Monitoring of oxidative stress and acute phase proteins following testes exposure with ultrasound waves in cats

Abstract

Backgrounds

Ultrasonic therapy has harmfully effects on the structure and function of the testis. Various mechanisms are involved in the impact of ultrasound waves on testes tissue. This study aimed to investigate the effects of ultrasound exposure on cat`s testes by monitoring acute phase proteins, oxidative stress indices, histopathology, and epididymal sperm evaluation over a period of 60 days. Ten adult domestic short hair (DSH) male cats were divided into two equal groups: one exposed to ultrasound and one control groups. The testes were exposed to ultrasonic therapy using 1 MHz, 1.5 W/cm², three times with a 48-hour interval. Blood samples were taken before treatment and at days 1, 3, and 6 after treatment. Histopathology of the testes and epididymal sperm evaluation were performed at day 60.

Results

The results showed that acute phase proteins, serum amyloid (SAA), C-reactive protein (CRP), and alpha-1 glycoprotein (AGP), significantly increased in the first three days after treatment and decreased significantly by day 6 in the treatment group. The increase in acute phase proteins was significantly higher in the treatment group compared to the control group. Malondialdehyde (MDA) and total antioxidant capacity (TAC) did not show significant change in the first week after treatment in the ultrasound exposure group compared to the control group. Testosterone concentration was significantly decreased in the ultrasound exposure group compared to the control group. The epididymal sperm and histopathology evaluation indicated no harmful effect of ultrasound in the treatment group compared to the control group.

Conclusions

Ultrasound exposure of cat`s testes induced an acute phase inflammatory response and decreased testosterone concentration in the first week after treatment, but it did not have harmful effect on testis tissue and epididymal sperm quality.

Background

Over population of stray cats is a critical problem worldwide due to zoonotic diseases, social interactions, and poor animal welfare conditions [1]. Cats are known to be stressful animals, and current programs for capturing, neutering, vaccinating, and releasing them are limited in their ability to neuter a large number of animals each year. The costs, trained personnel, and side effects of surgical neutering methods have reduced their effectiveness. Non-surgical methods are being developed to establish a cheap, widely available, and easy procedure for neutering and controlling the animal population [2, 3].

The surgical method of castration is the primary procedure used to sterilize male cats. This method requires anesthesia and veterinary care. There are also non-surgical methods with limited applications for male cat neutering, such as GnRH agonists, melatonin, GnRH-conjugated to keyhole limpet haemocyanin, intratesticular injection (calcium chloride, zinc gluconate, glycerol), and intra-epididymal injection [4].

The scientific prioritization of non-surgical sterilization methods specifically for male cats in population control programs is grounded in the biological and behavioral limitations of traditional Trap-Neuter-Return (TNR) approaches, which typically involve surgical castration. Castration eliminates testosterone production, causing neutered males to lose their sexual drive, territorial aggression, and social status within colony hierarchies, allowing intact males to move in and continue breeding, thus undermining population control efforts unless unrealistically high capture rates (> 57–82%) are achieved [5]. In contrast, hormone-sparing approaches like vasectomy preserve male social dominance and territorial behaviors, enabling vasectomized males to competitively exclude intact males from breeding while also inducing prolonged 45-day pseudopregnancy periods in females through non-productive copulation, resulting in superior population decline at significantly lower capture rates (≥ 35%) [5]. Computer simulation models demonstrate that Trap-Vasectomy-Hysterectomy-Release (TVHR) eliminates feral cat populations at a capture rate of 57% within 4,000 days, whereas traditional TNR requires > 82% capture for the same outcome, and TNR can be counterproductive by increasing adult and kitten survival rates, potentially leading to population growth rather than decline [5]. Furthermore, non-surgical methods offer practical advantages including reduced costs, improved scalability, enhanced feasibility in remote or resource-limited settings, and greater cultural acceptability, particularly in communities where surgical sterilization infrastructure is limited or where named animals cannot be euthanized [6, 7]. The evidence from population dynamics modeling, cost-effectiveness analyses, and stakeholder surveys collectively supports the integration of male-focused non-surgical sterilization techniques into comprehensive population management programs as a scientifically grounded, effective, and humane approach to controlling feral and free-roaming cat populations [8, 9].

The tissue of the testis is sensitive to changes in temperature, inflammation, and oxidative stress. Infections, trauma, and radiation increase intratesticular temperature, induce inflammation, and oxidation [10–12]. The protective mechanisms of the testes are active but have limited capability to reduce or overcome these harmful changes. If the damages surpasses these mechanisms, the seminiferous tubules and interstitial tissues begin to undergo apoptosis and necrosis overtime. Overall, these harmful changes lead to subfertility, infertility, and sterility, depending on the severity of damage to different compartments of the testes [13].

Therapeutic ultrasonography produces heat within the tissue that is different from the diagnostic ultrasonography. This method induces thermal coagulopathy necrosis with luminal occlusion within the epididym and necrosis in seminiferous tubules [14, 15]. The testes are affected by both the thermal and non-thermal mechanisms of ultrasound. However, the non-thermal mechanisms, particularly cavitation-induced oxidative stress and direct mechanical disruption of cell membrane integrity, are the dominant factors responsible for the profound and specific destruction of testicular tissue. The thermal effect contributes to the overall stress on spermatogenesis, but the unique, non-thermal actions of ultrasound are what make it a more effective contraceptive agent than simple heat application [14, 16, 17]. In dogs, testes are located in the inguinal region outside the abdominal cavity. Ultrasonic therapy is a non-surgical procedure that has been standardized for neutering male dogs. Repeated exposure of the testes to ultrasounds induces an acute inflammatory response, oxidative stress, tissue necrosis, stops spermatogenesis, and effectively spays male dogs [18, 19].

It has been confirmed that ultrasound waves penetrate to varying depths of tissues, depending on frequency, power, and wave length [20]. This can increase temperature, create microbubbles, inflammation, and oxidative stress [21]. As a result, non-surgical methods have been developed to induce sterility in animals using ultrasound. In recent years, Italian researchers have established and standardized this method for male dogs. They have studied different aspects of ultrasonic therapy of the testes in dogs and have suggested this method for neutering dogs [19, 22–25].

Research on the application of therapeutic ultrasound to cat testes has demonstrated its potential as a non-surgical method for suppressing spermatogenesis. Early studies by Fahim et al. (1977) showed that exposing cat testes to ultrasound at 1 W/cm² for 10 min significantly reduced sperm and spermatid counts, with two treatments administered 48 h apart completely abolishing spermatogenic stages, while Leydig cells and blood testosterone levels remained unaffected. The mechanism of action involves a combination of thermal effects, as ultrasound energy is absorbed and converted to heat, and non-thermal mechanical effects, including acoustic cavitation and alterations in cell membrane permeability that disrupt the ionic balance within the seminiferous tubules [14, 24]. However, this method has not been conclusively studied in male cats. The size, perineal location of testes, and hair coated of scrotum in cats are different from dogs. Therefore, the aim of this study is to expose the testes of cats to ultrasonic therapy waves and evaluate the acute inflammatory phase proteins, oxidative stress indices, testosterone concentration, and histopathology of testes tissue.

Results

Acute phase proteins

Alpha-1 Acid Glycoprotein (AGP)

Time and group factors significantly affected the concentration of AGP (P < 0.008). The AGP concentration was significantly increased in the treatment group on day 1 after ultrasonic exposure compared to before treatment (P < 0.05). Additionally, the AGP concentration was significantly higher in the treatment group compared to the control group on day 1 after ultrasound exposure (P < 0.05; see Table 1).

Table 1.

Changes in biochemical factors including CRP, SAA, AGP, TAC, MDA, and testosterone in the the blood serum of male cats in both the control and treatment groups on the day before (day 0) and on days 1, 3, and 6 after ultrasound exposure of the testes

		Days
Factors	Group	0	1	3	6
CRP (mg/dl)	U/S exposure	0.28 ± 0.08ac	2.98 ± 0.76b*	2.55 ± 0.63b*	2.05 ± 0.59bc*
	Control	0.29 ± 0.06	0.29 ± 0.06*	0.290.05*	0.28 ± 0.07*
SAA (µg/mL)	U/S exposure	0.02 ± 0.01ac	0.2 ± 0.03b*	0.18 ± 0.02b*	0.13 ± 0.02bc*
	Control	0.02 ± 0.01	0.016 ± 0.008*	0.016 ± 0.008*	0.018 ± 0.01*
AGP (µg/mL)	U/S exposure	1843 ± 585.83a	3656.2 ± 1163.49b*	3209.4 ± 1138.14	2978.2 ± 1104.02
	Control	1784.2 ± 465.19	1805 ± 532.22*	1849.4 ± 551.45	1883 ± 567.17
TAC (mmol/l)	U/S exposure	1.73 ± 0.21	1.43 ± 0.21	1.53 ± 0.17	1.6 ± 0.18
	Control	1.72 ± 0.18	1.73 ± 0.17	1.73 ± 0.21	1.74 ± 0.18
MDA (nmol/ml)	U/S exposure	30.23 ± 5.71	36.124 ± 6.01	33.61 ± 6.28	32.54 ± 5.98
	Control	30.43 ± 5.43	31.34 ± 5.44	29.39 ± 5.84	29.98 ± 5.93
Testosterone (ng/ml)	U/S exposure	17.19 ± 6.30a	7.30 ± 3.76b*	7.5 ± 3.01*	6.69 ± 3.33b*
	Control	17.87 ± 5.07	17.65 ± 5.08*	17.84 ± 5.54*	17.64 ± 5.11*

^abcsuperscript letters indicate significant difference between days of study in each row

*superscript astrics indicate significant difference between groups in each column

C-reactive protein (CRP)

Time and group factors had a significant impact on the concentration of CRP (P < 0.001). The CRP concentration significantly increased in the treatment group on days 1, 3 and 6 after ultrasonic exposure compared to before treatment (P < 0.05). Additionally, the CRP concentration was significantly lower in the treatment group on day 6 compared to days 1 and 3 (P < 0.05). Furthermore, the CRP concentration was significantly higher in the treatment group compared to the control group on days 1, 3 and 6 after ultrasound exposure (P < 0.05; see Table 1).

Serum amyloid A (SAA)

Time and group factors significantly affected the concentration of SAA (P < 0.0001). The SAA concentration showed a significant increase in the treatment group on days 1, 3 and 6 after ultrasonic exposure compared to before treatment (P < 0.05). Additionally, the SAA concentration was significantly lower in the treatment group on day 6 compared to days 1 and 3 (P < 0.05). Furthermore, the SAA concentration was significantly higher in the treatment group than in the control group on days 1, 3 and 6 after ultrasound exposure (P < 0.05; see Table 1).

Oxidative stress indices

Total antioxidant capacity (TAC)

Time and group factors did not affect the concentration of TAC (P > 0.05). The TAC concentration did not show significant change in the treatment and control groups during this study (P > 0.05). The TAC concentration was not significantly different between the treatment and control groups (P > 0.05; see Table 1).

Malondialdehyde (MDA)

Time and group factors did not affect the concentration of MDA (P > 0.05). The MDA concentration did not show significant change in either the treatment or control groups during the study (P > 0.05). Additionally, there was no significant difference in MDA concentration between the treatment and control groups (P > 0.05; see Table 1).

Testosterone

The group factor significantly affected the concentration of testosterone (P = 0.0001). Testosterone concentration was significantly decreased in the treatment group on days 1, and 6 after ultrasonic exposure compared to before treatment (P < 0.05). Additionally, testosterone concentration was significantly decreased in the treatment group compared to the control group on days 1, 3, and 6 after ultrasound exposure (P < 0.05; see Table 1).

Pathology

Interestitial and Leydig cell index

No significant changes was observed in interstitial tissue and Leydig cells 60 day after ultrasound exposure of cat`s testes (P > 0.05; see Table 2; Fig. 1).

Table 2.

Comparison of pathological indices of the testes between the ultrasound exposure group and the control group in male cats after 60 days

	Groups
	U/S Exposure	Control	P-value
JS	3.841 ± 0.99	3.814 ± 1.17	P = 0.74
MSTD	15.94 ± 2.67*	15.29 ± 2.12*	P = 0.04
GECT	5.242 ± 1.07*	4.735 ± 1.17*	P = 0.001

*superscript astrics indicate significant difference between groups in each row

Fig. 1.

Histopathological evaluation of cat testes 60 days after the ultrasonic exposure in treatment (B, n = 5) and control (A, n = 5) groups

Johansen-Stahle index (JS)

The extent of germ cell damage within the seminiferous tubules was not significantly different between the ultrasonic exposure and control groups (P > 0.05; see Table 2; Fig. 1).

Germinal epithelial cell thickness (GECT)

The thickness of germinal epithelial cells was significantly increased in the ultrasonic exposure group compared to the control group (P = 0.001; see Table 2; Fig. 1).

Mean seminiferous tubule diameter (MSTD)

Mean diameter of seminiferous tubules was significantly increased in the ultrasonic exposure group compared to the control group (P = 0.04; see Table 2; Fig. 1).

Epididymal sperm evaluation

Weight of epididym

The weight of the epididym was not significantly different between the ultrasound exposure and control groups (P > 0.05; see Table 3).

Table 3.

Comparison of epididymal sperm quality indices between the control group and the group exposed to ultrasound on testes in male cats after 60 days

	Groups
	U/S Exposure	Control	P-value
Weight of epididym (g)	0.17 ± 0.03	0.19 ± 0.01	P = 0.54
Sperm concentration (n/ml)	9,800,000 ± 5,032,395	8,740,000 ± 5,706,400	P = 0.76
Live/dead sperm (%)	67 ± 14.8	55 ± 21.21	P = 0.45
Normal sperm morphology (%)	96.67 ± 2.3	97.5 ± 0.7	P = 0.66

Sperm concentration

The sperm concentration did not show significant difference between the ultrasound exposure and control groups (P > 0.05; see Table 3).

Sperm viability

There was no significant difference in the percentage of live sperm between the ultrasound exposure and control groups (P > 0.05; see Table 3).

Sperm morphology

There was no significant difference in the percentage of normal sperm morphology between the ultrasound exposure and control groups (P > 0.05; see Table 3).

Discussion

In summary, ultrasound exposure of testes in cats led to an inflammatory response within the first week after treatment. Testosterone decreased significantly in treated cats compared with the control group. Changes in histopathology and epididymal sperm evaluation indicated that ultrasound exposure of the testes did not harmfully affect seminiferous tubule histology and sperm quality in the epididym.

Acute phase proteins increased during the first three days after ultrasound exposure of testes and their levels decresead by day 6 after treatment in cats. In our previous study in dogs, ultrasound exposure of testes led to acute inflammatory response on the first week [18]. The acute phase reponse and increasing of acute phase proteins occurred as a non-specific and as a part of the innate host defence in response to the irritation of the tissue [26]. In our study, we assayed the positive acute phase proteins CRP, SAA and AGP that increased within acute inflammatory response. The CRP and SAA are the main acute phase proteins in small animals and their changes are more significant [27]. Inflammatory responses are inextricabily linked to the oxidative stress and finally to the pathophysiological processes, apoptosis, protein oxidation, lipid peroxidation and sperm DNA damage in the male reproductive system [28, 29].

Inflammation in the testes and around the Leydig cells can reduced testosterone synthesis [30]. There are direct and indirect effects of inflammation on production of testosterone in the testes [31]. Inflammatory cytokines can directly interfere with Leydig cell function, reduce their ability to produce testosterone. Inflammatory cytokines like TNFα, IL-1β, and IL-6 can directly impair Leydig cell viability, and signaling pathways involved in testosterone synthesis. Induction of oxidative stress in Leydig cells following inflammation can directly damage their function [30, 32]. Indirectly, inflammation can negatively affect the hypothalamus-pituitory-testes axis, and disrupt the mechanisms involving in testosterone synthesis. This can lead to reduce LH concentration that is essential for Leydig cell stimulation and testosterone production [33]. In our study, testosterone concentration decreased significantly in ultrasound exposure group compared with control group at the first week of study. Acute phase proteins increased at the first three days. The inflammatory response and acute phase proteins may interfer production of testosterone and decrease its levels compared to control group.

The MDA concentration and total antioxidant capacity did not show significant change during the first week of the study. We observed a transient acute inflammatory response in the first three days after ultrasound exposure of the testes in the cats. However, the increase in acute phase proteins did not induce oxidative stress. Therefore, the detrimental effects of inflammation due to ultrasound exposure of the testes were not observed in histopathology or epididymal sperm quality at the end of the 60-day study. The power of ultrasound, exposure time, and intervals may influence the effects of ultrasound on the testes [18, 19]. We used ultrasonic therapy with a power of 1.5 W/cm², a frequency of 1 MHz, for five minutes, and with intervals of 48, repeated three times. This protocol differed from one used by Fahim et al., in 1977, which had a power of 1 W/cm², a duration of 10 min, and was repeated one or two times for male cats [24].

In spite of acute inflammatory response in the first week after ultrasonic exposure of testes, the seminiferous tubules and interstitial tissue and Leydig cells did not adversely affected in histopathology evaluation at day 60 of study. In contrast to our results, the previous study in 1977 indicated that exposure of cat`s testes with ultrasound waves, 1 W/cm² for 10 min and one or two times, led to reduction in sperm and spermatids and abolishment of different stages of spermatogenesis. But, testosterone concentration and Leydig cells did not affected [24]. The number of exposure times, power, and the time of treatment are different from our study. We were directly exposed the testes to the ultrasound probe and they used a plexiglass cup filled with water as a coupling gel. The size of testis may be different and we had shaved the srotum before applying coupling gel [34].

The epididymal sperm quality was not decreased in the ultrasound exposure group than in the control group. Transient acute phase inflammatory response and mild oxidative stress did not harmfully affect the tissue of the testis and epididymal sperm quality after 60 days. Ultrasound waves can affect the process of sperm production, but the effects can be complex and sometime contradictory. The ultrasound can stimulate spermatogenesis and improve sperm motility [35, 36]. Low-intensity pulsed ultrasound has been shown to enhance sperm swing velocity and motility characteristics [37]. Ultrasound exposure at 19.28 MHz and 2 W lead to a 34% increase of sperm swimming velocity [36]. Studies suggest that ultrasound can be used to activate and improve the viability of sperm. Intensity and frequency of ultrasound waves are crucial factors. Low-intensity ultrasound appears to have positive effects, while higher intensities may be detrimental. The duration of ultrasound exposure and the specific method of application can also influence the outcome. Individual responses to ultrasound exposure can vary, and factors like age, BMI, and pre-existing conditions may pay a role [18, 35–37]. On the other side, ultrasound can suppress spermatogenesis and ultimately sterilze the animals. Histological studies have shown that US exposure induces apoptosis primarily in pachytene spermatocytes and round spermatids [14, 16, 23]. The mitochondrial pathway of apoptosis, involving Bax and Bcl-2, appears to be activated, with increased Bax expression and decreased Bcl-2/Bax ratio [38]. Oxidative stress, as indicated by elevated MDA and decreased glutathione peroxidase, plays a central role [16, 39]. The inflammatory response, characterized by elevated CRP and SAA, peaks within the first week after exposure and subsides by day 21 [16]. Interestingly, Leydig cells are largely resistant to US damage, and testosterone production remains normal. This has been a consistent finding across species and exposure regimens [16, 17, 23, 40]. The preservation of androgen production is an advantage of US-based contraception, avoiding the hypogonadal side effects associated with hormonal contraceptives or surgical castration.

Ultrasound energy interacts with biological tissues through two primary mechanisms: thermal and non-thermal (mechanical). Both contribute to the observed effects on the testis and ovary, and their relative importance depends on exposure parameters and tissue properties. Absorption of US energy leads to localized heating. Because spermatogenesis requires a scrotal temperature 3–4 °C below core body temperature, even modest elevations can induce germ cell apoptosis and disrupt spermatogenesis [41–43]. In the ovary, pre-ovulatory follicles are normally cooler than surrounding stroma, and disruption of this temperature gradient can impair oocyte maturation and ovulation [44, 45]. The extent of tissue heating is determined by US frequency, intensity, duty cycle, and the absorption coefficient of the target tissue. Bone, which has a high absorption coefficient, can heat rapidly, posing a risk during trans-abdominal or trans-scrotal exposure [46]. In the rat, testicular temperatures rise by 1–2 °C during therapeutic US, and the combination of elevated baseline temperature (e.g., coupling medium at 37 °C) with US exposure is more effective than US alone at depleting germ cells [14, 47]. Mechanical effects include acoustic cavitation, acoustic radiation force, and microstreaming. Cavitation—the formation, oscillation, and collapse of gas bubbles—generates shock waves, micro-jets, and free radicals that can damage cell membranes and organelles [48, 49]. Stable cavitation produces microstreaming and shear stresses that alter membrane permeability and ion gradients [50, 51]. Fahim et al. proposed that US-induced changes in sodium and potassium concentrations within the seminiferous tubules and rete testis create an environment unfavorable for spermatogenesis [52, 53]. More recent studies have highlighted the role of oxidative stress. Exposure of dog testes to US (1 MHz, 1.5 W/cm², three 5-min treatments) resulted in a significant increase in serum malondialdehyde (MDA) and a decrease in antioxidant enzymes (superoxide dismutase, glutathione peroxidase, catalase) within the first week, indicating acute oxidative injury [16]. Similar findings were reported in rats [39]. The acute-phase response, marked by elevated C-reactive protein (CRP) and serum amyloid A (SAA), also accompanies US-induced testicular damage [16]. Thus, the contraceptive effect of US results from a combination of thermal stress, mechanical disruption, and oxidative damage, all of which converge on germ cell apoptosis.

Our finding that this ultrasound protocol did not adversely affect testicular histopathology or semen quality in cats directly contrasts with established literature. Fahim et al. (1977) achieved significant suppression of spermatogenesis in cats at a lower intensity (1 W/cm² for 10 min), with two treatments abolishing all spermatogenic stages [24]. More strikingly, Leoci et al. (2015) applied the exact same protocol we used (1.5 W/cm², 1 MHz, 5 min, 3 times at 48-hour intervals) to dogs and achieved azoospermia with irreversible tubular atrophy [17]. This suggests a pronounced species-specific sensitivity; cats may require a higher frequency (e.g., 3 MHz, as demonstrated by Tsuruta et al., 2012, for uniform rat germ cell depletion [14]) or a longer post-treatment observation period (Fahim assessed histology at 60 days) to manifest permanent damage. The lack of effect in our study implies that this specific dose triggers a sub-threshold injury in felines that is insufficient to permanently deplete the germinal epithelium.

Conclusions

The small sample size, lack of comparison of different ultrasound exposure protocols, lack of long-term follow-up, and absence of semen quality evaluation during the study have limited the results and conclusions. MDA and TAC were measured only during the first week. Late-phase oxidative stress was not evaluated, and more specific markers (SOD, GPx, catalase) were not used. Ultrasound exposure of cat testes with a 1 MHz frequency, 1.5 W/cm2 power, three times with 48-hour intervals induced an acute inflammatory response without oxidative stress within the first week after treatment. The structure of testes tissue, including seminiferous tubules and interstitials and epididymal sperm quality did not harmfully affect 60 days after exposure. The protocol was not effective as a sterilizing method in cats.

Methods

Experimental protocols were performed in accordance with the Iranian animal ethics framework under the supervision of the Iranian Society for the Prevention of Cruelty to Animals and Shiraz University Research Council (IACUC no: 4687/63). The recommendations of European Council Directive (2010/63/EU) of September 22, 2010, regarding the standards in the protection of animals used for experimental purposes, were also followed.

Animals

For this study, 10 adult, healthy, mixed-breed male cats (2–4 years oold and weighing 3.5–5 kg) were chosen from a shelter established to manage the cat population. The cats were housed in a cattery at Shiraz University School of Veterinary Medicine, fed a diet of commercially available dry food (100 g/cat/day; Fidar patira™; Iran), and had unlimited access to water. The light/dark cycles was adjusted 14/10 hours. The cats were housed in a cat well-designed furniture and products room and temperature setting up 25 °C. They were neutered and remained in the shelter at the end of the study. The cats` reproductive health was confirmed through palpation, ultrasound examination of the testes, and semen evaluation prior to the start of the experiment. In the first two weeks of acclimation to the new environment, the cats received anti-parasitic treatment with a medication (one tablet/10 kg; Endopet^®, Santavet, Turkey).

Experimental design

After confirming their health and completing of the habituation period (two weeks), the cats were randomly, using random number tables, divided into two equal groups (n = 5): a treatment and a control groups. The scrotal hairs were clipped before and during the study to establish reliable contact between the ultrasound probe and the scrotal skin. In the treatment group, the cats’ testes were directly exposed to ultrasound waves at an intensity of 1.5 W/cm2 and a frequency of 1 MHz, three times for five minutes, every 48 h [18]. The day before exposure was considered day 0, and the start of exposure was considered day 1 of the study. Blood sampling was conducted before exposure, and 1, 3, and 6 days after exposure. Epididymal semen collection was performed at the end of the study after surgical castration, and testis histopathology was conducted 60 days after exposure.

Ultrasound exposure

Testes exposure to therapeutic ultrasound waves was conducted using an ultrasonic physiotherapy machine (Unix U500; APC; Iran). The cats were restrained in a standing position and a water-soluble ultrasound gel was applied to their scrotums. The probe was then moved in circular motions over the skin of the testes. All cats in the treatment group were exposed to ultrasound waves at an intensity of 1.5 W/cm2 and a frequency of 1 MHz, three times for five minutes each, with sessions occurring every 48 h. The procedure was repeated in the control group with applying water soluble ultrasound gel to the scrotum and cicular motion of probe over the skin of testes for five minutes and three times every 48 h, but the device switched off.

Surgical castration

All cats were randomly selected for surgery, which was performed under general anesthesia. Premedication consisted of medetomidine hydrochloride (1 mg/mL; 20 µg/kg, IM; Dorbene Vet, Spain). Following sedation, a 24-gauge intravenous catheter was aseptically placed in the cephalic vein. Anesthesia was induced using ketamine hydrochloride 10% (5 mg/kg, IV; Alfasan, Woerden, The Netherlands) combined with midazolam (5 mg/mL; 0.25 mg/kg, IV; Caspian Pharmaceutical Co., Iran). Given the short duration of the surgical procedure, endotracheal intubation was not performed; instead, supplemental oxygen was administered via a facemask using an infant T-piece throughout the procedure. Anesthetic monitoring was limited to clinical assessment of vital parameters, including respiratory rate and pulse rate. If inadequate anesthetic depth was observed, additional doses of ketamine and midazolam were administered for maintenance of anesthesia. All animals received intravenous crystalloid fluids at a rate of 10 mL/kg/h during anesthesia and surgery. Prophylactic antibiotic therapy was provided with cefazolin (22 mg/kg, IV; Dana Pharmaceutics, Iran) administered at anesthetic induction [54]. Castration was performed on an operating table preheated to 37 °C. All surgical procedures were conducted by a single surgeon in a blinded manner, such that the surgeon was unaware of the animals’ group assignments. The scrotal hair was plucked, and the surgical site was aseptically prepared. Cats were positioned in lateral recumbency with the hind limbs extended cranially. A 1-cm scrotal incision was made over each testis. Following exteriorization of the testis and digital separation of its attachments, the spermatic cord was ligated using a closed technique without opening the vaginal tunic, employing 2 − 0 absorbable suture material (polyglycolate; Supa Medical Devices, Iran). The scrotal incisions were left to heal by second intention [55]. Upon completion of surgery, animals were transferred to a quiet environment and allowed to recover gradually. Immediately after surgery, all animals received meloxicam 0.5% (0.2 mg/kg; Rooyan Pharmaceutical Co., Iran) for postoperative analgesia.

Laboratory measurements

All laboratory measurements were validated for cat samples in the research clinical pathology laboratory at the School of Veterinary Medicine, Shiraz University. Laboratory personnel and histopathologists were blinded to group allocation.

Serum testosterone assay

Blood samples were collected from the jugular vein of cats before testes exposure and 1, 3, and 6 days after exposure, transferred into the tubes without an anticoagulant agent, and centrifuged at 750 ×g for 10 min for serum separation. Serum testosterone (ng/ml; Cat. no. 3725–300 A) was measured by the ELISA method (Monobind Inc, USA).

Acute phase proteins assay (SAA, AGP, CRP)

SAA (mg/ml), CRP (µg/L), and AGP (µg/ml) were measured in serum using the quantitative sandwich enzyme immunoassay included in the Shanghai Crystal Day Biotech commercial canine-specific kits (SAA; Cat. no. E0125CA, CRP; Cat. no. E0123CA, AGP; Cat. no. E0124CA). The sensitivity of the tests in the serum samples was determined by the manufacturer as 0.078 µg/ml for CRP and 0.3 µg/ml for SAA.

Oxidative stress indices (MDA, TAC)

Malondialdehyde (mmol/L; Cat. no. ZB-MDA-96 A) was measured using the ZellBio GmbH kit (Germany) based on its reaction with thiobarbituric acid in an acidic condition and high temperature; the color complex was measured colorimetrically at 535 nm. The values were finally expressed as mmol/L.

Histopathological evaluation

Histopathological and spermatogenesis evaluation was performed at the end of the study period (day 63). The cats were anesthetized and their testicles were removed by surgical castration. The specimens of the testicular tissues were immediately fixed in 10% neutral buffered formalin. After complete fixation and the routine tissue processing, all tissue samples were sectioned in 5 μm thickness, and stained with hematoxylin and eosin (H&E) method. Finally, the histopathological changes including germinal epithelial cell degeneration and necrosis of the seminiferous tubules, edema, hyperemia, hemorrhage and the presence of the acute inflammatory cells within the tubules and the interstitial tissue were evaluated by a pathologist who was blinded on sasmples using a light microscope (Olympus, Japan). For histomorphometric examination and analysis of spermatogenesis, mean seminiferous tubular diameter (MSTD) and germinal epithelial cell thickness (GECT) in µm unit were measured at least within 20 random circular seminiferous tubules per each tissue slide [56]. Also, at least 50 cross-sectioned seminiferous tubules were evaluated to measure the mean testicular biopsy score (MTBS) based on the Johnsen scoring (JS) system. In this scoring system, a score of 1–10 was assigned to each tubule per tissue slide based on the early mentioned histopathological changes [57, 58].

Statistical analysis

All data were summarized using Microsoft Excel version 2016 (Microsoft Corporation, Redmond, Washington, USA) and presented as mean and standard deviation. The normality and homogeneity of variance of data were confirmed using Shapiro-Wilk and Levene`s tests, respectively. The statistical analysis of the data was performed by GraphPad Prism Version 9.5 software (GraphPad Software, San Diego, California, USA), using two-way repeated measures ANOVA test. Tukey post-hoc test included for multiple comparisons. P values less than 0.05 were considered significant.

Abbreviations

DSH

Domestic short hair

SAA

Serum amyloid

CRP

C-reactive protein

AGP

Alpha-1 glycoprotein

MDA

Malondialdehyde

TAC

Total antioxidant capacity

JS

Johansen-Stahle index

GECT

Germinal epithelial cell thickness

MSTD

Mean seminiferous tubule diameter

Authors’ contributions

All authors contributed in all parts of study from designing study to writing and preparing of manuscript. AM, SN, MRD, ND, NA and ZH contributed in study design, performing study, sampling, data collection and analysis and preparing manuscript. AS, HB, EA, and AHH aimed in designing, surgery and performing study and laboratory analysis.

Funding

The study was financially supported by the School of Veterinary Medicine, Shiraz University (Grant No. 3GCB2M154630). There was no financial support for this work that could have influenced its outcome. The funding body had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

Data availability

The datasets generated and/or analysed during the current study are available from the corresponding author on request.

Declarations

Ethics approval and consent to participate

Our study was approved by the Iranian laboratory animal ethics framework under the supervision of the Iranian Society for the Prevention of Cruelty to Animals and Shiraz University Research Council (IACUC no: 4687/63). A written informed consent was obtained by the shelter. The recommendations of European Council Directive (2010/63/EU) of September 22, 2010, regarding the standards in the protection of animals used for experimental purposes, were also followed.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.