Open access publications in the SLU publication database

Abstract

Di(2-ethylhexyl) phthalate (DEHP), a plasticizer used in polyvinyl chloride (PVC) products, has been reported to have toxic effects on animal reproduction. However, these reports come from studies mostly using rodents as test species and using doses higher than the doses humans are, presumably, exposed to. In the present thesis work young boars were used as model animals to assess the effects of pre-pubertal DEHP exposure on the quality of fresh and cryopreserved spermatozoa post-puberty. Pairs (n=8) of cross-bred male boar siblings were used, with one brother per pair becoming, at random, the test animal while the other was the control. Test males were orally exposed to DEHP (300 mg/kg body weight (bw)) three times per week from 3 to 7 weeks of age, while controls were given placebo. Semen collections and analyses started when the boars were 6 months old and continued until they were 9 months old. Ejaculates collected between 8 and 9 months of age were also cryopreserved for analyses post-thaw. Ejaculates were evaluated for sperm numbers, motility, morphology and plasma membrane integrity (PMI). Post-thaw spermatozoa were assessed for sperm motility, PMI, the ability to capacitate in vitro when exposed to the effector bicarbonate, and to acrosome-react when exposed to calcium ionophore (Ca-ionophore), the ability of the nuclear deoxyribonucleic acid (DNA) to sustain denaturation in vitro using a sperm chromatin structure assay (SCSA), and the ability of post-thaw spermatozoa to in vitro penetrate homologous, in vitro-matured (IVM) oocytes. The spermiogram did not significantly differ between exposed and control boars, except for sperm morphology. Boars exposed to DEHP had fewer (p<0.05) spermatozoa with tailless, defective heads (at 7–8 months of age) and double-folded tails (at 6–7, 7–8 and 6–9 months) than did controls. Regarding post-thaw spermatozoa, there were no differences in PMI between groups, but the DEHP-exposed boars had significantly fewer linearly motile spermatozoa at 30 (p<0.05) and 120 (p<0.001) minutes post-thaw, depicting also a larger amplitude of lateral head displacement (LHD) 120 minutes post-thaw (p<0.05). Proportions of capacitated post-thaw spermatozoa were similar between groups (control: 3.7%; DEHP-exposed: 4.4%), and exposure to bicarbonate had similar effects on capacitation of stable spermatozoa (control: 24.0%; DEHP-exposed: 22.1%). Live post-thaw spermatozoa from either group were acrosome-reacted in vitro to similar rates after exposure to Ca-ionophore (control: 9.3%; DEHP-exposed: 8.9%). Chromatin structure stability was similar between groups, with low proportions of spermatozoa showing induced DNA-denaturation (DNA fragmentation index, DFI; control: 0.15; DEHP-exposed: 0.17). In vitro sperm penetration rate did not significantly differ between groups (control: 59%; DEHP-exposed: 50%), nor did the number of spermatozoa in the ooplasm (control: 1.7; DEHP-exposed: 1.5). In summary, the DEHP exposure in the present studies did not cause obvious adverse effects on sperm production or sperm quality in boars. Cryopreservation was only able to disclose minor post-thaw sperm kinematic deviations in DEHP-exposed boars. However, DEHP did not seem to affect the ability of spermatozoa to capacitate or acrosome-react or to damage the nuclear genome, nor did it seem to affect their in vitro fertilizing ability.