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A whole genome scanning for quantitative trait loci on traits related to sperm quality and ejaculation in pigs
Animal Reproduction Science 114 (2009) 210–218
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Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
A whole genome scanning for quantitative trait loci on traits related to sperm quality and ejaculation in pigs Yuyun Xing a, Jun Ren a, Dongren Ren a, Yuanmei Guo a, Yanbo Wu a, Guangcheng Yang a, Huirong Mao a, Bertram Brenig b, Lusheng Huang a,∗ a
Key Laboratory for Animal Biotechnology of Jiangxi Province and the Ministry of Agriculture of China, Jiangxi Agricultural University, Nanchang 330045, PR China Institute of Veterinary Medicine, Georg-August-University of Göttingen, Burckhardtweg 2, 37077 Göttingen, Germany
b
a r t i c l e
i n f o
Article history: Received 23 May 2008 Received in revised form 5 July 2008 Accepted 4 August 2008 Available online 8 August 2008 Keywords: QTL Semen quality Ejaculation Pig
a b s t r a c t To identify quantitative trait loci (QTL) for traits related to semen and ejaculation, phenotype data including semen volume, sperm concentration, total sperm per ejaculate, sperm motility, sperm abnormality rate, semen pH value, ejaculation times and ejaculation duration were measured on 206 F2 boar at 240 days in a White Duroc × Erhualian intercross. A genome-wide scan was performed and the entire White Duroc × Erhualian intercross was genotyped for 183 microsatellite markers covering the whole pig genome. QTL analysis was performed using a composite regression interval mapping method via QTLExpress. A total of 18 QTL were detected, including 4 genome-wide significant QTL each for semen pH on pig chromosome (SSC) 2 and SSC12, for semen volume on SSC15, and for ejaculation times on SSC17. Fourteen suggestive QTL were found on SSC1, 2, 3, 4, 6, 9, 17 and 18. To our knowledge, this is the first report about the QTL for semen and ejaculation traits in pigs, providing a start point to decipher the genetic basis of these complex traits. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Artificial insemination (AI) has been extensively used in the pig industry and has a significant influence on the swine production. It is well known that AI requires boars with excellent semen quality to
∗ Corresponding author. Tel.: +86 791 3805967; fax: +86 791 3805967. E-mail address: [email protected] (L. Huang). 0378-4320/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2008.08.008
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improve meat productivity. However, it is difficult to perform direct selection for semen quality because of their low heritabilities and sex-limited characteristics (Rothschild and Bidanel, 1998). Markerassisted selection on the basis of candidate gene or quantitative trait loci (QTL) could be an effective way to improve male reproductive traits. More than 60 QTL affecting at least 14 female reproductive traits have been identified across the pig genome (http://www.animalgenome.org/QTLdb/pig.html). In comparison, only about 20 QTL for male reproductive traits including testis and epididymis weight (size), seminal vesicles weight, seminiferous tubular diameter, length of bulbo-urethral gland, serum testosterone concentration and follicle-stimulating hormone (FSH) have been reported (Bidanel et al., 2001; Rohrer et al., 2001; Sato et al., 2003; Ren et al., 2008a). Furthermore, studies of candidate genes for semen quality are limited (Wimmers et al., 2005; Lin et al., 2006; Terman et al., 2006) and QTL for traits related to semen quality and ejaculation remain unexplored in pigs. Deciphering the genetic basis of porcine male reproductivity will not only benefit the pig industry but also human medicine. Human infertility is an important reproductive health problem that is implicated in about 50% of sterility cases (Griffin and Finch, 2005). About 15% of man infertility cases are attributable to genetic factors (Ørstavik, 2008). Pig is a good choice as animal model for human disease, and the genetic research for boar semen quality can provide referenced information for human infertility study. The Chinese Taihu pig is one of the most prolific pig breeds in the world. Boars from two subpopulations (Erhualian and Meishan) of the Taihu breed exhibit markedly higher concentration of serum FSH hormone, luteinizing hormone and androgens whereas lower testis size, total daily sperm number and ejaculate volume compared with European commercial boars (Borg et al., 1993; Lunstra et al., 1997). These particular attributes are useful for studies of the genetic basis of boar reproductive traits. We have established an F2 intercross between White Duroc boars and Chinese Erhualian sows and have recorded diverse phenotypic traits including those related to semen quality and ejaculation. The objective of this study was to identify QTL affecting semen quality and ejaculation traits by using the White Duroc-Erhualian resource population. 2. Materials and methods 2.1. Animals The White Duroc × Erhualian intercross resource population was developed and managed as described previously (Ren et al., 2006). Briefly, two White Duroc boars were mated to seventeen Erhualian sows that were selected from three breeding farms representing high reproductive performance in China. A total of 1912 F2 animals were generated in six batches by intercrossing of 9 F1 males and 59 F1 females avoiding full-sib mating. In this study, 206 boars at 280 days from 54 full-sib families were recorded for 8 traits related to semen quality and ejaculation including semen volume, sperm concentration, total sperm per ejaculate, sperm motility, sperm abnormality rate, semen pH value, ejaculation times and ejaculation duration. All boars were raised at the experimental research farm at Jiangxi Agriculture University in Nanchang (China). These boars were weaned at 46 days of age and moved into a nursery and were then housed individually after 120 days of age until phenotypic measurement. All the procedures involving animals followed the guidelines for the care and use of experimental animals established by the Ministry of Agriculture of China. 2.2. Phenotype measurement Semen collection and measurements of semen volume, sperm concentration and total sperm per ejaculate were described previously in Ren et al. (2008b). Briefly, each boar was trained to mount docile boars or receptive gilts that were positioned underneath a special dummy sow once a day from 240 days of age. After 280 days of age, each boar was collected for semen by glove-hand method 4 times at an interval of 4 days. To reduce personal error, all traits were measured by fixed personnel. Sperm motility was assessed with a light microscope (Chongqing, Jiangxi, China) at 200× magnification immediately after semen was collected. For the measurement of sperm abnormality rate, semen samples were
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Table 1 Phenotypic mean, standard error and range of F2 boars in a White Duroc × Erhualian resource population Trait
No.
Semen volume (ml) Sperm concentration (million/ml) Total sperm per ejaculate (109 ) Sperm motility Sperm abnormality rate pH value Ejaculation times Ejaculation duration (s)
177 176 176 175 173 177 177 177
Range 69.30–462.50 0.70–707.00 0.07–927.20 0.30–0.88 0.10–0.51 7.02–7.69 1.00–15.8 90.00–845.40
Mean ± S.E. 180.50 231.00 39.08 0.72 0.24 7.26 4.10 350.30
± ± ± ± ± ± ± ±
57.20 117.00 18.06 0.10 0.09 0.13 2.10 128.00
gently smeared with glass slides and then dried and stained in a phosphate-buffered Giemsa solution (Kovacs and Foote, 1992). Stained slides were evaluated by a Leica DMR microscope (Leica, Wetzlar, Germany) at 400× magnification and at least 200 sperms were examined in each assay. Semen pH value was measured by a Mp120 pH Meter (Mettler-Toledo, Greifensee, Switzerland) immediately after semen were collected. Ejaculation times were calculated on the basis of short interval of every ejaculation in the whole ejaculation process. A BD-TR118 stopwatch (Suzhou, China) was used to record the time of a whole ejaculation.
2.3. Genotyping and statistical analysis A total of 183 informative microsatellite markers across 18 autosomes and chromosome X were selected on the basis of their informativeness and locations. All founder animals, F1 animals and 206 F2 boars were genotyped for the 183 markers as described previously in Ren et al. (2008a). Genotypic data were analyzed with CRIMAP version 2.4 (Green et al., 1990) to construct a whole-genome linkage map. The PROC GLM procedure of SAS version 9.0 (SAS Institute Inc., Cary, NC, USA) was used to determine factors included in the subsequent QTL mapping analysis. Factors having effect above 5% significant level on trait of interest were kept as fixed effects or covariates in the QTL model. Family was included as a fixed effect for all traits. Farm was used as a fixed effect in the QTL model for semen volume and total number per ejaculate. Boar age was included as a covariate for semen volume. Semen temperature was a fixed effect for semen pH. Air temperature was fitted as a covariate for semen volume. The QTL analysis was performed with the web-accessible QTL Express at http://qtl.cap.ed.au.uk/, which was based on a least-squares method (Haley et al., 1994). The least-squares regression model was fitted at one centimorgan interval along each chromosome and the F-value for the QTL effect was calculated at each point. Empirical threshold values for QTL mapping were estimated by using the genomewide permutation test with 1000 random data shuffles as described by Churchill and Doerge (1994). The empirical 95% confidence intervals (CI) were evaluated by a bootstrapping approach with 2000 iterations (Visscher et al., 1996).
3. Results 3.1. Phenotype data Phenotypic mean, standard error and range of F2 boars are listed in Table 1. The White DurocErhualian crossbred boars showed considerable large variation and segregation in all traits measured (Table 1). The correlation coefficients among traits measured are shown in Table 2. Ejaculation duration had a strong positive correlation (r = 0.87) with ejaculation times, and total sperm per ejaculate had a strong positive correlation (r = 0.73) with sperm concentration. Ejaculation duration and ejaculation times had a medium positive correlation with semen volume whereas medium negative correlation with sperm concentration.
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Table 2 Pearson Product Moment Correlations among semen quality and ejaculation traits Trait
Semen volume
Total sperm per ejaculate
Sperm motility
Sperm abnormality rate
pH value
Ejaculation times
Ejaculation duration
Sperm concentration Semen volume Total sperm per ejaculate Sperm motility Sperm abnormality rate pH value Ejaculation times
−0.30**
0.73** 0.34**
−0.0006 −0.02 −0.09
−0.001 −0.08 −0.09 0.06
−0.21** −0.21** −0.37** −0.04 −0.07
−0.33** 0.48** −0.04 −0.21** −0.14 0.17*
−0.42** 0.49** −0.12 −0.08 −0.05 0.23** 0.87**
Significance level. * **
P < 0.05. P < 0.01.
3.2. QTL identification Details of QTL identified in this study are shown in Table 3. A total of 18 QTL affecting the 8 traits measured were detected on 11 chromosomes including 4 genome-wide significant QTL and 14 suggestive QTL. The F-value for thresholds of suggestive significance, 10%, 5% and 1% genome-wide significance was 4.39, 6.33, 6.94 and 8.47, respectively. A 10% genome-wide significant QTL for semen volume was mapped at 16 cM on pig chromosome (SSC) 15 (Fig. 1a). It explained 8.63% of the phenotypic variance, and the QTL allele increasing semen volume was inherited from the Erhualian breed. Moreover, two suggestive effect associated with semen volume were found at 101 cM on SSC3 and 69 cM on SSC18, respectively. At 33 cM on SSC1 and 37 cM on SSC2, two suggestive QTL for total sperm per ejaculate were found. The SWR100-SWR112 region (data not shown) on SSC17 had a suggestive significant effect on sperm concentration. A 5% genome-wide and a 10% genome-wide significant QTL for semen pH value was found on SSC2 and SSC12 (Fig. 1b, c), explaining 9.84% and 9.02% of the phenotypic variance, respectively. The alleles from the Erhualian breed at the two loci were associated with increased pH values. Two additional suggestive QTL for semen pH value were detected with peaks at 185 cM on SSC6 and 77 cM on SSC9, respectively. One 1% genome-wide significant QTL affecting ejaculation times with positive additive effect of Erhualian alleles was evidenced at 86 cM on SSC17 (Fig. 1d). This is the most significant QTL detected in this study, accounting for 11.83% of the phenotypic variation in the F2 population. This chromosomal region was also evidenced as a suggestive QTL for ejaculation duration. Another two suggestive QTL for ejaculation times were observed at 124 cM on SSC6 and 67 cM on SSC16. A suggestive QTL for ejaculation duration was found close the QTL for ejaculation times on SSC6. Two suggestive QTL for sperm abnormality were detected each on SSC4 and SSC9. A suggestive QTL affecting sperm motility was evidenced close to the QTL for total sperm per ejaculate on SSC1.
4. Discussion To our knowledge, this is the first report about QTL for sperm quality and ejaculation traits in pigs. In other mammals, only several QTL for sperm quality were reported in mice and rats (L’Hôte et al., 2007; Golas et al., 2008). Through pig–mouse comparative maps at http://www.ncbi.nlm.nih.gov/Homology/ and http://www.animalgenome.org/cgi-bin/QTLdb/SS/draw chromap, the QTL for sperm abnormality rate on SSC4 was homolog to the QTL for mouse sperm nucleus shape (L’Hôte et al., 2007). In this study, only 4 chromosomal regions showed genome-wide significant associations with the traits measured. The limited number of genome-wide significant QTL could be due to the relatively small size of the F2 population. We cautioned that those suggestive QTL could be interpreted as false positive results. As highly correlated traits, QTL for ejaculation times and ejaculation duration were found in the same regions on SSC6 and SSC17. It should be noted that QTL for sperm concentration and
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Table 3 QTL for traits related to semen and ejaculation in a White Duroc × Erhualian resource population Trait
Chr
Position (cM)
95% CI position
Additive effect ± S.E.
Dominant effect ± S.E.
Variance (%)b
Semen volume
15 18 3
16 69 101
0.0–21.5 2.0–69.0 1.5–120.0
6.55* 6.12 6.09
−25.21 ± 5.80 19.91 ± 5.71 −9.78 ± 6.23
−12.37 ± 8.87 −14.88 ± 8.41 20.64 ± 9.20
8.63 7.96 7.92
Total sperm per ejaculate
1 2 17
33 37 7
6.5–128.0 0–130.0 0.0–75.0
5.27 4.61 5.46
3.26 ± 7.74 −18.55 ± 7.24 −0.23 ± 0.13
−42.85 ± 11.45 30.12 ± 12.06 −0.65 ± 0.19
8.19 6.92 9.54
pH value
2 12 6 9
92 84 185 77
7.5–132.0 0.0–86.0 3.0–188.0 29.0–108.0
7.25** 6.57* 4.56 5.89
−0.04 0.04 −0.03 −0.03
Ejaculation times
6 16 17
124 67 86
60.0–176.0 18.5–91.5 31.5–88.0
4.89 4.81 8.85***
Ejaculation duration
6 17
124 70
40.0–184.0 20.5–88.0
4 9
131 21
1
41
Sperm concentration
Abnormality rate Sperm motility a * b
**
± ± ± ±
9.84 9.02 5.74 7.38
−0.16 ± 0.19 −0.41 ± 0.15 −0.79 ± 0.16
−0.37 ± 0.36 0.42 ± 0.24 0.03 ± 0.24
5.86 5.74 11.83
5.25 5.39
−0.33 ± 0.21 −0.86 ± 0.24
0.02 ± 0.42 0.29 ± 0.38
7.66 7.90
3.5–139.0 0.0–129.5
5.57 6.19
0.01 ± 0.01 0.03 ± 0.01
0.04 ± 0.01 0.01 ± 0.01
8.82 11.77
14.0–117.5
4.65
0.02 ± 0.01
0.05 ± 0.01
6.33
***
1% genome-wide significant level.
0.01 0.01 0.01 0.01
−0.07 0.08 0.04 0.06
0.02 0.02 0.02 0.02
10% genome-wide significant level, 5% genome-wide significant level,
Percentage of the phenotypic variance explained by QTL.
± ± ± ±
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F-ratioa
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total sperm per ejaculate were detected at distinct genomic regions in this study, although the two traits were highly correlated. A total of 16 QTL for male reproductive traits including testicular weight, epididymal weight, seminiferous tubular diameter, serum testosterone concentration have been identified in the White Duroc × Erhualian resource population (Ren et al., 2008a). In comparison, a suggestive QTL for epididymal weight at 300 days of age was close to the QTL for semen volume in this study. Another QTL for epididymal weight at 90 days of age overlapped with the QTL for semen pH value at 92 cM on SSC2. Bidanel et al. (2001) detected a suggestive QTL for weight of bulbo-urethral glands that overlapped
Fig. 1. The statistic F-curves indicating genome-wide significant QTL for semen characteristics on SSC15 (a), SSC2 (b), SSC15 (c) and SSC17 (d). Markers and distance in cM are given on the x-axis, and F-ratios are indicated on the y-axis.
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Fig. 1. ( Continued ).
with the QTL for abnormality rate on SSC4. However, no phenotypic correlation was reported among these traits mentioned above. Hence, these traits having overlapping QTL could be affected by different causal genes. Several QTL for porcine female reproductive traits have been reported close to some QTL in this study. The estrogen receptor locus associated with litter size (Rothschild et al., 1996) is located in the QTL for total sperm per ejaculate and near the QTL for sperm motility on SSC1. Overlapping regions between QTL for ovulation rate (Rohrer et al., 1999) and the QTL for pH value detected in this study were also found on SSC2 and SSC12. These indicate that these regions have significant effect on both male and female reproductive traits. However, it is difficult to assure whether these QTL were due to pleiotropic effects of a single locus or effects of closely linked multiple loci. Borg et al. (1993) reported that boars from the Taihu breed had lower total sperm per ejaculate than Duroc boars (P < 0.05). However, the Erhualian allele increasing sperm per ejaculate was observed at the QTL on SSC2, which is in contrast to the breed characteristic difference in this trait. Similar phenomena have also been observed in a Meishan × European intercross (Milan et al., 2002), and the reason for this remains unknown. It has been shown that boars carrying different estrogen receptor 1 (ESR1) genotypes have significant (P < 0.01) difference in semen traits including total sperm number per ejaculate and sperm motility (Terman et al., 2006). Moreover, reducing estrogen synthesis in developing boars increases total sperm production (At-Taras et al., 2006), and polymorphisms in the promoter of ESR1 are associated with men’s lower sperm count (Guarducci et al., 2006). ESR1 is localized on SSC1p24-25 (http://www.ag.unr.edu/beattie/research/first generation.htm), which was evidenced as the QTL for total sperm per ejaculate and close to the QTL for sperm motility in this study, providing an interesting candidate gene for the two traits. Several studies have shown the significant relationship of semen pH with semen quality or fertility. Reid et al. (1948) reported a significant correlation between the initial semen pH and sperm motility. Wichmann et al. (1994) found that semen pH was related to fertility (P = 0.045). Salsabili et al. (2006) showed that semen pH had a direct relation with sperm motility and an inverse relation with tail defects in humans (P < 0.05). However, semen pH did not show a high correlation with sperm motility and sperm abnormality rate in this study. Candidate genes associated with semen pH remain largely unknown. Although low pH value was significantly more frequent in patients with CFTR mutations (Von Eckardstein et al., 2000), the porcine CFTR gene is not located in the QTL region for semen pH in this study. It has been shown that ACTG2 mutations are significantly associated with semen volume in Pietrain boars (Wimmers et al., 2005). We mapped a QTL for semen volume on SSC3, but the peak of the QTL (101 cM) is far away from the location (at 30 cM) of the ACTG2 gene. This implies that ACTG2 could not be a positional candidate gene for semen volume in the White Duroc × Erhualian intercross.
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In summary, we detected 18 novel QTL for traits related to sperm quality and ejaculation in pigs. These results provide a start point to refine these QTL and ultimately identify causal genes and mutations underlying traits of interest. Acknowledgements This study was financially supported by National 973 program of China (2006CB102100) and National Natural Science Foundation of China (30425045). References At-Taras, E.E., Berger, T., McCarthy, M.J., Conley, A.J., Nitta-Oda, B.J., Roser, J.F., 2006. Reducing estrogen synthesis in developing boars increases testis size and total sperm production. J. Androl. 27, 552–559. Bidanel, J.P., Prunier, A., Iannuccelli, N., Milan, D., 2001. Detection of quantitative trait loci for male and female reproductive traits in Meishan × Large White F2 pigs. In: Proc. 52nd Annual Meeting of the E.A.A.P., Budapest, Hungary, p. 54. Borg, K.E., Lunstra, D.D., Christenson, R.K., 1993. Semen characteristics, testicular size, and reproductive hormone concentrations in mature Duroc, Meishan, Fengjing, and Minzhu boars. Biol. Reprod. 49, 515–521. Churchill, G.A., Doerge, R.W., 1994. Empirical threshold values for quantitative trait loci mapping. Genetics 138, 963– 971. Golas, A., Dzieza, A., Kuzniarz, K., Styrna, J., 2008. Gene mapping of sperm quality parameters in recombinant inbred strains of mice. Int. J. Dev. Biol. 52, 287–293. Green, P., Falls, K., Crook, S., 1990. Documentation for CRIMAP, Version 2.4. Washington University School of Medicine, St. Louis, MO. Griffin, D.K., Finch, K.A., 2005. The genetic and cytogenetic basis of male infertility. Hum. Fertil. 8, 19–26. Guarducci, E., Nuti, F., Becherini, L., Rotondi, M., Balercia, G., Forti, G., Krausz, C., 2006. Estrogen receptor alpha promoter polymorphism: stronger estrogen action is coupled with lower sperm count. Hum. Reprod. 21, 994–1001. Haley, C.S., Knott, S.A., Elsen, J.M., 1994. Mapping quantitative trait loci in crosses between outbred lines using least squares. Genetics 136, 1195–1207. Kovacs, A., Foote, R.H., 1992. Viability and acrosome staining of bull, boar and rabbit spermatozoa. Biotech. Histochem. 67, 119–124. L’Hôte, D., Serres, C., Laissue, P., Oulmouden, A., Rogel-Gaillard, C., Montagutelli, X., Vaiman, D., 2007. Centimorgan-range onestep mapping of fertility traits using interspecific recombinant congenic mice. Genetics 176, 1907–1921. Lin, C.L., Ponsuksili, S., Tholen, E., Jennen, D.G., Schellander, K., Wimmers, K., 2006. Candidate gene markers for sperm quality and fertility of boar. Anim. Reprod. Sci. 92, 349–363. Lunstra, D.D., Ford, J.J., Klindt, J., Wise, T.H., 1997. Physiology of the Meishan boar. J. Reprod. Fertil. Suppl. 52, 181–193. Milan, D., Bidanel, J.P., Iannuccelli, N., Riquet, J., Amigues, Y., Gruand, J., Le Roy, P., Renard, C., Chevalet, C., 2002. Detection of quantitative trait loci for carcass composition traits in pigs. Genet. Sel. Evol. 34, 705–728. Ørstavik, K.H., 2008. Genetic causes of male infertility. Tidsskr. Nor. Laegeforen. 128, 324–326. Reid, J.T., Ward, G.M., Salsbury, R.L., 1948. The relationship of the change in pH affected by incubation to other semen characteristics. J. Dairy Sci. 31, 383–388. Ren, D.R., Ren, J., Xing, Y.Y., Guo, Y.M., Wu, Y.B., Yang, G.C., Mao, H.R., Huang, L.S., 2008a. A genome scan for quantitative trait loci affecting male reproductive traits in a White Duroc × Chinese Erhualian resource population. J. Anim. Sci. (first published on July 3, 2008 as doi:10.2527/jas.2008-0923). Ren, D.R., Xing, Y.Y., Lin, M.J., Wu, Y.B., Li, K., Li, W.B., Yang, S.J., Guo, T.F., Ren, J., Ma, J.W., Lan, L.T., Huang, L.S., 2008b. Evaluations of the gonad development, spermatogenesis, semen characteristics, libido and serum testosterone levels in White Duroc × Chinese Erhualian crossbred boars. Reprod. Domest. Anim. (in press, doi:10.1111/j.1439-0531.2008.01117.x). Ren, J., Guo, Y.M., Ma, J.W., Huang, L.S., 2006. Growth and meat quality QTL in pigs with special reference to a very large White Duroc × Erhualian resource population. In: Proc. 8WCGALP, Brazil, Poster ID, pp. 11–13. Rohrer, G.A., Ford, J.J., Wise, T.H., Vallet, J.L., Christenson, R.K., 1999. Identification of quantitative trait loci affecting female reproductive traits in a multigeneration Meishan-White composite swine population. J. Anim. Sci. 77, 1385–1391. Rohrer, G.A., Wise, T.H., Lunstra, D.D., Ford, J.J., 2001. Identification of genomic regions controlling plasma FSH concentrations in Meishan-White Composite boars. Physiol. Genomics 6, 145–151. Rothschild, M.F., Bidanel, J.P., 1998. Biology and genetics of reproduction. In: Rothschild, M.F., Ruvinsky, A. (Eds.), The Genetics of the Pig. CAB International, Wallingford, Oxon, UK, pp. 313–343. Rothschild, M., Jacobson, C., Vaske, D., Tuggle, C., Wang, L., Short, T., Eckardt, G., Sasaki, S., Vincent, A., McLaren, D., Southwood, O., van der Steen, H., Mileham, A., Plastow, G., 1996. The estrogen receptor locus is associated with a major gene influencing litter size in pigs. Proc. Natl. Acad. Sci. U.S.A. 93, 201–205. Salsabili, N., Mehrsai, A., Jalalizadeh, B., Pourmand, G., Jalaie, S., 2006. Correlation of sperm nuclear chromatin condensation staining method with semen parameters and sperm functional tests in patients with spinal cord injury, varicocele, and idiopathic infertility. Urol. J. 3, 32–37. Sato, S., Oyamada, Y., Atsuji, K., Nade, T., Kobayashi, E., Mitsuhashi, T., Nirasawa, K., Komatsuda, A., Saito, Y., Terai, S., Hayashi, T., Sugimoto, Y., 2003. Quantitative trait loci analysis for growth and carcass traits in a Meishan × Duroc F2 resource population. J. Anim. Sci. 81, 2938–2949. Terman, A., Kmiec, M., Polasik, D., 2006. Estrogen receptor gene (ESR) and semen characteristics of boars. Arch. Tierz. Dummerstorf. 49, 71–76. Visscher, P.M., Thompson, R., Haley, C.S., 1996. Confidence intervals in QTL mapping by bootstrapping. Genetics 143, 1013– 1020.
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Von Eckardstein, S., Cooper, T.G., Rutscha, K., Meschede, D., Horst, J., Nieschlag, E., 2000. Seminal plasma characteristics as indicators of cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in men with obstructive azoospermia. Fertil. Steril. 73, 1226–1231. Wichmann, L., Isola, J., Tuohimaa, P., 1994. Prognostic variables in predicting pregnancy A prospective follow up study of 907 couples with an infertility problem. Hum. Reprod. 9, 1102–1108. Wimmers, K., Lin, C.L., Tholen, E., Jennen, D.G., Schellander, K., Ponsuksili, S., 2005. Polymorphisms in candidate genes as markers for sperm quality and boar fertility. Anim. Genet. 36, 152–155.
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A whole genome scanning for quantitative trait loci on traits related to sperm quality and ejaculation in pigs Yuyun Xing a, Jun Ren a, Dongren Ren a, Yuanmei Guo a, Yanbo Wu a, Guangcheng Yang a, Huirong Mao a, Bertram Brenig b, Lusheng Huang a,∗ a
Key Laboratory for Animal Biotechnology of Jiangxi Province and the Ministry of Agriculture of China, Jiangxi Agricultural University, Nanchang 330045, PR China Institute of Veterinary Medicine, Georg-August-University of Göttingen, Burckhardtweg 2, 37077 Göttingen, Germany
b
a r t i c l e
i n f o
Article history: Received 23 May 2008 Received in revised form 5 July 2008 Accepted 4 August 2008 Available online 8 August 2008 Keywords: QTL Semen quality Ejaculation Pig
a b s t r a c t To identify quantitative trait loci (QTL) for traits related to semen and ejaculation, phenotype data including semen volume, sperm concentration, total sperm per ejaculate, sperm motility, sperm abnormality rate, semen pH value, ejaculation times and ejaculation duration were measured on 206 F2 boar at 240 days in a White Duroc × Erhualian intercross. A genome-wide scan was performed and the entire White Duroc × Erhualian intercross was genotyped for 183 microsatellite markers covering the whole pig genome. QTL analysis was performed using a composite regression interval mapping method via QTLExpress. A total of 18 QTL were detected, including 4 genome-wide significant QTL each for semen pH on pig chromosome (SSC) 2 and SSC12, for semen volume on SSC15, and for ejaculation times on SSC17. Fourteen suggestive QTL were found on SSC1, 2, 3, 4, 6, 9, 17 and 18. To our knowledge, this is the first report about the QTL for semen and ejaculation traits in pigs, providing a start point to decipher the genetic basis of these complex traits. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Artificial insemination (AI) has been extensively used in the pig industry and has a significant influence on the swine production. It is well known that AI requires boars with excellent semen quality to
∗ Corresponding author. Tel.: +86 791 3805967; fax: +86 791 3805967. E-mail address: [email protected] (L. Huang). 0378-4320/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2008.08.008
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improve meat productivity. However, it is difficult to perform direct selection for semen quality because of their low heritabilities and sex-limited characteristics (Rothschild and Bidanel, 1998). Markerassisted selection on the basis of candidate gene or quantitative trait loci (QTL) could be an effective way to improve male reproductive traits. More than 60 QTL affecting at least 14 female reproductive traits have been identified across the pig genome (http://www.animalgenome.org/QTLdb/pig.html). In comparison, only about 20 QTL for male reproductive traits including testis and epididymis weight (size), seminal vesicles weight, seminiferous tubular diameter, length of bulbo-urethral gland, serum testosterone concentration and follicle-stimulating hormone (FSH) have been reported (Bidanel et al., 2001; Rohrer et al., 2001; Sato et al., 2003; Ren et al., 2008a). Furthermore, studies of candidate genes for semen quality are limited (Wimmers et al., 2005; Lin et al., 2006; Terman et al., 2006) and QTL for traits related to semen quality and ejaculation remain unexplored in pigs. Deciphering the genetic basis of porcine male reproductivity will not only benefit the pig industry but also human medicine. Human infertility is an important reproductive health problem that is implicated in about 50% of sterility cases (Griffin and Finch, 2005). About 15% of man infertility cases are attributable to genetic factors (Ørstavik, 2008). Pig is a good choice as animal model for human disease, and the genetic research for boar semen quality can provide referenced information for human infertility study. The Chinese Taihu pig is one of the most prolific pig breeds in the world. Boars from two subpopulations (Erhualian and Meishan) of the Taihu breed exhibit markedly higher concentration of serum FSH hormone, luteinizing hormone and androgens whereas lower testis size, total daily sperm number and ejaculate volume compared with European commercial boars (Borg et al., 1993; Lunstra et al., 1997). These particular attributes are useful for studies of the genetic basis of boar reproductive traits. We have established an F2 intercross between White Duroc boars and Chinese Erhualian sows and have recorded diverse phenotypic traits including those related to semen quality and ejaculation. The objective of this study was to identify QTL affecting semen quality and ejaculation traits by using the White Duroc-Erhualian resource population. 2. Materials and methods 2.1. Animals The White Duroc × Erhualian intercross resource population was developed and managed as described previously (Ren et al., 2006). Briefly, two White Duroc boars were mated to seventeen Erhualian sows that were selected from three breeding farms representing high reproductive performance in China. A total of 1912 F2 animals were generated in six batches by intercrossing of 9 F1 males and 59 F1 females avoiding full-sib mating. In this study, 206 boars at 280 days from 54 full-sib families were recorded for 8 traits related to semen quality and ejaculation including semen volume, sperm concentration, total sperm per ejaculate, sperm motility, sperm abnormality rate, semen pH value, ejaculation times and ejaculation duration. All boars were raised at the experimental research farm at Jiangxi Agriculture University in Nanchang (China). These boars were weaned at 46 days of age and moved into a nursery and were then housed individually after 120 days of age until phenotypic measurement. All the procedures involving animals followed the guidelines for the care and use of experimental animals established by the Ministry of Agriculture of China. 2.2. Phenotype measurement Semen collection and measurements of semen volume, sperm concentration and total sperm per ejaculate were described previously in Ren et al. (2008b). Briefly, each boar was trained to mount docile boars or receptive gilts that were positioned underneath a special dummy sow once a day from 240 days of age. After 280 days of age, each boar was collected for semen by glove-hand method 4 times at an interval of 4 days. To reduce personal error, all traits were measured by fixed personnel. Sperm motility was assessed with a light microscope (Chongqing, Jiangxi, China) at 200× magnification immediately after semen was collected. For the measurement of sperm abnormality rate, semen samples were
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Table 1 Phenotypic mean, standard error and range of F2 boars in a White Duroc × Erhualian resource population Trait
No.
Semen volume (ml) Sperm concentration (million/ml) Total sperm per ejaculate (109 ) Sperm motility Sperm abnormality rate pH value Ejaculation times Ejaculation duration (s)
177 176 176 175 173 177 177 177
Range 69.30–462.50 0.70–707.00 0.07–927.20 0.30–0.88 0.10–0.51 7.02–7.69 1.00–15.8 90.00–845.40
Mean ± S.E. 180.50 231.00 39.08 0.72 0.24 7.26 4.10 350.30
± ± ± ± ± ± ± ±
57.20 117.00 18.06 0.10 0.09 0.13 2.10 128.00
gently smeared with glass slides and then dried and stained in a phosphate-buffered Giemsa solution (Kovacs and Foote, 1992). Stained slides were evaluated by a Leica DMR microscope (Leica, Wetzlar, Germany) at 400× magnification and at least 200 sperms were examined in each assay. Semen pH value was measured by a Mp120 pH Meter (Mettler-Toledo, Greifensee, Switzerland) immediately after semen were collected. Ejaculation times were calculated on the basis of short interval of every ejaculation in the whole ejaculation process. A BD-TR118 stopwatch (Suzhou, China) was used to record the time of a whole ejaculation.
2.3. Genotyping and statistical analysis A total of 183 informative microsatellite markers across 18 autosomes and chromosome X were selected on the basis of their informativeness and locations. All founder animals, F1 animals and 206 F2 boars were genotyped for the 183 markers as described previously in Ren et al. (2008a). Genotypic data were analyzed with CRIMAP version 2.4 (Green et al., 1990) to construct a whole-genome linkage map. The PROC GLM procedure of SAS version 9.0 (SAS Institute Inc., Cary, NC, USA) was used to determine factors included in the subsequent QTL mapping analysis. Factors having effect above 5% significant level on trait of interest were kept as fixed effects or covariates in the QTL model. Family was included as a fixed effect for all traits. Farm was used as a fixed effect in the QTL model for semen volume and total number per ejaculate. Boar age was included as a covariate for semen volume. Semen temperature was a fixed effect for semen pH. Air temperature was fitted as a covariate for semen volume. The QTL analysis was performed with the web-accessible QTL Express at http://qtl.cap.ed.au.uk/, which was based on a least-squares method (Haley et al., 1994). The least-squares regression model was fitted at one centimorgan interval along each chromosome and the F-value for the QTL effect was calculated at each point. Empirical threshold values for QTL mapping were estimated by using the genomewide permutation test with 1000 random data shuffles as described by Churchill and Doerge (1994). The empirical 95% confidence intervals (CI) were evaluated by a bootstrapping approach with 2000 iterations (Visscher et al., 1996).
3. Results 3.1. Phenotype data Phenotypic mean, standard error and range of F2 boars are listed in Table 1. The White DurocErhualian crossbred boars showed considerable large variation and segregation in all traits measured (Table 1). The correlation coefficients among traits measured are shown in Table 2. Ejaculation duration had a strong positive correlation (r = 0.87) with ejaculation times, and total sperm per ejaculate had a strong positive correlation (r = 0.73) with sperm concentration. Ejaculation duration and ejaculation times had a medium positive correlation with semen volume whereas medium negative correlation with sperm concentration.
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Table 2 Pearson Product Moment Correlations among semen quality and ejaculation traits Trait
Semen volume
Total sperm per ejaculate
Sperm motility
Sperm abnormality rate
pH value
Ejaculation times
Ejaculation duration
Sperm concentration Semen volume Total sperm per ejaculate Sperm motility Sperm abnormality rate pH value Ejaculation times
−0.30**
0.73** 0.34**
−0.0006 −0.02 −0.09
−0.001 −0.08 −0.09 0.06
−0.21** −0.21** −0.37** −0.04 −0.07
−0.33** 0.48** −0.04 −0.21** −0.14 0.17*
−0.42** 0.49** −0.12 −0.08 −0.05 0.23** 0.87**
Significance level. * **
P < 0.05. P < 0.01.
3.2. QTL identification Details of QTL identified in this study are shown in Table 3. A total of 18 QTL affecting the 8 traits measured were detected on 11 chromosomes including 4 genome-wide significant QTL and 14 suggestive QTL. The F-value for thresholds of suggestive significance, 10%, 5% and 1% genome-wide significance was 4.39, 6.33, 6.94 and 8.47, respectively. A 10% genome-wide significant QTL for semen volume was mapped at 16 cM on pig chromosome (SSC) 15 (Fig. 1a). It explained 8.63% of the phenotypic variance, and the QTL allele increasing semen volume was inherited from the Erhualian breed. Moreover, two suggestive effect associated with semen volume were found at 101 cM on SSC3 and 69 cM on SSC18, respectively. At 33 cM on SSC1 and 37 cM on SSC2, two suggestive QTL for total sperm per ejaculate were found. The SWR100-SWR112 region (data not shown) on SSC17 had a suggestive significant effect on sperm concentration. A 5% genome-wide and a 10% genome-wide significant QTL for semen pH value was found on SSC2 and SSC12 (Fig. 1b, c), explaining 9.84% and 9.02% of the phenotypic variance, respectively. The alleles from the Erhualian breed at the two loci were associated with increased pH values. Two additional suggestive QTL for semen pH value were detected with peaks at 185 cM on SSC6 and 77 cM on SSC9, respectively. One 1% genome-wide significant QTL affecting ejaculation times with positive additive effect of Erhualian alleles was evidenced at 86 cM on SSC17 (Fig. 1d). This is the most significant QTL detected in this study, accounting for 11.83% of the phenotypic variation in the F2 population. This chromosomal region was also evidenced as a suggestive QTL for ejaculation duration. Another two suggestive QTL for ejaculation times were observed at 124 cM on SSC6 and 67 cM on SSC16. A suggestive QTL for ejaculation duration was found close the QTL for ejaculation times on SSC6. Two suggestive QTL for sperm abnormality were detected each on SSC4 and SSC9. A suggestive QTL affecting sperm motility was evidenced close to the QTL for total sperm per ejaculate on SSC1.
4. Discussion To our knowledge, this is the first report about QTL for sperm quality and ejaculation traits in pigs. In other mammals, only several QTL for sperm quality were reported in mice and rats (L’Hôte et al., 2007; Golas et al., 2008). Through pig–mouse comparative maps at http://www.ncbi.nlm.nih.gov/Homology/ and http://www.animalgenome.org/cgi-bin/QTLdb/SS/draw chromap, the QTL for sperm abnormality rate on SSC4 was homolog to the QTL for mouse sperm nucleus shape (L’Hôte et al., 2007). In this study, only 4 chromosomal regions showed genome-wide significant associations with the traits measured. The limited number of genome-wide significant QTL could be due to the relatively small size of the F2 population. We cautioned that those suggestive QTL could be interpreted as false positive results. As highly correlated traits, QTL for ejaculation times and ejaculation duration were found in the same regions on SSC6 and SSC17. It should be noted that QTL for sperm concentration and
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Table 3 QTL for traits related to semen and ejaculation in a White Duroc × Erhualian resource population Trait
Chr
Position (cM)
95% CI position
Additive effect ± S.E.
Dominant effect ± S.E.
Variance (%)b
Semen volume
15 18 3
16 69 101
0.0–21.5 2.0–69.0 1.5–120.0
6.55* 6.12 6.09
−25.21 ± 5.80 19.91 ± 5.71 −9.78 ± 6.23
−12.37 ± 8.87 −14.88 ± 8.41 20.64 ± 9.20
8.63 7.96 7.92
Total sperm per ejaculate
1 2 17
33 37 7
6.5–128.0 0–130.0 0.0–75.0
5.27 4.61 5.46
3.26 ± 7.74 −18.55 ± 7.24 −0.23 ± 0.13
−42.85 ± 11.45 30.12 ± 12.06 −0.65 ± 0.19
8.19 6.92 9.54
pH value
2 12 6 9
92 84 185 77
7.5–132.0 0.0–86.0 3.0–188.0 29.0–108.0
7.25** 6.57* 4.56 5.89
−0.04 0.04 −0.03 −0.03
Ejaculation times
6 16 17
124 67 86
60.0–176.0 18.5–91.5 31.5–88.0
4.89 4.81 8.85***
Ejaculation duration
6 17
124 70
40.0–184.0 20.5–88.0
4 9
131 21
1
41
Sperm concentration
Abnormality rate Sperm motility a * b
**
± ± ± ±
9.84 9.02 5.74 7.38
−0.16 ± 0.19 −0.41 ± 0.15 −0.79 ± 0.16
−0.37 ± 0.36 0.42 ± 0.24 0.03 ± 0.24
5.86 5.74 11.83
5.25 5.39
−0.33 ± 0.21 −0.86 ± 0.24
0.02 ± 0.42 0.29 ± 0.38
7.66 7.90
3.5–139.0 0.0–129.5
5.57 6.19
0.01 ± 0.01 0.03 ± 0.01
0.04 ± 0.01 0.01 ± 0.01
8.82 11.77
14.0–117.5
4.65
0.02 ± 0.01
0.05 ± 0.01
6.33
***
1% genome-wide significant level.
0.01 0.01 0.01 0.01
−0.07 0.08 0.04 0.06
0.02 0.02 0.02 0.02
10% genome-wide significant level, 5% genome-wide significant level,
Percentage of the phenotypic variance explained by QTL.
± ± ± ±
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F-ratioa
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total sperm per ejaculate were detected at distinct genomic regions in this study, although the two traits were highly correlated. A total of 16 QTL for male reproductive traits including testicular weight, epididymal weight, seminiferous tubular diameter, serum testosterone concentration have been identified in the White Duroc × Erhualian resource population (Ren et al., 2008a). In comparison, a suggestive QTL for epididymal weight at 300 days of age was close to the QTL for semen volume in this study. Another QTL for epididymal weight at 90 days of age overlapped with the QTL for semen pH value at 92 cM on SSC2. Bidanel et al. (2001) detected a suggestive QTL for weight of bulbo-urethral glands that overlapped
Fig. 1. The statistic F-curves indicating genome-wide significant QTL for semen characteristics on SSC15 (a), SSC2 (b), SSC15 (c) and SSC17 (d). Markers and distance in cM are given on the x-axis, and F-ratios are indicated on the y-axis.
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Fig. 1. ( Continued ).
with the QTL for abnormality rate on SSC4. However, no phenotypic correlation was reported among these traits mentioned above. Hence, these traits having overlapping QTL could be affected by different causal genes. Several QTL for porcine female reproductive traits have been reported close to some QTL in this study. The estrogen receptor locus associated with litter size (Rothschild et al., 1996) is located in the QTL for total sperm per ejaculate and near the QTL for sperm motility on SSC1. Overlapping regions between QTL for ovulation rate (Rohrer et al., 1999) and the QTL for pH value detected in this study were also found on SSC2 and SSC12. These indicate that these regions have significant effect on both male and female reproductive traits. However, it is difficult to assure whether these QTL were due to pleiotropic effects of a single locus or effects of closely linked multiple loci. Borg et al. (1993) reported that boars from the Taihu breed had lower total sperm per ejaculate than Duroc boars (P < 0.05). However, the Erhualian allele increasing sperm per ejaculate was observed at the QTL on SSC2, which is in contrast to the breed characteristic difference in this trait. Similar phenomena have also been observed in a Meishan × European intercross (Milan et al., 2002), and the reason for this remains unknown. It has been shown that boars carrying different estrogen receptor 1 (ESR1) genotypes have significant (P < 0.01) difference in semen traits including total sperm number per ejaculate and sperm motility (Terman et al., 2006). Moreover, reducing estrogen synthesis in developing boars increases total sperm production (At-Taras et al., 2006), and polymorphisms in the promoter of ESR1 are associated with men’s lower sperm count (Guarducci et al., 2006). ESR1 is localized on SSC1p24-25 (http://www.ag.unr.edu/beattie/research/first generation.htm), which was evidenced as the QTL for total sperm per ejaculate and close to the QTL for sperm motility in this study, providing an interesting candidate gene for the two traits. Several studies have shown the significant relationship of semen pH with semen quality or fertility. Reid et al. (1948) reported a significant correlation between the initial semen pH and sperm motility. Wichmann et al. (1994) found that semen pH was related to fertility (P = 0.045). Salsabili et al. (2006) showed that semen pH had a direct relation with sperm motility and an inverse relation with tail defects in humans (P < 0.05). However, semen pH did not show a high correlation with sperm motility and sperm abnormality rate in this study. Candidate genes associated with semen pH remain largely unknown. Although low pH value was significantly more frequent in patients with CFTR mutations (Von Eckardstein et al., 2000), the porcine CFTR gene is not located in the QTL region for semen pH in this study. It has been shown that ACTG2 mutations are significantly associated with semen volume in Pietrain boars (Wimmers et al., 2005). We mapped a QTL for semen volume on SSC3, but the peak of the QTL (101 cM) is far away from the location (at 30 cM) of the ACTG2 gene. This implies that ACTG2 could not be a positional candidate gene for semen volume in the White Duroc × Erhualian intercross.
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In summary, we detected 18 novel QTL for traits related to sperm quality and ejaculation in pigs. These results provide a start point to refine these QTL and ultimately identify causal genes and mutations underlying traits of interest. Acknowledgements This study was financially supported by National 973 program of China (2006CB102100) and National Natural Science Foundation of China (30425045). References At-Taras, E.E., Berger, T., McCarthy, M.J., Conley, A.J., Nitta-Oda, B.J., Roser, J.F., 2006. Reducing estrogen synthesis in developing boars increases testis size and total sperm production. J. Androl. 27, 552–559. Bidanel, J.P., Prunier, A., Iannuccelli, N., Milan, D., 2001. Detection of quantitative trait loci for male and female reproductive traits in Meishan × Large White F2 pigs. In: Proc. 52nd Annual Meeting of the E.A.A.P., Budapest, Hungary, p. 54. Borg, K.E., Lunstra, D.D., Christenson, R.K., 1993. Semen characteristics, testicular size, and reproductive hormone concentrations in mature Duroc, Meishan, Fengjing, and Minzhu boars. Biol. Reprod. 49, 515–521. Churchill, G.A., Doerge, R.W., 1994. Empirical threshold values for quantitative trait loci mapping. Genetics 138, 963– 971. Golas, A., Dzieza, A., Kuzniarz, K., Styrna, J., 2008. Gene mapping of sperm quality parameters in recombinant inbred strains of mice. Int. J. Dev. Biol. 52, 287–293. Green, P., Falls, K., Crook, S., 1990. Documentation for CRIMAP, Version 2.4. Washington University School of Medicine, St. Louis, MO. Griffin, D.K., Finch, K.A., 2005. The genetic and cytogenetic basis of male infertility. Hum. Fertil. 8, 19–26. Guarducci, E., Nuti, F., Becherini, L., Rotondi, M., Balercia, G., Forti, G., Krausz, C., 2006. Estrogen receptor alpha promoter polymorphism: stronger estrogen action is coupled with lower sperm count. Hum. Reprod. 21, 994–1001. Haley, C.S., Knott, S.A., Elsen, J.M., 1994. Mapping quantitative trait loci in crosses between outbred lines using least squares. Genetics 136, 1195–1207. Kovacs, A., Foote, R.H., 1992. Viability and acrosome staining of bull, boar and rabbit spermatozoa. Biotech. Histochem. 67, 119–124. L’Hôte, D., Serres, C., Laissue, P., Oulmouden, A., Rogel-Gaillard, C., Montagutelli, X., Vaiman, D., 2007. Centimorgan-range onestep mapping of fertility traits using interspecific recombinant congenic mice. Genetics 176, 1907–1921. Lin, C.L., Ponsuksili, S., Tholen, E., Jennen, D.G., Schellander, K., Wimmers, K., 2006. Candidate gene markers for sperm quality and fertility of boar. Anim. Reprod. Sci. 92, 349–363. Lunstra, D.D., Ford, J.J., Klindt, J., Wise, T.H., 1997. Physiology of the Meishan boar. J. Reprod. Fertil. Suppl. 52, 181–193. Milan, D., Bidanel, J.P., Iannuccelli, N., Riquet, J., Amigues, Y., Gruand, J., Le Roy, P., Renard, C., Chevalet, C., 2002. Detection of quantitative trait loci for carcass composition traits in pigs. Genet. Sel. Evol. 34, 705–728. Ørstavik, K.H., 2008. Genetic causes of male infertility. Tidsskr. Nor. Laegeforen. 128, 324–326. Reid, J.T., Ward, G.M., Salsbury, R.L., 1948. The relationship of the change in pH affected by incubation to other semen characteristics. J. Dairy Sci. 31, 383–388. Ren, D.R., Ren, J., Xing, Y.Y., Guo, Y.M., Wu, Y.B., Yang, G.C., Mao, H.R., Huang, L.S., 2008a. A genome scan for quantitative trait loci affecting male reproductive traits in a White Duroc × Chinese Erhualian resource population. J. Anim. Sci. (first published on July 3, 2008 as doi:10.2527/jas.2008-0923). Ren, D.R., Xing, Y.Y., Lin, M.J., Wu, Y.B., Li, K., Li, W.B., Yang, S.J., Guo, T.F., Ren, J., Ma, J.W., Lan, L.T., Huang, L.S., 2008b. Evaluations of the gonad development, spermatogenesis, semen characteristics, libido and serum testosterone levels in White Duroc × Chinese Erhualian crossbred boars. Reprod. Domest. Anim. (in press, doi:10.1111/j.1439-0531.2008.01117.x). Ren, J., Guo, Y.M., Ma, J.W., Huang, L.S., 2006. Growth and meat quality QTL in pigs with special reference to a very large White Duroc × Erhualian resource population. In: Proc. 8WCGALP, Brazil, Poster ID, pp. 11–13. Rohrer, G.A., Ford, J.J., Wise, T.H., Vallet, J.L., Christenson, R.K., 1999. Identification of quantitative trait loci affecting female reproductive traits in a multigeneration Meishan-White composite swine population. J. Anim. Sci. 77, 1385–1391. Rohrer, G.A., Wise, T.H., Lunstra, D.D., Ford, J.J., 2001. Identification of genomic regions controlling plasma FSH concentrations in Meishan-White Composite boars. Physiol. Genomics 6, 145–151. Rothschild, M.F., Bidanel, J.P., 1998. Biology and genetics of reproduction. In: Rothschild, M.F., Ruvinsky, A. (Eds.), The Genetics of the Pig. CAB International, Wallingford, Oxon, UK, pp. 313–343. Rothschild, M., Jacobson, C., Vaske, D., Tuggle, C., Wang, L., Short, T., Eckardt, G., Sasaki, S., Vincent, A., McLaren, D., Southwood, O., van der Steen, H., Mileham, A., Plastow, G., 1996. The estrogen receptor locus is associated with a major gene influencing litter size in pigs. Proc. Natl. Acad. Sci. U.S.A. 93, 201–205. Salsabili, N., Mehrsai, A., Jalalizadeh, B., Pourmand, G., Jalaie, S., 2006. Correlation of sperm nuclear chromatin condensation staining method with semen parameters and sperm functional tests in patients with spinal cord injury, varicocele, and idiopathic infertility. Urol. J. 3, 32–37. Sato, S., Oyamada, Y., Atsuji, K., Nade, T., Kobayashi, E., Mitsuhashi, T., Nirasawa, K., Komatsuda, A., Saito, Y., Terai, S., Hayashi, T., Sugimoto, Y., 2003. Quantitative trait loci analysis for growth and carcass traits in a Meishan × Duroc F2 resource population. J. Anim. Sci. 81, 2938–2949. Terman, A., Kmiec, M., Polasik, D., 2006. Estrogen receptor gene (ESR) and semen characteristics of boars. Arch. Tierz. Dummerstorf. 49, 71–76. Visscher, P.M., Thompson, R., Haley, C.S., 1996. Confidence intervals in QTL mapping by bootstrapping. Genetics 143, 1013– 1020.
218
Y. Xing et al. / Animal Reproduction Science 114 (2009) 210–218
Von Eckardstein, S., Cooper, T.G., Rutscha, K., Meschede, D., Horst, J., Nieschlag, E., 2000. Seminal plasma characteristics as indicators of cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in men with obstructive azoospermia. Fertil. Steril. 73, 1226–1231. Wichmann, L., Isola, J., Tuohimaa, P., 1994. Prognostic variables in predicting pregnancy A prospective follow up study of 907 couples with an infertility problem. Hum. Reprod. 9, 1102–1108. Wimmers, K., Lin, C.L., Tholen, E., Jennen, D.G., Schellander, K., Ponsuksili, S., 2005. Polymorphisms in candidate genes as markers for sperm quality and boar fertility. Anim. Genet. 36, 152–155.