• Korean Journal of Animal Reproduction
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• v.27 no.4
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• pp.339-347
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• 2003

The purpose of this study was to investigate the development of bovine nuclear transfer (NT) embryos cultured in serum-free conditions. Bovine NT embryos cultured in various culture conditions were compared blastocyst development, total cell number and apoptosis using TUNEL assay. In experiment 1, blastocyst rates of NT embryos were significantly higher (P<0.01) in FBS (22.0％) and BSA (26.6％) groups than in PVA (6.3％) group. Total cell number was significantly higher in FBS (78.4$\pm$19.4) and BSA (90.9$\pm$29.1) groups than in PVA group (46.0$\pm$0.0). Apoptotic cell number was significantly fewer in FBS (3.1$\pm$1.4) and BSA (1.7$\pm$1.4) groups than in PVA group (7.0$\pm$20.0) However, all of results were not different between the FBS and BSA group. In experiment 2, blastocyst rates of NT embryos were significantly higher (P<0.05) in fatty acid free-BSA (FAF-BSA) group (26.8％) than in fraction V-BSA group (11.2％). Total cell number were somewhat higher in FAF-BSA group (89.8$\pm$30.7) than in fraction V-BSA group (88.1$\pm$19.3). Apoptotic cell number were somewhat fewer in FAF-BSA (1.7$\pm$1.5) group than in fraction V-BSA group (4.2$\pm$2.9). These findings suggest that serum free condition were effective for the in vitro development of bovine NT embryos. Therefore, we concluded that fatty acid free-BSA has beneficial effect in development bovine NT embryos and can be use as a serum substitute.

• 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.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.19-19
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• 2003

소 난자 체외성숙에 있어 소 태아혈청 (fetal bovine serum: FBS)첨가시 높은 성숙율을 보이며 핵 이식 후 배 발달율에 있어서도 높다. 하지만 소 태아혈청은 hormone, growth factor, vitamine 그리고 다수의 어떤 잘 알려지지 않은 인자를 포함한 복잡한 배지로 알려져 있으며, 수정란 이식 후 태아의 발육중 및 태어난 직후에 발생되는 송아지에서 나타나는 몇몇 비정상적인 현상들의 원인인 것으로 보고되고 있다. 본 실험은 체외 배양시 소 태아혈청과 이의 대체물질로서 BSA 또는 PVA가 첨가된 배양조건에서의 복제 수정란의 배 발달율을 비교함과 동시에 배반포의 세포수 그리고 세포자연사 (Apoptosis)를 각각의 조건에서 비교함으로써 배발달에 미치는 효과를 알아보기 위하여 실시하였다. 핵이식은 소 체세포를 이용하였으며, 핵 이식 후 CR1aa 기본 배양액으로 FBS, BSA, 그리고 PVA를 첨가하여 5% $CO_2$, 5% $O_2$, 39$^{\circ}C$ 조건하에서 7~8일간 배양하였다.

• Applied Biological Chemistry
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• v.42 no.3
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• pp.229-234
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• 1999

The effects of linoleic acid(LA, 18 : 2) and/or bovine serum albumin(BSA) on the lipid metabolism in human hepatoma cell line HepG2 cells were evaluated. HepG2 cells were cultured in basal Dulbecco's modified Eagle's(DME) medium(Basal medium), DME medium containing 0.2 mM LA(LA medium), or DME medium containing both 0.2 mM LA and 0.2-1.0% BSA(LA+BSA medium). $[^{14}C]Acetate(0.3\;{\mu}Ci/ml\;medium)$ was added as a radioactive lipid precursor and the cells were incubated for 6 hours. An addition of LA to basal medium resulted in a decrease in the incorporation of $[^{14}C]acetate$ into total cholesterol fraction. In contrast, an addition of BSA to LA-containing medium tended to increase the incorporation of $[^{14}C]acetate$ into total cholesterol. The alteration of cholesterol metabolism in HepG2 cells incubated in LA+BSA medium was attributed by an increase in the incorporation of $[^{14}C]acetate$ into free cholesterol, but not cholesteryl ester fraction. In addition, the secretion of cholesterol was increased by LA+BSA medium, suggesting that BSA stimulates cholesterol secretion. No significant change in the incorporation of $[^{14}C]acetate$ into cellular total lipids was observed among the experimental groups. However, an increased incorporation of $[^{14}C]-labelled$ fatty acid into cellular triacylglycerol and decreased incorporation into phospholipid were observed in cells incubated with LA+BSA medium as compared to those of LA medium. The secretions of $[^{14}C]-labelled$ triacylglycerol, phospholipid, and free fatty acid were also stimulated in HepG2 cells incubated with LA+BSA medium. In conclusion, the present study suggests that in human hepatocytes, LA and BSA influence lipid metabolism, and BSA enhances the secretion of lipids.

• Asian-Australasian Journal of Animal Sciences
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• v.12 no.2
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• pp.169-173
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• 1999

Studies were made to evaluate the specific and combined effects of different fatty acids on the in vitro development of 8-cell rat embryo in culture media with and without carbohydrate substrate. Palmitic, oleic, linoleic and arachidonic acids were added singly and in combination to media which contained fatty acid-free BSA. Cell numbers in blastocysts cultured in the media were counted and compared with cell numbers in blastocysts at the corresponding stage collected from the uterus. Oleic, linoleic and arachidonic acids promoted the rat embryo development from 8-cell to the blastocysts. especially in the absence of carbohydrate substrates. Among these three, oleic acid was the most effective but embryo development was not accelerated by the addition of palmitic acid in either the presence or the absence of carbohydrate substrates. Addition of the mixture of four fatty acids was more effective for rat embryo development than single treatment with any of fatty acids tested. Cell numbers per blastocyst in the presence and absence of carbohydrate substrate were similar, and did not differ from those for blastocysts obtained from the uterus.

• Asian-Australasian Journal of Animal Sciences
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• v.9 no.5
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• pp.583-590
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• 1996

The purpose of this study was to evaluate some factors in the bovine embryonic development from one-cell to blastocyst using modified synthetic oviduct fluid medium (mSOFM), after maturation and in vitro fertilization of the oocytes. The embryonic development to the blastocyst stage was assessed at 7-10 days after in vitro fertilization, and the total cells in the blastocysts were counted by staining nuclei with fluorochrome. Some commercial calf sera (CS) and a superovulated cow serum had different effects on the embryonic development to the blastocyst stage (8.6-21.4%), dependent upon their product lots, although the development might not be affected at least by serum progesterone levels. ${\beta}$-Mercaptoethanol (${\beta}$-ME) supplemented into mSOFM was effective to the embryonic development (27.8%), as well as the co-culture system with cumulus cells (19.5%). In a serum- and feeder cell-free culture using mSOFM containing several growth factors and ${\beta}$-ME instead of CS plus co-cultured cumulus cells, bovine serum albumin (BSA, fraction V), but not polyvinyl alcohol (PVA), was highly effective in embryonic development to the blastocyst stage, almost comparable to CS in the serum-contained culture (CS, BSA and PVA; 27.8, 19.5 and 5.7%, respectively). However, fatty acid free BSA rather reduced the number of developed blastocysts, compared with fraction V BSA (7.3 vs 29.4%). In the serum- and feeder cell-free culture, supplement of glucose to the medium (final 2.0 mM) stimulated the cell proliferation of developing embryos 120 hr after in vitro fertilization. These results indicated that a serum-free medium supplemented with ${\beta}$-ME could successfully support the development of bovine one-cell embryos to the blastocyst stage. Moreover, supplement of glucose and fatty acids to the medium might support preferably the development and cell proliferation of embryos.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.3
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• pp.414-421
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• 2014

This study was conducted to determine the effects of saturated long-chain fatty acids (LCFA) on cell proliferation and triacylglycerol (TAG) content, as well as mRNA expression of ${\alpha}s1$-casein (CSN1S1) and genes associated with lipid and protein synthesis in bovine mammary epithelial cells (BMECs). Primary cells were isolated from the mammary glands of Holstein dairy cows, and were passaged twice. Then cells were cultured with different levels of palmitate or stearate (0, 200, 300, 400, 500, and 600 ${\mu}M$) for 48 h and fetal bovine serum in the culture solution was replaced with fatty acid-free BSA (1 g/L). The results showed that cell proliferation tended to be increased quadratically with increasing addition of stearate. Treatments with palmitate or stearate induced an increase in TAG contents at 0 to 600 ${\mu}M$ in a concentration-dependent manner, and the addition of 600 ${\mu}M$ was less effective in improving TAG accumulation. The expression of acetyl-coenzyme A carboxylase alpha, fatty acid synthase and fatty acid-binding protein 3 was inhibited when palmitate or stearate were added in culture medium, whereas cluster of differentiation 36 and CSN1S1 mRNA abundance was increased in a concentration-dependent manner. The mRNA expressions of peroxisome proliferator-activated receptor gamma, mammalian target of rapamycin and signal transducer and activator of transcription 5 with palmitate or stearate had no significant differences relative to the control. These results implied that certain concentrations of saturated LCFA could stimulate cell proliferation and the accumulation of TAG, whereas a reduction may occur with the addition of an overdose of saturated LCFA. Saturated LCFA could up-regulate CSN1S1 mRNA abundance, but further studies are necessary to elucidate the mechanism for regulating milk fat and protein synthesis.

• Asian-Australasian Journal of Animal Sciences
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• v.13 no.9
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• pp.1285-1289
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• 2000

This study investigated the lipogenesis capacity of hepatocytes and lipolysis rate of adipocytes of broilers as affected by different fatty acids (trial one) and different linoleic acid (C18:2) levels (trial two). Twenty 6-wk old broilers were used; their hepatocytes and adipocytes were isolated for the in vitro study. In trial one, four treatments were tested. The control group in which no fatty acid was added, and the test groups to which were added $300{\mu}M$ of C16:0, C18:1 and C18:2, respectively. For trial two, different levels (0, $300{\mu}M$ and 1 mM) of C18:2 combined to fatty acid-free bovine serum albumin (BSA) were added to the medium. According to results of trial one, added fatty acids significantly reduced the incorporation by hepatocytes of [U,$^{14}C$]glucose into total lipid (p<0.05); the lipogenesis capacity in C18:2 group was the lowest. Although a similar pattern was found with [l,$^{14}C$]acetate, the groups only slightly differed in terms of lipogenesis capacity (p=0.11). In addition, the C18:2 group had a significantly (p<0.05) greater lipolysis rate than the C16:0 and control groups. Results of trial two indicated that C18:2 significantly (p<0.05) reduced lipogenesis capacity both for [U,$^{14}C$]glucose and [l,$^{14}C$]acetate, and markedly stimulated the lipolysis rate (p<0.05), displaying a dose response. Results presented herein demonstrate that C18:2 can reduce lipogenesis capacity and stimulate the lipolysis rate in broilers.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.6
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• pp.842-849
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• 2007

The purpose of the present study was to produce live offspring from in vitro fertilized goat embryos. Oocytes were collected from abattoir ovaries and kept in oocyte collection medium. Oocytes were washed 4-5 times with maturation medium containing medium-199 with 5 ${\mu}g/ml$ FSH, 100 ${\mu}g/ml$ LH, 1 ${\mu}g/ml$ estradiol-$17{\beta}$ 50 ${\mu}g/ml$ gentamycin, 10% inactivated estrus goat serum, and 3% BSA (fatty acid free). Oocytes were placed in 100 ${\mu}l$ drops of maturation medium containing granulosa cell monolayer and incubated in a 5% $CO_2$ incubator at $38.5^{\circ}C$ for 27 h. For capacitation of spermatozoa fresh semen was processed and mixed in 3 ml fertilization TALP medium containing 50 ${\mu}g/ml$ heparin and kept in the above incubator for 2 h. The capacitated spermatozoa were coincubated with matured oocytes for fertilization. Cleaved embryos were separated and cultured in embryo development medium with oviductal cells and 494 embryos were produced. Recipient goats were synchronized with two injections of 15 mg $PGF_{{2}{\alpha}}$/goat 10 days apart. Eighty early stage embryos were transferred into the uterotubal junction of 14 surrogate mothers using laparoscopy techniques. One recipient delivered twin kids, whereas another two recipients each.delivered a single kid The parentage of these kids was evaluated using highly polymorphic co-dominant microsatellites markers. From the present study, it was concluded that live goat kids can be produced from in vitro matured and fertilized goat embryos, to the best of our knowledge for the first time in India.

• Korean Journal of Animal Reproduction
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• v.19 no.3
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• pp.227-234
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• 1995

This study was carried out to investigate the effects according to the time course of glucose exposure on the development of one-cell embryos beyond morula in CR$_laa$ medium. One-cell zygotes from B6CBA F$_1$ mice were recovered at 24 ~ 25h after hCG and cumulus cells were removed with 0.1% hyaluronidase. The embryos were pooled and subsequently divided into each groups and cultured in CR$_laa$ at 37$^{\circ}C$ in 5% CO$_2$ in aIr. The embryos were either, placed in CR$_laa$ containing various concentration (5.5, 16.5, 27.5 and 38.5 mM) of glucose for 1 min. and subsequently returned to the fresh culture medium (without glucose), or were transferred to the same media containing glucose at 72 h post hCG. The results obtained in these experiments were summarized as follows: 1. The development rates of zygotes, recovered from the oviducts in M2 and cultured in CR$_laa$ with 3mg/Im FAF-BSA, to expanded blastocysts (25.7%) and hatching bIastocysts (17.6%) were significantly higher than those of zygotes recovered in TL Hepes (0% and 0%, respectively). 2. The development rates of one-cell embryos exposed to 27.5 mM glucose at 72 h post hCG for 1 min, were 68.8% (CR$_1$+BSA) a and 77.1% (CR$_1$+FBS) of expanded blastocyst stage, but there were no significant differences between the embryos exposed for 1 min. or transferred at 72 h. 3. Regardless of glucose concentration (5.5, 16.5, 27.5 & 38.5mM), 45.7~61.5% of embryos developed to the blastocyst stage. There were no significant differences between any of the treatments on the devel-opment of one-cell embryos. Therefore, the detrimental effect of highly concentration was not appeared.