Acute Heat Stress Changes Protein Expression in the Testes of a Broiler-Type Strain of Taiwan Country Chickens.

Heat stress leads to decreased fertility in roosters. This study investigated the global protein expression in response to acute heat stress in the testes of a broiler-type strain of Taiwan country chickens (TCCs). Twelve 45-week-old roosters were randomly allocated to the control group maintained at 25°C, and three groups subjected to acute heat stress at 38°C for 4 h, with 0, 2, and 6 h of recovery, respectively. Testis samples were collected for hematoxylin and eosin staining, apoptosis assay, and protein analysis. The results revealed 101 protein spots that differed significantly from the control following exposure to acute heat stress. The proteins that were differentially expressed participated mainly in protein metabolism and other metabolic processes, responses to stimuli, apoptosis, cellular organization, and spermatogenesis. Proteins that negatively regulate apoptosis were downregulated and proteins involved in autophagy and major heat shock proteins (HSP90α, HSPA5, and HSPA8) were upregulated in the testes of heat-stressed chickens. In conclusion, acute heat stress causes a change in protein expression in the testes of broiler-type B strain TCCs and may thus impair cell morphology, spermatogenesis, and apoptosis. The expression of heat shock proteins increased to attenuate the testicular injury induced by acute heat stress.

Annotation of differential protein expression in the hypothalami of layer-type Taiwan country chickens in response to acute heat stress.

The hypothalamus is the coordinating center for maintaining temperature homeostasis. In this study, global protein expression in the hypothalami of layer-type Taiwan country chickens in response to acute heat stress was investigated. Twelve 30-week-old female TCCs were divided into three acute heat-stressed groups, namely acute heat stress at 36 °C for 4 h with 0 h (without recovery, H4R0), 2 h (H4R2), or 6 h (H4R6) of recovery. A control group was maintained at 25 °C. Hypothalamus samples were collected at the end of each time point for proteomic analysis. The analysis results revealed that 134 protein spots representing 118 distinct proteins exhibited differential expressions after acute heat stress treatment. Results of gene ontology analysis showed that most of the differentially expressed proteins are involved in carbohydrate metabolism, cellular processes, actin cytoskeleton organization, and responses to stimuli. Functional pathway analysis results suggested that the proteins are associated with networks of carbon metabolism, glycolysis, and gluconeogenesis. Upregulation of the expression of triosephosphate isomerase, phosphoglycerate kinase, pyruvate kinase, alpha-enolase, glycogen phosphorylase (brain form), phosphoglucomutase, L-lactate dehydrogenase A chain and downregulation of 6-phosphogluconolactonase expression indicated an increase in the glycolytic activity and glucose supply for ATP production in the hypothalami in response to heat stress. By contrast, upregulated expressions of heat shock protein 90 alpha, glutathione S-transferase 2s, peroxiredoxin-1, and dihydropyrimidinase-like 2 suggested that acute heat stress adversely affects the hypothalamus; thus, it induces mechanisms that prevent oxidative damage and endoplasmic reticulum stress. In conclusion, acute heat stress induces differential protein expression in the hypothalami of the L2 strain Taiwan country chickens, which may manifest detrimental effects. Furthermore, differential expression is a critical response in the hypothalamus for the regulation of thermotolerance.

Effects of Artemisia annua on experimentally induced leucocytozoonosis in chickens.

The biting midge Culicoides arakawae is the vector for the parasite Leucocytozoon caulleryi. Birds infected with L. caulleryi develop leucocytozoonosis. Given the food safety concern regarding drug residue in eggs, discovering a natural alternative to antibiotics is a worthy of exploration. Thus, we investigated the effects of the antimalarial herb Artemisia annua on experimentally induced leucocytozoonosis in chickens. We reared C. arakawae in the laboratory. Eggs were cultured, developing into larvae, pupae, and imagoes. Female midges sucked the blood of sick chickens and then were ground into a solution injected into healthy chickens. The control group was given empty capsules daily, whereas the 2 experimental groups were given 40 mg/kg sulfadimethoxine or 0.5 g of A. annua powder. Leucocytozoon gametocytes were detected in chicken blood through Giemsa staining. PCR detected the cytochrome b gene of L. caulleryi in the infected chickens. No significant among-group differences in body weight gain were observed before d 14 postinoculation (P > 0.05). Body weight gain in the control group was significantly lower from day 14 to 28 postinoculation (P < 0.05). After day 14, rectal temperature in the experimental groups decreased significantly compared with that in the control group. Lower rates of pale comb and green feces were observed in the animals receiving treatment from day 0. The experimental groups had a higher recovery rate and recovered earlier than did the control group. By day 31, all the animals had recovered. PCR detected L. caulleryi in the infected chickens with high sensitivity and accuracy. The animals receiving A. annua exhibited increased weight gain and reduced parasite concentrations in the blood. This in turn reduced mortality and the occurrence of pale comb and green feces. The findings are informative for research on leucocytozoonosis.

Performance comparison of dwarf laying hens segregating for the naked neck gene in temperate and subtropical environments.

This study compares laying performances between two environments of dwarf laying hen lines segregating for the naked neck mutation (NA locus), a selected dwarf line of brown-egg layers and its control line. Layers with one of the three genotypes at the NA locus were produced from 11 sires from the control line and 12 sires from the selected line. Two hatches produced 216 adult hens in Taiwan and 297 hens in France. Genetic parameters for laying traits were estimated in each environment and the ranking of sire breeding values was compared between environments. Laying performance was lower, and mortality was higher in Taiwan than in France. The line by environment interaction was highly significant for body weight at 16 weeks, clutch length and egg number, with or without Box-Cox transformation. The selected line was more sensitive to environmental change but in Taiwan it could maintain a higher egg number than the control line. Estimated heritability values in the selected line were higher in France than in Taiwan, but not for all the traits in the control line. The rank correlations between sire breeding values were low within the selected line and slightly higher in the control line. A few sire families showed a good ranking in both environments, suggesting that some families may adapt better to environmental change.

Proteomic Analysis of Thermal Regulation of Small Yellow Follicles in Broiler-Type Taiwan Country Chickens.

Heat stress hampers egg production and lowers fertility in layers. This study investigated global protein abundance in the small yellow follicles (SYFs, 6-8 mm diameter) of a broiler-type strain of Taiwan country chickens (TCCs) under acute heat stress. Twelve 30-week-old TCC hens were allocated to a control group maintained at 25°C, and to three acute heat-stressed groups subjected to 38°C for 2 h without recovery, with 2-h recovery, or with 6-h recovery. Two-dimensional difference gel electrophoresis analysis identified 119 significantly differentially expressed proteins after acute heat exposure. Gene ontology analysis revealed that most of these proteins are involved in molecular binding (34%), catalytic activity (23%), and structural molecule activity (11%), and participate in metabolic processes (20%), cellular processes (20%), and cellular component organization or biogenesis (11%). Proteins associated with stress response and survival (HSP25, HSP47, HSP70, HSC70, HSPA9), cytoskeleton remodeling, mitochondrial metabolic process of ATP production, antioxidative defense (peroxiredoxin-6), cargo lipid export and delivery (vitellogenin, apolipoprotein B and A1), and toxin/metabolite clearance and delivery (albumin) were upregulated after acute heat stress in the SYFs of TCCs. No overt cell death and atresia were observed in SYFs after acute heat stress. Collectively, these responses may represent a protective mechanism to maintain follicle cell integrity and survival, thereby ensuring a sufficient pool of SYFs for selection into the ovulation hierarchy for successful egg production.

Functional genomics study of acute heat stress response in the small yellow follicles of layer-type chickens.

This study investigated global gene and protein expression in the small yellow follicle (SYF; 6-8 mm in diameter) tissues of chickens in response to acute heat stress. Twelve 30-week-old layer-type hens were divided into four groups: control hens were maintained at 25 °C while treatment hens were subjected to acute heat stress at 36 °C for 4 h without recovery, with 2-h recovery, and with 6-h recovery. SYFs were collected at each time point for mRNA and protein analyses. A total of 176 genes and 93 distinct proteins with differential expressions were identified, mainly associated with the molecular functions of catalytic activity and binding. The upregulated expression of heat shock proteins and peroxiredoxin family after acute heat stress is suggestive of responsive machineries to protect cells from apoptosis and oxidative insults. In conclusion, both the transcripts and proteins associated with apoptosis, stress response, and antioxidative defense were upregulated in the SYFs of layer-type hens to alleviate the detrimental effects by acute heat stress. However, the genomic regulations of specific cell type in response to acute heat stress of SYFs require further investigation.

Proteomic analysis of the hypothalamus of broiler-type Taiwan country chickens in response to acute heat stress.

The hypothalamus is a critical center for regulating heat retention or dissipation. This study investigated global protein changes in the hypothalamus of broiler-type Taiwan country chickens (TCCs) after acute heat stress. Twelve TCC hens aged 30 weeks were allocated to groups subjected to acute heat stress at 38°C for 2 hr without recovery, with 2 hr of recovery, and with 6 hr of recovery; a control group was maintained at 25°C. Hypothalami were collected for protein expression analysis at the end of each time point. The results showed 114 protein spots differentially expressed after acute heat stress. Most of the differentially expressed proteins were involved in cellular processes, metabolism, transport, and cellular component organization. Functional annotation analysis suggested that these proteins were related to cellular defensive responses against heat and oxidative stress, detoxification and toxin export/delivery, cytoskeleton integrity, oxygen transport, and neural development. The results of this study suggest that acute heat stress damages the hypothalamus of broiler-type TCCs through oxidative stress and provokes a series of responses to stabilize protein structures, degrade misfolded proteins, and remodel cytoskeletons for attenuating the detrimental effects by acute heat stress.

Expression of 25 high egg production related transcripts that identified from hypothalamus and pituitary gland in red-feather Taiwan country chickens.

Expression levels of 33 high egg production candidate transcripts in Red-feather Taiwan country chickens (TCCs) were examined by quantitative reverse-transcription (RT) polymerase chain reactions (PCR) in this study. Candidate transcripts were previously identified from a L2-B (L2-subtract-B) hypothalamus/pituitary gland subtractive cDNA library. In this subtractive cDNA library, two divergently selected strains of TCCs, B and L2 were used. These two strains were originated from one single population and were further subjected (since 1982) to the selections of body weight/comb size (B) and eggs to 40wk of age (L2), respectively. Hypothalamuses and pituitary glands that sampled from Red-feather TCCs were previously grouped into high (Red-high; n=20) and low (Red-low; n=20) egg productions based on the rate of lay after 1st egg (hen-day laying rate; %). Rates of lay after 1st egg (mean+/-S.E.) in the Red-high and the Red-low subpopulations were 72.2+/-0.6 and 23.0+/-3.5, respectively (P<0.01). Quantitative RT-PCR validated that 25 candidate transcripts were significantly higher expressed in the Red-high than in the Red-low hens. These transcripts were ANP32A, BDH, CDC42, CNTN1, COMT, CPE, CTNNB1, DIO2, EIF4E, GARNL1, HSPCA, LAPTM4B, MBP, NAP1L4, NCAM1, PARK7, PCDHA@, PGDS, PLAG1, PRL, RAD21, SAR1A, SCG2, STMN1 and UFM1. Among these transcripts, 15 (79.0%), 13 (68.4%), and 12 (63.2%) genes were annotated to involve in cellular physiological process (GO:0050875), metabolism (GO:0008152) and cell communication (GO:0007154). Identified transcripts that related to high egg production are most active in focal adhesion, adherens junction, MAPK signaling, tight junction and cell adhesion pathways.

Differentially expressed transcripts in shell glands from low and high egg production strains of chickens using cDNA microarrays.

We have constructed a tissue-specific in-house cDNA microarray to identify differentially expressed transcripts in shell glands from low (B) and high (L2) egg production strains of Taiwanese country chickens during their egg-laying period. The shell gland cDNA library was constructed from the high egg production strain. cDNA clones (7680) were randomly selected and their 5'-end sequences characterized. After excluding overlapping sequences, an in-house cDNA microarray, representing 2743 non-redundant transcripts, was generated for functional genomic studies. Using our microarray, we have successfully identified 85 differentially expressed transcripts from the two different strains of chicken shell glands. In this study, 34 of these transcripts were associated with signal transduction, protein biosynthesis, cell adhesion, cellular metabolism, skeletal development, cell organization and biogenesis. We selected a number of the differentially expressed transcripts for further validation using semi-quantitative RT-PCR. These included elongation factor 2 (EEF2), ovocalyxin-32 (OCX-32) and annexin A2 (ANXA2) which were expressed at high levels in the chicken shell glands of the B strain and, in contrast, the coactosin-like protein (COTL1), transcription factor SOX18 and MX protein were more highly expressed in the L2 strain. Our results suggest that these differentially expressed transcripts may be suitable to use as molecular markers for high rates of egg production, and now need to be investigated further to assess whether they can be applied for use in breeding selection programs in Taiwanese country chickens.

Laying traits and underlying transcripts, expressed in the hypothalamus and pituitary gland, that were associated with egg production variability in chickens.

The objective was to characterize the potential laying traits and underlying transcripts expressed in the hypothalamus and pituitary gland that were associated with egg production variability in five genetic stocks of chickens: two commercial lines, Red- (n=12) and Black-feather (n=14) Taiwan country chickens (TCCs); two selected lines of TCCs, B (high body weight/comb size; n=17) and L2 (high-egg production; n=14); and a commercial single comb White Leghorn (WL; n=17). Six laying traits, age at first egg, clutch length, pause length, oviposition lag within clutch, follicle rapid growth period, and rate of yolk accumulation were measured. The significance of differential values among five chicken stocks and correlation coefficients between laying traits and number of eggs to 50 weeks of age or laying rate after first egg, and the expression level of 33 transcripts were determined. Longer clutch length and shorter oviposition lag within clutch contributed to a higher number of eggs to 50 weeks of age or laying rate after first egg in L2 (P<0.05) and WL strains (P<0.05). However, their rate of yolk accumulation (P<0.05) and follicle rapid growth period (P<0.05) were different, indicating the accumulation of different alleles after long-term, independent selection. Across all five strains, numbers of eggs to 50 weeks of age were positive correlated with average clutch length (P<0.05) as well as the rate of yolk accumulation (P<0.05). Expressions of PLAG1, STMN2, PGDS, PARK7, ANP32A, PCDHA@, SCG2, BDH and SAR1A transcripts contributed to number of eggs to 50 weeks of age (P<0.05) or laying rate after first egg (P<0.05). Analysis of correlation coefficients indicated that PLAG1 additionally played roles in decreasing average pause length. Two transcripts, PRL and GARNL1, specifically contributed to number of eggs to 50 weeks of age or laying rate after first egg by reducing oviposition lag within clutch (P<0.05) and/or increasing average clutch length (P<0.05), respectively. Expression level of NCAM1, contributed to laying rate after first egg by association with a shorter oviposition lag within clutch (P<0.05). The current study attributed egg production phenotype in five strains into several laying traits; correlations between these traits and expression levels of underlying transcripts expressed in the hypothalamus and pituitary gland were also established.

Identification of transcripts related to high egg production in the chicken hypothalamus and pituitary gland.

To identify transcripts related to high egg production expressed specifically in the hypothalamus and pituitary gland of the chicken, two subtracted cDNA libraries were constructed. Two divergently selected strains of Taiwan Country Chickens (TCCs), B (sire line) and L2 (dam line) were used; they had originated from a single population and were further subjected (since 1982) to selection for egg production to 40 wk of age and body weight/comb size, respectively. A total of 324 and 370 clones were identified from the L2-B (L2-subtract-B) and the B-L2 subtracted cDNA libraries, respectively. After sequencing and annotation, 175 and 136 transcripts that represented 53 known and 65 unknown non-redundant sequences were characterized in the L2-B subtracted cDNA library. Quantitative reverse-transcription (RT)-PCR was used to screen the mRNA expression levels of 32 randomly selected transcripts in another 78 laying hens from five different strains. These strains included the two original strains (B and L2) used to construct the subtracted cDNA libraries and an additional three commercial strains, i.e., Black- and Red-feather TCCs and Single-Comb White Leghorn (WL) layer. The mRNA expression levels of 16 transcripts were significantly higher in the L2 than in the B strain, whereas the mRNA expression levels of nine transcripts, BDH, NCAM1, PCDHA@, PGDS, PLAG1, PRL, SAR1A, SCG2 and STMN2, were significantly higher in two high egg production strains, L2 and Single-Comb WL; this indicated their usefulness as molecular markers of high egg production.

Profiling of differential gene expression in the hypothalamus of broiler-type Taiwan country chickens in response to acute heat stress.

Acute heat stress severely impacts poultry production. The hypothalamus acts as a crucial center to regulate body temperature, detect temperature changes, and modulate the autonomic nervous system and endocrine loop for heat retention and dissipation. The purpose of this study was to investigate global gene expression in the hypothalamus of broiler-type B strain Taiwan country chickens after acute heat stress. Twelve 30-week-old hens were allocated to four groups. Three heat-stressed groups were subjected to acute heat stress at 38 °C for 2 hours without recovery (H2R0), with 2 hours of recovery (H2R2), and with 6 hours of recovery (H2R6). The control hens were maintained at 25 °C. At the end, hypothalamus samples were collected for gene expression analysis. The results showed that 24, 11, and 25 genes were upregulated and 41, 15, and 42 genes were downregulated in H2R0, H2R2, and H2R6 treatments, respectively. The expressions of gonadotropin-releasing hormone 1 (GNRH1), heat shock 27-kDa protein 1 (HSPB1), neuropeptide Y (NPY), and heat shock protein 25 (HSP25) were upregulated at all recovery times after heat exposure. Conversely, the expression of TPH2 was downregulated at all recovery times. A gene ontology analysis showed that most of the differentially expressed genes were involved in biological processes including cellular processes, metabolic processes, localization, multicellular organismal processes, developmental processes, and biological regulation. A functional annotation analysis showed that the differentially expressed genes were related to the gene networks of responses to stress and reproductive functions. These differentially expressed genes might be essential and unique key factors in the heat stress response of the hypothalamus in chickens.

Proteomic analysis of hypothalamic proteins of high and low egg production strains of chickens.

Two slow-growth local chicken strains, derived from a common base population, were bi-directionally selected over twenty generations for carcass traits (B strain) and egg production (L2 strain). The objective of the present study was to identify hypothalamic proteins associated with high egg production (by taking advantage of the similar genetic background of these two strains). Prior to and during egg laying, hypothalamic proteins of B and L2 hens were analyzed with two-dimensional gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Approximately 430 well-resolved spots, ranging from 10 to 40 kDa, pH 5-9, were quantified by image processing. Eight protein spots differed in quantity between B and L2 strains at either stage. Using LC-MS/MS, we identified six of eight protein spots, including proteins known for regulating gene expression, signal transduction and lipid metabolism. The mRNA expression levels of these six proteins were then evaluated by quantitative RT-PCR in five strains of hens, including B, L2 and another three commercial strains; heterogeneous nuclear ribonucleoprotein H3 (HNRPH3) was higher in L2 than in the B strain (consistent with the findings in 2-DE). Increased levels of HNRPH3 mRNA were also present in the hypothalamus of high-egg-yield White Leghorn layers, but were absent in other domestic commercial strains with low egg production rates. In conclusion, the expression level of HNRPH3 may be a new molecular marker to screen for high egg production in slow-growth local chickens.

Differential protein expression in chicken spermatozoa before and after freezing-thawing treatment.

The biological characteristics of rooster sperm that has undergone freezing treatment remain elusive. This study analyzed the change in sperm proteins after freezing-thawing treatment by using a proteomic approach. Semen from three 36-wk-old L2 strain Taiwan country chickens were used. A qualifying ejaculate containing more than 80% motility and volume 200µL was used for cryopreservation. The proteomic analysis explored 55 protein spots that differed significantly before and after freezing-thawing treatment (P<0.05). Among the 55 protein spots, expression levels of 19 proteins decreased after treatment. Forty-five differentially expressed protein spots were identified and belong to 33 proteins. Results of gene ontology analysis revealed that most differentially expressed proteins were involved in molecular function of the cellular metabolism process (28%) and cellular carbohydrate metabolism process (15%), and were associated with molecular function of oxidoreductase activity (19%) and protein binding (18%). The differentially expressed proteins before and after freezing-thawing treatment, including fructose-bisphosphate aldolase C, triosephosphate isomerase, aconitate hydratase, tubulin and outer dense-fiber protein, are associated with sperm energy metabolism and flagellum structure. In conclusion, freezing-thawing treatment significantly affects the expression of proteins related to sperm metabolism and structure in chicken spermatozoa. The differing levels of these proteins could be valuable for further enhancing the fertility of frozen-thawed chicken spermatozoa.

Acute heat stress induces differential gene expressions in the testes of a broiler-type strain of Taiwan country chickens.

The expression of testicular genes following acute heat stress has been reported in layer-type roosters, but few similar studies have been conducted on broilers. This study investigated the effect of acute heat stress on the gene expression in the testes of a broiler-type strain of Taiwan country chickens. Roosters were subjected to acute heat stress (38°C) for 4 h, and then exposed to 25°C, with testes collected 0, 2, and 6 h after the cessation of heat stress, using non-heat-stressed roosters as controls (n = 3 roosters per group). The body temperature and respiratory rate increased significantly (p<0.05) during the heat stress. The numbers of apoptotic cells increased 2 h after the acute heat stress (79 ± 7 vs. 322 ± 192, control vs. heat stress; p<0.05), which was earlier than the time of increase in layer-type roosters. Based on a chicken 44 K oligo microarray, 163 genes were found to be expressed significantly different in the testes of the heat-stressed chickens from those of the controls, including genes involved in the response to stimulus, protein metabolism, signal transduction, cell adhesion, transcription, and apoptosis. The mRNA expressions of upregulated genes, including HSP25, HSP90AA1, HSPA2, and LPAR2, and of downregulated genes, including CDH5, CTNNA3, EHF, CIRBP, SLA, and NTF3, were confirmed through quantitative real-time polymerase chain reaction (qRT-PCR). Moreover, numerous transcripts in the testes exhibited distinct expressions between the heat-stressed broiler-type and layer-type chickens. We concluded that the transcriptional responses of testes to acute heat stress may differ between the broiler-type and layer-type roosters. Whether the differential expression patterns associate with the heat-tolerance in the strains require a further exploration.

Annotation of Differential Gene Expression in Small Yellow Follicles of a Broiler-Type Strain of Taiwan Country Chickens in Response to Acute Heat Stress.

This study investigated global gene expression in the small yellow follicles (6-8 mm diameter) of broiler-type B strain Taiwan country chickens (TCCs) in response to acute heat stress. Twelve 30-wk-old TCC hens were divided into four groups: control hens maintained at 25°C and hens subjected to 38°C acute heat stress for 2 h without recovery (H2R0), with 2-h recovery (H2R2), and with 6-h recovery (H2R6). Small yellow follicles were collected for RNA isolation and microarray analysis at the end of each time point. Results showed that 69, 51, and 76 genes were upregulated and 58, 15, 56 genes were downregulated after heat treatment of H2R0, H2R2, and H2R6, respectively, using a cutoff value of two-fold or higher. Gene ontology analysis revealed that these differentially expressed genes are associated with the biological processes of cell communication, developmental process, protein metabolic process, immune system process, and response to stimuli. Upregulation of heat shock protein 25, interleukin 6, metallopeptidase 1, and metalloproteinase 13, and downregulation of type II alpha 1 collagen, discoidin domain receptor tyrosine kinase 2, and Kruppel-like factor 2 suggested that acute heat stress induces proteolytic disintegration of the structural matrix and inflamed damage and adaptive responses of gene expression in the follicle cells. These suggestions were validated through gene expression, using quantitative real-time polymerase chain reaction. Functional annotation clarified that interleukin 6-related pathways play a critical role in regulating acute heat stress responses in the small yellow follicles of TCC hens.

Serum C-reactive protein in dairy herds.

The purpose of this study was to determine the relationship between the serum level of C-reactive protein (CRP) and lactation and health status. Blood samples were collected every 2 wk for 12 mo from 29 randomly selected dairy cattle on 3 farms. At the time the blood samples were collected, the stage of pregnancy, lactation status, breeding records, general health condition, reproductive status, and body condition score were recorded for each cow. Serum CRP was detected with sodium dodecyl sulfate polyacrylamide gel electrophoresis and western immunoblotting. C-reactive protein levels were measured with a densitometer and expressed as an optimal dose value. C-reactive protein levels were correlated with the body condition score, lactation status, and animal health (P < 0.05), but not with ambient temperature, animal age, or parity. C-reactive protein levels increased with milk production, peaking during high lactation (2 to 4 mo of pregnancy), and decreased when lactation ceased. In addition, the CRP level was highest during naturally occurring infections, such as mastitis and other tissue inflammation. Thus, the CRP level can confirm the presence of inflammation. The stress effect of taking blood samples as measured by the CRP level, was also examined. The CRP level became rapidly elevated 12 h after the blood samples were taken but returned to normal 36 h later. In conclusion, the stresses resulting from overall poor health, heavy lactation, and blood sampling caused the elevation of serum CRP. C-reactive protein is a marker or tool for evaluating the health status of a herd. C-reactive protein should also be considered as a useful criteria to assess the stress levels and may be useful in early surveillance of disease conditions in a dairy herd.

Changes in protein expression in testes of L2 strain Taiwan country chickens in response to acute heat stress.

Heat stress causes a decrease of fertility in roosters. Yet, the way acute heat stress affects protein expression remains poorly understood. This study investigated differential protein expression in testes of the L2 strain of Taiwan country chickens following acute heat stress. Twelve 45-week-old roosters were allocated into four groups, including control roosters kept at 25 °C, roosters subjected to 38 °C acute heat stress for 4 hours without recovery, with 2 hours of recovery, and with 6 hours of recovery. Testis samples were collected for morphologic assay and protein analysis. Some of the differentially expressed proteins were validated by Western blot and immunohistochemistry. Abnormal and apoptotic spermatogenic cells were observed at 2 hours of recovery after acute heat stress, especially among the spermatocytes. Two-dimensional difference gel electrophoresis revealed that 119 protein spots were differentially expressed in chicken testes following heat stress, and peptide mass fingerprinting revealed that these spots contained 92 distinct proteins. In the heat-stressed samples, the heat shock proteins, chaperonin containing t-complex, and proteasome subunits were downregulated, and glutathione S-transferase, transgelin, and DJ-1 were upregulated. Our results demonstrate that acute heat stress impairs the processes of translation, protein folding, and protein degradation, and thus results in apoptosis and interferes with spermatogenesis. On the other hand, the increased expression of antioxidant enzymes, including glutathione S-transferase and DJ-1, may attenuate heat-induced damage. These findings may have implications for breeding chickens that can tolerate more extreme conditions.

Differential gene expressions in testes of L2 strain Taiwan country chicken in response to acute heat stress.

Acute heat stress affects genes involved in spermatogenesis in mammals. However, there is apparently no elaborate research on the effects of acute heat stress on gene expression in avian testes. The purpose of this study was to investigate global gene expression in testes of the L2 strain of Taiwan country chicken after acute heat stress. Twelve roosters, 45 weeks old, were allocated into four groups, including control roosters kept at 25 °C, roosters subjected to 38 °C acute heat stress for 4 hours without recovery, with 2-hour recovery, and with 6-hour recovery, respectively. Testis samples were collected for RNA isolation and microarray analysis. Based on gene expression profiles, 169 genes were upregulated and 140 genes were downregulated after heat stress using a cutoff value of twofold or greater change. Based on gene ontology analysis, differentially expressed genes were mainly related to response to stress, transport, signal transduction, and metabolism. A functional network analysis displayed that heat shock protein genes and related chaperones were the major upregulated groups in chicken testes after acute heat stress. A quantitative real-time polymerase chain reaction analysis of mRNA expressions of HSP70, HSP90AA1, BAG3, SERPINB2, HSP25, DNAJA4, CYP3A80, CIRBP, and TAGLN confirmed the results of the microarray analysis. Because the HSP genes (HSP25, HSP70, and HSP90AA1) and the antiapoptotic BAG3 gene were dramatically altered in heat-stressed chicken testes, we concluded that these genes were important factors in the avian testes under acute heat stress. Whether these genes could be candidate genes for thermotolerance in roosters requires further investigation.

Different immune responses to three different vaccines following H6N1 low pathogenic avian influenza virus challenge in Taiwanese local chicken breeds.

BACKGROUND: H6N1 low pathogenic avian influenza virus (LPAIV) are frequently isolated in Taiwan and lead to significant economic losses, either directly or indirectly through association with other infectious diseases. This study investigates immune responses to three different vaccines following a H6N1 challenge in different local breeds. METHODS: Experimental animals were sampled from six local chicken breeds maintained at the National Chung-Hsing University, namely Hsin-Yi, Ju-Chi, Hua-Tung (Taiwan), Quemoy (Quemoy Island), Shek-Ki (China), Nagoya (Japan) and a specific pathogen free (SPF) White Leghorn line. A total number of 338 chickens have been distributed between a control and a challenge group, H6N1 challenge was performed at 7 weeks of age; vaccination against Newcastle Disease (ND), Infectious Bursal Disease (IBD) and Infectious Bronchitis (IB) was performed at 11 weeks. The anti-H6N1 LPAIV antibody titers were measured by ELISA at days 0, 7, 14 and 21 after challenge, and the anti-ND, anti-IBD and anti-IB antibody titers were measured by inhibition of hemagglutination test and ELISA at days 0, 14, 28 after vaccination. RESULTS: There was no effect of the H6N1 LPAIV challenge at 7 weeks of age on the subsequent responses to ND and IBD vaccine at 11 weeks of age, but, surprisingly, the H6N1 LPAIV challenge significantly affected antibody levels to IB vaccine in some breeds, since IB0 and IB14 antibody titers were lower in the challenge groups. However, there was no significant difference in IB28 antibody titers among the experimental groups. CONCLUSIONS: Local breeds have different immune response to H6N1 LPAIV challenge and subsequent vaccines. Differences dealt mainly with kinetics of response and with peak values. Quemoy exhibited higher antibody levels to H6N1, ND and IBD. The negative effect of the H6N1 LPAIV challenge on IB vaccine response may be related to the fact that both viruses target the lung tissues, and the type of local immune response induced by LPAIV challenge may not be favourable for birds to make optimum IB-specific antibody response.