The effects of dietary β-hydroxy-β-methylbutyrate (HMB) supplementation during gestation on bone, growth plate, and articular cartilage in newborns were determined. Thermal analysis of articular cartilage was performed to examine the structural changes in collagen. At day 70 of gestation, a total of 12 sows (Large White Polish breed, at the second parity) were randomly assigned to two groups, with each group receiving either a basal diet or the same diet supplemented with 0.2 g/day HMB until the 90th day. Maternal HMB supplementation enhanced body weight, bone length, and diameter in males. It also improved geometric and mechanical properties contributing to increased bone morphology and endurance. In turn, alteration of the length was only observed in females. The positive effects were mediated by increased serum concentrations of insulin-like growth factor-1 and leptin. HMB-treatment enhanced the concentration of FSH, LH, estradiol, and testosterone. Serum TAP was enhanced by the HMB-treatment by 34% in females and 138% in males. Beneficial effects of the HMB-treatment on trabecular bone and content of proteoglycans in articular cartilage were shown. The HMB-treatment significantly changed the collagen structure in cartilages, especially in the females, which was demonstrated by the PSR analysis. Differences between the HMB-supplemented and the control females in the calorimetric peak temperatures were presumably related to different collagen fibril density in the articular cartilage structure. In summary, maternal HMB supplementation in the mid-gestation period significantly improved general growth and mechanical endurance of long bones by the influence on the somatotropic and pituitary-gonadal axes in the offspring.

This study was designed to determine follicular atresia in the newborn and the prepubertal spiny mouse. We analyzed the processes of follicle loss using classical markers of apoptosis (TUNEL reaction, active caspase-3) and autophagy (Lamp1). Numerous small clear vacuoles and autophagosomes as well as strong Lamp1 staining were observed in dying oocytes of all follicle types, especially of the primordial and primary ones. Active caspase 3 and the TUNEL reaction were detected only in the granulosa cells of large secondary and antral follicles. The expression of apoptosis and autophagy markers was also changing during the prepubertal period. Western blot analysis indicated that at the moment of birth, females undergo an increased rate of follicular atresia mediated by autophagy, while apoptosis is the dominant form of ovarian atresia in consecutive postnatal days. On the basis of these observations, we concluded that apoptosis and autophagy are involved in follicular atresia and these processes are cell and developmental stage-specific.

This study was focused on analyzing the effects of dietary inclusion of raw chickpea seed as a replacement of soybean meal as a primary protein source on bone structure in broiler chickens. Broiler chickens (n = 160) received in their diet either soybean meal (SBM) or raw chickpea seeds (CPS) as a primary protein source throughout the whole rearing period (n = 80 in each group). On the 42th day randomly selected chickens from each group (n = 8) were slaughtered. Collected tibiotarsus were subjected to examination of the biomechanical characteristics of bone mid-diaphysis, microstructure of the growth plate and articular cartilages; the analysis of mineral content and crystallinity of mineral phase, and the measurements of thermal stability of collagen in hyaline cartilage were also carried out. The inclusion of chickpea seeds resulted in increase of bone osteometric parameters (weight, length and mid-diaphysis cross-sectional area) and mechanical endurance (yield load, ultimate load, stiffness, Young modulus). However, when loads were adjusted to bone shape (yield and ultimate stress) both groups did not differ. Mineral density determined by means of densitometric measurements did not differ between groups, however the detailed analysis revealed the differences in the macro- and microelements composition. The results of FT-IR and XRD analyses showed no effect of diet type on mineral phase crystallinity and hydroxyapatite nanocrystallites size. In trabecular bone, the increase of real bone volume (BV/TV) and number of trabeculae was observed in the CPS group. Total thickness of articular cartilage was the same in both groups, save the transitional zone, which was thicker in the SBM group. The total thickness of the growth plate cartilage was significantly increased in the CPS group. The area of the most intense presence of proteoglycans was wider in the SBM group. The structural analysis of fibrous components of bone revealed the increase of fraction of thin, immature collagen content in articular cartilage, trabeculae and compact bone in the CPS group. The dietary inclusion of CPS affected the thermal stability of collagen, as decrease of net denaturation enthalpy was observed. This study showed a beneficial effect of CPS on the skeletal development, improving the overall bone development and the microarchitecture of cancellous bone. It suggests that CPS can be a promising replacement for SBM in broilers feeding in the aspect of animal welfare related to the development of the skeletal system.

The aim of the study was to investigate the effect of 2-oxoglutaric acid (2-Ox) supplementation (a precursor of glutamine and hydroxyproline, the most abundant amino acid of collagen) on cartilage and bone in pigs after fundectomy. Pigs at the age of forty days were subjected to fundectomy and divided into two groups depending on 2-Ox supplementation (at the daily dosage of 0.4 g/kg of body weight). Other pigs were sham operated. Pigs were euthanized at the age of eight months. An analysis of the morphometry of trabeculae, growth plate and articular cartilage in fundectomy-induced osteopenic bone was performed. Moreover, the levels of expression of osteocalcin, osteopontin and osteoprotegerin in trabecular bone and osteocalcin in articular cartilage were evaluated. Articular cartilage was thinnest in fundectomized pigs and thickest in 2-Ox-supplemented animals after fundectomy. Moreover, 2-Ox supplementation after fundectomy enhanced the total thickness of the growth plate and trabeculae in fundectomized pigs. The most evident signal for osteocalcin and osteoprotegerin in trabecular bone was in sham-operated and 2-Ox-supplemented pigs; a low reaction was observed in the fundectomized group. Additionally, as a long-term postoperative consequence, a change was observed in the expression of osteocalcin in articular cartilage. It seems that 2-Ox is suitable for use in preventing the negative effects of fundectomy on cancellous bone and cartilage.

The polysaccharide-protein complex (PPC) isolated from metabolites of gut bacteria Raoultella ornithinolytica from Dendrobaena veneta earthworms exhibits activity against Candida albicans, in breast ductal carcinoma (line T47D) and in the endometrioid ovarian cancer line (TOV-112D) in vitro. The action against C. albicans was analyzed using light, SEM, TEM, and AFM microscopes. The changes observed indicated two directions of the action of the complex, that is, disturbance of metabolic activity and cell wall damage. The PPC is an adhesion-promoting complex inducing death of C. albicans cells by necrosis. Owing to its significant effect on C. albicans, the complex is a promising source of antifungal compounds. The PPC showed a minimal cytotoxic effect against human skin fibroblasts; however, the cytotoxicity against the T47D line was determined at 20% and 15% against the TOV-112D line. The action of the PPC against the T47D line exerted a cytopathic effect, whereas in the TOV-112D line, it caused a reduction in the cell number. The PPC induced death of tumor cells by apoptosis and necrosis. In view of the negligible cytotoxicity on fibroblasts, the PPC will be subjected to chemical modifications to increase its antitumor activity for prospective medical applications.