An immunocytochemical procedure for protein localization in various nematode life stages combined with plant tissues using methylacrylate-embedded specimens

Plant-parasitic nematodes possess a large number of proteins that are secreted in planta, allowing them to be successful parasites of plants. The majority of these proteins are synthesized mainly in the nematode subventral and dorsal glands as well as in other organs. To improve the immunovisualization of these proteins, we adapted a methacrylate embedding method for the localization of proteins inside nematode tissues, and extracellularly when secreted in planta or within plant cells. An important advantage is that the method is applicable for all nematode stages: preparasitic as well as parasitic stages, including large mature females. Herein, the method has been successfully applied for the localization of four nematode secreted proteins, such as Mi-MAP-1, Mi-CBM2-bearing proteins, Mi-PEL3, and Mi-6D4. In addition, we could also localize 14-3-3 proteins, as well as two cytoskeletal proteins, by double-immunolabeling on preparasitic juveniles. Superior preservation of nematode and plant morphology, allowed more accurate protein localization as compared with other methods. Besides excellent epitope preservation, dissolution of methacrylate from tissue sections unmasks target proteins and thereby drastically increases antibody access.     

Enhancing Embryo Yield in Superovulated Holstein Heifers by Immunization Against Inhibin

Eight heifers, aged 16–17 months and showing normal oestrous cycles, were immunized against a recombinant porcine inhibin α subunit immunogen, together with another 10 heifers of the same age as controls and treated with placebo immunogen. Primary (1 mg immunogen) and two booster (0.5 mg immunogen each) immunizations were administered at 28‐day intervals. Ten days after the second booster immunization, both groups of heifers underwent a superovulation treatment. Each animal was given an intravaginal progesterone releasing sponge, which was withdrawn 7 days following an i.m. injection of 0.5 mg cloprostenol. Heifers were treated with FSH for 4 days and artificially inseminated after oestrus occurred. The embryos were flushed and evaluated 7 days after insemination. Immunization significantly (p < 0.01) increased blood antibody titres against recombinant porcine inhibin α subunit, from pre‐immunizaion and control values of approximately 0.06 of ELISA 450 nm reading to 0.6 to 0.7 after two or three immunizations. The immunized heifers produced on average 15.8 ± 2.8 embryos, significantly (p < 0.05) higher than the yield of 8.3 ± 1.5 in the controls. The number of transferable embryos were non‐significantly higher in immunized than in control heifers (9.6 ± 3.1 vs 5.8 ± 1.6, p > 0.05). The peak plasma oestradiol concentrations were significantly higher in immunized than in control heifers, both immediately after FSH treatment and 20 days thereafter. Plasma P4 concentrations after superovulation were in the range of 20 ng / ml in the immunized heifers, significantly (p < 0.05) higher than the values approximately 15 ng / ml in control heifers. These results indicated that prior immunization against inhibin α subunit stimulated production of antibodies against inhibin, which enhanced follicular developmental response to superovulation and lead to higher yield of total and transferable embryos. Therefore immunization combined with the conventional superovulatory gonadotrophin treatment, can be a simple and efficient method to produce low cost bovine embryos.     

Exogenous DNA Uptake of Boar Spermatozoa by a Magnetic Nanoparticle Vector System

The sperm‐mediated gene transfer method is applicable to transgenesis in many species that use spermatozoa for reproduction recently, which has been shown various results. In the current study, we show that transgenic porcine embryos can be efficiently produced by employing a simple transfection method that uses magnetic nanoparticles (MNPs). The complexes formed between plasmid DNA and MNPs were bounded on ejaculated boar spermatozoa at a higher efficiency compared to methods using DNA alone or lipofection. Using confocal microscopy, rhodamine fluorophore‐labelled MNPs were detected on external surfaces of the spermatozoa membrane, which were bounded on zona pellucida of in vitro maturated oocyte during in vitro fertilization. Electron microscopy revealed that clusters of MNPs were detected in inside of plasma membrane and nucleus of the spermatozoa head. Additionally, we found that magnetofected boar spermatozoa could be fertilized with oocytes in vitro and that the resulting gene of green fluorescent protein was detected in fertilized eggs by genomic PCR analysis. Taken together, these results suggest that MNPs can be used to efficiently introduce a transgene into embryo via spermatozoa.     

Semen Collection, Examination and Spermiogram in Ostriches

The level of fertility in the male ostrich exerts considerable influence on the efficiency of the fertilization procedure, and thus also on reproductive performance. The determination of the reproductive capacity is of particular interest with regard to the selection of single individuals for optimizing reproduction ratios. Although the breeding and raising of ostriches has become increasingly important in many countries, little research has been completed on reproductive parameters and factors that may possibly influence them. This study presents observations made concerning the quantity and quality of sperm as found in the spermatological testing of 411 ejaculate samples taken from male ostriches on two farms in Namibia. The semen volume varied between 0.1 and 1.5 ml (mean, 0.64 ml). Normal ejaculate colours ranged from white to ivory; the consistency ranged from thin creamy to viscous. The measured pH values lay between 6.4 and 8.0 (mean, 7.3). Microscopic investigations revealed sperm concentrations of 8.9–78.1 million/μl and individual sperm motility from 42 to 96% (mean, 78%). No mass motility was detectable in 42% of the ejaculates; weak mass motility was found in 46%, and clear mass movements were to be found in only 12% of samples. Regarding the morphology of the sperm, 5 to 26% were abnormal (mean, 17%) and 4 to 28% (mean, 20%) were dead. Seasonal patterns of sperm concentration and the influence of frequency of semen collection were investigated in a group of 56 healthy male ostriches. Peak sperm concentrations were found at the beginning of the breeding season in spring; the lowest values were found at the end of the breeding season in autumn. The highest quality ejaculate was obtained from those males whose semen was collected once a week. The results of this study provide fundamental data for the establishment of minimum quality requirements for ostrich sperm to be met by individuals receiving certification as breeding animals and for the selection of suitable males for use in artificial insemination.     

低温胁迫下外源CaCl2对玉米种子萌发及幼苗生长的影响

以杂C-546为试材,采用10、20、40、80、160 mmol/L外源CaCl_2浸种和叶面喷施不同浓度处理种子,比较分析低温胁迫下玉米种子发芽率、发芽势、发芽指数和幼苗叶片相对电导率、可溶性蛋白、Pn、Fv/Fm、POD和SOD活性等生理生化指标的变化。结果表明,在低温抑制玉米幼苗生长条件下,一定浓度CaCl_2(10~80 mmol/L)处理可降低玉米幼苗生长伤害程度。80 mmol/L CaCl_2浸种处理对玉米低温胁迫的缓解效果最佳,与对照比较,发芽率提高8%,发芽势提高24.6%,发芽指数提高33.2,相对电导率降低51.8%,可溶性蛋白含量增加76.3%,SOD活性增加35.7%。适宜浓度的CaCl_2在低温条件下可促进玉米种子的萌发,降低低温对玉米幼苗造成的伤害。     

Assessment of Proliferative Activity by AgNOR and PCNA in Prostatic Tissues of Ram Lambs Implanted with Zeranol

The aim of this study was to assess cellular proliferation using silver-stained nucleolar organizer regions (AgNOR) and the proliferating cell nuclear antigen (PCNA) in various tissues in the prostate of ram lambs implanted with increasing zeranol doses and to compare the sensitivity of different tissues of lamb prostate to zeranol. Twenty-four Akkaraman lambs were implanted with increasing zeranol doses, including 12 mg (n = 8), 24 mg (n = 8) and 96 mg (n = 8), with eight lambs serving as controls. After 33 days, the prostate tissues of the lambs were stained using AgNOR and PCNA techniques. The prostate tissues were divided into two compartments--the epithelial tissues, including glandular acinus, collecting duct and penile urethra, and the non-epithelial tissues, including interstitial tissue and striated muscle. AgNOR dots and PCNA index on each prostatic tissue were counted under a light microscope and were evaluated statistically. AgNOR staining in the treatment groups showed a higher score in the non-epithelial tissues than the epithelial components, whereas the PCNA index was significant in the epithelial tissues and non-epithelial tissues had very low PCNA immunostaining. According to the PCNA index, collecting duct epithelium showed more sensitivity to increasing zeranol doses and according to AgNOR counts, there was no difference of sensitivity to zeranol among tissues of the same origin. Both AgNOR counts and PCNA indexes seem to be valuable proliferating markers for the epithelial components of ram prostate, but PCNA index had no significance in relation to the non-epithelial components in contrast to AgNOR counts. Therefore, the controversial results arising from the combined use of both techniques as proliferating markers for the ram prostate should be considered in further studies.     

Effectiveness of controlled internal drug release device treatment to alleviate reproductive seasonality in anestrus lactating or dry Barki and Rahmani ewes during non‐breeding season

This study aimed to evaluate the effectiveness of hormonal treatments on ovarian activity and reproductive performance in Barki and Rahmani ewes during non‐breeding season. Forty‐eight multiparous ewes, 24 Barki and 24 Rahmani ewes were divided into two groups, 12 lactating and 12 dry ewes for each breed. Controlled internal drug release (CIDR) device was inserted in all ewes for 14 days in conjunction with intramuscular 500 IU equine chronic gonadotrophin (eCG) at day of CIDR removal. Data were analysed using PROC MIXED of SAS for repeated measures. Breed, physiological status and days were used as fixed effects and individual ewes as random effects. Barki ewes recorded higher (p < .05) total number of follicles, number of large follicles, serum estradiol concentration and estradiol: progesterone (E₂:P₄) ratio compared to Rahmani ewes. Lactating ewes recorded higher (p < .05) number of small follicles and lower concentration of total antioxidant capacity (TAC) compared to dry ewes. Number and diameter of large follicles recorded the highest (p < .05) values accompanied with disappearance of corpora lutea at day of mating. Serum progesterone concentration recorded lower (p < .05) value at day of mating and the highest (p < .05) value at day 35 after mating. CIDR‐eCG protocol induced 100% oestrous behaviour in both breeds, but Rahmani ewes recorded longer (p < .05) oestrous duration compared to Barki. Conception failure was higher (p < .05) in Barki compared to Rahmani ewes. In conclusion, CIDR‐eCG protocol was more potent in improving ovarian activity in Barki compared to Rahmani ewes, but this protocol seems to induce hormonal imbalance in Barki ewes that resulted in increasing conception failure compared to Rahmani ewes.     

防治危害板蓝的小菜蛾和菜青虫的药剂筛选

通过４种杀虫剂田间药效试验,筛选出农地乐对小菜蛾防效最好：功夫、农地乐、敌敌畏对菜青虫有良好防效。选用农地乐可防治小菜蛾兼治菜青虫。     

Sarcomatoid renal cell carcinoma with scant epithelial components in an Angora cat

CASE HISTORY: A 6-year-old, neutered, female Angora cat presented with a history of lethargy and anorexia for 2 months and a clinically palpable and gradually enlarging, solid mass in the abdominal cavity extending from the last costal arch to the pelvic cavity.

CLINICAL FINDINGS: Examination of the cat revealed jaundice, dehydration and hypothermia. Haematological manifestations included lymphopenia and substantial decrease in haematocrit value. Biochemical analysis of the blood revealed hypoglycaemia, three-fold elevated blood urea nitrogen values, increased level of serum aspartate aminotransferase and increased total bilirubin while the creatinine level was normal. Ultrasonographic examination of the abdomen showed a disrupted and large hypoechoic area around the left kidney. The cat was anaesthetised and the left kidney was removed, but the cat died following surgery.

PATHOLOGICAL FINDINGS: On post-mortem examination, the left kidney was markedly enlarged and both the cortical and medullary parenchyma were replaced by confluent, multilobulated, pale tan-white, firm nodular masses protruding above the capsular surface. Metastasis was not observed. Cytological examination revealed a population of spindle-shaped cells of variable size, with abundant coarse chromatin and occasionally prominent nucleoli. Initial sections of the kidney were indicative of undifferentiated sarcoma confirmed by immunohistochemistry revealing vimentin-positive and cytokeratin-negative results in all tumour tissues. Additional sections showed very small amounts of both cytokeratin-positive and vimentin-positive areas.

DIAGNOSIS: Sarcomatoid renal cell carcinoma (SRCC) with scant epithelial components originating from left kidney.

CLINICAL RELEVANCE: Clinical and pathological features were similar to those of human SRCC, even though there was no evidence of metastases. Immunohistochemistry for vimentin and cytokeratin may be useful for definitive diagnosis of renal cell carcinoma with sarcomatoid differentiation, although staining of sections from several different parts of the tumour may be necessary. When a primary renal tumour is presented, SRCC should be considered as this diagnosis may influence treatment protocols and the clinical outcome.     

Identification of candidate miRNAs and expression profile of yak oocytes before and after in vitro maturation by high‐throughput sequencing

Small RNA represents several unique non‐coding RNA classes that have important function in a wide range of biological processes including development of germ cells and early embryonic, cell differentiation, cell proliferation and apoptosis in diverse organisms. However, little is known about their expression profiles and effects in yak oocytes maturation and early development. To investigate the function of small RNAs in the maturation process of yak oocyte and early development, two small RNA libraries of oocytes were constructed from germinal vesicle stage (GV) and maturation in vitro to metaphase II‐arrested stage (M II) and then sequenced using small RNA high‐throughput sequencing technology. A total of 9,742,592 and 12,168,523 clean reads were obtained from GV and M II oocytes, respectively. In total, 801 and 1,018 known miRNAs were acquired from GV and M II oocytes, and 75 miRNAs were found to be significantly differentially expressed: 47 miRNAs were upregulated and 28 miRNAs were downregulated in the M II oocytes compared to the GV stage. Among the upregulated miRNAs, miR‐342 has the largest fold change (9.25‐fold). Six highly expressed miRNAs (let‐7i, miR‐10b, miR‐10c, miR‐143, miR‐146b and miR‐148) were validated by real‐time quantitative PCR (RT‐qPCR) and consistent with the sequencing results. Furthermore, the expression patterns of two miRNAs and their potential targets were analysed in different developmental stages of oocytes and early embryos. This study provides the first miRNA profile in the mature process of yak oocyte. Seventy‐five miRNAs are expressed differentially in GV and M II oocytes as well as among different development stages of early embryos, suggesting miRNAs involved in regulating oocyte maturation and early development of yak. These results showed specific miRNAs in yak oocytes had dynamic changes during meiosis. Further functional and mechanistic studies on the miRNAs during meiosis may beneficial to understanding the role of miRNAs on meiotic division.