• Asian-Australasian Journal of Animal Sciences
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• 제28권12호
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• pp.1713-1720
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• 2015

The present work describes experiments undertaken to evaluate the usefulness of selected physicochemical indices of semen, cell membrane integrity and sperm chromatin structure for the assessment of boar semen sensitivity to processes connected with pre-insemination procedures. The experiments were carried out on 30 boars: including 15 regarded as providers of sensitive semen and 15 regarded as providers of semen that is little sensitive to laboratory processing. The selection of boars for both groups was based on sperm morphology analyses, assuming secondary morphological change incidence in spermatozoa as the criterion. Two ejaculates were manually collected from each boar at an interval of 3 to 4 months. The following analyses were carried out for each ejaculate: sperm motility assessment, sperm pH measurement, sperm morphology assessment, sperm chromatin structure evaluation and cell membrane integrity assessment. The analyses were performed three times. Semen storage did not cause an increase in the incidence of secondary morphological changes in the group of boars considered to provide sperm of low sensitivity. On the other hand, with continued storage there was a marked increase in the incidence of spermatozoa with secondary morphological changes in the group of boars regarded as producing more sensitive semen. Ejaculates of group I boars evaluated directly after collection had an approximately 6% smaller share of spermatozoa with undamaged cell membranes than the ejaculates of boars in group II ($p{\leq}0.05$). In the process of time the percentage of spermatozoa with undamaged cell membranes decreased. The sperm of group I boars was characterised with a lower sperm motility than the semen of group II boars. After 1 hour of storing diluted semen, the sperm motility of boars producing highly sensitive semen was already 4% lower ($p{\leq}0.05$), and after 24 hours of storage it was 6.33% lower than that of the boars that produced semen with a low sensitivity. Factors that confirm the accuracy of insemination male selection can include a low rate of sperm motility decrease during the storage of diluted semen, low and contained incidence of secondary morphological changes in spermatozoa during semen storage and a high frequency of spermatozoa with undamaged cell membranes.

• 대한생식의학회:학술대회논문집
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• 대한생식의학회 2007년도 제52차 춘계학술대회 초록집
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• pp.67-67
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• 2007

• Animal Bioscience
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• 제34권8호
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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.

• 한국수정란이식학회지
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• 제14권1호
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• pp.47-57
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• 1999

Among proteins separated from methanol extract of follicular fluid with superose column, the components inducing sperm swim-up separation through sucrose layer were analysed with superose column in Smart system and SDS-PAGE. And the results obtained were as follows; The fractions of retention volume (RV) 0.83ml and RV 1.36ml separated with superose column should stimulate sperm migration and movement. However, RV 0.83 fraction was consisted of complex materials containing RV 1.36 component. RV 1.36 fraction contained a BSA analogue of 67 kilodaltons (Kd) and showed identical peak pattern with BSA fraction V. In conclusion, the protein of 67 Kd in follicular fluid should stimulate sperm migration and movement.

• Asian-Australasian Journal of Animal Sciences
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• 제33권9호
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• pp.1411-1420
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• 2020

Objective: The aim of study was to investigate the effects of season and single layer centrifugation (SLC) before cryopreservation on post-thaw bull sperm quality in Thailand. Methods: Semen was collected from 6 bulls (Bos indicus) in summer, rainy season and winter 2014 through 2016. Semen characteristics, sperm morphology, sperm kinematics, viability, chromatin structure and mitochondrial membrane were evaluated. Meteorological data were available from the local meteorological station; Results: Season had an effect on semen characteristics in the raw ejaculate, with higher proportions of normal spermatozoa and lower abnormalities in winter than in the other two seasons. Sperm kinematics, viability, DNA fragmentation index, and mitochondrial membrane potential were not different between seasons. Sperm samples selected by SLC had greater normal morphology and a lower proportion with bent tails than controls and higher values of progressive motility (PRO), beat cross frequency, linearity, straightness, wobble (WOB), and lower values of slow motility, velocity average path (VAP), velocity curved line, and amplitude of lateral head displacement than controls. In addition, SLC-selection had a favorable effect on PRO, VAP, and WOB that differed among seasons. Conclusion: Our results suggested that these bulls were well adapted to their location, with season having an effect on sperm morphology. Moreover, SLC could be used prior to cryopreservation, regardless of season, to enhance normal morphology and kinematics of bull sperm samples without adversely affecting other parameters of sperm quality. However, there was considerable variation among bulls in DNA fragmentation index, mitochondrial membrane potential and sperm viability. In addition, SLC had a positive effect on sperm morphology and sperm kinematics, which could be expected to influence fertility.

• Clinical and Experimental Reproductive Medicine
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• 제18권1호
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• pp.95-99
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• 1991

Swim-up and Wang's tube system are known methods of motile sperm selection and aerobic bacterial removal from the raw semen. This study was designed to evaluate the recovery rate of motile sperm, % normal morphology of sperm, the efficiency of bacterial removal after sperm preparation by the above two methods. The results were as follows. 1. There was more significant increase of sperm concentration in preparation by swim-up than Wang's tube (p<0.05). The concentration of sperm by swim up was changed from $82.5{\times}10^6/ml$ to $6836{\times}10^6/ml$, and Wang's tube was changed from $82.5{\times}10^6/ml$ to $36.0{\times}10^6/ml$. 2. There was significant increase in sperm motility after preparation by two methods in comparison with initial sperm motility (p<0.05), but no statistical difference between two methods was noted. The % motility of sperm by swim-up was increased from 66.1% to 95.7% and Wang's tube from 66.1% to 98.1%. 3. There was significant increase of % normal morphology of sperm in the samples prepared by two methods (p<0.05), from 49.2% to 85.3% in swim-up and from 49.2% to 92.1% in Wang's tube, but there was no statistical significance between them. 4. There was no bacterial growth in aerobic culture after preparation by two methods.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.73-73
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• 2003

The present study examined the possibility of long term storage, by cryopreservation in liquid nitrogen, of the sperm of Filefish (Thamnaconus septentrionalis), and the changes in motility, survival rate and ultrastructure of the sperm after freezing and thawing. The sperm was collected by stripping and stored on ice until experiments. For selection of the immobilizing solution, diluted artificial seawater (ASW) of 20, 30 and 40% were tested. The sperm motility was significantly inhibited in 30% ASW, and restored entirely after 100% ASW was added again. Two cryoprotectants, dimethyl sulfoxide ($Me_2$SO) and glycerol, were added to 30% ASW to formulate the extenders at the concentrations between 5 to 20% by volume for freezing. The sperm was diluted at the ratio of 1 :6 with the extenders, inserted into 0.5ml plastic straws and frozen at a freezing rate of $50^{\circ}C$/min to $-100^{\circ}C$ after equilibration for 10 min at room temperature, followed by plunging into liquid nitrogen. The straws were thawed in a $30^{\circ}C$ water bath for 15 sec. The highest post-thawed sperm motility and survival rate were obtained with 5% glycerol Afterward, the effect of different freezing rates was examined using 5% glycerol as a cryoprotectant, and the rate of $20^{circ}C/min to $-80^{\circ}C$ showed the best result Some ultrastructural changes of sperm, such as the detachment of plasmatic and nuclear membranes, destruction of mitochondria, were observed after cryopreservation. Morphological normality of the sperm in 5% glycerol frozen at the ratio of 1$0^{\circ}C$/min to $-80^{\circ}C$ was better than that of others.

• 한국동물생명공학회지
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• 제35권1호
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• pp.58-64
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• 2020

This present study was conducted to investigate protective effect of discontinuous Percoll gradient containing alpha-linolenic acid (ALA) before freezing process on viability, acrosome damage, mitochondrial activity, and oxidative stress of frozen-thawed boar spermatozoa. The separation of spermatozoa by discontinuous Percoll gradient was performed by different concentration of Percoll solution (45/90%) containing ALA combined with bovine serum albumin (BSA), and collected sperm in each Percoll layer was cryopreserved. To evaluate viability, acrosome damage, mitochondrial activity, and reactive oxygen species (ROS) level of frozen-thawed sperm, flow cytometry was used. Morphological abnormalities were observed under light microscope. In results, viability of sperm from 90% Percoll layer was higher than control and 45% Percoll group (p < 0.05). Separated sperm in 90% Percoll layer had lower acrosome damage and morphological abnormalities than control as well as viability, whereas 45% Percoll group was higher (p < 0.05). Similar with acrosome damage and abnormalities, mitochondrial activity was slightly enhanced and the population of live sperm with high ROS level was decreased by 90% Percoll separation, however, there was no significant difference. Supplementation of 3 ng/mL ALA into Percoll solution increased sperm viability and decreased population of live sperm with high ROS compared to control (p < 0.05). In conclusion, discontinuous Percoll gradient before freezing process could improve efficiency of cryopreservation of boar sperm through selection of sperm with high freezing resistance, and supplement of ALA during Percoll gradient might contribute suppression of ROS generation via stabilizing of plasma membrane during cryopreservation.

• Clinical and Experimental Reproductive Medicine
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• 제27권3호
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• pp.267-273
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• 2000

Objectives: We have previous reported that thawed testicular sperm and sperm extracted from seminiferous tubule could achieved optimal fertilization and pregnancy in azoospermic patients. However, thawed testicular sperm did not show motility in many cases. Therefore we studied viability of immotile sperm extracted from frozen-thawed seminiferous tubule using hypo-osmotic swelling (HOS) test and eosin-Y test. Materials and Methods: After sperm extraction using for ICSI, the remained sections of seminiferous tubules were frozen with a computerized freezer. For thawing and preparation of testicular sperm, the seminiferous tubules were thawed by removing from $LN_2$ and letting them at room temperature for 10 min followed by %37^{\circ}C$ water bath for 10 min. The prepared samples were washed for free of preservation medium and sperm preparation method described previous. Sperm was suspended in 0.1 ml hypoosmotic solution. After 30 minutes, the type of distally coiled sperm were assessed. Results: In 44 cases of cryopreservation of seminiferous tubules in obstructive azoospennic patients, the fertilization rates with 2PN were 71.4% and pregnancy rates were 34.1%. The presence of motile spermatozoa on subsequent post-thaw testicular sperm remarked 15.1% and were increased to 77.3% just before ICSI. After sperm extracted from frozen-thawed seminiferous tubule, 3 hrs later in in vitro culture, the cases of presence of motile sperm, reaction of hypo-osmotic swelling test and viable sperm were 63.6% (28/44), 93.2% (41/44), and 77.3% (34/44), respectively. Conclusions: Just after post-thawed testicular sperm did not showed motility. Although motility was gained after in vitro culture, many cases showed non-motile sperm until optimal insemination time. However, HOS test showed positive reaction in non-motile sperm. Therefore, HOS test is an alternative method for the selection of viable sperm for ICSI.

• 한국수정란이식학회지
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• 제13권3호
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• pp.291-300
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• 1998

난포액에 함유되어 있는 steroids와 sterol이 수정에 참여하는 정자의 주화성에 미치는 영향을 밝히기 위하여, progesterone, estradiol 17 beta 및 cholesterol이 sucrose 층으로부터 정자의 swim-up 분리에 미치는 영향을 조사하였던 바 얻어진 결과는 다음과 같다. 1. Progesterone은 정자의 이동과 운동성을 억제하였으나 수정능획득한 정자를 유인하였으며, 특히 50$\mu$g/ml 수준의 progesterone은 수정능력을 획득한 정자의 이동을 유의하게 자극하였다. 2. Estradiol 17 beta의 첨가수준이 증가할수록 정자의 이동과 운동성이 억제되었으나, 10$\mu$g/ml 수준의 estradiol은 정자의 이동과 운동성 및 수정능획득정자의 이동에 영향을 미치지 않았다. 3. Cholesterol은 정자의 이동과 운동성을 자극하였으나, 수정능획득정자의 이동에 영향을 미치지 않았으며, 특히 50$\mu$g/ml 수준의 cholesterol은 정자의 이동과 운동성을 유의하게 자극하였다. 4. Progesterone, estradiol 및 cholesterol의 병용처리에서, cholesterol은 정자의 이동과 운동을 유의하게 자극하였으나, progesterone과 estradiol은 이러한 cholesterol의 효과를 감소시켰다. Progesterone은 수정획득 정자의 이동을 자극하였으나 estradiol이나 cholesterol은 progester-one의 이러한 효과를 억제하지 않았다. 결론적으로 progesterone은 50$\mu$g/ml 첨가수준에서 수정능력을 획득한 정자의 swim-up이동을 유의하게 자극하였으며, cholesterol은 50$\mu$g/m1 첨가수준에서 정자의 이동을 자극하였으나, estradiol은 10$\mu$g/m1 첨가수준에서 정자의 이동과 운동성에 영향을 미치지 않았다.