绵羊输卵管上皮细胞的分离与培养

通过建立输卵管上皮细胞的体外培养和传代的方法，进行形态学观察及冻存。取雌性绵羊输卵管上皮细胞，用胶原酶消化和不锈钢网过筛法分离输卵管内膜基质细胞和上皮细胞，进行单细胞原代及传代培养。在输卵管上皮细胞的培养可持续培养4-6周，传3-4代。原代培养细胞7d长成单层，传代细胞4d长成单层。该研究旨在建立一套输卵管上皮细胞体外培养和传代的方法，为进一步研究β-防御素在雌性生殖道的作用方面提供很好的研究模型。     

Three‐Dimensional Study of the Terminal Portion in Sprague‐Dawley Rat Ejaculatory Ducts

In mammals, a pair of ejaculatory ducts exists in the urethra at the seminal colliculus. The detailed anatomical structures of the distal end of the ejaculatory ducts of Sprague‐Dawley rats were investigated by the computer‐assisted three‐dimensional reconstruction analysis using light‐microscopic serial sections. A three‐dimensional reconstruction revealed that in adult rats, the ejaculatory sinus pair consists of two parts: the cranial section – a compartment region composed of a fusion of the ampullary gland duct and the seminal vesicle duct, and the caudal section – a grooved region composed of a long slitlike ejaculatory ostium that extends into the urethra on both sides of the seminal colliculus. But the sphincter structure was not observed. The long axis of the compartment region was approximately 58 μm in length, and that of the groove region was approximately 495 μm. Although many epithelial glands ducts were distributed throughout the ejaculatory sinuses, the prostate and coagulation gland ducts did not open in these sinuses. The urethra was composed of transitional epithelium, while the ejaculatory sinuses were composed of single to stratified cuboidal epithelium. The ejaculatory ducts continued to the ejaculatory ostium in male adult Sprague‐Dawley rat were composed of the seminal vesicle ducts received the ampullary gland ducts.     

Histological Characteristics of the Equine Oviductal Mucosa at Different Reproductive Stages

The objective of this work was to study cellular changes in the epithelium of the mare’s oviduct. Oviductal samples were taken from mares at different reproductive stages for optical microscopy and Hoechst 33258 staining. Glandular-like structures were observed in 100% of the oviducts. These structures were of the tubular type and were formed by ciliated and nonciliated epithelial cells arranged in a way similar to the epithelial surface. The amount of structures decreased progressively from the ampulla to the isthmus, but did not change through the different reproductive stages. Histological changes in the epithelium of the oviduct were observed associated with the reproductive stages. In the ampulla, the amount of ciliated cells decreased in the anovulatory phase compared with other reproductive stages. Cords of connective tissue lined by epithelium (trabeculae) and dividing completely the lumen of the oviduct were found in 50% of the oviducts. Epithelial cells projected toward the lumen as large vesicles of cytoplasm, sometimes containing a nucleus. The amount of cells presenting nuclear protrusion varied throughout the oviduct, with highest incidence in the ampulla, decreasing progressively toward the isthmus (P < .05). In addition, nuclear protrusions were higher in number during the anovulatory and luteal phases than in the other reproductive stages (P < .05). These nuclear protruding cells appeared to be extruding from the epithelium and showed no signs of apoptosis based on the histological and fluorescent stains used. The existence of these gland-like structures in the oviductal mucosa should be considered when studying the oviductal physiology in mares.     

Effect of Bovine Sperm‐Bound Antisperm Antibodies on Oviductal Binding Index

This study was designed to test the hypothesis that sperm‐bound IgG and IgA decrease binding of bull spermatozoa to oviductal epithelial cells in vitro. Three ejaculates were cryopreserved from each of four antisperm antibody (ASA)‐negative satisfactory breeder bulls. Bulls were then immunized with autologous spermatozoa, and three ASA‐positive ejaculates were cryopreserved from each bull post‐immunization. First, microscopy methods were compared to select the most appropriate assay for evaluation of oviductal binding index (BI). The BI did not differ when the evaluation was performed under fluorescence microscopy (131.1 sperm/mm²; 62.5–251.1 sperm/mm²), phase‐contrast microscopy (160.5 sperm/mm²; 56.8–397.4 mm²) or their combination (116.4 sperm/mm²; 56.8–249.6 sperm/mm²) (Median; IQR). The combination of microscopy methods was selected as it allowed better visualization of cells. Then, BI was compared between ASA‐negative and ASA‐positive ejaculates, and the association between BI and ASA binding was evaluated. The BI was less in ASA‐positive (114.9; 0 to 201.8 sperm/0.1 mm²) than ASA‐negative samples (218.9; 24.7 to 276.8 sperm/0.1 mm²) (P = 0.0002). This reduction in BI was significant in three of the four bulls. Regression analysis identified a negative association between BI and the percentage of IgG‐bound (p = 0.013) but not IgA‐bound spermatozoa. In conclusion, sperm‐bound IgG decreased the ability of bovine spermatozoa to bind to oviductal epithelial cells in vitro.     

Age‐ and Gender‐Dependent Changes of Bovine Myocardium Architecture

Growth, gravidity and lactation put high demands on the performance of the myocardium. The aim of this study, which was performed in 40 female and 20 male bovines ranging from 1 to 4.5 years old, was to determine gross and microscopic morphometric data of bovine myocardium to establish a comparative measure of myocardial growth during juvenile development. During the developmental stage of young adulthood, age‐related increases in female myocardial characteristics included cardiac mass, left and right ventricular mass and the ratio of cardiac mass to loose connective tissue. Age‐related decreases were observed in the number of myocyte nuclei per mm² and the thickness of the right ventricular wall. Sex differences in these parameters were found between 2‐year‐old bulls (N = 20) and 2‐year‐old heifers (N = 10), with males having heavier hearts, thicker ventricular walls, less myocytes in the left ventricle and less connective tissue in both ventricles. Age and sex had no influence on the ratio of capillaries to myocytes, estimated at 0.98 in the adult bovine. Capillary density does not change during juvenile development, but cross‐sectional capillary area does adapt to myocyte cross‐sectional area, accounting for this relatively constant ratio.     

Ovine sperm DNA oxidation quantification using an 8‐OHdG immunodetection assay

Our aim was to optimize 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) immunodetection in order to detect DNA damage caused by oxidative stress that may not be detected by other DNA integrity analysis techniques, especially due to the high compaction of DNA in ruminants. Semen samples from 6 rams were cryopreserved. After thawing, samples were subjected to the DNA oxidation quantification using an 8‐OHdG immunodetection assay by flow cytometry. We have evaluated two different incubation times (30 min vs. overnight) at 4°C of the primary antibody (monoclonal anti‐8‐OHdG antibody). We have also compared the results of this technique with the sperm chromatin structure assay (SCSA^®). The analysis revealed that there were no significant differences (p > .05) between different incubation times. However, overnight incubation seems to cause more non‐specific binding of the secondary antibody. Significant differences (p < .05) between subjects and oxidation controls (8 M H₂O₂/800 μM FeSO₄?7H₂O) were evident. We can conclude that the 8‐OHdG immunodetection assay for DNA oxidation quantification of ram sperm can be performed subjecting sperm samples to a very high oxidative treatment.     

Three‐dimensional T1‐weighted gradient echo is a suitable alternative to two‐dimensional T1‐weighted spin echo for imaging the canine brain

Volumetric imaging (VOL), a three‐dimensional magnetic resonance imaging (MRI) technique, has been described in the literature for evaluation of the human brain. It offers several advantages over conventional two‐dimensional (2D) spin echo (SE), allowing rapid, whole‐brain, isotropic imaging with submillimeter voxels. This retrospective, observational study compares the use of 2D T1‐weighted SE (T1W SE), with T1W VOL, for the evaluation of dogs with clinical signs of intracranial disease. Brain MRI images from 160 dogs who had T1W SE and T1W VOL sequences acquired pre‐ and postcontrast, were reviewed for presence and characteristics of intracranial lesions. Twenty‐nine of 160 patients were found to have intracranial lesions, all visible on both sequences. Significantly better grey‐white matter (GWM) differentiation was identified with T1W VOL (P < .001), with fair agreement between the two sequences (weighted κ = 0.35). Excluding a mild reduction in lesion intensity in three dogs precontrast on the T1W VOL images compared to T1W SE, and meningeal enhancement noted on the T1W VOL images in one dog, not identified on T1W SE, there was otherwise complete agreement between the two sequences. The T1W VOL sequence provided equivalent lesion evaluation and significantly improved GWM differentiation. Images acquired were of comparable diagnostic quality to those produced using a conventional T1W SE technique, for assessment of lesion appearance, number, location, mass effect, and postcontrast enhancement. T1W VOL, therefore, provides a suitable alternative T1W sequence for canine brain evaluation and can facilitate a reduction in total image acquisition time.     

Three‐dimensional CT observation of position and movability of the scapula in the horse using carcasses of Falabella

The three‐dimensionally real position and movement of the scapula in the lateral side of the ribcage could not be clarified in the horse, since the body size of the horse is too large to apply the CT scanning and image analysis methods. In this study, therefore, we examined the position and the movability of scapula using a carcass of the Falabella which is one of the smallest breeds of the horse. The whole skeletal system in thoracic part of the Falabella could be three‐dimensionally observed by CT scanning method. The three‐dimensional images show that the scapula cranially slides and the ventral part of the scapula dorso‐cranially rotates, when the shoulder joint moves to the most cranial position as simulation. The three‐dimensional rotation angle was approximately 10 degrees. As a result of comparative osteology of the scapula between Falabella and the large draft horse, the infraspinous fossa was caudally enlarged in the larger draft horse, whereas the Falabella had narrower infraspinous fossa. We suggest that it may be due to the adaptational morphological change in the scapula bearing various body weights among breeds. The three‐dimensional CT image analysis and the simulation in carcass contribute to the analysis of the bone movements of the horse during walking and running locomotion as well as the motion analysis from external functional‐morphological data.     

Three‐dimensional CT examination of the forefoot and hindfoot of the hippopotamus and tapir during a semiaquatic walking

Semiaquatic walking has resulted in the evolution of functional and morphological changes in various hoofed mammals, such as hippopotamus and Brazilian tapir. The biomechanics of skilful walking in wetlands or at the bottom of a waterbody involve the medio‐lateral opening and closing of the feet to effectively support and stabilize the body on soft ground and to reduce the water resistance during recovery stroke, respectively. We demonstrate that the opening and closing of the feet in hippopotamus and Brazilian tapir are mediated by the adduction and abduction of the most medial and lateral phalanges from the CT examination. The axial toes, metacarpals and metatarsals do not contribute to changes in the width and shape of the feet, unlike the medial and lateral toes. We suggest that this semiaquatic walking motion is derived from the original terrestrial mode of locomotion, in contrast to the highly functional swimming motion using webs or fins in morphologically modified feet and tail. From the present data we demonstrate that semiaquatic locomotion evolved due to the acquisition of adductor–abductor mobility in the phalanges of the most medial and lateral digits, as shown in hippopotamus and Brazilian tapir.