bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterisation of mouse interferon-induced transmembrane protein-1 gene expression in the mouse uterus during the oestrous cycle and pregnancy
Authors: Park Hyun Jung , Kuk In Sun , Kim Jin Hoi , Kim Jae Hwan , Song Sang Jin , Choi Bum Chae , Kim Bokyoung , Kim Nam Hyung , Song Hyuk
Published: November 2018

During the oestrous cycle, pregnancy and parturition, the uterus undergoes marked morphological, physiological and functional changes. Amid these changes, the Wnt/β-catenin signalling pathway has been identified as having a crucial role in regulating associated biological events. Recently, based on results from a mouse embryo study, interferon-induced transmembrane protein 1 (Ifitm1) was reported as a downstream molecule of the Wnt/β-catenin signalling pathway. Differential expression patterns of the Ifitm1 gene during the oestrous cycle, pregnancy and parturition were identified in the present study. Quantitative real-time polymerase chain reaction data from uterine samples of mice induced start the oestrous cycle by injection of human chorionic gonadotropin (hCG) and revealed that Ifitm1 mRNA expression increased from late pro-oestrus to metoestrus, and decreased during dioestrus and early pro-oestrus. During pregnancy, Ifitm1 gene expression was minimal until parturition, but increased markedly 2 days after parturition. This significant elevation in Ifitm1 gene expression at post partum stage was identical to Ifitm1 expression after the induction of abortion by injection of prostaglandin F2α. Interestingly, pregnant mare serum gonadotropin (PMSG) and oestrogen are also facilitates changes in Ifitm1 gene expression in an ovariectomised (OVX) mouse model. Expression of Ifitm1 mRNA was higher in response to PMSG than other hormones investigated. These results suggest that Ifitm1 may be involved in uteri physiology, although the mechanisms involved in the regulation of this gene expression and function in the uterus remain unknown. In the present study, differential expression patterns of the Ifitm1 gene were identified in the uteri of mice and the correlation between the patterns of Ifitm1 gene expression and Wnt/β-catenin signalling discussed.
117 Subclinical Mastitis Reduces Ovulation and Oocyte Quality in Milk-Producing Cows
Authors: Santos G. , Bottino M. P. , Ferreira M. B. D. , Silveira J. C. , Avila A. C. F. C. M. , Bridi A. , DeSouza J. C. , Sales J. N. S.
Published: November 2018

The aim was to evaluate the effect of subclinical mastitis by somatic cell count (SCC) on follicular dynamics, ovulation, oocyte and cumulus cell quality, exosome size and concentration in milk-producing cows. Crossbred cows (Bos taurus × Bos indicus; that is, Holstein × Gyr) were randomly allocated to control (SCC <200,000 cells mL−1] and mastitis (SCC >400,000 cells mL−1) groups. In experiment 1 (follicular dynamics), cows (n = 57) were submitted to ultrasonographic evaluations every 24 h, after removal of an intravaginal progesterone device (Day 8) up to Day 10. From Day 10, ultrasound evaluations were performed every 12 h, until ovulation or until 96 h after progesterone device withdrawal, in order to follow final dominant follicle growth and ovulation. In experiment 2 (oocyte, cumulus cells, and follicular fluid evaluation), cows (n = 23) were submitted to follicular aspirations, preceded by synchronization of the emergence of the follicular wave. The levels of target genes in cumulus cells (BCL2, BAX, PI3K, PTEN, FOXO3) were evaluated by RT-qPCR. In the follicular fluid, the exosomes were isolated for evaluation of particle size. Data were analysed by the Glimmix procedure of SAS (SAS Institute Inc., Cary, NC, USA). Ovulation rate (P = 0.09) was higher in control cows [control 77.42% (24/31) and mastitis 57.69% (15/26)]. Viable oocyte rate (P = 0.01) was also higher in control cows [control 59.1% (130/220) and mastitis 41.9% (125/298)]. The dynamics of follicular growth did not differ between groups. The number of degenerate oocytes (P = 0.001) was higher in cows of the mastitis group. In the evaluation of cumulus cell gene expression, there was a higher abundance of BAX transcripts (P = 0.003) in cells of mastitis cows. Additionally, the mean and mode of exosome diameter in mastitis cows were smaller (P = 0.03 and P = 0.02, respectively). In conclusion, ovulation rate, oocyte quality, and follicular fluid exosome diameter were lower in cows with subclinical mastitis, demonstrating a link between mammary gland sanitary status and reproduction.
130 In Vitro Production of Hybrid Desert Bighorn×Domestic Sheep Embryos Using Frozen–Thawed Epididymal Semen from a Hunter-Harvested Ram
Authors: Licea M. G. , Pichardo J. E. H. , Rodríguez J. L. , García-Contreras A. , Rosales B. C. , Palma-Irizarry M. , Romo S. , Kjelland M. E.
Published: November 2018

Although considered a species of least concern by the International Union for Conservation of Nature red list, the Desert Bighorn sheep (Ovis canadensis nelsoni) is listed in Appendix II of CITES (Convention on International Trade in Endangered Species of Wild Fauna and Flora) in Mexico, due to population size and the lack of protected areas. Postmortem epididymal sperm collected from a hunter-harvested Desert Bighorn sheep ram in Mexico, with an unofficial Safari Club International score of 197 2/8 and an estimated 5.5 years old, were used to evaluate the in vitro production (IVP) of embryos using postmortem-collected ram sperm. Testicles with epididymides were placed in the refrigerator ~45 min after harvest. Sperm were extracted from each epididymis and assessed separately for total motility (TM), progressive motility (PM), and membrane integrity using a phase contrast microscope. The sperm suspension was obtained from the distal end of both epididymides and cryopreserved 12 h postmortem using triladyl with egg yolk. Membrane integrity and morphology were evaluated using Eosin-Nigrosin stain. Sperm DNA fragmentation was analysed using the Halomax kit (Halosperm SL, Madrid, Spain) with fluorescence microscopy. Centrifugation with density gradient PureSperm (Nidacon International, Mölndal, Sweden) was used to remove dead sperm and debris before IVF. Ovaries were collected from Domestic sheep (Ovis aries) at a local slaughterhouse. The maturation medium was TCM-199 with Earle’s salts and a modified Tris-buffered medium was used for fertilization. Frozen straws of sperm from the Desert Bighorn ram were thawed for 45 s at 37°C. Sperm were diluted with modified Tween medium B with milk powder (mTBM) to a final concentration of 5 × 106 cells mL−1. The gametes were co-incubated for 18 h under previously described conditions. The cumulus cells were mechanically removed from zygotes and grown using a co-culture with granulosa cells in sequential media SOF1-SOF2. With regard to sperm collection, epididymis 1 produced 29 straws of sperm (0.25 mL, 136 × 106 sperm mL−1) and epididymis 2 produced 32 straws of sperm (0.25 mL, 68 × 106 sperm mL−1). The sperm sample used for IVF had TM of 60% and PM of 30%. Live dead staining of fresh sperm showed 68% live (i.e. intact cell membranes) and 28% post-thaw. Regarding DNA integrity, only 2% of sperm had DNA fragmentation at 0 h. Of 15 Grade 1 oocytes used for IVF, 4 cleaved (27%), with 1 developing to blastocyst stage (25%). The results show that frozen–thawed epididymal sperm collected from a recently deceased Desert Bighorn ram can provide a valuable source of sperm for IVP of embryos. These results also provide new information on Desert Bighorn sheep reproductive parameters for use in health assessment, or reproduction and conservation management through gene banking and assisted reproductive techniques.
138 Relationships Between Antral Follicle Count, Ovarian Volume, Pre-Antral Follicle Number, and Oocyte Quality
Authors: Vasconcelos G. L. , Maculan R. , Alves N. , Ribeiro A. L. A. P. L. , Silva A. W. B. , Cunha E. V. , Moreira G. M. , Viafara J. A. S. , Guimaraes A. S. B. , Ribeiro C. V. , Silva J. R. V. , Pinto T. L. C. , Sales J. N. S. , DeSouza J. C.
Published: November 2018

The objective was to evaluate the possible relationships between AFC, ovarian volume, ovarian follicle reserve and oocyte quality in abattoir-derived ovaries (experiment 1) and in cows (experiment 2) submitted to OPU. Antral follicle counts of ≥25, 16 to 24, and ≤ 16 were used to define AFC classes as high (HAFC), intermediate (IAFC), and low (LAFC) in both experiments. In experiment 1, after antral follicles were aspirated, abattoir ovaries (n = 10 per AFC class) were processed by conventional histology and pre-antral follicles were counted within primordial, primary, secondary, and tertiary classes and classified as either healthy or degenerate under regular microscopy (Cushman et al. 1999). In experiment 2, HAFC (n = 42), IAFC (n = 34), and LAFC (n = 29) cows were submitted to OPU and oocytes classified as grades 1, 2, and 3 or degenerate (IETS, 2010). Antral follicles (≥3 mm in diameter) were counted by ultrasonography. Data were analysed by GENMOD and GLM procedures of SAS (SAS Institute Inc., Cary, NC, USA) after transformations, when required. In experiment 1, mean normal primordial follicle number was higher (P < 0.001) in HAFC (137.0 ± 1.6)a compared with IAFC (52.6 ± 1.9)b and LAFC (20.2 ± 5.3)c ovaries. However, the mean number of degenerate primordial follicles was lower (P < 0.001) in low count ovaries (2.4 ± 0.6) compared with HAFC (19.0 ± 4.7) and IAFC (16.4 ± 1.5, P < 0.001). Normal primary follicle number was higher in the HAFC compared with IAFC and LAFC ovarian classes (86.2 ± 7.0a v. 34.6 ± 5.1b and 14.4 ± 3.3c, respectively; P < 0.01). Degenerate primary follicles were higher in the HAFC compared with LAFC ovarian class (16.8 ± 6.5 v. 5.2 ± 2.64; P < 0.05). Normal secondary follicle number was also higher in the HAFC compared to LAFC ovarian classes (25.2 ± 7.67 v. 2.4 ± 0.8; P < 0.05). The number of degenerate secondary follicles differed (P < 0.01) only between the IAFC and the LAFC ovarian classes (0.6 ± 0.4 and 7.2 ± 2.4, respectively), which were similar (P > 0.5) to the HAFC class (3.8 ± 1.0). In experiment 2, grade 1, 2, and 3 oocytes, viable oocytes, and ovarian volume (mm3) were higher (P < 0.001) in HAFC compared with IAFC and LAFC cows (grade 1: 7.9 ± 0.6a, 4.9 ± 0.7b and 3.3 ± 0.7c; grade 2: 4.0 ± 0.4a, 2.8 ± 0.4b and 1.2c; grade 3: 2.1 ± 0.4a, 2.5 ± 0.4a and 1.3 ± 0.5b, respectively; viable oocytes: 16.3 ± 1.1a, 13.1 ± 1.2b, and 8.1 ± 1.3c, respectively; (volumes: 12.6 ± 0.7a, 10.1 ± 0.8b, and 8.1 ± 0.9c, respectively). In conclusion, high AFC is linked to a higher follicular reserve, oocyte quality, and ovarian volume. It is safe to apply AFC in the selection of bovine females without compromising oocyte or pre-antral follicular population qualities.
157 Sodium Fluoride Exposure Exerts Toxic Effects on Porcine Oocyte Maturation
Authors: Nie Z.-W. , Liang S. , Cui X.-S.
Published: November 2018

Excessive long-term fluoride intake is associated with several health problems, including infertility. However, limited information is available on the toxic effects of fluoride exposure on the female reproductive system, especially oocyte maturation. In this study, we investigated the toxic effect of sodium fluoride (NaF) exposure on porcine oocyte maturation and its possible underlying mechanisms. The level of reactive oxygen species, glutathione (GSH), mitochondrial membrane potential, apoptosis, and DNA damage and cathepsin B in the blastocysts were observed by staining with 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA), 4-chloromethyl-6.8-difluoro-7-hydroxycoumarin (CMF2HC, Invitrogen, Carlsbad, CA, USA), 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1), annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit, anti-γH2A.X (Ser139) and Magic Red cathepsin B assay kit, respectively. Data obtained from the experimental groups were compared using Student’s t-test. Our results showed that treatment with 60, 100, and 150 μg mL−1 NaF significantly decreased the degree of cumulus cell expansion (CEI: 2.64 ± 0.27, 1.32 ± 0.52, and 0.57 ± 0.15 v. 3.58 ± 0.19; P < 0.05) and the rate of maturation (74.33% ± 4.32%, 57.83% ± 7.14%, 44.00% ± 5.93% v. 84.50% ± 3.62%; P < 0.05) in a dose-dependent manner during in vitro maturation for 44 h. Cell cycle analysis showed that NaF exposure blocked meiotic resumption, disturbed spindle dynamics, disrupted chromosome separation, and increased aneuploidy in porcine oocytes. Moreover, NaF exposure disturbed mitochondrial function, triggered DNA damage response, and induced early apoptosis in porcine oocytes. Exposure to NaF also induced oxidative stress, decreased GSH level, and increased cathepsin B activity in and impaired the further development potential of porcine oocytes, as indicated by a decrease in blastocyst formation rate, increase in apoptosis, and inhibition of cell proliferation. Together, these results indicate that NaF exposure impairs the maturation capacity of porcine oocytes by inhibiting cumulus cell expansion, disturbing cytoskeletal dynamics, and blocking nuclear and cytoplasmic maturation, thus decreasing the quality and affecting the subsequent embryonic development potential of porcine oocytes.
194 Usefulness of the Ultrasound Imaging Modalities B-Mode and Colour Doppler Sonography and Serum Progesterone Concentrations for Determining Superovulatory Ovarian Responses in Ewes
Authors: Ribeiro I. F. , Rodrigues M. G. K. , Maciel G. S. , Fonseca J. F. , Brandão F. Z. , Bartlewski P. M. , Oliveira M. E. F.
Published: November 2018

The main goal of this study was to assess the usefulness of the 2 imaging modalities—B-mode and colour Doppler sonography—for determining the superovulatory response in ewes. In addition, serum concentrations of progesterone (P4) were examined for correlations with the numbers of laparoscopically detected luteal structures. Oestrus was synchronized in 24 sexually mature Santa Inês ewes with intravaginal P4-releasing devices (CIDR®) inserted for 8 days on a random day of the oestrous cycle. An injection of 37.5 μg of prostaglandin F2α analogue (d-cloprostenol) was given at the time of CIDR® insertion and withdrawal. The superovulatory treatment consisting of 8 consecutive doses of porcine FSH (total dose of 200 or 133 or 100 mg of pFSH; n = 8/total dose) given at 12-h intervals began 48 h before CIDR® removal. All animals received a single injection of 300 IU of eCG concurrently with a first dose of pFSH. On the day of surgical embryo recovery (6 days after the onset of behavioural oestrus), the ovaries of all donor ewes were visualised and examined with transrectal ultrasonography and then with videoendoscopy to identify and enumerate corpora lutea (CL) and luteinized unovulated follicles (LUF). Jugular blood samples were collected just before ovarian imaging for the measurement of circulating P4 concentrations. Ovaries could be easily and repeatedly detected with both the transrectal ultrasonography and videoendoscopic technique. Prematurely regressing CL could not be distinguished from healthy CL using either B-mode or colour Doppler ultrasonography but they were identified by their pale color by using videoendoscopy. All animals responded to the superovulatory treatment and produced ≥4 CL; however, a wide variation in ovarian responses was observed among ewes (range: 4–24 CL/ewe). Three ewes failed to produce healthy CL and had prematurely regressing CL only. In general, LUF were observed in 20/24 ewes (1–5/ewe) and 14/24 ewes had regressing CL (1–22/ewe). The total number of CL (r = 0.78 and 0.83; P < 0.0001) and LUF (r = 0.74 and 0.90; P < 0.0001) enumerated using the B-mode and colour Doppler ultrasonographic technique, respectively, were correlated with that ascertained by videoendoscopy. The determination of the superovulatory response was better by colour Doppler ultrasonography in relation to the B-mode, because it allows a better delimitation of the luteal structures. Serum P4 concentrations were related inversely to the numbers of prematurely regressing CL (r = –0.70; P = 0.0004) and directly to the number of healthy CL (r = 0.69; P = 0.0005). The present results indicate that ultrasonographic imaging and serum P4 measurements on the day of embryo recovery are both very accurate indicators of ovarian responses in superovulated ewes.
20 Assessment of Motion and Kinematic Characteristics of Semen from Four Cattle Breeds Using Computer-Aided Sperm Analysis
Authors: Mphaphathi M. L. , Seshoka M. M. , Netshirovha T. R. , Raphalalani Z. C. , Bovula N. , Thandavhathu M. R. , Raito B. , Kanuya N. L. , Maqhashu A. , Nedambale T. L.
Published: November 2018

Subjective semen evaluation using standard optical microscopy is the most common practice. Semen parameters routinely assessed are volume, concentration, progressive motility, and morphology. However, computer-aided sperm analysis (CASA) represents an objective evaluation, sperm assessment that are reproducible and reliable. Such semen parameters have not been evaluated in Afrikaner, Brahman, and Bonsmara bulls’ semen. The present study evaluated the sperm motion and kinematics characteristics of semen from stud Afrikaner, Brahman, Bonsmara, and Nguni bulls using CASA technology. The electro-ejaculator was used for semen collection from Afrikaner (n = 11), Brahman (n = 7), Bonsmara (n = 10) and Nguni (n = 16) bulls of known and proven fertility. Semen was collected following 4 days of resting period. The bulls ranged between 5 and 6 years of age. After collection, the semen samples were immediately transferred to a thermo-flask and maintained at 37°C for further evaluation in the mobile laboratory (Nedambale, 2014). The CASA-Sperm Class Analyzer® system (Microptic, Barcelona, Spain) was used to evaluate sperm motion, velocity, and kinematic parameters or characteristics of raw/fresh semen from 4 cattle breeds. Data were analysed using GenStat® statistical programme (VSN International, Hemel Hempstead, United Kingdom). Treatment means were compared using one-way ANOVA. The total sperm motility rate was similar for all breeds: Afrikaner (92.2 ± 4.2), Brahman (90.7 ± 9.0), Bonsmara (93.9 ± 4.0), and Nguni (96.0 ± 2.7). However, Brahman and Afrikaner bull semen had higher sperm cells moving in a progressive motility of 57.3 and 45.6%, respectively, compared with other breeds (P < 0.05). Nguni, Afrikaner, and Bonsmara had the highest sperm cells moving in a rapid movement of 73.7, 72.4, and 67.4% (P > 0.05), respectively. The bulls sperm trajectories had a variation, as they were recorded to be irregular and not linear (P < 0.05). The straight-line sperm velocity (µm s−1), wobbling %, and amplitude of lateral head displacement % was similar for the 4 breeds (P > 0.05). In conclusion, CASA technology was a useful technique for assessing differences in sperm motion and kinematic (motility and velocity characteristics) among different bull breeds.
24 Evaluation of Latrunculin A for the Activation of Hand-Made Cloning (HMC) Porcine Embryos
Authors: Castañeda F. K. , Canel N. G. , Landschoot G. V. , De Stéfano A. , Bevacqua R. J. , Salamone D. F.
Published: November 2018

Somatic cell nuclear transfer (SCNT) is an important biotechnological tool. However, production rates of viable offspring remain low. One possible cause of this low efficiency is chromosomal losses during early activation process (Liu et al. 2015 Cell. Reprogram. 17, 463–471). The use of actin inhibitors that block second polar body extrusion during activation protocols might be a strategy to avoid such losses. The objective of this work was to compare the efficiency of the use of 2 actin inhibitors during the activation of hand-made cloning (HMC) porcine embryos. One of the compounds used was latrunculin A (LatA), which joins directly to actin monomers, preventing their assembly to the filaments. The other was cytochalasin B (CB), which is commonly used for activation protocols. It binds to the growing actin filaments and prevents their elongation. For this purpose, in vitro-matured cumulus–oocyte compexes were deprived of their cumulus and zonae pellucidae cells by mechanical and enzymatic treatments. Oocytes were randomly distributed in 2 experimental groups (HMC) and 2 parthenogenetic control groups (PA). For HMC groups, oocytes were bisected using a microblade and the resulting hemioocytes were stained with Hoechst 33342 and observed under UV light to identify those that had lost the metaphase II plate. Adult skin fibroblasts from primary cultures were used as nuclear donors. For nuclear transfer, 2 hemicytoplasts were fused to a donor cell by an electric pulse of 1.42 kV/cm for 30 μs. After 2 h of nuclear reprogramming, the reconstituted embryos were activated by an electric pulse of 1.2 kV/cm for 80 μs and incubated with cycloheximide (CHX, 10 μg mL−1 , 3 h) in combination with one of the actin inhibitors: LatA 2 μM (CHX-LatA goup) or CB 2.5 μg mL−1 (CHX-CB group). The PA groups were subjected to the same activation treatments (PA-CHX+LatA and PA-CHX+CB groups). All embryos were cultured in SOFaa medium, using an adaptation of the well-of-the-well (WOW) system (microwells), in a humidified atmosphere with 5% CO2 in air at 39°C. Cleavage, morulae, and blastocysts rates were evaluated at Days 2, 4, and 7-8, respectively. At least 3 replicates were performed per group. Results are presented in Table 1. Our results demonstrate that the production of embryos by HMC activated with CHX-LatA is as efficient as that with CHX-CB, the protocol currently used in SCNT protocols. Further research is needed to study its effect on chromosomal complements and long-term development.

Table 1.Effect of activation with cycloheximide (CHX) and latrunculin A (LatA) on in vitro development of hand-made cloning (HMC) porcine embryos (% ± SD in parentheses)
31 Overall Goat Cloning Efficiency Under Suboptimal Conditions — A 6-Year Experience
Authors: Aguiar L. H. , Méndez-Calderón C. E. , Ongaratto F. L. , Rossetto R. , Rondina D. , Rodrigues J. L. , Bertolini L. R. , Bertolini M.
Published: November 2018

Animal cloning involves a combination of several simple steps that need to be carried out at the highest efficacy to provide acceptable yet low overall cloning efficiency. Oocyte competence is key for proper somatic cell nucleus reprogramming, and technical elements must be refined to minimize interference in the overall outcome. The aim of this study was to compare the progress of a goat-cloning program over a 6-year period in which oocyte donors’ protocols and oocyte and embryo manipulation were continuously refined to cope with success under Brazilian semi-arid conditions. The cloning dataset was divided in 3 periods (P1, 2011-2012; P2, 2013-2014; P3, 2015-2016), using either in vivo- or in vitro-matured goat oocytes for cloning by nuclear transfer (NT) with different cell lines for subsequent transfer to synchronized recipients to produce transgenic liveborn kids. Over time, protocols for recovery of competent oocytes were adapted to existing conditions, also optimizing animal well being and nutritional status to attain better success. Data on total and viable oocytes, oocyte maturation, pregnancy, and cloning efficiency were analysed by the Chi-squared or t-test (P < 0.05). After 111 replicates, viable and matured oocyte rates were improved during the time, but mean number of total oocytes/donor were higher in the second period (25.4 ± 8.9 v. 18.5 ± 10.8 for P1 and 19.5 ± 6.1 for P3), whereas the mean of viable and matured oocytes were similar between P2 (19.4 ± 6.0 and 9.8 ± 4.6) and P3 (18.8 ± 7.0 and 9.6 ± 3.7), but higher than in P1 (12.8 ± 7.6 and 6.0 ± 4.1), respectively. Higher pregnancy rate was achieved in P1 and lower in P2, with both being similar to P3. However, the efficiency based on transferred embryos was similar between periods (Table 1). When compared with total oocytes, birth rate improved from the first to the last period (P1, 0.01%; P2, 0.02%; and P3, 0.09%). Cloning efficiency was measured by the number of transferred embryos compared with matured structures used for cloning, which showed an improvement over time (P1, 34.5%; P2, 43.9%; P3, 60.1%). A 5-fold enhancement was observed in the number of matured oocytes needed to produce a liveborn animal (P1, 2,779/1; P2, 1,892/1; P3, 545/1). Also, the number of donors (P1, 432; P2, 193.5; P3, 56.6) and recipients (P1, 114; P2, 57; P3, 27) needed to produce 1 animal was reduced by 7.6- and 4.2-fold between periods. In conclusion, complex and inefficient procedures such as cloning by NT require technical refinement and adjustments to existing conditions, including animal nutrition and welfare, and cumulative gain of expertise to attain successful results.

Table 1.Overall goat cloning efficiency over time.
46 Effect of Bioxcell® and Triladyl® Extenders on Washed Semen of South African Indigenous Bucks
Authors: Nethenzheni L. P. , Mphaphathi M. L. , Negota N. C. , Nedambale T. L.
Published: November 2018

Semen extenders and seminal plasma are vital for cryopreservation of buck semen. The objectives of the study were to evaluate the effect of 2 extenders: Triladyl® (Minitube, Tiefenbach, Bavaria) and Bioxcell® (IMV, L’Aigle, France) and the removal of seminal plasma on buck semen. Six indigenous bucks were used in this study and 6 ejaculates were collected from individual bucks. The semen was pooled and then randomly allocated into 6 groups: (1) raw-washed, (2) raw-non-washed, (3) Triladyl®-washed, 4) Triladyl®-non-washed, (5) Bioxcell®-washed, and (6) Bioxcell®-non-washed. Spermatozoa viability was assessed using Eosin-Nigrosin and morphology using Spermac® (Vitrolife, Göteborg, Sweden) stains. The washed semen samples were all diluted into (1:4 v/v) with PBS and centrifuged at 1500 × g for 10 min. Semen samples were then extended with Triladyl® or Bioxcell® per treatment groups and equilibrated for 2 h at 5°C. The semen samples were loaded into straws per treatment groups and placed 5 cm above a liquid nitrogen vapour for 10 min and then stored at –196°C until use. After 1 month of storage, frozen semen straws per treatment group were thawed at 37°C for 30 s, and spermatozoa parameters were analysed post-thaw. Significant differences among the mean values of semen parameters were determined by Tukey’s test using ANOVA, GLM procedure of SAS version 12.1 of 2010 (SAS Institute Inc., Cary, NC, USA). There was a higher (P < 0.05) live and normal spermatozoa percentage in non-washed semen extended with Bioxcell® (45.7 ± 21.2) than the semen extended with Triladyl® (24.5 ± 22.2%). Live and normal spermatozoa percentages were drastically reduced in the Bioxcell® (5.2 ± 4.9) and Triladyl® (6.9 ± 8.6%) washed semen groups. There was a higher (P < 0.05) percentage of spermatozoa with head abnormalities in non-washed semen extended with Triladyl® (20.4 ± 10.2), compared with the semen extended with Bioxcell® (18.3 ± 12.4%) following freeze-thawing. There was a higher (P < 0.05) percentage of spermatozoa with head abnormalities in washed semen samples extended with Triladyl® (34.0 ± 16.0) compared with the semen extended with Bioxcell® (10.1 ± 7.0%). There were higher (P < 0.05) percentages of spermatozoa with coiled tail abnormalities in washed semen extended with Bioxcell® (65.4 ± 25.0) compared with Triladyl® (35.9 ± 21.6%). In conclusion, the liveability of spermatozoa was negatively affected by washing of semen extended with Bioxcell® and Triladyl® extender. Bioxcell® significantly increased tail abnormalities and Triladyl® gave less protection against head abnormalities following cryopreservation of South African unimproved indigenous bucks’ semen.
49 Developmental Potential of Dromedary Camel Oocytes Vitrified at the Germinal Vesicle Stage: Effects of Different Cryoprotectant Combinations and Cryo-Carriers
Authors: Fathi M. , Moawad A. R. , Badr M. R.
Published: November 2018

Cryopreservation of oocyte would be an alternative to overcome the limited availability of dromedary camel oocytes and allow improvements in in vitro production in this species. Our aim was to develop a protocol for vitrification of dromedary camel oocytes at the germinal vesicle (GV) stage using various cryoprotectant combinations and cryo-carriers. In experiment 1, cumulus–ppcyte complexes (COC) obtained at slaughter were equilibrated in a solution composed of 10% ethylene glycol (EG) and 0.25 M trehalose. The oocytes were then exposed for 60 s to vitrification solutions (VS) composed of 20% EG and 20% dimethyl sulfoxide (DMSO; VS1) or 25% EG plus 25% DMSO (VS2) or 25% EG and 25% glycerol (VS3). The COC were then transferred into decreasing concentration of trehalose solution (toxicity test). In experiment 2, COC were randomly divided into 4 groups and vitrified by using straw or open pulled-straw (OPS) or solid surface vitrification (SSV) or cryotop in VS1 or VS2. Following vitrification and warming viable oocytes were matured in vitro for 30 h at 39°C in 5% CO2 in air. Matured oocytes were fertilized in vitro by epididymal spermatozoa of mature male camels and then cultured in modified KSOMaa medium for 7 days. Oocyte viability, maturation, fertilization, and embryo development were evaluated. Data were analysed using one-way ANOVA and t-test. Viability and nuclear maturation rates were significantly lower (P ≤ 0.05) in oocytes exposed to VS3 (44.8% and 34.0%) than those exposed to VS1 (68.2% and 48.0%) and VS2 (79.3% and 56.9%). Although recovery rates were significantly lower (P ≤ 0.05) in oocytes vitrified using SSV or cryotop in either VS1 or VS2 solutions (66.9% to 71.1%) than those vitrified by straws using VS1 or VS2 solutions (86.3% to 91.0%), survival rates were higher in SSV and cryotop groups (90.7% to 94.8%) than straw and OPS (68.2% to 86.5%) groups. Among vitrified groups, maturation and fertilization rates (51.8% and 39.2%, respectively) were the highest in the cryotop-VS2 group. Those values were comparable to those seen in the controls (59.2% and 44.6%, respectively). Cleavage (22.5% to 27.9%), morula (13.2% to 14.5%), and blastocyst (6.4% to 8.5%) rates were significantly higher (P ≤ 0.05) in SSV and cryotop groups than in straws. No significant differences were observed in these parameters between cryotop and control groups. Together, the results show that both vitrification solution and cryodevice affect viability and developmental competence of vitrified/warmed dromedary camel oocytes. We report for the first time that dromedary camel oocytes vitrified at the GV stage have the ability to be matured, fertilized, and subsequently develop in vitro to produce blastocyst embryos at frequencies comparable to those obtained using fresh oocytes.
55 The Role of Poly (ADP-Ribose) Polymerases in Porcine Cumulus - Oocyte Complex During In Vitro Maturation and Embryonic Development
Authors: Kim D. H. , Shin S. T. , Lee H. T.
Published: November 2018

Poly(ADP-ribosyl)ation (PARylation) is related to DNA repair, chromatin modification, and apoptosis and is catalyzed by PARylation polymerases (PARP). Previous studies have shown that PARylation regulates pre-implantation development and participates autophagy mechanism in mouse and pig. However, the involvement of PARylation and pro-survival autophagy in pre-implantation development from cumulus–oocyte complexes (COC) to the blastocyst stage has not yet been documented. Thus, we investigated the role of PARylation during in vitro maturation (IVM) of porcine COC and their embryonic development. To study the effect of PARylation, COC were cultured with 3-aminobenzamide (3-ABA, PARP inhibitor) during IVM. Nuclear maturation rates of oocytes were showed no significant differences between 2 groups in all stages (from GV to MII). However, the expansion rates of cumulus cells were significantly decreased in 3-ABA–treated COC compared with control (11.05 ± 1.09 v. 48.40 ± 0.67%). When we analysed mRNA levels of maturation- and expansion-related genes in cumulus cells, levels of PTX3, CX43, and COX-2 were increased but levels of HAS2 and TNFAIP6 were decreased in treatment group. In addition, expression levels of GDF9 and BMP15 in oocytes were up-regulated in treated group. Then, we examined the development of IVF embryos from IVM oocytes in the presence and absence of 3-ABA and their quality at the blastocyst stage. We found that the developmental rates of embryos were significantly decreased in 3-ABA-treated group. In particular, the proportion of expanded blastocysts was lower in the treated embryos (2.65 ± 1.53) compared with control embryos (12.27 ± 3.05). Furthermore, the transcript levels of autophagy-related genes (ATG5, BECLIN1, and LC3) in 3-ABA-treated embryos were lowered in all stages. In addition, we found a higher rate of apoptosis in treated blastocysts compared with the control (total apoptosis index; 15.65 ± 2.73 v. 4.89 ± 0.67). Finally, SQSTM1/p62 aggregate increased in 3-ABA-treated blastocysts, indicating that the inhibition of PARylation regulates selective autophagy pathways to utilise SQSTM1/p62. Therefore, these results indicate that PARylation by PARPs during IVM of COC is deeply involved in the pro-survival autophagy and influences the development and quality of porcine embryos.
This research was supported by a Grant from the Bio & Medical Technology Development Program (2015M3A9C7030091) of the National Research Foundation (NRF) funded by the Korean government.
7 Ovarian Follicular Dynamics and Conception Rate in Bos indicus Cows with Different Antral Follicle Counts Subjected to Timed Artificial Insemination
Authors: Morotti F. , Moretti R. , Gomes dos Santos G. M. , Silva-Santos K. C. , Seneda M. M.
Published: November 2018

Two experiments in Nelore cows subjected to conventional protocol for timed AI (TAI) were designed to compare the influence of antral follicle count (AFC; 1) on the ovarian follicular dynamics and (2) conception rates. First, multiparous cows with high (G-high, ≥45 follicles; n = 43) and low AFC (G-low, ≤15 follicles; n = 32) were selected to receive a conventional oestradiol/progesterone-based TAI protocol for monitoring ovarian follicular dynamics. Second, AFC from 962 multiparous suckling cows (40 ± 15 days postpartum) subjected to a conventional protocol for TAI were classified as G-high (≥45 follicles; n = 194), G-intermediate (≥20 and ≤40 follicles, n = 397), and G-low AFC (≤ 15 follicles; n = 243). Pregnancy status was evaluated 30 to 35 days later. Data were analysed using ANOVA followed by the Tukey test and model of logistic regression (P ≤ 0.05). In the first study, the AFC were greater (P ≤ 0.05) in G-high than in G-low, but remained stable in both groups during the evaluations. The ovarian diameter, perimeter, and area were greater (P ≤ 0.05) in G-high than in G-low (28.3 ± 3.9 v. 20.5 ± 3.2 mm, 100.9 ± 13.2 v. 73.8 ± 13.9 mm, and 67.5 ± 16.4 v. 37.7 ± 11.7 mm2; respectively). Follicular growth rate, time of ovulation, corpus luteum diameter, and progesterone concentration were similar (P > 0.05) for cows with low or high AFC. Dominant follicles showed greater (P ≤ 0.05) diameter in G-low than G-high at D4 (7.3 ± 2.2 v. 6.2 ± 1.4 mm; P = 0.069, tendency), D8 (11.2 ± 1.8v. 9.5 ± 1.8 mm), D9 (12.3 ± 1.7 v. 10.6 ± 1.7 mm), and D10 (13.4 ± 1.3 v. 12.2 ± 1.8 mm), and the estimated diameter of the ovulatory follicle tended (P = 0.08) to be higher in G-low than in G-high (14.4 ± 1.5 v. 13.4 ± 2.1 mm). In the second study, AFC of G-low cows showed higher (P ≤ 0.05) conception rate (61.7%) than those of G-intermediate (52.9%) and G-high (49.5%) cows. In Nelore cows subjected to ovulation synchronization protocol, cows with high AFC showed higher ovarian measurements, but those with low AFC had large follicular diameters. Additionally, the cows with low AFC showed higher conception rate than groups with intermediate and high counts.
Effects of grazing systems on herbage mass and liveweight gain of Tibetan sheep in Eastern Qinghai-Tibetan Plateau, China
Authors: Sun Y. , Angerer J. P. , Hou F. J.
Published: November 2018

Grazing strategies, consisting of grazing systems and stocking rate adjustments, have evolved from the need to sustain efficient use of the forage resources by livestock, increase animal performance and sustain forage production. A 3-year study was conducted with Tibetan sheep on the Eastern Qinghai-Tibetan Plateau, China to compare: (1) two grazing systems [season-long continuous (SLC; July to December) versus short duration with seasonal rotation (SDSR; July to September in growing-season pasture and October to December in cold-season pasture) with a stocking rate of 24 sheep months ha–1(SM ha–1)]; (2) SDSR system with 24, 36 and 48 SM ha–1; and (3) seasonal aspects of stocking rate under the SDSR system by comparing strategies of heavy stocking rate in the growing season and light stocking rate in the cold season (SDSR-HL) versus light stocking rate in the growing season and heavy stocking rate in the cold season (SDSR-LH). No differences were found between grazing systems in liveweight gain per head or per ha and in residual herbage mass. Liveweight gain per head for treatment SDSR24 was greater than for treatments SDSR36 and SDRS48, whereas liveweight gain per ha showed the opposite tendency. No differences were found between the SDSR-HL and SDSR-LH treatments in liveweight gain per head or per ha, whereas the ratio of residual herbage mass at the end of grazing the growing-season pasture to the cold pasture of treatment SDSR-LH was more than twice that of treatment SDSR-HL. Daily liveweight gain of Tibetan sheep decreased linearly with increasing grazing pressure in both growing and cold seasons. It was estimated that, at a grazing pressure index of 310 sheep days t–1 DM peak herbage mass, liveweight gain per head and ha appears to be optimised over the whole grazing period. Liveweight loss by Tibetan sheep during the cold season was apparent regardless of grazing pressure indicating that temperature had a stronger influence on sheep performance in the cold season than herbage availability.
Floristic composition and pasture condition of Aristida/Bothriochloa pastures in central Queensland. I. Pasture floristics
Authors: Silcock R. G. , Hall T. J. , Filet P. G. , Kelly A. M. , Osten D. , Schefe C. M. , Knights P. T.
Published: November 2018

A survey was conducted in central inland Queensland, Australia of 108 sites that were deemed to contain Aristida/Bothriochloa native pastures to quantitatively describe the pastures and attempt to delineate possible sub-types. The pastures were described in terms of their floristic composition, plant density and crown cover. There were generally ~20 (range 5–33) main pasture species at a site. A single dominant perennial grass was rare with three to six prominent species the norm. Chrysopogon fallax (golden-beard grass) was the perennial grass most consistently found in all pastures whereas Aristida calycina (dark wiregrass), Enneapogon spp. (bottlewasher grasses), Brunoniella australis (blue trumpet) and Panicum effusum (hairy panic) were all regularly present. The pastures did not readily separate into broad floristic sub-groups, but three groups that landholders could recognise from a combination of the dominant tree and soil type were identified. The three groups were Eucalyptus crebra (narrow-leaved ironbark), E. melanophloia (silver-leaved ironbark) and E. populnea (poplar box). The pastures of the three main sub-groups were then characterised by the prominent presence, singly or in combination, of Bothriochloa ewartiana (desert bluegrass), Eremochloa bimaculata (poverty grass), Bothriochloa decipiens (pitted bluegrass) or Heteropogon contortus (black speargrass). The poplar box group had the greatest diversity of prominent grasses whereas the narrow-leaved ironbark group had the least. Non-native Cenchrus ciliaris (buffel grass) and Melinis repens (red Natal grass) were generally present at low densities.
Describing pastures in terms of frequency of a few species or species groups sometimes failed to capture the true nature of the pasture but plant abundance for most species, as density, herbage mass of dry matter or plant crown cover, was correlated with its recorded frequency. A quantitative description of an average pasture in fair condition is provided but it was not possible to explain why some species often occur together or fail to co-exist in Aristida/Bothriochloa pastures, for example C. ciliaris and E. bimaculata rarely co-exist whereas Tragus australianus (small burrgrass) and Enneapogon spp. are frequently recorded together. Most crown cover was provided by perennial grasses but many of these are Aristida spp. (wiregrasses) and not regarded as useful forage for livestock. No new or improved categorisation of the great variation evident in the Aristida/Bothriochloa native pasture type can be given despite the much improved detail provided of the floristic composition by this survey.
Remote mining towns on the rangelands: determining dependency within the hinterland
Authors: Robertson Stuart , Blackwell Boyd
Published: November 2018

The literature is replete with examples of rangelands communities which have suffered significant impacts from nearby mine operations and their subsequent closure. Increasingly, there is awareness among the International and Australian mining peak bodies for the need to plan for enduring value for peripheral communities that are dependent on nearby mines at their ‘core’. We find a greater dependency in one of the case study locations, Leigh Creek, by hinterland communities. In contrast to Leigh Creek, hinterland communities of Roxby Downs are yet to develop as great a dependency on this major centre for services. The reported utilisation of the Port Augusta, (260 km from both centres), highlights the disproportionate costs for accessing services not available in Leigh Creek and Roxby Downs upon residents who are not in paid employment. The impending closure of the Leigh Creek mine and dependency upon Leigh Creek by its hinterland communities requires urgent planning to be undertaken to ensure that the services remain available in Leigh Creek, Whereas, in the case of Roxby Downs there is time for planning to diversify the economy to ensure it is able to withstand the cyclical nature of the resources cycle.
Supporting Indigenous rangers’ management of climate-change impacts on heritage sites: developing an effective planning
tool and assessing its value
Authors: Carmichael Bethune
Published: November 2018

Australian rangelands are rich in Indigenous cultural heritage sites and Indigenous rangers increasingly manage them. It is well documented that climate-change adaptation planning on a local scale benefits from a stakeholder-led or bottom-up process. However, to date, few bottom-up, practical adaptation pathways exist for Indigenous Australians. This paper describes the development of a planning tool that supports Indigenous rangers’ plan for climate-change impacts on cultural heritage sites. To date, a limited number of methodologies for managing climate-change impacts on heritage sites have been developed internationally. Importantly these are not geared to a bottom-up planning process. By contrast, many generic adaptation decision-support tools exist that support bottom-up planning. These tools commonly begin with a scoping phase. The scoping phase of a tool that supports Indigenous rangers manage climate-change impacts on heritage sites is described. A validation model, consisting of central assumptions behind each element of the scoping phase, is then set out. Future testing in the field would involve assessment of the tool through confirmation or otherwise of these assumptions. The first two assumptions in the validation model are then addressed: that Indigenous rangers perceive climate-change impacts on heritage sites and that planning for them is a priority need. Previous literature has not addressed these questions in detail. Only if positive responses are gained for these foundational assumptions can future testing of the tool be justified. Results from preliminary fieldwork undertaken in northern Australia found Indigenous rangers in two out of three case studies perceive impacts on heritage sites, and regard addressing these impacts as a priority.
Effects of fenced seed production areas and restoration treatments on the size and composition of the native grass seedbanks in moderately degraded rangelands in semiarid Australia
Authors: Bean Judith M. , Melville Gavin J. , Hacker Ronald B. , Anderson Sharon , Whittington Alicia , Clipperton Stephen P.
Published: November 2018

Seed production areas containing populations of native grasses were fenced on topographically high areas at two study sites in the semiarid rangelands of north-west New South Wales, Australia. The surrounding slopes were initially almost bare of pastorally preferred grasses. Three restoration treatments (control, mechanical pitting, and contour-aligned branch piles) were established on these surrounding slopes and the size and composition of the native grass seedbanks determined. A positive influence of the seed production areas mostly occurred within 15 m of the fence and was probably insignificant beyond 33 m at both sites. On a hard-setting red earth site, the size of the native grass seedbank in all three treatments increased over 2 years with the greatest increase under the piles of branches and smaller increases in the pitting and control treatments. The piles of branches preferentially incorporated seed of the pastorally preferred species Monachather paradoxus Steud into the seedbank whereas pits preferentially incorporated seed of the pastorally unpreferred species Aristida jerichoensis (Domin) Henrad. Evidence generally pointed to incoming seed rain rather than the seedbank as the main source of new seedlings. On a medium-textured lithosol site neither the pits nor the branches treatment was effective in enhancing either the size or composition of the seedbank; in fact the piles of branches resulted in a decrease in its size.
Cytogenetics and Reproductive Biology of some BELE (Abelmoschus manihot Linn., Medic Sub-Species manihot) Cultivars
Authors: Tyagi Anand P.
Published: November 2018

Four locally grown (from Fiji Islands) and three imported (from Papua New Guinea), a total of seven BELE (Abelmoschus manihot Linn., Medic sub-species manihot) cultivars were investigated for their cytogenetics and reproductive biology. Chromosome counting from flower buds and root tips showed that chromosome number in all the seven cultivars does not exceed 2n=66. Pollen viability in all the seven cultivars was high, exceeding 85% from pollen staining technique and 78% from pollen germination technique. All the seven Bele cultivars were fully self-compatible. Cross-compatibility among four local cultivars and three cultivars from Papua New Guinea was very high. However cross compatibility between local (Fijian) cultivars and cultivars from Papua New Guinea was partial indicating some degree of genetic difference between cultivars from two countries. This could be due to differences in compatibility alleles between cultivars from Fiji and Papua New Guinea. Procedures such as cutting the style shorter and placing pollen grains to enhance and affect fertilisation to get cross seed were suggested.
Pollen Fertility Status in Coastal Plant Species of Rotuma Island
Authors: Rigamoto Reijieli R. , Tyagi Anand P.
Published: November 2018

and Abstract The Island of Rotuma is facing widespread destruction and degradation of its flora. This investigation was conducted to determine the pollen fertility status in thirty two species of economic and environmentally important coastal species, which are reproducing freely thus most suitable to rehabilitate coastal areas. Pollen fertility status in these species varied from as low as 38% in Canavalia cathertica to as high as 100% in Hernandia nymphaeifolia and Vigna marina. It was interesting to note that species within same genus showed wide range of pollen viability. However, most of the species investigated had high pollen viability especially in those species, which grow close to sea. It was recorded that pollen fertility was low in small fragmented populations. This showed the effect of population size on reproduction. Larger sized populations showed high percentage of pollen fertility.