All animals used in this study were Indian-origin rhesus macaques housed indoors at the Oregon National Primate Research Center (ONPRC), an AAALAC-accredited facility. Monkeys were provided with a 12-h light cycle (07:00–19:00) year-round with natural humidity and temperatures were kept between 7 °C (45°F) and 30 °C (85°F), per the Animal Welfare Act. All rhesus macaques were fed a commercial monkey chow twice daily (6–7 biscuits/meal according to weight; LabDiet 5000, Purina Mills, Saint Louis, MS, USA) and given fresh produce or other food enrichment daily. Animals had ad libitum access to water. Additionally, the monkeys’ cognitive welfare was supported by the ONPRC Behavioral Management Plan which includes enrichment items such as toys, foraging devices and enrichment, frozen treats, TV, radio, mirrors, and removable verandas.
The study included 11 adult males (5 to 16 years old and weight 7.4–14.2 kg) that were part of the Assisted Reproductive Technologies (ART) Core pool of males. Therefore, these males had proven fertility through ongoing IVF studies. All macaques were born at the ONPRC and were considered specific pathogen free (SPF) for simian retrovirus, simian immunodeficiency virus, simian T-cell leukemia virus, and macacine herpesvirus 1. They were given a clinical health evaluation prior to sample collection and monitored throughout the study, with no evidence of pre-existing or acquired disease. All males were housed in pairs, unless the Behavioral Services Unit deemed them incompatible with other monkeys. All animal procedures were pre-approved by the Institutional Animal Care and Use Committee at the Oregon Health & Science University, followed the National Institute of Health’s Guide for the Care and Use of Laboratory Animals, and complied with the ARRIVE guidelines.
Semen collection and processing
All semen collections were performed by penile electroejaculation without sedation using a PTE 110 Volt AC electroejaculator (P–T Electronics, Model 303, Boring, OR) as previously described13. Ejaculate was collected directly into a wide mouth container and sat at 37 °C for 30 min to allow for liquefaction. The liquid portion of the ejaculate was transferred into a 15 mL conical tube. The coagulated plug was washed with 3 mL of TALP-HEPES (supplemented with 3 mg/ml of bovine serum albumin)14 to recover remaining spermatozoa then combined with the liquid fraction before adding additional TALP-HEPES Q.S. to 12 mL. Samples were washed twice by centrifugation at 300×g for 7 min at 37 °C. After removal of the supernatant (11 mL), the sperm pellet was resuspended in the remaining 1 mL of TALP-HEPES and analyzed by a computer assisted sperm analysis (CASA) system (IVOS II-Animal Motility software, version 1.11, Hamilton Thorne, Beverly, MA) for sperm concentration and motility13. After analysis, aliquots containing 250,000 spermatozoa were placed in separate 0.6 ml centrifuge tubes and snap frozen in liquid nitrogen for storage in a − 80 °C freezer until analysis. On the day of analysis, samples were thawed in a water bath at 37 °C for 30 s.
Validation of the assay
We initially ran a test to generate a validation curve as described previously15. Samples were processed as described above and aliquoted into four separate tubes—two tubes were processed as positive controls and the other two were processed as negative controls. The negative control samples underwent the standard treatment, as follows. Each sample was mixed with 150 µl of TNE buffer (Tris-Hydrochloric acid 0.01 M, NaCl 0.15 M, EDTA 1 mM; pH 7.4) and 300 µl of acid detergent (HCl 0.08 M, NaCl 0.15 M, Triton X-100 0.1%; pH 1.2), then incubated for 30 s before adding 900 µl of acridine orange solution (Citric acid 0.1 M, Na2HPO4 0.2 M, EDTA 0.001 M, NaCl 0.15 M, Acridine orange 6 µg/ml in Millipore water; pH 6.0). The positive control samples were initially treated with 300 µl of strong acid detergent (HCl 1.2 M, pH 0.1) for 1 min to induce 100% DFI, followed by 150 ul of TNE buffer. The samples were then centrifuged at 200×g for 5 min, after which the supernatant (350 µl) was removed and 900 µl of AO were added. The positive and negative control samples were mixed at three different ratios 1:3, 1:1, 3:1 to produce a curve; the assay was deemed valid only if R2 ≥ 0.95.
Percent DNA fragmentation index (% DFI)
After initial validation of the assay, semen samples were collected from each male (N = 11) to determine a reference range of DNA fragmentation index in rhesus macaques. The samples underwent the standard treatment described above for the negative control. The samples were run through a flow cytometer (BD LSR II Cell Analyzer, BD Biosciences, San Jose, CA 95,131) using a 488 nm excitation wavelength. Fluorescence of individual cells was collected using a FITC filter (505–525 nm) to measure green fluorescence (native or intact DNA) and an APC filter (650–670 nm) to measure red fluorescence (broken DNA). Raw data were exported to FlowJo (version 10.8.1; FlowJo, LLC; Ashland, OR 97,520). After gating out debris and dead cells, a frequency histogram (with red fluorescence, APC, on the X-axis) was used to determine the % DFI, the percent of total sperm cells that had high red fluorescence3.
Statistical analyses were conducted using Graphpad Prism version 9.3.1. Data were reported as mean ± standard deviation (SD). Linear regressions were performed on the validation curve data.