• Clinical and Experimental Reproductive Medicine
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• v.15 no.2
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• pp.149-155
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• 1988

To increase fertilization rate in vitro, separation of viable spermatozoa from the seminal plasma and its other components may be a useful procedure. Ejaculates from healthy men, whose semen analysis findings were normal in 19, and abnormal in 10, were filtered using the glass-wool filtration technique to yield a concentrated, viable sperm samples for IVF, and the usefulness and safety of this method were evaluated. The recovery rate of motile sperm in abnormal groups was 46.2% and 54.5% in normal group. The % motility was increased significantly compared with original sample after filtration, and the grade motility was improved, too. The sperm population with normal morphology was also increased significantly in both group. Using transmission electron microscopy, the ultrastructural integrity of acrosomal segment was examined in order to evaluate the potentially hazardous effect of glass-wool filtration to sperm head, however, sperm population with normal ultrastructure was increased compared with that of original ejaculate after separation. The filtered sperm was then processed for IVF, as the fertilizing capacity is the ultimate parameter of the sperm function. In abnormal group, the fertilization rate(41.5 %) and the ET rate per stimulated cycle were much lower than that of mormal group(69.6%). However, the cleavage rate and the number of embryos transfered per ET cycle were comparable with those of nomal group. The results suggest that the glass-wool filtration of sperm, particularly in oligo-asthenozoospsrmia, may be useful and safe method in the preparation of sperm for IVF.

• Journal of Embryo Transfer
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• v.24 no.4
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• pp.275-279
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• 2009

The beneficial effect of glycerol as a cryoprotectant, especially for sperm cryopreservation, has been shown in many studies. However, glycerol is toxic to living cells, and boar sperm in particular show greater sensitivity to glycerol than sperm from other domestic animals. Amides have been studied as alternative cryoprotectants for freezing stallion sperm. Sperm frozen in methylformamide or dimethylformamide as cryoprotectants show similar motility when thawed compared with sperm frozen in glycerol. We evaluated the cryoprotective effects of dimethylformamide on boar sperm freezing. To test the effect of amides, the concentration of boar semen was adjusted to $10^9sperm/mL$, and seminal plasma was removed using Hulsen solution. After centrifugation, the pellet was diluted in modified-Modena B extender. Lactose-egg yolk (LEY) extender was used as the cooling extender. The freezing extender was madeed aaddition of the optimal amount of glycerol and amides to LEY-Glycerol-Orvus ES Paste extender, and this extender was used for the second dilution. Diluted sperm were frozen in liquid nitrogen using the 0.5 mL straw method. Sperm frozen in extender with glycerol as a cderol were compared with those frozen in extender including the different amides. Sperm were tested for motility, viability, the sperm chromatin structure assay, and normal apical ridge after thawing. The percent of motile sperm diluted in glycerol was as high as that in the stallion study (61%). Dimethylformamide showed positive effects on sperm quality and was better than glycerol. Methylformamide provided similar sperm quality as glycerol. Therefore, dimethylformamide is useful for reducing cryoinjury in boar sperm and is expected to be useful as an alternative cryoprotectant.

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.1
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• pp.118-125
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• 2019

Objective: This study investigated the effects of soapnut (Sapindus mukorossi) shell powder (SSP) on serum hormone level, egg quality, semen characteristics and reproductive performance of broiler breeders fed with a maize-soybean meal based diet. Methods: Ninety six female and twenty four male CARIBRO-VISHAL broiler breeders, 38-week old, were individually caged and randomly allocated to four treatment groups (24 female breeders/treatment and 6 male breeders/treatment): an un-supplemented control (T1) and three groups with 0.0176% SSP (group T2), 0.026% SSP (group T3) and 0.0528% SSP (group T4), to have supplementary saponin at 0, 50, 75, and 150 ppm, respectively, for 42 days. Results: The results indicated that serum (p<0.001) and seminal plasma (p<0.05) testosterone level, semen volume (p<0.001), mass motility (p<0.001), and live spermatozoa count (p<0.001) was increased in groups T3 and T4 compared to T2 and control groups. Compared with control group, total sperm count was increased (p<0.001) and dead spermatozoa count was decreased (p<0.001) in SSP supplemented groups. Supplementation of SSP did not affected the quality of egg lay. Compared with control group, fertility (p<0.01) and hatchability (total eggs set and fertile eggs set) (p<0.001) were significantly improved in SSP supplemented groups with the highest improvement in T3 treatment group. Embryonic death was decreased (p<0.001) in SSP supplemented groups compared to control; lowest embryonic death was recorded in T3 treatment group. Conclusion: Thus, it was concluded that dietary supplementation of 0.026% SSP (saponin equivalent 75 ppm) improved the reproductive performance of broiler breeders.

• 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.

• Fisheries and Aquatic Sciences
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• v.24 no.2
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• pp.63-77
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• 2021

The aim of this research was to investigate different factors, including cryoprotective agents (CPAs), diluents, dilution ratios, equilibrium times, freezing rates, and thawing methods to optimize cryopreservation protocols for large yellow croaker (Larimichthys crocea). The parameters evaluated were sperm motility, sperm activity index (SAI), survival rate, and DNA damage. Different types of CPAs, such as dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), methanol, and glycerol, were tested for sperm preservation. The highest motility, SAI, and survival rate were observed when EG was used. Different diluents such as Stein's solution, Hank's balanced salt solution, marine fish Ringer's solution, artificial seminal plasma (ASP) of small yellow croaker, and Cortland solution were investigated. The highest post-thaw motility was observed upon using ASP as the diluent. Different concentrations of EG were then mixed with ASP to identify the optimal EG concentration. Experimental results showed that the motility (70.33 ± 1.20%), SAI (5), and survival rate (78.30 ± 0.42%) of post-thaw sperm were optimum when 10% EG and ASP were used as the CPA and diluent of cryopreservation, respectively. Post-thaw sperm motility was high at equilibration times below 150 s and at an optimum dilution ratio of 1:1 (sperm: CPA + diluent) and was not significantly different compared with fresh sperm motility. The freezing rate was found to be slow below -10℃/min. The thawing temperature of 45℃ was identified as ideal. The percentage of tail DNA in post-thaw sperm at 10% EG and ASP was also investigated and was found to have more significant DNA damage than that in fresh sperm but significantly lower damage than that in post-thaw sperm at EG concentrations of 5%, 15%, and 20% (p < 0.05). The cryopreservation protocols obtained in this study will be useful in large yellow croaker hatcheries.

• Korean Journal of Animal Reproduction
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• v.26 no.3
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• pp.229-234
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• 2002

This study was carried out to investigate the general characteristics such as concentration sperm motility and abnormality of sperm on the whole epididymal semen(EWS), RSP-S(removed seminal plasma by saline) and RSP-T(removed seminal plasma by tris-buffer) semen and survival rates after freezing on motility of whole and RSP-S and RSP-T semen and extender containing 2~8% glycerol, and ability of frozen-thawed sperm to penetrate homologous oocytes. 1. The concentration, motility and abnormality of epididymal WES, RSP-S and RSP-T sperm were 4.25 $\pm$ 0.25($\times$10$^{6}$ Cells/$m\ell$), 3.85$\pm$0.20($\times$10$^{6}$ Cells/$m\ell$), 4.05 $\pm$ 0.28($\times$10$^{6}$ Cells/$m\ell$), 50.55 $\pm$ 2.75%, 67.25 $\pm$ 2.55%, 78.75 $\pm$ 3.55 and 9.45 $\pm$ 2.25%, 37.75 $\pm$ 2.10%, 24.25 $\pm$ 1.55%, respectively. 2. The survival rates of slow and rapid frozen epididymal RSP-S and RSP-T sperm were 35.00 $\pm$ 2.35%, 45.50 $\pm$ 2.15％ and 16.50 $\pm$ 3.55%, 22.55 $\pm$ 3.95%, respectively. The survival rate of epididymal WES and RSP-T sperm after freezing following dilution with tris-buffer containing 2~8% glycerol were 9.25 $\pm$ 1.55%~17.50 $\pm$ 2.50%. 3. The percentage of capacitated and acrosome-reacted sperm prier to culture for fresh and frozen -thawed epididymal RSP-T semen were 45.25 $\pm$ 5.75%, 7.06 $\pm$ 0.25%, 48.20 $\pm$ 6.80% and 13.00 $\pm$ 2.35%, 3.55 $\pm$ 0.85%, 15.50 $\pm$ 1.90%, respectively. The penetration rate the number of sperm per penetrated for fresh and frozen-thawed epididymal RSP-T sperm were 39.25 $\pm$ 4.72%, 34.24 $\pm$ 3.93% and 1.30 $\pm$ 0.33, 1.10 $\pm$ 0.50., respectively.

• Journal of Aquaculture
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• v.8 no.3
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• pp.149-157
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• 1995

In order to obtain the basic knowledges concerned to the semen preservation of aquacultural fishes, studies on the physical and chemical properties of semen, and sperm motility with the different osmotic pressures making by adding $Na^+,\;K^+,\; Mg^{++},\;and\;Ca^{++}$ to artificial seawater (ASW) were conducted in black seabream, Acanthopagrus schlegeli. Average semen volume per fish in one strip was 1.97ml and sperm concentration was $2.33\pm1.30\times10^{10}$ sperm/ml. Spermatocrit and pH of semen were $90.6\pm5.0\;and\;8.3\pm0.1$, respectively, Osmotic pressures of rearing seawater, seminal fluid and plasma were $939\pm24,382\pm70\;and\;342\pm77$ mOsm/l, and $Na^+,\;K^+$ and $Cl^-$ concentrations of seminal fluid were $169.5\pm4.5,\;4.9\pm2.2,\;156.0\pm2.0\;mM/l$, respectively. When semen were diluted by using $Na^+,\;K^+,\;Mg^{++}\;and\;Ca^{++}$ free ASW, only $Na^+$ free ASW had no sperm motility. As raising osmotic pressure graduary by addition of 1M NaCl to the $Na^+$ free ASW, spermatozoa showed the high motilities in 457-1128 mOsm/l, but the low motilities in 1398-1736 mOsm/l. In the case of same treatments with 1M of KCl, $MgC1_2\;and\;CaC1_2$ to the $K^+,\;Mg^{++}\;and\;Ca^{++}$ free ASW, spermatozoa revealed the high motilities in $904\~1434,\;818\~1175\;and\;956\~1343$ mOsm/l, respectively.

• Korean Journal of Animal Reproduction
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• v.23 no.2
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• pp.127-132
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• 1999

This study was carried out to investigate the general characteristics such as volume, sperm concentration, sperm motility, sperm abnormality on whole semen, removed seminal plasma(RSP) semen and fractional semen of small dogs, and the effect of temperature and preservatio time and cryopreservation on motility of whole and removed seminal plasma semen. Multiple ejaculates were collected from small dogs by the digital manipulation of penis. 1. Average sperm concentration, sperm motility and abnormal sperm rates of whole semen and RSP semen were 5.07$\pm$2.32$\times$10$^{6}$ cells/$m\ell$, 95.42$\pm$2.65%, 4.42$\pm$0.157% and 4.69$\pm$3.27~4.25$\pm$3.65$\times$10$^{6}$ cells/$m\ell$, 91.17$\pm$3.85~88.52$\pm$3.85%, 6.57$\pm$0.43~5.54$\pm$0.52%, respectively. 2. Average semen volume per ejaculate, sperm concentration, sperm motility and abnormal sperm rate of 1st fractional semen were 0.92$\pm$0.7$m\ell$, 4.57$\pm$0.78$\times$10$^{6}$ cells/$m\ell$, 10.72$\pm$3.21% and 5.50$\pm$0.70%. Average semen volume per ejaculate, sperm concentration, sperm motility and abnormal sperm rate of 2nd fractional semen were 2. 14$\pm$0.19$m\ell$, 2.01$\pm$0.12$\times$10$^{6}$ cells/$m\ell$, 95.44$\pm$4.21% and 4.31$\pm$0.53%. Average semen volume per ejaculate, sperm concentration, sperm motility and abnormal sperm rate of 3rd fractional semen were 2.66$\pm$0.23$m\ell$, 2.35$\pm$0.21$\times$10$^{6}$ cells/$m\ell$, 90.71$\pm$2.63%, 6.33$\pm$0.91%, respectively. 3. Motility of whole semen and RSP semen were higher at 2$0^{\circ}C$ than at 4$^{\circ}C$ or 37$^{\circ}C$. When preservation temperature was 2$0^{\circ}C$, sperm motility were 98.32% at 1 hr, 92.15% at 5 hrs, 90.23% at 10 hrs 82.08% at 15 hrs 70.07% at 20 hrs 60.02% at 20 hrs 37.19% at 40 hrs respectively. 4. Average sperm motility of frozen 2nd fraction semen and RSP semen were 33.3$\pm$8.7, 54.7$\pm$9.5%, respectively. Sperm motility was significantly higher in frozen 2nd fraction semen and RSP semen compared with control group.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.2
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• pp.180-184
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• 2003

The intrauterine inseminator (IUI) was developed to provide the method of depositing dog semen into the uterine body instead of the vagina. The IUI consists of a vaginal endoscope, a balloon sheath, and injection catheter. When the endoscope is inserted into the vagina and the balloon expanded with air, the cervical os becomes visible so a injection catheter can be inserted through the cervix for deposition of the frozen-thawed semen. The efficacy of the IUI device was compared to intra-vaginal artificial insemination using semen that had been collected and frozen from pooled sperm-rich fraction of ejaculates collected from two Jindo dog donors. Aliquots of semen were extended with a Tris-egg yolk diluent, centrifuged, the seminal plasma removed, the pellet resuspended with the same diluent, and cooled to $5^{\circ}C$ over a 2 h period. A Tris-egg yolk-glycerol extender was added at $5^{\circ}C$; after 1 h, semen was loaded into 0.5 ml straws, and straws were frozen in LN vapor for 5 min, and immersed in LN for storage. The final sperm concentration for freezing was approximately $100{\times}10^{6}cells/ml$. The straws were thawed at $70^{\circ}C$ for precisely 6 sec, 1.5 ml Tris-egg yolk buffer at $38^{\circ}C$ added, and the 2 ml of thawed semen was used for a single insemination using the IUI device. Each bitch was inseminated at optimal insemination point, which was estimated by vaginal epithelial cells staining and progesterone concentration analysis. Use of the IUI device resulted in 21 of 26 females giving birth to 89 pups ($4.2{\pm}1.6$ pups per litter), while intra-vaginal AI resulted in 6 of 15 females whelping a total of 17 pups ($2.8{\pm}1.2$ pups per litter). We believe the IUI device is easier to use than previously described devices used for intrauterine insemination. In our experience the expansion of the balloon has a calming effect on the bitch that aids the inseminator. These results indicate that the IUI device was able to provide high fertility with 50 million frozen sperm per insemination and two inseminations.

• Reproductive and Developmental Biology
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• v.34 no.3
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• pp.271-274
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• 2010

This study was conducted to investigate the survival rate of frozen-thawed spermatozoa in equine by glycerol concentration and freezing speed Two stallions (1 Thoroughbred-13 year old and 1 Arab-7 year old) bred in Korea Racing authority was examined for 1 times in a couple of weeks. Semen was collected by condom method standing heated mare and were centrifuged 650 g for 15 min. and isolated the seminal plasma. Thick fraction of semen was diluted EDTA-Lactose-egg yolk diluents to 1:1 and contained in 0.5 ml straw as $6{\sim}14{\times}10^7\;cells/ml$. Final concentrations of glycerol were 3, 5 and 7% in cryopreseved diluents and added 4 times for 2 hours equilibration. For the freezing, equilibrated straws were located 3 or 5 em above $LN_2$ gas for 5 or 10 min. Survival rates of pre-frozen sperm were $65.0{\pm}13.2%$, $68.3{\pm}10.4%$, $66.7{\pm}11.5%$ and post-frozen were $53.3{\pm}23.1%$, $45.0{\pm}15.0%$, $50.0{\pm}18.0%$ in 3, 5, 7% glycerol concentration, respectively. There was no difference between glycerol concentrations. Survival rates of frozen-thawed sperm on freezing speed were $36.7{\pm}10.4%$, $40.0{\pm}7.1%$, $30.0{\pm}13.2%$ at 3 cm-5 min and $33.3{\pm}11.5%$, $31.7{\pm}2.9%$, $21.7{\pm}10.4%$ at 3 cm-10 min in 3, 5, 7% glycerol concentration, respectively. Survival rates of frozen-thawed sperm on freezing speed were $43.3{\pm}15.3%$, $32.0{\pm}17.9%$, $22.3{\pm}15.7%$ at 5cm-5 min and were $47.5{\pm}15.0%$, $43.3{\pm}12.6%$, $48.3{\pm}15.3%$ at 5cm-10 min in 3, 5, 7% glycerol concentration, respectively. There were significantly different between groups (p<0.05). These results suggest that glycerol concentration did not affect cryopreservation of stallion semen within 3~7% but freezing speed affects. In our experiment, the best cryopreservation condition was at 5 cm above $LN_2$ gas for 10 min for pre-freezing and 7% of glycerol concentration. These results lead to commercial AI with frozen-thawed stallion semen.