Eimeria maxima M6 oocysts were provided by Dr. John. R. Barta, University of Guelph, Canada. The methods for detecting and recovering oocysts from challenged chickens, oocyst sporulation, and the preparation of infective doses were conducted as described previously45,46.
Animal source and experimental design
Forty one-day-old male SPF chickens (ALPES® Tehuacan, Puebla, Mexico) were randomly allocated to one of two groups with four replicates per group (n=5 chickens/replicate). Chickens were placed in battery cages with a controlled age-appropriate environment at the diagnostic laboratory of the Avian Medicine Department of the Faculty of Veterinary Medicine and Zootechnics (FMVZ) at the National Autonomous University of Mexico (UNAM). Groups consisted of the Control (no challenge) or the Challenge group (E. maxima). Chickens were provided with ad libitum access to water and unmedicated feed. At day 28 of age, all chickens in the challenge group were orally gavaged with 40,000 sporulated E. maxima oocysts in a volume of 1 mL of sterile phosphate-buffered saline solution (PBS). The dose was selected based on a previous trial conducted to determine a challenge dose causing sub-clinical coccidiosis as described previously13. Negative control chickens were sham inoculated with 1 mL of PBS. Seven days after challenge, all chickens were bled, and half of them were euthanized to collect the second half of the jejunum to determine plasma and enteric concentrations of isoprostane 8‐iso‐PGF2α and PGF2α. At 9 days post-challenge, the rest of the chickens in both groups were bled and jejunum was collected to perform the evaluations. Oocysts per gram (OPG) of feces were evaluated at 7- and 9-days post-challenge.
The standards for 8‐iso‐PGF2α and 8‐iso‐PGF2α‐d4
The standards for 8‐iso‐PGF2α and 8‐iso‐PGF2α‐d4 (internal standard) were purchased from Cayman Chemicals (Ann Arbor, MI), while the standard for PGF2α was obtained from Sigma-Aldrich (St Louis, MO). Acetonitrile and methanol (HPLC grade) were purchased from JT Baker. Milli‐Q water (Millipore system) was used throughout the experiments. Formic acid (FA: 95%, reactive grade) and isopropanol (LC/MS grade) were purchased from Sigma-Aldrich (St Louis, MO). Ammonium hydroxide (NH4OH, reactive grade, 29.60%) and potassium hydroxide (KOH) were purchased from JT Baker. For solid‐phase microextraction (micro‐SPE), 96‐well Oasis® MAX μElution cartridges containing a water‐wettable reversed‐phase strong ammonium exchange mixed‐mode polymer, which is selective for acids and stable in organic eluents, were used. A Positive Pressure‐96 processor purchased from Waters was also used. Figure 1 shows the chromatograms of standards.
Procedure for the extraction of 8-iso-PGF2α and PGF2α in chicken plasma
Extraction of 8-iso-PGF2α and PGF2α were determined as previously described47. An aliquot of 500 µL chicken plasma was transferred to 2 mL vials, followed by the addition of 100 µL of 4 ng/mL 8‐iso‐PGF2α‐d4 as an internal standard and 500 µL of hydrolysis solution (KOH, 15%) to release 8-iso-PGF2α-esterified. The vials were mixed and incubated in an ultrasonic bath for 30 min at 40 ºC. Subsequently, the vials were cooled to room temperature and 225 µL of 6M formic acid (FA) was added, mixed, and centrifuged at 15000 rpm for 10min at 4 ºC. Solid‐phase microextraction using a 96‐well Oasis® MAX μElution plate conditioned with 500 μL of methanol and 500 μL of 20 mM FA was used. Finally, the cartridges were loaded with 350 μL of plasma and washed with 350 μL of 2% NH4OH. Samples were then eluted with 50 μL of a mixture of 5% FA in acetonitrile and isopropanol (40:60) and diluted with 150 μL of Milli‐Q water. Samples were analyzed (30 μL) using ultra‐performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS).
Procedure for the extraction of 8-iso-PGF2α and PGF2α in chicken intestine
For the extraction of 8-iso‐PGF2α and PGF2α, 0.1 g of homogenized second half of the jejunum (Meckel’s diverticulum to cecal tonsils) were transferred to 2 mL vials, followed by the addition of 100 µL of 4 ng/mL 8‐iso‐PGF2α‐d4 as the internal standard and 1.5mL of chloroform: methanol (80:20) mixture. The vials were mixed 30s by vortex and 15 min in an ultrasonic bath. Samples were then centrifuged at 15000 rpm for 20 min. The supernatant was evaporated and 500 µL of methanol and 500 µL of hydrolysis solution (KOH 15%) were added, mixed, and incubated in an ultrasonic bath for 30 min at 40 °C. Subsequently, the vials were cooled to room temperature and 225 µL of 6 M formic acid (FA) and 50 µL of 88% FA were added, mixed, and centrifuged at 15000 rpm for 10min at 4 ºC. Solid‐phase microextraction and analysis of samples were performed in the same way as for the determination of 8-iso-PGF2α and PGF2α in chicken plasma using a 96‐well Oasis® MAX μElution plate conditioned with 500 μL of methanol and 500 μL of 20 mM FA. Finally, the cartridges were loaded with 350 μL of jejunum sample and washed with 350 μL of 2% NH4OH. Samples were then eluted with 50 μL of a mixture of 5% FA in acetonitrile and isopropanol (40:60) and diluted with 150 μL of Milli‐Q water. The sample (30 μL) was injected into a UPLC-MS/MS system for analysis, under the chromatographic and mass spectrometric conditions described previously by Rodriguez Patiño et al47.
This study was carried out in accordance with the recommendations for the management of chickens as recommended by the Internal Committee for Care and Use of Experimental Animals (CICUAE, from its abbreviation in Spanish) of the National Autonomous University of Mexico (UNAM), Ethical approval code CICUAE: C20_06, and the study is in compliance with the ARRIVE guidelines where animals are involved.
Quantification of oocysts
The quantification of OPG from feces was performed at 7- and 9-days post-challenge by using the McMaster technique as previously described45.
Data and statistical analysis
PGF2α and 8-iso-PGF2α data are presented as means with standard deviation (S.D.). The number of samples per variable group was 20, implying a normal distribution (Shapiro-Wilk test), and the homoscedasticity was verified (Levene's test). Accordingly, the parametric test of one-tailed Student's t-test was performed, and the P value was established with an alpha level of P <0.01. OPG data are presented as means with median and variance. The number of samples per variable group was 20; however, the hypotheses of normal distribution (Shapiro-Wilk test) and homoscedasticity (Levene's test) were not confirmed. Consequently, non-parametric tests of the one-tailed Wilcoxon signed-rank test was applied with an alpha level P <0.0548.