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鸡胚盲肠上皮细胞原代培养与鉴定 总被引：2，自引：1，他引：1
为研究鸡E.tenella的宿主细胞--盲肠上皮细胞体外培养技术,为E.tenella损伤机制及抗球虫药的研究提供体外模型,分别用胰蛋白酶法、胶原酶Ⅰ法、嗜热菌蛋白酶法、嗜热菌蛋白酶+胶原酶Ⅰ法和中性蛋白酶Ⅰ+胶原酶Ⅺ法分离纯化原代鸡胚盲肠上皮细胞,通过测定细胞活力、细胞团块比例、总细胞产量和细胞团块产量,筛选出鸡胚盲肠上皮细胞最佳分离方法,并进行了纯化和培养,对培养细胞进行了鉴定.结果表明:嗜热菌蛋白酶消化、低速离心去除单细胞、差速贴壁除去成纤维细胞,为最佳分离和纯化盲肠上皮细胞的方法;分离纯化的细胞接种后分别于第3-5天、第10-11天进入对数生长期,可存活14 d以上;经形态学观察、碱性磷酸酶染色、扫描电镜鉴定为盲肠上皮细胞,所分离的细胞培养至第4、7、11天时上皮细胞比例分别为81.67%、84.33%和72.00%.用该法可获得纯度较高的原代鸡胚盲肠上皮细胞. 相似文献
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为明确鸡胚肠上皮细胞中Toll样受体(Toll-like receptors,TLR)的表达情况,本研究采用嗜热菌蛋白酶分离培养18日龄鸡胚的肠上皮细胞,在形态学观察及免疫细胞化学法鉴定基础上,采用RT-PCR方法检测其TLR表达谱,并对不同品种鸡胚肠上皮细胞TLR表达谱进行了比较研究。结果表明,应用嗜热菌蛋白酶消化法得到的鸡胚肠上皮细胞生长状况良好;采用低速离心和差速贴壁进行纯化,得到90%以上纯度的鸡胚肠上皮细胞;纯化后的细胞经形态学观察及细胞角蛋白单克隆抗体鉴定为阳性;10种TLR在鸡胚肠上皮细胞上均有mRNA表达,但其丰度存在明显差异;不同品种鸡胚肠上皮细胞TLR表达谱均无显著差异。 相似文献
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Chicken primary enterocytes: inhibition of Eimeria tenella replication after activation with crude interferon-gamma supernatants 总被引：1，自引：0，他引：1
A reproducible and original method for the preparation of chicken intestine epithelial cells from 18-day-old embryos for long-term culture was obtained by using a mechanical isolation procedure, as opposed to previous isolation methods using relatively high concentrations of trypsin, collagenase, or EDTA. Chicken intestine epithelial cells typically expressed keratin and chicken E-cadherin, in contrast to chicken embryo fibroblasts, and they increased cell surface MHC II after activation with crude IFN-gamma containing supernatants, obtained from chicken spleen cells stimulated with concanavalin A or transformed by reticuloendotheliosis virus. Eimeria tenella was shown to be able to develop until the schizont stage after 46 hr of culture in these chicken intestinal epithelial cells, but it was not able to develop further. However, activation with IFN-gamma containing supernatants resulted in strong inhibition of parasite replication, as shown by incorporation of [3H]uracil. Thus, chicken enterocytes, which are the specific target of Eimeria development in vivo, could be considered as potential local effector cells involved in the protective response against this parasite. 相似文献
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Youli Wang Qihang Hou Yuqin Wu Yanwei Xu Yan Liu Jing Chen Lingling Xu Yuming Guo Shuai Gao Jianmin Yuan 《动物营养（英文）》2022,8(1):38
Methionine and its hydroxy analogue(MHA)have been shown to benefit mouse intestinal regeneration.The intestinal organoid is a good model that directly reflects the impact of certain nutrients or chemicals on intestinal development.Here,we aimed to establish a chicken intestinal organoid culture method first and then use the model to explore the influence of methionine deficiency and MHA on intestinal organoid development.The results showed that 125-mm cell strainer exhibited the highest efficiency for chicken embryo crypt harvesting.We found that transforming growth factor-b inhibitor(A8301)supplementation promoted enterocyte differentiation at the expense of the proliferation of intestinal stem cells(ISC).The mitogen-activated protein kinase p38 inhibitor(SB202190)promoted intestinal organoid formation and enterocyte differentiation but suppressed the differentiation of enteroendocrine cells,goblet cells and Paneth cells.However,the suppression of enteroendocrine cell and Paneth cell differentiation by SB202190 was alleviated at the presence of A8301.The glycogen synthase kinase 3 inhibitor(CHIR99021),valproic acid(VPA)alone and their combination promoted chicken intestinal organoid formation and enterocyte differentiation at the expense of the expression of Paneth cells and goblet cells.Chicken serum significantly improved organoid formation,especially in the presence of A8301,SB202190,CHIR99021,and VPA,but inhibited the differentiation of Paneth cells and enteroendocrine cells.Chicken serum at a concentration of 0.25%meets the requirement of chicken intestinal organoid development,and the beneficial effect of chicken serum on chicken intestinal organoid culture could not be replaced by fetal bovine serum and insulin-like growth factor-1.Moreover,commercial mouse organoid culture medium supplemented with A8301,SB202190,CHIR99021,VPA,and chicken serum promotes chicken organoid budding.Based on the chicken intestinal organoid model,we found that methionine deficiency mimicked by cycloleucine suppressed organoid formation and organoid size,and this effect was reinforced with increased cycloleucine concentrations.Methionine hydroxy analogue promoted regeneration of ISC but decreased cell differentiation compared with the results obtained with L-methionine.In conclusion,our results provide a potentially excellent guideline for chicken intestinal organoid culture and insights into methionine function in crypt development. 相似文献
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用不同酶消化分离山羊小肠上皮细胞,从而找到最佳的分离方法,为进一步研究IEC提供合适的细胞模型.本研究分别用胶原酶Ⅰ、嗜热菌蛋白酶、胶原酶Ⅺ/中性蛋白酶Ⅰ消化分离刚出生未进奶的山羊小肠上皮细胞,通过观察IEC生长状态、形态特征、抗原鉴定比较哪种分离效果最佳.结果表明,联合用胶原酶Ⅺ/中性蛋白酶Ⅰ 1h可分离出大量健全绒毛隐窝单位,进一步培养获得的小肠粘膜上皮(IEC),上皮细胞膜抗原均呈阳性,用胶原酶Ⅰ和嗜热菌蛋白酶分离得到的大部分是成纤维细胞.选用用胶原酶Ⅺ/中性蛋白酶Ⅰ作为分离IEC的消化酶,可获得较纯的IEC. 相似文献
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Season Does Not Influence Embryo Recovery Rate and Conceptus Size Until Day 14 After Ovulation in the Horse 
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下载免费PDF全文 Although the horse is a seasonal breeding species, a considerable number of mares continue to cycle throughout autumn and winter. Slower equine embryo growth during the non‐breeding season has been hypothesized, and because smaller embryo size is beneficial for cryopreservation, embryo collection outside the breeding season could be an interesting approach for the production of frozen horse embryos. In the present retrospective study, we have therefore analysed embryo recovery rates and conceptus size in mares (n = 30) throughout the year. Conceptus diameter was either size determined after collection with a microscopic scale (day 7–10 after ovulation) or determined by transrectal ultrasound immediately before collection (day 11–14 after ovulation). In 19 of the 30 mares (63%), ovulatory cycles were detected throughout the year. A total of 352 embryo collections with a mean recovery rate of 64.2% were performed and not affected by season. The size was analysed in a total of 165 conceptuses. Conceptus diameter significantly increased (p < 0.001) with day of pregnancy (e.g. day 7: 0.3 ± 0.04, day 10: 4.1 ± 0.2, day 12: 10.1 ± 0.5, day 14: 17.4 ± 0.9 mm), but was not influenced by season. In conclusion, successful embryo collection is possible throughout the year in spontaneously cyclic mares. Under these conditions, neither collection rates nor embryo growth appeared to be affected by season. 相似文献
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Rebecca Ricci Bruce Hammerberg Judy Paps Barbara Contiero Hilary Jackson 《Veterinary dermatology》2010,21(4):358-366
Twenty‐six dogs with known adverse food reactions were fed whole chicken for 14 days. From this group, 12 dogs with cutaneous manifestations following exposure to chicken meat were selected and randomly divided into two groups (n = 6). Each group was then fed hydrolysed chicken or hydrolysed soy for 14 days in a blinded crossover design with a 17‐day washout period between each diet. Assessments of a CADESI (Canine Atopic Dermatitis Extent and Severity Index) score and pruritus were performed throughout the entire study, and combined in a global score (GS). Serum was collected weekly for the measurement of chicken‐ and soy‐specific IgG and IgE. Dogs displayed the most severe clinical response when eating whole chicken compared to baseline (P < 0.001). The GS was significantly reduced in 11 of the 12 dogs when fed hydrolysed chicken were compared to those fed whole chicken (3.58 ± 2.81 versus 20.38 ± 14.65, P < 0.01). Serum immunoglobulin G and E responses were variable and did not show relationship with specific dietary exposure. 相似文献
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CA Meza‐Herrera TT Ross DM Hallford DE Hawkins A Gonzalez‐Bulnes 《Reproduction in domestic animals》2010,45(4):723-728
The effects of supplemented protein level (PL) during the periconceptional period and their interaction with body condition were evaluated in sheep. Multiparous Rambouillet ewes (n = 12) received two PL of rumen undegradable protein (UIP) during a 30‐day pre‐mating and 15‐day post‐mating period: low [LPL, 24% crude protein (CP), 14 g UIP and 36 g/CP animal/day] and high [HPL, 44% CP, 30 g UIP and 50 g/CP animal/day]. While ovulation rate (OR) did not differ between treatments (1.6 ± 0.5, mean ± SEM), a lower fertility rate, a decreased embryo number and a reduced uterine pH (UpH) was observed in the HPL group (p < 0.05), irrespective of BC. Luteal tissue weight, volume and progesterone secretion did not differ among treatments. Sheep with lower UpH also had lower conceptus weight (Cwt; p < 0.05, r = 0.65) and conceptuses with lower mass tended to secrete less INF‐τ and IGF‐1, and the correspondent endometrial explants had a higher basal PGF2α release. Current study indicates that high protein diets during the periconceptional period in sheep modify uterine and embryonic relationships, increasing early pregnancy losses and inducing embryo growth retardation. Surviving embryos were affected by weight reductions, which could compromise later foetal growth and birth weight. Results evidence the key role of a balanced diet in reproductive success and indicate that the quality and nutrient composition of the maternal diet are essential for an adequate establishment of pregnancy, having paramount effects on the interplay of the embryo and the uterus. 相似文献
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Z. Y. Hu H. W. Su S. L. Li Z. J. Cao 《Journal of animal physiology and animal nutrition》2013,97(6):1007-1014
The objectives of this study were to determine the effects of an increased jugular supply of L‐Gln on post‐weaning growth, immune responses, intestinal morphology and autophagy of weaned calves. At 35 days of age, 24 Holstein calves (initial 50 ± 1.5 kg; 35 ± 2 day of age) were randomly allocated to four treatments, and each treatment included five male and one female calves. Holstein calves were assigned to treatments of (i) i.v. infusion of 2 l of 0.85% NaCl, Control group [C]; (ii) i.v. infusion of 8 g/day of L‐Gln mixed with 2 l of 0.85% NaCl solution [L]; (iii) i.v. infusion of 16 g/day of L‐Gln mixed with 2 l of 0.85% NaCl solution [M]; and (iv) i.v. infusion of 32 g/day of L‐Gln mixed with 2 l of 0.85% NaCl [H]; The infusion was 2 h/day for each of 14 consecutive days starting on day 1 after weaning. Feed and water were freely available to all calves. All calves were killed on the 14th day post‐weaning for measurements of autophagy of liver cell and intestinal morphology. Gln has no effect on dry matter intake (DMI) and average daily gain (ADG). Gln infusion increased quadratically the abundance of CD4+, monocyte and the ratio of CD4+/CD8+. The urea N, Gln and Glucose in plasma increased linearly with increasing Gln loads. Gln infusion increased quadratically villus height and crypt depth of intestine. The autophagy level of liver cell was improved with the Gln infusion dose increased. 相似文献
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Isolation of equine bone marrow‐derived mesenchymal stem cells: a comparison between three protocols
C. BOURZAC L. C. SMITH P. VINCENT G. BEAUCHAMP J.‐P. LAVOIE S. LAVERTY 《Equine veterinary journal》2010,42(6):519-527
Reason for performing the study: There is a need to assess and standardise equine bone marrow (BM) mesenchymal stem cell (MSC) isolation protocols in order to permit valid comparisons between therapeutic trials at different sites. Objective: To compare 3 protocols of equine BM MSC isolation: adherence to a plastic culture dish (Classic) and 2 gradient density separation protocols (Percoll and Ficoll). Materials and methods: BM aspirates were harvested from the sternum of 6 mares and MSCs isolated by all 3 protocols. The cell viability after isolation, MSC yield, number of MSCs attained after 14 days of culture and the functional characteristics (self‐renewal (CFU) and multilineage differentiation capacity) were determined for all 3 protocols. Results: The mean ± s.d. MSC yield from the Percoll protocol was significantly higher (6.8 ± 3.8%) than the Classic protocol (1.3 ± 0.7%). The numbers of MSCs recovered after 14 days culture per 10 ml BM sample were 24.0 ± 12.1, 14.6 ± 9.5 and 4.1 ± 2.5 × 10 6 for the Percoll, Ficoll and Classic protocols, respectively, significantly higher for the Percoll compared with the Classic protocol. Importantly, no significant difference in cell viability or in osteogenic or chondrogenic differentiation was identified between the protocols. At Passage 0, cells retrieved with the Ficoll protocol had lower self‐renewal capacity when compared with the Classic protocol but there was no significant difference between protocols at Passage 1. There were no significant differences between the 3 protocols for the global frequencies of CFUs at Passage 0 or 1. Conclusions and clinical relevance: These data suggest that the Percoll gradient density separation protocol was the best in terms of MSC yield and self‐renewal potential of the MSCs retrieved and that MSCs retrieved with the Ficoll protocol had the lowest self‐renewal but only at passage 0. Then, the 3 protocols were equivalent. However, the Percoll protocol should be considered for equine MSC isolation to minimise culture time. 相似文献
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Seyed Amin Kazemi Hamed Ahmadi Mohammad A. Karimi Torshizi 《Journal of animal physiology and animal nutrition》2019,103(5):1399-1407
An experiment was conducted to investigate the supplementation of two commercially available multistrain probiotics as an alternative to antibiotics on growth performance, intestinal morphology, lipid oxidation and ileal microflora in broiler chickens. A total of 280‐day‐old ROSS 308 mixed‐sex broiler chickens with an average initial body weight of 42 ± 0.5 g were randomly divided into four treatments with five replicate cages of 14 birds each cage in a completely randomized design and fed with the following diets for 42 day: (a) control (CON) (antibiotic‐free diet), (b) antibiotic (ANT) (CON + Avilamycin 150 g/ton feed), (c) probiotic A (CON + Protexin® 150 g/ton feed) and (d) probiotic B (CON + Bio‐Poul® 200 g/ton feed). The results showed the broilers fed the ANT diet had greater average daily gain than broilers fed the CON diet during day 1–14 (p < 0.05). At day 42, two birds were randomly selected per replicate for evaluation intestinal morphology, lipid oxidation and ileal microflora. birds fed diet supplemented with probiotic A and probiotic B increased villus height and goblet cells numbers in the jejunum and villus height to crypt depth ratio and villus height in the ileum as compared to birds fed CON diet (p < 0.05). The malondialdehyde value was reduced (p < 0.05) in the ANT, probiotic B and probiotic A groups compared with the CON group. The Lactobacillus population was increased and Clostridium spp. population decreased in the ileum of broilers fed diets containing the probiotic B and probiotic A compared with those fed CON diet (p < 0.05). The results from this study indicate that the probiotic A (Protexin®) and probiotic B (Bio‐Poul®) used in this trial may serve as alternatives to ANT. 相似文献
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陕西省鸡胚源性大肠杆菌的分离鉴定 总被引：9，自引：0，他引：9
从陕西关中六个种鸡场的１９８只死胚及出壳弱雏中分离出９８株大肠杆菌,平均分离率为４９．５％。通过小鸡致病力试验,筛选出６３株具有致病力的菌株,占分离菌的６４．３％。生化鉴定表明,这些致病菌均符合大肠埃希氏菌的生化特性。血清型鉴定可确定６种,其中以Ｏ１、Ｏ２、Ｏ７８为主,分别占鉴定菌的４４．４％（２８／６３）、３３．３％（２１／６３）和９．５％（６／６３）,另三种为Ｏ２２、Ｏ７５、Ｏ８９共占８．０％（５／６３）。有３株未定型,占４．８％（３／６３）。用１６种抗生素进行药敏试验,对先锋霉素Ｖ和恩诺沙星高度敏感,对羧苄青霉素、氨苄青霉素、氯霉素、卡那霉素、磺胺嘧啶、新霉素、链霉素中度敏感。 相似文献
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Gabriela Cardoso Dal Pont Emanuele Cristiny Goes Kariny Fonseca da Silva Simone Gisele de Oliveira Chayane da Rocha Alex Miorka 《Journal of animal physiology and animal nutrition》2019,103(5):1453-1461
Glycerol is one of the substrates used for glycogen production by the chicken embryo, which is the predominant energy source during the last days of incubation and during hatching. The objective of the present study was to evaluate the in ovo feeding (IOF) of glycerol in the light and heavy broiler eggs derived from breeders of two different ages. Two experiments, with 672 eggs each, were carried out. The only difference between the experiments was breeder age: 32 weeks old in Exp. I and 60 weeks old in Exp. II. A completely randomized experimental design in a 3 × 2 factorial arrangement was applied. Treatments consisted of three glycerol IOF doses (0, 6, or 12 mg/ml) and two egg weights (light or heavy). Incubation parameters, glycogen reserves and live performance parameters (1–7 days of age) were evaluated. Hatch of fertile eggs, embryo mortality after IOF and the number of early‐hatching chicks were not affected by the treatments in both experiments. Hatchlings from heavy eggs (68.03 ± 0.64 g) laid by young breeders and receiving 6 mg glycerol/ml showed higher liver glycogen levels than those injected with 0 or 12 mg/ml. Glycerol IOF of embryos from young breeders increased feed intake and weight gain at 7 days of age, independently of egg weight. However, different glycerol dosages had no effect on the performance of the progeny of 60‐week‐old breeders. These results show that glycerol may be used as an IOF ingredient without affecting incubation parameters. The chickens from young breeders had greater glycogen deposition with inoculation of 6 mg/ml of glycerol and better performance with glycerol administration. However, glycerol IOF did not improve the performance of the progeny of 60‐week‐old breeders. Therefore, glycogen IOF may be recommended for eggs laid by young breeders. 相似文献
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本研究旨在探索和建立昆明犬胎儿成纤维细胞体外分离、培养的技术方法并观察其生物学特性.试验利用剖腹手术法获得发育30 d的昆明犬胎儿12个,性别鉴定结果为7公5母.公、母胎儿体尺和体重分别为:体长1.90 cm±0.14 cm和2.00 cm±0.05 cm,体宽0.90 cm±0.13 cm和0.90 cm±0.15 cm;体重0.68 g±0.17 g和0.66 g±0.06 g,差异均不显著(P>0.05).以昆明犬胎儿组织为材料,采用胶原酶消化法和细胞冷冻保存技术建立了昆明犬胎儿成纤维细胞系,并对所构建的细胞系进行生物学特性研究.结果表明,分离的胎儿成纤维细胞呈典型的成纤维细胞形态,生长曲线呈S型,倍增时间约为36 h;冻存前和复苏后的存活率分别为96.1%和94.6%;染色体核型分析显示2n=78(XY),并在体外培养多代后仍能保持正常核型.本研究建立的昆明犬胎儿成纤维细胞系可在体外快速贴壁生长、增殖,进行稳定培养,这不仅使昆明犬遗传资源在细胞水平得以保存,且所获成纤维细胞能作为相关研究中所需细胞的来源. 相似文献
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ECR Leonel V Bento‐Silva KS Ambrozio HS Luna EV Costa e Silva CESN Zúccari 《Reproduction in domestic animals》2013,48(6):e85-e87
The aim of this study was to test the use of mechanical and mechanical‐enzymatic methods, saline solution (SS), and PBS solution for the manipulation and isolation of mare ovarian preantral follicles (PAFs). The ovaries were subjected to mechanical isolation (mixer) alone or in association with enzymatic digestion (collagenase). Incubation times of 10 and 20 min were employed. In the first group, 4.1 ± 4.9 PAFs were harvested with the mechanical‐enzymatic method vs 71.1 ± 19.2 with the mechanical procedure, showing a significant difference between methods; using SS and PBS, these numbers were 35.7 ± 34.3 and 39.6 ± 39.6, respectively, with no significant difference between solutions. In the second group, there was significant difference between methods, with 7.1 ± 10.6 follicles harvested with the mechanical‐enzymatic method vs 63.2 ± 22.9 with the mechanical procedure; using SS and PBS, means were 35.5 ± 36.4 and 34.9 ± 31.1, respectively. The mechanical method proved more effective than the mechanical‐enzymatic approach. Both SS and PBS can be used as a media for equine PAFs preparation. 相似文献
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To test whether undernutrition during foetal to pre‐pubertal life would have long lasting effects on testicular histology in adult male offspring, eleven adult Sprague–Dawley pregnant rats were divided into two groups: Control group, n = 4, fed ad libitum, during gestation and lactation (until 25 day post‐partum). Underfed group pregnant females (n = 7) were kept in cages where only dams had access to food (standard rat chow, 33.5% of ad libitum intake of Control group pregnant dams). After parturition, litters were adjusted to either 14 (Underfed group) or eight (Control group) pups. Mothers were weighed weekly. At 25 day of age pups were weaned, housed at four animals per cage, fed ad libitum and weighed weekly until euthanized at 100 day of age. Testes were processed for standard histology and morphometrical evaluation. At weaning, mother weight was lower in Underfedthan in Control group (mean ± SD): 214.1 ± 26.2 g vs 361.9 ± 33.1 g. Body weight at 100 days of age (254 ± 26.9 g vs 342.4 ± 10.2 g, p ≤ 0.001), testicular weight (1.29 ± 0.16 g vs 1.45 ± 0.13 g, p = 0.03), number of Sertoli cells per seminiferous tubule cross section (18.2 ± 1.2 vs 20.2 ± 1.3, p ≤ 0.01) and per testis (30.5 ± 4.2×106 vs 36.0 ± 5.4×106) were lower (p < 0.05) in Underfed than in Control group. This is the first report stating that foetal to pubertal subnutrition is accompanied by changes in testicular structure and lower Sertoli cell numbers in adult life, strongly suggesting lower daily sperm production. 相似文献
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利用从南宁市郊养鸡场球虫病鸡粪便中收集的球虫混合种卵囊感染小鸡,再应用单卵囊分离感染技术,从感染鸡盲肠中收集的卵囊分离纯化获得1株纯种球虫,经鸡体传代增殖,对该虫株的卵囊大小和卵形指数、潜在期、排卵高峰期、最短孢子化时间、寄生部位、致病性等指标进行观察和测定。结果测得该虫株卵囊的平均大小为(25.743±1.94126)μm×(21.4±1.85985)μm,平均卵型指数为1.2067±0.07;潜在期为140 h;其排卵囊峰期在第6～9天,最高峰在第7天;最短孢子化时间为19 h;寄生部位在盲肠;对两周龄的艾维茵鸡,当使用5×104的孢子化卵囊感染剂量时死亡率为7.5%。根据这些测定和观察到的指标综合鉴定该分离株球虫为柔嫩艾美耳球虫,并命名为柔嫩艾美耳球虫广西南宁株(Eimeria.tenella-GXNN),该研究结果为进一步研究本地区鸡球虫病的药物治疗和免疫预防等奠定了基础。 相似文献