Evaluating postoperative analgesics in mice using telemetry

The study examined the efficacy of preemptive or postoperative analgesia on surgical pain in the mouse. Radiotelemetry transmitters were surgically implanted in 28 female ICR mice. A mock ova implantation surgery was then performed. Mice were treated with a single dose of buprenorphine or flunixin meglumine prior to or after surgery, three doses of buprenorphine, or were untreated. Heart rate, blood pressure, home cage activity, food and water consumption, and body weight were measured. The no-analgesia group showed no significant differences between any parameters collected prior to surgery and those collected at similar times during the day of surgery. Significant increases in mouse activity on the day of surgery occurred with all analgesic treatments, compared with pre-surgical activity. There were no consistent significant changes in any other telemetry parameter after treatment with analgesics compared with no analgesia. Food consumption and body weight the day after surgery were reduced significantly in the animals treated with three doses of buprenorphine compared with untreated mice and mice given a single dose of buprenorphine. We conclude that the mock ova implant procedure does not induce sufficient pain to cause alterations in heart rate and blood pressure in the mouse. Activity was significantly reduced in the first 6 h after surgery in mice without analgesia, compared with activity prior to surgery. There were no significant differences between pre-emptive and postoperative analgesia. Body weight and food and water consumption were poor measures of pain because analgesia alone affected these parameters.     

Does preoperative administration of metamizol (Novalgin) affect postoperative body weight and duration of recovery from ketamine-xylazine anaesthesia in mice undergoing embryo transfer: a preliminary report

Thirty-two female mice used for embryo transfer or as controls received either metamizol or equal volumes of normal saline administered subcutaneously following induction of anaesthesia with ketamine-xylazine. Body weight was measured immediately before surgery, after 24 and after 48 h. The duration of the surgical anaesthesia was recorded and postoperative behavioural responses were measured. Comparison of the treatment groups revealed no significant differences in body weight and recovery times, nor were other signs of discomfort detected in either treatment group. It was concluded that administration of metamizol did not provide additional analgesia following embryo transfer in mice anaesthetized with ketamine-xylazine.     

Duration of effects on clinical parameters and referred hyperalgesia in rats after abdominal surgery and multiple doses of analgesic

This study evaluated the duration of clinical effects and referred hyperalgesia in rats (n = 10 per group) undergo ing abdominal surgery with analgesics (ketoprofen at 3 mg/kg and buprenorphine at 0.01 or 0.1 mg/kg) administered intramuscularly twice daily for 72 h beginning prior to surgery; no-surgery and no-analgesia control groups were included. Food and water consumption and body weight were monitored daily. As a measure of referred hyperalgesia, tail-flick latency was measured daily, before and 4 h after analgesia administration. Compared with those of the no-surgery controls, significant decreases in food consumption and body weight occurred 24 h after surgery without analgesics. There were nonsignificant reductions in these effects by analgesics, but the benefits were not significantly different than those of saline. These parameters continued to be decreased with variable significance in the buprenorphine groups at 48 and 72 h after surgery. In both buprenorphine-treated groups, water consumption was significantly increased at 24 h after surgery but not at 48 or 72 h. Tail-flick latency was not significantly different between the no-surgery and no-analgesia groups but was significantly increased 4 h after high-dose buprenorphine administration and declined nonsignificantly over time in the other groups. We conclude that painful effects from surgery are present primarily during the first 24 h after surgery. The analgesic regimens tested did not completely reduce these effects. Buprenorphine was associated with adverse effects for as long as 72 h after surgery. Referred hyperalgesia from this abdominal surgery could not be measured using the tail-flick assay.     

Evaluation of liposome-encapsulated oxymorphone hydrochloride in mice after splenectomy

The use of mice in biomedical research is increasing, largely due to the production and use of genetically engineered animals. Providing postoperative pain control in mice presents many challenges, and long-acting analgesic preparations would be advantageous for this species. A single subcutaneous injection of a liposome-encapsulated (LE) preparation of oxymorphone was compared with multiple injections of buprenorphine or saline in outbred mice undergoing splenectomy. Control groups were given isoflurane alone or isoflurane and an injection of LE oxymorphone but did not undergo surgery. The following parameters were evaluated for 5 days after surgery and were compared with presurgical baseline data for each group: food and water consumption, body weight, ethographic score, and voluntary exercise on a running wheel. Ethographic scores indicated less postsurgical pain in both groups of mice that received either analgesic preparation compared with mice that received only saline. However, mice given LE oxymorphone had superior postoperative recovery, as measured by wheel-running distance and body weight gain, compared with mice given buprenorphine or saline. Mice undergoing splenectomy had significant decreases in body weight, food and water consumption, voluntary exercise, and other normal behaviors. Administration of liposomal oxymorphone at the time of surgery improved postsurgical recovery as measured by these parameters compared with multiple injections of buprenorphine or saline alone. Administration of LE oxymorphone at the time of surgery improved postsurgical recovery, as measured by these parameters.     

Oral buprenorphine and aspirin analgesia in rats undergoing liver transplantation

The objective of this study was to establish effective postoperative analgesia for Dark Agouti rats undergoing liver transplantation with minimal additional stress due to handling and no adverse effect on transplant outcome. Oral administration of buprenorphine (0.5 mg/kg/dose) or aspirin (100 mg/kg/dose) in raspberry-flavoured gelatine were compared to controls receiving no treatment or plain gelatine. The drugs were presented five times: immediately on recovery from anaesthesia and at 12 h intervals thereafter. All rats underwent right nephrectomy and replacement of their liver by an arterialized liver isograft preserved optimally for 24 h. All groups had reversible hepatic damage, lost weight and demonstrated severely reduced dark cycle activity after surgery. Neither treatment appeared to ameliorate the loss of body weight that probably reflected hepatic insufficiency during the first week as well as pain and surgical stress. In the second week, when liver function was 'normal', rats began to regain weight at the pre-transplant rate. Aspirin treatment significantly increased activity during the first and second dark cycles after surgery, whereas buprenorphine significantly increased activity during the second dark cycle only. Neither drug had any apparent adverse effects on the rats or on graft function. Postoperative oral administration of aspirin should be incorporated into future programmes of liver transplantation in rodents. More effective treatment in the immediate postoperative period may require oral administration of analgesia prior to surgery or a single subcutaneous injection of an analgesic agent on completion of surgery in addition to postoperative oral administration of aspirin.     

Use of oral buprenorphine ('buprenorphine jello') for postoperative analgesia in rats--a clinical trial

Buprenorphine (0.1, 0.2, 0.3 or 0.4 mg/kg) in a flavoured gelatin base was administered preoperatively to rats undergoing a flank laparotomy. A control group of animals underwent surgery and received only flavoured gelatin. Body weight loss was significantly greater in the group which received no analgesia than in any of the analgesic-treated groups (P < 0.01). Food consumption was reduced significantly in all groups except in those animals which received 0.3 mg/kg buprenorphine. Water consumption was significantly reduced in the control (no analgesia) group (P < 0.001), but was not significantly depressed in the analgesic-treated groups (P > 0.05). Between-group comparisons did not show any significant difference between the different dose rates of analgesia used on either the change in body weight or the reduction in food or water consumption. The results of this study support the use of buprenorphine jelly for post-surgical analgesia in rats. This route of delivery is easy to use, and causes a minimum of stress to the rats.     

Influence of buprenorphine analgesia on post-operative recovery in two strains of rats.

The objective of this study was to establish an effective post-operative analgesic regimen for Sprague-Dawley (SD) and Dark Agouti (DA) rats. Buprenorphine (0.01 or 0.05 mg/kg), a partial mu opioid agonist, was administered subcutaneously immediately on completion of a standardized surgical procedure, involving anaesthesia, laparotomy and visceral manipulation. Two of the four treatment groups and the saline control group received a second injection 9 h later. Behavioural observations by three independent observers provided no information in assessing pain in this model. All rats lost weight, consumed less food and water after surgery. On the first day, both SD and DA rats receiving buprenorphine lost less weight than untreated control groups. Using weight loss as an efficacy criterion, low-dose buprenorphine, given once or twice, provided effective analgesia in SD rats. A higher single dose provided no additional benefit and a second dose was detrimental, reducing body weight and food intake. In DA rats, the high dose, given twice, appeared to be more effective than the lower dose. All DA cage cohorts consumed < 10% pre-operative food despite buprenorphine treatment, suggesting a higher dosage may be necessary. However, all SD and 80% DA rats who received no buprenorphine gained body weight on the second day, whereas most of the buprenorphine-treated rats continued to lose weight for another 2 days, despite increased food consumption by both strains. Buprenorphine may adversely affect intestinal function over a number of days due to its enterohepatic circulation; this effect may be more severe in DA rats. Adverse metabolic effects of buprenorphine and other opioids may preclude their use in the future if it can be shown that non-steroidal anti-inflammatory drugs (NSAIDs) provide equally effective analgesia.     

Comparison of buprenorphine and pethidine given intravenously on demand to relieve postoperative pain.

In a double-blind study of on-demand intravenous analgesia buprenorphine was found to be about 600 times as potent as pethidine. The incidence of side effects was similar with both drugs. The quality of analgesia, subjectively assessed, was good with both drugs using this method of administration. Provided that its low potential for abuse is substantiated, buprenorphine appears to be a powerful analgesic that may successfully be given intravenously on demand.     

Analgesic efficacy of orally administered buprenorphine in rats

The analgesic effect of orally administered buprenorphine was compared with that induced by a standard therapeutic injected dose (0.05 mg/kg of body weight, s.c.) in male Long-Evans rats. Analgesia was assessed by measuring pain threshold, using the hot-water tail-flick assay before and after administration of buprenorphine. The results suggest that a commonly used formula for oral buprenorphine in flavored gelatin, at a dose of 0.5 mg/kg, does not increase pain threshold in rats. Instead, oral buprenorphine doses of 5 and 10 mg/kg were necessary to induce significant increases in pain threshold. However, these doses had to be administered by orogastric infusion because the rats would not voluntarily eat flavored gelatin containing this much buprenorphine. The depth of analgesia induced by these infused doses was comparable to that induced by the clinically effective s.c. treatment (0.05 mg/kg).     

Pregnancy rates, prenatal and postnatal survival of offspring, and litter sizes after reciprocal embryo transfer in DBA/2JHd, C3H/HeNCrl and NMRI mice

Success of embryo transfer is often a limiting factor in transgenic procedures and rederivation efforts, and depends on the genetic background of the donor and recipient strains used. Here we show that embryo transfer to DBA/2J females is possible, and present data on pre- and postnatal success rates after reciprocal embryo transfer using the inbred DBA/2J and C3H/HeN, and outbred NMRI strains. The highest embryo yield was achieved in outbred NMRI females, but embryo yields were similar in DBA/2J and C3H/HeN mice following superovulation despite poor estrus cycle synchronization in DBA/2J females. In-strain transfer of DBA/2J blastocysts (transfer of embryos to recipients from the same strain) resulted in pregnancy rates (57.1%) similar to those obtained following in-strain transfer of C3H/HeN (60.0%) and NMRI mice (83.3%), although the prenatal survival rate of blastocysts was low. Moreover, from the pups born only half survived the postnatal period after transfer of DBA/2J and C3H/HeN blastocysts to DBA/2J recipients. These problems were not observed when transferring NMRI-blastocysts to C3H/HeN and DBA/2J mothers. The number of blastocysts transferred also had a positive effect on the success of embryo transfer. In conclusion, C3H/HeN and DBA/2J females can be used as recipients for embryo transfer procedures for certain donor strains like NMRI, as one major determinant seems to be the genetic background of the embryos transferred. We also recommend to increase the number of DBA/2J blastocysts transferred, and to foster the DBA/2J pups to other DBA/2J mothers postnatally for in-strain transfer of DBA/2J mice.     

Improved pregnancy and birth rates with routine application of nonsurgical embryo transfer

Nonsurgical embryo transfer (NSET) of blastocysts to pseudopregnant female recipients provides many benefits over surgical implantation with less distress for the mice, no anesthesia or analgesia required and a considerable reduction in implantation time per mouse. Although a disposable device to perform NSET is on the market since 2009, it is not generally used in transgenic facilities, most likely because surgical implantation is efficient and inexpensive. Here, we report that with several refinements to the original protocol, the NSET method becomes very attractive and outperforms the traditional surgical transfer on basis of pregnancy rate, birth rate and implantation-related discomfort. Furthermore, repeated use of the same NSET device on several recipient females reduces the costs to a reasonable level. The data presented covers all embryo transfers over the last 5 years at the transgenic facility of the Netherlands Cancer Institute, of which the last 2 years were performed exclusively with NSET.     

Buprenorphine does not affect acute murine toxoplasmosis and is recommended as an analgesic in Toxoplasma gondii studies in mice

Groups of mice were infected with tachyzoites of the RH strain of Toxoplasma gondii, treated with the opioid analgesic buprenorphine, sodium sulfadiazine, a combination of buprenorphine and sodium sulfadiazine, or nothing in the drinking water, on days -1 to 12 postinfection. Mice in the T. gondii-infected buprenorphine-treated group did not live significantly longer (P > 0.05) than mice given T. gondii and not treated with buprenorphine. Clinical observations of mice indicated that buprenorphine treatment reduced distress and pain in mice with acute toxoplasmosis. Mice treated with sodium sulfadiazine alone or sodium sulfadiazine combined with buprenorphine survived the 28-day study. Mice treated with buprenorphine and not infected with T. gondii also survived the 28 days. This study demonstrates that buprenorphine does not adversely interfere with acute T. gondii infection and indicates that buprenorphine can be given to mice to alleviate pain and distress associated with a T. gondii infection, and not adversely influence the results of toxoplasmosis studies. Analgesic (buprenorphine) treatment should now be the standard of care for mice in acute toxoplasmosis studies.     

Reversal of fentanyl/fluanisone neuroleptanalgesia in the rabbit using mixed agonist/antagonist opioids

The reversal of the neuroleptanalgesic combination of fentanyl/fluanisone using mixed agonist/antagonist opioids has been investigated in the rabbit. All of the compounds studied (naloxone, nalbuphine, meptazinol, butorphanol, buprenorphine, pentazocine, doxapram) reversed the respiratory depression and sedation produced by fentanyl/fluanisone. Fentanyl/fluanisone produced profound analgesia for 180 min, which was rapidly and completely antagonized by naloxone. The mixed agonist/antagonist opioids produced a reduction in the degree of analgesia but, in contrast to naloxone, analgesic activity persisted from 120 min (meptazinol) to 420 min (buprenorphine). Administration of buprenorphine to rabbits anaesthetized with fentanyl/fluanisone and midazolam confirmed that the reversal of respiratory depression was accompanied by the return of arterial pH, PCO2 and PCO2 to preanaesthetic values. The use of neuroleptanalgesic anaesthetic regimens, which have been shown to provide effective surgical anaesthesia, combined with reversal using a mixed agonist/antagonist opioid to provide postoperative analgesia, appears to be a valuable refinement of current laboratory animal anaesthetic practice.     

Production of identical twin and triplet mice by nuclear transplantation

Transplantation of a single nucleus from two- or four-cell embryos into one of the enucleated blastomeres of a two-cell embryo resulted in successful production of identical triplet and twin mice. The proportion of reconstituted embryos that developed in blastocysts was 71% (84/118) when four-cell embryos were used as donors of nuclei; 10 sets of quadruplet and nine sets each of triplet and twin blastocysts were obtained by this technique. After transfer to recipients, 30% (18/61) developed to term, and one set of identical triplet and four sets of identical twin mice were obtained. When two-cell embryos were used as donors of nuclei, 79 (95%) sets of twin embryos developed to blastocysts. Of 38 twin blastocysts transferred to recipients, 21 sets (55%) developed to term as identical twin mice. These results demonstrate that the enucleated two-cell embryo develops in vitro after transfer of a nucleus from a two- or four-cell embryo and the resultant blastocyst has high potential for development to term after transfer to a recipient.     

Correlation of increased concentration of ovine endometrial cyclooxygenase 2 with the increase in PGE2 and PGD2 in the late luteal phase

The effect of intraoviductal embryos on endometrial receptivity was studied by intraendometrial and intrauterine embryo transfer. Five-week-old female ICR mice were mated after superovulation; a vaginal plug confirmed day 1 of pregnancy. On day 4 (90 h after hCG injection), blastocysts were collected and transferred to pseudopregnant female mice and to recipient mice in which the uterotubal junction had been ligated bilaterally on day 1 of pregnancy. Three embryos per uterine horn, a total of six embryos per recipient mouse at days 1-6, were transferred to the endometrium or uterine cavity and implantation and pregnancy rates were calculated. The implantation rate for intraendometrial embryo transfer to recipients of days 3, 5 and 6 was significantly higher for uterotubal junction-ligated mice (72.2, 20.8 and 9.7%, respectively) than for pseudopregnant mice (55.0, 8.3 and 0.0%, respectively). The implantation rate for intrauterine embryo transfer to recipients at days 2, 5 and 6 was significantly higher for uterotubal junction-ligated mice (11.1, 25.0 and 8.3%, respectively) than for pseudopregnant mice (0.0, 3.3 and 0.0%, respectively). Uterotubal junction-ligated mice achieved implantation and bore neonates by intrauterine embryo transfer on days 2 and 6, whereas no implantation was achieved in pseudopregnant mice. The difference in implantation rate could not be explained by a difference in progesterone concentration between the groups. The distribution of proliferating cells in the endometrium was also studied immunohistochemically by use of anti-proliferating cell nuclear antigen (PCNA) antibody in the recipient mice. PCNA-positive cells were more abundant in uterotubal junction-ligated mice and demonstrated a marked extension from the epithelium to the stroma over time, in contrast to those in pseudopregnant mice. These findings indicate that an intraoviductal embryo exerts a biological effect by sending a signal to the endometrial epithelium and stroma, thus facilitating endometrial receptivity to the embryo and improving the rate of implantation.     

Are repeated doses of buprenorphine detrimental to postoperative recovery after laparotomy in rats?

Buprenorphine is a widely used analgesic for relief of postoperative pain in rats. The effect of repeated doses of buprenorphine throughout the postoperative pain and stress response is unknown. This investigation tested the hypotheses that (a) daily analgesic doses of buprenorphine for 7 d ameliorate the stress response after laparotomy in rats and (b) preoperative buprenorphine better ameliorates the response than do peri- and postoperative administration. Postoperative effects on body weight, daily food and water consumption, and daily fecal and urinary outputs were monitored in groups of rats treated for 7 d with analgesic doses of buprenorphine initiated at different time points relative to the time of laparotomy. Analgesic doses of buprenorphine had no effect on the study parameters in healthy unoperated rats. Daily injection of buprenorphine delayed the time at which the preoperative body weight was restored without decreasing the postoperative changes in daily food consumption, water intake, and fecal and urinary outputs in the operated rats. The effects of daily analgesic doses of buprenorphine for 7 d on body weight, daily food, and water consumption, and fecal and urinary outputs were minimal and less statistically significant than the changes caused by surgery itself. However, this dosing regimen seems to delay the restoration of body weight after abdominal surgery in rats.     

Hemodynamic and electroencephalographic effects of epidural buprenorphine during orthopedic hindlimb surgery in sheep: A comparison with intramuscular buprenorphine and epidural saline

Epidural opioids exert segmentally limited spinal analgesia by acting at dorsal horn neurons. While the spinal cord may be the predominant site of action for hydrophilic opioids such as morphine, initial supraspinal effects have been suggested for lipophilic opioids such as buprenorphine. In view of significant systemic effects, the value of epidural administration of lipophilic opioids in human patients has been questioned. Since epidural buprenorphine may be beneficial for hindlimb surgeries in sheep, intraoperative hemodynamic and central nervous effects were evaluated. In a prospective, randomized, and placebo-controlled study, 15 adult sheep anesthetized for cranial cruciate ligament reconstruction were treated with either epidural buprenorphine (5 μg/kg, n = 5), intramuscular buprenorphine (5 μg/kg, n = 5), or epidural saline (0.15 ml/kg, n = 5) preoperatively. Heart rate, arterial blood pressures and the electroencephalographic variables /δ ratio, α/δ ratio, β/δ ratio, median power frequency (MED), and 80% spectral edge frequency were recorded before and immediately after skin incision and during drilling a tunnel through the tibia bone. Arterial pressures after epidural buprenorphine were significantly lower compared with epidural saline but were quite similar to intramuscular buprenorphine before skin incision. Mean EEG /δ ratios and MED values following epidural and intramuscular buprenorphine were significantly lower compared with epidural saline but quite similar for the two buprenorphine groups during drilling. In conclusion, similar hemodynamic and electroencephalographic effects of epidural and intramuscular buprenorphine suggest systemic effects of epidural buprenorphine in sheep.     

多模式术后镇痛在腹腔镜结肠手术中的应用

目的:理论上联合使用不同机制镇痛药较镇痛药单独使用镇痛效果更完善,在妇科、骨科等手术中已有结论;笔者观察比较腹腔镜结肠手术术使用单一止痛药及联合使用不同机制镇痛药在术后镇痛的效果以及各自不良反应的发生率。方法:择期腹腔镜结肠手术患者90例,随机分为3组,每组30例。A组使用地佐辛+氟比洛酚酯行术后镇痛为多模式镇痛组;B组使用地佐辛行术后镇痛;C组使用氟比洛酚酯行术后镇痛。记录每组术后4、8、12、24 h视觉模糊评分(VAS)及术后不良反应包括嗜睡、躁动、恶心呕吐的发生率。结果:A组术后4 h、8 h的VAS评分低于B、C两组,差异有显著性,A组无嗜睡及躁动发生,发生呕吐1例,不良反应发生率A组低于B、C两组,差异有显著性。结论:地佐辛+氟比洛酚酯联合用药可安全有效应用于腹腔镜结肠手术术后镇痛,是一种有效的多模式术后镇痛方式,在减弱疼痛的放大效应及对中枢神经的作用两方面起效,因而较单独使用地佐辛及氟比洛酚酯有更好的镇痛效果,且不良反应低于单独使用地佐辛及氟比洛酚酯。     

Transdermal fentanyl compared with parenteral buprenorphine in post-surgical pain in swine: a case study

The use of pigs as research animals in survival surgery has increased greatly in the last 15 years. Personnel conducting pig research have been hampered by a lack of proven long-acting analgesics for treatment of surgical pain of longer duration, and by a lack of reliable non-subjective parameters for the assessment of pain relief. The efficacy of the mixed opioid agonist-antagonist buprenorphine hydrochloride 0.10 mg/kg pr (n = 2) in the treatment of post-thoracotomy pain was compared with that of a transdermal therapeutic system (TTS) delivering 25 microg/h (n = 3) or 50 microg/h (n = 2) of the mu opioid agonist fentanyl hydrochloride. Food consumption, pain score, activity level and rate of movement were assessed under four conditions: normal pre-operative control (24h), pre-operative with analgesic alone (24h) and post-operative days 1, 2, 3 (72h). Serum concentration-time curves for fentanyl in clinical cases revealed that female Yorkshire cross pigs weighing 26.2+ 2.1 kg achieved serum values in the recognized human therapeutic range when treated with TTS fentanyl at 50 microg/h and experienced adequate pain control. Pigs treated with 25 microg/h TTS fentanyl had serum levels below the human analgesic range, experienced less adequate analgesia, and required supplemental analgesia in some cases. Based on existing pharmacokinetic data for fentanyl in pigs, the rate of uptake of TTS fentanyl when attached on inter-scapular skin was lower than predicted. Clinical pain scores and time intervals between each major postural change were not affected by analgesics in the absence of pain, but increased in all groups after surgery regardless of treatment. Food consumption was unaffected by analgesic treatment alone but decreased in all groups after surgery regardless of treatment. Analgesic effects on postoperative activity level were variable. TTS fentanyl at appropriate doses is a cost effective means of delivering basal analgesia following major surgery in pigs.     

Adverse effects on growth rates in rats caused by buprenorphine administration

As a routine postoperative treatment, a single dose of buprenorphine was given to rats at a dose of 0.05 mg/kg subcutaneously. However, some rats developed abnormal secretions around the nose and mouth and some animals died 3-5 days after surgery and analgesic treatment. At autopsy a yellow fibrous mass was found in the stomach and intestines. Observations of animals given buprenorphine revealed an abnormal ingestion of bedding material. This caused a disturbance to normal digestion, with gastric distension, weight loss or decreased growth rate, constipation and occasionally death. In this study rats were monitored for 6 days following surgery and analgesic treatment. A comparison of growth rates was made between rats given saline and buprenorphine or nalbuphine and between animals kept on bedding or grid floors for the first 24h after treatment. Of the animals held on bedding, the buprenorphine-treated animals did not lose weight as the other animals did, but had on the other hand a decreased growth rate during the measuring period of 6 days after surgery. When denied access to bedding for the first 24 h after surgery, rats given saline or nalbuphine had a reduced weight gain over the first 24 h, similar to the groups held on bedding. Rats held on grid floors and given buprenorphine continued to gain weight for the first 24 h. From day 3, there was no significant difference between the groups, which all gained weight.