Acoustic radiation force impulse (ARFI) elastography to asses maternal and foetal structures in pregnant ewes

The aim of this study was to evaluate the stiffness of foetal lungs, liver, kidneys and placentomes by acoustic radiation force impulse (ARFI) elastography in ovine and to correlate obtained findings with foetal development. Twenty‐four ewes were included, and tissue stiffness of foetal lung, liver, kidney and placentome was measured beginning with 10th gestational week by qualitative–quantitative ARFI elastography. A total of 33 healthy lambs were born. Qualitative elastographic analysis permitted to classify maternal and foetal tissues elasticity in decreasing order as follows: placentome, kidney, liver and lung. Regarding quantitative ARFI elastography data, shear wave velocity (SWV) of foetal lung and liver varied. The lung SWV decreased gradually from the 16th to the 21st gestational week (R² = 0.80; p < 0.001), while liver SWV increased gradually from the 14th to 21st gestational week (R² = 0.80; p < 0.001). In contrast, the kidneys and placentomes SWV's remained constant through gestation (p = 0.076; and 0.34). ARFI elastography was shown feasible for evaluation of maternal and foetal tissues stiffness in the ovine model. It can be used to demonstrate pulmonary and hepatic stiffness modifications during foetal development.     

Growth of foetal bones and metabolic profile during gestation in primiparous ewes and multiparous ewes

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.     

Equine foetal gender determination in mid‐ to late gestational mares: A practical inquiry

In recent years, the interest in equine foetal gender determination (FGD) during gestation increased remarkably. Ultrasonographic FGD can be performed in two different periods during gestation. The earliest examination can take place at a gestational age of 60–70 days, whereby the genital tubercle is used to differentiate between male and female foeti. The time window of the second approach is wider (120–210 days), and there are more characteristics to take into consideration. In this article, the feasibility and accuracy of ultrasonographic FGD in mid‐ to late gestation are evaluated. One hundred twenty‐one mares from different breeds with a pregnancy stage between 120 and 270 days were examined once, using B‐scale ultrasonography (Esaote MyLab™ClassC). None of the mares were sedated nor shaved, and the procedure was completed within 15 min. Diagnosis was firstly based on the gonads. The final judgement was made based on all visible foetal reproductive organs. In three cases with a pregnancy stage beyond 257 days, FGD was not possible. All of the examined mares in which a FGD could be performed gave birth to a healthy foal. In 98% of the examinations (116/118), the diagnosis was correctly made. In both cases of misdiagnosis, only one characteristic was seen during the procedure and wrongly interpreted. Beyond 210 days of pregnancy, the extremities can preclude a good visualization of the inguinal region. In conclusion, equine FGD in mid‐ to late gestation is an accessible and accurate technique, although a good ultrasound device is a prerequisite and experience and expertise is necessary.     

Preliminary study on metabolic profile of pregnant and non‐pregnant ewes with high or low degree of behavioral lateralization

To evaluate the metabolic profile of pregnant and non‐pregnant ewes with high or low degree of behavioral lateralization, we used 57 clinically healthy Sardinian ewes. They were divided into three groups: Group A (nonpregnant ewes), Group B (pregnant ewes with one fetus) and Group C (pregnant ewes with twin fetuses). Using behavioral tests, in all groups lateralization was established and ewes were distinguished into ewes with high and low level of lateralization. Every 30 days for 4 months (at 35, 76, 103 and 145 days of gestation), blood samples were collected and total cholesterol, triglycerides, nonesterified fatty acids (NEFA), glucose, insulin, creatinine, urea, uric acid, total protein, total bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ‐glutamine transferase (γ‐GT), β‐hydroxybutyrate (β‐OHB), chloride (Cl), potassium (K), sodium (Na) and calcium (Ca) were assessed. The repeated measures multifactor analysis of variance (MANOVA) showed statistical differences of time, pregnancy and degree of lateralization on some studied parameters. These results confirm that pregnancy represents a very important metabolic moment and show the effect of the degree of behavioral lateralization on the assessment of metabolic parameters in non‐pregnant and pregnant ewes. Further studies are necessary to value the influence of these factors on other parameters such as hematological parameters and hormones.     

Impact of energy and protein restriction on energy expenditure of gestation in twin-bearing ewes

This study aimed to investigate the impact of energy and protein restriction on energy expenditure of gestation (EE_gest) in twin‐bearing ewes. Multiparous twin‐bearing ewes were fed either adequate (A: n = 10) or restricted to 60% of energy and protein requirements (R: n = 10) during the last 6 weeks of gestation. Whole‐body energy expenditure (EE) and retained energy (RE) were calculated from respiratory gaseous exchange combined with nitrogen balance at 7, 5 and 2 weeks prepartum. Twin lamb birth weight was lower in the R group compared to those in the A group (7.9 ± 0.31 vs 9.3 ± 0.19 kg, P < 0.01). The EE_gest was lower in the R group than the A group (4.6 vs 5.9 MJ/day, SE = 0.30, P < 0.01). Between 5 and 2 weeks prepartum, EE_gest contribution to the whole‐body EE significantly (P < 0.01) increased from 39% to 45% and from 34% to 40% in the A and R groups, respectively. Based on kg conceptus weight, both EE_homeorhetic (from 292 to 270 kJ/kg/day, SE = 6.2, P < 0.001) and EE_conceptus (from 259 to 177 kJ/kg/day, SE = 23.8, P = 0.02) decreased between weeks 5 and 2 prepartum. The EE_conceptus tended to be lower (P = 0.06) in the R group than the A group both at 5 weeks (219 vs 298 kJ/kg/day, SE = 32.8) and 2 weeks (from 138 to 217 kJ/kg/day, SE = 30.1) prepartum. In conclusion, energy and protein restriction reduced energy expenditure of gestation calculated per kg conceptus weight. The decrease may be associated with energy expenditure of conceptus growth and maintenance.     

Pharmacokinetics of chlortetracycline in maternal plasma and in fetal tissues following oral administration to pregnant ewes

The purpose of this study was to determine if concentrations of chlortetracycline could be detected in fetal plasma or tissues after administering an oral dose of chlortetracycline (CTC; 500 mg/head/day) reported to be effective in controlling Campylobacter spp. abortions. Five pregnant ewes were administered 250 mg/head twice a day (total dose 500 mg/hd/d) for 7 days. On the beginning of day 7, intravenous catheters were surgically implanted or inserted into the fetus and dam. Plasma samples were collected from the ewe and fetus at various time points before and up to 36 hr after the last dose of CTC. All ewes were then sacrificed, and tissues were harvested from the fetus for drug analysis. Concentrations of CTC in maternal plasma were consistent with our previous study and below the minimum inhibitory concentration of Campylobacter abortion isolates. Concentrations of CTC were below the limit of detection in three of five fetal plasma samples and all of the placenta, amniotic fluid, and fetal stomach contents. Low concentrations were detectable in fetal kidney and liver, suggesting that CTC reaches the fetus, although at a variable and low ratio when compared to maternal concentrations.     

实时B型超声扫描在绵羊妊娠监测中的应用

随着超声诊断技术的不断发展，它在兽医领域的应用也越来越广泛。目前，B型超声主要用于对动物妊娠期子宫和胎儿的监测，关于牛、羊、猪、马、犬、等动物都有应用B型超声监测妊娠的大量报道。本文仅就B型超声在绵羊早期妊娠诊断、怀胎数的判断及判断胎龄三方面进行综述。     

Activity of selected glycosidases and availability of their substrates in bovine placenta during pregnancy and parturition with and without retained foetal membranes

The activity of glycosidases is crucial for the function and biological activity of proteins conjugated with sugar moieties, which play an important role in adhesion of cells during attachment and detachment of the foetal membranes. The aim of study was to describe the ability of bovine placental tissues to break down O-glycosidic bonds in different glycoproteins by the determination of activity of β-galactosidase, α-l-fucosidase, β-N-acetyl-hexosaminidase and sialidase in early–mid-pregnancy as well as at parturition with released and retained foetal membranes. Moreover, the availability of substrates for these glycosidases in placental homogenates was evaluated. Placental samples were collected from pregnant (2–4 months) cows in slaughterhouse (n = 8) as well as during Caesarean section and divided into released foetal membranes (n = 8) and retained foetal membranes (n = 8). Tissue homogenates were subjected to spectrofluorimetric and spectrophotometric determinations of enzyme activities as well as electrophoretic separations. Enzyme activities expressed changes within examined time with significant (p < .05) differences between pregnancy and physiological parturition in β-N-acetyl-hexosaminidase and α-l-fucosidase in foetal part of placenta while in maternal part only in the latter one. Decreasing tendency in enzyme activity was noticed in foetal part of retained samples in comparison with released ones with significant (p < .05) differences in α-l-fucosidase activity. The analysis of staining of sugar moieties attached to selected proteins depicted availability of sugar molecules in examined tissues, but their patterns differed between samples. In conclusion, sugar moieties in conjugated proteins express changes in the course of pregnancy which is reflected by the alterations in activities of placental glycosidases.     

Effects of progestagen exposure duration on estrus synchronization and conception rates of crossbreed ewes undergoing fixed time artificial insemination

Synchronization of estrus and ovulation are of paramount importance in modern livestock improvement programs. These methods are critical for assisted reproduction technologies, including artificial insemination and embryo transfer, that can increase productivity. In the current study, subcutaneous implants containing norgestomet were placed for long (14 days), medium (9 days), and short (5 days) periods of time in 70 crossbred ewes undergoing fixed-time artificial insemination. The resulting effects on estrus synchronization and conception rates were subsequently evaluated. Among the synchronized ewes, 85.7% (60/70) underwent estrus over a period of 72 h after progestagen treatment ceased. The shortest mean interval between withdrawal of the device and onset of estrus (34.2 ± 8.9 h) was observed in the G14 days of P4 group (p < 0.05). The conception rate of the G14 days of P4 group was statistically higher than that of the other groups (83.3% vs. 60.9% vs. 47.8%; p < 0.05). In conclusion, 14 days of norgestomet treatment produced higher conception rates and a greater number of pregnancies at the beginning of the breeding season.     

Pharmacokinetics of tulathromycin in fetal sheep and pregnant ewes

Macrolides are important antimicrobials frequently used in human and veterinary medicine in the treatment of pregnant women and pregnant livestock. They may be useful for the control of infectious ovine abortion, which has economic, animal health, and human health impacts. In this study, catheters were surgically placed in the fetal vasculature and amnion of pregnant ewes at 115 (±2) days of gestation. Ewes were given a single dose of 2.5 mg/kg tulathromycin subcutaneously, and drug concentrations were determined in fetal plasma, maternal plasma, and amniotic fluid at 4, 8, 12, 24, 36, 48, 72, 144, and 288 hr after drug administration. Pharmacokinetic parameters in maternal plasma were estimated using noncompartmental analysis and were similar to those previously reported in nonpregnant ewes. Tulathromycin was present in fetal plasma and amniotic fluid, indicating therapeutic potential for use against organisms in these compartments, though concentrations were lower than those in maternal plasma. Time‐course of drug concentrations in the fetus was quite different than that in the ewe, with plasma concentrations reaching a plateau at 4 hr and remaining at this concentration for the remainder of the sampling period (288 hr), raising questions about how tulathromycin may be transported into or metabolized and eliminated by the fetus.     

Characterization of local and peripheral immune system in pregnant and nonpregnant ewes

Modulation of the immune system is known to be important for successful pregnancy but how immune function might differ between the lymph nodes draining the reproductive tract and peripheral lymph nodes is not well understood. Additionally, if immune system changes in response to the presence of an embryo during early pregnancy, and if this response differs in local versus peripheral immune tissue, has not been well characterized. To address these questions, we examined expression of genes important for immune function using NanoString technology in the ampulla and isthmus of the oviduct, endometrium, lymph nodes draining the reproductive tract (lumbo-aortic and medial iliac) as well as a peripheral lymph node (axillary), the spleen, and circulating immune cells from ewes on day 5 of the estrous cycle or pregnancy. Concentrations of estradiol and progesterone in plasma were also determined. Principal component analysis revealed separation of the local from the peripheral lymph nodes (MANOVA P = 3.245e-08, R² = 0.3) as well as separation of tissues from pregnant and nonpregnant animals [lymph nodes (MANOVA P = 2.337e-09, R² = 0.5), reproductive tissues (MANOVA P = 2.417e-14, R² = 0.47)]. Nine genes were differentially (FDR < 0.10) expressed between lymph node types, with clear difference in expression of these genes between the lumbo-aortic and axillary lymph nodes. Expression of these genes in the medial iliac lymph node was not consistently different to either the axillary or the lumbo-aortic lymph node. Expression of IL10RB was increased (FDR < 0.05) by 24% in the reproductive tissue of the pregnant animals compared to nonpregnant animals. Analysis of gene categories revealed that expression of genes of the T-cell receptor pathway in reproductive tract tissues was associated (P < 0.05) with pregnancy status. In conclusion, assessment of gene expression of reproductive and immune tissue provides evidence for a specialization of the local immune system around the reproductive tract potentially important for successful establishment of pregnancy. Additionally, differences in gene expression patterns in reproductive tissue from pregnant and nonpregnant animals could be discerned as early as day 5 of pregnancy. This was found to be associated with expression of genes important for T-cell function and thus highlights the important role of these cells in early pregnancy.     

Early luteal development in Santa Inês ewes superovulated with reduced doses of porcine follicle‐stimulating hormone

The aim was to compare the early luteal development in ewes superovulated with different doses of pFSH. Twenty‐nine Santa Inês ewes received a progesterone device (CIDR®) for 8 days. Gonadotrophic treatment started on Day 6: G200 (control, n = 9, 200 mg); G133 (n = 10, 133 mg); and G100 (n = 10, 100 mg of pFSH). On Day 6, all females received eCG (300 IU). B‐mode and spectral Doppler ultrasonography were performed daily during the early luteal phase (Days 11–15) to monitor the development of corpora lutea (CLs; dimensions) and ovarian arteries indices. CLs were also classified as normal or prematurely regressed (PRCL) on Day 15 by videolaparoscopy. Ewes from G100 and G133 showed gradual increase in luteal diameter during the early luteal phase (p < 0.001), whereas G200 animals presented increase from Day 11 to Day 13, and then decrease on Days 14 and 15 (p < 0.001). The G200 females showed greater percentage of PRCL (45.20%) than those of the other groups (p < 0.001). The normal CLs number was greater in G100 than in G133 (p = 0.04), while the PRCL number was greater in G200 than in the other groups (p = 0.03). Resistive index (RI) was greater in G200 than in G100 (p = 0.02). RI was lower in Day 12 than Day 15 (p = 0.02). Pulsatility index (PI) was greater on Days 14 and 15 (p < 0.01). In conclusion, the lowest dose of pFSH (100 mg) can be considered sufficient for an efficient superovulatory response in sheep, producing better CLs development dynamic in early luteal phase and ovarian blood perfusion and smaller number of PRCL than the traditional (200 mg) pFSH dose.     

Quantitative ultrasound elastography and biometry of the bitch uterus in the early puerperium after vaginal delivery and caesarean section

Elastography is an actual imaging method used to qualitatively and quantitatively evaluate the elastic properties of tissues. The aim of this study was to evaluate and compare uterine tissue biometry and stiffness during post-partum period in brachycephalic bitches (n = 12) after c-section (GCS; n = 8) or normal delivery (GNB; n = 4). These animals were evaluated daily by abdominal ultrasound from the day of delivery until the 10th day post-partum; measuring uterine diameter, myometrial and endometrial thickness (mm) and shear wave velocity (SWV; m/s), by B-mode and ARFI (acoustic radiation force impulse) elastography, respectively. Uterine diameter was higher (p = .012) in animals submitted to c-section (15.26 ± 4.73 mm) than in normal birth (12.53 ± 2.64 mm) during the first 7 days post-partum. Uterine thickness gradually involuted in both groups (p < .0001), the myometrium during the first 9 days (p = .005) and the endometrium during the first 6 days (p = .003). The myometrial and endometrial SWVs were similar between types of delivery (p = .7846 and .8273) and presented a gradual increase (p = .411; .0043, respectively), during the first 10 days post-partum. It was concluded that bitches with normal delivery had smaller uterine thickness and faster puerperal involution than submitted to c-section, while uterine tissue stiffness was similar between delivery types and increased gradually during post-partum.     

Effects of nutrient restriction and melatonin supplementation on maternal and foetal hepatic and small intestinal energy utilization

To determine how nutrient restriction and melatonin supplementation influence ewe and foetal hepatic and small intestinal energy use, 32 primiparous ewes on d 50 of gestation were fed 60% (RES) or 100% (ADQ) of NRC recommendations with 0 (CON) or 5 mg/d (MEL) of dietary melatonin. On d 130 of gestation, small intestine and liver were weighed and collected. Data were analysed as a completely randomized design with a 2 × 2 factorial arrangement of treatments. Liver weight (g/kg EBW) decreased (p = 0.02) in RES ewes. Jejunum weight (g/kg BW) increased (interaction p = 0.04) in ADQ‐MEL ewes compared with all other treatments. Total in vitro O₂ consumption (mol/min/tissue) and total citrate synthase activity (mol/min/tissue and mol/min/kg EBW) in liver decreased (p ≤ 0.03) in RES ewes. Oxygen consumption (mol/min/kg EBW) increased (interaction p = 0.02) in jejunum of ADQ‐CON versus RES‐MEL and ADQ‐CON. Citrate synthase activity (mol/min/kg of EBW) increased (interaction p = 0.03) in jejunum of ADQ‐MEL compared with RES‐MEL and ADQ‐CON. Foetal liver weight (g/kg BW) decreased (p = 0.02) in RES versus ADQ. Foetal small intestine weight (g/kg BW) decreased (interaction p = 0.05) in RES‐MEL versus ADQ‐MEL. Total O₂ consumption (mol/min/tissue) and total citrate synthase activity (mol/min/kg of BW) in foetal liver decreased (p ≤ 0.05) in RES versus ADQ. Foetal small intestinal O₂ consumption (mol/min/kg of BW) was greater (interaction p = 0.03) in RES‐CON and ADQ‐MEL than RES‐MEL and ADQ‐CON. Maternal nutrient restriction had a greater effect than melatonin supplementation on liver and jejunum mass and energy utilization in dams and foetuses. Because intestinal mass and energy utilization were more responsive to melatonin supplementation in ewes fed adequate nutrition compared with restricted ewes, melatonin may have limited use as a therapeutic supplement to help overcome potential negative effects of nutrient restriction.     

Comparative analysis between endometrial proteomes of pregnant and non-pregnant ewes during the peri-implantation period

Background

Early pregnancy failure has a profound impact on both human reproductive health and animal production. 2/3 pregnancy failures occur during the peri-implantation period; however, the underlying mechanism(s) remains unclear. Well-organized modification of the endometrium to a receptive state is critical to establish pregnancy. Aberrant endometrial modification during implantation is thought to be largely responsible for early pregnancy loss.

Result

In this study, using well-managed recipient ewes that received embryo transfer as model, we compared the endometrial proteome between pregnant and non-pregnant ewes during implantation period. After embryo transfer, recipients were assigned as pregnant or non-pregnant ewes according to the presence or absence of an elongated conceptus at Day 17 of pregnancy. By comparing the endometrial proteomic profiles between pregnant and non-pregnant ewes, we identified 94 and 257 differentially expressed proteins (DEPs) in the endometrial caruncular and intercaruncular areas, respectively. Functional analysis showed that the DEPs were mainly associated with immune response, nutrient transport and utilization, as well as proteasome-mediated proteolysis.

Conclusion

These analysis imply that dysfunction of these biological processes or pathways of DEP in the endometrium is highly associated with early pregnancy loss. In addition, many proteins that are essential for the establishment of pregnancy showed dysregulation in the endometrium of non-pregnant ewes. These proteins, as potential candidates, may contribute to early pregnancy loss.

Electronic supplementary material

The online version of this article (doi:10.1186/s40104-015-0017-0) contains supplementary material, which is available to authorized users.     

Qualitative and quantitative comparison of renal vs. hepatic ultrasonographic intensity in healthy dogs

A prospective, cross-sectional study was performed to qualitatively and quantitatively compare the echogenicity of the renal cortex relative to the liver in healthy dogs. Twenty-five normal adult dogs were examined ultrasonographically. Three standard B-mode images (8.0 MHz) and three tissue harmonic images of the cranial pole of the right kidney adjacent to the caudate lobe of the liver were obtained. Renal and hepatic echogenicities were qualitatively compared by two observers. Subsequently, regions of interest (ROIs) were drawn in the renal cortex and the adjacent liver parenchyma at equal depths on each image, using two different ROI geometries: deep adjacent half-annular ROIs centered at the focal zone and small superficial adjacent squares placed in the near field. Renal and hepatic mean pixel intensities were quantified and averaged for individual subjects. Qualitatively, the right renal cortex was more commonly hyperechoic to liver. Quantitatively, the renal cortical mean pixel intensity was significantly higher than that of liver using deep half-annular ROIs, but not superficial square ROIs, for both standard (P = 0.0007) and harmonic (P = 0.0107) tissue imaging. These findings suggest that the renal cortex can be slightly hyperechoic to adjacent liver. The framework within which the canine renal cortical parenchyma is routinely evaluated in abdominal ultrasonography should be reconsidered, and mild hyperechogenicity relative to the liver (at 8.0 MHz) interpreted as a normal finding.     

Colour Doppler ultrasound imaging of blood flows variations in neoplastic and non‐neoplastic testicular lesions in dogs

Testicular tumours are the most common neoplasms in male dogs accounting for approximately 90% of all tumours affecting the genitourinary tract. Gray‐scale ultrasonography in combination with colour and power Doppler imaging has been well accepted as an accurate technique for assessing scrotal lesions and vascularization of the testis. Colour Doppler sensitivity for low blood flows appears promising in the study of testicular disorders. The aim of this study was to assess if colour and power Doppler ultrasound is a good tool for the investigation of testicular lesions in dogs, to report the sonographic features of lesions and to measure colour and power Doppler parameters such as resistive index (RI), pulsatility index (PI), hypovascularization and hypervascularization (VI) determining if they can be used to distinguish testicular neoplasms from the wide spectrum of non‐neoplastic pathological findings. In this study, 50 male dogs of various breeds, aged between 7 and 14 years, presented with testicular disorders were selected. RI and PI were calculated. Mean RI values for neoplastic, inflammatory and degenerative lesions were 0.54, 0.45 and 0.58, respectively. Mean PI values were 0.62, 0.55 and 0.63, respectively. Hypovascularization and hypervascularization of the lesion were evaluated throughout the vascularity index (VI). Vascular signals in neoplasms were significantly intensified around and inside the mass if compared with those measured during inflammatory and degenerative lesions. VI markedly increased in solid tumours. Pathological testes were removed; macroscopical, histological and immunoistochemical evaluations were carried out. Colour Doppler showed increased intralesional and peripheral flows in all neoplastic lesions analysed. No flows were detected around cysts.