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W W W . LA C TAT I O N . L I F E . K U . D K
D E PAR T M E N T O F BAS I C AN I MAL AN D V E T E R I NA R Y S C I E N C E S
FACULTY OF LIF E SCIENCES
U N IVE R S ITY O F C O PE N HAG E N
Effect of continuous lactation in dairy goats on mammary histology,
cell turnover and expression of angiogenic factors
Sina Safayia,, Peter Kappel Theilb, Vibeke Sødring Elbrønda, Lei Houa, Marie Engbæka, Mette Olaf Nielsena
aDepartment
of Basic Animal and Veterinary Sciences, Faculty of LIFE Sciences, University of Copenhagen, Groennegaardsvej 7, DK-1870 Frederiksberg C, Denmark
bDepartment of Animal Health, Welfare and Nutrition, Faculty of Agricultural Sciences, University of Aarhus, P.O. Box 50, DK-8830 Tjele
Aims:
Results:
To test the hypotheses:
1) mammary remodelling during lactation involves vascular in addition to
alveolar remodelling,
2) continuous lactation negatively impacts mammary remodelling, and
3) angiogenic factors as well as factors involved in cell turnover could be
responsible for such changes.
A) GENE EXPRESSION: (Figure 3)
 No effect of continuous lactation (CL).
 Compared to other stages of lactation:
- TGFB1R1 was high in late lactation, & like BAX, peaked before dry period.
- BCL2, CCND1, IGF1 & IGFBP3 were high before and during the dry period.
- VEGF had its lowest level during dry period, & peaked immediately after
parturition.
40.14
38.68
10.0
10.0
 9 multiparous dairy goats!
37.77
35.62
A)
SL < 0.0001
CL = 0.90
SL*CL = 0.74
SL = 0.001
CL = 0.62
SL*CL = 0.52
 A complete block design (udder-half as block; one half was dried-off
approximately 6 weeks pre-partum, one was milked continuously until
parturition).
40.62
BCL2
VEGF
Material and Methods:
B) HISTOLOGY & IMMUNOHISTOCHEMISTRY: (Figure 4)
 During the dry period (compared to at parturition and early lactation):
Lumen density, epithelium (especially fully differentiated EPC) were at lowest.
Alveoli area, stroma, endothelial cells, proliferative and poorly differentiated
EPC were at highest.
 The quantity of the intermediately differentiated EPC was greater in normal
lactating glands than continuously lactating ones. Reversely, endothelial cells
had a tendency to be greater in continuously lactating glands.
34.44
32.92
31.79
28.31
26.30
26.94
26.38
1.0
-100
0
100
200
-100
20.94
20.69
17.95
22.21
1.0
0
100
18.03
200
19.40
15.77
15.52
14.66
15.45
14.81 14.82
12.65
 Two mammary biopsies obtained from each udderhalf at days -60, -15 (dryperiod), +1 (parturition), +3, +10, +60, and +180 (Figure 1).
9.77
-60
0.1
3 10
IGF1
CCND1
60
Dry Period
180
5.57
PR
APP
2.70
2.05
2.27
ALV LUM EPC
IT
ENC
PR
APP
ALV LUM EPC
-15
SL < 0.0001
CL = 0.95
SL*CL = 0.67
SL < 0.0001
CL = 0.53
SL*CL = 0.92
1.01
0.00
1.22
0.04
0.18 0.20
1.06
0.16
0.18
0.14
10.0
10.00
IT
ENC
PR
APP
ALV LUM EPC
1
B)
IT
APP
ALV LUM EPC
1.00
1.0
0
100
200
-100
0
100
200
29.09
20.18
8.91
5.54
2.38
6.22
0.1
P
INT
-15
TGFB1R1
IGFBP3
-100
 On the 2nd biopsy: (Figure 2)
Paraffin embedded and sliced to be studied for:
A) Histology:
PAS stained, 6 pictures (of 2 slices), a 30 points transparent grid used to
quantify the relative proportion of the alveoli (ALV), lumen (LUM), stroma
(STR), endothelial cells (ENC) and epithelial cells (EPC) (Classified in 3
types: poorly (P), intermediate (INT) and fully (F) differentiated EPC).
B) Immunohistochemistry:
I) TUNEL stained: 12 pictures (of 4 slices), to detect apoptotic cells,
II) Ki-67 stained: 12 pictures (of 4 slices), to detect proliferative cells.
0
100
200
F
P
INT
1
F
P
3.05
INT
10
F
P
INT
F
60
Figure 4- Charts show the relative proportions of: A) the tissue components, B)
3 EPC types (P: Poorly, INT: Intermediate, F: Fully differentiated); in the
mammary gland of multiparous dairy goats during continuous and normal
lactation at days -15, 1, 10 and 60 relative to parturition day (day 1). (Y axis =
LSMean estimates, and X axis = components within days relative to
parturition).
10.00
1.0
35.34
22.30
13.12
0.10
52.43
43.67
39.88
33.71
18.40
 On the 1st biopsy:
Relative expression was quantified using 18S rRNA as housekeeping gene for
the following genes:
A) Angiogenesis:
(VEGF, VEGFR1, VEGFR2, ANGPT1, ANGPT2 and RTK)
B) Cell turnover: (BAX, BCL2, CCND1, IGF1, IGF1R, IGFBP3, IGFBP5,
TGFB1, TGFB1R1 and TGFB1R2).
ENC
54.56
30.64
32.81
22.53
IT
60
58.01
56.8550.91
Figure 1- Different stages of lactation at which the samples have been taken.
Data were analyzed using SAS, mixed model procedure.
PR
43.29
-100

ENC
10
40.69
Lactation days
10.38
9.40
4.763.81
0.02
0.12
Parturition
(Day 1)
-15
10.42
10.50
11.33
16.04
13.57
13.88
12.65
7.57
0.1
-6 weeks
23.17
23.64
SL = 0.04
CL = 0.56
SL*CL = 0.23
1.00
-100
0
100
200
Conclusions:
SL < 0.0001
CL = 0.61
SL*CL = 0.97
0.1
Figure 2- A) PAS stained slide with a 30 points grid on, B) Slide stained with
TUNEL. C) Slide stained with Ki-67. (B &C : Using ImageJ software, all the
EPC in the red 25% grid were counted, considering two forbidden lines.)
0.10
Figure 3- Charts show the expression levels of some of target factors in the
mammary gland of multiparous dairy goats during continuous and normal
lactation, the P values of the stage of lactation (SL), continuous lactation (CL),
and their interaction (SL*CL). (Y axis = logarithmic scale of relative gene, and
X axis = days relative to parturition).
 Continuous lactation induces changes in mammary morphology which can
not be related to altered expression of cell turnover and angiogenic factors.
 Angiogenesis seems to be more abundant at and a few days after parturition.
Results are consistent with a role for VEGF in adjustment of vascular function.
 EPC number increases substantially with initiation of lactation. Postnatally,
however, changes in mammary secretory appear to be associated mainly with
differentiation of EPC in early lactation followed by loss of de-differentiation of
cells in mid lactation rather than changes in EPC number.
Abbreviations used: 18S-rRNA1: 18S Rebosomal RNA 1; ALV: Alveoli; ANGPT1: Angiopoietin 1; ANGPT2: Angiopoietin 2; BAX: Bcl2-Associated X Protein; BCL2: B-Cell CLL/Lymphoma 2; CCND1: Cyclin D1; IGF1: Insulin-like Growth Factor I; CL: Continuous Lactation; ENC: Endothelial cells; EPC: Epithelial Cells; F:Fully; IGF1-R: Insulin-like Growth Factor I Receptor; IGFBP3: Insulin-like Growth Factor Binding Protein 3; IGFBP5: Insulin-like Growth Factor Binding Protein 5; INT: Intermediate; Ki-67: Ki67 Nuclear Antigen; LUM: Lumen; P: Poorly; PAS: Periodic Acid-Schiff; RTK: Tyrosine Kinase Tie2 Receptor (TEK); RT-PCR: Reverse Transcript Polymerase Chain Reaction; SAS: Statistical Analysis System; SL: Stage of Lactation; STR: Stroma; TGFB1: Transforming Growth Factor B1; TGFB1R1: Transforming Growth Factor B1 Receptor 1; TGFB1R2: Transforming Growth Factor B1 Receptor 2; TUNEL: Terminal Deoxynucleotidyl Transferase (TdT)-mediated dUTP Nick End Labeling; VEGF: Vascular
Endothelial Growth Factor; VEGFR1: Vascular Endothelial Growth Factor Receptor 1 (fms-like tyrosine kinase); VEGFR2: Vascular Endothelial Growth Factor Receptor 2 (Fetal liver kinase 1).