Effect of different mini‐volume colloid centrifugation configurations on flow cytometrically sorted sperm recovery efficiency and quality using a computer‐assisted semen analyzer

Straws of sex‐sorted sperm are usually packaged at a low concentration (e.g., ~2.1 × 10⁶ sperm/ml) and cost significantly more than unsorted conventional semen from the same sire. In order to maximize the efficiency of using sex‐sorted sperm under in vitro fertilization conditions, the selection of an appropriate sperm separation technique is essential. In this study, the effect of using different silane‐coated silica colloid dilutions and layering configurations during centrifugation of sex‐sorted sperm was examined over an extended period of incubation time. Sperm recovery and viability after centrifugation using the colloid separation technique were measured along with several sperm motility parameters using CASA. For this purpose, frozen and thawed sex‐sorted sperm samples were centrifuged using mini‐volume single‐layer (40%, 60% and 80%) and mini‐volume two‐layer (45%/90%, 40%/80% and 30%/60%) separation configurations using PureSperm^®. A single layer of 40% PureSperm^® recovered significantly more sex‐sorted sperm (78.07% ± 2.28%) followed by a single layer of 80% PureSperm^® (68.43% ± 2.33%). The lowest sperm recovery was obtained using a two‐layer PureSperm^® dilution of 45%/90% (47.57% ± 2.33%). Single‐layer centrifugation recovered more sorted sperm (68.67% ± 1.74%) than two layer (53.74% ± 1.74%) (p < .0001). A single layer of 80% PureSperm^® exhibited the highest sorted sperm viability (72.01% ± 2.90%) after centrifugation (p < .05). The mini‐volume single layer of 80% PureSperm^® was determined to be an effective alternative to a two‐layer centrifugation configuration for sex‐sorted sperm selection. In addition, single‐layer colloid dilution of 80% performed either as well as or significantly outperformed the other treatments, as well as the control, with regard to motility (MOT) for all time periods of analysis.     

Characterisation of porcine haemophili isolated from Australian pigs between 1988 and 1992

SUMMARY A total of 362 haemophili, isolated from pigs throughout Australia, were characterised by phenotypic properties. Most were identified as Actinobacillus pleuropneumoniae (296 isolates) or Haemophilus parasuis (52 isolates). The remaining isolates were identified as Haemophilus Taxon ‘minor group’ (12 isolates) and Haemophilus Taxon D (two isolates). All 296 A pleuropneumoniae isolates were serotyped by slide agglutination and/or gel diffusion, using rabbit antisera against all 12 recognised serovars. Of these, only 156 (52.7%) could be assigned to a single serovar as follows: serovar 1–85 isolates, serovar 2–4 isolates, serovar 3–2 isolates, serovar 5–10 isolates, serovar 7–51 isolates, serovar 11–2 isolates and serovar 12–2 isolates. Of the remaining 140 isolates, 91 gave cross-reactions with serovars 3 and 6, one cross-reacted with serovars 9 and 10, one cross-reacted with serovars 9 and 11 whereas 47 gave no reaction with any of the antisera.     

Comparative Aspects of the Endotoxin- and Cytokine-Induced Endocrine Cascade Influencing Neuroendocrine Control of Growth and Reproduction in Farm Animals

Disease in animals is a well-known inhibitor of growth and reproduction. Earlier studies were initiated to determine the effects of endotoxin on pituitary hormone secretion. These studies found that in sheep, growth hormone (GH) concentration was elevated, whereas insulin-like growth factor-I (IGF-I) was inhibited, as was luteinizing hormone (LH). Examination of the site of action of endotoxin in sheep determined that somatotropes expressed the endotoxin receptor (CD14) and that both endotoxin and interleukin-Iβ activated GH secretion directly from the pituitary. In the face of elevated GH, there is a reduction of IGF-I in all species examined. As GH cannot activate IGF-I release during disease, there appears to be a downregulation of GH signalling at the liver, perhaps related to altered nitration of Janus kinase (JAK). In contrast to GH downregulation, LH release is inhibited at the level of the hypothalamus. New insights have been gained in determining the mechanisms by which disease perturbs growth and reproduction, particularly with regard to nitration of critical control pathways, with this perhaps serving as a novel mechanism central to lipopolysaccharide suppression of all signalling pathways. This pathway-based analysis is critical to the developing novel strategies to reverse the detrimental effect of disease on animal production.     

Development of a monoclonal antibody against deoxynivalenol for magnetic nanoparticle-based extraction and an enzyme-linked immunosorbent assay

Monoclonal antibody (mAb, NVRQS-DON) against deoxynivalenol (DON) was prepared. DON-Ag coated enzyme linked immunosorbent assay (ELISA) and DON-Ab coated ELISA were prepared by coating the DON-BSA and DON mAb. Quantitative DON calculation ranged from 50 to 4,000 ng/mL for DON-Ab coated ELISA and from 25 to 500 ng/mL for DON-Ag coated ELISA. 50% of inhibitory concentration values of DON, HT-2, 15-acetyl-DON, and nivalenol were 23.44, 22,545, 5,518 and 5,976 ng/mL based on the DON-Ab coated ELISA. Cross-reactivity levels of the mAb to HT-2, 15-acetyl-DON, and nivalenol were 0.1, 0.42, and 0.40%. The intra- and interassay precision coefficient variation (CV) were both <10%. In the mAb-coated ELISA, mean DON recovery rates in animal feed (0 to 1,000 µg/kg) ranged from 68.34 to 95.49% (CV; 4.10 to 13.38%). DON in a buffer solution (250, 500 and 1,000 ng/mL) was isolated using 300 µg of NVRQS-DON and 3 mg of magnetic nanoparticles (MNPs). The mean recovery rates of DON using this mAb-MNP system were 75.2, 96.9, and 88.1% in a buffer solution spiked with DON (250, 500, and 1,000 ng/mL). Conclusively we developed competitive ELISAs for detecting DON in animal feed and created a new tool for DON extraction using mAb-coupled MNPs.     

Preliminary Study in Immature Canine Oocytes Stained with Brilliant Cresyl Blue and Obtained From Bitches with Low and High Progesterone Serum Profiles

This study was conducted: (i) to observe the features and levels of blue colour impregnation in morphologically selected immature canine cumulus oocyte complexes (COCs) stained with the brilliant cresyl blue (BCB) dye, as indicators of quality, and integrity of nuclear oocyte chromatin configuration before in vitro maturation (IVM); (ii) to observe the relationship between the influence of serum progesterone (SP) concentrations from ovary donors and BCB staining of immature dog oocytes. The results showed that out of 138 canine COCs, germinal vesicle (GV) stage prevailed in BCB+ oocytes at percentages of 67.4% (60/89), which were statistically higher than those observed in BCB+/− (52.2%; 23/44) and BCB− (20%; 1/5) oocytes (p = 0.023). Oocytes BCB+ were interpreted as those having completed their growth and therefore possessing the capacity to mature and develop in vitro . Ooplasm and cumulus cells (CCs) of canine oocytes were BCB staining independent. Ooplasm blue colour staining reaction varied between grown oocytes, revealing different levels of glucose-6-phosphate dehydrogenase activity among and within oocytes. Additionally, SP profile of ovary donors was not a relevant indicator for selection of oocytes screened with the BCB stain. Similar numbers of high quality oocytes were observed to be BCB+, BCB+/− and BCB− between groups of females with SP varying from 0 to 2.5 ng/ml (n = 5), and those with SP varying from 2.6 to 16.7 ng/ml (n = 4) (p = 0.680). It may be inferred that bitches with low and high SP profiles have grown oocytes in their ovaries, as determined by the BCB absorbance in their ooplasms.     

Chemical Usurpation of a Nest by Paper Wasp Parasites

The paper wasp Polistes atrimandibularis is an obligatory social parasite of another Polistes species, P. biglumis bimaculatus. To control the host nest, the parasite sequentially changes the composition of its chemical signature, the cuticular hydrocarbons, during the colonial cycle. Gas chromatography-mass spectrometry of the cuticular hydrocarbons at every stage of the cycle showed that the parasite can switch on and off an entire chemical family, namely, the unsaturated hydrocarbons. In this way the parasite can match the host signature at a critical moment of the colonial cycle.     

Identification and Biochemical Characterization of Halisulfate 3 and Suvanine as Novel Inhibitors of Hepatitis C Virus NS3 Helicase from a Marine Sponge

Hepatitis C virus (HCV) is an important etiological agent that is responsible for the development of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV nonstructural protein 3 (NS3) helicase is a possible target for novel drug development due to its essential role in viral replication. In this study, we identified halisulfate 3 (hal3) and suvanine as novel NS3 helicase inhibitors, with IC₅₀ values of 4 and 3 µM, respectively, from a marine sponge by screening extracts of marine organisms. Both hal3 and suvanine inhibited the ATPase, RNA binding, and serine protease activities of NS3 helicase with IC₅₀ values of 8, 8, and 14 µM, and 7, 3, and 34 µM, respectively. However, the dengue virus (DENV) NS3 helicase, which shares a catalytic core (consisting mainly of ATPase and RNA binding sites) with HCV NS3 helicase, was not inhibited by hal3 and suvanine, even at concentrations of 100 µM. Therefore, we conclude that hal3 and suvanine specifically inhibit HCV NS3 helicase via an interaction with an allosteric site in NS3 rather than binding to the catalytic core. This led to the inhibition of all NS3 activities, presumably by inducing conformational changes.     

The Recipients' Parity Does Not Influence Their Reproductive Performance Following Non‐Surgical Deep Uterine Porcine Embryo Transfer

With the development of the non‐surgical deep uterine (NsDU) embryo transfer (ET) technology, the commercial applicability of ET in pigs is now possible. There are, nevertheless, many factors that influence NsDU‐ET effectiveness that need to be addressed. The aim of this study was to evaluate the effects of the weaned recipients' parity on fertility and prolificacy following NsDU‐ET. The recipients (n = 120) were selected based on their reproductive history and body condition and grouped into three categories according to their parity: primiparous sows, sows of parity 2 and sows of parities from 3 to 5. Thirty fresh embryos (morulae and unhatched blastocysts) were non‐surgically transferred into one uterine horn of each recipient. It was possible to insert the NsDU‐ET catheter through the cervix along a uterine horn in 98.3% of the recipients. The parity had no influence on the difficulty grade of the insertions or on the percentage of correct insertions. The cervix and uterine wall were not perforated during the insertions, and vaginal discharge was not observed after transfer in any of the recipients. There were no differences in the pregnancy rates (74.8%), farrowing rates (71.2%) or litter sizes (9.6 ± 3.3) between groups. Also, there were no differences between groups regarding to the piglets' birthweights or piglet production efficiency. In conclusion, these results demonstrate that weaned sows from parity 1 to 5 are appropriate to be used as recipients in NsDU‐ET programs, which increase the possibilities for the utilization of ET in the recipient farms.