bluuebunny/crossref_metadata_2025 huggingface dataset:
61 Extracellular vesicles from serum in culture media are internalized by bovine embryos produced in vitro
Authors: Melo-Baez B. , Mellisho E. , Rodriguez-Alvarez L.
Published: December 2018

Extracellular vesicles (EV) are currently considered a mechanism of cell communication. These are secreted from different cell types, including embryos, to serve as mediators of short and long distance signals. EV can be identified in vivo in different biological fluids, as well as in vitro embryo culture medium. Usually, media used for embryo in vitro culture are supplemented with serum or other protein sources that favour cell proliferation and development. Serum and protein sources contain EV, including microvesicles and exosomes that in principle can be internalized by embryonic cells. The aim of this study was to evaluate if serum-derived EV are internalized by the embryo at different stages of the early development, and if EV from the serum are required for in vitro bovine embryo development. For that, it was first evaluated if EV depleted culture media affect embryo development up to the blastocyst stage; oocytes were in vitro matured for 22 to 23h and in vitro fertilized for 18h. Posteriorly, presumptive zygotes were in vitro cultured in groups (25 embryos/well in 4-well plates) in SOF or SOF depleted of EV for 8 days. To evaluate EV internalization, culture media was supplemented with labelled EV and confocal imaging was performed. The EV were obtained by ultrafiltration (centrifugal filter devices 100 kDa, Amicon; Millipore, Billerica, MA, USA) for 15min at 3000 rpm. Then, EV were stained with PKH67 dye and washed 3 times with PBS by ultrafiltration to remove excess dye. The EV labelled with PKH67 were resuspended in SOFaa depleted of EV (3×109 particles per 500µL) and supplemented for 24h at the 1-cell stage (Day 1 post IVF), 16 cells (Day 4 post IVF), and early blastocyst (Day 6 post IVF) in 5% CO2, 5% O2, and 90% N2. PBS with PKH67 dye was used as a control treatment. Hoechst 33343 was used to label the nuclei before washing with PBS and fixation with 0.4% paraformaldehyde. Images were acquired on a Zeiss (Zeiss, Jena, Germany) LSM 780 confocal microscope. There were no statistical differences on blastocyst rate at Day 8 between embryos cultured in SOF depleted of EV (19.5%) and control group (SOF; 22.7%; P>0.05). We observed punctuated green fluorescence near the embryo nuclei in the 3 stages studied in embryos supplemented with EV but not in the control treatment, which indicates that EV from serum are uptaken by embryonic cells in early development. Therefore, we demonstrated uptake of EV from fetal calf serum added to culture media, although its absence does not affect embryo development.
Research was supported by FONDECYT, Chile (1170310).
66 Bovine corpus luteum affects embryo developmental competence and production
Authors: Alvarado J. M. , Tenemaza M. A. , Merchán S. L. , Argudo D. E. , Méndez M. S. , Soria M. E. , Galarza L. R. , Ayala L. , Hernández H. J. , Perea F. P.
Published: December 2018

This study was conducted with the aim to determine the effect of the bovine corpus luteum on in vitro embryo production. Immature cumulus-oocyte complexes (COC) were aspirated from abattoir ovaries from cows (mainly Holstein and dairy crossbred cows and heifers) with (ipsilateral; CL+) and without (contralateral; CL−) corpus luteum (CL), and from cows without CL in any of the ovaries. The average weight of the ovaries from each group was 10.4±0.25, 5.7±0.25, and 6.1±0.25g for CL+, CL−, and C, respectively. The experiment was completed within 12 replicates (100 ovaries per group). The COC were cultured in maturation medium (TCM-199 supplemented with 10% fetal bovine serum, 100µg mL−1 sodium pyruvate, 0.75mg mL−1 of L-glutamine, 4µg mL−1 of FSH-p, 100 µM cysteamine, and 250µg mL−1 of gentamicin) followed by IVF (synthetic oviducal fluid medium supplemented with 10µg mL−1 heparin) and in vitro culture (citrate synthetic oviducal fluid medium). On Day 7 after IVF, the embryos were evaluated and classified into morulae (M), early blastocysts (EB), regular blastocysts (RB), expanded blastocysts (ExB), and hatched blastocysts (HB), and the embryos with quality 1 (according to IETS criteria) were recorded. Data were analysed by logistic regression and general linear model of SAS (SAS Institute Inc., Cary, NC, USA), and means were compared by the least squares means method. Results of cleavage, embryo rate at Day 7, and rates of M+EB, RB, ExB, and HB are shown in Table 1. The number of embryos per ovary was greater (P<0.01) in CL+ (1.16±0.11) than in CL− (0.45±0.15) and C (0.55±0.15). Also, the number of embryos per cultured oocyte was significantly greater in CL+ than in CL− and C (0.27±0.02v. 0.14±0.03 and 0.15±0.03, respectively; P<0.01). The results of this study reveal that the presence of the corpus luteum in the ovary at the time of the oocyte recovery affects the developmental capacity of the bovine embryos, and such influence probably occur through intraovarian interactions.

Table 1.Effect of bovine corpus luteum on embryo production
67 Optimizing a protocol for isolating extracellular vesicles from medium conditioned by bovine embryos in vitro
Authors: Pavani K. C. , Hendrix A. , Leemans B. , Van Soom A.
Published: December 2018

In the absence of the maternal tract, pre-implantation bovine embryos cultured in group are able to promote their own development in vitro by releasing autocrine embryotropins. Recently we have identified extracellular vesicles (EV) among these embryotropins as one of the communication mechanisms among embryos. Extracellular vesicles are nano-sized (25-250nm), with a lipid bilayer, and are functionally active, since they contain proteins, lipids, and nucleic acids, including RNA and miRNA. However, one of the major challenges in isolating EV is an inadequate volume of medium conditioned by bovine embryo. As it requires larger volumes of conditioned medium to isolate EV, our study mainly focused on isolating high yields of functional EV from a minimal volume. There are 3 known isolation methods for EV: differential ultracentrifugation (DU), OptiPrep™ density gradient ultracentrifugation (ODGU), and size-exclusion chromatography (SEC). We have used these 3 protocols to determine the method that yielded the highest number of EV. We used routine in vitro maturation and fertilization methods, but for in vitro culture presumed zygotes were cultured until 8 days post-insemination (dpi) in medium (synthetic oviducal fluid supplemented with insulin, transferrin, selenium, and bovine serum albumin) that was ultracentrifuged to remove any possible contaminating EV. In vitro embryo culture took place in groups of 25 presumed zygotes in 50-mL drops, covered with mineral oil and incubated at 38°C in 5% CO2, 5% O2, and 90% N2. On 8 dpi, medium conditioned by bovine embryo was collected and pooled until 3mL. For each isolation method, 1mL of conditioned medium was used, and next, EV isolated from each isolation method were analysed with nanoparticle tracking, electron microscopy, and Western blot (CD9, Flotillin 1, and AGO 2). We observed higher concentrations (1.03×109 particles mL−1) of EV were isolated from the SEC compared with the other 2 methods (301.5×108 particles mL−1 and 64.5×108 particles mL−1 for DU and ODGU, respectively; P<0.05), whereas smaller size EV (20-50nm) were lost during the ultracentrifugation methods. Besides, it takes only 2h of time to perform size-exclusion chromatography for isolating EV, whereas it takes more than 1 day to perform ultracentrifugation methods. Therefore, we propose to use SEC for further downstream processing and sequencing of miRNA in isolated EV. We are currently focusing on optimizing an EV isolation protocol to extract EV from very low volumes of conditioned medium (less than 500 µL).
69 Cloned bovine embryonic development derived from interferon tau knockout cells
Authors: Kim K.-M. , Lee S.-J. , Yum S.-Y. , Kim H.-S. , Kim H.-J. , Park J.-H. , Lee J.-H. , Koo S.-H. , Lee W.-W. , Lee W.-S. , Jang G.
Published: December 2018

Interferon tau (IFNT), a type I interferon, is known as a key signal molecule during pregnancy in ruminants because of the necessity in maternal recognition of pregnancy. It is produced in trophectoderm cells of the elongation bovine conceptus at Day 13-21, and peak production is at Day 15 to 17 of pregnancy. In addition, other studies show that it can affect embryonic development and quality. In this study, the effect of IFNT knockout in donor cells to bovine cloned embryonic development by somatic cell nuclear transfer (SCNT) was investigated. To proceed with this experiment, immature oocytes from ovaries at a local slaughterhouse were matured in vitro for 22h. To prepare the donor cell with IFNT knockout, somatic cells were transfected with Cas9 and single guide RNA targeting IFNT, and several single derived colonies with high proliferation were isolated for mutation assay. Finally, one colony that had mono-allelic mutation (4bp deletion) was selected and used as the donor cell for SCNT. A donor cell was injected into an enucleated oocyte. Reconstructed oocytes with the donor cell were fused by electrical shock, activated by chemical stimulation, and cultured for 7 days in chemically defined medium. For this study, control (n=94) and IFNT knockout groups (n=140) were compared with 4 replications. The results showed no significant difference between control and IFNT knockout groups not only in cleavage rate, but also in blastocyst formation rate (control: 15.7±8.3%, IFNT knockout group: 26.3±13.1%). In addition, the number of blastocyst cell was not different between control (88.2±27.0) and IFNT knockout group (88.0±21.1). Some IFNT mutated blastocysts from SCNT were randomly selected for confirmation of the deletion of IFNT, and all samples were positive for mutation. In conclusion, these data demonstrated that the disruption of IFNT did not affect embryonic development. In future study, we would transfer these embryos and check the effect of IFNT during pregnancy status.
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.
7 Pregnancy rates in suckled beef cows synchronized with a shortened progesterone/oestradiol-based protocol (J-synch) and inseminated with conventional or sexed-sorted semen
Authors: Bo G. A. , Huguenine E. E. , de la Mata J. J. , de Carneiro R. L. R. , Menchaca A.
Published: December 2018

An experiment was designed to evaluate pregnancy rates (P/AI) in suckled beef cows synchronized with a progesterone (P4)/oestradiol-based protocol (J-Synch) and inseminated (AI) with sexed or nonsexed (conventional) semen. Angus or Angus crossbred cows (n=877), 60 to 90 days postpartum, with a body condition score of 2.5 to 3.5 (scale of 1 to 5) and a corpus luteum or a follicle >8mm in diameter detected by ultrasonography, were randomly allocated to be AI with sexed-sorted or nonsexed (conventional) semen. The experiment was performed in 4 farms, and the sexed-sorted (SexedUltra™ 4M, with 4 million sperm; ST Genetics, Navasota, TX, USA) and conventional (25 million sperm) semen were from split ejaculates from 5 Angus bulls. All cows received a P4 device (DIB 0.5, Zoetis, Villa Adelina, Argentina) and 2mg of oestradiol benzoate (Gonadiol, Zoetis) on Day 0. On Day 6, cows received 400IU of eCG (Novormon 5000, Zoetis) and 500µg of cloprostenol (Ciclase DL, Zoetis) and DIB was removed. All cows were also tail painted and observed for signs of oestrus (i.e. >30% of the tail paint rubbed off) at 60, 72, and 84h after DIB removal. Cows with the tail paint rubbed off by 60 or 72h after DIB removal were artificially inseminated at 72h with either sexed or conventional semen. Those not showing oestrus by 72h received 100µg of gonadorelin acetate (Gonasyn GDR, Zoetis) at that time and were artificially inseminated with either sexed-sorted or conventional semen 12h later (i.e. 84h). Pregnancy was diagnosed by ultrasonography 30 days after AI. Data were analysed by general linear model for binary data with a logit link. Overall, 76.4% (678/877) of cows showed oestrus 60 to 84h after DIB removal, and there were differences in P/AI between sexed-sorted and conventional semen (P<0.01; Table 1); however, there was no farm effect (range: 52-59%) and no interactions. Fertility differed (P<0.05) among bulls and ranged from 25.8 to 55.3% for sexed-sorted semen and from 53.1 to 73.9% for conventional semen. In cows artificially inseminated with sexed-sorted semen, P/AI was the highest in cows showing oestrus and AI at 84 h; however, no differences were detected among cows artificially inseminated with conventional semen (Table 1). Although P/AI was lower for sexed-sorted than conventional semen, tail painting for oestrus detection can be successfully applied to AI beef cows with sexed-sorted semen synchronized with a shortened P4/oestradiol-based protocol.

Table 1.Effect of semen type (sexed-sorted or conventional) and time of oestrus and AI on P/AI in suckled beef cows synchronized with a shortened P4/oestradiol-based protocol

Research was supported by Fondo Nacional de Ciencia y Tecnología (FONCYT PICT 2017-4550) and UNVM.
70 Does the addition of docosahexaenoic acid to in vitro systems during culture improve the quality of bovine embryos?
Authors: Sánchez Viafara J. A. , Lopez de Vasconcelos G. , Maculan R. , Gomes Alves N. , Camisão de Souza J.
Published: December 2018

The aims of this study were to decrease the apoptotic index and increase cryotolerance of bovine embryos produced in vitro with the addition of 1 µM docosahexaenoic acid (DHA). On Day 1, presumed zygotes were cultivated with 1µM DHA (Sigma, St. Louis, MO, USA; n=437), and without the agonist (control group; n=450). The cleavage rate (%) was evaluated on Day 2 and the development of blastocysts on Day 7. Embryos before and after vitrification were fixed for the TUNEL trial. After vitrification, the embryos were heated and re-cultivated to evaluate the hatching rate at 12, 24, 36, 48, 60, and 72h. A sample of re-cultivated embryos at 12h of DHA (n=5), and without the agonist (control group; n=6), was frozen for mass spectrometry (MALDI-MS). Statistical analyses of deviance were carried out considering generalized mixed linear models, and the effect of the collection day (block) was considered as random. For the count variables, the Poisson distribution and the log link function were considered. In the cases of variables represented by rates, binomial distribution and the logit link function were used. In the study of cryotolerance, ANOVA of the hatching rate for each one of the times evaluated was carried out. In cases of significance of the effect of treatments, the Dunnett test was applied to compare treatments. Multivariate and univariate statistical models were used for analysis of MALDI-MS. All analyses were made using the GLIMMIX procedure of the SAS software (SAS Institute Inc., Cary, NC, USA). The cleavage rate was not different between the groups (P>0.05) and the production of blastocysts was lower in the DHA group (P<0.05). The number of cells per embryo was higher (P<0.05) by the addition of 1μM DHA in blastocysts pre- and post-vitrification. The rate of total and internal cell mass apoptosis in fresh embryos (11.73 and 15.98%) increased compared with the control group (9.62% and 11.03%, respectively). The proportion of internal cell mass in fresh embryos decreased in the DHA group (39.93%) compared with the control group (57.00%). Hatching rates at 48, 60, and 72h after devitrification in the group treated with 1μM DHA were not different (P>0.05) compared with the control group. Phosphatidylcholine [phosphatidylcholine (32: 0)+H] was more abundant (P<0.05) in embryos cultured with DHA, and thus was considered as a negative apoptosis biomarker. In conclusion, the use of 1μM DHA in vitrification of bovine embryos impairs embryonic quality and development under the conditions of the present study.
Research was supported by CAPES, FAPEMIG, PPGCV/UFLA, EVUFMG, CENATTE EMBRIÕES.
75 Culture of isolated blastomeres supplemented with L-ascorbic acid 2-phosphate in a well-of-the-well culture dish
Authors: Hasiyada Y. , Matsuda H. , Aikawa Y. , Ohtake M. , Yamanouchi T.
Published: December 2018

We have reported monozygotic twin calves that can be produced efficiently by blastomere separation of 2-cell stage embryos and by the use of a commercially provided well-of-the-well culture dish (Hashiyada 2017 J. Reprod. Dev.). The present study was conducted to evaluate the effect of a culture supplement, l-ascorbic acid 2-phosphate (AA-2P), a sustained antioxidant substance that reduces reactive oxygen species. Embryos were produced using oocytes from ovaries collected at an abattoir by in vitro maturation, IVF, and in vitro culture (IVC). TCM199 supplemented with 5% calf serum, Brackett-Oliphant solution supplemented with 10mg mL−1 BSA, and CR1aa containing 5% calf serum were used for each culture step. Two-cell stage embryos were obtained 24 to 27h post-insemination (hpi). Zonae pellucidae were removed by exposure to 0.25% pronase. Then, embryos were separated into each blastomere by gentle pipetting in IVC medium. Each blastomere was introduced into a single conical micro-well of 25 wells, each having a diameter and depth of ~287 and 168µm (Dai Nippon Printing, Tokyo, Japan). Culture of blastomeres in wells was performed covered with a droplet of 2.5 µL/well IVC medium supplemented with 0 (n=212), 250 (n=214), 500 (n=206), and 750 µM (n=204) of AA-2P. The blastocyst formation rate at Day 8 after IVF, the quality of blastocysts assessed by morphological observation, and the cell numbers were compared among each concentration of AA-2P. In addition, the developmental speed to the blastocyst stage was analysed using time-lapse cinematography for 0 and 500 µM of AA-2P (n=40, respectively). Statistical analysis was performed using Fisher’s exact test and ANOVA. The blastocyst formation rate (32-40%), the total cell number (108-114), and inner cell mass cell number (26-28) did not differ among groups. The time to reach the 4-cell stage was significantly shorter in media supplemented with 0 µM (43 hpi) than with 500 µM (52 hpi); however, the time to reach the blastocyst stage did not differ (150 and 155 hpi, respectively). Regarding the proportion of quality grade 1 to 3 blastocysts and the developmental speed to the blastocyst stage, high-quality grade 1 embryos were significantly faster than those of middle and low-quality grade 2 and 3 ones in 0 (145 v. 154 hpi; P<0.05) and 500 µM (150v. 158 hpi; P<0.05) supplemented medium. In this experiment, no effect of AA-2P was observed for the culture of isolated blastomeres from 2-cell stage embryos, although it was suggested that blastomeres with high developmental competence reach the blastocyst stage faster, which might reflect the quality of the embryos.
76 Effects of serum type in maturation medium on in vitro development of bovine embryos
Authors: Mesalam A. , Zhang S. , Lee K.-L. , Song S.-H. , Xu L. , Joo M.-D. , Hwang J.-Y. , Kong I.-K.
Published: December 2018

This study investigated the effect of bovine serum albumin (BSA), charcoal:dextran stripped fetal bovine serum (CDS FBS), and heat-inactivated FBS (HI FBS) in maturation medium on their ability to support in vitro oocyte maturation, cumulus cell-oocyte gap junctional communication, and development of bovine embryos. Charcoal:dextran treatment of FBS removes lipophilic chemicals, certain steroid hormones, and certain growth factors; however, HI FBS have a lot-to-lot variation in steroid hormones level that can affect the reproducibility of experimental findings. Oocytes were cultured in TCM-199 supplemented with either 8% (w/v) BSA, 10% (v/v) CDS FBS, or 10% (v/v) HI FBS and 1µg mL−1 oestradiol-17β, 10µg mL−1 FSH, 10ng mL−1 epidermal growth factor, 0.6mM cysteine, 0.2mM sodium pyruvate, and followed by IVF, and the zygotes were cultured in SOF-BE1 medium. The developmental ability and quality of bovine embryos were determined by assessing their cell number, lipid content, mitochondrial activity, gene expression, immunocytochemistry, and cryo-tolerance. The differences in embryo development between experimental groups were analysed by 1-way ANOVA. The Duncan’s multiple range tests were used to test the differences between the treatments. The level of statistical significance was set at P<0.05. We have shown that CDS FBS significantly improved (P<0.05) the percentage of MII oocytes compared with that in the BSA supplemented group (77.2±1.0% v. 69.3%±2.3%, respectively). Moreover, CDS FBS had a higher significant (P<0.05) effect on the rate of blastocyst formation compared with HI FBS and BSA (45.2±0.7% v. 37.4±1.5% and 31.1±3.9%, respectively; 6 replicates were performed). Culture of oocytes with CDS FBS increased (P<0.05) the expression of gap junction proteins, CX37 and CX43, at both transcriptional and translation levels as determined by quantitative RT-PCR and immunofluorescence analysis, respectively. We also found that CDS FBS significantly increased total cell number and decreased the apoptotic index in Day-8 blastocysts compared with the BSA group. Furthermore, the beneficial effects of CDS FBS on embryos were associated with significantly reduced intracellular lipid content and increased mitochondrial activity in both oocytes and blastocysts as identified by Nile red and MitoTracker Green staining, respectively. Taken together, these data suggest that supplementation of maturation medium with CDS FBS, as an alternative to HI FBS, affected cumulus cell-oocyte gap junctional communication, and subsequently improved in vitro developmental competence of bovine oocytes and embryos.
Research was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through Agri-Bio industry Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (grant numbers: 117029-3 and 315017-5).
77 Development and quality of in vitro bovine hemi embryos produced by blastomere separation and embryo bisection
Authors: Ynsaurralde Rivolta A. E. , Suvá M. , Alberio V. , Vazquez Echegaray C. , Guberman A. , Bevacqua R. J. , Salamone D. F.
Published: December 2018

Bovine monozygotic production of twins became popular in the 1980s as a technique to multiply high value genetics. Moreover, it also became a powerful model for research. Different techniques have been used on bovine embryos obtained by superovulation. In this work, we compared the development rates and quality of monozygotic twin embryos produced by blastomere separation (BS) and embryo bisection (EB) of IVF embryos. To this aim, cumulus-oocytes complexes collected from slaughterhouse ovaries were in vitro matured in TCM 199 containing 10% fetal bovine serum, 10µg mL−1 FSH, 0.3mM sodium pyruvate, 100mM cysteamine, and 2% antibiotic-antimycotic for 24h, at 6.5% CO2 in humidified air and 38.5°C. The IVF was performed with 16×106 spermatozoa per mL for 5h. Afterward, presumptive zygotes were cultured in SOF medium for 7 days at 38.5°C and 5% O2. After 24h of culture, blastomeres of 2-cell stage embryos (N=114) were separated and each one was cultured individually in a microwell for 7 days. Embryo bisection (N=179) was performed manually on Day-7 blastocysts previously depleted of their zonae pellucidae, under stereoscopic microscope. Hemi embryos were cultured for 24h and then twins and single blastocyst rates were calculated. For quality assessment, diameter, total and inner cell mass (ICM) cell number of hemi embryos (BS: 6 couples; ES: 10 couples) and the control group (C: 11) were evaluated. The ICM cell number was measured by immunofluorescence staining using SOX2 antibody and the percentage of ICM and trophectoderm (TE) cells was calculated. The results were analysed using Fisher’s exact test and ANOVA with mean comparison using Tukey’s test (P=0.05). No statistical differences were found in blastocyst rates of twins and single hemi embryos produced by BS (28 and 25%) or EB (23 and 32%). Blastocyst diameter was similar between groups and control. Hemi embryos exhibited lower total and ICM cell number than control (BS: 43±18, EB: 57±14v. C: 93±35 and BS: 16±7, EB: 12±8v. C: 34±19). However, BS hemi embryos had higher ICM and lower TE percentage (40/60%) compared with the EB group (20/80%). The control group did not differ with hemi embryo treatments for ICM and TE (30/70%). Our preliminary results have indicated that although the development rates of hemi embryos produced in vitro were similar between both techniques, blastomere separation generates better quality embryos than blastocyst bisection.
8 Evaluation of puberty induction protocol in peripubertal beef heifers prior to fixed-time AI programs
Authors: Borges J. B. S. , Luiz D. S. V. , Aguiar P. R. L. , Berlitz C. G. B. , Velho G. S. , Oliveira C. R. , Guerreiro B. M. , Freitas B. G. , Dalto A. G. C.
Published: December 2018

Beef heifers that conceive early during their first breeding season calve earlier, wean heavier calves, and tend to continue doing so throughout the rest of their reproductive lives. Otherwise, under extensively managed systems in Southern Brazil, only a variable proportion of 2-year-old beef heifers reach puberty before the start of the breeding season. The aim of this study was to evaluate a protocol to induce puberty in peripubertal beef heifers using injectable progesterone (P4) and oestradiol cypionate (EC) to improve pregnancy per AI (P/AI) in fixed-time AI (FTAI) programs. A total of 311 Angus heifers, 24±3 months of age, 3.1±0.3 body condition score (1=emaciated; 5=obese), and 328±41kg of body weight (BW), were examined before FTAI protocol (Day −24) by transrectal ultrasonography to determine reproductive tract score (RTS; 1=prepubertal; 5=pubertal). Prepubertal heifers (RTS 1; 12.8%, 40/311) were excluded from the study. Heifers with RTS=2 (n=271) were assigned to 2 groups: P4 (RTS 2 or 3, n=100) and cyclic (RTS 4 and 5, n=171). Peripubertal heifers were treated on Day −24 with 150mg of injectable P4 IM (Sincrogest® injectable, Ourofino Animal Health, Cravinhos, Brazil) and on Day −12 received 1mg of EC IM (Sincro CP®, Ourofino Animal Health). Cyclic heifers received no hormonal treatment before FTAI protocol. On Day 0, heifers in the P4 group were evaluated again to determine RTS, body condition score, and BW. Induction of puberty was defined by the presence of a follicle >8.5mm (RTS=4) or a corpus luteum (RTS=5). All heifers received an FTAI protocol consisting of 2mg of oestradiol benzoate IM (Sincrodiol®, Ourofino Animal Health) and an intravaginal progesterone implant (1 g; Sincrogest®, Ourofino Animal Health). On Day 8, heifers had implants removed and were injected with 1mg of EC and 500µg of sodium cloprostenol IM (Sincrocio®, Ourofino Animal Health). After 48h, all heifers received an FTAI. Thirty days after AI, the diagnosis of gestation was performed by ultrasonography. Statistics included chi-square and linear regression with coefficients analysed as independent variables, considering a significant P-value of 0.05. On Day 0, percentages of RTS 5, 4, and 3 in the P4 heifers were 37, 27, and 36%, respectively, resulting in 64% of puberty induction. The P/AI was similar (P=0.14) in P4-treated and cyclic heifers (59v. 50.3%, respectively). The regression analysis presented a linear and positive effect of BW on P/AI (P<0.001) in cyclic heifers and a positive influence of BW on RTS (P=0.05) and RTS on P/AI (P=0.008) in P4-treated heifers. The application of RTS system associated with P4 treatment of peripubertal heifers before FTAI protocol improved ovarian activity and might have contributed to higher P/AI, comparable with that achieved in cyclic heifers.
84 Evaluation of indirect methods for pregnancy diagnosis at Day 21 in in vitro-produced embryo transfer recipient heifers
Authors: Garcia-Guerra A. , Monteiro Jr P. L. J. , Gamarra C. A. , Walleser E .A. , Prata A. , Mezera M. A. , Gennari R. , Sala R. V. , Moreno J. F. , Sartori R. , Wiltbank M. C.
Published: December 2018

Early pregnancy detection has the potential to improve reproductive efficiency of embryo transfer (ET) programs by identifying nonpregnant animals and allowing their prompt reutilisation. Although methods are available to determine pregnancy after ~28 days, recent studies have highlighted the potential of indirect measurements, such as colour Doppler ultrasonography of the corpus luteum (CL), to serve as early pregnancy diagnostic methods. The objective of this study was to evaluate the sensitivity, specificity, and positive (PPV) and negative predictive value (NPV) of multiple indirect methods for pregnancy diagnosis at Day 21 in in vitro-produced ET recipients. Dairy heifers (n=796) were synchronized using a modified 5-day CoSynch and received a fresh in vitro-produced embryo 7±1 day after gonadotropin-releasing hormone. Heifers were examined by transrectal ultrasound on Day 21 after gonadotropin-releasing hormone to determine uterine endometrial thickness (UET), CL volume, and CL blood flow. Blood flow to the CL was determined by colour Doppler ultrasonography (Sonovet Pico, 7.5-MHz linear array probe, PRF 1kHz). Blood samples were collected on Day 21 to determine serum progesterone (P4) concentrations. Cutoff points for UET, CL volume, and P4 were evaluated using receiver operating characteristic curves and cutoffs for pregnancy were determined to be <8.75mm, >1800mm3, and >2.65 ng/mL for UET, CL volume, and serum P4, respectively. Heifers were classified as pregnant by CL colour Doppler ultrasound if they exhibited colour pixels in more than 10% of the periphery or exhibited colour pixels internally. Heifers were evaluated by transrectal ultrasonography on Day 28 to determine true pregnancy status. Differences between measurements were determined by generalized estimating equations. Pregnancies per ET on Day 28 was 50.1% (399/796). Sensitivity was lowest for UET and was different (P<0.01) from the other three tests (Table 1). Specificity was greatest (P<0.01) for serum P4, lowest for UET, and intermediate for CL volume and colour Doppler (Table 1). Serum P4, CL volume, and CL colour Doppler showed excellent NPV, indicating that each of these values accurately predicted nonpregnant heifers, the main emphasis of this study. However, PPV was moderate, indicating the potential for false positive results either due to pregnancy loss between Day 21 and 28 or, alternatively, inaccuracy of the method. Thus, on Day 21 of pregnancy, an accurate nonpregnancy diagnosis can be performed in ET recipients by using CL volume with or without CL colour Doppler, allowing for improved reproductive efficiency.

Table 1.Sensitivity, specificity, and positive (PPV) and negative predictive value (NPV) for various indirect methods to determine pregnancy on Day 21 in embryo transfer (ET) recipient heifers
95 N-Acetyl cysteine as a potential treatment for equine persistent breeding-induced endometritis
Authors: Caissie M. , Chenier T. , Gartley C. , Scholtz E. , Johnson R. , Hewson J. , Bienzle D.
Published: December 2018

Persistent breeding-induced endometritis (PBIE) is a major cause of infertility in mares. Transient uterine inflammation is a normal response to breeding; however, PBIE-susceptible mares do not clear this inflammation in a timely fashion. Uterine inflammation at the time of embryonic descent from the oviducts ultimately results in early embryonic death and is seen clinically as infertility. Several risk factors for PBIE have been identified and include age, parity, anatomical conformation, such as poor perineal conformation and cervical fibrosis, as well as other reproductive tract abnormalities. N-Acetyl cysteine (NAC), a mucolytic used to treat endometritis in mares, has anti-inflammatory properties, affects inflammatory cytokines, and is a mild inhibitor of nitric oxide synthase. Increased nitric oxide, causing smooth muscle relaxation and alterations in inflammatory cytokines, has been documented in PBIE mares. The objective of our study was to determine if NAC treatment would lower nitric oxide and inflammatory cytokine levels, thereby resolving PBIE. A randomised, blinded, crossover design clinical trial was performed utilising PBIE-susceptible mares (n=10). Mares were screened for bacterial endometritis before enrolment in the study and only mares that had negative bacterial cultures were used. No other treatments were given to mares while they were enrolled in the study. Intrauterine infusion of 180mL of 3.3% NAC was performed 12h before insemination, when a follicle >35mm was present. Mares were sampled for endometrial cytology and endometrial fluid to determine inflammatory cytokine (ELISA) and nitric oxide (colourimetric assay) levels at 12 and 72h post-insemination. Endometrial biopsies were taken at the same time points post-insemination to determine gene expression of inflammatory cytokines by qPCR. Clinical signs of endometrial oedema and intrauterine fluid volumes were assessed at 12 and then every 24h post-breeding. Statistical assessment of the data was performed using a repeated-measures ANOVA. Uterine inflammation, as determined by percent number of neutrophils on endometrial cytology (P=0.0006), and interleukin 6 gene expression (P=0.003) were highest at 12h. Uterine oedema was greatest at 12 and 24h (P=0.02) and uterine fluid volumes were highest at 24h (P=0.02). Interestingly, interleukin-6 protein levels were not in accordance with gene expression, and were highest at 72h. In this clinical trial, pre-breeding intrauterine treatment with NAC did not affect nitric oxide levels, cytokine gene expression, or clinical signs of PBIE. However, the pattern of inflammation noted in this study is consistent with that described in PBIE mares. Nevertheless, the assessment of inflammatory cytokines, both at the gene and protein level at different time points post-AI, in combination with clinical signs will add to the understanding of the alterations in inflammatory cytokine levels in mares susceptible to PBIE.
97 Ovulation induction in anovulatory southern white rhinoceros (Ceratotherium simum simum)
Authors: Pennington P. , Capiro J. , Marshall K. , Felton R. , Durrant B.
Published: December 2018

The extant Rhinocerotidae family is experiencing threats in the wild, making captive populations important genetic reservoirs for species survival. Because each species faces distinct challenges in captivity, populations are not self-sustaining. Therefore, assisted reproductive technologies (ART) such as AI will be necessary to maintain or increase captive genetic variation. Captive-born female white rhinoceros (Ceratotherium simum simum) have low reproductive rates and apparent acyclicity is a common issue. Although females fail to ovulate and progesterone remains at baseline levels, follicle growth may occur and ovulation can be induced with exogenous hormones. Female southern white rhinos (n=6) housed as a bachelorette group were determined to be ovulatory (n=1) or anovulatory (n=5) by serial ultrasound and fecal progesterone (P4; ng g−1) analysis. However, all anovulatory females grew follicles beyond preovulatory size, which then regressed. At the time of study, when follicles were preovulatory size (35.4±1.2 mm; mean±SD), anovulatory females (n=4) were induced to ovulate (n=11) with a gonadotropin releasing hormone (GnRH) agonist (4.5mg, SucroMate™; Bioniche Animal Health, Bogart, GA, USA) in a single intramuscular injection. Nine treatments resulted in ovulation (81.8%), all between 36 and 48h post-treatment, while 2 hemorrhagic anovulatory follicles (18.2%; HAF) formed, both in the same female. Ovulations were confirmed by disappearance of the follicle by 48h and P4 elevation above baseline was coincident with corpus luteum (CL) and HAF formation. All resulting luteal structures were included in analysis. Follicle growth was permitted to proceed without GnRH treatment between inductions (n=6); dominant follicles grew beyond preovulatory size (43.8±6.1mm) followed by regression and growth of another preovulatory follicle that was subjected to GnRH treatment. Data were analyzed by R studio (ver. 1.1.383). Luteal phases were characterized as “short” (<50 days) or “long” (>50 days). Only P4 above baseline (days) was significantly different (P<0.05) between long and short cycles. Other cycle parameters, such as CL visibility (days), time from ovulation to P4 above baseline (days), maximum P4 (ng g−1), and maximum luteal size (mm), were not significantly different between cycle types. Both cycle types were observed following both spontaneous (short, n=5; long, n=3) and induced ovulations (short, n=8; long, n=3). These data provide additional insight into the differences between long and short cycles and that GnRH is a reliable and effective method to overcome anovulation in SWR. We also prove that long cycle lengths are not necessarily indicative of early pregnancy loss, as has been previously suggested. Taken together, this information can enhance captive breeding efforts and the genetic diversity of the ex situ, SWR insurance population.
98 Efficacy of commercial equine semen freezing extenders for cryopreservation of southern white rhinoceros (Ceratotherium simum simum) sperm
Authors: Young C. , Ravida N. , Pennington P. , Durrant B.
Published: December 2018

Once nearly extinct in the wild, the southern white rhinoceros is currently listed as near threatened by IUCN. This status is likely to change as poaching continues to escalate. To preserve the species’ current genetic diversity, cryopreserving and biobanking white rhinoceros sperm is imperative. The horse is the closest domestic relative of the rhinoceros and a useful model for the development of assisted reproductive technologies, including semen cryopreservation. Two equine semen cryopreservation protocols were compared to a common rhinoceros freezing method. Semen was collected from a single male on 3 occasions by electroejaculation. Initial semen parameters were 86% motility; speed 3.2 (scale 1-5); 89% plasma membrane integrity; and 95% intact acrosomes. Semen was extended 1:1 in INRA 96 (IMV Technologies, L’Aigle, France) before centrifugation at 400×g for 10min. Supernatant was removed and the sperm pellet was subjected to 1 of 2 treatments: resuspension in 500µL of either BotuCrio (Botupharma, Botucatu, Brazil) or Cryomax (ARS Inc., Chino, CA, USA), both containing a proprietary combination of glycerol and an amide as cryoprotectants. Following a 40-min cool at 4°C, extended semen was frozen in vials at a cooling rate of 30°C/min for 3min before LN submersion. Control semen was extended 1:1 in TEST-Y buffer without cryoprotectant and cooled for 2.5h before adding glycerol to a final concentration of 4%. Extended sperm (500µL) was frozen in vials at 12.5°C/min for 15min before LN submersion. Initial motility score (IMS;% motile×speed of progression2), plasma membrane integrity (IPL), and acrosome integrity (IAC) were recorded after extension. All vials were thawed at 37°C for 60s and the cryoprotectant was removed by centrifugation. Sperm pellets were resupended in M199+HEPES and sperm was evaluated for the characteristics described above at 37°C at 0, 30, and 60min (T0, T30, T60) post-thaw. All data are expressed as a percentage of initial (%IMS,%IPL, and%IAC) to account for the differences in sperm parameters between ejaculates. Cryopreservation protocol significantly affected%IMS at T0 (P=0.0131, Table 1). Although the differences were significant only at T0, sperm frozen in Botucrio or Cryomax tended to maintain a higher%IMS than the control freeze at all time points. However, sperm frozen in Cryomax lost a greater percentage of%IMS over time (67% from T0 to T60v. 44 and 46% for Botucrio and TEST-Y, respectively). Cryopreservation protocol did not affect%IAC or%IPL at any time point, but again Cryomax and Botucrio tended to be higher than TEST-Y. This study indicates that rhinoceros sperm may suffer less cryodamage in Botucrio or Cryomax frozen at 30°C/min than in the conventional TEST-Y frozen at 12.5°C/min.

Table 1.Percent of initial motility score (IMS), plasma membrane integrity (IPL), and acrosome integrity (IAC) at 0, 30, and 60min post-thaw (T0, T30, and T60, respectively)
Local community involvement in forest rangeland management: case study of compensation on forest area closed to grazing in Morocco
Authors: Moukrim Said , Lahssini Said , Naggar Mustapha , Lahlaoi Hicham , Rifai Nabil , Arahou Moustapha , Rhazi Laïla
Published: December 2018

Unmanaged livestock grazing is the greatest threat to the health and sustainability of forest rangelands in Morocco. Historically, communities have developed ingenious traditional systems in order to regulate natural resource uses. However, currently most of these principles have been undermined and have led to open access of a common pool resource. To achieve viable solutions to unmanaged livestock grazing in forestlands, local community involvement was introduced in Moroccan forestry early on. The main objective of this study was to show the importance of an original mechanism called compensation on forest area closed to grazing, carried out by the Forestry Department to involve communities that have the right of use in the restoration of forest rangeland ecosystems. It also aims to assess the mechanism’s technical and socioeconomic impacts. Analysis of the process of community participation in the case of Moroccan forest management revealed that it was perceived and implemented in different ways, and considered either as an end in itself or (rarely) as a means to an end. Forest managers and use-rights holders appreciate the mechanism of compensation for forest areas closed to grazing. Since the implementation of this program, the number of grazing association members has increased. This trend has been associated with a positive impact on the reduction in the number of offences and on improving reforestation success rates. In addition, remote sensing showed a positive trend in the relative density and the evolution of the health of vegetation in the areas covered by this mechanism. This program helped to develop consensus in forest ecosystem restoration that will help managers to break the vicious cycle of unmanaged grazing, and promote a new collective stewardship of this precious land. As a result of this success, this program should be replicated and valued. It should be presented in the future as a tool for natural resource conservation with unintended human capital development benefits.
The geosemiotics of tahrir square
Authors: Aboelezz Mariam
Published: December 2018

The year 2011 saw unprecedented waves of people occupying key locations around the world in a statement of public discontent. In Egypt, the protests which took place between 25 January and 11 February 2011 culminating in the ouster of former Egyptian president Hosni Mubarak have now come to be known as the Egyptian Revolution. Media reporting of the revolution often portrayed it as a ‘spectacle’ playing out on the stage of Tahrir Square which was dubbed ‘the symbolic heart of the Egyptian revolution’. Tahrir Square quickly became a space serving various functions and layered with an array of meanings. This paper explores the relationship between the discourse of protest messages and the space of Tahrir Square during the January 25 revolution, demonstrating how the two were mutually reinforcing. The messages are drawn from a corpus of approximately 2000 protest messages captured in Tahrir Square between 25 January and 11 February 2011. The analysis is presented in the form of six conceptualising frames for the space of Tahrir Square which take into account both its geographical and social context. The conceptualisation draws from the field of geosemiotics, which posits that all discourses are ‘situated’ both in space and time (Scollon & Scollon 2003), and on the Lefebvrian principles of the production of space which provide a useful framework for interpreting urban space (Lefebvre 1991).
Mobilities of a linguistic landscape at Los Angeles City Hall Park
Authors: Chun Christian W.
Published: December 2018

In this article, I expand a category of linguistic landscapes, the signs by individuals in public spaces, to include another form of linguistic landscape even more transgressive in nature and intent: the panoply of protest signs produced and mobilized by the Occupy Movement during the Fall of 2011 at Los Angeles City Hall Park. My data are drawn from the photographs I took of these signs at the Park and the near vicinity, a YouTube video of a protest sign, a blog commenting on this sign, and a political cartoon using the same image featured on two other signs. I explore how social actors drew upon and mediated specific discourses in their protest signs that became transportable across time and space, the role of these signs in transforming public space, and this linguistic landscape’s ensuing mobilities in its mediated relocations to online social media sites and blogs.
Counter-discourse corpora, ethical subjectivity and critique of argument
Authors: O’Halloran Kieran
Published: December 2018

In the article, I model an alternative critical discourse analysis (CDA) pedagogy which is based on an ethical subjectivity instead of a political subjectivity. Aimed at undergraduates, it facilitates critical purchase on arguments which attack the standpoint of relatively powerless groups/organizations (who seek political change). Via corpus linguistic analysis of appropriate web-based data, I show how the analyst can rigorously find out at scale the recurrent key concerns of a relatively powerless Other with whom they were previously unfamiliar. They use this counter-discourse information as a lens on an argument which criticises the relatively powerless group, ascertaining whether or not the argument has distorted the group’s key concerns. Should this be the case, I highlight how the analyst can go on to explore whether any mischaracterisation has implications for the argument’s credibility because it loses coherence relative to the outlook of the Other. The approach is grounded in Jacques Derrida’s ‘ethics of hospitality to the Other’. It is in being hospitable to the outlook of a relatively powerless Other, and adopting it for purposes of argument evaluation, that the analyst effectively creates an ethical subjectivity. That said, the ethical and political are, in principle, relatable with this method as I indicate.
Framing and blaming in times of economic crisis
Authors: Murray Jill C.
Published: December 2018

This article takes a critical approach to the language used by Australian politicians during the global financial crisis of 2007–8. Critical periods in history provide a rich substrate for the appearance of new expressions with the potential to frame the debate, influencing the ways events are interpreted and blame attributed. Passing unnoticed into usage, such memes have the potential to become part of unexamined background knowledge and covertly co-opt hearers and users into shared systems of value and belief. The study focusses on one specific neologism deployed by opposition politicians, firstly in an attempt to create the erroneous impression that a recession was occurring and secondly that it was the fault of the Australian Prime Minister, Kevin Rudd. Patterns of occurrence were tracked against local and international events, indicating a life cycle with several distinct phases: chance emergence, a strategic deployment, cross-genre diffusion, resistance and eventual rejection.