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Efficient production of pronuclear embryos in breeding and nonbreeding season for generating transgenic sheep overexpressing TLR4.

Li Y, Lian D, Deng S, Zhang X, Zhang J, Li W, Bai H, Wang Z, Wu H, Fu J, Han H, Feng J, Liu G, Lian L, Lian Z - J Anim Sci Biotechnol (2016)

Bottom Line: Our results indicated the no. of embryos recovered of donors and the rate of pronuclear embryos did not show any significant difference between breeding and non-breeding seasons (P > 0.05).The lambs overexpressing TLR4 had similar growth performance with non-transgenic lambs, and the blood physiological parameters of transgenic and non-transgenic were both in the normal range and did not show any difference.The over-expression of TLR4 had no adverse effect on the growth of the sheep.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, Beijing, 100193 China.

ABSTRACT

Background: Brucella is a zoonotic Gram-negative pathogen that causes abortion and infertility in ruminants and humans. TLR4 is the receptor for LPS which can recognize Brucella and initiate antigen-presenting cell activities that affect both innate and adaptive immunity. Consequently, transgenic sheep over-expressing TLR4 are an suitable model to investigate the effects of TLR4 on preventing Brucellosis. In this study, we generated transgenic sheep overexpressing TLR4 and aimed to evaluate the effects of different seasons (breeding and non-breeding season) on superovulation and the imported exogenous gene on growth.

Results: In total of 43 donor ewes and 166 recipient ewes in breeding season, 37 donor ewes and 144 recipient ewes in non-breeding season were selected for super-ovulation and injected embryo transfer to generate transgenic sheep. Our results indicated the no. of embryos recovered of donors and the rate of pronuclear embryos did not show any significant difference between breeding and non-breeding seasons (P > 0.05). The positive rate of exogenous TLR4 tested were 21.21 % and 22.58 % in breeding and non-breeding season by Southern blot. The expression level of TLR4 in the transgenic sheep was 1.5 times higher than in the non-transgenic group (P < 0.05). The lambs overexpressing TLR4 had similar growth performance with non-transgenic lambs, and the blood physiological parameters of transgenic and non-transgenic were both in the normal range and did not show any difference.

Conclusions: Here we establish an efficient platform for the production of transgenic sheep by the microinjection of pronuclear embryos during the whole year. The over-expression of TLR4 had no adverse effect on the growth of the sheep.

No MeSH data available.


Related in: MedlinePlus

Pregnancy and lambing. a The pregnancy rate of different no. of corpus luteum of recipients (For each no. of CL, N = 4). b The pregnancy rate of reuse of donors as recipients and ordinary recipient. c The lambing rate of different no. of embryos transfered (For each no. of embryos, N = 4). d The rate of multiplets of different no. of embryos transfered (For each no. of embryos, N = 4). CL = Corpus Luteum. Superscript letter (*) represents statistically significant difference (P < 0.05)
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Fig3: Pregnancy and lambing. a The pregnancy rate of different no. of corpus luteum of recipients (For each no. of CL, N = 4). b The pregnancy rate of reuse of donors as recipients and ordinary recipient. c The lambing rate of different no. of embryos transfered (For each no. of embryos, N = 4). d The rate of multiplets of different no. of embryos transfered (For each no. of embryos, N = 4). CL = Corpus Luteum. Superscript letter (*) represents statistically significant difference (P < 0.05)

Mentions: In total, 13 of 78 recipients with one corpus luteum, 25 of 94 recipients with two corpus luteum and 20 of 85 recipients with three corpus luteum were pregnant, the other 53 recipients had more than three corpus luteum and we didn’t analyzed them on account of the small quantity. The pregnancy rate of recipients were 18.23 ± 6.95 %, 26.25 ± 3.71 % and 23.09 ± 5.79 %, respectively, and there were no significant differences among them (P > 0.05, Fig. 3a). In this study, after superovulation, 30 donors turned to be recipients and 5 sheep were pregnant. The pregnancy rate of the reuse of donors as recipients was 16.67 %, and the pregnancy rate of non-donor recipient was 23.21 % (65/280), there were no significant difference between them (Fig. 3b). In this experiment, 90 recipients were transplanted two embryos and 18 sheep were born including one double-lamb; 106 recipients were transplanted three embryos and 28 sheep were born including three double-lamb; 114 recipients were transplanted four embryos and 34 sheep were born including six double-lamb. The lambing rate were 21.08 ± 7.13 %, 26.53 ± 6.48 % and 29.94 ± 4.46 %, respectively, and there was no difference between the recipients transplanting different no. of embryos (P > 0.05, Fig. 3c). The rate of multiplets were 0.83 ± 1.67 %, 2.78 ± 2.08 % and 5.47 ± 2.96 %, respectively, and transplanting four embryos got higher rate than transplanting three embryos (P < 0.05, Fig. 3d).Fig. 3


Efficient production of pronuclear embryos in breeding and nonbreeding season for generating transgenic sheep overexpressing TLR4.

Li Y, Lian D, Deng S, Zhang X, Zhang J, Li W, Bai H, Wang Z, Wu H, Fu J, Han H, Feng J, Liu G, Lian L, Lian Z - J Anim Sci Biotechnol (2016)

Pregnancy and lambing. a The pregnancy rate of different no. of corpus luteum of recipients (For each no. of CL, N = 4). b The pregnancy rate of reuse of donors as recipients and ordinary recipient. c The lambing rate of different no. of embryos transfered (For each no. of embryos, N = 4). d The rate of multiplets of different no. of embryos transfered (For each no. of embryos, N = 4). CL = Corpus Luteum. Superscript letter (*) represents statistically significant difference (P < 0.05)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4940989&req=5

Fig3: Pregnancy and lambing. a The pregnancy rate of different no. of corpus luteum of recipients (For each no. of CL, N = 4). b The pregnancy rate of reuse of donors as recipients and ordinary recipient. c The lambing rate of different no. of embryos transfered (For each no. of embryos, N = 4). d The rate of multiplets of different no. of embryos transfered (For each no. of embryos, N = 4). CL = Corpus Luteum. Superscript letter (*) represents statistically significant difference (P < 0.05)
Mentions: In total, 13 of 78 recipients with one corpus luteum, 25 of 94 recipients with two corpus luteum and 20 of 85 recipients with three corpus luteum were pregnant, the other 53 recipients had more than three corpus luteum and we didn’t analyzed them on account of the small quantity. The pregnancy rate of recipients were 18.23 ± 6.95 %, 26.25 ± 3.71 % and 23.09 ± 5.79 %, respectively, and there were no significant differences among them (P > 0.05, Fig. 3a). In this study, after superovulation, 30 donors turned to be recipients and 5 sheep were pregnant. The pregnancy rate of the reuse of donors as recipients was 16.67 %, and the pregnancy rate of non-donor recipient was 23.21 % (65/280), there were no significant difference between them (Fig. 3b). In this experiment, 90 recipients were transplanted two embryos and 18 sheep were born including one double-lamb; 106 recipients were transplanted three embryos and 28 sheep were born including three double-lamb; 114 recipients were transplanted four embryos and 34 sheep were born including six double-lamb. The lambing rate were 21.08 ± 7.13 %, 26.53 ± 6.48 % and 29.94 ± 4.46 %, respectively, and there was no difference between the recipients transplanting different no. of embryos (P > 0.05, Fig. 3c). The rate of multiplets were 0.83 ± 1.67 %, 2.78 ± 2.08 % and 5.47 ± 2.96 %, respectively, and transplanting four embryos got higher rate than transplanting three embryos (P < 0.05, Fig. 3d).Fig. 3

Bottom Line: Our results indicated the no. of embryos recovered of donors and the rate of pronuclear embryos did not show any significant difference between breeding and non-breeding seasons (P > 0.05).The lambs overexpressing TLR4 had similar growth performance with non-transgenic lambs, and the blood physiological parameters of transgenic and non-transgenic were both in the normal range and did not show any difference.The over-expression of TLR4 had no adverse effect on the growth of the sheep.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, Beijing, 100193 China.

ABSTRACT

Background: Brucella is a zoonotic Gram-negative pathogen that causes abortion and infertility in ruminants and humans. TLR4 is the receptor for LPS which can recognize Brucella and initiate antigen-presenting cell activities that affect both innate and adaptive immunity. Consequently, transgenic sheep over-expressing TLR4 are an suitable model to investigate the effects of TLR4 on preventing Brucellosis. In this study, we generated transgenic sheep overexpressing TLR4 and aimed to evaluate the effects of different seasons (breeding and non-breeding season) on superovulation and the imported exogenous gene on growth.

Results: In total of 43 donor ewes and 166 recipient ewes in breeding season, 37 donor ewes and 144 recipient ewes in non-breeding season were selected for super-ovulation and injected embryo transfer to generate transgenic sheep. Our results indicated the no. of embryos recovered of donors and the rate of pronuclear embryos did not show any significant difference between breeding and non-breeding seasons (P > 0.05). The positive rate of exogenous TLR4 tested were 21.21 % and 22.58 % in breeding and non-breeding season by Southern blot. The expression level of TLR4 in the transgenic sheep was 1.5 times higher than in the non-transgenic group (P < 0.05). The lambs overexpressing TLR4 had similar growth performance with non-transgenic lambs, and the blood physiological parameters of transgenic and non-transgenic were both in the normal range and did not show any difference.

Conclusions: Here we establish an efficient platform for the production of transgenic sheep by the microinjection of pronuclear embryos during the whole year. The over-expression of TLR4 had no adverse effect on the growth of the sheep.

No MeSH data available.


Related in: MedlinePlus