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The Effects of L-Ergothioneine, N-acetylcystein and Cystein on Freezing of Ram Semen [1][2]
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1 Kafkas Univ Vet Fak Derg 21 (1): 81-86, 2015 DOI: /kvfd Kafkas Universitesi Veteriner Fakultesi Dergisi Journal Home-Page: Online Submission: Research Article [1] [2] 1 2 The Effects of L-Ergothioneine, N-acetylcystein and Cystein on Freezing of Ram Semen [1][2] Savaş YILDIZ 1 Yavuz ÖZTÜRKLER 1 Umut Çağın ARI 1 Necdet Cankat LEHİMCİOĞLU 1 Emine ATAKİŞİ 2 Recai KULAKSIZ 1 This study was supported by the TÜBİTAK-Tovag (Project No: 109O236) This study was presented as poster at the VII. National Reproduction and Artificial Insemination Science Congress on 1-4 July 2013, Kars, Turkey Department of Artificial İnsemination, Faculty of Veterinary Medicine, University of Kafkas, TR36100 Kars - TURKEY Department of Biochemistry, Faculty of Veterinary Medicine, University of Kafkas, TR36100 Kars TURKEY Article Code: KVFD Received: Accepted: Published Online: Abstract The aim of this study was to determine the influence of different doses of L-Ergothioneine (LE), cystein and N-acetylcystein (NAC) on post-thawing semen parameters in rams. Ejaculates collected from four Tushin Rams were evaluated and pooled at 35 C. Semen samples were diluted with skim-milk based extender containing LE (5, 10 mm), Cystein (5, 10 mm), NAC (5, 10 mm) or not containing any antioxidant (control) and loaded to 0.25 ml French straws. Straws were cooled to 5 C for 2 h, frozen in liquid nitrogen vapour (aprox C) for 15 min and then being stored in liquid nitrogen until thawing process. Straws were thawed in water bath (37 C for 1 min). The percentages of motility, viability, abnormal acrosome, total abnormalities, membrane integrity (hypoosmatic swelling test, HOST) were statistically assessed. Also total antioxidant capacity (TAC) and total oxidatif stres (TOS) were evaluated in samples from replications. It was seen that LE was superior to N-acetylcystein and cystein in motility, viability, defected acrosome, total morphological abnormalities, HOST and TAC, (P<0.05) except cystein in motility. Neverthless, there was not any statistically difference between LE and control groups (P>0.05). It was conlcluded that there was not any beneficial or detrimental effects of LE on post thawing semen parameters in rams while it was determined that cystein and NAC may have been some detrimental effects on post thawing semen parameters in rams. These results warrants future scientific studies on LE, NAC and cystein in ram semen cryopreservation. Keywords: Ram semen, Cryopreservation, L-Ergothioneine, Cystein, N-acetylcystein, Tushin Özet Koç Spermasının Dondurulması Üzerine L-Ergothionin, N-asetil sistein ve Sisteinin Etkileri Bu çalışmanın amacı, koçlarda L-Ergothionin (LE), sistein ve N-asetil sisteinin (NAC) farklı dozlarının çözüm sonu sperma parametreleri üzerine etkisini belirlemekti. Dört Tuj ırkı koçtan alınan ejakülatlar değerlendirildi ve 35 C de karışım yapıldı. Sperma örnekleri, LE (5, 10 mm), Sistein (5, 10 mm), NAC (5, 10 mm) içeren veya antioksidan içermeyen (kontrol), yağsız süt temelli sulandırıcıyla sulandırılarak, 0.25 ml lik payetlere dolduruldu. Payetler 5 C ye 2 saat süreyle soğutuldu, sıvı azot buharında (yaklaşık -120 C) 15 dak. süre ile donduruldu ve çözme işlemine kadar sıvı azot içinde saklandı. Payetler su banyosunda (37 C de 1 dak.) çözüldü. Motilite, canlılık, anormal akrozom, toplam anormalite ve membran bütünlüğü (hipoozmotik şişme testi, HOST) yüzdeleri istatistiksel olarak değerlendirildi. Ayrıca, toplam antioksidan kapasite (TAK) ve toplam oksidatif stres (TOS), replikasyonlardan elde edilen örneklerde değerlendirildi. LE nin motilite (sistein hariç), canlılık, akrozom hasarı, total morfolojik anormalite, HOST ve TAK oranları bakımından, NAC ve sisteine üstünlük sağladığı görüldü (P<0.05). Yine de, LE ve kontrol grubu arasında istatistiksel bir fark yoktu (P>0.05). Sistein ve NAC ın koçlarda çözüm sonu spermatolojik parametreler üzerine bazı olumsuz etkileri belirlenirken, LE nin olumlu ya da olumsuz etkisinin olmadığı sonucuna varıldı. Bu sonuçlar, koç spermasının dondurulmasında, LE, NAC ve sistein kullanılması üzerine yeni bilimsel çalışmaların yapılması gerektiğini göstermektedir. Anahtar sözcükler: Koç sperması, Dondurma, L-Ergotiyonin, Sistein, N-asetilsistein, Tuj İletişim (Correspondence) /5247 GSM:
2 82 The Effects of L-Ergothioneine... INTRODUCTION Artificial insemination with frozen semen does not yet give satisfactory results in sheep. Therefore, for increasing fertility rates are needed in the development of new semen extenders or new compositions. In semen, cooling, freezing, thawing and premature activation in genital tract cause deterioration in membrane integrity and decreases the success of artificial insemination [ 1,2]. It is well known that ram semen is susceptible to freezing and thawing processes. So, in recent years, various antioxidants have been used in the freezing of ram semen. In ram semen, despite the positive effects of antioxidants on the motility and membrane integrity, previous results are not satisfactory to achieve in artificial insemination. Hence, the many studies have been continuing on this subject. Although there are many studies on effect of antioxidants, the avaliable information on the antioxidant capacity and lipid peroxidation in frozen ram semen is limited [1-3]. The investigations on the selection of the best diluent, antioxidant and dose have been continued by researchers [4-8]. Some literatures documented have shown that antioxidants supplemented to semen extenders have a positive effect on the success in cryopreservation [9-11]. Even synthetic antioxidants on sperm have been investigated [2]. Not only investigation and detection of ram semen characteristics, also the measuring the activity of antioxidant enzymes in ram semen has recently gained importance in researches. The measurement of antioxidant activity for better understanding of the effects of antioxidants gives important clues in terms of the success of freezing [9,10]. Recently, a thiol compound, LE, has been used to determine the antioxidant activity in in the storage of semen and shown to have positive effects on storage of ram semen [1]. However, the studies regarding use of antioxidants in the storage of semen is very limited. Nevertheless, it has been reported that N-acetylcysteine reduce reactive oxygen species and DNA damage in human sperm [11,12]. Also, It has been shown that L-Cystein hidrochloide, reducing oxidative stress in semen, is one of major thiol compounds which is involved in glutathione structure and decreases chromatin damage and also enhances the sperm viability [13]. The oxidative damage during freezing and thawing is induced by the reactive oxygen species (ROS) produced by cellular metabolism and sperm components. Hence, ROS is one of the main causes of decrease of motility and potential fertility in spermatozoa and comprises primary factor comprising oxidative stress. For these reasons, addition of antioxidant compounds to the culture media have become widespread in recent times [14,15]. However, according to current literature knowledged, it is seen that LE which has been much more important than the other antioxidants upto now and superior features of LE have not been effectively used in culture medium of oocytes and embryos. It has been known that LE is a fungal metabolite with antioxidant function in mammalian cells [16]. The preventing of oxidative stress induced by hydrogen peroxide in the presence of catalase and glutathione (GSH) consumption stability regulated by N-ethylmaleimide is realized by intracellular LE [17]. It had been investigated the effectiveness of LE on prevention of DNA damage and revention of cell deaths caused by hydrogen peroxyde. In this study, researches had concluded that LE could act as an in vivo non-toxic thiol buffer antioxidant and provide the oxidative stability in pharmaceutical preparations [18]. LE is known as an unique and single antioxidant amongst antioxidants which chelates heavy metals and protects of the cells (primarily erythrocytes) from ROS damage. Further, LE is shown as a single and powerful antioxidant which induces lipid peroxidation alike thiol antioksidants in the presence of ferric acid antioxidants and neutralizes the hydroxyl radicals, peroxynitrite hypochloroik acid and peroxynitrite [19-21]. In reproductive studies, the significant protective effects of LE has been demonstrated especially on semen. LE is the predominant sulphidryl in human, equine and porcine semen. It has been reported that LE explicitly protects the semen from oxidatif stress. As a result of the antioxidant properties of LE, while it increases sperm viability during storage, eliminates the harmful effects of hydrogen peroxide on viability of spermatozoa and vital impact [1,22]. The aim of in this study was to investigate the effects of LE, NAC and Cystein on the frozen ram semen. MATERIAL and METHODS The experiment was performed in accordance with guidelines for animal research from the National Instutites of Health and all procedures on animals were approved by the Kafkas University Ethics Committee on Animal Research in current study (approval date/number: 2009/02). Chemicals All chemicals were provided from Sigma-Aldrich (Germany) if not stated. Animals The animals were housed at the Kafkas University, Faculty Of Veterinary Medicine, Education Research and Practice Farm and kept under uniform feeding, housing and lighting conditions. Semen Extension, Freezing and Thawing Semen samples were obtained from four Tushin rams (2 and 4 years of age) and all of ejaculates were collected from the rams with artificial vagina twice a week during
3 83 YILDIZ, ÖZTÜRKLER, ARI LEHİMCİOĞLU, ATAKİŞİ, KULAKSIZ the breeding season. The ewes in oestrus were used as phantom. Ejaculates were evaluated. Only ejaculates with in volume, spermatozoa with >80% progressive motility and concentration higher than spermatozoa/ml were pooled, balancing the sperm output of each male to compansate individual differences [23-25]. Each pooled ejaculate was splitted into seven equal aliquots and diluted at 37 C with skimmed milk extender (prepared from 10% milk powder w/v, 0.9% glucose w/v; and added 10% egg yolk v/v, 5% glycerol v/v; ph adjusted to 6.8 with 1N NaOH) supplemented with LE (5 and 10 mm), Cystein (5 and 10 mm), N-acetylcystein (5 and 10 mm) and noantioxidant (control), respectively, and a total of seven experimental groups were established. After dilution, final concentration was adjusted to of approximately spermatozoa/ml. Diluted semen samples were aspirated into 0.25 ml French straws, sealed with polyvinyl alcohol powder and equilibrated at 5 C for 2 h. After equilibration, the straws were frozen in liquid nitrogen vapor, 4 cm above the liquid nitrogen (-120 C), for 15 min. Thereafter, the straws were plunged into the liquid nitrogen for storage. The frozen semen in straws were thawed at 37 C for 1 min in a water bath for post-thawing evaluation immediately after thawing. Analysis of Standard Semen Parameters Semen Volume: The volume of ejaculates was measured in a conical tube graduated at 0.1 ml intervals. Motility: Progressive spermozoa motility was estimated using phase-contrast microscopy (400 ; Nikon Eclipes e400, Japan), with a warm stage kept at 37 C. For motility assessment, one drop of semen placed directly on a microscope slide and covered by a cover slip. Spermmotility estimations were performed in three different microscopic fields for each semen sample. The mean (SEM ±) of the three successive estimations was recorded as final motility percentage (%). The Percentage of Live and Dead Sperm: The percentage of live sperm in the sample was assessed by means of a nigrosin eosin staining method [26]. In examination, the 2% solution of eosin stain powder prepared with 3% Sodium Citrate were used for staining. The stain fixation of sperm were prepared by mixing a drop of semen with two drops of the stain on a warm slide and spreading the stain immediately with a second slide. The viable sperm rate was assessed by counting 200 cells under bright field microscope (400 magnification; Olympus CX21, Japan). Spermatozoa showing partial or complete purple colorization was considered non-viable or dead and only sperm showing strict exclusion of the stain were considered to be alive. The Percentage of Sperm Abnormalities: For the assessment of sperm abnormalities, one drop of each sample were added to Eppendorf tubes containing 0.5 ml of Hancock solution (62.5 ml of 37%, formalin 150 ml sodium saline solution, 150 ml buffer solution and 500 ml double-distilled water) [27]. The mixing samples were stored at refrigerator (+4 o C). After providing fixation of samples, one drop of this mixture was put on a slide and covered with a cover slip. The percentage total sperm abnormalities were determined by counting a total of 400 spermatozoa under phase-contrast microscopy (magnification 1000 /oil immersion object). Further, sperm abnormalities were classified as acrosome, head, midpiece and tail (AHMT), and each category were recorded seperately. Sperm Concentration: The sperm concentration was measured by using a haemocytometer [28]. The obtained results were recorded as 10 9 /ml. The Hypo-osmotic Swelling Test (HOST): The hypoosmotic swelling test (HOST) was used to evaluate the functional integrity of the sperm membrane, basedon curled and swollen tails. This procedure was carried out by incubating 30 µl of semen with 300 µl of a 100 mosm hypo-osmotic solution (9.0 g fructose g sodium citrate in ml of distilled water) at 37 C for 60 min. After incubation, 0.2 ml of the mixture was spreaded with a cover slip on a warm slide. Four hundred sperms were evaluated (magnification 400 ) with bright-field microscopy (Olympus CX21, Japan). Sperm with coiled tails were recorded as membrane intact [29,30]. Biochemical Assays Biochemical assays were performed on the sperm samples immediately after thawing and without washing by using spesific kits. Total antioxidant capacity (TAC) in samples was assayed by following the procedures as described in recent years [31]. After semen samples were centrifuged in rapid of rpm/5 min, seminal plasma were obtained. TAC and TOC levels were measured by using kit (Rel Assay Diagnostic, Turkey). The test named as bluegreen ABTS radical cation which is based on the principle of color loss. In this method, 2,2 -azino-di (3-sulfonate to etlylbenzthiozol=abts) reacts with peroxidase and hydrogen peroxide. At the end of this reaction, radical cation ABTS positive (+) is formed and a blue-green color is obtained spectrophotometrically. This color was measured by spectrophotometry at 600 nm [32]. Statistical Analysis The study was replicated seven times. The means of results obtained from seven different experimental groups were analyzed. Results were expressed as the mean±s.e.m mean were analyzed using a one-way analysis of variance (ANOVA), followed by the Tukey s post hoc test to determine significant differences in all the parameters between groups using the SPSS/PC computer program (version 14.0, SPSS, Chicago, IL). Differences with values of P<0.01 were considered to be statistically significant [33].
4 84 The Effects of L-Ergothioneine... RESULTS The mean±s.e.m average of motility, viability, total sperm abnormalities, acrosomal anormalities and the values of HOST, TAC and TOS values in frozen ram semen dilueted skimmed milk powder with supplemented of different antioxidants in different doses are shown in Table 1. The results of spermatological parameters, the values of TAC and TOS in all experimental groups were not superior with the addition of the anti-oxidants, when compared to the control. However, LE supplemented groups were superior to the other antioxidant supplemented groups (P<0.01) (Table 1). DISCUSSION The evaluation of the data obtained from control, N-acetyl cystein, cystein and LE groups pointed out that motility rates of LE groups were statistically superior to the other experimental groups except control in current study. Viability rate in control and LE groups was found as numerically superior to other groups. Acrosome and total morphological defects rates in the control group was statistically significant lower than other groups, except LE group. On the other hand, the acrosomal defects in LE group were lower than the other groups, this lowerness was found as statically signficant compared to NAC5 and NAC10 groups, except control. From these results, acrosome defect rate and total morphological defect rates were understood to be parallel with each other. In present study, the best membrane integrity (HOS Test) results were obtained from groups of LE5 and LE10, similar to rates in other groups. These findings demonstrated that there was consistency among sperm parameters evaluated in current study. In the examinig of TAC, although the LE group was relatively more successful, there were no any differences between groups in TOS values. Considering all the assessments mentioned above, it might be suggested that LE tried for the first time, Cystein and N-acetylcystein in the freezing of semen did not give succesful results compared to control. Unlike, regarding with spermatological parameters (motility, viability, total morphological abnormalities, intact acrosome and HOST) LE group have been successfully compared to other antioxidant groups. According to the literatures documented, as it has not been found any literature except for two studies (Çoyan et al. [34] and Ari et al. [35] ) regarding with LE and the other antioxidants investigated in the present study, the main theme of the experiment could not be done subjectspecific discussion in detail. However, in different number of mammalian species, analyzing the results of different or similar antioxidants with some studies [36,37] especially, motility results obtained in the present study is lower and in those of some others is similar [38,39]. In the same study, in the group added 2 mm LE subjective, CASA and progressive motility values were 81.3%, 57.9% and 31.0%, while in the group added 4 mm, these scores were 80.6%, 65.2% and 32.4% respectively. It was observed that the percentage of motility in LE group was significantly higher cystein group and the more addition dose was increased the success of study increased in LE group. The motility percentages were significantly lower than those of the results in the study of Çoyan et al. [34] similarly, in the present study, LE group was higher than cystein group. Moreover, Ari et al. [35] determined that 10 mm LE improved motility and membrane integrity in replication with poor freezability compared with 0, 1, 2, 5 mm LE. However, they could not determine any beneficial effects of LE in replication with good freezabillity. In the present study, the viability percentages in the groups added LE and cystein were similar to the results obtained from some studies carried out in various animal species [39,40], while the results of the group supplemented with N-acetylcystein were lower than the results of some investigators [40,41]. On the other hand, the percentages Table 1. The mean±s.e.m average of motility, viability, total sperm abnormalities, acrosomal anormalities and the values of HOST, TAC and TOS values in frozen ram semen dilueted skimmed milk powder with supplemented of different antioxidants Tablo 1. Farklı antioksidanlar içeren yağsız süt tozu sulandırıcısında dondurulmuş koç spermasında ortalama motilite, canlılık, toplam anormalite, akrozom bozuklukları, HOST, TAK ve TOS değerleri Parameters Control LE5 LE10 NAC5 NAC10 C5 C10 Motility (%) 26.28±3.16 a 24.71±2.85 a 26.14±4.13 a 4.00±2.69 b 2.85±2.42 b 15.14±3.28 c 16.00±4.38 c Viability (%) 32.28±4.10 a 28.71±3.12 a 26.85±4.21 ab 17.57±3.56 bc 14.00±3.92 d 24.71±2.40 ab 25.00±3.40 ab Total Morphological Abnormalities (%) 46.14±3.48 a 49.42±2.56 ab 51.71±3.80 ab 78.42±2.92 c 82.57±3.24 c 57.00±3.47 b 59.28±3.65 b Acrosome anormalities (%) 42.00±3.92 a 45.85±2.74 ab 49.00±3.60 ab 73.14±3.45 c 77.57±4.08 c 53.71±4.50 b 55.42±3.62 b HOST (%) 30.28±3.25 a 29.57±2.62 a 28.57±2.66 a 15.50±3.80 b 14.80±4.31 b 24.14±3.13 a 23.00±3.75 a TAC (mm/lt) ±2.72 a ±5.09 a ±28.0 ab ±17.2 b ±17.8 b ±23.5 b ±19.2 b TOS (mm/lt) 66.61± ± ± ± ± ± ±11.9 a, b, c Different letters within the same column are significantly different (P<0.01) LE5: 5 mm L-Ergothionin supplementation, LE10: 10 mm L-Ergothionin supplementation, NAC5: 5 mm N-Acetyl Cystein supplementation, NAC10: 10 mm -Acetyl Cystein supplementation, C5: 5 mm Cystein supplementation, C10: 10 mm Cystein supplementation
5 85 YILDIZ, ÖZTÜRKLER, ARI LEHİMCİOĞLU, ATAKİŞİ, KULAKSIZ of viability in a study which has been added 6 mm N-acetylcystein to dog semen [37] had been higher than those of the present study s results. The percentages of acrosomal defects in the present study were lower than some studies s results [24] while were higher than those of some studies [37,38,40,41]. However, the percentages of acrosomal defects in a study [38] which was subjected to addition of cystein in bull semen were highly lower than those of the present study. It also suggests that bull semen is more resistance than ram semen to thawing procedure. On the other hand, the acrosomal defects rates of the groups supplemented with N-acetyl cystein and cystein in the present study were higher than those of the other experimental groups (LE and control groups) and some other studies [24,40,41]. In present study, it was seen that total morphological abnormalities showed similar pattern with the percentage of acrosome disorders, so in the same way, these rations has shown similar differences with other studies [24,37,38,40,41]. Compared with Çoyan et al. [34] study in ram semen, the findings obtained from control and LE groups of the present study were higher and similar to those of N-acetylcysteine and cysteine added. Our findings about LE doses was similar with findings from Ari et al. [35] when all replications considered. However, Ari et al. [35] pointed out that higher doses of LE (i.e. 10 mm) improved post-thaw motility and membrane integrity in poor freezability replications, while lower doses of LE (1, 2, 5 mm) did not show any beneficial effects on post thawing sperm parameters neither in poor nor in good freezability replications. Ari et al. [42] determined that higher doses of NAC (i.e mm) had some detrimental effect on post-thawing motility and other semen parameters on ram semen diluted with milk based extender. Similarly, we found that 5 mm and 10 mm doses of NAC detrimentally affected post thawing parameters in ram semen. Compared HOST results obtained from present study in all groups with other studies [35-37,39] are seen to below. TAC and TOS relevant results, obtained from the present study, have not been able to discuss in detail with the results from other studies because limited literatures found. However, Çoyan et al. [34] reported that LE and cystein did not affect significantly the superoxide dismutase (SOD) and gluthation peroxidase (GPx) activity. In TOS terms, similar situation was observed in the present study. On the other hand, in the same study, it was shown that the motility rates were positively affected in the groups of the addition of LE compared with control. Differences between results in our study and the other studies might have been orginated from different methods and sheep breeds or species. Because it has been pointed out that the different methods and breeds or species used may cause variations in sensitivity to oxidative stres in sperm [39]. In addition, it is also known that individual characteristics of rams, season, native quality of semen, extenders/diluents, cryoprotectants, procedure of cryopreservation and also thawing procedure can directly affect results of post-thawing semen quality in rams [40]. In conclusion, in this study, while the 5 and 10 mm doses of LE were superior to the 5 and 10 mm doses of NAC in the view of the percentages of motility, viability, acrosomal defects, total morphological abnormalities, membrane integrity (HOST) and TAC values, except motilty rates in the 5 and 10 mm doses of cystein. However, 5 mm dose of LE were more succesful than all doses of NAC and cystein, considered TAC values. 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Kids Taco Halloween Costume – Yo Quiero Taco Costume?
A Kids Taco Halloween Costume
Looking for a kids taco Halloween costume good enough to eat? Okay, not really, but a kids taco costume is a really cool idea for Halloween. One year we dressed up all of our kids as food, we thought it was silly and fun and they really enjoyed themselves. The thing about a food costume like this is that it can be saved and worn again by future generations or played with as a role play dress-up item. My kids love to wear their costumes for role play and a kids taco costume is certainly an exciting one to have in your dress-up trunk.
Taco Costume for Kids
You might be thinking why would anyone want taco Halloween costumes? For the same reason anyone would want a kids pizza costume, they're fun! Dressing up as food is unique and different from you average super hero or princess costume. Here are some of the most popular taco Halloween costumes for kids that I think you might enjoy. I like to shop the different online retailers when I'm looking for Halloween costumes for my kids because there is more of a selection. You're not likely going to find a kids taco Halloween costume in brick and mortar retail locations just because its not very common, which is great, who wants to be the same thing every other kid on the block is going to be?
Kids Taco Halloween CostumeTaco Child Halloween CostumeBaby Taco Costume
You Wanna Taco 'Bout It?
Wanna Taco Bout It Kids T-Shirt
Most Popular Kids Taco Costume
This is the most popular kids taco costume for Halloween by Rasta Imposta. This child size Halloween costume fits children sizes 7-10. This taco is a pull-over tunic, it will fit over your child's regular clothing. What's nice about the pull over tunic style costumes is that no matter what the weather is like on Halloween; warm or cold – your child is going to be comfortable. This particular kids taco Halloween costume is available from two online retailers. Depending on how close it is to Halloween, one or both might run out, so its best to order your costume early if you know you're going to want this one. The price fluctuates with the season, the closer we get to October, the more the costumes are going to cost.
Kids Taco Halloween CostumeTaco Child Halloween Costume
Cute Taco Costume for a Baby
This is a taco costume for a baby! It has a foam shell with foam fillings so it's comfortable and lightweight for baby to wear. This costume is for ages 6-12 months of age. It's definitely one of those costumes that will have people looking at your baby.
The taco shell of this baby costume is made of a soft foam. The lettuce, tomato and cheese fillings are detachable, you add them in after you put the baby taco suit on.
I think putting a little Senor mustache on your baby would be hilarious for Halloween. Give him an authentic Mexican flare. LOL Imagine seeing this little baby taco crawling around the Halloween party? Yea. That's deliciously cute on so many many levels!
DIY Baby Taco Costume Idea
Don't want the taco baby costume featured above? How about just a simple onesie, mustache and Mexican straw hat? Make your own baby taco costume with some simple prop ideas.
Do You Wanna Taco 'Bout It?Child's Mexican SombreroMustachifier Mustache Pacifier
Taco Halloween Costumes for the Family
There is an entire Taco Family line of costumes if you are interested in the whole family dressing up as a group. It's a pretty unique idea for families with a baby. Halloween doesn't have to be on Taco Tuesday for you to enjoy these taco costumes! There is practically a taco costume for everyone in the family. Dress up as your favorite Mexican food family with these adorable costumes. It's the perfect Halloween costume idea if you have a baby because there is a baby taco costume too!
Rasta Imposta Taco, Tan, StandardRasta Imposta Women's Foodies Taco DressTaco Mascot Costume
Halloween Food Costume Ideas
If Mexican food gives you the burps you could always try some of our other Halloween food costume ideas here at the Halloween Fun Shoppe! We've got lots of tasty favorites for you try! We don't just do lunch and dinner either, we have some cool ice cream costumes that are sweeter than sweet!
Peanut Butter and Jelly Halloween CostumesKids Pizza Halloween CostumesIce Cream Halloween Costumes
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Dog Boarding
Doggy Day Care
Hosting Preferences
Additional Preferences
No Females in Heat
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Natasha is a great pet sitter. My dog loved her an her children. She watched my dog every Friday, (because I have an extra long day.) untill he passes. Very caring and reliable. I suggest her for all your dog sitting needs
About Natasha
Tasha's pet paradise
Hi my name is Natasha and I am a big animal lover. I have 4 dogs, 2 cat and 15 chickens at home along with 3 wonderful children. My children are all in school now an I have spent my time volunteering ( 4Paws 4 Ability) and working in different pet stores. So I decided I was gonna start my own thing. I have a good sized side yard that is fenced in . I will also ...
What Natasha would like to know about your dog
Any food allergies? And socialization issues like.. doesn't like other dog, doesn't like to share toys. Talkative. Hyper...


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Did you know that video content makes up more than 70% of all content consumed on the internet and is growing every year. How about that YouTube is the 2nd largest search engine next to Google and the 3rd most visited website next to Google and Facebook?
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Wednesday, June 28, 2017
Polka dots denim wrap shirt - Lekala 4651
I had some real troubles putting together the previous Lekala pattern I had done and I still had not reviewed so I though I should take some step by step pictures this time just in case someone wanders how I put this together:
I 1st marked the back and bust darts and sewed that up, I cut in my paper to make it easier to transfer those marks:
7 comments:
It's such a fun style, it's a shame that the fit didn't turn out as you like. I think you should persevere and give it another try. It looks like you could take a dart out of the neckline tapering into the armhole. I've not tried Lekala patterns; they do always seem to have some great details, but the one size is a little intimidating.ReplyDeleteReplies
That dart sound like a really good idea to try but as much as I loved their sketch when I first see it nothing on this top felt right on me , except maybe the bust darts - love how they get some right every time... So I will probably just let this one go and try new ones!Delete
Did you tell Lekala there was a problem with this pattern? If you do their may be a chance they will fix it. I love the style of this top and you did a great job saving it.ReplyDeleteReplies
I am not sure if the problem is really the pattern! I measured the 2 sides and they seemed to have the same length... also there is no mark ( or at least I didn't find any ) as how those 2 side should overlap... To be honest I am at lost as what was really wrong here - the pattern , the measurements and alterations that I requested, the fabric, or just my personal taste....those this is my second deep V Lekala that is gaping badly so I think I should stay away from them !?!Delete
Hi GeorgiaSDelete
There is definitely problem with this pattern. You should report this to Lekala. I have the same problem with my project. Blouse and sleeves are too long. I cut all parts about 6 cm. V-neck is to wide, it start to overlap at the same point. They should fix it cause it's nice project, I'd like to sew this blouse once more but not with this pattern-it's damaged. It's seems to be for person about 4 inches taller! I'm going to tell Lekala about it.
I devoted for this pattern very rare fabric given by my mom, it could be so nice blouse:( I'm trying to fix it the same way as you did ;)
I love the top! It looks lovely on you, and fabric choice is nice! You did a beautiful job as usual.ReplyDelete


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We can help with towing (locally) or vehicle transport (anywhere in North America) to get your vehicle to our facility. We use a local towing service who will go to Springs, Cheyenne, Vail, etc and this tower has owned a Hummer H1 for 20 years (one of the only 1-owner Hummers around). The transport service we use to move vehicles here from elsewhere is based in Denver with a fleet of approx 80 semis and they have always taken good care of us moving these vehicles.


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Mike Murphy 703-259-1757
703-638-8547 email@example.com
May 5, 2017
Route 29 North in Gainesville Nighttime Lane Closures May 7-10
Drainage pipe replacement
GAINESVILLE – Single lane closures will occur along northbound Route 29 between Lucas Court and Carver Road the nights of Sunday, May 7 through Wednesday, May 10 from 8 p.m. to 4 a.m. each night for drainage pipe replacement, according to the Virginia Department of Transportation.
One lane will remain open to traffic during the work; however, drivers should expect delays and are advised to use alternate routes.
All work is weather permitting.
Follow VDOT Northern Virginia on Twitter: @vadotnova
Information in VDOT news releases was accurate at the time the release was published. For the most current information about projects or programs, please visit the project or program Web pages. You may find those by searching by keyword in the search Virginia DOT box above.


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Tuesday, December 31, 2013
Agricultural Exports Will Be Up For Many Commodities in 2014, But Down Overall From 2013Tweet
In the week of December 19th, 36 million bushels of corn were shipped, pushing the total to 70% above year ago levels. New export sales included 58 million bushels for the old crop and 20 million for the new crop with China continuing to take shipments and place new orders for US corn. Export inspections for soybeans were 55 million bushels in the week of December 19th, the 10th successive week with more than 55 million bushels of shipments recorded. New export sales were 27 million bushels for the old crop and 29 million bushels for the new crop. 821 million bushels have been shipped out so far from the 2013 crop, dominating corn shipments at many ports and the river system. The current pace puts soybean exports at 180 million bushels above USDA's target. China is taking two of every three bushels shipped, with Dalian Exchange prices back above the $20 mark. While global importers, and particularly China, have had a penchant for US grains and other commodities, the export trade volume and value will drop compared to fiscal 2013.
For fiscal 2014, US farm exports are now forecast at $137 billion, down $3.9 billion below the level of the prior year, due to lower values for wheat and some feed products. That is the analysis in USDA's Outlook for U S Agricultural Trade. One of the main drivers is world economic growth which is expected to rise from 2.1% in 2013 to 2.8% in 2014. Trade growth itself which was 2.5% in 2013 is projected to double in the coming year. While Asian GDP growth has been higher than the global average, it is expected to stay level. That includes some softening of the economies in some smaller Asian nations. Globally, world growth expansion is helped by the end to Europe's recession and a speed up of the economy in North and South America. While it is a low probability, economists say there is a potential for slower growth in Brazil, Russia, India, and China.
A result of the macro-economic dynamics is more demand for US agricultural commodities; however that will not be shared equally:
· Grain and feed exports will be down $700 million due to lower wheat and certain feed product values.
· Coarse grain exports will be $8.3 billion, down slight from lower unit values, but offset by sharply higher volume.
· Corn alone will see a 10% increase in volume exported.
· Wheat exports will be down $500 million, on lower volume and value, in part from Canadian competition.
· While oilseeds and their products will see a nearly 10% increase, improved soybean production will allow a $1.5 billion increase in soybeans and a $900 million rise in soybean meal exports.
· Overall livestock exports will be up $700 million to a record of $31.8 billion. Exports will be constrained by tight domestic supplies due to lower production.
· Pork exports will be up $400 million on higher prices.
· Dairy will be up $300 million on strong global demand.
· Beef exports will be up $100 million to $5 billion with higher prices and tight supplies of exporters.
Exports to China will be up $1 billion to $21.5 billion from the initial August forecast, with soybeans being a major contributor to that trend. However, that total is down from the $23.5 billion in 2013. Wheat shipments will fall due to less demand for imported wheat, and cotton exports to china will be down. USDA says China is the second largest buyer of US agricultural exports behind Canada. Canada will be buying $21.6 billion worth of farm commodities, a $100 million increase from 2013 based on higher pork prices.
The US will be importing $109.5 billion in food commodities, up $5.7 billion from 2013. While that is based on an improvement in the US economy, it means agriculture maintains its positive trade balance with a difference of $27.5 billion. However, that is the least since 2009, when it was $22.9 billion. The trade surplus hit a $42.9 billion high in 2011.
Agricultural exports continue to be important to the overall value of commodities, as well as to the overall US trade picture. Most Cornbelt commodities will see higher volumes and values being shipped abroad in 2014, except for wheat which will be lower on both value and volume. Canada is the top customer for agricultural exports, but only by $100 million over second place China.
Posted by Stu Ellis on 12/31 at 06:32 AM | Permalink
Posted by: nancy l.cuskaden at March 13, 2014 3:03PM


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Informatica Corporation (NASDAQ: INFA), the world's number one independent leader in data integration software, today announced that Earl Fry, executive vice president and chief financial officer (CFO), has won the prestigious title of Finance Executive of the Year at the 6th Annual International Business Awards in New York . These awards, also known as the International Stevie Awards, were created to honor the achievements of organizations and business people worldwide, raising their profile among the business community and general public. Winners were selected from more than 1,700 entries received from organizations and individuals in more than 40 countries.
Fry has been Informatica's CFO since January 2000 and is a 25-year Silicon Valley veteran. His continued financial stewardship and attention to sustained performance and ongoing company growth, has helped Informatica attain record results in the data integration and data quality markets.
This is the company's third Stevie in 2009. Earlier in the year Informatica was recognized with two other Stevie awards:
Fry was also recognized as the 2008 Bay Area CFO of the Year in the category of public companies with revenue up to $500 million.
About Informatica
Informatica Corporation (NASDAQ: INFA) is the world's number one independent provider in data integration software. The Informatica Platform provides organizations with a comprehensive, unified, open and economical approach to lower IT costs and gain competitive advantage from their information assets. More than 3,700 enterprises worldwide rely on Informatica to access, integrate and trust their information assets held in the traditional enterprise and in the internet cloud. For more information, call +1 650-385-5000 (1-800-653-3871 in the U.S.), or visit www.informatica.com.
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Note: Informatica is a registered trademark of Informatica Corporation in the United States and in jurisdictions throughout the world. All other company and product names may be trade names or trademarks of their respective owners.


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We get and hour and 15 minutes and no one cares what we do.
Last month I decided that we should take the kids outside before summer got here and it would be way too hot for any outside activities.
Joe and I decided that a nature walk would be fun. We live and work near a few parks and the "Tan Chan". It is a stream that flows through the city and there are walking/running paths along it as well as basketball courts, tennis courts, play equipment that encourages people to be active.
Every morning and evening we take Eddie for a walk along the Tan Chan. He gets to be off his leash and run around. There is a park with a soccer field and a few places where Eddie gets to jump across rocks in the stream. Eddie LOVES his Tan Chan walk. It's about 2 miles long and takes us about 30-40 minutes.
When Joe and I decided to take the kids on a nature walk we thought that the Eddie walk would be perfect. We would walk about a mile, stop on the soccer field to kick the ball around, then walk the other mile back to school.
For the record, my team won. 2 to 0. No big deal. It's just a game. But my team won.
The loosing team with their team captain, Mr. Joe.
One little girl needed a little help. She held Joe's little finger while she jumped from rock to rock. They were all really proud of themselves when the got to the other side.
Joe helped some of the slow movers across.
Sometimes I forget how lucky we are to be able to live in such a great location.
You can almost see our aprtment in the background. Its the building second from the right.
It was a great little adventure. The kids seemed to really enjoy the walk.
I think all of the kids said that their favorite part of the walk was jumping across the rocks.


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|Fuck you, 40!
1. I got injured and developed CRPS so exercise is almost completely out of the question. Fuck you, nerve disorder! I can blame CRPS for at least part of my fat ass can't I? I mean if I didn't have CRPS I would be able to do yoga like I used to and maybe I would be all svelt and stuff.
2. I <3 food way too much. Y'all if I could roll around in a pool filled with Key Lime Pie filling and eat my way out, I totally would. Then I'd jump into the pool filled with whipped cream. The real kind too, not that Cool Whip shit.
|Oh yeah. Source: Jason Merritt/Getty Images North America
5. Wait. What were we talking about?
6. I love lists.
|Gimme some of that yogurt or just John Stamos. Whatever.
8. Why does John Stamos look better than me? He probably works out huh? I wonder how old he is.
9. Holy shit! I just looked it up and he is 48! Now I'm really depressed. He's 8 years older than me and looks 10 years younger than me. Thanks a lot Uncle Jesse! Way to make a girl feel good. I hope you're proud of yourself sir.
10. I think you see now why I'm dreading my 40th birthday. I don't look like John Stamos. Fuck.


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To play in this exclusive online slots tournament, visit Caribbean Gold by way of the links within this blog (or on the banner below) and make a deposit into your account (or set up a new account and also take advantage of the 100% welcome bonus deposit matches they have going on right now). Check out the tournament section and, as long as you've entered through this blog or the Games and Casino site links, you'll see the $500 exclusive tournament page to get started.
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- The Games and Casino Vegas Tech Tournament Page. Here you'll find all the information you could ever want to know about the tournament, including the short list of other VT casinos that are also hosting the event. You'll also get re-buy information as well as the terms and conditions.
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This should be enough to get you to the start line. How you finish will be completely up to you. And, well, Lady Luck. Good Hunting!


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It was a crazy weekend/Monday. My parents asked me to 'babysit' for them while they went away for the weekend. Baby sitting for my parents involves dogsitting, catsitting, horse sitting (there's 2 quarter horses, 4 miniature horses, and one mini donkey) and housesitting. I was also working this weekend and trying to plan a surprise birthday party for my Mom's birthday (yesterday). Mothers are hard people to be sneaky around!
Anyways, my friend found this blog that I wanted to share with you. It's called "Stampin Pretty" and it is fantastic! The lady who owns the blog is a Stampin Up demonstrator; I have a lot of Stampin Up stuff and this blog is great for getting new ideas.


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This is just a super quick Sunday post to say I'm taking part in an amazing new read-a-thon called Tome Topple (created by the fantastic Sam from Thoughts on Tomes - go follow her right now)!
Tome Topple is read-a-thon running from June 5th (midnight) to June 19th (11.59pm) where you are.
It focuses on big books (over 500 pages), you can follow along on twitter and instagram using the hashtag (you guessed it) #tometopple. One of the best bits about this read-a-thon is Sam has organised lots co-hosts from around the world and on different platforms so wherever you are and however you're joining in, there should be sprints and chats etc happening!
There are 5 challenges that you can use to create your TBR (thought of course you don't have to follow them, it is fun!) and they are as follows;
1. Read more than one tome (500+ pages)
2. Take a graphic novel break (doesn't need to be over 500 pages!)
3. Read a tome that is part of a series
4. Read over 500 pages in one week
5. Read an adult novel
I did use the challenges to pick out my books, cos I like the added challenge and it's all a bit of fun. However, I'm really rubbish with following TBRs so I decided to pick out a few books to choose from so I don't get too bogged down and here they are!
I have to admit I am cheating a little bit with two of these. The Steel Kiss by Jeffery Deaver actually comes in at only 482 pages but it is the hardback edition so I do still think of that as a tome! Plus I got it from the library so I have to read it now PLUS I have been waiting a very long time for a new Lincoln Rhyme and somehow I missed this one coming out in March!!
Similarly the Sculptor comes in at around the same, however I have had this on my shelf for far too long, and I thought it was quite cool to have a graphic novel tome included.
The Name of the Wind was the original title that sprung to mind when I first saw this challenge (it was an obvious choice if you read my Intimidating Reads post a few weeks ago!), however then my copy of A Gathering of Shadows finally arrived (I mean by "finally arrived" I finally decided to face whatever tragedy happens and order it) but I don't know if I want to read two fantasy, but hey I might! I want to read these two equally as much, so let me know down below your vote for what to read first!
I thought The Unexpected Everything was a good choice for a little bit of levity, and I know Morgan Matson books are such quick reads it might feel like a bit of a break.
Lastly, I threw in a few shorter comic books (The Mighty Thor, Descender and Wayward - all volume 1) and graphic novels (The Babysitters Club) from my to be read shelves, I've been in a real comic book/graphic novel mood recently so I think it'll be very easy to polish these off. I also have a few on my iPad that I still need to get to so will throw them in if I need them. Ps. yes, that is a full colour Babysitters Club graphic novel and I feel NO SHAME. I can't wait to re-experience these amazing books in graphic novel form, especially with Raina at the helm!
With these choices I'm happily ticking all the boxes, and I feel so ready, let's DO THIS!
So will you be joining in? Please do, I need more read-a-thon buddies! and link me up below if you are!
You can check out how I'm doing over at Smoking Peaches Goodreads, Smoking Peaches Instagram and/or Smoking Peaches Twitter!


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David Dobbs recently suggested that the 'selfish gene is one of the most successful science metaphors ever invented; unfortunately, it's wrong.' He purports to uncover a scientific trend in genetics that trumps the understanding of the central role the gene plays in biological evolution. To parrot the author, unfortunately, he's wrong. Dobbs describes the real phenomena of gene expression, and the fact that genes do not have to change for an organism to change its behavior, something that he refers to as genetic accommodation. He promotes the idea that gene expression is the engine of evolution rather than inheritable genes, without acknowledging that the variability of gene expression can only be preserved through inheritable genes, or that the concept of the selfish gene encapsulates this relationship between the two.
Multi-cellular organisms develop tissues as they mature. The cells that form a tissue express specific genes that provide structure or enzymes particular to the function of the tissue; these same cells inhibit expression of all other genes that code for functions performed by other tissues. This is simply the genetic explanation for tissue differentiation, and is an example of gene expression. This process is controlled by regulatory genes, and these genes are passed on to future offspring, allowing the same organism to perform the same functions in the same tissues generation after generation. Differential gene expression is ubiquitous, and regulatory genes that control gene expression are in fact the phenotypical result of genes themselves.
Genetic accommodation is a highly similar process: Gene expression driven by environmental stress, as in the author's example, can create a worker, a drone or a queen bee; in another species, either a grasshopper or locust. This still requires genes to be turned on or off to stop expressing one set of genes and start expressing another, and such differential gene expression is again either controlled by other genes, or the genetic control includes some kind of environmental feedback. The function of environmental feedback in the control of gene expression has already been highly characterized. One of the earliest elucidations of this phenomenon is the lac operon, which is a set of related genes which control milk sugar metabolism. The lactase enzyme is constructed when milk sugar is present, because the milk sugar itself biochemically interacts with the gene control mechanism found in the lac operon to allow the lactase to be constructed; absence of milk sugar biochemically triggers inhibition of the same genes. In these gene expression mechanisms, any short-term response to the environment cannot be passed on to future individuals without changes to the genes coded in the germ cell DNA, in this example the genes for the lac operon, some of which are environmentally sensitive regulatory genes and one of which codes for the lactase enzyme.
Epigenetics is the study of gene expression controls outside of the DNA. Epigenetic control of gene expression is a recently established phenomenon, but inheritance of epigenetic changes across generations outside of the normal DNA replication mode is not. Again, epigenetic control of gene expression (via methylation mechanisms in control regions of DNA or on structural chromosomal histone proteins to help open unfold DNA to allow access for gene expression, or conversely to inhibit unfolding of DNA) itself requires biochemical reactions mediated by enzymes, coded for by . . . wait for it . . . DNA. There are some recent claims that epigenetic changes of somatic cells are being inherited by new generations, but that seems a huge stretch: Only germ cells create the DNA used to create new offspring, and there is no obvious mechanism that would propagate somatic changes to the germ cells. Arguing even more against this Lamarckian transformation is that during the replication of DNA for the construction of a germ cell, even the epigenetic markers on the chromosomes and the histones for the germ cells themselves are systematically removed. It is far more likely that any inheritance of epigenetic changes is in fact inheritance of germ cell DNA mutations or chromosomal rearrangements during meiosis of regulatory or control genes which epigenetically regulate or control the specific phenotype in the somatic tissue being observed.
For either epigenetics or genetic accommodation or other forms of gene expression to take the predominant role in evolution would first require that there be a demonstrated mechanism for passing these changes either outside of the well-defined DNA replication mode, or that these gene expressions somehow generated changes in the genes coded for in DNA such that they are passed on. The author presents no evidence or argument for an alternative mechanism.
There are other problems with the article; the only reason I bothered to comment on this article at all is that it addresses important recent discoveries and new understandings, but misunderstands or misconstrues their impact and how they fit into today's biology. As it turns out, I found two other responses to this article which do a much clearer job of making my argument, this one by the evolutionary biologist Jerry Coyne: David Dobbs mucks up evolution, and this one, the response by the author of the Selfish Gene, Richard Dawkins: Adversarial journalism and the selfish gene.


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A controversial technique for extracting oil and gas from California's shale reserves is facing new scrutiny in California. But it's easier to describe what current rules for fracking are not, rather than what they are.
Fracking, or more formally, hydraulic fracturing, is a practice by which oil and gas explorers drive fluid into fissures in sediment and rock in order to force the fossil fuels out. The fluid's usually water, at least 90 percent of it, mixed with a "proppant" – sand or grit to "prop" open the space in the rock – and chemicals usually making up less than 1 percent of the mix.
In California, oil and gas producers don't need a special permit to frack a well, just an initial permit to drill or rework a site. The state's Department of Conservation, Division of Oil, Gas & Geothermal Resources, asks companies to disclose the practice, voluntarily, but doesn't require that. Nor does DOGGR, as it's called, require companies to say where they're fracking, what chemicals they're using and in what volume, how much water the practice requires, or what happens with the water that's used.
Activists say that's shocking, especially since historic records show fracking has happened in California for 60 years.
Now DOGGR is circulating what it calls a "discussion draft" of regulations – an informal set of rules meant to prompt public discussion. The discussion draft is a precursor to draft regulations, which will undergo formal scrutiny in order to be approved.
Defenders of the practice point out that, after all, it's been happening in California since the 1960s. A limited industry-reported analysis of California fracking practices compared to those in the Marcellus Shale has found that less water is necessary for fracking in California. California's geology, in which seismic faults fold, raise and lower rock strata, may limit the kind of techniques that work.
Regulators are holding three public hearings, in Los Angeles, in Bakersfield, and in Monterey, to "workshop" the rules.


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Mary - posted on 09/20/2012 ( 1 mom has responded )
Help!! My 3yo and I are flying cross country for a wedding and Disneyland. I have flown before but not since being diagnosed with anxiety disorder and PTSD, so I am a complete mess trying to prepare.
I need to know what to expect for my 3yo on the flight, and what to pack, for both carry-ons and baggage. What do I need to do to prepare my child for the trip.
I know I can ask the airline for help on the day of but I need help with everything before I get to the airport. I'm going to be a complete mess for the next 3 weeks till we leave.


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Pinoy Parazzi Vol 6 Issue 46 April 03 – 04, 2013
Related Post
If I were Angelica, I will not expect anything from this relationship. SHe has to find herself and do some soul searching. She just came from a break up and she was able to rebound right away? The first guy that came along, agad agad? Ano 'to, parang trial and error? Same with JL. By the way, bakit anong reason ng mga pag hihiwalay ni JL sa mga past GF niya? Third party ba? In other words, hindi pa sigurado si JL sa kung ano at sino talaga ang gusto niya. He is still searching and I can almost see that this relationship is going to be another statistic. Kailangan pa ni Lloydie ng emotional stability. I do wish however na kung si ANgel man ang gusto niya, dapat lang panindigan niya at hwg saktan ang puso ni Angel kasi vulnreable pa siya. She is not completely healed from her breakup with someone she lived with for 5 or 6 years. She is trying though. Kung niligawan niya si JL, dapat mag propose na rin siya para siguradong walang kawala.


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