# Physiological Effects of Hydrolyzed Skim Milk and Probiotics on Osteoporosis Models
## 3 Results and Discussion
### Effect of Skin Milk and Probiotics on the Small Intestine  
According to our results, the consumption of both skim milk and probiotics had positive effects on weight loss and the prevention of osteoporosis. Several studies have reported that probiotics positively affect gut health [29]. Therefore, we analyzed the histological changes and lengths of the villi, crypts, and serosa. The lengths of the villi and crypts were significantly increased in the skim-milk and skim-milk-with-probiotic groups compared to those in the OVX and control groups (Figure 3A). In addition, the length of the serosa significantly increased in the skim-milk-with-probiotic group compared with that in the other groups (Figure 3B). A previous study revealed a significant decrease in intestinal villus height, density, and the villus height:crypt ratio in OVX rats compared with those in sham-operated rats, suggesting a compromised gut barrier [30]. However, our results did not reveal a significant difference in villus or crypt lengths between the control and OVX groups. Long villi are associated with an increased absorptive surface area and absorption capacity of the intestine [31]. In piglets, feeding with probiotics enhanced intestinal integrity, lengthened intestinal villi in the jejunum, and positively impacted the intestinal microbiota [32]. The addition of probiotics or synbiotics increased the villus-height-to-crypt-depth ratio and villus height in both the duodenum and ileum of broiler chickens [33]. The increase in villus height and villus-height-to-crypt-depth ratio was associated with an improvement in the growth performance of both synbiotics and probiotics. Our study revealed the positive effect of milk production and probiotics on OVX-induced osteoporosis in mice, indicating their beneficial effects in older women; this will have a different effect compared to the growth time window. We analyzed the gene expression of occludin and mucin 2 in the intestinal samples. The expression levels of occludin and mucin 2 significantly increased in the skim-milk and skim-milk-with-probiotic groups compared with those in the OVX groups (Figure 4A,B). Consistently, the immunostaining result showed that the mucin 2 positive area increased in the skim-milk and skim-milk-with-probiotic groups compared with that in the OVX group (Figure 4C). Quantification of the occludin protein also revealed a statistically significant increase in its level in both the skim-milk and skim-milk-with-probiotic groups compared with that in the OVX groups (Figure 4D). Figure 3: Analysis of the villi, crypt, and serosa lengths in the ovariectomy (OVX) and skim-milk-and-probiotic groups. Hematoxylin and eosin staining analysis of mice intestine from each experimental group (**A**) and the lengths of villi, crypt, and serosa. Lengths are presented as \(\upmu\)m (**B**). Data from the experimental group were compared with data from the OVX group. The results are expressed as mean \(\pm\) SD. * \(p<0.05\) and **\(p<0.01\)** were considered to indicate statistical significance. Data from the experimental group were compared with data from the OVX group. The intestinal epithelial tight junction (TJ) barrier controls the paracellular permeation of contents from the intestinal lumen into the intestinal tissue and systemic circulation [30]. Commensal bacteria and probiotics have been demonstrated to promote intestinal barrier integrity in vitro and in vivo. Probiotics were found to preserve the intestinal barrier in mouse models of colitis [34]. Mucin is a major component of the intestinal mucosal layer, plays an essential role in maintaining intestinal mucosal homeostasis, and has been detected in various organs, tissues, and body fluids. Mucin 2, the most abundant mucin family member in the small intestine and colon [35], is a key player during infection in the body. For instance, [PERSON] et al.