B2 Medium Research Articles

Effect of Periodontal Disease-associated Bacteria on the Formation of Dental Calculus: An In Vitro Study

Aim: To investigate whether bacteria that play a major role in periodontal disease pathology and in the formation of dental plaque also affect the formation of dental calculus, which is a predisposing factor for the initiation and progression of periodontal diseases. Materials and Methods: This was an in vitro study, and cultures of bacteria were obtained from the American Type Culture Collection and Department of Biology, Faculty of Science, Atatürk University. Young cultures of bacteria of Streptococcus mutans ( S. mutans), Streptococcus sanguinis ( S. sanguinis), Streptococcus gordonii ( S. gordonii), Aggregatibacter actinomycetemcomitans ( A. actinomycetemcomitans), Porphyromonas gingivalis ( P. gingivalis), Fusobacterium nucleatum ( F. nucleatum), and Corynebacterium matruchotii ( C. matruchotii) were prepared in media containing their specific enriching factors. B2 solid, B4 solid, and B2 liquid media were used to determine active calcification, whereas the mineral salt basal (MSB) medium was used to observe passive calcification. Calcification in the media was measured under light microscopy and in MSB using a spectrophotometer and was recorded as the percent transmittance. Results: S. mutans, S. sanguinis, and S. gordonii showed calcification in the B2 medium. S. mutans, S. sanguinis, S. gordonii, and C. matruchotii demonstrated calcification in MSB. A. actinomycetemcomitans, P. gingivalis, and F. nucleatum did not show any calcification. Conclusions: It was concluded that streptococci present in dental plaque take part in the formation of dental calculus, whereas periodontopathogens have no role in the formation of dental calculus.

The effect of storage time and egg weight at room temperature on interior quality of hatching egg in Magelang duck

The objective of this study was to examine the effect of storage time and egg weight at room temperature on the interior quality of Magelang duck. The material used were 120 eggs of Magelang duck. The parameters observed were albumen index, egg yolk index and Haugh unit. The 4 x 3 factorial experiment with 2 factors was applied. In which the first factor (A) was the length of egg storage of: a1 = 1 day, a2 = 3 days, a3 = 5 days and a4 = 7 days and the second factor (B) was egg weight of b1 (low = 48,4g – 63,1g) b2 (medium = 63,2g – 73,6g) and b3 (high 73,7g - 81g,). The data were analyzed by the General Linear Model (GLM) procedure of Statistical Analysis System (SAS). The result showed that there was no interaction between storage time and egg weight on quality interior. Storage time was significant different (P<0,05) on albumin index, egg yolk index and Haugh unit. Egg weight had no effect on index albumen, egg yolk index and Haugh unit. In conclusion, the quality of the interior began to decline after being saved 3 days and the optimal egg weight for hatching eggs is medium egg weight.

Evaluation of Pertussis Toxin Expression in B2 and THIJS Media.

Whole-cell pertussis vaccine (wP) has been imperative and highly effective in preventing childhood deaths due to pertussis. Pertussis toxin is one of the virulence factors of Bordetella pertussis in all available pertussis vaccines. wP production in Razi Vaccine and Serum Research Institute is according to bioreactor culture of B. pertussis strains in B2 medium. The aim of this study was to evaluate B. pertussis strain 509 PT production in B2 and Thalen-IJssel (THIJS) media by Chinese Hamster Ovary (CHO) cell and enzyme-linked immunosorbent assay methods (ELISA). In the current study, B. pertussis strain 509 was cultured in B2 and THIJS media. Six samples were taken during the log growth phase within 2-3 h intervals (triplicate). The growth rate was calculated using opacity and the quantification of cell-associated and released PT measured by ELISA and CHO cell assays. THIJS medium was significantly showed an increase in the bacterial growth rate. During the first 29 h, bacterial concentrations in B2 and THIJS culture medium were 19 and 29 IOU, respectively. In THIJS medium, greater amount of pertussistoxin production was cell-associated. In B2 medium, maximum cell-associated toxin by ELISA and CHO cell assays were in the ODs of 1.1 and 0.9 and for THIJS medium in the ODs of 1.6 and 1.1, respectively. B. pertussis strain 509 in THIJS medium produced higher cell mass and cell-associated pertussis toxin than that of B2. It can be used for the production of whole-cell vaccine with higher pertussis toxin and accordingly using lower biomass per dose leading to the reduction of vaccine toxicity.

Different pre-implantation phenotypes of bovine blastocysts produced in vitro.

Embryo transfer in cattle is performed with blastocysts produced in vivo or in vitro using defined media. However, outdated systems such as those that use serum and co-culture remain of interest for research purposes. Here, we investigated the effect of additional culture time on in vitro-produced embryos. Specifically, we compared embryos that formed a blastocoel at different times after fertilisation to those that stayed in culture for up to two additional days with respect to their development in vivo after temporary transfer to oestrus-synchronised recipients. A pre-transfer set (D6, D6+1, D6+2, D7, D7+1, D8) was examined using microarray analyses and correlated with a post-transfer set that included two different days of transfer (D6-T6, D6+2-T8, D7+1-T8, D8-T8). All surviving conceptuses reached primitive-streak stages and filamentous sizes similarly to in vivo (D18) or in vitro controls (D7/T7). The recovery rate differed between D6 and D8 embryos that were immediately transferred (58 vs 25%). With an intermediate survival rate (33%), the D6 embryos with two additional days in culture produced nine times more IFN-tau (IFNT) at D18 than the D6 embryos that were immediately transferred. At the end of culture, D6 and D6+2 embryos displayed the highest number of gene expression differences. Despite a mortality of 40–60%, no signature was detectable in any of the transferred groups that would account for the embryos’ fates. Initially reputed to be beneficial in producing more blastocysts, our culture system of B2 medium plus serum and co-culture generated blastocysts that were distinct from those developed in vivo (D7).

Electrical activation of rabbit oocytes increases fertilization and embryo development by intracytoplasmic sperm injection using sperm from deceased male

We investigated the effect of electrical stimulation on rabbit oocyte activation using intracytoplasmic sperm injection (ICSI) to determine whether viable offspring can be produced from deceased rabbit sperm using ICSI. Sperm were collected from a heterozygote GFP male rabbit 5h after sacrifice and cryopreserved in liquid nitrogen. Mature oocytes were fertilized using ICSI. A series of electrical pulse procedures were used to activate oocytes before and/or after ICSI. Following ICSI, zygotes were cultured in B2 medium for 4days or transferred into the oviducts of recipient rabbits at the 2- or 4-cell stage. The blastocyst formation rate was significantly greater in oocytes that received one or two pulses prior to ICSI compared to controls and other electrically stimulated groups. In the single pulse before ICSI group, 23% of the blastocysts expressed GFP, which was significantly greater than all other groups. However, those that received treatment before and after, or just following ICSI, showed a significant decrease in embryo survival. Finally, embryos from the single pulse before ICSI group were transferred into recipient female rabbits and a full-term kit was successfully delivered. One pulse of electrical stimulation prior to sperm injection was an effective method to activate rabbit oocytes for fertilization. Sperm collected from a deceased rabbit is able to produce viable embryos through ISCI that are capable of normal fetal and kit development.

222 EFFECT OF THE PRESENCE OR ABSENCE OF PERCOLL CENTRIFUGATION; PENICILLAMINE, HYPOTAURINE, AND EPINEPHRINE; AND HEPARIN ON IN VITRO PRODUCTION OF BOVINE EMBRYOS

Protocols for the production of bovine embryos in vitro routinely include Percoll centrifugation of semen, usually include heparin, and often include penicillamine, hypotaurine, and epinephrine (PHE) in the fertilization media. This study examined the contribution of each of these components to the success of in vitro fertilization of bovine oocytes and subsequent blastocyst development. Bovine oocytes were aspirated from 2- to 10-mm follicles within 5 h after slaughter of cattle at a local abattoir. Groups of 30 to 40 cumulus–oocyte complexes (COC) were matured in 0.5 mL of TCM-199 with 10% FCS, 4 µg mL–1 of FSH, and 6 µg mL–1 of LH (NOBL Laboratories, Sioux Center, IA, USA) for 24 h (39°C, 4% CO2 in air). The COC were then washed and placed in 0.5 mL of modified Tyrode-lactate medium for IVF with various combinations of 2 µg mL–1 of heparin, 20 µM penicillamine, 10 µM hypotaurine, and 1 µM epinephrine. Each group of COC was inseminated with 0.25 × 106 frozen–thawed sperm from a single bull after 30 min of centrifugation with (Exp. 1) or without (Exp. 2) a 45/90% Percoll gradient with sperm TALP. Oocytes were vortexed to remove the cumulus after 18 h and placed in co-culture wells containing a monolayer of buffalo rat liver cells and 0.5 mL of Menezo’s B2 medium supplemented with 10% FCS. On the fourth day of in vitro culture, cleavage was defined as 2 cells or greater and embryos were transferred to fresh co-culture wells. There were 4 replicates in the first experiment and 6 in the second. Data were analysed by ANOVA. In the first experiment, the use of a Percoll gradient during centrifugation for separation of viable sperm from seminal plasma and cryprotectants resulted in significantly higher cleavage and Day 8 blastocyst rates than did the absence of Percoll when PHE and heparin were used together, and both cleavage and blastocyst rates were lower when only PHE or heparin was used separately compared with when both were used together (Table 1). The absence of Percoll, PHE, and heparin resulted in the lowest rates of cleavage and development. In the second experiment, the absence of either PHE or heparin resulted in lower cleavage rates, but not blastocyst rates, compared with the use of both, and the absence of both resulted in the lowest cleavage and blastocyst rates in spite of the use of Percoll. Table 1.Effects of Percoll; penicillamine, hypotaurine, and epinephrine (PHE); and heparin on cleavage and subsequent embryo development per oocyte

Differential Effect of Medium on the Ratio of ICM/TE of Bovine Embryos in a Co-culture System

Background This study was undertaken to investigate the efficiency of two different embryo somatic cell co-culture conditions tissue culture medium 199 (TCM199)–vero cells and Menezo B2 (B2)-vero cells for the in vitro developmental quantity and quality of bovine embryos. Materials and methods Bovine oocytes were allowed to mature and subsequently undergo fertilization in vitro. Their presumptive zygotes were cultured in either TCM199 or B2 culture media in conjunction with vero cells for up to nine days. The culture media were refreshed every two days and the proportion of embryos that cleaved and further developed to the morula and blastocyst (early expand and hatched) stages were recorded. Hatched blastocysts underwent differential staining in order to determine the numbers of inner cell mass (ICM) and tropho ectoderm (TE) and total cell number (TCN). Results Of the two groups no significant difference was seen between the proportions of the presumptive zygotes cleaved those which developed to 8-16 cells morula and reached days 7or 8 blastocyst stage or hatched. However the values for TCN and TE of the TCM199- vero embryos were significantly greater than those of B2-vero embryos. The values for ICM/ TCN and ICM/TE were significantly greater in the B2-vero group versus the TCM199-vero group. Conclusion Both TCM199 and B2 culture media in conjunction with vero cells were of the same efficiency when used for in vitro development of bovine presumptive zygotes. However TCM199 was superior in providing embryos with more embryo cell numbers whereas B2 medium was superior in providing embryos with greater ICM/TE and ICM/ TCN ratios.

443 BIOLOGICAL FACTORS AFFECTING OOCYTE COLLECTION AND EMBRYOS PRODUCTION IN A COMMERCIAL OVUM PICKUP-IVP SYSTEM IN HOLSTEIN AND MONTBELIARD BREEDS

The aim of this work was to identify biological factors affecting oocyte collection, embryo production, and subsequent pregnancy rates through a retrospective study conducted from 381 commercial ovum pickup (OPU)-IVP sessions performed on high genetic Holstein and Montbeliard donors. Oocytes were retrieved using an ultrasound scanner SC 200 (Pie Medical, Maastricht, the Netherlands) equipped with a 7.5-MHz annular-array transducer. Donors were superovulated at Day 12 of presynchronized cycle with FSH (Stimufol®, Rhone-Merieux, Lyon, France) divided in 5 decreasing doses over 2.5 days. Cumulus-oocyte complexes (COCs) were collected 12h after last FSH injection and matured for 22 h in TCM-199 HEPES plus FCS, FSH, estradiol, and EGF They were then fertilized in fert-TALP with frozen-thawed semen. Zygotes were cultured for 6 days on a monolayer of Vero cells in B2 medium. Day 7 embryos were transferred as fresh into recipients. The effects of donor breed, dominant follicle puncture (DFP), and physiological state of females on oocyte and embryo production were analyzed by ANOVA (proc GLM, SAS Institute, Cary, NC, USA). From all collected animals, most (80.6%) of the donors were cows (55% collected as early pregnant and 31.9% previously infertile) and 19.4% were heifers (from those 86.5% pregnant). The mean number of collected COCs per session was 13.3 ± 8.3 (mean ± SD), 57.8% of which had ≥3 layers of cumulus cells (grades 1 and 2). A mean of 10.8 ± 6.9 COCs were subjected to IVM-IVF-IVC, which resulted in 3.5 ± 3.5 blastocysts per session (77% were graded as 1 and 2) whereas 2.8 ± 2.5 embryos were transferred into recipients. An overall pregnancy rate of 50.4% (544/1080) was obtained at Day 90: 59.1%, 50.4%, and 27.9% for grade 1, grade 2, and grade 3 blastocysts, respectively. The OPU sessions resulted in at least one Day 90 pregnancy in 61.2% of the cases. Breed had no effect on the number of collected COCs but a higher percentage of grade 1 and 2 COCs were observed for Montbeliard than for Holstein donors (60 v. 53%). Dominant follicle puncture 48 h prior to superovulation treatment significantly increased the number of COCs (15.7 ± 9.9 v. 10.9 ± 5.6; P < 0.05), the number of developed and transferred embryos and the number of pregnancies per session (4 ± 33.9 v. 7 ± 2.8; 3.3 ± 2.6 v. 2.4 ± 2.4; 1.7 ± 1.6 v. 1.2 ± 1.4, respectively). This effect was particularly impressive for Montbeliard heifers. Higher numbers of COCs were collected from infertile cows, nonpregnant cows as for early pregnant heifers (16.9 ± 10.4; 13.8 ± 7.6, and 13.8 ± 8.6, respectively) when compared with pregnant cows and nonpregnant heifers (11.0 ± 6.1; 10.5 ± 6.6; P < 0.05). The mean number of developed embryos was also influenced by parity of the donor and was 4.5 ± 4.5; 3.4 ± 3.7; 3.1 ± 2.8 for infertile, nonpregnant, and pregnant cows and 3.0 ± 3.4 and 2.5 ± 2.3 for pregnant and nonpregnant heifers, respectively. In conclusion, oocyte collection and embryo production were mainly influenced by parity and physiological status of Holstein and Montbeliard donors. Puncture of dominant follicle at the start of superovulation treatment increased the overall quantity of COCs and improved the efficiency of the IVP procedure.

98 EIGHT-CELL STAGE DELINEATES CRYOTOLERANCE OF VITRIFIED RABBIT EMBRYOS

A number of groups have successfully vitrified rabbit embryos at morula or blastocyst (BL) stage. For earlier stages (1- to 8-celled), however, there are very limited studies and the results are generally unsatisfactory. In this study, we examined the survival and developmental competence of rabbit embryos vitrified at different preimplantational stages. Sexually matured female New Zealand White (NZW) rabbits were superovulated with our standard protocols, followed by mating with NZW males. At 18h post-hCG treatment, viable fertilized embryos were collected and cultured in 2.5% FBS B2 medium (Laboratories CCD, Paris, France) in 5% CO2 with humidified air at 38.5°C. A total of 691 rabbit embryos at pronuclear, 2-celled, 4-celled, 8-celled, and morula/early blastocyst (BL; Day 3) stages were vitrified by the open pulled straw (OPS) method in HEPES buffered TCM-199 medium containing 20% fetal calf serum, ethylene glycol, and dimethyl sulfoxide. After stored in liquid nitrogen for at least 1 month, embryos were sequentially warmed, rehydrated, and washed before culture in B2 culture medium. Survival and developmental rates were analyzed by general linear model analysis (SPSS 11.0, SPSS Inc., Chicago, IL, USA). Embryos vitrified at 8-celled stage or beyond showed greater survival, and expanded BL and hatching rates than those at pronuclear, 2-celled and 4-celled embryos (Table 1). In particular, the expanded and hatched BL rates were significantly higher in the 8-celled group than those in 4-celled group, suggesting that the 8-celled is the threshold stage to tolerate the vitrification procedure in rabbit embryos. In addition, the rates of expanded and hatched blastocysts in morula/BL group were still significantly higher than those in the 8-celled group. Our results may provide the proper timing of cryopreserving fertilized, transgenic, and/or cloned rabbit embryos at early stages for biomedical research. Table 1.Survival and developmental competence of rabbit embryos vitrified at different stages after thawing This study was supported by NIH1R43 RR023774-01A1 and 5R44HL091605-03.

67 PRODUCTION OF CLONED TRANSGENIC RABBITS FROM MESENCHYMAL STEM CELLS

Mesenchymal stem cells (MSC) are adult stem cells with fibroblast-like morphology, which can be easily isolated from bone marrow and expanded in culture. Mesenchymal stem cells are able to grow from a single cell into a cell clone, which makes them potentially useful for gene targeting. In our recent study we investigated the dynamics of epigenetic reprogramming following nuclear transfer (NT) with MSC and found that these cells can support development of cloned embryos as good as genetically identical fibroblasts (Brero et al. 2009 Cloning Stem Cells 11, 319-329). In the present study we tested whether live cloned rabbits can be produced from MSC. Nuclear donor cells were isolated from a 6-week-old transgenic Ali/Bas rabbit, expanded in culture, and assessed for their differentiation potential. Mesenchymal stem cells were transfected with a green fluorescent protein (GFP) reporter gene construct and stable cell clones were selected (GFP-MSC). The MSC and GFP-MSC were used for NT at passage 3 to 7 after serum starvation for 2 to 4 days. Nuclear transfer was performed essentially as described previously (Yang et al. 2007 Reproduction 133, 219-320). To assess the development to blastocyst, reconstructed embryos were cultured in B2 medium for 5 to 6 days, whereas for in vivo development embryos were cultured only overnight and then transferred into recipients at the 4- to 8-cell stage. In the MSC group, 844 oocytes were used, 793 (94%) of them fused, 698/786 (89%) cleaved, and 48/128 (38%) developed to blastocyst. After transfer of 483 cloned embryos into 13 recipients, 2 from 8 pregnant recipients gave birth to 10 (2.4%) rabbits, from which 2 and 1 survived for more than 7 days and 3 months, respectively. In the GFP-MSC group, 444 oocytes were used, 412 (93%) of them fused, 377/409 (92%) cleaved, and 97/178 (55%) developed to blastocyst. Transfer of 216 cloned embryos into 8 recipients resulted in 4 pregnancies. One recipient gave birth to 6 (3.7%) live and 2 stillborn rabbits, from which 2 and 1 rabbits survived for more than 3 days and 2 weeks, respectively. All cloned rabbits carried a GFP gene, and green fluorescence could be detected in the follicles of the skin under a fluorescence microscope (Zeiss Axiovert200, Carl Zeiss, Germany). Our study demonstrates that live cloned rabbits can be produced from genetically modified MSC, thus paving the way to generate gene targeted animals. This work is supported by Roche Diagnostic GmbH.

81 THE EFFECT OF FOLLICULAR AND OVIDUCT OOCYTES ON THE DEVELOPMENT OF RABBIT NUCLEAR TRANSFERRED EMBRYOS IN VITRO

This study was designed to examine the effect of rabbit oocytes collected from oviducts v. follicles on the developmental potential of nuclear transplant (NT) embryos. Rabbit oocytes were flushed from the oviducts (oviduct oocytes) or collected from the ovarian Graafian follicles(follicular oocytes) of superovulated does at 12 h post-hCG injection (hpi). Cumulus cells were then removed from the oocytes by incubation in 0.5% hyaluronidase and pipetting. Oocyte enucleation was conducted in TCM-199 +10% fetal bovine serum (FBS) and confirmed under fluorescent microscopy. Skin fibroblasts from an adult rabbit were prepared and cultured to passage 8 to 10 before use as nuclear donors. A donor cell with a diameter of approximately 15 to 19 μm was transferred into the perivitelline space of an enucleated oocyte and subsequently fused with the recipient oocyte by applying 3 direct current pulses at 3.2 kV cm-1 for 20 μs per pulse. Fused oocytes were activated by the same electrical stimulation described above, and then cultured in TCM-199 + 10% FBS containing 2.0 mM 6-DMAP and 5 μg mL-1 cycloheximide for 1 h. Cloned embryos were cultured in 2.5% FBS B2 medium in 5% CO2 and 95% humidified air at 38.5°C for 3 d. Embryo development to cleavage (2- to 4-cell), 8-cell, and morula/blastocyst (Mor/BL) stages was evaluated. The data were analyzed by the General Linear Model procedure (SPSS 11.0, SPSS Inc., Chicago, IL, USA).The total number of oocytes collected per animal was 27.6 ± 1.3, with 47.8% from oviducts, and 52.2% from follicles. The percentage of oviduct oocytes that showed the first polar body was 98.3% (n = 150) at the time of collection, whereas follicular oocytes only had 54.8% at collection (n = 93), but it reached 92.4% when immature follicular oocytes were cultured for 3 h in vitro. The enucleation rates were similar between the follicular (82.7%) and the oviduct (79.1%) groups. Table 1 shows that a significantly higher fusion rate was found in follicular oocytes compared with that in the oviduct group (90.8 v. 63.4%; P < 0.05). There was no difference in the cleavage rate and Mor/BL development between the 2 groups, although the 8-cell(78.4 v. 63.9%; P = 0.11) and the overall efficiencies (30.6% v. 17.9%; P = 0.14) appeared higher in the follicular group. These results demonstrated that rabbit follicular oocytes at 12 hpi have potential equivalent or maybe better (fusion) than that with oviduct oocytes for promoting the preimplantational development of NT embryos. Table 1.The effect of follicular and oviduct oocytes on the development of rabbit NT embryos Supported by NIH1R43 RR023774-01A1 and 5R44HL091605-03.

333 EFFECTS OF BOVINE OOCYTE QUALITY ON KINETICS OF NUCLEAR MATURATION AND EMBRYONIC DEVELOPMENT AFTER IN VITRO FERTILIZATION

Many factors act together to prepare the immature oocyte for successful development to a competent embryo after fertilization. Defects in oocyte maturation and further development can possibly be caused by the oocyte quality or an inadequate nuclear maturation or even by a failure of both. In the present study, the effect of COCs quality on meiotic development and further embryo-development after in vitro fertilization was evaluated. A total of 3604 COCs was separated according to their morphological aspect and were classified as A, B, and C categories. Briefly, in class A, oocytes possessed compact layers of cumulus cells, being difficult to evaluate their number having a homogenous ooplasm with uniform color. In class B, oocytes show more or equal to five layers of cumulus cells, easily identifiable under a stereomicroscope and/or granulations in the ooplasm. In class C, some granulation was observed in oocytes with about three layers of cumulus cells. The total number of oocytes was divided into two groups (I and II) in which in the group I, COCs (n = 540) were fixed 0, 6, 12, 18, 24, and 30 h following ovarian aspiration, DNA was stained with aceto-orcein, and the nucleus were observed under a phase contrast microscope. In the Group II, COCs (n = 3064) were fertilized with frozen/thawed bull semen after 24 h of maturation, which was made in M199 medium (Sigma, St, Louis, MO, USA). The development of the embryos was evaluated on the third and seventh day after fertilization. Embryos were co-cultured with monolayers of granulosa cells in 45 μL droplets of B2 medium (CCD Laboratory, Paris, France), supplemented with 10% serum under mineral oil, at 39°C and 5% CO2 in air. It was observed that, other than the oocytes achieved metaphase II at 24 h was greater for the oocytes classified as A (65.4%), and B (61.0%) greater than C (51.2%), no statistical difference was observed between oocyte quality and capability to maturation. As far as the embryonic development is concerned, the same tendency was observed for the cleavage and for the morulae/blastocyst stage after 7 days after fertilization (P < 0.001). The percentages of cleaved oocytes classified as A, B, and C, were respectively 65.2%, 58.4%, and 48.0%. The development to the morulae/blastocyst stage of the cleaved embryos was A = 38.5%/27.4%, B = 33.6%/25.0%, and C = 30.9%/17.2% (Table 1). The results of our study clearly demonstrate that the morphology of the oocytes plays an important role on the in vitro embryonic developmental competence after fertilization. Table 1.Development of oocytes according to COCs quality, evaluated 3 and 7 days after fertilization The first author is supported by the Regional Foundation for Science and Technology of the Azores Government. This study was supported by the IBBA Institute grant number M2.1.2/I/022/2008 CITA-A is fully acknowledged.

Assessment of climate change impacts on the hydrology of Gilgel Abay catchment in Lake Tana basin, Ethiopia

AbstractIn this study, large‐scale atmospheric variables are downscaled to meteorological variables at local scale for the daily time step to assess hydrological impacts by climate changes. Large‐scale atmospheric modelling was by the HadCM3 General Circulation Model (GCM) while downscaling and water balance modelling was through the Statistical DownScaling Model and the HBV semi‐distributed rainfall‐runoff model, respectively. The area of study was the Gilgel Abay catchment that drains in Lake Tana. A selection of large‐scale atmospheric variables by the HadCM3 GCM are downscaled by a multiple linear regression model, were minimum and maximum temperature and precipitation for future time horizons are calculated. Climate scenarios as developed for the A2 (medium–high emission) and B2 (medium–low emission) scenarios for a 100‐year period based on the mean of 20 ensembles have been selected for this study. In addition, a synthetic incremental scenario was tested for a wide range of changes in climatic variables. Stream flow simulations by the HBV model were carried out for the 2020s (2011–2040), 2050s (2041–2070) and 2080s (2071–2099) to define hydrologic impacts. The result of downscaled precipitation reveals that precipitation does not manifest a systematic increase or decrease in all future time horizons for both A2 and B2 scenarios unlike that of minimum and maximum temperature and related evaporation. For the future horizons significant changes and variations in the seasonal and monthly flows are to be expected and for the 2080s the runoff volume in the rainy season will reduce by approximately 11·6 and 10·1% for the A2 and B2 scenarios. Results from synthetic incremental scenarios also indicate sensitivities to climate change. As much as 33% of the seasonal and annual runoff is expected to reduce when temperature increases by 2 °C and when rainfall decreases by approximately 20%. Copyright © 2009 John Wiley & Sons, Ltd.

Improved Efficiency of Bovine Somatic Cell Nuclear Transfer by Optimizing Operational Procedures

To improve bovine somatic cell nuclear transfer (SCNT) efficiency, we studied various aspects to optimize the experimental procedures. Firstly, donor cells were treated with pronase, which resulted in a higher fusion rate than that of cells without the pronase treatment (78.3 vs. 53.9%). Secondly, when fused embryos were activated either by chemical (ionomycin + cyclohemixide (CHX)) or electrical + CHX stimulation, the cleavage and blastocyst formation rates were comparable amongst these treatment groups (P>0.05); however, mortality following electrical + CHX activation was significantly higher than that observed with the chemical activation, regardless of the pronase treatment (P<0.05). Finally, we compared the culture conditions of the reconstructed embryos using ACM medium plus mouse embryonic fibroblasts (MEF) vs. B2 medium plus granulose cells (GC), and the results clearly demonstrated that the former culture conditions led to a higher blastocyst rate, 90-day pregnancy rate, and newborn rate, than that observed for culture in B2 medium plus GC (46.7 vs. 34.7%, 36.1 vs. 9.6% and 25.9 vs. 5.8% for the blastocyst, pregnancy and newborn rates, respectively). In summary, the efficiency of bovine SCNT can be greatly improved using optimized operational procedures, including treating the donor cells with pronase, activation of fused embryos by ionomycin + CHX and the culture of the reconstructed embryos in ACM + MEF media.

51 DEVELOPMENT OF EQUINE CLONED EMBRYOS DERIVED FROM IN VITRO-MATURED OOCYTES RECEIVING ADULT DERMAL FIBROBLAST CELL NUCLEI

The purpose of our study was to determine the in vitro developmental competences of equine NT embryos reconstructed with adult dermal fibroblast cells. Frozen/thawed fibroblast cells, whose mitotic cycle had been synchronized at G1/G0 stages through a contact inhibition of their migration and proliferative activity under total confluency, were used as a source of nuclear donor cells in the somatic cell cloning procedure. In vitro-matured oocytes were used as recipient cells for fibroblast cell nuclei. The compact cumulus–oocyte complexes (cpCOCs) were collected from abattoir-derived mare ovaries and selected for in vitro maturation. The cpCOCs were cultured in TC-199 medium supplemented with 5 mU mL–1 follicle-stimulating hormone (FSH), 10% fetal bovine serum (FBS) and 75 μg mL–1 kanamycin monosulfate (kanamycin A) for 30 h at 38.2°C in a 100% water-saturated atmosphere of 5% CO2 and 95% air. Cumulus-denuded in vitro-matured oocytes were incubated in the maturation medium supplemented with 0.4 μg mL–1 demecolcine for 40 min. The treated oocytes were subsequently transferred into TC-199 medium containing 4 mg mL–1 BSA-V and 5 μg mL–1 cytochalasin B. Metaphase chromosomes, which had been allocated into the chemically-induced protrusion of the plasma membrane, were removed microsurgically. The chemically-assisted enucleation was accomplished by gently aspirating the ooplasmic cone, which contained the condensed chromosome mass, with the aid of a beveled micropipette. The single nuclear donor cells were inserted into perivitelline space of previously enucleated oocytes. Fibroblast cell-ooplast couplets were fused with two consecutive DC pulses of 2.4 kV cm–1 for 30 μs. After a 1.5-h delay, nuclear transfer-derived oocytes were chemically activated by exposure to 5 μm L–1 calcium ionomycin for 5 to 7 min, followed by their incubation in B2 medium with addition of 2 mm L–1 6-dimethylaminopurine (6-DMAP) for 4 h. Reconstructed embryos were in vitro cultured in B2 medium for 2 days. Afterwards, cleaved embryos were co-cultured with Vero cells in B2 medium supplemented with 10% FBS for 5 to 6 days up to morula/blastocyst stages. From among 88 in vitro cultured cpCOCs, 55 (62.5%) acquired meiotic nuclear and cytoplasmic maturity state after reaching the Metaphase II stage. A total of 55 enucleated oocytes underwent reconstruction and 44/55 (80.0%) were successfully fused with nuclear donor cells. Out of 44 cultured NT embryos, 21 (47.7%) were cleaved. The frequencies of cloned embryos that reached the morula and blastocyst stages were 6/44 (13.6%) and 3/44 (6.8%), respectively. In conclusion, the cell nuclei of in vitro cultured adult dermal fibroblast cells, which had undergone the contact inhibition, were able to direct the preimplantation development of equine cloned embryos to morula and blastocyst stages. This work was supported by the Scientific Net of Animal Reproduction Biotechnology.

Analysis of bovine sexed sperm for IVF from sorting to the embryo

The objective of this study was to characterize bovine semen parameters and determine the best IVF conditions to produce a maximal percentage of blastocysts. Four types of semen were analyzed with CASA and flow cytometry: fresh and frozen non-sexed semen; fresh and frozen sexed semen. Semen was obtained from four Holstein bulls and two ejaculates from each bull were analyzed. Oocytes from slaughterhouse ovaries were matured and fertilized in vitro with all types of semen (for sexed semen, 2, 5 or 10 μg/mL heparin was added to the IVF media while for non-sexed semen, 10 μg/mL was added in the IVF medium). Presumptive zygotes were co-cultured with Buffalo rat liver cells in Menezo's B2 medium, and cleavage rates at Day 2, and blastocyst rates at Day 7 of culture, were recorded. Sexed semen resulted in fewer blastocysts than non-sexed semen ( P < 0.05), and certain bulls performed better in IVF. Freezing, and not sexing, had a more significant negative effect on semen quality. Compromised semen quality due to sexing and/or freezing can explain the reduced in vitro blastocyst rates when using frozen–thawed sexed semen. Sexed semen that appeared more capacitated seemed to require less heparin in IVF than sexed semen that appeared less capacitated to produce a maximal percentage of blastocyst. Flow cytometry sorting eliminates spermatozoa that possess compromised DNA, and therefore the reduced fertility seen in vitro is not due to an increased percentage of spermatozoa with compromised DNA. This study describes tools that can monitor semen parameters to optimize IVF conditions and thus obtain maximal blastocyst rates.

102 IDENTIFICATION OF CONTRASTED PHENOTYPES IN THE BOVINE FROM REPEATED IN VIVO AND IN VITRO EMBRYO PRODUCTION FOLLOWING SUPEROVULATION

This study was initiated to evaluate maternal influence on in vivo and in vitro bovine embryo production and identify animals with contrasted phenotypes for reproductive parameters. Nine Montbéliard cows raised on the same farm and with various genetic origins were included in the study. In vivo-derived embryos were collected nonsurgically from superovulated cows on day 7 after AI (34 collections). Immature oocytes were collected by ovum pickup from the same (superovulated) cows (36 sessions) then matured, fertilized (day 0) with the same bull, and cultured in vitro until day 7 on Vero cell monolayers in B2 medium. Grade 1 to 3 in vivo and grade 1 and 2 in vitro produced embryos deemed viable according to IETS criteria. The mean numbers of blastocysts and viable blastocysts per session per cow were, respectively, 8.3 ± 5.5 and 4.8 ± 3.6 in the in vivo system and 2.5 ± 2.6 and 1.8 ± 2.2 in the in vitro system. Individual cow data of in vivo and in vitro embryo production were analyzed by ANOVA (GLM program in SAS; SAS Institute Inc., Cary, NC, USA). Results are presented in Table 1: mean ± SD. Quantity and quality of produced embryos varied significantly among females, and production in vivo and in vitro was not systematically related. Contrasted phenotypes were identified according to their viable blastocyst rates in both systems (in vivo: no viable/recovered; in vitro: no viable/inseminated). Two females presented a relatively high percentage of viable blastocysts in both systems (over 30% in vitro and over 70% in vivo, Table 1). On the contrary, 2 females showed low percentages of blastocysts in the 2 systems (&lt;10% in vitro and &lt;50% in vivo). For most other females, the percentage of in vivo-produced blastocysts was relatively high (&gt;50%), but in vitro development rates were low. Only one female (C3) presented the inverse situation. Oocytes collected from animals with contrasted phenotypes will be analysed for gene expression to identify marker genes associated with oocyte developmental competence. Table 1. This study was conducted with financial support of ‘Genanimal’ – French Ministry of Research (#03P409) and Apis-Gene.

Parthenogenetic development of rabbit oocytes after electrical stimulation

The aim of this study was to determine the effect of electric pulses on the structural and functional condition of rabbit oocytes. The New Zealand White female rabbits at 3&amp;ndash;5 months of age and at 3&amp;ndash;4 kg body weight served as oocyte donors. Oocytes after flushing from the oviducts were placed between two electrodes in an electroporation chamber which was filled with a dielectric solution. Following a short incubation in B2 medium, oocytes were subjected to an electric pulse released by an electrical pulse generator. Oocytes were then incubated in 500 &amp;micro;l of B2 medium supplemented with 20% foetal calf serum (FCS) at 38&amp;deg;C in an atmosphere of 5% CO&lt;sub&gt;2 &lt;/sub&gt;in air. Oocytes were cultured until the morula/blastocyst stage (approx. 72 h). The experiment was conducted using 430 oocytes obtained post mortem. In vitro cultured oocytes not subjected to an electric pulse were the control. Each group was subdivided into replications according to electric current intensity. The analysis of experimental variants shows that in the first variant all embryos developed to the morula stage but only 10% of them continued to develop to the blastocyst stage. In the second variant we observed that 5&amp;ndash;10% of oocytes developed to the blastocyst stage after treatment with 2.0 and 2.5 kV/cm pulse but in the group of 1.0 kV/cm pulse 35% of oocytes developed only to the 2&amp;ndash;12 b stage. In the third variant only 1 oocyte (5%) continued to develop to the blastocyst stage, but in the fourth variant oocyte development stopped at the morula stage. In the fifth variant, called an &amp;ldquo;extreme&amp;rdquo; one, oocytes stopped to develop at the stage of 2&amp;ndash;12 b (about 25%) and the percentage of degenerated oocytes dramatically increased (about 60%). &amp;nbsp;

Transcriptional Regulation of Uterine Vascular Endothelial Growth Factor during Early Gestation in a Carnivore Model, Mustela vison

Vascular endothelial growth factor (VEGF) is an essential angiogenic signaling element that acts through its two tyrosine kinase receptors, inducing both proliferation of endothelial cells and vascular permeability. Given the importance of vasculogenesis and angiogenesis to early pregnancy, it is of interest to understand the mechanisms regulating vascular development at this stage. We previously demonstrated that VEGF and receptors are up-regulated during embryo implantation in an unique animal model, the mink, a species displaying obligate embryonic diapause. Herein we examined the role of prostaglandin E2 (PGE(2)) as a regulator of VEGF during early pregnancy and established the mechanisms of this regulation. We demonstrate that activated embryos secrete PGE(2) and that expression of PGE synthase protein in the uterus is dependent upon direct contact with invading trophoblast cells during implantation. Using mink uterine stromal cells transfected with mink VEGF promoter driving the luciferase reporter gene, we show that PGE(2) induces promoter transactivation and that this response can be eliminated by blockade of protein kinase A. Treatment with antagonists to PGE(2) receptors EP2 and EP4 eliminated the PGE(2)-induced response in transfected cells. Deletional studies of the promoter revealed that a region of 99 bp upstream of the transcription start site is required for PGE(2)-induced transactivation. Mutation of an AP2/Sp1 cluster, found within the 99 bp, completely eliminated the PGE(2) response. Furthermore, chromatin immunoprecipitation assays confirmed binding of the AP2 and Sp1 transcription factors to the endogenous mink VEGF promoter in uterine cells. PGE(2) stimulated acetylation of histone H3 associated with the promoter region containing the AP2/Sp1 cluster. Taken together, these results demonstrate that PGE(2) plays an important role in regulating uterine and thus placental vascular development, acting through its receptors EP2 and EP4, provoking protein kinase A activation of AP2 and Sp1 as well as acetylation of histone H3 to transactivate the VEGF promoter.

362 SEX RATIO OF IN VITRO-PRODUCED BLASTOCYSTS OBTAINED USING FROZEN SEMEN FROM A CLONED BULL OR ITS ORIGINAL CELL DONOR

Bulls obtained by somatic cell nuclear transfer (SCNT) have proved to develop normally, produce sperm, and be fertile (Shiga et al. 2005 Theriogenology 64, 334–343; Tecirlioglu et al. 2006 Theriogenology 65, 1783–1799). However, due to epigenetic variability encountered with clones, it cannot be excluded that major alterations may affect the X chromosome and especially the dosage of X inactivation in female embryos. This could result in a deviation of the sex ratio in offspring. To test this hypothesis, the sperm from a cloned bull was used in IVF to assess its ability to induce a different proportion of male and female embryos when compared to results obtained with the sperm of the original donor before cloning. In the present experiment, semen was collected twice weekly during 3 months from a 3-year-old cloned bull of the Charolais breed (H-2) and frozen-stored. Samples from 3 different ejaculates were used in comparison with control frozen straws from the original bull (H-0), prepared at the same age, to produce in vitro embryos. A total of 6 replicate IVF experiments were performed on the same batches of in vitro-matured oocytes (n = 654) using the standard swim-up and IVF technique in the laboratory. Twenty hours after insemination, presumptive zygotes were vortexed and cultured for 7 days in microdrops of B2 medium with Vero cells. Fertilization, cleavage, and blastocyst formation was assessed, and by Day 7, all of the blastocysts were individually frozen after grading. Two groups of 40 representative blastocysts derived from each bull (clone or donor) were used to determine the sex ratio using the sexing kit developed by UNCEIA R&amp;amp;D (Maisons-Alfort, France). Percentages between groups were compared by chi-square test. Semen from the cloned bull resulted in significantly lower fertilization and cleavage rates than that from the original donor (82.7% and 70% vs. 94.6% and 91%, respectively; P &amp;lt; 0.01), but further in vitro development was not different, as the proportions of blastocysts/cleaved were, respectively, 31.5% (74/235) vs. 38.7% (112/290). Sex determination was achieved in 79/80 of the in vitro-produced embryos and indicated that 55% of the blastocysts derived from the clone were male (22/40) and 45% female (18/40). This was not different from the proportion of male (61.5%, 24/39) and female (38.5%, 15/39) embryos in the group derived from semen of the original donor bull. In conclusion, these preliminary results indicate that the semen from this cloned Charolais bull has the same potential for in vitro embryo production as its original cell donor, and there is no evidence that SCNT could induce any deviation of the sex ratio. This observation needs to be confirmed with other sets of cloned bulls. (Shiga et al. 2005 Theriogenology 64, 334–343. Tecirlioglu et al. 2006 Theriogenology 65, 1783–1799.)