A. Franco

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

Iberian red deer need to be conserved for their economic role and for their genetic importance as an important component of the ecosystem. Modifications currently being made to traditional management systems require a better understanding of the structure, function and development of their alimentary system. Here we describe a histomorphometric and immunohistochemical analysis of the stomach of 25 red deer embryos and fetuses from 30 days of gestation until birth (235 days). Differentiation of the reticular compartment from the primitive gastric tube begins at 67 days, forming a three‐layered structure: epithelium, pluripotential blastemal tissue and serosa. The primitive reticular cells are initiated as small epithelial evaginations (primary ribs) at 117 days. At 142 days, lateral growths appear from the primary reticular ribs, forming the corneum papillae. The secondary reticular ribs form at 142 days as growths from the primary ribs. The uneven height of primary and secondary reticular ribs leads to the formation of cells of varying size. Growth of the reticular ribs involves the lamina propria but not the submucosa, so clear separation of these layers is maintained during histodifferentiation. Formation of the tunica muscularis from the pluripotential blastemal tissue begins at 67 days of intrauterine life, as two layers of longitudinally and circularly arranged myoblasts. Differentiation of the muscularis from the mucosa occurs at approximately 205 days, as longitudinal projections of the internal bundles of the tunica muscularis form the musculature of the primary ribs. The secretion of neutral and acid mucopolysaccharides by the reticular epithelial layer begins at 67 days, establishing the gradual adaptation of the mucosa to its protective function in postnatal life. Neuroendocrine (non‐neuron enolase) and glial cells (glial fibrillary acidic protein and vimentin) were detected by immunohistochemistry, in a similar localization and intensity to that reported in the rumen. The neuropeptides vasoactive intestinal peptide and neuropeptide Y showed a positive immunoreaction in the reticular epithelium from 142 days of prenatal life, again earlier than reported for the rumen. In comparison with domestic ruminants, deer were shown to be less precocious with regard to development of gastric tube, in their capacity to secrete neutral mucopolysaccharides, and in their neuroendocrine nature, as determined by the detection of positive neuroedocrine and/or glial cells.

Structure of the ovine pineal gland during prenatal development

Regodón S, Franco A, Masot J, Redondo E. Structure of the ovine pineal gland during prenatal development. J. Pineal Res. 1998; 25:229–239.

Histomorphometric and immunohistochemical study of the goat rumen during prenatal development.

This study sought to chart the ontogenesis of the goat rumen by histomorphometric examination, scanning electron microscopy and immunohistochemical analysis. A total of 140 goat embryos and fetuses were used, from the first stage of prenatal life until birth. The appearance of the rumen from the primitive gastric tube was observed at 35 days of prenatal life (CRL 3 cm, 23% gestation). By 38 days (CRL 4.3 cm CRL, 25% gestation) the ruminal wall comprised three layers: an internal epithelial layer, a middle layer of pluripotential blastemic tissue and an external layer or serosa. Ruminal pillars were visible at 46 days (CRL 6 cm, 30% gestation), and by 76 days (CRL 18 cm, 50% gestation) ruminal papillae were starting to appear. Under scanning electron microscopy, by 50 days (CRL 7.7 cm, 33% gestation) small ruminal papillae were observed protruding from the surface. Finally, neuroendocrine cells (synaptophysin, SYP) were detected at 53 days (CRL 9 cm CRL, 35% gestation), while glial cell markers (glial fibrillary acidic protein—GFAP, and vimentin‐VIM) were found at 108 days (CRL 31 cm, 72% gestation) and 39 days (CRL 4.4 cm, 26% gestation), respectively. Neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) were detected immunohistochemically at 113 days (CRL 33 cm, 75% gestation) and 120 days (CRL 35 cm, 80% gestation), respectively. In conclusion, histomorphogenesis of the rumen in goats was similar to that reported in deer, but rather slower than observed for sheep or cattle. Anat Rec, 2012.

Ontogenesis of the rumen: A comparative analysis of the Merino sheep and Iberian red deer

The aim of this study is to describe differences in the ontogenesis of the rumen in the sheep (domestic ruminant) and deer (wild ruminant). A total of 50 embryos and fetuses of Merino sheep and 50 of Iberian deer were used, from the first stages of prenatal life until birth. For the study, the animals were divided into five experimental groups according to the most relevant histological characteristics. The appearance of the rumen from the primitive gastric tube was earlier in the sheep (22% gestation, 33 days) than in the deer (25% gestation, 66 days). In both cases it displayed a primitive epithelium of a stratified, cylindrical, non-ciliary type. At around 28% gestation in the sheep (42 days) and 26% (67 days) in the deer, the rumen was configured of three clearly-differentiated layers: internal or mucosal, middle or muscular and external or serosal. In both species the stratification of the epithelial layer was accompanied by modifications in its structure with the appearance of the ruminal pillars and papillae. The pillars appeared before the papillae and the appearance of both structures was always earlier in the deer (pillars: 70 days, 27% gestation; papillae: 97 days, 36% gestation) than in the sheep (pillars: 42 days, 28% gestation; papillae: 57 days, 38% gestation). The outlines of the ruminal papillae appeared as evaginations of the basal zone toward the ruminal lumen, dragging in their formation the basal membrane, the lamina propria and the submucosa. The tegumentary mucosa of the rumen was without secretion capability in the first embryonic phases. From 67 days (26% gestation) the neutral mucopolysaccharides appeared in the deer and at 46 days (30% gestation) in the sheep. In both cases they continued to decrease until birth, this diminution being more pronounced in the deer. Finally, the presence of neuroendocrine and glial cells was detected in the deer at earlier stages than in the sheep.

A combined immunohistochemical and electron microscopic study of the second cell type in the developing sheep pineal gland

ABSTRACT: Ultrastructural and immunohistochemical techniques were used to study the second cell type in sheep embryo pineal glands. Thirty‐two embryos were studied from day 54 of development through birth. Specimens were arranged in four age groups, defined in terms of the most relevant histological features: Group 1 (54‐67 days of prenatal development), Group 2 (71‐92 days), Group 3 (98‐113 days), and Group 4 (118–150 days). At 98 days, a second cell type was observed which differed from pinealoblasts and showed uniform ultrastructural characteristics similar to those of astrocytes in the central nervous system. Ultrastructural homogeneity was not matched by the results of histochemical and immunohistochemical analysis: while all Type II cells stained positive to phosphotungstic acid hematoxylin, only 50% expressed glial fibrillary acidic protein. In the course of ovine intrauterine development, the vascular affinity of this second cell population, composed of glial‐like or astrocytic cells at varying stages of maturity, leads to the formation of a limiting pineal barrier. This barrier may constitute the morphological expression of a hypothetical functional involvement in the exchange of substances between blood and pineal parenchyma.

Prenatal development of the sheep pineal gland: An ultrastructural study

Abstract: The ultrastructure of the pineal gland of 32 sheep embryos was studied from day 54 of development through birth. Embryos were arranged in four age‐groups, defined in terms of the most relevant histological features: group 1 (54 to 67 days of prenatal development), group 2 (71 to 92 days), group 3 (98 to 113 days), and group 4 (118 to 150 days). A primary cell type, designated the pinealoblast, was observed from 54 days until birth; ultrastructurally, this cell was found to contain all the organelles required for hormone synthesis. A second cell population, classified as interstitial cells by virtue of their location among pinealoblasts, appeared at 78 days gestation and persisted until birth. Interstitial cells were scarce and exhibited tropism for the perivascular space. From 118 days gestation until birth, a third cell type, termed the pigmented cell, was visible. Pigmented cells, whose ultrastructural characteristics differed from those of pinealoblasts, contained a large number of pigment granules of varying size and shape. The pineal gland of developing sheep embryos showed considerable innervation and abundant vascularization; this, together with certain ultrastructural characteristics, suggests that the gland has a secretory function in uterine life.

Histomorphometric and immunohistochemical study of the goat reticulum during prenatal development.

This study sought to describe the morphological changes taking place in the goat reticulum during prenatal development, using histomorphometric and immunohistochemical techniques. A total of 140 goat embryos and foetuses were used, from the first stages of prenatal life until birth. Differentiation of the reticulum as a separate compartment of the primitive gastric tube was observed at 35 days of prenatal life (23% gestation). By 38 days (25% gestation) the reticular wall comprised three layers: an internal epithelial layer, a middle layer of pluripotential blastemic tissue and an external layer or serosa. Primary reticular crests were visible at 59 days (38% gestation) as evaginations of the epithelial stratum basale, marking the earliest histological differentiation of future reticular cells. Secondary reticular crests were observed at 87 days (61% gestation). Corneum papillae first became apparent on the lateral surface of primary reticular crests at 101 days (64% gestation). The muscularis mucosae was visible by 101 days (64% gestation) in primary reticular crests. Neuroendocrine cells were detected by synaptophysin at 64 days (43% gestation), while glial cell markers (glial fibrillary acidic protein and vimentin) were observed at 64 days (43% gestation) and 38 days (25% gestation), respectively. The peptidergic innervation markers such as neuropeptide Y and vasoactive intestinal polypeptide were detected at 75 days (50% gestation). In conclusion, prenatal development of the reticulum - like that of the rumen - appears to take place somewhat earlier in goats than in sheep or cattle, but at a similar rate to that reported in deer.

Ontogenesis of the Reticulum with Special Reference to Neuroendocrine and Glial Cells: A Comparative Analysis of the Merino Sheep and Iberian Red Deer

The present study was designed to compare the differences in the ontogenesis of the reticulum in sheep (domestic ruminant) and deer (wild ruminant). A total of 50 embryos and foetuses Merino sheep and 50 Iberian deer were used, from the first pre‐natal life until birth. The appearance of the reticulum from the primitive gastric tube was earlier in the sheep (22% gestation, 33 days) than in the deer (25% gestation, 66 days). In both cases, it displayed a primitive epithelium of a stratified, cylindrical, non‐ciliary type. At around 48% gestation in the sheep (72 days) and 36% (97 days) in the deer, the reticulum was configured of four clearly differentiated layers: mucosa (with epithelial layer and lamina propria), submucosa, tunica muscularis and serosa. The stratification of the epithelial layer was accompanied by modifications in its structure with the appearance of the primitive reticular ribs. The primary ribs began to be formed first in the deer, at 117 days of pre‐natal life (40% gestation) and later in the sheep (79 days, 53% gestation). The differentiation of the corneum papillae in the primary ribs coincided with the appearance of secondary reticular ribs. These structures began to be formed first in the deer, at 142 days of pre‐natal life (51% gestation) and later in the sheep (83 days, 55% gestation). The presence of neuroendocrine cells (non‐neuronal enolase‐positive cells) in the reticular mucosa was not detected until 97 days (36% gestation) in deer and 81 days (54% gestation) in sheep. The presence of glial cells (GFAP‐positive cells) occurred at around 142 days (51% gestation) in deer and at 112 days (75% gestation) in sheep. In conclusion, the presence of neuroendocrine and glial cells was detected in deer at earlier stages than sheep.

Immunohistochemical evaluation of the goat forestomach during prenatal development

Here we report the detection and distribution of synaptophysin (SPY), non-neuronal enolase (NNE), glial fibrillary acidic protein (GFAP), vimentin (VIM), neuropeptide Y (NPY), and vasoactive intestinal peptide (VIP) expression in the goat forestomach during prenatal development. A total of 140 embryos and fetuses were examined to evaluate protein expression from the first stage of prenatal life until birth. In all cases, SPY immunoreactivity was detected at 53 days gestation in the lamina propria-submucosa, tunica muscularis, serosa, and myenteric plexuses. Immunoreactivity to NNE was observed at 64 days gestation in the same locations as well as the epithelial layer. Glial cells were found at 64 days as indicated by signals corresponding to GFAP and VIM at 39 days. Positive staining for NPY and VIP was observed at 113, 75, and 95 days in the rumen, reticulum, and omasum, respectively, in the lamina propria-submucosa, tunica muscularis, and myenteric plexuses of each of these gastric compartments. These findings indicate possible preparation of the fetal goat forestomach for postnatal function. Compared to other ruminant species, neuroendocrine cells, glial cells and peptidergic innervations markers were detected earlier compared to sheep but at around the same stage as in deer.

Changes in concentrations of cortisol and melatonin in plasma, expression of synaptophysin, and ultrastructural properties of pinealocytes in goat kids in situations of stress due to early weaning: The effect of melatonin

Abstract AIM: To analyse the changes in some histophysiological parameters of the pineal gland of goat kids in situations of stress due to early weaning, and the effect of exogenous treatment with melatonin. METHODS: Twenty-four 6-day-old Verata goat kids were used; 12 suckled their dams throughout the study (non-weaned groups), and the other 12 were removed from their dams and fed a milk replacer (weaned groups). Six goat kids in each group were treated with melatonin, and the other six with doubledistilled pyrogen-free water (Day 0). On Days 28–29, blood samples were collected at 0600, 1000, 1400, 1800, 2200, 0200 and 0600 hours, to determine concentrations of cortisol and melatonin in plasma. On Days 29 and 30, six animals per group (three at 1400 and three at 0200 hours, respectively) were subject to euthanasia and the weight of their pineal glands determined. The structural immunocytochemistry, morphometric analysis, ultrastructural analysis and immunotransmission electron microscopy of the pineal glands were established. RESULTS: Concentrations of cortisol in plasma were significantly higher in weaned than in non-weaned goat kids (p<0.05), and treatment with melatonin reduced the concentrations in weaned kids (p<0.05). Concentrations of melatonin in plasma showed a similar pattern in the four groups, with peak values at 0200 and troughs at 1400 hours. Mean concentrations of melatonin in plasma in weaned goat kids were significantly lower than those in the other groups (p<0.05). In weaned goat kids not treated with melatonin, the weight and volume of the pineal gland, and number of pinealocytes, were significantly lower when compared with those from non-weaned kids (p<0.05). Quantitative ultrastructural analysis of pinealocytes showed the relative volume of mitochondria, rough endoplasmic reticulum and Golgi complex was significantly lower in weaned than nonweaned goat kids (p<0.05); treatment with melatonin significantly increased these parameters in weaned kids. CONCLUSIONS: Taken together, these results indicate that treatment with melatonin in goat kids in situations of stress due to premature weaning could play an important role in the improvement of histophysiological function of the pineal gland.