• Clinical and Experimental Reproductive Medicine
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• 제13권2호
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• pp.175-180
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• 1986

It has been reported that oral kallikrein therapy exerts a favourable effect on sperm motility in asthenozoospermic patients. In order to evaluate the efficacy of kallikrein on asthenozoospermia, a total of 20 subfertile male patients with varicocele, whose sperm counts were less than $40{\times}10^6/ml$ and sperm motility was less than 30%, was subjected to this clinical study (Table 1). They were divided into 2 study groups: 1) Varicocelectomy group consisted of 10 patients with varicocele (grade II-III) who underwent varicocelectomy. 2) Kallikrein group was composed of 10 patients with varicocele (grade I) who were given kallikrein orally 600 KU (kallikrein unit) daily divided 3 times after meal for 3 to 9 months. Semen analyses were repeated twice before the study, once a month during the study and twice after the study. Effective results designate that sperm parameters improved more than 30% from the basical levels after varicocelectomy or kallikrein exposure. Sperm counts increased from $32.5{\times}10^5/ml$ to $45.5{\times}10^6/ml$ after varicocelectomy in 3 patients and sperm motility increased from 25% to 38.5% after varicocelectomy in 3 patients. Pregnancy occurred in 2 patients of 3 responders and 1 patient of 7 non-responders 3 to 6 months after varicocelectomy in Varicocelectomy group. Sperm motility increased from 28% to 40.2% after kallikrein treatment in 3 patients. Pregnancy occurred in 2 patients of the 3 responders in Kallikrein group (Tables 2-3). There were no significant changes in volume and morphology in Varicocelectomy group before after varicocelectomy and no significant changes in volume, counts, and morphology before and after kallikrein exposure. No remarkable side effects were noted with kallikrein treatment.

• 농업과학연구
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• 제47권3호
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• pp.657-665
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• 2020

The objective of this study was to investigate whether supplementation of pentoxifylline (PTX; phosphodiesterase inhibitor) to thawed boar semen improves the post-thaw motility of sperm and affects the efficiency of artificial insemination (AI) and further development. To determine the concentration of PTX for AI, frozen-thawed semen was incubated with 0, 5, 10, and 20 mM PTX in an extender freezing medium, respectively, after thawing. Kinematic properties of sperm were examined with a computer-assisted semen analysis (CASA) system. In addition, viability and mitochondrial activity were also tested by LIVE/DEAD and a MitoTracker kit. There were no significant differences in the kinetic parameters of thawed sperm between control and treatment groups, but overall assessment parameters such as motility and rapid progressive were higher in the 10 mM PTX group. In the viability and mitochondrial assay, there were no significant differences observed in the PTX treatment, compared to the control. For further analysis, artificial inseminations were performed using frozen semen and 10 mM PTX treated cryopreserved semen, respectively. There were no differences in pregnancy rates and fetus weights among the groups until 30 and 40 days, but litter size was reduced and relatively low-birth weight was observed in the PTX group. In summary, our findings suggest that enhancement of in vitro sperm quality or non-toxicity supplemented by PTX may have detrimental effects on fetus development.

• 한국임상수의학회지
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• 제33권6호
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• pp.356-361
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• 2016

During storage, boar spermatozoa undergo several changes including diminished motility and viability and accumulated reactive oxygen species (ROS). In this study, we investigated the effects of green tea extract (GTE) supplementation in the Sui Dil extender on the sperm motility, viability, ROS and lipid peroxidation (LPO) of long-term preserved boar semen at $17^{\circ}C$. A total number of eight boars were used for this experiment. Pooled ejaculates were diluted to $20{\times}10^6sperm/ml$ in the Sui Dil extender containing 0 (control), 1, 10, 100 or 500 mg/l GTE and were preserved at $17^{\circ}C$ for 24, 72, 120 and 168 h, respectively. At each storage time, sperm motility and viability were estimated by microscopic examination and the fluorescent double stain $Fertilight^{(R)}$, respectively. Sperm ROS level and LPO were assessed using the 2', 7'-dichlorodihydrofluorescein diacetate ($H_2DCFDA$)/propidium iodide (PI) and C11-BODIPY581/591/PI with flow cytometry, respectively. Compared to that of the 500 mg group, there were higher sperm motility and viability in the 1, 10 and 100 mg GTE groups during the preservation from 24 to 168 h (p < 0.05). The ROS levels of the 10 and 100 mg groups during the 168 h preservation were lower than those of the 0, 1 and 500 mg groups (p < 0.05). There were no significant differences in LPO regardless of the preservation period or the GTE concentration. In conclusion, the optimal concentrations (10 and 100 mg/l) of GTE that led to lower ROS levels may be useful for liquid boar sperm preservation at $17^{\circ}C$ for a period of 168 h.

• 농업과학연구
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• 제49권1호
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• pp.103-112
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• 2022

Fipronil is a popular insecticide used in both agricultural and domestic fields. Factors that affect sperm and eggs have a direct influence on reproductive outcomes. This study was undertaken to assess the effect of varying concentrations (10 - 200 μM) of fipronil and incubation times (30 min and 2 hrs) on boar spermatozoa. Spermatozoa were evaluated for motility, motion kinematics, viability, chromatin stability, and for the generation of intracellular reactive oxygen species (ROS) and the results were compared to those from corresponding controls. The findings revealed a significant, dose-dependent reduction in sperm motility in all fipronil treatment groups at 30 min of incubation (p < 0.05). A similar dose-dependent reduction in sperm motility was observed subsequent to fipronil exposure for 2 hrs of incubation (p < 0.05). Groups treated with fipronil showed a gradual reduction in motion kinematics (p < 0.05). Moreover, a significantly higher percentage of dead sperm was observed at 200 μM fipronil, as compared to the highest live percentage obtained in controls (p < 0.05). Evaluating the sperm chromatin integrity revealed a significantly higher percentage of damaged chromatin in spermatozoa incubated with 200 μM of fipronil. Moreover, ROS production was significantly higher in fipronil-exposed sperm (p < 0.05). In conclusion, boar spermatozoa incubated with fipronil showed decreased levels of sperm motility and viability, weaker chromatin integrity, and increased levels of intracellular ROS generation, all of which indicate that exposure to fipronil potentially impairs the fertilization competence of boar spermatozoa.

• Asian-Australasian Journal of Animal Sciences
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• 제19권4호
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• pp.486-494
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• 2006

In order to protect the spermatozoa against cold shock, hen egg yolk is widely used as a cryoprotective agent in semen freezing extenders for domestic animals. The protective action of yolk is largely presumed to be due to low density lipoproteins (LDL). The effects of LDL on sperm quality of bull and northern pike (Esox lucius) after freezing-thawing have been reported, but no study has been made to evaluate the effect of LDL on boar sperm motility and other characteristics. The experiment was carried out to investigate the effect of LDL on the freezing of boar sperm in 0.25 ml straws. The aim was to evaluate the quality of boar spermatozoa cryopreserved in the presence of LDL. Motility of semen cryopreserved in LDL was analyzed and compared to semen cryopreserved with Tris-citric acid-glucose (TCG) and Tris-citric acid-fructose (TCF), two basic freezing extenders containing egg yolk. Similarly, acrosome and plasma membrane integrity were also evaluated and compared to semen cryopreserved with TCG and TCF. Analysis of sperm quality after freeze-thaw showed that the motility, acrosome and plasma membrane integrity were improved with LDL in the extender, as compared to the TCG and TCF. The highest post-thaw integrity of acrosome and plasma membrane and motility were obtained with 9% LDL (w/v). Consequently, the optimum LDL concentration in the extender was 9%. It is also suggested that the concentration of LDL addition is important for the effect on boar sperm protection during freezing and thawing. The percentage of motile spermatozoa was significantly higher after freezing in 9% LDL than in TCG and TCF 54.4% versus 30.4% and 30.1% (p<0.05), respectively. The integrity of acrosome and plasma membrane were also significantly higher at 70.3% and 50.5% respectively with semen frozen in 9% LDL extender compared to TCG at 37.8% and 30.3% and TCF at 36.4% and 29.9%, respectively (p<0.05),. In conclusion, we propose that extender containing LDL extracted from hen egg yolk could be used as a cryoprotective media with a better efficiency than TCG and TCF. LDL improved boar semen quality, allowing better spermatozoa motility, acrosome and plasma membrane integrity after the freeze-thaw process. Furthermore, we found out that the extender with 9% LDL concentration significantly enhanced motility, acrosome and plasma membrane integrity of boar sperm after freezing and thawing.

• Asian-Australasian Journal of Animal Sciences
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• 제24권2호
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• pp.181-189
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• 2011

Cryopreservation protocols induce partially irreversible damage to mammalian sperm plasma membranes. Previous studies have indicated that adding cholesterol to the plasma membrane, as cholesterol-loaded-cyclodextrins, improves cryosurvival of sperm. Therefore, the purpose of this study was to determine if treating sperm of Markhoz bucks with cholesterol-loaded-cyclodextrins (CLC) (0, 0.75, 1.5, 2.25 and 3 mg/ml diluted $240{\times}10^6$ sperm/ml) in Tris-citric acid-glucose diluents with and without egg yolk (containing 5% glycerol) would improve the post-thaw sperm quality. The motion characteristics were evaluated with a Computer Assisted System Analyzer (CASA); acrosome integrity and vitality were measured with the triple-stain technique. Samples were recovered before and after freezing by means of putting straws into $37^{\circ}C$ water for 30 sec and then parameters were assessed. The results showed that the treatments significantly affected motility, progressive motility, recovery rate, curvilinear velocity, beat cross frequency, live sperm with reacted acrosome, live sperm with unreacted acrosome, dead sperm with reacted acrosorne, and dead sperm with unreacted acrosome during freezing (p<0.05). However; no significant differences were found for average path velocity, straight line velocity, amplitude of lateral head displacement, straightness and linearity (p>0.05). The best results were observed for extender containing 2.25 mg/ml ($240{\times}10^6$ sperm/ml) CLC supplemented with 2.6% egg yolk. In conclusion, the findings of this study indicate improved Markhoz sperm viability and motility following treatment in the presence of egg yolk.

• Clinical and Experimental Reproductive Medicine
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• 제46권2호
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• pp.67-75
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• 2019

Objective: Sperm cryopreservation has been widely used in assisted reproductive technology, as it offers great potential for the treatment of some types of male infertility. However, cryopreservation may result in changes in membrane lipid composition and acrosome status, as well as reductions in sperm motility and viability. This study aimed to evaluate sperm DNA fragmentation damage caused by conventional freezing using the sperm chromatin dispersion test. Methods: In total, 120 fresh human semen samples were frozen by conventional methods, using SpermFreeze Solution as a cryoprotectant. Routine semen analysis and a Halosperm test (using the Halosperm kit) were performed on each sample before freezing and after thawing. Semen parameters and sperm DNA fragmentation were compared between these groups. Results: There was a significant decrease in sperm progressive motility, viability, and normal morphology after conventional freezing (32.78%, 79.58%, and 3.87% vs. 16%, 55.99%, and 2.55%, respectively). The sperm head, midpiece, and tail defect rate increased slightly after freezing. Furthermore, the DNA fragmentation index (DFI) was significantly higher after thawing than before freezing (19.21% prior to freezing vs. 22.23% after thawing). Significant increases in the DFI after cryopreservation were observed in samples with both normal and abnormal motility and morphology, as well as in those with normal viability. Conclusion: Conventional freezing seems to damage some sperm parameters, in particular causing a reduction in sperm DNA integrity.

• Animal Bioscience
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• 제35권9호
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• pp.1351-1359
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• 2022

Objective: The objective of this study was to analyse the differentially abundant proteins caused by freeze-thawing of ram sperm and explore candidate proteins of interest for their ability to improve ram sperm cryopreservation outcomes in vitro. Methods: Sperm were from three mature Dorper. Fresh and frozen sperm proteins were extracted, and the differentially abundant proteins were analysed by mass spectrometry. Among these proteins, lactoferrin (LTF) was selected to be added before cryopreservation. Next, sperm samples were diluted in Tris extender, with the addition of 0, 10, 100, 500, and 1,000 ㎍/mL of LTF. After thawing, sperm quality was evaluated by motility, plasma membrane integrity, mitochondrial activity and reactive oxygen species (ROS). Results: Cryopreservation significantly altered the abundance of 40 proteins; the abundance of 16 proteins was increased, while that of 24 proteins was decreased. Next, LTF was added to Tris extender applied to ram sperm. The results showed that sperm motility and plasma membrane integrity were significantly improved (p<0.05) by supplementation with 10 ㎍/mL LTF compared to those in the control group. There was no significant difference in mitochondrial activity between the 0 ㎍/mL group and other groups (p>0.05). Supplementation of the cryoprotective extender with 10 ㎍/mL LTF led to decreased ROS levels compared with those in the control and other groups (p<0.05). Conclusion: The LTF is an important protein during cryopreservation, and the addition of 10 ㎍/mL LTF to a cryoprotective extender can significantly improve the function of frozen ram sperm.

• Animal Bioscience
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• 제36권12호
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• pp.1821-1830
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• 2023

Objective: This study investigated the effect of adding seminal plasma to frozen-thawed semen on the quality of sperm and pregnancy following insemination in dromedary camels. Methods: In experiment 1, the frozen-thawed semen from 9 collections (3 bulls) was further diluted with either the base extender or homologous seminal plasma (HSP). In the second experiment, a pooled sample of frozen-thawed semen was diluted with either seminal plasma from another three bulls. Live percentage, total and progressive motility, functional and acrosome integrity, and sperm kinematics were evaluated at 15, 60, and 120 minutes post-thawing and compared to the non-treated control. In experiment 3, frozen semen was used to inseminate camels in the following experimental groups: 1-Single insemination with double dose undiluted frozen semen (n = 9); 2-Re-insemination in 6 hours with undiluted semen (n = 13); 3-Single insemination with HSP treated sperm (n = 14). Results: Frozen-thawed sperm diluted in HSP or the non-homologous seminal plasma from Bull C indicated an improvement in all parameters after 1 hour post-thawing incubation (p<0.05). The proportion of total and progressively motile sperm did not drop significantly at 60 minutes post-thawing when diluted with the seminal plasma of Bull C (p>0.05). Double insemination with nontreated sperm and single insemination with HSP-treated sperm resulted in similar pregnancy rates (15.3% vs 21.4%, p>0.05). None of the camels conceived with double-dose single insemination of nontreated sperm. Conclusion: Seminal plasma improves sperm longevity and motility after thawing in dromedary camel with a significant between-bull variation in effect. Low post-thaw sperm longevity might be the cause behind the low pregnancy rates in frozen semen insemination of dromedary camels.

• Animal Bioscience
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• 제37권4호
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• pp.631-639
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• 2024

Objective: This study evaluates goat sperm motility in response to metabolic substrates and various inhibitors, aiming to assess the relative contribution of glycolysis and mitochondrial oxidation for sperm movement and adenosine triphosphate (ATP) production. Methods: In the present study, two main metabolic substrates; 0 to 0.5 mM glucose and 0 to 30 mM pyruvate were used to evaluate their contribution to sperm movements of goats. Using a 3-chloro-1,2-propanediol (3-MCPD), a specific inhibitor for glycolysis, and carbonyl cyanide 3-chlorophenylhydrazone as an inhibitor for oxidative phosphorylation, cellular mechanisms into ATP-generating pathways in relation to sperm movements and ATP production were observed. Data were analysed using one-way analysis of variance for multiple comparisons. Results: Sperm motility analysis showed that either glucose or pyruvate supported sperm movement during 0 to 30 min incubation. However, the supporting effects were abolished by the addition of a glycolysis inhibitor or mitochondrial uncoupler, concomitant with a significant decrease in ATP production. Although oxidative phosphorylation produces larger ATP concentrations than those from glycolysis, sperm progressivity in relation to these two metabolic pathways is comparable. Conclusion: Based on the present study, we suggest that goat sperm use glucose and pyruvate to generate cellular energy through glycolysis and mitochondrial respiration pathways to maintain sperm movement.