• Korean Journal of Animal Reproduction
• /
• v.7 no.2
• /
• pp.27-40
• /
• 1983

Frozen boar semen should be utilized in swine production in order to get its advantages. Much studies were carried out for the practical use of frozen semen. Some of frozen boar semen are used in swine production and some companies are exporting frozen boar semen in U.S.A.. For the practical utilization of frozen semen in swine production in Korea, we have to get deep knowledge and understanding about frozen boar semen and studies on processing and conception of frozen semen.

• Journal of Embryo Transfer
• /
• v.11 no.3
• /
• pp.309-316
• /
• 1996

The aim of this study is to elucidate sperm chemotaxis and to set up the optirnal condition for selection of motile and capacitated sperm from hovine frozen-semen. Thus, the effects of semen-washing after thawing, concentrations of progesterone (P4) and bovine serum albumin (BSA), and sperm-washing frequency on sperm selection were examined. For evaluating their effects, number, viability and acrosome reaction of sperm swim-up seperated from semen, which were incubated for 30 minutes at 36$^{\circ}C$ in the M2 solution containing P4 and BSA, were investigated. For frozen-semen just after thawing, sperm recovery and viability were not significantly different between P4-treated and -untreated semen. However, washing frozen-semen decreased the number of sperm and increased the viability of sperm that were recovered from semen treated with P4. Progesterone affected the recovery rate, the viability and the acrosome-reaction rate of sperm recovered from washed frozen-semen. Especially, number of motile and capacitated sperm were highest in semen treated with 50$\mu$g /ml among 0, 20, 50 and 100$\mu$g /ml of P4 concentrations. BSA affected the recovery rate and the viability of sperm recovered from washed frozen-semen that were treated with 50$\mu$g /ml of P4. Especially, the percentage of viable sperm were highest in semen treated with 4mg /ml among 0, 2, 4, and 6mg /ml of BSA concentrations. Repeatedly sperm-washing did not affect the recovery rate and the viability of sperm recovered from washed frozen-semen that were treated with 50$\mu$g /ml of P4 and 4mg /ml of BSA In conclusion, using progesterone and BSA could efficiently make the selection of motile and capacitated sperm from washed frozen-semen.

• Korean Journal of Veterinary Research
• /
• v.24 no.2
• /
• pp.245-266
• /
• 1984

As a fundamental study to increase the reproductive efficiency in cattle, highly motile sperm were separated and collected from raw semen, extended semen and frozen semen by different methods using various concentrations of bovine serum albumin or Tyrode's solution. Various characteristics and light microscopic and electron-microscopic morphology of sperm separated by different methods were compared. The results obtained were as follows; 1. The sperm separated from raw semen using bovine serum albumin showed significantly high value in motility, motile sperm count, percent of normal sperm and progressive motility, as compared with control sperm and revealed the highest sperm recovery rate when separated with 6% bovine serum albumin. 2. The sperm motility, percent of normal sperm and progressive motility of the highly motile sperm frozen after being separated from raw semen with bovine serum albumin, showed significantly high value than those of a control sperm and especially found the highest value when separated with 20% bovine serum albumin. 3. Light-microscopic percent of abnormality was significantly low in the prefrozen and postfrozen highly motile sperm separated with bovine serum albumin, as compared with control sperm. 4. Electron-microscopic finding of the highly motile sperm separated with bovine serum albumin showed low percent of deformity in the dilatation and vesiculation of cell membrane, in dilatation and density loss of acrosome than in those of control sperm. 5. It was impossible to separate the highly motile sperm from frozen semen with bovine serum albumin, but it was possible with Tyrode's solution. 6. Recovery rate of highly motile sperm from raw semen extended semen and frozen semen was the highest when the sperm pellet stood in Tyrde's solution for 80 minutes. 7. The highly motile sperm separated from raw semen, extended semen and frozen semen with Tyrode's solution showed significantly high value in motility, progressive motility and percent of normal sperm, as compared with control sperm. 8. Highly motile sperm, when separated from raw semen, extended semen and frozen semen with Tyrode's solution, showed significantly low percent of microscopic abnormality as compared with control sperm.

• Journal of Embryo Transfer
• /
• v.23 no.3
• /
• pp.161-166
• /
• 2008

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 $104\times10^6/ml$ and $86\times10^6/ml$ 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 $5^{\circ}C$ was significantly lower than that at 0 hr ($30.0\pm4.1%\;vs.\;78.0\pm2.5%,\;p<0.05$), 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.

• Korean Journal of Animal Reproduction
• /
• v.10 no.1
• /
• pp.19-26
• /
• 1986

This experiment was carried out to investigate the effect of bovine serum ablumin (BSA), sugars, glycerol equilibration time, straw size and thawing method on the survival index and the morphology of frozen boar spermatozoa. The results obtained were summarized as follow: 1. When the semen frozen in BF5 dilutor as pellet form was thawed in BTS at 37$^{\circ}$and 50$^{\circ}C$, BF5 dilutor with fructose showed higher sperm survival index than that with dextrose, however, when the semen was thawed on dry test tube at 37$^{\circ}C$, BF5 dilutor with sucrose showed higher sperm survival index than with other sugars. 2 When the semen forzen in BF5 dilutor with straw and thawed at 37$^{\circ}C$, BF5 dilutor with dextrose showed higher sperm survival index than those with other sugars, and there was no difference in sperm survival index between 0.5 and 1.0 ml straws. 3. The sperm survival index of frozen sperm was significantly (P<0.05) improved due to addition of BSA (0.05%) to BF5 dilutor. 4. When the extended semen with BF5 dilutor contatining 0.01 to 0.05% of BSA was frozen in the straw, the semen without glycerol equilibration showed significantly (P<0.05) higher sperm survival index than those with 2, 4 and 6 hrs glycerol equilibration time. 5. The sperm frozen in BF5 dilutor with dextrose or fructose, sucrose and raffinose showed 77 to 88% in normal acrosome rate and no difference among sugars. 6. The frozen semen showed lower normal acrosome rate than the first and second diluted semen, whereas the frozen semen showed higher swollen, damaged and missing acrosome rate than the first and second diluted semen. 7. Damaged and missing acrosome rate of sperm head due to freezing was somewhat inhibited by addition of BSA (0.01 to 0.05) to the BF5 dilutor.

• Journal of Embryo Transfer
• /
• v.25 no.4
• /
• pp.267-272
• /
• 2010

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.

• Journal of Embryo Transfer
• /
• v.26 no.1
• /
• pp.13-17
• /
• 2011

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.

• Korean Journal of Veterinary Research
• /
• v.47 no.1
• /
• pp.135-138
• /
• 2007

A 3 year-old female Afghan hound came to the Veterinary Referral Hospital of the College of Veterinary Medicine, Seoul National University for artificial insemination (AI) with frozen semen. In order to inseminate, semen was frozen in USA 3 years ago. Frozen semen was sent by air from Santiago to Seoul for AI. The stud died 2 years ago, so we could only use a limited amount of frozen semen in that estrus cycle. The number of total motile spermatozoa was $59.4{\times}10^6$and the total volume was 1.2 ml. The frozen spermatozoa were thawed in $70^{\circ}C$ water for 8 sec, which were then deposited at the bilateral uterine horns by a surgical method. The number of corpus luteum was 6. Sixty days after artificial insemination resulted in the birth of 4 puppies, all of which are alive and healthy.

• Journal of Veterinary Clinics
• /
• v.18 no.1
• /
• pp.44-47
• /
• 2001

Semen were collected from 9 male dogs and frozen by liquid nitrogengas. Frozen semen were thawed at 7$0^{\circ}C$ for 8 seconds. About $2{\timss}10^8$ sperm per insemination were inseminated to 10 bitches (3 Retrievers, 4 Chihuahuas, 1 Yorkshire Terriers, 1 Maltese, and 1 Poodle) at three and six days after the estimated peak of luteinizing hormone. For small breed dogs, uretero-renoscope (Kahl Storz, Germany, 12.5 Fr) was used for trans-cervical insemination, whereas cystoscope(Kahl Storz, Germany, 22Fr) was used for large breeds (Retrievers). Pregnancy was diagnosed by ultrasonography at 30 days after insemination. All of 3 Retrievers (100.0%) and 3 bitches of 7 small breed dogs (42.9%) were conceived (60.0% in total). This result indicated that trans-cervical insemination using endoscope is an effective method for AI with frozen semen not only for large breed dogs such as Retriever but also for small breeds.

• Journal of Animal Science and Technology
• /
• v.56 no.3
• /
• pp.8.1-8.6
• /
• 2014

Background: Artificial insemination (AI) can serve as a powerful tool to the sheep owners for making rapid genetic progress of their flock. The AI in sheep is mostly performed using fresh semen with two reasons i) lambing rate following trans-cervical AI with frozen semen is limited by the inability of frozen-thawed sperm to transit the cervix and ii) the need of circumventing the cervical barrier through laparoscope aided intrauterine AI. Therefore, AI with frozen-thawed semen is not as widespread in sheep as it is in other domestic species. However, to get maximum benefits through the use of AI, frozen-thawed semen is a prerequisite because instead of high fertility, the short shelf life of fresh semen coupled with a limitation on the number of insemination doses achievable per unit time restricts the widespread use of individual sires. Therefore, in order to enhance lambing rate, a total of 240 trans-cervical artificial inseminations with frozen-thawed semen were performed in Bharat Merino ewes during autumn season either once in the evening (G-I, 10 h after onset of estrus, n = 100) or twice (G-II, 14 h and 22 h after onset of estrus, n = 140) i.e. once in the morning and again in the evening. Results: The pregnancy rate (proportion of pregnant ewes confirmed by ultrasonography at day 40) and lambing rate (proportion of ewes lambed) were higher in G-II as compared to G-I (26.4 vs 20% and 19.3 vs 10%, respectively). The difference in lambing rates was statistically (P < 0.05) significant. The depth of insemination within cervico-uterine tract had no significant effect on pregnancy and lambing rates. Conclusions: The results indicate that lambing rate in sheep following TCAI with frozen-thawed semen was significantly influenced by time of inseminations. Two inseminations after 14 and 22 h of onset of estrus enhanced the lambing rates of Bharat Merino sheep as compare to single insemination after 10 h of onset of estrus. The TCAI technique with frozen-thawed ram semen is promising and may serve as a valuable tool for genetic improvement of sheep breeds. Research efforts are going on worldwide to overcome the poor fertility following TCAI with frozen-thawed semen.