양돈산업은 돼지고기 내 웅취발생 예방을 위한 방법으 로 수컷 자돈에게 물리적 거세를 관행적으로 실시해왔다. 그러나 동물복지에 대한 관심이 고조됨에 따라 일부 국가 들에서는 고통 최소화 물리적 거세법 권장 또는 대체 방 법에 대해 연구를 수행하고 있다. 따라서 본 연구에서는 비거세돈(EM; entire male pigs) 및 거세돈(CM; castrated male pigs)에서 이눌린의 급여가 지방조직 내 웅취물질 농도에 미치는 영향을 구명하고자 실시하였다. 시험동물 은 총 26두의 3원 교잡돈(EM, n=18; CM, n=8)을 사용하 였다. 시험 처리구는 3% 이눌린의 급여와 비급여를 설정 하여 총 4개 처리구(EM0, EM3, CM0 및 CM3)로 구성하 였다. 웅취분석을 위해 지방조직은 등지방, 목지방 및 삼 겹지방을 수집하였다. 등지방 내 인돌 농도는 EM0에 비 해 CM3에서 감소하였고(p<0.05) 목지방 및 삼겹지방에서 는 처리구간 차이가 없었다. 지방조직 내 평균 인돌 농도 는 EM0에 비해 EM3, CM0 및 CM3에서 감소하였다 (p<0.05). 등지방 내 스카톨 농도는 CM0, CM3에서 EM0 보다 감소하였다(p<0.05). 또한, 목지방 내 스카톨 농도 및 지방조직 내 평균 스카톨 농도는 CM3에서 EM0과 EM3 보다 감소가 확인되었다(p<0.05). 그러나 안드로스테논 농 도는 분석된 지방조직들 모두에서 처리구간 통계적인 차 이가 없다. 이러한 결과들은 이눌린 급여에 의해 비거세 돈의 지방 내 웅취물질 저감에 부분적인 효과를 나타냈 을 뿐만 아니라 거세돈에서도 웅취물질 감소에 긍정적인 영향을 미쳤음을 시사한다. 따라서, 본 연구결과는 물리적 인 거세가 전면 금지된다면 동물복지형 사양관리 기술개 발의 기초자료로 활용할 수 있을 것이다.

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 study investigated the influence of sodium bicarbonate (NaHCO3) and progesterone on acrosome reaction and proportion of polyunsaturated fatty acid (PUFA) composition boar sperm. The sperm were diluted with semen extender and incubated with NaHCO3 and progesterone at 38℃, 5% CO2 for 6 h. Plasma membrane integrity and acrosome reaction were analyzed using SYBR14/propidium iodide (PI) and FITC-PNA/PI doubling staining method, and proportion of PUFA was analyzed using gas chromatography. In results, Plasma membrane integrity was significantly decreased in 50 mM NaHCO3 group and acrosome reaction was significantly increased by over the 100 mM NaHCO3 group compared to control group (p < 0.05). In addition, progesterone significantly increased decreased plasma membrane integrity at 100 mM progesterone and acrosome reaction at over the 5.0 µM progesterone (p < 0.05), but there was no difference among the 5.0 to 100 µM groups. PUFAs were significantly decreased in 100 mM NaHCO3 and 50 µM progesterone treatments compared to control group. In summary NaHCO3 and progesterone induce acrosome reaction and reduce PUFA composition in boar sperm, therefore, the results maybe help to understand basically knowledge for the acrosome reaction and PUFA composition in boar sperm.

Sperm cryopreservation is a fundamental process for the long-term conservation of livestock genetic resources. Yet, the packaging method has been shown, among other factors, to affect the frozen-thawed (FT) sperm quality. This study aimed to develop a new mini-straw for sperm cryopreservation. In addition, the kinematic patterns, viability, acrosome integrity, and mitochondrial membrane potential (MMP) of boar spermatozoa frozen in the developed 0.25 mL straw, 0.25 mL (minitube, Germany), or 0.5 mL (IMV technologies, France) straws were assessed. Postthaw kinematic parameters were not different (experiment 1: total motility (33.89%, 32.42%), progressive motility (19.13%, 19.09%), curvilinear velocity (42.32, 42.86), and average path velocity (33.40, 33.62) for minitube and the developed straws, respectively. Further, the viability (38.56%, 34.03%), acrosome integrity (53.38%, 48.88%), MMP (42.32%, 36.71%) of spermatozoa frozen using both straw were not differ statistically (p > 0.05). In experiment two, the quality parameters for semen frozen in the developed straw were compared with the 0.5 mL IMV straw. The total motility (41.26%, 39.1%), progressive motility (24.62%, 23.25%), curvilinear velocity (46.44, 48.25), and average path velocity (37.98, 39.12), respectively, for IMV and the developed straw, did not differ statistically. Additionally, there was no significant difference in the viability (39.60%, 33.17%), acrosome integrity (46.23%, 43.23%), and MMP (39.66, 32.51) for IMV and the developed straw, respectively. These results validate the safety and efficiency of the developed straw and highlight its great potential for clinical application. Moreover, both 0.25 mL and 0.5 mL straws fit the present protocol for cryopreservation of boar spermatozoa.

본 연구는 Yorkshire종, Landrace종 및 Duroc종에 대한 유전체자료를 이용하여 수퇘지의 웅취를 유발하는 세 가지 호르몬인 androstenone, indole 및 skatole 호르몬에 대한 유의적인 유전자영역, SNP 마커 및 후보유전자를 발굴하여 최종적으로 저웅취 종돈을 육종하는데 그 목적이 있다. Genomoe-Wide Association Study를 수행하기 위한 참조집단으로 수집한 유전체 정보는 Yorkshire, Landrace 및 Duroc종에서 각각 3,858 두, 472두 및 1,029두로 총 5,359두에 대한 유전체자료를 분석에 이용하였다. 추정되는 육종가의 정확도를 평가하기 위하여 REML방법을 ASREML 4.1 소프트웨어를 이용하여 분석하였고 세 가지 호르몬에 대하여 다형질 개체모형을 적용하였으며 추정된 육종가로부터 산출한 deregreessed DEBVincPA를 반응변수로 이용하여 연구를 수행하였다. 세 가지 호르몬에 대하여 BayesB와 C의 방법론을 통하여 분석한 결과 BayesB에서 세 가지 호르몬과 연관될 것으로 예상되는 SNP marker 9개, 즉 androstenone에서 3개, indole에서 1개 및 skatole에서 5개가 발굴되었다. BayesC에 서는 이보다 적은 SNP marker 3개가 발굴되었다. 수퇘지의 웅취 호르몬에 영향을 미칠 것으로 예상되는 후보유전자는 총 6개로 각각 LMAN2L, ABLI, NRG3, CDH12, TRAPPC9, MAN1A2로 나타났다

Cryopreservation is a widely-used efficient means of long-term sperm preservation. However, unlike other types of semen, cryopreserved boar semen has reduced fertility and the efforts continue to optimize post-thawing sperm recovery. In this study, we evaluated the effects of various washing solutions (Hulsen solution, labmade DPBS and commercial DPBS) on post-thawing porcine sperm kinematics (CASA system), viability (SYBR-14/PI) and acrosome integrity (PSA/FITC). We also examined the effect of washing-centrifugation on frozen-thawed semen kinematics. The results indicate that type of washing solution and post-thawing centrifugation alters parameters linked to sperm quality (total motility, progressive motility, viability and acrosome integrity). Significantly higher (p < 0.05) motility and progressive motility were obtained when cryopreserved semen was processed with Hulsen solution. The postthaw percentage of live and intact acrosomal sperm was significantly higher in group 1 (Hulsen solution) as compared to other groups. Following thawing-centrifugation, the results showed significantly higher motility and progressive motility in group 1 than other groups. However, the latter two DPBS groups did not differ statistically. Taken together, Frozen-thawed spermatozoa motility, acrosome integrity and viability can be affected by the type of washing solution used. Moreover, centrifugation of frozenthawed semen has an unfavorable effect on total motility and progressive motility.

The estimation of the postmortem interval (PMI, the time that has elapsed since the death) is a critical issue for the biosecurity enforcement officers who implement to the timely establishment of biosecurity zone for preventing susceptible animals from disease transmission given the rapid occurrence of an infectious disease. Increasing attention has been paid to PMI of wild boar (Sus scrofa) carcasses associated with African swine fever epidemics in Korea since October 2019 to explain the geographical transmission of the disease, as well as to provide potential target animals for prevention measures in terms of farm biosecurity. This study is, to the best of our knowledge, the first to describe the decomposition process of wild boars in different microhabitats in the country. In the present comparative study, we obtained field data from the decomposition process of the wild boar and domestic pig carcasses continuously exposed aboveground in natural environment settings allowing animal scavenging. This study compared the pattern of decomposition in 16 wild boar carrion and 10 domestic pig carcasses placed between August and November 2019. Quite differences in decomposition rate measured by total body score and tissue's gross morphology over time were observed between wild boars and domestic pigs. Overall, the wild boar decomposed much more slowly than the domestic pig throughout the end of the experimental period. In addition, color changes to the skin were easily seen in domestic pigs, whereas there is much variation in the wild boar, especially carcasses placed in late autumn. Moreover, some wild boar carcasses did not show any sign of bloating. These results indicate that decomposition rates derived from forensic taphonomic studies on domestic pigs may be not directly applicable to the wild boar, hence there is a need to develop regional decomposition models to be employed in different geographical situations to increase the accuracy of PMI of wild boar carcasses.

The distribution of wild boar (Sus scropa) in the Republic of Korea was forecasted using environmental factors. A species distribution model was applied with the standard normalized difference vegetation index (NDVI), enhanced vegetation index (EVI), solar zenith angle (SUNZ), daytime land surface temperature (dTemp), and nighttime land surface temperature (nTemp). Understanding wild boar distribution is important for controlling African swine fever (ASF) because the disease could be endemic in wild boar or spread from wild boars to domestic pigs. Among the five predictors, the NDVI was the most influencing factor for the wild boar distribution. The relative contributions of the predictors were 67.4 for NDVI, 16.9 for dTemp, 10.5 for SUNZ, 4.4 for EVI, and 0.8 for nTemp. The area size under the receiver-operating curve of the receiver-operating characteristics for the current model was 0.62, but the real wild boar observation data overlapped with the predicted high-density wild boar distribution area. The wild boar distribution density was relatively higher in Gangwon-do, Gyeongsangbuk-do, Gyeongsangnam-do, and Jeollanam-do. Given the ASF epidemics, contact between ASF-infected animals and ASF-susceptible animals in high-density wild boar distribution areas should be prevented by long-range fencing or active surveillance.

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.

웅취는 돼지고기의 이취로서 소비자의 소비성에 부정적인 영향을 미친다. 웅취는 거세를 하지 않은 수퇘지의 지방조직에 안드로스테 논, 스카톨 및 인돌과 같은 호르몬의 축적으로 인한 것이다. 본 연구는 웅취에 영향을 미치는 호르몬과 성장 및 번식 형질간의 유전상관도 를 추정하고 동물복지측면에서 웅취 호르몬이 낮은 수퇘지를 개발하기 위해 수행되었다. 본 연구를 위하여 3개 품종, Duroc, Landrace, Yorskhire 517두를 공시돈으로 수행하였다. 웅취 호르몬 분석을 위한 샘플채취는 수퇘지의 목부위 지방을 바이옵시 방식으로 채취하여 각 품종별 안드로스테논, 스카톨 및 인돌을 분석하였다. 안드로스테논 분석 결과, Duroc 품종이 1.684 ug/g으로 가장 높았고, 타 품종은 0.875-0.920 ug/g으로 상대적으로 낮았다. 스카톨의 농도는 Yorskhire가 0.095 ug/g으로 가장 높았고 인돌은 Duroc이 0.058 ug/g으로 가장 높았다. 품종과 개체에 따라서 3종류 웅취 호르몬의 농도 차이는 매우 컸다. 안드로스테논, 스카톨 및 인돌의 유전력은 0.40, 0.17 및 0.01으로, 안드로스테논의 유전력이 가장 높았다. 웅취 호르몬 농도와 다른 경제 형질(성장 및 번식 형질) 사이의 유전적 상관관계를 조사하였으나, 웅취 농도와의 상관관계는 매우 낮았다. 웅취 호르몬의 농도가 유전력도 높고 개체별 차이가 크고 다른 경제 형질과의 유전적 상관도가 낮기 때문에, 육종을 통한 저 웅취 부계 종돈 작출이 가능할 것으로 판단된다.

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.

Cryopreservation of boar semen is continually researched in reproductive technologies and genetic resource banking in breed conservation. For evaluating the boar semen quality, sperm motility (MOT) is an important parameter because the movement of spermatozoa indicates active metabolism, membrane integrity and fertilizing capacity. Recently, polymorphisms reported to be significant association with sperm MOT. This study was conducted to evaluate the SNP in the coding region of ESR1 (g.672C>T inexon 1) as a positional controlling for motility and kinematic characteristics of post-thawed boar semen. To results,The g.672C>T was significantly associated with frozen semen motility and kinematic characteristics. g.158 T>C SNP was high significantly associated with MOT, VCL, VSL and VAP Also, the SNP was low significantly associated with ALH.Therefore, we suggest that theSNP in the coding region of ESR1 (g.672C>T in exon 1) may be used as a molecular marker for Duroc boar Post-thawed semen quality.

Estrogen receptor 2 (ESR2) is involved in oestrogen-related apoptosis in cell cycle spermatogenesis but their effects have not yet confirmed in pig. Therefore, this study was performed to investigatetheir association with semen motility and kinematic characteristics. DNA samples from 126 Duroc pigs with records of sperm motility and kinematic characteristics [Total motile spermatozoa (MOT), Curvilinear velocity(VCL), Straight-line velocity(VSL), the ratio between VSL and VCL(LIN), Amplitude of Lateral Head displacement(ALH)] were used in present study. A single nucleotide polymorphism (g.35547A>G) was associated with MOT, VCL, VAP and ALH in Duroc population (p < 0.05). Therefore, we suggest that the porcine ESR2 may be used as a molecular marker for Duroc boar semen quality, although its functional effect was not clear yet. These results will improve the understanding of the functions of the ESR2 in spermatogenesis within the reproductive tracts, and will shed light on ESR2 as a candidate gene in the selection of good sperm quality boars.

During the freezing and thawing process, fatty acids in the plasma membrane of sperm are released, which results in a functional damage of sperm. Sperm with functional loss due to cryo-damage result in a decrease in fertility. Previous studies have shown that the addition of one of the fatty acid alpha-linolenic (ALA) with carrier proteins improves the stability of plasma membrane and reduces the damage. In this experiment, we focused on the functional aspects of the plasma membrane of sperm and experimented with motility and morphology. For preparation of ALA-carrier protein complex, 3 ng/ml ALA was mixed with 0.7 μg/ml bovine serum albumin (BSA) or 14 ng/ml methyl-β-cyclodextrin (MBCD) in distilled water. The boar semen was purchased from GUMBO Company. Boar semen was cryo-preserved in 20% egg yolk freezing extender containing ALA, BSA, MBCD, ALA+BSA, ALA+MBCD. The frozen boar sperm was thawed at 37.5 ℃ for 45 sec in water-bath. The sperm motility and morphological abnormalities were evaluated under a phase-contrast microscope at 200 × magnification and randomly counts of 200 sperm each sample. In results, motility of frozen-thawed sperm was increased in all treatment groups. In particular, there has been significant improvement in ALA+BSA and ALA+MBCD treatment groups than control (p<0.05). However, there was no significant difference in ALA, BSA and MBCD treatment groups. Morphological normalities in frozen-thawed sperm was reduced in complex treatment groups (p<0.05). However, there was no significant difference in single treatment groups. In both motility and morphology characteristics, ALA+BSA and ALA+MBCD treatment group was higher than all treatment groups. In conclusion, the addition of ALA with carrier proteins during cryopreservation has a positive effect in its functional aspect. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Education) (2016R1D1A1B03931746).

The aim of this study was performed to evaluate the effects of ice-binding protein from the arctic yeast Leucosporidium (LeIBP) supplementation on cryopreservation of boar sperm. The collected semen was diluted (1.5×108/ml) in lactose egg yolk (LEY) and cooled at 5°C for 3 h. The cooled semen was then diluted (1×108/ml) in LeIBP containing LEY with 9% glycerol and maintained at 5°C for 30 min. The semen was divided into six experimental groups (control, 0.001, 0.005, 0.01, 0.05 and 0.1 mg/ml of LeIBP). The straws were kept on above the liquid nitrogen (LN2) vapors for 20 minutes and then plunged into LN2. After thawing, computer-assisted sperm analysis was used for sperm motility and flow cytometry was performed to assess the viability, acrosome integrity (FITC-PSA/PI), ROS (DCF/PI), lipid peroxidation (BODIPY C11/PI) and apoptosis (Annexin V/PI), respectively. No significant responses were observed for sperm motility. However, sperm viability was significantly increased on 0.05 and 0.1 mg/ml of LeIBP groups compared to control (P < 0.05). In addition, acrosome integrity was significantly increases LeIBP groups (P < 0.05) and both ROS and lipid peroxidation level were lower in all LeIBP groups than those of control (P < 0.05). On the other hand, a significant higher apoptosis rate was observed in 0.05 and 0.1 mg/ml of LeIBP groups compared to control (P < 0.05). It can be assumed that a supplementation of LeIBP in boar sperm freezing extender is an effective method to increase the sperm qualities after cryopreservation.

최근 국내 멧돼지 문제는 농작물 피해 및 도심 출몰로 인하여 사회적으로 심각한 상황에 직면해 있다. 본 연구는 경남 거창군의 멧돼지에 의한 농작물 피해사례를 바탕으로 피해에 영향을 끼치는 환경 요인들에 대하여 파악하고자 2012년 5월부터 10월까지 수행 되었다. 농작물 피해 분석 결과 주로 8월과 9월 사이 피해가 가장 많이 발생하였고, 벼의 빈도가 가장 높았으며 피해 강도는 고구마가 가장 높은 것으로 나타났다. 이는 멧돼지의 농작물 선호성과 특정시기에 대한 가용성과 관련이 있는 것으로 판단된다. 또한 농작물 피해에 경사, 지형기복, 산림과의 거리, 수계로부터의 거리, 도로로부터의 거리, 주거지로부터의 거리의 요인들이 영향을 미치는 것으로 나타났다. 피해 강도에 따른 환경요인의 차이는 나타지 않아 멧돼지는 한번 피해를 끼친 농경지를 지속적으로 방문하여 피해를 누적 시키는 것으로 파악된다. 따라서 멧돼지에 의한 농작물 피해를 저감시키기 위해서는 피해가 발생할 수 있는 곳에 멧돼지가 비선호하는 작물로 대체 재배하며 전기펜스 설치, 엽사와 포획틀을 이용한 멧돼지 개체수 조절이 필요할 것으로 판단된다.

This study was to investigate effect of tunicamycin (TM) on sperm viability, mitochondrial activity and motility in boar semen. Collected sperm were incubated with semen extender containing 0, 1, 2, and 5 μM TM for 3, 6 and 9 h. Sperm viability was analyzed using SYBR14/PI doubling staining, and mitochondrial activity was detected using Rhodamine123/PI staining methods. Sperm viability, mitochondrial activity and motility were measured every 3 h during incubation. In results, boar sperm viability, mitochondrial activity and motility were significantly decreased in 2 and 5 μM TM groups compare to control group at all incubation time (p<0.05). In addition, mitochondrial activity and motility were significantly decreased in 1, 2, and 5 μM TM groups compare to control group at 9 h after incubation (p<0.05). These results suggest that TM can inhibit sperm viability, mitochondrial activity and motility in boar semen, and it may influence on the fertility of sperm.

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.