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1.
The present study aimed to establish an efficient system for bovine embryo production by in vitro fertilization (IVF) that can achieve stable normal fertilization and blastocyst developmental rates in any bull without optimization of the sperm concentration in IVF medium. We examined the effects of a PHE mixture (20 μM D-penicillamine, 10 μM hypotaurine and 1 μM epinephrine), theophylline (2.5 mM), and sperm concentration (1, 2 or 5 × 106 cells/ml) on fertilization and blastocyst developmental rates. High cleavage rates (78.3 to 92.4%) and blastocyst developmental rates (31.9 to 62.0%) at day 7 were obtained in the presence of PHE and theophylline in IVF medium with a sperm concentration of 2 × 106 cells/ml using sperm from 9 bulls. In addition, the synergistic effect of PHE and theophylline on normal fertilization (2 pronuclei) was clarified at 12 h after IVF with a sperm concentration of 1 × 106 cells/ml. Moreover, high linearity, high flagellar beat cross frequency, and low amplitude of lateral head of motile sperm were found by computer-assisted sperm analysis. In conclusion, the combination of the PHE mixture and theophylline synergistically accelerates sperm motility and sperm penetration of bovine oocytes. Theophylline activates sperm motility with increasing intracellular cAMP. However, PHE prevents an excessive increase of cAMP and maintains sperm motility without hyperactivation. When the combination of PHE and theophylline is added to IVF medium at a sperm concentration of 2 × 106 cells/ml, we can achieve stable normal fertilization and blastocyst development in any bull.  相似文献   

2.
This study examined the influence of EGF on the expression of EGF receptors (EGFR) and developmental competence of embryos cultured individually versus those cultured in groups. Cat oocytes were in vitro matured and fertilized (IVM/IVF), and cleaved embryos were randomly assigned to one of seven culture conditions: one group each in which embryos were subjected to group culture supplemented with or without 5 ng/ml EGF and five groups in which embryos were subjected to single-embryo culture supplemented with EGF (0, 5, 25, 50 or 100 ng/ml). Morulae, blastocysts and hatching blastocysts were assessed at days 5 and 7; post IVF, respectively, and total blastocyst cell numbers were assessed at day 7. Relative mRNA expressions of EGFR of 2–4-cell embryos, 8–16-cell embryos, morulae and blastocysts cultured in groups or singly with or without EGF supplementation were examined. OCT3/4 and Ki67 in blastocysts derived from the group or single-embryo culture systems with or without EGF supplementation were localized. A higher rate of embryos cultured in groups developed to blastocysts than individually incubated cohorts. Although EGF increased blastocyst formation in the single-embryo culture system, EGF did not affect embryo development in group culture. Expression levels of EGFR decreased in morulae and blastocysts cultured with EGF. An increased ratio of Ki67-positive cells to the total number of cells in the blastocyst was observed in singly cultured embryos in the presence of EGF. However, EGF did not affect the expression of OCT3/4. These findings indicate that EGF enhanced developmental competence of cat embryos cultured singly by stimulating cell proliferation and modulating the EGFR expression at various developmental stages.  相似文献   

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4.
The aim of this study was to compare the effects of spermatozoa separation techniques on sperm quality and in-vitro fertilization (IVF) results for cryopreserved bovine semen. Sephadex, glass wool and Percoll gradient separation techniques were used for sperm separation and sperm motility, morphology and membrane integrity were evaluated before and after separation. Also, cleavage and blastocyst developmental rate were investigated after IVF with sperm recovered by each separation technique. The motility of samples obtained by the three separation techniques were greater compared to the control samples (p < 0.05). The percentage of spermatozoa with intact plasma-membrane integrity, identified by 6-carboxyfluoresceindiacetate/propidium iodide fluorescent staining and the hypo-osmotic swelling test, was highest in the glass wool filtration samples (p < 0.05). The cleavage and blastocyst rate of total oocytes produced from glass wool filtration samples were also higher than the control and Sephadex filtration samples (p < 0.05), but were not significantly different from Percoll separation samples. However, a significantly greater number of cleaved embryos produced by glass wool filtration developed to blastocyst stage than those produced by Percoll separation (p < 0.05). These results indicate that spermatozoa with good quality can be achieved by these three separation techniques and can be used for bovine IVF. In particular, it suggests that glass wool filtration would be the most effective method of the three for improving sperm quality and embryo production for cryopreserved bovine spermatozoa.  相似文献   

5.
Effective preselection of sex has been accomplished in several species of livestock and also in humans using the flow cytometric sperm sorting method. A guaranteed high sorting accuracy is a key prerequisite for the widespread use of sperm sexing. The standard validation method is flow cytometric remeasurement of the DNA content of the sexed sperm. Since this method relies on the same instrument that produced the original sperm separation, it is not truly independent. Therefore, to be able to specifically produce either male or female offspring in the dog, we developed a method of direct visualization of sex chromosomes in a single sperm using fluorescence in situ hybridization (FISH) as a validation method. Denaturation of canine spermatozoa by immersion in 1 M NaOH for 4 min yielded consistent hybridization results with over 97% hybridization efficiency and a good preservation of sperm morphology. There was no significant difference between the theoretical ratio (50:50) and the observed ratio of X- and Y-chromosome-bearing spermatozoa in any of the three dogs. In addition, the mean purities of flow-sorted sex chromosomes in spermatozoa of the three dogs were 90.8% for the X chromosome fraction and 89.6% for the Y chromosome fraction. This sorting was evaluated by using the dual color FISH protocol. Therefore, our results demonstrated that the FISH protocol worked reliably for both unsorted and sexed sperm samples.  相似文献   

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7.
Acetamiprid (ACE) and imidacroprid (IMI) are known neonicotinoid insecticides with strong affinities for the insect-selective nicotinic acetylcholine receptor. These provide insect control by hyperstimulating insect nerves and are used for agricultural pest management. However, it has also been reported that ACE and IMI affect mammalian reproductive function. We determined the effects of ACE and IMI on the in vitro maturation of porcine oocytes. Significant decreases in nuclear maturation rates were observed in the ACE or IMI-exposed groups. Also, in matured oocytes from the ACE or IMI-exposed groups, irregular chromosomes were observed. Our results suggest that ACE and IMI exposure was detrimental to porcine oocytes and the extent of the effects depends on the concentration of exposure.  相似文献   

8.
The temperature-sensitive hemagglutinin (Tsh) expressed by strains of avian pathogenic Escherichia (E.) coli (APEC) has both agglutinin and protease activities. Tsh is synthesized as a 140 kDa precursor protein, whose processing results in a 106 kDa passenger domain (Tshs) and a 33 kDa β-domain (Tshβ). In this study, both recombinant Tsh (rTsh) and supernatants from APEC, which contain Tshs (106 kDa), caused proteolysis of chicken tracheal mucin. Both rTsh (140 kDa) and pellets from wild-type APEC, which contain Tshβ (33 kDa), agglutinated chicken erythrocytes. On Western blots, the anti-rTsh antibody recognized the rTsh and 106 kDa proteins in recombinant E. coli BL21/pET 101-Tsh and in the supernatants from APEC grown at either 37℃ or 42℃. Anti-rTsh also recognized a 33 kDa protein in the pellets from APEC13 cultures grown in either Luria-Bertani agar, colonization factor antigen agar, or mucin agar at either 26℃, 37℃, or 42℃, and in the extracts of outer membrane proteins of APEC. The 106 kDa protein was more evident when the bacteria were grown at 37℃ in mucin agar, and it was not detected when the bacteria were grown at 26℃ in any of the culture media used in this study. Chicken anti-Tsh serum inhibited hemagglutinating and mucinolytic activities of strain APEC13 and recombinant E. coli BL21/pET101-Tsh. This work suggests that the mucinolytic activity of Tsh might be important for the colonization of the avian tracheal mucous environment by APEC.  相似文献   

9.
Production of knockout mice using targeted embryonic stem cells (ESCs) is a powerful approach for investigating the function of specific genes in vivo. Although the protocol for gene targeting via homologous recombination (HR) in ESCs is already well established, the targeting efficiency varies at different target loci and is sometimes too low. It is known that knockdown of the Bloom syndrome gene, BLM, enhances HR-mediated gene targeting efficiencies in various cell lines. However, it has not yet been investigated whether this approach in ESCs is applicable for successful knockout mouse production. Therefore, we attempted to answer this question. Consistent with previous reports, Blm knockdown enhanced gene targeting efficiencies for three gene loci that we examined by 2.3–4.1-fold. Furthermore, the targeted ESC clones generated good chimeras and were successful in germline transmission. These data suggest that Blm knockdown provides a general benefit for efficient ESC-based and HR-mediated knockout mouse production.  相似文献   

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12.
Bacillus (B.) anthracis is the pathogen that causes fatal anthrax. Strain-specific detection of this bacterium using molecular approaches has enhanced our knowledge of microbial population genetics. In the present study, we employed molecular approaches including multiple-locus variable-number tandem repeat analysis (MLVA) and canonical single-nucleotide polymorphism (canSNP) analysis to perform molecular typing of B. anthracis strains isolated in Korea. According to the MLVA, 17 B. anthracis isolates were classified into A3a, A3b, and B1 clusters. The canSNP analyses subdivided the B. anthracis isolates into two of the three previously recognized major lineages (A and B). B. anthracis isolates from Korea were found to belong to four canSNP sub-groups (B.Br.001/2, A.Br.005/006, A.Br.001/002, and A.Br.Ames). The A.Br.001/002 and A.Br.Ames sub-lineages are closely related genotypes frequently found in central Asia and most isolates were. On the other hand, B. anthracis CH isolates were analyzed that belonged to the B.Br.001/002 sub-group which found in southern Africa, Europe and California (USA). B.Br.001/002 genotype is new lineage of B. anthracis in Korea that was not found before. This discovery will be helpful for the creation of marker systems and might be the result of human activity through the development of agriculture and increased international trade in Korea.  相似文献   

13.
本试验通过观察呼吸道疾病综合征（PRDC）病猪中性粒细胞（PMN）环磷酸腺苷磷酸二酯酶（cAMP-PDE）活性和血浆cAMP、白介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）含量的变化及自拟中药汤剂对其的影响与疗效,以期为呼吸道炎症的病理机制与中药防制积累资料。将PRDC病猪随机分组,治疗组灌服由芦根、黄芪、白芍、甘草组成的1 g生药/mL中药,1.5 mL/kg,每天2次,连续给药10 d。停药后取全血检测各组外周血白细胞数量;并分离PMN,HPLC法检测cAMP-PDE活性;ELISA试剂盒测定血浆中cAMP、IL-6、TNF-α的含量。与健康对照组相比,患病组白细胞数量显著升高（P<0.05）,cAMP-PDE活性和血浆TNF-α、IL-6含量均极显著升高（P<0.01）;治疗组TNF-α含量显著升高（P<0.05）。与患病组相比,治疗组的白细胞数量、cAMP-PDE活性和血浆TNF-α含量均显著降低（P<0.05）,IL-6极显著降低（P<0.01）。结果表明PRDC病猪存在外周血白细胞数量、PMN cAMP-PDE活性和血浆IL-6、TNF-α含量升高的异常变化,自拟中药汤剂能改善呼吸道病猪的临床症状,其作用机制与降低白细胞数量,抑制PMN cAMP-PDE活性和降低血浆IL-6、TNF-α含量相关。  相似文献   

14.

Background

Facing numerous challenges, such as illness, storms or human disturbance, some harbor seal (Phoca vitulina) pups lose contact to their dams and are found abandoned along the North Sea coast. In Schleswig-Holstein, pups with the prospect of surviving rehabilitation are admitted to the Seal Center Friedrichskoog. Despite elaborate clinical health assessments on admission, including differential hematology, in 2010, 17% of 108 admitted pups did not survive the first 20 days. The death rate during the years 2006 and 2009 varied between 9 and 19%. To broaden the spectrum of variables which could be predictive for survival, blood gas and serum analyses were performed for 99 pups using venous blood. Variables included total CO2, pH, partial CO2, HCO3–, base excess and anion gap as well as glucose, urea nitrogen, sodium, potassium and chloride. Moreover, total serum protein and fat (triglyceride) concentrations were measured for all pups on admission.

Results

Repeated measurements of 12 randomly selected individuals revealed a significant (p = 0.002) positive influence of time in rehabilitation on triglyceride concentrations. This trend probably shows the improvement of the pups’ nutritional status as a consequence of the shift from milk replacer formula to fish. No such positive influence was detected for total protein concentrations though. Hematologic values, including blood gases, were not predictive for survival.

Conclusions

For the first time blood gas values are reported in this study for a large sample size (N = 99) of seal pups (regardless of their health status). The ranges and medians calculated from the data can serve as a stepping stone towards the establishment of reference values for neonate harbor seals. However, future investigations on the development of blood gases in harbor seals with different health conditions and ages over time are necessary to allow for a better understanding of acid–base regulation in harbor seals.  相似文献   

15.
Babesia sp. (EU1), first characterized in 2003, has been implicated in human cases of babesiosis in Italy, Austria and Germany. It has been identified in roe deer and in its suspected tick vector, Ixodes ricinus, in several European countries. The aim of the present study was to validate the competence of I. ricinus as a vector of Babesia sp. (EU1) via experimental infections. For this purpose, a parasite strain isolated from roe deer was cloned in sheep erythrocytes. After experimental infections, parasite DNA was successfully amplified by PCR in both eggs and larvae originating from infected I. ricinus females and in the salivary glands of females exposed to Babesia sp. (EU1) as nymphs. We also demonstrate that infected females were able to transmit parasite DNA during a new blood meal. Together with previous epidemiological studies, these results validate I. ricinus as a competent vector for Babesia sp. (EU1).  相似文献   

16.
试验旨在探讨人工感染鸭源新城疫病毒（Newcastle disease virus,NDV）对绍鸭β-防御素（avian β-defensins,AvBDs）和细胞因子基因表达的影响。选用45只20日龄SPF绍鸭,随机分为攻毒组（27只）和对照组（18只）,攻毒组采用滴鼻点眼的途径（108.38 EID50）接种鸭源NDV强毒株（Md/CH/LGD/1/2005）,对照组接种磷酸盐缓冲液。在攻毒后24和48 h,从对照组及攻毒组各随机取6只鸭屠宰,分别采集肾脏、肺脏、气管、腺胃、骨髓、脾脏、盲肠扁桃体、哈德氏腺、法氏囊9个组织,运用实时荧光定量PCR法检测组织样品中9种AvBDs和细胞因子的表达量;剩余鸭每天持续观察,每隔4 d采血1次,直至24 d,检测血清抗体水平;于攻毒后24、48、72和120 h采集咽喉及泄殖腔拭子,以确定排毒周期。结果表明,感染该NDV毒株后,鸭没有临床发病症状和死亡情况发生;感染后鸭排毒开始于攻毒后第1天,到第5天停止,在攻毒组咽喉及泄殖腔拭子中均检测到NDV。抗体水平检测结果显示,该NDV能够诱导鸭体内产生抗NDV特异抗体。实时荧光定量PCR结果发现,与对照组相比,感染后24 h,部分鸭组织中AvBD1、AvBD2、AvBD5、AvBD6、AvBD9和AvBD16表达量均显著升高（P<0.05）;感染后48 h,AvBD1和AvBD6在部分鸭组织中表达量均显著上调（P<0.05）;感染后48 h法氏囊中白细胞介素6（IL-6）和脾脏中干扰素γ（IFN-γ）的表达量明显低于感染后24 h的表达量。综上所述,该鸭源NDV毒株感染可诱导绍鸭体内在早期产生天然免疫反应,这些反应可能与病毒在机体内复制有关。  相似文献   

17.
Epidemiological studies suggest that alcohol consumption increases the risk of developing colorectal cancer. However, the data are confounded by numerous cosegregating variables. To cast further light on the relationships between alcohol intake and colon cancer development, 21-day-old male F344/DuCrj rats were fed 200 ppm 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in their diet for 8 weeks and doses of 0, 0.1, 0.3, 1, 3, 10 and 20% of ethanol in their drinking water ad libitum for 16 weeks thereafter. The rats were sacrificed after 24 weeks of experiment, and aberrant crypt foci (ACF), surrogate lesions for colon cancer, were examined under a light microscope at low magnification. Ethanol was found not to affect the ACF formation at any dose compared with the initiated-controls. Furthermore, ethanol did not alter colon epithelial cell proliferation. These data, obtained by analysis of a colon cancer surrogate marker lesion, indicate that ethanol lacks promotion activity for MeIQx-initiated rat colon carcinogenesis.  相似文献   

18.
本试验旨在观察前列腺素E2受体激动剂(布他前列腺素(butaprost))与雌激素对奶牛输卵管上皮细胞中转化生长因子β3(TGFβ3)表达的影响,阐明butaprost和雌激素对奶牛输卵管上皮细胞TGFβ3有无协同调控作用.采用胰酶消化法及机械法分离培养奶牛输卵管上皮细胞,分别将butaprost和雌激素作用于体外培养的奶牛输卵管上皮细胞,采用实时荧光定量PCR技术检测butaprost和雌激素对奶牛输卵管上皮细胞中TGFβ3 mRNA表达的影响.结果显示,与0 h作用组相比,雌激素作用16、24和48 h时对奶牛输卵管上皮细胞TGFβ3的表达量均极显著升高(P< 0.01),4 h的表达量极显著降低(P< 0.01);且受体激动剂butaprost和雌激素有协同调控TGFβ3的效应;加入吲哚美辛后能有效抑制内源性前列腺素对TGFβ3表达的作用.结果表明,butaprost和雌激素可调控奶牛输卵管上皮细胞TGFβ3 mRNA 的表达.  相似文献   

19.
王晓梅  迟德富  宇佳 《草业学报》2018,27(9):95-109
匍枝筋骨草悬浮细胞中含有较高β-蜕皮甾酮,为了进一步提高其β-蜕皮甾酮含量,通过添加茉莉酸甲酯(MeJA)进行一系列试验研究。以4~10代筋骨草悬浮细胞为试验材料,测定不同处理浓度和处理时间的MeJA对细胞生长、β-蜕皮甾酮积累的影响,细胞生长量采用称量计数,β-蜕皮甾酮含量则使用高效液相进行检测。结果表明:匍枝筋骨草悬浮细胞生长曲线及β-蜕皮甾酮积累曲线符合Logistics模型。在细胞快速生长的初始期(第4天)或中期(第7天)添加不同浓度的MeJA,对细胞生长影响相对较小,生长曲线均有小高峰,分别出现在处理后第5天和第3天,干物质积累量分别达到0.60和0.62 g。而在细胞快速生长的高峰期添加MeJA,细胞生长曲线呈下降趋势,细胞鲜重和干重显著低于CK(P<0.05)。在细胞快速生长的初始期或中期添加MeJA后细胞鲜重与β-蜕皮甾酮积累量呈显著相关。在细胞快速生长的初始期或中期添加10~50 μmol·L-1 MeJA后,细胞鲜重与CK对比表现为显著增加,其中添加50 μmol·L-1 MeJA后细胞鲜重最佳,最高可达到35.90 g,显著高于其他处理(P<0.05)。而同样条件下β-蜕皮甾酮表现为积累量小幅增加,最高量为0.5095 mg·g-1。添加100~200 μmol·L-1 MeJA均会抑制细胞的生长,其中添加200 μmol·L-1 MeJA细胞鲜重与CK相比显著下降,抑制率最高可达到38.88%。而添加100~200 μmol·L-1 MeJA后β-蜕皮甾酮积累量表现为大幅提升,最高可达3.5315 mg·g-1,为同期CK的14.44倍(P<0.01)。在细胞快速生长的高峰期添加10~200 μmol·L-1 MeJA后,细胞鲜重与CK相比都表现为下降,说明在此时添加这些浓度MeJA可抑制细胞的生长,最高抑制率可达31.01%。而在细胞快速生长的高峰期添加10~50 μmol·L-1 MeJA后,β-蜕皮甾酮积累量可在短时间内大量激增,β-蜕皮甾酮积累量最高达到1.4136 mg·g-1,是CK的5.06倍(P<0.01)。添加100~200 μmol·L-1 MeJA则积累量较少。在细胞快速生长的中期添加100 μmol·L-1 MeJA条件下对细胞的刺激较小,β-蜕皮甾酮积累量最高,达到3.5315 mg·g-1。  相似文献   

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