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Genome-wide scan identifies genetic variants associated with reduced fertility in Hanoverian stallions
Abstracts / Journal of Equine Veterinary Science 43 (2016) S56eS82
12 Genome-wide scan identifies genetic variants associated with reduced fertility in Hanoverian stallions H. Sieme 1, R. Schrimpf 1,2, M. Gottschalk 2, J. Metzger 2, G. Martinsson 3, O. Distl 2 1 University of Veterinary Medicine Hannover, Unit for Reproductive Medicine, Clinic for Horses, Germany; 2 University of Veterinary Medicine Hannover, Institute for Animal Breeding and Genetics, Germany; 3 State Stud Celle of Lower Saxony, Celle, Germany Stallion sub- and infertility is of high economic importance for the breeding industry and is a pivotal criterion for genomic selection programs. The underlying mechanisms causing sub- and infertility in stallions are poorly understood. The objectives of the present study were to identify rare genetic variants using next generation sequence (NGS) data, and to demonstrate the extent to which these variants are associated with reduced fertility in stallions. Target traits employed were estimated breeding values for the paternal component of the pregnancy rate per estrus (EBV-PAT) in Hanoverian stallions. We performed whole genome sequencing for seven stallions and four mares using NGS-technology and filtered this data for genetic variants and insertion/deletion mutations based on the horse reference genome EquCab2.0. Gene ontology (GO) terms and search results from public databases were used to obtain a comprehensive list of human and mice genes predicted to participate in the regulation of male reproduction. The corresponding equine orthologs genes were searched in whole genome sequence data from the eleven horses and filtered for high-impact genetic variants using SnpEFF, SIFT and Polyphen-2 software. All genetic variants for which the homozygous mutant genotype was present in the data were filtered out. After this filtering process, 17 single nucleotide variants (SNVs) were left. All these 17 genetic variants with the missing homozygous mutant genotype were genotyped for 337 fertile stallions from 19 breeds using KASP genotyping assays or PCR-RFLP. Mixed linear model analysis for deregressed EBV-PAT in 216 Hanoverian stallions showed a genomewide significant association for the splice-site disruption variant g.37455302G>A in NOTCH1. For a further 9 high-impact variants within the genes CFTR, OVGP1, FBXO43, TSSK6, PKD1, FOXP1, TCP11, SPATA31E1 and NOTCH1 (g.37453246G>C) absence of the homozygous mutant genotype in the validation sample of all 337 fertile stallions was obvious. Therefore, these variants were considered as potentially deleterious for stallion fertility. Combining all genetic variants found to be associated with fertility in Hanoverian stallions, we constructed a fertility score comprising the genetic variants for PLCz1, FKBP6, NEURL1 and NOTCH1 which explained >20% of the variance of EBV-PAT. Key Words: infertility, notch I, genetic variants
13 Gonadotropin and testosterone concentrations in light breed stallions after administration of hCG and deslorelin acetate R.S. McCann 1, I.F. Canisso 1, D.L. Thompson 2, K.H. Kline 3 1 Department of Veterinary Clinical Medicine, University of Illinois, Urbana, IL, USA; 2 School of Animal Sciences, Louisiana State University, Baton Rouge, LA, USA; 3 Department of Animal Sciences, University of Illinois, Urbana, IL, USA Testing of the hypothalamic-pituitary gonadal axis (HPG) is important both in routine stallion breeding soundness examination as well as in the identification of potential cryptorchid stallions. One of the standard tests that is used to assess the HPG is a
S61
testosterone stimulation test using human chorionic gonadotropin (hCG) for its LH-like properties. Presumably hCG will be taken off the market; besides a recent study demonstrated that hCG administered to stallions may cause focal testicular necrosis upon histological evaluation. Thus, alternatives to hCG for testing HPG are warranted. An injectable preparation of deslorelin acetate, a GnRH agonist, is available but there are no published reports about its use in the stimulation of LH, FSH, or testosterone. Eleven sexually mature light breed stallions (5-17 yrs) were enrolled in the study. Over a 6-week period during the physiological breeding season, each stallion received three testosterone stimulation test protocols: (i) administration of 10,000 IU hCG IV, (ii) administration of 1.8 mg deslorelin acetate IM, and (iii) administration 3.6 mg deslorelin acetate IM. Baseline plasma samples were collected prior to administration. Plasma samples were collected 1, 2, 24 and 48 h post drug administration. Plasma concentrations of testosterone, FSH, and LH were determined by RIA. Hormone concentrations were expressed as means ± standard error of the mean and as mean percent change (MPC). The data were log-transformed and analyzed by mixed models. Posthoc comparison with Fisher’s LSD was performed with significance set at p<0.05. Both deslorelin acetate doses increased LH and FSH significantly: peak plasma concentrations were achieved at 2 h. Deslorelin, 1.8 mg, induced a peak MPC of 168% for LH and 116% for FSH. Similarly, 3.6 mg deslorelin caused a MPC of 168% for LH and 167% for FSH. Levels of FSH and LH diminished by 24 h and returned to baseline by 48 h. Administration of hCG caused significantly elevated plasma testosterone concentrations throughout the testing period. By 1 h the MPC of testosterone increased 294% from baseline and by 48 h the MPC was 608% above baseline. Deslorelin at doses of 1.8 mg and 3.6 mg caused testosterone to reach peak concentrations at 2 h, respectively, with levels returning to baseline within 24 h. Deslorelin of 1.8mg increased the MPC of testosterone to 84%, whereas 3.6 mg of deslorelin caused a 168% increase. This study demonstrates that deslorelin acetate is able to stimulate the release of FSH, LH, and testosterone and could be used to assess the HPA. However, deslorelin acetate induced a significantly smaller MPC in testosterone concentrations than hCG administration. Key Words: testosterone, FSH, LH
14 Identification of genetic variants associated with semen traits in warmblood stallions H. Sieme 1, M. Gottschalk 2, J. Rau 1, *, G. Martinsson 3, O. Distl 2 1 University of Veterinary Medicine Hannover, Unit for Reproductive Medicine, Clinic for Horses, Germany; 2 University of Veterinary Medicine Hannover, Institute for Animal Breeding and Genetics, Germany; 3 State Stud Celle of Lower Saxony, Celle, Germany Consistent high semen quality is a desirable feature for stallions used for in artificial insemination (AI). Considerable variation in semen quality has been shown among stallions within breeds. Several studies have confirmed that the stallion accounts for a significant proportion of the variance in semen quality. The objective of the current study was to estimate permanent environmental and genetic variances for semen quality traits of 241 fertile German Warmblood stallions routinely employed in AI. Estimated breeding values (EBVs) and their reliabilities should indicate whether semen quality in stallions could be improved through genetic selection. De-regressed EBVs were employed to
*
equal contribution
12 Genome-wide scan identifies genetic variants associated with reduced fertility in Hanoverian stallions H. Sieme 1, R. Schrimpf 1,2, M. Gottschalk 2, J. Metzger 2, G. Martinsson 3, O. Distl 2 1 University of Veterinary Medicine Hannover, Unit for Reproductive Medicine, Clinic for Horses, Germany; 2 University of Veterinary Medicine Hannover, Institute for Animal Breeding and Genetics, Germany; 3 State Stud Celle of Lower Saxony, Celle, Germany Stallion sub- and infertility is of high economic importance for the breeding industry and is a pivotal criterion for genomic selection programs. The underlying mechanisms causing sub- and infertility in stallions are poorly understood. The objectives of the present study were to identify rare genetic variants using next generation sequence (NGS) data, and to demonstrate the extent to which these variants are associated with reduced fertility in stallions. Target traits employed were estimated breeding values for the paternal component of the pregnancy rate per estrus (EBV-PAT) in Hanoverian stallions. We performed whole genome sequencing for seven stallions and four mares using NGS-technology and filtered this data for genetic variants and insertion/deletion mutations based on the horse reference genome EquCab2.0. Gene ontology (GO) terms and search results from public databases were used to obtain a comprehensive list of human and mice genes predicted to participate in the regulation of male reproduction. The corresponding equine orthologs genes were searched in whole genome sequence data from the eleven horses and filtered for high-impact genetic variants using SnpEFF, SIFT and Polyphen-2 software. All genetic variants for which the homozygous mutant genotype was present in the data were filtered out. After this filtering process, 17 single nucleotide variants (SNVs) were left. All these 17 genetic variants with the missing homozygous mutant genotype were genotyped for 337 fertile stallions from 19 breeds using KASP genotyping assays or PCR-RFLP. Mixed linear model analysis for deregressed EBV-PAT in 216 Hanoverian stallions showed a genomewide significant association for the splice-site disruption variant g.37455302G>A in NOTCH1. For a further 9 high-impact variants within the genes CFTR, OVGP1, FBXO43, TSSK6, PKD1, FOXP1, TCP11, SPATA31E1 and NOTCH1 (g.37453246G>C) absence of the homozygous mutant genotype in the validation sample of all 337 fertile stallions was obvious. Therefore, these variants were considered as potentially deleterious for stallion fertility. Combining all genetic variants found to be associated with fertility in Hanoverian stallions, we constructed a fertility score comprising the genetic variants for PLCz1, FKBP6, NEURL1 and NOTCH1 which explained >20% of the variance of EBV-PAT. Key Words: infertility, notch I, genetic variants
13 Gonadotropin and testosterone concentrations in light breed stallions after administration of hCG and deslorelin acetate R.S. McCann 1, I.F. Canisso 1, D.L. Thompson 2, K.H. Kline 3 1 Department of Veterinary Clinical Medicine, University of Illinois, Urbana, IL, USA; 2 School of Animal Sciences, Louisiana State University, Baton Rouge, LA, USA; 3 Department of Animal Sciences, University of Illinois, Urbana, IL, USA Testing of the hypothalamic-pituitary gonadal axis (HPG) is important both in routine stallion breeding soundness examination as well as in the identification of potential cryptorchid stallions. One of the standard tests that is used to assess the HPG is a
S61
testosterone stimulation test using human chorionic gonadotropin (hCG) for its LH-like properties. Presumably hCG will be taken off the market; besides a recent study demonstrated that hCG administered to stallions may cause focal testicular necrosis upon histological evaluation. Thus, alternatives to hCG for testing HPG are warranted. An injectable preparation of deslorelin acetate, a GnRH agonist, is available but there are no published reports about its use in the stimulation of LH, FSH, or testosterone. Eleven sexually mature light breed stallions (5-17 yrs) were enrolled in the study. Over a 6-week period during the physiological breeding season, each stallion received three testosterone stimulation test protocols: (i) administration of 10,000 IU hCG IV, (ii) administration of 1.8 mg deslorelin acetate IM, and (iii) administration 3.6 mg deslorelin acetate IM. Baseline plasma samples were collected prior to administration. Plasma samples were collected 1, 2, 24 and 48 h post drug administration. Plasma concentrations of testosterone, FSH, and LH were determined by RIA. Hormone concentrations were expressed as means ± standard error of the mean and as mean percent change (MPC). The data were log-transformed and analyzed by mixed models. Posthoc comparison with Fisher’s LSD was performed with significance set at p<0.05. Both deslorelin acetate doses increased LH and FSH significantly: peak plasma concentrations were achieved at 2 h. Deslorelin, 1.8 mg, induced a peak MPC of 168% for LH and 116% for FSH. Similarly, 3.6 mg deslorelin caused a MPC of 168% for LH and 167% for FSH. Levels of FSH and LH diminished by 24 h and returned to baseline by 48 h. Administration of hCG caused significantly elevated plasma testosterone concentrations throughout the testing period. By 1 h the MPC of testosterone increased 294% from baseline and by 48 h the MPC was 608% above baseline. Deslorelin at doses of 1.8 mg and 3.6 mg caused testosterone to reach peak concentrations at 2 h, respectively, with levels returning to baseline within 24 h. Deslorelin of 1.8mg increased the MPC of testosterone to 84%, whereas 3.6 mg of deslorelin caused a 168% increase. This study demonstrates that deslorelin acetate is able to stimulate the release of FSH, LH, and testosterone and could be used to assess the HPA. However, deslorelin acetate induced a significantly smaller MPC in testosterone concentrations than hCG administration. Key Words: testosterone, FSH, LH
14 Identification of genetic variants associated with semen traits in warmblood stallions H. Sieme 1, M. Gottschalk 2, J. Rau 1, *, G. Martinsson 3, O. Distl 2 1 University of Veterinary Medicine Hannover, Unit for Reproductive Medicine, Clinic for Horses, Germany; 2 University of Veterinary Medicine Hannover, Institute for Animal Breeding and Genetics, Germany; 3 State Stud Celle of Lower Saxony, Celle, Germany Consistent high semen quality is a desirable feature for stallions used for in artificial insemination (AI). Considerable variation in semen quality has been shown among stallions within breeds. Several studies have confirmed that the stallion accounts for a significant proportion of the variance in semen quality. The objective of the current study was to estimate permanent environmental and genetic variances for semen quality traits of 241 fertile German Warmblood stallions routinely employed in AI. Estimated breeding values (EBVs) and their reliabilities should indicate whether semen quality in stallions could be improved through genetic selection. De-regressed EBVs were employed to
*
equal contribution