bluuebunny/crossref_metadata_2025 huggingface dataset:
8 THE USE OF ANNEXIN V MAGNETIC-ACTIVATED CELL SORTING TO SEPARATE APOPTOTIC SPERM FROM THE EJACULATE OF STALLIONS
Authors: Coutinho da Silva M. A. , Pinto C. R. F. , Young J. M. , Cole K.
Published: August 2011

Magnetic-activated cell sorting (MACS) has been used successfully in humans to remove apoptotic sperm from the ejaculate. Annexin V-conjugated microbeads recognise sperm with externalized phosphatidylserine, which is considered one of the features of apoptosis, and the labelled sperm is separated by MACS. The goals of the study were to determine if MACS can be used to separate apoptotic sperm from the ejaculate of stallions; and to determine if removal of apoptotic sperm improves the quality of stallion sperm. Our hypothesis was that MACS would improve semen quality by removing apoptotic sperm, resulting in samples with higher motility and viability. Two ejaculates from three different stallions of good fertility were used. Sperm were diluted with Tyrode’s albumin lactate pyruvate (TALP) and incubated with annexin V-conjugated microbeads for 15 min at 37°C. Control samples were incubated in the absence of annexin V microbeads. The suspension was then loaded into the separation column containing iron globes, which were fitted in a magnet (MiniMACS; Miltenyi Biotec Inc., Auburn, CA, USA). The effluent sample containing annexin-negative sperm was collected and then, the column was removed from the magnetic field and rinsed with TALP to collect the annexin-positive cells. Sperm viability, motility, morphology and caspase activation were determined in all three samples: control, annexin-negative, and annexin-positive. Data were evaluated by ANOVA and individual comparisons were performed by Tukey’s hsd test. Significance was set at P < 0.05 and data is presented as means ± SEM (Table 1). The main effect of stallion was significant only for sperm motility parameters. Sperm recovery rate following MACS was 46 ± 3%. In conclusion, the use of MACS was effective in removing apoptotic sperm from the ejaculate. The annexin-positive population displayed a higher proportion of sperm with activated caspases and lower membrane integrity and motility. However, removal of apoptotic sperm from the ejaculate did not improve sperm parameters in the annexin-negative group compared to control group. In addition, sperm morphology was not affected by MACS. Further studies are necessary to determine if MACS could be used successfully to improve sperm quality from subfertile stallions and frozen semen.

Table 1.Sperm parameters following annexin V MACS (mean ± SEM)

The authors are thankful to Mark Williams at Miltenyi Biotec Inc. for providing supplies; and Dr Ashok Agarwal at The Center for Reproductive Medicine, Cleveland Clinic, for scientific input.
81 HIGH THROUGHPUT VITRIFICATION OF IN VIVO FERTILIZED AND IN VITRO CULTURED PORCINE EMBRYOS
Authors: Murphy C. N. , Spate L. D. , Bauer B. K. , Prather R. S.
Published: August 2011

One barrier to successfully making embryo transfer viable in the swine industry is an inability to consistently cryopreserve oocytes and embryos. This process is made difficult by the high lipid content of porcine oocytes and embryos. The objective of this study was to test the in vivo fertilized embryo’s sensitivity to vitrification. Gilts were inseminated on the first day of standing oestrus (Day 0) and then again 12 h later. On Day 2 the oviducts and tip of the uterine horns were flushed with PVA-treated TL-HEPES and 2-cell stage embryos were collected and placed into PVA-treated TL-HEPES and centrifuged at 17 000 × g. The treatment groups were 1) 300 mOsmo centrifuged for 6 min, 2) 500 mOsmo centrifuged for 6 min, 3) 500 mOsmo centrifuged for 12 min, and 4) 500 mOsmo centrifuged for 18 min. After centrifugation the embryos were transferred to Porcine Zygote Medium 3 (PZM3) and cultured to Day 6 or 7 at which point blastocysts were vitrified using 10% DMSO, 10% ethylene glycol in M199 supplemented with 20% FBS (holding medium) for 2 min. Embryos were transferred to holding media with 20% DMSO and 20% ethylene glycol and drawn into an open pulled straw via capillary reaction; it was then submerged into LN2. Embryos were thawed using a step down concentration of 0.33 mM and then 0.2 mM sucrose in holding media each for 6–7 min and then were moved to holding medium alone for 6 to 7 min. The embryos were washed in PZM3, then transferred to 500 μL of PZM3 and cultured for 18 h. Re-expanded embryos were observed, and the nuclei of all embryos were stained with Biz-benzimide and visualised with UV light to determine total cell number. After the embryos were centrifuged and cultured, there was no difference in development to blastocyst (SAS Institute, Cary, NC, USA; Proc GLM) with a mean percentage blastocyst of 85.1% and an N of 54, 51, 53, and 51, respectively, for each treatment. After thawing, percentage of embryos re-expanded was 23.5a, 26.4a,b, 43.2a,b, and 45.6b, respectively. Data was analysed using a PROC GLM in SAS (P < 0.05), with 37, 43, 30, and 36 embryos in each group, respectively. No difference in total cell number across treatments was detected after analysis using PROC GLM in SAS (P < 0.05) with a mean cell number of 29.0. These data suggest that in vivo matured and fertilized blastocysts can survive high osmolarity treatment, centrifugation, and vitrification. The data also show that a high osmolarity treatment centrifuged for 18 min leads to a greater number of re-expanded embryos post-thaw, which may be attributed to better separation of the lipid.

Funded by the NIH NCRR R21RR025879 and Food for the 21st Century.
83 EFFECTS OF CYTOCHALASIN B AND VITRIFICATION PROCEDURE ON SURVIVAL AND IN VITRO MATURATION OF SWAMP BUFFALO OOCYTES CRYOPRESERVED AT THE IMMATURE STAGE
Authors: Liang Y. Y. , Phermthai T. , Nagai T. , Somfai T. , Parnpai R.
Published: August 2011

Cryopreservation of oocytes from swamp buffaloes of high genetic and individual values is important for conservation of animal genetic resources. This study investigated the effect of cytochalasin B (CB) pretreatment and the efficiencies of solid surface vitrification (SSV) and cryotop (CT) vitrification methods to cryopreserve immature buffalo oocytes. Cumulus oocyte complexes harvested from abattoir-derived ovaries were randomly separated into 5 groups: 1) CT method with pretreatment by 7.5 μg mL–1 CB in base medium (BM, TCM-199 HEPES+20% FBS) for 15 min [CT(+)CB]; 2) CT method without CB pretreatment [CT(–)CB]; 3) SSV method with pretreatment by 7.5 μg mL–1 CB in BM for 15 min [SSV(+)CB]; 4) SSV method without CB pretreatment [SSV(–)CB]; and 5) fresh control. Immature oocytes were partially denuded, and those with 2 layers of cumulus cells were placed in 10% dimethylsulfoxide (DMSO) + 10% ethylene glycol (EG) for 1 min followed by exposure to 20% DMSO + 20% EG + 0.5 M sucrose in BM for 30 s. The oocytes were then vitrified either by SSV or CT method and stored in liquid nitrogen. Vitrified oocytes were thawed by placing them directly in warming solution (0.5 M sucrose in BM) for 5 min at 38.5°C and then washed in BM for 5 min. Oocyte viability was assessed by fluorescein diacetate staining. Surviving oocytes were in vitro matured (IVM) for 23 h (Parnpai et al. 1999 Buffalo J. 15, 371–384) and their meiotic progression was investigated by nuclear staining. Data were analysed by 1-way ANOVA. The survival rate of oocytes in CT groups [CT(+)CB, 82%; CT(–)CB, 82%] was significantly lower (P < 0.05) than that of the control group (100%), but significantly higher (P < 0.05) than those in the SSV groups [SSV(+)CB, 71%; SSV(–)CB, 72%]. After IVM, the proportions of viable oocytes were similar among vitrified groups [CT(+)CB, 84%; CT(–)CB, 86%; SSV(+)CB, 80%; SSV(–)CB, 79%], but significantly lower than those of the control group (96%). Pretreatment with CB did not increase oocyte survival. Among vitrified groups the highest maturation rate was obtained in CT(–)CB (32%), which was significantly higher than that of SSV(+)CB (13%). Vitrified oocytes in all groups reached metaphase-II at significantly lower rates compared with the control group (55%). In conclusion, CB did not improve the survival and maturation of vitrified oocytes. Higher survival and maturation rates in CT groups indicate the superiority of this method.

This work was supported by Thailand Research Fund, Suranaree University of Technology and Japan Society for the Promotion of Science – National Research Council of Thailand (JSPS-NRCT) scientific cooperation program.
84 COMPARISON BETWEEN IN VIVO AND IN VITRO PRODUCED EMBRYOS WITH FORSKOLIN AFTER VITRIFICATION
Authors: Paschoal D. M. , Sudano M. J. , Rascado T. S. , Magalhães L. C. O. , Crocomo L. F. , Lima-Neto J. F. , Martins Júnior A. , Landim-Alvarenga F. C.
Published: August 2011

The high concentration of lipids on embryos reduces their viability after cryopreservation. The use of drugs to modify their metabolism has been used to produce embryos with greater resistance to cryopreservation. The present experiment aimed to induce cytoplasmic lipolysis in in vitro produced (IVP) bovine embryos using forskolin (Sigma-Aldrich, St. Louis), which raises the levels of intracellular cAMP. Nelore cow cumulus–oocyte complexes (COC) were matured in TCM 199 and fertilized with frozen Nelore bull semen. Presumptive zygotes were cultured in SOFaa supplemented with BSA in the presence of 2.5% FCS. Embryos were kept in a humidified atmosphere with 5% CO2, 5% O2, and 90% N2 at 38.5°C. On Day 6, embryos were divided into 2 groups: 2.5% FCS (2.5% FCS without forskolin) and 2.5% FCS + F (2.5% FCS plus 10 μM forskolin). Embryo cleavage was recorded on Day 3 (IVF: D0), and blastocyst production on Day 7. Embryo viability was estimated by the index of total number of cells per embryos observed after the staining with propidium iodite and Hoechst 33342. In vitro produced embryos were compared with embryos obtained in vivo from Nelore cows. Embryos were vitrified using the protocol developed by Campos-Chillòn et al. (2006). After thawing, the re-expansion rate and cell number were again estimated (after 12 h). For statistical analysis, percentage cleaved and percentage blastocyst, percentage re-expansion, and total number of cells were transformed using the arcsine transformation (√y/100) and analysed using ANOVA followed by Tukey’s test. The level of significance adopted was 5%. No statistical differences were observed between IVP embryos concerning cleavage (2.5% FCS: 87.48 ± 9.52 and 2.5% FCS + F: 85.13 ± 7.57) and blastocyst formation rates (2.5% FCS: 46.8 ± 13.28 and 2.5% FCS + F: 46.31 ± 11.39). Also, no differences were detected in total number of cells per embryos (2.5% FCS: 162.4 ± 43.3; 2.5% FCS + F: 147.6 ± 35.3 and in vivo: 143.5 ± 11.5) when IVP and in vivo produced embryos were compared. After vitrification the re-expansion rate was similar between IVP and in vivo produced embryos (2.5% FCS: 75.07 ± 9.81; 2.5% FCS + F: 81.09 ± 10.90 and in vivo: 86.40 ± 18.62). But the total cell number of IVP embryos was significantly lower than the in vivo produced embryos [2.5% FCS: 42.6 ± 17.2a (P < 0.001); 2.5% FCS + F: 59.9 ± 46.2a (P < 0.05) and in vivo: 124.2 ± 12.9b]. If one side the viability cell between IVP and in vivo produced embryos is the same, after vitrification in vivo produced embryos showed greater resistance after the vitrification. The addition of forskolin did not interfere in embryo production or quality, but this drug was not efficient in increasing embryo tolerance after vitrification.

FAPESP 07/53505-1.
85 DOMESTIC CAT KITTENS BORN AFTER TRANSFER OF CRYOPRESERVED EMBRYOS PRODUCED BY IN VITRO FERTILIZATION OF OOCYTES WITH FLOW-SORTED SPERM
Authors: Pope C. E. , Crichton E. G. , Gómez M. C. , Dumas C. , Dresser B. L.
Published: August 2011

For successful application of sperm sorting technology to a particular species, the various assisted reproductive techniques required for in vivo or in vitro embryo production must be established. Previously, we have demonstrated the in vivo developmental competence of cat embryos derived by IVF 1) with presumptive X-chromosome bearing sperm and 2) after controlled-rate cryopreservation. Here, our intent was to determine in vivo viability by transfer to recipients of cryopreserved cat embryos produced by IVF with flow-sorted sperm. Semen collected from a single male using an artificial vagina was extended in electrolyte-free solution and shipped overnight (4°C) to the sorting facility. After sorting immediately upon arrival, sperm were concentrated by centrifugation, suspended in TEST yolk buffer, and return-shipped to the IVF laboratory at 4°C. At 48 h after semen collection, in vivo matured oocytes (n = 64) recovered from 2 gonadotropin-treated donors were inseminated in vitro with presumptive X-chromosome-bearing sperm. At 5 h post-insemination, oocytes were rinsed and placed in modified Tyrodes+BSA and NEAA (IVC-1) at 38°C in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2. At 27 h post-insemination (Day 1), 43 two-cell embryos (67% cleavage rate) were equilibrated (3 steps) in 1.4 M propylene glycol, 0.125 M sucrose, and 10% dextran 70, cooled at a slow controlled rate (0.3°C min–1) from –6°C to –30°C, and plunged into liquid nitrogen. After 26 to 43 months in cryostorage, 4 straws, each containing 8 or 9 embryos, were thawed in air (22°C) and cryoprotectant was removed from the embryos by a 5-step rinse. Then, embryos were cultured for 17 to 20 h in IVC-1, as described earlier. On Day 2, 35 embryos were transferred (8 or 9 per recipient) by laparoscopy to the oviduct of four Day 1 gonadotropin-treated recipients at 24 h after laparoscopic oocyte retrieval (16 to 25 oocytes/donor). Most embryos (83%) were at the four- to eight-cell stages at transfer. Three recipients established pregnancies and delivered, without assistance, litters of 1, 2, and 3 live kittens (27% embryo survival) between Day 64 and Day 67 of gestation. Four kittens were female, and two were male. Birth weights ranged from 73 to 133g (mean = 117 g). Overall, of 18 kittens [12 from fresh (2009 Theriogenology, 71, 864) +6 from frozen embryos] born after transfer of embryos produced by IVF with sex-sorted sperm, 16 (89%) have been female. The variation in sex ratios between kittens born from fresh v. cryopreserved embryos probably is a reflection of the sort purity of the different sperm samples used for IVF. The ability to produce kittens after transfer of cryopreserved embryos derived by in vitro insemination with sex-sorted sperm represents an expansion in the assortment of assisted reproductive techniques that may be useful for selective propagation of biomedically relevant and endangered felids.
86 EFFECT OF CRYOPROTECTANT EXPOSURE, VITRIFICATION, AND WARMING TIME OF BOVINE CUMULUS OOCYTE COMPLEXES ON IN VITRO FERTILIZATION AND EMBRYONIC DEVELOPMENT
Authors: Prentice J. R. , Singh J. , Mapletoft R. J. , Anzar M.
Published: August 2011

Despite the importance of cryoprotectants for avoiding ice crystal formation, the high concentrations required for vitrification may be toxic to bovine oocytes. During warming (thawing), the removal of permeating cryoprotectants from cells can lead to osmotic injury, and the most appropriate time interval for warming and cryoprotectant removal from vitrified oocytes is currently uncertain. The present study aimed to evaluate the effect of cryoprotectant exposure, vitrification, and warming time of bovine cumulus oocyte complexes (COC) on fertilization and ability to develop as embryos in vitro. Follicles <8 mm in diameter were aspirated from slaughterhouse-derived bovine ovaries. Cumulus oocyte complexes with ≥3 layers of cumulus cells and a uniform cytoplasm were selected, washed 3 times in Dulbecco’s PBS + 5% newborn calf serum (CS), and randomly divided into 4 groups: 1) control group: no treatment; 2) VS1 group: COC were exposed to vitrification solution 1 [VS1: 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) in TCM-199 + 20% CS] for 5 min; 3) VS1+VS2 group: COC were exposed to VS1 for 5 min followed by vitrification solution 2 (VS2: 15% EG, 15% DMSO, and 0.5 M sucrose in TCM-199 + 20% CS) for 30 s; and 4) vitrified group: COC were exposed to VS1 and VS2, and then vitrified in liquid nitrogen using cryotops. The COC in VS1, VS1+VS2, and vitrified groups were exposed to a warming solution (0.5 M sucrose in TCM-199) for 1 or 5 min. The COC from all groups were in vitro matured (IVM) for 22 h in TCM-199 containing 5% CS, 5 μg mL–1 LH, 0.5 μg mL–1 FSH, and 0.05 μg mL–1 gentamicin at 38.5°C, 5% CO2, and high humidity, incubated with frozen–thawed sperm in Brackett-Oliphant capacitating medium for 18 h, and the presumptive zygotes were cultured in Charles Rosenkrans 1 amino acids (CR1aa) + 5% CS for 9 days. Data were analysed using Proc Glimmix in SAS® 9.2 (SAS Institute Inc., Cary, NC, USA). Cleavage and blastocyst rates in the vitrified group (25 and 2%, respectively) were significantly lower (P < 0.0001) than in control (75 and 27%), VS1 (68 and 19%), or VS1+VS2 (63 and 22%) groups. Cleavage and blastocyst rates did not differ among non-vitrified groups (P > 0.05). In VS1, VS1+VS2, and vitrified groups, warming time had no effect on cleavage or blastocyst rates (P > 0.05). In conclusion, although cryoprotectant exposure and warming times had no apparent adverse effect, vitrification of bovine COC drastically reduced cleavage and blastocyst rates. Further studies are required to understand how vitrification of bovine COC affects subsequent fertilization and embryo development.

This study was supported by the Canadian Animal Genetic Resources Program, Agriculture and Agri-Food Canada.
87 EFFECT OF CRYOPROTECTANT CONCENTRATION IN THE VITRIFICATION SOLUTION ON THE ZONA PELLUCIDA HARDENING AND SPERMATOZOA PENETRATION OF BOVINE OOCYTES
Authors: Quiñones Martorello A. , Rios G. , Cano A. , Alberio R. H.
Published: August 2011

In the murine model, it has been shown that the high concentration of cryoprotectants required for vitrification can activate the oocytes through a process mediated by calcium influx. This activation induces the zona pellucida (ZP) hardening and affects the sperm penetration. This study aimed to evaluate the effect of exposure of bovine oocytes to the vitrification solutions (VS1 and VS2) in calcium-free medium with 3 concentrations of etilenglycol (EG) and dimetylsulfoxide (DMSO) on the oocyte activation. Cumulus oocyte complexes (COC) were matured in vitro (22 h), partially denuded through pipetting in medium with hyaluronidase, and subject to four treatments: T1, untreated (control); T2, exposed to 20% EG+0% DMSO (VS1) and then 40% EG+0% DMSO (VS2); T3, 10% EG+10% DMSO (VS1) and then 20% EG+20% DMSO (VS2); and T4, 0% EG+20% DMSO (VS1) and then 0% EG+40% DMSO (VS2). The contact with each VS was 3 min and 30 s, respectively. After this, the COC were matured up to 24 h. In Expt. 1, COC were denuded and placed in a solution of pronase E in PBS (1 mg mL–1) to determine the number of oocytes with ZP digested after 9 min of exposure to the enzyme. In Expt. 2, COC were fertilized in TALP medium with 50 mg mL–1 heparin and 1 million mL–1 sperm. After 12 h, COC were denuded and stained with bisbenzimide (Hoechst 33342) and examined under epi-fluorescence. The number of oocytes indicating spermatic penetration was determined by presence of intact sperm heads, spermatic pro-nucleus, or 2 polar bodies. Data were analysed by the PROC GENMOD (SAS; see Table 1). In Expt. 1, there were no differences in the percentage of oocytes without ZP after pronase treatment in groups T1, T2, and T3. The T4 group had the lowest percentage of digestion, and T3 was not different from T4. In Expt. 2 there were no differences in the percentage of sperm penetration between T2, T3, and T4. All treatments had lower values than T1. In conclusion, bovine oocytes undergo hardening of the ZP when put in contact with the cryoprotectants, and this effect was significantly increased with the use of DMSO. Moreover, there was a decrease in sperm penetration in all treated groups, indicating that the natural blocking of polyspermy depends not only on the hardening of the ZP, but another process that could act at the plasma membrane. It is possible that cryoprotectants, regardless of their concentration, may trigger this early block through a mechanism that would be independent of calcium.

Table 1.Effect of EG and DMSO concentration in the VS on the ZP hardening and sperm penetration of bovine oocytes exposed to these solutions

Acknowledgment: the National Research Agency through the grant PICT 2007/1205.
95 THE EFFECT OF FREEZING TECHNIQUES ON QUALITY OF CAT TESTICULAR SPERM
Authors: Techakumphu M. , Buarpung S. , Tharasanit T.
Published: August 2011

Cryopreservation of testicular tissue is beneficial for valuable animals that die unexpectedly or when elective castration is required. Until recently, knowledge regarding cryopreservation of testicular tissue/sperm in the domestic cat has been limited. The objective of this study was to determine the effects of freezing techniques and cryoprotectants on quality of testicular sperm. In Experiment 1, each testis was cut into 10 equal small pieces (∼2 × 3 × 5 mm) and cryopreserved in freezing medium containing with 5% (v/v) glycerol using conventional (10 min in liquid nitrogen vapors) or controlled-rate freezing techniques. In Experiment 2, testicular tissues were conventionally frozen with different types of 5% (v/v) cryoprotectants [glycerol (Gly), dimethylsulphoxide (DMSO), 1,2-propanediol (PrOH), or ethylene glycol (EG)]. Non-cryopreserved testicular sperm was used as a control. After thawing, testicular sperm were extracted and examined for viability and DNA integrity using non-membrane-permeable DNA staining (Ethidium homodimer-1) and TUNEL assay, respectively. Viability of testicular sperm cryopreserved by controlled-rate cryopreservation (45.9 ± 3.7) was significantly lower than non-frozen control (60.3 ± 0.9) and conventional freezing technique (55.0 ± 2.7). Gly (58.2 ± 2.6) and EG (53.3 ± 2.3) yielded a similar viability compared with non-frozen control (P > 0.05), whereas DMSO and PrOH demonstrated an inferior cryoprotectant for feline testicular sperm (% viability for DMSO and PrOH: 46.3 ± 3.3 and 44.3 ± 2.9, respectively). In both experiments, DNA integrity of frozen–thawed testicular sperm did not significantly differ from the control group. In conclusion, cat testicular tissue can be frozen as small pieces using both conventional technique and controlled rate freezing. However, freezing technique and type of cryoprotectant markedly affect the post-thawed quality of testicular sperm. Further study requires examination of the optimal cooling rate during cryopreservation and also the fertilizability of frozen–thawed testicular sperm.

This study was financially supported by CHE-TRF Senior Research Scholars RTA-5080010.
96 THE EFFECT OF CHEMICAL DELIPIDATION ON CRYOPRESERVABILITY OF CAT EMBRYOS
Authors: Tharasanit T. , Techakumphu M.
Published: August 2011

Embryo cryopreservation is a desired technique for long-term preservation of embryos. However, the success rate of cryopreserved in vitro produced cat embryos is currently poor. Until recently, the mechanism underlying the cause of cryoinjury that occurs during cooling and cryopreservation is not well understood. This study aimed to examine the effect of chemical delipidation (forskolin) before cryopreservation of 4- to 8-cell cat embryos on post-thaw embryo survival and in vitro developmental capability. A total of 333 cumulus oocyte complexes (COCs) were matured and fertilized in vitro. At 24 h post-IVF, the presumptive embryos were randomly assigned into one of the following groups: 1) non-frozen control (n = 63); 2) forskolin treatment without freezing (n = 52); 3) freezing without forskolin (n = 77); and 4) freezing after forskolin treatment (n = 89). The embryos were cryopreserved using a programmable controlled-rate freezer. After freezing and thawing, the embryos were subsequently cultured in vitro for a further 6 days. The development competence was assessed by morula and blastocyst rates on Days 5 and 8 of their development, respectively. Percentages of cleaved embryos on Day 2 (IVF = Day 0) did not significantly differ among groups, indicating that there was no adverse effect of forskolin on cleavage rates. Furthermore, blastocyst formation rates of cat embryos treated with forskolin (53.5 ± 3.1) did not significantly differ when compared with non-treated controls (54 ± 9.3). Forskolin-treated embryos survived after cryopreservation at a higher rate than non-forskolin treatment, in terms of survival (93.1 ± 2.6 v. 88.2 ± 1.4), morula (56.9 ± 7.6 v. 40.8 ± 5.7), and blastocyst formation (47.6 ± 6.4 v. 35.6 ± 3.6) rates. It is concluded that partial delipidation of cat embryos before cryopreservation improves the cryopreservability of cat embryos. This study demonstrates that intracellular lipid has an impact on cryopreservability of cat embryos. Further study is required to examine in utero development of these delipidated embryos after embryo transfer.

This study was financially supported by the Zoo organisation of Thailand and CHE-TRF Senior Research Scholars RTA-5080010.
Wet season resting - economic insights from scenario modelling
Authors: MacLeod N. D. , Nelson B. S. , McIvor J. G. , Corfield J. P.
Published: August 2011

Pasture degradation, particularly that attributable to overgrazing, is a significant problem across the northern Australian rangelands. Although grazing studies have identified the scope for wet season resting strategies to be used to rehabilitate degraded pastures, the economic outcome of these strategies has not been extensively demonstrated. An exploratory study of the prospective economic value of wet season resting is presented using an economic simulation model of a 28 000 ha beef enterprise located in the Charters Towers region of north-eastern Australia to explore seven hypothetical scenarios centred on the projected performance of a wet season resting strategy. A series of 20-year simulations for a range of pasture recovery profiles, stocking capacity, animal productivity responses, beef prices and agistment options are compared with a baseline scenario of taking no action. Estimates of the net present value of the 20-year difference in total enterprise gross margins between the various resting options and the ‘do nothing’ option identify that wet season resting can offer a positive economic return for the range of scenarios examined, although this is contingent on the assumptions that are made concerning the trajectories of change in carrying capacity and animal productivity. Some implications for management and policy making to support the practical implementation of wet season resting strategies are discussed.
A framework for assessing regional biodiversity condition under changing environments of the arid Australian rangelands
Authors: Smyth A. K. , Brandle R. , Chewings V. , Read J. , Brook A. , Fleming M.
Published: August 2011

Rangelands support many ecosystem services important to humans, including climate regulation. They also have a significant role to play in the mitigation of greenhouse gases. However, the capacity of any rangeland to do this depends foremost upon the condition of biodiversity, and the functioning of its ecosystems. Considerable research has been undertaken on rangeland condition but it has not yet included the assessment of biodiversity (plants, animals and microbes) as a primary focus. Rangeland managers have struggled to assess biodiversity condition because it is rarely defined, is everywhere (so what do you assess?), is always changing in response to natural and human disturbances (so how do you know when it has changed?) and what amount signals management action. Here we present a framework that addresses these issues, and apply it to select surrogates and indicators that are scientifically defensible in biological and planning terms for assessing biodiversity. An arid Australian rangeland region is used as a case study to develop and apply our approach. We were not able to illustrate interpretation of condition because of the absence of long-term monitoring data in Australian rangelands, but we do provide guiding principles about sampling design and analytical methods for interpretation that use raw data rather than multimetrics. We discovered that different management outcomes expected to be informed from assessing biodiversity condition affected surrogate and indicator choice, and that a number indicators were not robust when assessed on conceptual relevance, measurement qualities, feasibility of implementation and policy and management relevance for four different management outcomes. Our work highlights the importance of stating the expected outcomes of biodiversity condition assessments up front, so that indicators relevant to future management are chosen. It also shows that critical thought on the robustness of indicators is warranted, especially as condition assessments under climate change will require information on the functional traits of species. We conclude by assessing the strengths and weaknesses of our framework in relation to environmental planning.
Ecosystem services from tropical savannas: economic opportunities through payments for environmental services
Authors: Greiner Romy , Gordon Iain , Cocklin Chris
Published: August 2011

Economic activity in the tropical savannas of northern Australia, like rangeland regions across the globe, has traditionally been based on primary production – predominantly cattle grazing and mining. More recently, northern Australia has experienced an increase in the extent of the conservation estate and in tourism and associated service industries. These trends demonstrate an increasing recognition of the multifunctional character of the tropical savannas and business opportunities additional to the traditional primary production systems. The increasing recognition of the multifunctionality of landscapes and increasing demand for ecosystem services provides potential opportunities for economic returns for businesses and communities in the tropical savannas through the delivery of environmental services to sustain the region’s natural capital. This paper pursues two objectives. Firstly, it sets out to provide conceptual clarity around the notions of ecosystem services, environmental services and payments for environmental services (PES). Secondly, we sketch some of the opportunities associated with PES, with particular reference to the Gulf of Carpentaria in northern Australia.
The climate change risk management matrix for the grazing industry of northern Australia
Authors: Cobon David H. , Stone Grant S. , Carter John O. , Scanlan Joe C. , Toombs Nathan R. , Zhang Xike , Willcocks Jacqui , McKeon Greg M.
Published: August 2011

The complexity, variability and vastness of the northern Australian rangelands make it difficult to assess the risks associated with climate change. In this paper we present a methodology to help industry and primary producers assess risks associated with climate change and to assess the effectiveness of adaptation options in managing those risks. Our assessment involved three steps. Initially, the impacts and adaptation responses were documented in matrices by ‘experts’ (rangeland and climate scientists). Then, a modified risk management framework was used to develop risk management matrices that identified important impacts, areas of greatest vulnerability (combination of potential impact and adaptive capacity) and priority areas for action at the industry level. The process was easy to implement and useful for arranging and analysing large amounts of information (both complex and interacting). Lastly, regional extension officers (after minimal ‘climate literacy’ training) could build on existing knowledge provided here and implement the risk management process in workshops with rangeland land managers. Their participation is likely to identify relevant and robust adaptive responses that are most likely to be included in regional and property management decisions. The process developed here for the grazing industry could be modified and used in other industries and sectors.
By 2030, some areas of northern Australia will experience more droughts and lower summer rainfall. This poses a serious threat to the rangelands. Although the impacts and adaptive responses will vary between ecological and geographic systems, climate change is expected to have noticeable detrimental effects: reduced pasture growth and surface water availability; increased competition from woody vegetation; decreased production per head (beef and wool) and gross margin; and adverse impacts on biodiversity. Further research and development is needed to identify the most vulnerable regions, and to inform policy in time to facilitate transitional change and enable land managers to implement those changes.
The Australian Collaborative Rangelands Information System: preparing for a climate of change
Authors: Bastin G. N. , Smith D. M. Stafford , Watson I. W. , Fisher A.
Published: August 2011

Change is a constant in Australia’s rangelands. Appropriate management of this change requires a sound knowledge of drivers (e.g. climate variability, livestock grazing), their impacts on natural resources (state and trend), socio-economic outcomes, and how these feed back through learning and adaptive management to affect drivers and their impacts. Information is required at scales from enterprise to national, with regional and broader level information serving to influence rangelands governance through institutional arrangements, policy and funding programs. The Australian Collaborative Rangelands Information System (ACRIS) collates and analyses data from national sources and from its State and Territory jurisdictional partners to track and understand change at regional to national scales. ACRIS has recently reported changes between 1992 and 2005 in several biophysical and socio-economic themes at bioregional resolution. This paper describes the processes used to collate and analyse the often disparate data, to synthesise information across data types and to integrate emergent higher order information across drivers, impacts and outcomes to provide more complete understanding of change. Data gaps and inconsistencies were a major challenge, and we illustrate how some of these issues were addressed by using indicators to report changes in biodiversity. ACRIS now needs to foster increased coordinated monitoring activity and develop its reporting capacity to become the valued information system for Australia’s rangelands. We propose that future improvements will be best structured within a hierarchically nested framework that provides consistent overarching data at national scale relevant to the variety of rangeland values (e.g. change in ground cover) but focuses on regionally-relevant ecosystem services, and their appropriate measures, at the regional scale. A key challenge is to implement consistent and systematic methods for monitoring biodiversity within this hierarchical framework, given limited institutional resources. Finally, ACRIS needs to develop a dynamic web-based delivery system to enable more frequent and flexible reporting of interpreted change than is possible through periodic published reports.
First sexual experience and current sexual behaviour among older Thai men and women
Authors: Ford Kathleen , Chamratrithirong Aphichat
Published: August 2011

Background: The focus of most studies of sexual behaviour has been on younger adults and adolescents, because they are perceived as having higher levels of sexual activity than older persons, and a consequently higher risk of HIV infection. Much less attention has been paid to the sexual behaviour of men and women in their forties and fifties. Using a life course framework, the objective of the present study is to examine AIDS knowledge, HIV testing and sexual risk behaviours among Thai men and women aged 40–59 years. The study also examines the influence of initial sexual experiences on later sexual behaviour. Methods: Data for the study are from the 2006 Thai National Survey of Sexual Behaviour, a national probability sample of 6048 individuals. Results: Older Thai men and women were well informed about methods of HIV transmission, but many were unaware of antiretroviral (ARV) medications. Older adults were also less accepting of HIV positive persons than were younger adults. Fewer than half of the older adults had undergone HIV testing, with testing associated with medical checkups or undertaken before operations. Reported condom use was very low with regular partners, moderate with casual partners, and high with sex workers. While the age at first sex has not changed markedly, the type of partner has changed over time. Fewer men have their first sex with a commercial partner. In general, first sex with someone with strong ties to the respondent was related to lower levels of risk behaviour in late adulthood. Conclusions: Areas of concern for AIDS prevention programs include condom use with casual partners and paid partners, knowledge of ARV, and attitudes towards persons living with AIDS.
Effectiveness of premarital sexual counselling program on sexual satisfaction of recently married couples
Authors: Vural Bilgin Kıray , Temel Ayla Bayık
Published: August 2011

Background: Through its ability to address and remove fear and misunderstanding and the resulting sexual reluctance and related problems, pre-marital sexual education and counselling can contribute to sexual satisfaction. Methods: This quasi-experimental research conducted in a pre-test–post-test control group design aimed to examine the effectiveness of nursing interventions on a premarital counselling program and its impacts on the sexual satisfaction of couples (36 couples in the experimental group and 35 couples in the control group). Results: Although no difference was detected between the experimental and control groups in terms of the level of knowledge on pre-test point averages, the difference between them in terms of post-test knowledge gain averages was statistically significant. Approval rates for sexual myths in the pre-test were 27.87% in the experimental group and 37.03% in the control group; in the post-test they were 23.51% and 36.66% respectively. In the experimental group, 80.6% of the women and 63.9% of the men, and in the control group, 77.1% of the women and 71.4% of the men were established as having a problem-free sexual life. Conclusions: It was also discovered that levels of sexual satisfaction were shown to be higher among women and men in the experimental group who had attended premarital sexual counselling education than the women and men in the control group. A recommendation to encourage engaged couples to attend premarital sexual counselling is made based on the findings. It is thought that an intervention plan prepared within the framework of the Information, Motivation, Behavioural Skills theoretical model will help nurses guide recently-married couples to greater sexual satisfaction.
Sexual behaviour and knowledge among adolescents in Ireland
Authors: Drennan Jonathan , Hyde Abbey , Howlett Etaoine
Published: August 2011

Background: In the present study the reported sexual activity and aspects of sexual knowledge of adolescents in both rural and urban areas in Ireland was documented. Methods: A questionnaire was administered to secondary school pupils aged 14–19 years during 2003–04. Data were analysed using the software package SPSS version 15.0. Associations between boys and girls, younger (≤15 years) and older respondents (≥16 years), and students from urban and rural schools were measured using the χ2 statistic. Results: The findings indicated that over a third of respondents had experienced sexual intercourse. However, the consistent trend in all previous studies of a greater proportion of boys reporting that they had full sex was reversed in the present study, with a higher proportion of girls stating that they had experienced their sexual debut. With regard to sexual coercion, while only a tenth of boys reported that they felt pressured to have full sex, approximately a third of girls reported that they had at some time felt pressured. Boys manifested a poorer understanding of fertility than girls, with more than half believing that a pregnancy would not occur while a woman was menstruating. Conclusions: Endeavours designed to improve young people’s knowledge of reproductive physiology and the risks they pose to themselves by having sex without a condom need to be improved.
Role of the general practitioner in testing for genital Chlamydia trachomatis infection: an analysis of enhanced surveillance data
Authors: Shaw Kelly , Stephens Nicola , Coleman David , O'Sullivan Maree
Published: August 2011

Background: To examine the relationship between genital chlamydia testing by healthcare providers and patient demographic characteristics in Tasmania, Australia, from 2001 to 2007. Methods: Analysis of enhanced surveillance data for genital Chlamydia trachomatis infections notified to the Tasmanian Communicable Diseases Prevention Unit between 1 January 2001 and 31 December 2007. Results: General practitioners identify most cases of genital chlamydia infection, irrespective of patient age, gender, indigenous status or urban status. Tests that are performed for screening purposes identify the largest number of cases in females, particularly in very young females. In males, tests performed due to the presence of clinical symptoms identify the majority of cases. However, tests performed for the purposes of contact tracing also identify a substantial burden of genital chlamydia infection, particularly in males. Conclusions: The present study demonstrates the critical role the general practitioner has in the identification of genital chlamydia infection. Opportunistic screening for genital chlamydia, including in the context of contact tracing, is an essential clinical activity that results in the identification of substantial numbers of cases of infection. Policy makers and public health practitioners should support general practice screening initiatives and remove the barriers to genital chlamydia screening in general practice.
General Practice funding to improve provision of adolescent primary sexual health care in New Zealand: results from an observational intervention
Authors: Morgan Jane , Haar Jarrod
Published: August 2011

Background: Free general practice (GP) sexual health visits for registered adolescents have been introduced in parts of New Zealand with the aim of improving provision of primary sexual health care. Published evidence of the effectiveness of such health care interventions, particularly around any impact on uptake of testing and detection of Chlamydia trachomatis, is limited. Methods: In 2003–2004, additional funding enabled 20 practices in Waikato, New Zealand to offer free sexual health consultations for registered under-25 year olds. Practice selection was non-random and biased towards lower socioeconomic, Māori and rural populations. Registered population data were linked to laboratory testing for C. trachomatis from January 2003 to December 2005. Twenty-nine practices without additional funding served as controls. Results: Chlamydia testing among under-25 year olds at the 20 intervention practices increased over time, in contrast to non-intervention practices, with coverage of females aged 18–24 years within the intervention increasing from 13.9% in 2003, to 15.5% during the roll-out phase and to 16.8% in 2005. Intervention practices had higher test positivity rates than non-intervention practices (8.7% v. 5.9%, P < 0.01) with increases in test positivity, from 7.7% in 2003 to 10% in 2005, relating mainly to increases in positive tests among females aged less than 25 years. There was no increase in testing or detection among those aged 25 years and older at intervention practices. Conclusions: Introducing free GP visits for under-25 year olds living in rural and lower socioeconomic areas in New Zealand was associated with a significant increase in testing and detection for C. trachomatis in the target age group. This observational intervention supports the ongoing provision of free adolescent primary sexual health care.
Evaluation of self-collected urine dip swab method for detection of Chlamydia trachomatis
Authors: Costa Anna-Maria , Fairley Christopher K. , Garland Suzanne M. , Tabrizi Sepehr N.
Published: August 2011

Background: The present study is an evaluation of a self-collected urine dip (SCUD) swab as an alternative sampling method for the detection of Chlamydia trachomatis (CT) in urine samples that conforms to postal service regulations in Australia. Methods: Sixty urine samples, previously identified as CT positive were used to prepare SCUD swabs in vitro. In addition, replicate SCUD swabs were prepared from known CT positive urine samples and stored at room temperature, or sent through the postal system. All samples were tested for CT and an inhibition control using the Roche TaqMan 48 Real-time polymerase chain reaction system. Results: Overall, 58/60 (97%) SCUD swabs generated positive CT results. Triplicate SCUD swabs prepared from five known positive urine samples and stored up to 7 days at room temperature, showed positive results in all samples. Ten replicates of SCUD swabs from five known CT positive samples were also tested after being posted from different regions in Australia, with a transit time of 2–7 days, back to the Melbourne laboratory. There was 94% positivity of the SCUD swab samples. Conclusion: The present study demonstrated SCUD swabs to be a sensitive and robust method of self-collecting samples for detection of CT subsequent to sending the samples through the postal service.
Dyspareunia: a complex problem requiring a selective approach
Authors: Walid Mohammad Sami , Heaton Richard L.
Published: August 2011

Dyspareunia frequently has a multifactorial aetiology. The problem with the term is that it is not specific enough and does not allow for proper discussion of the very important problem of pain with sexual intercourse, a problem that can be very disturbing to a couple’s relationship. We present two cases of patients who had multiple potential anatomic reasons for dyspareunia. The clinical picture, treatment strategy and the complex nature of deep penetration pain was discussed. We also proposed a new way of defining dyspareunia to allow a more adequate way of studying and discussing the problem.
Ecological impacts of feral pig diggings in north Queensland rainforests
Authors: Mitchell J. , Dorney W. , Mayer R. , McIlroy J.
Published: August 2011

This two-year study examined the impacts of feral pig diggings on five ecological indicators: seedling survival, surface litter, subsurface plant biomass, earthworm biomass and soil moisture content. Twelve recovery exclosures were established in two habitats (characterised by wet and dry soil moisture) by fencing off areas of previous pig diggings. A total of 0.59 ha was excluded from further pig diggings and compared with 1.18 ha of unfenced control areas. Overall, seedling numbers increased 7% within the protected exclosures and decreased 37% within the unprotected controls over the two-year study period. A significant temporal interaction was found in the dry habitat, with seedling survival increasing with increasing time of protection from diggings. Feral pig diggings had no significant effect on surface litter biomass, subsurface plant biomass, earthworm biomass or soil moisture content.