• Korean Journal of Animal Reproduction
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• v.7 no.2
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• pp.57-71
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• 1983

1. Diluted chicken semen can be preserved at 2 to 5$^{\circ}C$ for 24 to 48 hr with resultant fertility of greater than 90% of that of fresh semen. Turkey semen can be preserved at 10 to 15$^{\circ}C$ for 6 to 24 hr and provide economical fertility. 2. Frozen chicken semen has given variable results; a 21 to 93% fertility ranges as compared to 92 to 94% expected with fresh semen. Highest fertility levels obtained with frozen turkey semen intravaginally inseminated have been 61 and 63% using DMSO and glycerol, respectively, as cryoprotectants. 3. The use of glycerol as a cryoprotectant reauires that its concentration in semen be reduced to less than 2% either by dialysis or centrifugation after thawing and before intravaginal insemination if optimal fertility is to be obtained. 4. The temperature at which cryoprotectants are added to semen and the time allowed for equilibration are important for subsequent fertility pre- and post-freezing. 5. The type of container used for packaging the semen, freeze or cooling rates, thaw rates and level of cryoprotectant all interact in affecting cell survival. 6. Plastic freeze straws as a packaging device for semen offers the following advantages: easy to handle, require minimal storage space, offer a wide range of freeze and thaw rates, and insemination can be made directly from them upon thawing. 7. Controlled slow cooling rates of 1 to 8$^{\circ}C$/min have thus far provided the best results for cooling chicken semen throught the transition phase change (liquid to solid) or critical temperature range of ＋5 to -20 or -35$^{\circ}C$. 8. Highest fertilities have been achieved with frozen chicken semen where a slow thaw rate (2。 to 5$^{\circ}C$) has been used regardless of the freeze rate. 9. To maintain a constant high level of fertility throughout a breeding season with frozen semen, a higher absolute number of spermatozoa must be inseminated (2 to 3 times as many) as compared to fresh semen since a, pp.oximately 50% are destroyed during processing and freezing. 10. The quality of semen may vary with season and age of the male. Such changes in sperm quality could be accentuated by storage effects. Thus, the correct number of spermatozoa may very well vary during the course of a breeding period. 11. As to time of insemination, it is best to avoid inseminating chicken hens within 1-2 hr after or 3-5 hr before oviposition; and turkey hens during or 7-10 hr before oviposition. 12. The physiological receptiveness of the oviduct at the time of insemination is a very important biological factor influencing fertility levels throughout the breeding season.

• Journal of Animal Science and Technology
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• v.65 no.2
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• pp.401-411
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• 2023

Many studies have been conducted to improve technology for semen cryopreservation in pigs. However, computer-assisted analysis of sperm motility and morphology is insufficient to predict the molecular function of frozen-thawed semen. More accurate expression patterns of boar sperm proteins may be derived using the isobaric tags for relative and absolute quantification (iTRAQ) technique. In this study, the iTRAQ-labeling system was coupled with liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis to identify differentially expressed CM10-fractionated proteins between fresh and frozen-thawed boar semen. A total of 76 protein types were identified to be differentially expressed, among which 9 and 67 proteins showed higher and lower expression in frozen-thawed than in fresh sperm samples, respectively. The classified functions of these proteins included oxidative phosphorylation, mitochondrial inner membrane and matrix, and pyruvate metabolic processes, which are involved in adenosine triphosphate (ATP) synthesis; and sperm flagellum and motile cilium, which are involved in sperm tail structure. These results suggest a possible network of biomarkers associated with survival after the cryopreservation of Duroc boar semen.

• Reproductive and Developmental Biology
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• v.38 no.4
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• pp.171-177
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• 2014

L-Carnitine is an antioxidant for the transport of fatty acids in mitochondria and breakdown of lipids for metabolic energy. Some studies have suggested that carnitine improves sperm motility in mammals. The objective of this study was to investigate the effect of L-carnitine on the characteristics in fresh semen of miniature pigs. The collected fresh semen was stored in modena B medium with L-carnitine (0, 1.0, 2.0, and 4.0 mg/ml) for 10 days at $18^{\circ}C$. The semen quality of viability, acrosome reaction and mitochondria integrity was analyzed on 0, 3, 7, and 10 day of semen storage. The percentages of live and dying sperm were not different among treatment groups with different concentrations of L-carnitine during the storage period. In acrosome reaction analysis, when the sperm stored for 7 day, the percentages of live sperm with acrosome reaction were significantly (p<0.05) lower in 1 ($9.0{\pm}0.9%$), 2 ($7.6{\pm}0.2%$) or 4 mg/ml ($7.9{\pm}0.8%$) L-carnitine-treated groups than the control group (0 mg/ml L-carnitine) ($11.12{\pm}0.2%$). However, there were no difference in percentages of live sperm with acrosome reaction for 3 and 10 days of storage with each concentrations of L-carnitine. When sperm was stored for 3 and 10 days, the percentages of live sperm with mitochondria integrity were significantly higher in 2 mg/ml of L-carnitine-treated group than control group (p<0.05). In conclusion, the L-carnitine has a positive effect on acrosome reaction and mitochondria integrity in liquid state of fresh semen in miniature pigs.

• Journal of Animal Reproduction and Biotechnology
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• v.36 no.2
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• pp.91-98
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• 2021

The objective of this study was to establish a selection process for high quality sperm in bovine semen using sperm separation by magnetic activation (MACS). For this, semen from 21 Nellore bulls was collected using an artificial vagina. To guarantee the presence of pathologies in the ejaculate, animals previously declassified in four consecutive spermiogram were used. Semen was analyzed in five statuses: (1) fresh semen (fresh); (2) density gradient centrifugation (DGC), percoll column; (3) non-apoptotic fraction after separation by MACS (MAC); (4) apoptotic fraction from the separation (MACPOOR); and (5) MAC followed by DGC (MACDGC). Using a computerized analysis system (CASA), motility was measured. The sperm morphology was evaluated by phase contrast, and the supravital test was completed with eosin/nigrosin staining. For DGC, 20 × 10⁶ cells were used in a gradient of 90% and 45% percoll. MACS used 10 × 10⁶ cells with 20 μL of nanoparticles attached to annexin V, and filtered through the MiniMACS magnetic separation column. Membrane integrity was assessed with SYBR-14/IP and mitochondrial potential with JC-1 by flow cytometry. Processing sperm by MACDGC, was more effective in obtaining samples with high quality sperm, verified by the total of abnormalities in the samples: 35.04 ± 2.29%, 21.50 ± 1.47%, 17.30 ± 1.10%, 30.68 ± 1.94% and 10.50 ± 1.46%, respectively for fresh, DGC, MAC, MACPOOR, and MACDGC. The subpopulation of non-apoptotic sperm had a high number of live cells (82.65%), membrane integrity (56.60%) and mitochondrial potential (83.98%) (p < 0.05). These findings suggest that this nanotechnological method, that uses nanoparticles, is efficient in the production of high-quality semen samples for assisted reproduction procedures in cattle.

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.227-227
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• 2004

The purpose of this study was to investigate the effect of taurine on sperm characteristics and gene expressions(bax and Gpx) in fresh boar semen during in vitro storage. The motility of spermatozoa in Modena, Modana plus taurine 25 mM, Modana plus taurine 50 mM, Modana plus taurine 75 mM and Modana plus taurine 100 mM were 63.1%, 65.1%, 65.3%, 82.5% and 80.8%, respectively. (omitted)

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.92-92
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• 2002

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 evaluate semen quality after cryopreservation and to evaluate the Pregnancy rate after insemination (AI). Fifty infertilie dogs (age 2∼3 years) were selected for the study and divided into three different estrus induction treatment groups. Group 1: dogs (n=15) were given clomifene (0.1 mg/kg) orally for five days at 12 hr intervals. Group 2: dogs (n=15) were given bromocriptine (50 $\mu\textrm{g}$/kg) orally for five days at 12 hr intervals, followed by single injection intravenously of 500 IU GnRH (Group 3, n=20) when pro-estrus occurred. 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. The ejaculated semen to freeze 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 cm above the surface of liquid nitrogen (LN$_2$) for 23 min. The motility of frozen-thawed spermatozoa was assessed by phase-contrast microscopy. To assess their viability and acrosome content, spermatozoa were stained with a vital stain and Fluorescence conjugated lectin Pisum Savitum Agglutinin (FITC/PAS), respectively. Pregnancy was confirmed by ultrasonograpy on day 25, 35 and 55 post insemination. 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, the pregnancy rate of dogs treated with a combination of GnRH and bromocriptine was more effective than use of clomifene or bromocriptine only. In addition, frozen-thawed semen can be used successfully far artificial insemination in dog.

• Journal of Embryo Transfer
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• v.27 no.1
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• pp.29-35
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• 2012

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 $in-vitro$ fertilization rate, and the artificial insemination pregnancy rate. The diluents used in this experiment were Triladyl$^{(R)}$ 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 ($p$ <0.05). The mean volume of semen collected from the 15-month-old bulls (2.3 ml) was significantly lower ($p$ <0.05) than that from the 36-month-old bulls (5.0 ml). The mean semen concentration was similar for the 15-month-old ($2.1{\times}10^9$ spermatozoa/ml) and 36-month-old ($1.8{\times}10^9$ 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 ($p$ <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 $in-vitro$ 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 $in-vitro$ 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.

• Animal Bioscience
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• v.37 no.2
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• pp.203-209
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• 2024

Objective: This study aimed to assess the impact of the dilution ratio of Tris diluent, storage at 0℃, and long-distance transportation on the spermatozoa of Simmental cattle. It also validated the feasibility of the regional distribution of fresh semen. Methods: In experiment 1, semen was diluted at four dilution ratios (1:6, 1:9, 1:12, and 1:15) to determine the optimal dilution ratio of Tris diluent. In experiment 2, we assessed sperm viability, progressive motility (objectively assessed by computer-assisted sperm analyzer), and acrosome intactness in Tris dilutions kept at constant 0℃ for 1, 3, 6, 9, and 12 days. We compared them to Tianshan livestock dilutions (Commercial diluent). In experiment 3, semen was diluted using Tris diluent, and sperm quality was measured before and after long-distance transport. Artificial insemination of 177 Simmental heifers compared to 156 using Tianshan Livestock dilution. Results: The outcomes demonstrated that 1:9 was the ideal Tris diluent dilution ratio. The sperm viability, Progressive Motility, and acrosome integrity of both Tris and Tianshan dilutions preserved at 0℃ gradually decreased over time. sperm viability was above 50% for both dilutions on d 9, with a flat rate of decline. The decrease in acrosome integrity rate was faster for Tianshan livestock dilutions than for Tris dilutions when stored at 0℃ for 1 to 6 days. There was no significant difference (p>0.05) in sperm viability between semen preserved in Tris diluent after long-distance transportation and semen preserved in resting condition. The conception rates for Tris dilution and Tianshan livestock dilution were 49.15% and 46.15% respectively, with no significant difference (p>0.05). Conclusion: This shows that Tris diluent is a good long-term protectant. It has been observed that fresh semen can be successfully preserved for long-distance transport when stored under 0℃ conditions. Additionally, it is feasible to distribute semen regionally.

• Korean Journal of Animal Reproduction
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• v.5 no.2
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• pp.43-48
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• 1981

This experiment was carried out with the purpose to get some information about the properties of Simmental bull semen during summer season. The results obtained were summarized as follows: 1. Semen volume per ejaculation, sperm concentration and sperm viability were averaged 5.16ml, 6.6billion and 65%, respectively. 2. Percentage of motile sperm after dilution in skmmilk solution and trisbuffer for 5 days were 34.16% and 35.0%, respectively. 3. Viability of spermatozoa frozen in skimmilk extender and trisbuffer for 5 days were 20. 83% and 25.66%, respectively. 4. Percentage of live sperm, MRT and pH value were 71.8∼72.1%, 8.40∼8.21 minutes and 6.78, respectively. 5. Diluted semen showed strong resistance to the cold shock than that of fresh semen. 6. Rscovery of sperm motility after freezing for 24 hours was relatively weak.

• Korean Journal of Poultry Science
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• v.44 no.1
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• pp.59-65
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• 2017

This study examined factors affecting the analysis of motility of chicken semen. The viability of spermatozoa was estimated using varying dilution ratios and supplementation with BSA or fatty acid free (FAF)-BSA as protein sources in semen diluent. Fresh semen was examined after preparing dilutions in beltsvile poultry semen extender (BPSE) of 1/8, 1/16 and 1/32 at $25^{\circ}C$. The motility of incubated semen at each dilution was observed at 3 min (89.9%, 69.9% and 53.2%), 30 min (86.7%, 71.4% and 51.7%), 1 h (89.5%, 74.0% and 53.5%) and 3 h (78.5%, 66.5% and 45.7%), respectively. The addition of BSA or FAF-BSA to BPSE diluent significantly increased the viability of semen in 1/32 dilution with results of 53.2% (control), 84.8% (BSA) and 92.9% (FAF-BSA) (p<0.05). This phenomenon was also observed in the dilution of frozen semen, where FAF-BSA treatment increased the viability of thawed semen from 17.6% to 34.0% in a 1/8 dilution (p<0.05). When the protein sources were used in the dilution, the survival rates of diluted chicken semen were also increased with time lapse. These results show that FAF-BSA may act to protect chicken semen and is suitable as a basic component of chicken semen diluent for the method of analyzing rooster semen after freezing.