Differential protein profiles in duck meat during the early postmortem storage period

The present study was designed to investigate proteomic differences in duck breast muscle during the early postmortem storage period. The meat quality was evaluated at 0 hr and 24 hr postmortem at 4°C in Pekin ducks, black Muscovy ducks and Mule ducks. Differentially expressed proteins were detected by two‐dimensional gel electrophoresis (2‐DE) and matrix‐assisted laser desorption ionization‐time‐of‐flight mass spectrometry (MALDI‐TOF/TOF MS) at 0 hr and 24 hr postmortem in the three duck breeds. The results showed that 53 proteins spots were differentially expressed at 0 hr and 24 hr postmortem at 4°C in Pekin ducks, 75 spots in black Muscovy ducks, and 72 spots in Mule ducks. A total of 30 (10 spots for each breed) were selected for identification by mass spectrometry. Seven proteins were identified in Pekin ducks, eight in black Muscovy ducks and seven in Mule ducks. Moreover, the above results obtained by 2‐DE and MALDI‐TOF/TOF MS were confirmed by western blotting. To our knowledge, this study is the first to provide insights into the protein profiles of ducks during postmortem storage and provides a better understanding of the biochemical processes that contribute to duck meat quality.     

乳蛋白组学研究进展

作者综述了蛋白质组学研究中的关键技术及蛋白质组学在乳蛋白组学研究中的应用。尤其强调了二维凝胶电泳结合各种质谱技术对乳蛋白组学研究的突出贡献。此外,就蛋白质组学新技术的引入对推进乳蛋白质更广泛、深入地研究进行了展望。     

早期妊娠诊断技术在夏南牛生产中的应用研究

摘 要：【目的】缩短母牛产犊间隔,提高母牛繁殖效率,探索母牛一年一胎的实现路径。【方法】随机挑选21头4-6岁,产后2--3个月未发情的空怀母牛,采取以下同期发情处理程序：第1d肌肉注射GnRH 100μg/头（戈那瑞林）,第8d肌肉注射PG 0.33mg/头（氯前列醇）,第10d肌肉注射GnRH 100μg/头,第11d配种,配种后30d左右采血样送检,检测孕情状况。【结果】经早孕检测,21头送检血样中大于或临近OD值弱阳性对照（0.315）的有14头,小于OD值弱阳性对照（0.315）有7头,检测怀孕率达到66.7%。随后经直肠检查和产犊验证,实际怀孕率66.7%、怀孕牛产犊率100%。【结论】(1)早期妊娠诊断技术已经成熟,该批次血样检测正确率达100%。（2）早期妊娠诊断技术在肉牛生产中的应用可有效解决母牛的隐性发情,缩短母牛产犊间隔,提高母牛繁殖效率。（3）为夏南牛一年一胎技术的实施探索了新途径。     

Endometrial transcript profile of progesterone‐regulated genes during early pregnancy of Water Buffalo (Bubalus bubalis)

Progesterone (P₄) plays a key role in the establishment and maintenance of pregnancy in most mammals. Unravelling the expression of progesterone‐regulated genes can expand the understanding of the embryonic mortality. Accordingly, we studied the relative mRNA expression of the P₄‐regulated genes in the buffalo. Uteri were collected from the abattoir and categorized into nonpregnant late luteal phase, stage I (28–38th days of gestation) and stage II (48–56th days of gestation) of pregnancy (n = 6/group). After extraction of total RNA from the endometrial tissues, we carried out qRT‐PCR for determining the relative mRNA expression of the P₄‐regulated genes using nonpregnant late luteal phase as calibrator group. The expression of LGALS3BP (essential for maternal recognition of pregnancy) gene was found to be significantly upregulated (p < 0.05), while MUC1 (important for embryo attachment) gene was downregulated in stage I and II of pregnancy. We observed no significant change in the expression of LGALS1, LGALS9 and CTSL genes. The SLC5A11 and SLC2A1 genes (involved in the transport of glucose to endometrium) in early pregnancy were upregulated in the pregnancy stage I (p < 0.05) relative to nonpregnant late luteal phase. The CST3 gene was significantly upregulated in pregnancy stage II (p < 0.01). These results provide molecular insights into the specific pathways involved in foeto‐maternal communication during early pregnancy in buffaloes.     

Proteomic analysis of Tianzhu White Yak (Bos grunniens) testis at different sexual developmental stages

To explore the protein expression profiles of white yak (Bos grunniens) testis at different sexual developmental stages. The protein profiles of yak testis were determined using two‐dimensional electrophoresis and the expression levels of 298 protein spots were analyzed. Mass spectrometry was performed to identify those significantly differential expressed proteins; Western blotting was used to confirm the results. During the developmental stages, 29 protein spots showed more than twofold differences (p < 0.05) at ≥1 time point and were successfully identified. Two proteins were upregulated with age (category 1), five proteins (17.2%) were downregulated with age (category 2), four proteins were upregulated before 4 years of age and downregulated thereafter (category 3), fifteen proteins were upregulated before 2 years of age and downregulated thereafter (category 4), and three proteins fluctuated with age (category 5). The expression patterns of regucalcin and heat shock 60 kDa protein in category 2 were confirmed. The 29 differentially expressed proteins from yak testes (some had more than one function) were categorized into binding (n = 15), catalytic activity (n = 13), molecular function regulator (n = 4), antioxidant (n = 4), molecular transducer (n = 2), transporter (n = 1), and structural molecule (n = 1). The identification and analysis of these testis proteins may assist in understanding the developmental biology of reproduction system in male yak.     

家蚕化蛹第7天卵巢组织蛋白质标准图谱的构建

家蚕蛹期是卵子发育的重要时期,对蛹期卵巢的蛋白质组学研究将有助于认识家蚕的卵子发育过程。获取处于卵黄生成期的家蚕化蛹第7天卵巢组织,提取蛋白样品进行双向电泳,经银染后检测到682个蛋白点,这些蛋白点主要分布于分子质量15-90 kD、等电点4-8之间。对其中较高丰度的蛋白点进行飞行时间质谱(MALDI-TOF-MS)鉴定,共分析得到40个可信蛋白点,其中包括4个在家蚕中新鉴定的蛋白。经基因功能分类分析,这些蛋白包括营养储存蛋白、应激相关蛋白、蛋白翻译相关蛋白及代谢相关蛋白等。     

Evaluation of a bovine visual pregnancy test for the detection of pregnancy‐associated glycoproteins in sheep

As sheep produce similar pregnancy‐associated glycoproteins (PAGs) to cattle, a commercially available bovine visual pregnancy test based on the detection of PAGs (visual‐PAG‐test) was evaluated in sheep. The test was performed with whole blood (WhB), plasma (P) and serum (S) of 163 pregnant and 153 non‐pregnant ewes. Additionally, 11 pregnant ewes were tested weekly from day 14 to 49 of gestation and monthly from day 60 to day 149. Ten ewes were sampled weekly from the date of lambing until day 63 post‐partum (p.p.). The sensitivity in mid‐pregnancy (n = 163) was 98.16% (WhB), 99.39% (P) and 99.39% (S) compared to transabdominal ultrasonography as the gold standard while the specificity (n = 153) was 94.12% (WhB), 76.47% (P) and 93.46% (S), respectively. During early pregnancy, all 11 ewes were correctly identified as pregnant on day 42 (100%); however, the test sensitivity decreased to 54.6% (WhB) and 63.6% (S and P, respectively) at day 49. The ewes were again consistently identified as pregnant on day 63 (P) or on day 119 (S, WhB). The test was consistently negative from day 42 p.p. onwards in eight out of ten ewes. Two ewes remained consistently positive until the last sample on day 63 p.p. In conclusion, the test could be used to accurately select pregnant ewes at day 42 with a drop in sensitivity at day 49. The sensitivity of the visual‐PAG‐test was good in mid to late pregnancy, and early detection of pregnancy was possible in individual animals. In some ewes, the PAGs were however detectable for more than 63 days p.p.—the previous breeding history should therefore always be taken into account for correct interpretation of the test results.     

Changes in the bovine whey proteome during the early lactation period

To investigate time‐dependent change in the bovine whey proteome during the early lactation period, a two‐dimensional gel‐based approach was used in this study. Milk samples were collected from five healthy Friesian‐Holstein dairy cows up to 10 days postpartum. Spot patterns of whey proteins varied drastically from immediately after parturition to 48 h, but no significant changes occurred thereafter. Protein identification by mass spectrometry revealed that the ratios of caseins and immunoglobulins drastically decreased during 48 h postpartum, while those of lower molecular mass proteins such as α‐lactalbumin and β‐lactoglobulin increased. More than 100 spots were detected, being much more abundant in colostral whey than in mature milk whey. Of a total of 25 proteins identified, four, viz. zinc‐α‐2‐glycoprotein, vitamin D‐binding protein, immunoglobulin G2 chain C and β2‐microglobulin, were detectable only in colostrum. Our results indicate that most of the minor whey proteins in colostrum relate to the passive immunity of newborn calves, but some of them play significant roles in nutritional supplementation of the neonate. The characteristics of whey proteins in transition imply that enhancement of innate immunity becomes more important than protection of the neonate against pathogens via passive immunity after 48 h postpartum.     

Proteome changes in renal cortex and medulla induced by dietary supplementation with inulin‐type fructans in growing pigs

The aim of the study was to evaluate the influence of dietary supplementation with inulin extract from chicory root and dried chicory root on the protein profile of the renal cortex and medulla of growing pigs. The experiment was carried out on renal cortex and medulla tissue collected from 24 50‐day‐old PIC x Penarlan P76 crossbred piglets (males). Animals were divided into three dietary groups (n = 8) and fed with a control diet, diet supplemented with 2% inulin extract from chicory root and a diet supplemented with 4% dried chicory root. Kidney samples were collected after 40 days of feeding, and renal cortex and medulla proteins were separated by two‐dimensional electrophoresis. Protein identification was performed using MALDI‐TOF mass spectrometry. The diet supplemented with 2% chicory inulin induced significant expression changes of 20 and 26 protein spots in the renal cortex and medulla respectively. Supplementation with 4% dried chicory root triggered changes in the expression of 44 and 24 proteins in the renal cortex and medulla respectively. Both forms of chicory inulin‐type fructans effectively affected the expression of proteins involved in energy metabolism, heat shock proteins and other chaperones, cytoskeletal and cytoskeleton‐related proteins, as well as other proteins. Additionally, changes in transferrin abundance in both experimental groups suggested the significance of chicory fructan supplementation for iron absorption and bioavailability. In conclusion, 2% inulin extract from chicory root and 4% dried chicory root exerted a similar effect on changes in renal protein expression; however, more pronounced alterations were induced by dried chicory root. Nevertheless, further studies are needed for better understanding the mechanism underlying the effect of chicory inulin‐type fructans and their fermentation end products on the kidneys of growing pigs.     

Proteomic analysis of urine from male dogs during early stages of tubulointerstitial injury in a canine model of progressive glomerular disease

Background: Sensitive and specific noninvasive biomarkers for tubulointerstitial injury are lacking, and proteomic techniques provide a powerful tool for biomarker discovery. Objective: The aim of this study was to identify novel urinary biomarkers of early tubulointerstitial injury in canine progressive renal disease using both 2‐dimensional differential in‐gel electrophoresis (2‐D DIGE), which identifies individual proteins, and surface‐enhanced laser desorption ionization time‐of‐flight mass spectrometry (SELDI‐TOF), which generates protein peak profiles. Methods: Urine was collected from 6 male dogs with X‐linked hereditary nephropathy (XLHN) at 2 time points (TP): 1) the onset of overt proteinuria (urine protein:creatinine ratio>2) and 2) the onset of azotemia (creatinine ≥1.2 mg/dL); corresponding renal biopsies were analyzed from 3 of the dogs. Urine samples from the 6 dogs were subjected to analysis by 2‐D DIGE and SELDI‐TOF. Urinary retinol‐binding protein (RBP) was evaluated in 25 male dogs with XLHN and normal control dogs by Western blot analysis. Results: Clinical data and histologic evaluation revealed reduced renal function and increased tubulointerstitial fibrosis at TP 2. A number of urine proteins and protein peaks were differentially present at the 2 time points, with several known biomarkers of renal disease identified in addition to several promising new biomarkers. RBP was first detected in urine approximately 2 months before onset of azotemia (TP 2), but after onset of overt proteinuria, and amounts increased with progression of disease. Conclusions: Proteomic techniques were successfully used to identify urinary biomarkers of renal disease in dogs with XLHN. Urinary RBP is a promising biomarker for early detection of tubulointerstitial damage and progression to end‐stage renal disease.     

Assessment of pregnancy‐associated glycoprotein (PAG) concentrations in swamp buffalo samples from fetal and maternal origins by using interspecies antisera

Pregnancy‐associated glycoproteins (PAG) constitute a large family of glycoproteins found in the outer placental epithelial cell layer of the placenta in Eutherian species. In ruminants, they are noted to be structurally closely related among the different species. This study was designed to determine PAG concentrations in maternal and fetal plasma, allantoic and amniotic fluids in buffalo species. Antisera (AS) generated in rabbits against distinct PAG molecules were used in three radioimmunoassay (RIA)‐PAG systems: RIA‐1 (antiserum raised against bovine PAG67kDa; AS#497), RIA‐2 (antiserum raised against caprine PAG55 + 62 kDa; AS#706) or RIA‐3 (antiserum raised against buffalo PAG; AS#859). Samples were collected at a slaughterhouse (n = 67). PAG concentrations determined by RIA‐2 gave significantly higher results in both allantoic and amniotic fluids (12.7 ± 2.1 ng/mL and 24.0 ± 7.3 ng/mL, respectively). Regarding maternal and fetal plasma, PAG concentrations obtained by RIA‐2 (21.8 ± 2.4 ng/mL and 20.2 ± 2.5 ng/mL, respectively) and RIA‐3 (25.0 ± 2.2 ng/mL and 21.9 ± 3.2 ng/mL, respectively) were higher than those obtained by RIA‐1 (15.5 ± 1.4 ng/mL and 16.1 ± 1.8 ng/mL, respectively). The correlation among the three systems was very high. The study clearly reveals the ability of different PAG‐RIA systems to measure PAG concentration in swamp buffalo samples.     

Comparison of transcutaneous ultrasound over the right flank with transrectal ultrasound for pregnancy diagnosis in the dairy cow

Objective   Evaluate the sensitivity and specificity of transrectal ultrasound and transcutaneous ultrasonography across the right flank between days 31 and 196 of pregnancy in the dairy cow.
Procedure   Pregnancy status and stage of gestation at date of pregnancy diagnosis by transcutaneous and transrectal ultrasonography were determined for 1570 dairy cattle. Sensitivity and specificity values and probabilities of a correct diagnosis for both techniques were determined and compared. Possible effects of gestational age, herd, method of transcutaneous ultrasound diagnosis, cow age, cow and bull breed, and calf sex on the determination of a correct diagnosis of pregnancy status were evaluated for both techniques.
Results   The overall sensitivity and probability of a correct diagnosis of pregnancy status from days 31 to 196 of gestation were significantly higher when using transrectal ultrasound relative to transcutaneous ultrasound over the right flank. However, the sensitivity of transcutaneous ultrasound changed over the course of pregnancy: it was low (7–8%) before day 84, increased to 89% between days 141 and 154, and was close to 100% thereafter. The specificity of both methods did not differ significantly. Herd and gestational age at date of pregnancy diagnosis were significantly associated with making a correct test result using transcutaneous ultrasound, although only the latter predictor was significantly associated with making a correct test result using transrectal ultrasound.
Conclusion   Transcutaneous ultrasound can not be recommended as an accurate method for early pregnancy diagnosis in the dairy cow, particularly on a whole-herd basis. However, if pregnancy testing is undertaken in mid to late gestation, this technique could provide an accurate and rapid alternative to transrectal ultrasound or manual palpation per rectum.     

三种抗原对家畜日本血吸虫病诊断价值的比较

以日本血吸虫基因重组抗原LHD-Sj23／pGEX、可溶性虫卵抗原（soluble egg antigen，SEA）及SEA经Sephadex G-200柱层析分离后得到的第一峰SEAl作为诊断抗原，应用ELISA检测人工感染日本血吸虫绵羊血清及自然感染日本血吸虫水牛血清。结果显示，三种抗原用于检测人工感染日本血吸虫绵羊血清的阳性符合率分剐为88．79％、98．15％、100％，阴性符合率均为100％；自然感染日本血吸虫水牛血清的阳性符合率分别为80％、80％、84％，阴性符合率分别为88．9％、93．3％、91．1％；三种抗原与伊氏锥虫病牛血清无交叉反应；检测感染日本血吸虫不同时间绵羊血清，发现感染6周后均被检出针对这三种抗原的特异性抗体，且SEA及SEAl在感染4周后即可检出特异性抗体。三种抗原的诊断效果差异不显著。     

Development of an in vitro oviduct epithelial explants model for studying sperm–oviduct binding in the buffalo

In this study, we developed an in vitro model for studying sperm–oviduct binding in the buffalo. Oviduct explants were prepared by overnight culture of epithelial cells in TCM‐199 medium under 5% CO₂ at 38.5 °C. Cryopreserved spermatozoa from buffalo bulls (n = 4) were incubated with the oviduct explants, and the sperm–oviduct explants complex was stained with JC‐1. The effect of sperm concentration (2, 3 and 4 million), size of the oviduct explants (<0.2, 0.2–0.3, 0.3–0.4 and >0.4 mm²) and time of incubation (1 hr and 4 hr) on binding index (BI—number of sperm bound to unit area of explants) was studied. No significant difference was observed in the BI among <0.2, 0.2–0.3 and 0.3–0.4 mm² size of explants; however, the BI decreased significantly (p < .05) when the size of explants exceeded 0.4 mm². The BI decreased significantly (p < .05) when the sperm concentration was increased to 4 million, while the duration of incubation did not have any significant effect on the BI. The interaction of bulls with explants size, sperm concentration and incubation time was not significant. The developed assay has the potential to be used as an in vitro model for studying sperm–oviduct binding in the buffalo.     

Dietary chicory root and chicory inulin trigger changes in energetic metabolism,stress prevention and cytoskeletal proteins in the liver of growing pigs – a proteomic study

Currently, a wide array of plant preparations exerting health‐promoting properties are commonly used as feed additives. Among them, Cichorium intybus L. have gained considerable attention as a source of compounds showing prebiotic character. Large body of evidence suggests that products of prebiotic fermentation (short‐chain fatty acids) may influence the expression of genes encoding liver enzymes involved in the regulation of energetic metabolism. Given the above, the present study was aimed at estimating the influence of a diet supplemented with chicory root or water extract of chicory inulin on liver proteome in growing pigs. The study was performed on 24 castrated male piglets (PIC × Penarlan P76). Animals were assigned to three equal groups (n = 8) and fed cereal‐based isoenergetic diets: control and supplemented with 2% of inulin extract from chicory root or 4% of dried chicory root. Liver proteins were separated using two‐dimensional electrophoresis, followed by the identification of statistically valid protein spots with the aid of MALDI‐TOF mass spectrometry. Both experimental factors significantly modulated the expression of liver proteins associated with energetic metabolism, particularly those involved in cholesterol and triglyceride metabolism. Additionally, both dietary additives induced increased expression of proteins involved in hepatocyte protection against oxidative stress. In the present study, we have shown for the first time that diet supplementation with dried chicory root or inulin caused significant changes in the expression of liver cytoskeletal proteins. Close attention should be paid to the downregulation of cytokeratin 18, hepatic acute phase protein that can enhance the anti‐inflammatory properties of inulin‐type fructans.     

HPLC法检测水牛血浆中吡喹酮的研究

建立了一种简便、灵敏、可重复的高效液相色谱法检测水牛血浆中吡喹酮(Praziquantel,PZQ)的含量。吡喹酮在C18(250 mm×4.0 mm,5μm)柱中可以很好地与杂质峰分离开。其保留时间、紫外检测波长、流动相、流速、色谱柱温度分别为10.6 min、220 nm、甲醇∶水(70∶30,V/V)、0.8 mL/min和40℃。水牛血浆样品(0.5 mL)经乙酸乙酯(6 mL)两步提取后,采用旋转蒸发仪蒸干,1 mL流动相溶解定容,取上清滤液20μL进样做HPLC分析。吡喹酮的血浆药物浓度在0.062 5~8 mg/L范围内,呈现良好的线性关系,相关系数r0.999。在0.062 5、1、16 mg/L的3个药物浓度水平下,吡喹酮的平均回收率均在90%以上。日内和日间变异系数小于5%。PZQ的LOD可达0.01 mg/L,LOQ为0.03mg/L。该方法的专属性强、灵敏度高,适用于水牛血浆中PZQ的药动学检测。