Determination of furaltadone and nifursol residues in poultry eggs by liquid chromatography-electrospray ionization tandem mass spectrometry

The use of nitrofurans as veterinary drugs has been banned from intensive animal production in the European Union (EU) since 1993. The objective of the present study was to evaluate the accumulation and depletion of furaltadone and nifursol and their side-chain metabolites 5-methylmorpholino-3-amino-2-oxazolidinone (AMOZ) and 3,5-dinitrosalicylic acid hydrazide (DNSAH) in eggs after administration of therapeutic and subtherapeutic doses of the drugs to laying hens during three consecutive weeks. LC-MS/MS, with positive and negative electrospray ionization methods, was used for the determination of parent compounds and metabolites in yolk and egg white and was validated according to criteria established by Commission Decision 2002/657/EC. The decision limit (CCα) and the detection capability (CCβ) of the analytical methodology for metabolites were 0.1 and 0.5 μg/kg for AMOZ and 0.3 and 0.9 μg/kg for DNSAH, respectively. For the parent compounds, CCα and CCβ were 0.9 and 2.0 μg/kg for furaltadone and 1.3 and 3.1 μg/kg for nifursol, respectively. The data obtained show that the parent compounds are much less persistent than their side-chain metabolites in either yolk or egg white. Between the studied metabolites, AMOZ is the most persistent and could be detected in either yolk or egg white three weeks following withdrawal from treatment.     

高效液相色谱法检测泰乐菌素在肉鸡组织中的残留

将肉鸡的肌肉、肝脏、肾脏和脂肪组织制成匀浆,用乙腈、石油醚和二氯甲烷萃取、净化后,用乙腈定容。以乙腈－ 甲醇－ ０．００５ ｍ ｏｌ／ Ｌ磷酸二氢铵溶液（６０∶３０∶１０）为流动相,用高效液相色谱检测。本法对溶解于乙腈中的泰乐菌素的最低检测限为 ０．０５ ｍ ｇ／ Ｌ,在肉鸡各种组织中的最低检测限为 ０．１０ ｍ ｇ／ Ｌ。标准曲线的线性范围为 ０．１０～６．４０ ｍ ｇ／ Ｌ。泰乐菌素质量浓度为 ０．１０、０．８０、６．４０ ｍ ｇ／ Ｌ 时的批间变异系数分别为（２．３±０．２）％ 、（１．９±１．３）％ 、（２．６±０．９）％ 。０．２０、０．４０、０．８０ ｍ ｇ／ Ｌ 的泰乐菌素在以上 ４ 种组织的回收率均符合残留检测的要求。结果表明,本法是一种灵敏、精密、可靠的检测肉鸡组织中泰乐菌素残留的分析方法。     

Bovine Oocyte Quality in Relation to Ultrastructural Characteristics of Zona Pellucida,Polyspermic Penetration and Developmental Competence

In the present study, the effect of bovine oocyte quality related to ultrastructural characteristics of zona pellucida (ZP), polyspermic penetration and embryo developmental competence was evaluated. Cumulus–oocyte complexes were punctioned from 453 ovaries, classified as 1, 2, 3 and 4 according to their morphological aspect, matured for 24 h and then divided into two groups. In group A, oocytes were fixed in 2.5% glutaraldehyde and 0.1 m sodium cacodylate and examined under a scanning electron microscope. Photomicrographs were taken and ZP’s pores were evaluated in squares of 6.4‐μm width. In group B, oocytes were fertilized in vitro. After 48 h, non‐cleaved oocytes were fixed for polyspermy evaluation. On days 7, 9 and 10, embryos were classified as developed (blastocysts and hatched blastocysts). Results showed that quality 1 oocytes revealed a ZP pore diameter of 0.50 ± 0.07 μm, which was smaller than the observed on oocytes of quality 2 (0.83 ± 0.10 μm), quality 3 (1.02 ± 0.22 μm) and quality 4 (1.38 ± 0.59 μm) (p ≤ 0.05). For In Vitro Fertilization (IVF), results showed that embryos originating from oocytes classed as 3 and 4 had lower cleavage rate (68.4% and 43.8%) than those belonging to class 1 and 2 (79.5% and 69.3%) (p ≤ 0.05). None oocyte classified as 3 and 4 developed to hatch blastocysts, while for oocytes belonging to quality 1 and 2, these values were, respectively, 15.2% and 12.5%. Concerning polyspermy, oocytes class 1 and 2 had lower polyspermic penetration than those belonging to class 3 and 4 (respectively 4.1%, 4.5%, 11.1% and 9.8%, for class 1, 2, 3 and 4). In conclusion, the present study demonstrated that oocytes with low qualities result in lower developmental competence and with high percentage of polyspermy after IVF, which can be the result of the ZP structure such as the number and the pore’s diameter.     

Transmission electron microscopy for characterization of acrosomal damage after Percoll gradient centrifugation of cryopreserved bovine spermatozoa

The objective of this study was to characterize acrosomal ultrastructure following discontinuous Percoll gradient centrifugation of cryopreserved bovine sperm. Semen was collected from six bulls of different breeds and three ejaculates per bull were evaluated. Frozen semen samples were thawed and the acrosomal region of sperm cells was evaluated by transmission electron microscopy (TEM) before (n = 18) and after (n = 18) Percoll centrifugation. The evaluation of 20 sperm heads from each of the 36 samples analyzed ensured that a large number of cells were investigated. The data were subjected to analysis of variance at a level of significance of 5%. Percoll centrifugation reduced the percentage of sperm exhibiting normal acrosomes (from 61.77 to 30.24%), reduced the percentage of sperm presenting atypical acrosome reactions (from 28.38 to 4.84%) and increased the percentage of sperm exhibiting damage in the acrosome (from 6.14 to 64.26%). The percentage of sperm with typical acrosome reactions was not significantly different before (3.70%) and after (0.67%) centrifugation. TEM distinguished four different types of acrosomal status and enabled ultrastructural characterization of acrosomal injuries. The percentage of sperm exhibiting normal acrosomes decreased and damage in the acrosome was the most frequent acrosomal injury with the Percoll gradient centrifugation protocol utilized.     

A longitudinal study of gastrointestinal parasites in Canadian dairy farms. The value of an indirect Ostertagia ostertagi ELISA as a monitoring tool

The general objective of this study was to evaluate a crude Ostertagia ostertagi antigen enzyme-linked immunosorbent assay (ELISA) for monitoring gastrointestinal parasites in lactating dairy cattle. A longitudinal study of gastrointestinal parasites in lactating dairy cows was carried out in 38 herds in four provinces of Canada (Prince Edward Island, Quebec, Ontario and Saskatchewan) from September 1999 to October 2000. Bulk tank milk, cow milk, serum and fecal samples were collected monthly or quarterly from all these farms. Information on herd management factors was collected by a standard questionnaire and individual cow production data were obtained from an electronic database. The overall mean optical density ratio (ODR) was 0.30 and ranged from -0.05 to 1.55. Although a clear seasonal pattern was not observed, the ODR values tended to decrease during the housing period and start increasing in the spring before the cows went out to pasture. The second and third or greater lactation cows had significantly higher ODR values compared with first lactation animals. The individual cow ODR had a very low correlation with individual squared root fecal egg counts but showed a reasonably high correlation when herd averages values were computed (r=0.73). A moderate correlation (r approximately 0.50) between the bulk tank and herd average ODR was observed. Milk yield was negatively associated with individual cow milk ODR and a quadratic effect on ODR was observed for days in milk. Twenty-eight of the herds participated in a clinical trial of eprinomectin (Ivomec Eprinex) treatment at calving. The cow level ODR values determined late in the previous lactation had a marginally significant effect (P=0.07) on treatment response, suggesting that high OD cows responded better to the anthelmintic treatment. However, because of the small sample size available in this model, more research is needed to better understand this relationship. In conclusion, the indirect ELISA using an O. ostertagi crude antigen appears useful as a technique for monitoring gastrointestinal parasite burdens in adult dairy cows and holds promise as a potential predictor of response to anthelmintic treatment.     

Application of a plant bioassay for the evaluation of ecotoxicological risks of heavy metals in sediments affected by mining activities

Purpose

The objective of this work was to evaluate the effectiveness of a plant bioassay (Phytotoxkit®) for screening ecotoxicological risks in sediments affected by mining activities.

Materials and methods

A total of 42 sediment samples affected by mining activities were studied, including 39 sediment samples from the Sierra Minera, Spain, an area affected by old extraction procedures, and three sediments from an area affected by opencast mining. These three samples were then mixed with limestone filler at 10, 20 and 30 %, providing nine stabilised samples. The total and soluble metal(loid) content (As, Cd, Cu, Fe, Pb and Zn) was determined in all samples, and the Phytotoxkit® bioassay was applied to determine the ecotoxicological effect of this procedure.

Results and discussion

The stabilised material had a neutral pH and low soluble metal(loid) concentration, similar to that of samples in which a natural attenuation process had taken place because of mixing with surrounding carbonate-rich materials. An ecotoxicological survey identified the low toxicity levels of the stabilised samples.

Conclusions

The applied bioassay is a good tool for screening metal(loid) contamination in areas affected by mining activities, since it provides information on both natural and simulated attenuation processes. The mixing of sediments with limestone filler could be applied to the remediation of zones affected by mining activities, because the toxicological effect on the tested organisms in the stabilised sediments was reduced significantly and the metal(loid) content was diminished.