Synchronization of development between the embryo and uterus is required for successful pregnancy establishment. Transfer of early embryos requires synchrony with the recipient uterus of 2 days or less in sheep, becau...Synchronization of development between the embryo and uterus is required for successful pregnancy establishment. Transfer of early embryos requires synchrony with the recipient uterus of 2 days or less in sheep, because asynchrony of 3 days or more results in failure of pregnancy recognition signaling for maintenance of corpus luteum （CL） and progesterone （P4） production and/or uterine support of the embryo. The objective was to determine if P4 treatment of recipient ewes would obviate the need for pregnancy recognition signaling and maintain a uterine environment conducive to embryo survival after asynchronous transfer, thereby establishing a universal recipient. Embryos （morulae/blastocysts） were recovered on day 6 from super-ovulated donor ewes. Recipient ewes received 25 mg P4 daily from day 6 post-estrus until 60 days after embryo transfer. Embryos were transferred into recipients on day 6,9, 12,18, or 30 post-estrus. The pregnancy rate on day 22 post-transfer was 60% for synchronous transfers to day 6 ewes, 44% and 22% for asynchronous transfers to day 9 and 12 ewes, and 0% for asynchronous transfers to day 18 and 30 ewes. On day 39 posttransfer ,pregnancy rates remained 60% for day 6 ewes,33% for day 9 ewes,and 0% for day 12,18, and 30 ewes. The P4 treatment did extend the window of uterine receptivity to early embryos in ewes by one day ,but did not create a universal recipient. Available results support the idea that a window of uterine receptivity to the conceptus exists in sheep that is independent of pregnancy recognition signaling.展开更多
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 a...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.展开更多
There is little information in the scientific literature concerning sheep pregnancy and lambing success with regard to the timeframe from when in vitro produced embryos are transported to the designated location for e...There is little information in the scientific literature concerning sheep pregnancy and lambing success with regard to the timeframe from when in vitro produced embryos are transported to the designated location for embryo transfer (ET). The aim of this study was to transfer in vitro produced embryos under two different conditions that could typically occur using the aforementioned assisted reproductive techniques (ARTs). Abattoir ovaries were used to procure oocytes for in vitro embryo production and subsequent transfer to synchronized ewes. The study consisted of two experiments: Experiment 1 (Exp1)—embryos taken from the laboratory to a nearby surgical room for immediate ET, and Experiment 2 (Exp2)—ET after 5 hours (h) of transport to a rural farm. Lambing in relation to detected pregnancies, births compared to pregnancies, and the proportion of twin offspring were all higher in Exp2. Notably, in both Exp1 and Exp2, there was not a significant difference (P > 0.05) between the number of embryos transferred, i.e., 3 versus 4, respectively, and the number of ewes that underwent parturition in each group. Also, in both experiments there was not a significant difference (P > 0.05) in the number of ewes that underwent parturition based on the number (i.e., ≥1) of corpora lutea present. The results of the present study demonstrate the importance of evaluating different conditions when applying ARTs, as there are many variables that can influence the outcome. Importantly, Exp2 results show that ovine ET in places located far away from the embryo production site can be useful and successful provided that embryo transport, ET, and recipient conditions are adequate.展开更多
This study was conducted to investigate the in-vitro production technology of embryos from young Dorper sheep, so as to provide technical support for the utilization of ovarian follicles in young Dorper sheep. Tests w...This study was conducted to investigate the in-vitro production technology of embryos from young Dorper sheep, so as to provide technical support for the utilization of ovarian follicles in young Dorper sheep. Tests were conducted from the induction of Dorper sheep of 4 to 8 weeks old using follicle stimulating hormone(FSH) and pregnant horse serum(PMSG), collection of oocytes, in-vitro oocyte maturation-fertilization-zygote cultivation and 2-4-cell-stage fertilized ovum transfer. The results showed that 585 oocytes were collected from eight Dorper sheep at the age of 4 and 8 weeks, with an average of 73.13 oocytes/sheep. 346 of the 2-4-cell-stage fertilized eggs were obtained, whose cleavage rate was 59.15%. 77 in-vitro fertilized eggs at 2-4-cell stage were transplanted into 17 recipient sheep, seven of which were pregnant and gave birth to 13 "test-tube sheep" with a conception rate of 41.18%. It is indicated that the hormone induction technique, in-vitro oocyte maturation-fertilization-zygote cultivation technique and 2-4-cell-stage fertilized ovum transfer technique used in this study can serve as effective techniques for the in-vitro production of embryos from Dorper sheep of 4-8 weeks old.展开更多
A series of experiments were conducted to study the major procedures in nuclear transplantation such as oocyte enucleation and activation, electrofusion and developnent of the nuclear transplant embryos in the mouse, ...A series of experiments were conducted to study the major procedures in nuclear transplantation such as oocyte enucleation and activation, electrofusion and developnent of the nuclear transplant embryos in the mouse, rabbits and sheep. The important results are as follows:11. In the mouse, only 35% of the oocytes collected 15～16 h after hCG had a notable first polar body (FPb) and those without FPb were enucleated by removing cytoplasm from the PVS-wider side and the enucleation rate was similar to that in the oocytes with FPb, and the enucleation rate of removing 1/3 cytoplasm was remarkably higher than that of removing 1/4 cytoplasm. 2. Among the three fusion media tested, mannitol and sucrose solutions produced better results than M2 in electrofusion of mouse 2-cell embryos. Under favorable pulse conditions, the osmotic pressure of fusion medium had no motable effect on electrofusion, but as the conditions became so unfavorable that some embryos began to lyse, the fusion rates in hypertonie mannitol solution were significantly higher than those in isotonic or hypotonic solutions. A wide range of pulse strengths (0.31～2.04 by/ cm) and durations(10～1280 μs) were used and 100% of fusion were obtained in many cases. Optimal pulse durations were plotted for field strengths to obtain high fusion rates (96%～ 100%) in mouse2-cell embryos. 3. With one pulse of 0.45 by / cm, satisfactory results of mouse oocyte activation were obtained only when the duration increased to 160 μs or longer. The activation rate increased as the oocytes got older. Some of the oocytes ar. rested at metaphase Ⅲ after electrical stimulation and their proportion to the number of oocytes not activated increased with egg age. 4. 10% and 31% of the nuclear transplant embryos developed to morula or blastocyst stage in sheep and rabbits, respectively, with Chinese-made hormones and chemicals.展开更多
文摘Synchronization of development between the embryo and uterus is required for successful pregnancy establishment. Transfer of early embryos requires synchrony with the recipient uterus of 2 days or less in sheep, because asynchrony of 3 days or more results in failure of pregnancy recognition signaling for maintenance of corpus luteum （CL） and progesterone （P4） production and/or uterine support of the embryo. The objective was to determine if P4 treatment of recipient ewes would obviate the need for pregnancy recognition signaling and maintain a uterine environment conducive to embryo survival after asynchronous transfer, thereby establishing a universal recipient. Embryos （morulae/blastocysts） were recovered on day 6 from super-ovulated donor ewes. Recipient ewes received 25 mg P4 daily from day 6 post-estrus until 60 days after embryo transfer. Embryos were transferred into recipients on day 6,9, 12,18, or 30 post-estrus. The pregnancy rate on day 22 post-transfer was 60% for synchronous transfers to day 6 ewes, 44% and 22% for asynchronous transfers to day 9 and 12 ewes, and 0% for asynchronous transfers to day 18 and 30 ewes. On day 39 posttransfer ,pregnancy rates remained 60% for day 6 ewes,33% for day 9 ewes,and 0% for day 12,18, and 30 ewes. The P4 treatment did extend the window of uterine receptivity to early embryos in ewes by one day ,but did not create a universal recipient. Available results support the idea that a window of uterine receptivity to the conceptus exists in sheep that is independent of pregnancy recognition signaling.
基金supported by grants from National Transgenic Creature Breeding Grand Project (2014ZX08008-005)
文摘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.
文摘There is little information in the scientific literature concerning sheep pregnancy and lambing success with regard to the timeframe from when in vitro produced embryos are transported to the designated location for embryo transfer (ET). The aim of this study was to transfer in vitro produced embryos under two different conditions that could typically occur using the aforementioned assisted reproductive techniques (ARTs). Abattoir ovaries were used to procure oocytes for in vitro embryo production and subsequent transfer to synchronized ewes. The study consisted of two experiments: Experiment 1 (Exp1)—embryos taken from the laboratory to a nearby surgical room for immediate ET, and Experiment 2 (Exp2)—ET after 5 hours (h) of transport to a rural farm. Lambing in relation to detected pregnancies, births compared to pregnancies, and the proportion of twin offspring were all higher in Exp2. Notably, in both Exp1 and Exp2, there was not a significant difference (P > 0.05) between the number of embryos transferred, i.e., 3 versus 4, respectively, and the number of ewes that underwent parturition in each group. Also, in both experiments there was not a significant difference (P > 0.05) in the number of ewes that underwent parturition based on the number (i.e., ≥1) of corpora lutea present. The results of the present study demonstrate the importance of evaluating different conditions when applying ARTs, as there are many variables that can influence the outcome. Importantly, Exp2 results show that ovine ET in places located far away from the embryo production site can be useful and successful provided that embryo transport, ET, and recipient conditions are adequate.
基金Supported by Earmarked Fund for Construction of National Wool Sheep Industry Technology Research System(CARS-39-24)Program for Science and Technology Development of Shanxi Province(20120311024-1)+1 种基金Fund for Science and Technology Innovation Team in Shanxi Province(201705D131028-20)Shanxi Agricultural Industry Development Technology Leading Fund(2017CYYL-08)
文摘This study was conducted to investigate the in-vitro production technology of embryos from young Dorper sheep, so as to provide technical support for the utilization of ovarian follicles in young Dorper sheep. Tests were conducted from the induction of Dorper sheep of 4 to 8 weeks old using follicle stimulating hormone(FSH) and pregnant horse serum(PMSG), collection of oocytes, in-vitro oocyte maturation-fertilization-zygote cultivation and 2-4-cell-stage fertilized ovum transfer. The results showed that 585 oocytes were collected from eight Dorper sheep at the age of 4 and 8 weeks, with an average of 73.13 oocytes/sheep. 346 of the 2-4-cell-stage fertilized eggs were obtained, whose cleavage rate was 59.15%. 77 in-vitro fertilized eggs at 2-4-cell stage were transplanted into 17 recipient sheep, seven of which were pregnant and gave birth to 13 "test-tube sheep" with a conception rate of 41.18%. It is indicated that the hormone induction technique, in-vitro oocyte maturation-fertilization-zygote cultivation technique and 2-4-cell-stage fertilized ovum transfer technique used in this study can serve as effective techniques for the in-vitro production of embryos from Dorper sheep of 4-8 weeks old.
文摘A series of experiments were conducted to study the major procedures in nuclear transplantation such as oocyte enucleation and activation, electrofusion and developnent of the nuclear transplant embryos in the mouse, rabbits and sheep. The important results are as follows:11. In the mouse, only 35% of the oocytes collected 15～16 h after hCG had a notable first polar body (FPb) and those without FPb were enucleated by removing cytoplasm from the PVS-wider side and the enucleation rate was similar to that in the oocytes with FPb, and the enucleation rate of removing 1/3 cytoplasm was remarkably higher than that of removing 1/4 cytoplasm. 2. Among the three fusion media tested, mannitol and sucrose solutions produced better results than M2 in electrofusion of mouse 2-cell embryos. Under favorable pulse conditions, the osmotic pressure of fusion medium had no motable effect on electrofusion, but as the conditions became so unfavorable that some embryos began to lyse, the fusion rates in hypertonie mannitol solution were significantly higher than those in isotonic or hypotonic solutions. A wide range of pulse strengths (0.31～2.04 by/ cm) and durations(10～1280 μs) were used and 100% of fusion were obtained in many cases. Optimal pulse durations were plotted for field strengths to obtain high fusion rates (96%～ 100%) in mouse2-cell embryos. 3. With one pulse of 0.45 by / cm, satisfactory results of mouse oocyte activation were obtained only when the duration increased to 160 μs or longer. The activation rate increased as the oocytes got older. Some of the oocytes ar. rested at metaphase Ⅲ after electrical stimulation and their proportion to the number of oocytes not activated increased with egg age. 4. 10% and 31% of the nuclear transplant embryos developed to morula or blastocyst stage in sheep and rabbits, respectively, with Chinese-made hormones and chemicals.
