Global genomic methylation linked to the degree of parasitism in cattle

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The experiment was conducted in accordance with the ethical guidelines for experimentation on animals of the Ethics and Animal Welfare Committee of the Faculty of Agricultural and Technological Sciences of the State University of São Paulo “Júlio de Mesquita Filho” (UNESP), Dracena (FCAT/UNESP) . The study was approved by the COMMISSION FOR ETHICS IN THE USE OF ANIMALS – CEUA of the UNESP College of Agriculture and Technology under registration number 13/2017.

Seventy-two first-cross ½ Angus × ½ Nellore heifers from the same homogeneous herd of Nellore cows artificially inseminated at a fixed time from the semen of a single Aberdeen Angus bull were used. This cross is currently the most used in Brazil, given the high demand for this type of animal due to its precocity, the quality of its carcass and its adaptation to the climatic conditions of the country.14.

The heifers remained in the same environment from birth to the end of the study, as well as their mothers, who had the same genetic characteristics and were kept in the same lot and under the same conditions throughout gestation and of lactation. The animals received only antiparasitic treatment two months before the start of the evaluations, started at weaning at the age of 8 months, comprising levamisole 18.8% injected subcutaneously at a dose of 4.7 mg /kg of body weight for the control of gastrointestinal helminths, and cypermethrin (7 mg/kg), applied to the dorsal line, for the control of ectoparasites; no further antiparasitic treatment was administered the following year.

The design used was completely randomized, considering each animal as a treatment (72 treatments) with 11 repetitions, referring to the number of samples taken. Subsequently, the number of eggs per gram of faeces (EPG), the number of ticks and the number of horn flies were subjected to statistical analysis to divide the heifers into three groups by statistical difference (p

Resistant animals are those that manage to eliminate or suppress the development of parasites, without presenting a parasite load or very low. Susceptible animals are animals with a high parasite load, whose performance is impaired by parasites. Resilient animals are those that have a parasite load but suffer little parasite damage5.

The degree of parasitism was assessed by collecting faeces and counting horn flies and ticks every 28 days. The faeces were taken directly from the rectum of the animals for coprological examinations, carried out at the Laboratory of Parasitology and Animal Health of the Faculty of Agronomic and Technological Sciences, by counting the eggs per gram of faeces (EPG) using a McMaster bedroom15. Stool culture and larval extraction were also performed16 for further identification of the genera present therein17.

The counting of horn flies consisted of driving the heifers into the corral for the quantification of their cervico-dorsal-lumbar region during the milder periods of the day (in the morning).18. Tick ​​assessment was performed by quantifying engorged females equal to or greater than 4.5 mm in size on one side of the animal and then multiplying by two19.

Global DNA extraction and methylation

Total genomic DNA was extracted from blood, collected during the last evaluation, using the EasyPureBlood Genomic DNA Kit (Transgen Biotch, Beijing, China). Sample quantification and quality assessment were performed using a spectrophotometer (NanoDrop2000 – Thermo Scientific).

Methylation analysis was performed using the Imprint DNA Methylation Quantification Kit (Sigma), using strips with pretreated wells containing methylated DNA binding reagent and using a capture antibody sensitive to DNA methylation and a detection antibody, allowing the colorimetric detection of the absolute amount of DNA methylation in each animal. The absorbance of the solution contained in the wells was measured on an ELISA spectrophotometer (Kasuaki-DR-200Bs-BI) at a wavelength of 450 nm.

statistical analyzes

Distributions of EPG, tick and horn fly numbers were verified by the Shapiro-Wilk test using SISVAR 5.420.

EPG, tick, and horn fly counts were analyzed separately in a fully randomized design, including 72 treatments and 11 replicates for each collection; means of each variable were subjected to analysis of variance (ANOVA) and subsequent statistical pooling analysis by Scott–Knott test (5%) using SISVAR 5.420. Thus, the animals of the herd were, according to the degree of parasitism based on the statistical difference of the means of each animal, grouped into three categories for each variable: resistant, resilient or susceptible.

General classification based on degree of parasitism was performed using Selegen software21, and a dendrogram was then created using the hierarchical clustering method (Ward’s method) of the 72 heifers by considering the three variables simultaneously (EPG, ticks and horn fly counts); genetic distances were calculated22. Thus, the animals were also classified into three groups: resistant, resilient and susceptible, and the mean counts of the three variables were analyzed by ANOVA using SISVAR 5.4.20, then the means were compared by Tukey’s test (p

Statistical analysis of global DNA methylation was performed in a fully randomized design, with three treatments (resistant, resilient, or susceptible) and 72 replicates, using absolute absorbance values ​​and mean difference values. of methylation between the animals transformed into (sqrt[2]{n+0.5})where n is the absolute absorbance values ​​of methylation, which were analyzed by ANOVA using SISVAR 5.420 assess the statistical significance between the different methylation values ​​of the three groups; the number of gastrointestinal helminths, ticks and horn flies was analyzed by Tukey’s test (5%).

Thanks to the study, it was possible to evaluate two different forms of classification by statistical analysis, the first classifying animals using the SISVAR program according to the degree of parasitism caused by helminths, ticks and flies horns separately, and the second classifying animals by the GENES program through a hierarchical grouping that considers the degree of parasitism by helminths, ticks and flies, correlating them with DNA methylation, simultaneously.

Compliance with ethical standards

The authors affirm that all procedures contributing to this work comply with the ethical standards of the Ethics and Animal Welfare Committee of the Faculty of Agricultural and Technological Sciences of the State University of São Paulo “Júlio de Mesquita Filho “, Dracena, SP (FCAT/UNESP), approved under registration number 13/2017. All animal experiments followed ARRIVE guidelines.

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