Effect of continuous lactation in dairy goats on mammary histology, cell turnover and expression of angiogenic factors Aims: 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. Material and Methods:  9 multiparous dairy goats!  A complete block design (udder-half as block; one half was dried-off approximately 6 weeks pre-partum, one was milked continuously until parturition).  Two mammary biopsies obtained from each udderhalf at days -60, -15 (dry- period), +1 (parturition), +3, +10, +60, and +180 (Figure 1). Sina Safayi a,, Peter Kappel Theil b, Vibeke Sødring Elbrønd a, Lei Hou a, Marie Engbæk a, Mette Olaf Nielsen a a Department of Basic Animal and Veterinary Sciences, Faculty of LIFE Sciences, University of Copenhagen, Groennegaardsvej 7, DK-1870 Frederiksberg C, Denmark b Department of Animal Health, Welfare and Nutrition, Faculty of Agricultural Sciences, University of Aarhus, P.O. Box 50, DK-8830 Tjele W W W. L A C T A T I O N. L I F E. K U. D K D E P A R T M E N T O F B A S I C A N I M A L A N D V E T E R I N A R Y S C I E N C E S F A C U L T Y O F L I F E S C I E N C E S U N I V E R S I T Y O F C O P E N H A G E N Conclusions:  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. Figure 1- Different stages of lactation at which the samples have been taken.  On the 1 st 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).  On the 2 nd 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.  Data were analyzed using SAS, mixed model procedure. Lactation days Parturition (Day 1) -6 weeks Dry Period Results: 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. 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: Ki- 67 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). 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.) 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). 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. 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). SL = CL = 0.62 SL*CL = 0.52 A)