Chronic dietary exposure to a glyphosate-based herbicide alters ovarian functions in young female broilers

Abstract

Glyphosate (GLY)-based herbicide (GBH) formulations are widely used pesticides in agriculture. The European Union recently decided to extend the use of GLY in Europe until 2034. Previously, we showed that chronic dietary GBH exposure in adult hens resulted in a reversible increase in early mortality in chicken embryos. In this present study, we investigated the GBH effects on metabolism and ovarian functions by using a transcriptomic approach in vivo in young female broilers and in vitro in ovarian explant cultures. We exposed 11-day-old female broilers to 13 mg GLY equivalent/kg body weight/d (GBH13, n = 20), 34 mg GLY equivalent/kg body weight/d (GBH34, n = 20), or a standard diet (control [CT], n = 20) for 25 d. These 2 GBH concentrations correspond to approximatively one-eighth and one-third of the no observed adverse effect level (NOAEL) as defined by European Food Safety Authority in birds. During this period, we evaluated body weight, fattening, food intake, and the weight of organs (including the ovaries). Chronic dietary GBH exposure dose dependently reduced food intake, body weight, and fattening, but increased oxidative stress and relative ovary weight. We analyzed the ovarian gene expression profile in CT, GBH13, and GBH34 broilers with RNA sequencing and showed that differentially expressed genes are mainly enriched in pathways related to cholesterol metabolism, steroidogenesis, and RNA processing. With quantitative polymerase chain reaction and western blotting, we confirmed that GBH decreased ovarian STAR and CYP19A1 messenger RNA and protein expression, respectively. Furthermore, we confirmed that GBH altered steroid production in ovarian explants. We have identified potential regulatory networks associated with GBH. These data provide valuable support for understanding the ovarian transcriptional regulatory mechanism of GBH in growing broilers.

Keywords: food intake; glyphosate; ovary; transcriptomic approach; young broiler.

Figure 1

Experimental design. Eleven-day-old female broilers were exposed to glyphosate (GLY)-based herbicide (GBH) in the diet for 25 d, corresponding to a dose of 13 mg GLY equivalent/kg body weight/d (the GBH13 group, n = 20) or 34 mg GLY equivalent/kg body weight/d (the GBH34 group, n = 20). The control group (CT, n = 20) received a normal diet. Food intake was determined each day. On d 1 (D1), D9, D16, and D25 of the experiment, the broilers were weighed; on D9 and D25, an ultrasonographic examination was used to estimate the abdominal adipose tissue thickness; and on D25, blood was collected for oxidative stress assessment and the GLY and aminomethylphosphonic acid (AMPA) assays. All the animals were euthanized to collect tissues on D25.

Figure 2

(A) The effect of chronic dietary exposure to 2 glyphosate (GLY)-based herbicide (GBH) concentrations on food intake in growing broilers. Eleven-day-old female broilers were exposed to GBH in the diet for 25 d, corresponding to a dose of 13 mg GLY equivalent/kg body weight/d (the GBH13 group, n = 20) or 34 mg GLY equivalent/kg body weight/d (the GBH34 group, n = 20). The control group (CT, n = 20) received a normal diet. The daily food consumption is shown as a percentage of the total food given (mean ± standard error of the mean). The P-values for the time (days), GBH exposure, and the time × GBH interaction are presented. The daily concentration of ingested equivalent (B) GLY and (C) AMPA (mg/kg body weight) in the CT, GBH13, and GBH34 broilers (n = 20 per group). The results are presented as mean ± the standard error of the mean. The P-values for the time (days), GBH exposure, and the time × GBH interaction are presented. The evolution of (D) body weight (g) on d 1 (D1), D9, D16, and D25 of the experiment; (E) fat thickness (mm) on D9 and D25; and (F) the feed conversion ratio (FCR) on D9, D16, and D25 in the CT, GBH13, and GBH34 broilers (n = 20 per group). The results are presented as the mean ± the standard error of the mean. For each day, different letters indicate significant differences determined with 1-way analysis of variance followed by Tukey's honestly significant difference test for pairwise comparisons (P < 0.05).

Figure 3

The plasma (A) glyphosate (GLY, ng/L) and (B) aminomethylphosphonic acid (AMPA, ng/L) concentrations and the thiobarbituric acid-reactive substances (TBARS) index in the control (CT), GBH13, and GBH34 broilers on d 25 of the experiment. Eleven-day-old female broilers were exposed to GBH in the diet for 25 d, corresponding to a dose of 13 mg GLY equivalent/kg body weight/d (the GBH13 group, n = 10) or 34 mg GLY equivalent/kg body weight/d (the GBH34 group, n = 10). The CT group (n = 10) received a normal diet. The results are presented as the mean ± standard error of the mean. Different letters indicate significant differences determined with one-way analysis of variance followed by Tukey's honestly significant difference test for pairwise comparisons (P < 0.05).

Figure 4

The weight of the (A) ovaries, (B) abdominal adipose tissue, and (C) spleen relative to the body weight in the control (CT), GBH13, and GBH34 broilers on d 25. Eleven-day-old female broilers were exposed to GBH in the diet for 25 d, corresponding to a dose of 13 mg GLY equivalent/kg body weight/d (the GBH13 group, n = 20) or 34 mg GLY equivalent/kg body weight/d (the GBH34 group, n = 20). The CT group (n = 20) received a normal diet. The results are presented as the mean ± the standard error of the mean. Different letters indicate significant differences determined with 1-way analysis of variance followed by Tukey's honestly significant difference test for pairwise comparisons (P < 0.05).

Figure 5

Venn diagram of differentially expressed genes in the ovaries of control (CT), GBH13, and GBH34 broilers on d 25. Eleven-day-old female broilers were exposed to GBH in the diet for 25 d, corresponding to a dose of 13 mg GLY equivalent/kg body weight/d (the GBH13 group) or 34 mg GLY equivalent/kg body weight/d (the GBH34 group). The CT group received a normal diet.

Figure 6

(A) The top 3 enriched gene ontology (GO) terms of differentially expressed genes (DEG) in the ovaries for the control (CT, n = 5) vs. GBH34 (n = 5) comparison on d 25 of the experiment. The size of the dots is positively correlated with the number of DEGs in the pathway. (B) Gene interaction network of the DEGs in the CT and GBH34 ovaries. The network nodes and edges represent gene and gene–gene associations, respectively. The red and purple nodes indicate the upregulated and downregulated genes, respectively. The size of the dots is positively correlated with the number of DEGs in the pathway.

Figure 7

(A) The top 6 enriched gene ontology (GO) terms of differentially expressed genes (DEG) in the ovaries of the control (CT, n = 5) vs. GBH13 (n = 5) comparison. The size of the dots is positively correlated to the number of DEGs in the pathway. (B) Gene interaction network of the DEGs in the CT and GBH13 ovaries. The network nodes and edges represent gene and gene–gene associations, respectively. The red and purple nodes indicate the upregulated and downregulated genes, respectively. The size of the dots is positively correlated with the number of DEGs in the pathway.

Figure 8

(A) The top 10 enriched gene ontology (GO) terms of differentially expressed genes (DEG) in the ovaries of the GBH13 (n = 5) vs. GBH34 (n = 5) comparison. The size of the dots is positively correlated to the number of DEGs in the pathway. (B) Gene interaction network of the DEGs in the CBH13 and GBH34 ovaries. The network nodes and edges represent gene and gene–gene associations, respectively. The red and purple nodes indicate the upregulated and downregulated genes, respectively. The size of the dots is positively correlated with the number of DEGs in the pathway.

Figure 9

The (A) messenger RNA (mRNA, n = 8 per group) and (B) protein (n = 6 per group) expression of steroidogenic factors (STAR, HSD3B1, CYP11A1 and CYP19A1) in control (CT), GBH13, and GBH34 ovaries on d 25. Eleven-day-old female broilers were exposed to GBH in the diet for 25 d, corresponding to a dose of 13 mg GLY equivalent/kg body weight/d (the GBH13 group) or 34 mg GLY equivalent/kg body weight/d (the GBH34 group). The CT group received a normal diet. The results are presented as the mean ± the standard error of the mean. Different letters indicate significant differences determined with 1-way analysis of variance followed by Tukey's honestly significant difference test for pairwise comparisons (P < 0.05).

Figure 10

The effect of glyphosate-based herbicide (GBH) on in vitro steroid production by chicken ovarian explants. Conditioned culture medium was collected after 24-h exposure to GBH (0.000036–36 g glyphosate/L), and the (A) progesterone (Pg) and (B) estradiol (E2) concentrations were measured with enzyme-linked immunosorbent assays. (C) Total RNA was extracted from ovarian explants, and HSD3B1, STAR, CYP11A1, and CYP19A1 messenger RNA (mRNA) expression was determined. The data are expressed as the mean ± the standard error of the mean of 5 replicates (one replicate is representative of approximately 2 ovaries for each condition). Bars with different letters are significantly different (P < 0.05).