The study aims to assess viability, acrosomal menbrane, integrity and mitochondria membrane potential of sperm separated using a percoll density gradient(45% and 90%) and swim-up methods using Hanwoo epididymal sperm frozen semen. Briefy, motile sperm separated using a percoll gradient and swim-up. 25 μl of sperm dilution from droplets were transferred to 1.5 mL tube and incubated with fluorescent probes at 39°C in dark as follows. After incubation, 75 μl of 5,5',6,6'-tetrachloro-1,1',3,3'- -JC-1; Mitochondrial Membrane Potential Detection kit, Cell Technology Inc., USA) working solution was mixed with sperm dilution and incubated for 30 min. One μl of Hoechst 33342 (H1339; Molecular Probe, Eugene, OR, USA) stock solution was mixed with sperm dilution and incubated for 10 min. And then, 0.5 μl of propidium iodide (PI) stock solution and 0.5 μl of fluorescein peanut agglutinin FITC conjugate (FITC-PNA; Vector Laboratories, FL-1071) were mixed with sperm dilution and incubated for 8 min. After mixing with fluorescent probes and sperm dilution, 5 μl of stained sperm dilution was mounted on a slide glass and covered with cover glass. More than 200 sperms in a slide glass were counted with × 400 magnification by fluorescent microscope (Eclipse Ci_L, Nikon, JAPAN) and evaluated viability, acrosomal membrane integrity, and mitochondrial membrane potential of sperm with triple band filter (DAPI/FITC/TRITC; Nikon, Tokyo, Japan). Live sperm were stained with Hoechst 33342(blue) and dead sperm were stained with PI (red). Damaged acrosomal membrane of sperm was stained with FITC-PNA (green) and intact acrosomal membrane of sperm was not stained. Both of sperm swim-up method with or without BSA separated to Live intact mitochondria (15.39±4.31 vs, 12.58±3.74, and) without significant difference. and percoll density gradient method also similar (7.29±6.54), swim-up method of sperm preparation appeared to be more efficacious in percentage recovery of motile sperm concentration compared to Density gradient method.

Sperm recovery from epididymis in animals considered as important tools to preserve high-value or endangered species. However, there are no appropriate castrating indicators of timing for recovery of sperm which can be available to artificial reproduction technologies such as artificial insemination (AI) and in vitro fertilization (IVF), particularly in young Hanwoo bull. Therefore, this study aimed to investigate semen volume, morphology and motility of sperm in epididymis of young Hanwoo bulls at 8, 13, 14, and 15 months of age. About 2 cm of the epididymal tail only was cut and minced using blades. Minced epididymal tail tissues were mixed with semen extender (OptixCell, France, IMV technologies) and sperm were recovered with a cell strainer (100 μm nylon mesh). The number of sperm at 8 months of age was lower than that at 13, 14, 15 months of age in bulls after collection (33.6±27.2 vs. 352.4±39.2, 320.4±113.6 and 422.8±252.4×107cells respectively; P<0.05). After the frozen-thawed sperm the the percentage of abnormal head, tail and dead damaged acrosome did not differ between the ages 13, 14 and 15 months of age in bulls (P>0.05), however, the dead sperm with intact acrosome (DIA), the numbers showed that more than 15 months in 8, 13, 14 months (8.7±4.1 vs. 47.3±12.2, 34.8±14.0, 28.8±8.5, P<0.05). In addition, frozen-thawed sperm at 8 months of age showed low total motile sperm compared to those at 13, 14 and 15 months of age (26.4±8.2 vs. 45.7±29.5, 62.3±41.0, 70.4±15.9%, respectively; P<0.05). In conclusion, sperm derived from epididymal tail at 8 months of age in Hanwoo bulls showed high abnormal morphology and poor motility, which is not adequate for artificial insemination(AI) and in vitro fertilization(IVF). On the other hand, sperm derived from epididymal tail at 13, 14, 15 months of age in bull showed high normal morphology and motility, which may be available for AI and IVF. Epididymal sperm collected from bulls over 13 months is needed for further study whether to use the actual in vitro fertilization.

Semen collection from epididymis has been used as an important alternative tool, particularly on abnormal animals including those with joint disorder, poor sex drive and poor libido. However, previous studies had been focused on only recovery of sperm derived from testes, regardless of fundamental parameters such as age, body weight, scrotal circumference, testicular size (weight, length, width and volume) and semen volume. In addition, there is no precise castrating periods to recover sperm from young Hanwoo bulls’ testis. Therefore, we investigated fundamental parameters in Hanwoo bulls at 7, 8, 14, 15, 16 and 17 months of age, which parameter can be used for sperm recovery from testis. Before castration, body weight and scrotal circumference were recorded. After castration, testes were transported to laboratory and testicular weight, length and width were recorded. About 2 cm of epididymal tail was collected and minced using blades on 100 mm petridish. Minced epididymal tail tissues were mixed with Optixcell (France, IMV technologies) and debris were removed by filteration with 100 um nylon mesh. Seven to 8 months of age in bulls showed low fundamental parameters compared to those of at 14 to 17 months of age in bulls (body weight, 218-258 vs. 330-429 kg; scrotal circumference, 23.4-24.6 vs. 28.8-32.8 cm; testicular weight, 92-104 vs. 222.3-269.9 g; testicular length, 10.9-11.6 vs. 13.4-15.6 cm; testicular width, 4.7 vs. 6.2-6.6; testicular volume, 126.4-136.4 vs. 268.9-357.6 cm3, semen volume, 3.0-4.8 vs. 60.6-68.3 ml, p<0.001). In conclusion, to recover semen derived from epididymis of testis in young Hanwoo bull, these fundamental parameters should be satisfied; 330 kg of body weight, 28.8 cm of scrotal circumference, 222.3 g of testicular weight, 13.4 cm of testicular length, 6.2 cm of testicular width, 268.9 cm3 of testicular volume. In further study, fundamental parameters of 9 to 13 months of age in bull should be investigated to determine more accurate castrating periods for sperm recovery from testis.

The study aims to assess the embryo development and survivability of bovine embryos cultured in vitro by addition of cysteine. The rates of metaphase II formation are not significantly different among the three groups(73.8% for TCM199, 76.9% for TCM199 with 0.3mM cysteine and 83.8% for TCM199 with 0.5mM cysteine). No differences on cleavage rate(70.6~74.6%) was observed among three culture medium(70.6% for TCM199, 71.3% for CR1aa, and 74.6% for SOF) with 0.5mM cysteine. However, significantly(P<0.05) higher development rate was obtained in the blastocyst stage by adding 0.5mM cysteine in SOF medium(35.6%) than in TCM199(27.6%) or CR1aa(26.6%). No significant differences in the cleavage rates were observed among the three culture. After freezing the blastocysts cultured with 0.5M cysteine, the re-expansion rates ranged from 61.3% to 86.4% among groups, and hatching rates are from 26.3% to 46.9% among groups. The rates of re-expansion and hatching are significantly(P<0.05) higher in SOF medium(86.4% and 46.9%, respectively) than those in TCM199(61.3% and 26.3%) and CR1aa medium(87.1 and 44.4%). After thawing, the blastocyst re-expansion rate become significantly(P<0.05) higher in in vivo (87.1%) and in vitro (70.3%) embryos. In conclusion, our results demonstrate that supplementation of IVM and IVC media with 0.5mM cysteine improved the quality of in vitro production of embryo and post-thawed embryo. Future studies comparing these culture systems in well-designed trials should be performed.

The recovery of epididymal sperm in animals is considered as one of the important tools to preserve high value or endangered species. However, there are no appropriate castrating indicators such as months of age in bull, sperm morphology, and motility, particularly in young Korean native bull (Hanwoo). Therefore, this study aimed to investigate sperm number, morphology, and motility of sperm in the epididymis tail of young Hanwoo bulls at 8 and 15 months of age. After castration, epididymal tails were collected and minced with blades to recover sperm. In experiments 1 and 2, sperm number, morphology, and motility were examined. Total number of sperm and percentage of normal sperm from bulls at 8 months of age was lower than that of bulls at 15 months of age after collection (P<0.05). Percentage of abnormal head, tail, proximal cytoplasmic droplet, dead and damaged acrosome of sperm from bulls at 8 months of age were higher than those of bulls at 15 months of age (P<0.05). In experiment 3, sperm motility from bulls at 8 and 15 months of age were examined before freezing and after thawing. Frozen-thawed sperm at 8 months of age showed low total motility and motile sperm with ≥ 25 μm/sec compared to those at 15 months of age and commercially-used sperm (P<0.05). In conclusion, sperm derived from the epididymal tail of bulls at 8 months of age showed high abnormal morphology and poor motility, which are not adequate for AI and IVF. On the other hand, sperm derived from the epididymal tail of bulls at 15 months of age showed high normal morphology and motility.

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 investigate the interrelationship of weight and hormonal changes through the administration of vitamin A to first time pregnant heifers. A total of 28 Korean heifers was used for this study. The heifers were divided into two groups - with vitamin A (n=14) and without vitamin A (control) (n=14) in the feed. Body weight increased in vitamin A treated heifers starting 9 months until 15 months. After pregnancy, vitamin A treated heifers were found to maintain higher body weights than the control group. Pre-pregnancy and post pregnancy progesterone levels were not different between the two groups. Serum estradiol levels of heifers at different growth stages showed relatively higher E2 levels than the control. Also, the control during pregnancy may show higher serum E2 levels than the vitamin A treated heifers. The growth phase serum estradiol levels in heifers may be relatively higher than the control. During pregnancy it showed a similar trend. Serum levels of vitamin A treated heifers did not differ from pregnant heifers at 5 months of age. However, after 5 months from conception until 8 months of treatment it showed a high level. Serum cholesterol in pregnant cows was higher in the control group than the treatment from beginning until the end of pregnancy. This is considered to be related to fetus development during pregnancy, as well as the mother's health.

The present study was performed on farm animals to test the effectiveness of progesterone-releasing intravaginal device (Cue-Mate® 1.56 g) and injection of prostaglandin F2α (PGF2α) for synchronization estrus in Hanwoo cattle. The cattle were at random stage of the estrus cycle. The cows were artificially inseminated at day 7 after Cue-Mate withdrawal, using commercial semen from Korean native bulls. There was a season effect on the estrus synchronization rate. It was higher in spring (94.3%) followed by winter (93.3%), autumn (90.4%) and summer (67.2%). In summary, The results of this study revealed that season has influences on estrus behavior of cattle with no significant effect on pregnancy rate. In summary, we suggest summer reproductive management to alleviate the effects of heat stress. It should be based on intensive cooling combined with hormonal treatment. Given that different subgroups of cows benefit differently from the treatments, selective hormonal administration should be considered.