Vasalgel devices consisted of 25% solutions by weight of SMA in dimethyl sulfoxide (DMSO). The average molecular weight (Mw) of the SMA anhydride was 330 kDa based on standardized gel permeation chromatography (GPC) methodology (Jordi Labs, Mansfield, MA, USA). The SMA acid was made by hydrolysis of the anhydride and had a Mw of 360 kDa. One device contained only SMA acid and is referred to as Vasalgel 100. A second device contained a mixture of 80% SMA acid and 20% SMA anhydride by weight and is referred to as Vasalgel 80. The final devices were prepared and packaged in a nitrogen atmosphere in 4 ml glass vials by Polysciences, Inc. (Warrington, PA, USA).
Subjects, housing and care
The study was performed using 7 mature azoospermic male New Zealand White rabbits (Harlan Laboratories, Oxford, MI) from the Vasalgel contraceptive efficacy study. The animals averaged 1.9 years of age (SD = 0.07 weeks) and weighed an average of 4.6 kg (SD = 0.49 kg) (see  for details of care and housing). Three mature females were used as teasers for the collection of semen. All animal procedures were approved by the Loyola University Medical Center, (Maywood, IL) Institutional Animal Care and Use Committee.
Baseline semen sample collections were obtained prior to implantation of the Vasalgel device during the contraceptive efficacy study . All rabbits received bilateral vas deferens implants of Vasalgel 100 (n = 2) or Vasalgel 80 (n = 5). Fourteen months (SD = 0.75 months) after implant the azoospermic males underwent a reversal procedure to remove the Vasalgel and return patency to the vasa deferentia. Semen collections began two weeks following the procedure and continued for six months to establish the presence and quality of spermatozoa.
Implantation of devices
Rabbits were injected with the devices as previously described . Briefly, the vasa deferentia of anesthetized rabbits were externalized and injected with approximately 100–120 μl of the device in about 30–40 s using a 24 gauge 1.6 cm catheter (Quik-Cath by Baxter, Deerfield, IL). 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.
Animals were weighed, administered 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 suture marking the site of implantation was identified.
A 19 gauge needle was used to enter the vasa deferentia approximately 0.3 cm from the Vasalgel injection site (towards the epididymis). A two lumen 24 gauge 15 cm in length catheter was then inserted approximately 0.5 cm into the vas deferens and advanced to a maximum distance of approximately 8 cm when resistance to further insertion occurred. The catheter was then withdrawn about 1 cm and a 20 ml syringe attached to the outer lumen of the catheter to flush the vasa with sodium bicarbonate solution (2 M, pH 8.0, Hospira, Lake Forest, IL, USA). The central lumen provided a path for any off gassing due to the reaction of Vasalgel and the bicarbonate as well as any excess fluid being instilled. Gentle pressure was initially applied to the syringe until resistance was encountered, the pressure stopped for about 15–30 s and then pressure again applied. This process was repeated several times until the gentle pressure on the syringe allowed unrestricted flow of the bicarbonate through the vas. The number of repeated cycles varied between vasa and the total volume required prior to initially obtaining unrestricted flow was about 2 to 5 ml of the bicarbonate solution. A maximum of 10 ml was injected.
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 . Semen specimens were evaluated for volume, total sperm count, motility and forward progression using manual methods.
Euthanasia and necropsy
Rabbits were euthanized using standard procedures at the conclusion of the study and necropsied with particular attention to the reproductive tract.
The vasa deferentia from treated animals were harvested and immersed in 10% neutral buffered formalin for fixation. The tissues were processed, sectioned and stained with hematoxylin and eosin utilizing standard methods for evaluation.
Data were summarized by subject, device group (Vasalgel 100 and Vasalgel 80) and condition (baseline and post-reversal) for each sperm measurement. The reversibility of the device was evaluated by comparing the mean sperm parameters (sperm concentration, forward progression and motility) for each subject by condition and by device. Initial evaluation of the data set revealed deviations from normal distribution. Thus, nonparametric tests were applied to determine any difference between the Vasalgel 100 and Vasalgel 80 groups (Mann Whitney U test) and evaluate any difference between the measures at baseline vs. post-reversal (Wilcoxon Matched Pairs test) using Statistica (StatSoft, Inc., Tulsa, OK, USA). A significance level of p < 0.05 was determined. Data are presented as mean ± standard deviation.