bluuebunny/crossref_metadata_2025 huggingface dataset:
151 Associations of Testicular Size and Arterial Blood Flow with Sexual Development and Semen Characteristics in Dorper Rams Raised in a Subtropical Climate
Authors: Camela E. S. C. , Nociti R. P. , Santos V. J. C. , Macente B. I. , Maciel G. S. , Feliciano M. A. R. , Vicente W. R. R. , Gill I. , Bartlewski P. M. , Oliveira M. E. F.
Published: November 2018

Spectral Doppler (velocimetric) indices are important indicators of blood perfusion and physiological status of internal organs. Doppler ultrasound is used in andrology to assess the blood flow in the testicular arteries, aiding in the diagnosis of scrotal disorders and abnormal spermatogenesis. It was proposed that blood flow indices measured in the testicular artery, namely, blood flow resistance and pulsatility index, were significant predictors of semen quality in clinically healthy dogs. There is a paucity of studies on the associations of testicular morphology and blood flow dynamics with spermatogenesis in growing rams. Forty-five Dorper rams (24 rams aged 8 to 11 months and 21 rams aged 12 to 24 months) were examined using a portable ultrasound scanner connectecd to a linear-array 7.5-MHz transducer. Measurements of the testes were taken with built-in electronic calipers on images containing the largest cross-sectional area of each testicle in 3 planes (coronal, sagittal, and transverse). Colour and spectral Doppler scans were performed immediately after scrotal ultrasonography to detect the testicular arteries and record the following velocimetric indices: peak systolic velocity (PSV, cm/s), end-diastolic velocity (EDV, cm/s), resistive index (RI = [PSV – EDV)/PSV], and pulsatility index [PI = (SPV – EDV)/mean velocity] of the blood vessels. The length (9.7 ± 0.3 v. 9.0 ± 0.2 cm; mean ± SD) of the testes and scrotal circumference (33.3 ± 0.5 v. 31.8 ± 0.4 cm) were greater (P < 0.05) in sexually mature rams than in peri-pubertal rams. There were no differences (P > 0.05) between the 2 age groups of Dorper rams in mean values for blood flow indices measured in testicular arteries. Semen was collected by ejaculation into the artificial vagina from 38% (8/21) of post-pubertal rams and 21% (5/24) of peri-pubertal animals (Chi-squared test; P < 0.05). Semen volume was negatively correlated with PI of the testicular arteries (r = –0.57, P = 0.04). In summary, the attainment of sexual maturity in Dorper rams maintained in a subtropical climate was associated with significant elongation of testes but not with the changes in testicular volume and blood perfusion. Testicular artery PI was a significant predictor of semen volume in peri-pubertal and sexually mature rams. Based on this and several previous studies, B-mode ultrasonography combined with echotextural analyses of the epididymides and accessory sex glands and serum testosterone measurements provide more useful information on the progression of sexual maturation and present semen quality in rams than the ultrasonographic assessment of testicular dimensions and an array of blood flow indices.
161 Cumulus Cell Luteinization is Enhanced During Aging of Bovine Oocytes Matured In Vitro
Authors: Lebedeva I. Y. , Singina G. N. , Uzbekova S. V. , Papillier P. , Shedova E. N. , Lopukhov A. V. , Mityashova O. S.
Published: November 2018

Maturing mammalian oocytes can prevent luteinization of cumulus cells (CC) both in vivo and in vitro by regulating CC steroidogenesis (Gilchrist et al. 2004 Anim. Reprod. Sci. 82-83, 431-446; Ramirez et al. 2016 Anim. Reprod. 14, 280). However, when the oocyte attains the metaphase II stage, aging processes are accelerated and may impair protective abilities of CC. The aim of the present research was to study the luteinization of CC surrounding aging bovine oocytes and its susceptibility to prolactin (PRL), which performs a luteotropic function in mammals. We analysed (1) the steroidogenic activity of CC during the prolonged culture of cumulus–oocyte complexes (COC), and (2) the expression of genes related to luteinization in CC. Bovine COC were matured in TCM-199 containing 10% fetal calf serum (FCS), 10 μg mL−1 porcine FSH, and 10 μg mL−1 ovine LH. After 22 h of IVM, COC were transferred to the aging medium consisting of TCM-199 supplemented with 10% FCS and cultured for 0, 12, or 24 h in the absence (Control) or presence of 50 ng mL−1 bovine PRL. Progesterone (P4) and oestradiol-17β (E2) levels in spent media were determined by ELISA. The expression of luteinization-related genes STAR (steroidogenic acute regulatory protein), HSD3B1 (3β-hydroxysteroid dehydrogenase type 1), PGR (genomic P4 receptor), and PGRMC1 and PGRMC2 (P4 receptor membrane components 1 and 2) was analysed by real-time RT-PCR. The data from 6 to 8 replicates (at least 170 oocytes for each condition) were analysed by ANOVA following by Tukey’s (steroidogenesis) or Dunn’s (gene expression) tests. In the control group, the level of P4 production exhibited by COC during 24-h aging was 5.5-fold higher than the level observed during in vitro maturation (1.50 ± 0.15 ng v. 0.27 ± 0.02 ng of P4/COC; P < 0.001). Conversely, E2 production by aging COC was 2.7 times lower than that by maturing COC (0.39 ± 0.05 pg v. 1.04 ± 0.05 pg of E2/COC; P < 0.001). The CC expression of genes responsible for P4 synthesis, STAR and HSD3B1, increased progressively by 24 h of aging (7.1- and 34.6-fold, respectively; P < 0.001). Meanwhile, the steroid secretion and expression of STAR and HSD3B1 genes were unaffected by PRL. The relative mRNA abundance of PGRMC1 increased 2.3-fold (P < 0.05) in the control (at 24 h) and PRL groups (at 12 h), while that of PGRMC2 did not change significantly in both groups. In addition, PGR transcript level decreased (P < 0.01) 9.9-fold at 12 h and 18.3-fold at 24 h aging of PRL-treated COC, whereas this decrease was only 5-fold (P < 0.05) at 24 h in Control. The findings indicate that aging of bovine oocytes matured in vitro enhances luteinization of surrounding CC. Prolactin does not affect steroidogenesis but may modulate the expression of P4 receptors in cumulus cells.
This research was supported partly by FASO Russia and INRA (France).
178 Differential Behavior of Porcine Mesenchymal Stem Cells from Bone Marrow and Adipose During Osteogenic Differentiation on a Glycosaminoglycan Hydrogel Scaffold for Bone and Cartilage Tissue Engineering
Authors: Womack S. A. , Milner D. J. , Weisgerber D. W. , Harley B. A. , Wheeler M. B.
Published: November 2018

The pig is an ideal species for use in tissue engineering studies targeted towards repair of bone and cartilage defects. Novel collagen-glycosaminoglycan hydrogel (CG) scaffolds have shown promise for supporting bone and cartilage growth from mesenchymal stem cells. In order to determine the suitability of these scaffolds for use in porcine model systems for bone and cartilage tissue engineering, we have begun to investigate the behaviour of porcine mesenchymal stem cells on this material. The purpose of this study was to determine whether mesenchymal stem cells from adipose (ASC) and bone marrow (BMSC) form bone on a CG scaffold material. Primary BMSC and ASC from 6-month-old Yorkshire pigs were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum. The ASC and BMSC were then trypsinized at passage 4 or 5 and used to seed ~4-mm-diameter CG scaffolds with 2 million cells/scaffold. Scaffolds were seeded by suspending the cells in medium that had been equilibrated for 30 min, and then placing the CG scaffold into the medium. This method of seeding was determined to be most effective in previous experiments. Scaffolds were then cultured for 7 days in DMEM followed by 21 days in osteogenic media. At the conclusion of the incubation period, the diameter of the scaffolds was measured, and they were fixed with 4% paraformaldehyde and cryosectioned. Then, 10-µm sections were stained with Alizarin Red to assay for mineralization, a hallmark of osteogenic differentiation. Both ASC- and BMSC-loaded scaffolds showed Alizarin Red staining throughout the section after incubation, demonstrating that both undergo osteogenesis on the scaffold material (n = 4). During osteogenic differentiation, scaffolds seeded with both ASC and BMSC showed a decrease in diameter. Unseeded scaffolds showed no decrease in size when in media. The BMSC scaffolds demonstrated a more extensive decrease in size than ASC. The average diameter of ASC loaded scaffolds after differentiation was 2.49 ± 0.39 mm, and that of BMSC-loaded scaffolds was 1.47 ± 0 0.18 mm (n = 3, P < 0.05, Student’s t-test). This suggests a differential ability of ASC and BMSC to break down and metabolize the scaffold matrix, and may indicate that one cell type may be preferable to the other for repairing osteogenic defects using these scaffolds. Current experiments underway will analyse expression of matrix-degrading enzymes to determine the source of the difference between cell types in scaffold shrinkage during differentiation. We will also quantify mineralization in ASC- v. BMSC-loaded scaffolds and assay gene expression of osteogenic markers to determine if there is a difference in osteogenic potential between sources of mesenchymal stem cells on these scaffolds.
180 Increasing GfrA1-Positive Spermatogonial Stem Cell Population of Goat
Authors: Sharma V. , Saini S. , Aneja B. , Kumar A. , Thakur A. , Bajwa K. K. , Kumar S. , Mohanty A. K. , Malakar D.
Published: November 2018

Spermatogonial stem cells (SSC) form the basis of spermatogenesis and continuous fertility in male. Their meagre population in the testis is a hindrance in the in vitro study of biological activity of these cells. The objective of the present study was to isolate and characterise goat SSC and increase their number during in vitro culture by different methods. Two goat testes (3 to 4 months of age) were collected from the slaughterhouse and transported to the laboratory. The testes were washed and seminiferous tubules were collected and minced in the laminar flow hood. The seminiferous tubules were washed twice with PBS to remove spermatozoa and subjected to double enzymatic digestion (collagenase, 1 mg mL−1, hyaluronidase, 1 mg mL−1, trypsin, 0.05%, and DNaseI, 10 µg mL−1 for 45 min and second digestion with same set of enzymes except trypsin for 30 min). The isolated cells were filtered sequentially through nylon mesh filters of pore size 70 and 40 µm. The cells were plated on DSA-lectin coated dishes for 4 h and the unattached cells were cultured on a Sertoli cell feeder layer prepared by treating with mitomycin-C for 3 h. The cells were cultured in DMEM/F-12 supplemented with human recombinant growth factors (glial cell-derived neurotrpic factor, 10 ng mL−1, fibroblast growth factor FGF, 10 ng mL−1, epidermal growth factor, 20 ng mL−1), 10% fetal bovine serum, and antibiotics. The expression of pluripotency markers (Oct4, Nanog, Sox2) and SSC-specific markers (Thy1, GfrA1, and Uchl1) in the SSC colonies was determined by RT-PCR and immunostaining, after in vitro culture of 3 weeks. The SSC population was enhanced by differential plating, Percoll density gradient (on Day 1) and SSC passaging (by passaging SSC colonies on Day 20). The cells were tagged with GfrA1 antibody and their population was tested by flow cytometry. The SSC colonies started appearing after 7 days and continued to grow in size and number until 3 weeks. The SSC colonies were positive for the pluripotency markers Oct4, Nanog, and Sox2 by RT-PCR and immunostaining. The SSC were also positive for the SSC-specific markers Thy1, GfrA1, and Uchl1 by RT-PCR and immunostaining. Flow cytometry showed that the GfrA1-positive population in the SSC enriched by the differential plating was 11.23%, Percoll density gradient was 23.57%, and by passaging of SSC colonies, after picking and trypsinising with 0.05% trypsin, was 91.23%. In vitro culture of the SSC enriched by these methods also revealed that the number of SSC colonies appearing in the cells enriched by passaging was higher than the other methods. From the results of present study, we conclude that SSC are positive for markers of pluripotency and SSC-specific markers. The SSC population can be enhanced to a very high level following SSC passaging, which is an inexpensive method and does not require expensive instruments like fluorescence- or magnetic-activated cell sorting.
181 Establishment of Induced Pluripotent Stem Cells from Fishing Cat and Clouded Leopard Using Integration-Free Method for Wildlife Conservation
Authors: Sukparangsi W. , Bootsri R. , Sikeao W. , Karoon S. , Thongphakdee A.
Published: November 2018

Fishing cat (Prionailurus viverrinus) and clouded leopard (Neofelis nebulosa) are wild felids, currently in vulnerable status according to the International Union for Conservation of Nature red list (2017). Several measures in assisted reproductive technology (e.g. AI, embryo transfer) have been used by the Zoological Park Organization of Thailand (ZPO) to increase their offspring in captivity. Recently, the generation of induced pluripotent stem cell (iPS cells) becomes popular and provides alternative way to preserve good genetics in the form of cell with diverse capacities. This great potential of iPS cells is unlimited self-renewal and pluripotency, similar to embryonic stem cells (ESC). Under the right cell culture conditions, pluripotent stem cells can differentiate into all cell types of the body. Here, we aimed to find the optimal condition to generate integration-free iPS cells from fishing cat and clouded leopard. At first, to obtain somatic cells for cellular reprogramming, adult dermal fibroblast cell lines from both species were established from belly skin tissues. Subsequently, several nucleofection programs of AmaxaTM 4D-nucleofectorTM (Lonza, Basel, Switzerland) were examined to introduce integration-free DNA vectors carrying reprogramming factors into the felid fibroblasts. The transfected cells were cultured under numerous conditions: (1) matrix/defined surface including irradiated mouse embryonic fibroblast, gelatin, vitronectin, and Geltrex® (Thermo Fisher Scientific, Waltham, MA, USA); (2) ESC/iPS cell medium including Essential 8TM (Thermo Fisher Scientific) DMEM containing KnockOutTM Serum Replacement (KOSR; Thermo Fisher Scientific) and/or fetal bovine serum (FBS); and (3) supplement including basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), l-ascorbic acid, nicotinamide, ALK5 inhibitor (A83-01) and RevitaCellTM (Thermo Fisher Scientific). We found that optimal nucleofection programs for human dermal fibroblast including FF-135 and EN-150 were able to transfer episomal vectors and excisable piggyBAC transposon carrying reprogramming factors into fishing cat and clouded leopard fibroblasts, respectively. The iPS-like colonies appeared around 26 to 30 days post-nucleofection. The culture of transfected cells on either Geltrex® or Vitronectin-coated surface supports the formation of iPS-like colonies with different derivation efficiency (0.01 and 0.005%, respectively). In addition, all colonies were formed under medium containing FBS, together with both bFGF and LIF supplements. Taken together, we have developed a platform to generate iPS cells from tissue collection to the establishment of iPS cell culture. This will further enable us to apply the technique to obtain iPS cells from other endangered and vulnerable felid species.
189 The Effect of Vitamin K on the Differentiation of Adipose-Derived Stem Cells into Osteoblasts
Authors: Bane T. A. , Bertels J. C. , Polkoff K. M. , Rubessa M. , Wheeler M. B.
Published: November 2018

Tissue engineering offers a viable alternative to bone grafts in repairing large bone defects. This involves using scaffolds of various sizes and shapes that contain stem cells and other osteoinductive molecules. The aim of this project was to evaluate the effects of vitamin K in osteogenic medium and its effect on the differentiation of adipose-derived stem cells (ASC) into osteoblasts. Vitamin K has been shown to increase bone mineral density by acting as a coenzyme in the γ-carboxylation of osteocalcin, a protein involved in bone mineralization (Weber 2001 Nutrition 11–12, 1024). Our hypothesis was that the presence of vitamin K in the osteogenic medium would positively influence the number of osteoblastic nodules formed. Swine ASC were isolated as described (Monaco et al. 2009 Open Tissue Eng. Regen. Med. J. 2, 20–33). The ASC were divided into 7 different treatments: 5 concentrations of vitamin K in the osteogenic medium (10, 50 100, 500, 1000 nM) plus 2 control treatments (osteogenic medium without vitamin K and a negative control, DMEM). The media was changed twice a week for 4 weeks. The experiment was replicated 6 times. At the end of the culture period, cells were stained with Alizarin Red S and Von Kossa. In each well, we counted the nodules and then divided them in 2 categories: formed and forming nodules. Data were analysed by analysis of variance using the generalized linear model (GLM) procedure of SPSS (IBM/SPSS, Armonk, NY, USA); the least significant difference (l.s.d.) post hoc test was used to perform statistical multiple comparison, and the α-level was set at 0.05. The results showed (in Table 1) that there was no positive effect on nodule formation when vitamin K was added to the medium; however, when 1000 nM vitamin K was added, nodule formation decreased. More experiments need to be conducted to determine if vitamin K can act synergistically with other vitamins to produce a significant role in ASC differentiation into osteoblasts. This preliminary experiment is the first step towards the analysis of the behaviour of ASC on scaffolds with vitamin K incorporated into their matrix.

Table 1.The average number of formed and forming osteoblast nodules compared between treatment groups (SD in parentheses)
2 Response to Treatment with Human Chorionic Gonadotropin on Pregnancy Rate of Heat-Stressed Lactating Cows: Interactions with Parity and Genotype
Authors: Zolini A. M. , Ortiz W. , Eliab E. , Serdal D. , Hansen P. J.
Published: November 2018

Reported effects of treatment of lactating dairy cattle with hCG after AI have been variable. The current objective was to evaluate whether fertility response to hCG in lactating Holsteins exposed to heat stress depends on parity or genotype. The experiment was conducted at a commercial dairy in northern Florida during the summer (June to September). Ovulation was synchronized using the Double Ovsynch protocol for 282 primiparous and 490 multiparous first-service lactating cows. Cows were randomly assigned to receive either 3,300 IU of hCG (Chorulon, MSD, Millsboro, DE, USA) or diluent, IM, on Day 5 after AI. Cows were genotyped by PCR-based KASP assay (LGC Genomics, Middlesex, United Kingdom) for 4 single nucleotide polymorphisms (SNP) previously associated with fertility, embryonic development, or stress tolerance. The SNP were as follows: heat shock protein A1L (HSPA1L, HSP70C895D), prostate androgen-regulated mucin-like protein 1 (PARM1, rs111027720), coenzyme Q9 (COQ9, rs109301586), and progesterone receptor (PGR, rs109506766). Pregnancy diagnosis was performed at Day 60 after AI by ultrasonography. Data were analysed using the GLIMMIX procedure of SAS (SAS Institute Inc., Cary, NC, USA). When genotype was not considered in the model, there was a tendency (P = 0.08) for a treatment × parity interaction, with hCG increasing pregnancy rate in primiparous cows (32.1% ± 0.04 v. 42.0% ± 0.04) but not multiparous cows (27.7% ± 0.02 v. 27.0% ± 0.03). When genotype for COQ9 was included in the model, the parity × treatment interaction was significant (P = 0.036). Moreover, the response to treatment was affected by COQ9 genotype (P = 0.023) and the 3-way interaction (P = 0.060). In cows treated with vehicle, pregnancy rate was greatest for the AA allele (40.2% ± 0.05 for AA, 21.5% ± 0.03 for AG, and 33.3% ± 0.05 for GG). However, for cows treated with hCG, pregnancy rate was lowest for AA (23.5% ± 0.05 for AA, 32.9% ± 0.04 for AG, and 34.0% ± 0.03 for GG). The 3-way interaction occurred because the negative effect of hCG on fertility in AA animals only occurred in multiparous cows. Pregnancy rate was also affected by genotype for HSPA1L (25.0% ± 0.02 for the major allele CC, 38.7% ± 0.04 for CD, and 27.5 ± 0.04 for the minor allele DD; P = 0.003) but there were no interactions with treatment. There was a tendency for pregnancy rate to be affected by genotype for PGR (P = 0.076) but there was no interaction with treatment. The PARM1 genotype was not associated with pregnancy rate. In conclusion, treatment with hCG 5 days after AI improved pregnancy rate in primiparous lactating cows under heat stress but had no effect on pregnancy rate of multiparous cows. Moreover, actions of hCG to improve fertility were associated with a SNP in COQ9. Thus, genotype can affect response of cows to a fertility-promoting drug.
204 Blood Analysis of Cas9-Expressing Cattle
Authors: Yum S.-Y. , Lee S.-J. , Hahn S.-E. , Lee C.-I. , Kim H.-S. , Kim H.-J. , Jin Y.-K. , Choi W.-J. , Lee J.-H. , Lee W.-S. , Jang G.
Published: November 2018

The CRISPR/Cas9 system has proved to be a powerful tool for knockout and knock-in in various species. When 2 components—Cas9 and single guide (sg)RNA—are delivered into cells or embryos, the events of gene editing occur. Because Cas9 is essential for gene editing in the CRISPR/Cas9 system, some studies have reported the production of Cas9-expressing animals, such as mice, which could be used to increase gene editing efficiency in subsequent experiments. In previous reports, we successfully produced 4 Cas9-expressing cattle via microinjection (Hahn et al. 2016 Reprod. Fertil. Dev. 29, 211). Primary cells from these calves had Cas9 activity because transfection of only sgRNA resulted in gene deletion. The aim of this study was to analyse the blood of the transgenic cattle to investigate the effect of Cas9 expression on health. Two of 4 transgenic calves died; one had severe ruminant tympany, failed to respond to treatment, and died at 4 months of age, and the other died at 5 months of age due to accidental ingestion of a needle from a feed bunk. Blood samples were obtained from the surviving 2 transgenic cattle (1 male and 1 female) at 7 and 12 months for blood analysis. Five milliliters of whole blood samples was collected from the jugular vein. Portions were used for CBC (Hemavet 950, Drew Scientific, Miami Lakes, FL, USA) and for serum chemistry analysis (BS-400, Mindray, Shenzhen, China). Average values for white blood cells (9600 and 1057/mm3), neutrophils (4590 and 3870/mm3), lymphocytes (4020 and 5910/mm3), red blood cells (732,000 and 798,000/mm3), hemoglobin (9.5 and 10.2 g dL−1), packed cell volume (24.3 and 25.3%), platelet (439,000 and 327,500/mm3), AST (76 and 104 IU), ALP (140 and 133 IU), BUN (7.5 and 10.5 mg dL−1), and creatinine (1.3 and 1.0 mg dL−1) of male and female transgenic calves were within the reference range. Additionally, there was no difference in general health information, including body temperature and feeding. In conclusion, we demonstrated that continuous Cas9 expression in transgenic cattle did not affect health status of the surviving calves in terms of blood analysis. They have grown up without any health issues and are currently 14 (female) and 15 (male) months old. In the near future, we will evaluate their germline transmission by natural breeding or in vitro fertilization.
This work was supported by BK21 PLUS Program for Creative Veterinary Science, NRF (NRF-2017R1A2B3004972), and Seoul Milk Coop (SNU 550–20160004).
208 Cryotolerance of In Vitro-Produced Cattle Embryos Produced with Different Maturation and Culture Media
Authors: Taylor M. A. , Owen C. M. , Barceló-Fimbres M. , Campos-Chillon L. F.
Published: November 2018

In vitro-produced (IVP) bovine embryos suffer from damage due to suboptimal culture conditions involving altered metabolism, reactive oxygen species generation and high lipid accumulation, which could be impacted by oocyte maturation × embryo culture interaction. We hypothesised that optimizing oocyte and embryo culture conditions would lead to a higher post-thaw embryo survival rate. The experiment was replicated 9 times in a factorial design with 3 oocyte maturation media: TCM-199 plus 10% serum and gonadotropins (control), BO-HEPES-IVM (BO, IVF Bioscience UK), and HMM (HEPES-buffered maturation medium) and 3 embryo culture media: BO-IVC (BOC), BBH7 (Cooley Biotech, Gainesville, FL, USA), and SCF1 (SOF for Conventional Freezing 1; Owen et al. 2017 Reprod Fertil Dev. 29, 129-130). Bovine oocytes (n = 2907) were aspirated from 2- to 8-mm follicles of abattoir ovaries, matured at 38.5°C for 22 to 24 h in 5% CO2 (control) or in BO and HMM in air, fertilized with semen from 1 of 3 bulls, and cultured in BOC, BBH7, and SCF1 at 38.5°C in 5.5% CO2, 5% O2, and 90% N2. Cleavage and blastocyst rates were evaluated at Day 3 and 7, respectively, after IVF. Selected stage 7 blastocysts were slow frozen using 1.5 M ethylene glycol supplemented with 1 mm l-ascorbic acid for 20 min (equilibration). The embryos were cooled to and seeded at –6°C, and then cooled to −32°C at 0.5°C/min. Embryos were thawed and re-expansion was assessed at 24 and 48 h. Data (Table 1) was arcsin transformed and analysed by ANOVA, and means separated by l.s.d. Results indicate that oocytes matured in BO and embryos cultured in SCF1 had a higher blastocyst rate than all oocytes matured in control medium and all embryos cultured in BBH7 (P < 0.05). However, oocytes matured in control medium and embryos cultured in BOC had lower post-thaw re-expansion rates than other treatment groups (P < 0.05). These results suggest that a combination of oocyte maturation and embryo culture media plays an important role in post-thaw embryo survival, al though interactions of present treatments need to be further evaluated.

Table 1.Effect of maturation and culture media on blastocyst rate and cryotolerance ± SEM)
29 Co-Incubation of Equine Cloned Embryos with Sialic Acid: Effect on Pregnancy Rate
Authors: Vichera D. , Olivera R. , Arnold V. , Vergara J. , Jordan R. , Vichera G.
Published: November 2018

In vitro-produced equine embryos have certain morphological characteristics that differ from embryos produced in vivo. One of them is the absence or inadequate formation of the embryo capsule. This capsule is composed of mucin-like glycoproteins produced by the trophectoderm with high proportion of sialic acid, which confers anti-adhesive properties. This characteristic is necessary for the intrauterine embryo migration process to occur, which is vital and fundamental for pregnancy recognition. For this reason, inadequate formation of the glycoprotein capsule could result in a lower pregnancy rate. In this study, we aimed to evaluate the effect of cloned equine embryo co-incubation with sialic acid on blastocyst and pregnancy rates. To achieve this, equine oocytes obtained from slaughterhouse ovaries were matured in TCM-199 HEPES medium with 2% fetal bovine serum, 2% antibiotics, and 1 μg mL−1 FSH, incubated at 39°C for 24 h. Matured oocytes were denuded with pronase, enucleated, and fused to donor bone marrow mesenchymal cells according to Olivera et al. (2016 PLoS One 11, e0164049, 10.1371/journal.pone.0164049). Chemical activation was induced using 8.7 μM ionomycin for 4 min and embryos were incubated with 1 mM 6-DMAP and 5 mg mL−1 cycloheximide for 4 h. Afterwards embryos were cultured in microwells for 8 days in DMEM-F12 medium. On Day 6, cloned equine embryos were exposed to 5 μM sialic acid for 48 h (SA group). On Day 8, blastocysts were transferred to recipient mares 5 days post-ovulation and pregnancy was confirmed 15 days post-transfer by transrectal ultrasound. Embryo clones generated without sialic acid exposure were used as a control (C) group. Fisher test was used to analyse both blastocyst and pregnancy rates. Blastocyst rates were 14% (46/328) and 15% (62/413) and pregnancy rates were 30.4% (7/23) and 19.4% (6/31) for SA and C groups, respectively. No statistical differences were observed between groups for the analysed parameters, even though pregnancy rates tended to be higher in the SA group. This effect could be a consequence of higher concentrations of the glycoprotein involved in the formation of the embryo capsule.
30 Vitrification of Equine Embryos: Application in a Commercial Cloning Program
Authors: Vichera G. , Jordan R. , Arnold V. , Dobler D. , Olivera R.
Published: November 2018

During a commercial horse cloning program, a critical point is the availability and maintenance of suitable recipient mares for a large quantity of embryo transfers. Usually, pregnancy rates and viable births off the breeding season decrease significantly, whereas the rate of in vitro embryo production remains constant. For this reason, an efficient vitrification system allows continuous embryo production throughout the year with the advantage of doing the transfers only during the breeding season. The vitrification technique evaluated in this study was the one described by Kuwayama et al. (2007 Theriogenology 67, 73-80). By using this method, we compared post-warming recovery efficiency, pregnancy rates, and viable foaling rates in 2 experimental groups: cloned blastocysts vitrified off-season and transferred in breeding season (VC, n = 337), and non-vitrified cloned blastocysts also transferred in breading season (no-VC, n = 516). To achieve this, equine oocytes were collected from slaughterhouse ovaries, matured, enucleated, and fused to a donor cell according to Olivera et al. (2016 PLoS One 11, e0164049, 10.1371/journal.pone.0164049). The reconstructed embryos (RE) were cultured in a well-of-the-well system by adding 3 RE per well for 7 to 8 days to reach the blastocyst stage, at which they were vitrified as mentioned above. During the breeding season, blastocysts were warmed and transferred in couples in a single cycling receptive mare. Pregnancies were confirmed by transrectal ultrasonography 15 days post-transfer. All variables were analysed by Fisher test (P < 0.05). The warming recovery rate was 91% (308/337) for cloned blastocysts. In addition, pregnancy and viable birth rates were similar for the VC and no-VC groups: 15.6% (24/154) v. 16.7% (43/258) for pregnancy rates, respectively, and 37.5% (9/24) v. 37.2% (16/43) for foaling rates, respectively. In summary, 9 viable cloned foals were obtained with off-season embryos warmed and transferred during the breeding season, showing that vitrification did not affect embryo quality. Hence, the proposed strategy provides the ability to maximize production efficiency of equine clones by generating a large number of pregnancies without stopping in vitro embryo production at any time of the year.
36 Transgenic Somatic Cell Nuclear Transfer Blastocyst Selection with Embryo Biopsying
Authors: Nõmm M. , Ivask M. , Pärn P. , Jaakma Ü. , Kõks S.
Published: November 2018

Somatic cell nuclear transfer (SCNT) is, to date, the most used technology producing transgenic (TG) cattle. Depending on the gene construct and transfection method, transfection efficiency may differ greatly. Applying a more intense selection regime after transfection may obliterate the cells. An extended selection affects the passage number and leads to genotypic and phenotypic drift of the cells. We used the pBC1 Milk Expression Vector Kit (cat. no. K270-01, Invitrogen Corp., Carlsbad, CA, USA) to make the expression vector of human FSH (hFSH). For TG fibroblast cell line, the AmaxaTM NucleofectorTM Kit for Primary Fibroblasts (cat. no. VPI-1002, Lonza Grouop, Basel, Switzerland) was used. For TG fibroblast selection, G418 (neomycin) was used for 21 days with a final concentration of 400 µg mL−1. The final passage number of the cell line was 6. The primers included in the pBC1 Milk Expression Vector Kit-BCF (GATTGACAAGTAATACGCTGTTTCCTC) and BCR (CATCAGAAGTTAAACAGCACAGTTAG)-were used to control the insert. The transgenesis of the cell line was confirmed by sequencing the PCR product and analysing it with the BlastN and Bioedit software to make sure the fibroblast cell line was hFSH-positive. These cells were thereafter randomly used for SCNT as donor cells. All the SCNT embryos were cultured for 4 days in IVF Bioscience (Falmouth, United Kingdom) culture media and then biopsied. After aspirating 1 blastomere from the 6- to 8-cell-stage embryo, the biopsied embryos were further individually cultured until Day 7 and blastocyst formation was recorded. Genomic DNA from the biopsies was isolated and amplified with REPLI-g Single Cell Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. The primers BCF and BCR were used to control the hFSH positivity of the embryos, and the PCR product was visualised on a 1% agarose gel. From 62 biopsied SCNT cloned embryos, 22 (35.48%) tested TG positive. The total blastocyst yield from biopsied embryos was 26 (41.93%), of which 12 (54.54%) were TG positive blastocysts and selected for transfer. Our hFSH TG fibroblast cell line demonstrated a low concentration of TG cells in its culture, despite the selection and verification methods applied. Based on the analysis of SCNT embryos, only 54.54% of the embryos developed were TG positive. The embryo biopsying technique enables us to use only TG-positive SCNT cloned embryos for transfer, therefore avoiding non-TG pregnancies.
This study was supported by Enterprise Estonia grant EU30020, Institutional research funding IUT 8-1 and Horizon 2020 Project SEARMET 692299.
93 Oestrous Synchronisation Studies in a Marsupial
Authors: Witt R. R. , Hinds L. A. , Rodger J. C.
Published: November 2018

There is no strategy to precisely synchronise and predict oestrus and ovulation in marsupials. This is the major limitation in applying assisted reproduction to marsupial conservation. Routine methods of female synchronization that target the ovary (effective in eutherians) are ineffective in marsupials because they do not disrupt the corpus luteum. Gondaotropin-releasing hormone (GnRH) agonists have been used to suppress female cycling in several marsupials and work by targeting GnRH action in the pituitary. After the GnRH agonist reaches its active endpoint, female cycling returns. This study investigated whether Lucrin Depot (Abbott Laboratories, Lake Bluff, IL, USA), a tailored 1-month microsphere GnRH agonist preparation, has the potential to first suppress female cycling, and then facilitate a synchronous return to female cycling in a model marsupial, the tammar wallaby (Macropus eugenii). Preliminary studies indicated that Lucrin could be used to regulate ovarian activity, but a dose >0.20 mg kg−1 was required. In this study, nonpregnant tammars (n = 18, 6 per group) had their pouch young removed (RPY) on Day 0; RPY suspends lactation and reactivates cycling, allowing for controlled assessment of oestrus and mating in treated and untreated females. On Day 0, an intramuscular (IM) injection of water (Control) or 1 mg kg−1 of Lucrin Depot (group A) was given after RPY. Group B also received 1 mg kg−1 of Lucrin Depot but this was not injected until Day 10 after RPY (a strategy to assess whether timing of Lucrin yields different results). Females were placed in breeding pens of 1 male to 3 females. Reproductive suppression and synchrony of return to female cycling was assessed by pouch and urogenital opening checks to detect newborn young and copulatory plugs. All data (time of mating/conception) were analysed using ANOVA (significance, P < 0.05). There was a significant difference between the first mating response in the Control compared with Groups A and B (P = 0.0003), but the time from Lucrin injection to mating, Day 0 or Day 10, was not significant (P = 0.1431). All control females mated before Day 32 RPY, whereas all Lucrin-treated females mated from Day 33 to 66. All Lucrin-treated females conceived as evidenced by pouch young. Further analysis revealed 2 Lucrin-injection to mating response intervals, those females delayed by ~10 days that conceived between 33 and 50 days (n = 7: 41 ± 2.1 days; mean ± SEM), and those females delayed by ~30 days that conceived between 61 and 66 days (n = 5: 62 ± 1.0 days; mean ± SEM) following injection. Thus, an IM injection of 1 mg kg−1 of Lucrin Depot is sufficient to delay oestrus in breeding tammar wallabies whether given at the time of RPY or 10 days later. This study confirmed that Lucrin Depot can be used to regulate ovarian activity in macropod marsupials, and has potential to form the basis of a GnRH agonist-based synchronisation strategy for use in assisted breeding for marsupial conservation.
This research was supported by Holsworth Wildlife Research Endowment.
98 Assessing Endangered Felid Puma concolor Sperm Fertility by In Vitro Fertilization with Domestic Cat Oocytes
Authors: Duque M. , Sestelo A. , Salamone D. F.
Published: November 2018

The Puma concolor population has been decreasing during the last 30 years. Semen cryopreservation of this species has been accomplished successfully and offers the possibility of preserving endangered species. We previously showed that fertilizing capability of wild felid spermatozoa can be evaluated using intracytoplasmic sperm injection (ICSI) with in vitro-matured domestic cat oocytes (Moro et al. 2014 Reprod. Domest. Anim. 49, 693-700). Due to the lack of homologous oocytes, we evaluated the capability of the Puma concolor sperm to induce domestic cat oocyte fertilization and subsequent pre-implantation embryo development. In the present study, cryopreserved sperm obtained by electroejaculation from five different males were used for IVF of in vitro-matured (IVM) domestic cat oocytes. Straws were thawed by exposing them to air for 10 s and then immersing in a 37°C water bath for 30 s. The contents of the straws were poured into a sterile 1.5-mL microtube pre-warmed to 37°C. The sperm suspension was diluted (1:3 v/v) by the slow (drop-by-drop) addition of a modified Tyrode’s solution. For IVF, IVM oocytes (n = 370) were co-incubated with 0.5 × 105 motile spermatozoa mL−1 in an atmosphere of 21% O2 in air at 38.5°C for 18 to 20 h. Presumptive zygotes were cultured in vitro in 50-μL drops of modified Tyrode’s medium on 6.5% CO2 in air at 38.5°C. Cleavage was determined at 48 h post-fertilization, and 5% FBS was added at Day 5 of in vitro culture. Blastocyst stage was evaluated at Day 8. Results (mean ± SEM) showed a high cleavage rate (179/370, 49.0 ± 4.0%), and a high development to morula stage (137/370, 34.4 ± 7.2%), and to blastocyst stage (94/370, 23.4 ± 4.7%) for all males. These results indicated that Puma concolor spermatozoa can induce domestic cat oocyte activation and development to blastocyst stage in similar rates to domestic cat homologous IVF: IVM oocytes (n = 291), cleavage rate (199/291, 67.1 ± 6.1%), development to morula stage (144/291, 47.8 ± 4.9%), and to blastocyst stage (86/291, 30.1 ± 1.6%). In conclusion, we demonstrated that domestic cat oocyte can be used to evaluated cryopreserve sperm samples from another felid species.
Climate changes during the past 31 years and their contribution to the changes in the productivity of rangeland vegetation in the Inner Mongolian typical steppe
Authors: Wu Xinhong , Li Peng , Jiang Chao , Liu Pengtao , He Jing , Hou Xiangyang
Published: November 2018

The objectives of this study were to explore the impact of climate change and human activities on the annual production of aboveground biomass of vegetation during the past 31 years at a county scale in the typical steppe region of Inner Mongolia. The changes in three banners in the region (Abag Banner, Xilinhaote City, and Xiwuzhumuqin Banner) were analysed. The changes in the annual potential grassland production (net primary productivity) and in the annual production of vegetation, as the sum of aboveground biomass and consumption by livestock, were estimated for each year. A comparison of the changing rates in net primary productivity and aboveground biomass of vegetation over the 31 years was used to distinguish the effects of climate change on grassland production from human activities. The results showed that the climate had become warmer and drier during the past 31 years and thus net primary productivity and annual production of vegetation decreased significantly. Climate change was a major factor for these decreases, while human activities were a minor factor in the decrease of grassland production in Xuwuzhumuqi Banner. The importance of human activities in reducing this decrease in grassland production during the last 31 years is in accordance with the changes in grassland-use policy that has encouraged destocking for grassland restoration in recent years.
Innovation in the rangelands: the role of people
Authors: Chaney F.
Published: November 2018

The importance of the rangelands, economically, environmentally, and culturally to Australia, is highlighted. Australians need to be more aware of and appreciate new ways of working in pastoralism, environmental management, with Indigenous communities and mining that point the way to better social, economic, cultural and environmental outcomes. Optimism about the future role of the rangelands stems from the changes in Australia that have occurred over the past 50 years, from a country that was legally and socially segregated. Changes started with advocacy of voting rights for Indigenous people in 1961 and continued with the establishment of Aboriginal legal services, the setting up of the National Native Title Tribunal and native title representative bodies, and the founding of Reconciliation Australia. Changes have occurred because people have tried to make things better, not just for themselves but for Australia. Leadership and tireless action from Indigenous people and non-Indigenous collaborators have been powerful forces for change. However, governments continue to often fail those who live and work in areas that are distant from cities. Change needs to continue and everyone who cares about rangelands has a role, in different ways, to nudge the world of the rangelands to a better place.
Comparing fixed and flexible stocking as adaptations to inter-annual rainfall variability in the extensive beef industry of northern Australia
Authors: Pahl Lester , Scanlan Joe , Whish Giselle , Cowley Robyn , MacLeod Neil
Published: November 2018

Many beef producers within the extensive cattle industry of northern Australia attempt to maintain a constant herd size from year-to-year (fixed stocking), whereas others adjust stock numbers to varying degrees annually in response to changes in forage supply. The effects of these strategies on pasture condition and cattle productivity cannot easily be assessed by grazing trials. Simulation studies, which include feedbacks of changes to pasture condition on cattle liveweight gain, can extend the results of grazing trials both spatially and temporally. They can compare a large number of strategies, over long periods of time, for a range of climate periods, at locations which differ markedly in climate.
This simulation study compared the pasture condition and cattle productivity achieved by fixed stocking at the long-term carrying capacity with that of 55 flexible stocking strategies at 28 locations across Queensland and the Northern Territory. Flexible stocking strategies differed markedly in the degree they increased or decreased cattle stocking rates after good and poor pasture growing seasons, respectively. The 28 locations covered the full range in average annual rainfall and inter-annual rainfall variability experienced across northern Australia.
Constrained flexibility, which limited increases in stocking rates after good growing seasons to 10% but decreased them by up to 20% after poor growing seasons, provides sustainable productivity gains for cattle producers in northern Australia. This strategy can improve pasture condition and increase cattle productivity relative to fixed stocking at the long-term carrying capacity, and its capacity to do this was greatest in the semiarid rangeland regions that contain the majority of beef cattle in northern Australia. More flexible stocking strategies, which also increased stocking rates after good growing seasons by only half as much as they decreased them after poor growing seasons, were equally sustainable and more productive than constrained flexibility, but are often impractical at property and industry scales. Strategies with the highest limits (e.g. 70%) for both annual increases and decreases in stocking rates could achieve higher cattle productivity, but this was at the expense of pasture condition and was not sustainable. Constrained flexible stocking, with a 10% limit for increases and a 20% limit for decreases in stocking rates annually, is a risk-averse adaptation to high and unpredictable rainfall variability for the extensive beef industry of northern Australia.
Innovation in Australian rangelands. A special issue from the 18th Biennial Conference of the Australian Rangeland Society
Authors: Davies Jocelyn , Race Digby , Wright Boyd
Published: November 2018

This special issue highlights organisational innovations and new insights about challenges of living successfully in Australian rangelands through a diverse set of papers from the 18th Biennial Conference of the Australian Rangeland Society. Key themes in the papers are collaborations for innovation and adapting to change. The papers indicate a shift to increased interdisciplinary and transdisciplinary research and greater diversity in the perspectives being voiced about rangeland futures.
Human papillomavirus prevalence to age 60 years among Australian women prevaccination
Authors: Brotherton Julia M. L. , Condon John R. , McIntyre Peter B. , Tabrizi Sepehr N. , Malloy Michael , Garland Suzanne M. , on behalf of the WHINURS study group
Published: November 2018

Background
The prevalence of human papillomavirus (HPV) at the cervix varies with age, peaking following sexual debut and declining thereafter in most populations. In some populations, a second peak is observed. Here we describe the prevalence of HPV at the cervix among Australian women before the commencement of the HPV vaccination program. Methods: Women aged 15 to 60 years attending health services for cervical screening between 2005 and 2008 were invited to participate. Liquid based cervical specimens were tested for 37 types of HPV using linear array. The percentage and 95% confidence interval of women with any type of HPV, any of 13 high risk HPV types, and with vaccine-preventable HPV types (types 6, 11, 16 and 18) were estimated in 5-year age bands. Results: Among 1929 women aged 15–60 years, HPV prevalence peaked at 64% at age 15–20 years, then declined gradually to 12% at age 41–45 years, whereafter it rose to 19% in women 51–55 years then returned to 14% in 56–60 year olds. Prevalence curves were similar for high-risk HPV types and vaccine-targeted HPV types 6, 11, 16 and 18 and when results were restricted to women with only normal cytology. Conclusions: The shape of the prevalence curve we observed is similar to those from other Western populations. Variation in prevalence curves is likely due to differences in sexual behaviour between populations and over time, reactivation of HPV during perimenopause, and possibly the presence of cervical screening programs. These data are the first such data from the Oceania region.