Cryoconservation of Chicken Blastodermal Cells: Effects of Slow Freezing, Vitrification, Cryoprotectant Type and Thawing Method during In Vitro Processing.

Cryoconservation of blastodermal cells (BCs) can preserve genetic material for the future reconstruction of poultry breeds. The aim of our study was to compare the effects of three slow freezing programs and vitrification, different cryoprotectants (5% DMSO, 10% DMSO, or multi-component cryoprotectant (MC) and two thawing methods on the viability of chicken BCs. Significant differences in the survival of slowly frozen BCs using program 3 (2°C/min. to 0.4°C/min.) compared with programs 1 (1°C/min. to 0.3°C/min.) and 2 (4°C/min. to 0.3°C/min.) were observed. The percentage of live BCs was significantly higher after slow freezing in the presence of the MC compared with DMSO. The thawing method did not have a significant effect on the percentage of live BCs. We also observed significant differences in the survival rate of BCs after vitrification (81%) and slow freezing in the presence of 10% DMSO using program 3 (60%). The highest percentage of viable BCs was achieved by slow freezing with the MC using program 2 and thawing with method 1 (94%). The most unfavorable combination for BCs survival was slow freezing in 5% DMSO using program 3 and thawing with method 2 (58.3%). This is the first study to apply MC to the slow freezing of BCs. We also showed successful BCs vitrification.

Influence of synbiotics delivered in ovo on immune organs development and structure.

Prebiotics and probiotics applied alone or together (synbiotics) can influence the intestinal microbiota and modulate the immune response. We analyzed the impact of in ovo administration of synbiotics on immune system development in Ross (broiler) and Green-legged Partridgelike (GP, dual-purpose fowl) chickens. For in ovo delivery on the 12th day of the eggs incubation, two strains of lactic acid bacteria (LAB) were used, i.e. Lactococcus lactis subsp. lactis IBB SL1 (S1) and Lactococcus lactis subsp. cremoris IBB SC1 (S2), combined with raffinose family oligosaccharides (RFO) prebiotic. Other treatments included in ovo delivery of commercial synbiotic (S3), RFO prebiotics alone (P) and physiological saline (C). Immune system development was analyzed by relative weight (indices) and histology of the lymphatic organs (bursa of Fabricius, thymus and spleen) at two time points (3rd and 6th week of life). The results indicate that the development of the lymphatic organs was significantly affected by in ovo treatment. The bursa and bursa to spleen index was higher in P and S2 groups of broilers (P < 0.05) when compared to S3. In GP at the 3rd week of age, the spleen index was significantly higher in S2 (P < 0.05). The histological image of the thymus displayed an increase of thymocytes in the cortex in all synbiotic-treated groups (S1, S2, S3). In ovo delivery of synbiotics is an efficient mode of immune system stimulation in chickens but its efficiency depends on chicken genotype.

Expression of myogenic genes in chickens stimulated in ovo with light and temperature.

Since chicken myogenesis is tightly controlled by myogenic regulatory factors (MRFs), the external stimuli (e.g. light or temperature) affecting the proliferation and differentiation of the muscle cells have a primary effect on the gene expression of MRFs. The aim of this study was to analyze the expression of some of MRF genes (MyoD1, myogenin and Myf5) in response to the stimulation of chicken embryos with green light (AL group) or increased temperature (38.5°C; AT group) on day 18 of embryo development (18ED) as well as on days 4 (4PHD) and 8 (8PHD) post hatch. To achieve this goal a quantitative reverse transcription polymerase chain reaction was used. The most prominent differences in gene expression were observed before hatching. Relative expression of MyoD1 on 18ED was higher (p<0.05) in AL and control (AC) groups in comparison to the AT group. Myogenin expression on 18ED was lower (p<0.05) in control chickens than in both treated groups. Light stimulation in ovo decreased (p<0.05) the Myf5 expression on 18ED in comparison to the control group. Green-light illumination applied during in ovo development had more pronounced effects on mRNA level of MRFs genes measured during both the pre- and post hatch development. The elevated temperature applied during embryonic development affected only the 18ED time point. This suggests that the effect of green-light illumination on chicken myogenesis was more prolonged than that of elevated temperature.

Cryoconservation of embryonic cells and gametes as a poultry biodiversity preservation method.

A report of the Food and Agriculture Organization of the United Nations (FAO) from 2000 claims that 9% of the global farm animal population is in a critical condition and 39% is threatened with extinction. Production efficiency, exploitation and conservation of animal genetic resources are crucial not only for the global economy, but also for the environment. As many as 30% of poultry breeds are threatened with extinction and 9% have already gone extinct. To preserve the genetic resources in situ methods are used, however, they need to be supported by an ex situ strategy. This includes the storage of genetic material in liquid nitrogen under a deep freeze. This process can be performed by using electronically-controlled programs or vitrification. Data shows that usage of electronically-controlled programs leads increases cell viability. A good technique of cell culture and freezing methods will give a broad perspective for unlimited storage of genetic recourses, which in the future can be useful for the restoration of extinct species/breeds.