Page 4 - 48_Freemartinism

Table 1. Female (F) and male (M) phenotypic sex, karyotype, percentages of female and male blood cells, and clinical observa-
tions in the ten river buffaloes (2 males and 8 females) found to be freemartin. Cases 1 and 2, as well 7 and 8 are co-twins.
Case Sex Karyotype F cells M cells Clinical observations
(%)
1 F 50 50 Normal body conformation, normal vulva, vagina and clitoris, atrophy of internal sex
2n = 5O,XX/XY
adducts, small ovaries, sterile.
2 M 2n = 5O,XX/XY 41 59 Normal body conformation and external genitalia.
3 F 2n = 5O,XX/XY 50 50 Body conformation with some male traits (tight pelvis, large horn base circumference),
normal vulva, vagina and clitoris, serious atrophy of Muller's ducts, small ovaries,
sterile.
4 F 2n = 5O,XX/XY 50 50 Normal body conformation, normal vulva, vagina and clitoris, atrophy of internal sex
adducts, ovaries not detectable, sterile.
5 F 2n = 5O,XX/XY 51 49 As in case 4.
6 F 2n = 5O,XX/XY 30 70 Normal body conformation, normal vulva, vagina and clitoris, closed vagina with lack
of internal sex adducts, sterile.
7 M 2n = 5O,XX/XY 89 11 Normal body conformation and penis, one testis much smaller than the other normal
one.
8 F 2n = 5O,XX/XY 96 4 As in case 6.
9 F 2n = 5O,XX/XY 50 50 As in case 1.
10 F 2n= 5O,XX/XY 63 37 Body conformation with some male traits (tight pelvis) and unusual horns (thin),
normal vulva, vagina and clitoris, lack of internal sex adducts, sterile.
Materials and methods tive) and late- (inactive) replicating X chromosome in female
cells (Fig. 1 b).
Forty phenotypic females with reproductive problems and two pheno-
Of the 42 animals we investigated, 10 (24 %)animals (8 phe-
typic male co-twins of females, raised in the provinces of Caserta and Salerno
notypic females and 2 phenotypic males) were found to be free-
(Campania, Southern Italy) underwent cytogenetic investigation from 1998
to 2003. Of the 42 investigated animals, cases 1 and 2, as well as 7 and 8 martin. The frequency of male and female blood cells (Table 1)
(Table 1) were co-twins. The former were cytogenetically studied at both a varied among the investigated animals, the most frequent cases
young age (about one month) and later (about one year). The male was not being close to 50 % male and female cells, although two females
eliminated at birth because of its high genealogy, but about one year later due (cases 6 and 8) had a very different percentage of male cells (70
to some observed hoof defects (shape not complying with the breed stan-
and 4 %,respectively).
dard). Also case 8 underwent cytogenetic investigation a second time during
reproductive age (2.5 years).
Concanavalin A-stimulated peripheral blood cells were incubated for Clinical observations
72 h at 37.8 ° C. Two types of cell cultures were performed: without (normal All freemartin females underwent clinical observation of
cultures) and with addition of both BrdU (15 gg/ml) and Hoechst 33258
body conformation, as well as the external and internal repro-
(30 wg/ml) during the last 6 b after thymidine (300 pg/ml) block for about
17 h. Slides from normal cultures were treated by the CBA-banding tech- ductive organs by means of both rectal palpation (5 females) and
nique (Iannuzzi, 2003), while those obtained from late incorporation of both direct observation after mating (3 females; cases 3, 6 and 10).
BrdU and Hoechst 33258 were RBA-banded (Iannuzzi, 2003). A fluores- Six freemartin phenotypic females showed normal body
cence Nikon E-1000 microscope, connected with a CCD camera (Coolsnap
CF, Photometrics) and equipped with Genicon (Nikon) software, was conformation, clitoris and vulva. In both cases 3 and 10 some
employed. One hundred (normal cultures) and 30 cells (RBA-banding) per male traits (tight pelvis) were observed, while a large base horn
animal were studied and chromosome identification followed the standard circumference (35 em), as in the males, was only observed in
karyotype (CSKBB, 1994). case 3. Case 10 showed horns, which were thin, recalling those
of swamp buffalo.
Rectal palpation revealed damage in the internal reproduc-
Results and discussion tive organs of all females, varying from complete lack of inter-
nal sex adducts (closed vagina, cases 6, 8 and 10) to hypoplasia
Cytogenetic analysis of Múllerian ducts and absence (or atrophy) of ovaries (Fig. 2).
The CBA-banding technique is the best and simplest band- A considerable reduction in uterus size and oviducts was
ing technique to clearly distinguish sex chromosomes from the observed in several freemartin cows (Khan and Foley, 1994). In
autosomes, as well as male and female cells in river buffalo our study, all females found freemartin were sterile, even when
(Fig. 1 a). Indeed, the X is the largest acrocentric chromosome the percentage of male blood cells was very low (4 %,case 8).
showing the largest heterochromatin (HC) block at the cen- The two phenotypic male co-twins showed no particular
tromere, in addition to a proximal C-positive band, while the Y problems, except for case 7 which had a smaller testis com-
chromosome is a small acrocentric with the centromere C-band pared to the other one and to those of males of the same age.
negative and a distal C-positive band (Fig. 1 a), although it may However, it is interesting to note that this animal was male with
often appear almost heterochromatic (C-band positive) de- only 11 % of male cells. Since the male river buffaloes are gener-
pending on the degree of chromosome denaturation (Di Meo et ally culled after birth or before they reach the reproductive life
al., 1995). The R-banding technique may also be useful to dis- (as in two male co-twins studied here), it is difficult to ascertain
tinguish sex chromosomes, especially to reveal the early- (ac- whether cell chimeric conditions may cause reproductive prob-
356 Cytogenet Genome Res 108:355-358 (2005)