Olfactory receptors (OR) are primarily responsible for the detection of odorant molecules. We previously demonstrated that OR7D4, an OR for androstenone, is expressed in the vomeronasal organ and olfactory epithelium tissue of stallions. Recently, the expression of OR1I1 in the human testes was reported and the possible roles of OR1I1 in the testicular cells were suggested. The objectives of this study were 1) to explore the expression of OR7D4 and OR1I1 in stallion testes, and 2) to define the specific localization of OR7D4 and OR1I1 in the testicular tissues. Stallion testicular tissue samples were used for this study. Western blot was performed to confirm the cross-reactivity of OR7D4 and OR1I1 antibody with stallion testicular tissue samples. OR7D4 and OR1I1 gene expressions were investigated using reverse transcriptionpolymerase chain reaction (RT-PCR) in stallion testes. Immunofluorescence was performed to investigate the expression of OR7D4 and OR1I1 in stallion testicular tissues. The protein bands for OR7D4 and OR1I1 from the testes were observed at approximately 38 kDa and 43 kDa, respectively. The mRNA of OR7D4 and OR1I1 were detected in stallion testes. Immunolabeling of OR7D4 and OR1I1 in the cytoplasm of both spermatogonia and Leydig cells was observed. In conclusion, androstenone and another odorant chemical, which is recognized by OR1I1, may play an important role in stallion testes.

국내 승용마 산업 발전과 더불어 인공수정 기술을 활용하여 생산된 승용마 두수가 증 가하고 있다. 말 인공수정용 정액은 신선정액, 냉장정액, 냉동정액의 형태로 활용된다. 말 동결정액은 타축종과는 달리 개체 차이가 크고 해동 후 운동성이 크게 낮아지는 문제 점 때문에 인공수정 시 신선정액 또는 냉장정액을 이용하는 비율이 높다. 본 연구에서는 정장 제거 후 정액 냉장보관 시 pentoxifylline이 미치는 효과를 구명하기 위하여 수행하 였다. 본 실험은 제주산마 3두를 공시하였으며, 정액채취는 예비 채취 후 일주일 간격으 로 각 2회 수행하였다. pentoxifylline 1mM, 2mM, 3mM, 4mM이 포함된 INRA96 희석제 에 희석 후 4℃에 보관하며 평가를 위해 정자의 운동성, 생존율, 원형질막 온전성을 24시 간 간격으로 조사하였다. 운동성과 직진 운동성은 CASA 분석 시스템을 이용하여 분석하 였고 생존율을 검사를 위하여 eosin-nigrosin 염색과 원형질막 온전성 검사를 위하여 HOSTest가 수행되었다. 본 실험에서는 대조군에 비해 pentoxifylline 1mM, 2mM 첨가군 이 48시간 이후 운동성과 직진 운동성에서 더 높은 경향을 보였으며, 생존율과 원형질막 온전성에서 24시간 측정 결과 대조군에 비해 pentoxifylline 2mM첨가군이 높은 경향을 보였다. 본 연구결과 향후 정자의 수정능력에 대한 연구가 추가적으로 이루어져야 할 것 으로 고려된다.

Endocrine system of hormones is the critical factor for the development of testes. The levels of hormones are orchestrated by a positive or negative feedback system controlled by the hyphothalamic-pituitary-gonad axis. The aim of this study was to investigate the effect of unbalanced endocrine system induced by the hemi-castration on testicular development in stallions. Four Thoroughbred stallions (age ranging from 3 to 5 yr) were used in this study. To disturb endocrine system, hemicastration has been performed on the stallions. Several parameters including testicular weight, volume, germ cell population on the cross-sections of round tubule, and the area of seminiferous tubules of stallion testes collected at the 1st hemi-castration and the 2nd hemi-castration (about 1 year after 1st hemi-castration) were compared. Testosterone levels were compared for 3 weeks before, after 1st castration, and before 2nd castration using enzyme-linked immunosorbent assay (ELISA) analysis. Immunohistochemistry (IHC) procedure was conducted to compare germ cell populations between after 1st and 2nd castration using VASA antibody. The VASA positive cell population per a cross section of round seminiferous tubule was obtained by monitoring 100 tubules. Interestingly, the weight of testes obtained at 2nd hemi-castration (384±14 g) were significantly higher compared to that of testes collected at the 1st hemi-castration (288±34 g). The volume of testes (306±34 ml) collected at the 2nd hemi-castration was higher than that of testes (169±18 ml) collected at the 1st castration. In contrast, VASA positive germ cell population on the cross section of round tubule (124.9±12.4 vs 142.9±21.6) and the area of round tubule (124±9.7 vs 122.9±1.7 mm2) were not different after 1st castration and 2nd castration. the testosterone levels in the blood collected before, after 1st castration, and before 2nd castration were not significantly different. In conclusion, the hemi-castration induces testicular development to maintain the normal reproductive systems in stallions.

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 efficiency of artificial insemination (AI) for horses remains unsatisfactory. It is mainly because each process of AI causes a detrimental effect on semen quality. To sustain quality of semen properly, several factors including libido of stallions and sperm damage during sperm processing and preservation should be considered. Stallions with decent libido produce a high ratio of sperm to seminal plasma in their ejaculates, which is the ideal semen composition for maintaining sperm quality. Thus, to maximize the fertility rate upon AI, stallions should be appropriately managed to enhance their libido. Seminal plasma should have a positive effect on horse fertility in the case of natural breeding, whereas the effects of seminal plasma on both sperm viability and quality in the context of AI remain controversial. Centrifugation of semen is performed during semen processing to remove seminal plasma and to isolate fine quality sperm from semen. However, the centrifugation process can also result in sperm loss and damage. To solve this problem, several different centrifugation techniques such as Cushion Fluid along with dual and single Androcoll-ETM were developed to minimize loss of sperm and to damage at the bottom of the pellet. Most recently, a new technique without centrifugation was developed with the purpose of separating sperm from semen. AI techniques have been advanced to deliver sperm to optimal region of female reproductive tract at perfect timing. Recombinant equine luteinizing hormone (reLH) and low dose insemination techniques have been developed to maximize both fertility rate and the efficiency of AI. Horse breeders should consider that the entire AI procedure should be optimized for each stallion due to variation in individual horses for a uniformed AI protocol.

The techniques for the collection, cooling and freezing of semen and artificial insemination of horses are not fully understood in Korea. We investigated the percentages of total motile (TM) and progressively motile (PM) sperms after the collection, cooling and freezing of stallion semen. The average volume of semen was 167 ml in Thoroughbred and 68 ml in Arab. The average numbers of spermatozoa in Thoroughbred and Arab were and respectively. The average percentages of TM and PM were 82.3% and 88.6% in Thoroughbred, and 61.4% and 82.6% in Arab, respectively. The average percentage of TM at 4 hr after cooling at was significantly lower than that at 0 hr (<), but the percentage of PM was similar between 66.5 and 73.2% at 0, 1, and 4hr. The average percentage of frozen-thawed Thoroughbred semen frozen in MFR5 extender was 56.2%, which was significantly higher than that of the semen frozen in LE extender (average 32.9%, p<0.05). The percentage of TM in Arab was similar for semen frozen in MFR5 extender and LE extender (18.2% and 21.2%, respectively), but the percentage of PM was significantly higher in sperm frozen in MFR5 extender than in sperm frozen in LE extender (69.0% vs. 36.4%, p<0.05). Four mares were artificially inseminated by Thoroughbred frozen-thawed semen and one of them fertilized at 11 day after artificial insemination. In this study, the collection, cooling and freezing of equine semen were possible under domestic conditions.