bluuebunny/crossref_metadata_2025 huggingface dataset:
202 EFFECT OF OSTEOPONTIN ON IN VITRO EMBRYO DEVELOPMENT IN CATTLE
Authors: Gasparrini B. , Monaco E. , Boccia L. , De Rosa A. , Attanasio L. , Killian G.
Published: January 2008

Osteopontin (OPN) is an acidic single-chain phosphorylated glycoprotein found both in the oviduct fluid (ODF) and oviductal epithelium in cattle, which is believed to facilitate fertilization. It was recently reported that addition of a rabbit polyclonal immunoglobulin G antibody against purified bovine milk OPN with sperm oocytes, bovine oocytes, or both decreased (P < 0.05) fertilization compared with the in vitro-fertilized control (Goncalves et al. 2007 Theriogenology 67, 468–74). The aim of the present work was to determine the effect of in vitro addition of OPN to the fertilization medium on both cleavage and postfertilization embryo development in cattle. In the first experiment, in vitro-matured oocytes were fertilized in modified TALP medium in the presence of 0.0, 0.1, 1.0, or 10 µg mL–1 of OPN. In a second experiment, matured oocytes were in vitro-fertilized in modified TALP medium in the presence of 0.0, 10, 20, or 40 µg mL–1 of OPN. In vitro fertilization was carried out with frozen–thawed spermatozoa from a bull previously tested for IVF. After 20 to 22 h of coincubation at 39�C, 5% in CO2 in air, presumptive zygotes were vortexed to remove cumulus cells, washed, and transferred, 30 to 50 per well, into 400 µL of SOF modified medium. Zygotes were incubated in a humidified mixture of 5% CO2, 7% O2, and 88% N2 in air at a temperature of 39�C. On Day 7 of development (Day 0 = day of insemination), cleavage and development rates into transferable embryos (TE)–tight morulae (TM) and blastocysts (Bl) of superior quality were recorded. Differences in the percentages of both cleavage and blastocyst rates among groups were analyzed by a chi-square test. In experiment 1, numerically higher percentages of TM–Bl (29.5, 29.5, 30.5, and 37.5%, respectively, in the control group and in the groups with 0.1, 1, and 10 µg mL–1 of OPN; P = 0.25) and Bl (28.6, 27.5, 29.1, and 36.7, respectively, in the control group and in the groups with 0.1, 1, and 10 µg mL–1 of OPN; P = 0.24) were observed with 10 µg mL–1 of OPN. In experiment 2, significantly more cleavage (80.0 v. 71.3%; P < 0.05) and higher percentages of TM–Bl (44.6 v. 34.5%; P < 0.05) and Bl (39.2 v. 30.6%; P = 0.06) were observed with 10 µg mL–1 of OPN v. the control. Combined analysis from both experiments showed an overall effect of 10 µg mL–1 of OPN v. the control in the percentages of TM–Bl and Bl (respectively, 41.1 v. 33.3% and 37.7 v. 30.5%; P < 0.05). These results indicate that it is possible to improve the efficiency of bovine in vitro embryo production by adding the oviductal protein OPN.
211 INTRODUCING NEW GENETICS INTO A CLOSED BIOSECURE HERD OF DAIRY GOATS
Authors: Melican D. , Blash S. , Gavin W.
Published: January 2008

Transgenic dairy goats expressing recombinant molecules in their milk have been validated as a viable method for producing human therapeutic proteins. Although maintaining a closed herd ensures biosecurity within a facility, the ability to introduce new genetics into the herd can be difficult. In this work we determined the ability to use cryopreserved caprine semen, imported from New Zealand into the United States, for IVF as a method to increase the genetic diversity of the GTC Biotherapeutics closed caprine herd. Semen was collected from bucks owned by GTC Biotherapeutics and maintained in New Zealand. The bucks were serologically screened for goat pathogens prior to collection, and were maintained in quarantine during semen collection. Separate single experiments were performed using cryopreserved semen from each of 2 different bucks (NZ1 and NZ2). One or 2 straws of semen (107/0.25 mL straw) from each buck were thawed and then purified using a Percoll gradient. Ovulated oocytes surgically collected from superovulated does were co-incubated with sperm (5 × 105 mL–1) in Brackett-Oliphant medium supplemented with 10% fetal bovine serum, 7.7 mm calcium lactate plus 2.5 μg mL–1 of heparin for 18 h at 38°C. Presumptive zygotes were transferred to equilibrated SOF plus 0.8% BSA and cultured in vitro for 24 h. On Day 2 cleavage was determined and, as an added precaution, embryos selected for transfer were washed per the IETS protocol for the sanitary handling of embryos. Five 2-cell to 8-cell embryos from individual donors were surgically transferred to a single oviduct of each synchronized surrogate recipient. Pregnancies were determined by ultrasonography. Pregnancy rates for recipients at Day 50 of gestation (71 v. 67% pregnant), at term (100 v. 100%), and the proportion of offspring born from total embryos transferred (17 v. 23% offspring) were comparable for buck NZ1 and buck NZ2, respectively (P > 0.05). A total of 13 offspring (6 bucks and 7 does) were produced from 9 different oocyte donors. These results demonstrate that cryopreserved caprine semen, imported from New Zealand into the United States, can be used for IVF to introduce new genetics into a closed biosecure caprine herd. The use of IVF, compared with AI, allows more offspring to be produced per straw of semen. In addition, IVF offers the advantage of accelerated genetic gain by producing multiple offspring from elite does with more desirable lactation, reproduction, and conformation traits. Beyond the new F1 animals produced by IVF, several techniques (natural mating, AI, or IVF) can then be used to quickly disseminate the new genetics into both the nontransgenic and transgenic herds. Finally, skin cells obtained from the female IVF offspring or fetal cells derived from any pregnancies of the initial IVF offspring could also be used to generate transfected cells as karyoplast donors for future somatic cell nuclear transfer work.

Table 1. Summary of caprine IVF
212 INSEMINATION OF OVUM PICKUP-DERIVED DAIRY COWS RESULTS IN OFFSPRING WITH NORMAL BIRTH WEIGHT
Authors: Mullaart E. , Landman B. , Merton J. S.
Published: January 2008

Animals derived by ovum pickup-in vitro production (OPU-IVP) have a higher birth weight compared with animals derived by AI (Wagtendonk et al. 2000 Theriogenology 53, 575–597). It has been suggested that this higher birth weight is the result of epigenetic changes such as aberrant methylation and gene expression pattern, which are caused by the presence of serum in the culture medium (Wrenzycki et al. 2004 Anim. Reprod. Sci. 82–83, 593–603). The present study aimed to investigate whether the higher birth weight, possibly caused by epigenetic changes, is a permanent characteristic that is transmitted to the offspring. We therefore monitored the birth weight of calves born after insemination of OPU-IVP-derived animals. Ovum pickup-IVP was performed according to routine procedures. Immature COC were recovered by OPU. The COC were matured in vitro in TCM-199 supplemented with fetal calf serum (FCS)/LH/FSH. Subsequently, matured oocytes were fertilized with frozen–thawed gradient-separated semen and further cultured for 7 days in TCM-199/10% FCS on a BRL monolayer (CoCul group) or in SOFaaBSA (SOF group). First-generation OPU-IVP animals were produced from oocytes collected by OPU of AI-derived animals. The second generation was produced by inseminating OPU-IVP animals. Calves generated by inseminating AI animals were used as a control group. Birth weights of control AI, first-generation, and second-generation calves were analyzed by using restricted maximum likelihood (Genstat 9.1). Model Birth Weight: *Fixed: Parity Recipient + Sex + Gestation Length + Year + Embryo Type (AI, first, or second generation) + Culture System (CoCul or SOF). *Random: Sire + Barn. The results (Table 1) clearly show that the first-generation (OPU-IVP) calves had, on average, a 3.4-kg greater birth weight than the AI calves. The second-generation calves, however, had approximately the same birth weight as the calves in the AI control group. Our results indicated that the high birth weight of OPU-IVP-derived calves is not a permanent characteristic that is transmitted to their offspring. Previous studies have demonstrated that the fertility of OPU-IVP-derived animals is in the normal range (Wagtendonk et al. 2000 Theriogenology 53, 575–597).

Table 1. Birth weight (least squares means ± SE) of AI calves (control), first generation OPU-IVP-derived calves, and second generation AI derived calves from OPU-IVP mothers
220 EFFECTS OF FOLLICLE-STIMULATING HORMONE AND 6-N-PROPYL-2-THIOURACIL ON BOAR SPERMATOGENESIS
Authors: Baldrighi J. , Averhart W. , Mello M. , Ford J. , Franca L. , Carnes K. , Hess R. , Clark S.
Published: January 2008

Currently, swine biotechnologies related to reproduction increase considerably. Investments are made in order to improve the reproductive rates and performance of breeding stock. Understanding the physiology of spermatogenesis will help increase sperm production and improve boar efficiency. Sertoli cells are the only somatic cells present in the seminiferous tubules. Their function is to guarantee proper sperm formation and maturation. Each Sertoli cell is responsible for nursing a finite number of spermatogonia. At puberty, Sertoli cell maturation and lumen formation have occurred within the seminiferous tubules and germ cells have proliferated rapidly followed by the onset of spermatogenesis. At least two hormones are known to play a role in Sertoli cell proliferation and maturation: follicle-stimulating hormone (FSH) and thyroid hormone. FSH secretion has been assumed to be the stimulus for proliferation. The thyroid hormone is responsible for normal postnatal growth and development. Alterations in thyroid activity have frequently been associated with changes in male reproductive functions, since hypothyroidism, induced with 6-N-propyl-2-thiouracil (PTU) soon after birth, is associated with a marked delay in sexual maturation and development. The goal of this study was to report the effect of FSH and PTU on the stages of sperm cell development of young pigs. Six piglets of 1, 7, 14, 25, and 55 days of age were castrated and their testes were sectioned to grafts of 5 mm3. The grafts were then transplanted subcutaneously into the dorsum of 12 castrated nude mice per age group. Two days post-surgery mice were randomly assigned to one of four treatment groups: control, FSH (5 IU rFSH), PTU (0.015% solution), and FSH + PTU. Following 14 days of treatment, testicular tissue pieces were allowed to grow for 2 additional weeks. Tissues were then harvested, immersion-fixed in neutral buffered formalin, and embedded in paraffin. Five-micron-thick sections were stained using hematoxylin and eosin. Slides were evaluated under light microscopy and the oldest germ cell type present in each section was recorded. Germ cell types were recorded as spermatogonium, spermatocyte, early spermatid, and late spermatid. Statistical differences between all groups were detected using paired Student t-tests. There were no differences noted between control groups and those treated with PTU or FSH alone. No effect concerning age of castration on grafts development was observed. There was a slightly significant increase (P = 0.05) in the number of spermatocytes observed in the groups treated with FSH+PTU. These data suggest that there is a potential synergistic effect of FSH and PTU on sperm cell development. Based on these results, further studies need to be performed to completely understand the effect of these two hormones on Sertoli cells.
232 PARTHENOGENETIC DEVELOPMENT AND PLOIDY OF BOVINE OOCYTES FOLLOWING VARIOUS CHEMICAL ACTIVATION REGIMENS
Authors: Ock S.-A. , Rho G.-J.
Published: January 2008

Bovine oocytes treated using various combinations of ionomycin (Ion), cycloheximide (CHX), and cytochalasin B (CCB) were evaluated for developmental rates and ploidy status. Metaphase II oocytes were allocated 5 treatment groups, and the groups were treated as follows: Group 1: 5 µm Ion for 5 min; Group 2: Ion + 10 µg mL–1 CHX for 5 h; Group 3: Ion + 10 µg mL–1 CHX + 5 µg mL–1 CCB for 1 h + 10 µg mL–1 CHX for 4 h; Group 4: Ion + 10 µg mL–1 CHX + 5 µg mL–1 CCB for 3 h + 10 µg mL–1 CHX for 2 h; and Group 5: Ion + 10 µg mL–1 CHX + 5 µg mL–1 CCB for 5. Difference among groups was analyzed using one-way ANOVA by SPSS 10.0 (SPSS, Inc., Chicago, IL, USA). In Experiment 1, 430 oocytes in 4 replicates were compared for the extrusion rate of the second polar body (PB) at 8 h after Ion treatment among groups. Group 5 exhibited significantly (P < 0.05) lower rates of second PB extrusion than did Groups 1–4 (22% v. 53–67%). Experiment 2 compared the rates of cleavage at 48 h and development to the blastocyst stage at 216 h after Ion treatment among groups. A total of 536 oocytes were used in 5 replicates. Parthenotes in Group 1 showed lower rates of cleavage and blastocyst development than those in other groups (20% and 1% v. 53–67% and 6–31%). Among the groups, parthenotes in Group 5 showed significantly (P < 0.05) higher blastocyst development. In Experiment 3, at 8 h after Ion treatment, oocytes from Groups 2, 3, and 5 were divided into two subgroups based on the presence or absence of the second PB, and assessed for cleavage rates and ploidy in 239 2-cell-stage parthenotes in 4 replicates, as described earlier by King et al. (1979 Vet. Sci. Commun. 3, 51–56). The cleavage rates did not differ among activation treatments, or by the presence or absence of the second PB in any activation group. The haploid rate was significantly (P < 0.05) higher in Group 2 than in Groups 3 and 5 (38% v. 19% and 0%, respectively). The diploid rate was significantly (P < 0.05) higher in Group 5 than in Groups 2 and 3 (88% v. 69% and 45%, respectively). In Experiment 4, the diploid rate of Group 2 blastocyst-stage parthenotes was 100% (4/4), whereas the diploid rates of Groups 3 and 5 blastocyst-stage parthenotes were 50% (6/12) and 71% (17/24), respectively, but the rates did not differ among groups. These results indicate that oocyte activation by CHX/CCB for 5 h after Ion treatment could enhance parthenogenetic development in bovines with higher rates of diploidy by preventing the extrusion of the second PB.
233 STUDY OF CORTICAL GRANULE CONTENT IN IN VITRO-MATURED PORCINE OOCYTES BY MEANS OF PNA LECTIN PRECIPITATION
Authors: Saavedra M. D. , Avils M. , Coy P. , Romar R.
Published: January 2008

Cortical granules (CG) are clue organelles in the oocyte since their content is released under oocyte activation (i.e. fertilization) modifying the zona pellucida and thus blocking polyspermy. Once released, CG are not renewed. Research on cortical reaction and putative CG enzymes has progressed slowly because mammalian eggs contain only picogram quantities of CG-derived proteins (Moller and Wassarman 1989 Dev. Biol. 132, 103–112; Green 1997 Rev. Reprod. 2, 147–156), so the protein(s) responsible for the physiological changes in ZP after cortical reaction are not well known. The objective of this project was to study porcine CG content in in vitro-matured porcine oocytes by means of lectin precipitation with peanut agglutinin (PNA), since this lectin binds to porcine CG (Yoshida et al. 1993 Mol. Reprod. Dev. 36, 462–468). Immature porcine cumulus–oocytes complexes (COCs) from Landrace � Large White gilts were in vitro-matured for 44 h in NCSU-37 medium. After IVM period, COCs were stripped of cumulus cells, washed in PBS, and quickly washed through purified water. Then oocytes were lysed in a fresh water droplet by gentle pipetting using a narrow-bore glass pipette. Once lysed, zonae pellucidae were removed and oocyte cytoplasmic content (lysate) collected. Lysate from 1000 IVM-oocytes was incubated under continuous shaking (2 h, room temperature) with 100 µL PNA-agarose (Sigma, St. Louis, MO, USA) so that proteins bound to PNA could be precipitated by centrifugation. After lectin precipitation, proteins were detached from PNA-agarose beads by boiling in reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (5 min, 100�C) (Laemmli 1970 Nature 227, 689). Samples were then centrifuged (5 min, 7000g), the pellet containing PNA-agarose beads was discarded, and the supernatant containing the proteins was collected and further separated by SDS-PAGE. The silver staining of electrophoresis gels revealed eleven bands from 37 to 180 kDa, so a second gel was electrotransferred to a polyvinylidene fluoride (PVDF) membrane (100V, 1 h) and incubated with PNA-horseradish peroxidase (PNA-HRP, 10 µg mL–1) for 1 h. Visualization was accomplished using the enhanced chemiluminiscence (ECL plus) method and Typhoon 9410 following the manufacturer's instructions (Amersham Biosciences, Freiburg, Germany); only four bands of approximately 57 kDa, 60 kDa, 70 kDa, and 80 kDa were observed. These bands will be cut and processed for proteomic analysis for further studies. Preliminary results show that porcine CG-derived proteins can be studied by PNA lectin precipitation. These results could be employed in the future to develop specific antibodies against porcine CG.
242 USE OF BRAIN-DERIVED NEUROTROPHIC FACTOR IN IN VITRO PREMATURATION OF BOVINE OOCYTES SUBJECTED TO PARTHENOGENETIC ACTIVATION
Authors: De Bem T. H. C. , Rochetti R. , Pires P. R. L. , Bressan F. F. , Adona P. R. , Leal C. L. V.
Published: January 2008

Prematuration provides an additional time for oocyte capacitation and maturation in an attempt to improve in vitro embryo production (IVP) rates and allows media supplementation during this period for IVP. The aim of this study was to use brain-derived neurotropic factor (BDNF) in prematuration to improve maturation of bovine oocytes subjected to parthenogenetic activation and cultured with different media. Oocytes were subjected to prematuration in TCM-199 medium supplemented with 10 µm butyrolactone I, 2.0 mm pyruvate, and 10 µg mL–1 gentamicin for 24 h in the absence of BDNF (control) or in the presence of 10 ng mL–1 BDNF (BD). Oocytes were then in vitro-matured (IVM) in TCM-199 medium supplemented with 10% FCS, 0.5 µg mL–1 FSH, 5.0 µg mL–1 LH, 2.0 mm pyruvate, and 10 µg mL–1 gentamicin at 38.5�C under 5% CO2 in air. After 19 h oocytes were denuded using hyaluronidase and vortexing for 3 min for the 1st polar body (1PB) selection. Those which extruded the 1PB were maintained in IVM until 26 h, when parthenogenetic activation was performed (5 min in 5 µm ionomycin, followed by 3 h in 2 mm 6-DMAP). Activated oocytes were then transferred to in vitro culture (IVC) for embryo development evaluation. Embryos from both groups were cultured in SOF medium with 2.5% FCS, 0.05 g mL–1 BSA, 0.2 mm pyruvate, and 10 mg mL–1 gentamicin. Cleavage rates on the second day of in vitro culture (D2), embryo production at Days 7 and 8 (D7 and D8), and hatching rate at Day 8 were evaluated. Data regarding 1PB extrusion, cleavage, blastocyst development on D7 and D8, and blastocyst D8 hatching rates of three replicates were analyzed by chi-square test at 5% significance using the BIOESTATS 4.0 software. Control and BD, respectively, did not show differences (P > 0.05) regarding 1PB extrusion (n = 164, 63.81%, and n = 175, 66.79%) or cleavage (n = 117, 71.34%, and n = 138, 78.86%). However, for control and BD, respectively, blastocyst development on D7 (n = 63, 38.41%, and n = 89, 50.86%), D8 (n = 63, 38.41%, and n = 91, 52.00%), and hatching on D8 (n = 22, 34.92%, and n = 39, 43.82%) were all significantly higher for BD when compared with control (P < 0.05). In conclusion, BDNF during prematuration improved in vitro embryo development by increasing blastocyst and hatching rates of parthenogenetic embryos.
244 AMPHIREGULIN CAN ASSIST IMMATURE PORCINE OOCYTES TO DEVELOP IN AN IN VITRO SYSTEM
Authors: Jeong S. H. , Lee J. H. , Jeung E. B. , Lee E. S. , Hyun S. H.
Published: January 2008

Porcine IVM-IVF technique has been used and improved. However, polyspermic penetration, low rate of blastocyst (BL) formation, and poor quality of BLs are induced by imperfect nuclear and cytoplasmic maturation. It has been reported that epidermal growth factor (EGF) is beneficial for oocyte maturation to improve the IVM system, and amphiregulin (AR) is a growth factor containing an EGF-like domain. Consequently, the present study was performed to investigate whether TCM199 supplemented with EGF and/or AR is profitable for improving oocyte maturation and embryo development. The experimental groups were control (A), EGF 15 ng mL–1 (B), AR 15 ng mL–1 (C), and EGF 15 ng mL + AR 15 ng mL–1 (D) in TCM199. In Experiment 1, oocytes were selected and maturated in each group. After 44 h, oocytes were stained with Hoechst, and metaphase II and development rate were evaluated. In Experiment 2, maturated oocytes were fertilized with proven sperm and stained with Hoechst; the penetration rate after 10 h of post-insemination was evaluated. In Experiment 3, after 168 h of in vitro culture, cleavage rate and BL formation rate were recorded. BLs were stained with differential stain and the cell numbers calculated. In Experiment 4, porcine oocytes were maturated for 44 h and denuded by hyaluronidase. Thereafter, oocytes were stained with fluorescein isothiocyanate-labeled peanut agglutinin and the distribution of cortical granules (CGs) was evaluated by laser confocal microscopy. All data was analyzed by a Duncan test using SPSS (SPSS, Inc., Chicago, IL, USA). In Experiment 1, the maturation rate into metaphase II stage in groups B, C, and D (71.0 � 15.5%, 75.8 � 9.8%, and 71.5 � 10.8%, respectively) was significantly different (P < 0.05) from that of group A (51.4 � 18.3%). In Experiment 2, there was no difference among all groups in relation to penetration rate. But polyspermy was decreased (P < 0.05) in group D (26.2 � 11.3%) compared with group A (42.3 � 13.0%). In Experiment 3, there was no significant difference among all groups regarding the cleavage rate. However, BL developmental rates in group D (17.3 � 11.5%) were increased (P < 0.05) compared with groups A and B (9.6 � 3.2% and 13.3 � 4.6%, respectively). In groups B, C, and D, the cell number in BL increased significantly (P < 0.05) compared with that of group A(81.4 � 32.0%, 79.7 � 27.3%, and 80.3 � 28.5% v. 56.3 � 25.5%). In Experiment 4, group D had the largest CG area among the groups. The results of this study indicate that the addition of EGF or AR or both to TCM199 promotes oocyte maturation and increases the total cell number. EGF plus AR increases development to the BL stage and decreases polyspermy. It is suggested that AR can assist immature porcine oocytes to the metaphase II stage, that AR may enhance developmental ability in the in vitro system, and that there is a synergistic effect between EGF and AR during the maturation period.
248 OPTIMAL IN VITRO MATURATION TIME DEPENDS ON MORPHOLOGICAL CHARACTERISTICS OF BOVINE CUMULUS - OOCYTE COMPLEXES
Authors: Morales-Pliego F. , Barceló-Fimbres M. , Amorim L. S. , Seidel Jr G. E.
Published: January 2008

Oocytes with compact cumulus cells are usually chosen for bovine in vitro maturation and IVF experiments, while oocytes that do not have compact cumulus cells are often discarded. In this experiment, we studied the interaction of maturation time and cumulus characteristics. Five cumulus classifications (compact, partially expanded, expanded, denuded, dark-coagulated) were studied using 3 maturation times. A total of 1923 oocytes were aspirated from 3- to 8-mm follicles; matured for 1, 4, or 23 h; and fertilized with sperm from 3 bulls (2 replicates per bull with one replicate using ovaries of cull cows and the other, ovaries of fattened heifers). IVF was done with sperm centrifuged through a 45/90% Percoll gradient. All media were chemically defined (CDM) plus 0.5% fatty acid-free BSA. Sperm were co-incubated with oocytes for 16–18 h; presumptive zygotes were then vortexed to remove cumulus cells and cultured for 7 days (De La Torre-Sanchez et al. 2006 Mol. Reprod. Dev. 18, 585–596). Overall blastocyst production was low, partly due to differences among bulls (P < 0.01) (2.8, 4.5, and 11.3% blastocysts per oocyte for semen of bulls A, B, and C), and partly because some ovaries were from fattened heifers fed a progestin. There was a significant interaction between type of cumulus and maturation time for development to the 8-cell stage (P < 0.01) and blastocysts (P < 0.07), but not for cleavage (data not presented). From Table 1, it is clear that 1 or 4 h is insufficient maturation time for oocytes from all cumulus classifications except for expanded cumulus. For that category, 1 h maturation resulted in blastocyst production equivalent to oocytes with compact cumulus matured 23 h. Standard timing of 23 h maturation was suboptimal for oocytes with expanded cumulus. For oocytes with compact cumulus, 23 h maturation was superior to shorter times (P < 0.05); oocytes with partially expanded cumulus cells responded similarly to those with compact cumulus except for a higher percent of 8-cell embryos with 23 h maturation. While 8-cell embryo production was above 40% per oocyte for all groups with 23 h of maturation, blastocyst production was very low for oocytes with dark cumulus, and also low when oocytes were denuded or had expanded cumulus. In conclusion, while 23 h maturation was optimal for oocytes with compact or partially expanded cumulus, similar blastocyst rates were obtained with oocytes with expanded cumulus if maturation time was shortened to 1 h.

Table 1. Mean percentage development of oocytes to 8-cell and blastocyst stages
253 LACK OF ABILITY TO REGULATE KINASE ACTIVITIES MAY BE RESPONSIBLE FOR MEIOTIC INCOMPETENCE IN THE DOG OOCYTE
Authors: Songsasen N. , Stein P. , Schultz R. M. , Travis A. J. , Wildt D. E. , Pukazhenthi B. P.
Published: January 2008

The size of donor follicles influences meiotic maturation of oocytes, including those of the domestic dog (Songsasen and Wildt 2005 Mol. Reprod. Dev. 72, 113–119). Maturation promoting factor (MPF, CDK1) and mitogen activating protein kinase (MAPK) play a pivotal role in regulating meiosis in several species (Abrieu et al. 2001 J. Cell Sci. 114, 257–267). Accordingly, we determined (1) MPF and MAPK activities, and (2) mRNA expression of cell cycle genes, including CDK1, CCNB1, and CDC25 in oocytes obtained from small (<1 mm diameter), medium (1 to 2 mm) and large (>2 mm) follicles. In Study 1, ovarian oocytes were classified into three groups (based on these sizes) and then cultured (38.5�C in 5% CO2) for 0, 24, or 48 h in TCM-199 (+25 µm β-mercaptoethanol, 10 ng mL–1 epidermal growth factor, 0.25 mm pyruvate, 2.0 mm glutamine, and 0.1% polyvinyl alcohol). Oocytes were denuded, assessed for nuclear status, and stored individually at –80�C until MPF and MAPK activities were assayed using a double kinase assay. Kinase activities of in vitro-matured (IVM) oocytes were expressed as a ratio of MPF and MAPK to that in metaphase I (MI) oocytes flushed from the oviducts of estrous bitches. In Study 2, oocytes (n = 20/follicular size class) were immediately frozen at –80�C; RNA was extracted, reverse transcribed, and subjected to quantitative real-time PCR analysis. Expression levels of each transcript were normalized to levels of endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Statistical analyses were performed using ANOVA followed by the Holm-Sidak test for multiple comparisons. Both MPF and MAPK activities varied among follicular size classes. Kinase activities increased in oocytes from large follicles upon meiotic resumption, with germinal vesicle (GV) oocytes expressing the lowest levels compared to their metaphase II (MII) counterpart (GV: 25.9 � 5.2% v. MII: 211.5 � 19.5% and 36.4 � 9.1% v. 150.7 � 22.9% for MPF and MAPK, respectively; P < 0.01). For medium follicles, MPF increased (P < 0.01) as oocytes progressed from GV (23.3 � 6.3%) to MII (243.4 � 55.5%). However, MAPK levels remained constant until the MI stage, and then increased (P < 0.01) in MII oocytes. For small follicles, MPF increased (P < 0.05) at the MI stage and then remained constant until meiosis was completed, whereas MAPK activities remained constant after GVBD. Kinase activity levels were not different (P < 0.05) between MI and MII oocytes harvested from the three follicular classes. Interestingly, follicular size had no effect (P > 0.05) on expression levels of cell cycle transcripts. These findings suggest that the compromised developmental competence of dog oocytes from small follicles likely is related to the oocytes' inability to regulate MAPK activity during meiotic resumption.
254 MOLECULAR AND SUBCELLULAR CHARACTERIZATION OF OOCYTES SCREENED FOR THEIR DEVELOPMENTAL COMPETENCE BASED ON G6PDH ACTIVITY
Authors: Torner H. , Ghanem N. , Ambros C. , Hoelker M. , Tomek W. , Phatsara C. , Alm H. , Kanitz W. , Sirard M. A. , Schellander K. , Tesfaye D.
Published: January 2008

Oocyte selection based on glucose-6-phosphate dehydrogenase (G6PDH) activity has been successfully used to differentiate between competent and incompetent bovine oocytes (Alm 2005 Theriogenology 63, 2194–2205). However, the intrinsic molecular and subcellular characteristics of these oocytes have not yet been investigated. Here we aim to compare the developmental, molecular, and subcellular characteristics of oocytes selected using brilliant cresyl blue (BCB) staining based on G6PDH activity. Immature compact cumulus–oocyte complexes (COCs) were stained with 26 µm BCB (B-5388, Sigma-Alderich, Taufenkirchen, Germany) for 90 min. Based on their coloration, oocytes were divided into BCB– (colorless cytoplasm, high G6PDH activity) and BCB+ (colored cytoplasm, low G6PDH activity). The chromatin configuration and the mitochondrial activity of oocytes were determined by fluorescence labelling and photometric measurement (n = 337). The abundance and phosphorylation pattern of protein kinases Akt and MAP kinase were estimated by western blot analysis (n = 500). A bovine cDNA microarray with 2000 clones was used to analyze the gene expression profiles of BCB+ and BCB– oocytes (n = 580). BCB+ oocytes were found to result in a higher blastocyst rate (33.1 � 3.1%) until Day 8 of in vitro culture compared to BCB– ones (12.1 � 1.5%). Moreover, BCB+ oocytes showed higher phosphorylation levels of Akt and MAP kinase compared to the BCB– oocytes. After array data analysis, BCB+ oocytes were found to be enriched with genes regulating transcription (SMARCA5), cell cycle (NASP), and protein biosynthesis (RPS274A and EEF1A1), while the BCB– oocytes had a higher level of genes involved in ATP synthesis (ATP5A1), mitochondrial electron transport (FL405), calcium ion binding (S100A10), and growth factor activity (BMP-15). Independent real-time quantitative PCR validated 90% (9/10) of the genes investigated to be in agreement with the array expression profile. The study has shown evidence of differences in molecular and subcellular organization of oocytes with different G6PDH activity.
265 DIFFERENTIATING X- AND Y-BEARING SPERMATOZOA ASSOCIATED WITH THE ZONA PELLUCIDA AT THE TIME OF FERTILIZATION
Authors: Mao J. , Davis A. M. , Fountain E. D. , Roberts R. M. , Rosenfeld C. S.
Published: January 2008

In this laboratory, we are studying why a nutritionally complete maternal diet of high energy content can skew the sex ratio toward males in mice, and why a diet low in energy favors female offspring. One possibility is that the oocyte is able to attract or be fertilized by one class, i.e., X- or Y-bearing sperm relative to the other as a result of maternal diet during oogenesis. Alternatively, maternal diet may result in a reproductive tract environment that provides a competitive advantage for one class of sperm over the other. The objective of current experiment was to establish a fluorescent in situ hybridization (FISH) method for determining relative zona pellucida (ZP)-associated X and Y sperm number in control mice. While our laboratory and many others have employed FISH-based approaches to distinguish epididymal X and Y sperm, such procedures cannot be applied directly to ZP-associated sperm. Herein, we describe a successful method that maintains the integrity of this association and differentiates the ZP-associated X- versus Y-bearing sperm by FISH. Cumulus-oocyte complexes (COCs) were collected from NIH Swiss females on a control diet. Spermatozoa were obtained from the caudal epididymis of stud male mice, capacitated, and used to inseminate COCs under standard culture conditions. After 5 h, sperm/eggs were washed with PBS, transferred to a positively charged glass slide, and treated with 0.25 µL of 0.01 m HCl/0.1% Tween 20 solution at room temperature. The remaining structures, including attached spermatozoa, were fixed in 80% methanol (20 min, –20�C), subjected to microwave heating for 105 s, and treated with 0.01% pepsin for 20 min at 37�C, washed in 1 � SSC, fixed in 80% methanol, and air-dried. Hybridization probes (concentrated FITC-labeled X, Cy3-labeled Y; CamBio, Cambridge, UK) were combined and held at 65�C for 10 min and 37�C for 35 min before application to the slides at 37�C. Slides were sealed with coverslips and rubber cement, incubated at 75�C for 3 min, transferred to a pre-warmed humidified chamber, and incubated for 20 h at 37�C. Specimens were washed successively for 8 min twice in each of stringent conditions (50% formamide + 50% 1 � SSC, 45�C), 1 � SSC, and detergent solution, and then mounted and examined under a fluorescence microscope. A total of 20 oocytes with 1539 zona-associated sperm were analyzed. The average numbers of X and Y sperm per oocyte were 40.65 � 4.23 and 36.30 � 3.78, respectively, values not statistically different from 1:1. This FISH procedure appears to provide a valid and effective way to assess ratios of X andY sperm associated with the ZP. This procedure will be applied in future studies to determine whether ZP-associated X- versus Y-sperm number varies according to maternal diet in mice.
This work was supported by NIH Grant HD 044042.
267 BIRTH OF DOMESTIC CAT KITTENS OF PREDETERMINED SEX AFTER TRANSFER OF EMBRYOS PRODUCED BY IN VITRO FERTILIZATION OF OOCYTES WITH FLOW-SORTED SPERMATOZOA
Authors: Pope C. E. , Crichton E. B. , Gmez M. C. , Dumas C. , Dresser B.
Published: January 2008

In cats, sex selection by fertilization of oocytes with sperm sorted into X- andY-chromosome-bearing populations has credible biomedical, commercial, and conservation connotations. Our objectives were (1) to evaluate the efficiency with which embryos could be produced by IVF of in vivo- and in vitro-matured oocytes with cooled sex-sorted sperm after overnight shipment to the sorting facility and overnight return delivery to an IVF laboratory, and (2) to determine if live kittens of predetermined sex (female) could be produced after transfer of embryos derived by IVF of in vivo-matured oocytes with X-chromosome-bearing sperm to recipient females. Semen samples (n = 5) collected from a single male using an artificial vagina were extended in electrolyte-free solution (glucose–BSA) and shipped overnight in an Equitainer (4�C) to the sorting facility. Upon arrival, sperm were stained (9 µm Hoechst 33 342; 75 � 106 sperm mL–1), adjusted to 50 � 106 sperm mL–1 with 4% egg yolk TALP containing 0.002% food dye, and sorted on an SX MoFlo� flow cytometer (Dako, Fort Collins, CO, USA). Purities from resort analysis averaged 94% (X) and 83% (Y). After sorting, sperm were concentrated by centrifugation, suspended in TEST-yolk buffer (Irvine Scientific, Santa Ana, CA, USA), and return-shipped to the IVF lab where they were received 48 h after collection. In vivo-matured oocytes recovered from gonadotropin-treated donors were inseminated in vitro with X-chromosome-bearing sperm. In vitro-matured oocytes obtained from ovaries donated by local clinics were inseminated in vitro with control (cooled/shipped/non-sorted) or X- orY-chromosome-bearing sperm. At 5 h or 18 h post-insemination, in vivo- and in vitro-matured oocytes, respectively, were rinsed and placed in IVC-1 medium (Pope et al. 2006 Theriogenology 66, 59–71). Embryos produced from in vitro-matured oocytes were allowed to develop until Day 8 in a three-step culture system (Pope et al. 2006). Cleavage frequency of in vitro-matured oocytes after insemination with X-, Y-, and control sperm was 33% (40/120), 35% (52/150), and 42% (48/115), and blastocyst development was 50% (11/22), 55% (23/42), and 53% (21/40), respectively (P > 0.05). Incidence of cleavage after insemination of in vivo-matured oocytes with X-sperm was 62% (54/87). On Day 2, 45 embryos (9–16 per recipient) produced by in vitro insemination of in vivo-matured oocytes with X-sperm were transferred by laparoscopy to the oviducts of four Day 1 gonadotropin-treated recipients. Three recipients established pregnancies and delivered litters of one, four, and seven female kittens between Day 62 and Day 66 of gestation. We have demonstrated that sperm-sorting technology can be applied and used effectively in domestic cats and, potentially, should be relevant to the selective breeding of endangered cats.
280 ISOLATION, PRESERVATION, AND CHARACTERIZATION OF EQUINE UMBILICAL CORD BLOOD STEM CELLS
Authors: De Schauwer C. , Piepers S. , Hoogewijs M. K. , Govaere J. L. J. , Rijsselaere T. , Demeyere K. , Meyer E. , Van Soom A.
Published: January 2008

The isolation, preservation, and identification of hematopoietic and mesenchymal stem cells from fresh umbilical cord blood (UCB) has been extensively reported in humans. Although both types of stem cells may be of therapeutic interest in horses, data on equine UCB cells are scarce. In the present study, two separation methods to isolate stem and progenitor cells from equine UCB and two cryoprotectant solutions for their subsequent freezing were compared. Characterization of the isolated cells was evaluated flow cytometrically, based on the presence of the cytosolic enzyme aldehyde dehydrogenase (ALDH), which has been shown to be highly expressed in primitive hematopoietic cells in a number of species. Cord blood was collected from 15 foals immediately after birth. While the placenta was still in utero, the umbilical cord was clamped and disinfected. A sterile blood bag collection system containing citrate-phosphate-dextrose-adenine anticoagulant was used to collect the UCB by gravity. The UCB units were stored at 4�C and processed within 36 h. Percoll density gradient separation and rouleaux formation induced by hydroxyethyl starch (HES) were tested in parallel on equal volumes of each UCB unit. The enriched progenitor cell fraction was cryopreserved at 10 � 106 nucleated cells mL–1 using two cryoprotectant solutions based on plasma or RPMI 1640, and both containing 10% DMSO and DNase I (20 IU mL–1). Before and after thawing, cells were labeled using a fluorescent ALDH substrate (Aldefluor�, StemCell Technologies SARL, Grenoble, France) including a negative control. Cell viability was simultaneously evaluated by means of exclusion of propidium iodide. Cryopreservation was performed using a programmable freezer (–1�C/min–1 until –70�C, then –10�C/min–1 until –140�C) prior to storage in liquid nitrogen. Results were analyzed statistically with a nonparametric Mann-Whitney test. The concentration of the isolated UCB cells ranged from 0.3 to 4 � 106 cells mL–1 for Percoll and from 0.4 to 7.3 � 106 cells mL–1 for HES. The average viability before and after freezing was 94% and 93% for Percoll-, and 93% and 94% for HES-separated cells, respectively. No significant differences in concentration or in viability were observed between both isolation procedures and both cryoprotectant solutions. Before freezing, the proportion of Aldefluor�-positive cells after Percoll and HES isolation ranged between 0.5 and 38% and between 1 and 60.5%, respectively. No significant differences were found. In conclusion, the percentage of ALDH-positive cells as determined by flow cytometry was highly variable between foals, but was independent of the isolation procedures used. Whether the isolated cells represent true progenitor cells remains to be confirmed. Ongoing, flow cytometrical experiments showed that the isolated cells are CD29+ and CD44+, which may be indicative for their mesenchymal origin.
281 USE OF MESENCHYMAL STEM CELLS COLLECTED FROM BONE MARROW OF ADULT DOGS FOR THE TREATMENT OF NON-UNION BONE FRACTURES
Authors: Dores C. B. , Lima-Neto J. F. , Pereira-Junior O. C. M. , Rascado T. S. , Passarelli D. , Lorena S. E. R. S. , Rahal S. C. , Landim-Alvarenga F. C.
Published: January 2008

The mesenchymal stem cells (MSC) are multipotent cells present in the bone marrow. The plasticity of these cells allows them to be used in cell therapy since they have the potential to replicate as undifferentiated cells and can be induced to differentiate into bone, fat, cartilage, tendon, muscle, and other tissues. The establishment of a pattern to culture MSC is the first step to start further experiments including MSC differentiation, cell therapy, and autologous transplants. In the present study, three dogs presenting non-union bone fractures, with one to two years of evolution, one in the middle third of the femur and the other two on the distal third of the radius and the ulna, were submitted to stem cell transplantation. After general anesthesia (IV) MSCs were aspirated from the head of the humerus from each dog and centrifuged at 1500 rpm for 10 minutes to eliminate the serum and the fat. The material was resuspended in a 1/1 proportion with DMEM High Glucose (GIBCO, Grand Island, NY, USA) and centrifuged with 7 mL of Ficoll-Paque (density 1.077 g mL–1; Amersham Biosciences, Sao Paulo, Brazil) at 1500 rpm for 40 min. The middle ring formed was aspirated and washed in DMEM High Glucose. The pellet was resuspended in DMEM High glucose with 20% fetal calf serum, penicillin, amphotericin B, and streptomycin. Primary cultures were established and subcultivated for as many as 4 passages. MSCs were cultured in humidified incubators with 5% CO2 in air and allowed to adhere for 120 h, followed by media change every 3 to 4 days. When cultures reached more than 90% confluence, adherent cells were detached with 0.05% trypsin-EDTA (GIBCO) and replanted (passage) at a density of 2 � 106 per 175 cm2 flask. To confirm the lineage of the MSCs, anti-vimentin immunocytochemistry was performed. After about 15 days of culture the cells were resuspended at a concentration of 2 � 107 cells mL–1 and prepared for transfer. The animals were subjected to a surgical procedure where the metallic implant (n = 3) and the fibrous tissue present in the non-union fracture site were removed and the fracture was stabilized with the use of steel bone plate and screws. At the end of the fracture stabilization, the cells were transferred directly into the fracture site. Radiographic exams were performed on the post-surgical site immediately after the surgery and monthly until complete bone healing, which was considered satisfactory 5 to 8 months after the surgery in all animals. The clinical results indicate that the therapy with homologous mesenchymal stem cells is a promising and efficient method to treat non-union bone fractures in dogs.
This work was supported by FAPESP (grants 06/54575-0 and 06/56738-4).
282 CHARACTERIZATION OF THE DEVELOPMENTAL POTENTIAL OF FETAL SOMATIC STEM CELLS ISOLATED FROM DIFFERENT TISSUES
Authors: Hornen N. , Kues W. A. , Niemann H.
Published: January 2008

Recently we discovered a novel type of stem cells which can be derived from primary cultures of fetal connective tissue using a high-density culture (HD) system (Kues et al. 2005 Biol. Reprod. 70, 1020–1028). This cell type shows most of the characteristics of true embryonic stem cells and is a promising alternative for developing therapeutically useful cells. The goal of the present study was to analyze and characterize stem cell populations obtained from five different tissues, i.e., brain, heart, lung, liver, and adrenal gland, using this HD culture system. Explant cultures of the double transgenic strain OG2/Rosa26 were established from mouse fetuses at Day 13.5 post-conception. In this strain, cells with pluripotent properties are characterized by the green fluorescence of the green fluorescent protein (GFP) which is under the transcriptional control of the Oct-4 promoter. In a total of five replicates, cells were subcultivated and reseeded at a concentration of 25 000 cells/cm–2. Cell culture conditions were as recently reported for fetal somatic stem cell derivation (Kues et al. 2005 Biol. Reprod. 72, 1020–1028). After colony formation and/or observation of green fluorescence under UV illumination, mRNA was isolated for RT-PCR analysis of the expression of genes known to be involved in pluripotency, i.e., Oct-4, Nanog, Stat3, TNAP, Rex1, and Sox2. Under these culture conditions, 12.5% of the liver and 80% of the brain explants showed senescence and could not be seeded at the required concentration. No colony formation was observed in brain and heart cultures. Cells from heart explants showed a three-dimensional growth over the whole surface of the attached cells and were difficult to separate. On average, colony formation was observed after 11 days � 8 (mean � SD) (fetal fibroblasts), 10 days � 7 (lung), 18 days � 11 (liver), and 4 days � 1 (adrenal gland). Green fluorescence was detected after 17 days � 9 (fetal fibroblasts), 17 days � 9 (lung), 35 days � 13 (heart, randomly distributed), 26 days � 10 (brain), 26 days � 8 (liver), and 8 days � 5 (adrenal gland). A single large clump of stem-like cells formed in 43% of the fetal fibroblast, 35% of the lung, and 41% of the liver derivatives. Oct-4 expression was not observed in any of the cultures. Nanog and Stat3 were expressed in all cell cultures, and TNAP was expressed in all cultures from heart, brain, lung, and adrenal gland. Rex1 was expressed in all cultures from brain and adrenal gland, and in 25% of the heart cultures and in 16.6% of the fetal fibroblasts. Sox2 was expressed in all cultures from brain and in 16.6% of fetal fibroblast cultures. These preliminary results show that cells with some of the typical features of stem cells can be derived from various tissues of the body, but that the efficiency is different among tissues. Only cells derived from the adrenal gland exhibited better colony formation than the original fibroblasts.
This work was funded by DFG grant Ni 256/28-1.