幼年牦牛松果体的光镜和电镜观察

利用光镜和电镜技术对幼年牦牛松果体的组织形态结构特征进行了研究。结果表明，光镜下，幼年牦牛松果体由松果体细胞、少量的神经胶质细胞、毛细血管和神经等组成。电镜下，松果体细胞的电子致密度低，细胞质内含有丰富的线粒体、粗面内质网、滑面内质网、高尔基复合体、微管、微丝和核糖体，典型异质细胞器突触带呈球形，多位于质膜附近。神经胶质细胞内的线粒体丰富，胞体突起呈球形膨大伸入到松果体细胞之间。松果体细胞以及神经胶质细胞间均存在突触和连接复合体。牦牛松果体内的毛细血管为连续型，远腹侧血管周围可见色素细胞。     

奶牛乳腺上皮细胞的原代培养及其生物学特性分析

旨在从奶牛乳腺组织中分离原代乳腺上皮细胞（bovine mammary epithelial cells,BMECs）并传代培养后探究其生物学特性。本研究从屠宰场采集健康泌乳奶牛乳腺并采用改进的酶消化法从乳腺中分离得到原代奶牛乳腺上皮细胞,通过形态学观察、免疫荧光以及染色体核型分析的方法对其进行鉴定。同时,研究第3、第6和第9代乳腺上皮细胞的生长曲线、群体倍增时间和冻存复苏活力,检测不同代次细胞分泌乳蛋白、乳脂、乳糖的功能及泌乳相关基因的表达。结果表明,所分离的奶牛乳腺上皮细胞纯度较好,细胞生长呈现S型,3个代次细胞的群体倍增时间依次为34.87、41.45和65.04 h,冻存复苏活力为88%~93%;在细胞分泌功能方面,诱导培养2 d后均能检测到酪蛋白、甘油三酯和乳糖,且各代次间无显著差异;此外,3个代次的细胞诱导后均能表达乳成分合成相关基因。本研究成功培养了原代奶牛乳腺上皮细胞,并证明直到第9代细胞仍然具有正常的生物学功能,为体外探究乳腺细胞增殖与分化机制提供了良好的试验材料和技术支撑。     

Effect of anaesthesia on cell-mediated immunity in dogs undergoing mastectomy for mammary cancer

ObjectiveTo investigate if anaesthesia for canine cancer mastectomy further influences host cell-mediated immunity (CMI) promoting cancer progression.Study designA randomized, controlled, blinded clinical study.AnimalsA total of 20 bitches with malignant mammary tumours of clinical stage II or III undergoing the same type of mastectomy (regional mastectomy).MethodsDogs were randomly allocated to one of two anaesthetic groups (10 per group). The anaesthetic protocol of group A used minimally immunosuppressive drugs (tramadol, robenacoxib, propofol), whereas that of group B (control) used more immunosuppressive drugs (morphine, fentanyl, thiopental, isoflurane). For each animal, measurements of white blood cells (WBCs), neutrophils and lymphocytes, and flow cytometric assessment of T cells (CD3⁺), helper T cells (CD4⁺), cytotoxic T cells (CD8⁺) and CD5^low+ T cells were performed prior to anaesthesia (day 0) and on days 3 and 10 postsurgery. Data were analysed using a General Linear Model for repeated measures and presented as mean ± standard deviation, p ≤ 0.05.ResultsIn all animals, on day 3, WBCs and neutrophils were significantly increased (p < 0.0005), while flow cytometry revealed significantly decreased relative percentages of T cells (CD3⁺) (p = 0.003) and their subpopulations CD4⁺ (p = 0.006), CD8⁺ (p = 0.029) and CD5^low+ (p = 0.031). Specifically, on day 3, the cytotoxic T cells (CD8⁺) were significantly decreased (p = 0.05) only in group B, whereas the CD4⁺ (p = 0.006) and CD5^low+ (p = 0.008) T cells in group A. The only significant difference between groups was found preoperatively in the CD4⁺/CD8⁺ ratio, which was higher in group A (p = 0.006).Conclusions and clinical relevanceIn dogs with mammary cancer undergoing regional mastectomy, a significant decrease in components of CMI was observed on day 3 postsurgery in both anaesthetic groups. Some indication, however, for better preserved cellular immunity by less immunosuppressive anaesthetic/analgesic drugs was detected, rendering their use advisable.     

ULTRASONOGRAPHIC EVALUATION OF ADRENAL GLAND SIZE COMPARED TO BODY WEIGHT IN NORMAL DOGS

The accepted cut‐off value for adrenal gland maximum diameter of 0.74 cm to distinguish adrenal gland enlargement in dogs regardless of body weight may not be appropriate for small to medium breed dogs. The purpose of the current retrospective study was to examine adrenal gland dimensions as a function of body weight in healthy dogs in three weight categories (< 10 kg, 10–30 kg, and > 30 kg) representing small, medium, and large breeds, respectively, to establish greater confidence in determining if adrenal gland size is abnormal. The measurements of length (sagittal plane), cranial and caudal pole thickness (sagittal and transverse planes), and caudal pole width (transverse plane) of both adrenal glands were obtained ultrasonographically in clinically healthy dogs (n = 45) with 15 dogs in each weight group. Findings support our hypothesis that adrenal gland size correlates with body weight in normal dogs, and more precise reference intervals should be created for adrenal gland size by categorizing dogs as small, medium, or large breed. The caudal pole thickness of either adrenal gland in a sagittal plane was the best dimension for evaluating adrenal gland size based on low variability, ease, and reliability in measurement.     

INTRAARTERIAL INJECTION OF IODINATED CONTRAST MEDIUM FOR CONTRAST ENHANCED COMPUTED TOMOGRAPHY OF THE EQUINE HEAD

Minimizing the volume of contrast administered for contrast‐enhanced computed tomography (CT) of the equine head is desirable for reducing costs and risks of adverse reactions, however evidence‐based studies on the effects of varying volumes on image quality are currently lacking. The objective of the current study was to determine whether low‐volume intraarterial administration of contrast medium would result in an equivalent image quality and tissue attenuation vs. high‐volume intravenous bolus administration. A prospective cross‐over experimental design was used in a sample of six horses. After anesthetic induction, the right carotid artery was exposed surgically and catheterized. Four CT scans of the cranium were performed for each horse: baseline, immediately following intraarterial contrast injection, five‐min postinjection (return to baseline) and a final scan after intravenous contrast administration. Soft tissue attenuation in predetermined regions of interest (ROI); and length, width, and height measurements of the pituitary gland were recorded at each time point. Horses were euthanized and measurements of the pituitary gland were repeated postmortem. No adverse reactions to contrast administration were observed. Intraarterial and intravenous administration of contrast medium resulted in significantly greater soft tissue enhancement of some brain ROI's and the pituitary gland vs. baseline values. Pituitary gland measurements made on postcontrast CT images did not differ from those obtained during postmortem examination. Findings indicated that low‐dose intraarterial administration of contrast material in the equine head resulted in comparable soft tissue enhancement vs. high volume intravenous administration.     

Spontaneous necrotizing sialometaplasia of the submandibular salivary gland in a Beagle dog

A single mass was found on the left submandibular salivary gland at necropsy of a 15-month-old male commercially bred laboratory Beagle dog from a control dose group from a repeat toxicity study. Microscopically, the mass was composed of a well-demarcated area of coagulative necrosis surrounded and separated from the normal salivary gland tissue by a thick fibrovascular capsule. Necrosis was admixed with areas of hemorrhage, fibrin, edema, fibrinoid necrosis of the vascular tunica media, and thrombosis of small and large vessels. Within the necrotic tissue, there was marked ductal hyperplasia, and squamous metaplasia of duct and acinar epithelium. The mass was diagnosed as necrotizing sialometaplasia of the submandibular gland. Hyperplastic ductal elements and squamous metaplasia can be mistaken microscopically with squamous cell carcinoma. Therefore, pathologists should be aware of this lesion as to avoid errors in the diagnosis of this benign pathologic condition.     

Relationship between three novel SNPs of BRCA1 and canine mammary tumors

The BRCA1 gene plays an important role in the development of human breast cancer, and recent research indicated that genetic variations of BRCA1 are also related to canine mammary tumors (CMTs). Here, using rapid amplification of cDNA ends (RACE), we cloned the 5′- and 3′-UTRs of BRCA1. By direct sequencing of the flanking sequences of the 5′- and 3′-UTRs of BRCA1, three previously unreported single-nucleotide polymorphisms (SNPs) were identified, two (−1228T >C, −1173C >T) in the putative promoter regions and one non-synonymous SNP (63449G >A) in exon 23. Compared with 16 normal samples, the sequences from 34 CMTs suggested that SNP (−1173C >T) was associated with the development of CMTs (odds ratio (OR)=2.57, 95% confidence interval (CI): 1.07–6.15).     

Alteration of somatostatin receptor 2 expression in canine mammary gland tumor

Somatostatin receptor 2 (SSTR2) is a negative regulator of cell proliferation in human breast cancer. Since there is little information about SSTR2 in canine mammary gland tumor (MGT), we clarified its distribution and expression level in normal mammary gland, benign MGT and malignant MGT. SSTR2 expression determined by immunohistochemical staining was observed in the cytoplasm of luminal epithelial cells. The intensity was negatively correlated with malignancy: normal tissues and some of the benign tumors had the highest levels, while the malignant tumors had little or no SSTR2 expression. As for the Western blotting, SSTR2 protein level in benign tumors was significantly lower than the normal mammary gland. On the other hand, SSTR2 protein levels in two of three malignant tumors were higher than the other groups. These results suggest that SSTR2 expression alters according to the malignancy of canine MGT.     

Glucose transport and milk secretion during manipulated plasma insulin and glucose concentrations and during LPS‐induced mastitis in dairy cows

In dairy cows, glucose is essential as energy source and substrate for milk constituents. The objective of this study was to investigate effects of long‐term manipulated glucose and insulin concentrations in combination with a LPS‐induced mastitis on mRNA abundance of glucose transporters and factors involved in milk composition. Focusing on direct effects of insulin and glucose without influence of periparturient endocrine adaptations, 18 dairy cows (28 ± 6 weeks of lactation) were randomly assigned to one of three infusion treatments for 56 h (six animals each). Treatments included a hyperinsulinemic hypoglycaemic clamp (HypoG), a hyperinsulinemic euglycaemic clamp (EuG) and a control group (NaCl). After 48 h of infusions, an intramammary challenge with LPS from E. coli was performed and infusions continued for additional 8 h. Mammary gland biopsies were taken before, at 48 (before LPS challenge) and at 56 h (after LPS challenge) of infusion, and mRNA abundance of genes involved in mammary gland metabolism was measured by RT‐qPCR. During the 48 h of infusions, mRNA abundance of glucose transporters GLUT1, 3, 4, 8, 12, SGLT1, 2) was not affected in HypoG, while they were downregulated in EuG. The mRNA abundance of alpha‐lactalbumin, insulin‐induced gene 1, κ‐casein and acetyl‐CoA carboxylase was downregulated in HypoG, but not affected in EuG. Contrary during the intramammary LPS challenge, most of the glucose transporters were downregulated in NaCl and HypoG, but not in EuG. The mRNA abundance of glucose transporters in the mammary gland seems not to be affected by a shortage of glucose, while enzymes and milk constituents directly depending on glucose as a substrate are immediately downregulated. During LPS‐induced mastitis in combination with hypoglycaemia, mammary gland metabolism was more aligned to save glucose for the immune system compared to a situation without limited glucose availability during EuG.     

Colostrogenesis during an induced lactation in dairy cattle

Colostrum immunoglobulin G (IgG) is of major importance for the newborn calf because epitheliochorial placentae do not provide transport in utero. The formation of colostrum occurs in the later stages of pregnancy. Our objectives were to induce lactation in non‐pregnant dairy cows and (i) to determine the changes of IgG in serum and mammary secretions during the induction process and (ii) to establish α‐lactalbumin (αLA) and prolactin (Prl) alterations to monitor the changing mammary epithelial tight junction status and development pattern. Estradiol‐17β (E2) and progesterone (P4) injections in a 1–7 days series were combined with a 3‐day injection series (day 21–23) of dexamethasone (DEX). Blood and both front quarter secretion samples were collected daily. Milking started 24 days after the start of the experiment. Results show that the mammary secretory IgG1 was increased at >7 days after the start of steroid injections and depicted a bimodal pattern reaching a high of 16 mg/ml at 21 day compared with 3.2 mg/ml in the serum. There was a small increase in secretory IgG2 that did not correlate with tight junction status, but never reached the serum concentration. The injections of DEX resulted in constriction of tight junctions. Secretory αLA was immediately increased with steroid injections, dropped precipitously after 7 days and then began a steady increase until the start of milking. Changes in serum αLA are related to mammary tight junctions while serum Prl gradually increased from 30 to >60 ng/ml after the steroid injections stopped. These results provide insights into the mechanisms and timing of colostrogenesis during an induced lactation protocol.