• Korean Journal of Animal Reproduction
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• v.26 no.3
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• pp.205-214
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• 2002

There are many methods to introduce exogenous DNA into embryo to produce transgenic animals. Exogenous gene can be integrated into oocyte by sperm vector. In this study, sperm was used as a vector for a transgene, which is encoding enhanced green fluorescent protein (EGFP). The objective of this study was to investigate the expression of exogenous gene in bovine embryos after injection of spermatozoa cocultured with EGFP DNA fragment. Spermatozoa were plunged into liquid nitrogen and thawed several times or shook in 0.2% Triton X-100 to remove sperm membrane followed by DTT treatment. The injected oocytes were co-cultured with vero cells in CR1aa, and expression of EGFP gene was observed under fluorescent microscope. Blastocyst formation rates of oocytes injected with sperm treated with DTT, DTT-freezing or DTT-Triton X-100 were 34.7, 39.4 and 31.9%, respectively. The rates of EGFP expression in oocytes injected with 54 ng DNA after DTT-treated, DTT-freezing and DTT-Triton X-100-treated sperm were 0, 19.1 and 13.9%. On the other hands, expression rate of oocytes injected with sperm cocultured with 13.5, 27 and 63.5 ng of EFGP DNA were 6.7, 9.0 and 5.1%, respectively. When intact sperm was mixed with 63.5 ng/${mu}ell$ EGFP DNA fragment, and then electroporated before injection, the expression rate of injected oocyte was 2%. Unexpectedly, electro-poration could not increase the expression rate. These results suggest that sperm can be used as a transgene vector, even if the efficiency was low (19.1%).

• Korean Journal of Veterinary Research
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• v.39 no.1
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• pp.189-203
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• 1999

To determine sex of bovine embryos using hamster histocompatibility Y(H-Y) antibodies, bovine compact morulae were incubated for 6 hours in TCM199 supplemented with 10% hamster H-Y antiserum and the embryos with developmental arrest were diagnosed as male embryos, while the embryos showing development during the incubation as female embryos. This presumptive embryo sexing was confirmed by polymerase chain reaction(PCR)method. 1. In the result of hamster sperm cytotoxicity test to measure H-Y antibody titer, the rate of dead sperm was considerably lower in H-Y antiserum absorbed with hamster male splenocytes than in H-Y antiserum absorbed with hamster female splenocytes or H-Y antiserum unabsorbed with splenocytes(p<0.01). 2. The rate of oocytes fertilized in vitro and the rate of blastocysts of the fertilized oocytes were 58.5% and 32.4%, respectively. The rate of blastocysts on day 8 was 15.9%, denoting the highest rate during whole culture period posterior to in vitro fertilization (IVF). 3. The bovine 16 cell and compact morulae embryos incubated in the medium supplemented with hamster H-Y antibodies showed 37.1% and 48.9% of developmental arrest which were diagnosed as male, respectively, and rates of redeveloped embryos from the arrested were 24.1% in 16 cell and 44.3% in compact morulae embryos, respectively, denoting higher rate of sex determination and rate of redevelopment in compact morulae than 16 cell embryos. 4. Bovine compact morulae of Korean cattle and Holstein were treated with hamster H-Y antibodies for sex determination and the rates of developmental arrest(diagnosed as male) were 48.4% for Korean cattle and 47.9% for Holstein, respectively. The rates of redeveloped embryos to blastocyst after treatment were 42.6% for Korean cattle and 41.8% for Holstein, respectively, showing no significant differences of sex determination and redevelopment between both breed. 5. The sex determination of bovine embryos(Korean cattle and Holstein) using hamster H-Y antibodies was diagnosed by PCR for confirmation, denoting the rates of 86.1% for Korean cattle and 85.9% for Holstein male embryos, respectively, and the rates of 91.9% for Korean cattle and 90.1% for Holstein female embryos, respectively, with no significant differences of sex determination between both breed. These results indicated that hamster H-Y antibodies can be usable for sex determination of bovine embryos of Korean cattle and Holstein, the viability of bovine embryos was sustained while being cultured in the medium supplemented with hamster H-Y antibodies of appropriate titer and sex determination of bovine embryos by PCR can be feasible for confirmation.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.1
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• pp.58-64
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• 2020

This present study was conducted to investigate protective effect of discontinuous Percoll gradient containing alpha-linolenic acid (ALA) before freezing process on viability, acrosome damage, mitochondrial activity, and oxidative stress of frozen-thawed boar spermatozoa. The separation of spermatozoa by discontinuous Percoll gradient was performed by different concentration of Percoll solution (45/90%) containing ALA combined with bovine serum albumin (BSA), and collected sperm in each Percoll layer was cryopreserved. To evaluate viability, acrosome damage, mitochondrial activity, and reactive oxygen species (ROS) level of frozen-thawed sperm, flow cytometry was used. Morphological abnormalities were observed under light microscope. In results, viability of sperm from 90% Percoll layer was higher than control and 45% Percoll group (p < 0.05). Separated sperm in 90% Percoll layer had lower acrosome damage and morphological abnormalities than control as well as viability, whereas 45% Percoll group was higher (p < 0.05). Similar with acrosome damage and abnormalities, mitochondrial activity was slightly enhanced and the population of live sperm with high ROS level was decreased by 90% Percoll separation, however, there was no significant difference. Supplementation of 3 ng/mL ALA into Percoll solution increased sperm viability and decreased population of live sperm with high ROS compared to control (p < 0.05). In conclusion, discontinuous Percoll gradient before freezing process could improve efficiency of cryopreservation of boar sperm through selection of sperm with high freezing resistance, and supplement of ALA during Percoll gradient might contribute suppression of ROS generation via stabilizing of plasma membrane during cryopreservation.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.6-6
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• 2001

Oocyte freezing has become a prevalent source for related reproductive technologies. This study was carried out to evaluate viability of post-thawed bovine oocyte injected DTT-treated sperm following by two different activation stimuli (Group 1, 5 M ionomycin, 5 min ＋ CR1aa, 3 h . 1.9 mM dimetylaminopurine (DMP), 3 h; group 2, ionomycin ＋ 10 $\mu\textrm{g}$/$m\ell$ cycloheximide(CHX), 5h). The techniques of ultra-rapid freezing used in this study were essentially similar to those of described by Vajta et al (Theriogenology 1999; 52:939-948), Denuded oocytes at 22 h of culture were exposed to cryoprotectant (3.2 M Ethylene glycol, 2.36 M DMSO, 0.6 M sucrose), and followed by freezing in electron microscopic grid. After thawing the oocytes were transferred back into the drop of maturation medium and cultured for additional 2 h before being subjected to ICSI. All eggs were then cultured in CRlaa medium, and transferred into M199+10% FCS on day 4. The culture was maintained until day 9. In Experiment 1, frozen-ICSI eggs were compared on development into blastocyst to those of unfrozen and IVF control. Those eggs were activated with the method of group 2. A higher proportion of unfrozen-ICSI and IVF eggs developed into cleavage and blastocysts than of frozen-ICSI eggs (65% and 13%; 71% and 23% vs. 39% and 8%; P<0.05). In Experiment 2, development and ploidy of embryos made from group 1 were compared to those from group 2. Between groups there did not differ on the rates of development, however, chromosomal abnormality in group 1 was significantly higher than in group 2 (49% vs. 30%; P<0.05). The present result suggests that frozen bovine oocytes can be used for ICSI.

• Reproductive and Developmental Biology
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• v.29 no.4
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• pp.235-239
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• 2005

This study was conducted to test whether the transgenic cattle pass the transgene to their progeny through germ cells, and whether the transgene is expressed in the mammary gland of ransgenic cows. Two male ransgenic calves were born from IVF-derived embryos injected with bovine $\beta-casein/human$ lactoferrin fusion gene and then grew up to be reproducible. Semen was collected from a transgenic bull after 18 mon of age and then frozen. Bovine oocytes matured in vitro were fertilized with spermatozoa of the transgenic bull and cultured in $50\;{\mu}L$ drops of CRlaa medium supplemented with 3 mg/mL BSA. After 48 h of culture, cleaved embryos were determined for the presence of transgenes by DNA polymerase chain reaction (PCR). Proportion of transgene positives among bovine embryos fertilized with sperm of the transgenic bull was $20.9\%$ (28/134). One of transgenic bulls did not produce transgenic sperm. Out of 34 calves produced from recipient heifers inseminated with semen of the other bull, 3 $(8.8\%)$ were transgenic animals (2 females and 1 male). Thus, one transgenic bull showed a low transmission frequency below Mendelian levels in both the IVF-derived embryos and his progeny. It was demonstrated by Southern blot that copy numbers of the transgene in the transgenic progeny enhanced about 1.8 times as compared to those of the founder bull The results demonstrate that the transgenic bull carrying human lactoferrin gene could pass his transgene to the progeny through germ cells, although he is a germ-line mosaic.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.133-133
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• 2003

Recently, sperm has been used as a vector to carry exogenous genes for the production of transgenic animals. However, the success in cattle is low, due to deficiencies in oocyte activation and sperm decondensation caused by high disulphide bond (S=S) content in mature sperm. This study was carried out to develop an effective method for producing transgenic animals with round spermatids (RS). Two methods of embryo production - electric fusion (EC) or intracyto-plasmic injection (IC) and three activation treatments were compared. RS were isolated from bull testes by Percoll density gradients (20, 35, 40, 45 and 90%). Fusion between ooplast and RS was performed with a single DC electric pulse (1.0 KV/cm, 45 sec) in 0.28 M mannitol solution supplemented with 100 M CaCl2 and 100 M MgCl$_2$. (중략)

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.8
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• pp.1127-1133
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• 2008

This study was designed to develop a method of liquid storage of boar sperm at $4^{\circ}C$ by using the modified Beltsville F5 (BF5) diluent with bovine serum albumin (BSA) and N-acetyl-D-glucosamine. Boar sperm were stored in lactose-egg yolk and N-acetyl-D-glucosamine (LEN), BF5 and Golden-Pig liquid 4 (GPL4) diluents at $4^{\circ}C$ for 5 days and were examined for sperm viability, adenosine triphosphate (ATP) and in vitro fertilization (IVF). The percentage of sperm viability in GPL4 diluent was higher than in LEN and BF5 diluent from 1 to 5 days of storage at $4^{\circ}C$. The percentage of sperm viability steadily declined from 1 to 5 days of storage in the three different diluents. Sperm ATP in GPL4 diluent was higher than in LEN and BF5 diluents from 1 to 5 days of storage. Sperm ATP rapidly declined after 5 days of storage in the three different diluents. Porcine oocytes matured in vitro were inseminated with different sperm concentrations of liquid semen stored for 3 days in GPL4 diluent. The percentage of monospermic oocytes did not show any differences from 2.5 to $20{\times}10^5$ sperm/ml. However, the percentage of polyspermic oocytes in the sperm concentration of $2.5{\times}10^5$ sperm/ml was lower than in concentrations of 5, 10 and $20{\times}10^5$5 sperm/ml. The percentage of blastocysts from the cleaved oocytes at $2.5{\times}10^5/ml$ sperm concentration was significantly lower than at 5, 10 and $20{\times}10^5sperm/ml$ concentrations. In conclusion, GPL4 diluent can be stored at $4^{\circ}C$ for 5 days and showed higher sperm viability and sperm ATP concentration compared with LEN and BF5 diluents. Also, we found that GPL4 diluent can be used for IVF of porcine oocytes.

• 대한생식의학회:학술대회논문집
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• 2001.11a
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• pp.118.2-118.2
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• 2001

• 대한생식의학회:학술대회논문집
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• 2002.05a
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• pp.86-86
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• 2002

• 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.