Anim. Blood G r p s biochem. Genet. 9 (1978)15-18
Serum alkaline phospha tase polymorphism in pigs' Danuta Kierek-Jaszczuk, Maciej hrkowski, Elibieta Skladanowska-Krzyzanowska and Krystyna Tomaszewska-Guszkiewicz Institute of Genetics and Animal Breeding Polish Academy of Sciences, Jastrzqbiec, Poland Received 30 June 1978; accepted 6 December 1978
Key-words: pig, serum, alkaline phosphatase, starch gel electrophoresis
Summary
Genetic variants of serum alkaline phosphatase were studied by the method of starch gel electrophoresis in the Zlotnicka Pstra breed of pigs. Two regions of alkaline phosphatase migration were observed. A single fraction in region I and four different phenotypes: AB, B, BC and BD in region 11, were found. For AB, B and BC phenotypes the genetic control by three alleles A k p A , AkpB and AkpC in suggested. The observed segregation ratios in some cases deviated significantly from the expected ones. Introduction
In pig serum Imlah (1970) found two electrophoretic forms of alkaline phosphatase, fast and slow. For the fast form he reported a typically ontogenetic variation. Saison (1970) examined pigs of different breeds and also reported fast and slow forms of alkaline phosphatase in serum. Dinklage (1970) found 5 fractions of alkaline phosphatase in the serum of German Improved Landrace and Gottingen Miniature pigs. On the basis of preliminary family studies he suggested that alkaline phosphatase was controlled by 5 alleles: A k p A , AkpB, AkpC, AkpD and AkpE. Later Verhorst (1973) from the same Department suggested the occurrence of only three alleles: A k p A , AkpB and AkpC. Materials and methods
The material consisted of Zlotnicka Pstra pigs from the Popielno farm. A total of Supported by the Polish Academy of Sciences within the project No 09.7.1.
15
DANUTA KIEREK-JASZCZUK ET AL. 26 sows and 5 boars and 573 offspring at 8 weeks of age were studied. Serum samples were analysed by horizontal starch gel electrophoresis. Hydrolysed starch was prepared at the Department of Animal Immunogenetics and was used at 14 % . A discontinous buffer system as described by Poulik (1957) was used: gel buffer - 0.076 M Tris, 0.005 M citric acid pH 8.65; electrode buffer - 0.3 M boric acid, 0.05 M sodium hydroxide, pH 8.05. The electrophoresis was conducted at 4 "Cfor three hours with stepwise potential increase: 0-30 min 10 V/cm, 30-60 rnin 15 V/cm, 60-90 min 17,5 V/cm and 90-180 min 20 V/cm. The starting line was 7 cm from the cathodic side of the gel. The samples were soaked on pieces of Whatman No 3 filter paper size 6 mm X 8 mm, and inserted into the gel. After electrophoresis the 6 mm thick gel was cut into two parts. The bottom part was incubated for 2 hours at 37 "C, according to the method proposed by Wilcox (1966) with some modifications. The staining mixture consisted of a buffer containing: 0.228 M Tris, 0.015 M citric acid, pH 8.9, 0.02 M MgCl,, 0.05 % Fast Blue BB or RR salt, 0.05 % of sodium a-naphthyl phosphate. After incubation the gels were stored in a mixture composed of 5 parts of ethyl alcohol, 5 parts of distilled water and 1 part of glacial acetic acid.
Results Alkaline phosphatase zones were observed in two redons of the stained gels. In region I a single fraction was observed near the starting line. This fraction stained strongly in samples from 8-week-old animals but was weaker and sometimes difficult to see in adults. Four phenotypes Akp B, Akp AB, Akp BC and Akp BD were observed in region I1 (Fig. 1).On the basis of the families examined and according to Verhorst (19731, it was suggested that the phenotypes B, AB and BC wzre controlled by three autosomal, codominant alleles: A k p A , AkpB and AkpC. T h e distribution of alkaline phosphatase phenotypes in the progeny from different types of mating is shown in Table 1. In all mating types, except Akp B X Akp B, significant deviations were found
Fig. 1. Starch gel zymogram of serum alkaline phosphatase phenotypes of region 11 in pigs. From left: Akp AB, Akp B, Akp BC, Akp BD.
16
Anim. Blood Grpr biochem. Genet. 9 (1978)
SERUM ALKALINE PHOSPHATASE POLYMORPHISM IN PIGS Table 1. Observed andexpected distribution of blood serum alkaline phosphatase types in the offspring of different mating types. Mating
Number Number oflitters ofpiglets
Phenotypes AB
ABxB
7
59
observed expected
x2 BxB
27
198
B
I7 19.5 5.3
3L
212
observed expected
7
61
x2
BC
x BC
5
33
126 106 3.8
15 15.25 0
86 106 3.8
29 17 15.25 15.25 12.4*** 0.2
observed expected
18 15 8.25 16.5 11.5*** 0.1
x?
** P < 0.01;
10.6"
0
observed expected observed expected
x2
C
198 198
x' AB x BC
AC
42 29.5 5.3
x2 BCxB
BC
7.5'.
0 15.25 15.2***
27.8***
0 8.25 8.2* *
19.9** *
*** P < 0.001.
in the observed segregation ratio in comparison to the expected one. In both the AB X B and BC X B mating types the observed numbers of prenotype Akp B were significantly more than expected. In the AB X BC matings, the number of animals observed with phenotype Akp AB and Akp BC agreed well with the expected number of animals. However, not a single Akp AC phenotype was observed alth,ough 15 were expected. The number of pigs with phenotype Akp B was almost twin the expected value. In the BC X BC types of mating no progeny with phenotype Akp C was observed while the expected Table 2. Matings in which the BD alkaline phosphatase phenotype was observed. Father
B No 1472
Mother
Number of piglets
Phenotype
B
BC
BD
2
1
B No 347
9
8 4
3
BC No 26
B No 157
9
BC No 26
B No 169
6
1
4
1
8
2
4
2
BC No 26
B No 169
BC No 26
B No 349
8
4
2
2
BC No 26
BC No 136
4
1
1
2
Anim. Blood Crps biochem. Genet. 9 (1978)
17
DANUTA KIEREK-JASZCZUK ET AL.
number was about 8. The results of the segregation ratios indicated an excess of alkaline phosphatase type Akp B in the progeny. Moreover, ten offspring of the phenotype Akp BD were observed from mating animals belonging to B and BC types (Table 2). The reason for this observation is unknown and therefore the animals with the Akp BD phenotype were excluded from the genetic and statistical analysis.
Discussion The results from the population of the Ztotnicka Pstra pigs studied demonstrated that blood serum alkaline phosphatase in this breed is a genetically controlled enzyme. Presumably, its synthesis is controlled by at least three alleles: A k p A , AkpB and AkpC. However, further investigations are needed in order to explain the observed unexpected excess of animals with Akp B phenotype and complete lack of animals with Akp BC and Akp C phenotypes. Also the Akp BD phenotype demands further investigations. At present, no progeny from matings involving the Akp BD phenotype are available. Therefore it was not possible to determine whether the phosphatase zone D has a separate genetic control or arises from physiological or pathological changes during life. References Dinklage, H.,1970. The alkaline phosphatase system in the pig. Proc. l l t h Eur. Conf. h i m . Blood Grps biochem. Polymorphism (Warsaw): 329-330. Imlah, P., 1970. Ontogenetic and familial variation in serum alkaline phosphatase of pigs. Proc. l l t h Eur. Conf. Anim. Blood Grps biochem. Polymorphism (Warsaw) 331-339. Poulik, M. D., 1957. Starch gel electrophoresis in a discontinous system of buffers. Nature 4600: 1477-1479. Saison, R., 1970. Serum and red cell enzyme systems in pigs. Proc. 11th Eur. Conf. Anim. Blood Grps biochem. Polymorphism (Warsaw) 321-328. Verhorst, D., 1973. Enzym- und Serumproteinpolymorphismus in Schweinezuchtlinien. Dissertation, Giittingen. Wilcox, F. H., 1966. A recessively inherited electrophoretic variant of alkaline phosphatase in chicken serum. Genetics 53: 799-805.
Anim. Blood Grps biochem. Genet. 9 (1978)
Serum alkaline phospha tase polymorphism in pigs' Danuta Kierek-Jaszczuk, Maciej hrkowski, Elibieta Skladanowska-Krzyzanowska and Krystyna Tomaszewska-Guszkiewicz Institute of Genetics and Animal Breeding Polish Academy of Sciences, Jastrzqbiec, Poland Received 30 June 1978; accepted 6 December 1978
Key-words: pig, serum, alkaline phosphatase, starch gel electrophoresis
Summary
Genetic variants of serum alkaline phosphatase were studied by the method of starch gel electrophoresis in the Zlotnicka Pstra breed of pigs. Two regions of alkaline phosphatase migration were observed. A single fraction in region I and four different phenotypes: AB, B, BC and BD in region 11, were found. For AB, B and BC phenotypes the genetic control by three alleles A k p A , AkpB and AkpC in suggested. The observed segregation ratios in some cases deviated significantly from the expected ones. Introduction
In pig serum Imlah (1970) found two electrophoretic forms of alkaline phosphatase, fast and slow. For the fast form he reported a typically ontogenetic variation. Saison (1970) examined pigs of different breeds and also reported fast and slow forms of alkaline phosphatase in serum. Dinklage (1970) found 5 fractions of alkaline phosphatase in the serum of German Improved Landrace and Gottingen Miniature pigs. On the basis of preliminary family studies he suggested that alkaline phosphatase was controlled by 5 alleles: A k p A , AkpB, AkpC, AkpD and AkpE. Later Verhorst (1973) from the same Department suggested the occurrence of only three alleles: A k p A , AkpB and AkpC. Materials and methods
The material consisted of Zlotnicka Pstra pigs from the Popielno farm. A total of Supported by the Polish Academy of Sciences within the project No 09.7.1.
15
DANUTA KIEREK-JASZCZUK ET AL. 26 sows and 5 boars and 573 offspring at 8 weeks of age were studied. Serum samples were analysed by horizontal starch gel electrophoresis. Hydrolysed starch was prepared at the Department of Animal Immunogenetics and was used at 14 % . A discontinous buffer system as described by Poulik (1957) was used: gel buffer - 0.076 M Tris, 0.005 M citric acid pH 8.65; electrode buffer - 0.3 M boric acid, 0.05 M sodium hydroxide, pH 8.05. The electrophoresis was conducted at 4 "Cfor three hours with stepwise potential increase: 0-30 min 10 V/cm, 30-60 rnin 15 V/cm, 60-90 min 17,5 V/cm and 90-180 min 20 V/cm. The starting line was 7 cm from the cathodic side of the gel. The samples were soaked on pieces of Whatman No 3 filter paper size 6 mm X 8 mm, and inserted into the gel. After electrophoresis the 6 mm thick gel was cut into two parts. The bottom part was incubated for 2 hours at 37 "C, according to the method proposed by Wilcox (1966) with some modifications. The staining mixture consisted of a buffer containing: 0.228 M Tris, 0.015 M citric acid, pH 8.9, 0.02 M MgCl,, 0.05 % Fast Blue BB or RR salt, 0.05 % of sodium a-naphthyl phosphate. After incubation the gels were stored in a mixture composed of 5 parts of ethyl alcohol, 5 parts of distilled water and 1 part of glacial acetic acid.
Results Alkaline phosphatase zones were observed in two redons of the stained gels. In region I a single fraction was observed near the starting line. This fraction stained strongly in samples from 8-week-old animals but was weaker and sometimes difficult to see in adults. Four phenotypes Akp B, Akp AB, Akp BC and Akp BD were observed in region I1 (Fig. 1).On the basis of the families examined and according to Verhorst (19731, it was suggested that the phenotypes B, AB and BC wzre controlled by three autosomal, codominant alleles: A k p A , AkpB and AkpC. T h e distribution of alkaline phosphatase phenotypes in the progeny from different types of mating is shown in Table 1. In all mating types, except Akp B X Akp B, significant deviations were found
Fig. 1. Starch gel zymogram of serum alkaline phosphatase phenotypes of region 11 in pigs. From left: Akp AB, Akp B, Akp BC, Akp BD.
16
Anim. Blood Grpr biochem. Genet. 9 (1978)
SERUM ALKALINE PHOSPHATASE POLYMORPHISM IN PIGS Table 1. Observed andexpected distribution of blood serum alkaline phosphatase types in the offspring of different mating types. Mating
Number Number oflitters ofpiglets
Phenotypes AB
ABxB
7
59
observed expected
x2 BxB
27
198
B
I7 19.5 5.3
3L
212
observed expected
7
61
x2
BC
x BC
5
33
126 106 3.8
15 15.25 0
86 106 3.8
29 17 15.25 15.25 12.4*** 0.2
observed expected
18 15 8.25 16.5 11.5*** 0.1
x?
** P < 0.01;
10.6"
0
observed expected observed expected
x2
C
198 198
x' AB x BC
AC
42 29.5 5.3
x2 BCxB
BC
7.5'.
0 15.25 15.2***
27.8***
0 8.25 8.2* *
19.9** *
*** P < 0.001.
in the observed segregation ratio in comparison to the expected one. In both the AB X B and BC X B mating types the observed numbers of prenotype Akp B were significantly more than expected. In the AB X BC matings, the number of animals observed with phenotype Akp AB and Akp BC agreed well with the expected number of animals. However, not a single Akp AC phenotype was observed alth,ough 15 were expected. The number of pigs with phenotype Akp B was almost twin the expected value. In the BC X BC types of mating no progeny with phenotype Akp C was observed while the expected Table 2. Matings in which the BD alkaline phosphatase phenotype was observed. Father
B No 1472
Mother
Number of piglets
Phenotype
B
BC
BD
2
1
B No 347
9
8 4
3
BC No 26
B No 157
9
BC No 26
B No 169
6
1
4
1
8
2
4
2
BC No 26
B No 169
BC No 26
B No 349
8
4
2
2
BC No 26
BC No 136
4
1
1
2
Anim. Blood Crps biochem. Genet. 9 (1978)
17
DANUTA KIEREK-JASZCZUK ET AL.
number was about 8. The results of the segregation ratios indicated an excess of alkaline phosphatase type Akp B in the progeny. Moreover, ten offspring of the phenotype Akp BD were observed from mating animals belonging to B and BC types (Table 2). The reason for this observation is unknown and therefore the animals with the Akp BD phenotype were excluded from the genetic and statistical analysis.
Discussion The results from the population of the Ztotnicka Pstra pigs studied demonstrated that blood serum alkaline phosphatase in this breed is a genetically controlled enzyme. Presumably, its synthesis is controlled by at least three alleles: A k p A , AkpB and AkpC. However, further investigations are needed in order to explain the observed unexpected excess of animals with Akp B phenotype and complete lack of animals with Akp BC and Akp C phenotypes. Also the Akp BD phenotype demands further investigations. At present, no progeny from matings involving the Akp BD phenotype are available. Therefore it was not possible to determine whether the phosphatase zone D has a separate genetic control or arises from physiological or pathological changes during life. References Dinklage, H.,1970. The alkaline phosphatase system in the pig. Proc. l l t h Eur. Conf. h i m . Blood Grps biochem. Polymorphism (Warsaw): 329-330. Imlah, P., 1970. Ontogenetic and familial variation in serum alkaline phosphatase of pigs. Proc. l l t h Eur. Conf. Anim. Blood Grps biochem. Polymorphism (Warsaw) 331-339. Poulik, M. D., 1957. Starch gel electrophoresis in a discontinous system of buffers. Nature 4600: 1477-1479. Saison, R., 1970. Serum and red cell enzyme systems in pigs. Proc. 11th Eur. Conf. Anim. Blood Grps biochem. Polymorphism (Warsaw) 321-328. Verhorst, D., 1973. Enzym- und Serumproteinpolymorphismus in Schweinezuchtlinien. Dissertation, Giittingen. Wilcox, F. H., 1966. A recessively inherited electrophoretic variant of alkaline phosphatase in chicken serum. Genetics 53: 799-805.
Anim. Blood Grps biochem. Genet. 9 (1978)
