Early femoral ossification in offspring as an indicator of linear growth following maternal protein restriction: Effects of protein repletion and spirulina supplementation

¹ Nutritional Science Study Program, Faculty of Medicine and Nutrition, IPB University, Bogor 16680, Indonesia
² Nutrition Study Program, Faculty of Science and Technology, Universitas Al-Azhar Indonesia, Jakarta 12110, Indonesia
³ Medicine Study Program, Faculty of Medicine and Nutrition, Bogor 16680, Indonesia
⁴ Division of Anatomy, Histology, and Embryology, School of Veterinary Medicine and Biomedical Sciences, IPB University, Bogor 16680, Indonesia

^* Corresponding author: This email address is being protected from spambots. You need JavaScript enabled to view it.

Abstract

Maternal protein deficiency can impair early postnatal growth, including skeletal development in offspring. This study evaluated the effects of pre-pregnancy maternal protein restriction and postnatal nutritional recovery, with or without spirulina, on postnatal bone growth in rat offspring. The experimental animal study was conducted at IPB University (2025). Pregnant rats (n=25) were assigned to five groups: normal, protein-restricted (pre-pregnancy), protein recovery, spirulina (400 mg/kg BW/day), and protein recovery + spirulina (until postnatal day 7). Femoral ossification was assessed at D-1 and D-8 using Alizarin Red S staining. Ossification length was quantified using ImageJ and analyzed using ANOVA. At D-1, femoral ossification length did not differ significantly among groups (p>0.05). By D-8, significant differences were observed (p<0.001). Offspring from protein-restricted dams exhibited the shortest femoral ossification length. Protein recovery during lactation significantly improved postnatal femoral growth, while spirulina supplementation alone resulted in partial improvement. The combination of protein recovery and spirulina produced femoral ossification lengths comparable to the normal protein group. These findings indicate that nutritional recovery, particularly when combined with spirulina supplementation, may help mitigate the adverse effects of maternal protein restriction on early skeletal development.