In the present study, we examined if deep uterine artificial insemination (DUAI) can improve the pregnancy rate of artificial insemination (AI) using epididymal spermatozoa (ES) in Hanwoo cattle. The estrus cycles of 88 Hanwoo cows were synchronized, and 17 cows were artificially inseminated using the DUAI method with ES, 20 cows were artificially inseminated via the uterine body (BUAI) method with ES, and as a control, 51 cows were inseminated by using the BUAI method with ejaculated spermatozoa from 1 proven bull after frozen thawing. The pregnancy rate of the DUAI method (58.8%) was higher than that of the BUAI method (25.0%, p = 0.0498). The motility of ES was examined immediately after thawing and after 3 and 6 h of incubation. The rapid progressive sperm motility of the control group was significantly higher than that of the ES group immediately after thawing and after 3 and 6 h of incubation (p < 0.05). The straight line velocity and average path velocity of the ES group after 6 h of incubation were significantly lower than those in the control group (p < 0.05). The linearity and amplitude of lateral head of ES were lower than those at 6 h (p < 0.05). The flagellar beat cross frequency and hyperactivation of ES were lower than the control spermatozoa immediately after thawing and at 3 h (p < 0.05). These motility parameters suggested that ES had a low motility and fertilization ability compared to the control spermatozoa. After frozen-thawing and 3 h of incubation, the percentage of live spermatozoa with intact acrosomes in the ES was significantly lower than that in ejaculated spermatozoa (p < 0.05). Our findings suggested that the DUAI method can overcome the low pregnancy rate of ES, despite the low motility, viability, and fertilization ability of ES.

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.

The purpose of this study was to establish an optimal storage temperature and characteristic analysis after frozen-thawed dairy goat sperm. Sperm was collected at Chojeong dairy goat farm using an electric stimulator and dilluted with semen washing media. The egg yolk-triladyl frozen solution was used for the freezing of Saanen dairy goat sperm and the freezing concentration was set to 1×108sperm/ml. The frozen sperm were thawed in water bath at 37.5℃ for 45 seconds and motility was measured after preservation for 0, 30, 60, 90 and 120 min at 4℃, 17℃ and 37℃, respectively. Sperm characteristic analysis was conducted by flow cytometry. In results, sperm motility at 30, 60, 90 and 120 min after thawing was significantly higher in 17℃ than 4℃ and 37℃ (p<0.05). On the other hand, the frozen-thawed sperm motility were gradually decreased with storage periods increased (30, 60, 90 and 120 min) at 4℃, 17℃ and 37℃. Viability(42%), acrosome damage(24%), mitochondrial damage(28%) and ROS level(4%) were analyzed by flow cytometry in frozen-thawed spermatozoa in 7 male dairy goats. In summary, the motility of frozen-thawed spermatozoa in Saanen dairy goat was more efficient for storage 17℃. The average of viability 42%(30%~54%), acrosome damage 24%(16%~33%), mitochondrial damage 28%(20%~54%) and ROS level 4%(3%~6%) were arranged as standard value by 7 male dairy goats. However, more researches are needed to establish the optimal conditions or proper supplementation for sperm preservation. This study was carried out with the support of research project on feasibility study of the research & development projects for activating the hillside livestock farming and the development of goat grazing program of Rural Development Administration by Korea government (2018PJ013546).

The aim of this study was to evaluate effect of α-linolenic acid (ALA) on viability, acrosome reaction and mitochondrial intact in frozen-thawed boar sperm. The boar semen was collected by gloved-hand method and cryopreserved in 20% egg yolk freezing extender containing ALA (0, 3, 5, and 10 ng/mL) with 0.05% ethanol. The frozen-boar spermatozoa were thawed at 37.5°C for 45 sec in water-bath. The spermatozoa samples were evaluated the plasma membrane integrity, acrosome reaction, and mitochondrial integrity using flow cytometry. In results, population of live sperm with intact plasma membrane was significantly higher in control and 3 ng/mL ALA treatment group than ethanol group (p<0.05). In contract, dying sperms were higher in ethanol group than 3 ng/mL ALA treatment (p<0.05). Acrosomal membrane damage in all sperm population was reduced in 3 ng/mL ALA groups compared with ethanol treatment (p<0.05). However, acrosome damage in live sperm population was no significant difference among the all treatment groups. Mitochondrial integrity was not influenced by ALA treatments in both of live and all sperm population. In conclusion, this results show that supplement of ALA during the cryopreservation process could reduce the membrane damages including plasma and acrosomal membrane, whereas ALA did not influence to mitochondria in boar spermatozoa. Therefore, these results suggest that ALA can protect against the membrane damage derived cryo-stress, and cryopreservation efficiency of boar semen would be improved by use of ALA.

The aim of the study was to investigate the ability of sperm derived from the epididymis in regard to sperm motility, sperm penetration to oocyte and subsequent development of the embryo. Frozen-thawed sperm from epididymis showed similar percentage of motile sperm (VSL ≥ 25 μm/sec) as compared to that of commercial sperm (control). Sperm penetration of frozen-thawed epididymal and commercial sperm was not significantly different. Moreover, cleavage and blastocyst rates were similar in both epididymal and control. Sperm derived from the epididymis also showed fertilizability and subsequent embryonic development

The objective of this study was to evaluate the characteristics of Korean Native Cattle sperm frozen-thawed with L-cysteine and/or catalase. The semen from bulls was collected by the artificial vagina method, and Triladyl containing 20% egg-yolk and/or L-cysteine (L), catalase (C) and L-cysteine + catalase was added to the diluted semen for cryopreservation. The results showed that sperm viability was significantly higher in the L-cysteine + catalase (69.49 ± 3.16%) group than in the control (60.5 ± 3.94%) group (p<0.05). Acrosome damage was significantly lower in the L-cysteine (17.12 ± 1.08%) group than in the control (21.46 ± 1.14%), catalase (20.54 ± 0.76%), and L-cysteine + catalase (19.29 ± 0.65%) groups (p<0.05). In addition, the level of intact mitochondria in the spermatozoa was significantly higher in the L-cysteine (58.65 ± 1.39%) group than in the control (50.63 ± 2.37%) group (p<0.05). The hydrogen peroxide level in the frozen-thawed sperm was significantly lower in the L-cysteine (3.74 ± 1.66%), catalase (4.65 ± 1.87%), and L-cysteine + catalase (8.11 ± 2.15%) groups than in the control (13.22 ± 1.6%) group (p<0.05). The glutathione level was significantly higher in the L-cysteine (1.33 ± 0.03%) group than in the control (1.08 ± 0.06 %), catalase (1.05 ± 0.02%) and L-cysteine + catalase (1.11 ± 0.03%) groups (p<0.05). In conclusion, L-cysteine and catalase could protect the membrane of Korean Native Cattle sperm from damage during sperm cryopreservation. Especially, L-cysteine was more effective for keeping acrosomes and mitochondria intactness during sperm cryopreservation.

Cryopreservation of epididymal spermatozoa offers a potential tool for rescuing genetic material from males of genetically elite populations. Castration, catastrophic injury, sudden death or any other event that makes semen collection or mating impossible may prematurely terminate a stallion reproduction. Stallion epididymal spermatozoa vary widely in the loss of progressive motility, acrosomal integrity, and viability during freezing and thawing. The objective of this work was to investigate the effect of (1) freezing package types on cryopreservation efficiency, (2) thawing temperatures (37, 56 or 70℃) on Computer Assisted Sperm Analysis (CASA) parameters and (3) post-thawing incubation time (0, 1, 2 or 4h) on castrated stallion epididymis. Post-thawed sperm motility ranged between 59.69% and 64.28% (56℃ and 37℃) in various thawing temperatures. When stallion epididymis sperm was frozen, straw was better than freezing tube on VCL (Velocity of Curvilinear Line) and VAP (Velocity of Average Path) parameter. Higher percentage of motility was observed at 37℃ thawing temperature even though no significant difference was observed among various temperatures. The motility, VCL, ALH (Amplitude of Lateral Head displacement), VAP, BCF (Beat-Cross Frequency) and STR (Straightness index) parameter of post-thawed sperm were significantly decreased by increasing the incubation time for all thawing temperatures. The present study showed that type of freezing package (Straw vs. Freezing tube) was not significantly different on cryopreservation efficiency. Furthermore, stallion epididymal spermatozoa frozen-thawed at 37℃ for 1 min resulted the highest proportion of motility and velocity movement. In addition, motility and viability of frozen-thawed stallion epididymal spermatozoa were also decreased by incubation.

The increase in the meat quality and milk production of cows, which breed Korean Native Cow (KNC) and Holstein cow, is not improving reproductive efficiency. In this study, we examined the effect of interferon (IFN) supplementation on motility of frozen-thawed semen and pregnancy rate after artificial insemination of KNC and Holstein cow. In experiment 1, we investigated the effect of IFN-tau concentration (10,000 IU and 20,000 IU) on the percentage of total motility (TM) and progressive motility (PM) of frozen-thawed spermatozoa. In experiment 2, KNC were infused 20,000 IU IFN-tau at insemination or after insemination. In experiment 3, KNC or Holstein cow were inseminated with frozen-thawed semen and infused 20,000 IU IFN-gamma or -tau after insemination. In experiment 1, the average of TM (23.9% to 30.9%) and PM (18.5% to 21.9%) were similar between control and treatments. In experiment 2, the pregnancy rates of IFN infusing times were not different from 45.8% to 53.6%. In experiment 3, the pregnancy rates of Holstein cow infused different kinds of IFN were similar (control, IFN-gamma, IFN-tau; 42.9%, 40.5%, 48.0%). In the case of KNC, however, the pregnancy rate of control was 55.6%, which was significantly lower than that of IFN-gamma (68.9%; p<0.05). Thus, IFN is effective on the improvement of reproductive efficiency, but further study is needed.

The objective of this study was to examine the effect of various discontinuous Percoll washing conditions on sperm capacitation status and sperm survival. Frozen epididymal sperm samples from 3 bulls (0.5 ml plastic straws, 6% glycerol in egg yolk- Tris-glycerol extender) were thawed in 37℃ water bath for 1 min. To rule out individual variation, 3 sperm samples were mixed after thawing. The mixed samples then 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 X g and 700 X 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. Sperm capacitation status and sperm survival were evaluated using combined Hoechst 33258 and chlortertracycline fluorescence staining assay. The acrosome reacted spermatozoa (AR pattern), uncapaciated spermatozoa (F pattern) and sperm survival were significantly correlated with centrifugation time (p< 0.01). Significantly decreased F pattern observed as centrifugal time increased. As centrifugal time increased, spermatozoa with F pattern decreased and spermatozoa showing AR pattern increased. Moreover, the dead spermatozoa were significantly stimulated in time-dependent manner. However, there were no significant differences in various force of centrifugation and Percoll volume. These results suggest that only centrifugation time significantly affects sperm capacitation status and sperm survival.

To evaluate the effect of spermatozoa culture on glycosidase activity of frozen-thawed spermatozoa in human, the spermatozoa were treated experimentally and assayed for activities of α-L-fucosidase, α-D-mannosidase, β-D-galactosidase and N-acetyl-β-D-glucosaminidase (β-GlcNAc’ase). The β-GlcNAc’ase activity was at least two-folds higher than other glycosidases regardless of spermatozoa incubation (p<0.05). The spermatozoa motility was decreased with incubation periods, but no effects by different glycosidases on the changes of spermatozoa motility during the various periods of incubation. In all glycosidases, the spermatozoa-zona binding rates in spermatozoa without incubation were higher than in spermatozoa incubated for 2 h (p<0.05). β-GlcNAc’ase is present mainly in the plasma membrane of spermatozoa frozen-thawed in human. It was also shown that the glycosidase activity was increased in all glycosidases in spite of lower sperm-zona binding by spermatozoa incubation.

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 6～14×107cells/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 cm above LN2 gas for 5 or 10 min. Survival rates of pre-frozen sperm were 65.0±13.2%, 68.3±10.4%, 66.7±11.5% and post-frozen were 53.3±23.1%, 45.0±15.0%, 50.0±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±10.4%, 40.0±7.1%, 30.0±13.2% at 3 cm－5 min and 33.3±11.5%, 31.7± 2.9%, 21.7±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±15.3%, 32.0±17.9%, 22.3±15.7% at 5cm－5 min and were 47.5±15.0%, 43.3±12.6%, 48.3±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 LN2 gas for 10 min for pre-freezing and 7% of glycerol concentration. These results lead to commercial AI with frozen-thawed stallion semen.

This studies were conducted to investigate the survival rate of frozen-thawed spermatozoa of Jindo Dog by monosaccharide and freezing rates. Experimental animals were prepared 12 males within 1~8 year's old and collected once in a couple of weeks by digital manuplation methods. Collected semen was diluted 1:1 with Tris-egg yolk extender and added 4, 6 or 8% of glycerol and none, 4 mM glucose or 4 mM fructose as cryoprotectant and was equilibrated for 2 hrs in . In monosaccharide groups, the freezing rate was 5 cm-5 min. above . The survival rates without monosaccharide were , , in 4, 6 or 8% glycerol, respectively. In addition of glucose, the survival rates were , , in 4, 6 or 8% glycerol, respectively and in fructose, were , , in 4, 6 or 8% glycerol, respectively. There showed significantly different between glycerol groups and monosaccharides groups (p<0.05). The survival rates of freezing rate in 5 cm-5 min. group was , , and in 10 cm-10 min. group was , , in 4, 6 or 8% glycerol, respectively. There were significantly different between freezing rates (p<0.05). These results suggest that the addition of fructose with 6%-glycerol and slow freezing improve the survival of frozen-thawed sperm in Jindo Dog.

This study was conducted to investigate the effects of type of the sugar supplemented to the extender on the vigor, viability and intact acrosomal rates of frozen-thawed dog spermatozoa. The ejaculated semen was diluted with TRIS-citric acid extender containing 200mM TRIS, 73mM citric acid, 6% (v/v) glycerol, 20% (v/v) egg yolk, 1% (v/v) antibiotics (streptomycin/penicillin), 44 mM sugar, which was either glucose, fructose or glucose-fructose combination, and distilled water to make the final volume of 100ml. Extended semen samples were cooled at for an hour, packaged in 0.25ml straws, equilibrated for 10 minutes in liquid nitrogen vapor, and frozen in liquid nitrogen. Samples were thawed by placing straws into water for 120 seconds. After thawing, vigor, viability and intact acrosomal rates of frozen-thawed semen were compared according to type of sugar. No significant differences were observed between glucose and fructose groups. In addition, combination of the 2 sugars also did not show any significant differences in the vigor, viability and intact acrosomal rates. In conclusion, glucose and fructose were equally efficient as sugar supplements for freezing extender.

Glycerol is the cryoprotectant most frequently used to freeze semen in several of species. The objective of the present study was to compare the effect of three different glycerol concentrations (4, 6 or 8%, v/v) on frozen-thawed dog sperm survival rate. Ejaculates from 9 dogs collected by digital manipulation were pooled and assessed by macroscopic and microscopic criteria. Semen was divided into 3 aliquots, which were centrifuged and the sperm pellets rediluted with first Tris-glucose-citric acid extender. After 1 h cooling at , second extender containing 4, 6 or 8% glycerol was added, respectively. The semen was loaded into 0.25 ml straws and frozen and stored in liquid nitrogen and thawed. Sperm vigor, live:dead spermatozoa ratio using HOS test, and sperm morphology using stain were evaluated. After thawing, there were no significant differences among groups in vigor, viability and morphology. In conclusion, the three glycerol concentrations (4, 6 or 8%) can be used successfully in cryopreservation of canine semen. Therefore the use of 4% glycerol in the extender has less toxic effect and reduces of freezing injuries.

This experiment was to investigate whether the mitochondria function assessment can be used for the prediction of sperm fertility through examining the correlation between mitochondria fluoromicroscopic frequency of frozen-thawed eight Hanwoo bull semen using rhodamine123 (R123) and in vitro embryo development following fertilization. Individual sperm were stained in 5ug/mL R123-added calcium-free Sp-TALP for 30 min at 0 h, 6 h, 12 h and 24 h after thawing and examined their mid-piece under an epifluorescence microscope using 495 nm excitation filter (x1,000). Three replications were taken, and at least 300 sperm per individual were examined. When semen samples were separated into two groups (good and poor) by sperm motility and fluorescent frequencies at just after thawing, average fluorescent frequencies were remarkably reduced as time going (0 h; 53.29~72.94%, 6 h; 21.40~58.90%, 12 h; 8.26~25.93%, 24 h; 1.00~13.78%, irrespective of selected group, and there were no differences at 6 h or 12 h after thawing between selected groups but indicated significant difference at 24 h after thawing (p<0.05). In vitro fertilization rates in good and poor groups ranging 70.8~77.8% and 52.1~84.5%, respectively, were not significantly different. However, in vitro development rates of the same groups ranging 25.7~40.0% and 12.9~1.8%, respectively, were significant different (p<0.05). These results demonstrate that mitochondria fluoromicroscopic assessment of frozen-thawed bovine sperm may be used as a criterion to select more fertile sperm.

Flow cytometry를 이용하여 개 정자의 생존율 평가를 수행하고자 2-4세의 수캐 5두가 이용되었고, 분석을 위해 PI염색을 실시하였다. Flow cytometry를 이용한 개 신선 정액의 생존율 평가는 생존 정자와 죽은 정자의 비율을 1:0, 1:1, 1:3으로 조성하여 이를 flow cytometry로 평가하고 광학현미경검사, CFDA/PI 염색검사, HOS test에 의한 생존율과 비교하여 flow cytometry와의 상관관계를 알아보았다. 또한 개 정액을 동결하여 응해 후의 개 정자의 생존율 평가에도 동일한 방법으로 상관관계를 조사하였다. 신선 정액에서 생존 정자와 죽은 정자의 비율이 1:0, 1:1, 1:3 모든 경우에서 flow cytometry를 이용한 생존율은 HOS test에 의한 생존율과 높은 상관관계를 나타내었다 (p<0.01). 신선 정액에서 생존 정자와 죽은 정자의 비율이 1:0과 1:3일 때 광학현미경적 검사에 의한 생존율은 flow cytometry 분석에 의한 생존율과 유의 적인 상관관계를 나타내었으나 (p<0.05), 1:1 비율의 경우 상관관계를 보이지 않았다. 신선 정액에 생존 정자와 죽은 정자의 비율이 1:0과 1:1일 때 CFDA/PI 염색 검사에 의한 생존율은 flow cytometry분석에 의한 생존율과 높은 상관관계를 보였으며(p<0.01), 1:3 비율에서는 유의적인 상관관계를 보였다 (p<0.05). 동결 및 응해 후의 개 정자의 생존율 평가에서 HOS test 결과는 flow cytometry분석에 의한 생존율과 높은 상관관계를 보였으며 (p<0.01), 광학현미경적 검사를 통한 생존율은 유의적인 상관관계를 보였으나 (p<0.05), CFDA/PI 염색 검사결과는 상관관계를 보이지 않았다. 이상의 결과 flow cytometry는 신선정액 및 동결ㆍ융해 후 개 정자에 대한 생존율 검사에 정확한 평가 방법 인 것으로 판단되었다.

본 연구는 돼지 동결-융해 정자의 배양에 의한 체외수정능력과 glycosidase activity를 평가하기 위하여 chlortetracycfine fluorescence분석법을 이용하여, glycosidase가 체외수정능력과 정자침입에 미치는 영향에 대하여 검토하였다. 또한 돼지난자의 투명대내에서 발견된 당잔기에 대한 정자의 glyco-sidase 특이성을 확인하기 위하여 α-L-fucosidase, α-D-mannosidase, β-D-galactosidase 및 N-acetyl-β-D-glucosaminidase (β-GlcNAc''''ase)의 activity를 분석하였다. 그 결과 glycosidase activity는 동결정자의 응해 후 배양하지 않았을 때보다 2시간 배양했을 때 더 높게 나타났다. β-GlcNAc''''ase의 activity 는 정자 배양 유무에 관계없이 다른 glycosidase 처리시보다 최소한 2배 이상 높게 나타났다. 또한 첨체반응이 유기된 정자의 비율은 glycosidase (α-D-mannosidase; P<0.05)에 의해 영향을 받았으며 자를 배양하지 않은 경우보다는 배양된 정자에서 높게 나타났다. 그러나 배양시간에 따른 정자의 존성에 대해 glycosidase의 종류에 따른 유의차는 인정되지 않았다. 한편 투명대내 정자의 접착과 입에 대한 또 다른 실험에서, 서로 다른 glyco-sidase가 첨가된 배양액내에서 수정된 정자가 배양시간이 길어짐에 따라 정자의 침입율은 낮아졌다 β-GlcNAc''''ase; P<0.05). 투명대내의 정자접착정도는 glycosidase의 첨가시에 무첨가시보다 접착정도가 더 높았으며, 가장 높은 접착율은 β-GlcNAc''''ase 첨가시 나타났다. 또한 모든 glycosidase 처리시 2시간 배양한 정자보다는 배양하지 않은 정자에서 투명대에 대한 접착정도가 높게 나타났으며, α-D-mannosidase의 처리시 유의적인 차이를 보였다 (P<0.05). 본 연구의 결과, β-GlcNAc''''ase가 주로 돼지정자의 원형질막내에 존재하는 것으로 추측되며, 배양된 정자에 의한 투명대 접착정도와 침입율이 낮았음에도 불구하고 glycosidase activity가 증가하는 것으로 나타났다.