• Journal of Embryo Transfer
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• v.26 no.3
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• pp.219-222
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• 2011

Eight female Himalayan tahrs (Hemitragus jemlahicus) were estrus-synchronized, and transcervically inseminated with frozen-thawed semen in September, 2009, about 2 to 3 months earlier than their natural breeding season. Intravaginal progesterone-releasing devices were inserted into vaginas of six Himalayan tahrs on September 7, and the other two on September 8 to suppress luteal function of ovaries. The devices had been placed deep inside the vagina prior to withdrawal on September 23. A day before CIDR removal, a combination of PMSG 400 IU and hCG 200 IU was intramuscularly injected. Forty hours later, frozen-thawed semen was transcervically inseminated. Pregnancy diagnosis was performed 39 days later by analyzing progesterone level of serum. Every treatment was done under anesthesia inducted by xylazine injection. In conclusion, vaginal discharge of cervical mucus, hormonal changes induced by implant-typed or muscularly injectable hormones and widening of cervix enough to insert an insemination gun into uterine body were achieved in non-breeding season. Moreover, the first inseminated Himalayan tahr, 36 hours after CIDR removal was assumed to be pregnant but the fetus may have been lost due to the use of anesthetic drug.

• Korean Journal of Veterinary Research
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• v.49 no.4
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• pp.285-290
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• 2009

This study was performed to investigate the possibility of superfecundation by surgical intrauterine artificial insemination in dogs of confirmed genetic pedigree. Artificial insemination was performed on 3 days after ovulation with $1.3{\times}$ $10^8$ spermatozoa. Five puppies were delivered on 60 days after insemination. The ratio of the number of newborns to the number of corpora lutea was 83.3% (5/6). Parentage analysis with 10 canine-specific microstatellite markers demonstrated that one puppy was genetically relative to the sire-A family and four puppies were genetically relative to the sire-B. The present study demonstrated that two kinds of puppies with different genetic pedigree can be produced by surgical uterine insemination of semen of individual dog into each uterine horn of a bitch.

• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.329-334
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• 2011

This study was conducted to establish a freezing method of miniature pig spermatozoa. The semen 더aculated from PWG M-type miniature pig was collected by gloved-hand method. The semen was diluted with same volume extender (m-Modena B). The frozen solution used frozen solution of four different (LEY, TCG, BF-5 and m-Modena+egg yolk) for find optimal frozen solution in miniature pig sperm. The diluted semen for frozen rate assay was added to LEY solution (solution I: 11% lactose+egg yolk; solution II: solution I+glycerol+OEP), and frozen depending on freezing rate by the three different freezing methods (A: until $5^{\circ}C$ for 1 hrs, holding at $-102^{\circ}C$ for 10 min; B: until $5^{\circ}C$ for 2 hrs, holding at $-102^{\circ}C$ for 10 min; C: until $5^{\circ}C$ for 3 hrs, holding at -80 and $-102^{\circ}C$ for 10 min). Semen cooled until $5^{\circ}C$ was added with glycerol 1, 3 and 5%, and take a equilibrium time for 0, 10 and 30min. Frozen-thawed sperm were evaluated for viability, acrosomal status and morphological abnormality. The results of frozen-thawed sperm ability by frozen solution, viability was higher in LEY solution compared to other three different frozen solution. AR pattern of LEY solution were lower than other three different frozen solution. The results of freezing rate, viability was higher in B method compared to other methods (p<0.05). Acrosomal statute was intacted in A and B methods than C method. The experiment for glycerol condition was showed that sperm viability was higher in extender with 1% and 3% glycerol and equilibrium time of 0 min. The acrosome damage was lower in extender with 1% glycerol and equilibrium time of 10 min than other conditions. In conclusion, the optimal conditions for cryopreservation of miniature pig spermatozoa obtained in LEY frozen solution, cooling rate of 1~2 hrs, 1~3% glycerol concentrations and glycerol equilibrium time of 0~10 min.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.4
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• pp.507-515
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• 2009

A series of experiments were conducted to determine the suitable freezing and thawing temperatures for the freezing of boar semen in 5 ml maxi-straws. The ultrastructure, in vitro fertilization (IVF) and artificial insemination (AI) of frozen-thawed semen were also be evaluated. The 5 cm freezing height gave the best results not only in post-thaw motility rate (54.00%), but also in normal acrosome morphology rate (NAR) (80.23%). There was no significant difference in the post-thaw motility between different thawing temperatures and corresponding thawing times (p>0.05); the group of $52^{\circ}C$ and 25 s gave the highest motility rate (45.00%). As a whole, not only from the motility but also the NAR, thawing at $42^{\circ}C$ was better than the other two treatments. In the freezing packages, 5 ml maxi-straw gave a little lower mobility (40%), viability rate (49.58%), plasma membrane integrity rate (53.91%) and NAR (52.65%) than the 0.25 ml straw, but there was no significant difference between the two straw volumes (p>0.05). The IVF capacity of frozen-thawed semen in this experiment was similar to fresh semen. From ultrastructure observation, the main damage to boar spermatozoa after freezing was seen in the acrosome, such as swelling and formation of vesicles. After AI in recipient Shanghai White sows, frozen-thawed semen from 5 ml maxi-straws and pellets produced 72.2% and 80% conception rate and 7.8 and 8 litter sizes, respectively, and there was no significant difference between the 5 ml maxi-straw and the pellet (p>0.05).

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.235-241
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• 2017

To preserve genetic materials, cryopreservation of the semen from live animals is the main technique to establish cryo-banking system which could be used for artificial insemination and embryo transfer. However, the population of Korean black goat (KBG) becomes to dwindle in number and is now faced genetic erosion by crossbreeding with non-native breeds in small KBG farms. In this study, simple freezing method was used to preserve frozen semen from KBG using spermatozoa of cauda epididymis (CE) and electro-stimulated semen (ES). The negative effects of seminal plasma on fresh sperm was confirmed using precipitation test of Triladyl egg yolk diluent and sperm viability after thawing was compared between CE and ES spermatozoa. When seminal plasma of fresh ES semen was washed with semen washing media (SWM), the rates of live sperm shown no significant difference between CE and ES spermatozoa before freezing. However, the survival rate of frozen/thawed CE sperm was higher than ES ($74.6{\pm}10.6%$ vs $53.8{\pm}5.2%$) with significant difference (p < 0.05). The results of longevity test on frozen/thawed sperm from CE showed healthier sperm than ES. Therefore, spermatozoa from CE could be used for cryo-banking system in KBG lines. The more studies are needed to increase survival rate of ES semen.

• Journal of Embryo Transfer
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• v.21 no.3
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• pp.263-271
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• 2006

The purpose of this study was undertaken to compare ability of frozen-thawed sperm characteristics between two strains (miniature pig and Duroc). The semen was collected by gloved-hand method into a pre-warmed ($37^{\circ}C$) thermos bottle. The semen was diluted with same volume extender and added to LEY solution for freezing. The diluted semen was placed in 0.5 ml straws, and freezing was initiated by exposing the straws to liquid nitrogen ($LN_2$) vapours for 10 min before placing them into $LN_2$ for cryopreservation. The frozen-semen straw were thawed at 20, 37 and $50^{\circ}C$ for 1 min, 45 sec and 10 sec within water-bath. The semen sample were evaluated at 0, 3, 6, 9, and 12 h after incubation at $37^{\circ}C$ for analysis of sperm ability. Abnormality of spermatozoa in miniature pig was significantly (p<0.05) higher than that in Duroc at 0, 9 and 12 h of post-thawing incubation after frozen-thawing. The percentage of F-patterned spermatozoa in miniature pig was significantly (p<0.05) lower, while the percentage of AR (acrosome reacted spermatozoa) pattern was higher in the miniature than in the Duroc. On the other hand, there was no significant difference in the viability of spermatozoa thawed at different temperature ($20^{\circ}C\;and\;37^{\circ}C$) between two species, but the viability in miniature pig was higher (p<0.05) than in Duroc when sperm was thawed at $50^{\circ}C$. In conclusion, this study suggest that suitable freezing method for miniature pig semen is required for increasing post-thawing viability and fertilization capacity.

• Journal of Veterinary Clinics
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• v.8 no.2
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• pp.183-190
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• 1991

Pellet and straw methods in canine semen freezing are compared with respect to motility, viability and acrosome demage of sperm during each of the two major processing steps, to prior-freezing and to frozen-thawing. Senen was extended with a tris-buffered egg yolk contained 4% glycero1 Pellet freezing in the hole of dry ice and straw freezing on the surface of liquid nitrogen were carried out, respectively. The frozen semen 10 days after storage in liquid nitrogen container. wao thawed. In the comparison of two freezing methods, the straw freezing method with 42.7% in motility. 49.2% in viability and 0.186 acrosome score after thawing seems to be superior to the pellet freezing method with 31.2%, 34.5% and 0.314%, respectively. Sperm motility of processing step to frozen-thawing against decrease rate 12.67% to Prior freezing appeared of 33.84% and 49.37% in straw and pellet freezing and increase of 0.02 in acrsomal score to prior freezing appeared of 0.08 and 0.21 in straw and pellet freezing method to frozen-thawing

• Journal of Embryo Transfer
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• v.18 no.1
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• pp.61-68
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• 2003

Considerable attention has been focused on the cryopreservation of semen and estrus induction in dog, as consequence of poor productivity caused by long anestrus period, in order to enhance the productivity of youngs and to preserve the breeds. The objectives of this study were to improve reproductive efficiency of artificial insemination with fresh- and frozen-semen following estrus induction in dog. Fifty infertilie dogs (age 2~3 years) were selected fur the study and divided into three different estrus induction treatment groups. Group 1 : dogs (n=15) were given clomifene (0.1 mg/kg) orally f3r five days at 12 hr intervals. Croup 2: dogs (n=15) were given bromocriptine (50 $\mu$g/kg) orally for five days at 12 hr intervals, followed by single injection intravenously of 500 IU GnRH (Croup 3, n=20) when pro-estrus occurred. After being treated, the dogs were evaluated fur the rates of estrus induction and time interval lapses from treatment to beginning of the pro-estrus. The rates of pregnancy in estrus inducted dogs mated naturally compared to those inseminated artificially with ejaculated fresh semen and frozen-thawed semen. Estrus detection was performed using the method of vaginal smear and confirmed by the plasma progesterone assay. Pregnancy was confirmed by ultrasonograpy on day 25, 35 and 55 post insemination. The ejaculated semen was exposed to a mixture of Tris extender with cryoprotectant (Trisma, 81 mM; TES, 209 mM; citric acid, 6 mM; glucose, 5 mM; glycerol, 8%) and cryopreserved gradually by slow-cooling at 17 co above the surface of liquid nitrogen (L$N_2$) for 23 min. The use of fresh semen, the pregnancy rates were observed 66.6, 66.6, 75.0 and 83.3% in natural estrus, clomifene induced, bromocriptine induced and a combination of GnRH and bromocriptine, respectively. The use of frozen-thawed semen, the pregnancy rates were observed 66.6, 33.3, 50.0 and 60.0% in natural estrus, clomifene induced, bromocriptine induced and a combination of GnRH and bromocriptine, respectively. No difference was observed in the number of offspring produced among natural estrus and treated groups inseminated with fresh or frozen-thawed semen. In conclusion, there was no significant differences in the pregnancy rate of dogs between group treated with a combination of GnRH and bromocriptine and group treated clomifene or bromocriptine only. However, frozen-thawed semen can be used successfully fur artificial insemination in dog.

• Reproductive and Developmental Biology
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• v.31 no.1
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• pp.43-48
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• 2007

This study was conducted to examine the characteristics of fresh and frozen semen, proliferating efficiency by AI, and the coat color of offsprings in Korean Native Striped Cattle (Bos namadicus Falconer, Chikso). Semen were collected from 6 heads of tiger-coated male Chikso. In vitro fertilization (IVF) was conducted with frozen-thawed semen and in vitro matured Korean native brown cattle (general Hanwoo) oocytes. Total 18 heads of Hanwoo and Chikso were inseminated using Chikso semen. Coat colors of total 40 offsprings produced by AI were evaluated. The characteristics of the fresh and frozen-thawed Chikso semen did not differ among individuals. In vitro fertilization rate of Chikso semen was not different from that of general Hanwoo semen. However, developmental rate to the blastocyst stage of IVF embryos was higher in Chikso semen (25.9%) than in general Hanwoo semen (p<0.05). There was no difference in conception rate after AI between Chikso and general Hanwoo. The coat colors of offsprings varied, only 42.5% (17/40 heads) of offsprings had tiger coat color. Futhermore, only 55% of offsprings produced from the tiger-coated recipients had tiger coat color. This result shows that proliferation of Chikso by AI is possible, but further research approaches may be needed to enhance the productivity of tiger-coated Chikso.

• Korean Journal of Animal Reproduction
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• v.25 no.4
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• pp.409-419
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• 2001

This study was to examine whether the in vitro friability, motility and intact acrosome of frozen-thawed bovine and human sperm can be improved by adding Pentoxifylline (PF) or Fertilization Promoting Peptide (FPP). Human semen was frozen ultra-rapidly using Test yolk-buffer (TYB) freezing medium. Additive (PF, FPP) effects in frozen-thawed bovine and human sperm were analyzed by microscopic count for sperm motility and coomassie brilliant blue staining method f3r sperm acrosome intact. The in vitro motility of frozen-thawed bovine sperm with 5 mM PF treatment group (50.0%) was significantly higher than that of control (34.0%) (P＜0.05). In the frozen-thawed bovine sperm was examined, the intact acrosome rate of 50 nM FPP treatment (49.0%) was significantly higher than those of control (30.0%) and 25 nM FPP (38.0%) treatment groups (P＜0.01). In human semen, when in vitro motility of sperm with PF addition prior to freezing was examined, the result of 5 mM treatment group (51.0%) was significantly higher than those of control and 2.5 mM treatment group (39.0, 40.0%) (P＜0.01). In addition, 50 nM (75.5%) FPP adding in all treatment procedures for human semen freezing (before freezing, freezing and after thawing) was significant effect on maintenance of the sperm intact acrosome percentage (control: 45.0; 25 nM: 53.0; 100 nM: 68.0%) (P＜0.01). Also, the intact acrosome rate of human sperm with FPP (65.0%) was significantly higher than that with PF (43.0%) (P＜0.05), although sperm motility was slightly higher in PF treatment group. These results suggest that improved sperm motility and intact acrosome of frozen thawed bovine and human sperm can be obtained by addition of PF or FPP, and that the enhanced in vitro viability, motility and intact acrosome can be obtained by addition of FPP in all semen freezing procedures.