Veterinary Research Forum

Abstract The effects of substituting whole egg yolk (WEY) in a tris-citrate-based extender with two derived fractions, including a buffer-soluble fraction (BSF) and an ammonium sulfate insoluble yolk fraction, on the freezability of bull sperm were investigated. The BSF and ammonium sulfate insoluble yolk fraction levels of egg yolk in the respective diluents were consistent with their extracted values from an equivalent volume (20.00%) of egg yolk in the control diluent. The extenders were then enriched with 0.00, 5.00, and 10.00% (v/v) of pasteurized skim milk. Semen samples were collected weekly over five consecutive weeks from six adult bulls, and the standard ejaculates were pooled. The pooled semen was subsequently divided into seven experimental extenders and frozen in 0.50 mL French straws. Various sperm quality parameters, including kinematics, acrosome integrity, capacitation, and DNA fragmentation, were evaluated post-thawing. Results indicated that sperm kinematics and the percentage of acrosome-reacted sperm in experimental extenders were not significantly different from those in the control group. The milk-free ammonium sulfate insoluble yolk fraction extender exhibited higher percentages of capacitated sperms, but lower percentages of spermatozoa with intact DNA compared to the WEY extender sperm. The addition of 10.00% milk into the BSF diluent resulted in a significant increase in the proportion of sperm with intact DNA and a notable decrease in the percentage of sperm with partially fragmented DNA compared to the control. In conclusion, a BSF extender enriched with 10.00% cow's skimmed milk is recommended as a substitute for WEY in the cryopreservation of bull semen.

Abstract During the freezing process of semen, due to the generating of significant amounts of free radicals, the quality of sperm changes. Epigallocatechin-3-gallate (EGCG) is a green tea catechin, which in this study was applied to investigate its effect on the quality of bulls' sperm. We collected semen samples with an artificial vagina from 12 Simmental bulls to evaluate the effect of EGCG (10.00 and 20.00 µmol) in two cryopreserving methods on the quality parameters of semen. We designed six groups including two control groups (method one and two) and four treatments (EGCG 10.00 µmol + method one; EGCG 20.00 µmol + method one; EGCG 10.00 µmol + method two; EGCG 20.00 µmol + method two). The 20.00 µmol EGCG and a method two significantly affected the amending oxidative conditions as well as an increase in total antioxidant capacity and a decrease in malondialdehyde. The effect of EGCG in both concentrations was more on method two. The desired impact on sperm motility, viability, inhibition of lipid peroxidation and sperm DNA damage was observed in EGCG groups compared to control groups. Among the two methods, the method two had fewer adverse effects on the plasma membrane, motility parameters, viability and DNA of sperm. The EGCG in the semen extender yielded a favorable impact on thawed sperm. This effect was prompted in combination with the method two.

Abstract Current study was aimed to evaluating protective effects of cornus mas fruit extract (CMFE) in mice treated with methotrexate (MTX). For this purpose, 48 young mature male mice were divided into 6 groups. Control group received only normal saline (0.1 mLper day, intraperitoneally), and the second group was administered MTX (20 mg kg^-1 perweek, intraperitoneally). The third, fourth and fifth groups received MTX daily oral doses of 250, 500 and 1000 mg kg^-1 CMFE as well as MTX. The sixth group was only given CMFE with a dose of 1000 mg kg^-1 perday, orally, for 35 days. Then, the animals were anesthetically euthanized and the sperms were separated from epididymis. DNA damage level, the amount of malondialdehyde (MDA) as well as in vitro fertility was evaluated. The number of sperms with damaged DNA and MDA level in MTX-treated group showed a significant increase compared to control group (p < 0.05). In groups receiving CMFE along with MTX, DNA damage level and MDA amount suggested a decrease in comparison with MTX group (p < 0.05). Also, in vitro fertilization and embryonic development in MTX-treated group was significantly lower than the control group, and the level of embryonic arresting was higher (p < 0.05). In groups which received CMFE along with MTX, in vitro fertility and embryonic development was higher than MTX group (p < 0.05) and the arrested embryos showed a decrease. This study suggested that cornus mas is able to ameliorate the side effects of MTX.

Abstract Cyclophosphamide (CP) is one of the anti-neoplastic drugs. Despite its numerous clinical applications, it has devastating effects on the testicles and declines the sperm quality in treated patients. This study was aimed to investigate the protective effect of crocin in improving the toxicity induced by CP in reproductive system. In this study, 24 male adult mice (6 to 8 weeks) were randomly divided into three groups, control group received normal saline (0.1 mL, IP, daily), the CP group received CP (15 mg kg-1, IP, weekly) and the CP + crocin group received CP along with crocin (200 mg kg-1, IP, daily). After 35 days of treatment, animals were sacrificed. The samples of epididymis in human tubal fluid medium incubated for 30 min in 5% CO2 for flotation of sperm. Sperm were obtained from caudal epididymis using dissecting method. Then, the parameters of sperm quality including sperm count, motility, viability, DNA damage, nuclear maturation, and sperm morphology were evaluated. In CP group, the sperm count, motility, viability, nuclear maturation and sperm morphology were significantly decreased compared to control group (p < 0.05) and in the CP + crocin group all of these parameters significantly increased compared to CP group (p < 0.05). The percentage of sperm with DNA damage in the CP group significantly increased compared to other groups (p < 0.05). The results of this study indicated that the crocin was able to suppress free radicals and enhance the quality of sperm in CP treated animals.

Abstract This study was aimed to assess the protective effects of Cornus mas fruit extract (CMFE) and vitamin E (Vit E) on sperm quality parameters in the methotrexate (MTX)-treated mice. Forty-eight young adult male mice (8-12 weeks) were randomly divided into six groups including control and test groups. The control group received normal saline orally , and the test groups were treated MTX (20 mg kg^-1, ip, once weekly), MTX + CMFE (250 mg kg^-1), MTX + CMFE (500 mg kg^-1), MTX + CMFE (1000 mg kg^-1), and MTX + Vit E (100 IU kg^-1, po) for 35 consecutive days. On day 35, after euthanasia the epididymal sperms were isolated. Then the total mean sperm count, sperm viability and motility were determined. The total antioxidant capacity (TAOC) of all experimental groups were also evaluated. The MTX-treated animals showed a significant changes in all parameters of sperm quality assessment compared to the control group. Both Vit E and CMFE were able to protect from MTX-induced effects on sperm maturity and DNA damage. Co-administration of MTX and CMFE and/or Vit E resulted in protection from MTX-reduced TAOC. In conclusion, these data suggested that MTX administration could adversely affect the sperm quality. Moreover, the protective effect of Vit E and CMFE on MTX-induced sperm toxicity was also documented.

Abstract The purpose of this study was to evaluate the probable effects of leptin addition in different levels to the semen extender on sperm quality (motility and motility parameters, viability, sperm membrane integrity, and DNA damage). Semen specimens were evaluated immediately after leptin addition, equilibration time and after thawing the frozen semen. Five healthy buffalo bulls (5 ejaculates from each bull) were used. Each ejaculate was diluted at 37 ˚C with tris-based extender containing 0 (control), 10, 20, 50, 100, and 200 ng mL^-1 leptin. The diluted semen was kept 4 hr in refrigerator to reach to the equilibration time and then packed in 0.5 mL French straws and frozen in liquid nitrogen. Our results showed that, in the fresh semen, no significant difference was observed in all sperm quality parameters evaluated among all of the examined leptin concentrations. Addition of 10 ng mL^-1 leptin into semen extender significantly preserved sperm motility, all of the motility parameters, and viability in equilibrated semen compared to that of control group. However, in vitro addition of 200 ng mL^-1 leptin, significantly decreased theses parameters. In the frozen thawed semen, all leptin concentrations decreased sperm motility and viability, but significant decrease was observed in concentrations of 100 and 200 ng mL^-1. Adding leptin to semen extender did not have any significant influence on sperm DNA damage and sperm membrane integrity in all examined groups. These findings suggest that in vitro addition of 10 ng mL^-1 leptin could preserve sperm motility and viability in cooled semen of buffaloes.

Abstract The objective of this study was to investigate whether or not the adverse effects of heat on sperm quality could be prevented by green tea extract (GTE) administration. Ninety adult male mice were randomly divided to two groups. The scrotum of each animal in the first group was immersed once for 20 min in a water bath maintained at 42 oC (heat group, H) and the second group (control group, C) was maintained at 23oC. Heat-treated and control groups were allocated randomly into three subgroups. The first subgroup from heat-treated mice was administered sterile saline (heat saline, HS) and the two other subgroups were administered orally with two different doses of GTE including 500 and 750 mg kg-1 (HG500 and HG750) for 49 consecutive days. Likewise, the first subgroup from control mice was administered sterile saline (control saline, CS) and the two other subgroups were administered orally with 500 and 750 mg kg-1 of GTE (CG500 and CG750), respectively. Heat stress significantly reduced (P < 0.05) sperm concentration, total sperm motility, progressive sperm motility and hypo-osmotic swelling-water test positive spermatozoa at the first 14 days after the heat treatment. However, a partial recovery was observed at the day 42, which was still significantly lower than that of the CS group. Administration of GTE in two doses (HG500 and HG750 treatment groups) following heat treatment could significantly recover adverse effects of heat on above-mentioned parameters during the first 28 days. The present study demonstrates that the adverse effects of hyperthermia on semen parameters might be recovered following administration of green tea extract in a short period of time.

Abstract The purpose of this study was to evaluate the probable effects of the vitamin E addition in different levels to the extender of chicken semen on spermatozoa quality during storage of semen at 4°C for 0, 3, 6, 10 and 24 hours. Eight young Ross broiler breeder strain 308 roosters were used in this experiment. The collected semen from all roosters was mixed together and diluted with modified a Ringer’s solution. The diluted pooled semen was divided into 5 treatments (T). T1 was a control group without any vitamin E addition. For T2 to T5 groups 0.5 %, 1 %, 2 % and 3 % vitamin E (w/v), were added respectively. Treatments were evaluated for sperm motility, sperm viability and probable morphological defects after 0, 3, 6, 10 and 24 hours of incubation at 4°C. The evaluations of spermatozoa immediately after semen collection, were revealed no significant differences among values of treatment groups, whereas after incubating the treatments for different spans of time, the sperm progressive motility and viability rates for groups supplemented with vitamin E were significantly (P < 0.05) higher than that of the control group. In addition, morphological defect rates of chicken spermatozoa in the groups supplemented with different levels of vitamin E were significantly (P < 0.05) lower than that in control group. According to the results of this study we conclude that, the most excellent level of vitamin E for supplementation to the extended semen of chicken in order to improve the sperm motility and viability plus to reduce the morphological defect rates of the spermatozoa up to 24 hours storage time at 4°C is 2 % (w/v).