Association of GH Gene Polymorphism with Semen Parameters of Boars

Kmieç M., A. Terman, H. Wierzbicki , S. Zych: Association of GH Gene Polymorphism with Semen Parameters of Boars. Acta Vet 2007, 76: 41-46. Relations between polymorphism of the Growth Hormone gene and semen characters were analyzed. The DNA for the purpose of examination was isolated from the peripheral blood of 173 boars. In the boar herd under study the frequency of allele occurrence for the GH/MspI was as follows: allele GHA 0.79 and allele GHB 0.21. As far as the GH/HaeII polymorphism is concerned, the relevant frequency was as follows: allele GHA 0.53 and allele GHB 0.47, respectively. The relationship between the GH genotypes and semen characteristic traits were analyzed. The study showed that boars with GHBGHB genotype of both polymorphous loci of the GH gene produced ejaculates of larger volume, higher percentage, number of normozosperms in the ejaculate and number of insemination as compared to GHA GHA and GHAGHB boars. Our current findings suggested that polymorphism of the GH/MspI and GH/HaeII might have potential effect for reproductive performance traits of boars. GH gene, polymorphism, PCR-RFLP, boars, semen characteristics Growth hormone is a protein produced in the anterior pituitary and its secretion varies throughout the entire life of an animal it peaks in puberty and then remains slightly lower (Machnik and Lechniak 2000) and depends on sex and physiological condition (Izadyar et al. 1999), e.g. in cows during lactation it is close to zero. The hormone concerned affects target cells in direct or indirect manner. Results of the study showed that the GH directly affects the steroidogenesis, gametogenesis and gonad differentiation (Zachman 1992; Franks 1998). Reportedly it also plays a major role in the growth and development of testis since the discovery of the relation between the GH deficiency and male hypogonadism in humans (Spi ter i -Grech and Nieschlag 1992). It has been proved that the GH and IGF-I under in vitro conditions influence the growth and division rate of the sustentacular (Sertoli) cells and interstitial (Leydig) cells and the IGF-II stimulates the differentiation of Sertoli cells. The testis performance and steroidogenesis and gametogenesis could simultaneously be affected by testicular and hypophyseal growth hormones (Hull and Harvey 2000). Clinical examinations in men showed that the GH is required to maintain the correct sperm morphology and sperm concentration (Gravance et al. 1997). In case of the hormone deficiency, the sperm movement in men was limited and restored only after the administration of exogenous GH (Gravance et al. 1997; Breier et al. 1998). Somatotropin also affects the prostate indirectly through the regulation of somatomedine C concentration and a number of receptors (Rei ter et al. 1999); it may also be an essential hormone during the period of penis development (Laron and Klinger 1998). The GH gene in pigs is located on the chromosome 12 (Yerle et al. 1993) and consists of 5 exons, the total length of which is 1.7 kb (Vize and Wells 1987). The genetic polymorphism of the GH gene in pigs was found and about 20 genetic variations of the GH gene in different breeds and lines of pigs were described ACTA VET. BRNO 2007, 76: 41–46; doi:10.2754/avb200776010041 Address for correspondence: Prof. Dr. Marek Kmieç Department of Genetics and Animal Breeding ul. Doktora Judyma 6, 71-466 Szczecin, Poland Phone/fax: +48 91 45 41 497 E-mail: m.kmiec@biot.ar.szczecin.pl http://www.vfu.cz/acta-vet/actavet.htm (Schel lander et al. 1994; Handler et al. 1995; Knorr et al. 1997). Relations between certain genetic variations of the GH gene in pigs and carcass fat deposition and meat quality (Gelderman et al. 1996) and the loin eye area and fat deposition were identified to show that the GH gene is a gene-candidate for the fat deposition in pigs (Knorr et al. 1997). Taking into account numerous reports on potential reproduction functions of the GH and relations between the GH genetic variations and meat performance in livestock, this study was aimed at the detection of the mutation in the GH gene and identification of the genetic structure of the insemination boar herd and establishment of relations between the GH genetic variations and qualitative and quantitative traits of the insemination boar semen. Materials and Methods The study included 173 breeding boars kept at the AI station that served to inseminate sows in the station operation area (Table 1). All the boars were kept in the same conditions and used for insemination purposes only. The data were collected on 19,379 ejaculates sampled from 173 boars between 1996 and 2002 and referred to ejaculate volume, sperm concentration, live sperm percentage, number of live sperm in ejaculate, number of insemination doses. In order to eliminate the age effect, only the data on 4,713 ejaculates sampled from boars at the age of 221 to 240 days were analyzed. The semen sampling period was divided into 12 months, and along with the year, breed and father’s effect were taken into consideration when defining the variation. The DNA for the examination was isolated from peripheral blood with Master PureTM Genomic DNA Purification Kit of Epicenter Technologies. Such isolation procedure produced the DNA of 7585 μg/ml concentration and over 85% purity. The PCR-RFLP method was applied to determine the GH genotypes. The PCR reaction was conducted in 20 μl mixture of the following thermal profile: 1) 94 °C/5 min, 2) 94 °C/45 sec, 3) 57 °C/45 sec, 4) 72 °C/45 sec, 5) 72 °C/5 min.; the reaction covered 35 cycles (stages 2 through 4). Starter sequences were used (forward primer: 5’GCC AAG TTT TAA ATG TCC CTG-3’ and reverse primer: 5’-CTG TCC CTC CGG GAT GTA G-3’) for the PCR reaction to identify the DNA fragment of 506 bp. The said amplified fragment of 506 bp was digested for 3 hours at 37 °C with 4 I.U. of restriction enzyme MspI or 4 I.U. of restriction enzyme HaeII, respectively. The restrictive fragments of the DNA were separated by means of the electrophoresis in 2% agarose gel stained with ethidium bromide in the 1 × TBE buffer. Then, the gels were visualized and analyzed in the UV rays and recorded with the use of the Vilber Lourmat system. The statistical analysis of relations was carried out by means of the SAS/STAT (General Linear Model Procedure) calculation package according to the following linear model: Yijkl = m + Gi + Bj + YSk + Sl + eijkl where: Yijklm character value for i-th genotype, j-th boar breed, k-th year season, l-th sire; μ population mean; Gi effect of i-th genotype (i = 1, 2, 3); Bj constant effect of j-th boar breed (j = 1, 2, ... , 5); YSk constant effect of k-th year season (k = 1,2,_, 28) (7 years*4 seasons); Sl fixed effect of the l-th sire (l = 1, 2, ... , 94); eijklm random residual term. 42 Table 1. The number and frequency of GH1/MspI genotype and alleles of boars under study Breed N GH genotype GH allele

Growth hormone is a protein produced in the anterior pituitary and its secretion varies throughout the entire life of an animal -it peaks in puberty and then remains slightly lower (Machnik and Lechniak 2000) and depends on sex and physiological condition (Izadyar et al. 1999), e.g. in cows during lactation it is close to zero.The hormone concerned affects target cells in direct or indirect manner.Results of the study showed that the GH directly affects the steroidogenesis, gametogenesis and gonad differentiation (Zachman 1992;Franks 1998).Reportedly it also plays a major role in the growth and development of testis since the discovery of the relation between the GH deficiency and male hypogonadism in humans (Spiteri-Grech and Nieschlag 1992).
It has been proved that the GH and IGF-I under in vitro conditions influence the growth and division rate of the sustentacular (Sertoli) cells and interstitial (Leydig) cells and the IGF-II stimulates the differentiation of Sertoli cells.The testis performance and steroidogenesis and gametogenesis could simultaneously be affected by testicular and hypophyseal growth hormones (Hull and Harvey 2000).Clinical examinations in men showed that the GH is required to maintain the correct sperm morphology and sperm concentration (Gravance et al. 1997).In case of the hormone deficiency, the sperm movement in men was limited and restored only after the administration of exogenous GH (Gravance et al. 1997;Breier et al. 1998).Somatotropin also affects the prostate indirectly through the regulation of somatomedine C concentration and a number of receptors (Reiter et al. 1999); it may also be an essential hormone during the period of penis development (Laron and Klinger 1998).
The GH gene in pigs is located on the chromosome 12 (Yerle et al. 1993) and consists of 5 exons, the total length of which is 1.7 kb (Vize and Wells 1987).
The genetic polymorphism of the GH gene in pigs was found and about 20 genetic variations of the GH gene in different breeds and lines of pigs were described (Schellander et al. 1994;Handler et al. 1995;Knorr et al. 1997).Relations between certain genetic variations of the GH gene in pigs and carcass fat deposition and meat quality (Gelderman et al. 1996) and the loin eye area and fat deposition were identified to show that the GH gene is a gene-candidate for the fat deposition in pigs (Knorr et al. 1997).
Taking into account numerous reports on potential reproduction functions of the GH and relations between the GH genetic variations and meat performance in livestock, this study was aimed at the detection of the mutation in the GH gene and identification of the genetic structure of the insemination boar herd and establishment of relations between the GH genetic variations and qualitative and quantitative traits of the insemination boar semen.

Materials and Methods
The study included 173 breeding boars kept at the AI station that served to inseminate sows in the station operation area (Table 1).All the boars were kept in the same conditions and used for insemination purposes only.
The data were collected on 19,379 ejaculates sampled from 173 boars between 1996 and 2002 and referred to ejaculate volume, sperm concentration, live sperm percentage, number of live sperm in ejaculate, number of insemination doses.In order to eliminate the age effect, only the data on 4,713 ejaculates sampled from boars at the age of 221 to 240 days were analyzed.The semen sampling period was divided into 12 months, and along with the year, breed and father's effect were taken into consideration when defining the variation.
The DNA for the examination was isolated from peripheral blood with Master Pure TM Genomic DNA Purification Kit of Epicenter Technologies.Such isolation procedure produced the DNA of 75-85 µg/ml concentration and over 85% purity.
The PCR-RFLP method was applied to determine the GH genotypes.The PCR reaction was conducted in 20 µl mixture of the following thermal profile: 1) 94 °C/5 min, 2) 94 °C/45 sec, 3) 57 °C/45 sec, 4) 72 °C/45 sec, 5) 72 °C/5 min.; the reaction covered 35 cycles (stages 2 through 4).Starter sequences were used (forward primer: 5'-GCC AAG TTT TAA ATG TCC CTG-3' and reverse primer: 5'-CTG TCC CTC CGG GAT GTA G-3') for the PCR reaction to identify the DNA fragment of 506 bp.The said amplified fragment of 506 bp was digested for 3 hours at 37 °C with 4 I.U. of restriction enzyme MspI or 4 I.U. of restriction enzyme HaeII, respectively.The restrictive fragments of the DNA were separated by means of the electrophoresis in 2% agarose gel stained with ethidium bromide in the 1 × TBE buffer.Then, the gels were visualized and analyzed in the UV rays and recorded with the use of the Vilber Lourmat system.
The statistical analysis of relations was carried out by means of the SAS/STAT (General Linear Model Procedure) calculation package according to the following linear model: where: Y ijklm -character value for i-th genotype, j-th boar breed, k-th year season, l-th sire; µ -population mean; G i -effect of i-th genotype (i = 1, 2, 3); B j -constant effect of j-th boar breed (j = 1, 2, ... , 5); YS k -constant effect of k-th year season (k = 1,2,_, 28) -(7 years*4 seasons); S l -fixed effect of the l-th sire (l = 1, 2, ... , 94); e ijklm -random residual term.3 and 4 show the number and characteristics of semen samples and mean values and standard deviations.

Results
Due to the application of starter sequences a product of 506 bp size that covered the range from +385 th base of intron I to + 889 th base of intron III of the certain GH gene of livestock was amplified.After the digestion with endonuclease MspI, that identified sequences 5'-CZCGG-3' and 3'-GGC-C-5' in the second intron of GH gene various arrays of bands were found in the agarose gel and thus two alleles GH A and GH B were found that affect the occurrence of three genotypes (GH A GH A -222, 147 and 137 bp; GH A GH B -284, 222, 147 and 137 bp and GH B GH B -284 and 222 bp compared to DNA pUC19/MspI molecular marker).Table 1 lists frequencies of identified GH/MspI genotypes and alleles in the boar herd under study.The frequency of GH A allele was 0.79 and that of GH B allele was 0.21.The frequency of the GH A GH A genotype in the boar herd under study was 0.61.The highest frequency was found in Polish Landrace boars (0.92), and the lowest in Hampshire × Pietrain boars (0.32).The frequency of the GH A GH B genotype was 0.36.In the boars herd under study the highest frequency of this genotype was found in Hampshire × Pietrain cross-breed boars (0.60).The frequency of the GH B GH B genotype in the insemination boars herd under study was 0.03.The highest value was found in Hampshire × Pietrain boars (0.08), whereas in Polish Landrace and PIC boars did not exhibit the genotype concerned (Table 1) .
The amplified product of 506 bp was subject to the digestion with endonuclease HaeII, that identified sequences 5'-RGCGCZY↓ 3' and 3'-Y↑CGCGR-5' (where R = G or A, Y = C or T) in the second exon of GH gene and various arrays of strips were found in the agarose gel and thus two alleles GH A and GH B were found that affect the occurrence of three genotypes (GH A GH A -337 and 173; GH A GH B -506, 333 and 173 bp and GH B GH B -506 bp compared to DNA pUC19/MspI molecular marker).
Table 2 lists frequencies of identified GH/HaeII genotypes and alleles in the boar herd under study.The frequency of GH A allele was 0.53 and that of GH B allele was 0.47.The frequency of the GH A GH A genotype in the boar herd under study was 0.27 and varied among breeds, from 0.06 in the Polish Landrace to 0.49 in Duroc × Pietrain pigs (Table 2).The frequency of the GH A GH B genotype was 0.51 and the values ranged from 0.39 to 0.64.The lowest frequency in the insemination boar herd under study was reported for the GH B GH B -0.22 (Table 2).
The analysis of relations between GH/MspI polymorphism showed that ejaculates of highest volume were produced by the boars of the GH B GH B (244.6 cm 3 ) whereas the boars of GH A GH A and GH A GH B genotypes produced smaller ejaculates: 215.5 cm 3 and 212.0 cm 3 , respectively (Table 3).The study showed significantly higher sperm concentration in boars of the GH A GH A and GH A GH B genotypes rather than the GH B GH B , and the difference in the sperm concentration between the boars of the GH A GH A and GH A GH B were small and statistically non-significant (Table 3).The relations between GH/MspI polymorphism and the average percentage of live sperms in the boar semen show that the percentage of live sperms in the semen of the GH B GH B genotype boars was significantly higher than in the semen of the GH A GH B and GH A GH A boars, and the differences were confirmed statistically (Table 3).It was also shown that the semen from the GH B GH B genotype boars contained a greater number of live sperms than ejaculates from the boars of the GH A GH B and GH A GH A genotypes.Ejaculates yielded from the GH A GH B genotype boars exhibited the smallest number of live sperms per ejaculate and the differences were confirmed statistically (Table 3).One ejaculate from the boar in the herd under study produced 23.9 insemination doses.The greatest number of insemination doses was produced from ejaculates of the GH A GH A genotype boars, and the smallest of the GH A GH B boars, the differences, however, were minor and not confirmed statistically (Table 3).
Table 4 lists average parameters of semen characters of insemination boars versus GH/HaeII polymorphism.It was shown that the GH B GH B genotype boars produced ejaculates of significantly greater volume than the boars of other GH/HaeII genotypes.The analysis of relations between GH/HaeII genotypes and sperm concentration showed the smallest value in boars with GH B GH B genotype and the differences were statistically confirmed.The highest percentage of live sperms and the greatest number of live sperms per Mean values in a line designated with the same letter differ significantly at P ≤ 0.01 ejaculate were found in ejaculates produced by the GH B GH B boars and the differences were statistically confirmed (Table 4).Ejaculates taken from the GH A GH A and GH A GH B boars produced 23.4 insemination doses on average, whereas ejaculates taken from the GH B GH B boars produced 25.6 doses on average, respectively.The ejaculates taken from the GH B GH B genotype boars produced by 2.2 insemination doses more than the ones from the GH A GH A and GH A GH B boars and the difference was confirmed statistically (Table 4).

Discussion
The higher frequency of the GH A GH A (GH/MspI) was reported by Schellander et al. (1994) -0.96 andHandler et al. (1995) -0.86.The extremely low frequency of this genotype, however, was reported by Kuciel et al. (1998) -0.27.The higher frequency of the GH A GH B genotype concerned was found by Kuciel et al. (1998) -0.58, whereas Handler et al. (1995) reported a much lower value of 0.12.The frequency of the GH B GH B genotype in herds of pigs researched by Handler et al. (1995) and Schellander et al. (1994) was low and amounted to: 0.02 and 0.01, respectively.Only Kuciel et al. (1998) reported the higher frequency of the genotype concerned, i.e. 0.15.
The same frequency of the above alleles of GH/HaeII was reported by Kuciel et al. (1998), and very similar values were reported by Puntova et al. (2001).High frequency of the GH A was reported by Kirkpatrick (1993) -0.82 whereas the analysis of the polymorphism by Handler et al. (1995) and Schellander et al. (1994) showed much lower values of the allele concerned, i.e. 0.24 and 0.17, respectively.Similar values of the GH A GH A were reported by Kuciel et al. (1998)  The analysis of ejaculate volume showed that the average of this trait in the boar herd was 21.6,4 cm 3 and by far exceeded the values given by Wierzbowski (1996) -200 cm 3 .The sperm concentration in the boar herd under study was 599.7 mln/cm 3 and exceeded the average value given by Wierzbowski (1996).The study showed also similar number of insemination doses per ejaculate as reported by Kondracki et al. (1998).
Our study showed that boars with GH B GH B genotype of both polymorphous loci of the GH gene (pGH/MspI and pGH/HaeII) produced ejaculates of larger volume, higher percentage, number of normozosperms in the ejaculate and number of insemination does compared to GH A GH A and GH A GH B boars.Statistically significant (P ≤ 0.01) differences were found between pGH/MspI and pGH/HaeII genotypes and qualitative and quantitative semen characters under study.
The study confirmed the occurrence of pGH/MspI and pGH/HaeII polymorphism in a selected sequence of the GH gene and the results of the analysis of relations suggest possibilities to use existing polymorphism in the GH gene to improve the insemination performance of the boars.

Table 1 .
The number and frequency of GH1/MspI genotype and alleles of boars under study

Table 2 .
The number and frequency of GH1/HaeII genotype and alleles of boars under study

Table 3 .
Values of studied semen traits in reference to GH/MspI genotype Mean values in a line designated with the same letter differ significantly at P ≤ 0.01

Table 4 .
Values of studied semen traits in reference to GH/HaeII genotype