• Journal of Embryo Transfer
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• v.31 no.4
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• pp.355-365
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• 2016

The cryopreservation of sperm has become the subject of research for successful artificial insemination technologies. Antifreeze proteins (AFPs), one of the factors necessary for effective cryopreservation, are derived from certain Antarctic organisms. These proteins decrease the freezing point of water within these organisms to below the temperature of the surrounding seawater to protect the organism from cold shock. Accordingly, a recent study found that AFPs can increase the motility and viability of spermatozoa during cryopreservation. To evaluate this relationship, we performed cryopreservation of boar sperm with AFPs produced in the Arctic yeast Leucosporidium sp. AFP expression system at four concentrations (0, 0.01, 0.1, and $1{\mu}g/ml$) and evaluated motility using computer assisted sperm analysis. DNA damage to boar spermatozoa was measured by the comet assay, and sperm membrane integrity and acrosome integrity were evaluated by flow cytometry. The results showed that motility was positively affected by the addition of AFP at each concentration except $1{\mu}g/ml$ (p<0.001). Although cryopreservation with AFP decreased the viability of the boar sperm using, the tail DNA analyses showed that there was no significant difference between the control and the addition of 0.1 or $0.01{\mu}g/ml$ AFP. In addition, the percentage of live sperm with intact acrosomes showed the least significant difference between the control and $0.1{\mu}g/ml$ AFP (p<0.05), but increased with $1{\mu}g/ml$ AFP (p<0.001). Our results indicate that the addition of AFP during boar sperm cryopreservation can improve viability and acrosome integrity after thawing.

• Molecules and Cells
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• v.26 no.6
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• pp.558-565
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• 2008

Although the fertilizing ability of spermatozoa is greatly reduced after freezing, complete understanding of alterations induced by cryopreservation has not been elucidated. The present study evaluates the effects of cryopreservation on the $Ca^{2+}$ handling of boar spermatozoa using several sperm activators. Intracellular $Ca^{2+}$ signals from single spermatozoa were measured using confocal $Ca^{2+}$ imaging of unfrozen samples and of other spermatozoa after having been frozen. Elevation of the external $K^{2+}$ concentration elicited a three times larger $Ca^{2+}$ increase in fresh spermatozoa than in cryopreserved spermatozoa. Caffeine elicited $Ca^{2+}$ transients with some oscillations in the fresh spermatozoa, but not in the thawed spermatozoa. Depletion of the $Ca^{2+}$ store with thapsigargin induced a rapid rise in $Ca^{2+}$ in the control but generated a smaller increase of $Ca^{2+}$ after thawing. Exposure to progesterone induced a biphasic rise of the $Ca^{2+}$ level in the fresh spermatozoa only. Sperm viability was reduced by cryopreservation. Resting $Ca^{2+}$ levels in fresh and cryopreserved spermatozoa were similar. Longer incubation (2.5 h) of thawed spermatozoa partly recovered the $Ca^{2+}$ response to the interventions. These results suggest that cryopreservation reduces the responsiveness of spermatozoa to depolarization, modulators of the internal $Ca^{2+}$ store and progesterone in terms of the $Ca^{2+}$ signal, thus providing a possible mechanism for reduced fertility observed in cryopreserved boar spermatozoa.

• Journal of Embryo Transfer
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• v.28 no.3
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• pp.265-271
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• 2013

Cryopreservation and in vitro fertilization (IVF) protocols are important in genetic studies and applications to transgenic animals. Various studies about boar sperm cryopreservation have been studied for a long time. Those were about the use of extenders, the choice of sugars, the cooling and warming rates. The factors that influence the boar sperm are the dramatic changes in temperatures, osmotic and toxic stresses, and reactive oxygen species (ROS) generation. Among these factors, ROS generation is the main damage to DNA which is a principal genetic material and the most important for the practical applications. So we wondered whether ROS generation could be reduced. In previous study, monothioglycerol (MTG) was essential for the culture of embryo stem cells. Therefore we added MTG in the freezing extender based on lactose-egg yolk (LEY) with trehalose. For the assessment of the frozen-thawed sperm, we focused onmotility, membrane integrity and DNA damage. First, we used a computer-aided sperm analysis system for overall conditions of sperm such as motility and viability. Then we performed the sperm chromatin structure assay for DNA integrity and hypo-osmotic swelling test for membrane integrity. And our result showed the existence of MTG in the freezing extender caused less damage to DNA and higher motility in frozen-thawed boar sperm. Also we checked a relative antioxidant activity of MTG in modified Modena B extender. We concluded that this reagent can activate sperm mitochondria at MTG $0.2{\mu}M$, contribute to sperm motility and DNA integrity but there was no significant difference on membrane integrity. Also antioxidant activity of MTG in modified Modena B extender was proved.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.11
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• pp.1501-1508
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• 2004

In this study the effect of extenders and temperatures on sperm viability and fertilizing capacity of boar sperm during long-term storage was investigated. Acrosomal integrity, membrane integrity, motility and hypo-osmotic resistance were evaluated by fluorescence and light microscopy. An in vitro fertilization test was performed to assess the fertilizing capacity of stored spermatozoa. The five diluents tested were ranked according to their ability to maintain sperm functional parameters and Zorlesco (ZO) extender with BSA or with PVA instead of BSA produced the best results. Zorlesco extender substituted with PVA (ZO+PVA) was found to maintain motility both at 15 and 20$^{\circ}C$. within 5 days of storage, but the quality of semen stored at 15$^{\circ}C$ decreased thereafter as compared to semen stored at 20$^{\circ}C$ Semen stored at 5$^{\circ}C$ demonstrated rapid loss of motility already within 24 h. Both fertilization and cleavage of semen stored at 20$^{\circ}C$ in ZO substituted with PVA instead of BSA did not change significantly until day 8 of storage. It is therefore concluded that PVA can be used to substitute for BSA and 20$^{\circ}C$ was more suitable than 15$^{\circ}C$ for boar semen storage, and in vitro fertilizing capacity of spermatozoa was maintained for at least 8 days in ZO+PVA at 20$^{\circ}C$.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.10
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• pp.1456-1463
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• 2017

Objective: To investigate the effect of host genetics on gut microbial diversity, we performed a structural survey of the fecal microbiota of four purebred boar pig lines: Duroc, Landrace, Hampshire, and Yorkshire. Methods: The V3-V4 regions of the 16S rRNA genes were amplified and sequenced. Results: A total of 783 operational taxonomic units were shared by all breeds, whereas others were breed-specific. Firmicutes and Bacteroidetes dominated the majority of the fecal microbiota; Clostridia, Bacilli, and Bacteroidia were the major classes. Nine predominant genera were observed in all breeds and eight of them can produce short-chain fatty acids. Some bacteria can secrete cellulase to aid fiber digestion by the host. Butyric, isobutyric, valeric, and isovaleric acid levels were highest in Landrace pigs, whereas acetic and propionic acid were highest in the Hampshire breed. Heatmap was used to revealed breed-specific bacteria. Principal coordinate analysis of fecal bacteria revealed that the Landrace and Yorkshire breeds had high similarity and were clearly separated from the Duroc and Hampshire breeds. Conclusion: Overall, this study is the first time to compare the fecal microbiomes of four breeds of boar pig by high-throughput sequencing and to use Spearman's rank correlation to analyze competition and cooperation among the core bacteria.

• Reproductive and Developmental Biology
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• v.32 no.1
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• pp.59-64
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• 2008

The objective of this study was to estimate modification of semen quality during storage. Liquid boar semen samples extended in Beltsville Thawing Solution were stored at $17^{\circ}C$ up to 5 days. While % motility and linearity significantly decreased eon day 3 in extender, the qualitative motility patterns were maintained satisfactorily. Also the storage of boar semen up to 5 days before insemination did not significantly changed the acrosome intactness. However, acrosome changed sperm significantly increased and capacitated sperm significantly decreased from day 4. No significant modifications in acrosome integrity were showed during sperm storage; these results suggest that liquid boar semen may keep the quality in extender for 3 days.

• Korean Journal of Animal Reproduction
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• v.21 no.1
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• pp.19-23
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• 1997

This present study was carried out to examine the effect of maturation media and liquid boar semen on in vitro maturation and feritilization of pig oocytes. The results obtained were as follows : When the oocytes were cultured for 36∼42 hours in mTCM-199, Waymouth MB 725/1 and mTLP-PVA medium, the maturation rates were 90%, 92% and 88%, respectively. The sperm penetration rates of pig oocyte matured in vitro were 87%(mTCM-199), 90%(Waymouth MB 725/1) and 86%(mTLP-PVA), respectively. The rates of nuclear maturation and fertilization of pig oocytes among three different media did not differ. However, the rate of male pronucleus formation of pig oocytes was significantly higher in pig oocytes matured in Waymouth MB 725/1(91%) than oocytes matured in mTCM-199(66%) and mTLP-PVA(62%) medium (P<0.05). When the collected sperm-rich fraction without diluent was used fro in vitro fertilization in mTCM-199 fertilization medium, the fertilization rate was 87.9%. However, when the liquid boar semen diluted with B tschwiler diluent was used at day 3 and 5 after dilution, the fertilization rate was 40.8% and 0.0%, respectively.

• Reproductive and Developmental Biology
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• v.29 no.4
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• pp.253-257
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• 2005

This study was carried out to evaluate the effects of washing medium, breed and washing temperature of fresh and frozen-thawed boar sperm on mitochondrial activity and membrane integrity by flow cytometry. More than $80\%$ of fresh sperm washed with mTLP-PVA medium at $20^{\circ}C$ exhibited an intact membrane and a functional mitochondrion. With frozen-thawed samples, a large number of sperm showed both damaged membrane $(36.4\~46.9\%)$ and nonfunctional mitochondrion $(55.1\~71.1\%)$ in the mTLP-PVA and BTS washing media at $20^{\circ}C$. There were no breed effects of fresh and frozen-thawed sperm on mitochondrial activity and membrane integrity. The percentages of damaged membrane of fresh and frozen sperm, respectively, were higher at $4^{\circ}C$ washing temperature than at $20^{\circ}C$ washing temperature in the mTLP-PVA medium. We found that washing medium and washing temperature of fresh and frozen-thawed boar sperm were important for the analyses of mitochondrial activity and membrane integrity by flow cytometry.

• Journal of Veterinary Clinics
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• v.21 no.4
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• pp.363-368
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• 2004

To investigate the effects of seeding during freezing procedure on post-thaw viability, motility, and acrosome integrity of boar spermatozoa, semen from 5 Yorkshire boars were collected for this experiment. Raw semen were diluted with Merck I, subsequently added with cooling diluent containing lactose and egg yolk and with freezing diluent containing glycerol. The diluted semen were frozen on the rack in the styrofoam box filled with liquid nitrogen at the distance of 5 cm or I cm above LN2 level. Seeding was performed to only a group of straws frozen at 5 cm away on the surface of LN2. The frozen semen were thawed in $50^{\circ}C$C water and the viability and local motility were analyzed by sperm analysis imaging system. A part of thawed semen was taken for the examination of morphology of apical ridge of the acrosome to compare with the effect of seeding between the seeding-treated and non treated groups. I. Post-thaw viability was considerably higher in seeding-treated sperm than non-seeding group (p<0.01), however, no difference of local motility was obtained among the groups. 2. At three hours after thawing, viability was also higher in seeding-treated group than non-treated group (p<0.05), along with no difference of motility among the groups. 3. Higher normal acrosome integrity was obtained in the seeding-treated sperm than non-treated groups (p<0.01). 4. Between non-seeded groups, higher normal acrosome integrity was obtained in the sperm group frozen at 5cm upper on the surface of LN2 than that frozen at 1cm away (p<0.01). These results indicated that seeding treatment during freezing boar spermatozoa was beneficial to post-thaw viability and normal acrosome integrity.

• Korean Journal of Veterinary Service
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• v.34 no.2
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• pp.111-116
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• 2011

Artificial insemination (AI) of swine is a very useful reproductive tool and that offers convenience in the Korean swine industry. Since many viruses have been reported to be excreted through boar semen, we investigated the presence of antibodies and antigens against viruses causing reproductive failure in semen of boar in 349 semen samples collected from six Korean AI centers. Viral antigens were detected by polymerase chain reaction (PCR) or reverse transcription-PCR predominantly. The results was as follows. The major reproductive failure causing factor was porcine circovirus type 2 (PCV2), followed by porcine reproductive and respiratory syndrome virus (PRRSV) ($X^2$=166.64, P<0.001). PCV2 and PRRSV, Japanese encephalitis virus (JEV), encephalomyocarditis virus (EMCV) was detected in 73 samples (20.9%), 44 samples (12.6%), 4 samples (1.1%), 3 samples (0.9%), respectively and porcine parvovirus in one sample (0.3%) Classical swine fever virus (CSFV), bovine viral diarrhea virus and Aujeszky's disease virus (ADV) were not detected. Enzyme-linked immunosorbent assay was carried out in 111 serum samples from three AI centers. In most pigs, antibodies response was showed prominently in CSFV (105 sera, 94.6%) ($X^2$=82.580, P<0.001), followed by, in PRRSV (100 sera, 90.1%), PCV2 (92 sera, 90.1%), and PPV (8 sera, 82.9%). ADV antibody was not detected. Thus, the experimental results will be used for the base data, with respect to the state of viral stillbirth in general pig farms, as well as AI centers and breeding farms in Korea.