• 한국동물생명공학회지
• /
• 제35권1호
• /
• pp.58-64
• /
• 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
• /
• 제21권3호
• /
• pp.261-266
• /
• 1994

Semen preparation for Assisted Reproductive Technology(ART) involves the separation of the motile sperm from non-motile, dead sperm, cellular debris and seminal fluid. The aim of this study is to compare the motile sperm recovery rate and motility index of different sperm preparation method(swim-up method, 80% isotonic continuous percoll method, two-layer discontinuous percoll method, mini-percoll method). In Mini-Percoll method, pellets from patients were suspended in 0.3ml of medium and layerd on a discontinuous percoll gradient consisting of 0.3ml each of 50, 70, 95% isotonic percoll. All semen samples are divided into normal and subnormal sperm group(oligo-, astheno-, oligoasthenozoospermia). Especially, we evaluated the effect of mini-percoll method in subnormal sperm group. In normal sperm group, mini-percoll method and two layer discontinuous percoll method (40%/80%) allowed increasing of motile sperm recovery rate. But motility index was higher in swimup method than the other methods. In subnormal sperm group, mini-percoll method has advantages as compare with the other methods in motile sperm recovery rate and motility index. These results suggest that modified mini-percoll method could be certainly a valuable tool in some cases of severe male factor sperm.

• Clinical and Experimental Reproductive Medicine
• /
• 제26권2호
• /
• pp.257-263
• /
• 1999

It is well known that discard of seminal plasma from the semen and separation of motile sperm should be preceded before insemination for IUI or IVF. Till now, more than ten kinds of semen treatment methods have been developed. Of those, swim-up and Percoll methods have been used widely in ART laboratories as a routine semen treatment methods because of its advantages. However, there are reports that Percoll can make a genetic trouble because of its chemical structure and therefore the necessity has been arisen to substitute Percoll for other equivalent materials. This study was performed to evaluate the effects of three different sperm preparation methods (swim-up, Percoll and Sil-Select) on sperm motility, sperm recovery rate and fertilization rate. Also, the feasibility of using Sil-Select instead of Percoll in ART was evaluated. Each semen samples were divided into three fractions and motile sperm were recovered by swim-up, Percoll and Sil-Select gradient centrifugation methods. Normal and sub-normal criteria of fifteen semen samples and seventeen IVF cycles were included in these study. As results, no significant difference was found in sperm recovery rate in normal semen treated by a Swim-up, Percoll and Sil-Select method ($13.2{\times}10^6,\;17.5{\times}10^6\;and\;17.7{\times}10^6$ respectively). The initial sperm motility was 61.9% and this increased to 87.1%, 92.6% and 89.5% through Swim-up, Percoll and Sil-Select treatment, respectively. Higher motility was observed in Percoll and Sil-Select treated groups (81.5%, 79.2%, respectively) than swim-up group (66.8%) after incubation for 24hrs. In sub-normal group, sperm recovery rates were higher in Sil-Select group $(2.9{\times}10^6)$ than Percoll gradients group $(1.8{\times}10^6)$. In IVF cycles, the outcomes of fertilization using sperm treated by swim-up and Sil-Select group were similar (82.2%, 79.7% respectively). In conclusion, our results indicate that Sil-Select can be used as a substitute material for sperm preparation instead of Percoll.

• Asian-Australasian Journal of Animal Sciences
• /
• 제12권5호
• /
• pp.702-707
• /
• 1999

A percoll density gradient technique was developed for producing high quality turkey semen and improving the fertility by removing deleterious cellular components, including spermiophages, bacteria, abnormal or dead spermatozoa, and other cellular debris. The combination of three different percoll densities, 1.05, 1.07, and 1.08 showed the best resolution and was selected to prepare a discontinuous percoll density gradient to obtain healthy spermatozoa from semen smples. Bacteria, spermiophages, and abnormal or dead spermatozoa were detected from the density range from 1.05, 1.05 to 1.07, and 1.07 to 1.08, respectively. Healthy spermatozoa were collected from the density greater than 1.08. Spermatozoa obtained from percoll density gradient centrifugation showed better sperm motility than those from unprocessed pooled semen. Bacteria including Escherichia coli, Staphylococcus aureus, and Proteus spp., were predominant contaminants in turkey semen, and the numbers of cells were approximately $5{\times}10^5$ to $1{\times}10^9cfu/ml$. The overall fertility rates in hens inseminated with processed percoll density gradient were higher than those in hens with unprocessed semen especially for unhealthy sperm. In conclusion, semen quality can be improved by percoll density gradient centrifugation, which augmented the fertility of turkey breeders.

• Journal of Microbiology and Biotechnology
• /
• 제9권4호
• /
• pp.522-524
• /
• 1999

A discontinuous Percoll gradient was used to separate islets from the collagenase-treated mouse pancreas easily and rapidly. Since the osmolality of Percoll is very low, adjustment of its osmolality to 340 mOs/kg $H_2$O was essential for securing the optimal separation. A discontinuous gradient layering with Percoll solution of 1.09 g, 1.07g, and1.05g/m, respectively, when centrifuged at 800$\times$g for 10 min, resulted in an optimal condition for separation and yielded a banding pattern with an even distribution of islet cells. No significant difference was observed in the morphological features between the Percoll-isolated and the manually-isolated islets. In conclusion, the discontinuous Percoll gradient can be effectively used to isolate the pancreatic islets from mice with four-fold higher efficiency compared to the handpicking method.

• Clinical and Experimental Reproductive Medicine
• /
• 제20권1호
• /
• pp.57-63
• /
• 1993

From September 1988 to August 1992, two different methods of preparing human sperm before intrauterine insemination(IUI) were compared using the semen samples of seventy-three infertile couples. The sperms were prepared by a swim-up after sperm washing or by a continuous percoll gradient technique. Fourteen of 35 women conceived during IUI cycles using a sperm washing and swim-up method (40%), and 12 of 38 women conceived during IUI cycles using a percoll gradient technique(31.6%). Among the group with male infertile etiologic factor only, one of 5 women conceived during sperm washing and swim-up cycles(20%); one of 4 women conceived during percoll gradient cycles(25%). On the contrary, among the group with cervical factor only, six of 10 women conceived during sperm washing and swim-up cycles (60%) ; Five of 17 women conceived during percoll gradient cycles(29.4%). It is suggested that sperm separation by sperm washing ar -up is a useful technique for intrauterine insemination in cervical infertility, and sperm separation in percoll gradient appears to be more valuable for intrauterine insemination of male subfertility.

• Clinical and Experimental Reproductive Medicine
• /
• 제27권4호
• /
• pp.339-344
• /
• 2000

Objective: To evaluate silane-coated silica particles (Sil-select) as an alternative to polyvinylpyrrolidone-coated particles (Percoll) for gradient separation of spermatozoa, for use in assisted reproduction. Methods: 20 normal semen based on WHO criteria were included in this study. Recovery of motile and morphologically normal spermatozoa after using two-layer Percoll and Sil-select gradient respectively was recorded. Motility, HOST (hypoosmotic swelling test) and the detection of malondialdehyde for LPO (lipid peroxidation) after 24 h of incubation at $37^{\circ}C$ in a 5% $CO_2$ incubator were compared. Results: Percoll (78.5%) and Sil-select (79.1%) showed a significant increase in the motility compared to ejaculate (60.9%) but no difference between Percoll and Sil-select. Normal sperm morphology significantly increased after Percoll (57.6%) and Sil-select (53.7%) compared to ejaculate (35.8%) but no difference between Percoll and Sil-select. No differences in the recovery of motile spermatozoa and motility, HOST and the production of malondialdehyde after 24 h incubation were found when comparing the use of Percoll and Sil-select. Conclusion: Sil-select seems to be an attractive alternative to Percoll for sperm separation in assisted reproduction.

• Reproductive and Developmental Biology
• /
• 제35권1호
• /
• pp.61-65
• /
• 2011

The objective of this study was to examine the effect of various discontinuous Percoll washing conditions on motile sperm recovery rate and motion kinematics. Frozen semen samples from 3 bulls (0.5 ml plastic straws, 6% glycerol in egg yolk-Tris-glycerol extender) were thawed in $37^{\circ}C$ water bath for 1 min. After thawing, the mixed semen samples were randomly allocated to 12 treatment groups. Briefly, the spermatozoa were centrifuged for three different time lengths (10, 20, and 30 min) at two gravities ($300{\times}g$ and $700{\times}g$) through two concentrations of discontinuous Percoll density gradient of 1 ml 90%: 1 ml 45% Percoll and 2 ml 90%: 2 ml 45% Percoll to remove extender, debris, and dead spermatozoa. Motile sperm recovery rate and motion kinematics were evaluated by computer assisted sperm analyzer using Makler counting chamber. Sperm motility (%) and motile sperm recovery rate showed similar pattern in all treatment groups. However, sperm motility (%) and motile sperm recovery rate were highest at $700{\times}g$ for 30 min through a discontionous Percoll density gradient of 1 ml 90%: 1 ml 45% Percoll. There were no significant differences in motion kinematics after various Percoll washings. These results suggest that force of centrifugation, centrifugation time, and Percoll volume significantly affect motile sperm recovery rate.

• 한국수정란이식학회지
• /
• 제30권3호
• /
• pp.121-128
• /
• 2015

The objective of this study was to evaluate the efficiency of sperm cryosurvival in boar sperm separated by Percoll containing antioxidant enzymes. The boar semen was collected into a pre-warmed ($37^{\circ}C$) thermos bottle by gloved-hand method and was separated by 65% Percoll with superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) before freezing. The frozen sperm was thawed at $38.5^{\circ}C$ for 45 sec in water-bath for sperm characteristic analysis. The sperm were estimated with SYBR14/PI double staining for viability, FITC-PNA/PI double staining for acrosome reaction, Rhodamine123/PI double staining for mitochondrial integrity and were analyzed using flow cytometry. In results, sperm viability, acrosome reaction and mitochondrial integrity were improved in separated sperm groups compared with unseparated sperm by Percoll (UP) group. Especially, viability was significantly higher in sperm separated by Percoll containing 400 IU CAT group compared with other groups (P<0.05). And acrosome reaction was decreased in sperm separated by Percoll with 300 IU SOD, 400 IU CAT and 0.5 mM GSH groups compared with other groups, however, there were no significantly difference mitochondrial integrity among sperm separated by Percoll with antioxidant enzymes. In conclusion, we suggest that use of Percoll containing antioxidant enzymes for sperm separation will be beneficial for sperm cryopreservation in pigs.

• Reproductive and Developmental Biology
• /
• 제35권1호
• /
• pp.85-91
• /
• 2011

The objective of this study was to evaluated the efficiency on sperm cryosurvival and ability of in vitro fertilization using Triladyl and Lactose Egg-Yolk(LEY) as extenders for cryopreservation of separated sperm by 65% percoll in miniature pig. Sperm viability was measured with SYBR-14/PI double stained sperm by flow cytometry. Ability on embryo cleavage rate and blastocyst development were observed by in vitro fertilization after frozen-thawing of sperm separated by 65% percoll. The experimental groups were designed that separated sperm by 65% percoll with Triladyl (ST) or LEY(SL) and unseparated sperm with Triladyl(UT) or LEY(UL) for cryopreservation. As a results, the viability was significantly(p<0.05) higher in ST(55.1%), SL(63.1%), UL(58.8%) than UT(38.2%) group. Sperm viability in SL(63.1%) group was significantly(p<0.05) higher than other experimental groups. On the other hand, embryo cleavage rate was significantly(p<0.05) higher in ST(79.1%), SL(83.2) than UT(74.1) and UL(75.7%) groups at 96h after in vitro fertilization. Blastocyst development was also significantly(p<0.05) higher in ST(21.5%), SL(20.9%) than UT(17.0%) and UL(18.8%) groups. In conclusion, cryopreservation of miniature boar sperm separated by 65% percoll were beneficial to viability and capacity on in vitro fertilization.