Morphometric and immunohistochemical study of the omasum of red deer during prenatal development

Abstract

The red deer is an important study species because of its value in the national economy and because it provides a wealth of genetic material. To date, there has been little research into the prenatal development of the stomach of ruminants, and none of the red deer. We therefore performed a histological evaluation of the ontogenesis of the omasum in the red deer. Histomorphometric and immunohistochemical analyses were carried out on 50 embryos and fetuses of deer from the initial stages of prenatal life until birth. For test purposes, the animals were divided into five experimental groups: Group I (1.4-3.6 cm crown-rump length, CRL; 30-60 days, 1-25% of gestation); Group II (4.5-7.2 cm CRL; 67-90 days, 25-35% of gestation); Group III (8-19 cm CRL; 97-135 days, 35-50% of gestation); Group IV (21-33 cm CRL; 142-191 days, 50-70% of gestation); and Group V (36-40 cm CRL; 205-235 days, 75-100% of gestation). At 67 embryonic days, the omasum wall was differentiated, and comprised three layers: the epithelial layer, pluripotential blastemic tissue and serosa. The stratification of the epithelial layer was accompanied by changes in its structure, with the appearance of four laminae of different sizes; in order of appearance these were: primary at 67 days, secondary at 90 days, tertiary at 97 days and quaternary at 135 days. At around mid-gestation, lateral evaginations were formed from the stratum basale of the primary and secondary smaller laminae. These were the primitive corneum papillae. From 205 days, the corneum papillae were present in all four sizes of laminae. The histodifferentiation of the lamina propia-submucosa, tunica muscularis and serosa showed patterns of development similar to those reported for the rumen and reticulum of red deer. The omasum of red deer during prenatal life, especially from 67 days of gestation, was shown to be an active structure with full secretory capacity. Its histological development, its secretory capacity (detected by the presence of neutral mucopolysaccharides) and its neuroendocrine nature (detected by the presence of positive non-neuronal enolase cells and the neuropeptides vasoactive intestinal peptide and neuropeptide Y) were parallel to the development of the rumen and the reticulum. However, its prenatal development was later than that of the omasum in sheep, goat and cow.

Fig. 1

a Photomicrograph of a transverse section of the undifferentiated stomach at 1.4 cm CRL, 30 days. The wall was composed of two layers: epithelium (e) and pluripotential blastemic tissue (pbt). H-E, ×250. b Photomicrograph of a transverse direction section of the omasal wall at 4.5 cm CRL, 67 days. Three layers are visible: epithelium (e), pluripotential blastemic tissue (pbt) and serosa (s). In the epithelial layer the primary smaller laminae can be seen (L1). H-E, ×180. c Photomicrograph of a transverse direction section of the omasal wall at 7.2 cm CRL, 90 days. Myoblastic fibres of the tunica muscularis (tm), infiltrating into the smaller laminae and constituting the muscularis mucosae (mm), were observed. The epithelium is stratified in two zones: a basal zone or stratum germinativum (g) and another apical zone or stratum granulosum (gr). Serosa is visible (s). H-E, ×250. d Photomicrograph of a transverse direction section of the omasal wall at 7.2 cm CRL, 97 days. The secondary smaller laminae (L2) can be observed in the spaces between the primary smaller laminae (L1). H-E, ×180. e Photomicrograph of a transverse direction section of the omasal wall at 8 cm CRL, 97 days. Four layers can be detected: epithelium, lamina propia-submucosa (Lp-Sb), tunica muscularis (tm) and serosa. Presence of the primary (L1), secondary (L2), tertiary (L3) and quaternary smaller laminae (L4). H-E, ×120. f Photomicrograph of a transverse direction section of the omasal wall at 8 cm CRL, 97 days. The epithelium is stratified in three zones: a basal zone or stratum germinativum (g), stratum granulosum (gr) and another apical zone or stratum corneum (c). H-E, ×350. g Photomicrograph of a transverse direction section of the omasal wall at 19 cm CRL, 135 days. Presence of corneum papillae (pc) in the primary smaller laminae with stratified epithelium: stratum germinativum (g), granulosum (gr) and corneum (c). VG, ×250. h Photomicrograph of a transverse direction section of the omasal wall at 19 cm CRL, 135 days. Muscularis mucosae (mm), constituting the muscularis of the smaller laminae. The stratified epithelium with a stratum germinativum (g), stratum granulosum (gr) and stratum corneum (c) is visible. H-E, ×250.

Fig. 2

a Photomicrograph of a transverse direction section of the omasal wall at 21 cm CRL, 142 days. Omasal wall with the four laminar generations: primary (L1), secondary (L2), tertiary (L3) and quaternary (L4). H-E, ×120. b Photomicrograph of a transverse direction section of the omasal wall at 21 cm CRL, 142 days. Internal fascicule of the tunica muscularis (tm) filling the width of the smaller laminae and forming the muscularis mucosae (mm). MT, ×350. c Photomicrograph of a transverse direction section of the omasal wall at 33 cm CRL, 191 days. Primary lamina with stratified epithelium: stratum germinativum (g), granulosum (gr), lucidum-spinosum (l-e) and corneum (c). Corneum papillae (pc) and muscularis mucosae (mm) can also be seen (pc). H-E, ×250. d Photomicrograph of a transverse direction section of the omasal wall at 33 cm CRL, 191 days. Presence of corneum papillae (pc) in the secondary smaller laminae. H-E, ×180. e Photomicrograph of a transverse direction section of the omasal wall at 36 cm CRL, 205 days. Omasal mucosa with the four sizes of smaller laminae: primary (L1), secondary (L2), tertiary (L3) and quaternary (L4). H-E, ×180. f Photomicrograph of a transverse direction section of the omasal wall at 36 cm CRL, 205 days. Presence of reticulin fibres in the interior of the corneum papillae (pc) of the primary smaller laminae. RG, ×350. g Photomicrograph of a transverse direction section of the omasal wall at 40 cm CRL, 235 days. Stratified epithelial layer: stratum germinativum (g), granulosum (gr), lucidum-spinosum (l-e) and corneum (c). Muscularis mucosae (mm) coming from the internal fascicule of the tunica muscularis (tm). VG, ×350. h Photomicrograph of a transverse direction section of the omasal wall at 40 cm CRL, 235 days. Abundant presence of corneum papillae (pc) in the omasal smaller laminae. A thick muscularis mucosae can also be observed (mm). TM, ×350.

Fig. 3

a Photomicrograph of a transverse direction section of the omasal wall at 4.5 cm CRL, 67 days. Presence of neuroendocrine cells (NNE) in the tunica muscularis (arrows). EAS, ×250. b Photomicrograph of a transverse direction section of the omasal wall at. 40 cm CRL, 235 days. Presence of neuroendocrine cells (NNE) in the lamina propia-submucosa (arrows). EAS, ×250. c Photomicrograph of a transverse direction section of the omasal wall at 8 cm CRL, 97 days. Presence of GFAP-positive cells (arrows) in lamina propia and submucosa. EAS, ×180. d Photomicrograph of a transverse direction section of the omasal wall at 40 cm CRL, 235 days. Presence of GFAP-positive cells (arrow) in tunica muscularis. EAS, ×350. e Photomicrograph of a transverse direction section of the omasal wall at 19 cm CRL, 135 days. Presence of VIM-positive cells (arrows) in lamina propia and submucosa. EAS, ×250. f Photomicrograph of a transverse direction section of the omasal wall at 40 cm CRL, 235 days. Presence of VIM-positive cells (arrow) in tunica muscularis. EAS, ×350. g Photomicrograph of a transverse direction section of the omasal wall at 36 cm CRL, 205 days. Positive immunodetection of VIP (arrows) in the lamina propia-submucosa (submucosus plexus). EAS, ×250. h Photomicrograph of a transverse direction section of the omasal wall at 36 cm CRL, 205 days. Positive immunoreaction for NPY (arrow) in the submucosus ganglion. EAS, ×250.

Fig 4

Mathematical model of omasum growth (epithelium).

Fig. 5

Mathematical model of omasum growth (lamina propia and submucosa).

Fig. 6

Mathematical model of omasum growth (tunica muscularis).

Fig. 7

Mathematical model of omasum growth (serosa).

Fig. 8

Mathematical model of omasum wall growth.