In 1951, Colin Russell Austin and Min Chueh Chang identified “capacitation”, a special process involving ejaculated spermatozoa in the female reproductive tract. Capacitation is a phenomenon that occurs in vivo , but almost all knowledge of capacitation has been obtained from in vitro studies. Therefore, numerous trials have been performed to establish in vitro capacitation methods for various studies on reproduction. Although a series of studies have been conducted to develop an optimal protocol for inducing capacitation, most have focused on identifying the appropriate chemical compounds to induce the capacitation of boar spermatozoa in vitro. Therefore, the purpose of this study was to identify the optimal incubation time for inducing capacitation in vitro. Duroc semen was incubated for various periods (60, 90, and 120 min) to induce capacitation. Sperm function (sperm motility, motion kinematic parameters, and capacitation status) was evaluated. The results showed that total sperm motility, rapid sperm motility, progressive sperm motility, curvilinear velocity, and average path velocity significantly decreased in a time-dependent manner. However, the capacitation status did not show any significant changes. Taken together, these results indicate that an incubation time of more than 60 min suppresses sperm motility and motion kinematic parameters. Therefore, we suggest that 60 min may be the best incubation time to induce capacitation without negative effects on sperm motility and motion kinematics in boar spermatozoa in vitro.

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 ability of conventional semen analysis to predict male fertility is questionable. Since the prediction of male fertility is extremely of importance for the artificial insemination and profitable farm managements in animals, the development of highly sensitive biomarker of male fertility is a prime concern. Porcine Seminal Protein I (PSP-I) and Porcine Seminal Protein II (PSP-II) have been known that they are related with motility, and viability of spermatozoa. Thus, we investigated PSP-I and PSP-II level in boar spermatozoa to predict boar’s fertility. The expressions of PSP-I and PSP-II in spermatozoa from 21 individual boars with different fertility and litter size (litter size ranges from 10.3 – 14.2) were examined using qRT-PCR. Litter size was determined in 530 saws after artificial insemination (AI). In addition, sperm motility, motion kinematics, and capacitation status were measured using computer-assisted sperm analysis and Hoechst 33258/chlortetracycline fluorescence staining, respectively. PSP-I and PSP-II showed significantly negative correlation with litter size (r=0.578; P=0.006 and r=0.456; P=0.038, respectively). Furthermore, receiver-operating curves (ROC) was used to determine the accuracy for the prediction of boar fertility. Therefore we divided into 2 groups based on the median value of litter size. When selecting higher litter size group, PSP-I can predict litter size with overall accuracy 90.48% (sensitivity 88.89, specificity 91.67, negative predictive value 91.67, and positive predictive value 88.89) and PSP-II can predict with overall accuracy 81.82% (sensitivity 55.56, specificity 100.00, negative predictive value 76.47, and positive predictive value 100.00). Interestingly, PSP-I and PSP-II were found to increase 0.76 pups than average litter size (average 12.48) in tested boars. To best of our knowledge, this study is the first trial to investigate the correlation between PSP-I, PSP-II, and litter size. Therefore, we suggest that PSP-I and PSP-II could be considered as promising biomarkers for predicting male fertility and litter size outcome in field condition.

Rubus coreanus is known to have diverse biological properties, such as free radical scavenging activity and antibacterial activity. In the present study, Rubus coreanus leaf and stem extract (RLSE) was used in boar semen preservation whether it has a beneficial effect on assisted reproductive technology (ART) in mammals. Boar spermatozoa were preserved in Beltsville thawing solution (BTS) in the presence of varying concentrations of RLSE (0-10 μg/mL). Sperm motility, sperm viability, and intracellular reactive oxygen species (ROS) levels were examined after 2 days of preservation. The percentage of total motile spermatozoa and progressive motile spermatozoa improved in the spermatozoa preserved with 0.5 μg/mL RLSE. Higher proportions of viable spermatozoa were seen in the presence of 0.5 and 1 μg/mL RLSE than in the control. Intracellular ROS levels decreased when the spermatozoa were preserved in BTS with 0.1–1 μg/mL RLSE. In order to examine the bacterial growth, E. coli was added to liquid semen diluted with antibiotics-free BTS in the presence or absence of RLSE. No anti-bacterial activity of RLSE against E. coli was observed during liquid semen preservation. Although there was no inhibition of E. coli growth, the addition of RLSE might help improve sperm motility and viability during boar semen preservation, suggesting it as a potential reagent for ART in mammals.

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 cold shock of spermatozoa is associated with oxidative stress induced by reactive oxygen species. This study was conducted to evaluate the toxicity of natural antioxidant green tea extract (GTE) in lactose-egg yolk (LEY) extender during boar sperm cooling prior to freezing. Spermatozoa were cooled to 5℃ for 3 h in LEY extender containing 0 (control), 1, 10, 100 or 1,000 mg/l of GTE, re-suspended with LEY-glycerol-Equex extender and cooled at 5℃ for 30 min. Sperm progressive motility, viability and phosphatidylserine (PS) translocation were evaluated. PS translocation was assayed by flow cytometry using Annexin V-FITC apoptosis detection kit. The sperm function including progressive motility, viability and PS translocation was not significantly different regardless of GTE concentrations (P>0.05). In conclusion, this study demonstrated non-toxicity of GTE supplement in LEY extender during sperm cooling.

The objective of this study was to compare the effect of semen extenders on the sperm motility, viability, acrosome integrity and functional integrity of plasma membrane (HOST: hypo-osmotic swelling test) during liquid preservation of Korean Native boar semen. In this experiment, semen was diluted in Androhep plus, Beltsville Thawing Solution (BTS), ModenaTM, Seminark and Vitasem LD. Sperm-rich fractions were collected from three Korean Native boars and sub-samples were diluted (30×106 spermatozoa/ml) in different semen extenders. Semen samples were stored at 17℃ for 96 hours. On everyday (0, 24, 48, 72, 96 h) after storage, the sperm characteristics relevant for fertility, such as sperm motility, viability, acrosome integrity and HOST positive were evaluated. The motility of spermatozoa stored in different extenders was no significantly different among other extenders (P>0.05). Also, no difference was observed among samples processed with different extenders in the percentage of sperm viability, acrosome integrity and HOST positive. All extenders maintained a high percentage (70%) of sperm motility, viability and acrosome integrity through 96 h of storage. The result of this study show that there was no significant differences among extenders in their capacity to preserve motility, viability and membrane integrity of spermatozoa from normal, fertile Korean Native boars for 96 h of liquid preservation at 17℃.

The objective of this study was to determine the effect of semen extenders on the motility, viability and fertility in vitro of spermatozoa during storage of fresh boar semen diluted in different commercial extenders used for pig artificial insemination (AI). In this experiment, semen were diluted in Androhep plus, Beltsville Thawing Solution (BTS), Modena, Seminark and Vitasem LD. Five ejaculates were collected from three Duroc boars and sub-samples were diluted (30×106 spermatozoa/ml) in different extenders. Semen was stored at 17℃ for 10 days. Sperm motility and viability was assessed using Computer-Assisted Semen Analysis (CASA) and flow-cytometry on 1, 3, 5 and 10 day post collection. The motility of spermatozoa stored in different extenders was gradually decreased by increasing the duration of storage of semen. However, there was not significantly different in the sperm motility and viability among other extenders. On the other hand, the in vitro-matured oocytes were fertilized and cultured in vitro to assess the fertility of boar spermatozoa stored for 3 and 10 days in different extenders. The percentage of morula and blastocyst were taken as indicators of fertility in vitro of spermatozoa. Therefore, there were no differences in the rate of embryos developed to the molular and blastocyst stage. There were no differences in the motility and fertility in vitro among 5 kinds of commercial boar semen extenders.

The ability to preselect the sex of piglets is advantageous in the pig industry. The objective of this study was to examine the feasibility of using intracytoplasmic sperm injection (ICSI) with sorted spermatozoa to produce piglets with a preselected sex. Pig embryos were produced by ICSI of frozen X- and Y-sperm that had been separated by flow cytometry. The developmental competence of the embryos was investigated in vitro and in vivo. The populations of X- and Y-spermatozoa were 52.7% and 47.3%, respectively in our samples. The in vitro development of ICSI embryos was enhanced by longer of in vitro maturation of oocytes ( vs. ). Their cleavage () and blastocyst formation () rates were not significantly different between male and female ICSI embryos, or between sorted and unsorted sperm-derived embryos. One pregnancy was established in a recipient that was transferred with 110 female ICSI embryos, but the pregnancy was terminated on Day 89 of gestation. Our results suggest that the separation X- and Y-spermatozoa by flow cytometric sorting can be a useful tool in combination with ICSI for the production of pig embryos and piglets of preselected sex.

The objective of this study was to examine the effect of caffeine and sodium bicarbonate in a fertilization medium on the fertilizability of boar spermatozoa that were frozen in straws. Boar spermatozoa were extended with Beltsville F5 extender and frozen in 0.25‐ml straws. In vitro matured porcine oocytes were fertilized in vitro (IVF) with frozen‐thawed boar spermatozoa for 6 h in a modified tris‐buffered medium (mTBM) or in its modified medium by substituting the tris with 25 mM sodium bicarbonate (modified bicarbonate‐buffered medium; mBBM). Some of inseminated oocytes were fixed and stained for examination of sperm penetration. IVF embryos were cultured in a North Carolina State University‐23 medium for embryo development. The percentage of live sperm was 47±4% and morphological abnormality of acrosome was found in 14±3% of spermatozoa. Optimal sperm concentration for IVF was 0.75～1.0×106 sperms/ml when mTBM containing 5 mM caffeine was used as the fertilization medium. Sperm penetration was significantly (p<0.05) stimulated by increasing caffeine concentration in the IVF medium. In addition, mBBM significantly (p<0.05) increased sperm penetration (92%) compared to mTBM (65%). More (p<0.05) blastocysts (22% vs. 32%) developed from the oocytes that were fertilized in mBBM containing 1 mM caffeine than from those fertilized in mTBM with 5 mM caffeine. Our results indicate that boar spermatozoa can be frozen successfully in straws with holding their normal fertilizability and that caffeine and sodium bicarbonate stimulates sperm penetration in vitro.

The objective of this study was to establish the optimal conditions for hypoosmotic swelling (HOS) test to assess the functional integrity of the membranes of boar fresh or frozen/thawed spermatozoa. When pooled semen sample was incubated for 30 min at 37℃ with different test solution of varied osmolarity, the highest percentage of HOS positive spermatozoa was observed in a 150 mOsmol fructose/Na-citrate solution (33.6%). Incubation time did not affect significantly the score of HOS positive spermatozoa observed in a 150 mOsmol fructose/Na-citrate solution at 37℃, but the osmolarity affected the score of HOS positive spermatozoa under the same condition above. Fresh semen was significantly better than frozen/thawed semen in semen parameters evaluated such as motility, viability, membrane integrity and lipid peroxidation (p<0.05). In the relationships of sperm parameters, motility vs viability, motility vs membrane integrity and viability vs membrane integrity were positively correlated (0.82～0.94) but lipid peroxidation vs other estimated factors was negatively correlated (－0.90～－0.98). Among the evaluation methods, motility vs viability, motility vs membrane integrity and lipid peroxidation vs other estimated factors were significantly correlated (p<0.05). These results of this study indicate that the optimal condition of HOST in boar spermatozoa is a 150 mOsmol fructose/Na-citrate solution for 30 min incubation at 37℃ and HOST can substitute the examination of motility, viability and lipid peroxidation.

This study was conducted to examine the effect of several saccharides on the induction of capacitation and acrosome reaction (AR) and to examine the effects of mono and polysaccharides on the penetration activity of boar spermatozoa. Spermatozoa were inseminated in medium with fucose, galactose and mannose as monosaccharide, and fucoidan, galactan and mannan as polysaccharide. The penetration rates were significantly (p<0.05) lower in medium with galactose (40.6%), mannose (38.1%), fucose (41.6%) and fucoidan (36.6%) compared with control (56.7%). The rates of AR were increased (40.7 to 59.8%) by the preincubation periods prolonged from 0 to 4 hr (p<0.05). Similar tendencies were observed in AR when spermatozoa were treated with monosaccharides, but not significantly differ among the groups treated with different time of preincubation with some exception of galactose. When spermatozoa were treated with polysaccharides, the rates of AR were significantly (p<0.05) increased by preincubation time prolonged from 0 to 4 hr with an exception of fucoidan. In conclusion, the present study suggests that penetration rate of spermatozoa is higher in presence of polysaccharides than monosaccharides. Also, it may resume that the comparing to control, the all saccharides (L-fucose, D-galactose, D-mannose, fucoidan, galactan and mannan)-treated groups slightly increase the AR pattern as preincubation time prolonged.