• Asian-Australasian Journal of Animal Sciences
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• v.23 no.7
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• pp.873-879
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• 2010

We investigated the effect of various concentrations of taurine during in vitro fertilization (IVF) on the embryonic development up to the blastocyst stage of bovine oocytes fertilized with three different Japanese Black bulls (Bull A, B and C). In vitro matured oocytes were fertilized with various concentrations of taurine (0, 1, 10, 50 and 100 mM) in the presence of 2.5 or 5.0 mM caffeine plus $25{\mu}g$/ml heparin (CH) for 6 hr or $100{\mu}g$/ml heparin (H) for $24{\pm}2$ h. After IVF, the cleavage rates from the 2 to 16 cell stage determined at 3 days and the development rates up to the blastocyst stage determined at 7-8 days from the onset of IVF were assessed. Although the cleavage rates for the taurine concentration groups were not significantly increased in any of the three bulls in the CH groups, the development rates up to the blastocyst stage of the 50 mM taurine group of Bulls A and B, and of the 1 to 50 mM groups of Bull C were increased (p<0.05) compared to those of the control (0 mM taurine) groups. On the other hand, none of the bulls in the H groups showed any significant increase either in the cleavage rates or blastocyst formation rates in any taurine concentrations groups compared with those of the control groups. These results indicate that the addition of 50 mM taurine to a fertilization medium containing caffeine and heparin may stimulate embryonic development up to the blastocyst stage when fertilized with different bull semen.

• Reproductive and Developmental Biology
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• v.29 no.3
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• pp.141-144
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• 2005

This experiment was to investigate whether the mitochondria function assessment can be used for the prediction of sperm fertility through examining the correlation between mitochondria fluoromicroscopic frequency of frozen-thawed eight Hanwoo bull semen using rhodamine123 (R123) and in vitro embryo development following fertilization. Individual sperm were stained in 5 ${\mu}g/mL$ R123-added calcium-free Sp-TALP for 30 min at 0 h, 6 h, 12 h and 24 h after thawing and examined their mid-piece under an epifluorescence microscope using 495 nm excitation filter (x1,000). Three replications were taken, and at least 300 sperm per individual were examined. When semen samples were separated into two groups (good and poor) by sperm motility and fluorescent frequencies at just after thawing, average fluorescent frequencies were remarkably reduced as time going (0 h; $53.29{\~}72.94\%$, 6 h; $21.40{\~}58.90\%$, 12 h; $8.26{\~}25.93\%$, 24 h; $1.00{\~}13.78\%$, irrespective of selected group, and there were no differences at 6 h or 12 h after thawing between selected groups but indicated significant difference at 24 h after thawing (p<0.05). In vitro fertilization rates in good and poor groups ranging $70.8{\~}77.8\%$ and $52.1{\~}84.5\%$, respectively, were not significantly different. However, in vitro development rates of the same groups ranging $25.7{\~}40.0\%$ and $12.9{\~}1.8\%$, respectively, were significant different (p<0.05). These results demonstrate that mitochondria fluoromicroscopic assessment of frozen-thawed bovine sperm may be used as a criterion to select more fertile sperm.

• Korean Journal of Animal Reproduction
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• v.10 no.1
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• pp.19-26
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• 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
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• v.12 no.3
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• pp.307-313
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• 1997

Experiments were conducted to assess the effect of quality and viability of bovine blastocysts derived from in-vitro culture(IVC) of in vitro matured and fertilized(IVM-IVF) oocytes during their transport 2 hours. Follicular oocytes were collected form ovaries obtained at a slaughterhouse and were cultured for 24 hours in TCM-199. The IVM oocytes were fertilized in vitro with caudal epididymis spermatozoa. Fertilized oocytes were cultured for 7 to 9 days, and embryos that developed to the blastocyst stage were used for the experiment. The blastocysts, packed in straws with storage medium that consisted TCM-199 with HEPES equilibratd in air and supplemented with 10% FCS were transported at 39~(2.0 h). The quality of blastocysts was assessed and ranked as A(excel-lent), B(Good), fair or poor after transportation. The percentages of A and B grade blastocysts after transport duration for < 1 hours(97.7%) were similar to the result from transport duration for 1~2 hours (92.9%) and 2~3 hours(89.6%), but significantly(P<0.05) higher than transpot duration for 3~4 hours(76.3%). The percentages of A and B grade blastocysts after transport duration for two hours from developed blastocyst at 7day(100%) and 8day(85.0%) were higher 9day(96.6%) and >9day (40.0%). And early to expanded blastocyst produced in vitro were transferred to recipient cow by additional embryos at 7 and 8th day after AI. Three of them were pregnant to term and produced four twin calves, and two calves was premature birth. The gestation lengths of male to female and female to female twin were 282 and 281 days, respectively. And birth weight of twin calves were male to female(22.Skg) and female to female twin(20.3Okg), respectively.

• Korean Journal of Veterinary Research
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• v.33 no.4
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• pp.757-762
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• 1993

The ovaries of Korean Native cows or heifers were obtained from an abattoir and kept on 20 to $25^{\circ}C$ and transported to laboratory within 2 hrs. The follicular oocystes were collected from 2~6mm follicles in diameter and classified into 3 grades by the morphology of cumulus cells attached. The oocytes were matured in vitro(IVM) for 24 hrs. in TCM-199 supplemented with $23{\mu}g/ml$ FSH, $10{\mu}g/ml$ LH, $1{\mu}g/ml$ estradio-17 ${\beta}$ and granulosa cells at $39^{\circ}C$ under 5% $CO_2$ in air. They were fertilized in vitro(IVF) by incubation for 12 hrs. of epididymal spermatozoa pretreated with heparin, and then the zygotes were co-cultured in vitro(IVC) with oviductal epithelial cells for 7 to 9 days. Assessment of maturation revealed that 93.0%(147/158) of grade I oocytes had expanded of cumulus cells, which was higher(p<0.05) than the 79.4%(85/107) of grade II oocytes. Compared to epididymal sperm(32.9%), the insemination with frozen and thawed sperm resulted in slightly lower(20.5%), but not significant, development to morulae and blastocysts from grade I oocytes. Co-culture of bovine IVF embryos with oviductal epithelial cells improved the development to transferable embryos significantly(38.1%), compared to co-culture with granulosa cells(20.0%). When VF bovine embryos were vitrified at blastocyst, the post-thaw survival rate was obtained higher resulf for 1 min. equilibration time(82.6%) or 2 min.(73.9%) than 3 min.(18.2%) in EFS solution.

• 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.

• 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.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 1997.05a
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• pp.27-34
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• 1997

Bovine oocytes surrounded with compact cumulus cells were cultured for 20 to 22 hours($38^{\circ}C$, 5% $CO_2$) in modified TCM-199 medium supplemented with 5% superovulated cow serum(SCS) and inseminated by in vitro capacitated spermatozoa. Day 7 to 8 embryos were equilibrated for 10 minutes in 1.3M methyl cellosolve(MC) <1.1M diethylene glycol(DEG), 1.8M ethylene glycol(EG), 1.6M propylene glycol(PG) and 1.1 M 1,3-butylene glycol(BG) solutions. They were then loaded into 0.25ml straws, placed into an alcohol bath freezer at $0^{\circ}C$, cooled from $0^{\circ}C$ to $-6^{\circ}C$ at $-1^{\circ}C$/minute, seeded, held for 10 minutes, and stored in liquid nitrogen. After thawing in $30^{\circ}C$ water, the embryos wee rehydrated in TCM-199 medium and then cultured for 48 hours in TCM-199 plus 5% SCS. Embryos were considered viable if they progressed to later developmental stages with a good morphology. Some of the embryos frozen in each cryoprotectant were thawed and transferred non-surgically without removing the cryoprotectant. Hatched embryos survived freezing and one-step dilution as follows : EG(50.0%), MC(53.6%), DEG(56.9%), PG(58.0%) and BG(11.5%). The survival rate of embryos cooled at -0.3^{\circ}C$ vs. $-0.5^{\circ}C$/minute was not significantly different(P<0.05), however, blastocysts hatched most often (P<0.01) in vitro when cooled at a rate of $0.3^{\circ}C$/minute(64.6%), 31/48) than at $-0.5^{\circ}C$/minute(22.6%, 12/53). Pregnancy rates resulting from embryos frozen in the different cryoprotectants were as follows : MC(48%, 10/21); DEG(30%, 3/10); EG(74%, 20/27); and PG(40%, 4/10). These results indicate that MC, DEG, EG and PG have utility as cryoprotectants for the freezing and thawing of IVF Bovine embryos.

• Journal of Embryo Transfer
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• v.11 no.3
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• pp.249-258
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• 1996

To improve the efficiency of in vitro production of embryos with follicular oocytes in Korean Native cows, the recovery rates, in vitro maturation, fertilization and development, and the time required for collecting and processing oocytes by aspiration with or without slicing were evaluated comparatively. The ovaries were obtained from a local abattoir and placed in physiological saline at 25~28$^{\circ}C$ and brought to the laboratory within 3 hrs. The oocytes were collected by aspiration of follicles(2~6mm) with or without slicing ovaries after aspiration, and classified into Grade I, Grade II, Denuded, Expanded oocytes by the morphology of cumulus cells attached and the homogeneity of cytoplasmic granules. Also the time required for each step of collecting and processing oocytes were measured. The cumulus cells were removed in some Grade I oocytes to measure their size and nuclear configuration before and after in vitro maturation. The Grade I oocytes were matured in vitro(IVM) for 24 hrs. in TGM-199 supplemented with 35$\mu$g /ml FSH, 10$\mu$g /ml LH, 1 $\mu$g /ml at 39$^{\circ}C$ under 5% C02 in air. They were fertilized in vitro(IVF) by epididymal spermatozoa treated with heparin for 24hrs. and then the zygotes were cocultured in vitro (IVC) with bovine oviductal epithelial cells for 10 days. The results obtained were as follows: The number of oocytes recovered per ovary was averaged 6.6 by aspiration and 11.2 by slicing post aspiration, which summed to 17.8. The number of Grade I oocytes recovered per ovary was averaged 3.1 by aspiration and 3.6 by slicing, which summed to 6.7. The percentage of Grade I to total oocytes recovered was significantly(P<0.05) higher as 48.0 % in aspiration than 31.6% in slicing post aspiration. The time requlred for recovering a Grade I oocyte by aspiration and slicing was 1.1 and 2.5 min, respectively. The mean diameter of Grade I oocytes by aspiration and slicing was similar as 148.7 and 151.5$\mu$m, respectively. The percentage of Metaphase II stage oocytes after IVM for 24 hours was significantly (P

• Journal of Embryo Transfer
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• v.13 no.3
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• pp.227-233
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• 1998

The aim of these experiments was to investigate the effects of oviduct epithelial cells on bovine in vitro fertilization. Oviduct epithelial cell monolayers (OEC) on the 4-well dish were prepared according to general procedures. Monolayers were formed within 5days. The medium for OEC culture (TCM199 with 10% FBS) was replaced with IVF-TALP 2h before each experiment. Macromolecules/proteins from oviductal conditioned medium (OM) were recovered by ultrafiltration, which desalted and concentrated macromolecules greater than 5kDa, and this OM were added to W medium (experiment 1). The cleavage rate in OM+OEC group was significantly higher than in OM group (p〈0.01). In this experiment 2, oocytes were inseminated on OEC with sperm which had been pre-incubated with OEC for 0 or 4h before insemination. In this experiment, oocytes were exposed to sperm only 8 h for clarifying the effect. After insemination, oocytes were cultured in CRlaa. At 42 h post insemination, oocytes were denuded and examined for evidence of cleavage. The cleavage rates of oocytes which were inseminated with OEC treated sperm for 4 h were significantly higher than those of the other group (p〈0.01). In conclusion, sperm released from OEC have more fertilizing ability than those before attachment.