Poultry Lines Research Articles

Humoral Immunity in chicken lines developed by Embrapa Suínos e Aves: Natural and specific antibodies

The antibodies produced in the first days of vertebrate life and are called natural antibodies (NAb). Other antibodies, produced in response to restricted contact with the antigen, are called specific antibodies (SpAb). To evaluate the production of NAb studies have used rabbit red blood cells (RRBC). On the other hand, evaluation of the production of specific antibodies can be performed with the use of sheep red blood cells (SRBC), a cell type that results in high production of SpAb. The aim of this study was to evaluate the production of NAb and SpAb in chicken lines developed by EMBRAPA Suínos e Aves. Animals of laying and poultry lines were inoculated intramuscularly with 5% of SRBC. The titers of NAb and SpAb were higher in laying hens than in broiler lines. The same results were obtained with anti-RRBC, the exception was males of the LLc lines, with lower antibody production compared to the other lines and females of the same line. These data show that the production of natural and specific antibodies is higher in laying hens compared to broilers, and that there is an effect of sex on antibody production.

Strategies to combat heat stress in poultry production-A review.

The effects of heat stress (HS) caused by high temperatures continue to be a global concern in poultry production. Poultry birds are homoeothermic, however, modern-day chickens are highly susceptible to HSdue to their inefficiency in dissipating heat from their body due to the lack of sweat glands. During HS, the heat load is higher than the chickens'ability to regulate it. This can disturb normal physiological functioning, affect metabolismand cause behavioural changes, respiratory alkalosis and immune dysregulation in birds. These adverse effects cause gut dysbiosis and, therefore, reduce nutrient absorption and energy metabolism. Thisconsequentlyreduces production performances and causes economic losses. Several strategies have been explored to combat the effects of HS. These include environmentally controlled houses, provision of clean cold water, low stocking density, supplementation of appropriate feed additives, dual and restricted feeding regimes, early heat conditioningand genetic selection of poultry lines to produce heat-resistant birds. Despite all these efforts, HS still remains a challenge in the poultry sector. Therefore, there is a need to explore effective strategies to address this long-lasting problem. The most recent strategy to ameliorate HS in poultry is early perinatal programming using the in ovo technology. Such an approach seems particularly justified in broilers because chick embryo development (21 days) equals half of the chickens'posthatch lifespan (42 days). As such, this strategy is expected to be more efficient and cost-effective to mitigate the effects of HS on poultry and improve the performance and health of birds. Therefore, this review discusses the impact of HS on poultry, the advantages and limitations of the different strategies. Finally recommend a promising strategy that could be efficient in ameliorating the adverse effects of HS in poultry.

Spermiogram, microbial contaminants and sensitivity to antibiotics in fresh semen of two poultry lines

This study aimed to isolate bacteria flora in fresh semen of helmeted guinea cock (Numidia meleagris) and Nigeria local turkeys (Meleagris gallopavo) and determine the sensitivity of the semen microbes to antibiotics with further assessment of sperm quality characteristics in the freshly collected semen. Five matured guinea cocks weighing approximately 3 – 4kg and five matured turkey toms of 10 – 14kg body weights were selected for the study as sperm donors. Semen collection was done twice a week for five weeks using a modified abdominal massage method and ejaculates were pooled independently based on the animal group. The pooled ejaculates in each species were further divided into aliquot A for spermiogram analysis and aliquot B for microbes and antimicrobial sensitivity analysis using standard methods. Results showed that freshly collected semen from both donor birds were contaminated; although, presented good qualities when evaluated for sperm characteristics such as progressive motility (%), livability (%), morphologic abnormalities (%) and concentration (cell×109/ml). Isolated contaminants included Escherichia coli, Shigella spp, Staphylococcus aureus and Bacillus spp in fresh semen of the helmeted guinea cocks, similar in turkeys except for Shigella spp. The organisms were all sensitive to streptomycin, gentamycin and pefloxacin except the Staph aureus which showed resistance to pefloxacin and Bacillus spp which was resistant to gentamycin. It was then concluded, that whereas freshly collected semen of guinea cock and turkey toms were contaminated during collection, they still possessed good quality traits when evaluated for semen characteristics and antibiotics like streptomycin, pefloxacin or gentamycin are recommended for use during in-vitro processing and extension of semen from both poultry species.

IMPORTANCE OF SELECTED PROTEINS OF COMPACT BONE TISSUE IN POULTRY

Within modern poultry lines, the integrity of the skeleton is subjected to increasing genetic and production stress, which in many cases leads to different health problems of the skeletal system, including problems with osteoporosis and the development of fractures. The role of genetics in bone integrity has been demonstrated by several studies, while the knowledge gained from the targeted study of genes, i.e., proteins that play an important role in bone metabolism, is of great value both for skeletal health and may provide new clues to the biological processes underlying diseases leading to the weakening of the bones. In addition to summarizing basic knowledge about bone metabolism, this review provides insight into the structure and function of proteins that are part of compact bone tissue, focusing on non-collagenous proteins and proteins that are encoded by genes involved in signaling pathways that play an important role in bone metabolism.

Research Note: Growth promoting potential in Mstn mutant quail dependent and independent of increased egg size

In avian species, positive relationships between egg weight (EW) and body weight (BW) have been reported. However, the correlation between the body growth rate and different weights of eggs from genetically mutated avian species was not studied yet. Myostatin (Mstn), an anti-myogenic factor, mutant quail were recently developed, and it was reported that EW produced from Mstn homozygous mutant quail (HO) was heavier compared to those from wild-type quail (WT). In the current study, distributions of pre-incubated EW and associations between EW and BW were compared between the Mstn mutant and WT quail lines. Average egg weight for the HO group was significantly heavier than the WT (P < 0.001) and the number of eggs having heavier EW (over 11 g) was higher in the HO compared to the WT (P < 0.01). BWs at wk (W) 0, 4, and 6 after hatch were also significantly greater in the HO (P < 0.001 in all groups). In addition, linear regression analyses revealed positive relationships between EW and BW from W0 to W6, regardless of sexes and genotypes. Furthermore, Mstn mutant quail were a heavier BW compared to the WT quail originated from eggs with similar weights. These data indicate that increased BW by Mstn mutation is contributed by increased EW and/or growth promoting activity of Mstn mutation independent of increasing egg sizes. These findings provide Mstn as a desirable genetic factor for selection of poultry breeds with superior growth. In addition, the knowledge gained from this study could inspire similar proof-of-concept studies involving standard and commercial lines of poultry.

Analysis of the accumulation of homozygosity regions in chickens of the Pushkin breed using data from whole genome genotyping

BACKGROUND: The search for genetic factors influencing the formation of the productive qualities of new breeding forms and populations of chickens is an important direction in the study of animal genomes. The knowledge gained can be in demand in projects to create modern domestic highly productive poultry lines.&#x0D; AIM: To conduct a comparative analysis of the localization of homozygous regions on chicken chromosomes in representatives of four breeds from the Genetic Collection of Rare and Endangered Breeds of Chickens to identify traces of selection and search for genes associated with productive traits.&#x0D; MATERIALS AND METHODS: Based on the bioinformatic analysis of 42275 SNP markers identified using the Illumina Chicken 60K SNP iSelect BeadChip microchip, data were obtained on the distribution of homozygous regions of four populations of chickens at the Genetic Collection of Rare and Endangered Breeds of Chickens (VNIIGRZH, St. Petersburg): Pushkinskaya (n = 20), Cornish (n = 22), Black-and-White Australorp (n = 20), Russian White (n = 23).&#x0D; RESULTS AND CONCLUSIONS: In chickens of the Pushkin breed, the observed heterozygosity was 0.372 0.004, which is higher than in other populations. According to the inbreeding index, based on the analysis of homozygous regions, the maximum level was found in Cornish chickens. For the Pushkin breed, the accumulation of homozygous regions on chromosomes 1, 2, 6, 8 was noted. The genes annotated in these loci are evidence of intensive selection.

ВЛИЯНИЕ РАЗЛИЧНЫХ КОРМОВЫХ ДОБАВОК НА АКТИВНОСТЬ ФЕРМЕНТОВ ХИМУСА И ПЛАЗМЫ КРОВИ МОЛОДНЯКА ИСХОДНЫХ ЛИНИЙ МЯСНЫХ КУР

The research aimed to determine the effect of a mixture of low molecular weight organic acids and complex phytobiotics when replacing a feed antibiotic with them on the activity of digestive enzymes in the duode-nal chyme and the activity of pancreatic enzymes in the blood plasma of young stock B5 and B9 meat chicken lines. The experiments were carried out on the original lines of meat poultry lines B5 (Cornish) and B9 (Plymouth rock). There was an operation to insert a cannula into the duodenum at the age of 6 weeks. The enzymatic activity of the duodenum chyme and the content of pancreatic enzymes in the blood plasma in the groups of chickens receiving antibiotics, low molecular weight organic acids, and phytobiotics with the diet. The data showed that the influence of feed additives on the physiological status of poultry was different. The use of low molecular weight organic acids of the B5 and B9 chicken lines had a significant effect on the production of digestive enzymes due to an increase in the activity of chyme lipase (by 98.3%) and blood plasma lipase (by 26.6%) in B9 chickens and an increase in chyme proteases (by 30.9%) in B5 chickens compared with the control group, where the antibiotic was used. The introduction of complex phytobiotics into the diet had a negative effect on chickens of the B5 line (Cornish), reducing the activity of amylase and lipase of the duodenal chyme (by 29.2 and 26.9%) compared with the control group. In B9 (Plymouth rock) chickens, only the chyme amylase activity increased by 30.8% that indicates an improvement in the availability of feed carbohydrates. These data confirm the need to take into account the different effects of feed additives on the digestion processes in different poultry crosses.

Effects of Maternal Stress on Measures of Anxiety and Fearfulness in Different Strains of Laying Hens.

Maternal stress can affect the offspring of birds, possibly due to hormone deposition in the egg. Additionally, phenotypic diversity resulting from domestication and selection for productivity has created a variety of poultry lines that may cope with stress differently. In this study, we investigated the effects of maternal stress on the behavior of different strains of laying hens and the role of corticosterone as its mediator. For this, fertilized eggs of five genetic lines—two brown (Brown 1 and 2), two white (White 1 and 2), and one pure line White Leghorn—were reared identically as four flocks of 27 birds (24F: 3M) per strain. Each strain was equally separated into two groups: Maternal Stress (“MS”), where hens were subjected to a series of daily acute psychological stressors for 8 days before egg collection, and “Control,” which received routine husbandry. Fertile eggs from both treatments were collected at three different ages forming different offspring groups that were treated as replicates; additional eggs from Control were injected either with corticosterone diluted in a vehicle solution (“CORT”) or just “Vehicle.” Eggs from each replicate were incubated and hatched, and offspring (N = 1,919) were brooded under identical conditions. To measure the effects of maternal stress on anxiety and fear-like behavior, offspring were subjected to a social isolation test (SI) between 5 and 10 days of age and a tonic immobility test (TI) at 9 weeks of age. Compared to Control, MS decreased the number of distress vocalizations emitted by White 2 in SI. No effects of MS were observed in TI, and no effects of CORT were observed in any tests. Overall, brown lines vocalized more in SI and remained in TI for a longer duration than white strains, suggesting genetic differences in fear behavior. Females vocalized more than males in TI and showed a trend toward significance for the same trait in SI. Overall, results suggest that the effects of maternal stress on fearfulness are not directly mediated by corticosterone. Moreover, it highlights behavioral differences across various strains of laying hens, suggesting that fear responses are highly dependent on genotype.

Genotype frequency distributions of 28 SNP markers in two commercial lines and five Chinese native chicken populations

BackgroundModern breeding in the poultry industry mainly aims to produce high-performance poultry lines and breeds in two main directions of productivity, meat and eggs. To understand more about the productive potential of lowly selected Chinese native chicken populations, we selected 14 representative SNP markers strongly associated with growth traits or carcass traits and 14 SNP markers strongly associated with egg laying traits through previous reports. By using the MassArray technology, we detected the genotype frequency distributions of these 28 SNP markers in seven populations including four lowly selected as well as one moderately selected Sichuan native chicken populations, one commercial broiler line and one commercial layer line.ResultsBased on the genotype frequency distributions of these 28 SNP markers in 5 native chicken populations and 2 commercial lines, the results suggested that these Chinese indigenous chicken populations have a relatively close relationship with the commercial broiler line but a marked distinction from the commercial layer line. Two native chicken breeds, Shimian Caoke Chicken and Daheng Broilers, share similar genetic structure with the broiler line.ConclusionsOur observations may help us to better select and breed superior domestic chickens and provide new clues for further study of breeding programs in local chicken populations.

221 Isolation of turkey spermatogonia

Across different types of spermatogenic epithelium, researchers have focused on the study and production of spermatogonial stem cells and their use as genetic material for creating gene pool cryobanks of valuable breeds and lines of poultry. The aim of our research was to identify optimal conditions for obtaining and culturing turkey spermatogonia. A series of experiments were carried out to determine the optimal age of males for isolating spermatogonia and optimize conditions for their isolation and cultivation. Age-related features of turkey spermatogenesis were assessed by testis histological studies. Statistical analysis was performed using SPSS v15.0 (t-test; SPSS Inc./IBM Corp.). Turkey testis tissue was mechanically crushed and treated with enzymes: 0.1% collagenase and 0.25% trypsin. Purification of spermatogonia from other types of cells was performed by adhesion and separation. The conditions and duration of spermatogenic cell cultures were selected experimentally. Primary Sertoli cells of turkeys, Sertoli cells of roosters, STO cell line, and transplanted Sertoli cells of pigs (PTP) were used as a feeder layer for the cultivation of spermatogonia. Spermatogonia culture medium consisted of Dulbecco's modified Eagle's medium (DMEM)-high glucose supplemented with 5% fetal calf serum, 2mM α-glutamine, 10µLmL−1 MEM, 100x antibiotics, 10µLmL−1 ITS solution, 5×10−5 M mercaptoethanol, 5mgmL−1 albumin, 1µLmL−1 DL-lactic acid, 20ngmL−1 epidermal growth factor, 10ngmL−1 basic fibroblast growth factor, and 2ngmL−1 leukemia inhibitory growth factor (LIF). The SSEA-1 antibody was used for identification of spermatogonia colonies. Histological study of the turkey testes at different ages showed that in animals under 12 weeks, the population of generative cells was represented mainly by spermatogonia (P&amp;lt;0.05). Thus, spermatogonia were isolated from the testes of 4-wk-old males. Isolated cells were cultured for 24h, after which the supernatant containing non-adherent cells (mainly spermatogonia) was transferred to a new dish for continued culture. Maximal homogeneity of the cell population was detected after transferring non-adherent cells 3 times at 24-h intervals. The best cell growth was observed when spermatogonia were cultivated on turkey Sertoli cells. The number of these cells in suspension reached 81% purity. Formation of spermatogonia colonies was observed on Days 7 to 8 of cultivation, the presence of which was confirmed by immunohistochemical staining with SSEA-1. This study was supported by the Russian Science Foundation within project no.16-16-04104.

The phenomenon of the monovular twinning in the endangered zatorska goose

The phenomenon of multiple embryos in birds and other oviparous vertebrates is very rare and thus poorly studied. Some populations of wild birds or lines of poultry, including the zatorska goose, may be particularly predisposed to developing monovular twin embryos (MTE). The aim of the study was to investigate the frequency of single yolk twin embryos in the zatorska goose and to establish whether these embryos are generated as a result of polyembryony or other developmental anomalies.Embryopathological analysis, which included identification of the developmental stages of all MTEs at the time of death, was carried out on 1,955 unhatched eggs obtained from 20 pedigree breeding flocks of the zatorska goose. Post-mortem examinations revealed as many as 13 MTEs (0.66% compared to embryonated eggs) at varying developmental stages.The results of genotyping of the microsatellite loci of 7 pairs of twins demonstrated genetic similarity in 6 of them; this suggests that they developed from 1 blastoderm, while differences in 2 loci of 1 pair show that the MTE originates from 2 blastodiscs. Pedigree analysis of the MTEs does not provide evidence that predisposition toward twinning results from the genetic background. However, more frequent MTE occurrence could be seen in eggs stored for more than 8 d. It seems that embryos of the zatorska goose are more susceptible to duplication under special conditions, such as prolonged egg storage.

Avian Biotechnology.

Primordial germ cells (PGCs) generate new individuals through differentiation, maturation and fertilization. This means that the manipulation of PGCs is directly linked to the manipulation of individuals, making PGCs attractive target cells in the animal biotechnology field. A unique biological property of avian PGCs is that they circulate temporarily in the vasculature during early development, and this allows us to access and manipulate avian germ lines. Following the development of a technique for transplantation, PGCs have become central to avian biotechnology, in contrast to the use of embryo manipulation and subsequent transfer to foster mothers, as in mammalian biotechnology. Today, avian PGC transplantation combined with recent advanced manipulation techniques, including cell purification, cryopreservation, depletion, and long-term culture in vitro, have enabled the establishment of genetically modified poultry lines and ex-situ conservation of poultry genetic resources. This chapter introduces the principles, history, and procedures of producing avian germline chimeras by transplantation of PGCs, and the current status of avian germline modification as well as germplasm cryopreservation. Other fundamental avian reproductive technologies are described, including artificial insemination and embryo culture, and perspectives of industrial applications in agriculture and pharmacy are considered, including poultry productivity improvement, egg modification, disease resistance impairment and poultry gene "pharming" as well as gene banking.

Effects of rearing environment on the gut antimicrobial responses of two broiler chicken lines

To reduce the risk of enteric disease in poultry, knowledge of how bird gut innate defences mature with age while also responding to different rearing environments is necessary. In this study the gut innate responses of two phylogenetically distinct lines of poultry raised from hatch to 35days, in conditions mimicing high hygiene (HH) and low hygiene (LH) rearing environments, were compared. Analyses focussed on the proximal gut antimicrobial activities and the duodenal and caecal AvBD1, 4 and 10 defensin profiles.Variability in microbial killing was observed between individual birds in each of the two lines at all ages, but samples from day 0 birds (hatch) of both lines exhibited marked killing properties, Line X: 19±11% (SEM) and Line Y: 8.5±12% (SEM). By day 7 a relaxation in killing was observed with bacterial survival increased from 3 (Line Y (LY)) to 11 (Line X (LX)) fold in birds reared in the HH environment. A less marked response was observed in the LH environment and delayed until day 14. At day 35 the gut antimicrobial properties of the two lines were comparable.The AvBD 1, 4 and 10 data relating to the duodenal and caecal tissues of day 0, 7 and 35 birds LX and LY birds revealed gene expression trends specific to each line and to the different rearing environments although the data were confounded by inter-individual variability. In summary elevated AvBD1 duodenal expression was detected in day 0 and day 7 LX, but not LY birds, maintained in LH environments; Line X and Y duodenal AvBD4 profiles were detected in day 7 birds reared in both environments although duodenal AvBD10 expression was less sensitive to bird age and rearing background. Caecal AvBD1 expression was particularly evident in newly hatched birds.These data suggest that proximal gut antimicrobial activity is related to the bird rearing environments although the roles of the AvBDs in such activities require further investigation.

Identification and characterisation of alternative transcriptional variants of PDGFRL in two lines of commercial poultry.

The platelet-derived growth factor (PDGF) family of genes and their receptors are involved in angiogenesis and steroid hormone production. A putative member of the family, platelet-derived growth factor receptor-like (PDGFRL), has been implicated in steroid-based feedback mechanisms within the chicken reproductive system. Three potential variants of PDGFRL were identified in the chicken, supported by in silico prediction and EST sequencing. The three potential transcripts have been further verified and the 5' terminal regions sequenced in this research. The sum of expression of all three transcripts in broiler breeders (the parents of broiler chickens) has been shown to be consistent with total expression of the gene. However, cumulative expression of the three transcripts in a range of tissues in egg layers was significantly short of total expression, indicating the existence of potential additional variants. Two additional variants were subsequently identified in egg layer cerebellum tissue and the 5' terminal regions sequenced. Sequence analysis of the three initial variants suggests that only one variant, which was the most abundant in broiler breeders and the majority of egg layer tissues, had a functional signal peptide. Although 5'RACE identified two additional transcripts in egg layers, the most likely protein translations indicated that these variants possessed no functional signal peptide, suggesting that, if they have a function, it is not a traditional one.

Current status and phenotypic characteristics of Bulgarian poultry genetic resources

SummaryPoultry biodiversity conservation is a great challenge for many countries. Within the last several years, the number of endangered local breeds has increased, leading to a considerable loss of genetic resources. A similar trend was observed among the poultry breeds, including chicken, local turkey and goose breeds/lines established in Bulgaria, part of which is definitely lost. Currently these breeds/lines are at risk and/or threatened with extinction. The information obtained by phenotypic characterization of these breeds is the first step for planning the management of poultry genetic resources through setting up improved selection schemes and conservation strategies. In this paper, we reviewed the current state of knowledge regarding the morphological and phenotypic diversity of local poultry breeds and some old productive poultry lines in Bulgaria.

한국재래닭(오계)의 원시 생식 세포의 냉동 보존에 있어서 여러 조건의 평가

동결 닭 원시 생식 세포의 생식계열 키메라를 이용한 생체에의 복원을 실용화 하기 위해서는, 닭 원시 생식 세포의 동결 보존 기술의 향상에 의해 동결 및 융해 후의 많은 생존세포를 확보하는 것이 반드시 필요하다. 닭 원시 생식 세포는 배양 5.5일령의 닭 원시 생식선으로부터 채취하고, MACS 방법에 의해서 순수 닭 원시 생식 세포를 분리했다. 15% 각각의 EG를 동결 보호제로 사용한 처리군이 각 군의 농도에 상관없이 유의적(p<0.05)으로 PG 처리군보다 동결 및 융해 후의 세포의 생존율이 높음을 확인하였다. 특히, 동결 보호제로 10% EG를 이용한 유리화 처리군에서 85.63%로 동일한 농도의 PG 처리군(66.81%)보다 유의적(p<0.05)으로 가장 높은 생존율을 보였다. 한편, 10% EG를 이용한 완만 동결 처리군에서 66.14%로 동일한 농도의 PG 처리군(50.11%)보다 유의적(p<0.05)으로 가장 높은 생존율을 보였다. 이상의 결과들로부터 유리화 동결에 있어서 가장 높은 생존율을 보인 10% EG이 최적의 동결 보호제로서 사용 가능함을 확인하였고, 이는 한국재래닭(오계)의 원시 생식 세포의 동결 보존의 실용화가 보다 더 향상될 수 있는 또 하나의 방법이 될 수 있음을 시사한다. Cryopreserving cells which are maintaining their viability are the very complex process. This study has been carried out in order to find the effects of cryopreservation steps and freezing media on the rates of viability of cryopreserved chicken primordial germ cells (PGCs). PGCs obtained from the germinal gonade of 5.5~6 day (stage 28) chick embryos of Korean Ogye (KO) and Commercial breeds (C), using the MACS method were suspended in a freezing medium containing a freezing and protecting agents (e.g. dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PG)). Gonads were harvested from stage 28 chick embryos and pooled in groups of 5, 10, 15, 20E embryos, contributing gonads to the cell suspension. The gonadal cells, including PGCs, were then frozen in 1 of the following cryoprotectant treatments : 2.5%, 5%, 10%, 15% and 0% cryoprotectant (DMSO, EG, PG) as a control. Effects of exposure to slow freezing and vitrification, with different concentrations of the cryoprotectant solution, were examined. After vitrification and slow freezing, survival rates of the frozen-thawed PGCs from the 10% EG plus FBS treatment were 85.63%, and 66.14% (p<0.05), respectively. The viability of PGCs after freeze-thawing was significantly higher for 10% EG plus FBS treatment than for 10% PG + FBS treatment (p<0.05) (85.63% vs 66.81%) by vitrification. This study established a method for preserving chicken PGCs that enables systematic storage and labeling of cryopreserved PGCs in liquid (<TEX>$LN_2$</TEX>) at a germplasm repository and ease of entry into a data base. In the future, the importance for this new technology is that poultry lines can be conserved while work is being conducted on improving the production of germline chimeras.

Genetic correlation between egg quality traits.

Genetic correlation coefficients between egg quality traits were studiedusing fifty- six harco pullets raised under standard management practices. Theestimates were carried out at 22-week, 26-week, 30-week and 32-week of age. Eggquality traits studied include egg weight (EW), shell thickness (ST), haughunit (HU), yolk index (YI) and shape index (SI). Zero to slight negative correlationcoefficients was observed between EW and HU, EW and YI, EW and SI, ST and SI,and HU and YI. This implies that selection for any of the character entailsminimal retrogressive response on corresponding trait. Conversely, zero toslight positive correlation coefficients was recorded between EW and SI, SI andYI, and HU and SI. Again, selection for any of this character will bring abouta slight genetic gain on the corresponding trait. Furthermore, the study maintainedthat the genetic correlation coefficients between egg quality traits areindependent of the laying age, and for faster genetic response in egg qualitytraits, independent culling method. Again, since genes governing the expressionof these egg quality traits appear to be independent of each other, it ispossible to localize, isolate and intersperse these genes simultaneously infoundational poultry lines using biotechnological tools for faster geneticgain.

63 THE EFFECT OF A MODIFIED CRYOPRESERVATION METHOD ON THE VIABILITY OF FROZEN–THAWED PRIMORDIAL GERM CELL ON THE KOREAN NATIVE CHICKEN (Ogye)

Cryopreservation of poultry semen has been reported, but preservation of female genetic material has not been possible because of the unique anatomical and physiological characteristics of the avian egg. Thus, conservation of genetic material in chickens was attempted by preserving primordial germ cells (PGC) in LN2. This study established a method for preserving chicken PGC that enables long-term storage in LN2 for preservation of species. The purpose of this study was to clarify the effects of fetal bovine serum (FBS) or chicken serum (CS) treatment on the viability of cryopreserved PGC in the Korean native chicken (Ogye). Primordial germ cells were separated from a germinal gonad using a fine glass micropipette under a microscope and were suspended in a freezing medium containing freezing and protecting agents [e.g. dimethyl sulfoxide (DMSO) and ethylene glycol (EG)]. The PGC were then purified using the magnetic-activated cell sorting (MACS) method. The viability of the PGC in both groups was determined by the trypan blue exclusion method. The values of the 0, 5, 10, and 15% DMSO plus FBS treatment were 21.6, 30.36, 36.42, 50.39, and 48.36%, respectively. The viability of PGC after freeze-thawing was significantly higher for the 10% EG plus FBS treatment than for the 10% EG plus CS treatment (P &lt; 0.05; 64.36 v. 50.66%). This study established a method for preserving chicken PGC that enables systematic storage and labelling of cryopreserved PGC in LN2 at a germplasm repository and ease of entry into a database. In the future, the importance of this new technology is that poultry lines can be conserved while work is being conducted on improving the production of germ line chimeras.

МЕТОДЫ ГЕНЕТИЧЕСКОЙ МОДИФИКАЦИИ И СЕЛЕКЦИЯ СЕЛЬСКОХОЗЯЙСТВЕННОЙ ПТИЦЫ (обзор)

The positive practical results in poultry breeding due to investigations on transgenesis and their prospects are discussed. The method for obtaining transgenic chickens and quail by microinjection of foreign DNA into eggs are described. It is based on a surgical operation, providing the access to the eggs to conduct microinjection of DNA and the natural formation of tertiary egg shells in the bird's genital tract. The microinjection into eggs at different stage of their development is considered in more detail. The transgenic chickens with humane growth hormone gene, b-galactosidase gene and humane b-interferon gene, and the transgenic quails with bovine growth hormone gene were produced by this technique. The quails with introduced bovine growth hormone gene and their offspring generations are characterized by an increased average egg mass and the higher immune status. Transgenesis used to transfer a target gene between two poultry lines, if compared with common breeding technologies, takes at least 7 to 8 years less and is probably less expensive, because there is no need in backcrosses to remove the unnecessary genes transmitted in the course of natural sexual hybridization. Nowadays, the transgenesis is very similar to directed, but indeterminate mutation. A transgene chances to insert genome, and that is why a lot of initial transgenic individuals are needed to achieve an inherited transgenesis.

028 Successful cryopreservation of avian germplasm: Why a multifaceted approach is required

The value of the ability to cryopreserve and store germplasm has long been recognized for indefinite preservation of genetic material, especially for “at-risk” populations. For domestic livestock species, options for preserving genetic material in frozen form range from sperm to oocytes to embryos. More than sixty years ago, the discovery of glycerol’s cryoprotective properties for rooster sperm pioneered the success of modern cryobiology and led to the development of semen cryopreservation for a wide range of species. Despite decades of research, the overall fertility rates of frozen/thawed poultry sperm are highly variable and not reliable enough for preservation of genetic stocks. The relatively low fertilizing ability most likely results from physiological sensitivity to the cryogenic process coupled with the requirement for prolonged sperm functionality in the hen reproductive tract. We have conducted systematic experiments to understand how and why poultry sperm lose functional competence after cryopreservation, and have shown that the glycocalyx, the carbohydrate-rich zone on the sperm surface, is altered by the cryogenic cycle. The amount of sialic acid, a terminal sugar critical for sperm transport in the female reproductive tract, was reduced after cryopreservation; however, we have shown that sperm cells can incorporate exogenous sialic acid, suggesting a strategy for sperm freezing protocols. Second, sperm lipids such as phosphotidylcholine also are altered during cold storage. The ratio of lipids in the sperm membrane determine the overall fluidity of the membrane and impact the ability of sperm to remain viable during the wide temperature fluctuations that are part of the cryogenic cycle. We have shown that supplemental lipids can circumvent the loss of phospholipids during cryopreservation. Finally, we have conducted the first proteomic analysis of poultry sperm and have identified three proteins not previously found in sperm from any species to date (dihydropyrimidinase, mitofilin and mitochondrial tri-functional protein). We currently are evaluating the proteomic profile of frozen/thawed sperm to determine the impact of cryopreservation on sperm proteins. Taken together, these data suggests innovative strategies for developing reliable poultry semen cryopreservation methods. For most species, sperm cryopreservation effectively captures the entire genome; however, in birds, the female is the heterogametic sex. Cryopreservation of avian oocytes and embryos is problematic because of the macrolecithal characteristics of the egg, where the surface-to-volume ratio poses a significant barrier to the movement of solutes/cryoprotectants and impedes effective protection of the cells from freeze/thaw damage. Recent advances for manipulation of poultry primordial germ cells and culture of embryonic stem cells suggests that cryopreservation of these cell types, followed by re-implantation in recipient embryos, could provide a mechanism to preserve the entire genome; albeit the efficiency rates of reconstitution associated with the current technologies are rather low. A novel innovation of cryopreservation and transplantation of ovarian tissue offers an alternative for whole genome preservation in birds (Song and Silversides, 2008); although chemical treatment to reduce the growth of host germ cells does not completely prevent the production of host-derived offspring. Reconstitution of poultry lines will most likely rely on more than one type of cryopreserved material. Source of funding: USDA ARS Project Number: 1245-31000-105-00. Conflict of interest: None declared. [email protected]