Zizyphi spinosi semen (Z. spinosi) has been used in traditional Chinese medicine for the treatment of rheumatoid arthritis and wounds. However, toxicity in high doses was often observed due to the presence of alkaloids. This study was conducted to investigate the potential genotoxicity of Z. spinosi in vitro and in vivo. This was examined by the Bacterial reverse mutation (Ames) test using Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2uvrA, Chromosomal aberration was investigated using Chinese hamster lung cells and the micronucleus test using mice. Z. Spinosi did not induce mutagenicity in the Ames test, and it did not produce chromosomal aberration in Chinese hamster lung cells with and without metabolic activation, nor in the micronucleated polychromatic erythrocytes in the bone marrow cells in mice. Based on these results, it is concluded that Z. spinosi does not have mutagenic potential under the conditions examined in each study.

Cryopreservation of avian semen is a useful tool to preserve genetic resource for aim of preventing extinction induced by infectious disease like avian influenza. Unlike those of mammals, data from chicken cryopreserved semen has not been showed feasible results. So, various cryoprotectants and diluents have been examined in many methods. In this report, as a major ingredient of avian seminal plasm, glutamine was substituted by alanyl glutamine to enhance physiological stability of chicken semen during freezing. We studied effect of glycerol and Dimethylacetamide(DMA) on motility and progressive motility of spermatozoa using glutamine diluent(EK-G) or alanyl glutamine diluent(EK-A) condition. The semen of Ogye was collected twice a week by the dorso-abdominimal massage method and diluted with same volume of EK-G or EK-A at 25℃ and stored for 10 min at 4℃ in cold chamber. Glycerol or DMA was added to diluted semen to reached 7% of final concentration at 4℃. After 3min of equilibration, the diluted semen was packed into 0.25ml straws and subjected to cryopreservation used freezing equipment. The packed straw were placed on height 5 cm above surface of liquid nitrogen(LN2) and held for 10min. After preserved for 2 weeks, the straw was thawed onto the 4℃ cooling bath. The images of motility and progressive motility spermatozoa were recorded by digital image recorder and analyzed by manual. The results showed 68.5% motility and 34.1% progressive motility in DMA/EKA diluent, 31.45% and 17.6% in glycerol/EKA, 45.4% and 8.6% in DMA/EKG, and 9.7% and 6.4% in glycerol/EKG. With these results, the alanyl glutamine and DMA could be used as a main composition of diluent and cryoprotectant for cryopreservation of chicken semen.

Cryopreservation of canine spermatozoa affords potential exchange of genetic material, and thus may lead to improvement in the breeding management. However, canine spermatozoa undergo many damages such as, cold shock, ice crystal formation, oxidative stress during cryopreservation. In this study used the CASA for investigating the effect of various trehalose concentrations and thawing temperatures on the sperm viability. In addition, the efficacy of the most optimal of the tested cryopreservation protocols in this study was verified by AI as the in vivo test. Also, this study evaluates the variation of frozen- thawed canine spermatozoa during different incubation condition. The addition of trehalose 25 mM was optimal concentration and frozen-thawed semen quality was significantly higher better than control (Glucose) and other concentration groups. In effect of thawing temperature on frozen-thawed sperm movement and intact acrosome evaluations, which result enhance the sperm motility and movement value depending on increase temperature condition at 36, 54 and 72℃. Also, in the effect of different incubation condition on frozen-thawed sperm after thawing at 36℃ for 60 sec, that the results trehalose 25 mM was significantly better (p<0.05) than glucose in general as well as, the post-thawed sperm motility and intact acrosome was reduced depending on increase the incubation time. Especially, incubation at 4 to 8 hour was rapidly depreciation of movement value and the rate of intact acrosome was dropped similar tendency. Thus, incubation 17℃ was better than other incubation groups on sperm motility and acrosome integrity. For the in vivo evaluate of spermatozoa survival and is the most definitive test of sperm function, we performed artificial insemination in estrous bitch. The semen was prepared for intrauterine insemination using the 25 mM trehalose freezing extender and thawing at 36℃, and 2 bitches were inseminated with 1×106 motile spermatozoa by surgical method. The results of AI, the pregnancy rates, mean litter size and oocyte fertilization rate were 16.6% (1/6), and 50% (2/4), respectively. In conclusion, based on the results of these experiments, the effect of addition of trehalose on extender improves the movement and intact acrosome of frozen-thawed semen. In particular, trehalose 25 mM groups was higher than other different concentration group on movement value and acrosome integrity of frozen-thawed sperm. Also, through incubation condition, this study identify the optimal incubation temperature after thawing was 17℃. Furthermore, the information will be contributed to develop the canine ART including AI, IVF and canine ICSI. * This research was supported by iPET (Grants 110056-3), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

The objective of this study was to assess the effect on post-thawed sperm motility, viability and acrosome integrity of boar semen frozen in the freezing extender with chicken or duck egg yolks. The Sperm rich fraction of ejaculates from three Duroc boars were collected by a glove-hand technique. Samples with more than 80% motile sperm were used for this experiment. Semen was diluted with freezing extender (LEY) containing 11% (v/v) lactose, 20% (v/v) hen egg yolk with 3.5% (v/v) glycerol, and 0.5% (v/v) Orvus Es Paste(OEP, Nova Chemical Sales Inc., Scituate, MA. USA) to yield a final sperm concentration of 5×108 cells/ml. Following complete dilution, semen samples were loaded in 0.5 ml French medium straws (IMV technologies, France) and transferred to programmable semen freezer (SY-LAB Gerate GmbH, Austria). For freezing the semen samples, each straw was cooled from 5℃ to — 5℃ at 6℃/min, auto-seeding at — 5℃ and held for 60sec, samples were then cooled from — 5 to — 80℃ at 40℃/min, and thereafter from — 80℃ to — 150℃ at 60℃/min. The yolks used were sourced from fresh chicken and duck eggs. To evaluate the post-thaw sperm quality, semen was thawed at 38℃ for 20 sec and sperm motility, viability and acrosome integrity were assessed. Motility was assessed for %motile cell characteristics using computer-assisted semen analysis (CASA; SAIS SI-100, Medical supply, Korea). The percentage of sperm viability was assessed using LIVE/DEAD® sperm viability kit (Molecular probes, Eugene, OR, USA). The acrosome integrity was assessed by FITC-PNA staining. Sperm quality in terms of motility, viability and acrosome integrity showed higher after freezing in medium containing duck yolk than chicken yolk. However, there was no significant difference in sperm quality for the different types of yolk(p>0.05). * The result of this study showed that there was no significant difference between the egg yolk types when considering the sperm motility, viability and acrosome integrity of boar semen frozen in the freezing extender with chicken or duck egg yolks.

A study was conducted on four crossbred bulls, used as artificial insemination (AI) sires, to correlate their semen quality with their non return rate (NRR). Semen was collected once a week via an artificial vagina, diluted in egg yolk-citrate and maintained at for three days. It was evaluated for sperm motility, viability, morphology immediately after collection and was examined daily for sperm motility, viability and morphology of acrosome, mid piece and tail for a total of three days. A total of 2016 cows were inseminated by two AI technicians. The proportions of sperm with normal heads were 83.4% (63.7~91.7%), the proportion of spermatozoa exhibiting normal morphology (acrosome, mid piece and tail), motility and viability were 89.2% (82.3~92.0%), 71.3% (61.7~75.0%) and 76.7% (65.7~85.0%), respectively in fresh ejaculates. Sperm motility and sperm viability was significantly ( <0.05) lower in Holstein-Friesian Local bull than in other bulls during all three days of storage. The overall NRR for four bulls was 82.7% (72.9-87.5%). Bulls with higher sperm motility, viability and normal morphology of spermatozoa of individual bull had significantly (each <0.05) higher NRR. The highest ( <0.01) NRR (87.5%) was observed in a Red Chittagong bull whose semen qualities were significantly ( <0.05) higher than Holstein-Friesian Local bull (NNR 72.9%). The results of the present study concluded that NRR at 56 days post AI is related to parameters of semen quality. Therefore, semen evaluation may allow the discarding of bulls with poor fertility in an AI program.

The objective of this study was to investigate the motility and kinematics of boar sperm that while stored at 4C. The samples of fresh boar semen were place into an extender, Androhep, and stored at . In three of these samples, cryoprotectants were added. The sperm's motilities and kinematics were evaluated by using microscope () and the viability status was evaluated by using with eosin staining method. The 5 sample groups are; Goup A:Androhep (extender), stored at . Group B:Androhep (extender), stored at . Group C:Androhep (extender), + 3% glycerol (cryoprotectant), stored at . Group D:Androhep (extender), + 3% DMSO (cryoprotectant), stored at . Group E:Androhep (extender), + 3% ethylene glycol (cryoprotectant), stored at . In group A, the sperm's motility was reduced. On day one the sperm's motility was () and day 5 the motility was (). In group B, C and D the sperm's motility were reduced to 0 on day 5. In group E the sperm's percentage of motility decreased. On day one the sperm's motility was () and day 5 the motility was (). When comparing cryoprotectant in samples of boar sperm there is a slight improvement in the results when the use of Androhep Lite (extender), + 3% ethylene glycol (cryoprotectant), stored at are used. Based on these results, ethylene glycol can protect sperm from heat shock at , but not satisfactory level. However, it showed the possibilities of liquid semen preservation at by using cryoprotectant.

The objective of this study is to estimate the effect of adding TES to LEY and FGE freezing extender for the sperm viability, acrosomal morphology and DNA fragmentation from miniature pig sperm, we evaluated sperm characteristics in TFGE, TLE and LEY with various thawing condition ( for 20 sec, 45 sec and for 5 sec, respectively), and in different concentration of glycerol at 1%, 1.5%, 3%. The sperm viability and normal acrosome intact(NAI) in TFGE (Viability : , NAI : ), TLE (, ) extender significantly(p<0.05) increased than that in LEY (, ) extender thawed at for 5 sec. According to the results from glycerol concentration, the viability and NAI of miniature pig sperm in 1.5% glycerol TLE (, ) was highest among the experimental groups. In accordance with this, DNA fragmentation rates was the lowest in TLE () while that in LEY () is the highest. Therefore, these results suggest that TLE extender method for freezing- thawing of miniature pig sperm increased the viability after thawing.

The purpose of this study was to examine the effect of growing stages of the Korean Native Striped Bull (KNSB) on the freezability and fertility of frozen-thawed semen. First, we investigated the total motility (TM) and progressive motility (PM) according to the diluent used for semen freezing. Second, we examined the effect of the age of KNSB on semen volume, TM and PM of fresh and frozen-thawed semen. Third, we examined the effect of frozen semen from the different age of KNSB on the fertilization rate, and the artificial insemination pregnancy rate. The diluents used in this experiment were Triladyl and Tris-egg yolk extender (EYE). Semen was collected from 5 KNSB in the growing stage (15 months) and 5 adult KNSB (36 months). When Triladyl or Tris-EYE extender was used for semen freezing, there was no difference of the mean TM and the mean PM. However, the mean TM was significantly higher in Bull No. 1885 than Bull No. 4283 ( <0.05). The mean volume of semen collected from the 15-month-old bulls (2.3 ml) was significantly lower ( <0.05) than that from the 36-month-old bulls (5.0 ml). The mean semen concentration was similar for the 15-month-old ( spermatozoa/ml) and 36-month-old ( spermatozoa/ml) bulls. For the 15-month-old and 36-month-old bulls, the mean TM of fresh semen were 93.7% and 88.3%, respectively, and the mean PM were 97.0% and 88.3%, respectively; the 15-month-old bulls showed a particularly high PM ( <0.05). For the 15-month-old and 36-month-old bulls, the mean TM (56.0% and 58.0%, respectively) and the mean PM (64.0% and 70.7%, respectively) of frozen-thawed semen did not differ. The development rates of embryos after fertilization and the pregnancy rate after artificial insemination using frozen-thawed semen did not differ according to the bull's age. In summary, semen volume differed according to the bull's age, but semen concentration and survival rate, the fertilization rate, and the pregnancy rate did not differ according to the stripe bull's age. Accordingly, semen from bulls in the growing stage can be collected and frozen for the preservation and multiplication of rare livestock.

This study was carried out to investigate artificial insemination (AI) failure status and frozen semen characteristics in Korean proven bulls‘ number (KPN) semen used for AI of Hanwoo cows in Gangwon East region (Gangneung, Donghae, Taebaek, Samcheok, Sokcho, Yangyang, Goseong). Among semen used for AI, AI failure rate showed lowest at KPN506 (27.6%), whereas highest at KPN593 (77.2%). Correlations of AI failure in between Korean proven bulls semen and cows was 0.2941, which means that AI failure rate of Korean proven bulls semen may have respectable effect on reproduction of Hanwoo cow. In addition, present study was conducted to investigate spermatozoal viability rate, ruptured acrosome rate and active mitochondria in frozen Korean proven bulls semen with flow cytometry. The semen of KPN593 showed significantly (p<0.05) higher viability rate in KPN593 (30.49%) than that in KPN637 (37.34%). Furthermore, percentage of ruptured acrosome was lower in KPN637 as 21.37% than in KPN637 (21.37%), but it was not statistically significant. In conclusion, these results indicate that choice of Korean proven bulls semen may correlate positively with conception rate in Hanwoo cow. Therefore, KPN with high AI failure rate might be avoid to increase conception rate and characteristics of frozen semen might be evaluated before its use for AI.

The objective of this study was to determine the effects of E. coli isolated from porcine semen on sperm viability, motility, and semen pH. Semen samples were prepared using commercial extender, SeminarkPro (Noahbio Tech, Korea) that did not contain antibiotics. And 4 different levels of E. coli were artificially innoculated to semen with following concentrations; 4,000 of sperms with 1 of E. coli (T1), 400 with 1 (T2), 40 with 1 (T3), and 4 with 1 (T4). Semen samples were preserved at 17℃ for 5 days in semen storage box until analyzed by flowcytometer. Aliquots were subjected to measure the sperm viability (Live/Dead® stain), motility (mitochondrial function), and semen acidity (pH) from day 0 (day of semen collection) to day 5. Sperm motility and viability were significantly decreased (p<0.05) on day 0 (4 hrs after preservation at 17℃) in T3 and T4 compared to control groups and were significantly decreased (p<0.05) in all groups from day 3. Sample pH was acidic in T3 (6.90～6.86) and T4 (6.86～6.65) from day 3 to day 5 (p<0.05). On the other hand, sample pH was maintained 7.0～7.1 in control, T1, and T2 during the experimental period. Sperm motility and viability were significantly decreased from day 0 to day 5 compared to control in samples contaminated with E. coli above a value of 40:1 (20×106 sperm cells/ml : 5×105 cfu/ml). Even on day 1 in T4 and on day 3 in T3, semen pH was acidic probably due to the acidification of dead spermatozoa. These results suggest that E. coli contamination has a concentration-dependent detrimental effect on extended porcine semen quality.

The aim of this study was to evaluate the post-thawed characteristics of leopard cat semen. In this experiment, semen was collected from two leopard cats (A and B) at wild animal center in Seoul Grand Park in Korea. After collection, the sperms were washed with D-PBS and diluted by the freezing medium (Irvine science, USA) and stored in liquid nitrogen. The post-thawed concentration was for A and for B. The viability of post-thawed sperm from A and B individual was 24.0% and 19.0%, respectively. Pre-freezing motility of A and B individual semen was 68.54% and 56.65. Leopard cat A had more normal sperm than that of B (69.5% vs. 54.5%). Acrosome integrity analysis detected live (14.5% vs. 9.0%), damage (39.0% vs. 44.0%) and dead (46.0% vs. 47.0%) in leopard cat A and B, respectively. The present results concluded that leopard cat semen can be collected successfully by electro-ejaculation method and cryopreserved successfullyfor future use in different assisted reproductive technologies. The cryopreservation protocol needs to be modified for increasing post-thawed viability of leopard cat spermatozoa.

Bacterial contamination reduces the semen quality, semen preservation, and cause of disease spread as well. Sperm fertility is essential factor of reproductive performance in swine. Sperm fertility is affected by semen quality such as sperm motility, abnormality, morphology, and rate of bacterial contamination. This study was conducted to determine the relationship between elapsed time after semen preservation on the changes of bacteria and semen quality. Semen was diluted with BTS extender without antibiotic for 7 days and sperm parameter and fertility were measured. Sperm motility was measured by CASA and total bacteria number was counted after 22 24 hr incubation from counting agar plate in which sperm dilute to 10 106 in 0.9% saline solution and inoculate to agar. Acrosomal integrity was measured by Chlortetracycline (CTC) staining. CTC patterns were uniform fluorescence over the whole head (pattern A), characteristic of uncapacitated acrosome-intact spermatozoa; fluorescence-free band in the post-acrosomal region (pattern B), characteristic of capacitated acrosome-intact spermatozoa; and almost no fluorescence over the whole head except for a thin band in the equatorial segment (pattern C), characteristic of acrosome reacted spermatozoa. Total number of bacteria was significantly increased (p<0.0001) 3 days after preservation. Sperm motility, viability, and morphological abnormality on elapsed time after preservation were lower from 5 (77.24±6.47, p<0.001) and 7 days (77.24±6.47, p< 0.001) after preservation compared to 1 (15.71±7.18) and 3 days(18.39±7.22) after preservation, respectively. Sperm viability was significantly lower (53.25±35.03, p<0.0001) at 7 days after preservation. Mohological abnormality of sperm was lower (p<0.001) at 1 (15.71±7.18) and 3 (18.39±7.22) days compared to (5 21.84±7.91) and 7 (22.59± 9.93) days after preservation. Acrosomal integrity and capacitation rate (pattern A) were significantly lower (p<0.001) from 5 days after preservation.

Sperm examination is an important tool in estimating the fertilizing capacity of an ejaculate. The number of spermatozoa in a semen dose, morphology and motility are important for the fertilization process. By evaluation of semen, artificial insemination (AI) using high quality of semen can increase fertilization rate. Boar semen is subject to contamination by various pathogens that can result in fertility disorders in sows. Among these pathogens, porcine reproductive and respiratory syndrome virus (PRRSV), porcine parvovirus (PPV), porcine circovirus-2 (PCV-2) are of particular importance and accurate monitoring prior to and during the presence of boars in AI stations is essential. Because of the high risk of dissemination of disease via AI, The absolute goal is to provide pathogen-free semen and this is feasible with the adequate measures. The disease affects boars semen causes a significant reduction quality. In this study we investigated the characterization boar semen in Jeju, interaction of pathogenic virus infection with characterization of boar semen. Forty-two boar semen from 13 farms were investigated. The semen were stored during 5 days at 17℃ and the sperm qualities in the stored semen were analysed. Visual-motility assessment is a tool (Computer- Assisted Semen Analysis) used to determine the quality of boar semen. Percentage of morphologically normal spermatozoa were assessed. PRRS ,PPV and PCV-2 were detected in boar semen using PCR. The motion characteristics in boar semen was showed 68.4±9.1% for motility, 48.6±7.1 μm/s for VAP, 45.3±7.0 μm/s for VSL, 79.1±8.7 μm/s for VCL, 1.3±0.2 μm/s for ALH, 8.3±0.4 Hz for BCF, 93.6±3.5% for STR, 57.9±6.4 % for LIN. The percentage of sperm with abnormal head, midepeice and tail were 0.3±0.7%, 14.4±12.5%, 4.9±6.6%, respectively. Based on the PCR method, PPV was detected in 20 samples (48%). However, PCV-2 and PRRSV were not detected in any cases. Marked differences in motility and morphology between PPV negative and PPV positive semen were not observed. Sperm cell production was not affected by PPV infection. However, slight increases in detached head, coiled tail after infection were observed (p<0.05). The motility of semen in Jeju is similar to case comparing with other regions in Korea. Although PPV in semen was not affected in semen quality, there is the high risk of virus excretion in the semen of Jeju boars. Therefore continuous screening tests for some particular pathogens in boar semen would be warranted.

Genetic resources of buffalo, cattle, goat, sheep, deer, rabbit, pig, chicken, duck, goose, turkey and swan in Taiwan are conserved in living form or frozen genetic materials. Preserving farm animals in living form must conserve a considerable number of mature individuals with reproductive potential, and continued funding and breeding space are necessary. Based on the afore‐mentioned restraints and consideration of risk diversification, living animals are preserved in Taiwan Livestock Research Institute (TLRI) branches and breeding stock reproduction grounds throughout Taiwan. Genetic materials preserved in frozen form include germ cell (sperm, egg and embryo), tissue, somatic cell, cell line, DNA and gene pool. Those preserved for long term more than 10 years are conserved in liquid nitrogen storage tank at ‒185 to ‒196℃. Animal industry on utilization of farm animal genetics emphasizes the importance of in situ conservation and considers ex situ conservation as an essential complementary activity to in situ. In situ and ex situ utilization are complementary, not mutually exclusive. The exact strategy of germplasm cryobanking will clearly depend on the conservation objectives of TLRI. In situ and ex situ strategies differ in their capacity to achieve the different bio‐utilization objectives. Based on the objectives for bio‐utilization and conservation, the existing national technical capacity and infrastructure for cryoconservation, and amount of capital to invest in developing and maintaining a gene bank for food animal genetic resources (FAnGR), each country should determine for which FAnGR, if any, national cryobanking program should be undertaken. The health and sanitary issues of animals that must be considered when establishing and operating gene banks for animal genetic resources to help prevent the conservation of potentially dangerous pathogens along with the valuable genetic material in the ultra‐low temperature cell repository. Bio‐utilization of material stored in the bio‐bank must eventually be thawed and used to create new animals and therefore, national organization and annotation of the stored material is critical to ensure its proper utilization. Stock animals are the source of semen, embryo and oocytes are usually privately owned. This ownership may or may not change during the gene banking process, but the terms of agreements between bio‐banks and donors must be explicitly defined. Taiwan Animal Germplasm Center now conserves genetic resources of 77 livestock breeds and strains, including 19 native and 38 foreign species as well as 20 new breeds produced by inbreeding in poultry or breed‐crossing with the artificial insemination in livestock. Cryobanking of semen of native animals is for genetic diversity but also for risk management to long‐term global food security. Ex situ conservation program involving in vitro storage of germplasm cryobanking can contribute to ensure that it will be available to allow livestock keepers and animal breeders to confront future changes in animal production and economic environments.

The objective of this study was to examine effect of ethylene glycol for semen cryopreservation in Korean Jeju Black Bull. The semen was cryopreserved with extenders containing cryoprotectants (7% glycerol and 3%, 5%, 7% ethylene glycol) and packed to 0.5 ml straws. The semen straws were located above 3 cm of liquid nitrogen for 5 min, 5 cm for 10 min and 8 cm for 10 min. And then frozen straw was plunged into LN2. Post-thawed sperm motility, viability and membrane integrity were significantly higher in 5% ethylene glycol (72.5±5.00%, 54.88±0.66% and 46.00±2.40%; p<0.05). Motility and viability were similar between 7% glycerol and 5% ethylene glycol. However, the membrane integrity was significantly higher in 5% ethylene glycol (34.69±4.64% vs 46.00±2.40%; p<0.05). The viability and membrane integrity were significantly higher in 5 cm for 10 min and 8 cm for 10 min than 3 cm for 5 min (viability: 55.81±2.94, 55.19±3.34 vs 47.94±3.48%; p<0.05 and membrane integrity: 44.94±3.51, 46.06±2.25 vs 40.38±1.03%; p<0.05). The percentage of capacitated sperm assessed by CTC staining, percentage of F pattern was higher in 7% glycerol, 5% and 7% ethylene glycol, and AR pattern was significantly higher in 3% ethylene glycol. F pattern was significantly increased in 5 cm for 10 min and 8 cm for 10 min (p<0.05), but AR pattern was significantly increased in 3 cm for 5 min (p<0.05).

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 objective of this study was to examine the effect of various discontinuous Percoll washing conditions on motile sperm recovery rate and motion kinematics. Frozen semen 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. After thawing, the mixed semen samples 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×g and 700×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. Motile sperm recovery rate and motion kinematics were evaluated by computer assisted sperm analyzer using Makler counting chamber. Sperm motility (%) and motile sperm recovery rate showed similar pattern in all treatment groups. However, sperm motility (%) and motile sperm recovery rate were highest at 700×g for 30 min through a discontionous Percoll density gradient of 1 ml 90%: 1 ml 45% Percoll. There were no significant differences in motion kinematics after various Percoll washings. These results suggest that force of centrifugation, centrifugation time, and Percoll volume significantly affect motile sperm recovery rate.

The objective of this study was carried out to evaluate the efficiency of sperm collection methods on the post-thaw viability of cat semen. The cat semen was collected by artificial virginal (AV) and electronic ejaculate (EE) methods. The composition of semen extender was consisted of Tris-buffer supplemented with 20% egg yolk and 1% P/S antibiotics in Ext I, and more added 8% glycerol, 1.0% Equex STM paste of total volume in Ext II. The collected semen was adjusted the concentration and then diluted in Ext I for optimal concentration. The diluted semen was cooling to 5℃ temperature in refrigerator for at least 2 hrs and then diluted stepwise with Ext II for at least 1 hrs. After an equilibration for 1 hrs, the cooled semen was packaged in 0.5 ml straw and then freezing on the LN2 vapor over 5 cm above from LN2 and then immersed directly in LN2 for cryopreservation. The frozen semen was thawed in 38℃ water for 15 sec and then evaluated the motility, viability, and morphology. Post-thaw semen were calculated the motility by SMI (sperm motility index). The live-dead sperm was evaluated by Eosin-B and morphological evaluation was by Diff-quik kit staining. The post-thaw concentration (89×106 /ml vs. 128×106 /ml), viability (22.6±10.6% vs. 37.1±26.1%), morphological normality (27.0±50.2% vs. 45.6±123.0%) of EE and AV groups were not significant different, but the post-thaw motility was significant lower in EE than that in AV group (53.1±3.6 vs. 73.6±5.7) (p<0.05). In conclusion, semen collection methods did not significant different between EE and AV groups except of post-thaw motility and so both semen collection methods could be applied in feline semen collection methods.

희소 한우인 칡소의 정액 동결을 위해서 레시딘을 기본 희석제로 하는 AndroMed와 Tris-egg yolk extender를 사용하여 정자의 생존율과 활력 조사를 위해서 본 연구를 수행하였다. AndroMed 희석제를 사용하였을 때 생존율과 활력은 와 의 결과를 보였다. 그리고 Tris-egg yolk extender의 경우는 각각 와 결과를 보여 생존율에서는 Tris-egg yolk 희석제가 AndroMed 희석제를 사용하였을 때보다 유의적으로(p

Research in the area of equine artificial insemination (AI) has led to its increased application in field trials. However, procedures for equine semen collection, cooling and freezing of semen and artificial insemination need further improvement. In experiment 1, we investigated the percentage of total motility (TM) and progressive motility (PM) of sperms at after-collection, cooled-diluted, cooled-transported or frozen-thawed semen. In experiment 2, mares were inseminated with either cooled-diluted, cooled-transported or frozen-thawed semen. In experiment 3, we examined the effect of buffer (skim-milk extender), which was infused into the uterus at the time of AI with frozen-thawed semen. In experiment 4, we compared AI pregnancy rates for mares ovulating spontaneously versus after treatment with hCG. In experiment 1, the average percentage of TM was decreased from 75.3% to 14.4% at the stage of after-collection to frozen-thawed semen (p<0.05). The average percentage of PM was 58.2% and 59.6% at after-collection and cooled-diluted, but it was significantly increased 71.7% after frozen-thawed (p<0.05). In experiment 2, the pregnancy rates after AI using cooled-diluted, cooled-transported and frozen-thawed semen were 60%, 50% and 37.5%, respectively, and similar among treatments. In experiment 3, the pregnancy rate of mares infused with buffer at AI was 40% which was higher than that with no buffer (10%). In experiment 4, the pregnancy rates of mares were similar between ovulated spontaneously (25%) and ovulated with hCG (50%). The results suggest that equine semen that has been cooled-diluted, cooled-transported or frozen can be successfully used to establish AI, pregnancy and foal production. Also, the pregnancy rates after AI can be increased by infusing buffer into the uterus at AI or by inducing ovulation with hCG, but further study is need.