The experiments described in this paper were conducted with the approval of the Monash University Department of Anatomy Ethics Committee and under the national animal in experimentation guidelines on the care and use of animals of the National Health and Medical Research Council of Australia. Also, Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines for reporting animal studies were adhered.
Animals
Fifteen mature male Sprague–Dawley rats (90 days old) were used for vas autograft surgery, and ten unoperated males served as controls. These animals were later mated with 30 female rats (two females: one male) to assess fertility (see “Fertility assessment” below).
Surgical procedures
Following the induction of general anesthesia using sodium pentobarbital (Nembutal 60 mg/ml, intraperitoneal), a 1-cm low, ventral, midline incision was used to expose the abdominal viscera. The testes and spermatic cords were delivered into the surgical field through the inguinal canals using gentle scrotal pressure to expose the vasa deferentia. A 2.5 cm segment of the right vas was gently freed from the surrounding tissues from the junction of the convoluted and straight vas distally (Figure 1a). Major vessels at the proximal and distal points of transection of the vas were cauterized with microforceps using bipolar cautery, and the vas, along with its associated neurovascular supply, was then severed with microscissors. The same procedure was applied to the distal end of the dissected portion of the vas to produce a 2.5-cm isolated vas segment for use as an autograft (Figure 1b). Care was taken to preserve the integrity of the testicular blood supply. Prior to excising the autograft, the deferential vessels were cauterized immediately proximal and distal to the proposed graft site. The graft segment was detached from the vas using microscissors and placed in Ringer’s solution at room temperature. The ischemic time in cold Ringer’s solution was less than 20 minutes. A similar segment from the left vas deferens was then excised for cross-grafting. The free portion of the right vas was then interposed as an autograft between the transected ends of the left vas. The proximal and distal anastomoses were performed under 9-25× magnification using a single layer of five full-thickness 10–0 monofilament nylon sutures (Figure 1c). These sutures were placed equidistant around the vas to ensure an exact approximation of the lumen at each end of the autograft and the two severed ends of the vas. The left vas and its associated testis and epididymis were then returned to the scrotum. The same procedure was performed on the right using the contralateral vas segment and the midline ventral incision was closed in a two-layer fashion using 6–0 nylon sutures for the body wall and 6–0 silk sutures for the skin.
The polarity of the vas autografts was reversed in a sub-group of five rats, such that the proximal end of the autograft was anastomosed to the distal end of the in situ vas, and the distal end of the graft was joined to the proximal in situ vas.
Fertility assessment
On the 20th postoperative day, two mature female rats were placed in the cage of each male rat. The abdomens of these females were gently palpated daily for evidence of pregnancy. At the first sign of pregnancy in either female, both female rats were isolated from their male consort. The first of the two female rats showing evidence of pregnancy by palpation of the abdomen was followed carefully throughout gestation. At the time of littering, the date and the number of offspring were noted. The date of conception was then calculated based on the 21-day gestational period for rats.
Control animals were exposed to females at the same time when the experimental rats were evaluated for establishment of pregnancies.
Three months after cross-grafting, those males that were unsuccessful in establishing a pregnancy were separated from their female consorts for autograft evaluation. A mature, male, unoperated, control rat was placed with the remaining nongravid females to eliminate the possibility of female infertility as a confounding factor.
Sperm motility studies
Sperm motility was assessed in all of the experimental animals after fertility assessments were completed. The right vas was cut 3 mm distal to the distal suture line, and spermatozoa were retrieved using a standard microaspiration technique [13]. The percentage of motile spermatozoa in each sample and their motility indices were calculated using the same routine visual analysis procedures recommended for human semen analysis. Sperm samples were also microaspirated from the convoluted vas at a level 3 mm proximal to the proximal suture line and analyzed as described above. Similar sample collection procedures and motility analyses were used to assess the motility of spermatozoa from the contralateral side. Similar sperm motility assessments were also performed on the ten unoperated control rats. Within-animal data were compared using Student’s t-test, and sperm motility data from the same anatomical levels in experimental and control rats were compared using a one-way ANOVA (Minitab Statistical Software Inc., Pennsylvania, USA).
Autograft evaluation
After 90 days, fertility assessments were completed and each rat was sacrificed. The viability of the vas grafts evidenced by the presence of spermatozoa inside each autograft and in the adjacent vas above and below the graft were examined under low power magnification (9×) in all 15 experimental animals. The patency of the autografts was evaluated under 9× magnification. The presence of a continuous white column of seminal fluid throughout the autograft and into the distal (abdominal) vas was indicative of graft patency. The presence of anastomotic leakage or granuloma was also documented. The autograft, including the proximal and distal anastomotic sites, was excised, preserved in Bouin’s solution, and prepared for light microscopy. Longitudinal sections were cut through the anastomoses for further study of the operative sites. Four-micron-thick transverse sections were cut through the center of each autograft and from regions adjacent to the proximal and distal anastomosis sites to examine the vas autografts for any obvious histological changes. Sections stained with hematoxylin and eosin and Masson’s trichrome were prepared from each paraffin block. Similar histological observations were made on vas samples obtained from similar regions in each of the ten unoperated control rats.A grading system was devised to objectively assess the microscopic changes in autograft histology. The extent of smooth muscle degeneration was the basis for this system (see Figure 2 for a drawing of the vas grading system (0–3) with an appropriate description in the legend).