Wistar albino male rats (Rattus norvegicus) aged 7–8 weeks, weighing 200–210 g, were obtained from the Animal Care Center, College of Science, King Saud University, Riyadh, Saudi Arabia. The Ethical Committee of the Experimental Animal Care Center approved the study. Animals were housed in a temperature-controlled room on a 12-h light/dark cycle and had access to water and normal chow diet ad libitum. B. papyrifera (crude incense) and B. carterii (refined incense) were obtained locally.

After 1 week of acclimatization period, the rats were randomly divided into three groups, viz. groups I, II and III, with each group consisting of 11 animals. Each group of rats was housed separately from the other groups to avoid the cross exposure to incense smoke. Rats in group I served as control and were kept in normal conditions, while rats in group II and III were exposed to B. papyrifera and B. carterii smoke respectively, in a smoking chamber as described by Wang et al. [12]. The rats were exposed daily to the smoke emanating from the burning of 4 g of each incense material for 4 months. Smoke exposure durations lasted for 30–35 min/day. The dose and duration of incense exposure followed in this study were based on the optimized conditions from our lab studies [10,11]. At the end of exposure duration, all animals were killed by cervical dislocation.

After 4 months of treatment, blood samples were collected from each group. They were kept at room temperature for approximately 30 min. Then, the tubes were centrifuged at 2500 rpm for 15 min, the supernatants were collected, and the serum was used for analyzing the hormone level. Serum concentrations of follicle stimulating, luteinizing and testosterone hormones (Diagnostic System Laboratories kits [DSL], USA) were measured following an immunoenzymatic method with an ELISA reader, according to the standard protocol given in the assay kit.

The epididymal plasma was collected by cutting the epididymides cauda in halves, and then by carefully scraping and aspirating with an insulin syringe without a needle, then by centrifugating the sample at 12,000 g for 30 min in an Eppendorf centrifuge 5415 C apparatus. The epididymal plasma was stored at −40 °C before the specific spectrophotometric determination of proteins, sialic acid, carnitine, and carbohydrates. The biochemical compositions were estimated according to Huacuja et al. [13].

The cauda epididymis was carefully squeezed into phosphate buffered glucose saline (PBGS); and a clear suspension was obtained. The epididymal plasma was used for total sperm count, sperm motility, forward velocity and relative percentage of abnormal sperms determination. The total sperm count and motility were calculated according to the method of Besley et al. [14], using a Neubauer hemocytometer. The spermatozoa were allowed to settle down in the hemocytometer for total sperm count by keeping them in a humid chamber (4 °C) for one hour. The sperm count was recorded in R.B.C. counting chambers [15]. Similarly, the total numbers of motile sperm cells were calculated immediately using a saline phosphate buffer instead of spermicidal solution and the time gap between counts were 0.5 to 10 s. The forward sperm velocity was calculated according to Ratnasooriya [16]. The assessment was made under the light microscope, fitted with a movable mechanical stage and a calibrated ocular micrometer, at 400× magnification. A drop of sperm suspension was transferred to a clean glass slide and the initial place and time of each sperm were recorded. The time taken for forward movement of the sperm from the initial place within the microscopic field was recorded using a stopwatch. The procedure was repeated for 10 spermatozoa for each sample and the average forward velocity of the sperm was calculated and expressed in μm/s. The relative proportions of abnormal sperms were analyzed by Bauer et al. [17]. Briefly, equal volumes of cauda epididymal plasma and 5% NaHCO₃ were placed in a centrifuge tube, mixed well and centrifuged for 5 min at 4000 rpm. The supernatant was discarded and 5 mL of normal saline solution was added to the precipitate, mixed well and centrifuged again. The procedure was repeated two to three times until a clear precipitate was obtained. To the final precipitate, a few drops of normal saline solution were added, mixed thoroughly and a smear was prepared on a clean slide. The smear was dried at room temperature, fixed by heating over the flame for two to three seconds. Then, the smear was flushed with 95% alcohol, drained, and dried. Smear was stained in Ziehl Neelson's Carbol Fuchsin diluted with equal volumes of 95% alcohol for 3 min and counter stained with a 1:3 (v/v) aqueous solution of Loeffer's methylene blue for 2 min. After staining, the smear was rinsed in water and dried in the air. The abnormal sperms were characterized based on the presence of double tails, detached heads, detached tails, midpiece bending, and irregular heads. The relative proportions of normal and abnormal sperm were expressed in terms of percentages.

Immediately after removal of cauda epididymis from the dissected rats, tissues were sliced into small-size pieces (1 mm³) and fixed in 3% buffered glutaraldehyde for 4 h at 4 °C. Tissue specimens were then post-fixed in 1% osmium tetraoxide (OsO₄) for 1 h 30. Dehydration of the fixed tissues was performed using ascending grades of ethanol and then the tissues were transferred to resin via propylene oxide. After impregnation with the pure resin, tissue specimens were embedded in the same resin mixture [18]. Ultra-thin sections (70–75 nm) were cut on an ultra-microtome (Leica, UCT) with a diamond knife (Diatome, Switzerland) and stained with uranyl acetate and lead citrate. Sections were observed under TEM (JEOL 1011 CX) operating at 80 kV.

Data were expressed as mean values ± SE. Student's t-test was applied to compare the significance of difference. A probability level P < 0.05 or less was accepted as significant.

No evident changes were noticed in the body weight of the reproductive organs in control as well as in treated groups (data not shown).

Table 1 showed the diminished levels (P < 0.001) of circulating follicle stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T) in the serum after exposure to B. papyrifera and B. carterii.

The biochemical composition of the cauda epididymal plasma displayed a significant decrease in proteins, sialic acid, and carnitine in B. papyrifera and B. carterii groups when compared to the control group (Table 2; P < 0.001). At the same time, no significant changes were observed in carbohydrates of all groups.

Sperm parameters including total sperm count, total number of motile sperm (usually motile sperm swim forward in an essentially straight line, whereas non-motile sperm swim, but following abnormal paths, such as circles), forward velocity and percentage of abnormal sperm, fructose content of epididymal and seminal plasma were compared between exposed and non-exposed control rats. Relative to the control group, the animals exposed to B. papyrifera and B. carterii showed a significant decrease in the total sperm count, the total number of motile sperm and the forward velocity of the sperm (Fig. 1, P < 0.001). The percentages of abnormal sperm were increased (P < 0.001).

In ultrastructural studies, control rats exhibited normal spermatozoa (Fig. 2A–D). The perforatorium and acrosome were enclosed with the plasma membrane (Fig. 2B and C). An intact acrosome was covered with an acrosomal membrane. The whole spermatozoon was intact with all the membranes and organelles (Fig. 2C). Conversely, animals exposed to B. papyrifera and B. carterii evidence a disruption in the plasma and acrosomal membranes. The surface became rough, with accumulated debris and vague materials (Figs. 3A–D and 4A and B). The tip of the sperm head showed a disruption of the plasma membrane as well as of the acrosome (Figs. 3A–C and 4A and B). The perforatorium was condensed and most of its surface was covered by a thin layer of acrosomal sac (Figs. 3A–C and 4A and B). Many serrations were observed in the head region of the spermatozoa. The shape and size of the sperm head has also changed considerably (Figs. 3A–C and 4A and B). There was a severe dorsoventrally constriction in the mid-head region in most sperms. The perforatorium was bulged/swelled (Fig. 3A–C). The anterior and caudal portions of the sperm heads revealed a loss of the plasma membrane and of the acrosome, and present small vesicles on the ventral surface of the perforatorium (Figs. 3A–C and 4A and B). The tail sections in sperms exposed to B. papyrifera and B. carterii showed disruption and complete degeneration of the mitochondrial sheath and a loss of plasma membrane (Figs. 3A and 4B). The commencements of degeneration of mitochondria were observed in almost all sheaths, either on one or both sides, and there was an abnormal pattern of outer dense fibers (Figs. 3B and C and 4B–D). Different parts of the tail region exhibit discontinuation of plasma membrane and fibrous sheath, respectively (Figs. 3A and 4B). Most of the spermatozoa showed a splitting of the tail and distinct visibility of balloon-like cytoplasmic droplets in the mid-region of the tail (Figs. 3D and 4C and D).