• 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.

• Asian-Australasian Journal of Animal Sciences
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• v.33 no.11
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• pp.1763-1769
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• 2020

Objective: This study was conducted to investigate the effect of L-carnitine on the pig semen characteristics during storage. Methods: Spermatozoa samples were examined for spermatozoa quality and then randomly divided into 5 groups: 0 (control), 12.5, 25, 50, and 100 mM L-carnitine. Sperm motility, plasma membrane integrity and antioxidant parameters (total reactive oxygen species, total antioxidant capacity, and malondialdehyde) were evaluated after 0, 3, 5, and 10 day cooled-storage at 17℃. Moreover, ATP content, mitochondria activity as well as sperm-binding and in vitro fertilizing ability of preserved boar sperm were also investigated. Results: Supplementation with 50 mM L-carnitine could effectively maintain boar sperm quality parameters such as sperm motility and membrane integrity. Besides, we found that L-carnitine had positive effects on boar sperm quality mainly through improving antioxidant capacities and enhancing ATP content and mitochondria activity. Interestingly, by assessing the effect of L-carnitine on sperm fertility and developmental potential, we discovered that the extender containing L-carnitine could improve sperm quality and increase the number of sperms bounding to zona pellucida, without improving in vitro fertility and development potential. Conclusion: These findings suggested that the proper addition of L-carnitine to the semen extender improved boar sperm quality during liquid storage at 17℃.

• Animal Bioscience
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• v.35 no.9
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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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• v.36 no.12
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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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• v.37 no.5
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• pp.852-861
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• 2024

Objective: The present study aimed to investigate the effect of β-nicotinamide mononucleotide (NMN) supplementation on ram sperm quality during storage at 4℃ in vitro. Methods: Tris-citric acid-glucose solution containing different doses of NMN (0, 30, 60, 90, and 120 µM) was used to dilute semen collected from rams and it was stored at 4℃. Sperm motility, plasma membrane integrity as well as acrosome integrity were evaluated at 0, 24, and 48 h time points after storage at 4℃. In addition, sperm mitochondrial activity, lipid peroxidation (LPO), malondialdehyde (MDA) content, reactive oxygen species (ROS) content, glutathione (GSH) content, superoxide dismutase (SOD) activity, and apoptosis were measured at 48 h time point after storage at 4℃. Results: Results demonstrate that the values obtained for sperm motility, acrosome integrity, and plasma membrane integrity in the NMN treatments were significantly higher than control (p<0.05). The addition of 60 µM NMN significantly improved ram sperm mitochondrial activity and reduced LPO, MDA content, and ROS content compared to control (p<0.05). Interestingly, sperm GSH content and SOD activity for the 60 µM NMN treatment were much higher than those observed for control. NMN treatment also decreased the level of Cleaved-Caspase 3, Cleaved-Caspase 9, and Bax while increasing Bcl-2 level in sperm at 48 h time point after storage at 4℃. Conclusion: Ram sperm quality can be maintained during storage at 4℃ with the addition of NMN at 60 µM to the semen extender. NMN also reduces oxidative stress and apoptosis. Overall, these findings suggest that NMN is efficient in improving the viability of ram sperm during storage at 4℃ in vitro.

• Korean Journal of Agricultural Science
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• v.51 no.2
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• pp.109-121
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• 2024

Mancozeb is a manganese and zinc-containing fungicide that belongs to the ethylene bisdithiocarbamate group and produces ethylene thiourea (ETU) after biotransformation or environmental degradation, which has toxicological hazard owing to its known antithyroid properties. Although mancozeb leads to negative changes in fertility capacity, the effects of ETU are less known. Therefore, this study examined the alteration of fertilization competence in boar spermatozoa exposed to ETU. The sperm motility, motion kinematics, viability, acrosome integrity, chromatin stability, and intracellular reactive oxygen species (ROS) production of sperm subjected to various ETU concentrations (10, 50, 100, and 200 µM) were evaluated after two different incubation times (30 min and 2 hrs). In addition, the relative mRNA expression of the sperm functional proteins was analyzed after exposure to ETU. A dose-dependent motility reduction was observed in sperm exposed to ETU during both incubation periods compared to the controls. The motion kinematics were reduced significantly in sperm incubated with ETU. Higher percentages of viable sperm were observed in the controls, while such viability was decreased significantly in sperm with 10 - 200 µM ETU. The acrosome integrity was particularly damaged on sperm incubated with 10 - 200 µM ETU for 30 min. Higher intracellular ROS levels were produced in sperm exposed to 200 µM ETU. In addition, lower relative levels of AKAP3, AKAP4, ODF2, and ZPBP2 expression were observed in sperm exposed to ETU compared to the controls. Mancozeb and ETU could adversely affect the reproductive functions of mammals. Hence, the effects of ETU on the reproductive system should be examined further.

• Proceedings of the Korean Aquaculture Society Conference
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• 2003.10a
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• pp.52-52
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• 2003

For the evaluation of hormonal control of spermiation in fish, a method to quanify the spermiation response or mature Rhynchocypris oxycephalus (Sauvage and Dabry) to hormonal therapy is described. Spermatocrit was determined after 7 min centrifugation at 18,000 $\times$ｇ and sperm density was estimated by a standard hemocytormer method. Sperm density can be predicted from spermatocrit since their relationship is linear as described by the regression equation, Y=3.68X-27,18 ($R^2$=0.82, N=50), where Y is spermatocrit and X is sperm density. Milt production by mature R. oxycephalus was highest at 24 h after injection of 1,000 IU human chorionic gonadotropin (HCG) and 50 $\mu\textrm{g}$ luteinizing hormone-releasing hormone analogue (LHRHa) per kg body weight. Increased milt production coincided with low spermotocrit and sperm density levels. These results demonstrate that spermition in mature R. oxycephalus can be reliably evaluated by a spermatocrit method and that HCG and LHRHa are effecove in stimulating of spermiation in this species.

• Asian-Australasian Journal of Animal Sciences
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• v.8 no.2
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• pp.123-127
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• 1995

In vitro embryo production (IVP) is affected by various factors during in vitro maturation, fertilization, and development. In this experiment, the effect of ovary type, quality of follicular oocyte, medium used for fertilization, presence of hormone in medium, sperm concentration on in vitro maturation and fertilization were examined for effective IVP. In vitro maturation was carried out using TCM-199 supplemented with 15% FCS and hormones in 5% $CO_2$ incubator for 24h. In vitro fertilization was performed with frozen-thawed sperm in modified mTALP medium containing 0.3% BSA, $10{\mu}g/ml$ heparin, and 5mM/ml caffeine for 24h. The fertilized embryos were co-cultured on monolayer of cumulus cells in TCM-199. When oocytes were collected from functionally active and inactive ovaries, maturation rate was 76.9 and 7.7%, respectively. When oocytes were classified morphologically to good and poor grades, maturation rate was 75 and 58.8%, respectively. FSH + LH + $E_2$ (86.4%) showed higher maturation rate than control (53.0%) and FSH (73%). The fertilization rate was 28.2, 100 and 91.7% in $1.6{\times}10^5$, $5.0{\times}10^5$ and $10.0{\times}10^5$ sperm concentration per ml. When oocytes were fertilized in mTALP and BO media, fertilization and cleavage rates of oocytes in mTALP were higher (84.3 and 56.9%) than those (67.4 and 23.3%) in BO medium. In this experiment, in vitro maturation, fertilization and development of oocytes were affected by type of ovary, grade of oocyte, hormones, sperm concentration and fertilization medium.

• Clinical and Experimental Reproductive Medicine
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• v.45 no.2
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• pp.88-93
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• 2018

Objective: The aim of this study was to measure reactive oxygen species (ROS) production and total antioxidant capacity (TAC) in the seminal fluid of the male partners in couples undergoing intrauterine insemination and to evaluate correlations between these values and their semen parameters. Methods: The study was conducted at Vamsam Fertility Center, Coimbatore, India and enrolled 110 male patients from whom semen samples were collected. ROS production was measured by a thiobarbituric acid reactive species assay, and TAC was measured by a 2,2-diphenyl-2-picrylhydrazyl free radical assay. The differences in the TAC and malondialdehyde (MDA) levels between the subfertile and fertile groups were analysed. Correlations between sperm parameters and TAC and MDA levels were statistically analysed, and cutoff values with respect to the controls were determined. All hypothesis tests used were two-tailed, with statistical significance assessed at the level of p< 0.05. Results: A total of 87 subfertile and 23 fertile men were included in the study. The mean MDA level was significantly higher in the subfertile subjects than in the fertile subjects, and the mean antioxidant level was significantly lower in the subfertile subjects than in the fertile subjects. Seminal MDA levels were negatively associated with sperm concentration, motility, and morphology, whereas the opposite was seen with TAC levels. Conclusion: Measurements of seminal TAC and ROS are valuable for predicting semen quality, and hence predicting the outcomes of fertility treatment.

• Korean Journal of Veterinary Research
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• v.37 no.4
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• pp.925-934
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• 1997

This study was directed at inducing the production of antibodies by immunizing heifers with bovine sperm antigen and on measuring the serum antibodies using indirect immunofluorescence assay(IFA) and agglutination test. The effect of antisperm antibodies on fertilizing capacity of bovine spermatozoa was evaluated. 1. Three heifers between 12- and 15- month old were immunized with bovine spermatozoa or phosphate-buffered saline. In heifers immunized with bovine spermatozoa serum IgG level was highest between 3 weeks and 5 weeks postimmunization detected by IFA. The antibody levels persisted through week 7 and slowly declined until week 20 and then antisperm antibodies were localized on spermatozoa. The fluorescent antisperm antibodies were detected at 2~20 weeks and at 6~9 weeks postinoculation on acrosome and tail, respectively. Among 21 sera from repeat breeder cows, only one cow has shown positive antisperm antibody response detected by IFA. 2. In spite of vital rate of bovine sperm after swim-up was not significantly affected by different concentration of antisperm antibodies in sera, the numbers of bovine sperm after swim-up were significantly reduced in proportion to the increased concentration of antibodies. Above 1/512 dilution of antibody neither influence on vital rate and numbers of bovine sperm nor sperm agglutination after swim-up. The study has also shown that the vital rate and number of sperm after swim-up and capacitation were also significantly reduced by the addition of antisperm antibodies. Although antisperm antibodies did not influence on the acrosome reaction rate of sperm during swim-up, did significantly reduce the sperm acrosome reaction rate after capacitation. The studies have resulted that the bovine antisperm antibodies can prevent the sperm motility by agglutination and block the capacitation and acrosome reaction of bovine sperm.