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Background: Türkiye is a country in the world ranking in terms of sheep breeding. Hamdani crossbred sheep breed is one of the sheep breeds that can adapt to the difficult conditions in our country. In addition, the sentence may be corrected as 'Especially in the southeastern part of the Türkiye, crossbreeding is preferred by breeders to increase the yield characteristics of sheep'., Objectives: In our study, it was aimed to perform a pelvimetric analysis of Hamdani crossbred sheep fetuses in the second and third trimesters by three-dimensional modelling method., Methods: For this purpose, a total of 40 second-trimester (10 females, 10 males) and third-trimester (10 females, 10 males) fetuses were used. The pelvises of the fetuses were imaged with CT and 3D models were created. Sixteen pelvimetric measurements were performed from the models. The results obtained were evaluated statistically., Results: The study found statistically significant differences between male and female fetuses in terms of vertical diameter (VD) and foramen obturatum width (FOW) measurement parameters in the second trimester. In the third trimester, there were statistically significant differences between genders in acetabulum (AC) (p < 0.01), medial ischial tuberosities (MIT), and ischiatic arch (IA) (p < 0.05) parameters. As a result of the correlation analysis, it was determined that the pelvis length (PL) measurement parameter was positively correlated with different parameters in varying degrees in both periods, while the IA parameter did not show significant correlations with the other parameter in the last two periods of pregnancy., Conclusion: In conclusion, the data obtained will be useful in pelvimetric evaluations of human and animal fetuses, anatomy education, zooarchaeology and taxonomy studies., (© 2024 The Author(s). Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

The present study was conducted to decipher the proteome of in vivo-produced pre-implantation ovine embryos. Ten locally adapted Morana Nova ewes received hormonal treatment and were inseminated 12 hr after ovulation. Six days later, 54 embryos (morula and blastocyst developmental state) were recovered from eight ewes and pooled to obtain sufficient protein for proteomic analysis. Extracted embryo proteins were analysed by LC-MS/MS, followed by identification based on four database searches (PEAKS, Proteome Discoverer software, SearchGUI software, PepExplorer). Identified proteins were analysed for gene ontology terms, protein clusters and interactions. Genes associated with the ovine embryo proteome were screened for miRNA targets using data sets of TargetScan (http://www.targetscan.org) and mIRBase (http://www.mirbase.org) servers. There were 667 proteins identified in the ovine embryos. Biological processes of such proteins were mainly related to cellular process and regulation, and molecular functions, to binding and catalytic activity. Analysis of the embryo proteins revealed 49 enriched functional clusters, linked to energy metabolism (TCA cycle, pyruvate and glycolysis metabolism), zona pellucida (ZP), MAPK signalling pathway, tight junction, binding of sperm to ZP, translation, proteasome, cell cycle and calcium/phospholipid binding. Sixteen miRNAs were related to 25 pre-implantation ovine embryo genes, all conserved in human, bovine and ovine species. The interaction network generated by miRNet showed four key miRNAs (hsa-mir-106b-5p; hsa-mir-30-5p; hsa-mir-103a-5p and hsa-mir-106a-5p) with potential interactions with embryo-expressed genes. Functional analysis of the network indicated that miRNAs modulate genes related to cell cycle, regulation of stem cell and embryonic cell differentiation, among others. Retrieved miRNAs also modulate the expression of genes involved in cell signalling pathways, such as MAPK, Wnt, TGF-beta, p53 and Toll-like receptor. The current study describes the first major proteomic profile of 6-day-old ovine embryos produced in vivo, setting a comprehensive foundation for our understanding of embryo physiology in the ovine species., (© 2021 Wiley-VCH GmbH.)

Microinjection of exogenous DNA into the cytoplasm of matured oocytes or zygotes is a promising technique to generate transgenic animals. However, the data about the microinjection time and procedure in sheep are limited and have not treated in detail. To obtain more in-depth information, the Sarda sheep oocytes from abattoir-derived ovaries were subjected to IVM and IVF. Then, the GFP plasmid as a reporter gene was injected into the cytoplasm of MII oocytes (n: 95) and zygotes at different post-insemination intervals (6-8 hpi, n: 120; 8-10 hpi, n: 122; 10-12 hpi, n: 110 and 12-14 hpi, n: 96). There were no significant differences in the cleavage rates between the groups. However, blastocyst rate of injected zygotes at all-time intervals was significantly lower than injected MII oocytes and control group (p < 0.05). Interestingly, the proportion of GFP-positive embryos was higher at 8-10 hpi compared with other injected groups (4 % versus 0 %, p  < 0.01). Among these, the proportion of mosaic embryos was high and two of those embryos developed to the blastocyst stage. In conclusion, we settled on the cytoplasmic microinjection of GFP plasmid at 8-10 hpi as an optimized time point for the production of transgenic sheep and subsequent experiments., (© 2020 Wiley-VCH GmbH.)

Primiparous ewes and multiparous ewes show physiological differences during pregnancy, which can have an impact on the development of their offspring. The objective of this study was to compare the changes in the metabolic profile and in the size of some foetal bones throughout gestation between primiparous and multiparous ewes. Twelve primiparous (PM) ewes and 14 multiparous (MT) ewes were used. According to the dates of lambing, two groups of ewes were formed: Group 1 (G1, n = 6 PM and n = 7 MT) and Group 2 (G2, n = 6 PM and n = 7 MT). The body weight, body condition score, metabolic and foetal morphometric parameters were determined from before conception until the end of gestation. After lambing, the body weight and survival rate during the first 72 hr of life of lambs, as well as the maternal behaviour score were recorded. The PM ewes were lighter (p < .01) and had a greater mobilization of body reserves during gestation, mainly evidenced by a greater serum concentration of NEFAs and lower serum concentration of total proteins (p < .05) compared with the MT ewes. The parity did not affect the foetal morphometric variables. The lambs of MT ewes were heavier at parturition (p = .002) and tended to have a greater survival rate than those lambs of PM ewes (p = .09). In conclusion, PM ewes and MT ewes differ in their metabolic profile throughout the gestation. However, in the present study, we did not find parity differences in the dimensions of foetal bones during growth in gestation., (© 2020 Blackwell Verlag GmbH.)

This study aimed to determine the optimum concentration of trehalose in solutions used for vitrification of in vitro matured (IVM) ovine oocytes. IVM oocytes were randomly divided into four experimental (vitrified) and one control (fresh) groups. Experimental groups were treated with different concentrations (0.0, 0.25, 0.5 and 1.0 M) of trehalose. After warming, some viable oocytes were exposed to 0.25% pronase to test zona pellucida hardening, whereas the others were fertilized and cultured in vitro for 8 days to evaluate their developmental competence. Blastocysts quality was assessed by differential staining and TUNEL test. Survival and developmental rates of oocytes vitrified in the presence of 0.5 M trehalose were significantly higher than those of the other vitrified groups. Furthermore, there was a significant difference between fresh and vitrified groups in total blastocyst rate. Analysis of blastocysts quality also revealed a significant difference between the group treated with 0.5 M trehalose and other groups in terms of apoptotic index. Furthermore,zona pellucida digestion time period was longer in trehalose-free (0.0 M) group compared to other groups. In conclusion, we found that IVM ovine oocytes vitrified in solutions containing 0.5 M trehalose are fertilization-competent and are able to produce good-quality blastocysts with an apoptotic index comparable to that of the fresh oocytes. Therefore, 0.5 M may be considered the optimum concentration of trehalose to be used in solutions prepared for vitrification of oocytes., (© 2018 Blackwell Verlag GmbH.)

Background: MicroRNAs (miRNAs) have been identified as important contributors to the regulation of early fetal cardiopulmonary development. However, miRNA expression profiles during late gestation and the early neonatal period are not fully elaborated in large mammals such as sheep (ovis aries). The aim of this study was to sequence miRNA from cardiopulmonary tissues in late gestation and neonate sheep to identify changes in miRNA expression., Methods: Illumina HiSeq next-generation deep sequencing (NGS) was performed on ovine tissues from the left (LV) and right ventricles (RV), lungs and pulmonary artery (PA) of preterm fetuses (128 days), near-term fetuses (140 days) (term = 148 days) and neonatal lambs (5 days). NGS reads were mapped to the sheep genome (OviAri) and published miRNA sequences., Results: Of 1345 cardiopulmonary miRNAs that were sequenced, relatively few major shifts in miRNA expression were detected with increased age from near term to neonates, and were confirmed by quantitative real-time PCR: bta-miR-146a (lung), bta-miR-22-3p (lung, LV), hsa-miR-335* (lung, PA), and miR-210 (lung, PA, LV)., Conclusions: Sequencing of miRNA led to identification of four predominant miRNA in ovine cardiopulmonary tissues which alter expression during late gestation and the early neonatal period, concurrent with important functional changes in heart and lungs., Competing Interests: The authors have declared that no competing interests exist.

The aim of this study was to examine foetal organs and placental tissue to establish a correlation between the changes in the composition of these structures associated with their maturation and the ultrasonographic characteristics of the images. Twenty-four pregnant ewes were included in the study. Ultrasonography assessments were performed in B-mode, from the ninth gestational week until parturition. The lungs, liver and kidneys of foetuses and placentomes were located in transverse and longitudinal sections to evaluate the echogenicity (hypoechoic, isoechoic, hyperechoic or mixed) and echotexture (homogeneous and heterogeneous) of the tissues of interest. For quantitative evaluation of the ultrasonographic characteristics, it was performed a computerized image analysis using a commercial software (Image ProPlus ® ). Mean numerical pixel values (NPVs), pixel heterogeneity (standard deviation of NPVs) and minimum and maximum pixel values were measured by selecting five circular regions of interest in each assessed tissue. All evaluated tissues presented significant variations in the NPVs, except for the liver. Pulmonary NPVmean, NPVmin and NPVmax decreased gradually through gestational weeks. The renal parameters gradually decreased with the advancement of the gestational weeks until the 17th week and later stabilized. The placentome NPVmean, NPVmin and NPVmax decreased gradually over the course of weeks. The hepatic tissue did not show echogenicity and echotexture variations and presented medium echogenicity and homogeneous echotexture throughout the experimental period. It was concluded that pixels numerical evaluation of maternal-foetal tissues was applicable and allowed the identification of quantitative ultrasonographic characteristics showing changes in echogenicity related to gestational age., (© 2018 Blackwell Verlag GmbH.)

BACKGROUNDWhite matter brain injury in preterm infants can induce neurodevelopmental deficits. Umbilical cord blood (UCB) cells demonstrate neuroprotective properties, but it is unknown whether cells obtained from preterm cord blood (PCB) vs. term cord blood (TCB) have similar efficacy. This study compared the ability of TCB vs. PCB cells to reduce white matter injury in preterm fetal sheep.METHODSHypoxia-ischemia (HI) was induced in fetal sheep (0.7 gestation) by 25 min umbilical cord occlusion. Allogeneic UCB cells from term or preterm sheep, or saline, were administered to the fetus at 12 h after HI. The fetal brain was collected at 10-day post HI for assessment of white matter neuropathology.RESULTSHI (n=7) induced cell death and microglial activation and reduced total oligodendrocytes and CNPase+myelin protein in the periventricular white matter and internal capsule when compared with control (n=10). Administration of TCB or PCB cells normalized white matter density and reduced cell death and microgliosis (P<0.05). PCB prevented upregulation of plasma tumor necrosis factor (TNF)-a, whereas TCB increased anti-inflammatory interleukin (IL)-10 (P<0.05). TCB, but not PCB, reduced circulating oxidative stress.CONCLUSIONSTCB and PCB cells reduced preterm HI-induced white matter injury, primarily via anti-inflammatory actions. The secondary mechanisms of neuroprotection appear different following TCB vs. PCB administration.

Background: Cathepsin B is a lysosomal cysteine protease involved in apoptosis and oocytes which have lower developmental competence show higher expression of Cathepsin B. Furthermore, expression of Cathepsin B show a decreasing trend from oocyte toward blastocyst stage., Results: Present study assessed the effect of cathepsin B inhibitor, E-64, on developmental competency and cryo-survival of pre-implantation ovine IVF derived embryos. Cathepsin B inhibitor was added during day 3 to 8 of development. One μM E-64 was defined as the optimal concentration required for improving blastocyst rate. This concentration also reduced DNA fragmentation and BAX as apoptotic markers while increasing total cell number per blastocyst and improving anti-apoptotic marker, the BCL2. We further showed that addition of 1.0 μM of E-64 during day 3 to 8 of development improved re-expansion and hatching rates of blastocysts post vitrification. E-64 also reduced rate of DNA fragmentation and BAX expression and increased total cell number per blastocyst and BCL2 expression post vitrification. However, addition of E-64 post vitrification reduced the hatching rate., Conclusion: Therefore, it can be concluded that inhibition of cathepsin B in IVC, not only improves quality and quantity of blastocysts but also improves the cryo-survival of in vitro derived blastocysts.

Previous studies in experimental animals have shown that maternal exposure to bisphenol A (BPA) during late pregnancy leads to high plasma concentrations of BPA glucuronide (BPAG) in fetus compared to mother due to the inability of BPAG to cross the placental barrier. A recent in vitro study has reported that BPAG can exert adipogenic effect underlining the need for characterization of the fetal disposition of BPAG. Experiments were conducted in chronically catheterized fetal sheep to determine the contribution of BPAG hydrolysis to BPA to the elimination of BPAG from the fetal compartment and its resulting effect on the overall fetal exposure to free BPA. Serial sampling of fetal arterial blood, amniotic fluid, maternal venous blood and urine was performed following separate single doses of BPA and BPAG administered intravenously to eight fetal/maternal pairs after cesarean section, and repeated BPAG doses given to two fetal sheep. On average 67% of the BPA entering the fetal circulation was rapidly eliminated through fetal to maternal clearance, with a very short half-life (20 min), while the remaining fraction (24%) was glucuronoconjugated. BPA conjugation-deconjugation cycling was responsible for a 43% increase of the overall fetal exposure to free BPA. A very specific pattern of fetal exposure to free BPA was observed due to its highly increased persistence with a hydrolysis-dependent plasma terminal free BPA half-life of several tens of hours. These findings suggest that although the high fetal to maternal clearance of free BPA protects the fetus from transient increases in free BPA plasma concentrations associated with maternal BPA intake, low but sustained basal free BPA concentrations are maintained in the fetus through BPA conjugation-deconjugation cycling. The potential health implications of these low but sustained basal concentrations of free BPA in fetal plasma should be addressed especially when considering time-dependent effects., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Septa from sheep hearts at 130 days gestation, term, and 14-day-old lambs were used to model the changes in gene expression patterns during the perinatal period using Agilent 15k ovine microarrays. We used Bioconductor for R to model five major patterns of coexpressed genes. Gene ontology and transcription factor analyses using Webgestalt modeled the biological significances and transcription factors of the gene expression patterns. Modeling indicated a decreased expression of genes associated with anatomical development and differentiation during this period, whereas those associated with increased protein synthesis and growth associated with maturation of the endoplasmic reticulum rose to term but did not further increase from the near term expression. Expression of genes associated with cell responsiveness, for example, immune responses, decreased at term but expression returned by postnatal day 14. Changes in genes related to metabolism showed differential substrate-associated patterns: those related to carbohydrate metabolism rose to term and remained stable thereafter, whereas those associated with fatty acid oxidation facility rose throughout the period. The timing of many of these maturational processes was earlier in relation to birth than in the rodent. The importance of the transcription factors, estrogen-related receptors, and v-myc avian myelocytomatosis viral oncogene homolog was also highlighted in the pattern of gene expression during development of the perinatal sheep heart., (Copyright © 2015 the American Physiological Society.)

This study evaluated whether the administration of 50 and 100mg bovine somatotropin (bST) at the start of synchronization and at the time of natural mating in ewes improves the ovulation rate, embryonic development and pregnancy rate of transferred embryos. Forty-eight donors were assigned to three treatments: the bST-100 treatment (n=15) received 100mg bST at the start of synchronization and at natural mating, the bST-50 treatment (n=15) received 50mg bST on the same schedule as the previous group, and the control (n=18) did not receive any bST. Two embryos were transferred to each recipient (n=121): 35 received embryos from bST-100; 50 received embryos from bST-50, and 36 received embryos from the control. The superovulatory rate, percentage of recovered structures, cleavage rate, percentage of transferable embryos, embryo quality and development and pregnancy rate were analyzed using the GENMOD procedure of SAS. The number of corpora lutea and the cell number were analyzed using the GLM procedure of SAS. The insulin and IGF-1 concentrations were analyzed with ANOVA for repeated measures. The bST application did not affect the superovulatory rate, number of corpora lutea and recovered structures (P>0.05). The numbers of transferable embryos and embryos reaching the blastocyst were higher (P≤0.01) in the bST-50 (96.4±3.6% and 69.0±7.8%) than the bST-100 (93.0±4.5% and 27.2±38.9%) and control (87.7±5.4% and 50.4±6.4%) groups. The insulin and IGF-1 concentrations were higher (P<0.05) in the bST-treated groups, but the insulin concentration was higher (P<0.05) in the bST-100 group than in the bST-50 group. The pregnancy rate was similar (P=0.21) in ewes receiving embryos from the two treatments [bST-50, (70.0%); bST-100, (62.5%), and control, (56.6%)]. The administration of 50mg bST at the start of synchronization and at natural mating in superovulated ewes was concluded to enhance the proportion and development of transferable embryos. However, bST did not affect the pregnancy rate of transferred embryos., (Copyright © 2014. Published by Elsevier B.V.)

Intrauterine growth restriction (IUGR) is a risk factor for obesity, particularly when offspring are born into an unrestricted nutritional environment. In this study, we investigated the impact of IUGR and gender on circulating lipids and on expression of adipogenic, lipogenic and adipokine genes in perirenal adipose tissue. Singleton lambs born to overnourished adolescent dams were normal birth weight (N) or IUGR (32% lower birth weight due to placental insufficiency). IUGR lambs exhibited increased fractional growth rates but remained smaller than N lambs at necropsy (d77). At 48 days, fasting plasma triglycerides, non-esterified fatty acids and glycerol were elevated predominantly in IUGR males. Body fat content was independent of prenatal growth but higher in females than in males. In perirenal fat, relative to male lambs, females had larger adipocytes; higher lipoprotein lipase, fatty acid synthase and leptin and lower IGF1, IGF2, IGF1R, IGF2R and hormone-sensitive lipase mRNA expression levels, and all were independent of prenatal growth category; peroxisome proliferator-activated receptor gamma and glycerol-3-phosphate dehydrogenase (G3PDH) mRNA expression were not affected by IUGR or gender. Adiposity indices were inversely related to G3PDH mRNA expression, and for the population as a whole the expression of IGF system genes in perirenal fat was negatively correlated with plasma leptin, fat mass and adipocyte size, and positively correlated with circulating IGF1 levels. Higher plasma lipid levels in IUGR males may predict later adverse metabolic health and obesity, but in early postnatal life gender has the dominant influence on adipose tissue gene expression, reflecting the already established sexual dimorphism in body composition., (© 2014 Society for Endocrinology.)

This study investigated the effects of maternal undernutrition during late pregnancy on the development and function of ovine fetal liver. Eighteen ewes with singleton fetuses were allocated to three groups at d 90 of pregnancy: Restricted Group 1 (RG1, 0.175MJMEkgBW(-0.75)d(-1), n=6), Restricted Group 2 (RG2, 0.33MJMEkgBW(-0.75)d(-1), n=6) and a Control Group (CG, ad libitum, 0.67MJMEkgBW(-0.75)d(-1), n=6). Fetuses were recovered at slaughter on d 140. Fetuses in the RG1 group exhibited decreased (P<0.05) liver weight, total antioxidant capacity (T-AOC), superoxide dismutase activity (SOD), cholinesterase (CHE), total protein (TP), globulin (GLB), and alanine transaminase (ALT). In addition, intermediate changes were found in the RG2 fetuses, including decreased liver weight, T-AOC and CHE (P<0.05). In contrast, increases in fetal hepatic collagen fibers and reticular fibers, glutathione peroxidase (GSH-Px), malondialdehyde (MDA), nitric oxide (NO), nitric oxide synthase (NOs), monoamine oxidase (MAO), albumin (ALB)/GLB, aspartate transaminase (AST), and AST/ALT were found in the RG1 fetuses (P<0.05). The RG2 fetuses had increased fetal hepatic collagen fibers, NOs and MAO (P<0.05) relative to the control fetuses. These results indicate that impaired fetal hepatic growth, fibrosis, antioxidant imbalance and dysfunction were associated with maternal undernutrition., (Copyright © 2014 Elsevier B.V. All rights reserved.)

After cryopreservation, embryos become sensitive to the oxidative stress, resulting in lipid peroxidation, membrane injury, and structural destruction. The present study aimed to assess the effect of increasing concentration of melatonin during postwarming culture on embryo's ability to restore its functions after cryopreservation. In vitro-produced blastocysts were vitrified, warmed, and cultured in vitro in TCM 199 with 5 different supplementations: control (CTR): 10% fetal calf serum; bovine serum albumin (BSA): 0.04% (wt/vol) BSA; and MEL(-3), MEL(-6), MEL(-9): BSA plus melatonin 10(-3), 10(-6), and 10(-9) M. The medium with the highest melatonin concentration had the highest trolox equivalent antioxidant capacity, whose values were comparable with those determined in plasma sampled from adult ewes (8.7 ± 2.4 mM). The other media had lower trolox equivalent antioxidant capacity values (P < 0.01), below the range of the plasma. At the same time, embryos cultured with the highest melatonin concentration reported a lower in vitro viability, as evaluated by lower re-expansion and hatching rates, and lower total cell number compared with the other groups (P < 0.05). Their metabolic status was also affected, as evidenced by higher oxidative and apoptotic index and lower ATP concentration. The beneficial effects of melatonin on embryo development during postwarming culture were observed only at low concentration (10(-9) M). These results suggest that melatonin at high concentration may exert some degree of toxic activity on pre-implantation embryos. Thus, the dose at which the embryos are exposed is pivotal to obtain the desiderate effect., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Fetal rivalry for space and nutrients compromises intrauterine environment and fetal growth, this leading to further consequences during adult life (i.e., fetal programming). Focusing on sheep, relevant fetal programming effects have been revealed on body composition and growth although little is known about their potential impact on the reproductive performance of adult ewes. This research focused on the analysis of fetal programming-related effects on 41,475 litter size (LS) records from 7,177 purebred Ripollesa ewes. Fetal programming sources of variation accounted for the linear and quadratic effect of absolute birth BW (ABBW), relative birth BW (RBBW) of twin-born ewes (i.e., both magnitude and direction of the birth BW difference between the ewe and its co-twin), and sex of twin ewe's littermate (SLM). More specifically, data were analyzed under a threshold mixed model and the statistical relevance of models accounting for different combinations of ABBW, RBBW, and SLM effects was compared by Bayes factors (BF; i.e., the ratio between the posterior probability of 2 competing models). The model accounting for RBBW and discarding both ABBW and SLM effects was clearly preferred; its posterior probability was 35.2 to 362.3 times higher than from remaining models and provided very strong (31.6 < BF < 100) and decisive evidences (BF > 100) supporting the relevance of RBBW and the negligibility of both ABBW and SLM. Single-born ewes were included as reference group and they reached a predicted LS of 1.189 lambs per lambing. Twin-born ewes being >600 g lighter than their co-twins suffered from an impaired reproductive ability with 1.162 lambs per lambing (95% credible interval [95CI], 1.147 to 1.179), and this estimate increased until ewes were 151 to 300 g lighter than their co-twins (1.226 lambs per lambing; 95CI, 1.208 to 1.244). Remaining categories (i.e., ewes being heavier or equal than their co-twins) did not provide significant differences and showed an enhanced reproductive ability of approximately 1.23 lambs per lambing. These significant differences in LS highlighted the influence of fetal programming in sheep under rangeland conditions, which implies decisive economic consequences worldwide. Moreover, these results could contribute additional information on twin biology, which could be useful in other mammalian species such as humans.

Perinatal hypoxia is a major cause of brain injury in preterm babies. Thus, neuro-protective treatments play a pivotal role during the first 6-8 hours post hypoxic-ischemic insult. However, at present it is not possible to determine which infants are suffering from hypoxic ischemia. Recent investigations suggest that there are high frequency micro-scale transients exist in the first 6-8 hours of a hypoxic ischemic EEG which could be utilized as the useful benchmarks for the prediction of hypoxia. Type-2 Fuzzy Logic Systems (Type-2 FLS) have the capability to handle inherent uncertainties in nonlinear signals. This paper describes the application of a Type-2 FLS to detect spikes in the preterm fetal sheep electroencephalogram (EEG) after asphyxia in utero. The Type-2 FLS differentiates each detected event in terms of its spikiness and specifies the potential events based on their degree of similarity to an EEG expert definition of a standard spike. An adaptive thresholding method has been employed in order to increase the spike detection ability of the purposed system. The sensitivity and selectivity verify enhanced performance of the Type-2 FLS for spike detection in fetal sheep EEG signals with a 98.1% and 93.7% respectively which are significantly improved in comparison to our previous methods.

Embryonic loss and intrauterine growth restriction (IUGR) are significant problems in humans and other animals. Results from studies involving pigs and sheep have indicated that limited uterine capacity and placental insufficiency are major factors contributing to suboptimal reproduction in mammals. Our discovery of the unusual abundance of the arginine family of amino acids in porcine and ovine allantoic fluids during early gestation led to the novel hypothesis that arginine plays an important role in conceptus (embryo and extra-embryonic membranes) development. Arginine is metabolized to ornithine, proline, and nitric oxide, with each having important physiological functions. Nitric oxide is a vasodilator and angiogenic factor, whereas ornithine and proline are substrates for uterine and placental synthesis of polyamines that are key regulators of gene expression, protein synthesis, and angiogenesis. Additionally, arginine activates the mechanistic (mammalian) target of rapamycin cell signaling pathway to stimulate protein synthesis in the placenta, uterus, and fetus. Thus, dietary supplementation with 0.83 % L-arginine to gilts consuming 2 kg of a typical gestation diet between either days 14 and 28 or between days 30 and 114 of pregnancy increases the number of live-born piglets and litter birth weight. Similar results have been reported for gestating rats and ewes. In sheep, arginine also stimulates development of fetal brown adipose tissue. Furthermore, oral administration of arginine to women with IUGR has been reported to enhance fetal growth. Collectively, enhancement of uterine as well as placental growth and function through dietary arginine supplementation provides an effective solution to improving embryonic and fetal survival and growth.

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., (Copyright © 2013 Wiley Periodicals, Inc.)

Proper establishment of the placenta is important for fetal survival; however, placental adaptations to inadequate maternal nutrition or other stressors are imperative for fetal growth to be optimal. The effects of maternal nutritional status and activity level on placental vascular function and uteroplacental blood flows are important to understand as improper placental function leads to reduced growth of the fetus. In environments where fetal growth can be compromised, potential therapeutics may augment placental function and delivery of nutrients to improve offspring performance during postnatal life. Factors that could enhance placental function include supplementation of specific nutrients, such as protein, hormone supplements, such as indolamines, and increased activity levels of the dam. To understand the mechanism of how the maternal environment can impact uterine or umbilical blood flows, assessment of placental vascular reactivity has been studied in several large animal models. As we begin to understand how the maternal environment impacts uterine and umbilical blood flows and other uteroplacental hemodynamic parameters, development of management methods and therapeutics for proper fetal growth can be achieved.

Study Question: Is DNA methyltransferase 1 (DNMT1) dysfunction involved in epigenetic deregulation of placentae from embryos obtained by assisted reproduction technologies (ARTs)?, Summary Answer: DNMT1 expression in growing placentae of in vitro produced (IVP) embryos is compromised and associated with pregnancy loss., What Is Known Already: DNMT1 maintains the methylation profile of genes during cell division. The methylation status of genes involved in placenta development is altered in embryos obtained in vitro. Disturbances in the epigenetic regulation of gene expression during placentogenesis could be involved in the frequent developmental arrest and loss of IVP embryos., Study Design, Size, Duration: Forty sheep were naturally mated (Group 1, CTR). IVP blastocysts (2-4 per ewe) were surgically transferred to the remaining 46 recipient sheep 6 days after oestrus (Group 2). Twenty-one recipients from Group 1 and 27 recipients from Group 2 were allowed to deliver in order to compare embryo survival in both groups at term (150 days). From the remaining recipients (n = 38), fetuses and placentae of both groups were recovered by paramedian laparotomy at Days 20, 22, 24, 26 and 28 of gestation., Materials, Setting, Methods: Immediately after collection, early placental tissues (chorion-allantois) were snap frozen in liquid nitrogen and DNMT1 expression and activity was evaluated. mRNA levels (for DNMT1, HDAC2, PCNA, DMAP1, MEST, IGF2, CDKN1C, H19) and the methylation status of H19 were also analyzed. Furthermore, embryo size and survival rate were measured., Main Results and the Role of Chance: Our study shows that DNMT1 expression was reduced in early placentae from sheep IVP embryos. This reduction was associated with growth arrest and subsequent death of the sheep embryos. Conversely, normal levels of DNMT1 and its cofactors were observed in placentae from IVP embryos that survived this developmental bottleneck. Although DNA methylation machinery was severely compromised in IVP placentae only up to Day 24, the low DNMT1 enzymatic activity that persisted after this stage in IVP placentae was not lethal for the developing embryos., Limitations, Reasons for Caution: The studied genes represent only a small fraction of genes regulating DNA methylation. Further studies are needed to evaluate changes in the expression and methylation status of other genes that may lead to developmental arrest of IVP embryos. As this is the only study evaluating the functionality of DNMT1 machinery in placentae from ART embryos, studies on other species are needed to confirm if our observation may be applicable to all mammalian embryos produced in vitro., Wider Implications of the Findings: The knowledge about compromised activity of DNMT1 in placentae obtained from IVP embryos should stimulate detailed studies on the metabolic requirements of oocytes and embryos in order to adequately enrich the culture media.

This study was designed to characterize development of the ovine conceptus throughout gestation to establish the temporal relationships in metabolites, electrolytes, fluid volumes within the placenta, and hormonal changes with fetal growth. Length and weight of placentae, weight of cotyledons, and uterine weight increased between d 25 and 80 of gestation in advance of increases in fetal growth between d 80 and 140 of gestation. Allantoic fluid volumes changed (P < 0.01) between d 25 (21 mL) and 40 (91 mL), decreased to d 70 (32 mL), and then increased to d 140 (438 mL). Concentrations and total amounts of proteins in allantoic fluid were reduced between d 25 and 50, but total protein increased (P < 0.01) from d 40 (63 mg) to d 140 (2,991 mg). Concentrations of fructose in allantoic fluid varied between 2 and 6 mg/mL throughout gestation, but total fructose increased (P < 0.01) between d 25 (46 mg) and d 120 (679 mg). Concentrations of glucose ranged from 0.1 to 0.3 mg/mL, and total glucose increased (P < 0.05) from d 25 (3 mg) to d 140 (63 mg) of gestation. Amniotic fluid volume increased (P < 0.01) between d 30 and 140. Concentrations of estrogens in allantoic fluid, maternal uterine artery, and uterine vein increased (P < 0.01) with advancing pregnancy, and concentrations of progesterone in allantoic fluid (P < 0.07) and plasma (P < 0.05) were affected by day of gestation. Concentrations of glucose were greater (P < 0.05) in uterine artery than uterine vein, but concentrations of electrolytes and osmolarity of plasma were not affected by day of gestation. Increases in weights of fetal organs were proportional to increases in fetal weight during gestation. Results of the present study of conceptus growth and development highlight areas of needed research and provide benchmarks for comparisons when evaluating effects of various treatments, environmental conditions, and epigenetics on successful outcomes of pregnancy in sheep.

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., (© 2010 The Authors. Journal compilation © 2010 Japanese Society of Animal Science.)

The objective of this study was to identify the migration period of the genital tubercle and the period of visualization of external genital structures in fetuses of the Dorper breed of sheep derived from natural mating and from fresh, frozen and vitrified embryo transfer. Transrectal ultrasound was performed using a double-frequency linear transducer (6.0 and 8.0 MHz) to monitor 130 ewe fetuses distributed in the four treatments regarding embryo origin. The accuracy of the ultrasound was 100% in this experiment. The fetuses originated from controlled natural mating (NM) and from fresh (FrE), frozen (FE) and vitrified (VE) embryo transfer, with embryos collected 7 days after breeding. Migration of the genital tubercle occurred earlier (P<0.05) in NM (42.21+/-2.86 days) than in FrE (43.98+/-3.00 days), FE (44.97+/-1.83 days) and VE (44.58+/-1.97 days). Visualization of the scrotal bag, prepuce and vulva occurred, respectively, earlier (P<0.05) in NM (45.22+/-1.25, 45.95+/-1.53 and 45.01+/-1.03 days) than in FrE (48.91+/-1.92, 48.52+/-1.41 and 47.41+/-1.41 days), FE (49.97+/-1.08, 49.18+/-2.00 and 47.64+/-1.82 days) and VE (50.12+/-1.66, 49.27+/-1.61 and 47.93+/-1.92 days). The results show that fetal sexing can be accomplished from the 50th day onward in fetuses produced by natural mating and from the 55th day onward in fetuses derived from fresh, frozen and vitrified embryos. It can also be concluded that real-time ultrasonography is a reliable tool for fetal sex determination in sheep taking into account both the location of the genital tubercle and the identification of external genital structures.

Establishment of pregnancy in ruminants requires blastocyst growth to form an elongated conceptus that produces interferon tau, the pregnancy recognition signal, and initiates implantation. Blastocyst growth and development requires secretions from the uterine endometrium. An early increase in circulating concentrations of progesterone (P4) stimulates blastocyst growth and elongation in ruminants. This study utilized sheep as a model to identify candidate genes and regulatory networks in the endometrium that govern preimplantation blastocyst growth and development. Ewes were treated daily with either P4 or corn oil vehicle from day 1.5 after mating to either day 9 or day 12 of pregnancy when endometrium was obtained by hysterectomy. Microarray analyses revealed many differentially expressed genes in the endometria affected by day of pregnancy and early P4 treatment. In situ hybridization analyses revealed that many differentially expressed genes were expressed in a cell-specific manner within the endometrium. The Database for Annotation, Visualization, and Integrated Discovery (DAVID) was used to identify functional groups of genes and biological processes in the endometrium that are associated with growth and development of preimplantation blastocysts. Notably, biological processes affected by day of pregnancy and/or early P4 treatment included lipid biosynthesis and metabolism, angiogenesis, transport, extracellular space, defense and inflammatory response, proteolysis, amino acid transport and metabolism, and hormone metabolism. This transcriptomic data provides novel insights into the biology of endometrial function and preimplantation blastocyst growth and development in sheep.

A classical paradigm in population genetics is that homozygosity or inbreeding affects individual fitness through increased disease susceptibility and mortality, and diminished breeding success. Using data from an insular population of mouflon (Ovis aries) founded by a single pair of individuals, we compare embryo number of ewes with different levels of inbreeding. Contrary to expectations, ewes with the highest levels of homozygosity showed the largest number of embryos. Using two different statistical approaches, we showed that this relationship is probably caused by heterozygosity at specific genes. The genetics of embryo number coupled with cyclic dynamics could play a central role in promoting genetic variation in this population.

Back and hind limb muscles of sheep paternally heterozygous for the callipyge single nucleotide polymorphism undergo extensive hypertrophy shortly after birth. We have established cell cultures from foetal semitendinosus and longissimus dorsi muscles of normal and callipyge animals. Cultures were assessed for rates of proliferation, cell death, myogenicity and DLK1 expression. Myoblasts from callipyge semitendinosus, but not longissimus dorsi muscles, proliferated faster than myoblasts isolated from normal semitendinosus muscle, and cells isolated from either callipyge muscle were more resistant to serum deprivation-induced apoptosis than equivalent cells isolated from normal individuals. These observations indicate that there are intrinsic differences in the behaviour of isolated myoblasts, which are associated with their muscle and genotype of origin. As myoblasts are the cells responsible for hypertrophy of muscle fibres, the observed differences in cell growth may play a role in the hypertrophy of certain muscles in callipyge animals., (Copyright 2007 S. Karger AG, Basel.)

To improve the viability of embryos produced in vitro from lamb oocytes, maturation medium was supplemented with insulin-like growth factor-I (IGF-I), epidermal growth factor (EGF), cysteamine, and combinations thereof. Experiment 1 examined the effects of IGF-I supplementation and duration of oocyte maturation on nuclear maturation and embryo development while Experiments 2 and 3 examined the effects of cysteamine and EGF supplementation respectively on embryo development. In Experiment 4, embryo development was examined after maturation with various combinations of supplements. IGF-I supplementation increased cleavage rate (P < 0.05) but its effect on the rate of blastocyst production from original oocytes was variable. Supplementation with IGF-I increased (P < 0.01) the proportion of oocytes at Metaphase II (MII) after 18 h of maturation but not at later times. EGF either alone or combined with IGF-I significantly (P < 0.05) increased cleavage rates compared with other treatment groups but EGF consistently failed to improve blastocyst production rates. Cysteamine improved hatching rates but only when supplemented alone. Maturation of lamb oocytes for 22 h in medium supplemented with 100 ng mL(-1) IGF-I and 100 microm cysteamine resulted in the production of 16.0 lambs per donor lamb after embryos were transferred to recipient ewes. It is concluded that EGF and, to a lesser extent, IGF-I, whilst beneficial to initial cleavage, can adversely influence subsequent embryo development. Improvements in embryo viability may more likely be obtained by addressing issues that influence fetal oocyte quality than by modifying in vitro methodology.

Mitochondria are the key generators of cellular ATP, and contain extranuclear genome-mitochondrial DNA (mtDNA). In the process of nuclear transfer (NT), heteroplasmic sources of mtDNA from a donor cell and a recipient oocyte are mixed in the cytoplasm of the reconstituted embryo. Previous studies showed inconsistent patterns of mtDNA inheritance in offspring and early fetuses generated through interspecies NT. The quantitative analysis of mitochondrial RNA (mtRNA) in interspecies cloned embryos is useful for better understanding the fate of two types of mitochondria. The components of nicotinamide adenine dinucleotide (NADH) dehydrogenase were coded by both nuclear DNA (nDNA) and mtDNA. The Subunit 1 (ND-1) is one of seven NADH dehydrogenase subunits coded by mtDNA. In present study, using real-time and reverse-transcription PCR, the copy number of species-specific ND-1 mRNA was examined in goat-sheep cloned embryos of various developmental stages, and was applied to evaluate the expression pattern of species-specific mtDNA. The results of showed that (1) the expression of mtDNA derived from goat fetal fibroblast (GFF) decreased from 1-cell stage (immediately after fused) to 2-cell stage, and could not be detected from 4-cell stage onward to blastocyst stage; (2) the expression of mtDNA derived from sheep oocyte was roughly constant from 1-cell stage to the 8-cell stage, increased gradually from 16-cell stage, and sharply at morula and blastocyst stage. Moreover, we strongly argued a mechanism, that is GFF-derived mitochondria were degraded for the depression of bioenergetic functions, and then selectively eliminated during the embryogenesis of goat-sheep cloned embryos., ((c) 2007 Wiley-Liss, Inc.)

Placental oestrogens play an important role as local regulators of placental growth and differentiation during gestation, and toward term they are also involved in the preparation of parturition. They are synthesized within the fetal cotyledons of placentomes by aromatase cytochrome P450 (P450arom; EC 1.14.14.1), the product of the Cyp 19 gene. The first step of regulation of P450arom expression, and hence enzyme activity and oestrogen production, takes place at the level of Cyp 19 transcription, which is driven by a proximal promoter region, P1.5/2, in the sheep placenta. The aim of the present study was to find out if different Cyp 19 expression levels, which previously had been observed in ovine placentome tissues, correlate with the tissue-specific chromatin structure of the promoter. To this end, we investigated the chromatin structure across the P1.5/2 region in caruncles and cotyledons from 100 and 125 days pregnant ewes, and in term placentae, respectively, by analyzing the DNA methylation and the accessibility to restriction digestion. Our data show that: (1) cotyledonal DNA was significantly lower methylated than caruncular DNA; (2) methylation of cotyledonal DNA was low at 100 and 125 days of pregnancy, and increased to a significant higher level in term placentae; and (3) concurrently, cotyledonal chromatin became inaccessible to restriction digestion at term of gestation. The results imply that DNA methylation and chromatin accessibility of the P1.5/2 promoter region correlate with expression levels of the Cyp 19 gene., ((c) 2007 Wiley-Liss, Inc.)

An interspecific embryo transfer program was conducted for genetic improvement and increasing the number of offspring from a flock of mouflon sheep in Argentina. The female donor mouflons were divided into three groups, G1 (n=5), G2 (n=4) and G3 (n=5). The total NIH-FSH-P1 dose given to each donor on the superovulatory treatment was 260, 200 and 160 mg for G1, G2 and G3, respectively. The mouflons in G3 were maidens, while the others were multiparous. Domestic Corriedale ewes (n=60) were synchronized and used as recipients. The embryo recovery and transfer was performed by a surgical method. Mouflons (n=13) responded to the superovulatory treatment with an average of 9.1+/-2.8 ovulations. A low incidence of early luteal regression was found (1 out of 14 donors). Embryo recovery rates were 60, 31 and 76% in groups G1, G2 and G3, respectively. The percentage of transferable embryos obtained in G1 and in G2 exceeded 80%. None of the embryos obtained from G3 were of transferable quality. In G1, 25 transferable embryos were recovered and transferred to 13 recipients, resulting in a pregnancy rate of 76.9% (10/13). In G2, 10 embryos were transferred to 5 recipients, resulting in a 60% pregnancy rate (3/5). Lambing rate was 60% (15/25) and 30% (3/10) for G1 and G2, respectively. Thirteen lambs were born to the 14 donors following natural service after the embryo recoveries. This study demonstrates that the application of IET technology would have great reproductive impact, especially when the donor mouflon hinds are selected according to age and reproductive history.

CCL25/TECK and CCL28/MEC are CC chemokines primarily expressed in thymic dendritic cells and mucosal epithelial cells. The cognate receptors of CCL25 and CCL28, CCR9 and CCR10, respectively, are mainly expressed on T and B lymphocytes. In human, mouse and pig, CCL25 and CCL28 play a key role in the segregation and the compartmentalization of the mucosal immune system through recruitment of immune cells to specific locations. However, little is known about their role in the ontogeny of the mucosal immune system during fetal development. In the present paper, we report the cloning and the sequencing of ovine CCL25, CCL28, CCR9 and CCR10 and the subsequent assessment of their mRNA expression by q-polymerase chain reaction in several tissues, including thymus, gut-associated lymphoid tissue and mammary gland, from young and adult sheep and in the fetal lamb during the development of the immune system. CCL25 mRNA was highly expressed in thymus and gut while CCL28 mRNA was more expressed in large intestine, trachea, tonsils and mammary gland, especially at the end of gestation. These results are consistent with observations in other species suggesting similar roles for these chemokines in sheep. In fetuses, mRNA of CCL25, CCL28 and their receptors are expressed early in the thymus and mucosal tissues, including the small intestine and the nasal mucosa. Furthermore, their expression increased towards the end of gestation. Consequently, we hypothesize that CCL25 and CCL28 play an important role in the lymphocyte colonization of fetal tissues, enabling the development of a functional immune system.

Evidence for the in vivo transmission of porcine endogenous retrovirus (PERV) from porcine xenografts to various recipient animals has been inconsistent. To characterize the contribution of the host immune system to the potential for PERV transmission from pig islet tissue xenografts to host tissues, we examined two immunoincompetent animal models, thymectomizsed fetal lambs and NODscid mice. Pig proislets were grafted into fetal lambs or adult NODscid mice. Conventional, nested and real-time PCR/RT-PCR tests were used to search for PERV and pig cell-specific sequences (porcine mitochondrial cytochrome oxidase II (COII) or mitochondrial ribosomal 12S) in pig proislets, host liver and spleen at 5-84 days (lambs) or 96 days (mice) after transplantation. Xenografts were harvested at the same time points. The copy number of PERV sequences and host cell-specific nuclear (palmitoylcarnitine transferase) sequences was assessed by real-time PCR to estimate the proportion of PERV-infected host cells. Pig proislets were shown to be PERV+ve by PCR and immunohistochemistry (PERV B env protein p15E). PERV transmission (PERV A, B or C DNA in the absence of porcine COII or 12S sequences) was detected by nested PCR and real-time PCR in 4/12 fetal lamb liver samples 5-23 days after transplantation; the maximum copy number of PERV B env sequences was found at day 5 (700 copies/1 x 10(6) lamb cells). A total of 4/12 fetal lambs demonstrated both PERV and 12S porcine sequences in liver samples (days 5-84) by real-time PCR, suggesting that pig cells had migrated to those tissues and established microchimerism; nested PCR showed evidence for microchimerism (porcine COII sequences alone) in 2/12 lambs (day 5). The incidence of PERV transmission and frequency of microchimerism was similar in host spleen analysed by real-time PCR. Histological examination showed complete xenograft rejection by 23 days after transplantation to fetal lambs. In contrast, pig proislet xenografts survived long term (> or =day 96) in NODscid mice but no PERV transmission was found. Both nested and real-time PCR assays revealed that 2/3 mice had become microchimeric. Long-term expression of PERV A, B and C as well as porcine 12S or COII RNAs was found at the graft site (day 96) only, indicating that PERV transcription and possibly replication, continued in the donor pig islet tissue after transplantation. Overall, detection of PERV transmission and microchimerism was limited by the sensitivity of the PCR assay and the primers chosen. The absence of stable PERV transmission and microchimerism in fetal lambs and the rejection of pig proislet xenografts correlated in time with the establishment of host immunocompetence. We therefore suggest that the frequent failure to identify PERV transmission late after transplantation could be due to the immunological destruction of PERV-infected host cells. Recipient NODscid mice demonstrated long-term microchimerism and intragraft PERV expression, which was consistent with their stable immunoincompetence.

In order to improve fetal sexing in the Dorper sheep breed, the objective of the present study was to determine, by repeated ultrasonographic examinations, the migration period of the genital tubercle (GT) in sheep fetuses derived from natural mating or embryo transfer and to compare the accuracy of a single examination with repeated examinations at short intervals. For this purpose, transrectal ultrasound was performed, using a double-frequency linear transducer (6.0 and 8.0 MHz) for monitoring 51 sheep fetuses distributed in three experimental groups (EI, EII and EIII). The fetuses in EI (n = 23) and EII (n = 18) derived, respectively, from natural mating and embryo transfer were monitored at 48-h intervals from the 30th to 60th day of gestation and sexed based on the final location of the GT. The fetuses in EIII (n = 10), which originated from embryo transfer, were examined only once on the 65th day of gestation and sexed taking into consideration the final position of the GT and/or by identification of anatomical structures of external genitalia. The accuracy of fetal sexing was 91.3% (21 fetuses sexed/23 quantified) in EI, 88.9% (16 sexed/18 quantified) in EII and 100% (10 sexed/10 quantified) in EIII, without significant difference (P > 0.05) between experiments. Migration of the GT occurred earlier (P < 0.05) in fetuses produced by natural mating (43.0 +/- 2.8 days) than in those derived from embryo transfer (46.1 +/- 4.7 days). The results show that fetal sexing can be done from the 50th day onward in fetuses produced by natural mating and from the 60th day onward in fetuses derived from frozen embryos. It can also be concluded that repeated ultrasonographic exams in short time intervals do not maximise the accuracy of fetal sexing. In addition, real-time ultrasonography is a reliable tool for fetal sex determination in sheep after Day 50 of gestation, taking into account both the location of the GT and the identification of external genital structures.

Fatty acid-free bovine serum albumin (BSA(FAF)) can be added to supplement medium used in the culture of sheep embryos. BSA(FAF) was able to support blastocyst and subsequent embryo development at rates equivalent to that of fetal calf serum (FCS)-supplemented medium when fresh embryos were transferred. Furthermore, culture with BSA(FAF) significantly increased development of vitrified blastocysts transferred into synchronized sheep. The addition of the glycosaminoglycan, hyaluronan (HA) to the culture medium in the third and fifth day also increased cryo-tolerance of blastocysts and in turn lambing rate was improved.

Transient expression of tyrosine hydroxylase (TH, the first enzyme in catecholamine synthesis) has been shown in different brain and peripheral structures of various species. TH-immunoreactive neurons have been reported in the nasal region of human and rat fetuses migrating to the forebrain with GnRH neurons during embryogenesis. In the present study, immunohistochemical analysis and in situ hybridization were performed in fetal sheep and in vitro sheep embryo olfactory placode cultures to confirm this population in this species. On embryonic days 33 to 35, TH-immunoreactive cells as well as TH cDNA-hybridized cells were found in the olfactory and respiratory epithelium and were spatially separated from GnRH-immunoreactive neurons. In days 40 to 44 of gestation, TH-immunoreactive neurons were no longer observed in the olfactory epithelium, and TH-immunoreactive fibers were found on the trajectories of the olfactory nerves. At this stage, some TH-immunoreactive fibers were also labeled for GnRH. TH-immunoreactive cells were also found in primary cultures of olfactory placodes of fetal sheep at 10 to 18 days in vitro. Some of them coexpressed GnRH. These results imply that olfactory epithelium is also able to give rise to TH expressing cells in fetal sheep, but this expression is suppressed earlier in ontogenesis than in humans due to some unidentified factors not present in the primary cultures of olfactory placode. The role of TH expression remains unclear as in other previously described examples.

In order to find out whether exposure to environmental pollutants (EP) present in sewage sludge can change the behaviour of sheep, we compared the behaviour of two groups of 5-month old lambs (Ovis aries) with respect to their emotional reactivity and exploratory behaviour. One group (treated, T) comprised the offspring of ewes who had been kept throughout their lives on pastures with slightly elevated, environmental levels of pollutants, as a result of the application of sewage sludge at rates used in normal practice. The other group (control, C) were the offspring of ewes whose pastures had been treated with inorganic fertiliser. During a 1-min period in the weigh crate, T lambs were less reactive than C lambs, but vocalised more. Exploratory behaviour showed a clear sex difference, with female C lambs exploring more than male C lambs. However, this difference was not found in the T lambs, where both males and females showed high levels of exploration. This points to a demasculinising effect of exposure to higher pollutant concentrations with respect to exploratory behaviour. These observations demonstrate the need to take account of the effects of combinations of pollutants, even at very low, environmental concentrations, and further highlight the usefulness of ethotoxicology for the study of biological effects of environmental pollutants.

We sequenced 2939 ESTs from fetal and adult sheep skin. Stages of gestation were picked to coincide with the major events in skin appendage (wool follicle) formation. Clustering analysis generated a nonredundant set of ESTs 2435 strong (83% nonredundant). Approximately 24% of these gave no hit to NCBI build 29 of the human genome, while 35% were tentatively classified by putative function based on BLASTX hits with a p(N) of <10(-4). In addition to bioinformatics analysis of our ESTs and gene mapping, we have generated a large EST spatial expression data set using in situ hybridization. One thousand one hundred forty-two ESTs have been used for in situ localization; about 31% are from adult sheep skin, 39% from late gestation fetal sheep skin, and 30% from midgestation fetal sheep skin. These probes have been used in over 3000 hybridization experiments. In this report, we summarize the results of in situs on adult sheep skin.

The importance of prostaglandins in the regulation of the renin-angiotensin system during development is not known. These experiments were conducted to examine the effects of prostaglandin synthesis inhibitors on basal and isoproterenol-induced plasma renin concentration and renin gene expression in the late-gestation fetal lamb. Eighteen lamb fetuses ranging in gestational age from 129 to 138 days underwent surgical insertion of femoral arterial and venous catheters under general endotracheal anesthesia. After a period of recovery, animals underwent an infusion of isoproterenol after administration of a saline bolus (control experiments); 24-48 h later a second study was performed after administration of NS-398, a cyclooxygenase (COX)-2 inhibitor, or saline for a second control study. Administration of COX-2 inhibitor significantly reduced baseline plasma renin levels and attenuated responses in fetal renin secretion to isoproterenol infusions. Renal cortical cells from animals receiving COX-2 inhibitor had significantly lower levels of renin mRNA compared with animals receiving only saline. Renal cortical cells in culture from animals receiving only saline exhibited increased levels of renin mRNA when treated with isoproterenol, forskolin, or IBMX. Only forskolin increased renin mRNA levels in renal cortical cells in culture from animals receiving COX-2 inhibitor. We conclude that prostaglandins play a stimulatory role in the regulation of the renin-angiotensin system and are necessary for beta-adrenergic stimulation of renin secretion and gene expression in the late-gestation fetal lamb.

Gonadotropin-releasing hormone (GnRH) neurons involved in controlling the reproductive function in vertebrates are derived from the olfactory placode. However, in the sheep and the rat species, GnRH-immunoreactive (GnRH-IR) neurons could not be detected in the olfactory region during the earliest phase of GnRH system development. Using in situ hybridization (ISH) and immunohistochemistry (IHC) in sheep embryos ranging from 26 to 53 days' gestational age (G26-G53), the present work confirmed that GnRH expression could not be detected during the earliest steps of migration. The first ISH+ cells were detected in the nasal septum and at the entrance of the telencephalon at G33 stage. [(3)H]-thymidine pulses applied to in vitro olfactory explant cultures showed that GnRH neuron precursor cells have an extended multiplication period corresponding to G26-G36 before entering the neuronal differentiation process. Therefore, the lack of GnRH neuron detection during the early phase of development in the sheep compared to the mouse and other vertebrates represents a major difference in the early development of GnRH neurons. In the mouse, GnRH neuron precursors have a limited multiplication period in the vomeronasal pit and only postmitotic neurons start migration, whereas in the sheep embryo, the multiplication period is extended to about 10 days as demonstrated in olfactory explant cultures., (Copyright 2003 S. Karger AG, Basel)

Corticotroph responsiveness to arginine vasopressin (AVP) increases during late gestation in fetal sheep. The mechanism of this increase in AVP responsiveness is currently unknown but could be related to an increase in vasopressin type 1b (V1b) receptor expression in the pituitary during development. To determine if there are ontogenic changes in V1b receptor expression that may help explain the changes in ACTH responses to AVP, we studied pituitaries from three groups of fetal sheep [100, 120, or 140 days gestational age (dGA)]. V1b receptor mRNA and protein significantly decreased by 140 dGA. Peak V1b mRNA levels were detected at 100 dGA, while peak V1b protein levels were detected at 120 dGA. The reduction in V1b receptor expression in late gestation may be due to the naturally occurring peripartum increase in fetal plasma cortisol because cortisol infusion at 122-130 dGA decreased V1b receptor mRNA. Thus there is a marked decrease in the expression of the V1b receptor in the pituitary during fetal development, leaving the role of the V1b receptor in increasing AVP responsiveness uncertain.

The functional dentition of the domestic sheep lacks all upper incisors and the upper canines. Nevertheless, occurrence of a dental lamina and rudimental tooth primordia had been described in the upper incisor region of the sheep. The aim of this study was to describe temporo-spatial pattern of origin and regression of these rudimental tooth primordia by light microscopy, computer-aided three-dimensional reconstruction and morphometry of the dental epithelium. Transient existence of a dental lamina in the upper incisor region of the sheep and three epithelial thickenings on its deep mesenchymal margin has been observed at day of ontogeny (DO) 48-53. They could not been identified as full-value tooth primordia, because they did not induce differentiation of tooth mesenchyme, but they could represent last remnants of functional upper incisors in early ancestors of ruminants. Additionally, a large rudimental upper canine primordium near the sutura maxilloincisiva occurred at DO43, reached early cap stage at DO52 and started to regress at DO53. Thus, our findings showed a discrepancy between the embryonic and adult dental pattern in the sheep. Similar molecular mechanisms as described for diastemal tooth rudiments in rodents could be involved during regression of rudimental tooth primordia in the upper incisor region of the sheep.