Veterinary Research Forum

Abstract This study evaluated the protective effects of intraperitoneal (IP) tannic acid (TA) against ischemia-reperfusion (IR) injury in a rat model of testicular torsion. Eighteen mature male Wistar rats were randomized into three groups, including sham (surgery without ischemia), IR (3 hr ischemia + 3 hr reperfusion), and IR + TA (50.00 mg kg^-1 TA; 100 µL intraperitoneally 30 min before reperfusion). Testicular tissues were sampled immediately after reperfusion for biochemical assays to measure malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GPx) levels. Epididymides were analyzed 60 days later for sperm count, motility, viability, morphology, and DNA integrity. The IR significantly elevated MDA and sperm DNA damage while reducing SOD, GPx, sperm parameters, testicular weight, and spermatogenesis. The TA administration reversed these changes and restored the parameters to levels close to those of the sham group. Overall, intraperitoneal TA mitigated IR-induced oxidative stress and preserved reproductive function, indicating its potential therapeutic value in testicular torsion.

Abstract Cholestasis, a multi-faceted liver complication arising from an imbalance in bile production and secretion, can lead to damage in extra-hepatic organs, including the reproductive system. This study aimed to investigate the therapeutic potential of Livergol^®, a silymarin-based drug, in the reproductive complications induced by experimental cholestasis in mature male rats. Twenty adults male Wistar rats were randomly divided into four groups (n = 5), including group 1 (control) remained untreated, group 2 (sham) underwent laparotomy and bile duct manipulation without ligation, group 3 (cholestasis) underwent bile duct ligation (BDL) to induce experimental cholestasis, and group 4 (cholestasis + Livergol^®) received oral Livergol^® (300 mg kg^-1) for 4 weeks post-BDL. After the study period, testicular histopathology was evaluated using Johnsen's score and the Cosentino criterion. Epididymal sperm parameters, including count, viability, and motility, were also assessed. The BDL-induced cholestasis caused significant testicular damage, evidenced by a decreased Johnsen's score and an increased Cosentino criterion. It also led to a significant reduction in sperm count, viability, and motility. Treatment with Livergol^® significantly ameliorated these effects, leading to a higher Johnsen's score, a lower Cosentino criterion, and marked improvements in all evaluated sperm parameters compared to the untreated cholestatic group. Oral administration of Livergol^® effectively attenuates testicular damage and improves spermatogenesis and sperm quality in rats with experimental cholestasis. These findings suggest Livergol^® as a promising protective agent against cholestasis-induced reproductive toxicity, likely due to the anti-oxidant properties of its active component, silymarin.

Abstract Testicular ischemia-reperfusion injury during testicular torsion is strongly influenced by oxidative stress caused by excessive accumulation of uncaptured reactive oxygen species. Kisspeptin-10 (Kiss-10), a biologically active fragment of kisspeptin peptide family, has demonstrated significant anti-oxidant and anti-apoptotic properties. Recent studies indicate that Kiss-10 can mitigate oxidative stress by reducing reactive oxygen species levels and enhancing the activity of endogenous anti-oxidant enzymes, such as superoxide dismutase and catalase. This study examined the effects of Kiss-10on ischemia-reperfusion injury in testicular torsion/detorsion (T/D) of male rats. Twenty male rats were divided into four groups, including the control group and three treatment groups (720° T/D, 720° T/D + 0.50 µg kg^-1 Kiss-10, and 720° T/D + 1.00 µg kg^-1 Kiss-10). After inducing 720° clockwise testicular torsion for 2 hr, sperm parameters, such as number, motility, viability, DNA damage, and hypo-osmotic test were evaluated. The results showed that 720° T/D can increase sperm DNA damage. Also, it also had negative effects on overall motility and other properties, such as viability and plasma membrane functionality. The results also showed that administration of Kiss-10to T/D rats can reduce DNA damage. It could also increase overall motility, viability, and plasma membrane functionality compared to the T/D group. Based on our results, Kiss-10 provides significant protection against acute T/D injury to the testis when administered after spermatic cord torsion in rat.

Abstract Cadmium (Cd) is a highly toxic environmental pollutant known to cause severe damage to the male reproductive system. This study aimed to investigate the protective effects of bee bread (BB), a natural product with anti-oxidant, anti-apoptotic, and anti-inflammatory properties, against Cd-induced testicular toxicity in male Wistar rats. A total number of 32 rats were divided into four groups, including control, BB (0.50 g kg^-1), Cd (5.00 mg kg^-1), and Cd + BB (5.00 mg kg^-1 and 0.50 g kg^-1, respectively) groups. Administrations via oral gavage were performed for 4 weeks. Semen analysis revealed significant reductions in sperm motility and density along with increases in abnormal and dead sperm ratios in the Cd and Cd + BB groups compared to controls. Histopathological examination showed severe degeneration and desquamation of germ cells, tubular atrophy, and a decrease in spermatozoa in the Cd-treated groups. Polymerase chain reaction analysis indicated up-regulation of apoptotic markers (caspase-3, -8, and -9) and oxidative stress enzymes (catalase and superoxide dismutase) in the Cd group, signifying disrupted testicular function. The BB administration partially mitigated Cd-induced damage as evidenced by less severe histopathological changes and moderated gene expression alterations. However, the protective effects of BB were not sufficient to completely counteract the toxic impact of Cd. The present study concluded that while BB had potential in reducing Cd-induced testicular toxicity, its protective efficacy was limited, warranting further research to explore its therapeutic potential in combination with other protective agents.

Abstract Buildup of reactive oxygen species during testicular torsion causes oxidative stress and ischemia-reperfusion (I/R) injury in testis. The purpose of this study was to investigate influence of β-cryptoxanthin (BCX) on I/R injury in testicular torsion/detorsion in mature rats. Thirty mature male Wistar rats were divided into five groups of six animals each, including sham group: In this group, midline incision of the scrotum was performed and the testicles were taken out for 2 hr with a 720-degree rotation, I/R group: In this group, midline incision of the scrotum was performed and the testicles were taken out and undergone ischemia for 2 hr with a 720-degree rotation, I/R/Oil group: In this group, a midline scrotum cut was performed, the testicles were taken out, ischemia was created for 2 hr with a 720-degree rotation, and at the end of ischemia 100 µL of corn oil (BCX solvent) was injected intraperitoneally, I/R/BCX10 group: The same as I/R/Oil group, as well as intraperitoneal administration of 100 µL of BCX (10.00 µg kg^-1) at the end of ischemia, and I/R/BCX40: The same as I/R/Oil group, as well as intraperitoneal administration of 100 µL of BCX (40.00 µg kg^-1) at the end of ischemia. Evaluations were based on histopathological and spermatological parameters and oxidative stress assessments. Histopathological spermatological and oxidative stress parameters values obtained from I/R/BCX40 were significantly different from those of other groups (p < 0.05). It could be concluded that BCX could ameliorate testicular injuries in acute testicular torsion/detorsion in mature rats.

Abstract Exposure to extreme temperature conditions such as occurs in certain occupations is known to induce male infertility. In humans and most of the mammals, it has been shown that whole-body heat stress (HS) decreases fertility and produces defective embryos. Hence, the present study aimed at gaining some insights into the mechanisms producing defects after HS. In the present study, 56 mature male Wistar rats were randomly categorized into eight groups (n = 7), including group 1: Control, groups 2: Bioactive peptides (BPs; 10.00 mg kg^-1), groups 3, 4, and 5: Heat-stressed (37.00, 39.00, and 43.00 ˚C for 20 min, respectively), and groups 6, 7, and 8: Heat-stressed along with BPs (10.00 mg kg^-1), respectively. All treatments were administered orally once per day. The HS was induced through the immersion of rat scrotums in a water bath. After 45 days, rats were sacrificed and left testes were removed, fixed, and used for histological and immunohistochemical studies. Harvested right testes were also used for oxidative stress assessments and molecular analyses. Heat stress increased testicular tissue damage, elevated oxidative stress and reactive oxygen species production, and increased germ cells apoptosis, p53 and caspase 3 expressions, and Bax/Bcl-2 ratio. Treatment with BPs as a substance with anti-oxidant properties ameliorated the damage caused by HS. The results of this study highlight the protective role of BPs in the reproductive tract under HS. Bioactive peptides may have potential function against testicular tissue oxidative stress and apoptosis.

Abstract Testicular ischemia-reperfusion (I/R) injury during testicular torsion is strongly influenced by oxidative stress caused by excessive accumulation of unscavenged reactive oxygen species. This study aimed to investigate the effects of intra-peritoneal administration of Mito-TEMPO (MT) on I/R injury in testicular torsion/detorsion (T/D) in mice. Forty-two male mice were divided into seven groups including 1 control and 6 treatment groups (360° T/D, 720° T/D, 360° T/D + 0.70 mg kg^-1 MT, 360° T/D + 1.00 mg kg^-1 MT, 720° T/D + 0.70 mg kg^-1 MT, and 720° T/D + 1.00 mg kg^-1 MT). After inducing 360° and 720° clockwise testicular torsions for 2 hr, sperm parameters, apoptosis-related genes expression, and in vivo fertility index were evaluated. The results showed that 720° T/D can lead to increased abnormal sperm morphology, sperm DNA damage, and Bax expression, while the Bcl-2 expression was reduced compared to the other groups. In addition, it also had negative effects on sperm total and progressive motilities as well as viability and plasma membrane functionality (PMF). The results also showed that administration of MT to T/D mice can result in a reduction in abnormal sperm morphology, DNA damage, and Bax expression. It could also increase sperm total and progressive motilities, viability and PMF, Bcl-2 expression, and in vivo fertility index. Based on our results, it is concluded that MT, when administered after spermatic cord torsion in mice, provides significant protection against acute testicular T/D injury.

Abstract About a third of human infertility is related to male factors. Of these, idiopathic-related infertility is not curable. Diabetes mellitus is a metabolic disorder affecting male impotence and fertility by increased production of free radicals and oxidative stress. Saponin, a glycosidic compound found in many plants, improves sperm parameters. The present study investigated the effect of saponin on sperm oxidative stress and testicular structure in streptozotocin (STZ)-induced diabetic mice. The diabetes was induced by the administration of 150 mg kg^-1 STZ via a single intra-peritoneal injection. All experimental mice were allocated to the following groups: Control group, diabetic control group, diabetic group administrated 100 mg kg^-1 saponin daily and one healthy group administrated saponin daily for 56 days. At the end of the treatment period, serum levels of insulin, glucose and oxidative stress markers were measured. A histological evaluation of testicles was performed. Treatment of diabetic mice with saponin ameliorated testicular tissue damage as well as serum glucose and insulin concentrations. Furthermore, in the diabetic group, the serum concentration of malondialdehyde was increased; while, the activity of superoxide dismutase and glutathione peroxidase enzymes was reduced. The mean Johnsen's score and the diameter and thickness of seminiferous tubules were lower in the diabetic mice than control ones. However, these parameters were higher in the saponin-treated mice than controls. Overall, saponin administration rectified all examined parameters. The anti-oxidant role of saponin improves sperm parameters and diabetes-induced testicular oxidative damage.

Abstract The torsion model of testis in a rat was adopted for evaluation of possible effects of propolis (Prop) on ischemia-reperfusion (IS/REP) injury. The healthy male Wistar rats (totally 24 animals) were randomized into four groups (n = 6) and animals experienced bilateral testicular torsions as follows: In sham group just, laparotomy was performed and in IS group, animals experienced a 3 hr period testicular IS. In IS/REP group, a 3 hr period of IS followed by a 3 hr period of testicular REP for left testis and a one-week testicular REP for right testis were done. In this group animals were gavaged by 1.00 mL normal saline 1 hr before the onset of IS. In IS/REP/ Prop group, the same procedures for IS/REP animals were followed as well as gavage of 1.00 mL Prop extract solution 1 hr before the onset of IS. Analyses of biochemistry, histology, inflammatory biomarkers and sperm parameters were carried out. In IS/REP/Prop group, nitric oxide synthase malondialdehyde, myeloperoxidase and 8-hydroxy-2 deoxyguanine in IS/REP/Prop group were significantly decreased and, superoxide dismutase, total glutathione, glutathione peroxidase, glutathione reductase and glutathione S-transferase were significantly increased compared to the other animals. In IS/REP/Prop group, seminiferous tubules (with normal spermatogenesis) showed all stages of spermatogenic cells with plentiful spermatozoa. Tubular deterioration and atrophy and spermatogenic cell loss in were seen in a limited extent. The mean concentrations of Interleukin-1 beta and tumor necrosis factor alpha in IS/REP/Prop were significantly decreased. Sperm quality was significantly improved by Prop in IS/REP/Prop group. It was concluded that Prop could be supportive in diminishing IS/REP injury in testicular tissue exposed to ischemia.

Abstract Fluoxetine is a selective serotonin reuptake inhibitor is commonly prescribed to treat maternal depression in pregnancy and lactation. This study aimed to investigate the effects of maternal exposure to fluoxetine via lactation on testicular tissue, sperm parameters including count, motility, viability, and normal morphology and testicular oxidative stress status in male mice offspring. Ten mice dams were divided into control and experimental groups. The control group received water and the experimental group received fluoxetine (20 mg kg^-1) by gavage daily from postnatal days of 0-21. Histology of testis, sperm parameters and oxidative stress in the testicular tissue were analyzed at 80 days after birth in their male offspring (n = 8). Significant reductions in the body and testes weights were observed in animals exposed to fluoxetine. Additionally, fluoxetine exposure significantly reduced all sperm parameters, tubular diameter and epithelial height of the seminiferous tubules as well as Leydig cells number. Significant increases in the testicular malondialdehyde levels and percentage of sperm with chromatin/DNA damage were observed in mice exposed to fluoxetine compared to control. These findings suggest that maternal exposure to fluoxetine during lactation in mice has a negative effect on the testicular tissue of their offspring and impairs the spermatogenesis process which in turn can induce infertility.

Abstract Methotrexate (MTX) as a chemotherapeutic agent, has adverse effects on reproductive organs by enhancing oxidative stress. In this study, the protective effects of Cornus mas fruit extract (CMFE) against MTX side effects were evaluated. Forty-eight mature male NMRI mice were divided into six groups: group 1 (control) received 0.10 mL per day of normal saline intraperitoneally (IP), group 2 received MTX (20 mg kg^-1 per week, IP), group 3 received MTX along with CMFE 250 mg kg^-1 per day by oral gavage, group 4 received MTX along with CMFE 500 mg kg^-1 per day by oral gavage, group 5 received MTX plus 1000 mg kg^-1 per day of CMFE by oral gavage, and group 6 received 1000 mg kg^-1 per day of CMFE extract, orally. All animals were treated for 35 consecutive days. Thickness of testicular capsule and germinal epithelium and diameter of seminiferous tubules were measured. Intra-cytoplasmic levels of carbohydrate, unsaturated fatty acid (UFA) and alkaline phosphatase were assessed. Serum level of testosterone and testicular total antioxidant capacity (TAC) were also evaluated. The results demonstrated that MTX administration caused morphometrical parameters except the thickness of testicular capsule were significantly different in comparison to control group and decreased cytoplasmic concentration of carbohydrate in the first three layers of germinal epithelium and increased the UFA levels. Contrarily, CMFE ameliorates the condition. Moreover, CMFE increased testosterone level and increased the MTX-reduced TAC level. In conclusion, it was revealed that CMFE decreased the cellular atrophy by controlling the energy substrate utilization based on lipids and carbohydrates via provoking the testicular antioxidant status.

Abstract Fertility reduction due to sub-fertile males is a major concern in breeder flocks. Phenotypic traits of broiler breeder males and their relationships with fertility can be used as reliable indicators for identification and removal of sub-fertile males from the breeder flocks. This study was conducted to investigate semen traits (semen volume, sperm motility, sperm viability and sperm count) and testes histomorphometric features including tubule differentiation index (TDI), spermiation index (SPI), Sertoli cell index (SCI) and mitotic index (MI) of broiler breeder males with the same age but different phenotypic traits. According to phenotypic traits, 12 broiler breeder males (Ross-308 strain) were classified into three equal groups. Group 1: roosters with fertile phenotypic traits (fertile), group 2: roosters with the lowest fertile phenotypic traits (sub-fertile) and group 3: roosters with moderate fertile phenotypic traits (moderate). The results confirmed potential relationship between phenotypic traits and fertility in broiler breeder males. Semen traits and histomorphometric features of broiler breeder males' testis of the group 3 were more similar to those of the fertile roosters. Therefore, it can be concluded that exclusion of these roosters from the breeder flock may have undesirable effects on flock fertility.

Abstract Unilateral orchidectomy (UO) is required when further breeding potential is important. It is sometimes necessary to remove a single testis in a matured stallion for therapeutic reasons. In this study, twelve donkeys were used to evaluate three techniques of unilateral castration, histological and morphological changes on the remaining testis. Results of the study showed that each of the surgical techniques used had its advantages and disadvantages in comparison with the other two techniques. Therefore the selection among the three techniques depends on the surgeon preferences and the environment in which the unilateral orchidectomy is performed. The volume of the remaining testis recorded at the end of the study was significantly greater than that estimated at the start of the study (p < 0.05). The percentage of sperm motility obtained from the remaining testis was significantly decreased (p < 0.05). Histological examination of the testis in open surgery (group I) (where the scrotum was left opened) revealed severe hemorrhages, edema and fibrosis. The testis showed degenerative changes in the seminiferous tubules and interstitial orchitis. Histological examination of the testes removed using a closed technique, (in groups II and III) where the scrotum wound was sutured, revealed hyperplasia of spermatogenic series and Leydig cells. In conclusion, unilateral orchidectomy had compensatory effects on the weight and volume of remaining testis. Adverse effects on sperm motility and viability can affect the fertility of the animal.

Abstract Systemic amyloidosis and testicular interstitial tumor are rare conditions in birds and this is the first report in Iran. A male zebra finch was presented because of white diarrhea, anorexia, loss of weight and lethargy. At necropsy, the small intestine was edematous and congested. The spleen appeared pale. The liver was large, firm and brown. One testis was cystic and neoplastic and the remaining testis was atrophic. Histologically, amyloid materials were seen predominantly in the liver and spleen. Hyaline substances were deposited in the Disse space and in the media of blood vessels of the liver. In spleen, marked deposits thickened the basement membranes of blood vessels and extended into the surrounding parenchyma. In addition, there were lesser degrees of amyloidosis in other organs such as small intestine. Amyloid stained positively with Congo red. In testis, there was encapsulated unilateral interstitial cell tumor, with multiple foci of necrosis and hemorrhage. The neoplastic cells were round to polyhedral, with small round hyperchromatic nuclei and finely vacuolated cytoplasm. Signs of feminization were observed. The cause of amyloidosis in this study was not conclusively identified.