• Animal Bioscience
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• v.34 no.8
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• pp.1253-1270
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• 2021

Assessment of male fertility is based on the evaluation of sperm. Semen evaluation measures various sperm quality parameters as fertility indicators. However, semen evaluation has limitations, and it requires the advancement and application of strict quality control methods to interpret the results. This article reviews the recent advances in evaluating various sperm-specific quality characteristics and methodologies, with the help of different assays to assess sperm-fertility status. Sperm evaluation methods that include conventional microscopic methods, computer-assisted sperm analyzers (CASA), and flow cytometric analysis, provide precise information related to sperm morphology and function. Moreover, profiling fertility-related biomarkers in sperm or seminal plasma can be helpful in predicting fertility. Identification of different sperm proteins and diagnosis of DNA damage has positively contributed to the existing pool of knowledge about sperm physiology and molecular anomalies associated with different infertility issues in males. Advances in methods and sperm-specific evaluation has subsequently resulted in a better understanding of sperm biology that has improved the diagnosis and clinical management of male factor infertility. Accurate sperm evaluation is of paramount importance in the application of artificial insemination and assisted reproductive technology. However, no single test can precisely determine fertility; the selection of an appropriate test or a set of tests and parameters is required to accurately determine the fertility of specific animal species. Therefore, a need to further calibrate the CASA and advance the gene expression tests is recommended for faster and field-level applications.

• Toxicological Research
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• v.11 no.1
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• pp.69-75
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• 1995

The present study was carried out to establish several spermatotoxicity test methods. For this purpose we investigated following parameters in the fertility study of DA-125, a new anticancer agent, in rats: testicular spermatid counts, epididymal sperm counts, daily sperm production rate, sperm morphology, and serum testosterone concentration. Motility and velocity of sperms were also measured using non-treated rats. At 0.3 mg DA-125/kg, spermatids per 1g testis and daily sperm production rate per 1g testis were significantly decreased, when compared with those of control group. Several types of abnormal sperms, such as no head, pin head, double head, hook at wrong angle, no tail, and small sperm, were found in both treated and control groups at a low frequency. Serum testosterone concentration at 0.3 mg DA-125/kg was close to the control value. Sperm motility and velocity measured with non-treated rats were in a good agreement with the results of other investigators. In our study established spermatotoxicity test methods can be used as a tool not only for the close examination of the cause of drug- or chemical-induced infertility, but also for the effective evaluation of reproductive toxicity.

• Journal of Veterinary Clinics
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• v.17 no.2
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• pp.431-437
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• 2000

Evaluation of viability and capacitation of canine sperm is of great importance to deter- mine good condition for freezing canine semen and consequently to improve conception rate by arti-ficial insemination. Semen were collected from nine male dots which had been proved to be fertile in the post and the semen were treaded for freezing procedure. Semen were thawed at 37$^{\circ}C$ for 30 seconds. In this study, hypoosmotic swelling(HOS) test and chlortetracycline(CTC) test were per- formed to evaluate post-thaw viability and capacitated status of sperm, respectively. In HOS test far canine sperm, the highest percentage of curled sperm was shown at 60 mOsm. In HOS test for canine semen, there were considerably significant correlation between HOS values and sperm motil- ity(r=0.9064, p<0.01) and converse correlation between HOS values and sperm abnormality(r=- 0.6905, p<0.05). The sperm viability and HOS-values for chilled extended semen were significantly decreased from 0 to 72 hours during storage at 5$^{\circ}C$ (p<0.05). Of the media added to canine semen after thawing, the most capacitated sperm were shown in CCM(p<0.01), and then This Fructose Cit- rate(TFC) medium with calcium from 3 hours after incubation with media. It was concluded that HOS test is of great value to determine the viability and motility of canine sperm, whereas CTC test is usable to determine the capacitated status. Consequently, both tests were thought to be useful as the additional tests to standard semen analysis.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.1
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• pp.81-86
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• 1996

The assessment of acrosomal status is important in evaluating the ability of sperm to fertilize the egg. The acrosomal status of sperm from 47 normal volunteers with proven fertility and 167 subfertile men with not to achieve pregnancy for at least 1 year were evaluated with Acrobeads test(FUSO Pharmaceutical Industries, Ltd, Japan) using immunobeads coated with MH61 monoclonal antibody, which is specific for acrosome-reacted sperm. The mean${\pm}$SD of acrobeads score in 47 volunteer group was $2.8{\pm}0.7$, of which 46(97.9%)cases were ${\geq}$ 2. The mean${\pm}$SD of acrobeads score in 167 subfertile group was $1.7{\pm}0.8$, of which 73(79.3%)cases were ${\leq}$ 1. The aerobe ads score in subfertile group were significantly lower(r=0.294, p<0.05) than those in volunteer group. In subfertile group, acrobeads score were well correlated with the sperm density and motility(r=0.275, r=0.281, p<0.01), but not with semen volume(r=0.16) and serum hormone level(FSH r=0.084, LH r=0.036, testosterone r=0.058, prolactin r=0.006 and estradiol r=0.060)(p>0.05). Of 63 subfertile cases with normozoospermia, 22(34.9%)cases showed 0 or 1 of acrobeads score, which means to accompany with a functional defect in spite of normal morphology. As a results, Acrobeads test is not only a technically simple sensitive procedure with good reproducibility in evaluating the sperm fertilizing capacity but also an useful in the evaluation of effectiveness in the treatment of infertility and the separation of acrosome-reacted sperm in the assisted reproductive technique.

• Journal of Embryo Transfer
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• v.25 no.4
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• pp.229-235
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• 2010

The MTT assay is one of superior evaluation methods widely used to analyze the viability of metabolically active cell. It can be used to determine the percentage of viable sperm through measurement of the reduction of MTT granules at mitochondria in sperm tail. The purpose of this study is to determine the optimal condition of a simple and easy MTT assay to validate boar sperm viability and compare the accuracy of this test with microscopic examination. The MTT reduction rate for sperm viability were analyzed in microtiter plates (96 well) from 1 hr to 5 hr incubation periods at $37^{\circ}C$ using spectrophotometer (microplate reader) at 550 nm wavelength. The remainder of semen sample was simultaneously examined to compare the correlation of accuracy between MTT assay and other sperm parameters. Those sperm parameters were included the motility, survival rates, membrane integrity, mitochondria activity and acrosome integrity. The OD values of MTT assay (MTT reduction rates) did not greatly change at 1 hr to 5 hr incubation periods in different proportion of live and freeze-killed sperms (dead sperm). The MTT reduction rates or survival rates were decreased according to the different concentration of live and dead sperm. The linear regression at 1 hr and 4 hr incubation periods in sperm MTT assay was y=291.55x-72.176 and y= 180.64x-44.569, respectively. There are high correlation between 1 hr and 4 hr incubation periods (p<0.001). The results of MTT assay and other sperm parameters has a positive correlation (p<0.01 or 0.05). The correlation coefficients for MTT assay was 0.88115 for motility, 0.89868 for survival rates, 0.91722 for membrane integrity and 0.77372 for acrosome integrity, respectively. In conclusion, the MTT assay can be used as a reliable and efficient evaluation method for boar sperm viability. It can be use practical means to evaluate the quality of boar sperm by a fast, inexpensive and easy method.

• Clinical and Experimental Reproductive Medicine
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• v.46 no.1
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• pp.8-13
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• 2019

Objective: Density gradient centrifugation (DGC) is frequently used to isolate high-motility fractions of spermatozoa. We compared the efficacy of four DGC media in terms of the percentage of morphologically normal spermatozoa, DNA fragmentation level, and hyaluronic acid (HA) binding ability. Methods: Thirty men with a total motile spermatozoa count > 80 million participated. Semen samples were divided into four aliquots, which were processed using PureSperm, PureCeption, Sidney, and SpermGrad media, respectively. The DNA fragmentation level was measured using the Halosperm assay kit and HA binding ability was measured using the HBA assay kit. Results: The mean percentage of morphologically normal spermatozoa was significantly enhanced after DGC using all four media (10.3%, 9.9%, 9.8%, and 10.7%, respectively; p< 0.05 for each when compared with 6.9% in raw semen). The DNA fragmentation level was significantly reduced after DGC using PureSperm, PureCeption, and SpermGrad media (6.0%, 6.5%, and 4.9%, respectively; p< 0.05 for each when compared with 11.2% in raw semen), but not after DGC using Sidney media (8.5%, p> 0.05). HA binding ability did not change after DGC using any of the four media. Conclusion: The four media were equally effective for obtaining a sperm fraction with highly motile, morphologically normal sperm. PureSperm, PureCeption, and SpermGrad media were equally effective for acquiring a sperm fraction with less DNA fragmentation.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.26-26
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• 2003

Hyaluronic acid (HA)-binding sites have been shown the diagnostic potential fur assessment of sperm maturity, which is related to male fertility. This study was designed to evaluate chromosomal patterns in porcine embryos produced by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) with non- or HA-binding sperm (HABS). For binding of sperm with HA, sperm incubated in 10 ${mu}ell$ drop containing HA (0.8 mg/ml)-agarose (0.8%) mixture for 15 min. IVF and ICSI with non- or HA-bound sperm examined with matured oocytes at 44 hr after in vitro maturation. Embryos were cultured in 50 ${mu}ell$ of NCSU 23 containing 0.5% BSA for 5 days and then in 50 ${mu}ell$ of NCSU 23 containing 10% FBS for 2 days. For the evaluation of chromosomal aneuploidies, chromosome 1 sub-metacentric specific probe was used in sperm and embryos by fluorescence in situ hybridization (FISH). The frequency of aneuploidy sperm for chromosome 1 was 6.25%. The significant differences following IVF and ICSI with non- or HA-bound sperm were not observed in blastocyst formation rates (18.6, 23.5, and 23.8%) and cell number (61.8 $\pm$ 12.5, 55.5 $\pm$ 7.3, and 59.3 $\pm$ 9.6). Moreover, the percentage of diploidy in 4-cell stage embryos was 57.1% (IVF), 68.8% (ICSI), and 76.3% (ICSI-HABS). These results suggest that HA-binding sites may be a material for selection of normal sperm for ICSI. Therefore HA selection of normal sperm may be reduce the loss to embryonic mortality prior to embryo transfer in pig.

• Clinical and Experimental Reproductive Medicine
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• v.42 no.3
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• pp.101-105
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• 2015

Objective: Several publications have established a relationship between sperm DNA damage and male factor infertility, based on data from America, Europe, and Asia. This study aimed to compare the extent of sperm DNA damage in sperm samples from Nigerian men with unexplained infertility and in sperm samples from a fertile group composed of sperm donors who had successfully impregnated a female partner naturally or through assisted conception. Methods: A total of 404 men underwent male fertility evaluation at Androcare Laboratories and Cryobank participated in this study. Semen analysis and a sperm chromatin structure assay (SCSA) were performed on all subjects. Results: The men in the unexplained infertility group were slightly older than the men in the fertile sperm group ($36{\pm}10$ years vs. $32{\pm}6$ years, p=0.051). No significant difference was observed between the two groups in semen analysis parameters ($p{\geq}0.05$). Men in the unexplained infertility group with normal semen parameters had a significantly higher DNA fragmentation index (DFI) than men in the fertile sperm group ($27.5%{\pm}7.0%$ vs. $14.1%{\pm}5.3%$, p<0.05). In the unexplained infertility group, 63% of the men had a DFI greater than 20%, compared to 4% in the fertile sperm group. In the unexplained infertility group, 15.2% of the subjects had a DFI greater than 30%, compared to 1% in the fertile sperm group. Conclusion: Our study showed that the SCSA may be a more reliable predictor of fertility potential than traditional semen analysis in cases of unexplained infertility.

• Development and Reproduction
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• v.21 no.3
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• pp.229-235
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• 2017

In the field of reproductive medicine, assessment of sperm motility is a key factor for achieving successful artificial insemination, in vitro fertilization, or intracellular sperm injection. In this study, the motility of boar sperms was estimated using real-time imaging via confocal microscopy. To confirm this confocal imaging method, flagellar beats and whiplash-like movement angles were compared between fresh and low-temperature-preserved ($17^{\circ}C$ for 24 h) porcine sperms. Low-temperature preservation reduced the number of flagellar beats from $11.0{\pm}2.3beats/s$ (fresh sperm) to $5.7{\pm}1.8beats/s$ and increased the flagellar bending angle from $19.8^{\circ}{\pm}13.8^{\circ}$ (fresh) to $30.6^{\circ}{\pm}15.6^{\circ}$. These data suggest that sperm activity can be assessed using confocal microscopy. The observed motility patterns could be used to develop a sperm evaluation index and automated confocal microscopic sperm motility analysis techniques.

• Journal of Animal Reproduction and Biotechnology
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• v.36 no.2
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• pp.106-115
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• 2021

Sperm cryopreservation is an important method of assisted reproductive techniques and storing genetic resources. It plays a vital role in genetic improvement, livestock industrial preservation of endangered species, and clinical practice. Consequently, the cryopreservation technique is well organized through various studies, especially on Korean native cattle (Hanwoo). However, the cryopreservation technique of Korean native brindled cattle, which is one of the native cattle species in Korea, is not well organized. Therefore, it is necessary to develop a Supplementary Table technique for the cryopreservation of Korean native brindled cattle. For this purpose, it is important to first evaluate the quality of the currently produced cryopreserved sperm of Korean native brindled cattle. In this study, we randomly selected 72 individual Korean native brindled cattle semen samples collected from 8 different region research centers and used them to evaluate sperm functions. We focused on the quality evaluation of cryopreserved Korean native brindled cattle semen following the measurement of motion kinematics, capacitation status, intracellular ATP level, sperm motility, and cell viability. Then, the values of each of the eight groups were derived from various sperm parameters of nine individual samples, including sperm motility, kinematics, cellular motility, and intracellular ATP levels, which were used to compare and evaluate sperm function. Overall, differences in various sperm parameters were observed between most of the research centers. Particularly, the deviations of motility and motion kinematics were high according to the sample. Therefore, we suggest that it is necessary to develop a standard method for the cryopreservation of Korean native brindled cattle semen. We also suggest the need for sperm quality evaluation of the cryopreserved semen of Korean native brindled cattle before using artificial insemination to attain a high fertility rate.