bluuebunny/crossref_metadata_2025 huggingface dataset:
53 IMPACTS OF USING PROCAINE AS A DNA-DEMETHYLATING AGENT IN IN VITRO CULTURE OF BOVINE CELLS
Authors: Michalczechen-Lacerda V. A. , Rodrigues F. C. , de Sousa R. V. , Rumpf R. , Franco M. M.
Published: August 2011

Euchromatin and heterochromatin organisation define the specificity of each cell type. This structure is controlled by epigenetic modifications and the DNA methylation is one of the best known for inducing transcriptional repression. Recently, procaine was uncovered as a DNA-demethylating agent, but there are few reports about its dynamic epigenetic action on somatic cells. Mono-allelic expression of imprinted genes is controlled by DNA methylation and inherited to somatic tissues of a sex-specific manner. The aim was to investigate the effects of using procaine, a DNA-demethylating agent, in in vitro culture of bovine (Bos taurus indicus) fibroblast for 72 h (passage 4). We have evaluated cell viability, chromosome integrity, and DNA methylation patterns. To evaluate cell viability, we have used trypan blue 0.4%. To evaluate chromosome integrity, we have used conventional cytogenetic analysis. To investigate DNA methylation patterns, we have analysed 2 differentially methylated regions (DMR) located into the exon 10 of IGF2 and exon 1 of XIST imprinted genes, using the bisulfite sequencing method (EZ DNA methylation kit, Zymo Research, Orange, CA, USA). After bisulfite treatment and nested-PCR, the amplicons were separated in agarose gel electrophoresis, purified with GenClean III kit (MP Biomedicals, Irvine, CA, USA), cloned in a pGEM-T easy vector system (Promega, Madison, WI), and sequenced. The DNA sequences were analysed using the BiQ Analyzer v. 2.0 (2008) software. The cell viability data were analysed using ANOVA and Tukey or Kruskal-Wallis and Mann-Whitney tests, and the methylation status were analysed using Student’s t-test or Mann-Whitney tests in the Prophet software (BBN Systems and Technologies). Cell culture using 0.1 mM or 0.5 mM of procaine were viable and the number of cells with intact membrane was higher than the control and 2.0 mM of procaine groups (P ≤ 0.05). The total number of cells was lower in the group with 2.0 mM of procaine (P ≤ 0.01). Cytogenetic analysis showed no differences among the groups, with no chromosome abnormalities detected. The methylation pattern was not different for both DMR evaluated among the groups. We have observed that there was a beneficial effect to the cells that have received supplementation with 0.1 mM or 0.5 mM of procaine, because there was an increase in the number of viable cells without chromosomal abnormalities. We cannot ignore that a global DNA demethylation may have occurred, which was not detected in the specific analysed regions. The results obtained here may contribute to improving the efficiency of animal cloning, transgenic animal production, and the knowledge about stem cells.

Supported by Embrapa Genetic Resources and Biotechnology and CAPES.
54 THE EFFECT OF TREATMENT OF BOVINE NUCLEAR TRANSFER EMBRYOS WITH VALPROIC ACID ON THEIR SUBSEQUENT IN VITRO DEVELOPMENT
Authors: Mizutani E. , Akagi S. , Kaneda M. , Somfai T. , Watanabe S. , Geshi M. , Nagai T.
Published: August 2011

Recently, trichostatin A (TSA) and some other histone deacetylase inhibitors (HDACi) were reported to enhance the development of mouse somatic cell nuclear transfer (SCNT) embryos. Previously, we have succeeded in improving in vitro development of bovine SCNT embryos significantly with inhibitors of class I, IIa, and IIb HDACs such as TSA or Scriptaid (SCR). In this study, we examined the effect of valproic acid (VPA), a selective inhibitor of class I and IIa HDACi, on in vitro development of bovine SCNT embryos. 3 cell lines (adult male, adult female, and fetal female fibroblast cells) were used as donor cells. SCNT was performed as previously described (Akagi et al. 2003 Mol. Reprod. Dev. 66, 264–272). Reconstructed embryos were chemically activated by treatments with 10 μM calcium ionophore for 5 min and 10 μg mL–1 cycloheximide for 5 h. Then SCNT embryos were cultured in serum-free medium for 7 days. Embryo development data were analyzed by chi-square test. Differences were considered significant at P < 0.05. Each experiment was replicated at least 3 times. At first, to determine the suitable concentration of VPA, bovine SCNT embryos derived from adult female fibroblast were treated with various concentrations of VPA (0, 0.01, 0.1, 1, 10 mM) for 20 h from the start of chemical activation and embryo development was examined. There was no difference in blastocyst formation rates based on the number of cleaved embryos among the groups [46% (166/357), 53% (49/92), 46% (51/112), 55% (56/102), and 44% (44/100), respectively], and the cell numbers of blastocysts were also similar. Next, we examined the effect of duration of VPA treatment on development of SCNT embryos obtained from adult male, female, and fetal female fibroblast cells. Based on the results of previous experiment, bovine SCNT embryos were treated with 1 mM VPA for 20 h or 40 h and those without treatment were used as control. Neither 20 h nor 40 h VPA treatment affected blastocyst formation rates of SCNT embryos from adult male, control: 38% (41/107), 20 h: 45% (47/104), 40 h: 46% (39/85); adult female, control: 47% (166/357), 20 h: 55% (56/102), and 40 h: 36% (12/33); and fetal female fibroblast cells, control: 7% (4/58), 20 h: 13% (7/54), and 40 h: 16% (8/51). In the present study, treatment of bovine SCNT embryos with VPA did not improve in vitro development significantly. Comparing these results with our previous results on TSA or SCR treatment, inhibition of HDAC class IIb may be a key factor to improve development of bovine SCNT embryos.
56 ESTABLISHMENT OF TRANSGENIC RED FLUORESCENCE PROTEIN (RFP) CLONE DOGS THROUGH A STABLE TRANSMISSION OF RFP GENE TO NEXT GENERATION
Authors: Oh H. J. , Park J. E. , Kim M. J. , Kim G. A. , Park E. J. , Hong S. G. , Jang G. , Lee B. C.
Published: August 2011

Somatic cell nuclear transfer (SCNT) technology has been spotlighted not only for its advantage in producing unlimited numbers of genetically identical animals, but also the possibility of producing complex genetic modifications in animals. However, a few reports showed that mosaic expression of transgene in transgenic animals produced by SCNT (Park et al. 2002) and down-regulated gene expression is sometimes irreversible in their offspring (Bordignon et al. 2003). Therefore, we investigated reproductive ability by a breeding between female transgenic beagles and wild-type beagles. When female transgenic beagles (R1, R2, R3, and R5) expressing red fluorescence protein (RFP) gene reached puberty at 373, 353, 283, and 354 days after birth, serum progesterone concentration was monitored for detecting timing of ovulation. Approximately 72 to 79 h after ovulation, the beagles were naturally mated or artificially inseminated. Pregnancy was confirmed by ultrasonography at Day 30 after insemination. The transgenic bitches (R1, R2, R3, and R5) were then bred with wild-type male dogs, became pregnant, and successfully delivered 13 puppies (9 female and 4 male). In order to prove integration of RFP gene in all offspring, DNA was extracted from the blood of pups on Day 7 after birth. For PCR analysis, a primer pair for the RFP gene, forward primer (5′CGTGAAGCTGAAGGTGA-3′) and reverse primer (5′-CTCGTACTGCTCCACGA-3′), were used to amplify a 517-bp DNA fragment. The initial denaturation was performed at 94°C for 5 min, followed by 30 cycles at 94°C for 40 s (denaturation), 58°C for 40 s (annealing), and 72°C for 40 s (extension), and a final incubation at 72°C for 10 min to ensure complete strand extension. Presence of the RFP transgene in 7 of the puppies was confirmed by PCR and the puppies expressed RFP upon UV illumination. It was not different from the 53.8% expected Mendelian ratio. The present result demonstrated a stable transmission of the RFP gene into 5 female and 2 male offspring in the second generation. Among the second generation, 2 female puppies integrated with the RFP gene were in heat at ∼1-year-old. They were then bred with the semen of a wild-type beagle and bore 6 puppies. In the third generation, 3 puppies carried the RFP gene and results showed the expected Mendelian ratio. In conclusion, the present study demonstrates that female transgenic beagles have normal reproductive ability and a stable insertion of the transgene to the next generation.

This study was financially supported by NRF (#M10625030005-508-10N25), SNU foundation (Benefactor; RNL BIO), BK 21 for Veterinary Science, and Purina Korea.
60 EFFECT OF CYTOPLASMIC VOLUME ON DEVELOPMENTAL COMPETENCE OF HAND-GUIDED CLONED BUFFALO (BUBALUS BUBALIS) EMBRYOS
Authors: Panda S. K. , George A. , Saha A. P. , Sharma R. , Kamble N. M. , Manik R. S. , Chauhan M. S. , Palta P. , Singla S. K.
Published: August 2011

Despite recent successes in the birth of buffalo calves cloned through SCNT or hand-guided cloning (HGC), the cloning efficiency is very low in this species because of lack of information on factors that influence it. The goal of this study was to examine the effects of cytoplasmic volume on the developmental competence of cloned buffalo embryos produced by HGC. In vitro matured oocytes were stripped of their cumulus investment and zona pellucida using hyaluronidase and pronase, respectively. Protrusion cone-guided bisection of zona-free oocytes was performed to remove the nucleus. For reconstructing control HGC embryos, 2 enucleated oocytes (demi-cytoplasts) were fused with a single somatic cell. For reconstruction of embryos with lower or higher cytoplasmic volume, 1 or 3 demi-cytoplasts were fused, respectively, with the donor somatic cell. 2 different cell types, i.e. buffalo fetal fibroblasts (BFF) between passage 10 and 15 and buffalo embryonic stem cell (ESC)-like cells between passage 22 and 25 were used as nuclear donors in 2 different experiments. Data were analysed by 1-way ANOVA after arcsine transformation of percentage values. For BFF, the blastocyst rate for doublet and triplet embryos were significantly higher (P ≤ 0.01) than that for singlet embryos despite the cleavage rate for the 3 groups being similar. For the ESC-like cells, the cleavage and the blastocyst rate were significantly lower (P ≤ 0.01) for the singlet than that for the doublet embryos. The pregnancies were established only in doublet and triplet embryo groups using BFF cells and in the doublet embryo group using ESC-like cells. These results indicate that increasing the cytoplasmic volume could be helpful in improving cloning efficiency in terms of blastocyst production rate in buffaloes.

Table 1.Effect of cytoplasmic volume on the developmental competence of cloned buffalo embryos

This work was funded by NAIP grant C 2-1-(5)/2007 to SKS.
67 PIG OOCYTES WITH A LARGE PERIVITELLINE SPACE MATURED IN VITRO HAVE GREATER DEVELOPMENTAL COMPETENCE AFTER PARTHENOGENESIS AND SOMATIC CELL NUCLEAR TRANSFER
Authors: You J. , Kim N. , Kang S. , Lee E.
Published: August 2011

The size of perivitelline space (PVS) is closely related with the frequency of polyspermic fertilization in pig oocytes. It has been reported that enlargement of PVS is attributed to accumulation of glycoproteins synthesised and secreted from cumulus cells and that culture of immature oocytes in low-salt medium enlarges PVS in pigs. This study examined the developmental competence of pig oocytes after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in relation to the size of the PVS of oocytes matured in vitro (IVM). Cumulus–oocyte complexes were matured in medium 199 (Experiment 1) or porcine zygote medium (PZM)-3 (Experiment 2) supplemented with pig follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones for the first 22 h and then cultured in hormone-free medium for an additional 22 h. IVM oocytes were activated electrically for PA or used as recipient cytoplasts for SCNT. PA and SCNT embryos were cultured for 7 days in PZM-3 medium supplemented with bovine serum albumin. The intracellular glutathione (GSH) level in IVM oocytes was determined by analysing the fluorescence intensity of oocytes after staining with CellTracker Blue CMF2HC. The expression of CDK1, PCNA, and ERK2 mRNA in IVM oocytes was analysed by RT-PCR. Data were analysed using a general linear model procedure followed by the least significant difference mean separation procedure when the treatments differed at P < 0.05. In Experiment 1, oocytes with a larger PVS had higher (P < 0.05) levels of intracellular GSH (1.0 pixels/oocyte v. 0.6 pixels/oocyte) and blastocyst formation (54% v. 37%) after PA than oocytes with smaller PVS. In Experiment 2, maturation culture of oocytes in PZM-3 with reduced (61.6 mM) NaCl concentration significantly increased (P < 0.05) the size of the PVS (5.2 μM v. 3.3 μM) compared with control oocytes that were matured in PZM-3 containing 108 mM NaCl, although the treatment did not alter the nuclear maturation. Moreover, oocytes with increased PVS expressed more CDK1, PCNA, and ERK2 mRNA and had higher (P < 0.05) intracellular GSH levels (1.6 pixels/oocyte v. 1.2 pixels/oocyte) and increased blastocyst formation after PA (52% v. 41%) and SCNT (32% v. 18%) compared with control oocytes. Our results demonstrate that pig oocytes with a large PVS have greater developmental competence after PA and SCNT, which is attributed to improved cytoplasmic maturation resulting from the enhanced GSH level and transcription factor expression and that enlargement of PVS by the culture in low-NaCl medium also improves developmental competence of pig oocytes.

This work was supported by grants (#20070301034040 and #20080401034072) from the BioGreen 21 Program (Rural Development Administration, Republic of Korea).
69 QUALITY OF IN VITRO PRODUCED AND CRYOPRESERVED PORCINE EMBRYOS ASSESSED BY CELL NUMBER, TUNEL-POSITIVE NUCLEI, AND CASPASE-3 ACTIVITY
Authors: Bryla M. , Trzcinska M. , Gajda B.
Published: August 2011

The aim of this experiment was to investigate the quality of in vitro cultured and cryopreserved porcine expanded blastocysts from Day 5, 6, and 7 of culture. The quality of the preimplantation embryos was determined by counting the number of cells, observing a TUNEL-positive reaction (TUNEL reagent; In Situ Cell Detection kit, Roche Diagnostics, Germany) and by caspase-3 labelling (PhiPhiLuxG2D2 Kit, Calbiochem, Germany). Embryos were collected from 32 superovulated donor gilts. All were crossbreds of Polish Landrace and Large White, age 6–8 months, weighing 90–100 kg. The experiment was done on 2–4 cell embryos produced in vivo and cultured in vitro for 7 days in NCSU-23 medium until expanding blastocyst stage. The embryos of this stage were obtained on Day 5, 6, and 7 of in vitro culture and divided into two groups: control-(1) 210 nonvitrified (NV) embryos and -(2) vitrified/thawed (VT) 169 embryos. The expanded blastocysts were vitrified the open pulled straw (OPS) method (Vajta 2000 Anim. Reprod. Sci. 60–61, 357–364). The results were analyzed by Student’s t-test, and all values were significant at P ≤ 0.05. The NV group of embryos showed significant differences in the number of cells (66.5 ± 24.0 v. 54.8 ± 15.9) and in TUNEL-positive nuclei (8.8 ± 12.5 v. 16.2 ± 14.9) between Day 5 and Day 7 of culture, respectively. Analysis of VT embryos also revealed significant differences in the number of cells (65.2 ± 17.4 v. 55.5 ± 14.3) and in TUNEL-positive nuclei (25.5 ± 16.4 v. 35.8 ± 19.3) between Day 5 and Day 7 of culture, respectively. Lower percentage of NT and VT blastocysts produced on Day 5 of culture revealed caspase-3 activity (51.3 v. 64.8%) compared with embryos on Day 7 (76.8 v. 89.3%), respectively. In conclusion, blastocysts cultured in vitro for 5 days consist of a high number of nuclei, have a low incidence of TUNEL-positive nuclei, and low caspase-3 activity compared with blastocysts cultured for 6 and 7 days in all analysed groups. Our results revealed that expanding blastocysts produced on Day 5 of in vitro culture had higher ability to survive vitrification/thawing procedure.

This work was supported by Grant NR 12 0036 06 from the National Centre of Research and Development, Poland.
76 EFFECT OF CULTURE METHOD ON THE mRNA EXPRESSION BEFORE AND AFTER CRYOPRESERVATION IN BOVINE BLASTOCYSTS
Authors: Kuzmany A. , Havlicek V. , Wrenzycki C. , Wilkening S. , Brem G. , Besenfelder U.
Published: August 2011

Blastocyst mRNA expression and cryopreservability are thought to be suitable indicators of embryo quality and developmental competence and have been shown to be affected by production methods and culture systems. The aim of the present study was to assess cryosurvival and levels of mRNA expression of selected genes [occludin, desmocollin 2, solute carrier family 2 member 3 (formerly glucose transporter 3), BAX, BCL xL, heat shock protein A1A (formerly heat shock protein 70.1), aquaporin 3, and DNA methyltransferase 1a] of bovine blastocysts derived by 4 different, established culture methods [in vitro production (IVP); multiple-ovulation embryo transfer (MOET); transfer into the heifer oviducts of gametes (GIFT); or in vitro derived cleaved stage embryos (Days 2–7)]. Linear models were used for the comparison of the relative abundances of the blastocyst mRNA transcripts. Separate 1-way ANOVA were used. The production methods were used as factors, except for the comparisons between pre- and post-cryopreservation, where 2-way ANOVA were used. The level of significance was set at P ≤ 0.05. A significant difference in re-expansion rates was found only at 24 h post-thawing, with significantly higher rates in blastocysts produced in vitro compared to embryos of the Days 2–7 group. Levels of mRNA expression were assessed using RT-qPCR. Before cryopreservation of embryos, no significant inter-group differences were seen. However, significantly more desmocollin 2 mRNA expression was detected in embryos of the MOET group compared with blastocysts derived by the other production methods. Post-cryopreservation, blastocysts of 3 embryo production groups (IVP, MOET, Days 2–7) were available for analysis. Compared with levels of mRNA expression before cryopreservation, re-expanded blastocysts after cryopreservation showed a significant up-regulation of heat shock protein A1A transcripts in all groups, and of solute carrier family 2 member 3 transcripts only in the IVP-derived group. The BAX, BCL-xL, occludin, and desmocollin 2 were significantly up-regulated in embryos of the MOET and IVP groups after cryopreservation, as compared with their counterparts before cryopreservation. None of the culture groups showed any pre- v. post-cryopreservation differences in the aquaporin 3 and the DNA methyltransferase 1 mRNA levels. Blastocysts derived by transfer of in vitro derived cleaved stage embryos into the oviduct of synchronised heifers (Days 2–7) did not show any pre- v. post-cryopreservation differences in the mRNA levels of any of the assessed genes. These results merit further investigation. After the process of cryopreservation and thawing, re-expanded embryos of the MOET and IVP groups do increase their mRNA levels to prepare for hatching and further development.
77 PREGNANCY RATES AFTER VITRIFICATION OF FRESH AND COOLED EQUINE EMBRYOS USING THE CRYOTOP METHOD
Authors: Baca Castex C. , Dalvit G. , Miragaya M. , Alonso A. , Pinto M. , Etcharren V. , Castaneira C. , Losinno L.
Published: August 2011

Cryopreservation of equine embryos is still not a routine procedure. Pregnancies have been obtained after transfer of vitrified embryos of less than 300 μm (Eldridge-Panuska et al. 2005). The aim of this study was to use the cryotop method (Kuwayama, 2007) to obtain pregnancies after transfer of vitrified thawed cooled and fresh embryos collected in our clinical embryo transfer programme. Embryos were assigned either to be vitrified within 3 h of collection or to be cooled for 18–24 h before vitrification. All embryos were vitrified and thawed by Cryotop Vitrification Kit® (Cryo Tech Laboratory®). Briefly, they were equilibrated in a solution containing ethylene glycol (EG), dimethylsulphoxide (DMSO) in TCM-199 for 10 to 25 min. Then they were moved to vitrification solution containing EG, DMSO, and sucrose in TCM-199 and loaded with a glass capillary onto the top of the film strip. After loading, almost all the solution was removed to leave only a thin layer covering the embryo, and the sample was quickly immersed into liquid nitrogen and covered with a protective cap. The time between entry to vitrification solution and nitrogen was from 1 to 3 min. At warming, the strip was immersed directly for 1 min into a 37°C medium containing sucrose in TCM-199. The embryo was incubated 3 min in a diluent solution, washed twice 5 min each in washing solution, and further cultured in DMEM F-12 with 10% FBS at 38.5°C 5% CO2 between 2 to 5 h. For transfer, the embryo was loaded in 0.5-mL straws. All recipient mares had ovulated 4 to 7 days before nonsurgical transfer. Pregnancies were detected 6 to 8 days later. A total of 15 embryos, grades 1 to 2, were obtained. Fresh embryos (n = 7) ranged between 250 and 800 μm, and refrigerated embryo (n = 8) diameter was between 130 and 550 μm. Pregnancy rates were 37.5% (3/8) for embryos cooled before vitrification and 28.6% (2/7) for embryos vitrified within 3 h. The overall pregnancy rate was 33.3% (5/15). Shipping cooled embryos allows maintaining a large number of recipients far away from donors, without decreasing pregnancy rate. It also makes it possible to send embryos to a specialised laboratory in order to be vitrified and preserved until recipients are available. Equine embryos collected 6 days after ovulation are generally smaller than 300 μm and have shown the highest survival rate after cryopreservation. However, the embryo recovery rate is higher when flushing is performed at Day 7 or 8. This cryopreservation protocol could provide a way to vitrify fresh and cooled embryos up to 550 μm, which would prevent the loss of valuable embryos collected in more advanced stages of development. In summary, pregnancies can be obtained after cooling for 18 to 24 h and vitrification of embryos collected 7 or 7.5 days after.
82 CRYOPRESERVATION OF BOAR EPIDIDYMAL SPERMATOZOA; ADDITION OF SEMINAL PLASMA TO THAWING SOLUTION IMPROVES REPRODUCTIVE PERFORMANCE BY ARTIFICIAL INSEMINATION
Authors: Okazaki T. , Akiyoshi T. , Kan M. , Teshima H. , Shimada M.
Published: August 2011

Epididymal spermatozoa are one of the available male germ cells for cryopreservation. It has been reported that frozen–thawed porcine epididymal spermatozoa have a high fertilization competence in vitro as compared with that in ejaculated one. However, there is little information about reproductive performance, such as conception rate or litter size, after artificial insemination (AI) using frozen–thawed epididymal spermatozoa. Recently, we demonstrated that the addition of seminal plasma to thawing solution improves membrane and acrosomal integrity, and enhanced both in vivo and in vitro fertilizing activity of frozen–thawed ejaculated spermatozoa. Moreover, the injection of seminal plasma to uterus with frozen–thawed spermatozoa significantly increased the number of implantation site (Okazaki et al. 2009 Theriogenology 71, 491–498). Thus, to apply those positive functions of seminal plasma to AI using frozen–thawed epididymal sperm, in this study, we added seminal plasma to thawing solution and then analysed the sperm functions including AI test using frozen–thawed epididymal spermatozoa. Epididymal spermatozoa collected by flushing caudal epididymis were frozen as described in our previous study (Okazaki et al. 2009). Frozen-spermatozoa were thawed in Modena solution with or without different percentages of seminal plasma. Protein tyrosine phosphorylation as a marker of capacitation was detected by western blotting. To examine the reproductive performance, the sows of natural oestrus were artificially inseminated two times (5 × 109 50 mL–1 per injection). When the frozen–thawed ejaculated or epididymal sperm was incubated up to 6 h, the motility of epididymal sperm was significantly higher than that of ejaculated sperm (19.6 v. 37.6%). However, the acrosomal membrane was damaged in epididymal sperm group at 3-h incubation period (15.2 v. 36.0%). The addition of seminal plasma [0, 10, 15, 20% (v/v)] in Modena solution protected the acrosomal injury (3 h; 35.2, 19.5, 15.6, 14.6%) and maintained high rate of motility (6 h; 38.8, 48.8, 62.5, 60.0%) in a dose-dependent manner. Furthermore, the addition of seminal plasma suppressed the expression of the 15 kDa phosphoprotein (early capacitation status), and the maximum effect was detected at 15% (v/v) seminal plasma. When the frozen–thawed epididymal spermatozoa with 15% (v/v) seminal plasma were artificially inseminated to swine (n = 15), the conception rate and the mean number of litter size were increased as compared with control (93 v. 43%, 10.0 v. 5.0). From these results, we concluded that the addition of seminal plasma to thawing solution was a beneficial method for artificial insemination using frozen–thawed epididymal spermatozoa in the pig.

This work was supported by the Programme for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry, and JST-Grant (No. 12-068 and No. 12-104).
88 EFFECT OF VITRIFICATION PROCEDURE ON SURVIVAL RATE OF BOVINE EMBRYOS PRODUCED IN VITRO
Authors: Herrera E. Y. , de Frutos C. , Laguna-Barraza R. , Gutierrez-Adan A. , Rizos D.
Published: August 2011

Vitrification as a cryopreservation method has many advantages compared with slow freezing. Many variables in the vitrification process exists that influence the survival rates of vitrified oocytes and embryos. These include the cryoprotectants (type, concentration, and duration of exposure), the temperature of the vitrification solution at exposure, the device used for vitrification, and the quality and developmental stage of embryos. It is worthwhile to mention that vitrification protocols successfully used in bovine oocytes and embryos have been used also with human oocytes and embryos. Vitrification is relatively simple, requires no freezing equipment, and relies on the placement of the embryos in a very small volume of vitrification medium that must be cooled at extreme rates not obtainable in regular enclosed straws. The aim of the present study was to evaluate the efficiency of 4 different vitrification protocols on the survival rate of in vitro produced (IVP) bovine embryos. Blastocysts were produced by a standard IVP procedure following in vitro maturation, fertilization, and culture in synthetic oviduct fluid supplemented with 5% fetal calf serum (FCS). On Day 7 (Day of IVF = Day 0), a total of 297 blastocysts were vitrified using (i) the open pulled straw (OPS) in 20% DMSO and 20% ethylene glycol (EG) in a basal medium of TCM-199 with HEPES supplemented with 20% FCS; (ii) the modified OPS, in 20% DMSO, 20% EG, and 0.5 M sucrose in a basal medium of phosphate buffer saline (PBS) supplemented with 20% FCS; (iii) the cryoloop, in 15% DMSO, 15% EG, 10 mg mL–1 Ficoll 70, and 0.65 M sucrose in a basal medium of PBS supplemented with 20% FCS; and (iv) in 0.25 straws in 20% glycerol, 20% EG, 0.3 M sucrose, 3% polyethylene glycol, and 0.3 M xylose in a basal medium of PBS. After warming, embryos were placed in culture for additional 24 h. Re-expansion and hatching rates were measured at 2 and 24 h after warming. Data were analysed by 1-way ANOVA. At 2 h post-warming, the re-expansion of blastocysts vitrified with cryoloop was significantly higher compared with OPS, modified OPS, and the 0.25 straw methods (54.08 ± 15.53 v. 10.40 ± 3.00, 22.67 ± 9.20, and 8.82 ± 2.15, respectively; P ≤ 0.028). At 24 h post-warming, only embryos from cryoloop and modified OPS were still alive with a survival rate of embryos vitrified with cryoloop significantly higher than that of those vitrified with modified OPS (48.45 ± 17.56 v. 3.75 ± 3.75, respectively; P ≤ 0.007). Hatching rates at 24 h post-warming were not different between cryoloop and modified OPS groups (5.63 ± 4.40 and 1.25 ± 1.25, respectively). These results clearly demonstrate that embryo cryotolerance is affected by the method used for cryopreservation. Moreover, cryoloop vitrification was found to be more effective than OPS and 0.25 straw methods for the cryopreservation of bovine embryos.
90 OPEN PULLED STRAW VITRIFICATION OF IN VITRO PORCINE BLASTOCYTS IN A CHEMICALLY DEFINED MEDIUM
Authors: Sanchez-Osorio J. , Cuello C. , Gomis J. , Maside C. , Gil M. A. , Parrilla I. , Roca J. , Vazquez J. M. , Martinez E. A.
Published: August 2011

The aim of this study was to design a chemically defined medium for the vitrification of in vitro produced porcine blastocysts avoiding the use of serum or serum components. Cumulus–oocyte complexes were matured in vitro in NCSU-23 for 44 h and were inseminated with frozen–thawed spermatozoa. Presumptive zygotes were cultured for 16 h to assess in vitro fertilization (IVF) parameters (N = 200) or for 6 days (N = 600) in order to obtain blastocysts. For chemically delipidation, 10 μM forskolin was added to the culture medium on Day 5 of in vitro culture. On Day 2, embryos were evaluated for cleavage rate. On Day 6, embryos were assessed for blastocyst formation; only those blastocysts showing excellent morphological appearance were selected for vitrification. Blastocysts were vitrified using as basic medium TCM-199 HEPES supplemented with 20% of newborn calf serum (NBCS; n = 65), with 0.1% of polyvinyl alcohol (PVA; n = 64) or without additives (WA; n = 65). The OPS-vitrification and warming were performed as described by (Sanchez-Osorio et al. 2010 Theriogenology 73, 300–308) using 16% of Etylenglycol and 16% of dimetyl sulfoxide as final concentrations of cryoprotectants. Vitrified blastocysts were warmed and cultured in vitro for 24 h to assess their viability. Blastocysts that totally reformed their blastocoel cavity showing a normal or excellent morphology were considered viable. In addition, after in vitro culture vitrified-warmed viable embryos were fixed in 4% paraformaldehyde in PBS medium and stained with Hoechst 33342 in order to assess the total number of cells. Data were analysed by using the MIXED procedure of SPSS. The threshold for significance was set at P < 0.05. Results are expressed as least squares means ± SEM. The maturation, penetration, and monospermy rates were 98.5 ± 1.2%, 85.3 ± 3.6%, and 48.8 ± 5%, respectively. The efficiency of IVF (defined as the ratio of monospermic oocytes to the total number of inseminated oocytes) was 41.0 ± 4.9%. The values of cleavage rate at Day 2 and blastocysts formation rate were 67.8 ± 1.4% and 37.3 ± 1.6%, respectively. After vitrification and warming, similar survival rates were observed for NBCS (33.8 ± 5.9) PVA (40.6 ± 6.0), and WA (30.8 ± 5.9) groups. No significant differences were found for the total number of cells (ranged from 35.4 ± 6.8 to 50.8 ± 8.3) among vitrification groups. In conclusion, in vitro derived porcine blastocysts can be vitrified in the absence of serum and serum components. Furthermore, PVA is a suitable substitute for serum in vitrification solutions with no detrimental effect on the viability of in vitro produced pig blastocysts.

This study was supported by the Seneca foundation of Murcia (GERM 04543/07).
97 IMPROVED CRYOPRESERVATION OF DOMESTIC CAT SPERMATOZOA IN A SOY LECITHIN-BASED EXTENDER
Authors: Vick M. M. , Bateman H. L. , Swanson W. F.
Published: August 2011

Development of a chemically defined, plant-based cryopreservation media would reduce extender variability and the potential for transmission of zoonotic pathogens compared with traditional egg-yolk-based extenders. The objective of this study was to compare effects of egg yolk- and soy lecithin-based cryopreservation media and the temperature of glycerol addition on sperm parameters following freezing and thawing of domestic cat spermatozoa. Fresh semen was collected by manual stimulation on 3 separate occasions from 4 adult male cats. Each ejaculate was washed to remove seminal plasma, divided into 4 equal aliquots, and extended at room temperature in one of the following treatments: 1) TEST-egg yolk (Irvine Scientific Inc., Santa Ana, CA, USA) medium with 4% glycerol (EYG); 2) TEST-egg yolk, with 4% glycerol added after cooling to 5°C (EY); 3) TES-Tris buffer with soy-lecithin (1%) and 4% glycerol (SLG); and 4) TES-Tris buffer with 1% soy-lecithin, and 4% glycerol added after cooling to 5°C (SL). Sperm progressive motility (%) and rate of progressive movement (scale of 0–5) were evaluated at 0, 1, 3, 6, and 24 h post-thaw. Sperm capacitation (chlortetracycline staining), acrosome integrity (FITC-PNA staining), and DNA integrity (acridine orange staining) were assessed at 15 min post-thaw. Data were exponentially transformed to achieve normal distribution and then subjected to GLM analysis to determine effects of media and temperature of glycerol addition on sperm traits. At 0 and 1 h post-thaw, acrosome integrity, DNA integrity and % sperm motility did not differ (P > 0.05) among treatments. However, % sperm motility was greater in the soy-based media compared to egg yolk-based media at 3, 6, and 24 h post-thaw (Table 1; P < 0.05). A higher percentage of uncapacitated spermatozoa were present in soy-based compared to egg-yolk based cryopreservation media (63.9 ± 9.3 v. 51.2 ± 11.5, respectively; P < 0.05), regardless of temperature of glycerol addition. Finally, addition of glycerol at 5°C resulted in higher % sperm motility compared to room temperature at 6 and 24 h post-thaw in both medium types (Table 1; P < 0.05). Our results suggest that use of a chemically defined, soy-based medium improves long-term motility and capacitation status of frozen–thawed domestic cat spermatozoa compared with cryopreservation in a traditional egg yolk-based extender.

Table 1.Motile spermatazoa and motility score at 3, 6, and 24 h
Can bare ground cover server as a surrogate for plant biodiversity in grazed tropical woodlands?
Authors: McCosker Juliana , Rolfe John , Fensham Rod
Published: August 2011

A bare ground index derived by remote sensing would provide a rapid methodology for assessing the biodiversity condition of an ecosystem, providing that ground cover is a satisfactory correlate with key biodiversity attributes. The relationship between plant species richness and the abundance of individual species was examined in relation to ground cover within silver-leaved ironbark (Eucalyptus melanophloia F. Muell.) woodlands in the Desert Uplands bioregion of north-eastern Australia. There was significant correlation between the bare ground index and ground cover and biomass measurements. Twenty-four species, including the perennial grasses Sehima nervosum (Rottler) Stapf, Themeda triandra Forssk. and Bothriocloa ewartiana (Domin) C.E. Hubb., were significantly and negatively correlated with bare ground. Scleroleana birchii (F. Muell.) Domin and Sida fibulifera Lindl.displayed significant positive relationships with increasing bare ground, and where abundant indicate heavy grazing in this land type. The study suggests that satellite-derived data does provide a meaningful methodology for assessing vegetation condition although it is strongly associated with seasonal conditions, but is only useful in relation to the regional average for a land type. The findings suggest that plant diversity is maintained at a relatively high level throughout most of these woodlands in the Desert Uplands.
A conservation auction for landscape linkage in the southern Desert Uplands, Queensland
Authors: Windle Jill , Rolfe John , McCosker Juliana , Lingard Andrea
Published: August 2011

Conservation auctions are a type of market-based instrument (MBI) that can achieve a more cost-efficient allocation of public funds than approaches such as devolved grants. In this paper, the conduct of a multiple round conservation auction to improve biodiversity management in a rangelands area is outlined. The auction was designed to develop a wildlife corridor across the southern Desert Uplands bioregion in Queensland and to improve management of rangelands areas.
The conservation auction incorporated two important new design features. First, there was a need to promote landholder cooperation so that proposed areas for better land management were aligned and connected across the region. The second innovative design feature was to hold multiple bidding (three) rounds, which differs from the standard application of a single bidding round.
The auction outcomes resulted in conservation contracts covering 85 000 ha of remnant vegetation awarded at an average cost of $2* per hectare per annum. Although complete landscape connectivity across the Desert Uplands was not achieved, over 70% of the successful bids, accounting for over 62 000 ha (77% of the total bid area), were part of a group that formed a distinct corridor or landscape linkage with only single or part-property gaps. The results also indicate that multiple bidding rounds improved auction efficiency (for the government), although there was little improvement in connectivity. Sixty-six percent more environmental benefit units could be purchased for the given budget of $350 000 between rounds one and three.
Kangaroos in the rangelands: opportunities for landholder collaboration
Authors: Baumber A. , Cooney R. , Ampt P. , Gepp K.
Published: August 2011

For 3 years, the Future of Australia’s Threatened Ecosystems (FATE) Program has been working towards achieving multiple benefits for rangelands by applying conservation through sustainable use (CSU) approaches to the kangaroo industry. A critical component of this work is landholder involvement in kangaroo management that results in commercial gain. We are developing strategies for landholders to add value to the harvest at the same time as achieving better control over the impact that kangaroos can have on their land.
This paper outlines FATE’s experiences with two related initiatives exploring landholder involvement in kangaroo harvest in the rangelands. First, a trial in the Barrier Ranges of north-western New South Wales demonstrates the potential benefits of collaboration for landholders in reducing their exposure to kangaroo harvest variability and the associated business risks. Second, an analysis of the various enterprise models which landholders could employ to enter the kangaroo industry identifies opportunities for landholders and kangaroo harvesters to collaborate for mutual benefit. Several challenges exist in bringing these potential benefits to fruition.
The paper includes: (1) analysis of harvest data across collaborating properties; (2) progress towards allocation of harvest tags on a group rather than an individual property basis; (3) results of discussions between key stakeholders; and (4) a description of models for landholder involvement and analysis of the extent to which they can achieve multiple benefits.
Defining and predicting the 'break of the season' for north-east Queensland grazing areas
Authors: Balston Jacqueline , English Bernie
Published: August 2011

For pasture growth in the semi-arid tropics of north-east Australia, where up to 80% of annual rainfall occurs between December and March, the timing and distribution of rainfall events is often more important than the total amount. In particular, the timing of the ‘green break of the season’ (GBOS) at the end of the dry season, when new pasture growth becomes available as forage and a live-weight gain is measured in cattle, affects several important management decisions that prevent overgrazing and pasture degradation. Currently, beef producers in the region use a GBOS rule based on rainfall (e.g. 40 mm of rain over three days by 1 December) to define the event and make their management decisions. A survey of 16 beef producers in north-east Queensland shows three quarters of respondents use a rainfall amount that occurs in only half or less than half of all years at their location. In addition, only half the producers expect the GBOS to occur within two weeks of the median date calculated by the CSIRO plant growth days model GRIM. This result suggests that in the producer rules, either the rainfall quantity or the period of time over which the rain is expected, is unrealistic. Despite only 37% of beef producers indicating that they use a southern oscillation index (SOI) forecast in their decisions, cross validated LEPS (linear error in probability space) analyses showed both the average 3 month July–September SOI and the 2 month August–September SOI have significant forecast skill in predicting the probability of both the amount of wet season rainfall and the timing of the GBOS. The communication and implementation of a rigorous and realistic definition of the GBOS, and the likely impacts of anthropogenic climate change on the region are discussed in the context of the sustainable management of northern Australian rangelands.
Critical success factors of a whole of business extension approach for increased capacity of beef producers and improved enterprise profit and sustainability
Authors: Nelson B. S. , Robinson E.
Published: August 2011

The ‘Research to Reality Project’ assisted beef producers in the Burdekin catchment of north Queensland to develop practical responses to a range of production and grazing land management challenges. The project involved three groups of beef producers encompassing 19 enterprises, 680 000 ha of land and the management of 162 000 cattle. The project was founded on a continuous improvement and innovation approach, and included an employed industry champion and multi-disciplinary project team who used a range of extension methods to identify, develop and implement on-property projects. Extension methods included one-on-one property planning activities, economic benchmarking, land condition assessments, on-property demonstrations, structured learning workshops and information products. The value of project evaluation to producers and the project staff is discussed. Further, the link between increased profitability and better land management was established for many producers. This learning is reflected in the uptake of recommended grazing practices and the decisions producer participants are now making about their grazing business.
Land condition monitoring information for reef catchments: a new era
Authors: Karfs R. A. , Abbott B. N. , Scarth P. F. , Wallace J. F.
Published: August 2011

Land condition monitoring information is required for the strategic management of grazing land and for a better understanding of ecosystem processes. Yet, for policy makers and those land managers whose properties are situated within north-eastern Australia’s vast Great Barrier Reef catchments, there has been a general lack of geospatial land condition monitoring information. This paper provides an overview of integrated land monitoring activity in rangeland areas of two major Reef catchments in Queensland: the Burdekin and Fitzroy regions. The project aims were to assemble land condition monitoring datasets that would assist grazing land management and support decision-makers investing public funds; and deliver these data to natural resource management (NRM) community groups, which had been given increased responsibility for delivering local environmental outcomes. We describe the rationale and processes used to produce new land condition monitoring datasets derived from remotely sensed Landsat thematic mapper (TM) and high resolution SPOT 5 satellite imagery and from rapid land condition ground assessment. Specific products include subcatchment groundcover change maps, regional mapping of indicative very poor land condition, and stratified land condition site summaries. Their application, integration, and limitations are discussed. The major innovation is a better understanding of NRM issues with respect to land condition across vast regional areas, and the effective transfer of decision-making capacity to the local level. Likewise, with an increased ability to address policy questions from an evidence-based position, combined with increased cooperation between community, industry and all levels of government, a new era has emerged for decision-makers in rangeland management.
Penile oil injection, penile implantation and condom use among Myanmar migrant fishermen in Ranong, Thailand
Authors: Ohnmar  , Geater Alan F. , Winn Than , Chongsuvivatwong Virasakdi
Published: August 2011

Background: The present study describes sexual risk behaviours including penile oil injection and implantation among migrant Myanmar fishermen in Thailand, and their determinants. Methods: A cross-sectional survey among 639 Myanmar fishermen in Ranong, Thailand was conducted in 1999 following in-depth interviews. Results: Prevalence of penile oil injection, penile implantation, and both practices were 48 (7.5%), 79 (12.4%) and nine (1.4%) respectively. Over one-third (38.6%) of those who had injected oil had various kinds of problems during sex. One-year period prevalence of sex with commercial sex workers (CSW) was higher in those with unusual practices than others (61.9% v. 22.8%, P < 0.0001). The condom use rate with CSW at all times was non-significantly lower among those with unusual practices (32.9%) than others (44.5%). From multivariate analysis, oil injections are associated with young age, low education, ethnicity, and longer stay in Thailand, whereas the duration of stay in Thailand is the only factor associated with the practice of penile implantation. Both oil injection (odds ratio 3.03, 95% confidence interval 1.39–6.60), and penile implantation (odds ratio 2.18, 95% confidence interval 1.19–3.99) have independent predictive risk of having sex with CSW in addition to other factors (younger age, single, and Walat boat type). Conclusion: These exorbitant practices were common, associated with not using condoms and probably resulted from the working environment in Thailand. Further study is needed to reduce this risk behaviour.
The incidence of genital warts in Australian women prior to the national vaccination program
Authors: Brotherton Julia M. L. , Heywood Anita , Heley Stella
Published: August 2011

The quadrivalent human papillomavirus (HPV) vaccine currently being delivered to Australian women aged 12–26 years under the National HPV Vaccination Program promises to substantially reduce the incidence of genital warts. We review what is known about the burden of genital warts among Australian women. Incidence appears to peak among women aged 20–24 years, of whom 1.4% report genital warts in the previous year and who are hospitalised for treatment at a rate of 26 per 100 000. A surveillance system capable of documenting any decrease in the incidence of genital warts and recurrent respiratory papillomatosis after vaccination is urgently required.
Sexual transmission of intestinal parasites in men who have sex with men
Authors: Abdolrasouli Alireza , McMillan Alexander , Ackers John P.
Published: August 2011

Direct oral-anal sexual contact is a common practice among men who have sex with men (MSM) and is implicated in the transmission of various enteric pathogens including intestinal parasites. The present study reviewed data on the sexual transmission of intestinal parasites among MSM, and highlighted advances in the diagnosis of such infections. The emergence and spread of intestinal parasites is of public health concern particularly in the homosexual community. Intestinal parasitic infection should be considered in the differential diagnosis of gastrointestinal disease in this population. Combination of traditional diagnostic procedures with implementation of testing based on novel molecular methods in the accurate identification of intestinal parasites is important so that early intervention and control of infection is facilitated.