Detection of Chronic Wasting Disease Prions in Fetal Tissues of Free-Ranging White-Tailed Deer.

The transmission of chronic wasting disease (CWD) has largely been attributed to contact with infectious prions shed in excretions (saliva, urine, feces, blood) by direct animal-to-animal exposure or indirect contact with the environment. Less-well studied has been the role that mother-to-offspring transmission may play in the facile transmission of CWD, and whether mother-to-offspring transmission before birth may contribute to the extensive spread of CWD. We thereby focused on a population of free-ranging white-tailed deer from West Virginia, USA, in which CWD has been detected. Fetal tissues, ranging from 113 to 158 days of gestation, were harvested from the uteri of CWD+ dams in the asymptomatic phase of infection. Using serial protein misfolding amplification (sPMCA), we detected evidence of prion seeds in 7 of 14 fetuses (50%) from 7 of 9 pregnancies (78%), with the earliest detection at 113 gestational days. This is the first report of CWD detection in free ranging white-tailed deer fetal tissues. Further investigation within cervid populations across North America will help define the role and impact of mother-to-offspring vertical transmission of CWD.

Amino acids activate mTORC1 to release roe deer embryos from decelerated proliferation during diapause.

Embryonic diapause in mammals leads to a reversible developmental arrest. While completely halted in many species, European roe deer (Capreolus capreolus) embryos display a continuous deceleration of proliferation. During a 4-mo period, the cell doubling time is 2 to 3 wk. During this period, the preimplantation blastocyst reaches a diameter of 4 mm, after which it resumes a fast developmental pace to subsequently implant. The mechanisms regulating this notable deceleration and reacceleration upon developmental resumption are unclear. We propose that amino acids of maternal origin drive the embryonic developmental pace. A pronounced change in the abundance of uterine fluid mTORC1-activating amino acids coincided with an increase in embryonic mTORC1 activity prior to the resumption of development. Concurrently, genes related to the glycolytic and phosphate pentose pathway, the TCA cycle, and one carbon metabolism were up-regulated. Furthermore, the uterine luminal epithelial transcriptome indicated increased estradiol-17ß signaling, which likely regulates the endometrial secretions adapting to the embryonic needs. While mTORC1 was predicted to be inactive during diapause, the residual embryonic mTORC2 activity may indicate its involvement in maintaining the low yet continuous proliferation rate during diapause. Collectively, we emphasize the role of nutrient signaling in preimplantation embryo development. We propose selective mTORC1 inhibition via uterine catecholestrogens and let-7 as a mechanism regulating slow stem cell cycle progression.

In Vitro Development and Mitochondrial Gene Expression in Brown Brocket Deer (Mazama gouazoubira) Embryos Obtained by Interspecific Somatic Cell Nuclear Transfer.

The genetic diversity of Neotropical deer is increasingly jeopardized, owing to declining population size. Thus, the formation of cryobanking of somatic cells is important for the preservation of these species using cloning. The transformation of these cells into viable embryos has been hampered by a lack of endangered species oocytes. Accordingly, the aim of this study was to produce brown brocket deer embryos by interspecific somatic cell nuclear transfer (iSCNT), using goat or cattle oocytes as cytoplasts, and to elucidate embryo mitochondrial activity by measuring the expression levels of ATP6, COX3, and ND5. Cattle embryos produced by in vitro fertilization (IVF) were used as a control. There were no differences in the development of embryos produced by traditional SCNT and iSCNT when using either the goat cytoplasts (38.4% vs. 25.0% cleaved and 40.0% vs. 50.0% morula rates, respectively) or cattle cytoplast (72.8% vs. 65.5% cleaved and 11.3% vs. 5.9% blastocyst rates, respectively). Concerning the gene expression, no significant difference was observed when goat oocytes were used as cytoplasts. However, when using cattle oocytes and 16S as a reference gene, the iSCNT upregulated COX3, when compared with SCNT group. In contrast, when GAPDH was used as a reference gene, all the evaluated genes were upregulated in the iSCNT group, when compared with the IVF group. When compared with the SCNT group, only the expression of ATP6 was statistically different. In conclusion, it was demonstrated that interspecific nuclear transfer is a potentially useful tool for conservation programs of endangered similar deer species.

Prenatal histomorphological development of the reticulum in fallow deer (Dama dama).

The histomorphological changes occurring in the Dama dama reticulum during prenatal development have been investigated. Twenty-five Dama dama embryos were used, from the first stages of prenatal life until birth. Differentiation of the reticulum was observed at 23% gestation. By 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 38% gestation. Secondary reticular crests were observed at 61% gestation. Neuroendocrine cells were detected by synaptophysin (SYP) at 35% gestation, in the lamina propria-submucosa, tunica muscularis, and serosa. Epithelial Cytokeratin-18 (CK-18) cells were observed at 35% gestation extended throughout the epithelial layers. The glial cells (vimentin -VIM- and glial fibrillary acidic protein-GFAP-markers) were discerned at 25% and 43% gestation, respectively, in myenteric and submucosal plexuses, lamina propria, muscularis mucosae, tunica muscularis, and perivascular connective tissue. The neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) markers were immunodetected at 75% and 80 gestation, respectively, in the lamina propria-submucosa, muscularis mucosae, tunica muscularis, serosa, and myenteric plexuses. The prenatal development of the fallow deer reticular mucosa evidenced a considerable precocity similar to that previously reported in goat and red deer.

Oxygen tension during in vitro oocyte maturation and fertilization affects embryo quality in sheep and deer.

Incubation gas atmosphere affects the development of in vitro produced embryos. In this study, there was examination of effects of two different oxygen (O2) tensions (5 % and 21 %) during in vitro maturation (M5 and M21) and/or fertilization (F5 and F21) on embryo production and quality in deer and sheep. There was assessment of the percentage of embryos with cell cleavage occurring, percentage that developed to the blastocyst stage, and analysis of the relative abundance of mRNA transcript for genes important for development to the blastocyst stage. The O2 tension treatment did not affect (P > 0.05) percentage cleavage or blastocyst development in either species. In sheep, there was a greater abundance of SHC1, GPX1, TP53, BAX and NRF1 mRNA transcript (P < 0.05) in M21 F5-derived embryos. In deer, there was a greater abundance of SOD2 mRNA transcript (P < 0.05) when oocytes had been matured under relatively lesser O2, regardless of the tension used during fertilization. There was a lesser abundance of SOX2 mRNA transcript (P < 0.05) in the M5F21 compared to the other three treatment groups. The AKR1B1 mRNA transcript was in greater abundance (P < 0.05) in M21 F21 as compared to M21 F5 and M5F21 group, and there was a greater abundance PLAC8 mRNA transcript (P < 0.05) in M21 F21, as compared to all other treatment groups. In conclusion, while O2 tension had no effect on developmental rates it did affect the relative abundance of mRNA transcript of multiple genes related to important cell functions during development.

Influence of foetal calf serum supplementation during in vitro embryo culture in Iberian red deer.

Nowadays, the use of foetal calf serum (FCS) during in vitro embryo culture is very controversial. Whilst some authors have encouraged its use, others reject it because of its harmful effects. Although in vitro embryo production in red deer is a promising assisted reproductive technique, it is still in its infancy and a great effort is needed to update the protocols used. The aim of this study was to assess whether FCS supplementation in red deer embryo culture medium is necessary to produce blastocyst and, if so, when is the best time to add it in terms of blastocyst production and quality. In vitro blastocysts were cultured with FCS added at 24, 48 or 96 hours post-insemination (hpi). In addition, a treatment without FCS was used as control. Six hundred and ninety-four cumulus-oocyte complexes were collected for in vitro fertilization. Cleavage rate was examined at 48 hpi, and blastocyst yield was recorded on days 6, 7 and 8. FCS had no influence on cleavage and blastocyst rate for any of the treatments studied. However, the number of cells was higher (p = .025) in those blastocysts cultured with FCS from 48 hpi compared with FCS-free culture media (93.88 ± 7.76 vs. 54.11 ± 8.36). In conclusion, the addition of FCS to the embryo culture medium at 48 hpi improves the quality of red deer blastocyst, although it does not affect the percentage of embryos obtained.

Embryonic and fetal development of the red brocket deer (Mazama americana).

The red brocket deer (Mazama americana), a medium-sized Neotropical ungulate, is one of the most hunted mammals in the Amazon. This study analyzes the intrauterine development in the red brocket deer through the description of the external and internal morphology in one embryo and 38 fetuses collected from animals hunted for subsistence in the Amazon. The chronological order of occurrence of external characteristics in relation to the total dorsal length (TDL) was: differentiated genitalia, limbs and eyelid buds (TDL = 3.9 cm), fusioned eyelids, outer ear and hooves (TDL ≥ 9.5 cm), skin (TDL ≥ 20.4 cm), tactile pelage and nasal pigmentation (TDL ≥ 21.5 cm), covering pelage and skin spots (TDL ≥ 31.3 cm), and teeth eruption and opened eyelids (TDL ≥ 34.2 cm). The formula of fetal age was ∛W = 0.072 (t - 42), with a high linear relationship between TDL and gestational age. Multiple linear and non-linear regressions showed strong positive associations between biometric measures and absolute visceral weights with TDL. The relative weight of the tubular gastrointestinal organs, spleen and thymus increased during the fetal development; in contrast, the liver and kidneys' relative weight diminished during the fetal development. Advanced fetuses (≥44.0 cm TDL) had lower proportion of liver and larger tubular gastrointestinal organs within the visceral weight than adults. The chronology of appearance of the main events of the fetal development suggests that the red brocket deer adopt some precocial features, such as the early development of the sensorial function, including the early development of eyelids, outer ear and tactile pelage, the early development of the covering pelage which acts in thermoregulation and the early teeth eruption which allows the early foraging. Nevertheless, the precocial level of the red brocket deer is apparently lower than other species more frequently predated by large felids, such as peccaries and the paca.

Prenatal histomorphological development of the rumen in Dama dama.

This work studies the morphological changes taking place in the Dama dama rumen during prenatal development using histomorphometrics, surface microstructure and immunohistochemistry analysis as well as carrying out a comparative analysis of this species with other wild (red deer) and domestic-type ruminants. A total of 25 fallow deer 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 51 days of prenatal life (CRL 3 cm, 21% gestation). By 57 days (CRL 4.3 cm, 24% 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 72 days (CRL 6 cm, 30% gestation), and by 85 days (CRL 7.2 cm, 35% gestation) ruminal papillae were starting to appear. Under scanning electron microscopy, by 80 days (CRL 7 cm, 33% gestation) small ruminal papillae were observed protruding from the surface. Morphometric results showed accelerated growth of the epithelial layer and the tunica muscularis at 180 days (75% gestation). By contrast, the growth-rate of the lamina propria and submucosa declined from the early embryonic stages until birth. The serosa maintained a steady rate of growth until birth. Neuroendocrine cells (synaptophysin) were detected at 85 days (CRL 7.2 cm CRL, 35% gestation), while glial cell markers (glial fibrillary acidic protein and vimentin) were found at 108 days (CRL 31 cm, 45% gestation) and 63 days (CRL 4.4 cm, 26% gestation) respectively. Neuropeptide Y and vasoactive intestinal polypeptide were detected immunohistochemically at 180 days (CRL 33 cm, 75% gestation) and 192 days (CRL 35 cm, 80% gestation) respectively. In comparison to other wild and domestic-type ruminants, histomorphogenesis of the rumen in Dama dama was similar to that reported in red deer and goats, but rather slower than that observed for sheep or cattle.

Description of post-implantation embryonic stages in European roe deer (Capreolus capreolus) after embryonic diapause.

The embryonic stage of development is defined as the period between fertilization and the establishment of most of the organ systems by the end of this period. Development in this stage is rapid. In many mammalian species, particularly in humans, the interval between fertilization and implantation is exactly determined and continuous without intermission. However, European roe deer (Capreolus capreolus) embryos undergo a reversible retardation of development. This interesting reproduction strategy is called embryonic diapause (delayed implantation). After this period of embryonic arrest, development continues without further interruption. The aim of this study was to investigate embryonic development after diapause in European roe deer. Because of the embryonic diapause and the unknown date of fertilization, it was impossible to assign the embryos to a certain gestational age (days). This study describes normal stages of embryonic development mainly based on the external morphological traits of 56 well-preserved post-implantation roe deer embryos and attempts to assign the embryos to certain development stages. Carnegie stages of human embryos were used as an orientation for staging roe deer embryos. We observed a considerable range of variation of embryonic stages investigated until the end of January. We found post-implantation stages of embryonic development already at the end of December and foetuses at the end of January. Moreover, assigning the embryos to a particular stage of development allows the comparison between pairs of twins and triplets. We showed that twins and triplets were always at the same development level, despite the discrepancy in inter-twin and inter-triplet size.

Comparative analysis of the merino sheep and Iberian red deer abomasum during prenatal development.

The aim of this study is to describe differences in the ontogenesis of the abomasum in sheep (domestic ruminant) and deer (wild ruminant). Histomorphometric and immunohistochemical analysis were carried out on 50 embryos and fetuses of the sheep and 50 red deer from the first prenatal stages until birth. To compare similar periods of gestation in both species, we calculate the percentages of gestation. The appearance of the abomasum was earlier in the red deer (22% gestation) than in the sheep (25% gestation). Throughout development the epithelium happened sequentially, being of the types pseudostratified to simple cylindrical. This important modification was earlier in the red deer than the sheep. At 46% gestation in red deer and 50% in sheep, gastric pits were observed on the surface of abomasal folds. Our studies suggest a close link between the initial formation of these pseudoglandular structures and the clear separation of lamina propria and submucosa separated by de muscularis mucosae. At 54% gestation in red deer and at 60% in sheep, in the bottom of these pits the first outlines of glands were distinguishable. Finally, the presence of neuroendocrine and glial cells were detected in deer at earlier stages than in sheep.

The impact of ovarian stimulation protocol on oocyte quality, subsequent in vitro embryo development, and pregnancy after transfer to recipients in Eld's deer (Rucervus eldii thamin).

Propagating genetically valuable individuals through oocyte collection, in vitro fertilization (IVF) and embryo transfer is critical to maintain sustainable populations of the endangered Eld's deer. The objectives of this study were to assess the impact of exogenous FSH injections on (1) the number and in vitro competence of oocytes collected and (2) the developmental potential of resulting IVF embryos after transfer into recipients during the breeding season (February-April). In a pilot experiment, estrus synchronization was conducted in three surplus females (using intravaginal progesterone-releasing devices, CIDRG for 14 days and injections of buserelin (a GnRH agonist). Five days after CIDR removal, ovaries were excised, minced and a total of 133 oocytes were recovered. Following in vitro maturation (IVM) and IVF, 63% of the oocytes formed embryos but only 5% reached the blastocyst stage. In a subsequent study, follicle numbers and diameters were compared between synchronized does stimulated with 0 or 80 mg FSH (-FSH and +FSH; n = 8 does in each group) and oocytes collected either by laparoscopic ovum pick-up or ovariectomy. FSH stimulation increased the main follicular diameter from 2-3 mm to 4-5 mm (P < 0.05) but not the oocyte number (∼20/donor) or the percentage of good quality oocytes (57%) regardless of the treatment. FSH stimulation did not either affect the percentage of cleaved embryos after IVF (25-35%; P > 0.05). Lastly, embryos at the 2-to 8-cell stage (from either + FSH or -FSH groups) were transferred into the oviducts of 11 synchronized recipients. With the +FSH embryos, three pregnancies failed between 90 and 120 days of gestation and two fawns that were born preterm (Days 215 and 224 of gestation) died at birth. In the -FSH group one healthy female fawn was born on Day 234 of gestation. This is the first report of a successful in vitro embryo production and subsequent birth of a live Eld's deer fawn. Further investigations are required to improve IVM/IVF success and the developmental potential of the embryos.

Congenital Filariasis Caused by Setaria bidentata (Nematoda: Filarioidea) in the Red Brocket Deer (Mazama americana).

The filarial nematode Setaria bidentata was found in 10 of 31 fetuses of the red brocket deer ( Mazama americana ) from the Loreto region of the Peruvian Amazon. A total of 25 specimens were collected and morphologically identified as S. bidentata. Filarial nematodes were found in the peritoneal cavity of 9 deer fetuses and the thoracic cavity of 1 fetus. Most specimens were adult stage. In this report, we provide morphometric data for these filarial specimens. This is the first study to demonstrate prenatal S. bidentata infection in cervid fetuses. Also, the finding of S. bidentata in Peru expands the geographic range of this parasite.

PCR-fingerprint profiles of mitochondrial and genomic DNA extracted from Fetus cervi using different extraction methods.

The use of Fetus cervi, which is derived from the embryo and placenta of Cervus Nippon Temminck or Cervs elaphus Linnaeus, has been documented for a long time in China. There are abundant species of deer worldwide. Those recorded by China Pharmacopeia (2010 edition) from all the species were either authentic or adulterants/counterfeits. Identification of their origins or authenticity became a key in the preparation of the authentic products. The traditional SDS alkaline lysis and salt-outing methods were modified to extract mt DNA and genomic DNA from fresh and dry Fetus cervi in addition to Fetus from false animals, respectively. A set of primers were designed by bioinformatics to target the intra-and inter-variation. The mt DNA and genomic DNA extracted from Fetus cervi using the two methods meet the requirement for authenticity. Extraction of mt DNA by SDS alkaline lysis is more practical and accurate than extraction of genomic DNA by salt-outing method. There were differences in length and number of segments amplified by PCR between mt DNA from authentic Fetus cervi and false animals Fetus. The distinctive PCR-fingerprint patterns can distinguish the Fetus cervi from adulterants and counterfeit animal Fetus.

[Morphometry in Development of Red Deer's Adrenal Glands].

Histological structures and morphometric and some histochemical indicators of elk's adrenal gland development as subspecies of red deer in prenatal and postnatal ontogenies stages was studied. It was found that the growth of the fetus adrenal glands weight and the thickness of the structures adrenal glands fragments continue throughout the prenatal period of ontogeny. The cells of androgenic zone with single wandering sympathogoniae are differentiated in the adrenal glands in the second month of development. The androgenic and definite zone and the adrenal medulla are differentiated by the third month of development. At the 4 months, adrenal gland cortex zona glomerulosa and zona fasciculate-reticularis are differentiated; zona reticularis is differentiated only by the seventh month. By the eighth month, the structure of adrenal glands corresponds to the adrenal glands of a newborn. Full structural formation of the adrenal glands takes place in young animals by age 1.5. Obvious structural changes were not found late in the postnatal stages of development.

In utero transmission and tissue distribution of chronic wasting disease-associated prions in free-ranging Rocky Mountain elk.

The presence of disease-associated prions in tissues and bodily fluids of chronic wasting disease (CWD)-infected cervids has received much investigation, yet little is known about mother-to-offspring transmission of CWD. Our previous work demonstrated that mother-to-offspring transmission is efficient in an experimental setting. To address the question of relevance in a naturally exposed free-ranging population, we assessed maternal and fetal tissues derived from 19 elk dam-calf pairs collected from free-ranging Rocky Mountain elk from north-central Colorado, a known CWD endemic region. Conventional immunohistochemistry identified three of 19 CWD-positive dams, whereas a more sensitive assay [serial protein misfolding cyclic amplification (sPMCA)] detected CWD prion seeding activity (PrPCWD) in 15 of 19 dams. PrPCWD distribution in tissues was widespread, and included the central nervous system (CNS), lymphoreticular system, and reproductive, secretory, excretory and adipose tissues. Interestingly, five of 15 sPMCA-positive dams showed no evidence of PrPCWD in either CNS or lymphoreticular system, sites typically assessed in diagnosing CWD. Analysis of fetal tissues harvested from the 15 sPMCA-positive dams revealed PrPCWD in 80 % of fetuses (12 of 15), regardless of gestational stage. These findings demonstrated that PrPCWD is more abundant in peripheral tissues of CWD-exposed elk than current diagnostic methods suggest, and that transmission of prions from mother to offspring may contribute to the efficient transmission of CWD in naturally exposed cervid populations.

The construction of cloned Sika deer embryos (Cervus nippon hortulorum) by demecolcine auxiliary enucleation.

The objective of our study was to establish the feasibility of experimental protocols for cloning sika deer. We performed auxiliary enucleation to improve the efficiency of nuclear transfer operation by optimizing the demecolcine concentration to induce cytoplasmic protrusions in the sika deer oocytes. In the present study,we had studied the impact of different demecolcine concentrations on cytoplasmic protrusions and enucleation rates. We determined that 95.9% of the sika deer oocytes formed cytoplasmic protrusions when treated for 1 h with 0.8 µg/ml demecolcine. The lowest observed rate of protrusion was 19.3% after overnight treatment with demecolcine. When the oocytes aged or had a poor cumulus expansion, they exhibited a significant decrease in the ability to form cytoplasmic protrusions. The rates of enucleation (94.9% vs 85.8%, p < 0.05), cell fusion (84.6% vs 70.1%, p < 0.05) and blastocyst formation (15.4% vs 10.9%, p < 0.05) using demecolcine auxiliary enucleation were significantly higher than those after blind enucleation. These results demonstrated that sika deer oocytes could be enucleated quickly and effectively using demecolcine auxiliary enucleation, which could enhance the enucleation rate, cell fusion rate and blastocyst rate of cloned embryos in vitro.

The entotympanic in late fetal Artiodactyla (Mammalia).

The entotympanic is a neomorphic component of the bulla tympanica of placental mammals. Ontogenetically, its rostral component seems to be derived from the tubal cartilage, whereas its caudal component is normally connected with the sheath of the tympanohyal; the present study indicates additional sources of the caudal entotympanic. The entotympanics develop in late fetal or early postnatal life as cartilaginous structures, but in most taxa they ossifiy endochondrally as "os bullae". This skeletal element is absent only in a few placental orders, among them the Artiodactyla. Because it is present in their sister taxa within the Scrotifera, it is likely to be reduced secondarily in the even-toed mammals. The study of histological serial sections of late fetal stages of several artiodactyl species shows that vestigial cartilaginous homologues of the entotympanics are invariably present, contrary to statements in the literature. In a few perinatal stages even secondary ossifications or calcifications of the entotympanic cartilages can be observed. The tubal cartilage of artiodactyls also continues into an anterior tegmen tympani (new term) that forms the floor of the fossa muscularis major.

Development of the permanent mandibular cheek teeth in fallow deer (Dama dama).

The study describes crown and root formation of the permanent mandibular cheek teeth of fallow deer from a gestational age of 22-23 weeks up to a post-natal age of 33 months. Tooth development was recorded using a scoring scheme based on morphological criteria ranging from crypt formation to completion of root growth. The morphological appearance of the enamel surface during three different stages (secretory-stage enamel, maturation-stage enamel and mature enamel) was described, and the approximate age at termination of the secretory stage of amelogenesis in the deciduous and permanent mandibular cheek teeth was determined. The data enable an age estimation of fallow deer up to 3 years of age and provide a basis for assessing the timing of stress episodes that affect tooth crown formation. This information is useful for the management of the species as well as in bioarchaeological and bioindication studies.

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.

Molecular cloning and gene expression analysis of Ercc6l in Sika deer (Cervus nippon hortulorum).

BACKGROUND: One important protein family that functions in nucleotide excision repair (NER) factors is the SNF2 family. A newly identified mouse ERCC6-like gene, Ercc6l (excision repair cross-complementing rodent repair deficiency, complementation group 6-like), has been shown to be another developmentally related member of the SNF2 family. METHODOLOGY/PRINCIPAL FINDINGS: In this study, Sika deer Ercc6l cDNA was first cloned and then sequenced. The full-length cDNA of the Sika deer Ercc6l gene is 4197 bp and contains a 3732 bp open reading frame that encodes a putative protein of 1243 amino acids. The similarity of Sika deer Ercc6l to Bos taurus Ercc6l is 94.05% at the amino acid sequence level. The similarity, however, is reduced to 68.42-82.21% when compared to Ercc6l orthologs in other mammals and to less than 50% compared to orthologs in Gallus gallus and Xenopus. Additionally, the expression of Ercc6l mRNA was investigated in the organs of fetal and adult Sika deer (FSD and ASD, respectively) by quantitative RT-PCR. The common expression level of Ercc6l mRNA in the heart, liver, spleen, lung, kidney, and stomach from six different developmental stages of 18 Sika deer were examined, though the expression levels in each organ varied among individual Sika deer. During development, there was a slight trend toward decreased Ercc61 mRNA expression. The highest Ercc6l expression levels were seen at 3 months old in every organ and showed the highest level of detection in the spleen of FSD. The lowest Ercc6l expression levels were seen at 3 years old. CONCLUSIONS/SIGNIFICANCE: We are the first to successfully clone Sika deer Ercc6l mRNA. Ercc6l transcript is present in almost every organ. During Sika deer development, there is a slight trend toward decreased Ercc61 mRNA expression. It is possible that Ercc6l has other roles in embryonic development and in maintaining the growth of animals.