bluuebunny/crossref_metadata_2025 huggingface dataset:
149 Preliminary Results of the Oxytocin Treatment on the Collection of Cat Semen by Urethral Catheterization After Pharmacological Induction
Authors: Cremonesi F. , Mussi M. , Perrini C. , Lange-Consiglio A.
Published: November 2018

Urethral catheterization after pharmacological induction (Ur.Ca.P.I.) has been developed as a method to collect semen from cats (Zambelli et al. 2008 Theriogenology 69, 485-490). This procedure is based on contraction of the vas deferens induced by the α-agonist medetomidine and the subsequent release of sperm into the urethra. To ensure adequate sedation, the medetomidine dosage is 130 µg kg−1. Usually, a single collection of semen is done by catheterization immediately after medetomidine takes effect. A 3.5 French (Fr) gauge urinary tom cat catheter with the tip cut is inserted 90 mm deep into the urethra. Even in the absence of clinical signs, the cut catheter tip may cause minor trauma to the urethral mucosa. Aims of this study were to test a new catheter and evaluate effects of oxytocin administration on quality and quantity of semen collected. The study was performed on 8 client-owned, pubertal domestic cats, 10 to 18 months old. These cats were randomly allocated into 2 groups: control and treatment, with 3 and 5 cats, respectively. In each group, 2 semen collections were performed. The catheter had a diameter of 4 Fr and a rounded tip without mandrel and it was inserted 90 mm deep. In both groups, the first collection was performed as effects of medetomidine were apparent. In the control group, the second collection was done 10 min later. However, in the treated group, 5 IU of oxytocin was injected intramuscularly immediately after semen collection, with the second collection performed 10 min later. Semen was evaluated for volume, concentration, rate of progressive motility, curvilinear speed (VCL), morphology, and vitality. Comparisons between collections and between groups were done with a Student’s t-test. Comparing the second versus first collection in the control group, there was only an increase in volume (9.33 v. 2.67 µL; P < 0.05). In contrast, in the oxytocin group, all parameters evaluated were better (P < 0.05) for the second collection. As expected, the first collection was not different between the 2 groups for any parameter. However, for the second collection, there were differences (P < 0.05) between the oxytocin and the control group for volume (12.6 v. 3.7 µL), concentration (1252.6 v. 858 × 106 sperm mL−1), progressive motility (90.2 v. 77.8%), VCL (137.71 v. 117.94 µm s−1), morphologically normal sperm (87.0 v. 81.4%), and vitality (86.4 v. 82.8%). Oxytocin may be involved in mobilization of sperm from the epididymis. We concluded that oxytocin improved recovery of sperm from epididymal reserves, resulting in sperm with better morphology and function than those present in the vas deferens.
165 Effect of Dimethyl Sulfoxide Supplementation During In Vitro Maturation on the Genetic Expression Pattern of Bovine Blastocyst
Authors: Ynsaurralde-Rivolta A. E. , Suvá M. , Bevacqua R. , Rodriguez-Alvarez L. , Velasquez A. , Briski O. , Salamone D.
Published: November 2018

Supplementation of bovine oocytes with 0.5% (v/v) dimethylsulfoxide (DMSO) during in vitro maturation (IVM) results in increased blastocysts rates (Ynsaurralde et al. 2016 Reprod. Fertil. Dev. 29, 201-202). Recently, an important role of DMSO in stem cell differentiation has been observed, attributed to modulation of gene expression. However, the effect of DMSO suplementation during in vitro maturation on gene expression profiles and embryo quality have not been evaluated so far. Thus, we examinated the effect of DMSO during IVM on the expression of some key genes (Sox2, Oct4, and Cdx2) and on the degree of DNA fragmentation at the blastocyst stage. To this aim, cumulus–oocyte complexes collected from slaughterhouse ovaries were matured in TCM-199 containing 10% fetal bovine serum, 10 µg mL−1 FSH, 0.3 mM sodium pyruvate, 100 mM cysteamine, and 2% antibiotic-antimycotic for 24 h, at 6.5% CO2 in humidified air and 38.5°C. Maturation media was supplemented with 0, 0.5, or 0.75% (v/v) DMSO. In vitro fertilization (IVF) was performed with 16 × 106 spermatozoa per mL for 5 h. Afterwards, presumptive zygotes were cultured in SOF for 7 days at 38.5°C and 5% O2. Three pools of 5 blastocysts were analysed for each treatment. Gene expression analysis was performed by real-time qPCR and DNA fragmentation of blastocysts was measured by TUNEL assay (n = 8, 7, and 14 blastocysts analysed for 0, 0.5, and 0.75% v/v DMSO, respectively). The results were statistically analysed using ANOVA with a completely randomised model by InfoStat software Version 1.1 (https://www.infostat.com.ar/). The pluripotency marker genes Sox2 and Oct4 were up-regulated in blastocysts only when the oocytes were matured in 0.75% DMSO, whereas the trophoblastic marker Cdx2 showed no differences among treatments. No differences were detected in the number of TUNEL-positive cells among treatments: 10/65 (15%) in 0%, 19/110 (18%) in 0.5%, and 18/98 (20%) in 0.75% (v/v) DMSO. In conclusion, supplementation with 0.5% (v/v) DMSO, as previously published, increases the production of blastocysts without disrupting the expression pattern of the evaluated genes.
203 Genome Modifications by Sleeping Beauty Transposition and CRISPR/Cas9 to Improve Cow Milk Composition for Human Consumption
Authors: Alessio A. , Pericuesta E. , Llamas-Toranzo I. , Forcato D. , Fili A. , Liaudat C. , Rodriguez N. , Kues W. , Bermejo-Álvarez P. , Bosch P.
Published: November 2018

Genome manipulation of cattle represents a powerful tool to increase the nutritional value and reduce allergenicity of cow milk for human consumption. This could be accomplished by improving the amount of polyunsaturated fatty acids (ω-3 and ω-6) and simultaneously abolishing β-lactoglobulin (BLG), a potent allergen for predisposed humans. The aim of this study was to introduce the sequence for a desaturase construct (mFAT-2, from C. elegans), which is able to catalyse the synthesis of ω-3 and ω-6 fatty acids, into the bovine genome by Sleeping Beauty (SB) transposition, and simultaneously knocking out the bovine β-lactoglubulin gene using CRISPR/Cas9 system. The sgRNA (AAGTGCCTCCTGCTTGCCC) targeted to BLG exon 1 was synthesised as an oligo linker and cloned into the px459-Cas9. The mutation activity of the designed sgRNA at the target locus was determined by T7 endonuclease assay I (T7EI) mismatch detection assay. Briefly, bovine fetal fibroblasts (BFF) were seeded at 0.5 × 105 cells per well of a 24-well plate in triplicate, when the cells reached 80% confluence (12–24 h), cultures were transfected with 1 μg of px459-Cas9::BLG plasmid co-expressing Cas9 and sgRNA using polyethylenimine reagent (PEI; 3 ng μL−1). After 3 days of puromycin selection, genomic DNA from transfected cells were extracted and the sequence of interest was PCR-amplified and digested by T7EI restriction enzyme. Digestion products showed a mutation efficiency at the target locus of 29%. Subsequently, we chemically cotransfected 0.5 × 105 BFF with 0.5 μg of knockout vector (px459-Cas9::BLG) and 0.5 μg of SB plasmids (carrying mFAT-2 cDNA for mammary gland-specific expression) using 3 ng μL−1 PEI in triplicate. At 48 h post-transfection, cell cultures were subjected to 3 days of puromycin and 21 days of neomycin selection. PCR analysis of antibiotic resistant colonies revealed the presence of mFAT-2 transgene in almost 70% of the analysed cells lines. Genotyping of BLG exon 1 was performed by direct sequencing of PCR amplicons using primers flanking the target site. Despite the appreciable gene mutation activity of the sgRNA sequence previously determined by T7EI assay (29%), none of the cell lines analysed showed modification in the BLG target locus. We speculate that the SB vector might have disrupted the activity of targeting vector. We are currently performing additional experiments to accomplish gene addition (mFAT) and gene knockout (BGL) in one step using these highly efficient and precise transgenic tools. Genetically modified cells will be used as nuclear donor to produce transgenic cows by somatic cells nuclear transfer.
The financial support of CONICET, UNRC and FONCYT is gratefully acknowledged.
206 Effect of L-Ascorbic Acid, Polydatin, and In-Straw Rehydration of Slow-Frozen In Vitro-Produced Bovine Embryos
Authors: Dinndorf C. M. , Owen C. M. , Barceló-Fimbres M. , Campos-Chillon L. F.
Published: November 2018

In vitro-produced (IVP) bovine embryos have poor cryotolerance due to high lipid content and reactive oxygen species levels that hinder post-thaw survival. We hypothesised that in-straw rehydration of slow-frozen embryos with sucrose and the addition of the antioxidant polydatin and l-ascorbic acid would increase post-thaw survival. The IVP embryos (n = 116) were generated in 7 replicates by aspirating oocytes from 2- to 8-mm follicles of abattoir ovaries, matured for 23 h, fertilized with semen from 1 of 3 bulls using standard procedures, and cultured in SCF1 medium for 7 days (Owen et al. 2017 Reprod. Fertil. Dev. 29, 129-130). Stage 7 embryos were slow-frozen using 1 of 4 protocols in a 2 × 2 factorial design: embryos were equilibrated in conventional slow-freezing media for 20 min [1.5 M ethylene glycol (EG) and 0.5 M sucrose] with 1 mm l-ascorbic acid or 1 μM polydatin, and loaded in the straw adjacent to columns of freezing medium or 0.75 M EG and 0.6 M sucrose and then seeded at −6°C, cooled at 0.5°C min−1, and plunged at –32°C. Embryos were thawed in air for 10 s followed by 30 s in 32 to 35°C water bath. Once straw columns were disrupted, embryos were allowed to equilibrate for 5 min. Subsequently, embryos were washed and placed back in culture and re-expansion was assessed at 24 and 48 h. Data (Table 1) were analysed by ANOVA with means separated by Tukey’s HSD. Results indicate that there was no main effect between the 2 antioxidants or the use of rehydration columns (P < 0.05); however, there was higher (P < 0.05) re-expansion for embryos frozen with polydatin and with rehydration column than embryos frozen with l-ascorbic acid and no rehydration column. This suggests that polydatin coupled with in-straw rehydration (0.75 M EG and 0.6 M sucrose) may improve post-thaw survival of IVP bovine embryos.

Table 1.Post-thaw re-expansion rates of embryos exposed to antioxidants and in-straw rehydration (± SEM)
209 Imaging the Molecular and Cell Dynamics that Form the Early Mouse Embryo
Authors: Plachta N.
Published: November 2018

Our goal is to reveal how mammalian cells resolve their fate, shape, and position in the body in real time. Understanding how these decisions are made is critical to realise how embryos form and what problems compromise human fertility. Yet, their real-time control in vivo remains unknown. Because fixed specimens cannot capture cell dynamics, we established imaging technologies to study cells directly in live mouse embryos. We recently showed how transcription factors search and bind to the DNA to determine the first cell fates of the embryo. We found that differences in the binding of the transcription factor Sox2 to DNA appear as early as the 4-cell stage of development and predict cell fate. We also discovered that as cell choose their initial fates, they extend long filopodia protrusions to pull their neighbour cells closer, revealing a mechanism for embryo compaction and polarization. Finally, we established techniques to image how dynamic changes in the organisation of the actin and microtubule cytoskeletons regulate the first spatial segregation of inner and outer cells to form the future pluripotent inner mass and placental tissues of the embryo. Together, our findings reveal some of the key dynamic mechanisms that pattern the early mouse embryo.
28 Generation of Immunoglobulin Heavy Constant Mu (IGHM) Knockout Goats Using CRISPR/Cas9 and Somatic Cell Nuclear Transfer
Authors: Fan Z. , Regouski M. , Van Wettere A. J. , Wang Z. , Sullivan E. , Polejaeva I. A.
Published: November 2018

Towards the goals of inactivating endogenous goat immunoglobulin (Ig) genes and producing fully human polyclonal antibodies in transchromosomal goats carrying a human artificial chromosome comprising the entire human Ig gene repertoire, we report here the successful generation of IgM heavy chain knockout (IGHM−/−) goats using CRISPR/Cas9 and somatic cell nuclear transfer (SCNT) techniques. Two single-guide RNAs (sgRNAs) were designed specific for exon 1 of the goat IGHM constant region (GenBank: EU182621.1). The gene-targeting vectors were constructed by using pX330 plasmid (Plasmid No. 42230, Addgene, Cambridge, MA, USA) and transfected into 2 × 105 goat fetal fibroblast cells. Gene-targeting efficiency of each targeting vector was determined by PCR-restriction fragment length polymorphism (RFLP) assay 3 days post-transfection. Our results showed that one of the sgRNAs, 5′-GAAAGGCGCTTGAGGAATGC-3′, was efficient in directing Cas9 to generate targeted cleavages in exon 1 of IGHM constant region, with a mutation efficiency of 20%. We established single cell-derived fetal fibroblast colonies by limiting dilution of the cells transfected with the targeting vector. Colony screening with the PCR-RFLP assay confirmed that we achieved targeted gene disruption in exon 1 in 11/49 (22.4%) of the colonies (7 colonies with biallelic and 4 with monoallelic gene disruption). Sanger sequencing analysis of genomic DNA isolated from cell colonies with biallelic mutations showed that typical nucleotide deletions and insertions (indels), caused by repairing double-strand DNA breaks during the error-prone non-homologous end joining (NHEJ) process, were generated at the targeting site of exon 1. One of the colonies harboring a 1-nucleotide (nt) deletion was used as nuclear donors for SCNT. A total of 102 1-cell-stage cloned embryos were generated and surgically transferred into 6 synchronized recipients. Three of the recipients (3/6, 50.0%) were confirmed pregnant by ultrasonography on Day 40 to 45 of gestation. Two pregnancies were sacrificed for IGHM−/− fetal fibroblast isolation and one pregnancy was allowed to go to term, which led to the birth of a live and seemingly healthy kid. This goat was reared conventionally and appeared healthy until 5 weeks of age when it was killed because of commensal virus infection. The PCR-RFLP assay and sequence analysis showed that this cloned goat carried a biallelic 1-nt deletion in exon 1 of IGHM, which was identical to the donor colony it was originated from. No lymphoid follicles were observed in lymph nodes and spleen by histology, and immunohistochemistry for B cells (CD20) and T cells (CD3) demonstrated a lack of B cells in lymph nodes and spleen but the presence of T cells, confirming that IGHM has been successfully knocked out.
33 Improved Dog Cloning Efficiency Using Post-Activation with Ro-3306, a Cdk1 Inhibitor
Authors: Kim M. J. , Oh H. J. , Setyawan E. M. N. , Lee S. H. , Lee B. C.
Published: November 2018

Inactivation of maturation promoting factor requires proteolytic destruction of cyclin B that results in the loss of cyclin-dependent kinase 1 (Cdk1) activity and exit from metaphase. The aim of this study was to investigate that treatment of Ro-3306, a Cdk1 inhibitor, during post-activation could increase the development of somatic cell nuclear transfer (SCNT) embryos in dogs. Mixed breed female dogs aged at 1 to 5 years and weighing 20 to 35 kg were used in this study (approval number: SNU-160602-14-1). Canine cumulus–oocyte complexes were collected surgically by flushing oviducts with HEPES-buffered TCM-199 medium ~72 h after ovulation, which was determined by serum progesterone concentration. After removal of cumulus cells from oocytes by repeated pipetting in hyaluronidase, matured oocytes were selected for the following experiment. In experiment I, oocytes were activated with (1) 10 μM calcium ionophore and then post-activated with 1.9 mM DMAP (control); (2) DMAP along with 10 μM Ro-3306 (10 μM group); or (3) DMAP along with 50 μM Ro-3306 (50 μM group). Parthenotes were cultured in the synthetic oviducal fluid (SOF) medium after post-activation, and in vitro development was evaluated at 48 h (2-4 cell) and 72 h (6-8 cell). In experiment II, SCNT embryos were produced after oocyte enucleation, donor cell injection, fusion, and activation. Only fused cytoplasts were activated with (1) 1.9 mM DMAP (control) or (2) DMAP along with 50 μM Ro-3306 (50 μM group) and transferred to the oviducts of recipients. The day of embryo transfer was regarded as Day 0. Pregnancy diagnosis was performed by ultrasonography after Day 28 and cloned puppies were delivered Day 58 to 60. Embryo developmental rates in experiment I and II were analysed by one-way ANOVA and t-test, respectively, and pregnancy and delivery rate were analysed by chi-squared test using Graph Prism software (GraphPad, San Diego, CA, USA). The significance level was P < 0.05. Results in experiment I showed that cleavage rate of parthenogenetic embryos in the 50 μM group (89.3 ± 6.8%) was significantly higher than that of 10 μM group or control (50.8 ± 9.9% and 55.4 ± 18.8%, respectively). However, embryonic development to 4 cells and 6-8 cells was not different between treatments. In experiment II, pregnancy rates of recipients receiving embryos in 50 μM group (3/5, 60.0%) were significantly higher than that of control (2/6, 33.3%), but the number of healthy cloned puppies delivered in the 50 µM group (n = 6) versus the control (n = 2) was not different. In conclusion, post-activation with 50 μM Ro-3306 may enhance nuclear reprogramming of dog cloned embryos.
This study was supported by RDA (#PJ010928032017), Korea IPET (#316002-05-2-SB010), Research Institute for Veterinary Science, Natural Balance Korea and the BK21 plus program.
37 Healthy Foals Produced Using Bone Marrow-Mesenchymal Stem Cells as Nuclear Donors in Horse Cloning
Authors: Olivera R. , Moro L. , Jordan R. , Luzzani C. , Miriuka S. , Vichera G.
Published: November 2018

Somatic cell nuclear transfer efficiency is based on the capacity of the donor cell to be reset and reprogrammed to an embryonic state. So, the less differentiated the donor cells are, the more easily they could be reprogrammed by a recipient cytoplasm. Failures on appropriate nuclear reprogramming frequently lead to abnormalities associated with the placenta, umbilical cord, birthweight, and limbs. In the present study, we evaluated the efficiency of bone marrow mesenchymal stem cells (BM-MSC) compared with adult fibroblasts (AF) as nuclear donors in horse cloning and evaluated both in vitro and in vivo development of the embryos generated. Moreover, we focused on comparing the health of the foals generated and on the presence of anatomical abnormalities in foals produced from the different treatments. Embryos produced by AI, recovered by uterine flushing, and transferred to recipient mares were used as controls. All variables were analysed by Fisher test (P < 0.05). The cloning procedure was performed according to Olivera et al. (2016 PLoS One 11, e0164049, 10.1371/journal.pone.0164049). Both cleavage and blastocyst rates were higher when MSC were used as nuclear donors (P < 0.05). Cleavage rates were 85.6% (3875/4527) v. 90.2% (3095/3432) and blastocyst rates were 10.9% (492/4527) and 18.1% (622/3432) for AF and MSC groups, respectively. In the AF group, 476 blastocysts were transferred to recipient mares (232 transfers), and in the MSC group, 594 blastocysts were transferred 297 transfers). In the AI control group, 88 embryos were transferred. Pregnancies were diagnosed by transrectal ultrasonography 15 days after embryo transfer in all the groups. Pregnancy rates were similar between both cloning groups (41/232, 17.7% and 37/297, 12.5%for AF and MSC, respectively), but higher in the AI group (71/88, 80.7%). However, significant differences were observed in the birth of viable offsprings among the cloning groups. Despite similar rates of foal delivery (AF, 17/41, 41.5%; MSC, 21/37, 56.7%), a higher proportion of viable foals were obtained from the MSC group (20/37, 54.1%) compared with the AF group (9/41, 22%; P < 0.05). Surprisingly, as in the AI group (63/63, 100%), all of the viable foals obtained using MSC (20/20, 100%) were considered normal and did not show abnormalities associated with cloning. In contrast, in the AF group, only 4/9 (44.4%) were considered normal foals. The defects present in the other 5 foals were related to flexural and angular limb deformities and umbilical cord malformations. These were corrected rapidly with standard treatments or, in the case of the umbilical cords, minor surgery. This study shows for the first time that BM-MSC can be used as nuclear donors in horse cloning and that the foals obtained are as healthy as those produced by AI, showing no abnormalities related to deficiencies in nuclear reprogramming.
45 DNA Fragmentation of Epididymal Freeze-Dried Ram Spermatozoa Impairs Embryo Development
Authors: Palazzese L. , Anzalone D. A. , Gosálvez´ J. , Loi P. , Saragusty J.
Published: November 2018

Sperm freeze-drying is a revolutionary technique that resolves many of the drawback of long-term storage under liquid nitrogen. The first significant result of this method was provided by Wakayama and Yanagimachi (1998 Nat. Biotechnol. 16, 639-641, 10.1038/nbt0798-639), demonstrating for the first time the birth of healthy offspring from epididymal freeze-dried (mouse) spermatozoa. Besides models in the mouse and rat, which are the first small mammals born from epididymal lyophilized sperm by intracytoplasmic sperm injection (ICSI), most studies in this field have used ejaculated sperm. In this work, aiming to repeat the result of Wakayama and Yanagimachi, we tried to apply this technique to epididymal spermatozoa from a large mammal (ram). Moreover, we checked the correlation between freeze-dried spermatozoa DNA integrity and embryo development. To do this, epididymal sperm from 4 rams was lyophilized in a medium containing trehalose, glucose, KCl, HEPES, and Trolox. To evaluate DNA damage and fragmentation at rehydration, part of the sperm was processed for sperm chromatin dispersion test (SCD) and two-tailed comet assay and the rest was used for ICSI. Compared with rams 1 and 3, rams 2 and 4 had higher rate of spermatozoa with intact DNA (median: 3.3% v. 16.5%, respectively), lower rate of single strand breaks (SSB; median: 94.2% v. 81.5%, respectively) and lower rate of double-strand breaks (DSB; median: 2.5% v. 2%, respectively). Embryo development following ICSI showed that blastocyst stage was reached only from rams that had sperm with more intact DNA: ram 2 (4.8%, n = 83) and ram 4 (6.3%, n = 64). Spermatozoa from rams 1 and 3 produced no blastocysts. This can be explained by the fact that rams 2 and 4 had higher rate of spermatozoa with intact DNA than rams 1 and 3. Definitively, the implication of sperm DNA damage in embryonic development should depend on the balance between the extent of sperm DNA fragmentation, the type of fragmentation (SSB or DSB), and the oocyte’s repair capacity. Rams 2 and 4 were the only rams that produced blastocyst probably because they had considerably more sperm with normal DNA; thus, it is important to select spermatozoa of the best quality to perform a good ICSI. Fragmentation of DNA due to the lyophilization process impairs embryonic development. To conclude, oocytes injected with epididymal freeze-dried ram spermatozoa can reach the blastocyst stage. These are preliminary results; more conclusive outcomes will be given following embryo transfer experiments that are now in progress.
52 Difference of Seminal Plasma Proteins in Good- and Poor-Freezability Boar Ejaculates
Authors: Rungruangsak J. , Suwimonteerabutr J. , Asawakarn S. , Buranaamnuay K. , Chantaravisoot N. , Pisitkun T. , Tummaruk P.
Published: November 2018

Seminal plasma is the semen components that maintain sperm metabolism, pH and osmolality. Fibronectin (FN1) and glutathione peroxidase (GPX5) are the seminal plasma proteins that play an important role on the boar sperm functions. The purpose of the present study was to determine the differences in GPX5 and FN1 contents in the boar semen having good, moderate, and poor freezability. A total of 38 ejaculates from 25 boars with proven fertility were included in the experiment. All the ejaculates included in the study had >70% subjective motility, >75% normal morphology, >75% sperm viability, and volume >100 mL per ejaculate. The semen was collected through semen collection bag with filter and split into 2 portions. The first portion was prepared for the evaluation of seminal plasma proteins (i.e. GPX5 and FN1) and the second portion was cryopreserved and evaluated for post-thaw semen qualities. The seminal plasma samples were collected in cryotube and plug into liquid nitrogen. The samples was stored at –80°C before protein extraction. After thawing, the ejaculates were classified into 3 groups according to their post-thawed sperm motility: poor (14.6 ± 3.9%), modersate (28.5 ± 4.1%), and good (64.0 ± 8.7%) freezability. The amounts of GPX5 and FN1 proteins were evaluated through Western blot analysis. The normalized quantity of proteins was compared among groups by one-way ANOVA. The normalized level of FN1 in seminal plasma was higher in good- than in poor-freezability groups (8.0 ± 0.8% v. 5.7 ± 0.7%, respectively; P < 0.05), but did not differ significantly compared with that of the moderate-freezability group (7.5 ± 0.8%; P > 0.05). The levels of GPX5 in good-, moderate-, and poor-freezability groups were 14.7 ± 3.0%, 16.8 ± 3.2%, and 10.9 ± 3.0%, respectively (P > 0.05). The level of FN1 in seminal plasma was significantly correlated with the post-thaw sperm progressive motility (r = 0.38, P = 0.01), total motility (r = 0.37, P = 0.02), and the proportion of bent tail sperm (r = –0.33, P = 0.04). The level of GPX5 was not correlated with any of the post-thaw sperm qualities (P > 0.05). However, the levels of GPX5 was positively correlated with FN1 (r = 0.40, P = 0.01, n = 38). It can be concluded that FN1 in seminal plasma can be used as a marker of sperm freezability in boar.
54 Effect of Different Cryopreservation Protocols for Sheep Embryonic Stem Cells
Authors: Ibraimova N. , Seisenbayeva A. , Toishibekov Y.
Published: November 2018

Particular attention is required to improve cryopreservation of embryonic stem cells (ESC) and study their characteristics. Stem cells were obtained from the inner cell mass of Day 5-6 blastocysts. The ESC were then cultured on mTeSR™1 medium (Stemcell Technologies, Cambridge, MA, USA). We studied the survival of ESC after slow freezing and vitrification. Slow freezing was carried out using a Planer Kryo 360-3.3 freezer (Planer plc, Sunbury-on-Thames, United Kingdom), using various cryoprotectants: 1.5 M dimethyl sulfoxide (Me2SO), 1.5 M ethylene glycol (EG), or 1.5 M propylene glycol (PG). Six vitrification solutions (VS) were used to vitrify ESC: VS1 = 20% Me2SO + 20% EG + 0.5 M sucrose; VS2 = 20% Me2SO + 20% PROH + 0.5 M sucrose; VS3 = 20% EG + 20% PG + 0.5 M sucrose; VS4 = 20% Me2SO + 20% EG + 0.5 M sucrose + 10% FCS; VS5 = 20% Me2SO + 20% PROH + 0.5 M sucrose + 10% FCS; and VS6 = 20% EG + 20% PG + 0.5 M sucrose + 10% FCS. For the dehydration of cells and the addition of vitrification solutions, a 3-step equilibration was used. The proliferative properties of the cells were determined using an Apel PD-303S spectrophotometer (Apel Co. Ltd., Kawaguchi, Japan), using an MTT test (staining with methylthiazolyl-diphenyl tetrazolium). After slow freezing, the highest percentage of frozen–thawed cells proliferating was observed when using 1.5 M EG (P > 0.05). At the same time, the highest cell doubling after thawing was observed when using 1.5 M EG, and 1.5 M Me2SO. After vitrification, the highest percentage of proliferation was observed in the VS2 and VS4 groups (49.7 ± 3.2% and 53.2 ± 3.8%, respectively). It should be noted that the addition of fetal calf serum to the vitrification solution also increased the proliferation of ESC after vitrification and thawing.
6 CRISPR-Generated ZP4 Knockout Rabbits Exhibit Impaired In Vivo Pre-Implantation Development
Authors: Lamas-Toranzo I. , Balvís N. F. , Lorenzo P. L. , Rebollar P. G. , Avilés M. , Bermejo-Álvarez P.
Published: November 2018

Mammalian oocytes are surrounded by a glycoprotein layer termed the zona pellucida (ZP), which acts as a protective barrier during pre-implantation embryo development and plays essential roles during follicular development and fertilization. This structure is composed of 4 different proteins in several species such as rabbits and humans. However, the oocytes of the only species for which knockout (KO) embryos are readily available, the laboratory mouse, lacks one of these proteins (ZP4), so its function remains elusive. To determine the role of ZP4, we have generated ZP4 KO rabbits by CRISPR and compared their reproductive function to that of heterozygous (Hz) and wild type (wt) rabbits. Following mating with wt males, female KO rabbits showed a clear impairment in reproductive performance compared with Hz or wt females, with only 1 out of 5 KO females producing a small litter of 4 pups, whereas all mated Hz and wt females exhibited normal litter sizes (wt 9.2 ± 0.6; Hz 10.6 ± 0.5; mean ± SEM). In order to elucidate the cause for the impaired delivery rate, ovulation rates and cleavage rates were initially assessed, with no differences observed between groups (oocytes ovulated: wt 11.7 ± 1; Hz 15 ± 2.9; KO 13.3 ± 2.9; cleavage rates (%): wt 81.7 ± 0.1; Hz 95.5 ± 0.1; KO 87.3 ± 0.1), which suggests that ZP4 does not play a critical role in folliculogenesis or fertilization. However, when expanded blastocysts were recovered at Day 6 after mating, embryo development was found to be clearly impaired in embryos lacking ZP4, as none of the embryos obtained from 2 KO females were able to undergo blastocyst expansion. Morphological differences were observed in the zona pellucida from KO rabbits, being irregular, noticeably less elastic and easier to deform, and significantly thinner (thickness: wt 15.2 ± 1.5 µm; Hz 15.3 ± 1.4 µm; KO 10.9 ± 0.7 µm; ANOVA P < 0.05). These morphological differences suggest that rabbit ZP without ZP4 may fail to protect the embryo during development or may physically impede blastocyst expansion.
This study is supported by the projects AGL2014-58739-R and RYC-2012-10193 (to PBA), AGL2015-70159-P (to MA) and AGL2015-65572-C2-1-R (to PGR and PL). ILT and NFB are supported by FPI grants.
63 Impact of Endometrial Biopsy on Corpus Luteum Function
Authors: Ramirez-Garzon O. , Satake N. , Hill J. , Holland M. K. , McGowan M.
Published: November 2018

Reproductive procedures involving uterine manipulation (UM), such as transcervical embryo transfer (TET) or the collection of an endometrial biopsy (EB) may cause trauma to the endometrium resulting in release of endogenous prostaglandin F2α (PGF2α) causing premature regression of the corpus luteum (CL). This study aimed to evaluate the effect of UM and EB on corpus luteum lifespan in tropically adapted beef heifers. In Experiment 1, pre-synchronised Droughtmaster heifers (n = 9; 25.8 ± 0.4 m, live weight 391 ± 21.5 kg) were randomly selected either at Day 4 (UM4 = 5) or at Day 7 (UM7 = 4) post-oestrus for transrectal uterine manipulation and transcervical passage of the biopsy Storz® device without sample collection. In Experiment 2, pre-synchronised Charbray heifers (n = 13, 2 to 3 years old, live weight 439.8 ± 17.2) were biopsied in the horn ipsilateral to the CL at Day 4 (B4 = 6) or at Day 7 (B7 = 7) post-oestrus. In both experiments, blood samples were taken from jugular/coccygeal vein throughout the oestrus cycle on Days 3, 4, 6, 7, 9, 11, 13, 16 and 19 (Day 0 = synchronised oestrous) for progesterone (P4) analysis. Signs of heat were visually detected daily and confirmed by ovarian ultrasound. For the PGF2α metabolite 13,14-dihydro-15-keto-PGF2α (PGFM) assessment, blood samples were collected before and after uterine trauma at –24, –12, 0 h and +6, +18, +24 h (Hour 0 = UM or EB either at Day 4 or Day 7). Progesterone was assessed by liquid chromatography-mass spectrometry (LC-MS) and PGFM using an enzyme immunoassay DetectX® kit (Arbor Assays, Ann Arbor, MI, USA). The intra- and interassay coefficients of variations were 3.7% and 12.8%, respectively. A series of generalized additive mixed effect models (GAMM) were created to quantify P4 differences for UM and EB. The results showed that oestrous cycle length was not affected by UM or EB conducted at Day 4 or at Day 7, because P4 concentration remained above 1 ng mL−1 after uterine trauma during dioestrus. Also, UM and EB did not induce the release of luteolytic pulses of PGF2α. Although P4 concentration followed the normal cycling pattern throughout the oestrus cycle, the shape of the curve in EB4 and UM4 heifers showed higher concentrations of P4 compared with EB7 and UM7 heifers. In conclusion, UM and EB with Storz® device during metoestrus or early dioestrus had minimal or nil effects on CL activity. Whether lower P4 profiles affect embryo development after uterine trauma needs to be further elucidated.
72 Protective Effects of C-Phycocyanin on the Developmental Competence of Porcine Parthenotes
Authors: Niu Y.-J. , Kim N.-H. , Cui X.-S.
Published: November 2018

C-Phycocyanin (CP) is a biliprotein enriched in blue-green algae that is known to possess antioxidant, anti-apoptosis, anti-inflammatory, and radical-scavenging properties in somatic cells. However, the protective effect of CP on porcine embryo developmental competence in vitro remains unclear. In the present study, we investigated the effect of CP on the development of porcine early embryos as well as its underlying mechanisms exposing them to H2O2 to induce oxidative stress. The levels of reactive oxygen species, mitochondrial membrane potential, apoptosis, DNA damage, and autophagy in the blastocysts were observed by staining with 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA), 5,5′,6,6’-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1), terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate (dUTP) nick-end labelling (TUNEL), anti-cytochrome c, and anti-γH2A.X (Ser139), respectively. Colocalization assay of mitochondria and cytochrome c of blastocysts were staining with MitoTracker Red CMXRos and anti-cytochrome c. All data were subjected to one-way ANOVA. Different concentrations of CP (1, 2, 5, 8, 10 µg mL−1) were added to porcine zygote medium 5 (PZM-5, l-glutamine concentration of PZM-3 was modified from 1 to 2 mM) during in vitro culture. The results showed that 5 µg mL−1 CP significantly increased blastocyst formation (62.5 ± 2.1 v. 52.7 ± 2.4; P < 0.05) and hatching rate (10.9 ± 1.9 v. 36.6 ± 5.2; P < 0.05) compared with controls. Blastocyst formation (53.1 ± 2.3 v. 40.1 ± 2.3; P < 0.05) and quality were significantly increased in the 50 µM H2O2 treatment group following 5 µg mL−1 CP addition. C-Phycocyanin prevented the H2O2-induced compromise of mitochondrial membrane potential, release of cytochrome c from the mitochondria, and generation of reactive oxygen species. Furthermore, apoptosis, DNA damage level, and autophagy in the blastocysts were attenuated by supplementation of CP in the H2O2-induced oxidative injury group compared with that in controls. These results suggest that CP has beneficial effects on the development of porcine parthenotes by attenuating mitochondrial dysfunction and oxidative stress.
88 Effect of Follicular Ablation and GnRH on Recipient Synchrony in Holstein Heifers
Authors: Palomino J. M. , Grand F.-X. , Vigneault C. , Blondin P. , Sirard M.-A.
Published: November 2018

In Holstein heifer recipients that are synchronized with a single dose of prostaglandin F2α (PGF2α), only 65% (on average) are eligible to receive an IVF embryo 7 ± 1 days after oestrus. The high variability in time to onset of oestrus after PGF2α treatment and the small size of the corpus luteum (CL) on the day of embryo transfer (ET) may explain these results. Synchronizing ovulation might decrease the variability of the interval from treatment to ovulation and maximize the size of the CL. For this purpose, Holstein heifers were randomly allocated (using the lottery method of sampling) to 3 groups: (1) Control (n = 88), a single dose of PGF2α (500 μg of cloprostenol, Merck, Darmstadt, Germany; Day 0) and transrectal ultrasonography of the ovaries on Day 0 to determine the diameter of the largest follicle, on Days 2, 3, 4, and 5 to determine occurrence of ovulation, and on Day 10 to determine presence and diameter of the CL; (2) follicular aspiration (n = 15), ultrasound-guided follicular aspiration (FA) 4 days before PGF2α administration and ovarian evaluation as described above; and (3) FA + GnRH (n = 19), FA at Day –4, gonadotropin-releasing hormone (GnRH, 100 µg of gonadorelin acetate, Vetoquinol USA, Fort Worth, TX, USA) on Day 2, and ovarian examination as described in the control group. Data were analysed by one-way ANOVA and proportions by Chi-squared. The interval (days) from PGF2α treatment to ovulation was not different among groups (3.2 ± 0.4, 3.1 ± 0.2, 3.0 ± 0.1, respectively). However, the variability (residuals) in the FA + GnRH group was the smallest (P < 0.05; high degree of synchrony) in the study (0.1 ± 0.1). Likewise, the FA group (0.5 ± 0.1) had a smaller interval (P < 0.05) compared with the Control group (0.8 ± 0.1). Interestingly, 86% of heifers from FA + GnRH group ovulated in a single day (Day 3; P < 0.05) compared with the FA and Control groups (55% and 53%, respectively). Considering the day of ovulation (only recipients that ovulated 2 to 4 days after PGF2α treatment) and CL diameter (>20 mm; Nogueira et al. 2012 R. Bras. Zootec. 1, 2129-2133; Yoshida et al. 2012 Anim. Sci. J. 83, 207-212), heifers in the FA and FA + GnRH groups had higher proportion (P < 0.05) of animals selected to be transferred at Day 10 compared with the Control group (93%, 95%, and 67%, respectively). In conclusion, FA induced a more synchronous ovulation and increased the number of recipients ready to be transferred at Day 10 (7 ± 1 days after ovulation). These effects were further improved with the administration of GnRH.
Floristic composition and pasture condition of Aristida/Bothriochloa pastures in central Queensland. II. Soil and pasture condition interactions
Authors: Silcock R. G. , Hall T. J. , Filet P. G. , Kelly A. M. , Osten D. , Graham T. W. G.
Published: November 2018

Sustainable management of native pastures requires an understanding of what the bounds of pasture composition, cover and soil surface condition are for healthy pastoral landscapes to persist. A survey of 107 Aristida/Bothriochloa pasture sites in inland central Queensland was conducted. The sites were chosen for their current diversity of tree cover, apparent pasture condition and soil type to assist in setting more objective bounds on condition ‘states’ in such pastures. Assessors’ estimates of pasture condition were strongly correlated with herbage mass (r = 0.57) and projected ground cover (r = 0. 58), and moderately correlated with pasture crown cover (r = 0.35) and tree basal area (r = 0.32). Pasture condition was not correlated with pasture plant density or the frequency of simple guilds of pasture species. The soil type of Aristida/Bothriochloa pasture communities was generally hard-setting, low in cryptogam cover but moderately covered with litter and projected ground cover (30–50%). There was no correlation between projected ground cover of pasture and estimated ground-level cover of plant crowns. Tree basal area was correlated with broad categories of soil type, probably because greater tree clearing has occurred on the more fertile, heavy-textured clay soils. Of the main perennial grasses, some showed strong soil preferences, for example Tripogon loliiformis for hard-setting soils and Dichanthium sericeum for clays. Common species, such as Chrysopogon fallax and Heteropogon contortus, had no strong soil preference. Wiregrasses (Aristida spp.) tended to be uncommon at both ends of the estimated pasture condition scale whereas H. contortus was far more common in pastures in good condition. Sedges (Cyperaceae) were common on all soil types and for all pasture condition ratings. Plants identified as increaser species were Tragus australianus, daisies (Asteraceae) and potentially toxic herbaceous legumes such as Indigofera spp. and Crotalaria spp. Pasture condition could not be reliably predicted based on the abundance of a single species or taxon but there may be scope for using integrated data for four to five ecologically contrasting plants such as Themeda triandra with daisies, T. loliiformis and flannel weeds (Malvaceae).
New partnerships for managing large desert landscapes: experiences from the Martu Living Deserts Project
Authors: Jupp Tony , Fitzsimons James , Carr Ben , See Peter
Published: November 2018

Native fauna in Australia’s arid zone has declined significantly since European settlement; however, Martu country in the Western Desert of Western Australia retains a diversity of iconic and threatened species that were once more widespread. An innovative partnership between The Nature Conservancy, BHP Billiton and the Martu people (represented by Kanyirninpa Jukurrpa – KJ) is achieving positive social, cultural, economic and environmental outcomes, which builds on funding from the Australian Government for land management on Martu country. The partners support Martu people in fulfilling their desire to conserve the cultural and natural values of their 13.7 million ha native title determination area. Through KJ as the local delivery partner, Martu people are returning to work on country to clean and protect waterholes; improve fire management; control feral herbivores and predators; manage cultural heritage; and actively manage priority threatened species (such as the Greater Bilby and the Black-flanked Rock-wallaby). The project provides significant employment opportunities for Martu men and women in ranger teams working throughout their country. It is also generating measurable social, cultural and economic benefits for Martu people and environmental benefits for part of the most intact arid ecosystem anywhere on Earth.
Rangeland pastoralism in northern Australia: change and sustainability
Authors: Holmes P. R.
Published: November 2018

Australian rangeland pastoralism is now a marginal economic activity. To survive as an industry, it must retain critical mass and to do that, the majority of producers need to be financially sustainable. Significant change has taken place in the past 40 years to facilitate this, but this change appears to have had limited enduring value for much of the industry. The productivity of the average northern rangeland beef herd in absolute terms is poor and is far from being competitive now, let alone in the future. If preservation of the natural resource base is included as a sustainability criterion, the industry outlook becomes even more uncertain. The conundrum is that it has never been more opportune to succeed in rangeland pastoralism, but the evidence is that most producers do not. The conclusion is that the lack of financial literacy and business skill remains the biggest impediment to most pastoralists achieving financial sustainability.
Cultural indicators, country and culture: the Arabana, change and water
Authors: Nursey-Bray Melissa , Arabana Aboriginal Corporation
Published: November 2018

Water is of paramount importance to all people who live in Kati Thanda-Lake Eyre, Australia, but to the Arabana people, one of the Indigenous peoples of the region, it also holds immense cultural significance. Having lived in the region for thousands of years, Arabana people have developed their own methods to assess the ecological condition of their water sites. This paper presents the results of a collaborative project designed to develop a suite of cultural indicators for water sites in the Kati Thanda region. Based on field trips and a comprehensive desktop review, this paper presents the indicators or ‘signs’ used by the Arabana to assess condition. Key results show that although water has multiple values for the Arabana, water itself is understood as a series of sites within one country rather than as a series of ecosystems or types of water body. Further, the cultural indicators or the ‘signs’ by which Arabana people assess conditions are potentially synergistic with scientific indicators and include fauna, flora, quality, soil, and climatic dimensions. However, Arabana people also assess a site according to its history and the level of cultural knowledge about the site. The paper concludes with a reflection on whether or not these indicators can be used in more generic ways across the region to assist in broader river assessment processes. A cultural indicator schemata is suggested as a starting mechanism for identifying cultural indicators in other parts of Kati Thanda-Lake Eyre, but the paper concludes by arguing that any effective implementation of such a schemata must involve Indigenous peoples at every level.
Sexual behaviour of inmates with Chlamydia trachomatis infection in the prisons of Catalonia, Spain
Authors: Corbeto Evelin L. , Carnicer-Pont Dolors , Lugo Rossie , Gonzalez Victoria , Bascuñana Elisabet , Lleopart Nuria , Barbero Luis , Humet Victoria , Casabona Jordi , Chlamydia Study Group in Catalonia Prisons
Published: November 2018

Objective
To determine the prevalence of Chlamydia trachomatis (CT) and high risk factors for acquisition in preventive prisoners in Catalonia.
Methods: Cross-sectional study of a convenience sample of 478 prisoners aged between 18 and 35 years was analysed using real-time polymerase chain reaction. A standardized questionnaire was used to collect behavioural data. Significant differences were analysed in the descriptive study using Pearson’s χ2. The association between CT and its determinants was analysed using the Mantel–Haenszel test and a multivariate logistic regression model.
Results: The overall prevalence of CT was 5.4%. The independent risk factors for infection by CT were as follows: foreign origin, having had concurrent sexual partners, and alcohol consumption.
Conclusions: This is the first study performed in prisons of Catalonia that shows the prevalence of CT in young prisoners. The high mobility of young detainees could explain the similarity in prevalence obtained about young people in Catalonia. Systematic monitoring of CT infection in young preventive prisoners is important in order to prevent further problems in themselves and in the general population, since they become a ‘bridge population’ in sexually transmissible infection spreading.