Effects of Ethanol on Synthesis of Prostaglandin F2α in Bovine Females

Ethanol stimulates the production of prostaglandins in many species. The purpose of this study was to verify the effect of ethanol on the production of prostaglandin F2α (PGF2α) and luteolysis in bovine females. In the first experiment, Holstein cows at day 17 of the oestrous cycle were treated with 100% ethanol (0.05 ml/kg of body weight, IV; n = 5), saline (0.05 ml/kg of body weight, IV; n = 4) or synthetic prostaglandin (150 μg of D‐cloprostenol/cow, IM; n = 4). The plasma concentrations of 13, 14‐dihydro‐15‐keto PGF2α (PGFM; the main metabolite of PGF2α measured in the peripheral blood) were assessed by radioimmunoassay (RIA). There was an acute release of PGFM in response to ethanol comparing to other treatments (p ≤ 0.05). However, only cows treated with PGF2α underwent luteolysis. In the second experiment, endometrial explants of cross‐bred beef cows (n = 4) slaughtered at day 17 of the oestrous cycle were cultured for 4 h. During the last 3 h, the explants were cultured with medium supplemented with 0, 0.1, 1, 10 or 100 μl of 100% ethanol/ml. Medium samples were collected at hours 1 and 4 and concentrations of PGF2α were measured by RIA. Ethanol did not induce PGF2α production by the endometrium. In conclusion, ethanol does not cause luteolysis in cows because it stimulates production of PGF2α in extra‐endometrial tissues.     

Effects of Prostaglandin E2, Prostaglandin F2α and Endotoxin on Plasma Calcium Levels in The Goat

Intravenous administration of 2.6–3.3 µg/kg of an endotoxin from Salmonella typhimurium to goats caused a marked drop in plasma Ca levels associated with an increase of prostaglandin synthesis and release measured as peripheral plasma levels of 15-keto-13,14-dihydro-PGF_{2 α}. This is one of the main metabolites of PGF_{2 α}, but also PGE_{2 α} is partly metabolised to this compound. The infusion of 10 mg of PGF_{2 α} lowered plasma Ca levels. Ten mg of PGE₂ did not change Ca concentrations significantly.     

Prostaglandin Endoperoxide Synthase 2 (PTGS2) and Prostaglandins F2α and E2 Synthases (PGFS and PGES) Expression and Prostaglandin F2α and E2 Secretion Following Oestrogen and/or Progesterone Stimulation of the Feline Endometrium

Sex steroids in synergy with prostaglandins (PG) are involved in the regulation of cyclic ovarian function. In this study, we investigated the mRNA expression of three genes involved in arachidonic acid (AA) metabolism and hence PG production in domestic cats: PG‐endoperoxide synthase (PTGS2), PGF_2α synthase (PGFS) and PGE₂ synthase (PGES). Feline endometria (n = 16) were collected at oestrus and mid and late phases of pseudopregnancy. In addition, the effects of E₂ and/or P₄ on PG secretion and gene expression on endometrial explants were studied in an in vitro culture system. Expression levels of all examined genes were up‐regulated at the mid phase of pseudopregnancy. The effects of E₂ and/or P₄ treatment on both PG secretion and expression of the genes were observed after 12 h of culture. Expression of PGES was significantly up‐regulated by E₂ plus P₄ at oestrus and the mid phase of pseudopregnancy and was also up‐regulated by a single treatment with P₄ at late pseudopregnancy (p < 0.05). Simultaneous incubation with E₂ and P₄ up‐regulated PTGS2 gene expression at oestrus and mid‐luteal phase (p < 0.05). Progesterone plus E₂ significantly increased PGE₂ secretion at oestrus and the mid phase of pseudopregnancy. However, treatment with E₂ and/or P₄ affected neither PGF_2α secretion nor PGFS expression at any phase after 12 h of culture. The overall findings indicate that genes involved in PG synthesis are up‐regulated at the mid phase of pseudopregnancy. An increase in PGE₂ secretion and up‐regulation of PGES and PTGS2 are the main responses of the endometrium to treatment with E₂ and P₄ at oestrus and the mid phase of pseudopregnancy in the cat. These data support the hypothesis that ovarian sex steroids via endometrial PGE₂ are involved in endocrine homoeostasis, especially at oestrus and the mid, but not the late, phase of pseudopregnancy in cats.     

Developmental Competence of Frozen-thawed Blastocysts from Fair-quality Bovine Embryos Cultured with β-Mercaptoethanol

Two-hundred-and-thirty-one fair-quality embryos at the compacted morula stage collected from 89 superovulated cows were cultured in TCM199 or Brinster's BMOC-3 medium with or without 100 microM beta-mercaptoethanol (beta-ME). After 24 h culture, a total of 142 fair-quality embryos developed to the blastocyst stage, of which 106 were subsequently frozen with 1.8 M ethylene glycol. The mean cell number and development rates of frozen-thawed blastocysts from the fair-quality embryos cultured in TCM199 containing beta-ME were higher than those of the fair-quality embryos directly frozen without culture. The pregnancy rates obtained with frozen blastocysts from fair-quality embryos tended to be lower than those of non-cultured fresh fair-quality embryos and cultured fresh blastocysts. These results indicate that the inclusion of beta-ME in pre-freezing culture media improve the development of frozen-thawed blastocysts from fair-quality embryos, but not the pregnancy rate.     

In Vitro Development of Bovine Embryos after Fertilization using Semen from Different Donors

Contents: The aim of this study was to determine whether the semen donor and/or heparin concentration influences the rate of fertilization of bovine follicular oocytes and their subsequent embryonic development in vitro. Frozen-thawed semen from five highly fertile bulls was treated with one of four concentrations of heparin (0.5,1.0, 2.0 and 5.0 μg/ml) on a 5 x4 factorial basis in an IVM-NF programme. Zygotes/oocytes were cultured in frozen-thawed bovine oviduct cell-conditioned medium for 6 days. The use of semen from different bulls resulted in significantly (P < 0.001) different rates offertilization, as judged by cleavage rates of the oocytes at 72 h post insemination, and subsequent embryonic development through the'8-cell-block'(P < 0.05) in vitro. Development up to the morula/blastocyst stage, however, did not differ significantly (P = 0.06) among groups of oocytes fertilized with spermatozoa from different bulls. Heparin levels ranging from 0.5 to 5.0 μg/ml did not differ in their effect on in vitro fertilization as judged by the rate of normally cleaved oocytes (P = 0.14). The overall parthenogenetic division rate at 72 h post insemination was 12.4% and was not influenced by the heparin concentration. There was a linear relationship (P < 0.001) between fertility estimates based on AI and the estimates basedon the first cleavage following in vitro fertilization.     

The Effect of Long‐term In Vivo Culture in Bovine Oviduct and Uterus on the Development and Cryo‐tolerance of In Vitro Produced Bovine Embryos

The objective of this study was to compare the embryo production and quality carried out entirely in vitro or partly in vitro combined with short‐ vs long‐term in vivo culture using the homologous cattle oviduct. The IVM oocytes were in vitro fertilized and cultured for 7 and 8 days (IVP‐Group), or after IVF and 2–3 days of IVC, 4–8 cell stage embryos were endoscopically transferred into oviducts of synchronized heifers (In Vivo‐Group) or IVM oocytes were co‐incubated with spermatozoa for 3–4 h and transferred into the oviducts of synchronized heifers (GIFT‐Group). Embryos of the In Vivo‐Group and the GIFT‐Group were recovered on day 7 from the oviducts and uterine horns. Embryos of all groups were either cryopreserved at day 7 (day 7 blastocysts) or cultured in vitro in CR1aa‐medium supplemented with 5% ECS for further 24 h and cryopreserved (day 8 blastocysts). The total blastocyst yield found in the in vivo cultured groups was similar to the results of the IVP‐Group. But the appearance of blastocysts was dependent on the duration of in vivo culture. The more time the embryos spent in the in vivo environment, the more blastocysts appeared at day 8. The quality of produced blastocysts assessed by cryo‐survival was also correlated to the culture conditions; the in vivo cultured embryos showed higher cryo‐tolerance. However, the duration of in vivo culture crucially influenced the cryo‐tolerance of produced blastocysts. It is concluded that tubal access is a promising tool to provide a further basis for studying embryo sensitivity to environmental changes.     

Estrus Synchronization in Sheep and Goats Using Combinations of GnRH,Progestagen and Prostaglandin F2α

The objective of this experiment was to evaluate the effect of GnRH, progestagen and prostaglandin F_2α on estrus synchronization in sheep and goats. Sixty Awassi ewes and 53 Damascus does were used in the study. The experiment started at the beginning of the breeding season (June/July). The same treatments were applied to sheep and goats as follows: no treatment (CON), 14‐day progestagen sponges and 600 IU equine chorionic gonadotropin (S), gonadotropin releasing hormone followed 5 days later by prostaglandin F_2α (GP) and gonadotropin releasing hormone, progestagen sponges for 5 days and prostaglandin F_2α on the day of sponge removal (GSP). None of the ewes in the S group lambed from mating during the induced cycle. A greater lambing rate (p < 0.05) was observed in the GSP group compared with the CON and S groups while the GP group was intermediate. The number of lambs born per lambed ewe was similar among the CON, GP and GSP groups. However, the number of lambs per exposed ewe was greater (p < 0.05) in the GSP than the remaining groups. The induced cycle kidding rate was 77% for all treatments combined. Similar kidding rate were observed among treatments. The numbers of kids born per kidded and exposed doe from mating during the induced estrus were also similar among treatments. Greater numbers of multiple births were observed in the GP and GSP than in the S group. In conclusion, a combination of GnRH, progestagen sponges and PGF_2α can be effective in synchronizing estrus and improving fecundity in sheep and goats. Although the use of GnRH–PGF_2α was effective, the addition of progestagen sponges at the time of GnRH administration appeared to improve reproductive parameters.     

紫外线照射时间对孤雌激活和体外受精胚胎发育的影响

用核荧光染料 Hoechst3 3 3 42对 5组牛成熟卵母细胞染色后 ,以紫外线 ( UV)分别照射0 ,10 ,2 0 ,3 0 s和 40 s。观察和分析对孤雌激活胚胎和体外受精胚胎卵裂及体外发育的影响。结果表明 ,经 UV照射 0 ,10 ,2 0 ,3 0 s和 40 s的卵母细胞孤雌激活后的卵裂率分别为 80 .8% ,77.9% ,72 % ,61.4%和 45 % ,囊胚发育率分别为 3 9.2 % ,3 6.4% ,19.4% ,14 .5 %和 11.1% ;UV照射 2 0 s以上的囊胚发育率都极显著低于UV未照射和照射 10 s的卵母细胞 ( P <0 .0 1) ,UV照射卵母细胞超过 2 0 s降低了孤雌激活胚胎的体外发育潜力 ;体外受精胚胎中 ,UV照射2 0 s以上与未照射和照射 10 s组相比 ,卵裂率和囊胚发育率显著差异 ( P >0 .0 5 ) ,U V照射卵母细胞超过 2 0 s时 ,显著降低了体外受精胚胎的发育潜力。由此可知 ,卵母细胞经染色后 ,UV照射时间应控制在 2 0 s以内 ,并以照射 10 s时 ,对孤雌激活胚和体外受精胚的体外发育影响最小     

Conversion of Cortisone to Cortisol and Prostaglandin F2α Production by the Reproductive Tract of Cows at the Late Luteal Stage In Vivo

Previous in vitro studies demonstrated that bovine endometrium has the capacity to convert inactive cortisone to biologically active cortisol (Cr) and that Cr inhibits cytokine‐stimulated prostaglandin F_2α (PGF) production. This study was carried out to test the hypothesis that bovine reproductive tract has the capacity to convert cortisone to Cr in vivo and to evaluate the effects of intravaginal application of exogenous cortisone on uterine PGF secretion during the late luteal stage. The temporal relationships between PGF and Cr levels in uterine plasma were also determined. Catheters were inserted into jugular vein (JV), uterine vein (UV), vena cava caudalis (VCC) and aorta abdominalis (AA) of six cows on Day 15 of the oestrous cycle (ovulation = Day 0) for frequent blood collection. On Day 16, the cows were divided randomly into two groups and infused intravaginally with vaseline gel (10 ml; control; n = 3) or cortisone dissolved in vaseline gel (100 mg; n = 3). Blood samples were collected at ?2, ?1, ?0.5, 0, 0.5, 1, 1.5, 2, 3, 4, 5 and 6 h after treatments (0 h). Intravaginal application of cortisone increased plasma concentrations of Cr between 0.5 and 1.5 h in UV, at 0.5 h in VCC, at 1 h in JV and at 1.5 h in AA. The plasma concentrations of PGF in UV and of PGF metabolite in JV were greater at 0.5 and 1 h in the cortisone‐treated animals than in control animals. The levels of PGF in UV blood plasma decreased after Cr reached its highest levels. The overall findings suggest that the female reproductive tract has the capacity to convert cortisone to Cr in vivo. Based on the temporal changes of PGF and Cr levels in the uterine plasma, a biphasic response in PGF secretion was found to be associated to the Cr increase induced by the cortisone treatment at the late luteal stage in non‐pregnant cows.     

氨基酸和牛磺酸对绵羊体外受精胚胎体外培养的影响

本文在KSOM培养液中添加牛磺酸和NEAA、EAA ,研究氨基酸对绵羊胚胎体外培养的影响。研究表明 :①牛磺酸、NEAA可以显著提高胚胎的桑椹胚和囊胚发育率 ;②EAA能促进胚胎由囊胚向孵化囊胚发育 ;③NEAA和EAA可促进胚胎的囊胚发育和孵化囊胚的发育。     

前列腺素E2和F2α对LPS刺激后奶牛子宫内膜上皮细胞COX-1和COX-2表达的影响

试验旨在阐明前列腺素E₂（prostaglandin E₂,PGE₂）和F_2α（prostaglandin F_2α,PGF_2α）对体外培养的奶牛子宫内膜上皮细胞中环氧合酶-1（cyclooxygenase-1,COX-1））与环氧合酶-2（cyclooxygenase-2,COX-2）表达的影响。培养奶牛子宫内膜上皮原代细胞和传代细胞,第4代细胞以1×10⁶个/孔接种于6孔板,以10^-7mol/L PGE₂和PGF_2α分别预处理细胞24 h,以100 ng/mL细菌脂多糖（lipopolysaccharides,LPS）刺激细胞4、8和12 h后分别提取RNA和总蛋白质,采用实时荧光定量PCR与Western blotting等技术检测COX-1与COX-2 mRNA和蛋白质的表达量。结果表明,与对照组相比,COX-1 mRNA表达量在PGE₂单独作用4、8和12 h后显著上调（P<0.05）;COX-2 mRNA表达量在PGE₂单独作用4和12 h后显著上调（P<0.05）,PGE₂单独处理使COX-1、COX-2蛋白表达量均显著上调（P<0.05）。与对照组相比,LPS刺激8和12 h时COX-1 mRNA表达量显著下调（P<0.05）,LPS刺激后COX-1蛋白表达量无显著变化（P>0.05）;LPS刺激后4、8和12 h时COX-2 mRNA表达量显著上调（P<0.05）,LPS刺激后COX-2蛋白表达量显著上调（P<0.05）。与LPS单独处理组相比,LPS+PGE₂处理组在8和12 h时COX-1和COX-2 mRNA表达量均显著上调（P<0.05）,同时COX-1和COX-2蛋白表达量也显著上调（P<0.05）。PGF_2α在LPS未刺激和刺激后对COX-1和COX-2 mRNA的表达无显著影响（P>0.05）,仅在PGF_2α单独处理8和12 h后COX-1 mRNA表达量上调（P<0.05）。两种激素联合处理与各自单独处理及LPS单独刺激相比,对COX-1和COX-2 mRNA表达具有一定的协同诱导作用。     

Beneficial Effect of Two Culture Systems with Small Groups of Embryos on the Development and Quality of In Vitro‐Produced Bovine Embryos

Currently, in vitro‐produced embryos derived by ovum pick up (OPU) and in vitro fertilization (IVF) technologies represent approximately one‐third of the embryos worldwide in cattle. Nevertheless, the culture of small groups of embryos from an individual egg donor is an issue that OPU‐IVF laboratories have to face. In this work, we tested whether the development and quality of the preimplantation embryos in vitro cultured in low numbers (five embryos) could be improved by the addition of epidermal growth factor, insulin, transferrin and selenium (EGF‐ITS) or by the WOW system. With this aim, immature oocytes recovered from slaughtered heifers were in vitro matured and in vitro fertilized. Presumptive zygotes were then randomly cultured in four culture conditions: one large group (LG) (50 embryos/500 μl medium) and three smaller groups [five embryos/50 μl medium without (control) or with EGF‐ITS (EGF‐ITS) and five embryos per microwell in the WOW system (WOW)]. Embryos cultured in LG showed a greater ability to develop to blastocyst stage than embryos cultured in smaller groups, while the blastocyst rate of WOW group was significantly higher than in control. The number of cells/blastocyst in LG was higher than control or WOW, whereas the apoptosis rate per blastocyst was lower. On the other hand, the addition of EGF‐ITS significantly improved both parameters compared to the control and resulted in similar embryo quality to LG. In conclusion, the WOW system improved embryo development, while the addition of EGF‐ITS improved the embryo quality when smaller groups of embryos were cultured.     

Prostaglandin F2α Metabolite Levels Following an Embryo Transfer Procedure in the Mare

Hormonal, chemical, and mechanical stimuli can activate the arachidonic acid cascade and result in formation of prostaglandins and related substances. These compounds can have a profound role in the initiation of the inflammatory process (Higgins & Lees 1984). Prostaglandin (PG) F_2α is the key hormone in reproductive physiology with well-known effects on reproductive performance e.g. luteolysis and abortion. An activation of the arachidonic acid cascade, caused by mechanical manipulation during an embryo transfer procedure, might be one explanation for early embryonic loss.     

氟尼辛葡甲胺对大鼠小鼠的抗炎镇痛作用研究

氟尼辛葡甲铵盐是一种动物专用的非甾体抗炎药,具有镇痛和解热功能,但抗炎是其主要功效。本试验通过对鸡蛋清诱导的大鼠踝关节炎性肿胀模型和冰醋酸所致小鼠疼痛扭体反应来研究其抗炎镇痛效果。与对照组相比,FM高中低各剂量组(0.55、1.1、2.2 m g/kg)对大鼠足关节炎症均有显著的对抗作用(P<0.05,P<0.01),呈明显的量效关系。高剂量组(2.2m g/kg)抗炎功效明显优于吲哚美辛组(2.25 m g/kg)和地塞米松组(2.52 m g/kg)。中剂量组(1.1 m g/kg)作用堪比吲哚美辛,而低剂量组(0.55 m g/kg)稍逊于地塞米松。与同类对照药双氯芳酸钠(16.25 m g/kg)和安乃近(32.5 m g/kg)相比,结果显示FM(2.5 m g/kg)对小鼠扭体抑制作用最强,抑制率高达82.69%。     

Evaluation of Trypsin Treatment on the Inactivation of Bovine Herpesvirus Type 1 on In Vitro Produced Pre-implantation Embryos

The aim of this study was to evaluate the efficiency of trypsin treatment on the inactivation of bovine herpesvirus type 1 (BoHV-1) on in vitro produced by fertilization and artificially infected bovine embryos. Bovine embryos on day 7 were exposed with 10 μl of BoHV-1, Los Angeles strain 10^7.5 TCID. These embryos and control embryos were divided in two groups: submitted to the sequential washes or to the trypsin treatment according to the International Embryo Transfer Society (IETS) guidelines. The embryos and the last washing drop of each group were used as inoculum to infect Madin Darby bovine kidney (MDBK) cells and submitted to nested PCR reaction using the primer that encodes the gene conserved region of virus glycoprotein gB. The data have shown that the control embryos and their last washing drop were negative. The exposed embryos that were treated with trypsin have shown positive results on the n-PCR and MDBK culture, and their last washing drop were negative. Our data have demonstrated that the trypsin treatment was not able to eliminate the BHV-1 of the embryos, suggesting an interaction between virus and embryo.