Veterinary Research Forum

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 Anti-oxidants are vital for protecting sperm and can mitigate the negative effects associated with cryopreservation. This study was conducted to evaluate the protective effects of adding anti-oxidants (vitamins and amino acids) to extender in sex-reversed rainbow trout (Oncorhynchus mykiss) sperm. The collected sperm was diluted at the ratio of 1 : 5 by the extenders supplemented with different anti-oxidants including 1.00 mM of ascorbic acid  and L-tryptophan, 2.00 mM of cysteine and α-tocopherol, and their combination. After dilution, the semen was aspirated into 0.50 mL straws, and the straws were placed on the tray, frozen for 10 min, and plunged into liquid nitrogen. Straws were thawed in a 30.00 ˚C water bath for 15 sec. The Sperm Class Analyzer System was used to evaluate sperm kinematics. The activity of anti-oxidant enzymes and lipid peroxidation were determined as oxidative stress indices. Our data indicated that the incorporation of anti-oxidants and amino acids increased sperm motility duration. The elevated activity of glutathione peroxidase, superoxide dismutase, and catalase in post-thaw samples indicates that the anti-oxidant system in sex-reversed rainbow trout sperm likely plays a crucial role in protecting membrane compounds from oxidation. In conclusion, the combination of 1.00 mM L-tryptophan and ascorbic acid to the extender media caused a prolonging effect in sperm motility after thawing and they have the potential to serve as effective agents for improving sperm cryosurvival.

Abstract Diabetes is a metabolic disorder characterized by hyperglycemia due to the defects in insulin secretion and function, or both. Glabridin (GBD) is one of the natural anti-oxidants used for infertility treatment. This study was planned to evaluate the effects of GBD on testicular oxidative status, sperm characteristics, and early embryo development in diabetic mature mice. Forty mature male mice were allotted to five equal groups, including control group received no treatment, diabetic group received intraperitoneal streptozotocin (50.00 mg kg^-1), and three experimental groups receiving 12.50, 50.00, and 200 mg kg^-1 GBD by gavage daily for 30 days, respectively. Serum levels of testosterone, sperm parameters, and testicular malondialdehyde, total anti-oxidant capacity, and catalase levels, as well as pre-implantation embryo development were determined. The diabetic group exhibited significantly reduced sperm motility, viability, and count, testosterone level, and testicular total anti-oxidant capacity and catalase levels, and increased testicular malondialdehyde level, and DNA-damaged and immature sperms along with poor in vitro fertilization outcomes compared to the control group. In contrast, the GBD administration, particularly at the highest dose, caused a pronounced improvement in the above-noted parameters. These findings suggest that GBD may play a role in impeding diabetes-induced male reproductive complications through oxidative stress repression, and sperms and early embryos protection.

Abstract This study examined the effects of alpha-ketoglutaric acid (AKG; 2.00, 4.00, and 8.00 mM L^-1) and folic acid (FA; 50.00 nM L^-1) on bull sperm cells under ammonia stress induced by 175 µM L^-1 ammonium chloride. Sperm parameters including kinematic motility, survival rate, membrane integrity, DNA integrity, superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), total antioxidant capacity (TAC) activities, and apoptosis were assessed in the groups. Sperm motility indices, survival rate, plasma membrane integrity, SOD, CAT, TAC, and GPx enzymes activity, DNA damage, and apoptosis in the treated groups were significantly differed from those in the control groups. The AKG concentrations of 4.00 and 8.00 mM L^-1 co-supplemented with 50.00 nM L^-1 FA mitigated the negative effects of ammonia on sperm cells. This study indicated that supplementation with AKG and FA at the desired concentrations counteracted the adverse effects of ammonia toxicity that preceded clinical signs. Further studies are needed to evaluate the fertility of these sperms, either in vitro or in vivo.

Abstract Chemotherapy agents impose harmful side effects beside their therapeutic properties. This study aimed to uncover the ameliorative effects of thiamine (VitB1) on sperm characteristics and in vitro fertilizing ability in methotrexate (MTX) exposed mice. For this purpose, 36 apparently healthy adult male mice were randomly divided into six groups (n = 6), including control (0.10 mL normal saline per day; intraperitoneally [IP]), sham (10.00 mg kg^-1 per week MTX; IP), positive control group (100 mg kg^-1 per day VitB1; IP), and three experimental groups (25.00, 50.00, and 100 mg kg^-1 per day; IP VitB1 plus MTX, respectively; IP). Then, sperm parameters and the in vitro fertilizing potentials were assessed. The sperm count, motility, and viability in the sham group showed a significant decrease in comparison with the VitB1-treated and control groups. In the VitB1 100 mg kg^-1 + MTX group, there was a significant increase in sperm count in comparison with the sham group. The sperms with DNA damages and immature sperms in the sham group significantly increased in comparison with the positive control and control groups. In vitro fertilization and embryonic development in the sham group were decreased in comparison with both the control and VitB1 groups. In VitB1 50.00 mg kg^-1 and 100 mg kg^-1 + MTX groups, there was an increase in the fertilization rate, along with embryonic development promotion. According to the results of this study, thiamine ameliorates toxic effects of MTX on sperm traits and in vitro fertilizing potentials by constraining oxidative stress.

Abstract Cyclophosphamide leaves an undesirable effect on testes. This study was conducted to explore the effects of the Phoenix dactylifera (DP) on testes following the cyclophosphamide treatment. Thirty-six male mice were divided into six groups, one control, one cyclophosphamide, two groups of cyclophosphamide with a dose of 200 mg kg^-1 and a dose of 400 mg kg^-1 DP, and two of only high and low doses of DP. All groups were gavaged daily for 28 days. The animals were euthanized 24 hr after implementing the last treatment. Then, the testes and epididymis samples were removed and weighed. The main sperm characteristics such as the number of sperm and sperm viability, the morphometric changes, biochemical analysis of testes and enzyme activity were investigated. With the cyclophosphamide group, only body weight, testes weight, epididymis weight, sperm viability and the fertilization percentage were decreased significantly compared to the control group. Moreover, the spermatogenesis indices and morphometric parameters in this group indicated a significant decrease. Furthermore, the morphological changes were observed in the testicular tissue, including seminiferous tubule atrophy, vacuolation, hyperemia of blood vessels and increased space in the interstitial tissue. In the biochemical study of cyclophosphamide group, the amount of glutathione peroxidase in serum was decreased, whereas, the amount of malondialdehyde in testicular tissue showed a significant increase. The DP group included the antioxidant and anti-apoptotic properties. It seemed that the compounds in the DP would lead to the inhibition of the production of active metabolites released from the cyclophosphamide.

Abstract The aim of this study was to determine the potential fertilizing of spermatozoa from the epididymal tail in different periods of time post-orchiectomy (P-OQ). Therefore, the study was approached in two stages. In the first stage, the orchiectomy was performed in 30 adult pigs. The testicles were stored at 5.00 ˚C in physiological saline solution for 5, 24, 48, 72, 96 and 120 hr. The spermatozoa were obtained by backflushing the vas deferens. The spermogram and fluorometric study were performed for each sample to evaluate the exposure of phosphatidyl-serine (PS) and acrosome reaction (AR). The second stage included the fertilization test, 16 prepubertal sows were selected, after synchronizing the oestrous cycle and the post-cervical artificial insemination was performed with the refrigerated sperm samples from each P-OQ time. The percentage of live sperm remained without significant changes until 96 hr P-OQ. An increase in the percentage of spermatozoa that showed a PS exposure was observed. The premature AR was evident after 72 hr. Considering that the artificial insemination was performed ensuring a minimum number of live sperms, no significant differences were observed in the number of embryos and corpora lutea. The results indicated that pig sperm collected from the epididymal tail P-OQ and stored for 5 and up to 72 hr at 5.00 ˚C had viable characteristics and maintained their fertilization ability. However, there was an increase in the loss of phospholipid asymmetry of the plasma membrane as time increased (72 and 96 hr), therefore, sperm viability was decreased.

Abstract This study evaluated the possible protective effect of royal jelly (RJ) on sperm parameters and sperm malondialdehyde (MDA) concentration and in vitro fertilizing potential in nicotine (NIC) exposed male mice. Thrtiy-six male BALB/c mice were randomly divided into six groups (n = 6). Group 1 received normal saline, group 2 received 100 mg kg^-1 per day RJ, groups 3 and 4 received NIC at doses of 0.50 and 1.00 mg kg^-1 per day, respectively and groups 5 and 6 received NIC at doses of 0.50 and 1.00 mg kg^-1 per day, respectively plus RJ. Caudal epididymal sperm characteristics, lipid peroxidation and in vitro fertilizing capacity and embryo development were evaluated after 35 days. The NIC treatment caused a significant decrease in sperm motility and viability and fertilization rate along with poor blastocyst formation and increased sperm DNA damage and MDA levels. Moreover, the incidences of chromatin abnormality in spermatozoa were significantly higher in NIC-exposed mice than those of control. Nevertheless, RJ treatment improved sperm parameters and in vitro fertilization outcome as well as sperm lipid peroxidation level. Data from the current study suggest that RJ has a potential repro-protective action against NIC-induced sperm abnormalities and embryotoxicity in mice.

Abstract This study was aimed to investigate the effects of 17 𝛽-estradiol (E2) and 𝛼-zearalenol (α-ZOL) on motility parameters, plasma membrane integrity, levels of produced nitric oxide (NO) and total antioxidant capacity of Ghezel ram sperm during the liquid storage at 4 ˚C, for various periods of time. Semen samples were collected from four rams and diluted with Tris–egg yolk extender and supplemented with E2 (100 µmol) or different concentrations of α-ZOL (100 pmol, 100 nmol and 100 µmol) at a final concentration of 200 × 10⁶ sperm per mL. We failed to show any significant effect of E2 at 100 µmol concentration on ram’s sperm parameters while α-ZOL resulted in a significant decrease of plasma membrane integrity at 100 µmol concentration (55.40% for α-ZOL vs 62.20% for control) after 96 hr incubation. Alpha-ZOL had decreasing effect on sperm motility parameters including curvilinear velocity and average path velocity at 100 µmol concentration after 96 hr storage. Although remarkable reduction of total antioxidant capacity at high concentration of α-ZOL and long incubation time was found, however no significant changes were recorded in NO level during storage time. It was concluded that the detrimental effect of α-ZOL on ram sperm might be attributed to its induced oxidative stress and damage to the plasma membrane.

Abstract Nicotine (NIC) adversely influences male reproductive system. Achillea millefolium (Achm) as a medicinal plant is highly regarded for its antioxidant and anti-inflammatory properties. The present study was conducted to assess whether Achminflorescences alcoholic extract could serve as a protective agent against reproductive toxicity in NIC-exposed male rats. Adult male rats were randomly divided into six groups. Two groups received NIC at doses of 0.20 and 0.40 mg kg^-1 per day in 0.50 mL sterile distilled water for 48 days intraperitoneally, respectively. The further two groups received NIC at doses of 0.20 and 0.40 mg kg^-1 per day in 0.50 mL sterile distilled water for intraperitoneally along with Achm extract at a dose of 1.20 g kg^-1 per day in 1 mL sterile distilled water orally for 48 days, respectively. A vehicle treated control group and an Achm-only treated group were also included. The NIC-exposed groups showed significant reductions in epididymal sperm count, motility, viability and serum levels of FSH, LH and testosterone as well as testicular antioxidant capacity. Moreover, the incidence of apoptosis and abnormality in spermatozoa along with testicular malondialdehyde and total nitrite levels were significantly higher in NIC-treated rats. The above-mentioned parameters were restored to near normal levels by Achm co-administration. These findings indicated thatAchmmay partially be protective against NIC-induced testicular toxicity.

Abstract Cryopreservation has the capacity to extend spermatozoa’s lifespan and viability. In addition, the semen samples can be collected, preserved and stored or sent to distant locations and still be used long after the death of the semen donor. In this study for the vitrification of dog sperm (fresh and swum-up sperm), different cryopreservation mediums on the basis of glycerol, milk and egg yolk were used. Then, all of the samples were vitrified in the liquid nitrogen and thawed at least 48 hr later for re-examination of sperm parameters. The sperm parameters before and after cryopreservation in all groups were compared. It was found that during vitrification process, spermatozoa were damaged by the mechanical blows in centrifugation during swim-up processing, so they had less resistance than fresh semen. The examination of different cryoprotectants revealed that milk has better effects on the cryopreservation of semen than glycerol and egg yolk. With the comparison of the effects of glycerol and egg yolk as cryoprotectants, it was found that glycerol had better effects than egg yolk on the cryopreservation of the semen. In conclusion, milk might be used as a cryoprotectant instead of glycerol for canine sperm cryopreservation.

Abstract Formalin is widely used in industry and in medicine (as tissue fixative and disinfectant).It contains reactive molecules which have been known for its cytotoxic effects. To evaluate the effect of formalin exposure on the testicular tissue and sperm parameter from neonatal period through physical and sexual maturity, 28 male Wister rats were assigned into two equal test and control groups. The test group was exposed to 1.5 ppm of the vapour of 10% formaldehyde in a special chamber for 2 hr per day at 20-26 ˚C and the air pressure of 760-763 atm. After 55 days, the tubular differentiation (TDI) and repopulation (RI) indexes in testicular tissue, sperm quality parameters, serum total antioxidant capacity and testosterone level were determined. The formaldehyde-exposed animals showed severe seminiferous tubules atrophy, edematous connective tissue, arrested spermatogenesis with negative TDI and RI and vascular thrombosis compared to control group. Histomorphological studies showed a high sperm mortality and abnormality associated with a remarkable decrease in sperm count. Formaldehyde-exposed animals revealed with decreased serum level of testosterone (p < 0.05) and down-regulated antioxidant status versus control group. In conclusion, the current data provide inclusive histological and biochemical information about the chronic exposure to formaldehyde with emphasizing on reproductive disorders including histological adverse effects on the testicular tissue, spermatogenesis, sperm viability, count and the abnormalities which can potentially cause infertility after sexual maturation.

Abstract Gibberellic acid (GA3) is a group of plant hormones identified in various plants. The aim of this study was to determine the effects of GA3 on sperm parameters and in vitro fertilization (IVF). Fifty six adult male rats were divided into seven groups as, control, treatment and sham. Following 15, 30 and 45 days of GA3 and methanol alcohol (MA) administration, rats were euthanized and epididymis tail was transferred to human tubular fluid (HTF) medium containing 4 mg mL^-1 bovine serum albumin (BSA) .Total number of sperms, the percentage of live sperms, immature sperms and sperms with damaged chromatin and IVF were examined. The oocytes were obtained from immature rats after the injection of pregnant mare's serum (PMSG) and human chorionic gonadotropin (HCG) hormones. Human tubular fluid was used as the fertilization medium and zygotes transferred to fresh 1-cell rat embryos culture medium (mR1ECM) to reach the blastocyst stage. This study showed that GA3 could decrease the number of total sperms on days 30 and 45 in treated group comparison with the control and sham groups. Additionally, GA3 increased the immature sperms and sperms with damaged chromatin. The percentage of fertilization, two-cell embryos and blastocyst resulting from the treatment group on days 30 and 45 also decreased and showed significant differences with the control and sham groups (p < 0.05). The results obtained from this study indicated that the oral use of GA3 could reduce the fertility in rats by influencing the sperm number and the quality of sperm’s chromatins.

Abstract Reproductive parameters evaluation is considered as helpful tool for gene bank formation in ecological and economically important animals species. Gloydius halys caucasicus is venomous, viviparous pit viper of northwest of Iran. In this research, the spermatic reproductive parameters of this taxon were studied. Twenty six male snakes were collected from Takht-e-Soleiman region between September and October, 2010. Findings revealed that male snakes with body length of 45.07 ± 2.83 cm and body weight of 51.50 ± 10.42 g, and right and left gonads volume of 0.12 ± 0.03 mL and 0.06 ± 0.01 mL are mature ones and sperms concentration in first, middle and final regions of vas deferens duct were, 22.30 ± 19.34 ×106 mL-1, 30.34 ± 11.55 ×106 mL-1, and 37.65 ± 16.46×106 mL-1, respectively. The sperms motility at three regions of duct were 60.53%, 62.07%, and 60.00% and percentage of immotile sperms in these regions were 39.46%, 37.92%, and 39.84%, respectively. Percentage of morphologically normal sperms was 69.23 ± 10.57% and abnormal sperms was 30.76 ± 10.57%; including 12.69 ± 5.25% spiral tailed, 7.33 ± 4.37% coiled tailed and 4.16 ± 2.51% folded tailed sperms. Percentage of live sperms in the first, middle and final regions of duct were 55.76 ± 10.77%, 58.84 ± 12.77%, and 57.69 ± 9.91%, respectively and percentage of dead sperm in these regions were 44.23 ± 10.77%, 41.15 ± 12.77%, and 42.30 ± 9.91%, respectively. Results suggested, mature sperms with acceptable reproductive quality could be collected from Gloydius halys caucasicus snake of Iran between September and October.

Abstract The objective of this study was to evaluate quality of frozen-thawed bull semen processed with extenders containing vitamin C and E as antioxidants. Pooled semen's were collected from 5 bulls and diluted to a concentration of 30 × 106 sperm/mL with citrate –egg yolk (CEY) or Tris – egg yolk (TEY) extenders. The diluted semen was divided to 5 aliquots including control and 4 experimental groups. Each aliquot was further diluted with an equal volume of CEY or a Tris (hydroxymethyl) aminomethane (TRIS) - based extender without (control) or containing vitamin C 1mM or 2mM and vitamin E 0.1mM or 0.2mM, and routine semen evaluations like sperm motility, viability and measurement of lipid peroxidation (LPO) were conducted. Significant reductions of LPO were achieved by addition of 1mM vitamin C and 0.1 mM vitamin E to CEY extender (P < 0.05). The highest sperm viability was achieved by addition of 0.1mM vitamin E to CEY extender. Supplementing CEY extender with 2mM vitamin C and 0.1mM vitamin E improved the sperm motility compared with the control group. On the basis of the present results it is concluded that vitamin C and E are very efficient antioxidants in CEY extender.