The interest in embryology, the science of the development of a zygote into a completely developed foetus, has increased greatly in recent years due to a number of studies involving embryonic and induced pluripotent stem cells. In addition, the development of techniques such as cloning has aided to understand the critical events that occur during embryonic development. In this study, we describe the morphology of two sheep embryos and one foetus using macroscopic and microscopic techniques. We investigated sheep without defined breed on days 24, 32, and 50 of gestation (estimated by crown-rump length [CR]). Macroscopically, we observed the development of E1 (24 days), with visible optic vesicle, but without retinal pigmentation and the forelimbs bud in development. In the E2 (32 days), we noticed the presence of optic retinal pigmentation and forelimbs more developed in comparison with E1. As expected, F1 revealed an eyeball already covered and the forelimbs developed. Meanwhile, microscopic analysis revealed somite, ventricle, atrium, and oral cavity in development in E1. However, in F1 we were able to identify more complex structures, such as ossification in the spine, ventricle, atrium, intraventricular septum, pericardial sac, and oral cavity with tongue. This work brings more precise and detailed data on the morphological characteristics of the major organ systems (nervous, circulatory, respiratory, digestive, and urinary) at each embryonic and foetal stage analysed.

Histomorphometric and scanning electron microscopic analyses were performed on 74 embryos and fetuses and 20 sheep (early postnatal to adult age). Histologic differentiation of the omasum took place at 33 days of fetal life, with the appearance of first-order laminae. Second-, third-, and fourth-order laminae appeared at 39, 50, and 59 days, respectively. Neutral mucopolysaccharides first appeared in epithelial cells at 46 days of fetal life, decreasing quantitatively until birth, before subsequently stabilizing in postnatal life. Acid mucopolysaccharides, mucins, and mucoid compounds were not detected. Growth curves and formulas were constructed for each tissue layer. Initial tests involved multiplicative (y = axb), exponential (y = EXP [a + bx]), linear (y = a + bx), and polynomial models [y = a + bx + cx(2) + dx(3)].

Histomorphometric and scanning electron microscopic analyses were carried out on 74 embryos and fetuses and 20 sheep (early postnatal to adult age). Histodifferentiation of the rumen took place at 33 days of fetal fife. Ruminal pillars were observed at 42 days, and at 61 days, ruminal papillae appeared as evaginations of the epithelial stratum basale. Neutral mucopolysaccharides first appeared in epithelial cells at 46 days of fetal life; thereafter, numbers decreased gradually and subsequently stabilized in postnatal life. Acid mucopolysaccharides, mucins, and mucoid compounds were not detected. Age and diet were recognized as factors that determine the structure of the ruminal mucosa. Growth curves and formulas were set out for each tissue layer.

Fetal infectivity of Ehrlichia risticii was investigated in 19 ponies that were E risticii negative on the basis of results of an indirect fluorescent antibody (IFA) test. Thirteen pregnant ponies were infected by IV administration of E risticii between 90 and 180 days of gestation. Six pregnant ponies served as noninfected controls. Each infected pony had clinical signs of equine monocytic ehrlichiosis, was confirmed to be ehrlichemic, and developed an IFA titer to E risticii. Two infected ponies became recumbent, were unresponsive to supportive care, and were euthanatized. After recovery from clinical illness, the remaining ponies were observed throughout gestation for reproductive abnormalities. On abortion, each fetus was necropsied and tissue specimens from the liver, bone marrow, spleen, colon, and mesenteric lymph nodes were inoculated into canine monocyte cell cultures. Six infected ponies aborted at a mean 217 days of gestation, which was between postinoculation days 65 and 111. Five fetuses were recovered for evaluation, and E risticii was isolated from 4 of them. All 5 fetuses recovered had similar histologic findings, including enterocolitis, periportal hepatitis, and lymphoid hyperplasia with necrosis of the mesenteric lymph nodes and spleen. All fetuses tested negative for IgG to E risticii, although 3 had low IgM titer to E risticii. The remaining 5 infected ponies had normal parturition. Presuckle IFA titer to E risticii was measured in 4 of the term foals, and results for 3 were positive. Two foals from infected ponies were monitored for 6 months and daily gain in body weight was comparable to that of a control foal. None of the control ponies became ill or seroconverted during the clinical illness phase, and none aborted throughout gestation. Two control ponies seroconverted to E risticii 6 weeks before parturition. Results of this study indicate that E risticii is a primary abortifacient under experimental conditions.

The left ovary of chicken embryos was removed and incubated in culture medium with a thymidine analogue, bromodeoxyuridine (BrdU), in vitro. In addition, fertile chicken eggs were injected with BrdU via the extraembryonic vessels and incubated for 24 hours. The ovaries were then processed for immunohistochemical localization of calbindin-D28K (a 28-kd vitamin D-dependent calcium-binding protein) and BrdU. Calbindin-D28K was detected in the germinal epithelium and in cells surrounding the oogonia and oocytes (future granulosa cells) of the embryonic chicken ovary. However, Brdu was observed in the nucleus of the oogonia and oocytes of the chicken embryonic ovaries. Comparison of the 2 adjacent sections, immunostained for calbindin-D28K and BrdU consecutively, indicated that BrdU, the marker for cell proliferation was not detected in calbindin-D28K-containing cells, namely, germinal epithelium and future granulosa cells, in the ovary of chicken embryos. These results suggested that calbindin-D28K-containing cells in the ovary were not in the process of cell division during the 24-hour incubation of chicken embryos.

Akabane virus (AKV) strain OBE-1 was inoculated IV into 17 pregnant sheep. Ten fetuses infected at 29 to 45 days of gestation and examined 29 to 30 days later had AKV antigen in the following groups of cells: neuroglial cells in the brain and spinal cord, ganglion cells in the cranial and abdominal ganglia, layer of ganglion cells in the retina, ganglion cells (Auerbach's plexus) in small intestine, hepatocytes, cells in the arterial wall of mesenteric membrane, and trophoblast cells in the placenta. Prior to detection of circulating virus-neutralizing antibody, immunoglobulin-containing cells were found initially at 59 days of gestation in the peripheral portion of white pulp tissue in the spleen. After that, numbers of immunoglobulin-containing cells gradually increased. These results indicated that AKV may have strong affinity for neuronal and ganglional cells in infected fetuses and immunoglobulin-containing cells might be considered the earliest immunologic response to AKV replication in the fetus.

Five cows in the last month of gestation, provided with uterine electrodes and in which catheters had been chronically installed in the fetal aorta, were used to study patterns of fetal heart rate (FHR) and the influence of periods of myometrial electrical activity during gestation (contractures) on FHR. The FHR was calculated by counting the number of blood pressure pulses on the tracings during alternate periods of 12 seconds. Three 1-hour recordings without contractures and 10 recordings during the time of a contracture were randomly selected for each cow. The calculated data points were plotted on a graph to display FHR patterns. In 41 periods associated with single contractures, FHR data points were taken every 72 seconds. Changes in absolute and relative FHR in these periods were determined to analyze a possible effect of contractures on FHR. Three types of variation in FHR patterns could be distinguished: a short-term, low-amplitude variation of basal FHR; a second type in which the duration was < 4 minutes and the amplitude was greater than or equal to 15 beats/min; and prolonged periods with increased or decreased FHR values (> 4 minutes and greater than or equal to 15 beats/min). The relationship between these types of variation and fetal activity states remains to be established for cows. During the 60 hours of recordings that were analyzed, a period of several minutes during which FHR values were extremely high (> 180 beats/min) was found 3 times. There were no significant differences in absolute or relative FHR before, during, or after a contracture.

Studies were undertaken to assess the chicken embryo and newly hatched chicken as models for studying the effects of bone-active agents. Initially, 1,25-dihydroxycholecaliferol (1,25[OH]2D3), sodium fluoride (NaF), parathyroid extract, epidermal growth factor, and prostaglandin E2, were tested for lethality over a broad dose range. One or 3 injections of 1,25(OH)2D3 into the yolk sac of chicken embryos resulted in death of embryos given greater than 0.1 ng/injection, whereas 0.01 ng was tolerated by the embryos. Administering 1,25(OH)2D3 intraperitoneally to newly hatched chickens as a single injection or weekly for 3 weeks resulted in no deaths at doses up to 50 ng. One or 3 IV injections of less than 400 micrograms were tolerated by the embryo. Giving chickens feed and water containing 2.4 g of NaF/kg was lethal but no deaths occurred when chickens were given feed containing less than 1.2 g of NaF/kg. Mortality associated with the administration of epidermal growth factor to embryos was inconsistent, in that death occurred in embryos given a single injection of greater than 250 ng, but no deaths occurred in embryos given 3 injections at similar doses. Parathyroid extract and prostaglandin F2 were not lethal when administered to embryos and chickens in a single-injection or multiple-injection regimen. Overall, lethality in chicken embryos given a particular agent reflected the dose of bone-active agent injected, rather than the number of injections. Three of the bone-active agents were selected to characterize their microscopic bone effects in chicken embryos and chickens. Administration of 1,25(OH)2D3 to embryos on day 14 at doses of 100, 10, 1, and 0.1 ng led to subperiosteal hyperosteoidosis in all 5 of the tibiotarsi examined from the high-dose (100 ng) group necropsied on day 18 of incubation. Three of 5 of the tibiotarsi from the 10-ng treatment group were similarly affected. Bone effects were noticed in chickens hatched from the aforementioned treatment groups or in chickens given 1,25(OH)2D3 intraperitoneally and examined at 3 and 6 weeks of age. Administration of NaF to chicken embryos on the 10, 12th, and 14th days of incubation via the IV route at doses of 160, 80, 40 and 20 micrograms/embryo led to subperiosteal hyperosteoidosis in tibiotarsi from 3 of 10 embryos (examined at 18 days of incubation) from the 2 high-dose groups. Tibiotarsi of chickens from this treatment group were microscopically normal at 3 weeks after hatching. When newly hatched chickens were given a diet containing NaF at dosages of 1.2 g/kg, 0.6 g/kg, and 0.3 g/kg, a dose-dependent increase in osteoid was seen at 3 and 6 weeks. In addition, cortical thinning and expansion of the medullary canal were observed only at 3 weeks. In contrast to the effects observed with 1,25(OH)2D3 and NaF, parathyroid extract caused no microscopic bone alterations when given to embryos or chickens. Overall, the bone alterations in the embryo were attributed to increased subperiosteal osteoid formation and defective mineralization. These findings were consistent with known effects of NaF and 1,25(OH)2D3 on bone, and they establish the chicken embryo as a sensitive model for studying bone-active agents.

Ascaris suum, swine, immune responses monitored by lymphocyte blastogenesis assays, indirect radioimmunoassays, peripheral eosinophil counts, and cutaneous delayed hypersensitivity reactions

Selenium concentration was measured in paired maternal blood samples and fetal liver specimens collected at a San Joaquin County, Calif, slaughterhouse (beef = 19, dairy = 54) and from bovine aborted fetuses submitted to the California Veterinary Diagnostic Laboratory System (CVDLS; beef = 20, dairy = 20). Of the slaughterhouse samples and specimens, dairy maternal blood selenium concentration was significantly (P < 0.001) higher (mean +/- SD; 0.22 +/- 0.056 micrograms/ml) than that for beef breeds (0.137 +/- 0.082 micrograms/ml). The CVDLS mean maternal blood selenium concentration for the dairy-breed samples (0.192 +/- 0.028 micrograms/ml) was similar to that for the slaughterhouse dairy-breed samples, but was greater than that for the slaughterhouse beef-breed samples. Slaughterhouse mean fetal liver selenium content also was higher (P < 0.001) for the dairy breeds (0.777 +/- 0.408 micrograms/g), compared with the beef breeds (0.443 +/- 0.038 micrograms/g). Mean fetal liver selenium content for slaughterhouse specimens was higher (P < 0.002) than that for the CVDLS specimens (beef, 0.244 +/- 0.149 micrograms/g; dairy, 0.390 +/- 0.165 micrograms/g). At the CVDLS, dairy fetal liver content was greater (P < 0.001) than that for beef breeds. Mean ratio of fetal liver selenium content to maternal blood selenium concentration was 3.53 +/- 1.89 for dairy breeds at the slaughterhouse (liver-to-blood correlation [r] = 0.38), and was 2.11 +/- 1.00 for dairy breeds at the CVDLS (r = 0.31) and 3.43 +/- 1.50 for beef breeds (r = 0.58). Both slaughterhouse breed ratios were significantly (P < 0.002) greater than the CVDLS dairy-breed ratio. On the basis of these results, breed and source location should be taken into account when interpreting selenium values. Fetal liver selenium content should only be used as a screening test and combined with whole blood selenium concentration from clinically normal herdmates to evaluate herd selenium status.