Effects of calcium and vitamin D Intakes In Post weaning Rats on Later Adiposity Outcome

Effects of calcium and vitamin D Intakes In Post weaning Rats on Later Adiposity Outcome

Gamila, M.S. Mohamed* - Ashraf.A.Abdel- Maged**- Rasha, S.A. Ismail**

*Graduate Student, Nutrition and Food Science Dept. Faculty of Home Economics, Helwan university Egypt.

** Department of Nutrition and Food science, Faculty of Home Economics, Helwan university Egypt.

Abstract

This study was carried out to investigate the effects of calcium (Ca) and vitamin D (V.D) intakes in post weaning rats on later adiposity outcomes. Feed intake (FI), body weight gain % (BWG%), organs weight (OW) / body weight (BW) %(OW/BW%), peritoneal fat pad% (PFP %) in post weaning rats, serum glucose ,and leptin level were studied. A total of 66 male albino rats of Sprague Dawley strain, 25 days of age weighing 40±5g were used in this study. The rats were divided into two main groups: The first main group (6 rats) was fed on abasal diet (BD) and used as a control negative group (-ve).

The second main group(60 rats) was divided into (10 subgroups), one of them (6rats fed on BD) and considered as control positive (+ve) group. The other (9 subgroups) were fed on (BD) containing different levels (50, 150, and 200%) from calcium, vitamin D, and calcium plus vitamin D, for 8 weeks, followed by second 8 weeks' rats were fed on a high- fat diet (HFD) as the following. The positive control group (+ve) shift up to feed only on (HFD), while the other (9subgroups) were fed on (HFD) containing the same previous levels (50, 150 & 200%) from Ca,Vit.D, and Ca plus Vit D.

At the end of the experiment, rats were sacrificed, and the peritoneal fat pad, liver, and kidney were then removed and weighed Blood samples were collected, then serum was separated, leptin and glucose levels were determined. The obtained results revealed that feeding the early post weaning rats for the first8 weeks on (BD) containing 50% of Ca, vitamin D & calcium plus vitamin D requirements  followed by second 8 weeks fed on (HFD) containing the same low-levels from Ca, Vit. D and Ca plus V.D caused a significant increase in feed intake, body weight gain % , peritoneal fat pad % , organs weight / body weight % , and a significant increase in Leptin and blood glucose level, while supplementation with Ca, Vit.D and Ca plus Vit. D showed a significant decrease in postweaning rats later adiposity outcome and a significant decrease in serum leptin hormone and glucose levels .

Introduction

Childhood obesity, the abnormal or excessive fat accumulation that may impair health has become a global problem.Epidemiological data show that the number of overweight or obese infants and young children (aged 0-5 years) increased from 32 million globally in 1990 to 42 million in 2013. If this trend will continue, the number of overweight or obese infants and young children is expected to spiral up to 70 million by 2025 ( WHO, 2016).

Although the prevalence of childhood obesity is estimated to be lower than the prevalence of adult obesity (5% against 13%), the rate of increase in childhood obesity in many countries is alarmingly greater than the rate of increase in adult obesity (Afshin et al., 2017).

There is evidence that on early adiposity rebound the point in life when body mass index(BMI) rises again after reaching a nadir, Predicts later obesity (Van Hoek et al., 2014). Hence obese children are more likely than children with normal BMI to remain obese as an adult (Middieron et al., 2016).

Nobre et al., (2012) reinforced that early weaning Leads to late development of some components of the metabolic syndrome and Leptin resistance. Many researchers have focused on the identification of the combination of macronutrients Capable of regulating body weight , but the effect of micronutrients still needs further explored (Malanson et al.,(2003) & Loos et al., (2004) and Gunther et al.,(2005). Cleared that minerals participate in the energy metabolism and in the secretion and action of insulin and may interfere in the control of obesity. The combination of low serum 25-hydroxy vitamin D and inadequate calcium intake has been associated with cardiovascular risk factors such as hypertension, obesity, metabolic syndromes and type 2 diabetes mellitus (Buyukinan et al., 2012).Therefore, the present study was carried out to investigate the relationship between a supplemented diet with different levels of calcium and vitamin D alone or in combination in post weaning rats and later adiposity outcome in male albino rats.

Material and Methods

Chemicals: Casein, vitamins, minerals, cellulose, choline chloride were obtained from El-Gomhoria company, Cairo, Egypt, Starch, and soy oil were obtained from local market.

Kits: Kits for biochemical analysis were obtained from Gamma Trade Co. For pharmaceutical and chemicals. Dokki, Egypt.

Rats:   Sixty-six male albino rats of Sprague Dawley strain, 25 days of age weighing (40 ± 5g), were obtained from the laboratory of the animal colony, Ministry of Health and population, Helwan, Cairo. Egypt.

Experimental Animal Design:

Rats were housed in individual Cages under hygienic laboratory conditions and were fed on a basal diet for one week for adaptation. After this week, the rats were divided into two main groups. The first main group (6 rats) fed on abasal diet (BD) as a control negative group (-ve). The second main group: Sixty rats were fed for 8 weeks on (BD) containing different levels (50,150 and 200%) from calcium, vitamin D, and calcium plus vitamin Das the following: Subgroup one, slat mix containing (100%) from calcium and vitamin D requirements and used as a positive control group (+ve). Subgroup two fed on (BD) while salt mix containing 50% from calcium requirement, subgroup three fed on (BD) salt mixture containing 150% from calcium requirement, while subgroup four Salt mixture containing 200% from calcium requirement. Subgroup five fed on (BD) containing vitamin mixture containing 50% from vitamin D requirement, while subgroups, six and seven fed on (BD) vitamin mixture containing 150 and 200% from vitamin D requirement. While subgroup eight fed on (BD) containing 50% from calcium plus 50% from vitamin D requirements, while nine and ten subgroups fed on (BD) containing 150, 200%respectively from their calcium and vitamin D requirements.

After the first eight weeks all animals of the second main group, followed by the second 8 weeks, rats were shift up to feed on a high-fat diet (HFD) as the following. The positive control group (+ve) shift up to feed on (HFD) containing (100%) from calcium and vitamin D requirements., while the other (9 subgroups) were fed on (HFD)containing the same previous different levels (50, 150& 200%) from calcium, vitamin D and calcium plus vitamin D.

The basal diet consists of 14% casein (protein 85%), corn oil 4%,saltmixture 3.5%, vitamin mixture 1% choline chloride 0.25%, cellulose 5% and (72.25%) cornstarch (Reeves et al. 1993).

The salt mixture and vitamin mixture were prepared according to Hegested et al (1941) and campbell (1963). The high fat diet, containing (14% protein form casein,20% fat (19% saturated fat and 1% unsaturated fat), 5% cellulose,3.5% salt mixture, 1% vitamin mixture, 10% sucrose, 0.25% choline chloride and the reminder was corn starch (Min et al., 2004). Calcium and Vit. D were calculated as a percentage of the standard requirements. Percentage between calcium and phosphorus was 1:1 on all experimental levels. During the experimental periods first (8 weeks) and second 8 weeks. Body weight and food consumption were measured twice a week and total food intake of the experimental period was calculated. Biological evaluation for different groups, body weight gain%, organs weight/body weight were determined according to Chapman et al. (1959).Determination of retroperitoneal fat pads according to (Azain et al.,2000).

Biochemical analysis of serum:

At the end of the experiment period, the rats were starved for 12h and then sacrificed under ether anesthetization. Blood samples were collected from the hepatic vein by the means of fine capillary glass tubes Schermer, (1967). Blood samples were received into clean dry centrifuged for 10 minutes at 3000 r.p.m. to separate serum. Serum was carefully separated into dry clean Wasserman tubes, using a Pasteur pipette and kept frozen at (-20 °c ) till estimation of some biochemical parameters.

Determination of leptin hormone by leptin ELISA Kit according to the method described by Guillaume and Bjorntorp (1996). Determination of serum glucose was carried out calorimetrically according to the method of Trinder, (1969). Statistical analysis was carried out using SPSS statistical software version 11 (SAS., 2004).

Results and Discussion

Effects of Basal Diet Containing Different Levels form Calcium and Vitamin Don Feed Intake and Body Weight Gain% in Early Post weaning Albino Rats:

            Table (1) shows the effect of the basal diet (BD) containing different levels(50,150&200%) from calcium (Ca), vitamin D & calcium plus vitamin D for the first 8 weeks of early post weaning rats and its effect on feed intake (FI) and body weight gain% (BWG%) of post weaning albino rats 25 days of age.

Results revealed that all groups fed on (BD) containing high level form calcium, vitamin D& calcium plus vitamin D (100, 150&200%) recorded a significant decrease (P < 0.05) in FI as compared to groups fed on (BD) containing 50% from calcium, vitamin D & Calcium plus vitamin D, on the other hand, results showed that groups fed on (BD) containing 50% form calcium, vitamin D & calcium plus vitamin D recorded a significant increase (P<0.05) in FI as compared to the control (-ve) group.

Concerning body weight gain% our results revealed that animal groups fed on (BD) containing 50% from Calcium or vitamin D & calcium plus vitamin D recorded a significant increase (P < 0.05) in BWG % as compared to the control group fed on (BD) containing 100% from calcium and vitamin D.

Our results showed that animal groups fed on (BD) containing 150 or 200% from calcium, vitamin D& calcium plus vitamin D recorded a significant decrease (P<0.05) in BWG% as compared to  control group fed on (BD) containing 100%From calcium and vitamin D requirements.

Our results indicate that supplementation with calcium plus vitamin D significantly reduced FI and BWG%.

In this respect (Zemel, 2005) suggested that dietary calcium and calcium supplements may increase fecal excretion of fat by forming insoluble complexes in the intestine. On the other side (Morris and Zemel, 2005) demonstrated that calcium intake may interfere with appetite regulation, however, this effect was assessed in only a few studies and hypothesis is not been confirmed (Dougkas et al., 2011).

Effects of (BD) Containing Different Levels from Calcium and Vitamin D for 8 Weeks Followed by Second 8 Weeks On (HFD) Containing The Same Levels from Ca & Vit.D On (FI) And (BWG%) In Post Weaning Rats.

Table (2) illustrates the effect of a high-fat diet (HFD) containing different levels from calcium and vitamin D (50,150 and 200%) on (FI) and (BWG%) of post-weaning rats after the second 8 weeks.

Results revealed that (FI) in post weaning albino rats, fed on (HFD) containing low level (50%) from calcium, vitamin D, and calcium plus vitamin D induced a significant increase (P < 0.05) in FI as compared to the control (+ve) group which fed on (HFD) containing 100% from calcium and vitamin D requirements, while other groups fed on (HFD) containing 150 or 200% from calcium, vit D & calcium plus vitamin D FI recorded a non-significant difference as compared to the control (-ve) group. Concerning body weight gain % of post weaning albino rats after the second 8 weeks which fed on (HFD) containing different levels from calcium. vitamin D and calcium plus vitamin D.

In conclusion, our results revealed that groups of post weaning rats fed on BD for 8 weeks followed by 8 weeks on (HFD) containing low level 50 % from calcium, vitamin D & calcium plus vitamin D recorded a significant increase (P < 0.05) in FI, BWG% as compared to groups fed on (BD) followed by (HFD) containing 150 or 200% from calcium, vitamin D & calcium plus vitamin D.

On the other hand, results revealed a non-significant difference in body weight gain% between (+ve) group fed on (HFD) containing 100% from calcium and vitamin D requirements and post weaning groups fed on (HFD) containing 50% from calcium, vitamin D, and calcium plus vitamin D. Results revealed that supplemented diet of post weaning rats with 150 or 200%from calcium, vitamin D & calcium plus vitamin D induced a significant decrease (P < 0.05) in FI and BWG %.

Our results revealed that the best result as anti-obesity greatest effect recorded by group fed on (BD) for 8 weeks followed by second 8 weeks fed on (HFD) supplemented with 200% from ca plus vitamin D followed by group fed on (BD) for 8 weeks followed by second 8 weeks on (HFD) supplemented with 150% form calcium plus vitamin D then 200% from calcium or vitamin D.

In this respect (Bruckbaueretal., 2009) suggested that Calcium attenuates the direct effects of vitamin D on adipocyte lipid metabolism, which accounts for at least part of the “antiobesity” effects of dietary calcium, an alteration in metabolic efficiency and increased core temperature and uncoupling Protein-2 expression, in addition to decreased efficiency of energy utilization.

Effects of (BD) for 8 Weeks Containing Different Levels Followed by Second 8 Weeks On (HFD) Containing Different Levels from Calcium and Vitamin D on Peritoneal Fat Pad % and Organs Weight / Body Weight % In Post weaning Albion Rats:

Table (3) shows the effect of (BD) containing different levels (50,150&200%) from calcium, vitamin D & calcium plus vitamin D for the first 8 weeks in early post weaning rats , followed by second 8 weeks shift up to feed on (HFD) containing the same levels (50, 150&200%) from calcium, vitamin D and calcium plus vitamin D on peritoneal fat pad %  (PFP%) and organs weight/body weight% (OW/BW%) in post weaning albino rats. Results revealed that control (+ve) post weaning rats fed on (HFD) containing 100% from Ca or vitamin D requirements recorded a significant increase (P<0.05) in (PFP%) and (OW/BW%) as compared to the control (-ve) group fed on (BD).

On the other side results revealed that all groups fed on (HFD) containing low levels (50%) from calcium, vitamin D& calcium  plus vitamin D recorded a significant increase (P < 0.05) in ( PFP%) and (OW/BW %) in post weaningrats. On the other hand, our results revealed that all groups fed on (HFD) supplemented with (150 or 200%) from calcium , vitamin D & calcium plus vitamin D induced a significant decrease (P<0.05) in (PFP%) and (OW/BW) in post weaning albino rats.

Results revealed a non-significant difference between (PFP% and OW/BW%) of control negative group fed on (BD) and groups fed on (HFD) supplemented with 200% from calcium plus vitamin D. In this concern Sergeev and song, 2014) demonstrated that the combination of D3 plus Ca was more effective than Ca alone in decreasing adiposity. Also cleared that modulation of apoptosis is emerging as a promising antiobesity strategy because removal of adipocytes through this process will result in reducing body fat.

Effects of (BD) Containing Different Levels from Calcium and Vitamin D for8 Weeks Followed by Second 8 Weeks On (HFD) Containing The Same Different Levels from Calcium and Vitamin D On Leptin Hormone Level in Post weaning Rats:

Results presented in table (4) illustrate the effect of feeding early post-weaning rats on (BD) containing different levels (50, 150&200%) from calcium, vitamin D, and calcium plus vitamin D, followed by second 8 weeks fed on (HFD) containing the same levels from Ca, Vit. D and Ca plus Vit. D. on serum leptin level.

Results revealed that the serum leptin in the control (+ve) group fed on (HFD) was (12.555 ± 0.994) while the control (-ve) group fed on (BD) recorded (4.767 ± 0.318).Statistical analysis showed that there was a significant increase (P< 0.05) in serum leptin level of the positive control group as compared to the control negative group fed on (BD).

Our results revealed that groups that fed on (HFD)containing 50% form calcium, Vit. D & Cal plus vitamin D recorded Leptin hormone levels as (13.833 ± 0.521, 13.050 ± 0.794 & 14.893 ±0.7,respectively). Statistically, this result revealed a significant increase in serum leptin hormone level as compared to the control (+ve) group fed on (HFD) containing 100% calcium and vitamin D Requirement.

On the other hand, results revealed that groups that fed on (HFD) containing 150 or 200% from calcium, vitamin D, and calcium plus vitamin D, recorded leptin values as (9.502 ±0.593, 8.886 ± 0.760 & 7.949 ± 0.456 and 6.819., respectively).

Statistically, results showed a significant decrease (P<0.05) in Leptin hormone as compared to the (+ve) control group fed on (HFD) containing 100% calcium and vitamin D requirements.

Concerning this respect (Pilcova et al, 2003) reported that high serum leptin level in obese patients could indicate leptin resistance. on the other hand, fat mass loss triggers a decrease in serum leptin level may reflect restoration of leptin sensitivity. changes in leptin levels were significantly correlated to the changes of percentage body fat (Reinehr et al 2005) .

Effects of (BD) Containing Different Levels from Calcium and Vitamin D for 8 Weeks Followed by Second 8 Weeks On (HFD) Containing The Same Different Levels from Calcium, Vitamin D On Serum Glucose Level in Post weaning Rates:

Table (5) shows the effect of feeding early post weaning rats on (BD) containing different levels (50,150&200%) from calcium, vitamin D& calcium plus vitamin D for 8 weeks followed by second 8 weeks fed on (HFD) containing the same different levels from calcium, vitamin D and calcium plus vitamin D on serum glucose level in post weaning rats. Results revealed that the serum glucose level of the control (+ve) group fed on (HFD) containing 100% from calcium and vitamin D requirement VS the (-ve) group fed on (BD) recorded (154.624 ± 3.115vs80.973 ± 2.993, respectively). Statistically, results showed a significant increase (P<0.05) in serum blood glucose in the (+ve) group as compared to the (-ve) control group fed on (BD).

On the other side, results revealed that groups which fed on (HFD) containing low levels 50% from calcium, vitamin D & calcium plus vitamin D recorded serum blood glucose levels as (157.139 ±3.490,162.651 ± 3.966&174.990 ±4.007, respectively). Statistically these results showed a significant increase (P<0.05) in serum blood glucose level as compared to the control (+ve) group and as compared to the group fed on (HFD) containing (150 or 200%) from calcium, vitamin D and calcium plus vitamin D.

Our results also cleared that groups that fed on (HFD) Containing 150 and 200% from calcium, vitamin D & calcium plus vitamin D recorded serum blood glucose levels as (136.706 ± 2.217, 134.333 ±2.652 and 119.136 ±2.221, 128.173 ±1.912,124,284 ±2.489 & 109.895 ±2.803, respectively).

Statistically results revealed a significant decrease (P< 0.05) in serum blood glucose level of groups fed on(BD) then on (HFD) containing supplementation with 150 or 200% from calcium, vitamin D, and calcium plus vitamin D, as compared to groups fed on (HFD) containing the low level 50% from calcium, vitamin D& calcium plus vitamin D.

The best results were recorded by groups fed on (BD) then (HFD) supplemented with 200% from calcium plus vitamin D followed by groups supplemented with 150% from calcium plus vitamin D.

In this concern (Pannu et al., 2016) explored a mechanistic framework that links calcium and vitamin D status, calcium increasing whole-body fat oxidation and increasing fecal fat excretion, while there is good cellular evidence for vitamin D reducing inflammation, an increase in insulin sensitivity following vitamin D.

Conclusion

In conclusion, the results indicated that at early post weaning age groups of rats fed for the first 8 weeks on (BD) followed by second 8 weeks fed on (HFD) containing low level 50% from calcium, vitamin D and calcium plus vitamin D induced a significant increase in FI, BWG%, and PFP % and elevated in Leptin and blood glucose level while groups fed for 8 weeks on (BD) supplemented with levels (150 or 200%) from calcium, vitamin D and calcium plus vitamin D, followed by second 8 weeks were fed on (HFD) supplemented with the same levels from calcium and vitamin D recorded a significant decrease in FI, BWG%, and Peritoneal Fat Pad (PFP) % and induced a significant decrease in Leptin and blood glucose levels, In conclusion, the results taken together suggested that, at early post weaning age first and second 8 weeks, supplemented diet with calcium and vitamin D maybe have a significant effect to compate for later adiposity outcome and its complications.

Table (1) :Effect of basal diet containing different levels from calcium and vitamin D on feed intake and body weight gain % in albino rats 25 age after the first 8 weeks:

*(BD) Basal Diet

*(BDC) Basal Diet containing different Levels from calcium and vitamin D.

***(LSD): least significant differences at p < 0.05

Values in each column which have different litters are significantly different (p < 0.05).

Table (2) :Effect of basal diet containing different levels from Ca and Vit.D on feed intake and body weight gain % of albino rats after the second 8 weeks. 

*(BD) Basal Diet

**(HFD) High fat Diet containing different Levels from calcium and vitamin D.

***(LSD) least significant differences at p< 0.05.

Values in each column which have different litters are significantly different (p < 0.05).

Table (3) : Effect of basal diet containing different levels from calcium and vitamin D on peritoneal fat pad % and organs weight / body weight % in post weaning albino rats after the second 8 weeks.

*(BD) Basal Diet

**(HFD) High Fat Diet containing different Levels from calcium and vitamin D.

***(LSD) least significant  differences at p< 0.05.

Values in each column which have different litters are significantly different (p < 0.05).

table (4): Effect of basal diet containing different levels from calcium and vitamin D on leptin hormone level in post weaning albino rats after the second 8 weeks:

*(BD) Basal Diet

**(HFD) High fat Diet containing different Levels from calcium and vitamin D.

***(LSD) least significant  differences at p< 0.05.

Values in each column which have different litters are significantly different (p < 0.05).

Table (5): Effect of basal diet containing different levels from calcium and vitamin D on serum blood glucose level in post weaning albino rats after the second 8 weeks.

*(BD) Basal Diet

**(HFD) High fat Diet containing different Levels from calcium and vitamin D.

***(LSD) least significant  differences at p< 0.05.

Values in each column which have different litters are significantly different (p < 0.05).

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تأثير المأخوذ من الکالسيوم وفيتامين د في الفئران بعد فطامها على حدوث السمنة فيما بعد.

  جميلة محمد سيد محمد*   ا.د. أشرف عبد العزيز عبد المجيد** ا.د. رشا صبحي أحمد إسماعيل**

*طالبة دراسات عليا بقسم التغذية وعلوم الأطعمة – کلية الاقتصاد المنزلي جامعة حلوان

**قسم التغذية وعلوم الأطعمة – کلية الاقتصاد المنزلي- جامعة حلوان

الملخص العربي

أجريت هذه الدراسة لمعرفة تأثير المأخوذ من الکالسيوم وفيتامين د في الفئران بعد  فطامها على حدوث السمنة فيما بعد وذلک عن طريق تقدير المأخوذ من الطعام، نسبة الزيادة في وزن الجسم، نسبة وزن الأعضاء لوزن الجسم، نسبة الدهون وقد استخدم في هذه الدراسة عدد 66 فأر من نوع الالبنيو (اسبراجودولي) عمر 25 يوم أوزانهم ( 40± 5 جرام)  .تم تقسيم الفئران إلى مجموعتين رئيسيتين المجموعة الرئيسية الأولى (6 فئران) تم تغذيتها على غذاء أساسي وتم استخدامها کمجموعة ضابطه سالبة. المجموعة الرئيسية الثانية مکونة من (60 فأر) تم تقسيمها إلى عشرة مجموعات فرعية واحده منهم ( 6 فئران) تم تغذيتها على الغذاء الأساسي وتم استخدامها کمجموعة ضابطة موجبة بينما التسعة مجموعات الأخرى تم تغذيتها على الغذاء الأساسي مع احتوائه على نسب (50 أو 150 أو 200% ) من الاحتياجات لکل من الکالسيوم، فيتامين د، الکالسيوم مع فيتامين د، وذلک لمدة 8 أسابيع أعقبها 8 أسابيع ثانية , تم تغذية العشرة مجموعات الفرعية على غذاء عالي الدهون يحتوي نفس الترکيزات من الکالسيوم، فيتامين د، الکالسيوم مع فيتامين د وفي نهاية مدة التجربة تم اخذ الأوزان ثم تم تخدير الفئران وتم تجميع عينات الدم، ثم تم فصل الدم لتقدير هرمون اللبتن، الجلوکوز کما تم الإزالة وتقدير الوزن لطبقة الدهون في الغشاء البريتوني، الکبد، الکلى وقد أظهرت النتائج أن إطعام الفئران في مرحلة الفطام المبکر لمدة 8 أسابيع على غذاء أساسي يحتوي على مستوى منخفض 50% من الاحتياجات من الکالسيوم، فيتامين د, تلاها التغذية لمدة 8 أسابيع ثانية تمت التغذية على غذاء عالي الدهون يحتوي على نفس النسب من فيتامين د والکالسيوم تسبب في حدوث زيادة في کلا من نسبة الطعام المأکول، نسبة الزيادة في وزن الجسم، نسبة وزن الأعضاء الداخلية بالنسبة لوزن الجسم، نسبة الدهون في العشاء البريتوني وارتفاع في هرمون الليبتن ومستوى سکر الدم بينما أظهرت النتائج أن المجموعات الأخرى التي تغذت على غذاء رئيسي مدعم بنسب 150 أو 200% من الکالسيوم، فيتامين د، لمدة 8 أسابيع تلاها التغذية لمدة 8 أسابيع ثانية على غذاء عالي الدهون ومدعم أيضا بنفس النسب من الکالسيوم، فيتامين د، الکالسيوم مع فيتامين د. قد  أدى ذلک إلى انخفاض معنوي في کلا من معدل تناول الطعام، نسبة الزيادة في وزن الجسم، نسبة وزن الأعضاء لوزن الجسم وأيضا انخفاض في مستوى هرمون الليبتن وسکر الدم. وقد استخلصت النتائج أن التدعيم بالکالسيوم وفيتامين د في مرحلة الفطام المبکر يمکن أن يکون له تأثير معنوي في التحکم في منع حدوث السمنة ومضاعفاتها.