bluuebunny/crossref_metadata_2025 huggingface dataset:
34 The effect of dilution method of beagle dog semen on the survival rate of cryopreserved spermatozoa after thawing
Authors: Kim S. W. , Kim C.-L. , Jeon I. S. , Ko Y. G. , Hwang I.-S.
Published: December 2018

The successful cryopreservation of spermatozoa of the beagle dog for AI is essential for the establishment of the genetic banks of drug detection dogs. The beagle dog is widely used for drug testing and chosen for breeding by breeders. However, the use of cryopreserved beagle semen is limited by the lower number of offspring of dog species. In this study, 3 highly trained beagle dogs were chosen and their semen was cryopreserved for the next generation. The effects of dilution methods of beagle semen were tested using a direct dilution method at RT and a 2-step dilution method at 5°C. As a control group, the effects of a direct dilution method of semen on the percentage of motile sperm and progressive motility were analysed by computer-assisted semen analysis system (SAIS, Korea), and abnormality of spermatozoa was examined by Diff Quik staining. A total of 9 samples from 3 dogs were extended in 4% glycerol containing Tris-egg yolk diluents at approximately 22 to 25°C. The diluted semen was cooled to 5°C within 2h. The packed 0.5-mL straws were placed 5cm above the surface of LN for 10min and then plunged in. A 2-step dilution method was conducted using the same procedures of freezing, but the first dilution was done with glycerol-free diluent. After cooling to 5°C within 2h, the second diluent with 8% glycerol was added to the same volume of diluted semen at 5°C and stabilised for 1h. After thawing for 45s at 37°C, the semen from the 2-step dilution method showed the higher percentage of motile sperm (65.4±6% v. 45.3±8%; P<0.05) and progressive motility (41.6±5.3% v. 32.3±3.7%; P<0.05). However, the abnormalities between groups showed no differences. The results suggest that the optimal method for freezing beagle dog spermatozoa is a 2-step dilution process that consists of the first dilution at RT and the second dilution with glycerol at 5°C into diluted semen.
37 Viability of sheep skin fibroblasts after vitrification
Authors: Toishibekov Y. , Asanova E. , Yermekova M. , Seisenbayeva A. , Toishybek D.
Published: December 2018

Both tissue and cell cryopreservation can be applied for biodiversity conservation. The proper preservation of tissues and cells from a wide range of animals of different species is of paramount importance because these cell samples could be used to reintroduce lost genes back into the breeding pool by somatic cell cloning. The aim of this work was to investigate the effect of vitrification on viability of vitrified sheep fibroblasts for conservation of biodiversity so that it might be used in the future to provide nuclear donors. Skin samples collected from 10 adult sheep were cut into small pieces (1×1mm), placed into culture Petri dishes containing DMEM supplemented with 20% (vol/vol) fetal bovine serum, and covered with coverslips followed by incubation at 5% CO2, 95% RH, and 37°C. During culture, fibroblasts left skin samples and proliferated. Culture medium was changed every 4 days. After 21 to 22 days of incubation, a fibroblast monolayer was observed, culture medium was removed, and cells were incubated for 7 to 10min in the presence of Dulbecco’s PBS+0.25% trypsin. Dissociated fibroblasts were washed with DMEM by centrifugation at 300×g for 10min. For vitrification, fibroblast samples were then diluted at a concentration of 2×106cellsmL−1 in DMEM+ 20% ethylene glycol, 20% dimethylsulfoxide, and 0.5molL−1 of sucrose. The fibroblasts were then exposed to 50 and 100% vitrification solution (VS) at 37°C for 5min and 30s, respectively. Fibroblasts after saturation in VS were transferred and placed into 0.25-mL plastic straws. Straws were sealed with modelling clay and plunged into LN. Viability of frozen-thawed fibroblast samples was detected using the Trypan Blue staining method (frozen-thawed: 53.0±2.6%; control (fresh): 98.5±1.2%). The values obtained are expressed as mean±standard error of the mean. Statistical analysis was done using Student’s t-test. Results indicated that there was a significant difference in viability between fresh and cryopreserved fibroblasts. Importantly, our data suggest that the use of vitrification reduced the toxic elements contained in the cryopreservation solution while maintaining a similar ability to produce viable fibroblasts after cryopreservation. Although further work on the viability of sheep skin fibroblast with the vitrification method is needed, these data suggest that with vitrification a faster cooling rate and high level of cryoprotectants are able to minimize ice crystal formation and should be further evaluated as a routine mechanism for cryopreserving sheep fibroblasts.
49 Collection of Day 7 equine embryos in aluteal cycles in mares
Authors: Mak C. K. , Medina V. , Markle M. , Pinto C. R. F.
Published: December 2018

Serial administration of prostaglandin F2α (PGF2) in early diestrus has been recently shown to prevent luteal function in mares and is associated with delayed embryo development in induced aluteal cycles (progesterone deprived environment; <1.0ng mL−1; Leisinger et al. 2018 Theriogenology 105, 178-183). We hypothesised that embryos collected on Day 7 during induced aluteal cycles would be developmentally delayed compared with those collected from control cycles (progesterone deprived environment but supplemented with exogenous progestogen). Mares were monitored until a preovulatory follicle=35mm and the presence of uterine oedema were detected by ultrasonography. Mares were treated IV once with 2000IU of human chorionic gonadotropin and artificially inseminated every other day with total motile spermatozoa from one stallion of known fertility until ovulation. Mares were examined twice daily to determine the occurrence of ovulation. After ovulation, mares were randomly assigned to the control group (serial PGF2 treatment+long-acting altrenogest) or AL group (serial PGF2 treatment only). Using a protocol to induce aluteal cycles (Leisinger et al. 2018 Theriogenology 105, 178-183), mares in the AL group (n=7) were treated twice daily with 10mg of PGF2 (Lutalyse, dinoprost tromethamine, Zoetis, Florham Park, NJ, USA) IM on Days 0, 1, 2, and then once daily on Days 3 and 4. Mares in the control group (n=4) were treated with serial PGF2 treatment as mentioned before, and treated with a single injection of 225mg altrenogest (BioRelease Altrenogest LA 150; BET Pharm, Lexington, KY, USA) IM at the time of ovulation. On Day 7 post-ovulation, embryo collection was performed by uterine flushing using lactated Ringer’s solution. The developmental stage of embryos, diameter, and quality were determined using a stereomicroscope and photographed. Embryos were washed 3 times in commercial embryo holding medium (EmCare™ Holding Solution, ICP Bio, Spring Valley, WI) and stained with 1µg mL−1 of 4’,6-diamidino-2-phenylindole at 38.5°C for 15min to determine the number of nonviable cells. Data were analysed using t-test or Mann-Whitney U test where appropriate. Statistical significance was set at P=0.05. Data are reported as mean±s.e.M. Overall, the developmental stage of control embryos differed from AL embryos (P<0.03). In the control group, all embryos (n=4) collected were classified as expanded blastocysts. In contrast, embryos (n=7) collected from the AL group were all early blastocysts. Furthermore, the mean embryo diameter differed in control v. AL groups, 401±8mm v. 166±2mm, respectively (P<0.001). The quality grades of control embryos (1.3±0.3) were not different from the AL group (1.3±0.2; P>0.05). All embryos of both groups had less than 10% of blastomeres stained with 4’,6-diamidino-2-phenylindole. This study showed that morphologically normal equine embryos of acceptable quality can be collected on Day 7 in aluteal cycles.
50 Equine uterine fluid proteome on the fifth day after ovulation
Authors: Lancheros-Buitrago D. J. , Rodriguez-Villamil P. , Gregory J. W. , Camacho-Rozo C. A. , Caballeros-Haeussler J. E. , Cazales N. , Bastos H. B. A. , Barros E. , Pimentel A. M. , Mattos R. C.
Published: December 2018

The characterisation of the uterine proteome before the entry of the embryo to the uterus is essential to know the factors involved in the physiological events of gestation. The objective of the study was to compare, by a proteomics approach, the uterine lumen of cyclic and inseminated mares on Day 5 after ovulation. Samples of endometrial secretion were collected over 2 cycles during Day 5 post-ovulation. The first cycle constituted the cyclic group (n=10), and in the following cycle, the same mares were inseminated and considered as the inseminated group (n=8). All samples were subjected to polyacrylamide gel electrophoresis with sodium sulfate in gels of 12.5%. A total of 107 spots were visualised by 2-dimensional polyacrylamide gel electrophoresis with sodium sulfate. Eight selected spots were extracted, digested by trypsin, and analysed by mass spectrometry for protein identification. Selected spots were considered representative of the differentially expressed proteins when it was shown that the intensity of the spots between the groups in the study was significantly different and considering that the protein was present in at least 50% of the gels (P<0.05). Student’s t-test was used to compare the relative percentage of expression of the spots. Four different proteins were identified by MASCOT (http://www.matrixscience.com/) and validated by the application SCAFFOLD, version 3.6.4 (http://www.proteomesoftware.com/products/scaffold/). In this study, ceruloplasmin was the main protein identified in the equine endometrium on Day 5 after ovulation. The results demonstrated statistically significant changes in ceruloplasmin expression for the inseminated group in comparison with the cyclic group (P=0.036). Our findings suggest that the increase of ceruloplasmin expression in the uterine fluid represents preparation of the endometrial for implantation and development of a potential equine embryo. The comparison of the proteic expression from the endometrium of cyclic and inseminated mares before the embryo moves into the uterus enhances understanding of the processes associated with conceptus-endometrium interaction during the maternal recognition period in the mare.
64 Culture of bovine oocytes and embryos with metabolic hormones concentrations associated with equine metabolic syndrome
Authors: Bresnahan D. , Carnevale E.
Published: December 2018

Maternal metabolic status could affect fertility and early embryo development due to altered concentrations of metabolic hormones. Equine metabolic syndrome (EMS) is a condition in horses associated with obesity and insulin resistance. Equine metabolic syndrome is accompanied by increased concentrations of insulin and leptin and decreased concentrations of adiponectin, in ovarian follicular fluid (FF) and in systemic circulation (SYST). We sought to determine how altered concentrations of insulin, leptin, and adiponectin (ILA), consistent with those in mares with EMS (EMS) or normal mares (normal), would affect blastocyst formation rates, blastocyst gene expression for metabolism and inner cell mass formation (OCT4, SOX2, COX2, DNMT3a1, HK2, LDH, PDH, and GLUT1), and metabolite uptake from culture media. Because equine oocytes are not available for large-scale study, a bovine model was used in this preliminary study to determine the impact of altered ILA on oocytes and embryos. Bovine ovaries were obtained from an abattoir and embryos produced as previously described using chemically defined media (CDM; Barcelo-Fimbres and Seidel 2007Mol. Reprod. Dev. 74, 1406-1418). Briefly, oocytes were cultured in in vitro maturation medium (IVM), fertilized in FCDM, presumptive zygotes were placed into CDM-1 for ~56h. Cleavage rates were assessed, and embryos were moved to CDM-2 for ~122 additional hours. Treatments consisted of 5 groups: (1) standard oocyte IVM, FCDM and embryo production (EP) system (control), (2) IVM with normal FF ILA and control FCDM and EP, (3) IVM with normal FF ILA and FCDM and EP with normal SYST ILA, (4) IVM with EMS FF ILA and control FCDM and EP, and (5) IVM with EMS FF ILA and FCDM and EP with EMS SYST ILA. Seven days after fertilization, blastocysts were pooled in groups of 5 and placed into 50mL of CDM-2 for 24h. Embryos were removed, and medium was frozen and stored at −80°C to determine metabolite usage via gas chromatography mass spectroscopy. Pooled embryos were washed and placed into RNA lysis solution for relative quantitative PCR. Statistical comparisons were performed using ANOVA with a post-hoc Tukey test. Blastocyst formation rates and gene expression of viability markers were not significantly different among groups. However, aspartate was lower (P=0.02) in spent media from Group 3 (normal FF and SYST ILA) and tended (P=0.09) to be lower in media from Group 5 (EMS FF and SYST ILA) when compared with controls (Group 1). The ILA during early embryo development but not oocyte maturation appeared to be associated with increased uptake of aspartate, a nonessential amino acid, thought to be involved in osmoregulation, cellular signalling, and in mouse embryos, facilitate the metabolism of lactate. In conclusion, the addition of ILA in concentrations observed in normal horses and EMS horses did not affect blastocyst formation rates or markers of embryo viability, although embryo metabolism could have been altered.
72 Effect of incubation temperature and of CO2 concentration during early cleavage on equine in vitro embryo production
Authors: Brom-de-Luna J. , Salgado R. , Canesin H. , Diaw M. , Hinrichs K.
Published: December 2018

Intracytoplasmic sperm injection (ICSI) is currently being used for equine in vitro embryo production for both research and clinical purposes. However, some basic parameters for in vitro embryo production, such as the optimum incubator temperature and the optimum CO2 concentration/pH of medium for early embryo development, have not yet been established in the horse. The incubation temperature used by many laboratories for equine in vitro embryo production is 38.2°C, whereas the range of normal rectal temperature in the horse is 37.2 to 38.3°C. In Exp. 1, we evaluated maturation, cleavage, and blastocyst rates under 3 different culture temperatures. Cumulus-oocyte complexes were recovered from slaughterhouse-derived ovaries and shipped overnight at room temperature. Oocyte maturation was performed concurrently in separate incubators set to 37.2, 37.7, or 38.2°C. Mature oocytes were subjected to ICSI, then cultured in mixed gas (6% CO2, 5% O2, and remainder N2) at the same temperature at which they were matured. Embryo culture media used were a commercial human medium (global) for Days 0 to 5, then DMEM/F-12 from Day 5 to 10, both with 10% fetal bovine serum (FBS). In Exp. 2, we evaluated 3 different CO2 concentrations (6, 6.5, or 7% CO2) in mixed gas for the Day 0 to 5 culture in global+FBS, at 38.2°C. Cumulus-oocyte complexes were recovered from live mares by transvaginal aspiration and held overnight at room temperature; all other parameters remained the same as for Exp. 1. Data were analysed by Fisher’s exact test. In Exp. 1, a total of 280 oocytes were utilised; the outcomes for the 37.2, 37.7, and 38.2°C treatments were, respectively: maturation rates of 33, 38, and 42%; cleavage rates of 84, 86, and 88%; and blastocyst rates per injected oocyte of 35, 44, and 44%. There were no significant differences among the 3 temperature treatments for any parameter (P>0.2). In Exp. 2, the pH of the global+FBS medium was 7.38, 7.35, and 7.3 for 6, 6.5, and 7% CO2, respectively. A total of 106 mature oocytes underwent ICSI; the outcomes for the 3 CO2 atmospheres, respectively, were cleavage rates of 68, 80, and 70% and blastocyst rates per injected oocyte of 42, 54, and 27%. The blastocyst rate for the 7% CO2 treatment was significantly lower than that for the 6.5% CO2 treatment (P<0.05). These results indicate that equine in vitro embryo production is equally effective throughout the range of normal equine body temperature, and that equine blastocyst production is sensitive to small changes in CO2 atmosphere/pH of medium during early cleavage-stage development.
Research was supported by the Clinical Equine ICSI Program, Texas A&M University, the Link Equine Research Endowment, Texas A&M University, and Fonds en Santé Équine (FSÉ), Faculté de Médecine Vétérinaire, and Université de Montréal.
82 Disruption of p53 in bovine somatic cells affected cloned embryonic development
Authors: Kim K.-M. , Lee S.-J. , Yum S.-Y. , Kim H.-S. , Kim H.-J. , Lee J.-H. , Koo S.-H. , Lee W.-W. , Lee W.-S. , Jang G.
Published: December 2018

The p53 gene is a tumour suppressor gene with many roles in cellular reprogramming, senescence, or cancers. Knockdown of p53 in somatic cells improves generation of induced pluripotent stem cells and developmental competence of cloned embryos. In this study, we hypothesised that disruption of p53 in bovine somatic cells using CRISPR-Cas9 may improve reprogramming and result in increased blastocyst formation. For the study, ovary-derived immature bovine cumulus-oocyte complexes were matured in vitro. To prepare the donor cells with disruption of p53, primary cells from the skin tissue were transfected with spCas9 and single guide RNA for bovine p53. Two days later, genomic DNA was extracted and the mutation was evaluated on the target region using the T7E1 enzyme. Several single cell colonies were isolated to investigate the mutation and sequencing for mutation positive colonies was performed. One colony with high proliferative activity and heterozygotic mutation (−7bp deletion) was selected and used as the donor cell for somatic cell nuclear transfer. The donor cell was microinjected into an enucleated oocyte. Reconstructed oocytes were fused by electrical pulse, activated by chemical stimulation, and cultured in chemically defined media for 7 days at 5% CO2 and 5% O2. Data were subjected to ANOVA and least significant different test to determine differences between experimental groups by using SAS software (SAS Institute Inc., Cary, NC, USA). Statistical significance was determined when the P-value was less than 0.05. Blastocyst formation rate (13.5±7.6%) from the mutated cell line was significantly lower than wild type cells (43.9±12.1%). Moreover, total cell number (115.4±13.3) of the blastocysts from knockout cells was significantly decreased compared with the control group (143.6±22.5). Some randomly selected blastocysts derived from mutated cells were subjected to mutation assay to confirm deletion of p53. As expected, all the samples were positive to mutation. These data did not support our hypothesis that p53 disruption could improve blastocyst formation competence of bovine cloned embryos. In the future, the reason for impaired embryonic development of clones of p53 deleted somatic cells will be further investigated.
This work was supported by BK21 PLUS Program for Creative Veterinary Science, the National Research Foundation of Korea (2017R1A2B3004972), and the Technology Development Program (S2566872) by MSS.
85 Elongation of trophoblastic vesicles between Days 15 and 18 in cattle
Authors: Richard C. , Degrelle S. , Gelin V. , Neveux A. , Chavatte-Palmer P. , Heyman Y. , Hue I.
Published: December 2018

Once formed, bovine blastocysts differentiate while growing exponentially from 150-200μm (Days 7 or 8) to 200-250mm (Days 17 to 18; Sandra et al. 2017 Annu. Rev. Anim. Biosci. 5, 205-228). Thus, the length of the conceptus doubles every day between Days 9 and 16 (Berg et al. 2010 Theriogenology 73, 250-260); however, this was observed on whole conceptuses. The objective of the current study was to test whether this elongation rate is similar when the embryonic disc has been excised. Six heifers were used to produce Day-15 conceptuses, either fully developed in vivo or developed in vivo for a week after embryo transfer of Day 8in vitro-produced blastocysts. Day 15 conceptuses were recovered, measured, and cut into pieces to produce trophoblastic vesicles (TV; Heyman et al. 1984 J. Reprod. Fertil. 70, 533-540) of 4±0.07 or 4.4±0.65mm long (mean±standard error of the means) for in vivo- or in vitro-produced TV, respectively. All TV were transferred into oestrus-synchronized recipients (5 heifers and 1 cow). Each female received 8-9 TV so that a total of 24in vivo-derived and 26in vitro-produced TV were transferred in utero for a period of 3 days. The TV originating from different Day-15 conceptuses were mixed at the time of transfer, so that each recipient received the TV from different origins (conceptus and donor cow). Transcervical collection was used at Day 15 and 18 for conceptus and TV recovery (Richard et al. 2015 Theriogenology 83, 1101-1109). At Day 18, TV elongation size was analysed (mean±standard error of the means) by unpaired t-test using GraphPad Prism software (GraphPad Inc., San Diego, CA, USA). At Day 15, conceptuses from the in vivo and in vitro groups displayed different sizes and length variabilities (24-32v. 2-24mm, respectively). At Day 18, TV recovery rate was 79% for the in vivo- v. 62% for the in vitro-derived group and mean elongation rate (over 3 days) was ×5.4 (minimum=2.5, maximum=10) v. ×7.6 (minimum=0.7, maximum=20.5), respectively. There was no significant difference for TV size between groups at Day 18 (21.75±2.24 mm v. 33.38±11.63mm, respectively). Altogether, the variability in length at Day 15 was previously reported, the difference in TV recovery between in vivo and in vitro groups reached 17% and was similar to the loss of 11% that occurs in the first week after classical embryo transfer. In opposition to studies where in vitro-produced conceptuses were shorter than in vivo-developed ones, in vivo and in vitro groups of TV likely followed similar growth. Whether this reflects a normal growth awaits further studies.
94 Impact of number of embryos transferred on the number of offspring produced in a commercial transgenic founder production operation
Authors: Buzzell N. , Blash S. , Miner K. , Schofield M. , Pollock J. , Hawkins N. , Hevy M. , Gavin W.
Published: December 2018

The production of transgenic founder dairy goats (cross-bred Saanens, Alpines, Toggenburgs, and Nubians) involves the collection, microinjection, and transfer of numerous embryos into suitable recipient goats to ultimately produce a transgenic founder(s). The objective of this study was to determine the most efficient number of microinjection embryos to transfer to suitable recipients for transgenic founder generation. This is critically important in a commercial production program, as it impacts the goal for the number of embryos collected from donors, number of recipients utilised, and, hence, the overall number of surgical procedures being performed. The entire embryo collection, transfer, and founder-generation process is continuously being evaluated for ways to become more efficient in producing transgenic animals. During LFB USA’s commercial founder-production campaigns over the years (1997-2017), pronuclear microinjection was performed and 3, 4, or 5 embryos were transferred to female goat recipients. The recipients were synchronized using a progesterone implant on Day 0, a prostaglandin injection at Day 7, an injection of 300-500IU of pregnant mare serum gonadotropin on Day 13, progesterone implant removal on Day 14, and surgical transfer of pronuclear microinjected 1- or 2-cell embryos into the oviduct on Day 17. The individual totals and calculation for offspring per embryos transferred was compared for 3, 4, and 5 embryos transferred per recipient and was determined to be (1659/8637) 0.19, (912/4548) 0.20, and (112/675) 0.17, respectively. These embryo efficacy ratios were not significantly different (P>0.05) using the Wald Chi-squared test under logistic regression, and suggests that the number of offspring born is not impacted by number of embryos transferred. Seasonality was also evaluated in this production environment located in North America, with in-season being considered September to December and out-of-season being January to July. Nulliparous recipients during in-season (September to December) embryo transfer operations produced a significant difference, with totals and calculation for (offspring per embryo transferred) of (470/2346) 0.20, (260/1088) 0.24, and (23/190) 0.12 for 3, 4, and 5 embryo transfers, respectively (Table 1). This data indicates that when using nulliparous recipients during the in-season, transferring 4 embryos is optimal for offspring produced.

Table 1.Comparison of the individual totals and the calculation of (offspring/embryo) by parity and season
99 Effect of application of seminal plasma on Day 0, 5, or 7 postmating on pregnancy rate and embryonic survival in alpacas (Lama pacos)
Authors: Cuya R. , Huanca W. , Medina G. , Sanchez R. , Huanca W. F.
Published: December 2018

Alpacas, similar to other camelids, are classified as induced ovulators, as an external stimulus is required for the occurrence of ovulation. A protein identified as β-nerve growth factor, present in the seminal plasma (SP), has the capacity to induce ovulation and corpus luteum formation. Alpacas exhibit poor reproductive efficiency, with birth rates below 50% due in part to high embryo mortality before 35 days post-mating. A study was carried out to evaluate the effect of the application of SP on Day 0, 5, or 7 post-mating on pregnancy rate and embryo survival, defined as the difference in the numbers of pregnant females between Day 35 and 25. Nonpregnant adult alpaca females (n=124) between 5 to 6 years old were evaluated by transrectal ultrasonography to determine presence of a follicle ≥7mm, and then 2 days later to confirm permanence of the follicle. Alpacas were then bred by natural mating and assigned randomly to 1 of 4 treatments: 1mL of SP IM at mating; 1mL of SP IM Day 5 post-mating; 1mL of SP IM Day 7 post-mating; and control. Semen was collected from adults male and ejaculates were diluted 1:1 with PBS and then centrifuged for 30min at 3000 rpm. Supernatant was separated and a drop evaluated to determine absence of spermatozoa and SP-free sperm was stored at −20°C. Twenty adult males with optimal reproductive performance were used for mating with females assigned to the different treatments. Animals were evaluated by ultrasound with an Aloka SSD 500 (Aloka, Tokyo, Japan) and 5.0-MHz linear transducer on Day 25 and 35 to determine pregnancy rate and embryonic survival. Data were analysed by chi-square. Results are present in Table 1. The results differ from our initial hypothesis and a possible explanations may be that additional application of SP IM could saturate receptors and block the action of the seminal plasma present in the ejaculate of males.

Table 1.Pregnancy rate and embryonic survival in alpacas with application of seminal plasma on Day 0, 5, or 7

Study was supported by project no. 405-PNICP-PIAP-UNMSM.
Testing the feasibility of wireless sensor networks and the use of radio signal strength indicator to track the movements of wild animals
Authors: Krull C. R. , McMillan L. F. , Fewster R. M. , van der Ree R. , Pech R. , Dennis T. , Stanley M. C.
Published: December 2018

Context
Wireless sensor networks (WSNs) are revolutionising areas of animal behaviour research and are advantageous based on their ability to be deployed remotely and unobtrusively, for long time periods in inaccessible areas.

Aims
We aimed to determine the feasibility of using a WSN to track detailed movement paths of small animals, e.g. rats (Rattus spp.) 100–400g, too small for current GPS technology, by calibrating active Radio Frequency Identification (RFID) tags and loggers using Radio Frequency Signal Strength Indicator (RSSI) as a proxy for distance. Active RFIDs are also called Wireless Identification (WID) tags.

Methods
Calibration tests were conducted using a grid of loggers (n=16) spaced at 45-m intervals in clear line-of-sight conditions. WID tags (n=16) were placed between the loggers at 45-m intervals. Eight ‘walks’ were also conducted through the grid using a single WID tag. This involved attaching the tag to a small bottle of water (to simulate the body of an animal), towed around the grid using a 1-m long tow line attached to a volunteer walker. The volunteer also held a GPS device that logged their track. Models were constructed to test the effects of distance, tag movement and individual differences in loggers and tags on the reliability of movement data.

Key results
Loggers were most successful at detecting tags at distances <50m. However, there was a significant difference in the detection probabilities of individual loggers and also the transmission performance of individual tags. Static tags were less likely to be detected than the mobile tag; and although RSSI was somewhat related to distance, the reliability of this parameter was highly variable.

Implications
We recommend caution in the future use of current radio frequency ID tags in wireless sensor networks to track the movement of small animals, and in the use of RSSI as an indicator of individual distance values, as extensive in situ calibration is required. ‘Off the shelf’ devices may vary in performance, rendering data unreliable. We emphasise the importance of calibrating all equipment in animal tracking studies to reduce data uncertainty and error.
Mechanisms of climate-change-induced species decline: spatial, temporal and long-term variation in the diet of an endangered marsupial carnivore, the eastern quoll
Authors: Fancourt Bronwyn A. , Hawkins Clare E. , Nicol Stewart C.
Published: December 2018

Context
Climate change is having significant impacts on species worldwide. The endangered eastern quoll (Dasyurus viverrinus) has recently undergone rapid and severe population decline, with no sign of recovery. Spatially and temporally-explicit weather modelling suggests a prolonged period of unfavourable weather conditions during 2001–03 as the proximate cause of decline. However, the mechanisms of this weather-induced decline are not currently understood.

Aims
The aim of this study was to investigate the hypothesis that changing weather conditions have altered the availability of key prey species, potentially contributing to the species’ decline.

Methods
We analysed 229 scats collected from 125 individual wild quolls across four sites between July 2011 and May 2012. Variation in dietary composition and niche breadth was compared across sites and seasons. We also compared contemporary dietary composition and niche breadth to historic dietary studies performed before the species’ decline, to identify any key changes in dietary composition over time.

Key results
Dietary composition and niche breadth were similar across sites but differed between seasons. Dietary niche contracted during winter (July) and early spring (September) when insect larvae formed the bulk of quoll diet, rendering the species vulnerable to weather-related fluctuations in food availability at that time. Large differences were also evident between current and historic dietary composition, with a marked shift from insect larvae to mammals, predominantly due to a reduction in corbie (Oncopera intricata) and southern armyworm (Persectania ewingii) moth larvae. Quoll abundance appears positively related to corbie larva abundance during winter, and both quoll and corbie larva abundance appear negatively related to winter rainfall.

Conclusions
The lower contribution of insects at sites with low quoll densities suggests that insects represent an important food item for eastern quolls during winter, when dietary niche is narrowest and energy demands are highest. Our findings suggest that weather-induced fluctuations in quoll abundance, including the significant statewide decline during 2001–03, are potentially driven by weather-induced fluctuations in corbie larva abundance.

Implications
Continued deterioration in climatic suitability with recent and predicted climate change could further threaten eastern quolls through reductions in the availability and stability of reliable food sources at critical life-history stages when dietary options are already limited.
Phosphorylation cascade regulates the formation and maturation of rotaviral replication factories
Authors: Criglar Jeanette M. , Anish Ramakrishnan , Hu Liya , Crawford Sue E. , Sankaran Banumathi , Prasad B. V. Venkataram , Estes Mary K.
Published: December 2018

The rotavirus (RV) genome is replicated and packaged into virus progeny in cytoplasmic inclusions called viroplasms, which require interactions between RV nonstructural proteins NSP2 and NSP5. How viroplasms form remains unknown. We previously found two forms of NSP2 in RV-infected cells: a cytoplasmically dispersed dNSP2, which interacts with hypophosphorylated NSP5; and a viroplasm-specific vNSP2, which interacts with hyperphosphorylated NSP5. Other studies report that CK1α, a ubiquitous cellular kinase, hyperphosphorylates NSP5, but requires NSP2 for reasons that are unclear. Here we show that silencing CK1α in cells before RV infection resulted in (
i
) >90% decrease in RV replication, (
ii
) disrupted vNSP2 and NSP5 interaction, (
iii
) dispersion of vNSP2 throughout the cytoplasm, and (
iv
) reduced vNSP2 protein levels. Together, these data indicate that CK1α directly affects NSP2. Accordingly, an in vitro kinase assay showed that CK1α phosphorylates serine 313 of NSP2 and triggers NSP2 octamers to form a lattice structure as demonstrated by crystallographic analysis. Additionally, a dual-specificity autokinase activity for NSP2 was identified and confirmed by mass spectrometry. Together, our studies show that phosphorylation of NSP2 involving CK1α controls viroplasm assembly. Considering that CK1α plays a role in the replication of other RNA viruses, similar phosphorylation-dependent mechanisms may exist for other virus pathogens that require cytoplasmic virus factories for replication.
Structural mechanism for HIV-1 TAR loop recognition by Tat and the super elongation complex
Authors: Schulze-Gahmen Ursula , Hurley James H.
Published: December 2018

Promoter-proximal pausing by RNA polymerase II (Pol II) is a key regulatory step in human immunodeficiency virus-1 (HIV-1) transcription and thus in the reversal of HIV latency. By binding to the nascent transactivating response region (TAR) RNA, HIV-1 Tat recruits the human super elongation complex (SEC) to the promoter and releases paused Pol II. Structural studies of TAR interactions have been largely focused on interactions between the TAR bulge and the arginine-rich motif (ARM) of Tat. Here, the crystal structure of the TAR loop in complex with Tat and the SEC core was determined at a 3.5-Å resolution. The bound TAR loop is stabilized by cross-loop hydrogen bonds. It makes structure-specific contacts with the side chains of the Cyclin T1 Tat-TAR recognition motif (TRM) and the zinc-coordinating loop of Tat. The TAR loop phosphate backbone forms electrostatic and VDW interactions with positively charged side chains of the CycT1 TRM. Mutational analysis showed that these interactions contribute importantly to binding affinity. The Tat ARM was present in the crystallized construct; however, it was not visualized in the electron density, and the TAR bulge was not formed in the RNA construct used in crystallization. Binding assays showed that TAR bulge-Tat ARM interactions contribute less to TAR binding affinity than TAR loop interactions with the CycT1 TRM and Tat core. Thus, the TAR loop evolved to make high-affinity interactions with the TRM while Tat has three roles: scaffolding and stabilizing the TRM, making specific interactions through its zinc-coordinating loop, and making electrostatic interactions through its ARM.
Microbiome interactions shape host fitness
Authors: Gould Alison L. , Zhang Vivian , Lamberti Lisa , Jones Eric W. , Obadia Benjamin , Korasidis Nikolaos , Gavryushkin Alex , Carlson Jean M. , Beerenwinkel Niko , Ludington William B.
Published: December 2018

Gut bacteria can affect key aspects of host fitness, such as development, fecundity, and lifespan, while the host, in turn, shapes the gut microbiome. However, it is unclear to what extent individual species versus community interactions within the microbiome are linked to host fitness. Here, we combinatorially dissect the natural microbiome of
Drosophila melanogaster
and reveal that interactions between bacteria shape host fitness through life history tradeoffs. Empirically, we made germ-free flies colonized with each possible combination of the five core species of fly gut bacteria. We measured the resulting bacterial community abundances and fly fitness traits, including development, reproduction, and lifespan. The fly gut promoted bacterial diversity, which, in turn, accelerated development, reproduction, and aging: Flies that reproduced more died sooner. From these measurements, we calculated the impact of bacterial interactions on fly fitness by adapting the mathematics of genetic epistasis to the microbiome. Development and fecundity converged with higher diversity, suggesting minimal dependence on interactions. However, host lifespan and microbiome abundances were highly dependent on interactions between bacterial species. Higher-order interactions (involving three, four, and five species) occurred in 13–44% of possible cases depending on the trait, with the same interactions affecting multiple traits, a reflection of the life history tradeoff. Overall, we found these interactions were frequently context-dependent and often had the same magnitude as individual species themselves, indicating that the interactions can be as important as the individual species in gut microbiomes.
Low-oxygen response is triggered by an ATP-dependent shift in oleoyl-CoA in
Arabidopsis
Authors: Schmidt Romy R. , Fulda Martin , Paul Melanie V. , Anders Max , Plum Frederic , Weits Daniel A. , Kosmacz Monika , Larson Tony R. , Graham Ian A. , Beemster Gerrit T. S. , Licausi Francesco , Geigenberger Peter , Schippers Jos H. , van Dongen Joost T.
Published: December 2018

Plant response to environmental stimuli involves integration of multiple signals. Upon low-oxygen stress, plants initiate a set of adaptive responses to circumvent an energy crisis. Here, we reveal how these stress responses are induced by combining (
i
) energy-dependent changes in the composition of the acyl-CoA pool and (
ii
) the cellular oxygen concentration. A hypoxia-induced decline of cellular ATP levels reduces LONG-CHAIN ACYL-COA SYNTHETASE activity, which leads to a shift in the composition of the acyl-CoA pool. Subsequently, we show that different acyl-CoAs induce unique molecular responses. Altogether, our data disclose a role for acyl-CoAs acting in a cellular signaling pathway in plants. Upon hypoxia, high oleoyl-CoA levels provide the initial trigger to release the transcription factor RAP2.12 from its interaction partner ACYL-COA BINDING PROTEIN at the plasma membrane. Subsequently, according to the N-end rule for proteasomal degradation, oxygen concentration-dependent stabilization of the subgroup VII ETHYLENE-RESPONSE FACTOR transcription factor RAP2.12 determines the level of hypoxia-specific gene expression. This research unveils a specific mechanism activating low-oxygen stress responses only when a decrease in the oxygen concentration coincides with a drop in energy.
Inherent auditory skills rather than formal music training shape the neural encoding of speech
Authors: Mankel Kelsey , Bidelman Gavin M.
Published: December 2018

Significance

Musicianship is widely reported to enhance auditory brain processing related to speech–language function. Such benefits could reflect true experience-dependent plasticity, as often assumed, or innate, preexisting differences in auditory system function (i.e., nurture vs. nature). By recording speech-evoked electroencephalograms, we show that individuals who are naturally more adept in listening tasks but possess no formal music training (“musical sleepers”) have superior neural encoding of clear and noise-degraded speech, mirroring enhancements reported in trained musicians. Our findings provide clear evidence that certain individuals have musician-like auditory neurobiological function and temper assumptions that music-related neuroplasticity is solely experience-driven. These data reveal that formal music experience is neither necessary nor sufficient to enhance the brain’s neural encoding and perception of sound.
Itinerant ferromagnetism of the Pd-terminated polar surface of PdCoO
2
Authors: Mazzola Federico , Sunko Veronika , Khim Seunghyun , Rosner Helge , Kushwaha Pallavi , Clark Oliver J. , Bawden Lewis , Marković Igor , Kim Timur K. , Hoesch Moritz , Mackenzie Andrew P. , King Phil D. C.
Published: December 2018

The ability to modulate the collective properties of correlated electron systems at their interfaces and surfaces underpins the burgeoning field of “designer” quantum materials. Here, we show how an electronic reconstruction driven by surface polarity mediates a Stoner-like magnetic instability to itinerant ferromagnetism at the Pd-terminated surface of the nonmagnetic delafossite oxide metal PdCoO
2
. Combining angle-resolved photoemission spectroscopy and density-functional theory calculations, we show how this leads to a rich multiband surface electronic structure. We find similar surface state dispersions in PdCrO
2
, suggesting surface ferromagnetism persists in this sister compound despite its bulk antiferromagnetic order.
Resource extraction and infrastructure threaten forest cover and community rights
Authors: Bebbington Anthony J. , Humphreys Bebbington Denise , Sauls Laura Aileen , Rogan John , Agrawal Sumali , Gamboa César , Imhof Aviva , Johnson Kimberly , Rosa Herman , Royo Antoinette , Toumbourou Tessa , Verdum Ricardo
Published: December 2018

Significance

While infrastructure expansion has been broadly investigated as a driver of deforestation, the impacts of extractive industry and its interactions with infrastructure investment on forest cover are less well studied. These challenges are urgent given growing pressure for infrastructure investment and resource extraction. We use geospatial and qualitative data from Amazonia, Indonesia, and Mesoamerica to explain how infrastructure and extractive industry lead directly and indirectly to deforestation, forest degradation, and increasingly precarious rights for forest peoples. By engaging in explicit analyses of community rights, the politics of development policy, and institutions for transparency, anticorruption, and the defense of human rights, Sustainability Science could be more effective in examining deforestation and related climate-change impacts and in contributing to policy innovation.
Ferredoxin-linked flavoenzyme defines a family of pyridine nucleotide-independent thioredoxin reductases
Authors: Buey Rubén M. , Fernández-Justel David , de Pereda José M. , Revuelta José L. , Schürmann Peter , Buchanan Bob B. , Balsera Monica
Published: December 2018

Ferredoxin-dependent thioredoxin reductase was identified 35 y ago in the fermentative bacterium
Clostridium pasteurianum
[Hammel KE, Cornwell KL, Buchanan BB (1983)
Proc Natl Acad Sci USA
80:3681–3685]. The enzyme, a flavoprotein, was strictly dependent on ferredoxin as reductant and was inactive with either NADPH or NADH. This early work has not been further pursued. We have recently reinvestigated the problem and confirmed that the enzyme, here designated ferredoxin-dependent flavin thioredoxin reductase (FFTR), is a flavoprotein. The enzyme differs from ferredoxin−thioredoxin reductase (FTR), which has a signature [4Fe−4S] cluster, but shows structural similarities to NADP-dependent thioredoxin reductase (NTR). Comparative amino acid sequence analysis showed that FFTR is present in a number of clostridial species, some of which lack both FTR and an archetypal NTR. We have isolated, crystallized, and determined the structural properties of FFTR from a member of this group,
Clostridium acetobutylicum
, both alone and in complex with Trx. The structures showed an elongated FFTR homodimer, each monomer comprising two Rossmann domains and a noncovalently bound FAD cofactor that exposes the isoalloxazine ring to the solvent. The FFTR structures revealed an alternative domain organization compared with NTR that enables the enzyme to accommodate Fdx rather than NADPH. The results suggest that FFTR exists in a range of conformations with varying degrees of domain separation in solution and that the stacking between the two redox-active groups for the transfer of reducing equivalents results in a profound structural reorganization. A mechanism in accord with the findings is proposed.
MRI demonstrates glutamine antagonist-mediated reversal of cerebral malaria pathology in mice
Authors: Riggle Brittany A. , Sinharay Sanhita , Schreiber-Stainthorp William , Munasinghe Jeeva P. , Maric Dragan , Prchalova Eva , Slusher Barbara S. , Powell Jonathan D. , Miller Louis H. , Pierce Susan K. , Hammoud Dima A.
Published: December 2018

Significance

Cerebral malaria (CM) is the deadliest complication ofPlasmodium falciparuminfection, resulting in a 15 to 25% mortality rate in African children despite antimalarial chemotherapy. Tragically, nearly a fourth of pediatric CM survivors suffer long-term neurological sequelae. There is an urgent public health and humanitarian need for therapies for CM. In a mouse model of CM, we used magnetic resonance imaging (MRI) to monitor infected mice longitudinally as CM progressed and noninvasively demonstrate that the edema and blood–brain barrier dysfunction, which ultimately result in death, are rapidly reversed by treatment with the glutamine antagonist JHU-083. The similarities between CM MRI shown in mice and those reported in children and adults suggest that glutamine antagonists may be effective CM therapies.
Synergistic neuroprotection by coffee components eicosanoyl-5-hydroxytryptamide and caffeine in models of Parkinson's disease and DLB
Authors: Yan Run , Zhang Jie , Park Hye-Jin , Park Eun S. , Oh Stephanie , Zheng Haiyan , Junn Eunsung , Voronkov Michael , Stock Jeffry B. , Mouradian M. Maral
Published: December 2018

Significance

Coffee consumption is linked with reduced risk of Parkinson’s disease (PD), and caffeine is generally believed to be the protective agent. However, several lines of evidence suggest the presence of additional compound(s) in coffee that can be protective as well. Here we show that eicosanoyl-5-hydroxytryptamide, which we purified from coffee as an agent that leads to enhanced enzymatic activity of the specific phosphatase PP2A that dephosphorylates the pathogenic protein α-synuclein, works in synergy with caffeine in protecting against mouse models of PD and Dementia with Lewy bodies. The mechanism of this synergy is also through enhancing PP2A, which is dysregulated in the brains of individuals with these α-synucleinopathies.
Basophils trigger emphysema development in a murine model of COPD through IL-4–mediated generation of MMP-12–producing macrophages
Authors: Shibata Sho , Miyake Kensuke , Tateishi Tomoya , Yoshikawa Soichiro , Yamanishi Yoshinori , Miyazaki Yasunari , Inase Naohiko , Karasuyama Hajime
Published: December 2018

Significance

Chronic obstructive pulmonary disease has generally been considered a non-Th2-type lung disease, in contrast to allergic asthma. We identified unexpected roles for basophils, Th2 cytokine IL-4, and interstitial macrophages in triggering emphysema formation in spite of the fact that basophils account for less than 1% of cellular infiltrates in the lung. Our observations in a series of genetically engineered mice illustrated that basophil-derived IL-4 acts on lung-infiltrating monocytes to promote their differentiation into interstitial macrophages that in turn produce a pathogenic enzyme, matrix metalloproteinase-12 (MMP-12), resulting in the destruction of alveolar walls and leading to emphysema. Thus, the present study has identified a previously unappreciated role for the basophil-derived IL-4/monocyte–derived interstitial macrophage/MMP-12 axis at the initiation phase of emphysema development in mice.