Braz . ./. vet. Res. anim. Sci.,
São Paulo, v.33, n.3, p. 176-1SO, 1996.
Possible association between hem oglobin types and
reproductive disorders in Brazilian Mangalarga mares*
Possível associação entre tipos de hemoglobina e problemas
reprodutivos em éguas Mangalarga brasileiras
Rejane de Lima e SILVA
Paulo R oberto R odrigu es R A M O S 2;
Miirio R ubens Holtz de A L M E I D A 2; Jehud B O R T O L O Z Z I
Alicio M A R TINS JU N IO R J; Edson Marcelo B R U D E R 5
CORRESPONDENCE TO:
Paulo Roberto Rodrigues Ramos
Departamento de Física e Biofísica
Instituto de Biociências da UNESP
Distrito de Rubião Júnior - Campus Universitário
Caixa Postal 510
18618-000 - Botucatu - SP - Brasil
1 - Instituto de Biociências da UNESP- Botucatu - SP
2 - Instituto de Biociências da UNESP - Botucatu - SP
3 - Faculdade de Ciências da UNESP - Bauru - SP
4 - Faculdade de Odontologia da UNESP Araçatuba - SP
5 - Instituto de Biociências da UNESP - Botucatu - SP
SUMMARY
In the present report the biochemical polymorphism of Mangalarga mares hemoglobin, in reproductive age, from Santa
Fé Farm, Botucatu, São Paulo, was studied. Animals were classified in two groups, according to reproductive history
of each mare; the first group was performed by normal mares (control group) and the second one by animals with
reproductive disorders (barren mares). From each animal, around 15 ml of vessel blood were collected. Hemoglobins
were typed by polyacrylamide gel electrophoresis, 7% of concentration in the resolving gel, in a discontinuous
alkaline (pH 8.6) buffer system. The following hemoglobins phenotypes were found in the control group, with the
respective frequencies: Ar ~ (2.0%), A1A2m+m+(21.0%) and A1A2m+m (27.0%). To the group performed by reproductive
disorders carrier animals the following results were obtained A-,—(10.0%), A1A2m+m+(12.0%) and A1A2m+m (28.0%).
The difference observed in the Ar -- phenotype between the groups may be due to a probable liaison with hemo­
globin locus and another one related with reproductive traits. Besides this fact, tropical environment effects may be act­
ing on this locus, thus leading to obtained results.
UNITERMS: Hemoglobin; Electrophoresis; Mares.
INTRODUCTION
angalarga Horse breed has as main former the
Andalusian. In the beginning of this century other
breeds were introduced, like Arab, Anglo-Arab
(Arab x Thoroughbred), Thoroughbred and Am erican
Saddle Horse, according to Simões20 ( 1978). In São Paulo it
is possible to note three lineages originated in the south of
Minas Gerais. Presently, the Mangalarga breed is mainly
based in four breeds. Although this equine breed has been
diffused into almost all the Brazilian country, hence
becoming a national breed and no more a regional breed, a
high uniformity degree among the animals is noted. An
important cause of infertility or subfertility, especially in
older, multiparous mares is the condition of persistent
endometritis, which represents an economic impact on the
horse-breeding industry. Endometritis can be defined as an
infection or inflammation of the endometrium which may
not be discernible on rectal examination, as stated by
Threlfall22 (1980). It is peipetuated by anatomic, physiologic
and immunologic failures in mares, as stated by A sbury1
M
* S u p o rte fin a n c e iro - F U N D U N E S P - C A P E S .
176
(1983). Most of the bacteria associated with uterine disease
are opportunistic pathogens normally found on the external
genitalia of clinically normal mares and stallions. Probably,
they are transported to uterus during coitus. Most mares are
resistant to intrauterine infection and rapidly eliminate
bacteria without treatment, while others apparently are
unable to eliminate bacteria quickly, and so develop a
chronic endometritis, according to Hughes; Loy14 (1975).
This increasing susceptibility to bacterial colonization of the
uterus seems to be more common in older mares, but it is
caused by an individual variation, as affirmed by Blue et al?
(1982). Blood proteins show a polymorphism due to genetical
variance. Such variants are transmitted by simple Mendelian
heritage, with no dominance. To explain the hemoglobin
bands intensity variation in horses, the existence of a locus
that would control its synthesis is admitted. Generally, the
variants are detected by electrophoretic methods because
aminoacids change and/or deletions modify molecules
structure and/or charges. The first genetical variation in
horses hemoglobin was described by Cabannes; Serain*
(1955). Bangham; Lehm ann2 (1958), employing paper
electrophoresis, found two fractions in 64 animals and one
horse showing only the fast component, confirming data
SILVA, R.L.; R A M O S, P.R.R.; A L M E ID A , M .R .H .; B O R TO LO ZZI, J.; M A R T IN S JU N IO R , A.; B R U D E R , E.M . Possible asso ciatio n betw een h em oglobin types and reproductive
disorders in Brazilian M angalarga m ares. Bra/,. J. vet. Res. aniin. Sci. São Paulo, v.33, n.3, p. 176-180, 1996.
obtained by Cabannes; Serain8 (1955). Meanwhile, the same
authors used starch gel electrophoresis and observed that the
same animal had two hemoglobin fractions, in a ratio of
0.25:1, which led the authors to conclude that there would be
more than one locus controlling hemoglobins expression.
Braend; Stormont6 (1964) observed that 183 horses’ blood
samples showed the same type of hem oglobins; two
com ponents, the fast one moved with the same mobility of
the B bovine fraction and the slow was intermediary
between A and B bovine fractions. Kilmartin; C leggls
(1967) investigated by chromatography the aminoacid
sequence in the equine hemoglobin types. They observed
that the difference in electrophoretic mobilities was due to
aminoacid changes at position 60 in a chain. Glutamine, a
neutral aminoacid, appeared in the fast component, while it
was changed by lysine in the slow one, which shows positive
charge in an alkaline pH. This fact would explain the difference
in mobility. Braend4 (1967) analyzed horse hemoglobin
samples by electrophoresis and quantified components A,
and A,. In horses from various races three phenotypes
appeared, as previously described by Braend; Efremov5
(1964). One of them showed only the fast component (A r —).
The others showed two bands (A ,A 2), but with quantitative
differences among them. Component A2 was slower in alkaline
pH. A group had around 38% of A2, that varied from 34% to
41%; another class showed around 18% of A2 varying from 15
to 23%. With the aim of explaining such variations, authors
admitted that another locus would control horse hemoglobin
synthesis. This locus was named as modulator locus Hbm,
which would influence the slow hemoglobin component
(A2) by the inhibition of a structural gene. This gene would be
responsible for the synthesis of one polypeptidic chain from
Hb A t. If there is homozygosity in the locus (Hb"’ Hb1" ), and in
such a way that a complete inhibition occurs, the slow
component will not appear. Only 20% of the slow component
will appear if only one of the alleles is active (Hbm,Hbm+). The
present work was done with the aim of studying the genetical
polymorphism of hemoglobins in blood samples from normal
mares and from mares Mangalarga breed with reproductive
disorders. A probable association between phenotypes and
reproductive disorders was also investigated and studied.
Piemedical, Netherlands); the other group (G2) constituted
by 50 mares with reproductive disorders, which were bred at
least in two cycles during the same breeding season, but did
not conceive. Such animals were submitted to clinical
examination and to cytological and microbiological test to
determine the ethiology of the disorder. From each animal
15 ml of blood sample were obtained by venobleeding.
Erythrocytes were obtained by three consecutive isosmotic
saline washing. Hemoglobin solution was obtained by
hiposmotic cell rupture as stated by Ram os18 (1986), and
was kept under refrigeration (-20°C) until use. Hemoglobins
were submitted to alkaline vertical polyacrylamide gel
electrophoresis, in a discontinuous buffer system, 7% of
concentration in the resolving gel according with Davis11
(1964). Slabs were stained with Amido Black (0.5% w/v in
acetic acid 7% v/v) and submitted to densitometry (Zeiss
MD 100). Mares were classified according to electrophoretic
patterns described by Braend4 (1967). Descriptive statistic
was employed as recommended by Zar24 (1984). Groups
were compared by means of X2, in basis of Hardy-Weinberg,
as recommended by Crow; K im ura10 (1970). Possible
associations were analyzed by Variance Analysis as
described by Brow; Forsyte7 (1974), and Tuckey test.
RESULTS
Hemoglobins showed patterns of one or two bands and
the slow one presented differences in staining intensity, as
may be seen in Fig. I. Phenotypes’ frequencies are described
in Tab. 1 showing that only 4% of healthy mares had phenotype
A ,— , while 20% of problem animals showed the same
MATERIAL AND METHOD
In the present report, 100 Mangalarga mares, from one
farm located in Botucatu (Santa Fé), São Paulo, Brazil,
aging from 4 to 27 years, with different numbers of birth
supplied by the Brazilian Association of Mangalarga Horses
breeders (ABCCM). Animals were classified in two groups,
one constituted by 50 healthy considered mares, through
reproductive history and clinical general examination (G t).
They were considered able to mating and conceiving, and
gestation diagnose was confirmed by rectal evaluation and
by consecutive ultrasonography (scanners 450 VET,
Figure 1
Electropherogram showing obtained results in alkaline
vertical slab polyacrilamide gel electrophoresis 7%,
pH 8.6, phenotypes Ar - , A] A2mtmt and A] A2m 'm .
177
SILVA, R.L.; RA M O S, P R .R.; A LM EID A , M .R.H .; B O R TO LO ZZI, J.; M A R TIN S JU N IO R , A.; B R U D E R , E.M . P ossible asso ciatio n b etw een hem oglobin types and reproductive
disorders in Brazilian M angalarga mares. Braz. J. vet. Res. anim. Sei. São Paulo, v.33. n.3, p. 17 6 - 180, 1996.
phenotype. Tab. 2 shows there were differences (X2)
between the considered groups. According to information
from ABCCM relative to number of births until 1992 and
ages of mares, in the group performed by normal animals
ages varied from 4 to 27 years, and in the unhealthy mares
from 4 to 25 years. Tab. 3 describes the number of colts by
mare in the following age classes: 4 to 7 years, 8 to 14 years
and 15 to 27 years.
D ISC U SSIO N AND C O N C L U S IO N S
Results relative to electrophoretical fractioning of
hemoglobins were in agreement with Cabanes; Serains
(1955); Bangham; Lehmann2 (1958); Braend; Efremov5
(1964); Braend; Storm ont6 (1964); Braend4 (1967);
Sandberg; Bengtsson19 (1972) and Ezcurra; M itat12 (1973).
Hemoglobins were named according to nom enclature
proposed by Braend4 (1967), which considers the action of
modulatior genes on synthesis of the slow component a
chain. Clegg9 (1970) proposed another nom enclature,
employing aminoacid sequence, determining the ones which
occupied positions 24 and 60. However, such determination
Table 1
Horse hemoglobin patterns phenotypic frequencies in
reproductively healthy mares (Gi) and in unhealthy
mares (G 2) of Mangalarga breed. Botucatu - SP,
1991/1992.
Groups
Phenotypes
Number
G1
Ar ~
A1A2m,m*
A1A2m,m
10
20
28
Phenotypic
frequencies
0.20
0.40
0.56
g2
Ar ~
A1A2nrmt
A1A2m*m
10
12
28
0.20
0.24
0.56
was not possible to obtain. Obtained results showed similar
patterns to the published by Lucas; Becari16 (1986) and
Singhvi; Khanna21 (1987). Analyzing the hemoglobin locus
all the population as a single group too, it was observed that
the population is under Hardy-Weinberg equilibrium. It
wouldn't have to occur, because in the beginning of the
breed formation there was a certain inbreeding, as stated in
Simoes20 (1978). This fact drives to the conclusion that
elected characteristics to the improvement of this race had no
sufficient influence on the hemoglobin locus equilibrium.
When the population was divided in two groups according to
reproductive traits, data displayed in Tab. 2 show that there
are differences between healthy an unhealthy mares. Such
differences occur due to the high frequency of A ,— phenotype
in the unhealthy group, and to the A |A 2m 'm f phenotype in
the group of healthy mares, according to Tab. I. There are
two hypotheses to explain such high frequencies. The first
one could be a linkage between Hb locus (a genetical
marker) and another, at least related to some reproduction
characteristics. The second, heterosis, where heterozigote
mares to the A locus (A ^ n T m * ) could show a positive
effect that would improve reproductive traits. These
hypotheses may be enforced by results obtained by
Velhankar et a 1.23 (1977), who observed that hemoglobin
heterozygote of Gyr heifers became able to reproduction earlier
than homozygote ones. In contrast, homozygote lambs to
hem oglobin show ed b etter rep roductive index than
heterozygote according to Hanrahan et al,13 (1978) and
M ilewski17 (1983). This controversy may be explained by
the fact that Gyr and M angalarga mares, studied in the
present report, were developed under tropical conditions.
Environment, then, brought some influence over genome,
selecting a more adequate phenotype group. The same may be
said about lambs which showed controversial data when
compared with the ones presented here. Those animals
formed a group developed and reared different environmental
conditions, which exercised different selective forces and led to
different phenotypic hemoglobins frequencies.
Table 2
Observed and expected distributions of hemoglobins phenotypes in reproductive normal mares (Gj) and barren mares
(G2). Botucatu - SP, 1991/1992.
A y-
OBS.
2.0
EXP.
6.0
178
Gl
Phenotypes
A 1A2m+mt
A 1A2m+m“
20.0
28.0
16.5
27.5
g2
Phenotypes
A]—
A lA2m+m*
10.0
12.0
A ^ nvm
28.0
6.0
16.5
27.5
SILVA, R.L.; R A M O S, P.R.R.; A L M E ID A , M .R .H .; B O R T O L O Z Z I, J.; M A RT IN S JU N IO R , A.; B R U D E R , E.M . P ossible asso ciatio n betw een h em oglobin types and reproductive
disorders in Brazilian M angalarga m ares. Bra/.. J . vet. Res. anim . Sci. São Paulo, v.33, n.3, p. 176-180, 1996.
Table 3
Number of colts by mare distribution under three age classes in reproductive normal mares (Gj) and barren mares (G2),
in Mangalarga breed. Botucatu - SP, 1991/1992.
Number
Number of
Number of
Number of
Age classes
mares
colts
mares
colts
12
36
15
1(4-7 years)
33
27
98
75
II (8-14 years)
15
72
02
31
08
III (15-27 years)
RESUMO
Foram estudadas as hemoglobinas de 100 éguas da raça Mangalarga, em idade de reprodução, provenientes da
Fazenda Santa Fé, situada na região de Botucatu. Esses animais foram divididos em 2 grupos, de acordo com o
histórico reprodutivo de cada animal, sendo um formado por éguas reprodutivamente normais e o segundo por éguas
portadoras de problemas reprodutivos. Foram colhidas amostras de 15 ml de sangue com anticoagulante. As
hemoglobinas foram identificadas por meio de eletroforese em gel de poliacrilamida em placa vertical, a 7% em pH
8.6, segundo Davis11 (1964). Quanto ao sistema de hemoglobinas, foram encontrados os seguintes fenótipos para o
grupo de éguas reprodutivamente normais: Ar - (2,0%), A1A2m+m+ (21,0%) e A ^ rrrm (27,0%); para o grupo de
éguas com problemas reprodutivos: Ar - (10,0%), A ^ n rn v (12,0%) e A ^ n v m (28,0%). A diferença na freqüência
do fenótipo Ar ~ entre os grupos pode ter ocorrido devido ã existência da ligação do loco hemoglobina a outro que
controlaria características de produção. Além disso, pode estar ocorrendo influência do tipo de clima existente nos
trópicos.
REFERENCES
1-A SB U R Y , A .C . B a c te ria l e n d o m e tritis . In: R O B IN S O N , E. C u r r e n t
te r a p y in eq u in e m ed icin e. P h ilad e lp h ia , W .B. S au n d ers, p.410-4.
2-B A N G H A M , A .D .: L E H M A N N , H. M ultiple h aem o g lo b in s in horse.
N a tu re, v.181, p .2 6 7 -8 , 1958.
3-B L U E , M .G .; B RA D Y . A .A .; D A V ID SO N , J.N .; K E N N E Y , R .M . S tudies
on the co m p o sitio n a n d a n tib a cte rial a ctiv ity o f u terin e flu id from
m ares. J o u r n a l o f R e p ro d u ctiv e and F ertility , v.32, p . 143, 1982.
4 -B R A E N D , M. G en etic v ariatio n o f h orse h e m o g lo b in . H ered ita s, v.58,
n.25, p .3 8 5 -9 2 , 1967.
1 l-D A V IS , B.J. D isc electro p h o resis. 11. M ethod and ap p licatio n to hum an
serum proteins. A m erican N ew York A cad em ic Science, v. 121,
p.404, 1964.
12-E Z C U R R A , L.; MITAT, J. E lectro p h o retic stu d y o f h a em o g lo b in in the
A m erican Q u a rte r H orse. R evista C u b a n a de C ien cias
V eterin arias, v.4, n. 1/2, p.93-5, 1973.
13-H A N R A H A N , J.P.; Q U IR K E , J.F.; G O S L IN G . E .M .; W IL K IN S , N.P.
E ffect o f h a em o g lo b in g e n o ty p e on the re sp o n se o f Finn e w es to
P M S G . A n im a l B reed in g A b stract, v.46, n .l I, 1978 (A bstract,
5321).
14-H U G H E S . J.P.; LOY, R .G . T h e relatio n to infertility in the m are and
stallion. E q u in e V eterin ary Jo u r n a l, v.7. p. 155-9. 1975.
5-B R A E N D , M.; E F R E M O V , G . I h e m o g lo b in s , h ap to g lo b in and a lb u m in s o f
ho rses, In: E U R O P E A N A N IM A L B L O O D G R O U P C O N F E R E N .C E ,
9., P rag u e, 1964. P ro ceed in g s, p .253-9.
15 -K IL M A R T IN , J.V.; C L E G G . J.B . A m in o -acid re p la c em e n ts in horse
h a em o g lo b in . N atu re, v.21, p .2 6 9 -7 1, 1967.
6 -B R A E N D , M .; S T O R M O N T , C. S tu d ies on h e m o g lo b in an d transferrin
types o f h o rses. N o rd isk V eterin a rm ed icin . v. 16, p .3 1-7, 1964.
16-L U C A S, G .; B E C C A R L M. L’e m o g lo b in a d ell cav allo . N o ta II.
C arac tte ristic h e dei geni glo b in ici e v arianti po lim o rfich e. A rch ivio
V eterin ário Italian o, v.37, n .3 16, p. 154-62, 1986.
7-B R O W , M .B.; F O R S Y T E . A .B . T h e a n o v a an d m u ltip le co m p a riso n s fo r
da ta w ith h ete ro g e n o u s varian a. B io m etrics, A le x an d ria , v.30, p .719-24,
1974.
8 -C A B A N N E S , R.; S E R A IN , C. É tude é le ctro p h o ré tiq u e des h e m oglobins
des m a m m ifè re s d o m e stiq u es d ’A lgérie. C o m p tes R en d u s d es S éan ces
de la S o ciété d e B io lo g ie, v. 149, p .l 193-7, 1955.
9-C L E G G , J.B . H orse h a em o g lo b in p o ly m o rp h ism : E v id en c e fo r tw o linked
n o rm allelic alfa -c h a in g en es. P r o c e ed in g s o f th e R oyal S o ciety.
B, v. 176, p .2 3 5 -4 6 , 1970.
10-CROW , J.F.; K IM U R A , M. An introd uction to pop ulation s genetics
th eory. N ew York, H arp er Row, 1970.
17-M IL E W S K I, S. T he effect o f d ifferent diets and haem oglobin and tran s­
ferrin types on the re p ro d u c tiv e e ffic ie n c y o f ew es. A nim al
B reed in g A b stract, v .5 l, n.K ), 1983 (A bstract. 6059).
18-R A M O S , P.R.R. (ir u p o s sa n g u ín eo s, p o lim o r fism o b io q u ím ico das
h e m o g lo b in a s e h è m a tim etria em B ú falos. B otucatu, 1986.
D issertação (M estrado). U n iv e rsid ad e E stadual Paulista Ju lio de
M esq u ita Filho.
19-S A N D B E R G , K.; B E N G T S S O N . P o ly m o rp h ism o f h e m og lobin and
6-P h o sp h o g lu c o n a te d eh y d ro g e n ase in horses erith ro cy tes. In:
E U R O P E A N C O N F E R E N C E A N IM A L B L O O D G R O U P S
B IO C H E M IS T R Y , 12.. B u d ap e ste , 1972. P ro ceed in g s, p.527-31.
179
SILVA, R.L.: K AM OS. P.R.R.; A LM EID A , M .R.H .: B O R TO LO ZZI. J.: M AR TIN S JU N IO R , A.; B R U D E R , E.M . P ossible asso ciatio n b etw een h em oglobin types and reproductive
disorders in Brazilian M angalarga m ares. B ra /. J . vet. Res. an im . Sei. São Paulo, v.33, n.3, p. 176-180. 1996.
20 -S IM Õ E S , F. M a n g a la rg a c o c a v a lo do sela b ra sileiro . 2 .ed ., S ã o P aulo,
Ed. do s C riad o res L td a., 1978, p.223.
2 1-SIN G H VI, N.; K H A N N A , N .D . E lec tro p h o retic stu d ies on h a em o g lo b in
p o ly m o rp h ism in Indian h o rses, d o n k e y s and m ules. Ind ian V eterin ary
J o u rn a l, v.(S4, n.9, p .7 4 8 -5 0 , 1987.
22-T H R E L F A L L , W .R. D iag n o sis an d tre a tm e n t o f in fertility in m are.
V eterin ary M ed icin e Sm all A n im a l C lin icia n , v.75, p .4 8 3-6, 1980.
23-V E L H A N K A R , D.; T A D PA T R IK A R , N .S.; SA N E , C .R . H a em oglobin
p o ly m o rp h ism in relatio n to p u b erty and sexual m a tu rity in G ir heifers.
Indian J o u r n a l o f D a iry S c ie n c e , v.3(), n.2. p. 134-7, 1977.
24-Z A R . J.ll. B io esta tistica l a n a ly sis. 2.ed. E n g lew o o d C liffs, Hall
In tern atio n al, 1984.
R ecebido para publicação: 25/05/95
A provado para publicação: 18/09/95
180