Physicochemical and Biological Characterization of rhC1INH Expressed in CHO Cells

The disfunction or deficiency of the C1 esterase inhibitor (C1INH) is associated with hereditary or acquired angioedema (HAE/AAE), a rare life-threatening condition characterized by swelling in the skin, respiratory and gastrointestinal tracts. The current treatment options may carry the risks of either viral infection (plasma-derived Berinert®) or immune reaction (human recombinant C1INH from rabbit milk, Ruconest®). This study describes the physicochemical and biological characterization of a novel recombinant human C1 esterase inhibitor (rhC1INH) from Chinese hamster ovary (CHO) cells for the treatment of hereditary angioedema compared to the marketed products Berinert® and Ruconest®. The mass spectrometry results of total deglycosylated rhC1INH revealed a protein with a molecular mass of 52,846 Da. Almost full sequence coverage (98.6%) by nanoLC-MS/MS peptide mapping was achieved. The purity and C1s inhibitory activity of rhC1INH from CHO cells are comparable with Ruconest®, although we found differences in charge isoforms distribution, intact mass values, and N-glycans profile. Comparison of the specific activity (IC50 value) of the rhC1INH with human C1 esterase inhibitor from blood serum showed similar inhibitory properties. These data allow us to conclude that the novel rhC1INH molecule could become a potential therapeutic option for patients with HAE/AAE.

Reproductive Performance of Hycole Rabbit Does, Growth of Kits and Milk Chemical Composition during Nine Consecutive Lactations under Extensive Rhythm

The goal of this study was to analyze the reproductive performance of does, growth of their kits, and chemical composition of their milk over nine consecutive parities in order to indicate the boundary of female reproductive profitability. The novelty of this study results from the combinations of three factors: extensive reproductive rhythm, commercial farming conditions, and a period of nine consecutive parities, showing the actual lifespan of a rabbit doe on commercial farms. The data was collected on 60 Hycole females kept at a commercial rabbit farm. Throughout the study, 32 does were excluded due to different reasons (e.g., excluded by means of selection—43.8% and mortalities—25.0%). The does were first inseminated at 28 weeks of age. Following artificial inseminations were conducted 14–15 days after each parturition. All kits were weaned at the age of 35 days. The following characteristics were analysed: body weight of rabbit does at artificial insemination, milk production per lactation, litter size, litter weight, average kit weight, and milk chemical composition. Rabbit does had a significant decrease in kindling rate between the eighth and the ninth parity (by 10.0 percentage points; p = 0.039). The litter size at weaning in the ninth parity was significantly lower to litters weaned at other analysed parities. The amount of milk produced per lactation was affected by the parity order (6.31–6.76 kg; p = 0.042). The litter weights on day 21 and 35 were the lowest at ninth parity. The content of total solids (TS), solids-not-fat, and fat was affected by the parity order on both analysed lactation days. The content of TS and fat in rabbit milk was characterized with a decreasing trend over the analysed period, on both lactation days. The results clearly indicate that rabbit does under extensive reproductive cycles characterize with a very good reproductive performance and can be successfully used for reproduction even up to the eighth parity. However, further research is needed if keeping them longer will not be profitable.

Comparison of Rabbit, Kitten and Mammal Milk Replacer Efficiencies in Early Weaning Rabbits

Early weaned rabbits should be fed using a milk replacer in order to survive. Therefore, a rabbit milk replacer (RMR) was developed and compared with a kitten milk replacer (KMR®: KMR) and a mammal milk replacer (Zoologic® Milk matrix 30/52: MMR). Thirty-six native crossbred rabbits aged 18 days were divided into three experimental groups (six replicates/group, two rabbits/replicate), fed RMR, KMR or MMR daily until they were 36 days old and euthanized at 38 days, while a complete pelleted diet and water were provided ad libitum. No statistically significant differences were observed in growth performance parameters, water intake, faecal weight, nutrient digestibility, internal organ weight, caecal pH, caecal cellulose activity, number of faecal pellets and amount of crude protein intake (p > 0.05). Caecal amylase activity in the KMR group and caecal protease activity in the RMR group were higher than in the MMR group (p < 0.05). The villus height and crypt depth of the MMR group were greater than in the RMR and KMR group (p < 0.05). In conclusion, it is possible to feed RMR to early weaning rabbits without serious adverse effects. However, probiotics and/or prebiotics should be supplemented in milk replacers and their benefits studied.

Hycole Doe Milk Properties and Kit Growth

The level of production and the physiochemical traits of rabbit milk affect the growth and the mortality of bunnies during lactation. The goal of the study was to analyze the effect of litter size and day of lactation on the quality traits of rabbit milk, milk production, and associative traits. The study was conducted on 32 Hycole does and their litters. The rabbit milk pH ranged from 6.61 to 7.46. The colostrum was characterized by the highest content of total solids (31.54 and 31.80 g kg−1) and fat content (15.73 and 15.9 g kg−1). The milk from the beginning of the lactation was characterized by the highest level of somatic cell count (SCC) (523.67 and 536.57 103 mL−1), which gradually decreased to reach the lowest level on days 17 and 21 of lactation. The daily milk production was greater for does nursing 10 kits per litter compared to those nursing eight kits per litter (p < 0.001). The peak of milk production occurred on day 17 postpartum. To conclude, the litter size has a clear effect on milk production as well as litter weight and litter weight gains. It is also important to note that the day of lactation affected the physiochemical traits of rabbit milk.

Green fluorescent protein gene for site-specific integration into the locus of the rabbit whey acidic protein gene

The high content of whey acidic protein in rabbit milk makes the gene of this protein a promising candidate for its replacement by the gene of pharmacologically active protein using the CRISPR/Cas9 system. The plasmid that contains 5’ and 3’ arms of homology to the rabbit WAP gene was created. A fragment containing a green fluorescent protein gene under the CMV promoter has been integrated into this site. A strategy of making double-stranded cuts in the gene WAP and receiving four pX330 plasmids encoding the endonuclease Cas9 and guide RNAs was developed. The plasmid containing a fragment cmvEGFP was designed for site-specific integration by homologous recombination into the gene WAP to assess the effectiveness of site-specificity of components of the CRISPR/Саѕ9 in vitro.

Recombinant Human Tissue Non-Specific Alkaline Phosphatase Successfully Counteracts Lipopolysaccharide Induced Sepsis in Mice

Sepsis is a life threatening condition that arises when the body's response to an infection injures its own tissues and organs. Sepsis can lead to shock, multiple organ failure and death especially if not recognized early and treated promptly. Molecular mechanisms underlying the systemic inflammatory response syndrome associated with sepsis are still not completely defined and most therapies developed to target the acute inflammatory component of the disease are insufficient. In this study we investigated a possibility of combating sepsis in a mouse model by intravenous treatment with recombinant human tissue non-specific alkaline phosphatase (rhTNAP) derived from transgenic rabbit milk. We induced sepsis in mice by intraperitoneal injection of LPS and three hours later treated experimental group of mice by intravenous injection with rhTNAP derived from transgenic rabbits. Such treatment was proved to be physiologically effective in this model, as administration of recombinant rhTNAP successfully combated the decrease in body temperature and resulted in increased survival of mice (80 % vs. 30 % in a control group). In a control experiment, also the administration of bovine intestinal alkaline phosphatase by intravenous injection proved to be effective in increasing survival of mice treated with LPS. Altogether, present work demonstrates the redeeming effect of the recombinant tissue non-specific AP derived from milk of genetically modified rabbits in combating sepsis induced by LPS.