Вплив Репродуктивного Сезону На Спермопродуктивність Гермінтативних Химер Селезнів
Abstract: У статті наведені результати дослідження спермопродуктивності селезнів порід шаосінь, шанма та герментативних химер. Було встановлено, що за об’ємом еякуляту, концентрацією та активністю сперматозоїдів герментативні химери переважали селезнів порід шаосінь (Р < 0,05) та шанма (Р < 0,01). Результати дисперсійного аналізу свідчать, що частка впливу породи на об’єм еякуляту становить 25%, а періоду репродуктивного сезону – 13%. Спермопродуктивність гермінативних химер показує, що їх можливо використивувати … Show more
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Cited by 3 publications
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The consequences of chimerization and its possible influence on the productivity of chimera offspring remain poorly understood. The objects of research were ducks (Anas platyrhynchos) of the Shanma (Shan partridge duck) and Shaoxing breeds kept at the Zhuji Guowei Poultry Development Co, Ltd, P.R.China. The study was conducted in the poultry genetics laboratory of the Zhejiang Academy of Agricultural Sciences on a duck farm of Zhejiang Generation Biological Science and Technology Co., Ltd. (Zhejiang Province, PRC). To create chimeras of ducks, the method described by Aige-Gil, Simkiss, 1991; M.T. Tagirov, 2010 was used. Blastodiscs have been isolated from freshly hatched fertilized eggs using a filter paper ring. Shanma duck embryos have been used as recipients, and Shaoxing duck embryos, homozygous for plumage color gene allele (wild type), have been used as donors. Busulfan (SigmaAldrich, United States) have been used as a chemical agent that suppresses a division of primary germ cells (PGC) of recipient embryos. A hole in an eggshell (window) of recipients (Shanma breed) have been made between a blunt and sharp ends of eggs. (This reduced a distance between an injector and an embryo needle). The recipients havebeen incubated for 8–10 hours at a temperature of 38 °C. After recipient eggs incubation for 8 hours, the windows were opened in them. Busulfan was injected into the subgerminal cavity of the embryo with a micropipette (1.5–3 μl of liquid). After busulfan injection, the empty cavity was filled with culture medium (RPMI-1640) supplemented with antibiotics (ampicillin, streptomycin), the hole was closed by plastic wrap and adhesive tape. The eggs have been incubated at a reduced temperature (+32 °C) for 24 hours with the aim of prolong the duration of busulfan action on the PGC (primary germ cells). More than 50% of embryos have been died in the first 2–3 days (after an incubation start). Head and neck disorders have been observed in the 1.2% of embryos. Busulfan injection at a concentration of 300 ng per egg have been leads to 95.0–96.3% mortality of duck embryos, concentration of 150 ng per egg, a mortality rate of 33.3–75.3% have been observed, concentration to 75 ng led to 18.75–38.5% of embryonic mortality. Analysis of the age of puberty (laying of the first egg) indicates that the chimeras matured later. If in the control group the average age of puberty was 139 ± 9 days, in the group of chimeras - 148 ± 13 days. Thus, we can attest that in our experiment, the chimeras matured later than the control animals, which may be due to the effect of busulfan in the sterilization of recipient embryos. The average weight of ducks in the control group was lower, and the group itself was more consolidated. Thus, in the control ducks weighed 1422.40 ± 57.00 g, the chimeras 1608.80 ± 94.76 g. The advantage of live weight chimeras over the control group may be due to the fact that the control group consisted of recipients served by Shanma animals. Egg production of ducks for the entire study period was 87.5 ± 0.05 % (control) 79.5±0.12 % (busulfan). The weight of eggs of ducks of two groups for the entire period was 70.62±0.199 g (control) and 71.15±0.157 g (p˂0.001). The eggs morphometric parameters of the studied ducks groups were: the average values of egg length were 6.056±0.0564 cm (control) and 6.269±0.1341cm (busulfan); egg breadth were 4.520±0.0053 cm (control) and 4.529±0.004 cm (busulfan). There were no statistical intergroup differences in the morphometric parameters of the eggs of the studied groups. In fact, we obtained results similar to the previous ones, which concerned the egg production of daughters of drake chimeras.
The consequences of chimerization and its possible influence on the productivity of chimera offspring remain poorly understood. The objects of research were ducks (Anas platyrhynchos) of the Shanma (Shan partridge duck) and Shaoxing breeds kept at the Zhuji Guowei Poultry Development Co, Ltd, P.R.China. The study was conducted in the poultry genetics laboratory of the Zhejiang Academy of Agricultural Sciences on a duck farm of Zhejiang Generation Biological Science and Technology Co., Ltd. (Zhejiang Province, PRC). To create chimeras of ducks, the method described by Aige-Gil, Simkiss, 1991; M.T. Tagirov, 2010 was used. Blastodiscs have been isolated from freshly hatched fertilized eggs using a filter paper ring. Shanma duck embryos have been used as recipients, and Shaoxing duck embryos, homozygous for plumage color gene allele (wild type), have been used as donors. Busulfan (SigmaAldrich, United States) have been used as a chemical agent that suppresses a division of primary germ cells (PGC) of recipient embryos. A hole in an eggshell (window) of recipients (Shanma breed) have been made between a blunt and sharp ends of eggs. (This reduced a distance between an injector and an embryo needle). The recipients havebeen incubated for 8–10 hours at a temperature of 38 °C. After recipient eggs incubation for 8 hours, the windows were opened in them. Busulfan was injected into the subgerminal cavity of the embryo with a micropipette (1.5–3 μl of liquid). After busulfan injection, the empty cavity was filled with culture medium (RPMI-1640) supplemented with antibiotics (ampicillin, streptomycin), the hole was closed by plastic wrap and adhesive tape. The eggs have been incubated at a reduced temperature (+32 °C) for 24 hours with the aim of prolong the duration of busulfan action on the PGC (primary germ cells). More than 50% of embryos have been died in the first 2–3 days (after an incubation start). Head and neck disorders have been observed in the 1.2% of embryos. Busulfan injection at a concentration of 300 ng per egg have been leads to 95.0–96.3% mortality of duck embryos, concentration of 150 ng per egg, a mortality rate of 33.3–75.3% have been observed, concentration to 75 ng led to 18.75–38.5% of embryonic mortality. Analysis of the age of puberty (laying of the first egg) indicates that the chimeras matured later. If in the control group the average age of puberty was 139 ± 9 days, in the group of chimeras - 148 ± 13 days. Thus, we can attest that in our experiment, the chimeras matured later than the control animals, which may be due to the effect of busulfan in the sterilization of recipient embryos. The average weight of ducks in the control group was lower, and the group itself was more consolidated. Thus, in the control ducks weighed 1422.40 ± 57.00 g, the chimeras 1608.80 ± 94.76 g. The advantage of live weight chimeras over the control group may be due to the fact that the control group consisted of recipients served by Shanma animals. Egg production of ducks for the entire study period was 87.5 ± 0.05 % (control) 79.5±0.12 % (busulfan). The weight of eggs of ducks of two groups for the entire period was 70.62±0.199 g (control) and 71.15±0.157 g (p˂0.001). The eggs morphometric parameters of the studied ducks groups were: the average values of egg length were 6.056±0.0564 cm (control) and 6.269±0.1341cm (busulfan); egg breadth were 4.520±0.0053 cm (control) and 4.529±0.004 cm (busulfan). There were no statistical intergroup differences in the morphometric parameters of the eggs of the studied groups. In fact, we obtained results similar to the previous ones, which concerned the egg production of daughters of drake chimeras.
Transgenesis biotechnological procedures influence on domestic duck embryos survival
Due to its high reproductive potential, short interval between generations and embryonic development outside the mother's body, the bird provides unique opportunities for its use in fundamental and applied biological research. The creation of a transgenic bird is complicated by the structure of its opaque egg cell with a large yolk and a unique reproductive system of this class. Direct microinjection of DNA into an oocyte, which is often used in mammals, is practically impossible for birds, since fertilization occurs in the infudibulum of the reproductive tract and can be polyspermic. Therefore, manipulations with the zygote turned out to be difficult for their use in creating a transgenic bird. Over the past decades, some alternative strategies have been developed for producing transgenic poultry using bizarre animals created by transferring blastodermal cells. However, to date, the efficiency of creating transgenic poultry in many cases remains very low, and the technique of using ducks to create transgenic poultry is practically not developed. Busulfan is used to suppress cell proliferation. Injection of busulfan into the pidembryonic cavity is one of the methods that increases the number of donor cells when creating chimeras. However, until now, methods of creating hermentative ducks chimeras face difficulties associated with the structure of the shell of waterfowl. Therefore, the aim of the work was to establish the effect of factors influencing the survival of transgenic embryos when using various methods of introducing a DNA construct into the duck genome. The objects of the study were ducks (Anas platyrhynchos) of the Shan partridge duck and Shaoxing breeds kept at the duck farm of Zhuji Guowei Poultry Development Co., Ltd, China. The studies were carried out in the poultry genetics laboratory of the Zhejiang Academy of Agricultural Sciences and on the duck farm of Zhejiang Generation Biological Science and Technology Co., Ltd. (Zhejiang Province, PRC). For the analysis of survival, we used embryos obtained by using various methods of introducing the DNA (insertion of the EGFP gene, mediated by homologous repair (HDR)) 1) direct injection of the DNA construct into the sub-embryonic cavity; 2) transfection of DNA with sperm; 3) injection of transfected donor blastomeres into recipient embryos after exposure to busulfan or ultraviolet radiation. A total more than 1100 eggs were examined. As a result of the direct injection of a transgenic DNA construction ( sub-embryonic cavity of 300 embryos, 35.7% of embryos did not develop after injection, 36% stopped developing at the time of the first ovoscopy (day 9 of incubation), 8% died within 10-15 days, 17, 3% - 16-25 days. In total, after direct injections, 9 live ducklings were received (the survival rate was 3%), of which 4 were transgenic. After insemination of ducks transfected with sperm, 292 eggs were laid for incubation. After the first ovoscopy, 51.4% of the eggs were unfertilized; 0.7% of embryos stopped developing at the time of the first ovoscopy (9 day of incubation), 1.0% died within 10-15 days, 17.8% - 16-25 days, 6.2% suffocated during hatching. In total, after using the transfected sperm, 67 live ducklings were obtained (the survival rate of embryos from fertilized eggs was 47.2%). Among 31 adult animals, 19 were transgenic. To sterilize recipient cells for the use of busulfan at a concentration of 300 ng per egg, followed by injection of blastodermal transfected donor cells, 200 embryos were examined, among which 61.0% of embryos developed after injection, 17.0% stopped in development at the time of the first ovoscopy (day 9 of incubation ), 12.5% of those died in the period of 10-15 days, 9.0% - 16-25 days. In total, after injections of busulfan at a concentration of 300 ng per egg, 1 live duckling was obtained (the survival rate was 0.5%). Using busulfan at a concentration of 150 ng per egg, 100 embryos were examined, among which 68.0% of embryos developed after injection, 11.0% stopped developing at the time of the first ovoscopy (day 9 of incubation), 5% died within 10-15 days, 14.0% - 16-25 days. In total, after injections of busulfan at a concentration of 150 ng per egg, 2 live ducklings were obtained (the survival rate was 0.5%). Using busulfan at a concentration of 75 ng per egg, 100 embryos were examined, among which 12.0% of embryos developed after injection, 27.0% stopped developing at the time of the first ovoscopy (9 day of incubation), 14.0% died in the period 10-15 days, 42.0% - 16-25 days. In total, after injections of busulfan at a concentration of 75 ng per egg, 5 live ducklings were obtained (the survival rate was 5%). Ultraviolet irradiation of 200 embryos for 1 hour followed by injection of blastodermal transfected donor cells resulted in death after injection of 20%, stopped developing 27.5% (9 days of incubation), 7.5% died within 10-15 days , 35.0% - 16-25 days. A total of 20 live ducklings were obtained using ultraviolet radiation (survival rate was 10%). Among 13 adult animals gave offspring, 7 were transgenic chimeras. The use of ultraviolet light has reduced the impact of egg infection due to the structure of waterfowl shells. Thus, the safest for the survival of embryos was the method of insemination of ducks with transfected sperm, using which 47.2% of embryos survived.
Оптимізація Біотехнологічних Процедур Підготовки Сперми До Трансфекції Днк З Ліпофектаміном
З метою аналізу впливу трансфекції з ліпофектаміном на запліднювальну здатність сперміїв качурів (Anas platyrhynchos Linnaeus, 1758) породи Shaoxing проведено аналіз яєць та ембріонів 11 качок породи Shaoxing, які були запліднені спермою 13 качурів, обробленою ліпофектаміном. Птиця відповідала стандарту породи Shaoxing. Качок у віці десяти місяців відбирали за умови їх несучості ≥ 90% та плодючості ≥ 90% після штучного осіменіння. Качурів відбирали зі стабільним рефлексом спермовіддачі при стимуляції методом масажу поперекової частини тулуба. Сперму збирали в конічні полістирольні чашки з подальшим розведенням до 1: 1 середовищем OPTI-MEM (Invitrogen, США) та транспортуванням до лабораторії (протягом 15–20 хвилин після збору) для оцінки якості та трансфекції сперми. Рухливість і концентрацію сперматозоїдів оцінювали за стандартними методиками під оптичним мікроскопом. Для трансфекції сперматозоїдів 300 мкл плазмідної ДНК (25 нг/мл кожного вектора) змішували з 1 мл середовища OPTI-MEM. Тим часом 300 мкл Lipofectamine® 2000 (Invitrogen, США) змішували з 1 мл середовища OPTI-MEM. Після того, як два розчини інкубували протягом 5 хвилин при кімнатній температурі, їх об'єднували та інкубували при кімнатній температурі ще 20 хвилин. Сперму двічі центрифугували (1000g, 10 хв), надосадову рідину видаляли і проводили розведення осаджених клітин сперми середовищем OPTI-MEM 1: 1. Комплекс ДНК-Lipofectamine® 2000 додавали до клітин сперми після другого центрифугування, та видалення надосадової рідини, потім його перемішували та інкубували при кімнатній температурі протягом години. Після трансфекції рухливість сперматозоїдів знову оцінювали для визначення якості сперми. Трансфіковані клітини сперми використовували для глибокого штучного запліднення. За одне запліднення було взято п’ятсот мільйонів сперматозоїдів. Після осіменіння качок трансфікованими сперматозоїдами яйця збирали і інкубували протягом 10 або 28 днів, потім виділяли ембріони або вирощували каченят. Для контролю впливу проведених маніпуляцій зі спермою під час проведення трансфекції на її запліднюючу здатність, експеримент побудували методом груп-аналогів, було відібрано 11 качок яких осіменяли інтактною спермою. В дослідній групі заплідненість становила 52,2±4,97%, що достовірно відрізнялось від групи контролю 84.1±5.83% (p < 0.01). Виводимість становила 47.18% (67 каченят з 142 запліднених яєць)
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