To edit the duck genome by HDR-directed integration of the EGFP gene into the duck host genome in combination with SMGT using CRISPR/Cas9. Methods. HDR-mediated gene of green fluorescent protein (EGFP) was crried out by the combined action of four plasmids. The pX330 contained the Cas9 gene. Two plasmids contained sgRNA genes: pBR322-sgRNA1 and pBR322-sgRNA2. The pBR322-HDR-EGFP plasmid was constructed to contain the DNA vector with left homologous sequence part(LHP), the EGFP gene coding sequences and the right homologous sequence part(RHP). The DNA sequence data for designing the HDR-EGFPinsert and sgRNA 1 and sgRNA 2 were taken from the genome DNA sequence of Anas platyrhynchos Spindlin 1 (SPIN1) gene. Twenty four ducks (13 males and 11 females) of the Shaoxing breed were used for this experiment. The sperm transfection was performed using Lipofectamine 2000. Results. Thirty one ducks were obtained, 19 of which carried the EGFP gene. F2 analysis revealed that 16 ducks (F1) (14 females and 2 males) transmitted the transgene DNA to their offsprings. Thus 27.6 % (56/203) of F2 descendants were positive for the transgene DNA construct. Conclusions. Exogenous DNA was successfully inserted into the duck genome.
The purpose of this study was to monitor the egg productivity of the Shaoxing breed ducks from the age of 24 (beginning of egg laying) and 75 (end of egg laying) weeks. According to the results of a study of over 2385 eggs, a significant difference was found between the weight indicators (57.93 g and 70.81 g; p˂0.01), the shape index (73.71% and 75.35%, p˂0.01), the strength of the shell (4.92kg and 4.28kg; p˂0.01), the thickness of the shell of the egg (0.48mm and 0.45mm; p˂0.01) in ducks at the age of 24 and 75 weeks. It also shows changes in the weight and shape of the egg with the age of the bird. The necessity of further studying the genetic diversity of birds, which causes the separate variability of indices of individual ducks, is substantiated.
The goose is an economically important poultry species and was one of the first to be domesticated. However, studies on population genetic structures and domestication in goose are very limited. Here, we performed whole genome resequencing of geese from two wild ancestral populations, five Chinese domestic breeds, and four European domestic breeds. We found that Chinese domestic geese except Yili geese originated from a common ancestor and exhibited strong geographical distribution patterns and trait differentiation patterns, while the origin of European domestic geese was more complex, with two modern breeds having Chinese admixture. In both Chinese and European domestic geese, the identified selection signatures during domestication primarily involved the nervous system, immunity, and metabolism. Interestingly, genes related to vision, skeleton, and blood-O2 transport were also found to be under selection, indicating genetic adaptation to the captive environment. A forehead knob characterized by thickened skin and protruding bone is a unique trait of Chinese domestic geese. Interestingly, our population differentiation analysis followed by an extended genotype analysis in an additional population suggested that two intronic SNPs in EXT1, an osteochondroma-related gene, may plausibly be sites responsible for knob. Moreover, CSMD1 and LHCGR genes were found to be significantly associated with broodiness in Chinese domestic geese and European domestic geese, respectively. Our results have important implications for understanding the population structure and domestication of geese, and the selection signatures and variants identified in this study might be useful in genetic breeding for forehead knob and reproduction traits.
The main biological functions of eggs include its ability to create optimal conditions for embryos, which, accordingly, contributes to the preservation and reproduction of the species. It is recommended for incubation to take eggs from physiologically healthy, mature birds, because the quality of the incubation eggs depends on the yield of the young, the vitality and productivity of the poultry. A prerequisite for a successful incubation is the assessment of eggs for a variety of morphological characteristics, but this can not be done without taking into account the biological features of incubation eggs associated with breed, crossbreed and bird age. It is known that with the age of the bird, the morphological parameters of the egg and its incubation capacity are changing. In particular, the weight and shape of the egg vary during the period of increasing the bird's egg-laying. The chicken egg has been extensively studied in the world and national scientific literature, its quality and composition, but over the past decades eggs of various types of poultry have been entered into the field of research: turkeys, guinea fowl, quails, ostriches and ducks. The Shaoxing Species (Shaoxing) belongs to the main Chinese egg rocks. Ducks of this breed are characterized by high performance, such as early maturity (130–140 days), long-term peak period of laying eggs (8–10 months) and good breeding (290–310 eggs for 500 days). Such indicators are some of the best for egg bird. That is why the purpose of the work was to determine the effect of the physico-morphological indices of the egg, the age of the bird and the color of the shell on the incubation properties of the eggs of the ducks of the Shaoxing breed. Three comparative analyzes of morphological parameters and their influence on the incubation capacity of eggs were selected from three experimental groups of ducks of the Shaoxing of different ages. Group number 1 carried eggs from females age 41 week, group number 2–63 weeks, and females group number 3 had an age of 71 weeks. The study of changes in the physico-morphological parameters of eggs and incubation ability was investigated within three months. A total of 360 eggs were analyzed. As a result of the experiment, the mass of experimental eggs ranged from 65.83 g (group 1) to 71.13 g (group 3). Between these indicators, a significant difference was found (p ˂ 0,01). Comparison of the studied physico-morphological characteristics of the groups number 1, number 2 and number 3 showed that there is a significant difference between the indices of longitudinal and transverse diameters in the eggs of the studied groups (p ˂ 0,01). Investigation of the thickness of the egg shell has shown that, according to the results, this ostentatious decreases with age (p ˂ 0,05). The obtained data coincide with the results of other researchers. After analyzing the data of the egg shape index, it was found that in the birds of the three studied groups, the average figures were in line with the norm. However, a significant difference was found between the characteristics of groups 1 and 3 (p ˂ 0.1). After analyzing the results of the incubation of eggs from the experimental groups of ducks, we found that the highest fertility of the eggs of group 3 was 92.5%, compared with the group 2–89.16% and 1–87.5%. According to the index of egg output and ducklings, the leader, the group was 3 (75.0%). The results obtained fully correspond to the data obtained from these physical and morphological studies of these eggs. The worst indices of both egg and duckin removal were in group 1. It should also be noted that this group had the largest number of choked (6.67%) and unfertilized eggs (15 pcs.), But these parameters decreased with the age of bird. Consequently, we have established that with the age of birds there is a change in the physical and morphological parameters of eggs (increase in weight, decrease in the thickness of the shell and increase in the index of suitability of eggs for incubation). The general tendency to reduce the embryonic viability of the duckies associated with the mass (group 1) and the form index (group 2) has also shown that eggs of medium size that are not below the standards for the breed standard by mass and index should be selected for incubation forms (group 3). There was a correlation between the incidence of egg incubation and the index of form in group 3, which had a direct impact on incubation and egg output.
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.
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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