than for the control group. The blastocyst rate on Day 8 was significantly higher for the 0.25 M group than for any other experimental group but still significantly lower than for the control. In conclusion, this study suggests that with this vitrification/warming procedure the optimum concentration of sucrose in the dilution solution is 0.25 M.

Table 1.
Oocyte survival after vitrification/warming and subsequent embryo development

The authors would like to thank Ms Colleen Shaffer for the preparation of bovine oocytes.
83 CRYOPRESERVATION OF IN VITRO PORCINE OOCYTES BY SOLID SURFACE VITRIFICATION (SSV)
Authors: Dinnyes A. , Somfai T. , Sage D. , Marosan M. , Carnwath J. , Niemann H.
Published: December 2004

The effects of the cytosceletal inhibitor cytochalasin in solid surface vitrification (SSV; Dinnyes et al. 2000 Biol. Reprod. 63, 513–518) were investigated on in vitro matured (IVM) porcine oocytes. Cumulus-free IVM oocytes were subjected to one of the following: SSV (equilibration in 4% ethylene glycol (EG) for 10 min followed by vitrification in 35% EG, 5% polyvinyl pirrolidone, and 0.3 M trehalose on a cold (about −150°C) surface; warming in 0.4 M trehalose at 37°C), SSV pre-treatment with 5 μg/mL cytochalasin B (SSV + CB), or the steps of SSV without cooling, i.e. toxicity control (TC). Non-lysed oocytes together with the non-treated controls were subjected to parthenogenetic activation and then in vitro cultured (IVC) for six days. The proportion of non-lysed oocytes was higher when pre-treatment with CB was performed compared to SSV. However, both results were significantly lower than that of the TC. After parthenogenetic activation via a combination of a direct current electric pulse (1 kV/cm for 45 μs) followed by 3 h treatment with 2 mM 6-dimethylaminopurine, the proportion of cleaved embryos was higher in the SSV + CB than in the SSV. Nevertheless, significantly more oocytes cleaved in the TC and control groups. On Day 6 no blastocyst, were determined in the SSV group, and one blastocyst was obtained in the SSV + CB, while significantly more blastocysts developed in both the TC and the control (Table 1). There was no significant difference in blastocyst rates and in the number of blastomere nuclei/embryo between the TC and the control. These results indicate that the high concentration of cryoprotectants per se applied in SSV were not detrimental for in vitro development and that CB pre-treatment increased survival and further development of SSV vitrified pig oocytes resulting in one parthenogenetic blastocyst from vitrified pig oocytes.

Table 1.
Parthenogenetic development of vitrified IVM porcine oocytes

This research was supported by a Bilateral Scientific and Technological Collaboration (TET, No. D6/01) grant between Germany and Hungary and by the Hungarian National Office of Research and Technology (OM-KMUFA; BIO-00086/2002).
94 COMPARING DIFFERENT LEVELS OF OSMOLARITY AND pH OF LACTOSE EXTENDER ON THE VIABILITY OF SPERMATOZOA IN THE BACTRIAN CAMEL (CAMELUS BACTRIANUS)
Authors: Mosaferi S. , Niasari-Naslaji A. , Gharahdaghi A.A. , Abarghani A. , Ghanbari A. , Gerami A.
Published: December 2004

Lactose has been used widely as a semen extender for camels although in the absence of evidence illustrating its suitablility. Considering the osmolarity (316.1 ± 1.48 mOsm/kg) and pH (7.4 ± 0.03) of Bactrian camel semen (Mosaferi S et al. 2004 Theriogenology, in press), the objective of this study was to investigate the effect of osmolarity and pH of lactose extender on the viability of Bactrian camel spermatozoa. In Experiment I, with pH adjusted to 6.9, the effect of lactose concentrations of 9, 10, 11, 12, and 13% with an osmolarity of 290, 333, 350, 376, and 419 mOsm/kg, respectively, on the viability of spermatozoa was investigated. In Experiment II, with lactose fixed at 10%, the effect of extender with pH of 5.9, 6.9, 7.5, 7.9, and 8.9 on the viability of spermatozoa was examined. All extenders contained 20% egg yolk. In both experiments, semen was collected from camels with a sound history of semen quality and fertility (n = 3), using a modified artificial vagina, and divided into different treatments after mechanical removal of semen viscosity (Mosaferi et al. 2004). Progressive forward motility of spermatozoa was examined at the time of semen collection and at 4, 12, and 24 h after incubation at 4°C. Data were analyzed using the GLM procedure in SAS/STAT (SAS Institute, Inc., Cary, NC, USA) after arcsine transformation. At the time of semen dilution, the progressive forward motility of spermatozoa was greater at osmolarity of 290 (28.5%), 333 (34%), and 350 (31%) compared to 376 (13.5%) and 419 (1%) mOsm/kg (P < 0.05). The same trend in the progressive forward motility of spermatozoa was noticed after 4 h of incubation at 4°C; although a significant decrease (P < 0.05) occurred at 290 (11%), 333 (18 %) and 350 (16%). After 12 and 24 h of incubation at 4°C, the progressive forward motility of spermatozoa was less than 10% at 333 and 350 mOsm/kg (P < 0.05). At the time of semen dilution, the progressive forward motility of spermatozoa was greater (P < 0.05) at pH 6.9 (35.5%) and 7.5 (18%) compared to pH 5.9 (0%), 7.9 (7.5%) and 8.9 (2.5%). The same trend in the progressive forward motility of spermatozoa was observed after 4 h incubation at 4°C; although, a significant decrease (P < 0.05) occurred at pH 6.9 (15%) and 7.5 (9%) at this time. After 12 h incubation at 4°C, the progressive forward motility of spermatozoa was less than 5% at pH 6.9 and 7.5 (P < 0.05). In conclusion, 10% (333 mOsm/kg) and 11% (350 mOsm/kg) lactose, at the adjusted pH of 6.9, were the most suitable concentrations of lactose extender for preserving Bactrian camel semen for less than 4 h after which the viability of spermatozoa deteriorated significantly in this extender.
The authors wish to thank the director and station staff of Bactrian Camel Research Center at Jahadabad, Meshkinshahr, Ardabil, for kind provision of facilities and assistance throughout the experiment.
Scattered paddock trees, litter chemistry, and surface soil properties in pastures of the New England Tablelands, New South Wales
Authors: Graham S. , Wilson B. R. , Reid N. , Jones H.
Published: December 2004

Scattered paddock trees are widespread throughout rural Australia but their effect on soil conditions has received only limited research attention. This study investigated the influence of 3 Eucalyptus species on surface soil properties on different parent materials at both stocked and unstocked sites on the Northern Tablelands of New South Wales. Mineral soil samples to a depth of 5 cm were collected at intervals up to twice the canopy radius away from tree trunks and litter samples were collected at corresponding points. Mineral soils were analysed for pH (CaCl2), organic carbon (C), and extractable phosphorus (P) concentration, while for the litter samples, P, sulfur, cations, and ash alkalinity were determined. Stocking with sheep and cattle increased surface soil acidity and C and P concentrations at each location. However, soils under E. melliodora and E. viminalis showed higher pH and increased C and P concentrations close to the tree stem irrespective of grazing. Soils under E. caliginosa, while having similar patterns of C and P, showed variable acidity patterns with instances of lower pH close to the tree stem. Spatial patterns in soil acidity were associated with the ash alkalinity of litter, indicating litter as a source of alkalinity addition to the soil surface, although different patterns of soil pH could not be fully explained by litter ash alkalinity alone. The close correlation of litter Ca content with ash alkalinity suggests that this element might be a suitable indicator of the acid amelioration capacity of different tree species.
Response of excluder, indicator, and hyperaccumulator plants to nickel availability in soils
Authors: Massoura Stamatia Tina , Echevarria Guillaume , Leclerc-Cessac Elisabeth , Morel Jean Louis
Published: December 2004

Availability is a key property for the assessment of soil-to-plant transfer of heavy metals. This work was conducted to determine whether the available pool of Ni differs according to the ability of plants to take up and accumulate the metal. An excluder plant species (Triticum aestivum L.), an indicator (Trifolium pratense L.), and 3 populations of the Ni-hyperaccumulator Alyssum murale (Waldst. & Kit.) were grown for 90 days on 4 soils with a gradient of concentrations of total and available Ni. Isotopic exchange methods with 63Ni ions were used to measure the exchangeable soil Ni (E-value, intensity, and capacity factors), to monitor its uptake by plants and to determine the size of the available pool (L-value). Results showed that, for a given soil, the L-values were similar for all plant species, showing that they all access the same Ni exchangeable pool regardless of their Ni uptake capacity. Also, L-values for a given soil were equal to the E-value calculated for a 90-day period, demonstrating that plant Ni originated from the isotopically exchangeable soil Ni. This pool can be accurately and simply determined with the isotopic exchange kinetic methods run on soil–solution batch systems without plants. Moreover, the results indicate that the plant species take up Ni as a response to ‘intensity’, ‘capacity’, and ‘quantity’ soil factors and that E-value alone is not enough to predict plant uptake. This work suggests a uniform behaviour of the plants tested towards soil Ni and may have practical applications in phytoextraction and phytomining, as the plants removed Ni exclusively from the exchangeable pool.