• Reproductive and Developmental Biology
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• v.40 no.4
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• pp.33-37
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• 2016

This study was conducted to evaluate effect of ${\alpha}$-linolenic acid (ALA) and bovine serum albumin (BSA) on viability, acrosome reaction and mitochondrial intact in frozen-thawed boar sperm. The boar semen was collected by gloved-hand method and cryopreserved using freezing extender containing 3 ng/mL ALA and/or $20\;{\mu}g/mL$ BSA. Cryo-preserved boar sperms were thawed in $37^{\circ}C$ water-bath for 45 sec to analysis. Viability, acrosome reaction, and mitochondrial intact were analyzed using flow cytometry. In results, viability of frozen-thawed boar sperm was significantly higher in only ALA+BSA supplement group than control group (p<0.05), whereas there was no difference either in ALA or BSA supplement. However, acrosome reacted sperm in both of live and all sperm population were significantly decreased in all treatment groups than control (p<0.05). Interestingly, mitochondrial intact of boar sperm was enhanced in ALA and ALA+BSA groups compared with control (p<0.05). In this study, we showed that supplementation of ALA and BSA in freezing extender enhanced the sperm viability, mitochondrial intact and decrease acrosomal membrane damage. In conclusion, our findings suggest that quality of frozen-thawed sperm in mammalians could improve by using of ALA and BSA.

• Korean Journal of Animal Reproduction
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• v.14 no.4
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• pp.309-313
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• 1990

This stduy was carried out to investigate the effect of concentration of PC12 and washing of sperm of sperm motility and acrosome reaction, and the effect of sperm incubated in mTALP solution containing PC12 112.5$\mu\textrm{g}$/ml on development of follicular oocytes matured in vitro. The results obtained were as follows. 1. When fresh sperm was once washed and then incubated in mTALP solution containing 0, 75, 112.5 and 225$\mu\textrm{g}$/ml PC12 for 15minutes, 225$\mu\textrm{g}$/ml showed significantly higher percent of acrosome reacted sperm than 0, 75, 112.5 and 225$\mu\textrm{g}$/ml. 2. When once or twice washed fresh sperm was cultured in mTALP containing 112.5$\mu\textrm{g}$/ml PC12 for 15minutes, no-washing showed significantly higher percent of motile sperm than that once- and twice-washing showed significantly higher percent of acrosome reacted seprm than no- and once-washing. 3. When sperm was cultrued in mTALP containing PC12, 112.5$\mu\textrm{g}$/ml for 15minutes and then was cocultured with bovine follicular oocytes matured in vitro, 11.2 to 22.4% of the oocytes were coleaved to more than 2cell stage.

• Asian-Australasian Journal of Animal Sciences
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• v.12 no.1
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• pp.22-27
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• 1999

Bovine oocytes with compact and complete cumulus cells were cultured in 6 groups for up to 24h in TCM199 buffered with 25 mmol/1 HEPES and supplemented with 10% FCS, 1 mg/ml $17{\beta}$-estradiol, 20 IU/ml hCG. Half of the oocytes at each group cultured in the presence of $25{\mu}g/ml$ cycloheximide at different times during maturation (0, 6, 12, 18, 20, 22 h) were fixed at 24 h of maturation to examine the nuclear progression. The rests of them were inseminated with frozen-thawed spermatozoa in medium BO with 10 mg/ml BSA and 10 mg/ml heparin and fixed after additional 18-20 h culture to evaluate the sperm head transformation. When a protein synthesis inhibitor was added at the onset of the maturation, the oocytes were prevented to proceed GVBD. A few of the oocytes (16%) were able to be penetrated and sperm head decondensation was inhibited either. Addition of cycloheximide after 6-12 h of culture resulted in an increasing percentage of GVBCD (more than 80%), but the oocytes became arrested in M-I (69.2%). More than half of the oocytes was penetrated with a decondensing sperm head. Formation of male pronucleus was first obtained at 12 h of culture in the presence of cycloheximide. When cycloheximide was added from 18 h of culture onwards, nuclear progression to M-II was increasingly restored (80.4-85.5%). The proportion of male and female pronuclear formation increased from 17.9% to 46.2%. It is concluded that protein synthesis is necessary not only for GVBD and development from M-I to M-II, but also for sperm head decendensation and male pronuclear formation in bovine oocytes.

• Asian-Australasian Journal of Animal Sciences
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• v.24 no.10
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• pp.1358-1364
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• 2011

The sex-determining region on the Y (SRY) gene is important in mammalian sex determination and differentiation. We report a study of the abundance of SRY gene products in bovine ejaculate. RT-PCR experiments using RNA extracted from bovine spermatozoa with SRY-specific primers yielded a 456 bp product, but the amount of SRY mRNA in sperm was lower than that in the testes (p<0.01). A protein of approximately 27 KDa was detected by western blotting. The SRY transcript was detected in the midpiece of approximately half the spermatozoa by in situ hybridization, and the SRY protein was detected in the heads of half the spermatozoa by immunofluorescence, indicating that SRY mRNA and protein may only be present in Y-bearing spermatozoa. These results suggest that the SRY transcript and protein are present in bovine ejaculated Y-sperm. The roles of the SRY gene in spermatogenesis, sperm motility, and the sperm-oocyte interaction merit further investigation.

• Asian-Australasian Journal of Animal Sciences
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• v.26 no.2
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• pp.178-188
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• 2013

The glycosaminoglycans (GAGs) present in the female reproductive tract promote sperm capacitation. When bovine sperm were exposed to 10 ${\mu}g/ml$ of one of four GAGs (Chondroitin sulfate, CS; Dermatan sulfate, DS; Hyaluronic acid, HA; Heparin, HP) for 5 h, the total motility (TM), straight-line velocity (VSL), and curvilinear velocity (VCL) were higher in the HP- or HA-treated sperm, relative to control and CS- or DS-treated sperm. HP and HA treatments increased the levels of capacitated and acrosome-reacted sperm over time, compared to other treatment groups (p<0.05). In addition, sperm exposed to HP or HA for 1 h before IVF exhibited significantly improved fertilizing ability, as assessed by 2 pronucleus (PN) formation and cleavage rates at d 2. Exposure to these GAGs also enhanced in vitro embryo development rates and embryo quality, and increased the ICM and total blastocyst cell numbers at d 8 after IVF (p<0.05). A real-time PCR analysis showed that the expression levels of pluripotency (Oct 4), cell growth (Glut 5), and anti-apoptosis (Bax inhibitor) genes were significantly higher in embryos derived from HA- or HP-treated sperm than in control or other treatment groups, while pro-apoptotic gene expression (caspase-3) was significantly lower in all GAG treatment groups (p<0.05). These results demonstrated that exposure of bovine sperm to HP or HA positively correlates with in vitro fertilizing ability, in vitro embryo developmental potential, and embryonic gene expression.

• Journal of Embryo Transfer
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• v.14 no.1
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• pp.31-37
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• 1999

The objective of this study was to optimize the selection of sperm sources, optimal culture systems and vitrification method depends on sperm sources. The oocytes were inseminated with either KPN 105, 114, 191, SNU 101, 102, 103 or epididymis and then embryos inseminated were cultured in oviductal cell co-culture or HECM-6 as defined me dium. The blastocysts produced were pooled according to sperm sources as KPN, SNU or epididymis and then vitrified by OPP vitrification method. The results obtained were as follows: 1. The cleavage(86.2 or 84.7%) and development rates to blastocyst (30.6 or 32.0%) were not significantly different between oviductal cell co-culture or HECM-6 culture systems(P<0.05). 2. To determine the optimal sperm sources for using IVF in this system, cleavage rates in KPN 191 and SNU 101 (74.2, 55.8%) were significantly lower rather than those in KPN 105, 114, SNU 102, 103 or epididymis (86.7, 85.1, 89.8, 85.5 or 81.2%), but development rates to blastocyst in KPN 114, SNU 103 or epididymis sperm (30.0, 33.0 or 28.6%) were significantly higher rater than those in KPN 105, 191, SNU 101, 102(21.4, 15.4, 14.9 or 25.4%), respectively (P<0.05). 3. The blastocysts produced were pooled according to sperm sources as KPN, SNU or epididymis and then vitrified by OPP vitrification method. The survival rates were not significantly different among sperm sources (89.6%: 43/48 ; 90.1%: 46/51 ; 83.3% : 20/24). These results obtained indicate that the defined medium, HECM-6, could be use to produce of IVP bovine embryos. Since the frozen semen must be required to maintain of unvariation data in IVP embryo production system, KPN 114 and SNU 103 produced in our laboratory were useful for this purpose. The blastocysts produced by different sperm sources as KPN, SNU or epididymis were vitrified by OPP vitrification method and survived very high rates. The OPP vitrification method could be susceptibility to use of IVP bovine blastocyst embryos.

• Reproductive and Developmental Biology
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• v.37 no.4
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• pp.169-174
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• 2013

Sperm capacitation refers to polymerization of filamentous (F)-actin from globular (G)-actin. While the role of actin-related protein 2/3 (Arp2/3) complex in actin polymerization is well appreciated, the underlying mechanism(s) and its relationship with capacitation are poorly understood. Therefore, to evaluate the potential role of Arp2/3 complex on capacitation, bovine spermatozoa were incubated with multiple doses (1, 10 and $100{\mu}M$) of CK-636, an inhibitor of Arp2/3 complex with heparin. The cellular localization of the Arp2/3 complex in spermatozoa was identified by immunohistochemistry, whereas western blot was also applied to detect the protein tyrosine phosphorylation of sperm proteins. Additionally, sperm motility and kinematic parameters were evaluated using a computer-assisted sperm analysis system. CK-636 resulted in significant changes in the ratio of Arp2/3 complex localization between acrosome and equatorial region of the spermatozoa. Short-term exposure of spermatozoa to $100{\mu}M$ of CK-636 significantly decreased sperm motility, however a non-detectable effect on protein tyrosine phosphorylation was observed during capacitation. On the basis of these results, we propose that Arp2/3 complex is associated with morphological changes during capacitation and compromised sperm motility.

• Development and Reproduction
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• v.23 no.1
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• pp.11-19
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• 2019

The study was conducted to investigate the effects of alpha-linolenic acid (ALA) combined with bovine serum albumin (BSA) or methyl-beta-cyclodextrin (MBCD) on plasma and acrosomal membrane damages, mitochondrial activity, morphological abnormality, motility, and oxidative stress in frozen-thawed boar sperm. In previous our study, 3 ng/mL ALA had been shown protective effect during freezing process of boar sperm. Therefore, we used 3 ng/mL ALA in present study and ALA was combined with same molar ratio of BSA or MBCD (ALA+BSA and ALA+MBCD, respectively). To confirm the effect of two carrier proteins, same volume of BSA and MBCD without ALA were added during cryopreservation. Membrane damage, mitochondrial activity, reactive oxygen species (ROS) and lipid peroxidation (LPO) levels were measured using flow cytometry, and movement of sperm tail as motility parameter and morphological abnormality were observed under light microscope. In results, all of sperm parameters were enhanced by ALA combined with BSA or MBCD compared to control groups (p<0.05). Mitochondrial activity, morphological abnormality, ROS and LPO levels in ALA+BSA or MBCD groups were no significant difference compared with ALA, BSA and MBCD treatment groups. On the other hand, plasma and acrosomal membrane intact, and sperm motility in ALA+MBCD group were higher than single treatment groups (p<0.05), whereas ALA+BSA did not differ. Our findings indicate that carrier proteins such as BSA and MBCD could improve the effect of ALA during cryopreservation of boar sperm, and treatment of ALA with carrier proteins enhance membrane integrity, mitochondrial activity through reduction of ROS-induced LPO.

• Journal of Embryo Transfer
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• v.11 no.3
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• pp.309-316
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• 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 Animal Reproduction
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• v.17 no.3
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• pp.233-242
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• 1993

The effects of in vitro maturation and sperm treatment condition on the in vitro fertilization (IVF) and developmental capacity of bovine oocytes were investigated and the development of embryos was compared under the 2 different co-culture system, with GC or BOEC. The cultured embryo to 16 cell or morula wre transferred into recipients or frozen by 2 different freezing method. The results obtained were summarized as follows; 1. In vitro maturation rates of vovine follicular oocytes cultrued in TCM199 with 10% FCS or ECS were 64.0% and 72.7%, but the case of addition of 10% FCS or ECS to TCM199 co-cultured with granulosa cells were 81.3% and 84.0%, respectively. IVM rate of three TCM199 added to granulosa cells was higher than that of media without granulosa cells. 2. When bovine follicular oocytes were matured in TCM199 with 10% FCS and GC and then fertilized in vitro by sperm treated with caffeine, embryo developments of bovine oocytes co-cultured with BOEC were 38.4% and 51.4%, respectively. But those of bovine oocytes co-cultured with GC were 52.2% by sperm treated with caffeine-heparin. 3. Cleavage rates of bovine oocytes cultured with 10% FCS alone and fertilized in vitro by sperm treated with caffeine-heparin was 33.0%. 4. When bovine follicular oocytes were matured in TCM199 with 10% FCS and GC, embryo developments of bovine ooctyes co-cultured with BOEC of GC were 46.0% and 50.2%, respectively. 5. When bovine follicular oocytes were matured in TCM199 with 10% ECS and GC, embryo developments co-cultured with BOEC or GC were 45.2% and 51.4%, respectively. 6. When Korean Native cow's follicular oocytes matured in TCM199 with 10% FCS and GC, embryo developed co-cultured with BOEC or GC were 45.2% and 51.4%, respectively. 6. When Korean Native cow's follicular oocytes matured in TCM199 with 10% FCS and GC, embryo developments of the bovine oocyte co-cultured with BOEC and GC were 41.8% and 60.1%. But with FCS 10% those of the bovine oocytes co-cultured with BOEC and GC were 42.0% and 48.4%, respectively. 7. When Holstein's follicular oocytes were matured in TCM199 with 10% ECS and GC, embryo developments fo the bovine oocytes co-cultured with BOEC and GC were 50.0% and 57.7%, but with ECS 10% those of the bovine oocytes co-cultured with BOEC and GC were 52.2% and 56.5%, respectively. 8. The viability of frozen-thawed embryos ranged from 60~80% and those of frozen-thawed embryos from vitrification was lower than that from conventional metiod. 9. The selected fresh embryos were transferred nonsurgically to 7 recipients but did not result in pregnancy.