Test Subjects

The experiment involved the participation of 23 self-identified heterosexual men between the ages of 18 and 23 years old. Each subject provided seven ejaculate samples over the course of 15 days. The recruitment and procedures involving collection of semen samples were all in accordance with the College of Wooster’s Human Subjects Research Committee guidelines. Although we knew the identities of the participants, which remained confidential, all other information, including personal health issues and semen parameters, was anonymous. Prior to beginning the experiment, each subject was assessed for healthiness and history of sexual dysfunction, as defined by the World Health Organization (2010), using a questionnaire. Men were only included in the study if they self-reported to have no history of sexual dysfunction, no conditions affecting testicular health, and no sexually transmitted infections and were not taking any medications, were not regular smokers of tobacco or any other substance, and did not chew tobacco regularly. Prior to beginning the experiment, each participant refrained from alcohol consumption and sexual activity for 48 h. Before each ejaculate collection, participants completed a survey self-reporting the number of alcoholic drinks they had consumed and whether they had ejaculated since the last collection. If they reported having more than four alcoholic drinks or they ejaculated between collections, the participants’ data were not included in the study. One participant chose to leave the study and his data were excluded. Another participant’s data were excluded because he ejaculated between collections, thus reducing our sample size to 21.

Collection Procedure

We used segments of sexually explicit films involving one actor and one actress as the stimuli for the experiment. Each participant viewed the films in the same private room, at approximately the same time of day every 48 to 72 h. For each participant, all sessions were scheduled in advance at the beginning of his participation period. Each film segment consisted of a 3-min section from a longer ~20-min film. This 3-min segment was played on repeat for as long as necessary to achieve ejaculation. Of the seven film segments each participant viewed, the first six comprised the habituation period. Each of these six film segments featured the same man and woman but differed in respect to the sexual acts performed. The seventh film segment comprised the novel female treatment. This film segment featured the same man, but with a different woman who differed distinctly from the first woman in facial and body features, hair color, and tattoos. Across the study, four different 20-min films were used. Each man was assigned to a particular pair of 20-min films, and the segments he viewed were extracted from these films. The specific films used as the habituation film and the novel stimulus film for each participant were randomly assigned. The specific segments from those films that the participant viewed on a particular collection day were also randomized.

Each participant was left alone in a private room to watch the provided film and masturbate into a wide-mouth sterile collection cup. He was instructed to record what time he began watching the film, what time he ejaculated, and whether all of the ejaculate was deposited in the collection cup. If some ejaculate failed to be deposited into the cup, the data from the ejaculate were not used for calculations of volume-based measures (see below).

Semen Assays

We performed all semen assays blind to the identity and the treatment group (familiar or novel stimulus) of the participant and within an hour after ejaculation. Ejaculate volume was measured to the nearest 0.1 mL using a 15 mL graduated conical tube. Once in the tube, the sample was gently vortexed and placed into 1.5 mL Eppendorf tubes. The samples were then allowed to liquefy for 20 min. After vortexing one of the 1.5 mL tubes again to make the sample homogenous, 5 μL of the sample were loaded on a Microcell counting chamber (Vitrolife, San Diego, CA) to record sperm concentration and motility. Using a phase contrast microscope at 20× objective and an eyepiece video camera, we filmed five arbitrarily chosen fields of view close to the center of the counting chamber slide for a minute each.

To determine sperm concentration and percent motility, we counted total sperm number and total immotile sperm. We used Adobe Illustrator and Adobe Photoshop to place a 10 × 10 grid on top of the sperm videos. For each of the five fields of view of each sample that were videotaped, we counted the total number of sperm and the number of immotile sperm within 1–10 rows (10–100 squares) of the grid. The number of rows counted depended on how concentrated the sample was with sperm. We only counted one to two rows for each field of view for highly concentrated samples (average of 16–32 sperm cells per row). If samples were moderately concentrated, five rows were counted (average of eight sperm cells per row). If there were fewer than five sperm cells in a row, all ten rows were counted. The number of rows counted for each of the five fields of view for a given sample remained consistent.

Sperm concentration was calculated by multiplying the average total sperm counts for the five fields of view by the row factor (1 row = 10; 2 rows = 5; 5 rows = 2; 10 rows = 1) and the magnification factor of the microscope (MF = 20) and then dividing by 100, the total number of grid cells. Total sperm count was calculated as the product of sperm concentration and ejaculate volume. Sperm motility for each of the five fields of view was calculated as the total number of motile sperm divided by the total number of sperm, multiplied by 100. The average of these five values was used as the percent sperm motility measure for the ejaculate sample. Total motile sperm was calculated as the product of total sperm count and the percent sperm motility.

Statistical Analyses

We tested for differences between the initial trial and the habituated trial (trials 1 and 6) and between the habituated trial and the novel stimulus trial (trials 6 and 7) for all of the measured variables using either one-tailed paired t tests (after natural log transformation for time to ejaculation) or one-tailed Wilcoxon signed-rank tests (total motile sperm and concentration) in SPSS version 22. For all ejaculate measures (sperm parameters and volume), we predicted a decrease with habituation and an increase upon exposure to a novel stimulus. For time until ejaculation, we predicted an increase with habituation and a decrease upon exposure to a novel stimulus. Data from some participants were excluded from the volume, total motile sperm, and total sperm comparisons because some of their ejaculate missed the collection container (four men excluded in the trial 1 vs. 6 comparison, two men excluded in the trial 6 vs. 7 comparison). Data from one additional participant was excluded from all analyses due to extremely low sperm concentration (<1 million sperm per mL). Additionally, we compared each ejaculate parameter across trials 2 through 6 to determine whether there were any changes across the habituation period. To do so, we used a repeated-measures ANOVA for the parameters that were normally distributed and had homogeneity of variances across these trials (volume and natural log transformed time to ejaculation) and pairwise Wilcoxon tests (trial 2 vs. trial 3, trial 3 vs. trial 4, etc.) for all of the other parameters (reported in Supplemental Tables 1 and 2).