The ability of animals to adapt to stress is not only an animal health and welfare concern, but also influences reproduction potential and robustness. An important pathway involved in the stress response is the hypothalamic-pituitary-adrenal axis (HPAA) that results in the release of cortisol from the adrenal gland. In this study the cortisol responses of South African Merinos were measured to assess HPAA responsiveness to stress and relate it to behavioural stress responses to

flock-isolation. The experiment was structured according to a 2×2 statistical design, with CYP17 genotype (WT1/WT1 vs. WT1/WT2) and selection line (H-line vs. L-line) as factors. Selection line criteria was based on divergent selection for (H-line) or against (L-line) maternal multiple rearing ability, where the H-line generally outperformed the L-line in terms of reproduction, animal

welfare and resistance to certain pathogens. The CYP17 genotype is involved in the biosynthesis pathway of cortisol. In the present study the CYP17 genotype showed a significant influence on behavioural stress responses, where three parameters of the flock-isolation test were affected (P&#60;0.05), namely the number of bleats uttered, the urinating frequency and the average distance from a human operator. It is suggested that the CYP17 genotype affects behavioural responses via its effects on cortisol production, and that the SNPs located within the CYP17 genotype may have application in marker-assisted selection of sheep

Cloete, S.W.P.

Hough, D.

Storbeck, K.

Swart, P.

English

Enlighten: Publications

  
 
 
Hough, D., Cloete, S.W.P., Swart, P., and Storbeck, K. (2010) Improving stress 
coping ability: comparison between the CYP17 genotype Of Ovis Aries And 
Capra Hircus. In: 9th World Congress on Genetics Applied to Livestock 
Production, 1-6 Aug 2010, Leipzig, Germany. 
 
 
Copyright © 2010 9th World Congress on Genetics Applied to Livestock 
Production 
 
 
A copy can be downloaded for personal non-commercial research or 
study, without prior permission or charge 
 
Content must not be changed in any way or reproduced in any format 
or medium without the formal permission of the copyright holder(s) 
 
 
When referring to this work, full bibliographic details must be given 
 
 
  
http://eprints.gla.ac.uk/96851 
 
 
 
Deposited on:  12 September 2014 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Enlighten – Research publications by members of the University of Glasgow 
http://eprints.gla.ac.uk 
Improving Stress Coping Ability:  Comparison 
Between The CYP17 Genotype Of Ovis Aries And 
Capra Hircus 
D. Hough*, K. Storbeck*, S.W.P. Cloete† and P. Swart* 
 
 
Introduction 
In mammals, physiological stress stimulates the release of glucocorticoids (cortisol and 
corticosterone) from the adrenal cortex that results in glucose production at the expense of 
glycolysis (Munch (1971)). Cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17) plays a 
critical role in the production of these glucocorticoids. Abnormalities in the CYP17 gene or 
protein can result in various mild to lethal disorders (Payne and Hales (2004)). In the case of 
the South African Angora goat (Capra hircus), the activities of the two CYP17 isoforms was 
found to be the main cause for hypocortisolism that results in an increased susceptibility to 
cold stress (Engelbrecht et al., (2000); Storbeck et al. (2007)). In addition, Engelbrecht et al. 
(2000) showed that the stimulation of the hypothalmo-pituitary-adrenal (HPA) axis with 
insulin challenge in vivo resulted in less cortisol being produced in Angora goats when 
compared to South African Boer goats (Capra hircus) and Merino sheep (Ovis aries).  
 
Two CYP17 genetic sequences have been identified for Angora goats, namely ACS- 
(GenBank accession no. EF524063) and CYP17 ACS+ (GenBank accession no. EF524064) 
that is 100% homologous with Boer goat CYP17 (GenBank accession no. AF251387) 
(Storbeck et al. (2007)). These sequences differ with four single nucleotide polymorphisms. 
In Merino sheep, two genetic sequences have been identified, namely CYP17 WT1 
(GenBank accession no. L40335) and WT2 (GenBank accession no. AF251388) (Storbeck et 
al. (2007)) that differ with two single nucleotide polymorphisms. 
 
This study investigates the importance of the CYP17 genotype in cortisol production in the 
Merino sheep and Angora goat adrenal gland as potential genetic marker for stress coping 
ability in selection programs to ultimately improve livestock fitness. 
Material and methods 
Animals. Merino sheep from a breeding program were used, where divergent selection was 
based on maternal ranking values for number of lambs weaned per mating. At present, both 
reproduction (Cloete et al. (2004)) and stress coping ability, in terms of cortisol production in 
response to insulin challenge (Van der Walt et al. (2009)), is higher in the positive selected 
                                                 
*
 Biochemistry Department, Private Bag X1, Stellenbosch University, Stellenbosch 7602, South Africa 
†
 Department of Animal Sciences, University of Stellenbosch, Stellenbosch 7602, South Africa 
(H-line) than the negative selected line (L-line). South African Angora, respectively, were 
randomly selected from the same flock. 
 
CYP17 genotyping with real-time polymerase chain reaction (PCR). Blood samples were 
collected from the jugular vein of adult sheep and goats, or from the heart chamber of dead 
lambs (2008 lambing season). The DNA isolation kit for mammalian blood (Roche, 
Germany) was used to isolate genomic DNA, which was then genotyped using the real-time 
PCR method developed by Storbeck et al. (2008).  
 
CYP17 copy number determination. Relative copy number determinations were performed 
on the three goat genotypes (Ho, Hu and He) and heterozygous (WT1/WT2) sheep using 
quantitative PCR (Storbeck et al. (2008)). Fold change values were calculated relative to an 
Ho genotype calibrator using the ∆∆Ct method (Livak and Schmittgen (2001)).  
 
Stress test. Merino sheep in the above mentioned breeding program (17 H-line and 21 L-line 
rams, 2-6 years of age) and a group of 30 Angora goats (3 groups of 10 goats for each 
CYP17 genotype: 5 ewes and 5 rams, 14 months of age) were injected intravenously with 
human insulin at a dose of 0.1 IU/kg body mass after which blood samples were collected at 
times: 0, 15, 30, 60, 90 and 120 min. Blood plasma glucose and free cortisol was determined 
with RIA. Ethics approval was obtained from the Departmental Ethics Committee for 
Research on Animals (DECRA ref: R08/21). 
 
Statistical analyses. The Chi-square test was used to analyze CYP17 genotype frequencies. 
For the stress test, the total HPA axis response to insulin induced hypoglycemia was 
calculated from the area under the curve that represents the amount of plasma cortisol 
produced (nmol/L) per plasma glucose drop (mmol/L) at times 0, 15, 30, 60, 90 and 120 min 
post insulin challenge. ANOVA tests were done for these responses, as well as for the 
relative DNA copy number determinations, followed by Bonferonni’s post-test. Graphs 
represent the mean and standard error of the mean. GraphPad Prism (version 4) software 
(GraphPad Software, US) was used for all analysis with α=0.05 for statistical significance. 
Results and discussion 
Merino CYP17 genotyping. The real-time PCR method developed by Storbeck et al. (2008) 
to genotype Angora CYP17 (ACS- and ACS+), was also suitable to genotype the two Merino 
sheep CYP17 sequences: WT1 and WT2. The CYP17 frequency distribution of 144 adult 
Merino sheep (112 H-line and 32 L-line) in the breeding program is 86.1% heterozygous 
WT1/WT2 and 13.9% homozygous WT1/WT1. No significant association (P=0.6722, Chi-
square=0.7944, df=2) was found between CYP17 genotypes and designation of population 
sample (H-line, L-line or lamb mortalities). Similar CYP17 frequencies were observed in the 
case of Angora goats (Storbeck et al. (2008)). Interestingly, the homozygous WT2/WT2 
genotype was not detected in Merino sheep, while in the case of Angora goats, the 
homozygous ACS+/ACS+ genotype was also not detected (Storbeck et al. (2008)). 
 
Relative CYP17 copy number determination. The absence of a homozygous genotype in 
both Angora and Merino suggests that either the homozygous genotype is lethal, or that there 
is an underlying genetic aberration for the CYP17 gene. Storbeck et al. (2008) did a relative 
CYP17 copy number determination for Angora goats with Boer goats and Merino sheep as 
controls. Their data revealed that three CYP17 genotypes exist in the case of the Angora 
goat, namely Ho, Hu and He. The Ho genotype has only one CYP17 gene, namely ACS-. In 
contrast, the He genotype has two CYP17 genes, where the ACS- locus is always present 
together with the ACS+ locus. This is the reason why the homozygote for ACS+ is never 
detected. Crossing Ho and He goats would yield the proposed intermediate Hu genotype. This 
genotype would receive both ACS- and ACS+ loci from the He parent, but only the ACS- 
locus from the Ho parent. The ACS- : ACS+ ratio in the Hu genotype would therefore be 2:1. 
 
Storbeck et al. (2008) also confirmed that there is only one CYP17 gene in sheep with two 
alleles: WT1 and WT2. Given this data, three genotypes would be expected in the 
population: homozygous WT1/WT1; heterozygous WT1/WT2 and homozygous WT2/WT2. 
However, no homozygous WT2/WT2 genotype was detected (n=180). The possibility that 
this genotype is lethal is therefore considered. 
 
The CYP17 genotype was thus determined for lambs that died during the peri-parturition 
period. However, the WT2/WT2 genotype remained undetected. It is suggested that the 
earlier stages in fetal or embryonic development be tested for the WT2/WT2 CYP17 
genotype. In addition, the CYP17 genotyping test should be optimized for Merino sheep 
CYP17 detection, since it was originally designed for goat CYP17 genotyping. 
 
Angora goat stress test. The total response of Angora goats to insulin challenge is depicted 
in Figure 1A. One-way ANOVA indicated that the CYP17 genotype affected the ability of 
Angora goats to respond to insulin-induced hypoglycemia. The Bonferonni’s post-test 
showed a reduced response in the Ho group compared to the Hu and He groups. No significant 
difference in total response was observed between the latter groups. These results suggest 
that the presence of CYP17 ACS+ in the He and Hu genotypes is more advantageous for 
cortisol production than the presence of CYP17 ACS-. 
 
Merino sheep stress test. When the Merino sheep responses to insulin challenge were 
grouped according to CYP17 genotype, the CYP17 WT1/WT1 group had an improved 
response compared to the CYP17 WT1/WT2 group (data not shown). Interesting results were 
obtained where H- and L-line Merino sheep in the breeding program were subdivided into 
CYP17 genotypes (Fig. 1B). Two-way ANOVA indicated that the interaction between 
selection line and CYP17 genotype is significant. Bonferonni’s post-test revealed that the 
response was the same for all groups, except for L-line heterozygous WT1/WT2 sheep where 
a lower response was observed. These results indicate that the presence of CYP17 WT1 is 
more advantageous for cortisol production than the presence of CYP17 WT2 in the L-line 
where there is arguable an accumulation of low fitness alleles. This finding suggests that the 
effect of CYP17 genotype is dependent on the interaction with other factors along the HPA 
axis. CYP17 can thus be used as a genetic marker (selection for WT1) to improve fitness in 
selection lines with an inherent low ability to cope with stress.  
 
 Figure 1: The total cortisol response to glucose decrease of (A) Angora goats and (B) 
Merino sheep (H- and L-line) grouped according CYP17 genotype. 
Conclusion 
The CYP17 genotype is a factor that exerts its effect on stress coping ability via cortisol 
production in the adrenal cortex. This effect of the CYP17 genotype seems to be dependent 
on the fitness of the sheep, or more specifically, the accumulation of other traits that also 
affect cortisol production. Furthermore, genetic variation in CYP17 exists among species 
which results in inherently different abilities to produce cortisol. The unique genotypes in the 
South African Angora goat differ not only in terms of the genetic sequences encoding for 
CYP17, but also in copy number. The genotypes in the Merino sheep, however, differ only in 
terms of the genetic sequences encoding the one CYP17 gene. It is suggested that the missing 
WT2/WT2 genotype in sheep may be lethal or that the CYP17 genotyping test needs to be 
optimised for Merino sheep. This study showed that the CYP17 genotype is a relevant 
genetic marker to include in selection criteria to improve stress coping ability of livestock. 
Further research is needed for the better understanding of the impact of CYP17 genotypes on 
stress in livestock. 
References 
Cloete, S.W.P., Gilmour, A.R., Olivier, J.J. and Van Wyk, J.B. (2004). Aus. J. Exp. Agr., 
44: 745-754. 
Engelbrecht, Y. and Swart, P. (2000). J. Anim. Sci., 78: 1036-1046. 
Livak, K.J. and Schmittgen, T.D. (2001). Methods, 25: 4402–4408. 
Munch, A. (1971). Perspect. Biol. Med., 14:265. 
Payne, A.H. and Hales, D.B. (2004). Endo. Rev., 25: 947-970. 
Storbeck, K., Swart, A.C., Slabbert, J.T. and Swart, P. (2007). Drug Metabol. Rev., 39: 
467-480. 
Storbeck, K., Swart, A.C., Snyman, M.A. and Swart, P. (2008). FEBS J., 275: 3934-3943. 
Van der Walt, D., Cloete, S.W.P., Storbeck, K. and Swart, P. (2009) In Proc. Assoc. 
Advmt. Anim. Breed. Genet., volume 18, pages 100-103. 


Improving stress coping ability: comparison between the CYP17 genotype Of Ovis Aries and Capra Hircus

Enlighten

    Hough, D., Cloete, S.W.P., Swart, P., and Storbeck, K. (2010) Improving stress coping ability: comparison between the CYP17 genotype Of Ovis Aries And Capra Hircus. In: 9th World Congress on Genetics Applied to Livestock Production, 1-6 Aug 2010, Leipzig, Germany.   Copyright © 2010 9th World Congress on Genetics Applied to Livestock Production   A copy can be downloaded for personal non-commercial research or study, without prior permission or charge  Content must not be changed in any way or reproduced in any format or medium without the formal permission of the copyright holder(s)   When referring to this work, full bibliographic details must be given     http://eprints.gla.ac.uk/96851    Deposited on:  12 September 2014                 Enlighten – Research publications by members of the University of Glasgow http://eprints.gla.ac.uk Improving Stress Coping Ability:  Comparison Between The CYP17 Genotype Of Ovis Aries And Capra Hircus D. Hough*, K. Storbeck*, S.W.P. Cloete† and P. Swart*   Introduction In mammals, physiological stress stimulates the release of glucocorticoids (cortisol and corticosterone) from the adrenal cortex that results in glucose production at the expense of glycolysis (Munch (1971)). Cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17) plays a critical role in the production of these glucocorticoids. Abnormalities in the CYP17 gene or protein can result in various mild to lethal disorders (Payne and Hales (2004)). In the case of the South African Angora goat (Capra hircus), the activities of the two CYP17 isoforms was found to be the main cause for hypocortisolism that results in an increased susceptibility to cold stress (Engelbrecht et al., (2000); Storbeck et al. (2007)). In addition, Engelbrecht et al. (2000) showed that the stimulation of the hypothalmo-pituitary-adrenal (HPA) axis with insulin challenge in vivo resulted in less cortisol being produced in Angora goats when compared to South African Boer goats (Capra hircus) and Merino sheep (Ovis aries).   Two CYP17 genetic sequences have been identified for Angora goats, namely ACS- (GenBank accession no. EF524063) and CYP17 ACS+ (GenBank accession no. EF524064) that is 100% homologous with Boer goat CYP17 (GenBank accession no. AF251387) (Storbeck et al. (2007)). These sequences differ with four single nucleotide polymorphisms. In Merino sheep, two genetic sequences have been identified, namely CYP17 WT1 (GenBank accession no. L40335) and WT2 (GenBank accession no. AF251388) (Storbeck et al. (2007)) that differ with two single nucleotide polymorphisms.  This study investigates the importance of the CYP17 genotype in cortisol production in the Merino sheep and Angora goat adrenal gland as potential genetic marker for stress coping ability in selection programs to ultimately improve livestock fitness. Material and methods Animals. Merino sheep from a breeding program were used, where divergent selection was based on maternal ranking values for number of lambs weaned per mating. At present, both reproduction (Cloete et al. (2004)) and stress coping ability, in terms of cortisol production in response to insulin challenge (Van der Walt et al. (2009)), is higher in the positive selected                                                  * Biochemistry Department, Private Bag X1, Stellenbosch University, Stellenbosch 7602, South Africa † Department of Animal Sciences, University of Stellenbosch, Stellenbosch 7602, South Africa (H-line) than the negative selected line (L-line). South African Angora, respectively, were randomly selected from the same flock.  CYP17 genotyping with real-time polymerase chain reaction (PCR). Blood samples were collected from the jugular vein of adult sheep and goats, or from the heart chamber of dead lambs (2008 lambing season). The DNA isolation kit for mammalian blood (Roche, Germany) was used to isolate genomic DNA, which was then genotyped using the real-time PCR method developed by Storbeck et al. (2008).   CYP17 copy number determination. Relative copy number determinations were performed on the three goat genotypes (Ho, Hu and He) and heterozygous (WT1/WT2) sheep using quantitative PCR (Storbeck et al. (2008)). Fold change values were calculated relative to an Ho genotype calibrator using the ∆∆Ct method (Livak and Schmittgen (2001)).   Stress test. Merino sheep in the above mentioned breeding program (17 H-line and 21 L-line rams, 2-6 years of age) and a group of 30 Angora goats (3 groups of 10 goats for each CYP17 genotype: 5 ewes and 5 rams, 14 months of age) were injected intravenously with human insulin at a dose of 0.1 IU/kg body mass after which blood samples were collected at times: 0, 15, 30, 60, 90 and 120 min. Blood plasma glucose and free cortisol was determined with RIA. Ethics approval was obtained from the Departmental Ethics Committee for Research on Animals (DECRA ref: R08/21).  Statistical analyses. The Chi-square test was used to analyze CYP17 genotype frequencies. For the stress test, the total HPA axis response to insulin induced hypoglycemia was calculated from the area under the curve that represents the amount of plasma cortisol produced (nmol/L) per plasma glucose drop (mmol/L) at times 0, 15, 30, 60, 90 and 120 min post insulin challenge. ANOVA tests were done for these responses, as well as for the relative DNA copy number determinations, followed by Bonferonni’s post-test. Graphs represent the mean and standard error of the mean. GraphPad Prism (version 4) software (GraphPad Software, US) was used for all analysis with α=0.05 for statistical significance. Results and discussion Merino CYP17 genotyping. The real-time PCR method developed by Storbeck et al. (2008) to genotype Angora CYP17 (ACS- and ACS+), was also suitable to genotype the two Merino sheep CYP17 sequences: WT1 and WT2. The CYP17 frequency distribution of 144 adult Merino sheep (112 H-line and 32 L-line) in the breeding program is 86.1% heterozygous WT1/WT2 and 13.9% homozygous WT1/WT1. No significant association (P=0.6722, Chi-square=0.7944, df=2) was found between CYP17 genotypes and designation of population sample (H-line, L-line or lamb mortalities). Similar CYP17 frequencies were observed in the case of Angora goats (Storbeck et al. (2008)). Interestingly, the homozygous WT2/WT2 genotype was not detected in Merino sheep, while in the case of Angora goats, the homozygous ACS+/ACS+ genotype was also not detected (Storbeck et al. (2008)).  Relative CYP17 copy number determination. The absence of a homozygous genotype in both Angora and Merino suggests that either the homozygous genotype is lethal, or that there is an underlying genetic aberration for the CYP17 gene. Storbeck et al. (2008) did a relative CYP17 copy number determination for Angora goats with Boer goats and Merino sheep as controls. Their data revealed that three CYP17 genotypes exist in the case of the Angora goat, namely Ho, Hu and He. The Ho genotype has only one CYP17 gene, namely ACS-. In contrast, the He genotype has two CYP17 genes, where the ACS- locus is always present together with the ACS+ locus. This is the reason why the homozygote for ACS+ is never detected. Crossing Ho and He goats would yield the proposed intermediate Hu genotype. This genotype would receive both ACS- and ACS+ loci from the He parent, but only the ACS- locus from the Ho parent. The ACS- : ACS+ ratio in the Hu genotype would therefore be 2:1.  Storbeck et al. (2008) also confirmed that there is only one CYP17 gene in sheep with two alleles: WT1 and WT2. Given this data, three genotypes would be expected in the population: homozygous WT1/WT1; heterozygous WT1/WT2 and homozygous WT2/WT2. However, no homozygous WT2/WT2 genotype was detected (n=180). The possibility that this genotype is lethal is therefore considered.  The CYP17 genotype was thus determined for lambs that died during the peri-parturition period. However, the WT2/WT2 genotype remained undetected. It is suggested that the earlier stages in fetal or embryonic development be tested for the WT2/WT2 CYP17 genotype. In addition, the CYP17 genotyping test should be optimized for Merino sheep CYP17 detection, since it was originally designed for goat CYP17 genotyping.  Angora goat stress test. The total response of Angora goats to insulin challenge is depicted in Figure 1A. One-way ANOVA indicated that the CYP17 genotype affected the ability of Angora goats to respond to insulin-induced hypoglycemia. The Bonferonni’s post-test showed a reduced response in the Ho group compared to the Hu and He groups. No significant difference in total response was observed between the latter groups. These results suggest that the presence of CYP17 ACS+ in the He and Hu genotypes is more advantageous for cortisol production than the presence of CYP17 ACS-.  Merino sheep stress test. When the Merino sheep responses to insulin challenge were grouped according to CYP17 genotype, the CYP17 WT1/WT1 group had an improved response compared to the CYP17 WT1/WT2 group (data not shown). Interesting results were obtained where H- and L-line Merino sheep in the breeding program were subdivided into CYP17 genotypes (Fig. 1B). Two-way ANOVA indicated that the interaction between selection line and CYP17 genotype is significant. Bonferonni’s post-test revealed that the response was the same for all groups, except for L-line heterozygous WT1/WT2 sheep where a lower response was observed. These results indicate that the presence of CYP17 WT1 is more advantageous for cortisol production than the presence of CYP17 WT2 in the L-line where there is arguable an accumulation of low fitness alleles. This finding suggests that the effect of CYP17 genotype is dependent on the interaction with other factors along the HPA axis. CYP17 can thus be used as a genetic marker (selection for WT1) to improve fitness in selection lines with an inherent low ability to cope with stress.    Figure 1: The total cortisol response to glucose decrease of (A) Angora goats and (B) Merino sheep (H- and L-line) grouped according CYP17 genotype. Conclusion The CYP17 genotype is a factor that exerts its effect on stress coping ability via cortisol production in the adrenal cortex. This effect of the CYP17 genotype seems to be dependent on the fitness of the sheep, or more specifically, the accumulation of other traits that also affect cortisol production. Furthermore, genetic variation in CYP17 exists among species which results in inherently different abilities to produce cortisol. The unique genotypes in the South African Angora goat differ not only in terms of the genetic sequences encoding for CYP17, but also in copy number. The genotypes in the Merino sheep, however, differ only in terms of the genetic sequences encoding the one CYP17 gene. It is suggested that the missing WT2/WT2 genotype in sheep may be lethal or that the CYP17 genotyping test needs to be optimised for Merino sheep. This study showed that the CYP17 genotype is a relevant genetic marker to include in selection criteria to improve stress coping ability of livestock. Further research is needed for the better understanding of the impact of CYP17 genotypes on stress in livestock. References Cloete, S.W.P., Gilmour, A.R., Olivier, J.J. and Van Wyk, J.B. (2004). Aus. J. Exp. Agr., 44: 745-754. Engelbrecht, Y. and Swart, P. (2000). J. Anim. Sci., 78: 1036-1046. Livak, K.J. and Schmittgen, T.D. (2001). Methods, 25: 4402–4408. Munch, A. (1971). Perspect. Biol. Med., 14:265. Payne, A.H. and Hales, D.B. (2004). Endo. Rev., 25: 947-970. Storbeck, K., Swart, A.C., Slabbert, J.T. and Swart, P. (2007). Drug Metabol. Rev., 39: 467-480. Storbeck, K., Swart, A.C., Snyman, M.A. and Swart, P. (2008). FEBS J., 275: 3934-3943. Van der Walt, D., Cloete, S.W.P., Storbeck, K. and Swart, P. (2009) In Proc. Assoc. Advmt. Anim. Breed. Genet., volume 18, pages 100-103. 

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Improving stress coping ability: comparison between the CYP17 genotype Of Ovis Aries and Capra Hircus

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