High throughput phenotyping for aphid resistance in large plant collections

ABSTRACT: BACKGROUND: Phloem-feeding insects are among the most devastating pests worldwide. They not only cause damage by feeding from the phloem, thereby depleting the plant from photo-assimilates, but also by vectoring viruses. Until now, the main way to prevent such problems is the frequent use of insecticides. Applying resistant varieties would be a more environmental friendly and sustainable solution. For this, resistant sources need to be identified first. Up to now there were no methods suitable for high throughput phenotyping of plant germplasm to identify sources of resistance towards phloem-feeding insects. RESULTS: In this paper we present a high throughput screening system to identify plants with an increased resistance against aphids. Its versatility is demonstrated using an Arabidopsis thaliana activation tag mutant line collection. This system consists of the green peach aphid Myzus persicae (Sulzer) and the circulative virus Turnip yellows virus (TuYV). In an initial screening, with one plant representing one mutant line, 13 virus-free mutant lines were identified by ELISA. Using seeds produced from these lines, the putative candidates were re-evaluated and characterized, resulting in nine lines with increased resistance towards the aphid. CONCLUSIONS: This M. persicae-TuYV screening system is an efficient, reliable and quick procedure to identify among thousands of mutated lines those resistant to aphids. In our study, nine mutant lines with increased resistance against the aphid were selected among 5160 mutant lines in just 5 months by one person. The system can be extended to other phloem-feeding insects and circulative viruses to identify insect resistant sources from several collections, including for example genebanks and artificially prepared mutant collections.     

The pre-emptive effect of epidural ketamine on wound sensitivity in horses tested by using von Frey filaments

Objective To evaluate the pre‐emptive analgesic effect of pre‐incisional epidural ketamine. Study Design A blinded, randomized experimental study. Animals Sixteen mixed breed mares, 7.6 ± 2.8 years old, weighing 352 ± 32 kg. Methods In a pilot study, an incision was made on one lateral thigh using a lidocaine block and no further analgesics, and it was verified that the nociceptive threshold was lower on the incised side than nonincised side (p ≤ 0.05), and that von Frey filaments evoked a pain response. The 16 animals were divided into group A (ketamine, n = 9) and B (saline, n = 7). An epidural catheter was inserted 24 hours before the trials. The thigh was shaved bilaterally, and the right side was blocked (incised side) using lidocaine. Twenty‐five minutes later, ketamine (A) or saline (B) was administered epidurally. Five minutes later, a 10‐cm skin incision was made on the right side, and then sutured. Nociceptive threshold was determined with von Frey filaments at 1, 3, and 5 cm around the incision at 15‐minute intervals for 2 hours, then at 4, 6, and 8 hours. Behavioral alterations, heart and respiratory rates were recorded. Nociceptive thresholds from these points were averaged to obtain mean values at each time, converted to a logarithmic scale, and submitted to a nonparametric analysis (Mann–Whitney and one‐way repeated measures anova test, p ≤ 0.05). Results After 8 hours, the global range score revealed reduced hyperalgesia (p < 0.01) around the incision in 92% (4.65–4.27) of evaluated intervals in group A (ketamine). There were no significant changes in behavior, heart and respiratory rates. Conclusions It was concluded that pre‐emptive epidural ketamine reduced post‐incisional pain in the horse, and that von Frey filaments were able to quantify cutaneous sensitivity after tissue damage. Clinical relevance Epidural ketamine injection can reduce post‐incisional sensitivity in the horse.     

Postpartum Variations of Plasma IGF and IGFBPs,Oocyte Production and Quality in Dairy Cows: Relationships With Parity and Subsequent Fertility

The aim of this study was to determine whether postpartum variations of plasma IGF‐1 and IGFBP concentrations, oocyte production and quality were related to parity and subsequent conception rate in Holstein dairy cows. Holstein dairy cows [10 primiparous (PP) and 22 multiparous (MP)] were allotted in six batches and sampled once weekly between calving and oestrous synchronization treatment started at 71.2 ± 2.0 days postpartum. During the 3 weeks before treatment, ovum pick‐up (OPU) was performed twice weekly. Oocytes were scored on a 4‐point scale, and oocytes from OPU1, 3 and 5 were fertilized in vitro. Seventeen cows became pregnant after first and second AI and were considered as fertile (F), while the others were considered to be subfertile (SF). Logistic regression was carried out to investigate the relationships between repeated measurements and fertility including parity and batch effects in the models. Likelihood of fertility significantly increased when plasma urea and IGFBP‐3 concentrations decreased and was higher in PP compared with MP cows. There was a trend for fertility to increase when plasma IGF‐1 concentrations increased (p = 0.07). In vitro cleavage and development rates were similar between SF and F cows (46.4% and 28.3% in SF vs 55.0% and 22.1% in F). Parity had an effect on plasma IGF‐1 concentrations (PP: 61.65 ± 2.67 vs MP: 41.63 ± 5.81 ng/ml, p < 0.001), mean number of follicles aspirated per session (PP: 5.7 ± 1.3 vs MP: 9.5 ± 0.8, p < 0.05) and fertility (PP: 8/10 = 80% vs MP: 9/22 = 41%, p < 0.05) but not on the number of oocytes recovered per session nor their quality. In conclusion, postpartum plasma urea and IGFBP‐3 concentrations, but not oocyte production and quality before breeding, were related to subsequent conception rate in our experimental design. Parity had a significant effect on energy status, follicular growth and fertility and needs to be considered when investigating relationships between nutrition and reproduction.     

Environmental mastitis in intensive high‐producing dairy herds in New South Wales

Objective To determine the prevalence of mastitis pathogens in high‐producing intensive dairy herds in New South Wales. Design Field survey. Procedure Milk samples from the mastitis‐affected quarter were collected from cows on five high‐producing dairy farms in NSW. The 820 samples were cultured using standard microbiological culture techniques. Results Bacteria or fungi were isolated from 83.3% of samples (683/820). More than two colony types were isolated from 16.7% of samples (137/820), two types from 6.6% (54/820), and one type from 52.3% (429/820). No bacteria were isolated from 24.4% (200/820) of the primary cultures, but enrichment cultures of these samples yielded single colony type bacterial isolates from 36.5% (73/200) of samples. Environmental pathogens, including coliforms, environmental Streptococcus and Staphylococcus spp., made up 91% (555/610) of isolates and accounted for 33.6% (205/610), 41.6% (254/610) and 15.7% (96/610), respectively, of isolates. Escherichia coli accounted for 76.1% (156/205) of the coliform isolates, Streptococcus uberis and Streptococcus dysgalactiae accounted for 32.3% (82/254) and 28.0% (71/254), respectively, of the environmental streptococcal isolates. Contagious pathogens were uncommon, comprising only 2.5% (15/610) of the total isolates. Conclusion The incidence and causes of mastitis are largely influenced by farm management. The relatively high prevalence of coliform mastitis in the intensive high‐producing herds in this survey contrasts with the low incidence reported in surveys of pasture‐based herds in Victoria. If the Australian dairy industry continues its current trend of intensification, coliform intra‐mammary infections may emerge as an increasingly important cause of mastitis.     

Effect of Pre‐Treatment of Ovine Sperm on Male Pronuclear Formation and Subsequent Embryo Development Following Intracytoplasmic Sperm Injection

The objective of this study was to determine the effects of various methods of sperm pre‐treatment on male pronuclear (MPN) formation and subsequent development of ovine embryos derived from in vitro‐matured oocytes and intracytoplasmic sperm injection (ICSI). The effect of treatment of injected oocytes with dithiothreitol (DTT) on embryo development was also assessed. In Exp. 1, the injected oocytes with non‐treated sperm were activated with three different procedures. The cleavage and blastocyst rates in those activated with DTT was lower (p < 0.05) than those activated with either ionomycin (Io) + 6‐dimethylaminopurine (6‐DMAP) or DTT + I + 6‐DMAP. In Exp. 2, the effects of sperm pre‐incubated with DTT, sodium dodecyl sulphate (SDS) or DTT + SDS as well as two‐time frozen/thawed sperm (without cryoprotectant) on MPN formation and oocyte activation were examined. The non‐treated sperm served as controls. The MPN formation in DTT + SDS group was higher (p < 0.05) than other groups except for freeze–thaw group. No difference in the rate of activated ICSI oocytes was observed among groups. In Exp. 3, the effect of pre‐treatment of sperm on subsequent development of ICSI embryos and blastocyst cell numbers were examined. The rates of cleavage and blastocyst formation as well as the blastocyst cell numbers were similar among the pre‐treated and control groups. In conclusion, pre‐treatment of sperm with DTT + SDS positively affected MPN formation, although the subsequent development capacity of the resulting embryos remained limited. Moreover, DTT was not effective on oocyte activation compared with Io + 6‐DMAP after ICSI.     

Black soil blindness: A new mycotoxicosis of cattle grazing Corallocytostroma-infected Mitchell grass (Astrebla spp)

A new, fatal mycotoxicosis of cattle has been recognised in north-western Australia. A feeding trial confirmed the toxicity of a previously unknown species of Corallocytostroma that grows on Mitchell grass (Astrebla spp). The disease has been colloquially named ‘black soil blindness’ because its most prominent features are its confinement to pastures on black soil, and blindness and death of affected animals. Over 500 cattle have died and considerable subclinical disease is present. Above average wet season rainfall and extended growing seasons may explain the emergence of the fungus. The disease is important because cattle production in large areas of Australia utilise Mitchell grass pastures.     

Vascular leakage induced by histamine, bradykinin, serotonin and prostaglandin E2 in Greyhounds

SUMMARY Vascular leakage induced by intradermal injection of histamine, bradykinin and serotonin alone and co-injected with prostaglandin E₂ was measured in Greyhounds using ¹²⁵Iodine-labelled human serum albumin (¹²⁵I-HSA) as a marker in the blood. Histamine and bradykinin produced dose-dependent vascular leakage. At equimolar concentrations, histamine was more than twice as potent as bradykinin. Serotonin did not induce vascular leakage and was irritant. Prostaglandin E₂ did not induce significant vascular leakage (maximum 5μL) when injected alone, but when co-injected with histamine and bradykinin, the vascular leakage of both histamine and bradykinin was increased. This effect was more pronounced if lower concentrations of histamine and bradykinin were injected. The induced vascular leakage was greatest during the first five minutes of lesion development for histamine, during the second five minutes of lesion development for bradykinin, and the synergistic effect of prostaglandin E₂ was maximal during the third five minute period of lesion development.     

Effect of oestrous synchrony between embryo donors and recipients,embryo quality and state on the pregnancy rate in beef cattle

This study aimed to evaluate the effect of oestrous synchrony between donors and recipients and the embryo quality on the pregnancy rate in beef cow recipients. The experiment was performed over two years at an embryo transfer (ET ) centre in Southern Brazil. Ninety Aberdeen Angus cows were subjected to superovulation (SOV ) protocols, resulting in the recovery of 1,048 transferable embryos. Eleven groups were formed with intervals of 6 hr, from ?30 to +30 hr, with respect to recipient versus donor oestrous detection. Evaluation of embryo quality was according to the IETS guidelines. The overall pregnancy rate was 52%. Effects related to donor and recipient oestrous synchronization on pregnancy rate were observed (p  =  .01), ranging from 36% to 50%. The embryo quality rate affected the pregnancy rate, where Grade I resulted in 57% and Grade III in 43% of pregnancy (p  <  .001). The embryonic state (frozen or fresh) showed no (p  >  .05) effect on pregnancy rate: 53% for fresh embryos and 44% for frozen embryos. The odds ratio for explanatory variables causing pregnancy indicated that Grade III embryos had 31% less chance of conception compared to Grade I. Thus, oestrous synchrony between donor and recipient, considering ±30 hr apart, can affect the pregnancy rate along with embryo quality.