Male exposure to environmental toxicants can disrupt spermatogenesis and impair sperm function. However, the consequences of environmentally relevant levels of toxicants to ejaculated mammalian spermatozoa on sperm function and male fertility are not well studied. Tributyltin chloride (TBT) is an organotin with historical use as an antifouling agent in paints and is a contaminant of soil and groundwater in the United States. Tributyltin chloride is an endocrine disruptor, is detectable in human cord blood, and has negative effects on female reproduction. We hypothesised that TBT could affect sperm function and thereby affect male fertility. To test our hypothesis, we exposed frozen-thawed bull sperm to environmentally relevant doses of TBT (0, 0.1, 1.0, 10, and 100nM) for 90min and then measured sperm motility parameters, fertilisation, and embryo development by IVF. Briefly, frozen-thawed sperm from two bulls were isolated through a 45:90 Percoll gradient, pooled, and then maintained in noncapacitating conditions at 37°C in Tyrode's albumin lactate pyruvate medium devoid of bovine serum albumin and HCO3
− for 90min. Vehicle control (VC) samples consisted of 0.1% MeOH. Sperm motility kinematics were objectively measured after the addition of treatment and every 30min thereafter using computer-aided sperm analysis (IVOS System, Hamilton Thorne). Five replicates were evaluated, and differences in motility kinematics were analysed by analysis of variance using SAS statistical software (SAS Institute Inc.). Sperm treated with 100nM TBT displayed decreased total motility (88 vs. 79%), progressive motility (80 vs. 70%), curvilinear velocity (100 vs. 88 µ/s), and beat-cross frequency (38 vs. 34Hz) over 90min compared with the VC samples (P<0.05). No differences (P>0.05) were detected among any other treatments. Following 90min of exposure to TBT 100nM, sperm were washed twice by centrifugation and re-extended in fertilisation medium. Abattoir-derived bovine oocytes were fertilised with 100nM TBT and VC-exposed sperm. Embryo cleavage and 8- to 16-cell embryos were quantified at 48 and 72h, respectively, in three replicates, and results were assessed using chi-square analysis. Embryos fertilised by TBT-exposed sperm had reduced cleavage to 2-cell (80 vs. 62%) and 8- to 16-cell morulae stages (56 vs. 24%, respectively; P<0.05). In summary, although sperm kinematics were decreased in TBT-exposed sperm, gross motility parameters remained within acceptable ranges for IVF, suggesting that sperm motility alone is not a sufficient measure of sperm function or indicator of male fertility. In conclusion, ejaculated bull sperm exposed to environmentally relevant levels of TBT for 90min had reduced sperm motility parameters, impaired sperm function, and reduced embryo development potential.
Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under award number T32HD087166. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Objective assessment of bull sperm motility parameters using computer vision algorithms