Pregnant Sheep Research Articles

Anaemia and Hypoproteinaemia in Pregnant Sheep during Anaesthesia.

Simple SummaryAnaemia during pregnancy is commonly reported in humans and is considered a significant health issue as it is associated with adverse neonatal outcomes including low birth weight, preterm birth and possible perinatal mortality. Pregnant sheep are a common model for research investigating the causes and consequences of preterm birth, but the incidence and significance of anaemia during pregnancy is not known. However, intraoperative anaemia in pregnant sheep has been reported previously. In addition, it has been demonstrated that the cause of intraoperative anaemia is not pregnancy alone as pregnant sheep in a farm environment did not develop anaemia. The aim of this study was to document the red blood cell and protein content of maternal blood before, during and after anaesthesia and surgery to elucidate the cause and duration of intraoperative anaemia in pregnant sheep in a research context. The concentration of red blood cells and protein decreased during anaesthesia and were attributed to the use of anaesthetic drugs and fluids as opposed to the pregnancy status of the sheep.The aim of this study was to document the packed cell volume (PCV), haemoglobin concentration and total protein concentration of maternal blood before, during and after anaesthesia. Six singleton Merino-cross pregnant ewes at 116-117 days of gestation were premedicated with intramuscular acepromazine (0.02 mg/kg) and buprenorphine (0.01 mg/kg), and anaesthesia was induced with intravenous midazolam and ketamine. Anaesthesia was maintained with isoflurane in 100% oxygen. Serial blood samples were collected the day before anaesthesia (baseline), immediately prior to induction of anaesthesia (pre-op), at the end of the procedure (intra-op) and the following day (post-op). There was a significant change in the PCV during the study (p = 0.003) with an initial decrease of 12.5% from the baseline (0.36 (0.36–0.4) to 0.315 (0.29–0.34), p = 0.044), a further intraoperative decrease of 41.7% from the baseline (0.21 (0.195–0.245), p = 0.002) and an increase the day afterwards (0.3 (0.285–0.35), p > 0.99 compared with baseline). The haemoglobin concentration also changed (p < 0.0001) (baseline: 114 (111.8–123); pre-op: 97 (77.25–104.5), 14.9% decrease, p = 0.022; intra-op: 70 (61.5–83.25), 38.5% decrease, p = 0.0009; post-op: 101.5 (96.25–114) g/L, p > 0.99). Likewise the change in total protein during the study was significant (p = 0.0003) and decreased from the baseline [70 (67.25–70.75) g/L] prior to anaesthesia (61 (58.25–64.5) g/L, 12.9% decrease, p = 0.0437) and further during anaesthesia (55.5 (53.75–63.25) g/L, 20.7% decrease, p = 0.0021) with an increase [63 (61.25–67) g/L, p > 0.99] on the first post-op day. In conclusion, intraoperative anaemia and hypoproteinaemia occurred in this study. These alterations are attributed to a combination of the side effects of acepromazine and haemodilution.

Ruminant models of prenatal growth restriction

Intrauterine growth restriction (IUGR) is a significant health issue that not only affects infant mortality and morbidity, but may also predispose individuals to coronary heart disease, diabetes, hypertension and stroke as adults. The majority of IUGR pregnancies in humans are characterized by asymmetric fetal growth, resulting from inadequate nutrient transfer to the fetus. Furthermore, most of these pregnancies involve functional placental insufficiency, and may also show altered umbilical velocimetry. As the severity of IUGR increases, the fetus becomes increasingly hypoxic, hypoglycaemic and acidotic. In addition, placental transfer or utilization of some amino acids is known to be altered in IUGR pregnancies. Although a great deal has been learned from clinical studies of human IUGR, appropriate animal models are required to define completely the mechanisms involved in the development of IUGR. The pregnant sheep is a long-standing model for placental-fetal interactions, and fetal growth restriction can be induced in pregnant sheep by maternal nutrient restriction, maternal nutrient excess, administration of glucocorticoid, utero-placental embolization, carunclectomy and maternal hyperthermia. Although all of these sheep models are capable of inducing fetal growth restriction, the degree of restriction is variable. This review compares these sheep models of IUGR with the characteristics of human IUGR.

The polypeptides and genes for ovine and bovine trophoblast protein-1

Summary. Ovine and bovine trophoblast protein-1 (oTP-1 and bTP-1) have been strongly implicated as antiluteolytic agents and responsible for maternal recognition of pregnancy in sheep and cattle, respectively. Both are interferons (IFN) belonging to the IFN-α family, but their length (172 residues versus 166 for most IFN-α) places them in an unusual subclass (the IFN-αII). The various isoforms of oTP-1 and bTP-1 produced by trophoblast tissue appear to arise in part from translation of multiple mRNAs which are themselves the products of distinct genes. These genes, like those for other IFN-α, are without introns. However, the genes for oTP-1 and bTP-1 form a distinct subgroup within the IFN-αII on the basis of their overall primary sequences and the high conservation of the 3'-untranslated ends of their transcription units. The bTP-1 genes also differ from the bovine IFN-αII in the organization of the promoter regions upstream from the transcription start site. Nevertheless, computer-aided analysis of the primary polypeptide sequences of oTP-1 and bTP-1 indicates that the molecules are likely to have approximately the same shapes and dimensions as all other IFN-α moleculcs. It remains to be determined whether they have unique biological properties which distinguish them from other IFN-α moleculcs.

Control of luteal function during early pregnancy in sheep

Prolactin and LH are required for the maintenance of normal luteal structures during early pregnancy in the ewe, whilst luteolysis, induced by prostaglandin F-2 alpha is inhibited by the presence of the conceptus. The trophoblast also secretes a local anti-luteolytic factor, trophoblastin, which is most probably a protein because it is thermolabile and inactivated by pronase. It is soluble at pH 9.6 and can be separated on Ultrogel. Trophoblastin is present for a short time (12th-22nd day of pregnancy) but its effect may last several months.

Physiological mechanisms of pregnancy recognition in ruminants

Maternal recognition of pregnancy in sheep, cattle and goats involves physiological mechanisms that result in protection of corpora lutea from luteolysis by modification or inhibition of uterine production of luteolytic pulses of prostaglandin (PG) F-2 alpha. Ovine, bovine and caprine luteal cells release oxytocin in a pulsatile manner during late dioestrus. Oxytocin then binds to its endometrial receptors and initiates luteolytic pulses of PGF-2 alpha. Ovine, bovine and caprine trophoblast protein-1 (oTP-1, bTP-1 and cTP-1) are secreted by the trophectoderm of conceptuses between Days 10 and 21-24 of pregnancy. These antiluteolytic proteins (oTP-1 and bTP-1) are primarily responsible for inhibiting uterine production of luteolytic amounts of PGF-2 alpha. During early pregnancy, the numbers of endometrial receptors for oxytocin are significantly lower in ewes and cows, and stimulatory effects of exogenous oxytocin on uterine production of PGF-2 alpha are correspondingly reduced or absent for ewes, cows and goats. Exogenous oestrogens can, through a uterine-dependent mechanism, stimulate synthesis of endometrial receptors for oxytocin and uterine production of PGF-2 alpha; an effect which is significantly attenuated during early pregnancy. These results suggest that oTP-1, bTP-1 and possibly cTP-1 exert their antiluteolytic effect(s) by: (1) inhibiting effects of oestrogen and/or progesterone necessary for synthesis of endometrial receptors for oxytocin; (2) inhibiting endometrial synthesis and/or recycling of oxytocin receptors directly; or (3) inducing the endometrium to synthesize an inhibitor of an enzyme(s) necessary for synthesis of PGF-2 alpha.

In-vitro studies of the effects of interferons on endometrial metabolism in sheep

Primary cultures of ovine epithelial and stromal cells have been used to examine paracrine interactions between the endometrium and the preimplantation sheep blastocyst, and in particular the actions of the blastocyst alpha-interferon, ovine trophoblast protein-1 (oTP-1), on endometrial cell metabolism. The synthesis and secretion of several 'pregnancy-related' acidic proteins with molecular weights in the range 70,000-120,000 can be induced by addition of oTP-1 or human recombinant interferon (IFN) to cultured enriched epithelial endometrial cells. Consistent with the antiluteolytic role of oTP-1, dose-dependent attenuation of both PGE and PGF-2 alpha release has been demonstrated. Arachidonic acid added to the cells increased overall PG release but the inhibitory effects of interferons were still apparent. Immunocytochemical analysis of PG synthase demonstrated marked cyclic variation of its localization within the endometrium, but no differences in distribution or intensity of staining were apparent in endometrium of early pregnancy (Day 15) compared with that of the cycle (Day 15). It appears that conceptus-induced changes in PG release do not occur via changes in concentration or localization of PG synthase but rather by modifying its activity. Highly purified epithelial cells cultured on matrigel-coated millicell inserts retain important morphological features seen in vivo. Under such conditions, PGF-2 alpha and PGE release into the basal compartment was greater than that into the apical compartment. Stromal fibroblasts cultured under similar conditions secreted less PGF-2 alpha but more PGE than epithelial cells. Whilst the limitations of in-vitro studies are acknowledged, these findings are compatible with and markedly extend what is known of the action of embryonic interferons in vivo in the establishment of pregnancy in sheep.

Assessing gene function in the ruminant placenta

The placenta provides the means for nutrient transfer from the mother to the fetus, waste transfer from the fetus to the mother, protection of the fetus from the maternal immune system, and is an active endocrine organ. While many placental functions have been defined and investigated, assessing the function of specific genes expressed by the placenta has been problematic, since classical ablation-replacement methods are not feasible with the placenta. The pregnant sheep has been a long-standing animal model for assessing in vivo physiology during pregnancy, since surgical placement of indwelling catheters into both maternal and fetal vasculature has allowed the assessment of placental nutrient transfer and utilization, as well as placental hormone secretion, under unanesthetized-unstressed steady state sampling conditions. However, in ruminants the lack of well-characterized trophoblast cell lines and the inefficiency of creating transgenic pregnancies in ruminants have inhibited our ability to assess specific gene function. Recently, sheep and cattle primary trophoblast cell lines have been reported, and may further our ability to investigate trophoblast function and transcriptional regulation of genes expressed by the placenta. Furthermore, viral infection of the trophoectoderm layer of hatched blastocysts, as a means for placenta-specific transgenesis, holds considerable potential to assess gene function in the ruminant placenta. This approach has been used successfully to knockdown gene expression in the developing sheep conceptus, and has the potential for gain-of-function experiments as well. While this technology is still being developed, it may provide an efficient approach to assess specific gene function in the ruminant placenta.

The effects of maternal nutrition around the time of conception on the health of the offspring

The incidence of prematurity, diabetes and cardiovascular disease have been increasing in both the developed and developing world. Increasing numbers of human studies suggest that these serious health outcomes may have developmental origins originating from nutritional deficits in the periconceptional period, with maternal nutrition around the time of conception now shown to have important effects on the length of gestation, trajectory of fetal growth and on postnatal growth and health. Biomedical research using the pregnant sheep has been widely employed to gain a deeper understanding of the underlying mechanisms involved. There is growing awareness that this field of research has major implications for the livestock production industry. From our own studies on sheep we have evidence that maternal undernutrition during the periconceptional period results in altered fetal hypothalamic-pituitary-adrenal axis (HPAA) development, an increased rate of premature birth, altered fetal pancreatic function, insulin signalling and amino acid metabolism, and also alterations in maternal adaptation to pregnancy. We are currently studying the postnatal consequences of these changes. Other research groups have shown that restricted nutrition of sheep in the early part of pregnancy alters postnatal muscle development, fat deposition, cardiovascular regulation and HPAA function. One aim of this review is to illustrate how biomedical research using animals such as the sheep has been used to gain a better understanding of the consequences of reduced maternal nutrition during the periconceptional period. We suggest that there are equally important consequences of this research for the livestock production industries.

Discovery of Bacteria in Meconium of Normal, Uncatheterized, and Uninfected Late‐Gestation Fetal Sheep

Fetal development is thought to proceed in a sterile environment. Recent reports of the presence of bacterial DNA in human placenta, the transfer of live bacteria from mother to fetus after hypoxia in the pregnant sheep, and the presence of bacteria in the meconium of newborn infants have suggested that the fetus might be exposed to bacteria in utero. We performed the present studies to build on our discovery of bacterial rRNA in the meconium of normal fetal sheep. Specifically, the present experiments were designed to test the hypothesis that intact bacteria could be visualized histologically in meconium from normal, uncatheterized, and uninfected fetal sheep. Pregnant ewes, uncatheterized and free of clinical disease, were euthanized in late gestation. At necropsy, meconium was isolated from fetal colon using RNAse and DNAse‐free instruments and snap‐frozen, then stored at −80C until analyzed. Approximately 150 mg of meconium was homogenized and both RNA and DNA extracted in Trizol. RNA was isolated, converted to cDNA using random hexamers, then subjected to endpoint PCR using primers 341F and 806R that amplify a 465 bp region of bacterial 16S rRNA. All assays were performed with both positive and negative controls. In meconium, 7/7 fetuses (143 days gestation; term=147 days) harbored bacterial RNA. Smears of meconium were stained using Gram stain or hematoxylin stain. Gram stain revealed Gram positive and Gram negative organisms with morphologies that are consistent with the genus/species of bacteria that we have previously identified in meconium using 16S rRNA gene sequencing in DNA isolated from meconium. Hematoxylin staining confirmed the morphology of bacteria observed in the Gram‐stained samples. Bacteria in meconium appear in clusters that are consistent with live, replicating, organisms. The present results support previous work from this laboratory demonstrating that bacteria gain access to the normal fetal sheep. These results are consistent with our observations that bacteria are present in low numbers in amniotic fluid. It is possible that amniotic fluid containing bacteria is swallowed by the fetus, that bacteria survive the transit through the immature fetal gastrointestinal tract, and that the bacterial population expands in the nutrient‐rich environment of the fetal colon.Support or Funding InformationThis work was supported by HD033053 and AI120195.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Proof of Principle: Transfer of Small Numbers of Bacteria from Mother to Fetus

The fetus is thought to develop in a sterile environment in utero. Long standing dogma that “the uterus and the feto‐placental unit is “sterile” is based primarily on microbiological culture‐based techniques that were unsuccessful in growing “culture resistant” bacteria or intracellular bacteria. We have reported the presence of low numbers of bacteria in tissues from normal sheep fetuses in pregnancies not complicated by infection. The exposure of the fetus to bacteria might aid neonatal survival by informing fetal immune development. We propose that the fetus is not sterile and that bacteria or fragments of bacteria can be transferred from mother to fetus. We hypothesize that inoculation in the maternal mouth results in the appearance of bacteria in the fetus. We used S. aureus containing green (GFP), red (RFP), or far‐red (FRFP) fluorescent protein‐expressing plasmids to inoculate late‐gestation pregnant sheep (gestational age= 130–135 days, n=7) intravenously (GFP, 104 cfu), into maternal mouth (RFP, 104 cfu) and vagina (FRFP, 104 cfu). These were small doses of bacteria which did not cause physiological (no fever) or psychological (no anorexia) signs of infection. Five to seven days post maternal inoculation, animals were humanely sacrificed, and fetal tissues were collected, and DNA was extracted from placenta and fetal liver, spleen, and cerebral cortex. We probed for GFP plasmid using several primer pairs for endpoint PCR. While detection of whole‐length plasmids was not successful, PCR reactions probing for smaller fragments of plasmid were successful. We found GFP plasmid DNA in all tissues in 10/10 fetal livers and RFP in 10/10 livers. The appearance of GFP and RFP‐labelled bacteria in fetal liver (p=7.497e‐06) was statistically significant as tested by Chi‐Square analysis. We did not detect significant FRFP plasmid DNA in liver. Analysis of tissues by immunohistochemistry revealed GFP and RFP‐expressing bacteria in fetal tissues, although most appeared to be in aggregates. We conclude that S. aureus, introduced in small numbers in maternal mouth and bloodstream, appear in the fetus and placenta. We were unable to determine whether these bacteria were alive in the fetus.Support or Funding InformationThis work was supported by HD033053, AI120195, and HL083810This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hepatic GSTZ1 Expression in Pregnant Ewes and Their Offspring: Influence of Treatment with Dichloroacetate

Dichloroacetate (DCA) is an investigational drug that can stimulate mitochondrial energy metabolism by inhibiting pyruvate dehydrogenase kinase. We hypothesize that DCA could be used in high‐risk pregnancy to reduce perinatal mitochondrial deficiency and resulting cardiac dysfunction in at‐risk fetuses and are testing this postulate in a pregnant sheep model. DCA is metabolized by the enzyme glutathione transferase zeta1 (GSTZ1) to glyoxylate. GSTZ1 is not expressed in human fetal liver, but expression rises after birth. Furthermore, DCA inactivates GSTZ1, resulting in altered pharmacokinetics with repeated dosing. The objectives of this research were (1) to investigate the relative expression of GSTZ1 in maternal and fetal sheep liver from untreated controls close to full term (140 days gestation) and (2) to determine if treatment of sheep with DCA resulted in reduced expression of GSTZ1. We administered DCA intravenously to either the ewe or the fetus at daily divided doses of 25 mg/kg for three to five days prior to sacrifice 24 h after the last dose. We used samples of maternal and fetal liver to prepare cytosol and mitochondria and employed a custom‐made polyclonal antibody to rat GSTZ1 to measure expression relative to a single rat hepatic cytosol standard by Western blot. The sheep liver samples exhibited good cross‐reactivity to the rat antibody. Surprisingly, expression of GSTZ1 in the full‐term fetal liver cytosol fractions from controls was similar to that in the control maternal liver. Treatment with DCA resulted in reduced expression in both maternal and fetal liver cytosol fractions to less than 10% of the control values. In control fetuses, mitochondrial expression of GSTZ1 was 17.6 ± 7.6% of the cytosolic expression. In the DCA‐treated group, mitochondrial expression decreased and levels were undetectable in one‐half of the fetal sheep. These studies demonstrate that (1) GSTZ1 expression in liver cytosol and mitochondria in the late fetal period varies with animal species and (2) repeated treatment with DCA results in reduced GSTZ1 expression, which is likely to alter DCA pharmacokinetics.Support or Funding InformationSupported in part by the US Public Health Service, grant R21HD91599This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Maternal Supplementation with Antioxidant Vitamins in Sheep Results in Increased Transfer to the Fetus and Improvement of Fetal Antioxidant Status and Development.

Twinning and maternal nutritional restriction leads to fetal hypoxia, oxidative stress, and intrauterine growth restriction (IUGR) in near-term sheep pregnancies. Our aim was to determine the effect of oral supplementation of vitamins C and E in pregnant sheep on maternal and umbilical cord blood concentrations of vitamins C and E and the effects on fetal antioxidant status, growth, and placental efficiency. Sixteen single- and sixteen twin-bearing ewes, grazing natural Patagonian prairies, were selected after transrectal ultrasound at day 30 after mating. Half of ewes from each pregnancy rank were supplemented daily with vitamins C and E, administered orally, from 30 to 140 days of gestation, when maternal jugular and fetal venous cord blood samples were obtained during cesarean section. Fetuses were weighed and sexed. Placental weight in each fetus was also obtained. Blood plasma was harvested for measurements of maternal and fetal vitamins concentration and fetal antioxidant capacity. Maternal administration of vitamin C and E was associated with increased fetal cord levels of both vitamins, improved antioxidant status, and enhanced fetal growth in both singleton and twin pregnancies associated with increased placental efficiency. These results highlight the potential of vitamin C and E supplementation to reduce the impact of IUGR in both livestock and humans.

Pregnancy Increases Ca2+ Sparks/Spontaneous Transient Outward Currents and Reduces Uterine Arterial Myogenic Tone.

Spontaneous transient outward currents (STOCs) at physiological membrane potentials of vascular smooth muscle cells fundamentally regulate vascular myogenic tone and blood flow in an organ. We hypothesize that heightened STOCs play a key role in uterine vascular adaptation to pregnancy. Uterine arteries were isolated from nonpregnant and near-term pregnant sheep. Ca2+ sparks were measured by confocal microscopy, and STOCs were determined by electrophysiological recording in smooth muscle cells. Percentage of Ca2+ spark firing myocytes increased dramatically at the resting condition in uterine arterial smooth muscle of pregnant animals, as compared with nonpregnant animals. Pregnancy upregulated the expression of RyRs (ryanodine receptors) and significantly boosted Ca2+ spark frequency. Ex vivo treatment of uterine arteries of nonpregnant sheep with estrogen and progesterone imitated pregnancy-induced RyR upregulation. STOCs occurred at much more negative membrane potentials in uterine arterial myocytes of pregnant animals. STOCs in uterine arterial myocytes were diminished by inhibiting large-conductance Ca2+-activated K+ (BKCa) channels and RyRs, thus functionally linking Ca2+ sparks and BKCa channel activity to STOCs. Pregnancy and steroid hormone treatment significantly increased STOCs frequency and amplitude in uterine arteries. Of importance, inhibition of STOCs with RyR inhibitor ryanodine eliminated pregnancy- and steroid hormone-induced attenuation of uterine arterial myogenic tone. Thus, the present study demonstrates a novel role of Ca2+ sparks and STOCs in the regulation of uterine vascular tone and provides new insights into the mechanisms underlying uterine vascular adaptation to pregnancy.

The development of ovarian tissue cryopreservation in Edinburgh: Translation from a rodent model through validation in a large mammal and then into clinical practice.

Ovarian tissue cryopreservation has developed rapidly from its origins in experiments in sheep in the 1990s, and is now becoming recognized as a standard, rather than experimental, procedure. This review summarizes the origins of the technique, and key milestones in its development since the first reported sheep pregnancy in 1994 to the first successful human pregnancy in 2004, and now to the first baby born following cryopreservation of prepubertal ovarian tissue. Many challenges remain to optimize this technique, to improve the survival of follicles within the reimplanted ovarian tissue, to improve its reproducibility and hence the success rate and the lifespan of the graft. The other key area remains the possibility of the grafted tissue containing malignant cells, most importantly in leukemia.

Ovine uterine artery hydrogen sulfide biosynthesis in vivo: effects of ovarian cycle and pregnancy†.

Uterine vasodilation dramatically increases during the follicular phase of the estrous cycle and pregnancy, which are estrogen-dominant physiological states. Uterine vasodilation is believed to be mainly controlled by local uterine artery (UA) production of vasodilators and angiogenic factors. The extremely potent vasodilator and proangiogenic hydrogen sulfide (H2S) is synthesized via metabolizing L-cysteine by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH). This study was designed to determine if UA H2S production increases with augmented expression and/or activity of CBS and/or CTH during the ovarian cycle and pregnancy in sheep. Uterine arteries from intact nonpregnant (NP) luteal and follicular phase and late (130-135 days, term≈145 days) pregnant (P) ewes were collected; endothelium-enriched proteins (UAendo) and endothelium-denuded smooth muscle (UAvsm) were mechanically prepared for accessing CBS and CTH proteins by immunoblotting; their cellular localization was determined by semi-quantitative immunofluorescence microscopy. H2S production was measured by the methylene blue assay. Immunoblotting revealed that CBS but not CTH protein was greater in P>>>NP follicular>luteal UAendo and UAvsm (P<0.001). H2S production was greater in P>>>NP UAendo and UAvsm (P <0.01). Pregnancy-augmented UAendo and UAvsm H2S production was inhibited by the specific CBS but not CTH inhibitor. CBS and CTH proteins were localized to both endothelium and smooth muscle; however, only CBS protein was significantly greater in P vs NP UA endothelium and smooth muscle. Thus, ovine UA H2S production is significantly augmented via selectively upregulating endothelium and smooth muscle CBS during the follicular phase and pregnancy in vivo.

Perfil hematológico durante la gestación de Ovinos de Pelo Criollos (Ovis aries) en el departamento de Córdoba, Colombia.

La etapa de gestación en los animales de producción es fundamental, puesto que es aquí donde comienza la línea de producción y donde se ve un incremento en las necesidades de algunos factores, los cuales pueden intervenir directamente en el mantenimiento de la preñez. Por la importancia de establecer valores de laboratorio de referencia para cada especie y sus grupos, además de poder utilizarlos como una herramienta diagnóstica que permita mejorar los índices productivos de las explotaciones, el presente estudio tuvo como objetivo principal determinar valores hematológicos en ovinos criollos de pelo durante la fase de gestación en el departamento de Córdoba. Fueron realizados hemogramas con un analizador automático a 74 ovejas gestantes divididas en 4 grupos (&lt;31, 31 a 60, 61 a 90 y &gt;91 días de gestación). Los resultados del presente estudio para las variables hematológicas de ovinos criollos colombianos no presentaron diferencias entre los grupos de gestación, en su mayoría, concuerdan con los reportados por otros investigadores en razas criollas tanto de ovinos como de caprinos de otros países. Se halló diferencia significativa (p≤0.05) entre el porcentaje de monocitos con relación al periodo de gestación en ovejas criollas, por lo cual este trabajo podría servir como ayuda clínica para determinar en cualquier tiempo de la gestación si el porcentaje de monocitos se encuentra acorde o no con el momento de la preñez.

Differential expression of T helper cytokines in the liver during early pregnancy in sheep.

Liver plays important roles in the innate and adaptive immunity, and contributes to the maternal immune adjustments during pregnancy in mice and rats. T helper 1 (Th1) and Th2 cytokines are related to immune response. However, expression of Th1 and Th2 cytokines in maternal livers is unclear during early pregnancy in sheep. In this study, livers were collected on day 16 of the estrous cycle and on days 13, 16 and 25 of pregnancy (n = 6 for each group) in ewes, and qRT-PCR, western blot and immunohistochemistry were used to analyze the expression of Th1 and Th2 cytokines in the livers. Our results showed that interferon-gamma (IFN-γ), interleukin (IL)-2, IL-4, IL-6 and IL-10 were downregulated, and IL-5 was upregulated in the livers during early pregnancy. Furthermore, there was no effect for early pregnancy on expression of TNF-β in the livers, and the IFN-γ protein was limited to the endothelial cells of the proper hepatic arteries and portal veins. In conclusion, early pregnancy exerted its effect on the liver to regulate the Th cytokines expression, but there was no evident shift from Th1 to Th2 cytokines, which may be necessary for the maternal hepatic immune adjustments during early pregnancy in sheep.

Characterization of the Th cytokines profile in ovine spleen during early pregnancy

ABSTRACT The spleen combines the innate and adaptive immune systems, and the early conceptus must regulate the maternal intrauterine immune and systemic immune response during early pregnancy in sheep. However, it is unclear whether early pregnancy exerts its effects on the characterization of the T helper (Th) cytokines profile in the spleen during early pregnancy in ewes. In this study, spleens were obtained at day 16 of the oestrous cycle and at days 13, 16 and 25 of pregnancy (n = 6 for each group) from ewes, and qRT-PCR, western blot and immunohistochemistry analysis were used to analyze the Th cytokines profile of the spleens. Our results showed that there was down-regulation of IFN-γ at days 13 and 16 of pregnancy, but there was upregulation of IL-2, IL-5 and IL-6 at day 25 of pregnancy, TNF-β at days 16 and 25 of pregnancy, and IL-4 and IL-10 in all pregnant groups. Immunohistochemistry results showed that the IL-6 protein was limited to the capsule, trabeculae and splenic cords. This paper reported, for the first time, that characterization of the Th cytokines profile varied in the maternal spleen during early pregnancy, which may be essential for successful pregnancy in sheep.

Immunogenicity and efficacy of Schmallenberg virus envelope glycoprotein subunit vaccines.

The Schmallenberg virus (SBV) is an orthobunyavirus that causes abortions, stillbirths, and congenital defects in pregnant sheep and cattle. Inactivated or live attenuated vaccines have been developed in endemic countries, but there is still interest in the development of SBV vaccines that would allow Differentiating Infected from Vaccinated Animals (DIVA). Therefore, an attempt was made to develop novel DIVA-compatible SBV vaccines using SBV glycoproteins expressed in baculovirus. All vaccines and phosphate buffered saline (PBS) controls were prepared with adjuvant and administered subcutaneously to cattle at 6 month of age. The first trial included 2 groups of animals vaccinated with either carboxyl-terminus glycoprotein (Gc) or PBS and boosted after 2 weeks. In the second trial, 3 groups of cattle were administered either Gc, Gc and amino-terminus glycoprotein (Gn), or PBS with a booster vaccination after 3 weeks. The animals were challenged with SBV 9 days after the booster vaccination in the first study, and 3 weeks after the booster vaccination in the second study. Using a SBV Gc-specific enzyme-linked immunosorbent assay, antibodies were first detected in serum samples 14 days after the first vaccination in both trials, and peaked on days 7 and 9 after the booster in the first and second trials, respectively. Low titers of neutralizing antibodies were detected in serum from only 3/6 and 2/4 animals in the first and second trial, respectively, at 14 days after the first vaccination. The titers increased 2 to 3-fold after the booster vaccination. SBV-specific RNA was detected in the serum and selective tissues in all animals after SBV challenge independent of vaccination status. The SBV candidate vaccines neither prevented viremia nor conferred protection against SBV infection.

The effect of consecutive pregnancies on the ovine pelvic soft tissues: Link between biomechanical and histological components

BackgroundPelvic organ prolapse, various types of incontinence (urinary incontinence, defecatory dysfunction), chronic cystourethritis, and sexual dysfunctions remain between the most common disorders in urogynecology. Currently, it is believed that the nature and number of births plays a major role in their development. Moreover, after these events, pelvic floor tissues may not recover to their original statuses. The close anatomical relationship among the vaginal wall, bladder and rectum often contribute to the emergence of anatomical-functional failure of adjacent organs and systems. Basic proceduresThe aim of this study was to investigate the effect of consecutive pregnancies on pelvic floor soft tissues, conducting biomechanical and histological analysis. Fifteen Swifter ewes: virgins, parous and pregnant were used. Samples, for uniaxial tension tests and histological analysis, were cut out from fresh tissue. A description of the mechanical properties of native tissue was obtained from the stress-strain curve. Histological samples were stained with Miller’s Elastica staining and analyzed using ImageJ software. Collagen, elastin, and smooth muscle contents (%) were analyzed along the full wall thickness of the selected organs. The links between mechanical properties of the soft tissues and histological parameters were analyzed. Main findingsMechanically, vaginal wall tissue and cervix of pregnant sheep were more compliant. In contrast, bladder and rectum became stiffer and had the highest total collagen content. Parous sheep rectum and bladder were stiffer, compared to virgin sheep. Principal conclusionsTensile strength appears to be linked to total collagen content. Elastin and smooth muscle show a direct influence on tissue compliance.