bluuebunny/crossref_metadata_2025 huggingface dataset:
4 IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF HEAT SHOCK PROTEIN 40 IN PIG OVARY
Authors: Pennarossa G. , Maffei S. , Rahman M. M. , Vanelli A. , Berruti G. , Brevini T. A. L. , Gandolfi F.
Published: August 2011

Decreased fertility during the hot season is a common problem in pigs. Maternal hyperthermia reduces oocyte fertilizability and increases embryonic mortality. Cell biochemical thermoprotection mechanisms involve members of the heat shock protein (Hsp) family. Hsp40, also known as Mammalian Relative of DnaJ (MRJ) protein, plays a pivotal role as co-chaperone after heat shocks. This protein binds Hsp70 and, through ATP hydrolysis, induces the conformational changes needed by Hsp70 to bind and release heat unfolded proteins. However, no information is available on the role of Hsp40 in mammalian ovary. Here we investigate a) the expression and localization of Hsp40 in pig ovaries; b) its response to heat stress in oocytes and cumulus cells. To identify Hsp40 in pig, we extracted RNA from ovaries, granulosa cells and from pools of 5 oocytes. cDNA was amplified using primers specifically designed for Hsp40, based on sequence data available in other species. The amplified products were sequenced and aligned using ClustalW. The nucleotide sequence showed an homology of 95% with the human, 92% with the bovine and 84% with the mouse orthologs. A polyclonal antibody was raised against the mouse GST/MSJ1(145–242) homologue protein in New Zealand rabbits and serum was affinity-purified using a GST-coupled Affigel-10 column. Whole ovary proteins were separated by SDS PAGE, immunoblotted and stained with the Hsp40 antibody. The protein displayed a MW of 38 KDa, in agreement with results obtained in other species. Immunofluorescence studies showed that Hsp40 is found in oocytes, granulosa and theca cells of follicle at all developmental stages. Pig ovaries, collected at the slaughterhouse, were exposed to 42°C for 1 h, to verify whether Hsp40 has a functional response to heat stress in this specie. Transcript level was compared with the control group, maintained at 38.5°C. mRNA was extracted from cumulus cells and pools of 5 oocytes, isolated from follicles of 3 to 5 mm. Semi-quantitative analysis was performed in the exponential phase of PCR amplification, using 28S as endogenous control. Data were analyzed with Quantity One (Bio-Rad) and Student-t statistical analysis was performed. Exposure of porcine ovaries to 42°C for 1 h resulted in a significant increase (P ≤ 0.05) of Hsp40 mRNA levels (2.4 ± 0.35 fold) in oocytes, while no significant raise was detected in cumulus cells. To our knowledge, this is the first demonstration that Hsp40 is expressed and responds to a thermal stress in pig ovary. Since this co-chaperone acts upstream to other heat shock protein-such as Hsp70- and it is specifically up regulated in the oocytes, our findings suggest that it may play an important protective role against heat stress infertility.
43 SHORT- AND LONG-LASTING EFFECTS OF TRICHOSTATIN A TREATMENT OF SCNT EMBRYOS IN CATTLE
Authors: Lagutina I. , Duchi R. , Colleoni S. , Lazzari G. , Galli C.
Published: August 2011

Both preimplantation and full-term development of mouse somatic cell nuclear transfer (SCNT) embryos are significantly enhanced by treatment with trichostatin A (TSA), an inhibitor of histone deacetylase (Kishigami et al. 2006 Biochem. Biophys. Res. Commun. 340, 183–189; Rybouchkin et al. 2006 Biol. Reprod. 74, 1083–1089). The present study was designed to examine the effect of TSA treatment on preimplantation and full-term development of bovine cloned embryos. To investigate the effect of TSA on bovine NT embryos development, we treated them with 50 nM TSA during the first 10 h after activation. Bovine NT-embryos were reconstructed using adult fibroblasts of 2 female donors (A and B) with significantly different in vitro cloning efficiency (respectively, 84/245; 34.3% v. 155/298; 52.1% blastocyst D7, P ≤ 0.05, chi-square test). TSA treatment significantly improved blastocyst rate in A, however did not affect development in B (56.3% and 50.5%, respectively). The level of acetylated histone H3K9 10 h after activation detected by anti-acH3K9 antibody was significantly increased after TSA-treatment in A (P ≤ 0.05, Student’s t-test) but did not change in B, thus demonstrating that the levels of histone acetylation in cloned embryos correlate with their in vitro developmental potential. To evaluate the long-lasting effect of TSA-treatment on the full-term development of cloned embryos, SCNT embryos derived from 4 female donor animals were reconstructed. 196 TSA-treated embryos at the blastocyst stage were transferred into 98 recipients and 2 calves (2%) were born. In the control group, 167 embryos were transferred into 141 recipients and 3 calves (2.1%) were born. Our data show that cell lines demonstrate different susceptibility to TSA that may affect reprogramming of the somatic genome with low level of acetylation resulting in higher in vitro embryo development. However, TSA does not improve overall cloning efficiency in cattle, measured as full-term development.

Project partly supported by EU grants Plurisys (n 22348), Xenome (LSHB-CT-2006-037377) and Regione Lombardia.
44 IN VITRO DEVELOPMENT OF INTERSPECIES NUCLEAR TRANSFER EMBRYOS GENERATED WITH BOVINE OOCYTES AND EQUINE SKIN FIBROBLASTS
Authors: Lee J. , Park J. , Chun Y. , Lee W. , Song K.
Published: August 2011

Study for equine somatic cell nuclear transfer (SCNT) is an attractive field for research, but it has not been a major field of study because it is hard to obtain a sufficient number of ovaries and it takes a lot of time and effort for the recovery of oocytes matured in vivo by ovum pickup. It was reported that the bovine cytoplast could support the remodelling of equine donor cells (Zhou et al. 2007 Reprod. Domest. Anim. 42, 243–247). The objectives of this study are 1) to monitor the early events of equine SCNT by interspecies SCNT (isSCNT) between bovine cytoplast and equine donor cell, and 2) to investigate the developmental competence of isSCNT embryos. Bovine oocytes were recovered from the follicles of slaughtered ovaries, and matured in TCM-199 supplemented with 10 mU mL–1 FSH, 50 ng mL–1 EGF, and 10% FBS at 39°C under 5% CO2 in air for 22 h. Fibroblasts derived from bovine or equine skin tissues were synchronized at G0/G1 stage by contact inhibition for 72 h. After IVM, oocytes with polar body were enucleated and electrically fused with equine or bovine skin fibroblasts (1.0 kV cm–1, 20 μs, 2 pulses). Fused couplets were activated with 5 μM ionomycin for 4 min followed by 5 h culture in 10 μg mL–1 cycloheximide (CHX) and/or 2 mM 6-DMAP, and cultured in modified synthetic oviduct fluid (mSOF) at 39°C under 5% CO2, 5% O2, and 90% N2 for 7 days. All analyses were performed using SAS (version 9.1; SAS Institute, Cary, NC, USA). The cleavage rate of isSCNT embryos derived from equine cell was not different (252/323, 78.7%; P = 0.94) from that of SCNT embryos derived from bovine cell (230/297, 79.2%). However, the rate of isSCNT embryos developed to over 8-cell stage was lower (3.3%; P < 0.0001) than that of bovine SCNT embryos (39.4%), and total cell number of isSCNT embryos developed to over 8-cell stage was lower (17.5, n = 12; P < 0.0001) than that (80.8, n = 110) of bovine SCNT embryos. Also, the rate of blastocyst formation of isSCNT embryos (0/323; 0.0%) was lower (P < 0.0001) than that of bovine SCNT embryos (83/297; 29.3%). Meanwhile, reconstructed oocytes for isSCNT were fixed at 8 h after activation to investigate the formation of pseudo-pronucleus (PPN) after post-activation treatment with CHX or CHX+6-DMAP. The ratio of oocytes with single PPN after treatment with CHX+6-DMAP (26/35; 74.3%) was not different (P = 0.63) from that of oocytes treated with CHX (24/36; 68.1%). Although isSCNT embryos derived from bovine cytoplast and equine donor cell could not develop to more than the 16-cell stage, it is believed that the results of this isSCNT study could be used for the preliminary data regarding the reprogramming of donor cell in equine SCNT.
45 IN VITRO DEVELOPMENT OF BOVINE TRANSGENIC NUCLEAR TRANSFER EMBRYOS IN SERUM-FREE AND SERUM-SUPPLEMENTED MEDIA
Authors: Lee J. Y. , Lee S. G. , Jung E. J. , Jeong S. H. , Yang C. J. , Jeong Y. W. , Hyun S. H. , Kim Y. W. , Shin T. , Jeung E.-B. , Hwang W. S.
Published: August 2011

Novel serum-free media (IVD101) has been shown to be effective for the production of in vitro-produced embryos for subsequent implantation into cows (Hoshi 2003 Theriogenology 59, 675–685). The objective of the present study was to determine whether serum-free embryo cultivation during preimplantation stage could be used for the production of bovine transgenic nuclear transfer embryos. Somatic cell nuclear transfer (SCNT) embryos were produced by using donor cells containing a vector to induce the production of human erythropoietin in cow's milk. αS1-casein was selected as the promoter to be used in this study through the specific promoter activity test, and enhanced green fluorescent protein(EGFP) gene was attached to the CMV promoter to allow observation of the donor cell during the experiment. Adult fibroblast cells were transfected with lipofectamine. After G418 selection, the transfected cells were injected to the enucleated oocytes, and injected embryos were accomplished by cell-to-cell fusion. These embryos were then activated with calcium ionomycin and 6-dimethylaminopurine. The reconstructed embryos were cultured in IVD101 and mSOF media at 38.5°C, in a 5% CO2, 5% O2, and 90% N2 atmosphere. Embryos were cultured for 4 days, followed by addition of FBS in case of mSOF media. On day-7, the developmental ability and the number of cells in the reconstructed embryos were determined. Statistical analysis of embryo development data was carried out using unpaired t-test, or ANOVA. There were no significant differences in the cleavage rate (69.6 ± 3.2% v. 64.5 ± 5.0%), blastocyst rate (18.7 ± 1.3% v. 22.0 ± 1.6%), and cell number (113.9 ± 7.5 v. 103.6 ± 7.9) between IVD101 and mSOF+FBS cultured embryos. These results indicated that serum-free media did not reduce the developmental competence of SCNT embryos compared with serum-supplemented media. Further studies are required to investigate whether this serum-free transgenic embryo cultivation could be used for developmental potential in terms of full-term development after embryo transfer.
46 THREE STAGES ESTABLISHMENT OF IN VITRO MATURATION OF PORCINE OOCYTES USED AS RECIPIENT CYTOPLASTS FOR SOMATIC CELL NUCLEAR TRANSFER
Authors: Lee M. R. , Park S. H. , Kim T. S. , Kim S. Y. , Eun H. J. , Park C. S. , Lee J. H.
Published: August 2011

Oocytes at either anaphase/telophase of the first meiotic division (AI/TI) or metaphase of the second meiotic division (MII) are potential candidates used as recipient cytoplasts for somatic cell nuclear transfer (SCNT) because they contain active maturation-promoting factor (MPF), which causes nuclear membrane breakdown (NEBD) and premature chromosome condensation (PCC) in the transferred nucleus and may be essential for nuclear reprogramming (Campbell and Alberio 2003 Reprod. Suppl. 61, 477–494). In vitro maturation of porcine oocytes spends longer times (44 to 48 h), progresses asynchronously from immature stage and presents insufficient MPF for NEBD and PCC at AI/TI or MII. Here we have developed 3 stages establishment of in vitro oocyte maturation to select good quality of porcine oocytes used as recipient cytoplasts for SCNT. First, porcine oocytes were assessed by the activity of glucose-6-phosphate dehydrogenase (G6PD) before in vitro oocyte maturation. These oocytes with loss of expression of G6PD failed to enzymatically break down the dye, brilliant cresyl blue (BCB) and thus stain positively (Roca et al. 1998 Reprod. Fertil. Dev. 10, 479–485). Positively BCB stained oocytes reached to MII (67.0% v. 58.4%) and produced more parthenogenetic blastocysts (50.2% v. 29.6%) in comparison with negatively BCB stained oocytes (P < 0.05). Second, positively BCB stained oocytes were treated with 5 μg mL–1 cycloheximide (CHXM, a nonspecific protein-synthesis inhibitor) to block meiotic progression for synchronizing the cell cycle of oocytes for 16 h. All of the oocytes (130/130) were efficiently synchronized at the germinal vesicle (GV) stage by treatment with CHXM, whereas control oocytes (w/o CHXM) were passed through GV stage (80/176). Retrieval from treatment of CHXM could reversibly induce meiotic resumption and progresses synchronously in porcine oocytes. Following incubation with gonadotropin hormones [epidermal growth factor (EGF) 10 ng mL–1, LH 5μg mL–1, FSH 1 μg mL–1], treatment with 5 mM caffeine (a phosphatase inhibitor) for 12 h significantly increased the activity of MPF in porcine oocytes (P < 0.05). However, there was no difference between CHXM treated and nontreated group (21.9% v. 22.3%) in the developmental rate to the blastocyst stage of parthenogenetic embryos. At least 3 replicates were performed. These data may contribute to an improved nuclear reprogramming in SCNT.
49 IMPROVED PORCINE CLONING EFFICIENCY WITH CELLS CULTURED FOR SEVERAL GENERATIONS AFTER A SINGLE TREATMENT WITH XENOPUS EGG EXTRACT
Authors: Liu Y. , Østrup O. , Li J. , Vajta G. , Kragh P. M. , Purup S. , Li R. , Callesen H.
Published: August 2011

Extracts from eggs of Xenopus laevis frogs can induce nuclear remodelling or increase transcriptional reprogramming in somatic cells. However, it is not known if this effect is passed on from one cell generation to another, or how it affects somatic cell nuclear transfer in porcine cells. This study aimed to investigate the effect of extract-treated cells over several generations on porcine cloning. Extracts were prepared from 2 frogs (B1 and B2) by the same protocol (Higa et al. 2006 Methods 39, 284–290). Fetal fibroblasts grown on poly-L-lysine coated coverslips were permeabilized by digitonin (7 μg mL–1, 2 min, 4°C) and incubated with 1 extract batch at 37°C for 30 min. After resealing the membrane in DMEM supplemented with 2 mM CaCl2 at 37°C for 2 h, the remaining cells were cultured in ES medium (Vejlsted et al. 2005 Mol. Reprod. Dev. 70, 445–454) for 7 to 8 days when they formed colonies. The colonies were trypsinized and divided onto 2 coverslips for subculture, defined as Experimental Passage 1 (XP1). New subcultures were made every 7 to 8 days when 70 to 80% clusters become colonies until XP15. Colonies from XP3, 8 and 15 were isolated and trypsinized before being used in handmade cloning. Nontreated cells grown in DMEM were used as controls (no colony formation was observed). On each cloning day, cells from different XP number and controls were used. Rates of cleavage (Day 2) and blastocyst development (Day 6) were analysed with chi-square test (SAS version 9.2, SAS Institute Inc., Cary, NC, USA). Results are summarised in Table 1. No difference was observed in cleavage rate between groups. Blastocyst rates of all XP colony cells were significantly higher than their controls. For the same XP number and their controls, blastocyst rates were similar between the colony cells from the 2 extract batches, and there was no difference between their controls, either. In conclusion, the cloning efficiency in porcine cells could be increased with extract-treated cells used for several generations, and this effect was present at XP3, 8, and 15.

Table 1.Developmental competence of cloned porcine embryos with extract-treated cells from different batches of extract (B1 and B2) and Experimental Passage (XP) numbers
5 PRODUCTION OF TRANSGENIC LIVESTOCK USING A LENTIVIRUS EXPRESSING MULTIPLE SHORT INTERFERING RNAs TARGETING FOOT AND MOUTH DISEASE VIRUS
Authors: Peoples M. , Westhusin M. , Tessanne K. , Long C.
Published: August 2011

One goal of transgenic livestock production is developing animals with enhanced production characteristics. Transgenic animals with resistance to viral disease could greatly reduce economic losses. The use of short interfering RNA (siRNA) or short hairpin RNA (shRNA) targeting viral genomes have shown great promise in vitro for both human and animal applications. However, because of the rapid mutation rate, viruses are able to escape single siRNA inhibition. One method to reduce the chances of a functional escape virus is to target its genome with multiple shRNAs simultaneously. The goal of this research project was to produce a recombinant lentiviral vector that expresses three unique shRNAs targeting different regions of the foot and mouth disease virus (FMDV) and use it to produce transgenic livestock. In these initial experiments we used the goat as our model system. Previously, we confirmed that three distinct siRNAs individually could reduce the ability of the FMDV virion to replicate in vitro. Based upon these results we produced a recombinant lentiviral vector that utilised three bovine Pol III promoters (7sk, H1, U6-2), each transcribing a different effective shRNA targeting FMDV. In addition, the vector also contained the fluorescent marker zsGreen and an antibiotic resistance gene. The lentiviral vector was co-transfected with pCMV and pMDG into 293T cells to produce replication incompetent retroviral particles. The supernatant was collected and ultra-centrifuged (50 000 × g for 1.5 h) to concentrate the viral particles resulting in a high-titer viral preparation (>109 mL–1 infective viral particles). To produce the transgenic caprine offspring, three embryo donors were superovulated and naturally bred. Nineteen zygotes were surgically collected from the oviduct 24 h after mating. Recombinant lentivirus was microinjected into the zygote perivitelline space. Immediately following the injections, four goat embryos were surgically transferred into the oviduct of each synchronized recipient. Pregnancy was determined by ultrasound at Day 30 in 2 of 5 recipients that received embryos. One pregnancy was carried to term resulting in triplets; 1 live birth, and 2 stillborn. The placenta and tissue sample of the live goat both contained a subpopulation of zsGreen positive cells when analysed with fluorescent microscopy. A fibroblast cell line was derived from the tissue sample and placed under antibiotic selection. Results indicate that only the fluorescent cells also expressed a resistance to antibiotic selection. RNA was collected from the fibroblast cells and mature shRNA production was confirmed using the QuantiMir kit (System Biosystems). Expression of all 3 mature shRNAs was verified in these cells. This data further supports that the entire transgene was integrated into the genome. This is the first report of transgenic livestock produced that expresses multiple shRNAs targeting a viral genome.
51 SCRIPTAID AND MG132 IMPROVE DEVELOPMENT OF PORCINE EMBRYOS RECONSTRUCTED BY SOMATIC CELL NUCLEAR TRANSFER
Authors: Mao J. , Tracy L. , Zhao J. , Whitworth K. M. , Spate L. , Walters E. M. , Prather R. S.
Published: August 2011

Nuclear transfer efficiency in pigs and other large animal species is low. Previous studies have shown that histone deacetylase inhibitor (Scriptaid) and proteasomal inhibitor (MG132) treatment of somatic cell nuclear transfer (SCNT) pig embryos enhances blastocyst formation and pregnancy. The current experiment was carried out to determine the effects of combined MG132 and Scriptaid treatment on early development of cloned pig embryos reconstituted by SCNT. A total of 328 sow oocytes procured from ART (Madison, WI, USA) were reconstructed using α-1,3-Galactosyltransferase knockout hDAF transgenic pig fetal-derived fibroblast cells. Immediately after electrofusion and activation, SCNT oocytes were treated with 0, 1, or 10 μM MG132 for 2 h and then treated with 500 nM Scriptaid for another 16 h. The SCNT embryos were washed and cultured in Porcine Zygote Medium 3 for 7 days. Percent cleavage was determined on Day 2, and blastocyst formation and cell number were determined on Day 7. The experiment was repreated 8 times. There was no difference (P > 0.05) in percent cleavage (57.9 to 66.7%), or cell number (25.5 to 30.6) among the 3 groups. Interestingly, while there was no difference in the percent blastocyst between the 1 μM and 0 μM MG132 treatment groups, more oocytes from the 1 μM MG132 group developed into blastocysts than in the 10 μM MG132 group (25.1 ± 4.6% v. 12.9 ± 3.3%; P = 0.045). Further research will be conducted to transfer these embryos to surrogate gilts to determine the true developmental competence of these embryos.

Supported by the National Institutes of Health National Center for Research Resources (RR018877 and RR013438), and Food for the 21st Century.
57 GENE EXPRESSION PATTERN OF MINIATURE PIG SOMATIC CELL NUCLEAR TRANSFER EMBRYOS TREATED WITH THE HISTONE DEACETYLASE INHIBITOR SCRIPTAID
Authors: Park C. H. , Lee S. G. , Lee H. J. , Jung T. K. , Jeong Y. H. , Jeong Y. W. , Hyun S. H. , Kim Y. W. , Shin T. , Jeung E.-B. , Hwang W. S.
Published: August 2011

It was recently shown that treatment of cloned embryos with histone deacetylase inhibitors improves efficiency for the success rate of developmental potential to term in several species. The objective of the present study was to investigate the influence of the histone deacetylase inhibitor Scriptaid (Sc) on in vitro development in early porcine SCNT embryos and on their gene expression pattern. Based on the findings of previous porcine studies (Zhao et al. 2009), the reconstructed oocytes were treated with 500 nM Scriptaid for 14 to 16 h after post-fusion activation (6-DMAP/demecolcine). In our preliminary study, blastocyst rate significantly increased in the Sc-treated group, compared with the control group (25.1 ± 2.8% and 13.8 ± 1.9%, respectively, P < 0.05). We determined gene expression using quantitative real-time RT-PCR. The results showed that OCT3/4 gene was expressed at a similar level in in vivo and SCNT blastocysts with/without Sc. IGF2 and H19 genes tended to be highly expressed in both SCNT blastocysts with (1.6-fold and 3.1-fold, respectively) and without (2.0-fold and 5.8-fold, respectively) Sc than that of the in vivo blastocysts. We found differences in imprinted gene expression patterns between in vivo and cloned blastocysts. Expression of H19 and IGF2 in SCNT blastocysts after Scriptaid treatment decreased towards the expression levels of in vivo blastocysts. These results indicated that Scriptaid treatment in SCNT embryos may also have beneficial effects on in vitro developmental competence as well as their gene expression pattern.
58 ISOLATION OF BOVINE TROPHOBLAST AND ITS REPROGRAMMING BY NUCLEAR TRANSFER
Authors: Saadeldin I. M. , Kim B. H. , Roibas da Torre B. , Koo O. J. , Jang G. , Lee B. C.
Published: August 2011

Nuclear transfer (NT) has been used to produce many cloned offspring using several types of cells, including embryonic cells. Even though inner cell mass cells have been used as donor karyoplast for producing cloned animals, there are few studies using trophoblast. In mice, clones were born by nuclear transfer of trophoblasts from the expanded blastocyst into enucleated oocytes as a trial to show the totipotency of both inner cell mass and trophectoderm cells isolated from blastocysts (Tsunoda and Kato 1998 J. Reprod. Fertil. 113, 181–184). However, bovine trophoblast cell (TC) lines have not been used in NT to date. The purpose of this study was to elucidate whether TC as donor cell can be reprogrammed in bovine enucleated oocyte and determine the relative abundance of interferon tau (IFNτ) expression in the resulting cloned preimplantational embryos. Hatched blastocysts produced by IVF were used to isolate TCs on mouse embryonic fibroblasts treated with mitomycin C as feeder cells. TCs and adult fibroblasts (AF, control group for NT) were microinjected to perivitelline space of in vitro mature enucleated oocytes and electrically fused. Reconstructed embryos were chemically activated and cultured in a 2-step chemically defined medium. Levels of IFNτ expression in IVF-, TC-, and AF-derived blastocysts were analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). IVF produced embryos were used as reference to analyze the linear progressive expression of IFNτ through mid-, expanded, and hatching blastocysts. As a result, TCs expressing IFNτ were successfully isolated and cultured on feeder layers. It grew as cell sheets of cuboidal epithelium with high proliferation capacity as a single colony originated from a small clump of cells measured 0.5 cm within 7 days of culture. TCs were reprogrammed in the enucleated oocytes to blastocyst with similar efficiency to AF (14.5% and 15.6%, respectively; P ≤ 0.05). RT-qPCR studies showed that IFNτ expression was higher in TC-derived blastocysts than IVF- and AF-derived blastocysts. Both IVF- and TC-derived blastocysts, showed progressive increase of IFNτ expression through the advancement of blastocyst development when it was compared to AF-derived blastocysts. In conclusion, using TCs expressing IFNτ as donor cell for bovine NT could increase the developmental competence of cloned embryos as indicated by progressive linear increase in IFNτ expression.

This study was supported by grants from IPET (#109023-05-1-CG000), NRF (#M10625030005-10N250300510), MKE (#2009-67-10033839, #2009-67-10033805), and BK21 program. Saadeldin I. M. is supported by Islamic Development Bank (IDB) merit scholarship, Jeddah, Saudi Arabia.
59 SYNCHRONIZATION OF CELL CYCLE STAGE OF BUFFALO (BUBALUS BUBALIS) FETAL FIBROBLAST CELLS BY DIFFERENT TREATMENTS
Authors: Selokar N. L. , George A. , Saha A. P. , Sharma R. , Muzaffar M. , Palta P. , Chauhan M. S. , Manik R. S. , Singla S. K.
Published: August 2011

Cell cycle stage of donor cells significantly influences the cloning efficiency during SCNT. Donor cells in G1/G0 stage have better capability to undergo nuclear reprogramming following transfer to an unfertilized oocyte. The lack of availability of cells synchronized at G1/G0 stage is one of the major factors limiting cloning efficiency in buffalo. The aim of this study was to compare the efficacy of various methods for cell cycle synchronization of buffalo fetal ﬁbroblast cells for SCNT. Cells isolated from fetus, 2 to 3 months old, were cultured in DMEM + 10% FBS. The primary culture was sub-cultured 8 to 10 times. For cell cycle synchronization, the cells were cultured to 1) 60 to 70% confluence (controls), 2) 60 to 70% confluence followed by serum starvation (DMEM + 0.5% FBS) for 24 h (serum starved), 3), full confluence followed by culture for additional 3 to 5 days (full confluent), 4) full confluence followed by serum starvation (DMEM + 0.5% FBS) for 24 h (full confluent+serum starved) and 5) 60 to 70% confluence followed by treatment with roscovitine (10, 20, or 30 μM) for 24 h. The synchronization efficiency was examined by propidium iodide staining followed by analysis of DNA content using flow cytometry and the data were analysed by 1-way ANOVA followed by Fisher’s l.s.d. test after arcsine transformation. The percentage of cells in G0/G1 phase of cell cycle was significantly higher (P < 0.05) in the full confluent+serum starved and roscovitine treated (20 or 30 μM) groups than that in the full confluent group and that treated with 10 μM roscovitine which, in turn, was higher (P < 0.05) than that in the serum starved and control groups. These results suggest that buffalo fetal fibroblast cells can be synchronized by roscovitine treatment or by serum starvation of fully confluent cell cultures to obtain a high proportion of cells in G0/G1 stage for SCNT.

Table 1.Buffalo skin fibroblast cells at various stages following different treatments for cell cycle synchronization

Supported by grant No. 1(5)/2007-NAIP from ICAR, India.
62 COMPARATIVE PROTEOMIC ANALYSIS OF LIVER MITOCHONDRIA DERIVED FROM DECEASED NEWBORN CLONED CALVES AND ADULT CLONES BY TWO-DIMENSIONAL DIFFERENTIAL GEL ELECTROPHORESIS
Authors: Takeda K. , Tasai M. , Akagi S. , Watanabe S. , Oe M. , Chikuni K. , Ohnishi-Kameyama M. , Nakamura Y. , Tagami T. , Hanada H. , Nirasawa K.
Published: August 2011

Aberrant reprogramming of donor somatic cell nuclei may result in many severe problems in animal cloning. The inability to establish functional interactions between donor nucleus and recipient mitochondria is also likely responsible for developmental deficiency. However, an understanding of the expressed proteins in cattle is lacking. In the present study, alterations in mitochondrial protein levels between somatic cell nuclear transferred (SCNT) and control animals (mostly produced by AI) were investigated. Nuclear transfer was performed using donor cells prepared from cumulus cells (B1), ear skin, or skeletal muscle from adult Japanese Black cattle, and enucleated in vitro matured oocytes (Holstein or Japanese Black) as previously reported (Akagi et al. 2003). Liver samples were collected from postmortem SCNT calves (CB1-3; 0, 1, and 9 days postnatally) and adult SCNT cattle (CA1-4; 6, 6, 6, and 5 years of age) produced from the same cell line (B1) and preserved at –80°C. Mitochondrial fractions were prepared from the frozen–thawed liver samples by mechanical homogenization and differential centrifugation, and subjected to two-dimensional difference in gel electrophoresis (2D-DIGE) using CyDye™ dyes (Cy2, Cy3, Cy5; GE Healthcare) for specific labelling. Protein expression changes were confirmed by ImageMaster 2D Platinum software with a volume ratio greater than 2.0 (Student’s t-test; P < 0.05). The expression of 5 proteins were up-regulated in SCNT calves compared to control calves (n = 6; Day 250 fetus, 0, 4, 8, 8, and 8 days after birth; P < 0.05). Expressed protein patterning compared to control groups was different among SCNT calves. The protein spots of CB-1 showed great differences compared with other SCNT calves; 13 spots were up-regulated, and 18 spots were down-regulated. In adult SCNT cattle, the concentrations of 3 proteins were higher when compared to control cattle (n = 4; 2, 2, 6, and 8 years of age; P < 0.05). Protein expression was different among individual SCNT animals even if they were produced from the same donor cell source. For example, 9 spots were up-regulated and 7 spots were down-regulated in CA-1. In contrast, no differences were detected in 2 of the SCNT cattle (CA-3 and 4; P < 0.05). Novel proteins were not identified in any of the SCNT cattle or calves. In conclusion, alteration of mitochondrial protein expression levels were observed in non-viable neonatal SCNT calves and varied among SCNT individuals; suggesting that mitochondrial related gene expression may be implicated in early losses. Comparative proteomic analysis represents an important tool for further studies on SCNT animals.

We thank Dr. C. A. Pinkert (Auburn Univ.) and Dr. Somfai (NARO) for their assistance. This work was supported by a grant from the NARO, Japan.
66 DNA METHYLOME ANALYSIS IN NUCLEAR TRANSFER DONOR CELLS AND ITS RELATIONSHIP TO CLONING EFFICIENCY IN SWINE
Authors: Whyte J. J. , Isom S. C. , Spollen W. G. , Blake S. M. , Walters E. M. , Prather R. S.
Published: August 2011

The success of cloning by somatic cell nuclear transfer is unpredictable due in part to abnormal patterns of DNA methylation and histone modifications in the donor genome. The state of these epigenetic markers appears to regulate the timing and degree of embryonic gene expression by altering chromatin structure. In this study, we sought to determine the pattern of DNA methylation in genetically distinct clonal populations of swine fetal fibroblast donor cells that have previously been demonstrated to have either consistently high or consistently low success at producing healthy cloned pigs. To accomplish this goal, we combined methylated DNA immunoprecipitation using antibodies to 5-methylcytosine with Illumina high throughput sequencing (MeDIP-seq) to detect broad regions of methylated and unmethylated DNA in the 2 populations of donor cells (termed high success v. low success). Size-selected immunoprecipitated DNA samples each had matching input controls. Model-based analysis of ChIP-Seq revealed a total of 272 598 and 261 339 genomic regions enriched for methylated cytosine in high v. low success donor cells, respectively. The majority of these enriched regions (peaks) in high and low success samples coincided in genomic position, but 12.9% of the peaks were unique in high success cells, while 20.4% were unique in low success cells. When intersected with 38 778 predicted CpG islands in the swine genome, 3129 of the total MeDIP peaks (1.1%) overlapped for high success cells as compared to 1836 peaks (0.7%) for the low success cells. Of these intersecting MeDIP-CpG peaks for both groups, 1505 peaks were located in unique locations for high success cells and 205 were unique for low success cells. The chromosomal location of the CpG intersections differed between each cell populations, with the greatest number of overlaps on chromosomes 1 and 3 for high and low success cells, respectively, and the least number of overlaps on chromosomes 9 and 13, respectively. Of the total MeDIP peaks, 6632 (2.4%) from the high success cell population overlapped with predicted swine genes (UCSC Genome Database/Ensembl) compared to 4041 (1.5%) from low success donor cells. Less similar was the percentage of the predicted genes in these methylated regions that were unique to each cell group: 2875 (43.4%) for high success cells v. 338 (8.3%) for low success cells. These results highlight differences in the degree of DNA methylation and the pattern of methylated genes in NT donor cells that produce healthy clones v. those that do not. Such comprehensive genomic analyses of epigenetic markers may provide predictive data to screen for optimal NT donor cells, thereby improving cloning efficiency.

This work was supported by the National Institutes of Health NIH R01 RR013438 and Food for the 21st Century at the University of Missouri.
70 REFREEZING POST-THAWED GOAT SEMEN
Authors: Carwell D. B. , Scott B. R. , Gentry G. T. , Bondioli K. R. , Godke R. A.
Published: August 2011

The ability to successfully refreeze caprine sperm could provide a means of salvaging semen that was mistakenly thawed. The objective of this study was to compare treatment post-thaw semen parameters of twice-frozen caprine semen. Frozen semen from six mature Boer bucks (range in age from 2 to 6 years) was utilised for this experiment. Semen from each buck was extended in an egg yolk-based extender and packaged in 0.5-mL plastic straws before freezing and stored in liquid nitrogen. Three units of frozen semen from each buck was randomly allotted to each of four treatments as follows: (A) thaw and evaluate (control), (B) thaw, then plunge into liquid nitrogen, thaw, and evaluate, (C) thaw, incubate for 3 min at 37°C, slow cool and freeze, thaw, and evaluate, and (D) thaw, incubate for 5 min at 37°C, slow cool and freeze, thaw, and evaluate. Post-thaw parameters included total motility (TM), progressive motility (PM), membrane integrity (MI), and sperm abnormalities (AB). To obtain MI and AB, samples were stained with an eosin-nigrosin stain. A computerized programmable freezer was used to refreeze semen samples in treatment (Trt) C and Trt D. During the slow cooling portion of the protocol, samples were allowed to equilibrate at 38°C, then cooled to 4°C at a rate of 0.30°C min–1, and then held for 5 min. Samples were then cooled to –8°C at a rate of 15°C min–1, seeded, and cooled to –10°C at 15°C min–1, samples were then ramped to –80°C at 30°C min–1 before plunging into liquid nitrogen. Results indicate that post-thaw TM was significantly greater for Trt A (60%) when compared with Trt B, C, and D (0.05, 35, and 39%, respectively). Mean TM were not different between Trt C (35%) and Trt D (39%) but were greater than that for Trt B (0.05%). The PM for post-thaw semen in Trt A was also significantly greater (P < 0.05) when compared with that for Trt B and C (0.05 and 25%); however, no difference was found for mean PM for Trt A (47%) and Trt D (30%), nor were differences found between Trt C (25%) or Trt D (30%). Membrane integrity was higher in Trt A (27%) when compared to Trt B (2.2%). No differences in membrane integrity where found between Trt A, C, and D (27, 13, and 14%, respectively). Additionally, no differences were found between Trt B, C, and D for membrane integrity. Sperm morphology were not different were found with across all treatment groups. These results (i.e. Trt C and D) indicate that semen from mature Boer bucks can undergo a second freeze thaw cycle and still retain motility without dramatically affecting sperm morphology and membrane integrity. These findings indicate that directly plunging recently thawed semen back into liquid nitrogen should not be used for artificial insemination.
71 WILL DEHYDRATION BY EXPOSURE TO SUCROSE IMPROVE POST-VITRIFICATION SURVIVAL OF MURINE EMBRYOS VITRIFIED BY THE OPEN PULLED STRAW METHOD?
Authors: El-Gayar M. , Reischl J. , Gauly M. , Holtz W.
Published: August 2011

Vitrification of mammalian embryos comprises suspension in highly concentrated solutions of penetrating cryoprotectants. These cryoprotectants are known to be extremely cytotoxic to cells in an unfrozen state. In the present experiment, it was attempted to reduce the amount of cryoprotectants entering the cells to a minimum by at least partially dehydrating the blastomeres before exposing them to vitrification solutions. Sucrose is known to be a non-permeating osmotically active agent and is, therefore, suited to serve this purpose. It has been shown that concentrations of up to 1.1 M sucrose are well tolerated (Krag et al. 1985 Theriogenology 23, 199). Morphologically intact mouse blastocysts collected from superovulated 5- to 8-week-old virgin female NMRI mice were randomly allocated to 4 treatment groups (100 embryos/group). A control group (Group 1) was vitrified according to the slightly modified (El-Gayar et al. 2008 Cryobiology 57, 191–194) protocol of (Vajta et al. 1998 Mol. Reprod. Dev. 51, 53–58). The protocol involves exposure to 10% dimethyl-sulfoxide (Me2SO) + 10% ethylene glycol (EG) for 1 min; 20% Me2SO + 20% EG for 20 s; loading into straws and plunging directly into liquid nitrogen. In embryos of Group 2, the procedure mentioned above was preceded by exposure to a 1 M sucrose solution for 1 min. In Group 3, the procedure of Group 2 was followed; however, vitrification solution no. 1 (10% Me2SO + 10% EG) was omitted. In Group 4, sucrose was added to both vitrification solutions at a concentration of 0.2 mol with the 10% Me2SO + 10% EG-solution and of 0.4 mol with the 20% Me2SO + 20% EG-solution. After warming, embryos of all groups were cultured in vitro in microdrops of M16 medium for 48 h. Differences between means were tested for significance by the chi-square test. All embryos were recovered after warming. In the control group, 97% of the embryos had continued development to the expanded blastocyst stage and 89% had proceeded to hatch. The corresponding values for Groups 2, 3, and 4 were 81 and 64%; 60 and 29%, and 93 and 88%, respectively. Earlier studies have shown that in vitro hatching is closely correlated with in vivo survival (El-Gayar et al. 2008 Cryobiology 57, 191–194; Cryoletters 2010, in print). The differences between the control group and Groups 2 and 3 were significant (P < 0.01), whereas with Group 4 it was not (P > 0.05). Thus, exposure to sucrose before vitrification (Groups 2 and 3) compromised the viability of murine blastocysts rather than protecting them, whereas addition of sucrose to vitrification solutions did not. Because embryo survival after cryopreservation by the standard OPS procedure was exceptionally high, it was impossible to detect a potential improvement.

This study was partly supported by a grant from the Egyptian government.
72 SUPERIOR PREGNANCY RATES WITH THE TRANSFER OF OPEN PULLED STRAW-VITRIFIED V. CONVENTIONALLY CRYOPRESERVED EMBRYOS IN GOATS
Authors: El-Gayar M. , Al Yacoub A. N. , Gauly M. , Holtz W.
Published: August 2011

Two separate studies were conducted in the course of which goat embryos were cryopreserved either by the open pulled straw (OPS) procedure of (Vajta et al. 1998 Mol. Reprod. Dev. 51, 53–58) or by conventional slow freezing. Embryos of each doe were assigned to both methods. A total of 94 morphologically intact blastocysts, collected by transcervical flushing from superovulated pluriparous Boer goat does, were involved. For OPS-vitrification the method of Vajta et al. (1998) was slightly modified (El-Gayar et al. 2008 Cryobiology 57, 191–194). It involves exposure of the embryos to a medium containing 10% dimethyl-sulfoxide (Me2SO) + 10% ethylene glycol (EG) for 1 min, followed by 20 s in medium containing 20% Me2SO + 20% EG. After aspiration into thinly drawn-out straws, the straws were plunged directly into liquid nitrogen. Conventional freezing comprises equilibration in M2 medium containing 1.4 M glycerol and cooling in 0.25-mL straws at a rate of 0.3°C min–1 as described in (Nowshari and Holtz 1993 J. Anim. Sci. 71, 3403–3408). Embryos of both treatment groups were transferred to synchronized recipients (2/recipient) within 2 h after thawing. The surrogate mothers were diagnosed for pregnancy 5 weeks later and, if gravid, permitted to carry to term. Differences between means were tested for significance by the chi-square test. All embryos were recovered after thawing. Within the 2-h period from thawing to transfer, OPS-vitrified embryos had the appearance of flawless, expanded blastocysts, whereas conventionally frozen–thawed embryos had not quite reached full re-expansion. Of 25 recipients receiving OPS-vitrified blastocysts, 22 (88%) kidded with average litter size of 1.5. Of 22 recipients receiving conventionally frozen–thawed blastocysts, 10 (45%) kidded with average litter size of 1.8. Embryo survival in vitrified blastocysts amounted to 67%, and in conventionally frozen–thawed blastocysts 42%. The difference in kidding rate between the two methods was statistically significant (P < 0.01). Regardless of the cryopreservation method all kids born were viable, their birth weight being within the range typical for the population. The results indicate that, for the cryopreservation of caprine blastocysts, OPS vitrification is a more suitable technique than conventional freezing and should be routinely employed.

The senior author owes thanks to the Arab Fund for Economic and Social Development for the financial support.
73 LIPID CONTENT AND CRYOTOLERANCE OF PORCINE EMBRYOS CULTURED WITH PHENAZINE ETHOSULFATE
Authors: Gajda B. , Romek M. , Grad I. , Krzysztofowicz E. , Bryla M. , Smorag Z.
Published: August 2011

In this study, the addition of phenazine ethosulfate (PES) to culture medium was investigated for its effect on cytoplasmic lipid content in cultured pig embryo and survival after open pulled straw (OPS) vitrification (Vajta et al. 1997 Acta Vet. Scand. 38, 363–366). In addition, in cultured blastocysts, the total cell number per blastocyst and the degree of apoptosis were assessed. Porcine zygotes were cultured up to the blastocyst stage in NCSU-23 medium supplemented with 0 (control, n = 146) or 0.05 μM PES (n = 150). To evaluate the lipid content in embryos, we employed Nile Red (NR), a fluorescent dye specific for intracellular lipids (Genicot et al. 2005 Theriogenology 63, 1181–1194). We measured the amount of fluorescence originating from NR using LSM 510 Meta Zeiss confocal microscope and ImageJ version 1.38x software (National Institutes of Health, Bethesda, MD, USA) and an Integrated Density (ID) parameter. The total amount of fluorescence per embryo (TF), proportional to the amount of lipids, was calculated as the sum of ID measured for all optical slices in each individual z-stack. Blastocysts that were cultured with (n = 48) or without PES (n = 34) were vitrified using OPS technology. Results were analysed using chi-square, Fisher, and Student’s t-tests. The total number of cells and the percentage of TUNEL-positive nuclei of PES-treated blastocysts were significantly different than for the control group (43.6 v. 37.6; P < 0.05 and 1.6 v. 2.9; P < 0.01, respectively). Blastocysts stained with Nile Red fluorescent dye showed intracellular lipid mainly localised to the lipid droplets. They were present both in the embryoblast and trophoblast cells. Mean values of TF estimated for the experimental group was lower by ∼23% than those of the control group. Thereby, blastocysts of the control group possess a higher content of lipids then those found in the experimental group cultured in medium with 0.05 μM PES (P < 0.001). The survival rate of vitrified blastocysts was slightly enhanced, although not significantly, in the presence of PES compared to the PES-free group (44.8 and 37.1%, respectively). These results showed that culturing porcine embryos in medium with phenazine ethosulfate supplementation increased the total cell number per blastocyst and reduced the index of DNA fragmentation of cultured blastocysts. Use of PES in porcine culture medium reduced the cytoplasmic lipid content, as measured by fluorescence of blastocysts stained with Nile Red. However, the use of PES during in vitro culture had a limited effect on porcine blastocyst survival after vitrification.

This study was partially supported by Grant NR 12 0036 06 from NCBiR, Poland.
78 EFFECT OF EXTENDER AND STORAGE PERIOD ON THE SOUTH AFRICAN INDIGENOUS KOLBROEK BOAR SPERM CELL MOTILITY RATES FOLLOWING ANALYSIS BY COMPUTER-ASSISTED SPERM ANALYSIS
Authors: Masenya M. B. , Mphaphathi M. L. , Munyai P. H. , Egerszegi I. , Umesiobi D. O. , Dinnyes A. , Nedambale T. L.
Published: August 2011

The choice of the short-term extender is important to preserve the quality of the diluted South African indigenous Kolbroek boar semen during short-term storage prior to artificial insemination. The aim of this study was to identify the suitable extender for short-term storage of Kolbroek boar semen at 17°C. Five ejaculates were collected separately from five Kolbroek boars using the gloved-hand technique in a 300-mL glass beaker. The filtered sperm fraction were sealed with a gauze filter inside a prewarmed (39°C) insulated thermos flask. Following semen collection, the semen was evaluated for macroscopic characteristics; semen volume, pH, and concentration. Sperm cells morphology was evaluated using eosin-nigrosin staining solution using fluorescent microscope system. Only ejaculates with >75% motile sperm and <15 abnormalities were used. The semen was pooled and diluted with five different short-term extenders, namely: Beltsville Thawing Solution (BTS), Kobidil+, Hungarian Standard, Citrate, and Tris-based extenders at a ratio of 1:1 (v/v). Sperm cells motility parameters were evaluated using a sperm class analyzer (SCA) at 0, 3, 24, and 48 h intervals. The data were analysed by SPSS. The results demonstrated that sperm cell motility rate did not differ when stored at 0 (88.9 ± 8.3) and 3 (93.8 ± 4.4) h irrespective of extender. However, semen that was diluted with Hungarian extender and Tris-based extender maintained higher proportion of sperm cells motility (90.0 ± 2.5 and 87.8 ± 4.8, respectively) for a longer period during storage for 24 h as compared with the other extenders. In conclusion, the study demonstrated that prolonged storage of boar sperm cells reduces motility rates. However; Hungarian Standard and Tris-based extenders preserved sperm motility rate better than the other extenders when stored up to 48 h at 17°C. Thus, this study suggests that Kolbroek sperm cells can be transported within 24 to 48 h in South African regions without adversely affecting motility rates when diluted with Hungarian Standard and Tris-based extender at 17°C without cryopreserving them.

This study was funded by the Department of Agriculture Forestry and Fishery, ARC, DST (RT19000), and National Research Foundation (NRF, Grant. No. RT21 and 24000).