• Animal Bioscience
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• v.37 no.4
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• pp.640-654
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• 2024

Objective: The purpose of this study was to explore the effect of sodium salicylate (SS) on semen preservation and metabolic regulation in goats. Methods: Under the condition of low temperature, SS was added to goat semen diluent to detect goat sperm motility, plasma membrane, acrosome, antioxidant capacity, mitochondrial membrane potential (MMP) and metabonomics. Results: The results show that at the 8th day of low-temperature storage, the sperm motility of the 20 μM SS group was 66.64%, and the integrity rates of the plasma membrane and acrosome were both above 60%, significantly higher than those of the other groups. The activities of catalase and superoxide dismutase in the sperm of the 20 μM SS group were significantly higher than those of the control group, the contents of reactive oxygen species and malondialdehyde were significantly lower than those in the control group, the MMP was significantly higher than that in the control group, and the contents of Ca²⁺ and total cholesterol were significantly higher than those in the control group. Through metabonomics analysis, there were significant metabolic differences between the control group and the 20 μM SS group. Twenty of the most significant metabolic markers were screened, mainly involving five metabolic pathways, of which nicotinic acid and nicotinamide metabolic pathways were the most significant. Conclusion: The results indicate that SS can effectively improve the low-temperature preservation quality of goat sperm.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.5
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• pp.622-637
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• 2017

Fatty acids such as n-3 and n-6 polyunsaturated fatty acids (PUFA) are critical nutrients, used to improve male reproductive performance through modification of fatty acid profile and maintenance of sperm membrane integrity, especially under cold shock or cryopreservation condition. Also, PUFA provide the precursors for prostaglandin synthesis and can modulate the expression patterns of many key enzymes involved in both prostaglandin and steroid metabolism. Many studies carried out on diets supplemented with PUFA have demonstrated their capability to sustain sperm motility, viability and fertility during chilling and freezing as well as improving testis development and spermatogenesis in a variety of livestock species. In addition to the type and quantity of dietary fatty acids, ways of addition of PUFA to diet or semen extender is very crucial as it has different effects on semen quality in male ruminants. Limitation of PUFA added to ruminant ration is due to biohydrogenation by rumen microorganisms, which causes conversion of unsaturated fatty acids to saturated fatty acids, leading to loss of PUFA quantity. Thus, many strategies for protecting PUFA from biohydrogenation in rumen have been developed over the years. This paper reviews four aspects of PUFA in light of previous research including rumen metabolism, biological roles, influence on reproduction, and strategies to use in male ruminants.

• Clinical and Experimental Reproductive Medicine
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• v.36 no.1
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• pp.45-53
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• 2009

Objectives: To determinate the optimal culture condition to maintain lifespan in human sperm, we evaluated the effect of different temperature on sperm motility and viability up to 5 days in normal specimens. Methods: Ejaculated semen samples with normal semen parameters were gently washed in HEPES buffered Tyrod's-Albumin-Lactate-Pyruvate (HTALP) media. Each 5 ml of HTALP + 0.3% albumin with $1{\times}10^6$ sperm/ml was incubated for 5 days in $37^{\circ}C$, $22^{\circ}C$, and $4^{\circ}C$. The sperm motility and kinematics were analyzed using computer assisted sperm analysis (CASA), membrane integrity was assessed by hypoosmotic swelling test (HOST), and capacitation status was evaluated by chlorotetracycline (CTC) fluorescence pattern. Each parameter was measured on day 1, 3, and 5, respectively. Results: The motility, viability and live/uncapacitated pattern were demonstrated significantly in temperature- and time-dependent difference (p<0.05). While the sperm cultured for 1 day in each temperature was not significantly different, the sperm cell kept in $22^{\circ}C$ after 3 days were preserved sperm motility, viability, membrane integrity, and F pattern better than in other culture temperatures. Conclusions: HTALP can be used a basic medium for culture and longevity preservation, and sperm cell kept at $22^{\circ}C$ is beneficial for assisted reproductive techniques.

• Korean Journal of Agricultural Science
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• v.46 no.1
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• pp.195-204
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• 2019

In this study, we examined the effect of a liposome-based extender (Optixcell) and a tris-citric egg-yolk extender (Triladyl) on the frozen-thawed spermatozoa characteristics and the calving rate. The percentages for the total motility of the frozen-thawed spermatozoa were similar in the Optixcell and Triladyl groups. However, among the motile spermatozoa with a straight line velocity (VSL) ${\geq}25{\mu}m/sec$, the curvilinear velocity (VCL, ${\mu}m/sec$), VSL (${\mu}m/sec$), average path velocity (VAP, ${\mu}m/sec$), amplitude of lateral head displacement (ALH, ${\mu}m$), beat cross frequency (BCF, Hz), and plasma membrane integrity of the frozen-thawed spermatozoa for the Optixcell group were significantly higher than those for the Triladyl group. Furthermore, the calving rate in the Optixcell group (79.0%) was higher than that of the Triladyl group (62.8%). However, the acrosomal membrane integrity of the frozen-thawed spermatozoa in the Optixcell and Triladyl groups was not significantly different. These results indicate that semen freezing with Optixcell improved the motility and plasma membrane integrity of frozen-thawed spermatozoa and the calving rate of Hanwoo cows (native Korean cattle). In conclusion, our results suggest that semen freezing with the liposome-based extender Optixcell is more efficient than with the tris-citric egg-yolk extender Triladyl for improved offspring production.

• Clinical and Experimental Reproductive Medicine
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• v.28 no.4
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• pp.295-300
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• 2001

Objective : To assess the effects of progesterone and acetyl-L-carnitine used after treated with Isolate�� gradient before semen cryopreservation-thawing on sperm parameters and membrane integrity. Material and Methods : From April 2001 to July 2001, ten normal male partner of couples who were visited in vitro fertilization (IVF) clinics. the semens were treated with $Isolate^{(R)}$ gradient before cryopreservation, spermatozoa was incubated with progesterone (1, 5 and $10{\mu}M$), acetyl-L-carnitine (2.5, 5 and $10{\mu}M$), or both (progesterone, $1{\mu}M$; and acetyl-L-carnitine, $5{\mu}M$) for 30 min. Results: There were no differences in sperm parameters and vital stain among isolate only treated group, progesterone (1, 5 and $10{\mu}M$), acetyl-L-carnitine (2.5, 5 and $10{\mu}M$) and both (progesterone, $1{\mu}M$; and acetyl-L-carnitine, $5{\mu}M$). But, in high concentration of acetyl-L-carnitine ($10{\mu}M$) treated group, sperm parameters and vital stain were decreased. The statistical method was used ANOVA (Kruskal-Wallis test) and p value was <0.01. Conclusions : Neither progesterone nor acetyl-L-carnitine show to be protective effect on the cryodamage assessed by sperm parameters and vital stain (eosin-Y stain) in normal sperm. High concentration of acetyl-L-carnitine ($10{\mu}M$), however, was harmful effect on cryoprevention.

• Asian-Australasian Journal of Animal Sciences
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• v.15 no.10
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• pp.1412-1421
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• 2002

To examine the feasibility of using a sperm vector system for gene transfer, we have investigated the binding and the uptaking of foreign DNA into the sperm nucleus by PCR, in situ hybridization and LSC. We have also examined the transportation of exogenous DNA into oocytes by immunofluorescene via PCR. Sperm cells were incubated with DNA/liposome complexes (1:4 ratio) in fertilization medium with BSA or without BSA. In situ hybridization demonstrated that the transfection rate of sperm cells with and without BSA was 41 and 68% respectively, when the cells were treated with liposome/DNA complexes and 13% for DNA alone. LSC analysis showed that the binding of exogenous DNA was greatly reduced by DNase I treatment which digests DNA bound onto spermatozoa, suggesting that some of the DNA was internalized into the sperm membrane. To find out whether transfected DNA was internalized into sperm intracytomembrane, sperm DNA was amplified by inverse PCR. No PCR products were detected from sperm cells, indicating that the foreign DNA was simply bound onto the sperm membrane. To investigate transfer rates of exogenous DNA into oocytes via sperm cells, we used immunofluorescene method to follow the distribution of foreign DNA via spermatozoa: a few exogenous DNA was located in the cytoplasm of early embryos (13/60, 21.7% for DNA+/liposome+/BSA) and was not located in the pronucleus and/or nucleus. These results suggest that most of the transfected sperm cells could carry the foreign DNA into the egg by in vitro fertilization, but that the transferred DNA is degraded in the developing embryos without stable integration into the zygote genome. Therefore, we have directly injected with transfected sperm cell into oocyte cytoplasm and observed that some of the exogenous DNA was detected in preimplantation embryonic cytoplasm and expressed at preimplantation stages, suggesting that exogenous DNA in early zygote has their integrity. In this study, we have not identified a noble mechanism that interfering transportation of foreign DNA into zygote genome via spermatozoa. Our data, however, demonstrated that inverse PCR and immunofluorescene methods would be used as a new tool for follow-up of gene distribution in oocyte via sperm cells.

• Reproductive and Developmental Biology
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• v.36 no.3
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• pp.199-206
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• 2012

Cryopreservation induces sublethal damage to the spermatozoa, which leads to their reduced fertile life. This study was designed to determine effect of glycerol and ethylene glycol as cryoprotectant in extender on improve the freezability of Jeju horse semen. The semen was cryopreserved with glucose-EDTA extender containing each 5% glycerol, 5% ethylene glycol, 8% glycerol or 8% ethylene glycol, respectively. Post-thawed sperm were evaluated motility, viability, Membrane integrity and acrosome integrity. Post-thawed sperm motility were not significantly differences among treatments. However, sperm viability were significantly higher (p<0.05) in 8% glycerol ($39.85%{\pm}11.41$) than in 5% glycerol treatment ($18.08%{\pm}1.61$). In membrane integrity, swelling sperm ratio was significantly higher (p<0.05) in 8% glycerol ($34.12%{\pm}11.02$) than other treatments. In the percentage of capacitated sperm assessed by CTC staining, F pattern was significantly higher in 8% ethylene glycol than 5% glycerol and 5% ethylene glycol (p<0.05). B pattern ratio was significantly increased in 5% ethylene glycol compared with 8% glcerol and 8% ethylene glycol (p<0.05). Moreover, 8% ethylene glycol treatment was significantly decreased AR pattern ratio compared with other treatments (p<0.05). It is concluded that treatment of 8% glycerol was improved the sperm viability and 8% ethylene glycol was improved the sperm ascrosome integrity after thawing. However, they were not significantly difference between 8% glycerol and 8% ethylene glycol on post-thawed sperm viability. Therefore, 8% ethylene glycol was more effective sperm cryoprotectant than 8% glycerol in Jeju Horse.

• Journal of Embryo Transfer
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• v.26 no.3
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• pp.187-193
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• 2011

This study was carried out to establish most suitable freezing condition, to evaluate the different glycerol concentration of freezing and thawing rates on motility, viability, membrane integrity and acrosome intecrity of frozen Korean Jeju Black Bull spermatozoa, Semen was collected from a Korean Jeju Black Bull using an artificial vagina and transported to the laboratory. The semen was extended gradually 1:5 then cooled slowly for 2 hrs to 4$^{\circ}C$. The semen was diluted 1:1 with cryoprotectant extenders (3%, 5% and 7% glycerol) and equilibrated for 2 hrs at cold chamber and packed to 0.5 ml straws. The semen straws were located above 3 cm of liquid nitrogen for 5 minutes, above 5 cm for 10 min and above 8 cm for 10 min. And then the frozen straw was plunged into LN$_2$. The presented straws were examined the viability and motility after thawed at 37$^{\circ}C$ water bath. The viability and membrane integrity immediately post-thawing were significantly higher in samples frozen in 7% glycerol than 3% and 5% glycerol (p<0.05). After CTC staining to assess acrosome integrity, F pattern was significantly increased, but B pattern was significantly decreased in 7% glycerol (p<0.05). Freezing distance of 5 cm from liquid nitrogen and pre-cooling for 10 min yield better survival and membrane integrity, but not significant difference. However, AR pattern according to CTC staining was significantly decreased in 3 cm for 5 min.

• Asian-Australasian Journal of Animal Sciences
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• v.24 no.2
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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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• v.46 no.2
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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.