Vasalgel test articles consisted of 25 % solutions by weight of SMA in DMSO. The average molecular weight (Mw) of the SMA anhydride (Poly(Styrene-co-Maleic Anhydride, CAS Registry Number: 9011-13-6) was 330 kDa according to standardized gel permeation chromatography (GPC) methodology (Jordi Labs, Mansfield, MA, USA). The SMA acid (Poly(styrene-alt-maleic acid, CAS Registry Number: 25736-61-2) was made by hydrolysis of the anhydride and had a Mw of 360 kDa. Test article #1 contained only SMA acid (referred to as “100 % acid”). Test article #2 was prepared by weighing appropriate amounts of the SMA acid and SMA Anhydride and to achieve a mixture of 80 % SMA acid and 20 % SMA anhydride by weight (referred to as “80:20 mix”) in DMSO. The final test articles were prepared and packaged in a nitrogen atmosphere in 2 ml glass vials by Polysciences, Inc. (Warrington, PA, USA). The 80:20 mix was selected to provide a comparison with the 100 % SMA Acid. This was based upon an evaluation of the material responses in the presence in saline. An increase in the amount of anhydride in the mix resulted in a less flexible and harder material which was not desired within the vas.
Subjects, housing and care
The studies were performed using 15 mature male and three mature female New Zealand White rabbits that averaged 26.2 weeks of age (SD = 0.37 weeks) and weighed an average of 3.7 kg (SD = 0.10 kg) on arrival from Harlan Laboratories, Oxford, MI. The animals were screened for health conditions and acclimated for 7 days. Housing and care protocols used recommendations of the National Centre for the Replacement, Refinement, and Reduction of Animals in Research . Rabbits were individually housed in stainless steel cages in temperature- and light- controlled rooms meeting the requirements specified in the FELASA publication entitled Euroguide  and fed Purina® Rabbit Chow™ (Purina Mills, St. Louis, MO, USA). The rabbits also had regular access to a play area within the same room, where they could socialize and exercise. All animals were checked at least twice daily for viability and general health during the entire period of the study. The female rabbits were only used as teaser animals when collecting semen specimens. They were housed in the same room as the male rabbits and were placed for adoption at the end of the study. All animal procedures were approved by the Loyola University Chicago Institutional Animal Care and Use Committee.
Baseline semen sample collection was attempted for all 15 male rabbits for an average of 6 weeks (SD = 2.7 weeks) prior to implantation of the Vasalgel device. All rabbits then received bilateral vas deferens implants of 100 % acid (n = 8) or 80:20 mix (n = 7). Semen collection began again at an average of 10.8 weeks (SD = 4.9 weeks) post implantation. Semen collection continued approximately weekly until azoospermia was observed. Semen was then collected twice monthly until approximately 12 months post injection.
Implantation of test articles
Animals were weighed, given an antibiotic (Baytril® [Bayer Healthcare, KS, USA] 5 mg/kg) and then anesthetized with an intramuscular injection of xylazine HCl (4 mg/kg) and ketamine HCl (50 mg/kg) and a subcutaneous injection of acepromazine maleate (1.0 mg/kg). A 1 cm suprapubic transverse incision was made in the midline approximately 2 cm cephalad to the pubic symphysis. The spermatic cords were brought up through the incision and isolated. The cremasteric fascia was incised in a longitudinal fashion and the vas deferens isolated with its blood supply.
The isolated right and left vasa deferentia were elevated and injected with approximately 100–120 μl of test article in about 30–40 seconds using a 24 gauge 1.6 cm catheter (Quik-Cath by Baxter, Deerfield, IL). This volume of material was expected to fill the vas deferens to an approximate length of 2 cm based on preliminary studies in euthanized rabbits. The catheter was then removed, the vasa deferentia gently compressed for about 30 s and the vasal muscularis at the site of injection identified with a 6-0 Prolene suture. The vas deferens was returned to the spermatic cord and the site closed with 4-0 nylon sutures. The rabbits were given antibiotic Baytril® (5 mg/kg) (Bayer Healthcare, KS, USA) once per day for 7 days post-operatively and pain medication buprenorphine HCl (0.2 ml) every eight hours for 72 h post-operatively. All surgical procedures were performed by the same surgical team.
Semen collection and evaluation
Semen collections were performed using a warmed artificial vagina semen-collection device designed for use with the rabbit and a “teaser” female to encourage mounting (e.g., ). Semen specimens were evaluated for volume, total sperm count, sperm motility and forward progression using standard manual methods. Additionally, any observations of possible sperm were recorded when there were too few sperm to count using standard methods.
Euthanasia and necropsy
Rabbits that died during the 23 month study received a necropsy following standard protocol with particular attention to the reproductive tract.
The vas deferens from six animals were harvested and immersed in 10 % neutral buffered formalin for fixation. The vasa were from one animal that died three days after implant, three animals necropsied during the course of the study due to not providing semen samples or behavioral issues (Days 189, 214 and 248) and two after extended exposure to Vasalgel (Days 572 and 538). The tissues were processed, sectioned and stained with hematoxylin and eosin utilizing standard methods for evaluation.
Data were summarized by subject. The efficacy of the test article to block sperm was evaluated by comparing the mean sperm parameters (sperm concentration, forward progression and motility) for each subject by condition (baseline vs. post-implant) and by test article (100 % acid, 80:20 mix). Repeated measures analysis of variance using a 2 × 2 design was used to statistically test the condition x test article significance using the program Statistica (StatSoft, Inc. Tulsa, OK, USA). T-test for independent samples was conducted to determine any difference in sperm parameters at baseline. A significance level of p < 0.05 was determined. Data are presented as mean ± standard deviation.