Yen-Pai Lee

Using molecular markers and multivariate methods to study the genetic diversity of local European and Asian chicken breeds

French and Asian subsets of chicken breeds were first analysed using 22 microsatellites and then compared to the AVIANDIV European set using 14 loci. Positive correlations were observed between F(IT) or F(ST) and typological values or variance of markers using the multivariate analysis mcoa. The first axis of the multivariate representation separated Asian from European breeds, revealing breeds with Asian ancestor. Using all or 14 loci, correct assignation rate was always higher than 93%. The Weitzman index and the aggregate diversity D were calculated using 22 loci within French and Asian breeds. The French breed Coucou de Rennes and the Hua-Tung breed seemed to contribute the most to the global diversity of each subset. This approach on French-only breeds and then on French with AVIANDIV domestic breeds (14 loci) showed that the Marans breed contributed the most. The AVIANDIV framework could be useful to evaluate the genetic diversity of local breeds and to help in connecting national and regional conservation policies.

Substantial decrease of heat-shock protein 90 precedes the decline of sperm motility during cooling of boar spermatozoa.

The decline in boar semen quality after cryopreservation may be attributed to changes in intracellular proteins. Thus, the aim of the present study was to evaluate the change of protein profiles in boar spermatozoa during the process of cooling and after cryopreservation. A total of 9 sexually mature boars (mean age = 25.5+/-12.3 mo) was used. Samples for protein analysis were collected before chilling, after cooling to 15 degrees C, after cooling to 5 degrees C, following thawing after freezing to -100 degrees C, and following thawing after 1 wk of cryopreservation at -196 degrees C. Semen characteristics evaluated included progressive motility and the percentage of morphologically normal spermatozoa. Total proteins from 5x10(6) spermatozoa were separated and analyzed by SDS-PAGE. The results revealed that there was a substantial decrease of a 90 kDa protein in the frozen-thawed spermatozoa. Western blot analysis demonstrated that this protein was 90 kDa heat-shock protein (HSP90). Time course study showed that the decrease of HSP90 in spermatozoa initially occurred in the first hour during cooling to 5 degrees C. When compared with the fresh spermatozoa before chilling, there was a 64% decrease of HSP90 in spermatozoa after cooling to 5 degrees C. However, the motility and percentage of normal spermatozoa did not significantly decrease during this period of treatment. Both declined substantially as the semen was thawed after freezing from -100 degrees C. The results indicated that the decrease of HSP90 precedes the decline of semen characteristics. The length of time between a decrease of HSP90 and the decline in sperm motility was estimated to be 2 to 3 h. Taken together, the above results suggested that a substantial decrease of HSP90 might be associated with a decline in sperm motility during cooling of boar spermatozoa.

The decline of porcine sperm motility by geldanamycin, a specific inhibitor of heat-shock protein 90 (hsp90)

Sperm motility is an important parameter for fertility. The molecular mechanisms of mammalian sperm motility are still largely undefined. Our previous observations suggested that heat shock protein 90 (HSP90) may be associated with porcine sperm motility. The aim of the present study was to further characterize the plausible novel function of HSP90 on sperm motility. Semen from normal, sexually mature boars with sperm motility higher than 80% was used. An HSP90-specific inhibitor, geldanamycin (GA), was added to diluted semen at 0.5, 1.0, 2.5 or 5.0 microg/mL and the semen was then incubated at 37 degrees C for 15, 30, 45 or 60 min. Sperm motility was determined by using computer-assisted semen analyzer at the end of incubation. The results indicated that GA significantly reduced sperm motility in a dose and time dependent manner. Moreover, incubation of semen with 5.0 microg/mL GA for 15 min completely stopped sperm motility. To test the reversibility of the GA effect on sperm motility, GA was removed after 30 min incubation and was replaced with fresh extender alone or with extender plus 5 mM caffeine, then incubated for another 15, 30, 45 or 60 min. The results showed that simply removing GA did not reverse the inhibitory effect on sperm motility, while adding caffeine partially reversed this inhibitory effect. However, the effect of 2.5 or 5.0 microg/mL GA was not reversed by caffeine. Considering the specificity of GA targeting to HSP90, the above observations suggested that HSP90 may play a crucial role in regulating porcine sperm motility.

A global analysis of molecular markers and phenotypic traits in local chicken breeds in Taiwan

Molecular and phenotypic data have been combined to characterize the genetic diversity of six local chicken breeds maintained with a long-term conservation programme. Hua-Tung, Hsin-Yi, Ju-Chi and Quemoy originated from Taiwan, Shek-Ki is from South China, and Nagoya is from Japan. Molecular tools included 24 microsatellite markers, melanocortin 1 receptor (alpha melanocyte stimulating hormone receptor) (MC1R), the LEI0258 marker located within the major histocompatibility complex (MHC), and mitochondrial DNA. Performance was recorded on the same individuals for body weight, panting rate in summer and antibody response (antigens: Newcastle disease virus and sheep red blood cells). A multivariate method previously proposed for taxonomy was used to combine the different data sets. Melanocortin 1 receptor (alpha melanocyte stimulating hormone receptor) and the MCW330 marker contributed the most to the first axis of the multiple coinertia analysis of molecular markers. Melanocortin 1 receptor (alpha melanocyte stimulating hormone receptor) showed evidence of selection, probably related to its effect on feather colour. The MHC exhibited a large diversity, with 16 alleles of the LEI0258 marker. Immune response traits contributed the most to the principal component analysis of phenotypic data. Eight mitochondrial DNA haplotypes related to clades A, B, C and E were distributed across breeds and revealed an important contribution of Indian and European breeds to Ju-Chi, Quemoy and Hsin-Yi. Phenotypic data contributed less than molecular data to the combined analysis, and two markers, LEI0258 and LEI0228, contributed the most. The combined analysis could clearly discriminate all breeds, except Ju-Chi, which was similar to Quemoy for many criteria, except immune response.

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.

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.

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.

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.

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.

Prostaglandin-D synthetase induces transcription of the LH beta subunit in the primary culture of chicken anterior pituitary cells via the PPAR signaling pathway

Downstream effects of prostaglandin-D synthetase (PGDS) in a primary culture of chicken (Gallus gallus) anterior pituitary cells were investigated to study how PGDS regulated laying in hens. Either PGDS downstream metabolite, PGD(2) or PGJ(2), elevated LHB mRNA and LHB protein levels in dose- and time-dependent manners, and treatment with arachidonic acid (1 microM) alone upregulated 15-deoxy-Delta(12,14)-PGJ(2) (15-d-PGJ(2); derived from PGJ(2))/PGJ(2), LHB mRNA, and LHB protein levels (P<0.05) in the primary culture of chicken pituitary anterior cells. Transfection of the plasmid Enhanced Green Fluorescent Protein-PGDS plasmid into these cells in medium containing 1 microM arachidonic acid additionally increased 15-d-PGJ(2)/PGJ(2), LHB mRNA, and LHB protein levels (P<0.05). In the hypothalamus/pituitary gland of laying hens, there was a high correlation (r=0.64; P<0.05) between PGDS and the peroxisome proliferator-activated receptor A (PPARA) mRNA level in a high egg production strain, the L2 Taiwan country chickens. In commercial Single-Comb White Leghorn layers, there were high correlation coefficients between PGDS and PPARA (r=0.65; P<0.05) and between PGDS and PPARG (r=0.67; P<0.01) mRNA levels. A broad-range PPARs agonist, GW9578 (5 to 500 nM), enhanced LHB mRNA and LHB protein levels (P<0.05). The PPARA-specific (GW6471) and PPARG-specific (T0070907) antagonists suppressed endogenous LHB mRNA and LHB protein levels (P<0.05); in addition, both antagonists attenuated arachidonic acid-induced LHB mRNA levels (P<0.05) and PGDS-induced (in the presence of 1 microM arachidonic acid) LHB mRNA and LHB protein (P<0.05) levels in the primary culture of chicken anterior pituitary cells. Higher LHB mRNA/LHB protein ratios in PGD(2)-, PGJ(2)-, arachidonic acid-, PGDS-, and GW9578-induced as well as GW6471- and T0070907-suppressed anterior pituitary cells suggested that LHB transcription occurred before translation. In conclusion, PGDS induced LHB transcription and subsequent translation via the PPAR signaling pathway.