Effects of Di-(2-Ethylhexyl) Phthalate (DEHP) on Gamete Quality Parameters of Male Koi Carp (Cyprinus carpio)

Abstract

In this study, we evaluated gamete quality parameters of mature male koi carp (Cyprinus carpio) exposed to three different concentrations (1, 10, and 100 µg/L) of di-(2-ethylhexyl) phthalate (DEHP). After 60 days of exposure, there was a significant decrease in the gonadosomatic index (GSI) of males exposed to 10 and 100 µg/L of DEHP. Histological analysis of the testes revealed impaired histoarchitecture, including inflammatory cells, intratubular vacuoles, and swollen seminiferous tubules in treatment groups. Gamete quality parameters like sperm production, motility, spermatocrit, and sperm density values were significantly decreased at the 10 and 100 µg/L concentrations. Biochemical compositions, including glucose, cholesterol, and total protein levels, were significantly changed in the treatment groups. Similarly, the ionic compositions of seminal fluid (Na, K, Ca, and Mg) also varied in the treatment groups. Furthermore, the 11-ketotestosterone levels were decreased, and the 17-β estradiol levels were increased in the DEHP-treated groups. The mRNA expression levels of reproduction-related genes, including Fshr, Lhr, Ar, Erα, and Erβ, were significantly changed in the DEHP-treated males in a dose-dependent manner. In conclusion, the findings of this study confirmed that environmentally relevant exposure to DEHP may contribute to a decline in the gamete quality of male fishes.

Keywords: di-(2-ethylhexyl) phthalate (DEHP); gamete quality; gene expression; koi carp (Cyprinus carpio); sex steroids.

Figure 1

GSI (%) of male mature koi carp exposed to nominal 1, 10, and 100 µg/L of DEHP for 60 days. Data are expressed as mean ± SEM (n = 6), and values with different superscript letters (a denotes the highest value, followed by b and c) significantly differ at 0.05 level.

Figure 2

Histopathological changes in testes of male mature koi carp exposed to nominal 1, 10, and 100 µg/L of DEHP for 60 days. Control group (a) shows intact seminiferous tubules (ST) with primary spermatocyte (PS), secondary spermatocyte (SS), and spermatids (S). Group treated with 1 µg/L of DEHP (b) shows intratubular vacuoles (IV) and inflammatory cells (IC). Group treated with 10 µg/L of DEHP (c) shows vacuolization (V) and inflammation (I). Group treated with 100 µg/L of DEHP (d) shows enhanced inflammation (EI) and necrosis (N).

Figure 3

Accumulation of DEHP (µg/g) in pooled samples of liver and testes tissue of male mature koi carp exposed to nominal 1, 10, and 100 µg/L of DEHP for 60 days. Data are expressed as mean ± SEM (n = 6), and values with different superscript letters (a denotes the highest value, followed by b, c, and d) significantly differ at the 0.05 level.

Figure 4

Semen volume (mL) of male mature koi carp exposed to nominal 1, 10, and 100 µg/L of DEHP for 60 days. Data are expressed as mean ± SEM (n = 6), and values with different superscript letters (a denotes the highest value, followed by b, c, and d) significantly differ at the 0.05 level.

Figure 5

pH value in semen samples of male mature koi carp exposed to nominal 1, 10, and 100 µg/L of DEHP for 60 days. Data are expressed as mean ± SEM (n = 6), and values with superscript letters (a) do not significantly differ at the 0.05 level.

Figure 6

Sperm motility (%) as well as motility durations (s) of male mature koi carp exposed to nominal 1, 10, and 100 µg/L of DEHP for 60 days. Data are expressed as mean ± SEM (n = 6), and values with different superscript letters (a denotes the highest value, followed by b, c, and d) significantly differ at the 0.05 level.

Figure 7

Spermatocrit values (%) of male mature koi carp exposed to nominal 1, 10, and 100 µg/L of DEHP for 60 days. Data are expressed as mean ± SEM (n = 6), and values with different superscript letters (a denotes the highest value, followed by b, c, and d) significantly differ at the 0.05 level.

Figure 8

Sperm density (× 10⁶/µL) of male mature koi carp exposed to nominal 1, 10, and 100 µg/L of DEHP for 60 days. Data are expressed as mean ± SEM (n = 6), and values with different superscript letters (a denotes the highest value, followed by b, c, and d) significantly differ at the 0.05 level.

Figure 9

Sex steroid levels of 17-β estradiol (a) and 11-keto testosterone (b) (pg/mL) of male mature koi carp exposed to nominal 1, 10, and 100 µg/L of DEHP for 60 days. Data are expressed as mean ± SEM (n = 6), and values with different superscript letters (a denotes the highest value, followed by b, c, and d) significantly differ at the 0.05 level.

Figure 10

mRNA expression levels of Fshr (a), Lhr (b), Ar (c), Erα (d), and Erβ (e) of male mature koi carp exposed to nominal 1, 10, and 100 µg/L of DEHP for 60 days. Data are expressed as mean ± SEM (n = 6), and values with different superscript letters (a denotes the highest value, followed by b and c) significantly differ at the 0.05 level.