Development of innovative marker detection methods for high-fertility ducks (Anas plastyrhynchos).

We previously analyzed the genome-wide gene expression at the transcription level in pre-hierarchical ovarian follicles (approximate 5 mm in diameter) between two groups of ducks representing high and low fertility. Orthodenticle homeobox 2 (otx2) was identified with significantly differential expression in the high-fertility group versus the low-fertility group. To identify the relationships between genotypes and phenotypes, we recorded the reproductive performance in advance, including fertility, hatchability, and fertile period of female ducks. To ensure coverage of the entire duration of the fertile period, we extended the egg collection period after artificial insemination. Naturally, sperm cannot survive after a certain period of time in the female reproductive tract (sperm is not immortal); therefore, lower average values for fertility were observed in this study than that observed after a normal egg collection period, i.e., the lower average values of fertility (18 days after artificial insemination), were not due to the effect of otx2. The otx2 genomic sequence of Tsaiya ducks was firstly amplified with a primer pair of i3F and i3R for polymerase chain reaction based on Pekin duck sequence and a resultant 444-base pair fragment was obtained for DNA sequencing. Using multiple sequence alignment, new single-nucleotide polymorphisms g.366T > C and g.182G > T were discovered in the otx2 gene. With respect to g.366T > C, ducks were classified into CC, CT, and TT genotypes. For g.182G > T, three genotypes (GG, GT, and TT) were identified. Ducks were genotyped using novel specific primers and probes to rapidly screen their single-nucleotide polymorphisms. The results indicated that ducks with the CC genotype of g.366T > C exhibited the highest fertility among the CC, CT, and TT genotypes (p < 0.05). No significant difference was found in the fertile period and hatchability among three genotypes of g.366T > C. Moreover, no association was found between g.182G > T genotypes and the three reproductive phenotypes examined in this study. Collectively, the otx2 g.366T > C genotype is associated with duck females, and can be used as a marker for farming a flock of ducks with high fertility, as well as for genetic selection of breeders.

A new DNA marker of the TMIGD1 gene used to identify high fertilization rates in Tsaiya ducks (Anas platyrhynchos).

In a prior study, comparisons of individuals of Anas platyrhynchos with higher/lower reproductive performances showed that the expression of the transmembrane and immunoglobulin domain containing 1 (TMIGD1) gene significantly differed between the two groups. Here, we demonstrate that ducks with the TMIGD1 GG genotype have a significantly higher fertilization rate than other TMIGD1 genotypes. Primers designed based on the TMIGD1 sequence of Pekin duck were able to successfully amplify a TMIGD1 fragment from Tsaiya ducks, and sequencing results indicated that a single nucleotide polymorphism (SNP) of the TMIGD1 gene existed. We also developed a cost-effective method of restriction fragment length polymorphism. Using the above methods, ducks were classified into three genotypes. To identify the relationships between genotypes and traits, we recorded the ducks' performance; to ensure the coverage of the entire duration of the fertile period, the egg collection period was extended to 18 days, and therefore, lower than usual fertilization rates were observed. Further assessment using a high-throughput system showed that the ducks with the GG genotype exhibited the highest fertilization rates among genotypes (P < 0.05). We suggest that TMIGD1 may affect the release of sperm protection factors from the female genital tract, and thus alter fertilization rate. In conclusion, the results of this study demonstrate that the TMIGD1 GG genotype can be used as a new DNA marker to identify animals with high fertilization rates at a young age, a process which could improve farming efficiency.

A new method for detection of single nucleotide polymorphism in a female reproduction-associated gene, tmigd1, of Anas platyrhynchos using a strip biosensor with gold nanoparticles.

In this study, we first reported a lateral flow assay combined with primer extension (PEXT) and gold nanoparticles for single-nucleotide polymorphism (SNP) genotyping of the tmigd1 gene of the Tsaiya ducks (Anas platyrhynchos), which has the advantages of simplicity of operation, cost-effectiveness, and time-saving. Gold nanoparticles were tailed with thiol-thymine oligodeoxyribonucleotides (thiol-(dT)30) using the salt-aging method at 25°C and used as a label in a lateral flow assay. The lateral flow device was composed of test and control zones on a nitrocellulose membrane containing streptavidin and adenosine oligodeoxyribonucleotides ((dA)30), respectively. When the specific SNP existed, the corresponding primers were extended, and the reaction product was captured by streptavidin at the test zone owing to the introduction of biotin-deoxyuridine triphosphate (biotin-dUTP) into the reaction product during PEXT. Gold nanoparticles hybridized with the reaction product to render it visible. Here, we developed a new system for detection of single nucleotide polymorphism in a female reproduction-associated gene, tmigd1, of Anas platyrhynchos using the strip biosensor, and identified the optimized parameters for the concentration of Mg2+ in the PEXT reaction and the amount of streptavidin used on membranes for signal specificity.

Global transcriptional expression in ovarian follicles from Tsaiya ducks (Anas platyrhynchos) with a high-fertilization rate.

Novel candidates for biomarkers of a high-fertilization rate were identified here through global transcriptional profiling of ovarian follicles. Some other differentially expressed candidate genes were first noted to influence animal reproduction in our previous cDNA microarray analysis and are now recognized as markers for marker-assisted selection. In the present study, we compared gene expression in ovarian follicles from animals with high- and low-fertilization rates using an oligonucleotide array. On the basis of a fold change of greater than 1.2 and less than -1.2, a difference of >100 Affymetrix arbitrary units between the two groups, and a P value of less than 0.05, 47 genes were found to be associated with fertilization rate. GOEAST and MetaCore software were further used to identify the functional categories of genes that were differentially expressed. Then, we focused on three interesting genes associated with a high-fertilization rate: one of these genes was discovered to participate in signaling pathways of fertilization, and two genes take roles in lipid metabolism. An oligonucleotide array showed that the levels of orthodenticle homeobox 2 (OTX2) and lecithin:cholesterol acyltransferase (LCAT) gene expression were 1.62-fold and 1.95-fold higher in the high-fertilization rate group than in the low-fertilization rate group, respectively (P < 0.05). The level of apolipoprotein A-I (APOA1) gene expression was also higher in the high-fertilization rate group, with a difference of 2.31-fold (P < 0.05). The data were validated through quantitative polymerase chain reaction analysis. These results confirm the usefulness of the array technique and data mining methods in the discovery of new biomarkers and add knowledge to our understanding of the factors affecting fertilization rates in ovarian follicles.

A novel minisequencing single-nucleotide polymorphism marker of the lysozyme gene detects high hatchability of Tsaiya ducks (Anas platyrhynchos).

Lysozyme, one of the major albumen antimicrobials, can break down the polysaccharide walls of a broad spectrum of bacteria. This study presents a novel lysozyme marker of high hatchability in the form of minisequencing single-nucleotide polymorphisms (SNPs). Recently, lysozyme was identified by complementary DNA microarray analysis as one of several differentially expressed genes noted to influence hatchability and recognized as a marker candidate for animal marker-assisted selection. Higher levels (P < 0.05) of lysozyme mRNA (via real-time polymerase chain reaction analysis) and protein (in Western blotting results) were found to be associated with a high-hatchability phenotype. In the preliminary sequence analysis of this study, TsLy1-1 and TsLy1-2 primer pairs, designed according to the lysozyme sequence, were used to amplify small-scale genomic DNA samples from animals in two extreme groups of hatchability. Sequence analysis of the amplified 763-bp DNA products clearly showed that AA and GG genotypes of SNP g.390A > G were from the ducks of the low- and high-hatchability groups, respectively. The SNP g.390A > G also created a new specificity protein 1 transcription factor binding site in the lysozyme gene. Primer pairs of TsLy2-1 and TsLy2-2 then probed the amplified 763-bp DNA products to produce a shorter fragment for easier minisequencing analysis to divide 114 ducks into GG, GA, and AA genotypes. The GG ducks had the highest hatchability, representing that a new lysozyme SNP marker of good hatchability performance can be used for the purpose of marker-assisted selection in Tsaiya ducks.

A novel SNP marker of ovalbumin gene in association with duck hatchability.

Our previous transcriptome analysis using a cDNA microarray identified differentially-expressed transcripts in Tsaiya ducks (Anas platyrhynchos); we concluded that the ovalbumin gene might be involved in duck hatchability. In the present study, associations of single nucleotide polymorphism (SNP) genotypes of the duck ovalbumin gene with hatchability were investigated. To confirm the cDNA microarray analysis, real-time polymerase chain reaction (PCR) and Western blot analysis were used to validate ovalbumin gene expression. The messenger RNA and protein expression of the ovalbumin gene were higher (P < 0.05) in the low-hatchability group (1.00 ± 0.19; 30.36 ± 3.51 arbitrary units) than in high-hatchability counterparts (0.56 ± 0.07; 8.53 ± 2.97 arbitrary units), consistent with the previous cDNA microarray analysis. The PCR products (506 base pairs) of ovalbumin gene amplified by the primer pair of TovaF and TovaR from the genomic DNA templates of 10 ducks were sequenced and a g.385 C>T SNP site in the 506-base pair sequence of the ovalbumin gene identified. Genotyping of SNP of 187 ducks was then carried out by PCR restriction fragment length polymorphism and minisequencing methods. Based on SNP genotypes of the duck ovalbumin gene, there were three types: CC, TT, and CT. Birds with the CC and TT genotypes had higher hatchability (79.59 ± 3.40, 76.35 ± 1.77) (P < 0.05) than those with a CT genotype (65.77 ± 2.07). In conclusion, the ovalbumin gene was an important candidate gene that can be used for marker-assisted selection to increase hatchability in Tsaiya ducks.

Association of prolactin haplotypes with reproductive traits in Tsaiya ducks.

A previous cDNA microarray study showed that the prolactin (PRL) gene may be involved in the duck ovarian follicle development and egg formation process. The purpose of this study was to investigate the relationship between PRL genotypes of single nucleotide polymorphism (SNP) and reproductive traits of Tsaiya ducks. Primer pairs for the coding regions in the PRL were designed based on the duck genomic sequence database. Polymorphisms were detected by polymerase chain reaction (PCR)-single strand polymorphism (SSCP) and were verified by DNA sequencing. Six novel SNPs (T233C, T295C, G309T, C381A, G3941T and A3975C) were identified in the 1972 bp region of duck PRL gene, and all of them were located in non-coding regions. Single SNP-trait association analysis showed that each SNP was associated with at least one duck reproductive trait (P<0.05). Haplotype combinations constructed on these SNPs were associated with egg weight at 40 weeks of age (EW40), fertility rate (FR) and maximum duration of fertility (MDF) (P<0.0001). In particular, diplotype H1H2 had positive effect on EW40, whereas it had negative effect on FR and MDF (P<0.05). Positive effects of the diplotype H1H5 were observed for FR and MDF, but a negative effect was observed for EW40 (P<0.05). This suggested that the PRL gene plays an important role in the regulation of egg weight and fertility-related traits and could be a potential marker in a marker assisted selection program during duck balancing selection. Further investigations on more duck populations with large sample sizes are needed to confirm this finding.

A novel non-synonymous SNP of the COLX gene and its association with duck reproductive traits.

Type X collagen (COLX) is a known marker of chondrocyte hypertrophy, which is exclusively expressed in hypertrophic chondrocytes and is reported to be involved in the process of mineralization. The purpose of this study was to investigate the relationship between COLX genotypes of single nucleotide polymorphism (SNP) and reproductive traits of Tsaiya ducks. A total of 336 Brown Tsaiya ducks from two lines, the control line (CL) with no selection and the selected line (SL), were employed for testing. We employed polymerase chain reaction -single strand conformation polymorphism and DNA sequencing to screen the polymorphisms of the COLX gene. One novel non-synonymous SNP in coding region (T74C: Val24Ala) of the COLX gene was found, and resulted in 3 genotypes TT, TC, and CC. The frequencies of genotype TT and allele T were high in both lines. Regarding egg weight at 40 weeks of age (EW40), based on SNP-trait association analysis, ducks with the CC genotype had a 4.09 and 4.15 g/egg lower EW40 as compared with ducks with the TT and TC genotypes in the CL, respectively (P < 0.05). In addition, significant positive dominance effect of 2.10 ± 1.05 g/egg for EW40 was detected (P = 0.0481). This finding indicated that selection for the genotype TT and TC ducks might contribute to an improved egg weight in the Tsaiya ducks. Further investigations on more duck populations with large sample sizes are needed to confirm.

Preliminary evaluation of growth and conformation traits of local goats and Nubians upgraded by a black Boer line in Taiwan.

The goal of this research, conducted in the most southern part of Taiwan, was to create a new genotype: the "Hengchun Black Goat" (HB). Nubian (NU) goats were first crossed with a local breed, the Taiwan native (TN), then the F1 females were crossed with the imported black Boer (BO) bucks. The upgraded genotypes were then compared with the parental breeds and Kinmen (KM), another local breed, for growth traits and body conformation. The study concerned 1,136 kids born between 2005 and 2007. The analysed traits were body weight (BW), average daily gain and three linear measurements, namely height at withers, body length and chest girth. The results indicated that environmental factors, sex, birth and rearing type, dam parity and birth year had significant effects from birth to 6 months of age. The same differences persisted to 1 year. At 6 months of age, the least square means of BW were 16.2, 19.2, 25.1, 32.0, 23.9, 23.8, 23.0 and 23.9 kg, for KM, TN, NU, 1/2BO, 3/4BO, 7/8BO, BO and HB, respectively. These first results also indicate that the growth performances of the newly created line, Hengchun Black, were equivalent to those of Boer goats.

The SNP genotypes of growth hormone gene associated with reproductive traits in Tsaiya ducks.

Our previous cDNA microarray study showed that the growth hormone (GH) gene may involve in the duck egg formation process. The purpose of this study was to investigate the relationship between GH genotypes of single nucleotide polymorphisms (SNPs) and reproductive traits of Tsaiya ducks. Primer pairs for the coding region in the GH were designed based on the duck genomic sequence. Polymorphisms were detected by polymerase chain reaction (PCR)-single strand polymorphism (SSCP) and were verified by DNA sequencing. Nineteen SNPs were identified in the duck GH gene, of which three coding SNPs (C3169T, C3700T and C5058G) were genotyped to investigate the associations with reproductive traits. The results showed that each SNP was associated with at least one duck fertility-related trait (p < 0.05). Haplotypes constructed on these three SNPs were associated with fertility rate (FR) and maximum duration of fertility (MDF) (p < 0.05). In particular, diplotype H1H1 was dominant for FR and MDF. This suggested that GH gene polymorphisms are associated with duck fertility-related traits. The SNPs in this gene may be used as potential markers for marker-assisted selection.

Genome-wide transcript expression analysis in the uterovaginal junction in association with fertile period in Tsaiya ducks.

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.

Eleven generations of selection for the duration of fertility in the intergeneric crossbreeding of ducks.

A 12-generation selection experiment involving a selected line (S) and a control line (C) has been conducted since 1992 with the aim of increasing the number of fertile eggs laid by the Brown Tsaiya duck after a single artificial insemination (AI) with pooled Muscovy semen. On average, 28.9% of the females and 17.05% of the males were selected. The selection responses and the predicted responses showed similar trends. The average predicted genetic responses per generation in genetic standard deviation units were 0.40 for the number of fertile eggs, 0.45 for the maximum duration of fertility, and 0.32 for the number of hatched mule ducklings' traits. The fertility rates for days 2-8 after AI were 89.14% in the S line and 61.46% in the C line. Embryo viability was not impaired by this selection. The largest increase in fertility rate per day after a single AI was observed from d5 to d11. In G12, the fertility rate in the selected line was 91% at d2, 94% at d3, 92% at days 3 and 4 then decreased to 81% at d8, 75% at d9, 58% at d10 and 42% at d11. In contrast, the fertility rate in the control line showed an abrupt decrease from d4 (74%). The same tendencies were observed for the evolution of hatchability according to the egg set rates. It was concluded that selection for the number of fertile eggs after a single AI with pooled Muscovy semen could effectively increase the duration of the fertile period in ducks and that research should now be focused on ways to improve the viability of the hybrid mule duck embryo.

Duck (Anas platyrhynchos) linkage mapping by AFLP fingerprinting.

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.

Selection responses for the number of fertile eggs of the Brown Tsaiya duck (Anas platyrhynchos) after a single artificial insemination with pooled Muscovy (Cairina moschata) semen.

A seven-generation selection experiment comprising a selected (S) and a control (C) line was conducted with the objective of increasing the number of fertile eggs (F) of the Brown Tsaiya duck after a single artificial insemination (AI) with pooled Muscovy semen. Both lines consisted of about 20 males and 60 females since parents in each generation and each female duck was tested 3 times, at 26, 29 and 32 weeks of age. The fertile eggs were measured by candling at day 7 of incubation. The selection criterion in the S line was the BLUP animal model value for F. On average, 24.7% of the females and 15% of the males were selected. The direct responses to the selection for F, and correlated responses for the number of eggs set (Ie), the number of total dead embryos (M), the maximum duration of fertility (Dm) and the number of hatched mule ducklings (H) were measured by studying the differences across the generations of selection between the phenotypic value averages in the S and C lines. The predicted genetic responses were calculated by studying the differences between the S and C lines in averaged values of five traits of the BLUP animal model. The selection responses and the predicted responses showed similar trends. There was no genetic change for Ie. After seven generations of selection, the average selection responses per generation were 0.40, 0.33, 0.42, 0.41 genetic standard deviation units for F, M, Dm, and H respectively. Embryo viability was not impaired by this selection. For days 2-8 after AI, the fertility rates (F/Ie) were 89.2% and 63.8%, the hatchability rates (H/F) were 72.5% and 70.6%, and (H/Ie) were 64.7% and 45.1% in the S and C lines respectively. It was concluded that upward selection on the number of fertile eggs after a single AI with pooled Muscovy semen may be effective in ducks to increase the duration of the fertile period and the fertility and hatchability rates with AI once a week instead of twice a week.