Subcutaneous application of hyperimmune serum against Histophilus somni recombinant proteins affects serum antibody reactivity in beef calves.

BACKGROUND: Respiratory tract diseases cause significant economic loss in beef cattle. This study aimed to determine whether the application of hyperimmune serum (HS) containing antibodies against selected antigens of Gram-negative bacteria would improve the health and growth of different breeds of beef calves kept on three farms. Two recombinant protein antigens (Histophilus somni rHsp60 and rOMP40) were used to immunize four cows to produce HS. Eighty seven beef calves (Charolaise n = 36, Limousine n = 34, and crossbreed n = 17) were included into study. One hundred milliliters of serum were administered subcutaneously to 43 beef calves (Charolaise n = 18, Limousine n = 17, and crossbreed n = 8) twice, between 1 and 5 and 21-28 days of life. Calves were examined three times, and blood samples were taken to evaluate immunoglobulin M, G1, and G2, fibrinogen, serum amyloid A, and haptoglobin concentrations and reactivity of these Ig classes of antibodies against H. somni rHsp60 and rOMP40. Average daily weight gain during the first month and until weaning was calculated. RESULTS: HS showed higher (p ≤ 0.05) reactivity in calf sera against H. somni rHsp60 and OMP40 in IgG1 and IgG2. In experimental calves, compared to control calves, the reactivity of IgG1 against rOMP40 in the second sampling was higher in Limousine calves (p ≤ 0.001) and in the other two herds (p ≤ 0.05). Serum IgG2 antibody activity against H. somni rHsp60 in the second sampling was higher in experimental calves than in control calves in charolaise (p ≤ 0.05) and limousine (p ≤ 0.001) herds. The reactivity of IgG2 against rOMP40 in the second sampling of experimental calves was higher in herds with Charolaise and Limousine calves (p ≤ 0.001) and in crossbred calves (p ≤ 0.05). In the third sampling, serum IgG1 antibody reactivity against rOMP40 in Limousine calves was higher (p ≤ 0.05) in the experimental group. Among the other evaluated parameters, only SAA in the second sampling in the herd with Charolaise calves and heart rate in the herd with Limousine calves were significantly higher in the control calves (p ≤ 0.05). CONCLUSION: The application of HS to calves in all herds had an impact on specific reactivity in IgG1 and IgG2 classes against H. somni rOMP40 and rHsp60, antigens which were used for serum production.

Impact of early posthatch feeding on the immune system and selected hematological, biochemical, and hormonal parameters in broiler chickens.

Under commercial conditions, chicks hatch within a 24 to 48 h window, a period known as the hatching window. Subsequently, they undergo various treatments before finally being transported to the broiler farm. These procedures may delay the chicks' access to food and water, sometimes receiving them as late as 72 h after hatching. Previous studies have indicated that fasting during this initial period is detrimental, leading to impaired body growth, compromised immune system response, and hindered muscle development. The objective of this study was to assess the impact of early posthatch feeding on immune system organs and selected hematological, biochemical, and hormonal parameters. The experiment utilized Ross 308 broiler eggs incubated under typical commercial hatchery conditions. The experimental group's eggs were hatched in HatchCare hatchers (HC) with immediate access to feed and water, while the control group's eggs were hatched under standard conditions (ST). Thirty chickens from each group were assessed on the 1st (D1), 7th (D7), 21st (D21), and 35th (D35) day after hatching. On D1, the HC group exhibited lower hemoglobin, hematocrit, and total serum protein values, suggesting that early access to water prevents initial dehydration in newborn chicks. Conversely, the ST group showed a stress reaction on D1 due to feed deprivation, leading to an almost 2-fold higher serum corticosterone concentration compared to the HC group. However, this increase did not result in a significant change in the heterophil/lymphocyte ratio. Furthermore, the HC group displayed an increase in triglyceride concentration and a decrease in HDL concentration on D1. On D7, the HC group exhibited an increased relative weight of the bursa and a higher CD4+ cell number in the cecal tonsil (CT), indicating a more rapid development of these organs resulting from early stimulation of the gastrointestinal tract. However, early feeding did not influence the numbers of Bu-1+, CD4+, and CD8+ cells or the germinal center (GC) areas in the spleen. In conclusion, early feeding contributes to the welfare of newborn chicks by reducing dehydration and stress levels and stimulating the development of gut-associated lymphoid tissue.

Evaluation of the immunogenic properties of the recombinant Histophilus somni outer membrane protein 40 kDa (rOMP40).

BACKGROUND: Gram-negative bacterial infections are a serious problem in beef and dairy cattle. Bacterial outer membrane proteins (OMPs) play a pivotal role in cellular survival and the host-bacterium interaction. Histophilus somni OMP40 was identified as a porin with homology between its N-terminal amino acid sequence and the sequences of porins of other gram-negative bacteria The aim of this study was to produce recombinant H. somni OMP40 (rOMP40), optimize its production and evaluate its immunogenic properties in calves. The cross-reactivity of anti-rOMP40 antibodies were also checked. RESULTS: The highest overexpression of rOMP40 was demonstrated by Escherichia coli C41 using the autoinduction process. Double immunization of calves (20 µg rOMP40 per animal) induced a significant increase of anti-rOMP40 antibodies in the IgG1 (P ≤ 0.01) and IgG2 (P ≤ 0.01, after first immunization only) subclasses, but not IgM. ELISA revealed increased reactivity of the IgG against surface antigens of E. coli and Pasteurella multocida after the second immunization (P < 0.01). Cross reactivity of anti-rOMP40 antibodies with ~ 40 kDa antigens of most common gram-negative pathogens was shown by Western blotting. CONCLUSION: Immunization with H. somni rOMP40 induced a humoral response in cattle with broad cross-reactivity with similar antigens of other species of Pasteurellaceae and Enterobacteriaceae families and the delayed-type hypersensitivity reaction. The obtained results encourage further study to evaluate the protective effect of the produced protein as a subunit vaccine in cattle.

Homeostasis of the Intestinal Mucosa in Healthy Horses-Correlation between the Fecal Microbiome, Secretory Immunoglobulin A and Fecal Egg Count.

The defensive function of the intestinal mucosa depends both on the ability to secrete immunoglobulin A and communication with the mucus microbiome. In horses, the functioning of this system is also influenced by the presence of nematode eggs. Feces collected from healthy horses were examined to determine the fecal egg count, immunoglobulin A level (ELISA), microbiome composition (Next-Generation Sequencing, NGS, V3−V4 and V7−V9 hypervariable regions of the 16S rRNA gene analysis and short-chain fatty acid (SCFA) production ((high-performance liquid chromatography, HPLC). In the taxonomic analysis within the phylum, the following order of dominance was found: Firmicutes, Bacteroidota, Verrucomicrobiota and Fibrobacterota. The coefficient of phylogenetic diversity of the microbiome positively correlated with both secretory immunoglobulin A (SIgA) [µg/g of feces] (p = 0.0354, r = 0.61) and SIgA [µg/mg of fecal protein] (p = 0.0382, r = 0.6) and with the number of Cyathostomum eggs (p = 0.0023, r = 0.79). Important components of the key microbiome in horses, such as phylum Proteobacteria and species Ruminococcus flavefaciens, were positively correlated with the fecal SIgA (p < 0.05). All the obtained results indicate the existence of significant relationships between the host response (SIgA production) and composition and SCFA production in the microbiome as well as the presence of small strongyles in the digestive tract of horses.

The Dynamics of Circulating Immune Complexes in Horses with Severe Equine Asthma.

Non-invasive diagnostic biomarkers of equine asthma syndrome (EAS) from blood or urine are sought. The aim of this study was to assess the absorbance of circulating immune complexes (CICs) during the exacerbation, remission, and treatment of an asthma episode and assess the potential usefulness of CIC levels in the diagnosis and monitoring of the disease. The control group, asthma group, and treated asthma group each contained six horses. Following an initial examination and group classification, the horses were kept in a dusty environment for seven days and then moved to an asthma-friendly environment for three weeks (the treated group received injections of glucocorticoids). Blood was collected at baseline and on the 1st, 2nd, 3rd, 7th, 14th and 30th days. CIC was measured using the modified Haskova method. The time points did not show significant statistical differences. There was a significant decrease in CIC in the treated group, and a significant increase in CIC in the non-treated group on day 30. CIC did not support the EAS diagnosis, although it may help in monitoring patients. To the best of the authors' knowledge, this is the first study to analyse the dynamics of CIC during environmental challenge, remission, and treatment.

Ubiquitin-specific protease 7 as a potential therapeutic target in dogs with hematopoietic malignancies.

BACKGROUND: Ubiquitin-specific protease 7 (USP7) belongs to the group of deubiquitinating enzymes (DUBs), which remove ubiquitin which controls various cellular processes such as chromosome segregation, DNA repair, gene expression, protein localization, kinase activity, protein degradation, cell cycle progression, and apoptosis. It is critical for several important functions in the cell, and therefore dysregulation of USP7 can contribute to tumorigenesis. OBJECTIVES: Alterations in the USP7 protein have been identified in various malignancies of humans. Our aim was to examine whether USP7 could be a potential therapeutic target in hematopoietic cancers of dogs. METHODS: The expression level of USP7 in lymphocytes from healthy dogs and canine lymphoma cells was determined, and the effect of USP7 inhibition on the vital functions of canine cancer cells was examined. RESULTS: We showed that USP7 was overexpressed in lymphomas in dogs. The USP7 inhibitor P5091 has selective cytotoxic activity in canine lymphoma and leukemia cell lines. Our results indicate that inhibition of USP7 leads to a disruption of cell cycle progression, and triggers DNA damage and apoptosis. The observed proapoptotic effect of the USP7 inhibitor most likely is not dependent on the p53 pathway. CONCLUSIONS AND CLINICAL IMPORTANCE: Our results suggest that USP7 could be explored as a potential therapeutic target in dogs with lymphoma. The effectiveness of USP7 inhibition in malignant cells is predicted to be independent of their p53 status.

Changes of plasma fibronectin and fibronectin-fibrin complexes in dams of stillborn dairy calves.

BACKGROUND: Fibronectin (FN) is a large (450-500 kDa), multidomain and multifunctional glycoprotein existing in mammalian tissues. Some fibronectin (FN) molecular forms might be involved in biological processes occurring within the perinatal period, such as tissue remodeling, coagulation, and repair. RESULTS: In this study fibronectin (FN) and fibrinogen (Fb) concentrations and FN-fibrin complexes occurrence and its relative amounts with increasing high molecular masses were respectively determined by ELISA, heat precipitation, and SDS-agarose-immunoblotting methods. Plasma samples from three groups of dams with: 1) singleton stillborn calf without or with negligible autolytic changes in internal organs (DSBn), 2) singleton stillborn calf with advanced autolytic changes in internal organs (DSBa), 3) singleton live-born control calf (DC), and 4) a group of cows during mid to late lactation (LC) were analyzed. Maternal plasma FN concentration in the DSBn and DSBa groups was significantly lower than in the LC group. The plasma samples of DSBa showed a significantly lower FN concentration than in the DC group. Plasma Fb concentration was significantly higher in the DSBa and DSBn, than in the LC group. FN immunoblotting of the cow plasma samples revealed, besides an FN-dimer band, the presence of supramolecular FN-fibrin bands corresponding to FN-fibrin complexes with increasing molecular masses: up to 5 bands from 750 kDa to 1900 kDa in the DSBn and DSBa plasma samples, two bands of 750 and 1000 kDa in the DC group, and only the smallest one of 750 kDa in the LC group. CONCLUSIONS: The observed low FN concentration and occurrence of supramolecular FN-fibrin complexes (1000 kDa and more) in the maternal plasma comparing to cows in lactation might have been associated with periparturient changes in tissues. The presence in maternal plasma of high-molecular FN-fibrin complexes (1300-1900 kDa) arouse the question if this is the consequence of calf perinatal mortality.

Reduced IgE and IgG antigenic response to milk proteins hydrolysates obtained with the use of non-commercial serine protease from Yarrowia lipolytica.

The aim of the study was to investigate the use of serine protease from Yarrowia lipolytica yeast for reduction of milk proteins allergenicity. Whey protein concentrate (WPC-80), αs-casein and their hydrolysates were analyzed for the capacity to bind IgE and IgG antibodies present in sera from patients with cow milk protein allergy using a competitive ELISA. The hydrolysis of αs-casein and whey protein concentrate contributed to a significant reduction of their immunoreactive epitopes. In case of IgE antibodies, the lowest binding capacity was detected in the 24 h hydrolysates of both proteins in which the inhibition of the reaction was ≤20 and ≤68% for αs-casein and whey protein concentrate respectively. One hour hydrolysis of WPC-80 reduced the protein antigenicity, while the longer time (5 h) might lead to the exposure of new IgE - reactive epitopes.

Evaluation of the protective effect of immunization spf DBA/2J mice with selected bacterial, recombinant Hsp60 antigens during Salmonella Enteritidis challenge.

Salmonella Enteritidis is one of the most common causes of food poisoning in humans. Many attempts have been made to develop an effective vaccine against S. Enteritidis for use in poultry, but experiments aimed at the complete elimination of this pathogen from poultry farms have not provided satisfactory results. The development of new generation vaccines against salmonellosis, such as subunit vaccines based on heat shock proteins (HSPs), is strongly justified. The high immunogenicity of Hsp60 isolated from Procaryota, including Salmonella, has been suggested by the presence of IgG anti-Hsp60 antibodies in mice immunized with these proteins. The aim of the studies was to evaluate the protective effects of immunization with recombinant Hsp60 from selected gram-negative bacteria (S. Enteritidis, Escherichia coli, Pasteurella multocida, Histophilus somni) in spf DBA/2 J mice experimentally infected with S. Enteritidis. The study demonstrated that double subcutaneous immunization of mice with a dose of 10 µg rHsp60 induced a specific immune response of IgG antibodies in tested animals. The median lethal dose (LD50) for the murine model spf DBA/2 J orally infected with S. Enteritidis was estimated at 6.84 × 105 cfu/animal. Mice immunized with rHsp60 from gastrointestinal pathogens (S. Enteritidis and E. coli) showed better survival after experimental infection with a 3 × LD50 dose from S. Enteritidis, compared to animals immunized with proteins obtained from respiratory pathogens (P. multocida and H. somni). However, the log-rank analysis did not show significant differences in the survival rates between rHsp60-immunized mice and controls. S. Enteritidis was not isolated any less frequently from internal organs and faeces of rHsp60-immunized mice than from controls. Nevertheless, the level of haptoglobin (but not IL-6) was increased in all mice in which the presence of the pathogen was observed. Bacterial Hsp60 is an interesting candidate for a subunit vaccine, but its use in livestock animals must be further investigated.

The evaluation of immunogenic impact of selected bacterial, recombinant Hsp60 antigens in DBA/2J mice.

Heat Shock Proteins (HSP) are highly conserved proteins that are widely spread throughout all organisms. They function in the cytoplasm as chaperones; however, they could be expressed on the cell surface. It has been shown that Hsp60 obtained from gram-negative bacteria are able to stimulate cells of the acquired and innate immune system. The aim of this study was the evaluation of the immunogenic properties of recombinant Hsp60 proteins derived from four common pathogenic bacteria: Escherichia coli, Histophilus somni, Pasteurella multocida and Salmonella Enteritidis. The analysis of the humoral immune response in DBA/2J mice hyperimmunized with selected rHsp60 revealed high levels of IgG rHsp60-antibody with the predominance of the IgG1 subclass, in the reaction with both homologous and heterologous antigens. The presence of IgG2a and IgG2b was also observed; however, no antibodies of subclass IgG3 were detected. The comparison of plasma IgG antibody reactivity of mice immunized with two different doses of rHsp60 (10/20 µg) showed that the lower dose was sufficient to induce a strong humoral response. The reactivity of the IgG rHsp60-antibody with whole bacterial cells showed a significantly higher reaction with H. somni compared with other pathogens. It was demonstrated that the addition of all rHsp60 with polymyxin B to the culture medium stimulated splenocytes isolated from hyperimmunized mice to release IL-1ß and IL-6. As a strong stimulator of the immune system, bacterial-origin Hsp60 seems to be an interesting potential component of subunit vaccines aimed at the development of protection for animals during infections caused by gram-negative bacteria.

The Effect of L-Carnitine, Hypotaurine, and Taurine Supplementation on the Quality of Cryopreserved Chicken Semen.

The objective of this study was to investigate the effect of L-carnitine (LC), hypotaurine (HT), and taurine (T) on the quality of frozen-thawed chicken semen. Pooled semen samples were divided into seven aliquots (control, 1 mM LC, 5 mM LC, 1 mM HT, 10 mM HT, 1 mM T, and 10 mM T) and subjected to cryopreservation. Postthaw sperm motility was determined by IVOS system and sperm characteristics were assessed with fluorochromes and flow cytometry. The highest sperm motility and the highest percentage of viable sperm were in the HT1 group (P < 0.01 and P < 0.05) following cryopreservation. After thawing, we observed a higher percentage of sperm without apoptosis and membrane reorganization changes in the LC1 and T1 group when compared to the control (P < 0.05). There was a higher percentage of live sperm without lipid peroxidation (LPO) in all treatments (P < 0.01; P < 0.05), when compared to the control group. The percentage of sperm with high mitochondrial potential significantly increased with LC1, T1, and T10 (P < 0.05). Supplementation of the diluent with LC1, LC5, and T1 significantly (P < 0.05) reduced DNA susceptibility to fragmentation, compared to the control and HT1 groups. These results indicate that the addition of examined antioxidants improves the quality of cryopreserved chicken semen.

Molecular identification of Giardia duodenalis isolates from domestic dogs and cats in Wroclaw, Poland.

INTRODUCTION: Giardia duodenalis (G. intestinalis) is a common protozoan causing gastrointestinal disorders in many species of mammals. The genus of Giardia has high molecular diversity. Dogs and cats, in addition to their typical infection with assemblages C, D and F, may be a reservoir of zoonotic assemblages (A and B). OBJECTIVE: The aim of this study was a genetic characteristic of Giardia isolates of dogs and cats from the area of Wroclaw (Poland). MATERIALS AND METHOD: A total of 128 and 33 faecal samples from dogs and cats, respectively, were analyzed by routine coprological methods. The animals were diagnosed on the presence of G. duodenalis antigens in faeces soluble with the use of SNAP Giardia (IDEXX Laboratories) immunosorbent assay. 27 DNA isolates of Giardia were subjected to molecular identification (PCR-RFLP). RESULTS AND CONCLUSIONS: The prevalence of G. duodenalis was 21.1% (27/128) in dogs and 15.1% (5/33) in cats. In dogs, C assemblage was present in 18 (81%) positive stool samples, D assemblage in 2 (9%) samples, B assemblage present in one (4.5%), and mixed assemblages (C and D) occurred in one (4.5%) sample. F assemblage was found in 4 (80%) cats' positive stool samples and A assemblage occurred in one case (20%). Confirmation of the presence of A and B zoonotic assemblages suggests that infected pets can be a threat to human health. This study describes for the first time the presence of mixed infections within host-specific C and D assemblages in dogs in Poland.

Antlerogenic stem cells: molecular features and potential in rabbit bone regeneration.

AIM: (i) To assess the expression profiles of stem cell-associated markers including Oct4, Sox2, Klf4, Nanog, C-myc, Stat3 and Cd9, (ii) analyze the nanotopography of the MIC-1 stem cells and (iii) evaluate the efficiency of live stem cell implants and stem cell culture derivatives on the regeneration of bone deficiencies in rabbit mandibles. MATERIALS AND METHODS: The expression profiles of stem cell-associated genes, including Oct4, Sox2, Klf4, Nanog, C-myc, Stat3 and CD9 were assessed using reverse transcription polymerase chain reaction and flow cytometry. Nanotopography of the antlerogenic MIC-1 cell lineage was analyzed using atomic force microscopy. The effect of MIC-1 stem cells, their homogenate and supernatant on the regeneration of bone deficiencies in rabbit mandibles was evaluated using histological analysis. The effect of MIC-1 stem cells and stem cell-based derivatives on the immune responses of the animals was assessed by analyses of acute phase protein levels (haptoglobin and fibrinogen). RESULTS: We found that the MIC-1 cells isolated from the apical regions of growing antlers exhibited molecular features that were characteristics of pluripotent stem cells. Using atomic force microscopy, we determined the details of the cell surface morphologies with a particular emphasis on the patterns of formation of plasma extensions for interlinking adjacent cells. We also demonstrated that not only implanted stem cells but also cell homogenates and cell post-culture supernatants have potential in the regeneration of bone deficiencies in the rabbit mandible. CONCLUSIONS: Our findings indicate that the use of both antlerogenic stem cell implants and the preparations derived from the cells offer alternative approaches to those based on autologous stem cells in the biological stimulation of osteogenesis and in bone regeneration.

[Heat shock protein HSP60 and the perspective for future using as vaccine antigens]. / Bialka szoku cieplnego HSP60 i perspektywy ich wykorzystania jako antygenów szczepionkowych.

Heat Shock Proteins (HSPs) are widely spread in nature, highly conserved proteins, found in all prokaryotic and eukaryotic cells. HSPs have been classified in 10 families, one of them is the HSP60 family. HSP60 function in the cytoplasm as ATP-dependent molecular chaperones by assisting the folding of newly synthesised polypeptides and the assembly of multiprotein complexes. There is a large amount of evidence which demonstrate that HSP60 is expressed on the cell surface. Especially in bacteria the expression on the surface occurs constitutively and increases remarkably during host infection. HSP60 also play an important role in biofilm formation. In the extracellular environment, HSP60 alone or with self or microbial proteins can acts not only as a link between immune cells, but also as a coordinator of the immune system activity. This protein could influence the immune system in a different way because they act as an antigen, a carrier of other functional molecules or as a ligand for receptor. They are able to stimulate both cells of the acquired (naïve, effector, regulatory T lymphocyte, B lymphocyte) and the innate (macrophages, monocytes, dendritic cells) immune system. HSPs have been reported to be potent activators of the immune system and they are one of the immunodominant bacterial antigens they could be a good candidate for a subunit vaccine or as an adjuvant.

Hydrolysis with Cucurbita ficifolia serine protease reduces antigenic response to bovine whey protein concentrate and αs-casein.

In the present study the effect of hydrolysis with non-commercial Cucurbita ficifolia serine protease on a reduction of the IgE and IgG binding capacity of whey protein concentrate and αs-casein was investigated. The intensity of the protein degradation was analyzed by the degree of hydrolysis, the free amino groups content and RP-HPLC. The ability to bind the antibodies by native proteins and their hydrolysates was determined using a competitive ELISA test. Deep hydrolysis contributed to a significant reduction of immunoreactive epitopes present in WPC. In the case of IgE and IgG present in the serum pool of children with CMA, the lowest binding capacity was detected in the 24 h WPC hydrolysate, where the inhibition of the reaction with native WPC was ≤23 and ≤60 %, respectively. The analysis of the IgG reactivity in the antiserum of the immunized goat showed that the lowest antibody binding capacity was exhibited also by 24 h WPC hydrolysate at a concentration of 1000 µg/ml where the inhibition of the reaction with nWPC was ≤47 %. One-hour hydrolysis of α-casein was sufficient to significant reduction of the protein antigenicity, while the longer time (5 h) of hydrolysis probably lead to the appearance of new epitopes reactive with polyclonal.

The effect of cysteine and superoxide dismutase on the quality of post-thawed chicken sperm.

The study was conducted to determine the influence of N-acetyl-l-cysteine (NAC) and superoxide dismutase (SOD) on chicken sperm motility, plasma membrane and acrosome integrity, mitochondrial activity, lipid peroxidation (LPO) and apoptotic changes after freezing-thawing process. Semen samples from fifteen Greenlegged Partridge roosters were pooled, diluted with EK extender without antioxidants (control), or supplemented with 5 mM NAC, or 200 U/mL SOD. Samples were subjected to cryopreservation. After thawing, sperm parameters evaluated by using CASA system and flow cytometry were assessed. The extender supplemented with NAC and SOD caused the increase (P < 0.01) in sperm motility and provided the higher percentage of rapid sperm (P < 0.01) compared to control. The addition of NAC increased the progressive motility of cells (P < 0.01). In NAC and SOD groups post-thaw plasma membrane integrity was higher (P < 0.05) and less spermatozoa showed apoptotic changes (P < 0.01, P < 0.05). Post-thaw percentage of sperm with high mitochondrial activity was the greatest (P < 0.05) with NAC addition. The SOD supplementation only reduced (P < 0.05) the percentage of sperm with LPO, following the cryopreservation. These results indicate that the addition of NAC (5 mM) and SOD (200 U/mL) is beneficial for the function of frozen-thawed chicken spermatozoa. The antioxidants prevented the reduction in motility, viability and mitochondrial membrane potential, as well as protected from apoptotic changes in sperm. Lipid peroxidation in sperm plasma membrane was decreased by SOD supplementation. Therefore, these antioxidants can be recommended as an additional component of chicken freezing extender.