This study was undertaken to determine whether the presence of fertility-associated antigen (FAA) in semen would influence semen characteristics and conception rate of artificial insemination in Hanwoo. The response to FAA of 36 heads of proven bull, 7 heads of young bull, and 27 heads of performance-tested bull was that one proven bull was FAA-negative and the others were FAA-positive, therefore FAA-negative bull was 1.4%. FAA-negative bull was lower in first and second semen concentrations than those of FAA-positive bull in 5,301 semen of 21 heads of proven bull, then FAA-negative bull was fewer as 11.5% in total sperm counts. The estrus of 22 heads was 70d-nonreturned in 36 cows first inseminated with frozen semen of FAA-negative bull, but that of 249 heads in 378 cows first inseminated with frozen semen of FAA-positive bull. Each conception rate was 61.1% and 65.9%, respectively. The difference of conception rates was 4.8%. These results indicate that the response of FAA to semen were influenced semen characteristics and conception rate of artificial insemination, but further investigations are needed to confirm the results.

Four estrus-induced Himalayan tahrs (Hemitragus jemlahicus) were inseminated with frozen-thawed semen by laparoscopic or transcervical insemination techniques with no regard to the site of ovulation in non-breeding season. In June and July, 2009, estrus was synchronized by Eazi-Breed (Controlled internal drug release; Pfizer Animal Health, New Zealand) insertion for 16 days and PG 600 (PMSG 400IU, hCG 200 IU; Intervet, Netherlands) injection (IM) a day before removing . Forty eight hours later, laparoscopic or transcervical insemination was done to each of two tahrs under anesthetic condition inducted by ketamine (1.5 mg/kg) and medetomidine (0.09 mg/kg). For examination of estradiol and progesterone, blood was collected right before insertion, PG 600 injection, removal and insemination. Estradiol levels of four tahrs (No. 1, 2, 3, 4) before insertion and insemination were 13.3, 8.8, 14.3, 12 pg/ml and 23.5, 25.5, 21.1, 11.5 pg/ml, respectively. Progesterone levels of four tahrs (No. 1, 2, 3, 4) before insertion and insemination were 1.8, 0.05, 0.63, 0.61 ng/ml and 1.03, 0.37, 1.48, 2.12 ng/ml. Except for No. 4 tahr, cervices showed cervical mucus and opened enough to penetrate with embryo transfer gun sheet usually used for cows. Therefore, No.4 was laparoscopically inseminated together with No. 1. In conclusion, none of four Himalayan tahrs was pregnant. However, we proved that estrus could be induced by CIDR and PG 600 injection in non-breeding season, and laparoscopic or transcervical insemination with frozen-thawed semen could be one of assisted reproductive techniques in Himalayan Tahr.

The present study was to investigate the source of contamination during semen processing for in vitro uses. In the present study, frozen semen was prepared from liquid semen in our laboratory for in vitro fertilization (IVF) experiments due to lack of fresh semen. Antibiotics were added in the frozen semen extender (kanamycin and gentamicin) and in vitro culture (IVC) medium (gentamicin) for further inhibiting growth of microorganisms. Nevertheless, proliferations of microorganisms were observed in IVC culture drop during culturing of IVF embryos using frozen semen. Randomly 3 samples were taken from the liquid semen, frozen semen and egg yolk. Contaminated IVC medium, frozen-thawed semen, liquid semen and egg yolk were cultured in de Man, Rogosa and Sharpe (MRS) agar medium. Whitish colonies were detected in contaminated IVC drop, frozen-thawed semen samples and egg yolk but no colonies were formed in liquid semen samples. Gram-negative and rod-shaped identical bacteria were found in both frozen-thawed semen sample and contaminated IVC drop and egg yolk samples. Enterobacter cloacae were confirmed by API 20E kit according to manufacturer's instruction with identification value (% ID) 94.3% and T index 0.88. Antibiotic susceptibility tests were done according to Clinical and Laboratory Standards Institute (CLSI) by using ampicillin, amikacin, cephalothin, gentamicin, kanamycin, tetracycline, oxytetracycline, sulfamethoxazole trimethoprim, norfloxacin and ciprofloxacin test. Among them Enterobacter cloacae were resistant to ampicillin, amikacin, cephalothin, gentamicin, kanamycin but susceptible to tetracycline, oxytetracycline, sulfamethoxazole trimethoprim, norfloxacin and ciprofloxacin. From these findings it could be suggested that this contamination sources might be from egg yolk.

Only an optimum number of viable spermatozoa in a frozen-thawed insemination dose can ensure conception at artificial insemination (AI). We report here the percentages of normal, abnormal and viable spermatozoa present in the frozen-thawed semen of 20 Black Bengal bucks used for commercial AI. Bucks in this experiment were of 19.3~46.1 months old and 25~42 kg body weight. Four semen straws (0.25 ml) from each buck were collected for evaluation of their kinetic parameters. Scrotal circumference was measured by using a scrotal tape, sperm motility was estimated on eye estimation and sperm concentration was determined by using a haemocytometer. Sperm morphology was studied in paraformaldehyde fixed spermatozoa under differential interference contrast (DIC) microscope. To determine the proportion of live (plasma membrane intact) spermatozoa, semen was stained with SYBR-14 and propidium iodide and examined under fluorescent microscope. Scrotal circumference, post-thaw sperm motility, sperm concentration per insemination dose and proportion of normal spermatozoa were , , million and , respectively. The percentages of spermatozoa with head shape and acrosome abnormalities were lower ( and , respectively), whereas higher percentages of abnormalities () were observed in mid piece and tail portion. The proportion of live spermatozoa was . It is concluded that although a good number of morphologically normal spermatozoa are present in the insemination dose, the proportion of live spermatozoa is low, which warrants further improvements of buck semen freezing procedures to ensure good quality at AI.

The MTT assay is one of superior evaluation methods widely used to analyze the viability of metabolically active cell. It can be used to determine the percentage of viable sperm through measurement of the reduction of MTT granules at mitochondria in sperm tail. The purpose of this study is to determine the optimal condition of a simple and easy MTT assay to validate boar sperm viability and compare the accuracy of this test with microscopic examination. The MTT reduction rate for sperm viability were analyzed in microtiter plates (96 well) from 1 hr to 5 hr incubation periods at using spectrophotometer (microplate reader) at 550 nm wavelength. The remainder of semen sample was simultaneously examined to compare the correlation of accuracy between MTT assay and other sperm parameters. Those sperm parameters were included the motility, survival rates, membrane integrity, mitochondria activity and acrosome integrity. The OD values of MTT assay (MTT reduction rates) did not greatly change at 1 hr to 5 hr incubation periods in different proportion of live and freeze-killed sperms (dead sperm). The MTT reduction rates or survival rates were decreased according to the different concentration of live and dead sperm. The linear regression at 1 hr and 4 hr incubation periods in sperm MTT assay was y=291.55x-72.176 and y= 180.64x-44.569, respectively. There are high correlation between 1 hr and 4 hr incubation periods (p<0.001). The results of MTT assay and other sperm parameters has a positive correlation (p<0.01 or 0.05). The correlation coefficients for MTT assay was 0.88115 for motility, 0.89868 for survival rates, 0.91722 for membrane integrity and 0.77372 for acrosome integrity, respectively. In conclusion, the MTT assay can be used as a reliable and efficient evaluation method for boar sperm viability. It can be use practical means to evaluate the quality of boar sperm by a fast, inexpensive and easy method.

The objective of this study was to investigate the effect of storage time on fresh boar semen in Androhep and Beltsville Thawing Solution (BTS). Boar semen samples extended in each extender were stored at 17℃ up to 4 days. Sperm motility kinematics was evaluated by computer assisted sperm analyzer (CASA) and capacitation status by chlortetracycline (CTC)/Hoechst 33258 staining. Sperm motility (%) was not decreased during storage in BTS and Androhep. No significant difference between extenders was observed. Only significant differences in kinematic parameters on linearity during storage were found. The percentage of dead sperm significantly decreased during storage (p<0.05). Also the percentage of noncapacitated, capacitated, and acrosome-reacted sperm significantly modified during storage (p<0.05). However, there was no significant difference between extenders except proportion of capacitated sperm. This finding supported that modification in these parameters was not significantly different between extenders during this short-term storage. Our finding strongly indicated that both Androhep and BTS maintained favorable conditions for motility, motility kinematics, and capacitation status during short-term storage. Despite modifications in some parameters were apparent during sperm storage in extenders, these may not affect the fertilizing capacity of boar semen.

We report herein the successful results of estrus induction, sperm cryopreservation and kids born by transcervical insemination of frozen-thawed semen in a Saanen goat. Flugestone acetate (FGA: 60 mg) was inserted into vagina for 15 days. The goat was intramuscularly injected with 400 IU PMSG and 200 IU hCG (: Intervet, Korea) a day before withdrawal of the FGA sponge. Follicles and corpora lutea were identified on both ovaries by laparoscopy. Artificial insemination was performed 46 hours after removal of FGA sponge. The concentration of frozen-thawed semen was and 0.5 ml of frozen-thawed semen was transcervically inseminated into uterine body under anesthesia. Three kids, all females, were born 144 days after artificial insemination. This is the first report producing kids by transcervical insemination of frozen-thawed semen in a Saanen goat of which the estrus was induced by FGA sponges, PMSG and hCG during non-breeding season in Korea.

The relationships of scrotal circumference (SC) to semen characteristics and the conception rate (70 days-nonreturn rate) of artificial insemination in farm were studied with 137 heads of bull in Hanwoo. The average and range of SC were cm and 26.0~52.5 cm, respectively. Hanwoo bulls were classified with SC, divided into 34 cm below group, 34~39 cm group, and 39 cm over group. The 5,487 semen records of 43 heads of bull from July. 1. 2007 to June. 30. 2008. were used to determine the relationships between SC and semen characteristics. The semen concentration and total sperm number of each group were 11.18, 16.68, and , and 69.83, 101.64 and /ejaculate. The bulls with 34 cm or more SC were higher than the bulls with 34 cm below in semen concentration and total sperm number (p<0.01). But between SC and semen volume have no significant relationship (p>0.05). The 9,862 mating records of 44 farm with 137 heads of bull were used to determine the relationships between SC and conception rate. The conception rate of 1st artificial insemination were 73.31, 74.16, and 77.33 % in each group. Also SC was positively correlated with pregnancy rate (r=0.12, p=0.17). These results indicate that SC correlates positively with semen characteristics, and maybe with pregnancy rate in Hanwoo.

The aim of present experiment was to examine commercial synthetic extender(AndroMed) for semen cryopreservation of Korean Black Bull. Semen was collected from a Korean Black Bull using an artificial vagina and transported to the laboratory. The semen was diluted 1:1 by AndroMed. The pellect was diluted to final sperm concentration of by doubling in every 10 minutes at cold chamber. The semen was equilibrated for 1 hr at cold chamber and packed to 0.5 ml straw. The semen straws were located above 5 cm of liquid nitrogen for 5 minutes, above 5 cm for 10 minutes and above 10 cm for 10 min. And then the frozen straw was plunged to . The presented straws were examined the viability and motility after thawed at water bath. Hanwoo semen was used as KPN (Korea Proven Bull Number) in this experiment. The survival rates was significantly higher in fresh semen than frozen semen (). However, the motility rates was similar (80.7% and 66.4%). The survival and motility rates were higher in 5cm, 10 min treatment group than the other two groups in straw-located height and duration above ( and 70.7% vs, 33.18% and vs, 30.14% and 65.7%, respectively). The development rates to cleavage was higher in Black Cow than Hanwoo semen (62.2%, 64.4%), However, The development rates to blastocyst was higher in Hanwoo than Black cow semen (25.9%, 23.0%). In conclusion. The present results that acceptable fertilization and cryopreservation could be obtained by in vitro fertilization with frozen-thawed semen using a synthetic semen extender (AndroMed).