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In order to breed a strain that has heat tolerance and meat productivity, the commercial red-feathered Taiwan native chickens were male (F group), and heat stress resistant strain Taiwan native chickens (Taishu-9, bred by the Taiwan Livestock Research Institute) were female (TR9 group) to hybridize to generate offspring (F9 group). Three breeds of birds (male) were conducted to compare acute heat stress and meat quality. At 12 weeks of age, TR9 group showed the significantly lowest activity of plasma creatine kinase upon acute heat stress which indicated heat stress resistant in TR9 group as expected. In addition, only limited thermoregulation was obtained in F9 group, while F group exhibited almost no acute heat stress tolerance ability. After slaughtered at 16 weeks of age, the F group revealed poor meat quality in breast meat as pale, soft, and exudative (PSE)-like muscle samples according to CIE L* and pH value. The F9 group was an offspring of TR9 group with heat tolerance, but it only demonstrated limitation of heat resistance. However, the improve meat quality was obtained in F9 group compared to F group, and that may be contributed from better anti-stress as like as TR9 group during slaughtering process.

Elongator complexes are well known to be involved in a wide variety of cellular processes; however, their functions in mammalian oocytes have not been characterized. Here, we demonstrated in mice that specific deletion of one of the core subunits, Ikbkap/Elp1, in oocytes resulted in spindle defects and chromosome disorganization without affecting folliculogenesis. In accordance with these findings, we observed that Ikbkap mutant female mice are subfertile. Further analyses uncovered that kinetochore–microtubule attachments are severely compromised in Ikbkap-deficient oocytes. Moreover, we revealed that Ikbkap modulates the acetylation status of α-tubulin in oocytes, which may at least in part mediate the meiotic phenotypes described above by affecting microtubule dynamics and kinetochore function. Finally, we showed that embryos derived from Ikbkap-deficient oocytes exhibit an increased frequency of aneuploidy, digyny, progressive delays in preimplantation development, and severe degeneration before reaching the blastocyst stage. In summary, we identify Ikbkap as an important player in regulating oocyte meiosis by modulating tubulin acetylation for chromosome/spindle organization.

We report for the first time the subcellular localization of endogenous Aurora-C and examine its roles during female mouse meiosis. The most dramatic effect observed in the oocyte injected with kinase-deficient Aurora-C mRNA is cytokinesis failure in meiosis I, resulting in production of large polyploid oocytes., We previously isolated Aurora-C/Aie1 in a screen for kinases expressed in mouse sperm and eggs. Here, we show the localization of endogenous Aurora-C and examine its roles during female mouse meiosis. Aurora-C was detected at the centromeres and along the chromosome arms in prometaphase I–metaphase I and was concentrated at centromeres at metaphase II, in which Aurora-C also was phosphorylated at Thr171. During the anaphase I–telophase I transition, Aurora-C was dephosphorylated and relocalized to the midzone and midbody. Microinjection of the kinase-deficient Aurora-C (AurC-KD) mRNA into mouse oocytes significantly inhibited Aurora-C activity and caused multiple defects, including chromosome misalignment, abnormal kinetochore–microtubule attachment, premature chromosome segregation, and cytokinesis failure in meiosis I. Furthermore, AurC-KD reduced Aurora-C and histone H3 phosphorylation and inhibited kinetochore localization of Bub1 and BubR1. Similar effects also were observed in the oocytes injected with INCNEP-delIN mRNAs, in which the Aurora-C binding motif was removed. The most dramatic effect observed in AurC-KD–injected oocytes is cytokinesis failure in meiosis I, resulting in producing large polyploid oocytes, a pattern similar to Aurora-C deficiency human spermatozoa. Surprisingly, we detected no Aurora-B protein in mouse oocytes. We propose that Aurora-C, but not Aurora-B, plays essential roles in female mouse meiosis.

The purpose of this study was to characterize differentially expressed transcripts associated with varying rates of egg production in Taiwan country chickens. Ovarian follicles were isolated from two strains of chicken which showed low (B) or high (L2) rates of egg production, then processed for RNA extraction and cDNA library construction. Three thousand and eight forty clones were randomly selected from the cDNA library and amplified by PCR, then used in microarray analysis. Differentially expressed transcripts (P0.05, log(2)or = 1.75) were sequenced, and aligned using GenBank. This analysis revealed 20 non-redundant sequences which corresponded to known transcripts. Eight transcripts were expressed at a higher level in ovarian tissue prepared from chicken strain B, and 12 transcripts were expressed at a higher level in L2 birds. These differential patterns of expression were confirmed by semi-quantitative RT-PCR. We show that transcripts of cyclin B2 (cycB2), ferritin heavy polypeptide 1 (FTH1), Gag-Pol polyprotein, thymosin beta4 (TB4) and elongation factor 1 alpha1 (EEF1A1) were enriched in B strain ovarian follicles. In contrast, thioredoxin (TXN), acetyl-CoA dehydrogenase long chain (ACADL), inhibitor of growth family member 4 (ING4) and annexin II (ANXA2) were expressed in at higher levels in the L2 strain. We suggest that our approach may lead to the isolation of effective molecular markers that can be used in selection programs in Taiwan country chickens.

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.

That most Columbidae birds have no conspicuous sexual dimorphism often makes it difficult to identify their sex on the basis of external morphology. In the present study, we report a novel sex-specific DNA marker in Columbidae birds. DNA was extracted from one member of this bird group, Streptopelia orientalis (S. orientalis, oriental turtle dove), and used to identify a female-specific DNA marker using a random amplified polymorphic DNA (RAPD) fingerprinting. One hundred and sixty random primers were used for the RAPD-PCR reactions. When using the OPAV17 primer, a novel 902 bp sex-specific PCR product was amplified from known female birds. This fragment of DNA was cloned and sequenced. Two primers, TurSexOPAV17-F and TurSexOPAV17-R, were designed from the cloned sex-specific sequence, and were successfully used to amplify a 777 bp female-specific fragment using PCR from S. orientalis DNA. This sex-specific marker was also amplified from genomic DNA samples of two other female Columbidae, S. chinensis and Columba livia. Sequence analysis showed that this novel sex-specific marker was highly conserved amongst these three bird species. In contrast, the PCR product was not amplified from male DNA of these species, nor from either sex of the S. chinensis formosa birds. Therefore, we concluded that our novel marker can be used to rapidly and accurately identify the sex of birds from three species of Columbidae.

We previously isolated Aurora-C (Aurkc/Aie1) in a screen for kinases expressed in mouse sperm and eggs. Aurora-C kinase was reported to be a chromosomal passenger protein that plays critical roles in chromosome alignment, segregation, kinetochore-microtubule attachment, and cytokinesis in female mouse meiosis. This chapter describes experimental approaches for examining the subcellular localization and function of Aurora-C kinase during female mouse meiosis, presenting detailed methods for introducing exogenous Aurora-C wild-type and kinase-dead mutant mRNAs into mouse oocytes by cytosolic microinjection, and preparing whole-mount meiotic oocytes and chromosome spreads for confocal immunofluorescence microscopy.

We performed the first genome-wide expression analysis to compare the differences in gene expression in the female sperm reservoir of the duck reproductive tract between two groups with long and short fertile periods to identify factors that may be associated with the fertile period using an oligonucleotide microarray. RNA was extracted from the uterovaginal junction (UV junction) of the two groups. Affymetrix chips containing comprehensive coverage of 32773 transcripts were hybridized with biotin-labeled cRNA, and three biological repeats were performed. We identified 27 transcripts as being differentially regulated. Interestingly, by mapping the differentially expressed transcripts to annotated pathways, we found that Neuropeptide Y (NPY), the RNA expression of which was increased by 2.96-fold in the short-fertile-period group as compared with the long-fertile-period group in our experiment, has been shown to reduce blood flow and substance supply to local tissues. Enah/Vasp-like (EVL), the RNA expression of which was significantly increased by 1.77-fold in the short-fertile-period group as compared with the long-period group, has been demonstrated to be important in activated T-cells. In contrast, trafficking kinesin-binding protein 1 (TRAK1), the expression of which was increased by 2.33-fold in the long-period group as compared with its counterparts, has been suggested to inhibit precocious activation of sperm and prolong sperm life in the female sperm reservoir. The results of real-time PCR confirmed the data obtained by microarray analysis. Our study demonstrated that combining global gene expression investigation with annotated pathway resources contributes to the understanding of sperm life when sustained in the UV junction.

Amplified fragment length polymorphism (AFLP) with multicolored fluorescent molecular markers was used to analyze duck (Anas platyrhynchos) genomic DNA and to construct the first AFLP genetic linkage map. These markers were developed and genotyped in 766 F2 individuals from six families from a cross between two different selected duck lines, brown Tsaiya and Pekin. Two hundred and ninety-six polymorphic bands (64% of all bands) were detected using 18 pairs of fluorescent TaqI/EcoRI primer combinations. Each primer set produced a range of 7 to 29 fragments in the reactions, and generated on average 16.4 polymorphic bands. The AFLP linkage map included 260 co-dominant markers distributed in 32 linkage groups. Twenty-one co-dominant markers were not linked with any other marker. Each linkage group contained three to 63 molecular markers and their size ranged between 19.0 cM and 171.9 cM. This AFLP linkage map provides important information for establishing a duck chromosome map, for mapping quantitative trait loci (QTL mapping) and for breeding applications.

1. The accuracy and reproducibility of AFLP fingerprinting was investigated in the duck (Anas Platyrhynchos), using a multicolour fluorescent labeling technique. The fluorescent labelling fragments were separated on a capillary electrophoresis-base ABI PRISM 3100 Genetic Analyzer. 2. A total of 337 AFLP peaks with 103 of them being polymorphic markers were generated by 16 sets consisting of EcoRI/TaqI primer pair combinations. The number and size range of AFLP polymorphisms detected per primer pair varied from 3 to 11 and 58 to 290 bp, respectively. About 30.6% (103/337) of AFLP peaks were detected polymorphisms, with an average of 6.4 polymorphic markers per primer pair. 3. The clear polymorphic peaks were amplified with EcoR+AC/Taq+AC primer combinations. The AFLP peaks showed high reproducibility. From the family testing, we found that the fingerprints of all the offspring were derived from one or other parent. Therefore, we conclude that AFLP fingerprinting might be a suitable method for duck paternity testing.

Ostrich absence of heteromorphic sex chromosomes, unique sequences or markers located in the ostrich W-chromosome. Random amplified polymorphic DNA (RAPD) fingerprinting was carried out to investigate the sex-specific DNA sequence for sexing in ostrich. One hundred and forty random primers were used for random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). One of these primers, OPAJ-13, produced a sex-specific band only found in tested females, which was isolated and constructed into plasmids for nucleotide sequencing. A 760 bp novel female-specific sequence was obtained. Two primers (OstSexOPAJ13-F and -R) were designed according to the cloned female sequence to amplify the female-specific fragment from genomic DNA of ostriches for sexing by PCR. The sex-specific band was represented in females but none were found in the males. This result showed that the sex of ostrich could be easily and effectively identified using the female-specific primers for PCR technique.