In general, in our experiment, different levels of dietary VAL were effective on the histology of quail liver and testis. With increasing VAL level, the highest diameter of liver cell nucleus and hepatocytes, were observed with the consumption of 1.05% and 1.15% of VAL in both female and male, and the percentage of these changes versus control were high, which means a decrease in the hepatocytes’ functional activity through the reduction of nuclear plasma ratio in higher levels of VAL in diet. At high levels of VAL consumption (1.05 and 1.15% VAL), bile duct hyperplasia and mild hepatosteatosis were also observed. Pathomorphological liver changes consist in dystrophic and necrotic changes in hepatocytes and bile duct epithelial hyperplasia (Zao et al., 2010). In broilers, hyperplasia and fibroplasia of the bile ducts are classified as nonspecific lesions and are associated with changes in hepatic metabolism that occur systematically after lesion of the liver parenchyma (Hochleithner et al., 2005). Altogether, the use of high levels of VAL (more than 0.85%) in a low protein diet can lead to histological damage in the liver of quails.
Liver hepatocytes are any of the polygonal epithelial parenchymatous cells of the liver that secrete bile. The BCAAs enhanced hepatocyte regeneration in a rat hepatectomy model (Kim et al., 2011) and were shown to increase the secretion of hepatocyte growth factor (Tomiya et al., 2004). BCAA has been associated with cell proliferation through activation of mechanistic target of rapamycin complex 1 (mTORC1). BCAAs are also shown to suppress oxidative stress by stimulating the expression of Peroxisome proliferator-activated receptor-gamma coactivator (PGC)-α or Sirtuin-1 (SIRT-1), or by activating the genes involved in antioxidant defenses (Tajiri & Shimizu, 2018). Those mechanisms could also contribute to promote hepatocyte proliferation (Tajiri & Shimizu, 2018). It is surprising to note that among BCAA, VAL stimulates the lymphogenesis of granular and agranular lymphocytes as well as increases natural killer cells (Kim et al., 2022). In this regard, the highest level of superoxide dismutase was observed by 0.1 and 0.2% VAL with 18% CP diet in quail, which may result in a superior antioxidative status (Hanafy & Attia., 2018). Based on our findings, it seems that the level of 0.85% VAL with 17% CP diet was suitable for improving the functional activity of the liver, without any negative histological effects. Since BCAA administration is used in the treatment of hepatic encephalopathy, the results of the present experiment can also be proposed to investigation in human.
In our experiment, differences between the two sexes were also observed. The diameter of liver cell nucleus and the diameter of hepatocytes of male were higher than the female quails. While the volume of sinusoids was no different among treatments, in female was higher than male. Sinusoids, small blood vessels between the radiating rows of hepatocytes, convey oxygen-rich hepatic arterial blood and nutrient-rich portal venous blood to the hepatocytes and eventually drain into the central vein, which drains into the hepatic vein. In general, in adulthood, there is a weight difference between two sexes of quails. Female quail birds attained higher weight than the male quails at 4-weeks (Khaldari et al., 2010). Body weight is one of the main factors that affects the birds’ maintenance requirements (NRC, 1994). Therefore, it is assumed that the maintenance requirements for males may also be lower than those of females. Thus, the use of similar diets for both sexes could exceed the nutrient needs for males (Retes et al., 2019). Therefore, the difference between the two sexes may be due to the difference in their body size and, as a result, the difference in their maintenance requirements and body metabolism.
With increasing VAL level in the diet, TDI and SI increased linearly. TDI is the percentage of seminiferous tubules containing at least three differentiated germ cells and SI is the number of spermatozoa per 100 spermatogenic cells. According to the Zamir-Nasta et al. (2021) a reduction in the percentage of spermatogenic tubules with TDI, tube replacement (RI) and negative spermatogenesis coefficient (SPI) in testicular tissue, disrupted the process of cell division, which in turn could disrupt the operation of spermatogenesis. Based on the fact that fertilization in avian species depends on the release of spermatozoa from sperm storage tubules (SST), active ciliary movement as well as structural integrity of glandular cells are important (Kimaro, 2016). Our study showed that the release of spermatogenic cells into the lumen of the seminiferous tubule was observed in treatment 1.15% VAL; and also, in the germinal epithelium of the groups treated with the levels of 0.95%, 1.05% and 1.15% VAL, vacuole formation was observed between the germ cells (Fig. 2). In fact, these vacuoles can indicate the loss of cell connections or the reduction of adhesive molecules such as cadherins and can be considered as one of the signs of apoptosis (MohamadGhasemi, et al., 2010). In the present study, the use of high levels of VAL (0.95, 1.05 and 1.15%) with 17% CP in the diet lead to histological damage in the testis of quails.
In our experiment, the effect of VAL level on testicular capsule diameter, epithelium height and Sertoli cell number at 42 d of age were not significant between treatments. Retes et al. (2021) showed that the Sertoli cell number at 60 days of age increased linearly with increasing dietary CP. Increases in the number of Sertoli cells and spermatogonia were not associated with increases in the sperm concentration of the birds (Retes et al., 2022). Also, they showed that the testis size, seminiferous tubular area, number of spermatogonia, and germinal epithelial height at 35 days of age increased linearly with increasing dietary CP, while the number of Leydig cells decreased. Hanafy & Attia (2018) indicated that dietary 18% CP with 0.2% VAL was suitable for breeder quails at 14–28 weeks of age. They showed that cloacal gland area and semen ejaculate volume of male quail were significantly improved by 18% CP level. Dietary protein concentration affected body and testicular development in male Japanese quails but did not affect reproductive efficiency (Retes et al., 2022). During the growth phase, rapid body development is directly related to reproductive organ development (Sarabia et al., 2013). Thus, the supply of amino acids during this phase may affect bird growth. Based on the obtained results, it could be concluded that the dietary protein requirements for breeder quails could be reduced during 1 to 42 d of age. Supplementation of low protein diet (17% CP) with extra VAL (0.85% of diet) can improve reproductive performance of male quails by increasing TDI and SI, without any negative effect on testicular histology.