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.

Microsatellite markers are now been widely used for the detection and description of micropopulation processes occurring in the populations of domestic animals for the effects of various factors of breeding pressure. Microsatellite loci distributed throughout eukaryotic genomes, making them the preferred genetic marker for high resolution genetic mapping. In recent years, rapid advances have been made in the development of molecular genetic maps. High-density linkage maps are now available for many farm animals, such as cattle, pigs, and goats. In contrast, mapping studies in avian species are much less advanced except in the chicken. According to FAO about 70% of ducks are bred in China. This country is a leader in growing ducks. The Shaoxing breed is one of the three major duck breeds in China. Ducks of this breed are characterized by high performance. According to the Bureau of Product Quality, the age of maturity (the beginning of egg laying) in these birds occurs at 130-140 days. The characteristics of the Shaoxing breed include the fact that the peak period of laying eggs lasts from eight to ten months. On average, one duck in 500 days gives from 290 to 310 eggs, which is one of the highest rates for egg breeds. That is why the purpose of our study was the microsatellite analysis of two populations of Shaoxing breed with 9 locuses was conducted. The selection of birds for the study were carried out on a duck farms in Zhejiang Generation Biological Science and Technology Co., Ltd. and Zhuji Guowei Poultry Development Co, Ltd., and at the laboratory of the Jjejiang Academy of Sciences Institute. Samples collection and DNA preparation: Venous blood samples were collected from 480 ducks (240 ducks of population I and 240 ducks of population II of the Shaoxing breeds) of both populations into 3 ml tubes containing EDTA as anticoagulant agent. In total of 9 investigated loci in the Shaoxing breed population, only one locus was monomorphic (SMO10). The number of different alleles (Na) for each polymorphic locus ranged from 2 (SMO12) to 13 (APL79, CMO11) in population I and from 2 (APL78, SMO12) to 7 (APL79) in population II. On average, one locus had 5.889 alleles in population I and 3.889 of alleles in the population II. The effective number of alleles (Nе) was 1.735 in population I and 1.599 in population II. The number of alleles and the expected heterozygosity (Hexp) values can provide important information for the discrimination of individuals and breeds. The index of expected heterozygosity in population I was 0.336 and 0.307 in population II. The information index (I) was 0,702 in population I and 0,576 in population II. For each population was found private alleles, in population I 6 alleles and in population II just 4 alleles. The results show high level of polymorphism of the studied populations of ducks. The obtained results can be used in the creation of new lines of ducks.

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 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.

No 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 у біотехнологічних процедурах по створенню трансгенних качок.