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    To confirm the development of calcium deficiency, blood ionized calcium levels were measured on Days 10 and 17, at ~7h after the start of the light period. Approximately 70μl of blood was collected from the tip of the tail into a heparin-coated capillary tube (RAPIDLyte Multicap, Siemens) and within 15sec analyzed using a Rapidlab 348 blood gas analyzer (Siemens, Germany) located in the vivarium. For this and subsequent tests, care was taken to collect blood samples from the rats in an order that controlled for disturbances and any delays between sampling and assaying. To do this, the samples were collected successively from rats in tetrads containing one representative of each of the four diet groups, and the order within each tetrad was counterbalanced.

    Fasting blood glucose, insulin and triglyceride (TG) levels were measured in blood samples collected on Days 11, 18 and 42, at ~7h after the start of the light period. Taking into account that young growing rats were relatively small at the beginning of the experiment we opted to use a morning fast instead of more severe overnight food deprivation: In small rodents prolonged overnight fasting induces a catabolic state, and along with standard housing conditions at subthermoneutral temperatures it can decrease metabolic rate [ native Mens Howard Boat Shoe Shell White/Begonia Orange 100% authentic low price for sale discount low shipping fee izrgZF8
    ]. In our experiment, we aimed to collect measurements under less extreme physiological conditions. Rats were fasted for 5–6h and then a blood sample was collected from the tip of the tail into an EDTA-treated tube. The tubes were centrifuged and plasma glucose and TG levels were analyzed using enzyme-based colorimetric assays (Cayman Chemical, MI). Insulin levels were measured using ELISA (Rat insulin kit # EZRMI-13K, EMD Millipore, MA). Sometimes when the rats were young, insufficient blood was collected to conduct all the measurements. In these cases, insulin measurements were given priority.

    An oral glucose tolerance test was given between Days 23–25, at ~7h after the start of the light period. After an overnight fast, each rat was gavaged with 8ml/kg of 25% glucose solution (2g/kg). Blood was collected at 0, 15, 30, 60 and 120min. Plasma glucose and insulin levels were measured as described above.

    An insulin tolerance test (ITT) was given on either Day 36 or 37, at ~7h after the start of the light period. After a 5–6h fast, each rat received insulin [0.75IU/kg, 0.75ml/kg, i.p. of 1IU insulin (HumilinR, Eli Lilly) solution prepared in isotonic saline]. For this test, glucose levels were measured using an Accu-Check Aviva Plus blood glucose meter (Roche Diagnostics).

    Body weights were analyzed using weekly measurements taken on the day of the body composition assessment. Daily food intakes were averaged into weekly blocks, based on the sum of daily measurements divided by the number of days; days when rats were fasted overnight were excluded from analysis.

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    The progestogen is injected into a muscle or under the skin and then is gradually released into the bloodstream. It works mainly by stopping the release of the egg from the ovary (ovulation). It also thickens the mucus made by the neck of the womb (cervix) which forms a mucous plug. This stops sperm getting through to the womb (uterus) to fertilise an egg. It also makes the lining of the womb thinner. This makes it unlikely that any egg that has managed to be fertilised will be able to implant in the womb.

    Dr Rosemary Leonard

    It is very effective. Between 2-60 women in every 1,000 using it will become pregnant after two years. Compare this to when no contraception is used: more than 800 in 1,000 sexually active women who do not use contraception become pregnant within one year.

    Some women find that having unpredictable or irregular periods can be a nuisance. However, if you do develop irregular bleeding while receiving the injection then you should inform your doctor or nurse. You may be able to have your next injection early or be given other treatment to stop the bleeding. Irregular bleeding can sometimes be due to another reason, such as an infection. This may need to be treated.

    Apart from changes to periods, side-effects are uncommon. If one or more should occur, they often settle down over a couple of months or so. One possible side-effect is weight gain. This seems to be a particular problem for young women (under 18 years) who are already overweight when they start the injection. Some women also report fluid retention, worsening acne, headaches and breast discomfort. However, there is little evidence to say that the injection causes these symptoms.

    The most common reason for women to stop having the injections is because of irregular bleeding.

    The injection can lead to some 'thinning' of the bones. This does not usually cause any problems and the bones go back to normal when the injections are stopped. Using injectable contraception for many years might lead to more bone thinning. It is therefore recommended that you have a review every two years with your doctor or nurse. They will discuss if this method is still the best one for you.

    Very occasionally, the injection can cause some pain or swelling at the site where the injection was given, particularly with Sayana Press®. You should see your doctor or nurse if you have any signs or symptoms of infection at the site of injection (for example, redness or swelling).

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